Regulatory effects of arachidonate 5-lipoxygenase on hepatic microsomal TG transfer protein activity and VLDL-triglyceride and apoB secretion in obese mice

As 5-lipoxygenase (5-LO) is an emerging target in obesity and insulin resistance, we have investigated whether this arachidonate pathway is also implicated in the progression of obesity-related fatty liver disease. Our results show that 5-LO activity and 5-LO-derived product levels are significantly elevated in the liver of obese ob/ob mice with respect to wild-type controls. Treatment of ob/ob mice with a selective 5-LO inhibitor exerted a remarkable protection from hepatic steatosis as revealed by decreased oil red-O staining and reduced hepatic triglyceride (TG) concentrations. In addition, 5-LO inhibition in ob/ob mice downregulated genes involved in hepatic fatty acid uptake (i.e., L-FABP and FAT/CD36) and normalized peroxisome proliferator-activated receptor alpha (PPARalpha) and acyl-CoA oxidase expression, whereas the expression of lipogenic genes [i.e., fatty acid synthase (FASN) and SREBP-1c] remained unaltered. Furthermore, 5-LO inhibition restored hepatic microsomal TG transfer protein (MTP) activity in parallel with a stimulation of hepatic VLDL-TG and apoB secretion in ob/ob mice. Consistent with these findings, 5-LO products directly inhibited MTP activity and triggered cytosolic TG accumulation in CC-1 cells, a murine hepatocyte cell line. Taken together, these findings identify a novel steatogenic role for 5-LO in the liver through mechanisms involving the regulation of hepatic MTP activity and VLDL-TG and apoB secretion.     

Hepatic triglyceride synthesis and nonalcoholic fatty liver disease

PURPOSE OF REVIEW: Nonalcoholic fatty liver disease is a spectrum of diseases ranging from simple steatosis to cirrhosis. The hallmark of nonalcoholic fatty liver disease is hepatocyte accumulation of triglycerides. We will review the role of triglyceride synthesis in nonalcoholic fatty liver disease progression and summarize recent findings about triglyceride synthesis inhibition and prevention of progressive disease. RECENT FINDINGS: Attempts to inhibit triglyceride synthesis in animal models have resulted in improvement in hepatic steatosis. Studies in animal models of nonalcoholic fatty liver disease demonstrate that inhibition of acyl-coenzyme A:diacylglycerol acyltransferase, the enzyme that catalyzes the final step in triglyceride synthesis, results in improvement in hepatic steatosis and insulin sensitivity. We recently confirmed that hepatic specific inhibition of acyl-coenzyme A:diacylglycerol acyltransferase with antisense oligonucleotides improves hepatic steatosis in obese, diabetic mice but, unexpectedly, exacerbated injury and fibrosis in that model of progressive nonalcoholic fatty liver disease. When hepatocyte triglyceride synthesis was inhibited, free fatty acids accumulated in the liver, leading to induction of fatty acid oxidizing systems that increased hepatic oxidative stress and liver damage. These findings suggest that the ability to synthesize triglycerides may, in fact, be protective in obesity. SUMMARY: Nonalcoholic fatty liver disease is strongly associated with obesity and peripheral insulin resistance. Peripheral insulin resistance increases lipolysis in adipose depots, promoting increased free fatty acid delivery to the liver. In states of energy excess, such as obesity, the latter normally triggers hepatic triglyceride synthesis. When hepatic triglyceride synthesis is unable to accommodate increased hepatocyte free fatty acid accumulation, however, lipotoxicity results. Thus, rather than being hepatotoxic, liver triglyceride accumulation is actually hepato-protective in obese, insulin-resistant individuals.     

