Maladaptive response following glucose overload in GLUT4-overexpressing H9C2 cardiomyoblasts.

BACKGROUND: Glucose overload drives diabetic cardiomyopathy by affecting the tricarboxylic acid pathway. However, it is still unknown how cells could overcome massive chronic glucose influx on cellular and structural level. METHODS/MATERIALS: Expression profiles of hyperglycemic, glucose transporter-4 (GLUT4) overexpressing H9C2 (KE2) cardiomyoblasts loaded with 30 mM glucose (KE230L) and wild type (WT) cardiomyoblasts loaded with 30 mM glucose (WT30L) were compared using proteomics, real-time polymerase quantitative chain reaction analysis, or Western blotting, and immunocytochemistry. RESULTS: The findings suggest that hyperglycemic insulin-sensitive cells at the onset of diabetic cardiomyopathy present complex changes in levels of structural cell-related proteins like tissue inhibitor of metalloproteases-1 (1.3 fold), intercellular adhesion molecule 1 (1.8 fold), type-IV-collagen (3.2 fold), chaperones (Glucose-Regulated Protein 78: 1.8 fold), autophagy (Autophagosome Proteins LC3A, LC3B: 1.3 fold), and in unfolded protein response (UPR; activating transcription factor 6α expression: 2.3 fold and processing: 2.4 fold). Increased f-actin levels were detectable with glucose overload by immnocytochemistry. Effects on energy balance (1.6 fold), sirtuin expression profile (Sirtuin 1: 0.7 fold, sirtuin 3: 1.9 fold, and sirtuin 6: 4.2 fold), and antioxidant enzymes (Catalase: 0.8 fold and Superoxide dismutase 2: 1.5 fold) were detected. CONCLUSION: In conclusion, these findings implicate induction of chronic cell distress by sustained glucose accumulation with a non-compensatory repair reaction not preventing final cell death. This might explain the chronic long lasting pathogenesis observed in developing heart failure in diabetes mellitus.

Mosaic vs. Single Image Analysis with Confocal Microscopy of the Corneal Nerve Plexus for Diagnosis of Early Diabetic Peripheral Neuropathy.

INTRODUCTION: The assessment of the corneal nerve fibre plexus with corneal confocal microscopy (CCM) is an upcoming but still experimental method in the diagnosis of early stage diabetic peripheral neuropathy (DPN). Using an innovative imaging technique-Heidelberg Retina Tomograph equipped with the Rostock Cornea Module (HRT-RCM) and EyeGuidance module (EG)-we were able to look at greater areas of subbasal nerve plexus (SNP) in order to increase the diagnostic accuracy. The aim of our study was to evaluate the usefulness of EG instead of single image analysis in diagnosis of early stage DPN. METHODS: This prospective study was performed on 60 patients with type 2 diabetes mellitus, classified equally into two subgroups based on neuropathy deficient score (NDS): patients without DPN (group 1) or with mild DPN (group 2). The following parameters were analysed in the two subgroups: corneal nerve fibre length (CNFL; mm/mm2), corneal nerve fibre density (CNFD; no./mm2), corneal nerve branch density (CNBD; no./mm2). Furthermore, we compared the data calculated with the novel mosaic, EG-based method with those received from single image analysis using different quantification tools. RESULTS: Using EG we did not find a significant difference between group 1 and group 2: CNFL (16.81 ± 5.87 mm/mm2 vs. 17.19 ± 7.19 mm/mm2, p = 0.895), CNFD (254.05 ± 115.36 no./mm2 vs. 265.91 ± 161.63 no./mm2, p = 0.732) and CNBD (102.68 ± 62.28 no./mm2 vs. 115.38 ± 96.91 no./mm2, p = 0.541). No significant difference between the EG method of analysing the SNP and the single image analysis of 10 images per patient was detected. CONCLUSION: On the basis of our results it was not possible to differentiate between early stages of large nerve fibre DPN in patients with type 2 diabetes mellitus via SNP analysis. To improve sensitivity and specificity of this method newer technologies are under current evaluation. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT05326958.

