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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."

Background: Type 2 diabetes patients and individuals at risk of developing diabetes are characterized by metabolic inflexibility and disturbed glucose homeostasis. Low carnitine availability may contribute to metabolic inflexibility and impaired glucose tolerance. Here, we investigated whether carnitine supplementation improves metabolic flexibility and insulin sensitivity in impaired glucose tolerant (IGT) volunteers., Methods: Eleven IGT- volunteers followed a 36-day placebo- and L-carnitine treatment (2 g/day) in a randomised, placebo-controlled, double blind crossover design. A hyperinsulinemic-euglycemic clamp (40 mU/m 2 /min), combined with indirect calorimetry (ventilated hood) was performed to determine insulin sensitivity and metabolic flexibility. Furthermore, metabolic flexibility was assessed in response to a high-energy meal. Skeletal muscle acetylcarnitine concentrations were measured in vivo using long echo time proton magnetic resonance spectroscopy ( 1 H-MRS, TE=500 ms) in the resting state (7:00AM and 5:00PM) and after a 30-min cycling exercise. Twelve normal glucose tolerant (NGT) volunteers were included without any intervention as control group., Results: Metabolic flexibility of IGT-subjects completely restored towards NGT control values upon carnitine supplementation, measured during a hyperinsulinemic-euglycemic clamp and meal test. In muscle, carnitine supplementation enhanced the increase in resting acetylcarnitine concentrations over the day (delta 7:00 AM versus 5:00 PM) in IGT-subjects. Furthermore, carnitine supplementation increased post-exercise acetylcarnitine concentrations and reduced long-chain acylcarnitine species in IGT-subjects, suggesting the stimulation of a more complete fat oxidation in muscle. Whole-body insulin sensitivity was not affected., Conclusion: Carnitine supplementation improves acetylcarnitine formation and rescues metabolic flexibility in IGT-subjects. Future research should investigate the potential of carnitine in prevention/treatment of type 2 diabetes., (Copyright © 2019 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rodent studies have indicated that physical exercise may improve adipose tissue function. We investigated the effects of a 12-wk supervised, progressive exercise training program on adipocyte morphology and abdominal subcutaneous adipose tissue function in metabolically well-phenotyped subjects with obesity. Men with obesity ( n = 21) participated in a 12-wk supervised, progressive, combined exercise training program consisting of aerobic exercise (30 min at 70% of maximal power output 2 times/wk) and resistance exercise (3 × 10 repetitions at 60% of 1 repeated maximum 1 time/wk), with adjustment of exercise intensity every 4 wk. At baseline and after intervention, abdominal subcutaneous adipose tissue biopsies were collected to determine 1) adipocyte morphology, 2) gene expression of markers for lipolysis, inflammation, browning, adipokines, and mitochondrial biogenesis/function, 3) protein expression of mitochondrial oxidative phosphorylation (OXPHOS) complexes, and 4) ex vivo basal and β 2 -adrenergic stimulated lipolysis. The exercise training program, which increased maximal aerobic capacity ( P < 0.001) and muscle strength ( P < 0.001), slightly reduced adipose tissue mass (~0.7 kg, P = 0.021) but did not affect abdominal subcutaneous adipocyte size ( P = 0.744), adipose tissue gene expression of markers for mitochondrial biogenesis and function, browning, lipolysis, inflammation and adipokines, total OXPHOS protein content ( P = 0.789), or β 2 -adrenergic sensitivity of lipolysis ( P = 0.555). A 12-wk supervised, progressive exercise training program did not alter abdominal subcutaneous adipocyte morphology and adipose tissue gene/protein expression of markers related to adipose tissue function or β 2 -adrenergic sensitivity of lipolysis in male subjects with obesity. NEW & NOTEWORTHY Studies that investigated the effects of exercise training on adipose tissue function in well-phenotyped humans are scarce. We demonstrate that 12 wk of supervised exercise training improved physical fitness and peripheral insulin sensitivity but did not alter abdominal subcutaneous adipocyte morphology, adipose tissue gene and protein expression of markers related to adipose tissue function, or β 2 -adrenergic receptor-mediated lipolysis in men with obesity. A prolonged and/or more intense training program may be required to improve human adipose tissue function.

Metabolic stresses such as dietary energy restriction or physical activity exert beneficial metabolic effects. In the liver, endospanin-1 and endospanin-2 cooperatively modulate calorie restriction-mediated (CR-mediated) liver adaptations by controlling growth hormone sensitivity. Since we found CR to induce endospanin protein expression in skeletal muscle, we investigated their role in this tissue. In vivo and in vitro endospanin-2 triggers ERK phosphorylation in skeletal muscle through an autophagy-dependent pathway. Furthermore, endospanin-2, but not endospanin-1, overexpression decreases muscle mitochondrial ROS production, induces fast-to-slow fiber-type switch, increases skeletal muscle glycogen content, and improves glucose homeostasis, ultimately promoting running endurance capacity. In line, endospanin-2-/- mice display higher lipid peroxidation levels, increased mitochondrial ROS production under mitochondrial stress, decreased ERK phosphorylation, and reduced endurance capacity. In conclusion, our results identify endospanin-2 as a potentially novel player in skeletal muscle metabolism, plasticity, and function.

Purpose: To assess if short-duration (5 to ~300 s) high-power performance can accurately be predicted using the anaerobic power reserve (APR) model in professional cyclists., Methods: Data from 4 professional cyclists from a World Tour cycling team were used. Using the maximal aerobic power, sprint peak power output, and an exponential constant describing the decrement in power over time, a power-duration relationship was established for each participant. To test the predictive accuracy of the model, several all-out field trials of different durations were performed by each cyclist. The power output achieved during the all-out trials was compared with the predicted power output by the APR model., Results: The power output predicted by the model showed very large to nearly perfect correlations to the actual power output obtained during the all-out trials for each cyclist (r = .88 ± .21, .92 ± .17, .95 ± .13, and .97 ± .09). Power output during the all-out trials remained within an average of 6.6% (53 W) of the predicted power output by the model., Conclusions: This preliminary pilot study presents 4 case studies on the applicability of the APR model in professional cyclists using a field-based approach. The decrement in all-out performance during high-intensity exercise seems to conform to a general relationship with a single exponential-decay model describing the decrement in power vs increasing duration. These results are in line with previous studies using the APR model to predict performance during brief all-out trials. Future research should evaluate the APR model with a larger sample size of elite cyclists.

Purpose: The primary aims of this study were (1) to evaluate whole-body mechanical efficiency (ME) in a large group of chronic obstructive pulmonary disease (COPD) patients with a wide range of degrees of illness and (2) to examine how ME in COPD is related to absolute work rate and indices of disease severity during exercise testing., Methods: A total of 569 patients (301 male patients; GOLD stage I: 28, GOLD stage II: 166, GOLD stage III: 265, and GOLD stage IV: 110) with chronic obstructive pulmonary disease (COPD) were included in the data analysis. Individual maximal workload (watt), peak minute ventilation ((Equation is included in full-text article.)E, L/min body temperature and pressure, saturated), and peak oxygen uptake ((Equation is included in full-text article.)O2, mL/min standard temperature and pressure, dry) were determined from a maximal incremental cycle ergometer test. Ventilatory and metabolic response parameters were collected during a constant work rate test at 75% of the individual maximal workload. From the exercise responses of the constant work rate test, the gross ME was calculated., Results: The mean whole-body gross ME was 11.0 ± 3.5% at 75% peak power. The ME declined significantly (P < .001) with increasing severity of the disease when measured at the same relative power. Log-transformed absolute work rate (r = .87, P < .001) was the strongest independent predictor of gross ME. Body mass was the single other variable that contributed significantly to the linear regression model., Conclusions: Gross ME in COPD was largely predicted by the absolute work rate (r = .87; P < .001) while indices of the severity of the disease did not predict ME in COPD.

