MicroRNA‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression

Recently, it is implicated that aberrant expression of microRNAs (miRs) is associated with insulin resistance. However, the role of miR‐17 family in hepatic insulin resistance and its underlying mechanisms remain unknown. In this study, we provided mechanistic insight into the effects of miR‐20a‐5p, a member of miR‐17 family, on the regulation of AKT/GSK pathway and glycogenesis in hepatocytes. MiR‐20a‐5p was down‐regulated in the liver of db/db mice, and NCTC1469 cells and Hep1‐6 cells treated with high glucose, accompanied by reduced glycogen content and impaired insulin signalling. Notably, inhibition of miR‐20a‐5p significantly reduced glycogen synthesis and AKT/GSK activation, whereas overexpression of miR‐20a‐5p led to elevated glycogenesis and activated AKT/GSK signalling pathway. In addition, miR‐20a‐5p mimic could reverse high glucose‐induced impaired glycogenesis and AKT/GSK activation in NCTC1469 and Hep1‐6 cells. P63 was identified as a target of miR‐20a‐5p by bioinformatics analysis and luciferase reporter assay. Knockdown of p63 in the NCTC1469 cells and the Hep1‐6 cells by transfecting with siRNA targeting p63 could increase glycogen content and reverse miR‐20a‐5p inhibition‐induced reduced glycogenesis and activation of AKT and GSK, suggesting that p63 participated in miR‐20a‐5p‐mediated glycogenesis in hepatocytes. Moreover, our results indicate that p63 might directly bind to p53, thereby regulating PTEN expression and in turn participating in glycogenesis. In conclusion, we found novel evidence suggesting that as a member of miR‐17 family, miR‐20a‐5p contributes to hepatic glycogen synthesis through targeting p63 to regulate p53 and PTEN expression.     

Enhanced peripheral blood miR-324-5p is associated with the risk of metabolic syndrome by suppressing ROCK1

The current study aims to evaluate whether peripheral blood miR-324-5p could be used to differentiate patients with metabolic disorders and healthy controls. Our data showed that miR-324-5p levels were elevated in the peripheral blood of patients with hyperglycemia or hyperlipidemia. In addition, the expression of miR-324-5p was enhanced in the peripheral blood and liver of db/db mice. Further study indicated that overexpression of miR-324-5p reduced the activation of the AKT/GSK pathway and increased lipid accumulation, while the inhibition of miR-324-5p activated the AKT/GSK pathway and decreased lipid accumulation. A dual luciferase assay revealed that Rho-associated coiled-coil containing protein kinase 1 (ROCK1) was a target gene of miR-324-5p. In addition, silencing ROCK1 deteriorated lipid and glucose metabolism. More importantly, knockdown of ROCK1 reversed the miR-324-5p inhibitor-induced improvement of glucose and lipid metabolism. In summary, miR-324-5p plays a regulatory role in glucose and lipid metabolism by targeting ROCK1, which is involved in metabolic disorders. The use of miR-324-5p in diagnosing metabolic syndrome is worth investigating and may benefit patients.     

miR-200s Contribute to Interleukin-6 (IL-6)-induced Insulin Resistance in Hepatocytes

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 μg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.     

Glycogen synthase kinase 3beta is a novel regulator of high glucose- and high insulin-induced extracellular matrix protein synthesis in renal proximal tubular epithelial cells

High glucose (30 mM) and high insulin (1 nM), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininbeta1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3beta (GSK3beta) by high glucose and high insulin induces increase in synthesis of laminin beta1 via activation of eIF2Bepsilon. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3beta at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bepsilon and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3beta or constitutively active kinase led to increased and diminished laminin beta1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin beta1 synthesis and phosphorylation of GSK3beta were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3beta phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3beta. Status of GSK3beta was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3beta and decreased phosphorylation of eIF2Bepsilon, which correlated with renal hypertrophy at 2 weeks, and increased laminin beta1 and fibronectin protein content at 2 months. GSK3beta and eIF2Bepsilon play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes.     

Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.     

