Untargeted metabolomics identifies a plasma sphingolipid-related signature associated with lifestyle intervention in prepubertal children with obesity.

OBJECTIVE: Childhood obesity is a strong risk factor for adult obesity and metabolic diseases, including type 2 diabetes and cardiovascular disease. Early lifestyle intervention in children with obesity reduces future disease risk. The objective of this study is to identify metabolic signatures associated with lifestyle intervention in prepubertal children with obesity. METHODS: Thirty-five prepubertal children (7-10 years) with obesity (body mass index (BMI)>2 standard deviations) were enrolled in the study and participated in a 6-month-long lifestyle intervention program. Physiological and biochemical data and blood samples were collected both at baseline and after the intervention. A liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach was applied to obtain a comprehensive profiling of plasma samples, identifying 2581 distinct metabolite. Principal component analysis (PCA) was performed to consolidate all features into 8 principal components. Associations between metabolites and physiological and biochemical variables were investigated. RESULTS: The intervention program significantly decreased mean (95% CI) BMI standard deviation score from 3.56 (3.29-3.84) to 3.11 (2.88-3.34) (P<0.001). PCA identified one component (PC1) significantly altered by the intervention (Bonferroni adjusted P=0.008). A sphingolipid metabolism-related signature was identified as the major contributor to PC1. Sphingolipid metabolites were decreased by the intervention, and included multiple sphingomyelin, ceramide, glycosylsphingosine and sulfatide species. Changes in several sphingolipid metabolites were associated with intervention-induced improvements in HbA1c levels. CONCLUSIONS: Decreased circulating sphingolipid-related metabolites were associated with lifestyle intervention in prepubertal children with obesity, and correlated to improvements in HbA1c.

Influence of catch up growth on spatial learning and memory in a mouse model of intrauterine growth restriction.

BACKGROUND: Intrauterine growth restriction (IUGR) and rapid postnatal weight gain or catch up growth (CUG) increase the susceptibility to metabolic syndrome during adult life. Longitudinal studies have also revealed a high incidence of learning difficulties in children with IUGR. The aim of the present study was to investigate the effect of nutrition and CUG on learning memory in an IUGR animal model. We hypothesized that synaptic protein expression and transcription, an essential mechanism for memory consolidation, might be affected by intrauterine undernutrition. METHODS: IUGR was induced by 50% maternal caloric undernutrition throughout late gestation. During the suckling period, dams were either fed ad libitum or food restricted. The pups were divided into: Normal prenatal diet-Normal postnatal diet (NN), Restricted prenatal diet- Normal postnatal diet + catch up growth (RN+), Normal prenatal diet-Restricted postnatal diet (NR) and Restricted prenatal diet-Restricted postnatal diet (RR). At 4 weeks of age, memory was assessed via a water maze test. To evaluate synaptic function, 2 specific synaptic proteins (postsynaptic density-95 [PSD95], synaptophysin) as well as insulin receptors (IR) were tested by Western Blot and quantitative polymerase chain reaction (qPCR). Brain-derived neurotrophic factor and serum insulin levels were also studied. RESULTS AND CONCLUSIONS: The RN+ group presented a learning curve similar to the NN animals. The RR animals without CUG showed learning disabilities. PSD95 was lower in the RR group than in the NN and RN+ mice. In contrast, synaptophysin was similar in all groups. IR showed an inverse expression pattern to that of the PSD95. In conclusion, perinatal nutrition plays an important role in learning. CUG after a period of prenatal malnutrition seems to improve learning skills. The functional alterations observed might be related to lower PSD95 activity and a possible dysfunction in the hormone regulation of synaptic plasticity.

In utero undernutrition reduces diabetes incidence in non-obese diabetic mice.

AIMS/HYPOTHESIS: Observational studies in humans suggest that low birthweight may decrease the risk of type 1 diabetes, but the mechanism is unknown. We hypothesised that antenatal undernutrition would decrease the incidence of type 1 diabetes in non-obese diabetic (NOD) mice. MATERIALS AND METHODS: A 40% restriction of energy intake was applied to pregnant NOD dams from day 12.5 to day 18.5 of gestation, resulting in intrauterine growth retardation of offspring. All mice were fed a standard diet after weaning. Control and undernourished female offspring were followed to assess diabetes incidence. Male NOD mice were treated with cyclophosphamide to accelerate development of diabetes. Glucose homeostasis, body composition and pancreatic histology were compared in control and undernourished offspring. RESULTS: Mean birthweight was lower in undernourished than in control mice (p = 0.00003). At 24 weeks of age, the cumulative incidence of spontaneous diabetes in female mice was 73% in control and 48% in undernourished mice (p = 0.003). In cyclophosphamide-treated male mice, antenatal undernutrition also tended to reduce the development of diabetes (p = 0.058). Maternal leptin levels were lower in undernourished dams on day 18.5 of pregnancy (p = 0.039), while postnatal leptin levels were significantly higher in undernourished offspring at 4, 20 and 27 weeks of life (p < 0.05). Beta cell mass was similar in both groups (control = 0.4 mg; undernourished = 0.54 mg; p = 0.24). Histological evidence of apoptosis at 20 weeks was greater in control than in undernourished mice (control = 6.3 +/- 1.4%; undernourished = 4.2 +/- 0.3%, p = 0.05). CONCLUSIONS/INTERPRETATION: Antenatal undernutrition reduces the incidence of diabetes in NOD mice, perhaps via alterations in apoptosis.

