Caveolin-1 Deficiency Leads to Increased Susceptibility to Cell Death and Fibrosis in White Adipose Tissue: Characterization of a Lipodystrophic Model

Caveolin-1 (CAV1) is an important regulator of adipose tissue homeostasis. In the present study we examined the impact of CAV1 deficiency on the properties of mouse adipose tissue both in vivo and in explant cultures during conditions of metabolic stress. In CAV1^−/− mice fasting caused loss of adipose tissue mass despite a lack of hormone-sensitive lipase (HSL) phosphorylation. In addition, fasting resulted in increased macrophage infiltration, enhanced deposition of collagen, and a reduction in the level of the lipid droplet protein perilipin A (PLIN1a). Explant cultures of CAV1^−/− adipose tissue also showed a loss of PLIN1a during culture, enhanced secretion of IL-6, increased release of lactate dehydrogenase, and demonstrated increased susceptibility to cell death upon collagenase treatment. Attenuated PKA-mediated signaling to HSL, loss of PLIN1a and increased secretion of IL-6 were also observed in adipose tissue explants of CAV1^+/+ mice with diet-induced obesity. Together these results suggest that while alterations in adipocyte lipid droplet biology support adipose tissue metabolism in the absence of PKA-mediated pro-lipolytic signaling in CAV1^−/− mice, the tissue is intrinsically unstable resulting in increased susceptibility to cell death, which we suggest underlies the development of fibrosis and inflammation during periods of metabolic stress.     

Liver-Specific Deletion of Phosphatase and Tensin Homolog Deleted on Chromosome 10 Significantly Ameliorates Chronic EtOH-Induced Increases in Hepatocellular Damage

Alcoholic liver disease is a significant contributor to global liver failure. In murine models, chronic ethanol consumption dysregulates PTEN/Akt signaling. Hepatospecific deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN^LKO) mice possess constitutive activation of Akt(s) and increased de novo lipogenesis resulting in increased hepatocellular steatosis. This makes PTEN^LKO a viable model to examine the effects of ethanol in an environment of preexisting steatosis. The aim of this study was to determine the impact of chronic ethanol consumption and the absence of PTEN (PTEN^LKO) compared to Alb-Cre control mice (PTEN^f/f) on hepatocellular damage as evidenced by changes in lipid accumulation, protein carbonylation and alanine amino transferase (ALT). In the control PTEN^f/f animals, ethanol significantly increased ALT, liver triglycerides and steatosis. In contrast, chronic ethanol consumption in PTEN^LKO mice decreased hepatocellular damage when compared to PTEN^LKO pair-fed controls. Consumption of ethanol elevated protein carbonylation in PTEN^f/f animals but had no effect in PTEN^LKO animals. In PTEN^LKO mice, overall hepatic mRNA expression of genes that contribute to GSH homeostasis as well as reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were significantly elevated compared to respective PTEN^f/f counterparts. These data indicate that during conditions of constitutive Akt activation and steatosis, increased GSH homeostasis assists in mitigation of ethanol-dependent induction of oxidative stress and hepatocellular damage. Furthermore, data herein suggest a divergence in EtOH-induced hepatocellular damage and increases in steatosis due to polyunsaturated fatty acids downstream of PTEN.     

PPARδ activation attenuates hepatic steatosis in Ldlr?/? mice by enhanced fat oxidation,reduced lipogenesis,and improved insulin sensitivity

