Identification and characterization of murine SCD4, a novel heart-specific stearoyl-CoA desaturase isoform regulated by leptin and dietary factors

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids. Thus far, three isoforms of SCD (SCD1, SCD2, and SCD3) have been identified and characterized. Regulation of the SCD1 isoform has been shown to be an important component of the metabolic actions of leptin in liver, but the effects of leptin on SCD isoforms in other tissues have not been investigated. We found that although the mRNA levels of SCD1 and SCD2 were not affected by leptin deficiency in the hearts of ob/ob mice, the SCD activity and levels of monounsaturated fatty acids were increased, implying the existence of another SCD isoform. This observation has led to the cDNA cloning and characterization of a fourth SCD isoform (SCD4) that is expressed exclusively in the heart. SCD4 encodes a 352-amino acid protein that shares 79% sequence identity with the SCD1, SCD2, and SCD3 isoforms. Liver X receptor alpha (LXR alpha) agonists and a high carbohydrate fat-free diet induced SCD4 expression, but unlike SCD1, SCD4 expression was not repressed by dietary polyunsaturated fatty acids. SCD4 mRNA levels were elevated 5-fold in the hearts of leptin-deficient ob/ob mice relative to wild type controls. Treatment of ob/ob mice with leptin decreased mRNA levels of SCD4, whereas levels of SCD1 and SCD2 were not affected. Furthermore, in the hearts of SCD1-deficient mice, SCD4 mRNA levels were induced 3-fold, whereas the levels of SCD2 were not altered. The current studies identify a novel heart-specific SCD isoform that demonstrates tissue-specific regulation by leptin and dietary factors.     

Regulation of stearoyl-CoA desaturases and role in metabolism

Stearoyl-CoA desaturase (SCD) is the rate-limiting enzyme catalyzing the synthesis of monounsaturated fatty acids, mainly oleate (18:1) and palmitoleate (16:1). These represent the major monounsaturated fatty acids of membrane phospholipids, triglycerides, wax esters and cholesterol esters. The ratio of saturated to monounsaturated fatty acids affects phospholipid composition and alteration in this ratio has been implicated in a variety of disease states including cardiovascular disease, obesity, diabetes, neurological disease, and cancer. For this reason, the expression of SCD is of physiological significance in both normal and disease states. Several SCD gene isoforms (SCD1, SCD2, SCD3) exist in the mouse and one SCD isoform that is highly homologous to the mouse SCD1 is well characterized in human. The physiological role of each SCD isoform and the reason for having three or more SCD gene isoforms in the rodent genome are currently unknown but could be related the substrate specificities of the isomers and their regulation through tissue-specific expression. The recent studies of asebia mouse strains that have a natural mutation in the SCD1 gene and a mouse model with a targeted disruption of the SCD1 gene have provided clues concerning the role that SCD1 and its endogenous products play in the regulation of metabolism.     

Comparison of pig, sheep and chicken SCD5 homologs: Evidence for an early gene duplication event

Stearoyl-CoA desaturase (SCD) catalyzes the desaturation of saturated fatty acyl-CoA substrates at the delta-9 position. Multiple SCD isoforms are well characterized in rodents, especially in mice, where four isoforms have been described. In humans and cows, two SCD isoforms have been described: SCD1, which is a homolog of murine SCD1, and SCD5, which appears to be a distinct SCD gene rather than an ortholog of any of the four murine isoforms. In this paper, we describe for the first time SCD5 homologs in sheep, pigs and chickens. The SCD5 nucleotide sequences have notably higher GC content than SCD1 sequences, and the predicted protein sequences lack N-terminal PEST sequences typically found in SCD1 proteins. Similar to humans and bovines, the mRNA expression of sheep and pig SCD5 is greatest in the brain, and the mRNA expression of chicken SCD5 is greatest in the pancreas and brain. In contrast, SCD1 expression was found to be highest in adipose tissue in pigs and sheep, and liver in the chicken. This is the first report of an SCD5 homolog in a non-mammalian species, and suggests that SCD5 may be the result of an early gene duplication event that occurred before the divergence of mammals.     

