Henk M. De Feyter

13C MRS studies of neuroenergetics and neurotransmitter cycling in humans.

In the last 25 years, 13 C MRS has been established as the only noninvasive method for the measurement of glutamate neurotransmission and cell‐specific neuroenergetics. Although technically and experimentally challenging, 13 C MRS has already provided important new information on the relationship between neuroenergetics and neuronal function, the energy cost of brain function, the high neuronal activity in the resting brain state and how neuroenergetics and neurotransmitter cycling are altered in neurological and psychiatric disease. In this article, the current state of 13 C MRS as it is applied to the study of neuroenergetics and neurotransmitter cycling in humans is reviewed. The focus is predominantly on recent findings in humans regarding metabolic pathways, applications to clinical research and the technical status of the method. Results from in vivo 13 C MRS studies in animals are discussed from the standpoint of the validation of MRS measurements of neuroenergetics and neurotransmitter cycling, and where they have helped to identify key questions to address in human research. Controversies concerning the relationship between neuroenergetics and neurotransmitter cycling and factors having an impact on the accurate determination of fluxes through mathematical modeling are addressed. We further touch upon different 13C‐labeled substrates used to study brain metabolism, before reviewing a number of human brain diseases investigated using 13 C MRS. Future technological developments are discussed that will help to overcome the limitations of 13 C MRS, with special attention given to recent developments in hyperpolarized 13 C MRS. Copyright

Increased intramyocellular lipid content but normal skeletal muscle mitochondrial oxidative capacity throughout the pathogenesis of type 2 diabetes

Currently inherited or acquired skeletal muscle mitochondrial dysfunction is linked to dysregulated fatty acid metabolism, resulting in increased levels of intramyocellular lipids (IMCLs) and lipid intermediates, inducing insulin resistance. The present study aimed to clarify the order of changes in IMCL levels and skeletal muscle mitochondrial function during the development of type 2 diabetes in Zucker diabetic fatty (ZDF) rats. IMCL levels and skeletal muscle oxidative capacity were determined in vivo, using localized 1H magnetic resonance spectroscopy (MRS) and dynamic 31P MRS, respectively. In parallel, in vitro activities were measured from enzymes involved in fatty acid oxidation, the tricarboxylic acid cycle and the electron transport chain. Fa/fa ZDF rats were studied at 3 different ages corresponding to different stages of type 2 diabetes, whereas fa/ + rats served as controls. Fa/fa ZDF rats had higher IMCL contents than controls throughout the duration of the study. In vivo muscle oxidative capacity was not different in fa/fa animals compared to controls, and in vitro enzyme activity data suggested improved functionality of enzymes involved in fat oxidation in type 2 diabetic animals. Accordingly, we can conclude that in the ZDF rat model, type 2 diabetes develops in the absence of skeletal muscle mitochondrial dysfunction.— De Feyter, H. M., Lenaers, E., Houten, S. M., Schrauwen, P., Hesselink, M. K., Wanders, R. J. A., Nicolay, K., Prompers, J. J. Increased intramyocellular lipid content but normal skeletal muscle mitochondrial oxidative capacity throughout the pathogenesis of type 2 diabetes. FASEB J. 22, 3947–3955 (2008)

2-Hydroxyglutarate produced by neomorphic IDH mutations suppresses homologous recombination and induces PARP inhibitor sensitivity

The oncometabolite 2-hydroxyglutarate renders IDH1/2 mutant cancer cells deficient in homologous recombination and confers vulnerability to synthetic lethal targeting with PARP inhibitors. Target 2HG or not 2HG, that is the question Mutations in isocitrate dehydrogenase 1 and 2, which result in overproduction of 2-hydroxyglutarate (2HG), are observed in multiple tumor types, including gliomas and acute myelogenous leukemia. An additional form of 2HG is produced under hypoxia, which is also frequent in tumors. 2HG is considered to be an oncometabolite, or a metabolite that promotes carcinogenesis, and inhibitors of mutant isocitrate dehydrogenase are in development to target this process. However, Sulkowski et al. found that it may be possible to take advantage of 2HG overproduction instead. The authors discovered that 2HG overproduction impairs homologous recombination used in DNA repair and sensitizes cancer cells to treatment with PARP inhibitors, another class of cancer drugs that are already in clinical use. 2-Hydroxyglutarate (2HG) exists as two enantiomers, (R)-2HG and (S)-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)–dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase 1 (IDH1) and IDH2 mutations, whereas the latter is produced under pathologic processes such as hypoxia. We report that IDH1/2 mutations induce a homologous recombination (HR) defect that renders tumor cells exquisitely sensitive to poly(adenosine 5′-diphosphate–ribose) polymerase (PARP) inhibitors. This “BRCAness” phenotype of IDH mutant cells can be completely reversed by treatment with small-molecule inhibitors of the mutant IDH1 enzyme, and conversely, it can be entirely recapitulated by treatment with either of the 2HG enantiomers in cells with intact IDH1/2 proteins. We demonstrate mutant IDH1–dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells in culture and genetically matched tumor xenografts in vivo. These findings provide the basis for a possible therapeutic strategy exploiting the biological consequences of mutant IDH, rather than attempting to block 2HG production, by targeting the 2HG-dependent HR deficiency with PARP inhibition. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair, and genetic instability.

