Invasive and noninvasive markers of human skeletal muscle mitochondrial function.

Mitochondria are organelles that fuel cellular energy requirements by ATP formation via aerobic metabolism. Given the wide variety of methods to assess skeletal muscle mitochondrial capacity, we tested how well different invasive and noninvasive markers of skeletal muscle mitochondrial capacity reflect mitochondrial respiration in permeabilized muscle fibers. Nineteen young men (mean age: 24 ± 4 years) were recruited, and a muscle biopsy was collected to determine mitochondrial respiration from permeabilized muscle fibers and to quantify markers of mitochondrial capacity, content such as citrate synthase (CS) activity, mitochondrial DNA copy number, TOMM20, VDAC, and protein content for complex I-V of the oxidative phosphorylation (OXPHOS) system. Additionally, all participants underwent noninvasive assessments of mitochondrial capacity: PCr recovery postexercise (by 31 P-MRS), maximal aerobic capacity, and gross exercise efficiency by cycling exercise. From the invasive markers, Complex V protein content and CS activity showed the strongest concordance (Rc = 0.50 to 0.72) with ADP-stimulated coupled mitochondrial respiration, fueled by various substrates. Complex V protein content showed the strongest concordance (Rc = 0.72) with maximally uncoupled mitochondrial respiration. From the noninvasive markers, gross exercise efficiency, VO2max , and PCr recovery exhibited concordance values between 0.50 and 0.77 with ADP-stimulated coupled mitochondrial respiration. Gross exercise efficiency showed the strongest concordance with maximally uncoupled mitochondrial respiration (Rc = 0.67). From the invasive markers, Complex V protein content and CS activity are surrogates that best reflect skeletal muscle mitochondrial respiratory capacity. From the noninvasive markers, exercise efficiency and PCr recovery postexercise most closely reflect skeletal muscle mitochondrial respiratory capacity.

Postexercise changes in myocellular lipid droplet characteristics of young lean individuals are affected by circulatory nonesterified fatty acids.

Intramyocellular lipid (IMCL) content is an energy source during acute exercise. Nonesterified fatty acid (NEFA) levels can compete with IMCL utilization during exercise. IMCL content is stored as lipid droplets (LDs) that vary in size, number, subcellular distribution, and in coating with LD protein PLIN5. Little is known about how these factors are affected during exercise and recovery. Here, we aimed to investigate the effects of acute exercise with and without elevated NEFA levels on intramyocellular LD size and number, intracellular distribution and PLIN5 coating, using high-resolution confocal microscopy. In a crossover study, 9 healthy lean young men performed a 2-h moderate intensity cycling protocol in the fasted (high NEFA levels) and glucose-fed state (low NEFA levels). IMCL and LD parameters were measured at baseline, directly after exercise and 4 h postexercise. We found that total IMCL content was not changed directly after exercise (irrespectively of condition), but IMCL increased 4 h postexercise in the fasting condition, which was due to an increased number of LDs rather than changes in size. The effects were predominantly detected in type I muscle fibers and in LDs coated with PLIN5. Interestingly, subsarcolemmal, but not intermyofibrillar IMCL content, was decreased directly after exercise in the fasting condition and was replenished during the 4 h recovery period. In conclusion, acute exercise affects IMCL storage during exercise and recovery, particularly in type I muscle fibers, in the subsarcolemmal region and in the presence of PLIN5. Moreover, the effects of exercise on IMCL content are affected by plasma NEFA levels.NEW & NOTEWORTHY Skeletal muscle stores lipids in lipid droplets (LDs) that can vary in size, number, and location and are a source of energy during exercise. Specifically, subsarcolemmal LDs were used during exercise when fasted. Exercising in the fasted state leads to postrecovery elevation in IMCL levels due to an increase in LD number in type I muscle fibers, in subsarcolemmal region and decorated with PLIN5. These effects are blunted by glucose ingestion during exercise and recovery.

Circadian misalignment disturbs the skeletal muscle lipidome in healthy young men.

Circadian misalignment, as seen in shift work, is associated with an increased risk to develop type 2 diabetes. In an experimental setting, we recently showed that a rapid day-night shift for 3 consecutive nights leads to misalignment of the core molecular clock, induction of the PPAR pathway, and insulin resistance in skeletal muscle of young, healthy men. Here, we investigated if circadian misalignment affects the skeletal muscle lipidome and intramyocellular lipid droplet characteristics, explaining the misalignment-induced insulin resistance. Fourteen healthy men underwent one aligned and one circadian misalignment period, both consisting of ~3.5 days. In the misaligned condition, day and night were rapidly shifted by 12 hours leading to opposite eating, sleep, and activity times compared with the aligned condition. For each condition, two muscle biopsies were taken from the m. vastus lateralis in the morning and evening and subjected to semi-targeted lipidomics and confocal microscopy analysis. We found that only 2% of detected lipids were different between morning and evening in the aligned condition, whereas 12% displayed a morning-evening difference upon misalignment. Triacylglycerols, in particular species of a carbon length ≥55, were the most abundant lipid species changed upon misalignment. Cardiolipins were decreased upon misalignment, whereas phosphatidylcholines consistently followed the same morning-evening pattern, suggesting regulation by the circadian clock. Cholesteryl esters adjusted to the shifted behavior. Lipid droplet characteristics remained unaltered upon misalignment. Together, these findings show that simulated shift work disturbs the skeletal muscle lipidome, which may contribute to misalignment-induced insulin resistance.

