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1.
Cardiovasc Diabetol ; 23(1): 258, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-39026321

RESUMO

BACKGROUND: Insulin signaling regulates cardiac substrate utilization and is implicated in physiological adaptations of the heart. Alterations in the signaling response within the heart are believed to contribute to pathological conditions such as type-2 diabetes and heart failure. While extensively investigated in several metabolic organs using phosphoproteomic strategies, the signaling response elicited in cardiac tissue in general, and specifically in the specialized cardiomyocytes, has not yet been investigated to the same extent. METHODS: Insulin or vehicle was administered to male C57BL6/JRj mice via intravenous injection into the vena cava. Ventricular tissue was extracted and subjected to quantitative phosphoproteomics analysis to evaluate the insulin signaling response. To delineate the cardiomyocyte-specific response and investigate the role of Tbc1d4 in insulin signal transduction, cardiomyocytes from the hearts of cardiac and skeletal muscle-specific Tbc1d4 knockout mice, as well as from wildtype littermates, were studied. The phosphoproteomic studies involved isobaric peptide labeling with Tandem Mass Tags (TMT), enrichment for phosphorylated peptides, fractionation via micro-flow reversed-phase liquid chromatography, and high-resolution mass spectrometry measurements. RESULTS: We quantified 10,399 phosphorylated peptides from ventricular tissue and 12,739 from isolated cardiomyocytes, localizing to 3,232 and 3,128 unique proteins, respectively. In cardiac tissue, we identified 84 insulin-regulated phosphorylation events, including sites on the Insulin Receptor (InsrY1351, Y1175, Y1179, Y1180) itself as well as the Insulin receptor substrate protein 1 (Irs1S522, S526). Predicted kinases with increased activity in response to insulin stimulation included Rps6kb1, Akt1 and Mtor. Tbc1d4 emerged as a major phosphorylation target in cardiomyocytes. Despite limited impact on the global phosphorylation landscape, Tbc1d4 deficiency in cardiomyocytes attenuated insulin-induced Glut4 translocation and induced protein remodeling. We observed 15 proteins significantly regulated upon knockout of Tbc1d4. While Glut4 exhibited decreased protein abundance consequent to Tbc1d4-deficiency, Txnip levels were notably increased. Stimulation of wildtype cardiomyocytes with insulin led to the regulation of 262 significant phosphorylation events, predicted to be regulated by kinases such as Akt1, Mtor, Akt2, and Insr. In cardiomyocytes, the canonical insulin signaling response is elicited in addition to regulation on specialized cardiomyocyte proteins, such as Kcnj11Y12 and DspS2597. Details of all phosphorylation sites are provided. CONCLUSION: We present a first global outline of the insulin-induced phosphorylation signaling response in heart tissue and in isolated adult cardiomyocytes, detailing the specific residues with changed phosphorylation abundances. Our study marks an important step towards understanding the role of insulin signaling in cardiac diseases linked to insulin resistance.


Assuntos
Insulina , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos , Fosfoproteínas , Proteômica , Transdução de Sinais , Animais , Miócitos Cardíacos/metabolismo , Masculino , Insulina/metabolismo , Fosforilação , Fosfoproteínas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Ativadoras de GTPase/genética , Receptor de Insulina/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos
3.
Artigo em Inglês | MEDLINE | ID: mdl-38820114

RESUMO

CONTEXT: Given the promising effects of prolonged treatment with beta2-agonist on insulin sensitivity in animals and non-diabetic individuals, the beta2-adrenergic receptor has been proposed as a target to counter peripheral insulin resistance. On the other hand, rodent studies also reveal that beta2-agonists acutely impair insulin action, posing a potential caveat for their use in treating insulin resistance. OBJECTIVE: To assess the impact of beta2-agonist on muscle insulin action and glucose metabolism and identify the underlying mechanism(s) in 10 insulin-resistant subjects. METHODS AND PARTICIPANTS: In a cross-over design, we assessed the effect of beta2-agonist on insulin-stimulated muscle glucose uptake during a 3-h hyperinsulinemic isoglycemic clamp with and without intralipid infusion in 10 insulin-resistant overweight subjects. Two hours into the clamp, we infused beta2-agonist. We collected muscle biopsies before, two hours into and by the end of the clamp and analyzed them using metabolomic and lipidomic techniques. RESULTS: We establish that beta2-agonist, independently from and additively to intralipid, impairs insulin-stimulated muscle glucose uptake via different mechanisms. In combination, beta2-agonist and intralipid nearly eliminates insulin-dependent muscle glucose uptake. While both beta2-agonist and intralipid elevated muscle glucose-6-phosphate, only intralipid caused accumulation of downstream muscle glycolytic intermediates, whereas beta2-agonist attenuated incorporation of glucose into glycogen. CONCLUSIONS: Our findings suggest that beta2-agonist inhibits glycogenesis while intralipid inhibits glycolysis in skeletal muscle of insulin-resistant individuals. These results should be addressed in future treatment of insulin resistance with beta2-agonist.

