Metabolic programming in the offspring after gestational overfeeding in the mother: toward neonatal rescuing with metformin in a swine model.

OBJECTIVES: Maternal overfeeding during gestation may lead to adverse metabolic programming in the offspring mediated by epigenetic alterations. Potential reversal, in early life, of these alterations may help in the prevention of future cardio-metabolic conditions. In this context, our aims were: (1) to study the effects of maternal overfeeding on the metabolic and epigenetic programming of offspring's adipose tissue; and (2) to test the potential of postnatal metformin treatment to reverse these changes. METHODS: We used a swine animal model where commercial production sows were either overfed or kept under standard diet during gestation, and piglets at birth were randomly assigned to metformin (n = 16 per group) or vehicle treatment during lactation (n = 16 per group). RESULTS: Piglets born to overfed sows showed a worse metabolic profile (higher weight, weight gain from birth and abdominal circumference; all p < 0.05) together with altered serological markers (increased HOMA-IR, fructosamine, total cholesterol, C-Reactive Protein and lower HMW adiponectin; all p < 0.05). The visceral adipose tissue also showed altered morphology (increased adipocyte area, perimeter and diameter; all p < 0.05), as well as changes in gene expression (higher CCL2 and INSR, lower DLK1; all p < 0.05), and in DNA methylation (96 hypermethylated and 99 hypomethylated CpG sites; FDR < 0.05). Metformin treatment significantly ameliorated the abnormal metabolic profile, decreasing piglets' weight, weight gain from birth, abdominal circumference and fructosamine (all p < 0.05) and reduced adipocyte area, perimeter, and diameter in visceral adipose tissue (all p < 0.05). In addition, metformin treatment potentiated several associations between gene expression in visceral adipose tissue and the altered metabolic markers. CONCLUSIONS: Maternal overfeeding during gestation leads to metabolic abnormalities in the offspring, including adipose tissue alterations. Early metformin treatment mitigates these effects and could help rescue the offspring's metabolic health.

Catch-up growth in juvenile rats, fat expansion, and dysregulation of visceral adipose tissue.

BACKGROUND: Accelerated catch-up growth following intrauterine restriction increases the risk of developing visceral adiposity and metabolic abnormalities. However, the underlying molecular mechanisms of such metabolic programming are still poorly understood. METHODS: A Wistar rat model of catch-up growth following intrauterine restriction was used. A gene expression array was performed in the retroperitoneal adipose tissue sampled at postnatal day (PD) 42. RESULTS: Five hundred and forty-six differentially expressed genes (DEGs) were identified (adjusted p value < 0.05). Gene ontology enrichment analysis identified pathways related to immune and lipid metabolic processes, brown fat cell differentiation, and regulation of PI3K. Ccl21, Npr3, Serpina3n, Pnpla3, Slc2a4, and Serpina12 were validated to be upregulated in catch-up pups (all p < 0.01) and related to several fat expansion and metabolic parameters, including body weight at PD42, postnatal body weight gain, white and brown adipose tissue mass, plasma triglycerides, and insulin resistance index (all p < 0.05). CONCLUSIONS: Genes related to immune and metabolic processes were upregulated in retroperitoneal adipose tissue following catch-up growth in juvenile rats and were found to be associated with fat expansion and metabolic parameters. Our results provide evidence for several dysregulated genes in white adipose tissue that could help develop novel strategies to prevent the metabolic abnormalities associated with catch-up growth. IMPACT: Catch-up growth presents several dysregulated genes in white adipose tissue related to metabolic abnormalities. Ccl21, Npr3, Serpina3n, Pnpla3, Slc2a4, and Serpina12 were validated to be upregulated in catch-up pups and related to visceral fat expansion and metabolic parameters. Profiling and validation of these dysregulated genes in visceral adipose tissue could help develop novel strategies to prevent the metabolic abnormalities associated with catch-up growth.

Circulating IGF-1 Independently Predicts Blood Pressure in Children With Higher Calcium-Phosphorus Product Levels.

