RESUMO
Hyperthyroidism causes increased energy intake and expenditure, although anorexia and higher weight loss have been reported in elderly individuals with hyperthyroidism. To determine the effect of age on energy homeostasis in response to experimental hyperthyroidism, we administered 200 µg tri-iodothyronine (T3) in 7- and 27-mo-old rats for 14 d. T3 increased energy expenditure (EE) in both the young and the old rats, although the old rats lost more weight (147 g) than the young rats (58 g) because of the discordant effect of T3 on food intake, with a 40% increase in the young rats, but a 40% decrease in the old ones. The increased food intake in the young rats corresponded with a T3-mediated increase in the appetite-regulating proteins agouti-related peptide, neuropeptide Y, and uncoupling protein 2 in the hypothalamus, but no increase occurred in the old rats. Evidence of mitochondrial biogenesis in response to T3 was similar in the soleus muscle and heart of the young and old animals, but less consistent in old plantaris muscle and liver. Despite the comparable increase in EE, T3's effect on mitochondrial function was modulated by age in a tissue-specific manner. We conclude that older rats lack compensatory mechanisms to increase caloric intake in response to a T3-induced increase in EE, demonstrating a detrimental effect of age on energy homeostasis.
Assuntos
Fatores Etários , Metabolismo Energético , Homeostase , Hormônios Tireóideos/administração & dosagem , Animais , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , DNA Mitocondrial/metabolismo , Ingestão de Alimentos , Hipertireoidismo/metabolismo , Hipotálamo/fisiologia , Masculino , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos F344RESUMO
Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3ß phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3ß when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Insulina/metabolismo , Insulina/farmacologia , Músculo Esquelético/metabolismo , Esfingolipídeos/metabolismo , Animais , Ceramidas/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Frações Subcelulares/metabolismoRESUMO
CD36 expression in both immune and non-immune cells is known to be directly involved in cancer metastasis. Extracellular vesicles (EVs) secreted by malignant melanocytes play a vital role in developing tumor-promoting microenvironments, but it is unclear whether this is mediated through CD36. To understand the role of CD36 in melanoma, we first analyzed the SKCM dataset for clinical prognosis, evaluated the percentage of CD36 in lymphatic fluid-derived EVs (LEVs), and tested whether melanoma-derived EVs increase CD36 expression and induce M2-macrophage-like characteristics. Furthermore, we performed a multiplex immunofluorescence (MxIF) imaging analysis to evaluate the CD36 expression and its colocalization with various other cells in the lymph node (LN) of patients and control subjects. Our findings show that cutaneous melanoma patients have a worse clinical prognosis with high CD36 levels, and a higher percentage of CD36 in total LEVs were found at baseline in melanoma patients compared to control. We also found that monocytic and endothelial cells treated with melanoma EVs expressed more CD36 than untreated cells. Furthermore, melanoma-derived EVs can regulate immunosuppressive macrophage-like characteristics by upregulating CD36. The spatial imaging data show that cells in tumor-involved sentinel LNs exhibit a higher probability of CD36 expression than cells from control LNs, but this was not statistically significant. Conclusively, our findings demonstrated that CD36 plays a vital role in controlling the immunosuppressive microenvironment in the LN, which can promote the formation of a protumorigenic niche.
Assuntos
Antígenos CD36 , Vesículas Extracelulares , Melanoma , Microambiente Tumoral , Humanos , Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Antígenos CD36/metabolismo , Neoplasias Cutâneas/patologia , Neoplasias Cutâneas/metabolismo , Linhagem Celular Tumoral , Macrófagos/metabolismo , Macrófagos/patologia , Prognóstico , Feminino , Melanoma Maligno Cutâneo , Linfonodos/patologia , Linfonodos/metabolismo , MasculinoRESUMO
PURPOSE: AB160 is a 160-nm nano-immunoconjugate consisting of nab-paclitaxel (ABX) nanoparticles noncovalently coated with bevacizumab (BEV) for targeted delivery into tissues expressing high levels of VEGF. Preclinical data showed that AB160 resulted in greater tumor targeting and tumor inhibition compared with sequential treatment with ABX then BEV. Given individual drug activity, we investigated the safety and toxicity of AB160 in patients with gynecologic cancers. PATIENTS AND METHODS: A 3+3 phase I trial was conducted with three potential dose levels in patients with previously treated endometrial, cervical, and platinum-resistant ovarian cancer to ascertain the recommended phase II dose (RP2D). AB160 was administered intravenously on days 1, 8, and 15 of a 28-day cycle (ABX 75-175 mg/m2, BEV 30-70 mg/m2). Pharmacokinetic analyses were performed. RESULTS: No dose-limiting toxicities (DLT) were seen among the three dose levels tested. Grade 3/4 toxicities included neutropenia, thromboembolic events, and leukopenia. DL2 (ABX 150 mg/m2, BEV 60 mg/m2) was chosen as the RP2D. Seven of the 19 patients with measurable disease (36.8%) had confirmed partial responses (95% confidence interval, 16.3%-61.6%). Pharmacokinetic analyses demonstrated that AB160 allowed 50% higher paclitaxel dosing and that paclitaxel clearance mirrored that of therapeutic antibodies. CONCLUSIONS: The safety profile and clinical activity of AB160 supports further clinical testing in patients with gynecologic cancers; the RP2D is DL2 (ABX 150 mg/m2, BEV 60 mg/m2).
