Maternal obesity increases hypothalamic miR-505-5p expression in mouse offspring leading to altered fatty acid sensing and increased intake of high-fat food.

In utero exposure to maternal obesity programs increased obesity risk. Animal models show that programmed offspring obesity is preceded by hyperphagia, but the mechanisms that mediate these changes are unknown. Using a mouse model of maternal obesity, we observed increased intake of a high-fat diet (HFD) in offspring of obese mothers that precedes the development of obesity. Through small RNA sequencing, we identified programmed overexpression of hypothalamic miR-505-5p that is established in the fetus, lasts to adulthood and is maintained in hypothalamic neural progenitor cells cultured in vitro. Metabolic hormones and long-chain fatty acids associated with obesity increase miR-505-5p expression in hypothalamic neurons in vitro. We demonstrate that targets of miR-505-5p are enriched in fatty acid metabolism pathways and overexpression of miR-505-5p decreased neuronal fatty acid metabolism in vitro. miR-505-5p targets are associated with increased BMI in human genetic studies. Intra-cerebroventricular injection of miR-505-5p in wild-type mice increased HFD intake, mimicking the phenotype observed in offspring exposed to maternal obesity. Conversely, maternal exercise intervention in an obese mouse pregnancy rescued the programmed increase of hypothalamic miR-505-5p in offspring of obese dams and reduced HFD intake to control offspring levels. This study identifies a novel mechanism by which maternal obesity programs obesity in offspring via increased intake of high-fat foods.

Programming of cardiac metabolism by miR-15b-5p, a miRNA released in cardiac extracellular vesicles following ischemia-reperfusion injury.

OBJECTIVE: We investigated the potential involvement of miRNAs in the developmental programming of cardiovascular diseases (CVD) by maternal obesity. METHODS: Serum miRNAs were measured in individuals from the Helsinki Birth Cohort (with known maternal body mass index), and a mouse model was used to determine causative effects of maternal obesity during pregnancy and ischemia-reperfusion on offspring cardiac miRNA expression and release. RESULTS: miR-15b-5p levels were increased in the sera of males born to mothers with higher BMI and in the hearts of adult mice born to obese dams. In an ex-vivo model of perfused mouse hearts, we demonstrated that cardiac tissue releases miR-15b-5p, and that some of the released miR-15b-5p was contained within small extracellular vesicles (EVs). We also demonstrated that release was higher from hearts exposed to maternal obesity following ischaemia/reperfusion. Over-expression of miR-15b-5p in vitro led to loss of outer mitochondrial membrane stability and to repressed fatty acid oxidation in cardiomyocytes. CONCLUSIONS: These findings suggest that miR-15-b could play a mechanistic role in the dysregulation of cardiac metabolism following exposure to an in utero obesogenic environment and that its release in cardiac EVs following ischaemic damage may be a novel factor contributing to inter-organ communication between the programmed heart and peripheral tissues.

The metabolic response of human trophoblasts derived from term placentas to metformin.

