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Hypoxia plays a role in many diseases and can have a wide range of effects on cardiac metabolism depending on the extent of the hypoxic insult. Non-invasive imaging methods could shed valuable light on the metabolic effects of hypoxia on the heart in vivo. Hyperpolarized carbon-13 magnetic resonance spectroscopy (HP 13C MRS) in particular is an exciting technique for imaging metabolism that could provide such information. The aim of our work was, therefore, to establish whether hyperpolarized 13C MRS can be used to assess the in vivo response of cardiac metabolism to systemic acute and chronic hypoxic exposure. Groups of healthy male Wistar rats were exposed to either acute (30 minutes), one week or three weeks of hypoxia. In vivo MRS of hyperpolarized [1-13C] pyruvate was carried out along with assessments of physiological parameters and ejection fraction. No significant changes in heart rate, respiration rate, or ejection fraction were observed at any timepoint. Haematocrit was elevated after one week and three weeks of hypoxia. Thirty minutes of hypoxia resulted in a significant reduction in pyruvate dehydrogenase (PDH) flux, whereas one or three weeks of hypoxia resulted in a PDH flux that was not different to normoxic animals. Conversion of hyperpolarized [1-13C] pyruvate into [1-13C] lactate was elevated following acute hypoxia, suggestive of enhanced anaerobic glycolysis. Elevated HP pyruvate to lactate conversion was also seen at the one-week timepoint, in concert with an increase in lactate dehydrogenase (LDH) expression. Following three weeks of hypoxic exposure, cardiac metabolism was comparable to that observed in normoxia. We have successfully visualized of the effects of systemic hypoxia on cardiac metabolism using hyperpolarized 13C MRS, with differences observed following 30 minutes and 1 week of hypoxia. This demonstrates the potential of in vivo hyperpolarized 13C MRS data for assessing the cardiometabolic effects of hypoxia in disease.

Dysregulation in NAD+/NADH levels is associated with increased cell division and elevated levels of reactive oxygen species in rapidly proliferating cancer cells. Conversion of the ketone body acetoacetate (AcAc) to β-hydroxybutyrate (β-HB) by the mitochondrial enzyme β-hydroxybutyrate dehydrogenase (BDH) depends upon NADH availability. The β-HB-to-AcAc ratio is therefore expected to reflect mitochondrial redox. Previous studies reported the potential of hyperpolarized 13C-AcAc to monitor mitochondrial redox in cells, perfused organs and in vivo. However, the ability of hyperpolarized 13C-AcAc to cross the blood brain barrier (BBB) and its potential to monitor brain metabolism remained unknown. Our goal was to assess the value of hyperpolarized [1,3-13C2]AcAc in healthy and tumor-bearing mice in vivo. Following hyperpolarized [1,3-13C2]AcAc injection, production of [1,3-13C2]β-HB was detected in normal and tumor-bearing mice. Significantly higher levels of [1-13C]AcAc and lower [1-13C]β-HB-to-[1-13C]AcAc ratios were observed in tumor-bearing mice. These results were consistent with decreased BDH activity in tumors and associated with increased total cellular NAD+/NADH. Our study confirmed that AcAc crosses the BBB and can be used for monitoring metabolism in the brain. It highlights the potential of AcAc for future clinical translation and its potential utility for monitoring metabolic changes associated with glioma, and other neurological disorders.

Hypoxia activates the hypoxia-inducible factor (HIF), promoting glycolysis and suppressing mitochondrial respiration. In the type 2 diabetic heart, glycolysis is suppressed whereas fatty acid metabolism is promoted. The diabetic heart experiences chronic hypoxia as a consequence of increased obstructive sleep apnoea and cardiovascular disease. Given the opposing metabolic effects of hypoxia and diabetes, we questioned whether diabetes affects cardiac metabolic adaptation to hypoxia. Control and type 2 diabetic rats were housed for 3 weeks in normoxia or 11% oxygen. Metabolism and function were measured in the isolated perfused heart using radiolabelled substrates. Following chronic hypoxia, both control and diabetic hearts upregulated glycolysis, lactate efflux and glycogen content and decreased fatty acid oxidation rates, with similar activation of HIF signalling pathways. However, hypoxia-induced changes were superimposed on diabetic hearts that were metabolically abnormal in normoxia, resulting in glycolytic rates 30% lower, and fatty acid oxidation 36% higher, in hypoxic diabetic hearts than hypoxic controls. Peroxisome proliferator-activated receptor α target proteins were suppressed by hypoxia, but activated by diabetes. Mitochondrial respiration in diabetic hearts was divergently activated following hypoxia compared with controls. These differences in metabolism were associated with decreased contractile recovery of the hypoxic diabetic heart following an acute hypoxic insult. In conclusion, type 2 diabetic hearts retain metabolic flexibility to adapt to hypoxia, with normal HIF signalling pathways. However, they are more dependent on oxidative metabolism following hypoxia due to abnormal normoxic metabolism, which was associated with a functional deficit in response to stress.

AIMS: The aim of this work was to use hyperpolarized carbon-13 ((13)C) magnetic resonance (MR) spectroscopy and cine MR imaging (MRI) to assess in vivo cardiac metabolism and function in the 15-week-old spontaneously hypertensive rat (SHR) heart. At this time point, the SHR displays hypertension and concentric hypertrophy. One of the cellular adaptations to hypertrophy is a reduction in β-oxidation, and it has previously been shown that in response to hypertrophy the SHR heart switches to a glycolytic/glucose-oxidative phenotype. METHODS AND RESULTS: Cine-MRI (magnetic resonance imaging) was used to assess cardiac function and degree of cardiac hypertrophy. Wistar rats were used as controls. SHRs displayed functional changes in stroke volume, heart rate, and late peak-diastolic filling alongside significant hypertrophy (a 56% increase in left ventricular mass). Using hyperpolarized [1-(13)C] and [2-(13)C]pyruvate, an 85% increase in (13)C label flux through pyruvate dehydrogenase (PDH) was seen in the SHR heart and (13)C label incorporation into citrate, acetylcarnitine, and glutamate pools was elevated in proportion to the increase in PDH flux. These findings were confirmed using biochemical analysis of PDH activity and protein expression of PDH regulatory enzymes. CONCLUSIONS: Functional and structural alterations in the SHR heart are consistent with the hypertrophied phenotype. Our in vivo work indicates a preference for glucose metabolism in the SHR heart, a move away from predominantly fatty acid oxidative metabolism. Interestingly, (13)C label flux into lactate was unchanged, indicating no switch to an anaerobic glycolytic phenotype, but rather an increased reliance on glucose oxidation in the SHR heart.

Although diabetic cardiomyopathy is widely recognized, there are no specific treatments available. Altered myocardial substrate selection has emerged as a candidate mechanism behind the development of cardiac dysfunction in diabetes. As pyruvate dehydrogenase (PDH) activity appears central to the balance of substrate use, we aimed to investigate the relationship between PDH flux and myocardial function in a rodent model of type 2 diabetes and to explore whether or not increasing PDH flux, with dichloroacetate, would restore the balance of substrate use and improve cardiac function. All animals underwent in vivo hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopy and echocardiography to assess cardiac PDH flux and function, respectively. Diabetic animals showed significantly higher blood glucose levels (10.8 ± 0.7 vs. 8.4 ± 0.5 mmol/L), lower PDH flux (0.005 ± 0.001 vs. 0.017 ± 0.002 s-1), and significantly impaired diastolic function (transmitral early diastolic peak velocity/early diastolic myocardial velocity ratio [E/E′] 12.2 ± 0.8 vs. 20 ± 2), which are in keeping with early diabetic cardiomyopathy. Twenty-eight days of treatment with dichloroacetate restored PDH flux to normal levels (0.018 ± 0.002 s-1), reversed diastolic dysfunction (E/E′ 14 ± 1), and normalized blood glucose levels (7.5 ± 0.7 mmol/L). The treatment of diabetes with dichloroacetate therefore restored the balance of myocardial substrate selection, reversed diastolic dysfunction, and normalized blood glucose levels. This suggests that PDH modulation could be a novel therapy for the treatment and/or prevention of diabetic cardiomyopathy.