Chronic Hyperglycaemia Inhibits Tricarboxylic Acid Cycle in Rat Cardiomyoblasts Overexpressing Glucose Transporter Type 4.

An oversupply of nutrients with a loss of metabolic flexibility and subsequent cardiac dysfunction are hallmarks of diabetic cardiomyopathy. Even if excess substrate is offered, the heart suffers energy depletion as metabolic fluxes are diminished. To study the effects of a high glucose supply, a stably glucose transporter type 4 (GLUT4)-overexpressing cell line presenting an onset of diabetic cardiomyopathy-like phenotype was established. Long-term hyperglycaemia effects were analysed. Rat cardiomyoblasts overexpressing GLUT4 (H9C2KE2) were cultured under normo- and hyperglycaemic conditions for long-term. Expression profiles of several proteins were compared to non-transfected H9C2 cells (H9C2) using RT-qPCR, proteomics-based analysis, or Western blotting. GLUT4 surface analysis, glucose uptake, and cell morphology changes as well as apoptosis/necrosis measurements were performed using flow cytometry. Additionally, brain natriuretic peptide (BNP) levels, reactive oxygen species (ROS) formation, glucose consumption, and lactate production were quantified. Long-term hyperglycaemia in H9C2KE2 cells induced increased GLUT4 presence on the cell surface and was associated with exaggerated glucose influx and lactate production. On the metabolic level, hyperglycaemia affected the tricarboxylic acid (TCA) cycle with accumulation of fumarate. This was associated with increased BNP-levels, oxidative stress, and lower antioxidant response, resulting in pronounced apoptosis and necrosis. Chronic glucose overload in cardiomyoblasts induced by GLUT4 overexpression and hyperglycaemia resulted in metabolically stimulated proteome profile changes and metabolic alterations on the TCA level.

Enhanced growth factor expression in chronic diabetic wounds treated by cold atmospheric plasma.

AIMS: Cold atmospheric plasma (CAP) has been proven to enhance wound healing in superficial, chronically infected, diabetic foot ulcers. We aimed to investigate the molecular drivers responsible for this macroscopically observed improvement in diabetic wound healing. METHODS: Wound exudate was available from each change of dressing within a prospective, randomised, patient-blinded clinical trial. Specific protein level analyses were conducted via multiplex ELISA for wound samples of a representative subcohort (placebo: n = 13; CAP: n = 14). Expression of fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor A (VEGF-A), cytokines and matrix metalloproteinases (MMPs) were evaluated over a treatment period of about 14 days. RESULTS: Analysis revealed increased levels of the growth factors FGF-2 (placebo: median 46.9 range [32.0-168.6] AU vs. CAP: 113.7[55.8-208.1] AU) and VEGF-A (placebo: 79.7 [52.4-162.7] AU vs. CAP: 120.8 [51.1-198.1] AU) throughout the treatment period and in head-to-head comparison in a daily assessment. CAP-treated wounds showed increased levels of tumour necrosis factor-alpha, interleukins 1α and 8. However, the total protein amounts were not significantly elevated. The total protein amounts of MMPs were not altered by CAP. CONCLUSIONS: Induction of crucial growth factors, like FGF-2 and VEGF-A, and interleukins appears to be an important component of CAP-mediated promotion of granulation, vascularisation and reepithelialisation in the diabetic foot. These findings demonstrate for the first time that CAP-mediated growth factor induction also occurs in persons with diabetes, as previously described only in several in vitro and rodent experiments. Clinical Trial registration KPWTRIAL: NCT04205942, ClinicalTrials.gov.

Peripheral oscillometric arterial performance does not depict coronary status in patients with type 2 diabetes mellitus.

BACKGROUND: Arterial stiffness is associated with cardiovascular events. Matrix metalloproteases (MMPs), their tissue inhibitors (TIMPs) and galectin-3 are involved in the pathogenesis of end organ damage. This study aimed to evaluate the contribution of arterial stiffness, MMPs, TIMPs and galectin-3 with the current vascular status in type 2 diabetes mellitus (T2DM). METHODS: 74 patients with T2DM, 36 with coronary heart disease (CHD) (T2DM + CHD) and 38 without CHD (T2DM - CHD) were included. Aortic pulse wave velocity (PWVao), aortic and brachial augmentation indices (AIx aortic and AIx brachial) and central-aortic blood pressure values were determined by non-invasive arteriography. MMPs, TIMPs and galectin-3 plasma concentrations were analysed by ELISA. RESULTS: Patients with T2DM and CHD presented with significantly increased arterial stiffness determined as AIx and significantly elevated values for TIMP-4 and galectin-3. Heterogeneous peripheral vascular status regardless of the CHD status was observed, and increasing severity of CHD was associated with an increased arterial stiffness. TIMP-4 correlated significantly with an elevated PWVao in the whole cohort independently from CHD status. CONCLUSION: Determination of arterial stiffness is an effective and, compared to laboratory markers, more reliable method for determining the peripheral vascular situation in patients with T2DM, but it does not clearly depict coronary situation.

Metformin Is Associated with Reduced Tissue Factor Procoagulant Activity in Patients with Poorly Controlled Diabetes.

PURPOSE: Metformin is the first-line antidiabetic drug and shown to reduce cardiovascular risk independent from its glucose lowering action. Particularly in poorly controlled diabetes, tissue factor (TF) is expressed in the vasculature and accounts for thromboembolic complications. Here, we aimed to assess the effect of metformin on TF activity and markers of vascular inflammation in poorly controlled type 2 diabetes. METHODS: In a cohort of patients with uncontrolled type 2 diabetes (glycosylated hemoglobin 8.39 ± 0.24%, 68.1 ± 2.6 mmol/mol, n = 46) of whom half of the individuals were treated with metformin and the other half did not receive metformin as part of an anti-diabetic combination therapy, we assessed TF activity and markers of vascular inflammation. In vitro, human monocytic cells (THP-1) were exposed to metformin and TF expression measured in the presence and absence of the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide riboside (AICAR) or the AMPK inhibitor compound C. RESULTS: In the patients, metformin treatment was associated with lower levels of TF protein (241.5 ± 19 vs. 315.4 ± 25 pg/mL, p = 0.03) and reduced TF activity (408.9 ± 49 vs. 643.8 ± 47 U/mL, p = 0.001) compared with controls. Moreover, the patients on metformin showed lower levels of vascular cell adhesion molecule (VCAM)1 (26.6 ± 1.4 vs. 35.03 ± 3.1 ng/mL, p = 0.014) and higher expression of miR-126-3p/U6sno (11.39 ± 2.8 vs. 4.26 ± 0.9, p = 0.006), a known post-transcriptional down regulator of TF and VCAM1. In vitro, metformin dose-dependently reduced lipopolysaccharide (LPS)-induced TF expression in THP-1 cells. The AMPK activator AICAR alone lowered TF expression in THP-1, while the AMPK inhibitor compound C abrogated the metformin-dependent reduction in TF expression. CONCLUSIONS: Our data are the first to report that metformin is associated with reduced plasma TF procoagulant activity possibly explaining-at least in part-the vasculoprotective properties of metformin.

Effect of Cold Atmospheric Plasma Therapy vs Standard Therapy Placebo on Wound Healing in Patients With Diabetic Foot Ulcers: A Randomized Clinical Trial.

