Minggu, 03 Juli 2016

Regulation of Fat Synthesis and Adipogenesis


Adipocytes are highly specialized cells that play a crucial role in the energy balance of
most vertebrates. Adipocytes convert excess energy to triacylglycerol and deposit it
during feeding in preparation for periods of food deprivation when energy intake is low.
Adipocytes may become enlarged by increased fat storage. Moreover, precursor cells
present in the stromal vascular fraction of adipose tissue can differentiate into adipocytes
even in mature animals.These two processes, fat synthesis and adipogenesis, are under
tight hormonal and nutritional control. In this review, we have summarized our work on
the regulation of fat synthesis. We have focused specifically on the transcriptional
activation of the fatty acid synthase (FAS) gene and on the inhibitory role of two
secretory factors, preadipocyte-specific preadipocyte factor-1 (Pref-1) and adipose tissuespecific
adipocyte differentiation-specific factor (ADSF), in adipose differentiation.


2.1. Nutritional and Hormonal Regulation of Lipogenic Enzymes
Fatty acid and triacylglycerol synthesis is regulated in response to the
nutritional/hormonal state in animals. Subjecting rodents to fasting causes a decrease in
lipogenesis; when fasted animals are subsequently refed a diet high in carbohydrate and
low in fat, there is a prompt and drastic rise in the production of fatty acids and
triacylglycerol to levels well above those observed in normally fed rats. Under lipogenic
conditions, excess glucose is converted to acetyl-CoA, which is used for the synthesis of
long-chain fatty acids. By the action of its seven active sites, fatty and synthase (FAS)
catalyzes all of the reaction steps in the conversion of acetyl-CoA and malonyl-CoA to
palmitate. The fatty acids produced are then used for esterification of glycerol-3-
phosphate to generate triacylglycerol. Mitochondrial glycerol-3-phosphate
acyltransferase (GPAT) catalyzes the first committed step in glycerophospholipid
biosynthesis by catalyzing acylation of glycerol-3-phosphate using fatty acyl-CoA to
generate l-acylglycerol-3-phosphate. The concentrations of many of the key enzymes in
this pathway, including FAS and mitochondrial GPAT, are decreased during fasting and
subsequently “superinduced” during the refeeding period. Induction of these enzymes is
highly coordinated and these inducible genes may be regulated via common mechanisms

It is generally accepted that insulin in the circulation, along with glucose, is elevated
during feeding of a high carbohydrate diet and induces enzymes involved in fatty acid
and triacylglycerol synthesis. Glucagon, on the other hand, is elevated during starvation
and suppresses activities of enzymes in fatty acid and fat synthesis by increasing
intracellular cyclic adenosine monophosphate (cAMP). In our early studies, we showed
that transcription of the FAS and mitochondrial GPAT genes increased when previously
fasted mice were refed a high carbohydrate diet (2, 3). There was no detectable
transcription of FAS or mitochondrial GPAT genes in fasted or fasted-refed
streptozotocin-diabetic mice, indicating that insulin is required for transcriptional
induction by fasting/refeeding. Administration of cAMP at the start of feeding in normal
mice prevented an increase in the transcription of these genes by feeding. Furthermore,
there was a rapid and marked increase in the transcription rates of the FAS and GPAT
genes when insulin was given to diabetic mice (2, 3). Overall, these genes are regulated at
the transcriptional level by nutritional and hormonal stimuli. The molecular mechanisms
underlying transcriptional regulation of these genes need to be elucidated.

2.2. Regulation of Fatty Acid Synthase Gene Transcription
To study the molecular mechanisms by which lipogenic enzymes such as FAS and
GPATare regulated, we employed 3T3-L1 adipocytes in culture. These cells provide a
good model system for studying lipogenic gene transcription since these genes are highly
induced during the differentiation process and are sensitive to hormones. In these cells
the regulation of FAS and GPAT mimics regulation in vivo (2). We identified an E-box
motif (5′-CATGTG-3′) at position-65 that is a binding site for upstream stimulating factor
(USF) (4), a ubiquitous member of the bHLH leucine zipper family of transcription
factors implicated in glucose control of L-type pyruvate kinase gene transcription (5).
Both USF-1 and USF-2 occupy the-65 complex (4); dominant negative mutants of USF-1
and USF-2 inhibited insulin stimulation of the FAS promoter (6), demonstrating that
these proteins are required for insulin stimulation of FAS gene transcription. We also
found that insulin regulation of the FAS promoter occurs via the PI3-kinase/Akt pathway