AIMS/HYPOTHESIS: Metformin is increasingly used therapeutically during pregnancy worldwide, particularly in the treatment of gestational diabetes, which affects a substantial proportion of pregnant women globally. However, the impact on placental metabolism remains unclear. In view of the association between metformin use in pregnancy and decreased birthweight, it is essential to understand how metformin modulates the bioenergetic and anabolic functions of the placenta. METHODS: A cohort of 55 placentas delivered by elective Caesarean section at term was collected from consenting participants. Trophoblasts were isolated from the placental samples and treated in vitro with clinically relevant doses of metformin (0.01 mmol/l or 0.1 mmol/l) or vehicle. Respiratory function was assayed using high-resolution respirometry to measure oxygen concentration and calculated [Formula: see text]. Glycolytic rate and glycolytic stress assays were performed using Agilent Seahorse XF assays. Fatty acid uptake and oxidation measurements were conducted using radioisotope-labelled assays. Lipidomic analysis was conducted using LC-MS. Gene expression and protein analysis were performed using RT-PCR and western blotting, respectively. RESULTS: Complex I-supported oxidative phosphorylation was lower in metformin-treated trophoblasts (0.01 mmol/l metformin, 61.7% of control, p<0.05; 0.1 mmol/l metformin, 43.1% of control, p<0.001). The proton efflux rate arising from glycolysis under physiological conditions was increased following metformin treatment, up to 23±5% above control conditions following treatment with 0.1 mmol/l metformin (p<0.01). There was a significant increase in triglyceride concentrations in trophoblasts treated with 0.1 mmol/l metformin (p<0.05), particularly those of esters of long-chain polyunsaturated fatty acids. Fatty acid oxidation was reduced by ~50% in trophoblasts treated with 0.1 mmol/l metformin compared with controls (p<0.001), with no difference in uptake between treatment groups. CONCLUSIONS/INTERPRETATION: In primary trophoblasts derived from term placentas metformin treatment caused a reduction in oxidative phosphorylation through partial inactivation of complex I and potentially by other mechanisms. Metformin-treated trophoblasts accumulate lipids, particularly long- and very-long-chain polyunsaturated fatty acids. Our findings raise clinically important questions about the balance of risk of metformin use during pregnancy, particularly in situations where the benefits are not clear-cut and alternative therapies are available.

Large-scale exome sequence analysis identifies sex- and age-specific determinants of obesity.

Obesity contributes substantially to the global burden of disease and has a significant heritable component. Recent large-scale exome sequencing studies identified several genes in which rare, protein-coding variants have large effects on adult body mass index (BMI). Here we extended such work by performing sex-stratified associations in the UK Biobank study (N∼420,000). We identified genes in which rare heterozygous loss-of-function increases adult BMI in women (DIDO1, PTPRG, and SLC12A5) and in men (SLTM), with effect sizes up to ∼8 kg/m2. This is complemented by analyses implicating rare variants in OBSCN and MADD for recalled childhood adiposity. The known functions of these genes, as well as findings of common variant genome-wide pathway enrichment analyses, suggest a role for neuron death, apoptosis, and DNA damage response mechanisms in the susceptibility to obesity across the life-course. These findings highlight the importance of considering sex-specific and life-course effects in the genetic regulation of obesity.

Sex-specific effects of maternal metformin intervention during glucose-intolerant obese pregnancy on body composition and metabolic health in aged mouse offspring.

AIMS/HYPOTHESIS: Metformin is increasingly used to treat gestational diabetes (GDM) and pregnancies complicated by pregestational type 2 diabetes or polycystic ovary syndrome but data regarding long-term offspring outcome are lacking in both human studies and animal models. Using a mouse model, this study investigated the effects of maternal metformin intervention during obese glucose-intolerant pregnancy on adiposity, hepatic steatosis and markers of metabolic health of male and female offspring up to the age of 12 months. METHODS: C57BL/6J female mice were weaned onto either a control diet (Con) or, to induce pre-conception obesity, an obesogenic diet (Ob). The respective diets were maintained throughout pregnancy and lactation. These obese dams were then randomised to the untreated group or to receive 300 mg/kg oral metformin hydrochloride treatment (Ob-Met) daily during pregnancy. In male and female offspring, body weights and body composition were measured from 1 month until 12 months of age, when serum and tissues were collected for investigation of adipocyte cellularity (histology), adipose tissue inflammation (histology and quantitative RT-PCR), and hepatic steatosis and fibrosis (histochemistry and modified Folch assay). RESULTS: At 12 months of age, male Ob and Ob-Met offspring showed increased adiposity, adipocyte hypertrophy, elevated expression of proinflammatory genes, hyperleptinaemia and hepatic lipid accumulation compared with Con offspring. Male Ob-Met offspring failed to show hyperplasia between 8 weeks and 12 months, indicative of restricted adipose tissue expansion, resulting in increased immune cell infiltration and ectopic lipid deposition. Female Ob offspring were relatively protected from these phenotypes but Ob-Met female offspring showed increased adiposity, adipose tissue inflammation, hepatic lipid accumulation, hyperleptinaemia and hyperinsulinaemia compared with Con female offspring. CONCLUSIONS/INTERPRETATION: Maternal metformin treatment of obese dams increased offspring metabolic risk factors in a sex- and age-dependent manner. These observations highlight the importance of following up offspring of both sexes beyond early adulthood after interventions during pregnancy. Our findings illustrate the complexity of balancing short-term benefits to mother and child vs any potential long-term metabolic effects on the offspring when prescribing therapeutic agents that cross the placenta.

