The novel adrenergic agonist ATR-127 targets skeletal muscle and brown adipose tissue to tackle diabesity and steatohepatitis.

OBJECTIVE: Simultaneous activation of ß2- and ß3-adrenoceptors (ARs) improves whole-body metabolism via beneficial effects in skeletal muscle and brown adipose tissue (BAT). Nevertheless, high-efficacy agonists simultaneously targeting these receptors whilst limiting activation of ß1-ARs - and thus inducing cardiovascular complications - are currently non-existent. Therefore, we here developed and evaluated the therapeutic potential of a novel ß2-and ß3-AR, named ATR-127, for the treatment of obesity and its associated metabolic perturbations in preclinical models. METHODS: In the developmental phase, we assessed the impact of ATR-127's on cAMP accumulation in relation to the non-selective ß-AR agonist isoprenaline across various rodent ß-AR subtypes, including neonatal rat cardiomyocytes. Following these experiments, L6 muscle cells were stimulated with ATR-127 to assess the impact on GLUT4-mediated glucose uptake and intramyocellular cAMP accumulation. Additionally, in vitro, and in vivo assessments are conducted to measure ATR-127's effects on BAT glucose uptake and thermogenesis. Finally, diet-induced obese mice were treated with 5 mg/kg ATR-127 for 21 days to investigate the effects on glucose homeostasis, body weight, fat mass, skeletal muscle glucose uptake, BAT thermogenesis and hepatic steatosis. RESULTS: Exposure of L6 muscle cells to ATR-127 robustly enhanced GLUT4-mediated glucose uptake despite low intramyocellular cAMP accumulation. Similarly, ATR-127 markedly increased BAT glucose uptake and thermogenesis both in vitro and in vivo. Prolonged treatment of diet-induced obese mice with ATR-127 dramatically improved glucose homeostasis, an effect accompanied by decreases in body weight and fat mass. These effects were paralleled by an enhanced skeletal muscle glucose uptake, BAT thermogenesis, and improvements in hepatic steatosis. CONCLUSIONS: Our results demonstrate that ATR-127 is a highly effective, novel ß2- and ß3-ARs agonist holding great therapeutic promise for the treatment of obesity and its comorbidities, whilst potentially limiting cardiovascular complications. As such, the therapeutic effects of ATR-127 should be investigated in more detail in clinical studies.

A conserved complex lipid signature marks human muscle aging and responds to short-term exercise.

Studies in preclinical models suggest that complex lipids, such as phospholipids, play a role in the regulation of longevity. However, identification of universally conserved complex lipid changes that occur during aging, and how these respond to interventions, is lacking. Here, to comprehensively map how complex lipids change during aging, we profiled ten tissues in young versus aged mice using a lipidomics platform. Strikingly, from >1,200 unique lipids, we found a tissue-wide accumulation of bis(monoacylglycero)phosphate (BMP) during mouse aging. To investigate translational value, we assessed muscle tissue of young and older people, and found a similar marked BMP accumulation in the human aging lipidome. Furthermore, we found that a healthy-aging intervention consisting of moderate-to-vigorous exercise was able to lower BMP levels in postmenopausal female research participants. Our work implicates complex lipid biology as central to aging, identifying a conserved aging lipid signature of BMP accumulation that is modifiable upon a short-term healthy-aging intervention.

The effect of preprandial versus postprandial physical activity on glycaemia: Meta-analysis of human intervention studies.