Another transcription factor found to play a key role in FAS gene transcription is
sterol regulatory element binding protein-1 (SREBP-1). This protein recognizes a sterol
regulatory element (SRE) (5′-TCACNCCAC-3′) sequence (8–12), but can also bind to Eboxes
due to the presence of an atypical tyrosine residue in the DNA-binding domain
(13). A major role for SREBP in transcriptional regulation of FAS was first suggested
when Goldstein and Brown demonstrated that overexpression of the truncated active
form of SREBP-1 in liver causes a large accumulation of triacylglycerol and the
induction of a battery of lipogenic genes including FAS and mitochondrial GPAT (14).
Others have shown that induction of FAS and other lipogenic enzymes by
fasting/refeeding is severely impaired in SREBP-1 knockout mice (15). It has also been
shown that SREBP-1c, one of two isoforms of SREBP-1, is highly induced by refeeding
a carbohydrate-enriched diet and that SREBP can transactivate the FAS promoter by binding to the -65 E-box (16). However, as described earlier, our in vitro data strongly
indicated to us that the critical factor functioning through the-65 E-box was USF, not
SREBP (Fig. 1A). In addition, we identified a canonical SRE, at150 (5′-ATCACCCCAC-
3′) in the FAS promoter suggesting a potential role of SREBP in regulation of the FAS
gene (17). To determine whether SREBP functions through this site, we cotransfected a
truncated active form of SREBP-1a into 3T3-L1 cells along with various FAS-LUC
reporter plasmids (17)FAS-luciferase(FASLUC).

InductionoftheFASreportergenebySREBP in vitro was reduced when sequences
between −136 and −19 were deleted (not shown), suggesting the presence of a binding
site for SREBP in this region, probably the −65 E-Box. However, when binding of
SREBP to the −65 E-box was prevented by mutation, deletion of sequences between
−184 and −136 abolished transactivation by SREBP-1 (Fig. 1B), indicating the presence
of an SREBP-responsive element in this region. In vitro, SREBP may activate and
increase FAS promoter activity if any single putative SREBP binding site is present,
regardless of its true physiological relevance. In support of this, only when both the −150
SRE and −65 E-box were mutated was trans-activation of the FAS promoter prevented by
SREBP in vitro (Fig. 1C).
FIGURE 1 Constructs of FAS promoter and localization of the FAS promoter region mediating FAS transactivation by SREBP-1a. (A) Schematic of putative USF and SREBP binding site in the FAS promoter. The diagram represents the proximal 444 bp of the FAS promoter. Genomics and proteomics in nutrition 80(B) Localization of the FAS promoter region mediating FAS transactivation
by SREBP-1a. Five micrgrams of −2100, −2100 (−65), −444 (−65), − 184, and −136 (−65) FAS-LUC plasmids were cotransfected with 25 ng of an expression vector for SREBP- 1a into 3T3-L1 fibroblasts. The values represent the mean± standard deviation. (C) Role of the −150 SRE in
activation of the FAS promoter by SREBP Five micrograms of each of the indicated constructs containing −444 bp of the 5′-flanking sequence of the FAS gene bearing mutations at the indicated positions were cotransfected with 25 ng of an expression vector for SREBP-1a into 3T3-L1 fibroblasts.The values represent the mean ± standard deviation.