OBJECTIVE: To study the association between insulin-like growth factor 1 (IGF-1) and blood pressure in children, in particular, the potential interaction with the serum calcium-phosphorus product (Ca*P). METHODS: A longitudinal study included 521 children (age 8.8 ± 0.1) from northeastern Spain, of whom 158 were followed-up after 5 years. IGF-1, insulin-like growth factor-binding protein 3 (IGFBP-3), and serum calcium and phosphorus were measured at baseline. Anthropometric (body-mass index [BMI] and waist) and cardiometabolic variables (systolic [SBP] and diastolic blood pressure), pulse pressure, insulin, homeostatic model assessment of insulin resistance [HOMA-IR], high-density lipoprotein [HDL]-cholesterol, and triglycerides) were assessed at baseline and at the end of follow-up. Statistical analysis included Pearson correlations followed by multivariable linear regression analyses. RESULTS: Baseline IGF-1 and IGF-1/IGFBP-3 molar ratio positively correlated with baseline and follow-up BMI, waist, SBP, pulse pressure, insulin, HOMA-IR and triglycerides (r 0.138-0.603; all P < 0.05). The associations with SBP were stronger with increasing Ca*P (r 0.261-0.625 for IGF-1; and r 0.174-0.583 for IGF-1/IGFBP-3). After adjusting for confounding variables, baseline IGF-1 and IGF-1/IGFBP-3 remained independently associated with both baseline and follow-up SBP in children in the highest Ca*P tertile (ß = 0.245-0.381; P < 0.01; model R2 = 0.246-0.566). CONCLUSIONS: Our results suggest that IGF-1 in childhood is an independent predictor of SBP in apparently healthy children, especially in those with high Ca*P levels.

Metabolic syndrome, clustering of cardiovascular risk factors and high carotid intima-media thickness in children and adolescents.

OBJECTIVE: The clinical utility of screening for pediatric metabolic syndrome (MetS) in children and adolescents is still controversial. We examined the performance of pediatric MetS vs. clustering of cardiovascular risk factors (which are the components of MetS) for predicting high carotid intima-media thickness (cIMT) in children and adolescents. METHODS: Participants included 2427 children and adolescents aged 6-17 years from population-based studies in three countries (Brazil, China and Italy). Pediatric MetS was defined using either the modified National Cholesterol Education Program Adult Treatment Panel III criteria or the modified International Diabetes Federation criteria. Clustering of cardiovascular risk factors was calculated as the sum of five components of MetS (i.e. central obesity, elevated blood pressure, elevated triglycerides, reduced HDL-cholesterol and elevated fasting blood glucose). High cIMT was defined as cIMT at least 95th percentile values for sex and age developed from European children. RESULTS: Presence of one, two or at least three cardiovascular risk factors (using the National Cholesterol Education Program Adult Treatment Panel III criteria), as compared with none, was associated with gradually increasing odds of high cIMT [odds ratios (95% confidence intervals): 1.60 (1.29-1.99), 2.89 (2.21-3.78) and 4.24 (2.81-6.39), respectively]. High cIMT was also associated with presence (vs. absence) of MetS (odds ratio = 2.88, 95% confidence interval = 1.95-4.26). However, clustering of cardiovascular risk factors predicted high cIMT markedly better than MetS (area under the curve of 0.66 vs. 0.54, respectively). Findings were similar using the International Diabetes Federation criteria for pediatric MetS. CONCLUSION: In children and adolescents, a graded score based on five cardiovascular risk factors (used to define MetS) predicted high cIMT markedly better than MetS. These findings do not support the clinical utility of MetS for screening youth at increased cardiovascular risk, as expressed in this study by high cIMT.

Dlk1 expression relates to visceral fat expansion and insulin resistance in male and female rats with postnatal catch-up growth.

BACKGROUND: Although prenatal and postnatal programming of metabolic diseases in adulthood is well established, the mechanisms underpinning metabolic programming are not. Dlk1, a key regulator of fetal development, inhibits adipocyte differentiation and restricts fetal growth. METHODS: Assess DLk1 expression in a Wistar rat model of catch-up growth following intrauterine restriction. Dams fed ad libitum delivered control pups (C) and dams on a 50% calorie-restricted diet delivered pups with low birth weight (R). Restricted offspring fed a standard rat chow showed catch-up growth (R/C) but those kept on a calorie-restricted diet did not (R/R). RESULTS: Decreased Dlk1 expression was observed in adipose tissue and skeletal muscle of R/C pups along with excessive visceral fat accumulation, decreased circulating adiponectin, increased triglycerides and HOMA-IR (from p < 0.05 to p < 0.0001). Moreover, in R/C pups the reduced Dlk1 expression in adipose tissue and skeletal muscle correlated with visceral fat (r = -0.820, p < 00001) and HOMA-IR (r = -0.745, p = 0.002). CONCLUSIONS: Decreased Dlk1 expression relates to visceral fat expansion and insulin resistance in a rat model of catch-up growth following prenatal growth restriction. Modulation of Dlk1 expression could be among the targets for the early prevention of fetal programming of adult metabolic disorders.

