The Role Of Mitochondria And Its Regulatory Genes In The Pathophysiology Of Impaired Glycolipids Metabolism

ObjectiveMitochondrial DNA (mtDNA) mutation and reduced mitochondrial DNA content both involved in the mitochondrial dysfunction. Reduced mitochondrial DNA content plays a driving force in age-related regenerative diseases such as type 2 diabetes. The study will carry on from mitochondrial diabetes, and further to investigate the association of mitochondrial DNA content and impaired glucose metabolism in type 2 diabetes. By building high-fat induced UCP2 gene (Uncoupling protein 2, a vital gene regulates oxidative phosphorylation) knockout mice, to explore UCP2 and its upstream regulatory genes in the glucolipids metabolism and to analyze these genes polymorphisms in Chinese population with variant plasma glucose status (ranging from normal glucose tolerance, pre-diabetes to diabetes). Furthermore, to study the influence of dietary structure on the mitochondrial function and telomere length (Telomeres are essential and dynamic regulators of cellular life span and chromosome integrity in eukaryocyte), analyzing the role of diet in mitochondria mediated abnormality of glucolipids metabolism. We aim to comprehensively investigate the pathophysiology of type 2 diabetes from the aspects of genetic and environmental factors. Moreover, we also aim to explore practical predictors used for assessment of insulin resistance and islet β cell function and to assess whether the blood lipid ratio (TG/HDL-C) could be a useful markers for assessment of insulin resistance and islet P cell function.MethodsSubjects in the study including: ①Rixteen mitochondrial diabetes patients with mitochondrial DNA 3243 A>G mutation from department of endocrinology in Peking Union Medical College Hospital (PUMCH) between 2007 and 2015; ②Five hundred and ninety-nine subjects with varied glucose tolerance were recruited from a type 2 diabetes project in a Beijing suburb in China between March 2014 and January 2015; ③Seventy-six subjects with type 2 diabetes in 6-years cohort study were recruited from outpatient department of endocrinology in PUMCH between April 2005 and April 2006; We collected demographic, clinical feature, biochemical data and dietary intake in population. The PCR assay was designed to detect the mt 3243A to G mutation using a direct PCR. The peak height G/A ratio was defined as the peak height A was divided by the peak height G. The relative mtDNA copy numbers and telomere length were measured by quantitative real-time PCR (qPCR) and corrected by simultaneous measurement of nuclear DNA. Gene polymorphism analysis using Sequenom mass spectrum detection platform, to test 8 UCP2 function polymorphism loci (rs660339, rs659366, rs649446, rs586773, rs34408426, rs7109266, rs3019463, rs591758) and 7 PPARy function polymorphism Ioci(rs3856806, rs2920502, rs17029007, rs73021485, rs73813168, rs2920503, rs79310821). The oxidative stress indicators(SOD, GR,8-oxo-dG) and inflammation factors(IL-6, TNF-a) were measured by Elisa kits. Animals model:male UCP2-/-mice with C57BL/6 backgrounds and the control UCP2+/+mice with the same C57BL/6 backgrounds fed by high-fat diet for 16 weeks. The gene expression profiles in the mouse liver tissues were determined using genome-wide gene expression arrays. The liver tissues were analyzed by an Affymetrix Mouse Gene ST 1.0 array.Results1. Analysis of association among clinical features, mutation heterogeneity and disease spectrum in mitochondrial diabetes with m.3243A>G mitochondrial DNA mutationThe patients with mitochondrial diabetes all had definite maternally inherited history, normal BMI (19.5±2.36 kg/m2), early onset of diabetes (35.0±14.6 years) and deafness. The peak height G/A ratio was negatively associated with the age at the diagnosis of diabetes after adjusting the age, BMI (r=-0.841; P=0.001).2.Reduced peripheral blood mitochondrial DNA content associates with impaired glucose-stimulated islet β cell functionPeripheral blood mtDNA content was positively associated with DI30, DI120 and was negatively related with 30min,60min,120min plasma glucose after oral glucose. But there was no correlation between mt DNA content and the index the basal insulin secretion, serum lipid. Multiple linear regression showed that reduced peripheral blood mt DNA content increases the risk of impaired glucose-stimulated β cell function (DI30:0=0.104, P=0.019; DI120:(3=0.116, P=0.009). Multiple logistic regression indicated that peripheral mtDNA content negatively associated with the risk of type 2 diabetes (OR:0.468,95% CI:0.245-0.893, P=0.021).3.Effects of UCP2 on glucolipids metabolism and related signaling pathway expression profiling analysisThe insulin sensitivity and β cell function were improved in UCP2-/-group on high-fat diet. The serum total cholesterol, triglyceride and free fatty acid concentrations were significantly lower in the UCP2-/-group on high-fat diet than in the UCP2+/+group on high-fat diet. According to gene profiling analysis, we concentrated on the "PPAR signaling pathway", because it is closely associated with the regulation of glucose and lipid profiles.In PPAR signaling pathway,seven genes (Ppary,Dbi,Acsl3,Lp1,Mel,Scdl,Fads2) in the UCP2-/-mice were significantly up-regulated. The expression of other PPAR isoforms, Ppara and PparS were not significantly