Stearoyl-CoA Desaturase-2 in Murine Development, Metabolism, and Disease.

Stearoyl-CoA Desaturase-2 (SCD2) is a member of the Stearoyl-CoA Desaturase (SCD) family of enzymes that catalyze the rate-limiting step in monounsaturated fatty acid (MUFA) synthesis. The MUFAs palmitoleoyl-CoA (16:1n7) and oleoyl-CoA (18:1n9) are the major products of SCD2. Palmitoleoyl-CoA and oleoyl-CoA have various roles, from being a source of energy to signaling molecules. Under normal feeding conditions, SCD2 is ubiquitously expressed and is the predominant SCD isoform in the brain. However, obesogenic diets highly induce SCD2 in adipose tissue, lung, and kidney. Here we provide a comprehensive review of SCD2 in mouse development, metabolism, and various diseases, such as obesity, chronic kidney disease, Alzheimer's disease, multiple sclerosis, and Parkinson's disease. In addition, we show that bone mineral density is decreased in SCD2KO mice under high-fat feeding conditions and that SCD2 is not required for preadipocyte differentiation or the expression of PPARγ in vivo despite being required in vitro.

Factors to consider in using [U-C]palmitate for analysis of sphingolipid biosynthesis by tandem mass spectrometry.

This study describes the use of a stable-isotope labeled precursor ([U-¹³C]palmitate) to analyze de novo sphingolipid biosynthesis by tandem mass spectrometry. It also describes factors to consider in interpreting the data, including the isotope's location (¹³C appears in three isotopomers and isotopologues: [M + 16] for the sphingoid base or N-acyl fatty acid, and [M + 32] for both); the isotopic enrichment of palmitoyl-CoA; and its elongation, desaturation, and incorporation into N-acyl-sphingolipids. For HEK293 cells incubated with 0.1 mM [U-¹³C]palmitic acid, ∼60% of the total palmitoyl-CoA was ¹³C-labeled by 3 h (which was near isotopic equilibrium); with this correction, the rates of de novo biosynthesis of C16:0-ceramide, C16:0-monohexosylceramide, and C16:0-sphingomyelins were 62 ± 3, 13 ± 2, and 60 ± 11 pmol/h per mg protein, respectively, which are consistent with an estimated rate of appearance of C16:0-ceramide using exponential growth modeling (119 ± 11 pmol/h per mg protein). Including estimates for the very long-chain fatty acyl-CoAs, the overall rate of sphingolipid biosynthesis can be estimated to be at least ∼1.6-fold higher. Thus, consideration of these factors gives a more accurate picture of de novo sphingolipid biosynthesis than has been possible to-date, while acknowledging that there are inherent limitations to such approximations.

Hormonal and nutritional regulation of SCD1 gene expression.

Stearoyl-CoA Desaturase 1 (SCD1) is the rate limiting enzyme catalyzing the biosynthesis of monounsaturated fatty acids preferentially from palmitoyl-CoA and stearoyl-CoA forming respectively palmitoleyl-CoA and oleyl-CoA. These monounsaturated fatty acids are the key components of triglycerides and membrane phospholipids. Studying the regulation of SCD1 is of particular interest since alterations in phospholipids composition have been implicated in a variety of diseases including cancers, diabetes and cardiovascular disorders. Furthermore, oleic acid, the main product of SCD1 reaction, is the predominant fatty acid of human adipose tissue triacylglycerols, associating SCD1 with the development of obesity and the metabolic syndrome. In light of the key role of SCD1 in general metabolism, it is not surprising to observe a very tight and complex regulation of SCD1 gene expression in response to various parameters including hormonal and nutrient factors. In this review we analyze the anatomy and index the transcription factors that have been characterized to bind the SCD1 promoter. Then we present the current knowledge on how hormones regulate SCD1 expression with a particular interest on the role of insulin and leptin. We also describe how nutrients especially polyunsaturated fatty acids and carbohydrates modulate SCD1 gene expression.

Identification and characterization of a novel bovine stearoyl-CoA desaturase isoform with homology to human SCD5.

