Research Notes: Hyperlipogenesis and Hypolipolysis

From the OMIM entry on PWS:

DNA Cell Biol. 2007 Sep 15.
Gene Set Enrichment Analysis Reveals Several Globally Affected Pathways due to SKI-1/S1P Inhibition in HepG2 Cells.
De Windt A, Rai M, Bernier L, Thelen K, Soini J, Lefebvre C, Chintawar S, Lavigne J, Saarinen L, Kytömäki L, Munzer JS, Lütjohann D, Pandolfo M, Davignon J, Seidah NG, Laaksonen R.
Department of Neurology, Free University of Brussels, Brussels, Belgium.

Sterol regulatory element-binding proteins (SREBPs) are transcription factors governing transcription of genes related to cholesterol and fatty acid metabolism. To become active, SREBPs must undergo a proteolytic cleavage to allow an active NH(2)-terminal segment to translocate into the nucleus. SKI-1/S1P is the first protease in the proteolytic activation cascade of SREBPs. SREBP inhibition may be useful, for example, in the treatment of liver steatosis caused by homocysteine-induced lipid synthesis. Accordingly, we overexpressed inhibitory prodomains (proSKI) of SKI-1/S1P in HepG2 cells to block SREBP activation to evaluate potential of SKI-1/S1P in controlling cellular cholesterol synthesis. SKI-1/S1P inhibition resulted in reduced cholesterol synthesis and mRNA levels of the rate-limiting enzymes, HMG-CoA reductase and squalene epoxidase, in the cholesterol synthetic pathway. The inhibitory effect was maintained also in the presence of homocysteine-induced endoplasmic reticulum stress. A gene set enrichment analysis was performed to elucidate other metabolic effects caused by SKI-1/S1P inhibition. SKI-1/S1P inhibition was observed to affect a number of other metabolic pathways, including glycolysis and citric acid cycle. These results demonstrate that inhibition of SREBPs decreases cholesterol synthesis in HepG2 cells both in the absence and presence of homocysteine. SKI-1/S1P inhibition may cause widespread changes in other key metabolic pathways.

J Lipid Res. 2005 Apr.
Regulation of SREBP-1 expression and transcriptional action on HKII and FAS genes during fasting and refeeding in rat tissues.
Gosmain Y, Dif N, Berbe V, Loizon E, Rieusset J, Vidal H, Lefai E.
Unité Mixte de Recherche Institut National de la Santé et de la Recherche Médicale-U449/Institut National de la Recherche Agronomique-U1235, Institut, Lyon, France.
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The sterol regulatory element binding protein 1 (SREBP-1) is regarded as a major factor involved in the nutritional regulation of lipogenesis. The aim of the present work was to demonstrate its involvement in the response of key genes of glucose and lipid metabolism in liver, adipose tissue, and skeletal muscle during fasting and refeeding. The regulation of hexokinase-2 (HKII) was investigated as a marker of the glucose metabolic pathway and that of FAS was investigated as a marker of the lipogenic pathway. The in vivo association of SREBP-1 with the promoter regions of these genes was determined in the different tissues using chromatin immunoprecipitation assays. Fasting decreased, and refeeding restored, FAS and HKII mRNA and protein levels in each tissue. The concomitant measurement of SREBP-1a and SREBP-1c mRNA levels, of mature SREBP-1 protein abundance in nuclear extracts, and of SREBP-1 interaction with target promoters led to the conclusion that SREBP-1 plays a major role in the response of FAS and HKII genes to nutritional regulation in rodents. These data elucidate the important role of SREBP-1 not only in the regulation of lipid metabolism but also of glucose metabolism and energy homeostasis.

From the full text article:

Sterol regulatory element binding proteins (SREBPs) are membrane-bound transcription factors of the basic-helix-loop-helix-leucine zipper family that have been shown to regulate gene expression of several enzymes implicated in cholesterol, lipid, and glucose metabolism (1, 2). Three members of the SREBP family have been identified and characterized (3). SREBP-1a and -1c are derived from a single gene through the use of alternative transcription start sites that produce different types of exon 1 (4).The third protein, SREBP-2, is derived from a separate gene (5). All SREBPs are synthesized as 1,150 amino acid precursors bound to the endoplasmic reticulum and nuclear envelope (3). For its activation, the SREBPs undergo a sequential two-step cleavage process to release their NH2-terminal segments that can then translocate to the nucleus (6). The mature form of SREBPs bind to sterol regulatory elements (SREs) in the promoter region of target genes to modulate their transcription (4, 7).

SREBP-1 has been implicated in the effect of insulin on the expression of key genes of lipid and glucose metabolism in different cell models, including hepatocytes, adipocytes, and muscle cells (8–12). Moreover, the expression of SREBP-1c was also found to be regulated by insulin (13–15), and SREBP-1 abundance is tightly related to the nutritional state in liver and adipose tissue (12, 16–18). These data suggested that SREBP-1, and mostly SREBP-1c, might directly be involved in the integration of nutritional changes and of insulinemia variations at the level of gene transcription (1, 2). This physiological role of SREBP-1 in response to nutrition was initially proposed for the control of lipogenic genes such as those coding for FAS and acetyl coenzyme-A carboxylase (ACC) in the liver and adipose tissue (1). Using in vivo chromatin immunoprecipitation (ChIP) assay, direct evidence for the involvement of SREBP-1 was recently provided through the demonstration of a diet-related modulation of its binding to the promoter region of FAS and ACC-2 genes in rodent liver (19–21). In addition to lipogenic tissues, SREBP-1 mRNAs and proteins are expressed in skeletal muscle (14, 22), and recent work supports its role in the regulation of glucose metabolism-related genes, such as hexokinase-2 (HKII) (10, 11). Because SREBP-1 expression is also modulated by nutrition in muscle, with decreased levels in the fasted state and increased expression with feeding (23, 24), the central role of this transcription factor in the integration of nutritional changes might extend from lipogenesis to additional pathways, such as glucose metabolism in skeletal muscle.

Biochimie. 2004 Nov.
SREBP transcription factors: master regulators of lipid homeostasis.
Eberlé D, Hegarty B, Bossard P, Ferré P, Foufelle F.
Inserm U465, Université Pierre et Marie Curie, 15, rue de l'Ecole de médecine, 75270 Paris cedex 06, France.

Sterol regulatory element binding proteins (SREBPs) are a family of transcription factors that regulate lipid homeostasis by controlling the expression of a range of enzymes required for endogenous cholesterol, fatty acid (FA), triacylglycerol and phospholipid synthesis. The three SREBP isoforms, SREBP-1a, SREBP-1c and SREBP-2, have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1c is involved in FA synthesis and insulin induced glucose metabolism (particularly in lipogenesis), whereas SREBP-2 is relatively specific to cholesterol synthesis. The SREBP-1a isoform seems to be implicated in both pathways. SREBP transcription factors are synthetized as inactive precursors bound to the endoplasmic reticulum (ER) membranes. Upon activation, the precursor undergoes a sequential two-step cleavage process to release the NH(2)-terminal active domain in the nucleus (designated nSREBPs). SREBP processing is mainly controlled by cellular sterol content. When sterol levels decrease, the precursor is cleaved to activate cholesterogenic genes and maintain cholesterol homeostasis. This sterol-sensitive process appears to be a major point of regulation for the SREBP-1a and SREBP-2 isoforms but not for SREBP-1c. Moreover, the SREBP-1c isoform seems to be mainly regulated at the transcriptional level by insulin. The unique regulation and activation properties of each SREBP isoform facilitate the co-ordinate regulation of lipid metabolism; however, further studies are needed to understand the detailed regulation pathways that specifically regulate each SREBP isoform.

