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Background and Aim: Plasma circulating microRNA (miRNA)-126, -145, and -155 are associated with vascular remodeling, atherosclerotic lesion formation, and plaque vulnerability. In this study, we evaluated the levels of plasma circulating miRNAs in patients with stable coronary artery disease (CAD), different cardiovascular risk profiles, and different glomerular filtration rates (GFR). Methods and Results: Forty patients with stable CAD admitted for elective percutaneous coronary intervention (PCI) were enrolled in a prospective study. Before PCI, fasting blood samples were obtained to evaluate clinical parameters and miRNA-126 and miRNA-155 expression. The GFR was calculated by the MDRD and CKD-EPI formulas, and the severity of CAD was calculated according to the SYNTAX score. All these parameters were correlated with miRNAs. The association between miRNA levels and clinical characteristics was evaluated. The expression of miRNA-126 positively correlated with a higher SYNTAX score (r = 0.337; p=0.034); however, no significant correlations between miR-126, GFR, and clinical characteristics were observed. Higher plasma levels of miRNA-155 correlated with increased levels of triglycerides (r = 0.317; P = 0.049), C-peptide (r = 0.452; P = 0.011), and the HOMA index (r = 0.447; P = 0.012) and a higher body mass index (BMI) (r = 0.385; P = 0.015). GFR and miRNA-155 (MDRD – Rho=0.353; P = 0.027. CKD-EPI – Rho=0.357; P = 0.026) were found to have a moderate correlation, although miRNA-155 had no correlation with the SYNTAX score. Conclusion: Plasma circulating miRNA-126 levels were increased in patients with severe atherosclerosis as determined by the SYNTAX score. Elevated miRNA-155 expression was observed in patients with Stage 1 GFR but was lower in patients with Stages 2 and 3 GFR. Plasma circulating miRNA-155 had positive correlations with higher levels of BMI, HOMA index, C-peptide, and triglycerides. Relevance for Patients: Although further investigations are needed to confirm the role of miRNA-155 and miRNA-126, they may serve as potential biomarkers detecting severity of CAD, lowering of kidney function and metabolic syndrome.

Additional file 2: Quantile-quantile plots for GWAS of T2DM complications.

Additional file 1. Timeline following the CARE guidelines. Additional clinical data. A detailed description of Materials and Methods used in the study.

Additional file 3. Document file (30 pages) describing alignment quality metrics for each sample (10 pages per sample).

