Deficiency of liver adipose triglyceride lipase in mice causes progressive hepatic steatosis

Accumulation of cytoplasmic triacylglycerol (TG) underlies hepatic steatosis, a major cause of cirrhosis. The pathways of cytoplasmic TG metabolism are not well known in hepatocytes, but evidence suggests an important role in lipolysis for adipose triglyceride lipase (ATGL). We created mice with liver-specific inactivation of Pnpla2, the ATGL gene. These ATGLLKO mice had severe progressive periportal macrovesicular and pericentral microvesicular hepatic steatosis (73, 150, and 226 μmol TG/g liver at 4, 8, and 12 months, respectively). However, plasma levels of glucose, TG, and cholesterol were similar to those of controls. Fasting 3-hydroxybutyrate level was normal, but in thin sections of liver, beta oxidation of palmitate was decreased by one-third in ATGLLKO mice compared with controls. Tests of very low-density lipoprotein production, glucose, and insulin tolerance and gluconeogenesis from pyruvate were normal. Plasma alanine aminotransferase levels were elevated in ATGLLKO mice, but histological estimates of inflammation and fibrosis and messenger RNA (mRNA) levels of tumor necrosis factor-α and interleukin-6 were similar to or lower than those in controls. ATGLLKO cholangiocytes also showed cytoplasmic lipid droplets, demonstrating that ATGL is also a major lipase in cholangiocytes. There was a 50-fold reduction of hepatic diacylglycerol acyltransferase 2 mRNA level and a 2.7-fold increase of lipolysosomes in hepatocytes (P < 0.001), suggesting reduced TG synthesis and increased lysosomal degradation of TG as potential compensatory mechanisms. CONCLUSION: Compared with the hepatic steatosis of obesity and diabetes, steatosis in ATGL deficiency is well tolerated metabolically. ATGLLKO mice will be useful for studying the pathophysiology of hepatic steatosis.     

Adipose triglyceride lipase expression in human adipose tissue and muscle. Role in insulin resistance and response to training and pioglitazone

Adipose triglyceride lipase (ATGL) catalyzes the first step in adipocyte and muscle triglyceride hydrolysis, and comparative gene identification-58 (CGI-58) is an essential cofactor. We studied the expression of ATGL and CGI-58 in human adipose and muscle and examined correlations with markers of muscle fatty acid oxidation. Nondiabetic volunteers were studied. Subjects with impaired glucose tolerance were treated with pioglitazone or metformin for 10 weeks. Subjects with normal glucose tolerance underwent a 12-week training program. We examined changes in ATGL and CGI-58 with obesity and insulin resistance, and effects of exercise and pioglitazone. Adipose triglyceride lipase messenger RNA (mRNA) expression showed no correlation with either body mass index or insulin sensitivity index in either adipose or muscle. However, adipose ATGL protein levels were inversely correlated with body mass index (r = −0.64, P < .02) and positively correlated with insulin sensitivity index (r = 0.67, P < .02). In muscle, ATGL mRNA demonstrated a strong positive relationship with carnitine palmitoyltransferase I mRNA (r = 0.82, P < .0001) and the adiponectin receptors AdipoR1 mRNA (r = 0.71, P < .0001) and AdipoR2 mRNA (r = 0.74, P < .0001). Muscle CGI-58 mRNA was inversely correlated with intramyocellular triglyceride in both type 1 (r = −0.35, P < .05) and type 2 (r = −0.40, P < .05) fibers. Exercise training resulted in increased muscle ATGL, and pioglitazone increased adipose ATGL by 31% (P < .05). Pioglitazone also increased ATGL in adipocytes. Adipose ATGL protein is decreased with insulin resistance and obesity; and muscle ATGL mRNA is associated with markers of fatty acid oxidation in muscle, as is CGI-58. The regulation of ATGL and CGI-58 has important implications for the control of lipotoxicity.     

Anti-angiogenic pigment epithelium-derived factor regulates hepatocyte triglyceride content through adipose triglyceride lipase (ATGL)