PPARδ regulates systemic lipid homeostasis and inflammation, but its role in hepatic lipid metabolism remains unclear. Here, we examine whether intervening with a selective PPARδ agonist corrects hepatic steatosis induced by a high-fat, cholesterol-containing (HFHC) diet. Ldlr^−/− mice were fed a chow or HFHC diet (42% fat, 0.2% cholesterol) for 4 weeks. For an additional 8 weeks, the HFHC group was fed HFHC or HFHC plus GW1516 (3 mg/kg/day). GW1516-intervention significantly attenuated liver TG accumulation by induction of FA β-oxidation and attenuation of FA synthesis. In primary mouse hepatocytes, GW1516 treatment stimulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation in WT hepatocytes, but not AMPKβ1^−/− hepatocytes. However, FA oxidation was only partially reduced in AMPKβ1^−/− hepatocytes, suggesting an AMPK-independent contribution to the GW1516 effect. Similarly, PPARδ-mediated attenuation of FA synthesis was partially due to AMPK activation, as GW1516 reduced lipogenesis in WT hepatocytes but not AMPKβ1^−/− hepatocytes. HFHC-fed animals were hyperinsulinemic and exhibited selective hepatic insulin resistance, which contributed to elevated fasting FA synthesis and hyperglycemia. GW1516 intervention normalized fasting hyperinsulinemia and selective hepatic insulin resistance and attenuated fasting FA synthesis and hyperglycemia. The HFHC diet polarized the liver toward a proinflammatory M1 state, which was reversed by GW1516 intervention. Thus, PPARδ agonist treatment inhibits the progression of preestablished hepatic steatosis.     

Absence of adipose differentiation related protein upregulates hepatic VLDL secretion,relieves hepatosteatosis,and improves whole body insulin resistance in leptin-deficient mice

We previously showed that adipose differentiation related protein (Adfp)-deficient mice display a 60% reduction in hepatic triglyceride (TG) content. In this study, we investigated the role of ADFP in lipid and glucose homeostasis in a genetic obesity model, Lep^ob/ob mice. We bred Adfp^−/− mice with Lep^ob/ob mice to create Lep^ob/ob/Adfp^−/− and Lep^ob/ob/Adfp^+/+ mice and analyzed the hepatic lipids, lipid droplet (LD) morphology, LD protein composition and distribution, lipogenic gene expression, and VLDL secretion, as well as insulin sensitivity of the two groups of mice. Compared with Lep^ob/ob/Adfp^+/+ mice, Lep^ob/ob/Adfp^−/− mice displayed an increased VLDL secretion rate, a 25% reduction in hepatic TG associated with improvement in fatty liver grossly and microscopically with a change of the size of LDs in a proportion of the hepatocytes and a redistribution of major LD-associated proteins from the cytoplasmic compartment to the LD surface. There was no detectable change in lipogenic gene expression. Lep^ob/ob/Adfp^−/− mice also had improved glucose tolerance and insulin sensitivity in both liver and muscle. The alteration of LD size in the liver of Lep^ob/ob/Adfp^−/− mice despite the relocation of other LDPs to the LD indicates a nonredundant role for ADFP in determining the size and distribution of hepatic LDs.     

Fasting Enhances TRAIL-Mediated Liver Natural Killer Cell Activity via HSP70 Upregulation

Acute starvation, which is frequently observed in clinical practice, sometimes augments the cytolytic activity of natural killer cells against neoplastic cells. In this study, we investigated the molecular mechanisms underlying the enhancement of natural killer cell function by fasting in mice. The total number of liver resident natural killer cells in a unit weight of liver tissue obtained from C57BL/6J mice did not change after a 3-day fast, while the proportions of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL)⁺ and CD69⁺ natural killer cells were significantly elevated (n = 7, p <0.01), as determined by flow cytometric analysis. Furthermore, we found that TRAIL⁻ natural killer cells that were adoptively transferred into Rag-2^−/− γ chain^−/− mice could convert into TRAIL⁺ natural killer cells in fasted mice at a higher proportion than in fed mice. Liver natural killer cells also showed high TRAIL-mediated antitumor function in response to 3-day fasting. Since these fasted mice highly expressed heat shock protein 70 (n = 7, p <0.05) in liver tissues, as determined by western blot, the role of this protein in natural killer cell activation was investigated. Treatment of liver lymphocytes with 50 µg/mL of recombinant heat shock protein 70 led to the upregulation of both TRAIL and CD69 in liver natural killer cells (n = 6, p <0.05). In addition, HSP70 neutralization by intraperitoneally injecting an anti- heat shock protein 70 monoclonal antibody into mice prior to fasting led to the downregulation of TRAIL expression (n = 6, p <0.05). These findings indicate that acute fasting enhances TRAIL-mediated liver natural killer cell activity against neoplastic cells through upregulation of heat shock protein 70.     