Glutamate Metabolism in Major Depressive Disorder

Research on novel treatments for major depressive disorder focuses quite deeply on glutamate function, and this research would benefit from a brain-imaging technique that precisely quantified glutamate function. Signs of a specific form of glutamate-related dysfunction that could be targeted by novel therapies were found using novel, state-of-the-art techniques to address this issue.

Increased brain uptake and oxidation of acetate in heavy drinkers.

When a person consumes ethanol, the body quickly begins to convert it to acetic acid, which circulates in the blood and can serve as a source of energy for the brain and other organs. This study used 13C magnetic resonance spectroscopy to test whether chronic heavy drinking is associated with greater brain uptake and oxidation of acetic acid, providing a potential metabolic reward or adenosinergic effect as a consequence of drinking. Seven heavy drinkers, who regularly consumed at least 8 drinks per week and at least 4 drinks per day at least once per week, and 7 light drinkers, who consumed fewer than 2 drinks per week were recruited. The subjects were administered [2-13C]acetate for 2 hours and scanned throughout that time with magnetic resonance spectroscopy of the brain to observe natural 13C abundance of N-acetylaspartate (NAA) and the appearance of 13C-labeled glutamate, glutamine, and acetate. Heavy drinkers had approximately 2-fold more brain acetate relative to blood and twice as much labeled glutamate and glutamine. The results show that acetate transport and oxidation are faster in heavy drinkers compared with that in light drinkers. Our finding suggests that a new therapeutic approach to supply acetate during alcohol detoxification may be beneficial.

Adaptations in mitochondrial function parallel, but fail to rescue, the transition to severe hyperglycemia and hyperinsulinemia: a study in Zucker diabetic fatty rats.

Cross‐sectional human studies have associated mitochondrial dysfunction to type 2 diabetes. We chose Zucker diabetic fatty (ZDF) rats as a model of progressive insulin resistance to examine whether intrinsic mitochondrial defects are required for development of type 2 diabetes. Muscle mitochondrial function was examined in 6‐, 12‐, and 19‐week‐old ZDF (fa/fa) and fa/+ control rats (n = 8–10 per group) using respirometry with pyruvate, glutamate, and palmitoyl‐CoA as substrates. Six‐week‐old normoglycemic–hyperinsulinemic fa/fa rats had reduced mitochondrial fat oxidative capacity. Adenosine diphosphate (ADP)‐driven state 3 and carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP)‐stimulated state uncoupled (state u) respiration on palmitoyl‐CoA were lower compared to controls (62.3 ± 9.5 vs. 119.1 ± 13.8 and 87.8 ± 13.3 vs. 141.9 ± 14.3 nmol O2/mg/min.). Pyruvate oxidation in 6‐week‐old fa/fa rats was similar to controls. Remarkably, reduced fat oxidative capacity in 6‐week‐old fa/fa rats was compensated for by an adaptive increase in intrinsic mitochondrial function at week 12, which could not be maintained toward week 19 (140.9 ± 11.2 and 57.7 ± 9.8 nmol O2/mg/min, weeks 12 and 19, respectively), whereas hyperglycemia had developed (13.5 ± 0.6 and 16.1 ± 0.3 mmol/l, weeks 12 and 19, respectively). This mitochondrial adaptation failed to rescue the progressive development of insulin resistance in fa/fa rats. The transition of prediabetes state toward advanced hyperglycemia and hyperinsulinemia was accompanied by a blunted increase in uncoupling protein‐3 (UCP3). Thus, in ZDF rats insulin resistance develops progressively in the absence of mitochondrial dysfunction. In fact, improved mitochondrial capacity in hyperinsulinemic hyperglycemic rats does not rescue the progression toward advanced stages of insulin resistance.

Regional variations in intramyocellular lipid concentration correlate with muscle fiber type distribution in rat tibialis anterior muscle

1H MR spectroscopy (MRS) has proved to be a valuable noninvasive tool to measure intramyocellular lipids (IMCL) in research focused on insulin resistance and type II diabetes in both humans and rodents. An important determinant of IMCL is the muscle fiber type, since oxidative type I fibers can contain up to three times more IMCL than glycolytic type II muscle fibers. Because these different muscle fiber types are inhomogeneously distributed in rodent muscle, in the present study we investigated the distribution of IMCL within the rat tibialis anterior muscle (TA) in vivo using single‐voxel 1H MRS along with the muscle fiber distribution in the TA ex vivo determined from immunohistological assays. IMCL levels in the TA differed by up to a factor of 3 depending on the position of the voxel. The distribution of IMCL over the TA cross section was not random, but emerged in a pattern similar to the distribution of the predominantly oxidative muscle fiber types. Dietary interventions, such as high‐fat feeding and 15 hr of fasting, did not significantly change this typical fiber type‐dependent pattern of IMCL content. These results stress the importance of voxel positioning when single‐voxel 1H MRS is used to study IMCL in rodent muscle. Magn Reson Med, 2006.

