Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human.

Brown fat generates heat via the mitochondrial uncoupling protein UCP1, defending against hypothermia and obesity. Recent data suggest that there are two distinct types of brown fat: classical brown fat derived from a myf-5 cellular lineage and UCP1-positive cells that emerge in white fat from a non-myf-5 lineage. Here, we report the isolation of "beige" cells from murine white fat depots. Beige cells resemble white fat cells in having extremely low basal expression of UCP1, but, like classical brown fat, they respond to cyclic AMP stimulation with high UCP1 expression and respiration rates. Beige cells have a gene expression pattern distinct from either white or brown fat and are preferentially sensitive to the polypeptide hormone irisin. Finally, we provide evidence that previously identified brown fat deposits in adult humans are composed of beige adipocytes. These data provide a foundation for studying this mammalian cell type with therapeutic potential. PAPERCLIP:

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.

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.

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.

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).

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.

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.

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.

Prolonged ß2-adrenergic agonist treatment improves glucose homeostasis in diet-induced obese UCP1-/- mice.

Prolonged supplementation with the ß2-agonist clenbuterol improves glucose homeostasis in diabetic rodents, likely via ß2-adrenoceptor (ß2-AR)-mediated effects in the skeletal muscle and liver. However, since rodents have, in contrast to-especially diabetic-humans, substantial quantities of brown adipose tissue (BAT) and clenbuterol has affinity to ß1- and ß3-ARs, the contribution of BAT to these improvements is unclear. Therefore, we investigated clenbuterol-mediated improvements in glucose homeostasis in uncoupling protein 1-deficient (UCP1-/-) mice, lacking thermogenic BAT, versus wild-type (WT) mice. Anesthetized WT and UCP1-/- C57Bl/6 mice were injected with saline or clenbuterol and whole body oxygen consumption was measured. Furthermore, male WT and UCP1-/- C57Bl/6 mice were subjected to 17-wk of chow feeding, high-fat feeding, or high-fat feeding with clenbuterol treatment between weeks 13 and 17. Body composition was measured weekly with MRI. Oral glucose tolerance and insulin tolerance tests were performed in week 15 and 17, respectively. Clenbuterol increased oxygen consumption approximately twofold in WT mice. This increase was blunted in UCP1-/- mice, indicating clenbuterol-mediated activation of BAT thermogenesis. High-fat feeding induced diabetogenic phenotypes in both genotypes. However, low-dose clenbuterol treatment for 2 wk significantly reduced fasting blood glucose by 12.9% in WT and 14.8% in UCP1-/- mice. Clenbuterol treatment improved glucose and insulin tolerance in both genotypes compared with HFD controls and normalized to chow-fed control mice independent of body mass and composition alterations. Clenbuterol improved whole body glucose homeostasis independent of UCP1. Given the low human abundancy of BAT, ß2-AR agonist treatment provides a potential novel route for glucose disposal in diabetic humans.NEW & NOTEWORTHY Improvements in whole body glucose homeostasis of rodents upon prolonged ß2-adrenergic agonist supplementation could potentially be attributed to UCP1-mediated BAT thermogenesis. Indeed, we show that acute injection with the ß2-AR agonist clenbuterol induces BAT activation in mice. However, we also demonstrate that prolonged clenbuterol supplementation robustly improves whole body glucose and insulin tolerance in a similar way in both DIO WT and UCP1-/- mice, indicating that ß2-AR agonist supplementation improves whole body glucose homeostasis independent of UCP1-mediated BAT thermogenesis.

The importance of 24-h metabolism in obesity-related metabolic disorders: opportunities for timed interventions.