Cardiovascular disease is the leading cause of mortality in type 2 diabetic patients. Metabolic changes and abnormal hypoxia signalling in the diabetic heart are hypothesised to decrease recovery following a myocardial infarction. Therefore the aim of this study was to investigate the cardiac metabolic adaptation to physiological hypoxia in diabetic rats. Type 2 diabetes was generated in rats using a high fat diet and low dose streptozotocin. Both control and diabetic rats were housed in hypoxia (11% oxygen) or normoxia for three weeks. Following hypoxia, control hearts had upregulated anaerobic glycolysis, increased glycogen content and suppressed fatty acid oxidation. Conversely, these hypoxia-induced metabolic changes were decreased in diabetic hearts, in that glycolysis and glycogen content remained lower, while fatty acid oxidation rates remained higher than in control hearts. Peroxisome proliferator-activated receptor α (PPARα) is a transcription factor that positively regulates fatty acid metabolism, and PPARα-regulated targets were downregulated following hypoxia in the control hearts. PPARα target proteins were upregulated in normoxic diabetic rat hearts and remained elevated following hypoxia. We found that cardiac mRNA expression of the hypoxia-inducible factor (HIF) target genes prolyl hydroxylase 3, heme oxygenase 1 and VEGF were regulated in control and diabetic hearts to the same extent, showing that there were no defects in hypoxia signalling or HIF in diabetic hearts. In conclusion, the abnormal metabolic adaptation to hypoxia in diabetes is associated with an inability of hypoxia to suppress PPARα activation, and occurs independently of changes in hypoxia and HIF signalling pathways. Thus fat metabolism is maintained to the detriment of glucose metabolism.

Introduction In the healthy heart, approximately 30% of the cells’ energy comes from glucose oxidation and the remaining 70% from fatty acids. On exposure to hypoxia, the balance is altered, with increased reliance on glycolysis due to reduced oxygen availability. Pyruvate dehydrogenase (PDH) is the enzyme that stands as the gatekeeper between glycolysis and the Krebs’ cycle. It has previously been demonstrated that hypoxia causes increased expression of pyruvate dehydrogenase kinase (PDK) 1, an enzyme that inhibits PDH. Upregulation of PDK1 enhances inhibition, and it can therefore be hypothesised that PDH flux will be decreased. In the diabetic heart there is a preference for fatty acid use even in the presence of elevated circulating glucose. Exposure to hypoxia and the associated upregulation of glycolysis, may to some extent rebalance fuel selection. However, it is not known if this would be the case as it has also been suggested that the diabetic heart is compromised in terms of its ability to respond to hypoxia. The advent of hyperpolarized carbon-13 ( 13 C) magnetic resonance spectroscopy (MRS) has enabled the visualisation of metabolism in real-time and in vivo . 13 C-labelled pyruvate can be injected into an anaesthetised animal in an MRS system, and spectra acquired (one per second over one minute) to show the metabolism of pyruvate and therefore the flux through PDH. Using hyperpolarized 13 C MRS, this study has investigated in vivo alterations in glucose metabolism in hypoxia in comparison with normoxia, and how these alterations are affected by diabetes. Methods A rat model of late-stage type 2 diabetes was induced by high fat feeding and injection of streptozotocin (25mg/kg). Diabetic and control (chow-fed) animals (n=8) were housed for 3 weeks either in hypoxia (one week gradual acclimatisation and two further weeks at 11% oxygen) or normoxia. Animals were then anaesthetised and scanned in a 7T MRS system, with an injection of hyperpolarized [1- 13 C]-labelled pyruvate and cardiac spectra acquired over ∼1 min. Animals were terminated after scanning, blood samples taken and tissue samples freeze-clamped for later analysis. Data were analysed using jMRUI and the AMARES package, and the conversion of pyruvate into bicarbonate taken as a measure of PDH flux. Results There were no alterations in cardiac PDH flux (Kpyr-bic) observed in hypoxic control animals when compared to normoxic controls. There were also no changes in 13 C label transfer to lactate (Kpyr-lac). Diabetic animals in both normoxia and hypoxia demonstrated decreased PDH flux when compared to controls, however there was no interaction in terms of PDH flux between the hypoxic and diabetic conditions (Figure 1). Conclusions We have observed no alteration in cardiac PDH flux caused by three weeks of chronic hypoxia. In addition, whilst there was a significant reduction in PDH flux in diabetes, exposure to chronic hypoxia had no further metabolic effect on PDH flux.