This meta-analysis aims to investigate the effect of preprandial physical activity (PA) versus postprandial PA on glycaemia in human intervention studies. Medline and Embase.com were searched until February 2023 for intervention studies in adults, directly comparing preprandial PA versus postprandial PA on glycaemia. Studies were screened using ASReview (34,837) and full texts were read by two independent reviewers (42 full text, 28 included). Results were analysed using pooled mean differences in random-effects models. Studies were either acute response studies (n = 21) or Randomized Controlled Trials (RCTs) over multiple weeks (n = 7). In acute response studies, postprandial outcomes followed the expected physiological patterns, and outcomes measured over 24 h showed no significant differences. For the RCTs, glucose area under the curve during a glucose tolerance test was slightly, but not significantly lower in preprandial PA vs postprandial PA (-0.29 [95 %CI:-0.66, 0.08] mmol/L, I2 = 64.36 %). Subgroup analyses (quality, health status, etc.) did not significantly change the outcomes. In conclusion, we found no differences between preprandial PA versus postprandial PA on glycaemia both after one PA bout as well as after multiple weeks of PA. The studies were of low to moderate quality of evidence as assessed by GRADE, showed contradictive results, included no long-term studies and used various designs and populations. We therefore need better RCTs, with more similar designs, in larger populations and longer follow-up periods (≥12 weeks) to have a final answer on the questions eat first, then exercise, or the reverse?

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.

The impact of prolonged fasting on 24h energy metabolism and its 24h rhythmicity in healthy, lean males: A randomized cross-over trial.

OBJECTIVE: Human energy expenditure and substrate oxidation are under circadian control and food intake is a time cue for the human biological clock, leading to 24h feeding-fasting cycles in energy and substrate metabolism. In recent years, (intermittent) fasting protocols have also become popular to improve metabolic health. Here, we aimed to investigate the impact of food intake on the 24h patterns of energy metabolism as well as to provide data on the timeline of changes in energy metabolism that occur upon an extended period of fasting. RESEARCH DESIGN AND METHODS: In a randomized, cross-over design, twelve healthy males underwent a 60h fast which was compared to a 60h fed condition. In the fed condition meals were provided at energy balance throughout the study. Conditions were separated by a two week period of habitual diet. Volunteers resided in a respiration chamber for the entire 60h to measure energy expenditure and substrate oxidation hour by hour. Volunteers performed a standardized activity protocol while in the chamber. Blood samples were drawn after 12, 36 and 60h. RESULTS: Immediately following the breakfast meal (in the fed condition), fat oxidation became higher in the fasted condition compared to the fed condition and remained elevated throughout the study period. The initial rapid increase in fat oxidation corresponded with a decline in the hepatokine activin A (r = -0.86, p = 0.001). The contribution of fat oxidation to total energy expenditure gradually increased with extended abstinence from food, peaking after 51h of fasting at 160 mg/min. Carbohydrate oxidation stabilized at a low level during the second day of fasting and averaged around 60 mg/min with only modest elevations in response to physical activity. Although 24h energy expenditure was significantly lower with prolonged fasting (11.0 ± 0.4 vs 9.8 ± 0.2 and 10.9 ± 0.3 vs 10.3 ± 0.3 MJ in fed vs fasting, day 2 and 3 respectively, p < 0.01), the 24h fluctuations in energy expenditure were comparable between the fasted and fed condition. The fluctuations in substrate oxidation were, however, significantly (p < 0.001 for both carbohydrate and fat oxidation) altered in the fasted state, favouring fat oxidation. CONCLUSIONS: Energy expenditure displays a day-night rhythm, which is independent of food intake. In contrast, the day-night rhythm of both carbohydrate and fat oxidation is mainly driven by food intake. Upon extended fasting, the absolute rate of fat oxidation rapidly increases and keeps increasing during a 60h fast, whereas carbohydrate oxidation becomes progressively diminished. TRIAL REGISTRATION: www.trialregister.nl NTR 2042.

SIRT3 overexpression in rat muscle does not ameliorate peripheral insulin resistance.