Stearoyl-CoA desaturase (SCD) is an enzyme responsible for the production of cis-9, trans-11 conjugated linoleic acid in ruminant fats, and for the synthesis of palmitoleoyl-CoA and oleoyl-CoA. To date, only one SCD isoform has been described in ruminant species, although multiple isoforms have been found in many other mammalian species. In this paper, we describe for the first time a second SCD isoform in cattle, which appears to be an ortholog of human SCD5 rather than a homolog of bovine SCD1 or any of the described murine SCD isoforms. As described in other SCD proteins, the predicted amino acid sequence of bovine SCD5 includes four transmembrane domains and three conserved histidine motifs. The amino-terminus of the predicted protein sequence of SCD5 lacks the PEST sequences typically found in SCD1 homologs, which are thought to target proteins for rapid degradation. Similar to human SCD5, the bovine SCD5 gene is organized into five exons and four introns, and is highly expressed in the brain. In other tissues examined, mRNA expression of SCD5 was minimal. Furthermore, the expression levels of SCD5 between brain gray and white matter are not different. This is the first description of a homolog of human SCD5 in a non-primate species.

The carcinogenicity of dichloroacetic acid in the male Fischer 344 rat.

The chlorinated acetic acids, in particular dichloroacetic acid (DCA), are found as chlorine disinfection by-products in finished drinking water supplies. DCA has previously been demonstrated to be a mouse liver carcinogen. Chronic studies are described in which male Fischer (F344) rats were exposed to DCA in their drinking water. In the first study, 28 day old rats were exposed to a regimen of 0.05, 0.5 and 5.0 g/l DCA. When animals in the high dose group began to exhibit peripheral hind leg neuropathy, the dose was lowered in stages to 1 g/l. These animals were sacrificed at 60 weeks due to the severe, irreversible neuropathy and were not included in this analysis. The remaining groups of animals were treated for 100 weeks. In the second study, rats were initially exposed to 2.5 g/l DCA which was lowered to 1 g/l after 18 weeks. The mean daily concentration (MDC) of 1.6 g/l was calculated over the 103 week exposure period. Time-weighted mean daily doses (MDD) based on measured water consumption were 3.6, 40.2 and 139 mg/kg bw/day for the 0.05, 0.5 and 1.6 g/l DCA respectively. Based upon the pathologic examination, DCA induced observable signs of toxicity in the nervous system, liver and myocardium. However, treatment related neoplastic lesions were observed only in the liver. A statistically significant increase of carcinogenicity (hepatocellular carcinoma) was noted at 1.6 g/l DCA. Exposure to 0.5 g/l DCA increased-hepatocellular neoplasia, (carcinoma and adenoma) at 100 weeks. These data demonstrate that DCA is an hepatocarcinogen to the male F344 rat. Calculation of the MDD at which 50% of the animals exhibited liver neoplasia indicated that the F344 male rat (approximately 10 mg/kg bw/day) is ten times more sensitive than the B6C3F1 male mouse (approximately 100 mg/kg bw/day). A "no observed effects level' (NOEL) of 0.05 g/l (3.6 mg/kg/day) was the same as for the mouse (3-8 mg/kg/day).

Lack of stereoselectivity of the peroxisome proliferation induced by 2-phenylpropionic acid: evidence against a role for lipid disturbance in peroxisome proliferation.

The significance of disturbances of lipid metabolism caused by xenobiotic acyl-CoAs as possible causes of peroxisomal proliferation has been studied with the enantiomers of 2-phenylpropionic acid (2-PPA), the (R)-enantiomer of which is converted to the acyl-CoA in rats while its (S)-antipode is not. rac-2-PPA (250 mg/kg/day ip x 3) was shown to be an hepatic peroxisomal proliferator in male Sprague-Dawley rats on the basis of increases in microsomal cytochrome P-450 content and lauric acid hydroxylation and hepatic CN(-)-insensitive palmitoyl-CoA oxidation, a peroxisomal marker activity, while electron microscopy revealed a rise in the peroxisome/mitochondria ratio in hepatocytes. Further studies established the dose-response relationships for these biochemical changes. The (R)- and (S)-enantiomers were administered at a dose of 50 mg/kg/day ip x 3 and both were peroxisome proliferators of very similar potency. The effects of 100 mg/kg/day ip x 3 of the racemate, a dose giving ca. 75% of maximal response, were essentially additive of those of 50 mg/kg/day ip x 3 of its two component isomers. The stereoselectivity of acyl-CoA formation from the enantiomers of 2-PPA was confirmed by their differential inhibition of microsomal palmitoyl-CoA synthesis. Taken together, these data indicate that it is very unlikely that the acyl-CoA of 2-PPA plays any role in the peroxisomal proliferation which this compound causes in the rat.