Thioesterase Superfamily Member 2 (Them2) and Phosphatidylcholine Transfer Protein (PC-TP) Interact To Promote Fatty Acid Oxidation and Control Glucose Utilization

Thioesterase superfamily member 2 (Them2) is a mitochondrion-associated long-chain fatty acyl coenzyme A (CoA) thioesterase that is highly expressed in the liver and oxidative tissues. Them2 activity in vitro is increased when it interacts with phosphatidylcholine transfer protein (PC-TP), a cytosolic lipid binding protein. Them2^−/− and Pctp^−/− mice exhibit enhanced hepatic insulin sensitivity and increased adaptive thermogenesis, and Them2^−/− mice are also resistant to diet-induced hepatic steatosis. Although we showed previously that a Them2–PC-TP complex suppresses insulin signaling, the enzymatic activity of Them2 suggests additional direct involvement in regulating hepatic nutrient homeostasis. Here we used cultured primary hepatocytes to elucidate biochemical and cellular mechanisms by which Them2 and PC-TP regulate lipid and glucose metabolism. Under conditions simulating fasting, Them2^−/− and Pctp^−/− hepatocytes each exhibited decreased rates of fatty acid oxidation and gluconeogenesis. In results indicative of Them2-dependent regulation by PC-TP, chemical inhibition of PC-TP failed to reproduce these changes in Them2^−/− hepatocytes. In contrast, rates of glucose oxidation and lipogenesis in the presence of high glucose concentrations were decreased only in Them2^−/− hepatocytes. These findings reveal a primary role for Them2 in promoting mitochondrial oxidation of fatty acids and glucose in the liver.     

CD36 Protein Influences Myocardial Ca2+ Homeostasis and Phospholipid Metabolism: CONDUCTION ANOMALIES IN CD36-DEFICIENT MICE DURING FASTING*

Sarcolemmal CD36 facilitates myocardial fatty acid (FA) uptake, which is markedly reduced in CD36-deficient rodents and humans. CD36 also mediates signal transduction events involving a number of cellular pathways. In taste cells and macrophages, CD36 signaling was recently shown to regulate store-responsive Ca²⁺ flux and activation of Ca²⁺-dependent phospholipases A₂ that cycle polyunsaturated FA into phospholipids. It is unknown whether CD36 deficiency influences myocardial Ca²⁺ handling and phospholipid metabolism, which could compromise the heart, typically during stresses. Myocardial function was examined in fed or fasted (18–22 h) CD36^−/− and WT mice. Echocardiography and telemetry identified conduction anomalies that were associated with the incidence of sudden death in fasted CD36^−/− mice. No anomalies or death occurred in WT mice during fasting. Optical imaging of perfused hearts from fasted CD36^−/− mice documented prolongation of Ca²⁺ transients. Consistent with this, knockdown of CD36 in cardiomyocytes delayed clearance of cytosolic Ca²⁺. Hearts of CD36^−/− mice (fed or fasted) had 3-fold higher SERCA2a and 40% lower phospholamban levels. Phospholamban phosphorylation by protein kinase A (PKA) was enhanced after fasting reflecting increased PKA activity and cAMP levels in CD36^−/− hearts. Abnormal Ca²⁺ homeostasis in the CD36^−/− myocardium associated with increased lysophospholipid content and a higher proportion of 22:6 FA in phospholipids suggests altered phospholipase A₂ activity and changes in membrane dynamics. The data support the role of CD36 in coordinating Ca²⁺ homeostasis and lipid metabolism and the importance of this role during myocardial adaptation to fasting. Potential relevance of the findings to CD36-deficient humans would need to be determined.     

