NMR-Based Metabolomic Approach Tracks Potential Serum Biomarkers of Disease Progression in Patients with Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia associated with alterations in carbohydrate, lipid, and protein metabolism. The prognosis of T2DM patients is highly dependent on the development of complications, and therefore the identification of biomarkers of T2DM progression, with minimally invasive techniques, is a huge need. In the present study, we applied a H-1-Nuclear Magnetic Resonance (H-1-NMR)-based metabolomic approach coupled with multivariate data analysis to identify serum metabolite profiles associated with T2DM development and progression. To perform this, we compared the serum metabolome of non-diabetic subjects, treatment-naive non-complicated T2DM patients, and T2DM patients with complications in insulin monotherapy. Our analysis revealed a significant reduction of alanine, glutamine, glutamate, leucine, lysine, methionine, tyrosine, and phenylalanine in T2DM patients with respect to non-diabetic subjects. Moreover, isoleucine, leucine, lysine, tyrosine, and valine levels distinguished complicated patients from patients without complications. Overall, the metabolic pathway analysis suggested that branched-chain amino acid (BCAA) metabolism is significantly compromised in T2DM patients with complications, while perturbation in the metabolism of gluconeogenic amino acids other than BCAAs characterizes both early and advanced T2DM stages. In conclusion, we identified a metabolic serum signature associated with T2DM stages. These data could be integrated with clinical characteristics to build a composite T2DM/complications risk score to be validated in a prospective cohort

Leukocyte telomere length and mortality risk in patients with type 2 diabetes

Leukocyte telomere length (LTL) shortening is found in a number of age-related diseases, including type 2 diabetes (T2DM). In this study its possible association with mortality was analyzed in a sample of 568 T2DM patients (mean age 65.9 \ub1 9 years), who were followed for a median of 10.2 years (interquartile range 2.2). A number of demographic, laboratory and clinical parameters determined at baseline were evaluated as mortality risk factors. LTL was measured by quantitative real-time PCR and reported as T/S (telomere-to-single copy gene ratio). Age, gender, creatinine, diabetes duration at baseline, and LTL were significantly different between T2DM patients who were dead and alive at follow-up. In the Cox regression analysis adjusted for the confounding variables, shorter LTL, older age, and longer disease duration significantly increased the risk of all-cause mortality (HR = 3.45, 95%CI 1.02-12.5, p = 0.004). Kaplan-Maier analysis also found a different cumulative mortality risk for patients having an LTL shorter than the median (T/S 64 0.04) and disease duration longer than the median (>10 years) (log-rank = 11.02, p = 0.011). Time-dependent mortality risk stratification showed that T2DM duration and LTL combined was a fairly good predictor of mortality over the first 76 months of follow-up. In conclusion, LTL combined with clinical parameters can provide additive prognostic information on mortality risk in T2DM patients

MiR-21-5p and miR-126a-3p levels in plasma and circulating angiogenic cells: relationship with type 2 diabetes complications

Innovative biomarkers are required to manage type 2 diabetic patients (T2DM). We focused our study on miR-126-3p and miR-21-5p levels, as biomarkers of endothelial function and inflammation. MiRNAs levels were measured in plasma from 107 healthy subjects (CTR) and 193 diabetic patients (T2DM), 76 without (T2DM NC) and 117 with (T2DM C) complications. When diabetic complication were analysed as a whole, miR-126-3p and miR-21-5p levels declined significantly from CTR to T2DM NC and T2DM C patients. When miRNAs levels were related to specific complications, significantly higher miR-21-5p levels (0.46 \ub1 0.44 vs. 0.26\ub10.33, p < 0.001) and significant lower miR-126-3p levels (0.21\ub10.21 vs. 0.28\ub10.22, p = 0.032) were found in T2DM with previous major cardiovascular events (MACE) vs. all the others T2DM patients. To confirm these results we focused on circulating angiogenic cells (CACs) from a subgroup of 10 CTR, 15 T2DM NC and 15 T2DM patients with MACE. CACs from T2DM patients expressed higher miR-21-5p and lower miR-126-3p levels than CACs from CTR. Furthermore, CACs from T2DM + MACE showed the highest levels of miR-21-5p. Circulating miR-21-5p and miR-126-3p emerge as dynamic biomarkers of systemic inflammatory/angiogenic status. Their expression levels in CACs from T2DM with MACE suggest a shift from a proangiogenic to a proinflammatory profile

Small extracellular vesicles deliver miR-21 and miR-217 as pro-senescence effectors to endothelial cells

The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40-100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication