Exercise training modifies skeletal muscle clock gene expression but not 24-hour rhythmicity in substrate metabolism of men with insulin resistance.

Twenty-four hour rhythmicity in whole-body substrate metabolism, skeletal muscle clock gene expression and mitochondrial respiration is compromised upon insulin resistance. With exercise training known to ameliorate insulin resistance, our objective was to test if exercise training can reinforce diurnal variation in whole-body and skeletal muscle metabolism in men with insulin resistance. In a single-arm longitudinal design, 10 overweight and obese men with insulin resistance performed 12 weeks of high-intensity interval training recurrently in the afternoon (between 14.00 and 18.00 h) and were tested pre- and post-exercise training, while staying in a metabolic research unit for 2 days under free-living conditions with regular meals. On the second days, indirect calorimetry was performed at 08.00, 13.00, 18.00, 23.00 and 04.00 h, muscle biopsies were taken from the vastus lateralis at 08.30, 13.30 and 23.30 h, and blood was drawn at least bi-hourly over 24 h. Participants did not lose body weight over 12 weeks, but improved body composition and exercise capacity. Exercise training resulted in reduced 24-h plasma glucose levels, but did not modify free fatty acid and triacylglycerol levels. Diurnal variation of muscle clock gene expression was modified by exercise training with period genes showing an interaction (time × exercise) effect and reduced mRNA levels at 13.00 h. Exercise training increased mitochondrial respiration without inducing diurnal variation. Twenty-four-hour substrate metabolism and energy expenditure remained unchanged. Future studies should investigate alternative exercise strategies or types of interventions (e.g. diet or drugs aiming at improving insulin sensitivity) for their capacity to reinforce diurnal variation in substrate metabolism and mitochondrial respiration. KEY POINTS: Insulin resistance is associated with blunted 24-h flexibility in whole-body substrate metabolism and skeletal muscle mitochondrial respiration, and disruptions in the skeletal muscle molecular circadian clock. We hypothesized that exercise training modifies 24-h rhythmicity in whole-body substrate metabolism and diurnal variation in skeletal muscle molecular clock and mitochondrial respiration in men with insulin resistance. We found that metabolic inflexibility over 24 h persisted after exercise training, whereas mitochondrial respiration increased independent of time of day. Gene expression of Per1-3 and Rorα in skeletal muscle changed particularly close to the time of day at which exercise training was performed. These results provide the rationale to further investigate the differential metabolic impact of differently timed exercise to treat metabolic defects of insulin resistance that manifest at a particular time of day.

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.

PCr/ATP ratios and mitochondrial function in the heart. A comparative study in humans.

Cardiac energy status, measured as phosphocreatine (PCr)/adenosine triphosphate (ATP) ratio with 31P-Magnetic Resonance Spectroscopy (31P-MRS) in vivo, is a prognostic factor in heart failure and is lowered in cardiometabolic disease. It has been suggested that, as oxidative phosphorylation is the major contributor to ATP synthesis, PCr/ATP ratio might be a reflection of cardiac mitochondrial function. The objective of the study was to investigate whether PCr/ATP ratios can be used as in vivo marker for cardiac mitochondrial function. We enrolled thirty-eight patients scheduled for open-heart surgery in this study. Cardiac 31P-MRS was performed before surgery. Tissue from the right atrial appendage was obtained during surgery for high-resolution respirometry for the assessment of mitochondrial function. There was no correlation between the PCr/ATP ratio and ADP-stimulated respiration rates (octanoylcarnitine R2 < 0.005, p = 0.74; pyruvate R2 < 0.025, p = 0.41) nor with maximally uncoupled respiration (octanoylcarnitine R2 = 0.005, p = 0.71; pyruvate R2 = 0.040, p = 0.26). PCr/ATP ratio did correlate with indexed LV end systolic mass. As no direct correlation between cardiac energy status (PCr/ATP) and mitochondrial function in the heart was found, the study suggests that mitochondrial function might not the only determinant of cardiac energy status. Interpretation should be done in the right context in cardiac metabolic studies.

Twenty-four hour rhythmicity in mitochondrial network connectivity and mitochondrial respiration; a study in human skeletal muscle biopsies of young lean and older individuals with obesity.

OBJECTIVE: Mitochondrial network dynamics may play role in metabolic homeostasis. Whether mitochondrial network dynamics are involved in adaptations to day-night fluctuations in energy supply and demand is unclear. Here we visualized and quantified the mitochondrial network morphology in human skeletal muscle of young healthy lean and older individuals with obesity over the course of 24 h METHODS: Muscle biopsies taken at 5 timepoints over a 24-hour period obtained from young healthy lean and older metabolically impaired obese males were analyzed for mitochondrial network integrity with confocal laser scanning microscopy. Variation of level of fragmentation over the course of the day were aligned with variation of mitochondrial respiration over the day RESULTS: Young healthy lean individuals displayed a day-night rhythmicity in mitochondrial network morphology, which aligned with the day-night rhythmicity of mitochondrial respiratory capacity, with a more fused network coinciding with higher mitochondrial respiratory capacity. In the older individuals with obesity, the mitochondrial network was more fragmented overall compared to young healthy lean individuals and completely lacked 24 h rhythmicity, which was also true for the mitochondrial respiratory capacity CONCLUSIONS: Our data shows a paralleled rhythmicity between mitochondrial network morphology and mitochondrial oxidative capacity, which oscillates over the course of a mimicked real-life day in human skeletal muscle of young, healthy lean individuals. In older individuals with obesity, the lack of a 24-hour rhythmicity in mitochondrial network connectivity was also aligned with a lack in respiratory capacity. This suggests that 24-hour rhythmicity in mitochondrial network connectivity is a determinant of rhythmicity in mitochondrial respiratory capacity. Thus, restoring mitochondrial network integrity may promote mitochondrial respiratory capacity and hence contribute to blunting the metabolic aberrations in individuals with a disturbed 24-hour rhythmicity in metabolism, like older individuals with obesity.

Effect of ß2-agonist treatment on insulin-stimulated peripheral glucose disposal in healthy men in a randomised placebo-controlled trial.

ß2-agonist treatment improves skeletal muscle glucose uptake and whole-body glucose homeostasis in rodents, likely via mTORC2-mediated signalling. However, human data on this topic is virtually absent. We here investigate the effects of two-weeks treatment with the ß2-agonist clenbuterol (40 µg/day) on glucose control as well as energy- and substrate metabolism in healthy young men (age: 18-30 years, BMI: 20-25 kg/m2) in a randomised, placebo-controlled, double-blinded, cross-over study (ClinicalTrials.gov-identifier: NCT03800290). Randomisation occurred by controlled randomisation and the final allocation sequence was seven (period 1: clenbuterol, period 2: placebo) to four (period 1: placebo, period 2: clenbuterol). The primary and secondary outcome were peripheral insulin-stimulated glucose disposal and skeletal muscle GLUT4 translocation, respectively. Primary analyses were performed on eleven participants. No serious adverse events were reported. The study was performed at Maastricht University, Maastricht, The Netherlands, between August 2019 and April 2021. Clenbuterol treatment improved peripheral insulin-stimulated glucose disposal by 13% (46.6 ± 3.5 versus 41.2 ± 2.7 µmol/kg/min, p = 0.032), whereas skeletal muscle GLUT4 translocation assessed in overnight fasted muscle biopsies remained unaffected. These results highlight the potential of ß2-agonist treatment in improving skeletal muscle glucose uptake and underscore the therapeutic value of this pathway for the treatment of type 2 diabetes. However, given the well-known (cardiovascular) side-effects of systemic ß2-agonist treatment, further exploration on the underlying mechanisms is needed to identify viable therapeutic targets.