Importance: Diabetic foot ulcers are a common complication of diabetes and require specialized treatment. Cold atmospheric plasma (CAP) has been associated with benefits in wound infection and healing in previous smaller series of case reports. Yet the effect of CAP compared with standard care therapy in wound healing in diabetic foot ulcers remains to be studied. Objective: To determine whether the application of CAP accelerates wound healing in diabetic foot ulcers compared with standard care therapy. Design, Setting, and Participants: A prospective, randomized, placebo-controlled, patient-blinded clinical trial was conducted at 2 clinics with recruitment from August 17, 2016, to April 20, 2019. Patients were scheduled to remain in follow-up until April 30, 2024. Patients with diabetes and diabetic foot ulcers described using the combined Wagner-Armstrong classification of 1B or 2B (superficial or infected diabetic foot ulcers extending to tendon) were eligible. A patient could participate with 1 or more wounds in both groups in both intervention and control groups. Wounds were randomized separately, allowing a participant to be treated several times within the study following a 2 × 2 × 2 randomization strata considering sex, smoking status, and age (≤68 years and >68 years). Interventions: Standard care treatment with 8 applications of either CAP generated from argon gas in an atmospheric pressure plasma jet or 8 applications of placebo treatment in a patient-blinded manner. Main Outcomes and Measures: Primary end points were reduction in wound size, clinical infection, and microbial load compared with treatment start. Secondary end points were time to relevant wound reduction (>10%), reduction of infection, parameters of patient's well-being, and treatment-associated adverse events. Results: Of 65 diabetic foot ulcer wounds from 45 patients assessed for study, 33 wounds from 29 patients were randomized to CAP and 32 wounds from 28 to placebo, with 62 wounds from 43 patients (31 wounds per group) included for final evaluation (mean [SD] age, 68.5 [9.1] years for full sample). Four patients with 5 wounds of 31 (16.1%) wounds in the CAP group and 3 patients with 4 wounds of 31 (13%) wounds in the placebo group were active smokers. CAP therapy yielded a significant increase in wound healing, both in total mean (SD) area reduction (CAP vs placebo relative units, -26.31 [11.72]; P = .03) and mean (SD) time to relevant wound area reduction (CAP vs placebo relative units, 10% from baseline, 1.60 [0.58]; P = .009). Reduction of infection and microbial load was not significantly different between CAP and placebo. No therapy-related adverse events occurred during therapy; patient's perceptions during therapy were comparable. Conclusions and Relevance: In this randomized clinical trial, CAP therapy resulted in beneficial effects in chronic wound treatment in terms of wound surface reduction and time to wound closure independent from background infection. Trial Registration: ClinicalTrials.gov Identifier: NCT04205942.

Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes.

BACKGROUND: Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. METHODS: In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. RESULTS: In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS. CONCLUSIONS: miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.

Exenatide Delays Gastric Emptying in Patients with Type 2 Diabetes Mellitus but not in Those with Gastroparetic Conditions.

The effect of the treatment with glucagon-like peptide (GLP)-1 receptor agonists on gastric emptying in patients with diabetes with and without gastroparesis is analysed. Patients with type 2 diabetes mellitus subjected to GLP-1 receptor agonist therapy with exenatide were examined before and shortly after initiation of treatment. Gastric half-emptying time was determined by 13C-octanoic breath test; routine laboratory parameter as well as active GLP-1, ghrelin, leptin, insulin, proinsulin and C-peptide levels were determined in fasting state as well as postprandial secretion within 1 h after a standardised meal. Thirty patients' data sets were available for evaluation, of those 20 patients had no gastroparesis and 10 patients showed pathological results following the breath test. Gastric half-emptying time was prolonged in nearly all patients who presented without gastroparesis at initiation of treatment with GLP-1 receptor agonists, only 2 patients with pre-existing mild gastroparesis had worsening of gastric emptying. No effect was detected on leptin and ghrelin levels. Postprandial GLP-1 concentrations measured as AUC after meal decreased significantly. Fasting insulin and C-peptide levels increased significantly without effect on postprandial levels. Proinsulin levels - fasting as well as AUC - decreased non-significantly. Patients reported comparable perception of therapeutic effects. Treatment with GLP-1 receptor agonists may be applied in patients with pre-existing gastroparesis; no effect in terms of worsening of symptoms compared to those without gastroparesis was detected. Patients reported outcome was independent from underlying gastroparesis. Negative effects on gastric emptying were only detected in patients without or with mild gastroparesis.

