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.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Early nutrition and ageing: can we intervene?

Ageing, a complex process that results in progressive decline in intrinsic physiological function leading to an increase in mortality rate, has been shown to be affected by early life nutrition. Accumulating data from animal and epidemiological studies indicate that exposure to a suboptimal nutritional environment during fetal life can have long-term effects on adult health. In this paper, we discuss the impact of early life nutrition on the development of age-associated diseases and life span. Special emphasis is given to studies that have investigated the molecular mechanisms underlying these effects. These include permanent structural and cellular changes including epigenetics modifications, oxidative stress, DNA damage and telomere shortening. Potential strategies targeting these mechanisms, in order to prevent or alleviate the detrimental effects of suboptimal early nutrition on lifespan and age-related diseases, are also discussed. Although recent reports have already identified effective therapeutic interventions, such as antioxidant supplementation, further understanding of the extent and nature of how early nutrition influences the ageing process will enable the development of novel and more effective approaches to improve health and extend human lifespan in the future.

SILAC Mass Spectrometry Profiling: A Psychiatric Disorder Perspective.

Stable isotope labelling by amino acids in cell culture (SILAC) is a technique that allows proteomic profiling of cells. In this chapter we describe a protocol for the identification and quantification of newly synthesised proteins. The methodology can be applied to any cultured cell system with relevance to schizophrenia, affective disorders and autism spectrum conditions including those addressing responses to pharmacological stimuli.

Diacylglycerol kinase-δ regulates AMPK signaling, lipid metabolism, and skeletal muscle energetics.

Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase-δ (DGKδ), an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here, we tested the hypothesis that DGKδ plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGKδ(+/-) mice, but glycogen content and mitochondrial markers were unaltered, suggesting that DGKδ deficiency affects skeletal muscle energetics but not mitochondrial protein abundance. We next determined the role of DGKδ in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling were reduced in DGKδ(+/-) mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGKδ deficiency impaired work performance, as evident by altered force production and relaxation dynamics in response to repeated contractions. In conclusion, DGKδ deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGKδ deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.

Autocrine role of interleukin-13 on skeletal muscle glucose metabolism in type 2 diabetic patients involves microRNA let-7.

Low-grade inflammation associated with type 2 diabetes (T2DM) is postulated to exacerbate insulin resistance. We report that serum levels, as well as IL-13 secreted from cultured skeletal muscle, are reduced in T2DM vs. normal glucose-tolerant (NGT) subjects. IL-13 exposure increases skeletal muscle glucose uptake, oxidation, and glycogen synthesis via an Akt-dependent mechanism. Expression of microRNA let-7a and let-7d, which are direct translational repressors of the IL-13 gene, was increased in skeletal muscle from T2DM patients. Overexpression of let-7a and let-7d in cultured myotubes reduced IL-13 secretion. Furthermore, basal glycogen synthesis was reduced in cultured myotubes exposed to an IL-13-neutralizing antibody. Thus, IL-13 is synthesized and released by skeletal muscle through a mechanism involving let-7, and this effect is attenuated in skeletal muscle from insulin-resistant T2DM patients. In conclusion, IL-13 plays an autocrine role in skeletal muscle to increase glucose uptake and metabolism, suggesting a role in glucose homeostasis in metabolic disease.

Exercise alters the molecular pathways of insulin signaling and lipid handling in maternal tissues of obese pregnant mice.

Obesity during gestation adversely affects maternal and infant health both during pregnancy and for long afterwards. However, recent work suggests that a period of maternal exercise during pregnancy can improve metabolic health of the obese mother and her offspring. This study aimed to identify the physiological and molecular impact of exercise on the obese mother during pregnancy that may lead to improved metabolic outcomes. To achieve this, a 20-min treadmill exercise intervention was performed 5 days a week in diet-induced obese female mice from 1 week before and up to day 17 of pregnancy. Biometric, biochemical and molecular analyses of maternal tissues and/or plasma were performed on day 19 of pregnancy. We found exercise prevented some of the adverse changes in insulin signaling and lipid metabolic pathways seen in the liver, skeletal muscle and white adipose tissue of sedentary-obese pregnant dams (p110ß, p110α, AKT, SREBP). Exercise also induced changes in the insulin and lipid signaling pathways in obese dams that were different from those observed in control and sedentary-obese dams. The changes induced by obesity and exercise were tissue-specific and related to alterations in tissue lipid, protein and glycogen content and plasma insulin, leptin and triglyceride concentrations. We conclude that the beneficial effects of exercise on metabolic outcomes in obese mothers may be related to specific molecular signatures in metabolically active maternal tissues during pregnancy. These findings highlight potential metabolic targets for therapeutic intervention and the importance of lifestyle in reducing the burden of the current obesity epidemic on healthcare systems.

