Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13 C]glucose, [U-13 C]lactate, [2-13 C]-pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13 C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented Ͼ95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between ϳ10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism. substrate metabolism; carbohydrates; fatty acids THE PREVAILING VIEW of cardiac energy production is that long-chain fatty acids are the primary oxidative energy source, followed to a lesser extent by glucose use, with other substrates such as lactate playing a much smaller role. This is despite the fact that the myocardium is capable of utilizing a wide variety of fuels for oxidative energy production. At rest, circulating lactate concentrations are ϳ1 mM (Table 1) and during moderate exercise can easily reach up to 3-5 mM (37). More than 20 years ago, Drake et al. (20) showed that the uptake of lactate by the heart in vivo is directly proportional to its serum concentration. In addition, it has been shown that in the isolated perfused heart, lactate contributes significantly to acetyl-CoA formation, often contributing more than glucose (9,10,35). In addition, serum pyruvate is typically in the range of 0.1-0.2 mM (Table 1), and, while this is relatively low, pyruvate is readily taken up and oxidized by the heart (36, 40, 45) and thus could contribute significantly to overall oxidative energy production in vivo.There is a resurgence of interest in the regulation of cardiac metabolism, driven, at least in part, by the manipulation of cardiac metabolism as a potential ...

Changes in the levels of O-linked N-acetylglucosamine (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.

Laczy B, Marsh SA, Brocks CA, Wittmann I, Chatham JC. Inhibition of O-GlcNAcase in perfused rat hearts by NAG-thiazolines at the time of reperfusion is cardioprotective in an O-GlcNAc-dependent manner. Acute increases in O-linked ␤-N-acetylglucosamine (O-GlcNAc) levels of cardiac proteins exert protective effects against ischemiareperfusion (I/R) injury. One strategy to rapidly increase cellular O-GlcNAc levels is inhibition of O-GlcNAcase (OGA), which catalyzes O-GlcNAc removal.Here we tested the cardioprotective efficacy of two novel and highly selective OGA inhibitors, the NAGthiazoline derivatives NAG-Bt and NAG-Ae. Isolated perfused rat hearts were subjected to 20 min global ischemia followed by 60 min reperfusion. At the time of reperfusion, hearts were assigned to the following four groups: 1) untreated control; 2) 50 M NAG-Bt; 3) 100 M NAG-Bt; or 4) 50 M NAG-Ae. All treatment groups significantly increased total O-GlcNAc levels (P Ͻ 0.05 vs. control), and this was significantly correlated with improved contractile function and reduced cardiac troponin I release (P Ͻ 0.05). Immunohistochemistry of normoxic hearts showed intense nuclear O-GlcNAc staining and higher intensity at Z-lines with colocalization of OGlcNAc and the Z-line proteins desmin and vinculin. After I/R, there was a marked loss of both cytosolic and nuclear O-GlcNAcylation and disruption of normal striated Z-line structures. OGA inhibition largely preserved structural integrity and attenuated the loss of OGlcNAcylation; however, nuclear O-GlcNAc levels remained low. Immunoblot analysis confirmed ϳ50% loss in both nuclear and cytosolic O-GlcNAcylation following I/R, which was significantly attenuated by OGA inhibition (P Ͻ 0.05). These data provide further support for the notion that increasing cardiac O-GlcNAc levels by inhibiting OGA may be a clinically relevant approach for ischemic cardioprotection, in part, by preserving the integrity of O-GlcNAcassociated Z-line protein structures.is increasingly recognized as a critical signaling mechanism regulating a diverse range of biological processes in mammalian cells (3,19,44). This attachment of O-GlcNAc to Ser/Thr residues of proteins is frequently considered to be analogous to protein phosphorylation in that it is a highly dynamic, reversible, and tightly regulated enzyme-catalyzed process. Sustained activation of the hexosamine biosynthesis pathway (HBP) and the resulting increase in O-GlcNAcylation has been implicated in the etiology of glucotoxicity and insulin resistance. However, there is rapidly emerging evidence demonstrating that acute activation of these pathways affords protection against a wide range of injury, including cardioprotection against ischemia-reperfusion (I/R) injury in different biological systems (4, 5, 17, 21).We have previously reported that pretreatment with high glucose or glucosamine attenuated cell death following hypoxia-reoxygenation in isolated cardiomyocytes (4, 5). We also demonstrated that administration of glucosamine and glutamine before ischemia resul...

Objective We have previously shown that increasing protein O-linked β-N-acetylglucosamine (O-GlcNAc) levels by different mechanisms reduced inflammatory responses and improved organ function 2 hours after trauma-hemorrhage (T-H). The aim of the study was to evaluate the effects of O-GlcNAc levels on survival, inflammation and organ damage 24 hours after T-H. Design Prospective, randomized, controlled study. Setting Animal research laboratory. Subjects Male, adult Sprague-Dawley rats. Interventions Overnight fasted animals were subjected to either sham surgery (SH) or (T-H) and during the resuscitation phase received glucosamine (270 mg/Kg, GlcN) to increase O-GlcNAc synthesis or O-(2-Acetamido-2-deoxy-D-glucopyranosylidene)amino N-phenyl Carbamate, (7mg/Kg, PUGNAc) to inhibit O-GlcNAc removal, or mannitol as control (CON). Measurements and main results Survival was followed up for 24 hours. Surviving rats were euthanized and inflammatory responses, and end organ injuries were assessed. Both GlcN and PUGNAc increased 24 hours survival compared to controls (CON: 53%, GN: 85%, PUGNAc: 86%, logrank test, p<0.05). PUGNAc attenuated the T-H induced increase in serum IL-6 (SH: 8±6, CON: 181±36, PUGNAc: 42±22 pg/mL, p<0.05), ALT (SH: 95±14, CON: 297±56, PUGNAc: 126±21 IU, p<0.05), AST (SH: 536±110, CON: 1661±215, PUGNAc: 897±155 IU, p<0.05) and LDH (SH: 160±18, CON: 1499±311, PUGNAc: 357±99 IU, p<0.05); however, GlcN had no effect on these serum parameters. Furthermore, PUGNAc but not GlcN maintained O-GlcNAc levels in liver and lung and significantly attenuated the NF-κB DNA activation in the liver. In the liver and heart, increased iNOS expression was also attenuated in the PUGNAc treated group. Conclusions These results demonstrate that increasing O-GlcNAc with either GlcN or PUGNAc improved 24 hour survival after T-H. However, only PUGNAc treatment attenuated significantly the subsequent tissue injury and inflammatory responses, suggesting that inhibition of O-GlcNAc removal may represent a new therapeutic approach for the treatment of hypovolemic shock.

