Deborah Szalkowski

Insulin- and Mitogen-activated Protein Kinase-mediated Phosphorylation and Activation of Peroxisome Proliferator-activated Receptor γ

Peroxisome proliferator-activated receptor (PPAR) γ plays an important role in adipocyte differentiation and the regulation of adipocyte gene expression. Insulin also serves to promote adipogenesis. We report that insulin and a PPARγ ligand (thiazolidinedione (TZD)) stimulate in a synergistic manner the expression of an adipocyte-specific gene (aP2) in rat adipocytes and 3T3-L1 cells. Potential cross-talk between insulin signaling and PPARγ was studied in Chinese hamster ovary cells expressing insulin receptors (CHO.T), PPARγ, and reporter genes. Both TZD and insulin independently stimulated PPARγ-mediated transactivation of aP2 promoter-luciferase reporter genes; both agents combined resulted in a synergistic effect. Co-transfection of CHO.T cells with dominant-negative mitogen-activated protein (MAP) kinase-kinase (MKK1) abrogated both insulin- and TZD-mediated activation of PPARγ; transactivation was markedly increased in cells co-transfected with constitutively active MKK1. Both insulin and constitutively active MKK1 also stimulated 32P incorporation into PPARγ in vivo. The conclusions are: 1) Insulin synergizes with a PPARγ ligand and can activate the receptor in a ligand-independent fashion. 2) PPARγ is phosphorylated in vivo by insulin stimulation or activation of the MAP kinase pathway. 3) MAP kinase is an important mediator of cross-talk between insulin signal transduction pathways and PPARγ function.

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.

Activation of Insulin Signal Transduction Pathway and Anti-diabetic Activity of Small Molecule Insulin Receptor Activators

We recently described the identification of a non-peptidyl fungal metabolite (l-783,281, compound 1), which induced activation of human insulin receptor (IR) tyrosine kinase and mediated insulin-like effects in cells, as well as decreased blood glucose levels in murine models of Type 2 diabetes (Zhang, B., Salituro, G., Szalkowski, D., Li, Z., Zhang, Y., Royo, I., Vilella, D., Diez, M. T., Pelaez, F., Ruby, C., Kendall, R. L., Mao, X., Griffin, P., Calaycay, J., Zierath, J. R., Heck, J. V., Smith, R. G. & Moller, D. E. (1999) Science 284, 974–977). Here we report the characterization of an active analog (compound 2) with enhanced IR kinase activation potency and selectivity over related receptors (insulin-like growth factor I receptor, epidermal growth factor receptor, and platelet-derived growth factor receptor). The IR activators stimulated tyrosine kinase activity of partially purified native IR and recombinant IR tyrosine kinase domain. Administration of the IR activators to mice was associated with increased IR tyrosine kinase activity in liver.In vivo oral treatment with compound 2 resulted in significant glucose lowering in several rodent models of diabetes. In db/db mice, oral administration of compound 2 elicited significant correction of hyperglycemia. In a streptozotocin-induced diabetic mouse model, compound 2 potentiated the glucose-lowering effect of insulin. In normal rats, compound 2 improved oral glucose tolerance with significant reduction in insulin release following glucose challenge. A structurally related inactive analog (compound 3) was not effective on insulin receptor activation or glucose lowering in db/db mice. Thus, small molecule IR activators exert insulin mimetic and sensitizing effects in cells and in animal models of diabetes. These results have implications for the future development of new therapies for diabetes mellitus.

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.

Regulation of insulin signal transduction pathway by a small-molecule insulin receptor activator.

Insulin regulates cellular metabolism and growth through activation of insulin receptors (IRs). We recently identified a non-peptide small-molecule IR activator (compound 2), which induced human IR tyrosine kinase activity in Chinese-hamster ovary cells expressing human IR [Qureshi, Ding, Li, Szalkowski, Biazzo-Ashnault, Xie, Saperstein, Brady, Huskey, Shen et al. (2000) J. Biol. Chem. 275, 36590-36595]. Oral treatment with this compound resulted in correction of hyperglycaemia, hypertriacylglycerolaemia and hyperinsulinaemia in several rodent models of diabetes. In the present study, we have found that this compound increased tyrosine phosphorylation of the IR beta-subunit and IR substrate 1 in primary rat adipocytes as well as induced phosphorylation of Akt, the 70 kDa ribosomal protein S6 kinase and glycogen synthase-3 (deactivation) in Chinese-hamster ovary cells expressing human IR. Similar to insulin, compound 2 stimulated glucose uptake, glycogen synthesis and inhibited isoprenaline-stimulated lipolysis in adipocytes. A structurally related analogue (compound 3) was devoid of the above activities suggesting that the activity of compound 2 is specifically mediated by targeted IR activation. The effects of compound 2 on stimulation of glucose uptake, glycogen synthesis and inhibition of lipolysis were blocked by wortmannin, consistent with the involvement of a phosphoinositide 3-kinase-dependent pathway. In addition, compound 2, but not compound 3, exhibited additive or synergistic effects with sub-maximal concentrations of insulin in rat adipocytes. Thus the IR activator was capable of activating insulin-mediated signalling and metabolic pathways in primary adipocytes. These results demonstrate that IR activators have implications for the future development of new therapeutic approaches to Type I and Type II diabetes.

The basal SAR of a novel insulin receptor activator.

The synthesis and SAR of analogues prepared from novel insulin receptor activator 1 are described. Changes to the dihydroxyquinone core were not tolerated while functionalization of the two indoles contained in 1 resulted in little effect upon activation of the insulin receptor.

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 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.