Effects of the sodium-glucose cotransporter 2 inhibitor dapagliflozin on substrate metabolism in prediabetic insulin resistant individuals: A randomized, double-blind crossover trial.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter 2 inhibitor (SGLT2i) treatment in type 2 diabetes mellitus patients results in glucosuria, causing an energy loss, and triggers beneficial metabolic adaptations. It is so far unknown if SGLT2i exerts beneficial metabolic effects in prediabetic insulin resistant individuals, yet this is of interest since SGLT2is also reduce the risk for progression of heart failure and chronic kidney disease in patients without diabetes. METHODS: Fourteen prediabetic insulin resistant individuals (BMI: 30.3 ± 2.1 kg/m2; age: 66.3 ± 6.2 years) underwent 2-weeks of treatment with dapagliflozin (10 mg/day) or placebo in a randomized, placebo-controlled, cross-over design. Outcome parameters include 24-hour and nocturnal substrate oxidation, and twenty-four-hour blood substrate and insulin levels. Hepatic glycogen and lipid content/composition were measured by MRS. Muscle biopsies were taken to measure mitochondrial oxidative capacity and glycogen and lipid content. RESULTS: Dapagliflozin treatment resulted in a urinary glucose excretion of 36 g/24-h, leading to a negative energy and fat balance. Dapagliflozin treatment resulted in a higher 24-hour and nocturnal fat oxidation (p = 0.043 and p = 0.039, respectively), and a lower 24-hour carbohydrate oxidation (p = 0.048). Twenty-four-hour plasma glucose levels were lower (AUC; p = 0.016), while 24-hour free fatty acids and nocturnal ß-hydroxybutyrate levels were higher (AUC; p = 0.002 and p = 0.012, respectively) after dapagliflozin compared to placebo. Maximal mitochondrial oxidative capacity was higher after dapagliflozin treatment (dapagliflozin: 87.6 ± 5.4, placebo: 78.1 ± 5.5 pmol/mg/s, p = 0.007). Hepatic glycogen and lipid content were not significantly changed by dapagliflozin compared to placebo. However, muscle glycogen levels were numerically higher in the afternoon in individuals on placebo (morning: 332.9 ± 27.9, afternoon: 368.8 ± 13.1 nmol/mg), while numerically lower in the afternoon on dapagliflozin treatment (morning: 371.7 ± 22.8, afternoon: 340.5 ± 24.3 nmol/mg). CONCLUSIONS/INTERPRETATION: Dapagliflozin treatment of prediabetic insulin resistant individuals for 14 days resulted in significant metabolic adaptations in whole-body and skeletal muscle substrate metabolism despite being weight neutral. Dapagliflozin improved fat oxidation and ex vivo skeletal muscle mitochondrial oxidative capacity, mimicking the effects of calorie restriction. TRIAL REGISTRATION: ClinicalTrials.gov NCT03721874.

Effects of SGLT2 inhibitor dapagliflozin in patients with type 2 diabetes on skeletal muscle cellular metabolism.

OBJECTIVE: SGLT2 inhibitors increase urinary glucose excretion and have beneficial effects on cardiovascular and renal outcomes; the underlying mechanism may be metabolic adaptations due to urinary glucose loss. Here, we investigated the cellular and molecular effects of 5 weeks of dapagliflozin treatment on skeletal muscle metabolism in type 2 diabetes patients. METHODS: Twenty-six type 2 diabetes mellitus patients were randomized to a 5-week double-blind, cross-over study with 6-8-week wash-out. Skeletal muscle acetylcarnitine levels, intramyocellular lipid (IMCL) content and phosphocreatine (PCr) recovery rate were measured by magnetic resonance spectroscopy (MRS). Ex vivo mitochondrial respiration was measured in skeletal muscle fibers using high resolution respirometry. Intramyocellular lipid droplet and mitochondrial network dynamics were investigated using confocal microscopy. Skeletal muscle levels of acylcarnitines, amino acids and TCA cycle intermediates were measured. Expression of genes involved in fatty acid metabolism were investigated. RESULTS: Mitochondrial function, mitochondrial network integrity and citrate synthase and carnitine acetyltransferase activities in skeletal muscle were unaltered after dapagliflozin treatment. Dapagliflozin treatment increased intramyocellular lipid content (0.060 (0.011, 0.110) %, p = 0.019). Myocellular lipid droplets increased in size (0.03 µm2 (0.01-0.06), p < 0.05) and number (0.003 µm-2 (-0.001-0.007), p = 0.09) upon dapagliflozin treatment. CPT1A, CPT1B and malonyl CoA-decarboxylase mRNA expression was increased by dapagliflozin. Fasting acylcarnitine species and C4-OH carnitine levels (0.4704 (0.1246, 0.8162) pmoles∗mg tissue-1, p < 0.001) in skeletal muscle were higher after dapagliflozin treatment, while acetylcarnitine levels were lower (-40.0774 (-64.4766, -15.6782) pmoles∗mg tissue-1, p < 0.001). Fasting levels of several amino acids, succinate, alpha-ketoglutarate and lactate in skeletal muscle were significantly lower after dapagliflozin treatment. CONCLUSION: Dapagliflozin treatment for 5 weeks leads to adaptive changes in skeletal muscle substrate metabolism favoring metabolism of fatty acid and ketone bodies and reduced glycolytic flux. The trial is registered with ClinicalTrials.gov, number NCT03338855.

