Sam J. Bhathena

Identification of Human Mononuclear Leukocytes Bearing Receptors for Somatostatin and Glucagon

Mononuclear leukocytes (MNL) were isolated from human blood by Ficoll-Hypaque. These cells were further separated into lymphocyte (L) and monocyte (M) enriched fractions. L contained 99% lymphocytes and M contained 74% monocytes, a threefold enrichment over MNL. Specific binding of somatostatin, glucagon, and insulin was measured in the three fractions. Binding of all three hormones in the M fraction was increased by a factor of 3 compared with MNL and was linear with cell number. Binding of glucagon and insulin to the L fraction was very low while, in contrast, somatostatin binding was substantial and linear with lymphocyte number. Autoradiography confirmed the binding of glucagon to monocytes and of somatostatin to both monocytes and lymphocytes. Somatostatin is the first of the peptide hormones shown to bind to both types of circulating mononuclear cells, perhaps complicating quantification of somatostatin binding in disease states in which differential alteration of binding to lymphocytes or monocytes might occur.

Dietary phytoestrogens: A possible role in renal disease protection

There is growing evidence that dietary phytoestrogens have a beneficial role in chronic renal disease. This review summarizes the recent findings from dietary intervention studies performed in animals and humans suggesting that consumption of soy-based protein rich in isoflavones and flaxseed rich in lignans retards the development and progression of chronic renal disease. In several animal models of renal disease, both soy protein and flaxseed have been shown to limit or reduce proteinuria and renal pathological lesions associated with progressive renal failure. In studies of human subjects with different types of chronic renal disease, soy protein and flaxseed also appear to moderate proteinuria and preserve renal function. However, most of these clinical trials were of relatively short duration and involved a small number of patients. Furthermore, it is not clear whether the renal protective effects of soy protein and flaxseed are caused by the isoflavones (daidzein and genistein) and lignans (matairesinol and secoisolariciresinol) or some other component. The biochemistry, metabolism, and mechanisms of actions of isoflavones and lignans are discussed. Isoflavones and lignans appear to act through various mechanisms that modulate cell growth and proliferation, extracellular matrix synthesis, inflammation, and oxidative stress. Some of these actions have been shown in vitro, but studies of the mechanisms operative in vivo are lacking. The diversity of cellular actions of isoflavones and lignans supports their protective effects in a variety of experimental and human types of chronic renal disease. Further investigations are needed to evaluate their long-term effects on renal disease progression in patients with chronic renal failure.

Chromium improves insulin response to glucose in rats

The effects of chromium (Cr) supplementation on insulin secretion and glucose clearance (KG) during intravenous glucose tolerance tests (IVGTTS) were assessed in rats with impaired glucose tolerance due to dietary Cr deficiency. Male Wistar rats were maintained after weaning on a basal low-Cr diet containing 55% sucrose, 15% lard, 25% casein. American Institute of Nutrition (AIN)-recommended levels of vitamins, no added Cr, and an altered mineral content as required to produce Cr deficiency and impaired glucose tolerance. The Cr-supplemented group ([+Cr] n = 6) were provided with 5 ppm Cr as CrCl3 in the drinking water, and the Cr-deficient group ([-Cr]n = 5) received purified drinking water. At 12 weeks on the diet, both groups of rats were hyperinsulinemic (+Cr, 103 +/- 13; -Cr, 59 +/- 12 microU/mL) and normoglycemic (+Cr, 127 +/- 7; -Cr, 130 +/- 4 mg/dL), indicating insulin resistance. After 24 weeks, insulin levels were normal (+Cr, 19 +/- 5; -Cr, 21 +/- 3 microU/mL) and all rats remained normoglycemic (+Cr, 124 +/- 8; -Cr, 131 +/- 6 mg/dL). KG values during IVGTTS were lower in -Cr rats (KG = 3.58%/min) than in +Cr rats (KG = 5.29%/min), correlating with significantly greater 40-minute glucose areas in the -Cr group (P < .01). Comparisons of 40-minute insulin areas indicated marked insulin hyperresponsiveness in the -Cr group, with insulin-secretory responses increased nearly twofold in -Cr animals (P < .05). Chromium deficiency also led to significant decreases in cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activity in spleen and testis (P < .01). In these studies, Cr deficiency was characterized by both beta-cell hypersecretion of insulin and tissue insulin resistance that were associated with decreased tissue levels of cAMP PDE activity.