4.
Diabetes ; 73(7): 1072-1083, 2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38608261

RESUMO

Insulin resistance is a risk factor for type 2 diabetes, and exercise can improve insulin sensitivity. However, following exercise, high circulating fatty acid (FA) levels might counteract this. We hypothesized that such inhibition would be reduced by forcibly increasing carbohydrate oxidation through pharmacological activation of the pyruvate dehydrogenase complex (PDC). Insulin-stimulated glucose uptake was examined with a crossover design in healthy young men (n = 8) in a previously exercised and a rested leg during a hyperinsulinemic-euglycemic clamp 5 h after one-legged exercise with 1) infusion of saline, 2) infusion of intralipid imitating circulating FA levels during recovery from whole-body exercise, and 3) infusion of intralipid + oral PDC activator, dichloroacetate (DCA). Intralipid infusion reduced insulin-stimulated glucose uptake by 19% in the previously exercised leg, which was not observed in the contralateral rested leg. Interestingly, this effect of intralipid in the exercised leg was abolished by DCA, which increased muscle PDC activity (130%) and flux (acetylcarnitine 130%) and decreased inhibitory phosphorylation of PDC on Ser293 (∼40%) and Ser300 (∼80%). Novel insight is provided into the regulatory interaction between glucose and lipid metabolism during exercise recovery. Coupling exercise and PDC flux activation upregulated the capacity for both glucose transport (exercise) and oxidation (DCA), which seems necessary to fully stimulate insulin-stimulated glucose uptake during recovery.


Assuntos
Exercício Físico , Insulina , Músculo Esquelético , Complexo Piruvato Desidrogenase , Humanos , Masculino , Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Músculo Esquelético/efeitos dos fármacos , Insulina/metabolismo , Insulina/sangue , Complexo Piruvato Desidrogenase/metabolismo , Adulto , Adulto Jovem , Técnica Clamp de Glucose , Estudos Cross-Over , Ácido Dicloroacético/farmacologia , Resistência à Insulina/fisiologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Óleo de Soja/farmacologia , Recuperação após o Exercício , Emulsões , Fosfolipídeos
5.
Diabetologia ; 67(7): 1386-1398, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38662135

RESUMO

AIMS/HYPOTHESIS: Exercise has a profound effect on insulin sensitivity in skeletal muscle. The euglycaemic-hyperinsulinaemic clamp (EHC) is the gold standard for assessment of insulin sensitivity but it does not reflect the hyperglycaemia that occurs after eating a meal. In previous EHC investigations, it has been shown that the interstitial glucose concentration in muscle is decreased to a larger extent in previously exercised muscle than in rested muscle. This suggests that previously exercised muscle may increase its glucose uptake more than rested muscle if glucose supply is increased by hyperglycaemia. Therefore, we hypothesised that the exercise-induced increase in muscle insulin sensitivity would appear greater after eating a meal than previously observed with the EHC. METHODS: Ten recreationally active men performed dynamic one-legged knee extensor exercise for 1 h. Following this, both femoral veins and one femoral artery were cannulated. Subsequently, 4 h after exercise, a solid meal followed by two liquid meals were ingested over 1 h and glucose uptake in the two legs was measured for 3 h. Muscle biopsies from both legs were obtained before the meal test and 90 min after the meal test was initiated. Data obtained in previous studies using the EHC (n=106 participants from 13 EHC studies) were used for comparison with the meal-test data obtained in this study. RESULTS: Plasma glucose and insulin peaked 45 min after initiation of the meal test. Following the meal test, leg glucose uptake and glucose clearance increased twice as much in the exercised leg than in the rested leg; this difference is twice as big as that observed in previous investigations using EHCs. Glucose uptake in the rested leg plateaued after 15 min, alongside elevated muscle glucose 6-phosphate levels, suggestive of compromised muscle glucose metabolism. In contrast, glucose uptake in the exercised leg plateaued 45 min after initiation of the meal test and there were no signs of compromised glucose metabolism. Phosphorylation of the TBC1 domain family member 4 (TBC1D4; p-TBC1D4Ser704) and glycogen synthase activity were greater in the exercised leg compared with the rested leg. Muscle interstitial glucose concentration increased with ingestion of meals, although it was 16% lower in the exercised leg than in the rested leg. CONCLUSIONS/INTERPRETATION: Hyperglycaemia after meal ingestion results in larger differences in muscle glucose uptake between rested and exercised muscle than previously observed during EHCs. These findings indicate that the ability of exercise to increase insulin-stimulated muscle glucose uptake is even greater when evaluated with a meal test than has previously been shown with EHCs.


