Membrane topology of mouse stearoyl-CoA desaturase 1

Stearoyl-CoA desaturase (SCD) is an integral membrane protein anchored in the endoplasmic reticulum. It catalyzes the biosynthesis of monounsaturated fatty acids that are required for the synthesis of triglycerides, cholesteryl esters, and phospholipids. Four mouse isoforms of SCD (SCD1-4) and two human isoforms have been characterized. In the current study, we characterize the topology of the mouse SCD1 isoform. Hydropathy analysis of the 355-amino acid mouse SCD1 protein predicts that the protein contains four transmembrane domains (TMDs) and three loops connecting the membrane-spanning domains. To define the topology of the protein, recombinant SCD1 constructs containing epitope tags were transiently expressed in HeLa cells and analyzed by indirect immunofluorescence and cysteine derivatization. Our data provide evidence that the N and C termini of SCD1 are oriented toward the cytosol with four transmembrane domains separated by two very short hydrophilic loops in the ER lumen and one large hydrophilic loop in the cytosol. In addition, based on the previous observation that SCD is a thiol enzyme, we sought to investigate whether the cysteine residues were essential for enzyme activity through mutagenesis studies, and our data suggest that the cysteines in SCD are not catalytically essential.     

Discovery of piperidine-aryl urea-based stearoyl-CoA desaturase 1 inhibitors

A series of structurally novel stearoyl-CoA desaturase1 (SCD1) inhibitors has been identified via molecular scaffold manipulation. Preliminary structure–activity relationship (SAR) studies led to the discovery of potent, and orally bioavailable piperidine-aryl urea-based SCD1 inhibitors. 4-(2-Chlorophenoxy)-N-[3-(methyl carbamoyl)phenyl]piperidine-1-carboxamide 4c exhibited robust in vivo activity with dose-dependent desaturation index lowering effects.     

Lack of stearoyl-CoA desaturase-1 function induces a palmitoyl-CoA Delta6 desaturase and represses the stearoyl-CoA desaturase-3 gene in the preputial glands of the mouse

The mouse preputial gland (PG), a specialized sebaceous structure, is rich in wax esters, triglycerides, and alkyl-2,3-diacylglycerol. We have found that the mouse PG expresses the three gene isoforms (SCD1, SCD2, and SCD3) of the Delta9 stearoyl-CoA desaturase enzyme that catalyzes the biosynthesis of monounsaturated fatty acids mainly, C16:1n-7 and C18:1n-9. However, mice with a targeted disruption in the SCD1 isoform (SCD1(-/-)) have undetectable SCD3 mRNA expression in the PG while the expression of SCD2 isoform was not altered. The levels of C16:1n-7 were reduced by greater than 70% while that of C18:1n-9 were reduced by 28%. The content of the C16:1n-10 (Delta6 hexadecenoic acid) isomer and a major fatty acid of the PG was increased by greater than 2-fold, mainly in the wax ester fraction of the SCD1(-/-) mouse. We demonstrate that the increase in C16:1n-10 is due to induction of a specific palmitoyl-CoA Delta6 desaturase activity. Testosterone administration to the SCD1(-/-) mouse induced SCD3 mRNA expression and resulted in an increase in the Delta9 desaturation of 16:0-CoA, but not of 18:0-CoA. These observations demonstrate that loss of SCD1 function alters the expression of SCD3 and reveal for the first time the presence and regulation of a palmitoyl-CoA Delta6 desaturase enzyme in mammals.     

Discovery of liver-targeted inhibitors of stearoyl-CoA desaturase (SCD1)

Inhibitors based on a benzo-fused spirocyclic oxazepine scaffold were discovered for stearoyl-coenzyme A (CoA) desaturase 1 (SCD1) and subsequently optimized to potent compounds with favorable pharmacokinetic profiles and in vivo efficacy in reducing the desaturation index in a mouse model. Initial optimization revealed potency preferences for the oxazepine core and benzylic positions, while substituents on the piperidine portions were more tolerant and allowed for tuning of potency and PK properties. After preparation and testing of a range of functional groups on the piperidine nitrogen, three classes of analogs were identified with single digit nanomolar potency: glycine amides, heterocycle-linked amides, and thiazoles. Responding to concerns about target localization and potential mechanism-based side effects, an initial effort was also made to improve liver concentration in an available rat PK model. An advanced compound 17m with a 5-carboxy-2-thiazole substructure appended to the spirocyclic piperidine scaffold was developed which satisfied the in vitro and in vivo requirements for more detailed studies.     

Loss of stearoyl-CoA desaturase 1 rescues cardiac function in obese leptin-deficient mice

The heart of leptin-deficient ob/ob mice is characterized by pathologic left ventricular hypertrophy along with elevated triglyceride (TG) content, increased stearoyl-CoA desaturase (SCD) activity, and increased myocyte apoptosis. In the present study, using an ob/ob;SCD1^−/− mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle (LV) function in leptin deficiency. We show that disruption of the SCD1 gene improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction without affecting levels of plasma TG and FFA. The improvement is associated with reduced expression of genes involved in FA transport and lipid synthesis in the heart, as well as reduction in cardiac FFA, diacylglycerol, TG, and ceramide levels. The rate of FA β-oxidation is also significantly lower in the heart of ob/ob;SCD1^−/− mice compared with ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be one of the main mechanisms responsible for improved LV function in ob/ob mice caused by SCD1 deficiency.     