Evaluation of 1,5-anhydro-d-glucitol in clinical and forensic urine samples.

Because of the lack of characteristic morphological findings post mortem diagnosis of diabetes mellitus and identification of diabetic coma can be complicated. 1,5-Anhydroglucitol (1,5-AG), the 1-deoxy form of glucose, competes with glucose for renal reabsorption. Therefore low serum concentrations of 1,5-AG, reflect hyperglycemic excursions over the prior 1-2 weeks in diabetic patients. Next to clinical applications determination of 1,5-AG can also be used in forensic analysis. To investigate the elimination of 1,5-AG, a liquid chromatographic-mass spectrometric method for the determination of 1,5-AG and creatinine in urine was developed and validated according to international guidelines. To evaluate ante mortem concentrations of 1,5-AG spot urine samples of 30 healthy subjects, 46 type 1 and 46 type 2 diabetic patients were analyzed. 1,5-AG urine concentrations of diabetic patients were significantly (p<0.001) lower (mean: 1.54µg/ml, n=92) compared to concentrations of healthy subjects (mean: 4.76µg/ml, n=30) which led to the idea that 1,5-AG urine concentrations post mortem might help in the interpretation of a diabetic coma post mortem. Urine of 47 deceased non-diabetics, 37 deceased diabetic and 9 cases of diabetic coma were measured. Comparison of blood and urine 1,5-AG concentrations in clinic samples (linear, R2=0.13) and forensic samples (linear, R2=0.02) showed no correlation. Urinary levels of 1,5-AG in deceased diabetic (mean 6.9µg/ml) and in non-diabetic patients (mean 6.3µg/ml) did not show a significant difference (p=0.752). However, urinary 1,5-AG concentrations in deceased due to diabetic coma (mean: 1.7µg/ml) were significantly lower than in non-diabetic (mean: 6.3µg/ml, p=0.039) and lower than in diabetic cases (mean: 4.7µg/ml, p=0.058). The determination of a reliable cut-off for the differentiation of diabetic to diabetic coma cases was not possible. Normalization of urinary 1,5-AG concentrations with the respective creatinine concentrations did not show any gain of information. In clinical (serum) and forensic blood samples a significant difference between all groups could be detected (p<0.05). Comparison of blood and urine 1,5-AG concentrations in clinical samples (linear, R2=0.13) and forensic samples (linear, R2=0.02) showed no correlation.

Effects of a hypoenergetic diet rich in α-linolenic acid on fatty acid composition of serum phospholipids in overweight and obese patients with metabolic syndrome.

OBJECTIVES: Plant-derived α-linolenic acid (ALA) may exert cardioprotective effects. Dietary ALA can undergo desaturation and elongation to form long-chain ω-3 polyunsaturated fatty acids, but the extent to which this occurs in humans is unclear. The aim of the study was to examine the effects of an energy-restricted diet enriched with ALA on fatty acid composition of serum phospholipids in patients with metabolic syndrome. METHODS: The present analysis compared the effects of a hypoenergetic diet high in ALA (3.4 g/d) with a control diet low in ALA (0.9 g/d) on fatty acid composition of serum phospholipids in 81 overweight or obese patients with features of metabolic syndrome. RESULTS: After a 26-wk intervention, concentration of ALA in serum phospholipids remained constant in both diet groups. The control group had a significant decrease in serum phospholipid eicosapentaenoic acid concentration, although no significant intergroup difference was observed. Serum phospholipid docosahexaenoic acid concentration significantly decreased to a similar extent with both interventions. Additionally, both interventions significantly decreased serum phospholipid concentrations of palmitic acid, stearic acid, total saturated fatty acids, linoleic acid, total ω-6 and ω-3 polyunsaturated fatty acids, with no effect of diet group on these changes. Compared with the ALA diet, the control diet led to a significant increase in serum phospholipid oleic acid concentration. CONCLUSION: Daily intake of 3.4 g of ALA during a 26-wk energy-restricted diet did not lead to an enrichment of serum phospholipids with ALA and did not increase eicosapentaenoic acid due to conversion. Additionally, dietary ALA was unable to compensate for a decrease in serum phospholipid docosahexaenoic acid.

