Deletion of the gene encoding G0/G1 switch protein 2 (G0s2) alleviates high-fat-diet-induced weight gain and insulin resistance, and promotes browning of white adipose tissue in mice

Conclusions/interpretation: Our data show that G0s2 is a physiological regulator of adiposity and energy metabolism and is a potential target in the treatment of obesity and insulin resistance.Results: We report that G0s2 inhibits ATGL and regulates lipolysis and energy metabolism in vivo. G0s2−/− mice are lean, resistant to weight gain induced by a high-fat diet and are glucose tolerant and insulin sensitive. The white adipose tissue of G0s2−/− mice has enhanced lipase activity and adipocytes showed enhanced stimulated lipolysis. Energy metabolism in the G0s2−/− mice is shifted towards enhanced lipid metabolism and increased thermogenesis. G0s2−/− mice showed enhanced cold tolerance and increased expression of thermoregulatory and oxidation genes within white adipose tissue, suggesting enhanced ‘browning’ of the white adipose tissue.Aims/hypothesis: Obesity is a global epidemic resulting from increased energy intake, which alters energy homeostasis and results in an imbalance in fat storage and breakdown. G0/G1 switch gene 2 (G0s2) has been recently characterised in vitro as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting step in fat catabolism. In the current study we aim to functionally characterise G0s2 within the physiological context of a mouse model.Methods: We generated a mouse model in which G0s2 was deleted. The homozygous G0s2 knockout (G0s2−/−) mice were studied over a period of 22 weeks. Metabolic variables were measured including body weight and body composition, food intake, glucose and insulin tolerance tests, energy metabolism and thermogenesis

Identification of a protein, G0S2, that lacks Bcl-2 homology domains and interacts with and antagonizes Bcl-2

The Bcl-2 family of proteins consists of both antiapoptotic and proapoptotic factors, which share sequence homology within conserved regions known as Bcl-2 homology domains. Interactions between Bcl-2 family members, as well as with other proteins, regulate apoptosis through control of mitochondrial membrane permeability and release of cytochrome c. Here we identify a novel regulator of apoptosis that lacks Bcl-2 homology domains but acts by binding Bcl-2 and modulating its antiapoptotic activity. To identify regulators of apoptosis, we performed expression profiling in human primary fibroblasts treated with tumor necrosis factor-alpha (TNF-alpha), a potent inflammatory cytokine that can regulate apoptosis and functions, at least in part, by inducing expression of specific genes through NF-kappaB. We found that the gene undergoing maximal transcriptional induction following TNF-alpha treatment was G(0)-G(1) switch gene 2 (G0S2), the activation of which also required NF-kappaB. We show that G0S2 encodes a mitochondrial protein that specifically interacts with Bcl-2 and promotes apoptosis by preventing the formation of protective Bcl-2/Bax heterodimers. We further show that ectopic expression of G0S2 induces apoptosis in diverse human cancer cell lines in which endogenous G0S2 is normally epigenetically silenced. Our results reveal a novel proapoptotic factor that is induced by TNF-alpha through NF-kappaB and that interacts with and antagonizes Bcl-2

Saturated fatty acids synergize with elevated glucose to cause pancreatic beta-cell death

We have proposed the "glucolipotoxicity" hypothesis in which elevated free fatty acids (FFAs) together with hyperglycemia are synergistic in causing islet β-cell damage because high glucose inhibits fat oxidation and consequently lipid detoxification. T

Ceramide generation by two distinct pathways in tumor necrosis factor α-induced cell death

AbstractCeramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor α. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action

Deletion of the gene encoding G0/G 1 switch protein 2 (G0s2) alleviates high-fat-diet-induced weight gain and insulin resistance, and promotes browning of white adipose tissue in mice

