Norbert Fülöp

Aging leads to increased levels of protein O-linked N-acetylglucosamine in heart, aorta, brain and skeletal muscle in Brown-Norway rats

Changes in the levels of O-linked N-acetyl-glucosamine (O-GlcNAc) on nucleocytoplasmic protein have been associated with a number of age-related diseases such as Alzheimer’s and diabetes; however, there is relatively little information regarding the impact of age on tissue O-GlcNAc levels. Therefore, the goal of this study was to determine whether senescence was associated with alterations in O-GlcNAc in heart, aorta, brain and skeletal muscle and if so whether there were also changes in the expression of enzymes critical in regulating O-GlcNAc levels, namely, O-GlcNAc transferase (OGT), O-GlcNAcase and glutamine:fructose-6-phosphate amidotransferase (GFAT). Tissues were harvested from 5- and 24-month old Brown-Norway rats; UDP-GlcNAc, a precursor of O-GlcNAc was assessed by HPLC, O-GlcNAc and OGT levels were assessed by immunoblot analysis and GFAT1/2, OGT, O-GlcNAcase mRNA levels were determined by RT-PCR. In the 24-month old animals serum insulin and triglyceride levels were significantly increased compared to the 5-month old group; however, glucose levels were unchanged. Protein O-GlcNAc levels were significantly increased with age (30–107%) in all tissues examined; however, paradoxically the expression of OGT, which catalyzes O-GlcNAc formation, was decreased by ∼30% in the heart, aorta and brain. In the heart increased O-GlcNAc was associated with increased UDP-GlcNAc levels and elevated GFAT mRNA while in other tissues we found no difference in UDP-GlcNAc or GFAT mRNA levels. These results demonstrate that senescence is associated with increased O-GlcNAc levels in multiple tissues and support the notion that dysregulation of pathways leading to O-GlcNAc formation may play an important role in the development of age-related diseases.

Acute regulation of cardiac metabolism by the hexosamine biosynthesis pathway and protein O-GlcNAcylation.

Objective The hexosamine biosynthesis pathway (HBP) flux and protein O-linked N-acetyl-glucosamine (O-GlcNAc) levels have been implicated in mediating the adverse effects of diabetes in the cardiovascular system. Activation of these pathways with glucosamine has been shown to mimic some of the diabetes-induced functional and structural changes in the heart; however, the effect on cardiac metabolism is not known. Therefore, the primary goal of this study was to determine the effects of glucosamine on cardiac substrate utilization. Methods Isolated rat hearts were perfused with glucosamine (0–10 mM) to increase HBP flux under normoxic conditions. Metabolic fluxes were determined by 13C-NMR isotopomer analysis; UDP-GlcNAc a precursor of O-GlcNAc synthesis was assessed by HPLC and immunoblot analysis was used to determine O-GlcNAc levels, phospho- and total levels of AMPK and ACC, and membrane levels of FAT/CD36. Results Glucosamine caused a dose dependent increase in both UDP-GlcNAc and O-GlcNAc levels, which was associated with a significant increase in palmitate oxidation with a concomitant decrease in lactate and pyruvate oxidation. There was no effect of glucosamine on AMPK or ACC phosphorylation; however, membrane levels of the fatty acid transport protein FAT/CD36 were increased and preliminary studies suggest that FAT/CD36 is a potential target for O-GlcNAcylation. Conclusion/Interpretation These data demonstrate that acute modulation of HBP and protein O-GlcNAcylation in the heart stimulates fatty acid oxidation, possibly by increasing plasma membrane levels of FAT/CD36, raising the intriguing possibility that the HBP and O-GlcNAc turnover represent a novel, glucose dependent mechanism for regulating cardiac metabolism.

Evidence of O-linked N-acetylglucosamine in diabetic nephropathy

AIMS There is increasing evidence that O-linked N-acetylglucosamine (O-GlcNAc) plays an important role in cell signaling pathways. It has also been reported that increases in O-GlcNAc contribute to the development of diabetes and diabetic complications; however, little is known about O-GlcNAc levels in diabetic nephropathy (DNP). Therefore the goal of this study was to determine whether O-GlcNAc could be detected in human kidney biopsy specimens, and if so to examine whether O-GlcNAc levels were increased in the kidneys of patients with DNP compared to the non-diabetic individuals. MAIN METHODS Kidney biopsy specimens were obtained from type-2 diabetic patients (n=6) and patients diagnosed with thin basement membrane nephropathy (n=7) were used as non-diabetic controls. O-GlcNAc levels were assessed by immunohistochemistry using the anti-O-GlcNAc antibody CTD110.6. KEY FINDINGS We show that O-GlcNAc modification of proteins can be detected in the human kidney biopsy specimens. Furthermore, in diabetic patients, we found significantly increased numbers of O-GlcNAc positive cells in the glomeruli and significantly elevated staining in the tubuli (both in the nucleus and in the cytosol). In addition we also observed an intense, granular O-GlcNAc staining specifically in diabetic tubuli. SIGNIFICANCE In light of the increase in O-GlcNAc staining in the diabetic patients, we propose that increased O-GlcNAc levels might contribute to the development of diabetic nephropathy.

Glucosamine administration improves survival rate after severe hemorrhagic shock combined with trauma in rats.

We have previously shown that glucosamine administration resulted in higher cardiac output and improved tissue perfusion after trauma-hemorrhage with resuscitation in rats, which was associated with the increased levels of protein O-linked-N-acetylglucosamine (O-GlcNAc). The purpose of the study was to evaluate the effect of glucosamine on the survival, without resuscitation, in rats. Adult male rats underwent midline laparotomy and 55% of total blood volume was withdrawn for 25 min under isoflurane anesthesia. At the end of the hemorrhage period, 2.5 mL of 150 mM glucosamine or equivalent osmolarity of mannitol solution was injected intravenously for 10 min. The survival time, mean blood pressure, heart rate, and central body temperature were monitored continuously; then, the O-GlcNAc levels in heart, brain, liver, and muscle were measured by means of Western blot analysis. Glucosamine administration significantly increased the survival rate in comparison with mannitol administration (percentage of survival after 2 h, 47% vs. 20%; P < 0.05). The mean arterial pressure was significantly higher in the glucosamine group for 18 min after treatment. The protein O-GlcNAc levels, assessed 30 min after glucosamine treatment, were significantly increased in the heart, brain, and liver. These data demonstrate that i.v. glucosamine administration improves the survival rate after trauma-hemorrhage without resuscitation; this effect may be related to the glucosamine-induced increase in protein O-glycosylation. Furthermore, the increase in mean arterial pressure may suggest a vasoactive and/or positive inotropic effect of glucosamine in hypovolemic shock.