The interaction of bioactive peptides with an immobilized phosphatidylcholine monolayer.
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The interaction of three bioactive peptides, bombesin, beta-endorphin, and glucagon with a phosphatidylcholine monolayer that was immobilized to porous silica particles and packed into a stainless steel column cartridge, has been studied using dynamic elution techniques. This immobilized lipid monolayer provides a biophysical model system with which to study the binding of peptides to a lipid membrane. In particular, the influence of temperature and methanol concentration on the affinity of each peptide for the immobilized lipid surface was assessed. For all test peptides, nonlinear retention plots were observed at all temperatures that contrasted sharply with the simple linear plots observed for the small unstructured control molecules N-acetyltryptophanamide and diphenylalanine. An analysis of the thermodynamics of the interaction of peptides with the immobilized monolayer was also carried out. The results revealed that while the peptides interacted with the monolayer predominantly through hydrophobic interactions, the relative contribution of DeltaH(assoc)(O) and DeltaS(assoc)(O) to the overall free energy of association was dependent on the temperature and methanol concentration. In particular, it was evident that under most conditions, the binding of the peptides to the immobilized lipid monolayer was enthalpy-driven, i.e., mediated by nonclassical hydrophobic interactions. Significant band-broadening and asymmetric and split peaks were also observed for bombesin, beta-endorphin, and glucagon at different temperatures and methanol concentrations. These changes in affinity and peak shape are consistent with the formation of multiple conformational species during the interaction of these peptides with the lipid monolayer. In addition, the binding behavior of the three test peptides on an n-octylsilica surface that lacked the phospho headgroups of the phospholipid was significantly different from that observed with the immobilized phosphatidylcholine surface, indicating a specificity of interaction between the peptides and the lipid surface. Overall, these experimental results demonstrate that the biomimetic phosphatidylcholine monolayer provides a stable and sensitive system with which to explore the molecular mechanism of peptide conformational changes during membrane interactions. (+info)
Diadenosine polyphosphates and the control of cyclic AMP concentrations in isolated rat liver cells.
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Extracellular diadenosine polyphosphates (Ap(n)A), through their interactions with appropriate P(2) receptors, influence a diverse range of intracellular activities. In particular, Ap(4)A stimulates alterations in intracellular calcium homeostasis and subsequent activation of glycogen breakdown in isolated liver cells. Here we show that, like ATP, Ap(4)A and other naturally occurring diadenosine polyphosphates attenuate glucagon-stimulated accumulation of cyclic AMP in isolated rat liver cells. The characteristics of Ap(4)A- and ATP-dependent modulation of glucagon-stimulated cyclic AMP accumulation are similar. These results are discussed in the context of the repertoire of intracellular signalling processes modulated by extracellular nucleotides. (+info)
Functional studies of a glucagon receptor isolated from frog Rana tigrina rugulosa: implications on the molecular evolution of glucagon receptors in vertebrates.
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In this report, the first amphibian glucagon receptor (GluR) cDNA was characterized from the liver of the frog Rana tigrina rugulosa. Functional expression of the frog GluR in CHO and COS-7 cells showed a high specificity of the receptor towards human glucagon with an EC(50) value of 0.8+/-0.5 nM. The binding of radioiodinated human glucagon to GluR was displaced in a dose-dependent manner only with human glucagon and its antagonist (des-His(1)-[Nle(9)-Ala(11)-Ala(16)]) with IC(50) values of 12.0+/-3. 0 and 7.8+/-1.0 nM, respectively. The frog GluR did not display any affinity towards fish and human GLP-1s, and towards glucagon peptides derived from two species of teleost fishes (goldfish, zebrafish). These fish glucagons contain substitutions in several key residues that were previously shown to be critical for the binding of human glucagon to its receptor. By RT-PCR, mRNA transcripts of frog GluR were located in the liver, brain, small intestine and colon. These results demonstrate a conservation of the functional characteristics of the GluRs in frog and mammalian species and provide a framework for a better understanding of the molecular evolution of the GluR and its physiological function in vertebrates. (+info)
Inhibition of glycogenolysis in primary rat hepatocytes by 1, 4-dideoxy-1,4-imino-D-arabinitol.
