The details of the hexosamine biosynthesis pathway and its role in metabolism and development can be found in the Glycoproteins page.
Numerous proteins involved in insulin signaling and the downstream targets of these signaling cascades have been shown to be O-GlcNAcylated. With respect to insulin receptor signaling proteins, IRS-1, PI3K, PKB/AKT, PDK1, and GSK3β are all known to be O-GlcNAcylated. These modifications have all been observed in adipocytes which are a major target for the actions of insulin. Insulin-stimulated glucose uptake into adipocytes occurs via insulin-mediated mobilization of GLUT4 to the plasma membrane. Increased glucose uptake, in response to insulin, can therefore, significantly modify the rate of flux through the HBP. Evidence linking the correlation between the HBP and insulin resistance in adipocytes was demonstrated at least 20 years ago. Using cultured rat adipocytes experiments demonstrated that chronic exposure to both insulin and glucose was required for the adipocytes to become insulin-resistant. This is now a common theme underlying insulin resistance in other insulin-sensitive tissues such as skeletal muscle. In these early experiments is was shown that the impairment in insulin-stimulated glucose uptake, under hyperglycemic and hyperinsulinemic conditions, was exclusively dependent on the presence of the amino acid glutamine. Remember that glutamine is required as a substrate for GFAT, the rate-limiting enzyme in the HBP. Inhibition of GFAT activity was observed in the hyperglycemic and hyperinsulinemic conditions likely due to feedback inhibition by UDP-GlcNAc as the HBP product was shown to accumulate in the treated cells. However, if GFAT was inhibited with the use of various amidotransferase inhibitors the hyperglycemia-induced insulin resistance was prevented. Additionally, if cells are treated with glucosamine, which enters the HBP after the GFAT catalyzed reaction, there was a greater reduction in insulin-mediated glucose uptake compared to the hyperglycemic condition. As expected, since GFAT is bypassed, the glucosamine-induced insulin resistance does not require glutamine. Although glucose and glutamine metabolism are key inducers of the flux through the HBP, free fatty acids (FFA) and uridine are also potent modulators of the HBP.
Utilizing experiments in whole animals, as opposed to cell culture, has provided additional direct evidence that excess flux through the HBP leads to modulation of insulin sensitivity in adipocytes. When GFAT is overexpressed in mice under the control of a GLUT4 promoter the animals develop classical insulin-resistant phenotype with hyperinsulinemia and reduction in whole-body glucose disposal rate. Because GLUT4 is highly expressed in adipose tissue and skeletal muscle, two major insulin-responsive tissues, it is not surprising that defective whole-body glucose disposal was observed. Elevation in serum leptin level was also observed in these GFAT overexpressing mice. Interestingly, muscle explants from GLUT4-GFAT mice showed normal insulin-stimulated glucose uptake. This latter observation is strong evidence that adipocytes play a major regulatory role in the HBP-mediated whole-body insulin resistance.
Another strain of mice has been utilized for studies on the role of HBP in insulin sensitivity that express GFAT specifically in adipose tissue by the use of an aP2 (adipocyte lipid binding protein) promoter driving its expression. Adipose tissue-restricted elevations in O-GlcNAc levels are detected in these mice and this is associated the development of whole-body insulin resistance. The results in these animals is characterized by a reduction in both glucose disposal rate and skeletal muscle glucose uptake. An increase in serum leptin and a decrease in serum adiponectin levels were also found in these mice.