Physiological and pathological changes in glucose regulate brain Akt and glycogen synthase kinase-3

Insulin regulates the phosphorylation and activities of Akt and glycogen synthase kinase-3 (GSK3) in peripheral tissues, but in the brain it is less clear how this signaling pathway is regulated in vivo and whether it is affected by diabetes. We found that Akt and GSK3 are sensitive to glucose, because fasting decreased and glucose administration increased by severalfold the phosphorylation of Akt and GSK3 in the cerebral cortex and hippocampus of non-diabetic mice. Brain Akt and GSK3 phosphorylation also increased after streptozotocin administration (3 days), which increased blood glucose and depleted blood insulin, indicating regulation by glucose availability even with deficient insulin. Changes in Akt and GSK3 phosphorylation and activities in epididymal fat were opposite to those of brain after streptozotocin treatment. Streptozotocin-induced hyperglycemia and increased brain Akt and GSK3 phosphorylation were reversed by lowering blood glucose with insulin administration. Long term hyperglycemia also increased brain Akt and GSK3 phosphorylation, both 4 weeks after streptozotocin and in db/db insulin-resistant mice. Thus, the Akt-GSK3 signaling pathway is regulated in mouse brain in vivo in response to physiological and pathological changes in insulin and glucose.     

Acute selective glycogen synthase kinase-3 inhibition enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle

Glycogen synthase kinase-3 (GSK3) has been implicated in the multifactorial etiology of skeletal muscle insulin resistance in animal models and in human type 2 diabetic subjects. However, the potential molecular mechanisms involved are not yet fully understood. Therefore, we determined if selective GSK3 inhibition in vitro leads to an improvement in insulin action on glucose transport activity in isolated skeletal muscle of insulin-resistant, prediabetic obese Zucker rats and if these effects of GSK3 inhibition are associated with enhanced insulin signaling. Type I soleus and type IIb epitrochlearis muscles from female obese Zucker rats were incubated in the absence or presence of a selective, small organic GSK3 inhibitor (1 microM CT118637, Ki < 10 nM for GSK3alpha and GSK3beta). Maximal insulin stimulation (5 mU/ml) of glucose transport activity, glycogen synthase activity, and selected insulin-signaling factors [tyrosine phosphorylation of insulin receptor (IR) and IRS-1, IRS-1 associated with p85 subunit of phosphatidylinositol 3-kinase, and serine phosphorylation of Akt and GSK3] were assessed. GSK3 inhibition enhanced (P <0.05) basal glycogen synthase activity and insulin-stimulated glucose transport in obese epitrochlearis (81 and 24%) and soleus (108 and 20%) muscles. GSK3 inhibition did not modify insulin-stimulated tyrosine phosphorylation of IR beta-subunit in either muscle type. However, in obese soleus, GSK3 inhibition enhanced (all P < 0.05) insulin-stimulated IRS-1 tyrosine phosphorylation (45%), IRS-1-associated p85 (72%), Akt1/2 serine phosphorylation (30%), and GSK3beta serine phosphorylation (39%). Substantially smaller GSK3 inhibitor-mediated enhancements of insulin action on these insulin signaling factors were observed in obese epitrochlearis. These results indicate that selective GSK3 inhibition enhances insulin action in insulin-resistant skeletal muscle of the prediabetic obese Zucker rat, at least in part by relieving the deleterious effects of GSK3 action on post-IR insulin signaling. These effects of GSK3 inhibition on insulin action are greater in type I muscle than in type IIb muscle from these insulin-resistant animals.     

TFE3 transcriptionally activates hepatic IRS-2, participates in insulin signaling and ameliorates diabetes

Using an expression cloning strategy, we have identified TFE3, a basic helix-loop-helix protein, as a transactivator of metabolic genes that are regulated through an E-box in their promoters. Adenovirus-mediated expression of TFE3 in hepatocytes in culture and in vivo strongly activated expression of IRS-2 and Akt and enhanced phosphorylation of insulin-signaling kinases such as Akt, glycogen synthase kinase 3beta and p70S6 kinase. TFE3 also induced hexokinase II (HK2) and insulin-induced gene 1 (INSIG1). These changes led to metabolic consequences, such as activation of glycogen and protein synthesis, but not lipogenesis, in liver. Collectively, plasma glucose levels were markedly reduced both in normal mice and in different mouse models of diabetes, including streptozotocin-treated, db/db and KK mice. Promoter analyses showed that IRS2, HK2 and INSIG1 are direct targets of TFE3. Activation of insulin signals in both insulin depletion and resistance suggests that TFE3 could be a therapeutic target for diabetes.     