AIMS/HYPOTHESIS: Obesity is a global epidemic resulting from increased energy intake, which alters energy homeostasis and results in an imbalance in fat storage and breakdown. G0/G1 switch gene 2 (G0s2) has been recently characterised in vitro as an inhibitor of adipose triglyceride lipase (ATGL), the rate-limiting step in fat catabolism. In the current study we aim to functionally characterise G0s2 within the physiological context of a mouse model. METHODS: We generated a mouse model in which G0s2 was deleted. The homozygous G0s2 knockout (G0s2 (-/-)) mice were studied over a period of 22 weeks. Metabolic variables were measured including body weight and body composition, food intake, glucose and insulin tolerance tests, energy metabolism and thermogenesis. RESULTS: We report that G0s2 inhibits ATGL and regulates lipolysis and energy metabolism in vivo. G0s2 (-/-) mice are lean, resistant to weight gain induced by a high-fat diet and are glucose tolerant and insulin sensitive. The white adipose tissue of G0s2 (-/-) mice has enhanced lipase activity and adipocytes showed enhanced stimulated lipolysis. Energy metabolism in the G0s2 (-/-) mice is shifted towards enhanced lipid metabolism and increased thermogenesis. G0s2 (-/-) mice showed enhanced cold tolerance and increased expression of thermoregulatory and oxidation genes within white adipose tissue, suggesting enhanced \u27browning\u27 of the white adipose tissue. CONCLUSIONS/INTERPRETATION: Our data show that G0s2 is a physiological regulator of adiposity and energy metabolism and is a potential target in the treatment of obesity and insulin resistance

PACE4 cleaves PRR intracellularly in prostate cancer cells.

(A) Western blot analysis showing Prorenin Receptor (PRR), furin, and PACE4 expression, sPRR secretion, and cell lysate and conditioned media total lane protein (CLTLP, CMTLP) in LNCaP cells infected with a control non-target shRNA (NT), Furin shRNA (shfurin), or PACE4 shRNA (shPACE4). (B) Western Blot analysis demonstrating the expression of PRR and secretion of sPRR in LNCaP cells infected with an empty pLenti6 vector or with pLenti6-PACE4 to overexpress PACE4. (C) Quantification of sPRR levels in pLenti6 and pLenti-PACE4-infected LNCaP cells (*PD) Western blot showing the reduction in PRR processing resembled as a ratio of HA-tagged M8.9 (M8.9-HA) to HA-tagged full-length PRR (PRR-HA) in cellular extract of LNCaP cells after a 50 μM PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23) treatment. (E) Corresponding quantification of the ratio of M8.9-HA to PRR-HA standardized over total lane protein (TLP) (*PF) Analysis of PRR peptide cleavage by recombinant PACE4 (rPACE4) or recombinant furin (rfurin) monitored after a 2-hour incubation by high pressure liquid chromatography (HPLC). Mass spectrometry was done to confirm identity of peptide after cleavage. Cleavage site is underlined on the peptide sequence. Western blot analysis of PRR expression and sPRR secretion and quantification of sPRR secretion after DMSO (Vehicle), 50 μM multi-Leucine peptide (ML) PACE4 inhibitor, or 50 μM PEGylated cell-impermeable ML (PEG8-ML) treatment of DU145 (***PG, H) or LNCaP (I, J) (**P<0.01, n = 3) cells, respectively. Beta-Actin (β-actin) and TLP were used as loading controls. Data are presented as the mean ± SEM. Statistical tests were conducted using Student’s t test.</p

PACE4 inhibition, like PRR knockdown, reduces V-ATPase activity.

(A) Representative proliferation images of LNCaP exposed to both DMSO or a 100 μM of PACE4 inhibitor [33] LLLRVK-amidinobenzylamide (Amba) (C23) over 0 and 64 hours. (B) Western blot analysis of soluble prorenin receptor (sPRR) secretion in conditioned media of LNCaP exposed to DMSO or a 100 μM C23. (C) Representative LysoTracker images of LNCaP cells transfected with non-silencing control siRNA (NSC), PRR siRNA 1 and 2, PACE siRNA, or treated with 100 nM Bafilomycin A1. (D) Quantification of LysoTracker signal intensity relative to number of cells in brightfield images treated with 1% DMSO (Vehicle), 50 μM C23, 100 nM Bafilomycin A1 (BafA1), or transfected with PRR siPRR 1 or siPRR 2. (E) Schematic showing the PRR-HA, sPRR-HA, and M8.9-HA vectors used in panels F, G and H. (F) Representative LysoTracker images of LNCaP cells transfected with empty vector (EV) and treated with DMSO, treated with 100 μM C23, transfected with PRR-HA and treated with 100 μM C23, transfected with M8.9-HA and treated with 100 μM C23, transfected with sPRR-HA and treated with 100 μM C23, or treated with 100 nM BafA1. (G) Corresponding quantification of LysoTracker intensity relative to cell number in the same treatments (**P ≤ 0.01, ***P ≤ 0.001, **** P H) Western blot analysis of PRR and HA expression in the same treatments. Total lane protein (TLP) was used as loading control. Data are presented as the mean ±SEM. Statistical tests were conducted using Student’s t test. Scale bar measures 100.</p