Cardiac responses to hypoxia: the role of pyruvate dehydrogenase complex in carbohydrate utilisation

Accelerated carbohydrate utilisation and suppressed lipid oxidative metabolism are hallmarks of heart failure (HF). Hypoxia mimics failing heart metabolic reprogramming and has been suggested to play a role in cardiac metabolic switch. One aspect of the regulation of cardiac energy metabolism is the pyruvate dehydrogenase complex (PDC). Hypoxia inducible factor (HIF) signalling is thought regulate hypoxia-induced adaptations. HIF transcriptional activity is controlled by prolyl hydroxylase domain (PHD) protein and factor inhibiting HIF (FIH-1). In chapter 3 revealed that relative to baseline, acute hypoxia increased cardiac lactate efflux and suppressed fatty acid oxidation (FAO) rates in non-treated isolated mouse hearts with final cardiac recovery being 63% of baseline values. Hypoxic and post-hypoxic PDC activation, via dichloroacetate (DCA), decreased cardiac lactate release and FAO during reoxygenation, but failed to improve cardiac recovery relative to control hearts. Chapter 4 sought to establish how chronic hypoxia (11%) upregulates cardiac glycolytic flux, determined via 3H-glucose. Findings of this chapter indicate that of four enzymes considered to set the pace of glycolysis, upregulated pyruvate kinase (PK) flux, appears to explain accelerated hypoxia-induced cardiac glycolytic flux. Western blotting analysis revealed increased PK M2 protein isoform. Sustained hypoxia increased pentose phosphate pathway (PPP) flux, but left lactate accumulation unaltered. Chapter 5 examined the role of sustained in vivo hypoxia in modulating cardiac tolerance to subsequent acute H/R injury and chronic PDC activation in modifying hypoxic heart tolerance to acute injury. Chronic hypoxia reduced cardiac tolerance to H/R injury accompanied by increased glycolytic flux and lactate efflux during reoxygenation injury. Chronic PDC activation improved hypoxic heart tolerance to the acute injury and normalized cardiac metabolic flux and reduced tissue lactate accumulation during reoxygenation, indicative of increased carbohydrate oxidation. Collectively, the data appear to imply that forced carbohydrate oxidation normalizes hypoxic heart recovery from acute injury. In chapter 6 we demonstrated that global FIH-1 deletion increased isolated heart glycolytic flux at baseline and during reoxygenated. FIH-1 KO hearts displayed increased reoxygenated hexokinase (HK) and PK activities, but no changes in PK protein isoforms. Functional analysis revealed that FIH-1 deficiency does not affect isolated heart function at baseline and in response to acute injury. Acute PDC activation does not appear to improve cardiac function during acute hypoxic stress. Conversely, chronic PDC activation normalized, via restored metabolic flux, cardiac tolerance to acute injury following sustained in vivo hypoxia. Furthermore, the present thesis revealed increased PPP flux following sustained in vivo hypoxia, and proposed a pivotal role PKM2 may play in the regulation of hypoxic heart carbohydrate metabolism. In addition, we identified FIH-1 as a novel regulator of cardiac carbohydrate metabolism at baseline and following acute hypoxic injury

Likely additive ergogenic effects of combined pre-exercise dietary nitrate and caffeine ingestion in trained cyclists.

Aims. To evaluate the possible additive effects of beetroot juice plus caffeine on exercise performance. Methods. In a randomized, double-blinded study design, fourteen healthy well-trained men aged 22 ± 3 years performed four trials on different occasions following preexercise ingestion of placebo (PLA), PLA plus 5mg/kg caffeine (PLA+C), beetroot juice providing 8mmol of nitrate (BR), and beetroot juice plus caffeine (BR+C). Participants cycled at 60% maximal oxygen uptake (VO2max) for 30min followed by a time to exhaustion (TTE) trial at 80% VO2max. Saliva was collected before supplement ingestion, before exercise, and after the TTE trial for salivary nitrate, nitrite, and cortisol analysis. Results. In beetroot trials, saliva nitrate and nitrite increased >10-fold before exercise compared with preingestion ( ≤ 0.002). TTE in BR+C was 46% higher than in PLA ( = 0.096) and 18% and 27% nonsignificant TTE improvements were observed on BR+C compared with BR and PLA+C alone, respectively. Lower ratings of perceived exertion during TTE were found during 80% VO2max on BR+C compared with PLA and PLA+C ( < 0.05 for both). Conclusions. Acute preexercise beetroot juice coingestion with caffeine likely has additive effects on exercise performance compared with either beetroot or caffeine alone

