Cardiometabolic Changes in Sirtuin1-Heterozygous Mice on High-Fat Diet and Melatonin Supplementation.

A hypercaloric fatty diet predisposes an individual to metabolic syndrome and cardiovascular complications. Sirtuin1 (SIRT1) belongs to the class III histone deacetylase family and sustains anabolism, mitochondrial biogenesis, and fat distribution. Epididymal white adipose tissue (eWAT) is involved in inflammation, whilst interscapular brown adipose tissue (iBAT) drives metabolism in obese rodents. Melatonin, a pineal indoleamine, acting as a SIRT1 modulator, may alleviate cardiometabolic damage. In the present study, we morphologically characterized the heart, eWAT, and iBAT in male heterozygous SIRT1+/- mice (HET mice) on a high-fat diet (60%E lard) versus a standard rodent diet (8.5% E fat) and drinking melatonin (10 mg/kg) for 16 weeks. Wild-type (WT) male C57Bl6/J mice were similarly fed for comparison. Cardiomyocyte fibrosis and endoplasmic reticulum (ER) stress response worsened in HET mice on a high-fat diet vs. other groups. Lipid peroxidation, ER, and mitochondrial stress were assessed by 4 hydroxy-2-nonenal (4HNE), glucose-regulated protein78 (GRP78), CCAA/enhancer-binding protein homologous protein (CHOP), heat shock protein 60 (HSP60), and mitofusin2 immunostainings. Ultrastructural analysis indicated the prevalence of atypical inter-myofibrillar mitochondria with short, misaligned cristae in HET mice on a lard diet despite melatonin supplementation. Abnormal eWAT adipocytes, crown-like inflammatory structures, tumor necrosis factor alpha (TNFα), and iBAT whitening characterized HET mice on a hypercaloric fatty diet and were maintained after melatonin supply. All these data suggest that melatonin's mechanism of action is strictly linked to full SIRT1 expression, which is required for the exhibition of effective antioxidant and anti-inflammatory properties.

Insulin Modulates the Bioenergetic and Thermogenic Capacity of Rat Brown Adipocytes In Vivo by Modulating Mitochondrial Mosaicism.

The effects of insulin on the bioenergetic and thermogenic capacity of brown adipocyte mitochondria were investigated by focusing on key mitochondrial proteins. Two-month-old male Wistar rats were treated acutely or chronically with a low or high dose of insulin. Acute low insulin dose increased expression of all electron transport chain complexes and complex IV activity, whereas high dose increased complex II expression. Chronic low insulin dose decreased complex I and cyt c expression while increasing complex II and IV expression and complex IV activity. Chronic high insulin dose decreased complex II, III, cyt c, and increased complex IV expression. Uncoupling protein (UCP) 1 expression was decreased after acute high insulin but increased following chronic insulin treatment. ATP synthase expression was increased after acute and decreased after chronic insulin treatment. Only a high dose of insulin increased ATP synthase activity in acute and decreased it in chronic treatment. ATPase inhibitory factor protein expression was increased in all treated groups. Confocal microscopy showed that key mitochondrial proteins colocalize differently in different mitochondria within a single brown adipocyte, indicating mitochondrial mosaicism. These results suggest that insulin modulates the bioenergetic and thermogenic capacity of rat brown adipocytes in vivo by modulating mitochondrial mosaicism.

Melatonin Efficacy in Obese Leptin-Deficient Mice Heart.

Cardiomyocytes are particularly sensitive to oxidative damage due to the link between mitochondria and sarcoplasmic reticulum necessary for calcium flux and contraction. Melatonin, important indoleamine secreted by the pineal gland during darkness, also has important cardioprotective properties. We designed the present study to define morphological and ultrastructural changes in cardiomyocytes and mainly in mitochondria of an animal model of obesity (ob/ob mice), when treated orally or not with melatonin at 100 mg/kg/day for 8 weeks (from 5 up to 13 week of life). We observed that ob/ob mice mitochondria in sub-sarcolemmal and inter-myofibrillar compartments are often devoid of cristae with an abnormally large size, which are called mega-mitochondria. Moreover, in ob/ob mice the hypertrophic cardiomyocytes expressed high level of 4hydroxy-2-nonenal (4HNE), a marker of lipid peroxidation but scarce degree of mitofusin2, indicative of mitochondrial sufferance. Melatonin oral supplementation in ob/ob mice restores mitochondrial cristae, enhances mitofusin2 expression and minimizes 4HNE and p62/SQSTM1, an index of aberrant autophagic flux. At pericardial fat level, adipose tissue depot strictly associated with myocardium infarction, melatonin reduces adipocyte hypertrophy and inversely regulates 4HNE and adiponectin expressions. In summary, melatonin might represent a safe dietary adjuvant to hamper cardiac mitochondria remodeling and the hypoxic status that occur in pre-diabetic obese mice at 13 weeks of life.

Hepatic Macrosteatosis Is Partially Converted to Microsteatosis by Melatonin Supplementation in ob/ob Mice Non-Alcoholic Fatty Liver Disease.

BACKGROUND: Obesity is a common risk factor for non-alcoholic fatty liver disease (NAFLD). Currently, there are no specific treatments against NAFLD. Thus, examining any molecule with potential benefits against this condition emerged melatonin as a molecule that influences metabolic dysfunctions. The aim of this study was to determine whether melatonin would function against NAFDL, studying morphological, ultrastuctural and metabolic markers that characterize the liver of ob/ob mice. METHODS: Lean and ob/ob mice were supplemented with melatonin in the drinking water for 8 weeks. Histology and stereology were performed to assess hepatic steatosis and glycogen deposition. Ultrastructural features of mitochondria, endoplasmic reticulum (ER) and their juxtapositions were evaluated in livers of all experimental groups. Furthermore, hepatic distribution and expression of markers of ER and mitochondria (calnexin, ATP sintase ß, GRP78 and CHOP) and metabolic dysfunction (RPB4, ß-catenin) and cellular longevity (SIRT1) were analyzed. RESULTS: Melatonin significantly reduced glycemia, identified also by a decrease of hepatic RBP4 expression, reversed macrosteatosis in microsteatosis at the hepatic pericentral zone, enlarged ER-mitochondrial distance and ameliorated the morphology and organization of these organelles in ob/ob mouse liver. Furthermore, in ob/ob mice, calnexin and ATP synthase ß were partially restored, GRP78 and CHOP decreased in periportal and midzonal hepatocytes and ß-catenin expression was, in part, restored in peripheral membranes of hepatocytes. Melatonin supplementation to ob/ob mice improves hepatic morphological, ultrastructural and metabolic damage that occurs as a result of NAFLD. CONCLUSIONS: Melatonin may be a potential adjuvant treatment to limit NAFLD and its progression into irreversible complications.

Long-term dietary L-arginine supplementation increases endothelial nitric oxide synthase and vasoactive intestinal peptide immunoexpression in rat small intestine.

BACKGROUND AND AIMS: Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term L-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. METHODS: Animals (six per group) acclimated to room temperature (22 ± 1 °C) and cold (4 ± 1 °C), respectively, were treated with 2.25% L-arginine, a substrate for NOSs, or with 0.01% N(ω)-nitro-L-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. RESULTS: Long-term dietary L-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. CONCLUSION: Our results demonstrate that long-term dietary L-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP.