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Calorie restriction reduces liver steatosis in liver specific Pten deleted mouse model

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Calorie restriction reduces liver steatosis in liver specific Pten deleted mouse model

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CALORIE RESTRICTION REDUCES LIVER STEATOSIS IN LIVER SPECIFIC
Pten DELETED MOUSE MODEL

by

Ajeetha Josephrajan

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2010

Copyright 2010 Ajeetha Josephrajan

ii

Acknowledgements:

I am grateful to my mentor and advisor, Dr. Bangyan Stiles, for teaching me various
things about research and its aspects. She was also very supportive and provided with me
guidelines regarding the experiments and lab work and in thesis writing. I also want to
thank Dr. Zoltan Tokes and Dr. Tobias Ulmer for being my committee members and
providing me with their support and advice. Finally, I want to thank all my laboratory
colleagues for helping me with experiments.

iii

Table of Contents:

Acknowledgements ……………………………………………………………….… ii
List of Figures ………..………………………………………………………...…… iv
Abstract ……………………………………………………………………………… v
Introduction …………………………………………………………………………. 1
Hypothesis …………………………………………………………………………… 7
Materials and Methods ……………………………………………………………… 8
Results ……………………………………………………………………………….. 12
Discussion ………………………………………………………………………….... 31
Summary …………………………….………………………………………..…….... 34
References …………………………….………………………………………..…….. 35

iv

List of Figures:

Figure 1: Body weight comparison among the groups ………………………………….12

Figure 2: Blood glucose comparison among the groups ……………......……………… 14

Figure 3: CR reduces liver weight and Liver to Body weight ratio in mutant mice …… 15

Figure 4: CR reduces steatosis and lipid deposition ………………………………….... 17

Figure 5: CR shows reduces triglyceride level ………………………………………… 19

Figure 6: Induction of lipogenesis genes under CR ………………………………….... 22

Figure 7: Effect of CR on the expression levels of lipogenesis genes …………………. 24

Figure 8: Fatty acid breakdown is increased when under CR ……………........………. 30

Figure 9: Fatty acid oxidation pathway regulation by SCD and MCAD ………… … 32
according to the conclusion from the expression levels of SCD and MCAD.

v

Abstract:
Calorie restriction (CR) is an approach wherein there is moderate restriction of
food intake while providing sufficient amount of essential nutrients.CR is beneficial in
decreasing tumor progress, physiological aging and increasing the life span of laboratory
animals. Previously our study showed that Pten deletion in liver resulted in fatty liver
phenotype due to excess accumulation of lipid in liver. In this study, we tested the
hypothesis that CR can reduce the burden of fatty liver using our liver Pten model. We
showed that CR blocked the triglyceride accumulation in the liver of the Pten null mice.
In this study we also investigated the pathway/mechanism involved in this change in
phenotype when mice were under calorie restriction. We observed the expression patterns
of genes involved in lipogenesis and fatty acid oxidation. Our analysis showed that CR
treatment did not alter lipogenesis although lipogenesis expressing genes were induced
by CR treatment in Pten control mice. We showed that induction of β –oxidation pathway
is likely the reason for the inhibitory effect of CR on liver steatosis in the Pten model.

1

Introduction:
1.1 Introduction to PTEN
PTEN (Phosphatase and Tensin homolog deleted on chromosome 10) is a lipid
and protein phosphatase. Pten which was originally identified as a tumor suppressor gene
is the second most commonly mutated gene only next to p53. (Chow & Baker,
2006)Mutation or deletion of PTEN has been identified in many cancers including
prostate, brain, breast, liver etc (Guldberg et al., 1997; Risinger, Hayes, Berchuck, &
Barrett, 1997; Stiles et al., 2004). The main function of PTEN is its phosphoinositide
dephosphorylating activity, which dephosphorylate PtdIns (3, 4, 5) P3

at the 3′-position of
the inositol ring, to generate PtdIns (4, 5) P2 and therefore acts as an antagonist by
terminating the downstream signaling of PI3K/AKT signaling pathway. (Maehama &
Dixon, 1998; M. P. Myers et al., 1998).

1.1.1 PTEN signaling in PI3K/AKT pathway:

Phosphoinositide 3-kinase (PI3K) responds to extracellular signals and acts in
ways to bring about many cellular functions, the important downstream effector of which
being AKT, a serine- threonine kinase. The PI3Ks can be activated by a large number of
membrane receptors by recruitment to the membrane and phosphorylation. The activated
PI3Ks bring about the conversion of Phosphatidylinositol-4, 5 bisphosphate (PIP2) to
phosphatidylinositol-3, 4, 5-trisphosphate (PIP3). The PIP3 acts as a secondary
messenger to recruit Pleckstrin homology containing proteins such as AKT to the plasma
2

membrane. The activated AKT regulates a variety of cell functions including
proliferation, growth and survival. The PI3K/PKB signaling is also important in
maintaining the glucose homeostasis. (Lizcano & Alessi, 2002)

PTEN is an antagonist of this PI3k/AKT signaling pathway, it acts by
dephosphorylating PIP3 and PIP2, resulting in PIP2 and PIP respectively. It has also been
shown that PTEN-deficient cells have elevated levels of intracellular PIP
3
(Stambolic et
al., 1998; Sun et al., 1999). The tumor suppression activity of PTEN is most likely due to
these antagonistic effects (Yuan & Cantley, 2008). PTEN was also shown to be altered in
a number of cancers including prostate carcinoma cells.

