sq-23377 and Diabetes-Mellitus--Type-2

The excessive amount of reactive species under chronic inflammation, which are accompanied by an increase body temperature, lead to diabetic complications. Phagocyte NADPH oxidase is the key enzyme in these processes. The role of high temperature in its regulation in diabetes is not clear. The aim was to investigate the effect of high temperature on NADPH-oxidase-dependent generation of reactive species in diabetic patients. Chemiluminescent method was applied to assess respiratory burst kinetics initiated by opsonized zymosan in blood or phorbol ester in isolated granulocytes. Analyzing ROC curves, the main predictors and changes in stages of activation of NADPH oxidase were determined. Phosphoisoforms of p47

Topics: Diabetes Mellitus, Type 2; Humans; Inflammation; Ionomycin; NADP; NADPH Oxidases; Neutrophils; Phorbol Esters; Phosphoproteins; Temperature; Zymosan

MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus.. This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function.. Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins.. Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.

Topics: Adult; Animals; Blood Platelets; Calcium; Calpain; Cytoskeletal Proteins; DEAD-box RNA Helicases; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Factor XIII; Female; Humans; Ionomycin; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Microsatellite Repeats; Middle Aged; Neoplasm Proteins; Platelet Aggregation; Proteome; Ribonuclease III

Irisin, myokine secreted by skeletal muscle, was suggested to mediate some of exercise health benefits via "browning" of white adipose tissue. However, mounting evidence contradicts the regulatory role of exercise for muscle irisin production/secretion in humans. Thus, we explored the direct effect of exercise-mimicking treatment on irisin in human primary muscle cells in vitro. Human primary muscle cell cultures were established from lean, obese prediabetic and type-2-diabetic individuals. Complex metabolic phenotyping included assessment of insulin sensitivity (euglycemic hyperinsulinemic clamp) and adiposity content&distribution (MRI&MRS). In vitro exercise-mimicking treatment (forskolin+ionomycin) was delivered in 1-h pulse/day during differentiation. Fndc5 mRNA (qRT-PCR) and secreted irisin (ELISA) were determined in cells and media. Exercise-mimicking treatment more than doubled Pgc1α mRNA in differentiated muscle cells. Nevertheless, Fndc5 mRNA was reduced by 18% and irisin in media by 20%. Moreover, Fncd5 mRNA was increased in myotubes derived from individuals with type-2-diabetes, independent on exercise-mimicking treatment. Fndc5 mRNA in cells was positively related to fasting glycemia (p=0.0001) and negatively to whole-body insulin sensitivity (p<0.05). Collectively, our data do not support the role of exercise-related signaling pathways in irisin regulation in human skeletal muscle and confirm our previous observations on increased Fndc5 expression in muscle cells from individuals with type-2-diabetes.

Topics: Cells, Cultured; Colforsin; Diabetes Mellitus, Type 2; Exercise; Fibronectins; Humans; Ionomycin; Muscle Fibers, Skeletal; Muscle, Skeletal; Obesity; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Prediabetic State; RNA, Messenger; Transcription Factors

Intracellular Ca2+ homeostasis in platelets of patients with non-insulin-dependent diabetes mellitus (NIDDM) has been reported to be altered, leading to an increased adhesiveness and spontaneous aggregation. Among the disturbed Ca2+ mechanism in platelets from NIDDM subjects, a reduced Ca2+ extrusion by the plasma membrane Ca2+-ATPase (PMCA) is especially relevant, maintaining an elevated cytosolic free Ca2+ concentration that results in platelet hypersensitivity. Here we show that treatment of platelets from NIDDM patients with 300 U/mL catalase or 5 mM D-mannitol, which prevent H2O2- and hydroxyl radicals-mediated oxidative stress, respectively, increases Ca2+ extrusion after treatment with thapsigargin (TG) plus ionomycin (Iono). In contrast, 1 mM trolox, a scavenger of ONOO-, did not alter TG + Iono-induced response. Catalase and D-mannitol reversed the enhanced tyrosine phosphorylation of PMCA induced by TG + Iono in NIDDM patients. These findings open up new horizon for the development of therapeutic strategies to palliate cardiovascular disorders in NIDDM.