Aims/hypothesis: We aimed to examine the effects of breaking sitting with standing and light-intensity walking vs an energy-matched bout of structured exercise on 24 h glucose levels and insulin resistance in patients with type 2 diabetes., Methods: In a randomised crossover study, 19 patients with type 2 diabetes (13 men/6 women, 63 ± 9 years old) who were not using insulin each followed three regimens under free-living conditions, each lasting 4 days: (1) Sitting: 4415 steps/day with 14 h sitting/day; (2) Exercise: 4823 steps/day with 1.1 h/day of sitting replaced by moderate- to vigorous-intensity cycling (at an intensity of 5.9 metabolic equivalents [METs]); and (3) Sit Less: 17,502 steps/day with 4.7 h/day of sitting replaced by standing and light-intensity walking (an additional 2.5 h and 2.2 h, respectively, compared with the hours spent doing these activities in the Sitting regimen). Blocked randomisation was performed using a block size of six regimen orders using sealed, non-translucent envelopes. Individuals who assessed the outcomes were blinded to group assignment. Meals were standardised during each intervention. Physical activity and glucose levels were assessed for 24 h/day by accelerometry (activPAL) and a glucose monitor (iPro2), respectively. The incremental AUC (iAUC) for 24 h glucose (primary outcome) and insulin resistance (HOMA2-IR) were assessed on days 4 and 5, respectively., Results: The iAUC for 24 h glucose (mean ± SEM) was significantly lower during the Sit Less intervention than in Sitting (1263 ± 189 min × mmol/l vs 1974 ± 324 min × mmol/l; p = 0.002), and was similar between Sit Less and Exercise (Exercise: 1383 ± 194 min × mmol/l; p = 0.499). Exercise failed to improve HOMA2-IR compared with Sitting (2.06 ± 0.28 vs 2.16 ± 0.26; p = 0.177). In contrast, Sit Less (1.89 ± 0.26) significantly reduced HOMA2-IR compared with Exercise (p = 0.015) as well as Sitting (p = 0.001)., Conclusions/interpretation: Breaking sitting with standing and light-intensity walking effectively improved 24 h glucose levels and improved insulin sensitivity in individuals with type 2 diabetes to a greater extent than structured exercise. Thus, our results suggest that breaking sitting with standing and light-intensity walking may be an alternative to structured exercise to promote glycaemic control in patients type 2 diabetes., Trial Registration: Clinicaltrials.gov NCT02371239 FUNDING: : The study was supported by a Kootstra grant from Maastricht University Medical Centre + , and the Dutch Heart Foundation. Financial support was also provided by Novo Nordisk BV, and Medtronic and Roche made the equipment available for continuous glucose monitoring.

A 5-day High-Fat High-Calorie diet (HFHC-diet) reduces insulin-stimulated glucose disposal (Rd) in South Asian, but not Caucasian healthy lean males. We aimed to investigate if differences in myocellular lipid handling are underlying this differential response. A two-step hyperinsulinemic-euglycemic clamp and muscle biopsies were performed in 12 healthy lean Caucasian and South Asian males (BMI < 25 kg/m 2 , 19-25 years) before and after a 5-day HFHC-diet (regular diet + 375 mL cream/day; 1275 kcal/day; 94% fat). Triglyceride extractions and Western Blots for lipid droplet and mitochondrial proteins were performed. Intramyocellular lipid content and HFHC-diet response were similar between ethnicities (group effect: P = 0.094; diet effect: +~30%, P = 0.044). PLIN5 protein content increased upon the HFHC-diet (P = 0.031) and tended to be higher in South Asians (0.87 ± 0.42 AU vs. 1.35 ± 0.58 AU, P = 0.07). 4-HNE tended to increase in South Asians upon the HFHC-diet (interaction effect: P = 0.057). In Caucasians ΔPLIN5 content correlated with ΔR d (Caucasians: r = 0.756, P = 0.011; South Asians: r = -0.085, P = 0.816), while in South Asians Δ4-HNE associated with ΔPLIN5 content (Caucasians: r = 0.312, P = 0.380; South Asians: r = 0.771, P = 0.003). These data indicate that in Caucasians, PLIN5 may be protective against HFHC-diet induced insulin resistance, which for reasons not yet understood is not observed in South Asians, who possess increased lipid peroxidation levels., Competing Interests: The authors declare no competing financial interests.

Purpose: Acetylcarnitine formation is suggested to be crucial in sustaining metabolic flexibility and glucose homeostasis. Recently, we introduced a method to detect acetylcarnitine in vivo with long TE 1 H-MRS. Differences in T 1 relaxation time between lipids and acetylcarnitine can be exploited for additional lipid suppression in subjects with high myocellular lipid levels., Methods: Acquisition of spectra with an inversion recovery sequence was alternated with standard signal acquisition to suppress short T 1 metabolite signals. A proof of principle experiment was conducted in a lean subject and the new approach was subsequently tested in four overweight/obese subjects., Results: Using the new T 1 editing approach, lipid signals in spectra of skeletal muscle can be (additionally) suppressed by a factor of 10 using a TI of 900 ms. Combination of the long TE protocol with the T 1 editing resulted in a well-resolved acetylcarnitine peak in the obese subjects., Conclusion: The T 1 editing approach suppresses short T 1 metabolites and offers a new contrast in 1 H-MRS. The approach should be used in combination with a long TE in subjects with high lipid contamination for accurate quantification of the acetylcarnitine concentration. Magn Reson Med 77:505-510, 2017. © 2016 International Society for Magnetic Resonance in Medicine., (© 2016 International Society for Magnetic Resonance in Medicine.)

Objective: To determine whether resveratrol supplementation can improve insulin sensitivity and promote overall metabolic health on top of standard diabetes care., Research Design and Methods: Seventeen subjects with well-controlled type 2 diabetes (T2D) were treated with placebo and 150 mg/day resveratrol (resVida) in a randomized double-blind crossover study for 30 days. The main outcome measure was insulin sensitivity by the hyperinsulinemic-euglycemic clamp technique., Results: Hepatic and peripheral insulin sensitivity were not affected by resveratrol treatment. Intrahepatic lipid content also remained unaffected by resveratrol; however, the change in intrahepatic lipid content correlated negatively with plasma resveratrol levels (R = -0.68, P = 0.03). Intramyocellular lipid content increased in type 2 muscle fibers (P = 0.03), and systolic blood pressure tended to decrease (P = 0.09) upon resveratrol treatment. In addition, resveratrol significantly improved ex vivo mitochondrial function (state 3 and state U respiration upon malate with octanoyl-carnitine, P < 0.005). Intriguingly, a correlation was found between plasma levels of a metabolite of resveratrol (dihydroresveratrol) and the metformin dose used by the patients (R = 0.66, P = 0.005), suggesting an interaction between metformin and resveratrol. It could be speculated that the lack of a resveratrol-induced insulin-sensitizing effect is caused by this interaction., Conclusions: Resveratrol supplementation does not improve hepatic or peripheral insulin sensitivity. Our results question the generalized value of resveratrol as an add-on therapy in the treatment of T2D and emphasize the need to perform studies in drug-naive patients with T2D or subjects with prediabetes., (© 2016 by the American Diabetes Association.)

