SUMMARY Lipid droplets (LDs) store metabolic energy and membrane lipid precursors. With excess metabolic energy, cells synthesize triacylglycerol (TG) and form LDs that grow dramatically. It is unclear how TG synthesis relates to LD formation and growth. Here, we identify two LD subpopulations: smaller LDs of relatively constant size, and LDs that grow larger. The latter population contains isoenzymes for each step of TG synthesis. Glycerol-3-phosphate acyltransferase 4 (GPAT4), which catalyzes the first and rate-limiting step, relocalizes from the endoplasmic reticulum (ER) to a subset of forming LDs, where it becomes stably associated. ER-to-LD targeting of GPAT4 and other LD-localized TG synthesis isozymes is required for LD growth. Key features of GPAT4 ER-to-LD targeting and function in LD growth are conserved between Drosophila and mammalian cells. Our results explain how TG synthesis is coupled with LD growth and identify two distinct LD subpopulations based on their capacity for localized TG synthesis.
Liver fatty acid-binding protein (L-Fabp
The importance of cholesterol ester synthesis by acyl CoA:cholesterol acyltransferase (ACAT) enzymes in intestinal and hepatic cholesterol metabolism has been unclear. We now demonstrate that ACAT2 is the major ACAT in mouse small intestine and liver, and suggest that ACAT2 deficiency has profound effects on cholesterol metabolism in mice fed a cholesterol-rich diet, including complete resistance to diet-induced hypercholesterolemia and cholesterol gallstone formation. The underlying mechanism involves the lack of cholesterol ester synthesis in the intestine and a resultant reduced capacity to absorb cholesterol. Our results indicate that ACAT2 has an important role in the response to dietary cholesterol, and suggest that ACAT2 inhibition may be a useful strategy for treating hypercholesterolemia or cholesterol gallstones.
Dietary triacylglycerols are a major source of energy for animals. The absorption of dietary triacylglycerols involves their hydrolysis to free fatty acids and monoacylglycerols in the intestinal lumen, the uptake of these products into enterocytes, the resynthesis of triacylgylcerols, and the incorporation of newly synthesized triacylglycerols into nascent chylomicrons for secretion. In enterocytes, the final step in triacylglycerol synthesis is believed to be catalyzed primarily through the actions of acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. In this study, we analyzed intestinal triacylglycerol absorption and chylomicron synthesis and secretion in DGAT1-deficient (Dgat1 ؊/؊ ) mice. Surprisingly, DGAT1 was not essential for quantitative dietary triacylglycerol absorption, even in mice fed a high fat diet, or for the synthesis of chylomicrons. However, Dgat1 ؊/؊ mice had reduced postabsorptive chylomicronemia (1 h after a high fat challenge) and accumulated neutrallipid droplets in the cytoplasm of enterocytes when chronically fed a high fat diet. These results suggest a reduced rate of triacylglycerol absorption in Dgat1 mice. Analysis of intestine from Dgat1؊/؊ mice revealed activity for two other enzymes, DGAT2 and diacylglycerol transacylase, that catalyze triacylglycerol synthesis and apparently help to compensate for the absence of DGAT1. Our findings indicate that multiple mechanisms for triacylglycerol synthesis in the intestine facilitate triacylglycerol absorption.The absorption of triacylglycerols by the intestine is highly efficient, and more than 95% of dietary triacylglycerols is absorbed, even if the diet is rich in fat. By comparison, only 30 -70% of dietary cholesterol is absorbed in most animals (1). The high efficiency of triacylglycerol absorption is likely due to an evolutionary pressure that maximized the ability to absorb rich sources of energy (such as fat) when food sources were scarce.Intestinal triacylglycerol absorption occurs by a series of steps in which dietary triacylglycerols are first hydrolyzed in the intestinal lumen and then resynthesized within enterocytes. In the lumen, dietary triacylglycerols are hydrolyzed by lipases to generate free fatty acids and monoacylglycerols. These molecules are taken up by enterocytes and then enter the triacylglycerol biosynthesis pathways. The triacylglycerol products are incorporated into nascent chylomicrons, which are subsequently secreted from enterocytes and enter the lymphatic system.Triacylglycerol biosynthesis in the intestine is believed to occur mainly through the monoacylglycerol pathway. In this pathway, monoacylglycerol and fatty acyl-CoA are covalently joined to form diacylglycerol in a reaction catalyzed by monoacylglycerol acyltransferase (MGAT) 1 (2). Diacylglycerol and fatty acyl-CoA are then used to synthesize triacylglycerol in a reaction catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. High levels of DGAT activity are present in the small intestine (3-5), and both known DGAT genes, Dgat1...