Reduced expression of the NAD+-dependent deacetylase, SIRT3, has been associated with insulin resistance and metabolic dysfunction in humans and rodents. In this study, we investigated whether specific overexpression of SIRT3 in vivo in skeletal muscle could prevent high-fat diet (HFD)-induced muscle insulin resistance. To address this, we used a muscle-specific adeno-associated virus (AAV) to overexpress SIRT3 in rat tibialis and extensor digitorum longus (EDL) muscles. Mitochondrial substrate oxidation, substrate switching and oxidative enzyme activity were assessed in skeletal muscles with and without SIRT3 overexpression. Muscle-specific insulin action was also assessed by hyperinsulinaemic-euglycaemic clamps in rats that underwent a 4-week HFD-feeding protocol. Ex vivo functional assays revealed elevated activity of selected SIRT3-target enzymes including hexokinase, isocitrate dehydrogenase and pyruvate dehydrogenase that was associated with an increase in the ability to switch between fatty acid- and glucose-derived substrates in muscles with SIRT3 overexpression. However, during the clamp, muscles from rats fed an HFD with increased SIRT3 expression displayed equally impaired glucose uptake and insulin-stimulated glycogen synthesis as the contralateral control muscle. Intramuscular triglyceride content was similarly increased in the muscle of high-fat-fed rats, regardless of SIRT3 status. Thus, despite SIRT3 knockout (KO) mouse models indicating many beneficial metabolic roles for SIRT3, our findings show that muscle-specific overexpression of SIRT3 has only minor effects on the acute development of skeletal muscle insulin resistance in high-fat-fed rats.

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.

A metabolomic signature of decelerated physiological aging in human plasma.

The degenerative processes that occur during aging increase the risk of disease and impaired health. Meanwhile, interventions that target aging to promote healthy longevity are gaining interest, both academically and in the public. While nutritional and physical interventions exist, efficacy is often difficult to determine. It is therefore imperative that an aging score measuring the biological aging process is available to the wider public. However, simple, interpret, and accessible biological aging scores are lacking. Here, we developed PhysiAge, a physiological aging score based on five accessible parameters that have influence on or reflect the aging process: (1) average daily step count, (2) blood glucose, (3) systolic blood pressure, (4) sex, and (5) age. Here, we found that compared to calendar age alone, PhysiAge better predicts mortality, as well as established muscle aging markers such as decrease in NAD+ levels, increase in oxidative stress, and decline in physical functioning. In order to demonstrate the usefulness of PhysiAge in identifying relevant factors associated with decelerated aging, we calculated PhysiAges for a cohort of aged individuals and obtained mass spectrometry-based blood plasma metabolomic profiles for each individual. Here, we identified a metabolic signature of decelerated aging, which included components of the TCA cycle, including malate, citrate, and isocitrate. Higher abundance of these metabolites was associated with decelerated aging, in line with supplementation studies in model organisms. PhysiAge represents an accessible way for people to track and intervene in their aging trajectories, and identifies a metabolic signature of decelerated aging in human blood plasma, which can be further studied for its causal involvement in human aging.

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.

Physiological and molecular mechanisms of cold-induced improvements in glucose homeostasis in humans beyond brown adipose tissue.

Exposure to low ambient temperatures has previously been demonstrated to markedly improve glucose homeostasis in both rodents and humans. Although the brown adipose tissue is key in mediating these beneficial effects in rodents, its contribution appears more limited in humans. Hence, the exact tissues and underlying mechanisms that mediate cold-induced improvements in glucose homeostasis in humans remain to be fully established. In this review, we evaluated the response of the main organs involved in glucose metabolism (i.e. pancreas, liver, (white) adipose tissue, and skeletal muscle) to cold exposure and discuss their potential contribution to cold-induced improvements in glucose homeostasis in humans. We here show that cold exposure has widespread effects on metabolic organs involved in glucose regulation. Nevertheless, cold-induced improvements in glucose homeostasis appear primarily mediated via adaptations within the skeletal muscle and (presumably) white adipose tissue. Since the underlying mechanisms remain elusive, future studies should be aimed at pinpointing the exact physiological and molecular mechanisms involved in humans. Nonetheless, cold exposure holds great promise as a novel, additive lifestyle approach to improve glucose homeostasis in insulin resistant individuals. Parts of this graphical abstract were created using (modified) images from Servier Medical Art, licensed under the Creative Commons Attribution 3.0 Unported License. TG = thermogenesis, TAG = triacylglycerol, FFA = free fatty acid, SLN = sarcolipin, UCP3 = uncoupling protein 3, ß2-AR = beta-2 adrenergic receptor, SNS = sympathetic nervous system.

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.

Skeletal muscle mitochondrial inertia is associated with carnitine acetyltransferase activity and physical function in humans.

