Heckmann, Bradlee L.

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 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

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.

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.

Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr372 resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr372 eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr372 to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr372 as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.

Recent biochemical and cell-based studies identified G0/G1 switch gene 2 (G0S2) as an inhibitor of adipose triglyceride lipase (ATGL), a key mediator of intracellular triacylglycerol (TG) mobilization. Here, we show that upon fasting, G0S2 protein expression exhibits an increase in liver and a decrease in adipose tissue. Global knockout of G0S2 in mice enhanced adipose lipolysis and attenuated gain of body weight and adiposity. More strikingly, G0S2 knockout mice displayed a drastic decrease in hepatic TG content and were resistant to high-fat diet (HFD)-induced liver steatosis, both of which were reproduced by liver-specific G0S2 knockdown. Mice with hepatic G0S2 knockdown also showed increased ketogenesis, accelerated gluconeogenesis, and decelerated glycogenolysis. Conversely, overexpression of G0S2 inhibited fatty acid oxidation in mouse primary hepatocytes and caused sustained steatosis in liver accompanied by deficient TG clearance during the fasting-refeeding transition. In response to HFD, there was a profound increase in hepatic G0S2 expression in the fed state. Global and hepatic ablation of G0S2 both led to improved insulin sensitivity in HFD-fed mice. Our findings implicate a physiological role for G0S2 in the control of adaptive energy response to fasting and as a contributor to obesity-associated liver steatosis.