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.

Increased O‐linked‐N‐acetylglucosamine (O‐GlcNAc) levels of cardiac proteins afford protection against ischemia‐reperfusion (I/R) injury. One strategy to increase cellular O‐GlcNAc levels is inhibition of O‐GlcNAcase, which catalyzes O‐GlcNAc removal. Here we tested the cardioprotective efficacy of two novel and highly selective O‐GlcNAcase inhibitors, NAG‐Bt (Bt) and NAG‐Ae (Ae). Isolated perfused rat hearts were subjected to 20 min no‐flow ischemia followed by 60 min reperfusion. At the time of reperfusion hearts were assigned to 4 groups 1) untreated control (CON); 2) 50µM Bt; 3) 100µM Bt or 50µM Ae. All treatment groups significantly increased O‐GlcNAc levels and improved functional recovery compared to CON group. O‐GlcNAc increased in a dose dependent manner with Bt and was highest in Ae group. There was a strong correlation between O‐GlcNAc, improved functional recovery and reduced troponin I release. Immunohistochemistry of non‐ischemic hearts showed higher O‐GlcNAc intensity at the Z‐lines and intense nuclear staining. There was a marked loss of nuclear O‐GlcNAc and disruption of the striated Z‐line structures following I/R. Bt treated hearts exhibited less structural disruption and higher overall O‐GlcNAc. These data further support the notion that increasing cardiac O‐GlcNAc by inhibition of O‐GlcNAcase may be a clinically relevant approach for ischemic cardioprotection. (NIH HL079364)

We have shown that O‐GlcNAc (N‐acetyl‐glucosamine) agonists improve survival and reduce inflammatory response 2 hours after trauma‐hemorrhage (T‐H). The aim of the study was to evaluate the effects of O‐GlcNAc levels on survival, inflammation and organ damage 24 hours after T‐H. Fasted male rats were subjected to either sham surgery (SH) or (T‐H) and during the resuscitation phase received glucosamine (0.64g/kg, GN) or O‐(2‐Acetamido‐2‐deoxy‐D‐glucopyranosylidene)amino N‐phenyl Carbamate, (7mg/kg , PUGNAc) to increase O‐GlcNAc levels, or mannitol as control (CON). Survival was followed up for 24 hours. Both GN and PUGNAc increased 24 hours survival compared to controls (CON: 53%, GN: 85%, PUGNAc: 86%, logrank test, p<0.05). PUGNAc attenuated the T‐H induced increase in serum IL‐6 (SH: 8±6, CON: 181±36, PUGNAc: 42±22 pg/mL, p<0.05), ALT (SH: 89±19, CON: 297±56, PUGNAc: 125±21 IU, p<0.05), AST (SH: 503±218, CON: 1661±215, PUGNAc: 897±155 IU, p<0.05) and LDH (SH: 129±26, CON: 1499±311, PUGNAc: 357±99 IU, p<0.05); however, GN had no effect on serum parameters. PUGNAc but not GN maintained O‐GlcNAc levels in liver and lung and also attenuated the enhanced NF‐κB DNA binding and iNOS expression in the liver. These results demonstrate that increasing O‐GlcNAc levels with GN and PUGNAc improves 24 hr survival after T‐H; the different effects of GN and PUGNAc on tissue injury require further studies. NIH grant HL076165.

We have shown that increasing the level of O‐linked‐N‐acetylglucosamine (O‐GlcNAc) on proteins protects the heart against ischemia‐reperfusion injury; however, the specific proteins altered by O‐GlcNAc have yet to be determined. Isolated perfused hearts from male rats (n=5–9/groups) were subjected to 10 min no‐flow ischemia (ISC) or ISC followed by 60 min reperfusion (I/R). Overall protein O‐GlcNAc levels, determined by anti‐O‐GlcNAc immunoblots, were increased following ISC (p<0.05) compared to normoxic controls. Using MALDI‐TOF we identified proteins in two bands in which O‐GlcNAc levels significantly increased during ISC and subsequently decreased following I/R and one band that increased only following I/R. The proteins in the O‐GlcNAc positive bands that increased during ISC were identified as glycogen phosphorylase b and mitochondrial aconitase. The protein in the O‐GlcNAc band that increased with I/R was identified as the cytoskeletal protein vinculin. Vinculin and aconitase were subsequently immunoprecipitated and both were found to be O‐GlcNAc positive by immunoblot analysis. Further studies are needed to determine the effect of O‐GlcNAc on the function of these proteins and how this influences the response to I/R. NIH grants: HL067464 and HL079364.