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The pivotal role of AMP-activated protein kinase in the regulation of fatty acid metabolism in skeletal muscle

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The pivotal role of AMP-activated protein kinase in the regulation of fatty acid metabolism in skeletal muscle

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THE PIVOTAL ROLE OF AMP-ACTIVATED PROTEIN KINASE IN THE
REGULATION OF FATTY ACID METABOLISM IN SKELETAL MUSCLE

by

Marcia Jeanine Abbott

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(INTEGRATIVE AND EVOLUTIONARY BIOLOGY)

August 2010

Copyright 2010 Marcia Jeanine Abbott
ii

ACKNOWLEDGEMENTS

I would like to thank the following people for their help and support in completing this
degree:

Dr. Lorraine Turcotte for her infinite guidance, support, and her assistance in helping me
to become a successful scientist; James T. Schurman for his unending patience and for
sticking by me when times were tough; my family for all of their love and support.
iii

TABLE OF CONTENTS

Acknowledgements ii
List of Tables iv
List of Figures v
Abstract viii
I. Introduction 1
II. Background and Significance 4

III. Experiment 1: 8
CaMKK is an Upstream Signal of AMP-activated Protein Kinase
in Regulation of Substrate Metabolism in Contracting Skeletal Muscle

IV. Experiment 2: 34
AMPKα2 deficiency uncovers time-dependency in the regulation of
contraction-induced palmitate and glucose uptake in mouse muscle
V. Experiment 3: 63
AMPKα2 is an Essential Signal in the Regulation of Insulin-Stimulated
Fatty Acid Uptake in Control-Fed and High Fat-Fed Mice

VI. Experiment 4: 86
Exercise training-induced restoration of insulin sensitivity in skeletal
muscle is dependent on AMPKα2 activity in high fat fed mice

VII. Summary 112

VIII. Conclusions 116

References 118

Appendix A: 135
Overall Conclusions from Experiment 1 and Experiment 2

Appendix B: 136
Overall Conclusions from Experiment 3 and Experiment 4

Appendix C: 137
Power Point Presentation

iv

LIST OF TABLES

Table 1: Perfusion characteristics of rat hindlimbs perfused 27
with caffeine, AICAR or during moderate intensity
muscle contraction

Table 2: Effect of AMPKα2 DN transgene on muscle protein 55
expression

Table 3: Effect of AMPKα2 DN transgene on hindlimb perfusion 56
characteristics during rest, caffeine treatment, or muscle
contraction

Table 4: Changes in body weight, blood glucose, and plasma 78
insulin

Table 5: Perfusion characteristics of hindlimbs 79

Table 6: Body weight, blood glucose, and plasma insulin in 104
control diet mice

Table 7: Body weight, blood glucose, and plasma insulin in 105
high fat diet mice

Table 8: Perfusion characteristics of hindlimbs 106

v

LIST OF FIGURES

Figure 1: Proposed role of AMPK in the regulation of 3
fatty acid metabolism during both acute and chronic experiments

Figure 2: Effect of CaMKK inhibition on glucose uptake, palmitate 28
uptake, and oxidation in perfused rat hindlimbs during caffeine
or AICAR treatment, or moderate intensity muscle contraction

Figure 3: The effect of STO-609 on CaMKK  activity in perfused 29
rat hindlimbs during caffeine or AICAR treatment, or moderate
intensity muscle contraction

Figure 4: Effect of CaMKK inhibition on AMPKα1 and AMPKα2 30
activity in perfused rat hindlimbs during caffeine or AICAR
treatment, or moderate intensity muscle contraction

Figure 5: Effect of CaMKK inhibition on ACC-Ser
79
31
phosphorylation and total ACC expression in perfused rat
hindlimbs during caffeine or AICAR treatment, or moderate
intensity muscle contraction

Figure 6: Effect of CaMKK inhibition on plasma membrane (PM) 32
and total content of CD36 and plasma membrane content of
FABPpm in perfused rat hindlimbs during caffeine or AICAR
treatment or moderate intensity muscle contraction

Figure 7: Effect of CaMKK inhibition on plasma membrane (PM) 33
and total content of GLUT4 in perfused rat hindlimbs during
caffeine or AICAR treatment or moderate intensity muscle
contraction

Figure 8: Effect of AMPKα2DN transgene on skeletal muscle 57
protein expression of multiple signaling intermediates in rest
groups from a mixed gastrocnemius-soleus-plantaris muscle
preparation

Figure 9: Mean force production (g) over the 20 minute contraction 58
period in WT (open circles) and DN (closed circles) hindlimb
muscles

vi

Figure 10: Effect of AMPKα2 DN transgene on glycogen content in 59
a mixed gastrocnemius-soleus-plantaris muscle preparation
(A), time effects for glucose uptake in muscle contraction groups
(B) and caffeine groups (D), and mean glucose uptake (C) in
perfused mouse hindlimbs during caffeine treatment or
moderate intensity muscle contraction

Figure 11: Effect of AMPKα2 DN transgene on time effects for 60
palmitate uptake in muscle contraction groups (A), mean
palmitate uptake (B), time effects for palmitate oxidation (C),
time effects for palmitate uptake in caffeine groups (D), mean
palmitate oxidation (E), and time effects for palmitate oxidation
in caffeine groups in perfused mouse hindlimbs during caffeine
treatment or moderate intensity muscle contraction

Figure 12: The effect of AMPKα2 DN transgene on AMPKα2 61
activity (A), AMPKα1 activity (B), ACC phosphorylation
(C), and SIRT1 expression and activity (D) in perfused mouse
hindlimbs during caffeine treatment or moderate intensity muscle
contraction

Figure 13: The effect of AMPKα2 DN transgene on CaMKI (A), 62
ERK1/2 (B), AS160/TBC1D4 (C), and AS150/TBC1D1
(D) protein expression and phosphorylation state in perfused
mouse hindlimbs during caffeine treatment or moderate intensity
muscle contraction

Figure 14: Effect of AMPKα2 DN transgene and insulin on glucose 80
uptake (A and B) and palmitate uptake (C and D) in perfused
hindlimbs of CD and HFD mice

Figure 15: Effect of AMPKα2 DN transgene and insulin on 81
palmitate oxidation in perfused hindlimbs of CD and HFD mice

Figure 16: Effect of AMPKα2 DN transgene on AMPKα2 (A) and 82
AMPKα1 (B) activity on non-perfused basal hindlimbs muscle
of CD and HFD mice

Figure 17: Effect of AMPKα2 DN transgene on SIRT1 expression 83
(A) from non-perfused basal hindlimb muscle of CD and HFD
(B) mice and PTP1b expression in CD mice (C) and HFD mice
(D) in non-perfused basal hindlimb muscle

Figure 18: Effect of AMPKα2 DN transgene on PGC1α expression 84
(A), CPT1 expression (B), and CD36 expression (C) from
non-perfused basal hindlimbs muscle of CD and HFD mice
vii

Figure 19: Effect of AMPKα2 DN transgene on IL6 (A) and 85
TNFα (B) expression in skeletal muscle from non-perfused
basal hindlimbs muscle of CD and HFD mice

Figure 20: Effect of AMPKα2 DN transgene on time spent 107
running in control diet mice (A) and high fat diet mice (B),
distance ran in control diet mice (C) and high fat diet mice (D),
and citrate synthase activity in sedentary and trained mice under
control diet conditions (E) and sedentary and trained mice under
high fat diet conditions (F)

Figure 21: Effect of AMPKα2 DN transgene and training on 108
AMPKα1 activity in control diet mice (A) and high fat diet
mice (B), AMPKα2 activity in control diet mice (C) and high
fat diet mice (D), and phosphorylation of ACC in control diet
mice (E) and high fat diet mice (F) in perfused hindlimbs

Figure 22: Effect of AMPKα2 DN transgene and training on glucose 109
uptake in control diet mice (A) and high fat diet mice (B),
palmitate uptake in control diet mice (C) and high fat diet mice
(D), and palmitate oxidation in control diet mice (E) and high fat
diet mice (F) in perfused hindlimbs

Figure 23: Effect of AMPKα2 DN transgene and training on 110
phosphorylated ERK1/2 in control diet mice (A) and high fat
diet mice (B), and on phosphorylated JNK1/2 in control diet
mice (C) and high fat diet mice (D) in perfused hindlimbs

Figure 24: Effect of AMPKα2 DN transgene and training on CPT1 111
expression in control diet mice (A) and high fat diet mice
(B), and CD36 expression in control diet mice (C) and high fat
diet mice (D) in perfused hindlimbs

viii

ABSTRACT

The contents of this dissertation contain four experiments aimed at determining
the role of AMP-activated protein kinase (AMPK) in the regulation of fatty acid (FA)
metabolism in skeletal muscle.
The findings in the first study suggest a link between Ca
2+
signaling, via CaMKK,
and AMPKα2 activation in the regulation of glucose uptake and FA uptake and oxidation
in perfused rat skeletal muscle contracting at moderate intensity. Additionally, the data
obtained with the AICAR groups provide further evidence for the presence of a Ca
2+
-
independent AMPK-dependent signaling cascade in the regulation of glucose uptake and
FA uptake and oxidation. Furthermore, the GLUT4 and CD36 data indicate that the
translocation of these transport proteins may be regulated via Ca
2+
-dependent and Ca
2+
-
independent signaling. Together, these results suggest that during muscle contraction,
AMP- and Ca
2+
-induced signaling cascades regulate muscle metabolism in part by
converging at AMPK.
The data from the second study provide further evidence for the involvement of
AMPKα2 in the regulation of substrate use during muscle contraction. Additionally, the
results of this study suggest that while AMPKα2 is activated by caffeine-induced
increases in intracellular [Ca
2+
], this activation is not essential to observe increases in
substrate uptake and FA oxidation during caffeine treatment. These data also suggest a
role for AMPK-independent Ca
2+
-dependent signaling in the regulation of substrate
metabolism in skeletal muscle. However, physiologically active AMPKα2 appears to be
necessary to record time-dependent changes in FA uptake and glucose uptake, that match
levels of wild type mice, in contracting skeletal muscle. Indeed, one of the more
ix

interesting findings of this study is that AMPKα2 deficiency led to a reciprocal switch in
FA and glucose uptake during the 20-min contraction protocol.
The data from the third study suggest an involvement of AMPKα2 in the
regulation of insulin stimulated fatty acid metabolism both in control diet (CD) and high
fat diet (HFD) mice. In contrast, it does not appear that AMPKα2 is necessary in the
regulation of basal and/or insulin stimulated glucose uptake in CD and HFD mice.
Additionally, these data suggest that AMPKα2 may be involved in the expression and
activation of proteins shown to be associated with the regulation of substrate metabolism
and insulin signaling, such as CPT1, Akt, SIRT1, and PTP1b. Finally, a role for
AMPKα2, in skeletal muscle, in the regulation of IL6 and TNFα protein expression in
adipose tissue was established, suggesting the existence of “cross-tissue talk.”
The data from the final study provide evidence for the involvement of AMPKα2
activity in the regulation of fatty acid metabolism following exercise training in control
fed mice. In contrast, it does not appear that AMPKα2 activity is necessary for the
alterations in fatty acid metabolism that occur with exercise training in high fat-fed mice.
However, it does appear that AMPKα2 activity is necessary for the effects of training,
while under high fat-fed conditions, on glucose uptake. Additionally, these data suggest
that AMPKα2 may be involved in the activation of ERK1/2 but not JNK1/2 in high fat-
fed mice under sedentary and endurance training conditions.
Taken together, the results from these four experiments solidify the role of AMPK
signaling in the regulation of FA metabolism in skeletal muscle. Additionally, it was
shown that AMPK may have alternative upstream activators, such as CaMKK during
caffeine treatment. Further, AMPK signaling does appear to be involved in the
x

development of insulin resistance under high fat feeding conditions in mice and AMPK
appears to be essential in the exercise training effects that occur while on a high fat diet
in mice.

1

I. INTRODUCTION

Obesity has been linked to numerous diseases and disorders, including type 2
diabetes (40) resulting in health care costs upwards of $117 billion a year (8). A
precursor and a detrimental indicator of type II diabetes is insulin resistance in the
skeletal muscle (9; 29). While the mechanisms leading to the development of insulin
resistance have not been completely identified, there is evidence that its progression is
linked to alterations in lipid metabolism leading to the accumulation of lipids and lipid
intermediates (1; 25; 71). Furthermore insulin resistance has been shown to be associated
with an inflammatory response associated with increased circulating fatty acids and or
obesity (11; 88). Therefore it is of tremendous importance to determine the mechanisms
by which lipids are metabolized in the skeletal muscle in order to identify alterations due
to insulin resistance. There are multiple signaling pathways that have been implicated in
the regulation of lipid metabolism; however their relative role remains unknown. An
essential signaling molecule, AMP-activated protein kinase (AMPK), has been identified
and shown to be involved in the regulation of fatty acid (FA) metabolism in skeletal
muscle, during acute stimulation, such as exercise or pharmacological activation (82;
139). Indeed, it has been determined that AMPK is acutely activated by muscle
contraction (48) and this has been associated with increases in FA uptake and oxidation
(12; 48; 52; 82). However the upstream regulators of contraction-induced AMPK
activation have yet to be fully defined. Since intracellular calcium levels are known to
increase with muscle contraction, it has been suggested that calcium-induced signaling
intermediates may regulate AMPK activation upstream of AMPK. Similarly, the long
term effects of repeated AMPK activation or inhibition, induced by physiological stimuli

2

such as high fat diet or exercise training, on the activity and or gene expression of AMPK
in skeletal muscle and the relative impact on FA metabolism have not been established.
Furthermore, if genetic manipulations are utilized to inactivate AMPK it is not clear what
effects chronic high fat feeding or exercise training would have on FA metabolism and
insulin sensitivity in skeletal muscle. Therefore the goal of the proposed project is to
determine the pivotal role of AMPK in the regulation of FA metabolism. Specifically it
is essential to determine the importance of AMPK in calcium-mediated signaling, chronic
exercise training and the development of insulin resistance via high fat feeding. AMPK
will be altered pharmacologically and genetically in acute and chronic experiments in
order to investigate the role it may play in the regulation of FA metabolism in skeletal
muscle.
In order to better define the pathways regulating FA metabolism in skeletal
muscle, the following Specific Aims are proposed:
To determine, in acute experiments, if Ca
2+
signaling is involved in the regulation of
FA metabolism and if Ca
2+
signaling regulates FA metabolism through AMPK activation
To determine, in chronic experiments, the role of AMPK in the development of insulin
resistance, the role of AMPK in exercise training, and if AMPK is necessary for the
protective effects of endurance training in the development of insulin resistance.

3

AMPK
FA Metabolism
Acute
Muscle
Contraction
Chronic
FA Uptake
FA Oxidation
Gene
Expression
Ca
2+
?
Ca
2+
?
Exercise Training High Fat Diet
Inflammation
?

Figure 1. Proposed role of AMPK in the regulation of fatty acid metabolism during both acute
and chronic experiments

4

II. BACKGROUND AND SIGNIFICANCE

It has become clear that obesity is a growing and serious epidemic in our modern
society. Obesity has been linked to numerous diseases and disorders including type II
diabetes (9). In turn, type II diabetes is known to be caused in part by alterations in fatty
acid and glucose metabolism (35). It is of extreme importance to further our knowledge
in the area of insulin resistance, as a precursor to type II diabetes, in order to provide
possible therapies to individual’s suffering from this disorder. Fatty acids (FA) are an
important fuel source for skeletal muscle at rest and during muscle contraction.
Dysregulation of FA metabolism may lead to the development of insulin resistance. In
particular, accumulation of lipid metabolites and intermediates, such as fatty acyl CoA,
diacylgylcerides and ceramides, have been shown to contribute to the development of
insulin resistance (5; 11; 50; 65). The inflammatory response that arises as a result of a
high fat diet or lipid infusion studies have also been implicated in the development of
insulin resistance (10; 99). It is clear that insulin resistance is a multifactorial process
that requires further investigation. Insulin resistance results in an increase in FA
transport into the muscle cell and an increase in FA oxidation when compared to an
insulin sensitive muscle cell (130). It is not clear if the development of insulin resistance
is due solely to an increase in lipid supply or alterations in the molecular signaling
mechanisms that regulate lipid metabolism within the skeletal muscle. Of particular
interest with regards to lipid utilization is the regulatory mechanism by which FA are
taken up and oxidized in the skeletal muscle. It has been shown that muscle contractions
result in an increase in the use of FA as a fuel source (100). It was a previous belief that
FA entered the muscle cell by diffusing through the lipid bilayer of the sarcolemmal

5

membranes of the skeletal muscle (41). Data has shown that CD36, FATP and FABP
PM

are Transport proteins responsible for the transport of FA into the skeletal muscle from
the interstitial space (4) and during muscle contraction CD36 has been shown to
translocate from an intracellular pool to the plasma membrane (13). The signaling
molecules that promote the translocation of CD36 to the plasma membrane, resulting in
FA uptake, are not entirely known.
Recently there has been an increased knowledge of two specific signaling
pathways that may play a role in the regulation of FA metabolism in skeletal muscle:
AMP-activated protein kinase pathway (AMPK) and Ca
2+
-calmodulin dependent (CaM)
pathways. These pathways have been implicated in regulating substrate uptake and
oxidation in skeletal muscle (46; 60). AMPK has been proposed to be a master fuel
sensor of the cell (137). Once AMPK is phosphorylated, acetyl-CoA carboxylase (ACC)
is in turn phosphorylated and its activity is decreased (96), resulting in an increase in FA
oxidation via a decrease in malonyl CoA concentrations (111; 136). However, a recent
study in our lab has shown that AMPK may not be the predominate signaling
intermediate regulating FA uptake and oxidation during muscle contraction (96).
Therefore, additional signals must exist during muscle contraction to regulate the increase
in FA uptake and oxidation that is measured with muscle contraction. Ca
2+
-calmodulin-
dependent protein kinase (CaMKII) has recently been identified as an additional
signaling molecule that is activated as a result of muscle contraction (26; 102; 115).
Interestingly, recent data from our lab has shown that CaMKII is in fact involved in the
regulation of FA uptake and oxidation during increased intracellular calcium levels,
stimulated by caffeine, and during moderate intensity muscle contraction (95).

6

Additionally, Ca
2+
-calmodulin-dependent protein kinase kinase (CaMKK) has
been shown to activate AMPK (47; 60; 143) and pharmacological inhibition of CaMKK
nearly eliminated AMPK activity (143). However the regulatory role of CaMKK in FA
uptake and oxidation is not clear. Therefore, it is of importance to further study the role
of the CaMKK signaling pathway and if it acts solely through activation of AMPK in the
regulation of FA uptake and oxidation.
Along with acute regulation of FA metabolism; accumulation of FA intermediates
within the muscle cell have proven to contribute to insulin resistance following high fat
feeding (11; 25). High fat feeding has become an increasingly utilized tool to induce
insulin resistance in rodents (19; 140). There is contradicting data as to the implications
of high fat feeding on the content and activity of particular enzymes associated with FA
metabolism. It has been shown that high fat feeding in rats for four months results in a
decrease in expression of AMPKα2 as well as a decrease in basal levels of
phosphorylated AMPKα when compared to normal diet rats (74). Therefore it can be
postulated that AMPK is in fact impaired as a result of high fat feeding which may lead
to detrimental dysregulation of fuel sources. In contrast an additional study found that
there was not a decrease in total AMPKα2 content following 12 weeks of high fat feeding
in mice (80). Furthermore this study found that AICAR, an AMP analogue known to
activate AMPK, was able to phosphorylate AMPKα following the high fat feeding (80).
Therefore it is not clear what regulatory role, if any, AMPK plays in regulating fuel
metabolism following high fat feeding. Additionally it is not known if enzymes involved
in acute activation of AMPK are affected by high fat feeding, such as CaMKK or
CaMKII.

7

It has also been demonstrated that exercise training may have positive effects in
regards to treating insulin resistance (16; 17). Voluntary wheel running in a mouse
model has proven to be an effective mode of exercise training (28). However it has yet to
be determined what effects voluntary wheel running may have on the expression and
activity of AMPK in regards to acute FA uptake and oxidation (61). Therefore by
combining exercise training with high fat diet it will be possible to determine the central
role of AMPK in the regulation of insulin resistance. Insight into these specific signaling
pathways may provide information in regards to the mechanism by which FA is brought
into the skeletal muscle and utilized for fuel during muscle contraction.

8

III. EXPERIMENT 1

CaMKK is an Upstream Signal of AMP-activated Protein Kinase in Regulation of
Substrate Metabolism in Contracting Skeletal Muscle
1

CHAPTER ABSTRACT

There are multiple signals that have been shown to be involved in the regulation
of fatty acid (FA) and glucose metabolism in contracting skeletal muscle. The purpose

of
this study was to determine whether a Ca
2+
-stimulated kinase, CaMKK, is involved in

the
regulation of contraction-induced substrate metabolism

and whether it does so in an

AMPK-dependent manner. Rat hindlimbs were perfused either at rest (n=16), 3 mM
caffeine (n=15), 2 mM AICAR (n=16), or during moderate intensity muscle contraction
(MC; n=14), and with or without 5µM STO-609, a CaMKK inhibitor. FA uptake and
oxidation increased (P<0.05) 64 and 71% by caffeine, 42 and 93% by AICAR and 65 and
143% by MC. STO-609 abolished (P<0.05) caffeine- and MC-induced FA uptake and
oxidation, but had no effect with AICAR treatment. Glucose uptake increased (P<0.05)
104% by caffeine, 85% by AICAR, and 130% by MC and STO-609 prevented the
increase in glucose uptake in the caffeine and muscle contraction groups. CaMKKβ
activity increased (P<0.05) 113% by caffeine treatment and 145% by MC, but was not
affected by AICAR treatment. STO-609 prevented the caffeine- and MC-induced
increase in CaMKKβ activity. Caffeine, AICAR and MC increased (P<0.05) AMPKα2
activity by 295%, 11-fold and 7-fold, but did not affect AMPKα1

activity. STO-609
decreased (P<0.05).

