Protein Tyrosine Phosphatase 1B Levels Research Articles

Protein tyrosine phosphatase 1B contributes to neuropathic pain by aggravating NF-κB and glial cells activation-mediated neuroinflammation via promoting endoplasmic reticulum stress.

Neuropathic pain is a prevalent and highly debilitating condition that impacts millions of individuals globally. Neuroinflammation is considered a key factor in the development of neuropathic pain. Accumulating evidence suggests that protein tyrosine phosphatase 1B (PTP1B) plays a crucial role in regulating neuroinflammation. Nevertheless, the specific involvement of PTP1B in neuropathic pain remains largely unknown. This study aims to examine the impact of PTP1B on neuropathic pain and unravel the underlying molecular mechanisms implicated. In the current study, we evaluated the paw withdrawal threshold (PWT) of male rats following spared nerve injury (SNI) to assess the presence of neuropathic pain. To elucidate the underlying mechanisms, western blotting, immunofluorescence, and electron microscopy techniques were employed. Our results showed that SNI significantly elevated PTP1B levels, which was accompanied by an increase in the expression of endoplasmic reticulum (ER) stress markers (BIP, p-PERK, p-IRE1α, and ATF6) and phosphorylated NF-κB in the spinal dorsal horn. SNI-induced mechanical allodynia was impaired by the treatment of intrathecal injection of PTP1B siRNA or PTP1B-IN-1, a specific inhibitor of PTP1B. Moreover, the intrathecal administration of PTP1B-IN-1 effectively suppressed the expression of ER stress markers (BIP, p-PERK/p-eIF2α, p-IRE1α, and ATF6), leading to the inhibition of NF-κB, microglia, and astrocytes activation, as well as a decrease in pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β. However, these effects were reversed by intrathecal administration of tunicamycin (Tm, an inducer of ER stress). Additionally, intrathecal administration of Tm in healthy rats resulted in the development of mechanical allodynia and the activation of NF-κB-mediated neuroinflammatory signaling. The upregulation of PTP1B induced by SNI facilitates the activation of NF-κB and glial cells via ER stress in the spinal dorsal horn. This, in turn, leads to an increase in the production of pro-inflammatory cytokines, thereby contributing to the development and maintenance of neuropathic pain. Therefore, targeting PTP1B could be a promising therapeutic strategy for the treatment of neuropathic pain.

CD36 gene deletion reduces muscle insulin sensitivity in mice by up-regulating PTP1B expression

To investigate the effect CD36 deficiency on muscle insulin signaling in mice fed a normal-fat diet and explore the possible mechanism. Wild-type (WT) mice and systemic CD36 knockout (CD36-/-) mice with normal feeding for 14 weeks (n=12) were subjected to insulin tolerance test (ITT) after intraperitoneal injection with insulin (1 U/kg). Real-time PCR was used to detect the mRNA expressions of insulin receptor (IR), insulin receptor substrate 1/2 (IRS1/2) and protein tyrosine phosphatase 1B (PTP1B), and Western blotting was performed to detect the protein expressions of AKT, IR, IRS1/2 and PTP1B in the muscle tissues of the mice. Tyrosine phosphorylation of IR and IRS1 and histone acetylation of PTP1B promoter in muscle tissues were detected using co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP), respectively. CD36-/- mice showed significantly lowered insulin sensitivity with obviously decreased area under the insulin tolerance curve in comparison with the WT mice (P < 0.05). CD36-/- mice also had significantly higher serum insulin concentration and HOMA-IR than WT mice (P < 0.05). Western blotting showed that the p-AKT/AKT ratio in the muscle tissues was significantly decreased in CD36-/- mice as compared with the WT mice (P < 0.01). No significant differences were found in mRNA and protein levels of IR, IRS1 and IRS2 in the muscle tissues between WT and CD36-/- mice (P>0.05). In the muscle tissue of CD36-/- mice, tyrosine phosphorylation levels of IR and IRS1 were significantly decreased (P < 0.05), and the mRNA and protein levels of PTP1B (P < 0.05) and histone acetylation level of PTP1B promoters (P < 0.01) were significantly increased as compared with those in the WT mice. Intraperitoneal injection of claramine, a PTP1B inhibitor, effectively improved the impairment of insulin sensitivity in CD36-/- mice. CD36 is essential for maintaining muscle insulin sensitivity under physiological conditions, and CD36 gene deletion in mice causes impaired insulin sensitivity by up-regulating muscle PTP1B expression, which results in detyrosine phosphorylation of IR and IRS1.

