The tyrosine kinase activity of the human insulin receptor (HIR) is essential for its signalling function [1]. Insulin-induced autophosphorylation of the receptor b-subunit at tyrosine residues in the kinase domain and in the juxtamembrane domain of the receptor b-subunit is critical for autoactivation of the receptor and for substrate phosphorylation [2,3]. After the rapid insulin-stimulated tyrosine phosphorylation of the receptor b-subunit, there is a delayed insulinstimulated increase of serine and threonine phosphorylation of the receptor b-subunit in intact cells [4±7]. Serine phosphorylation occurs as well on the next level of the insulin signalling chain, i. e. receptor sub- Diabetologia (2000) 43: 443±449 Serine residues 994 and 1023/25 are important for insulin receptor kinase inhibition by protein kinase C isoforms b2 and v

The role of leptin in the pathogenesis of obesity has been intensively studied in recent years [1]. Many studies in obese patients found an apparent discrepancy between circulating leptin levels and leptin effects, suggesting that leptin resistance might be a common phenomenon in obese individuals [1]. The mechanisms causing leptin resistance are not, however, clear. Structural defects of the leptin receptor, associated with a loss of function which might cause cellular leptin resistance, do not seem to be a common cause in human beings [2] indicating that abnormalities in postreceptor signalling elements, either genetically determined or caused by regulatory mecha- Diabetologia (2001) Abstract Aims/hypothesis. Leptin resistance in obese humans seems to be predominantly caused by signalling abnormalities at the post receptor level. Leptin resistance in obese individuals is frequently associated with insulin resistance and pronounced hyperinsulinaemia indicating a negative crosstalk of the insulin and leptin signalling chain.Methods. This hypothesis was tested using a cell model of peripheral leptin signalling, i. e. insulin-secreting cell lines (RINr1046±38). Mechanisms for a crosstalk between the insulin and leptin signalling pathway were also studied in rat-1 and HEK293 cells overexpressing elements of the insulin and leptin signalling chain.Results. The effects of leptin on insulin secretion are completely cancelled by a 4-h preincubation with 1 nmol/l insulin, supporting the hypothesis of a negative crosstalk of insulin and leptin signalling. We investigated the potential molecular mechanisms in more detail in HEK293 cells and Rat-1 fibroblasts that overexpressed proteins of the insulin and leptin signalling chain. Leptin (60 ng/ml) stimulated autophosphorylation of JAK-2 in HEK 293 cells. This leptin effect could be inhibited by simultaneous treatment of cells with insulin. Furthermore, overexpression of the insulin receptor in HEK 293 cells clearly reduced JAK-2 phosphorylation and led further downstream to a diminished phosphatidylinositol 3-kinase activity. The inhibitory effect of the insulin signal could be partially prevented by transfection of the cells with an inactive mutant of the tyrosine phosphatase SHP-1. Conclusion/interpretation. In summary, our data suggest that the insulin receptor signalling pathway interferes with leptin signalling at the level of JAK-2. Inhibition of JAK-2 phosphorylation might occur through SHP-1-dependent pathways, indicating that hyperinsulinaemia contributes to the pathogenesis of leptin resistance. [Diabetologia (2001

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Aims/hypothesis: New insulin analogues have been created by amino-acid exchange to provide an improved pharmacokinetic profile. However, safety issues have been raised regarding their use, as amino-acid exchange of insulin may induce altered metabolic and mitogenic effects. For example, the insulin analogue Asp(B10) causes breast cancer in rodents. B10). Materials and methods:We analysed insulin receptor binding characteristics and dissociation kinetics, as well as insulin-induced receptor auto-and dephosphorylation kinetics, in rat-1 fibroblasts overexpressing the human insulin receptor isoform B. Mitogenic activity was tested in the non-malignant cell line MCF10. Results: Regular insulin, HMR1964 and HMR1423 showed no significant differences in receptor association, dissociation and receptor binding affinity, while Asp(B10) displayed markedly increased insulin receptor affinity. All of the analogues induced rapid insulin receptor autophosphorylation, reaching a maximum 10 min after stimulation (10 −9 mmol/l insulin). In contrast, Asp(B10) induced a prolonged phosphorylation and dephosphorylation state of the 95 kDa insulin receptor β-subunit. With respect to [ 3 H]thymidine incorporation, the new analogues had similar (HMR1423) or even lower (HMR1964) effects than regular insulin in the mammary epithelial cell line MCF10, while Asp(B10) showed increased [ 3 H]thymidine incorporation. Conclusions/interpretation: HMR1964 and HMR1423 displayed the same association, dissociation and insulin receptor affinity kinetics as regular insulin, and might therefore be useful for the treatment of diabetes.