Assuntos
Glicemia , Exercício Físico , Técnica Clamp de Glucose , Resistência à Insulina , Insulina , Refeições , Músculo Esquelético , Humanos , Masculino , Exercício Físico/fisiologia , Músculo Esquelético/metabolismo , Resistência à Insulina/fisiologia , Adulto , Glicemia/metabolismo , Insulina/metabolismo , Insulina/sangue , Adulto Jovem , Refeições/fisiologia
6.
Diabetologia ; 67(6): 1095-1106, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38427076

RESUMO

AIMS/HYPOTHESIS: As a result of early loss of the glucagon response, adrenaline is the primary counter-regulatory hormone in type 1 diabetes. Diminished adrenaline responses to hypoglycaemia due to counter-regulatory failure are common in type 1 diabetes, and are probably induced by exposure to recurrent hypoglycaemia, however, the metabolic effects of adrenaline have received less research attention, and also there is conflicting evidence regarding adrenaline sensitivity in type 1 diabetes. Thus, we aimed to investigate the metabolic response to adrenaline and explore whether it is modified by prior exposure to hypoglycaemia. METHODS: Eighteen participants with type 1 diabetes and nine healthy participants underwent a three-step ascending adrenaline infusion during a hyperinsulinaemic-euglycaemic clamp. Continuous glucose monitoring data obtained during the week before the study day were used to assess the extent of hypoglycaemia exposure. RESULTS: While glucose responses during the clamp were similar between people with type 1 diabetes and healthy participants, plasma concentrations of NEFAs and glycerol only increased in the group with type 1 diabetes (p<0.001). Metabolomics revealed an increase in the most common NEFAs (p<0.01). Other metabolic responses were generally similar between participants with type 1 diabetes and healthy participants. Exposure to hypoglycaemia was negatively associated with the NEFA response; however, this was not statistically significant. CONCLUSIONS/INTERPRETATION: In conclusion, individuals with type 1 diabetes respond with increased lipolysis to adrenaline compared with healthy participants by mobilising the abundant NEFAs in plasma, whereas other metabolic responses were similar. This may suggest that the metabolic sensitivity to adrenaline is altered in a pathway-specific manner in type 1 diabetes. TRIAL REGISTRATION: ClinicalTrials.gov NCT05095259.


Assuntos
Glicemia , Diabetes Mellitus Tipo 1 , Epinefrina , Técnica Clamp de Glucose , Hipoglicemia , Adulto , Feminino , Humanos , Masculino , Adulto Jovem , Glicemia/metabolismo , Glicemia/efeitos dos fármacos , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/sangue , Epinefrina/sangue , Epinefrina/administração & dosagem , Ácidos Graxos não Esterificados/sangue , Glucagon/sangue , Glicerol/sangue , Glicerol/administração & dosagem , Hipoglicemia/sangue , Insulina/administração & dosagem , Estudos de Casos e Controles
7.
Nat Metab ; 6(4): 764-777, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38429390

