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Effects of high carbohydrate and high long chain fatty acid availability on skeletal muscle fatty acid metabolism and regulation of substrate utilization

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Effects of high carbohydrate and high long chain fatty acid availability on skeletal muscle fatty acid metabolism and regulation of substrate utilization

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EFFECTS OF HIGH CARBOHYDRATE AND
HIGH LONG CHAIN FATTY ACID AVAILABILITY ON
SKELETAL MUSCLE FATTY ACID METABOLISM AND
REGULATION OF SUBSTRATE UTILIZATION
by
Michele L. Sacman
A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(KINESIOLOGY)
May 2002
Copyright 2002 Michele Sacman
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UMI Number: 1411802
Copyright 2002 by
Sacman, Michele L.
All rights reserved.
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U N IV ERSITY O P S O U T H E R N C A L IFO R N IA
THE GRADUATE SCHOOL.
UNIVERSITY PARK
LOS ANGELES. CA LIFO RN IA SOOOT
This thesis, written by
MICHELE L. SACMAN
under the direction of h33. Thesis Committee,
and approved by all its members, has been pre
sented to and accepted by the Dean of The
Graduate School, in partial fulfillment of the
requirements for the degree of
MASTER OF SCIENCE IN KINESIOLOGY
Utmm
D a te May 10 * 2002
OMMITTEE
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TABLE OF CONTENTS
List of Tables................................................................................................................ ii
List of Figures ...................................................................................................... iii
I. INTRODUCTION............................................................................................1
II. LIMITATIONS................................................................................................ 4
HI. BACKGROUND............................................................................................. 7
IV. EXPERIMENT.............................................................................................. 33
Title: High carbohydrate and high LCFA availability increase LCFA
uptake and oxidation in perfused muscle.......................................... 33
V. SUMMARY AND CONCLUSIONS........................................................... 75
VI. THESIS REFERENCES............................................................................... 77
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LIST OF TABLES
Table I. Effects of high carbohydrate availability on palmitate metabolism
in resting hindquarters perfused with high LCFA availability 41
Table 2. Effects of high carbohydrate availability on muscle triglyceride
concentration and synthesis rate in resting hindquarters perfused
with high LCFA availability..............................................................52
iii
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LIST OF FIGURES
Figure I . Effects of high carbohydrate availability on fractional and
total palmitate uptake in resting hindquarters perfused with
high LCFA availability............................................................... 42
Figure 2. Effects of high carbohydrate availability on percent and total
palmitate oxidation in resting hindquarters perfused with
high LCFA availability............................................................... 43
Figure 3. Effects of high carbohydrate availability on arterial glucose
concentration and glucose uptake in resting hindquarters
perfused with high LCFA availability........................................45
Figure 4. Effects of high carbohydrate availability on arterial glucose
concentration and glucose uptake in resting hindquarters
perfused with high LCFA availability........................................46
Figure 5. Effects of high carbohydrate availability on glycolytic rate
and glucose oxidation in resting hindquarters perfused with
high LCFA availability............................................................... 47
Figure 6. Effects of high carbohydrate availability on lactate
concentration and lactate release in resting hindquarters
perfused with high LCFA availability........................................49
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Figure 7. Effects of high carbohydrate availability on lactate
concentration and lactate release in resting hindquarters
perfused with high LCFA availability........................................50
Figure 8. Effects of high carbohydrate availability on glycerol release
in resting hindquarters perfused with high LCFA availability...51
Figure 9. Effects of high carbohydrate availability on palmitate
accumulation into triglyceride and diglyceride in resting
hindquarters perfused with high LCFA availability...................53
Figure 10. Effects of high fuel availability on total palmitate uptake
in resting hindquarters perfused with either high carbohydrate
availability, high LCFA availability, or both............................. 56
Figure 11. Possible mechanisms for changes in fatty acid oxidation
with excess substrate availability............................................... 58
Figure 12. Metabolic pathway for triacylglycerol synthesis in skeletal
muscle..........................................................................................64
v
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I. INTRODUCTION
The relative contribution of long chain fatty acids (LCFA) to muscle
oxidative metabolism is regulated by many factors, which include carbohydrate and
LCFA availability. Experiments in perfused rat muscle, and in human whole body,
demonstrated that high carbohydrate availability in the presence of physiologic
levels of LCFA were associated with an increase in LCFA uptake and a decrease in
LCFA oxidation (Turcotte 2002, Sidossis 1996). Conversely, rat hindlimb perfusion
studies and human whole body experiments demonstrated that high LCFA
availability in the presence of physiologic levels of glucose were associated with an
increase in both LCFA uptake and oxidation (Turcotte 1998, Wolfe 1988).
While the independent effects of high carbohydrate and LCFA availability on
LCFA metabolism has been considered, the combined effects of carbohydrate and
LCFA excess on LCFA metabolism have not been studied. Additionally, because of
the recognized interactions between carbohydrate and lipid fuels in the regulation of
skeletal muscle oxidative metabolism, experiments on the combined effects of high
carbohydrate and high LCFA availability would allow further exploration into fuel
interactions and their link to the regulation of oxidative metabolism.
This research was undertaken to elucidate the effects of superphysiologic
levels of carbohydrate and LCFA on skeletal muscle LCFA metabolism. The
hindlimb perfusion system allows the study of substrate exchange across the
hindlimb in an in situ model for skeletal muscle metabolism (Ruderman 1971). The
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hindlimb perfusion system permits the use of fixed concentrations of substrates and
hormones, while allowing observation of various physiologic parameters, such as
blood flow, blood hematocrit, blood hemoglobin. This experimental system also
allows for collection of arterial and venous perfusate samples, to measure substrate
exchange, as well as muscle samples, to measure muscle metabolites.
Experiment Effects o f high carbohydrate availability on LCFA metabolism in
resting hindquarters perfused with high LCFA availability.
Rationale and Significance. Hindlimb perfusion experiments were performed
to determine if LCFA uptake, LCFA oxidation, glucose oxidation, and muscle
triglyceride levels are altered with substrate availability. The purpose of the
experiment was two-fold: I) To determine the effect of combined high carbohydrate
and high LCFA availability on LCFA metabolism, and 2) To determine, under
conditions of excess fuel availability, the metabolic fate of carbohydrate and LCFA
fuels. Our findings revealed a significant increase in LCFA uptake and oxidation
with excess carbohydrate and LCFA fuels. However, the relative contribution of
palmitate to substrate oxidation did not change. Furthermore, this study combined
with previous results suggests that the effects of high carbohydrate availability on
LCFA uptake may be independent of those of high LCFA availability, and that
separate cellular mechanisms of regulation exist depending on which fuel is in
excess.
2
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Experimental Protocol. Male Wistar rats were prepared for in situ hindlimb
perfusion. The perfusate consisted of Krebs-Henseleit solution, 1-day old washed
bovine erythrocytes (hematocrit, 30%), 4% bovine serum albumin, 1500 pM
albumin-bound palmitate, and albumin-bound [l-l4C]palmitate, [3-3 H]glucose, and
either low glucose availability with 6 mM glucose and 10 pU/mL insulin, or high
glucose availability with 20 mM glucose and 1000 pU/mL insulin. Following an
equilibration period of 20 minutes, the right leg was perfused at rest. Arterial and
venous perfusate samples were taken at 10 minute intervals for analysis of
concentrations of glucose, lactate, glycerol, and LCFA, as well as for [l4C]LCFA,
[1 4 ]C02 , and 3 H20 activities. Muscle samples were taken at both pre- and post
perfusion timepoints and were analyzed for muscle triglyceride (TG) content,
[1 4 C]palmitate incorporation into muscle TG, [3 H]gIucose incorporation into
glycogen, and malonyl-CoA content.
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n . LIMITATIONS
Hindlimb Perfusion System
The hindlimb perfusion system is employed in our lab to study substrate
exchange across the hindlimb. Because the muscle is isolated prior to perfusion with
pre-determined substrates, this system is a suitable in situ model to study skeletal
muscle metabolism (Ruderman 1971). Furthermore, the hindlimb perfusion system
permits the use of fixed concentrations of substrates and hormones, while allowing
observation of various physiologic parameters, such as blood flow, blood hematocrit,
and blood hemoglobin. This experimental system also allows for collection of
arterial and venous perfusate samples, to measure substrate exchange, as well as
muscle samples, to measure muscle metabolites.
A disadvantage of the hindlimb perfusion system is the contribution of non
muscle sources, such as adipose tissue and skin, to metabolism. Furthermore, the
pre-perfusion anesthesia and surgery may create an environment that does not
accurately reflect normal physiologic conditions. To ascertain that the hindlimb
remained close to physiologic conditions, oxygen uptake and blood pressure were
closely monitored.
Perfusate Concentrations
Perfusate concentrations were selected to reflect superphysiologic conditions
of LCFA, and either physiologic or superphysiologic conditions of glucose and
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insulin. The glucose and insulin concentrations were set to obtain maximal glucose
uptake at either basal or superphysiologic levels. Similarly, concentrations of LCFA
were set at levels previously shown in perfused skeletal muscle to be the Vmax of
the saturation curve for palmitate uptake (Turcotte 1991).
Tracer methodology
The tracer [I4 C]palmitate was used to trace LCFA metabolism. Palmitate is a
characteristic LCFA and its use permits comparison with other studies performed in
our lab. Use of [l4C]palmitate allows sensitive determination of plasma palmitate
uptake, oxidation, and incorporation into muscle TG. An acetate correction factor is
used to correct for the loss o f l4C 02 derived from palmitate oxidation that may
become trapped in bicarbonate and acetate pools. The tracer [3 H]glucose was used
to trace glucose incorporation into muscle glycogen and to measure glycolytic rates,
which allowed for an estimation of glucose oxidation.
Assays
The assay for muscle triglycerides was performed in an attempt to determine
the fate of LCFA under conditions of high substrate availability. The assay for
malonyl-CoA was performed to determine possible regulatory mechanisms that exist
when carbohydrate and LCFA are in excess. Assay results showed some intra- and
inter-subject variability likely due to the variation in muscle samples since the entire
hindlimb was freeze-clamped in situ without fiber typing. Additionally, despite a
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concerted effort to separate the muscle tissue from other non-muscle components,
such as tendon or ligament, trace amounts may have been present in varying amounts
in the samples.
In order to elucidate the mechanisms by which high fuel availability impacted
LCFA metabolism at the cellular level, determinations of mitochondrial content and
enzyme activities would have been invaluable. Additionally, because of the known
association between LCFA availability and LC-CoA, coupled with the role of LC-
CoA as a key regulator in many metabolic pathways, determination of LC-CoA
levels may have helped to clarify the role of LC-CoA in skeletal muscle metabolism
in the presence of high carbohydrate availability. However, limited quantities of
muscle tissue prevented the performance of such studies.