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.

Pulsed SILAC as a Approach for miRNA Targets Identification in Cell Culture.

Pulsed stable isotope labeling by amino acids in cell culture (pSILAC) comprises a variation of the classical SILAC proteomic methodology that enables the identification of short-term proteomic responses such as those elicited by micro RNAs (miRNAs). Here, we describe a detailed pSILAC protocol for global identification and quantification of protein translation alterations induced by a miRNA using 3T3-L1 pre-adipocytes as a model system.

A post-weaning obesogenic diet exacerbates the detrimental effects of maternal obesity on offspring insulin signaling in adipose tissue.

Previous studies have shown that maternal diet-induced obesity leads to increased risk of type 2 diabetes in offspring. The current study investigated if weaning onto an obesogenic diet exaggerated the detrimental effects of maternal diet-induced obesity in adipose tissue. Maternal obesity and offspring obesity led to reduced expression of key insulin signalling proteins, including insulin receptor substrate-1 (IRS-1). The effects of maternal obesity and offspring obesity were, generally, independent and additive. Irs1 mRNA levels were similar between all four groups of offspring, suggesting that in both cases post-transcriptional regulation was involved. Maternal diet-induced obesity increased miR-126 expression however levels of this miR were not influenced by a post-weaning obesogenic diet. In contrast, a post-weaning obesogenic diet was associated with increased levels of suppressor of cytokine signaling-1, implicating increased degradation of IRS-1 as an underlying mechanism. Our results suggest that whilst programmed reductions in IRS-1 are associated with increased levels of miR-126 and consequently reduced translation of Irs1 mRNA, the effects of a post-weaning obesogenic diet on IRS-1 are mediated by miR-126 independent mechanisms, including increased IRS-1 protein degradation. These divergent mechanisms explain why the combination of maternal obesity and offspring obesity leads to the most pronounced effects on offspring metabolism.

Exercise rescues obese mothers' insulin sensitivity, placental hypoxia and male offspring insulin sensitivity.

The prevalence of obesity during pregnancy continues to increase at alarming rates. This is concerning as in addition to immediate impacts on maternal wellbeing, obesity during pregnancy has detrimental effects on the long-term health of the offspring through non-genetic mechanisms. A major knowledge gap limiting our capacity to develop intervention strategies is the lack of understanding of the factors in the obese mother that mediate these epigenetic effects on the offspring. We used a mouse model of maternal-diet induced obesity to define predictive correlations between maternal factors and offspring insulin resistance. Maternal hyperinsulinemia (independent of maternal body weight and composition) strongly associated with offspring insulin resistance. To test causality, we implemented an exercise intervention that improved maternal insulin sensitivity without changing maternal body weight or composition. This maternal intervention prevented excess placental lipid deposition and hypoxia (independent of sex) and insulin resistance in male offspring. We conclude that hyperinsulinemia is a key programming factor and therefore an important interventional target during obese pregnancy, and propose moderate exercise as a promising strategy to improve metabolic outcome in both the obese mother and her offspring.

Prolonged fasting elicits increased hepatic triglyceride accumulation in rats born to dexamethasone-treated mothers.

We investigated the effect of dexamethasone during the last week of pregnancy on glucose and lipid metabolism in male offspring. Twelve-week old offspring were evaluated after fasting for 12-hours (physiological) and 60-hours (prolonged). Physiological fasting resulted in glucose intolerance, decreased glucose clearance after pyruvate load and increased PEPCK expression in rats born to dexamethasone-treated mothers (DEX). Prolonged fasting resulted in increased glucose tolerance and increased glucose clearance after pyruvate load in DEX. These modulations were accompanied by accumulation of hepatic triglycerides (TG). Sixty-hour fasted DEX also showed increased citrate synthase (CS) activity, ATP citrate lyase (ACLY) content, and pyruvate kinase 2 (pkm2), glucose transporter 1 (slc2a1) and lactate dehydrogenase-a (ldha) expressions. Hepatic AKT2 was increased in 60-hour fasted DEX, in parallel with reduced miRNAs targeting the AKT2 gene. Altogether, we show that metabolic programming by prenatal dexamethasone is characterized by an unexpected hepatic TG accumulation during prolonged fasting. The underlying mechanism may depend on increased hepatic glycolytic flux due to increased pkm2 expression and consequent conversion of pyruvate to non-esterified fatty acid synthesis due to increased CS activity and ACLY levels. Upregulation of AKT2 due to reduced miRNAs may serve as a permanent mechanism leading to increased pkm2 expression.

