G0S2 Protein Research Articles

Loss of carnitine palmitoyltransferase 1a reduces docosahexaenoic acid-containing phospholipids and drives sexually dimorphic liver disease in mice

Background and aimsGenome and epigenome wide association studies identified variants in carnitine palmitoyltransferase 1a (CPT1a) that associate with lipid traits. The goal of this study was to determine the role of liver-specific CPT1a on hepatic lipid metabolism. Approach and resultsMale and female liver-specific knockout (LKO) and littermate controls were placed on a low-fat or high-fat diet (60% kcal fat) for 15 weeks. Mice were necropsied after a 16 h fast, and tissues were collected for lipidomics, matrix-assisted laser desorption ionization mass spectrometry imaging, kinome analysis, RNA-sequencing, and protein expression by immunoblotting. Female LKO mice had increased serum alanine aminotransferase levels which were associated with greater deposition of hepatic lipids, while male mice were not affected by CPT1a deletion relative to male control mice. Mice with CPT1a deletion had reductions in DHA-containing phospholipids at the expense of monounsaturated fatty acids (MUFA)-containing phospholipids in whole liver and at the level of the lipid droplet (LD). Male and female LKO mice increased RNA levels of genes involved in LD lipolysis (Plin2, Cidec, G0S2) and in polyunsaturated fatty acid metabolism (Elovl5, Fads1, Elovl2), while only female LKO mice increased genes involved in inflammation (Ly6d, Mmp12, Cxcl2). Kinase profiling showed decreased protein kinase A activity, which coincided with increased PLIN2, PLIN5, and G0S2 protein levels and decreased triglyceride hydrolysis in LKO mice. ConclusionsLiver-specific deletion of CPT1a promotes sexually dimorphic steatotic liver disease (SLD) in mice, and here we have identified new mechanisms by which females are protected from HFD-induced liver injury.

Loss of G0/G1 switch gene 2 (G0S2) promotes disease progression and drug resistance in chronic myeloid leukaemia (CML) by disrupting glycerophospholipid metabolism.

Tyrosine kinase inhibitors (TKIs) targeting BCR::ABL1 have turned chronic myeloid leukaemia (CML) from a fatal disease into a manageable condition for most patients. Despite improved survival, targeting drug-resistant leukaemia stem cells (LSCs) remains a challenge for curative CML therapy. Aberrant lipid metabolism can have a large impact on membrane dynamics, cell survival and therapeutic responses in cancer. While ceramide and sphingolipid levels were previously correlated with TKI response in CML, the role of lipid metabolism in TKI resistance is not well understood. We have identified downregulation of a critical regulator of lipid metabolism, G0/G1 switch gene 2 (G0S2), in multiple scenarios of TKI resistance, including (1) BCR::ABL1 kinase-independent TKI resistance, (2) progression of CML from the chronic to the blast phase of the disease, and (3) in CML versus normal myeloid progenitors. Accordingly, CML patients with low G0S2 expression levels had a worse overall survival. G0S2 downregulation in CML was not a result of promoter hypermethylation or BCR::ABL1 kinase activity, but was rather due to transcriptional repression by MYC. Using CML cell lines, patient samples and G0s2 knockout (G0s2-/- ) mice, we demonstrate a tumour suppressor role for G0S2 in CML and TKI resistance. Our data suggest that reduced G0S2 protein expression in CML disrupts glycerophospholipid metabolism, correlating with a block of differentiation that renders CML cells resistant to therapy. Altogether, our data unravel a new role for G0S2 in regulating myeloid differentiation and TKI response in CML, and suggest that restoring G0S2 may have clinical utility.

Bioinformatic Analysis and Biophysical Characterization Reveal Structural Disorder in G0S2 Protein.

