In Vitro Metabolic and Mitogenic Signaling of Insulin Glargine and Its Metabolites

with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities.The affinity of human insulin, insulin glargine and its metabolites to the IR isoforms A and B or IGF1R was analyzed in a competitive binding assay using SPA technology. Receptor autophosphorylation activities were studied via In-Cell Western in CHO and MEF cells overexpressing human IR-A and IR-B or IGF1R, respectively. The metabolic response of the insulins was studied as stimulation of lipid synthesis using primary rat adipocytes. Thymidine incorporation in Saos-2 cells was used to characterize the mitogenic activity. value for autophosphorylation of the receptor and a more potent stimulation of thymidine incorporation in Saos-2 cells. In contrast, the metabolites M1 and M2 were significantly less active in binding to and activation of the IGF1R and their mitogenicity in Saos-2 cells was equal to human insulin. These findings strongly support the idea that insulin glargine metabolites contribute with the same potency as insulin glargine to blood glucose control but lead to significantly reduced growth-promoting activity

Identification of Ser-1275 and Ser-1309 as autophosphorylation sites of the insulin receptor 1This paper is dedicated to Prof. Günter Legler on the occasion of his 70th birthday.1

AbstractWe have identified Ser-1275 and Ser-1309 as novel serine autophosphorylation sites by direct sequencing of HPLC-purified tryptic phosphopeptides of the histidine-tagged insulin receptor kinase IRKD-HIS. The corresponding peptides (Ser-1275, amino acids 1272–1292; Ser-1309, amino acids 1305–1313) have been detected in the HPLC profiles of both the soluble kinase IRKD, which contains the entire cytoplasmic domain of the insulin receptor β-subunit, and the insulin receptor purified from human placenta. In contrast, a kinase negative mutant, IRKD-K1018A, did not undergo phosphorylation at either the tyrosine or serine residues, strongly suggesting that insulin receptor kinase has an intrinsic activity to autophosphorylate serine residues

9-PAHSA displays a weak anti-inflammatory potential mediated by specific antagonism of chemokine G protein-coupled receptors

Introduction: 9-PAHSA belongs to a class of endogenous mammalian bioactive lipids, fatty acid esters of hydroxy fatty acids (FAHFA), that are present in circulation at nanomolar concentrations in mice and humans. Published preclinical data suggest beneficial effects of 9-PAHSA treatment on glucose metabolism as well as modulation of immune function. However, receptor molecules with high affinity towards these lipids have not been identified so far.Methods: In a broad screen of a panel of G protein-coupled receptors (GPCRs) we discovered that 9-PAHSA displays antagonist activity with an IC50 in the micromolar range on selected chemokine receptors, namely, CCR6, CCR7, CXCR4, and CXCR5. The potential immunomodulatory activities in a human cellular model of innate immunity were then investigated.Results and discussion: In our in vitro experiments, a weak anti-inflammatory potential for high concentrations of 9-PAHSA (10–100 µM) could be detected, as treatment reduced the LPS-induced secretion of certain chemokines, such as CXCL10, MIP-1 beta and MCP. Regarding metabolic effects, we re-investigated 9-PAHSA on glucose metabolism and insulin sensitivity in vitro and in mice confirming conclusions from our earlier study that FAHFAs lack glucoregulatory activity following an acute treatment. In conclusion, the specific interactions with a subset of chemokine receptors may contribute to weak anti-inflammatory properties of 9-PAHSA, but further studies are needed to confirm its in anti-inflammatory potential in vivo

Reminiscences of Some Models of Japanese Style Vessels

We investigated 1) the ability of purified glargine (GLA), metabolites 1 (M1) and 2 (M2), IGF-I, and NPH insulin to activate the insulin receptor (IR)-A and IR-B and IGF-I receptor (IGF-IR) in vitro; 2) plasma concentrations of GLA, M1, and M2 during longterm insulin therapy in type 2 diabetic patients; and 3) IR-A and IR-B activation in vitro induced by serum from patients treated with GLA or NPH insulin. A total of 104 patients (age 56.3 ± 0.8 years, BMI 31.4 ± 0.5 kg/m2, and A1C 9.1 ± 0.1% [mean ± SE]) were randomized to GLA or NPH insulin therapy for 36 weeks. Plasma concentrations of GLA, M1, and M2 were determined by liquid chromatography- tandem mass spectrometry assay. IR-A, IR-B, and IGF-IR autophosphorylation was induced by purified hormones or serum by kinase receptor activation assays. In vitro, M1 induced comparable IR-A, IR-B, and IGF-IR autophosphorylation (activation) as NPH insulin. After 36 weeks, M1 increased from undetectable