BACKGROUNDAt the onset of exercise, the speed at which phosphocreatine (PCr) decreases toward a new steady state (PCr on-kinetics) reflects the readiness to activate mitochondrial ATP synthesis, which is secondary to Acetyl-CoA availability in skeletal muscle. We hypothesized that PCr on-kinetics are slower in metabolically compromised and older individuals and are associated with low carnitine acetyltransferase (CrAT) protein activity and compromised physical function.METHODSWe applied 31P-magnetic resonance spectroscopy (31P-MRS) to assess PCr on-kinetics in 2 cohorts of volunteers. Cohort 1 included patients who had type 2 diabetes, were obese, were lean trained (VO2max > 55 mL/kg/min), and were lean untrained (VO2max < 45 mL/kg/min). Cohort 2 included young (20-30 years) and older (65-80 years) individuals with normal physical activity and older, trained individuals. Previous results of CrAT protein activity and acetylcarnitine content in muscle tissue were used to explore the underlying mechanisms of PCr on-kinetics, along with various markers of physical function.RESULTSPCr on-kinetics were significantly slower in metabolically compromised and older individuals (indicating mitochondrial inertia) as compared with young and older trained volunteers, regardless of in vivo skeletal muscle oxidative capacity (P < 0.001). Mitochondrial inertia correlated with reduced CrAT protein activity, low acetylcarnitine content, and functional outcomes (P < 0.001).CONCLUSIONPCr on-kinetics are significantly slower in metabolically compromised and older individuals with normal physical activity compared with young and older trained individuals, regardless of in vivo skeletal muscle oxidative capacity, indicating greater mitochondrial inertia. Thus, PCr on-kinetics are a currently unexplored signature of skeletal muscle mitochondrial metabolism, tightly linked to functional outcomes. Skeletal muscle mitochondrial inertia might emerge as a target of intervention to improve physical function.TRIAL REGISTRATIONNCT01298375 and NCT03666013 (clinicaltrials.gov).FUNDINGRM and MH received an EFSD/Lilly grant from the European Foundation for the Study of Diabetes (EFSD). VS was supported by an ERC starting grant (grant 759161) "MRS in Diabetes."

Divergent remodeling of the skeletal muscle metabolome over 24 h between young, healthy men and older, metabolically compromised men.

24 h whole-body substrate metabolism and the circadian clock within skeletal muscle are both compromised upon metabolic disease in humans. Here, we assessed the 24 h muscle metabolome by serial muscle sampling performed under 24 h real-life conditions in young, healthy (YH) men versus older, metabolically compromised (OMC) men. We find that metabolites associated with the initial steps of glycolysis and hexosamine biosynthesis are higher in OMC men around the clock, whereas metabolites associated with glutamine-alpha-ketoglutarate, ketone, and redox metabolism are lower in OMC men. The night period shows the largest number of differently expressed metabolites. Both groups demonstrate 24 h rhythmicity in half of the metabolome, but rhythmic metabolites only partially overlap. Specific metabolites are only rhythmic in YH men (adenosine), phase shifted in OMC men (cis-aconitate, flavin adenine dinucleotide [FAD], and uridine diphosphate [UDP]), or have a reduced 24 h amplitude in OMC men (hydroxybutyrate and hippuric acid). Our data highlight the plasticity of the skeletal muscle metabolome over 24 h and large divergence across the metabolic health spectrum.

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.

Three weeks of time-restricted eating improves glucose homeostasis in adults with type 2 diabetes but does not improve insulin sensitivity: a randomised crossover trial.

