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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.

Pancreatic β-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify β-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 β-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 β-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 β-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced β-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in β-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify β-cell dysfunction and loss.

Salmonella serotype (ser.) Enteritidis infection in broilers is a main foodborne illness that substantially threatens food security. This study aimed to examine the effects of a novel polysaccharide isolated from alfalfa (APS) on the intestinal microbiome and systemic health of S. ser. Enteritidis-infected broilers. The results indicated that broilers receiving the APS-supplemented diet had the improved (P

Background & aimsAlcohol-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.MethodsTo 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.ResultsWe 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.ConclusionsThese 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.

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.

In the normal physiological state, intestinal epithelial cells act as a defensive frontline of host mucosal immunity to tolerate constant exposure to external stimuli. In this study, we investigated the potential anti-inflammatory and gut permeability protective effects of Laminaria japonica (LJ) water extract (LJE) and three types of fermented Laminaria japonica water extracts (LJE-F1, LJE-F2, and LJE-F3) in lipopolysaccharide (LPS)-stimulated Caco-2, human intestinal epithelial cells. All four extracts significantly decreased the production of nitric oxide and interleukin-6 induced by LPS stimulus. In addition, LJE and the three types of LJE-Fs also inhibited LPS-induced loss of monolayer permeability, as assessed by changes in transepithelial electrical resistance. All four LJ extracts significantly prevented the inhibition of the protein levels of occludin, whereas LJE, LJE-F1, and LJE-F3 significantly attenuated the reduction in phosphorylation of adenosine monophosphate-activated protein kinase compared with the LPS-treated group in Caco-2 cells. In conclusion, LJE and its fermented water extracts appear to have potential gut health-promoting effects by reducing inflammation and partially regulating the tight junction-related proteins in human intestinal epithelial cells. Thus, additional studies are warranted to evaluate Laminaria japonica as a therapeutic agent for inflammatory bowel diseases.

Increased permeability of the intestinal barrier is proposed as an underlying factor for obesity-associated pathologies. Consumption of high fat diets (HFD) is associated with increased intestinal permeabilization and increased paracellular transport of endotoxins which can promote steatosis and insulin resistance. This study investigated whether dietary (-)-epicatechin (EC) supplementation can protect the intestinal barrier against HFD-induced permeabilization and endotoxemia, and mitigate liver damage and insulin resistance. Mechanisms leading to loss of integrity and function of the tight junction (TJ) were characterized. Consumption of a HFD for 15 weeks caused obesity, steatosis, and insulin resistance in male C57BL/6J mice. This was associated with increased intestinal permeability, decreased expression of ileal TJ proteins, and endotoxemia. Supplementation with EC (2-20mg/kg body weight) mitigated all these adverse effects. EC acted modulating cell signals and the gut hormone GLP-2, which are central to the regulation of intestinal permeability. Thus, EC prevented HFD-induced ileum NOX1/NOX4 upregulation, protein oxidation, and the activation of the redox-sensitive NF-κB and ERK1/2 pathways. Supporting NADPH oxidase as a target of EC actions, in Caco-2 cells EC and apocynin inhibited tumor necrosis alpha (TNFα)-induced NOX1/NOX4 overexpression, protein oxidation and monolayer permeabilization. Together, our findings demonstrate protective effects of EC against HFD-induced increased intestinal permeability and endotoxemia. This can in part underlie EC capacity to prevent steatosis and insulin resistance occurring as a consequence of HFD consumption.

BackgroundDiabetic 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 methodsMice 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.ResultssEH 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.ConclusionsThese findings establish sEH in podocytes as a significant contributor to renal function under hyperglycemia.General significanceThese data suggest that sEH is a potential therapeutic target for podocytopathies.

ObjectiveDiabetic 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.MethodsTo 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.ResultsPTP1B 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.ConclusionsThese findings demonstrate that podocyte PTP1B deficiency attenuates hyperglycemia-induced renal damage and suggest that PTP1B may present a therapeutic target in renal injury.

