Simultaneous extraction of beta-endorphin and leu- and met-enkephalins from human and rat plasma

A simple, rapid and reliable procedure is described to simultaneously concentrate and purify beta-endorphin, leu- and met-enkephalins from small volumes of human and rat plasma before radioimmunoassay is performed. It uses C18 Sep-Pak reverse phase cartridges. The effectiveness of different protease inhibitors in preventing degradation of opiates by plasma and different solvent systems for eluting opiates is also evaluated.     

Half life of injected 125I-insulin in control and ob/ob mice

The t-1/2 of moniodo 125I-insulin in ob/ob mice and their lean litter mates is 10 min. No difference was found between the two groups. Further, 48 hr of fasting did not alter the t-1/2 in ob/ob mice. In view of the markedly enlarged insulin pool in ob/ob mice, one must conclude that the mass of insulin degraded in unit time is increased. However, these findings indicate that the cause of hyperinsulinism in ob/ob mice is unrelated to rates of insulin degradation.     

Comparison of effects of decapitation and anesthesia on metabolic and hormonal parameters in Sprague-Dawley rats.

The modes of euthanasia by either anesthesia or by decapitation were compared by assessing several metabolic and hormonal parameters from plasma and hormone receptors from liver plasma membranes. Two different anesthetics were used. Compared to decapitation, euthanasia by anesthesia significantly increased plasma glucose and triglyceride levels but not plasma cholesterol. Plasma insulin was also significantly increased by anesthetics. No significant differences were observed in plasma glucagon levels or insulin and glucagon receptors from liver plasma membranes between rats euthanized by decapitation and anesthesia. Glucagon receptors were however, affected by dietary carbohydrates. It is concluded that in studies involving measurements of metabolic and hormonal parameters, the use of anesthesia is to be avoided for euthanasia and that decapitation should be the method of choice.     

Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes

Soybean and its isoflavones have been shown to have beneficial effects on carbohydrate and lipid metabolism and on renal function. Probiotics may potentiate the beneficial effects of isoflavones by converting the inactive isoflavone glycoside to aglycones, which are biologically active, thereby producing a synergistic effect. We therefore studied the effects of soybean isoflavones in the presence and absence of probiotics on glucose and triglyceride metabolism and the peptide hormones involved in their metabolism. Lean and obese SHR/N-cp rats were fed AIN-93 diets containing 0.1% soybean isoflavone mixture, 0.1% probiotics mixture or both. Plasma was analyzed for glucose, triglycerides, parameters of renal function and peptide hormones -- insulin, leptin, glucagon and ACTH -- that are involved in glucose and lipid metabolism. Isoflavones given alone lowered plasma glucose in both phenotypes while triglyceride was decreased only in lean animals. Isoflavones also lowered aspartate amino transferase and alanine amino transferase in both phenotypes. Isoflavones had significant effect on plasma insulin, leptin and glucagon in lean rats but not in obese rats. Thus, our data show that in lean animals, isoflavones have hypoglycemic and hypolipidemic effect, and the effect is mediated by changes in peptide hormones. When lipid levels are very high as in obese rats, isoflavones fail to lower plasma triglyceride levels. Probiotics do not appear to enhance the effect of isoflavones.     

Proinflammatory cytokines and the hypermetabolism of children with sickle cell disease

Sickle cell anemia (HbSS) includes chronic inflammation, but the origin is unclear. We hypothesized that in stable HbSS patients the inflammation was associated with hypermetabolism. We compared selected hypermetabolic and key immunomodulator indicators in HbSS versus control children and examined associations between measures of hypermetabolism and inflammation. Twelve fasting asymptomatic HbSS children 6-12 years and 9 controls matched for age, gender and fat mass (FM) were studied. Proportional reticulocyte count (retic%) and resting energy expenditure (REE) represented hypermetabolism, and C-reactive protein (CRP) indicated inflammation. Proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6), chemokine monocyte chemoattractant protein-1 (MCP-1), and energy balance cytokine leptin were measured. Methods were indirect calorimetry, enzyme-linked immunosorbent assay, and radioimmunoassay. Statistical analysis included simple correlation and regression analysis. REE (51 +/- 6 vs. 43 +/- 12 kcal/kg per fat-free mass (FFM), mean +/- SD), retic% (12 +/- 4 vs. 0.7 +/- 0.3%), CRP (5 +/- 3 vs. 0.3 +/- 0.4 mg/liter), and IL-6 (71 +/- 40 vs. 20 +/- 7 pg/ml) were significantly higher for HbSS than controls (P < 0.05). Conversely, leptin (0.1 +/- 0.1 vs. 2 +/- 1 microg/liter per kgFM) and MCP-1 (34 +/- 5 vs. 41 +/- 4 pg/ml) were significantly lower for the HbSS subjects (P < 0.01). TNF-alpha was not significantly different. There were no significant associations between REE or retic% and any cytokine measured. However, CRP was significantly associated with REE in HbSS (r = 0.8, P = 0.003) and an important predictor of REE/FFM. We provide new evidence for low circulating levels of inflammatory chemokine MCP-1 in stable HbSS children, confirm mostly low cytokine levels, inflammation, and hypermetabolism and demonstrate association of hypermetabolism with inflammation via CRP but not via cytokines.     