Synthesis of docosahexaenoyl coenzyme A in human spermatozoa.

The synthesis of docosahexaenoyl coenzyme A (22:6-CoA) was studied in a long-chain fatty acid: CoASH ligase (AMP)-enriched fraction from human spermatozoa and was compared to palmitoyl CoA (16:0-CoA) synthesis. The pH optimum for 22:6 activation was 8.4, which was identical to the value obtained with 16:0. The Km for ATP was 0.5 mM when 22:6 was the acyl substrate; however, when 16:0 was incubated with the ligase preparation, the Km for ATP was 2.9 mM. When CoASH was varied and 22:6 was the fatty acyl acceptor, a pattern of negative cooperatively was observed. This was confirmed by a downwardly concave double-reciprocal plot, a Hill coefficient of 0.63, and an Rs in excess of 150. The Hill coefficient with 16:0 and CoASH was 0.94. Palmitic acid was demonstrated to be a competitive inhibitor of 22:6-CoA synthesis. Based upon these data, we conclude that the kinetics of spermatozoan ligase are complex, and, in addition, these data support the hypothesis that 22:6 may regulate ligase activity, and therefore free fatty acid utilization, in sperm.

Inhibition kinetics of hepatic microsomal long chain fatty acid-CoA ligase by 2-arylpropionic acid non-steroidal anti-inflammatory drugs.

Microsomal long chain fatty acid CoA ligase (EC 6.2.1.3) has been implicated in the formation of CoA thioesters of xenobiotics containing a carboxylic acid moiety. In this study we have demonstrated that the microsomal enzyme from rat liver exhibits biphasic kinetics for the formation of palmitoyl-CoA, i.e. there are high affinity low capacity Kmhigh, 1.6 microM, Vmaxhigh, 12.9 nmol/mg/min) and low affinity high capacity (Kmlow, 506 microM, Vmaxlow, 58.3 nmol/mg/min) components. Inhibition of the high affinity isoform was studied using the R and S enantiomers of ibuprofen, fenoprofen, ketoprofen and naproxen. The high affinity component of palmitoyl-CoA formation was competitively inhibited by R-fenoprofen (Ki 15.4 microM) while R-ibuprofen exhibited mixed inhibition kinetics. In contrast the R and S enantiomers of ketoprofen and naproxen were non-competitive inhibitors. This diversity of inhibition kinetics observed argues in favour of a binding site in addition to the catalytic site. A competitive interaction with the high affinity form correlated with literature evidence of enantiospecific chiral inversion and "hybrid" triglyceride formation for the R enantiomers of fenoprofen and ibuprofen. Paradoxically, R-ketoprofen which is extensively inverted in rats was a non-competitive inhibitor of palmitoyl-CoA formation by the high affinity isoform suggesting that it may not act as an alternate substrate. The results of this study clearly indicate that formation of R-2-arylpropionate-CoAs is not fully explained by interaction with the high affinity isoform of a microsomal long chain (palmitoyl) CoA ligase and therefore the involvement of other isoforms cannot be discounted.

Hepatic peroxisomal and microsomal enzyme induction by citral and linalool in rats.

The short-term effects of oral administration of citral and linalool to rats have been compared. Male Wistar rats were given, by gastric intubation, 1.5 g citral or linalool/kg body weight/day for 5 days. Citral caused peroxisome proliferation as indicated by induction of cyanide-insensitive palmitoyl-CoA oxidation and bifunctional enzyme; levels of microsomal cytochrome P-450 IVA1 were also raised. Linalool caused induction of the peroxisomal enzymes but not of cytochrome P-450 IVA1, indicating that it possesses activity somewhat different from that of citral. These results suggest that the mechanisms of peroxisome proliferation may be independent of induction of cytochrome P-450 IVA1.

Arachidonoyl-coenzyme A synthetase and nonspecific acyl-coenzyme A synthetase activities in purified rat brain microvessels.