Various metabolic processes in the body oscillate throughout the natural day, driven by a biological clock. Circadian rhythms are also influenced by time cues from the environment (light exposure) and behaviour (eating and exercise). Recent evidence from diurnal- and circadian-rhythm studies indicates rhythmicity in various circulating metabolites, insulin secretion and -sensitivity and energy expenditure in metabolically healthy adults. These rhythms have been shown to be disturbed in adults with obesity-related metabolic disturbances. Moreover, eating and being (in)active at a time that the body is not prepared for it, as in night-shift work, is related to poor metabolic outcomes. These findings indicate the relevance of 24-h metabolism in obesity-related metabolic alterations and have also led to novel strategies, such as timing of food intake and exercise, to reinforce the circadian rhythm and thereby improving metabolic health. This review aims to deepen the understanding of the influence of the circadian system on metabolic processes and obesity-related metabolic disturbances and to discuss novel time-based strategies that may be helpful in combating metabolic disease.

Circadian misalignment induces fatty acid metabolism gene profiles and compromises insulin sensitivity in human skeletal muscle.

Circadian misalignment, such as in shift work, has been associated with obesity and type 2 diabetes. However, direct effects of circadian misalignment on skeletal muscle insulin sensitivity and the muscle molecular circadian clock have never been studied in humans. Here, we investigated insulin sensitivity and muscle metabolism in 14 healthy young lean men [age 22.4 ± 2.8 years; body mass index (BMI) 22.3 ± 2.1 kg/m2 (mean ± SD)] after a 3-d control protocol and a 3.5-d misalignment protocol induced by a 12-h rapid shift of the behavioral cycle. We show that short-term circadian misalignment results in a significant decrease in muscle insulin sensitivity due to a reduced skeletal muscle nonoxidative glucose disposal (rate of disappearance: 23.7 ± 2.4 vs. 18.4 ± 1.4 mg/kg per minute; control vs. misalignment; P = 0.024). Fasting glucose and free fatty acid levels as well as sleeping metabolic rate were higher during circadian misalignment. Molecular analysis of skeletal muscle biopsies revealed that the molecular circadian clock was not aligned to the inverted behavioral cycle, and transcriptome analysis revealed the human PPAR pathway as a key player in the disturbed energy metabolism upon circadian misalignment. Our findings may provide a mechanism underlying the increased risk of type 2 diabetes among shift workers.

Treatment with a ß-2-adrenoceptor agonist stimulates glucose uptake in skeletal muscle and improves glucose homeostasis, insulin resistance and hepatic steatosis in mice with diet-induced obesity.

AIMS/HYPOTHESIS: Chronic stimulation of ß2-adrenoceptors, opposite to acute treatment, was reported to reduce blood glucose levels, as well as to improve glucose and insulin tolerance in rodent models of diabetes by essentially unknown mechanisms. We recently described a novel pathway that mediates glucose uptake in skeletal muscle cells via stimulation of ß2-adrenoceptors. In the current study we further explored the potential therapeutic relevance of ß2-adrenoceptor stimulation to improve glucose homeostasis and the mechanisms responsible for the effect. METHODS: C57Bl/6N mice with diet-induced obesity were treated both acutely and for up to 42 days with a wide range of clenbuterol dosages and treatment durations. Glucose homeostasis was assessed by glucose tolerance test. We also measured in vivo glucose uptake in skeletal muscle, insulin sensitivity by insulin tolerance test, plasma insulin levels, hepatic lipids and glycogen. RESULTS: Consistent with previous findings, acute clenbuterol administration increased blood glucose and insulin levels. However, already after 4 days of treatment, beneficial effects of clenbuterol were manifested in glucose homeostasis (32% improvement of glucose tolerance after 4 days of treatment, p < 0.01) and these effects persisted up to 42 days of treatment. These favourable metabolic effects could be achieved with doses as low as 0.025 mg kg-1 day-1 (40 times lower than previously studied). Mechanistically, these effects were not due to increased insulin levels, but clenbuterol enhanced glucose uptake in skeletal muscle in vivo both acutely in lean mice (by 64%, p < 0.001) as well as during chronic treatment in diet-induced obese mice (by 74%, p < 0.001). Notably, prolonged treatment with low-dose clenbuterol improved whole-body insulin sensitivity (glucose disposal rate after insulin injection increased up to 1.38 ± 0.31%/min in comparison with 0.15 ± 0.36%/min in control mice, p < 0.05) and drastically reduced hepatic steatosis (by 40%, p < 0.01) and glycogen (by 23%, p < 0.05). CONCLUSIONS/INTERPRETATION: Clenbuterol improved glucose tolerance after 4 days of treatment and these effects were maintained for up to 42 days. Effects were achieved with doses in a clinically relevant microgram range. Mechanistically, prolonged treatment with a low dose of clenbuterol improved glucose homeostasis in insulin resistant mice, most likely by stimulating glucose uptake in skeletal muscle and improving whole-body insulin sensitivity as well as by reducing hepatic lipids and glycogen. We conclude that selective ß2-adrenergic agonists might be an attractive potential treatment for type 2 diabetes. This remains to be confirmed in humans. Graphical abstract.