J Lipid Res. 2002 May.
Suppression of fatty acid synthase promoter by polyunsaturated fatty acids.
Moon YS, Latasa MJ, Griffin MJ, Sul HS.
Department of Nutritional Sciences, University of California, Berkeley, CA, USA.
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Dietary polyunsaturated fat is known to suppress expression of fatty acid synthase (FAS), a central enzyme in de novo lipogenesis. The sterol regulatory element-binding protein (SREBP) has recently been shown to be involved in this suppression. We previously reported that the first 2.1 kb of the FAS promoter are sufficient for transcriptional induction by a high carbohydrate diet as well as suppression by polyunsaturated fat in transgenic mice. Here, we first examined the DNA sequences responsible for SREBP-mediated suppression of FAS promoter activity by polyunsaturated fatty acids (PUFA) in vivo. Feeding polyunsaturated fat prevented both the low-level activation of the -278 FAS promoter which contains the -150 sterol response element (SRE), as well as the maximal activation of the longer -444 FAS promoter. We observed that ectopic expression of the activated form of SREBP in liver prevented PUFA-mediated suppression of both the endogenous FAS and FAS promoter-reporter transgene expression. We also found that the promoter region required for PUFA suppression in vivo is located between -278 to -131, where SREBP functions. Using HepG2 cells, we further examined the specific FAS promoter elements required for PUFA suppression. We found that the -150 SRE, as well as the -65 E-Box, contribute to PUFA suppression of the FAS promoter, at least in vitro.

Am J Physiol Endocrinol Metab. 2002 Jan.
Acetyl-coenzyme A synthetase is a lipogenic enzyme controlled by SREBP-1 and energy status.
Sone H, Shimano H, Sakakura Y, Inoue N, Amemiya-Kudo M, Yahagi N, Osawa M, Suzuki H, Yokoo T, Takahashi A, Iida K, Toyoshima H, Iwama A, Yamada N.
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Japan.
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DNA microarray analysis on upregulated genes in the livers from transgenic mice overexpressing nuclear sterol regulatory element-binding protein (SREBP)-1a, identified an expressed sequence tag (EST) encoding a part of murine cytosolic acetyl-coenzyme A synthetase (ACAS). Northern blot analysis of the livers from transgenic mice demonstrated that this gene was highly induced by SREBP-1a, SREBP-1c, and SREBP-2. DNA sequencing of the 5' flanking region of the murine ACAS gene identified a sterol regulatory element with an adjacent Sp1 site. This region was shown to be responsible for SREBP binding and activation of the ACAS gene by gel shift and luciferase reporter gene assays. Hepatic and adipose tissue ACAS mRNA levels in normal mice were suppressed at fasting and markedly induced by refeeding, and this dietary regulation was nearly abolished in SREBP-1 knockout mice, suggesting that the nutritional regulation of the ACAS gene is controlled by SREBP-1. The ACAS gene was downregulated in streptozotocin-induced diabetic mice and was restored after insulin replacement, suggesting that diabetic status and insulin also regulate this gene. When acetate was administered, hepatic ACAS mRNA was negatively regulated. These data on dietary regulation and SREBP-1 control of ACAS gene expression demonstrate that ACAS is a novel hepatic lipogenic enzyme, providing further evidence that SREBP-1 and insulin control the supply of acetyl-CoA directly from cellular acetate for lipogenesis. However, its high conservation among different species and the wide range of its tissue distribution suggest that this enzyme might also play an important role in basic cellular energy metabolism.

From the full text article:

Intracellular cholesterol and fatty acid synthesis are regulated at the transcriptional level, mainly by sterol regulatory element-binding proteins (SREBPs), transcription factors belonging to the basic-helix-loop-helix leucin zipper family (2-4, 28). SREBPs are synthesized in a membrane-bound form. Upon sterol deprivation, nuclear SREBPs are cleaved to enter the nucleus and activate the transcription of genes involved in cholesterol and fatty acid synthesis by binding to sterol regulatory elements (SREs) or to palindromic sequences called E boxes within their promoter regions (3, 5, 26, 28). SREBPs consist of three isoforms (SREBP-1a, SREBP-1c, and SREBP-2), where SREBP-1a and -1c are generated from a single gene through alternative splicing (14, 31).

Cumulative lines of evidence, including normal, transgenic, and knockout mice on diet studies, established that SREBP-1 plays a role in regulating the transcription of genes involved in fatty acid synthesis, whereas SREBP-2 is actively involved in the transcription of cholesterogenic enzymes (13, 24). SREBP-1a is a stronger activator than SREBP-1c because of a longer transactivation domain, and it has a wider range of target genes involved in both cholesterol and fatty acid synthesis (22, 23). Transgenic mice overexpressing nuclear SREBP-1a in the liver demonstrated a marked induction of cholesterogenesis and lipogenesis resulting in engorged fatty livers (22).

Lipogenic enzymes, which are involved in energy storage through synthesis of fatty acids and triglycerides, are coordinately regulated at the transcriptional level during different metabolic states (9, 11). Recent in vivo studies demonstrated that SREBP-1c plays a crucial role in the dietary regulation of most hepatic lipogenic genes. These include studies of the effects of the absence or overexpression of SREBP-1 on hepatic lipogenic gene expression (22-24), as well as physiological changes of SREBP-1c protein in normal mice after dietary manipulations, such as placement on high carbohydrate diets, polyunsaturated fatty acid-enriched diets, and fasting-refeeding regimens (12, 15, 25, 29, 30). Recent studies suggest that insulin or insulin-facilitated glucose uptake mediates lipogenesis through SREBP-1c induction (7, 10, 18).

Acetyl-CoA synthetase (ACAS) is an intracellular enzyme that catalyzes the formation of acetyl-coenzyme A (acetyl-CoA) from coenzyme A and acetate (17). ACAS is known to be involved in ethanol and acetate metabolism of bacteria, and its molecular characterization has been well described from a microbiological point of view (8, 27). ACAS activity has also been well known among researchers in ruminology to play a crucial role in energy production of ruminants, because volatile fatty acids (also known as short-chain fatty acids), produced through fermentation of cellulose and other fibers in rumen, are their main source of energy. Even in other mammals, including rodents and humans, acetate, a major component of volatile fatty acids, can contribute considerably as an energy source as a result of fermentation of dietary fibers (20).

Beyond this limited information, the molecular characterization of ACAS in mammals has not been well understood. Because ACAS produces acetyl-CoA, which is a key branching molecule for different metabolic pathways, it could play an important role in energy metabolism in mammals. In a search for new targets of SREBP-1, we cloned the murine ACAS cDNA and analyzed its gene promoter. Investigation of the tissue-specific expression profile and nutritional regulation demonstrated a new aspect of this gene as a lipogenic enzyme.

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Effects of fasting and refeeding on ACAS expression in mice. Recently, SREBP-1 has been reported to play a crucial role in hepatic expression of lipogenic enzyme genes, especially in nutritional regulation, as was observed in fasted and refed mice (24). As shown by Northern blot analysis in Fig. 8, ACAS expression was significantly downregulated in a fasted state and markedly upregulated by refeeding in both liver and adipose tissue. This nutritional change is similar to changes of other lipogenic enzymes that are controlled by SREBP-1 (24). This refeeding induction of the ACAS gene was nearly abolished in the livers and adipose tissue of SREBP-1 knockout (KO) mice (Fig. 8), indicating that lipogenic induction of the ACAS gene by refeeding is controlled mainly by SREBP-1. At the same time, a slight but significant increase in ACAS RNA was seen in the SREBP-1 KO mice as well as wild-type mice, suggesting that other factors contribute to expression to a lesser degree.

Effect of insulin-depleted diabetes on ACAS expression. Because insulin has also been known to be important for lipogenic enzyme expression, we estimated the effects of insulin depletion and its supplementation on the hepatic mRNA level of ACAS. As shown in Fig. 9, STZ-induced diabetic mice showed markedly decreased ACAS expression in the livers compared with normal control mice, expression that was totally restored by insulin administration.

We also investigated the consequence of ACAS expression after overloading the substrate of the enzyme in drinking water. Acetic acid loading resulted in a significant decrease of ACAS expression in both fasted and fed mice (Fig. 10). A dose-dependent suppression was observed for acetate concentrations between 0 and 10% in fasted animals. In fed animals, acetate overloading also suppressed ACAS expression. There was no significant difference in either food or water consumption that might have affected SREBP-1c expression.