1.1.2 Pten loss in NAFLD and cancer:

Nonalcoholic fatty liver disease (NAFLD) refers to the diseases in liver
caused due to reasons which doesn’t involve excessive alcohol consumption (Adams,et
al.,2005). Fatty liver (Steatosis), NASH (Nonalcoholic Steatohepatitis) and cirrhosis
(Contos et al., 2004) (irreversible, advanced scarring of the liver) all fall under this
category. The common stages of these diseases involve accumulation of fat in
hepatocytes mostly leading to varying degrees of inflammation and scarring (fibrosis) of
the liver. PTEN downregulation or deletion leads to the development of steatosis
(Vinciguerra et al., 2007); this was an evident phenotype in liver-specific PTEN knockout
mice model. The model with genetic deletion of PTEN also showed steatohepatitis at
early stages of development and Hepatomegaly (swelling of liver) as well as HCC
3

(Hepatocellular carcinoma) later in life.(Horie et al., 2004; Stiles et al., 2004; Xu et al.,
2006,)
1.1.2.1 PTEN and fatty liver:

In liver, insulin signaling helps bring about glycogen synthesis and
glycolysis and fatty acid synthesis. Insulin binding to its receptor signals the activation of
PI (3)K. Inhibitors of PI(3)K are found to block the insulin signaling actions like
stimulation of glucose transport, glycogen and lipid synthesis ( Myers et al., 1992) This
PI(3)K signals the production of PIP3 which in turn binds to the PH domains of many
other signaling molecules. One of them is the PDK (Phosphoinositide dependant kinase)
which phosphorylates and activates AKT. AKT is also directly activated by PIP3 through
its PH domain.

The AKT being downstream of Insulin signaling pathway plays an important
role in the transmission of insulin signal through GSK3, forkhead receptors and cAMP
response element binding proteins.(Cross, et al., 1995; Nakae, Park, & Accili, 1999) It
has also been shown that deletion of AKT2 produces hepatic insulin resistance in mice
(Cho et al., 2001) and AKT is a requisite for insulin dependent regulation of lipid
metabolism during insulin resistance (Leavens et al.,2009).Thus in the case of increased
lipogenesis and fatty liver, insulin signaling plays a major role through this PI3K/AKT
signaling pathway. PTEN acts as a negative regulator of this insulin signaling pathway
(Nakashima et al., 2000) by converting PIP3 to PIP2.Inactivation of PTEN resulted in
elevated expression levels of PIP3 and dose dependent increase in the activity of AKT.
4

(Sun et al., 1999). PTEN deletion thus causes increase in insulin action and increased
AKT expression, resulting in the regulation of key enzymes regulating fatty acid and
glycogen synthesis.

Pten deletion in the liver results in fatty liver phenotype along with
hepatomegaly.(Horie et al., 2004; Stiles et al., 2004; Wu et al., 2003; Xu et al., 2006)
This is due to the increase in lipogenesis .Along with this increased glucose tolerance
and enhanced insulin action was also observed.

Fatty liver caused by Pten deletion showed (1)Increased glycogen synthesis
due to AKT activation (2) Increased De Novo lipogenesis ,that is increased levels of
FAS (Fatty acid Synthase) which plays a role in fatty acid synthesis (3) Decreased whole
body fat was observed, caused due to the redistribution of fat from other tissues to liver.
(4) Fasting blood glucose levels were low and increased glucose tolerance was shown. (5)
Glycogen Synthase (GS) is required for the conversion of glucose to glycogen. GSK-3 is
a negative regulator, which regulates this pathway by phosphorylating GS to inactivate
the kinase. The GSK - 3 is in turn inactivated by phosphorylation of AKT. In fatty liver
hyperphosphorylation of GSK-3 is observed which could account for increase in
glycogen accumulation. (6) Down-regulation of G6P (Glucose 6 phosphatase) and
PEPCK was also shown, both of which are gluconeogenic enzymes.

5

1.2 Calorie restriction:

Calorie restriction (CR) refers to the moderate restriction of food intake
while providing sufficient amount of essential nutrients. Calorie restriction has shown to
(1) decreases tumor progress, (2) physiological aging and (3) increase the life span of
laboratory animals when compared to the laboratory animals which had free access to
food. (Weindruch, 1996) ; (Masoro, 2003).When dietary restriction with 10-50 %
decrease in calorie intake, was done on laboratory animals with tumor, they showed
incidences where the growth rate/progressiveness of the tumors is reduced. (Klurfeld et
al., 1989; Tannenbaum & Silverstone, 1953).