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.

Effect of L-arginine on energy metabolism, skeletal muscle and brown adipose tissue in South Asian and Europid prediabetic men: a randomised double-blinded crossover study.

AIMS/HYPOTHESIS: Individuals of South Asian origin are at increased risk of developing type 2 diabetes mellitus and associated comorbidities compared with Europids. Disturbances in energy metabolism may contribute to this increased risk. Skeletal muscle and possibly also brown adipose tissue (BAT) are involved in human energy metabolism and nitric oxide (NO) is suggested to play a pivotal role in regulating mitochondrial biogenesis in both tissues. We aimed to investigate the effects of 6 weeks of supplementation with L-arginine, a precursor of NO, on energy metabolism by BAT and skeletal muscle, as well as glucose metabolism in South Asian men compared with men of European descent. METHODS: We included ten Dutch South Asian men (age 46.5 ± 2.8 years, BMI 30.1 ± 1.1 kg/m2) and ten Dutch men of European descent, that were similar with respect to age and BMI, with prediabetes (fasting plasma glucose level 5.6-6.9 mmol/l or plasma glucose levels 2 h after an OGTT 7.8-11.1 mmol/l). Participants took either L-arginine (9 g/day) or placebo orally for 6 weeks in a randomised double-blind crossover study. Participants were eligible to participate in the study when they were aged between 40 and 55 years, had a BMI between 25 and 35 kg/m2 and did not have type 2 diabetes. Furthermore, ethnicity was defined as having four grandparents of South Asian or white European origin, respectively. Blinding of treatment was done by the pharmacy (Hankintatukku) and an independent researcher from Leiden University Medical Center randomly assigned treatments by providing a coded list. All people involved in the study as well as participants were blinded to group assignment. After each intervention, glucose tolerance was determined by OGTT and basal metabolic rate (BMR) was determined by indirect calorimetry; BAT activity was assessed by cold-induced [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography-computed tomography scanning. In addition, a fasting skeletal muscle biopsy was taken and analysed ex vivo for respiratory capacity using a multisubstrate protocol. The primary study endpoint was the effect of L-arginine on BAT volume and activity. RESULTS: L-Arginine did not affect BMR, [18F]FDG uptake by BAT or skeletal muscle respiration in either ethnicity. During OGTT, L-arginine lowered plasma glucose concentrations (AUC0-2 h - 9%, p < 0.01), insulin excursion (AUC0-2 h - 26%, p < 0.05) and peak insulin concentrations (-26%, p < 0.05) in Europid but not South Asian men. This coincided with enhanced cold-induced glucose oxidation (+44%, p < 0.05) in Europids only. Of note, in skeletal muscle biopsies several respiration states were consistently lower in South Asian men compared with Europid men. CONCLUSIONS/INTERPRETATION: L-Arginine supplementation does not affect BMR, [18F]FDG uptake by BAT, or skeletal muscle mitochondrial respiration in Europid and South Asian overweight and prediabetic men. However, L-arginine improves glucose tolerance in Europids but not in South Asians. Furthermore, South Asian men have lower skeletal muscle oxidative capacity than men of European descent. FUNDING: This study was funded by the EU FP7 project DIABAT, the Netherlands Organization for Scientific Research, the Dutch Diabetes Research Foundation and the Dutch Heart Foundation. TRIAL REGISTRATION: ClinicalTrials.gov NCT02291458.