Role of the PDK1-PKB-GSK3 pathway in regulating glycogen synthase and glucose uptake in the heart

In order to investigate the importance of the PDK1-PKB-GSK3 signalling network in regulating glycogen synthase (GS) in the heart, we have employed tissue specific conditional knockout mice lacking PDK1 in muscle (mPDK1-/-), as well as knockin mice in which the protein kinase B (PKB) phosphorylation site on glycogen synthase kinase-3alpha (GSK3alpha) (Ser21) and GSK3beta (Ser9) is changed to Ala. We demonstrate that in hearts from mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, insulin failed to stimulate the activity of GS or induce its dephosphorylation at residues that are phosphorylated by GSK3. We also establish that in the heart, both GSK3 isoforms participate in the regulation of GS, with GSK3beta playing a more prominent role. This contrasts with skeletal muscle where GSK3beta is the major regulator of insulin-induced GS activity. Despite the inability of insulin to stimulate glycogen synthesis in hearts from the mPDK1-/- or double GSK3alpha/GSK3beta knockin mice, these animals possessed normal levels of cardiac glycogen, demonstrating that total glycogen levels are regulated independently of insulin's ability to stimulate GS in the heart and that mechanisms such as allosteric activation of GS by glucose-6-phosphate and/or activation of GS by muscle contraction, could operate to maintain normal glycogen levels in these mice. We also demonstrate that in cardiomyocytes derived from the mPDK1-/- hearts, although the levels of glucose transporter type 4 (GLUT4) are increased 2-fold, insulin failed to stimulate glucose uptake, providing genetic evidence that PDK1 plays a crucial role in enabling insulin to promote glucose uptake in cardiac muscle.     

miR-335-5p induces insulin resistance and pancreatic islet β-cell secretion in gestational diabetes mellitus mice through VASH1-mediated TGF-β signaling pathway

Multiple studies have reported different methods in treating gestational diabetes mellitus (GDM); however, the relationship between miR-335-5p and GDM still remains unclear. Here, this study explores the effect of miR-335-5p on insulin resistance and pancreatic islet β-cell secretion via activation of the TGFβ signaling pathway by downregulating VASH1 expression in GDM mice. The GDM mouse model was established and mainly treated with miR-335-5p mimic, miR-335-5p inhibitor, si-VASH1, and miR-335-5p inhibitor + si-VASH1. Oral glucose tolerance test (OGTT) was conducted to detect fasting blood glucose (FBG) fasting insulin (FINS). The OGTT was also used to calculate a homeostasis model assessment of insulin resistance (HOMA-IR). A hyperglycemic clamp was performed to measure the glucose infusion rate (GIR), which estimated β-cell function. Expressions of miR-335-5p, VASH1, TGF-β1, and c-Myc in pancreatic islet β-cells were determined by RT-qPCR, western blot analysis, and insulin release by ELISA. The miR-335-5p mimic and si-VASH1 groups showed elevated blood glucose levels, glucose area under the curve (GAUC), and HOMA-IR, but a reduced GIR and positive expression of VASH1. Overexpression of miR-335-5p and inhibition of VASH1 contributed to activated TGFβ1 pathway, higher c-Myc, and lower VASH1 expressions, in addition to downregulated insulin and insulin release levels. These findings provided evidence that miR-335-5p enhanced insulin resistance and suppressed pancreatic islet β-cell secretion by inhibiting VASH1, eventually activating the TGF-β pathway in GDM mice, which provides more clinical insight on the GDM treatment.     