The arachidonic acid cascade is arguably the most widely known biologic regulatory pathway. Decades after the seminal discoveries involving its cyclooxygenase and lipoxygenase branches, studies of this cascade remain an active area of research. The third and less widely known branch, the cytochrome P450 pathway leads to highly active oxygenated lipid mediators, epoxy fatty acids (EpFAs) and hydroxyeicosatetraenoic acids (HETEs), which are of similar potency to prostanoids and leukotrienes. Unlike the COX and LOX branches, no pharmaceuticals currently are marketed targeting the P450 branch. However, data support therapeutic benefits from modulating these regulatory lipid mediators. This is being approached by stabilizing or mimicking the EpFAs or even by altering the diet. These approaches lead to predominantly beneficial effects on a wide range of apparently unrelated states resulting in an enigma of how this small group of natural chemical mediators can have such diverse effects. EpFAs are degraded by soluble epoxide hydrolase (sEH) and stabilized by inhibiting this enzyme. In this review, we focus on interconnected aspects of reported mechanisms of action of EpFAs and inhibitors of soluble epoxide hydrolase (sEHI). The sEHI and EpFAs are commonly reported to maintain homeostasis under pathological conditions while remaining neutral under normal physiological conditions. Here we provide a conceptual framework for the unique and broad range of biological activities ascribed to epoxy fatty acids. We argue that their mechanism of action pivots on their ability to prevent mitochondrial dysfunction, to reduce subsequent ROS formation and to block resulting cellular signaling cascades, primarily the endoplasmic reticulum stress. By stabilizing the mitochondrial - ROS - ER stress axis, the range of activity of EpFAs and sEHI display an overlap with the disease conditions including diabetes, fibrosis, chronic pain, cardiovascular and neurodegenerative diseases, for which the above outlined mechanisms play key roles.

Calorie restriction, without malnutrition, has been shown to increase lifespan and is associated with a shift away from glycolysis toward beta-oxidation. The objective of this study was to mimic this metabolic shift using low-carbohydrate diets and to determine the influence of these diets on longevity and healthspan in mice. C57BL/6 mice were assigned to a ketogenic, low-carbohydrate, or control diet at 12 months of age and were either allowed to live their natural lifespan or tested for physiological function after 1 or 14 months of dietary intervention. The ketogenic diet (KD) significantly increased median lifespan and survival compared to controls. In aged mice, only those consuming a KD displayed preservation of physiological function. The KD increased protein acetylation levels and regulated mTORC1 signaling in a tissue-dependent manner. This study demonstrates that a KD extends longevity and healthspan in mice.

Atherosclerosis impacts arteries where disturbed blood flow renders the endothelium susceptible to inflammation. Cytokine activation of endothelial cells (EC) upregulates VCAM-1 receptors that target monocyte recruitment to atherosusceptible regions. Endoplasmic reticulum (ER) stress elicits EC dysregulation in metabolic syndrome. We hypothesized that ER plays a central role in mechanosensing of atherosusceptible shear stress (SS) by signaling enhanced inflammation. Aortic EC were stimulated with low-dose TNFα (0.3 ng/ml) in a microfluidic channel that produced a linear SS gradient over a 20mm field ranging from 0-16 dynes/cm2. High-resolution imaging of immunofluorescence along the monolayer provided a continuous spatial metric of EC orientation, markers of ER stress, VCAM-1 and ICAM-1 expression, and monocyte recruitment. VCAM-1 peaked at 2 dynes/cm2 and decreased to below static TNFα-stimulated levels at atheroprotective-SS of 12 dynes/cm2, whereas ICAM-1 rose to a maximum in parallel with SS. ER expansion and activation of the unfolded protein response also peaked at 2 dynes/cm2, where IRF-1-regulated VCAM-1 expression and monocyte recruitment also rose to a maximum. Silencing of PECAM-1 or key ER stress genes abrogated SS regulation of VCAM-1 transcription and monocyte recruitment. We report a novel role for ER stress in mechanoregulation at arterial regions of atherosusceptible-SS inflamed by low-dose TNFα.

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.EnzymesSoluble epoxide hydrolase: EC 3.3.2.10.

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid derived from the cytochrome P450 enzymes, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties, and inhibition of sEH might provide protective effects against inflammatory bone loss. Thus, in the present study, we tested the selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in a mouse model of periodontitis induced by infection with Aggregatibacter actinomycetemcomitans Oral treatment of wild-type mice with TPPU and sEH knockout (KO) animals showed reduced bone loss induced by A. actinomycetemcomitans This was associated with decreased expression of key osteoclastogenic molecules, receptor activator of nuclear factor-κB/RANK ligand/osteoprotegerin, and the chemokine monocyte chemotactic protein 1 in the gingival tissue without affecting bacterial counts. In addition, downstream kinases p38 and c-Jun N-terminal kinase known to be activated in response to inflammatory signals were abrogated after TPPU treatment or in sEH KO mice. Moreover, endoplasmic reticulum stress was elevated in periodontal disease but was abrogated after TPPU treatment and in sEH knockout mice. Together, these results demonstrated that sEH pharmacological inhibition may be of therapeutic value in periodontitis.