Calorie restriction was also found to decrease the GH-IGF signaling which
might cause these effects on aging (Higami et al., 2006) Also CR rats were shown to have
2 phases of metabolism namely the fasted phase and fed phase. During the fasted phase
reserve energy like proteins and lipids were used as the main source of energy. During
the fed phase, which occurs immediately after the food is provided; carbohydrates are the
main source of energy. Thus during the fed phase when carbohydrates are metabolized
and used as energy source, other metabolic processes like lipogenesis also takes place and
they are stored. These observations made from the CR rodents show that anabolic
processes like lipogenesis takes place when food is available and when food is restricted
gluconeogenesis processes are activated. This pattern was not observed in control rats
which had free access to food.(Duffy et al., 1989; McCarter & Palmer, 1992)

6

These cyclic changes in the metabolic pattern and macronutrient utilization
especially in the case of fatty acid (FA) synthesis and oxidation might be the metabolic
mediator for the decrease in tumor progression in CR. There was a proposed reduced rate
of FA synthesis in CR (Cao, Dhahbi, Mote, & Spindler, 2001; Chen et al., 2008;
Mulligan, Stewart, & Saupe, 2008). Also CR showed increase in the expression of FA
oxidation genes. Since there is a metabolic shift between the carbohydrate and fatty acid
as energy sources, the entry points for each metabolic pathway into the electron transport
chain varies and this may reduce the production of reactive oxygen species (ROS)
(Guarente, 2008). These reasons might account for the health benefits of CR.

7

Hypothesis:

According to the previous studies calorie restriction played an important role in the fatty
acid metabolism.CR has been shown to inhibit fatty liver phenotype. In the liver specific
Pten deleted null mouse model, fatty liver phenotype (Liver Steatosis) was observed due
to increased fatty acid synthesis. Hence we wanted to observe if the calorie restriction
will have any effect in this fatty liver caused due to genetic deletion of Pten. The
hypothesis that calorie restriction reduces the liver steatosis in this particular mouse
model was based on this rationale.

8

Materials and Methods:

Animals:
Liver specific Pten deletion in mice was brought about by breeding Pten
loxp/loxp
mice with Alb-Cre
+
mice (Stiles et al., 2004) .The control mice are Pten
loxp/loxp
, Alb-Cre
-
.
The animals were housed in humidity, temperature and light controlled (12hr light/ dark
cycle) room .All experiments were conducted in accordance with Institutional Animal
Care and Use Committee of the University of Southern California research guidelines.

Animals used for the experiments were all 3 months old male mice. Blood
glucose was measured after having the animals fasted overnight. The liver tissues
collected from the mice after overnight fasting was perfused with fresh PBS. Part of the
liver tissue was taken in formalin for histology, and the rest was flash-frozen in liquid
nitrogen for RNA, protein and triglyceride analyses and for frozen sectioning.

Calorie restriction:
The animals were split into four groups to study the effect of Calorie restriction
(CR). Two groups which were under regular/normal diet with free access to food
(Labdiet, PMI Nutrition International, LLC, USA) and water. And the other two groups
were under restricted diet; with 60% of the diet consumed by control mice. Amongst the
two groups which were under CR one group carries the Pten
loxp/loxp
, Alb-Cre
-
(Con/CR+)
and the other has Pten
loxp/loxp
, Alb-Cre
+
(Mut/CR+) genotype.
9

The same pattern was followed in the groups under regular diet also, namely
Pten
loxp/loxp
, Alb-Cre
-
(Con/CR-) and Pten
loxp/loxp
, Alb-Cre
+
(Mut/CR-), thus comprising
the four groups. The animals were put under calorie restriction when they were exactly
one month old. The diet was provided at the same time (15:00 p.m PST) everyday for a
period of 2 months.

Immunohistochemistry:

H&E Staining:
Liver tissue which was collected earlier in formalin solution was fixed
overnight and then embedded in paraffin and then later sectioned into slices. The sections
were then stained with hematoxylin and eosin (H&E) for histopathological examination.

Oil Red O Staining:
The frozen liver tissue collected for frozen sectioning was briefly fixed in
cold 10% formalin after sectioning them. The slides were then stained in Oil Red O
solution.

Serum Metabolic Indices quantification:
The blood of the animals was collected via cardiac puncture after having
them fasted overnight. The serum plasma was obtained from the blood by centrifuging
them in plasma separator tubes with Lithium heparin. The serum was then used for
10

analysis of Serum TG, cholesterol and NEFA (Non Esterified fatty acids) by using kits
from Wako chemicals.

Liver TG quantification:
The lipid/triglyceride was extracted from liver by homogenizing them
in 2:1 chloroform/methanol solution according to Folch method. The supernatant was
used for TG assay, which was performed by using Triglyceride (GPO) Reagent set from
Thermo. The pellets were then used for extraction of DNA.