Umbilical Cord miRNAs in Small-for-Gestational-Age Children and Association With Catch-Up Growth: A Pilot Study.

CONTEXT: Catch-up growth in infants who are small for gestational age (SGA) is a risk factor for the development of cardiometabolic diseases in adulthood. The basis and mechanisms underpinning catch-up growth in newborns who are SGA are unknown. OBJECTIVE: To identify umbilical cord miRNAs associated with catch-up growth in infants who are SGA and study their relationship with offspring's cardiometabolic parameters. DESIGN: miRNA PCR panels were used to study the miRNA profile in umbilical cord tissue of five infants who were SGA with catch-up (SGA-CU), five without catch-up (SGA-nonCU), and five control infants [appropriate for gestational age (AGA)]. The miRNAs with the smallest nominal P values were validated in 64 infants (22 AGA, 18 SGA-nonCU, and 24 SGA-CU) and correlated with anthropometric parameters at 1 (n = 64) and 6 years of age (n = 30). RESULTS: miR-501-3p, miR-576-5p, miR-770-5p, and miR-876-3p had nominally significant associations with increased weight, height, weight catch-up, and height catch-up at 1 year, and miR-374b-3p, miR-548c-5p, and miR-576-5p had nominally significant associations with increased weight, height, waist, hip, and renal fat at 6 years. Multivariate analysis suggested miR-576-5p as a predictor of weight catch-up and height catch-up at 1 year, as well as weight, waist, and renal fat at 6 years. In silico studies suggested that miR-576-5p participates in the regulation of inflammatory, growth, and proliferation signaling pathways. CONCLUSIONS: Umbilical cord miRNAs could be novel biomarkers for the early identification of catch-up growth in infants who are SGA. miR-576-5p may contribute to the regulation of postnatal growth and influence the risk for cardiometabolic diseases associated with a mismatch between prenatal and postnatal weight gain.

Renal size and cardiovascular risk in prepubertal children.

Renal size is an important parameter for the evaluation and diagnosis of kidney disease and has been associated with several cardiovascular risk factors in patients with kidney failure. These results are however discordant and studies in healthy children are lacking. We aimed to study the association between renal size (length and volume) and cardiovascular risk parameters in healthy children. Clinical, analytical and ultrasound parameters [renal length, renal volume, perirenal fat and carotid intima-media thickness (cIMT)] were determined in 515 healthy prepubertal children (176 lean, 208 overweight and 131 obese). Renal length and volume associated significantly and positively with several anthropometric and cardiovascular risk parameters including cIMT and systolic blood pressure (SBP) (all p < 0.001). Renal length and volume associated with cIMT and SBP in all study subgroups, but these associations were predominant in obese children, in whom these associations were independent after adjusting for age, gender and BSA (all p < 0.05). In multivariate analyses in the study subjects as a whole, renal length was an independent predictor of cIMT (ß = 0.310, p < 0.0001) and SBP (ß = 0.116, p = 0.03). Renal size associates with cIMT and SBP, independent of other well-established cardiovascular risk factors, and may represent helpful parameters for the early assessment of cardiovascular risk in children.

SRp55 Regulates a Splicing Network That Controls Human Pancreatic ß-Cell Function and Survival.

Progressive failure of insulin-producing ß-cells is the central event leading to diabetes, but the signaling networks controlling ß-cell fate remain poorly understood. Here we show that SRp55, a splicing factor regulated by the diabetes susceptibility gene GLIS3, has a major role in maintaining the function and survival of human ß-cells. RNA sequencing analysis revealed that SRp55 regulates the splicing of genes involved in cell survival and death, insulin secretion, and c-Jun N-terminal kinase (JNK) signaling. In particular, SRp55-mediated splicing changes modulate the function of the proapoptotic proteins BIM and BAX, JNK signaling, and endoplasmic reticulum stress, explaining why SRp55 depletion triggers ß-cell apoptosis. Furthermore, SRp55 depletion inhibits ß-cell mitochondrial function, explaining the observed decrease in insulin release. These data unveil a novel layer of regulation of human ß-cell function and survival, namely alternative splicing modulated by key splicing regulators such as SRp55, that may cross talk with candidate genes for diabetes.

Neuron-enriched RNA-binding Proteins Regulate Pancreatic Beta Cell Function and Survival.

Pancreatic beta cell failure is the central event leading to diabetes. Beta cells share many phenotypic traits with neurons, and proper beta cell function relies on the activation of several neuron-like transcription programs. Regulation of gene expression by alternative splicing plays a pivotal role in brain, where it affects neuronal development, function, and disease. The role of alternative splicing in beta cells remains unclear, but recent data indicate that splicing alterations modulated by both inflammation and susceptibility genes for diabetes contribute to beta cell dysfunction and death. Here we used RNA sequencing to compare the expression of splicing-regulatory RNA-binding proteins in human islets, brain, and other human tissues, and we identified a cluster of splicing regulators that are expressed in both beta cells and brain. Four of them, namely Elavl4, Nova2, Rbox1, and Rbfox2, were selected for subsequent functional studies in insulin-producing rat INS-1E, human EndoC-ßH1 cells, and in primary rat beta cells. Silencing of Elavl4 and Nova2 increased beta cell apoptosis, whereas silencing of Rbfox1 and Rbfox2 increased insulin content and secretion. Interestingly, Rbfox1 silencing modulates the splicing of the actin-remodeling protein gelsolin, increasing gelsolin expression and leading to faster glucose-induced actin depolymerization and increased insulin release. Taken together, these findings indicate that beta cells share common splicing regulators and programs with neurons. These splicing regulators play key roles in insulin release and beta cell survival, and their dysfunction may contribute to the loss of functional beta cell mass in diabetes.

Papel del péptido insulinotrópico dependiente de glucosa en la programación nutricional del síndrome metabólico / Role of glucose-dependent insulinotropic peptide in the nutritional programming of metabolic syndrome

La restricción nutricional precoz ha sido asociada con una mayor incidencia de patologías relacionadas con el síndrome metabólico durante la edad adulta. Sin embargo, los mecanismos subyacentes que determinan el desarrollo de dichas patologías aún no se conocen en su totalidad. En el presente trabajo, se analizó el papel del péptido insulinotrópico dependiente de glucosa (GIP) en el desarrollo dichas patologías en un modelo de rata Wistar. Las ratas gestantes fueron alimentadas ad libitum (C) o sometidas a restricción nutricional (S) durante el embarazo y la lactancia, al final de la cual las crías fueron realimentadas con dieta grasa (CR, SR) durante 22 semanas. Tanto los machos como las hembras SR mostraron un fenotipo obesogénico caracterizado por hiperfagia, acumulación de grasa visceral e hipertrofia adipocitaria, de manera más pronunciada que la población CR. Los test de tolerancia oral a la glucosa mostraron que las hembras SR experimentaron intolerancia a la glucosa e hipersecreción de insulina y GIP. La administración del antagonista del receptor de GIP, (Pro3)GIP, a las hembras SR dio lugar a una significativa reducción del tejido adiposo y del tamaño adipocitario, junto a una mejora de la tolerancia a la glucosa y de la sensibilidad a la insulina. En conclusión, la exacerbada secreción de GIP parece representar el estímulo para la hipersecreción de insulina y el desarrollo de resistencia a la misma en las hembras SR, lo que sugiere que GIP jugaría un papel esencial en el desarrollo de alteraciones metabólicas asociadas a la rehabilitación nutricional

Early nutritional restriction has been associated with increased incidence of metabolic syndrome-associated pathologies in adulthood. However, the underlying mechanisms that determine the development of these diseases are not yet fully known. In the present work, we explored the relevance of glucose-dependent insulinotropic polypeptide (GIP) in the development of these pathologies in a model of Wistar rats. Two groups of dams were fed ad libitum (C) or food-restricted (U) during pregnancy and suckling. At that time, rats were refed a high-fat diet (HFD; CHF and UHF) for 22 weeks. Both male and female UHF rats showed an obese phenotype characterized by hyperphagia, visceral fat accumulation and adipocyte hypertrophy, which was more pronounced than in CHF rats. Oral glucose tolerance tests showed that female UHF rats experienced glucose intolerance, insulin hypersecretion and an exacerbated GIP secretion. Administration of the GIP receptor antagonist, (Pro3)GIP, to UHF female rats markedly reduced visceral fat mass and adipocyte hypertrophy, and these changes were accompanied by improvement of glucose tolerance and insulin sensitivity. In conclusion, the exacerbated production and secretion of GIP seems to represent the stimulus for insulin hypersecretion and insulin resistance shown by UHF female rats, suggesting that GIP may play a critical role in the development of metabolic disturbances related to nutritional rehabilitation

Early and Long-term Undernutrition in Female Rats Exacerbates the Metabolic Risk Associated with Nutritional Rehabilitation.