Assuntos
Albuminas , Protocolos de Quimioterapia Combinada Antineoplásica , Bevacizumab , Neoplasias dos Genitais Femininos , Paclitaxel , Humanos , Feminino , Paclitaxel/administração & dosagem , Paclitaxel/efeitos adversos , Paclitaxel/farmacocinética , Pessoa de Meia-Idade , Albuminas/administração & dosagem , Albuminas/efeitos adversos , Idoso , Neoplasias dos Genitais Femininos/tratamento farmacológico , Neoplasias dos Genitais Femininos/patologia , Bevacizumab/administração & dosagem , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacocinética , Imunoconjugados/uso terapêutico , Resultado do Tratamento , Dose Máxima TolerávelRESUMO
Omega-3 polyunsaturated fatty acids (n-3 PUFAs) enhance insulin sensitivity and glucose homeostasis in rodent models of insulin resistance. These beneficial effects have been linked with anti-inflammatory properties, but emerging data suggest that the mechanisms may also converge on mitochondria. We evaluated the influence of dietary n-3 PUFAs on mitochondrial physiology and muscle lipid metabolites in the context of high-fat diet (HFD) in mice. Mice were fed control diets (10% fat), HFD (60% fat), or HFD with fish oil (HFD+FO, 3.4% kcal from n-3 PUFAs) for 10 wk. Body mass and fat mass increased similarly in HFD and HFD+FO, but n-3 PUFAs attenuated the glucose intolerance that developed with HFD and increased expression of genes that regulate glucose metabolism in skeletal muscle. Despite similar muscle triglyceride levels in HFD and HFD+FO, long-chain acyl-CoAs and ceramides were lower in the presence of fish oil. Mitochondrial abundance and oxidative capacity were similarly increased in HFD and HFD+FO compared with controls. Hydrogen peroxide production was similarly elevated in HFD and HFD+FO in isolated mitochondria but not in permeabilized muscle fibers, likely due to increased activity and expression of catalase. These results support a hypothesis that n-3 PUFAs protect glucose tolerance, in part by preventing the accumulation of bioactive lipid mediators that interfere with insulin action. Furthermore, the respiratory function of skeletal muscle mitochondria does not appear to be a major factor in sphingolipid accumulation, glucose intolerance, or the protective effects of n-3 PUFAs.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Óleos de Peixe/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Peso Corporal/fisiologia , Dieta Hiperlipídica , Gorduras na Dieta/farmacologia , Metabolismo Energético/fisiologia , Intolerância à Glucose/metabolismo , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Distribuição AleatóriaRESUMO
Hyperandrogenism and chronic low-grade inflammation are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate inflammation. We determined the effect of oral androgen administration on fasting and glucose-stimulated nuclear factor-κB (NF-κB) activation and expression and related markers of inflammation in mononuclear cells (MNC) of lean reproductive-age women. Sixteen lean, ovulatory reproductive-age women were treated with 130 mg of DHEA or placebo (n = 8 each) for 5 days in a randomized, controlled, double-blind fashion. Nuclear activation of NF-κB, p65 and p105 NF-κB subunit RNA, TNFα and IL-1ß mRNA, and NF-κB p65 and inhibitory-κB (IκB) protein were quantified from MNC obtained while fasting and 2 h after glucose ingestion, before and after DHEA or placebo administration. Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any inflammatory markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-S, increased the percent change in fasting and glucose-challenged activated NF-κB, p65, p105, TNFα, and IL-1ß RNA and p65 protein, and decreased the percent change in fasting and glucose-challenged IκB protein. We conclude that elevation of circulating androgens to the range observed in PCOS upregulates the NF-κB inflammation pathway in lean reproductive-age women. Thus, hyperandrogenemia activates and sensitizes MNC to glucose in this population.