Suppression of diacylglycerol acyltransferase-2 (DGAT2), but not DGAT1, with antisense oligonucleotides reverses diet-induced hepatic steatosis and insulin resistance

Nonalcoholic fatty liver disease (NAFLD) is a major contributing factor to hepatic insulin resistance in type 2 diabetes. Diacylglycerol acyltransferase (Dgat), of which there are two isoforms (Dgat1 and Dgat2), catalyzes the final step in triglyceride synthesis. We evaluated the metabolic impact of pharmacological reduction of DGAT1 and -2 expression in liver and fat using antisense oligonucleotides (ASOs) in rats with diet-induced NAFLD. Dgat1 and Dgat2 ASO treatment selectively reduced DGAT1 and DGAT2 mRNA levels in liver and fat, but only Dgat2 ASO treatment significantly reduced hepatic lipids (diacylglycerol and triglyceride but not long chain acyl CoAs) and improved hepatic insulin sensitivity. Because Dgat catalyzes triglyceride synthesis from diacylglycerol, and because we have hypothesized that diacylglycerol accumulation triggers fat-induced hepatic insulin resistance through protein kinase C epsilon activation, we next sought to understand the paradoxical reduction in diacylglycerol in Dgat2 ASO-treated rats. Within 3 days of starting Dgat2 ASO therapy in high fat-fed rats, plasma fatty acids increased, whereas hepatic lysophosphatidic acid and diacylglycerol levels were similar to those of control rats. These changes were associated with reduced expression of lipogenic genes (SREBP1c, ACC1, SCD1, and mtGPAT) and increased expression of oxidative/thermogenic genes (CPT1 and UCP2). Taken together, these data suggest that knocking down Dgat2 protects against fat-induced hepatic insulin resistance by paradoxically lowering hepatic diacylglycerol content and protein kinase C epsilon activation through decreased SREBP1c-mediated lipogenesis and increased hepatic fatty acid oxidation.     

Liver triacylglycerol lipases

The hallmark of obesity and one of the key contributing factors to insulin resistance, type 2 diabetes and cardiovascular disease is excess triacylglycerol (TG) storage. In hepatocytes, excessive accumulation of TG is the common denominator of a wide range of clinicopathological entities known as nonalcoholic fatty liver disease, which can eventually progress to cirrhosis and associated complications including hepatic failure, hepatocellular carcinoma and death. A tight regulation between TG synthesis, hydrolysis, secretion and fatty acid oxidation is required to prevent lipid accumulation as well as lipid depletion from hepatocytes. Therefore, understanding the pathways that regulate hepatic TG metabolism is crucial for development of therapies to ameliorate pathophysiological conditions associated with excessive hepatic TG accumulation, including dyslipidemias, viral infection and atherosclerosis. This review highlights the physiological roles of liver lipases that degrade TG in cytosolic lipid droplets, endoplasmic reticulum, late endosomes/lysosomes and along the secretory route. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

Liver triacylglycerol lipases

The hallmark of obesity and one of the key contributing factors to insulin resistance, type 2 diabetes and cardiovascular disease is excess triacylglycerol (TG) storage. In hepatocytes, excessive accumulation of TG is the common denominator of a wide range of clinicopathological entities known as nonalcoholic fatty liver disease, which can eventually progress to cirrhosis and associated complications including hepatic failure, hepatocellular carcinoma and death. A tight regulation between TG synthesis, hydrolysis, secretion and fatty acid oxidation is required to prevent lipid accumulation as well as lipid depletion from hepatocytes. Therefore, understanding the pathways that regulate hepatic TG metabolism is crucial for development of therapies to ameliorate pathophysiological conditions associated with excessive hepatic TG accumulation, including dyslipidemias, viral infection and atherosclerosis. This review highlights the physiological roles of liver lipases that degrade TG in cytosolic lipid droplets, endoplasmic reticulum, late endosomes/lysosomes and along the secretory route. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.     

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.     

Sixteen hours of fasting differentially affects hepatic and muscle insulin sensitivity in mice

Fasting readily induces hepatic steatosis. Hepatic steatosis is associated with hepatic insulin resistance. The purpose of the present study was to document the effects of 16 h of fasting in wild-type mice on insulin sensitivity in liver and skeletal muscle in relation to 1) tissue accumulation of triglycerides (TGs) and 2) changes in mRNA expression of metabolically relevant genes. Sixteen hours of fasting did not show an effect on hepatic insulin sensitivity in terms of glucose production in the presence of increased hepatic TG content. In muscle, however, fasting resulted in increased insulin sensitivity, with increased muscle glucose uptake without changes in muscle TG content. In liver, fasting resulted in increased mRNA expression of genes promoting gluconeogenesis and TG synthesis but in decreased mRNA expression of genes involved in glycogenolysis and fatty acid synthesis. In muscle, increased mRNA expression of genes promoting glucose uptake, as well as lipogenesis and beta-oxidation, was found. In conclusion, 16 h of fasting does not induce hepatic insulin resistance, although it causes liver steatosis, whereas muscle insulin sensitivity increases without changes in muscle TG content. Therefore, fasting induces differential changes in tissue-specific insulin sensitivity, and liver and muscle TG contents are unlikely to be involved in these changes.     