BACKGROUND/AIMS: Anti-angiogenic pigment epithelium-derived factor (PEDF) is a 50 kDa secreted glycoprotein that is highly expressed in hepatocytes. Adipose triglyceride lipase (ATGL), a novel lipase critical for triglyceride metabolism, is a receptor for PEDF. We postulated that hepatocyte triglyceride metabolism was dependent on interactions between PEDF and ATGL, and loss of PEDF would impair mobilization of triglycerides in the liver. METHODS: Immunoprecipitation studies were performed in PEDF null and control hepatocytes with recombinant PEDF (rPEDF) as bait. Immunofluorescent microscopy was used to localize ATGL. Triglyceride content was analyzed in hepatocytes and in whole liver with and without rPEDF. ATGL was blocked using an inhibitor, (R)-bromoenol lactone. RESULTS: PEDF co-immunoprecipitated with ATGL in hepatic and HCC lysates. All PEDF deficient livers demonstrated steatosis. Triglyceride content was significantly increased in PEDF null livers compared to wildtype (p<0.05) and in isolated hepatocytes (p<0.01). Treatment of PEDF null hepatocytes with rPEDF decreased TG content (p<0.05) and this activity was dependent on ATGL. CONCLUSIONS: Our results identify a novel role for PEDF in hepatic triglyceride homeostasis through binding to ATGL and demonstrate that rPEDF and ATGL localize to adiposomes in hepatocytes. Dysregulation of this pathway may be one mechanism underlying fatty liver disease.     

Aromatase-deficient (ArKO) mice are retrieved from severe hepatic steatosis by peroxisome proliferator administration

Tamoxifen is a potent antagonist of estrogen, and hepatic steatosis is a frequent complication in adjuvant tamoxifen for breast cancer. Recently, aromatase-deficient (ArKO, Ar−/−) mice lacking intrinsic estrogen was developed and the molecular mechanism involved in progression of massive hepatic steatosis in estrogen-deficiency was elucidated; impairment in hepatic fatty acid β-oxidation of peroxisomes, microsomes and mitochondria. This impairment is latent, but is potentially serious, because hepatic energy supply depends greatly on fatty acid β-oxidation. Therefore in the present study, we tried to conquer impaired hepatic fatty acid β-oxidation by administrating bezafibrate, a potent peroxisome proliferator, to Ar−/− mice through activating fatty acid β-oxidation via the peroxisome proliferator activated receptor-α mediated signaling pathway. Northern blot analysis of Ar−/− mice liver revealed a significant restoration of mRNA expression of very long fatty acyl-CoA synthetase in peroxisome, peroxisomal fatty acyl-CoA oxidase, and medium-chain acyl-CoA dehydrogenase in mitochondria, essential enzymes in fatty acid β-oxidation by administration of bezafibrate. Severe hepatic steatosis observed in Ar−/− mice regressed dramatically. Consistent findings were obtained in the in vitro assays of fatty acid β-oxidation activity. These findings demonstrate that bezafibrate is capable of restoring impaired fatty acid β-oxidation in vivo via the peroxisome proliferator-activated receptor-α mediated signaling pathway and is potent enough to regress severe hepatic steatosis in mice deficient in intrinsic estrogen.     

Dissociation between fatty liver and insulin resistance: the role of adipose triacylglycerol lipase

Non-alcoholic fatty liver disease (NAFLD) is strongly associated with insulin resistance and type 2 diabetes in humans. Ongoing research aims to clarify the mechanisms involved in this relationship. Studying pathways that are involved in the dissociation between fatty liver and insulin resistance may help to achieve this goal. Among several enzymes that regulate the fate of hepatic lipids, adipose triacylglycerol lipase (ATGL) is of interest. This article briefly summarises novel information about the impact of ATGL in this process.     

Adipose triglyceride lipase gene expression in human visceral obesity.

In comparison to subcutaneous (SC) fat, visceral adipose tissue is more sensitive to catecholamine-induced lipolysis and less sensitive to the antilipolytic effects of insulin. Variation in the expression of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) have been reported. We therefore hypothesized that expression of adipose triglyceride lipase (ATGL) is different in visceral and SC depot and investigated whether ATGL mRNA expression is related to obesity, fat distribution and insulin sensitivity. ATGL, LPL, and HSL mRNA expression was measured in 85 paired samples of omental and subcutaneous adipose tissue in normal glucose tolerant lean and obese individuals. In addition, we included a subgroup of obese (BMI >30 kg/m2) individuals with either impaired or preserved insulin sensitivity determined by euglycemic-hyperinsulinemic clamps. ATGL mRNA levels are significantly decreased in insulin resistant obese subjects. Independently of body fat mass, omental ATGL mRNA correlates with fasting insulin concentration, glucose uptake during the steady state of the clamp and HSL mRNA expression. In obese, but not in lean subjects, LPL and HSL mRNA expression was significantly higher in omental compared to SC fat. In both depots, HSL mRNA was significantly lower in obese individuals. Visceral HSL mRNA expression is closely related to adipocyte size and fasting plasma insulin concentrations, whereas visceral fat area significantly predicts visceral LPL mRNA expression. ATGL mRNA expression is not significantly different between omental and SC fat. HSL, but not ATGL mRNA expression is closely related to individual and regional differences in adipocyte size. Impaired insulin sensitivity was associated with decreased ATGL and HSL mRNA expression, independently of body fat mass and fat distribution.     