Maternal diet-induced obesity during pregnancy alters lipid supply to mouse E18.5 fetuses and changes the cardiac tissue lipidome in a sex-dependent manner.

Maternal obesity during pregnancy has immediate and long-term detrimental effects on the offspring heart. In this study, we characterized the cardiac and circulatory lipid profiles in late gestation E18.5 fetuses of diet-induced obese pregnant mice and established the changes in lipid abundance and fetal cardiac transcriptomics. We used untargeted and targeted lipidomics and transcriptomics to define changes in the serum and cardiac lipid composition and fatty acid metabolism in male and female fetuses. From these analyses we observed: (1) maternal obesity affects the maternal and fetal serum lipidome distinctly; (2) female fetal heart lipidomes are more sensitive to maternal obesity than males; (3) changes in lipid supply might contribute to early expression of lipolytic genes in mouse hearts exposed to maternal obesity. These results highlight the existence of sexually dimorphic responses of the fetal heart to the same in utero obesogenic environment and identify lipids species that might mediate programming of cardiovascular health.

Effects of maternal diet-induced obesity on metabolic disorders and age-associated miRNA expression in the liver of male mouse offspring.

OBJECTIVE: This study investigated the effect of maternal obesity on aged-male offspring liver phenotype and hepatic expression of a programmed miRNA. METHODS: A mouse model (C57BL/6 J) of maternal diet-induced obesity was used to investigate fasting-serum metabolites, hepatic lipid content, steatosis, and relative mRNA levels (RT-PCR) and protein expression (Western blotting) of key components involved in hepatic and mitochondrial metabolism in 12-month-old offspring. We also measured hepatic lipid peroxidation, mitochondrial content, fibrosis stage, and apoptosis in the offspring. To investigate potential mechanisms leading to the observed phenotype, we also measured the expression of miR-582 (a miRNA previously implicated in liver cirrhosis) in 8-week-old and 12-month-old offspring. RESULTS: Body weight and composition was similar between 8-week-old offspring, however, 12-month-old offspring from obese mothers had increased body weight and fat mass (19.5 ± 0.8 g versus 10.4 ± 0.9 g, p < 0.001), as well as elevated serum levels of LDL and leptin and hepatic lipid content (21.4 ± 2.1 g versus 12.9 ± 1.8 g, p < 0.01). This was accompanied by steatosis, increased Bax/Bcl-2 ratio, and overexpression of p-SAPK/JNK, Tgfß1, Map3k14, and Col1a1 in the liver. Decreased levels of Bcl-2, p-AMPKα, total AMPKα and mitochondrial complexes were also observed. Maternal obesity was associated with increased hepatic miR-582-3p (p < 0.001) and miR-582-5p (p < 0.05). Age was also associated with an increase in both miR-582-3p and miR-582-5p, however, this was more pronounced in the offspring of obese dams, such that differences were greater in 12-month-old animals (-3p: 7.34 ± 1.35 versus 1.39 ± 0.50, p < 0.0001 and -5p: 4.66 ± 1.16 versus 1.63 ± 0.65, p < 0.05). CONCLUSION: Our findings demonstrate that maternal diet-induced obesity has detrimental effects on offspring body composition as well as hepatic phenotype that may be indicative of accelerated-ageing phenotype. These whole-body and cellular phenotypes were associated with age-dependent changes in expression of miRNA-582 that might contribute mechanistically to the development of metabolic disorders in the older progeny.

Clinical Utility of microRNAs in Exhaled Breath Condensate as Biomarkers for Lung Cancer.