AIMS/HYPOTHESIS: Time-restricted eating (TRE) is suggested to improve metabolic health by limiting food intake to a defined time window, thereby prolonging the overnight fast. This prolonged fast is expected to lead to a more pronounced depletion of hepatic glycogen stores overnight and might improve insulin sensitivity due to an increased need to replenish nutrient storage. Previous studies showed beneficial metabolic effects of 6-8 h TRE regimens in healthy, overweight adults under controlled conditions. However, the effects of TRE on glucose homeostasis in individuals with type 2 diabetes are unclear. Here, we extensively investigated the effects of TRE on hepatic glycogen levels and insulin sensitivity in individuals with type 2 diabetes. METHODS: Fourteen adults with type 2 diabetes (BMI 30.5±4.2 kg/m2, HbA1c 46.1±7.2 mmol/mol [6.4±0.7%]) participated in a 3 week TRE (daily food intake within 10 h) vs control (spreading food intake over ≥14 h) regimen in a randomised, crossover trial design. The study was performed at Maastricht University, the Netherlands. Eligibility criteria included diagnosis of type 2 diabetes, intermediate chronotype and absence of medical conditions that could interfere with the study execution and/or outcome. Randomisation was performed by a study-independent investigator, ensuring that an equal amount of participants started with TRE and CON. Due to the nature of the study, neither volunteers nor investigators were blinded to the study interventions. The quality of the data was checked without knowledge on intervention allocation. Hepatic glycogen levels were assessed with 13C-MRS and insulin sensitivity was assessed using a hyperinsulinaemic-euglycaemic two-step clamp. Furthermore, glucose homeostasis was assessed with 24 h continuous glucose monitoring devices. Secondary outcomes included 24 h energy expenditure and substrate oxidation, hepatic lipid content and skeletal muscle mitochondrial capacity. RESULTS: Results are depicted as mean ± SEM. Hepatic glycogen content was similar between TRE and control condition (0.15±0.01 vs 0.15±0.01 AU, p=0.88). M value was not significantly affected by TRE (19.6±1.8 vs 17.7±1.8 µmol kg-1 min-1 in TRE vs control, respectively, p=0.10). Hepatic and peripheral insulin sensitivity also remained unaffected by TRE (p=0.67 and p=0.25, respectively). Yet, insulin-induced non-oxidative glucose disposal was increased with TRE (non-oxidative glucose disposal 4.3±1.1 vs 1.5±1.7 µmol kg-1 min-1, p=0.04). TRE increased the time spent in the normoglycaemic range (15.1±0.8 vs 12.2±1.1 h per day, p=0.01), and decreased fasting glucose (7.6±0.4 vs 8.6±0.4 mmol/l, p=0.03) and 24 h glucose levels (6.8±0.2 vs 7.6±0.3 mmol/l, p<0.01). Energy expenditure over 24 h was unaffected; nevertheless, TRE decreased 24 h glucose oxidation (260.2±7.6 vs 277.8±10.7 g/day, p=0.04). No adverse events were reported that were related to the interventions. CONCLUSIONS/INTERPRETATION: We show that a 10 h TRE regimen is a feasible, safe and effective means to improve 24 h glucose homeostasis in free-living adults with type 2 diabetes. However, these changes were not accompanied by changes in insulin sensitivity or hepatic glycogen. TRIAL REGISTRATION: ClinicalTrials.gov NCT03992248 FUNDING: ZonMW, 459001013.

A randomized placebo-controlled clinical trial for pharmacological activation of BCAA catabolism in patients with type 2 diabetes.

Elevations in plasma branched-chain amino acid (BCAA) levels associate with insulin resistance and type 2 diabetes (T2D). Pre-clinical models suggest that lowering BCAA levels improve glucose tolerance, but data in humans are lacking. Here, we used sodium phenylbutyrate (NaPB), an accelerator of BCAA catabolism, as tool to lower plasma BCAA levels in patients with T2D, and evaluate its effect on metabolic health. This trial (NetherlandsTrialRegister: NTR7426) had a randomized, placebo-controlled, double-blind cross-over design and was performed in the Maastricht University Medical Center (MUMC+), the Netherlands, between February 2019 and February 2020. Patients were eligible for the trial if they were 40-75years, BMI of 25-38 kg/m², relatively well-controlled T2D (HbA1C < 8.5%) and treated with oral glucose-lowering medication. Eighteen participants were randomly assigned to receive either NaPB 4.8 g/m²/day and placebo for 2 weeks via controlled randomization and sixteen participants completed the study. The primary outcome was peripheral insulin sensitivity. Secondary outcomes were ex vivo muscle mitochondrial oxidative capacity, substrate oxidation and ectopic fat accumulation. Fasting blood samples were collected to determine levels of BCAA, their catabolic intermediates, insulin, triglycerides, free fatty acids (FFA) and glucose. NaPB led to a robust 27% improvement in peripheral insulin sensitivity compared to placebo (ΔRd:13.2 ± 1.8 vs. 9.6 ± 1.8 µmol/kg/min, p = 0.02). This was paralleled by an improvement in pyruvate-driven muscle mitochondrial oxidative capacity and whole-body insulin-stimulated carbohydrate oxidation, and a reduction in plasma BCAA and glucose levels. No effects were observed on levels of insulin, triglycerides and FFA, neither did fat accumulation in muscle and liver change. No adverse events were reported. These data establish the proof-of-concept in humans that modulating the BCAA oxidative pathway may represent a potential treatment strategy for patients with T2D.