Influence of CMV/EBV Serostatus on Respiratory Infection Incidence During 4 Months of Winter Training in a Student Cohort of Endurance Athletes

The purpose of this study was to examine the influence of previous infection with cytomegalovirus (CMV) or Epstein Barr virus (EBV) on the incidence, severity and duration of upper respiratory tract illness (URTI) episodes in endurance athletes during a 4-month winter training period. Blood samples were obtained from 236 healthy subjects (186 males, 70 females) who were engaged in regular sports training (predominantly endurance-based activities such as running, cycling, swimming, triathlon, team games and racquet sports) at the start of the study period for CMV and EBV serostatus analysis. Their baseline characteristics were (mean ± SD) age: 21 ± 2 years, body mass: 73.5 ± 11.2 kg, height: 176.5 ± 9.3 cm, body mass index 23.6 ± 2.2 kg/m2. Weekly training and daily illness logs were kept. Self-reported weekly training duration averaged 9.6 ± 5.2 h/week and 4.0 ± 1.6% of the cohort experienced a URTI episode each week. Twenty-five percent of the subject cohort were CMV positive with a similar proportion in males (24%) and females (26%) whereas 84% of the subject cohort were EBV positive with a similar proportion in males (84%) and females (83%). In addition, 21% of the subject cohort were both CMV and EBV positive (CMV+EBV+) whereas 13% of the subject cohort had no prior CMV or EBV infection (CMV-EBV-). With regard to CMV/EBV serostatus, the results indicated that there was no difference in the proportion of subjects who presented with symptoms of infection between CMV/EBV positive and negative groups. Athletes with previous CMV infection had fewer URTI symptom days during the study period than those with no previous infection [mean and interquartile range (IQR), positives 2 (0-7) days, negatives 4 (1-9) days, P = 0.033] and EBV serostatus had no influence on URTI episode incidence, severity or duration. Moreover, we found that athletes with prior infection of both CMV and EBV had fewer URTI episodes and symptom days than athletes who were seronegative for both CMV and EBV [mean (IQR), URTI episodes: positives 0 (0-1), negatives 1 (0-2), P = 0.04; symptom days: positives 2 (0-7), negatives 8 (2-12), P = 0.01]. The reasons for this are still unclear but could be related to the previously reported elevated T cytotoxic cell response to exercise in individuals with positive CMV serostatus. Previous coinfection with CMV and EBV might promote protective immune surveillance to lower the risk of URTI

The influence of exercise training status on antigen-stimulated IL-10 production in whole blood culture and numbers of circulating regulatory T cells

Highly trained athletes are associated with high resting antigen-stimulated whole blood culture interleukin (IL)-10 production. The purpose of the present study was to examine the effects of training status on resting circulating T regulatory (T) cell counts and antigen-stimulated IL-10 production and the effect of acute bout of exercise on the T response. Forty participants volunteered to participate and were assigned to one of the four groups: sedentary (SED), recreationally active (REC), sprint-trained athletes and endurance-trained athletes (END). From the resting blood sample, CD4CD25CD127 T cells and in vitro antigen-stimulated IL-10 production were assessed. Ten REC subjects performed 60 min cycling at 70 % of maximal oxygen uptake and blood samples for T analysis were collected post- and 1 h post-exercise. IL-10 production was greater in END compared with the other groups (P  0.05). Our results demonstrate that high training loads in END are associated with greater resting IL-10 production and T cell count and suggest a possible mechanism for depression of immunity commonly reported in athletes engaged in high training loads. © 2013 Springer-Verlag Berlin Heidelberg

Sex differences in upper respiratory symptoms prevalence and oral-respiratory mucosal immunity in endurance athletes

