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The c-Jun N-terminal kinases (JNKs) have been implicated in the development of insulin resistance, diabetes, and obesity. Genetic disruption of JNK1, but not JNK2, improves insulin sensitivity in diet-induced obese (DIO) mice. We applied RNA interference to investigate the specific role of hepatic JNK1 in contributing to insulin resistance in DIO mice. Adenovirus-mediated delivery of JNK1 short-hairpin RNA (Ad-shJNK1) resulted in almost complete knockdown of hepatic JNK1 protein without affecting JNK1 protein in other tissues. Liver-specific knockdown of JNK1 resulted in significant reductions in circulating insulin and glucose levels, by 57 and 16%, respectively. At the molecular level, JNK1 knockdown mice had sustained and significant increase of hepatic Akt phosphorylation. Furthermore, knockdown of JNK1 enhanced insulin signaling in vitro. Unexpectedly, plasma triglyceride levels were robustly elevated upon hepatic JNK1 knockdown. Concomitantly, expression of proliferator-activated receptor gamma coactivator 1 beta, glucokinase, and microsomal triacylglycerol transfer protein was increased. Further gene expression analysis demonstrated that knockdown of JNK1 up-regulates the hepatic expression of clusters of genes in glycolysis and several genes in triglyceride synthesis pathways. Our results demonstrate that liver-specific knockdown of JNK1 lowers circulating glucose and insulin levels but increases triglyceride levels in DIO mice.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. The purpose of this study was to evaluate the differences in insulin sensitivity between neonate and adult hepatocytes lacking PTP1B. Immortalized neonatal hepatocytes and primary neonatal and adult hepatocytes have been generated from PTP1B(-/-) and wild-type mice. PTP1B deficiency in immortalized neonatal hepatocytes prolonged insulin-induced tyrosine phosphorylation of the insulin receptor (IR) and IR substrates (IRS) -1, -2 compared with wild-type control cells. Endogenous IR and IRS-2 were down-regulated, whereas IRS-1 was up-regulated in PTP1B(-/-) neonatal hepatocytes and livers of PTP1B(-/-) neonates. Insulin-induced activation of phosphatidylinositol 3-kinase/Akt pathway was prolonged in PTP1B(-/-) immortalized neonatal hepatocytes. However, insulin sensitivity was comparable to wild-type hepatocytes. Rescue of PTP1B in deficient cells suppressed the prolonged insulin signaling, whereas RNA interference in wild-type cells promoted prolonged signaling. In primary neonatal PTP1B(-/-) hepatocytes, insulin prolonged the inhibition of gluconeogenic mRNAs, but the sensitivity to this inhibition was similar to wild-type cells. By contrast, in adult PTP1B-deficient livers, p85alpha was down-regulated compared with the wild type. Moreover, primary hepatocytes from adult PTP1B(-/-) mice displayed enhanced Akt phosphorylation and a more pronounced inhibition of gluconeogenic mRNAs than wild-type cells. Hepatic insulin sensitivity due to PTP1B deficiency is acquired through postnatal development. Thus, changes in IR and IRS-2 expression and in the balance between regulatory and catalytic subunits of phosphatidylinositol 3-kinase are necessary to achieve insulin sensitization in adult PTP1B(-/-) hepatocytes.

Protein-tyrosine phosphatase (PTP)1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. In this study, we have assessed the role of PTP1B in the insulin sensitivity of skeletal muscle under physiological and insulin-resistant conditions. Immortalized myocytes have been generated from PTP1B-deficient and wild-type neonatal mice. PTP1B(-/-) myocytes showed enhanced insulin-dependent activation of insulin receptor autophosphorylation and downstream signaling (tyrosine phosphorylation of insulin receptor substrate [IRS]-1 and IRS-2, activation of phosphatidylinositol 3-kinase, and serine phosphorylation of AKT), compared with wild-type cells. Accordingly, PTP1B(-/-) myocytes displayed higher insulin-dependent stimulation of glucose uptake and GLUT4 translocation to the plasma membrane than wild-type cells. Treatment with tumor necrosis factor-alpha (TNF-alpha) induced insulin resistance on glucose uptake, impaired insulin signaling, and increased PTP1B activity in wild-type cells. Conversely, the lack of PTP1B confers protection against insulin resistance by TNF-alpha in myocyte cell lines and in adult male mice. Wild-type mice treated with TNF-alpha developed a pronounced hyperglycemia along the glucose tolerance test, accompanied by an impaired insulin signaling and increased PTP1B activity in muscle. However, mice lacking PTP1B maintained a rapid clearance of glucose and insulin sensitivity and displayed normal muscle insulin signaling regardless the presence of TNF-alpha.

