Norbert Tennagels

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

Background Insulin glargine (Lantus®) is a long-acting basal insulin analog that demonstrates effective day-long glycemic control and a lower incidence of hypoglycemia than NPH insulin. After subcutaneous injection insulin glargine is partly converted into the two main metabolites M1 ([GlyA21]insulin) and M2 ([GlyA21,des-ThrB30]insulin). The aim of this study was to characterize the glargine metabolites in vitro with regard to their insulin receptor (IR) and IGF-1 receptor (IGF1R) binding and signaling properties as well as their metabolic and mitogenic activities. Methods 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. Conclusions The binding of insulin glargine and its metabolites M1 and M2 to the IR were similar and correlated well with their corresponding autophosphorylation and metabolic activities in vitro. No differences were found towards the two IR isoforms A or B. Insulin glargine showed a higher affinity for IGF1R than insulin, resulting in a lower EC50 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.

Functional annotation of the human fat cell secretome.

Context: Recent secretome analyses suggest that human fat cells secrete hundreds of proteins (adipokines). Objective: We made an overall analysis of their potential functional importance. Materials and methods: A secretome of 347 adipokines was evaluated by in silico analysis of their expression during adipocyte differentiation, regulation by obesity and adipose region. The gene expression in human adipose tissue was investigated in microarray studies using samples from different adipose depots from lean or obese patients. Results: 60% of the adipokines were regulated by obesity and 50% between visceral and subcutaneous adipose region. Eight adipokines, all novel, scored particularly high in the in silico analysis. Among those, four were both regulated by obesity and adipose region, namely WNT1-inducible-signaling pathway protein 2, transmembrane glycoprotein NMB, inter-alpha-trypsin inhibitor heavy chain H5, and complement C4-A. Furthermore, many adipokines were extracellular matrix proteins. Conclusion: Several novel adipokines have potential important functional features warranting in depth analysis.

Effect of Insulin Analogues on Insulin/IGF1 Hybrid Receptors: Increased Activation by Glargine but Not by Its Metabolites M1 and M2

Background In diabetic patients, the pharmacokinetics of injected human insulin does not permit optimal control of glycemia. Fast and slow acting insulin analogues have been developed, but they may have adverse properties, such as increased mitogenic or anti-apoptotic signaling. Insulin/IGF1 hybrid receptors (IR/IGF1R), present in most tissues, have been proposed to transmit biological effects close to those of IGF1R. However, the study of hybrid receptors is difficult because of the presence of IR and IGF1R homodimers. Our objective was to perform the first study on the pharmacological properties of the five marketed insulin analogues towards IR/IGF1R hybrids. Methodology To study the effect of insulin analogues on IR/IGF1R hybrids, we used our previously developed Bioluminescence Resonance Energy Transfer (BRET) assay that permits specific analysis of the pharmacological properties of hybrid receptors. Moreover, we have developed a new, highly sensitive BRET-based assay to monitor phophatidylinositol-3 phosphate (PIP3) production in living cells. Using this assay, we performed a detailed pharmacological analysis of PIP3 production induced by IGF1, insulin and insulin analogues in living breast cancer-derived MCF-7 and MDA-MB231 cells. Results Among the five insulin analogues tested, only glargine stimulated IR/IGF1R hybrids with an EC50 that was significantly lower than insulin and close to that of IGF1. Glargine more efficiently stimulated PIP3 production in MCF-7 cells but not in MDA-MB231 cells as compared to insulin. In contrast, glargine metabolites M1 and M2 showed lower potency for hybrid receptors stimulation, PIP3 production, Akt and Erk1/2 phosphorylation and DNA synthesis in MCF-7 cells, compared to insulin. Conclusion Glargine, possibly acting through IR/IGF1R hybrids, displays higher potency, whereas its metabolites M1 and M2 display lower potency than insulin for the stimulation of proliferative/anti-apoptotic pathways in MCF-7 cells.

