Pharmacological inhibition of the Src homology phosphatase 2 confers partial protection in a mouse model of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is a leading factor of liver-related death worldwide. ALD has various manifestations that include steatosis, hepatitis, and cirrhosis and is currently without approved pharmacotherapies. The Src homology phosphatase 2 (Shp2) is a drug target in some cancers due to its positive regulation of Ras-mitogen-activated protein kinase signaling and cell proliferation. Shp2 pharmacological inhibition yields beneficial outcomes in animal disease models, but its impact on ALD remains unexplored. This study aims to investigate the effects of Shp2 inhibition and its validity using a preclinical mouse model of ALD. We report that the administration of SHP099, a potent and selective allosteric inhibitor of Shp2, partially ameliorated ethanol-induced hepatic injury, inflammation, and steatosis in mice. Additionally, Shp2 inhibition was associated with reduced ethanol-evoked activation of extracellular signal-regulated kinase (ERK), oxidative, and endoplasmic reticulum (ER) stress in the liver. Besides the liver, excessive alcohol consumption induces multi-organ injury and dysfunction, including the intestine. Notably, Shp2 inhibition diminished ethanol-induced intestinal inflammation and permeability, abrogated the reduction in tight junction protein expression, and the activation of ERK and stress signaling in the ileum. Collectively, Shp2 pharmacological inhibition mitigates the deleterious effects of ethanol in the liver and intestine in a mouse model of ALD. Given the multifactorial aspects underlying ALD pathogenesis, additional studies are needed to decipher the utility of Shp2 inhibition alone or as a component in a multitherapeutic regimen to combat this deadly malady.

Effects of beef fat enriched with trans vaccenic acid and cis9, trans11-CLA on glucose homoeostasis and hepatic lipid accumulation in high-fat diet-induced obese mice.

Trans vaccenic acid (TVA, trans11-18 : 1) and cis9, trans11-CLA (also known as rumenic acid; RA) have received widespread attention as potentially beneficial trans-FA due to their putative health benefits, including anti-diabetic properties. The objective of this study was to determine the effects of beef fat naturally enriched with TVA and RA on parameters related to glucose homoeostasis and associated metabolic markers in diet-induced obese (DIO) mice. Thirty-six male C57BL/6J mice (8 weeks old) were fed for 19 weeks with either a control low-fat diet (CLF), a control high-fat diet (CHF), or a TVA+RA-enriched high-fat diet (EHF). Compared with CLF, feeding either CHF or EHF resulted in adverse metabolic outcomes associated with high-fat diets, including adiposity, impaired glucose control and hepatic steatosis. However, the EHF diet induced a significantly higher liver weight TAG content and elevated plasma alanine transaminase levels compared with the CHF diet. Collectively, the findings from this study suggest that EHF does not improve glucose tolerance and worsens liver steatosis in DIO mice. However, the adverse effects of EHF on the liver could be in part related to the presence of other trans-FA in the enriched beef fat.

Hepatic protein tyrosine phosphatase Shp2 disruption mitigates the adverse effects of ethanol in the liver by modulating oxidative stress and ERK signaling.

AIMS: Chronic excessive alcohol intake is a significant cause of alcohol-associated liver disease (ALD), a leading contributor to liver-related morbidity and mortality. The Src homology phosphatase 2 (Shp2; encoded by Ptpn11) is a widely expressed protein tyrosine phosphatase that modulates hepatic functions, but its role in ALD is mostly uncharted. MAIN METHODS: Herein, we explore the effects of liver-specific Shp2 genetic disruption using the established chronic-plus-binge mouse model of ALD. KEY FINDINGS: We report that the hepatic Shp2 disruption had beneficial effects and partially ameliorated ethanol-induced injury, inflammation, and steatosis in the liver. Consistently, Shp2 deficiency was associated with decreased ethanol-evoked activation of extracellular signal-regulated kinase (ERK) and oxidative stress in the liver. Moreover, primary hepatocytes with Shp2 deficiency exhibited similar outcomes to those observed upon Shp2 disruption in vivo, including diminished ethanol-induced ERK activation, inflammation, and oxidative stress. Furthermore, pharmacological inhibition of ERK in primary hepatocytes mimicked the effects of Shp2 deficiency and attenuated oxidative stress caused by ethanol. SIGNIFICANCE: Collectively, these findings highlight Shp2 as a modulator of hepatic oxidative stress upon ethanol challenge and suggest the evaluation of this phosphatase as a potential therapeutic target for ALD.