Activation of PKCtheta is associated with lipid-induced insulin resistance and PKCtheta knockout mice are protected from the lipid-induced defects. However, the exact mechanism by which PKCtheta contributes to insulin resistance is not known. To investigate whether an increase in PKCtheta expression leads to insulin resistance, C2C12 skeletal muscle cells were transfected with PKCtheta DNA and treated with different concentrations of insulin for 10 min. PKCtheta overexpression induced reduction of IRS-1 protein levels with a decrease in insulin-induced p85 binding to IRS-1, phosphorylation of PKB and its substrates, p70 and GSK3. Pretreatment of these cells with GF-109203X (a non-specific PKC inhibitor, IC50 for PKCtheta = 10 nM) recovered insulin signaling. PKCtheta was found to be expressed in liver and treatment of human hepatoma cells (HepG2) with high insulin and glucose resulted in an increase in PKCtheta expression that correlated with a decrease in IRS-1 protein levels and the development of insulin resistance. Reduction of PKCtheta expression using RNAi technology significantly inhibited the degradation of IRS-1 and enhanced insulin-induced IRS-1 tyrosine phosphorylation, p85 association to IRS-1 and PKB phosphorylation. In conclusion, by overexpressing PKCtheta or using RNAi technology to downregulate PKCtheta, we have demonstrated that PKCtheta has a key role in the development of insulin resistance. These findings suggest that PKCtheta mediates not only insulin resistance in muscle but also in liver, which may contribute to the development of whole body insulin resistance and diabetes.

Obesity and insulin resistance are strongly associated with systemic markers of inflammation and endoplasmic reticulum stress. c-Jun N-terminal kinases (JNK) are activated by inflammatory cytokines and have a key role in beta-cell apoptosis and in negative regulation of insulin signaling. JNK1-deficient mice are protected from diet-induced obesity and insulin resistance, while genetically obese mice with targeted mutations in JNK1 are leaner and have reduced insulin and blood glucose levels. These studies validate JNK as a link between inflammation and metabolic diseases and as a promising drug target. This review highlights recent advances in small-molecule inhibitors of JNK that have also been targeted for other diseases with an inflammatory component such as stroke, rheumatoid arthritis, and Alzheimer's and Parkinson's diseases.

Protein-tyrosine phosphatase-1B (PTP1B) has been implicated as a negative regulator of insulin signaling. PTP1B dephosphorylates the insulin receptor and insulin receptor substrates (IRS-1/2), inhibiting the insulin-signaling pathway. PTP1B has been reported to be elevated in diabetes and insulin-resistant states. Conversely, PTP1B null mice have increased insulin sensitivity. To further investigate the effect of PTP1B reduction on insulin signaling, FAO rat hepatoma cells were transfected, by electroporation, with a specific PTP1B antisense oligonucleotide (ASO), or a control oligonucleotide. The PTP1B ASO caused a 50-70% reduction in PTP1B protein expression as measured by Western blot analysis. Upon insulin stimulation, an increase in the phosphorylation of the insulin receptor and insulin receptor substrates was observed, without any change in protein expression levels. Reduction of PTP1B expression in FAO cells also caused an increase in insulin-stimulated phosphorylation of PKB and GSK3, without any change in protein expression. These results demonstrate that reduction of PTP1B can modulate key insulin signaling events downstream of the insulin receptor.

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin receptor (IR) signal transduction and a drug target for treatment of type 2 diabetes. Using PTP1B antisense oligonucleotides (ASOs), effects of decreased PTP1B levels on insulin signaling in diabetic ob/ob mice were examined. Insulin stimulation, prior to sacrifice, resulted in no significant activation of insulin signaling pathways in livers from ob/ob mice. However, in PTP1B ASO-treated mice, in which PTP1B protein was decreased by 60% in liver, similar stimulation with insulin resulted in increased tyrosine phosphorylation of the IR and IR substrate (IRS)-1 and -2 by threefold, fourfold, and threefold, respectively. IRS-2-associated phosphatidylinositol 3-kinase activity was also increased threefold. Protein kinase B (PKB) serine phosphorylation was increased sevenfold in liver of PTP1B ASO-treated mice upon insulin stimulation, while phosphorylation of PKB substrates, glycogen synthase kinase (GSK)-3alpha and -3beta, was increased more than twofold. Peripheral insulin signaling was increased by PTP1B ASO, as evidenced by increased phosphorylation of PKB in muscle of insulin-stimulated PTP1B ASO-treated animals despite the lack of measurable effects on muscle PTP1B protein. These results indicate that reduction of PTP1B is sufficient to increase insulin-dependent metabolic signaling and improve insulin sensitivity in a diabetic animal model.

