Liu, Jun

The interplay between intracellular and intravascular lipolysis is crucial for maintaining circulating lipid levels and systemic energy homeostasis. Adipose triglyceride lipase (ATGL) and lipoprotein lipase (LPL), the primary triglyceride (TG) lipases responsible for these two spatially separate processes, are highly expressed in adipose tissue. Yet the mechanisms underlying their coordinated regulation remain undetermined. Here, we demonstrate that genetic ablation of G0S2, a specific inhibitory protein of ATGL, completely abolished diet-induced hypertriglyceridemia and significantly attenuated atherogenesis in mice. These effects were attributable to enhanced whole-body TG clearance, not altered hepatic TG secretion. Specifically, G0S2 deletion increased circulating LPL concentration and activity, predominantly through LPL production from white adipose tissue (WAT). Strikingly, transplantation of G0S2-deficient WAT normalized plasma TG levels in mice with hypertriglyceridemia. In conjunction with improved insulin sensitivity and decreased ANGPTL4 expression, the absence of G0S2 enhanced the stability of LPL protein in adipocytes, a phenomenon that could be reversed upon ATGL inhibition. Collectively, these findings highlight the pivotal role of adipocyte G0S2 in regulating both intracellular and intravascular lipolysis, and the possibility of targeting G0S2 as a viable pharmacological approach to reducing levels of circulating TGs.

Evidence from our laboratory has established a role for the G0/G1 Switch Gene 2 (G0S2) as the primary inhibitor of adipose lipolysis through inhibition of adipose triglyceride lipase (ATGL), the rate‐limiting enzyme in triglyceride hydrolysis. We have demonstrated that differential expression of G0S2 plays a crucial role in regulating hepatic lipid content during fasting and feeding. Hepatic G0S2 expression is also significantly increased in a pathological condition in mice with obesity‐induced hepatic steatosis or non‐alcoholic fatty liver disease (NAFLD), major metabolic health issues in today's society. Based on preliminary evidence we hypothesized there is a dynamic relationship between adipose lipolysis and hepatic G0S2 expression during physiological fasting, a relationship that has not been previously described. To better understand this relationship we evaluated the effect of free fatty acids (FFA) on G0S2 expression in liver. Interestingly, hepatic G0S2 expression during fasting results from increased free fatty acid (FFA) delivery to the liver from adipose tissue. Moreover, we identified that increases in hepatic FFA lead to increased G0S2 expression in a bi‐mechanistic manner. We investigated and subsequently determined that both transcriptional activation of G0S2 and inhibition of protein degradation are occurring in response to increased hepatic FFA levels. Utilizing a luciferase based reporter system and in vivo chromatin immunoprecipitation we found transcriptional activation of G0S2 in liver occurs in response to activation of liver‐X‐receptor α (LXRα). LXRα is a known regulator of pro‐lipogenic signaling in liver and plays a major role in cholesterol homeostasis. It has also been shown that LXRα can be activated in response to FAs most likely through its dimerization partner RXRα that is a known FA responsive transcription factor. We demonstrate G0S2 is a direct downstream target gene for LXRα. Additionally, the direct repeat 4 (DR4) LXR response element (LXRE) found 2kb upstream of the transcriptional start site in the G0S2 promoter is required for G0S2/ LXRα binding. This DR4 binding site is conserved between the human and mouse isoforms of G0S2 and is required in each for LXRα binding. Furthermore, we found that G0S2 is subject to ubiquitin modification and is degraded in a proteasome dependent pathway. Mutation analysis of the six‐lysine residues found in G0S2 provided key evidence that the lysine at position 25 is the prominent site for ubiquitylation. The G0S2 K25 mutant is more stable than wild‐type G0S2, which is consistent with the accompanying decrease in ubiquitylation. Additionally, fatty acids were able to abrogate G0S2 degradation and increase protein stability. These findings indicate that two mechanisms of expression regulation in combination with adipose lipolysis are functioning concomitantly to regulate hepatic G0S2 expression under physiological conditions. Additionally, aberrant G0S2 expression may contribute to the pathological setting with metabolically linked diseases including NAFLD. Support or Funding Information This work was supported by NIDDK grants DK089178 and 2R56DK089178 from the National Institutes of Health. Pre‐doctoral funding support for B.H. was generously provided by the Mayo Graduate School and Mayo Foundation for Medical Education & Research.

