| Research Backgrounds and AimsAs we all know, type2diabetes (T2D) is a chronic complex disorder that is affected by multiple genetic and environmental factors. The clinical picture of T2D is formed by impairment in insulin secretion and resistance to insulin action. However, the mechanisms that underlie individual differences in the predisposition to T2D remain obscure. Although multiple susceptibility loci have been identified for this disease, the reported genetic variants account for only a small proportion of the heritability of T2D. The identification of genetic variants influencing disease predisposition will, it is hoped, deliver clues to the processes involved in disease pathogenesis. Therefore, extensive efforts have been made to identify the disease-affecting genes to better understand the disease pathogenesis, find new targets for clinical therapy, and allow prediction of disease.Accumulating evidence indicates a key role for serine phosphorylation of IRS-1in the regulation of insulin action. Recent studies suggest that Rho-kinase (ROCK) is an important regulator of insulin signaling and glucose metabolism, via interaction with IRS-1. Moreover, it has also been reported that marked increase of insulin gene transcription and glucose-stimulated insulin secretion by suppression of the Rho/Rho-kinase pathway in vitro and in vivo. Taking these tips into account, Rho/Rho-kinase pathway may be considered one of the major determinants of individual susceptibility to insulin resistance, derangement of glucose metabolism and T2D.In this study, we screened for the genetic variants by direct sequencing of all exons of ROCK gene. Genomic DNA was extracted from peripheral leukocytes in type2diabetic patients. Genetic association analyses were performed in a T2D case-control study. Meanwhile, we cloned a sequence, comprising the predicted binding sequence around SNP, into the3’UTR of a luciferase reporter vector for functional analysis. We aimed to determine whether ROCK funcational genetic variants confer significant susceptibility to T2D.Methods and ResultsWe looked for potential functional genetic variants by direct sequencing of all exons of the ROCK gene in48type2diabetic patients. Fluorescent dye-terminator cycle sequencing was performed, and the products were analysed using an Applied Biosystems3130xl capillary sequencer (Applied Biosystems, Foster City, CA, USA). By sequencing, we identified eight variants. All of them are rare except three variants, which had a frequency more than0.05for the minor allele. For these three variants, one of them led to non-synonymous polymorphisms (rs2230774, Thr431Asn), and the others in the3’ untranslated region (3’-UTR)(rs978906and rs71932930). On the basis of sequencing data, we found that rs978906was in strong linkage disequilibrium with rs71932930. Eventually, only2variants (rs978906and rs2230774) were selected for genotyping in1920type2diabetic patients and1920age-and sex-frequency-matched controls from an unrelated Chinese Han population. Unconditional logistic regression showed that no significant association was observed between common variants in the coding region of ROCK gene and T2D in our study (all P values>0.05). We also tested the association between these2variants and diabetes-related traits in384non-diabetic participants. However, no significant association was detected for rs978906, rs2230774and diabetes-related traits (all P values>0.05). Considering the possibility that3’-UTR variants may have an effect on the regulation of gene expression, various in silico analyses were undertaken. Bioinformatics tools showed that the rs978906variant located in the miRNA-33a and miRNA-33b target sites in the3’-UTR of the ROCK gene. We cloned sequence, comprising the predicted binding sequence around rs978906, into the3’-UTR of a luciferase reporter vector, pMIR-REPORT, carrying the wild-type (pMIR-A, with the A allele) and mutant (pMIR-G, with the G allele) sequence of rs978906, respectively.293T cells were co-transfected with these reporter constructs and miRNA (miR-33a or miR-33b). No significant difference was found in luminescence signal among all groups (all P values>0.05).ConclusionsWith the current sample size, we did not find any mutation in the coding sequence of the ROCK gene that confers a genetic risk for T2D in a Chinese population. Research Backgrounds and AimsRho is one of the best-known members of the small guanosine triphosphate (GTP)-binding protein superfamily that, in addition to its effect on actin organization or through this effect mediates various cellular physiologic functions such as contraction, migration, proliferation, and apoptosis. Once Rho is activated by agonists of receptors coupled to cell membrane G protein (such as endothelin, angiotensin Ⅱ and noradrenalin) and et al., it translocates to the cell membrane where it activates Rho-associated kinase (ROCK). ROCK, one of the first and the main downstream targets of the small GTP-binding protein Rho, activates myosin light chain kinase by phosphorylation of the myosin binding subunit in myosin light chain phosphatase, which is the main downstream target of ROCK, contributing to the pathogenesis and progression of endothelial dysfunction and several cardiovascular diseases. For years, investigation of the physiological roles of Rho/ROCK has largely focused on its association with the pathogenesis of hypertension, hypercholesterolemia, coronary artery diseases, pulmonary hypertension, heart failure, myocardial hypertrophy and stroke. Several lines of evidence have shown that increased ROCK activity may play a critical role in cardiovascular physiology and pathophysiology. Rho/ROCK, therefore, is becoming a new therapeutic target in cardiovascular disease.Recently, a role for Rho/ROCK in the pathogenesis of insulin resistance and diabetes has emerged. Considerable evidence suggests that ROCK is substantially involved in the pathogenesis of diabetes and subsequent complications via a direct interaction with IRS-1. RhoA/ROCK activation in diabetic rodent models has been shown in earlier studies. However, clinical studies is lacking showing that ROCK activity is increased in type2 diabetic patients. Such information may be clinically useful in developing novel therapies for T2D. The purpose of this study, therefore, is to determine whether ROCK activity is increased in T2D and whether this correlates with diabetes-related traits.Methods and ResultsVenous blood samples were drawn from41type2diabetic patients (56.1%men, mean age51.3±6.0years) and37control subjects (51.4%men, mean age50.4±4.1years). A total of7patients were followed up for a median of8weeks. All participants’ demographics were recorded. Rho-kinase activity in peripheral leukocytes was examined by determining the ratio of phosphorylated/total forms of myosin-binding subunit (MBS) of myosin light chain phosphatase. The expressions of total MBS were normalized to beta-actin in all participants. Rho-kinase activity in circulating neutrophils was significantly higher in the type2diabetic patients overall compared with control subjects (phosphorylated MBS/total MBS ratio0.80±0.11vs.0.71±0.08, P=0.01). There was also a significant correlation between p-MBS/total-MBS and HbAlc in all participants (P<0.05). No correlation was noted between Rho-kinase activity and age, body mass index, fasting glucose, systolic blood pressure or diastolic blood pressure in all subjects (all P values>0.05). In the T2D group, significant correlation was noted between Rho-kinase activity and HbAlc (R=0.52, P=0.01). In the control group, there was no significant correlation between Rho-kinase activity and diabetes-related traits (all P values>0.05). After the8-week medical treatment, Rho-kinase activity in the follow up of type2diabetic patients were significantly decreased (P=0.04).ConclusionsThese findings provide the first clinical evidence that Rho-kinase activity in peripheral leukocytes is enhanced in type2diabetic patients and may be a useful biomarker for the severity of T2D. |
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