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1.
Diabetologia ; 2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34510226

RESUMO

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

2.
Adipocyte ; 10(1): 408-411, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34402717

RESUMO

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


Assuntos
Tecido Adiposo/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Resveratrol/administração & dosagem , Tecido Adiposo/citologia , Enzima de Conversão de Angiotensina 2/genética , COVID-19/patologia , COVID-19/virologia , Estudos Cross-Over , Suplementos Nutricionais , Método Duplo-Cego , Regulação para Baixo/efeitos dos fármacos , Humanos , Leptina/genética , Leptina/metabolismo , Masculino , Pessoa de Meia-Idade , Obesidade/tratamento farmacológico , Obesidade/patologia , Efeito Placebo , Receptor Tipo 1 de Angiotensina/genética , Receptor Tipo 1 de Angiotensina/metabolismo , Resveratrol/farmacologia , SARS-CoV-2/isolamento & purificação
3.
Obesity (Silver Spring) ; 29(9): 1423-1426, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33955183

RESUMO

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

4.
J Clin Endocrinol Metab ; 106(5): 1437-1447, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33524145

RESUMO

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


Assuntos
Tecido Adiposo Marrom/efeitos dos fármacos , Niacinamida/análogos & derivados , Compostos de Piridínio/farmacologia , Receptores Adrenérgicos/metabolismo , Adipócitos/metabolismo , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Marrom/fisiologia , Adrenérgicos/farmacologia , Idoso , Animais , Células Cultivadas , Estudos Cross-Over , Método Duplo-Cego , Metabolismo Energético/efeitos dos fármacos , Feminino , Humanos , Lipólise/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Niacinamida/farmacologia , Cultura Primária de Células , Termogênese/efeitos dos fármacos
5.
Diabetologia ; 64(2): 424-436, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33258025

RESUMO

AIMS/HYPOTHESIS: Mitochondria operate in networks, adapting to external stresses and changes in cellular metabolic demand and are subject to various quality control mechanisms. On the basis of these traits, we here hypothesise that the regulation of mitochondrial networks in skeletal muscle is hampered in humans with compromised oxidative capacity and insulin sensitivity. METHODS: In a cross-sectional design, we compared four groups of participants (selected from previous studies) ranging in aerobic capacity and insulin sensitivity, i.e. participants with type 2 diabetes (n = 11), obese participants without diabetes (n = 12), lean individuals (n = 10) and endurance-trained athletes (n = 12); basal, overnight fasted muscle biopsies were newly analysed for the current study and we compared the levels of essential mitochondrial dynamics and quality control regulatory proteins in skeletal muscle tissue. RESULTS: Type 2 diabetes patients and obese participants were older than lean participants and athletes (58.6 ± 4.0 and 56.7 ± 7.2 vs 21.8 ± 2.5 and 25.1 ± 4.3 years, p < 0.001, respectively) and displayed a higher BMI (32.4 ± 3.7 and 31.0 ± 3.7 vs 22.1 ± 1.8 and 21.0 ± 1.5 kg/m2, p < 0.001, respectively) than lean individuals and endurance-trained athletes. Fission protein 1 (FIS1) and optic atrophy protein 1 (OPA1) protein content was highest in muscle from athletes and lowest in participants with type 2 diabetes and obesity, respectively (FIS1: 1.86 ± 0.79 vs 0.79 ± 0.51 AU, p = 0.002; and OPA1: 1.55 ± 0.64 vs 0.76 ± 0.52 AU, p = 0.014), which coincided with mitochondrial network fragmentation in individuals with type 2 diabetes, as assessed by confocal microscopy in a subset of type 2 diabetes patients vs endurance-trained athletes (n = 6). Furthermore, lean individuals and athletes displayed a mitonuclear protein balance that was different from obese participants and those with type 2 diabetes. Mitonuclear protein balance also associated with heat shock protein 60 (HSP60) protein levels, which were higher in athletes when compared with participants with obesity (p = 0.048) and type 2 diabetes (p = 0.002), indicative for activation of the mitochondrial unfolded protein response. Finally, OPA1, FIS1 and HSP60 correlated positively with aerobic capacity (r = 0.48, p = 0.0001; r = 0.55, p < 0.001 and r = 0.61, p < 0.0001, respectively) and insulin sensitivity (r = 0.40, p = 0.008; r = 0.44, p = 0.003 and r = 0.48, p = 0.001, respectively). CONCLUSIONS/INTERPRETATION: Collectively, our data suggest that mitochondrial dynamics and quality control in skeletal muscle are linked to oxidative capacity in humans, which may play a role in the maintenance of muscle insulin sensitivity. CLINICAL TRIAL REGISTRY: numbers NCT00943059, NCT01298375 and NL1888 Graphical abstract.