G0S2 is a small protein of 103 residues in length that is involved in multiple cellular processes. To date, several reports have shown that G0S2 functions by making direct protein–protein interactions with key proteins. In lipolysis, G0S2 specifically interacts with adipose triglyceride lipase, inhibiting its activity and resulting in lipolysis being downregulated. In a similar way, G0S2 also participates in the regulation of apoptosis, cell proliferation, and oxidative phosphorylation; however, information regarding G0S2 structural and biophysical properties is limited. In this work, we conducted a comparative structural analysis of human and mouse G0S2 proteins. Bioinformatics suggests the presence of a disordered C-terminal region in human G0S2. Experimental characterization by size-exclusion chromatography and dynamic light scattering showed that human and mouse G0S2 have different hydrodynamic properties. In comparison to the mouse G0S2, which behaves similar to a globular protein, the human G0S2 shows an elongated conformation, most likely by displaying a disordered C-terminal region. Further analysis of hydrodynamic properties under denaturing conditions suggests the presence of a structural element in the mouse protein that undergoes an order to disorder transition at low urea concentration. Structural analysis by circular dichroism revealed that in native conditions, both proteins are mainly unstructured, showing the presence of beta sheet structures. Further analysis of CD data suggests that both proteins belong to the premolten globule family of intrinsically disordered proteins. We suggest that the intrinsic disorder observed in the G0S2 protein may facilitate its interaction with multiple partners in the regulation of cellular metabolism.

A molecular triage process mediated by RING finger protein 126 and BCL2-associated athanogene 6 regulates degradation of G0/G1 switch gene 2

Oxidative phosphorylation generates most of the ATP in respiring cells. ATP is an essential energy source, especially in cardiomyocytes because of their continuous contraction and relaxation. Previously, we reported that G0/G1 switch gene 2 (G0S2) positively regulates mitochondrial ATP production by interacting with FOF1-ATP synthase. G0S2 overexpression mitigates ATP decline in cardiomyocytes and strongly increases their hypoxic tolerance during ischemia. Here, we show that G0S2 protein undergoes proteasomal degradation via a cytosolic molecular triage system and that inhibiting this process increases mitochondrial ATP production in hypoxia. First, we performed screening with a library of siRNAs targeting ubiquitin-related genes and identified RING finger protein 126 (RNF126) as an E3 ligase involved in G0S2 degradation. RNF126-deficient cells exhibited prolonged G0S2 protein turnover and reduced G0S2 ubiquitination. BCL2-associated athanogene 6 (BAG6), involved in the molecular triage of nascent membrane proteins, enhanced RNF126-mediated G0S2 ubiquitination both in vitro and in vivo Next, we found that Glu-44 in the hydrophobic region of G0S2 acts as a degron necessary for G0S2 polyubiquitination and proteasomal degradation. Because this degron was required for an interaction of G0S2 with BAG6, an alanine-replaced G0S2 mutant (E44A) escaped degradation. In primary cultured cardiomyocytes, both overexpression of the G0S2 E44A mutant and RNF126 knockdown effectively attenuated ATP decline under hypoxic conditions. We conclude that the RNF126/BAG6 complex contributes to G0S2 degradation and that interventions to prevent G0S2 degradation may offer a therapeutic strategy for managing ischemic diseases.

Hepatic G0S2 Exacerbates Hepatic Insulin Resistance in Estrogen-Deficient Female Wistar Rats