Assuntos
Citocinas/metabolismo , Regulação da Expressão Gênica , Hiperandrogenismo/imunologia , Hiperglicemia/etiologia , Leucócitos Mononucleares/imunologia , Adulto , Índice de Massa Corporal , Núcleo Celular/metabolismo , Citocinas/sangue , Citocinas/genética , Desidroepiandrosterona , Método Duplo-Cego , Feminino , Humanos , Hiperandrogenismo/sangue , Hiperandrogenismo/etiologia , Hiperandrogenismo/metabolismo , Proteínas I-kappa B/metabolismo , Leucócitos Mononucleares/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Síndrome do Ovário Policístico/fisiopatologia , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Congêneres da Testosterona/sangue , Adulto JovemRESUMO
Systemic insulin administration causes hypoaminoacidemia by inhibiting protein degradation, which may in turn inhibit muscle protein synthesis (PS). Insulin enhances muscle mitochondrial PS and ATP production when hypoaminoacidemia is prevented by exogenous amino acid (AA) replacement. We determined whether insulin would stimulate mitochondrial PS and ATP production in the absence of AA replacement. Using l-[1,2-¹³C]leucine as a tracer, we measured the fractional synthetic rate of mitochondrial as well as sarcoplasmic and mixed muscle proteins in 18 participants during sustained (7-h) insulin or saline infusion (n = 9 each). We also measured muscle ATP production, mitochondrial enzyme activities, mRNA levels of mitochondrial genes, and phosphorylation of signaling proteins regulating protein synthesis. The concentration of circulating essential AA decreased during insulin infusion. Mitochondrial, sarcoplasmic, and mixed muscle PS rates were also lower during insulin (2-7 h) than during saline infusions despite increased mRNA levels of selected mitochondrial genes. Under these conditions, insulin did not alter mitochondrial enzyme activities and ATP production. These effects were associated with enhanced phosphorylation of Akt but not of protein synthesis activators mTOR, p70(S6K), and 4EBP1. In conclusion, sustained physiological hyperinsulinemia without AA replacement did not stimulate PS of mixed muscle or protein subfractions and did not alter muscle mitochondrial ATP production in healthy humans. These results support that insulin and AA act in conjunction to stimulate muscle mitochondrial function and mitochondrial protein synthesis.
Assuntos
Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Insulina/metabolismo , Proteínas Mitocondriais/biossíntese , Músculo Esquelético/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Adulto , Aminoácidos/administração & dosagem , Isótopos de Carbono , Feminino , Regulação da Expressão Gênica , Humanos , Hiperinsulinismo/metabolismo , Infusões Intravenosas , Insulina/administração & dosagem , Insulina Regular Humana/administração & dosagem , Leucina/administração & dosagem , Leucina/metabolismo , Masculino , Mitocôndrias Musculares/enzimologia , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Músculo Esquelético/enzimologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/metabolismo , Serina-Treonina Quinases TOR/genética , Adulto JovemRESUMO
Introduction: Immune cell infiltration into the tumor microenvironment is generally associated with favorable clinical outcomes in solid tumors. However, the dynamic interplay among distinct immune cell subsets within the tumor-immune microenvironment as it relates to clinical responses to immunotherapy remains unresolved. In this study, we applied multiplex immunofluorescence (MxIF) to spatially characterize tumor-immune interactions within the metastatic melanoma lymph node. Methods: Pretreatment, whole lymph node biopsies were evaluated from 25 patients with regionally metastatic melanoma who underwent subsequent anti-PD1 therapy. Cyclic MxIF was applied to quantitatively and spatially assess expression of 45 pathologist-validated antibodies on a single tissue section. Pixel-based single cell segmentation and a supervised classifier approach resolved 10 distinct tumor, stromal and immune cell phenotypes and functional expression of PD1. Results: Single cell analysis across 416 pathologist-annotated tumor core regions of interest yielded 5.5 million cells for spatial evaluation. Cellular composition of tumor and immune cell subsets did not differ in the tumor core with regards to recurrence outcomes (p>0.05) however spatial patterns significantly differed in regional and paracrine neighborhood evaluations. Specifically, a regional community cluster comprised of primarily tumor and dendritic cells was enriched in patients that did not experience recurrence (p=0.009). By an independent spatial approach, cell-centric neighborhood analyses identified an enrichment for dendritic cells in cytotoxic T cell (CTL) and tumor cell-centric neighborhoods in the no recurrence patient response group (p<0.0001). Further evaluation of these neighborhoods identified an enrichment for CTL-dendritic cell interactions in patients that did not experience recurrence (p<0.0001) whereas CTL-macrophage interactions were more prevalent in CTL-centric neighborhoods of patients who experienced recurrence (p<0.0001). Discussion: Overall, this study offers a more comprehensive evaluation of immune infiltrates and spatial-immune signatures in the metastatic tumor-immune microenvironment as it informs recurrence risk following immunotherapy.
