Bruno G. Teodoro

Melatonin prevents mitochondrial dysfunction and insulin resistance in rat skeletal muscle

Melatonin has a number of beneficial metabolic actions and reduced levels of melatonin may contribute to type 2 diabetes. The present study investigated the metabolic pathways involved in the effects of melatonin on mitochondrial function and insulin resistance in rat skeletal muscle. The effect of melatonin was tested both in vitro in isolated rats skeletal muscle cells and in vivo using pinealectomized rats (PNX). Insulin resistance was induced in vitro by treating primary rat skeletal muscle cells with palmitic acid for 24 hr. Insulin‐stimulated glucose uptake was reduced by palmitic acid followed by decreased phosphorylation of AKT which was prevented my melatonin. Palmitic acid reduced mitochondrial respiration, genes involved in mitochondrial biogenesis and the levels of tricarboxylic acid cycle intermediates whereas melatonin counteracted all these parameters in insulin‐resistant cells. Melatonin treatment increases CAMKII and p‐CREB but had no effect on p‐AMPK. Silencing of CREB protein by siRNA reduced mitochondrial respiration mimicking the effect of palmitic acid and prevented melatonin‐induced increase in p‐AKT in palmitic acid‐treated cells. PNX rats exhibited mild glucose intolerance, decreased energy expenditure and decreased p‐AKT, mitochondrial respiration, and p‐CREB and PGC‐1 alpha levels in skeletal muscle which were restored by melatonin treatment in PNX rats. In summary, we showed that melatonin could prevent mitochondrial dysfunction and insulin resistance via activation of CREB‐PGC‐1 alpha pathway. Thus, the present work shows that melatonin play an important role in skeletal muscle mitochondrial function which could explain some of the beneficial effects of melatonin in insulin resistance states.

Hydrogen peroxide production regulates the mitochondrial function in insulin resistant muscle cells: Effect of catalase overexpression

The mitochondrial redox state plays a central role in the link between mitochondrial overloading and insulin resistance. However, the mechanism by which the ROS induce insulin resistance in skeletal muscle cells is not completely understood. We examined the association between mitochondrial function and H2O2 production in insulin resistant cells. Our hypothesis is that the low mitochondrial oxygen consumption leads to elevated ROS production by a mechanism associated with reduced PGC1α transcription and low content of phosphorylated CREB. The cells were transfected with either the encoded sequence for catalase overexpression or the specific siRNA for catalase inhibition. After transfection, myotubes were incubated with palmitic acid (500μM) and the insulin response, as well as mitochondrial function and fatty acid metabolism, was determined. The low mitochondrial oxygen consumption led to elevated ROS production by a mechanism associated with β-oxidation of fatty acids. Rotenone was observed to reduce the ratio of ROS production. The elevated H2O2 production markedly decreased the PGC1α transcription, an effect that was accompanied by a reduced phosphorylation of Akt and CREB. The catalase transfection prevented the reduction in the phosphorylated level of Akt and upregulated the levels of phosphorylated CREB. The mitochondrial function was elevated and H2O2 production reduced, thus increasing the insulin sensitivity. The catalase overexpression improved mitochondrial respiration protecting the cells from fatty acid-induced, insulin resistance. This effect indicates that control of hydrogen peroxide production regulates the mitochondrial respiration preventing the insulin resistance in skeletal muscle cells by a mechanism associated with CREB phosphorylation and β-oxidation of fatty acids.

Creatine supplementation prevents fatty liver in rats fed choline-deficient diet: a burden of one-carbon and fatty acid metabolism.