Low levels of physical activity and the presence of obesity are associated with mitochondrial dysfunction. In addition, mitochondrial dysfunction has been associated with the development of insulin resistance and type 2 diabetes mellitus (T2DM). Although the evidence for a causal relationship between mitochondrial function and insulin resistance is still weak, emerging evidence indicates that boosting mitochondrial function might be beneficial to patient health. Exercise training is probably the most recognized promoter of mitochondrial function and insulin sensitivity and hence is still regarded as the best strategy to prevent and treat T2DM. Animal data, however, have revealed several new insights into the regulation of mitochondrial metabolism, and novel targets for interventions to boost mitochondrial function have emerged. Importantly, many of these targets seem to be regulated by factors such as nutrition, ambient temperature and circadian rhythms, which provides a basis for nonpharmacological strategies to prevent or treat T2DM in humans. Here, we will review the current evidence that mitochondrial function can be targeted therapeutically to improve insulin sensitivity and to prevent T2DM, focusing mainly on human intervention studies.

Cell and animal studies have demonstrated that circadian rhythm is governed by autonomous rhythmicity of clock genes. Although disturbances in circadian rhythm have been implicated in metabolic disease development, it remains unknown whether muscle circadian rhythm is altered in human models of type 2 diabetes. Here we used human primary myotubes (HPM) to investigate if rhythmicity of clock- and metabolic gene expression is altered in donors with obesity or type 2 diabetes compared to metabolically healthy donors. HPM were obtained from skeletal muscle biopsies of four groups: type 2 diabetic patients and their BMI- and age-matched obese controls and from lean, healthy and young endurance trained athletes and their age-matched sedentary controls. HPM were differentiated for 7 days before synchronization by serum shock followed by gene expression profiling over the next 72 hours. HPM display robust circadian rhythms in clock genes, but REVERBA displayed dampened rhythmicity in type 2 diabetes. Furthermore, rhythmicity in NAMPT and SIRT1 expression was only observed in HPM from trained athletes. Rhythmicity in expression of key-regulators of carbohydrate and lipid metabolism was modest. We demonstrate that in human skeletal muscle REVERBA/B, NAMPT and SIRT1 circadian rhythms are affected in donors of sedentary life style and poor health status.

Non-alcoholic fatty liver (NAFL) is the most common liver disorder in western society. Various factors may play a role in determining hepatic fat content, such as delivery of lipids to the liver, de novo lipogenesis, hepatic lipid oxidation, secretion of intrahepatic lipids to the circulation or a combination of these. If delivery of lipids to the liver outweighs the sum of hepatic lipid oxidation and secretion, the intrahepatic lipid (IHL) content starts to increase and NAFL may develop. NAFL is closely related to obesity and insulin resistance and a fatty liver increases the vulnerability to type 2 diabetes development. Exercise training is a cornerstone in the treatment and prevention of type 2 diabetes. There is a large body of literature describing the beneficial metabolic consequences of exercise training on skeletal muscle metabolism. Recent studies have started to investigate the effects of exercise training on liver metabolism but data is still limited. Here, first, we briefly discuss the routes by which IHL content is modulated. Second, we review whether and how these contributing routes might be modulated by long-term exercise training. Third, we focus on the effects of acute exercise on IHL metabolism, since exercise also might affect hepatic metabolism in the physically active state. This will give insight into whether the effect of exercise training on IHL could be explained by the accumulated effect of acute bouts of exercise, or whether adaptations might occur only after long-term exercise training. The primary focus of this review will be on observations made in humans. Where human data is missing, data obtained from well-accepted animal models will be used., Competing Interests: Funding This work is supported by the Dutch Diabetes Research Foundation (grant no.: 2009.60.003). Duality of interest The authors declare that there is no duality of interest associated with this manuscript. Contribution statement BB undertook the literature research and drafted and wrote the article. MKCH, PS and VBS-H wrote the article and revised it critically for important intellectual content. All authors approved the version to be submitted.

The prevalence of fatty liver reaches alarming proportions. Fatty liver increases the risk for insulin resistance, cardiovascular disease, and nonalcoholic steatohepatitis (NASH). Although extensively studied in a preclinical setting, the lack of noninvasive methodologies hampers our understanding of which pathways promote hepatic fat accumulation in humans. Dietary fat retention is one of the pathways that may lead to fatty liver. The low (1.1%) natural abundance (NA) of carbon-13 ( 13 C) allows use of 13 C-enriched lipids for in vivo MR studies. Successful implementation of such methodology, however, is challenging due to low sensitivity of 13 C-magnetic resonance spectroscopy ( 13 C-MRS). Here, we investigated the use of 1-dimensional gradient enhanced heteronuclear single quantum coherence (ge-HSQC) spectroscopy for the in vivo detection of hepatic 1 H-[ 13 C]-lipid signals after a single high-fat meal with 13 C-labeled fatty acids in 5 lean and 6 obese subjects. Postprandial retention of orally administered 13 C-labeled fatty acids was significant ( P < 0.01). Approximately 1.5% of the tracer was retained in the liver after 6 hours, and retention was similar in both groups ( P = 0.92). Thus, a substantial part of the liver fat can originate directly from storage of meal-derived fat. The ge-HSQC can be used to noninvasively reveal the contribution of dietary fat to the development of hepatic steatosis over time.

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.

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.

Genistein, a natural food compound mainly present in soybeans, is considered a potent antioxidant and to improve glucose homeostasis. However, its mechanism of action remains poorly understood. Here, we analyzed whether genistein could antagonize the progression of the hyperinsulinemic normoglycemic state (pre-diabetes) toward full-blown T2DM in Zucker Diabetic Fatty (ZDF) rats by decreasing mitochondrial oxidative stress and improving skeletal muscle oxidative capacity. Rats were assigned to three groups: (1) lean control (CNTL), (2) fa/fa CNTL, and (3) fa/fa genistein (GEN). GEN animals were subjected to a 0.02% (w/w) genistein-enriched diet for 8 weeks, whereas CNTL rats received a standard diet. We show that genistein did not affect the overall response to a glucose challenge in ZDF rats. In fact, genistein may exacerbate glucose intolerance as fasting glucose levels were significantly higher in fa/fa GEN (17.6 ± 0.7 mM) compared with fa/fa CNTL animals (14.9 ± 1.4 mM). Oxidative stress, established by electron spin resonance (ESR) spectroscopy, carbonylated protein content and UCP3 levels, remained unchanged upon dietary genistein supplementation. Furthermore, respirometry measurements revealed no effects of genistein on mitochondrial function. In conclusion, dietary genistein supplementation did not improve glucose homeostasis, alleviate oxidative stress, or augment skeletal muscle metabolism in ZDF rats.