Assuntos
Melanoma , Segunda Neoplasia Primária , Humanos , Melanoma/tratamento farmacológico , Linfócitos T Citotóxicos , Imunoterapia , Linfonodos/patologia , Microambiente TumoralRESUMO
We investigated whether previously reported muscle mitochondrial dysfunction and altered gene transcript levels in type 2 diabetes might be secondary to abnormal blood glucose and insulin levels rather than an intrinsic defect of type 2 diabetes. A total of 13 type 2 diabetic and 17 nondiabetic subjects were studied on two separate occasions while maintaining similar insulin and glucose levels in both groups by 7-h infusions of somatostatin, low- or high-dose insulin (0.25 and 1.5 mU/kg of fat-free mass per min, respectively), and glucose. Muscle mitochondrial DNA abundance was not different between type 2 diabetic and nondiabetic subjects at both insulin levels, but the majority of transcripts in muscle that are involved mitochondrial functions were expressed at lower levels in type 2 diabetes at low levels of insulin. However, several gene transcripts that are specifically involved in the electron transport chain were expressed at higher levels in type 2 diabetic patients. After the low-dose insulin infusion, which achieved postabsorptive insulin levels, the muscle mitochondrial ATP production rate (MAPR) was not different between type 2 diabetic and nondiabetic subjects. However, increasing insulin to postprandial levels increased the MAPR in nondiabetic subjects but not in type 2 diabetic patients. The lack of MAPR increment in response to high-dose insulin in type 2 diabetic patients occurred in association with reduced glucose disposal and expression of peroxisome proliferator-activated receptor-gamma coactivator 1alpha, citrate synthase, and cytochrome c oxidase I. In conclusion, the current data supports that muscle mitochondrial dysfunction in type 2 diabetes is not an intrinsic defect, but instead a functional defect related to impaired response to insulin.
Assuntos
Glicemia/metabolismo , DNA Mitocondrial/genética , Diabetes Mellitus Tipo 2/genética , Perfilação da Expressão Gênica , Insulina/sangue , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/citologia , Transcrição Gênica , Biópsia , Glicemia/efeitos dos fármacos , Índice de Massa Corporal , Humanos , Infusões Intravenosas , Insulina/administração & dosagem , Insulina/farmacologia , Pessoa de Meia-Idade , Mitocôndrias Musculares/patologia , Músculo Esquelético/patologia , Valores de ReferênciaRESUMO
BACKGROUND: Hyperthyroidism causes a hypermetabolic state and skeletal muscle dysfunction, but the underlying mechanism remains incompletely defined. OBJECTIVE: The objective of the study was to determine whether treatment of hyperthyroidism causes changes in amino acid fluxes, synthesis rates of muscle proteins, and expression of muscle myosin heavy chain (MHC) that may impact skeletal muscle function and metabolic rate. METHODS: Eight hyperthyroid patients were studied (TSH 0.008 +/- 0.001 mU/liter) before treatment and at least 9 months after correction of hyperthyroidism (TSH 2.3 +/- 0.4) (P < 0.03). Fluxes of leucine and phenylalanine as well as muscle protein synthesis rates were measured using L[1,2 13C] leucine and L(15N) phenylalanine as tracers. mRNA levels of selected genes were measured in muscle biopsy samples. RESULTS: Treatment decreased resting metabolic rate that paralleled changes in fluxes of leucine and phenylalanine accompanied by improved muscle strength and mass. Synthesis rates of mixed muscle proteins (P = 0.01), sarcoplasmic (P = 0.04), and mitochondrial (P = 0.08) proteins decreased, whereas MHC synthesis was unchanged. Selective increases in mRNA abundance of muscle MHC1 isoform (P = 0.04) and decrease of MHCIIA (P = 0.007) and MHCIIx (P = 024) were observed. Muscle mitochondrial oxidative enzymes and mRNA levels of mitochondrial proteins were unchanged, but uncoupling protein2 and uncoupling protein3 mRNA levels (P = 0.02) decreased. CONCLUSION: Increased amino acid flux, mixed muscle protein synthesis, and synthesis of sarcoplasmic proteins are consistent with the hypermetabolic state in hyperthyroidism. After treatment, MHC synthesis rates were unchanged, but mRNA levels of isoforms of MHC found in slow-twitch and fast-twitch fibers increased and decreased, respectively. These results offer a mechanistic explanation for posttreatment improvement in muscle functions in hyperthyroidism.
Assuntos
Hipertireoidismo/tratamento farmacológico , Músculo Esquelético/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Antagonistas Adrenérgicos beta/uso terapêutico , Aminoácidos/sangue , Composição Corporal , Humanos , Radioisótopos do Iodo/uso terapêutico , Cinética , Leucina/sangue , Proteínas Musculares/biossíntese , Força Muscular , Fenilalanina/sangue , RNA Mensageiro/metabolismo , Radioisótopos/farmacocinética , Tiroxina/uso terapêuticoRESUMO
Insulin resistance increases and muscle oxidative capacity decreases during aging, but lifestyle changes-especially physical activity-may reverse these trends. Here we report the effect of a 16-week aerobic exercise program (n = 65) or control activity (n = 37) performed by men and women aged 21-87 years on insulin sensitivity and muscle mitochondria. Insulin sensitivity, measured by intravenous glucose tolerance test, decreased with age (r = -0.32) and was related to abdominal fat content (r = -0.65). Exercise increased peak oxygen uptake (VO(2peak); 10%), activity of muscle mitochondrial enzymes (citrate synthase and cytochrome c oxidase, 45-76%) and mRNA levels of mitochondrial genes (COX4, ND4, both 66%) and genes involved in mitochondrial biogenesis (PGC-1alpha, 55%; NRF-1, 15%; TFAM, 85%). Exercise also increased muscle GLUT4 mRNA and protein (30-52%) and reduced abdominal fat (5%) and plasma triglycerides (25%). None of these changes were affected by age. In contrast, insulin sensitivity improved in younger people but not in middle-aged or older groups. Thus, the muscle mitochondrial response to 4 months of aerobic exercise training was similar in all age-groups, although the older people did not have an improvement in insulin sensitivity.