Protein tyrosine phosphatase 1B is a regulator of alpha-actinin4 in the glomerular podocyte.

Glomerular podocytes are instrumental for the barrier function of the kidney, and podocyte injury contributes to proteinuria and the deterioration of renal function. Protein tyrosine phosphatase 1B (PTP1B) is an established metabolic regulator, and the inactivation of this phosphatase mitigates podocyte injury. However, there is a paucity of data regarding the substrates that mediate PTP1B actions in podocytes. This study aims to uncover novel substrates of PTP1B in podocytes and validate a leading candidate. To this end, using substrate-trapping and mass spectroscopy, we identified putative substrates of this phosphatase and investigated the actin cross-linking cytoskeletal protein alpha-actinin4. PTP1B and alpha-actinin4 co-localized in murine and human glomeruli and transiently transfected E11 podocyte cells. Additionally, podocyte PTP1B deficiency in vivo and culture was associated with elevated tyrosine phosphorylation of alpha-actinin4. Conversely, reconstitution of the knockdown cells with PTP1B attenuated alpha-actinin4 tyrosine phosphorylation. We demonstrated co-association between alpha-actinin4 and the PTP1B substrate-trapping mutant, which was enhanced upon insulin stimulation and disrupted by vanadate, consistent with an enzyme-substrate interaction. Moreover, we identified alpha-actinin4 tandem tyrosine residues 486/487 as mediators of its interaction with PTP1B. Furthermore, knockdown studies in E11 cells suggest that PTP1B and alpha-actinin4 are modulators of podocyte motility. These observations indicate that PTP1B and alpha-actinin4 are likely interacting partners in a signaling node that modulates podocyte function. Targeting PTP1B and plausibly this one of its substrates may represent a new therapeutic approach for podocyte injury that warrants additional investigation.

Deficiency of the Src homology phosphatase 2 in podocytes is associated with renoprotective effects in mice under hyperglycemia.

Diabetic nephropathy (DN) is a significant complication of diabetes and the leading cause of end-stage renal disease. Hyperglycemia-induced dysfunction of the glomerular podocytes is a major contributor to the deterioration of renal function in DN. Previously, we demonstrated that podocyte-specific disruption of the Src homology phosphatase 2 (Shp2) ameliorated lipopolysaccharide-induced renal injury. This study aims to evaluate the contribution of Shp2 to podocyte function under hyperglycemia and explore the molecular underpinnings. We report elevated Shp2 in the E11 podocyte cell line under high glucose and the kidney under streptozotocin- and high-fat diet-induced hyperglycemia. Consistently, Shp2 disruption in podocytes was associated with partial renoprotective effects under hyperglycemia, as evidenced by the preserved renal function. At the molecular level, Shp2 deficiency was associated with altered renal insulin signaling and diminished hyperglycemia-induced renal endoplasmic reticulum stress, inflammation, and fibrosis. Additionally, Shp2 knockdown in E11 podocytes mimicked the in vivo deficiency of this phosphatase and ameliorated the deleterious impact of high glucose, whereas Shp2 reconstitution reversed these effects. Moreover, Shp2 deficiency attenuated high glucose-induced E11 podocyte migration. Further, we identified the protein tyrosine kinase FYN as a putative mediator of Shp2 signaling in podocytes under high glucose. Collectively, these findings suggest that Shp2 inactivation may afford protection to podocytes under hyperglycemia and highlight this phosphatase as a potential target to ameliorate glomerular dysfunction in DN.

Genetic deficiency or pharmacological inhibition of soluble epoxide hydrolase ameliorates high fat diet-induced pancreatic ß-cell dysfunction and loss.

Pancreatic ß-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify ß-cell dysfunction and loss are essential to combat this deadly malady. In the current study, we sought to delineate the role of soluble epoxide hydrolase (sEH) in ß-cells under diet-induced metabolic stress. The expression of sEH was upregulated in murine and macaque diabetes models and islets of diabetic human patients. We postulated that hyperglycemia-induced elevation in sEH leads to a reduction in its substrates, epoxyeicosatrienoic acids (EETs), and attenuates the function of ß-cells. Genetic deficiency of sEH potentiated glucose-stimulated insulin secretion in mice, likely in a cell-autonomous manner, contributing to better systemic glucose control. Consistent with this observation, genetic and pharmacological inactivation of sEH and the treatment with EETs exhibited insulinotropic effects in isolated murine islets ex vivo. Additionally, sEH deficiency enhanced glucose sensing and metabolism with elevated ATP and cAMP concentrations. This phenotype was associated with attenuated oxidative stress and diminished ß-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced ß-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in ß-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify ß-cell dysfunction and loss.

