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author:("hoek's, Joris")
1.  A genistein-enriched diet neither improves skeletal muscle oxidative capacity nor prevents the transition towards advanced insulin resistance in ZDF rats 
Scientific Reports  2016;6:22854.
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.
PMCID: PMC4789602  PMID: 26973284
2.  Lack of UCP3 does not affect skeletal muscle mitochondrial function under lipid-challenged conditions, but leads to sudden cardiac death 
Basic research in cardiology  2014;109(6):447.
UCP3's exact physiological function in lipid handling in skeletal and cardiac muscle remains unknown. Interestingly, etomoxir, a fat oxidation inhibitor and strong inducer of UCP3, is proposed for treating both diabetes and heart failure. We hypothesize that the upregulation of UCP3 upon etomoxir serves to protect mitochondria against lipotoxicity. To evaluate UCP3's role in skeletal muscle (skm) and heart under lipid-challenged conditions, the effect of UCP3 ablation was examined in a state of dysbalance between fat availability and oxidative capacity. Wild type (WT) and UCP3−/− mice were subjected to high-fat feeding for 14 days. From day 6 onwards, they were given either saline or etomoxir. Etomoxir treatment induced an increase in markers of lipotoxicity in skm compared to saline. This increase upon etomoxir was similar for both, WT and UCP3−/− mice, suggesting that UCP3 does not play a role in protection against lipotoxicity. Interestingly, we observed 25 % mortality in UCP3−/−s upon etomoxir administration vs. 11 % in WTs. This increased mortality in UCP3−/− compared to WT mice could not be explained by differences in cardiac lipotoxicity, apoptosis, fibrosis (histology, immunohisto-chemistry), oxidative capacity (respirometry) or function (echocardiography). Electrophysiology demonstrated, however, prolonged QRS and QTc intervals and greater susceptibility to ventricular tachycardia upon programmed electrical stimulation in etomoxir-treated UCP3−/−s versus WTs. Isoproterenol administration after pacing resulted in 75 % mortality in UCP3−/−s vs. 14 % in WTs. Our results argue against a protective role for UCP3 on skm metabolism under lipid overload, but suggest UCP3 to be crucial in prevention of arrhythmias upon lipid-challenged conditions.
PMCID: PMC4329241  PMID: 25344084
Arrhythmia; Metabolism; Mitochondria; Muscle; Uncoupling protein
4.  Long–echo time MR spectroscopy for skeletal muscle acetylcarnitine detection 
The Journal of Clinical Investigation  2014;124(11):4915-4925.
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.
PMCID: PMC4347229  PMID: 25271624
5.  Calorie restriction-like effects of 30 days of Resveratrol (resVida™) supplementation on energy metabolism and metabolic profile in obese humans 
Cell metabolism  2011;14(5):10.1016/j.cmet.2011.10.002.
Resveratrol is a natural compound that affects energy metabolism and mitochondrial function and serves as a calorie restriction mimetic, at least in animal models of obesity. Here we treated 11 healthy, obese men with placebo and 150 mg/day resveratrol in a randomized double-blind cross-over study for 30 days. Resveratrol significantly reduced sleeping- and resting metabolic rate. In muscle, resveratrol activated AMPK, increased SIRT1 and PGC-1α protein levels, increased citrate synthase activity without change in mitochondrial content, and improved muscle mitochondrial respiration on a fatty acid-derived substrate. Furthermore, resveratrol elevated intramyocellular lipid levels, and decreased intrahepatic lipid content, circulating glucose, triglycerides, alanine-aminotransferase, and inflammation markers. Systolic blood pressure dropped and HOMA index improved after resveratrol. In the postprandial state, adipose tissue lipolysis and plasma fatty acid and glycerol decreased. In conclusion, we demonstrate that 30 days of resveratrol supplementation induces metabolic changes in obese humans, mimicking the effects of calorie restriction.
PMCID: PMC3880862  PMID: 22055504
6.  High Oxidative Capacity Due to Chronic Exercise Training Attenuates Lipid-Induced Insulin Resistance 
Diabetes  2012;61(10):2472-2478.
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/m2) and 10 untrained subjects (age = 21.9 ± 0.9 years; BMI = 22.8 ± 0.6 kg/m2) 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.
PMCID: PMC3447923  PMID: 22787138
7.  Beige Adipocytes are a Distinct Type of Thermogenic Fat Cell in Mouse and Human 
Cell  2012;150(2):366-376.
Brown fat defends against hypothermia and obesity through thermogenesis mediated by mitochondrial UCP1. 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 cloning 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 show that deposits of brown fat previously observed in adult humans are composed of beige adipose cells. These data illustrate a new cell type with therapeutic potential in mouse and human.
PMCID: PMC3402601  PMID: 22796012
8.  Cold acclimation recruits human brown fat and increases nonshivering thermogenesis 
The Journal of Clinical Investigation  2013;123(8):3395-3403.