RESUMO

Surviving long periods without food has shaped human evolution. In ancient and modern societies, prolonged fasting was/is practiced by billions of people globally for religious purposes, used to treat diseases such as epilepsy, and recently gained popularity as weight loss intervention, but we still have a very limited understanding of the systemic adaptions in humans to extreme caloric restriction of different durations. Here we show that a 7-day water-only fast leads to an average weight loss of 5.7 kg (±0.8 kg) among 12 volunteers (5 women, 7 men). We demonstrate nine distinct proteomic response profiles, with systemic changes evident only after 3 days of complete calorie restriction based on in-depth characterization of the temporal trajectories of ~3,000 plasma proteins measured before, daily during, and after fasting. The multi-organ response to complete caloric restriction shows distinct effects of fasting duration and weight loss and is remarkably conserved across volunteers with >1,000 significantly responding proteins. The fasting signature is strongly enriched for extracellular matrix proteins from various body sites, demonstrating profound non-metabolic adaptions, including extreme changes in the brain-specific extracellular matrix protein tenascin-R. Using proteogenomic approaches, we estimate the health consequences for 212 proteins that change during fasting across ~500 outcomes and identified putative beneficial (SWAP70 and rheumatoid arthritis or HYOU1 and heart disease), as well as adverse effects. Our results advance our understanding of prolonged fasting in humans beyond a merely energy-centric adaptions towards a systemic response that can inform targeted therapeutic modulation.


Assuntos
Restrição Calórica , Jejum , Proteoma , Humanos , Proteoma/metabolismo , Feminino , Masculino , Adulto , Redução de Peso , Proteômica/métodos , Adaptação Fisiológica
8.
J Clin Endocrinol Metab ; 109(7): 1754-1764, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38242693

RESUMO

CONTEXT: Regular exercise is a key prevention strategy for obesity and type 2 diabetes (T2D). Exerkines secreted in response to exercise or recovery may contribute to improved systemic metabolism. Conversely, an impaired exerkine response to exercise and recovery may contribute to cardiometabolic diseases. OBJECTIVE: We investigated if the exercise-induced regulation of the exerkine, growth differentiation factor 15 (GDF15) and its putative upstream regulators of the unfolded protein response (UPR)/integrated stress response (ISR) is impaired in skeletal muscle in patients with T2D compared with weight-matched glucose-tolerant men. METHODS: Thirteen male patients with T2D and 14 age- and weight-matched overweight/obese glucose-tolerant men exercised at 70% of VO2max for 1 hour. Blood and skeletal muscle biopsies were sampled before, immediately after, and 3 hours into recovery. Serum and muscle transcript levels of GDF15 and key markers of UPR/ISR were determined. Additionally, protein/phosphorylation levels of key regulators in UPR/ISR were investigated. RESULTS: Acute exercise increased muscle gene expression and serum GDF15 levels in both groups. In recovery, muscle expression of GDF15 decreased toward baseline, whereas serum GDF15 remained elevated. In both groups, acute exercise increased the expression of UPR/ISR markers, including ATF4, CHOP, EIF2K3 (encoding PERK), and PPP1R15A (encoding GADD34), of which only CHOP remained elevated 3 hours into recovery. Downstream molecules of the UPR/ISR including XBP1-U, XBP1-S, and EDEM1 were increased with exercise and 3 hours into recovery in both groups. The phosphorylation levels of eIF2α-Ser51, a common marker of unfolded protein response (UPR) and ISR, increased immediately after exercise in controls, but decreased 3 hours into recovery in both groups. CONCLUSION: In conclusion, exercise-induced regulation of GDF15 and key markers of UPR/ISR are not compromised in patients with T2D compared with weight-matched controls.


Assuntos
Diabetes Mellitus Tipo 2 , Exercício Físico , Fator 15 de Diferenciação de Crescimento , Músculo Esquelético , Resposta a Proteínas não Dobradas , Humanos , Fator 15 de Diferenciação de Crescimento/metabolismo , Fator 15 de Diferenciação de Crescimento/sangue , Fator 15 de Diferenciação de Crescimento/genética , Masculino , Diabetes Mellitus Tipo 2/metabolismo , Resposta a Proteínas não Dobradas/fisiologia , Músculo Esquelético/metabolismo , Exercício Físico/fisiologia , Pessoa de Meia-Idade , Adulto , Estudos de Casos e Controles , Estresse Fisiológico/fisiologia
9.
Diabetes ; 72(10): 1397-1408, 2023 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-37506328