The deleterious consequences of fatty acid (FA) and neutral lipid accumulation in nonadipose tissues, such as the heart, contribute to the pathogenesis of type 2 diabetes. To elucidate mechanisms of FA-induced cell death, or lipotoxicity, we generated Chinese hamster ovary (CHO) cell mutants resistant to palmitate-induced death and isolated a clone with disruption of eukaryotic elongation factor (eEF) 1A-1. eEF1A-1 involvement in lipotoxicity was confirmed in H9c2 cardiomyoblasts, in which small interfering RNA-mediated knockdown also conferred palmitate resistance. In wild-type CHO and H9c2 cells, palmitate increased reactive oxygen species and induced endoplasmic reticulum (ER) stress, changes accompanied by increased eEF1A-1 expression. Disruption of eEF1A-1 expression rendered these cells resistant to hydrogen peroxide-and ER stress-induced death, indicating that eEF1A-1 plays a critical role in the cell death response to these stressors downstream of lipid overload. Disruption of eEF1A-1 also resulted in actin cytoskeleton defects under basal conditions and in response to palmitate, suggesting that eEF1A-1 mediates lipotoxic cell death, secondary to oxidative and ER stress, by regulating cytoskeletal changes critical for this process. Furthermore, our observations of oxidative stress, ER stress, and induction of eEF1A-1 expression in a mouse model of lipotoxic cardiomyopathy implicate this cellular response in the pathophysiology of metabolic disease. INTRODUCTIONThe increased prevalence of obesity worldwide has contributed to the emergence of a disease cluster, the metabolic syndrome, that includes insulin resistance, type 2 diabetes, and cardiovascular disease. Elevated serum triglyceride and fatty acid (FA) levels associated with this syndrome contribute to lipid accumulation in many nonadipose tissues, including the heart. This inappropriate accumulation of excess lipid can lead to cellular dysfunction and cell death-a process called lipotoxicity (Unger, 2003).Lipotoxic cardiomyocyte death has been proposed to play a central role in heart failure associated with diabetes and obesity in animal models and in humans. Although FAs are the principal source of energy for cardiomyocytes, high serum triglyceride and FA levels result in FA uptake by the heart that exceeds the anabolic and catabolic needs of the tissue. Triglyceride accumulation in cardiomyocytes of leptin-or leptin receptor-deficient obese diabetic animal models is associated with cardiomyocyte apoptosis (Zhou et al., 2000) and contractile dysfunction (Zhou et al., 2000;Aasum et al., 2002;Christoffersen et al., 2003), suggesting that lipotoxic cell death in the heart may be important in the genesis of diabetic cardiomyopathy. Recently, similar observations were reported in patients with metabolic syndrome and nonischemic heart failure (Sharma et al., 2004). Consistent with this apparent cardiac lipotoxicity, cardiomyocyte-specific increases in FA uptake in mice with cardiac-restricted overexpression of long-chain acyl-CoA synthetase 1 (ACS1), li...