This study represents a novel proof of concept of the clinical utility of miRNAs from exhaled breath condensate (EBC) as biomarkers of lung cancer (LC). Genome-wide miRNA profiling and machine learning analysis were performed on EBC from 21 healthy volunteers and 21 LC patients. The levels of 12 miRNAs were significantly altered in EBC from LC patients where a specific signature of miR-4507, miR-6777-5p and miR-451a distinguished these patients with high accuracy. Besides, a distinctive miRNA profile between LC adenocarcinoma and squamous cell carcinoma was observed, where a combined panel of miR-4529-3p, miR-8075 and miR-7704 enabling discrimination between them. EBC levels of miR-6777-5p, 6780a-5p and miR-877-5p predicted clinical outcome at 500 days. Two additional miRNA signatures were also associated with other clinical features such as stage and invasion status. Dysregulated EBC miRNAs showed potential target genes related to LC pathogenesis, including CDKN2B, PTEN, TP53, BCL2, KRAS and EGFR. We conclude that EBC miRNAs might allow the identification, stratification and monitorization of LC, which could lead to the development of precision medicine in this and other respiratory diseases.

Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark.

AIMS/HYPOTHESIS: Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. METHODS: miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic-hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. RESULTS: The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. CONCLUSIONS/INTERPRETATION: Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target.

Impact of maternal obesity on placental transcriptome and morphology associated with fetal growth restriction in mice.

BACKGROUND: In utero exposure to obesity is consistently associated with increased risk of metabolic disease, obesity and cardiovascular dysfunction in later life despite the divergence of birth weight outcomes. The placenta plays a critical role in offspring development and long-term health, as it mediates the crosstalk between the maternal and fetal environments. However, its phenotypic and molecular modifications in the context of maternal obesity associated with fetal growth restriction (FGR) remain poorly understood. METHODS: Using a mouse model of maternal diet-induced obesity, we investigated changes in the placental transcriptome through RNA sequencing (RNA-seq) and Ingenuity Pathway Analysis (IPA) at embryonic day (E) 19. The most differentially expressed genes (FDR < 0.05) were validated by Quantitative real-time PCR (qPCR) in male and female placentae at E19. The expression of these targets and related genes was also determined by qPCR at E13 to examine whether the observed alterations had an earlier onset at mid-gestation. Structural analyses were performed using immunofluorescent staining against Ki67 and CD31 to investigate phenotypic outcomes at both timepoints. RESULTS: RNA-seq and IPA analyses revealed differential expression of transcripts and pathway interactions related to placental vascular development and tissue morphology in obese placentae at term, including downregulation of Muc15, Cnn1, and Acta2. Pdgfb, which is implicated in labyrinthine layer development, was downregulated in obese placentae at E13. This was consistent with the morphological evidence of reduced labyrinth zone (LZ) size, as well as lower fetal weight at both timepoints irrespective of offspring sex. CONCLUSIONS: Maternal obesity results in abnormal placental LZ development and impaired vascularization, which may mediate the observed FGR through reduced transfer of nutrients across the placenta.

Maternal exercise intervention in obese pregnancy improves the cardiovascular health of the adult male offspring.

OBJECTIVE: Obesity during pregnancy is associated with an elevated risk of cardiovascular disease in the offspring. With increased numbers of women entering pregnancy overweight or obese, there is a requirement for targeted interventions to reduce disease risk in future generations. Using an established murine model of maternal obesity during pregnancy, we investigated if a treadmill exercise intervention in the mother could improve offspring cardiac health and explored potential underlying mechanisms. METHODS: A 20-minute treadmill exercise intervention protocol was performed 5 days a week in diet-induced obese female C57BL/6 mice 1 week prior to, and up to E17 of pregnancy. All male offspring were weaned onto a control diet and studied at 8 weeks of age when their cardiovascular physiology was assessed by in vivo echocardiography and non-invasive tail cuff plethysmography. Cardiomyocyte cell area, re-expression of fetal genes and the expression of calcium handling and sympathetic activation proteins were determined. RESULTS: At 8 weeks, there was no difference in bodyweight or fat mass between groups. Offspring of obese dams developed pathologic cardiac hypertrophy, hypertension and cardiac dysfunction characterized by reduced ejection fraction (p < 0.001). Maternal exercise prevented cardiac hypertrophy and dysfunction but failed to prevent hypertension. These offspring of exercised dams also had enhanced (p < 0.001) levels of calcium handling proteins and a sympathetic-activated inotropic response. CONCLUSIONS: Exercise in obese pregnancy was beneficial to offspring cardiac function and structure but did not influence hypertension suggesting they are programmed by separate mechanistic pathways. These data suggest combination interventions in obese pregnancies will be required to improve all aspects of the cardiovascular health of the next generation.