Reversible mutations in gliding motility and virulence genes: A flexible and efficient phage defence mechanism in Flavobacterium psychrophilum.

Flavobacteria are among the most important pathogens in freshwater salmonid aquaculture worldwide. Due to concerns regarding development of antibiotic resistance, phage therapy has been proposed as a solution to decrease pathogen load. However, application of phages is challenged by the development of phage resistance, and knowledge of the mechanisms and implications of phage resistance is therefore required. To study this, 27 phage-resistant isolates of F. psychrophilum were genome sequenced and characterized to identify genetic modifications and evaluate changes in phenotypic traits, including virulence against rainbow trout. Phage-resistant isolates showed reduction or loss of gliding motility, proteolytic activity, and adhesion to surfaces, and most isolates were completely non-virulent against rainbow trout fry. Genomic analysis revealed that most phage-resistant isolates had mutations in genes associated with gliding motility and virulence. Reversal of these mutations in a sub-set of isolates led to regained motility, proteolytic activity, virulence and phage susceptibility. Although costly, the fast generation of phage resistance driven by single, reversible mutations likely represents a flexible and efficient phage defence mechanism in F. psychrophilum. The results further suggest that phage administration in aquaculture systems to prevent F. psychrophilum outbreaks selects for non-virulent phage-resistant phenotypes.

In Vitro Evolution of Specific Phages Infecting the Fish Pathogen Flavobacterium psychrophilum.

Background: Flavobacterium psychrophilum is the causative agent of the bacterial cold-water disease and rainbow trout fry syndrome. Owing to the issues associated with increasing use of antibiotics to control the diseases, phage therapy has been proposed as an alternative method to control Flavobacterium infection within the industry. Materials and Methods: We explored two simple and fast in vitro strategies for the isolation of evolved F. psychrophilum phages, using three well-characterized phages FpV4, FpV9, and FPSV-S20. Results: During in vitro serial transfer experiments, 12 evolved phages were selected 72-96 h after phage exposure in the first or second week. Phenotype analysis showed improvement of host range and efficiency of plating and adsorption constants. Comparative genomic analysis of the evolved phages identified 13 independent point mutations causing amino acid changes mostly in hypothetical proteins. Conclusions: These results confirmed the reliability and effectivity of two strategies to isolate evolved F. psychrophilum phages, which may be used to expand phage-host range and target phage-resistant pathogens in phage therapy applications against Flavobacterium infections.

Sitting less elicits metabolic responses similar to exercise and enhances insulin sensitivity in postmenopausal women.

