Effect of glucagon antiserum on somatostatin,glucagon and insulin release from the isolated perfused rat pancreas

In order to elucidate the effect of glucagon antiserum on the endocrine pancreas, the release of somatostatin, glucagon, and insulin from the isolated perfused rat pancreas was studied following the infusion of arginine both with and without pretreatment by glucagon antiserum. Various concentrations of arginine in the presence of 5.5 mM glucose stimulated both somatostatin and glucagon secretion. However, the responses of somatostatin and glucagon were different at different doses of arginine. The infusion of glucagon antiserum strongly stimulated basal secretion in the perfusate total glucagon (free + antibody bound glucagon) and also enhanced its response to arginine, but free glucagon was undetectable in the perfusate during the infusion. On the other hand, the glucagon antiserum had no significant effect on either insulin or somatostatin secretion. Moreover, electron microscopic study revealed degrannulation and vacuolization in the cytoplasm of the A cells after exposure to glucagon antiserum, suggesting a hypersecretion of glucagon, but no significant change was found in the B cells or the D cells. We conclude that in a single pass perfusion system glucagon antiserum does not affect somatostatin or insulin secretion, although it enhances glucagon secretion.     

A model of insulin distribution and uptake in the perfused rat liver

Indicator dilution curves of 51Cr-EDTA and 125I-insulin injected into perfusate entering the rat liver in vivo are used as a basis for developing a mathematical model of insulin distribution and uptake within the organ. EDTA is not taken up by liver cells and therefore serves as a "volume marker" whose dilution curve reflects the characteristics of perfusate flow through the organ and through the cannulae. These two components are modelled separately but the same approach is used in each case, that is, the minimum number of delayed exponential terms sufficient to reproduce the dilution curves is determined and used as the basis for modelling. This allows the cannula, vascular and sinusoidal volumes to be identified and described as corresponding compartmental configurations. The shape of the insulin dilution curve is additionally influenced by binding to, and uptake by, liver cells. Binding of insulin to receptors on hepatocyte plasma membranes and subsequent internalization of the insulin-receptor complex is modelled by the introduction of additional compartments but this is found to be insufficient unless non-specific binding is also taken into account. The accuracy of determination of the rate constants for insulin-receptor dissociation and for endocytosis is improved by a sudden reduction in the pH of the perfusate about 100 sec after injection of the insulin bolus. This releases any residual receptor bound insulin and is modelled by a sudden shift in the insulin-receptor dissociation rate constant. Matching of the complete model to individual pairs of 51Cr-EDTA and 125I-insulin dilution curves allows vascular and sinusoidal volumes to be determined together with the binding and endocytic rate constants. Use of the model to investigate the effect of substances that modify these rate constants is briefly illustrated in the case where the liver is preperfused with 5 mM indomethacin. The model can also be used to simulate the internal distribution and uptake of insulin with any nominated input function and any set of parameters; this is illustrated by comparing the impulse response in the normal case and that in which indomethacin has been preperfused. Although the present study is confined to insulin, the model and methodology that is developed should be applicable to other ligands for which the hepatocyte carries specific receptors.     

Release of carnitine from the perfused rat liver

Perfused rat liver was shown to be the proper model for studies on hepatic cellular transport of carnitine. During recirculating perfusion the livers kept equilibrium with 45 nmol/ml total carnitine in perfusate, exhibited concentrative uptake and there was no sign of artificial leakage. The release side of the carnitine transport was characterized by utilizing outflow perfusions. The livers from fed rats exported daily 9.93 mumol per 100 g body weight total carnitine. This release rate is 4- or 10-fold higher than the estimated daily turnover in vivo or the measured urinary excretion. Therefore, the major part of the released carnitine has to re-enter the liver. The outward carnitine transport does not depend on energy or the Na+-K+ pump, since it did not respond to metabolic poisons and ouabain. However, the release rate was strongly inhibited by mersalyl and showed saturability in function of tissue carnitine levels. The Vmax of the saturable outward transport system was 2.47 nmol . min-1 . g-1 liver, the apparent Km was 0.27 mM tissue level (both as compared to total carnitine). These data showed the outward transport of carnitine from the liver to be protein mediated. The contribution of a diffusion (nonsaturable) component was estimated to be 20-25% in the range of tissue levels occurring in vivo. The rate of carnitine release from the liver decreased as an effect of 24 h starvation from the daily 9.92 mumol release to 6.55 mumol on 100 g body weight basis. This decrease is more pronounced when the release rates are expressed on the basis of tissue carnitine levels. The resulting value can be called rate constant (at the linear part of the saturation curve, Fig. 5) and it decreased to 5.00 min-1 from 8.41 min-1 as an effect of starvation. We have concluded that the altered parameters of carnitine transport across the liver cell is decisive in developing the higher hepatic carnitine concentration in the fasted state.     

