Discovery of N-aryl-2-acylindole human glucagon receptor antagonists.

A novel class of N-aryl-2-acylindole human glucagon receptor (hGCGR) antagonists is reported. These compounds demonstrate good pharmacokinetic profiles in multiple preclinical species. One compound from this series, indole 33, is orally active in a transgenic murine pharmacodynamic model. Furthermore, a 1mg/kg oral dose of indole 33 lowers ambient glucose levels in an ob/ob/hGCGR transgenic murine diabetes model. This compound was deemed suitable for preclinical safety studies and was found to be well tolerated in an 8-day experimental rodent tolerability study. The combination of preclinical efficacy and safety observed with compound 33 highlights the potential of this class as a treatment for type 2 diabetes.

Discovery of cyclic guanidines as potent, orally active, human glucagon receptor antagonists.

In the course of the development of an aminobenzimidazole class of human glucagon receptor (hGCGR) antagonists, a novel class of cyclic guanidine hGCGR antagonists was discovered. Rapid N-dealkylation resulted in poor pharmacokinetic profiles for the benchmark compound in this series. A strategy aimed at blocking oxidative dealkylation led to a series of compounds with improved rodent pharmacokinetic profiles. One compound was orally efficacious in a murine glucagon challenge pharmacodynamic model and also significantly lowered glucose levels in a murine diabetes model.

Discovery of novel, potent, selective, and orally active human glucagon receptor antagonists containing a pyrazole core.

A novel class of 1,3,5-pyrazoles has been discovered as potent human glucagon receptor antagonists. Notably, compound 26 is orally bioavailable in several preclinical species and shows selectivity towards cardiac ion channels, other family B receptors such hGIP and hGLP1, and a large panel of enzymes and additional receptors. When dosed orally, compound 26 is efficacious in suppressing glucagon induced plasma glucose excursion in rhesus monkey and transgenic murine pharmacodynamic models at 1 and 10 mpk, respectively.

Development of a novel GLUT4 translocation assay for identifying potential novel therapeutic targets for insulin sensitization.

GLUT4 (glucose transporter 4) plays important roles in glucose homoeostasis in vivo. GLUT4 expression and function are diminished in diabetic human and animal subjects. The goal of the present study is to develop a cell-based assay for identifying negative regulators of GLUT4 translocation as potential targets for the treatment of Type 2 diabetes. Traditional GLUT4 translocation assays performed in differentiated myocytes or adipocytes are difficult to perform, particularly in HTS (high-throughput screening) mode. In the present study, we stably co-expressed c-Myc and eGFP [enhanced GFP (green fluorescent protein)] dual-tagged recombinant GLUT4 with recombinant IRS1 (insulin receptor substrate 1) in HEK-293 cells (human embryonic kidney cells) (HEK-293.IRS1.GLUT4 cells). Insulin treatment stimulated both glucose uptake and GLUT4 translocation in these cells. GLUT4 translocation is quantified by a TRF (time-resolved fluorescence) assay in a 96-well HTS format. TRF assays confirmed insulin-stimulated GLUT4 translocation, which can be inhibited by PI3K (phosphoinositide 3-kinase) or Akt [also called PKB (protein kinase B)] inhibitors. Treatment with palmitate increased IRS1 serine phosphorylation and reduced insulin-stimulated Akt phosphorylation and GLUT4 translocation, indicating insulin resistance. Knockdown of PTEN (phosphatase and tensin homologue deleted on chromosome 10) and PTP1B (protein tyrosine phosphatase 1B) gene expression by siRNA (small interfering RNA) treatment significantly increased GLUT4 translocation only in cells treated with palmitate but not in untreated cells. Similar results were obtained on treatment with siRNA of JNK1 (c-Jun N-terminal kinase 1), S6K1 (ribosomal protein S6 kinase, 70 kDa, polypeptide 1) and PKC(theta) (protein kinase C theta). In summary, we have established and validated a novel GLUT4 translocation assay that is optimal for identifying negative regulators of GLUT4 translocation. In combination with more physiologically relevant secondary assays in myotubes and adipocytes, this assay system can be used to identify potential novel therapeutic targets for the treatment of Type 2 diabetes.

Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1.

