Telomere shortening: a marker of atherosclerosis?

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Differential gene expression of NADPH oxidase (p22phox) and hemoxygenase-1 in patients with Type 2 diabetes and microangiopathy

Aims: While the downstream effects of increased reactive oxygen species (ROS) in the pathogenesis of diabetes were well studied, only a few studies have explored the cellular sources of ROS. We examined whether protection against oxidative stress is altered in patients with diabetes and microangiopathy by examining changes in NADPH oxidase (p22phox) and hemoxygenase‐1 (HO‐1) levels. Methods: NADPH oxidase (p22phox) and HO‐1 gene expression were probed by RT‐PCR using leucocytes from patients with Type 2 diabetes without (n = 19) and with microangiopathy (n = 20) and non‐diabetic subjects (n = 17). Levels of lipid peroxidation as measured by thiobarbituric reactive substances (TBARS) and protein carbonyl content (PCO) were determined by fluorimetric and spectrophotometric methods, respectively. Results: p22phox gene expression (mean ± se) was significantly (P < 0.05) higher in diabetic patients with (0.99 ± 0.04) and without microangiopathy (0.86 ± 0.05) compared with control subjects (0.66 ± 0.05). Consistent with the mRNA data, the p22phox protein expression and NADPH oxidase activity was also increased in cells from diabetic patients compared with control subjects. However, HO‐1 gene expression was significantly (P < 0.05) lower in patients with (0.73 ± 0.03) and without microangiopathy (0.85 ± 0.02) compared with control subjects (1.06 ± 0.03). The mean (± se) levels of TBARS were significantly (P < 0.05) higher in diabetic patients with (14.36 ± 1.3 nm/ml) and without microangiopathy (12.20 ± 1.3 nm/ml) compared with control subjects (8.58 ± 0.7 nm/ml). The protein carbonyl content was also significantly (P < 0.05) higher in diabetic patients with (1.02 ± 0.04 nmol/mg protein) and without microangiopathy (0.84 ± 0.06 nmol/mg protein) compared with control subjects (0.48 ± 0.02 nmol/mg protein). In diabetic subjects, increased p22phox gene expression was negatively correlated with HO‐1 and positively correlated with TBARS, PCO, HbA1c and diabetes duration. In contrast, HO‐1 gene expression was correlated negatively with p22phox, TBARS, PCO, HbA1c and diabetes duration. Conclusion: Our results indicate that increased oxidative damage is seen in Asian Indians with Type 2 diabetes and microangiopathy and is associated with increased NADPH oxidase (p22phox) and decreased HO‐1 gene expression

Telomere shortening & metabolic/vascular diseases

Telomeres are specialized DNA-protein structures located at the ends of eukaryotic chromosomes whose length is progressively reduced in most somatic cells during ageing. Over the past decade, emerging evidence has shown that the telomeres are essential regulators of cellular life span and chromosome integrity in a dynamic fashion. By inducing genomic instability, replicative senescence and apoptosis, shortening of telomeres is thought to contribute to organismal ageing. While the aetiology of cardiovascular diseases and diabetes represent a complex interaction between various risk factors overlaid on different genetic backgrounds, the conventional risk factors often did not explain the inter-individual variability related to predisposition of disease states. This underscores the need for biological indicators of ageing in evaluating the aetiology of several age-related disorders, and recent studies indicate that telomere length could qualify as an ideal marker of biological ageing. Short telomeres have been detected in senescent endothelial cells and vascular smooth muscle cells from human atherosclerotic plaque as well as in myocardial tissue from patients with end-stage heart failure and cardiac hypertrophy. In addition, telomere shortening has been demonstrated in WBCs from patients with coronary heart disease, premature myocardial infarction, hypertension and diabetes mellitus. In this review, we discuss the telomere hypothesis of ageing as well as human studies that address the role of telomeres in cardiovascular, diabetes and other cardio-metabolic pathologies

Low vitamin B12 and lipid metabolism: evidence from pre-clinical and clinical studies

Obesity is a worldwide epidemic responsible for 5% of global mortality. The risks of developing other key metabolic disorders like diabetes, hypertension and cardiovascular diseases (CVDs) are increased by obesity, causing a great public health concern. A series of epidemiological studies and animal models have demonstrated a relationship between the importance of vitamin B12 (B12) and various components of metabolic syndrome. High prevalence of low B12 levels has been shown in European (27%) and South Indian (32%) patients with type 2 diabetes (T2D). A longitudinal prospective study in pregnant women has shown that low B12 status could independently predict the development of T2D five years after delivery. Likewise, children born to mothers with low B12 levels may have excess fat accumulation which in turn can result in higher insulin resistance and risk of T2D and/or CVD in adulthood. However, the independent role of B12 on lipid metabolism, a key risk factor for cardiometabolic disorders, has not been explored to a larger extent. In this review, we provide evidence from pre-clinical and clinical studies on the role of low B12 status on lipid metabolism and insights on the possible epigenetic mechanisms including DNA methylation, micro-RNA and histone modifications. Although, there are only a few association studies of B12 on epigenetic mechanisms, novel approaches to understand the functional changes caused by these epigenetic markers are warranted