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.     

Degradation of Stearoyl-Coenzyme A Desaturase: Endoproteolytic Cleavage by an Integral Membrane Protease

Stearoyl-coenzyme A desaturase (SCD) is a key regulator of membrane fluidity, turns over rapidly, and represents a prototype for selective degradation of resident proteins of the endoplasmic reticulum. Using detergent-solubilized, desaturase-induced rat liver microsomes we have characterized a protease that degrades SCD. Degradation of SCD in vitro is highly selective, has a half-life of 3–4 h, and generates a 20-kDa C-terminal fragment of SCD. The N terminus of the 20-kDa fragment was identified as Phe¹⁷⁷. The cleavage site occurs in a conserved 12-residue hydrophobic segment of SCD flanked by clusters of basic residues. The SCD protease remains associated with microsomal membranes after peripheral and lumenal proteins have been selectively removed. SCD protease is present in normal rat liver microsomes and cleaves purified SCD. We conclude that rapid turnover of SCD involves a constitutive microsomal protease with properties of an integral membrane protein.     

Involvement of mouse nucleoplasmin 2 in the decondensation of sperm chromatin after fertilization

The tightly condensed chromatin of spermatozoa is rapidly decondensed after the spermatozoa enter oocytes. Although no factor involved in sperm chromatin decondensation (SCD) has been identified in mammals, it has been suggested that a factor related to SCD activity is present in the germinal vesicle (GV) of oocytes. Here, we found that the nucleolus-like body (NLB), which is a component of the GV, is involved in SCD in murine oocytes. When NLBs were microsurgically removed from GV-stage oocytes, SCD was significantly retarded in the paternal genome after fertilization following meiotic maturation. We found that the retardation of SCD in the NLB-removed oocytes was restored by the microinjection of mRNA encoding nucleoplasmin 2 (NPM2), a component of NLBs. Furthermore, SCD was retarded in the fertilized oocytes from Npm2-knockout females, and recombinant NPM2 alone could induce the SCD in vitro. These data provide evidence that NPM2 is involved in sperm chromatin remodeling in mammals.     

Metabolic reprogramming of the heart through stearoyl-CoA desaturase

Stearoyl-CoA desaturase (SCD), a central enzyme in lipid metabolism that synthesizes monounsaturated fatty acids, has been linked to tissue metabolism and body adiposity regulation. Recent studies showed that SCD has the ability to reprogram cardiac metabolism, thereby regulating heart function. In the heart, the lack of SCD1 enhances glucose transport and metabolism at the expense of fatty acid (FA) uptake and oxidation. The metabolic changes associated with SCD1 deficiency protect cardiac myocytes against both necrotic and apoptotic cell death and improve heart function. Furthermore, SCD4, a heart-specific isoform of SCD, is specifically repressed by leptin and the lack of SCD1 function in leptin-deficient ob/ob mice results in a decrease in the accumulation of neutral lipids and ceramide and improves the systolic and diastolic function of a failing heart. Large-population human studies showed that the plasma SCD desaturation index is positively associated with heart rate, and cardiometabolic risk factors are modulated by genetic variations in SCD1. The current findings indicate that SCD may be used to reprogram myocardial metabolism to improve cardiac function. Here, we review recent advances in understanding the role of SCD in the control of heart metabolism and its involvement in the pathogenesis of lipotoxic cardiomyopathies.     