In recent years, it has been shown that humans have active brown adipose tissue (BAT) depots, raising the question of whether activation and recruitment of BAT can be a target to counterbalance the current obesity pandemic. Here, we show that a 10-day cold acclimation protocol in humans increases BAT activity in parallel with an increase in nonshivering thermogenesis (NST). No sex differences in BAT presence and activity were found either before or after cold acclimation. Respiration measurements in permeabilized fibers and isolated mitochondria revealed no significant contribution of skeletal muscle mitochondrial uncoupling to the increased NST. Based on cell-specific markers and on uncoupling protein-1 (characteristic of both BAT and beige/brite cells), this study did not show “browning” of abdominal subcutaneous white adipose tissue upon cold acclimation. The observed physiological acclimation is in line with the subjective changes in temperature sensation; upon cold acclimation, the subjects judged the environment warmer, felt more comfortable in the cold, and reported less shivering. The combined results suggest that a variable indoor environment with frequent cold exposures might be an acceptable and economic manner to increase energy expenditure and may contribute to counteracting the current obesity epidemic.
PMCID: PMC3726172  PMID: 23867626
9.  Relationship of C5L2 Receptor to Skeletal Muscle Substrate Utilization 
PLoS ONE  2013;8(2):e57494.
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.
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.
The results suggest that insulin sensitivity may be permissive for coupling of C5L2 levels to lipid storage and utilization.
PMCID: PMC3583831  PMID: 23460866
10.  High Fat Diet-Induced Changes in Mouse Muscle Mitochondrial Phospholipids Do Not Impair Mitochondrial Respiration Despite Insulin Resistance 
PLoS ONE  2011;6(11):e27274.
Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.
C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.
Principal Findings
At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.
Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.
PMCID: PMC3225362  PMID: 22140436
11.  Prolonged Fasting Identifies Skeletal Muscle Mitochondrial Dysfunction as Consequence Rather Than Cause of Human Insulin Resistance 
Diabetes  2010;59(9):2117-2125.
Type 2 diabetes and insulin resistance have been associated with mitochondrial dysfunction, but it is debated whether this is a primary factor in the pathogenesis of the disease. To test the concept that mitochondrial dysfunction is secondary to the development of insulin resistance, we employed the unique model of prolonged fasting in humans. Prolonged fasting is a physiologic condition in which muscular insulin resistance develops in the presence of increased free fatty acid (FFA) levels, increased fat oxidation and low glucose and insulin levels. It is therefore anticipated that skeletal muscle mitochondrial function is maintained to accommodate increased fat oxidation unless factors secondary to insulin resistance exert negative effects on mitochondrial function.
While in a respiration chamber, twelve healthy males were subjected to a 60 h fast and a 60 h normal fed condition in a randomized crossover design. Afterward, insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp, and mitochondrial function was quantified ex vivo in permeabilized muscle fibers using high-resolution respirometry.
Indeed, FFA levels were increased approximately ninefold after 60 h of fasting in healthy male subjects, leading to elevated intramuscular lipid levels and decreased muscular insulin sensitivity. Despite an increase in whole-body fat oxidation, we observed an overall reduction in both coupled state 3 respiration and maximally uncoupled respiration in permeabilized skeletal muscle fibers, which could not be explained by changes in mitochondrial density.
These findings confirm that the insulin-resistant state has secondary negative effects on mitochondrial function. Given the low insulin and glucose levels after prolonged fasting, hyperglycemia and insulin action per se can be excluded as underlying mechanisms, pointing toward elevated plasma FFA and/or intramuscular fat accumulation as possible causes for the observed reduction in mitochondrial capacity.
PMCID: PMC2927932  PMID: 20573749
12.  Lower Intrinsic ADP-Stimulated Mitochondrial Respiration Underlies In Vivo Mitochondrial Dysfunction in Muscle of Male Type 2 Diabetic Patients 
Diabetes  2008;57(11):2943-2949.
OBJECTIVE—A lower in vivo mitochondrial function has been reported in both type 2 diabetic patients and first-degree relatives of type 2 diabetic patients. The nature of this reduction is unknown. Here, we tested the hypothesis that a lower intrinsic mitochondrial respiratory capacity may underlie lower in vivo mitochondrial function observed in diabetic patients.
RESEARCH DESIGN AND METHODS—Ten overweight diabetic patients, 12 first-degree relatives, and 16 control subjects, all men, matched for age and BMI, participated in this study. Insulin sensitivity was measured with a hyperinsulinemic-euglycemic clamp. Ex vivo intrinsic mitochondrial respiratory capacity was determined in permeabilized skinned muscle fibers using high-resolution respirometry and normalized for mitochondrial content. In vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time after exercise using 31P-magnetic resonance spectroscopy.
RESULTS—Insulin-stimulated glucose disposal was lower in diabetic patients compared with control subjects (11.2 ± 2.8 vs. 28.9 ± 3.7 μmol · kg−1 fat-free mass · min−1, respectively; P = 0.003), with intermediate values for first-degree relatives (22.1 ± 3.4 μmol · kg−1 fat-free mass · min−1). In vivo mitochondrial function was 25% lower in diabetic patients (P = 0.034) and 23% lower in first-degree relatives, but the latter did not reach statistical significance (P = 0.08). Interestingly, ADP-stimulated basal respiration was 35% lower in diabetic patients (P = 0.031), and fluoro-carbonyl cyanide phenylhydrazone–driven maximal mitochondrial respiratory capacity was 31% lower in diabetic patients (P = 0.05) compared with control subjects with intermediate values for first-degree relatives.
CONCLUSIONS—A reduced basal ADP-stimulated and maximal mitochondrial respiratory capacity underlies the reduction in in vivo mitochondrial function, independent of mitochondrial content. A reduced capacity at both the level of the electron transport chain and phosphorylation system underlies this impaired mitochondrial capacity.
PMCID: PMC2570390  PMID: 18678616

Results 1-12 (12)