Terence E. Ryan

Adipose Mitochondrial Biogenesis Is Suppressed in db/db and High-Fat Diet–Fed Mice and Improved by Rosiglitazone

The objective of this study was to further establish and confirm the relationship of adipose mitochondrial biogenesis in diabetes/obesity and the effects of rosiglitazone (RSG), a peroxisome proliferator–activated receptor (PPAR) γ agonist, by systematically analyzing mitochondrial gene expression and function in two mouse models of obesity and type 2 diabetes. Using microarray technology, adipose mitochondrial gene transcription was studied in db/db, high-fat diet–fed C57BL/6 (HFD) and respective control mice with or without RSG treatment. The findings were extended using mitochondrial staining, DNA quantification, and measurements of citrate synthase activity. In db/db and HFD mice, gene transcripts associated with mitochondrial ATP production, energy uncoupling, mitochondrial ribosomal proteins, outer and inner membrane translocases, and mitochondrial heat-shock proteins were decreased in abundance, compared with db/+ and standard-fat diet–fed control mice, respectively. RSG dose-dependently increased these transcripts in both db/db and HFD mice and induced transcription of mitochondrial structural proteins and cellular antioxidant enzymes responsible for removal of reactive oxygen species generated by increased mitochondrial activity. Transcription factors, including PPAR coactivator (PGC)-1β, PGC-1α, estrogen-related receptor α, and PPARα, were suppressed in both models and induced by RSG. The effects of RSG on adipose mitochondrial genes were confirmed by quantitative RT-PCR and further supported by mitochondrial staining, mitochondrial DNA quantification, and citrate synthase activity. Adipose mitochondrial biogenesis was overwhelmingly suppressed in both mouse models of diabetes/obesity and globally induced by RSG. These findings suggest an important role of adipose mitochondria in diabetes/obesity and the potential for new treatment approaches targeting adipose mitochondria.

Noninvasive evaluation of skeletal muscle mitochondrial capacity with near-infrared spectroscopy: correcting for blood volume changes

Near-infrared spectroscopy (NIRS) is a well-known method used to measure muscle oxygenation and hemodynamics in vivo. The application of arterial occlusions allows for the assessment of muscle oxygen consumption (mVo(2)) using NIRS. The aim of this study was to measure skeletal muscle mitochondrial capacity using blood volume-corrected NIRS signals that represent oxygenated hemoglobin/myoglobin (O(2)Hb) and deoxygenated hemoglobin/myoglobin (HHb). We also assessed the reliability and reproducibility of NIRS measurements of resting oxygen consumption and mitochondrial capacity. Twenty-four subjects, including four with chronic spinal cord injury, were tested using either the vastus lateralis or gastrocnemius muscles. Ten healthy, able-bodied subjects were tested on two occasions within a period of 7 days to assess the reliability and reproducibility. NIRS signals were corrected for blood volume changes using three different methods. Resting oxygen consumption had a mean coefficient of variation (CV) of 2.4% (range 1-32%). The recovery of oxygen consumption (mVo(2)) after electrical stimulation at 4 Hz was fit to an exponential curve, which represents mitochondrial capacity. The time constant for the recovery of mVo(2) was reproducible with a mean CV of 10% (range 1-22%) only when correcting for blood volume changes. We also examined the effects of adipose tissue thickness on measurements of mVo(2). We found the mVo(2) measurements using absolute units to be influenced by adipose tissue thickness (ATT), and this relationship was removed when an ischemic calibration was performed, supporting its use to compare mVo(2) between individuals of varying ATT. In conclusion, in vivo oxidative capacity can be assessed using blood volume-corrected NIRS signals with a high degree of reliability and reproducibility.

Isocitrate-to-SENP1 signaling amplifies insulin secretion and rescues dysfunctional β cells

Insulin secretion from β cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing β cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues β cell function in T2D.