Reductions in caloric intake and early postnatal growth prevent glucose intolerance and obesity associated with low birthweight.

AIMS/HYPOTHESIS: Low birthweight (LBW) and rapid postnatal weight gain, or catch-up growth, are independent risk factors for the development of obesity and diabetes during adult life. Individuals who are both small at birth and have postnatal catch-up growth are at the highest risk. We hypothesised that dietary interventions designed to attenuate catch-up growth in LBW subjects may have long-term beneficial consequences. MATERIALS AND METHODS: We used our previously described mouse model of LBW-associated diabetes, created by restricting maternal food intake to 50% during the last week of gestation. Control (C) dams and dams that had been subjected to undernutrition (U) were then provided either chow ad libitum after delivery or 50% food restriction on a per-day basis from delivery until weaning. We designated the resulting four groups control-control (CC), undernutrition-control (UC), control-undernutriton (CU) and undernutrition-undernutrition (UU), indicating the prenatal and postnatal experimental conditions, respectively. Carbohydrate metabolism and adiposity were assessed prospectively in offspring until age 6 months. RESULTS: Males that were small at birth and exhibited early postnatal catch-up growth developed glucose intolerance and obesity by age 6 months. In contrast, LBW mice without catch-up growth (UU) remained smaller than controls (CC), and glucose intolerance and obesity was prevented. Similarly, mice with normal birthweight that had blunted catch-up growth (CU) were leaner and had better tolerance test than CC mice. Catch-up growth during the first week of life correlated better than birthweight with glucose, fat mass and glucose tolerance up to 6 months of age. CONCLUSIONS/INTERPRETATION: Prevention of early catch-up growth reversed the development of glucose intolerance and obesity in our mouse model of LBW-associated diabetes.

Increased glucose disposal induced by adenovirus-mediated transfer of glucokinase to skeletal muscle in vivo.

In non-insulin-dependent diabetes mellitus, insulin-stimulated glucose uptake is impaired in muscle, contributing in a major way to development of hyperglycemia. We previously showed that expression of the glucose phosphorylating enzyme glucokinase (GK) in cultured human myocytes improved glucose storage and disposal, suggesting that GK delivery to muscle in situ could potentially enhance glucose clearance. Here we have tested this idea directly by intramuscular delivery of an adenovirus containing the liver GK cDNA (AdCMV-GKL) into one hind limb. We injected an adenovirus containing the beta-galactosidase gene (AdCMV-lacZ) into the hind limb of newborn rats. beta-Galactosidase activity was localized in muscle for as long as 1 month after delivery, with a large percentage of fibers staining positive in the gastrocnemius. Using the same approach with AdCMV-GKL, GK protein content was increased from zero to 50-400% of the GK in normal liver sample, and total glucose phosphorylating activity was increased in GK-expressing muscles relative to the counterpart uninfected muscle. Expression of GK in muscle improved glucose tolerance rather than changing basal glycemic control. Glucose levels were reduced by approximately 35% 10 min after administration of a glucose bolus to fed animals treated with AdCMV-GKL relative to AdCMV-lacZ-treated controls. The enhanced rate of glucose clearance was reflected in increases in muscle 2-deoxy glucose uptake and blood lactate levels. We conclude that restricted expression of GK in muscle leads to an enhanced capacity for muscle glucose disposal and whole body glucose tolerance under conditions of maximal glucose-insulin stimulation, suggesting that under these conditions glucose phosphorylation becomes rate-limiting. Our findings also show that gene delivery to a fraction of the whole body is sufficient to improve glucose disposal, providing a rationale for the development of new therapeutic strategies for treatment of diabetes.-Jiménez-Chillarón, J. C., Newgard, C. B., Gómez-Foix, A. M. Increased glucose disposal induced by adenovirus-mediated transfer of glucokinase to skeletal muscle in vivo.

Reduction of islet amylin expression and basal secretion by adenovirus-mediated delivery of amylin antisense cDNA.

Reduction of amylin content and secretion in rat islets was attempted by transduction with an adenovirus bearing a 0.2-kb fragment of rat amylin cDNA inserted in the antisense orientation (AdCMV-alpha amylin). Exposure of islets to AdCMV-alpha amylin at a multiplicity of infection (moi) of 200 (1.2 x 10(7) pfu/ml) reduced amylin mRNA levels by 37 +/- 5% (p < 0.005), whereas infection with an adenovirus expressing the reporter gene of beta-galactosidase (AdCMV-lacz) did not modify amylin expression. Transduction with the antisense construct was specifically associated with the decrease (30 +/- 6%; p < 0.001) in the amylin content. Insulin content was unaltered in AdCMV-alpha amylin islets compared to AdCMV-lacz-transduced or untransduced cells. Basal amylin secretion (2.8 mM glucose) was 36 +/- 3% (p < 0.005) lower in AdCMV-alpha amylin islets than in untransduced or AdCMV-lacz-transduced islets. In contrast, no difference in amylin secretion in response to high glucose concentrations (16.7 mM) was detected in AdCMV-alpha amylin-transduced islets. Thus, a reduction of amylin content and basal secretion in islet cells can be achieved by the adenovirus-mediated expression of antisense RNA.