RESUMO

Exercise increases muscle glucose uptake independently of insulin signaling and represents a cornerstone for the prevention of metabolic disorders. Pharmacological activation of the exercise-responsive AMPK in skeletal muscle has been proven successful as a therapeutic approach to treat metabolic disorders by improving glucose homeostasis through the regulation of muscle glucose uptake. However, conflicting observations cloud the proposed role of AMPK as a necessary regulator of muscle glucose uptake during exercise. We show that glucose uptake increases in human skeletal muscle in the absence of AMPK activation during exercise and that exercise-stimulated AMPKγ3 activity strongly correlates to muscle glucose uptake in the postexercise period. In AMPKγ3-deficient mice, muscle glucose uptake is normally regulated during exercise and contractions but impaired in the recovery period from these stimuli. Impaired glucose uptake in recovery from exercise and contractions is associated with a lower glucose extraction, which can be explained by a diminished permeability to glucose and abundance of GLUT4 at the muscle plasma membrane. As a result, AMPKγ3 deficiency impairs muscle glycogen resynthesis following exercise. These results identify a physiological function of the AMPKγ3 complex in human and rodent skeletal muscle that regulates glucose uptake in recovery from exercise to recapture muscle energy stores. ARTICLE HIGHLIGHTS: Exercise-induced activation of AMPK in skeletal muscle has been proposed to regulate muscle glucose uptake in recovery from exercise. This study investigated whether the muscle-specific AMPKγ3-associated heterotrimeric complex was involved in regulating muscle glucose metabolism in recovery from exercise. The findings support that exercise-induced activation of the AMPKγ3 complex in human and mouse skeletal muscle enhances glucose uptake in recovery from exercise via increased translocation of GLUT4 to the plasma membrane. This work uncovers the physiological role of the AMPKγ3 complex in regulating muscle glucose uptake that favors replenishment of the muscle cellular energy stores.


Assuntos
Proteínas Quinases Ativadas por AMP , Exercício Físico , Glucose , Animais , Humanos , Camundongos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glicogênio/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Exercício Físico/fisiologia
10.
Proc Natl Acad Sci U S A ; 120(27): e2211041120, 2023 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-37364105

RESUMO

The molecular events governing skeletal muscle glucose uptake have pharmacological potential for managing insulin resistance in conditions such as obesity, diabetes, and cancer. With no current pharmacological treatments to target skeletal muscle insulin sensitivity, there is an unmet need to identify the molecular mechanisms that control insulin sensitivity in skeletal muscle. Here, the Rho guanine dissociation inhibitor α (RhoGDIα) is identified as a point of control in the regulation of insulin sensitivity. In skeletal muscle cells, RhoGDIα interacted with, and thereby inhibited, the Rho GTPase Rac1. In response to insulin, RhoGDIα was phosphorylated at S101 and Rac1 dissociated from RhoGDIα to facilitate skeletal muscle GLUT4 translocation. Accordingly, siRNA-mediated RhoGDIα depletion increased Rac1 activity and elevated GLUT4 translocation. Consistent with RhoGDIα's inhibitory effect, rAAV-mediated RhoGDIα overexpression in mouse muscle decreased insulin-stimulated glucose uptake and was detrimental to whole-body glucose tolerance. Aligning with RhoGDIα's negative role in insulin sensitivity, RhoGDIα protein content was elevated in skeletal muscle from insulin-resistant patients with type 2 diabetes. These data identify RhoGDIα as a clinically relevant controller of skeletal muscle insulin sensitivity and whole-body glucose homeostasis, mechanistically by modulating Rac1 activity.


Assuntos
Diabetes Mellitus Tipo 2 , Resistência à Insulina , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho , Animais , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Músculo Esquelético/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidor alfa de Dissociação do Nucleotídeo Guanina rho/metabolismo
11.
Diabetes ; 72(7): 857-871, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37074686

RESUMO

The ability of insulin to stimulate glucose uptake in skeletal muscle is important for whole-body glycemic control. Insulin-stimulated skeletal muscle glucose uptake is improved in the period after a single bout of exercise, and accumulating evidence suggests that phosphorylation of TBC1D4 by the protein kinase AMPK is the primary mechanism responsible for this phenomenon. To investigate this, we generated a TBC1D4 knock-in mouse model with a serine-to-alanine point mutation at residue 711 that is phosphorylated in response to both insulin and AMPK activation. Female TBC1D4-S711A mice exhibited normal growth and eating behavior as well as intact whole-body glycemic control on chow and high-fat diets. Moreover, muscle contraction increased glucose uptake, glycogen utilization, and AMPK activity similarly in wild-type and TBC1D4-S711A mice. In contrast, improvements in whole-body and muscle insulin sensitivity after exercise and contractions were only evident in wild-type mice and occurred concomitantly with enhanced phosphorylation of TBC1D4-S711. These results provide genetic evidence to support that TBC1D4-S711 serves as a major point of convergence for AMPK- and insulin-induced signaling that mediates the insulin-sensitizing effect of exercise and contractions on skeletal muscle glucose uptake.