Effective therapies for the treatment of obesity, a key element of metabolic syndrome, are urgently needed but currently lacking. Stearoyl-CoA desaturase-1 (SCD1) is the rate-limiting enzyme catalyzing the conversion of saturated long-chain fatty acids into monounsaturated fatty acids, which are major components of triglycerides. In the current study, we tested the efficacy of pharmacological inhibition of SCD1 in controlling lipogenesis and body weight in mice. SCD1-specific antisense oligonucleotide inhibitors (ASOs) reduced SCD1 expression, reduced fatty acid synthesis and secretion, and increased fatty acid oxidization in primary mouse hepatocytes. Treatment of mice with SCD1 ASOs resulted in prevention of diet-induced obesity with concomitant reductions in SCD1 expression and the ratio of oleate to stearoyl-CoA in tissues and plasma. These changes correlated with reduced body adiposity, hepatomegaly and steatosis, and postprandial plasma insulin and glucose levels. Furthermore, SCD1 ASOs reduced de novo fatty acid synthesis, decreased expression of lipogenic genes, and increased expression of genes promoting energy expenditure in liver and adipose tissues. Thus, SCD1 inhibition represents a new target for the treatment of obesity and related metabolic disorders.

Discovery of potent, orally active benzimidazole glucagon receptor antagonists.

The discovery and optimization of potent and selective aminobenzimidazole glucagon receptor antagonists are described. One compound possessing moderate pharmacokinetic properties in multiple preclinical species was orally efficacious at inhibiting glucagon-mediated glucose excursion in transgenic mice expressing the human glucagon receptor, and in rhesus monkeys. The compound also significantly lowered glucose levels in a murine model of diabetes.

A glucose-responsive insulin therapy protects animals against hypoglycemia.

Hypoglycemia is commonly associated with insulin therapy, limiting both its safety and efficacy. The concept of modifying insulin to render its glucose-responsive release from an injection depot (of an insulin complexed exogenously with a recombinant lectin) was proposed approximately 4 decades ago but has been challenging to achieve. Data presented here demonstrate that mannosylated insulin analogs can undergo an additional route of clearance as result of their interaction with endogenous mannose receptor (MR), and this can occur in a glucose-dependent fashion, with increased binding to MR at low glucose. Yet, these analogs retain capacity for binding to the insulin receptor (IR). When the blood glucose level is elevated, as in individuals with diabetes mellitus, MR binding diminishes due to glucose competition, leading to reduced MR-mediated clearance and increased partitioning for IR binding and consequent glucose lowering. These studies demonstrate that a glucose-dependent locus of insulin clearance and, hence, insulin action can be achieved by targeting MR and IR concurrently.

Potentiation of insulin signaling in tissues of Zucker obese rats after acute and long-term treatment with PPARgamma agonists.

Thiazolidinediones (TZDs), agonists of peroxisome proliferator-activated receptor-gamma (PPARgamma), improve insulin sensitivity in vivo, and the mechanism remains largely unknown. In this study, we showed that, in Zucker obese (fa/fa) rats, acute (1-day) treatment with both rosiglitazone (a TZD) and a non-TZD PPARgamma agonist (nTZD) reduced plasma free fatty acid and insulin levels and, concomitantly, potentiated insulin-stimulated Akt phosphorylation at threonine 308 (Akt-pT308) in adipose and muscle tissues. A similar effect on Akt was observed in liver after a 7-day treatment. The increase in Akt-pT308 was correlated with an increase in Akt phosphorylation at serine 473 (Akt-pS473), tyrosine phosphorylation of insulin receptor beta subunit and insulin receptor substrate-1, and serine phosphorylation of glycogen synthase kinase-3alpha/beta. The agonists appeared to potentiate Akt1 phosphorylation in muscle and liver and both Akt1 and Akt2 in adipose. Finally, potentiation of insulin signaling was also observed in isolated adipose tissue ex vivo and differentiated 3T3 L1 adipocytes in vitro, but not in rat primary hepatocytes in vitro. These results suggest that 1) PPARgamma agonists acutely potentiate insulin signaling in adipose and muscle tissues and such regulation may be physiologically relevant to insulin sensitization in vivo; 2) the agonists directly target adipose tissues; and 3) the metabolic and signaling effects of the agonists are mediated by structurally distinct PPARgamma agonists.

Rosiglitazone, an agonist of peroxisome-proliferator-activated receptor gamma (PPARgamma), decreases inhibitory serine phosphorylation of IRS1 in vitro and in vivo.