Discussion

ACAS is a new member of the family of lipogenic enzymes. The current study clearly demonstrates that expression of the mouse acetyl-CoA synthetase gene is nutritionally regulated in the same fashion as all other known lipogenic enzymes. Hepatic/adipose ACAS expression was suppressed by fasting and highly induced by refeeding, which is a typical feeding response of lipogenic genes. It was also suppressed in a state of insulin depletion by administration with STZ and was restored by insulin supplement, also a well known response of lipogenic enzymes in a diabetic state. Therefore, nutritional regulation of ACAS followed a lipogenic pattern. This is not surprising, as ACAS is one of the enzymes responsible for the production of acetyl-CoA, an initial substrate for lipogenesis. In a nutritional state favorable for lipogenesis, acetyl-CoA is produced from glycolysis and transported from mitochondria to the cytosol through a sequence of steps. However, lipogenic induction of cytosolic ACAS suggests that direct production of acetyl-CoA from free acetate in the cytosol might play a role in lipogenesis. The relative contribution of this pathway to lipogenesis remains unknown, and it awaits gene KO mice of this gene to estimate this. This enzyme could be more important in ruminants in which glycolytic activity is low and acetate is a main source of energy.

The ACAS gene is a target of SREBPs. The expression of the EST clone from the ACAS gene was upregulated by SREBP-1a, which led us to clone this gene. Recently, we reported that SREBP-1c is a dominant factor for the expression of most lipogenic genes in the liver (24). Absence of hepatic/adipocytic induction of the ACAS gene in refed SREBP-1 KO mice in the current study supports the notion that ACAS is another target of SREBP-1 as a lipogenic enzyme. Upregulation of the ACAS gene by SREBPs has already been shown in the first report of this gene (19). The SRE sequence was found in the promoter region of the ACAS gene and was confirmed to be responsible for SREBP activation by promoter analysis. Luciferase assays showed that the ACAS promoter was activated by SREBP-1a, -1c, or -2, consistent with the observation that the ACAS mRNA was increased in livers from transgenic mice overexpressing any of the SREBPs. The relative activity of each SREBP isoform for the ACAS SRE as estimated by Northern blot analysis of transgenic livers (Fig. 1) was similar to that of classic SRE: SREBP-1a > SREBP-2 > SREBP-1c (21). This is presumably due to a high similarity between the SRE in the ACAS promoter and the classic SRE originally found in the low-density lipoprotein receptor promoter.

The current studies with STZ-induced diabetic mice demonstrated that insulin regulates ACAS gene expression. This is consistent with the previous report on changes in hepatic ACAS enzyme activity in STZ-induced rats (21). Because insulin is important for SREBP-1c expression, insulin-dependent ACAS expression can be explained at least partially by its activation of SREBP-1c.

Physiological roles of ACAS gene in mammals. The decreased ACAS expression in the mouse liver by oral administration of acetate is implicative. The suppression of the enzyme expression by excess substrate is a good contrast to the regular mechanism of lipogenic enzyme regulation, in which conversion of excess energy to lipids is free from a negative feedback control. There may be a regulatory system for cytosolic production of acetyl-CoA by excess exogenous acetate. In addition, this gene is highly expressed in many other tissues, as well as in lipogenic organs. We also observed a considerable expression of this gene in cultured cells such as 293 cells (data not shown). The ACAS gene expression in the cultured cells is reported to be partially under sterol regulation, as predicted from the control by SREBPs (19). These observations suggest that ACAS might have some physiological roles other than in lipogenesis. From this standpoint, it is important to identify and clone a mitochondrial ACAS. This enzyme produces acetyl-CoA in mitochondria and would be involved in ketogenesis or ATP production in the tricarboxylic acid cycle and should be regulated in a different way from the cytosolic enzyme.

Crabtree et al. (6) have proposed futile cycling of acetate between free acetate and acetyl-CoA though cytoplasmic and mitochondrial pathways. One hypothesis of why such a pathway exists is to provide a means by which free acetate levels can be controlled (i.e., buffered). This hypothesis is attractive when one considers that ACAS is expressed in all tissues studied. There could be other functions for ACAS as well. Further studies are needed to clarify the physiological roles and regulation of both enzymes in cellular energy metabolism.

In the current studies, we cloned and identified the murine ACAS gene as a target of SREBPs and a new member of the lipogenic enzyme family. Acetyl-CoA plays a pivotal role in cellular fuel metabolism. Further studies on ACAS might open up a new aspect of glucose and fatty acid metabolism and have therapeutic implications, because acetate is known to be a better fuel source than glucose, especially for individuals with impaired glucose tolerance and diabetes.

Vitam Horm. 2002.
Sterol regulatory element-binding protein family as global regulators of lipid synthetic genes in energy metabolism.
Shimano H.
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

Sterol regulatory element-binding proteins (SREBPs) have been established as lipid synthetic transcription factors for cholesterol and fatty acid synthesis. SREBPs are synthesized as membrane-bound precursors with their N-terminal active portions entering the nucleus to activate target genes after proteolytic cleavage in a sterol-regulated manner. This cleavage step is regulated by a putative sterol-sensing molecule, SREBP-activating protein (SCAP), that forms a complex with SREBPs and traffics between the rough endoplasmic reticulum and Golgi. DNA cis-elements that SREBPs bind, originally identified as sterol-regulatory elements (SREs), now expands to a variety of SRE-like sequences and some of E-boxes, which makes SREBPs eligible to regulate a wide range of lipid genes. Animal experiments including transgenic and knockout mice suggest that three isoforms, SREBP-1a, -1c, and -2, have different roles in lipid synthesis. In differentiated tissues and organs, SREBP-1c is involved in fatty acid, whereas SREBP-2 plays a major role in regulation of cholesterol synthesis. SREBP-1a is expressed in growing cells, providing both cholesterol and fatty acids that are required for membrane synthesis. SREBP-1c seems to be a mediator for insulin/glucose signaling to lipogenesis, and could be involved in insulin resistance, remnant lipoproteins, and fatty livers. Future studies in this field will certainly focus on understanding the molecular mechanisms sensing cellular sterol and energy states leading to the activation of SREBP-mediated gene transcription.

Prog Lipid Res. 2001 Nov.
Sterol regulatory element-binding proteins (SREBPs): transcriptional regulators of lipid synthetic genes.
Shimano H.
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki, Japan.

Roles of sterol regulatory element-binding proteins (SREBPs) have been established as lipid synthetic transcription factors especially for cholesterol and fatty acid synthesis. SREBPs have unique characteristics. Firstly, they are membrane-bound proteins and the N-terminal active portions enter nucleus to activate their target genes after proteolytic cleavage, which requires sterol-sensing molecule, SREBP-activating protein (SCAP) and is crucial for sterol-regulation. Secondly, they bind and activate sterol-regulatory (SREs) containing promoters as well as some E-boxes, which makes SREBPs eligible to regulate a wide range of lipid genes. Finally, three isoforms, SREBP-1a-1c, and have different roles in lipid synthesis. In vivo studies using transgenic and knockout mice suggest that SREBP-1 seems to be involved in energy metabolism including fatty acid and glucose/insulin metabolism, whereas SREBP-2 is specific to cholesterol synthesis. Future studies will be focused on understanding molecular mechanisms sensing cellular sterol and energy states where SREBPs are deeply involved.

J Nutr. 2001 Sep.
Exercise down-regulates hepatic fatty acid synthase in streptozotocin-treated rats.
Fiebig RG, Hollander JM, Ji LL.
Interdepartmental Graduate Program in Nutritional Science and Department of Kinesiology, University of Wisconsin-Madison, USA.
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An acute bout of prolonged exercise has been shown to decrease hepatic fatty acid synthase (FAS) mRNA and activity induced by high carbohydrate diets. The purpose of the current study was to examine the role of insulin in this exercise down-regulation of FAS. Sixty-four male Wistar rats were randomly divided into normal and streptozotocin (STZ)-treated diabetic groups. After being starved for 48 h and refed a high cornstarch (C) or fructose (F) diet for 10 h, one half of each group of rats was killed after an acute bout of prolonged exercise (E), while the other half of the group was killed in the rested state. STZ treatment suppressed plasma insulin and elevated plasma glucagon levels along with a severe hyperglycemia. FAS mRNA levels decreased by 60% (P < 0.05) with STZ treatment but were 250% higher in F-fed versus C-fed rats. E abolished F-induced FAS mRNA levels in both normal and STZ rats and decreased plasma glucose concentration in STZ rats (P < 0.05). F-fed normal rats showed twofold higher hepatic FAS activity than did C-fed normal rats and this dietary induction was abolished by STZ (P < 0.05). FAS activity in normal rats was not affect by E and was increased with E in STZ rats. Nuclear protein binding to the insulin response sequence was not affected by STZ or diet and increased with E (P < 0.05). Carbohydrate response element binding was greater with F- versus C-feeding (P < 0.05) but unaffected by E. E enhanced inverted CCAAT-box element binding regardless of diet and STZ. We conclude that although insulin status had a great influence on FAS gene expression, E-induced down-regulation of FAS mRNA was not mediated by altered insulin response sequence binding but primarily by increased inverted CCAAT-box element binding to the FAS promoter and/or decreased concentration of carbohydrate metabolites.