6.
Mol Metab ; 41: 101050, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32659272

RESUMO

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

7.
J Cell Physiol ; 235(12): 9851-9863, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32452584

RESUMO

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


Assuntos
MicroRNAs/genética , Mitocôndrias/genética , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/metabolismo , Animais , Autofagia/genética , Biópsia , Humanos , Camundongos , Mitocôndrias Musculares/genética , Mitocôndrias Musculares/metabolismo , Mitofagia/genética , Biogênese de Organelas , Oxirredução , Estresse Oxidativo/genética
8.
Diabetologia ; 63(6): 1211-1222, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32185462

RESUMO

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

9.
Front Microbiol ; 10: 1711, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31396199

RESUMO

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

10.
FEMS Microbiol Ecol ; 95(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30624625

RESUMO

Vibrio anguillarum is a marine bacterium that can cause vibriosis in many fish and shellfish species. Although phage therapy has been proposed as an alternative treatment, the defense mechanisms against phage infection in V. anguillarum and their impact on host function are not fully understood. Here, we examined phage defense strategies in four V. anguillarum strains during exposure to the broad-host-range bacteriophage KVP40. Whole-genome sequences of phage-resistant V. anguillarum isolates showed mutations causing premature stop codons, frameshifts and amino acid changes in the OmpK phage receptor. Moreover, certain phage-resistant variants recovered susceptibility to phage infection following re-culturing, suggesting alternative protection mechanisms, such as formation of biofilm, receptor downregulation and phage inactivation by proteases. Also, the lack of phage production by some strains despite strong phage control suggested an abortive infection mechanism was in play. In addition, examination of the virulence properties and extracellular enzyme secretion of the phage-resistant variants suggested that phage resistance was associated with reduced virulence in V. anguillarum. Altogether, the results identified a variety of phage resistance mechanisms in V. anguillarum including both mutational and non-mutational defenses and demonstrated a significant fitness loss associated with mutational changes, which may explain the selection for alternative defense mechanisms.


Assuntos
Bacteriófagos/fisiologia , Doenças dos Peixes/microbiologia , Vibrioses/veterinária , Vibrio/patogenicidade , Vibrio/virologia , Animais , Proteínas de Bactérias/genética , Peixes/microbiologia , Genoma Bacteriano/genética , Mutação , Vibrio/genética , Vibrioses/microbiologia , Virulência/genética
11.
J Cell Physiol ; 234(5): 6601-6610, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30417335

RESUMO

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


Assuntos
MicroRNAs/genética , Mitocôndrias Musculares/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Animais , Camundongos , Músculo Esquelético/metabolismo , Proteínas Ribossômicas/metabolismo , Resposta a Proteínas não Dobradas/genética
12.
Mol Metab ; 17: 71-81, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30174227

RESUMO

OBJECTIVE: Intramyocellular lipid (IMCL) storage negatively associates with insulin resistance, albeit not in endurance-trained athletes. We investigated the putative contribution of lipid droplet (LD) morphology and subcellular localization to the so-called athlete's paradox. METHODS: We performed quantitative immunofluorescent confocal imaging of muscle biopsy sections from endurance Trained, Lean sedentary, Obese, and Type 2 diabetes (T2DM) participants (n = 8/group). T2DM patients and Trained individuals were matched for IMCL content. Furthermore we performed this analysis in biopsies of T2DM patients before and after a 12-week exercise program (n = 8). RESULTS: We found marked differences in lipid storage morphology between trained subjects and T2DM: the latter group mainly store lipid in larger LDs in the subsarcolemmal (SS) region of type II fibers, whereas Trained store lipid in a higher number of LDs in the intramyofibrillar (IMF) region of type I fibers. In addition, a twelve-week combined endurance and strength exercise program resulted in a LD phenotype shift in T2DM patients partly towards an 'athlete-like' phenotype, accompanied by improved insulin sensitivity. Proteins involved in LD turnover were also more abundant in Trained than in T2DM and partly changed in an 'athlete-like' fashion in T2DM patients upon exercise training. CONCLUSIONS: Our findings provide a physiological explanation for the athlete's paradox and reveal LD morphology and distribution as a major determinant of skeletal muscle insulin sensitivity.


Assuntos
Exercício Físico/fisiologia , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Adulto , Atletas , Biópsia por Agulha/métodos , Estudos Transversais , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Proteínas de Ligação ao GTP , Humanos , Resistência à Insulina/fisiologia , Metabolismo dos Lipídeos/fisiologia , Masculino , Pessoa de Meia-Idade , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Países Baixos , Obesidade/metabolismo , Sobrepeso/metabolismo , Resistência Física/fisiologia
13.
J Physiol ; 596(5): 857-868, 2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29110300

RESUMO

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


Assuntos
Atletas , Diabetes Mellitus Tipo 2/fisiopatologia , Resistência à Insulina , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Músculo Esquelético/fisiopatologia , Perilipina-5/metabolismo , Adulto , Estudos de Casos e Controles , Exercício Físico , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Resistência Física , Adulto Jovem
14.
Obesity (Silver Spring) ; 26(1): 135-140, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29178600

RESUMO

OBJECTIVE: Human brown adipose tissue (BAT) activity decreases with age and obesity. In addition to uncoupling protein 1 (UCP1), several genetic markers of BAT in humans have been published. However, the link between human BAT activity and genetic markers has been inadequately explored. METHODS: White adipose tissue (WAT) and BAT biopsies were obtained from 16 patients undergoing deep neck surgery. In vitro differentiated adipocytes were used to measure norepinephrine-stimulated mitochondrial uncoupling as a measure of in vitro BAT activity. Gene expression was determined in adipose tissue biopsies. RESULTS: Norepinephrine increased in vitro BAT activity in adipocytes derived from human BAT, and this increase was abolished by propranolol. Furthermore, in vitro BAT activity showed a negative correlation to age and BMI. UCP1 messenger RNA (mRNA) expression showed a positive correlation to in vitro BAT activity, while zinc finger protein of cerebellum 1 (ZIC1) mRNA showed a negative correlation to in vitro BAT activity. In human BAT biopsies, UCP1 mRNA showed negative correlations to age and BMI, while ZIC1 mRNA showed positive correlations to age and BMI. CONCLUSIONS: Differentiated adipocytes derived from human BAT maintain intrinsic characteristics of the donor. High ZIC1 mRNA does not necessarily reflect high BAT activity.


Assuntos
Tecido Adiposo Marrom/metabolismo , Marcadores Genéticos/genética , Adulto , Idoso , Animais , Diferenciação Celular , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
15.
Mol Metab ; 6(11): 1429-1442, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-29107290