The purpose of this study was to examine sex differences in oral-respiratory mucosal immunity and the incidence, severity and duration of upper respiratory symptoms (URS) episodes in endurance athletes during a 16-week winter training period. Blood was collected from 210 subjects (147 men and 63 women) at the start and end of the study for determination of differential leukocyte counts. Timed collections of unstimulated saliva were obtained at the start and at 4-week intervals during the study period. Saliva samples were analysed for salivary antimicrobial peptides and proteins (AMPs). Weekly training and daily illness logs were kept using validated questionnaires. Training loads averaged 11 h/week of moderate-vigorous physical activity and were not different for males and females. The salivary concentration of lysozyme and lactoferrin (both P<0.04) but not salivary immunoglobulin A (SIgA) or amylase were higher in males than females. Saliva flow rates were significantly higher in males than females (P < 0.03) and consequently so were the salivary secretion rates of lysozyme, lactoferrin and amylase (all P<0.01) but not SIgA (P = 0.097). Total blood leukocyte, monocyte and lymphocyte counts were not different between the sexes but females had higher numbers of circulating neutrophils (P = 0.040). The average number of URS episodes was 0.6 ± 0.8 (mean ± SD) in males and 0.8 ± 1.0 in females (P = 0.103) and the number of URS days was higher in females (4.7 vs 6.8 days, P < 0.02). The duration of URS episodes was longer in females (11.6 vs 15.5 days, P < 0.03). The findings of this study concur with recent reports of illness incidence at major competitive games indicating that female athletes may be more susceptible than their male counterparts to URS and that lower oral-respiratory mucosal immunity may, in part, account for this

Potential role for pyruvate kinase M2 in the regulation of murine cardiac glycolytic flux during in vivo chronic hypoxia

Carbohydrate metabolism in heart failure shares similarities to that following hypoxic exposure, and is thought to maintain energy homeostasis in the face of reduced O2 availability. As part of these in vivo adaptations during sustained hypoxia, the heart upregulates and maintains a high glycolytic flux, but the underlying mechanism it is still elusive. We followed the cardiac glycolytic responses to a chronic hypoxic (CH) intervention using [5-3H]-glucose labelling in combination with detailed and extensive enzymatic and metabolomic approaches to provide evidence of the underlying mechanism that allows heart survivability. Following three weeks of in vivo hypoxia (11% oxygen), murine hearts were isolated and perfused in a retrograde mode with function measured via an intraventricular balloon and glycolytic flux quantified using [5-3H]-glucose labelling. At the end of perfusion, hearts were flash-frozen and central carbon intermediates determined via liquid chromatography tandem mass spectrometry (LC-MS/MS). The maximal activity of glycolytic enzymes considered rate-limiting was assessed enzymatically, and protein abundance was determined using Western blotting. Relative to normoxic hearts, CH increased ex vivo cardiac glycolytic flux 1.7-fold with no effect on cardiac function. CH upregulated cardiac pyruvate kinase (PK) flux 3.1-fold and cardiac pyruvate kinase M2 (PKM2) protein content 1.4-fold compared to normoxic hearts. CH also augmented cardiac pentose phosphate pathway flux, reflected by higher ribose-5-phosphate content. These findings support an increase in the covalent (protein expression) and allosteric (flux) control of PKM2 as being central to the sustained upregulation of the glycolytic flux in the chronically hypoxic heart

Divergent amino acid and sphingolipid metabolism in patients with inherited neuro-retinal disease

OBJECTIVES: The non-essential amino acids serine, glycine, and alanine, as well as diverse sphingolipid species, are implicated in inherited neuro-retinal disorders and are metabolically linked by serine palmitoyltransferase (SPT), a key enzyme in membrane lipid biogenesis. To gain insight into the pathophysiological mechanisms linking these pathways to neuro-retinal diseases we compared patients diagnosed with two metabolically intertwined diseases: macular telangiectasia type II (MacTel), hereditary sensory autonomic neuropathy type 1 (HSAN1), or both. METHODS: We performed targeted metabolomic analyses of amino acids and broad sphingolipids in sera from a cohort of MacTel (205), HSAN1 (25) and Control (151) participants. RESULTS: MacTel patients exhibited broad alterations of amino acids, including changes in serine, glycine, alanine, glutamate, and branched-chain amino acids reminiscent of diabetes. MacTel patients had elevated 1-deoxysphingolipids but reduced levels of complex sphingolipids in circulation. A mouse model of retinopathy indicates dietary serine and glycine restriction can drive this depletion in complex sphingolipids. HSAN1 patients exhibited elevated serine, lower alanine, and a reduction in canonical ceramides and sphingomyelins compared to controls. Those patients diagnosed with both HSAN1 and MacTel showed the most significant decrease in circulating sphingomyelins. CONCLUSIONS: These results highlight metabolic distinctions between MacTel and HSAN1, emphasize the importance of membrane lipids in the progression of MacTel, and suggest distinct therapeutic approaches for these two neurodegenerative diseases

The influence of exercise training status on antigen-stimulated IL-10 production in whole blood culture and numbers of circulating regulatory T cells