Soluble Epoxide Hydrolase Hepatic Deficiency Ameliorates Alcohol-Associated Liver Disease.

BACKGROUND & AIMS: Alcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored. METHODS: To decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model. RESULTS: We observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding. CONCLUSIONS: These findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

Hepatic protein-tyrosine phosphatase 1B disruption and pharmacological inhibition attenuate ethanol-induced oxidative stress and ameliorate alcoholic liver disease in mice.

Alcoholic liver disease (ALD) is a major health problem and a significant cause of liver-related death. Currently, the mainstay for ALD therapy is alcohol abstinence highlighting the need to develop pharmacotherapeutic approaches. Protein-tyrosine phosphatase 1B (PTP1B) is an established regulator of hepatic functions, but its role in ALD is mostly unexplored. In this study, we used mice with liver-specific PTP1B disruption as well as pharmacological inhibition to investigate the in vivo function of this phosphatase in ALD. We report upregulation of hepatic PTP1B in the chronic plus binge mouse model and, importantly, in liver biopsies of alcoholic hepatitis patients. Also, mice with hepatic PTP1B disruption attenuated ethanol-induced injury, inflammation, and steatosis compared with ethanol-fed control animals. Moreover, PTP1B deficiency was associated with decreased ethanol-induced oxidative stress in vivo and ex vivo. Further, pharmacological modulation of oxidative balance in hepatocytes identified diminished oxidative stress as a contributor to the salutary effects of PTP1B deficiency. Notably, PTP1B pharmacological inhibition elicited beneficial effects and mitigated hepatic injury, inflammation, and steatosis caused by ethanol feeding. In summary, these findings causally link hepatic PTP1B and ALD and define a potential therapeutic target for the management of this disease.

Protein tyrosine phosphatase 1B deficiency in podocytes mitigates hyperglycemia-induced renal injury.

OBJECTIVE: Diabetic nephropathy is one of the most devastating complications of diabetes, and growing evidence implicates podocyte dysfunction in disease pathogenesis. The objective of this study was to investigate the contribution of protein tyrosine phosphatase 1B (PTP1B) in podocytes to hyperglycemia-induced renal injury. METHODS: To determine the in vivo function of PTP1B in podocytes we generated mice with podocyte-specific PTP1B disruption (hereafter termed pod-PTP1B KO). Kidney functions were determined in control and pod-PTP1B KO mice under normoglycemia and high-fat diet (HFD)- and streptozotocin (STZ)-induced hyperglycemia. RESULTS: PTP1B expression increased in murine kidneys following HFD and STZ challenges. Under normoglycemia control and pod-PTP1B KO mice exhibited comparable renal functions. However, podocyte PTP1B disruption attenuated hyperglycemia-induced albuminuria and renal injury and preserved glucose control. Also, podocyte PTP1B disruption was accompanied with improved renal insulin signaling and enhanced autophagy with decreased inflammation and fibrosis. Moreover, the beneficial effects of podocyte PTP1B disruption in vivo were recapitulated in E11 murine podocytes with lentiviral-mediated PTP1B knockdown. Reconstitution of PTP1B in knockdown podocytes reversed the enhanced insulin signaling and autophagy suggesting that they were likely a consequence of PTP1B deficiency. Further, pharmacological attenuation of autophagy in PTP1B knockdown podocytes mitigated the protective effects of PTP1B deficiency. CONCLUSIONS: These findings demonstrate that podocyte PTP1B deficiency attenuates hyperglycemia-induced renal damage and suggest that PTP1B may present a therapeutic target in renal injury.

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.

Protein tyrosine phosphatase Shp2 deficiency in podocytes attenuates lipopolysaccharide-induced proteinuria.