Aim: We performed genome-wide and transcriptome-wide profiling to identify genes and single nucleotide polymorphisms (SNPs) associated with the response of triglycerides (TG) to exercise training., Methods: Plasma TG levels were measured before and after a 20-week endurance training programme in 478 white participants from the HERITAGE Family Study. Illumina HumanCNV370-Quad v3.0 BeadChips were genotyped using the Illumina BeadStation 500GX platform. Affymetrix HG-U133+2 arrays were used to quantitate gene expression levels from baseline muscle biopsies of a subset of participants (N=52). Genome-wide association study (GWAS) analysis was performed using MERLIN, while transcriptomic predictor models were developed using the R-package GALGO., Results: The GWAS results showed that eight SNPs were associated with TG training-response (ΔTG) at p<9.9×10(-6), while another 31 SNPs showed p values <1×10(-4). In multivariate regression models, the top 10 SNPs explained 32.0% of the variance in ΔTG, while conditional heritability analysis showed that four SNPs statistically accounted for all of the heritability of ΔTG. A molecular signature based on the baseline expression of 11 genes predicted 27% of ΔTG in HERITAGE, which was validated in an independent study. A composite SNP score based on the top four SNPs, each from the genomic and transcriptomic analyses, was the strongest predictor of ΔTG (R(2)=0.14, p=3.0×10(-68))., Conclusions: Our results indicate that skeletal muscle transcript abundance at 11 genes and SNPs at a number of loci contribute to TG response to exercise training. Combining data from genomics and transcriptomics analyses identified a SNP-based gene signature that should be further tested in independent samples., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Objective: Parental high-fat feeding was proposed to negatively impact metabolic health in offspring. Here, the ectopic fat storage in heart and liver in offspring was investigated, and the effects on mitochondrial function, de novo lipogenesis, and postprandial lipid metabolism were explored in detail., Methods: Male and female mice received either a high-fat (HF) or standard chow (LF) diet during mating, gestation and lactation. All offspring animals received the HF diet., Results: Abdominal visceral adipose tissue tended to be higher in HF/HF mice. Cardiac lipid content was also higher in the HF/HF mice (LF/HF vs., Hf/hf: 1.03% ± 0.08% vs. 1.33% ± 0.07% of water signal, P = 0.01). In contrast, hepatic lipid content tended to be lower in HF/HF mice compared to LF/HF mice. A severely disturbed postprandial lipid clearance was revealed in HF/HF mice by the results from the triglyceride (TG) tolerance tests (LF/HF vs., Hf/hf: 6,753 ± 2,213 vs. 14,367 ± 1,978 mmol l(-1)  min(-1) , P = 0.01) and (13) C-fatty acid retention test (LF/HF vs., Hf/hf: 2.73% ± 0.85% vs. 0.89% ± 0.26% retention from bolus, P = 0.04), which may underlie the lower hepatic lipid content., Conclusions: Here it is shown that HF diet negatively impacts postprandial TG clearance in offspring and results in an overall metabolic unfavorable phenotype and ectopic lipid deposition in the heart and in visceral storage sites., (© 2015 The Obesity Society.)

Brown adipose tissue (BAT) activation via cold exposure is increasingly scrutinized as a potential approach to ameliorate cardio-metabolic risk. Transition to cold temperatures requires changes in the partitioning of energy substrates, re-routing fatty acids to BAT to fuel non-shivering thermogenesis. However, the mechanisms behind the redistribution of energy substrates to BAT remain largely unknown. Angiopoietin-like 4 (ANGPTL4), a protein that inhibits lipoprotein lipase (LPL) activity, is highly expressed in BAT. Here, we demonstrate that ANGPTL4 is part of a shuttling mechanism that directs fatty acids derived from circulating triglyceride-rich lipoproteins to BAT during cold. Specifically, we show that cold markedly down-regulates ANGPTL4 in BAT, likely via activation of AMPK, enhancing LPL activity and uptake of plasma triglyceride-derived fatty acids. In contrast, cold up-regulates ANGPTL4 in WAT, abolishing a cold-induced increase in LPL activity. Together, our data indicate that ANGPTL4 is an important regulator of plasma lipid partitioning during sustained cold.

Cold exposure may be a potential therapy for diabetes by increasing brown adipose tissue (BAT) mass and activity. Here we report that 10 d of cold acclimation (14-15 °C) increased peripheral insulin sensitivity by ∼43% in eight type 2 diabetes subjects. Basal skeletal muscle GLUT4 translocation markedly increased, without effects on insulin signaling or AMP-activated protein kinase (AMPK) activation and only a minor increase in BAT glucose uptake.

Elevated hepatic lipid content (IntraHepatic Lipid, IHL) increases the risk of metabolic complications. Although prolonged exercise training lowers IHL, it is unknown if acute exercise has the same effect. Furthermore, hepatic ATP content may be related to insulin resistance and IHL. We aimed to investigate if acute exercise leads to changes in IHL and whether this is accompanied by changes in hepatic ATP. Twenty-one men (age 54.8 ± 7.2 years, BMI 29.7 ± 2.2 kg/m(2)) performed a 2 h cycling protocol, once while staying fasted and once while ingesting glucose. IHL was determined at baseline, 30 min post-exercise and 4 h post-exercise. Additionally ATP/Total P ratio was measured at baseline and 4 h post-exercise. Compared with baseline values we did not observe any statistically significant changes in IHL within 30 min post-exercise in neither the fasted nor the glucose-supplemented condition. However, IHL was elevated 4 h post-exercise compared with baseline in the fasted condition (from 8.3 ± 1.8 to 8.7 ± 1.8%, p = 0.010), an effect that was blunted by glucose supplementation (from 8.3 ± 1.9 to 8.3 ± 1.9%, p = 0.789). Acute exercise does not decrease liver fat in overweight middle-aged men. Moreover, IHL increased 4 h post-exercise in the fasted condition, an increase that was absent in the glucose-supplemented condition. These data suggest that a single bout of exercise may not be able to lower IHL.

Accumulation of fat in muscle tissue as intramyocellular lipids (IMCLs) is closely related to the development of insulin resistance and subsequent type 2 diabetes. Most IMCLs organize into lipid droplets (LDs), the fates of which are regulated by lipid droplet coat proteins. Perilipin 5 (PLIN5) is an LD coating protein, which is strongly linked to lipid storage in muscle tissue. Here we employ a tandem in vitro/ex vivo approach and use chemical imaging by label-free, hyperspectral coherent Raman microscopy to quantify compositional changes in individual LDs upon PLIN5 overexpression. Our results directly show that PLIN5 overexpression in muscle alters individual LD composition and physiology, resulting in larger LDs with higher esterified acyl chain concentration, increased methylene content, and more saturated lipid species. These results suggest that lipotoxic protection afforded by natural PLIN5 upregulation in muscle involves molecular changes in lipid composition within LDs.

Recent preclinical studies showed the potential of nicotinamide adenine dinucleotide (NAD(+)) precursors to increase oxidative phosphorylation and improve metabolic health, but human data are lacking. We hypothesize that the nicotinic acid derivative acipimox, an NAD(+) precursor, would directly affect mitochondrial function independent of reductions in nonesterified fatty acid (NEFA) concentrations. In a multicenter randomized crossover trial, 21 patients with type 2 diabetes (age 57.7 ± 1.1 years, BMI 33.4 ± 0.8 kg/m(2)) received either placebo or acipimox 250 mg three times daily dosage for 2 weeks. Acipimox treatment increased plasma NEFA levels (759 ± 44 vs. 1,135 ± 97 μmol/L for placebo vs. acipimox, P < 0.01) owing to a previously described rebound effect. As a result, skeletal muscle lipid content increased and insulin sensitivity decreased. Despite the elevated plasma NEFA levels, ex vivo mitochondrial respiration in skeletal muscle increased. Subsequently, we showed that acipimox treatment resulted in a robust elevation in expression of nuclear-encoded mitochondrial gene sets and a mitonuclear protein imbalance, which may indicate activation of the mitochondrial unfolded protein response. Further studies in C2C12 myotubes confirmed a direct effect of acipimox on NAD(+) levels, mitonuclear protein imbalance, and mitochondrial oxidative capacity. To the best of our knowledge, this study is the first to demonstrate that NAD(+) boosters can also directly affect skeletal muscle mitochondrial function in humans., (© 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.)

