Overexpression of UCP3 decreases mitochondrial efficiency in mouse skeletal muscle in vivo.

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 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H 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.

Proton magnetic resonance spectroscopy in skeletal muscle: Experts' consensus recommendations.

1 H-MR spectroscopy of skeletal muscle provides insight into metabolism that is not available noninvasively by other methods. The recommendations given in this article are intended to guide those who have basic experience in general MRS to the special application of 1 H-MRS in skeletal muscle. The highly organized structure of skeletal muscle leads to effects that change spectral features far beyond simple peak heights, depending on the type and orientation of the muscle. Specific recommendations are given for the acquisition of three particular metabolites (intramyocellular lipids, carnosine and acetylcarnitine) and for preconditioning of experiments and instructions to study volunteers.

Dissociation of Muscle Insulin Resistance from Alterations in Mitochondrial Substrate Preference.

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.

Reduced cognitive function, increased blood-brain-barrier transport and inflammatory responses, and altered brain metabolites in LDLr -/-and C57BL/6 mice fed a western diet.

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 ß-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.

Assessment of Hepatic Mitochondrial Oxidation and Pyruvate Cycling in NAFLD by (13)C Magnetic Resonance Spectroscopy.

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 (13)C labeling in hepatic [5-(13)C]glutamate and [1-(13)C]glutamate by (13)C MRS during an infusion of [1-(13)C]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-(13)C]lactate by monitoring the (13)C label in hepatic [2-(13)C]alanine and [2-(13)C]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.

Hypophosphatemia promotes lower rates of muscle ATP synthesis.

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 [31P]-magnetic resonance spectroscopy. By using this approach, we found that basal and insulin-stimulated rates of muscle ATP synthetic flux (VATP) and plasma inorganic phosphate (Pi) were reduced by 50% in mice with diet-induced hypophosphatemia as well as in sodium-dependent Pi transporter solute carrier family 34, member 1 (NaPi2a)-knockout (NaPi2a-/-) mice compared with their wild-type littermate controls. Rates of VATP normalized in both hypophosphatemic groups after restoring plasma Pi concentrations. Furthermore, VATP was directly related to cellular and mitochondrial Pi 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 Pi content and muscle VATP After oral Pi repletion and normalization of serum Pi levels, muscle VATP 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 Pi 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.

Direct assessment of hepatic mitochondrial oxidative and anaplerotic fluxes in humans using dynamic 13C magnetic resonance spectroscopy.

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.

High-intensity interval training increases in vivo oxidative capacity with no effect on P(i)→ATP rate in resting human muscle.

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.

³¹P-magnetization transfer magnetic resonance spectroscopy measurements of in vivo metabolism.

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 (P(i)) → 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 P(i) → 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 P(i) → ATP flux is a viable biomarker of metabolic function in vivo.

Regulation of hepatic fat and glucose oxidation in rats with lipid-induced hepatic insulin resistance.

UNLABELLED: 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 (V(PDH) ) to tricarboxylic acid flux (V(TCA) ) in livers of awake rats with lipid-induced hepatic insulin resistance. V(PDH) /V(TCA) flux was estimated from the [4-(13) C]glutamate/[3-(13) C]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 V(PDH) /V(TCA) 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 V(PDH) /V(TCA) 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 V(PDH) /V(TCA) 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 V(PDH) /V(TCA) flux. Only under conditions of combined hyperglycemia and hyperinsulinemia did V(PDH) /V(TCA) 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 V(PDH) /V(TCA) flux in vivo and that this flux is severely diminished in rats with chronic lipid-induced hepatic insulin resistance.

Intracellular energetics and critical PO2 in resting ischemic human skeletal muscle in vivo.

During ischemia and some types of muscular contractions, oxygen tension (Po(2)) declines to the point that mitochondrial ATP synthesis becomes limited by oxygen availability. Although this critical Po(2) has been determined in animal tissue in vitro and in situ, there remains controversy concerning potential disparities between values measured in vivo and ex vivo. To address this issue, we used concurrent heteronuclear magnetic resonance spectroscopy (MRS) to determine the critical intracellular Po(2) in resting human skeletal muscle in vivo. We interleaved measurements of deoxymyoglobin using (1)H-MRS with measures of high-energy phosphates and pH using (31)P-MRS, during 15 min of ischemia in the tibialis anterior muscles of 6 young men. ATP production and intramyocellular Po(2) were quantified throughout ischemia. Critical Po(2), determined as the Po(2) corresponding to the point where PCr begins to decline (PCr(ip)) in resting muscle during ischemia, was 0.35 ± 0.20 Torr, means ± SD. This in vivo value is consistent with reported values ex vivo and does not support the notion that critical Po(2) in resting muscle is higher when measured in vivo. Furthermore, we observed a 4.5-fold range of critical Po(2) values among the individuals studied. Regression analyses revealed that time to PCr(ip) was associated with critical Po(2) and the rate of myoglobin desaturation (r = 0.83, P = 0.04) but not the rate of ATP consumption during ischemia. The apparent dissociation between ATP demand and myoglobin deoxygenation during ischemia suggests that some degree of uncoupling between intracellular energetics and oxygenation is a potentially important factor that influences critical Po(2) in vivo.

Resistance to thyroid hormone is associated with raised energy expenditure, muscle mitochondrial uncoupling, and hyperphagia.

Resistance to thyroid hormone (RTH), a dominantly inherited disorder usually associated with mutations in thyroid hormone receptor beta (THRB), is characterized by elevated levels of circulating thyroid hormones (including thyroxine), failure of feedback suppression of thyrotropin, and variable tissue refractoriness to thyroid hormone action. Raised energy expenditure and hyperphagia are recognized features of hyperthyroidism, but the effects of comparable hyperthyroxinemia in RTH patients are unknown. Here, we show that resting energy expenditure (REE) was substantially increased in adults and children with THRB mutations. Energy intake in RTH subjects was increased by 40%, with marked hyperphagia particularly evident in children. Rates of muscle TCA cycle flux were increased by 75% in adults with RTH, whereas rates of ATP synthesis were unchanged, as determined by 13C/31P magnetic resonance spectroscopy. Mitochondrial coupling index between ATP synthesis and mitochondrial rates of oxidation (as estimated by the ratio of ATP synthesis to TCA cycle flux) was significantly decreased in RTH patients. These data demonstrate that basal mitochondrial substrate oxidation is increased and energy production in the form of ATP synthesis is decreased in the muscle of RTH patients and that resting oxidative phosphorylation is uncoupled in this disorder. Furthermore, these observations suggest that mitochondrial uncoupling in skeletal muscle is a major contributor to increased REE in patients with RTH, due to tissue selective retention of thyroid hormone receptor alpha sensitivity to elevated thyroid hormone levels.

Assessment of in vivo mitochondrial metabolism by magnetic resonance spectroscopy.

Magnetic resonance spectroscopy (MRS), a companion technique to the more familiar MRI scan, has emerged as a powerful technique for studying metabolism noninvasively in a variety of tissues. In this article, we review two techniques that we have developed which take advantage of the unique characteristics of (31)P and (13)C MRS to investigate two distinct parameters of muscle mitochondrial metabolism; ATP production can be estimated by using the (31)P saturation-transfer technique, and oxidation via the TCA cycle can be modeled from (13)C MRS data obtained during the metabolism of a (13)C-labeled substrate. We will also examine applications of the techniques to investigate how these parameters of muscle mitochondrial metabolism are modulated in insulin resistant and endurance trained individuals.

Intramyocellular oxygenation during ischemic muscle contractions in vivo.

There is some evidence that the fall in intramyocellular oxygen content during ischemic contractions is less than during ischemia alone. We used proton magnetic resonance spectroscopy to determine whether peak deoxy-myoglobin (dMb) obtained during ischemic ankle dorsiflexion contractions attained the maximal dMb level observed during a separate trial of ischemia alone (resting max). In six healthy young men, the rate of myoglobin desaturation was rapid at the onset of ischemic contractions and then slowed as contractions continued, attaining only 75 +/- 3.3% (mean +/- SE) of resting max dMb by the end of contractions (p = 0.03). Myoglobin continued to desaturate while ischemia was maintained following contractions, reaching 98 +/- 1.8% of resting max within 10 min (p = 0.03 vs. end of contractions). Notably, contractions performed after 10 min of ischemia did not affect dMb (dMb = 100 +/- 1.5% of resting max, p > 0.99), suggesting that full desaturation had already been achieved. The blunting of desaturation during ischemic contractions is likely a result of slowed mitochondrial oxygen consumption due to limited oxygen availability.

Paradoxical effects of increased expression of PGC-1alpha on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism.

Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha has been shown to play critical roles in regulating mitochondria biogenesis, respiration, and muscle oxidative phenotype. Furthermore, reductions in the expression of PGC-1alpha in muscle have been implicated in the pathogenesis of type 2 diabetes. To determine the effect of increased muscle-specific PGC-1alpha expression on muscle mitochondrial function and glucose and lipid metabolism in vivo, we examined body composition, energy balance, and liver and muscle insulin sensitivity by hyperinsulinemic-euglycemic clamp studies and muscle energetics by using (31)P magnetic resonance spectroscopy in transgenic mice. Increased expression of PGC-1alpha in muscle resulted in a 2.4-fold increase in mitochondrial density, which was associated with an approximately 60% increase in the unidirectional rate of ATP synthesis. Surprisingly, there was no effect of increased muscle PGC-1alpha expression on whole-body energy expenditure, and PGC-1alpha transgenic mice were more prone to fat-induced insulin resistance because of decreased insulin-stimulated muscle glucose uptake. The reduced insulin-stimulated muscle glucose uptake could most likely be attributed to a relative increase in fatty acid delivery/triglyceride reesterfication, as reflected by increased expression of CD36, acyl-CoA:diacylglycerol acyltransferase1, and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase, that may have exceeded mitochondrial fatty acid oxidation, resulting in increased intracellular lipid accumulation and an increase in the membrane to cytosol diacylglycerol content. This, in turn, caused activation of PKC, decreased insulin signaling at the level of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and skeletal muscle insulin resistance.

