Hexosamine biosynthesis pathway ( HBP )

27 There is increasing evidence that endoplasmic reticulum (ER) stress contributes to the 28 development atherosclerosis in diabetes mellitus. The purpose of this study was to determine the effects 29 of increased hexosamine biosynthesis pathway (HBP) flux on ER stress levels and the complications of 30 ER stress associated with diabetes and atherosclerosis in hepatic cells. Glutamine:fructose-6-phosphate 31 amidotransferase (GFAT), the rate limiting enzyme of the HBP, was overexpressed in HepG2 cells using 32 an adenoviral expression system. The ER stress response and downstream effects, including activation of 33 lipid and inflammatory pathways, were determined using real-time PCR, immunoblot analysis, and cell 34 staining techniques. GFAT overexpression resulted in increased expression of ER stress markers 35 including Grp78, Grp94, calreticulin, and GADD153, relative to cells infected with an empty adenoviral 36 vector. In addition, GFAT overexpression promoted lipid, but not cholesterol, accumulation in hepatic 37 cells as well as inflammatory pathway activation. Treatment with 6-diazo-5-oxo norleucine (DON), a 38 GFAT antagonist, blocked the effects of GFAT overexpression. Consistent with our in vitro data, 39 hyperglycemic mice presented with elevated markers of hepatic ER stress, glucosamine and lipid 40 accumulation. Together, these data suggest that HBP flux-induced ER stress plays a role in the 41 development of hepatic steatosis and atherosclerosis under conditions of hyperglycemia. 42