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SCAP plays a central role in controlling lipid homeostasis by activating SREBP-1, a master transcription factor in controlling fatty acid (FA) synthesis. However, how SCAP expression is regulated in human cancer cells remains unknown. Here, we revealed that STAT3 binds to the promoter of SCAP to activate its expression across multiple cancer cell types. Moreover, we identified that STAT3 also concurrently interacts with the promoter of SREBF1 gene (encoding SREBP-1), amplifying its expression. This dual action by STAT3 collaboratively heightens FA synthesis. Pharmacological inhibition of STAT3 significantly reduces the levels of unsaturated FAs and phospholipids bearing unsaturated FA chains by reducing the SCAP-SREBP-1 signaling axis and its downstream effector SCD1. Examination of clinical samples from patients with glioblastoma, the most lethal brain tumor, demonstrates a substantial co-expression of STAT3, SCAP, SREBP-1, and SCD1. These findings unveil STAT3 directly regulates the expression of SCAP and SREBP-1 to promote FA synthesis, ultimately fueling tumor progression., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Membrane biophysical properties are critical to cell fitness and depend on unsaturated phospholipid acyl tails. These can only be produced in aerobic environments since eukaryotic desaturases require molecular oxygen. This raises the question of how cells maintain bilayer properties in anoxic environments. Using advanced microscopy, molecular dynamics simulations, and lipidomics by mass spectrometry we demonstrated the existence of an alternative pathway to regulate membrane fluidity that exploits phospholipid acyl tail length asymmetry, replacing unsaturated species in the membrane lipidome. We show that the fission yeast, Schizosaccharomyces japonicus, which can grow in aerobic and anaerobic conditions, is capable of utilizing this strategy, whereas its sister species, the well-known model organism Schizosaccharomyces pombe, cannot. The incorporation of asymmetric-tailed phospholipids might be a general adaptation to hypoxic environmental niches., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families., Competing Interests: Conflict of interest A. D. N., A. D. U., and C. B. N. are members of the Consejo Nacional de Investigaciones Científicas y Técnicas. The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Browning of adipose tissue is induced by specific stimuli such as cold exposure and consists of up-regulation of thermogenesis in white adipose tissue. Recently, it has emerged as an attractive target for managing obesity in humans. Here, we performed a comprehensive analysis to identify genes associated with browning in murine adipose tissue. We focused on glycerol kinase (GYK) because its mRNA expression pattern is highly correlated with that of uncoupling protein 1 (UCP1), which regulates the thermogenic capacity of adipocytes. Cold exposure-induced Ucp1 up-regulation in inguinal white adipose tissue (iWAT) was partially abolished by Gyk knockdown (KD) in vivo Consistently, the Gyk KD inhibited Ucp1 expression induced by treatment with the β-adrenergic receptors (βAR) agonist isoproterenol (Iso) in vitro and resulted in impaired uncoupled respiration. Gyk KD also suppressed Iso- and adenylate cyclase activator-induced transcriptional activation and phosphorylation of the cAMP response element-binding protein (CREB). However, we did not observe these effects with a cAMP analog. Therefore Gyk KD related to Iso-induced cAMP products. In Iso-treated Gyk KD adipocytes, stearoyl-CoA desaturase 1 (SCD1) was up-regulated, and monounsaturated fatty acids such as palmitoleic acid (POA) accumulated. Moreover, a SCD1 inhibitor treatment recovered the Gyk KD-induced Ucp1 down-regulation and POA treatment down-regulated Iso-activated Ucp1 Our findings suggest that Gyk stimulates Ucp1 expression via a mechanism that partially depends on the βAR-cAMP-CREB pathway and Gyk -mediated regulation of fatty acid metabolism., (© 2020 Iwase et al.)

Increased carbohydrate consumption increases hepatic de novo lipogenesis, which has been linked to the development of chronic metabolic diseases, including obesity, hepatic steatosis, and insulin resistance. Stearoyl CoA desaturase 1 (SCD1) is a critical lipogenic enzyme that catalyzes the synthesis of two monounsaturated fatty acids, oleate and palmitoleate, from the saturated fatty acids stearate and palmitate, respectively. SCD1-deficient mouse models are protected against diet-induced adiposity, hepatic steatosis, and hyperglycemia. However, the mechanism of this protection by SCD1 deficiency is unclear. Using liver-specific SCD1 knockout (LKO) mice fed a high-carbohydrate, low-fat diet, we show that hepatic SCD1 deficiency increases systemic glucose uptake. Hepatic SCD1 deficiency enhanced glucose transporter type 1 (GLUT1) expression in the liver and also up-regulated GLUT4 and adiponectin expression in adipose tissue. The enhanced glucose uptake correlated with increased liver expression and elevated plasma levels of fibroblast growth factor 21 (FGF21), a hepatokine known to increase systemic insulin sensitivity and regulate whole-body lipid metabolism. Feeding LKO mice a triolein-supplemented but not tristearin-supplemented high-carbohydrate, low-fat diet reduced FGF21 expression and plasma levels. Consistently, SCD1 inhibition in primary hepatocytes induced FGF21 expression, which was repressed by treatment with oleate but not palmitoleate. Moreover, deletion of the transcriptional coactivator PPARγ coactivator 1α (PGC-1α) reduced hepatic and plasma FGF21 and white adipocyte tissue-specific GLUT4 expression and raised plasma glucose levels in LKO mice. These results suggest that hepatic oleate regulates glucose uptake in adipose tissue either directly or partially by modulating the hepatic PGC-1α-FGF21 axis., (© 2019 Aljohani et al.)

The skin is the single largest organ in humans, serving as a major barrier to infection, water loss, and abrasion. The functional diversity of skin requires the synthesis of large amounts of lipids, such as triglycerides, wax esters, squalene, ceramides, free cholesterol, free fatty acids, and cholesterol and retinyl esters. Some of these lipids are used as cell membrane components, signaling molecules, and a source of energy. An important class of lipid metabolism enzymes expressed in skin is the Δ(9)-desaturases, which catalyze the synthesis in Δ(9)-monounsaturated lipids, primarily oleoyl-CoA (18:1n-9) and palmitoyl-CoA (16:1n-7), the major monounsaturated fatty acids in cutaneous lipids. Mice with a deletion of the Δ(9)-desaturase-1 isoform (SCD1) either globally (Scd1(-/-)) or specifically in the skin (skin-specific Scd1-knockout; SKO) present with marked changes in cutaneous lipids and skin integrity. Interestingly, these mice also exhibit increased whole body energy expenditure, protection against diet-induced adiposity, hepatic steatosis, and glucose intolerance. The increased energy expenditure in skin-specific Scd1-knockout (SKO) mice is a surprising phenotype, as it links cutaneous lipid homeostasis with whole body energy balance. This minireview summarizes the role of skin SCD1 in regulating skin integrity and whole body energy homeostasis and offers a discussion of potential pathways that may connect these seemingly disparate phenotypes.

