Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by P-31 magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-C-13]acetate incorporation into muscle [4-C-13]-, [3-C-13]-glutamate, and [4-C-13]-glutamine by high-resolution C-13/H-1 MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.

Noninvasive techniques to quantify metabolites in skeletal muscle provide unique insight into human physiology and enable the translation of research into practice. Proton magnetic resonance spectroscopy (1H-MRS) permits the assessment of several abundant muscle metabolites in vivo, including carnosine, a dipeptide composed of the amino acids histidine and beta-alanine. Muscle carnosine loading, accomplished by chronic oral beta-alanine supplementation, improves muscle function and exercise capacity and has pathophysiological relevance in multiple diseases. Moreover, the marked difference in carnosine content between fast-twitch and slow-twitch muscle fibers has rendered carnosine an attractive candidate to estimate human muscle fiber type composition. However, the quantification of carnosine with 1H-MRS requires technical expertise to obtain accurate and reproducible data. In this review, we describe the technical and physiological factors that impact the detection, analysis, and quantification of carnosine in muscle with 1H-MRS. We discuss potential sources of error during the acquisition and preprocessing of the 1H-MRS spectra and present best practices to enable the accurate, reliable, and reproducible application of this technique.

Alterations in muscle mitochondrial substrate preference have been postulated to play a major role in the pathogenesis of muscle insulin resistance. In order to examine this hypothesis, we assessed the ratio of mitochondrial pyruvate oxidation (VPDH) to rates of mitochondrial citrate synthase flux (VCS) in muscle. Contrary to this hypothesis, we found that high-fat-diet (HFD)-fed insulin-resistant rats did not manifest altered muscle substrate preference (VPDH/VCS) in soleus or quadriceps muscles in the fasting state. Furthermore, hyperinsulinemic-euglycemic (HE) clamps increased VPDH/VCS in both muscles in normal and insulin-resistant rats. We then examined the muscle VPDH/VCS flux in insulin-sensitive and insulin-resistant humans and found similar relative rates of VPDH/VCS, following an overnight fast (∼20%), and similar increases in VPDH/VCS fluxes during a HE clamp. Altogether, these findings demonstrate that alterations in mitochondrial substrate preference are not an essential step in the pathogenesis of muscle insulin resistance.

Obese adolescents with PCOS are at high risk of nonalcoholic Fatty Liver Disease (NAFLD) which contributes to the risk of diabetes. Impaired hepatic energy metabolism is associated with diabetes and characterized by decreased inorganic phosphates (Pi) and ATP hepatic content. Membrane phospholipids measured by magnetic resonance spectroscopy (MRS) may reflect endoplasmic reticulum (ER) stress (phosphodiesters; PDE) or turnover/composition of membrane phospholipids: precursors (phosphomonoesters (PME): phosphoethanolamine (PE) and phosphocholine (PC)) and degradation products (PDE). It is not known if adolescents with PCOS and NAFLD have impaired hepatic energy metabolism or MRS signature associated with insulin sensitivity (Si). Obese adolescents with PCOS (n=25) underwent fasted hepatic 31phosphorus-MRS to measure ATP, Pi, PME and PDE relative to total phosphates (TP). Liver stiffness was measured with MR elastography and hepatic fat fraction (HFF) by MRI-proton density, with an HFF >5.5% classified as NAFLD. A 6h oral sugar tolerance test (OSTT, 25g fructose+75g glucose) was performed. Insulin sensitivity (Si) was calculated with the oral minimal model. 12 girls (BMI 36.4 ±7.3 kg/m2, age 16 (15-17)) had NAFLD and 13 did not (BMI 34.0±4.9 kg/m2, age 16 (14-17)). PME/TP were lower in girls with NAFLD (p=0.007) as well as the ratio of PME/PDE (p=0.002). A lower PE/TP was associated with higher liver stiffness (r= -0.55; p=0.009) and lower Si (r=0.57; p= 0.03). Total ATP/TP, y-ATP/TP and Pi/TP were similar between groups (p>0.15). Obese adolescents with PCOS and NAFLD have a distinct signature in hepatic 31P-MRS that is associated with Si suggesting alterations in membrane phospholipids. However, changes in energetics seen in adults with NAFLD are not present. Further work is needed to understand how these changes in hepatic phospholipids relates to insulin sensitivity and risk for diabetes. Disclosure A. Carreau: Advisory Panel; Self; Pfizer Inc. Y. Garcia Reyes: None. B. Newcomer: None. D.E. Befroy: None. M.S. Brown: None. M. Cree-Green: None. Funding National Institutes of Health (K23DK107871); Doris Duke Charitable Foundation; National Center for Advancing Translational Sciences (UL1TR002535); Diabetes Canada