Coordinated defects in hepatic long chain fatty acid metabolism and triglyceride accumulation contribute to insulin resistance in non-human primates

Non-Alcoholic fatty liver disease (NAFLD) is characterized by accumulation of triglycerides (TG) in hepatocytes, which may also trigger cirrhosis. The mechanisms of NAFLD are not fully understood, but insulin resistance has been proposed as a key determinant.

Aims

To determine the TG content and long chain fatty acyl CoA composition profile in liver from obese non-diabetic insulin resistant (IR) and lean insulin sensitive (IS) baboons in relation with hepatic and peripheral insulin sensitivity.

Methods

Twenty baboons with varying grades of adiposity were studied. Hepatic (liver) and peripheral (mainly muscle) insulin sensitivity was measured with a euglycemic clamp and QUICKI. Liver biopsies were performed at baseline for TG content and LCFA profile by mass spectrometry, and histological analysis. Findings were correlated with clinical and biochemical markers of adiposity and insulin resistance.

Results

Obese IR baboons had elevated liver TG content compared to IS. Furthermore, the concentration of unsaturated (LC-UFA) was greater than saturated (LC-SFA) fatty acyl CoA in the liver. Interestingly, LC-FA UFA and SFA correlated with waist, BMI, insulin, NEFA, TG, QUICKI, but not M/I. Histological findings of NAFLD ranging from focal to diffuse hepatic steatosis were found in obese IR baboons.

Conclusion

Liver TG content is closely related with both hepatic and peripheral IR, whereas liver LC-UFA and LC-SFA are closely related only with hepatic IR in non-human primates. Mechanisms leading to the accumulation of TG, LC-UFA and an altered UFA: LC-SFA ratio may play an important role in the pathophysiology of fatty liver disease in humans.     

Inhibition of intestinal bile acid transporter Slc10a2 improves triglyceride metabolism and normalizes elevated plasma glucose levels in mice

Interruption of the enterohepatic circulation of bile acids increases cholesterol catabolism, thereby stimulating hepatic cholesterol synthesis from acetate. We hypothesized that such treatment should lower the hepatic acetate pool which may alter triglyceride and glucose metabolism. We explored this using mice deficient of the ileal sodium-dependent BA transporter (Slc10a2) and ob/ob mice treated with a specific inhibitor of Slc10a2. Plasma TG levels were reduced in Slc10a2-deficient mice, and when challenged with a sucrose-rich diet, they displayed a reduced response in hepatic TG production as observed from the mRNA levels of several key enzymes in fatty acid synthesis. This effect was paralleled by a diminished induction of mature sterol regulatory element-binding protein 1c (Srebp1c). Unexpectedly, the SR-diet induced intestinal fibroblast growth factor (FGF) 15 mRNA and normalized bile acid synthesis in Slc10a2-/- mice. Pharmacologic inhibition of Slc10a2 in diabetic ob/ob mice reduced serum glucose, insulin and TGs, as well as hepatic mRNA levels of Srebp1c and its target genes. These responses are contrary to those reported following treatment of mice with a bile acid binding resin. Moreover, when key metabolic signal transduction pathways in the liver were investigated, those of Mek1/2-Erk1/2 and Akt were blunted after treatment of ob/ob mice with the Slc10a2 inhibitor. It is concluded that abrogation of Slc10a2 reduces hepatic Srebp1c activity and serum TGs, and in the diabetic ob/ob model it also reduces glucose and insulin levels. Hence, targeting of Slc10a2 may be a promising strategy to treat hypertriglyceridemia and diabetes.     