Krüppel-like factor 15 (KLF15), a member of the Krüppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15 Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic β cells mediated through modulation of oxidative stress.

Vascular calcification is recognized as an independent predictor of cardiovascular mortality, particularly in subjects with chronic kidney disease. However, the pathways by which dysregulation of lipid and mineral metabolism simultaneously occur in this particular population remain unclear. We have shown that activation of the farnesoid X receptor (FXR) blocks mineralization of bovine calcifying vascular cells (CVCs) and in ApoE knock-out mice with 5/6 nephrectomy. In contrast to FXR, this study showed that liver X receptor (LXR) activation by LXR agonists and adenovirus-mediated LXR overexpression by VP16-LXRα and VP16-LXRβ accelerated mineralization of CVCs. Conversely, LXR inhibition by dominant negative (DN) forms of LXRα and LXRβ reduced calcium content in CVCs. The regulation of mineralization by FXR and LXR agonists was highly correlated with changes in lipid accumulation, fatty acid synthesis, and the expression of sterol regulatory element binding protein-1 (SREBP-1). The rate of lipogenesis in CVCs through the SREBP-1c dependent pathway was reduced by FXR activation, but increased by LXR activation. SREBP-1c overexpression augmented mineralization in CVCs, whereas SREBP-1c DN inhibited alkaline phosphatase activity and mineralization induced by LXR agonists. LXR and SREBP-1c activations increased, whereas FXR activation decreased, saturated and monounsaturated fatty acids derived from lipogenesis. In addition, we found that stearate markedly promoted mineralization of CVCs as compared with other fatty acids. Furthermore, inhibition of either acetyl-CoA carboxylase or acyl-CoA synthetase reduced mineralization of CVCs, whereas inhibition of stearoyl-CoA desaturase induced mineralization. Therefore, a stearate metabolite derived from lipogenesis might be a risk factor for the development of vascular calcification.

The endoplasmic reticulum-associated NADH cytochrome b(5) oxidoreductase (Ncb5or) is widely distributed in animal tissues. Ncb5or(-/-) mice develop diabetes at age 7 weeks and have increased susceptibility to the diabetogenic oxidant streptozotocin. Ncb5or deficiency also results in lipoatrophy and increased hepatocyte sensitivity to cytotoxic effects of saturated fatty acids. Here we investigate the mechanisms of these phenomena in prediabetic Ncb5or(-/-) mice and find that, despite increased rates of fatty acid uptake and synthesis and higher stearoyl-CoA desaturase (SCD) expression, Ncb5or(-/-) liver accumulates less triacylglycerol (TAG) than wild type (WT). Increased fatty acid catabolism and oxidative stress are evident in Ncb5or(-/-) hepatocytes and reflect increased mitochondrial content, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) expression, fatty acid oxidation rates, oxidative stress response gene expression, and oxidized glutathione content. Ncb5or(-/-) hepatocytes readily incorporate exogenous fatty acids into TAG but accumulate more free fatty acids (FFA) and have greater palmitate-induced oxidative stress responses and cell death than WT, all of which are alleviated by co-incubation with oleate via TAG channeling. A high fat diet rich in palmitate and oleate stimulates both lipogenesis and fatty acid catabolism in Ncb5or(-/-) liver, resulting in TAG levels similar to WT but increased intracellular FFA accumulation. Hepatic SCD-specific activity is lower in Ncb5or(-/-) than in WT mice, although Ncb5or(-/-) liver has a greater increase in Scd1 mRNA and protein levels. Together, these findings suggest that increased FFA accumulation and catabolism and oxidative stress are major consequences of Ncb5or deficiency in liver.

De novo lipogenesis (DNL) is paradoxically up-regulated by its end product, saturated fatty acids (SAFAs). We tested the hypothesis that SAFA-induced up-regulation of DNL reflects coordinate up-regulation of elongation and desaturation pathways for disposal of SAFAs and production of monounsaturated fatty acids to protect cells from SAFA toxicity. Human preadipocytes were differentiated in vitro for 14 days with [U-(13)C]palmitate (0-200 microM) to distinguish exogenous fatty acids from those synthesized by DNL. Exogenous palmitate up-regulated DNL (p < 0.001) concomitantly with SCD and elongation (each p < 0.001). Adipocytes from some donors were intolerant to high palmitate concentrations (400 microM). Palmitate-intolerant cells showed lower TG accumulation. They had lower expression of SCD mRNA and less monounsaturated fatty acids in TG, emphasizing the importance of desaturation for dealing with exogenous SAFAs. There was greater [U-(13)C]palmitate incorporation in phospholipids. SCD knockdown with small interfering RNA caused down-regulation of DNL and of expression of DNL-related genes, with reduced membrane fluidity (p < 0.02) and insulin sensitivity (p < 0.01), compared with scrambled small interfering RNA controls. There was preferential channeling of DNL-derived versus exogenous palmitate into elongation and of DNL-derived versus exogenous stearate into desaturation. DNL may not act primarily to increase fat stores but may serve as a key regulator, in tandem with elongation and desaturation, to maintain cell membrane fluidity and insulin sensitivity within the human adipocyte.

Stearoyl-CoA desaturase-1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids and is an important regulator of whole body energy homeostasis. Severe cutaneous changes in mice globally deficient in SCD1 also indicate a role for SCD1 in maintaining skin lipids. We have generated mice with a skin-specific deletion of SCD1 (SKO) and report here that SKO mice display marked sebaceous gland hypoplasia and depletion of sebaceous lipids. In addition, SKO mice have significantly increased energy expenditure and are protected from high fat diet-induced obesity, thereby recapitulating the hypermetabolic phenotype of global SCD1 deficiency. Genes of fat oxidation, lipolysis, and thermogenesis, including uncoupling proteins and peroxisome proliferator-activated receptor-gamma co-activator-1alpha, are up-regulated in peripheral tissues of SKO mice. However, unlike mice globally deficient in SCD1, SKO mice have an intact hepatic lipogenic response to acute high carbohydrate feeding. Despite increased basal thermogenesis, SKO mice display severe cold intolerance because of rapid depletion of fuel substrates, including hepatic glycogen, to maintain core body temperature. These data collectively indicate that SKO mice have increased cold perception because of loss of insulating factors in the skin. This results in up-regulation of thermogenic processes for temperature maintenance at the expense of fuel economy, illustrating cross-talk between the skin and peripheral tissues in maintaining energy homeostasis.