Negative regulation of glucose metabolism in human myotubes by supraphysiological doses of 17ß-estradiol or testosterone.

OBJECTIVE: Exposure of skeletal muscle to high levels of testosterone or estrogen induces insulin resistance, but evidence regarding the direct role of either sex hormone on metabolism is limited. Therefore, the aim of this study was to investigate the direct effect of acute sex hormone exposure on glucose metabolism in skeletal muscle. MATERIALS/METHODS: Differentiated human skeletal myotubes were exposed to either 17ß-estradiol or testosterone and metabolic characteristics were assessed. Glucose incorporation into glycogen, glucose oxidation, palmitate oxidation, and phosphorylation of key signaling proteins were determined. RESULTS: Treatment of myotubes with either 17ß-estradiol or testosterone decreased glucose incorporation into glycogen. Exposure of myotubes to 17ß-estradiol reduced glucose oxidation under basal and insulin-stimulated conditions. However, testosterone treatment enhanced basal palmitate oxidation and prevented insulin action on glucose and palmitate oxidation. Acute stimulation of myotubes with testosterone reduced phosphorylation of S6K1 and p38 MAPK. Exposure of myotubes to either 17ß-estradiol or testosterone augmented phosphorylation GSK3ß(Ser9) and PKCδ(Thr505), two negative regulators of glycogen synthesis. Treatment of myotubes with a PKC specific inhibitor (GFX) restored the effect of either sex hormone on glycogen synthesis. PKCδ silencing restored glucose incorporation into glycogen to baseline in response to 17ß-estradiol, but not testosterone treatment. CONCLUSION: An acute exposure to supraphysiological doses of either 17ß-estradiol or testosterone regulates glucose metabolism, possibly via PKC signaling pathways. Furthermore, testosterone treatment elicits additional alterations in serine/threonine kinase signaling, including the ribosomal protein S6K1 and p38 MAPK.

Downregulation of IRS-1 in adipose tissue of offspring of obese mice is programmed cell-autonomously through post-transcriptional mechanisms.

We determined the effects of maternal diet-induced obesity on offspring adipose tissue insulin signalling and miRNA expression in the aetiology of insulin resistance in later life. Although body composition and glucose tolerance of 8-week-old male offspring of obese dams were not dysregulated, serum insulin was significantly (p<0.05) elevated. Key insulin signalling proteins in adipose tissue were down-regulated, including the insulin receptor, catalytic (p110ß) and regulatory (p85α) subunits of PI3K as well as AKT1 and 2 (all p<0.05). The largest reduction observed was in IRS-1 protein (p<0.001), which was regulated post-transcriptionally. Concurrently, miR-126, which targets IRS-1, was up-regulated (p<0.05). These two features were maintained in isolated primary pre-adipocytes and differentiated adipocytes in-vitro. We have therefore established that maternal diet-induced obesity programs adipose tissue insulin resistance. We hypothesise that maintenance of the phenotype in-vitro strongly suggests that this mechanism is cell autonomous and may drive insulin resistance in later life.

Nutritional programming of insulin resistance: causes and consequences.

Strong evidence indicates that adverse prenatal and early postnatal environments have a significant long-term influence on risk factors that result in insulin resistance, type 2 diabetes (T2D), and cardiovascular disease later in life. Here we discuss current knowledge of how maternal and neonatal nutrition influence early growth and the long-term risk of developing insulin resistance in different organs and at the whole-body level. Accumulating evidence supports a role for epigenetic mechanisms underlying this nutritional programming, consisting of heritable changes that regulate gene expression which in turn shapes the phenotype across generations. Deciphering these molecular mechanisms in key tissues and discovering key biological markers may provide valuable insight towards the development of effective intervention strategies.

Altered response of skeletal muscle to IL-6 in type 2 diabetic patients.