The final publication is available at springerlink.com.Highly trained athletes are associated with high resting antigen-stimulated whole blood culture interleukin (IL)-10 production. The purpose of the present study was to examine the effects of training status on resting circulating T regulatory (T) cell counts and antigen-stimulated IL-10 production and the effect of acute bout of exercise on the T response. Forty participants volunteered to participate and were assigned to one of the four groups: sedentary (SED), recreationally active (REC), sprint-trained athletes and endurance-trained athletes (END). From the resting blood sample, CD4CD25CD127 T cells and in vitro antigen-stimulated IL-10 production were assessed. Ten REC subjects performed 60 min cycling at 70 % of maximal oxygen uptake and blood samples for T analysis were collected post- and 1 h post-exercise. IL-10 production was greater in END compared with the other groups (P < 0.05). END had a higher T percentage of total lymphocyte count compared with SED (P < 0.05). A smaller proportion of T CD4 cells were observed in SED compared with all other groups (P < 0.05). IL-10 production significantly correlated with the proportion of T within the total lymphocyte population (r = 0.51, P = 0.001). No effect of acute exercise was evident for T cell counts in the REC subjects (P > 0.05). Our results demonstrate that high training loads in END are associated with greater resting IL-10 production and T cell count and suggest a possible mechanism for depression of immunity commonly reported in athletes engaged in high training loads. © 2013 Springer-Verlag Berlin Heidelberg

Cardiac responses to hypoxia: the role of pyruvate dehydrogenase complex in carbohydrate utilisation

Accelerated carbohydrate utilisation and suppressed lipid oxidative metabolism are hallmarks of heart failure (HF). Hypoxia mimics failing heart metabolic reprogramming and has been suggested to play a role in cardiac metabolic switch. One aspect of the regulation of cardiac energy metabolism is the pyruvate dehydrogenase complex (PDC). Hypoxia inducible factor (HIF) signalling is thought regulate hypoxia-induced adaptations. HIF transcriptional activity is controlled by prolyl hydroxylase domain (PHD) protein and factor inhibiting HIF (FIH-1). In chapter 3 revealed that relative to baseline, acute hypoxia increased cardiac lactate efflux and suppressed fatty acid oxidation (FAO) rates in non-treated isolated mouse hearts with final cardiac recovery being 63% of baseline values. Hypoxic and post-hypoxic PDC activation, via dichloroacetate (DCA), decreased cardiac lactate release and FAO during reoxygenation, but failed to improve cardiac recovery relative to control hearts. Chapter 4 sought to establish how chronic hypoxia (11%) upregulates cardiac glycolytic flux, determined via 3H-glucose. Findings of this chapter indicate that of four enzymes considered to set the pace of glycolysis, upregulated pyruvate kinase (PK) flux, appears to explain accelerated hypoxia-induced cardiac glycolytic flux. Western blotting analysis revealed increased PK M2 protein isoform. Sustained hypoxia increased pentose phosphate pathway (PPP) flux, but left lactate accumulation unaltered. Chapter 5 examined the role of sustained in vivo hypoxia in modulating cardiac tolerance to subsequent acute H/R injury and chronic PDC activation in modifying hypoxic heart tolerance to acute injury. Chronic hypoxia reduced cardiac tolerance to H/R injury accompanied by increased glycolytic flux and lactate efflux during reoxygenation injury. Chronic PDC activation improved hypoxic heart tolerance to the acute injury and normalized cardiac metabolic flux and reduced tissue lactate accumulation during reoxygenation, indicative of increased carbohydrate oxidation. Collectively, the data appear to imply that forced carbohydrate oxidation normalizes hypoxic heart recovery from acute injury. In chapter 6 we demonstrated that global FIH-1 deletion increased isolated heart glycolytic flux at baseline and during reoxygenated. FIH-1 KO hearts displayed increased reoxygenated hexokinase (HK) and PK activities, but no changes in PK protein isoforms. Functional analysis revealed that FIH-1 deficiency does not affect isolated heart function at baseline and in response to acute injury. Acute PDC activation does not appear to improve cardiac function during acute hypoxic stress. Conversely, chronic PDC activation normalized, via restored metabolic flux, cardiac tolerance to acute injury following sustained in vivo hypoxia. Furthermore, the present thesis revealed increased PPP flux following sustained in vivo hypoxia, and proposed a pivotal role PKM2 may play in the regulation of hypoxic heart carbohydrate metabolism. In addition, we identified FIH-1 as a novel regulator of cardiac carbohydrate metabolism at baseline and following acute hypoxic injury