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m . BACKGROUND
Introduction
Long chain fatty acids (LCFA) are molecules composed of long hydrocarbon
chains attached to a carboxyl group. While LCFA have significant physiological
roles in phospholipid synthesis, protein targeting, and hormone action, they are also
an important fuel source (Stryer 1995). The storage form of this fuel is the
triglyceride molecule, which consists of three fatty acids attached to a glycerol
backbone. When fuel sources are in limited supply or in high demand, as occurs
during exercise or starvation, adipose tissue triglycerides can be mobilized as fatty
acids to be used as an energy source (Salway 1994). However, from the initial
mobilization step to the oxidative pathways, many sites of regulation exist. Potential
regulatory steps in skeletal muscle LCFA metabolism can occur in delivery of
LCFA, LCFA transport across the plasma membrane, cytosolic transport of LCFA,
and cellular mechanisms involved in the regulation of LCFA oxidation, which
include in part oxidative capacity, mitochondrial transport, and interactions between
carbohydrate and LCFA fuels.
Fatty Acid Delivery
In order to be utilized in the body, triglycerides must first be broken down
into their constituent parts, namely three fatty acids and a glycerol residue. The
availability of these fatty acids determines in part the utilization of LCFA by skeletal
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muscle. Availability of LCFA to skeletal muscle or any other tissue is dependent on
the mobilization of lipids from adipose tissue triglyceride stores and blood flow.
LCFA mobilization from adipose tissue results from the occurrence of higher rates of
lipolysis than re-esterification (Turcotte 1999). Re-esterification refers to the
formation of new triglycerides after mobilization of LCFA.
The rate-limiting enzyme of adipose tissue lipolysis is hormone sensitive
lipase (HSL). The conversion of HSL into its active form involves phosphorylation
by protein kinase A. Protein kinase A is activated by cAMP, the formation of which
is aided by the enzyme adenylyl cyclase and regulated by G-proteins. G-proteins are
coupled to hormone receptors, making them highly sensitive to the presence of
hormones. Hormones, therefore, are potent regulators of adipose tissue lipolysis.
For example, lipolysis can be stimulated by catecholamine release, as occurs during
exercise and periods of stress, and inhibited by high insulin levels, as occurs in the
post-prandial state (Carey 1998).
Coppack et al (Coppack 1999) studied the relative rates of LCFA and
glycerol release from adipose tissue during the postabsorptive state after an overnight
fast. They found that rates of release from adipose tissue match the 3:1 ratio of
LCFA to glycerol in a triglyceride molecule. Their findings found a net uptake of
both LCFA and glycerol in skeletal muscle. This is in contrast to their findings in
adipose tissue, where there was no detectable uptake of either LCFA or glycerol
during the same postabsorptive period. However, with glucose infusion, LCFA and
glycerol uptake into adipose tissue increased significantly. These findings suggest
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that an increase in fuel availability increases uptake of LCFA and glycerol into the
adipocytes for storage (Coppack 1999).
When there is an increased demand to use fat as an energy source, as occurs
during exercise or periods of stress, LCFA availability should increase. As expected,
Wolfe et al (Wolfe 1990) showed that lipolysis increases during exercise, while re-
esterification into triglycerides decreases. Both effects act to increase LCFA
availability when there is an increased energy demand. Additionally, their finding is
in support of the triglyceride-fatty acid (TG-FA) cycle and its ability to regulate flux
of LCFA between lipolysis and re-esterification during either excessive or limited
fuel availability (Wolfe 1990). The existence of the TG-FA cycle is physiologically
advantageous in allowing us to mobilize LCFA quickly (Tagliaferro 1989).
Due to their hydrophobic nature, LCFA after they are released from
adipocyte stores, must be transported through the bloodstream to other tissues, bound
to albumin (Van der Vusse 1996). Albumin is a soluble protein that has sites that
bind to LCFA with high affinity (Turcotte 1999). Bound to albumin, LCFA can be
transported in the plasma to skeletal muscle or liver to be used as an energy source.
Plasma Membrane Transport
Traditionally, transport of LCFA across the plasma membrane has been
thought to be a passive diffusional process (Turcotte 199S). Physiological
considerations, however, contradict the notion that LCFA can only diffuse through
the plasma membrane (Turcotte 1995). For example, the anionic nature of LCFA in
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plasma at physiologic pH coupled with the negative charge on the cytosolic side of
the plasma membrane, defies basic tenets of the process of diffusion. Additionally,
the low concentration of unbound LCFA is inconsistent with the high rates of FA
influx into the cell. Structurally, the tight phospholipid bilayer is not conducive to
permeation by LCFA. These discrepancies were the basis for investigations into a
possible carrier-mediated transport system for LCFA across the plasma membrane.
Indeed, much evidence has shown that transport of LCFA across the plasma
membrane may be mediated by a transsarcolemmal LCFA transport system.
Specific binding of LCFA to proteins isolated from plasma membranes has been
shown to be saturable, inhibitable, pH and temperature dependent (Abumrad 1998,
Turcotte 1991, Stremmel 2000). Under controlled experimental conditions in
isolated rat adipocytes, where LCFA uptake was not limited by supply of unbound
LCFA and LCFA was not esterified by the cell, LCFA transport saturation kinetics
was not linear as would be expected in a diffusion-driven transport process
(Abumrad 1984). Furthermore, in perfused rat skeletal muscle, LCFA uptake
displayed saturation kinetics when plotted against unbound LCFA concentration,
indicating that a carrier mediated system is likely (Turcotte 1991). Other evidence
shows that LCFA transport in rat adipocytes can be reversibly inhibited by phloretin,
a known inhibitor of glucose and anion transport systems, and irreversibly inhibited
by LCFA derivatives (Abumrad 1998). Perhaps most convincing are studies
performed in giant sarcolemmal vesicles, which allows the examination of LCFA
transport without the influence of LCFA metabolism since they lack mitochondria
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and other intracellular organelles. Studies in vesicles prepared from rat hindlimb
demonstrated that LCFA uptake was associated with the presence of transport
proteins (Bonen 1998). This suggests that LCFA transport across the plasma
membrane involves these proteins, and that transport was not driven by metabolism
and esterification of LCFA. These studies further support the notion that a carrier-
mediated system exists for LCFA transport. To date, several plasma membrane
transport proteins have been identified and characterized (Abumrad 1993).
Experiments using transgenic animal models and transfected cells provide the
most compelling evidence for the ability of these transport proteins to mediated
LCFA transport into the cell. A peripheral membrane protein located on the plasma
membranes of muscle cells, plasma membrane fatty acid binding protein (FA BP pm),
is thought to be involved in LCFA transport across the sarcolemma (Turcotte 2000).
FA BPpm was discovered to have a homologous amino acid sequence to the
mitochondrial isoform of aspartate aminotransferase (m Asp At) (Stump 1993). A
study performed in 3T3 fibroblasts transfected with cDNA for mAspAt found that
the transfected cells expressed FA B P pm and demonstrated a 3.5 fold increase in
LCFA uptake (Isola 1995). This study supports the involvement of FA B P pm in
mediating transport of LCFA into cells.
Studies have also attempted to examine the association between FA B P pm and
LCFA oxidation. Evidence suggests that chronic conditions can give rise to changes
in FA B P pm content. Conditions such as endurance training, fasting, caloric
restriction, and obesity have been associated with increased levels of FA B P pm
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(Turcotte 1997). Different muscle fiber types have also been shown to have varying
contents of FA B P pm, likely related to the contribution of each fiber type to oxidative
metabolism. In slow-twitch oxidative fibers, FA BPpm was found to be significantly
higher, suggesting an involvement of this protein in transport of LCFA for aerobic
metabolism (Bonen 1998). In endurance-trained rats, it was also shown that
FA B P pm content was higher in red muscle than in white muscle (Turcotte 1999).
Furthermore, the high FABPpm content was associated with increased plasma LCFA
uptake both at rest and during muscle contraction, and increased plasma LCFA
oxidation in contracting muscle, providing additional evidence for the role of
FA B P pm as a LCFA transport protein and its importance in the regulation of
oxidative metabolism (Turcotte 1999).
A second membrane protein, fatty acid translocase (FAT) has been associated
with LCFA transport into the cell. Overexpression of FAT in transgenic mice was
shown to enhance LCFA oxidation in contracting skeletal muscle (Ibrahimi 1999).
Fibroblast cells transfected with clones containing a CD36 gene, which is a homolog
of FAT, demonstrated a 2-4-fold increase in FA uptake that was proportional to the
amount of protein expressed.
FAT is an integral membrane protein, but has also been found to exist
intracellularly (Bonen 2000). It has been suggested that FAT translocation from
inside the cell to the plasma membrane can occur, similar to translocation of GLUT-
4 to the plasma membrane (Bonen 2000). A recent study in giant sarcolemmal
vesicles obtained from rat hindlimb showed that with muscle contraction, the
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presence of intracellular FAT was significantly reduced while FAT at the surface of
the vesicles increased (Bonen 2000). In electronically stimulated muscle, FAT was
significantly higher, indicating that increases in oxidative capacity, such as those that
occur in endurance trained muscle, are linked to the expression of the transport
protein (Bonen 1999). FAT content has also been shown to be higher in red
oxidative than in white glycolytic muscle fibers, suggesting its probable role in
oxidative metabolism (Bonen 1999). Results from each of these studies support the
association between oxidative metabolism and the FAT protein.
Finally, another integral membrane protein present in skeletal muscle that is
thought to be involved in mediating intracellular transport of LCFA is fatty acid
transport protein (FATP) (Van der Vusse 1996). FATP has not been studied as
extensively as FA BPpm or FAT. Transfection experiments in 3T3 fibroblasts have
shown increased expression of the FATP protein, which was associated with
increased LCFA uptake (Chen 2000). However, LCFA transport studies from
different tissues show conflicting results (Schaffer 1994, Luiken 1999). In rat
adipocytes, increased expression o f FATP correlated with increased LCFA uptake
rate (Schaffer 1994). This is in contrast to a study in giant sarcolemmal vesicles
isolated from white skeletal muscle, in which a negative correlation was found
between FATP expression and LCFA uptake (Luiken 1999). Interestingly,
expression of FATP protein is present in high concentrations in white skeletal
muscle, compared to red skeletal muscle. FATP, therefore, may play a role in
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mediating intracellular FA transport; however, its exact role has yet to be
determined.
Cytosolic Transport
Upon entry of LCFA into the cell, whether by diffusion or with the aid of
transport proteins, LCFA must bind to a soluble protein in order to move through the
cytosol. A cytosolic fatty acid binding protein (FABPc) is believed to bind to LCFA
in the cytosol (Glatz 1990). The configuration of FABPc allows two LCFA
molecules to bind, and unsaturated LCFA have been shown to bind with greater
affinity than saturated LCFA (Van der Vusse 1996). FABPc expression has been
shown to be regulated at either the transcription step, or at the level of mRNA
stability (Carey 1994). A study in rats found that under conditions associated with
increased LCFA oxidation, namely streptozotocin-induced diabetes and fasting,
FABPc concentration and mRNA concentration were increased 2-fold (Carey 1994).
Rate of transcription increased with fasting, and was higher in red skeletal muscle
than in white skeletal muscle. This experiment provided evidence for FABPc
regulation under conditions of increased LCFA need. However, the effects of
exercise training on FABPc are conflicting. In one study, exercise training increased
FABPc content in plantaris muscles (Van der Vusse 1996). Similarly, in rat tibialis
anterior, chronic electrical stimulation increased content of FABPc. However, all
other studies failed to detect a training effect on changes in FABPC content (Van der
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Vusse 1996). Therefore, the exact role of FABPc on LCFA oxidation has yet to be
determined.