The influence of bright and dim light on substrate metabolism, energy expenditure and thermoregulation in insulin-resistant individuals depends on time of day.

AIMS/HYPOTHESIS: In our modern society, artificial light is available around the clock and most people expose themselves to electrical light and light-emissive screens during the dark period of the natural light/dark cycle. Such suboptimal lighting conditions have been associated with adverse metabolic effects, and redesigning indoor lighting conditions to mimic the natural light/dark cycle more closely holds promise to improve metabolic health. Our objective was to compare metabolic responses to lighting conditions that resemble the natural light/dark cycle in contrast to suboptimal lighting in individuals at risk of developing metabolic diseases. METHODS: Therefore, we here performed a non-blinded, randomised, controlled, crossover trial in which overweight insulin-resistant volunteers (n = 14) were exposed to two 40 h laboratory sessions with different 24 h lighting protocols while staying in a metabolic chamber under real-life conditions. In the Bright day-Dim evening condition, volunteers were exposed to electric bright light (~1250 lx) during the daytime (08:00-18:00 h) and to dim light (~5 lx) during the evening (18:00-23:00 h). Vice versa, in the Dim day-Bright evening condition, volunteers were exposed to dim light during the daytime and bright light during the evening. Randomisation and allocation to light conditions were carried out by sequential numbering. During both lighting protocols, we performed 24 h indirect calorimetry, and continuous core body and skin temperature measurements, and took frequent blood samples. The primary outcome was plasma glucose focusing on the pre- and postprandial periods of the intervention. RESULTS: Spending the day in bright light resulted in a greater increase in postprandial triacylglycerol levels following breakfast, but lower glucose levels preceding the dinner meal at 18:00 h, compared with dim light (5.0 ± 0.2 vs 5.2 ± 0.2 mmol/l, n = 13, p=0.02). Dim day-Bright evening reduced the increase in postprandial glucose after dinner compared with Bright day-Dim evening (incremental AUC: 307 ± 55 vs 394 ± 66 mmol/l × min, n = 13, p=0.009). After the Bright day-Dim evening condition the sleeping metabolic rate was identical compared with the baseline night, whereas it dropped after Dim day-Bright evening. Melatonin secretion in the evening was strongly suppressed for Dim day-Bright evening but not for Bright day-Dim evening. Distal skin temperature for Bright day-Dim evening was lower at 18:00 h (28.8 ± 0.3°C vs 29.9 ± 0.4°C, n = 13, p=0.039) and higher at 23:00 h compared with Dim day-Bright evening (30.1 ± 0.3°C vs 28.8 ± 0.3°C, n = 13, p=0.006). Fasting and postprandial plasma insulin levels and the respiratory exchange ratio were not different between the two lighting protocols at any time. CONCLUSIONS/INTERPRETATION: Together, these findings suggest that the indoor light environment modulates postprandial substrate handling, energy expenditure and thermoregulation of insulin-resistant volunteers in a time-of-day-dependent manner. TRIAL REGISTRATION: ClinicalTrials.gov NCT03829982. FUNDING: We acknowledge the financial support from the Netherlands Cardiovascular Research Initiative: an initiative with support from the Dutch Heart Foundation (CVON2014-02 ENERGISE).