From the full text article:

In mammalian species, high carbohydrate (CHO)/low fat diets result in de novo lipogenesis in the liver and adipose tissue (1). This is caused primarily by the induction of lipogenic enzymes, including fatty acid synthase (FAS), the key regulatory enzyme for hepatic fatty acid synthesis (1 2 3). The CHO-induced up-regulation of FAS is mediated by both metabolic and hormonal mechanisms (4). Insulin is the major hormone promoting hepatic lipogenesis via the stimulation of gene expression of FAS and several other lipogenic enzymes primarily by transcriptional activation, although alterations in FAS mRNA stability may also play a role (4 ,5). The stimulatory effect of insulin has been shown to result from the binding of upstream stimulatory factors to the insulin response sequence (IRS/A) in the promoter region of the FAS gene (6 ,7). Furthermore, hepatic FAS gene has been reported to contain an inverted CCAAT-box element (ICE) located adjacent to IRS/A. This region can bind the transcription factor NF-Y in response to cAMP and its occupancy can attenuate up-regulation of FAS by insulin in vitro (8).

Although CHO feeding has consistently been shown to induce hepatic lipogenic enzymes, fructose (F) ingestion in particular results in a greater effect than glucose or complex CHO, such as cornstarch (C), despite a lower insulin response (3 ,9 10 11 12). Therefore, CHO induction of FAS must be attributed at least partially to mechanisms independent of insulin. For example, the CHO response element (ChoRE) found within the first intron of the FAS gene has sequence similarity to known glucose/CHO response elements in other lipogenic and glycolytic genes and its binding has been shown to increase with a high F diet (13 ,14).

Endurance exercise causes various hormonal and metabolic changes that alter the cellular milieu in a number of tissues, including liver (15 16 17). Prolonged exercise consistently results in a decrease in insulin secretion and an elevation of plasma glucagon and catecholamine levels, both of which could decrease lipogenesis (8 ,19). In addition, endurance exercise decreases liver glycogen and blood glucose levels, which could attenuate lipogenic precursors and glycolytic metabolites in the liver. We have previously reported that an acute bout of exercise suppresses induction of both FAS mRNA and enzyme activity normally observed in rats starved and refed a high CHO diet (3). We also demonstrated that exercise attenuated nuclear protein binding to the IRS/A and ChoRE of the FAS gene in liver extracts, which was postulated to be partially responsible for the down-regulation of FAS enzyme activity (20). However, the effect of exercise on ICE binding has never been examined.

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F-fed rats showed 2.5-fold higher FAS mRNA and 2-fold higher FAS activity than C-fed rats. Furthermore, in the STZ-treated diabetic rats wherein FAS gene expression was heavily suppressed, FAS mRNA and activity were induced by F to a greater extent than by C and reached the same levels as C-fed normal rats. These data, consistent with many previous studies, clearly demonstrated the prominent lipogenic potential of F (3 ,11 ,38 39 40). F-fed normal and diabetic rats consumed 40% and 60% more food than their C-fed counterparts, which could play an important role in explaining the greater FAS induction. However, the mechanism underlying the observed fructose effect cannot be entirely due to greater energy intake because previous studies using a pair-feeding or meal-feeding regimen revealed a greater lipogenic effect with fructose feeding despite isocaloric intake (3 ,39 ,40). FAS up-regulation by fructose feeding has been shown to coincide with increased IRS/A binding in rat and mouse liver (28, 41). Therefore, a direct involvement of fructose in promoting FAS gene transcription was suggested. These studies were echoed by our recent experiment showing that refeeding a high F diet for 6 h to food-deprived rats significantly increased both IRS/A and ChoRE binding, along with a dramatic elevation (50-fold) of FAS gene transcription rate (20). In the present study, we observed an increased transcription factor binding to FAS-ChoRE in the F-fed rats, but IRS/A binding was not altered. There are two possible explanations for the discrepancy. There might have been a transient increase in IRS/A binding during the early phase of refeeding. By studying the time course of FAS induction by F, Katsurada et al. (2) revealed a peak FAS transcription rate at 4 h of refeeding, whereas FAS mRNA levels did not reach the maximal value until 16 h. Because increased nuclear protein binding is the overture of transactivation of FAS gene, we speculate that IRS/A binding might indeed have increased but then returned to normal at 9–10 h when rats were killed. Alternatively, F induction of FAS might have resulted primarily from increased mRNA stability, whereas increased transcription due to IRS/A and/or ChoRE binding might only play a minor role. F metabolites stimulate lipogenesis in rat liver more effectively than glucose (4). Incubating HepG2 cells with D-glucose, lactate and citrate, but not L-glucose, increased the half-life of FAS mRNA from 4.4 to 30 h, suggesting that the observed effects were mediated by glycolytic intermediates (5). In a previous study, we observed a greater hepatic pyruvate content in rats starved and refed a F diet for 12 and 24 h, which coincided with a greater FAS mRNA abundance (3). However, the importance of these potential regulatory mechanisms has not been confirmed under physiological conditions.

Biochim Biophys Acta. 2000 May 6.
Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver.
Ide T, Kobayashi H, Ashakumary L, Rouyer IA, Takahashi Y, Aoyama T, Hashimoto T, Mizugaki M.
Laboratory of Nutrition Biochemistry, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba Science City, Japan.

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in alpha-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary alpha-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.

J Nutr Sci Vitaminol (Tokyo). 1999 Jun.
Dietary phospholipid-dependent reductions in gene expression and activity of liver enzymes in fatty acid synthesis in fasted-refed rats.
Rouyer IA, Takahashi Y, Ide T.
Laboratory of Nutrition Biochemistry, Ministry of Agriculture, Forestry and Fisheries, Tsukuba Science City, Ibaraki, Japan.

The effects of dietary soybean phospholipid, its hydrogenation product and safflower phospholipid on gene expression and the activity of hepatic enzymes in fatty acid biosynthesis were examined in fasted-refed rats. Phospholipid composition of soybean phospholipid and its hydrogenation product were the same, but the hydrogenation product contained negligible amounts of unsaturated fatty acids. Among phospholipid classes, lysophosphatidylcholine and phosphatidylinositol proportions were slightly higher in safflower phospholipid than in soybean phospholipid or its hydrogenation product. Rats were fasted for 2 d and refed a fat-free diet or a diet containing 4% fatty acids either as soybean oil or various phospholipid preparations for 3 d. Compared to the fat-free diet, the soybean oil diet only slightly decreased specific, but not total hepatic fatty acid synthetase and malic enzyme activity, and it was totally ineffective in modulating glucose 6-phosphate dehydrogenase and pyruvate kinase activity under our experimental conditions. The diets containing phospholipids, however, markedly decreased the activity of these enzymes. The extent of reduction was somewhat attenuated with hydrogenated soybean phospholipid as compared with soybean and safflower phospholipids. Dot and Northern blot hybridization using specific cDNA probes showed that, compared to a fat-free diet, diets containing phospholipids profoundly decreased the hepatic mRNA levels of enzymes in fatty acid synthesis. Soybean oil, however, only marginally affected these parameters. Hepatic mRNA levels for enzymes correlated well with enzyme activity. Dietary phospholipids therefore appear to have decreased enzyme activity in fatty acid synthesis primarily by suppressing the mRNA levels of these enzymes. Compared to soybean oil, hydrogenated soybean phospholipid is still effective in decreasing the activity and mRNA level of enzymes in fatty acid synthesis. Therefore, it is difficult to ascribe the potent physiological activity of phospholipid in reducing fatty acid synthesis entirely to polyunsaturated fatty acid moiety.

Bull Mem Acad R Med Belg. 1997.
Molecular endocrinology of hereditary obesity. [Article in French]
Christophe J.
Laboratoire de Biochimie générale et humaine, Institut de Pharmacie, Université libre de Bruxelles.

