GCK exonic mutations induce abnormal biochemical activities and result in GCK-MODY.

Objective: Glucokinase-maturity-onset diabetes of the young (GCK-MODY; MODY2) is a rare genetic disorder caused by mutations in the glucokinase (GCK) gene. It is often under- or misdiagnosed in clinical practice, but correct diagnosis can be facilitated by genetic testing. In this study, we examined the genes of three patients diagnosed with GCK-MODY and tested their biochemical properties, such as protein stability and half-life, to explore the function of the mutant proteins and identify the pathogenic mechanism of GCK-MODY. Methods: Three patients with increased blood glucose levels were diagnosed with MODY2 according to the diagnostic guidelines of GCK-MODY proposed by the International Society for Pediatric and Adolescent Diabetes (ISPAD) in 2018. Next-generation sequencing (whole exome detection) was performed to detect gene mutations. The GCK gene and its mutations were introduced into the pCDNA3.0 and pGEX-4T-1 vectors. Following protein purification, enzyme activity assay, and protein immunoblotting, the enzyme activity of GCK was determined, along with the ubiquitination level of the mutant GCK protein. Results: Genetic testing revealed three mutations in the GCK gene of the three patients, including c.574C>T (p.R192W), c.758G>A (p.C253Y), and c.794G>A (p.G265D). The biochemical characteristics of the protein encoded by wild-type GCK and mutant GCK were different, compared to wild-type GCK, the enzyme activity encoded by the mutant GCK was reduced, suggesting thermal instability of the mutant GST-GCK. The protein stability and expression levels of the mutant GCK were reduced, and the enzyme activity of GCK was negatively correlated with the levels of fasting blood glucose and HbA1c. In addition, ubiquitination of the mutant GCK protein was higher than that of the wild-type, suggesting a higher degradation rate of mutant GCK than WT-GCK. Conclusion: GCK mutations lead to changes in the biochemical characteristics of its encoded proteins. The enzyme activities, protein expression, and protein stability of GCK may be reduced in patients with GCK gene mutations, which further causes glucose metabolism disorders and induces MODY2.

Vitamin D inhibits palmitate-induced macrophage pro-inflammatory cytokine production by targeting the MAPK pathway.

Vitamin D insufficiency is associated with chronic inflammatory diseases. However, the mechanism by which vitamin D reduces obesity-related inflammation remains poorly understood. In this study, we investigated the inhibitory effects of vitamin D on palmitate-induced inflammatory response in macrophages and explored the potential mechanisms of vitamin D action. The effect of vitamin D on the expression of inflammatory factors induced by palmitate, a saturated fatty acid, was investigated using human THP-1 macrophages and murine RAW 264.7 cells. To elucidate the mechanism by which vitamin D affects palmitate-induced inflammatory cytokine production, we investigated the activity of stress kinase-related proteins. Palmitate significantly increased TNF-α and IL-6 expression and secretion in THP-1 and RAW 264.7 macrophages. Treatment with the active form of vitamin D inhibited palmitate-induced TNF-α and IL-6 production in macrophages. Furthermore, vitamin D significantly reduced palmitate-stimulated activation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase 1/2 (ERK1/2). The mitogen-activated protein kinase signaling pathway partly accounts for the induction of pro-inflammatory cytokines by palmitate. Our data suggest that the attenuation of palmitate-induced TNF-α and IL-6 gene expression and protein secretion by vitamin D are associated with reduced activation of JNK and ERK1/2.

Folic acid supplementation alters the DNA methylation profile and improves insulin resistance in high-fat-diet-fed mice.

Folic acid (FA) supplementation may protect from obesity and insulin resistance, the effects and mechanism of FA on chronic high-fat-diet-induced obesity-related metabolic disorders are not well elucidated. We adopted a genome-wide approach to directly examine whether FA supplementation affects the DNA methylation profile of mouse adipose tissue and identify the functional consequences of these changes. Mice were fed a high-fat diet (HFD), normal diet (ND) or an HFD supplemented with folic acid (20 µg/ml in drinking water) for 10 weeks, epididymal fat was harvested, and genome-wide DNA methylation analyses were performed using methylated DNA immunoprecipitation sequencing (MeDIP-seq). Mice exposed to the HFD expanded their adipose mass, which was accompanied by a significant increase in circulating glucose and insulin levels. FA supplementation reduced the fat mass and serum glucose levels and improved insulin resistance in HFD-fed mice. MeDIP-seq revealed distribution of differentially methylated regions (DMRs) throughout the adipocyte genome, with more hypermethylated regions in HFD mice. Methylome profiling identified DMRs associated with 3787 annotated genes from HFD mice in response to FA supplementation. Pathway analyses showed novel DNA methylation changes in adipose genes associated with insulin secretion, pancreatic secretion and type 2 diabetes. The differential DNA methylation corresponded to changes in the adipose tissue gene expression of Adcy3 and Rapgef4 in mice exposed to a diet containing FA. FA supplementation improved insulin resistance, decreased the fat mass, and induced DNA methylation and gene expression changes in genes associated with obesity and insulin secretion in obese mice fed a HFD.

