| Diabetes is a common nutrition-related metabolic disease. Type 2 diabetes mellitus (T2DM) accounts for approximately 90-95% of those with diabetes. In the natural history of T2DM progression, prediabetes, also called impaired glucose regulation (IGR) including impaired fasting glucose (IFG) and impaired glucose tolerance (IGT), has been recognized as metabolic intermediate states between a "normal" state and frank T2DM.With the rapid development of social and economic status and changes in lifestyle, incidence rate and prevalence of diabetes has been increasing at an alarming rate. According to the latest estimation by International Diabetes Federation (IDF), the global number of diabetes cases is approximately 285 million people or 6.6% in the adult group (aged 20-79 years), accounting for 11.6% of the total healthcare expenditure in the world in 2010 (at least USD 376 billion). This number is expected to increase by more than 50%, to 438 million or 7.8% of the adult population in 2030. In China, the prevalence rates of total diabetes and prediabetes were 9.7% and 15.5%, respectively, accounting for 92.4 million adults with diabetes and 148.2 million adults with prediabetes. Thus, T2DM has no doubt become a worldwide epidemic and a leading threat to human health and development.As a complex disease, the pathological mechanisms of T2DM remain far to be clearly understand. Increasing evidence shows that oxidative stress, induced by hyperglycemia and possibly by free fatty acid (FFA), has been implicated in insulin resistance and beta-cell dysfunction, as well as the development of T2DM. Our group has previously found that oxidative stress markers were elevated in newly-diagnosed T2DM patients, manifesting increased plasma MDA concentration and DNA damage, decreased total antioxidant capacity and SOD activity in erythrocytes. In pancreatic beta cells, although high glucose exposure failed to induce the classical antioxidant enzymes, such as superoxide dismutase, catalase and glutathione peroxidase, it resulted in a marked rise in both heme oxygenase-1 (HO-1) gene expression and enzyme activities in the islets, in parallel with hyperglycemia-induced intracellular peroxide levels, which suggested that HO-1 might be early indicator for oxidative damage in pancreatic beta cells.Heme oxygenase-1 (HO-1), also known as heat shock protein 32 (Hsp32), is the inducible isoform of heme oxygenase that catalyzes the NADPH-dependent decomposition of heme to carbon monoxide (CO), ferrous iron, and biliverdin, which can be rapidly converted to bilirubin by biliverdin reductase. HO-1 has been emerged as a vital stress-responsive protein participating in the process of oxidative stress, inflammation, apoptosis, etc., and a potential novel target for anti-diabetic therapy.In STZ-induced diabetic mouse, our group has found that HO-1 expression in spleen was reduced. In addition, HO-1 expression in renal mitochondria was changed with time, showing an elevated level at the fourth week and reduced level at the eighth week. On the other side, our group have observed a (GT)n repeats microsatellite polymorphism and a single nucleotide polymorphism (SNP), T(-413)A, in the promoter region of HO-1 gene in a Chinese population comprising 1103 newly-diagnosed T2DM cases and 1615 controls. Moreover, allele L (> 25 repeats) has been suggested as a risk allele for T2DM, and individuals with LL genotype conveyed higher risk of developing T2DM compared with those with SS genotype (OR=1.37, P=0.007).Recently, it has been found that plasma or serum HO-1 is detectable and can serve as a novel biomarker for oxidative stress in chronic silicosis, Parkinson's disease and hemophagocytic syndrome. However, the association between circulating HO-1 concentration and T2DM has not been reported.Therefore, on the basis of our previous studies, we will focus on the relationship between plasma HO-1, HO-1 associated microRNA profiles in plasma, and type 2 diabetes, and we will also explore the impact of HO-1 gene polymorphisms on plasma HO-1 levels and ferritin levels. The main contents were shown as follows: Part One Association between plasma HO-1 levels and T2DM as well as impaired glucose regulationSection 1 Association between plasma HO-1 levels and T2DMObjective:To compare plasma HO-1 levels in T2DM patients and non-diabetic individuals, to assess the correlation between plasma HO-1 concentration and glucose and lipid indicators, and to explore the association between plasma HO-1 and T2DM.Methods:We performed a case-control study comprising T2DM patients who took oral glucose tolerance test (OGTT) at the outpatient clinics of Department of Endocrinology of Tongji Hospital affiliated to Tongji Medical College, and controls from an unselected population that underwent for a routine health examination in the same hospital during the period of December 2004 to December 2007. The diagnosis of T2DM was established according to the WHO 1999 criteria. Basic information was collected by questionnaire or anthropometric measurements. Plasma glucose, insulin, lipid profiles and HO-1 concentration was detected.Results:(1) The individuals with T2DM, compared to non-diabetic controls, had higher BMI, higher prevalence of alcohol drinking, hypertension, family history of diabetes, and higher levels of