Intracellular Metabolism
Aside from factors related to delivery and transport of LCFA, many other
factors regulate muscle LCFA metabolism inside the cell. Two distinct pathways are
involved in intracellular LCFA metabolism; namely, oxidative metabolism in which
LCFA are oxidized for production of energy, and triglyceride metabolism which is
the synthesis of LCFA into storage triglycerides. While many factors are known to
affect oxidative metabolism, the factors that will be considered in this review are
oxidative capacity, mitochondrial transport, fuel availability, and metabolic rate.
Three factors affecting triglyceride metabolism, namely, oxidative capacity, fuel
availability, and hormone effects will also be discussed.
Oxidative Metabolism
Oxidative capacity. The capacity to oxidize LCFA is dependent, in part, on
the activity of enzymes involved in the pathways of 13-oxidation, Krebs Cycle, and
electron transport chain. It has been shown that certain key enzymes can be
upregulated during times of increased LCFA requirements. Fatty acyl-CoA
dehydrogenase, for example, has been shown to increase in skeletal muscle of
trained rats compared to the sedentary controls (Mole 1971). Similarly, training has
been shown to increase the activity of B-hydroxyacyl-CoA dehydrogenase in human,
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rat, and horse muscle (Van der Vusse 1996, Kiens 1997, Helge 1997). Furthermore,
humans placed on a high fat diet during their training period had greater increases in
B-hydroxyacyl-CoA dehydrogenase enzyme activity, than their carbohydrate fed
counterparts (Helge 1997). It has also been shown that with training there is
increased activity of citrate synthase and carnitine palmitoyl transferase I (CPT-I),
rate limiting enzymes of the Krebs Cycle and transmitochondrial membrane
transport, respectively (Garrido 1996). Similarly, enzymes involved in LCFA
metabolism, LCFA-CoA synthetase, CPT-I, and LCFA-CoA dehydrogenase were
higher in trained rats than in their sedentary counterparts (Mole 1971). CPT-I
activity also differs between muscle fiber types, with red oxidative fibers having
nearly double the activity of the white glycolytic fibers (Baldwin 1972). Taken
together, these studies suggest the presence of cellular factors that act to increase
activity of key LCFA oxidation enzymes during times of higher LCFA availability
and demand.
The end product of 13-oxidation, acetyl-CoA is then fed into the Krebs Cycle
and electron transport chain to generate ATP for energy. Key enzymes in these
metabolic pathways have also been shown to increase with training. Malate
dehydrogenase has been shown to increase with training in both male and female
runners (Costill 1979). Additionally, while male runners have an overall higher
activity of the enzyme succinate dehydrogenase than female runners, both genders
show increases in enzyme activity with training. The greater enzyme activity is
likely a result of the higher content of oxidative muscle fibers in male runners
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(Costill 1976). It is logical that the oxidative fibers have higher activities of the
enzymes involved in oxidation, and that the activities of these enzymes are yet
higher in trained muscle.
Mitochondrial transport From transport in the plasma as albumin bound
LCFA to their entry in the mitochondria, there exists another transport step for
LCFA. Whereas short and medium chain LCFA can easily diffuse through the
mitochondrial membrane, mitochondrial LCFA transport occurs via the carnitine
shuttle (Salway 1994). Before transport occurs, LCFA must first be linked to the
sulfhydryl group of coenzyme A (CoA) (Stryer 1995). This reaction is catalyzed by
the enzyme acyl-CoA synthetase in an ATP-requiring reaction. Activity o f the acyl-
CoA synthetase enzyme has been shown to be higher in tibialis anterior, EDL, and
gastrocnemius, which are characteristic fast twitch glycolytic and fast twitch
oxidative/glycolytic fibers, compared to slow twitch oxidative soleus muscle fibers
(Alam 1998). Acyl-CoA synthetase activity has also been shown to respond to
exercise training, with higher enzyme activities in mixed hindlimb of trained rats
than their sedentary counterparts (Mole 1971).
Once formed, long chain fatty acyl-CoA (LC-CoA) can be transported
through the mitochondrial membrane by transfer of the acyl group from CoA to the
hydroxyl group of carnitine to form acyl-camitine (Salway 1994). This reaction is
catalyzed by enzyme carnitine palmitoyl transferase I (CPT-I). CPT-I is the first
enzyme o f the carnitine shuttle, a carrier-mediated system to transport LC-CoA into
the mitochondria. CPT-I converts LC-CoA into an acyl-camitine, which can cross
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the mitochondrial inner membrane via antiport with a free carnitine molecule, aided
by a translocase enzyme. The enzyme carnitine palmitoyl transferase II (CPT-II),
located on the surface of the inner membrane exchanges the carnitine with CoA, to
reproduce a LC-CoA. While each of the enzymes involved in the carnitine shuttle
system is critical to transport of LC-CoA into the mitochondria, regulation of the
CPT-I enzyme has been shown to be key in the control of LCFA oxidation.
CPT-I is the rate-limiting enzyme involved in mitochondrial uptake of LC-
CoA (Winder 1998). Studies have demonstrated that LCFA oxidation increases with
increased LCFA concentration, and that both CoA and carnitine are required for
optimal rates of LCFA oxidation (Winder 1998). In vitro studies in mitochondria
taken from rat hindlimb showed that with increasing carnitine concentration,
palmitate oxidation increased (Mole 1971). In vivo studies performed by McGarry
et al (1983) in rat and dog skeletal muscle and in dog heart, found that increases in
carnitine concentration paralleled increases in mitochondrial CPT-1 activity.
Carnitine, therefore, is a necessary substrate for CPT-1.
Studies have also shown that CPT-1 activity can be regulated through both
chronic and acute changes. In skeletal muscle homogenate taken from trained rats,
CPT-I activity was significantly higher, resulting in palmitate oxidation that was
nearly double that of their sedentary counterparts (Mole 1971). Endurance training
has also been shown to increase CPT-I activity in rat heart, skeletal muscle, and liver
mitochondria (Guzman 1988). This study also showed the acute effect of pH on
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CPT-I activity, with enzyme activity increasing in both the trained and control
groups as experimental pH was increased from 6.8 to 7.4.
Perhaps the most important and well-studied regulator of this mitochondrial
transport step is malonyl-CoA. Malonyl-CoA, the first intermediate in the synthesis
of LCFA, is a known inhibitor of CPT-I (Ruderman 1998). Malonyl-CoA competes
with LC-CoA for the CPT-I binding sites. When malonyl-CoA is present, entry of
LCFA into the mitochondria is greatly reduced, and subsequently oxidation of LCFA
is diminished (Winder 1998). Malonyl-CoA concentrations have been shown to
differ between muscle fiber types. In quadriceps muscle separated by fiber type,
malonyl-CoA concentrations in the red muscle fibers were more than three times
higher than in the white (Winder 1990). In addition, malonyl-CoA content in the
soleus muscle, a slow oxidative fiber, was closer to that of red quadriceps, while the
fast glycolytic gastrocnemius muscle had a lower malonyl-CoA content. The
reasons for these fiber type differences have not been studied.
In rats, malonyl-CoA concentration can be regulated acutely as demonstrated
by decreases in malonyl-CoA content in response to exercise and short-term fasting
(Winder 1990), both of which are conditions of decreased carbohydrate availability.
In rats, a 24 hour fast decreased malonyl-CoA content to 0.7 nmol/g, compared to
2.1 nmol/g in the fed state. A 48-hour fast resulted in even lower malonyl-CoA
content (McGarry 1983). Similarly, a significant decrease in malonyl-Co A during
exercise occurred in untrained rats after a 30-minute treadmill run (Winder 1989). In
this study, significant decreases in malonyl-CoA concentrations occurred after only 5
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minutes of running in the red quadriceps muscle, while malonyl-CoA did not
decrease in white quadriceps until 30 minutes of treadmill running.
Since prolonged exercise is associated with decreased glucose availability, it
is not surprising that glucose infusion prevented the exercise-induced drop in
malonyl-Co A concentrations (Elayan 1991). It was later shown in incubated rat
soleus that while increasing glucose concentrations prevented malonyl-CoA
concentrations from decreasing, the effect was even more pronounced in media
containing both glucose and insulin, suggesting the existence of a “fuel-sensing
mechanism” in muscle that involves malonyl-Co A (Saha 1995). Furthermore,
studies utilizing a euglycemic-hyperinsulinemic clamp demonstrated that sustained
levels of glucose and insulin were instrumental in increasing malonyl-CoA
concentrations, likely by a mechanism implicating acetyl-CoA carboxylase (ACC),
the rate-limiting enzyme in the formation of malonyl-CoA (Saha 1999). Possible
substrate interactions between carbohydrate and LCFA fuels and the involvement of
malonyl-CoA in the regulation of substrate utilization will be discussed in the next
section.
The presence of malonyl-CoA has been shown to decrease CPT-I activity in
rat skeletal muscle mitochondria, with higher concentrations of malonyl-CoA
resulting in increased inhibition of CPT-I activity (McGarry 1983, McGarry 1978).
Manipulation o f malonyl-CoA levels with different concentrations of palmitate in rat
perfused hindlimb, showed that reduction of muscle malonyl-CoA levels resulted in
increased LCFA oxidation at all concentrations of palmitate, providing evidence for
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the role of malonyl-CoA in preventing palmitate entry into the mitochondria (Merrill
1998).
Fuel availability. The interaction between carbohydrates and fats to regulate
oxidative metabolism is a valuable physiological adaptation. When looking at each
substrate in terms of fuel storage capacity, whole body glycogen stores account for a
mere 2% of total triglyceride stores (Newsholme 1983). Because of the efficiency
by which glucose units can be rapidly released from glycogen, it is reasonable that
during the early stages of exercise or starvation, glycogen is the primary fuel used.
But, as glycogen stores become depleted, fatty acids are mobilized and there is a
necessary shift to the use of fatty acids as the fuel source. Interactions between
carbohydrate and lipid fuel sources must, therefore, be involved in the control of fuel
utilization.
The existence of some type of interaction between carbohydrate and lipid
fuels, and the regulation of each one’s contribution to muscle oxidative metabolism,
is clearly evident. Carbohydrate availability has been shown to alter LCFA
utilization in perfused and incubated muscle as well as in whole body experiments
(Saha 1999, Sidossis 1996, Duan 1993, Turcotte 2002). We have previously shown
that perfusion with high carbohydrate availability in the presence of physiologic
levels of LCFA was associated with an increase in LCFA uptake and a decrease in
LCFA oxidation, which could be explained by an increase in malonyl-CoA levels.
Similarly, in humans infused with low LCFA concentrations during a
hyperinsulinemic, hyperglycemic clamp, plasma LCFA oxidation decreased
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significantly (Wolfe 1988). Taken together, these results demonstrate the
importance of carbohydrate availability in the regulation of LCFA utilization in
muscle.