Dietary fatty acids modulate antigen presentation to hepatic NKT cells in nonalcoholic fatty liver disease

Dietary fatty acids are major contributors to the development and progression of insulin resistance and nonalcoholic fatty liver disease (NAFLD). Dietary fatty acids also alter hepatic NKT cells that are activated by antigens presented by CD1d. In the current study, we examine the mechanism of dietary fatty acid induced hepatic NKT cell deficiency and its causal relationship to insulin resistance and NAFLD. We discover that dietary saturated fatty acids (SFA) or monounsaturated fatty acids (MUFA), but not polyunsaturated fatty acids (PUFA), cause hepatic NKT cell depletion with increased apoptosis. Dietary SFA or MUFA also impair hepatocyte presentation of endogenous, but not exogenous, antigen to NKT cells, indicating alterations of the endogenous antigen processing or presenting pathway. In vitro treatment of normal hepatocytes with fatty acids also demonstrates impaired ability of CD1d to present endogenous antigen by dietary fatty acids. Furthermore, dietary SFA and MUFA activate the NFκB signaling pathway and lead to insulin resistance and hepatic steatosis. In conclusion, both dietary SFA and MUFA alter endogenous antigen presentation to hepatic NKT cells and contribute to NKT cell depletion, leading to further activation of inflammatory signaling, insulin resistance, and hepatic steatosis.     

Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis

Hepatic steatosis is often associated with insulin resistance and obesity and can lead to steatohepatitis and cirrhosis. In this study, we have demonstrated that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for lipolysis in adipose tissues, also contribute to lipolysis in the liver and can mobilize hepatic triglycerides in vivo and in vitro. Adenoviral overexpression of HSL and/or ATGL reduced liver triglycerides by 40-60% in both ob/ob mice and mice with high fat diet-induced obesity. However, these enzymes did not affect fasting plasma triglyceride and free fatty acid levels or triglyceride and apolipoprotein B secretion rates. Plasma 3-beta-hydroxybutyrate levels were increased 3-5 days after infection in both HSL- and ATGL-overexpressing male mice, suggesting an increase in beta-oxidation. Expression of genes involved in fatty acid transport and synthesis, lipid storage, and mitochondrial bioenergetics was unchanged. Mechanistic studies in oleate-supplemented McA-RH7777 cells with adenoviral overexpression of HSL or ATGL showed that reduced cellular triglycerides could be attributed to increases in beta-oxidation as well as direct release of free fatty acids into the medium. In summary, hepatic overexpression of HSL or ATGL can promote fatty acid oxidation, stimulate direct release of free fatty acid, and ameliorate hepatic steatosis. This study suggests a direct functional role for both HSL and ATGL in hepatic lipid homeostasis and identifies these enzymes as potential therapeutic targets for ameliorating hepatic steatosis associated with insulin resistance and obesity.     

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase α (ACCα), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.     

Angiotensin II type 1 receptor blocker ameliorates overproduction and accumulation of triglyceride in the liver of Zucker fatty rats

The effects of angiotensin II type 1 receptor blocker (ARB) on triglyceride (TG) metabolism associated with insulin resistance were explored in Zucker fatty (ZF) rats. Olmesartan medoxomil, a newly developed ARB, was given as a 0.01% drinking solution ad libitum to ZF and Zucker lean (ZL) rats for 4 wk. Olmesartan lowered blood pressure in both strains to the same extent. ZF rats had a markedly low insulin sensitivity index (SI) and glucose effectiveness (SG), together with significantly increased glucose levels. Olmesartan treatment substantially elevated both SI and SG. The ZF rats were hyperlipidemic, with plasma TG levels sixfold higher than those of the ZL rats. Olmesartan remarkably decreased the plasma free fatty acid level in the ZF rats, but it did not exert a significant effect on the plasma TG level. The TG secretion rate assessed by the Triton WR-1339 technique was almost six times higher in the ZF than in the ZL rats, and olmesartan treatment suppressed this TG overproduction by one-half. The TG content in the liver was ten times higher in the ZF than in the ZL rats, and olmesartan halved this high hepatic TG content without affecting the cholesterol content. The fatty liver developed in the ZF rats was ameliorated by olmesartan treatment. Olmesartan treatment had no significant effects on TG metabolism or insulin sensitivity in the ZL rats. Taken in sum, ARB improves the overproduction and accumulation of TG in the liver associated with insulin resistance, and it does so through mechanisms independent of its hypotensive action.