ABSTRACT G0S2 and HIG2 are two selective inhibitors of ATGL (also known as PNPLA2), the key enzyme for intracellular lipolysis. Whereas G0S2 regulates triglyceride (TG) mobilization in adipocytes and hepatocytes, HIG2 functions to enhance intracellular TG accumulation under hypoxic conditions. A homologous hydrophobic domain (HD) is shared by G0S2 and HIG2 (also known as HILPDA) for binding to ATGL. However, the determinants of their lipid droplet (LD) localization are unknown. Here, we study how G0S2 and HIG2 are targeted to LDs, and identify both ATGL-independent and -dependent mechanisms. Structural prediction and studies in cells reveal that ATGL-independent localization of G0S2 to both the endoplasmic reticulum (ER) and LDs is mediated by a hairpin structure consisting of two hydrophobic sequences. Positively charged residues in the hinge region play a crucial role in sorting G0S2, which initially localizes to ER, to LDs. Interestingly, the role of these positive charges becomes dispensable when ATGL is co-expressed. In comparison, HIG2, which lacks a similar hairpin structure, is dependent on ATGL for its full LD targeting. Thus, our studies identify specific structural features and mechanisms for mediating accumulation of these two ATGL inhibitors on LDs.

Integrins are heterodimeric (α/β) membrane proteins that play fundamental roles in many biological processes, e.g. cell adhesion and spreading, which are important for platelet function and hemostasis. Integrin function is modulated by bi-directional transmembrane signaling: inside-out and outside-in, which is mediated through the interactions between integrin cytoplasmic tails and intracellular, regulatory proteins. Here, we show that VPS33B, a member of the Sec1/Munc18 (SM) family and component of the Class C core of the CORVET/HOPS sorting complexes, binds directly to the β subunit. Overexpression of VPS33B in CHO cells potentiated α IIb β 3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice we generated, had normal morphology yet their spreading on fibrinogen was impaired and they failed to support clot retraction. Platelet aggregation and ATP secretion in response to low-dose thrombin were reduced in the VPS33B knockout mice. α IIb β 3 -mediated endocytosis of fibrinogen was also defective. Tail bleeding times were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1 dependent pathways that leads to clot retraction and cell spreading, respectively. Our work demonstrates that vesicular trafficking complexes are a novel class of modifiers of integrin function and in hemostasis. Our data also provide insights into the molecular mechanism and treatment of ARC syndrome.

ABSTRACT G 0 /G 1 switch gene 2 (G0S2) is a specific inhibitor of adipose triglyceride lipase (ATGL), the rate‐limiting enzyme for intracellular lipolysis. Recent studies show that G0S2 plays a critical role in promoting triacylglycerol (TG) accumulation in the liver, and its encoding gene is a direct target of a major lipogenic transcription factor liver X receptor (LXR)α. Here we sought to investigate a lipolysis‐independent role of G0S2 in hepatic triglyceride synthesis. Knockdown of G0S2 decreased hepatic TG content in mice with ATGL ablation. Conversely, overexpression of G0S2 promoted fatty acid incorporation into TGs and diacylglycerols in both wild‐type and ATGL‐deficient hepatocytes. Biochemical characterization showed that G0S2 mediates phosphatidic acid synthesis from lysophosphatidic acid (LPA) and acyl‐coenzyme A. In response to a high‐sucrose lipogenic diet, G0S2 is up‐regulated via LXRα and required for the increased TG accumulation in liver. Furthermore, deletion of a distinct 4‐aa motif necessary for the LPA‐specific acyltransferase (LPAAT) activity impaired G0S2's ability to mediate TG synthesis both in vitro and in vivo . These studies identify G0S2 as a dual‐function regulator of lipid metabolism as well as a novel mechanism whereby hepatic TG storage is promoted in response to lipogenic stimulation. In addition to its role as a lipolytic inhibitor, G0S2 is capable of directly promoting TG synthesis by acting as a lipid‐synthesizing enzyme.—Zhang, X., Xie, X., Heckmann, B. L., Saarinen, A. M., Gu, H., Zechner, R., Liu, J. Identification of an intrinsic lysophosphatidic acid acyltransferase activity in the lipolytic inhibitor G 0 /G 1 switch gene 2 (G0S2). FASEB J. 33, 6655–6666 (2019). www.fasebj.org