Lipocalin-2 (LCN2) regulates PLIN5 expression and intracellular lipid droplet formation in the liver

Lipocalin-2 (LCN2) belongs to the superfamily of lipocalins and plays critical roles in the control of cellular homeostasis during inflammation and in responses to cellular stress or injury. In the liver, LCN2 triggers protective effects following acute or chronic injury, and its expression is a reliable indicator of liver damage. However, little is known about LCN2's functions in the homeostasis and metabolism of hepatic lipids or in the development of steatosis. In this study, we fed wild type (WT) and LCN2-deficient (Lcn2^−/−) mice a methionine- and choline-deficient (MCD) diet as a nutritional model of non-alcoholic steatohepatitis, and compared intrahepatic lipid accumulation, lipid droplet formation, mitochondrial content, and expression of the Perilipin proteins that regulate cellular lipid metabolism. We found that Lcn2^−/− mice fed an MCD diet accumulated more lipids in the liver than WT controls, and that the basal expression of the lipid droplet coat protein Perilipin 5 (PLIN5, also known as OXPAT) was significantly reduced in these animals. Similarly, the overexpression of LCN2 and PLIN5 were also found in animals that were fed with a high fat diet. Furthermore, the loss of LCN2 and/or PLIN5 in hepatocytes prevented normal intracellular lipid droplet formation both in vitro and in vivo. Restoration of LCN2 in Lcn2^−/− primary hepatocytes by either transfection or adenoviral vector infection induced PLIN5 expression and restored proper lipid droplet formation. Our data indicate that LCN2 is a key modulator of hepatic lipid homeostasis that controls the formation of intracellular lipid droplets by regulating PLIN5 expression. LCN2 may therefore represent a novel therapeutic drug target for the treatment of liver diseases associated with elevated fat accumulation and steatosis.     

Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes

Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.     

Liver Clock Protein BMAL1 Promotes de Novo Lipogenesis through Insulin-mTORC2-AKT Signaling

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1^−/− and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1^−/− primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1^−/− hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.     

Pantothenate Kinase 1 Is Required to Support the Metabolic Transition from the Fed to the Fasted State

Coenzyme A (CoA) biosynthesis is regulated by the pantothenate kinases (PanK), of which there are four active isoforms. The PanK1 isoform is selectively expressed in liver and accounted for 40% of the total PanK activity in this organ. CoA synthesis was limited using a Pank1 ^−/− knockout mouse model to determine whether the regulation of CoA levels was critical to liver function. The elimination of PanK1 reduced hepatic CoA levels, and fasting triggered a substantial increase in total hepatic CoA in both Pank1 ^−/− and wild-type mice. The increase in hepatic CoA during fasting was blunted in the Pank1 ^−/− mouse, and resulted in reduced fatty acid oxidation as evidenced by abnormally high accumulation of long-chain acyl-CoAs, acyl-carnitines, and triglycerides in the form of lipid droplets. The Pank1 ^−/− mice became hypoglycemic during a fast due to impaired gluconeogenesis, although ketogenesis was normal. These data illustrate the importance of PanK1 and elevated liver CoA levels during fasting to support the metabolic transition from glucose utilization and fatty acid synthesis to gluconeogenesis and fatty acid oxidation. The findings also suggest that PanK1 may be a suitable target for therapeutic intervention in metabolic disorders that feature hyperglycemia and hypertriglyceridemia.     

Mtu1-Mediated Thiouridine Formation of Mitochondrial tRNAs Is Required for Mitochondrial Translation and Is Involved in Reversible Infantile Liver Injury

Reversible infantile liver failure (RILF) is a unique heritable liver disease characterized by acute liver failure followed by spontaneous recovery at an early stage of life. Genetic mutations in MTU1 have been identified in RILF patients. MTU1 is a mitochondrial enzyme that catalyzes the 2-thiolation of 5-taurinomethyl-2-thiouridine (τm⁵s²U) found in the anticodon of a subset of mitochondrial tRNAs (mt-tRNAs). Although the genetic basis of RILF is clear, the molecular mechanism that drives the pathogenesis remains elusive. We here generated liver-specific knockout of Mtu1 (Mtu1^LKO) mice, which exhibited symptoms of liver injury characterized by hepatic inflammation and elevated levels of plasma lactate and AST. Mechanistically, Mtu1 deficiency resulted in a loss of 2-thiolation in mt-tRNAs, which led to a marked impairment of mitochondrial translation. Consequently, Mtu1^LKO mice exhibited severe disruption of mitochondrial membrane integrity and a broad decrease in respiratory complex activities in the hepatocytes. Interestingly, mitochondrial dysfunction induced signaling pathways related to mitochondrial proliferation and the suppression of oxidative stress. The present study demonstrates that Mtu1-dependent 2-thiolation of mt-tRNA is indispensable for mitochondrial translation and that Mtu1 deficiency is a primary cause of RILF. In addition, Mtu1 deficiency is associated with multiple cytoprotective pathways that might prevent catastrophic liver failure and assist in the recovery from liver injury.     