triglyceride, total cholesterol, FPG, and OGTT 2h. When looking at the insulin sensitivity indexes, we observed a lower HOMA-beta but a higher HOMA-IR in diabetes cases than that in the controls. (2) Plasma HO-1 concentrations were significantly increased in patients with New-T2DM compared with controls (P<0.001). (3) Plasma HO-1 concentrations were significantly correlated with plasma glucose concentrations (including FPG and OGTT2h), HOMA-beta, and HOMA-IR. (4) We observed increased ORs for New-T2DM associated with higher level of the plasma HO-1 concentration-likely a dose-response trend of this association. Participants in the highest quartile of plasma HO-1 concentrations, compared with the lowest, had a significantly increased risk for New-T2DM. After adjustment for age, sex, BMI and family history of diabetes, the adjusted OR was 8.23 (95% CI 5.55-12.21; P for trend <0.001). When stratified by age, sex and BMI, the association of plasma HO-1 concentrations and New-T2DM remained significant in all subgroups.Conclusion:Elevated plasma HO-1 concentrations were associated with an increased OR for New-T2DM, with strong dose-response trend. The association between plasma HO-1 concentrations and New-T2DM remained significant even after adjustment for known risk factors for T2DM.Section 2 Association between plasma HO-1 levels and impaired glucose regulationObjective:To examine the association between plasma HO-1 concentration and impaired glucose regulation (IGR), a generally regarded prediabetic state, in a non-diabetic Chinese population.Methods:Subject recruitment was described in section 1. According to the WHO 1999 diagnostic criteria, they were divided into IGR group (including IFG and IGT) and NGT control group. Basic information was collected by questionnaire or anthropometric measurements. Plasma glucose, insulin, lipid profiles, HO-1 and C-reactive protein (CRP) concentration were detected.Results:(1) The individuals with IGR, compared to NGT controls, had higher BMI, higher prevalence of hypertension, family history of diabetes, higher levels of FPG, OGTT 2h, triglyceride, CRP and HOMA-IR, and lower levels of HOMA-beta. (2) Plasma HO-1 concentrations were significantly increased in individuals with IGR, compared with controls (P<0.001). (3) We observed increased ORs for IGR associated with higher level of the plasma HO-1 concentration. Participants in the highest quartile of plasma HO-1 concentrations, compared with the lowest, had a significantly increased risk for IGR. After adjustment for age, sex, BMI, smoking, alcohol drinking, hypertension, family history of diabetes, the adjusted OR was 3.39 (95% CI 2.08-5.54; P for trend <0.001). Additional adjustment for TC, TG, HDL-C and CRP did not significantly alter the association (OR= 3.12,95% CI 1.89-5.16; P for trend <0.001). (4) When plasma HO-1 concentration was added to an established conventional risk model (including age, sex and BMI, smoking, alcohol drinking, hypertension, family history of diabetes, TC, TG, HDL-C and CRP), the area under the receiver-operating characteristic curves was increased (0.73 (0.70-0.77) vs. 0.75 (0.72-0.79); P for difference=0.010).Conclusion:Elevated plasma HO-1 concentration is significantly associated with increased ORs for IGR. The association between plasma HO-1 concentration and IGR retained rather consistent under adjustment for established factors, lipid profiles and inflammation marker CRP.Part Two Relationship among HO-1 gene polymorphisms, plasma ferritin concentration and T2DMSection 1 HO-1 gene polymorphisms and risk of T2DM:a systematic review and meta-analysisObjective:To integrate previous findings and summarize the effect size of HO-1 gene promoter polymorphisms associated with susceptibility of T2DM.Methods:We retrieved all studies matched to the search terms from PubMed/MEDLINE, EMBASE and ISI Web of Science databases published up to April 30, 2011. After data extraction and quality evaluation, odds ratios (ORs) were pooled using either fixed-effects or random-effects models for all alleles and genotypes, according to the results from heterogeneity analysis. Pooled effect and publication bias were visualized using forest plots and funnel plots, respectively.Results:A total of five eligible studies, comprising 1751 cases and 2902 controls, were included the final analysis. Allele L (≥25 or 27 GT repeats) is the risk locus for T2DM. Increased odds ratio (OR) for T2DM was observed in individuals with (GT)n L (long) allele compared to those with (GT)n S (short) allele (OR 1.12 95% CI[1.02-1.24], P=0.02). Furthermore, OR for T2DM in individuals with LL genotype, compared to those with SS genotype, was significantly increased (OR 1.25 95% CI[1.04-1.50] for LL vs SS genotype, P=0.02). No publication bias was found in the included studies. Unlike (GT)n repeats length polymorphism, T(-413)A SNP did not show any significant differences among subjects with T2DM, impaired glucose regulation and controls for either allelic or genotypic frequencies. Conclusion:Individuals carrying longer (GT)n repeats in HMOX1 gene promoter may have higher risk of T2DM, while no significant association was found between T(-413)A SNP and T2DM.Section 2 Impact of HO-1 gene polymorphisms on plasma HO-1 and plasma ferritin concentrationsObjective:To explore the impact