Recent work suggests that diet affects brain metabolism thereby impacting cognitive function. Our objective was to determine if a western diet altered brain metabolism, increased blood-brain barrier (BBB) transport and inflammation, and induced cognitive impairment in C57BL/6 (WT) mice and low-density lipoprotein receptor null (LDLr -/-) mice, a model of hyperlipidemia and cognitive decline. We show that a western diet and LDLr -/- moderately influence cognitive processes as assessed by Y-maze and radial arm water maze. Also, western diet significantly increased BBB transport, as well as microvessel factor VIII in LDLr -/- and microglia IBA1 staining in WT, both indicators of activation and neuroinflammation. Interestingly, LDLr -/- mice had a significant increase in 18F- fluorodeoxyglucose uptake irrespective of diet and brain 1H-magnetic resonance spectroscopy showed increased lactate and lipid moieties. Metabolic assessments of whole mouse brain by GC/MS and LC/MS/MS showed that a western diet altered brain TCA cycle and β-oxidation intermediates, levels of amino acids, and complex lipid levels and elevated proinflammatory lipid mediators. Our study reveals that the western diet has multiple impacts on brain metabolism, physiology, and altered cognitive function that likely manifest via multiple cellular pathways.

sought to assess adipose, hepatic, and peripheral insulin sensitivity in adolescents with and without type 2 diabetes. Methods used Forty-eight overweight/obese 12–19 year-old, pubertal, sedentary adolescents (27 with type 2 diabetes). Insulin action was measured with a four-phase hyperinsulinemic euglycemic clamp (basal, 10, 16 and 80 mU/m/min) with glucose and glycerol stable isotope tracers. Mean pubertal stage, habitual physical activity and BMI were similar between those with and without diabetes. Summary of results Adolescents with type 2 diabetes had a higher rate of lipolysis (p=0.012) endogenous glucose release (p<0.0001) and a lower clearance rate of glucose (p=0.002) during hyperinsulinemia than obese controls. In type 2 diabetes youth only, peripheral IR was related to FFA concentrations, hepatic fat and central obesity and inflammatory markers; hepatic IR related to central obesity and adipose IR. Conclusions Adolescents with type 2 diabetes have adipose, hepatic and peripheral IR compared to well-matched controls. Youth with type 2 diabetes may benefit from interventions directed at improving IR in these tissues, an area in need of further research.

Hypophosphatemia can lead to muscle weakness and respiratory and heart failure, but the mechanism is unknown. To address this question, we noninvasively assessed rates of muscle ATP synthesis in hypophosphatemic mice by using in vivo saturation transfer [P-31]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (V-ATP) and plasma inorganic phosphate (P-i) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent P-i transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a(-/-)) mice compared with their wild-type littermate controls. Rates of V-ATP normalized in both hypophosphatemic groups after restoring plasma P-i concentrations. Furthermore, V-ATP was directly related to cellular and mitochondrial P-i uptake in L6 and RC13 rodent myocytes and isolated muscle mitochondria. Similar findings were observed in a patient with chronic hypophosphatemia as a result of a mutation in SLC34A3 who had a 50% reduction in both serum P-i content and muscle V-ATP. After oral P-i repletion and normalization of serum P-i levels, muscle V-ATP completely normalized in the patient. Taken together, these data support the hypothesis that decreased muscle ATP synthesis, in part, may be caused by low blood P-i concentrations, which may explain some aspects of muscle weakness observed in patients with hypophosphatemia.-Pesta, D. H., Tsirigotis, D. N., Befroy, D. E., Caballero, D., Jurczak, M. J., Rahimi, Y., Cline, G. W., Dufour, S., Birkenfeld, A. L., Rothman, D. L., Carpenter, T. O., Insogna, K., Petersen, K. F., Bergwitz, C., Shulman, G. I. Hypophosphatemia promotes lower rates of muscle ATP synthesis.