Resveratrol-induced remodelling of myocellular lipid stores: A study in metabolically compromised humans.

In non-athletes, insulin sensitivity correlates negatively with intramyocellular lipid (IMCL) content. In athletes, however, a pattern of benign IMCL storage exists, which is characterized by lipid storage in type I muscle fibres, in small and numerous lipid droplets (LDs) preferable coated with PLIN5, without affecting insulin sensitivity. Administration of resveratrol has been promoted for its beneficial effects on glucose homeostasis. We observed that 30 days of oral resveratrol administration (150 mg/day) in metabolically compromised individuals showed a 33% increase in IMCL (placebo vs. resveratrol; 0.86 ± 0.090 AU vs. 1.14 ± 0.11 AU, p = 0.003) without impeding insulin sensitivity. Thus, the aim of the present study was to examine if a resveratrol-mediated increase in IMCL content, in metabolically compromised individuals, changes the LD phenotype towards the phenotype we previously observed in athletes. For this, we studied IMCL, LD number, LD size, subcellular distribution and PLIN5 coating in different fibre types using high-resolution confocal microscopy. As proof of concept, we observed a 2.3-fold increase (p = 0.038) in lipid accumulation after 48 h of resveratrol incubation in cultured human primary muscle cells. In vivo analysis showed that resveratrol-induced increase in IMCL is predominantly in type I muscle fibres (placebo vs. resveratrol; 0.97 ± 0.16% vs. 1.26 ± 0.09%; p = 0.030) in both the subsarcolemmal (p = 0.016) and intermyofibrillar region (p = 0.026) and particularly in PLIN5-coated LDs (p = 0.024). These data indicate that administration of resveratrol augments IMCL content in metabolically compromised individuals towards a LD phenotype that mimics an 'athlete like phenotype'.

Human skeletal muscle mitochondrial dynamics in relation to oxidative capacity and insulin sensitivity.

AIMS/HYPOTHESIS: Mitochondria operate in networks, adapting to external stresses and changes in cellular metabolic demand and are subject to various quality control mechanisms. On the basis of these traits, we here hypothesise that the regulation of mitochondrial networks in skeletal muscle is hampered in humans with compromised oxidative capacity and insulin sensitivity. METHODS: In a cross-sectional design, we compared four groups of participants (selected from previous studies) ranging in aerobic capacity and insulin sensitivity, i.e. participants with type 2 diabetes (n = 11), obese participants without diabetes (n = 12), lean individuals (n = 10) and endurance-trained athletes (n = 12); basal, overnight fasted muscle biopsies were newly analysed for the current study and we compared the levels of essential mitochondrial dynamics and quality control regulatory proteins in skeletal muscle tissue. RESULTS: Type 2 diabetes patients and obese participants were older than lean participants and athletes (58.6 ± 4.0 and 56.7 ± 7.2 vs 21.8 ± 2.5 and 25.1 ± 4.3 years, p < 0.001, respectively) and displayed a higher BMI (32.4 ± 3.7 and 31.0 ± 3.7 vs 22.1 ± 1.8 and 21.0 ± 1.5 kg/m2, p < 0.001, respectively) than lean individuals and endurance-trained athletes. Fission protein 1 (FIS1) and optic atrophy protein 1 (OPA1) protein content was highest in muscle from athletes and lowest in participants with type 2 diabetes and obesity, respectively (FIS1: 1.86 ± 0.79 vs 0.79 ± 0.51 AU, p = 0.002; and OPA1: 1.55 ± 0.64 vs 0.76 ± 0.52 AU, p = 0.014), which coincided with mitochondrial network fragmentation in individuals with type 2 diabetes, as assessed by confocal microscopy in a subset of type 2 diabetes patients vs endurance-trained athletes (n = 6). Furthermore, lean individuals and athletes displayed a mitonuclear protein balance that was different from obese participants and those with type 2 diabetes. Mitonuclear protein balance also associated with heat shock protein 60 (HSP60) protein levels, which were higher in athletes when compared with participants with obesity (p = 0.048) and type 2 diabetes (p = 0.002), indicative for activation of the mitochondrial unfolded protein response. Finally, OPA1, FIS1 and HSP60 correlated positively with aerobic capacity (r = 0.48, p = 0.0001; r = 0.55, p < 0.001 and r = 0.61, p < 0.0001, respectively) and insulin sensitivity (r = 0.40, p = 0.008; r = 0.44, p = 0.003 and r = 0.48, p = 0.001, respectively). CONCLUSIONS/INTERPRETATION: Collectively, our data suggest that mitochondrial dynamics and quality control in skeletal muscle are linked to oxidative capacity in humans, which may play a role in the maintenance of muscle insulin sensitivity. CLINICAL TRIAL REGISTRY: numbers NCT00943059, NCT01298375 and NL1888 Graphical abstract.

The effect of diet and exercise on lipid droplet dynamics in human muscle tissue.

The majority of fat in the human body is stored as triacylglycerols in white adipose tissue. In the obese state, adipose tissue mass expands and excess lipids are stored in non-adipose tissues, such as skeletal muscle. Lipids are stored in skeletal muscle in the form of small lipid droplets. Although originally viewed as dull organelles that simply store lipids as a consequence of lipid overflow from adipose tissue, lipid droplets are now recognized as key components in the cell that exert a variety of relevant functions in multiple tissues (including muscle). Here, we review the effect of diet and exercise interventions on myocellular lipid droplets and their putative role in insulin sensitivity from a human perspective. We also provide an overview of lipid droplet biology and identify gaps for future research.