Borapetoside C from Tinospora crispa improves insulin sensitivity in diabetic mice

Diabetes mellitus (DM) often leads to disability from vascular complications and neurological complications. Tinospora crispa has been widely used in Asia and Africa as a remedy for diabetes and other diseases. In this study, we investigated the hypoglycemic actions of borapetoside C isolated from T. crispa, and the mechanisms underlying its actions. Acute treatment with borapetoside C (5mg/kg, i.p.) attenuated the elevated plasma glucose induced by oral glucose in normal and type 2 DM (T2DM) mice. Compared to the effect of injected insulin (0.5 IU/kg), borapetoside C caused a more prominent increase of glycogen content in skeletal muscle of T2DM mice, but a less increase in type 1 DM (T1DM) mice. Combined treatment of a low dose borapetoside C (0.1mg/kg, i.p.) plus insulin enhanced insulin-induced lowering of the plasma glucose level and insulin-induced increase of muscle glycogen content. Continuous treatment with 5mg/kg borapetoside C (twice daily) for 7 days increased phosphorylation of insulin receptor (IR) and protein kinase B (Akt) as well as the expression of glucose transporter-2 (GLUT2) in T1DM mice. Combined treatment of a low dose borapetoside C (0.1mg/kg, twice daily) plus insulin for 7 days enhanced insulin-induced IR and Akt phosphorylation and GLUT2 expression in the liver of T1DM mice. This study proved that borapetoside C can increase glucose utilization, delayed the development of insulin resistance and enhanced insulin sensitivity. The activation of IR-Akt-GLUT2 expression and the enhancement of insulin sensitivity may contribute to the hypoglycemic action of borapetoside C in diabetic mice.     

Endurance exercise training increases APPL1 expression and improves insulin signaling in the hepatic tissue of diet-induced obese mice, independently of weight loss

Hepatic insulin resistance is the major contributor to fasting hyperglycemia in type 2 diabetes. The protein kinase Akt plays a central role in the suppression of gluconeogenesis involving forkhead box O1 (Foxo1) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), and in the control of glycogen synthesis involving the glycogen synthase kinase beta (GSK3β) in the liver. It has been demonstrated that endosomal adaptor protein APPL1 interacts with Akt and blocks the association of Akt with its endogenous inhibitor, tribbles-related protein 3 (TRB3), improving the action of insulin in the liver. Here, we demonstrated that chronic exercise increased the basal levels and insulin-induced Akt serine phosphorylation in the liver of diet-induced obese mice. Endurance training was able to increase APPL1 expression and the interaction between APPL1 and Akt. Conversely, training reduced both TRB3 expression and TRB3 and Akt association. The positive effects of exercise on insulin action are reinforced by our findings that showed that trained mice presented an increase in Foxo1 phosphorylation and Foxo1/PGC-1α association, which was accompanied by a reduction in gluconeogenic gene expressions (PEPCK and G6Pase). Finally, exercised animals demonstrated increased at basal and insulin-induced GSK3β phosphorylation levels and glycogen content at 24 h after the last session of exercise. Our findings demonstrate that exercise increases insulin action, at least in part, through the enhancement of APPL1 and the reduction of TRB3 expression in the liver of obese mice, independently of weight loss.     

Pseudogene PTENP1 sponges miR-214 to regulate the expression of PTEN to modulate osteoclast differentiation and attenuate osteoporosis

