High glucose, high insulin, and their combination rapidly induce laminin-β1 synthesis by regulation of mRNA translation in renal epithelial cells

Laminin is a glycoprotein that contributes to renal extracellular matrix expansion in diabetes. We investigated regulation of laminin-β1 synthesis in murine renal proximal tubular epithelial cells by 30 mmol/l glucose (high glucose), 1 nmol/l insulin (high insulin), and their combination (high glucose+high insulin), simulating conditions observed during progression of type 2 diabetes. Compared with 5 mmol/l glucose and no insulin (control), high glucose alone, high insulin alone, or high glucose+high insulin together increased laminin-β1 chain protein synthesis within 5 min, lasting for up to 60 min with no change in laminin-β1 mRNA levels. Cycloheximide, but not actinomycin-D, abrogated increased laminin-β1 synthesis. High glucose, high insulin, and high glucose+high insulin stimulated phosphorylation of 4E-BP1, a repressor binding protein for eukaryotic initiation factor 4E (eIF4E), that was dependent on activation of phosphatidylinositol 3-kinase, Akt, and mammalian target of rapamycin. High glucose, high insulin, and high glucose+high insulin also promoted release of eIF4E from 4E-BP1, phosphorylation of eIF4E, and increase in eIF4E association with eIF4G, critical events in the initiation phase of mRNA translation. High glucose, high insulin, and high glucose+high insulin increased Erk phosphorylation, which is an upstream regulator of eIF4E phosphorylation, and PD098059, which is a MEK inhibitor that blocks Erk activation, abolished laminin-β1 synthesis. This is the first demonstration of rapid increment in laminin-β1 synthesis by regulation of its mRNA translation by cells exposed to high glucose, high insulin, or high glucose+high insulin.