In 1963, the glucose-fatty acid cycle was first proposed by Randle, et al to
explain the reciprocal relationship between the rates of glucose oxidation and fat
oxidation. The glucose fatty acid cycle maintains that the rate of glucose oxidation is
determined by the rate of LCFA oxidation (Randle 1963); that is, when there is
increased LCFA oxidation, glucose oxidation rates decrease, and when LCFA
oxidation is low, glucose oxidation rates increase. Thus, under conditions of
increased energy need, when LCFA are used as the primary substrate, the rate of
glucose oxidation decreases.
In his original paper describing the glucose-fatty acid cycle, Randle proposed
that high plasma concentration of LCFA was the cause of a number of abnormalities
related to carbohydrate metabolism (Randle 1963). The cycle as originally
conceived stated that increased LCFA release from muscle or adipose tissue leading
to increased availability of LCFA for oxidation, inhibited glucose uptake and
metabolism (Randle 1963). Then, upon removal of the carbohydrate stress, the rates
of LCFA release decrease, and subsequently oxidation of LCFA decreased while
glucose metabolism increases. In the original 1963 article, little was known about
allosteric regulation of enzymes involved in metabolism. Evidence for the existence
of such a cycle came from studies of glycolytic flux in rat diaphragm and heart
muscle, under conditions of increased plasma LCFA concentrations, and in states of
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diabetes and starvation (Randle 1963). However, since then, further research has
suggested that enzymes involved in several metabolic pathways may regulate the
interaction between carbohydrate and fatty acid fuels (Randle 1964, Randle 1988).
Increased oxidation of LCFA initiates the cascade of events proposed by
Randle et al that lead to a shift in substrate utilization from carbohydrate to LCFA
fuels. Higher rates of LCFA oxidation lead to an increase in production of acetyl-
CoA from 3-oxidation. An increased ratio of mitochondrial acetyl-CoA to CoA
inhibits the activity of pyruvate dehydrogenase, the enzyme that converts pyruvate,
the product of glycolysis, to acetyl-CoA. This occurs through acetyl-CoA activation
of PDH kinase, which phosphorylates PDH, rendering it inactive. A second
regulatory pathway involving acetyl-CoA is in its conversion to citrate, a Krebs cycle
intermediate. Increased levels of mitochondrial citrate inhibit the activity of
phosphofructokinase (PFK), the rate limiting enzyme in glycolysis (Randle 1964,
Randle 1988). Inhibition of PFK causes accumulation of glucose-6-phosphate.
Increased levels of glucose-6-phosphate result in decreased hexokinase activity,
which is the enzyme that initiates glycolysis with the conversion of glucose to
glucose-6-phosphate (G6P). Acting in concert, the inhibition of these enzymes leads
to a decrease in glycolytic flux and glucose uptake (Randle 1964, Randle 1988).
The existence of the glucose-fatty acid cycle has become widely accepted,
despite the fact that studies in rat and human skeletal muscle have been unable to
show definitively the relationship between increased fat oxidation and glycolytic flux
(Sidossis 1996, Boden 1994, Hargreaves 1991, Putman 1993). Specifically, in
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healthy human subjects infused with elevated LCFA levels, concentrations of citrate
and G6P were not always increased (Boden 1994, Hargreaves 1991). Similarly, in a
study in young male subjects, where LCFA availability was increased through
alterations in dietary intake, citrate levels were unchanged and G6P levels were
significantly lower than in the control subjects (Putman 1993). These studies suggest
that another mechanism may be involved in the reciprocal regulation of carbohydrate
and LCFA metabolism.
In 1996, Sidossis and Wolfe proposed an alternative hypothesis for the
regulation of fuel selection in oxidative metabolism (Sidossis 1996). Termed the
reverse cycle, they proposed a mechanism that states the reverse of the glucose-fatty
acid cycle. They suggested that carbohydrate availability dictates oxidation of fat
and carbohydrate fuels. Their study, which utilized a hyperinsulinemic,
hyperglycemic clamp with maintenance of physiologic levels of LCFA via Intralipid
infusion, demonstrated an increase in glucose oxidation during the clamp, and a
subsequent decrease in LCFA oxidation. Sidossis and Wolfe, therefore, concluded
that it is the availability of intracellular glucose and/or insulin that determines fuel
utilization, and not the availability of intracellular LCFA, as suggested by the
glucose-fatty acid cycle.
In order to elucidate on the mechanism by which the reverse cycle occurs,
they performed a second study in humans (Sidossis 1996), again utilizing a
hyperinsulinemic, hyperglycemic clamp with Intralipid infusion. In this experiment,
they also infused tracers of both a medium chain LCFA, [l-l4C]octanoate, and a long
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chain LCFA, [l-l3C]oleate. Because long chain LCFA must be transported into the
mitochondria via the CPT shuttle, any effects of glucose or insulin on the transport
step would be denoted by changes in oleate, but not octanoate, oxidation. As
expected, oleate oxidation decreased by nearly 50%, while octanoate oxidation
remained unchanged (Sidossis 1996). This finding provided a possible mechanism
for the reverse cycle, likely through inhibition of CPT-1 (Sidossis 1996).
Early studies on CPT-I activity found that malonyl-CoA was a potent
inhibitor of CPT-I in liver, heart, and skeletal muscle (McGarry 1983, McGarry
1978). An obvious mechanism for the inhibition of LCFA oxidation as occurs in the
reverse cycle, therefore, was inhibition of CPT-I by malonyl-Co A. Many studies
have examined the role of malonyl-Co A in the regulation of LCFA oxidation in the
presence of high carbohydrate availability (Turcotte 2002, Duan 1993, Elayan 1991,
Saha 1999). Elevated levels of both glucose and insulin resulted in high intracellular
concentrations of malonyl-CoA. In addition, a series of experiments by Alam and
Saggerson in incubated rat soleus muscle found that manipulation of glucose and
pyruvate levels by insulin and dichloroacetate, respectively, also increased malonyl-
Co A content (Alam 1998). In our lab, we have demonstrated that high carbohydrate
availability was associated with increased LCFA uptake and decreased LCFA
oxidation in rat perfused hindlimb (Turcotte 2002). This decrease in LCFA
oxidation corresponded to an increase in malonyl-CoA levels, supporting the notion
that malonyl-CoA may be an important modulator of substrate oxidation in skeletal
muscle (Turcotte 2002). Conversely, it has been shown that under conditions of
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increased need to use LCFA as a fuel, such as occurs during exercise, malonyl-Co A
levels decrease (Winder 1989). It appears, therefore, that changes in malonyl-Co A
content are related to changes in energy or fuel demand. However, in humans,
malonyl-CoA measurements in muscle were not found to correlate with changes in
fat oxidation during exercise (Odland 1996, Odland 1998).
A possible mechanism for the alterations in malonyl-CoA content was
suggested by Winder and Hardie (1996). Their study in rat hindlimb found that
treadmill running dramatically decreased malonyl-CoA levels. The decrease in
malonyl-CoA was associated with a 2.4-fold activation of AMP-activated protein
kinase, AMPK, and a subsequent decrease in acetyl-CoA carboxylase (ACC)
activity, the enzyme involved in the synthesis of malonyl-CoA from acetyl-CoA. A
later study found that the increase in AMPK activity was associated with high levels
of free AMP (Hutber 1997). The mechanism proposed suggested that a high
AMP/ATP ratio, indicative of decreased fuel availability and increased energy
demand, resulted in activation of AMPK. The subsequent phosphorylation of ACC
by AMPK resulted in a decreased formation of malonyl-CoA, relieving inhibition of
CPT-I and increasing rates of LCFA oxidation. On the other hand, under conditions
of high fuel availability, such as in the sedentary or postprandial state, high
concentrations of circulating glucose causes increased levels of mitochondrial citrate,
the penultimate precursor of malonyl-CoA. Under these conditions, CPT-I is
inhibited and rates of LCFA oxidation decrease (Winder 1996).
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In addition to the effects imposed by carbohydrate availability on LCFA
metabolism, LCFA availability is also implicated in the regulation of LCFA
metabolism. Whole body human studies and rat hindlimb perfusion studies have
shown that high LCFA availability is associated with an increase in LCFA uptake
and oxidation, suggesting the importance of LCFA availability in the regulation of
LCFA utilization in muscle (Wolfe 1988, Turcotte 1998). Conditions of high LCFA
availability have also been associated with high muscle LC-CoA levels (Chien
2000). Chien et al have shown that increases in malonyl-CoA in starved-refed rats
resulted in increased RQ, suggesting a decrease in fat oxidation with increasing
malonyl-CoA concentrations. Interestingly, their findings show that the increases in
malonyl-CoA after refeeding were not associated with an increase in acetyl-CoA
carboxylase (ACC), the enzyme that carboxylates acetyl-CoA to form malonyl-CoA
nor was it associated with a decrease in malonyl-CoA decarboxylase (MCD), the
enzyme that decarboxylates malonyl-CoA to reform acetyl-CoA. They suggest that
a key factor in the mechanism of increased malonyl-CoA levels is related to
cytosolic LC-CoA levels (Chien 2000).
Indeed, LC-CoA have been reported to play an important role in the
regulation of muscle oxidative metabolism through their effects on enzyme activity
(Faergeman 1997). LC-CoA inhibits ACC activity through its activation of AMPKK
and subsequently AMPK. Phosphorylation of ACC by AMPK reduces ACC-
catalyzed formation of malonyl-CoA (Faergeman 1997). The findings of Chien et al
show that the increase in insulin and glucose concentrations that occur after
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refeeding following a 48 hour fast is associated with decreases in the cytosolic LC-
CoA levels, likely as a result of decreased LCFA availability and decreased rates of
hydrolysis of intramuscular triglycerides. They suggest that as LC-CoA levels
decrease, ACC activity increases causing formation of malonyl-Co A. LC-CoA have
also been shown to decrease activity of the mitochondrial citrate transporter
(Faergeman 1997) resulting in decreased availability of cytosolic citrate, the
penultimate precursor of malonyl-CoA production and a known allosteric activator
of ACC. Clearly, then, LC-CoA levels are key in the regulation o f LCFA entry into
the mitochondria for 3-oxidation. Furthermore, glucose and insulin availability are
determinants of LC-CoA levels suggesting interactions between carbohydrate and
lipid fuel sources.
Metabolic rate has also been shown to be key in the regulation of muscle
oxidative metabolism. Palmitate oxidation has been shown to increase in contracting
muscle of both untrained and trained rats, with significantly higher oxidation rates in
the trained rats (Dyck 2000). Similarly, in rat perfused hindlimb, palmitate oxidation
was significantly higher during muscle contraction than at rest, and that the V m ax for
LCFA uptake and oxidation was increased in contracting muscle (Turcotte 1998).
These findings suggest that increases in LCFA metabolism can be induced by muscle
contraction, and that alterations in cellular transport mechanisms occur as a result of
increased metabolic rate.