Background. Insulin signaling is involved in the regulation of both carbohydrate and lipid turnover and metabolism. The glucose tolerance test is routinely applied as a diagnostic method, and in addition to glucose measurements, assessment of fatty acid (FA) intermediates in the fasted state and 2 h after the glucose administration are feasible in clinical conditions. The aim of this study was to confirm the validity of measurements of FFAs and acylcarnitine levels during the glucose tolerance and mixed meal test as indicators of insulin sensitivity and tissue-specific insulin resistance.Methods Twenty three adult volunteers with a body mass index >25 kg/m2 were enrolled in the study. Twenty male C57bl/6N mice (7 weeks old, Envigo, Netherlands), 10 male diabetic db/db (BKS.Cg-+Lepr/+Lepr/OlaHsd) mice and age-matched 10 control db/L mice were used in experiments. To determine the insulin-sensitive glucose uptake in tissues, 1 u00b5Ci of 2-[1,2-3H]-deoxy-D-glucose ([3H]-DOG, specific activity, 60 Ci/mmol) was administered subcutaneously to the mice at 1 h 50 min after glucose administration. Insulin resistance was calculated from the fasting plasma C-peptide and glucose concentrations using the HOMA2-insulin resistance (IR) model. Insulin resistance was defined as fasting HOMA2-IR >1.8. The concentrations of acylcarnitines in the plasma samples were determined with a Waters UPLC MS/MS system.Results. In humane samples the mean postprandial decrease in the total concentration of long-chain plasma acylcarnitines the insulin resistant group was significantly 3-fold lower than that in the non-insulin resistant group (p=0.014). HOMA2-IR negatively correlated with the postprandial decrease of C14 acylcarnitine (Spearmanu2019s correlation (r) =-0.61, p=0.007), C16 (r=-0.67, p=0.002), C18:0 (r=-0.77, p=0.0002), C18:1 (r=-0.71, p=0.01), and C18:2 (r =-0.79, p=0.001) and the body mass index negatively correlated with the postprandial decrease of C14 (r=-0.47, p=0.048), C16 (r=-0.50, p=0.035). Insulin release in response to glucose administration decreased both FFA and long-chain acylcarnitine levels in plasma in healthy control animals by 30% (120 min). The glucose tolerance test and [3H]-deoxy-D-glucose uptake in tissues revealed that high fat diet-induced lipid overload in C57bl/6N mice evoked only adipose tissue insulin resistance, and plasma levels of FFAs did not decrease after glucose administration. In comparison, db/db mice developed type 2 diabetes with severely impaired insulin sensitivity and up to 70% lower glucose uptake in both adipose tissues and muscles (skeletal muscle and heart), and both plasma concentrations of FFAs and long-chain acylcarnitines did not decrease in response to glucose administration.Discussion. In summary, in the transition from a fasted to fed state, the decrease in concentrations of long-chain acylcarnitines indicate insulin sensitivity. The decrease in plasma FFA concentrations from the fasted state to the postprandial state after glucose administration reflects even early changes in adipose tissue insulin sensitivity, while an insufficient decrease in long-chain acylcarnitine levels is associated with muscle and heart insulin resistance. The data of this study indicate that the measurements of changes in plasma FFA and long-chain acylcarnitine concentrations at 2 h after glucose load in fasted subjects are useful as diagnostic markers for adipose and muscle tissue-specific insulin resistance.

Type 2 diabetes (T2D) is a common metabolic disorder with a prevalence of more than 8% and is the cause of serious health complications that represents a major healthcare burden associated with the treatment of diabetes. While the risk for T2D is one of the most studied traits in the genetics of common diseases, the genetic association studies on its complications are lagging behind. There is some number of genome-wide association studies (GWASs) involving diabetic microvascular complications of retinopathy, nephropathy, and neuropathy however not all of them have reported consistent results. Here we report a GWAS of different T2D complications in patients from Genome Database of Latvian Population.Our study consisted of 648 T2D patients for whom genotypes from an Illumina Global screening SNP array were available at the beginning of 2019. We used the data from 5 different national health registries including T2D register, death register, outpatient health records, hospitalized patient records, and State covered medication prescription registry in order to acquire data on the type and time of onset of T2D complication for these patients. Following cases and controls were identified for a number of different complications: 203 cases and 398 controls for retinopathy, 517 cases and 84 controls for nephropathy, 218 cases and 382 controls for neuropathy, 226 cases and 333 controls for macrovascular complications.Imputation was performed using the 1000 Genome Projects data set. Plink was used to perform logistic regression based association testing with adjustment for covariates (sex, age, BMI, duration of T2D, and the median value of HbA1c). No significant associations at the genome level (lowest P value =8.05x10-07) were found in case of retinopathy and nephropathy, however, some previously reported loci displayed a significant association (p value >5x10-6). In case of neuropathy, we identified 2 new significant loci: GYPA (SNPfreq=0.14, p=2.44x10-10, OR =2.44) and MEDAG (SNPfreq=0.37, p=2.35x10-8, OR =2.14). GYPA has been shown to be upregulated in dorsal root ganglia of the mouse model of T2DM and the metabolic syndrome as well as in dorsal root ganglia of mice model of type I diabetes, both with diabetic polyneuropathy. In case of macrovascular complications, we identified 1 locus including PDE4DIP gene (SNPfreq=0.36, p=1.01x10-9, OR =2.51). Another SNP in the PDE4DIP was previously associated with an increased risk of ischemic stroke and a primary interacting partner of PDE4DIP is PDE4D, originally identified as linked to ischemic stroke in Icelandic families.Despite the small sample size, we were able to replicate some previously reported associations with diabetic retinopathy and nephropathy and identify new loci in case of relatively less studied diabetic complication neuropathy.