different between UCP2-/-group and UCP2+/+group.4.Association between UCP2-PPARy polymorphism and impaired glucolipids metabolismThere was no significant difference among allele frequencies, genotype in every UCP2 gene SNP loci. The G allele, GG genotype of rs2920502 in PPARy were associated with decreased risk of impaired glucose tolerance(G allele:OR:0.818,95% CI:0.526-0.969, P=0.042; GG genotype:OR:0.715,95% CI:0.527-0.97, P=0.031). The T allele, TT genotype of rs3856806 in PPARy were associated with increased risk of impaired glucose tolerance (T allele:OR:1.46,95%CI:1.055-2.017, P=0.022; TT genotype:OR:1.58,95%CI:1.104-2.761,p=0.032).5.Telomere length among mitochondrial diabetes, type 2 diabetes and normal controlPeripheral blood leukocyte DNA telomere length was significantly shorter in patients with mitochondrial diabetes and type 2 diabetes than in healthy controls; however, there was no significant difference between those with mitochondrial diabetes and those with type 2 diabetes (Mitochondrial diabetes vs type 2 diabetes vs normal control:1.28±0.54 vs 1.14±0.43 vs 1.63±0.61, P=0.000). There was no significant relationship between peripheral blood leukocyte DNA telomere length and the peak height G/A ratio (r=-0.156, P=0.646).6.1nfluence of Diet on Leukocyte Telomere Length and glucose metabolismDiabetes group had the shortest telomere length and normal glucose group had the longest telomere length (Control vs Pre-diabetes vs Diabetes:2.01±0.03 vs 1.97±0.03 vs 1.89±0.03, P= 0.005). TL was not associated with daily energy intake, diet fat, carbohydrates and protein proportions. Multiple linear regression analysis indicated that legumes, nuts, fish and seaweeds were protective factors for TL shortening, and sweetened carbonated beverage was a risk factor for LTL shortening (legumes: β=0.105, P=0.018; nuts:β=0.110, P=0.011; fish:β=0.118, P=0.007; seaweeds: β=0.116, P=0.009; sweetened carbonated beverage:β=-0.120, P=0.004). Seaweeds and dairy intake were negatively associated with 8-oxo-dG (seaweed:r=-0.496, P =0.001; dairy:r=-0.246, P=0.046), vegetables and fruits were positively associated with GR (vegetables:r=0.101, P=0.034; fruits:r=0.125, P=0.045).7.The predictive role of TG/HDL-C ratio in islet P function and insulin resistanceIn population with varied glucose tolerance, TG/HDL, TG were significantly associated with IR. The AUROCs of TG/HDL-C and TG were 0.71(95% CI: 0.66-0.75) and 0.71(95% CI:0.65-0.75), respectively. The optimal cut-offs of TG/HDL-C and TG for IR diagnosis were 1.11 and 1.33 mmol/L, respectively. TG/HDL-C, TG were negatively associated with HOMA-P,but AUROCs were less than 0.50;therefore,the lipid ratios could not be predictors of basal β cell dysfunction. In the following 6-years cohort study, subjects were divided into slow decrease of beta cell function group and fast decrease group according to median of Delta CP AUC. Baseline log (TG)/HDL-C was positively correlated with Delta CP AUC (r=0.306, P =0.027); log (TG)/HDL-C in fast decrease group was higher than that in slow decrease group (0.103±0.033 vs 0.083±0.030, P=0.027). Baseline HDL-C at the 5 time points before and the 3h AUC in slow decrease group were all higher than those in fast decrease group. Baseline TG at the 5 time points and the 3 h AUC in Delta CP fast decrease group were higher than those in slow decrease group. Spearman correlation analysis showed that baseline HDL-C were negatively correlated with Delta CP AUC; triglyceride were positively correlated with Delta CP AUC.Conclusion1. Mitochondrial DNA 3243 A>G mutation causes the onset of mitochondrial diabetes, Deafness with definite maternal inheritance and normal/lower BMI, for the most part, suggest the diagnosis of mitochondrial diabetes. According to Sanger sequencing, the peak height G/A ratio could predict the onset age of the disease and might therefore provide a prediction regarding the age of onset in particular cases.2. The study first found that in population with varied glucose tolerance, decreased peripheral blood mtDNA content was probably more closely associated with glucose-stimulated insulin secretion than with basal secretion. Peripheral blood mtDNA content was positively associated with (3 cell function at early-phase insulin release and upon total-phases insulin release.3.3. UCP2 deficiency led to the amelioration of lipid metabolism and improved blood glucose by simultaneously promoting insulin sensitivity and β cell function through PPAR signaling pathway. The gene polymorphism of PPARy (rs2920502, rs3856806) closely related to glucolipid metabolism. In the liver the PPARy is important in regulating UCP2 under high-fat diet.4. Telomere length shortening is likely involved in the pathogenesis of diabetes; however, it was not specific for mitochondrial diabetes. Diet ingredients significantly impacted on markers of inflammation and oxidative stress, which probably had an effect on LTL.5. In a Chinese population with different levels of glucose tolerance, we first found that TG/HDL-C could be the predictors of IR. TG/HDL-C was significantly negative associated with β cell function in the population. In type 2 diabetes patients, high baseline log (TG)/HDL-C ratio predicts fast progression of islet beta cell dysfunction. It may be a simple index to predict progression speed of islet beta cell dysfunction.