MicroRNA-19a contributes to the epigenetic regulation of tissue factor in diabetes.

BACKGROUND: Diabetes mellitus is characterized by chronic vascular disorder and presents a main risk factor for cardiovascular mortality. In particular, hyperglycaemia and inflammatory cytokines induce vascular circulating tissue factor (TF) that promotes pro-thrombotic conditions in diabetes. It has recently become evident that alterations of the post-transcriptional regulation of TF via specific microRNA(miR)s, such as miR-126, contribute to the pathogenesis of diabetes and its complications. The endothelial miR-19a is involved in vascular homeostasis and atheroprotection. However, its role in diabetes-related thrombogenicity is unknown. Understanding miR-networks regulating procoagulability in diabetes may help to develop new treatment options preventing vascular complications. METHODS AND RESULTS: Plasma of 44 patients with known diabetes was assessed for the expression of miR-19a, TF protein, TF activity, and markers for vascular inflammation. High miR-19a expression was associated with reduced TF protein, TF-mediated procoagulability, and vascular inflammation based on expression of vascular adhesion molecule-1 and leukocyte count. We found plasma expression of miR-19a to strongly correlate with miR-126. miR-19a reduced the TF expression on mRNA and protein level in human microvascular endothelial cells (HMEC) as well as TF activity in human monocytes (THP-1), while anti-miR-19a increased the TF expression. Interestingly, miR-19a induced VCAM expression in HMEC. However, miR-19a and miR-126 co-transfection reduced total endothelial VCAM expression and exhibited additive inhibition of a luciferase reporter construct containing the F3 3'UTR. CONCLUSIONS: While both miRs have differential functions on endothelial VCAM expression, miR-19a and miR-126 cooperate to exhibit anti-thrombotic properties via regulating vascular TF expression. Modulating the post-transcriptional control of TF in diabetes may provide a future anti-thrombotic and anti-inflammatory therapy.

Intracellular Accumulation of Methylglyoxal by Glyoxalase 1 Knock Down Alters Collagen Homoeostasis in L6 Myoblasts.

Hyperglycemia results in accumulation of the reactive dicarbonyl methylglyoxal (MG). Methylglyoxal is detoxified by the glyoxalase system (glyoxalase 1 and 2). The influence of glyoxalase 1 knockdown on expression of collagens 1, 3, 4, and 5 in L6 myoblasts under hyperglycemic conditions was investigated. Increased biosynthesis of collagens 1, 3, 4, and 5 was detected at mRNA-level following knockdown of glyoxalase 1 (GLO1). At the protein level a significant elevation of the concentration of collagen 1 and 4 was shown, whereas no increase of collagen 5 and a non-significant increase in collagen 3 were detectable. These results could partially explain MG-induced changes in the extracellular matrix (ECM) which account for increased fibrosis and impaired function in myocytes. The mechanisms by which reactive glucose metabolites influence ECM composition deserve further investigation.

Metabolomic Signature of Coronary Artery Disease in Type 2 Diabetes Mellitus.

Coronary artery disease (CAD) is a common complication of type 2 diabetes mellitus (T2D). This case-control study was done to identify metabolites with different concentrations between T2D patients with and without CAD and to characterise implicated metabolic mechanisms relating to CAD. Fasting serum samples of 57 T2D subjects, 26 with (cases) and 31 without CAD (controls), were targeted for metabolite profiling of 163 metabolites. To assess the association between metabolite levels and CAD, partial least squares (PLS) analysis and multivariate logistic regression analysis with adjustment for CAD risk factors and medications were performed. We observed a separation of cases and controls with two classes of metabolites being significantly associated with CAD, including phosphatidylcholines, and serine. Four metabolites being independent from the common CAD risk factors displaying best separation between cases and controls were further selected. Addition of the metabolite concentrations to risk factor analysis raised the area under the receiver-operating-characteristic curve from 0.72 to 0.88 (p = 0.020), providing improved sensitivity and specificity for CAD classification. Serum phospholipid and serine levels independently discriminate T2D patients with and without CAD. Oxidative stress and reduced antioxidative capacity lead to lower metabolite concentrations probably due to changes in membrane composition and accelerated phospholipid degradation.