Peroxisome-proliferator-activated receptor gamma agonists such as rosiglitazone, a thiazolidinedione, improve insulin sensitivity in vivo, but the underlying mechanism(s) remains unclear. Phosphorylation of IRS1 (insulin receptor substrate protein 1) on certain serine residues, including S307 and S612 in rodent IRS1 (equivalent to S312 and S616 in human IRS1), has been shown to play a negative role in insulin signalling. In the present study, we investigated whether rosiglitazone improves insulin sensitivity by decreasing IRS1 inhibitory serine phosphorylation. In HEK-293 (human embryonic kidney 293) cells stably expressing recombinant IRS1 and in 3T3L1 adipocytes, rosiglitazone attenuated PMA-induced IRS1 S307/S612 phosphorylation and decreased insulin-stimulated Akt phosphorylation. We observed increased IRS1 S307 phosphorylation and concomitant decrease in insulin signalling as measured by insulin-stimulated IRS1 tyrosine phosphorylation, and Akt threonine phosphorylation in adipose tissues of Zucker obese rats compared with lean control rats. Treatment with rosiglitazone at 30 mg/kg body weight for 24 and 48 h increased insulin signalling and decreased IRS1 S307 phosphorylation concomitantly. Whereas the 48 h treatment reversed hyper-phosphorylation (and activation) of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase, the 24 h treatments only decreased hyper-phosphorylation of p38 mitogen-activated protein kinase. The treatment of the Zucker obese rats with rosiglitazone also reversed the high circulating levels of non-esterified fatty acids, which have been shown to be correlated with increased IRS1 serine phosphorylation in other animal models. Taken together, these results suggest that IRS1 inhibitory serine phosphorylation is a key component of insulin resistance and its reversal contributes to the insulin sensitizing effects by rosiglitazone.

Hepatic glucagon receptor binding and glucose-lowering in vivo by peptidyl and non-peptidyl glucagon receptor antagonists.

Glucagon receptor antagonists have been actively pursued as potential therapeutics for the treatment of type 2 diabetes. Peptidyl and non-peptidyl glucagon receptor antagonists have been shown to block glucagon-induced blood glucose elevation in both animals and humans. How the antagonists and the glucagon receptor interact in vivo has not been reported and is the subject of the current study. Using (125)I-labeled glucagon as a radiotracer, we developed an in vivo glucagon receptor occupancy assay in mice expressing a human glucagon receptor in place of the endogenous mouse glucagon receptor (hGCGR mice). Using this assay, we first showed that the glucagon receptor is expressed predominantly in liver, to a much lesser extent in kidney, and is below detection in several other tissues/organs in the mice. We subsequently showed that, at 2 mg/kg body weight (mg/pk) dosed intraperitoneally (i.p.), peptidyl glucagon receptor antagonist des-His-glucagon binds to approximately 78% of the hepatic glucagon receptor and blocks an exogenous glucagon-induced blood glucose elevation in the mice. Finally, we also showed that, at 10 and 30 mg/kg dosed orally (p.o.), compound A, a non-peptidyl small molecule glucagon receptor antagonist, occupied 65-70% of the hepatic glucagon receptor, and significantly diminished exogenous glucagon-induced blood glucose elevation in the mice. At 3 mg/kg, however, compound A occupied only approximately 39% of the hepatic glucagon receptor and did not affect exogenous glucagon-induced blood glucose elevation in the mice. Taken together, the results confirmed previous reports that glucagon receptors are present predominantly in the liver, and provide the first direct evidence that peptidyl and non-peptidyl glucagon receptor antagonists bind to the hepatic glucagon receptor in vivo, and that at least 60% receptor occupancy correlates with the glucose lowering efficacy by the antagonists in vivo.

FGF21 suppresses hepatic glucose production through the activation of atypical protein kinase Cι/λ.