Increased substrate oxidation and mitochondrial uncoupling in skeletal muscle of endurance-trained individuals.

Endurance exercise training is accompanied by physiological changes that improve muscle function and performance. Several studies have demonstrated that markers of mitochondrial capacity are elevated, however, these studies tend to be performed ex vivo under conditions that yield maximal enzyme activities or in vivo but monitoring the response to exercise. Therefore, it is unclear whether basal mitochondrial metabolism is affected by exercise training. To explore whether resting muscle metabolism was altered in trained individuals in vivo, two independent parameters of metabolic function-tricarboxylic acid (TCA) cycle flux (V(TCA)), and ATP synthesis (V(ATP))-were assessed noninvasively by using magnetic resonance spectroscopy in a cohort of young endurance trained subjects (n = 7) and a group of matched sedentary subjects (n = 8). V(TCA) was 54% higher in the muscle of endurance trained compared with sedentary subjects (91.7 +/- 7.6 vs. 59.6 +/- 4.9 nmol/g/min, P < 0.01); however, V(ATP) was not different between the trained and sedentary subjects (5.98 +/- 0.43 vs. 6.35 +/- 0.70 mumol/g/min, P = 0.67). The ratio V(ATP)/V(TCA) (an estimate of mitochondrial coupling) was also significantly reduced in trained subjects (P < 0.04). These data demonstrate that basal mitochondrial substrate oxidation is increased in the muscle of endurance trained individuals yet energy production is unaltered, leading to an uncoupling of oxidative phosphorylation at rest. Increased mitochondrial uncoupling may represent another mechanism by which exercise training enhances muscle insulin sensitivity via increased fatty acid oxidation in the resting state.

Contraction frequency modulates muscle fatigue and the rate of myoglobin desaturation during incremental contractions in humans.

The metabolic cost of force production, and therefore the demand for oxygen, increases with intensity and frequency of contraction. This study investigated the interaction between fatigue and oxygenation, as reflected by deoxymyoglobin (dMb), during slow and rapid rhythmic isometric contractions having the same duty cycles and relative force-time integrals (FTIs). We used 1H magnetic resonance spectroscopy and measures of dorsiflexor muscle force to compare dMb and fatigue (fall of maximal voluntary force, MVC) in 11 healthy adults (29 +/- 7 y) during 16 min of slow (4 s contraction, 6 s relaxation) and rapid (1.2 s, 1.8 s) incremental (10%-80% MVC) contractions. We tested the hypotheses that (i) the rate of Mb desaturation would be faster in rapid than in slow contractions and (ii) fatigue, Mb desaturation, and the fall in FTI would be greater, and PO2 (oxygen tension) lower, at the end of rapid contractions than at the end of slow contractions. Although dMb increased more quickly during rapid contractions (p = 0.05), it reached a plateau at a similar level in both protocols (approximately 42% max, p = 0.49), likely due to an inability to further increase force production and thus metabolic demand. Despite the similar dMb at the end of both protocols, fatigue was greater in rapid (56.6% +/- 2.7% baseline) than in slow (69.5% +/- 4.0%, p = 0.01) contractions. These results indicate that human skeletal muscle fatigue during incremental isometric contractions is in part a function of contraction frequency, possibly due to metabolic inhibition of the contractile process.

Comparative MR study of hepatic fat quantification using single-voxel proton spectroscopy, two-point dixon and three-point IDEAL.

Hepatic fat fraction (HFF) was measured in 28 lean/obese humans by single-voxel proton spectroscopy (MRS), a two-point Dixon (2PD), and a three-point iterative decomposition of water and fat with echo asymmetry and least-squares estimation (IDEAL) method (3PI). For the lean, obese, and total subject groups, the range of HFF measured by MRS was 0.3-3.5% (1.1 +/- 1.4%), 0.3-41.5% (11.7 +/- 12.1), and 0.3-41.5% (10.1 +/- 11.6%), respectively. For the same groups, the HFF measured by 2PD was -6.3-2.2% (-2.0 +/- 3.7%), -2.4-42.9% (12.9 +/- 13.8%), and -6.3-42.9% (10.5 +/- 13.7%), respectively, and for 3PI they were 7.9-12.8% (10.1 +/- 2.0%), 11.1-49.3% (22.0 +/- 12.2%), and 7.9-49.3% (20.0 +/- 11.8%), respectively. The HFF measured by MRS was highly correlated with those measured by 2PD (r = 0.954, P < 0.001) and 3PI (r = 0.973, P < 0.001). With the MRS data as a reference, the percentages of correct differentiation between normal and fatty liver with the MRI methods ranged from 68-93% for 2PD and 64-89% for 3PI. Our study demonstrates that the apparent HFF measured by the MRI methods can significantly vary depending on the choice of water-fat separation methods and sequences. Such variability may limit the clinical application of the MRI methods, particularly when a diagnosis of early fatty liver needs to be performed. Therefore, protocol-specific establishment of cutoffs for liver fat content may be necessary.