Paweł Maćkowiak

Acute orexin effects on insulin secretion in the rat: in vivo and in vitro studies

Orexin-A and orexin-B are members of a family of newly described orexigenic hypothalamic neuropeptides. Scanty data are available suggesting the involvement of orexins in regulation of the secretion of pituitary hormones and in control of energy homeostasis. Present studies aimed to explain whether orexins affect blood insulin concentration and insulin secretion in the rat. To check this possibility, adult female rats were subcutaneously injected with different doses (1 or 2 nmol) of orexin-A or orexin-B. A bolus administration of orexin-A resulted in an increase in blood insulin (up to min 120) and glucose (60 min after injection) concentration. The higher dose of orexin-B, on the other hand, exerted effect on insulin secretion only at min 60 of experiment and neither doses changed blood glucose level. Only orexin-A stimulated insulin secretion in an in vitro perfusion system of the rat pancreas preparation, while orexin-B was less effective. The results demonstrate that orexins belong to a group of neuropeptides influencing insulin secretion and acting directly on the pancreas. Direct, at least partial, effect of orexin on insulin secretion may be connected with the regulation of metabolism by this peptide.

Genistein-Induced Changes in Lipid Metabolism of Ovariectomized Rats

The effect of the isoflavone, genistein, on the lipid metabolism of ovariectomized rats was studied. Three types of experiments were performed. In the first one, the rats were fed diets supplemented with 0.01 or 0.1% of genistein for 14 days. In the second and third experiments, the direct effect of genistein on the liver and fat tissue were measured respectively by means of liver perfusion or incubation of isolated adipocytes with the isoflavone. Genistein in food significantly decreased blood serum and muscle triglyceride concentrations and increased the level of free fatty acids in serum. Serum free cholesterol was diminished and liver cholesterol was enhanced after genistein ingestion. When genistein acted directly on the liver during perfusion, a smaller incorporation of 14C-glucose into lipids was observed, and in parallel a greater output of free fatty acids into the medium was noticed. These changes were accompanied by diminution of the liver triglyceride contents. Genistein, acting on the adipocytes strongly depressed both basal and insulin-induced lipid synthesis, when glucose was used as a substrate. The effect of the isoflavone alone on the lipolysis in the adipocytes was negligible. However, it intensified lipolysis induced by epinephrine. The results obtained let us conclude that genistein in food can reduce the fattening processes in ovariectomized rats. This effect of genistein may be attributed, at least in part, to its direct influence on lipid metabolism in the liver and adipose tissue.

In vivo and in situ action of melatonin on insulin secretion and some metabolic implications in the rat.

Introduction The role of melatonin in human insulin regulation is poorly understood. Aim To investigate the influence of melatonin supplementation on glucose and insulin levels and on lipid metabolism in blood serum and the liver. Methodology The acute melatonin effects on insulin secretion in male Wistar rats were investigated. In addition, carbohydrate and lipid metabolism was studied. In in vivo experiments, melatonin was administered subcutaneously in two different doses (0.5 and 1.0 mg/kg body weight, respectively), and animals were decapitated after 1 hour. Results The higher dose of the hormone increased insulin level in blood. The applied pancreas perfusion technique allowed us to confirm a direct mechanism of melatonin action on the pancreas. The ability of melatonin to stimulate insulin output was dose dependent. The highest effect was noticed for 100 nmol/L, whereas 1 nmol/L did not influence this process. Conclusion Melatonin treatment in vivo caused many biochemical consequences. The hormone augmented significantly the concentrations of total, free, and esterified cholesterol, as well as high-density lipoprotein cholesterol in blood. Together with the enhanced insulin secretion observed in the in vivo experiment, the level of free fatty acids in blood decreased and, surprisingly, glucose concentration was significantly elevated.

Effect of Isoflavone Genistein on Insulin Receptors in Perfused Liver of Ovariectomized Rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein, insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd). BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610 +/- 77 x 10(-15) (no genistein) to 238 +/- 72 x 10(-15) mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372 +/- 59 x 10(-15) mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421 +/- 46 x 10(-15) mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM--1.44 +/- 0.18 x 10(-9) mol/l; perfusion with BMP + genistein--0.83 +/- 0.20 x 10(-9) mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.