of HO-1 gene polymorphisms (GT repeats length polymorphism and SNP) on plasma HO-1 and plasma ferritin concentrations.Methods:Subject recruitment was described in Part One. According to the WHO 1999 diagnostic criteria, they were divided into T2DM group, IGR group (including IFG and IGT) and NGT control group. Basic information was collected by questionnaire or anthropometric measurements. Plasma glucose, insulin, lipid profiles, HO-1 and ferritin concentration were detected. GT repeats length polymorphism and T(-413)A SNP were genotyped using genomic DNA extracted from peripheral blood mononuclear cells.Results:A total of 1475 subjects, including 529 T2DM patients,217 individuals with IGR and 729 NGT controls, were included in this study. The basic characteristics of the study population were similar with those in Part One. Plasma HO-1 and plasma ferritin levels showed a significant increasing trend among NGT controls, IGR and T2DM cases (P =0.0001 and P<0.0001, respectively). For GT repeats length polymorphism, plasma HO-1 and plasma ferritin levels differ significantly among various genotypes in the total population (P=0.0052 and P=0.0180, respectively). In addition, GT repeats length polymorphisms have significant impact on plasma HO-1 levels (P=0.0048) in T2DM patients in subgroup analysis. However, no significant association was observed between T(-413)A genotypes on either plasma HO-1 or plasma ferritin levels.Conclusion:GT repeats length polymorphisms have significant impact on plasma ferritin levels, which may be mediated through its impact on plasma HO-1 levels. T(-413)A genotypes showed no significant impact on either plasma HO-1 or plasma ferritin levels. Part Three HO-1-associated microRNAs in plasma and T2DMObjective:To examine the relationship between HO-1-associated microRNAs (miRNAs) in plasma and T2DM.Methods:The study population was recruited from individuals who took OGTT test at the outpatient clinics of Department of Endocrinology of Tongji Hospital affiliated to Tongji Medical College during the period of February 2011 to April 2011. The diagnosis of T2DM was established according to the WHO 1999 criteria. Basic information was collected by questionnaire or anthropometric measurements. Blood glucose (including FPG and OGTT2h) and insulin was measured using heparin-treated plasma. Total RNA (including microRNAs) was extracted in EDTA-treated plasma. Hsa-miR-122, hsa-miR-217 and hsa-miR-377 were chosen as candidate miRNAs, through comprehensive bioinformatics analysis and literature search. Plasma miRNAs were quantified with TaqMan probe-based RT-PCR kit, and the results were normalized using a commonly used endogenous control, U6 snRNA.Results:A total of 49 subjects, including 25 T2DM patients and 24 non-diabetic controls, were included in this study. The basic characteristics of the study population were similar with those in Part One. U6 snRNA showed stable and consistent amplification among samples, thus it could be considered as a suitable endogenous control in our study. Among the candidate miRNAs, hsa-miR-122 also was well amplified, while hsa-miR-217 and hsa-miR-377 were poorly amplified. Plasma hsa-miR-122 level in T2DM patients was significantly higher than that in non-diabetic controls (P=0.0406).Conclusion:Plasma hsa-miR-122 level was significantly increased in T2DM patients compared with non-diabetic controls. Innovation aspects of the current study:1. To explore the relationship between plasma HO-1 concentration and T2DM for the first time, as well as the relationship between plasma HO-1 concentration and IGR for the first time. We found that elevated plasma HO-1 concentration was significantly associated with increased risk of T2DM and IGR.2. To quantify HO-1-associated microRNAs in plasma for the first time. We found that plasma hsa-miR-122 level was significantly increased in T2DM patients compared with non-diabetic controls.3. To systematically evaluate the impact of HO-1 gene polymorphisms (GT repeats length polymorphism and SNP) on plasma HO-1 and plasma ferritin concentrations, which might help establish individualized circulating ferritin levels for different population.Limitations and future directions of the current study: 1. In the current study, we have found the associations between plasma HO-1 levels and T2DM as well as impaired glucose regulation; however, there are several questions to be addressed. First, the tissue source of plasma HO-1 remains unknown. Second, whether elevated plasma HO-1 is a pathologic cause or simply a biomarker for pathologic changes in T2DM remains unclear. Third, it is interesting to investigate the prospective trend of plasma HO-1 levels in newly-diagnosed T2DM, which may help demonstrate whether plasma HO-1 could serve as a prognostic biomarker in T2DM patients.2. Since HO-1 is highly responsive to oxidative stress; it is intriguing to examine the potential interaction in HO-1 gene polymorphisms and antioxidant nutrients, such as selenium, zinc and carotenoids, etc., in pathogenesis of T2DM. 3. Although we have found that plasma hsa-miR-122 level was significantly increased in T2DM patients compared with non-diabetic controls in this study. The result should be confirmed in further studies with larger sample size.
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