Purified rat brain microvessels were prepared to demonstrate the occurrence of acyl-CoA (EC 6.2.1.3) synthesis activity in the microvasculature of rat brain. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities showed an absolute requirement for ATP and CoA. This activity was strongly enhanced by magnesium chloride and inhibited by EDTA. The apparent Km values for acyl-CoA synthesis by purified rat brain microvessels were 4.0 microM and 5.8 microM for palmitic acid and arachidonic acid, respectively. The apparent Vmax values were 1.0 and 1.5 nmol X min-1 X mg protein-1 for palmitic acid and arachidonic acid, respectively. Cross-competition experiments showed inhibition of radiolabelled arachidonoyl-CoA formation by 15 microM unlabelled arachidonic acid, with a Ki of 7.1 microM, as well as by unlabelled docosahexaenoic acid, with a Ki of 8.0 microM. Unlabelled palmitic acid and arachidic acid had no inhibitory effect on arachidonoyl-CoA synthesis. In comparison, radiolabelled palmitoyl-CoA formation was inhibited competitively by 15 microM unlabelled palmitic acid, with a Ki of 5.0 microM and to a much lesser extent by arachidonic acid (Ki, 23 microM). The Vmax of palmitoyl-CoA formation obtained on incubation in the presence of the latter fatty acids was not changed. Unlabelled arachidic acid and docosahexaenoic acid had no inhibitory effect on palmitoyl-CoA synthesis. Both arachidonoyl-CoA and palmitoyl-CoA synthesis activities were thermolabile. Arachidonoyl-CoA formation was inhibited by 75% after 7 min at 40 degrees C whereas a 3-min heating treatment was sufficient to produce the same relative inhibition of palmitoyl-CoA synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

A high-performance liquid chromatographic method for the enantiomeric analysis of the enzymatically synthesized coenzyme A ester of 2-tetradecylglycidic acid.

The enantiomeric composition of an enzymatically synthesized sample of the coenzyme A ester of 2-tetradecylglycidic acid (TDGA-CoA) was determined by the use of high-performance liquid chromatography with a chiral stationary phase. The stationary phase was commercially available and consisted of (R)-N-(3,5-dinitrobenzoyl)phenylglycine covalently bonded to aminopropyl silica gel. Analysis was performed using the phenacyl derivative of 2-tetradecylglycidic acid (TDGA), obtained by mild hydrolysis of the TDGA-CoA followed by reaction of the extracted TDGA with phenacyl chloride. Chromatography showed the enantiomeric purity of TDGA-CoA, synthesized in a rat liver microsomal enzyme mixture over a 2-h period, to be a 15.6:1 ratio of the R:S enantiomers (88% ee). The result demonstrates the stereoselectivity of the long-chain fatty acid-coenzyme A synthetase for chiral fatty acid epoxide, TDGA.

Differential effects of phosphatidylcholine and cardiolipin on carnitine palmitoyltransferase activity.

Rates of carnitine palmitoyltransferase-catalyzed conversion of palmitoylcarnitine to palmitoyl-CoA are markedly decreased with the progress of this reaction presumably owing to the build up of inhibitory palmitoyl-CoA in the enzyme vicinity. High, above micellar, concentrations of palmitoylcarnitine, phosphatidylcholine liposomes and high KCl concentrations increased the activity, apparently by facilitating the removal of palmitoyl-CoA from the enzyme surface. The presence of cardiolipin was found to be inhibitory. The enzyme activity followed in the direction of palmitoylcarnitine formation with low palmitoyl-CoA concentration as substrate, was inhibited by phosphatidylcholine, but stimulated by cardiolipin. Both of these lipids markedly stimulated the enzyme activity followed by the isotope exchange procedure which requires progression of both the forward and the backward reactions. The results indicate that one of the effects of phospholipids on carnitine palmitoyltransferase activity is exerted from the ability of these substances to bind the amphipathic reactants of this enzyme, particularly long-chain acyl-CoA. The possibility that the activity of the membrane-bound carnitine palmitoyltransferase may at times be affected by changes in the concentrations and composition of the various phospholipids in the enzyme's vicinity is raised by these findings.

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A.

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.