Pyruvate dehydrogenase complex and nicotinamide nucleotide transhydrogenase constitute an energy-consuming redox circuit

Cellular proteins rely on reversible redox reactions to establish and maintain biological structure and function. How redox catabolic (NAD+/NADH) and anabolic (NADP+/NADPH) processes integrate during metabolism to maintain cellular redox homoeostasis, however, is unknown. The present work identifies a continuously cycling mitochondrial membrane potential (ΔΨm)-dependent redox circuit between the pyruvate dehydrogenase complex (PDHC) and nicotinamide nucleotide transhydrogenase (NNT). PDHC is shown to produce H2O2 in relation to reducing pressure within the complex. The H2O2 produced, however, is effectively masked by a continuously cycling redox circuit that links, via glutathione/thioredoxin, to NNT, which catalyses the regeneration of NADPH from NADH at the expense of ΔΨm. The net effect is an automatic fine-tuning of NNT-mediated energy expenditure to metabolic balance at the level of PDHC. In mitochondria, genetic or pharmacological disruptions in the PDHC-NNT redox circuit negate counterbalance changes in energy expenditure. At the whole animal level, mice lacking functional NNT (C57BL/6J) are characterized by lower energy-expenditure rates, consistent with their well-known susceptibility to diet-induced obesity. These findings suggest the integration of redox sensing of metabolic balance with compensatory changes in energy expenditure provides a potential mechanism by which cellular redox homoeostasis is maintained and body weight is defended during periods of positive and negative energy balance.

Skeletal muscle metabolism in individuals with spinal cord injury

With increasing survival rates in people with spinal cord injuries (SCI), detection and prevention of metabolic and cardiovascular disease have become increasingly important. Few studies have evaluated in vivo mitochondrial function in paralyzed skeletal muscle. The purpose of this study was to compare oxidative muscle metabolism using the rate of phosphocreatine (PCr) resynthesis measured by magnetic resonance spectroscopy (MRS) in people with SCI and able-bodied (AB) controls. Eight subjects with complete SCI (American Spinal Injury Association Impairment Scale A, levels T3-T12, injury duration 2-13 years) were compared with 12 AB controls. T1-weighted (1)H MR images of the thigh were taken to identify skeletal muscle. Phosphorous MRS was performed with a 13 × 13-cm(2) surface coil placed on the right vastus lateralis in a 3 Tesla clinical MRI scanner. PCr resynthesis was measured after electrical stimulation for 60 s at 4 Hz in SCI and AB and in AB subjects after 39 s of voluntary isometric contractions. Resting metabolites were not different between SCI and AB, except for an elevated phosphodiester peak. PCr recovery was slower in AB subjects using electrical stimulation compared with voluntary exercise (28.4 ± 6.1 vs. 41.5 ± 4.3 s; P < 0.05). PCr recovery rates and calculated muscle maximum oxidative capacity in SCI were both 52% of electrically stimulated AB (P < 0.001). In vivo oxidative metabolism was reduced in paralyzed muscle to a similar extent as seen in people with mitochondrial myopathies and heart failure.

Assessment of in vivo skeletal muscle mitochondrial respiratory capacity in humans by near-infrared spectroscopy: a comparison with in situ measurements.

In vivo skeletal muscle mitochondrial respiratory capacity was determined from the post‐exercise recovery kinetics of muscle oxygen consumption ( mVO2 ) measured using near‐infrared spectroscopy (NIRS) in humans. NIRS recovery rates were compared with the in situ gold standard of high‐resolution respirometry measured in permeabilized muscle fibre bundles prepared from muscle biopsies taken from the same participants. NIRS‐measured recovery kinetics of mVO2 were well correlated with maximal ADP‐stimulated mitochondrial respiration in permeabilized fibre bundles. NIRS provides a cost‐effective, non‐invasive means of assessing in vivo mitochondrial respiratory capacity.

Skeletal muscle metabolism in endurance athletes with near-infrared spectroscopy.

PURPOSE To determine whether near-infrared spectroscopy (NIRS) measurements of muscle mitochondrial function could detect the expected differences between endurance-trained athletes (n = 8) and inactive subjects (n = 8). METHODS Muscle oxygen consumption (mV˙O2) of the vastus lateralis was measured with continuous-wave NIRS using transient arterial occlusions. The recovery rate of mV˙O2 after electrical stimulation was fit to an exponential curve, with the time constant (Tc) used as an index of mitochondrial capacity. Whole-body peak oxygen uptake was determined by indirect calorimetry during a continuous ramp protocol on a cycle ergometer. RESULTS Whole-body peak oxygen uptake values for endurance-trained and inactive controls were 73.5 ± 9.1 and 33.7 ± 5.9 mL·kg·min, respectively (P < 0.001). The recovery rates of mV˙O2 after exercise for endurance training were 18.4 ± 3.2 and 18.8 ± 2.5 s, whereas those for inactive controls were 32.4 ± 5.2 and 34.9 ± 5.9 s for the shallow and deep channels, respectively (P < 0.001 for comparison between groups). Resting mV˙O2 was 0.52%·s ± 0.22%·s for endurance athletes and 0.77%·s ± 0.82%·s for inactive controls (P = 0.42). CONCLUSIONS The recovery rates of mV˙O2 after exercise in endurance athletes were almost twofold faster than inactive subjects measured with NIRS, consistent with previous studies using muscle biopsies and magnetic resonance spectroscopy. Our results support the use of NIRS measurements of the recovery of oxygen consumption to assess muscle oxidative capacity.