Biomarkers of oxidative stress: methods and measures of oxidative DNA damage (COMET assay) and telomere shortening

Oxidative stress is fast becoming the nutritional and medical buzzword for the twenty-first century. The theoretical importance of oxidative stress in diabetes is highlighted by its potential double impact on metabolic dysfunction on one hand and the vascular system on the other hand. The new concept of oxidative stress, being an important trigger in the onset and progression of diabetes and its complications, emphasizes the need for measurement of markers of oxidation to assess the degree of oxidative stress. While we have been routinely measuring biomarkers in our molecular epidemiology projects, here we discuss the utility of two assays, (a) DNA damage assessment by COMET measurement and (b) telomere length measurement. As DNA damage is efficiently repaired by cellular enzymes, its measurement gives a snapshot view of the level of oxidative stress. The protocol allows for measurement of oxidative DNA damage (FPG-sensitive DNA strand breaks). Telomere length measured by Southern blotting technique allows one to estimate the chronic burden of oxidative stress at the molecular level and is now considered as biomarker of biological aging

Oxidative DNA damage and augmentation of poly(ADP-ribose) polymerase/nuclear factor-kappa B signaling in patients with Type 2 diabetes and microangiopathy

Although oxidative stress and the subsequent DNA damage is one of the obligatory signals for poly(ADP-ribose) polymerase (PARP) activation and nuclear factor-kappa B (NFκB) alterations, these molecular aspects have not been collectively examined in epidemiological and clinical settings. Therefore, this study attempts to assess the oxidative DNA damage and its downstream effector signals in peripheral blood lymphocytes from Type 2 diabetes subjects without and with microangiopathy along with age-matched non-diabetic subjects. The basal DNA damage, lipid peroxidation and protein carbonyl content were significantly (p < 0.05) higher in patients with and without microangiopathy compared to control subjects. Formamido Pyrimidine Glycosylase (FPG)-sensitive DNA strand breaks which represents reliable indicator of oxidative DNA damage were also significantly (p < 0.001) higher in diabetic patients with (19.41 ± 2.5) and without microangiopathy (16.53 ± 2.0) compared to control subjects (1.38 ± 0.85). Oxidative DNA damage was significantly correlated to poor glycemic control. PARP mRNA expression and PARP activity were significantly (p < 0.05) increased in cells from diabetic patients with (0.31 ± 0.03 densitometry units; 0.22 ± 0.02 PARP units/mg protein, respectively) and without (0.35 ± 0.02; 0.42 ± 0.05) microangiopathy compared to control (0.19 ± 0.02; 0.11 ± 0.02) subjects. Diabetic subjects with and without microangiopathy exhibited a significantly (p < 0.05) higher (80%) NFκB binding activity compared to control subjects. In diabetic patients, FPG-sensitive DNA strand breaks correlated positively with PARP gene expression, PARP activity and NFκB binding activity. This study provides a comprehensive molecular evidence for increased oxidative stress and genomic instability in Type 2 diabetic subjects even prior to vascular pathology and hence reveals a window of opportunity for early therapeutic intervention

Association of telomere shortening with impaired glucose tolerance and diabetic macroangiopathy

Objective: Shortening of telomere length has been reported in several conditions including Type 2 diabetes and atherosclerosis. The aims of this study were (1) to assess whether telomere shortening occurs at the stage of pre-diabetes, i.e., impaired glucose tolerance (IGT) and (2) whether telomere shortening was greater in Type 2 diabetic subjects with atherosclerotic plaques. Methods: Subjects with impaired glucose tolerance (IGT) (n = 30), non-diabetic control subjects (n = 30), Type 2 diabetic patients without (n = 30) and with atherosclerotic plaques (n = 30) were selected from the Chennai Urban Rural Epidemiology Study (CURES), an ongoing epidemiological population-based study. Southern-blot analysis was used to determine mean terminal restriction fragment (TRF) length, a measure of average telomere size, in leukocyte DNA. Levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCO) and high sensitive C-reactive protein (hs-CRP) were measured by standard methodologies. Carotid intima-media thickness (IMT) was assessed by high resolution B-mode ultrasonography. Results: The mean (±S.E.) TRF lengths were significantly lower in IGT subjects (6.97 ± 0.3 kb; p = 0.002) and lower still in Type 2 diabetic subjects without plaques (6.21 ± 0.2; p = 0.0001) and lowest in Type 2 diabetic subjects with atherosclerotic plaques (5.39 ± 0.2; p = 0.0001) when compared to control subjects (8.7 ± 0.5). In IGT subjects, TRF length was positively correlated to HDL cholesterol and negatively correlated to glycated hemoglobin (HbA1c), TBARS, PCO, HOMA-IR and IMT. In multiple linear regression analysis, presence of diabetes, HDL cholesterol and increased TBARS levels appear as significant determinants of telomere shortening. Conclusion: Telomere shortening is seen even at the stage of IGT. Among subjects with Type 2 diabetes, those with atherosclerotic plaques had greater shortening of telomere length compared to those without plaques

Low vitamin B12 in pregnancy is associated with adipose derived circulating miRs targeting PPARγ and insulin resistance