Decreased Glucagon Receptors in Diabetic Rat Hepatocytes: EVIDENCE FOR REGULATION OF GLUCAGON RECEPTORS BY HYPERGLUCAGONEMIA

The effects of endogenous and exogenous hyperglucagonemia on the specific binding of glucagon to hepatocyte receptors was studied, as was the response of cAMP to glucagon. In streptozotocin diabetic rats, blood glucose and plasma glucagon increased and plasma insulin decreased as compared with controls. Insulin treatment in diabetic rats restored blood glucose and plasma glucagon toward normal and elevated plasma insulin. Specific binding of (125)I-glucagon to isolated hepatocytes (10(6) cells) decreased in diabetic rats (8.17+/-0.38%) compared to controls (14.05+/-0.87%) and was restored by insulin treatment (12.25+/-0.93%). Specific binding of (125)I-insulin in controls was 7.30+/-10.16%; it increased in diabetic rats to 12.50+/-0.86%, and decreased in diabetic rats after insulin treatment (9.08+/-0.87%). Scatchard analysis and the competition plots of the data indicate that decreased glucagon binding and increased insulin binding in diabetes were due to change in the number of receptors rather than a change in their affinity. Hepatocyte cAMP response to glucagon (0.25-5.0 ng/ml) was almost abolished in diabetic rats and was restored with insulin treatment. Specific glucagon binding by hepatocytes from chronically hyperglucagonemic (glucagon injected) rats was decreased (P < 0.005) to 8.76+/-0.61% compared with controls (13.20+/-0.74%) and acutely hyperglucagonemic animals (13.53+/-1.33%). The decreased binding was associated with a 70% decrease in hepatocyte cAMP response to glucagon compared with a normal response in acutely hyperglucagonemic rats.These data appear to support the concept of receptor regulation by ambient hormone level. In both endogenous and exogenous hyperglucagonemia, however, there was a disproportionately large decrease in cAMP response to glucagon compared to the decrease in glucagon binding.

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 Immuno-modulator 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-α (TNF-α) and interleukin-6 (IL-6), chemokine monocyte chemoattractant proteln-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 µg/liter per kgFM) and MCP-1 (34 ± 5 vs. 41 ± 4 pg/ml) were significantly lower for the HbSS subjects (P < 0.01). TNF-α 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.

Dietary Flaxseed Meal is More Protective Than Soy Protein Concentrate Against Hypertriglyceridemia and Steatosis of the Liver in an Animal Model of Obesity

Objective: Soy protein and flaxseed meal have been reported to have beneficial effects on many chronic diseases in humans and animals. The primary objective of the study was to evaluate the beneficial effects of soy protein and flaxseed meal on hypertriglyceridemia and liver steatosis associated with obesity and diabetes. We compared the effects of dietary soy protein and flaxseed meal with that of casein on plasma and liver lipids in a genetic model of obesity, type II diabetes and insulin resistance, namely the SHR/N-cp rat. Methods: Lean and obese phenotypes of SHR/-cp rats were fed AIN 93 diets containing 20% of energy from casein (control), soy protein concentrate or flaxseed meal for six months. Plasma was analyzed for total cholesterol, LDL cholesterol, triglyceride and total protein. Liver was analyzed for steatosis by light microscopy after staining samples with Hematoxylin-Eosin and Oil-Red-O. Results: In lean rats soy protein and flaxseed meal significantly decreased plasma total cholesterol (26.0% and 20.3% respectively) compared to casein. In obese rats flaxseed meal had significant cholesterol lowering effect compared to control rats (41%). Soy protein significantly lowered both plasma LDL-cholesterol and HDL-cholesterol in lean phenotypes while in obese phenotypes flaxseed meal significantly lowered LDL-cholesterol and HDL-cholesterol compared to casein-fed rats. Flaxseed meal also significantly lowered plasma triglyceride in both lean and obese rats compared to casein fed rats (33.7% and 37% respectively). There was significantly greater fat accumulation in livers of obese rats than lean rats (200%) regardless of dietary protein type. Flaxseed meal significantly lowered fat deposition in livers of both lean and obese rats compared to rats fed casein or soy protein. Dietary component(s) present in flaxseed meal or soy protein responsible for hypolipidemic effects is not clear. Conclusions: The marked hypotriglyceridemic and hypocholesterolemic effects of flaxseed meal may have important therapeutic implications in patients with hypertriglyceridemia and hypercholesterolemia and deserve further study in humans with these disorders. Flaxseed meal supplementation may provide a new therapeutic strategy to reduce hypertriglyceridemia and fatty liver.