The mRNA expression of lipogenic genes Scd-1 and Fas is regulated partly by the insulin-sensitive transcription factor SREBP-1c and liver X receptor alpha (LXRalpha). Compared with normal mice, the increase in the mRNA expression of hepatic Scd-1, Fas, and Srebp-1c was severely attenuated in peroxisome proliferator-activated receptor alpha (PPARalpha)-deficient mice during the transition from the starved to the re-fed states. The concentration of the membrane-bound form of SREBP-1c was also lower in the livers of the PPARalpha-deficient mice during re-feeding but there was little difference in the concentration of the active, nuclear form, or in the abundance of Insig-2a mRNA. The response of plasma insulin to starvation and re-feeding was normal in the PPARalpha-deficient mice. Rat hepatocytes transfected with an adenovirus encoding a dominant negative form of PPARalpha were resistant to the stimulatory effects of insulin on Fas and Scd-1 mRNA expression in vitro. When LXRalpha was activated in vivo by inclusion of a non-steroidal ligand in the diet, the expression of the mRNA for hepatic Srebp-1c, Fas, and Scd-1 was increased severalfold in mice of both genotypes and resistance associated with PPARalpha deficiency was abolished during re-feeding. However, although re-feeding the LXRalpha ligand induced the immature form of SREBP-1c equally in the livers of both genotypes, the concentration of the nuclear form remained relatively low in the livers of the PPARalpha-deficient mice. We conclude that intact PPARalpha is required to mediate the response of Scd-1 and Fas gene expression to insulin and that this is normally achieved directly by activation of LXRalpha.

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells.

c-Jun is a transcription factor activated by phosphorylation by the stress-activated protein kinase/c-Jun N-terminal kinase pathway in response to extracellular signals and cytokines. We show that adenovirus-mediated gene transfer of the dominant negative form of c-Jun (dn-c-Jun) in C57BL/6 mice increased greatly apoE hepatic mRNA and plasma levels, increased plasma cholesterol, triglyceride, and very low density lipoprotein levels, and resulted in the accumulation of discoidal high density lipoprotein particles. A similar but more severe phenotype was generated by overexpression of the mouse apoE in C57BL/6 mice, suggesting that dyslipidemia induced by dn-c-Jun was the result of apoE overexpression. Unexpectedly, infection of apoE(-/-) mice with adenovirus expressing dn-c-Jun reduced plasma cholesterol by 70%, suggesting that dn-c-Jun affected other genes that control plasma cholesterol levels. To identify these genes, we performed whole genome expression analysis (34,000 genes) of isolated livers from two groups of five apoE(-/-) mice, infected with adenoviruses expressing either the dn-c-Jun or the green fluorescence protein. Bioinformatic analysis and Northern blotting validation revealed that dn-c-Jun increased 40-fold the apoE mRNA and reduced by 70% the Scd-1 (stearoyl-CoA-desaturase 1) mRNA. The involvement of Scd-1 in lowering plasma cholesterol was confirmed by restoration of high cholesterol levels of apoE(-/-) mice following coinfection with adenoviruses expressing dn-c-Jun and Scd-1. In conclusion, dn-c-Jun appears to trigger two opposing events in mice that affect plasma cholesterol and triglyceride levels as follows: one results in apoE overexpression and triggers dyslipidemia and the other results in inhibition of Scd-1 and offsets dyslipidemia.

Dietary saturated fats have often been implicated in the promotion of obesity and related disorders. It has been shown recently that saturated fats act through the transcription factor SREBP-1c (sterol regulatory element-binding protein-1c) and its requisite coactivator, peroxisome proliferator-activated receptor-gamma coactivator-1beta (PGC-1beta), to exert their pro-lipogenic effects. We show here that a diet high in the saturated fat stearate induces lipogenic genes in wild-type mice, with the induction of the Scd1 (stearoyl-CoA desaturase-1) gene preceding that of other lipogenic genes. However, in Scd1-/- mice, stearate does not induce lipogenesis, and Srebp-1c and Pgc-1beta levels are markedly reduced. Instead, genes of fatty acid oxidation such as Cpt-1 (carnitine palmitoyltransferase-1) as well as Pgc-1alpha are induced. Mitochondrial fatty acid oxidation is increased, and white adipose tissue and hepatic glycogen stores are depleted in stearate-fed Scd1-/- mice. Furthermore, AMP-activated protein kinase is also induced by stearate feeding in Scd1-/- mice. These results indicate that the desaturation of saturated fats such as stearate by SCD is an essential step mediating their induction of lipogenesis. In the absence of SCD1, stearate promotes oxidation, leading to protection from saturated fat-induced obesity. SCD1 thus serves as a molecular switch in the promotion or prevention of lipid-induced disorders brought on by consumption of excess saturated fat.

Stearoyl-CoA desaturase catalyzes the rate-limiting step in the biosynthesis of monounsaturated fatty acids, which are required for normal rates of synthesis of triglycerides, cholesterol esters, and phospholipids. Mice with a targeted disruption of the stearoyl-CoA desaturase 1 (SCD1) isoform are protected against diet and leptin deficiency-induced adiposity, have increased energy expenditure, and have up-regulated expression of hepatic genes encoding enzymes of fatty acid beta-oxidation. Because peroxisome proliferator-activated receptor-alpha (PPARalpha) is a key transcription factor that induces the transcription of fatty acid beta-oxidation and thermogenic genes, we hypothesized that the increased fatty acid oxidation observed in SCD1 deficiency is dependent on activation of the PPARalpha pathway. Here we show that mice nullizygous for SCD1 and PPARalpha are still protected against adiposity, have increased energy expenditure, and maintain high expression of PPARalpha target genes in the liver and brown adipose tissue. The SCD1 deficiency rescued hepatic steatosis of the PPARalpha(-/-) mice. The SCD1 mutation increased the phosphorylation of both AMP-activated protein kinase and acetyl-CoA carboxylase, thereby increasing CPT activity and stimulating the oxidation of liver palmitoyl-CoA in the PPARalpha null mice. The findings indicate that the reduced adiposity, reduced liver steatosis, increased energy expenditure, and increased expression of PPARalpha target genes associated with SCD1 deficiency are independent of activation of the PPARalpha pathway.

