High-performance liquid chromatography method for quantifying sphingomyelin in rat brain.

A rapid, reproducible and accurate high-performance liquid chromatographic (HPLC) method for the quantitative determination of sphingomyelin in rat brain was developed and validated using normal-phase silica gel column, acetonitrile-methanol-water (65:18:17 (v/v)) at a flow rate of 1 ml/min, isocratic elution, UV detection at 207 nm and 1,2-dimyristoyl-sn-glycero-3-phosphocholine as an internal standard. Total run time was 10.0 min. The calibration curve was linear over the range of 0.025-0.4 mg/ml sphingomyelin (R2>0.99). The intra-day coefficient of variation ranged from 1.4% to 2.2%. The average inter-day coefficient of variation over a period of 4 days was 3.1%. The practical limit of detection was 0.005 mg/ml with a quantification limit of 0.01 mg/ml.

Effect of type-1 diabetes mellitus on the regulation of insulin and endothelin-1 receptors in rat hearts.

This project assesses the treatment role with insulin and (or) angiotensin II receptor subtype-1 (AT1-R) blocker (ARB) on insulin receptor and endothelin-1 receptor subtype (ETA-R and ETB-R) regulation in rat hearts suffering from insulin-dependent diabetes mellitus (IDDM). Animals were divided into 6 groups: groups 1, 3, and 5 were controls consisting of normal, diabetic (streptozotocin-treated, once at 0 time), and diabetic supplemented daily with insulin, respectively, whereas groups 2, 4, and 6 were the controls treated daily with losartan. One month after enrollment, rats were sacrificed and samples of cardiac tissue were snapped frozen for immunostaining and Western blotting. Insulin receptor density was observed to be upregulated in the cardiomyocytes of diabetic animals, but downregulated with insulin supplementation alone. Cotreatment with insulin and an ARB resulted in drastic increase in insulin-receptor density in the diabetic rats. In addition, expression of ETA-R in cardiomyocytes was upregulated and was consistently maintained within the various treatment modalities. However, ETB-R expression was significantly reduced in the diabetic group treated with both insulin and an ARB. The changes in the expression of the insulin, the ETA-Rs, and the ETB-Rs at the various sites of the myocardium and the effect of both insulin treatment and blockade of the AT1-R explain the new benefits related to the halting of myocardial remodeling in IDDM rats.

Effect of insulin and angiotensin II receptor subtype-1 antagonist on myocardial remodelling in rats with insulin-dependent diabetes mellitus.

OBJECTIVES: To assess the role of insulin or an angiotensin II receptor antagonist (losartan), or both, in preventing cardiomyocyte damage in rats suffering from insulin-dependent diabetes mellitus (IDDM), and to correlate it with insulin receptor modulation at the cardiomyocyte, coronary endothelium and skeletal muscle cell level. DESIGN: Animals were divided into groups of normal rats, diabetic rats, and diabetic rats given insulin, each subdivided into a control group and an experimental group treated with losartan. METHODS: The animals were killed 1 month after enrollment to the study. Perfusion of the heart with iodine-125-labelled insulin was carried out for all the groups and the binding kinetics of insulin to its receptors on the coronary endothelial cells and the cardiomyocytes were determined using a physical/mathematical model. In addition, tissue samples from the heart and intercostal skeletal muscle were snap frozen and used for histological, indirect immunofluorescence and western blot analysis. RESULTS: Cardiac muscle from diabetic animals exhibited diffuse cardiomyopathic changes consisting of widespread vacuolation, loss of striation and cellular hypertrophy, which were reduced and even prevented by treatment with insulin and losartan. In addition, losartan seemed to mediate the upregulation of insulin receptor density on cardiomyocytes and skeletal muscle, and increase insulin receptor affinity at the coronary endothelial site. Finally, treatment with losartan induced a significant decrease in glucose concentrations in the diabetic group compared with the appropriate controls. CONCLUSIONS: Addition of losartan to the standard insulin treatment in non-hypertensive animals with IDDM offers new benefits concerning cardiac protection and prevention of damage. This may be attributed, in part, to insulin receptor density and sensitization.

Effect of systemic insulin and angiotensin II receptor subtype-1 antagonist on endothelin-1 receptor subtype(s) regulation and binding in diabetic rat heart.

This study reports on the regulation and remodeling role of endothelin-1 (ET-1) and its receptor subtypes, ET(A)-Rs/ET(B)-Rs, at the coronary endothelium (CE) and cardiomyocyte (CM) sites. It is carried out in normal and normotensive rats with streptozotocin-induced diabetes mellitus receiving different treatment modalities. Normal rats were divided into two groups, namely a placebo (N) and a losartan-treated (NL), and diabetic rats into four groups receiving placebo (D), insulin-treated (DI), losartan-treated (DL), and insulin/losartan-treated (DIL) respectively. Binding kinetics of ET-1 to ET(A)-Rs/ET(B)-Rs on CE and CMs were assessed in the above groups to try to explain the effect of therapeutic doses of an angiotensin II receptor subtype-1 blocker on the dynamics of this ligand and its receptor in insulin supplemented diabetic animals. Each group was divided into two subgroups: CHAPS-untreated and CHAPS-treated rat hearts perfused with [125I]ET-1 to respectively estimate ET-1 binding affinity (tau = 1/k-n) to its receptor subtype(s) on CE and CMs using mathematical modeling describing a 1:1 reversible binding stoichiometry. Heart perfusion results revealed that insulin treatment significantly decreased tau on CE but not on CMs in diabetic rats. In diabetics treated with losartan, an increase in tau value on CE but not on CMs was noted. Cotreatment of diabetic rats with insulin and losartan normalized tau on CE but decreased it on CMs. Western blot, using snap-frozen heart tissues, revealed increase in ET(A)-R density in all diabetic groups. However, significant decrease in ET(B)-R density was observed in all groups compared to the normal, and was reconfirmed by immunohistochemical analysis. In conclusion, coadministration of insulin and losartan in nonhypertensive animals suffering from diabetes type 1 may offer new cardiac protection benefits by improving coronary blood flow and cardiomyocyte contractility through modulating ET-1 receptor subtypes density and affinity at CE and CM sites.