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BACKGROUND: The diaphragm is the primary muscle of inspiration, and its dysfunction is frequent during sepsis. However, the mechanisms associated with sepsis and diaphragm dysfunction are not well understood. In this study, we evaluated the morphophysiological changes of the mitochondrial diaphragm 5 days after sepsis induction. METHODS: Male C57Bl/6 mice were divided into two groups, namely, cecal ligation and puncture (CLP, n = 26) and sham-operated (n = 19). Mice received antibiotic treatment 8 h after surgery and then every 24 h until 5 days after surgery when mice were euthanized and the diaphragms were collected. Also, diaphragm function was evaluated in vivo by ultrasound 120 h after CLP. The tissue fiber profile was evaluated by the expression of myosin heavy chain and SERCA gene by qPCR and myosin protein by using Western blot. The Myod1 and Myog expressions were evaluated by using qPCR. Diaphragm ultrastructure was assessed by electron microscopy, and mitochondrial physiology was investigated by high-resolution respirometry, Western blot, and qPCR. RESULTS: Cecal ligation and puncture mice developed moderated sepsis, with a 74% survivor rate at 120 h. The diaphragm mass did not change in CLP mice compared with control, but we observed sarcomeric disorganization and increased muscle thickness (38%) during inspiration and expiration (21%). Septic diaphragm showed a reduction in fiber myosin type I and IIb mRNA expression by 50% but an increase in MyHC I and IIb protein levels compared with the sham mice. Total and healthy mitochondria were reduced by 30% in septic mice, which may be associated with a 50% decrease in Ppargc1a (encoding PGC1a) and Opa1 (mitochondria fusion marker) expressions in the septic diaphragm. The small and non-functional OPA1 isoform also increased 70% in the septic diaphragm. These data suggest an imbalance in mitochondrial function. In fact, we observed downregulation of all respiratory chain complexes mRNA expression, decreased complex III and IV protein levels, and reduced oxygen consumption associated with ADP phosphorylation (36%) in CLP mice. Additionally, the septic diaphragm increased proton leak and downregulated Sod2 by 70%. CONCLUSION: The current model of sepsis induced diaphragm morphological changes, increased mitochondrial damage, and induced functional impairment. Thus, diaphragm damage during sepsis seems to be associated with mitochondrial dysfunction.
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Background: Sepsis can cause the nonthyroidal illness syndrome (NTIS), resulting in perturbed thyroid hormone (TH) signaling and reduced thyroxine (T4) levels. TH is a major regulator of muscle function, via its influence on mitochondria. This study aimed at evaluating the relationship between TH signaling, mitochondrial function, and the antioxidant defense system in the diaphragms of septic mice. Methods: Male C57Bl/6 mice were divided into two groups: cecal ligation and puncture (CLP) and sham. Twenty-four hours after surgery, plasma, diaphragms, and livers were collected. TH metabolism and responses were analyzed by measuring messenger RNA (mRNA) expression of Dio1 in the liver, and Thra, Thrb, Dio2, Slc16a10, and Slc16a2 (encodes MCT 10 and 8), in the diaphragm. T4 plasma levels were measured by radioimmunoassay. Damage to diaphragm mitochondria was assessed by electron microscopy and real-time polymerase chain reaction (qPCR), and function with oxygraphy. The diaphragm antioxidative defense system was examined by qPCR, analyzing superoxide dismutase (SOD) 1 (Sod1), mitochondrial superoxide dismutase (SOD 2; Sod2), extracellular superoxide dismutase (SOD 3; Sod3), glutathione peroxidase 1 (Gpx1), and catalase (Cat) expression. The effect of TH replacement was tested by treating the mice with T4 and triiodothyronine (T3) (CLP+TH) after surgery. Results: CLP mice presented reduced total plasma T4 concentrations, downregulated Dio1, and upregulated Il1b mRNA expression in the liver. CLP mice also displayed downregulated Thra, Thrb, Slc16a10, and Slc16a2 expression in the diaphragm, suggesting that TH signaling was compromised. The expression of Ppargc1a (encoding PGC1a) was downregulated, which correlated with the decrease in the number of total mitochondria, increase in the percentage of injured mitochondria, downregulation of respiratory chain complex 2 and 3 mRNA expression, and reduced maximal respiration. In addition, septic animals presented a three-fold increase in Ucp3 and G6pdh expression; downregulated Sod3, Gpx1, and Cat expression; and upregulated Sod2 expression, potentially due to elevated reactive oxygen species levels. The mitochondrial number and the percentage of injured mitochondrial were similar between sham and CLP+TH mice. Conclusions: Sepsis induced responses consistent with NTIS, resulted in mitochondrial damage and functional impairment, and modulated the expression of key antioxidant enzymes in the diaphragm. Thus, impaired diaphragm function during sepsis seems to involve altered local TH signaling, mitochondrial dysfunction, and oxidative stress defense.
