Lei O. Li

Adipose Acyl-CoA synthetase-1 directs fatty acids toward β-oxidation and is required for cold thermogenesis

Long-chain acyl-CoA synthetase-1 (ACSL1) contributes 80% of total ACSL activity in adipose tissue and was believed to be essential for the synthesis of triacylglycerol. We predicted that an adipose-specific knockout of ACSL1 (Acsl1(A-/-)) would be lipodystrophic, but compared to controls, Acsl1(A-/-) mice had 30% greater fat mass when fed a low-fat diet and gained weight normally when fed a high-fat diet. Acsl1(A-/-) adipocytes incorporated [(14)C]oleate into glycerolipids normally, but fatty acid (FA) oxidation rates were 50%-90% lower than in control adipocytes and mitochondria. Acsl1(A-/-) mice were markedly cold intolerant, and beta(3)-adrenergic agonists did not increase oxygen consumption, despite normal adrenergic signaling in brown adipose tissue. The reduced adipose FA oxidation and marked cold intolerance of Acsl1(A-/-) mice indicate that normal activation of FA for oxidation in adipose tissue in vivo requires ACSL1. Thus, ACSL1 has a specific function in directing the metabolic partitioning of FAs toward beta-oxidation in adipocytes.

Acyl-CoA synthesis, lipid metabolism and lipotoxicity

Although the underlying causes of insulin resistance have not been completely delineated, in most analyses, a recurring theme is dysfunctional metabolism of fatty acids. Because the conversion of fatty acids to activated acyl-CoAs is the first and essential step in the metabolism of long-chain fatty acid metabolism, interest has grown in the synthesis of acyl-CoAs, their contribution to the formation of signaling molecules like ceramide and diacylglycerol, and their direct effects on cell function. In this review, we cover the evidence for the involvement of acyl-CoAs in what has been termed lipotoxicity, the regulation of the acyl-CoA synthetases, and the emerging functional roles of acyl-CoAs in the major tissues that contribute to insulin resistance and lipotoxicity, adipose, liver, heart and pancreas.

Rat long-chain acyl-CoA synthetase mRNA, protein, and activity vary in tissue distribution and in response to diet

Distinct isoforms of long-chain acyl-CoA synthetases (ACSLs) may partition fatty acids toward specific metabolic cellular pathways. For each of the five members of the rat ACSL family, we analyzed tissue mRNA distributions, and we correlated the mRNA, protein, and activity of ACSL1 and ACSL4 after fasting and refeeding a 69% sucrose diet. Not only did quantitative real-time PCR analyses reveal unique tissue expression patterns for each ACSL isoform, but expression varied markedly in different adipose depots. Fasting increased ACSL4 mRNA abundance in liver, muscle, and gonadal and inguinal adipose tissues, and refeeding decreased ACSL4 mRNA. A similar pattern was observed for ACSL1, but both fasting and refeeding decreased ACSL1 mRNA in gonadal adipose. Fasting also decreased ACSL3 and ACSL5 mRNAs in liver and ACSL6 mRNA in muscle. Surprisingly, in nearly every tissue measured, the effects of fasting and refeeding on the mRNA abundance of ACSL1 and ACSL4 were discordant with changes in protein abundance. These data suggest that the individual ACSL isoforms are distinctly regulated across tissues and show that mRNA expression may not provide useful information about isoform function. They further suggest that translational or posttranslational modifications are likely to contribute to the regulation of ACSL isoforms.

Diabetes promotes an inflammatory macrophage phenotype and atherosclerosis through acyl-CoA synthetase 1

The mechanisms that promote an inflammatory environment and accelerated atherosclerosis in diabetes are poorly understood. We show that macrophages isolated from two different mouse models of type 1 diabetes exhibit an inflammatory phenotype. This inflammatory phenotype associates with increased expression of long-chain acyl-CoA synthetase 1 (ACSL1), an enzyme that catalyzes the thioesterification of fatty acids. Monocytes from humans and mice with type 1 diabetes also exhibit increased ACSL1. Furthermore, myeloid-selective deletion of ACSL1 protects monocytes and macrophages from the inflammatory effects of diabetes. Strikingly, myeloid-selective deletion of ACSL1 also prevents accelerated atherosclerosis in diabetic mice without affecting lesions in nondiabetic mice. Our observations indicate that ACSL1 plays a critical role by promoting the inflammatory phenotype of macrophages associated with type 1 diabetes; they also raise the possibilities that diabetic atherosclerosis has an etiology that is, at least in part, distinct from the etiology of nondiabetic vascular disease and that this difference is because of increased monocyte and macrophage ACSL1 expression.

Long-chain acyl-CoA synthetases and fatty acid channeling

Thirteen homologous proteins comprise the long-chain acyl-CoA synthetase (ACSL), fatty acid transport protein (FATP), and bubblegum (ACSBG) subfamilies that activate long-chain and very-long-chain fatty acids to form acyl-CoAs. Gain- and loss-of-function studies show marked differences in the ability of these enzymes to channel fatty acids into different pathways of complex lipid synthesis. Further, the ability of the ACSLs and FATPs to enhance cellular FA uptake does not always require these proteins to be present on the plasma membrane; instead, FA uptake can be increased by enhancing its conversion to acyl-CoA and its metabolism in downstream pathways. Since altered fatty acid metabolism is a hallmark of numerous metabolic diseases and pathological conditions, the ACSL, FATP and ACSBG isoforms are likely to play important roles in disease etiology.