MicroRNA-382 silencing induces a mitonuclear protein imbalance and activates the mitochondrial unfolded protein response in muscle cells.

Proper mitochondrial function plays a central role in cellular metabolism. Various diseases as well as aging are associated with diminished mitochondrial function. Previously, we identified 19 miRNAs putatively involved in the regulation of mitochondrial metabolism in skeletal muscle, a highly metabolically active tissue. In the current study, these 19 miRNAs were individually silenced in C2C12 myotubes using antisense oligonucleotides, followed by measurement of the expression of 27 genes known to play a major role in regulating mitochondrial metabolism. Based on the outcomes, we then focused on miR-382-5p and identified pathways affected by its silencing using microarrays, investigated protein expression, and studied cellular respiration. Silencing of miRNA-382-5p significantly increased the expression of several genes involved in mitochondrial dynamics and biogenesis. Conventional microarray analysis in C2C12 myotubes silenced for miRNA-382-5p revealed a collective downregulation of mitochondrial ribosomal proteins and respiratory chain proteins. This effect was accompanied by an imbalance between mitochondrial proteins encoded by the nuclear and mitochondrial DNA (1.35-fold, p < 0.01) and an induction of HSP60 protein (1.31-fold, p < 0.05), indicating activation of the mitochondrial unfolded protein response (mtUPR). Furthermore, silencing of miR-382-5p reduced basal oxygen consumption rate by 14% ( p < 0.05) without affecting mitochondrial content, pointing towards a more efficient mitochondrial function as a result of improved mitochondrial quality control. Taken together, silencing of miR-382-5p induces a mitonuclear protein imbalance and activates the mtUPR in skeletal muscle, a phenomenon that was previously associated with improved longevity.

Dissociation of intramyocellular lipid storage and insulin resistance in trained athletes and type 2 diabetes patients; involvement of perilipin 5?

KEY POINTS: Intramyocellular lipid storage is negatively associated with insulin sensitivity. However, endurance trained athletes and type 2 diabetes mellitus (T2DM) patients store similar amounts of lipids in their muscle; the so-called athlete's paradox. Compared to T2DM, trained athletes possess higher levels of perilipin 5 (PLIN5), a lipid droplet (LD) coating protein. We examined whether coating LD with PLIN5 affects the pattern of muscle lipid (LD size and number) in relation to the athlete's paradox. Despite differences in PLIN5 protein content, we observed that coating the LD with PLIN5 could not explain the observed differences in LD size and number between athletes and T2DM. PLIN5-coated LDs were positively associated with oxidative capacity but not with insulin sensitivity. We conclude that coating of LDs with PLIN5 cannot causally explain the athlete's paradox. ABSTRACT: Intramyocellular lipid (IMCL) hampers insulin sensitivity, albeit not in endurance-trained athletes (Trained). Compared to type 2 diabetes mellitus (T2DM) patients, Trained subjects have high levels of perilipin 5 (PLIN5). In the present study, we tested whether the fraction of PLIN5-coated lipid droplets (LDs) is a determinant of skeletal muscle insulin sensitivity and contributes to the athlete's paradox. Muscle biopsies were taken from eight Trained, Lean sedentary, Obese and T2DM subjects. Trained, Obese and T2DM subjects were matched for total IMCL content. Confocal images were analysed for lipid area fraction, LD size and number and PLIN5+ and PLIN5- LDs were measured. A stepwise linear regression was performed to identify factors explaining observed variance in glucose infusion rate (GIR). Trained and T2DM subjects stored IMCL differently; Trained subjects had a higher number of LDs compared to T2DM subjects (0.037 ± 0.004 µm-2 vs. 0.023 ± 0.003 µm-2 , P = 0.024) that were non-significantly smaller (0.27 ± 0.01 µm2 vs. 0.32 ± 0.02 µm2 , P = 0.197, Trained vs. T2DM). Even though total PLIN5 protein content was almost double in Trained vs. T2DM subjects (1.65 ± 0.21 AU vs. 0.89 ± 0.09 AU, P = 0.004), PLIN5 coating did not affect LD number or size significantly. Of the observed variance in GIR, the largest fraction by far (70.2%) was explained by maximal oxygen uptake. Adding PLIN5 protein content or PLIN5+ LDs increased the explained variance in GIR (74.7% and 80.7% for PLIN5 protein content and PLIN5+ LDs, respectively). Thus, the putative relationship between PLIN5 and insulin sensitivity is at best indirect and is apparent only in conjunction with maximal oxygen uptake. Hence, PLIN5 abundance cannot be causally linked to the athlete's paradox.