Glyoxalase 1-knockdown in human aortic endothelial cells - effect on the proteome and endothelial function estimates.

Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage.

Real-World Data Collection Regarding Titration Algorithms for Insulin Glargine in Patients With Type 2 Diabetes Mellitus.

The primary objective of this study was to collect data regarding the effectiveness of different dose titration algorithms (TAs) for optimization or initiation of basal insulin supported oral therapy (BOT) in patients with type 2 diabetes. A total of 50 patients were enrolled in this trial (17 women, 33 men, age 63 ± 8 years, HbA1c 7.9 ± 0.8%). The investigator decided on an individual basis to apply any of 4 standard TAs: standard (S: fasting glucose target 90-130 mg/dL, n = 39), standard-fast titration (S-FT: 90-130 mg/dL, larger dose increments at FBG < 180 mg/dl, n = 1), less tight (LT: 110-150 mg/dL, n = 5), and tight (T: 70-100 mg/dL, n = 5). During the next 30 days daily contacts were used to adapt the insulin dose. The majority of all patients (70%) achieved a stable insulin glargine dose within 5 ± 6 days after initiation of the dose titration. HbA1c improved from 7.9 ± 0.8% to 7.5 ± 0.7% (P < .001). In total, 1300 dose decisions were made (1192 according to the TA and 108 by the physicians independently from the TA in 29 patients [58% of study population]). Reasons for TA-overruling dosing decisions were hypoglycemic events (14 mild/4 moderate) in 9 patients. In the majority of these cases (89.8%), the physician recommended continuation of the previous dose or a higher dose. The majority of FBG values were within the respective target range after 4 weeks. In conclusion, the insulin glargine TAs delivered safe dose recommendations with a low risk of hypoglycemia, which successfully led to a stable dose in the vast majority of patients.

Micro-RNA-126 Reduces the Blood Thrombogenicity in Diabetes Mellitus via Targeting of Tissue Factor.

OBJECTIVE: Diabetes mellitus involves vascular inflammatory processes and is a main contributor to cardiovascular mortality. Notably, heightened levels of circulating tissue factor (TF) account for the increased thrombogenicity and put those patients at risk for thromboembolic events. Here, we sought to investigate the role of micro-RNA (miR)-driven TF expression and thrombogenicity in diabetes mellitus. APPROACH AND RESULTS: Plasma samples of patients with diabetes mellitus were analyzed for TF protein and activity as well as miR-126 expression before and after optimization of the antidiabetic treatment. We found low miR-126 levels to be associated with markedly increased TF protein and TF-mediated thrombogenicity. Reduced miR-126 expression was accompanied by increased vascular inflammation as evident from the levels of vascular adhesion molecule-1 and fibrinogen, as well as leukocyte counts. With optimization of the antidiabetic treatment miR-126 levels increased and thrombogenicity was reduced. Using a luciferase reporter system, we demonstrated miR-126 to directly bind to the F3-3'-untranslated region, thereby reducing TF expression both on mRNA and on protein levels in human microvascular endothelial cells as well as TF mRNA and activity in monocytes. CONCLUSIONS: Circulating miR-126 exhibits antithrombotic properties via regulating post-transcriptional TF expression, thereby impacting the hemostatic balance of the vasculature in diabetes mellitus.

Impairment of Wound Healing in Patients With Type 2 Diabetes Mellitus Influences Circulating MicroRNA Patterns via Inflammatory Cytokines.