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and there is great interest in understanding the potential role of alterations in mitochondrial metabolism in its pathogenesis. To address this question, we assessed rates of hepatic mitochondrial oxidation in subjects with and without NAFLD by monitoring the rate of 13C labeling in hepatic [5-13C]glutamate and [1-13C]glutamate by 13C MRS during an infusion of [1-13C]acetate. We found that rates of hepatic mitochondrial oxidation were similar between NAFLD and control subjects. We also assessed rates of hepatic pyruvate cycling during an infusion of [3-13C]lactate by monitoring the 13C label in hepatic [2-13C]alanine and [2-13C]glutamate and found that this flux was also similar between groups and more than 10-fold lower than previously reported. Contrary to previous studies, we show that hepatic mitochondrial oxidation and pyruvate cycling are not altered in NAFLD and do not account for the hepatic fat accumulation.

Burgess et al.1 comment that the modeling approach used in our recent article2 may have led to an incorrect estimate of hepatic anaplerosis because we did not explicitly include a term for pyruvate cycling. Although we agree that a pyruvate substrate cycle is a feature of hepatic metabolism, we contend that under most conditions, the flux through this cycle is low, relative to the TCA cycle (VTCA), and therefore does not substantially affect the rates of anaplerosis (VANA) that we reported. Furthermore, our measurements of VTCA depend primarily on the kinetics of enrichment of glutamate at C5, which are independent of VANA and pyruvate cycling via pyruvate kinase (VPK). Several independent lines of evidence support these conclusions. Hepatic glutamate enrichment during these experiments is a complex function of the enrichments in acetyl-CoA, pyruvate and carbon dioxide, and the metabolic reactions that interact with these substrates, including VTCA, gluconeogenesis from phosphoenolpyruvate and entry of pyruvate–carbon-dioxide through pyruvate carboxylase and malic enzyme. Under fasting conditions, malic enzyme activity is low3,4, therefore pyruvate entry is almost exclusively through pyruvate carboxylase flux (VPC). VPC incorporates components owing to VANA and VPK, which we consider to be metabolically distinct processes. Anaplerotic pyruvate entry is required under gluconeogenic conditions to replenish the pool of TCA cycle intermediates that would otherwise be depleted due to the consumption of oxaloacetate (OAA) to form glucose via phosphoenolpyruvate (PEP), whereas pyruvate cycling involves the futile consumption and regeneration of pyruvate. Under this rationale, VPC = [VANA + VPK]. Because the total metabolite mass remains constant, VANA is also equivalent to the gluconeogenic flux from PEP. An earlier version of our model of hepatic metabolism included a discrete term for VPK to account for pyruvate cycling. Probability distribution analysis of Monte Carlo simulations of this model determined that VPK/VTCA was most likely to be <<0.49, with upper bound estimates (the 66th and 95th percentile of all runs) of 0.99 and 2.19, well below the values reported by Burgess et al.5,6. More importantly, simulations demonstrated that pyruvate cycling had no significant impact on our estimates of VANA and VTCA. Although our analysis indicated that PK flux must be low, owing to uncertainty in establishing an absolute rate of pyruvate cycling we felt that including it in our article would be misleading. In support of their hypothesis, Burgess et al.1 applied a steady-state model7 to simulate the ratio of glutamate C1 to glutamate C5 under a range of PK flux conditions. Unfortunately, their model is not suitable for examining our data because the system was not at isotopic steady state—enrichment of bicarbonate and glutamate C1 continue to increase throughout the 2-h study—and this leads to erroneous interpretations. In contrast, our analysis is based on the kinetics of plasma acetate and hepatic bicarbonate and glutamate 13C enrichment, where each time point contributes to establishing the