sq-23377 and Diabetes-Mellitus

In the current study, we examined whether diabetes affected the ability of HDL to stimulate nitric oxide (NO) production. Using HDL isolated from both diabetic humans and diabetic mouse models, we found that female HDL no longer induced NO synthesis, despite containing equivalent amounts of estrogen as nondiabetic controls. Furthermore, HDL isolated from diabetic females and males prevented acetylcholine-induced stimulation of NO generation. Analyses of both the human and mouse diabetic HDL particles showed that the HDLs contained increased levels of myristic acid. To determine whether myristic acid associated with HDL particles was responsible for the decrease in NO generation, myristic acid was added to HDL isolated from nondiabetic humans and mice. Myristic acid-associated HDL inhibited the generation of NO in a dose-dependent manner. Importantly, diabetic HDL did not alter the levels of endothelial NO synthase or acetylcholine receptors associated with the cells. Surprisingly, diabetic HDL inhibited ionomycin-induced stimulation of NO production without affecting ionomycin-induced increases in intracellular calcium. Further analysis indicated that diabetic HDL prevented calmodulin from interacting with endothelial NO synthase (eNOS) but did not affect the activation of calmodulin kinase or calcium-independent mechanisms for stimulating eNOS. These studies are the first to show that a specific fatty acid associated with HDL inhibits the stimulation of NO generation. These findings have important implications regarding cardiovascular disease in diabetic patients.

Topics: Acetylcholine; Animals; Calcium; Calmodulin; Cells, Cultured; Diabetes Mellitus; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Endothelial Cells; Estrogens; Female; Humans; Ionomycin; Ionophores; Lipoproteins, HDL; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myristic Acid; Nitric Oxide; Nitric Oxide Synthase Type III; Receptors, Cholinergic; Receptors, Leptin; Signal Transduction

Diabetic nephropathy is associated with progressive renal damage, leading to impaired function and end-stage renal failure. Secondary hypertension stems from a deranged ability of cells within the kidney to resolve and appropriately regulate sodium resorption in response to hyperglycaemia. However, the mechanisms by which glucose alters sodium re-uptake have not been fully characterised.. Here we present RT-PCR, western blot and immunocytochemistry data confirming mRNA and protein expression of the serum and glucocorticoid inducible kinase (SGK1) and the alpha conducting subunit of the epithelial sodium channel (ENaC) in a model in vitro system of the human cortical collecting duct (HCD). We examined changes in expression of these elements in response to glucose challenge, designed to mimic hyperglycaemia associated with type 2 diabetes mellitus. Changes in Na+ concentration were assessed using single-cell microfluorimetry.. Incubation with glucose, the Ca2+-ionophore ionomycin and the cytokine TGF-beta1 were all found to evoke significant and time-dependent increases in both SGK1 and alphaENaC protein expression. These molecular changes were correlated to an increase in Na+-uptake at the single-cell level.. Together these data offer a potential explanation for glucose-evoked Na+-resorption and a potential contributory role of SGK1 and ENaCs in development of secondary hypertension, commonly linked to diabetic nephropathy.

Topics: Blotting, Western; Cells, Cultured; Diabetes Mellitus; Epithelial Sodium Channels; Glucose; Humans; Hyperglycemia; Hypertension; Immediate-Early Proteins; Ionomycin; Kidney Tubules, Collecting; Protein Serine-Threonine Kinases; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sodium; Transforming Growth Factor beta1

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