Effect of exercise training on insulin and glucagon release from perfused rat pancreas

The effect of physical training on insulin and glucagon release in perfused rat pancreas was examined in the spontaneously exercised group running in a wheel cage an average of 1.4 km/day for 3 weeks and in the sedentary control group kept in the cage whose rotatory wheel was fixed on purpose. Pancreatic immunoreactive insulin (IRI) responses to glucose and arginine were reduced by 28% and 47.8% respectively in trained rats compared with untrained rats, while IRI content of the pancreas was similar in these two groups. The demonstrated decrease in insulin secretion of the beta-cell of the trained rats, in response to the glucose and arginine stimulations, may be functional in nature. On the other hand, neither pancreatic glucagon immunoreactivity (GI) response to glucose and arginine nor GI content of the pancreas was modified by exercise training. These results demonstrate that exercise training reduces IRI responses to glucose as well as to arginine stimulations, but does not modify any secretory response of pancreatic GI.     

Effect of beta-hydroxybutyrate and acetoacetate on insulin and glucagon secretion from perfused rat pancreas

To elucidate the physiological significance of ketone bodies on insulin and glucagon secretion, the direct effects of beta-hydroxybutyrate (BOHB) and acetoacetate (AcAc) infusion on insulin and glucagon release from perfused rat pancreas were investigated. The BOHB or AcAc was administered at concentrations of 10, 1, or 0.1 mM for 30 min at 4.0 ml/min. High-concentration infusions of BOHB and AcAc (10 mM) produced significant increases in insulin release in the presence of 4.4 mM glucose, but low-concentration infusions of BOHB and AcAc (1 and 0.1 mM) caused no significant changes in insulin secretion from perfused rat pancreas. BOHB (10, 1, and 0.1 mM) and AcAc (10 and 1 mM) infusion significantly inhibited glucagon secretion from perfused rat pancreas. These results suggest that physiological concentrations of ketone bodies have no direct effect on insulin release but have a direct inhibitory effect on glucagon secretion from perfused rat pancreas.     

Effect of insulin and glucagon on aldolase turnover in rat liver

The effects of exogenous and endogenous insulin and glucagon on aldolase turnover in rat liver and blood were studied. Some effects of these hormones on the biosynthesis and degradation of hepatic aldolase were specified. The rate of the "de novo" synthesis of aldolase was investigated in hepatocyte mitochondria and in blood plasma. The exogenous and endogenous hormones were shown to produce different effects on the biosynthesis and spontaneous degradation of rat liver aldolase.     

Effect of obestatin on insulin, glucagon and somatostatin secretion in the perfused rat pancreas

Obestatin is a 23-amino acid peptide derived from preproghrelin, purified from stomach extracts and detected in peripheral plasma. In contrast to ghrelin, obestatin has been reported to inhibit appetite and gastric motility. However, these effects have not been confirmed by some groups. Obestatin was originally proposed to be the ligand for GPR39, a receptor related to the ghrelin receptor subfamily, but this remains controversial. Obestatin and GPR39 are expressed in several tissues, including pancreas. We have investigated the effect of obestatin on islet cell secretion in the perfused rat pancreas. Obestatin, at 10 nM, inhibited glucose-induced insulin secretion, while at 1 nM, it potentiated the insulin response to glucose, arginine and tolbutamide. The potentiated effect of obestatin on glucose-induced insulin output was not observed in the presence of diazoxide, an agent that activates ATP-dependent K(+) channels, thus suggesting that these channels might be sensitive to this peptide. Obestatin failed to significantly modify the glucagon and somatostatin responses to arginine, indicating that its stimulation of insulin output is not mediated by an alpha- or delta-cell paracrine effect. Our results allow us to speculate about a role of obestatin in the control of beta-cell secretion. Furthermore, as an insulinotropic agent, its potential antidiabetic effect may be worthy of investigation.     

In vitro effects of glycosylated insulin and glucagon in perfused liver of the rat.