Background: Fat accumulation in nonadipose tissue is linked to insulin resistance and metabolic diseases. Earlier studies have shown that hepatic lipid accumulation can occur after 4 d of a high-fat diet in humans, and this fat accumulation can be blunted by the ingestion of additional proteins., Objectives: In this study, we explored whether a single high-fat meal increased the lipid content in liver and skeletal muscle as measured by using in vivo proton magnetic resonance spectroscopy (¹H-MRS) and whether the addition of protein can modulate the postprandial ectopic lipid storage., Design: Intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) concentrations were determined by using ¹H-MRS before and 3 and 5 h after a high-fat with added protein meal (61.5% of energy from fat) or a high-fat without added protein meal (mean ± SEM: 51.1 ± 7.9 g of protein; 191.9 ± 9.9 kcal added) in a randomized crossover study. IHL and IMCL concentrations were converted to absolute concentrations (g/kg wet weight) by using water as an internal reference., Results: Nine lean, healthy subjects [6 men and 3 women; mean (±SD) age: 22.7 ± 3.0 y; mean body mass index (in kg/m²): 21.8 ± 1.8] were included in this study. IHL concentrations increased ∼20% (P < 0.01) at 3 h after the meal and did not further increase after 5 h. In contrast, IMCL concentrations were not altered during the postprandial period (P = 0.74). The addition of protein to a single high-fat meal did not change the postprandial accumulation of fat in the liver (P = 0.93) or skeletal muscle (P = 0.84)., Conclusions: In this study, we showed that a single energy-dense, high-fat meal induced net lipid accumulation in the liver, which was detected by using in vivo ¹H-MRS. This noninvasive approach might bring new opportunities to study postprandial hepatic lipid dynamics. The addition of protein did not change the ectopic lipid retention after a single high-fat meal., (© 2015 American Society for Nutrition.)

Animal models suggest that acetylcarnitine production is essential for maintaining metabolic flexibility and insulin sensitivity. Because current methods to detect acetylcarnitine involve biopsy of the tissue of interest, noninvasive alternatives to measure acetylcarnitine concentrations could facilitate our understanding of its physiological relevance in humans. Here, we investigated the use of long-echo time (TE) proton magnetic resonance spectroscopy (1H-MRS) to measure skeletal muscle acetylcarnitine concentrations on a clinical 3T scanner. We applied long-TE 1H-MRS to measure acetylcarnitine in endurance-trained athletes, lean and obese sedentary subjects, and type 2 diabetes mellitus (T2DM) patients to cover a wide spectrum in insulin sensitivity. A long-TE 1H-MRS protocol was implemented for successful detection of skeletal muscle acetylcarnitine in these individuals. There were pronounced differences in insulin sensitivity, as measured by hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial function, as measured by phosphorus-MRS (31P-MRS), across groups. Insulin sensitivity and mitochondrial function were highest in trained athletes and lowest in T2DM patients. Skeletal muscle acetylcarnitine concentration showed a reciprocal distribution, with mean acetylcarnitine concentration correlating with mean insulin sensitivity in each group. These results demonstrate that measuring acetylcarnitine concentrations with 1H-MRS is feasible on clinical MR scanners and support the hypothesis that T2DM patients are characterized by a decreased formation of acetylcarnitine, possibly underlying decreased insulin sensitivity.

Physical activity increases energy metabolism in exercising muscle. Whether acute exercise elicits metabolic changes in nonexercising muscles remains unclear. We show that one of the few genes that is more highly induced in nonexercising muscle than in exercising human muscle during acute exercise encodes angiopoietin-like 4 (ANGPTL4), an inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance. Using a combination of human, animal, and in vitro data, we show that induction of ANGPTL4 in nonexercising muscle is mediated by elevated plasma free fatty acids via peroxisome proliferator-activated receptor-δ, presumably leading to reduced local uptake of plasma triglyceride-derived fatty acids and their sparing for use by exercising muscle. In contrast, the induction of ANGPTL4 in exercising muscle likely is counteracted via AMP-activated protein kinase (AMPK)-mediated down-regulation, promoting the use of plasma triglycerides as fuel for active muscles. Our data suggest that nonexercising muscle and the local regulation of ANGPTL4 via AMPK and free fatty acids have key roles in governing lipid homeostasis during exercise.

The accumulation of lipids in non-adipose tissues is attracting increasing attention due to its correlation with obesity. In muscle tissue, ectopic deposition of specific lipids is further correlated with pathogenic development of insulin resistance and type 2 diabetes. Most intramyocellular lipids are organized into lipid droplets (LDs), which are metabolically active organelles. In order to better understand the putative role of LDs in pathogenesis, insight into both the location of LDs and nearby chemistry of muscle tissue is very useful. Here, we demonstrate the use of label-free coherent anti-Stokes Raman scattering (CARS) microscopy in combination with multivariate, chemometric analysis to visualize intracellular lipid accumulations in ex vivo muscle tissue. Consistent with our previous results, hyperspectral CARS microscopy showed an increase in LDs in tissues where LD proteins were overexpressed, and further chemometric analysis showed additional features morphologically (and chemically) similar to mitochondria that colocalized with LDs. CARS imaging is shown to be a very useful method for label-free stratification of ectopic fat deposition and cellular organelles in fresh tissue sections with virtually no sample preparation.

The nuclear receptor Rev-erb-α modulates hepatic lipid and glucose metabolism, adipogenesis and the inflammatory response in macrophages. We show here that Rev-erb-α is highly expressed in oxidative skeletal muscle and that its deficiency in muscle leads to reduced mitochondrial content and oxidative function, as well as upregulation of autophagy. These cellular effects resulted in both impaired mitochondrial biogenesis and increased clearance of this organelle, leading to compromised exercise capacity. On a molecular level, Rev-erb-α deficiency resulted in deactivation of the Lkb1-Ampk-Sirt1-Ppargc-1α signaling pathway. These effects were recapitulated in isolated fibers and in muscle cells after knockdown of the gene encoding Rev-erb-α, Nr1d1. In complementary experiments, Rev-erb-α overexpression in vitro increased the number of mitochondria and improved respiratory capacity, whereas muscle overexpression or pharmacological activation of Rev-erb-α in vivo increased exercise capacity. This study identifies Rev-erb-α as a pharmacological target that improves muscle oxidative function by modulating gene networks controlling mitochondrial number and function.

The number of people suffering from metabolic disorders is dramatically increasing worldwide. The need for new therapeutic strategies to combat this growing epidemic of metabolic diseases is therefore also increasing. In 2003, resveratrol was discovered to be a small molecule activator of sirtuin 1 (SIRT1), an important molecular target regulating cellular energy metabolism and mitochondrial homeostasis. Rodent studies have clearly demonstrated the potential of resveratrol to improve various metabolic health parameters. To date, however, only limited clinical data are available that have systematically examined the health benefits of resveratrol in metabolically challenged humans. This short review will give an overview of the currently available clinical studies examining the effects of resveratrol on obesity and type 2 diabetes from a human perspective., (© 2013 New York Academy of Sciences.)

Context: Type 2 diabetes (T2D) has features of disordered lipid and glucose metabolism, due in part to reduced mitochondrial content., Objective: Our objective was to investigate effects of different types of exercise on mitochondrial content and substrate oxidation in individuals with T2D (ancillary study of the randomized controlled trial Health Benefits of Aerobic and Resistance Training in Individuals with Type 2 Diabetes, HART-D)., Intervention: T2D individuals were randomized to aerobic training (AT, n = 12), resistance training (RT, n = 18), combination training (ATRT, n = 12), or nonexercise control (n = 10). Blood draws, peak oxygen consumption tests, dual-energy x-ray absorptiometry scans and muscle biopsies of vastus lateralis were performed before and after 9 months. Ex vivo substrate oxidations ((14)CO2), mitochondrial content, and enzyme activities were measured. Glycated hemoglobin A1c and free fatty acids were also determined., Results: Mitochondrial content increased after RT and ATRT. Octanoate oxidation increased after AT and ATRT, whereas palmitate, pyruvate, and acetate oxidations increased in all exercise groups. Exercise-induced responses in mitochondrial DNA were associated with improvements in peak oxygen consumption, β-hydroxyacyl-coenzyme A dehydrogenase activity, and palmitate oxidation., Conclusions: Nine months of AT and RT significantly improved most aspects of skeletal muscle mitochondrial content and substrate oxidation, whereas the combination improved all aspects. These exercise responses were associated with clinical improvements, indicating that long-term training, especially combination, is an effective lifestyle therapy for individuals with T2D by way of improving muscle substrate metabolism.