Decreased Glucocorticoid Signaling Potentiates Lipid-Induced Inflammation and Contributes to Insulin Resistance in the Skeletal Muscle of Fructose-Fed Male Rats Exposed to Stress.

The modern lifestyle brings both excessive fructose consumption and daily exposure to stress which could lead to metabolic disturbances and type 2 diabetes. Muscles are important points of glucose and lipid metabolism, with a crucial role in the maintenance of systemic energy homeostasis. We investigated whether 9-week fructose-enriched diet, with and without exposure to 4-week unpredictable stress, disturbs insulin signaling in the skeletal muscle of male rats and evaluated potential contributory roles of muscle lipid metabolism, glucocorticoid signaling and inflammation. The combination of fructose-enriched diet and stress increased peroxisome proliferator-activated receptors-α and -δ and stimulated lipid uptake, lipolysis and β-oxidation in the muscle of fructose-fed stressed rats. Combination of treatment also decreased systemic insulin sensitivity judged by lower R-QUICKI, and lowered muscle protein content and stimulatory phosphorylations of insulin receptor supstrate-1 and Akt, as well as the level of 11β-hydroxysteroid dehydrogenase type 1 and glucocorticoid receptor. At the same time, increased levels of protein tyrosine phosphatase-1B, nuclear factor-κB, tumor necrosis factor-α, were observed in the muscle of fructose-fed stressed rats. Based on these results, we propose that decreased glucocorticoid signaling in the skeletal muscle can make a setting for lipid-induced inflammation and the development of insulin resistance in fructose-fed stressed rats.

\u03b2A1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

βA3/A1-crystallin, a lens protein that is also expressed in astrocytes, is produced as βA3 and βA1-crystallin isoforms by leaky ribosomal scanning. In a previous human proteome high-throughput array, we found that βA3/A1-crystallin interacts with protein tyrosine phosphatase 1B (PTP1B), a key regulator of glucose metabolism. This prompted us to explore possible roles of βA3/A1-crystallin in metabolism of retinal astrocytes. We found that βA1-crystallin acts as an uncompetitive inhibitor of PTP1B, but βA3-crystallin does not. Loss of βA1-crystallin in astrocytes triggers metabolic abnormalities and inflammation. In CRISPR/cas9 gene-edited βA1-knockdown (KD) mice, but not in βA3-knockout (KO) mice, the streptozotocin (STZ)-induced diabetic retinopathy (DR)-like phenotype is exacerbated. Here, we have identified βA1-crystallin as a regulator of PTP1B; loss of this regulation may be a new mechanism by which astrocytes contribute to DR. Interestingly, proliferative diabetic retinopathy (PDR) patients showed reduced βA1-crystallin and higher levels of PTP1B in the vitreous humor.

MiR-34c inhibits proliferation of glioma by targeting PTP1B.

Glioma is one of the most pervasive and invasive primary malignancies in the central nervous system. Due to its abnormal proliferation, glioma remains hard to cure at present. Protein tyrosine phosphatase 1B (PTP1B) has been proved to be involved in the process of proliferation in many malignancies. However, whether PTP1B is involved in the proliferation of glioma and how it acts are still unclear. In this study, the PTP1B expressions in glioma tissues and cells were determined by quantitative real-time PCR and western blot analysis. The effects of PTP1B on the proliferation characteristics of glioma were explored using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation assay, and tumor xenografts in mice. We found that the protein and mRNA levels of PTP1B in glioma tissues were significantly higher than those in paired nontumor tissues. MTT and clone formation assays showed that PTP1B is closely related to human glioma cell proliferation. In addition, TargetScan revealed that miR-34c regulates PTP1B. Mechanistically, we proved that miR-34c negatively regulates PTP1B and then participates in the regulation of glioma cell proliferation in vivo. Collectively, these results suggested that miR-34c inhibits the proliferation of human glioma cells by targeting PTP1B, which will provide a potential target for the treatment of glioma.