Selective mutagenesis of lysyl residues leads to a stable and active form of delta 9 stearoyl-CoA desaturase

Stearoyl-CoA desaturase (SCD) is a short-lived integral membrane protein of the endoplasmic reticulum (ER) that catalyzes the insertion of a double bond in the delta 9 position of saturated fatty acids. Its expression has been difficult in heterologous systems. In this study, recombinant adenovirus vector was used to express both wild-type (wt) and engineered forms of rat SCD in human transformed kidney cells. In the engineered form of SCD, lysyl residues at positions 33, 35, and 36 were mutated to alanine (SCD K/A). The recombinant adenovirus also contains a cDNA encoding the green fluorescent protein (GFP). The stable reporter GFP was used to analyze the efficiency of transfection and the stability of expressed SCDs. The wt SCD was unstable upon expression, whereas expression of SCD K/A resulted in the stabilization of the protein. The proteasome inhibitor MG132 did not affect the rapid degradation of expressed wt SCD, implying that proteasome is not involved in this degradation. Functional analysis of microsomes from infected cells expressing SCD K/A resulted in the formation of holoenzyme with desaturase activity. Here we report engineering a stabilized form of a rapidly degraded membrane protein for production of an active mutant form of SCD. The adenovirus transformed cells may provide a model for the study of the effects of positive SCD expression.     

Lack of stearoyl-CoA desaturase 1 upregulates basal thermogenesis but causes hypothermia in a cold environment

Stearoyl-CoA desaturase (SCD) is a microsomal enzyme involved in the biosynthesis of oleate and palmitoleate. Mice with a targeted disruption of the SCD1 isoform (SCD1-/-) exhibit reduced adiposity and increased energy expenditure. To address whether the energy expenditure is attributable to increased thermogenesis, we investigated the effect of SCD1 deficiency on basal and cold-induced thermogenesis. SCD1-/- mice have increased expression of uncoupling proteins in brown adipose tissue (BAT) relative to controls. The beta3-adrenergic receptor (beta3-AR) expression was increased and the phosphorylation of cAMP response element binding protein and the protein level of peroxisome proliferator-activated receptor-gamma coactivator-1alpha were increased in the SCD1-/- mice. Both lipolysis and fatty acid oxidation were increased in the SCD1-/- mice. When exposed to 4 degrees C, SCD1-/- mice showed hypothermia, hypoglycemia, and depleted liver glycogen. High levels of dietary oleate partially compensated for the hypothermia and rescued plasma glucose and liver glycogen. These results suggest that SCD1 deficiency stimulates basal thermogenesis through the upregulation of the beta3-AR-mediated pathway and a subsequent increase in lipolysis and fatty acid oxidation in BAT. The hypothermia and hypoglycemia in cold-exposed SCD1-/- mice and the compensatory recovery by oleate indicate an important role of SCD1 gene expression in thermoregulation.     

Identification of mouse palmitoyl-coenzyme A Delta9-desaturase

Stearoyl-coenzyme A desaturase (SCD) catalyzes the desaturation of saturated fatty acids to monounsaturated fatty acids in mammalian cells. Currently, there are four known enzymatic isoforms (SCD1-SCD4) in the mouse genome. The physiological roles for multiple SCD isoforms and their substrate specificities are unknown at present. We report here distinct substrate specificities for the mouse SCD isoforms. Each SCD isoform was able to complement the ole1 mutation in Saccharomyces cerevisiae through heterologous expression of transgenic SCD. Fatty acid analysis showed that mouse SCD1, SCD2, and SCD4 desaturate both C18:0 and C16:0, whereas mouse SCD3 uses C16:0 but not C18:0. We identify SCD3 as a mammalian palmitotyl-CoA Delta9-desaturase, and its existence in mouse helps explain distinct physiological roles for each SCD isoform.     

Plasma-based approach to measure target engagement for liver-targeting stearoyl-CoA desaturase 1 inhibitors

A positive correlation between stearoyl-CoA desaturase (SCD)1 expression and metabolic diseases has been reported in rodents and humans. These findings indicate that SCD1 is a promising therapeutic target for the chronic treatment of diabetes and dyslipidemia. The SCD1 enzyme is expressed at high levels in several human tissues and is required for the biosynthesis of monounsaturated fatty acids, which are involved in many biological processes. Liver-targeted SCD inhibitors were designed to pharmacologically manipulate SCD1 activity in the liver to avoid adverse events due to systemic inhibition. This article describes the development of a plasma-based SCD assay to assess the level of SCD inhibition, which is defined in this article as target engagement. Essentially, animals are dosed with an exogenous deuterated tracer (d7-stearic acid) as substrate, and the converted d7-oleic acid product is measured to monitor SCD1 inhibition. This study reveals that this plasma-based assay correlates with liver SCD1 inhibition and can thus have clinical utility.     

