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.

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.

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.

A novel protein complex facilitates lipid droplet autophagy.

Background— Integrins are heterodimeric (α/β) membrane proteins that play fundamental roles in many biological processes, for example, cell adhesion and spreading, which are important for platelet function and hemostasis. The molecular mechanism that regulates integrin activation is not completely understood. Methods and Results— Here, we show that VPS33B, a member of the Sec1/Munc18 family, binds directly to the integrin β subunit. Overexpression of VPS33B in Chinese hamster ovary cells potentiated αIIbβ3 outside-in signaling but not inside-out signaling. Platelets, from megakaryocyte- and platelet-specific VPS33B conditional knockout mice, 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 agonists were reduced in the VPS33B knockout mice. αIIbβ3-mediated endocytosis of fibrinogen was also defective. Tail bleeding times and times to occlusion in an FeCl 3 -induced thrombosis model were prolonged in the VPS33B knockout mice. Furthermore, VPS33B acted upstream of the RhoA-ROCK-MLC and Rac1-dependent pathways that lead to clot retraction and cell spreading, respectively. Conclusions— Our work demonstrates that vesicular trafficking complexes, containing VPS33B, are a novel class of modifiers of integrin function. Our data also provide insights into the molecular mechanism and treatment of arthrogryposis, renal dysfunction, and cholestasis syndrome.

Fat-specific protein 27 (FSP27) plays a pivotal role in controlling the formation of large lipid droplet and energy metabolism. The cellular levels of FSP27 are tightly regulated through the proteasomal ubiquitin-mediated degradation. However, the upstream signals that trigger FSP27 degradation and the underlying mechanism(s) have yet to be identified. Here we show that AMP-activated protein kinase (AMPK) activation by AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide) or phenformin induced the ubiquitination of FSP27 and promoted its degradation in 3T3-L1 adipocytes. The levels of FSP27 protein could be maintained by either knocking down AMPKα1 or blocking proteasomal pathway. Moreover, AICAR treatment induced multilocularization of LDs in 3T3-L1 adipocytes, reminiscent of the morphological changes in cells depleted of FSP27. Furthermore, mass spectrometry-based proteomic analysis identified heat shock cognate 70 (HSC70) as a novel binding protein of FSP27. The specific interaction was confirmed by co-immunoprecipitation of both ectopically expressed and endogenous proteins. Importantly, knockdown of HSC70 by small interference RNA resulted in increased half-life of FSP27 in cells treated with a protein synthesis inhibitor cycloheximide (CHX) or AICAR. However, silencing of the E3 ubiquitin ligase CHIP (COOH terminus of HSC70-interacting protein) failed to alter the stability of FSP27 protein under both conditions. Taken together, our data indicate that AMPK is a negative regulator of FSP27 stability through the proteasomal ubiquitin-dependent protein catabolic process. Promotion of FSP27 degradation may be an important factor responsible for the beneficial effect of AMPK activators on energy metabolism.