1
Paper published in American Journal of Physiology Regulatory, Integrative and
Comparative Physiology-297: R1724-R1732, 2009

9

AMPKα2 activity by 60 and 61% by caffeine treatment and MC, but did not
affect AICAR-induced activity. Plasma membrane transport protein content of CD36 and
GLUT4 increased (P<0.05) with caffeine, AICAR, and MC and STO-609 prevented the
caffeine- and MC-induced increases in protein content. These results show the
importance of Ca
2+
-dependent signaling via CaMKK activation in the regulation of
substrate uptake and FA oxidation in contracting rat skeletal muscle. Furthermore, our
data agree with the notion that CaMKK is an upstream kinase of AMPK in the regulation
of substrate metabolism in skeletal muscle.
CHAPTER INTRODUCTION
Obesity is a growing and serious epidemic in our modern society which leads to
the development of multiple detrimental pathologies. Sustained obesity has been linked
to numerous diseases and disorders including Type 2 diabetes (9). In turn, Type 2
diabetes is known to be caused in part by the development of insulin resistance which is
due in part to alterations in fatty acid (FA) metabolism (5; 11). FA are an important fuel
source for skeletal muscle at rest and during muscle contraction when FA uptake and
oxidation rates can increase up to 3-4 times the basal rates (124; 129). Evidence suggests
that multiple cellular signals regulate this increase in FA metabolism with muscle
contraction, including the AMP-activated protein kinase (AMPK) signaling cascade (13;
94; 95; 138). Although strong evidence indicates that AMPK is a key signaling
intermediate that regulates changes in substrate use in contracting skeletal muscle (46;
73; 142; 146), data also show that AMPK may not be the sole signal mediating
contraction-induced changes in substrate uptake and FA oxidation (95; 96). In addition,
it has become apparent that there may be multiple upstream regulators of AMPK in the

10

regulation of substrate metabolism (54; 95; 144). Mounting evidence suggests that
increases in intracellular [Ca
2+
] may play a key role in the regulation of substrate
utilization in skeletal muscle and that this may occur in part via AMPK activation (20;
81; 135; 141; 146; 148).
Ca
2+
-calmodulin-dependent protein kinase kinase (CaMKK) activates AMPK in
vitro in cells lacking the known upstream kinase of AMPK, LKB1, and in yeast (47; 53;
55; 143). Furthermore, pharmacological inhibition of CaMKK nearly eliminated AMPK
activity in mouse embryonic fibroblasts from LKB1 ‾/‾ mice (143). Despite this strong
evidence that CaMKK acts as an upstream kinase to AMPK in cells, findings in skeletal
muscle are conflicting (60; 81; 141). Furthermore, the role of CaMKK in the regulation
of FA uptake and oxidation in skeletal muscle during rest and contraction is unknown. To
improve treatment modalities of metabolic disorders, such as Type 2 diabetes, it is
essential that we fully understand the cellular mechanisms that regulate FA metabolism in
skeletal muscle.
Caffeine has been utilized by us and others to study the effects of Ca
2+
signaling
on skeletal muscle fuel metabolism (59; 91; 95; 145; 148). At low concentrations (3
mM) caffeine has been determined to stimulate Ca
2+
release from the sarcoplasmic
reticulum without establishing muscle contraction (92; 148). This release of Ca
2+

provides a tool to study Ca
2+
-dependent signaling without the effects of muscle
contraction. Likewise, AICAR may be utilized in a similar manner to activate AMPK
without employing muscle contraction. AICAR is an AMP analogue and has been used
extensively to study the AMP-dependent activation of AMPK in various tissues and
models (27; 82). Use of these pharmacological activators of muscle metabolism may

11

provide insight into the regulation of substrate use in skeletal muscle during muscle
contraction. Therefore, the purpose

of this study was to determine whether CaMKK
activation is one of the signaling mechanisms that regulates the contraction-induced
increase in glucose uptake, FA uptake and FA oxidation

and, if it is involved, whether
AMPK is a downstream intermediate of CaMKK-induced signaling. We hypothesized
that glucose uptake, FA uptake and FA oxidation in skeletal muscle would increase with
caffeine, AICAR, and with muscle contractions and that this would be accompanied by
increases in CaMKK and AMPK activity. We further

hypothesized that pharmacological
inhibition of CaMKK would partially decrease Ca
2+
-induced AMPK activation and
substrate uptake and FA oxidation.
MATERIALS AND METHODS
Animal Preparation. Male Wistar rats (285-365 g, 2-3 months old) were kept on a
12:12-h light-dark cycle and housed in pairs. Standard rat chow and water were provided
ad libitum to the rats. Animals were randomly divided into four experimental groups:
rest (R, n=16), caffeine (C, n=14), AICAR (A, n=16) and muscle contraction (MC, n=14)
of moderate intensity (64; 94). Each group of animals was further divided into treatment
with dimethyl sulfoxide (DMSO) as control vehicle or STO-609 (Calbiochem,
Gibbstown, NJ), an inhibitor of CaMKK (120), dissolved in DMSO. All procedures for
the present study were approved by the Institutional Animal Care and Use Committee at
the University of Southern California.
Hindquarter Perfusion. On the day of the experiment, rats were anesthetized by the
administration of an intraperitoneal injection of ketamine/xylazine cocktail (80 and 12
mg/kg body weight, respectively). Surgical preparation for the hindquarter perfusion

12

was performed as previously described (105; 129; 130). Before placement of the
perfusion catheters, 150 IU of heparin was injected into the inferior vena cava. The rats
were then euthanized with an intracardial injection of pentobarbital sodium (0.4 mg/g
body weight), and catheters were immediately inserted into the descending aorta and
vena cava. The prepared rat was then placed in a perfusion apparatus and the left iliac
vessels were tied off and a clamp was placed tightly around the proximal portion of the
leg to prevent bleeding (130).
The perfusate consisted of Krebs-Henseleit solution, 5% bovine serum albumin
(Millipore, Billerica, MA), 600 M albumin-bound palmitate, 8 µCi of albumin-bound
[1-
14
C] palmitate, 8 mM glucose, either 3 mM caffeine (Sigma, St. Louis, MO) or 2 mM
5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) (Sigma, St. Louis,
MO) dissolved in Krebs-Henseleit buffer in the C and A groups, respectively. The
hindlimbs were perfused with or without 5 µM STO-609, dissolved in DMSO. At this
concentration, STO-609 has been shown to inhibit both CaMKK isoforms and to have
limited effects on alternate kinases (47; 60; 120). The perfusate was kept at 37°C and
was continuously gassed with a mixture of 95% O
2
-5% CO
2
with arterial pH levels
between 7.2-7.7 and arterial P
O2
and P
CO2
values were between 235-450 and 29-54
mmHg, respectively. Perfusion pressures were not affected by treatment with STO-609
and averaged 115.5 ± 9.9, 98.8 ± 6.0, 115.2 ± 9.0, and 131.9 ± 10.4 mmHg in the R, C,
A, and MC groups, respectively (P > 0.05).
The first 25 ml of perfusate that passed through the right hindquarter was
discarded whereupon the preparation was equilibrated for 20 min. Perfusion flow rate
was maintained at 15 ml/min for all groups (average: 0.80 ± 0.01 ml/min/g of perfused

13

muscle). Arterial and venous samples were taken following the 20 min equilibration
period at 5, 10, 15, and 20 min for further analyses. Following the completion of the 20
min experimental perfusion period, the gastrocnemius-soleus-plantaris muscle group was
freeze-clamped in situ with pre-cooled aluminum clamps, removed and stored in liquid
N
2
for further analyses. In the MC group, the right tibiopatellar ligament was stabilized
for the recording of force production and a hook electrode was placed around the isolated
sciatic nerve. The gastrocnemius-soleus-plantaris muscle group length was adjusted at
the initiation of electrical stimulation to obtain maximal active tension. A S48 Grass
stimulator (Grass Telefactor, West Warwick, RI) was used to induce isometric muscle
contractions via delivery of 15-V trains of 100 Hz lasting 50 ms with impulse duration of
1 ms. This moderate intensity protocol has been shown to maximize FA metabolism
(94). A modular chart recorder (Cole Parmer, Vernon Hills, IL) was used to measure the
tension developed by the gastrocnemius-soleus-plantaris muscle group during the 20 min
muscle stimulation protocol. The decrease in tension development over the stimulation
period was used as an indicator of performance.
Blood sample analyses. Plasma samples collected during the perfusion were analyzed to
determine FA, glucose, and lactate concentrations as well as radioactive [
14
C]FA and
14
CO
2
contents. A WAKO NEFA HC kit (WAKO Chemicals, Richmond, VA) was used
to measure plasma FA concentrations spectrophotometrically. An YSI-1500 (Yellow
Springs Instruments, Yellow Springs, OH) analyzer was used to measure glucose and
lactate concentrations in the collected plasma samples. Plasma [
14
C]FA and
14
CO
2
radioactivities were measured as previously described (125; 127; 129). P
CO2
, P
O2
, and pH
were determined by utilizing an ABL-5 analyzer (Radiometer America, Westlake, OH).

14

Muscle sample preparation. For western blot analysis, frozen muscle samples (90 mg)
were powdered under liquid N
2
and homogenized in 1 ml of ice-cold RIPA buffer as
previously described (94; 96). The total cell homogenate was then transferred to a
microcentrifuge tube and vortexed frequently for 1 h whereupon the samples were
centrifuged at 4,500 g at 4ºC, for 1 h. For plasma membrane (PM) isolation, membrane
fractions were prepared as previously described (128). Briefly, 350 mg of frozen muscle
sample was homogenized with Tris-15% sucrose buffer and centrifuged at 100,000 g for
1 h. The pellets were suspended in Tris-15% sucrose buffer and centrifuged at 120,000 g
in a continuous sucrose gradient (35-70%) to separate the membrane fractions. The
plasma membrane layer was harvested, washed in Tris buffer and spun for an additional 1
h at 100,000g. The final pellet was re-suspended in Tris buffer and stored at -80ºC. For
immunoprecipitation procedures, approximately 400 mg of powdered muscle samples
were homogenized in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants
(400 μg) were incubated with antibodies for AMPKα1, AMPKα2, CaMKII, CaMKKα, or
CaMKKβ (Santa Cruz Biotechnology, Santa Cruz, CA) for 2 h at 4ºC with gentle
agitation (95). Following the incubation, Protein A/G agarose (Santa Cruz, SC-2003)
was added to the tubes and incubated overnight at 4ºC with gentle agitation. The
immunoprecipitates were collected by centrifugation. Pellets were washed with PBS
buffer and the final supernatants were re-suspended in sucrose homogenizing buffer and
stored at -80ºC until analysis. Protein concentrations were determined with the Bradford
protein assay (BioRad, Hercules, CA).
Western blot analysis. Approximately 20 µg of protein from the PM preparations and 40
µg of protein from the total cell homogenate preparations were separated on a 12% gel

15

via SDS-PAGE. Proteins were transferred onto Immobilon-P polyvinylidene difluoride
(PVDF) membranes and blocked with 5% BSA in Tween-TBS for 1 h. The membrane
was then incubated (4ºC) in 5% BSA in Tween-TBS with antibodies (1:1000) against
either phospho-ACC-Ser
79
, total ACC, or phospho-CaMKII-Thr
287
, total CaMKII, (Cell
Signaling, Danvers, MA), or phospho-CaMKI-Thr
177
, total CaMKI , CD36 (Santa Cruz
Biotechnology, Santa Cruz, CA), or FABPpm (131). Positive controls (50 µg), rat
cerebellum extracts, for CaMKKα and CaMKKβ (Santa Cruz Biotechnology, Santa
Cruz, CA) were used to identify the proteins in perfused hindlimb muscle. Following this
overnight incubation, the membranes were probed with a secondary antibody (anti-rabbit
IgG; 1:25,000) raised in goats (Pierce, Rockford, IL). Blots were then washed and
subjected to enhanced chemiluminescence (Pierce, Rockford, IL). Band density was
quantified using Scion Image (NIH, Bethesda, MD). All bands were compared to the
band obtained for a control sample of non-perfused muscle and expression was expressed
as percent control. A ponceau S total protein stain (Sigma, St. Louis, MO) was used on
the membranes as a loading control.
Activity assays. AMPKα1 and α2 activities were measured using
32
P-ATP incorporation
into SAMS peptide (Upstate Signaling, Lake Placid, NY) as described (96). Briefly,
immunoprecipitates were added to an assay cocktail containing
32
P-ATP and SAMS
peptide. Post-incubation, an aliquot was spotted onto a piece of Whatman filter paper
and treated as described below. For the measurement of CaMKII activity, an assay kit
(Upstate Cell Signaling, Lake Placid, NY) was used as described (95). Briefly, the
immunoprecipitated CaMKII protein was incubated with assay reagents and
32
P-ATP.
Post-incubation period, an aliquot was spotted onto a Whatman filter paper and treated as

16

described below. CaMKKα and β activities were measured using a similar method as
described (47; 120). The CaMKK assay incorporated active immunoprecipitates into
bacterially expressed and purified GST-CaMKI (93). Immunoprecipitates were incubated
with GST-CaMKI in HEPES buffer and with
32
P-ATP. After incubation in a shaking
water bath (37ºC), aliquots were spotted on a Whatman filter paper. All paper samples
were washed with phosphoric acid followed by an acetone wash. Sample papers were
analyzed for radioactivity in a Packard scintillation counter and counts were used to
calculate phosphotransferase activity.
Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and
percent and total palmitate oxidation were calculated, as described previously in detail
(125; 129). Both percent and total FA oxidation were corrected for label fixation by
using acetate correction factors (129). The specific activity for palmitate in the arterial
samples was not different between groups and did not vary over time, averaging 52.9 ±
3.6, 53.5 ± 4.1, 63.5 ± 4.7, and 60.4 ± 6.8 µCi/mmol, for the R, C, A, and MC groups,
respectively (P >0.05). Oxygen uptake, glucose uptake, and lactate release were
calculated as described (129). All uptake and release rates are expressed per gram of
perfused unilateral hindquarter muscle, which has been previously determined to be 5.6%
of total body weight (132). Time effects for glucose, lactate, and FA concentrations and
FA kinetic data were analyzed using a two-way ANOVA with repeated measures in each
of the experimental groups. Since there were no significant differences in the values
measured after 10, 15, or 20 minutes of perfusion, mean values were used for subsequent
analysis. The effects of perfusion treatment with caffeine, AICAR or muscle contraction
(rest, caffeine, AICAR, muscle contraction), and of CaMKK inhibition with STO-609

17

(without vs with STO) were the two factors analyzed using a two-way ANOVA
(Statview 5.0). Scheffe’s test for post-hoc multiple comparisons was performed when
appropriate. A significance level of 0.05 was chosen for all statistical methods.
RESULTS
Muscle performance and oxygen uptake. To verify physiological perfusion conditions
oxygen uptake was measured during the experimental perfusion period. There were no
differences in oxygen uptake over time in any of the experimental groups (P>0.05).
Neither caffeine nor AICAR had any effect on oxygen uptake compared with rates
measured in the resting group (P>0.05, Table 1). Muscle contraction resulted in an
increase in oxygen uptake when compared to the resting group (P<0.05). STO-609 had
no effect on oxygen uptake in any of the groups (P>0.05). Initial force development was
not affected by treatment with STO-609 (P>0.05, Table 1). Muscle tension development
decreased rapidly during the first 10 min of electrical stimulation and this was followed
by a more gradual decrease in tension development. After 10 min of electrical
stimulation, muscle force development had decreased by 65% of initial tension
development in both MC groups.
Substrate exchange across the hindlimb. Palmitate concentration and delivery were not
significantly different at any time point in any of the groups, as dictated by the protocol
(P>0.05, Table 1). Arterial perfusate lactate concentration was not different in the
caffeine and AICAR groups but was increased in the muscle contraction group when
compared to rest (P<0.05, Table 1). Lactate release was increased (P<0.05, Table 1) by
49% during caffeine treatment and 133% during muscle contraction but was not affected
by AICAR treatment. STO-609 treatment did not affect lactate release in any of the

18

groups (P>0.05). Arterial perfusate glucose concentration did not vary during the
experimental period in any of the groups and mean glucose concentration was not
different in any of the groups (P>0.05, Table 1). Glucose uptake (nmol∙min
-1
∙g
-1
) did not
vary over time in any of the groups (P>0.05) but was significantly increased (P<0.05,
Fig.2) 104% by caffeine, 85% by AICAR, and 130% by muscle contraction when
compared with the resting condition. STO-609 prevented the increase in glucose uptake
in the caffeine and muscle contraction groups but not in the AICAR group (Fig.2A).

Palmitate metabolism. FA uptake (nmol∙min
-1
∙g
-1
, Fig. 2B) increased (P<0.05) by 64%
with caffeine treatment, 42% with AICAR treatment and 65% with muscle contraction
(16.4 ± 1.2, 14.2 ± 0.6, and 16.5 ± 0.3 vs. 10.0 ± 0.9), respectively. FA oxidation
(nmol∙min
-1
∙g
-1
, Fig. 2C) increased by 71% with caffeine treatment, 93% with AICAR
treatment, and 143% with muscle contraction (2.4 ± 0.1, 2.7 ± 0.3, and 3.4 ± 0.2 vs. 1.4 ±
0.2). STO-609 abolished (P<0.05) the increase in caffeine- and contraction-induced FA
uptake (Fig. 2B) and oxidation (Fig. 2C), but had no effect on the increase in FA uptake
and oxidation induced by AICAR treatment (P>0.05, Fig.2).
Enzyme activities. CaMKKβ activity (pmol∙min
-1
∙g
-1
) was increased (P<0.05, Fig. 3A)
by 113% with caffeine treatment and by 145% with muscle contraction (C: 498.8 ± 98.1
and MC: 574.4 ± 120.0 vs. R: 234.1 ± 22.0), but was not affected by AICAR treatment
(A: 269.0 ± 50.0 vs. R: 234.1 ± 22.0, P>0.05). STO-609 prevented (P<0.05) the caffeine-
and contraction-induced increase in CaMKKβ activity. CaMKKα activity could not be
measured because, contrary to CaMKKβ, it lacks autonomous activity (121). As such, the
activity of CaMKKα is dependent on Ca
2+
/calmodulin binding to the kinase and this is
washed away in the immunoprecipitation process. It is also important to note that the

19

CaMKKβ activity reported here is not indicative of the maximal activation of the kinase
because a portion of the kinase’s activation state is dependent on Ca
2+
/calmodulin
binding as well (6). To verify the presence of CaMKKα and/or CaMKKβ in rat skeletal
muscle (66), western blots were performed. Western blotting showed that both enzymes
are present in rat hindquarter skeletal muscle (Fig. 3B). Lastly, to provide further
evidence of CaMKK activation, we determined whether CaMKI, a known downstream
target of CaMKK (33; 119), was phosphorylated at Thr
177
and activated with CaMKK
activation. Caffeine and MC increased (P<0.05, Fig. 3C) phosphorylated CaMKI-Thr
177

by 109% and 223%, respectively. There were no changes in phosphorylation state with
AICAR. STO-609 prevented (P<0.05) the caffeine- and contraction-induced increase in
phosphorylated CaMKI-Thr
177
. Caffeine, AICAR and muscle contraction increased
(P<0.05, Fig. 4B) AMPKα2 activity (pmol∙min
-1
∙g
-1
) by 295%, 11-fold and 7-fold (C:
752.2 ± 78.0, A: 2320.2 ± 320.8, MC: 1651.8 ± 142.9, vs. R: 190.3 ± 33.7, P <0.05)
respectively, but did not affect (P>0.05) AMPKα1

activity (Fig. 4A). STO-609 partially
and completely prevented the increase in AMPKα2 activity in the muscle contraction and
caffeine groups, respectively (MC: 1651.8 ± 142.9 vs. MC + STO-609: 651.7 ± 64.8 and
C: 752.2 ± 78.0 vs. C + STO-609: 302.1 ± 37.4), but did not affect (P>0.05) the AICAR-
induced increase in AMPKα2 activity. ACC protein expression was not different
(P>0.05) between groups and STO-609 treatment had no effect on ACC protein
expression (P>0.05) in any of the groups (Fig. 5). Caffeine, AICAR and muscle
contraction increased (P<0.05, Fig. 5) phosphorylated ACC-Ser
79
. In line with the results
obtained for AMPKα2 activity, STO-609 prevented the increase in phosphorylated ACC
in the caffeine and muscle contraction groups.

20

Transport Proteins. Plasma membrane (PM) CD36 content was increased by 42%, 53%,
and 72% in the caffeine, AICAR and muscle contraction groups, respectively (P<0.05,
Fig. 6A). CaMKK inhibition prevented the increase in PM CD36 content induced by
caffeine and muscle contraction (P<0.05 vs. control group, Fig. 6A) but did not affect the
AICAR-induced increase. Because total protein expression of CD36 was not different
between groups and STO-609 had no effect on the expression of the protein (P>0.05, Fig.
6A), the increase in PM CD36 provides evidence for the translocation of CD36 to the PM
with our three experimental treatments. Plasma membrane FABPpm content was not
different between any of the groups and STO-609 had no effect on its expression
(P>0.05, Fig. 6B). PM GLUT4 content was increased by 53%, 58%, and 104% in the
caffeine, AICAR and muscle contraction groups, respectively (P<0.05, Fig. 7). CaMKK
inhibition prevented the increase in PM GLUT4 content induced by caffeine and muscle
contraction (P<0.05 vs control group, Fig. 7) but had no effect on the AICAR-induced
increase. As shown for CD36, total protein expression of GLUT4 was not different in any
of the experimental groups and STO-609 had no effect on its expression (P>0.05, Fig. 7).
CaMKII activation. It has been previously demonstrated that CaMKK does not directly
activate CaMKII (119) and that STO-609 may inhibit CaMKII directly (47). In order to
verify the specificity of the CaMKK inhibitor STO-609 and that our results are based on
inhibition of CaMKK, the activity and phosphorylation state of CaMKII (data not shown)
were measured. CaMKII activity (pmol∙min
-1
∙g
-1
) was increased (P<0.05) by 144%
during caffeine stimulation and by 88% during muscle contraction (C: 3481.5 ± 482.4
and MC: 2685.4 ± 219.9 vs. R: 1425.5 ± 110.5). There were no changes in CaMKII
activity with AICAR treatment and STO-609 did not affect the caffeine- and contraction-

21

induced increase in CaMKII (P>0.05). Changes in the phosphorylation state of CaMKII
followed the same patterns as those measured for the activity of the enzyme.
DISCUSSION
The findings in this study suggest a link between Ca
2+
signaling, via CaMKK, and
AMPKα2 activation in the regulation of glucose uptake and FA uptake and oxidation in
perfused rat skeletal muscle contracting at moderate intensity. Additionally, the data
obtained with the AICAR groups provide further evidence for the presence of a Ca
2+
-
independent AMPK-dependent signaling cascade in the regulation of glucose uptake and
FA uptake and oxidation. Furthermore, our GLUT4 and CD36 data indicate that the
translocation of these transport proteins can be regulated via Ca
2+
-dependent and Ca
2+
-
independent signaling. Together, our results indicate that during muscle contraction,
AMP- and Ca
2+
-induced signaling cascades regulate muscle metabolism in part by
converging at AMPK.
As stated in the introduction, our objective was to determine the role played by
CaMKK activation in the regulation of muscle metabolism during muscle contraction.
We were especially interested in determining whether AMPK regulation was CaMKK-
dependent and/or CaMKK-independent. The results obtained from the MC groups show
convincingly that when CaMKK is inhibited, regulation of muscle metabolism in
response to caffeine or contraction is deleteriously altered. CaMKK inhibition completely
prevented the contraction-induced increase in glucose uptake and FA uptake and
oxidation. Furthermore, our data show that with CaMKK inhibition, AMPK 2 activation
induced by MC is significantly decreased. As expected, the caffeine group was similarly
affected by CaMKK inhibition. High intracellular calcium levels induced by caffeine or

22

agonist treatment have been shown to stimulate glucose and FA uptake in a variety of
isolated muscle preparations (49; 59; 95; 135; 146). Furthermore, our caffeine-induced
increase in FA oxidation agrees with the fact that caffeine has been shown to decrease
malonyl-CoA levels in perfused muscle (79). Similarly, in agreement with some but not
other data, we observed a caffeine-induced increase in AMPK 2 but not 1 activity and
in ACC phosphorylation (42; 59; 95; 146). Perfusion with STO-609 completely inhibited
the caffeine-induced increases in glucose uptake, FA uptake and oxidation and in
AMPK 2 activity. Overall, the changes in glucose uptake and FA uptake and oxidation
induced by caffeine or MC reflected AMPK 2 activation patterns being increased with
caffeine or MC and inhibited with STO-609. Thus, our results suggest that CaMKK
activation is critical for normal regulation of muscle metabolism during muscle
contraction. Our data further suggest that caffeine- and MC-induced Ca
2+
signaling and
MC-induced AMP signaling converge at AMPK. This is in agreement with the
suggestion made by Jensen and others (55; 59; 143) that intracellular Ca
2+
signaling may
act via a CaMKK-AMPK pathway.
To ensure that our conclusions are based on valid data, we measured CaMKK 
activity and verified the presence of CaMKK  and CaMKK  in rat skeletal muscle. In
line with several other studies (59; 60; 120), our CaMKK  activity results confirm that
the inhibitory agent STO-609 inhibited the contraction and caffeine-induced rise in
CaMKK  activity. Because CaMKK  lacks autonomous activity (121), it was not
feasible to measure its activity. In that sense, we can not confirm whether or not
CaMKK  activity was completely inhibited by STO-609 and/or involved in the
regulation of substrate metabolism. There has been some controversy regarding the

23

possible presence of the CaMKK  and/or  isoforms in rat and mouse skeletal muscle.
Whereas some have shown CaMKK  mRNA and protein to be expressed in rat
cardiomyocytes (63), others have been able to demonstrate the expression of CaMKK 
and  only occasionally in mouse skeletal muscle (60). In line with our data, McGee et al
were able to show expression of CaMKK  and  in overloaded plantaris muscle (81) and
others have shown that overexpression of CaMKK  increased glucose uptake in mouse
muscle (141). Thus, our western blot results agree with some published data (63; 81) and
show that CaMKK  and  are present in perfused rat skeletal muscle. It is important to
note that the perfused hindquarter preparation is a mixed muscle preparation which is
made up mostly of fast-twitch glycolytic and fast-twitch oxidative glycolytic fibers but
very little pure slow-twitch oxidative fibers (7). This characteristic mixture of fiber types
in the hindquarter preparation may account for the similarity of results between our data
and those of McGee et al who used the plantaris muscle, a mixed fiber made up of 50%
fast-twitch oxidative glycolytic fibers and 41% fast-twitch glycolytic fibers (7; 81).
Additionally, our CaMKI data provide further evidence for the activation and/or
inhibition of CaMKK, since it has been previously established that phosphorylation of
CaMKI is dependent on CaMKK (33; 119). Furthermore, our data showed that STO-609
had no effect on CaMKII activity supporting the specificity of pharmacological inhibition
of CaMKK via STO-609 when the concentration is below the threshold shown to directly
inhibit other enzymes (47; 143). It has been suggested that STO-609 may directly inhibit
AMPK in vitro (47; 143). Despite this, when AMPK was directly activated via AICAR
and STO-609 was present there was no evidence of inhibition of AMPKα1 or α2 activity.