AIM To examine the effects of creatine (Cr) supplementation on liver fat accumulation in rats fed a choline-deficient diet. METHODS Twenty-four rats were divided into 3 groups of 8 based on 4 weeks of feeding an AIN-93 control diet (C), a choline-deficient diet (CDD) or a CDD supplemented with 2% Cr. The CDD diet was AIN-93 without choline. RESULTS The CDD significantly increased plasma homocysteine and TNFα concentration, as well as ALT activity. In liver, the CDD enhanced concentrations of total fat (55%), cholesterol (25%), triglycerides (87%), MDA (30%), TNFα (241%) and decreased SAM concentrations (25%) and the SAM/SAH ratio (33%). Cr supplementation prevented all these metabolic changes, except for hepatic SAM and the SAM/SAH ratio. However, no changes in PEMT gene expression or liver phosphatidylcholine levels were observed among the three experimental groups, and there were no changes in hepatic triglyceride transfer protein (MTP) mRNA level. On the contrary, Cr supplementation normalized expression of the transcription factors PPARα and PPARγ that were altered by the CDD. Further, the downstream targets and fatty acids metabolism genes, UCP2, LCAD and CPT1a, were also normalized in the Cr group as compared to CDD-fed rats. CONCLUSION Cr supplementation prevented fat liver accumulation and hepatic injures in rats fed with a CDD for 4 weeks. Our results demonstrated that one-carbon metabolism may have a small role in mitigating hepatic fat accumulation by Cr supplementation. The modulation of key genes related to fatty acid oxidation pathway suggests a new mechanism by which Cr prevents liver fat accumulation.

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

Long‐chain acyl‐CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1‐α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism.

The combination of conjugated linoleic acid (CLA) and extra virgin olive oil increases mitochondrial and body metabolism and prevents CLA-associated insulin resistance and liver hypertrophy in C57Bl/6 mice

Clinical conditions associated with obesity can be improved by daily intake of conjugated linoleic acid (CLA) or extra virgin olive oil (EVOO). Here we investigated whether dietary supplementation with CLA and EVOO, either alone or in combination, changes body metabolism associated with mitochondrial energetics. Male C57Bl/6 mice were divided into one of four groups: CLA (1:1 cis-9, trans-11:trans-10, cis-12; 18:2 isomers), EVOO, CLA plus EVOO or control (linoleic acid). Each mouse received 3 g/kg body weight of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA, alone or in combination with EVOO: (a) reduced the white adipose tissue gain; (b) increased body VO2 consumption, VCO2 production and energy expenditure; (c) elevated uncoupling protein (UCP)-2 expression and UCP activity in isolated liver mitochondria. This organelle, when energized with NAD(+)-linked substrates, produced high amounts of H2O2 without inducing oxidative damage. Dietary supplementation with EVOO alone did not change any metabolic parameter, but supplementation with CLA itself promoted insulin resistance and elevated weight, lipid content and acetyl-CoA carboxylase-1 expression in liver. Interestingly, the in vivo antioxidant therapy with N-acetylcysteine abolished the CLA-induced rise of body metabolism and liver UCP expression and activity, while the in vitro antioxidant treatment with catalase mitigated the CLA-dependent UCP-2 expression in hepatocytes; these findings suggest the participation of an oxidative-dependent pathway. Therefore, this study clarifies the mechanisms by which CLA induces liver UCP expression and activity, and demonstrates for the first time the beneficial effects of combined CLA and EVOO supplementation.

Attenuation of obesity and insulin resistance by fish oil supplementation is associated with improved skeletal muscle mitochondrial function in mice fed a high-fat diet.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported to improve insulin sensitivity and glucose homeostasis in animal models of insulin resistance, but the involved mechanisms still remain unresolved. In this study, we evaluated the effects of fish oil (FO), a source of n-3 PUFAs, on obesity, insulin resistance and muscle mitochondrial function in mice fed a high-fat diet (HFD). C57Bl/6 male mice, 8 weeks old, were divided into four groups: control diet (C), high-fat diet (H), C+FO (CFO) and H+FO (HFO). FO was administered by oral gavage (2 g/kg b.w.), three times a week, starting 4 weeks before diet administration until the end of the experimental protocol. HFD-induced obesity and insulin resistance associated with impaired skeletal muscle mitochondrial function, as indicated by decreased oxygen consumption, tricarboxylic acid cycle intermediate (TCAi) contents (citrate, α-ketoglutarate, malate and oxaloacetate), oxidative phosphorylation protein content and mitochondrial biogenesis. These effects were associated with elevated reactive oxygen species production, decreased PGC1-a transcription and reduced Akt phosphorylation. The changes induced by the HFD were partially attenuated by FO, which decreased obesity and insulin resistance and increased mitochondrial function. In the H group, FO supplementation also improved oxygen consumption; increased TCAi content, and Akt and AMPK phosphorylation; and up-regulated mRNA expression of Gpat1, Pepck, catalase and mitochondrial proteins (Pgc1α, Pparα, Cpt1 and Ucp3). These results suggest that dietary FO attenuates the deleterious effects of the HFD (obesity and insulin resistance) by improving skeletal muscle mitochondrial function.

