Effect of Denervation on XBP1 in Skeletal Muscle and the Neuromuscular Junction.

Denervation of skeletal muscle is a debilitating consequence of injury of the peripheral nervous system, causing skeletal muscle to experience robust atrophy. However, the molecular mechanisms controlling the wasting of skeletal muscle due to denervation are not well understood. Here, we demonstrate that transection of the sciatic nerve in Sprague-Dawley rats induced robust skeletal muscle atrophy, with little effect on the neuromuscular junction (NMJ). Moreover, the following study indicates that all three arms of the unfolded protein response (UPR) are activated in denervated skeletal muscle. Specifically, ATF4 and ATF6 are elevated in the cytoplasm of skeletal muscle, while XBP1 is elevated in the nuclei of skeletal muscle. Moreover, XBP1 is expressed in the nuclei surrounding the NMJ. Altogether, these results endorse a potential role of the UPR and, specifically, XBP1 in the maintenance of both skeletal muscle and the NMJ following sciatic nerve transection. Further investigations into a potential therapeutic role concerning these mechanisms are needed.

Inhibiting the Hexosamine Biosynthetic Pathway Lowers O-GlcNAcylation Levels and Sensitizes Cancer to Environmental Stress.

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

Azide- and Alkyne-Bearing Metabolic Chemical Reporters of Glycosylation Show Structure-Dependent Feedback Inhibition of the Hexosamine Biosynthetic Pathway.

Metabolic chemical reporters (MCRs) of protein glycosylation are analogues of natural monosaccharides that bear reactive groups, like azides and alkynes. When they are added to living cells and organisms, these small molecules are biosynthetically transformed into nucleotide donor sugars and then used by glycosyltransferases to modify proteins. Subsequent installation of tags by bioorthogonal chemistries can then enable the visualization and enrichment of these glycoproteins. Although this two-step procedure is powerful, the use of MCRs has the potential to change the endogenous production of the natural repertoire of donor sugars. A major route for the generation of these glycosyltransferase substrates is the hexosamine biosynthetic pathway (HBP), which results in uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Interestingly, the rate-determining enzyme of the HBP, glutamine fructose-6-phosphate amidotransferase (GFAT), is feedback inhibited by UDP-GlcNAc. This raises the possibility that a build-up of UDP-MCRs would block the biosynthesis of UDP-GlcNAc, resulting in off target effects. Here, we directly test this possibility with recombinant human GFAT and a small panel of synthetic UDP-MCRs. We find that MCRs with larger substitutions at the N-acetyl position do not inhibit GFAT, whereas those with modifications of the 2- or 6-hydroxy group do. These results further illuminate the considerations that should be applied to the use of MCRs.

Hexosamine biosynthesis pathway flux promotes endoplasmic reticulum stress, lipid accumulation, and inflammatory gene expression in hepatic cells.

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