Fibroblast growth factor 21 (FGF21) has been identified as a potent and robust metabolic regulator. Administration of recombinant FGF21 protein to rodents and rhesus monkeys exerts strong anti-diabetic effects. Previous studies have demonstrated that FGF21 inhibits glucose output in the rat H4IIE hepatoma cell line. We performed pharmacological studies to investigate the mechanisms by which FGF21 regulates glucose production in these cells. We found that both insulin and FGF21 suppressed gene expression of G6Pase and PEPCK. Accordingly, glucose production was inhibited. The FGF21 effects were phosphoinositide 3-kinase (PI3K)-dependent, and, unlike insulin, Akt-independent. Additionally, we found that FGF21 induced PKCι/λ phosphorylation in a PI3K-dependent manner; and that a non-isoform selective PKC inhibitor blocked FGF21 inhibition of glucose production, while an inhibitor of classical and novel PKC isoforms had no effect on FGF21 inhibitory activity. Furthermore, hepatic PKCι/λ phosphorylation was upregulated in FGF21-treated diabetic db/db mice.These data support the proposition that FGF21 inhibits hepatic glucose production by the PI3K-dependent activation of PKCι/λ.

Downstream signaling pathways in mouse adipose tissues following acute in vivo administration of fibroblast growth factor 21.

FGF21 is a novel secreted protein with robust anti-diabetic, anti-obesity, and anti-atherogenic activities in preclinical species. In the current study, we investigated the signal transduction pathways downstream of FGF21 following acute administration of the growth factor to mice. Focusing on adipose tissues, we identified FGF21-mediated downstream signaling events and target engagement biomarkers. Specifically, RNA profiling of adipose tissues and phosphoproteomic profiling of adipocytes, following FGF21 treatment revealed several specific changes in gene expression and post-translational modifications, specifically phosphorylation, in several relevant proteins. Affymetrix microarray analysis of white adipose tissues isolated from both C57BL/6 (fed either regular chow or HFD) and db/db mice identified over 150 robust potential RNA transcripts and over 50 potential secreted proteins that were changed greater than 1.5 fold by FGF21 acutely. Phosphoprofiling analysis identified over 130 phosphoproteins that were modulated greater than 1.5 fold by FGF21 in 3T3-L1 adipocytes. Bioinformatic analysis of the combined gene and phosphoprotein profiling data identified a number of known metabolic pathways such as glucose uptake, insulin receptor signaling, Erk/Mapk signaling cascades, and lipid metabolism. Moreover, a number of novel events with hitherto unknown links to FGF21 signaling were observed at both the transcription and protein phosphorylation levels following treatment. We conclude that such a combined "omics" approach can be used not only to identify robust biomarkers for novel therapeutics but can also enhance our understanding of downstream signaling pathways; in the example presented here, novel FGF21-mediated signaling events in adipose tissue have been revealed that warrant further investigation.

Anti-diabetic efficacy and impact on amino acid metabolism of GRA1, a novel small-molecule glucagon receptor antagonist.

Hyperglucagonemia is implicated in the pathophysiology of hyperglycemia. Antagonism of the glucagon receptor (GCGR) thus represents a potential approach to diabetes treatment. Herein we report the characterization of GRA1, a novel small-molecule GCGR antagonist that blocks glucagon binding to the human GCGR (hGCGR) and antagonizes glucagon-induced intracellular accumulation of cAMP with nanomolar potency. GRA1 inhibited glycogenolysis dose-dependently in primary human hepatocytes and in perfused liver from hGCGR mice, a transgenic line of mouse that expresses the hGCGR instead of the murine GCGR. When administered orally to hGCGR mice and rhesus monkeys, GRA1 blocked hyperglycemic responses to exogenous glucagon. In several murine models of diabetes, acute and chronic dosing with GRA1 significantly reduced blood glucose concentrations and moderately increased plasma glucagon and glucagon-like peptide-1. Combination of GRA1 with a dipeptidyl peptidase-4 inhibitor had an additive antihyperglycemic effect in diabetic mice. Hepatic gene-expression profiling in monkeys treated with GRA1 revealed down-regulation of numerous genes involved in amino acid catabolism, an effect that was paralleled by increased amino acid levels in the circulation. In summary, GRA1 is a potent glucagon receptor antagonist with strong antihyperglycemic efficacy in preclinical models and prominent effects on hepatic gene-expression related to amino acid metabolism.

FGF21 analogs of sustained action enabled by orthogonal biosynthesis demonstrate enhanced antidiabetic pharmacology in rodents.