Astragaloside IV inhibits protein tyrosine phosphatase 1B and improves insulin resistance in insulin-resistant HepG2 cells and triglyceride accumulation in oleic acid (OA)-treated HepG2 cells

Ethnopharmacological relevanceAstragaloside IV (AST IV) is the active component of Astragalus membranaceus (Fisch.) Bunge, which regulates lipid and carbohydrate metabolism and improves insulin resistance. In this study, we investigated the effects of AST IV on insulin resistant cells and a non-alcoholic fatty liver disease (NAFLD) model induced by high-concentration insulin or oleic acid (OA) in HepG2 cells, as well as the associated regulatory markers. MethodsFirst, the target of AST IV was predicted via pharmacophore model matching and molecular docking. Then, enzyme kinetics experiments were conducted in vitro to determine the effect of AST IV on the target protein. Next, AST IV's toxicity was tested on HepG2 cells in vitro, through an insulin resistance model and an NAFLD model, by high-concentration insulin or OA, respectively. To explore the effects of AST IV on insulin resistance and lipid metabolism, we detected the related indexes of glucose and lipid metabolism through commercially available kits. Relevant proteins were also detected by Western blot to provide future direction for study. ResultsOur preliminary results of pharmacophore model matching and molecular docking suggested that AST IV and protein tyrosine phosphatase 1B (PTP1B) can be well-combined through hydrogen bonding. Further, the enzyme kinetics experiment showed that AST IV was an effective and specific inhibitor to PTP1B. We found that the protein level of PTP1B in HepG2 cells was significantly increased after treating with high-concentration insulin or OA. Additionally, the intervention of AST IV significantly increased glucose consumption in an insulin resistance model and reduced the content of triglyceride (TG), total cholesterol (TC), and free fatty acid (FFA) in the NAFLD model. Moreover, the 2-N-(7-nitrobenze-2-oxa-1, 3 diazol-4-yl) (2-NBDG) uptake rate in the NAFLD model was also greatly improved. These results validated the effects of AST IV on improving insulin resistance and lipid accumulation. Furthermore, Western blot results illustrated that AST IV suppressed PTP1B and increased levels of phosphorylated insulin receptor (p-IR) and phosphorylated insulin receptor substrate-1 (p-IRS-1) in insulin-resistant HepG2 cells, while also decreasing protein levels of PTP1B and sterol element regulatory binding protein-1c (SREBP-1c) in the NAFLD model. ConclusionThis study demonstrated that AST IV inhibited PTP1B and effectively improved insulin resistance in insulin-resistant HepG2 cells and triglyceride accumulation in OA-treated HepG2 cells.

Roux-en-Y gastric bypass surgery suppresses hypothalamic PTP1B protein level and alleviates leptin resistance in obese rats.

The present study aimed to explore the effect of Roux-en-Y gastric bypass (RYGB) surgery on protein tyrosine phosphatase 1B (PTP1B) expression levels and leptin activity in hypothalami of obese rats. Obese rats induced by a high-fat diet (HFD) that underwent RYGB (n=11) or sham operation (SO, n=9), as well as an obese control cohort (Obese, n=10) and an additional normal-diet group (ND, n=10) were used. Food efficiency was measured at 8 weeks post-operation. Plasma leptin levels were evaluated and hypothalamic protein tyrosine phosphatase 1B (PTP1B) levels and leptin signaling activity were examined at the genetic and protein levels. The results indicated that food efficiency was typically lower in RYGB rats compared with that in the Obese and SO rats. In the RYGB group, leptin receptor expression and proopiomelanocortin was significantly higher, while Neuropeptide Y levels were lower than those in the Obese and SO groups. Furthermore, the gene and protein expression levels of PTP1B in the RYGB group were lower, while levels of phosphorylated signal transducer and activator of transcription 3 protein were much higher compared with those in the Obese and SO groups. In conclusion, RYGB surgery significantly suppressed hypothalamic PTP1B protein expression. PTP1B regulation may partially alleviate leptin resistance.

Effect of rutin from tartary buckwheat sprout on serum glucose-lowering in animal model of type 2 diabetes.

