Samah Chahed

Differential Regulation of Endoplasmic Reticulum Stress by Protein Tyrosine Phosphatase 1B and T Cell Protein Tyrosine Phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.

Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates Diet-induced Endoplasmic Reticulum Stress in Liver and Adipose Tissue

Background: Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose pharmacological inhibition or targeted deletion in mice has beneficial effects, including improved insulin signaling in liver and adipose tissue. Results: sEH inhibition or deficiency attenuates high fat diet- and chemical-induced endoplasmic reticulum (ER) stress in mice and cells, respectively. Conclusion: sEH modulates ER stress in a cell-autonomous manner. Significance: sEH may be a therapeutic target for mitigating complications associated with the metabolic syndrome. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition has beneficial effects in cardiovascular, inflammatory, and metabolic diseases in murine models. Mice with targeted deletion or pharmacological inhibition of sEH exhibit improved insulin signaling in liver and adipose tissue. Herein, we assessed the role of sEH in regulating endoplasmic reticulum (ER) stress in liver and adipose tissue. We report that sEH expression was increased in the livers and adipose tissue of mice fed a high fat diet, the adipose tissue of overweight humans, and palmitate-treated cells. Importantly, sEH deficiency or inhibition in mice attenuated chronic high fat diet-induced ER stress in liver and adipose tissue. Similarly, pharmacological inhibition of sEH in HepG2 cells and 3T3-L1 adipocytes mitigated chemical-induced ER stress and activation of JNK, p38, and cell death. In addition, insulin signaling was enhanced in HepG2 cells treated with sEH substrates and attenuated in cells treated with sEH products. In summary, these findings demonstrate that sEH is a physiological modulator of ER stress and a potential target for mitigating complications associated with obesity.

Effects of soluble epoxide hydrolase deficiency on acute pancreatitis in mice

Background Acute pancreatitis (AP) is a frequent gastrointestinal disorder that causes significant morbidity, and its incidence has been progressively increasing. AP starts as a local inflammation in the pancreas that often leads to systemic inflammatory response and complications. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose inhibition in murine models has beneficial effects in inflammatory diseases, but its significance in AP remains unexplored. Methodology/Principal Findings To investigate whether sEH may have a causal role in AP we utilized Ephx2 knockout (KO) mice to determine the effects of sEH deficiency on cerulein- and arginine-induced AP. sEH expression increased at the protein and messenger RNA levels, as well as enzymatic activity in the early phase of cerulein- and arginine-induced AP in mice. In addition, amylase and lipase levels were lower in cerulein-treated Ephx2 KO mice compared with controls. Moreover, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B and IL-6 were lower in cerulein-treated Ephx2 KO mice compared with controls. Further, Ephx2 KO mice exhibited decreased cerulein- and arginine-induced NF-κB inflammatory response, MAPKs activation and decreased cell death. Conclusions -These findings demonstrate a novel role for sEH in the progression of cerulein- and arginine-induced AP.

Soluble Epoxide Hydrolase Pharmacological Inhibition Ameliorates Experimental Acute Pancreatitis in Mice

Acute pancreatitis (AP) is an inflammatory disease, and is one of the most common gastrointestinal disorders worldwide. Soluble epoxide hydrolase (sEH; encoded by Ephx2) deficiency and pharmacological inhibition have beneficial effects in inflammatory diseases. Ephx2 whole-body deficiency mitigates experimental AP in mice, but the suitability of sEH pharmacological inhibition for treating AP remains to be determined. We investigated the effects of sEH pharmacological inhibition on cerulein- and arginine-induced AP using the selective sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which was administered before and after induction of pancreatitis. Serum amylase and lipase levels were lower in TPPU-treated mice compared with controls. In addition, circulating levels and pancreatic mRNA of the inflammatory cytokines tumor necrosis factor-α, interleukin Il-1β, and Il-6 were reduced in TPPU-treated mice. Moreover, sEH pharmacological inhibition before and after induction of pancreatitis was associated with decreased cerulein- and arginine-induced nuclear factor-κB inflammatory response, endoplasmic reticulum stress, and cell death. sEH pharmacological inhibition before and after induction of pancreatitis mitigated cerulein- and arginine-induced AP. This work suggests that sEH pharmacological inhibition may be of therapeutic value in acute pancreatitis.

Adipose-specific deletion of Src homology phosphatase 2 does not significantly alter systemic glucose homeostasis

The SH2 domain-containing protein-tyrosine phosphatase Src homology phosphatase 2 (Shp2) has been implicated in a variety of growth factor signaling pathways, but its metabolic role in some peripheral insulin-responsive tissues remains unknown. To address the metabolic function of Shp2 in adipose tissue, we generated mice with adipose-specific Shp2 deletion using adiponectin-Cre transgenic mice. We then analyzed insulin sensitivity, glucose tolerance, and body mass in adipose-specific Shp2-deficient and control mice on regular chow and high-fat diet (HFD). Control mice on HFD exhibited increased Shp2 expression in various adipose depots compared with those on regular chow. Adiponectin-Cre mice enabled efficient and specific deletion of Shp2 in adipose tissue. However, adipose Shp2 deletion did not significantly alter body mass in mice on chow or HFD. In addition, mice with adipose Shp2 deletion exhibited comparable insulin sensitivity and glucose tolerance compared with controls. Consistent with this, basal and insulin-stimulated Erk and Akt phosphorylations were comparable in adipose tissue of Shp2-deficient and control mice. Our findings indicate that adipose-specific Shp2 deletion does not significantly alter systemic insulin sensitivity and glucose homeostasis.