Folic acid prevents cardiac dysfunction and reduces myocardial fibrosis in a mouse model of high-fat diet-induced obesity.

BACKGROUND: Folic acid (FA) is an antioxidant that can reduce reactive oxygen species generation and can blunt cardiac dysfunction during ischemia. We hypothesized that FA supplementation prevents cardiac fibrosis and cardiac dysfunction induced by obesity. METHODS: Six-week-old C57BL6/J mice were fed a high-fat diet (HFD), normal diet (ND), or an HFD supplemented with folic acid (FAD) for 14 weeks. Cardiac function was measured using a transthoracic echocardiographic exam. Phenotypic analysis included measurements of body and heart weight, blood glucose and tissue homocysteine (Hcy) content, and heart oxidative stress status. RESULTS: HFD consumption elevated fasting blood glucose levels and caused obesity and heart enlargement. FA supplementation in HFD-fed mice resulted in reduced fasting blood glucose, heart weight, and heart tissue Hcy content. We also observed a significant cardiac systolic dysfunction when mice were subjected to HFD feeding as indicated by a reduction in the left ventricular ejection fraction and fractional shortening. However, FAD treatment improved cardiac function. FA supplementation protected against cardiac fibrosis induced by HFD. In addition, HFD increased malondialdehyde concentration of the heart tissue and reduced the levels of antioxidant enzyme, glutathione, and catalase. HFD consumption induced myocardial oxidant stress with amelioration by FA treatment. CONCLUSION: FA supplementation significantly lowers blood glucose levels and heart tissue Hcy content and reverses cardiac dysfunction induced by HFD in mice. These functional improvements of the heart may be mediated by the alleviation of oxidative stress and myocardial fibrosis.

Characterization and predicted role of microRNA expression profiles associated with early childhood obesity.

MicroRNAs (miRNAs) are implicated in the pathogenesis of obesity. The aim of the present study was to characterize the miRNA profile associated with early childhood obesity in peripheral blood mononuclear cells (PBMCs). A total of 12 children (6 obese and 6 lean controls) aged 36 months old to 48 months old were recruited. The miRNA expression profile from PBMCs was detected using the multiplexed NanoString nCounter system. Bioinformatics was employed to detect target genes and miRNA­regulated biological function. A total of 9 differentially expressed miRNAs were identified in obese children compared with lean children (P<0.05). Among the 9 miRNAs, miR­199a­3p/miR­199b­3p and miR­4454 presented at least a 1.5­fold change in expression. A total of 643 potential target genes were regulated by the three miRNAs, and 291 of the potential genes were involved in a protein interaction network. Gene ontology annotation indicated that 291 potential genes were enriched in 14 biological process annotations and 2 molecular function annotations. miRNA dysregulation may be involved in early childhood obesity.

miR-206-3p Inhibits 3T3-L1 Cell Adipogenesis via the c-Met/PI3K/Akt Pathway.

MicroRNAs (miRNAs) are important post-transcriptional regulators during adipocyte adipogenesis. MiR-206-3p, a tissue-specific miRNA, is absent in white adipocytes. In this study, we examined the roles of mmu-miR-206-3p in the adipogenic differentiation of 3T3-L1 preadipocytes. The miR-206-3p expression has shown an apparent decreasing trend after induction, and sustained low expression throughout the differentiation of 3T3-L1 cells. miR-206-3p blocked the adipogenic differentiation of 3T3-L1 cells by attenuating c-Met expression; the inhibition effect of miR-206 to the adipogenic differentiation can be counteracted by restoring c-Met expression. In addition, miR-206-3p decreased the phosphorylation of Akt, which is the downstream effector of c-Met in the PI3K/Akt signaling pathway. These data indicate that miR-206-3p inhibits adipocyte adipogenesis through silencing c-Met and subsequently inactivating the PI3K/Akt signaling pathway.

Long Non-Coding RNA Expression Profiling in Obesity Mice with Folic Acid Supplement.

