In vivo and in vitro evidence that chronic activation of the hexosamine biosynthetic pathway interferes with leptin-dependent STAT3 phosphorylation.

We previously reported that a 2-day peripheral infusion of glucosamine caused leptin resistance in rats, suggesting a role for the hexosamine biosynthetic pathway (HBP) in the development of leptin resistance. Here we tested leptin responsiveness in mice in which HBP activity was stimulated by offering 30% sucrose solution in addition to chow and water or by infusing glucosamine. Mice were leptin resistant after 33 days of access to sucrose. Resistance was associated with increased activity of the HBP and with phosphorylation of transcription factor signal transducer and activator of transcription-3 Tyr705 [pSTAT3(Y705)] but inhibition of suppressor of cytokine signaling 3 in the liver and hypothalamus. Intravenous infusion of glucosamine for 3 h stimulated pSTAT3(Y705) but prevented leptin-induced phosphorylation of STAT3(S727). In an in vitro system, glucose, glucosamine, and leptin each dose dependently increased O-linked β-N-acetylglucosamine (O-GlcNAc) protein and pSTAT3(Y705) in HepG2 cells. To test the effect of glucose on leptin responsiveness cells were incubated in 5.5 mM (LG) or 20 mM (HG) glucose for 18 h and were treated with 0 or 50 ng/ml leptin for 15 min. HG alone and LG + leptin produced similar increases in O-GlcNAc protein, glutamine fructose-6-phosphate amidotransferase (GFAT), and pSTAT3(Y705) compared with LG media. Leptin did not stimulate these proteins in HG cells, suggesting leptin resistance. Leptin-induced pSTAT3(S727) was prevented by HG media. Inhibition of GFAT with azaserine prevented LG + leptin and HG stimulation of pSTAT3. These data demonstrate development of leptin resistance in sucrose-drinking mice and provide new evidence of leptin-induced stimulation of the HBP.