Assuntos
Envelhecimento/fisiologia , Exercício Físico/fisiologia , Intolerância à Glucose/prevenção & controle , Resistência à Insulina/fisiologia , Proteínas Mitocondriais , Proteínas Musculares , Músculo Esquelético/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Glicemia , Composição Corporal , Citrato (si)-Sintase/genética , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Mellitus Tipo 2/prevenção & controle , Feminino , Expressão Gênica , Intolerância à Glucose/fisiopatologia , Transportador de Glucose Tipo 4 , Humanos , Insulina/sangue , Lipídeos/sangue , Masculino , Pessoa de Meia-Idade , Mitocôndrias/enzimologia , Proteínas de Transporte de Monossacarídeos/genética , Fator 1 Relacionado a NF-E2 , Proteínas Nucleares/genética , Fator 1 Nuclear Respiratório , Fatores Nucleares Respiratórios , Oxirredução , Prostaglandina-Endoperóxido Sintases/genética , Transativadores/genética , Fatores de Transcrição/genéticaRESUMO
Aging is associated with reduced muscle strength and atrophy of type II muscle fibers. Muscle fiber type and contractile function are primarily determined by myosin heavy chain (MHC) isoforms. There are few data available on the effects of aging on MHC isoform expression in humans. In the present study, we tested the hypothesis that MHC isoform protein composition and mRNA abundance would favor a fast-to-slow isoform shift with aging and in response to endurance exercise training. Muscle biopsies were obtained from previously sedentary, healthy men and women, aged 21-87 yr before (n = 77) and after (n = 65) 16 wk of bicycle training (up to 45 min at 80% peak heart rate, 3-4 days/wk). At baseline, MHC I mRNA was unchanged with age, whereas IIa and IIx declined by 14 and 10% per decade, respectively (P < 0.001). MHC IIa and IIx protein declined by 3 and 1% per decade with a reciprocal increase in MHC I (P < 0.05). After training, MHC I and IIa mRNA increased by 61 and 99%, respectively, and IIx decreased by 50% (all P < 0.001). The increase in MHC I mRNA was positively associated with age, whereas the changes in MHC IIa and IIx mRNA were similar across age. MHC I protein increased by 6% and was positively related to age, whereas IIx decreased by 5% and was inversely related to age. These results suggest that the altered expression of MHC isoforms with aging is transcriptionally regulated. In response to endurance exercise, regulation of MHC isoform transcripts remains robust in older muscle, but this did not result in corresponding changes in MHC protein expression.
Assuntos
Envelhecimento/fisiologia , Exercício Físico/fisiologia , Regulação da Expressão Gênica/fisiologia , Fibras Musculares Esqueléticas/fisiologia , Músculo Esquelético/fisiologia , Cadeias Pesadas de Miosina/metabolismo , Resistência Física/fisiologia , Adaptação Fisiológica/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/metabolismo , Aptidão Física/fisiologia , RNA Mensageiro/metabolismo , Ativação Transcricional/fisiologiaRESUMO
Acute aerobic exercise increases reactive oxygen species and could potentially damage proteins, but exercise training (ET) enhances mitochondrial respiration irrespective of age. Here, we report a differential impact of ET on protein quality in young and older participants. Using mass spectrometry we measured oxidative damage to skeletal muscle proteins before and after 8 weeks of ET and find that young but not older participants reduced oxidative damage to both total skeletal muscle and mitochondrial proteins. Young participants showed higher total and mitochondrial derived semitryptic peptides and 26S proteasome activity indicating increased protein degradation. ET however, increased the activity of the endogenous antioxidants in older participants. ET also increased skeletal muscle content of the mitochondrial deacetylase SIRT3 in both groups. A reduction in the acetylation of isocitrate dehydrogenase 2 was observed following ET that may counteract the effect of acute oxidative stress. In conclusion aging is associated with an inability to improve skeletal muscle and mitochondrial protein quality in response to ET by increasing degradation of damaged proteins. ET does however increase muscle and mitochondrial antioxidant capacity in older individuals, which provides increased buffering from the acute oxidative effects of exercise.