While it has been reported that G0/G1 switch gene 2 (G0S2) inhibited the lipolytic activity of adipose triglyceride lipase, previously, we demonstrated that hepatic G0S2 promotes hepatic insulin resistance by the exacerbation of hepatic steatosis in the high fat diet (HF) fed male Wistar rats. However, the physiological role of hepatic G0S2 is still unknown. Estrogen suppresses gluconeogenesis and de novo lipogenesis in the liver and a decrease in serum estrogen levels in menopause is closely associated with the insulin resistance. These findings indicated that postmenopausal hepatic steatosis and insulin resistance are strongly associated with reductions in estrogen levels. To investigate the involvement of hepatic G0S2 in postmenopausal insulin resistance, we studied the effect of G0S2 on insulin sensitivity in HF-fed ovariectomized female Wistar rats by overexpressing G0S2 protein using an adenovirus (Ad). Six weeks female Wistar rats were ovariectomized (OVX) or sham operated (OVI), and maintained on 60% HF for a total of 8 weeks. Ad-G0S2 or control Ad-GFP were injected to these rats after 7 weeks diet challenge. On day 7 post Ad injection, euglycemic-hyperinsulinemic clamp studies were performed after a 12-hour fast. During the clamp studies on the OVI rats, the glucose infusion rate (Ginf), the insulin stimulated glucose disposal rate (IS-GDR), and the insulin suppression of HGO were not significantly different between the G0S2 and GFP groups. On the other hand, the OVX-G0S2 rats exhibited increased clamp HGO (∼36%) and decreased suppression of HGO (∼43%) but no significant change in ISGDR compared to OVX-GFP rats. Consist with the clamp data, the insulin-stimulated phosphorylation of Akt was significantly decreased only in the liver of OVX-G0S2 rats. In conclusion, hepatic G0S2 exacerbates hepatic insulin resistance only in the estrogen deficient status. The expression level of hepatic G0S2 could affect the development of hepatic insulin resistance in the postmenopausal stage. Disclosure H. Satoh: None. Y. Sugaya: None. C.T. Moriya: None. T. Miyatsuka: Research Support; Self; Eli Lilly and Company, Japan Society for the Promotion of Science, Daiichi Sankyo Company, Limited, Novartis Pharma K.K., Astellas Pharma Inc., MSD K.K., Takeda Pharmaceutical Company Limited. H. Watada: Advisory Panel; Self; AstraZeneca. Consultant; Self; Astellas Pharma US, Inc., AstraZeneca, Boehringer Ingelheim GmbH, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Novartis AG, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Development Center Asia, Pte. Ltd.. Research Support; Self; Abbott, Astellas Pharma US, Inc., AstraZeneca, Bayer AG, Benefit One Health Care Co., Ltd., Boehringer Ingelheim GmbH, Bristol-Myers Squibb Company, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Johnson & Johnson Diabetes Institute, LLC., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nitto Boseki Co., Ltd., Novartis AG, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Terumo Medical Corporation.

Palmitate induces fat accumulation by activating C/EBPβ-mediated G0S2 expression in HepG2 cells.

AIMTo determine the role of G0/G1 switch gene 2 (G0S2) and its transcriptional regulation in palmitate-induced hepatic lipid accumulation.METHODSHepG2 cells were treated with palmitate, or palmitate in combination with CCAAT/enhancer binding protein (C/EBP)β siRNA or G0S2 siRNA. The mRNA expression of C/EBPβ, peroxisome proliferator-activated receptor (PPAR)γ and PPARγ target genes (G0S2, GPR81, GPR109A and Adipoq) was examined by qPCR. The protein expression of C/EBPβ, PPARγ, and G0S2 was determined by Western blotting. Lipid accumulation was detected with Oil Red O staining and quantified by absorbance value of the extracted Oil Red O dye. Lipolysis was evaluated by measuring the amount of glycerol released into the medium.RESULTSPalmitate caused a dose-dependent increase in lipid accumulation and a dose-dependent decrease in lipolysis in HepG2 cells. In addition, palmitate increased the mRNA expression of C/EBPβ, PPARγ, and PPARγ target genes (G0S2, GPR81, GPR109A, and Adipoq) and the protein expression of C/EBPβ, PPARγ, and G0S2 in a dose-dependent manner. Knockdown of C/EBPβ decreased palmitate-induced PPARγ and its target genes (G0S2, GPR81, GPR109A, and Adipoq) mRNA expression and palmitate-induced PPARγ and G0S2 protein expression in HepG2 cells. Knockdown of C/EBPβ also attenuated lipid accumulation and augmented lipolysis in palmitate-treated HepG2 cells. G0S2 knockdown attenuated lipid accumulation and augmented lipolysis, while G0S2 knockdown had no effects on the mRNA expression of C/EBPβ, PPARγ, and PPARγ target genes (GPR81, GPR109A and Adipoq) in palmitate-treated HepG2 cells.CONCLUSIONPalmitate can induce lipid accumulation in HepG2 cells by activating C/EBPβ-mediated G0S2 expression.