A recently studied regulator of muscle LCFA metabolism is the enzyme
malonyl-Co A decarboxylase (MCD), the enzyme responsible for degrading malonyl-
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CoA. It has recently been shown under conditions of high fat feeding, fasting, and
streptozocin-induced diabetes, which were all associated with high plasma LCFA
concentrations, that MCD expression and activity in rat heart and skeletal muscle
increased (Young 2001). Previous studies in rat heart muscle have shown that LCFA
oxidation increased with contractile stimulation, and that this increase was associated
with higher MCD activity than in the unstimulated hearts (Goodwin 1999). Thus, it
appears that MCD may be an important regulator of muscle LCFA metabolism
through its role in regulation of malonyl-CoA levels; however, its exact role remains
to be elucidated.
Triglyceride Metabolism
Enzymatic capacity. If LCFA are not required for oxidation, an alternative
fate is their esterification into intramuscular triglycerides (IMTG). Before
triglyceride (TG) synthesis can occur, LCFA must be converted to fatty acyl-CoA
by the enzyme acyl-CoA synthetase, in a reaction similar to that which precedes
LCFA transport into the mitochondria via the carnitine shuttle, as previously
described. The fatty acyl-CoA are then esterified to glycerol-3-phosphate to form
lysophosphatidic acid, and then phosphatidic acid. They are then hydrolyzed to form
diacylglycerol, and acylated to form the triacylglycerol molecule (Coleman 2000).
The rate-limiting step in the de novo synthesis of TG involves the enzyme glycerol-
3-phosphate acyltransferase (GPAT), the enzyme that aids in the acylation of
glycerol-3-phosphate to form lysophosphatidate (Coleman 2000).
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GPAT activity has been shown to correlate inversely with CPT-I activity
(Beauseigneur 1999). This supports the reciprocal regulation between LCFA
oxidation and LCFA esterification into triglycerides. Additional evidence for the
interrelation between oxidation and esterification is a study that provides evidence
that muscle GPAT is a target for AMPK (Muoio 1999). In isolated rat skeletal
muscle, with increasing concentrations of AIC AR, which mimics AMPK activation
by its metabolism to an AMP analogue, an increase in LCFA oxidation and a
corresponding decrease in TG esterification was observed (Muoio 1999). This
finding also provides evidence for a possible link between increased energy demand
and a subsequent decrease in TG synthesis via a decrease in GPAT activity. The
association between energy demand and TG synthesis rates has been shown in a
study in fiber-typed skeletal muscle, which found a strong correlation between TG
synthesis rates and oxidative capacity for each of the fiber types examined
(Budohoski 1996). Synthesis rates were highest in oxidative muscle, intermediate in
mixed muscle, and lowest in glycolytic fibers. In line with these findings, IMTG
content is higher in oxidative than in glycolytic muscle fibers (Essen 1977),
suggesting an increased reliance on IMTG as a fuel source in oxidative muscle. No
changes in GPAT activity were observed with training or high CHO diet (Garrido
1996).
Fuel availability. Synthesis of TG is dependent in part on the exogenous
supply of fatty acids, fuel requirements, and dietary composition (Van der Vusse
1996). Indeed, we have demonstrated that under conditions of high carbohydrate
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availability and basal levels of LCFA, there was a decrease in LCFA oxidation that
was associated with increased muscle triglyceride concentrations (Turcotte 2002).
This finding may be related to either an increased activity in enzymes of TG
synthesis, or a decreased activity in enzymes involved in TG degradation. Indeed, it
has been shown that GPAT activity increased in fasted animals refed a high
carbohydrate diet (Coleman 2000). Similarly, GPAT activity was higher in animals
fed a 3-week high fat diet compared to the starch-fed controls (Lawson 1981). This
data suggests that conditions of excess fuel availability may stimulate TG synthesis
in part through its action on the GPAT enzyme. Conversely, lipoprotein lipase in
heart muscle has been shown to decrease during feeding of a carbohydrate rich diet
(Schoonderwoerd 1989). In com-oil and tallow fed animals, lipoprotein lipase
activity in adipose tissue and heart was significantly lower than in sucrose-fed
animals, who had significantly lower activity than in their starch-fed controls
(Lawson 1981). Therefore, as expected under conditions of fuel excess, activity of a
key enzyme involved in TG synthesis increases while the activity of an enzyme
involved in TG degradation is reduced.
Hormone effects. The enzymes involved in TG synthesis have been shown
to be regulated hormonally. In incubated myocytes and myocardial tissue, it has
been shown that insulin increases activity of GPAT, and that the activation is dose-
dependent (Vila 1990, Vila 1991, Schoonderwoerd 1989). In contrast, type I
diabetes which is characterized by insulin deficiency has been associated with
decreased activity of adipose tissue fatty acid synthase, GPAT, and PPH, three
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enzymes involved in the synthesis of TG (Saggerson 1987). Epinephrine has also
been shown to have various effects on the enzymes of TG synthesis in myocardial
tissue, decreasing GPAT and DGAT activity, while increasing activity of PPH
(Schoonderwoerd 1989).
Enzymes involved in the degradation of TG may also be modulated by
catecholamines. Hormone sensitive lipase was shown to have 10 times greater
activity in soleus muscle than in adipose tissue, and this activity increased in
response to epinephrine (Langfort 1999). Similarly, glucagon has been shown to
increase lipase activity in myocardial tissue (Schoonderwoerd 1989). It is clear,
therefore, that cAMP-mediated protein phosphorylation plays a role in the regulation
of enzymes involved in TG degradation.
Conclusions
Oxidation of fatty acids in skeletal muscle, from the initial mobilization step
in the adipose tissue triglyceride stores to the final role in oxidative metabolism in
skeletal muscle mitochondria, is regulated by numerous factors. Alterations in
LCFA delivery, transport across the plasma membrane and through the cytosol, and
intracellular mechanisms related to oxidative capacity, mitochondrial transport, and
fuel interactions can substantially alter rates of fatty acid oxidation. The obvious
need for LCFA as a fuel source in skeletal muscle underlies the need for continued
research in this area, to clearly elucidate the fate of LCFA on their path to 1 3 -
oxidation in skeletal muscle.
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IV. EXPERIMENT
High CHO and high LCFA availability increase
LCFA uptake and oxidation in perfused muscle
MICHELE L. SACMAN, ALICE J. YEE,
MARK K. TODD, AND LORRAINE P. TURCOTTE
Department of Kinesiology
University of Southern California, Los Angeles, CA
Running head: LCFA metabolism with high CHO and LCFA availability
Corresponding address:
Lorraine P. Turcotte, Ph.D.
Department of Kinesiology
University of Southern California
3560 Watt Way, PED 107
Los Angeles, CA 90089-0652
Tel.: (213) 740-8527
Fax: (213)740-7909
E-mail: turcotte@usc.edu
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ABSTRACT
To evaluate the effects of combined high carbohydrate and high long chain
fatty acid (LCFA) availability on skeletal muscle LCFA metabolism, rat hindquarters
were perfused at rest with 1S00 pM albumin-bound palmitate, albumin-bound
[1 4 C]palmitate, [3 H]glucose, and either 6 mM glucose and 10 nU/mL insulin (LG) or
20 mM glucose and 1000 |j.U/mL insulin (HG). For the same high plasma palmitate
delivery, high CHO availability was associated with an increased rate of palmitate
uptake, but no change in the relative contribution of palmitate to substrate oxidation.
Fractional and total rates of palmitate uptake were 38% and 55% higher,
respectively, in the HG group than in the LG (0.066 ± 0.006 vs 0.091 ±0.015 and
28.7 ± 5.1 nmol*min'1 -g*1 vs 18.6 ± 1.4 nmol-min'^g'1 , p<0.05). While the
percentage of palmitate oxidized was not significantly different between the two
groups, total palmitate oxidation was 39% higher in the HG group than in the LG
group (1.1 ± 0.2 nmol,min'1 *g‘l vs 1.8 ± 0.2 nmol-min'^g'1 , p<0.05). The increased
uptake of palmitate was not associated with a higher rate of triglyceride (TG)
synthesis, resulting in no change in TG concentration over the perfusion time. These
results demonstrate that conditions of high carbohydrate and high LCFA availability
are associated with increases in LCFA uptake and but no change in the relative
contribution o f LCFA to oxidative metabolism.
Keywords: triglyceride synthesis, LCFA metabolism, glucose uptake, intramuscular
triglyerides, skeletal muscle
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INTRODUCTION
The relative contribution of long chain fatty acids (LCFA) to muscle
oxidative metabolism is regulated by many factors, which include carbohydrate and
FA availability. Carbohydrate availability has been shown to alter LCFA utilization
in perfused muscle as well as in whole body experiments (39,49). In rat skeletal
muscle, we have shown that perfusion with high carbohydrate availability in the
presence of basal LCFA levels was associated with an increase in malonyl-CoA
levels, supporting the notion that malonyl-CoA may be an important modulator of
substrate oxidation in skeletal muscle (49). Similarly, in humans infused with low
LCFA concentrations, plasma LCFA oxidation decreased significantly during a
hyperinsulinemic, hyperglycemic clamp (39). These results demonstrate the
importance of carbohydrate availability in the regulation of LCFA utilization in
muscle.
It is also well accepted that LCFA availability is a key factor that determines
LCFA utilization in skeletal muscle. In human whole body experiments (54) and in
rat hindlimb perfusion studies (45), results have shown that high LCFA availability
is associated with an increase in LCFA uptake and oxidation. High long chain fatty
acyl-CoA (LC-CoA) levels, which have been associated with high LCFA levels (5),
have been linked to inhibition of enzymes involved in LCFA uptake and oxidation
(10). LCFA availability, therefore, has a demonstrated direct effect on LCFA
metabolism, but may also act indirectly through effects of LC-CoA on enzymes
involved in LCFA metabolism.
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While the independent effects of high carbohydrate availability and high
LCFA availability have been considered, the combined effects of high LCFA and
high carbohydrate availability have not been well characterized. Thus, it is not
known what effects high LCFA availability in combination with high CHO
availability would have on LCFA uptake and oxidation in skeletal muscle.
Therefore, the purpose of this study was to determine whether high
carbohydrate availability in the presence of high LCFA availability would alter the
rate of LCFA uptake by muscle, and the relative contribution of LCFA to total
oxidative metabolism in muscle. LCFA kinetics was measured in muscle perfused
under pre-determined conditions of high LCFA availability with either high or low
carbohydrate availability. Glucose kinetics, substrate exchange across the
hindquarter, and muscle triglyceride concentrations were also evaluated to examine
the impact of high substrate availability on regulation of substrate metabolism.
MATERIALS AND METHODS
Animals. Male Wistar rats were housed in pairs and maintained on a 12:12-h
light-dark cycle. They received regular rat chow and water ad libitum. Rats were
randomly assigned to the high glucose (HG: n=8, BW=307.3 ± 8.3 g) or low glucose
group (LG: n=12, BW=334.3 ±9.4 g, p>0.05).