Hepatic Gluconeogenesis Is Enhanced by Phosphatidic Acid Which Remains Uninhibited by Insulin in Lipodystrophic Agpat2?/? Mice

In this study we examined the role of phosphatidic acid (PA) in hepatic glucose production (HGP) and development of hepatic insulin resistance in mice that lack 1-acylglycerol-3-phosphate O-acyltransferase 2 (AGPAT2). Liver lysophosphatidic acid and PA levels were increased ∼2- and ∼5-fold, respectively, in male Agpat2^−/− mice compared with wild type mice. In the absence of AGPAT2, the liver can synthesize PAs by activating diacylglycerol kinase or phospholipase D, both of which were elevated in the livers of Agpat2^−/− mice. We found that PAs C16:0/18:1 and C18:1/20:4 enhanced HGP in primary WT hepatocytes, an effect that was further enhanced in primary hepatocytes from Agpat2^−/− mice. Lysophosphatidic acids C16:0 and C18:1 failed to increase HGP in primary hepatocytes. The activation of HGP was accompanied by an up-regulation of the key gluconeogenic enzymes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This activation was suppressed by insulin in the WT primary hepatocytes but not in the Agpat2^−/− primary hepatocytes. Thus, the lack of normal insulin signaling in Agpat2^−/− livers allows unrestricted PA-induced gluconeogenesis significantly contributing to the development of hyperglycemia in these mice.     

Hepatic trans-Golgi action coordinated by the GTPase ARFRP1 is crucial for lipoprotein lipidation and assembly

The liver is a major organ in whole body lipid metabolism and malfunctioning can lead to various diseases including dyslipidemia, fatty liver disease, and type 2 diabetes. Triglycerides and cholesteryl esters are packed in the liver as very low density lipoproteins (VLDLs). Generation of these lipoproteins is initiated in the endoplasmic reticulum and further maturation likely occurs in the Golgi. ADP-ribosylation factor-related protein 1 (ARFRP1) is a small trans-Golgi-associated guanosine triphosphatase (GTPase) that regulates protein sorting and is required for chylomicron lipidation and assembly in the intestine. Here we show that the hepatocyte-specific deletion of Arfrp1 (Arfrp1^liv−/−) results in impaired VLDL lipidation leading to reduced plasma triglyceride levels in the fasted state as well as after inhibition of lipoprotein lipase activity by Triton WR-1339. In addition, the concentration of ApoC3 that comprises 40% of protein mass of secreted VLDLs is markedly reduced in the plasma of Arfrp1^liv−/− mice but accumulates in the liver accompanied by elevated triglycerides. Fractionation of Arfrp1^liv−/− liver homogenates reveals more ApoB48 and a lower concentration of triglycerides in the Golgi compartments than in the corresponding fractions from control livers. In conclusion, ARFRP1 and the Golgi apparatus play an important role in lipoprotein maturation in the liver by influencing lipidation and assembly of proteins to the lipid particles.     

N-Glycan Remodeling on Glucagon Receptor Is an Effector of Nutrient Sensing by the Hexosamine Biosynthesis Pathway