Stearoyl-CoA desaturase (SCD) synthesizes oleate necessary for the biosynthesis of triglycerides and other lipids. Mice with a targeted disruption of the SCD1 gene are deficient in tissue oleate and have reduced expression of the sterol regulatory element-binding protein (SREBP) and its target genes. The SREBP-1c isoform is a known mediator of insulin action on hepatic gene expression, but its transcriptional effects due to glucose or fructose are still unclear. We found that fructose compared with glucose is a stronger inducer of SREBP-1c and lipogenic gene expression, causing a dramatic increase in hepatic triglyceride levels. However, when fed to the SCD1-/- mice, fructose failed to induce SREBP-1 or lipogenic genes and the triglyceride levels were not increased. Instead fructose feeding caused a decrease in hepatic glycogen and plasma glucose levels. The induction of SREBP-1 and lipogenic gene expression as well as the levels of liver triglycerides, glycogen, and plasma glucose was partially restored when the fructose diet was supplemented with very high levels of oleate (20% by weight) but not with palmitate, stearate, or linoleate. Fructose in a long term feeding induced the expression of SCD1 and that of other lipogenic genes in the liver of SREBP-1c-/- mice, and a further increase in expression of these genes occurred when the fructose diet was supplemented with oleate. Our observations demonstrated that oleate produced by SCD is necessary for fructose-mediated induction of lipogenic gene expression through SREBP-1c-dependent and -independent mechanisms and suggested that SCD1 gene expression is important in lipid and carbohydrate homeostasis.

Liver X receptor/retinoid X receptor (LXR/RXR) transcription factors have been found to induce a number of genes involved in the regulation of cellular cholesterol efflux, including the ATP-binding cassette transporter A1 (ABCA1), which mediates the active efflux of cellular cholesterol and phospholipids to extracellular acceptors, such as apolipoprotein A-I (apoA-I). In a screen for macrophage LXR/RXR target genes, we identified stearoyl-CoA desaturases 1 and 2 (Scd1 and Scd2), and subsequently tested the hypothesis that SCD activity might modulate cellular cholesterol efflux. In HEK 293 cells co-transfection of ABCA1 with either SCD1 or SCD2 inhibited ABCA1-mediated cholesterol efflux but not phospholipid efflux. In Chinese hamster ovary (CHO) cells with moderate stable overexpression of SCD1, cholesterol efflux to apoA-I was inhibited by 73%, whereas phospholipid efflux and ABCA1 protein levels were unchanged. In contrast, cholesterol efflux to HDL(2), which is not dependent on ABCA1, was increased 2-fold in CHO-SCD1 cells. The effect of SCD on cholesterol efflux to apoA-I was independent of acyl-CoA:cholesterol acyltransferase (ACAT) activity. SCD activity led to an increased content of plasma membrane monounsaturated fatty acids (18:1) at the expense of saturated fatty acids (18:0). As shown by confocal microscopy, SCD overexpression led to a decrease of Triton X-100-resistant domains in the plasma membrane, indicating a decrease in membrane-ordered regions. The data suggest that SCD changes membrane organization and depletes a specific pool of membrane cholesterol supporting ABCA1-mediated efflux, whereas increasing availability of cholesterol for passive efflux by HDL(2). ABCA1-mediated cholesterol and phospholipid efflux may be uncoupled in pathological states associated with high SCD activity, as in hyperinsulinemic obese mice, or in animals treated with LXR activators.

The regulation of stearoyl-CoA desaturase (SCD), a rate-limiting enzyme in the synthesis of unsaturated fatty acids, is physiologically important because the ratio of saturated to unsaturated fatty acids is thought to control cellular functions by modulating the structural integrity and fluidity of cell membranes. Transforming growth factor-beta (TGF-beta), a multifunctional cytokine, increased SCD mRNA expression in cultured human retinal pigment epithelial cells. This response was elicited by all three TGF-beta isoforms, beta1, beta2, and beta3. However, SCD mRNA expression was not increased either by other members of the TGF-beta family or by other growth factors or cytokines. TGF-beta also increased SCD mRNA expression in several other cell lines tested. The increase in SCD mRNA expression was preceded by a marked increase in Smad2 phosphorylation in TGF-beta-treated human retinal pigment epithelial cells. TGF-beta did not induce SCD mRNA expression in a Smad4-deficient cell line. However, Smad4 overexpression restored the TGF-beta effect in this cell line. Moreover, TGF-beta-induced SCD mRNA expression was effectively blocked by the overexpression of Smad7, an inhibitory Smad. Thus, a TGF-beta signal transduction pathway involving Smad proteins appears to regulate the cellular expression of the SCD gene, and this regulation may play an important role in lipid metabolism.

1-Alkyl-2,3-diacylglycerol (ADG) is a unique neutral lipid found in the eyeball-associated Harderian gland (HG) of the mouse and acts as a lubricant to facilitate eyelid movement. We found that the HG of the mice with a disruption in the gene for stearoyl-CoA desaturase 1 (SCD1) (SCD1-/-) is deficient in ADG. The amount of C20:1n-9, which is a major fatty acid of ADG, was reduced by greater than 90% despite normal elongase enzyme activity proposed to elongate it from C18:1n-9. HG from SCD1-/- mice exhibited high desaturase activity toward C16:0-CoA as substrate but had very low desaturase activity toward C18:0-CoA. Feeding diets containing high levels of oleate to the SCD1-/- mice did not increase the levels of C18:1n-9 or C20:1n-9 in the HG and failed to restore the ADG to the levels found in the HG of the wild-type mouse. De novo ADG synthesis as measured by the incorporation of [(3)H]glycerol and [(14)C]glucose was high in the SCD1+/+ mouse but was reduced by greater than 90% in the HG of SCD1-/- mouse. The deficiencies in the levels of ADG and C20:1n-9 were not compensated for by the expression of SCD2 and SCD3 isoforms in the HG of the SCD1-/- mouse. These observations demonstrate that SCD1-synthesized oleoyl-CoA is a major substrate required for the biosynthesis of normal levels of ADG and that the SCD isoforms present in the HG have different substrate specificity.