Hindquarter perfusion. Rats were anesthetized with ketamine/xylazine (80
mg and 12 mg/kg BW, respectively) and prepared surgically for hindquarter
perfusion as previously described (33). The initial perfusate (200 mL) consisted of
36
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Krebs-Henseleit solution, I-day old washed bovine erythrocytes (hematocrit, 30%),
4% bovine serum albumin, 1500 pM albumin-bound palmitate, and albumin-bound
[l-l4C]palmitate (ICN Pharmaceuticals, Costa Mesa, CA), [3-3 H]glucose (NEN Life
Science Products, Boston, MA), and either 6 mM glucose and 10 pU/mL insulin
(LG) or 20 mM glucose and 1000 pU/mL insulin (HG). This concentration of
palmitate was chosen because it has been shown that, at concentrations of 1500 pM,
the maximal velocity of palmitate uptake and oxidation are attained in the perfused
hindquarter preparation (44). The perfusate (37°) was continuously gassed with a
mixture of 95% 02-5% CO2, which yielded arterial pH values of 7.3-7.4 and arterial
PCO2 and p02 values that were typically 38-43 and 250-350 Torr, respectively.
The first 25 mL of perfusate that passed through the hindquarter was
discarded. The left gastrocnemius-soleus-plantaris muscle group was freeze-
clamped in situ and stored in liquid N2. The left iliac vessels were then tied off, and
a clamp was fixed tightly around the proximal part of the leg to prevent bleeding.
Following an equilibration period of 20 min, the right leg was perfused at rest for 40
min at a perfusate flow of 5 mL-min'1 (LG: 0.27 mL-min^g'1 ; HG: 0.29 mL-min*l-g l
perfused muscle, p>0.05). Arterial and venous perfusate samples were taken at 10,
20, 30, and 40 minutes. Arterial and venous perfusate samples for determinations of
PCO2, p C > 2, pH, and hemoglobin were taken after 15-20 minutes at rest.. Mean
perfusion pressures were 46±6 and 55±4 mmHg, for the HG and LG groups,
respectively, during unilateral hindquarter perfusion at rest. At the end of the
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perfusion period, the right gastrocnemius-soleus-plantaris muscle group was sampled
as described above.
Blood and muscle sample analysis. Arterial and venous perfusate samples
were analyzed for glucose, lactate, glycerol, and FA concentrations as well as for
[l4C]FA, l4C 02, and 3 H2 0 radioactivities. Samples for glucose and lactate were
analyzed using the YSI 1500 glucose and lactate analyzer (Yellow Springs
Instrument, Yellow Springs, OH). Samples for FA and glycerol were put in 200 pM
ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid (pH 7) and
centrifuged. The supernatant was frozen until analyzed spectrophotometrically using
the WAKO NEFA-C test (Biochemical Diagnostics, Edgewood, NY). Because the
FA concentration was low in the absence of added palmitate (<40 |iM) and because
palmitate was the only FA added, measured FA concentrations were taken to equal
palmitate concentrations.
To determine plasma palmitate and glucose radioactivity, duplicate 100 pL
aliquots of the perfiisate plasma were mixed with liquid scintillation fluid
(BudgetSolve; Research Product International, Mount Prospect, IL) and counted in a
Tri-carb scintillation counter with dual channel (model 4000CA; United
Technologies Packard, Downers Grove, IL). The liberation and collection of l4C02
from the blood were performed within 4-5 min of anaerobic collection (2 mL) as
previously described (47). Perfusate samples for the determination of pC02, p0 2 ,
pH, and hemoglobin were collected anaerobically, placed on ice, and measured
within 5 min of collection with an ABL-5 acid-base laboratory (Radiometer
38
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America, Westlake, OH) and spectrophotometrically (Sigma Chemical),
respectively. To determine plasma 3 H20 radioactivity, 100 jiL of perfusate plasma
were precipitated with Ba(OH) 2 and ZnS0 4 , and centrifuged, as previously described
(47). Half of the supernatant was evaporated to dryness prior to counting. The
plasma 3 H2 0 radioactivity was determined as the difference between the evaporated
and nonevaporated counts.
Muscle triglyceride concentration was determined as glycerol residues after
extraction and separation o f the muscle samples, as previously described (47). The
final supernatant was analyzed spectrophotometrically for glycerol by the enzymatic
glycerol kinase method (Sigma Chemical, St. Louis, MO). To measure the
incorporation of [1 4 C] palmitate into muscle triglycerides, lipids from the extracted
organic layer were separated by liquid chromatography as previously described (47).
Calculations and statistics. Fractional and total uptake, and percent and total
oxidation of palmitate were calculated as previously described (47). Both percent
and total palmitate oxidation were corrected for label fixation by using acetate
correction factors determined previously (49). Uptake and release of substrates and
uptake of oxygen across the hindquarter were calculated by multiplying perfusate
flow by the arteriovenous difference in concentration and were expressed per gram
of perfused muscle. Rates o f muscle glycolysis were calculated from the increment
per unit time in 3 H2 0 (dpm-mL ^m in1 ) x muscle water mass (mL)/[3-3 H]glucose
specific activity (dpnvmg'1 ). Glucose oxidation was calculated as the rate of
glycolysis minus lactate release and expressed in pmol*min‘1 -g'1 . Palmitate
39
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incorporation into muscle triglycerides was calculated as the radioactivity
accumulated in the triglyceride fraction and was expressed per milligram of wet
muscle weight. Triglyceride synthesis rate was calculated as the amount of
[l4C]palmitate incorporated into the triglyceride fraction divided by the specific
activity of the perfusate plasma [1 4 C]palmitate and corrected for time. Data were
analyzed using a one-way or two-way analysis of variance with repeated measures,
as needed (Statistica, Tulsa, OK). The significance level was set at p<0.05.
RESULTS
Palmitate metabolism (Table 1). As dictated by the experimental protocol, arterial
perfusate palmitate concentration and perfusate palmitate delivery to the hindquarter
were not significantly different between the LG and the HG group (p>0.05).
Palmitate fractional uptake was 38% higher in the HG group, and this was associated
with a significantly higher total rate of palmitate uptake in the HG group than in the
LG group (p<0.05) (Fig. 1). While the percentage of palmitate oxidized was not
significantly different between the two groups (p>0.05), total palmitate oxidation
was 39% higher in the HG group than in the LG group (p<0.05) (Fig.2).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Table 1. Effects of high carbohdrate availability on palmitate metabolism in
resting hindquarters perfused with high LCFA availability.
LG HG
Palmitate concentration, limol'L1 1551.2 ±49.7 1525.4 ±66.4
Palmitate delivery, nmol-min^-g1 293.4 ±12.2 311.6 ± 16.9
Palmitate fractional uptake 0.066 ±0.006 0.091 ±0.015
Palmitate uptake, nmohmin^g1 18.6 ±1.4 28.7 ±5.1*
Palmitate oxidation, % 9.0 ± 1.3 6.5 ±2.0
Palmitate oxidation, nmol-min ^ g1 1.1 ±0.2 1.8 ±0.2*
Values are means ± SEM; n=12 and n=8 for the LG and HG groups, respectively.
Because there were no significant changes over time in any of the variables, the
average of values measured after 10, 20, 30, and 40 minutes of perfusion were used
for each animal. Percent and total palmitate oxidation values were corrected for
label fixation, as described in METHODS. *Significantly different compared to LG
group, p<0.05.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A
0.15
□ LG
■ HG
i
3
m
e
o
s
o
C
0.10
0.05
0.00
B
Figure 1. Effects of high carbohydrate availability on fractional (A) and total
(B) palmitate uptake in resting hindquarters perfused with high LCFA
availability. Values are means ± SEM; n=12 and n=8 for the LG and the HG
groups, respectively. Because there was no significant change over time in any of
the variables, the average of values measured after 10, 20, 30, and 40 min of
perfusion were used for each animal. * Significantly different compared to LG
group, p<0.05.
42
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A
15
I 10
%
■ Q
'5
O
a? 5
□ LG
g HG
f t
T
f t
_______1
Figure 2. Effects of high carbohydrate availability on percent (A) and total (B)
palmitate oxidation in resting hindquarters perfused with high LCFA
availability. Values are means ± SEM; n=12 and n=8 for the LG and the HG
groups, respectively. Because there was no significant change over time in any of
the variables, the average of values measured after 10, 20, 30, and 40 min of
perfusion were used for each animal. Percent and total palmitate oxidation were
corrected for label fixation as described in MATERIALS AND METHODS. *
Significantly different compared to LG group, p<0.05
43
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Glucose metabolism. As dictated by the experimental protocol, arterial perfusate
glucose and insulin concentrations were significantly higher in the HG group than in
the LG group (Fig.3, Fig.4). Similarly, glucose uptake did not vary over time and
was significantly higher in the HG group than in the LG group (27.0 ± 1.6 vs 3 .9 ±
0.3 pmol’g'^hr'1 , respectively; p<0.05). Glycolytic rate and glucose oxidation were
significantly higher (p<0.05) in the HG group (190 ± 43 and 136 ± 47 nmol-min'1 -g I,
respectively) than in the LG group (82 ± 12 and 35 ± 13 nmol-min'l-g'1 , respectively)
(Fig. 5).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A
25
S
t 20
0
1
I 15
8 10
3 5
O
□ LG
■ HG
»
a
»
»
_______________ 1 _______________
B
30
js
| 20
< ■
z.
10
□ LG
■ HG
Figure 3. EfTects of high carbohydrate availability on arterial glucose
concentration (A) and glucose uptake (B) in resting hindquarters perfused with
high LCFA availability. Values are means ± SEM; n=12 and n=8 for the LG and
the HG groups, respectively. * Significantly different compared to LG group,
p<0.05.
45
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A
40
s
E
- LO
■ HO
e
0
1
e
s
§
o
8 1
o
u
3
(9
10 20 30 40
B
Time (minutes)
S t
3
o
75
• LG
« HG
5 0
25
0
10 20 30 40
Time (minutes)
Figure 4. Effects of high carbohydrate availability on arterial glucose
concentration and glucose uptake in resting hindquarters perfused with high
LCFA availability. Values are means ± SEM; n=12 and n=8 for the LG and the HG
groups, respectively. Becdause there was no significant change over time in any of
the variables, the average of values measured after 10,20, 30, and 40 min of
perfusion were used for each animal. * Significantly different compared to LG
group,, p<0.05.
46
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A
0.3
f
o 0.2
i
o
s.
£ 0.1
o
□ LG
■ HG
B
0.3
f
1
o
E
o
%
2
0
1
3
o
0.2
0.1
□ LG
■ HG
Figure 5. Effects of high carbohydrate availability on glycolytic rate (A) and
glucose oxidation (B) in resting hindquarters perfused with high LCFA
availability. Values are means ± SEM; n=12 and n=8 for the LG and the HG
groups, respectively. Because there was no significant change over time in any of
the variables, the average of values measured after 10, 20, 30, and 40 min of
perfusion were used for each animal. * Significantly different compared to LG
group, p<0.05.
47
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Substrate exchange across the hindquarter. Resting oxygen uptake did not vary
overtime and was not significantly different (p>0.05) between the HG and the LG
group (29.2 ± 3.2 vs. 27.6 ± 5.7 pmol*g'l*hr'1, respectively; p<0.05). Perfusate
lactate concentration was not significantly different between the two groups, but
increased by 46-64% (p<0.05) over the perfusion period in both groups (LG: 1.3 ±
0.1 to 1.9 ± 0.2 raM; HG: 1.1 ±0.1 to 1.8 ± 0.2 mM, respectively). Lactate release
did not vary over time (p>0.05), and was not significantly different between groups
(HG: 6.2± 1.2 pmol*g*l*hr*1 ; LG: 5.7 ± 1.0 pmol*g'l* h r* 1 ; p>0.05) (Fig.6, Fig.7).
