AIMS/HYPOTHESIS: We examined the properties of a mutant insulin receptor (IR) with an Arg(252) to Cys (IR(R252C)) substitution in the alpha-subunit originally identified in a patient with extreme insulin resistance and acanthosis nigricans. METHODS: We studied IR cell biology and signalling pathways in Chinese Hamster Ovary cells overexpressing this IR(R252C). RESULTS: Our investigation showed an impairment in insulin binding to IR(R252C) related mostly to a reduced affinity of the receptor for insulin and to a reduced rate of IR(R252C) maturation; an inhibition of IR(R252C)-mediated endocytosis resulting in a decreased insulin degradation and insulin-induced receptor down-regulation; a maintenance of IR(R252C) on microvilli even in the presence of insulin; a similar autophosphorylation of mutant IR(R252C) followed by IRS 1/IRS 2 phosphorylation, p85 association with IRS 1 and IRS 2 and Akt phosphorylation similar to those observed in cells expressing wild type IR (IRwt); and finally, a reduced insulin-induced Shc phosphorylation accompanied by decreased ERK1/2 phosphorylation and activity and of thymidine incorporation into DNA in cells expressing IR(R252C) as compared to cells expressing IRwt. CONCLUSION/INTERPRETATION: These observations suggest that: parameters other than tyrosine kinase activation participate in or control the first steps of IR internalisation or both; IR-mediated IRS 1/2 phosphorylation can be achieved from the cell surface and microvilli in particular; Shc phosphorylation and its subsequent signalling pathway might require IR internalisation; defective IR endocytosis correlates with an enhancement of some biological responses to insulin and attenuation of others.

Immortalized brown adipocyte cell lines have been generated from fetuses of mice deficient in the insulin-like growth factor I receptor gene (IGF-IR(-/-)), as well as from fetuses of wild-type mice (IGF-IR(+/+)). These cell lines maintained the expression of adipogenic- and thermogenic-differentiation markers and show a multilocular fat droplets phenotype. IGF-IR(-/-) brown adipocytes lacked IGF-IR protein expression; insulin receptor (IR) expression remained unchanged as compared with wild-type cells. Insulin-induced tyrosine autophosphorylation of the IR beta-chain was augmented in IGF-IR--deficient cells. Upon insulin stimulation, tyrosine phosphorylation of (insulin receptor substrate-1) IRS-1 was much higher in IGF-IR(-/-) brown adipocytes, although IRS-1 protein content was reduced. In contrast, tyrosine phosphorylation of IRS-2 decreased in IGF-IR--deficient cells; its protein content was unchanged as compared with wild-type cells. Downstream, the association IRS-1/growth factor receptor binding protein-2 (Grb-2) was augmented in the IGF-IR(-/-) brown adipocyte cell line. However, SHC expression and SHC tyrosine phosphorylation and its association with Grb-2 were unaltered in response to insulin in IGF-IR--deficient brown adipocytes. These cells also showed an enhanced activation of mitogen-activated protein kinase (MAPK) kinase (MEK1/2) and p42/p44 mitogen-activated protein kinase (MAPK) upon insulin stimulation. In addition, the lack of IGF-IR in brown adipocytes resulted in a higher mitogenic response (DNA synthesis, cell number, and proliferating cell nuclear antigen expression) to insulin than wild-type cells. Finally, cells lacking IGF-IR showed a much lower association between IR or IRS-1 and phosphotyrosine phosphatase 1B (PTP1B) and also a decreased PTP1B activity upon insulin stimulation. However, PTP1B/Grb-2 association remained unchanged in both cell types, regardless of insulin stimulation. Data presented here provide strong evidence that IGF-IR--deficient brown adipocytes show an increased insulin sensitivity via IRS-1/Grb-2/MAPK, resulting in an increased mitogenesis in response to insulin.

