Novel effectors of directed and Ngn3-mediated differentiation of mouse embryonic stem cells into endocrine pancreas progenitors.

The delineation of regulatory networks involved in early endocrine pancreas specification will play a crucial role in directing the differentiation of embryonic stem cells toward the mature phenotype of beta cells for cell therapy of type 1 diabetes. The transcription factor Ngn3 is required for the specification of the endocrine lineage, but its direct targets and the scope of biological processes it regulates remain elusive. We show that stepwise differentiation of embryonic stem cells using successive in vivo patterning signals can lead to simultaneous induction of Ptf1a and Pdx1 expression. In this cellular context, Ngn3 induction results in upregulation of its known direct target genes within 12 hours. Microarray gene expression profiling at distinct time points following Ngn3 induction suggested novel and diverse roles of Ngn3 in pancreas endocrine cell specification. Induction of Ngn3 expression results in regulation of the Wnt, integrin, Notch, and transforming growth factor beta signaling pathways and changes in biological processes affecting cell motility, adhesion, the cytoskeleton, the extracellular matrix, and gene expression. Furthermore, the combination of in vivo patterning signals and inducible Ngn3 expression enhances ESC differentiation toward the pancreas endocrine lineage. This is shown by strong upregulation of endocrine lineage terminal differentiation markers and strong expression of the hormones glucagon, somatostatin, and insulin. Importantly, all insulin(+) cells are also C-peptide(+), and glucose-dependent insulin release was 10-fold higher than basal levels. These data suggest that bona fide pancreas endocrine cells have been generated and that timely induction of Ngn3 expression can play a decisive role in directing ESC differentiation toward the endocrine lineage.

Differential phosphorylation of IRS-1 and IRS-2 by insulin and IGF-I receptors.

The specific contribution of insulin and IGF-I receptors to IRS-protein activation remains elusive. We studied the signalling properties of AspB10-insulin, an analog with enhanced affinity for the IGF-I receptor, in comparison to native insulin using primary human skeletal muscle cells. In myoblasts regular insulin and AspB10-insulin were equipotent in stimulating the IRS cascade, whereas this analog induced a significantly higher Shc phosphorylation. Phosphorylation of IRS-1 in response to insulin was inhibited equally by blocking either the insulin or the IGF-I receptor. IRS-1 activation by AspB10-insulin was only inhibited by blocking the IGF-I receptor. IRS-2 phosphorylation induced by both insulin and AspB10-insulin was nearly insensitive to blocking the insulin receptor, being predominantly mediated by the IGF-I receptor. We conclude that in myoblasts IRS-2, but not IRS-1, functions as preferred substrate for the IGF-I receptor. These data suggest a specific role for IRS-2 in growth and differentiation of human skeletal muscle.

[LysB3, GluB29] insulin: a novel insulin analog with enhanced beta-cell protective action.

Insulin receptor substrate (IRS)-2 has been implicated in the promotion of beta-cell survival. Here we tested the hypothesis that the novel analog [LysB3, GluB29] insulin (insulin glulisine, IG) might mediate an enhanced beta-cell protective effect due to its unique property of preferential IRS-2 phosphorylation. We assessed IRS activation by IG and its anti-apoptotic activity against cytokines or palmitic acid in comparison to insulin, insulin analogs, and insulin-like growth factor (IGF)-I using INS-1 cells. IG induced a prominent IRS-2 activation without significant IRS-1 stimulation. The marked cytokine- and fatty acid-induced apoptosis was strongly (55-60%) inhibited by IG both at the level of caspase 3 activation and nucleosomal release, with only 15% inhibition of apoptosis by regular insulin. At 1nM, insulin, insulin aspart, and insulin lispro were much less effective compared to IG. In conclusion, the prominent anti-apoptotic activity of insulin glulisine might serve to counteract autoimmune- and lipotoxicity-induced beta-cell destruction.

A novel insulin analog with unique properties: LysB3,GluB29 insulin induces prominent activation of insulin receptor substrate 2, but marginal phosphorylation of insulin receptor substrate 1.

The potentially enhanced mitogenic activity of insulin analogs represents a safety risk that requires detailed analysis of new analogs considered for therapeutic applications. We assessed the signaling properties and mitogenic potency of two novel rapid-acting insulin analogs, Lys(B3),Glu(B29) insulin (HMR 1964) and Lys(B3),Ile(B28) insulin (HMR 1153) using myoblasts and cardiomyocytes. In myoblasts, both binding and internalization were two- to threefold higher for Asp(B10) insulin and HMR 1153 when compared with HMR 1964 and regular insulin. This finding correlated with a prominent Shc/IGF-I receptor interaction, tyrosine phosphorylation of Shc, activation of extracellular signal-regulated protein kinase (ERK)-1 and -2, and stimulation of DNA synthesis by HMR 1153 and Asp(B10) insulin. In contrast, HMR 1964 produced a marginal activation of the Shc/ERK kinase cascade and was equipotent to insulin in stimulating DNA synthesis in myoblasts. Further, the in vivo growth-promoting activity of this analog was found to be identical to that of regular human insulin. In myoblasts, HMR 1964 produced a minor activation of insulin receptor substrate (IRS)-1 tyrosine phosphorylation, but a prominent activation of IRS-2, with a significantly stronger effect than insulin in human myoblasts. Predominant activation of IRS-2 was also observed in adult cardiomyocytes where HMR 1964 increased 3-O-methylglucose transport and the activation of Akt and glycogen synthase kinase-3 to the same extent as human insulin. We concluded that 1) the mitogenic properties of insulin analogs may result from a series of initial receptor interactions, including internalization and phosphorylation; 2) the mitogenic and metabolic potential of HMR 1964 is identical to that of insulin; and 3) predominant activation of IRS-2 may open new avenues for optimized insulin therapies.

Insulin receptor substrate-4 is expressed in muscle tissue without acting as a substrate for the insulin receptor.

Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.

Eicosanoids and the regulation of cardiac glucose transport.

Intact actin microfilaments are necessary for insulin-regulated GLUT4 translocation from intracellular pools to the plasma membrane. Products of the lipoxygenase (LO) pathway were shown to be implicated in the regulation of actin cytoskeleton rearrangement. The aim of this study was to examine the role of these LO products for cardiac insulin signaling and glucose uptake, GLUT4 translocation, and actin-based cytoskeleton structure. Exposure of cardiomyocytes to esculetin or NDGA, two structurally different LO inhibitors, induced a complete inhibition of insulin-stimulated glucose uptake, whereas control cells showed a threefold stimulation by insulin. Addition of 12(S)-HETE rendered the NDGA-treated cells insulin-sensitive. Early insulin signaling was not changed in cells exposed to LO inhibitors. Cell surface biotinylation of control cells showed a twofold increase of GLUT4 at the cell surface after insulin stimulation. In contrast, the LO inhibitors induced a complete inhibition of insulin-stimulated GLUT4 translocation. Labeling of the F-actin cytoskeleton revealed a prominent disassembly of actin fibers in cells exposed to the LO inhibitors. In conclusion, we show here that products of the LO reaction participate in the organization of the actin network in ventricular cardiomyocytes. Inhibition of LO blocks GLUT4 translocation without affecting insulin signaling events. These data suggest that products of the LO reaction participate in the regulation of glucose transport by contribution to a rearrangement of actin cytoskeletal elements.