Comparative sequence analysis and adipose-specific expression of G0S2 and ATGL in sheep

Lipolysis is a catabolic reaction that is mediated by adipose triglyceride lipase (ATGL) and its inhibitor such as G0/G1 switch gene 2 (G0S2) protein during the first step of hydrolysis. The hydrophobic domain of G0S2 protein interacts and binds to the patatin-like domain in ATGL. Although conservation of the hydrophobic domain has been reported across species, sheep G0S2 amino acids and its hydrophobic domain have not been sequenced and studied. In this study, the hydrophobic region of G0S2 which plays an important role in inhibition of ATGL-mediated lipolysis was highly conserved as shown in other species. Phylogenetic analysis revealed that ruminant G0S2 proteins showed greater homologies within the ruminant species compared to other mammalian species. Moreover, adipose-specific expression of G0S2 for sheep was observed. Together with an abundant expression of ATGL protein in adipose tissue of sheep, G0S2 protein may play a critical role in inhibiting ATGL-mediated triacylglycerol (TAG) hydrolysis in the adipose tissue.

Liver-specific G0 /G1 switch gene 2 (G0s2) expression promotes hepatic insulin resistance by exacerbating hepatic steatosis in male Wistar rats.

Hepatic steatosis is strongly associated with insulin resistance. It has been reported that G0 /G1 switch gene 2 (G0s2) inhibits the lipolytic activity of adipose triglyceride lipase, which is a major lipase in the liver as well as in adipocytes. Moreover, G0s2 protein content is increased in the livers of high-fat diet (HFD)-fed rats. In the present study, we investigated the effect of hepatic G0s2 on insulin sensitivity in male Wistar rats. Male Wistar rats were fed a 60% HFD for 4 weeks. After 3 weeks of feeding, rats were injected with adenovirus-expressing green fluorescent protein (Ad-GFP; control) or adenovirus-expressing mouse G0s2 (Ad-G0s2). On Day 7 after injection, a euglycemic-hyperinsulinemic clamp study was performed in rats fasted for 8 h. Body weight and fasting glucose levels were not significantly different between the Ad-GFP and Ad-G0s2 groups. During the clamp study, the glucose infusion rate required for euglycemia decreased significantly by 16% in the Ad-G0s2 compared with Ad-GFP group. The insulin-suppressed hepatic glucose output increased significantly in the Ad-G0s2 group, but the insulin-stimulated glucose disposal rate was not significantly different between the two groups. Consistent with the clamp data, insulin-stimulated phosphorylation of Akt decreased significantly in livers of rats injected with Ad-G0s2. Furthermore, Oil Red O-staining indicated that overexpression of G0s2 protein in the liver promoted hepatic steatosis by 2.5-fold in HFD-fed rats. The results of the present study indicate that hepatic G0s2 protein may promote hepatic insulin resistance by exacerbating hepatic steatosis.

Regulation of G0/G1 Switch Gene 2 (G0S2) Protein Ubiquitination and Stability by Triglyceride Accumulation and ATGL Interaction.

Intracellular triglyceride (TG) hydrolysis or lipolysis is catalyzed by the key intracellular triglyceride hydrolase, adipose triglyceride lipase (ATGL). The G0/G1 Switch Gene 2 (G0S2) was recently identified as the major selective inhibitor of ATGL and its hydrolase function. Since G0S2 levels are dynamically linked and rapidly responsive to nutrient status or metabolic requirements, the identification of its regulation at the protein level is of significant value. Earlier evidence from our laboratory demonstrated that G0S2 is a short-lived protein degraded through the proteasomal pathway. However, little is currently known regarding the underlying mechanisms. In the current study we find that 1) protein degradation is initiated by K48-linked polyubiquitination of the lysine- 25 in G0S2; and 2) G0S2 protein is stabilized in response to ATGL expression and TG accumulation. Mutation of lysine-25 of G0S2 abolished ubiquitination and increased protein stability. More importantly, G0S2 was stabilized via different mechanisms in the presence of ATGL vs. in response to fatty acid (FA)-induced TG accumulation. Furthermore, G0S2 protein but not mRNA levels were reduced in the adipose tissue of ATGL-deficient mice, corroborating the involvement of ATGL in the stabilization of G0S2. Taken together our data illustrate for the first time a crucial multifaceted mechanism for the stabilization of G0S2 at the protein level.