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1,4-Dideoxy-1,4-imino-d-arabinitol (DAB) was identified previously as a potent inhibitor of both the phosphorylated and non-phosphorylated forms of glycogen phosphorylase (EC 2.4.1.1). In the present study, the effects of DAB were investigated in primary cultured rat hepatocytes. The transport of DAB into hepatocytes was dependent on time and DAB concentration. The rate of DAB transport was 192 pmol/min per mg of protein per mM DAB(medium-concentration). In hepatocytes, DAB inhibited basal and glucagon-stimulated glycogenolysis with IC(50) values of 1.0+/-0.3 and 1.1+/-0.2 microM, respectively. The primary inhibitory effect of DAB on glycogenolysis was shown to be due to inhibition of glycogen phosphorylase but, at higher concentrations of DAB, inhibition of the debranching enzyme (4-alpha-glucanotransferase, EC 2.4.1.25) may have an effect. No effects on glycogen synthesis were observed, demonstrating that glycogen recycling does not occur in cultured hepatocytes under the conditions tested. Furthermore, DAB had no effects on phosphorylase kinase, the enzyme responsible for phosphorylation and thereby activation of glycogen phosphorylase, or on protein phosphatase 1, the enzyme responsible for inactivation of glycogen phosphorylase through dephosphorylation. (+info)
A new mouse model of spontaneous diabetes derived from ddY strain.
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By the selective breeding of obese male mice of the ddY strain and using indices of the heavy body weight and appearance of urinary glucose, we established two inbred strains in 1992: one with obesity and urinary glucose (Tsumura, Suzuki, Obese Diabetes: TSOD) and the other without them (Tsumura, Suzuki, Non Obesity: TSNO). The male TSOD mice constantly showed signs of obesity and urinary glucose with increases in food and water intake, body weight and some fat weight. The body mass index (BMI) clearly showed moderate obesity. Increases in the levels of diabetic blood parameters (glucose, insulin and lipids) were also found in males, in which the levels of blood glucose and insulin were high to the ages past the growth peak. In the histological studies, pancreatic islets of the TSOD males were found hypertrophic without any signs of insulitis or fibrous formation. Among these diabetic characteristics, some of which were similar to the reported models of non-insulin-dependent diabetes mellitus (NIDDM), the stable appearances of the hyperglycemia, the hyperinsulinemia and the hypertrophy of pancreatic islets to the ages past the growth peak were the prominent features. In these respect the TSOD mouse may be a useful model for researching the mechanisms of human diabetes and its complications. (+info)
Pancreatic alpha cell function in the fetal foal during late gestation.
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Plasma glucagon concentrations were measured in chronically catheterized fetal ponies and their mothers between 260 days of gestation and term (approximately 335 days). Fetal alpha cell responses to arginine and variations in fetal glycaemia were also examined during late gestation. Immunoreactive glucagon was present in fetal plasma at 260 days of gestation and its concentration in utero increased after 320 days and then again at birth. Maternal plasma glucagon concentrations were higher after 300 days than earlier in gestation but were lower than the corresponding fetal value throughout the period of gestation studied. Fetal alpha cells responded rapidly to intravenous arginine infusion but not to changes in the fetal glucose level induced by maternal fasting for 36 h or by intrafetal infusion of glucose. The maximal increment in fetal plasma glucagon in response to arginine occurred at the end of the 5 min infusion and was positively correlated to the basal pre-infusion plasma glucagon concentrations. Fetal plasma glucagon concentrations were unaffected by either hyper- or hypoglycaemia. In contrast, maternal plasma glucagon levels were significantly increased by fasting. These observations indicate that equine pancreatic alpha cells are functional in utero but that they are unresponsive to variations in glycaemia until after birth. (+info)
GLP-1 slows solid gastric emptying and inhibits insulin, glucagon, and PYY release in humans.