Glycerol release did not vary over time (p>0.05), and was not significantly different
between groups (HG: 0.297± 0.07 |imol-g’l-hr; LG: 0.182 ± 0.08 |a.mol-g'1 -hr‘l;
p>0.05) (Fig.8).
Reproduced with permission of the copyright owner Further reproduction prohibited without permission
Figure 7. Effects of high carbohydrate availability on lactate concentration and
lactate release in resting hindquarters perfused with high LCFA availability.
Values are means ± SEM; n=12 and n=8 for the LG and the HG groups, respectively.
50
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0.75
LO
m — ho
o »
i 0.5
9
0
0
g 0.25
8
0
o
> *
O
10 20 30 40
Time (minutes)
Figure 8. Effect of high carbohydrate availability on glycerol release in resting
hindquarters perfused with high LCFA availability. Values are means ± SEM;
n=6 for both the LG and the HG groups.
51
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Muscle metabolites (Table 2). Pre- and post-perfusion triglyceride (TG)
concentration was not significantly different between the two groups and averaged
3.87 ± 0.35 pmol'g ww'1 . There were no significant changes in muscle TG content
during the perfusion period in either group. Palmitate incorporation into diglycerides
and triglycerides was not different between the two groups. The rate of TG synthesis
was not significantly different between the two groups (Fig.9).
Table 2. Effects of high carbohydrate availability on muscle triglyceride
concentration and synthesis rate in resting hindquarters perfused with high
LCFA availability.
LG HG
TG-pre, pmol’g ww'1 3.93 ±0.42 3.80 ±0.62
TG-post, pmol'g ww'1 4.09 ±0.41 3.53 ±0.59
TG synthesis, nmohmin ^ g1 1.72 ±0.33 1.89 ±0.61
Values are means ± SEM; n=10 and n=8 for the LG and HG groups, respectively.
TG, triglyceride; pre, pre-perfusion; post, post-perfusion.
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
A
1 3
2
3
i ^
u
o
« s
O 1
S
* □ LG
■ HG
»
•
•
T
»
i
B
i.
i
□ LG
B HG
»
T
i
Figure 9. Effects of high carbohydrate availability on palmitate accumulation
into triglyceride (A) and diglyceride (B) in resting hindquarters perfused with
high LCFA availability. Values are means ± SEM; n=8 for both the LG and the
HG groups. TG, triglyceride; DG, diglyceride.
53
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DISCUSSION
Our results show that, in perfused resting muscle high carbohydrate
availability in the presence of high long chain fatty acid (LCFA) availability alters
LCFA utilization through its effect on both LCFA uptake and cellular LCFA
disposal. Thus, high carbohydrate availability was associated with an increased rate
of palmitate uptake, but no change in the relative contribution of palmitate to
substrate oxidation. The increased uptake of palmitate was not associated with a
higher rate of TG synthesis, suggesting that maximal rates of TG synthesis may be
achieved in the presence of high LCFA availability alone. Additionally, our data
coupled with our previous results (49) demonstrate that increased carbohydrate or
LCFA availability is associated with independent increases in LCFA uptake, and
suggest that separate cellular mechanisms of regulation may exist depending on
which fuel is in excess.
With the use of the hindlimb-perfusion system, plasma LCFA availability,
blood flow, and capillary density are all factors that could impact changes in muscle
LCFA metabolism (45). In this experiment, plasma LCFA availability and blood
flow were not different between groups. Thus, the calculated rate of plasma LCFA
delivery to the hindquarter was not different between groups and was not a limiting
factor in this experiment. This suggests that under conditions imposed by our
protocol, changes in LCFA metabolism occurred as a result of cellular-level
modifications elicited by high fuel availability.
54
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Under our experimental conditions of high palmitate availability, the
elevation in LCFA uptake associated with the high carbohydrate condition could be
attributed in part to an alteration in plasma membrane LCFA transport capacity.
LCFA transport across the plasma membrane has been shown to be partially
dependent on LCFA transport proteins (1, 20, 23). Indeed, expression of FAT and
FA B Ppm, two LCFA transporters found in skeletal muscle, has been shown to
correlate with LCFA uptake (20, 48). While FA B P pm content has been shown to
vary following chronic exposure to physiological conditions associated with changes
in LCFA metabolism, such as short-term fasting (46) and endurance training (17),
FAT content has been shown to vary following both acute and chronic physiological
manipulations. Furthermore, evidence exists to suggest that acute regulation of FAT
content at the plasma membrane occurs via translocation of FAT from an
intracellular location to the plasma membrane in a manner similar to GLUT-4 (2).
While the exact role of FAT or FABPpm on the changes in LCFA uptake induced by
high fuel availability has not been studied, our demonstrated increase in LCFA
uptake may be an acute effect of high fuel availability on FAT or any of the LCFA
transport proteins.
Alternatively, the increase in LCFA uptake could have been due in part to
alterations in intracellular metabolism imposed by high carbohydrate availability. As
shown in previous studies and in line with our present results, high carbohydrate
availability has been associated with increased glycolytic flux (6, 19). As glycolysis
proceeds at higher rates, intracellular glycerol-3-phosphate (G3P) levels would likely
55
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accumulate, providing ample amounts of the glycerol backbone required for
glycerolipid synthesis. Accumulation of G3P, therefore, may stimulate LCFA
uptake to drive the initial step in the de novo synthesis of cellular phospholipids by
the enzyme glycerol-3-phosphate acyltransferase (GPAT).
Combined with previous results from our lab, the present data also show that
high carbohydrate availability and high LCFA availability may increase LCFA
uptake by independent cellular mechanisms. Interestingly, the LCFA uptake rate
measured in this study in surfeit of both carbohydrate and LCFA fuels, is essentially
a summation of the LCFA uptake values resulting from an excess of each fuel
individually (Figure 10).
50
HG/LF
LG/HF
LG /H F HG/LF HG/HF H G /LF
LG /H F
Figure 10. Effects of high fuel availability on total palmitate uptake in resting
hindquarters perfused with either high glucose availability, high LCFA availability, or
both. Values are means ± SEM; n=l2, n=6, and n=8 for the LG/HF, HG/LF, and HG/HF
groups, respectively. Dashed bar indicates summation of uptake values for LG/HF and
HG/LF groups. LG/HF, low glucose and high LCFA availability; HG/LF, high glucose and
low LCFA availability; HG/HF, high glucose and high LCFA availability.
56
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These uptake values demonstrate that when both carbohydrate and LCFA fuels are in
excess, there is an additivity of palmitate uptake and, more importantly, suggests that
separate cellular mechanisms may be involved in the regulation of LCFA uptake
depending on which fuel is in excess. Indeed, we have shown that high LCFA
availability in the presence of low carbohydrate availability is associated with a
LCFA uptake value similar to that measured under conditions of high CHO
availability and low LCFA availability. Thus, under our conditions of combined
high carbohydrate and LCFA availability, LCFA uptake was ~29 nmol-min'1 * g * 1 , a
value equal to the sum of LCFA uptake rates when the separate fuels are in excess.
It has been proposed that long chain fatty acyl-CoA (LC-CoA) levels may
play an important role in the regulation of muscle oxidative metabolism in part
through their effects on enzyme activity (10, 28, 32). Because muscle LC-CoA
levels have been shown to increase under conditions of high LCFA availability (9,
43), our experimental protocol was likely associated with an increase in LC-CoA
levels. Among their many actions on metabolic enzymes, LC-CoA have been shown
to increase the activity of AMP-activated protein kinase kinase (AMPKK) (3). This
would lead to an increase in AMP-activated protein kinase (AMPK) activity.
Because AMPK decreases acetyl-CoA carboxylase (ACC) and malonyl-CoA levels,
it would ultimately release the inhibition of carnitine palmitoyltransferase I (CPT-I)
activity imposed by malonyl-CoA, and hence allow LCFA oxidation to proceed.
LC-CoA have also been shown to have a direct inhibitory effect on ACC, allowing
yet another mechanism by which LC-CoA may affect LCFA oxidation (27).
57
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Additionally, high LC-CoA levels have been associated with a decrease in the
activity of the mitochondrial citrate transporter (IS), resulting in a decreased
availability of cytosolic citrate, the ultimate precursor for malonyl-CoA production
and a known allosteric activator of ACC (14). Therefore, under our experimental
conditions, the presence of high LC-CoA levels by its action on AMPKK, ACC, and
the citrate transporter, would likely reduce malonyl-CoA levels, release inhibition of
CPT-I, and allow LCFA oxidation to proceed (Figure 11).
Another explanation could be related to the activity of malonyl-CoA
decarboxylase (MCD), the enzyme responsible for the degradation of malonyl-Co A
t citrate
Hieh CHO
t glycolytic flux
I citrate
t AMPK
t AMPKK
t LC-CoA
Hieh LCFA
1 citrate transporter
f iMal-CoAl |
J. LCPT-I I f
i _______
4 I LCFA Oxidation I f
Figure 11. Possible mechanisms for changes in FA oxidation with excess
substrate availability. CHO, carbohydrate, LCFA, long chain fatty acid, LC-CoA,
long chain fatty acyl-CoA, AMPKK, 5'AMP-activated protein kinase kinase,
AMPK, 5 'AMP-activated protein kinase, ACC, acetyl-CoA carboxylase, Mal-CoA,
malonyl-CoA, CPT-I, carnitine palmitoyltransferase-I.
58
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(8). It has been shown in perfused heart that an increase in LCFA oxidation was
associated with low malonyl-CoA levels and a parallel increase in MCD activity
(14), suggesting that MCD may play an important role in regulating LCFA
oxidation. Another recent finding showed that high plasma FA levels induced by
high fat diet resulted in increased expression of MCD in rat heart, EDL, and soleus
muscle (55). Our experimental protocol of high LCFA availability may have been
associated with increased MCD activity, and a subsequent decrease in malonyl-CoA
levels which could explain in part the increase in LCFA uptake and oxidation.
Conversely, high carbohydrate availability has been associated with increased
glycolytic flux (6, 19) and accumulation of citrate levels (36). In the absence of LC-
Co A high circulating citrate levels have been shown to be associated with
stimulation of ACC leading to an increase in malonyl-CoA levels (36). Conditions
of high carbohydrate availability, therefore, must be associated with decreases in
LCFA oxidation (Figure 11). Indeed, we have previously shown that under
conditions of high carbohydrate availability and basal levels of LCFA malonyl-CoA
levels were higher and this was associated with a decrease in the percent and total
rate of LCFA oxidized (49). Similarly, in rat heart, stimulation of glucose oxidation
by addition of dichloroacetate was associated with high malonyl-CoA levels and
decreased LCFA oxidation, which was shown to be associated with rising ACC
activity (34). Insulin has also been shown to activate ACC in liver (21) and heart
(13), providing another means by which LCFA oxidation may be acutely regulated.