Aims/hypothesis: While lipid deposition in the skeletal muscle is considered to be involved in obesity-associated insulin resistance, neutral intramyocellular lipid (IMCL) accumulation per se does not necessarily induce insulin resistance. We previously demonstrated that overexpression of the lipid droplet coat protein perilipin 2 augments intramyocellular lipid content while improving insulin sensitivity. Another member of the perilipin family, perilipin 5 (PLIN5), is predominantly expressed in oxidative tissues like the skeletal muscle. Here we investigated the effects of PLIN5 overexpression - in comparison with the effects of PLIN2 - on skeletal muscle lipid levels, gene expression profiles and insulin sensitivity., Methods: Gene electroporation was used to overexpress PLIN5 in tibialis anterior muscle of rats fed a high fat diet. Eight days after electroporation, insulin-mediated glucose uptake in the skeletal muscle was measured by means of a hyperinsulinemic euglycemic clamp. Electron microscopy, fluorescence microscopy and lipid extractions were performed to investigate IMCL accumulation. Gene expression profiles were obtained using microarrays., Results: TAG storage and lipid droplet size increased upon PLIN5 overexpression. Despite the higher IMCL content, insulin sensitivity was not impaired and DAG and acylcarnitine levels were unaffected. In contrast to the effects of PLIN2 overexpression, microarray data analysis revealed a gene expression profile favoring FA oxidation and improved mitochondrial function., Conclusions/interpretation: Both PLIN2 and PLIN5 increase neutral IMCL content without impeding insulin-mediated glucose uptake. As opposed to the effects of PLIN2 overexpression, overexpression of PLIN5 in the skeletal muscle promoted expression of a cluster of genes under control of PPARα and PGC1α involved in FA catabolism and mitochondrial oxidation., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Intramuscular accumulation of triacylglycerol, in the form of lipid droplets (LD), has gained widespread attention as a hallmark of metabolic disease and insulin resistance. Paradoxically, LDs also amass in muscles of highly trained endurance athletes who are exquisitely insulin sensitive. Understanding the molecular mechanisms that mediate the expansion and appropriate metabolic control of LDs in the context of habitual physical activity could lead to new therapeutic opportunities. Herein, we show that acute exercise elicits robust upregulation of a broad program of genes involved in regulating LD assembly, morphology, localization, and mobilization. Prominent among these was perilipin-5, a scaffolding protein that affects the spatial and metabolic interactions between LD and their surrounding mitochondrial reticulum. Studies in transgenic mice and primary human skeletal myocytes established a key role for the exercise-responsive transcriptional coactivator PGC-1α in coordinating intramuscular LD programming with mitochondrial remodeling. Moreover, translational studies comparing physically active versus inactive humans identified a remarkably strong association between expression of intramuscular LD genes and enhanced insulin action in exercise-trained subjects. These results reveal an intimate molecular connection between intramuscular LD biology and mitochondrial metabolism that could prove relevant to the etiology and treatment of insulin resistance and other disorders of lipid imbalance.

Objective: To investigate the role of Acylation Stimulating Protein (ASP) receptor C5L2 in skeletal muscle fatty acid accumulation and metabolism as well as insulin sensitivity in both mice and human models of diet-induced insulin resistance., Design and Methods: Male wildtype (WT) and C5L2 knockout (KO) mice were fed a low (LFD) or a high (HFD) fat diet for 10 weeks. Intramyocellular lipid (IMCL) accumulation (by oil red O staining) and beta-oxidation HADH enzyme activity were determined in skeletal muscle. Mitochondria were isolated from hindleg muscles for high-resolution respirometry. Muscle C5L2 protein content was also determined in obese type 2 diabetics and age- and BMI matched men., Results: IMCL levels were increased by six-fold in C5L2KO-HFD compared to WT-HFD mice (p<0.05) and plasma insulin levels were markedly increased in C5L2KO-HFD mice (twofold, p<0.05). Muscle HADH activity was elevated in C5L2KO-LFD mice (+75%, p<0.001 vs. WT-LFD) and C5L2KO-HFD displayed increased mitochondrial fatty acid oxidative capacity compared to WT-HFD mice (+23%, p<0.05). In human subjects, C5L2 protein content was reduced (-48%, p<0.01) in type 2 diabetic patients when compared to obese controls. Further, exercise training increased C5L2 (+45%, p = 0.0019) and ASP (+80%, p<0.001) in obese insulin-resistant men., Conclusion: The results suggest that insulin sensitivity may be permissive for coupling of C5L2 levels to lipid storage and utilization.

Introduction: Mitochondrial dysfunction, lipid accumulation, insulin resistance and metabolic inflexibility have been implicated in the etiology of type 2 diabetes (T2D), yet their interrelationship remains speculative. We investigated these interrelationships in a group of T2D and obese normoglycemic control subjects., Methods: 49 non-insulin dependent male T2D patients and 54 male control subjects were enrolled, and a hyperinsulinemic-euglycemic clamp and indirect calorimetry were performed. A muscle biopsy was taken and intramyocellular lipid (IMCL) was measured. In vivo mitochondrial function was measured by PCr recovery in 30 T2D patients and 31 control subjects., Results: Fasting NEFA levels were significantly elevated in T2D patients compared with controls, but IMCL was not different. Mitochondrial function in T2D patients was compromised by 12.5% (p<0.01). Whole body glucose disposal (WGD) was higher at baseline and lower after insulin stimulation. Metabolic flexibility (ΔRER) was lower in the type 2 diabetic patients (0.050±0.033 vs. 0.093±0.050, p<0.01). Mitochondrial function was the sole predictor of basal respiratory exchange ratio (RER) (R(2) = 0.18, p<0.05); whereas WGD predicted both insulin-stimulated RER (R(2) = 0.29, p<0.001) and metabolic flexibility (R(2) = 0.40, p<0.001)., Conclusions: These results indicate that defects in skeletal muscle in vivo mitochondrial function in type 2 diabetic patients are only reflected in basal substrate oxidation and highlight the importance of glucose disposal rate as a determinant of substrate utilization in response to insulin.

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)-coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation-related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet-induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid β-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Fat accumulation in skeletal muscle combined with low mitochondrial oxidative capacity is associated with insulin resistance (IR). Endurance-trained athletes, characterized by a high oxidative capacity, have elevated intramyocellular lipids, yet are highly insulin sensitive. We tested the hypothesis that a high oxidative capacity could attenuate lipid-induced IR. Nine endurance-trained (age = 23.4 ± 0.9 years; BMI = 21.2 ± 0.6 kg/m(2)) and 10 untrained subjects (age = 21.9 ± 0.9 years; BMI = 22.8 ± 0.6 kg/m(2)) were included and underwent a clamp with either infusion of glycerol or intralipid. Muscle biopsies were taken to perform high-resolution respirometry and protein phosphorylation/expression. Trained subjects had ~32% higher mitochondrial capacity and ~22% higher insulin sensitivity (P < 0.05 for both). Lipid infusion reduced insulin-stimulated glucose uptake by 63% in untrained subjects (P < 0.05), whereas this effect was blunted in trained subjects (29%, P < 0.05). In untrained subjects, lipid infusion reduced oxidative and nonoxidative glucose disposal (NOGD), whereas trained subjects were completely protected against lipid-induced reduction in NOGD, supported by dephosphorylation of glycogen synthase. We conclude that chronic exercise training attenuates lipid-induced IR and specifically attenuates the lipid-induced reduction in NOGD. Signaling data support the notion that high glucose uptake in trained subjects is maintained by shuttling glucose toward storage as glycogen.