24

Together our data strongly suggest that STO-609 exerted its observed effects on muscle
metabolism via inactivation of CaMKK.
To separate the effects of AMP- and Ca
2+
-mediated signaling cascades, we also
compared our muscle contraction groups to AICAR groups. Interestingly, lactate release
was increased in the caffeine and MC groups but not in the AICAR groups. These data
suggest that Ca
2+
-mediated signaling cascades may preferentially activate glycolytic
pathways, while AMP-mediated signaling pathways may preferentially activate oxidative
pathways. Furthermore, our results collected from the AICAR groups show that CaMKK
activation is not involved in the regulation of muscle metabolism when AMP-mediated
signaling cascades are induced by AICAR alone. In view of the fact that AICAR did not
increase CaMKK  activity, it was not surprising to find that CaMKK inhibition did not
affect the AICAR-induced increase in glucose uptake, FA uptake and oxidation,
AMPK 2 activity and ACC phosphorylation. Results on the effects of STO-609 on
AMPK activation are contradictory. In NIH3T3 cells and LKB1
-/-
cells, STO-609
decreased AICAR-induced AMPK activity (143) and reduced ionomycin-induced AMPK
activity (55). On the other hand, STO-609 had no effect on phenformin-induced AMPK
activity in rat brain slices but decreased AMPK activation induced by the calcium
ionophore A23187 in HeLa cells lacking LKB1 (47). Together, our results and those of
others (145; 146; 148) suggest that, contrary to muscle contraction, AICAR may activate
AMPK via a Ca
2+
-independent cascade.
Results from our study agree with data indicating that GLUT4 and CD36
translocation to the plasma membrane mediate the rise in glucose and FA uptake in
skeletal muscle induced by AICAR and muscle contraction (13; 24; 70; 76). In line with

25

these data, our results provide new evidence demonstrating that intracellular [Ca
2+
] may
similarly regulate GLUT4 and CD36 translocation to the plasma membrane. This agrees
with some data showing an increase in plasma membrane CD36 content in cardiac
myocytes incubated with phorbol 12-myristate 13-acetate (PMA), a phorbol ester that
activates the novel and conventional PKCs partially via a rise in intracellular [Ca
2+
] (75).
Additionally, it has recently been determined that GLUT4 mRNA and protein expression
may be regulated in a Ca
2+
-dependent manner in cultured muscle cells (87; 91); however,
plasma membrane protein content was not measured in these studies. Our data show that
translocation of CD36 and GLUT4 to the plasma membrane may be regulated via
CaMKK activation during caffeine treatment and muscle contraction. In line with our
kinetic data, CaMKK inhibition did not prevent the AICAR-induced increase in plasma
membrane CD36 and GLUT4 content providing support for the possible presence of
CaMKK-independent AMPK-dependent regulatory cascades. We were unable to measure
any increases in plasma membrane content of FABPpm in any of the treatment groups.
This supports our previous data in which increases in FABPpm on the plasma membrane
were measured after endurance training and not acute muscle contraction (62; 133).
Others have shown (14) increases in plasma membrane FABPpm content following acute
in situ muscle contraction, however these measurements were performed in giant
sarcolemma vesicles which may account for these discrepancies. Our data suggest that
translocation of the FA transport proteins CD36 and FABPpm may be regulated via
different mechanisms, either by acute muscle contraction or after long term treatment
imposed on the muscle such as exercise training. The current data present further
verification for CD36 and GLUT4 translocation, as a result of AICAR treatment and

26

moderate intensity muscle contraction, and for the first time provide evidence for Ca
2+
-
induced CaMKK-dependent regulation of this translocation.
PERSPECTIVES AND SIGNIFICANCE
The induction of signaling cascades by muscle contraction is known to be a
complex physiological process. Although regulation of glucose uptake, FA uptake and
FA oxidation during muscle contraction has traditionally been ascribed to AMPK, the
present study has expanded this view to include a role for CaMKK signaling, via both
AMPK-dependent and -independent cascades, in this regulation. The present data also
provide new evidence for a link between Ca
2+
-dependent signaling and CD36 and
GLUT4 translocation to the plasma membrane during muscle contraction. This last
observation suggests that increased transporter translocation induced by a rise in Ca
2+
-
signaling is one of the cellular mechanisms by which FA and glucose uptake are up-
regulated during muscle contraction. Collectively, these findings are important because
dysregulation of FA metabolism is a critical factor in the development of metabolic
disorders such as obesity, Type 2 diabetes and insulin resistance. As such, these results
provide valuable information about the mechanisms involved in the regulation of glucose
uptake, FA uptake and FA oxidation possibly providing targets for future therapies for
individuals suffering from metabolic disorders. The novel finding of this study is the
possibility of Ca
2+
, via CaMKK activation, as an upstream activator of AMPKα2

in the
regulation of glucose and FA metabolism in rat skeletal muscle during muscle
contraction. In order to define the roles of Ca
2+
signaling in the regulation of glucose and
FA metabolism and to determine whether AMPK is essential for this regulation,
additional studies will need to be performed.

27

Rest Caffeine AICAR MC

Vehicle
(n=8)
STO-609
(n=8)
vehicle
(n=7)
STO-609
(n=7)
Vehicle
(n=8)
STO-609
(n=8)
vehicle
(n=7)
STO-609
(n=7)
O2 uptake
µmol∙g
-1
∙h
-1

14.15±2.1 14.0±1.1 11.7 ± 1.6 13.9±1.2 14.4±1.3 15.6±1.2 21.6±0.8† 21.7±1.2†
Initial
force g
N/A N/A N/A N/A N/A N/A 693 ± 42.3 611 ± 62.5
[FA] µmol/l 569.2±16.9 594.8±37.6 583.8±27.2 636.2±31.4 578.7±43.5 592.1±30.2 613.6±47.5 573.2±34.2
FA delivery
nmol∙min
-1
∙g
-1

461.3±19.6 512.5±37.2 496.8±34.1 507.2±34.1 509.0±41.9 499.2±30.9 515.03±39.5 461.5±26.5
Glucose
concentration
mmol/l
6.9±0.5 7.6±0.5 7.7±0.5 7.3±0.3 7.5±0.4 7.6±0.4 6.9±0.5 7.6±0.7
Lactate
concentration
mmol/l
0.71±0.06 0.92±0.08 0.82±0.15 1.08±0.15 0.82 ±0.06 0.90±0.08 1.37±0.17† 1.51±0.15†
Lactate release
umol∙g
-1
∙h
-1

12.9±0.8 14.4±1.3 19.2±1.8† 20.9±1.1† 13.4±1.4 11.4±1.7 30.1±1.3† 25.5±1.3†
Table 1. Perfusion characteristics of rat hindlimbs perfused with caffeine, AICAR, or during moderate
intensity muscle contraction Values are expressed as means ± SE; n= number of rats. Rats were perfused with
either DMSO vehicle control or STO-609 dissolved in DMSO at rest, during caffeine, or AICAR treatment, or
during moderate intensity muscle contraction (MC). * P< 0.05 compared with respective vehicle control, † P<
0.05 compared with rest group.

28

0
5
10
15
20
25
30
nmol / hr / g
Control STO-609
R C
*
*
MC
#
#
#
#
A
G lucose Uptake
A
*
0
5
10
15
20
25
nmol / min / g
Control STO-609
R C
*
*
MC
#
#
#
#
A
P
a
l
m
i
t
a
t
e

U
p
t
a
k
e
B
0
1
2
3
4
nmol / min / g
Control STO-609
R C
*
*
MC
#
#
#
#
†
A
P
a
l
m
i
t
a
t
e

O
x
i
d
a
t
i
o
n
C
Figure 2. Effect of CaMKK inhibition on glucose uptake (A) palmitate uptake (B) and oxidation
(C) in perfused rat hindlimbs during caffeine or AICAR treatment, or moderate intensity muscle
contraction. Values are means ± SE for R (n= 8), R + STO-609 (n=8), C (n=7), C + STO-609
(n=7), A (n=8), A + STO-609 (n=8), MC (n=7), and MC +STO-609 (n=7). R, rest; C, caffeine;
A, AICAR; MC, muscle contraction. Open bars represent rats perfused with DMSO vehicle
control and solid bars represent rats perfused with the CaMKK inhibitor STO-609. Since there
were no differences between 10, 15, and 20 min data points, an average value was used for each
perfusion. # P<0.05 compared with rest group similarly treated; * P<0.05 compared with
respective control group; † P<0.05 compared with caffeine group similarly treated.

29

0
200
400
600
800
1000
pmol / min / g
Control STO-609
R C
*
*
MC
#
#
A
C
a
M
K
K

A
c
t
i
v
i
t
y
A
66 kDa
CaMKKa
6 3 k Da
CaMKKß
+ Control
+ Control
50 µg of rat hindlim b skeletal muscle
R C MC
R C MC
B
0
100
200
300
400
Arbitrary Units
Control STO-609
R C MC
*
*
#
#
A
CaMKI~pThr
177
CaMKI
Phosphorylated CaMKI
STO-609 5 µM - - + - + - + +
R C MC A
C
Figure 3. (A) The effect of STO-609 on CaMKK  activity in perfused rat hindlimbs during
caffeine or AICAR treatment, or moderate intensity muscle contraction. CaMKK  activity is
calculated per gram of muscle present in assay preparation. (B) Identification of CaMKKα and 
in rat hindquarter muscle. Rat cerebellum extracts (50 µg) were used as positive controls. R, rest;
C, caffeine; MC, muscle contraction. (C) Effect of CaMKK inhibition on CaMKI-Thr
177

phosphorylation and total CaMKI expression in perfused rat hindlimbs during caffeine or AICAR
treatment, or moderate intensity muscle contraction. Results are expressed after standardization
using non-perfused hindquarter muscle as control. Values are means ± SE for R (n= 8), R +
STO-609 (n=8), C (n=7), C + STO-609 (n=7), A (n=8), A + STO-609 (n=8), MC (n=7), and MC
+STO-609 (n=7). R, rest; C, caffeine; A, AICAR; MC, muscle contraction. Open bars represent
rats perfused with DMSO vehicle control and solid bars represent rats perfused with the CaMKK
inhibitor STO-609. # P<0.05 compared with rest group similarly treated; * P<0.05 compared
with respective control group.

30

0
400
800
1200
1600
2000
2400
2800
pmol / min / g
Control STO-609
R C
*
*
MC
†
#
#
#
#
†
A
A
M
P
K
a
2

A
c
t
i
v
i
t
y
B
0
200
400
600
800
pmol / min / g
Co ntrol STO -609
R C MC A
A
M
P
K
a
1

A
c
t
i
v
i
t
y
A
Figure 4. Effect of CaMKK inhibition on AMPKα1 (A) and AMPK α2 (B) activity in perfused
rat hindlimbs during caffeine or AICAR treatment, or moderate intensity muscle contraction.
Activity values are calculated per gram of muscle present in assay preparation. Values are
means ± SE for R (n= 8), R + STO-609 (n=8), C (n=7), C + STO-609 (n=7), A (n=8), A + STO-
609 (n=8), MC (n=7), and MC +STO-609 (n=7). R, rest; C, caffeine; A, AICAR; MC, muscle
contraction. Open bars represent rats perfused with DMSO vehicle control and solid bars
represent rats perfused with the CaMKK inhibitor STO-609. # P<0.05 compared with rest group
similarly treated; * P<0.05 compared with respective control group; † P<0.05 compared with
caffeine group similarly treated.

31

0
50
100
150
200
250
Arbitrary Units
Control STO-609
R C MC
*
*
#
#
A
#
#
ACC~p Ser
79
ACC
Phosphorylated ACC
STO-609 5µM - - + - + - + +
R C MC A
Figure 5. Effect of CaMKK inhibition on ACC-Ser
79
phosphorylation and total ACC expression
in perfused rat hindlimbs during caffeine or AICAR treatment, or moderate intensity muscle
contraction. Results are expressed after standardization using non-perfused hindlimb muscle as
control. Values are means ± SE for R (n= 8), R + STO-609 (n=8), C (n=7), C + STO-609 (n=7),
A (n=8), A + STO-609 (n=8), MC (n=7), and MC +STO-609 (n=7). R, rest; C, caffeine; A,
AICAR; MC, muscle contraction. Open bars represent rats perfused with DMSO vehicle control
and solid bars represent rats perfused with the CaMKK inhibitor STO-609. # P<0.05 compared
with rest group similarly treated; * P<0.05 compared with respective control group.

32

0
50
100
150
200
250
Arbitrary Units
Control STO-609
R C MC
*
*
#
#
A
# #
PM CD36
CD36
PM CD36 Content
STO-609 5µM - - + - + - + +
R C MC A
A
0
50
100
150
Arbitrary Units
Control STO-609
R C MC A
PM FABPpm
PM FABPpm Content
STO-609 5 µM
- - + - + - + +
R C MC A B
Figure 6. Effect of CaMKK inhibition on plasma membrane (PM) and total content of CD36 (A)
and plasma membrane content of FABPpm (B) in perfused rat hindlimbs during caffeine or
AICAR treatment or moderate intensity muscle contraction. Results are expressed after
standardization using non-perfused hindquarter muscle as control. Values are means ± SE for R
(n= 8), R + STO-609 (n=8), C (n=7), C + STO-609 (n=7), A (n=8), A + STO-609 (n=8), MC
(n=7), and MC +STO-609 (n=7). R, rest; C, caffeine; A, AICAR; MC, muscle contraction.
Open bars represent rats perfused with DMSO vehicle control and solid bars represent rats
perfused with the CaMKK inhibitor STO-609. # P<0.05 compared with rest group similarly
treated; * P<0.05 compared with respective control group.

33

0
50
100
150
200
250
Arbitrary Units
Control STO-609
R C MC
*
*
#
#
A
# #
P M GLUT4
GLUT4
P
M
G
L
U
T
4

C
o
n
t
e
n
t
STO-609 5 µM - - + - + - + +
R C MC A
Figure 7. Effect of CaMKK inhibition on plasma membrane (PM) and total content of GLUT4 in
perfused rat hindlimbs during caffeine or AICAR treatment or moderate intensity muscle
contraction. Results are expressed after standardization using non-perfused hindquarter muscle as
control. Values are means ± SE for R (n= 8), R + STO-609 (n=8), C (n=7), C + STO-609 (n=7),
A (n=8), A + STO-609 (n=8), MC (n=7), and MC +STO-609 (n=7). R, rest; C, caffeine; A,
AICAR; MC, muscle contraction. Open bars represent rats perfused with DMSO vehicle control
and solid bars represent rats perfused with the CaMKK inhibitor STO-609. # P<0.05 compared
with rest group similarly treated; * P<0.05 compared with respective control group.

34

IV. EXPERIMENT 2

AMPKα2 deficiency uncovers time-dependency in the regulation of contraction-
induced palmitate and glucose uptake in mouse muscle
2

CHAPTER ABSTRACT
AMPK has been shown to be a master fuel sensor in skeletal muscle with multiple
downstream signaling targets which may triggered by an increase in intracellular [Ca
2+
].
The purpose

of this study was to determine whether increases in intracellular [Ca
2+
]
induced by caffeine act solely via AMPKα2 and whether AMPKα2 is essential to
increase substrate uptake and oxidation in contracting skeletal muscle. Mouse (C57BL/6)
hindlimbs were perfused at rest (n=11), with 3 mM caffeine (n=10), or during moderate
intensity muscle contraction (n=11); mice were either wild type (WT) or possessed an
AMPKα2 dominant negative (DN) transgene. Time-dependent effects in glucose and FA
uptake were uncovered throughout the 20-min muscle contraction perfusion period
(P<0.05). Glucose uptake rates did not increase in DN mice during muscle contraction
until the last 5 min of the protocol (P<0.05). In contrast, FA uptake rates were elevated
at the onset of muscle contraction and diminished by the end of the protocol in DN mice
(P<0.05). Increases in FA oxidation rates were abolished in the DN mice during muscle
contraction (P<0.05). The DN transgene had no effect on caffeine-induced FA uptake
and oxidation (P>0.05). However glucose uptake rates were blunted in caffeine-treated
DN mice (P<0.05).

2
Paper accepted with revisions American Journal of Physiology Regulatory, Integrative
and Comparative Physiology

35

The DN transgene did not alter caffeine- or contraction-mediated changes in the
phosphorylation of CaMKI, ERK1/2, AS160/TBC1D4 or AS150/TBC1D1 (P>0.05).
SIRT1 activity was elevated with caffeine and muscle contraction in WT mice (P<0.05).
These data suggest that AMPKα2 is involved in the regulation of substrate uptake in a
time-dependent manner during muscle contraction but is not necessary for appropriate
regulation of FA uptake and oxidation during caffeine treatment.
Key Words: AS160, ERK1/2, SIRT1, Caffeine, CaMKK
CHAPTER INTRODUCTION
Evidence suggests that multiple cellular signals regulate the changes in substrate
metabolism with muscle contraction, including the AMP-activated protein kinase
(AMPK) signaling cascade (13; 94; 95; 138). During states of low energy, such as muscle
contraction or exercise, it is widely accepted that liver kinase B1 (LKB1) acts as an
upstream AMPK kinase that phosphorylates and activates AMPK and initiates a
multitude of signaling events to maintain energy homeostasis (108; 110; 117). Although
strong evidence indicates that the LKB1-AMPK cascade is a key signaling pathway that
regulates changes in substrate use in contracting skeletal muscle (46; 73; 142; 146), data
also show that AMPK may not be the sole signal mediating contraction-induced changes
in substrate uptake and oxidation (95; 96; 146).
Along with AMPK, mounting data suggest that increases in intracellular [Ca
2+
]
may play a key role in the regulation of substrate utilization in skeletal muscle and that
this may occur in part via AMPK activation (20; 81; 135; 141; 146; 148). There is
evidence that Ca
2+
-calmodulin-dependent protein kinase kinase (CaMKK) activates
AMPK in vitro in cells, in whole skeletal muscle, in yeast (47; 53; 55; 143), and we have

36

recently shown that activation of CaMKK and CaMKII leads to an activation of AMPK
in perfused rat hindlimbs (2; 95). Additionally, we have shown that inhibition of
CaMKK or CaMKII reduces glucose uptake, FA uptake and FA oxidation (2; 95).
However it is unclear whether Ca
2+
-signaling relies extensively on the activation of
AMPK to mediate its metabolic effects or whether LKB1 regulation of AMPK is central
to AMPK activation.
Recent data have suggested a link between AMPK and sirtuin1 (SIRT1), an
NAD
+
dependent deacetylase that has been implicated in the regulation of energy
metabolism (21; 54; 106). Both molecules have been examined independently and recent
data have suggested that activation of both intermediates may be related (21; 54). It is
unclear whether or not AMPK acts upstream of SIRT1 during muscle contraction.
Furthermore it is unclear whether or not AMPK acts upstream of alternative signaling
pathways that include extracellular signal-regulated kinases 1 and 2 (ERK1/2) or Akt
substrate 160 kd (AS160/TBC1D4) and Akt substrate 150 kd (AS150/TBC1D1) in the
regulation of contraction-induced substrate metabolism.
To study the role of AMPKα2 in the regulation of substrate metabolism in
skeletal muscle during increased intracellular [Ca
2+
]

induced by caffeine and muscle
contraction, we employed a transgenic mouse model possessing a dominant negative
(DN) α2 isoform of AMPK in skeletal muscle and heart muscle (86). There have been
multiple studies which have sought to determine the importance of AMPK in the
regulation of substrate metabolism. However, studies utilizing transgenic mouse models
where AMPK is either inactive or knocked out reveal conflicting results (72; 73; 78; 84).
Some studies suggest that AMPK is absolutely essential in the regulation of substrate

37

metabolism, whereas other studies show that AMPK is not necessary to measure
increases in substrate metabolism during exercise or other metabolic challenges.
Therefore, the purpose

of this study was to determine (1) whether a caffeine-
induced increase in intracellular [Ca
2+
] regulates muscle substrate use solely via
AMPKα2 activation and (2) whether the α2 subunit of AMPK is essential to increase
substrate uptake and oxidation in contracting skeletal muscle in a physiologically relevant
model such as a hindlimb perfusion system. We hypothesized that AMPKα2 is necessary
to measure increases in substrate uptake and FA oxidation in skeletal muscle during a
caffeine-induced increase in intracellular [Ca
2+
]. We further hypothesized that AMPKα2
plays a role in substrate use in contracting skeletal muscle but that it is not the sole signal
involved in the aforementioned regulation.