G0/G1 Switch Gene 2 controls adipose triglyceride lipase activity and lipid metabolism in skeletal muscle.

ObjectiveRecent data suggest that adipose triglyceride lipase (ATGL) plays a key role in providing energy substrate from triglyceride pools and that alterations of its expression/activity relate to metabolic disturbances in skeletal muscle. Yet little is known about its regulation. We here investigated the role of the protein G0/G1 Switch Gene 2 (G0S2), recently described as an inhibitor of ATGL in white adipose tissue, in the regulation of lipolysis and oxidative metabolism in skeletal muscle.MethodsWe first examined G0S2 protein expression in relation to metabolic status and muscle characteristics in humans. We next overexpressed and knocked down G0S2 in human primary myotubes to assess its impact on ATGL activity, lipid turnover and oxidative metabolism, and further knocked down G0S2 in vivo in mouse skeletal muscle.ResultsG0S2 protein is increased in skeletal muscle of endurance-trained individuals and correlates with markers of oxidative capacity and lipid content. Recombinant G0S2 protein inhibits ATGL activity by about 40% in lysates of mouse and human skeletal muscle. G0S2 overexpression augments (+49%, p < 0.05) while G0S2 knockdown strongly reduces (−68%, p < 0.001) triglyceride content in human primary myotubes and mouse skeletal muscle. We further show that G0S2 controls lipolysis and fatty acid oxidation in a strictly ATGL-dependent manner. These metabolic adaptations mediated by G0S2 are paralleled by concomitant changes in glucose metabolism through the modulation of Pyruvate Dehydrogenase Kinase 4 (PDK4) expression (5.4 fold, p < 0.001). Importantly, downregulation of G0S2 in vivo in mouse skeletal muscle recapitulates changes in lipid metabolism observed in vitro.ConclusionCollectively, these data indicate that G0S2 plays a key role in the regulation of skeletal muscle ATGL activity, lipid content and oxidative metabolism.

Integrated Regulation of Hepatic Lipid and Glucose Metabolism by Adipose Triacylglycerol Lipase and FoxO Proteins.

Metabolism is a highly integrated process that is coordinately regulated between tissues and within individual cells. FoxO proteins are major targets of insulin action and contribute to the regulation of gluconeogenesis, glycolysis, and lipogenesis in the liver. However, the mechanisms by which FoxO proteins exert these diverse effects in an integrated fashion remain poorly understood. We report that FoxO proteins also exert important effects on intrahepatic lipolysis and fatty acid oxidation via the regulation of adipose triacylglycerol lipase (ATGL), which mediates the first step in lipolysis, and its inhibitor, the G0/S1 switch 2 gene (G0S2). We also find that ATGL-dependent lipolysis plays a critical role in mediating diverse effects of FoxO proteins in the liver, including effects on gluconeogenic, glycolytic, and lipogenic gene expression and metabolism. These results indicate that intrahepatic lipolysis plays a critical role in mediating and integrating the regulation of glucose and lipid metabolism downstream of FoxO proteins.

Characterization of lipolytic inhibitor G(0)/G(1) switch gene-2 protein (G0S2) expression in male Sprague-Dawley rat skeletal muscle compared to relative content of adipose triglyceride lipase (ATGL) and comparitive gene identification-58 (CGI-58).