Effects of dietary fats on red blood cell membrane insulin receptor in normo- and hypercholesterolemic miniature swine

It has been demonstrated that the type of dietary fat affects insulin receptors in various tissues in normal humans and animals by altering membrane fluidity. This study compares the effects of n-3 fatty acids from fish oil and n-6 fatty acids from corn oil on red blood cell membrane insulin receptors in normal and hypercholesterolemic minipigs. A group of minipigs were made hypercholesterolemic by feeding cholesterol and lard for 2 months; the other group served as controls and was fed stock diet. Both groups were then fed experimental diets containing either corn oil or menhaden oil or a mixture of the two for 23 additional weeks. Blood was collected at 0, 2, 12 and 23 weeks after the start of the experimental diets and membranes were prepared from the red blood cells. Insulin binding to red blood cell membranes was measured by radioreceptor assay. Plasma insulin was measured by radioimmunoassay. Insulin binding to red blood cell membrane was compared with the fluidity of the membrane measured and reported earlier. There was no significant effect of cholesterol feeding on plasma insulin concentrations. After 23 weeks on experimental diet plasma insulin was significantly higher in minipigs fed menhaden oil compared to those fed corn oil. No such effect was observed in hypercholesterolemic minipigs. No significant effect of either hypercholesterolemia or fish oil was observed on red blood cell insulin binding. A significant negative relationship was observed between insulin binding and anisotropy at 4°C for all probes but at 37°C significant negative relationship was observed only with polar probes. The data suggest that n-3 fatty acids from fish oil significantly increases plasma insulin in minipigs compared to n-6 fatty acids from corn oil. However, the unsaturation has no significant effect on insulin receptors on erythrocytes. Similarly, prior hypercholesterolemic state also has no effect on plasma insulin levels or the insulin binding to red blood cell membranes.     

Copper deficiency in rats increases pancreatic enkephalin-containing peptides and insulin

Free enkephalins (enk) and higher molecular weight enkephalin-containing peptides (enk-c-p) are present in the endocrine pancreas of rats, presumably in B cells. To determine whether these opioid peptides show dynamic alterations as insulin content of pancreas changes, we utilized a copper deficient rat model, in which the exocrine pancreas atrophies and the endocrine pancreas is “intact” and insulin (IRI) content increases. Dietary copper deficiency (−C) was produced in weanling male rats for 4 and 7 weeks. The deficient and copper supplemented (+C) groups were further subdivided to receive all dietary carbohydrate as either 62% fructose (F) or 62% starch (S). −CF rats showed the most severe deficiency. After 7 weeks, total units of pancreatic IRI in −CF were 7.5, +CF 2.1, −CS 7.9 and in +CS 2.8 (p<0.001). Pancreatic content of Met⁵- and Leu⁵-enk was measured in extracts which were purified on C-18 Seppaks with and without prior treatment with trypsin and carboxypeptidase B. −C animals showed progressive, significant increases in pancreatic content of Leu-enk-c-p, with a decrease in free Leu- and Met-enk (p<0.02-0.01). The pancreatic findings are compatible with a co-localization of enkephalins and insulin in the endocrine pancreas and are suggestive of co-regulation.     

Insulin, glucagon, and somatostatin secretion by cultured rat islet cell tumor and its clones

Cells derived from rat islet tumor and grown in culture (parent cells-RIN-m) and two clones obtained from them were used to study the effect of various secretagogues on insulin, glucagon, and somatostatin secretion. Parent cells secreted all three hormones in various quantities, while clone 5F secreted predominantly insulin and clone 14B secreted predominantly somatostatin. The secretory behavior of these cells were compared to each other and to that of normal islets. In general, as in the case of normal islets, insulin secretion was stimulated by calcium, potassium, tolbutamide, theophylline, and glucagon. It was inhibited by somatostatin. Glucagon secretion was stimulated by calcium, arginine, and theophylline. Somatostatin secretion was stimulated in clone 14B by arginine, tolbutamide, theophylline, and insulin. These cells differ from normal islets, in that they do not respond to glucose or arginine with increased insulin secretion. Also somatostatin failed to inhibit glucagon secretion. The similarity in insulin secretory responses of parent cells and clone 5F suggests that local or paracrine islet hormone secretion plays only a negligible role in the control of other hormone secretion in these cells.