Adipose-specific deletion of stearoyl-CoA desaturase 1 up-regulates the glucose transporter GLUT1 in adipose tissue

Stearoyl-CoA desaturase 1 (SCD1) deficiency protects mice from diet-induced obesity and insulin resistance. To understand the tissue-specific role of SCD1 in energy homeostasis, we have generated mice with an adipose-specific knockout of Scd1 (AKO), and report here that SCD1 deficiency increases GLUT1 expression in adipose tissue of AKO mice, but not global SCD1 knockout (GKO) mice. In 3T3-L1 adipocytes treated with an SCD inhibitor, basal glucose uptake and the cellular expression of GLUT1 were significantly increased while GLUT4 expression remained unchanged. Consistently, adipose-specific SCD1 knockout (AKO) mice had significantly elevated GLUT1 expression, but not GLUT4, in white adipose tissue compared to Lox counterparts. Concurrently, adiponectin expression was significantly diminished, whereas TNF-α expression was elevated. In contrast, in adipose tissue of GKO mice, GLUT4 and adiponectin expression were significantly elevated with lowered TNF-α expression and little change in GLUT1 expression, suggesting a differential responsiveness of adipose tissue to global- or adipose-specific SCD1 deletion. Taken together, these results indicate that adipose-specific deletion of SCD1 induces GLUT1 up-regulation in adipose tissue, associated with decreased adiponectin and increased TNF-α production, and suggest that GLUT1 may play a critical role in controlling glucose homeostasis of adipose tissue in adipose-specific SCD1-deficient conditions.     

Increased expression of the gene encoding stearoyl-CoA desaturase 1 in human bladder cancer

Bladder cancer is a common disease and a significant cause of death worldwide. There is thus great interest in identifying a diagnostic and prognostic biomarker, as well as gaining an understanding of the molecular basis of bladder cancer. Stearoyl-CoA desaturase 1 gene (SCD1) is highly overexpressed in many human cancers. However, the expression of SCD1 has not yet been investigated in patients with bladder cancer. Here, we document that (a) the SCD1 is highly overexpressed in human bladder cancer; (b) high expression of SCD1 is more frequently observed in the late stage of disease and patients with lymph node metastasis; (c) bladder cancer patients with a higher SCD1 mRNA level have a poorer survival rate than those with normal SCD1 expression. Overall, this is the first report to indicate an association between SCD1 mRNA level and clinical indicators of human bladder cancer. Our study has provided evidence supporting the potential role of SCD1 as a biomarker for human bladder cancer prognosis.     

The role of stearoyl-CoA desaturase in the control of metabolism

Since obesity is becoming increasingly prevalent worldwide, much effort is being devoted to understanding its pathogenesis and treatment. In recent years, several candidate genes have been proposed as therapeutic targets. However, stearoyl-CoA desaturase 1 (SCD1) is of special significance, because it is the major gene target of leptin-a central mediator of energy homeostasis. There is evidence that SCD1 deficiency activates metabolic pathways that promote beta-oxidation and decrease lipogenesis in liver and skeletal muscles. One mechanism is via increased activation of AMP-activated protein kinase. SCD1 mutation results also in global changes in expression of genes involved in lipid metabolism. SCD1 deficient mice have increased energy expenditure, reduced body adiposity, and are resistant to diet-induced obesity. In this review, we examine data from our laboratory and others suggesting that SCD1 is an important component in the regulation of body metabolism.     

Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP-dependent cell death in human cancer cells

Background

Cancer cells present a sustained de novo fatty acid synthesis with an increase of saturated and monounsaturated fatty acid (MUFA) production. This change in fatty acid metabolism is associated with overexpression of stearoyl-CoA desaturase 1 (Scd1), which catalyses the transformation of saturated fatty acids into monounsaturated fatty acids (e.g., oleic acid). Several reports demonstrated that inhibition of Scd1 led to the blocking of proliferation and induction of apoptosis in cancer cells. Nevertheless, mechanisms of cell death activation remain to be better understood.

Principal Findings

In this study, we demonstrated that Scd1 extinction by siRNA triggered abolition of de novo MUFA synthesis in cancer and non-cancer cells. Scd1 inhibition-activated cell death was only observed in cancer cells with induction of caspase 3 activity and PARP-cleavage. Exogenous supplementation with oleic acid did not reverse the Scd1 ablation-mediated cell death. In addition, Scd1 depletion induced unfolded protein response (UPR) hallmarks such as Xbp1 mRNA splicing, phosphorylation of eIF2α and increase of CHOP expression. However, the chaperone GRP78 expression, another UPR hallmark, was not affected by Scd1 knockdown in these cancer cells indicating a peculiar UPR activation. Finally, we showed that CHOP induction participated to cell death activation by Scd1 extinction. Indeed, overexpression of dominant negative CHOP construct and extinction of CHOP partially restored viability in Scd1-depleted cancer cells.

