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.This work received funding from the British Heart Foundation, the European Union’s Seventh Framework Programme , MRC Metabolic Diseases Unit award and British Heart Foundation Studentship. Wellcome Trust

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

Abstract: 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. Graphical abstract

Insulin-resistant skeletal muscle cell shows altered response to IL-6 on carbohydrate metabolism but not on lipid metabolism.

A interleucina-6 (IL-6) é uma citocina com dupla função na sensibilidade à insulina. O objetivo desse estudo foi investigar o efeito da IL-6 durante 1 e 24 horas no metabolismo de células musculares resistentes à insulina. Em células de indivíduos saudáveis, 1 hora de exposição à IL-6 aumentou a síntese de glicogênio e a captação de glicose além de pJAK2 e pSTAT3, mas não em células de diabéticos tipo 2. Diferentemente, a IL-6 aumentou a oxidação de ácido graxo em ambos os grupos. Por outro lado, em células musculares da linhagem L6, a IL-6, em 24 horas, reduziu a síntese de glicogênio e a captação de glicose em condições normais, mas não em células resistentes à insulina. Além de reduzir pJak2, pStat3 e pAkt. A IL-6 aumentou oxidação de ácido graxo e pACC em ambos os grupos. Sendo assim, células musculares resistentes à insulina apresentam uma resposta alterada ao efeito da IL-6. Nessas células, o efeito dessa citocina está abolido no metabolismo de carboidratos mas permanece efetivo no lipídico.Interleukin-6 (IL-6) is a cytokine with a dual role in modulating insulin sensitivity. The aim of this study was to investigate the effect of IL-6 exposure for 1 hour and 24 hours on metabolism of insulin-resistant skeletal muscle cells. In muscle cells from healthy subjects, 1 hour of exposure to IL-6 increased glycogen synthesis, glucose uptake, pJAK2 and pSTAT3, which were not observed in muscle cells of type 2 diabetic subjects. In contrast, IL-6 increased fatty acid oxidation in cells from both groups. On the other hand, in skeletal muscle cell line L6, IL-6 for 24 hours reduced glycogen synthesis and glucose uptake in normal conditions, which were not observed in insulin-resistant L6 cells. In addition, pJak2, pStat3 and pAkt were reduced in insulin-resistant cells. In contrast, IL-6 increased fatty acid oxidation and pACC in both groups. Therefore, insulin resistant skeletal muscle cells have an altered response to the effect of IL-6. On these cells, the effect of IL-6 is abolished on carbohydrate metabolism, while remaining effective on lipid metabolism

Insulin-resistant skeletal muscle cell shows altered response to IL-6 on carbohydrate metabolism but not on lipid metabolism.

A interleucina-6 (IL-6) é uma citocina com dupla função na sensibilidade à insulina. O objetivo desse estudo foi investigar o efeito da IL-6 durante 1 e 24 horas no metabolismo de células musculares resistentes à insulina. Em células de indivíduos saudáveis, 1 hora de exposição à IL-6 aumentou a síntese de glicogênio e a captação de glicose além de pJAK2 e pSTAT3, mas não em células de diabéticos tipo 2. Diferentemente, a IL-6 aumentou a oxidação de ácido graxo em ambos os grupos. Por outro lado, em células musculares da linhagem L6, a IL-6, em 24 horas, reduziu a síntese de glicogênio e a captação de glicose em condições normais, mas não em células resistentes à insulina. Além de reduzir pJak2, pStat3 e pAkt. A IL-6 aumentou oxidação de ácido graxo e pACC em ambos os grupos. Sendo assim, células musculares resistentes à insulina apresentam uma resposta alterada ao efeito da IL-6. Nessas células, o efeito dessa citocina está abolido no metabolismo de carboidratos mas permanece efetivo no lipídico.Interleukin-6 (IL-6) is a cytokine with a dual role in modulating insulin sensitivity. The aim of this study was to investigate the effect of IL-6 exposure for 1 hour and 24 hours on metabolism of insulin-resistant skeletal muscle cells. In muscle cells from healthy subjects, 1 hour of exposure to IL-6 increased glycogen synthesis, glucose uptake, pJAK2 and pSTAT3, which were not observed in muscle cells of type 2 diabetic subjects. In contrast, IL-6 increased fatty acid oxidation in cells from both groups. On the other hand, in skeletal muscle cell line L6, IL-6 for 24 hours reduced glycogen synthesis and glucose uptake in normal conditions, which were not observed in insulin-resistant L6 cells. In addition, pJak2, pStat3 and pAkt were reduced in insulin-resistant cells. In contrast, IL-6 increased fatty acid oxidation and pACC in both groups. Therefore, insulin resistant skeletal muscle cells have an altered response to the effect of IL-6. On these cells, the effect of IL-6 is abolished on carbohydrate metabolism, while remaining effective on lipid metabolism

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

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

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

Abstract: 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. Graphical abstract