OBJECTIVE: MicroRNAs (miRNA/miR) are stably present in body fluids and are increasingly explored as disease biomarkers. Here, we investigated influence of impaired wound healing on the plasma miRNA signature and their functional importance in patients with type 2 diabetes mellitus. APPROACH AND RESULTS: miRNA array profiling identified 41 miRNAs significantly deregulated in diabetic controls when compared with patients with diabetes mellitus-associated peripheral arterial disease and chronic wounds. Quantitative real-time polymerase chain reaction validation confirmed decrease in circulating miR-191 and miR-200b levels in type 2 diabetic versus healthy controls. This was reverted in diabetic subjects with associated peripheral arterial disease and chronic wounds, who also exhibited higher circulating C-reactive protein and proinflammatory cytokine levels compared with diabetic controls. miR-191 and miR-200b were significantly correlated with C-reactive protein or cytokine levels in patients with diabetes mellitus. Indeed, proinflammatory stress increased endothelial- or platelet-derived secretion of miR-191 or miR-200b. In addition, dermal cells took up endothelial-derived miR-191 leading to downregulation of the miR-191 target zonula occludens-1. Altered miR-191 expression influenced angiogenesis and migratory capacities of diabetic dermal endothelial cells or fibroblasts, respectively, partly via its target zonula occludens-1. CONCLUSIONS: This study reports that (1) inflammation underlying nonhealing wounds in patients with type 2 diabetes mellitus influences plasma miRNA concentrations and (2) miR-191 modulates cellular migration and angiogenesis via paracrine regulation of zonula occludens-1 to delay the tissue repair process.

Impact of diabetes on epidemiology, treatment, and outcomes of patients with heart failure.

The prevalence of patients with concomitant heart failure (HF) and diabetes mellitus (DM) continues to increase with the general aging of the population. In patients with chronic HF, prevalence of DM is 24% compared with 40% in those hospitalized with worsening HF. Patients with concomitant HF and DM have diverse pathophysiologic, metabolic, and neurohormonal abnormalities that potentially contribute to worse outcomes than those without comorbid DM. In addition, although stable HF outpatients with DM show responses that are similar to those of patients without DM undergoing evidence-based therapies, it is unclear whether hospitalized HF patients with DM will respond similarly to novel investigational therapies. These data support the need to re-evaluate the epidemiology, pathophysiology, and therapy of HF patients with concomitant DM. This paper discusses the role of DM in HF patients and underscores the potential need for the development of targeted therapies.

Effects of an energy-restricted diet rich in plant-derived α-linolenic acid on systemic inflammation and endothelial function in overweight-to-obese patients with metabolic syndrome traits.

Plant-derived α-linolenic acid (ALA) may reduce the risk of CVD, possibly by decreasing systemic inflammation and improving endothelial function. In the present study, the effects of a hypoenergetic diet rich in ALA (3·4 g/d) on the biomarkers of systemic inflammation and vascular function were investigated in eighty-one overweight-to-obese patients with metabolic syndrome traits in comparison with a hypoenergetic diet low in ALA (0·9 g/d, control). After a 6-month dietary intervention, there were significant decreases in the serum concentrations of C-reactive protein (CRP), TNF-α, IL-6, soluble intercellular adhesion molecule-1 (sICAM-1), soluble endothelial selectin (sE-selectin) and asymmetric dimethylarginine in both dietary groups. However, no inter-group differences were observed for all these changes. The serum concentration of YKL-40 (human cartilage glycoprotein 39 or chitinase-3-like protein 1) decreased after the ALA diet when compared with the control diet (P< 0·05 for time × treatment interaction). Plasma concentrations of fibrinogen did not significantly change in the two dietary groups. The decreases in the serum concentrations of sICAM-1, sE-selectin, CRP and YKL-40 were significantly correlated with the decreases in body fat mass. In conclusion, the present study indicates that in overweight-to-obese patients with metabolic syndrome traits, both vascular function and inflammation are improved during body-weight loss. The high ALA intake led to a more pronounced reduction in the serum concentration of YKL-40 compared with the intake of the low-ALA control diet, indicating the existence of independent favourable physiological effects of ALA during weight loss.