Deficiency of acyl CoA:cholesterol acyltransferase 2 (ACAT2) in mice results in a reduction in cholesterol ester synthesis in the small intestine and liver, which in turn limits intestinal cholesterol absorption, hepatic cholesterol gallstone formation, and the accumulation of cholesterol esters in the plasma lipoproteins. Here we examined the contribution of ACAT2-derived cholesterol esters to atherosclerosis by crossing ACAT2-deficient (ACAT2 -/-) mice with apolipoprotein (apo) E-deficient (ApoE -/-) mice, an atherosclerosissusceptible strain that has impaired apoE-mediated clearance of apoB-containing lipoproteins. ACAT2 -/-ApoE -/-mice and ACAT2 ؉/؉ ApoE -/-(control) mice had similar elevations of plasma apoB and total plasma lipids; however, the lipid cores of the apoB-containing lipoproteins in ACAT2 -/-ApoE -/-mice contained primarily triglycerides rather than cholesterol esters. At 30 wk of age, only the control mice had significant atherosclerosis, which was nearly absent in ACAT2 -/-ApoE -/-mice. ACAT2 deficiency in the apoE-deficient background also led to a compensatory increase in the activity of lecithin͞cholesterol acyltransferase, the major plasma cholesterol esterification enzyme, which increased highdensity lipoprotein cholesterol esters. Our results demonstrate the crucial role of ACAT2-derived cholesterol esters in the development of atherosclerosis in mice and suggest that triglyceride-rich apoBcontaining lipoproteins are not as atherogenic as those containing cholesterol esters. Our results also support the rationale of pharmacological inhibition of ACAT2 as a therapy for atherosclerosis.
The absorptive cells of the small intestine, enterocytes, are not generally thought of as a cell type that stores triacylglycerols (TGs) in cytoplasmic lipid droplets (LDs). We revisit TG metabolism in enterocytes by ex vivo and in vivo coherent anti-Stokes Raman scattering (CARS) imaging of small intestine of mice during dietary fat absorption (DFA). We directly visualized the presence of LDs in enterocytes. We determined lipid amount and quantified LD number and size as a function of intestinal location and time post-lipid challenge via gavage feeding. The LDs were confirmed to be primarily TG by biochemical analysis. Combined CARS and fluorescence imaging indicated that the large LDs were located in the cytoplasm, associated with the tail-interacting protein of 47 kDa. Furthermore, in vivo CARS imaging showed real-time variation in the amount of TG stored in LDs through the process of DFA. Our results highlight a dynamic, cytoplasmic TG pool in enterocytes that may play previously unexpected roles in processes, such as regulating postprandial blood TG concentrations. With obesity and cardiovascular disease being worldwide health issues, the impetus of understanding the parameters that govern energy intake and blood lipid concentration has emerged. Blood lipid concentration and a major portion of energy intake are regulated through the highly efficient process of dietary fat absorption (DFA) by the small intestine. Greater than 95% of dietary fat consumed is absorbed whether a low-or high-fat diet is consumed (1), as evidenced by the small amount of fat that is excreted in feces. In the small intestine lumen, dietary fat in the form of triacylglycerol (TG) is hydrolyzed to generate FFAs and monoacylglycerols (MGs) by pancreatic lipase. These products are then emulsified with the help of phospholipids and bile acids present in bile to form micelles. FFAs and MGs are then taken up by the absorptive cells of the small intestine, enterocytes, where they are resynthesized into TGs and incorporated into the core of chylomicrons (CMs), which are secreted via the lymphatic system into the circulation (2, 3).Most current models of the synthesis of CMs show the resynthesized TG in this process entering the lumen of the endoplasmic reticulum (ER) where the assembly of CMs begins and do not highlight the potential for the resynthesized TG to enter a cytoplasmic TG storage pool unless there is a defect in CM synthesis or secretion (1,(3)(4)(5)(6)(7)(8). In fact, wild-type mice fed a high-fat diet, or challenged with an oil bolus by oral gavage, are commonly reported as having no lipid droplet (LD) accumulation in enterocytes (9-11). Nevertheless, both indirect and direct evidence exists supporting the presence of a cytoplasmic storage pool in enterocytes. In humans, sequential meal tests demonstrated that CMs secreted after a second meal carried TG ingested in the first meal (12, 13). In rats, TG is synthesized within 30 s after an intraduodenal fat infusion (14); however, the secretion of TG into the lymph does n...
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