Insulin rapidly stimulates the tyrosine kinase activity of its receptor, resulting in the phosphorylation of insulin receptor substrates (IRS), which in turn associates and activates PI 3-kinase, leading to an increase in glucose uptake. Phosphorylation of IRS proteins and activation of downstream kinases by insulin are transient and the mechanisms for the subsequent downregulation of their activity are largely unknown. We report here that the insulin-induced IRS-1 tyrosine phosphorylation and PI 3-kinase association to IRS-1 were strongly sustained by the proteasome inhibitors, MG132 and lactacystin. In contrast, no effect was detected on the insulin receptor and IRS-2 tyrosine phosphorylation. Interestingly, lactacystin also preserved PKB activation and insulin-induced glucose uptake. In contrast, calpeptin, a calpain inhibitor, was ineffective. Tyrosine phosphatase assays were also performed, showing that lactacystin was not functioning directly as a tyrosine phosphatase inhibitor "in vitro." In conclusion, proteasome inhibitors can regulate the tyrosine phosphorylation of IRS-1 and the downstream insulin signaling pathway, leading to glucose transport.

Protein tyrosine phosphatase 1B (PTP1B) has been implicated as a negative regulator of insulin action. Overexpression of PTP1B protein has been observed in insulin-resistant states associated with obesity. Mice lacking a functional PTP1B gene exhibit increased insulin sensitivity and are resistant to weight gain. To investigate the role of PTP1B in adipose tissue from obese animals, hyperglycemic obese (ob/ob) mice were treated with PTP1B antisense oligonucleotide (ISIS-113715). A significant reduction in adiposity correlated with a decrease of PTP1B protein levels in fat. Antisense treatment also influenced the triglyceride content in adipocytes, correlating with a downregulation of genes encoding proteins involved in lipogenesis, such as sterol regulatory element-binding protein 1 and their downstream targets spot14 and fatty acid synthase, as well as other adipogenic genes, lipoprotein lipase, and peroxisome proliferator-activated receptor gamma. In addition, an increase in insulin receptor substrate-2 protein and a differential regulation of the phosphatidylinositol 3-kinase regulatory subunit (p85alpha) isoforms expression were found in fat from antisense-treated animals, although increased insulin sensitivity measured by protein kinase B phosphorylation was not observed. These results demonstrate that PTP1B antisense treatment can modulate fat storage and lipogenesis in adipose tissue and might implicate PTP1B in the enlargement of adipocyte energy stores and development of obesity.

Chronic insulin exposure induces serine/threonine phosphorylation and degradation of IRS-1 through a rapamycin-sensitive pathway, which results in a down-regulation of insulin action. In this study, to investigate whether rapamycin (an mTOR inhibitor) could prevent insulin resistance induced by hyperinsulinemia, 3T3-L1 adipocytes were incubated chronically in the presence of insulin with or without the addition of rapamycin. Subsequently, the cells were washed and re-stimulated acutely with insulin. Chronic insulin stimulation caused a reduction of GLUT-4 and IRS-1 proteins with a correlated decrease in acute insulin-induced PKB and MAPK phosphorylations as well as a reduction in insulin-stimulated glucose transport. Rapamycin prevented the reduction of IRS-1 protein levels and insulin-induced PKB Ser-473 phosphorylation with a partial normalization of insulin-induced glucose transport. In contrast, rapamycin had no effect on the decrease in insulin-induced MAPK phosphorylation or GLUT-4 protein levels. These results suggest that chronic insulin exposure leads to a down-regulation of PKB and MAPK pathways through different mechanisms in adipocytes., ((c) 2002 Elsevier Science (USA).)

Protein-tyrosine phosphatase (PTP)1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. In this study, we have assessed the role of PTP1B in the insulin sensitivity of skeletal muscle under physiological and insulin-resistant conditions. Immortalized myocytes have been generated from PTP1B-deficient and wild-type neonatal mice. PTP1B-/- myocytes showed enhanced insulin-dependent activation of insulin receptor autophosphorylation and downstream signaling (tyrosine phosphorylation of insulin receptor substrate [IRS]-1 and IRS-2, activation of phosphatidylinositol 3-kinase, and serine phosphorylation of AKT), compared with wild-type cells. Accordingly, PTP1B-/- myocytes displayed higher insulin-dependent stimulation of glucose uptake and GLUT4 translocation to the plasma membrane than wild-type cells. Treatment with tumor necrosis factor-α (TNF-α) induced insulin resistance on glucose uptake, impaired insulin signaling, and increased PTP1B activity in wild-type cells. Conversely, the lack of PTP1B confers protection against insulin resistance by TNF-α in myocyte cell lines and in adult male mice. Wild-type mice treated with TNF-α developed a pronounced hyperglycemia along the glucose tolerance test, accompanied by an impaired insulin signaling and increased PTP1B activity in muscle. However, mice lacking PTP1B maintained a rapid clearance of glucose and insulin sensitivity and displayed normal muscle insulin signaling regardless the presence of TNF-α. Diabetes 56:404-413, 2007 [ABSTRACT FROM AUTHOR]