Role of stearoyl-coenzyme A desaturase in lipid metabolism

Stearoyl-CoA desaturase (SCD) (EC 1.14.99.5) is an endoplasmic reticulum-bound enzyme that catalyzes the delta9-cis desaturation of saturated fatty acyl-CoAs, the preferred substrates being palmitoyl- and stearoyl-CoA, which are converted to palmitoleoyl- and oleoyl-CoA, respectively. These monounsaturated fatty acids are used as substrates for the synthesis of triglycerides, wax esters, cholesteryl esters and membrane phospholipids. The saturated to monounsaturated fatty acid ratio affects membrane phospholipid composition and alteration in this ratio has been implicated in a variety of disease states including cardiovascular disease, obesity, diabetes, neurological disease, skin disorders and cancer. Thus, the expression of SCD is of physiological importance in normal and disease states. Several mammalian SCD genes have been cloned. A single human, three mouse and two rat are the best characterized SCD genes. The physiological role of each SCD isoform and the reason for having three or more SCD gene isoforms in the rodent genome are currently unknown. A clue as to the physiological role of the SCD, at least SCD1 gene and its endogenous products came from recent studies of asebia mouse strains that have a natural mutation in the SCD1 gene and a mouse model with a targeted disruption of the SCD1 gene. In this review we discuss our current understanding of the physiological role of SCD in lipid synthesis and metabolism.     

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.     

Stearoyl-CoA desaturase 1 coding sequences and antisense RNA affect lipid secretion in transfected chicken LMH hepatoma cells

Hepatic stearoyl CoA desaturase (SCD) activity in chickens from a fat line is higher than that of chickens from a lean line and correlates with plasma triacylglycerol concentrations. Furthermore, in these lines, the hepatic SCD1 mRNA level is positively correlated with the adipose tissue weight. To analyze the contribution of the SCD1 gene in the regulation of adiposity in the early stages of triacylglycerol secretion, SCD1 coding sequence and antisense RNA expression vectors were transfected in LMH cells. After selection, these cells were analyzed with regard to SCD1 expression and lipid secretion. The amounts of secreted triacylglycerols and phospholipids were shown to be higher in LMH cells transfected with the SCD1 gene, but reduced in those transfected with the SCD1 antisense sequences when compared to cells transfected with the vector alone (without SCD1 sequences). These results provide direct evidence that the expression of the SCD1 gene plays a major role in the triacylglycerol and phospholipid secretion process.     

Combined deletion of SCD1 from adipose tissue and liver does not protect mice from obesity

Stearoyl-CoA desaturase 1 (SCD1) catalyzes the synthesis of monounsaturated fatty acids (MUFA) from saturated FA. Mice with whole-body or skin-specific deletion of SCD1 are resistant to obesity. Here, we show that mice lacking SCD1 in adipose and/or liver are not protected from either genetic- (agouti; A(y)/a) or diet-induced obesity (DIO) despite a robust reduction in SCD1 MUFA products in both subcutaneous and epididymal white adipose tissue. Adipose SCD1 deletion had no effect on glucose or insulin tolerance or on hepatic triglyceride (TG) accumulation. Interestingly, lack of SCD1 from liver lowered the MUFA levels of adipose tissue and vice versa, as reflected by the changes in FA composition. Simultaneous deletion of SCD1 from liver and adipose resulted in a synergistic lowering of tissue MUFA levels, especially in the A(y)/a model in which glucose tolerance was also improved. Lastly, we found that liver and plasma TG show nearly identical genotype-dependent differences in FA composition, indicating that FA composition of plasma TG is predictive for hepatic SCD1 activity and TG FA composition. The current study suggests that SCD1 deletion from adipose and/or liver is insufficient to elicit protection from obesity, but it supports the existence of extensive lipid cross-talk between liver and adipose tissue.