Identification of a novel AS160 splice variant that regulates GLUT4 translocation and glucose-uptake in rat muscle cells

AS160 (AKT substrate of 160 kDa) is an important mediator of GLUT4 (glucose transporter 4) translocation and glucose-uptake in adipocytes and muscle cells. In our study we have identified a novel splice variant of AS160 (variant 2 of AS160, AS160_v2) that lacks exon 11 and 12. The protein is phosphorylated in response to insulin via the PI3K/AKT pathway. Expression of this splice variant in human tissues from different donors was examined with quantitative RT-PCR. Our data reveal a tissue specific distribution pattern of both isoforms with highest overall expression of AS160_v2. To investigate the function of the novel splice variant we established the doxycycline-inducible expression of the protein in a rat myoblast cell line co-expressing GLUT4-myc. In contrast to data reported for the full-length AS160 protein, over expression and activation of transcript variant 2 in this cell line increased GLUT4 translocation and glucose-uptake rates in response to insulin and IGF-1 but not in response to AICAR or metformin. Immunofluorescence based studies indicated a direct association of AS160_v2 with GLUT4 under basal but not under insulin-stimulated conditions. Additionally, over expression of AS160_v2 slightly improved glucose-uptake rates in a model of insulin resistance but was not able to fully prevent induction of insulin resistance. This was accompanied with decreased phosphorylation of AS160_v2 and AKT. Taken together, our data suggest a tissue specific distribution of full-length AS160 and the novel AS160 splice variant (AS160_v2) indicating different functions. In contrast to full-length AS160, transcript variant 2 of AS160 seems to be a novel regulator of glucose transport that positively influences glucose-uptake rates.

Chitinase-3-like protein 1 protects skeletal muscle from TNFα-induced inflammation and insulin resistance

CHI3L1 (chitinase-3-like protein 1) is a glycoprotein consisting of 383 amino acids with a molecular mass of 40 kDa, and its serum level is elevated in inflammatory diseases. Although CHI3L1 is described as a biomarker of inflammation, the function of this protein is not completely understood. In the present study, we examined the regulation of CHI3L1 in primary human skeletal muscle cells. Moreover, we analysed potential autocrine effects of CHI3L1. We show that myotubes express CHI3L1 in a differentiation-dependent manner. Furthermore, pro-inflammatory cytokines up-regulate CHI3L1 expression (6-fold) and release (3-fold). Importantly, CHI3L1 treatment blocked TNFα (tumour necrosis factor α)-induced inflammation by inhibiting NF-κB (nuclear factor κB) activation in skeletal muscle cells. We show that this effect is mediated via PAR2 (protease-activated receptor 2). In addition, CHI3L1 treatment diminished the TNFα-induced expression and secretion of IL (interleukin)-8, MCP1 (monocyte chemoattractant protein 1) and IL-6. In addition, impaired insulin action at the level of Akt and GSK3α/β (glycogen synthase kinase 3α/β) phosphoryl-ation and insulin-stimulated glucose uptake was normalized by CHI3L1. In conclusion, the novel myokine CHI3L1, which is induced by pro-inflammatory cytokines, can counteract TNFα-mediated inflammation and insulin resistance in human skeletal muscle cells, potentially involving an auto- and/or para-crine mechanism.

The metabolic and mitogenic properties of basal insulin analogues

Context: Retrospective, observational studies have reported an association between diabetes treatment with insulin and a higher incidence of cancer. Objective: Overview the literature for in vitro and in vivo studies of the metabolic and mitogenic properties of basal insulin analogues and assess the implications for clinical use. Methods: Relevant studies were identified through PubMed and congress abstract database searches; data on metabolic and mitogenic signalling in relation to insulin treatment of diabetes are included in this review. Results: The balance of evidence shows that although some analogues have demonstrated mitogenic potency in some in vitro studies in cancer cell lines, these findings do not translate to the in vivo setting in animals or to the clinical setting in humans. Conclusions: The current consensus is that there is no clinical or in vivo evidence to indicate that any commercially available insulin analogue has carcinogenic effects. Large-scale, prospective clinical and observational studies will further establish any potential link.

A robust assay measuring GLUT4 translocation in rat myoblasts overexpressing GLUT4-myc and AS160_v2

Muscle and fat cells translocate GLUT4 (glucose transporter 4) to the plasma membrane when stimulated by insulin. Usually, this event is measured in differentiated adipocytes, myotubes, or cell lines overexpressing tagged GLUT4 by immunostaining. However, measurement of the translocation in differentiated adipocytes or myotubes or GLUT4 overexpressing cell lines is difficult because of high assay variability caused by either the differentiation protocol or low assay sensitivity. We recently reported the identification of a novel splice variant of AS160 (substrate of 160kDa), namely AS160_v2, and showed that its coexpression with GLUT4 in L6 myoblasts increased the insulin-stimulated glucose uptake rate due to an increased amount of GLUT4 on the cell surface. L6 cells, which coexpress myc-tagged GLUT4 and AS160_v2, can be efficiently used to generate an assay useful for identifying compounds that affect cellular responses to insulin. We compared the EC(50) values for radioactive glucose uptake and GLUT4 translocation of different insulins and several small molecules to validate the assay. The use of L6 cells overexpressing AS160_v2 can be considered as a novel tool for the characterization of molecules modulating insulin signaling and GLUT4 translocation, and an image-based assay increases our confidence in the mode of action of the compounds identified.

