Skeletal muscle mitoflashes, pH, and the role of uncoupling protein-3.

Mitochondrial reactive oxygen species (ROS) are important cellular signaling molecules, but can cause oxidative damage if not kept within tolerable limits. An important proximal form of ROS in mitochondria is superoxide. Its production is thought to occur in regulated stochastic bursts, but current methods using mitochondrial targeted cpYFP to assess superoxide flashes are confounded by changes in pH. Accordingly, these flashes are generally referred to as 'mitoflashes'. Here we provide regulatory insights into mitoflashes and pH fluctuations in skeletal muscle, and the role of uncoupling protein-3 (UCP3). Using quantitative confocal microscopy of mitoflashes in intact muscle fibers, we show that the mitoflash magnitude significantly correlates with the degree of mitochondrial inner membrane depolarization and ablation of UCP3 did not affect this correlation. We assessed the effects of the absence of UCP3 on mitoflash activity in intact skeletal muscle fibers, and found no effects on mitoflash frequency, amplitude or duration, with a slight reduction in the average size of mitoflashes. We further investigated the regulation of pH flashes (pHlashes, presumably a component of mitoflash) by UCP3 using mitochondrial targeted SypHer (mt-SypHer) in skeletal muscle fibers. The frequency of pHlashes was significantly reduced in the absence of UCP3, without changes in other flash properties. ROS scavenger, tiron, did not alter pHlash frequency in either WT or UCP3KO mice. High resolution respirometry revealed that in the absence of UCP3 there is impaired proton leak and Complex I-driven respiration and maximal coupled respiration. Total cellular production of hydrogen peroxide (H2O2) as detected by Amplex-UltraRed was unaffected. Altogether, we demonstrate a correlation between mitochondrial membrane potential and mitoflash magnitude in skeletal muscle fibers that is independent of UCP3, and a role for UCP3 in the control of pHlash frequency and of proton leak- and Complex I coupled-respiration in skeletal muscle fibers. The differential regulation of mitoflashes and pHlashes by UCP3 and tiron also indicate that the two events, though may be related, are not identical events.

IL-15 improves skeletal muscle oxidative metabolism and glucose uptake in association with increased respiratory chain supercomplex formation and AMPK pathway activation.

BACKGROUND: IL-15 is believed to play a role in the beneficial impact of exercise on muscle energy metabolism. However, previous studies have generally used supraphysiological levels of IL-15 that do not represent contraction-induced IL-15 secretion. METHODS: L6 myotubes were treated acutely (3 h) and chronically (48 h) with concentrations of IL-15 mimicking circulating (1-10 pg/ml) and muscle interstitial (100 pg/ml -20 ng/ml) IL-15 levels with the aim to better understand its autocrine/paracrine role on muscle glucose uptake and mitochondrial function. RESULTS: Acute exposure to IL-15 levels representing muscle interstitial IL-15 increased basal glucose uptake without affecting insulin sensitivity. This was accompanied by increased mitochondrial oxidative functions in association with increased AMPK pathway and formation of complex III-containing supercomplexes. Conversely, chronic IL-15 exposure resulted in a biphasic effect on mitochondrial oxidative functions and ETC supercomplex formation was increased with low IL-15 levels but decreased with higher IL-15 concentrations. The AMPK pathway was activated only by high levels of chronic IL-15 treatment. Similar results were obtained in skeletal muscle from muscle-specific IL-15 overexpressing mice that show very high circulating IL-15 levels. CONCLUSIONS: Acute IL-15 treatment that mimics local IL-15 concentrations enhances muscle glucose uptake and mitochondrial oxidative functions. That mitochondria respond differently to different levels of IL-15 during chronic treatments indicates that IL-15 might activate two different pathways in muscle depending on IL-15 concentrations. GENERAL SIGNIFICANCE: Our results suggest that IL-15 may act in an autocrine/paracrine fashion and be, at least in part, involved in the positive effect of exercise on muscle energy metabolism.

Diet-resistant obesity is characterized by a distinct plasma proteomic signature and impaired muscle fiber metabolism.

BACKGROUND/OBJECTIVES: Inter-individual variability in weight loss during obesity treatment is complex and poorly understood. Here we use whole body and tissue approaches to investigate fuel oxidation characteristics in skeletal muscle fibers, cells and distinct circulating protein biomarkers before and after a high fat meal (HFM) challenge in those who lost the most (obese diet-sensitive; ODS) vs the least (obese diet-resistant; ODR) amount of weight in a highly controlled weight management program. SUBJECTS/METHODS: In 20 weight stable-matched ODS and ODR women who previously completed a standardized clinical weight loss program, we analyzed whole-body energetics and metabolic parameters in vastus lateralis biopsies and plasma samples that were obtained in the fasting state and 6 h after a defined HFM, equivalent to 35% of total daily energy requirements. RESULTS: At baseline (fasting) and post-HFM, muscle fatty acid oxidation and maximal oxidative phosphorylation were significantly greater in ODS vs ODR, as was reactive oxygen species emission. Plasma proteomics of 1130 proteins pre and 1, 2, 5 and 6 h after the HFM demonstrated distinct group and interaction differences. Group differences identified S-formyl glutathione hydratase, heat shock 70 kDA protein 1A/B (HSP72), and eukaryotic translation initiation factor 5 (eIF5) to be higher in ODS vs ODR. Group-time differences included aryl hydrocarbon interacting protein (AIP), peptidylpropyl isomerase D (PPID) and tyrosine protein-kinase Fgr, which increased in ODR vs ODS over time. HSP72 levels correlated with muscle oxidation and citrate synthase activity. These proteins circulate in exosomes; exosomes isolated from ODS plasma increased resting, leak and maximal respiration rates in C2C12 myotubes by 58%, 21% and 51%, respectively, vs those isolated from ODR plasma. CONCLUSIONS: Findings demonstrate distinct muscle metabolism and plasma proteomics in fasting and post-HFM states corresponding in diet-sensitive vs diet-resistant obese women.

Lactobacillus-produced exopolysaccharides and their potential health benefits: a review.

Lactic acid bacteria, such as those of the Lactobacillus genus, naturally reside within the microbiota of the human body and have long been used as starter cultures and probiotic enhancers in fermented foods, such as fermented drinks, yoghurts and cheeses. Many of the beneficial qualities of these bacteria have traditionally been associated with the bacteria themselves, however, a recent spate of studies have demonstrated a wide variety of biological effects exhibited by lactobacilli-produced exopolysaccharides which could, theoretically, confer a range of local and systemic health benefits upon the host. In this review, we discuss the production of exopolysaccharides within the Lactobacillus genus and explore their potential as beneficial bioactive compounds.