Decoration of intramyocellular lipid droplets with PLIN5 modulates fasting-induced insulin resistance and lipotoxicity in humans.

AIMS/HYPOTHESIS: In contrast to insulin-resistant individuals, insulin-sensitive athletes possess high intramyocellular lipid content (IMCL), good mitochondrial function and high perilipin 5 (PLIN5) levels, suggesting a role for PLIN5 in benign IMCL storage. We hypothesised a role for PLIN5 in modulating fasting-mediated insulin resistance. METHODS: Twelve men were fasted for 60 h, before and after which muscle biopsies were taken and stained for lipid droplets (LDs), PLIN5 and laminin. Confocal microscopy images were analysed for LD size, number, PLIN5 association and subcellular distribution. RESULTS: Fasting elevated IMCL content 2.8-fold and reduced insulin sensitivity (by 55%). Individuals with the most prominent increase in IMCL showed the least reduction in insulin sensitivity (r = 0.657; p = 0.028) and mitochondrial function (r = 0.896; p = 0.006). During fasting, PLIN5 gene expression or PLIN5 protein content in muscle homogenates was unaffected, microscopy analyses revealed that the fraction of PLIN5 associated with LDs (PLIN5+) increased significantly (+26%) upon fasting, suggesting PLIN5 redistribution. The significant increase in LD number (+23%) and size (+23%) upon fasting was entirely accounted for by PLIN5+ LDs, not by LDs devoid of PLIN5. Also the association between IMCL storage capacity and insulin resistance and mitochondrial dysfunction was only apparent for PLIN5+ LDs. CONCLUSIONS/INTERPRETATION: Fasting results in subcellular redistribution of PLIN5 and promotes the capacity to store excess fat in larger and more numerous PLIN5-decorated LDs. This associates with blunting of fasting-induced insulin resistance and mitochondrial dysfunction, suggesting a role for PLIN5 in the modulation of fasting-mediated lipotoxicity. TRIAL REGISTRATION: trialregister.nl NTR 2042.

ANT1-mediated fatty acid-induced uncoupling as a target for improving myocellular insulin sensitivity.