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-1β, 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.

ObjectiveVertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible remain incompletely defined. VSG increases circulating bile acid concentrations and bile acid signalling through TGR5 improves glucose homeostasis. Therefore, we investigated the role of TGR5 signalling in mediating the glucoregulatory benefits of VSG.DesignVSG or sham surgery was performed in high-fat-fed male Tgr5+/+ (wild type) and Tgr5-/- (knockout) littermates. Sham-operated mice were fed ad libitum or food restricted to match their body weight to VSG-operated mice. Body weight, food intake, energy expenditure, insulin signalling and circulating bile acid profiles were measured and oral glucose tolerance testing, islet immunohistochemistry and gut microbial profiling were performed.ResultsVSG decreased food intake and body weight, increased energy expenditure and circulating bile acid concentrations, improved fasting glycaemia, glucose tolerance and glucose-stimulated insulin secretion, enhanced nutrient-stimulated glucagon-like peptide 1 secretion and produced favourable shifts in gut microbial populations in both genotypes. However, the body weight-independent improvements in fasting glycaemia, glucose tolerance, hepatic insulin signalling, hepatic inflammation and islet morphology after VSG were attenuated in Tgr5-/- relative to Tgr5+/+ mice. Furthermore, VSG produced metabolically favourable alterations in circulating bile acid profiles that were blunted in Tgr5-/- relative to Tgr5+/+ mice. TGR5-dependent regulation of hepatic Cyp8b1 expression may have contributed to TGR5-mediated shifts in the circulating bile acid pool after VSG.ConclusionsThese results suggest that TGR5 contributes to the glucoregulatory benefits of VSG surgery by promoting metabolically favourable shifts in the circulating bile acid pool.

Our objective is to identify genes that influence the development of any phenotypes of type 2 diabetes (T2D) or kidney disease in obese animals. We use the reproductively isolated UC Davis fatty Zucker strain rat model in which the defective chromosome 4 leptin receptor (LeprfaSte/faSte) results in fatty obesity. We previously produced a congenic strain with the distal half of chromosome 1 from the Brown Norway strain (BN) on a Zucker (ZUC) background (BN.ZUC-D1Rat183-D1Rat90). Previously published studies in males showed that the BN congenic donor region protects from some phenotypes of renal dysfunction and T2D. We now expand our studies to include females and expand phenotyping to gene expression. We performed diabetes and kidney disease phenotyping in chow-fed females of the BN.ZUC-D1Rat183-D1Rat90 congenic strain to determine the specific characteristics of the UC Davis model. Fatty LeprfaSte/faSte animals of both BN and ZUC genotype in the congenic donor region had prediabetic levels of fasting blood glucose and blood glucose 2 hours after a glucose tolerance test. We observed significant congenic strain chromosome 1 genotype effects of the BN donor region in fatty females that resulted in decreased food intake, urine volume, glucose area under the curve during glucose tolerance test, plasma triglyceride levels, and urine glucose excretion per day. In fatty females, there were significant congenic strain BN genotype effects on non-fasted plasma urea nitrogen, triglyceride, and creatinine. Congenic region genotype effects were observed by quantitative PCR of mRNA from the kidney for six genes, all located in the chromosome 1 BN donor region, with potential effects on T2D or kidney function. The results are consistent with the hypothesis that the BN genotype chromosome 1 congenic region influences traits of both type 2 diabetes and kidney function in fatty UC Davis ZUC females and that there are many positional candidate genes.