RESUMO

OBJECTIVE: Strategies improving skeletal muscle mitochondrial capacity are commonly paralleled by improvements in (metabolic) health. We and others previously identified microRNAs regulating mitochondrial oxidative capacity, but data in skeletal muscle are limited. Therefore, the present study aimed to identify novel microRNAs regulating skeletal muscle mitochondrial metabolism. METHODS AND RESULTS: We conducted an unbiased, hypothesis-free microRNA silencing screen in C2C12 myoblasts, using >700 specific microRNA inhibitors, and investigated a broad panel of mitochondrial markers. After subsequent validation in differentiated C2C12 myotubes, and exclusion of microRNAs without a human homologue or with an adverse effect on mitochondrial metabolism, 19 candidate microRNAs remained. Human clinical relevance of these microRNAs was investigated by measuring their expression in human skeletal muscle of subject groups displaying large variation in skeletal muscle mitochondrial capacity. CONCLUSION: The results show that that microRNA-320a, microRNA-196b-3p, microRNA-150-5p, and microRNA-34c-3p are tightly related to in vivo skeletal muscle mitochondrial function in humans and identify these microRNAs as targets for improving mitochondrial metabolism.


Assuntos
MicroRNAs/genética , Mitocôndrias Musculares/metabolismo , Mioblastos de Músculo Liso/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Humanos , Masculino , Camundongos , MicroRNAs/metabolismo , Mitocôndrias Musculares/genética , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo
16.
Front Physiol ; 8: 711, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28983252

RESUMO

In recent years, several microRNAs (miRNAs)-post-transcriptional regulators of gene expression-have been linked to the regulation of peripheral insulin sensitivity. Many of these studies, however, have been conducted in cell or animal models and the few human studies available lack adequate measurements of peripheral insulin sensitivity. In the present study, we examined the expression of 25 miRNAs, putatively involved in (peripheral) insulin sensitivity, in skeletal muscle biopsies from extensively phenotyped human individuals, widely ranging in insulin sensitivity. To identify miRNAs expressed in skeletal muscle and associated with insulin sensitivity and type 2 diabetes, a comprehensive PubMed-based literature search was performed. Subsequently, the expression of selected miRNAs was determined by RT-qPCR using predesigned 384-well Pick-&-Mix miRNA PCR Panel plates in muscle biopsies from type 2 diabetes patients, non-diabetic obese/overweight individuals, lean sedentary individuals and endurance-trained athletes. In all subjects, peripheral insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp. The literature search resulted in 25 candidate miRNAs, 6 of which were differentially expressed in human type 2 diabetes compared to non-diabetic obese/overweight individuals. In turn, four of these miRNAs, i.e., miRNA27a-3p (r = -0.45, p = 0.0012), miRNA-29a-3p (r = -0.40, p = 0.0052), miRNA-29b-3p (r = -0.70, p < 0.0001) and miRNA-29c-3p (r = -0.50, p = 0.0004) demonstrated strong negative correlations with peripheral insulin sensitivity across all four subject groups. We identified miR-27a-3p and all members of the miRNA-29 family as potential regulatory players in insulin sensitivity in humans. These miRNA's may represent interesting novel targets for maintaining or improving insulin sensitivity.

17.
Viruses ; 9(5)2017 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-28531104

RESUMO

Nineteen Vibrio anguillarum-specific temperate bacteriophages isolated across Europe and Chile from aquaculture and environmental sites were genome sequenced and analyzed for host range, morphology and life cycle characteristics. The phages were classified as Siphoviridae with genome sizes between 46,006 and 54,201 bp. All 19 phages showed high genetic similarity, and 13 phages were genetically identical. Apart from sporadically distributed single nucleotide polymorphisms (SNPs), genetic diversifications were located in three variable regions (VR1, VR2 and VR3) in six of the phage genomes. Identification of specific genes, such as N6-adenine methyltransferase and lambda like repressor, as well as the presence of a tRNAArg, suggested a both mutualistic and parasitic interaction between phages and hosts. During short term phage exposure experiments, 28% of a V. anguillarum host population was lysogenized by the temperate phages and a genomic analysis of a collection of 31 virulent V. anguillarum showed that the isolated phages were present as prophages in >50% of the strains covering large geographical distances. Further, phage sequences were widely distributed among CRISPR-Cas arrays of publicly available sequenced Vibrios. The observed distribution of these specific temperate Vibriophages across large geographical scales may be explained by efficient dispersal of phages and bacteria in the marine environment combined with a mutualistic interaction between temperate phages and their hosts which selects for co-existence rather than arms race dynamics.


