Douglas G. Mashek

The role of lipid droplets in metabolic disease in rodents and humans

Lipid droplets (LDs) are intracellular organelles that store neutral lipids within cells. Over the last two decades there has been a dramatic growth in our understanding of LD biology and, in parallel, our understanding of the role of LDs in health and disease. In its simplest form, the LD regulates the storage and hydrolysis of neutral lipids, including triacylglycerol and/or cholesterol esters. It is becoming increasingly evident that alterations in the regulation of LD physiology and metabolism influence the risk of developing metabolic diseases such as diabetes. In this review we provide an update on the role of LD-associated proteins and LDs in metabolic disease.

Adipose triglyceride lipase is a major hepatic lipase that regulates triacylglycerol turnover and fatty acid signaling and partitioning

Despite advances in our understanding of the ways in which nutrient oversupply and triacylglycerol (TAG) anabolism contribute to hepatic steatosis, little is known about the lipases responsible for regulating hepatic TAG turnover. Recent studies have identified adipose triglyceride lipase (ATGL) as a major lipase in adipose tissue, although its role in the liver is largely unknown. Thus, we tested the contribution of ATGL to hepatic lipid metabolism and signaling. Adenovirus‐mediated knockdown of hepatic ATGL resulted in steatosis in mice and decreased hydrolysis of TAG in primary hepatocyte cultures and in vitro assays. In addition to altering TAG hydrolysis, ATGL was shown to play a significant role in partitioning hydrolyzed fatty acids between metabolic pathways. Although ATGL gain and loss of function did not alter hepatic TAG secretion, fatty acid oxidation was increased by ATGL overexpression and decreased by ATGL knockdown. The effects on fatty acid oxidation coincided with decreased expression of peroxisome proliferator‐activated receptor α (PPAR‐α) and its target genes in mice with suppressed hepatic ATGL expression. However, PPAR‐α agonism was unable to normalize the effects of ATGL knockdown on PPAR‐α target gene expression, and this suggests that ATGL influences PPAR‐α activity independently of ligand‐induced activation. Conclusion: Taken together, these data show that ATGL is a major hepatic TAG lipase that plays an integral role in fatty acid partitioning and signaling to control energy metabolism. (HEPATOLOGY 2011)

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.

Mammalian Triacylglycerol Metabolism: Synthesis, Lipolysis, and Signaling

Triacylglycerol (TAG) is the primary unit of energy storage in eukaryotic cells. TAG comprises more than 90% of the content of white adipocytes, and TAG synthesis is essential in enterocytes for the absorption of dietary lipids, in hepatocytes for the synthesis and transport of VLDL, and in mammary epithelial cells for the production of milk. TAG is critical for the formation of the water barrier of skin, it acts as a mechanical cushion within joints and around internal organs, and it also serves an important role in insulation. In virtually every type of cell, stored TAG sequesters essential fatty acids (FAs) and precursors of eicosanoids, as well as the DAG precursor of phospholipids. Storage of FAs in TAG protects cells from the potential detergent-like properties of FAs or their acyl-CoA derivatives which may injure cellular membranes. In addition, recent studies suggest that cellular stores of TAG in lipid droplets release FAs that are channeled selectively to β-oxidation and that act as signals to influence the transcriptional control of gene expression. Additional studies suggest that the process of synthesizing or degrading TAG produces lipid intermediates like lysophosphatidic acid (LPA), phosphatidic acid (PA), and diacylglycerol (DAG) that may serve as activators or inhibitors of signaling pathways controlled by peroxisome proliferator-activated receptor-γ (PPARγ), the mammalian target of rapamycin (mTOR), or protein kinase C (PKC) isoforms. These signaling pathways may link excess intracellular TAG storage with insulin resistance. It is the goal of this review to provide an overview of TAG metabolism as a dynamic process that allows its lipid participants to play numerous inter-related roles within cells. Thus, we will focus on both the synthesis and degradation of TAG, the enzymes involved and their regulation, and the overall physiological regulation of these dynamic processes. In addition to the lipolytic enzymes, this review will also highlight co-regulatory proteins that affect the activity of specific lipases. We have chosen to focus primarily on information reported during the past 10 years and recommend to readers several excellent reviews that present more detailed information from older studies: for the synthetic pathway as a whole,1 for GPAT,2 for GPAT, AGPAT and PAP,3–4 for DGAT,5 for the lipolytic pathway and ATGL,6 for HSL,7 and for MGL.8

Cellular fatty acid uptake: The contribution of metabolism

Purpose of review The aim of this review is to highlight the importance of fatty acid metabolism as a major determinant in fatty acid uptake. In particular, we emphasize how the activation, intracellular transport and downstream metabolism of fatty acids influence their uptake into cells. Recent findings Studies examining fatty acid entry into cells have focused primarily on the roles of plasma membrane proteins or the question of passive diffusion. Recent studies, however, strongly suggest that a driving force governing fatty acid uptake is the metabolic demand for fatty acids. Both gain and loss-of-function experiments indicate that fatty acid uptake can be modulated by activation at both the plasma membrane and internal sites, by intracellular fatty acid binding proteins, and by enzymes in synthetic or degradative metabolic pathways. Although the mechanism is not known, it appears that converting fatty acids to acyl-CoAs and downstream metabolic intermediates increases cellular fatty acid uptake, probably by limiting efflux. Summary Altered fatty acid metabolism and the accumulation of triacylglycerol and lipid metabolites has been strongly associated with insulin resistance and diabetes, but we do not fully understand how the entry of fatty acids into cells is regulated. Future studies of cellular fatty acid uptake should consider the influence of fatty acid metabolism and the possible interactions between fatty acid metabolism or metabolites and fatty acid transport proteins.