Protein tyrosine phosphatase 1B (PTP1B) inhibition increases insulin sensitivity and normalizes blood glucose levels in animals. The molecular events associated with PTP1B inhibition that increase insulin sensitivity remain controversial. Insulin resistant, diabetic ob/ob mice, dosed with PTP1B antisense for 3 weeks exhibited a decrease in PTP1B protein levels and a change in the expression level of p85α isoforms in liver, characterized by a reduction in p85α and an upregulation of the p50α and p55α isoforms. Transfection of mouse hepatocytes with PTP1B antisense caused a downregulation PTP1B and p85α protein levels. Furthermore, transfection of mouse hepatocytes with PTP1B siRNA downregulated p85α protein expression and enhanced insulin-induced PKB phosphorylation. Treatment of mouse hepatocytes with p85α antisense oligonucleotide caused a reduction of p85α and an increase in p50α and p55α isoforms and enhanced insulin-stimulated PKB activation. These results demonstrate that PTP1B inhibition causes a direct differential regulation of p85α isoforms of PI3-kinase in liver and that reduction of p85α may be one mechanism by which PTP1B inhibition improves insulin sensitivity and glucose metabolism in insulin-resistant states. [Copyright &y& Elsevier]

Insulin receptor substrate (IRS)-1 protein expression is markedly reduced in many insulin-resistant states, although the mechanism for this downregulation is unclear. In this study, we have investigated the early events in the insulin pathway that trigger the degradation of IRS-1. Incubation of the adipocytes with insulin induced a fast electrophoretic mobility shift of IRS-1 and a subsequent degradation of the protein. Wortmannin and rapamycin blocked this mobility shift of IRS-1, maintained the insulin-induced tyrosine phosphorylation of IRS-1, and blocked its degradation. In contrast, a glycogen synthase kinase 3 inhibitor, a mitogen-activated protein kinase/extracellular-regulated kinase inhibitor, and various protein kinase C inhibitors had no effect. Incubation with okadaic acid increased the serine/threonine phosphorylation of IRS-1 and its degradation, mimicking insulin, and its effect was prevented by the proteasome inhibitor lactacystin, as well as by rapamycin. Treatment of the cells with the tyrosine phosphatase inhibitor orthovanadate in the presence of insulin or okadaic acid partially inhibited the degradation of IRS-1. We propose that a rapamycin-dependent pathway participates as a negative regulator of IRS-1, increasing its serine/threonine phosphorylation, which triggers degradation. Thus, regulation of serine/threonine versus tyrosine phosphorylation may modulate IRS-1 degradation, affecting insulin sensitivity. [ABSTRACT FROM AUTHOR]

In a serum-free medium addition of insulin-like growth factor-1 (IGF-1) consistently enhanced lymphocyte proliferation response to PHA in a dose-dependent fashion. This effect was produced by an acceleration in the expression of clone expansion and not in the number of proliferating cells. This was documented by kinetics data obtained from the first proliferation round of PHA-stimulated lymphocytes, in which addition of IGE-1 reduced G₁-phase length, without changing Gophase, S-phase or cloned size. The data were confirmed in 10-day culture of stimulated lymphocytes where IGF-1 only accelerated cell proliferation without modifying the area enclosed by the proliferation curve. As IGF-1 is under the control of growth hormone, our results suggest that some of the immunoregulation effects ascribed to growth hormone in vivo could be produced by IGF-I. [ABSTRACT FROM AUTHOR]

Insulin signaling can be negatively regulated by phosphorylation of serine 307 of the insulin receptor substrate (IRS)-1. Rapamycin, an inhibitor of the kinase mTOR, can prevent serine 307 phosphorylation and the development of insulin resistance. We further investigated the role of mTOR in regulating serine 307 phosphorylation, demonstrating that serine 307 phosphorylation in response to insulin, anisomycin, or tumor necrosis factor was quantitatively and temporally associated with activation of mTOR and could be inhibited by rapamycin. Amino acid stimulation activated mTOR and resulted in IRS-1 serine 307 phosphorylation without activating PKB or JNK. Okadaic acid, an inhibitor of the phosphatase PP2A, activated mTOR and stimulated the phosphorylation of serine 307 in a rapamycin-sensitive manner, indicating serine 307 phosphorylation requires mTOR activity but not PP2A, suggesting that mTOR itself may be responsible for phosphorylating serine 307. Finally, we demonstrated that serine 307 phosphorylated IRS-1 is detected primarily in the cytosolic fraction. [Copyright &y& Elsevier]