This study investigates the anti-diabetic effects of rutin from tartary buckwheat sprout in type 2 diabetes mouse model. The rutin content in tartary buckwheat sprout (TBS) is five times higher than that found in common buckwheat sprout (CBS) as evident from high-performance liquid chromatography analysis. Administration of either rutin or TBS ethanolic extract to diabetes mice decreased the serum glucose level significantly. Rutin down-regulated the expression levels of protein-tyrosine phosphatase 1B; it is negative regulator of insulin pathway, both transcriptionally and translationally in myocyte C2C12 in a dose-dependent manner. In conclusion, rutin can play a critical role in down-regulation of serum glucose level in type 2 diabetes.

Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats

Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague–Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases.

Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway.

To evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process. ARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for α-smooth muscle actin (α-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay. The mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. α-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, α-SMA expression and cell migration. PTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

Sepsis-induced brain mitochondrial dysfunction is associated with altered mitochondrial Src and PTP1B levels

Sepsis-induced brain dysfunction (SIBD) is often the first manifestation of sepsis, and its pathogenesis is associated with mitochondrial dysfunction. In this study, we investigated the roles of the tyrosine kinase Src and protein tyrosine phosphatase 1B (PTP1B) in brain mitochondrial dysfunction using a rat model of lipopolysaccharide (LPS)-induced sepsis. We found that there was a gradual and significant increase of PTP1B levels in the rat brain after sepsis induction. In contrast, brain Src levels were reduced in parallel with the PTP1B increase. Sepsis led to significantly reduced tyrosine phosphorylation of mitochondrial oxidative phosphorylation (OXPHOS) complexes I, II and III. Pretreatment of mitochondrial proteins with active PTP1B significantly inhibited complexes I and III activities in vitro, whereas Src enhanced complexes I, II, and III activities. PTP1B and Src were each co-immunoprecipitated with OXPHOS complexes I and III, suggesting direct interactions between both proteins and complexes I and III. Src also directly interacted with complex II. Furthermore, pretreatment of mitochondrial proteins with active PTP1B resulted in overproduction of reactive oxygen species and decreased mitochondrial membrane potential. Pretreatment with active Src produced the opposite effect. These results suggest that brain mitochondrial dysfunction following LPS-induced sepsis in rats is partly attributed to PTP1B and Src mediated decrease in mitochondrial protein tyrosine phosphorylation.

Nck1 depletion induces activation of the PI3K/Akt pathway by attenuating PTP1B protein expression.

BackgroundActivation of the PI3K/Akt pathway mediates crucial cellular functions regulated by receptor tyrosine kinases, such as cell growth, proliferation, survival and metabolism. Previously, we reported that the whole-body knockout of the Src homology domain-containing adaptor protein Nck1 improves overall glucose homeostasis and insulin-induced activation of the PI3K/Akt pathway in liver of obese mice. The aim of the current study is to elucidate the mechanism by which Nck1 depletion regulates hepatic insulin signaling.ResultsHere, we demonstrate that Nck1 regulates the activation of the PI3K/Akt pathway in a protein tyrosine phosphatase 1B (PTP1B)-dependent mechanism. Indeed, depletion of Nck1 by siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation. In accordance, primary hepatocytes isolated from Nck1−/− mice also display enhanced Akt activation in response to insulin. Activation of the PI3K/Akt pathway in Nck1-depleted HepG2 cells relies on higher levels of tyrosine-phosphorylated proteins and correlates with decreased PTP1B levels. Interestingly, Nck1 and PTP1B in cells are found in a common molecular complex and their interaction is dependent on the SH3 domains of Nck1. Finally, Nck1 depletion in HepG2 cells neither affects PTP1B gene transcription nor PTP1B protein stability, suggesting that Nck1 modulates PTP1B expression at the translational level.ConclusionOur study provides strong evidence supporting that the adaptor protein Nck1 interacts with PTP1B and also regulates PTP1B expression. In this manner, Nck1 plays a role in regulating the PI3K/Akt pathway.