MATERIALS AND METHODS

Animal Preparation. Male C57BL/6 (24.3±0.80g, 2-3 months old) mice expressing an
AMPKα2 dominant negative transgene (DN; kindly provided by M.J. Birnbaum,
University of Pennsylvania, Philadelphia, PA) in cardiac and skeletal muscle (86) and
their wild type (WT) litter mates were kept on a 12:12-h light-dark cycle and standard rat
chow and water were provided ad libitum to the mice. Animals were randomly divided
into three experimental groups: rest (R, n=11), caffeine (C, n=10) and muscle contraction
(MC, n=11). All procedures for the present study were approved by the Institutional
Animal Care and Use Committee at the University of Southern California.
Hindlimb Perfusion. On the day of the experiment, mice were anesthetized by the
administration of an intraperitoneal injection of ketamine/xylazine cocktail (20 mg/kg
body weight). Surgical preparation for the hindlimb perfusion was performed as

38

previously described (105; 129; 130). Before placement of the perfusion catheters, 15 IU
of heparin was injected into the inferior vena cava. The mice were then euthanized with
an intracardial injection of pentobarbital sodium (0.4 mg/g body weight), and catheters
were immediately inserted into the descending aorta and ascending vena cava. The
hindlimbs were then washed extensively with saline and placed in a perfusion apparatus
for the equilibration (20 min) and experimental (20 min) perfusion periods.
The perfusate consisted of Krebs-Henseleit solution, 5% bovine serum albumin
(BSA; Millipore, Billerica, MA), 550 M albumin-bound palmitate, 24 µCi of albumin-
bound [1-
14
C] palmitate, 6 mM glucose, and 3 mM caffeine (Sigma, St. Louis, MO)
dissolved in Krebs-Henseleit buffer in the C groups. The perfusate was kept at 37°C and
was continuously gassed with a mixture of 95% O
2
-5% CO
2
. Perfusion flow rate was
maintained at 1.5 ml/min for all groups (average: 0.57 ± 0.02 ml/min/g of perfused
muscle). Arterial pH levels between 7.20-7.65 and arterial P
O2
and P
CO2
values were
between 200-455 and 22-48 mmHg, respectively. Perfusion pressures were not affected
by any of the experimental conditions and averaged 66.6±16.1, 42.0±12.1, 66.2±7.2
mmHg in the R, C, and MC groups, respectively (P > 0.05). Following the 20 min
equilibration period, arterial and venous samples were taken at 5, 10, 15, and 20 min of
the experimental period for further analysis. At the end of the experimental period (20
min), the gastrocnemius-soleus-plantaris muscle groups of both legs were freeze-clamped
in situ with pre-cooled aluminum clamps, removed and stored in liquid N
2
for further
analysis.
Muscle Contraction Protocol. Force production measurements were made in
anesthetized WT and DN mice to determine whether AMPK 2 deficiency would affect

39

(37) or not (83) force production with this protocol (44; 45). The right tibiopatellar
ligament was stabilized for the recording of force production and a modular chart
recorder (Cole Parmer, Vernon Hills, IL) was used to measure the tension developed by
the gastrocnemius-soleus-plantaris (GSP) muscle group during the 20-min muscle
stimulation protocol. The GSP muscle group length was adjusted at the initiation of
electrical stimulation to obtain maximal active tension and was connected to a S48 Grass
stimulator (Grass Telefactor, West Warwick, RI) to induce isometric muscle contractions
via delivery of 15-V trains of 100 Hz lasting 50 ms with impulse duration of 1 ms with 30
trains/min. This moderate intensity protocol has been shown to maximize FA
metabolism (94).
Perfusate sample analyses. Perfusate samples collected during the perfusion were
analyzed to determine FA, glucose, and lactate concentrations as well as radioactive
[
14
C]FA and
14
CO
2
contents. A WAKO NEFA HC kit (WAKO Chemicals, Richmond,
VA) was used to measure perfusate FA concentrations spectrophotometrically. An YSI-
1500 (Yellow Springs Instruments, Yellow Springs, OH) analyzer was used to measure
glucose and lactate concentrations in the collected perfusate samples. Perfusate [
14
C]FA
and
14
CO
2
radioactivities were measured as previously described (125; 127; 129). P
CO2
,
P
O2
, and pH were determined by utilizing an ABL-5 analyzer (Radiometer America,
Westlake, OH).
Muscle sample preparation. For western blot analysis, frozen gastrocnemius-soleus-
plantaris muscle samples (40 mg) were powdered under liquid N
2
and homogenized in
500 µl of ice-cold RIPA buffer as previously described (94; 96). The total cell
homogenate was then transferred to a microcentrifuge tube and vortexed frequently for 1

40

h whereupon the samples were centrifuged at 4,500 g at 4ºC, for 1 h. For
immunoprecipitation procedures, approximately 90 mg of powdered muscle samples
were homogenized in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants
(200 μg) were incubated with antibodies for AMPKα1 or AMPKα2 (Santa Cruz
Biotechnology, Santa Cruz, CA) for 2 h at 4ºC with gentle agitation (95). Following the
incubation, Protein A/G agarose (Santa Cruz, SC-2003) was added to the tubes which
were gently agitated overnight (4°). The immunoprecipitates were collected by
centrifugation. Pellets were washed with phosphate-buffered saline (PBS) buffer and the
final pellets were re-suspended in sucrose homogenizing buffer and stored at -80ºC until
analysis. Protein concentrations were determined with the Bradford protein assay
(BioRad, Hercules, CA). For nuclear extraction procedures, a nuclear extraction kit was
used (Pierce, Rockford, IL) and the manufacturer’s instructions were followed. Briefly,
approximately 40 mg of muscle samples were homogenized in a cytoplasmic extraction
buffer (CERI). The homogenate was vortexed and incubated on ice for 10 min at which
time an additional cytoplasmic extraction buffer (CERII) was added to the tube.
Following a 5 min centrifugation step (16,000 g), the recovered pellet was resuspended in
a nuclear extraction buffer (NER). The suspension was incubated on ice (40 min) with
intermittent vortexing. The tubes were then centrifuged for 10 min (16,000 g). The
nuclear extract was decanted and stored at -80°C until analysis. Muscle glycogen content
was determined as glucose residues after hydrolysis of the muscle samples in 1 M HCl
(100°C, 2 h) and was corrected for free glucose in the sample (123). For ADP
measurements, frozen gastrocnemius-soleus-plantaris muscle samples (10 mg) were
powdered under liquid N
2
and homogenized as described above. The total cell

41

homogenates were used to measure ADP levels using a colorimetric assay kit and the
manufacturer’s instructions (Abcam, Cambridge, MA).
Western blot analysis. Approximately 20 µg of protein from the total cell homogenate
preparations were separated on a 10% gel via SDS-PAGE. Proteins were transferred onto
Immobilon-P polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA
in Tween-Tris-buffered saline (TBS) for 1 h. The membrane was then incubated (4ºC) in
5% BSA in Tween-TBS with antibodies (1:1000) against either phospho-ACC-Ser
79
,
total ACC, (Cell Signaling, Danvers, MA), or phospho-CaMKI-Thr
177
, total CaMKI,
(Santa Cruz Biotechnology, Santa Cruz, CA), or phospo-ERK1/2, total ERK1/2, or total
AS160/TBC1D4 (Cell Signaling, Danvers, MA), or CTP1, or CD36, or PGC1α, or
SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA). As previously determined (39), the
Akt substrate at 160 kd corresponds to AS160/TBC1D4 and the Akt substrate at 150 kd
corresponds to AS150/TBC1D1 and PAS-AS160/TBC1D4 and PAS-AS150/TBC1D1
(#9611, Cell Signaling, Danvers, MA) were quantified. Following this overnight
incubation, the membranes were probed with a secondary antibody (anti-rabbit IgG;
1:25,000) raised in goats (Pierce, Rockford, IL). Blots were then washed and subjected to
enhanced chemiluminescence (Pierce, Rockford, IL). Band density was quantified using
Scion Image (NIH, Bethesda, MD). All bands were compared to the band obtained for
the resting group WT mice. A ponceau S total protein stain (Sigma, St. Louis, MO) was
used on the membranes as a loading control.
Activity assays. AMPKα1 and α2 activities were measured using
32
P-ATP incorporation
into SAMS peptide (Upstate Signaling, Lake Placid, NY) as described (96). Briefly,
immunoprecipitates were added to an assay cocktail containing
32
P-ATP and SAMS

42

peptide. Post-incubation, an aliquot was spotted onto a piece of Whatman filter paper
and all paper samples were washed with phosphoric acid followed by an acetone wash.
Sample papers were analyzed for radioactivity in a Packard scintillation counter and
counts were used to calculate phosphotransferase activity. SIRT1 activity was measured
on nuclear extracts with a commercially available histone deactelyase (HDAC)
colometric activation kit (Active Motif, Carlsbad, CA) as previously described (98).
Briefly, nuclear extracts were added to assay buffers with assay substrates into a 96 well
microplate. Trichostatin A was added to the wells to inhibit class I, II, and IV HDAC and
250 µM NAD+ (Sigma) was added to activate SIRT1 which is NAD-dependent.
Following incubation (37°C, 60 min), the reaction was stopped with the addition of a
developing solution. The samples were read in a microplate reader at 405 nm.
Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and
percent and total palmitate oxidation were calculated, as described previously in detail
(125; 129). Both percent and total FA oxidation were corrected for label fixation by
using acetate correction factors (129). The specific activity for palmitate in the arterial
samples was not different between groups and did not vary over time, averaging
153.7±9.8, 160.2±12.6, and 141.9 ± 16.8 µCi/mmol, for the R, C, and MC groups,
respectively (P >0.05). Oxygen uptake, glucose uptake, and lactate release were
calculated as described (129). All uptake and release rates are expressed per gram of
perfused hindlimb muscles of both legs, which we determined to be 7% of total body
weight for WT and DN mice. Time effects for glucose, lactate, and FA concentrations
and FA kinetic data were analyzed using a two-way ANOVA with repeated measures for
each experimental condition (R, C, and MC). If there were no significant differences in

43

the values measured after 5, 10, 15, or 20 min of perfusion, mean values were used for
subsequent analysis. The effects of the experimental condition (R, C, and MC) and of
genotype (WT vs DN) were the two factors analyzed using a two-way ANOVA (Statview
5.0). Tukey-Kramer test for post-hoc multiple comparisons was performed when
appropriate. A significance level of 0.05 was chosen for all statistical methods.
RESULTS
Effects of DN transgene on basal protein expression and force production. In the rest
condition, hindlimb skeletal muscle was used to determine whether expression of the DN
transgene led to differences in the expression of key proteins implicated in the regulation
of substrate use in skeletal muscle. There were no alterations in protein content of SIRT1,
PGC1α, ACC, CPT1, CD36, ERK1/2, CaMKI, or AS160/TBC1D4 between WT and DN
mice (Table 2, Fig. 8). Force production by the gastrocnemius-soleus-plantaris muscle
group was not different (P>0.05) between the WT and DN mice (Fig. 9). No force
production was recorded for the rest or caffeine-treated groups.
Perfusion Characteristics. To verify physiological perfusion conditions oxygen uptake
was measured during the experimental perfusion period. There were no differences in
oxygen uptake over time in any of the experimental groups (P>0.05). Caffeine had no
effect on oxygen uptake compared with rates measured in the resting group (P>0.05,
Table 3). Muscle contraction resulted in an increase in oxygen uptake when compared to
the rest and caffeine conditions (P<0.05). For all experimental conditions, oxygen uptake
was not significantly different between WT and DN mice (P>0.05). Palmitate
concentration and delivery were not significantly different at any time point in any of the
groups, as dictated by the protocol (P>0.05, Table 3). In both WT and DN mice, muscle

44

ADP levels were not affected (P>0.05) by caffeine treatment or muscle contraction.
Similarly, the DN transgene had no effect on muscle ADP levels (P>0.05).
Substrate exchange across the hindlimb. Lactate release was increased (178%) during
muscle contraction (P<0.05, Table 3). The DN transgene did not affect lactate release in
any of the groups (P>0.05). Arterial perfusate glucose concentration did not vary over
time in any of the groups and mean glucose concentration was not different between any
of the groups (P>0.05, Table 3). Mean glucose uptake (nmol∙min
-1
∙g
-1
) was significantly
increased (P<0.05, Fig. 10C) by caffeine treatment (168%) but the DN transgene blunted
the caffeine-induced increase in glucose uptake by 34% (P<0.05, Fig. 10C). During
muscle contraction time-dependent differences were uncovered between the WT and DN
mice. Within 5 min of initiating the muscle contraction protocol, glucose uptake had
increased by 156% in the WT mice and this elevated glucose uptake rate was maintained
throughout the rest of the contraction period (P>0.05, Fig. 10A). In contrast, glucose
uptake rates were not elevated in the DN mice until the last 5 min (200% increase vs.
rest+DN condition at same time point) of muscle contraction (P<0.05, Fig. 10A). At rest
and during caffeine treatment glucose uptake did not vary over time (Fig. 10B). In the
rest condition, muscle glycogen content was 29% lower in the DN group when compared
to the WT group (P<0.05, Fig. 10D). In the WT mice, caffeine treatment and muscle
contraction resulted in a 33-38% reduction in muscle glycogen content (P<0.05, Fig.
10D). In the DN mice, muscle glycogen content was not affected by caffeine treatment
or muscle contraction (P>0.05).

Palmitate metabolism. At rest and during caffeine treatment palmitate uptake (pmol∙min
-
1
∙g
-1
) did not vary over time (P>0.05, Fig. 11B and 11E). Mean palmitate uptake was

45

significantly increased (P<0.05, Fig 11E) by caffeine treatment (54%) but there were no
DN effects. As shown for glucose uptake, time-dependent differences were found
between the WT and DN mice during muscle contraction. Within 5 min of initiating the
muscle contraction protocol palmitate uptake had increased 76% and 54% in the WT and
DN mice, respectively, over their resting condition counterparts and the elevated
palmitate uptake remained in the WT mice (P<0.05, Fig 11A). However, the contraction-
induced elevation in palmitate uptake had dropped off after 10 min in the DN mice and
palmitate uptake was not different from the resting condition for the remaining 10 min of
the muscle contraction protocol (P>0.05, Fig. 11A). In WT mice, mean palmitate
oxidation (nmol∙min
1
∙g
1
, Fig. 11F) increased by 180% with caffeine treatment and 260%
with muscle contraction. In the resting condition, palmitate oxidation was higher (120%)
in the DN group when compared to the WT group (P<0.05, Fig. 11F). Unlike glucose
uptake and palmitate uptake, there were no time-dependent effects found in the caffeine
or muscle contraction condition (P>0.05, Fig. 11C and 11D). Mean contraction-mediated
palmitate oxidation was blunted in the DN mice and not significantly different from the
resting condition (P>0.05, Fig 11F).
Enzyme activities. As expected, AMPKα2 activity (pmol∙min
-1
∙g
-1
) was decreased by the
DN transgene (P<0.05, Fig. 12A). Caffeine treatment and muscle contraction increased
(P<0.05, Fig. 12A) AMPKα2 activity by 81% and 161%, respectively, but did not affect
(P>0.05) AMPKα1

activity (Fig. 12B). The DN transgene completely prevented the
increase in AMPKα2 activity with muscle contraction and caffeine treatment (Fig. 12A),
but did not affect (P>0.05) AMPKα1 activity (Fig. 12B). Total protein expression of
ACC was not affected by either caffeine treatment or muscle contraction and there were

46

no effects of the DN transgene (P>0.05, Fig. 12C). In the WT mice, caffeine treatment
and muscle contraction increased (P <0.05, Fig. 12C) phosphorylation of ACC by 32%
and 64% respectively. In line with the results obtained for AMPKα2 activity, the DN
transgene prevented the increase in phosphorylated ACC with caffeine treatment and
muscle contraction. In the rest condition, phosphorylated ACC was 11% lower in the DN
mice when compared to the WT mice.
Total protein expression of SIRT1 was not changed by either caffeine treatment or
muscle contraction and there were no effects of the DN transgene (P>0.05, Fig. 12D). In
the resting condition, SIRT1 activity was 44% higher in the DN mice when compared to
the WT mice (P<0.05, Fig. 12D). Caffeine treatment resulted in a 58% increase in
SIRT1 activity in the WT mice. Although there were no effects of the DN transgene in
the caffeine group there was not a subsequent increase in SIRT1 activity in the DN mice
with caffeine treatment over the already elevated SIRT1 activity observed at rest (Fig.
12D). The muscle contraction protocol yielded a 40% increase in SIRT1 activity in the
WT mice but no increase was observed in the DN mice (P<0.05, Fig. 12D).
Activation of Signaling Intermediates. Total protein expression of CaMKI, ERK1/2,
and AS160/TBC1D4 was not different between any of the groups and there no effects of
the DN transgene for any of the proteins (Fig. 13A, 13B, and 13C). Phosphorylation of
CaMKI
Thr177
was increased 150 and 104% with caffeine treatment and muscle
contraction, respectively (P<0.05, Fig. 13A). ERK1/2 phosphorylation was not affected
by caffeine treatment (P>0.05, Fig. 13B). However, with muscle contraction there was a
38% increase in ERK1/2 phosphorylation (P<0.05, Fig. 13B). Additionally,
phosphorylated AS160/TBC1D4 was increased 18 and 43% with caffeine treatment and

47

muscle contraction, respectively (P<0.05, Fig. 13C). AS150/TBC1D1 phosphorylation
was increased by 51% with muscle contraction (P<0.05, Fig. 13D) but there was no
effect of caffeine treatment (P>0.05, Fig. 13D).
DISCUSSION
Our data provide further evidence for the involvement of AMPKα2 in the
regulation of substrate use during muscle contraction. Additionally the results of this
study suggest that while AMPKα2 is activated by caffeine-induced increases in
intracellular [Ca
2+
], this activation is not essential to observe increases in substrate uptake
and FA oxidation during caffeine treatment. Additionally, these data suggest a role for
AMPK-independent Ca
2+
-dependent signaling in the regulation of substrate metabolism
in skeletal muscle. However, physiologically active AMPKα2 appears to be necessary to
record healthy time-dependent changes in FA uptake, glucose uptake, and FA oxidation
in contracting mouse skeletal muscle. Indeed, one of the more interesting findings of this
study is that AMPKα2 deficiency led to a reciprocal switch in FA and glucose uptake
during the 20-min contraction protocol.
Interestingly, during resting perfusion conditions FA oxidation rates were
elevated in mice that possessed the AMPKα2 DN transgene. These results were
surprising to us since AMPKα2 has been repeatedly shown to be an upstream regulator of
ACC activation, which in turn has been shown to modulate malonyl-CoA content
resulting in increases FA oxidation rates (97; 138). We hypothesized that low AMPKα2
activity measured in DN mice would result in lower or equivalent resting FA oxidation
rates as well as lower ACC phosphorylation when compared to WT mice. In line with
our hypothesis, we measured a decrease in ACC phosphorylation during the resting

48

condition in the DN mice, which is consistent with previous findings (78). But this
decrease in ACC phosphorylation state was not accompanied by a decrease in FA
oxidation rate. Furthermore, the higher FA oxidation rate at rest could not be explained
by higher levels of proteins known to be associated with the regulation of FA oxidation in
skeletal muscle, such as CD36, CPT1, and PGC1α. We did however measure an increase
in SIRT1 activity in the DN mice during the resting condition. Therefore, based on our
results, we hypothesize that low AMPKα2 activity resulted in the upregulation of
alternative signaling pathways which may involve SIRT1 activity. To date there is no
evidence to suggest that SIRT1 may directly be responsible for the increase in FA
oxidation observed in DN mice at rest. However, the possibility remains that in DN mice
higher activity of the nuclear factor SIRT1 may have led to alterations in the expression
or activation of as yet unidentified signaling intermediates involved in the regulation of
FA oxidation and/or in the gene expression of downstream targets of AMPKα2. Given
that AMPKα2 is known to regulation gene expression (REF), the deficiency in AMPKα2
activity associated with our model was likely associated with alterations in the expression
of some genes in DN mice when compared to their WT littermates. This conclusion is
consistent with the possibility that there are multiple redundant pathways responsible for
regulating substrate metabolism in skeletal muscle.
It has been shown by us and others that caffeine-induced increases in Ca
2+

signaling regulates substrate metabolism possibly via AMPK activation (2; 34; 59; 95).
Indeed, we have shown that when either CaMKII or CaMKK were inhibited during
caffeine stimulation there were subsequent decreases in AMPKα2 activity along with
decreases in the rates of FA uptake, glucose uptake, and FA oxidation (2; 95). In line

49

with these previous results, we measured an increase in AMPKα2 activity in caffeine-
treated WT mice in this study. Because caffeine-induced AMPKα2 activity was reduced
in the DN mice, we expected to measure coincident decreases in FA uptake and oxidation
with caffeine treatment. However, there were no measurable differences between the DN
and WT mice in FA metabolism during caffeine treatment. In contrast, caffeine-induced
glucose uptake was blunted in the DN mice. This reduction in glucose uptake is
consistent with the view that increases in intracellular [Ca
2+
]-induced signals that are
independent of AMPK activation may play a significant role in the regulation of skeletal
muscle glucose uptake (59; 141). Our results further suggest that AMPKα2 activation
may be more critical to the regulation of glucose metabolism during states of increased
intracellular [Ca
2+
] induced by caffeine than to the regulation of FA metabolism in
skeletal muscle. It has also been postulated by some that AMPK activation is associated
with increased expression of a downstream target of the CaMKK pathway such as
CaMKIV during low energy states (150). However, we did not measure any effects of
the DN transgene on the expression or phosphorylation state of CaMKI, an additional
downstream target of CaMKK (33; 119) in any of the experimental conditions. These
data suggest that CaMKK signaling either lies upstream of AMPKα2 or is not affected by
low AMPKα2 activity. While no caffeine-induced force production was observed in our
study, the increase in Ca
2+
release from the sarcoplasmic reticulum induced by caffeine
treatment may have been associated with a small twitch potentiation that was
unrecordable with our system. We consider this possibility to be unlikely because
caffeine-induced twitch potentiation and force production have not been measured at
concentration lower than 3.5 mM in mixed skeletal muscle (148). Taken together, our

50

results suggest that caffeine-induced AMPKα2 activation is important in the regulation of
glucose uptake, but is not required to measure increases in FA uptake and oxidation in
skeletal muscle during caffeine treatment.
Because significant metabolic shifts were observed during muscle contraction in
the DN mice, our data suggest that appropriate AMPKα2 activation is necessary to
measure a healthy response in glucose and FA uptake and in FA oxidation during muscle
contraction. These conclusions are in line with previous studies in which AMPKα2
appears to be a necessary signal in the regulation of substrate metabolism during muscle
contraction or treadmill exercise (72; 73). However, our data are not consistent with
some additional reports in which low AMPKα2 activity did not result in a decrease in
glucose uptake (78) or FA oxidation following treadmill exercise, or FA oxidation in
incubated EDL and soleus muscles during AICAR and muscle contractions (32; 84). One
explanation for this discrepancy could be due to the mode of exercise. In the current
study, we utilized an in situ electrical stimulation protocol and measured substrate uptake
and FA oxidation during moderate intensity muscle contraction. In contrast, in one of the
aforementioned studies (84), FA oxidation was measured in incubated muscle strips
following a low intensity treadmill exercise protocol. As such, data collected in that
particular study might be more appropriately viewed as post-exercise data. Further, ex
vivo incubated muscle were shown to have no difference in FA oxidation in the same
mouse that was employed in the current study during AICAR stimulation and muscle
contraction (32). Since our data is dramatically different from these studies it leads us to
conclude that the model in which substrate metabolism is studied is very important.
Additionally, in a recent study in which glucose uptake was measured in vivo in