Assuntos
Exercício Físico/fisiologia , Mitocôndrias Musculares/fisiologia , Proteínas Mitocondriais/fisiologia , Músculo Esquelético/fisiologia , Estresse Oxidativo/fisiologia , Resistência Física/fisiologia , Acetilação , Adolescente , Adulto , Fatores Etários , Idoso , Feminino , Humanos , Masculino , Proteólise , Comportamento Sedentário , Adulto JovemRESUMO
CONTEXT: Insulin and essential amino acids (EAAs) regulate skeletal muscle protein synthesis, yet their independent effects on mitochondrial protein synthesis (MiPS) and oxidative function remain to be clearly defined. OBJECTIVE: The purpose of this study was to determine the effects of high or low insulin with or without EAAs on MiPS. DESIGN: Thirty participants were randomized to 3 groups of 10 each with each participant studied twice. Study groups comprised (1) low and high insulin, (2) low insulin with and without EAAs, and (3) high insulin with and without EAAs. SETTING: The study was conducted in an in-patient clinical research unit. PARTICIPANTS: Eligible participants were 18 to 45 years old, had a body mass index of <25 kg/m(2), and were free of diseases and medications that might impair mitochondrial function. INTERVENTION: Low (â¼ 6 µU/mL) and high (â¼ 40 µU/mL) insulin levels were maintained by iv insulin infusion during a somatostatin clamp while maintaining euglycemia (4.7-5.2 mM) and replacing GH and glucagon. The EAA infusion was 5.4% NephrAmine. l-[ring-(13)C6]Phenylalanine was infused, and muscle needle biopsies were performed. MAIN OUTCOMES: Muscle MiPS, oxidative enzymes, and plasma amino acid metabolites were measured. RESULTS: MiPS and oxidative enzyme activities did not differ between low and high insulin (MiPS: 0.07 ± 0.009 vs 0.07 ± 0.006%/h, P = .86) or between EAAs and saline during low insulin (MiPS: 0.05 ± 0.01 vs 0.07 ± 0.01, P = .5). During high insulin, EAAs in comparison with saline increased MiPS (0.1 ± 0.01 vs 0.06 ± 0.01, P < .05) and cytochrome c oxidase activity (P < .05) but not citrate synthase (P = .27). EAA infusion decreased (P < .05) the glucose infusion rates needed to maintain euglycemia during low (â¼ 40%) and high insulin (â¼ 24%). CONCLUSION: EAAs increased MiPS and oxidative enzyme activity only with high insulin concentrations.
Assuntos
Aminoácidos Essenciais/farmacologia , Hipoglicemiantes/farmacologia , Resistência à Insulina/fisiologia , Insulina/farmacologia , Mitocôndrias Musculares/metabolismo , Proteínas Musculares/biossíntese , Adolescente , Adulto , Aminoácidos Essenciais/metabolismo , Glicemia/metabolismo , Feminino , Humanos , Hipoglicemiantes/metabolismo , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Mitocôndrias Musculares/efeitos dos fármacos , Músculo Esquelético/metabolismo , Fenilalanina/sangue , Fenilalanina/farmacologia , Somatostatina/farmacologia , Adulto JovemRESUMO
CONTEXT: Hyperandrogenism and oxidative stress are related in polycystic ovary syndrome (PCOS), but it is unknown whether hyperandrogenemia can activate oxidative stress. OBJECTIVE: The purpose of this study was to determine the effect of oral androgen administration on fasting and glucose-stimulated leukocytic reactive oxygen species (ROS) generation, reduced nicotinamide adenine dinucleotide phosphate oxidase p47(phox) subunit gene expression, and plasma thiobarbituric acid-reactive substances (TBARS) in lean healthy reproductive-age women. PARTICIPANTS, DESIGN, AND SETTING: Sixteen lean healthy ovulatory reproductive-age women were treated with 130 mg dehydroepiandrosterone (DHEA) or placebo (n = 8 each) for 5 d in this randomized, controlled, double-blind study that was performed at an an academic medical center. MAIN OUTCOME MEASURES: Leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS were quantified in the fasting state and 2 h after glucose ingestion, before and after treatment. RESULTS: Before treatment, subjects receiving DHEA or placebo exhibited no differences in androgens or any prooxidant markers while fasting and after glucose ingestion. Compared with placebo, DHEA administration raised levels of testosterone, androstenedione, and DHEA-sulfate, increased the percent change in glucose-challenged p47(phox) RNA content, and increased the percent change in fasting and glucose-challenged ROS generation from mononuclear cells and polymorphonuclear cells, p47(phox) protein content, and plasma TBARS. CONCLUSION: Elevation of circulating androgens comparable to what is present in PCOS increases leukocytic ROS generation, p47(phox) gene expression, and plasma TBARS to promote oxidative stress in lean healthy reproductive-age women. Thus, hyperandrogenemia activates and sensitizes leukocytes to glucose in this population.