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose- and time-dependent manner; an approximately 7-fold increase was observed with 1 microm all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR alpha-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR alpha in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RAR-independent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters. Two well characterized isoforms of SCD, SCD1 and SCD2, exist in the mouse. Most mouse tissues express SCD1 and 2 with the exception of the liver, which expresses mainly the SCD1 isoform. We found that asebia mice homozygous for a natural mutation of the gene for SCD1 (SCD-/-) are deficient in hepatic cholesterol esters and triglycerides despite the presence of normal activities of acyl-CoA:cholesterol acyltransferase and glycerol phosphate acyltransferase, the enzymes responsible for cholesterol ester and triglyceride synthesis, respectively, in the liver of these mice. Feeding diets supplemented with triolein or tripalmitolein to the SCD-/- mice resulted in an increase in the levels of 16:1 and 18:1 in the liver but failed to restore the 18:1 and 16:1 levels of the cholesterol ester and triglycerides to the levels found in normal mice. The SCD-/- mouse had very low levels of triglycerides in the VLDL and LDL lipoprotein fractions compared with the normal animal. Transient transfection of an SCD1 expression vector into Chinese hamster ovary cells resulted in increased SCD activity and esterification of cholesterol to cholesterol esters. Taken together, our observations demonstrate that the oleoyl-CoA and palmitoleyl-CoA produced by SCD1 are necessary to synthesize enough cholesterol esters and triglycerides in the liver and suggest that regulation of SCD1 activity plays an important role in mechanisms of cellular cholesterol homeostasis.

To elucidate the physiological role of sterol regulatory element-binding protein-1 (SREBP-1), the hepatic mRNA levels of genes encoding various lipogenic enzymes were estimated in SREBP-1 gene knockout mice after a fasting-refeeding treatment, which is an established dietary manipulation for the induction of lipogenic enzymes. In the fasted state, the mRNA levels of all lipogenic enzymes were consistently low in both wild-type and SREBP-1(-/-) mice. However, the absence of SREBP-1 severely impaired the marked induction of hepatic mRNAs of fatty acid synthetic genes, such as acetyl-CoA carboxylase, fatty acid synthase, and stearoyl-CoA desaturase, that was observed upon refeeding in the wild-type mice. Furthermore, the refeeding responses of other lipogenic enzymes, glycerol-3-phosphate acyltransferase, ATP citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase, and S14 mRNAs, were completely abolished in SREBP-1(-/-) mice. In contrast, mRNA levels for cholesterol biosynthetic genes were elevated in the refed SREBP-1(-/-) livers accompanied by an increase in nuclear SREBP-2 protein. When fed a high carbohydrate diet for 14 days, the mRNA levels for these lipogenic enzymes were also strikingly lower in SREBP-1(-/-) mice than those in wild-type mice. These data demonstrate that SREBP-1 plays a crucial role in the induction of lipogenesis but not cholesterol biosynthesis in liver when excess energy by carbohydrates is consumed.

Dietary polyunsaturated fatty acids (PUFA) are negative regulators of hepatic lipogenesis that exert their effects primarily at the level of transcription. Sterol regulatory element-binding proteins (SREBPs) are transcription factors responsible for the regulation of cholesterol, fatty acid, and triglyceride synthesis. In particular, SREBP-1 is known to play a crucial role in the regulation of lipogenic gene expression in the liver. To explore the possible involvement of SREBP-1 in the suppression of hepatic lipogenesis by PUFA, we challenged wild-type mice and transgenic mice overexpressing a mature form of SREBP-1 in the liver with dietary PUFA. In the liver of wild-type mice, dietary PUFA drastically decreased the mature, cleaved form of SREBP-1 protein in the nucleus, whereas the precursor, uncleaved form in the membranes was not suppressed. The decreases in mature SREBP-1 paralleled those in mRNAs for lipogenic enzymes such as fatty acid synthase and acetyl-CoA carboxylase. In the transgenic mice, dietary PUFA did not reduce the amount of transgenic SREBP-1 protein, excluding the possibility that PUFA accelerated the degradation of mature SREBP-1. The resulting sustained expression of mature SREBP-1 almost completely canceled the suppression of lipogenic gene expression by PUFA in the SREBP-1 transgenic mice. These results demonstrate that the suppressive effect of PUFA on lipogenic enzyme genes in the liver is caused by a decrease in the mature form of SREBP-1 protein, which is presumably due to the reduced cleavage of SREBP-1 precursor protein.

We previously identified stearoyl-CoA desaturase 2 (SCD2) as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear sterol regulatory element binding proteins (SREBPs) or adipocyte determination and differentiation factor 1 (ADD1). A novel sterol regulatory element (SRE) (5'-AGCAGATTGTG-3') identified in the proximal promoter of the mouse SCD2 gene is required for induction of SCD2 promoter-reporter genes in response to cellular sterol depletion (Tabor, D. E., Kim, J. B., Spiegelman, B. M., and Edwards, P. A. (1998) J. Biol. Chem. 273, 22052-22058). In this report, we demonstrate that this novel SRE is both present in the promoter of the SCD1 gene and is critical for the sterol-dependent transcription of SCD1 promoter-reporter genes. Two conserved cis elements (5'-CCAAT-3') lie 5 and 48 base pairs 3' of the novel SREs in the promoters of both the SCD1 and SCD2 murine genes. Mutation of either of these putative NF-Y binding sites attenuates the transcriptional activation of SCD1 or SCD2 promoter-reporter genes in response to cellular sterol deprivation. Induction of both reporter genes is also attenuated when cells are cotransfected with dominant-negative forms of either NF-Y or SREBP. In addition, we demonstrate that the induction of SCD1 and SCD2 mRNAs that occurs during the differentiation of 3T3-L1 preadipocytes to adipocytes is paralleled by an increase in the levels of ADD1/SREBP-1c and that the SCD1 and SCD2 mRNAs are induced to even higher levels in response to ectopic expression of ADD1/SREBP-1c. We conclude that transcription of both SCD1 and SCD2 genes is responsive to cellular sterol levels and to the levels of nuclear SREBP/ADD1 and that transcriptional induction requires three spatially conserved cis elements, that bind SREBP and NF-Y. Additional studies demonstrate that maximal transcriptional repression of SCD2 reporter genes in response to an exogenous polyunsaturated fatty acid is dependent upon the SRE and the adjacent CCAAT motif.