51

chronically cannulated mice during treadmill exercise and low AMPKα2 activity did not
affect the exercise-induced increases in glucose uptake (78). While in the current study it
appears that AMPKα2 activation plays a key role in the time-dependent changes in
glucose and FA uptake during muscle contraction. Time-dependent analysis revealed
that decreased AMPKα2 activity impaired the ability of the contracting muscle to
increase glucose uptake during the early phase of the stimulation protocol and to maintain
higher rates of FA uptake throughout the stimulation protocol. Thus, differences in the
timing of metabolic measurements may also significantly affect the measurement of
metabolic results. This may explain why there are reports that AMPK activation is not
necessary to measure increases in glucose uptake following muscle contraction (37).
These previous results would be consistent with our data if the last 5 min of our
contraction protocol were examined, in that the skeletal muscle maintains the ability to
increase glucose uptake with muscle contractions. In this study, there appears to be a
complementary switch from FA uptake to glucose uptake throughout the 20 min muscle
contraction protocol. This suggests that there are alternative pathways that may be
activated in the absence of AMPKα2 activity in skeletal muscle. Therefore, although
there are overall decreases in FA and glucose uptake in the DN mice, there are
compensatory mechanisms which are activated in a time-dependent manner aimed at
maintaining substrate use in the contracting muscle.
Consistent with previous data in the same mouse model, DN mice had lower
resting muscle glycogen content and did not utilize glycogen to the same extent as the
WT mice during muscle contraction (85). However, this is not consistent with recent
reports in which the same mouse model utilized more relative muscle glycogen during

52

treadmill running in the quadriceps muscle (78). It is important to note that in our model
we freeze-clamp the entire hindlimb muscles consisting of gastrocnemius-soleus-
plantarius. As such differences in the muscle mixture may contribute to measured
differences in muscle glycogen content. Additionally in our study, during caffeine
treatment the DN mice did not utilize glycogen as the WT mice did. These data suggest
that AMPKα2 may be more involved in maintaining carbohydrate metabolism during
metabolic challenges such as caffeine treatment or muscle contraction.
We did not measure any transgenic specific decreases in the phosphorylation state
of additional signaling molecules implicated in the regulation of substrate use in skeletal
muscle. Others have shown that AS160/TBC1D4 is activated during muscle contraction
and that it may be a convergence point between insulin and muscle contraction signaling
(18). It has also been postulated that AMPK may be responsible for the increases in
AS160/TBC1D4 activation during muscle contraction (122). It was surprising to us that
AMPKα2 does not appear to be necessary to increase AS160/TBC1D4 or
AS150/TBC1D1 phosphorylation during muscle contraction, as it has been previously
reported that phosphorylation of these proteins may be dependent on AMPK (69; 116;
122). The major difference between our results and previous reports may be explained
by fiber type differences. The notion of fiber type specific regulation parallels previous
reports of the greatest increase in AICAR stimulated AS160/TBD1D4 phosphorylation
being present in the tibialis anterior (TA) muscle and AS150/TBC1D1 was found to be
most abundant, and its phosphorylation regulated by AICAR, in the TA (116), which was
not included in our mixed muscle group. Likewise an additional study used the TA
muscle for their 5 or 10 minute contraction protocols and found evidence for AMPK-

53

dependent phosphorylation of AS160/TBC1D4 (69). In another report the EDL muscle
was subjected to muscle contraction for a 10 min protocol and AMPKα2 was suggested
to be involved in the regulation of AS160/TBC1D4 phosphorylation (122). However, our
protocol utilized the gastrocnemius-soleus-plantaris muscle group for 20 minutes of
muscle contraction and it is a possibility that variations in the AMPKα2-dependent
phosphorylation of AS160/TBC1D4 and AS150/TBC1D1 may either be dependent on
fiber types or the timing of the muscle contraction protocol.
In line with our previous results, we measured increases in ERK1/2
phosphorylation during muscle contraction but not during caffeine treatment (95; 126).
Given that there were no effects of the DN transene on AS160/TBC1D4 and/or ERK1/2
phosphorylation, our results suggest that AMPKα2 activation is not necessary for the
increases in AS160 or ERK1/2 phosphorylation during moderate intensity muscle
contraction. Furthermore we cannot exclude the possibility that the measured increase in
AS160 and/or ERK1/2 phosphorylation may be responsible for the increase in glucose
uptake during the last 10 min of the muscle contraction protocol or the increase in FA
uptake at the onset of muscle contraction in the transgenic mice.
In summary, our results suggest that AMPKα2 activation is a necessary
component of metabolic signaling during muscle contraction especially as it relates to
time-dependent changes in the regulation of contraction-induced metabolism. Our data in
DN mice, also provide evidence for the notion that Ca
2+
-induced regulation of substrate
use can be maintained in an AMPK-independent manner in skeletal muscle during
caffeine treatment. The data from this study also bring to light the importance of the
timing of a muscle contraction protocol when measuring the effects of AMPK signaling

54

on the regulation of substrate utilization in skeletal muscle. It appears that when
AMPKα2 is inactive, in the skeletal muscle, in order to maintain muscle contraction there
is a switch FA and glucose uptake. Additional studies must be employed to examine the
contribution of alternative signaling molecules to AMPK at various time points of muscle
contraction in the regulation of fuel use. Overall, our data suggest that while AMPKα2 is
a critical signaling intermediate in the regulation of substrate utilization, it is not the sole
signal responsible for regulating skeletal muscle metabolism.
Acknowledgements
The authors would like to thank Emily Kallen, Matthew Burkhard, and Nadia El-Fakih
for their valuable technical expertise. The present study was supported by grants from
the University of Southern California Women in Science and Engineering (WiSE)
program, and by fellowships from the Integrative and Evolutionary Biology Program and
the Gold Family Foundation.

55

WT

DN

SIRT1 106.0±3.0 109.5±4.6
PGC1α 103.8±3.6 114.4±14.7
ACC 110.1±10.1 101.5±7.7
CPT1 101.3±4.6 100.4±1.5
CD36 102.3±0.2 107.6±5.0
ERK1/2 106.3±5.6 94.0±3.1
CaMKI 123.4±18.2 136.4±11.8
AS160

94.2±5.6 108.6±5.5
Table 2. Effect of AMPKα2 DN Transgene on Muscle Protein
Expression Values are expressed as means ± SE and are
expressed as percent of control WT muscle. Western blotting
was performed using homogenate samples prepared from
hindlimbs of WT (n=5) or AMPKα2 DN (n=5) mice.

56

Rest Caffeine MC

WT
(n=5)
DN
(n=6)
WT
(n=5)
DN
(n=5)
WT
(n=6)
DN
(n=5)
Oxygen uptake,
µmol∙g
-1
∙h
-1

5.8±1.5 6.6±1.3 5.5 ± 1.1 6.0±0.7 11.4±0.8
*#
10.3±1.6
*#

FA concentration,
µmol/l
543.4±37.0 512.7±31.1 597.5±20.1 552.2±24.1 604.3±47.5 541.8±34.8
FA delivery,
nmol∙min
-1
∙g
-1

337.0±52.2 301.6±93.9 375.3±32.3 391.1±60.3 413.7±66.0 337.8±28.7
Glucose
concentration,
mmol/l
7.0±0.4 6.9±0.2 7.1±0.2 7.4±0.2 7.2±0.1 6.7±0.3
Lactate release,
µmol∙g
-1
∙h
-1

13.2±1.8 14.3±1.9 10.3±1.2 14.3±1.3 36.7±4.9
*#
30.3±5.2
*#

ADP,
µmol/g
0.75±0.04 0.77±0.10 0.82±0.05 0.81±0.10 0.88±0.10 0.75±0.03
Table 3. Effect of AMPKα2 DN Transgene on Hindlimb Perfusion Characteristics during Rest,
Caffeine Treatment or Muscle Contraction. Values are expressed as means ± SE; n= number of
mice. Wild-type (WT) or AMPKα2 DN mice were perfused during rest, caffeine treatment or
moderate intensity muscle contraction (MC). * P< 0.05 compared with respective rest group, #
P<0.05 compared with caffeine group.

57

CD36
SIRT1
PGC1 α
CPT1
ACC
ERK1/2
CaMKI
AS160
WT DN
Figure 8. Effect of AMPKα2 DN transgene on skeletal muscle protein expression of multiple
signaling intermediates in R groups from a mixed gastrocnemius-soleus-plantaris muscle
preparation. Western blots represent total protein content of SIRT1, PCG1α, ACC, CPT1, CD36,
ERK1/2, CaMKI, and AS160/TBC1D4. Values are means ± SE for WT (n= 5) and DN (n=6)
mice.

58

Figure 9. Effect of AMPKα2 DN transgene on force production over the 20-min contraction
period. Force produced by hindlimb muscles of WT (n=3; open circles) and DN (n=3; closed
circles) mice.

59

Figure 10. Effect of AMPKα2 DN transgene on time effects for glucose uptake in muscle
contraction groups (A) and caffeine groups (B) mean glucose uptake (C) and glycogen content
(D) in perfused mouse hindlimbs during caffeine treatment or moderate intensity muscle
contraction. In figure 3A, open squares represent the Rest + WT group, closed squares represent
Rest + DN group, open circles represent MC + WT group, and closed circles represent MC + DN
group. In figure 3B, open squares represent the Rest + WT group, closed squares represent Rest
+ DN group, open triangles represent Caff + WT group, and closed triangles represent Caff + DN
group. In figures 3C and 3D, open bars represent wild type (WT) mice and solid bars represent
AMPKα2 DN mice. Values are means ± SE for Rest + WT (n= 5), Rest + DN (n=6), Caffeine +
WT (n=5), Caffeine + DN (n=5), MC (n=6), and MC + DN (n=5). *P<0.05 compared with
respective rest group; # P<0.05 compared with respective WT group; & P<0.05 compared with
rest + WT group; ## P<0.05 compared with caffeine groups; ** P<0.05 compared with MC + DN
group; † P<0.05 compared with 5-min time point; *** P <0.05 compared with 10-min time point.

60

Figure 11. Effect of AMPKα2 DN transgene on time effects for palmitate uptake in muscle
contraction groups (A) and caffeine groups (B), time effects for palmitate oxidation in muscle
contraction groups (C) and caffeine groups (D) and mean palmitate uptake (E) and palmitate
oxidation (F) in perfused mouse hindlimbs during caffeine treatment or moderate intensity
muscle contraction. In figures 4A and 4C, open squares represent the Rest + WT group, closed
squares represent Rest + DN group, open circles represent MC + WT group, and closed circles
represent MC + DN group. In figures 4B and 4D, open squares represent the Rest + WT group,
closed squares represent Rest + DN group, open triangles represent Caff + WT group, and closed
triangles represent Caff + DN group. In figures 4E and 4F, open bars represent wild type (WT)
mice and solid bars represent AMPKα2 DN mice. Values are means ± SE for Rest + WT (n= 5),
Rest + DN (n=6), Caffeine + WT (n=5), Caffeine + DN (n=5), MC + WT (n=6), and MC + DN
(n=5). *P<0.05 compared with respective rest group.

61

Figure 12. Effect of AMPKα2 DN transgene on AMPKα2 activity (A), AMPKα1 activity (B),
ACC phosphorylation (C), and SIRT1 expression and activity (D) in perfused mouse hindlimbs
during caffeine treatment or moderate intensity muscle contraction. Activity values are
calculated per gram of muscle present in assay preparation (AMPK) or per µg of protein present
in assay preparation from nuclear extracts of perfused muscle samples (SIRT1). Preparations are
from the mixed gastrocnemius-soleus-plantaris muscle group. Values are means ± SE. Open bars
represent wild type (WT) mice and solid bars represent AMPKα2 DN mice. * P<0.05 compared
with rest + WT group; # P<0.05 compared with respective WT group.

62

Figure 13. Effect of AMPKα2 DN transgene on CaMKI (A), ERK1/2 (B), AS160/TBC1D4 (C),
and AS150/TBC1D1(D) protein expression and phosphorylation state in perfused mouse
hindlimbs during caffeine treatment or moderate intensity muscle contraction. Preparations are
from the mixed gastrocnemius-soleus-plantaris muscle group. Values are means ± SE. Open bars
represent wild type (WT) mice and solid bars represent AMPKα2 DN mice. * P<0.05 compared
with respective rest group; # P<0.05 compared with respective WT group; † P<0.05 compared
with respective caffeine group.

63

V. EXPERIMENT 3

AMPKα2 is an essential signal in the regulation of insulin-stimulated fatty acid
uptake in control-fed and high fat-fed mice
CHAPTER ABSTRACT
There have been multiple proposed targets in the treatment of insulin resistance,
however, AMP-activated protein kinase (AMPK) has been of central focus. The purpose
of the current study was to determine the role of AMPKα2 activity in the regulation of
FA metabolism in insulin resistant skeletal muscle. Male C57BL/6 mice were randomly
divided into control diet or high fat diet fed groups for six weeks and were either wild
type (WT) or possessed an AMPKα2 dominant negative transgene (DN). After 6 wks,
hindlimbs of CD (n=10) and HFD (n=10) mice were perfused (550µM palmitate, 6mM
glucose, [1-
14
C]palmitate) ±450µU insulin (IS). CD (n=8) and HFD (n=8) muscles were
used for basal protein expression. In CD, AMPKα2 deletion did not affect basal FA
uptake (FAU), but it increased basal FA oxidation (FAO) by 81% and α2 deletion
prevented the IS increase in FAU and decrease in FAO. HFD abolished the IS increase in
FAU and decrease in FAO in WT. In HFD, α2 deletion increased basal FAU by 139%
(P<0.05) but did not affect IS FAU nor FAO (P>0.05). IS SIRT1 activity was higher in
HFD-DN than HFD-WT (P<0.05). Neither DN nor HFD affected PGC1α content. HFD
increased (P<0.05) PTP1b, SIRT1, and CD36 content and α2 deletion prevented the
HFD-induced increases in PTP1b and SIRT1. In HFD, α2 deletion decreased (P<0.05)
CPT1 content. The data from this study provide evidence for the involvement of
AMPKα2 in the regulation of insulin stimulated fatty acid metabolism both in CD and

64

HFD mice. In contrast, it does not appear that AMPKα2 is necessary in the regulation of
basal and/or insulin stimulated glucose uptake in CD and HFD mice.
CHAPTER INTRODUCTION
A precursor and a detrimental indicator of type 2 diabetes is insulin resistance in
the skeletal muscle (9; 29). While the mechanisms leading to the development of insulin
resistance have not been completely identified, there is evidence that its progression is
linked to alterations in fatty acid (FA) metabolism (1; 25; 71). Furthermore insulin
resistance has been shown to be associated with an inflammatory response associated
with increased circulating fatty acids and or obesity (11; 88). There have been multiple
proposed targets in the treatment of insulin resistance, however, AMP-activated protein
kinase (AMPK) has been of central focus (103; 139).
AMPK has been studied extensively for its role as an energy sensor during states
of low energy balance, such as muscle contraction and hypoxia (56; 86). Additionally,
there have been multiple studies linking a high fat diet to alterations in AMPK activity
(74; 80; 113). A recent study has linked the deletion AMPKα2 activity to an attenuation
of high fat diet induced insulin resistance as it pertains to glucose metabolism (38).
However, it still remains to be elucidated what contributions AMPKα2 has on the
regulation of FA metabolism in the development of insulin resistance in skeletal muscle.
Further, the mechanisms by which alterations in substrate metabolism occur, following
high fat feeding, has not yet been identified.
The development of insulin resistance in skeletal muscle, as a result high fat
feeding, is a complex process. It has been demonstrated that adipose tissue is responsible
for increasing pro-inflammatory proteins in the circulation as a result of high fat feeding

65

(147). It has also been shown that increases in pro-inflammatory molecules in the
circulation, such as IL6, maybe a link between adipose tissue and other tissues following
high fat feeding and result in insulin resistance (67). Other pro-inflammatory molecules
expressed in adipose tissue, such as TNFα, have been implicated in regulating insulin
signaling in other insulin sensitive tissues following high fat feeding (147). However, the
role of cross tissue talk originating in the skeletal muscle not been extensively examined.
The purpose of the current study was to determine the role of AMPKα2 activity in
the regulation of FA metabolism in insulin resistant skeletal muscle. Furthermore, we
sought to determine which signaling intermediates may lie downstream of AMPKα2
signaling in the development of insulin resistance in skeletal muscle. Finally, we wanted
to establish if inactivating AMPKα2 in skeletal muscle would play a role in the regulation
of protein expression of inflammatory markers in the adipose tissue during high fat
feeding. We utilized a transgenic mouse strain expressing a dominant negative α2
subunit in skeletal muscle and cardiac muscle to determine the role of AMPK in skeletal
muscle insulin resistance (86). We hypothesized that the lack of AMPKα2 activity would
result in significant alterations in FA metabolism as well as glucose uptake in skeletal
muscle following a high fat diet in mice.
MATERIALS AND METHODS
Animal Preparation. Male C57BL/6 (2-3 months old) mice were kept on a 12:12-h
light-dark cycle and animals were randomly divided into control diet (CD, n=20) or high
fat diet (60% fat Bio-Serv; HFD, n=20) fed groups for six weeks. Mice were either wild
type (WT) or possessed an AMPKα2 dominant negative transgene (DN) kindly provided
by M.J. Birnbaum (University of Pennsylvania, Philadelphia, PA). Food intake and body

66

weight were monitored every week and blood glucose and plasma insulin were measured
every two weeks by tail vein puncture at 5-6 hours after the active feeding period. Blood
glucose was measured using a One-Touch Ultra Glucometer (Lifescan, Milpitas, CA) and
an ELISA kit was used to measure plasma insulin (ALPCO, Salem, NH). At the end of
the six weeks of feeding CD (n=10) and HFD (n=10) mice were randomly selected to
undergo hindlimb perfusions while the remaining CD (n=10) and HFD (n=10) mice were
used for basal muscle preparations. All procedures for the present study were approved
by the Institutional Animal Care and Use Committee at the University of Southern
California.
Hindlimb Perfusion. On the day of the experiment, mice were anesthetized by the
administration of an intraperitoneal injection of ketamine/xylazine cocktail (20 mg/kg
body weight). Epididymal adipose tissues were freeze-clamped in situ with pre-cooled
aluminum clamps, removed and stored in liquid N
2
for further analyses. If animals were
selected for basal muscle preparation then the gastrocnemius-soleus-plantaris muscle
groups were freeze-clamped in situ with pre-cooled aluminum clamps, removed and
stored in liquid N
2
for further analyses. Then the mice were then euthanized with an
intracardial injection of pentobarbital sodium (0.4 mg/g body weight).
Otherwise, surgical preparation for the hindlimb perfusion was performed as previously
described (105; 129; 130). Before placement of the perfusion catheters, 15 IU of heparin
was injected into the inferior vena cava. The mice were then euthanized with an
intracardial injection of pentobarbital sodium (0.4 mg/g body weight), and catheters were
immediately inserted into the descending aorta and ascending vena cava, the hindlimbs

67

were then washed extensively with saline. The prepared mouse was then placed in a
perfusion apparatus for the experimental perfusion periods.
The perfusate consisted of Krebs-Henseleit solution, 5% bovine serum albumin
(Millipore, Billerica, MA), 550 M albumin-bound palmitate, 24 µCi of albumin-bound
[1-
14
C] palmitate, 6 mM glucose. The perfusate was kept at 37°C and was continuously
gassed with a mixture of 95% O
2
-5% CO
2
with arterial pH levels between 7.08-7.65 and
arterial P
O2
and P
CO2
values were between 171-390 and 27-42 mmHg, respectively.
Perfusion pressures were not affected by any of the experimental conditions and averaged
66.6 ± 14.5, 64.0 ± 22.5, 35.8 ± 10.8, and 48.6 ± 7.9 mmHg in the CD + WT, CD + DN,
HFD + WT, and HFD + DN groups, respectively (P > 0.05).
The perfusion preparation was equilibrated for 20 minutes. Perfusion flow rate
was maintained at 1.5 ml/min for all groups (average: 0.46 ± 0.02 ml/min/g of perfused
muscle). A 15 minute basal perfusion, no insulin, ensued and arterial and venous samples
were taken at 5, 10, and 15 min. Then 450 µU of insulin was added and an additional
equilibration period of 15 minutes took place. An insulin stimulated perfusion period
followed for 25 minutes and arterial and venous samples were taken at 35, 40, 45, 50, and
55 minutes for further analyses. Following the completion of the 55 min experimental
perfusion period, the gastrocnemius-soleus-plantaris muscle groups were freeze-clamped
in situ with pre-cooled aluminum clamps, removed and stored in liquid N
2
for further
analyses. This muscle was considered insulin-stimulated muscle and was used in
appropriate preparations.
Plasma sample analyses. Plasma samples collected during the perfusion were analyzed
to determine FA, glucose, and lactate concentrations as well as radioactive [
14
C]FA and

68

14
CO
2
contents. A WAKO NEFA HC kit (WAKO Chemicals, Richmond, VA) was used
to measure plasma FA concentrations spectrophotometrically. An YSI-1500 (Yellow
Springs Instruments, Yellow Springs, OH) analyzer was used to measure glucose and
lactate concentrations in the collected plasma samples. Plasma [
14
C]FA and
14
CO
2
radioactivities were measured as previously described (125; 127; 129). P
CO2
, P
O2
, and pH
were determined by utilizing an ABL-5 analyzer (Radiometer America, Westlake, OH).
Tissue sample preparation. For western blot analysis, frozen muscle samples and
adipose tissue samples (40 mg) were powdered under liquid N
2
and homogenized in 500
µl of ice-cold RIPA buffer as previously described (94; 96). The total cell homogenate
was then transferred to a microcentrifuge tube and vortexed frequently for 1 h whereupon
the samples were centrifuged at 4,500 g at 4ºC, for 1 h. For immunoprecipitation
procedures, approximately 90 mg of powdered basal muscle samples were homogenized
in HEPES buffer and centrifuged at 15,000 g for 5 min. Supernatants (200 µg) were
incubated with antibodies for AMPKα1 or AMPKα2 (Santa Cruz Biotechnology, Santa
Cruz, CA) for 2 h at 4ºC with gentle agitation (95). Following the incubation, Protein
A/G agarose (Santa Cruz, SC-2003) was added to the tubes and incubated overnight at
4ºC with gentle agitation. The immunoprecipitates were collected by centrifugation.
Pellets were washed with phosphate-buffered saline (PBS) buffer and the final
supernatants were re-suspended in sucrose homogenizing buffer and stored at -80ºC until
analysis. Protein concentrations were determined with the Bradford protein assay
(BioRad, Hercules, CA). For nuclear extraction procedures, a nuclear extraction kit was
used (Pierce, Rockford, IL) and the manufacturer’s instructions were followed. Briefly,
approximately 40 mg of muscle samples from the insulin-stimulated perfusion were