Assuntos
Hiperandrogenismo/metabolismo , Hiperglicemia/metabolismo , Leucócitos/metabolismo , Estresse Oxidativo , Adulto , Glicemia/análise , Composição Corporal , Desidroepiandrosterona/farmacologia , Sulfato de Desidroepiandrosterona/sangue , Método Duplo-Cego , Feminino , Humanos , NADPH Oxidases/genética , Síndrome do Ovário Policístico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Testosterona/sangueRESUMO
CONTEXT: Both training and normal body mass index are associated with high insulin sensitivity, but the mechanism may be different. OBJECTIVE: The aim of the study was to examine whether lean trained humans may be protected from acute free fatty acid (FFA)-induced insulin resistance compared with lean sedentary humans. DESIGN AND SETTING: We conducted an interventional trial using either a 6-h lipid (20% Intralipid at 90 ml/h) or glycerol (2.25 g/100 ml at 90 ml/h) infusion along with a concurrent hyperinsulinemic-euglycemic clamp and serial muscle biopsies (0, 120, 360 min) at a clinical research unit at the University of Minnesota. PATIENTS OR PARTICIPANTS: The study included lean endurance-trained (n = 14) and sedentary (n = 14) individuals matched for age, gender, and body mass index. MAIN OUTCOME MEASURES: We measured the decline in glucose infusion rate (GIR) during the hyperinsulinemic-euglycemic clamp. RESULTS: The trained group had higher baseline mitochondrial DNA copy number, mRNA of cytochrome C oxidase subunit 3, and insulin sensitivity (as measured by GIR) compared with the sedentary group. When FFA was acutely elevated to the upper physiological range (0.6-0.7 mEq/liter) by lipid infusion, the GIR in both activity groups declined similarly compared with their respective glycerol controls, although insulin signaling, as measured by Ser 473 pAKT/AKT, remained comparable. Specific to the trained group, the stimulatory effect of hyperinsulinemia on mitochondrial mRNA levels during the glycerol infusion was absent during the lipid infusion. CONCLUSIONS: Elevated FFA had similar effects in reducing insulin sensitivity in trained and sedentary humans. In trained participants, this decline was associated with alterations in the skeletal muscle mitochondrial mRNA response to hyperinsulinemia.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Resistência à Insulina , Resistência Física/fisiologia , Adulto , Feminino , Técnica Clamp de Glucose , Humanos , Insulina/sangue , Masculino , Músculo Esquelético/metabolismo , Consumo de Oxigênio , RNA Mensageiro/análise , Adulto JovemRESUMO
Caloric restriction (CR) mitigates many detrimental effects of aging and prolongs life span. CR has been suggested to increase mitochondrial biogenesis, thereby attenuating age-related declines in mitochondrial function, a concept that is challenged by recent studies. Here we show that lifelong CR in mice prevents age-related loss of mitochondrial oxidative capacity and efficiency, measured in isolated mitochondria and permeabilized muscle fibers. We find that these beneficial effects of CR occur without increasing mitochondrial abundance. Whole-genome expression profiling and large-scale proteomic surveys revealed expression patterns inconsistent with increased mitochondrial biogenesis, which is further supported by lower mitochondrial protein synthesis with CR. We find that CR decreases oxidant emission, increases antioxidant scavenging, and minimizes oxidative damage to DNA and protein. These results demonstrate that CR preserves mitochondrial function by protecting the integrity and function of existing cellular components rather than by increasing mitochondrial biogenesis.
Assuntos
Restrição Calórica , Mitocôndrias/metabolismo , Renovação Mitocondrial/fisiologia , Envelhecimento , Animais , DNA Mitocondrial/metabolismo , Regulação para Baixo , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Perfilação da Expressão Gênica , Camundongos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Estresse Oxidativo , Proteômica , TranscriptomaRESUMO
OBJECTIVE: To determine biological mechanisms involved in posttransplantation diabetes mellitus caused by the immunosuppressant tacrolimus (FK506). METHODS: INS-1 cells and isolated rat islets were incubated with vehicle or FK506 and harvested at 24-hr intervals. Cells were assessed for viability, apoptosis, proliferation, cell insulin secretion, and content. Gene expression studies by microarray analysis, quantitative polymerase chain reaction, and motifADE analysis of the microarray data identified potential FK506-mediated pathways and regulatory motifs. Mitochondrial functions, including cell respiration, mitochondrial content, and bioenergetics were assessed. RESULTS: Cell replication, viability, insulin secretion, oxygen consumption, and mitochondrial content were decreased (P<0.05) 1.2-, 1.27-, 1.77-, 1.32-, and 1.43-fold, respectively, after 48-hr FK506 treatment. Differences increased with time. FK506 (50 ng/mL) and cyclosporine A (800 ng/mL) had comparable effects. FK506 significantly decreased mitochondrial content and mitochondrial bioenergetics and showed a trend toward decreased oxygen consumption in isolated islets. Cell apoptosis and proliferation, mitochondrial DNA copy number, and ATP:ADP ratios were not significantly affected. Pathway analysis of microarray data showed FK506 modification of pathways involving ATP metabolism, membrane trafficking, and cytoskeleton remodeling. PGC1-α mRNA was down-regulated by FK506. MotifADE identified nuclear factor of activated T-cells, an important mediator of ß-cell survival and function, as a potential factor mediating both up- and down-regulation of gene expression. CONCLUSIONS: At pharmacologically relevant concentrations, FK506 decreases insulin secretion and reduces mitochondrial density and function without changing apoptosis rates, suggesting that posttransplantation diabetes induced by FK506 may be mediated by its effects on mitochondrial function.