Background aimsOsteoporosis (OP) is a common bone metabolic disease with a high incidence. Our study aimed to explore the pseudogene PTENP1/miR-214/PTEN axis to modulate the osteoclast differentiation in osteoporosis.MethodsPatients with osteoporosis were recruited in our study, and RANKL-induced osteoclast differentiation and ovariectomy-induced osteoporosis mouse model were established in vitro and in vivo, respectively.ResultsPseudogene PTENP1 and PTEN were significantly down-regulated and miR-214 was up-regulated in osteoporosis patients. In addition, overexpression of PTENP1 or silence of miR-214 inhibited the expression levels of osteoclast specific markers and osteoclast differentiation induced by RANKL. Overexpression of PTENP1 or silence of miR-214 also inhibited the levels of phosphorylation of PI3K and AKT, p65 nuclear translocation, IκBα degradation and the expression level of NFATc1. AlsoSilence of PTENP1 or overexpression of miR-214 induced the osteoclast differentiation under normal physiological condition. Pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN.ConclusionsIn an ovariectomy-induced osteoporosis mouse model, obvious pathological changes in bone tissues were found, and bone marrow mononuclear cells in this group were more likely to differentiate into osteoclasts. Therefore, pseudogene PTENP1 sponged miR-214 to regulate the expression of PTEN to inhibit osteoclast differentiation and attenuate osteoporosis by suppressing the PI3K/AKT/NF-κB signaling pathway.     

Effects and mechanism of miR-23b on glucose-mediated epithelial-to-mesenchymal transition in diabetic nephropathy

MicroRNAs (miRNAs) play important roles in epithelial-to-mesenchymal transition (EMT). Moreover, hyperglycaemia induces damage to renal tubular epithelial cells, which may lead to EMT in diabetic nephropathy. However, the effects of miRNAs on EMT in diabetic nephropathy are poorly understood. In the present study, we found that the level of microRNA-23b (miR-23b) was significantly decreased in high glucose (HG)-induced human kidney proximal tubular epithelial cells (HK2) and in kidney tissues of db/db mice. Overexpression of miR-23b attenuated HG-induced EMT, whereas knockdown of miR-23b induced normal glucose (NG)-mediated EMT in HK2 cells. Mechanistically, miR-23b suppressed EMT in diabetic nephropathy by targeting high mobility group A2 (HMGA2), thereby repressing PI3K-AKT signalling pathway activation. Additionally, HMGA2 knockdown or inhibition of the PI3K-AKT signalling pathway with LY294002 mimicked the effects of miR-23b overexpression on HG-mediated EMT, whereas HMGA2 overexpression or activation of the PI3K-AKT signalling pathway with BpV prevented the effects of miR-23b on HG-mediated EMT. We also confirmed that overexpression of miR-23b alleviated EMT, decreased the expression levels of EMT-related genes, ameliorated renal morphology, glycogen accumulation, fibrotic responses and improved renal functions in db/db mice. Taken together, we showed for the first time that miR-23b acts as a suppressor of EMT in diabetic nephropathy through repressing PI3K-AKT signalling pathway activation by targeting HMGA2, which maybe a potential therapeutic target for diabetes-induced renal dysfunction.     

Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells

We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.     

Ebselen enhances insulin sensitivity and decreases oxidative stress by inhibiting SHIP2 and protects from inflammation in diabetic mice

Ebselen, a multifunctional organoselenium compound, has been recognized as a potential treatment for diabetes-related disorders. However, the underlying mechanisms whereby ebselen regulates metabolic pathways remain elusive. We discovered that ebselen inhibits lipid phosphatase SHIP2 (Src homology 2 domain-containing inositol-5-phosphatase 2), an emerging drug target to ameliorate insulin resistance in diabetes. We found that ebselen directly binds to and inhibits the catalytic activity of the recombinant SHIP2 phosphatase domain and SHIP2 in cultured cells, the skeletal muscle and liver of the diabetic db/db mice, and the liver of the SHIP2 overexpressing (SHIP2-Tg) mice. Ebselen increased insulin-induced Akt phosphorylation in cultured myotubes, enhanced insulin sensitivity and protected liver tissue from lipid peroxidation and inflammation in the db/db mice, and improved glucose tolerance more efficiently than metformin in the SHIP2-Tg mice. SHIP2 overexpression abrogated the ability of ebselen to induce glucose uptake and reduce ROS production in myotubes and blunted the effect of ebselen to inhibit SHIP2 in the skeletal muscle of the SHIP2-Tg mice. Our data reveal ebselen as a potent SHIP2 inhibitor and demonstrate that the ability of ebselen to ameliorate insulin resistance and act as an antioxidant is at least in part mediated by the reduction of SHIP2 activity.