69

homogenized in a cytoplasmic extraction buffer (CERI). The homogenate was vortexed
and incubated on ice for 10 min at which time and additional cytoplasmic extraction
buffer (CERII) was added to the tube. Following a 5 min centrifugation step (16,000 g),
the recovered pellet was resuspended in a nuclear extraction buffer (NER). The
suspension was incubated on ice (40 min) with intermittent vortexing. The tubes were
then centrifuged for 10 min (16,000 g). The nuclear extract was decanted and stored at -
80°C until analysis.
Western blot analysis. Approximately 20 µg of protein from the total cell homogenate
preparations were separated on a 10% gel via SDS-PAGE. Proteins were transferred onto
Immobilon-P polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA
in Tween-Tris Buffered Saline (TBS) for 1 h. The membrane was then incubated (4ºC) in
5% BSA in Tween-TBS with antibodies (1:1000) against either phospho-ACC-Ser
79
,
total ACC, (Cell Signaling, Danvers, MA), PGC1α, CPT1, CD36, phospho-Akt-Thr
308
,
total Akt, IL6, or TNFα (Santa Cruz Biotechnology, Santa Cruz, CA). Following this
overnight incubation, the membranes were probed with a secondary antibody (anti-rabbit
IgG; 1:25,000) raised in goats (Pierce, Rockford, IL). Blots were then washed and
subjected to enhanced chemiluminescence (Pierce, Rockford, IL). Band density was
quantified using Scion Image (NIH, Bethesda, MD). All bands were compared to the
band obtained for a control sample of non-perfused muscle and expression was expressed
as percent control. A ponceau S total protein stain (Sigma-Aldrich, St. Louis, MO) was
used on the membranes as a loading control.
Activity assays. AMPKα1 and α2 activities were measured using
32
P-ATP incorporation
into SAMS peptide (Upstate Signaling, Lake Placid, NY) as described (96). Briefly,

70

immunoprecipitates were added to an assay cocktail containing
32
P-ATP and SAMS
peptide. Post-incubation, an aliquot was spotted onto a piece of Whatman filter paper
and all paper samples were washed with phosphoric acid followed by an acetone wash.
Sample papers were analyzed for radioactivity in a Packard scintillation counter and
counts were used to calculate phosphotransferase activity. SIRT1 activity was measured
on nuclear extracts with a commercially available histone deacetylase (HDAC)
colometric activation kit (Active Motif, Calrsbad, CA). Nuclear extracts were added to
assay buffers with assay substrates in a 96 well microplate. Trichostatin A was added to
the wells to inhibit class I, II, and IV HDAC’s and 250 µM NAD+ (Sigma-Aldrich, St.
Louis, MO) was added to activate SIRT1 which is NAD-dependent. Following
incubation (37°C, 60 min), the reaction was stopped with the addition of a developing
solution. The samples were read in a microplate reader at 405 nm.
Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and
percent and total palmitate oxidation were calculated, as described previously in detail
(125; 129). Both percent and total FA oxidation were corrected for label fixation by
using acetate correction factors (129). The specific activity for palmitate in the arterial
samples was not different between groups and did not vary over time, averaging 149.2 ±
13.6, 189.2 ± 27.8, 128.8 ± 6.7, and 152.9 ± 10.2 µCi/mmol, for the CD + WT, CD +
DN, HFD + WT, and HFD + DN groups, respectively (P >0.05). Oxygen uptake,
glucose uptake, and lactate release were calculated as described (129). All uptake and
release rates are expressed per gram of perfused hindlimb muscles of both legs, which
has been previously determined to be 11% of total body weight (132). Time effects for
glucose, lactate, and FA concentrations and FA kinetic data were analyzed using a two-

71

way ANOVA with repeated measures in each of the experimental groups. If there were
no significant differences in the values measured after 5, 10, 15 or 35, 40, 45, 50, 55
minutes of perfusion, mean values were used for subsequent analysis. Data for CD and
HFD conditions were analyzed separately using a two-way ANOVA’s with perfusion
treatment (Basal vs. Insulin) and genotype (WT vs. DN) as the two analysis factors
(Statview 5.0). Tukey-Kramer test for post-hoc multiple comparisons was performed
when appropriate. A significance level of 0.05 was chosen for all statistical methods.
RESULTS
Effects of DN transgene on physiological characteristics. Body weight, blood glucose,
and plasma insulin are summarized in Table 4. There were no time effects present in the
CD mice for body weight, blood glucose, or plasma insulin (Table 4, P>0.05). HFD
mice had significantly higher body weight and plasma insulin by week 4 over the start of
the feeding protocol (Table 4, P<0.05). Blood glucose tended to be higher by week 4 but
was not significantly elevated (P<0.05) until week 6 of the high fat feeding protocol.
There were no genotype effects measured in any of the groups throughout the six week
feeding period. There were no differences in the amount (g) of food that was eaten
throughout the 6 weeks of feeding in either the CD or HFD groups and the average food
consumed was 25.2± 0.5 and 21.3±0.4, respectively (data not shown). However, high fat
fed mice consumed 18% more (P<0.05) kcal when compared to the CD group with no
measured genotype effects (data not shown). There were no effects of the DN transgene
on any of the parameters.
Perfusion Characteristics. To verify physiological perfusion conditions oxygen uptake
was measured during the experimental perfusion period. There were no differences in

72

oxygen uptake over time in any of the experimental conditions (P>0.05, Table 5).
Palmitate concentration and delivery were not significantly different at any time point in
any of the groups or experimental conditions, as dictated by the protocol (P>0.05, Table
5). Arterial perfusate glucose concentration did not vary over time in any of the groups
and mean glucose concentration was not different between any of the experimental
conditions (P>0.05, Table 5).
Substrate Exchange Across the Hindlimb. Lactate release was not different throughout
the perfusion period or between any of the experimental conditions (P>0.05, Table 5).
Glucose uptake (nmol•min
-1
•g
-1
) did not vary over time throughout the perfusion and was
not different in any of the experimental conditions. In the CD group, mean glucose
uptake increased 3-fold and 2-fold in WT and DN mice, respectively, with the addition of
insulin to the perfusate (P<0.05, Fig.14A). In the HFD group there were no effects of
insulin on glucose uptake and there were no genotype effects in either CD or HFD groups
(P>0.05, Fig. 14A and 14B). In the CD group, palmitate uptake (nmol•min
-1
•g
-1
)
increased 35% (P<0.05, Fig. 14C) when insulin was added to the perfusate in WT mice
however, there was no measureable increase in palmitate uptake in DN mice when insulin
was added (P>0.05, Fig. 14C). There was no insulin effect in HFD WT mice (P>0.05,
Fig. 1D). Palmitate uptake was 137% higher in HFD DN mice in the basal condition
when compared to the HFD WT basal mice (P<0.05, Fig. 14D). Once insulin was added
to the perfusate this increase was abolished in the HFD DN mice (P<0.05, Fig. 14D). As
we have previously reported (3), there was an 83% increase in palmitate oxidation rates
(nmol•min
-1
•g
-1
) in CD DN mice when compared to CD WT mice (P<0.05, Fig. 15A).
There were no significant effects of insulin on the rate of palmitate oxidation in any of

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the CD mice, however there was a 26% decrease in oxidation rates when insulin was
added to the perfusion in WT mice, suggesting a trend towards a decrease in palmitate
oxidation (Fig. 15A). The addition of insulin resulted in 93% and 64% higher rates of
palmitate oxidation in the HFD WT and DN, respectively, mice (P<0.05, Fig 15B).
Enzyme Activities. As expected, AMPKα2 activity (pmol•min
-1
•g
-1
) was decreased by
the DN transgene in CD and HFD mice (P<0.05, Fig. 16A and 16B). However, in the
HFD WT mice there was 39% lower AMPKα2 activity when compared to CD WT mice
(P<0.05, Fig 16B). There were no measured differences in AMPKα1 activity in any of
the experimental conditions (P>0.05, Fig. 16C and 16D). Total protein expression of
ACC was not different in any of the experimental groups (Fig. 16E and 16F). In line
with results obtained for AMPKα2 activity there was significantly reduced
phosphorylated ACC content in the DN mice in both CD and HFD (Fig. 17E and 17F).
SIRT1 protein expression was 82% higher in HFD WT mice when compared to CD WT
mice (P<0.05, Fig. 18A and 18B). However, insulin stimulated SIRT1 activity was not
significantly higher in HFD WT mice but HFD WT mice had 111% higher SIRT1
activity when compared to the CD WT mice (P<0.05, Fig. 18E and 18F).
Skeletal Muscle Protein Expression. PTP1b expression was 50% lower in HFD DN
mice when compared to HFD WT mice (P<0.05, Fig. 18C and 18D) and there were no
differences in the CD mice. There were no measured differences in PGC1α protein
expression in any of the experimental conditions (P>0.05, Fig. 18A and 18B). CPT1
protein expression was 21% lower in the HFD DN mice when compared to the HFD WT
and mice (P<0.05, Fig. 18C and 18D). CD36 protein expression was 46% higher with
HFD with no genotype effects (P<0.05, Fig. 18E and 18F.) Phosphorylation of Akt on

74

Thr308 was 41% lower in HFD DN mice when compared to HFD WT mice (P<0.05,
Fig. 19G and 19H). IL6 protein expression was unchanged due to any of the
experimental conditions in skeletal muscle (Fig. 19A and 19B). TNFα expression was
17% lower in the CD DN mice when compared to the CD WT mice in skeletal muscle
(P<0.05, Fig. 19C).
Adipose Tissue Protein Expression. There were no differences in IL6 and TNFα
expression in the CD mice in adipose tissue (P>0.05, Fig. 19E and 19G). IL6 expression
was 2 fold lower in HFD DN mice when compared to the HFD WT mice (P<0.05, Fig.
19F). TNFα expression was 30% lower in HFD DN mice when compared to HFD WT
mice (P<0.05, Fig. 19H).
DICUSSION
The data from this study provide evidence for the involvement of AMPKα2 in the
regulation of insulin stimulated fatty acid metabolism both in CD and HFD mice. In
contrast, it does not appear that AMPKα2 is necessary in the regulation of basal and/or
insulin stimulated glucose uptake in CD and HFD mice. Additionally, these data suggest
that AMPKα2 may be involved in the expression and activation of proteins shown to be
associated with the regulation of substrate metabolism and insulin signaling, such as
CPT1, Akt, SIRT1, and PTP1b. Finally, we have also shown a role for AMPKα2, in
skeletal muscle, in the regulation of IL6 and TNFα protein expression in adipose tissue,
suggesting the existence of “cross-tissue talk.”
In CD mice it appears that AMPKα2 plays a role in the regulation of insulin
stimulated FA metabolism. Surprisingly when insulin was added to the perfusion in CD
DN mice FA uptake rates decreased as compared to the physiological increase that was

75

measured in CD WT mice. This suggests that the muscle is able to be stimulated by
insulin but in a negative manner when AMPKα2 activity is low in skeletal muscle.
Furthermore, in the HFD DN mice there was an increase in FA uptake basally when
compared to HFD WT mice, which was dramatically reduced once insulin was added to
the perfusion. If the muscle were insulin resistant we could postulate that there would be
no effect of insulin on FA or glucose uptake. Further, glucose uptake rates in the CD DN
mice were similar to the CD WT mice, in that, there was a subsequent increase in glucose
uptake with the addition of insulin to the perfusion in both groups. These results are in
line with a previous study that examined the role of AMPKα2 in insulin stimulated
glucose transport (38). In that study there was a nearly significant increase in glucose
uptake following 30 weeks of control diet and no insulin effect following 30 weeks of
HFD in a similar mouse model. However following the 30 week control diet feeding
protocol insulin was still able to increase glucose uptake in skeletal muscle of WT mice.
This discrepancy could be due to the background of mouse strain, in that particular study
the mice were FVB and the mice in the current study are C57BL6. It has been previously
determined that there may be a tendency towards a resistance to high fat feeding induced
insulin resistance in FVB mice (43). Our data from the current study suggest that intact
AMPKα2 activity is a necessary component in the regulation of insulin stimulated FA
uptake in skeletal muscle in both control fed and high fat fed mice.
Although it appears that AMPKα2 is crucial to the regulation of insulin stimulated
FA uptake in skeletal muscle this cannot be explained by alterations in the transport
protein CD36. There were no alterations in the protein expression of CD36 between WT
and DN mice in either CD or HFD conditions. However, we cannot exclude the

76

possibility of an increase or decrease of CD36 on the plasma membrane as a result of the
diet or insulin stimulation. It has been previously shown that CD36 translocates to the
plasma membrane with insulin stimulation, however, it has been shown that insulin and
AMPK act to regulate this translocations through different mechanisms (24; 77). It is a
possibility that a downstream target in the insulin signaling cascade may play a more
central role to the alterations in FA uptake with insulin stimulation when AMPKα2
activity is low than FA transport proteins. In line with this there were genotype specific
decreases in Akt phosphorylation and PTP1b expression following high fat feeding.
Taken together these results suggest that low AMPKα2 activity in skeletal muscle may
result in decreased insulin signaling in high fat feeding induced insulin resistance.
It is also important to note that there were no measureable differences in FA
oxidation with HFD between the WT and DN mice. This can lead to the conclusion that
AMPKα2 is not involved in alterations that occur in FA oxidation as a result of high fat
feeding. However, in line with our previous data (unpublished observations) we
measured an increase in FA oxidation in CD DN mice over the WT mice. We cannot
conclude that these increases in FA oxidation are due to an increase in ACC activity or an
increase in CPT1 content since we were unable to measure any differences in either of
these proteins in the CD condition. Interestingly in this study insulin stimulated SIRT1
activity is not increased in the DN mice when compared with the WT mice. But in the
HFD mice there is an extreme increase in SIRT1 activity in the DN mice, which does not
appear to correspond with any of our metabolic measurements.
Finally, another interesting finding of the current study is the alterations in
adipose tissue proteins with high fat feeding in the skeletal muscle specific AMPKα2 DN

77

mice. We measured an increase in IL6 and TNFα expression in adipose tissue following
six weeks of high fat diet, which has been shown previously (15; 90; 107). The increase
in protein expression of TNFα in adipose tissue corresponded to increases in protein
expression in skeletal muscle as well, which has been previously reported (15).
However, IL6 protein expression was not increased in skeletal muscle in any of the
experiment conditions, suggesting an alternative “signal” from the skeletal muscle.
AMPKα2 is thought to modulate energy balance in a positive manner and possibly
protect against insulin resistance (139), but the subsequent decreases in inflammatory
markers in the adipose tissue, in the DN mice, point to a negative effect of AMPKα2 in
skeletal muscle during high fat feeding on the inflammatory response. Taken together,
these results suggest a link for AMPKα2 signaling between skeletal muscle and adipose
tissue during high fat feeding.
In summary, the data presented in this study have provided novel information
regarding the role AMPKα2 in the regulation of FA metabolism in skeletal muscle. In
view of this data it appears that AMPKα2 is not necessary in the regulation of alterations
that occur in FA oxidation following high fat feeding in skeletal muscle. In contrast, the
current data suggests a role for AMPKα2 in the regulation of insulin stimulated FA
uptake in control fed and high fat fed mice. Finally, AMPKα2 appears to be a mediator
in cross talk that may occur between skeletal muscle and adipose tissue during high fat
feeding. It is important to elucidate the role of AMPKα2 in the regulation on of FA
metabolism as well as in the regulation of inflammatory marker expression in adipose
tissue. Insight into this regulation may provide important information for the prevention
of insulin resistance and type 2 diabetes.

78

Week 0

Week 2

Week 4

Week 6

WT DN WT DN WT DN WT DN
Control Diet
Body Weight
(g)

25.7±0.7

24.2±1.8

24.3±0.8

24.7±1.0

26.1±2.1

27.2±1

27.1±0.2

31.2±01.6

Blood Glucose
(mg/dl)
140.7±14.5 124.0±13.1 146.2±6.4 126.5±7.5 145.8±14.1 140.5±8.1 122.7±6.9 131.0±3.0

Plasma Insulin
(ng/ml)
0.9±0.1 0.8±0.1 0.9±0.6 0.9±0.1 1.0±0.2 1.1±0.1 0.8±0.1 0.6±0.1
High Fat Diet

Body Weight
(g)

25.0±2.3

23.1±2.2

27.1±3.2

28.4±2.2

39.4±3.3*

32.4±2.9*

35.6±0.7*

41.8±3.1*

Blood Glucose
(mg/dl)
139.0±11.9 152.9±7.5 159.2±9.8 158.9±7.8 162.9±11.4 180.4±16.0 215.6±14.3* 190.6±24.9*
Plasma Insulin
(ng/ml)

1.5±0.3

1.0±0.2

2.8±0.6

2.8±0.5

4.4±0.9*

4.1±0.9*

5.5±1.2*

5.6±1.7*
Table 4. Changes in body weight, blood glucose, and plasma insulin. Values are expressed as means ± SE.
n = 9-10 for all points. Blood glucose and plasma insulin were measured 5-6 hours after the dark period.
* P< 0.05 time effect.

79

Control Diet High Fat Diet

WT
(n=6)
DN
(n=6)
WT
(n=5)
DN
(n=5)
Oxygen uptake,
µmol∙g
-1
∙h
-1

7.1±1.5 7.0±2.1 4.8±0.7 5.0±0.5
FA concentration,
µmol/l
542.9±14.6 514.8±28.8 534.8±19.8 536.7±30.0
FA delivery,
nmol∙min
-1
∙g
-1

248.8±13.1 225.4±21.2 223.7±20.9 206.7±23.9
Glucose
concentration,
mmol/l
6.6±0.1 6.9±0.3 6.8±0.4 7.2±0.3
Lactate release,
umol∙g
-1
∙h
-1

11.6±0.5 12.1±0.9 9.9±1.2 11.2±1.5
Table 5. Perfusion characteristics of hindlimbs. Values are expressed as
means ± SE; n= number of mice. Mice perfused with either wild type
(WT) or AMPKα2 dominant negative transgene (DN).

80

Figure 14. Effect of AMPKα2 DN transgene and insulin on glucose uptake (A and B) and
palmitate uptake (C and D) in perfused hindlimbs of CD and HFD mice. Open bars represent
wild type basal perfusion period and closed bars represent insulin-stimulated perfusion period.
Values are means ± SE for CD WT (n=5), CD DN (n=5), HFD WT (n=5), and HFD DN (n=5). *
P<0.05 insulin effect, # P<0.05 DN effect, & P<0.05 compared with all other groups.

81

Figure 15. Effect of AMPKα2 DN transgene and insulin on palmitate oxidation in perfused
hindlimbs of CD (A) and HFD (B) mice. Open bars represent wild type basal perfusion
period and closed bars represent insulin-stimulated perfusion period. Values are means ± SE
for CD WT (n=5), CD DN (n=5), HFD WT (n=5), and HFD DN (n=5). * P<0.05 insulin
effect, # P<0.05 DN effect.

82

Figure 16. Effect of AMPKα2 DN transgene on AMPKα2 activity in CD (A) and HFD (B) mice,
AMPKα1 activity in CD (C) and HFD (D) mice, and phosphorylated ACC in CD (E) and HFD
(F) mice from non-perfused basal hindlimbs. Open bars represent wild type (WT) mice and
closed bars represent dominant negative (DN) mice. Values are means ± SE for CD WT (n=5),
CD DN (n=5), HFD WT (n=5), and HFD DN (n=5). # P<0.05 DN effect.

83

Figure 17. Effect of AMPKα2 DN transgene on SIRT1 expression in CD (A) and HFD (B) mice
and PTP1b expression in CD mice (C) and HFD mice (D) in non-perfused basal hindlimb
muscles. SIRT1 activity was measured on insulin-stimulated muscle from perfused hindlimbs of
CD (E) and HFD (F) mice. Open bars represent wild type (WT) mice and closed bars represent
dominant negative (DN) mice. Values are means ± SE for Basal Muscle (A): CD WT (n=5), CD
DN (n=5), HFD WT (n=5), and HFD DN (n=5). For Insulin-Stimulated muscle (B): CD WT
(n=5), CD DN (n=5), HFD WT (n=5), and HFD DN (n=5). # P<0.05 DN effect.

84

Figure 18. Effect of AMPKα2 DN transgene on PGC1α expression in CD (A) and HFD (B)
mice, CPT1 expression in CD (C) and HFD (D) mice, and CD36 expression in CD (E) and HFD
(F) mice from non-perfused basal hindlimbs muscle. Akt protein expression and phosphorylation
state was measured on insulin-stimulated muscle from perfused hindlimbs of CD (G) and HFD
(H) mice. Open bars represent wild type (WT) mice and closed bars represent dominant negative
(DN) mice. Values are means ± SE for Basal Muscle (A): CD WT (n=5), CD DN (n=5), HFD
WT (n=5), and HFD DN (n=5). For Insulin-Stimulated muscle (B): CD WT (n=5), CD DN
(n=5), HFD WT (n=5), and HFD DN (n=5). # P<0.05 DN effect.

85

Figure 19. Effect of AMPKα2 DN transgene on IL6 expression in CD (A) and HFD (B) mice
and TNFα expression in CD (C) and HFD (D) mice in skeletal muscle from perfused insulin
stimulated hindlimb muscles. Effect of AMPKα2 DN transgene on IL6 expression in CD (E)
and HFD (F) mice and TNFα expression in CD (G) and HFD (H) mice in adipose tissue. Open
bars represent wild type (WT) mice and closed bars represent dominant negative (DN) mice.
Values are means ± SE for Basal Muscle (A and B): CD WT (n=5), CD DN (n=5), HFD WT
(n=5), and HFD DN (n=5). For Adipose Tissue (C and D): CD WT (n=5), CD DN (n=5), HFD
WT (n=5), and HFD DN (n=5). #P<0.05 DN effect.