The rate-limiting enzyme in lipolysis, adipose triglyceride lipase (ATGL), is activated by comparative gene identification-58 (CGI-58) and inhibited by the G(0)/G(1) switch gene-2 (G0S2) protein. It is speculated that inhibition of ATGL is through a dose dependent manner of relative G0S2 protein content. There is little work examining G0S2 expression in lipolytic tissues, and the relative expression across oxidative tissues such as skeletal muscle has not yet been described. Three muscles, soleus (SOL), red gastrocnemius (RG), and white gastrocnemius (WG) were excised from 57-day old male Sprague-Dawley rats (n = 9). QRT-PCR was used for mRNA analysis, and western blotting was conducted to determine protein content. ATGL and G0S2 protein content were both greatest in the lipolytic SOL, with the least amount of both ATGL and G0S2 protein content found in the WG. CGI-58 protein content however did not mirror ATGL and G0S2 protein content, since the RG had the greatest CGI-58 protein content when compared to the SOL and WG. When comparing our tissues based on CGI-58-to-ATGL ratio and G0S2-to-ATGL ratio, it was discovered that contrary to oxidative demand, the glycolytic WG had the greatest activator CGI-58-to-ATGL ratio with the oxidative SOL having the least, and no differences in G0S2-to-ATGL across the three muscle types. These data suggest that the content of G0S2 relative to the lipase in skeletal muscle would not predict lipolytic potential.

Abstract 12840: In vivo Visualization of ATP Dynamics Under Hypoxia Reveals That G0/G1 Switch Gene 2 Provides Ischemic Tolerance Through the Increase of ATP Production

Introduction: We have recently established the method for the selective measurement of intra-mitochondrial ATP levels ([ATP]mito) and have identified the hypoxia-inducible protein G0/G1 switch gene 2 (G0s2) as a positive regulator of mitochondrial oxidative phosphorylation by cultured cardiomyocyte-based experiments. However, the energy metabolism in cultured cells may be much different from that in the living tissue. In this study, we examined the in vivo role of G0s2 under hypoxia by using a novel real-time in vivo ATP imaging technique in zebrafish heart. Methods and Results: We first established the in vivo ATP imaging technique by introducing a FRET-based ATP biosensor named Mit-ATeam into zebrafish heart (Mit-ATeam zebrafish). This system also allows us the simultaneous evaluation of cardiac function. Using Mit-ATeam zebrafish, we successfully observed a decline in [ATP]mito and cardiac function under hypoxic stress and the recovery of these parameters by sequential re-oxygenation. Cardiac specific G0s2 transgenic zebrafish had significantly stronger tolerance against hypoxic stress than wild type zebrafish, while mutant G0s2 transgenic zebrafish did not. In addition, we generated a chimeric zebrafish model in which G0s2 was regionally overexpressed to examine whether focal overexpression of G0s2 could preserve regional cardiac function under hypoxia. Only G0s2-overexpressing cardiomyocyte populations showed enhanced contractility with increased [ATP]mito in hypoxia compared to the control region (i.e. non-G0s2-overexpressing cardiomyocyte populations). Furthermore, we detected enhanced protein expression of G0s2 protein in the risk area in canine ischemic preconditioned heart, suggesting the possible involvement of G0s2 in ischemic preconditioning mediated cardioprotection. Conclusions: These results suggest that G0s2 functions as a guardian of ischemic myocardium and could become a therapeutic target for ischemic heart diseases. Additionally, this study is the first to connect cardiac energy metabolism and function in living animal in real-time.

Targeted Disruption of G0/G1 Switch Gene 2 Enhances Adipose Lipolysis, Alters Hepatic Energy Balance, and Alleviates High-Fat Diet–Induced Liver Steatosis

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.

G0S2–A new player in leukemia

G0S2–A new player in leukemia

Abstract 4125: The retinoid-induced G0S2 protein undergoes ISG15ylation and destabilization.

