Influence of Glucosamine on Glomerular Meseangial Cell 3 Turnover: Implications for Hyperglycemia and Hexosamine Pathway

22 Cells exposed to high glucose may undergo hypertrophy, proliferation, and apoptosis, but 23 the role of hexosamine flux in mediating these effects has not been fully elucidated. Accordingly, 24 we studied the effects of glucose and glucosamine on rat glomerular mesangial cells (MC) 25 turnover. In comparison with physiologic glucose (5.6mM), treatment with high glucose (25mM) 26 for 24 hours stimulated MC proliferation, an effect that was mimicked by exposure to low 27 concentrations of glucosamine (0.05mM). The percentage of cells in G0/G1 phase of the cell cycle 28 was reduced with a concomitant increase of the number of cells in G2/M phase. Proliferating cell 29 nuclear antigen (PCNA), phosphorylated mammalian target of rapamycin (phospho-mTOR 30 [ser2448]) and total regulatory associated protein of mTOR (Raptor) were increased by high 31 glucose and glucosamine treatment. Inhibition of glutamine:fructose-6-phosphate amidotransferase 32 (GFAT), the rate-limiting enzyme for hexosamine flux, with 6-diazo-5-oxonorleucine (DON, 33 10μM) and of mTOR with rapamycin both attenuated glucose-mediated MC proliferation. Higher 34 glucosamine concentrations (0.25mM to 10mM) caused MC apoptosis after 48 hours and, in 35 addition, GFAT overexpression also increased MC apoptosis (TUNEL-positive cells: 3.8±0.3 % 36 versus 1.1±0.2% for empty vector; p<0.001). Hence, hexosamine flux is an important determinant 37 of MC proliferation and apoptosis. The proliferative response to high glucose and hexosamine flux 38 is rapamycin-sensitive, suggesting that this effect is associated with signaling through rapamycin39 sensitive mTOR complex 1 (mTORC1). 40 41 42