AIMS/HYPOTHESIS: In our current society sedentary behaviour predominates in most people and is associated with the risk of developing type 2 diabetes. It has been suggested that replacing sitting time by standing and walking could be beneficial for individuals with type 2 diabetes but the underlying mechanisms are unknown and direct comparisons with exercise are lacking. Our objective was to directly compare metabolic responses of either sitting less or exercising, relative to being sedentary. METHODS: We performed a randomised, crossover intervention study in 12 overweight women who performed three well-controlled 4 day activity regimens: (1) sitting regimen (sitting 14 h/day); (2) exercise regimen (sitting 13 h/day, exercise 1 h/day); and (3) sitting less regimen (sitting 9 h/day, standing 4 h/day and walking 3 h/day). The primary outcome was insulin sensitivity measured by a two-step hyperinsulinaemic-euglycaemic clamp. We additionally performed metabolomics on muscle biopsies taken before the clamp to identify changes at the molecular level. RESULTS: Replacing sitting time by standing and walking over 4 days resulted in improved peripheral insulin sensitivity, comparable with the improvement achieved by moderate-to-vigorous exercise. Specifically, we report a significant improvement in peripheral insulin sensitivity in the sitting less (~13%) and the exercise regimen (~20%), compared with the sitting regimen. Furthermore, sitting less shifted the underlying muscle metabolome towards that seen with moderate-to-vigorous exercise, compared with the sitting regimen. CONCLUSIONS/INTERPRETATIONS: Replacing sitting time by standing and walking is an attractive alternative to moderate-to-vigorous exercise for improving metabolic health. TRIAL REGISTRATION: ClinicalTrials.gov NCT03912922.

Resveratrol supplementation reduces ACE2 expression in human adipose tissue.

Angiotensin converting enzyme-2 (ACE2) is the cell-surface receptor enabling cellular entry of SARS-CoV-2. ACE2 is highly expressed in adipose tissue (AT), rendering AT a potential SARS-CoV-2 reservoir contributing to massive viral spread in COVID-19 patients with obesity. Although rodent and cell studies suggest that the polyphenol resveratrol alters ACE2, human studies are lacking. Here, we investigated the effects of 30-days resveratrol supplementation on RAS components in AT and skeletal muscle in men with obesity in a placebo-controlled cross-over study. Resveratrol markedly decreased ACE2 (~40%) and leptin (~30%), but did neither alter angiotensinogen, ACE and AT1R expression in AT nor skeletal muscle RAS components. These findings demonstrate that resveratrol supplementation reduces ACE2 in AT, which might dampen SARS-CoV-2 spread in COVID-19.

Comparative Genomic Analyses of Flavobacterium psychrophilum Isolates Reveals New Putative Genetic Determinants of Virulence Traits.

The fish pathogen Flavobacterium psychrophilum is currently one of the main pathogenic bacteria hampering the productivity of salmonid farming worldwide. Although putative virulence determinants have been identified, the genetic basis for variation in virulence of F. psychrophilum is not fully understood. In this study, we analyzed whole-genome sequences of a collection of 25 F. psychrophilum isolates from Baltic Sea countries and compared genomic information with a previous determination of their virulence in juvenile rainbow trout. The results revealed a conserved population of F. psychrophilum that were consistently present across the Baltic Sea countries, with no clear association between genomic repertoire, phylogenomic, or gene distribution and virulence traits. However, analysis of the entire genome of four F. psychrophilum isolates by hybrid assembly provided an unprecedented resolution for discriminating even highly related isolates. The results showed that isolates with different virulence phenotypes harbored genetic variances on a number of consecutive leucine-rich repeat (LRR) proteins, repetitive motifs in gliding motility-associated protein, and the insertion of transposable elements into intergenic and genic regions. Thus, these findings provide novel insights into the genetic variation of these elements and their putative role in the modulation of F. psychrophilum virulence.

The angiotensin II type 1 receptor blocker valsartan in the battle against COVID-19.

OBJECTIVE: Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) uses the host's angiotensin-converting enzyme 2 (ACE2) as a cellular entry point. Therefore, modulating ACE2 might impact SARS-CoV-2 viral replication, shedding, and coronavirus disease 2019 (COVID-19) severity. Here, it was investigated whether the angiotensin II type 1 receptor blocker valsartan alters the expression of renin-angiotensin system (RAS) components, including ACE2, in human adipose tissue (AT) and skeletal muscle. METHODS: A randomized, double-blind, placebo-controlled clinical trial was performed, in which 36 participants (BMI 31.0 ± 0.8 kg/m2 ) with impaired glucose metabolism received either valsartan or placebo for 26 weeks. Before and after 26 weeks' treatment, abdominal subcutaneous AT and skeletal muscle biopsies were obtained, and gene expression of RAS components was measured by quantitative reverse transcription polymerase chain reaction. RESULTS: Valsartan treatment did not significantly impact the expression of RAS components, including ACE2, in AT and skeletal muscle. CONCLUSIONS: Given the pivotal role of ACE2 in SARS-CoV-2 spread and the clinical outcomes in COVID-19 patients, the data suggest that the putative beneficial effects of angiotensin II type 1 receptor blockers on the clinical outcomes of patients with COVID-19 may not be mediated through altered ACE2 expression in abdominal subcutaneous AT.