Abstract The interferon stimulated protein ISG15 was one of the first ubiquitin-like modifiers discovered. Similar to ubiquitin, ISG15 is conjugated to cellular proteins through a three-step enzymatic cascade involving an activating enzyme E1 (UBE1L), a conjugating enzyme E2 and a ligase E3. Conjugated ISG15 is removed from substrate proteins by the UBE1L-ISG15 deconjugase Usp18 (UBP43). To date, few direct target proteins of the ISG15 system were found and the biologic implications of their ISG15ylation are actively under study. We previously reported that the G0/G1 switch gene 2 (G0S2) was a direct target of all-trans-retinoic acid (RA) treatment in acute promyelocytic leukemia (APL). Prior work by others found that G0S2 was able to regulate lipolysis by inhibiting adipose triglyceride lipase (ATGL). The current study extends prior work by reporting that G0S2 was induced in APL cells by RA-treatment coordinately with UBE1L, ISG15 and the deconjugase Usp18. G0S2 was also shown to be a previously unrecognized target of ISG15ylation. G0S2 species were co-immunoprecipitated with ISG15. The functional consequence of this modification was confirmed by finding that G0S2 protein stability was directly affected by the ISG15 deconjugase Usp18. Loss of Usp18 expression by transfection of individual siRNAs into studied cells led to G0S2 protein destabilization. In contrast, engineered gain of Usp18 expression in the same cells stabilized G0S2 protein. Agents that can inhibit specific protein degradation pathways were also examined. Findings revealed that G0S2 protein was stabilized by treatment with proteasome or caspase inhibitors, but was not affected by lysosome or aggresome inhibitors. Thus, these findings indicate that G0S2 is a novel ISG15 substrate and that this modification substantially reduces G0S2 protein stability. Taken together, these results provide a mechanistic basis for cross-talk between interferon and retinoid signaling at the level of the G0S2 protein that undergoes ISG15ylation. This provides evidence for a role of lipolysis regulation in both interferon and retinoid signaling. The functional consequences of this are under study in mouse models. Citation Format: Yun Lu, Lisa Maria Mustachio, Fadzai Chinyengetere, Wei Yang, Tian Ma, Jessica Dong, David J. Sekula, Sarah J. Freemantle, Ethan Dmitrovsky. The retinoid-induced G0S2 protein undergoes ISG15ylation and destabilization. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4125. doi:10.1158/1538-7445.AM2013-4125

Abstract 4732: Repression of transcriptional activity by the retinoid target gene G0S2

Abstract Acute promyelocytic leukemia (APL) cases that express the PML/RARα fusion protein block maturation of myeloid precursors. All-trans retinoic acid (RA)-based differentiation therapy of these APL cases causes clinical remissions. Yet, clinical use of RA is associated with toxicities and drug resistance. In the search for RA target genes that can serve as alternative therapeutic targets in APL, G0S2 was found as one of the most rapidly and prominently RA-induced species. G0S2 is a 103 amino acid protein without apparent homology domains to other species. It was recently shown to inhibit adipose triglyceride lipase (ATGL) activity, leading to repression of lipolysis. G0S2 is abundantly expressed in adipose tissues, which is consistent with its role in regulating lipolysis. It is also induced in response to inflammatory stimuli in other cell types, indicating it has additional roles beyond fat storage. Here, we report a new G0S2 function uncovered after its transient transfection that led to substantial repression of reporter activities of multiple constructs (including RA-regulated species RARα, UBE1L and G0S2 itself). This repression was antagonized by siRNA-mediated G0S2 knockdown. Notably, an engineered form of G0S2 that lacked the internal hydrophobic domain did not exhibit these repressive effects. Inhibitors of either the proteasomal or lysosomal pathways did not overcome this suppression by G0S2. Thus, these repressive activities were unlikely due to enhanced proteasome or lysosome activities. The reported inhibition of ATGL activity by G0S2 causes an increase in lipid droplet size, which can also be achieved by treating cells with the neutral lipid oleic acid. Yet, this treatment did not impact reporter activity, indicating that changes in lipid droplet size did not affect this transcriptional repression. Intriguingly, G0S2 expression did not reduce endogenous expression of RARα, UBE1L or G0S2 (each RA-regulated species), indicating that only exogenous genes were affected by G0S2 transient transfection. Subcellular localization of endogenous G0S2 was examined in RA-treated NB4 APL cells and G0S2 was markedly expressed in the cytosol and membranes of these cells. Transiently transfected G0S2 protein was also detected in the cytoplasm. In summary, we report a previously unrecognized function for the RA target gene G0S2: it repressed exogenous reporter activity, but not endogenous gene expression. Both endogenous and exogenous G0S2 proteins were detected in the cytoplasm. Mechanistic studies that engage this G0S2 repressive effect are now underway. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4732. doi:1538-7445.AM2012-4732