The PPARβ/δ activator GW501516 prevents the down-regulation of AMPK caused by a high-fat diet in liver and amplifies the PGC-1α-Lipin 1-PPARα pathway leading to increased fatty acid oxidation

Metabolic syndrome-associated dyslipidemia is mainly initiated by hepatic overproduction of the plasma lipoproteins carrying triglycerides. Here we examined the effects of the peroxisome proliferator-activated receptors (PPAR)-β/δ activator GW501516 on high-fat diet (HFD)-induced hypertriglyceridemia and hepatic fatty acid oxidation. Exposure to the HFD caused hypertriglyceridemia that was accompanied by reduced hepatic mRNA levels of PPAR-γ coactivator 1 (PGC-1)-α and lipin 1, and these effects were prevented by GW501516 treatment. GW501516 treatment also increased nuclear lipin 1 protein levels, leading to amplification in the PGC-1α-PPARα signaling system, as demonstrated by the increase in PPARα levels and PPARα-DNA binding activity and the increased expression of PPARα-target genes involved in fatty acid oxidation. These effects of GW501516 were accompanied by an increase in plasma β-hydroxybutyrate levels, demonstrating enhanced hepatic fatty acid oxidation. Moreover, GW501516 increased the levels of the hepatic endogenous ligand for PPARα, 16:0/18:1-phosphatidilcholine and markedly enhanced the expression of the hepatic Vldl receptor. Interestingly, GW501516 prevented the reduction in AMP-activated protein kinase (AMPK) phosphorylation and the increase in phosphorylated levels of ERK1/2 caused by HFD. In addition, our data indicate that the activation of AMPK after GW501516 treatment in mice fed HFD might be the result of an increase in the AMP to ATP ratio in hepatocytes. These findings indicate that the hypotriglyceridemic effect of GW501516 in HFD-fed mice is accompanied by an increase in phospho-AMPK levels and the amplification of the PGC-1α-lipin 1-PPARα pathway.     

Adipose triacylglycerol lipase is a major regulator of hepatic lipid metabolism but not insulin sensitivity in mice

Aims/hypothesis  

Hepatic steatosis is characterised by excessive triacylglycerol accumulation and is strongly associated with insulin resistance. An inability to efficiently mobilise liver triacylglycerol may be a key event mediating hepatic steatosis. Adipose triacylglycerol lipase (ATGL) is a key triacylglycerol lipase in the liver and we hypothesised that liver-specific overproduction of ATGL would reduce steatosis and enhance insulin action in obese rodents.     

Fasting energy homeostasis in mice with adipose deficiency of desnutrin/adipose triglyceride lipase

Adipose triglyceride lipase (ATGL) catalyzes the first step of lipolysis of cytoplasmic triacylglycerols in white adipose tissue (WAT) and several other organs. We created adipose-specific ATGL-deficient (ATGLAKO) mice. In these mice, in vivo lipolysis, measured as the increase of plasma nonesterified fatty acid and glycerol levels after injection of a β3-adrenergic agonist, was undetectable. In isolated ATGLAKO adipocytes, β3-adrenergic-stimulated glycerol release was 10-fold less than in controls. Under fed conditions, ATGLAKO mice had normal viability, mild obesity, low plasma nonesterified fatty acid levels, increased insulin sensitivity, and increased daytime food intake. After 5 h of fasting, ATGLAKO WAT showed phosphorylation of the major protein kinase A-mediated targets hormone-sensitive lipase and perilipin A and ATGLAKO liver showed low glycogen and triacylglycerol contents. During a 48-h fast, ATGLAKO mice developed striking and complex differences from controls: progressive reduction of oxygen consumption, high respiratory exchange ratio, consistent with reduced fatty acid availability for energy production, lethargy, hypothermia, and undiminished fat mass, but greater loss of lean mass than controls. Plasma of 48 h-fasted ATGLAKO mice had a unique pattern: low 3-hydroxybutyrate, insulin, adiponectin, and fibroblast growth factor 21 with elevated leptin and corticosterone. ATGLAKO WAT, liver, skeletal muscle, and heart showed increased levels of mRNA related to autophagy and proteolysis. In murine ATGL deficiency, adipose lipolysis is critical for fasting energy homeostasis, and fasting imposes proteolytic stress on many organs, including heart and skeletal muscle.     

Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides

Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

Branched esters of palmitic acid and hydroxy stearic acid (PAHSAs) are antiinflammatory and antidiabetic lipokines (1–3). PAHSA serum and adipose tissue levels correlate with insulin sensitivity and are decreased in insulin-resistant humans (2, 4). PAHSAs increase glucose-stimulated insulin secretion by enhancing the production of the gut-derived incretin glucagon-like peptide-1 (5, 6). The antiinflammatory effects of PAHSA isomers (2, 7, 8) are mediated via free fatty acid receptor 4 (FFAR4, GPR120) and modulate both innate and adaptive immune responses in a mouse colitis model (1) and type-1 diabetes (6). Therefore, PAHSAs have beneficial effects on both metabolism and the immune system (9).PAHSAs belong to the family of fatty acid (FA) esters of hydroxy FAs (HFAs) called FAHFAs, which are part of a much larger family of mono- or oligomeric FAHFA esters named estolides. Since FAHFAs contain only a single ester bond of one FA with one HFA (the estolide bond), they represent monoestolides (10). The position of the branching carbon atom defines the regioisomer (e.g., 5-PAHSA or 9-PAHSA). PAHSAs and other less-well-studied FAHFAs such as the oleic acid esters of hydroxy palmitic acid (OAHPAs) or the docosahexaenoic acid ester of 13-hydroxy linoleic acid (13-DHAHLA) derive from either dietary sources or de novo synthesis in adipose tissue and other organs (2, 11, 12). Nonesterified, free FAHFAs (free mono-estolides) can be esterified to glycerol to form FAHFA acylglycerols, which in combination with other FAs result in the formation of triacylglycerol (TAG) estolides, diacylglycerol (DAG) estolides, or monoacylglycerol (MAG) estolides. TAG estolides represent a major storage form of bioactive free FAHFAs and are present in plant oils (e.g., castor oil) (13, 14) and adipose tissue of mice (3, 15) and humans (16).Both the synthetic and catabolic pathways of FAHFAs and TAG estolides are insufficiently understood. The hydrolytic catabolism of FAHFAs and TAG estolides results in the generation of highly bioactive and physiologically relevant FAHFAs, HFAs, FAs, and DAGs. Given the structural and metabolic similarity between TAGs and TAG estolides, it seemed reasonable to suspect that canonical TAG lipases will be involved in FAHFA and TAG estolide degradation. Generally, the catabolism of TAGs in cells occurs in the cytosol (neutral lipolysis) or in lysosomes (acidic lipolysis). Neutral lipolysis represents the predominant pathway for the hydrolysis of lipid droplet-associated TAGs in adipocytes involving three major enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL). ATGL catalyzes the initial step of TAG hydrolysis, generating DAG and one FA (3, 17, 18). The enzyme belongs to the patatin-like phospholipase domain-containing (PNPLA) family of proteins comprising a number of lipid hydrolases (3, 15). ATGL is the most potent TAG hydrolase within this family but also exhibits some phospholipase, retinylesterase, and transacylase activities of undefined physiological relevance (17, 19, 20). For full TAG hydrolase activity, ATGL requires a coactivator named comparative gene identification-58 (CGI-58; also called α/β-hydrolase domain containing 5, ABHD5) (21–23). CGI-58 features α/β-hydrolase folds and also exerts some acyltransferase and protease activities (23–25). Yet, the physiological role of these activities remains elusive. ATGL exhibits unique regioselectivity for TAG substrates and preferentially hydrolyzes the sn-2 position of the glycerol chain of TAGs (26). Upon stimulation of ATGL by CGI-58 this regioselectivity broadens to the sn-1 but not the sn-3 position (26).HSL is rate-limiting for the second step of TAG lipolysis converting DAG to one FA and MAG (27). The enzyme preferentially catalyzes DAGs at the sn-3 position and cholesteryl esters (28, 29) but also cleaves TAGs (sn-1 and sn-3 position), retinylesters (30), or medium- and short-chain carboxylic acid glycerol esters (29). The enzyme is structurally unrelated to ATGL and does not require enzyme coactivators. Hormonal stimulation of neutral lipolysis by β-adrenoreceptor agonists such as catecholamines activates both ATGL and HSL by promoting the molecular interaction of ATGL with CGI-58 on the surface of TAG-containing lipid droplets (21, 31) and the translocation of HSL from the cytoplasm to lipid droplets. These processes involve the protein kinase A-dependent phosphorylation of perilipin-1, CGI-58, and HSL (31–33).Previous studies by Tan et al. (15) and our laboratory (3) demonstrated that ATGL and HSL are both able to hydrolyze FAHFA–glycerol ester bonds of TAG estolides. However, enzyme preferences for this reaction, the substrate requirements, or the contribution of these enzymes to hydrolyze the FA–HFA ester bond (estolide bond) in TAG estolides as well as in free FAHFAs remained unaddressed. Using a newly generated library of TAG estolides, we now show that ATGL and HSL play distinct roles in the formation of TAG estolides by transesterification reactions and the degradation of (TAG) estolides by hydrolysis reactions.