Synthesis of docosahexaenoyl-, arachidonoyl- and palmitoyl-coenzyme A in ocular tissues.

The synthesis of long-chain acyl coenzyme A (CoA) was studied in the cornea, lens, vitreous, retina and pigment epithelium (PE) in the rat using [14C]-labeled palmitic, arachidonic and docosahexaenoic acids as substrates. Except for retina and PE, the ocular tissues studied showed relatively little enzyme activity with the fatty acid substrates. In addition, the enzyme activities were studied in homogenates and microsomal fractions from retina, pigment epithelial cells and choroid of frog, bovine and human eyes. Long-chain acyl CoA synthetase from the microsomal fraction exhibited three- to fivefold greater activity than homogenates in retina and PE. The enzyme activity was highest with palmitic acid, followed by arachidonic acid and docosahexaenoic acid. There were significant differences in enzyme activity between the species. The apparent Km (microM) and Vmax [nmol min-1 (mg protein)-1] values for the enzyme in bovine retinal microsomes were 7.91 +/- 0.39 (S.E.) and 21.6 +/- 1.04, respectively, for palmitic acid substrate and 5.88 +/- 0.25 and 4.58 +/- 0.21, respectively, for docosahexaenoic acid substrate. These values for bovine pigment epithelial microsomes were 13.0 +/- 0.27 and 36.9 +/- 1.18, respectively, for palmitic acid and 15.8 +/- 0.40 and 13.2 +/- 0.56, respectively, for docosahexaenoic acid. The synthesis of acyl CoA may play a central role in controlling the availability of free arachidonic acid for eicosanoid formation and in the retention of polyunsaturated fatty acid families (18:2, n-6 and 18:3, n-3) within cells of ocular tissues, particularly retina and retinal PE.

Lipogenesis in human adipose tissue in vitro: effect of fat cell size on some enzymatic activities.

The incorporation of [1-C14] palmitate into palmitoyl CoA and triglycerides by homogenates of human adipose tissue have been studied. Adipose tissue samples were taken from three sites varying in adipocytes size (omentum, subcutaneous abdominal wall, buttock). The donors were normal weight women of constant weight. A significant positive correlation was found between initial velocity of palmitoyl CoA synthetase (EC 6.2.1.3) and total [1-C14] palmitate incorporation into triglycerides on one hand and adipocyte cell size on the other hand : these relations with cell size were apparent both within and between individuals. The mechanism of this "size effect" which is unrelated to the higher protein content of larger cells, is still unexplained. Lipogenesis, like most of the metabolic activities of adipose tissue increases with enlarging cell size. Acceleration of a lipogenesis-lipolysis cycle could constitute an energy wasting system able to limit the volume of the adipocytes.

On the capacity of the beta-oxidation of palmitate and palmitoyl-esters in rat liver mitochondria.

The beta-oxidation of palmitate, palmitoyl-CoA and palmitoyl-L-carnitine proceeded at a high rate in isolated rat liver mitochondria. At high concentrations (100 nmol/mg protein) the oxidation of palmitate and palmitoyl-CoA was only partly carnitine dependent. All substrates were most rapidly oxidized in the presence of oxaloacetate and state 3 conditions. Succinate inhibited beta-oxidation especially in state 4 conditions. beta-Oxidation was faster in hypotonic than in isotonic medium both in state 3 and state 4 conditions. Hypertonicity inhibited beta-oxidation. The initial formation of palmitoyl-CoA from palmitate, CoA and ATP was faster than the oxidation of palmitate under identical conditions. The presence of bovine serum albumin inhibited the beta-oxidation, especially with palmitoyl-CoA or free palmitate as the substrates. Mitochondria contain a palmitoyl-CoA hydrolase which may influence the available intramitochondrial palmitoyl-CoA. The present results demonstrate no single rate limiting step in the beta-oxidation in vitro. Both the NADH/NAD ratio, competition for the respiratory chain, the level of ADP, binding of palmitoyl-CoA to extramitochondrial protein, and possibly intramitochondrial hydrolysis of palmitoyl-CoA all seem to influence the rate of beta-oxidation in vitro. It is suggested that in vivo the most important factor is the availability of acyl-CoA to the outer carnitine palmitoyl-transferase of the mitochondria.