Maternal Obesity in Pregnancy Developmentally Programs Adipose Tissue Inflammation in Young, Lean Male Mice Offspring.

Obesity during pregnancy has a long-term effect on the health of the offspring including risk of developing the metabolic syndrome. Using a mouse model of maternal diet-induced obesity, we employed a genome-wide approach to investigate the microRNA (miRNA) and miRNA transcription profile in adipose tissue to understand mechanisms through which this occurs. Male offspring of diet-induced obese mothers, fed a control diet from weaning, showed no differences in body weight or adiposity at 8 weeks of age. However, offspring from the obese dams had up-regulated cytokine (Tnfα; P < .05) and chemokine (Ccl2 and Ccl7; P < .05) signaling in their adipose tissue. This was accompanied by reduced expression of miR-706, which we showed can directly regulate translation of the inflammatory proteins IL-33 (41% up-regulated; P < .05) and calcium/calmodulin-dependent protein kinase 1D (30% up-regulated; P < .01). We conclude that exposure to obesity during development primes an inflammatory environment in adipose tissue that is independent of offspring adiposity. Programming of adipose tissue miRNAs that regulate expression of inflammatory signaling molecules may be a contributing mechanism.

Selective regulation of hepatic lipid metabolism by the AMP-activated protein kinase pathway in late-pregnant rats.

The liver plays an essential role in maternal metabolic adaptation during late pregnancy. With regard to lipid metabolism, increased secretion of very low-density lipoprotein (VLDL) is characteristic of late pregnancy. Despite this well-described metabolic plasticity, the molecular changes underlying the hepatic adaptation to pregnancy remain unclear. As AMPK is a key intracellular energy sensor, we investigated whether this protein assumes a causal role in the hepatic adaptation to pregnancy. Pregnant Wistar rats were treated with vehicle or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide) for 5 days starting at gestational day 14. At the end of treatment, the rats were subjected to an intraperitoneal pyruvate tolerance test and in situ liver perfusion with pyruvate. The livers were processed for Western blot analysis, quantitative PCR, thin-layer chromatography, enzymatic activity, and glycogen content measurements. Blood biochemical profiles were also assessed. We found that AMPK and ACC phosphorylation were reduced in the livers of pregnant rats in parallel with a reduced level of hepatic gluconeogenesis of pyruvate. This effect was accompanied by both a reduction in the levels of hepatic triglycerides (TG) and an increase in circulating levels of TG. Treatment with AICAR restored hepatic levels of TG to those observed in nonpregnant rats. Additionally, AMPK activation reduced the upregulation of genes related to VLDL synthesis and secretion observed in the livers of pregnant rats. We conclude that the increased secretion of hepatic TG in late pregnancy is concurrent with a transcriptional profile that favors VLDL production. This transcriptional profile results from the reduction in hepatic AMPK activity.

Oral supplementations with free and dipeptide forms of L-glutamine in endotoxemic mice: effects on muscle glutamine-glutathione axis and heat shock proteins.