Context: Low vitamin B12 (B12) during pregnancy is associated with higher maternal obesity, insulin resistance(IR) and gestational diabetes(GDM). B12 is a key co-factor in 1-carbon metabolism. Objective: We hypothesize that B12 plays a role in epigenetic regulation by altering circulating miRNAs(miRs) during adipocyte differentiation and results in an adverse metabolic phenotype. Design, settings and main-outcome measure: Human pre-adipocyte cell-line(Chub-S7) were differentiated in various B12 concentrations: Control(500nM), LowB12(0.15nM) and NoB12(0nM). Maternal blood samples(n=91) and subcutaneous adipose tissue (SAT)(n=42) were collected at delivery. Serum B12, folate, lipids, plasma 1-carbon metabolites, miR profiling, miR expression and gene expression were measured. Results: Our in vitro model demonstrated that adipocytes in B12 deficient conditions accumulated more lipids, had higher triglyceride levels and increased gene expression of adipogenesis and lipogenesis. MiR array screening revealed differential expression of 133miRs involving several metabolic pathways (adjusted p<0.05). Altered miR expression were observed in 12miRs related to adipocyte differentiation and function in adipocytes. Validation of this data in pregnant women with low B12, confirmed increased expression of adipo/lipogenic genes and altered miRs in SAT, and altered levels of 11 of the 12miRs in circulation. After adjusting for other possible confounders, multiple regression analysis revealed an independent association of B12 with BMI (β: -0.264; 95% CI: -0.469, -0.058; p=0.013) and was mediated by four circulating miRs targeting PPARγ and IR. Conclusions: Low B12 levels in pregnancy alters adipose derived circulating miRs, which may mediate an adipogenic and IR phenotype leading to obesity

Metformin inefficiency to lower lipids in vitamin B12 deficient HepG2 cells is alleviated via adiponectin-AMPK axis

Background: Prolonged metformin treatment decreases vitamin B12 (B12) levels, whereas low B12 is associated with dyslipidaemia. Some studies have reported that metformin has no effect on intrahepatic triglyceride (TG) levels. Although AMP-activated protein kinase (AMPK) activation via adiponectin lowers hepatic TG content, its role in B12 deficiency and metformin has not been explored. We investigated whether low B12 impairs the beneficial effect of metformin on hepatic lipid metabolism via the AMPK-adiponectin axis. Methods: HepG2 was cultured using custom-made B12-deficient Eagle's Minimal Essential Medium (EMEM) in different B12-medium concentrations, followed by a 24-h metformin/adiponectin treatment. Gene and protein expressions and total intracellular TG were measured, and radiochemical analysis of TG synthesis and seahorse mitochondria stress assay were undertaken. Results: With low B12, total intracellular TG and synthesized radiolabelled TG were increased. Regulators of lipogenesis, cholesterol and genes regulating fatty acids (FAs; TG; and cholesterol biosynthesis were increased. FA oxidation (FAO) and mitochondrial function were decreased, with decreased pAMPKα and pACC levels. Following metformin treatment in hepatocytes with low B12, the gene and protein expression of the above targets were not alleviated. However, in the presence of adiponectin, intrahepatic lipid levels with low B12 decreased via upregulated pAMPKα and pACC levels. Again, combined adiponectin and metformin treatment ameliorated the low B12 effect and resulted in increased pAMPKα and pACC, with a subsequent reduction in lipogenesis, increased FAO and mitochondrion function. Conclusions: Adiponectin co-administration with metformin induced a higher intrahepatic lipid-lowering effect. Overall, we emphasize the potential therapeutic implications for hepatic AMPK activation via adiponectin for a clinical condition associated with B12 deficiency and metformin treatment

Low maternal vitamin B12 status is associated with lower cord blood HDL cholesterol in white Caucasians living in the UK

Background and Aims: Studies in South Asian population show that low maternal vitamin B12 associates with insulin resistance and small for gestational age in the offspring. Low vitamin B12 status is attributed to vegetarianism in these populations. It is not known whether low B12 status is associated with metabolic risk of the offspring in whites, where the childhood metabolic disorders are increasing rapidly. Here, we studied whether maternal B12 levels associate with metabolic risk of the offspring at birth. Methods: This is a cross-sectional study of 91 mother-infant pairs (n = 182), of white Caucasian origin living in the UK. Blood samples were collected from white pregnant women at delivery and their newborns (cord blood). Serum vitamin B12, folate, homocysteine as well as the relevant metabolic risk factors were measured. Results: The prevalence of low serum vitamin B12 (<191 ng/L) and folate (<4.6 μg/L) were 40% and 11%, respectively. Maternal B12 was inversely associated with offspring’s Homeostasis Model Assessment 2-Insulin Resistance (HOMA-IR), triglycerides, homocysteine and positively with HDL-cholesterol after adjusting for age and BMI. In regression analysis, after adjusting for likely confounders, maternal B12 is independently associated with neonatal HDL-cholesterol and homocysteine but not triglycerides or HOMA-IR. Conclusions: Our study shows that low B12 status is common in white women and is independently associated with adverse cord blood cholesterol