Interleukin-6 (IL-6) has a dual role in modulating insulin sensitivity, with evidence for this cytokine as both an enhancer and inhibitor of insulin action. We determined the effect of IL-6 exposure on glucose and lipid metabolism in cultured myotubes established from people with normal glucose tolerance or type 2 diabetes. Acute IL-6 exposure increased glycogen synthesis, glucose uptake, and signal transducer and activator of transcription 3 (STAT3) phosphorylation in cultured myotubes from normal glucose tolerant subjects. However, in type 2 diabetic patients, IL-6 was without effect on glucose metabolism and STAT3 signaling, concomitant with increased suppressor of cytokine signaling 3 (SOCS3) expression. IL-6 increased fatty acid oxidation in myotubes from type 2 diabetic and normal glucose tolerant subjects. Expression of IL-6, IL-6 receptor (IL-6R), or glycoprotein 130, as well as IL-6 secretion, was unaltered between cultured myotubes from normal glucose tolerant or type 2 diabetic subjects. Circulating serum IL-6 concentration was unaltered between normal glucose tolerant and type 2 diabetic subjects. In summary, skeletal muscle cells from type 2 diabetic patients display selective IL-6 resistance for glucose rather than lipid metabolism. In conclusion, IL-6 appears to play a differential role in regulating metabolism in type 2 diabetic patients compared with normal glucose tolerant subjects.

Endothelin-1 reduces glucose uptake in human skeletal muscle in vivo and in vitro.

OBJECTIVE: Endothelin (ET)-1 is a vasoconstrictor and proinflammatory peptide that may interfere with glucose uptake. Our objective was to investigate whether exogenous ET-1 affects glucose uptake in the forearm of individuals with insulin resistance and in cultured human skeletal muscle cells. RESEARCH DESIGN AND METHODS: Nine male subjects (aged 61 ± 3 years) with insulin resistance (M value <5.5 mg/kg/min or a homeostasis model assessment of insulin resistance index >2.5) participated in a protocol using saline infusion followed by ET-1 infusion (20 pmol/min) for 2 h into the brachial artery. Forearm blood flow (FBF), endothelium-dependent vasodilatation, and endothelium-independent vasodilatation were assessed. Molecular signaling and glucose uptake were determined in cultured skeletal muscle cells. RESULTS: ET-1 decreased forearm glucose uptake (FGU) by 39% (P < 0.05) after the 2-h infusion. ET-1 reduced basal FBF by 36% after the 2-h infusion (P < 0.05) and impaired both endothelium-dependent vasodilatation (P < 0.01) and endothelium-independent vasodilatation (P < 0.05). ET(A) and ET(B) receptor expression was detected on cultured skeletal muscle cells. One-hour ET-1 incubation increased glucose uptake in cells from healthy control subjects but not from type 2 diabetic patients. Incubation with ET-1 for 24 h reduced glucose uptake in cells from healthy subjects. ET-1 decreased insulin-stimulated Akt phosphorylation and increased phosphorylation of insulin receptor substrate-1 serine 636. CONCLUSIONS: ET-1 not only induces vascular dysfunction but also acutely impairs FGU in individuals with insulin resistance and in skeletal muscle cells from type 2 diabetic subjects. These findings suggest that ET-1 may contribute to the development of insulin resistance in skeletal muscle in humans.

Early and prolonged intake of partially hydrogenated fat alters the expression of genes in rat adipose tissue.

OBJECTIVE: Our previous study indicated that partially hydrogenated fat (PHF) diets, rich in trans-isomers, alter plasma lipids and increase the lipogenesis rate on adipose tissue in rats at a young age. In the present study we investigated the effects of dietary PHF on the expression of genes associated with glucose and lipid metabolism in rat adipose tissue. METHODS: Female Wistar rats were fed normolipidic diets containing PHF (rich in trans-fatty acids and poor in polyunsaturated fatty acids [PUFAs]), soy oil (rich in omega-6 PUFAs), and fish oil (rich in omega-3 PUFAs) during gestation and lactation; young male pups were fed the same diets from weaning until 120 d of life. The mRNA expression of peroxisome proliferator-activated receptor-gamma, tumor necrosis factor-alpha, resistin, adiponectin, and leptin were analyzed in retroperitoneal adipose tissue (RET) using real time polymerase chain reaction. RESULTS: The PHF group showed the highest triacylglycerol, glucose, and insulin levels and the lowest plasma adiponectin level. The RET of PHF incorporated trans-fatty acids, whereas fish oil and soy oil groups had increased omega-3 and omega-6 PUFAs, respectively. In the RET the PHF group had the highest resistin and tumor necrosis factor-alpha levels and the lowest adiponectin and peroxisome proliferator-activated receptor-gamma gene expressions, whereas the fish oil group had the highest peroxisome proliferator-activated receptor-gamma and the lowest leptin gene expression. CONCLUSION: Prolonged intake of PHF has a negative effect on the expression of genes in RET when compared with diets with omega-6 and omega-3 PUFAs. These changes may be an effect of the smaller proportions of PUFAs in this fat, instead of being only caused by trans-fatty acids.