Sepsis is a leading cause of death in intensive care units worldwide. Low availability of glutamine contributes to the catabolic state of sepsis. L-Glutamine supplementation has antioxidant properties and modulates the expression of heat shock proteins (HSPs). This study investigated the effects of oral supplementation with L-glutamine plus L-alanine (GLN+ALA), both in the free form and L-alanyl-L-glutamine dipeptide (DIP), on glutamine-glutathione (GSH) axis and HSPs expression in endotoxemic mice. B6.129F2/J mice were subjected to endotoxemia (lipopolysaccharides from Escherichia coli, 5 mg.kg(-1), LPS group) and orally supplemented for 48 h with either L-glutamine (1 g.kg(-1)) plus L-alanine (0.61 g.kg(-1)) (GLN+ALA-LPS group) or 1.49 g.kg(-1) of DIP (DIP-LPS group). Endotoxemia reduced plasma and muscle glutamine concentrations [relative to CTRL group] which were restored in both GLN+ALA-LPS and DIP-LPS groups (P<.05). In supplemented groups were re-established GSH content and intracellular redox status (GSSG/GSH ratio) in circulating erythrocytes and muscle. Thiobarbituric acid reactive substance was 4-fold in LPS treated mice relative to the untreated CTRL group, and plasma TNF-α and IL-1ß levels were attenuated by the supplements. Heat shock proteins 27, 70 and 90 (protein and mRNA) were elevated in the LPS group and were returned to basal levels (relative to CTRL group) in both GLN+ALA-LPS and DIP-LPS groups. Supplementations to endotoxemic mice resulted in up-regulation of GSH reductase, GSH peroxidase and glutamate cysteine ligase mRNA expression in muscle. In conclusion, oral supplementations with GLN+ALA or DIP are effective in reversing the conditions of LPS-induced deleterious impact on glutamine-GSH axis in mice under endotoxemia.

Long-term disruption of maternal glucose homeostasis induced by prenatal glucocorticoid treatment correlates with miR-29 upregulation.

Excess of glucocorticoids (GCs) during pregnancy is strongly associated with the programming of glucose intolerance in the offspring. However, the impact of high GC levels on maternal metabolism is not clearly documented. This study aimed to test the hypothesis that mothers exposed to elevated levels of GCs might also display long-term disturbances in glucose homeostasis. Dexamethasone (DEX) was administered noninvasively to the mothers via drinking water between the 14th and the 19th days of pregnancy. Mothers were subjected to glucose and insulin tolerance tests at 1, 2, 3, 6, and 12 mo postweaning. Pregnant rats not treated with DEX and age-matched virgin rats were used as controls. Pancreatic islets were isolated at the 20th day of pregnancy and 12 mo postweaning in order to evaluate glucose-stimulated insulin secretion. The expression of the miR-29 family was also studied due to its responsiveness to GCs and its well-documented role in the regulation of pancreatic ß-cell function. Rats treated with DEX during pregnancy presented long-term glucose intolerance and impaired insulin secretion. These changes correlated with 1) increased expression of miR-29 and its regulator p53, 2) reduced expression of syntaxin-1a, a direct target of miR-29, and 3) altered expression of genes related to cellular senescence. Our data demonstrate that the use of DEX during pregnancy results in deleterious outcomes to the maternal metabolism, hallmarked by reduced insulin secretion and glucose intolerance. This maternal metabolic programming might be a consequence of time-sustained upregulation of miR-29s in maternal pancreatic islets.

Maternal postnatal high-fat diet, rather than gestational diet, affects morphology and mTOR pathway in skeletal muscle of weaning rat.

The positive regulation of insulin pathway in skeletal muscle results in increased activity of the mammalian target of rapamycin (mTOR), a positive effector of mRNA translation rate and protein synthesis. Studies that assess the activity of this protein in response to chronic high-fat diet (HFD) are scarce and controversial, and to date, there are no studies evaluating the mTOR pathway in infants exposed to gestational and postgestational HFD. This study investigated the effect of maternal HFD on skeletal muscle morphology and on phosphorylation of proteins that comprise the intracellular mTOR signaling pathway in soleus muscle of offspring at weaning. For this purpose, 10 days prior to conception, 39 female Wistar rats were randomly assigned to either control diet (CTL) or HFD. Later, rats were distributed into four groups according to gestational and postpregnancy diet: CTL/CTL (n=10), CTL/HF (n=11), HF/HF (n=10) and HF/CTL (n=8). After 21 days of lactation, pups were killed, and blood samples and soleus and gastrocnemius skeletal muscle were collected for analysis. We observed an influence of maternal postgestational diet, rather than gestational diet, in promoting an obese phenotype, characterized by body fat accumulation, insulin resistance and high serum leptin, glucose, triglycerides and cholesterol levels (P<.05). We have also detected alterations on skeletal muscle morphology--with reduced myofiber density--and impairment on S6 kinase 1 and 4E binding protein-1 phosphorylation (P<.05). These results emphasize the importance of maternal diet during lactation on muscle morphology and on physiological adaptations of infant rats.