Assuntos
Glucose , Insulina , Feminino , Camundongos , Animais , Insulina/farmacologia , Insulina/metabolismo , Glucose/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Músculo Esquelético/metabolismo , Insulina Regular Humana/farmacologia , Fosforilação , Contração Muscular
12.
Elife ; 122023 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-37073948

RESUMO

Microtubules serve as tracks for long-range intracellular trafficking of glucose transporter 4 (GLUT4), but the role of this process in skeletal muscle and insulin resistance is unclear. Here, we used fixed and live-cell imaging to study microtubule-based GLUT4 trafficking in human and mouse muscle fibers and L6 rat muscle cells. We found GLUT4 localized on the microtubules in mouse and human muscle fibers. Pharmacological microtubule disruption using Nocodazole (Noco) prevented long-range GLUT4 trafficking and depleted GLUT4-enriched structures at microtubule nucleation sites in a fully reversible manner. Using a perifused muscle-on-a-chip system to enable real-time glucose uptake measurements in isolated mouse skeletal muscle fibers, we observed that Noco maximally disrupted the microtubule network after 5 min without affecting insulin-stimulated glucose uptake. In contrast, a 2-hr Noco treatment markedly decreased insulin responsiveness of glucose uptake. Insulin resistance in mouse muscle fibers induced either in vitro by C2 ceramides or in vivo by diet-induced obesity, impaired microtubule-based GLUT4 trafficking. Transient knockdown of the microtubule motor protein kinesin-1 protein KIF5B in L6 muscle cells reduced insulin-stimulated GLUT4 translocation while pharmacological kinesin-1 inhibition in incubated mouse muscles strongly impaired insulin-stimulated glucose uptake. Thus, in adult skeletal muscle fibers, the microtubule network is essential for intramyocellular GLUT4 movement, likely functioning to maintain an insulin-responsive cell surface recruitable GLUT4 pool via kinesin-1-mediated trafficking.


Assuntos
Resistência à Insulina , Insulina , Adulto , Animais , Humanos , Camundongos , Ratos , Glucose/metabolismo , Insulina/metabolismo , Cinesinas/metabolismo , Microtúbulos/metabolismo , Proteínas de Transporte de Monossacarídeos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Transporte Proteico , Transportador de Glucose Tipo 4
13.
J Gerontol A Biol Sci Med Sci ; 78(2): 177-185, 2023 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-36269629

RESUMO

AMP-activated protein kinase (AMPK), a highly conserved, heterotrimeric serine/threonine kinase with critical sensory and regulatory functions, is proposed to induce antiaging actions of caloric restriction (CR). Although earlier studies assessed CR's effects on AMPK in rodent skeletal muscle, the scope of these studies was narrow with a limited focus on older animals. This study's purpose was to fill important knowledge gaps related to CR's influence on AMPK in skeletal muscle of older animals. Therefore, using epitrochlearis muscles from 24-month-old ad-libitum fed (AL) and CR (consuming 65% of AL intake for 8 weeks), male Fischer-344 × Brown Norway F1 rats, we determined: (a) AMPK Thr172 phosphorylation (a key regulatory site) by immunoblot; (b) AMPKα1 and AMPKα2 activity (representing the 2 catalytic α-subunits of AMPK), and AMPKγ3 activity (representing AMPK complexes that include the skeletal muscle-selective regulatory γ3 subunit) using enzymatic assays; (c) phosphorylation of multiple protein substrates that are linked to CR-related effects (acetyl-CoA carboxylase [ACC], that regulates lipid oxidation; Beclin-1 and ULK1 that are autophagy regulatory proteins; Raptor, mTORC1 complex protein that regulates autophagy; TBC1D1 and TBC1D4 that regulate glucose uptake) by immunoblot; and (d) ATP and AMP concentrations (key AMPK regulators) by mass spectrometry. The results revealed significant CR-associated increases in the phosphorylation of AMPKThr172 and 4 AMPK substrates (ACC, Beclin-1, TBC1D1, and TBC1D4), without significant diet-related differences in ATP or AMP concentration or AMPKα1-, AMPKα2-, or AMPKγ3-associated activity. The enhanced phosphorylation of multiple AMPK substrates provides novel mechanistic insights linking AMPK to functionally important consequences of CR.