A low fat oxidative capacity has been linked to muscle diacylglycerol (DAG) accumulation and insulin resistance. Alternatively, a low fat oxidation rate may stimulate glucose oxidation, thereby enhancing glucose disposal. Here, we investigated whether an ethyl-2-[6-(4-chlorophenoxy)hexyl]-oxirane-2-carboxylate (etomoxir)-induced inhibition of fat oxidation leads to muscle fat storage and insulin resistance. An intervention in healthy male subjects was combined with studies in human primary myotubes. Furthermore, muscle DAG and triacylglycerol (TAG), mitochondrial function, and insulin signaling were examined in etomoxir-treated C57bl6 mice. In humans, etomoxir administration increased glucose oxidation at the expense of fat oxidation. This effect was accompanied by an increased abundance of GLUT4 at the sarcolemma and a lowering of plasma glucose levels, indicative of improved glucose homeostasis. In mice, etomoxir injections resulted in accumulation of muscle TAG and DAG, yet improved insulin-stimulated GLUT4 translocation. Also in human myotubes, insulin signaling was improved by etomoxir, in the presence of increased intramyocellular lipid accumulation. These insulin-sensitizing effects in mice and human myotubes were accompanied by increased phosphorylation of AMP-activated protein kinase (AMPK). Our results show that a reduction in fat oxidation leading to accumulation of muscle DAG does not necessarily lead to insulin resistance, as the reduction in fat oxidation may activate AMPK.

Context: Reduction of 50% excess body weight, using a very low-calorie diet (VLCD; 450 kcal/d) improves insulin sensitivity in obese type 2 diabetes mellitus patients., Objective: The objective of the study was to evaluate whether adding exercise to the VLCD has additional benefits., Design: This was a randomized intervention study., Setting: The study was conducted at a clinical research center in an academic medical center., Subjects: Twenty-seven obese [body mass index 37.2 ± 0.9 kg/m(2) (mean ± sem)] insulin-treated type 2 diabetes mellitus patients., Intervention: Patients followed a 16-wk VLCD. Thirteen of them simultaneously participated in an exercise program (E) consisting of 1-h, in-hospital training and four 30-min training sessions on a cycloergometer weekly., Outcome Measures: Insulin resistance was measured by a hyperinsulinemic euglycemic clamp. Insulin signaling, mitochondrial DNA (mtDNA) content, and intramyocellular lipid content was measured in skeletal muscle biopsies., Results: Baseline characteristics were identical in both groups. Substantial weight loss occurred (-23.7 ± 1.7 kg VLCD-only vs. -27.2 ± 1.9 kg VLCD+E, P = NS within groups). The exercise group lost more fat mass. Insulin-stimulated glucose disposal increased similarly in both study groups [15.0 ± 0.9 to 39.2 ± 4.7 μmol/min(-1) · kg lean body mass (LBM(-1)) VLCD-only vs. 17.0 ± 1.0 to 37.5 ± 3.5 μmol/min(-1) · kg LBM(-1) in VLCD+E], as did phosphorylation of the phosphatidylinositol 3-kinase-protein kinase B/AKT insulin signaling pathway. In contrast, skeletal muscle mtDNA content increased only in the VLCD+E group (1211 ± 185 to 2288 ± 358, arbitrary units, P = 0.016 vs. 1397 ± 240 to 1196 ± 179, P = NS, VLCD-only group). Maximum aerobic capacity also only increased significantly in the VLCD+E group (+6.6 ± 1.7 ml/min(-1) · kg LBM(-1) vs. +0.7 ± 1.5 ml/min(-1) · kg LBM(-1) VLCD-only, P = 0.017)., Conclusion: Addition of exercise to a 16-wk VLCD induces more fat loss. Exercise augments maximum aerobic capacity and skeletal muscle mtDNA content. These changes are, however, not reflected in a higher insulin-stimulated glucose disposal rate.

Adipose triglyceride lipase (ATGL) is a lipolytic enzyme that is highly specific for triglyceride hydrolysis. The ATGL-knockout mouse (ATGL(-/-)) accumulates lipid droplets in various tissues, including skeletal muscle, and has poor maximal running velocity and endurance capacity. In this study, we tested whether abnormal lipid accumulation in skeletal muscle impairs mitochondrial oxidative phosphorylation, and hence, explains the poor muscle performance of ATGL(-/-) mice. In vivo ¹H magnetic resonance spectroscopy of the tibialis anterior of ATGL(-/-) mice revealed that its intramyocellular lipid pool is approximately sixfold higher than in WT controls (P = 0.0007). In skeletal muscle of ATGL(-/-) mice, glycogen content was decreased by 30% (P < 0.05). In vivo ³¹P magnetic resonance spectra of resting muscles showed that WT and ATGL(-/-) mice have a similar energy status: [PCr], [P(i)], PCr/ATP ratio, PCr/P(i) ratio, and intracellular pH. Electrostimulated muscles from WT and ATGL(-/-) mice showed the same PCr depletion and pH reduction. Moreover, the monoexponential fitting of the PCr recovery curve yielded similar PCr recovery times (τPCr; 54.1 ± 6.1 s for the ATGL(-/-) and 58.1 ± 5.8 s for the WT), which means that overall muscular mitochondrial oxidative capacity was comparable between the genotypes. Despite similar in vivo mitochondrial oxidative capacities, the electrostimulated muscles from ATGL(-/-) mice displayed significantly lower force production and increased muscle relaxation time than the WT. These findings suggest that mechanisms other than mitochondrial dysfunction cause the impaired muscle performance of ATGL(-/-) mice.

Background: Detailed knowledge about physical activity energy expenditure (PAEE) can guide the development of school interventions aimed at reducing overweight in adolescents. However, relevant components of PAEE have never been objectively quantified in this population. This study investigated the contribution of active transport to and from school, physical education (PE), and leisure time activities to total PAEE during a regular school week in adolescents., Methods: Seventy-three adolescents (mean age: 15.7 years) wore an individually calibrated combined heart rate-acceleration monitor and kept an activity diary during a regular school week. Branched equation modeling was used to calculate PAEE of the specific activity categories, and their relative contribution to total PAEE was determined., Results: Active transport and PE contributed 30.0% and 17.4%, respectively, to school-related PAEE. Active transport to and from school contributed 15% to total PAEE. Youth with a high physical activity level (PAL) spent 4 hours less in sedentary behavior than subjects with a medium or low PAL (F = 77.415 (2.70), p < .001) and had higher PAEE during leisure time sports (F = 9.135 (2.70), p < .001) and work-related activities (F = 10.583 (2.70), p < .001) than youth with medium or low PAL values., Conclusions: Active transport and PE contribute significantly to PAEE during school hours in adolescents. To achieve an increase in total PAEE in the least active group of adolescents, promising strategies might be to reduce inactive behavior, increase participation in leisure time sports, and possibly to replace inactive for active jobs., (© 2012, American School Health Association.)