The hypothalamic disorders of obesity include hyperphagia, a low central orthosympathetic tone (with reduced thermogenesis), vagal hyperinsulinism, low serotonin efficacy, a hyperactive hypothalamo-hypophyseal-adrenal axis, a hypoactive GHRH-GH-IGF axis and hypogonadism of central origin. Hyperlipogenesis, glucose intolerance and excessive gluconeogenesis are secondary features. Most frequently the hypothalamic ARC reacts poorly to the leptin hypersecreted by adipose tissue, so that the local synthesis of NPY is unchecked. Fortunately, two prostaglandins derived from dietary arachidonic acid bind fat cell PPAR gamma and hepatic PPAR alpha. Both nuclear proteins are phosphorylated through an insulin pathway, thereby inhibiting the expression of genes favoring obesity and stimulating that of genes accelerating fatty acid oxidation. The array of dietetic and pharmacologic tools considered today is analyzed.

Biochim Biophys Acta. 1996 Nov 22.
Activity of hepatic fatty acid oxidation enzymes in rats fed alpha-linolenic acid.
Kabir Y, Ide T.
Laboratory of Nutrition Biochemistry, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Ibaraki, Japan.

The activity of hepatic fatty acid oxidation enzymes in rats fed linseed and perilla oils rich in alpha-linolenic acid (alpha-18:3) was compared to that in rats fed safflower oil rich in linoleic acid (18:2) and a saturated fat (palm oil). Palm and safflower oils were essentially devoid of alpha-18:3. The palmitoyl-CoA oxidation rates both in mitochondrial and peroxisomal pathways in liver homogenates were significantly higher in rats fed linseed oil than in those fed palm and safflower oils. Among rats fed diets containing palm oil, safflower oil, fat mixtures composed of safflower and perilla oils (2:1, w/w and 1:2, w/w), and perilla oil, mitochondrial and peroxisomal fatty oxidation rates increased with increasing dietary levels of perilla oil. Compared to palm and safflower oils, dietary alpha-18:3 either in the form of linseed or perilla oils profoundly increased the activity of carnitine palmitoyltransferase, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and 2,4-dienoyl-CoA reductase. Smaller but significant increases by dietary alpha-18:3 of the activity of acyl-CoA dehydrogenase, enoyl-CoA hydratase, and delta 3, delta 2-enoyl-CoA isomerase were also observed. Unexpectedly, dietary alpha-18:3 greatly reduced the activity of 3-hydroxy-acyl-CoA dehydrogenase. Compared to palm oil, dietary polyunsaturated fats significantly reduced the activity of fatty acid synthetase and glucose-6-phosphate dehydrogenase to the same levels. The activity of pyruvate kinase was significantly higher in rats fed palm oil than in those fed polyunsaturated fats. The extent of reduction was more prominent with polyunsaturated fats containing alpha-18:3 than with safflower oil devoid of alpha-18:3. Thus, compared to linoleic acid and saturated fatty acids, dietary alpha-18:3 caused characteristic changes in the activity of hepatic enzymes in fatty acid and glucose metabolism in rats.

J Nutr. 1992 Jan.
Regulation of hepatic lipogenic enzyme gene expression by diet quantity in rats fed a fat-free, high carbohydrate diet.
Iritani N, Nishimoto N, Katsurada A, Fukuda H.
Tezukayama Gakuin College, Osaka, Japan.
[ Free full text ]

This investigation concerns the effects of the level of intake of a high carbohydrate diet on transcriptional rate, mRNA concentration and enzyme induction for lipogenic enzymes in rat liver. Six hours after refeeding fasted rats, the transcriptional rates in livers reached low maximum levels with small quantities of diet, but the mRNA concentrations continued to increase as diet intake increased. Greater diet intake primarily increased transcriptional rates and mRNA concentrations of lipogenic enzymes. After refeeding for 16 h, the mRNA concentrations were sigmoidly increased relative to the diet quantity and reached maximum levels of 20-, 110-, 22- and 16-fold above each fasted level for acetyl-CoA carboxylase, fatty acid synthase, malic enzyme and glucose-6-phosphate dehydrogenase, respectively. After 3 d of refeeding (in a steady state of lipogenic enzyme activities), however, the transcriptional rates, mRNA concentrations and activity inductions of all the enzymes were sigmoidly increased relative to diet quantity, but were not different among the enzymes. Consequently, fatty acid synthesis and triglyceride levels in the liver were not increased by feeding less than 70% of ad libitum intake but were greatly increased by feeding greater than 70% of ad libitum intake.

Biull Eksp Biol Med. 1988 May.
Enzyme systems of the substrate and cofactor supply of hyperlipogenesis in non-insulin-dependent diabetes. [Article in Russian]
Obrosova IG, Efimov AS, Velikiĭ NN, Zimatkina TI, Moiseenok AG.

Enzymatic systems of hepatic hyperlipogenesis supply by substrate (acetyl-CoA) and cofactors (NADPH and ATP) were studied in experiments on diabetic C57Bl/Ks J mice (db/db) that served as a model of non-insulin dependent diabetes. The rise in acetyl-CoA synthetase activity catalyzing the primary step of lipogenesis from acetate has been found, while pyruvate dehydrogenase complex activity did not differ from the control and ATP-citrate lyase activity was lowered. Hyperlipogenesis in non-insulin dependent diabetes was induced by the activation of cellular energy supply revealed in enhanced 2-oxoglutarate dehydrogenase activity and elevated ATP level, as well as changes in the activity ratio of NADPH supply and utilization and the rise in fructose-1,6-diphosphate, allosteric effector of fatty acid synthetase, which resulted in the increase of the enzyme activity and created wider potentials of NADPH utilization as a reducing equivalent in lipogenesis.

Probl Endokrinol (Mosk). 1987 Nov-Dec.
CoA biosynthesis and the structure of its reserve in the liver in mice with diabetes (db/db) after administration of pantothenic acid derivatives. [Article in Russian]
Moiseenok AG, Efimov AS, Sheĭbak VM, Obrosova IG, Gurinovich VA.

In the liver of genetically diabetic mice (db/db) a rise of CoA and alterations in the structure of its moiety (an increase in CoA/short-chain fatty acyl-CoA and CoA/long-chain fatty acyl-CoA ratios) were found being one of the hyperlipogenesis-providing factors. A rise of the content of CoA in diabetes was caused by the activation of its biosynthesis from vitamin-containing precursors; an increase in the deposition of the latter in panthotenate-protein complexes was also noted. Panthetine and 4'-phosphopanthotenate administration to diabetic animals returned to normal the level of total and free CoA and the ratios of separate components in the structure of coenzyme moiety, and the content of CoA precursors (phosphopantheteine and dephospho-CoA) in the liver.

Biull Eksp Biol Med. 1987 May.
Changes in the content and structure of the coenzyme A moiety in the liver of diabetic mice (db/db) administered a regimen of nicotinamide. [Article in Russian]
Obrosova IG, Moĭseenok AG, Efimov AS, Sheĭbak VM, Chiger VB.

Alterations in the content and structure of CoA moiety typical of hyperlipogenesis (a rise in total and free CoA levels, a drop in short-chained fatty acyl-CoA/CoA and long-chained fatty acyl-CoA/CoA ratios) were found in the liver of obese mice with non-insulin-dependent diabetes (db/db). The treatment of diabetic mice with nicotinamide, an antilipemic drug, was accompanied by a decrease in total and free CoA levels and a rise in short-chained fatty acyl-CoA content and short-chained fatty acyl-CoA/CoA and long-chained fatty acyl-CoA/CoA ratios, probably leading to the inhibition of the enzymes of primary lipogenesis steps. It is suggested that CoA moiety structure is essential as an integral index regulating the rate of fatty acid biosynthesis in diabetes mellitus.

Vopr Pitan. 1986 Nov-Dec.
Proteinization and biotransformation of pantothenic acid in the liver during the activation of lipogenesis. [Article in Russian].
Gurinovich VA, Moĭseenok AG.