Interactions between Rainbow Trout Eyed Eggs and Flavobacterium spp. Using a Bath Challenge Model: Preliminary Evaluation of Bacteriophages as Pathogen Control Agents.

The microbial community surrounding fish eyed eggs can harbor pathogenic bacteria. In this study we focused on rainbow trout (Oncorhynchus mykiss) eyed eggs and the potential of bacteriophages against the pathogenic bacteria Flavobacterium psychrophilum and F. columnare. An infection bath method was first established, and the effects of singular phages on fish eggs was assessed (survival of eyed eggs, interaction of phages with eyed eggs). Subsequently, bacteria-challenged eyed eggs were exposed to phages to evaluate their effects in controlling the bacterial population. Culture-based methods were used to enumerate the number of bacteria and/or phages associated with eyed eggs and in the surrounding environment. The results of the study showed that, with our infection model, it was possible to re-isolate F. psychrophilum associated with eyed eggs after the infection procedure, without affecting the survival of the eggs in the short term. However, this was not possible for F. columnare, as this bacterium grows at higher temperatures than the ones recommended for incubation of rainbow trout eyed eggs. Bacteriophages do not appear to negatively affect the survival of rainbow trout eyed eggs and they do not seem to strongly adhere to the surface of eyed eggs either. Finally, the results demonstrated a strong potential for short term (24 h) phage control of F. psychrophilum. However, further studies are needed to explore if phage control can be maintained for a longer period and to further elucidate the mechanisms of interactions between Flavobacteria and their phages in association with fish eggs.

Nicotinamide Riboside Enhances In Vitro Beta-adrenergic Brown Adipose Tissue Activity in Humans.

CONTEXT: Elevating nicotinamide adenine dinucleotide (NAD+) levels systemically improves metabolic health, which can be accomplished via nicotinamide riboside (NR). Previously, it was demonstrated that NR supplementation in high-fat-diet (HFD)-fed mice decreased weight gain, normalized glucose metabolism, and enhanced cold tolerance. OBJECTIVE: Because brown adipose tissue (BAT) is a major source of thermogenesis, we hypothesize that NR stimulates BAT in mice and humans. DESIGN AND INTERVENTION: HFD-fed C56BL/6J mice were supplemented with 400 mg/kg/day NR for 4 weeks and subsequently exposed to cold. In vitro primary adipocytes derived from human BAT biopsies were pretreated with 50 µM or 500 µM NR before measuring mitochondrial uncoupling. Human volunteers (45-65 years; body mass index, 27-35 kg/m2) were supplemented with 1000 mg/day NR for 6 weeks to determine whether BAT activity increased, as measured by [18F]FDG uptake via positron emission tomography-computed tomography (randomized, double blinded, placebo-controlled, crossover study with NR supplementation). RESULTS: NR supplementation in HFD-fed mice decreased adipocyte cell size in BAT. Cold exposure further decreased adipocyte cell size on top of that achieved by NR alone independent of ex vivo lipolysis. In adipocytes derived from human BAT, NR enhanced in vitro norepinephrine-stimulated mitochondrial uncoupling. However, NR supplementation in human volunteers did not alter BAT activity or cold-induced thermogenesis. CONCLUSIONS: NR stimulates in vitro human BAT but not in vivo BAT in humans. Our research demonstrates the need for further translational research to better understand the differences in NAD+ metabolism in mouse and human.

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.

Day-night rhythm of skeletal muscle metabolism is disturbed in older, metabolically compromised individuals.