Obesity and type 2 diabetes (T2D) are major public health concerns. Visceral adipose tissue inflammation is considered a significant contributor to obesity-associated T2D development. We previously showed that the flavan-3-ol (-)-epicatechin (EC) can mitigate insulin resistance in mice fed a high fat diet (HFD). This study investigated the capacity of EC to inhibit visceral adipose tissue inflammation occurring as a consequence of HFD consumption in C57BL/6J mice, and characterized the underlying mechanisms. In association with the development of obesity and insulin resistance, HFD consumption caused inflammation in the visceral adipose tissue as evidenced by activation of the pro-inflammatory transcription factor NF-κB and increased tissue levels of the macrophage marker F4/80, tumor necrosis factor alpha (TNFα), and the chemokine MCP-1. EC supplementation mitigated all these events. In addition, we observed activation of the three branches of the unfolded protein response (UPR), and upregulation of NADPH oxidases NOX4 and NOX2 in visceral fat of mice fed HFD. These can account, at least in part, for the associated oxidative stress and activation of the redox sensitive NF-κB. Notably, EC supplementation mitigated this and the release of pro-inflammatory proteins from metabolically stressed adipocytes. Attenuation of adipocyte endoplasmic reticulum (ER) and oxidative stress by EC could contribute to decreased inflammation and improved visceral adipose tissue insulin sensitivity. Our results support the concept that consumption of EC-rich foods could mitigate obesity-associated insulin resistance through attenuation of adipose tissue inflammation.

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.

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.

Obesity constitutes a major public health concern, being frequently associated with type 2 diabetes (T2D). Evidence from studies in humans and experimental animals suggest that consumption of the flavan-3-ol (-)-epicatechin (EC) and of EC-rich foods may improve insulin sensitivity. To further understand the potential benefits of dietary EC consumption on insulin resistance, this study investigated the capacity of EC supplementation to prevent high fat diet (HFD)-induced insulin resistance in mice. To assess the underlying mechanisms, the effects of HFD and EC consumption on the activation of the insulin cascade and of its negative modulators were evaluated. HFD consumption for 15 w caused obesity and insulin resistance in C57BL/6J mice as evidenced by high fasted and fed plasma glucose and insulin levels, and impaired ITT and GTT tests. This was associated with alterations in the activation of components of the insulin-triggered signaling cascade (insulin receptor, IRS1, ERK1/2, Akt) in adipose and liver tissues. EC supplementation prevented/ameliorated all these parameters. EC acted improving insulin sensitivity in the HFD-fed mice in part through a downregulation of the inhibitory molecules JNK, IKK, PKC and protein tyrosine phosphatase 1B (PTP1B). Thus, the above results suggest that consumption of EC-rich foods could constitute a dietary strategy to mitigate obesity-associated insulin resistance.

ObjectiveObesity 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.MethodsThe metabolic effects of shikonin treatment on mice fed regular chow or challenged with a high-fat diet (HFD) were determined.ResultsShikonin treated mice fed regular chow exhibited improved glucose tolerance compared with controls. In addition, shikonin treated mice fed HFD displayed decreased weight gain and resistance to HFD-induced glucose intolerance. Further, shikonin treatment decreased HFD-induced hepatic dyslipidemia. These findings correlated with enhanced hepatic insulin signaling in shikonin treated mice as evidenced by increased tyrosyl phosphorylation of the insulin receptor and enhanced downstream signaling.ConclusionsThese studies identify shikonin as a potential regulator of systemic glucose tolerance, energy balance, and adiposity in vivo.

Background & aimsReactive oxidative species (ROS) are believed to be involved in the progression of nonalcoholic steatohepatitis (NASH). However, little is known about the sources of ROS in hepatocytes or their role in disease progression. We studied the effects of nicotinamide adenine dinucleotide phosphate reduced oxidase 4 (NOX4) in liver tissues from patients with NASH and mice with steatohepatitis.MethodsLiver biopsy samples were obtained from 5 patients with NASH, as well as 4 patients with simple steatosis and 5 patients without steatosis (controls) from the University of California, Davis Cancer Center Biorepository. Mice with hepatocyte-specific deletion of NOX4 (NOX4(hepKO)) and NOX4(floxp+/+) C57BL/6 mice (controls) were given fast-food diets (supplemented with high-fructose corn syrup) or choline-deficient l-amino acid defined diets to induce steatohepatitis, or control diets, for 20 weeks. A separate group of mice were given the NOX4 inhibitor (GKT137831). Liver tissues were collected and immunoblot analyses were performed determine levels of NOX4, markers of inflammation and fibrosis, double-stranded RNA-activated protein kinase, and phospho-eIF-2α kinase-mediated stress signaling pathways. We performed hyperinsulinemic-euglycemic clamp studies and immunoprecipitation analyses to determine the oxidation and phosphatase activity of PP1C.ResultsLevels of NOX4 were increased in patients with NASH compared with controls. Hepatocyte-specific deletion of NOX4 reduced oxidative stress, lipid peroxidation, and liver fibrosis in mice with diet-induced steatohepatitis. A small molecule inhibitor of NOX4 reduced liver inflammation and fibrosis and increased insulin sensitivity in mice with diet-induced steatohepatitis. In primary hepatocytes, NOX4 reduced the activity of the phosphatase PP1C, prolonging activation of double-stranded RNA-activated protein kinase and phosphorylation of extracellular signal-regulated kinase-mediated stress signaling. Mice with hepatocyte-specific deletion of NOX4 and mice given GKT137831 had increased insulin sensitivity.ConclusionsNOX4 regulates oxidative stress in the liver and its levels are increased in patients with NASH and mice with diet-induced steatohepatitis. Inhibitors of NOX4 reduce liver inflammation and fibrosis and increase insulin sensitivity, and might be developed for treatment of NASH.