To examine regulation of the FAS promoter in a physiological context in vivo, we
generated transgenic mice carrying the 2.1-kb 5′-flanking promoter region of the rat FAS
gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene (18). The
transgene was expressed strongly only in lipo-genic tissues, liver, and white adipose
tissue, and was drastically induced by feeding and insulin. Overall, the studies from these
transgenic mice demonstrated that the first 2.1-kb 5′-flanking sequence of the FAS gene
is sufficient for tissue-specific and hormonal/nutritional regulation. To further define the
FAS promoter sequences required for transcriptional activation by nutrients and
hormones in vivo, we generated several additional lines of transgenic mice (19), each
carrying different 5′-deletion constructs: −644, −444, −278, and −131 FAS-CAT (Fig.
2A). As shown in Fig. 2B, both the −644 and −444 constructs behaved in a manner
similar to that in the −2.1 kb transgenic mice, indicating that the region between −444
and −2.1 kb does not contain any sequences necessary for activation of FAS by fasting/
refeeding. However, when the same experiment was conducted on −278 FAS-CAT
transgenic mice, the induction of CAT, although detectable, was severely decreased. This
indicates that the region between −444 and −278 contains one or more elements required
for transcriptional activation of FAS. Furthermore, no CAT expression was detectable in
fasted/refed −131 FAS-CAT transgenic mice, indicating that the region between −278
and −131 contains additional element(s) required for basal transcription of FAS. Similar
results were obtained upon insulin administration to streptozotocin diabetic transgenic
mice. We concluded that two major regions of the FAS promoter are required for
transcriptional activation by refeeding/insulin: one between −278 and −131, which we

showed to be required for low-level activation of the promoter, and a second region
between −278 and −444, required for maximal “superinduction” of the gene. We also
showed through gel shift assays that a second E-box present at position −332 is a binding
site for USF-1 in vitro, and that this site may play an important role for the high-level
induction of FAS that is observed with feeding/insulin (19).
To determine the regions in the FAS promoter through which SREBP-1 functions in
vivo, we employed mice transgenic for the truncated, nuclear-active form of human
SREBP-1a (amino acids 1–460) under the control of the PEPCK promoter (14, 17). In
these mice, the transgenic SREBP-1a is induced by fasting and repressed by refeeding,
whereas endogenous

SREBP-1c is absent in the fasted state but induced by refeeding (17). The level of hepatic
FAS mRNA was found to be high whether these animals were fasted or refed (Fig. 2B).
We mated our five 5′-deletion FAS-CAT transgenic mice to SREBP transgenic mice and
subjected them to fasting/refeeding. As with the single FAS-CAT transgenic mice
described earlier, the −644 and −444 kb double transgenic mice both showed high CAT
expression in the refed state (Fig. 2B). Notably, CAT expression was also high in the
fasted state in all three constructs, indicating that the region between −2100 and −444
does not contain the putative site(s) for binding and function of SREBP-1. However, in
the −278 FAS-CAT transgenic mice, CAT induction was reduced whether the animals
were fasted or refed, and was completely absent in the −131 FAS-CAT double transgenic
mice, suggesting that the region between −131 and −278 contains at least one element
responsible for induction of FAS by SREBP. An increase in SREBP and its binding to the
− 150 SRE may be the major limiting mechanism for activation of FAS gene
transcription by fasting/refeeding in vivo.

2.3. Fatty Acid Synthase Promoter Occupancy and Function of USF and SREBP In Vivo
Our in vitro experiments clearly established the importance of the cis-acting elements in
the proximal FAS promoter required for insulin regulation in vitro. A critical remaining
question was whether the −150 SRE and −65 E-box each were required elements in vivo;
in our in vitro experiments, mutation of the −65 E-box in the context of the largest
−2.1kb promoter prevented induction by insulin (6), but had no effect on activation by
cotransfected SREBP-1 (17). To address these questions, we introduced mutations into
the −150 SRE and −65 E-box in the context of the −444 FAS-CAT transgenic construct
(20). We chose the −444 promoter fragment for these experiments as it was the shortest
5′-deletion construct that conferred maximal expression of CAT. As shown in Fig. 3A, no
expression of CAT was detected in three transgenic lines carrying a mutation at the −150
SRE, indicating that this element indeed is required for induction in vivo. Similar results
were obtained when the −444 (−65 mut) mice were fasted and refed: no significant
expression of CAT was detected, whereas the control −444 FAS-CAT mice showed a
strong induction by refeeding, and endogenous FAS expression was high in all mice (Fig.
3B). Moreover, CAT expression was still not detected when −444 (−150 mut) FASCAT/
PEPCK-SREBP-1 double transgenic mice were fasted or refed, strongly suggesting
that SREBP-1 functions directly through the −150 SRE in vivo. We concluded that both
the −150 SRE and −65 E-boxes are required for induction by fasting/refeeding.

Author: Kee-Hong Kim, Michael J.Griffin, Josep A. Villena, and Hei Sook Sul
Department of Nutritional Sciences and Toxicology, University of
California, Berkeley, California, U.S.A.

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