Liver-specific Loss of Long Chain Acyl-CoA Synthetase-1 Decreases Triacylglycerol Synthesis and β-Oxidation and Alters Phospholipid Fatty Acid Composition

In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25–35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1L−/− liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [14C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [14C]oleate suggested that hepatic ACSL1 is important for mitochondrial β-oxidation of long chain fatty acids. Because the Acsl1L−/− mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance.

Acyl-coenzyme A synthetases in metabolic control

Purpose of review The 11 long-chain (ACSL) and very long chain acyl-coenzyme A (acyl-CoA) synthetases [(ACSVL)/fatty acid transport protein] are receiving considerable attention because it has become apparent that their individual functions are not redundant. Recent findings Recent studies have focused on the structure of the acyl-CoA synthetases, their post-translational modification, their ability to activate fatty acids of varying chain lengths, and their role in directing fatty acids into different metabolic pathways. An unsettled controversy focuses on the ACSVL isoforms and whether these have both enzymatic and transport functions. Another issue is whether conversion of a fatty acid to an acyl-CoA produces an increase in the AMP/ATP ratio that is sufficient to activate AMP-activated kinase. Summary Future studies are required to determine the subcellular location of each ACSL and ACSVL isoform and the functional importance of phosphorylation and acetylation. Purification and crystallization of mammalian ACSL and ACSVL isoforms is needed to confirm the mechanism of action and discover how these enzymes differ in their affinity for fatty acids of different chain lengths. Functionally, it will be important to learn how the ACSL isoforms can direct their acyl-CoA products toward independent downstream pathways.

Overexpression of Rat Long Chain Acyl-CoA Synthetase 1 Alters Fatty Acid Metabolism in Rat Primary Hepatocytes

Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 μm [1-14C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, β-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and β-oxidation. The increase in [1,2,3-3H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [14C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [14C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.

Ribosomal protein-Mdm2-p53 pathway coordinates nutrient stress with lipid metabolism by regulating MCD and promoting fatty acid oxidation.

Significance Although progress has been made in the characterization of p53 in regulating metabolism, very little is known about the signaling pathways involved in this regulation in response to stress in vivo. Here we show that p53 controls hepatic fatty acid oxidation in mice in response to fasting. Disruption of ribosome protein (RP)-mouse double minute (Mdm)2 binding in Mdm2C305F mice results in fasting-induced hepatosteatosis. A full-dosage of p53 and an intact RP-Mdm2-p53 pathway are required for the induction of malonyl coA decarboxylase (MCD), a critical regulator of fatty acid oxidation. Thus, the RP-Mdm2-p53 pathway functions as a key regulator of hepatic lipid homeostasis in response to nutrient deprivation stress, a function that has implications in organismal survival and tumor suppression. The tumor suppressor p53 has recently been shown to regulate energy metabolism through multiple mechanisms. However, the in vivo signaling pathways related to p53-mediated metabolic regulation remain largely uncharacterized. By using mice bearing a single amino acid substitution at cysteine residue 305 of mouse double minute 2 (Mdm2C305F), which renders Mdm2 deficient in binding ribosomal proteins (RPs) RPL11 and RPL5, we show that the RP–Mdm2–p53 signaling pathway is critical for sensing nutrient deprivation and maintaining liver lipid homeostasis. Although the Mdm2C305F mutation does not significantly affect growth and development in mice, this mutation promotes fat accumulation under normal feeding conditions and hepatosteatosis under acute fasting conditions. We show that nutrient deprivation inhibits rRNA biosynthesis, increases RP–Mdm2 interaction, and induces p53-mediated transactivation of malonyl-CoA decarboxylase (MCD), which catalyzes the degradation of malonyl-CoA to acetyl-CoA, thus modulating lipid partitioning. Fasted Mdm2C305F mice demonstrate attenuated MCD induction and enhanced malonyl-CoA accumulation in addition to decreased oxidative respiration and increased fatty acid accumulation in the liver. Thus, the RP–Mdm2–p53 pathway appears to function as an endogenous sensor responsible for stimulating fatty acid oxidation in response to nutrient depletion.

Early Hepatic Insulin Resistance in Mice: A Metabolomics Analysis

When fed with a high-fat safflower oil diet for 3 wk, wild-type mice develop hepatic insulin resistance, whereas mice lacking glycerol-3-phosphate acyltransferase-1 retain insulin sensitivity. We examined early changes in the development of insulin resistance via liver and plasma metabolome analyses that compared wild-type and glycerol-3-phosphate acyltransferase-deficient mice fed with either a low-fat or the safflower oil diet for 3 wk. We reasoned that diet-induced changes in metabolites that occurred only in the wild-type mice would reflect those metabolites that were specifically related to hepatic insulin resistance. Of the identifiable metabolites (from 322 metabolites) in liver, wild-type mice fed with the high-fat diet had increases in urea cycle intermediates, consistent with increased deamination of amino acids used for gluconeogenesis. Also increased were stearoylglycerol, gluconate, glucarate, 2-deoxyuridine, and pantothenate. Decreases were observed in S-adenosylhomocysteine, lactate, the bile acid taurocholate, and 1,5-anhydroglucitol, a previously identified marker of short-term glycemic control. Of the identifiable metabolites (from 258 metabolites) in plasma, wild-type mice fed with the high-fat diet had increases in plasma stearate and two pyrimidine-related metabolites, whereas decreases were found in plasma bradykinin, alpha-ketoglutarate, taurocholate, and the tryptophan metabolite, kynurenine. This study identified metabolites previously not known to be associated with insulin resistance and points to the utility of metabolomics analysis in identifying unrecognized biochemical pathways that may be important in understanding the pathophysiology of diabetes.