Three sterol regulatory element-binding proteins (SREBP-1a, -1c, and -2) stimulate transcription of genes involved in synthesis and receptor-mediated uptake of cholesterol and fatty acids. Here, we explore the individual roles of each SREBP by preparing lines of Chinese hamster ovary (CHO) cells that express graded amounts of nuclear forms of each SREBP (designated nSREBPs) under control of a muristerone-inducible nuclear receptor system. The parental hamster cell line (M19 cells) lacks its own nSREBPs, owing to a deletion in the gene encoding the Site-2 protease, which releases nSREBPs from cell membranes. By varying the concentration of muristerone, we obtained graded expression of individual nSREBPs in the range that restored lipid synthesis to near physiologic levels. The results show that nSREBP-2 produces a higher ratio of synthesis of cholesterol over fatty acids than does nSREBP-1a. This is due in part to a selective ability of low levels of nSREBP-2, but not nSREBP-1a, to activate the promoter for squalene synthase. nSREBP-1a and -2 both activate transcription of the genes encoding stearoyl-CoA desaturase-1 and -2, thereby markedly enhancing the production of monounsaturated fatty acids. nSREBP-1c was inactive in stimulating any transcription at the concentrations achieved in these studies. The current data support the emerging view that the nSREBPs act in complementary ways to modulate the lipid composition of cell membranes.

To identify genes that are transcriptionally activated by sterol regulatory element-binding proteins (SREBPs), we utilized mRNA differential display and mutant cells that express either high or low levels of transcriptionally active SREBP. This approach identified stearoyl-CoA desaturase 2 (SCD2) as a new SREBP-regulated gene. Cells were transiently transfected with reporter genes under the control of different fragments of the mouse SCD2 promoter. Constructs containing >199 base pairs of the SCD2 proximal promoter were activated following incubation of cells in sterol-depleted medium or as a result of co-expression of SREBP-1a, SREBP-2, or rat adipocyte determination and differentiation factor 1 (ADD1). Electromobility shift assays and DNase I footprint analysis demonstrated that recombinant SREBP-1a bound to a novel cis element (5'-AGCAGATTGTG-3') in the proximal promoter of the SCD2 gene. The finding that the endogenous SCD2 mRNA levels were induced when wild-type Chinese hamster ovary fibroblasts were incubated in sterol-deficient medium is consistent with a role for SREBP in regulating transcription of the gene. These studies identify SCD2 as a new member of the family of genes that are transcriptionally regulated in response to changing levels of nuclear SREBP/ADD1. In addition, the sterol regulatory element in the SCD2 promoter is distinct from all previously characterized motifs that confer SREBP- and ADD1-dependent transcriptional activation.

The effects of arachidonic acid (20:4, n-6) and other fatty acids on the expression of stearoyl-CoA desaturase gene 1 were investigated in fully differentiated 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with arachidonic acid resulted in a decrease in stearoyl-CoA desaturase (Scd) enzyme activity and scd1 mRNA. Arachidonic acid did not alter the transcription of the scd1 gene, whereas the half-life of the scd1 mRNA was reduced from 25.1 to 8.5 h. Blocking the conversion of arachidonic acid to eicosanoids by pretreatment of the cells with cyclooxygenase, lipoxygenase, or cytochrome P-450 epoxygenase inhibitors did not reverse the inhibition caused by arachidonic acid, indicating that eicosanoid synthesis is not necessary for the repression of scd1 mRNA expression. Treatment of adipocytes with linoleic (18:2, n-6) and linolenic (18:3, n-3) acids also resulted in inhibition of scd1 mRNA accumulation. By contrast, oleic acid (18:1, n-9) and stearic acid (18:0) had no effect on scd1 mRNA levels. Taken together, these results suggest that polyunsaturated fatty acids repress the expression of the scd1 gene in mature adipocytes by reducing the stability of scd1 mRNA.

Stearoyl-CoA desaturase gene 1 (scd1) mRNA levels were induced during the 1st day of 3T3-L1 differentiation. scd1 expression had previously been observed only in late-stage differentiation, during the period of triacylglycerol accumulation. The induction of 3T3-L1 differentiation requires treatment with insulin, dexamethasone, and an agent that increases intracellular cAMP levels. Treatment of preadipocytes with cAMP-elevating agents caused an increase in scd1 mRNA concentrations. Insulin and dexamethasone had no effect on scd1 mRNA expression in preadipocytes. The increase in mRNA resulted from transcriptional activation of the scd1 gene. 8-Bromo-cAMP treatment of differentiated adipocytes had no effect on scd1 mRNA levels, suggesting a preadipocyte-specific effect. The increase in scd1 mRNA occurred maximally after 6 h of cAMP treatment and was shown to require protein synthesis. Deletion and mutagenesis analyses have localized the cAMP response to sequence within the first 250 base pairs of the 5'-flanking region of the scd1 gene. Treatment with phorbol ester enhanced the induction of scd1 mRNA by cAMP, suggesting the involvement of AP-2 as a mediator of the cAMP response. The induction of scd1 expression by cAMP during early differentiation was distinct from that observed during late adipocyte development. This early expression presents a novel regulated function of scd1 during the differentiation process, independent of its lipogenic role in late adipocyte development. The induction of scd1 also introduces a role for cAMP in 3T3-L1 differentiation.

The Saccharomyces cerevisiae OLE1 gene encodes the Delta-9 fatty acid desaturase, a highly regulated integral membrane enzyme involved in the formation of unsaturated fatty acids from saturated acyl-coenzyme A precursors. The mRNA levels of the OLE1 gene are regulated by at least two independent control systems that respond to nutrient fatty acids. One involves the unsaturated fatty acid repression of OLE1 transcription; the second, described in this report, involves unsaturated fatty acid-responsive changes in the half-life of the OLE1 mRNA. Measurements of OLE1 mRNA half-life indicate that it is a moderately stable species (t1/2 = 10 +/- 1.5 min) in cells grown in medium without exogenous fatty acids. Its half-life is drastically reduced (t1/2 < 2.5 min), in a time-dependent manner, following the addition of unsaturated fatty acids to the growth medium. Saturated fatty acids that have previously been shown to increase activation of OLE1 transcription do not regulate its mRNA stability. Inhibition of translation, by the addition of cycloheximide, slows the nucleolytic degradation of the OLE1 mRNA and blocks the unsaturated fatty acid-triggered reduction in its half-life. This suggests an intimate link between the two processes of mRNA decay and protein synthesis. A chimeric mRNA, produced by replacing the upstream activation and fatty acid-regulated regions of the OLE1 promoter with the GAL1 promoter sequences is destabilized by exogenous unsaturated fatty acids. A similar chimera under GAL1 control that replaces the OLE1 mRNA 5'-untranslated region with GAL1 sequences is not regulated by unsaturated fatty acids. These results suggest that the 5'-untranslated region of the OLE1 mRNA contains sequence elements required for fatty acid-triggered destabilization. Disruption of the XRN1 gene, which encodes a 5' --> 3'-exoribonuclease, results in an approximate 4-fold increase in OLE1 mRNA half-life in the absence of fatty acids. Its half-life is reduced when those cells are exposed to unsaturated fatty acids, indicating that the 5'-exoribonuclease encoded by the XRN1 gene is required for the rapid degradation of the OLE1 transcript but is not required for fatty acid-induced destabilization.