Fibroblast growth factor 21 (FGF21) mitigates many of the pathogenic features of type 2 diabetes, despite a short circulating half-life. PEGylation is a proven approach to prolonging the duration of action while enhancing biophysical solubility and stability. However, in the absence of a specific protein PEGylation site, chemical conjugation is inherently heterogeneous and commonly leads to dramatic loss in bioactivity. This work illustrates a novel means of specific PEGylation, producing FGF21 analogs with high specific activity and salutary biological activities. Using homology modeling and structure-based design, specific sites were chosen in human FGF21 for site-specific PEGylation to ensure that receptor binding regions were preserved. The in vitro activity of the PEGylated FGF21 ana-logs corresponded with the site of PEG placement within the binding model. Site-specific PEGylated analogs demonstrated dramatically increased circulating half-life and enhanced efficacy in db/db mice. Twice-weekly dosing of an optimal FGF21 analog reduced blood glucose, plasma lipids, liver triglycerides, and plasma glucagon and enhanced pancreatic insulin content, islet number, and glucose-dependent insulin secretion. Restoration of insulin sensitivity was demonstrated by the enhanced ability of insulin to induce Akt/protein kinase B phosphorylation in liver, muscle, and adipose tissues. PEGylation of human FGF21 at a specific and preferred site confers superior metabolic pharmacology.

Discovery of a novel glucagon receptor antagonist N-[(4-{(1S)-1-[3-(3, 5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-ß-alanine (MK-0893) for the treatment of type II diabetes.

A potent, selective glucagon receptor antagonist 9m, N-[(4-{(1S)-1-[3-(3,5-dichlorophenyl)-5-(6-methoxynaphthalen-2-yl)-1H-pyrazol-1-yl]ethyl}phenyl)carbonyl]-ß-alanine, was discovered by optimization of a previously identified lead. Compound 9m is a reversible and competitive antagonist with high binding affinity (IC(50) of 6.6 nM) and functional cAMP activity (IC(50) of 15.7 nM). It is selective for glucagon receptor relative to other family B GPCRs, showing IC(50) values of 1020 nM for GIPR, 9200 nM for PAC1, and >10000 nM for GLP-1R, VPAC1, and VPAC2. Compound 9m blunted glucagon-induced glucose elevation in hGCGR mice and rhesus monkeys. It also lowered ambient glucose levels in both acute and chronic mouse models: in hGCGR ob/ob mice it reduced glucose (AUC 0-6 h) by 32% and 39% at 3 and 10 mpk single doses, respectively. In hGCGR mice on a high fat diet, compound 9m at 3, and 10 mpk po in feed lowered blood glucose levels by 89% and 94% at day 10, respectively, relative to the difference between the vehicle control and lean hGCGR mice. On the basis of its favorable biological and DMPK properties, compound 9m (MK-0893) was selected for further preclinical and clinical evaluations.

Evidence that inhibition of insulin receptor signaling activity by PC-1/ENPP1 is dependent on its enzyme activity.

Plasma cell membrane glycoprotein-1 or ectonucleotide pyrophosphatase/phosphodiesterase (PC-1/ENPP1) has been shown to inhibit insulin signaling, and its genetic polymorphism or increased expression is associated with type 2 diabetes in humans. Therefore, PC-1 inhibition represents a potential strategy in treating diabetes. Since patients with phosphodiesterase/pyrophosphatase deficient PC-1 manifest abnormal calcification, enhancing insulin signaling by inhibiting PC-1 for the treatment of diabetes will be feasible only if PC-1 phosphodiesterase/pyrophosphatase activity needs not be significantly diminished. However, whether inhibition of insulin receptor signaling by PC-1 is dependent upon its phosphodiesterase/pyrophosphatase activity remains controversial. In this study, the extracellular domain of the human PC-1 in its native form or with a T256A or T256S mutation was overexpressed and purified. Enzymatic assays showed that both mutants have less than 10% of the activity of the wild-type protein. In HEK293 cells stably expressing recombinant insulin receptor or insulin-like growth factor 1 (IGF1) receptor, transient expression of wild-type full length PC-1 (PC-1.FL.WT) but not the T256A or T256S mutants inhibits insulin signaling without affecting IGF1 signaling. Western blot and FACS analysis showed that the wild-type and mutant full length PC-1 proteins are expressed at similar levels in the cells, and were localized to the similar levels on the cell surface. Overexpression of PC-1.FL.WT did not affect insulin receptor mRNA level, total protein and cell surface levels. Together, these results suggest that the inhibition of insulin signaling by PC-1 is somewhat specific and is dependent upon the enzymatic activity of the phosphodiesterase/pyrophosphatase.

