Oleoyl-CoA is the major de novo product of stearoyl-CoA desaturase 1 gene isoform and substrate for the biosynthesis of the Harderian gland 1-alkyl-2,3-diacylglycerol.

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.

Polyunsaturated fatty acid regulation of gene expression.

Polyunsaturated fatty acids (PUFAs), specifically the n-3 and n-6 series, play a key role in the progression or prevention of human diseases such as obesity, diabetes, cancer, neurological and heart disease, mainly by affecting cellular membrane lipid composition, metabolism, signal-transduction pathways, and by direct control of gene expression. PUFAs show regulation of gene expression in several tissues, including brain, liver, heart, and adipose. Most recently, research has focused on identifying the mechanisms by which PUFAs regulate lipogenic gene expression. Research to date indicates that PUFA-mediated regulation of the genetic expression and proteolytic maturation of a group of transcription factors termed sterol regulatory element binding proteins (SREBPs) accounts for the suppression of hepatic lipogenic gene expression. However, our recent studies on the transcriptional regulation of the stearoyl-coenzyme A (CoA) desaturase gene, encoding a key enzyme in the cellular synthesis of monounsaturated fatty acids from saturated fatty acids indicates that PUFA can suppress gene transcription by a mechanism independent of SREBP maturation.

Regulation of stearoyl-CoA desaturase activity by the trans-10,cis-12 isomer of conjugated linoleic acid in HepG2 cells.

Stearoyl-CoA desaturase (SCD) catalyzes the rate-limiting step in the cellular synthesis of monounsaturated fatty acids mainly oleate (C18:1) and palmitoleate (C16:1) which are the major monounsaturated fatty acids of membrane phospholipids, cholesterol esters, waxes, and triglycerides. Several SCD isoforms exist in the mouse whereas the human has one well-characterized SCD gene. The trans-10,cis-12 isomer of conjugated linoleic acid has been previously shown to repress the expression of the mouse SCD1 gene isomer by decreasing SCD gene expression as well as by direct inhibition of SCD enzyme activity. We studied the regulation of human stearoyl-CoA desaturase (SCD) expression by conjugated linoleic acid (CLA) in cultured human hepatoblastoma cell line, HepG2. Treatment of the cells with the trans-10,cis-12 CLA isomer did not cause changes in the SCD gene transcription, mRNA and protein levels. However, this isomer decreased both the SCD activity as well as the levels of monounsaturated fatty acids. The other major CLA isomer, cis-9,trans-11 CLA, had no effect on SCD gene expression and activity. These results suggest that in HepG2 cells the trans-10,cis-12 CLA isomer regulates human SCD activity mainly by a posttranslational mechanism.

Regulation of stearoyl-CoA desaturase genes: role in cellular metabolism and preadipocyte differentiation.

The degree of fatty acid unsaturation in cell membrane lipids determines membrane fluidity, whose alteration has been implicated in a variety of disease states including diabetes, obesity, hypertension, cancer, and neurological and heart diseases. Stearoyl-CoA desaturase (SCD) is a key rate-limiting enzyme in the synthesis of unsaturated fatty acids by insertion of a cis-double bond in the Delta9 position of fatty acid substrates. Palmitate and stearate are the preferred substrates, which are converted to palmitoleate and oleate, respectively. These monounsaturated fatty acids are the major constituents of cellular membrane phospholipids and triacylglycerol stores found in adipose tissue. Two mouse and rat SCD genes (SCD1 and SCD2) have been cloned and characterized. During the differentiation of 3T3-L1 preadipocytes into adipocytes, SCD1 and SCD2 mRNAs are induced concomitant with increased de novo synthesis of palmitoleate and oleate. The physiological significance of expressing the two isoforms in the adipocytes is currently unknown. In this review we discuss the role of the SCD isoforms in metabolism and the recent findings on the differential regulation of mouse SCD genes by the antidiabetic thiazolidinediones (TZDs), during preadipocyte differentiation.

Regulation of stearoyl-CoA desaturase by polyunsaturated fatty acids and cholesterol.

The lipid composition of cellular membranes is regulated to maintain membrane fluidity. A key enzyme involved in this process is the membrane-bound stearoyl-CoA desaturase (SCD) which is the rate-limiting enzyme in the cellular synthesis of monounsaturated fatty acids from saturated fatty acids. A proper ratio of saturated to monounsaturated fatty acids contributes to membrane fluidity. Alterations in this ratio have been implicated in various disease states including cardiovascular disease, obesity, non-insulin-dependent diabetes mellitus, hypertension, neurological diseases, immune disorders, and cancer. The regulation of stearoyl-CoA desaturase is therefore of considerable physiological importance and its activity is sensitive to dietary changes, hormonal imbalance, developmental processes, temperature changes, metals, alcohol, peroxisomal proliferators, and phenolic compounds. Two mouse and rat SCD genes (SCD1 and SCD2) and a single human SCD gene have been cloned and characterized. In the past several years we have studied the dietary influences on the genetic expression of the mouse stearoyl-CoA desaturase. The expression of the mouse SCD genes is regulated by polyunsaturated fatty acids and cholesterol at the levels of transcription and mRNA stability. Promoter elements that are responsible for the polyunsaturated fatty acid repression colocalize with the promoter elements for SREBP-mediated regulation of the SCD genes. It is the goal of this review to provide an overview of the genetic regulation of the stearoyl-CoA desaturase in response to dietary polyunsaturated fatty acids and cholesterol.

cAMP activates the expression of stearoyl-CoA desaturase gene 1 during early preadipocyte differentiation.

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.

Role of Ca2+ in the early stages of murine adipocyte differentiation as evidenced by calcium mobilizing agents.

We have investigated the effect of the endoplasmic reticulum Ca(2+)-ATPase inhibitor thapsigargin and the calcium ionophore A23187 on the differentiation of 3T3-L1 preadipocytes. Treatment of 3T3-L1 preadipocytes with either agent resulted in a dose-dependent inhibition of adipocyte differentiation. The cells accumulated neither fat droplets nor the adipocyte-specific mRNAs encoding stearoyl-CoA desaturase 1 (SCD1) and adipocyte-P2 (aP2). These late markers of differentiation were specifically affected, because thapsigargin and A23187 did not inhibit the expression of beta-tubulin mRNA. No inhibition of differentiation or the expression of the mRNAs occurred when the drugs were added either prior to or 2 days after the initiation of differentiation. Thapsigargin and A23187 were also shown to dramatically block cell proliferation and DNA replication, which occur early in differentiation. Furthermore, during the first 48 h, thapsigargin and A23187 mediated an elevated and prolonged expression of the immediate-early gene corresponding to c-myc, and altered intracellular levels of calcium. Our results suggests that changes in intracellular calcium levels elicited by thapsigargin and A23187 prevent differentiation of 3T3-L1 preadipocytes into adipocytes by blocking the postconfluent mitotic phase of the differentiation process and also by mediating c-myc gene expression.

Insulin and dietary fructose induce stearoyl-CoA desaturase 1 gene expression of diabetic mice.

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.