Based on the phenotypes of knockout mice and cell lines, as well as pathway-specific analysis, the insulin receptor substrates IRS-1, IRS-2, IRS-3, and IRS-4 have been shown to play unique roles in insulin signal transduction. To investigate possible functional complementarity within the IRS family, we generated mice with double knockout of the genes for IRS-1/IRS-3 and IRS-1/IRS-4. Mice with a combined deficiency of IRS-1 and IRS-4 showed no differences from Irs1(-/-) mice with respect to growth and glucose homeostasis. In contrast, mice with a combined deficiency of IRS-1 and IRS-3 developed early-onset severe lipoatrophy associated with marked hyperglycemia, hyperinsulinemia, and insulin resistance. However, in contrast to other models of lipoatrophic diabetes, there was no accumulation of fat in liver or muscle. Furthermore, plasma leptin levels were markedly decreased, and adenovirus-mediated expression of leptin in liver reversed the hyperglycemia and hyperinsulinemia. The results indicate that IRS-1 and IRS-3 play important complementary roles in adipogenesis and establish the Irs1(-/-)/Irs3(-/-) double knockout mouse as a novel model of lipoatrophic diabetes.

OBJECTIVE: The development of type 2 diabetes is linked to insulin resistance coupled with a failure of pancreatic beta-cells to compensate by adequate insulin secretion. DESIGN: Here, we review studies obtained from genetically engineered mice that provide novel insights into the pathophysiology of insulin resistance. RESULTS: Knockout models with monogenic impairment in insulin action have highlighted the potential role for insulin signalling molecules in insulin resistance at a tissue-specific level. Polygenic models have strengthened the idea that minor defects in insulin secretion and insulin action, when combined, can lead to diabetes, emphasizing the importance of interactions of different genetic loci in the production of diabetes. Knockout models with tissue-specific alterations in glucose or lipid metabolism have dissected the individual contributions of insulin-responsive organs to glucose homeostasis. They have demonstrated the central role of fat as an endocrine tissue in the maintenance of insulin sensitivity and the development of insulin resistance. Finally, these models have shown the potential role of impaired insulin action in pancreatic beta-cells and brain in the development of insulin deficiency and obesity.

Phosphatidylinositol 3-kinase is a key step in the metabolic actions of insulin. Two amino acid substitutions have been identified in the gene for the regulatory subunit of human p85alpha, Met-326Ile, and Asn-330Asp, and the former has been associated with alterations in glucose/insulin homeostasis. When the four human p85alpha proteins were expressed in yeast, a 27% decrease occurred in the level of protein expression of p85alpha(Ile/Asp) (P = 0.03) and a 43% decrease in p85alpha(Ile/Asn) (P = 0.08) as compared with p85alpha(Met/Asp). Both p85alpha(Ile/Asp) and p85alpha(Ile/Asn) also exhibited increased binding to phospho-insulin receptor substrate-1 by 41% and 83%, respectively (P

Generalised lipodystrophy of the Berardinelli-Seip type (BSCL) is a rare autosomal recessive human disorder with severe adverse metabolic consequences. A gene on chromosome 9 (BSCL1) has recently been identified, predominantly in African-American families. More recently, mutations in a previously undescribed gene of unknown function (BSCL2) on chromosome 11, termed seipin, have been found to be responsible for this disorder in a number of European and Middle Eastern families. We have studied the genotype/phenotype relationships in 70 affected subjects from 44 apparently unrelated pedigrees of diverse ethnic origin. In all subjects, hepatic dysfunction, hyperlipidaemia, diabetes mellitus, and hypertrophic cardiomyopathy were significant contributors to morbidity with no clear differences in their prevalence between subjects with BSCL1 or BSCL2 and those with evidence against cosegregation with either chromosome 9 or 11 (designated BSCLX). BSCL2 appears to be a more severe disorder than BSCL1 with a higher incidence of premature death and a lower prevalence of partial and/or delayed onset of lipodystrophy. Notably, subjects with BSCL2 had a significantly higher prevalence of intellectual impairment than those with BSCL1 or BSCLX (p