Podocyte‐specific soluble epoxide hydrolase deficiency in mice attenuates acute kidney injury

Podocytes play an important role in maintaining glomerular function, and podocyte injury is a significant component in the pathogenesis of proteinuria. Soluble epoxide hydrolase (sEH) is a cytosolic enzyme whose genetic deficiency and pharmacological inhibition have beneficial effects on renal function, but its role in podocytes remains unexplored. The objective of this study was to investigate the contribution of sEH in podocytes to lipopolysaccharide (LPS)‐induced kidney injury. We report increased sEH transcript and protein expression in murine podocytes upon LPS challenge. To determine the function of sEH in podocytes in vivo we generated podocyte‐specific sEH‐deficient (pod‐sEHKO) mice. Following LPS challenge, podocyte sEH‐deficient mice exhibited lower kidney injury, proteinuria, and blood urea nitrogen concentrations than controls suggestive of preserved renal function. Also, renal mRNA and serum concentrations of inflammatory cytokines IL‐6, IL‐1β, and TNFα were significantly lower in LPS‐treated pod‐sEHKO than control mice. Moreover, podocyte sEH deficiency was associated with decreased LPS‐induced NF‐κB and MAPK activation and attenuated endoplasmic reticulum stress. Furthermore, the protective effects of podocyte sEH deficiency in vivo were recapitulated in E11 murine podocytes treated with a selective sEH pharmacological inhibitor. Altogether, these findings identify sEH in podocytes as a contributor to signaling events in acute renal injury and suggest that sEH inhibition may be of therapeutic value in proteinuria.

Soluble epoxide hydrolase in podocytes is a significant contributor to renal function under hyperglycemia

BACKGROUND Diabetic nephropathy (DN) is the leading cause of renal failure, and podocyte dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH, encoded by Ephx2) is a conserved cytosolic enzyme whose inhibition has beneficial effects on renal function. The aim of this study is to investigate the contribution of sEH in podocytes to hyperglycemia-induced renal injury. MATERIALS AND METHODS Mice with podocyte-specific sEH disruption (pod-sEHKO) were generated, and alterations in kidney function were determined under normoglycemia, and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. RESULTS sEH protein expression increased in murine kidneys under HFD- and STZ-induced hyperglycemia. sEH deficiency in podocytes preserved renal function and glucose control and mitigated hyperglycemia-induced renal injury. Also, podocyte sEH deficiency was associated with attenuated hyperglycemia-induced renal endoplasmic reticulum (ER) stress, inflammation and fibrosis, and enhanced autophagy. Moreover, these effects were recapitulated in immortalized murine podocytes treated with a selective sEH pharmacological inhibitor. Furthermore, pharmacological-induced elevation of ER stress or attenuation of autophagy in immortalized podocytes mitigated the protective effects of sEH inhibition. CONCLUSIONS These findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia. GENERAL SIGNIFICANCE These data suggest that sEH is a potential therapeutic target for podocytopathies.

Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice

Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis.

Decreased adiposity and enhanced glucose tolerance in shikonin treated mice.

Obesity represents a major public health problem, and identifying natural compounds that modulate energy balance and glucose homeostasis is of interest for combating obesity and its associated disorders. The naphthoquinone shikonin has diverse beneficial properties including anti‐inflammatory, anti‐oxidant, and anti‐microbial effects. The objective of this study is to investigate the effects of shikonin on adiposity and glucose homeostasis.

Pancreatic T cell protein–tyrosine phosphatase deficiency affects beta cell function in mice

Aims/hypothesisT cell protein tyrosine phosphatase (TCPTP, encoded by PTPN2) regulates cytokine-induced pancreatic beta cell apoptosis and may contribute to the pathogenesis of type 1 diabetes. However, the role of TCPTP in pancreatic endocrine function and insulin secretion remains largely unknown.MethodsTo investigate the endocrine role of pancreatic TCPTP we generated mice with pancreas Ptpn2/TCPTP deletion (panc-TCPTP KO).ResultsWhen fed regular chow, panc-TCPTP KO and control mice exhibited comparable glucose tolerance. However, when challenged with prolonged high fat feeding panc-TCPTP KO mice exhibited impaired glucose tolerance and attenuated glucose-stimulated insulin secretion (GSIS). The defect in GSIS was recapitulated in primary islets ex vivo and after TCPTP pharmacological inhibition or lentiviral-mediated TCPTP knockdown in the glucose-responsive MIN6 beta cells, consistent with this being cell autonomous. Reconstitution of TCPTP in knockdown cells reversed the defect in GSIS demonstrating that the defect was a direct consequence of TCPTP deficiency. The reduced insulin secretion in TCPTP knockdown MIN6 beta cells was associated with decreased insulin content and glucose sensing. Furthermore, TCPTP deficiency led to enhanced tyrosyl phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT 1/3), and substrate trapping studies in MIN6 beta cells identified STAT 1/3 as TCPTP substrates. STAT3 pharmacological inhibition and small interfering RNA-mediated STAT3 knockdown in TCPTP deficient cells restored GSIS to control levels, indicating that the effects of TCPTP deficiency were mediated, at least in part, through enhanced STAT3 phosphorylation and signalling.Conclusions/interpretationThese studies identify a novel role for TCPTP in insulin secretion and uncover STAT3 as a physiologically relevant target for TCPTP in the endocrine pancreas.