Attenuation of Ca2+ homeostasis, oxidative stress, and mitochondrial dysfunctions in diabetic rat heart: insulin therapy or aerobic exercise?

We tested the effects of swimming training and insulin therapy, either alone or in combination, on the intracellular calcium ([Ca(2+)]i) homeostasis, oxidative stress, and mitochondrial functions in diabetic rat hearts. Male Wistar rats were separated into control, diabetic, or diabetic plus insulin groups. Type 1 diabetes mellitus was induced by streptozotocin (STZ). Insulin-treated groups received 1 to 4 UI of insulin daily for 8 wk. Each group was divided into sedentary or exercised rats. Trained groups were submitted to swimming (90 min/day, 5 days/wk, 8 wk). [Ca(2+)]i transient in left ventricular myocytes (LVM), oxidative stress in LV tissue, and mitochondrial functions in the heart were assessed. Diabetes reduced the amplitude and prolonged the times to peak and to half decay of the [Ca(2+)]i transient in LVM, increased NADPH oxidase-4 (Nox-4) expression, decreased superoxide dismutase (SOD), and increased carbonyl protein contents in LV tissue. In isolated mitochondria, diabetes increased Ca(2+) uptake, susceptibility to permeability transition pore (MPTP) opening, uncoupling protein-2 (UCP-2) expression, and oxygen consumption but reduced H2O2 release. Swimming training corrected the time course of the [Ca(2+)]i transient, UCP-2 expression, and mitochondrial Ca(2+) uptake. Insulin replacement further normalized [Ca(2+)]i transient amplitude, Nox-4 expression, and carbonyl content. Alongside these benefits, the combination of both therapies restored the LV tissue SOD and mitochondrial O2 consumption, H2O2 release, and MPTP opening. In conclusion, the combination of swimming training with insulin replacement was more effective in attenuating intracellular Ca(2+) disruptions, oxidative stress, and mitochondrial dysfunctions in STZ-induced diabetic rat hearts.

Body energy metabolism and oxidative stress in mice supplemented with conjugated linoleic acid (CLA) associated to oleic acid.

INTRODUCTION Some fatty acids may play an important role in regulating metabolism through PPARs activation. Conjugated linoleic acid (CLA) has been shown to reduce body fat accumulation and increase body metabolism; this effect has been associated with up-regulation of mitochondrial uncoupling proteins (UCPs) and PPARalfa activation. Oleic acid has shown beneficial effects on health, decreasing oxidative stress and improving clinical conditions related to obesity. Therefore, in this work, we addressed the effects of a oleic plus CLA-supplemented murine diet on body metabolism, mitochondrial energetics and oxidative stress in the liver, as well as on other associated morphological and functional parameters in C57BL/6 mice. METHOD The diet was supplemented with 2% CLA mixture (cis-9, trans-10 and trans-10, cis-12 isomers; 45% of each isomer) and/or 0.7% olive oil on alternating days (60 days) by gavage. RESULTS The results showed that diet supplementation with CLA increases body metabolism and reduces lipid accumulation in adipose tissues. Groups that received oleic acid (oleic and CLA oleic) showed decreased levels of total cholesterol and cholesterol non-HDL, and increased levels of HDL-cholesterol. Livers of mice fed a diet supplemented with CLA showed high levels UCP2 mRNA, and the isolated hepatic mitochondria showed indications of UCP activity and increased ROS generation. Oleic acid partially reversed the lower lipid accumulation increasing PPARgamma content, reversed the higher ROS generation by liver mitochondria and improved liver oxidative status. CONCLUSIONS These results indicate a beneficial and secure dose of CLA and oleic acid for diet supplementation in mice, which increases body metabolism inducing UCP2 overexpression/activity in liver while preserving the redox state of the liver. Therefore, diet supplementation with CLA associated to oleic acid may be regarded as a potential strategy for controlling obesity and oxidative stress. Supported by FAPESP.