Assuntos
Proteínas Quinases Ativadas por AMP , Restrição Calórica , Ratos , Masculino , Animais , Fosforilação , Proteínas Quinases Ativadas por AMP/metabolismo , Proteína Beclina-1/metabolismo , Músculo Esquelético/metabolismo , Ratos Endogâmicos F344 , Ratos Endogâmicos BN , Acetil-CoA Carboxilase/metabolismo , Acetil-CoA Carboxilase/farmacologia , Trifosfato de Adenosina/metabolismo
14.
J Clin Endocrinol Metab ; 108(3): 653-664, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36240323

RESUMO

CONTEXT: Exogenous ketone body administration lowers circulating glucose levels but the underlying mechanisms are uncertain. OBJECTIVE: We tested the hypothesis that administration of the ketone body ß-hydroxybutyrate (ßOHB) acutely increases insulin sensitivity via feedback suppression of circulating free fatty acid (FFA) levels. METHODS: In a randomized, single-blinded crossover design, 8 healthy men were studied twice with a growth hormone (GH) infusion to induce lipolysis in combination with infusion of either ßOHB or saline. Each study day comprised a basal period and a hyperinsulinemic-euglycemic clamp combined with a glucose tracer and adipose tissue and skeletal muscle biopsies. RESULTS: ßOHB administration profoundly suppressed FFA levels concomitantly with a significant increase in glucose disposal and energy expenditure. This was accompanied by a many-fold increase in skeletal muscle content of both ßOHB and its derivative acetoacetate. CONCLUSION: Our data unravel an insulin-sensitizing effect of ßOHB, which we suggest is mediated by concomitant suppression of lipolysis.


Assuntos
Hormônio do Crescimento Humano , Resistência à Insulina , Corpos Cetônicos , Humanos , Masculino , Ácido 3-Hidroxibutírico/farmacologia , Ácidos Graxos não Esterificados , Glucose , Técnica Clamp de Glucose , Hormônio do Crescimento , Hormônio do Crescimento Humano/farmacologia , Insulina/farmacologia , Resistência à Insulina/fisiologia , Corpos Cetônicos/farmacologia , Corpos Cetônicos/uso terapêutico , Lipólise/efeitos dos fármacos , Lipólise/fisiologia
15.
Diabetes ; 71(11): 2237-2250, 2022 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-36265014

RESUMO

Exercise profoundly influences glycemic control by enhancing muscle insulin sensitivity, thus promoting glucometabolic health. While prior glycogen breakdown so far has been deemed integral for muscle insulin sensitivity to be potentiated by exercise, the mechanisms underlying this phenomenon remain enigmatic. We have combined original data from 13 of our studies that investigated insulin action in skeletal muscle either under rested conditions or following a bout of one-legged knee extensor exercise in healthy young male individuals (n = 106). Insulin-stimulated glucose uptake was potentiated and occurred substantially faster in the prior contracted muscles. In this otherwise homogenous group of individuals, a remarkable biological diversity in the glucometabolic responses to insulin is apparent both in skeletal muscle and at the whole-body level. In contrast to the prevailing concept, our analyses reveal that insulin-stimulated muscle glucose uptake and the potentiation thereof by exercise are not associated with muscle glycogen synthase activity, muscle glycogen content, or degree of glycogen utilization during the preceding exercise bout. Our data further suggest that the phenomenon of improved insulin sensitivity in prior contracted muscle is not regulated in a homeostatic feedback manner from glycogen. Instead, we put forward the idea that this phenomenon is regulated by cellular allostatic mechanisms that elevate the muscle glycogen storage set point and enhance insulin sensitivity to promote the uptake of glucose toward faster glycogen resynthesis without development of glucose overload/toxicity or feedback inhibition.


Assuntos
Resistência à Insulina , Insulina , Humanos , Masculino , Insulina/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Resistência à Insulina/fisiologia , Insulina Isófana Humana , Músculo Esquelético/metabolismo , Glucose/metabolismo , Insulina Regular Humana
16.
EMBO J ; 41(17): e111650, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-35899396

RESUMO

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAKß is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKß's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.


