Our aim was to investigate the cellular effects of high glucose, high insulin and their combination on laminin beta-1 synthesis in MCT cells, which form the bulk of the kidney cortex. The three incubation conditions mimic their respective levels observed during the progression of type II diabetes. High glucose (30 mM) alone, high insulin (1 nM) alone, and high glucose and high insulin together significantly increased laminin beta1 protein in both cell lysates (p < .05) and medium (p < .05) within 5 minutes, the effect lasting for up to 30 minutes as compared to the cells in low glucose (5 mM). High mannitol (30 mM) did not have any effect. Actinomycin D and DRB, two inhibitors of transcription, did not affect laminin response to glucose or insulin, suggesting a nontranscriptional mechanism, i.e., translation. Protein translation is promoted by mRNA cap binding protein, eukaryotic initiation factor 4E (eIF4E), which is normally held inactive by its binding protein 4E-BP1 and released after phosphorylation of the latter. High glucose and high insulin, alone or in combination, stimulated phosphorylation of 4E-BP1, coinciding with laminin synthesis. This was associated with dephosphorylation of eEF2, which promotes elongation of nascent peptide chain. The three conditions activated Akt, an upstream regulator of mTOR and p70 S6 kinase, an mTOR substrate. mTOR is the upstream kinase that regulates 4E-BP1 phosphorylation. LY294002, a PI3-kinase inhibitor, and rapamycin, an mTOR inhibitor, blocked laminin synthesis induced by high glucose, high insulin or both together. Transfection of MCT cells with dominant negative constructs of Akt and mTOR abolished laminin synthesis. Glucose, insulin and their combination increased phosphorylation of Erk1/2 MAP kinase, which regulates eIF4E phosphorylation, and PD098059, a MEK inhibitor, abolished laminin synthesis induced by the three conditions. Our data show that exposure of MCT cells to high glucose, high insulin or both together (1) rapidly increases laminin beta-1 synthesis, (2) activates factors involved in initiation and elongation phase of protein translation, (3) recruits PI 3-kinase-Akt-mTOR pathway for regulating 4E-BP1, and (4) stimulates Erk phosphorylation required for eIF4E phosphorylation. These observations project for the first time the involvement of translation as a mechanism for glucose-, insulin- and their combination-induced rapid phase laminin synthesis in MCT cells.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.