Essential Role of Protein-tyrosine Phosphatase 1B in the Modulation of Insulin Signaling by Acetaminophen in Hepatocytes

Many drugs are associated with the development of glucose intolerance or deterioration in glycemic control in patients with pre-existing diabetes. We have evaluated the cross-talk between signaling pathways activated by acetaminophen (APAP) and insulin signaling in hepatocytes with or without expression of the protein-tyrosine phosphatase 1B (PTP1B) and in wild-type and PTP1B-deficient mice chronically treated with APAP. Human primary hepatocytes, Huh7 hepatoma cells with silenced PTP1B, mouse hepatocytes from wild-type and PTP1B-deficient mice, and a mouse model of chronic APAP treatment were used to examine the mechanisms involving PTP1B in the effects of APAP on glucose homeostasis and hepatic insulin signaling. In APAP-treated human hepatocytes at concentrations that did not induce death, phosphorylation of JNK and PTP1B expression and enzymatic activity were increased. APAP pretreatment inhibited activation of the early steps of insulin signaling and decreased Akt phosphorylation. The effects of APAP in insulin signaling were prevented by suramin, a PTP1B inhibitor, or rosiglitazone that decreased PTP1B levels. Likewise, PTP1B deficiency in human or mouse hepatocytes protected against APAP-mediated impairment in insulin signaling. These signaling pathways were modulated in mice with chronic APAP treatment, resulting in protection against APAP-mediated hepatic insulin resistance and alterations in islet alpha/beta cell ratio in PTP1B(-/-) mice. Our results demonstrate negative cross-talk between signaling pathways triggered by APAP and insulin signaling in hepatocytes, which is in part mediated by PTP1B. Moreover, our in vivo data suggest that chronic use of APAP may be associated with insulin resistance in the liver.

Effect of Hypericum perforatum L. extract on insulin resistance and lipid metabolic disorder in high-fat-diet induced obese mice.

Natural product Hypericum perforatum L. has been used in folk medicine to improve mental performance. However, the effect of H. perforatum L. on metabolism is still unknown. In order to test whether H. perforatum L. extract (EHP) has an effect on metabolic syndrome, we treated diet induced obese (DIO) C57BL/6J mice with the extract. The chemical characters of EHP were investigated with thin-layer chromatography, ultraviolet, high-performance liquid chromatography (HPLC), and HPLC-mass spectrometry fingerprint analysis. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and the glucose infusion rate (GIR) in hyperinsulinemic–euglycemic clamp test were performed to evaluate the glucose metabolism and insulin sensitivity. Skeletal muscle was examined for lipid metabolism. The results suggest that EHP can significantly improve the glucose and lipid metabolism in DIO mice. In vitro, EHP inhibited the catalytic activity of recombinant human protein tyrosine phosphatase 1B (PTP1B) and reduced the protein and mRNA levels of PTP1B in the skeletal muscle. Moreover, expressions of genes related to fatty acid uptake and oxidation were changed by EHP in the skeletal muscle. These results suggest that EHP may improve insulin resistance and lipid metabolism in DIO mice. © 2014 The Authors. Phytotherapy Research published by John Wiley & Sons Ltd.

Beneficial effects of Ginkgo biloba extract on insulin signaling cascade, dyslipidemia, and body adiposity of diet-induced obese rats.

Ginkgo biloba extract (GbE) has been indicated as an efficient medicine for the treatment of diabetes mellitus type 2. It remains unclear if its effects are due to an improvement of the insulin signaling cascade, especially in obese subjects. The aim of the present study was to evaluate the effect of GbE on insulin tolerance, food intake, body adiposity, lipid profile, fasting insulin, and muscle levels of insulin receptor substrate 1 (IRS-1), protein tyrosine phosphatase 1B (PTP-1B), and protein kinase B (Akt), as well as Akt phosphorylation, in diet-induced obese rats. Rats were fed with a high-fat diet (HFD) or a normal fat diet (NFD) for 8 weeks. After that, the HFD group was divided into two groups: rats gavaged with a saline vehicle (HFD+V), and rats gavaged with 500 mg/kg of GbE diluted in the saline vehicle (HFD+Gb). NFD rats were gavaged with the saline vehicle only. At the end of the treatment, the rats were anesthetized, insulin was injected into the portal vein, and after 90s, the gastrocnemius muscle was removed. The quantification of IRS-1, Akt, and Akt phosphorylation was performed using Western blotting. Serum levels of fasting insulin and glucose, triacylglycerols and total cholesterol, and LDL and HDL fractions were measured. An insulin tolerance test was also performed. Ingestion of a hyperlipidic diet promoted loss of insulin sensitivity and also resulted in a significant increase in body adiposity, plasma triacylglycerol, and glucose levels. In addition, GbE treatment significantly reduced food intake and body adiposity while it protected against hyperglycemia and dyslipidemia in diet-induced obesity rats. It also enhanced insulin sensitivity in comparison to HFD+V rats, while it restored insulin-induced Akt phosphorylation, increased IRS-1, and reduced PTP-1B levels in gastrocnemius muscle. The present findings suggest that G. biloba might be efficient in preventing and treating obesity-induced insulin signaling impairment.

