Activation of hexosamine pathway impairs nitric oxide (NO)-dependent arteriolar dilations by increased protein O-GlcNAcylation

Abstract

We hypothesized that under high glucose conditions, activation of the hexosamine pathway leads to impaired nitric oxide (NO)-dependent arteriolar dilation. Skeletal muscle arterioles (diameter: ~160μm) isolated from male Wistar rats were exposed to normal glucose (NG, 5.5mmol/L) or high glucose concentrations (HG, 30mmol/L, for 2h) and agonist-induced diameter changes were measured with videomicroscopy. Western blots were performed to identify the vascular levels of protein O-linked-N-acetyl-glucosamine (O-GlcNAc) and phosphorylated endothelial NO synthase (eNOS). In arterioles exposed to HG, dilations to histamine were abolished compared to those exposed to NG (max: -6±6% and 69±9%, respectively), while acetylcholine-induced responses were not affected. Inhibition of NO synthesis with N(G)-nitro-l-arginine methyl ester (L-NAME) reduced histamine-induced dilations in NG arterioles, but it had no effect on microvessels exposed to HG. Dilations to the NO donor, sodium nitroprusside and constrictions to norepinephrine and serotonin were similar in the two groups. In the presence of the inhibitor of hexosamine pathway, azaserine, histamine-induced dilations were significantly augmented in arterioles exposed to HG (max: 67±2%). Moreover, exposure of vessels to glucosamine (5mmol/L, for 2h) resulted in reduced histamine-induced arteriolar dilations (max: 26±3%). The level of protein O-GlcNAcylation was increased, whereas the P-eNOS (Ser-1177) was decreased in HG exposed vessels. These findings indicate that a high concentration of glucose may lead to glucosamine formation, which impairs histamine-induced, NO-mediated arteriolar dilations. We propose that interfering with the hexosamine pathway may prevent microvascular complications in diabetes.

Fig. 1

Percent changes in diameter of gracilis muscle arterioles in response to cumulative concentration of histamine (1 nmol/L–10 μmol/L, Panels A, C and E) and acetylcholine (ACh, 1 nmol/L–1 μmol/L, Panels B, D and F) in the presence of normal (5.5 mmol/L) or high glucose concentrations (30 mmol/L) and in the absence or in the presence of NO synthase inhibitor L-NAME (100 μM). Data are in means ± S.E.M, significant differences: * P≤0.05.

Fig. 2

Percent changes in diameter of gracilis muscle arterioles in response to cumulative concentration of sodium nitroprusside (SNP, 1 nmol/L–1 μmol/L) (Panel A), norepinephrine (NE, 0.3 nmol/L–0.1 μmol/L) (Panel B) and serotonine (0.1 nmol/L–1 μmol/L) (Panel C) in the presence of normal (5.5 mmol/L) or high glucose concentration (30 mmol/L). Data are in means ± S.E.M, significant differences: * P<0.05.

Fig. 3

Percent changes in diameter of gracilis muscle arterioles in response to cumulative concentration of histamine (1 nM–10 μM) in the presence of high glucose concentration before and after incubations with GFAT inhibitor, azaserine (20 μmol/L) or in combination with NO synthase inhibitor, L-NAME (Panel A). Percent changes in diameter of gracilis muscle arterioles in the presence of glucosamine (5 mmol/L) and in the simultaneous presence of glucosamine and the GFAT inhibitor, azaserine (Panel B). Data are in means±S.E.M, significant differences: * P<0.05.

Fig. 4

Representative Western blots (Panel A) and summary data (Panel B) show O-GlcNac formation in skeletal muscle arteries exposed to normal (5.5 mmol/L), high glucose concentration (30 mmol/L) and also to glucosamine (5 mmol/L). Western blots (Panel C) and summary data (Panel D and E) of normalized densitometry ratios from four independent experiments show O-GlcNac, eNOS and P-eNOS-Ser1177 levels. β-actin was used for normalizing of loading variations.

Fig. 5

Schematic draw demonstrates that increased flux through the hexosamine biosynthetic pathway leads to enhanced O-GlcNAcylation of eNOS in condition of high glucose, which interferes with eNOS-Ser1177 phosphorylation by Akt to reduce histamine-induced, NO-dependent dilation of skeletal muscle arterioles. GFAT: glutamine fructose-6-phosphate amidotransferase, O-GlcNAc: O-linked attachment of N-acetyl-glucosamine, OGT: O-linked N-acetylglucosaminyl-transferase.