Topics: Antioxidants; Blood Platelets; Calcium; Calcium-Transporting ATPases; Catalase; Cell Membrane; Diabetes Mellitus, Type 2; Enzyme Inhibitors; Humans; Ionomycin; Ionophores; Phosphotyrosine; Platelet Activation; Reactive Oxygen Species; Thapsigargin

Cytosolic Ca2+ mobilization, especially Ca2+ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca2+ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca2+ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca2+ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H2O2 and ONOO- are likely involved in the enhanced Ca2+ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability.

Topics: Calcium; Case-Control Studies; Catalase; Chromans; Diabetes Mellitus, Type 2; Humans; Hydrogen Peroxide; Ionomycin; Mannitol; Peroxynitrous Acid; Platelet Aggregation; Reactive Oxygen Species; Thapsigargin

We clearly show that plasma membrane Ca2+ ATPase (PMCA) activity is lower in platelets from patients with non-insulin-dependent diabetes mellitus (NIDDM) than in those from healthy controls. The lower activity is likely due to reduced PMCA expression and increased tyrosine phosphorylation. These findings provide an explanation for the cellular ionic defects occurring in insulin resistant conditions.

Topics: Adult; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cation Transport Proteins; Diabetes Mellitus, Type 2; Female; Humans; Insulin Resistance; Ionomycin; Male; Phosphorylation; Phosphotyrosine; Plasma Membrane Calcium-Transporting ATPases; Platelet Activation; Protein Processing, Post-Translational; Thapsigargin

We sought to determine the mechanisms for hyperactivity and abnormal platelet Ca(2+) homeostasis in diabetes. The glycosylated Hb (HbA(1c)) level was used as an index of glycemic control. Human platelets were loaded with Ca- green-fura red, and cytosolic Ca(2+) ([Ca(2+)](i)) and aggregation were simultaneously measured. In the first series of experiments, the platelets from diabetic and normal subjects were compared for the ability to release Ca(2+) or to promote Ca(2+) influx. A potent and relatively specific inhibitor of Na(+)/Ca(2+) exchange, 5-(4-chlorobenzyl)-2',4'-dimethylbenzamil (CB-DMB), increased the second phase of thrombin-induced Ca(2+) response, suggesting that the Na(+)/Ca(2+) exchanger works in the forward mode to mediate Ca(2+) efflux. In contrast, in the platelets from diabetics, CB-DMB decreased the Ca(2+) response, indicating that the Na(+)/Ca(2+) exchanger works in the reverse mode to mediate Ca(2+) influx. In the second series of experiments we evaluated the direct effect of hyperglycemia on platelets in vitro. We found that thrombin- and collagen-induced increases in [Ca(2+)](i) and aggregation were not acutely affected by high glucose concentrations of 45 mM. However, when the platelet-rich plasma was incubated with a high glucose concentration at 37 degrees C for 24 h, the second phase after thrombin activation was inhibited by CB-DMB. In addition, collagen-stimulated [Ca(2+)](i) response and aggregation were also increased. Thus in diabetes the direction and activity of the Na(+)/Ca(2+) exchanger is changed, which may be one of the mechanisms for the increased platelet [Ca(2+)](i) and hyperactivity. Prolonged hyperglycemia in vitro can induce similar changes, suggesting hyperglycemia per se may be the factor responsible for the platelet hyperactivity in diabetes.

Topics: Amiloride; Blood Glucose; Blood Platelets; Calcium; Collagen; Cytosol; Diabetes Mellitus; Diabetes Mellitus, Type 1; Diabetes Mellitus, Type 2; Homeostasis; Humans; In Vitro Techniques; Ionomycin; Platelet Aggregation; Reference Values; Sodium-Calcium Exchanger; Thrombin