The biosynthesis of glucagon in perfused rat pancreas

The biosynthesis of glucagon was studied by using the recirculated, isolated perfused rat pancreas. [³H]Tryptophan was initially incorporated into acid–ethanol-extractable protein, which on gel filtration was eluted with a molecular weight of about 9000 and contained a small amount of glucagon immunoreactivity. With longer incubation [³H]tryptophan incorporation into a second peak was obtained in an identical position with that of the majority of rat glucagon immunoreactivity. This peak of labelled protein exhibited migration characteristics on polyacrylamide-gel electrophoresis identical with those of rat glucagon and was identified as newly synthesized glucagon by demonstration of specific binding and dissociation behaviour with glucagon antibodies. The incorporation of [³H]tryptophan into acid–ethanol-extractable protein was inhibited by cycloheximide. High concentrations of glucose increased [³H]tryptophan incorporation into high-molecular-weight protein but decreased incorporation into proteins smaller than cytochrome c. The pattern of [³H]leucine incorporation into protein was similar to that of [³H]tryptophan.     

Secretion of the arginine-rich and A-I apolipoproteins by the isolated perfused rat liver.

Rates of secretion of the arginine-rich and A-I apolipoproteins into perfusates of rat livers were measured by specific radioimmunoassays. Livers were perfused for 6 hr in a recirculating system in the presence or absence of 5,5'-dithionitrobenzoic acid, an inhibitor of lecithin-cholesterol acyltransferase. Arginine-rich apoprotein (ARP) was secreted at a constant or increasing hourly rate of about 40 micro g/g liver, whereas the rate of accumulation of apoprotein A-I decreased progressively from about 12 to less than 5 micro g/g liver. These rates were not affected by inhibition of lecithin-cholesterol acyltransferase. The distribution of these two apolipoproteins was also measured in ultracentrifugally separated lipoprotein fractions from perfusates and blood plasma. Apoprotein A-I was mainly in high density lipoproteins, with the remainder in proteins of density > 1.21 g/ml. The percent of apoprotein A-I in the latter fraction was lowest in plasma (5%); in perfusates it was greater when the enzyme inhibitor was present (33%) than in its absence (11%). By contrast much less ARP was in proteins of d > 1.21 g/ml in perfusates than in blood plasma. Discoidal high density lipoproteins, recovered from perfusates in which lecithin-cholesterol acyltransferase was inhibited, contained much more arginine-rich apoprotein than apoprotein A-I (ratio = 10:1). The ratio in spherical plasma HDL was 1:7 and that in perfusate high density lipoproteins obtained in the absence of enzyme inhibitor was intermediate (2:1). It is concluded that: 1) the arginine-rich apoprotein is a major apolipoprotein whereas apoprotein A-I is a minor apolipoprotein secreted by the perfused rat liver; 2) the properties of the high density lipoproteins produced in this system are remarkably similar to those found in humans with genetically determined deficiency of lecithin-cholesterol acyltransferase.     

Secretion of lysosomal hydrolases by cultured human amnion epithelial cells

Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-beta-glucosaminidase, alpha-fucosidase, beta-glucuronidase, alpha-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies.     

Inhibitory effect of galanin on pancreatic polypeptide release by the perfused rat pancreas

We have investigated the effect of galanin infusion on unstimulated pancreatic polypeptide (PP) release as well as on the PP response to arginine by the perfused rat pancreas. Galanin significantly reduced unstimulated PP output. Addition of arginine to the perfusate evoked a biphasic pattern of PP release; the second phase of this PP response was delayed when galanin was simultaneously infused. These findings point to a regulatory role of galanin in the control of PP secretion.     

Secretion and elimination of insulin by the in vitro perfused pancreas and liver of rats with thioacetamide-induced liver cirrhosis

Pancreatic secretion and hepatic removal of insulin have been measured in thioacetamide (TAA)-induced compensated rat liver cirrhosis in perfusion experiments. Peripheral plasma concentrations of glucose and insulin were slightly decreased in TAA-treated rats. Pancreatic secretion and hepatic removal of insulin remained unchanged by the TAA-treatment. Thus, even in morphologically and biochemically proven experimental liver cirrhosis, insulin secretion and removal may not be disturbed.     