White female rats received a balanced synthetic ration (control) or a ration devoid of pantothenic acid (PAA) during 3 weeks. After 36-hour fasting adaptive hyperlipogenesis was induced by feeding the animals with a high-carbohydrate ration, then [114-C]-PAA (sodium salt, 182 nmol/kg) was administered with intervals of 3, 6, 24 hours up to 1 hour before decapitation. Radioactivity of the rats' boiled liver extracts depended on the hyperlipogenesis stage, its level rose progressively, in the control and reached the maximum in PAA-deficient animals by hour 6 after the feeding resumption. The PAA-deficient animals possessed a high PAA-accumulating capacity of the liver and cytosole of the liver including non-covalent radionuclide binding by protein complexes. CoA-synthesizing capacity of the liver in the control animals, evaluated by the biotransformation of the labeled vitamin with CoA precursors of CoA, was intensified with the lipogenesis activation; in vitamin-deficiency CoA biosynthesis was accelerated more than two-fold as compared to the control at the initial and extended periods of hyperlipogenesis (3.6 h). The differences in proteinization and biotransformation of PAA in the liver of control and PAA-deficient animals disappeared by 25 h of adaptive hyperlipogenesis.

Biochim Biophys Acta. 1986 Sep 12.
Effects of dietary nutrients on substrate and effector levels of lipogenic enzymes, and lipogenesis from tritiated water in rat liver.
Katsurada A, Fukuda H, Iritani N.

When fasted rats were refed for 4 days with a carbohydrate and protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate), the effects of dietary nutrients on the fatty acid synthesis from injected tritiated water, the substrate and effector levels of lipogenic enzymes and the enzyme activities were compared in the livers. In the carbohydrate diet group, although acetyl-CoA carboxylase was much induced and citrate was much increased, the activity of acetyl-CoA carboxylase extracted with phosphatase inhibitor and activated with 0.5 mM citrate was low in comparison to the carbohydrate and protein diet group. The physiological activity of acetyl-CoA carboxylase seems to be low. In the protein diet group, the concentrations of glucose 6-phosphate, acetyl-CoA and malonyl-CoA were markedly higher than in the carbohydrate and protein group, whereas the concentrations of oxaloacetate and citrate were lower. The levels of hepatic cAMP and plasma glucagon were high. The activities of acetyl-CoA carboxylase and also fatty acid synthetase were low in the protein group. By feeding fat, the citrate level was not decreased as much as the lipogenic enzyme inductions. Comparing the substrate and effector levels with the Km and Ka values, the activities of acetyl-CoA carboxylase and fatty acid synthetase could be limited by the levels. The fatty acid synthesis from tritiated water corresponded more closely to the acetyl-CoA carboxylase activity (activated 0.5 mM citrate) than to other lipogenic enzyme activities. On the other hand, neither the activities of glucose-6-phosphate dehydrogenase and malic enzyme (even though markedly lowered by diet) nor the levels of their substrates appeared to limit fatty acid synthesis of any of the dietary groups. Thus, it is suggested that under the dietary nutrient manipulation, acetyl-CoA carboxylase activity would be the first candidate of the rate-limiting factor for fatty acid synthesis with the regulations of the enzyme quantity, the substrate and effector levels and the enzyme modification.

J Nutr. 1986 Aug.
Inhibition of liver lipogenesis by dietary polyunsaturated fat in severely diabetic rats.
Wilson MD, Hays RD, Clarke SD.
[ Free full text ]

Diabetic and nondiabetic rats were used to ascertain if dietary polyunsaturated fats inhibited hepatic acetyl-CoA carboxylase and fatty acid synthetase in insulin-insufficient rats as had been previously shown for normal rats. Male rats were rendered diabetic (400-600) mg glucose/100 mL blood) with streptozotocin and were fed a high fructose fat-free diet. Safflower oil or palmitate (or tallow) was added to the basal fructose diet at a level to supply 12,24 or 36% additional digestible energy. Compared with normal rats, diabetic rats had significantly lower hepatic fatty acid biosynthesis, but the proportion of acetyl-CoA carboxylase expressing catalytic activity as determined by the avidin-inactivation technique was unaffected by diabetes. Diabetes did not lower the maximal maximal activities of carboxylase and fatty acid synthetase. Moreover, the activities of these enzymes greatly exceeded the rate of fatty acid synthesis. At all levels of fat supplementation, the high linoleate safflower oil consistently resulted in a 50% lower rate of fatty acid biosynthesis than did comparable levels of tallow or palmitate. Safflower oil was also a more effective suppressor of the activities of acetyl-CoA carboxylase and fatty acid synthetase than the saturated fats. Our data suggest that the greater inhibition of hepatic fatty acid biosynthesis by polyenoic fatty acids is an insulin-independent mechanism.

Fed Proc. 1986 Aug.
Regulation of hepatic gene expression by lipogenic diet and thyroid hormone.
Towle HC, Mariash CN.

Adaptive hyperlipogenesis in the liver occurs after feeding rats a high-carbohydrate, fat-free diet or after treatment with thyroid hormone. This phenomenon is accompanied by the induction of a family of enzymes and proteins involved in various aspects of lipogenesis, resulting largely from alterations in the rates of protein synthesis. The changes in protein production, in turn, are the result of increased or decreased cellular concentrations of specific mRNAs in response to carbohydrate feeding or hyperthyroidism. There is a large degree of overlap between the mRNA species induced by thyroid hormone and carbohydrate feeding in rat liver. One such mRNA species, spot 14, has several attractive features as a potential model for exploring the molecular mechanisms involved in the regulation of gene expression. Most importantly, the mRNA for spot 14 increases with a lag time of less than 20 min after treatment with thyroid hormone or sucrose gavage and thus may represent a primary response to the hormonal and dietary stimuli. Initial studies to elucidate the cellular site of action of thyroid hormone and dietary carbohydrate indicate that changes in both the rate of gene transcription and the stability of the nuclear RNA precursor are involved in spot 14 mRNA induction.

J Nutr. 1986 Feb.
Effects of dietary proteins on lipogenic enzymes in rat liver.
Iritani N, Nagashima K, Fukuda H, Katsurada A, Tanaka T.
[ Free full text ]

When fasted rats were fed fat-free diets containing various sources of protein for 3 d, the activities of liver glucose-6-phosphate dehydrogenase, malic enzyme, acetyl-CoA carboxylase and fatty acid synthetase were markedly lower in rats fed soybean protein or gluten than in those fed casein or fish protein. Since malic enzyme mRNA activity was not low in the soybean protein-fed animals, the translation of malic enzyme appears to be suppressed by dietary soybean protein. The incorporation of tritiated water into liver fatty acids was significantly lower in animals fed soybean protein than in those fed casein. The triglyceride levels in plasma and especially in liver were also lower in the groups fed soybean and gluten than in the groups fed casein and fish. In addition, when dietary soybean protein was replaced with amino acids to simulate casein or soybean protein, the effects on the levels of lipogenic enzymes were still found but were not as great. Thus, some effects can be ascribed to the protein itself and some to the amino acid composition of the diet.

Ukr Biokhim Zh. 1984 Jul-Aug.
Nicotinamide nucleotides as factors of lipogenesis regulation. [Article in Russian]
Velikiĭ NN.

Data are analyzed on a regulatory effect of the redox state of NAD- and NADP-couples (the free NAD+-/NADH, NADP+/NADPH ratios) on certain enzymic links of lipogenesis. A concept is formulated on coordination of the activity of lipogenesis key enzymes by a common signal, supposedly by changes in the NAD+/NADH and NADP+/NADPH values in cytoplasm and mitochondria of the rat liver cells. High values of the NAD- and NADP-couples ratios, activation of the citrate transport from mitochondria to cytoplasm and of enzymic systems supplying lipogenesis with a substrate - acetyl-CoA, reducing equivalents (NADPH) determine the maximal lipid synthesis rate observed in adaptive hyperlipogenesis. The inhibitory action of nicotinamide on lipogenesis is realized at the level of systems providing a high metabolic pool of acetyl-CoA and dehydrogenases, producing NADPH in cytoplasm of liver cells.