Human studies have suggested that early undernutrition increases the risk of obesity, thereby explaining the increase in overweight among individuals from developing countries who have been undernourished as children. However, this conclusion is controversial, given that other studies do not concur. This study sought to determine whether rehabilitation after undernutrition increases the risk of obesity and metabolic disorders. We employed a published experimental food-restriction model. Wistar female rats subjected to severe food restriction since fetal stage and controls were transferred to a moderately high-fat diet (cafeteria) provided at 70 days of life to 6.5 months. Another group of undernourished rats were rehabilitated with chow. The energy intake of undernourished animals transferred to cafeteria formula exceeded that of the controls under this regime and was probably driven by hypothalamic disorders in insulin and leptin signal transduction. The cafeteria diet resulted in greater relative increases in both fat and lean body mass in the undernourished rats when compared with controls, enabling the former group to completely catch up in length and body mass index. White adipose tissues of undernourished rats transferred to the high-lipid regime developed a browning which, probably, contributed to avoid the obesigenic effect observed in controls. Nevertheless, the restricted group rehabilitated with cafeteria formula had greater accretion of visceral than subcutaneous fat, showed increased signs of macrophage infiltration and inflammation in visceral pad, dyslipidemia, and ectopic fat accumulation. The data indicate that early long-term undernutrition is associated with increased susceptibility to the harmful effects of nutritional rehabilitation, without causing obesity.

Early undernutrition induces glucagon resistance and insulin hypersensitivity in the liver of suckling rats.

Developing brains are vulnerable to nutritional insults. Early undernutrition alters their structure and neurochemistry, inducing long-term pathological effects whose causal pathways are not well defined. During suckling, the brain uses glucose and ketone bodies as substrates. Milk is a high-fat low-carbohydrate diet, and the liver must maintain high rates of gluconeogenesis and ketogenesis to address the needs of these substrates. Insulin and glucagon play major roles in this adaptation: throughout suckling, their blood concentrations are low and high, respectively, and the liver maintains low insulin sensitivity and increased glucagon responsiveness. We propose that disturbances in the endocrine profile and available plasma substrates along with undernutrition-related changes in brain cortex capacity for ketone utilization may cause further alterations in some brain functions. We explored this hypothesis in 10-day-old suckling rats whose mothers were severely food restricted from the 14th day of gestation. We measured the plasma/serum concentrations of glucose, ketone body, insulin and glucagon, and hepatic insulin and glucagon responses. Undernutrition led to hypoglycemia and hyperketonemia to 84% (P < 0.001) and 144% (P < 0.001) of control values, respectively. Liver responsiveness to insulin and glucagon became increased and reduced, respectively; intraperitoneal glucagon reduced liver glycogen by 90% (P < 0.01) in control and by 35% (P < 0.05) in restricted. Cortical enzymes of ketone utilization remained unchanged, but their carrier proteins were altered: monocarboxylate transporter (MCT) 1 increased: 73 ± 14, controls; 169 ± 20, undernourished (P < 0.01; densitometric units); MCT2 decreased: 103 ± 3, controls; 37 ± 4, undernourished (P < 0.001; densitometric units). All of these changes, coinciding with the brain growth spurt, may cause some harmful effects associated with early undernutrition.

Early undernutrition increases glycogen content and reduces the activated forms of GSK3, AMPK, p38 MAPK, and JNK in the cerebral cortex of suckling rats.

Exposure to maternal undernutrition during development increases the risk for neurological and cognitive defects. However, little is known about the underlying mechanisms involved. Peripheral responses to insulin are increased following food-restriction, thus the possibility arises that brain insulin actions are affected by undernutrition, causing damages to the higher cerebral functions. In this study, we examined the effects of early undernutriton on molecular targets of insulin actions such as glucose transporters, glycogen, glycogen synthase kinase-3 (GSK3) and mitogen-activated protein kinases, as well as proteins involved in apoptosis in the cortex from 10-day-old rats. We show that undernutrition results in an enhanced glycogen content which is confined to astrocytes, according to our histochemical approaches. Cortical phospho-GSK3 is also increased. In addition to glycogen synthesis, GSK3 regulates crucial cellular processes. Therefore, its elevated degree of phosphorylation may have an impact on these processes and, consequently, on the cortical development. Phospho-p38 and both total JNK and phospho-JNK, which regulate apoptosis, are reduced following undernutrition. However, cleaved caspase 3 is not altered, which suggests that this condition does not induce extensive modifications to the cortical apoptosis. Thus, our results indicate that undernutrition gives rise to molecular alterations that may have repercussions on cerebral cortex development and functions.