Assuntos
Imunossupressores/toxicidade , Células Secretoras de Insulina/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Tacrolimo/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Linhagem Celular Tumoral , Ciclosporina/toxicidade , DNA Mitocondrial/análise , Perfilação da Expressão Gênica , Insulina/metabolismo , Secreção de Insulina , Mitocôndrias/fisiologia , RatosRESUMO
OBJECTIVE: To determine whether dehydroepiandrosterone (DHEA) replacement therapy in hypoadrenal women improves performance, muscle protein accretion, and mitochondrial functions. PARTICIPANTS AND METHODS: Thirty-three hypoadrenal women were enrolled in the study from May 1, 2002, through May 31, 2003. Twenty-eight completed a 12-week, prospective, randomized, placebo-controlled, crossover study with either daily placebo or 50 mg of DHEA with a 2-week washout period and then crossed over to the other treatment. Body composition, physical performance, whole-body and muscle protein metabolism, and mitochondrial functions were determined. RESULTS: Administration of DHEA significantly increased plasma levels of DHEA sulfate, testosterone, and androstenedione but did not change body composition, muscle strength, peak aerobic capacity, and whole-body protein turnover or synthesis rates of mitochondrial, sarcoplasmic, or mixed muscle proteins. Muscle mitochondrial oxidative enzymes and messenger RNA (mRNA) levels of genes encoding mitochondrial proteins and nuclear transcription factors did not change after DHEA administration. However, mRNA levels of muscle myosin heavy chain 1 (P=.004), which determines muscle fiber type, and those of insulinlike growth factor binding proteins 4 and 5 significantly decreased (P=.02 and P=.03, respectively). CONCLUSION: Three months of DHEA administration increased DHEA sulfate and androgen levels but had no effect on physical performance, body composition, protein metabolism, or muscle mitochondrial biogenesis in hypoadrenal women. However, lowering of mRNA levels of binding proteins of insulinlike growth factor 1 and myosin heavy chain 1 suggests potential effects of longterm treatment with DHEA on muscle fiber type.
Assuntos
Insuficiência Adrenal/tratamento farmacológico , Desidroepiandrosterona/uso terapêutico , Terapia de Reposição Hormonal , Músculo Esquelético/efeitos dos fármacos , Proteínas/efeitos dos fármacos , Androstenodiona/sangue , Composição Corporal/efeitos dos fármacos , Estudos Cross-Over , Sulfato de Desidroepiandrosterona/sangue , Feminino , Humanos , Pessoa de Meia-Idade , Mitocôndrias Musculares/efeitos dos fármacos , Mitocôndrias Musculares/fisiologia , Proteínas Mitocondriais/efeitos dos fármacos , Fibras Musculares Esqueléticas/efeitos dos fármacos , Proteínas Musculares/efeitos dos fármacos , Força Muscular/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Cadeias Pesadas de Miosina/efeitos dos fármacos , Oxirredutases/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Placebos , Estudos Prospectivos , Proteínas/metabolismo , RNA Mensageiro/efeitos dos fármacos , Retículo Sarcoplasmático/efeitos dos fármacos , Somatomedinas/efeitos dos fármacos , Testosterona/sangue , Fatores de Transcrição/efeitos dos fármacosRESUMO
OBJECTIVE: Type 2 diabetes has become a global epidemic, and Asian Indians have a higher susceptibility to diabetes than Europeans. We investigated whether Indians had any metabolic differences compared with Northern European Americans that may render them more susceptible to diabetes. RESEARCH DESIGN AND METHODS: We studied 13 diabetic Indians, 13 nondiabetic Indians, and 13 nondiabetic Northern European Americans who were matched for age, BMI, and sex. The primary comparisons were insulin sensitivity by hyperinsulinemic-euglycemic clamp and skeletal muscle mitochondrial capacity for oxidative phosphorylation (OXPHOS) by measuring mitochondrial DNA copy number (mtDNA), OXPHOS gene transcripts, citrate synthase activity, and maximal mitochondrial ATP production rate (MAPR). Other factors that may cause insulin resistance were also measured. RESULTS: The glucose infusion rates required to maintain identical glucose levels during the similar insulin infusion rates were substantially lower in diabetic Indians than in the nondiabetic participants (P < 0.001), and they were lower in nondiabetic Indians than in nondiabetic Northern European Americans (P < 0.002). mtDNA (P < 0.02), OXPHOS gene transcripts (P < 0.01), citrate synthase, and MAPR (P < 0.03) were higher in Indians irrespective of their diabetic status. Intramuscular triglyceride, C-reactive protein, interleukin-6, and tumor necrosis factor-alpha concentrations were higher, whereas adiponectin concentrations were lower in diabetic Indians. CONCLUSIONS: Despite being more insulin resistant, diabetic Indians had similar muscle OXPHOS capacity as nondiabetic Indians, demonstrating that diabetes per se does not cause mitochondrial dysfunction. Indians irrespective of their diabetic status had higher OXPHOS capacity than Northern European Americans, although Indians were substantially more insulin resistant, indicating a dissociation between mitochondrial dysfunction and insulin resistance.