Using perfused liver of the rat, the hepatic uptake of glycosylated insulin (GI) and glucagon (GG) and its effects on hepatic glucose output were investigated. Insulin and glucagon were glycosylated in ambient high glucose concentration, and GI80 or GG80 (insulin or glucagon incubated with 0.08% glucose), GI350 or GG350 (incubated with 0.35% glucose), and GI1000 or GG1000 (incubated with 1% glucose) were prepared. The liver was perfused with the medium containing 1000 microU/ml insulin and 200 pg/ml glucagon or 200 microU/ml insulin and 1000 pg/ml glucagon. The fractional uptake of insulin or glucagon by perfused liver was not significantly altered by the glycosylation. In the liver perfused with 1000 microU/ml insulin and 200 pg/ml glucagon, glucose output was not changed by the glycosylation of the hormones, while in the liver perfused with 200 microU/ml insulin and 1000 pg/ml glucagon, GI1000 reduced its biological activity, as reflected by insulin-mediated decrease in glucose output. These results suggest that in the liver insulin incubated with markedly high concentration of glucose reduces its biological activity at a physiological concentration in the presence of high concentration of glucagon.     

Effects of salicylate on insulin and glucagon secretion by the isolated and perfused rat pancreas

The effects of sodium salicylate, a prostaglandin synthesis inhibitor, on glucose-induced secretion of insulin and glucagon by the isolated perfused rat pancreas have been studied. Sodium salicylate inhibited both basal (2.8 mM glucose) and stimulated (16.7 mM glucose) insulin release in a dose dependent manner (1, 5 and 10 mM). This inhibition is not interpretable in terms of a simple inhibition of cyclooxygenase by sodium salicylate. Basal glucagon release was not changed by 1 mM sodium salicylate but the latter partially blocked its inhibition by 16.7 mM glucose. Higher doses of sodium salicylate (5 and 10 mM) inhibited basal glucagon secretion without affecting its response to 16.7 mM glucose. These findings suggest a predominant stimulatory action of endogenous prostaglandins on glucagon release.     

Phosphate and calcium uptake by mitochondria and by perfused rat liver induced by the synergistic action of glucagon and vasopressin.

Co-administration of glucagon and vasopressin to rat liver perfused with buffer containing 1.3 mM-Ca2+ induces a 4-fold increase in Pi in the subsequently isolated mitochondria (from approx. 9 to approx. 40 nmol/mg of mitochondrial protein). This increase is not attributable to PPi hydrolysis, and is not observed if the perfusate Ca2+ is lowered from 1.3 mM to 50 microM. The increase in mitochondrial Pi closely parallels that of mitochondrial Ca2+; when the increase in Pi and Ca2+ accumulation is maximal, the molar ratio is close to that in Ca3(PO4)2. Measurement of changes in the perfusate Pi revealed that, whereas administration of glucagon or vasopressin alone brought about a rapid decline in perfusate Pi, the largest decrease (reflecting net retention of Pi by the liver) was observed when the hormone was co-administered in the presence of 1.3 mM-Ca2+. The synergistic action of glucagon plus vasopressin was nullified by lowering the perfusate Ca2+ to 50 microM. The data provide evidence that, whereas glucagon may be able to alter Pi fluxes directly in intact liver, any alterations induced by vasopressin are indirect and result only from its action of mobilizing Ca2+.     

Effects of pancreastatin on insulin, glucagon and somatostatin secretion by the perfused rat pancreas

Pancreastatin is a novel peptide, isolated from porcine pancreatic extracts, which has been shown to inhibit glucose-induced insulin release "in vitro". To achieve further insight into the influence of pancreastatin on pancreatic hormone secretion, we have studied the effects of this peptide on unstimulated insulin, glucagon and somatostatin output, as well as on the responses of these hormones to glucose and to tolbutamide in the perfused rat pancreas. Pancreastatin strongly inhibited unstimulated insulin release as well as the insulin responses to glucose and to tolbutamide. It did not significantly affect glucagon or somatostatin output under any of the above-mentioned conditions. These findings suggest that pancreastatin inhibits B-cell secretory activity directly, and not through an A-cell or D-cell paracrine effect.     

Somatostatin, insulin and glucagon secretion by the perfused pancreas from the cysteamine-treated rat

In rats, administration of a single dose of cysteamine (300 mg/kg, intragastrically) induces a depletion of pancreatic somatostatin content (approximately 60%) without modifying pancreatic insulin or glucagon content. In perfused pancreases from cysteamine-treated rats, there was a lack of somatostatin response to glucose, arginine or tolbutamide. In the absence of stimulated somatostatin release, the secretory responses of insulin and glucagon to glucose, to arginine, and to tolbutamide were not significantly different from those observed in pancreases from control rats. Our data do not support the concept that pancreatic somatostatin plays a major role in the control of insulin and glucagon release.