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Diafragma/metabolismo , Mitocôndrias/metabolismo , Sepse/metabolismo , Transdução de Sinais/fisiologia , Hormônios Tireóideos/metabolismo , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Fígado/metabolismo , Camundongos , Estresse Oxidativo/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Estradiol has been used to prevent metabolic diseases, bone loss and menopausal symptoms, even though it might raise the risk of cancer. Metformin is usually prescribed for type 2 diabetes mellitus and lowers food intake and body mass while improving insulin resistance and the lipid profile. Ovariectomized rats show increased body mass, insulin resistance and changes in the lipid profile. Thus, the aim of this work was to evaluate whether metformin could prevent the early metabolic dysfunction that occurs early after ovariectomy. Female Wistar rats were divided into the following groups: SHAM-operated (SHAM), ovariectomized (OVX), ovariectomized + estradiol (OVX + E2) and ovariectomized + metformin (OVX + M). Treatment with metformin diminished approximately 50% of the mass gain observed in ovariectomized animals and reduced both the serum and hepatic triglyceride levels. The hepatic levels of phosphorylated AMP-activated protein kinase (pAMPK) decreased after OVX, and the expression of the inactive form of hepatic acetyl-CoA carboxylase (ACC) was also reduced. Metformin was able to increase the levels of pAMPK in the liver of OVX animals, sustaining the balance between the inactive and total forms of ACC. Estradiol effects were similar to those of metformin but with different proportions. Our results suggest that metformin ameliorates the early alterations of metabolic parameters and rescues hepatic AMPK phosphorylation and ACC inactivation observed in ovariectomized rats.
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NEW FINDINGS: What is the central question of this study? Is there sexual dimorphism in the occurrence of hepatic endoplasmic reticulum stress? What is the main finding and its importance? The transition from prepubertal to the adult age is associated with an increase in the unfolded protein response markers in the liver of male rats, which is probably due to an increase in serum testosterone levels. ABSTRACT: Male rodents present a higher predisposition to obesity and insulin resistance than females. These disorders have been associated with endoplasmic reticulum (ER) stress. To investigate a possible sexual dimorphism in the hepatic occurrence of ER stress, we evaluated the expression of ER stress markers in the livers of male and female rats in two phases of sexual development. In the first experimental model, male and female prepubertal and adult Wistar rats were used. Adult males presented higher body mass and greater mass of the adipose tissue and liver than adult females. Prepubertal animals presented no differences in these parameters between males and females. Despite this finding, the hepatic expression levels of Bip, Ire1α and Xbp1s mRNA were lower in prepubertal males than in females, while in adult animals, they did not differ between sexes. In the second experimental model, we anticipated the sexually mature phase by daily injections of testosterone propionate for 10 days in prepubertal males or by daily injections of oestradiol benzoate for 7 days in prepubertal females. Oestradiol administration in prepubertal females did not change any of the parameters evaluated. Testosterone administration to prepubertal males led to a higher body mass and greater expression of Bip, Ire1α, Atf4 and Xbp1s in the liver. These findings suggest that the increased ER stress predisposition observed in males during puberty is due to an increase in testosterone levels, indicating that ER stress is sexually dimorphic before puberty due to the lack of testosterone in males.