Relative Contribution of Adipose Triglyceride Lipase and Hormone-sensitive Lipase to Tumor Necrosis Factor-α (TNF-α)-induced Lipolysis in Adipocytes

TNF-α potently stimulates basal lipolysis in adipocytes, which may contribute to hyperlipidemia and peripheral insulin resistance in obesity. Recent studies show that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) act sequentially in catalyzing the first two steps of adipose lipolysis in response to β-adrenergic stimulation. Here, we sought to determine their functional roles in TNF-α-induced lipolysis. Silencing of ATGL expression in adipocytes almost completely abolished basal and TNF-α-induced glycerol release. In comparison, the glycerol release under the same conditions was only partially decreased upon reduction in expression of either HSL or the ATGL coactivator CGI-58. Interestingly, overexpression of ATGL restored the lipolytic rates in cells with silenced HSL or CGI-58, indicating a predominant role for ATGL. While expression of ATGL, HSL and CGI-58 remains mostly unaffected, TNF-α treatment caused a rapid abrogation of the ATGL inhibitory protein G0S2. TNF-α drastically decreased the level of G0S2 mRNA, and the level of G0S2 protein could be maintained by inhibiting proteasomal protein degradation using MG-132. Furthermore, coexpression of G0S2 was able to significantly decrease TNF-α-stimulated lipolysis mediated by overexpressed ATGL or CGI-58. We propose that the early reduction in G0S2 content is permissive for TNF-α-induced lipolysis.

Fasting, But Not Exercise, Increases Adipose Triglyceride Lipase (ATGL) Protein and Reduces G(0)/G(1) Switch Gene 2 (G0S2) Protein and mRNA Content in Human Adipose Tissue

Fasting and exercise are characterized by increased lipolysis, but the underlying mechanisms are not fully understood. The study was designed to test whether fasting and exercise affect mRNA and protein levels of adipose triglyceride lipase (ATGL) and G(0)/G(1) switch gene 2 (G0S2), a recently discovered ATGL inhibitor, in humans. We studied eight healthy men (age, 25.5 ± 4.3 yr) for 6 h (a 4-h basal and a 2-h clamp period) on three occasions in a randomized crossover design: 1) in the basal state and after; 2) 72-h fasting; and 3) 1-h exercise (65% VO(2max)). Subcutaneous abdominal adipose tissue (AT) biopsies were taken at t = 30 and 270 min. The study was conducted at a university hospital research unit. Circulating free fatty acids and GH were increased, and C-peptide was decreased by both fasting and exercise. During fasting, insulin failed to suppress free fatty acid levels, suggesting AT insulin resistance. ATGL protein was increased 44% (P < 0.001), and G0S2 mRNA and protein were decreased 56% (P = 0.02) and 54% (P = 0.01), respectively, after fasting, but both ATGL and G0S2 were unaffected by exercise. Protein levels of hormone-sensitive lipase and comparative gene identification-58 were unaffected throughout. We found increased AT content of ATGL and decreased protein and mRNA content of the ATGL inhibitor G0S2, suggesting increased ATGL activity during fasting, but not after short-term exercise. These findings are compatible with the notion that the ATGL-G0S2 complex is an important long-term regulator of lipolysis under physiological conditions such as fasting in humans.

Expression profiling of endometrium from women with endometriosis reveals candidate genes for disease-based implantation failure and infertility.

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.