metabolic rates that best fit the raw data. Here we demonstrate the effects of pyruvate cycling on the kinetics and magnitude of glutamate C1 enrichment (Fig. 1); low rates of VPK clearly fit the experimental data better than the higher values (VPK/VTCA > 3.5) suggested by Burgess et al.1,5,6. Figure 1 The effect of pyruvate cycling, expressed as VPK/VTCA, on the kinetics of hepatic [1-13C]glutamate (13C1-Glu) enrichment during a 2-h infusion of [1-13C]acetate. Raw data (black diamonds) are reproduced from Befroy et al.2. Low rates of VPK clearly fit ... Low rates of pyruvate cycling are supported by other experimental evidence. We observed minimal labeling at C1 of pyruvate in liver tissue extracts from our studies of rats infused with [1-13C] acetate2, suggesting that PK flux is very low. We have also estimated V(PK + ME) / V(PC + PDH) from the ratio of alanine C2 to glucose C5 enrichment in rat liver during an infusion of [3-13C]alanine3,4. Hepatic V(PK + ME) in these experiments was estimated to be <27% of V(PC + PDH), more than an order of magnitude lower than that reported by Sunny et al.5. Furthermore, during an infusion of [3-13C] lactate in humans who had fasted overnight, we observed negligible 13C labeling of C2 in hepatic alanine and C2 of lactate pools using our in vivo 13C magnetic resonance spectroscopy technique (Fig. 2). The shift in 13C label from the C3 to the C2 position requires an isotopic equilibration that occurs owing to the rapid interconversion of OAA-malate-fumarate followed by the regeneration of pyruvate via VPK. Rapid rates of pyruvate cycling would lead to the appearance of alanine C2 and lactate C2 peaks of similar magnitude to their C3 counterparts, which we did not observe (Fig. 2). In contrast, we observed substantial 13C enrichment of hepatic glutamate C2 and glucose C6 owing to anaplerosis and gluconeogenesis, respectively. Figure 2 13C spectra acquired from human liver at baseline (gray) and after an infusion of [3-13C]lactate (black). Negligible enrichment at alanine C2 and lactate C2 compared to their C3 counterparts (inset, acquired during the same study under identical conditions ... Finally, pyruvate cycling and gluconeogenesis from PEP require obligate energy consumption. On the basis of the data from Sunny et al.5, where gluconeogenesis is approximately equivalent to TCA flux and pyruvate cycling is ~3.5 times TCA flux, the energy produced by the TCA cycle would be almost entirely consumed by these processes, a situation that is energetically unsustainable. In conclusion, the rates of pyruvate cycling reported by Burgess et al.1,5,6 are incompatible with our human data2 and inconsistent with animal studies that have directly measured this flux in vitro3,8,9 and in vivo4. This difference may arise because [U-13C]propionate, the tracer used by Burgess et al.5,6, probably promotes pyruvate cycling anaplerosis, and gluconeogenesis by mass action effects and/or by its rapid conversion to propionyl-CoA and subsequent activation of pyruvate carboxylase10. Future studies should be able to assess this possibility directly.

Despite the central role of the liver in the regulation of glucose and lipid metabolism, there are currently no methods to directly assess hepatic oxidative metabolism in humans in vivo. By using a new (13)C-labeling strategy in combination with (13)C magnetic resonance spectroscopy, we show that rates of mitochondrial oxidation and anaplerosis in human liver can be directly determined noninvasively. Using this approach, we found the mean rates of hepatic tricarboxylic acid (TCA) cycle flux (VTCA) and anaplerotic flux (VANA) to be 0.43 ± 0.04 μmol g(-1) min(-1) and 0.60 ± 0.11 μmol g(-1) min(-1), respectively, in twelve healthy, lean individuals. We also found the VANA/VTCA ratio to be 1.39 ± 0.22, which is severalfold lower than recently published estimates using an indirect approach. This method will be useful for understanding the pathogenesis of nonalcoholic fatty liver disease and type 2 diabetes, as well as for assessing the effectiveness of new therapies targeting these pathways in humans.