Complement-mediated glomerular injury is reduced by inhibition of protein-tyrosine phosphatase 1B.

The unfolded protein response and endoplasmic reticulum-associated degradation (ERAD) contribute to injury in renal glomerular diseases, including those mediated by complement C5b-9. In the present study, we address the role of protein-tyrosine phosphatase 1B (PTP1B) in complement-mediated glomerular injury and ERAD. In glomerular epithelial cells (GECs)/podocytes and PTP1B-deficient mouse embryonic fibroblasts exposed to complement, inhibition/deletion of PTP1B reduced ERAD, as monitored by the ERAD reporter CD3δ. Overexpression of PTP1B produced an effect similar to PTP1B deficiency on ERAD in complement-treated GECs. Complement-mediated cytotoxicity was reduced after PTP1B overexpression and tended to be reduced after PTP1B inhibition. PTP1B enhanced the induction of certain ERAD components via the inositol-requiring-1α branch of the unfolded protein response. PTP1B knockout mice with anti-glomerular basement membrane glomerulonephritis had decreased proteinuria and showed less podocyte loss and endoplasmic reticulum dysfunction compared with wild-type littermates. These results imply that endogenous levels of PTP1B are tightly regulated and that both overexpression and inhibition can affect ERAD. The cytoprotective effects of PTP1B deletion in cultured cells and in anti-glomerular basement membrane nephritis suggest that PTP1B may potentially be a therapeutic target in complement-mediated diseases.

Mitochondrial Complex I Deficiency Enhances Skeletal Myogenesis but Impairs Insulin Signaling through SIRT1 Inactivation

To address whether mitochondrial biogenesis is essential for skeletal myogenesis, C2C12 myogenesis was investigated after knockdown of NADH dehydrogenase (ubiquintone) flavoprotein 1 (NDUFV1), which is an oxidative phosphorylation complex I subunit that is the first subunit to accept electrons from NADH. The NDUFVI knockdown enhanced C2C12 myogenesis by decreasing the NAD(+)/NADH ratio and subsequently inactivating SIRT1 and SIRT1 activators (pyruvate, SRT1720, and resveratrol) abolished the NDUFV1 knockdown-induced myogenesis enhancement. However, the insulin-elicited activation of insulin receptor β (IRβ) and insulin receptor substrate-1 (IRS-1) was reduced with elevated levels of protein-tyrosine phosphatase 1B after NDUFV1 knockdown in C2C12 myotubes. The NDUFV1 knockdown-induced blockage of insulin signaling was released by protein-tyrosine phosphatase 1B knockdown in C2C12 myotubes, and we found that NDUFV1 or SIRT1 knockdown did not affect mitochondria biogenesis during C2C12 myogenesis. Based on these data, we can conclude that complex I dysfunction-induced SIRT1 inactivation leads to myogenesis enhancement but blocks insulin signaling without affecting mitochondria biogenesis.