Liver tissue whole protein quantification:
The protein was extracted by homogenizing the liver tissue in SDS cell
lysis buffer containing protease. The protein lysates were obtained from the supernatant.
They were then quantified by Bio-Rad Dc protein Assay reagents (Bio-Rad laboratories,
Hercules, CA).

Western Blotting:
Protein lysates (40µg) were loaded in the SDS PAGE for protein
electrophoresis. The primary antibodies were diluted and used to probe the membranes
and incubated overnight at 4
o
C.Primary antibodies for PTEN, pAKT, FAS, ACC and
pAMPK. β-Actin (Sigma) and Vinculin were used as loading controls. The secondary
antibodies were diluted at the concentration of 1:3000 (Mouse) or 1:5000 (Rabbit). The
signals from the antigen-antibody interaction were detected by chemiluminescence using
the ECL Western Blotting Substrate (Pierce, Rockford, IL). The membranes were
11

incubated with ECL substrate mixture solution for one minute before exposure. The
signals were captured on X-Ray films (Bioland) when placed for exposure in
autoradiography cassettes. The films were then developed for the results.

Quantitative PCR:
RNA was extracted from liver samples using TRIZOL reagent
(Molecular Research Center, Inc.).From the total RNA, cDNA was synthesized by
reverse transcription according to the conditions provided by Promega RT system.
Random primers were used for this reaction. The cDNA thus obtained was used for
quantitative PCR which were all performed on ABI 7900HT Fast Real-Time PCR
System, Maxima SYBR Green qPCR Master Mix (Fermentas) was used for the qPCR
reaction .The primers used in generating qPCR were: ACC, FAS, SREBP, GK, G6P,
GAPDH, PEPCK, SCD, and MCAD. Quantification was determined by using the ΔΔ Ct
method.

12

Results:

1. CR reduces the body weight of mice :
To study the effects of calorie restriction, the body weights of the animals
were measured over the period of 2 months to observe the change they bring about to the
Pten deleted mouse model and also the controls. The body weights of the four groups of
mice were compared (Figure 1).

Figure 1: Body weight comparison among the groups: Shows body weight
comparison among the four groups of male mice (n=2).The body weight changes over a period of
3 months are observed here. In the 3rd month, Mut/CR+ showed statistically significant reduction
(p ≤ 0.05) in body weight when compared to Mut/CR- while Con/CR+ mice also showed
decrease in body weight though not significant statistically.

0
5
10
0
0
15
5
5
5
20
0
25
1 month 2 month 3 month
Con/CR-
Con/CR+
Mut /CR-
Mut/CR+
*
Age in months
[
Body Weight in g
13

Before CR was started at the 1
st
month we observed a significant difference in
body weight between the Pten
loxp/loxp
, Alb-Cre
-
(Con) and Pten
loxp/loxp
, Alb-Cre
+
(Mut)
groups. The body weight of the mutant mice is 57.89% higher than that of the controls.
This pattern in body weight was also observed in the 2
nd
month. At three months of age,
no difference was observed between the control and mutant mice with or without diet
treatment.

During the third month, there was a statistically significant difference between
the body weights of Mut CR- and CR+ (p≤ 0.05), and also lower body weight in CR+
mice in control when compared to the Con/CR- , though it was not statistically
significant. The mice under calorie restriction, has reduced body weight. Previous studies
show that CR is associated with reduced body weight. Our observation shows similar
results in Pten null mice also.

2. Effect of CR on blood glucose :
The effect of CR on blood glucose was studied by measuring the blood
glucose levels at regular intervals (Figure 2). Our observations show that long term CR
most likely has no effect on blood glucose. After 2 months of CR, we didn’t observe a
measurable difference in the fasting glucose levels between CR- and CR+ groups in
either control or Pten mutant mice. Though in earlier stages like in 2
nd
month there was
increase in blood glucose which might be due to adaptation to the new diet regimen and
there could have been increased blood glucose in the blood stream due to the increased
glucose output from the liver.
14

Figure 2: Blood glucose comparison among the groups: Shows the comparison of the fasting
blood glucose levels in male mice between the groups in 3months (n=2).

3. CR reduces the liver weight and Liver/Body weight ratio in mutant mice:
In observance to the previous study, the liver weight was significantly
higher in case of Pten mutant mice (p ≤ 0.05).A 3-fold increase was observed with
mutant when compared to the control mice. The control mice which were under CR
(Con/CR+) showed a slightly increased liver weight when compared to the control mice
under regular diet (Con/CR-).But in the case of mutant CR+ mice (Mut/CR+), the weight
of the liver was found to be decreased significantly (p ≤ 0.05) when compared to the CR-
mutant (Mut/CR-) (Figure 3A).
0
50
100
150
200
250
300
1 month 2 month
momont
hmonth
3 month
monthmo
mmmMom
onth
Con / CR-
Con / CR+
Mut/ CR-
Mut/ CR+
Fasting Blood glucose level in mg/dL
Age in months
15

In Figure 3B as in accordance to previous studies there is an increase
in liver to body weight percentage when the control (Con/CR-) and mutant (Mut/CR-)
mice which were under regular diet were compared, significantly (p ≤ 0.05).