The transcription and mRNA levels of murine liver stearoyl-CoA desaturase 1 (SCD1) are induced 11- and 45-fold, respectively, by feeding fasted normal mice with a fat-free, high carbohydrate diet (Ntambi, J. M. (1992) J. Biol. Chem. 267, 10925-10930). In this study, we used streptozotocin-induced diabetic mice to study the regulatory role of carbohydrate and insulin on expression of the SCD1 gene in liver. Fructose administration to fasted diabetic mice induced a 2-fold increase in SCD1 mRNA within 6 h and a 23-fold increase within 24 h. Similarly, insulin administration to diabetic mice induced SCD1 mRNA from 4-fold within 4 h to 22-fold within 24 h. Insulin plus fructose, however, achieved full induction, with a 45-fold increase of SCD1 mRNA and a 10-fold increase in SCD1 transcription within 24 h. Additionally, the effect of insulin on SCD1 mRNA was inhibited 75% with dibutyryl-cAMP and theophylline administration and 70% by cycloheximide administration. Synthesis of liver albumin mRNA showed little change upon dietary manipulation or insulin treatment. Our data demonstrate that insulin and dietary fructose or a metabolite of fructose positively regulate the expression of the SCD1 gene in mouse liver.

Expression of the murine liver stearoyl-CoA desaturase gene (SCD1) is induced upon feeding fasted mice a fat-free, high carbohydrate diet (Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., and Lane, M.D. (1988) J. Biol. Chem. 263, 17291-17300). In the present study, SCD1-specific RNA riboprobes and cDNA probes were used to study the mechanism for the induction of SCD1 mRNA. Based on the time course of induction, the SCD1 mRNA increased from 2-fold within 6 h to 45-fold within 36 h. Nuclear run-on transcription studies showed that the accumulation of SCD1 mRNA after refeeding starved mice a fat-free, high carbohydrate diet was a consequence of the transcriptional activation of the SCD1 gene. The SCD1 mRNA level decreased rapidly (t1/2 = approximately 4 h) within 24 h when mice fed the fat-free, high carbohydrate diet were switched to a regular chow diet. Furthermore, when the fat-free diet was supplemented with triacylglycerides containing polyunsaturated fatty acids, the transcription of the SCD1 gene and the induction of the SCD1 mRNA were significantly blunted. Triacylglycerides containing saturated and monounsaturated fatty acids were without dramatic effects. In contrast, synthesis of liver albumin mRNA was little affected by any of these dietary variations, indicating that the observed changes in the transcription of the SCD1 gene and mRNA levels were specific. These data demonstrate that both dietary carbohydrates and polyunsaturated fatty acids or their metabolites directly or indirectly regulate the expression of the SCD1 gene in mouse liver.

Insulin and tumor necrosis factor alpha (TNF alpha) produce potent and opposing physiological signals in adipocytes. However, genes that are co-regulated by the hormone and cytokine during and after adipocyte differentiation have not been characterized. Using 3T3-L1 cells, we have studied the regulation of the expression of genes encoding acyl-CoA synthetase (ACS), and stearoyl CoA desaturase-1 (SCD-1), two enzymes that play key roles in the metabolism of long chain fatty acids. Insulin is required for triggering the transcriptional activation of the ACS and SCD-1 genes at an early stage in adipocyte differentiation. In mature adipocytes insulin elicits a 4-fold increase in the rates of transcription of the two genes. However, when 3T3-L1 adipocytes are treated with TNF alpha the cytokine causes a 75-90% decrease in the levels of ACS and SCD-1 mRNAs. The decline in mRNA content is associated with similar decrements in the rates of transcription of the ACS and SCD-1 genes. Thus, the ACS and SCD-1 genes are subject to stimulation and counter-regulation (at the transcriptional level) by insulin and TNF alpha, respectively. The opposing effects of insulin and TNF alpha are observed in developing and terminally differentiated adipocytes. Unlike the ACS and SCD-1 genes, the genes that encode the lipogenic enzymes lipoprotein lipase and malic enzyme are not subject to counter-regulation by insulin and TNF alpha at the transcriptional level in 3T3-L1 adipocytes. These observations on the control of ACS and SCD-1 expression suggest possible mechanisms by which adipocytes can markedly adjust their capacity for long chain fatty acid metabolism in response to external stimuli.

Strains of Saccharomyces cerevisiae bearing the ole1 mutation are defective in unsaturated fatty acid (UFA) synthesis and require UFAs for growth. A previously isolated yeast genomic fragment complementing the ole1 mutation has been sequenced and determined to encode the delta 9 fatty acid desaturase enzyme by comparison of primary amino acid sequence to the rat liver stearoyl-CoA desaturase. The OLE1 structural gene encodes a protein of 510 amino acids (251 hydrophobic) having an approximate molecular mass of 57.4 kDa. A 257-amino acid internal region of the yeast open reading frame aligns with and shows 36% identity and 60% similarity to the rat liver stearoyl-CoA desaturase protein. This comparison disclosed three short regions of high consecutive amino acid identity (greater than 70%) including one 11 of 12 perfect residue match. The predicted yeast enzyme contains at least four potential membrane-spanning regions and several shorter hydrophobic regions that align exactly with similar sequences in the rat liver protein. An ole1 gene-disrupted yeast strain was transformed with a yeast-rat chimeric gene consisting of the promoter region and N-terminal 27 codons of OLE1 fused to the rat desaturase coding sequence. Fusion gene transformants displayed near equivalent growth rates and modest lipid composition changes relative to wild type yeast control implying a significant conservation of delta 9 desaturase tertiary structure and efficient interaction between the rat desaturase and yeast cytochrome b5.