Activity-induced changes in skeletal muscle metabolism measured with optical spectroscopy.

PURPOSE Previous studies have used near-infrared spectroscopy (NIRS) to measure skeletal muscle mitochondrial capacity. This study tested the hypothesis that NIRS-measured mitochondrial capacity would improve with endurance exercise training and decline with detraining. METHODS Nine young participants performed 4 wk of progressively increasing endurance exercise training of the wrist flexor muscles followed by approximately 5 wk of inactivity. The rate of recovery of muscle oxygen consumption (mV(˙)O₂) was measured with NIRS every 3-7 d, indicating mitochondrial oxidative capacity. RESULTS A linear increase in mitochondrial capacity (NIRS rate constant) was found with a group average of 64% ± 37% improvement after 4 wk of exercise training (P < 0.05). Mitochondrial capacity declined exponentially upon cessation of exercise training, with a mean half-time of approximately 7.7 d. CONCLUSIONS Both the magnitude and the time course of mitochondrial adaptations to exercise training and detraining measured with NIRS was consistent with previous studies using both in vitro and in vivo techniques. These findings show that NIRS-based measurements can detect meaningful changes in mitochondrial capacity.

A Direct Comparison of Metabolic Responses to High-Fat Diet in C57BL/6J and C57BL/6NJ Mice

Although nicotinamide nucleotide transhydrogenase (NNT)–deficient C57BL/6J (6J) mice are known to be highly susceptible to diet-induced metabolic disease, this notion stems primarily from comparisons of 6J mice to other inbred strains. To date, very few studies have directly compared metabolic disease susceptibility between NNT-deficient 6J mice and NNT-competent C57BL/6 substrains. In this study, comprehensive profiling of the metabolic response to a high-fat/high-sucrose diet (HFD) were compared across time in 6J and C57BL/6NJ (6N) mice. Given that increased peroxide exposure drives insulin resistance, coupled with the fact that NNT regulates peroxide detoxification, it was hypothesized that 6J mice would experience greater derangements in redox homeostasis/metabolic disease upon HFD exposure. Contrary to this, both lines were found to be highly susceptible to diet-induced metabolic disease, as evidenced by impairments in glucose tolerance as early as 24 h into the HFD. Moreover, various markers of the metabolic syndrome, as well as peroxide stress, were actually blunted, rather than exacerbated, in the 6J mice, likely reflecting compensatory increases in alterative redox-buffering pathways. Together, these data provide evidence that the susceptibility to HFD-induced metabolic disease is similar in the 6J and 6N substrains. Given the numerous genetic variances in the 6J stain, including loss of NNT function, these findings suggest that the 6N substrain is the more logical and representative genetic background model for metabolic studies.

Rosiglitazone Induces Mitochondrial Biogenesis in Differentiated Murine 3T3-L1 and C3H/10T1/2 Adipocytes

Growing evidence indicates that PPARγ agonists, including rosiglitazone (RSG), induce adipose mitochondrial biogenesis. By systematically analyzing mitochondrial gene expression in two common murine adipocyte models, the current study aimed to further establish the direct role of RSG and capture temporal changes in gene transcription. Microarray profiling revealed that in fully differentiated 3T3-L1 and C3H/10T1/2 adipocytes treated with RSG or DMSO vehicle for 1, 2, 4, 7, 24, and 48 hrs, RSG overwhelmingly increased mitochondrial gene transcripts time dependently. The timing of the increases was consistent with the cascade of organelle biogenesis, that is, initiated by induction of transcription factor(s), followed by increases in the biosynthesis machinery, and then by increases in functional components. The transcriptional increases were further validated by increased mitochondrial staining, citrate synthase activity, and O2 consumption, and were found to be associated with increased adiponectin secretion. The work provided further insight on the mechanism of PPARγ-induced mitochondrial biogenesis in differentiated adipocytes.