As pointed out above, numerous proteins downstream of the insulin receptor that are critical to insulin-mediated signal transduction are known to be O-GlcNAcylated. Therefore, it is not difficult to assume that HBP-mediated glucose desensitization will occur at multiple stages, in particular through insulin-mediated signal transduction. Under high glucose-induced insulin resistance, there is a reduction in insulin-stimulated phosphorylation of PKB/AKT. There has been some discrepancy in determining precisely how HBP flux affects PKB/AKT phosphorylation in response to insulin binding its receptor. Recent research has shown that when cells are exposed to chronically high glucose and insulin there is a concomitant reduction in PIP3 which is a product of activated PI3K, a target of the activated insulin receptor. This reduction in PIP3 levels is correlated with an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10) levels. PTEN is a known inhibitor of PI3K. In addition, it was shown that there is an increase in IRS-1 phosphorylation on Ser636 and Ser639. Since rapamycin treatment inhibits the alteration of PIP3 and PTEN levels under insulin-resistant conditions, it is believed that mammalian target of rapamycin complex 1 (mTORC1) is involved in negatively regulating the IRS-1/PI3K/Akt signaling cascade downstream of the insulin receptor. The sites on IRS-1 seen to be phosphorylated by chronic hyperglycemic and hypeinsulinemic conditions (S636/S639) are known to be substrates of mTORC1.
The regulation of insulin-stimulated GLUT4 translocation is also affected by changes in the flux rate through the HBP. Several cytoskeletal proteins involved in mobilization of GLUT4 to the plasma membrane are known to be O-GlcNAcylated. In addition, several of the proteins involved in the translocation process are targets of signaling proteins downstream of the insulin receptor. In cell culture models of both glucose- and glucosamine-induced insulin-resistance a reduction in the acute insulin-stimulated GLUT4 translocation is associated with a significant alteration in membrane redistribution of vesicle proteins such as t-(target membrane) SNARE, v-(vesicle membrane) SNARE and Munc18c (mammalian uncoordinated). SNARE stands for soluble-N-ethylmaleimide-sensitive factor attachment protein receptor. Munc18c is a negative regulator of both t- and v-SNAREs. Munc18c is known to be a target for O-GlcNAcylation. These results suggest a direct involvement of excess HBP flux in desensitizing the fusion between GLUT4-containing intracellular vesicles and the plasma membrane.
In addition to GLUT4 translocation, insulin-mediated PI3K and PKB/AKT activation also stimulates glycogen synthesis. The net effect is to balance the level of glucose metabolism in response to excess glucose influx. Insulin-dependent glycogen synthesis is mediated via the activation of of glycogen synthase (GS). Like other downstream targets of the insulin receptor, GS regulation involves a PKB/AKT-mediated inhibition of GSK3β which normally phosphorylates and inhibits GS. The insulin-stimulated increase in glycogen synthesis decreases the pool of G6P and subsequently F6P, thereby restricting flux through the HBP. PKB/AKT activation also leads to reduced dephosphorylation of GS via protein phosphatase 1 (PP1). Exposing cells to either high glucose or glucosamine results in a reduction in insulin-stimulated GS activity. Additionally, GS is a known O-GlcNAcylated protein and as might be expected it has been shown that GS becomes more resistant to dephosphorylation by PP1 under conditions of excess HBP flux.
While increased global O-GlcNAc levels are implicated in the development of insulin resistance, OGT is also regulated by insulin in adipocyte cell cultures. OGT is tyrosine-phosphorylated by the insulin receptor upon acute insulin stimulation and this phosphorylation increases the activity of the enzyme. In addition there is an observed shift in OGT localization from the nucleus to the cytosol in response to insulin stimulation. This OGT translocation to the plasma membrane is PI3K-dependant in response to acute insulin stimulation.
In summary, given that genetic and pharmacologic elevation in O-GlcNAc levels in cultured adipocytes and mouse models is associated with insulin-resistant phenotypes, it is likely that reducing O-GlcNAc levels in adipocytes should reverse the HBP-induced insulin resistance. A proof-of-concept experiment in transgenic mice (the insulin-resistant db/db mouse model which harbors a mutated leptin receptor) showed that overexpression of OGA, which reduces the level of O-GlcNAcylation, significantly improves whole-body glucose tolerance and insulin sensitivity. This result suggests that lowering O-GlcNAc levels in vivo should be of significant clinical beneficial.