Acute leptin action on insulin blood level and liver insulin receptor in the rat.

Aim of the study was to investigate acute leptin effect on insulin blood level and liver insulin binding in the rat. The administration of leptin induced time and dose dependent decrease in the insulin level, which was statistically significant in comparison to the control animals 120 min after administration of higher dose of peptide (0.30 +/- 0.05 vs 0.14 +/- 0.01 nmol/l, respectively). Simultaneously, we have shown the attenuation of liver sensitivity to insulin 2 hours after higher leptin dose injection. This phenomenon was caused by the decrease of binding capacity of high affinity insulin receptor sites (HAIR), which was statistically significant after higher leptin dose administration at both time points (0.54 +/- 0.13 vs 0.26 +/- 0.03 and 0.71 +/- 0.12 vs 0.40 +/- 0.05 pmol/mg protein for 1 and 2 h, respectively). The present study provides evidence that leptin, in addition to its inhibitory effect on insulin secretion, acts as a modulator of insulin receptor, through the decrease of binding capacity. It seems legitimate to suggest that leptin-induced decrease of insulin receptor binding capacity may be one of several causes of insulin resistance.

Evaluation of insulin binding and signaling activity of newly synthesized chromium(III) complexes in vitro.

In the present study, the influence of chromium(III) complexes (acetate, chloride, glycinate, histidinate, lactate and propionate) on insulin binding and signal transduction [phosphorylation of tyrosine and serine in the insulin receptor substrate (IRS)-1] was investigated in vitro using three experimental models: isolated rat liver membranes and cultured mouse C2C12 myoblasts or 3T3-L1 preadipocytes. The examined complexes did not elevate the binding of insulin to the liver membranes. Moreover, chromium histidinate, lactate, acetate and propionate complexes diminished the specific binding of insulin. Simultaneously, chromium chloride, which did not significantly elevate insulin binding, increased the number of membrane accessible particles of the insulin receptors. However, it was accompanied by slightly diminished affinity of the receptor to the hormone. Chromium acetate and propionate significantly diminished the binding capacity of the low-affinity insulin receptor class. Investigations with the myoblast cell line C2C12 and preadipocyte cell line 3T3-L1 did not allow differentiation of the influence of the examined complexes on insulin binding. Immunodetection of phosphorylated forms of IRS-1 showed that the chromium compounds modulated the transduction of the insulin signal. Chromium glycinate, acetate and propionate decreased the amount of IRS-1 phosphorylated at serine. Since it is generally thought that phosphorylation of serine in IRS-1 may moderate insulin action, the above mentioned chromium complexes may, in this way, enhance insulin effects inside target cells. Phosphorylation of tyrosine in IRS-1, which acts as a stimulatory signal for further steps of insulin action, was elevated after the incubation of 3T3-L1 cells with insulin. Chromium supplementation did not additionally intensify this process. However, in the absence of insulin, chromium glycinate and acetate slightly elevated the level of IRS-1 phosphorylated at tyrosine. This fact may be important in vivo at low levels of insulin in blood. The results indicate that the action of chromium(III) complexes involves a direct effect on the number of receptors accessible to insulin, their affinity to the hormone and the modulation of the signal multiplying proteins by their phosphorylation.

The influence of coumestrol, zearalenone, and genistein administration on insulin receptors and insulin secretion in ovariectomized rats.