Figure 3: CR reduces the Liver weight and Liver/Body weight ratio in mutant mice: A,
Shows liver weight in the four groups (n=2).CR+ mutant mice show decrease in liver weight
*(p≤0.05) when compared to CR- mutant mice. B, shows comparison of liver/BW% among the
four groups where n=2. Decrease in liver/BW % is seen though not significant.
A
B
*
16

When CR+ and CR- mice were compared in mutant groups, there was a decrease
in liver to body weight ratio in CR+ animals though it was not statistically significant
likely due to the fact that CR induced a body weight loss in Pten mutant mice.

4. Effect of CR in mutant liver pathology:
To explore the reason for the change in liver weight and to evaluate our
hypothesis that CR reduces steatosis in Pten deleted mouse model, we studied the
morphology of the liver of the various groups of animals. (Figure 4A). Both Con/CR- and
Con/CR+ show normal morphology. The CR treatment showed no change in the control
CR+ mice. As observed previously mice on regular diet showed increased deposition of
lipid in the liver seen by swollen hepatocytes. When the Pten mutant was treated by CR
the Mut/CR+ showed decreased lipid deposition compared to Mut/CR- mice. The
morphology of the Mut/CR+ mice is similar to control mice on CR or normal diet.

The histological sections from Oil Red O staining (ORO), (which indicates
lipid deposition) in Figure 4B, shows normal morphology in Con/CR- and Con/CR+ .In
the Pten Mut CR- mice, ORO staining is very strong, since the hepatocytes swollen
morphology in the Pten mutant mice is largely due to the lipid accumulation in the
hepatocytes. When the mutant mice were on CR diet, Mut/CR+ group, the lipid
accumulation decreased dramatically as seen by ORO staining. The intensity of ORO
staining is almost similar to control mice, collectively; the morphology and ORO data
indicate that CR indeed reduces lipid deposition and hence liver steatosis.

17

Control CR- (Con/CR-) Control CR+ (Con/CR+)

Mutant CR- (Mut/CR-) Mutant CR+ (Mut/CR+)

Figure 4: CR reduces steatosis and lipid deposition: A, Showing morphology of liver tissue
of 3 month old mice under H&E staining. Top two panels show control groups. Left top panel
shows control CR- (Con/CR-) and right top panel showing control CR+ mice liver (Con/CR-).
Bottom left panel shows liver steatosis in Pten mutant CR- (Mut/CR-) and bottom right panel
shows decrease in the level of Steatosis even when it is mutant since it is under CR(Mut/CR+).
The morphology is similar to wild type.

H&E
A
18

Control CR- (Con/CR-) Control CR+ (Con/CR+)

Mutant CR- (Mut/CR-) Mutant CR+ (Mut/CR+)

Figure 4B: CR reduces Steatosis and lipid deposition: B, Showing morphology of liver
tissue of 3 month old mice under Oil Red O staining. Top two panels show wild type groups. Left
top panel shows control CR- (Con/CR-) and right top panel showing control CR+ mice liver
(Con/CR-). Bottom left panel shows liver lipid accumulation as red droplets in Pten mutant CR-
(Mut/CR-) and bottom right panel shows decrease in the level of Steatosis even when it is Pten
mutant since it is under CR(Mut/CR+).The morphology is similar to wild type.

Oil Red O
B
19

5. CR reduces liver triglyceride level:
To quantify the severity of liver steatosis in various groups, we decided to study
the triglyceride levels in the liver since they are directly proportional to the amount of
lipid accumulation in liver. The quantification of the liver triglyceride showed a 59.75%
increase in TG in mutant CR- when compared to Control CR- (p=0.055); which is typical
of fatty liver phenotype wherein accumulation of fat is seen.

Figure 5: CR shows reduced triglyceride level: Shows a decrease in TG concentration in
mutant CR mice though not statistically significant. While statistically significant increase was
observed in mutant Pten null model when compared to control.