Increased brain lactate concentrations without increased lactate oxidation during hypoglycemia in type 1 diabetic individuals.

Previous studies have reported that brain metabolism of acetate is increased more than twofold during hypoglycemia in type 1 diabetic (T1D) subjects with hypoglycemia unawareness. These data support the hypothesis that upregulation of blood-brain barrier monocarboxylic acid (MCA) transport may contribute to the maintenance of brain energetics during hypoglycemia in subjects with hypoglycemia unawareness. Plasma lactate concentrations are ∼10-fold higher than acetate concentrations, making lactate the most likely alternative MCA as brain fuel. We therefore examined transport of [3-13C]lactate across the blood-brain barrier and its metabolism in the brains of T1D patients and nondiabetic control subjects during a hypoglycemic clamp using 13C magnetic resonance spectroscopy. Brain lactate concentrations were more than fivefold higher (P < 0.05) during hypoglycemia in the T1D subjects compared with the control subjects. Surprisingly, we observed no increase in the oxidation of blood-borne lactate in the T1D subjects, as reflected by similar 13C fractional enrichments in brain glutamate and glutamine. Taken together, these data suggest that in addition to increased MCA transport at the blood-brain barrier, there may be additional metabolic adaptations that contribute to hypoglycemia unawareness in patients with T1D.

Increased Brain Transport and Metabolism of Acetate in Hypoglycemia Unawareness

CONTEXT Intensive insulin therapy reduces the risk for long-term complications in patients with type 1 diabetes mellitus (T1DM) but increases the risk for hypoglycemia-associated autonomic failure (HAAF), a syndrome that includes hypoglycemia unawareness and defective glucose counterregulation (reduced epinephrine and glucagon responses to hypoglycemia). OBJECTIVE The objective of the study was to address mechanisms underlying HAAF, we investigated whether nonglucose fuels such as acetate, a monocarboxylic acid (MCA), can support cerebral energetics during hypoglycemia in T1DM individuals with hypoglycemia unawareness. DESIGN Magnetic resonance spectroscopy was used to measure brain transport and metabolism of [2-(13)C]acetate under hypoglycemic conditions. SETTING The study was conducted at the Yale Center for Clinical Investigation Hospital Research Unit, Yale Magnetic Resonance Research Center. PATIENTS AND OTHER PARTICIPANTS T1DM participants with moderate to severe hypoglycemia unawareness (n = 7), T1DM controls without hypoglycemia unawareness (n = 5), and healthy nondiabetic controls (n = 10) participated in the study. MAIN OUTCOME MEASURE(S) Brain acetate concentrations, (13)C percent enrichment of glutamine and glutamate, and absolute rates of acetate metabolism were measured. RESULTS Absolute rates of acetate metabolism in the cerebral cortex were 1.5-fold higher among T1DM/unaware participants compared with both control groups during hypoglycemia (P = .001). Epinephrine levels of T1DM/unaware subjects were significantly lower than both control groups (P < .05). Epinephrine levels were inversely correlated with levels of cerebral acetate use across the entire study population (P < .01), suggesting a relationship between up-regulated brain MCA use and HAAF. CONCLUSION Increased MCA transport and metabolism among T1DM individuals with hypoglycemia unawareness may be a mechanism to supply the brain with nonglucose fuels during episodes of acute hypoglycemia and may contribute to the syndrome of hypoglycemia unawareness, independent of diabetes.

Characterization of cerebral glutamine uptake from blood in the mouse brain: implications for metabolic modeling of 13C NMR data

13C Nuclear Magnetic Resonance (NMR) studies of rodent and human brain using [1-13C]/[1,6-13C2]glucose as labeled substrate have consistently found a lower enrichment (~25% to 30%) of glutamine-C4 compared with glutamate-C4 at isotopic steady state. The source of this isotope dilution has not been established experimentally but may potentially arise either from blood/brain exchange of glutamine or from metabolism of unlabeled substrates in astrocytes, where glutamine synthesis occurs. In this study, the contribution of the former was evaluated ex vivo using 1H-[13C]-NMR spectroscopy together with intravenous infusion of [U-13C5]glutamine for 3, 15, 30, and 60 minutes in mice. 13C labeling of brain glutamine was found to be saturated at plasma glutamine levels > 1.0 mmol/L. Fitting a blood–astrocyte–neuron metabolic model to the 13C enrichment time courses of glutamate and glutamine yielded the value of glutamine influx, VGln(in), 0.036 ± 0.002 μmol/g per minute for plasma glutamine of 1.8 mmol/L. For physiologic plasma glutamine level (~0.6 mmol/L), VGln(in) would be ~0.010 μmol/g per minute, which corresponds to ~6% of the glutamine synthesis rate and rises to ~11% for saturating blood glutamine concentrations. Thus, glutamine influx from blood contributes at most ~20% to the dilution of astroglial glutamine-C4 consistently seen in metabolic studies using [1-13C]glucose.