Conclusion

These results suggest that inhibition of de novo MUFA synthesis by Scd1 extinction could be a promising anti-cancer target by inducing cell death through UPR and CHOP activation.     

Elevated stearoyl-CoA desaturase in brains of patients with Alzheimer's disease

The molecular bases of Alzheimer's disease (AD) remain unclear. We used a lipidomic approach to identify lipid abnormalities in the brains of subjects with AD (N = 37) compared to age-matched controls (N = 17). The analyses revealed statistically detectable elevations in levels of non-esterified monounsaturated fatty acids (MUFAs) and mead acid (20:3n-9) in mid-frontal cortex, temporal cortex and hippocampus of AD patients. Further studies showed that brain mRNAs encoding for isoforms of the rate-limiting enzyme in MUFAs biosynthesis, stearoyl-CoA desaturase (SCD-1, SCD-5a and SCD-5b), were elevated in subjects with AD. The monounsaturated/saturated fatty acid ratio ('desaturation index')--displayed a strong negative correlation with measures of cognition: the Mini Mental State Examination test (r = -0.80; P = 0.0001) and the Boston Naming test (r = -0.57; P = 0.0071). Our results reveal a previously unrecognized role for the lipogenic enzyme SCD in AD.     

Discovery of 1-(4-phenoxypiperidin-1-yl)-2-arylaminoethanone stearoyl-CoA desaturase 1 inhibitors

A series of novel stearoyl-CoA desaturase 1 (SCD1) inhibitors were identified by scaffold design based on known SCD1 inhibitors. Large structural changes were made leading to multiple analogs with comparable or improved potency. This approach is valuable for generation of proprietary compounds without conducting a costly high-throughput screening.     

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.     

Advances in regulation and function of stearoyl-CoA desaturase 1 in cancer,from bench to bed

Stearoyl-CoA desaturase 1(SCD1) converts saturated fatty acids to monounsaturated fatty acids. The expression of SCD1 is increased in many cancers, and the altered expression contributes to the proliferation, invasion, stemness and chemoresistance of cancer cells. Recently, more evidence has been reported to further support the important role of SCD1 in cancer, and the regulation mechanism of SCD1 has also been focused. Multiple factors are involved in the regulation of SCD1, including metabolis...     

Differential regulation of the stearoyl-CoA desaturase genes by thiazolidinediones in 3T3-L1 adipocytes

Two stearoyl-CoA desaturase (SCD) isoforms can be expressed during the differentiation of 3T3-L1 preadipocytes into adipocytes. Here we report on the effects of the peroxisome proliferator-activated receptor gamma ligand troglitazone (TRO) on scd1 and scd2 mRNA levels as determined by Northern blotting, on SCD protein expression as determined by Western blotting, and on total lipid composition as determined by GC during differentiation. In preadipocytes, scd1 mRNA and SCD protein were not detected, whereas scd2 mRNA was detected. These cells have high levels of palmitate (16:0), stearate (18:0), and monounsaturated oleate (Delta(9)-18:1) and low levels of monounsaturated palmitoleate (Delta(9)-16:1). In MDI (methylisobutylxanthine, dexamethasone, and insulin)-treated cells, scd1 mRNA and SCD protein were increased approximately 100-fold relative to preadipocyte levels, the scd2 mRNA level was increased 2-fold, Delta(9)-16:1 was increased approximately 20-fold, and 18:0 was decreased approximately 3-fold. In TRO-treated cells, the scd1 mRNA level was lower than that observed in preadipocytes, while the scd2 mRNA level was similar. TRO also decreased scd1 mRNA in primary adipocytes. The TRO-treated cells contained a Delta(9)-18:1 level typical of MDI-treated cells whereas, conversely, these cells also contained a low Delta(9)-16:1 level typical of preadipocytes. The implications of these correlations for the regulatory and enzymatic mechanism(s) used to establish and maintain lipid composition are discussed.     

Characterization of HSCD5, a novel human stearoyl-CoA desaturase unique to primates

Stearoyl-CoA desaturase (SCD) is an integral membrane protein of the endoplasmic reticulum (ER) that catalyzes the formation of monounsaturated fatty acids from saturated fatty acids. Recent studies suggest that SCD is a key regulator of energy metabolism and has implications in dislipidemia and obesity. Four SCD isoforms (SCD1-4) have been identified in mouse. In human, only one SCD isoform has been characterized so far. Here we report that the previously reported human ACOD4 gene encodes a distinct stearoyl-CoA desaturase, hSCD5. GenBank database mining revealed orthologues of hSCD5 in the primates, but not in the rodents. In transiently transfected 293 cells, hSCD5 co-localized with calnexin on ER membrane. Microsome fractions prepared from hSCD1 and hSCD5 transfected cells displayed similar delta 9 desaturase activity. Quantitative real-time RT-PCR analysis suggested that hSCD5 was abundantly expressed in adult brain and pancreas. These data suggested that hSCD5 plays a role distinct from that of hSCD1 during development and in normal physiological conditions.