Consistent with the normal morphology observed in the Con/CR- and
Con/CR+ mice, we didn’t observe a clear difference in liver TG between those groups.
On the other hand, Pten mutant mice under CR (Mut/CR+) showed a 43.87% reduction
20

in the liver TG when compared to the Mut/CR- group. The TG level in Mut CR+ group is
comparable to the Con CR- and Con CR+ groups. (Figure 5).
6. CR induces genes for lipogenesis:
The TG content, the histological sections and the liver/BW data clearly
show that overall lipid deposition in the liver is reduced as a result of calorie restriction in
the Pten mutant mice, since lipogenesis is the major cause for lipid accumulation in the
livers of Pten mutant mice, we decided to determine if CR decreased the de novo
synthesis of lipid in the liver.
FAS (Fatty acid Synthase) levels are an indication of the amount of
lipogenesis occurring in liver. Earlier studies show that FAS is found to be markedly
increased in Pten mutant mice. ACC is downstream of AMPK and plays a role in lipid
synthesis.ACC was also slightly increased in Pten deleted mouse models according to
previous studies (Gonzalez et al., 2004). Since these 2 genes play an important role in
lipid accumulation when Pten is lost, we decided to study the effect of CR on FAS and
ACC.
We evaluated FAS and ACC using qPCR. As shown in previous studies
there was a ~5 fold increase in FAS and ~2.3 fold increase in ACC mRNA expression in
Pten mutant mice versus Pten control mice on regular diet.
When under CR, in the control mice we observed a ≈ 7 fold increase in
FAS in Con/CR+ when compared to Con/CR- while in the case of ACC there was nearly
a 2.2 fold increase. This is consistent with the previous studies showing increased
21

expression of FAS and ACC when the animals were under calorie restriction.(To et al.,
2007). In mutant mice also a similar trend was observed with a < 2 and < 3 fold increase
in FAS and ACC respectively when Mut/CR+ mice were compared with Mut/CR-mice.
Pten mutant mice already showed an increase in FAS and ACC levels, CR caused an
increase, even more than the levels in Pten null mice in these Con/CR+ and Mut/CR+
mice. But the increase was not statistically significant (Figure 6).
We then determined SREBP, another one of the lipogenic genes (Sterol
regulatory element binding protein) which controls the expression of nearly all genes
integral to fatty acid biosynthesis including FAS and ACC. Previous studies have shown
that increased expression of SREBP in turn increased the expression of FAS and ACC
(Robert et al., 2003; Jay et al., 2002). However our data indicated that SREBP was
decreased in Con/CR+ and increased in Mut/CR+ when compared to Con/CR- and
Mut/CR- respectively. And hence it didn’t show any pattern that might explain the
increase in the production of ACC and FAS. This was surprising as SREBP is a major
transcriptional factor in the pathway which induces FAS and ACC.
That led us to examine LXR (Liver X Receptor) which was another
transcription factor that regulates FAS, ACC through SREBP and also independently. We
found that the expression levels were corresponding to that of FAS and ACC. The
expression was increased 1.4 fold in Con/CR+ and ~2.3 fold in Mut/CR+ when compared
to their CR- counterparts.

22

Figure 6: Induction of lipogenesis genes under CR, Continued.

Figure 6: Induction of lipogenesis genes under CR: shows hepatic gene expression as measured
by real time quantitative PCR of 3 months old mice. The data represents mRNA expression
relative to that of GAPDH and normalized to the expression in that of wild type control, which is
set as 1 using the ∆∆Ct method. The values are represented as mean ± SEM with n=2.

23

Figure 6: Induction of lipogenesis genes under CR, Continued

Another gene which is involved in lipogenesis is SCD, Steroyl CoA
desaturase. SCD acts as a rate limiting step that catalyses the biosynthesis of the
monounsaturated FAs from saturated FAs by introducing a cis-double bond to a fatty
acyl-CoAs .The preferred substrates are palmitate and stearate, which yield palmitoleate
and oleate (Miyazaki et al., 2007) . SCD was studies using qPCR (Figure 6).
This SCD was found to be decreased when under CR. In the case of control,
the CR showed a ~3.5 fold decrease, when compared to Con/CR-.While the mutant CR+
showed a 2.3 fold decrease than Mut/CR-, the CR+ mice showed a statistically
24

significantly decrease in SCD levels. As a result of which there might be accumulation of
medium chain saturated fatty acid.

Figure 7: Effect of CR on the expression levels of lipogenesis genes, Continued.

Figure 7: Effect of CR on the expression levels of lipogenesis genes: A, Shows the
western blotting results of PTEN, pAKT which correspond with the genotype .B, shows the
western blotting results of FAS, pAMPK, ACC from proteins extracted from 3M old mice. Actin
and Vinculin are the loading controls. C shows the quantification results of proteins FAS,
pAMPK and ACC expressed as mean± SEM from 2 determinations.

Con / CR-
Mut /CR-
Con / CR+
Mut /CR+
PTEN
p AKT
Actin
A
25

Figure 7: Effect of CR on the expression levels of lipogenesis genes, Continued.

FAS
p AMPK
ACC
Vinculin

C
B
Con / CR-
Con / CR+
Mut /CR-
Mut /CR+
26

Figure 7: Effect of CR on the expression levels of lipogenesis genes, Continued.