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Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/fisiologia , Hormônios Esteroides Gonadais/farmacologia , Fígado/metabolismo , Animais , Endorribonucleases/metabolismo , Estradiol/farmacologia , Feminino , Glucose/metabolismo , Proteínas de Choque Térmico/metabolismo , Fígado/efeitos dos fármacos , Masculino , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Wistar , Caracteres Sexuais , Maturidade Sexual , Testosterona/farmacologia , Proteína 1 de Ligação a X-Box/metabolismoRESUMO
Mitochondria play an important role in providing ATP for muscle contraction. Muscle physiology is compromised in Duchenne muscular dystrophy (DMD) and several studies have shown the involvement of bioenergetics. In this work we investigated the mitochondrial physiology in fibers from fast-twitch muscle (EDL) and slow-twitch muscle (soleus) in the mdx mouse model for DMD and in control C57BL/10J mice. In our study, multiple mitochondrial respiratory parameters were investigated in permeabilized muscle fibers from 12-week-old animals, a critical age where muscle regeneration is observed in the mdx mouse. Using substrates of complex I and complex II from the electron transport chain, ADP and mitochondrial inhibitors, we found in the mdx EDL, but not in the mdx soleus, a reduction in coupled respiration suggesting that ATP synthesis is affected. In addition, the oxygen consumption after addition of complex II substrate is reduced in mdx EDL; the maximal consumption rate (measured in the presence of uncoupler) also seems to be reduced. Mitochondria are involved in calcium regulation and we observed, using alizarin stain, calcium deposits in mdx muscles but not in control muscles. Interestingly, more calcium deposits were found in mdx EDL than in mdx soleus. These data provide evidence that in 12-week-old mdx mice, calcium is accumulated and mitochondrial function is disturbed in the fast-twitch muscle EDL, but not in the slow-twitch muscle soleus.
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Cálcio/metabolismo , Mitocôndrias/metabolismo , Contração Muscular/fisiologia , Fibras Musculares de Contração Rápida/metabolismo , Fibras Musculares de Contração Lenta/metabolismo , Distrofia Muscular Animal/patologia , Trifosfato de Adenosina/biossíntese , Animais , Complexo I de Transporte de Elétrons/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/patologia , Consumo de Oxigênio/fisiologia , Regeneração/fisiologiaRESUMO
The thyroid hormones (THs), triiodothyronine (T3) and thyroxine (T4), are very important in organism metabolism and regulate glucose utilization. Hexokinase (HK) is responsible for the first step of glycolysis, catalyzing the conversion of glucose to glucose 6-phosphate. HK has been found in different cellular compartments, and new functions have been attributed to this enzyme. The effects of hyperthyroidism on subcellular glucose phosphorylation in mouse tissues were examined. Tissues were removed, subcellular fractions were isolated from eu- and hyperthyroid (T3, 0.25 µg/g, i.p. during 21 days) mice and HK activity was assayed. Glucose phosphorylation was increased in the particulate fraction in soleus (312.4% ± 67.1, n = 10), gastrocnemius (369.2% ± 112.4, n = 10) and heart (142.2% ± 13.6, n = 10) muscle in the hyperthyroid group compared to the control group. Hexokinase activity was not affected in brain or liver. No relevant changes were observed in HK activity in the soluble fraction for all tissues investigated. Acute T3 administration (single dose of T3, 1.25 µg/g, i.p.) did not modulate HK activity. Interestingly, HK mRNA levels remained unchanged and HK bound to mitochondria was increased by T3 treatment, suggesting a posttranscriptional mechanism. Analysis of the AKT pathway showed a 2.5-fold increase in AKT and GSK3B phosphorylation in the gastrocnemius muscle in the hyperthyroid group compared to the euthyroid group. Taken together, we show for the first time that THs modulate HK activity specifically in particulate fractions and that this action seems to be under the control of the AKT and GSK3B pathways.
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Hexokinase (HK) is the first enzyme in the glycolytic pathway and is responsible for glucose phosphorylation and fixation into the cell. HK (HK-II) is expressed in skeletal muscle and can be found in the cytosol or bound mitochondria, where it can protect cells against insults such as oxidative stress. 4-Phenyl butyric acid (4-PBA) is a chemical chaperone that inhibits endoplasmic reticulum stress and contributes to the restoring of glucose homeostasis. AIMS: Here, we decided to investigate whether HK activity and its interaction with mitochondria could be a target of 4-PBA action. MAIN METHODS: L6 myotubes were treated with 1mM 4-PBA for 24, 48 or 72h. We evaluated HK activity, glucose and oxygen consumption, gene and protein expression. KEY FINDINGS: We found that L6 myotubes treated with 4-PBA presented more HK activity in the particulate fraction, increased glucose consumption and augmented Glut4, Hk2 and Vdac1 mRNA expression. Moreover, 4-PBA prevented the deleterious effect of antimycin-A on HK particulate activity. SIGNIFICANCE: Together, these results suggest a new role of 4-PBA in glucose metabolism that includes HK as a potential target of beneficial effect of 4-PBA.