Insulin, Glucagon, and Somatostatin Receptors on Cultured Cells and Clones from Rat Islet Cell Tumor

SUMMARY Cells grown in culture from rat islet cell tumor (parent cells) and clones obtained from them were used in this study. Parent cells secreted primarily insulin and somatostatin with very small quantities of glucagon. The clones, based on hormone content and secretion, were divided into three phenotypic groups: insulin secreting, somatostatin secreting, and nonsecreting clones. Specific receptors for insulin, glucagon, and somatostatin were demonstrated on parent cells and clones. Parent cells bound 4.12 ± 0.46% insulin, 2.11 ± 0.29% glucagon, and 2.49 ± 1.24% somatostatin per 2 × 106 cells. Characteristic hormone binding patterns were observed in insulin secreting versus somatostatin secreting clones. Insulin secreting clones bound less insulin than somatostatin and other noninsulin- secreting clones (P < 0.02). In contrast, somatostatin secreting clones bound more somatostatin than non-somatostatin-secreting clones (P < 0.05). Somatostatin-secreting clones had a significantly greater number of receptors for all three hormones. The difficulties involved in the interpretation of the quantitative aspects of binding in the presence of continued hormone secretion are discussed. Nonetheless, the presence of receptors on the cells for hormones secreted by the same cells strongly suggests autoregulation. The apparent low affinity of some of these receptors and the presence of receptors for all three islet cell hormones on all islet cells supports the likelihood of paracrine controls.

Differential effects of dietary flaxseed protein and soy protein on plasma triglyceride and uric acid levels in animal models.

The effect of dietary soy protein and flaxseed meal on metabolic parameters was studied in two animal models, F344 rats with normal lipid levels and obese SHR/N-cp rats with elevated levels of cholesterol and triglyceride. The rats were fed AIN 93 diet differing only in the source of protein. The rats were fed either 20% casein, 20% soy protein or 20% flaxseed meal. Plasma was analyzed for cholesterol, triglyceride, uric acid, blood urea nitrogen (BUN), creatinine and total protein. In both strains of rats, flaxseed meal significantly decreased plasma cholesterol and triglyceride concentrations. The effect of soy protein on lipids was not as striking as that of flaxseed meal. Flaxseed meal also lowered uric acid in F344 rats and BUN in SHR/N-cp rats. Since cholesterol, triglyceride and uric acid are independent risk factors for cardiovascular disorders, our data show that both flaxseed meal and soy protein may have beneficial effects. Which chemical constituent(s) of flaxseed meal or soybean is (are) responsible for the beneficial effects need to be identified.

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

Abstract 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.

Effects of omega-3 fatty acid and vitamin E supplementation on erythrocyte membrane fluidity, tocopherols, insulin binding, and lipid composition in adult men

Abstract Dietary supplementation with an omega-3 fatty acid preparation (fish oil) together with pharmacologic doses of vitamin E increased both insulin binding and membrane fluidity in erythrocytes from human adult males. Supplementation with fish oil alone induced significant increases in the α- and γ-tocopherol contents of the red blood cell membranes. Forty healthy men were given controlled diets and supplements, which together provided 40% of energy from fat (polyunsaturated:monosaturated:saturated ratio of 0.8:1:1), 360 mg cholesterol/day, and a minimum of 22 mg α-tocopherol (αT)/day for three successive experimental periods of 10, 10, and 8 weeks, during which they were given capsules containing 15 g of a placebo oil/day, 15 g fish oil concentrate (FOC)/day, and 15 g fish oil + 200 IU αT (FOC + αT)/day, respectively. Erythrocyte ghost insulin binding (IB) and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence-determined fluidity were significantly increased following the FOC + αT period, however FOC alone had no effect. At the end of each experimental period, IB values, as percentage bound/100 μg ghost protein at 4° C, were 0.96, 0.91, and 1.35, and DPH steady state fluorescence anisotropies were 0.311, 0.303, and 0.296, at 4° C, respectively. Small but statistically significant decreases in fluorescence lifetimes further indicated increased fluidity. FOC supplementation resulted in significantly lower membrane cholesterol:phospholipid ratios and increased membrane tocopherols despite daily vitamin E consumption of only 22 mg as in the placebo period. Membrane incorporation of n-3 fatty acids was, however, limited. Thus, dietary polyunsaturated fatty acids exerted substantial effects on erythrocyte membranes by affecting membrane contents of lipid molecules other than the fatty acids.