We also decided to study the protein expression levels of FAS and ACC.
(Figure 7).Consistent with previous finding mutant mice showed increase in FAS and
ACC when compared to the control mice. However to our surprise, CR had no significant
27

effect on either FAS or ACC. A very slight increase in FAS is observed in the protein
level. This is in contrast to what we observed in the mRNA level. In the case of mRNA,
in FAS, we saw a 7-fold and < 2 -fold increase in the case of control and mutant
respectively in CR+ mice over CR- mice and ACC showed 2.2- fold and < 3 fold increase
in control and mutant mice under CR+.

ACC, one of the lipogenic gene is regulated post translationally by
AMPK through direct phosphorylation and inhibition. Phosphorylation of ACC by
AMPK results in increase in glucose transport, fatty acid oxidation and gene
transcription. It has been reported that AMPK may mediate the effects of CR under
certain CR regimen since AMPK is an energy related biochemical pathway. And hence
we wanted to evaluate pAMPK (Figure 7B&C).
The pAMPK didn’t show any observable change among the four groups of
mice which were 3 months old. Together, these data suggest that long term CR might
play a role in regulating the expression of lipogenic genes such as FAS and ACC through
LXR. However, lipogenesis is unlikely to play a role in the reduction of lipid
accumulation effect of CR as the changes due to lipogenesis are not consistent with the
decrease of liver TG.

28

7. CR shows no changes in blood metabolic indices.
To explore the potential involvement of other processes in CR induced
reduction of lipid accumulation in Pten null mice, we evaluated the serum profile of TG
and NEFA. The serum shows no change in triglyceride and NEFA (Non Esterified Fatty
Acids) levels when compared among the four groups. This might show that there is not
much change in the circulating lipid levels. In the case of animals under CR also there
were no statistically significant change in TG and NEFA.

The reason why we studied TG and NEFA, the neutral lipids in the serum,
is to study the fluctuation that might occur in TG and NEFA levels in serum while under
CR diet. These fluctuations might be due to 1) Less amount of free fatty acid (FFA)
flowing into the liver.2) More amount of FFA flowing out of the liver. Since there was no
change in the levels of TG and NEFA, it was concluded that FFA transport is not
involved here. There is no change in circulating levels of TG and NEFA but there is a
decrease in lipid deposition in liver CR mice. This might imply that there was more FA
burned up in liver.

29

Table 1. Serum measurements of the mutant and control groups with/without CR:
Serum
Metabolic
indices
Con / CR- Con / CR+ Mut / CR- Mut / CR+

TG (mg/dl)

168.77 ± 5.2

173.61 ± 2.97

187.73 ± 18.22

169.89 ± 1.49

NEFA (mg/dl) 18.83 ± 6.45

18.50 ± 3.42

19.02 ± 1.88

17.29 ± 1.7

The cholesterol levels did not show a particular pattern when observed
which might be due to the small sample number.

8. CR increases fatty acid oxidation:
There was no change in circulating levels of TG and NEFA. This led to the idea that
there was increased oxidation in liver in CR wherein here is less lipid accumulation. Therefore
decreased steatosis in mice may be due to increased β –oxidation which leads to
decreased steatosis seen in Pten null mouse. So we decided to study if CR regulates the
genes involved in β-oxidation pathway.

MCAD (Medium-chain acyl-coenzyme A dehydrogenase) is an enzyme
complex which oxidizes medium chain fatty acids and produces energy and hence is
involved in β -oxidation. We show here that there is an increase in the level of MCAD
(Figure 8), indicating that β – oxidation maybe increased. The increase was statistically

30

significant in CR+/mutant mice, with a ~2-fold increase and a ~1.2- fold increase was
seen in Con/CR+.

Figure 8: Fatty acid breakdown is increased when under CR. Shows the qPCR results of MCAD.
The values are represented as Mean± SEM.

The increase in MCAD might also have been mediated by an increase in the
amount of substrate which is medium chain fatty acids available for them. This could
have been provided by decrease in the lipogenetic gene SCD.

31

Discussion:
PTEN is a tumor suppressor which negatively regulates PI3K/AKT signaling.
When this Pten is lost, lipid accumulation is seen in hepatocytes and steatosis occurs.
This present study shows the effect of calorie restriction on such Pten deleted mouse
model. Calorie restriction which is moderate reduction in calorie intake was shown to
have various benefits, one of which was reduced carcinogenesis. We wanted to observe if
this calorie restriction had any effect on the steatosis causing Pten deletion in liver.
CR mice usually showed a fed and fasted phase (Duffy et al., 1989; McCarter
& Palmer, 1992) and accordingly during each phase the hepatic gene expression was also
altered. During the fasted phase when the allotment of food was all consumed, there is
increase in fatty acid oxidation, gluconeogenesis and ketosis in the liver. And during the
fed phase there was increase in the amount of lipogenesis. In this study, though the two
phases weren’t separately studied due to a different type of CR regimen followed, the
hepatic gene expression of increased fatty acid synthesis and oxidation was still the same
as studied previously even when there is Pten deletion.
The main finding of the present study is that under Pten deletion also we still
observed that CR acts in such a way that it reduces the steatosis which would not have
occurred if the mice were under regular diet. The decreased steatosis is due to the
decrease in deposition of excess lipid in the liver hepatocytes; this can be due to the
decrease in the synthesis of TG and increase in β–oxidation as indicated by the increase
of MCAD and decrease of SCD.
32