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Glucose/metabolismo , Hexoquinase/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fenilbutiratos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Consumo de Oxigênio/efeitos dos fármacos , Ratos , Fatores de TempoRESUMO
Uncoupling proteins (UCP) are able to increase H(+) leakage across the inner mitochondrial membrane, thus dissipating the membrane potential and increasing oxygen consumption. Despite the identification of several UCP orthologs in birds, reptiles, amphibians and fish, little is known about their functional properties in fish. The aim of this work was to identify and characterize a UCP in mitochondria found in goldfish white skeletal muscle. Western blot analysis, using a polyclonal antibody raised against mammalian UCP3, showed a single band at approximately 32 kDa. During non-phosphorylating respiration, we observed that palmitate promoted a dose-dependent increase in oxygen consumption that is abolished by addition of BSA (fatty acid chelator). Interestingly, this palmitate-induced increase in oxygen consumption was not inhibited by GDP, a well-known UCP inhibitor. In phosphorylating mitochondria, palmitate lowered both ADP/O ratio (number of atoms of phosphorus incorporated as ATP per molecule of O2 consumed) and the respiratory control ratio. Moreover, we found that different fatty acids can modulate mitochondrial membrane potential. In conclusion, our results suggest that goldfish UCP is functionally similar to the UCP found in other species, including mammals.
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Proteínas de Peixes/metabolismo , Canais Iônicos/metabolismo , Mitocôndrias Musculares/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxigênio/fisiologia , Animais , Inibidores Enzimáticos/farmacologia , Proteínas de Peixes/antagonistas & inibidores , Proteínas de Peixes/química , Carpa Dourada , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/química , Mitocôndrias Musculares/química , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/química , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/química , Músculo Esquelético/química , Consumo de Oxigênio/efeitos dos fármacos , Ácido Palmítico/farmacologia , Proteína Desacopladora 3RESUMO
Goldfish have been used for cold acclimation studies, which have focused on changes in glycolytic and oxidative enzymes or alterations in lipid composition in skeletal muscle. Here we examine the effects of cold acclimation on the functional properties of isolated mitochondria and permeabilized fibers from goldfish white skeletal muscle, focusing on understanding the types of changes that occur in the mitochondrial respiratory states. We observed that cold acclimation promoted a significant increase in the mitochondrial oxygen consumption rates. Western blot analysis showed that UCP3 was raised by â¼1.5-fold in cold-acclimated muscle mitochondria. Similarly, we also evidenced a rise in the adenine nucleotide translocase content in cold-acclimated muscle mitochondria compared to warm-acclimated mitochondria (0.96±0.05 vs 0.68±0.02â nmol carboxyatractyloside mg(-1) protein). This was followed by a 2-fold increment in the citrate synthase activity, which suggests a higher mitochondrial content in cold-acclimated goldfish. Even with higher levels of UCP3 and ANT, the effects of activator (palmitate) and inhibitors (carboxyatractyloside and GDP) on mitochondrial parameters were similar in both warm- and cold-acclimated goldfish. Thus, we propose that cold acclimation in goldfish promotes an increase in functional oxidative capacity, with higher mitochondrial content without changes in the mitochondrial uncoupling pathways.
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HK (hexokinase) is an enzyme involved in the first step in the glucose metabolism pathway, converting glucose into G6P (glucose 6-phosphate). Owing to the importance of skeletal muscle for fish swimming and acclimation processes, we used goldfish (Carassius auratus L.) white muscle in order to investigate subcellular distribution and kinetics of HK. In this study, we report that HK activity is predominantly localized in the mitochondrial fraction [NC-HK (non-cytosolic HK)] in goldfish white muscle. Studies of the kinetic parameters revealed that the Km (Michaelis-Menten constant) for glucose was 0.41±0.03 mM and that for mannose was 3-fold lower, whereas the affinity for fructose was too low to be measured. The Km for ATP was 0.88±0.05 mM, whereas no activity was observed when either GTP or ITP was used as a phosphate donor. A moderate inhibition (20-40%) was found for ADP and AMP. Similar to mammalian HK, G6P and glucose analogues were able to promote an inhibition of between 85 and 100% of activity. Here, we found that acclimation of goldfish at 5°C promoted a 2.5-fold increase in NC-HK compared with its counterpart acclimated at 25°C. However, cytosolic HK activity was not altered after thermal acclimation. In summary, our results suggest that the goldfish has a constitutive NC-HK that shows some similarities to mammalian HK-II and, curiously, may play a role in the broad metabolic changes required during the cold acclimation process.