AIMS/HYPOTHESIS: Dissipating energy via mitochondrial uncoupling has been suggested to contribute to enhanced insulin sensitivity. We hypothesised that skeletal muscle mitochondria of endurance-trained athletes have increased sensitivity for fatty acid (FA)-induced uncoupling, which is driven by the mitochondrial protein adenine nucleotide translocase 1 (ANT1). METHODS: Capacity for FA-induced uncoupling was measured in endurance-trained male athletes (T) and sedentary young men (UT) in an observational study and also in isolated skeletal muscle mitochondria from Zucker diabetic fatty (ZDF) rats and C2C12 myotubes following small interfering RNA (siRNA)-mediated gene silencing of ANT1. Thus, fuelled by glutamate/succinate (fibres) or pyruvate (mitochondria and myotubes) and in the presence of oligomycin to block ATP synthesis, increasing levels of oleate (fibres) or palmitate (mitochondria and myotubes) were automatically titrated while respiration was monitored. Insulin sensitivity was measured by hyperinsulinaemic-euglycaemic clamp in humans and via insulin-stimulated glucose uptake in myotubes. RESULTS: Skeletal muscle from the T group displayed increased sensitivity to FA-induced uncoupling (p = 0.011) compared with muscle from the UT group, and this was associated with elevated insulin sensitivity (p = 0.034). ANT1 expression was increased in T (p = 0.013). Mitochondria from ZDF rats displayed decreased sensitivity for FA-induced uncoupling (p = 0.008). This difference disappeared in the presence of the adenine nucleotide translocator inhibitor carboxyatractyloside. Partial knockdown of ANT1 in C2C12 myotubes decreased sensitivity to the FA-induced uncoupling (p = 0.008) and insulin-stimulated glucose uptake (p = 0.025) compared with controls. CONCLUSIONS/INTERPRETATION: Increased sensitivity to FA-induced uncoupling is associated with enhanced insulin sensitivity and is affected by ANT1 activity in skeletal muscle. FA-induced mitochondrial uncoupling may help to preserve insulin sensitivity in the face of a high supply of FAs. TRIAL REGISTRATION: www.trialregister.nl NTR2002.

Mitochondrial dynamics, quality control and miRNA regulation in skeletal muscle: implications for obesity and related metabolic disease.

The western dietary habits and sedentary lifestyle largely contributes to the growing epidemic of obesity. Mitochondria are at the front line of cellular energy homoeostasis and are implicated in the pathophysiology of obesity and obesity-related metabolic disease. In recent years, novel aspects in the regulation of mitochondrial metabolism, such as mitochondrial dynamics, mitochondrial protein quality control and post-transcriptional regulation of genes coding for mitochondrial proteins, have emerged. In this review, we discuss the recent findings concerning the dysregulation of these processes in skeletal muscle in obesogenic conditions.

Glucose uptake in human brown adipose tissue is impaired upon fasting-induced insulin resistance.

AIMS/HYPOTHESIS: Human brown adipose tissue (BAT) has recently emerged as a potential target in the treatment of type 2 diabetes, owing to its capacity to actively clear glucose from the circulation­at least upon cold exposure. The effects of insulin resistance on the capacity of human BAT to take up glucose are unknown. Prolonged fasting is known to induce insulin resistance in peripheral tissues in order to spare glucose for the brain. METHODS: We studied the effect of fasting-induced insulin resistance on the capacity of BAT to take up glucose during cold exposure as well as on cold-stimulated thermogenesis. BAT glucose uptake was assessed by means of cold-stimulated dynamic 2-deoxy-2-[(18)F]fluoro-D-glucose positron emission tomography/computed tomography ([(18)F]FDG-PET/CT) imaging. RESULTS: We show that a 54 h fasting period markedly decreases both cold-induced BAT glucose uptake and nonshivering thermogenesis (NST) during cold stimulation. In vivo molecular imaging and modelling revealed that the reduction of glucose uptake in BAT was due to impaired cellular glucose uptake and not due to decreased supply. Interestingly, decreased BAT glucose uptake upon fasting was related to a decrease in core temperature during cold exposure, pointing towards a role for BAT in maintaining normothermia in humans. CONCLUSIONS/INTERPRETATION: Cold-stimulated glucose uptake in BAT is strongly reduced upon prolonged fasting. When cold-stimulated glucose uptake in BAT is also reduced under other insulin-resistant states, such as diabetes, cold-induced activation of BAT may not be a valid way to improve glucose clearance by BAT under such conditions. TRIAL REGISTRATION: www.trialregister.nl NTR3523 FUNDING: This work was supported by the EU FP7 project DIABAT (HEALTH-F2-2011-278373 to WDvML) and by the Netherlands Organization for Scientific Research (TOP 91209037 to WDvML).