Andrzej S. Januszewski

The impact of glycation on apolipoprotein A-I structure and its ability to activate lecithin:cholesterol acyltransferase.

Aims/hypothesisHyperglycaemia, one of the main features of diabetes, results in non-enzymatic glycation of plasma proteins, including apolipoprotein A-I (apoA-I), the most abundant apolipoprotein in HDL. The aim of this study was to determine how glycation affects the structure of apoA-I and its ability to activate lecithin:cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport.Materials and methodsDiscoidal reconstituted HDL (rHDL) containing phosphatidylcholine and apoA-I ([A-I]rHDL) were prepared by the cholate dialysis method and glycated by incubation with methylglyoxal. Glycation of apoA-I was quantified as the reduction in detectable arginine, lysine and tryptophan residues. Methylglyoxal-AGE adduct formation in apoA-I was assessed by immunoblotting. (A-I)rHDL size and surface charge were determined by non-denaturing gradient gel electrophoresis and agarose gel electrophoresis, respectively. The kinetics of the LCAT reaction was investigated by incubating varying concentrations of discoidal (A-I)rHDL with a constant amount of purified enzyme. The conformation of apoA-I was assessed by surface plasmon resonance.ResultsMethylglyoxal-mediated modifications of the arginine, lysine and tryptophan residues in lipid-free and lipid-associated apoA-I were time- and concentration-dependent. These modifications altered the conformation of apoA-I in regions critical for LCAT activation and lipid binding. They also decreased (A-I)rHDL size and surface charge. The rate of LCAT-mediated cholesterol esterification in (A-I)rHDL varied according to the level of apoA-I glycation and progressively decreased as the extent of apoA-I glycation increased.Conclusions/interpretationIt is concluded that glycation of apoA-I may adversely affect reverse cholesterol transport in subjects with diabetes.

Increased serum pigment epithelium‐derived factor is associated with microvascular complications, vascular stiffness and inflammation in Type 1 diabetes

Aims  To determine in Type 1 diabetes patients if levels of pigment epithelium‐derived factor (PEDF), an anti‐angiogenic, anti‐inflammatory and antioxidant factor, are increased in individuals with complications and positively related to vascular and renal dysfunction, body mass index, glycated haemoglobin, lipids, inflammation and oxidative stress.

Multigenerational Undernutrition Increases Susceptibility to Obesity and Diabetes that Is Not Reversed after Dietary Recuperation

People in developing countries have faced multigenerational undernutrition and are currently undergoing major lifestyle changes, contributing to an epidemic of metabolic diseases, though the underlying mechanisms remain unclear. Using a Wistar rat model of undernutrition over 50 generations, we show that Undernourished rats exhibit low birth-weight, high visceral adiposity (DXA/MRI), and insulin resistance (hyperinsulinemic-euglycemic clamps), compared to age-/gender-matched control rats. Undernourished rats also have higher circulating insulin, homocysteine, endotoxin and leptin levels, lower adiponectin, vitamin B12 and folate levels, and an 8-fold increased susceptibility to Streptozotocin-induced diabetes compared to control rats. Importantly, these metabolic abnormalities are not reversed after two generations of unrestricted access to commercial chow (nutrient recuperation). Altered epigenetic signatures in insulin-2 gene promoter region of Undernourished rats are not reversed by nutrient recuperation, and may contribute to the persistent detrimental metabolic profiles in similar multigenerational undernourished human populations.

Non-invasive measures of tissue autofluorescence are increased in Type 1 diabetes complications and correlate with a non-invasive measure of vascular dysfunction

Diabet. Med. 29, 726–733 (2012)

Increased serum kallistatin levels in type 1 diabetes patients with vascular complications

BackgroundKallistatin, a serpin widely produced throughout the body, has vasodilatory, anti-angiogenic, anti-oxidant, and anti-inflammatory effects. Effects of diabetes and its vascular complications on serum kallistatin levels are unknown.MethodsSerum kallistatin was quantified by ELISA in a cross-sectional study of 116 Type 1 diabetic patients (including 50 with and 66 without complications) and 29 non-diabetic controls, and related to clinical status and measures of oxidative stress and inflammation.ResultsKallistatin levels (mean(SD)) were increased in diabetic vs. control subjects (12.6(4.2) vs. 10.3(2.8) μg/ml, p = 0.007), and differed between diabetic patients with complications (13.4(4.9) μg/ml), complication-free patients (12.1(3.7) μg/ml), and controls; ANOVA, p = 0.007. Levels were higher in diabetic patients with complications vs. controls, p = 0.01, but did not differ between complication-free diabetic patients and controls, p > 0.05. On univariate analyses, in diabetes, kallistatin correlated with renal dysfunction (cystatin C, r = 0.28, p = 0.004; urinary albumin/creatinine, r = 0.34, p = 0.001; serum creatinine, r = 0.23, p = 0.01; serum urea, r = 0.33, p = 0.001; GFR, r = -0.25, p = 0.009), total cholesterol (r = 0.28, p = 0.004); LDL-cholesterol (r = 0.21, p = 0.03); gamma-glutamyltransferase (GGT) (r = 0.27, p = 0.04), and small artery elasticity, r = -0.23, p = 0.02, but not with HbA1c, other lipids, oxidative stress or inflammation. In diabetes, geometric mean (95%CI) kallistatin levels adjusted for covariates, including renal dysfunction, were higher in those with vs. without hypertension (13.6 (12.3-14.9) vs. 11.8 (10.5-13.0) μg/ml, p = 0.03). Statistically independent determinants of kallistatin levels in diabetes were age, serum urea, total cholesterol, SAE and GGT, adjusted r2 = 0.24, p < 0.00001.ConclusionsSerum kallistatin levels are increased in Type 1 diabetic patients with microvascular complications and with hypertension, and correlate with renal and vascular dysfunction.