Tumor tissues are chronically exposed to hypoxia owing to aberrant vascularity. Lipid droplet (LD) accumulation is a hallmark of hypoxic cancer cells, yet how LDs form and function during hypoxia remains poorly understood. Herein, we report that in various cancer cells upon oxygen deprivation, HIF-1 activation down-modulates LD catabolism mediated by adipose triglyceride lipase (ATGL), the key enzyme for intracellular lipolysis. Proteomics and functional analyses identified hypoxia-inducible gene 2 (HIG2), a HIF-1 target, as a new inhibitor of ATGL. Knockout of HIG2 enhanced LD breakdown and fatty acid (FA) oxidation, leading to increased ROS production and apoptosis in hypoxic cancer cells as well as impaired growth of tumor xenografts. All of these effects were reversed by co-ablation of ATGL. Thus, by inhibiting ATGL, HIG2 acts downstream of HIF-1 to sequester FAs in LDs away from the mitochondrial pathways for oxidation and ROS generation, thereby sustaining cancer cell survival in hypoxia.

Melanocortin 2 receptor accessory protein (MRAP) is highly expressed in adrenal gland and adipose tissue. In adrenal cells, MRAP is essential for adrenocorticotropic hormone (ACTH)–induced activation of the cAMP/protein kinase A (PKA) pathway by melanocortin 2 receptor (MC2R), leading to glucocorticoid production and secretion. Although ACTH was known to stimulate PKA-dependent lipolysis, the functional involvement of MRAP in adipocyte metabolism remains incompletely defined. Herein, we found that knockdown or overexpression of MRAP in 3T3-L1 adipocytes reduced or increased ACTH-induced lipolysis, respectively. Moreover, an unbiased proteomics screen and coimmunoprecipitation analysis identified Gαs as a novel interacting partner of MRAP. An MRAP mutant disabled in Gαs association failed to augment the activation of PKA and lipolytic response to ACTH. Furthermore, compared with wild-type mice, transgenic mice (aP2-MRAP) overexpressing MRAP fat specifically exhibited increased lipolytic response to ACTH. When fed a high-fat diet (HFD), the transgenic mice displayed a significant decrease in the gain of adiposity and body weight as well as an improvement in glucose and insulin tolerance. These phenotypes were accompanied by increased adipose expression of genes for mitochondrial fatty acid oxidation and thermogenesis, and overall energy expenditure. Collectively, our data strongly suggest that MRAP plays a critical role in the regulation of ACTH-induced adipose lipolysis and whole-body energy balance.

Liver X receptors (LXRs) are transcription factors essential for cholesterol homeostasis and lipogenesis. LXRα has been implicated in regulating hepatic triglyceride (TG) accumulation upon both influx of adipose-derived fatty acids (FAs) during fasting and stimulation of de novo FA synthesis by chemical agonism of LXR. However, whether or not a convergent mechanism is employed to drive deposition of FAs from these 2 different sources in TGs is undetermined. Here, we report that the G0/G1 Switch Gene 2 (G0S2), a selective inhibitor of intracellular TG hydrolysis/lipolysis, is a direct target gene of LXRα. Transcriptional activation is conferred by LXRα binding to a direct repeat 4 (DR4) motif in the G0S2 promoter. While LXRα –/– mice exhibited decreased hepatic G0S2 expression, adenoviral expression of G0S2 was sufficient to restore fasting-induced TG storage and glycogen depletion in the liver of these mice. In response to LXR agonist T0901317, G0S2 ablation prevented hepatic steatosis and hypertriglyceridemia without affecting the beneficial effects on HDL. Thus, the LXRα-G0S2 axis plays a distinct role in regulating hepatic TG during both fasting and pharmacological activation of LXR.