BACKGROUND/AIMS: Obesity is a major contributor to the growing prevalence of metabolic and cardiovascular diseases. This study was designed to investigate the effect of folic acid (FA) on obese mice by detecting the genome-wide expression profile of lncRNAs and mRNAs in the heart. METHODS: Heart samples were collected from mice fed with standard diet (SD), high-fat diet (HFD) and high-fat diet with FA intake (HFDF). LncRNAs and mRNAs between HFD and HFDF group were analyzed by lncRNA microarray. Nine lncRNAs and mRNAs were validated using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Bioinformatics prediction was used to investigate the potential function of these differentially expressed lncRNAs. Co-expresson analysis was used to determine the transcriptional regulatory relationship of differentially expressed lncRNAs and mRNAs between two groups. RESULTS: The expression of 58,952 lncRNAs and 20,145 mRNAs in HFD and HFDF groups was profiled by using microarrays. Gene Ontology and pathway analyses indicated that the biological functions of differentially expressed mRNAs were related to inflammation, energy metabolism, and cell differentiation. Co-expression networks composed of lncRNAs and mRNAs were also constructed to investigate the potential regulatory roles of differentially expressed lncRNAs on mRNAs. LncRNAs, namely, NONMMUT033847, NONMMUT070811, and NONMMUT015327, were validated through qRT-PCR, and these lncRNAs may be important factors regulating inflammation, energy metabolism, and cell differentiation. The expression levels of Dnajb1, Egr2, Hba-a1, Il1ß, Cxcl2, and Tnfsf9 were significantly different between HFD and HFDF. CONCLUSIONS: Results suggested that FA may improve the cardiovascular function of obesity and contribute to those lncRNAs associated with inflammation and cell differentiation. In a nutshell, the present study identified a panel of lncRNAs and mRNAs that may be potential biomarkers or drug targets relevant to the high-fat diet related obesity.

Hypermethylation of AKT2 gene is associated with neural-tube defects in fetus.

INTRODUCTION: Neural-tube defects (NTDs) are common birth defects of complex etiology. Although many studies have confirmed a genetic component, the exact mechanism between DNA methylation and NTDs remains unclear. METHODS: In this work, we investigated the alteration of methylation from placental tissues obtained from 152 normal infants or with NTDs in 130 children with neural-tube defects. Genome-wide changes in DNA methylation were measured using the NimbleGen microarray. The expression levels of 12 genes were also determined, and two genes (AKT2 and CDC25C) showed low expression in NTDs by quantitative real-time PCR analysis. Then, the methyhlated region of AKT2 promoter sequences were confirmed by massARRAY. RESULTS: A total of 150 differentially methylated regions (81 low methylated regions and 69 high methylated regions) were selected by microarray. The expression levels of AKT2 and CDC25C showed lower expression in NTDs. And the percentage of methyhlated region of AKT2 promoter were increased in NTDs. CONCLUSIONS: DNA mythelation was one of the possible epigenetic variations correlated with the occurrence of NTDs, and AKT2 may be a candidate gene for NTDs.

[Lifestyle-related factors and cancer incidence in men: a public health assessment based on a 'healthy lifestyle score'].

OBJECTIVE: To evaluate the effects of combined lifestyle-related factors and risk of cancer incidence among adult men in urban Shanghai. METHODS: Information was obtained from 60 817 men in the Shanghai Men's Health Study (2002 - 2006) program and 2033 incident cancers who were confirmed at the end of 2009. A healthy lifestyle score (HLS) system was developed, based on five lifestyle-related factors, and participants were scored one point for each of the healthy behaviour: never smoked, alcohol intake less than 1 drink/day, under normal weight range (18.5 - 27.9 kg/m(2)), physical activity (≥ 54 Met-hours/week), fruit and vegetable intake ≥ 451 g/day, the else would score as zero. The total score would range from zero to five. Cox regression model was used to evaluate the associations between combined lifestyle-related factors and the cancer incidence. RESULTS: Compared to men having scores as zero or one, the hazard ratios (95% confidence intervals) for men with two, three, four, five health behaviour scores were 0.77 (95%CI: 0.66, 0.90), 0.67 (95%CI: 0.58, 0.78), 0.56 (95%CI: 0.47, 0.65), 0.51 (95%CI: 0.42, 0.62), respectively. The population attributable risks for cancer incidence was 10.4% for those having scores less than 3 items. CONCLUSION: Never smoked, moderate alcohol intake, maintaining normal weight, being physically fit, and having enough daily fruit and vegetable intake were associated with lower risk of total cancer incidence in men. Our data showed that healthy lifestyle could significantly benefit the public health programs of the population.