J Nutr. 1983 Nov.
Interaction of glucocorticoid and thyroxine in the responses of rats to starvation-refeeding.
Nessmith S, Baltzell J, Berdanier CD.
[ Free full text ]

The interaction of glucocorticoid (GC) and thyroxine (T4) in the generation of the hepatic enzyme overshoot and lipid response to starvation-refeeding was studied. Male Sprague-Dawley rats were either left intact, or treated with propylthiouracil (PTU), or adrenalectomized (ADX), or ADX and/or PTU treated and treated with GC and/or T4. One-half of each of these treatment groups was fed a 65% glucose diet while the remaining rats were starved for 48 hours and refed the glucose diet for 48 hours. After decapitation, hepatic lipid and glucose-6-phosphate dehydrogenase (G6PD) activity were determined. Rats treated with only PTU had less of an enzyme overshoot than nontreated rats, and the full overshoot response was restored with T4 treatment. ADX rats did not have the typical enzyme overshoot response to starvation-refeeding. However, ADX rats had their overshoot response restored with GC. PTU-treated ADX rats had more of an overshoot response than did ADX rats. When T4 was administered to PTU-treated ADX rats there was less of an enzyme overshoot; however, when both T4 and GC were administered to the PTU-treated ADX rats, the overshoot response was fully restored. The liver lipid response to starvation-refeeding followed a similar pattern except that in PTU-treated rats the liver lipid levels were significantly higher in the starved-refed rats than in the ad libitum-fed rats. These results indicate that T4 and GC play a role in the G6PD and liver lipid response to starvation-refeeding.

J Nutr. 1982 Jun.
Suppression of rat liver fatty acid synthesis by eicosa-5,8,11,14-tetraynoic acid without a reduction in lipogenic enzymes.
Clarke BA, Clarke SD.
[ Free full text ]

In meal-fed rats supplementation of safflower oil (5 g per 100 g diet) to a fat-free basal diet resulted in the characteristic suppression of liver fatty acid synthetase and acetyl-CoA carboxylase activities, which was accompanied by a 60% decrease in the rate of hepatic fatty acid synthesis. The decline in activity of these lipogenic enzymes was completely prevented by adding 0.05% eicosa-5,8,11,14-tetraynoic acid (TYA) to the safflower oil diet. Fatty acid analysis of the livers indicated that TYA significantly impaired the conversion of linoleate to arachidonate. Apparently the selective suppression of lipogenic enzymes by dietary linoleate is not caused by linoleate per se but requires its conversion to longer-chain fatty acids and/or protaglandins. In spite of high activities of fatty acid synthetase and acetyl-CoA carboxylase, liver fatty acid synthesis continued to be inhibited by the safflower oil + TYA dietary regimen. This continued inhibition of lipogenesis was due to the TYA, because addition of TYA to the fat-free diet precipitated a significant decline in liver fatty acid synthesis without a drop in lipogenic enzymes. Inhibition of fatty acid synthesis by TYA could not be attributed to a decrease in liver glucose utilization based on hepatic glycogen concentration, nor was it due to a reduction in the fraction of catalytically active polymeric acetyl-CoA carboxylase based on sensitivity of the enzyme activity to avidin.

J Nutr. 1982 Mar.
Effects of diet composition and adrenalectomy on the lipogenic responses of rats to starvation-refeeding.
Williams BH, Berdanier CD.
[ Free full text ]

The interacting effects of diet and glucocorticoid (GC) on the tritium incorporation into lipid and glucose-6-phosphate dehydrogenase activity in starved-refed rats was studied. Male Sprague-Dawley rats were intact, adrenalectomized (ADX), or ADX and given GC and fed either ad libitum or not fed for 48 hours amd refed either a 65% glucose diet, 65% sucrose diet, 65% starch diet, 65% protein diet or a 40% fat diet. No diet differences in rates of 3HOH incorporation into total lipids were observed in ad libitum-fed rats. ADX lowered lipogenesis and this effect was diet dependent. Sucrose-fed, glucose-fed and protein-fed ADX rats had lower rates of lipogenesis than their intact controls. Starvation-refeeding increased lipogenesis in all groups of intact rats except those fed the 40% fat diet. The magnitude of the response was diet dependent. Sucrose-fed rats had greater responses than fat-fed rats. The diet effect was dependent on the presence of the adrenals and GC. Thus, the large increase in liver lipid associated with starvation-refeeding is contingent on the composition of the diet and the presence of the adrenals.

Biokhimiia. 1981 Nov.
Control of lipogenesis in rat liver by changing the enzyme activity of the citrate-pyruvate system of acetyl coenzyme A transport. [Article in Russian]
Kaliman PA, Mishchenko VP.

The effects of insulin and tyroxin on the activities of pyruvate kinase, pyruvate dehydrogenase, ATP-citrate-lyase, NADP-malate dehydrogenase (decarboxylase), lactate dehydrogenase and on that of pyruvate carrier in rat liver were investigated. Insulin increased the activities of all the enzymes tested. The total activity of lactate dehydrogenase was not altered thereby; however, a redistribution of isoenzymes towards an increase in LDH1 and a decrease of LDH4 was observed. No increase of the pyruvate kinase, ATP-citrate-lyase and NADP-malate dehydrogenase activities took place, when actinomycin D was injected simultaneously with insulin. Tyroxin decreased the activities of pyruvate kinase, pyruvate dehydrogenase and ATP-citrate-lyase and increased that of NADP-malate dehydrogenase. The role of induction by insulin and inhibition by tyroxin of the enzyme activity in the citrate-pyruvate system of CoASA transport in lipogenesis control in rat liver is discussed. It is assumed that when lipogenesis is stimulated by insulin, the malate oxidized by the malate dehydrogenase reaction is formed in the cytoplasm, while under tyroxin action it is produced by the mitochondria.

J Nutr. 1981 Jan.
Coordinate suppression of liver acetyl-CoA carboxylase and fatty acid synthetase by polyunsaturated fat.
Toussant MJ, Wilson MD, Clarke SD.
[ Free full text ]

Polyunsaturated fats (PUFA) suppressed hepatic fatty acid synthesis and the activities of lipogenic enzymes more effectively than did saturated fats. The activity of glycolytic enzymes - glucokinase, phosphofructokinase and pyruvate kinase - were not affected by PUFA. The absolute rate of liver fatty acid synthesis after meal ingestion was very similar to the maximal activities of acetyl-CoA carboxylase and fatty acid synthetase. When PUFA was supplemented to a fat-free diet, the activities of carboxylase and synthetase decreased similarly over 3 days. During the 3 days, the concentration of liver malonyl-CoA (after meal ingestion) did not significantly differ between the fat-free and PUFA dietary treatments. Apparently PUFA feeding caused a coordinate decrease in the utilization and production of malonyl-CoA which resulted in no net change in malonyl-CoA pool size. Thus the mechanism by which PUFA suppresses fatty acid synthesis appears to be by coordinately and specifically reducing the amount of carboxylase and fatty acid synthetase.

Biokhimiia. 1981 Jan.
Possible mechanisms of the inhibiting effect of nicotinamide on lipogenesis in rat liver. [Article in Russian]
Mogilevich SE, Velikiĭ NN, Khalmurodov AG.

The activity of some NAD- and NADP-dependent dehydrogenases involved in generation of the reducing equivalents for lipogenesis and the activity and some kinetic parameters of ATP-citrate (pro-3S)-lyase from rat liver, i.e. the enzyme involved in the formation of CoASAc, the primary substrate of fatty acid biosynthesis, were studied. The changes in the activity of NADP-dependent dehydrogenase and ATP-citrate(pro-3S)-lyase, as well as the affinity of the latter for citrate and CoA and the rate of lipogenesis in starved rats and in rats kept on a carbohydrate-rich diet after starvation appeared to be parallel. Nicotinamide decreased the activity of all NADP-dependent dehydrogenases under study, which was especially well-pronounced after nicotinamide addition against increased lipogenesis. The affinity of ATP-citrate(pro-3S)-lyase for citrate and CoA decreased simultaneously with the decrease in the concentration of the latter. These changes can possibly induce the decrease of lipogenesis rate in rat liver after addition of nicotinamide.

Metabolism. 1980 Mar.
The mechanism of adaptive hyperlipogenesis: insulin receptor binding and glucokinase activity in rat liver during fasting and refeeding.
Field J, O'Dea K.

