Renal hypertrophy is the first renal structural abnormality in diabetes and involves both glomerular and tubular cells. The mechanism of high glucose-induced hypertrophy of GECs is not well understood. We examined the hypothesis that AMP kinase, a cellular energy sensor, is involved in glucose-induced hypertrophy of the GECs. Incubation of GECs with 30 mM glucose, but not equimolar mannitol, resulted in significant increase in de novo protein synthesis on days 3 and 6, as measured by incorporation of 35S-methionine into protein. High glucose also increased protein content per cell, indicating hypertrophy. Pre-incubation with LY294002, a PI 3-kinase inhibitor, inhibited high glucose-induced protein synthesis. As the initiation phase of protein translation is the rate-limiting step in protein synthesis, we examined phosphorylation of eukaryotic initiation factor 4E (eIF4E) binding protein (4E-BP1), an important step regulated by PI 3-kinase. Phosphorylation of 4E-BP1 is necessary to release eIF4E from its complex with BP-1 so that the latter can bind to mRNA cap and promote translation. In association with glucose-induced hypertrophy on days 3 and 6, there was a significant increase in phosphorylation of 4E-BP1. We also examined if high glucose regulated the elongation phase of protein translation during which amino acids are added in accordance with the codon sequences on the mRNA. This is under the control of eukaryotic elongation factor (eEF2) that is active in the dephosphorylated state. Immunoblotting revealed a reduction in phosphorylation of eEF2 on days 3 and 6, coinciding with high glucose-induced protein synthesis. Glucose regulates the energy state of the cell (AMP and ATP content); reduction in AMP reduces phosphorylation of AMP kinase. Phosphorylation of AMP kinase was reduced in association with high glucose-induced hypertrophy. Metformin induced phosphorylation of AMP kinase and fully prevented high glucose-induced protein synthesis when incubated for the last 24 hours on day 3 and 6 of high glucose exposure. We conclude (1) high glucose promotes protein synthesis and hypertrophy of GECs that is dependent on PI 3-kinase activity; (2) high glucose regulates both the initiation and elongation phases of protein translation; (3) high glucose-induced protein synthesis is associated with reduction in AMP kinase phosphorylation; stimulation of AMP kinase with metformin inhibits high glucose-induced increase in protein synthesis. AMP kinase, an energy sensor of the cell, modulates high glucose-induced protein synthesis and hypertrophy in the GECs.