A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy

MicroRNAs are now increasingly recognized as biomarkers of disease progression. Several quantitative real-time PCR (qPCR) platforms have been developed to determine the relative levels of microRNAs in biological fluids. We systematically compared the detection of cellular and circulating microRNA using a standard 96-well platform, a high-content microfluidics platform and two ultra-high content platforms. We used extensive analytical tools to compute inter- and intra-run variability and concordance measured using fidelity scoring, coefficient of variation and cluster analysis. We carried out unprejudiced next generation sequencing to identify a microRNA signature for Diabetic Retinopathy (DR) and systematically assessed the validation of this signature on clinical samples using each of the above four qPCR platforms. The results indicate that sensitivity to measure low copy number microRNAs is inversely related to qPCR reaction volume and that the choice of platform for microRNA biomarker validation should be made based on the abundance of miRNAs of interest.

Muscle grip strength predicts incident type 2 diabetes: Population-based cohort study.

OBJECTIVES To determine the longitudinal relationship of muscle mass and strength with incident type 2 diabetes, and previously unstudied mediating effects of testosterone and inflammation. METHODS Community-dwelling male participants (aged ≥35years) of the Men Androgen Inflammation Lifestyle Environment and Stress (MAILES) Study underwent biomedical assessment in 2002-2006 and 2007-2010, including hand grip strength (dynamometer), testosterone and inflammatory markers. Body composition (dual-energy X-ray absorptiometry) was assessed at baseline only. Incident type 2 diabetes was defined as a self-reported doctor diagnosis, diabetes medication use, fasting plasma glucose ≥7.0mmol/L, or glycated haemoglobin ≥6.5% (48mmol/mol) at follow-up, that was not present at baseline. RESULTS Of n=1632 men, incident type 2 diabetes occurred in 146 (8.9%). Muscle mass was not associated with incident type 2 diabetes. Grip strength was inversely associated with incident type 2 diabetes [unadjusted odds ratio (OR) per 5kg: 0.87, 95% confidence interval (CI): 0.80-0.95; adjusted OR, 95% CI: 0.87, 0.78-0.97]. Arm muscle quality (grip strength divided by arm lean mass) was similarly associated with incident type 2 diabetes. Testosterone, IL-6 and TNF-α did not significantly mediate the associations. The population attributable fraction of type 2 diabetes from low grip strength was 27% (13-40%), assuming intervention could increase strength by 25%. CONCLUSIONS Reduced muscle strength, but not reduced muscle mass, is a risk factor for incident type 2 diabetes in men. This is not mediated by testosterone or inflammation. Intervention could prevent a substantial proportion of disease.

Increased coated-platelet levels in chronic haemodialysis patients

Aim:  To determine if levels of coated‐platelets, which are potentially pro‐thrombotic, are increased in end‐stage renal disease patients on haemodialysis, a condition associated with high cardiovascular disease risk.

Plasma 1,5 anhydroglucitol levels, a measure of short-term glycaemia: Assay assessment and lower levels in diabetic vs. non-diabetic subjects

An assay of plasma 1,5-anhydroglucitol was evaluated. Assay CVs, effects of four plasma freeze-thaw cycles, glucose up to 80 mmol/L and triglycerides up to 20 mmol/L were acceptable. 1,5-anhydroglucitol levels were significantly lower in diabetic vs. non-diabetic subjects and correlated inversely with renal function, but not with HbA1c.

Plasma Low‐Molecular Weight Fluorescence in Type 1 Diabetes Mellitus

Abstract: Characteristic tissue fluorescence is associated with advanced glycation end product (AGE) accumulation in experimental diabetes models, but its utility in patients with type 1 diabetes remains to be established. We studied 148 patients with type 1 diabetes and 77 healthy age‐matched control subjects. Low‐molecular weight (LMW) fluorophore levels were estimated in plasma samples obtained after an overnight fast. Intra‐ and interassay coefficients of variation were 4.7% and 6.4%, respectively. LMW fluorophore levels were significantly higher in patients with diabetes than in control subjects (6.3 ± 0.6 AU/mL vs. 4.1 ± 0.3; P= 0.007). However, all of this difference came from patients with microvascular complications (n= 67, 7.5 ± 1.3). There was no significant difference in LMW fluorescence between complication‐free patients (4.4 ± 0.2) and control subjects (P > 0.05). On multivariate analysis, LMW fluorophores correlated with measures of renal function (P < 0.05) but not with diabetes per se. In addition, there was no correlation between LMW fluorophores and the markers of oxidative stress or systemic inflammation. Longitudinal and interventional studies are required to determine whether the association between LMW fluorophores and nephropathy is cause or effect.