86

VI. EXPERIMENT 4

Exercise training-induced restoration of insulin sensitivity in skeletal muscle is
dependent on AMPKα2 activity in high fat fed mice
CHAPTER ABSTRACT
AMP-activated protein kinase (AMPK) has been studied extensively and
postulated to be a target for the treatment and/or prevention of metabolic disorders such
as insulin resistance. Exercise training has been deemed a beneficial treatment for
obesity and insulin resistance and may be sufficient to combat these disorders while on a
high fat diet. The purpose of this study was to determine if AMPKα2 activity in skeletal
muscle is required to gain the beneficial effects of exercise training while under high fat-
fed conditions. Wild type (WT) and AMPKα2 dominant negative (DN) male C57BL/6
were randomly divided into control diet (CD, n=10), training (TR, n=10), high fat diet
(60% fat Bio-Serv; HFD, n=12), or training with high fat diet (60 % fat Bio-Serv, TR +
HFD, n=12). Mice were either wild type (WT) or possessed an AMPKα2 dominant
negative transgene (DN) kindly provided by M.J. Birnbaum (University of Pennsylvania,
Philadelphia, PA). After 6 wks, hindlimbs of the mice were perfused (550µM palmitate,
6mM glucose, [1-
14
C]palmitate, and 450µU insulin). In the HFD group TR resulted in a
40% increase in glucose uptake in WT mice (P<0.05). In the CD group palmitate uptake
was 43% lower in the DN mice when compared to the WT mice and TR resulted in a
46% increase in palmitate uptake rates (P<0.05). In the HFD TR resulted in a 93 and
108% increase in palmitate uptake rates the WT and DN mice, respectively, over the
sedentary mice (P<0.05). In the CD group TR resulted in 2-fold higher palmitate
oxidation rates in the WT mice and there was a 30% increase in palmitate oxidation in the

87

TR DN (P<0.0). AMPKα2 signaling appears to be required for alterations in glucose
uptake in high fat fed endurance trained mice it does not appear to be required for
alterations that occur in FA metabolism under these conditions.
CHAPTER INTRODUCTION
Obesity is a growing epidemic in our modern society, resulting in the contribution
and development of numerous diseases and disorders. It is widely accepted that exercise
training is beneficial in treating obesity in humans (30; 89; 109). Furthermore, it has
been shown that exercise training, while on a high fat diet, has the ability to restore
insulin sensitivity in rodent models (58; 68). It is not clear however, which signaling
molecules are responsible for the improved insulin sensitivity that occurs with exercise
training in either obese or high fat fed models.
AMP-activated protein kinase (AMPK) has been studied extensively in both acute
and chronic exercise studies (31; 56; 112). AMPK is an energy-sensing enzyme that
responds to an increase in the AMP:ATP ratio that occurs with physical stress, such as
hypoxia or muscle contractions (86). Recently a great deal of data has suggested a role
for AMPK in the prevention and/or treatment of insulin resistance (36; 104; 139; 149).
We have shown (unpublished observations) that AMPKα2 signaling may be involved in
the regulation of the development of insulin resistance and these data are in line with
other reports (38). In contrast, it has also been suggested that AMPKα2 may not be
necessary for metabolic adaptations that occur with exercise training (61). Therefore, the
exact role AMPK signaling in the regulation of insulin sensitivity in skeletal muscle must
be elucidated.

88

If AMPK is essential to obtain the beneficial effects of exercise training while on
a high fat diet it is unclear which mechanism or signaling pathways it may act through.
Recently there has been extensive speculation about the relationship of Sirtuin 1 (SIRT1)
and AMPK (21; 22; 54; 106). It has been hypothesized that AMPK may exert its
beneficial effects of exercise training through SIRT1 (21). Furthermore, we have shown
alterations in SIRT1 activity as a result of acute muscle stimulation and high fat feeding
(unpublished observations) in an AMPKα2 DN model specific to skeletal and heart
muscle (86). Beyond the relationship with SIRT1, it is yet to be elucidated if AMPK
signaling acts through stress kinase signaling pathways to regulate exercise adaptations to
chronic endurance training and what effects this may play in the prevention of insulin
resistance (51; 118).
The purpose of this study was to determine if AMPKα2 activity is a necessary
signal in the regulation of improved insulin sensitivity that occurs with endurance
training while under high fat feeding conditions. An AMPKα2 dominant negative (DN)
transgenic mouse model was subjected to six weeks of voluntary wheel running while on
either a control or high fat diet (86). Hindlimb perfusion procedures were utilized to
analyze substrate kinetics in the skeletal muscle as a result of the experimental
procedures. We hypothesized that AMPKα2 is a necessary signal in the restoration of
insulin sensitivity that occurs with endurance training under high fat feeding conditions.
MATERIALS AND METHODS
Animal Preparation. Male C57BL/6 (2-3 months old) were kept on a 12:12-h light-dark
cycle and animals were randomly divided into control diet (CD, n=10), training (TR,
n=10), high fat diet (60% fat Bio-Serv; HFD, n=12), or training with high fat diet (60 %

89

fat Bio-Serv, TR + HFD, n=12) groups and were monitored for six weeks. Mice were
either wild type (WT) or possessed an AMPKα2 dominant negative transgene (DN)
kindly provided by M.J. Birnbaum (University of Pennsylvania, Philadelphia, PA). Food
intake and body weight were monitored every week and blood glucose and plasma
insulin were measured every two weeks by tail vein puncture at 5-6 hours after the active
feeding period. Blood glucose was measured using a One-Touch Ultra Glucometer
(Lifescan, Milpitas, CA) and an ELISA kit was used to measure plasma insulin (ALPCO,
Salem, NH). Voluntary running wheels (Bio-Serv) were placed in the TR and TR + HFD
groups and activity was monitored using a Sigma Sport Bicycle odometer (Sigma Sport).
At the end of the six weeks mice underwent hindlimb perfusion experiments. All
procedures for the present study were approved by the Institutional Animal Care and Use
Committee at the University of Southern California.
Hindlimb Perfusion. On the day of the experiment, mice were anesthetized by the
administration of an intraperitoneal injection of ketamine/xylazine cocktail (20 mg/kg
body weight). Surgical preparation for the hindlimb perfusion was performed as
previously described (105; 129; 130). Before placement of the perfusion catheters, 15 IU
of heparin was injected into the inferior vena cava. The mice were then euthanized with
an intracardial injection of pentobarbital sodium (0.4 mg/g body weight), and catheters
were immediately inserted into the descending aorta and ascending vena cava, the
hindlimbs were then washed extensively with saline. The prepared mouse was then
placed in a perfusion apparatus for the experimental perfusion periods.
The perfusate consisted of Krebs-Henseleit solution, 5% bovine serum albumin
(Millipore, Billerica, MA), 550 M albumin-bound palmitate, 24 µCi of albumin-bound

90

[1-
14
C] palmitate, 6 mM glucose, and 450 µU insulin. The perfusate was kept at 37°C
and was continuously gassed with a mixture of 95% O
2
-5% CO
2
with arterial pH levels
between 7.15-7.68 and arterial P
O2
and P
CO2
values were between 246-421 and 22-49
mmHg, respectively. Perfusion pressures were not affected by any of the experimental
conditions and averaged 50.4±8.6, 72.9±16.0, 40.3±6.0, and 67.6±8.7 mmHg in the CD,
TR, HFD and TR + HFD groups, respectively (P > 0.05).
The perfusion preparation was equilibrated for 20 minutes. Perfusion flow rate
was maintained at 1.5 ml/min for all groups (average: 0.49 ± 0.01 ml/min/g of perfused
muscle). Arterial and venous samples were taken at 5, 10, 15, 20, and 25 minutes for
further analyses. Following the completion of the 25 min experimental perfusion period,
the gastrocnemius-soleus-plantaris muscle groups were freeze-clamped in situ with pre-
cooled aluminum clamps, removed and stored in liquid N
2
for further analyses.
Plasma sample analyses. Plasma samples collected during the perfusion were analyzed
to determine FA, glucose, and lactate concentrations as well as radioactive [
14
C]FA and
14
CO
2
contents. A WAKO NEFA HC kit (WAKO Chemicals, Richmond, VA) was used
to measure plasma FA concentrations spectrophotometrically. An YSI-1500 (Yellow
Springs Instruments, Yellow Springs, OH) analyzer was used to measure glucose and
lactate concentrations in the collected plasma samples. Plasma [
14
C]FA and
14
CO
2
radioactivities were measured as previously described (125; 127; 129). P
CO2
, P
O2
, and pH
were determined by utilizing an ABL-5 analyzer (Radiometer America, Westlake, OH).
Tissue sample preparation. For western blot analysis, frozen muscle samples (40 mg)
were powdered under liquid N
2
and homogenized in 500 µl of ice-cold RIPA buffer as
previously described (94; 96). The total cell homogenate was then transferred to a

91

microcentrifuge tube and vortexed frequently for 1 h whereupon the samples were
centrifuged at 4,500 g at 4ºC, for 1 h. For immunoprecipitation procedures,
approximately 90 mg of powdered muscle samples were homogenized in HEPES buffer
and centrifuged at 15,000 g for 5 min. Supernatants (200 µg) were incubated with
antibodies for AMPKα1 or AMPKα2 (Santa Cruz Biotechnology, Santa Cruz, CA) for 2
h at 4ºC with gentle agitation (95). Following the incubation, Protein A/G agarose (Santa
Cruz, SC-2003) was added to the tubes and incubated overnight at 4ºC with gentle
agitation. The immunoprecipitates were collected by centrifugation. Pellets were
washed with phosphate-buffered saline (PBS) buffer and the final supernatants were re-
suspended in sucrose homogenizing buffer and stored at -80ºC until analysis. Protein
concentrations were determined with the Bradford protein assay (BioRad, Hercules, CA).
For nuclear extraction procedures, a nuclear extraction kit was used (Pierce, Rockford,
IL) and the manufacturer’s instructions were followed. Briefly, approximately 40 mg of
muscle samples were homogenized in a cytoplasmic extraction buffer (CERI). The
homogenate was vortexed and incubated on ice for 10 min at which time and additional
cytoplasmic extraction buffer (CERII) was added to the tube. Following a 5 min
centrifugation step (16,000 g), the recovered pellet was resuspended in a nuclear
extraction buffer (NER). The suspension was incubated on ice (40 min) with intermittent
vortexing. The tubes were then centrifuged for 10 min (16,000 g). The nuclear extract
was decanted and stored at -80°C until analysis.
Western blot analysis. Approximately 20 µg of protein from the total cell homogenate
preparations were separated on a 10% gel via SDS-PAGE. Proteins were transferred onto
Immobilon-P polyvinylidene difluoride (PVDF) membranes and blocked with 5% BSA

92

in Tween-TBS for 1 h. The membrane was then incubated (4ºC) in 5% BSA in Tween-
TBS with antibodies (1:1000) against either phospho-ACC-Ser
79
, total ACC, (Cell
Signaling, Danvers, MA), CPT1, CD36, phospho-JNK, total JNK, and phospho-ERK1/2,
and total ERK1/2 (Santa Cruz Biotechnology, Santa Cruz, CA). Following this overnight
incubation, the membranes were probed with a secondary antibody (anti-rabbit IgG;
1:25,000) raised in goats (Pierce, Rockford, IL). Blots were then washed and subjected to
enhanced chemiluminescence (Pierce, Rockford, IL). Band density was quantified using
Scion Image (NIH, Bethesda, MD). All bands were compared to the band obtained for a
control sample of non-perfused muscle and expression was expressed as percent control.
A ponceau S total protein stain (Sigma, St. Louis, MO) was used on the membranes as a
loading control.
Activity assays. Citrate synthase was measured as previously described with some
modifications (57; 123). Muscle homogenates (20 mg) were added to a 96-well plate
containing 100 µM 5, 5’-dithio-bis(2-nitrobenzoic acid) and 250 µM acetyl-CoA. The
reaction was initiated with the addition of 500 µM oxaloacetate and was monitored in a
micro-plate reader for 5 min. The specific activity was calculated as the absorbance rate
per minute divided by the mercaptide extinction coefficient and expressed per muscle
weight. AMPKα1 and α2 activities were measured using
32
P-ATP incorporation into
SAMS peptide (Upstate Signaling, Lake Placid, NY) as described (96). Briefly,
immunoprecipitates were added to an assay cocktail containing
32
P-ATP and SAMS
peptide. Post-incubation, an aliquot was spotted onto a piece of Whatman filter paper
and all paper samples were washed with phosphoric acid followed by an acetone wash.
Sample papers were analyzed for radioactivity in a Packard scintillation counter and

93

counts were used to calculate phosphotransferase activity. SIRT1 activity was measured
on nuclear extracts with a commercially available histone deacetylase (HDAC)
colometric activation kit (Active Motif, Carlsbad, CA). Briefly, nuclear extracts were
added to assay buffers with assay substrates in a 96 well microplate. Trichostatin A was
added to the wells to inhibit class I, II, and IV HDAC’s and 250 µM NAD+ (Sigma) was
added to activate SIRT1 with is NAD-dependent. Following incubation (37°C, 60 min),
the reaction was stopped with the addition of a developing solution. The samples were
read in a microplate reader at 405 nm.
Calculations and statistics. Palmitate delivery, fractional and total palmitate uptake, and
percent and total palmitate oxidation were calculated, as described previously in detail
(125; 129). Both percent and total FA oxidation were corrected for label fixation by
using acetate correction factors (129). The specific activity for palmitate in the arterial
samples was not different between groups and did not vary over time, averaging 0.167 ±
0.02, 0.140 ± 0.01, 0.167 ± 0.01 and 0.171 ± 0.01 µCi/µmol, for the CD, TR, HFD, and
TR + HFD groups, respectively (P >0.05). Oxygen uptake, glucose uptake, and lactate
release were calculated as described (129). All uptake and release rates are expressed per
gram of perfused hindquarter muscles of both legs, which has been previously
determined to be 7% of total body weight. Time effects for glucose, lactate, and FA
concentrations and FA kinetic data were analyzed using a two-way ANOVA with
repeated measures in each of the experimental groups. If there were no significant
differences in the values measured after 5, 10, 15, 20, or 25 minutes of perfusion, mean
values were used for subsequent analysis. CD and HFD conditions were analyzed in two
separate two-way ANOVA’s and the effects of training and of genotype (WT vs. DN)

94

were the two factors analyzed using a two-way ANOVA (Statview 5.0). Tukey-Kramer
test for post-hoc multiple comparisons was performed when appropriate. A significance
level of 0.05 was chosen for all statistical methods.
RESULTS
Effects of DN transgene on physiological characteristics. Body weight, blood glucose,
and plasma insulin are summarized in Table 6 and Table 7. There were no differences in
body weight, plasma insulin, or blood glucose in the TR groups when compared to the
CD group (Table 6, P>0.05). By week 4 of the feeding period animals fed a HFD had
significantly (P<0.05) increased body weights over the TR + HFD group (Table 7).
Plasma insulin was elevated (P<0.05) in the high fat fed group by week 4 and remained
elevated through week 6 when compared to the TR + HFD group. Blood glucose tended
to be higher by week 4 but was not significantly elevated over the TR + HFD group
(P<0.05) until week 6 of the high fat feeding protocol (Table 7). There were no genome
effects measured in any of the groups (Table 6 and Table 7).
Voluntary Wheel Running. At the start of the six weeks of voluntary wheel running the
control fed WT mice averaged 2.6±0.5 km and spent 3.9±0.6 hrs running and the DN
mice averaged 1.4±0.2 km and spent 2.8±0.6 hrs per night wheel running (P<0.05, Fig.
20A and 20C). By the end of the six week protocol the WT mice averaged 4.9±0.5 km
and 5.9±0.4 hrs running and the DN mice averaged 3.4±0.5 km and 4.6±0.6 hrs per night
of wheel running (P<0.05, Fig. 20A and 20C). The high fat-fed WT mice averaged
2.6±0.4 km and 3.7±0.4 hrs per night running while the high fat-fed DN mice averaged
1.6±0.2 km and 3.7±0.7 hrs per night at the start of the six weeks of voluntary wheel
running (P<0.05, Fig. 20B and 20D). At the end of the six weeks of voluntary wheel

95

running the high fat-fed WT mice averaged 4.4±0.7 km and 5.1±0.1 hrs per night running
and the DN mice averaged 3.4±0.6 km and 4.5±0.6 hrs per night running (P<0.05, Fig.
20B and 20D). In the control fed mice there was a 38% increase in citrate synthase
activity following training in both the WT and DN mice (P<0.05, Fig. 20E). In the high
fat-fed DN mice there was 56% lower citrate synthase activity when compared to the
high fat-fed WT mice (P<0.05, Fig. 20F). The high fat-fed WT mice had a 48% increase
in citrate synthase activity as a result of training (P<0.05, Fig. 20F). The high fat-fed DN
mice there was a 74% increase in citrate synthase activity when compared to the
sedentary high fat-fed DN mice as a result of training (P<0.05, Fig. 20F). However, this
increase in citrate synthase activity was still 48% lower than the trained high fat-fed WT
mice (P<0.05, Fig. 20F).
Perfusion Characteristics. To verify physiological perfusion conditions oxygen uptake
was measured during the experimental perfusion period. There were no differences in
oxygen uptake over time in any of the experimental conditions (P>0.05, Table 8).
Palmitate concentration and delivery were not significantly different at any time point in
any of the groups or experimental conditions, as dictated by the protocol (P>0.05, Table
8). Arterial perfusate glucose concentration did not vary over time in any of the groups
and mean glucose concentration was not different between any of the experimental
conditions (P>0.05, Table 8). Additionally, lactate release was not different across time
or between any of the experimental conditions during the perfusion (P>0.05, Table 8).
Enzyme Activities. There were no measured differences in AMPKα1 activity in any of
the experimental conditions (P>0.05, Fig. 21A and 21B). As expected, AMPKα2 activity
(pmol•min
-1
•g
-1
) was decreased by the DN transgene in the CD group (P<0.05, Fig. 21C).

96

However, in the TR WT mice there was a 66% increase in AMPKα2 activity when
compared to CD WT mice (P<0.05, Fig 21C). In the HFD group there was a 169%
increase in AMPKα2 activity in the TR WT mice (P<0.05, Fig. 21D). Total protein
expression of ACC was not altered in any of the conditions (P>0.05, Fig. 21E and 21F).
In line with results obtained for AMPKα2 activity there was significantly reduced
phosphorylated ACC content in the DN mice in both CD and HFD and there were no
alterations with TR (P<0.05, Fig. 21E and 21F).
Substrate Exchange Across the Hindlimb. Glucose uptake (nmol•min
-1
•g
-1
) did not
vary over time in any of the experimental conditions throughout the perfusion
procedures. Mean glucose uptake was not different in any of the CD groups and TR had
no effect on glucose uptake (P>0.05, Fig. 22A). In the HFD group TR resulted in a 40%
increase in glucose uptake in WT mice (P<0.05, Fig. 22B). In the CD group palmitate
uptake (nmol•min
-1
•g
-1
) was 43% lower in the DN mice when compared to the WT mice
(P<0.05, Fig. 22C). Palmitate uptake increased 46% (P<0.05) with TR in the WT mice
and there were no measured increases in palmitate uptake in the TR DN mice when
compared to the CD DN mice (P>0.05, Fig. 22C). In the HFD group there were no
differences in palmitate uptake between WT and DN mice (P>0.05, Fig. 22D). TR
resulted in a 93 and 108% increase in palmitate uptake rates the WT and DN mice,
respectively, over the sedentary HFD groups (P>0.05, Fig. 22D). Palmitate oxidation
was 2-fold higher in the CD DN group when compared to the CD WT group, which we
have previously shown (unpublished observations, P<0.05, Fig. 22E). TR resulted in 2-
fold higher palmitate oxidation rates in the WT mice when compared to the CD WT mice
and there was a 30% increase in palmitate oxidation in the TR DN mice when compared

97

to the CD DN mice (P<0.05, Fig. 22E). In the HFD groups there were no alterations in
palmitate oxidation rates (P>0.05, Fig. 22F).
Activation and Expression of Signaling Molecules. There was a 121% increase in
phosphorylated ERK1/2 content as a result of training in the control diet condition
(P<0.05, Fig. 23A). Following high fat feeding the DN mice had 30% higher
phosphorylated ERK1/2 content when compared to the high fat-fed WT mice (P<0.05,
Fig. 23B). The trained high fat-fed DN mice also had 60% greater phosphorylated
ERK1/2 content when compared to the trained high fat-fed WT mice (P<0.05, Fib. 23B).
JNK1/2 phosphorylated content was 26% higher in both the WT and DN mice with
training when compared to the control fed sedentary mice (P<0.05, Fig. 23C). In the
trained high fat-fed mice there was 45% lower phosphorylated JNK1/2 in both the WT
and DN mice when compared to the sedentary high fat-fed mice (P<0.05, Fig. 23D).
There were no differences in SIRT1 activity (pmol•min
-1
•g
-1
) with training in the control
diet mice (P>0.05, Fig. 23E). There was a 176% increases in SIRT1 activity in the
sedentary DN group in the high fat fed mice (P<0.05, Fig. 23F). There were no
alterations in CPT1 expression as a result of training in either the WT or DN mice
(P>0.05, Fig. 24A). As we have previously reported, unpublished observations, there
was 30% lower CPT1 expression in the high fat-fed DN mice when compared to the high
fat-fed WT mice (P<0.05, Fig. 24B). In both the WT and DN trained high fat-fed mice
there was 15% lower protein expression than in the WT high fat-fed mice (P<0.05, Fig.
24B). There were no alterations in CD36 expression as a result of training in either the
WT or DN mice (P>0.05, Fig. 24C). The trained high fat-fed WT mice had 28% higher
CD36 expression than the trained high fat-fed DN mice (P<0.05, Fig. 24D).