OBJECTIVE: Skeletal muscle mitochondrial function and energy metabolism displays day-night rhythmicity in healthy, young individuals. Twenty-four-hour rhythmicity of metabolism has been implicated in the etiology of age-related metabolic disorders. Whether day-night rhythmicity in skeletal muscle mitochondrial function and energy metabolism is altered in older, metabolically comprised humans remains unknown. METHODS: Twelve male overweight volunteers with impaired glucose tolerance and insulin sensitivity stayed in a metabolic research unit for 2 days under free living conditions with regular meals. Indirect calorimetry was performed at 5 time points (8 AM, 1 PM, 6 PM, 11 PM, 4 AM), followed by a muscle biopsy. Mitochondrial oxidative capacity was measured in permeabilized muscle fibers using high-resolution respirometry. RESULTS: Mitochondrial oxidative capacity did not display rhythmicity. The expression of circadian core clock genes BMAL1 and REV-ERBα showed a clear day-night rhythm (p < 0.001), peaking at the end of the waking period. Remarkably, the repressor clock gene PER2 did not show rhythmicity, whereas PER1 and PER3 were strongly rhythmic (p < 0.001). On the whole-body level, resting energy expenditure was highest in the late evening (p < 0.001). Respiratory exchange ratio did not decrease during the night, indicating metabolic inflexibility. CONCLUSIONS: Mitochondrial oxidative capacity does not show a day-night rhythm in older, overweight participants with impaired glucose tolerance and insulin sensitivity. In addition, gene expression of PER2 in skeletal muscle indicates that rhythmicity of the negative feedback loop of the molecular clock is disturbed. CLINICALTRIALS. GOV ID: NCT03733743.

MicroRNA-204-5p modulates mitochondrial biogenesis in C2C12 myotubes and associates with oxidative capacity in humans.

Using an unbiased high-throughput microRNA (miRNA)-silencing screen combined with functional readouts for mitochondrial oxidative capacity in C2C12 myocytes, we previously identified 19 miRNAs as putative regulators of skeletal muscle mitochondrial metabolism. In the current study, we highlight miRNA-204-5p, identified from this screen, and further studied its role in the regulation of skeletal muscle mitochondrial function. Following silencing of miRNA-204-5p in C2C12 myotubes, gene and protein expression were assessed using quantitative polymerase chain reaction, microarray analysis, and western blot analysis, while morphological changes were studied by confocal microscopy. In addition, miRNA-204-5p expression was quantified in human skeletal muscle biopsies and associated with in vivo mitochondrial oxidative capacity. Transcript levels of PGC-1α (3.71-fold; p < .01), predicted as an miR-204-5p target, as well as mitochondrial DNA copy number (p < .05) and citrate synthase activity (p = .06) were increased upon miRNA-204-5p silencing in C2C12 myotubes. Silencing of miRNA-204-5p further resulted in morphological changes, induced gene expression of autophagy marker light chain 3 protein b (LC3B; q = .05), and reduced expression of the mitophagy marker FUNDC1 (q = .01). Confocal imaging revealed colocalization between the autophagosome marker LC3B and the mitochondrial marker OxPhos upon miRNA-204-5p silencing. Finally, miRNA-204-5p was differentially expressed in human subjects displaying large variation in oxidative capacity and its expression levels associated with in vivo measures of skeletal muscle mitochondrial function. In summary, silencing of miRNA-204-5p in C2C12 myotubes stimulated mitochondrial biogenesis, impacted on cellular morphology, and altered expression of markers related to autophagy and mitophagy. The association between miRNA-204-5p and in vivo mitochondrial function in human skeletal muscle further identifies miRNA-204-5p as an interesting modulator of skeletal muscle mitochondrial metabolism.

One-leg inactivity induces a reduction in mitochondrial oxidative capacity, intramyocellular lipid accumulation and reduced insulin signalling upon lipid infusion: a human study with unilateral limb suspension.