Cachexia is a prevalent phenomenon of non-small cell lung cancer (NSCLC) which is responsible for increased mortality and deterioration of physical performance. Preclinical research indicates that systemic inflammation induces cachexia-related muscle wasting through muscular Nuclear Factor-kappa B (NF-κB) signaling and subsequent ubiquitin proteasome system (UPS)-mediated proteolysis. As these pathways could be a target for early intervention strategies, it needs to be elucidated whether increased activation of these pathways is already present in early stage NSCLC cachexia. The aim of the present study was therefore to assess muscular NF-κB and UPS activation in patients with NSCLC pre-cachexia. Sixteen patients with newly diagnosed stages I-III NSCLC having <10% weight loss and ten healthy controls were studied. Body composition, systemic inflammation and exercise capacity were assessed in all subjects and NF-κB and UPS activity in vastus lateralis muscle biopsies in a subset. Patients showed increased plasma levels of C-reactive protein (CRP) (P<0.001), soluble Tumor Necrosis Factor receptor 1 (sTNF-R1) (P<0.05), fibrinogen (P<0.001) and decreased levels of albumin (P<0.001). No changes in fat free body mass or skeletal muscle NF-κB and UPS activity were observed, while peak oxygen consumption ( [Formula: see text] ) was significantly decreased in patients compared with healthy controls. In conclusion, this exploratory study demonstrates significantly reduced exercise capacity in NSCLC pre-cachexia despite maintenance of muscle mass and unaltered indices of UPS activation. The absence of muscular NF-κB-dependent inflammatory signaling supports the notion that transition of systemic to local inflammation is required to initiate UPS-dependent muscle wasting characteristic for (experimental) cachexia., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Skeletal muscle mitochondrial dysfunction has been linked to several disease states as well as the process of aging. A possible factor involved is the peroxisome proliferator-activated receptor (PPAR) γ co-activator 1α (PGC-1α), a major player in the regulation of skeletal muscle mitochondrial metabolism. However, it is currently unknown whether PGC-1α, besides stimulating mitochondrial proliferation, also affects the functional capacity per mitochondrion. Therefore, we here tested whether PGC-1α overexpression, besides increasing mitochondrial content, also leads to intrinsic mitochondrial adaptations. Skeletal muscle mitochondria from 10 male, muscle-specific PGC-1α overexpressing mice (PGC-1αTg) and 8 wild-type (WT) mice were isolated. Equal mitochondrial quantities were then analyzed for their oxidative capacity by high-resolution respirometry, fuelled by a carbohydrate-derived (pyruvate) and a lipid (palmitoyl-CoA plus carnitine) substrate. Additionally, mitochondria were tested for reactive oxygen species (superoxide) production and fatty acid (FA)-induced uncoupling. PGC-1αTg mitochondria were characterized by an improved intrinsic mitochondrial fat oxidative capacity as evidenced by pronounced increase in ADP-stimulated respiration (P < 0.001) and maximal uncoupled respiration (P < 0.001) upon palmitoyl-CoA plus carnitine. Interestingly, intrinsic mitochondrial capacity on a carbohydrate-derived substrate tended to be reduced. Furthermore, the sensitivity to FA-induced uncoupling was diminished in PGC-1αTg mitochondria (P = 0.02) and this was accompanied by a blunted reduction in mitochondrial ROS production upon FAs in PGC-1αTg versus WT mitochondria (P = 0.04). Uncoupling protein 3 (UCP3) levels were markedly reduced in PGC-1αTg mitochondria (P < 0.001). Taken together, in addition to stimulating mitochondrial proliferation in skeletal muscle, we show here that overexpression of PGC-1α leads to intrinsic mitochondrial adaptations that seem restricted to fat metabolism., (Copyright © 2011 Wiley Periodicals, Inc.)

Perilipin 5 (PLIN5/OXPAT) is a lipid droplet (LD) coat protein mainly present in tissues with a high fat-oxidative capacity, suggesting a role for PLIN5 in facilitating fatty acid oxidation. Here, we investigated the role of PLIN5 in fat oxidation in skeletal muscle. In human skeletal muscle, we observed that PLIN5 (but not PLIN2) protein content correlated tightly with OXPHOS content and in rat muscle PLIN5 content correlated with mitochondrial respiration rates on a lipid-derived substrate. This prompted us to examine PLIN5 protein expression in skeletal muscle mitochondria by means of immunogold electron microscopy and Western blots in isolated mitochondria. These data show that PLIN5, in contrast to PLIN2, not only localizes to LD but also to mitochondria, possibly facilitating fatty acid oxidation. Unilateral overexpression of PLIN5 in rat anterior tibialis muscle augmented myocellular fat storage without increasing mitochondrial density as indicated by the lack of change in protein content of five components of the OXPHOS system. Mitochondria isolated from PLIN5 overexpressing muscles did not possess increased fatty acid respiration. Interestingly though, (14)C-palmitate oxidation assays in muscle homogenates from PLIN5 overexpressing muscles revealed a 44.8% (P = 0.05) increase in complete fatty acid oxidation. Thus, in mitochondrial isolations devoid of LD, PLIN5 does not augment fat oxidation, while in homogenates containing PLIN5-coated LD, fat oxidation is higher upon PLIN5 overexpression. The presence of PLIN5 in mitochondria helps to understand why PLIN5, in contrast to PLIN2, is of specific importance in fat oxidative tissues. Our data suggests involvement of PLIN5 in directing fatty acids from the LD to mitochondrial fatty acid oxidation.

MRI has been proven to be an accurate method for noninvasive assessment of cardiac function. One of the current limitations of cardiac MRI is that it is time consuming. Therefore, various geometrical models are used, which can reduce scan and postprocessing time. It is unclear how appropriate their use is in rodents. Left ventricular (LV) volumes and ejection fraction (EF) were quantified based on 7.0 Tesla cine-MRI in 12 wild-type (WT) mice, 12 adipose triglyceride lipase knockout (ATGL(-/-)) mice (model of impaired cardiac function), and 11 rats in which we induced cardiac ischemia. The LV volumes and function were either assessed with parallel short-axis slices covering the full volume of the left ventricle (FV, gold standard) or with various geometrical models [modified Simpson rule (SR), biplane ellipsoid (BP), hemisphere cylinder (HC), single-plane ellipsoid (SP), and modified Teichholz Formula (TF)]. Reproducibility of the different models was tested and results were correlated with the gold standard (FV). All models and the FV data set provided reproducible results for the LV volumes and EF, with interclass correlation coefficients ≥0.87. All models significantly over- or underestimated EF, except for SR. Good correlation was found for all volumes and EF for the SR model compared with the FV data set (R(2) ranged between 0.59-0.95 for all parameters). The HC model and BP model also predicted EF well (R(2) ≥ 0.85), although proved to be less useful for quantitative analysis. The SP and TF models correlated poorly with the FV data set (R(2) ≥ 0.45 for EF and R(2) ≥ 0.29 for EF, respectively). For the reduction in acquisition and postprocessing time, only the SR model proved to be a valuable method for calculating LV volumes, stroke volume, and EF.

Context: Animal studies revealed that medium-chain fatty acids (MCFA), due to their metabolic characteristics, are not stored in skeletal muscle and may therefore not give rise to potentially hazardous lipid species impeding insulin signaling., Objective: We here hypothesized that infusion of medium-chain triacylglycerols (MCT) in healthy lean subjects does not lead to ectopic fat accumulation and hence does not result in lipid-induced insulin resistance., Design and Methods: Nine healthy lean male subjects underwent a 6-h hyperinsulinemic-euglycemic clamp with simultaneous infusion of 1) a 100% long-chain triacylglycerols (LCT) emulsion, 2) a 50/50% MCT/LCT emulsion, or 3) glycerol in a randomized crossover design. Muscle biopsies were taken before and after each clamp., Results: MCT/LCT infusion raised plasma free fatty acid levels to a similar level compared with LCT infusion alone. Despite elevated free fatty acid levels, intramyocellular triacylglycerol (IMTG) levels were not affected by the MCT/LCT emulsion, whereas LCT infusion resulted in an approximately 1.6-fold increase in IMTG. These differences in muscle fat accumulation did not result in significant differences in lipid-induced insulin resistance between LCT (-28%, P = 0.003) and MCT/LCT (-20%, P < 0.001). Total skeletal muscle ceramide content as well as lactosyl- and glucosylceramide levels were not affected by any of the interventions. In addition, the distribution pattern of all ceramide species remained unaltered., Conclusions: Although we confirm that MCFA do not lead to ceramide and IMTG accumulation in skeletal muscle tissue in humans, they do induce insulin resistance. These results indicate that, in humans, MCFA may not be beneficial in preventing peripheral insulin resistance.