Expression change of SH2B1, SOCS3, PTP1B and NPY in mice hypothalamus and its relation with obesity

To investigate the expression pattern of adapter protein with a Src-homology 2 domain (SH2B1), the suppressor of cytokine signaling-3 (SOCS3), protein-tyrosine phosphatase 1B (PTP1B) and neturopetide Y (NPY) in obese and normal mice hypothalamus and its relation with serum leptin and insulin levels. The obesity animal model was prepared with healthy C57/bl6 mice. Lee's index and Homeostasis model assessment-insulin resistance (HOMA-IR) were calculated. The mRNA levels of SH2B1, SOCS3, PTP1B and NPY were measured by fluorescent quantitation RT-PCR. The SH2B1 and NPY protein expressions were detected by Western blot. Compared with the normal mice of the same age, SH2B1 mRNA expression in the obese mice hypothalamus decreased. SOCS3 and PTP1B mRNA expression increased. Western blot showed that SH2B1 protein expression decreased, while NPY protein expression increased in the obese mice. Linear correlation analysis showed that the serum leptin and fasting insulin levels were negatively correlated with SH2B1mRNA expression and positively correlated with SOCS3 and PTP1B mRNA expression. SH2B1, SOCS3, PTP1B and NPY are key factors for obesity development.

Nck1 depletion induces activation of the PI3K/Akt pathway by attenuating PTP1B protein expression

Activation of the PI3K/Akt pathway mediates crucial cellular functions regulated by receptor tyrosine kinases, such as cell growth, proliferation, survival and metabolism. Previously, we reported that the whole-body knockout of the Src homology domain-containing adaptor protein Nck1 improves overall glucose homeostasis and insulin-induced activation of the PI3K/Akt pathway in liver of obese mice. The aim of the current study is to elucidate the mechanism by which Nck1 depletion regulates hepatic insulin signaling. Here, we demonstrate that Nck1 regulates the activation of the PI3K/Akt pathway in a protein tyrosine phosphatase 1B (PTP1B)-dependent mechanism. Indeed, depletion of Nck1 by siRNA in HepG2 cells enhances PI3K-dependent basal and growth factor-induced Akt activation. In accordance, primary hepatocytes isolated from Nck1 -/- mice also display enhanced Akt activation in response to insulin. Activation of the PI3K/Akt pathway in Nck1-depleted HepG2 cells relies on higher levels of tyrosine-phosphorylated proteins and correlates with decreased PTP1B levels. Interestingly, Nck1 and PTP1B in cells are found in a common molecular complex and their interaction is dependent on the SH3 domains of Nck1. Finally, Nck1 depletion in HepG2 cells neither affects PTP1B gene transcription nor PTP1B protein stability, suggesting that Nck1 modulates PTP1B expression at the translational level. Our study provides strong evidence supporting that the adaptor protein Nck1 interacts with PTP1B and also regulates PTP1B expression. In this manner, Nck1 plays a role in regulating the PI3K/Akt pathway.

Protein Tyrosine Phosphatase 1B Deficiency Ameliorates Murine Experimental Colitis via the Expansion of Myeloid-Derived Suppressor Cells

Protein tyrosine phosphatase 1B (PTP1B) is a key molecule in modulating low-degree inflammatory conditions such as diabetes. The role of PTP1B in other chronic inflammations, however, remains unknown. Here, we report that PTP1B deficiency ameliorates Dextran Sulfate Sodium (DSS)-induced murine experimental colitis via expanding CD11b+Gr-1+ myeloid-derived suppressor cells (MDSCs). Employing DSS-induced murine experimental colitis as inflammatory animal model, we found that, compared with wild-type littermates, PTP1B-null mice demonstrated greater resistance to DSS-induced colitis, as reflected by slower weight-loss, greater survival rates and decreased PMN and macrophage infiltration into the colon. The evidence collectively also demonstrated that the resistance of PTP1B-null mice to DSS-induced colitis is based on the expansion of MDSCs. First, PTP1B-null mice exhibited a greater frequency of MDSCs in the bone marrow (BM), peripheral blood and spleen when compared with wild-type littermates. Second, PTP1B levels in BM leukocytes were significantly decreased after cells were induced into MDSCs by IL-6 and GM-CSF, and the MDSC induction occurred more rapidly in PTP1B-null mice than in wild-type littermates, suggesting PTP1B as a negative regulator of MDSCs. Third, the adoptive transfer of MDSCs into mice with DSS-colitis significantly attenuated colitis, which accompanies with a decreased serum IL-17 level. Finally, PTP1B deficiency increased the frequency of MDSCs from BM cells likely through enhancing the activities of signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2 (JAK2). In conclusion, our study provides the first evidences that PTP1B deficiency ameliorates murine experimental colitis via expanding MDSCs.