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The aim of the present study was to assess the effect of glucagon-like peptide-1 (GLP-1) on solid gastric emptying and the subsequent release of pancreatic and intestinal hormones. In eight men [age 33.6 +/- 2.5 yr, body mass index 24.1 +/- 0.9 (means +/- SE)], scintigraphic solid gastric emptying during infusion of GLP-1 (0.75 pmol. kg(-1). min(-1)) or saline was studied for 180 min. Concomitantly, plasma concentrations of C- and N-terminal GLP-1, glucose, insulin, C-peptide, glucagon, and peptide YY (PYY) were assessed. Infusion of GLP-1 resulted in a profound inhibition of both the lag phase (GLP-1: 91.5, range 73.3-103.6 min vs. saline: 19. 5, range 10.2-43.4 min) and emptying rate (GLP-1: 0.34, range 0.06-0. 56 %/min vs. saline: 0.84, range 0.54-1.33 %/min; P < 0.01 for both) of solid gastric emptying. Concentrations of both intact and total GLP-1 were elevated to supraphysiological levels. Plasma glucose and glucagon concentrations were below baseline during infusion of GLP-1 in contrast to saline infusion, where concentrations were elevated above baseline (both P < 0.001). The insulin and C-peptide responses were lower during infusion with GLP-1 than with saline (P < 0.004 and P < 0.001, respectively). Plasma PYY concentrations decreased below baseline during GLP-1 infusion in contrast to saline, where concentrations were elevated above baseline (P = 0.04). Infusion of GLP-1 inhibits solid gastric emptying with secondary effects on the release of insulin, C-peptide, and glucagon, resulting in lower plasma glucose concentrations. In addition, the release of PYY into the circulation is inhibited by GLP-1 infusion, suggesting a negative feedback of GLP-1 on the function of the L-cell. (+info)
Metabolic and performance responses to constant-load vs. variable-intensity exercise in trained cyclists.
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We studied glucose oxidation (Glu(ox)) and glycogen degradation during 140 min of constant-load [steady-state (SS)] and variable-intensity (VI) cycling of the same average power output, immediately followed by a 20-km performance ride [time trial (TT)]. Six trained cyclists each performed four trials: two experimental bouts (SS and VI) in which muscle biopsies were taken before and after 140 min of exercise for determination of glycogen and periodic acid-Schiff's staining; and two similar trials without biopsies but incorporating the TT. During two of the experimental rides, subjects ingested a 5 g/100 ml [U-(14)C]glucose solution to determine rates of Glu(ox). Values were similar between SS and VI trials: O(2) consumption (3.08 +/- 0.02 vs. 3.15 +/- 0.03 l/min), energy expenditure (901 +/- 40 vs. 904 +/- 58 J x kg(-1) x min(-1)), heart rate (156 +/- 1 vs. 160 +/- 1 beats/min), and rating of perceived exertion (12.6 +/- 0.6 vs. 12.7 +/- 0.7). However, the area under the curve for plasma lactate concentration vs. time was significantly greater during VI than SS (29.1 +/- 3.9 vs. 24.6 +/- 3. 7 mM/140 min; P = 0.03). VI resulted in a 49% reduction in total muscle glycogen utilization vs. 65% for SS, while total Glu(ox) was higher (99.2 +/- 5.3 vs. 83.9 +/- 5.2 g/140 min; P < 0.05). The number of glycogen-depleted type I muscle fibers at the end of 140 min was 98% after SS but only 59% after VI. Conversely, the number of type II fibers that showed reduced periodic acid-Schiff's staining was 1% after SS vs. 10% after VI. Despite these metabolic differences, subsequent TT performance was similar (29.14 +/- 0.9 vs. 30.5 +/- 0.9 min for SS vs. VI). These results indicate that whole body metabolic and cardiovascular responses to 140 min of either SS or VI exercise at the same average intensity are similar, despite differences in skeletal muscle carbohydrate metabolism and recruitment. (+info)