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.

Rat long chain acyl-CoA synthetase 5 increases fatty acid uptake and partitioning to cellular triacylglycerol in McArdle-RH7777 cells.

Long chain acyl-CoA synthetase (ACSL) catalyzes the initial step in long chain fatty acid metabolism. Of the five mammalian ACSL isoforms cloned and characterized, ACSL5 is the only isoform found to be located, in part, on mitochondria and thus was hypothesized to be involved in fatty acid oxidation. To elucidate the specific roles of ACSL5 in fatty acid metabolism, we used adenoviral-mediated overexpression of ACSL5 (Ad-ACSL5) in rat hepatoma McArdle-RH7777 cells. Confocal microscopy revealed that Ad-ACSL5 colocalized to both mitochondria and endoplasmic reticulum. When compared with cells infected with Ad-GFP, Ad-ACSL5-infected cells at 24 h after infection had 2-fold higher acyl-CoA synthetase activities and 30% higher rates of fatty acid uptake when incubated with 500 μm [1-14C]oleic acid. Metabolism of [1-14C]oleic acid to cellular triacylglycerol (TAG) increased 42% in Ad-ACSL5-infected cells, but when compared with control cells, metabolism to acid-soluble metabolites, phospholipids, and medium TAG did not differ substantially. The incorporation of [1-14C]oleate and [1,2,3-3H]glycerol into TAG was similar in Ad-ACSL5-infected cells, thus indicating that Ad-ACSL5 increased TAG synthesis through both de novo and reacylation pathways. However, [1-14C]acetic acid incorporation into cellular lipids showed that, when compared with control cells, Ad-ACSL5-infected cells did not increase the metabolism of fatty acids that were derived from de novo synthesis. These results suggest that uptake of fatty acids into cells is regulated by metabolism and that overexpressed ACSL5 partitions exogenously derived fatty acids toward TAG synthesis and storage.

Palmitoleate induces hepatic steatosis but suppresses liver inflammatory response in mice.

The interaction between fat deposition and inflammation during obesity contributes to the development of non-alcoholic fatty liver disease (NAFLD). The present study examined the effects of palmitoleate, a monounsaturated fatty acid (16∶1n7), on liver metabolic and inflammatory responses, and investigated the mechanisms by which palmitoleate increases hepatocyte fatty acid synthase (FAS) expression. Male wild-type C57BL/6J mice were supplemented with palmitoleate and subjected to the assays to analyze hepatic steatosis and liver inflammatory response. Additionally, mouse primary hepatocytes were treated with palmitoleate and used to analyze fat deposition, the inflammatory response, and sterol regulatory element-binding protein 1c (SREBP1c) activation. Compared with controls, palmitoleate supplementation increased the circulating levels of palmitoleate and improved systemic insulin sensitivity. Locally, hepatic fat deposition and SREBP1c and FAS expression were significantly increased in palmitoleate-supplemented mice. These pro-lipogenic events were accompanied by improvement of liver insulin signaling. In addition, palmitoleate supplementation reduced the numbers of macrophages/Kupffer cells in livers of the treated mice. Consistently, supplementation of palmitoleate decreased the phosphorylation of nuclear factor kappa B (NF-κB, p65) and the expression of proinflammatory cytokines. These results were recapitulated in primary mouse hepatocytes. In terms of regulating FAS expression, treatment of palmitoleate increased the transcription activity of SREBP1c and enhanced the binding of SREBP1c to FAS promoter. Palmitoleate also decreased the phosphorylation of NF-κB p65 and the expression of proinflammatory cytokines in cultured macrophages. Together, these results suggest that palmitoleate acts through dissociating liver inflammatory response from hepatic steatosis to play a unique role in NAFLD.

Hepatic triacylglycerol hydrolysis regulates peroxisome proliferator-activated receptor α activity

Recent evidence suggests that fatty acids generated from intracellular triacylglycerol (TAG) hydrolysis may have important roles in intracellular signaling. This study was conducted to determine if fatty acids liberated from TAG hydrolysis regulate peroxisome proliferator-activated receptor &agr; (PPAR&agr;). Primary rat hepatocyte cultures were treated with adenoviruses overexpressing adipose differentiation-related protein (ADRP) or adipose triacylglycerol lipase (ATGL) or treated with short interfering RNA (siRNA) targeted against ADRP. Subsequent effects on TAG metabolism and PPAR&agr; activity and target gene expression were determined. Overexpressing ADRP attenuated TAG hydrolysis, whereas siRNA-mediated knockdown of ADRP or ATGL overexpression resulted in enhanced TAG hydrolysis. Results from PPAR&agr; reporter activity assays demonstrated that decreasing TAG hydrolysis by ADRP overexpression resulted in a 35–60% reduction in reporter activity under basal conditions or in the presence of fatty acids. As expected, PPAR&agr; target genes were also decreased in response to ADRP overexpression. However, the PPAR&agr; ligand, WY-14643, was able to restore PPAR&agr; activity following ADRP overexpression. Despite its effects on PPAR&agr;, overexpressing ADRP did not affect PPAR&ggr; activity. Enhancing TAG hydrolysis through ADRP knockdown or ATGL overexpression increased PPAR&agr; activity. These results indicate that TAG hydrolysis and the consequential release of fatty acids regulate PPAR&agr; activity.

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.