Metabolic effect and receptor signalling profile of a non-metabolisable insulin glargine analogue.

Abstract Context: Insulin glargine (GLA) is rapidly metabolized in vivo to metabolite M1, which has in vitro metabolic and mitogenic profiles comparable with human insulin (HI). Objective: To investigate the pharmacologic and signalling profiles of a non-metabolizable analogue (A21Gly,DiD-Arg) insulin (D-GLA). Methods: Rats were injected s.c. with 1, 12.5 or 200 U/kg of GLA or D-GLA; blood glucose and phosphorylation status of the insulin receptor (IR), Akt and IGF-1 receptor (IGF1R) in tissue samples were investigated after 1 h. Plasma samples were analysed for insulin by LC-MS/MS. Results: Blood glucose lowering was prolonged with D-GLA. D-GLA comprised ≥98% of insulin after D-GLA injection; M1 comprised 76–92% after GLA injection. IR and Akt phosphorylation were comparable with GLA and D-GLA. Neither analogue stimulated IGF1R phosphorylation. Conclusions: Suprapharmacological doses of D-GLA did not activate IGF1R in vivo. Mitogenic effects of insulin and insulin analogues might be solely based on IR growth-promoting activity.

Effect of insulin analogues on phosphatidyl inositol-3 kinase/Akt signalling in INS-1 rat pancreatic derived β-cells

Abstract Context: Insulin analogues are largely used for the treatment of diabetic patients, but concerns have been raised about their mitogenic/anti-apoptotic potential. It is therefore important to evaluate these analogues in different cell systems. Objective: The aim of this work was to establish the pharmacological profiles of insulin analogues towards PI-3 kinase/Akt pathway in INS-1 β-pancreatic cells. Methods: Bioluminescence Resonance Energy Transfer (BRET), in cell western and caspase 3/7 assays, was used to study the effects of ligands. Results: Among the five analogues evaluated, only glargine stimulated PI-3 kinase/Akt pathway with higher efficiency than insulin, whereas glargine’s metabolite M1 was less efficient. However, glargine did not show higher anti-apoptotic efficiency than insulin. Conclusion: Glargine was more efficient than insulin for the activation of PI-3 kinase/Akt pathway, but not for the inhibition of caspase 3/7 activity. Moreover, glargine’s metabolite M1 displayed lower efficiency than insulin towards PI-3 kinase/Akt activation and caspase 3/7 inhibition.

Evaluation of the innate immunostimulatory potential of originator and non-originator copies of insulin glargine in an in vitro human immune model

Background The manufacture of insulin analogs requires sophisticated production procedures which can lead to differences in the structure, purity, and/or other physiochemical properties of resultant products that can affect their biologic activity. Here, we sought to compare originator and non-originator copies of insulin glargine for innate immune activity and mechanisms leading to differences in these response profiles in an in vitro model of human immunity. Methods An endothelial/dendritic cell-based innate immune model was used to study antigen-presenting cell activation, cytokine secretion, and insulin receptor signalling pathways induced by originator and non-originator insulin glargine products. Mechanistic studies included signalling pathway blockade with specific inhibitors, analysis of the products in a Toll-like receptor reporter cell line assay, and natural insulin removal from the products by immunopurification. Findings All insulin glargine products elicited at least a minor innate immune response comparable to natural human insulin, but some lots of a non-originator copy product induced the elevated secretion of the cytokines, IL-8 and IL-6. In studies aimed at addressing the mechanisms leading to differential cytokine production by these products, we found (1) the inflammatory response was not mediated by bacterial contaminants, (2) the innate response was driven by the native insulin receptor through the MAPK pathway, and (3) the removal of insulin glargine significantly reduced their capacity to induce innate activity. No evidence of product aggregates was detected, though the presence of some high molecular weight proteins argues for the presence of insulin glargine dimers or others contaminants in these products. Conclusion The data presented here suggests some non-originator insulin glargine product lots drive heightened in vitro human innate activity and provides preliminary evidence that changes in the biochemical composition of non-originator insulin glargine products (dimers, impurities) might be responsible for their greater immunostimulatory potential.