ABSTRACT The influence of phytoestrogens (genistein and coumestrol) and mycoestrogen (zearalenone) on insulin secretion, liver insulin receptors and some aspects of lipid and carbohydrate metabolism were investigated in this study. Ovariectomized rats were injected s.c. with the above mentioned compounds in the amount of 1 mg for three days. Coumestrol and zearalenone caused a significant increase in uterus weight, similar to the effects observed after estrone action, while this effect was not observed after the genistein injection. Blood insulin level was not changed after phyto- or mycoestrogen treatment. However, coumestrol and genistein significantly decreased the binding capacity of liver insulin receptors. These changes corresponded with alterations in glucose and free fatty acids profiles in blood, as well as with glycogen content in liver. The effects observed after genistein and coumestrol injections differed from those noticed in rats treated with zearalenone or estrone. On the basis of these results we conclude that metabolic effects of high doses of coumestrol and genistein in ovariectomized rats are partly mediated by changes in insulin sensitivity of the liver and that the action of plant estrogens on metabolism is, at least to the some degree, independent of their estrogen activity.

Changes in obestatin gene and GPR39 receptor expression in peripheral tissues of rat models of obesity, type 1 and type 2 diabetes.

Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM.

Resistin is produced by rat pancreatic islets and regulates insulin and glucagon in vitro secretion

ABSTRACT Resistin participates in the regulation of energy homeostasis, insulin resistance, and inflammation. The potential expression in pancreas, and modulation of the endocrine pancreas secretion by resistin is not well characterized, therefore, we examined it on several levels. We examined the localization of resistin in rat pancreatic islets by immunohistochemistry and immunofluorescence, and the potential presence of resistin mRNA by RT-PCR and protein by Western Blot in these structures. In addition, we studied the regulation of insulin and glucagon secretion by resistin in pancreatic INS-1E β- and InR-G9 α-cell lines as well as isolated rat pancreatic islets. We identified resistin immunoreactivity in the periphery of rat pancreatic islets and confirmed the expression of resistin at mRNA and protein level. Obtained data indicated that resistin is co-localized with glucagon in pancreatic α-cells. In addition, we found that in vitro resistin decreased insulin secretion from INS-1E cells and pancreatic islets at normal (6 mM) and high (24 mM) glucose concentrations, and also decreased glucagon secretion from G9 cells and pancreatic islets at 1 mM, whereas a stimulation of glucagon secretion was observed at 6 mM glucose. Our results suggest that resistin can modulate the secretion of insulin and glucagon from clonal β or α cells, and from pancreatic islets.

Influence of short-term L-arginine supplementation on carbohydrate balance in rats with ischemia-reperfusion syndrome

BACKGROUND There are studies showing stimulative effect of arginine on insulin secretion. This mechanism is not fully explained. The effects of the impact of arginine on carbohydrate balance under the conditions of ischemia and reperfusion remain to be determined. The aim of this study is the evaluation of the influence of short-term L-arginine supplementation on the concentration of glucose and insulin in blood and insulin binding in rat skeletal muscle under the conditions of ischemia and reperfusion. METHODS The study was conducted on male Wistar rats with average body mass 250 ± 30 g. Animals were divided into four groups: Group I - control, Group II - placebo, Group III - L-arginine 500 mg/kg/24 h for 5 days, Group IV - L-arginine and L-NAME (75 μmol/rat/24 h) for 5 days. Each group was divided into subgroups depending on duration of ischemia and reperfusion. Acute ischemia of hind limb was induced in each group by putting pneumatic tourniquet on the thigh. Blood samples and skeletal muscles were collected from the rats. Plasma concentrations of glucose and insulin were measured. Insulin binding to insulin receptors was determined in skeletal muscle. RESULTS A clear reduction of insulin binding to receptor was found in the group of animals without ischemia and the group supplemented with L-arginine and subjected to 4-h ischemia and 30- and 120-min reperfusion. A significant increase in insulin level was found in groups of animals with L-arginine and/or L-NAME subjected to 4-h ischemia at all times of reperfusion. Supplementation with L-arginine and/or L-NAME decreased levels of glucose in blood serum of animals undergoing ischemia-reperfusion syndrome compared to the control and placebo groups. CONCLUSION Under conditions of ischemia-reperfusion, short-term administration of L-arginine causes a decrease in insulin binding capacity of insulin receptors in skeletal muscle, an increase in insulin level and a decrease in the concentration of glucose in blood serum.