Suppression of PC-1/ENPP-1 expression improves insulin sensitivity in vitro and in vivo.

Plasma cell membrane glycoprotein-1, or ectonucleotide pyrophosphatase/phosphodieterase (PC-1/ENPP1) has been shown to inhibit insulin signaling in cultured cells in vitro and in transgenic mice in vivo when overexpressed. Furthermore, both genetic polymorphism and increased expression of PC-1 have been reported to be associated with type 2 diabetes in humans. Thus it was proposed that PC-1 inhibition represents a potential strategy for the treatment of type 2 diabetes. However, it has not been proven that suppression of PC-1 expression or inhibition of its function will actually improve insulin sensitivity. We show in the current study that transient overexpression of PC-1 inhibits insulin-stimulated insulin receptor tyrosine phosphorylation in HEK293 cells, while knockdown of PC-1 with siRNA significantly increases insulin-stimulated Akt phosphorylation in HuH7 human hepatoma cells. Adenoviral vector expressing a short hairpin RNA against mouse PC-1 (PC-1shRNA) was utilized to efficiently knockdown PC-1 expression in the livers of db/db mice. In comparison with db/db mice treated with a control virus, db/db mice treated with the PC-1shRNA adenovirus had approximately 80% lower hepatic PC-1 mRNA levels, approximately 30% lower ambient fed plasma glucose, approximately 25% lower fasting plasma glucose, and significantly improved oral glucose tolerance. Taken together, these results demonstrate that suppression of PC-1 expression improves insulin sensitivity in vitro and in an animal model of diabetes, supporting the proposition that PC-1 inhibition is a potential therapeutic approach for the treatment of type 2 diabetes.

Discovery of novel, potent, and orally active spiro-urea human glucagon receptor antagonists.

A novel class of spiro-ureas has been discovered as potent human glucagon receptor antagonists in both binding and functional assays. Preliminary studies have revealed that compound 15 is an orally active human glucagon receptor antagonist in a transgenic murine pharmacodynamic model at 10 and 30 mpk. Compound 15 is orally bioavailable in several preclinical species and shows selectivity toward cardiac ion channels and other family B receptors, such as hGIP1 and hGLP.

Salicylic acid reverses phorbol 12-myristate-13-acetate (PMA)- and tumor necrosis factor alpha (TNFalpha)-induced insulin receptor substrate 1 (IRS1) serine 307 phosphorylation and insulin resistance in human embryonic kidney 293 (HEK293) cells.

Salicylates, including aspirin, have been shown to improve insulin sensitivity both in human and animal models. Although it has been suggested that salicylates sensitize insulin action by inhibiting IkappaB kinase beta (IKKbeta), the detailed mechanisms remain unclear. Protein kinase C isoforms and tumor necrosis factor alpha (TNFalpha) signaling pathways are well described mediators of insulin resistance; they are implicated in the activation of IKKbeta and the subsequent inhibition of proximal insulin signaling via insulin receptor substrate 1 (IRS1) and Akt. This study investigated the effect of salicylic acid on phorbol 12-myristate 13-acetate (PMA)- and TNFalpha-induced insulin resistance in a human embryonic kidney 293 (HEK293) cell line stably expressing recombinant human IRS1. The results showed that both PMA and TNFalpha inhibited insulin-induced Akt phosphorylation and promoted IRS1 phosphorylation on Ser-307. Salicylic acid pretreatment completely reversed the effects of PMA and TNFalpha on both Akt and IRS1. Whereas PMA activated protein kinase C isoforms and IKKbeta, TNFalpha activated neither. On the other hand, both PMA and TNFalpha activated the c-Jun N-terminal kinase (JNK), which has been reported to directly phosphorylate IRS1 Ser-307. SP600125, a JNK inhibitor, prevented PMA and TNFalpha-induced IRS1 Ser-307 phosphorylation. Finally, salicylic acid inhibited JNK activation induced by both PMA and TNFalpha. Taken together, these observations suggest that salicylic acid can reverse the inhibitory effects of TNFalpha on insulin signaling via an IKKbeta-independent mechanism(s), potentially involving the inhibition of JNK activation. The role of JNK in salicylic acid-mediated insulin sensitization, however, requires further validation because the JNK inhibitor SP600125 appears to have other nonspecific activity in addition to inhibiting JNK activity.