AIMS/HYPOTHESIS: Physical inactivity, low mitochondrial function, increased intramyocellular lipid (IMCL) deposition and reduced insulin sensitivity are common denominators of chronic metabolic disorders, like obesity and type 2 diabetes. Yet, whether low mitochondrial function predisposes to insulin resistance in humans is still unknown. METHODS: Here we investigated, in an intervention study, whether muscle with low mitochondrial oxidative capacity, induced by one-legged physical inactivity, would feature stronger signs of lipid-induced insulin resistance. To this end, ten male participants (age 22.4 ± 4.2 years, BMI 21.3 ± 2.0 kg/m2) underwent a 12 day unilateral lower-limb suspension with the contralateral leg serving as an active internal control. RESULTS: In vivo, mitochondrial oxidative capacity, assessed by phosphocreatine (PCr)-recovery half-time, was lower in the inactive vs active leg. Ex vivo, palmitate oxidation to 14CO2 was lower in the suspended leg vs the active leg; however, this did not result in significantly higher [14C]palmitate incorporation into triacylglycerol. The reduced mitochondrial function in the suspended leg was, however, paralleled by augmented IMCL content in both musculus tibialis anterior and musculus vastus lateralis, and by increased membrane bound protein kinase C (PKC) θ. Finally, upon lipid infusion, insulin signalling was lower in the suspended vs active leg. CONCLUSIONS/INTERPRETATION: Together, these results demonstrate, in a unique human in vivo model, that a low mitochondrial oxidative capacity due to physical inactivity directly impacts IMCL accumulation and PKCθ translocation, resulting in impaired insulin signalling upon lipid infusion. This demonstrates the importance of mitochondrial oxidative capacity and muscle fat accumulation in the development of insulin resistance in humans. TRIAL REGISTRATION: ClinicalTrial.gov NCT01576250. FUNDING: PS was supported by a 'VICI' Research Grant for innovative research from the Netherlands Organization for Scientific Research (Grant 918.96.618).

Phenotypic and Genetic Predictors of Pathogenicity and Virulence in Flavobacterium psychrophilum.

Flavobacterium psychrophilum causes bacterial cold-water disease (BCWD) in farmed rainbow trout (Oncorhynchus mykiss), with the multilocus sequence typing (MLST) clonal complex (CC) CC-ST10 accounting for the majority of outbreaks globally. The development of alternative strategies to antibiotic treatment of BCWD using bacteriophage-based control of F. psychrophilum, or virulence factors as targets for therapy, requires knowledge of the phage-sensitivity of outbreak strains and of universal traits contributing to their pathogenicity. To examine the association between virulence and both genetic (MLST sequence type (ST) and PCR-serotype) and phenotypic characteristics (adherence, antibiotic resistance, colony spreading motility, hemolytic and proteolytic activity), the median lethal dose (LD50) of 26 geographically disparate F. psychrophilum isolates was determined in rainbow trout. Furthermore, the in vitro sensitivity of the isolates against five bacteriophages was determined by the efficiency of plating (EOP). The tested F. psychrophilum isolates were mainly represented by CC-ST10 genotypes (22 out of 26) and showed up to 3-log differences in LD50 (8.9 × 103 to 3.1 × 106 CFU). No association between MLST ST and virulence was found because of a high variation in LD50 within STs. All identified serotypes (0, 1, and 2) were pathogenic, but ten most virulent isolates belonged to serotype 1 or 2. Isolates of high (LD50 < 105 CFU), moderate (LD50 = 105-106 CFU), and weak (LD50 > 106 CFU) virulence were similar in phenotypic characteristics in vitro. However, the only non-virulent CC-ST10 isolate was deficient in spreading motility and proteolytic activity, indicating that the characteristics are required for pathogenicity in F. psychrophilum. Univariate correlation studies found only non-significant associations between LD50 and the measured phenotypic characteristics, and the multivariable analysis did neither reveal any significant predictors of virulence. The majority of isolates (16 out of 26) were sensitive to at least four bacteriophages, with up to a 6-log variation in the EOP. Most CC-ST10 isolates (16 out of 22) were sensitive to the examined phages, including 5 out of the 7 most virulent isolates represented by prevalent and antibiotic-resistant STs. Our findings suggest that control of BCWD using lytic phages or interventions targeting shared characteristics of pathogenic F. psychrophilum strains should be further explored.