However, based on our findings, when both LCFA and carbohydrate fuels are in
59
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excess, it appears that inhibition of ACC through phosphorylation by AMPK may
have prevailed over stimulation of ACC by citrate or insulin. Therefore, under our
conditions of both high LCFA and high carbohydrate availability, our data on
percent and total LCFA oxidation suggest that malonyl-CoA levels may not have
been increased sufficiently to suppress CPT-I activity, ultimately leading to the lack
of change in percent LCFA oxidation. In line with this notion, it has been shown in
rat perfused hindlimb that under conditions of increased glucose uptake via
stimulation of insulin, the expected decrease in palmitate oxidation did not occur
(53). High rates of insulin-stimulated glucose uptake were associated with an
activation of AMPK, which prevented high glucose uptake rates and increased
glycolytic flux from inhibiting palmitate oxidation (53). In our experimental
protocol, which was likely associated with high LC-CoA levels and a subsequent
increase in AMPK activity, the activation of AMPK may have prevented the
decrease in palmitate oxidation normally expected with high carbohydrate
availability. During hindlimb perfusion with combined high LCFA and carbohydrate
availability, it appears that the effects of high LC-CoA supersede the effects of high
carbohydrate availability on LCFA oxidation.
We have previously shown that resting muscle perfused with high
concentrations of carbohydrate and insulin in the presence of low LCFA availability
resulted in a decrease in LCFA oxidation that was associated with increased muscle
triglyceride concentrations (49). In accordance with this result, others have shown
that high concentrations of insulin have been associated with higher GPAT activity,
60
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which were associated with higher rates of triglyceride synthesis (7, 38). Our current
study, however, found no difference in either muscle triglyceride accumulation or
triglyceride synthesis. Because both groups in our experimental protocol were
perfused with high LCFA availability, it is likely that the multifarious effects of
LCFA on the various steps of the glycerolipid synthesis pathway outweighed those
of insulin and high carbohydrate availability. Indeed, it has been shown in rat liver
that increasing concentrations of palmitate up to 700 pM led to increased formation
of phosphatidate (50), an intermediate of triglyceride production. Furthermore, it has
been demonstrated in microsomal preparations from rat livers that 50% of the LCFA
was diverted to glyceride formation, which was composed of more than 65%
phosphatidate, and only 5 and 20% recovery in diglycerides and triglycerides,
respectively (50). This partitioning of LCFA to the different pathways of
phospholipid formation can be rationalized by observing the specific activities of the
enzymes involved in the pathway of triglyceride synthesis. A study in rat
epididymal fat pads has shown that diacylglycerol acyltransferase (DGAT), the
enzyme in the ultimate step of triglyceride synthesis has significantly lower activity
than all other enzymes in this pathway (35). Specifically, DGAT specific activity is
five times lower than glycerol phosphate acyltransferase (GPAT), ten times lower
than phosphatidate phosphohydrolase (PPH), and nearly fifty times lower than
lysophosphatidate acyltransferase (LP AAT). It is likely that our imposed high
concentrations of LCFA alone, coupled with the rate-limiting nature of the DGAT
enzyme that ultimately leads to triglyceride formation, led to a maximum rate of
61
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triglyceride synthesis and forced the accumulating LCFA to other pathways
preceding formation of triglycerides. Therefore, the addition of high carbohydrate
availability did not result in a higher rate of either triglyceride concentration or
synthesis.
In line with the notion that carbohydrate availability dictates the relative
contribution of fuel sources to oxidation (39), our results show that high
carbohydrate availability is associated with an increase in glycolytic flux and glucose
oxidation. This may appear contradictory since muscle pyruvate dehydrogenase
(PDH) activity has been shown to be significantly lower under conditions of high
LCFA availability induced by lipid infusion (18, 26), high fat diet (31), and fasting
(56), or under conditions of high LC-CoA availability (24). However, the effect of
LCFA on pyruvate dehydrogenase (PDH) activity is reversible, as was shown in
perfused rat hearts in which PDH inhibition by LCFA was reversed by addition of
dichloroacetate (51), an inhibitor of pyruvate dehydrogenase kinase (PDHK) (11,
52). Similarly, it has been shown that pyruvate, a potent allosteric inhibitor of PDHK
(4, 12, 30), can override the inhibition of PDH by high LCFA availability (40).
Thus, in situations of high pyruvate availability, such as those produced by our
experimental conditions of high carbohydrate availability, the role of pyruvate as a
stimulator of PDH activity may take precedence over the inhibitory action of LC-
CoA on PDH.
Our experimental conditions of high carbohydrate availability were also
associated with superphysiologic insulin concentrations. Evidence in rat cardiac
62
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muscle has shown that increased concentrations of insulin reverse the inhibitory
effect of LC-CoA on PDH activity (41). Similarly, it has been shown that
hyperglycemia increased muscle glucose oxidation both by activating PDH and by
routing glucose towards oxidation, as demonstrated through no significant change in
lactate release between hyperglycemic and euglycemic clamp (22). Furthermore,
hyperglycemia coupled with hyperinsulinemia caused an even greater activation of
PDH (22). It is likely, therefore, that under our experimental conditions, the actions
of pyruvate and insulin on PDH activity prevailed over the inhibitory effect of high
LC-CoA levels on the enzyme, and that the observed increase in glucose oxidation is
a result of the presence of high insulin and glucose concentrations.
While the concept that high rates of glycolysis result in decreased LCFA
oxidation has become well accepted, our results show that in hindlimb perfused with
high carbohydrate and high LCFA concentrations, both glucose and LCFA oxidation
increase. Because resting hindlimb likely utilizes little muscle glycogen as a fuel
source, our data suggests that with increased oxidation of both circulating glucose
and LCFA fuels, the contribution of muscle TG to muscle oxidative metabolism is
greatly diminished. Although we did not detect a difference in muscle TG
accumulation, our uptake and oxidation results suggest that the amount of LCFA
entering the phospholipid biosynthesis pathways was 53% higher under conditions of
high carbohydrate and high LCFA availability. The spectrophotometric assay for
muscle TG, however, was not sensitive enough to detect this difference.
63
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Additionally, because of the many pathways available to LC-CoA in phospholipid
formation (Figure 12), the fate of LC-CoA is not limited to muscle TG.
Under the imposed conditions of excess fuel availability, therefore, the high LC-CoA
may have been directed to pathways leading to formation of a number of glycerolipid
intermediates.
/ . # s / . . v . ... ... v & A
L o n g c h a in f a t t y a c id s
4
LC-CoA +GLYCEROL-3-PHOSPHATE
4
L y s o p h o s p h a t i d a t e --------- > Anionic Phospholipids
4
P h o s p h a t id a t e
4
D i a c y l g l y c e r o l -------- > Phosphatidylethanolamine,
Phosphatidylserine
| Phosphatidylcholine
T r ia c y l g l y c e r o l
Figure 12. Metabolic pathway for triacylglycerol synthesis in skeletal muscle. Triacylglycerol
synthesis pathway is preceded by many intermediate steps, catalyzed by enzymes: ACS, acyl-CoA
synthetase, GPAT, glycerol-3-phosphate acyltransferase, LPAAT, lysophosphatidate acyltransferase,
PPH-1, phosphatidate phosphohydrolase, DGAT, diacylglycerol acyltransferase.
In summary, the present study has shown that conditions of high
carbohydrate and high LCFA availability are associated with changes in LCFA
uptake and cellular LCFA disposal. High carbohydrate availability in the presence
of high LCFA availability was associated with an increased rate of palmitate uptake
64
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that was not accompanied by a higher rate of TG synthesis. The findings
demonstrate that while the oxidation of blood-borne fuels increases under conditions
of excess fuel availability, the percentage of LCFA oxidized does not change. This
suggests a possible regulatory role of malonyl-CoA in LCFA oxidation in surfeit of
both carbohydrate and LCFA fuels. The increase in total LCFA oxidation, even
under conditions of extreme carbohydrate availability, suggests that LC-CoA may be
a key determinant in the regulation of substrate metabolism through its action on
ACC. Finally, under conditions of high carbohydrate or high LCFA availability,
palmitate uptake values appear to be additive, suggesting that separate cellular
mechanisms of regulation exist, depending on which substrate is in excess.
ACKNOWLEDGEMENTS
The authors wish to thank Michael Huoh for expert technical assistance.
The present study was supported by a grant from the National Institute of
Arthritis and Musculoskeletal and Skin Diseases (AR45168).
65
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ARTICLE REFERENCES
1. Abumrad NA, Harmon C, and Ibrahimi A. Membrane transport of long-
chain fatty acids: evidence for a facilitated process. J Lipid Res 39:2309-
2318, 1998.
2. Bonen A, Luiken JJFP, Arumugam Y, Glatz JFC, and Tandon NN. Acute
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V. SUMMARY AND CONCLUSIONS
The purpose of this study was to elucidate the effect of combined high
carbohydrate and high LCFA availability on LCFA and carbohydrate metabolism.
Under conditions of basal and superphysiologic levels of carbohydrate and high
levels of LCFA, LCFA uptake, LCFA oxidation, and glucose oxidation were
measured. The study was also designed to determine under conditions of excess fuel
availability, the metabolic fate of carbohydrate and LCFA fuels, and possible fuel
interactions that were involved in the regulation of skeletal muscle oxidative
metabolism.
The himdlimb perfusion system was utilized since it allowed the study of
substrate exchange across the hindlimb in an in situ model for skeletal muscle
metabolism. The experimental protocol utilized perfusate containing pre-determined
concentrations of substrates and hormones to set glucose uptake under either basal or
superphysiologic conditions. Perfusate samples were analyzed for plasma
concentrations of glucose, lactate, glycerol, palmitate, as well as for l-[l4C]palmitate,
[l4]C02, and 3 H2 0 activities. Muscle samples were analyzed for muscle triglyceride
(TG) content, l-[l4C]palmitate incorporation into muscle TG, 3-[3 H]glucose
incorporation into glycogen, and malonyl-CoA content.
The present study has demonstrated that conditions of high carbohydrate and
high LCFA availability are associated with changes in LCFA uptake and cellular
LCFA disposal. High carbohydrate availability in the presence of high LCFA
availability was associated with an increased rate of palmitate uptake that was not
75
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accompanied by a higher rate of TG synthesis. The findings demonstrate that while
the total oxidation of blood-borne fuels increases under conditions of excess fuel
availability, the percentage of LCFA oxidized does not change. The increase in total
LCFA oxidation, even under conditions of extreme carbohydrate availability,
suggests that LC-CoA may be a key determinant in the regulation of substrate
metabolism through its action on ACC, which suggests a possible regulatory role of
malonyl-CoA in LCFA oxidation. Finally, under conditions of high carbohydrate or
high LCFA availability, palmitate uptake values appear to be additive, suggesting
that separate cellular mechanisms of regulation exist, depending on which substrate
is in excess.
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Asset Metadata

Creator Sacman, Michele L. (author)

Core Title Effects of high carbohydrate and high long chain fatty acid availability on skeletal muscle fatty acid metabolism and regulation of substrate utilization

School Graduate School

Degree Master of Science

Degree Program Kinesiology

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

Tag biology, animal physiology,chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Turcotte, Lorraine (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-295793

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