Combined effect of AMPK/PPAR agonists and exercise training in mdx mice functional performance.

The present investigation was undertaken to test whether exercise training (ET) associated with AMPK/PPAR agonists (EM) would improve skeletal muscle function in mdx mice. These drugs have the potential to improve oxidative metabolism. This is of particular interest because oxidative muscle fibers are less affected in the course of the disease than glycolitic counterparts. Therefore, a cohort of 34 male congenic C57Bl/10J mdx mice included in this study was randomly assigned into four groups: vehicle solution (V), EM [AICAR (AMPK agonist, 50 mg/Kg-1.day-1, ip) and GW 1516 (PPARδ agonist, 2.5 mg/Kg-1.day-1, gavage)], ET (voluntary running on activity wheel) and EM+ET. Functional performance (grip meter and rotarod), aerobic capacity (running test), muscle histopathology, serum creatine kinase (CK), levels of ubiquitined proteins, oxidative metabolism protein expression (AMPK, PPAR, myoglobin and SCD) and intracellular calcium handling (DHPR, SERCA and NCX) protein expression were analyzed. Treatments started when the animals were two months old and were maintained for one month. A significant functional improvement (p<0.05) was observed in animals submitted to the combination of ET and EM. CK levels were decreased and the expression of proteins related to oxidative metabolism was increased in this group. There were no differences among the groups in the intracellular calcium handling protein expression. To our knowledge, this is the first study that tested the association of ET with EM in an experimental model of muscular dystrophy. Our results suggest that the association of ET and EM should be further tested as a potential therapeutic approach in muscular dystrophies.

Leucine supplementation improves adiponectin and total cholesterol concentrations despite the lack of changes in adiposity or glucose homeostasis in rats previously exposed to a high-fat diet.

BACKGROUND: Studies suggest that leucine supplementation (LS) has a therapeutic potential to prevent obesity and to promote glucose homeostasis. Furthermore, regular physical exercise is a widely accepted strategy for body weight maintenance and also for the prevention of obesity. The aim of this study was to determine the effect of chronic LS alone or combined with endurance training (ET) as potential approaches for reversing the insulin resistance and obesity induced by a high-fat diet (HFD) in rats. METHODS: Forty-seven rats were randomly divided into two groups. Animals were fed a control diet-low fat (n = 10) or HFD (n = 37). After 15 weeks on HFD, all rats received the control diet-low fat and were randomly divided according to treatment: reference (REF), LS, ET, and LS+ET (n = 7-8 rats per group). After 6 weeks of treatment, the animals were sacrificed and body composition, fat cell volume, and serum concentrations of total cholesterol, HDL-cholesterol, triacylglycerol, glucose, adiponectin, leptin and tumor necrosis factor-alpha (TNF-α) were analyzed. RESULTS: At the end of the sixth week of treatment, there was no significant difference in body weight between the REF, LS, ET and LS+ET groups. However, ET increased lean body mass in rats (P = 0.019). In addition, ET was more effective than LS in reducing adiposity (P = 0.019), serum insulin (P = 0.022) and TNF-α (P = 0.044). Conversely, LS increased serum adiponectin (P = 0.021) levels and reduced serum total cholesterol concentration (P = 0.042). CONCLUSIONS: The results showed that LS had no beneficial effects on insulin sensitivity or adiposity in previously obese rats. On the other hand, LS was effective in increasing adiponectin levels and in reducing total cholesterol concentration.