98

DISCUSSION
The data from this study provide evidence for the involvement of AMPKα2 activity in
the regulation of fatty acid metabolism following endurance training in control fed mice.
In contrast, it does not appear that AMPKα2 activity is necessary for the alterations in
fatty acid metabolism that occur with endurance training in high fat-fed mice. However,
it does appear that AMPKα2 activity is necessary for the effects of training, while under
high fat-fed conditions, on glucose uptake. Additionally, these data suggest that
AMPKα2 may be involved in the activation of ERK1/2 but not JNK1/2 in high fat-fed
mice under sedentary and endurance training conditions.
In the control diet mice our data suggests that AMPKα2 is a necessary component
in the regulation of FA uptake in skeletal muscle in both sedentary and endurance trained
mice. We have previously observed a down regulation of FA uptake in the presence of
insulin in our hindlimb perfusion model in the AMPKα2 DN mice (unpublished
observations from our laboratory) that was further supported in this study. Additionally
in this study we measured a decrease in FA uptake in the DN endurance trained mice
when compared to the increase in FA uptake that was measure in their WT counterparts.
Taken together these results suggest that AMPKα2 activity is necessary to maintain and
increase FA uptake rates in insulin stimulated conditions in both sedentary and trained
conditions. Additionally, we measured increased rates of FA oxidation in the WT trained
mice which was not apparent in the DN trained mice. Therefore, it does not appear that
AMPKα2 is a necessary signal in the regulation of FA oxidation following endurance
training. Thus, AMPKα2 is necessary for the downstream regulation of the insulin

99

signaling pathway as it pertains to FA uptake but it not involved in the up-regulation of
FA oxidation following endurance training.
In the high fat fed mice our data suggest that AMPKα2 is not necessary for any
alterations that occur in FA metabolism with endurance training. The voluntary wheel
running resulted in an increase in FA uptake in both the WT and DN mice suggesting.
However, there was no effect of training on FA oxidation rates while under high fat
feeding conditions. Interestingly, the sedentary DN mice had lower citrate synthase
activity when compared to the sedentary WT mice in the high fat fed group but not in the
control-fed mice. It has been previously shown that in basal muscle citrate synthase
activity was lower in an AMPKα2 KO and AMPKα2iTG mouse models while fed control
diets (61; 101). These differences in basal citrate synthase activity could be due to the
differences in the mouse models that were utilized, the α2 subunit was completely
knocked out one study (61), FVB mice were used as the α2 kinase dead mice in the other
study (101), and the model used here was a kinase dead model on a C57/BL background
(61; 86; 134). As demonstrate previously, there was an increase in citrate synthase
activity in both the WT and DN mice with training (61; 101), but training in the DN
mice fed a high fat diet resulted in a 26% greater increase in citrate synthase activity,
over the sedentary high fat fed mice, than in the WT mice. Beyond citrate synthase
activity, there are additional differences between our training study and other studies
examining the role of AMPKα2 with voluntary wheel running protocols (61; 101). In
that study the AMPKα2 transgenic and knockout mice spent the same time participating
in voluntary wheel running as the WT mice (61; 101). The DN mice in our study spend
significantly less time running than the WT mice, which is line with studies that

100

characterized this mouse model (86). Despites this, the increase in citrate synthase
activity indicate that training effects are occurring in the DN mice following the
voluntary wheel running. Therefore, it appears that this form of voluntary wheel running
was successful at restoring FA uptake rates, increasing citrate synthase activity, but may
not be sufficient to increase rates of FA oxidation while under high fat feeding conditions
in both WT and DN mice.
Although, AMPKα2 does not appear to be a necessary signal in the regulation of
alterations in FA metabolism as a result of endurance training in high fat fed mice, it does
appear to be necessary for the restoration of glucose uptake under these conditions. In
the high fat-fed WT mice glucose uptake rates were increased with the six weeks of
voluntary wheel running. These data are in line with previous reports of endurance
training improving insulin sensitivity under high fat feeding conditions (68). However in
the DN trained mice, fed the high fat diet, there was no increase in glucose uptake rates.
These data support previous reports that low AMPKα2 may exacerbate insulin resistance
with a high fat diet (38). Taken together this data suggests that AMPKα2 is necessary for
improvements in insulin sensitivity with voluntary wheel running in high fat-fed mice.
The voluntary wheel running did not result in any changes in AMPKα1 activity in
both the control fed and high fat fed mice and there was no effect of the DN transgene.
As expected there was a decrease in AMPKα2 activity in the DN groups under both diet
conditions and in both the sedentary and trained mice. However, the voluntary wheel
running was sufficient to increase AMPKα2 activity in both the control fed and high fat
fed mice. ACC phosphorylation followed the same activation pattern as AMPKα2 in the
DN mice in both the control fed and high fat-fed conditions but there was no effect of

101

training. The activation of ACC did not correspond to the alterations that were measured
in FA oxidation as a result of training in both the diet conditions. This provides further
evidence for the effectiveness of the voluntary wheel running protocol that was used in
this study and that ACC phosphorylation is not the only signal involved in the regulation
of FA oxidation.
It has been previously postulated that there is a possible link between AMPK
activity and SIRT1 activity (21). We have previously shown that SIRT1 expression and
activity may be dependent on AMPKα2 activity, which is in line with growing evidence
of a synergy between these two enzymes (22; 106). Here we show that there are no
alterations in SIRT1 activity under sedentary and trained conditions in the WT and DN
control fed mice. This is not consistent with previous reports of increases in SIRT1
activity with exercise training (114) and we have previously shown that acute muscle
contraction will result in increases in SIRT1 activity (unpublished observations).
However, that study utilized a forced treadmill running program, and in line with our
current results another study utilizing voluntary wheel running did not measure increases
in SIRT1 activity (23). Therefore, these reports suggest that the mode of exercise is
essential to measure increases in SIRT1 activity either with acute exercise or chronic
training. These results are not consistent with our previous data that showed an increase
in SIRT1 activity in DN mice. Consistent with our previous reports, we measured a
dramatic increase in SIRT1 activity in DN mice under high fat feeding conditions.
Although there is no evidence to suggest that SIRT1 may directly regulate FA oxidation,
it is a possibility that SIRT1 has unknown effects on mitochondrial enzymes.

102

Stress related kinases ERK1/2 and JNK1/2 activation was increased as a result if
the voluntary wheel running in both the WT and DN mice in the control fed condition.
Despite these increases in the control feeding condition the high fat diet did not produce
the same activation pattern in ERK1/2 and JNK1/2. ERK1/2 phosphorylation was
increased in the DN sedentary and trained high fat-fed mice over the WT mice. This
suggests that ERK1/2 activation under high fat feeding conditions may be dependent on
AMPKα2 activity. In contrast, JNK1/2 phosphorylation was lower as a result of training
in the high fat-fed mice and was not dependent on AMPKα2 activation. Taken together
these data provide evidence that JNK1/2 activation is not dependent on AMPKα2 activity
and that ERK1/2 is upregulated in the absence of AMPKα2 activity in sedentary and
trained high fat-fed mice.
Finally, the voluntary wheel running did not result in any alterations in protein
expression of CD36 or CPT1 in the WT mice in both diet conditions. However, with
high fat feeding CPT1 expression was higher than the DN mice or the training condition.
Interestingly this increase in CPT1 expression did not correspond to any changes in FA
oxidation rates. Additionally, CD36 expression was lower in the trained DN mice in the
high fat fed condition. This decrease in expression did not correspond to any changes in
FA uptake rates. These data provide evidence for alterations in CPT1 and CD36
expression as a result of either genotype or training, but the data is limited, in that it
would be important to measure the plasma membrane content of CD36 or the
mitochondrial content of CPT1. Further studies should be employed to measure the
various cell fraction content of these proteins to elucidate their mechanistic role as a
result of the experimental conditions in the current study.

103

In summary, the data in the current study support the notion that AMPKα2
activity is necessary for restoration of insulin sensitivity as a result of endurance training
in high fat fed mice. Although AMPKα2 signaling appears to be required for alterations
in glucose uptake in high fat fed endurance trained mice it does not appear to be required
for alterations that occur in FA metabolism under these conditions. AMPKα2 signaling
appears to regulate FA metabolism under control fed conditions but not under high fat
feeding conditions. These data provide a role for AMPKα2 signaling in the regulation of
glucose uptake following endurance training in high fat fed mice.

104

Week 0

Week 2

Week 4

Week 6

WT DN WT DN WT DN WT DN
Body Weight
(g),
Sedentary

24.7±0.8

25.2±1.9

24.0±0.8

24.6±1.1

25.9±1.9

26.2±0.7

28.5±0.5

30.1±1.5

Trained

25.0±2.3 23.0±2.3 24.1±0.5 24.0±1.1 26.5±0.9 25.8±0.9 26.9±0.9 25.2±0.2
Blood Glucose,
(mg/dL)
Sedentary

140.7±14.5

124.0±13.1

146.2±6.4

126.5±7.5

122.7±14.0

131.0±8.0

151.3±6.9

157.0±3.0
Trained 139.0±11.9 152.9±7.5 159.2±9.8 158.9±7.8 102.5±18.5 102.7±11.9 122.7±10.0 112.7±5.5
Plasma Insulin,
(ng/ml)
Sedentary

0.8±0.1

0.7±0.1

0.9±0.6

1.1±0.1

0.9±0.2

1.0±0.1

0.8±0.1

0.6±0.1
Trained 1.0±0.1 0.7±0.1 1.3±0.1 0.8±0.2 1.4±0.2 0.7±0.1 0.8±0.1 0.7±0.1
Table 6. Body weight, blood glucose, and plasma insulin in control diet mice Values are expressed as
means ± SE. n = 5-6 for all time points. Blood glucose and plasma insulin were measured 5-6 hours
after the dark period

105

Week 0

Week 2

Week 4

Week 6

WT DN WT DN WT DN WT DN
Body Weight
(g),
Sedentary

25.1±0.8

24.2±1.2

27.4±3.2

28.4±2.2

39.4±3.3
*

32.4±2.9
*

35.6±0.7
*

41.8±3.2
*

Trained

24.4±1.5 24.0±1.7 24.2±1.2 24.0±1.3 25.2±1.1 25.4±0.7 26.7±0.8 26.2±0.7
Blood Glucose,
(mg/dL)
Sedentary

139.0±11.9

152.9±7.6

159.0±9.8

158.9±7.7

162.9±11.4
*

180.4±16.0
*

215.6±14.3
*

190.6±24.9
*

Trained

139.2±18.2

155.6±6.6

152.1±8.0

162.0±21.5

126.0±10.1

116.2±11.3

102.5±10.4

133.7±17.5
Plasma Insulin,
(ng/ml)
Sedentary

0.9±0.2

1.1±0.3

2.9±0.6
*

2.4±0.4
*

4.0±1.0
*

3.5±0.9
*

5.2±1.2
*

6.1±1.8
*

Trained

1.0±0.2

1.0±0.2

1.4±0.2

1.6±0.2

2.0±0.4

3.1±0.6

1.7±0.4

2.5±0.3

Table 7. Body weight, blood glucose, and plasma insulin in high fat diet mice Values are expressed as
means ± SE. n = 5-6 for all time points. Blood glucose and plasma insulin were measured 5-6 hours after
the dark period. * P< 0.05 compared with trained high fat diet group.

106

Control Diet Trained High Fat Diet Trained + High Fat Diet

WT
(n=5)
DN
(n=5)
WT
(n=5)
DN
(n=5)
WT
(n=6)
DN
(n=6)
WT
(n=6)
DN
(n=6)
Oxygen
uptake,
µmol∙g
-1
∙h
-1

6.5 ± 0.7 5.3 ± 0.8 6.6 ± 0.3 5.7 ± 0.2 5.2 ± 0.5 5.8 ± 0.4 6.3 ± 0.2 5.6 ± 0.3
FA
concentration,
µmol/l
583.1±26.0 524.8±28.8 499.9±26.8 503.9±11.2 534.8±19.8 536.7±33.0 519.5±34.8 548.8±18.8
FA delivery,
nmol∙min
-1
∙g
-1

248.8±13.1 225.4±21.2 248.8±13.1 225.4±21.2 223.7±20.9 206.7±23.9 223.7±20.9 206.7±23.9
Glucose
concentration,
mmol/l
6.6±0.1 6.9±0.3 6.7±0.1 6.6±0.7 6.8±0.4 7.2±0.3 6.7±0.1 6.6±0.1
Lactate
release,
umol∙g
-1
∙h
-1

0.55±0.1 0.47±0.4 0.56±0.1 0.49±0.4 0.42±0.1 0.39±0.1 0.43±0.1 0.44±0.1
Table 8. Perfusion characteristics of hindlimbs Values are expressed as means ± SE; n= number of mice.
Mice were perfused with 450μU insulin and were either wild type (WT) or AMPKα2 dominant negative
transgene (DN).

107

Figure 20. Effect of AMPKα2 DN transgene on time spent running in control diet mice (A) and
high fat diet mice (B), distance ran in control diet mice (C) and high fat diet mice (D), and citrate
synthase activity in sedentary and trained mice under control diet conditions (E) and sedentary
and trained mice under high fat diet conditions (F). In figures A-D open circles represent WT
mice and closed circles represent DN mice. In figures E and F open bars represent WT mice and
closed bars represent DN mice. Values are means ± SE for sedentary WT (n=5) and DN (n=5)
mice and trained WT (n=5) and DN (n=5) mice fed control diet; sedentary WT (n=6) and DN
(n=6) mice and trained WT (n=6) and DN (n=6) mice fed high fat diet. *P<0.05 compared with
sedentary WT mice, **P<0.05 compared with sedentary DN mice, and #P<0.05 compared with
trained WT mice.

108

Figure 21. Effect of AMPKα2 DN transgene and training on AMPKα1 activity in control diet
mice (A) and high fat diet mice (B), AMPKα2 activity in control diet mice (C) and high fat diet
mice (D), and phosphorylation of ACC in control diet mice (E) and high fat diet mice (F) in
perfused hindlimbs (450µU insulin). Open bars represent WT mice and closed bars represent DN
mice. Values are means ± SE for sedentary WT (n=5) and DN (n=5) mice and trained WT (n=5)
and DN (n=5) mice fed control diet; sedentary WT (n=6) and DN (n=6) mice and trained WT
(n=6) and DN (n=6) mice fed high fat diet. *P<0.05 compared with sedentary WT mice and
#P<0.05 DN effect.

109

Figure 22. Effect of AMPKα2 DN transgene and training on glucose uptake in control diet mice
(A) and high fat diet mice (B), palmitate uptake in control diet mice (C) and high fat diet mice
(D), and palmitate oxidation in control diet mice (E) and high fat diet mice (F) in perfused
hindlimbs (450µU insulin). Open bars represent wild type (WT) mice and closed bars represent
dominant negative (DN) mice. Values are means ± SE for sedentary WT (n=5) and DN (n=5)
mice and trained WT (n=5) and DN (n=5) mice fed control diet; sedentary WT (n=6) and DN
(n=6) mice and trained WT (n=6) and DN (n=6) mice fed high fat diet. *P<0.05 compared with
sedentary WT mice and #P<0.05 DN effect.

110

Figure 23. Effect of AMPKα2 DN transgene and training on phosphorylated ERK1/2 in control
diet mice (A) and high fat diet mice (B), and on phosphorylated JNK1/2 in control diet mice (C)
and high fat diet mice (D) in perfused hindlimbs (450µU insulin). Open bars represent wild type
(WT) mice and closed bars represent dominant negative (DN) mice. Values are means ± SE for
sedentary WT (n=5) and DN (n=5) mice and trained WT (n=5) and DN (n=5) mice fed control
diet; sedentary WT (n=6) and DN (n=6) mice and trained WT (n=6) and DN (n=6) mice fed high
fat diet. *P<0.05 training effect and #P<0.05 DN effect.

111

Figure 24. Effect of AMPKα2 DN transgene and training on CPT1 expression in control diet
mice (A) and high fat diet mice (B), and CD36 expression in control diet mice (C) and high fat
diet mice (D) in perfused hindlimbs (450µU insulin). Open bars represent wild type (WT) mice
and closed bars represent dominant negative (DN) mice. Values are means ± SE for sedentary
WT (n=5) and DN (n=5) mice and trained WT (n=5) and DN (n=5) mice fed control diet;
sedentary WT (n=6) and DN (n=6) mice and trained WT (n=6) and DN (n=6) mice fed high fat
diet. *P<0.05 training effect and #P<0.05 DN effect.

112

VII. SUMMARY

It has been determined that AMPK signaling is an important factor in the
regulation of substrate metabolism in skeletal muscle. Furthermore, AMPK has been
proposed to be a therapeutic target for the treatment of metabolic disorders. Therefore
the current experiments sought to determine upstream regulators of AMPK such as acute
muscle contraction, CaMKK signaling, and chronic manipulations through high fat
feeding and/or exercise training. Additionally, these studies were designed to determine
the downstream targets of AMPK signaling and these effects on the regulation of
substrate metabolism in skeletal muscle.
To study the kinetics of fatty acid metabolism and glucose uptake, hindlimb
perfusion procedures were utilized in rats and mice. Electrical stimulation was utilized to
induce acute muscle contraction in the gastrocnemious-soleus-plantaris muscle groups
via sciatic nerve stimulation in rats and surface stimulation in mice. AICAR and caffeine
were utilized for pharmacological activation of calcium-dependent signaling and AMP-
dependent signaling cascades. Genetic deletion of AMPKα2 in skeletal and heart muscle
was used to inactivate AMP signaling and a chemical inhibitor of CaMKK was used to
determine the contribution of these separate signaling pathways in the regulation of
substrate metabolism in skeletal muscle.
Experiment 1 utilized a rat hindlimb perfusion model to examine the role of
calcium-dependent and AMP-dependent signaling in the regulation of substrate
metabolism in skeletal muscle. Hindlimbs were perfused with either 3mM caffeine,
2mM AICAR, or during moderate intensity muscle contraction. STO-609, a potent
CaMKK inhibitor, was used to inhibit calcium-dependent signaling. This study provided

113

evidence for CaMKK-dependent activation of AMPK and that this activation was
responsible for the regulation of fatty acid uptake and oxidation and glucose uptake in
skeletal muscle. Furthermore, data from this study showed a role for calcium-dependent
regulation of translocation of CD36 and GLUT4 to the plasma membrane during caffeine
treatment and muscle contraction. However, the use of AICAR indicated that there is
AMPK-dependent calcium-independent signaling involved in the regulation of substrate
metabolism in skeletal muscle. It is clear from this study that CaMKK signaling acts
upstream of AMPK and this signaling is involved in the regulation of substrate
metabolism during moderate intensity muscle contraction.
In the second experiment a mouse hindlimb model was utilized to determine the
role of AMPKα2 signaling in the regulation of substrate use during caffeine treatment
(3mM) and moderate intensity muscle contraction. This study is one of the first studies,
in this country, to utilize this perfusion model in the mouse. We were able to obtain a
mouse model that contained an inactive AMPKα2 subunit in skeletal and heart muscle.
This experimental model allowed us to study AMPK signaling on a genetic level rather
than utilizing a chemical inhibitor. The results from this study provide evidence for a
role of AMPKα2 signaling in the regulation of substrate metabolism during muscle
contraction in a time-dependent manner. Furthermore, data from this study suggest a
possible link between AMPKα2 signaling and SIRT1 activity in skeletal muscle during
rest and muscle contraction conditions. Finally, these data show that AMPKα2 signaling
is not necessary in the regulation of FA metabolism during caffeine treatment. Overall,
the data suggest that while AMPKα2 is a critical signaling intermediate in the regulation

114

of substrate utilization, it is not the sole signal responsible for regulating skeletal muscle
metabolism.
Experiment 3 utilized the same AMPKα2 dominant negative mouse model and we
induced insulin resistance by 6 weeks of high fat feeding in these mice. Following the
feeding period hindlimbs were perfused under both basal and insulin stimulated (450 µU)
conditions to determine if low AMPKα2 activity would exacerbate the development of
insulin resistance in skeletal muscle. This data suggested that AMPKα2 is not necessary
in the regulation of alterations that occur in FA oxidation following high fat feeding in
skeletal muscle. Likewise, the lack of AMPKα2 activity did not alter glucose uptake
under both basal and insulin stimulated conditions when compared to wild type mice in
both control fed and high fat fed mice. In contrast, the results suggested a role for
AMPKα2 in the regulation of insulin stimulated FA uptake in control fed and high fat fed
mice. Finally, AMPKα2 appears to be a mediator in “cross talk” that may occur between
skeletal muscle and adipose tissue during high fat feeding. A role for AMPKα2 signaling
in the regulation of Akt phosphorylation, SIRT1 activity, PTP1b expression, and CPT1
expression under high fat feeding conditions was apparent from this study. The data
provided from this experiment suggest that AMPKα2 signaling is involved in the
regulation of fatty acid uptake following high fat feeding as well as a role in mediating
the expression and activity of various molecules associated with insulin signaling in
skeletal muscle and adipose tissue.
In the last experiment exercise training was included with high fat feeding in the
AMPKα2 dominant negative mouse model. The aim of this study was to determine if
AMPKα2 activity in skeletal muscle is necessary to regain insulin sensitivity that occurs

115

with exercise training under high fat feeding conditions. Hindlimbs of the mice were
perfused with insulin (450 µU) to measure substrate kinetics following the 6 weeks of
high fat feeding and voluntary wheel running. The results from this study suggested that
AMPKα2 is necessary to restore glucose uptake to levels that occur with exercise training
under high fat feeding conditions. It also appears that AMPKα2 signaling is involved in
insulin stimulated FA uptake under control diet conditions which was also shown in the
third experiment. However, there were no effects of low AMPKα2 activity on the rates
of FA uptake or oxidation under high fat feeding conditions in either sedentary or
exercise trained mice. Under high fat feeding conditions, is appears that AMPKα2
signaling may act upstream of ERK1/2 signaling in sedentary and trained conditions and
upstream of CD36 expression with training. However, AMPKα2 signaling does not
appear to be involved in alterations in JNK1/2 activation in either sedentary or exercise
trained mice under control diet or high fat diet conditions. Finally, although we have
previously shown a possible link between AMPKα2 activation and SIRT1 activity it does
not appear that AMPKα2 is involved in the regulation of SIRT1 activity following
exercise training.

116

VIII. CONCLUSIONS

Data collected in these experiments has provided insight into the role of AMPK
signaling in the regulation of substrate metabolism in skeletal muscle. The data provides
evidence for a link between Ca
2+
mediated signaling in the regulation of substrate
metabolism in skeletal muscle. However, it appears that this signaling may rely on
AMPKα2 activity in the regulation of glucose uptake and not fatty acid uptake and
oxidation in skeletal muscle. Finally a role AMPKα2 signaling in the regulation of
substrate metabolism in skeletal muscle under high fat feeding and exercise training
conditions has been established.
These experiments have utilized novel techniques to examine substrate
metabolism in skeletal muscle. The hindlimb perfusion model has been utilized
extensively in a rat model and by transferring the rat model to the mouse model we were
able to take advantage of a genetically modified mouse model. These methods allow for
the examination of fatty acid kinetics in an in situ model with intact circulation and
connective tissue, which is not possible in ex vivo muscle studies. Now that we have
established this new model it may be utilized in the future to examine alternative proteins
of interest other than AMPK.
The data from these experiments solidify the role of AMPK signaling in the
regulation of FA metabolism in skeletal muscle. Additionally, it was shown that AMPK
may have alternative activators, such as CaMKK during caffeine treatment. Further,
AMPK signaling does appear to be involved in the development of insulin resistance
under high fat feeding conditions in mice and AMPK appears to be essential in the
exercise training effects that occur while on a high fat diet in mice.

117

The results from these studies have provided contributions to the understanding of
substrate metabolism in skeletal muscle. Since insulin resistance has been linked to
dysregulation of FA metabolism, these results provide valuable information into the
regulators and mechanisms involved in FA uptake and oxidation; possibly providing
targets for future therapies for individuals suffering from metabolic disorders. These data
will help to further establish the importance of AMPK signaling in skeletal muscle and
provide insight into AMPK as a potential target for therapies in metabolic disorders.

118

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APPENDIX A

Overall Conclusions from Experiment 1 and Experiment 2
Proposed signaling pathways during muscle contraction. Calcium signaling does not
appear to be dependent on AMPK signaling in the regulation of FA metabolism but
does appear to be dependent on AMPK signaling in the regulation of glucose uptake.
There are AMPK independent effects in the regulation of both FA metabolism and
glucose uptake during muscle contraction.

136

APPENDIX B

Overall Conclusions of Experiment 3 and Experiment 4
Proposed signaling pathways following high fat feeding, exercise training, and high
fat feeding combined with exercise training. AMPK appears to be involved in the
regulation of FA metabolism following high fat feeding and exercise training alone.
However when high fat feeding is combined with exercise training it appears
alternative signals are involved in the regulation of FA metabolism, while AMPK is
necessary for alterations in glucose uptake under these conditions.

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APPENDIX C

Power Point Presentation

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Abstract (if available)

Abstract The contents of this dissertation contain four experiments aimed at determining the role of AMP-activated protein kinase (AMPK) in the regulation of fatty acid (FA) metabolism in skeletal muscle.

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Creator Abbott, Marcia Jeanine (author)

Core Title The pivotal role of AMP-activated protein kinase in the regulation of fatty acid metabolism in skeletal muscle

School College of Letters, Arts and Sciences

Degree Doctor of Philosophy

Degree Program Integrative and Evolutionary Biology

Degree Conferral Date 2010-08

Publication Date 07/27/2010

Defense Date 05/24/2010

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag fatty acid metabolism,OAI-PMH Harvest,skeletal muscle metabolism

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Turcotte, Lorraine P. (committee chair), Donovan, Casey M. (committee member), McDonough, Alicia A. (committee member), McNitt-Gray, Jill L. (committee member)

Creator Email mabbott78@gmail.com,mjabbott@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3222

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