Effects of intermittent dietary supplementation with conjugated linoleic acid and fish oil (EPA/DHA) on body metabolism and mitochondrial energetics in mice

Understanding the mitochondrial processes that contribute to body energy metabolism may provide an attractive therapeutic target for obesity and co-morbidities. Here we investigated whether intermittent dietary supplementation with conjugated linoleic (CLA, 18:2n-6), docosahexaenoic (22:6n-3, DHA) and eicosapentaenoic (20:5n-3, EPA) acids, either alone or in combination, changes body metabolism associated with mitochondrial functions in the brain, liver, skeletal muscle and brown adipose tissue (BAT). Male C57Bl/6 mice were divided into groups: CLA (50% cis-9, trans-11; 50% trans-10, cis-12), EPA/DHA (64% EPA; 28% DHA), CLA plus EPA/DHA or control (linoleic acid). Each mouse received 3 g/kg b.w. of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA or EPA/DHA increased body VO2 consumption, VCO2 production and energy expenditure, being fish oil (FO) the most potent even in combination with CLA. Individually, both oils reduced mitochondrial density in BAT. CLA supplementation alone also a) elevated the expression of uncoupling proteins in soleus, liver and hippocampus and the uncoupling activity in the last two, ad this effect was associated with reduced hydrogen peroxide production in hippocampus; b) increased proteins related to mitochondrial fission in liver. EPA/DHA supplementation alone also a) induced mitochondrial biogenesis in liver, soleus and hippocampus associated with increased expression of PGC1-α; b) induced proteins related to mitochondrial fusion in the liver, and fission and fusion in the hippocampus. Therefore, this study shows changes on mitochondrial mechanisms induced by CLA and/or EPA/DHA that can be associated with elevated body energy expenditure.

Efeito da dieta hiperlipídica e do treinamento aeróbico na aterosclerose em camundongos apoE

INTRODUCAO: A populacao brasileira tem seguido a tendencia dos paises desenvolvidos, cuja caracteristica e o aumento do consumo de gorduras e sedentarismo. OBJETIVO: Investigar os efeitos da dieta hiperlipidica e do treinamento sobre o peso corporal, o consumo alimentar, a massa corporal magra, os lipidios plasmaticos, o peso do figado e a aterosclerose em camundongos com deficiencia de apolipoproteina E (apoE-/-). METODOS: vinte e seis camundongos foram divididos em grupos/dieta: NS (normolipidica e sedentario), HS (hiperlipidica e sedentario), NT (normolipidica e treinamento) e HT (hiperlipidica e treinamento). Aferiu-se peso corporal (PC), consumo alimentar (CA) e peso relativo do figado (PRF). Calculou-se a agua da carcaca (A%) pela diferenca do peso pre e pos-secagem, gordura (G%) por Soxhlet e proteina (P%) por Kjeldahl. Determinaram-se colesterol total (CT), triglicerideos (TG) e HDL por metodo enzimatico-colorimetrico e LDL e VLDL pela formula de Friedewald. Analisou-se a aterosclerose por fotomicrografia. Utilizou-se ANOVA e o metodo de Duncan, com P 0,05). A dieta hiperlipidica aumentou o CA (365,3 ± 5,09 vs. 340,16 ± 6,32) e LDL (452,20 ± 114,63 vs. 329,60 ± 77,54) nos animais sedentarios e o CT (858,75 ± 140,84 vs. 320,87 ± 157,81) e o LDL (839,36±139,94 vs. 282,66±166,92) nos treinados (P < 0,05). Ja o treinamento aumentou o CA (350,4 ± 7,81 vs. 340,16 ± 6,32) e A% (64,23 ± 2,46 vs. 62,62 ± 1,47) nos camundongos normolipidicos e diminuiu o CA (336,68 ± 6,23 vs. 365,3 ± 5,09), TG (22,75 ± 12,36 vs. 66,00 ± 21,88), HDL (14,83 ± 3,95 vs. 37,60 ± 13,12), VLDL (4,55 ± 2,47 vs. 13,20 ± 4,37) e aterosclerose (0,35 ± 0,13 vs. 0,56 ± 0,12) nos hiperlipidicos (P < 0,05). CONCLUSAO: A dieta hiperlipidica pode desencadear disturbios no metabolismo lipidico e, com isso, desencadear doencas cardiovasculares; porem, associada ao treinamento, pode diminuir a aterosclerose.