Glucose homeostasis in mammals is dependent on the opposing actions of insulin and glucagon. The Golgi N-acetylglucosaminyltransferases encoded by Mgat1, Mgat2, Mgat4a/b/c, and Mgat5 modify the N-glycans on receptors and solute transporter, possibly adapting activities in response to the metabolic environment. Herein we report that Mgat5^−/− mice display diminished glycemic response to exogenous glucagon, together with increased insulin sensitivity. Glucagon receptor signaling and gluconeogenesis in Mgat5^−/− cultured hepatocytes was impaired. In HEK293 cells, signaling by ectopically expressed glucagon receptor was increased by Mgat5 expression and GlcNAc supplementation to UDP-GlcNAc, the donor substrate shared by Mgat branching enzymes. The mobility of glucagon receptor in primary hepatocytes was reduced by galectin-9 binding, and the strength of the interaction was dependent on Mgat5 and UDP-GlcNAc levels. Finally, oral GlcNAc supplementation rescued the glucagon response in Mgat5^−/− hepatocytes and mice, as well as glycolytic metabolites and UDP-GlcNAc levels in liver. Our results reveal that the hexosamine biosynthesis pathway and GlcNAc salvage contribute to glucose homeostasis through N-glycan branching on glucagon receptor.     

Preferential lipolysis of DGAT1 over DGAT2 generated triacylglycerol in Huh7 hepatocytes

Two distinct diacylglycerol acyltransferases (DGAT1 and DGAT2) catalyze the final committed step of triacylglycerol (TG) synthesis in hepatocytes. After its synthesis in the endoplasmic reticulum (ER) TG is either stored in cytosolic lipid droplets (LDs) or is assembled into very low-density lipoproteins in the ER lumen. TG stored in cytosolic LDs is hydrolyzed by adipose triglyceride lipase (ATGL) and the released fatty acids are converted to energy by oxidation in mitochondria. We hypothesized that targeting/association of ATGL to LDs would differ depending on whether the TG stores were generated through DGAT1 or DGAT2 activities. Individual inhibition of DGAT1 or DGAT2 in Huh7 hepatocytes incubated with oleic acid did not yield differences in TG accretion while combined inhibition of both DGATs completely prevented TG synthesis suggesting that either DGAT can efficiently esterify exogenously supplied fatty acid. DGAT2-made TG was stored in larger LDs, whereas TG formed by DGAT1 accumulated in smaller LDs. Inactivation of DGAT1 or DGAT2 did not alter expression (mRNA or protein) of ATGL, the ATGL activator ABHD5/CGI-58, or LD coat proteins PLIN2 or PLIN5, but inactivation of both DGATs increased PLIN2 abundance despite a dramatic reduction in the number of LDs. ATGL was found to preferentially target to LDs generated by DGAT1 and fatty acids released from TG in these LDs were also preferentially used for fatty acid oxidation. Combined inhibition of DGAT2 and ATGL resulted in larger LDs, suggesting that the smaller size of DGAT1-generated LDs is the result of increased lipolysis of TG in these LDs.     

The Lipid Droplet Protein Hypoxia-inducible Gene 2 Promotes Hepatic Triglyceride Deposition by Inhibiting Lipolysis

The liver is a major site of glucose, fatty acid, and triglyceride (TG) synthesis and serves as a major regulator of whole body nutrient homeostasis. Chronic exposure of humans or rodents to high-calorie diets promotes non-alcoholic fatty liver disease, characterized by neutral lipid accumulation in lipid droplets (LD) of hepatocytes. Here we show that the LD protein hypoxia-inducible gene 2 (Hig2/Hilpda) functions to enhance lipid accumulation in hepatocytes by attenuating TG hydrolysis. Hig2 expression increased in livers of mice on a high-fat diet and during fasting, two states associated with enhanced hepatic TG content. Hig2 expressed in primary mouse hepatocytes localized to LDs and promoted LD TG deposition in the presence of oleate. Conversely, tamoxifen-inducible Hig2 deletion reduced both TG content and LD size in primary hepatocytes from mice harboring floxed alleles of Hig2 and a cre/ERT2 transgene controlled by the ubiquitin C promoter. Hepatic TG was also decreased by liver-specific deletion of Hig2 in mice with floxed Hig2 expressing cre controlled by the albumin promoter. Importantly, we demonstrate that Hig2-deficient hepatocytes exhibit increased TG lipolysis, TG turnover, and fatty acid oxidation as compared with controls. Interestingly, mice with liver-specific Hig2 deletion also display improved glucose tolerance. Taken together, these data indicate that Hig2 plays a major role in promoting lipid sequestration within LDs in mouse hepatocytes through a mechanism that impairs TG degradation.