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.

Despite intensive effort and resulting gains in understanding the mechanisms underlying neuropathic pain, limited success in therapeutic approaches have been attained. A recently identified, nonchannel, nonneurotransmitter therapeutic target for pain is the enzyme soluble epoxide hydrolase (sEH). The sEH degrades natural analgesic lipid mediators, epoxy fatty acids (EpFAs), therefore its inhibition stabilizes these bioactive mediators. Here we demonstrate the effects of EpFAs on diabetes induced neuropathic pain and define a previously unknown mechanism of pain, regulated by endoplasmic reticulum (ER) stress. The activation of ER stress is first quantified in the peripheral nervous system of type I diabetic rats. We demonstrate that both pain and markers of ER stress are reversed by a chemical chaperone. Next, we identify the EpFAs as upstream modulators of ER stress pathways. Chemical inducers of ER stress invariably lead to pain behavior that is reversed by a chemical chaperone and an inhibitor of sEH. The rapid occurrence of pain behavior with inducers, equally rapid reversal by blockers and natural incidence of ER stress in diabetic peripheral nervous system (PNS) argue for a major role of the ER stress pathways in regulating the excitability of the nociceptive system. Understanding the role of ER stress in generation and maintenance of pain opens routes to exploit this system for therapeutic purposes.

Liver fibrosis is a pathological condition in which chronic inflammation and changes to the extracellular matrix lead to alterations in hepatic tissue architecture and functional degradation of the liver. Inhibitors of the enzyme soluble epoxide hydrolase (sEH) reduce fibrosis in the heart, pancreas and kidney in several disease models. In this study, we assess the effect of sEH inhibition on the development of fibrosis in a carbon tetrachloride (CCl4)-induced mouse model by monitoring changes in the inflammatory response, matrix remolding and endoplasmic reticulum stress. The sEH inhibitor 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) was administered in drinking water. Collagen deposition in the liver was increased five-fold in the CCl4-treated group, and this was returned to control levels by TPPU treatment. Hepatic expression of Col1a2 and 3a1 mRNA was increased over fifteen-fold in the CCl4-treated group relative to the Control group, and this increase was reduced by 50% by TPPU treatment. Endoplasmic reticulum (ER) stress observed in the livers of CCl4-treated animals was attenuated by TPPU treatment. In order to support the hypothesis that TPPU is acting to reduce the hepatic fibrosis and ER stress through its action as a sEH inhibitor we used a second sEH inhibitor, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB), and sEH null mice. Taken together, these data indicate that the sEH may play an important role in the development of hepatic fibrosis induced by CCl4, presumably by reducing endogenous fatty acid epoxide chemical mediators acting to reduce ER stress.

ScopeThis study evaluated the capacity of dietary catechin (C), quercetin (Q), and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNF-α) in 3T3-L1 adipocytes.Methods and resultsIn rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/day of C, Q, and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNF-α-induced elevated protein carbonyls, increased proinflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to (i) decrease the activation of the mitogen-activated kinases (MAPKs) JNK and p38; and (ii) prevent the downregulation of PPAR-γ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose proinflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNF-α, MCP-1, resistin) insulin sensitivity.ConclusionTogether, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS-associated adipose inflammation, oxidative stress, and insulin resistance.