There is strong evidence that greater resistance to muscle fatigue in older adults, most often observed during low‐velocity or isometric contractions, has a basis in the bioenergetic response to contractions. However, the role of age‐related changes in motor unit (MU) behavior has not been evaluated in this context. To test the hypothesis that fatigue resistance in old resuls from adaptations in both MU behavior and energetics, we quantified dorsiflexor muscle fatigue (torque‐time integral, %initial); mean MU discharge rate (MUDR) by in‐dwelling electrode and cytosolic pH and inorganic phosphate (Pi) by magnetic resonance spectroscopy during parallel experiments using a protocol of 12 maximal isometric contractions (12s on, 8s off) in 10 young (35±4 yr, mean±SD) and 8 older (74±5) women. Older fatigued less than young (68±7%initial vs 59.7 ±9, p<0.05), had a smaller decrease in MUDR (Δ= −20±10 %initial vs −34±18, p=0.08), and tended to have less acidosis at fatigue (6.88±0.13 vs 6.77±0.16, p=0.18), with no difference in end‐exercise Pi. These data provide the first in vivo evidence of the combined roles of MUDR and energetic responses as mechanisms of fatigue resistance in older adults. NIH AG21094

Mitochondrial ATP production is vital for meeting cellular energy demand at rest and during periods of high ATP turnover. We hypothesized that high-intensity interval training (HIT) would increase ATP flux in resting muscle (VPi→ATP) in response to a single bout of exercise, whereas changes in the capacity for oxidative ATP production (Vmax) would require repeated bouts. Eight untrained men (27 ± 4 yr; peak oxygen uptake = 36 ± 4 ml·kg(-1)·min(-1)) performed six sessions of HIT (4-6 × 30-s bouts of all-out cycling with 4-min recovery). After standardized meals and a 10-h fast, VPi→ATP and Vmax of the vastus lateralis muscle were measured using phosphorus magnetic resonance spectroscopy at 4 Tesla. Measurements were obtained at baseline, 15 h after the first training session, and 15 h after completion of the sixth session. VPi→ATP was determined from the unidirectional flux between Pi and ATP, using the saturation transfer technique. The rate of phosphocreatine recovery (kPCr) following a maximal contraction was used to calculate Vmax. While kPCr and Vmax were unchanged after a single session of HIT, completion of six training sessions resulted in a ∼14% increase in muscle oxidative capacity (P ≤ 0.004). In contrast, neither a single nor six training sessions altered VPi→ATP (P = 0.74). This novel analysis of resting and maximal high-energy phosphate kinetics in vivo in response to HIT provides evidence that distinct aspects of human skeletal muscle metabolism respond differently to this type of training.

Figure 2 : A) Relative contribution (% total ATP production), of Creatine Kinase reaction (ATP PCr),glycolysis activity (ATP gly) and oxidative metabolism (ATP ox) to the total energy demand. B) Metabolic economy (ME) of the total contraction, in young (dark bars) and older (open bar) women.. B Glycogen concentration and bioenergetics in young and older women Anne TONSON, Jacob DEBLOIS, Jessica FAY, Steve FOULIS, Douglas BEFROY, and Jane Kent-Braun Department of Kinesiology, University of Massachusetts, Amherst, MA, United States, Magnetic Resonance Research Center, Yale University, New Haven, CT, United States

Magnetic resonance spectroscopy offers a broad range of noninvasive analytical methods for investigating metabolism in vivo. Of these, the magnetization-transfer (MT) techniques permit the estimation of the unidirectional fluxes associated with metabolic exchange reactions. Phosphorus (31P) MT measurements can be used to examine the bioenergetic reactions of the creatine-kinase system and the ATP synthesis/hydrolysis cycle. Observations from our group and others suggest that the inorganic phosphate (Pi) → ATP flux in skeletal muscle may be modulated by certain conditions, including aging, insulin resistance, and diabetes, and may reflect inherent alterations in mitochondrial metabolism. However, such effects on the Pi → ATP flux are not universally observed under conditions in which mitochondrial function, assessed by other techniques, is impaired, and recent articles have raised concerns about the absolute magnitude of the measured reaction rates. As the application of 31P-MT techniques becomes more widespread, this article reviews the methodology and outlines our experience with its implementation in a variety of models in vivo. Also discussed are potential limitations of the technique, complementary methods for assessing oxidative metabolism, and whether the Pi → ATP flux is a viable biomarker of metabolic function in vivo.