Insulin-like growth factor-1 (IGF-1) and insulin are known to prevent apoptosis. The signaling network of IGF-1 and insulin occurs via multiple pathways involving different insulin receptor substrates (IRSs). To define their roles in the anti-apoptotic function of IGF-1 and insulin, we established brown pre-adipocyte cell lines from wild-type and IRS knockout (KO) animals. In response to 16 h of serum deprivation, IRS-1-deficient cells showed a significant decrease in response to IGF-1 protection from apoptosis, whereas no changes were observed in the IRS-2, IRS-3, or IRS-4 KO cells. Five hours after serum withdrawal, cells already began to undergo apoptosis. At this early time point, IGF-1 and insulin were able to protect both wild-type and IRS-1 KO cells from death by 85-90%. After a longer period of serum deprivation, the protective ability of insulin and IGF-1 was decreased, and this was especially reduced in the IRS-1 KO cells. Reconstitution of these cells with IRS-1, IRS-2, IRS-3, or IRS-1/IRS-2 chimeras restored the anti-apoptotic effects of IGF-1, whereas overexpression of IRS-4 had no effect at long time points and actually reduced the effect of IGF-1 at the short time point. The biochemical basis of the defect in anti-apoptosis was not dependent on phosphorylation of mitogen-activated protein kinase; whereas phosphoinositide 3-kinase activity was decreased by 30% in IRS-1 KO cells. Akt phosphorylation was slightly reduced in these cells. Phosphorylation of the transcription factors cAMP response element-binding protein and FKHR by IGF-1 and insulin was markedly reduced in IRS-1 KO cells. In addition, both IGF-1 and insulin prevented caspase-3 cleavage in the wild-type cells, and this effect was greatly reduced in the IRS-1-deficient cells. These findings suggest that the IRS proteins may play differential roles in the anti-apoptotic effects of IGF-1 and insulin in brown pre-adipocytes, with IRS-1 being predominant, possibly acting through caspase-3-, CREB-, and FKHR-dependent mechanisms.

L-783,281, an antidiabetic fungal metabolite that has previously been shown to activate insulin signaling in CHO cells, was tested for its effect on intracellular Ca(2+) ([Ca(2+)](i)) and insulin secretion in single mouse pancreatic beta-cells. Application of 10 micromol/l L-783,281 for 40 s to isolated beta-cells in the presence of 3 mmol/l glucose increased [Ca(2+)](i) to 178 +/- 10% of basal levels (n = 18) as measured by fluo-4 fluorescence. L-767,827, an inactive structural analog of the insulin mimetic, had no effect on beta-cell [Ca(2+)](i). The L-783,281-evoked [Ca(2+)](i) increase was reduced by 82 +/- 4% (n = 6, P

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.

Cross talk between adrenergic and insulin signaling systems may represent a fundamental molecular basis of insulin resistance. We have characterized a newly established beta(3)-adrenoceptor-deficient (beta(3)-KO) brown adipocyte cell line and have used it to selectively investigate the potential role of novel-state and typical beta-adrenoceptors (beta-AR) on insulin signaling and action. The novel-state beta(1)-AR agonist CGP-12177 strongly induced uncoupling protein-1 in beta(3)-KO brown adipocytes as opposed to the beta(3)-selective agonist CL-316,243. Furthermore, CGP-12177 potently reduced insulin-induced glucose uptake and glycogen synthesis. Neither the selective beta(1)- and beta(2)-antagonists metoprolol and ICI-118,551 nor the nonselective antagonist propranolol blocked these effects. The classical beta(1)-AR agonist dobutamine and the beta(2)-AR agonist clenbuterol also considerably diminished insulin-induced glucose uptake. In contrast to CGP-12177 treatment, these negative effects were completely abrogated by metoprolol and ICI-118,551. Stimulation with CGP-12177 did not impair insulin receptor kinase activity but decreased insulin receptor substrate-1 binding to phosphatidylinositol (PI) 3-kinase and activation of protein kinase B. Thus the present study characterizes a novel cell system to selectively analyze molecular and functional interactions between novel and classical beta-adrenoceptor types with insulin action. Furthermore, it indicates insulin receptor-independent, but PI 3-kinase-dependent, potent negative effects of the novel beta(1)-adrenoceptor state on diverse biological end points of insulin action.