Aims Cardiovascular disease is the leading cause of mortality in people with type 2 diabetes. Following a myocardial infarction, the heart must adapt to the decreased oxygen availability by switching to more oxygen-efficient metabolism. We hypothesise that adaptation to hypoxia is impaired in type 2 diabetes. Methods Type 2 diabetes was induced in rats by high fat feeding combined with a low dose of streptozotocin (25mg/kg i.p). Following induction of diabetes, control and diabetic rats were housed in a hypoxia chamber for three week at 11% oxygen, to study chronic adaptation to hypoxia. Subsequently, hearts were isolated and perfused for measurement of substrate metabolism, or mitochondria were isolated and respiration was measured. In a separate study, hearts from normoxic diabetic and control rats were perfused ex vivo with hypoxic buffer, to study the acute response to hypoxia. Results Under normoxic conditions, diabetic hearts had a 30% decrease in glycolysis, a 14% decrease in pyruvate oxidation and a 34% increase in fatty acid oxidation, compared with normoxic control rats. When control rats were housed in hypoxia they adapted their cardiac metabolism to be more oxygen efficient, by increasing anaerobic glycolysis by 18%, increasing glycogen reserves by 24%, decreasing fatty acid oxidation by 18% and decreasing mitochondrial respiration by 26%. In contrast, when diabetic rats were housed in hypoxia they were unable to adapt metabolism to the same extent, being significantly different to hypoxic control rats. Glycolytic rates and glycogen content was significantly lower in hypoxic diabetic hearts compared with hypoxic controls, similarly fatty acid oxidation rates and mitochondrial oxygen consumption were both significantly higher in hypoxic diabetic hearts compared with hypoxic control hearts . Metabolic rates and mitochondrial oxygen consumption from hypoxic diabetic rat hearts were comparable to those in normoxic control hearts. Normoxic control and diabetic rat hearts were perfused with hypoxic buffer to study the functional and metabolic response to acute hypoxia. During acute hypoxia, diabetic hearts had 58% lower heart rates and 68% higher end-diastolic pressures compared to control hearts, associated with 55% lower rate of hypoxia-induced glycolysis. During the subsequent reoxygenation period, diabetic hearts had a 34% decrease in recovery of cardiac function compared with control hearts. Conclusions Adaptation to chronic hypoxia is limited in type 2 diabetic rat hearts, with anaerobic metabolism lower and oxidative metabolism higher than control heart. This increased dependence on oxidative metabolism under oxygen-limited conditions was associated with contractile dysfunction in the diabetic heart. This may contribute to the decreased recovery of the diabetic heart following a myocardial infarction.

Myocardial infarction results in the formation of a hypoxic scar region. Resident stem cells have been discovered in the adult heart that may be expanded in vitro via the formation of cardiospheres. Administration of these cardiosphere-derived cells (CDC) to the infarcted heart has been …

Peripheral neuropathy is a rare, yet treatable manifestation of sarcoidosis, a multisystem disorder characterized by the presence of non-caseating granulomas that are seldom found in nerve biopsy specimens. In order to learn more about the subject, we reviewed our clinical and pathological findings in a series of 11 patients (six men and five women aged 26-83 years) with symptomatic neuropathy associated with characteristic granulomas in nerve biopsy specimens. Only two patients were known to have sarcoidosis before the occurrence of the neuropathy. The neuropathy was focal or multifocal in six patients, including one with a multifocal neuropathy associated with conduction blocks, and one with a multifocal axonal motor deficit. Four patients had a distal symmetrical deficit and one patient had a Guillain-Barre-like syndrome with facial diplegia and respiratory failure. Serum angiotensin-converting enzyme concentration was elevated in only two patients. Epineurial granulomas and perineuritis were present in all nerve specimens. The inflammatory infiltrates invaded the endoneurium, following connective tissue septae and blood vessels, in five patients. Multinucleated giant cells were found in eight patients and necrotizing vasculitis in seven. Inflammatory lesions were associated with variable, asymmetrical involvement of nerve fascicles and axon loss. A muscle specimen was sampled during the same procedure in 10 patients. It showed inflammatory infiltrates and granulomas in nine patients and necrotizing vasculitis in two. Immunolabeling showed a mixed inflammatory infiltrate of T cells (predominantly CD4+ cells) and macrophages, in keeping with a delayed hypersensitivity reaction. In addition to nerve involvement, all patients had at least one other tissue or organ affected, including muscle in nine patients, lungs and/or intrathoracic lymph nodes in eight, skin in three, arthritis in two, and peripheral lymph nodes, stomach and eye in one patient each. Most patients improved on corticosteroids. Two patients remain free of symptoms after 7 years. Severe side-effects of long-term treatment with corticosteroids occurred in two patients, leading to death in one. This study illustrates the wide range of clinical manifestations of sarcoid neuropathy and the frequent association of granulomatous inflammatory infiltrates with necrotizing vasculitis and with silent or symptomatic involvement of other organs.