MCAD is the key enzyme involved in the fatty acid oxidation pathway. An
increase of MCAD expression suggests that there is increase in fatty oxidation. This
could have been driven by the decrease in expression of SCD in CR mice.SCD
downregulation leads to accumulation of low carbon chain fatty acid. In normal cases
such accumulation of low carbon chain fatty acid would have resulted in conversion of
these saturated fatty acids into triglycerides. This suggests that the lipogenesis pathway
don’t proceed all the way to synthesize TG and get stored or deposited in the liver rather
they are accumulated as short chain fatty acids. These short chain FA serves as the
substrate for β–oxidation and might have stimulated the expression of MCAD (Figure 9).
The observations that there is an increase in SCD and no change in FAS and ACC
expression levels leads to the accumulation of FA substrate available for β–oxidation.

Acetyl CoA
Mitochondrial matrix
Malonyl CoA
Palmitoyl CoA β-Oxidation

Palmitate
Oleoyl CoA Oleate

Figure 9: Fatty acid oxidation pathway regulation by SCD and MCAD according to the
conclusion from the expression levels of SCD and MCAD.
MCAD
SCD
FAS
ACC
TG synthesis
33

CR may also alter the energy metabolism by affecting the mechanism of the
nutrient sensing signaling pathways (Dilova, Easlon, & Lin, 2007).One of the major
enzymes which acts as a fuel sensing enzyme is the AMPK since it is dependent on the
AMP/ATP ratio in the cell. They normally act in such a way that when ATP is reduced
they restore it by stimulating processes which restore ATP levels like fatty acid oxidation
and glycolysis. And suppress lipogenesis genes. However our results obtained here it is
clear that there is no change in the levels of pAMPK when provided with 60% CR till the
mice were 3 months old.

Studies from Jiang, (2008) ; Bordone (2005); To (2007) ; Al-Regaiey, and
Gonzalez, (2004) show varying cases of upregulation and downregulation of pAMPK
when the animals were under CR. Same is true in the case of FAS and ACC. (Cao et al.,
2001; Chen et al., 2008; Mulligan et al., 2008; Tsuchiya et al., 2004).All these variation
across these studies may be attributed to the varying times, at which the gene expression
patterns were measured, the CR regimen that was followed which ranged from alternate
day feeding, overnight fasting to a daily calorie restricted diet. So the time dependant and
the metabolic cyclic dependant gene expression pattern were varied in each study.

34

Summary:
Calorie restriction was found to decrease the progression of
carcinogenesis, decrease physiological aging and increase the life span of lab animals. In
this study, we performed calorie restriction on liver specific Pten deleted mouse model.
Specific deletion of Pten in liver results in a fatty liver phenotype. Our study shows that
there is decrease in steatosis or abnormal accumulation of lipid in liver in Pten deleted
mouse model when under 60% calorie restriction on a daily CR regimen. This might be
due to increased expression of key gene MCAD observed in CR mice. Further studies are
needed to analyze the metabolic pathway, gene expression patterns occurring in such CR
mice and how it helps reduce the steatosis that is observed in NAFLD.

35

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Abstract (if available)

Abstract Calorie restriction (CR) is an approach wherein there is moderate restriction of food intake while providing sufficient amount of essential nutrients. CR is beneficial in decreasing tumor progress, physiological aging and increasing the life span of laboratory animals. Previously our study showed that Pten deletion in liver resulted in fatty liver phenotype due to excess accumulation of lipid in liver. In this study, we tested the hypothesis that CR can reduce the burden of fatty liver using our liver Pten model. We showed that CR blocked the triglyceride accumulation in the liver of the Pten null mice. In this study we also investigated the pathway/mechanism involved in this change in phenotype when mice were under calorie restriction. We observed the expression patterns of genes involved in lipogenesis and fatty acid oxidation. Our analysis showed that CR treatment did not alter lipogenesis although lipogenesis expressing genes were induced by CR treatment in Pten control mice. We showed that induction of β -oxidation pathway is likely the reason for the inhibitory effect of CR on liver steatosis in the Pten model.

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Asset Metadata

Creator Josephrajan, Ajeetha (author)

Core Title Calorie restriction reduces liver steatosis in liver specific Pten deleted mouse model

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-08

Publication Date 08/10/2010

Defense Date 06/22/2010

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

Tag calorie restriction,fatty acid oxidation,livers,MCAD,OAI-PMH Harvest,PTEN,steatosis

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Stiles, Bangyan L. (committee member), Ulmer, Tobias (committee member)

Creator Email ajosephr@usc.edu,j.ajeetha@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3385

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