Previously we isolated and characterized a differentially expressed gene from mouse 3T3-L1 preadipocytes that encodes stearoyl-CoA desaturase (SCD1; Ntambi, J. M., Buhrow, S. A., Kaestner, K. H., Christy, R. J., Sibley, E., Kelly, T. J., Jr., and Lane, M. D. (1988) J. Biol. Chem. 263, 17291-17300). Genomic Southern blot analysis indicated the existence of another closely related gene. Here we report the isolation and characterization of this gene and the corresponding cDNA which encode a second stearoyl-CoA desaturase, SCD2, 3T3-L1 adipocytes. SCD2 cDNA is 5 kilobase pairs in length and encodes a protein of 358 amino acids with greater than 87% amino acid sequence identity to SCD1. RNase protection analysis reveals a 10-fold increase in the expression of SCD2 mRNA during 3T3-L1 preadipocyte differentiation. SCD2 mRNA is expressed constitutively at a high level in brain, is not expressed in liver, and its expression in kidney, adipose, and lung tissue is increased greatly by shifting mice from a diet containing unsaturated fatty acids to a diet devoid of fat. The tissue distribution and the dietary alteration of SCD1 mRNA expression differs markedly from that of SCD2 mRNA being absent from brain, constitutive in adipose tissue, and subject to negative control in liver by feeding a diet containing unsaturated fatty acids. The SCD2 gene spans approximately 15 kilobase pairs and consists of six exons and five introns, with intron/exon junctions similar to those of SCD1. As determined by primer extension analysis the start site of transcription maps 300 nucleotides upstream of the initiator methionine codon. Unlike the SCD1 gene, SCD2 lacks a typical "TATA" box in the 5'-flanking region, but has two "CCAAT" boxes at positions -90 and -135 relative to the transcription initiation site. The SCD2 promoter contains a 140-base pair sequence (located between nucleotides -54 and -201) which possesses 77% sequence identity to a region (located between nucleotides -472 and -325) in the SCD1 promoter. There is a GC-rich sequence in the SCD2 promoter (at nucleotide -175) similar to the binding site for the nuclear transcription factor Sp1 as well as an element with homology to the core consensus sequence for the glucocorticoid regulatory element position -500 and a potential CCAAT box/enhance binding protein sequence at position -540. The SCD gene family provides a new model system for the study of differentiation-induced as well as tissue-specific metabolite controlled gene expression.

Previous studies have shown that differentiation of 3T3-L1 preadipocytes leads to the activation of transcription of an unidentified gene which encodes a 4.9-kilobase (kb) mRNA. Several cDNAs that include the complete sequence of this mRNA were obtained and used to isolate and characterize the gene. Analysis of the nucleotide and amino acid sequences of both cDNA and genomic clones revealed that the gene encodes the mouse stearoyl-CoA desaturase (SCD), an enzyme known to be expressed upon differentiation of 3T3-L1 preadipocytes. The predicted amino acid sequence (355 residues) of the mouse 3T3-L1 adipocyte SCD exhibits 92% identity to that of the rat liver SCD. There is also a high degree of nucleotide sequence identity between the mouse and rat mRNAs in their unusually long approximately 3.5-kb 3'-untranslated regions. Mice fed a diet containing unsaturated triacylglycerides express SCD mRNA only in adipose tissue, whereas mice starved and refed a fat-free diet, express SCD mRNA in both liver and adipose tissue. The mouse gene for the desaturase spans approximately 15 kb and contains 6 exons and 5 introns with all intron-exon junctions conforming to the GT/AG splicing rule. As determined by S1 nuclease mapping and primer extension analysis, the transcriptional initiation site maps 152 nucleotides upstream from the initiation methionine codon. A canonical promoter "TATA" box is located 30 base pairs upstream of the Cap site. A typical "CCAAT" box sequence is not present in the adjacent 5'-flanking region; however, there is a GC-rich sequence (at nucleotide -215) similar to the binding site for the nuclear transcription factor Sp1. Upstream from the transcriptional initiation site are elements with homology (approximately 75%) to the putative fat-specific transcriptional element FSE2 and core consensus sequences for cAMP and glucocorticoid regulatory elements. A chimeric construct, containing 363 base pairs of 5'-flanking sequence and 30 nucleotides of 5'-untranslated sequence of the mouse SCD gene ligated to the bacterial chloramphenicol acetyltransferase gene, was transfected into 3T3-L1 cells. When cells were induced to differentiate into adipocytes, expression of the SCD chloramphenicol acetyltransferase gene increased approximately 63-fold, suggesting that the SCD promoter region contains elements that mediate the response to adipogenic agents which induce differentiation.

Poly(A+) RNA isolated from livers of rats induced for stearyl-CoA desaturase contains elevated levels of mRNA for this enzyme which is translated in a rabbit reticulocyte system. The protein is immunologically and by peptide fingerprinting following Staphylococcus aureus V8 protease digestion identical to the isolated enzyme and, therefore, not synthesized in a detectable larger precursor form. The desaturase mRNA is selectively translated on free cytoplasmic polysomes from rat liver and represents at least a 40-fold increase in translatable mRNA in livers of induced animals. Northern blot analysis, using a cDNA probe complementary to rat liver desaturase mRNA, demonstrated that the desaturase is encoded by a 4900-base mRNA which is elevated approximately 50-fold in induced liver.

Hepatic poly(A+) RNA from rats induced for stearyl-CoA desaturase was used for primer-extension of cDNA coding for stearyl-CoA desaturase. Previously, Northern blot analysis showed that translatable desaturase mRNA is 4,900 nucleotides in length (Thiede, M. A., and Strittmatter, P. (1985) J. Biol. Chem. 260, 14459-14463). Six overlapping cDNAs, ranging from 850 to 1450 bases, were used to compile the 4,689-nucleotide sequence. The cDNA includes a 1,074-base open reading frame coding for 358 amino acids, corresponding to a molecular mass of 41,400 daltons. Positive identification of this open reading frame was accomplished by matching the amino acid sequence of both amino-terminal and cyanogen bromide peptides of the purified enzyme with regions of the sequence deduced from the cDNA. Amino acid composition data from the cDNA compares well with that from the desaturase. The protein contains 62% hydrophobic amino acids. An interesting feature of this mRNA is the 3,500-base 3' noncoding region, which has been localized on a single 3' exon by Southern blot analysis.