Pyruvate dehydrogenase plays a critical role in the regulation of hepatic glucose and fatty acid oxidation; however, surprisingly little is known about its regulation in vivo. In this study we examined the individual effects of insulin and substrate availability on the regulation of pyruvate dehydrogenase flux (VPDH) to tricarboxylic acid flux (VTCA) in livers of awake rats with lipid‐induced hepatic insulin resistance. VPDH/VTCA flux was estimated from the [4‐13C]glutamate/[3‐13C]alanine enrichments in liver extracts and assessed under conditions of fasting and during a hyperinsulinemic‐euglycemic clamp, whereas the effects of increased plasma glucose concentration on VPDH/VTCA flux was assessed during a hyperglycemic clamp in conjunction with infusions of somatostatin and insulin to maintain basal concentrations of insulin. The effects of increases in both glucose and insulin on VPDH/VTCA were examined during a hyperinsulinemic‐hyperglycemic clamp. The effects of chronic lipid‐induced hepatic insulin resistance on this flux were also examined by performing these measurements in rats fed a high‐fat diet for 3 weeks. Using this approach we found that fasting VPDH/VTCA was reduced by 95% in rats with hepatic insulin resistance (from 17.2 ± 1.5% to 1.3 ± 0.7%, P < 0.00001). Surprisingly, neither hyperinsulinemia per se or hyperglycemia per se were sufficient to increase VPDH/VTCA flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did VPDH/VTCA flux increase (44.6 ± 3.2%, P < 0.0001 versus basal) in low‐fat fed animals but not in rats with chronic lipid‐induced hepatic insulin resistance. Conclusion: These studies demonstrate that the combination of both hyperinsulinemia and hyperglycemia are required to increase VPDH/VTCA flux in vivo and that this flux is severely diminished in rats with chronic lipid‐induced hepatic insulin resistance. (HEPATOLOGY 2011.)

Magnetic resonance spectroscopy (MRS) is a companion technique to the more familiar magnetic resonance imaging (MRI) scan. Whereas MRI determines the spatial distribution of water (and lipid) protons across a region of interest, MRS measures the chemical content of MR-visible nuclei, which include the metabolically relevant elements of hydrogen (1H), carbon (13C), and phosphorus (31P). MRS is particularly advantageous for assessing metabolism because the chemical properties and environment of each nucleus determine the frequency at which it appears in the MR spectrum, giving rise to peaks corresponding not only to specific metabolites but also to the constituent nuclei of each metabolite (Fig. 1). It is therefore a multimodal, noninvasive technique capable of measuring a broad range of biological compounds across a variety of tissues. Repeated measures of metabolite content, metabolic fluxes, and their response to an intervention are possible, characteristics that make MRS ideally suited for in vivo studies of human metabolism. An additional advantage is that MRI scans can be obtained concurrently to provide structural/anatomical information that can guide data acquisition and assist in data analysis and interpretation. In this review, we will highlight some of the applications by which MRS can be used to investigate metabolism, focusing on its application to in vivo human studies. FIG. 1. MRS measures the chemical content of MR-visible nuclei across a region of interest and provides positional information as well as content. The 13C-MRS spectrum of glutamate displays peaks corresponding to each carbon atom of the molecule. The versatility of MRS provides a hugely flexible technique capable of probing a broad range of metabolic applications across a variety of tissues. Despite its many advantages, particularly for studies of human metabolism where noninvasive techniques are at a premium, effective implementation of MRS in vivo requires consideration of its methodological limitations and …