In the present study, rats were fasted for 3 days and subsequently refed for 1, 3, or 5 days. Measurements of insulin binding to its receptors on liver plasma membranes were carried out in conjunction with measurements of the activity of an insulin-regulated enzyme from liver cytosol, glucokinase. In response to the 3-day fast (chronic hypoinsulinemia), the insulin receptor number almost tripled, whereas the glucokinase activity was halved. The insulin receptor number slowly fell to control values during the 5 days of refeeding. In contrast, glucokinase activity rose to levels 2.5 times higher than control (5 times higher than the fasting values) after 1 day of refeeding. Altough the activity fell off somewhat during refeeding it was still dobule control values after 5 days refeeding. It was concluded that in the fasted rat there was a dissociation between insulin receptor concentration and the activity of the insulin-regulated enzyme glucokinase. However, the fasting-induced increase in receptor concentration appeared to play a permissive role in the rapid overshoot of glucokinase activity observed in the early stages of refeeding. Such a scheme would explain the metabolic changes occurring in the fasted-refed rat.

J Nutr. 1980 Feb.
Role of glucocorticoid in adaptive hyperlipogenesis in the rat.
Bouillon DJ, Berdanier CD.
[ Free full text ]

The effect of glucocorticoid (GC) on the enzyme overshoot response to starvation-refeeding (S-R) and on tritium incorporation into lipids was studied. Long-term effects of hypercortisolism on carcass and liver lipids were also determined. In the first series of experiments, intact, adrenalectomized (ADX) and ADX, GC-replaced rats were either ad libitum fed or starved and refed a 65% glucose diet. Glucose-6-phosphate dehydrogenase and malic enzyme activities in both liver and adipose tissue were determined as were the liver and fat pad lipid levels, hepatic and muscle glycogen content, and in vivo incorporation of 3H (from 3HOH, 1 mCi/100 g b.w.) into liver, adipose tissue and plasma lipids. The role for GC in the enzyme overshoot in S-R rats was reaffirmed as was the effect of ADX on enzyme activity and adipose tissue lipid. Hepatic glycogen content was reduced by adrenalectomy and not reversed by GC replacement in the ad libitum-fed animals. S-R reduced liver glycogen in the intact rats, did not affect liver glycogen in ADX-GC replaced rats and increased liver glycogen in the untreated ADX animals. S-R increased hepatic and adipose tissue lipid synthesis as measured by 3H incorporation. This effect was reduced by ADX and the ADX effect was reversed by GC. Prolonged administration of GC had no effect on increasing hepatic or carcass lipid content of ad libitum-fed animals. Results of these experiments suggest that while ineffective in inducing lipogenesis in ad libitum-fed animals, GC plays a role in the lipogenic response to starvation-refeeding and that this effect is apart from its role in the induction of the enzyme overshoot.

Fed Proc. 1979 Nov.
Regulation of fatty acid synthesis.
Donaldson WE.

Acetyl-CoA carboxylase and fatty acid synthetase are the two major enzymes involved in the synthesis of fatty acids in animals. The activities of both enzymes are affected by nutritional manipulations. Although acetyl-CoA carboxylase is considered generally to be the rate-limiting step in lipogenesis, there is evidence that suggests that fatty acid synthetase may become rate limiting under certain conditions. The principal support for the view that acetyl-CoA carboxylase is the rate-limiting enzyme for lipogenesis is that the activity of the enzyme is controlled by allosteric effectors that change the catalytic efficiency of the enzyme. Until recently, the only known control of fatty acid synthetase was through changes in rate of enzyme synthesis. Data are reviewed that show that fatty acid synthetase can exist in forms possessing different catalytic activities. Thus fatty acid synthetase appears to be subject to the type of control necessary for an enzyme to serve as a regulator of the rate of a biological process over a short term.

Eur J Biochem. 1975 May 6.
Modulation of the activity of insulin-dependent enzymes of lipogenesis by glucocorticoids.
Diamant S, Shafrir E.

Administration of triamcinolone or dexamethasone to rats led to a prompt, marked and persistent rise in liver acetyl-CoA carboxylase activity. The activity of fatty acid synthetase increased to a lesser extent and after a more prolonged glucocorticoid treatment, whereas the changes in that of NADP-malate dehydrogenase and ATP-citrate lyase were not appreciable. The overall channeling of [1-14-C]acetyl-CoA to fatty acids was enhanced. The triamcinolone effect on acetyl-CoA carboxylase activity appeared to be dependent on the coincident hyperinsulinemia since it was not obtained in alloxan-diabetic rats, whereas the alanine-aminotransferase-inducing effect of this hormone was additive to that of insulin deficiency. In adipose tissue triamcinolone treatment caused a reduction in the activity of all lipogenesis enzymes and blunted their response to insulin administration. The antagonism of glucocorticoids toward insulin, selectively modulating the responses of the insulin-sensitive enzymes in liver and adipose tissue is discussed. The rise in hepatic lipogenic capacity, through the retention of the ability of insulin to induce acetyl-CoA carboxylase, may be physiologically important in restraining the ketogenesis from acetyl-CoA despite the increased fat utilization during glucocorticoid excess.

Biochim Biophys Acta. 1975 Mar 24.
Hormonal regulation of fatty acid synthetase, acetyl-CoA carboxylase and fatty acid synthesis in mammalian adipose tissue and liver.
Volpe JJ, Marasa JC.

The major objectives of this study were to define the roles of adrenal glucocorticoids and glucagon in the long-term regulation of fatty acid synthetase and acetyl-CoA carboxylase of mammalian adipose tissue and liver. Particular emphasis was given to elucidation of the mechanisms whereby these hormones produce their regulatory effects on enzymatic activity. To dissociate mental manipulation, nutritional conditions were ridgidly controlled in the experiments described. Administration of glucocorticoids to adult rats led to a marked reductionin activities of fatty acid synthetase and carboxylase in adipose in adipose tissue but no change occurred in liver. Adrenalectomy produced an increase in activities of these lipogenic enzymes in adipose tissure, but, again, no change was noted in liver. The decrease in enzymatic activities in adipose tissue with glucocorticoid administration correlated well with a decrease in fatty acid synthesis, determined in vivo by the 3-H2O method. The mechanisms whereby glucocorticoids led to a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the decrease in fatty acid synthetase activity observed in adipose tissue was shown to reflect a decrease in content of enzyme, and not a change in catalytic efficiency. The mechanism underlying the decrease in enzyme content is a decrease in synthesis of the enzyme. The relation of the effects of glucocorticoids to the effects of certain other hormones involved in regulation of lipogenesis was investigated in hypophysectomized and in diabetic animals. Thus, the observation that the glucocorticoid effect on synthetase and carboxylase occurred in adipose tissue of hypophysectomized rats indicated that alterations in levels of other pituitary-regulated hormones were not necessary for the effect. That glucocorticoids play some role in regulation of synthetase and carboxylase in liver, at lease in the diabetic state, was shown by the observation that the low activities of these enzymes in diabetic animals could be restored to normal by adrenalectomy. An even more pronounced restorative effect was apparent in adipose tissue of adrenalectomized, diabetic animals. Administration of glucagon during the refeeding of starved rats resulted in a marked reduction in the induction of fatty acid synthetase, acetyl-CoA carboxylase and in the rate of incorporation of 3-H from 3-H2O into fatty acids in liver, but no change in these parameters occurred in adipose tissue. Administration of theophylline resulted in intermediate reduction in liver. The mechanisms whereby glucagon led tto a decrease in fatty acid synthetase activity were elucidated by the use of immunochemical techniques. Thus, the changes in fatty acid synthetase activity were shown to reflect reductions in content of enzyme. The mechanism underlying these reductions in content is reduced synthesis of enzyme.

Journal of Lipid Research. Jan 1965 Fatty acid synthesis during fat-free refeeding of starved rats.
David W. Allmann , Dorothy D. Hubbard , and David M. Gibson.
Department of Biochemistry, Indiana University School of Medicine, Indianapolis, Indiana.

Refeeding starved rats a fat-free diet over a 48 hr period brings about a marked elevation in the activity of the enzymes in liver cytoplasm which catalyze the synthesis of saturated fatty acids from acetyl CoA and malonyl CoA. Acetate incorporation into palmitoleic and oleic acid is also accelerated during this period. Enhanced capability for the synthesis of these fatty acids is reflected in the net accumulation of saturated and monounsaturated fatty acids, as well as the triglyceride fraction of the liver lipids. Coincident with these events the relative amount of linoleic acid among liver fatty acids rapidly falls. These changes are substantially the same as those observed in early linoleic acid deficiency.