Podocytes are specialized epithelial cells that play a significant role in maintaining the integrity of the glomerular filtration barrier and preventing urinary protein leakage. We investigated the contribution of protein tyrosine phosphatase Shp2 to lipopolysaccharide (LPS)-induced renal injury. We report increased Shp2 expression in murine kidneys and cultured podocytes following an LPS challenge. To determine the role of podocyte Shp2 in vivo, we generated podocyte-specific Shp2 knockout (pod-Shp2 KO) mice. Following administration of LPS, pod-Shp2 KO mice exhibited lower proteinuria and blood urea nitrogen concentrations than controls indicative of preserved filter integrity. In addition, renal mRNA and serum concentrations of inflammatory cytokines IL-1ß, TNFα, INFγ and IL-12 p70 were significantly decreased in LPS-treated knockout mice compared with controls. Moreover, the protective effects of podocyte Shp2 deficiency were associated with decreased LPS-induced NF-κB and MAPK activation, nephrin phosphorylation and attenuated endoplasmic reticulum stress. These effects were recapitulated in differentiated E11 murine podocytes with lentiviral-mediated Shp2 knockdown. Furthermore, Shp2 deficient podocytes displayed reduced LPS-induced migration in a wound healing assay. These findings identify Shp2 in podocytes as a significant contributor to the signaling events following LPS challenge and suggest that inhibition of Shp2 in podocytes may present a potential therapeutic target for podocytopathies.

S-nitrosylation of endogenous protein tyrosine phosphatases in endothelial insulin signaling.

Nitric oxide (NO) exerts its biological function through S-nitrosylation of cellular proteins. Due to the labile nature of this modification under physiological condition, identification of S-nitrosylated residue in enzymes involved in signaling regulation remains technically challenging. The present study investigated whether intrinsic NO produced in endothelium-derived MS-1 cells response to insulin stimulation might target endogenous protein tyrosine phosphatases (PTPs). For this, we have developed an approach using a synthetic reagent that introduces a phenylacetamidyl moiety on S-nitrosylated Cys, followed by detection with anti-phenylacetamidyl Cys (PAC) antibody. Coupling with sequential blocking of free thiols with multiple iodoacetyl-based Cys-reactive chemicals, we employed this PAC-switch method to show that endogenous SHP-2 and PTP1B were S-nitrosylated in MS-1 cells exposed to insulin. The mass spectrometry detected a phenylacetamidyl moiety specifically present on the active-site Cys463 of SHP-2. Focusing on the regulatory role of PTP1B, we showed S-nitrosylation to be the principal Cys reversible redox modification in endothelial insulin signaling. The PAC-switch method in an imaging format illustrated that a pool of S-nitrosylated PTP1B was colocalized with activated insulin receptor to the cell periphery, and that such event was endothelial NO synthase (eNOS)-dependent. Moreover, ectopic expression of the C215S mutant of PTP1B that mimics the active-site Cys215 S-nitrosylated form restored insulin responsiveness in eNOS-ablated cells, which was otherwise insensitive to insulin stimulation. This work not only introduces a new method that explores the role of physiological NO in regulating signal transduction, but also highlights a positive NO effect on promoting insulin responsiveness through S-nitrosylation of PTP1B's active-site Cys215.

Enhancement of insulin responsiveness by nitric oxide-mediated inactivation of protein-tyrosine phosphatases.

NO synthesis is a prerequisite for proper insulin sensitivity in insulin-targeted tissues; however, the molecular basis for this process remains unclear. Using a gain-of-function model of endothelial nitric-oxide synthase (eNOS)-transfected COS-7 cells, we have shown a critical role of NO in insulin responsiveness, as evidenced by an NO-dependent increase of tyrosine phosphorylation levels of the insulin receptor and its downstream effectors insulin receptor substrate-1 and PKB/AKT. We hypothesized that NO-induced inactivation of endogenous protein-tyrosine phosphatases (PTPs) would enhance insulin receptor-mediated signaling. To test this hypothesis, we devised a new method of the PTP labeling using a cysteine sulfhydryl-reacted probe. Under the acidic conditions employed in this study, the probe recognized the reduced and active forms but not the S-nitrosylated and inactive forms of endogenous PTPs. Our data suggest that phosphatases SHP-1, SHP-2, and PTP1B, but not TC-PTP, are likely S-nitrosylated at the active site cysteine residue concomitantly with a burst of NO production in signaling response to insulin stimulation. These results were further confirmed by phosphatase activity assays. We investigated further the role of NO as a regulator of insulin signaling by RNA interference that ablates endogenous eNOS expression in endothelial MS-1 cells. We have shown that eNOS-dependent NO production is essential for the activation of insulin signaling. Our findings demonstrate that NO mediates enhancement of insulin responsiveness via the inhibition of insulin receptor phosphatases.