Assuntos
Proteínas Quinases Ativadas por Mitógeno , Músculo Esquelético , Animais , MAP Quinase Quinase Quinases , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Contração Muscular/fisiologia , Músculo Esquelético/metabolismo , Fosforilação , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/genética
18.
Diabetes ; 71(5): 906-920, 2022 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-35192682

RESUMO

Insulin-stimulated muscle glucose uptake is a key process in glycemic control. This process depends on the redistribution of glucose transporters to the surface membrane, a process that involves regulatory proteins such as TBC1D1 and TBC1D4. Accordingly, a TBC1D4 loss-of-function mutation in human skeletal muscle is associated with an increased risk of type 2 diabetes, and observations from carriers of a TBC1D1 variant associate this protein to a severe obesity phenotype. Here, we identified interactors of the endogenous TBC1D4 protein in human skeletal muscle by an unbiased proteomics approach. We detected 76 proteins as candidate TBC1D4 interactors. The binding of 12 of these interactors was regulated by insulin, including proteins known to be involved in glucose metabolism (e.g., 14-3-3 proteins and α-actinin-4 [ACTN4]). TBC1D1 also coprecipitated with TBC1D4 and vice versa in both human and mouse skeletal muscle. This interaction was not regulated by insulin or exercise in young, healthy, lean individuals. Similarly, the exercise- and insulin-regulated phosphorylation of the TBC1D1-TBC1D4 complex was intact. In contrast, we observed an altered interaction as well as compromised insulin-stimulated phosphoregulation of the TBC1D1-TBC1D4 complex in muscle of obese individuals with type 2 diabetes. Altogether, we provide a repository of TBC1D4 interactors in human and mouse skeletal muscle that serve as potential regulators of TBC1D4 function and, thus, insulin-stimulated glucose uptake in human skeletal muscle.


Assuntos
Diabetes Mellitus Tipo 2 , Insulina , Animais , Diabetes Mellitus Tipo 2/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Insulina/farmacologia , Insulina Regular Humana , Iluminação , Camundongos , Músculo Esquelético/metabolismo , Fosforilação
19.
Acta Physiol (Oxf) ; 234(2): e13766, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34981891

RESUMO

Exercise activates a plethora of metabolic and signalling pathways in skeletal muscle and other organs causing numerous systemic beneficial metabolic effects. Thus, regular exercise may ameliorate and prevent the development of several chronic metabolic diseases. Skeletal muscle is recognized as an important endocrine organ regulating systemic adaptations to exercise. Skeletal muscle may mediate crosstalk with other organs through the release of exercise-induced cytokines, peptides and proteins, termed myokines, into the circulation. Importantly, other tissues such as the liver and adipose tissue may also release cytokines and peptides in response to exercise. Hence, exercise-released molecules are collectively called exerkines. Moreover, extracellular vesicles (EVs), in the form of exosomes or microvesicles, may carry some of the signals involved in tissue crosstalk. This review focuses on the role of factors potentially mediating crosstalk between muscle and other tissues in response to exercise.


Assuntos
Exercício Físico , Vesículas Extracelulares , Tecido Adiposo/metabolismo , Citocinas/metabolismo , Exercício Físico/fisiologia , Vesículas Extracelulares/metabolismo , Humanos , Músculo Esquelético/metabolismo
20.
Nat Biotechnol ; 40(4): 576-584, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34857927

RESUMO

Protein phosphorylation dynamically integrates environmental and cellular information to control biological processes. Identifying functional phosphorylation amongst the thousands of phosphosites regulated by a perturbation at a global scale is a major challenge. Here we introduce 'personalized phosphoproteomics', a combination of experimental and computational analyses to link signaling with biological function by utilizing human phenotypic variance. We measure individual subject phosphoproteome responses to interventions with corresponding phenotypes measured in parallel. Applying this approach to investigate how exercise potentiates insulin signaling in human skeletal muscle, we identify both known and previously unidentified phosphosites on proteins involved in glucose metabolism. This includes a cooperative relationship between mTOR and AMPK whereby the former directly phosphorylates the latter on S377, for which we find a role in metabolic regulation. These results establish personalized phosphoproteomics as a general approach for investigating the signal transduction underlying complex biology.


Assuntos
Fenômenos Biológicos , Fosfoproteínas , Fosfoproteínas/genética , Fosforilação , Proteômica/métodos , Transdução de Sinais/fisiologia
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