Assuntos
Bacteriófagos/classificação , Bacteriófagos/genética , Peixes/microbiologia , Siphoviridae/genética , Vibrio/virologia , Animais , Bacteriófagos/isolamento & purificação , Bacteriófagos/fisiologia , Sequência de Bases , Biodiversidade , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA Viral/análise , Genes Virais/genética , Variação Genética/genética , Genoma Viral , Geografia , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Lisogenia/fisiologia , Microscopia Eletrônica de Transmissão , Filogenia , Polimorfismo de Nucleotídeo Único , Prófagos/genética , Prófagos/isolamento & purificação , Água do Mar/virologia , Alinhamento de Sequência , Siphoviridae/classificação , Siphoviridae/isolamento & purificação , Siphoviridae/fisiologia , Vibrio/fisiologia , Integração Viral
18.
Mol Metab ; 5(8): 635-645, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27656401

RESUMO

OBJECTIVE: A disturbed day-night rhythm is associated with metabolic perturbations that can lead to obesity and type 2 diabetes mellitus (T2DM). In skeletal muscle, a reduced oxidative capacity is also associated with the development of T2DM. However, whether oxidative capacity in skeletal muscle displays a day-night rhythm in humans has so far not been investigated. METHODS: Lean, healthy subjects were enrolled in a standardized living protocol with regular meals, physical activity and sleep to reflect our everyday lifestyle. Mitochondrial oxidative capacity was examined in skeletal muscle biopsies taken at five time points within a 24-hour period. RESULTS: Core-body temperature was lower during the early night, confirming a normal day-night rhythm. Skeletal muscle oxidative capacity demonstrated a robust day-night rhythm, with a significant time effect in ADP-stimulated respiration (state 3 MO, state 3 MOG and state 3 MOGS, p < 0.05). Respiration was lowest at 1 PM and highest at 11 PM (state 3 MOGS: 80.6 ± 4.0 vs. 95.8 ± 4.7 pmol/mg/s). Interestingly, the fluctuation in mitochondrial function was also observed in whole-body energy expenditure, with peak energy expenditure at 11 PM and lowest energy expenditure at 4 AM (p < 0.001). In addition, we demonstrate rhythmicity in mRNA expression of molecular clock genes in human skeletal muscle. CONCLUSIONS: Our results suggest that the biological clock drives robust rhythms in human skeletal muscle oxidative metabolism. It is tempting to speculate that disruption of these rhythms contribute to the deterioration of metabolic health associated with circadian misalignment.

19.
Sci Rep ; 6: 22854, 2016 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-26973284

RESUMO

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.


Assuntos
Dieta , Genisteína/farmacologia , Resistência à Insulina , Músculo Esquelético/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Animais , Glicemia/metabolismo , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Genisteína/administração & dosagem , Glucose/metabolismo , Teste de Tolerância a Glucose , Homeostase/efeitos dos fármacos , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Oxirredução/efeitos dos fármacos , Fitoestrógenos/administração & dosagem , Fitoestrógenos/farmacologia , Ratos Zucker
20.
Diabetologia ; 59(5): 1030-9, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26886198

RESUMO

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.


Assuntos
Translocador 1 do Nucleotídeo Adenina/metabolismo , Ácidos Graxos/farmacologia , Músculo Esquelético/metabolismo , Translocador 1 do Nucleotídeo Adenina/genética , Animais , Humanos , Técnicas In Vitro , Insulina/genética , Insulina/metabolismo , Resistência à Insulina/genética , Masculino , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/metabolismo , Translocases Mitocondriais de ADP e ATP/genética , Translocases Mitocondriais de ADP e ATP/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/efeitos dos fármacos , Ácido Oleico/farmacologia , Ácido Palmítico/farmacologia , Ratos , Ratos Zucker
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