Secretion of apolipoproteins in very low density and high density lipoproteins by perfused rat liver

The incorporation of labeled amino acids into the peptides of very low density lipoproteins (VLDL) and high density lipoproteins (HDL) secreted by perfused rat liver was studied using a Ringer-albumin solution in the perfusate in place of serum to diminish exchange of peptides between VLDL and HDL. Among the lipoproteins, the greatest release of protein, greatest incorporation of amino acid, and highest specific activity were found in VLDL. After separation of the delipidated peptides by electrophoresis on polyacrylamide gel, the incorporation into VLDL peptides was found to be 5-10 times as great as into HDL peptides. There was virtually no incorporation into the peptides of low density lipoproteins (LDL). Approximately 25% of the radioactivity incorporated into perfusate VLDL failed to enter the 13% polyacrylamide gel. The remaining radioactivity was distributed primarily among three peptide bands; one, found in the upper portion of the gel, contained 45% of the total, most of the remainder being found in two rapidly migrating bands. These three peptides appear to approximate those of human apo-C in relative electrophoretic mobility. Most of the HDL peptide radioactivity entering the running gel was found in a band that migrates slightly faster than the main VLDL band. A portion of the radioactivity of this major HDL band did not enter the running gel unless beta-mercaptoethanol was present. Greater separation of these two bands by polyacrylamide gel electrophoresis for 24 hr confirmed that the major bands in VLDL and in HDL were different. The rapidly moving peptides of HDL were found to contain very little radioactivity. Determination of the intensity of staining of carrier-free perfusate VLDL and HDL peptides produced a pattern similar to the incorporation of labeled amino acids. It is concluded that the rapidly moving peptides, which may contain activators of lipoprotein lipase, are only secreted as part of the VLDL.     

The distribution of some hydrolases in glomeruli and tubular fragments prepared from rat kidney by zonal centrifugation

1. A collagenase digest of rat kidney cortex was separated into four bands by zonal centrifugation. 2. Two of these bands were shown by light-microscopy to contain glomeruli and tubular fragments, which were free from each other and well separated from other renal material. 3. Protein, N-acetyl-beta-glucosaminidase, 5'-nucleotidase, l-leucine beta-naphthylamidase, leucine aminopeptidase, acid phosphatase and alkaline phosphatase were assayed across the gradient. 4. The greater proportion of these enzyme activities was recovered in the tubular fragments and acid phosphatase was the only enzyme detected in significant amounts in the glomeruli. 5. Tubular fragments and glomeruli were sedimented and multiple forms of beta-naphthylamidase, N-acetyl-beta-glucosaminidase, acid phosphatase and alkaline phosphatase were investigated by starch-gel electrophoresis.     

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.     

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.     

Glucose dependent stimulation by prostaglandin D2 of glucagon and insulin in perfused rat pancreas

Effects of prostaglandin D2 on pancreatic islet function in perfused rat pancreas were examined in comparison with those of prostaglandin E2, which has hitherto been suggested to be a modifier of pancreatic hormone release. In the presence of 2.8 mM glucose, only glucagon release was strongly stimulated by 14 microM of prostaglandin D2, while release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2. When the glucose concentration was elevated to 11.2 mM, insulin release was accelerated by 14 microM of prostaglandin D2 but there was no effect upon glucagon release. Again, release of both glucagon and insulin was augmented by 14 microM of prostaglandin E2 in the presence of 11.2 mM of glucose. The regulation of glucagon and insulin release through prostaglandin D2 is apparently adapted to glycemic changes, and may be a physiological modulator of pancreatic islet function.     

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.     

The effects of prostaglandins on secretion of glucagon and insulin by the perfused rat pancreas.

The secretion of both glucagon and insulin by the isolated perfused rat pancreas was significantly stimulated by 10(-7) M PGH2. Experiments to show that the stimulated secretion was mediated by conversion of PGH2 to TXA2 or TXB2 revealed no correlation between the amount of secretion and the amount of thromboxane formed. Conversion of PGH2 with a crude platelet thromboxane synthase preparation caused a progressive loss of ability to secret insulin, whereas the capacity to stimulate release of glucagon remained at about one-half the maximal level. This relatively stable and selective secretagogue action on the alpha-cells appeared to be due to the formation of PGD2 by the platelet preparation. Direct administration of PGD2 confirmed this interpretation and showed clearly that this prostaglandin is a potent secretagogue for glucagon with little activity in stimulating the release of insulin. Our results have shown high and relatively equal stimulation of secretion by alpha- and beta-cells with exogenous PGE2, PGF2 alpha, and PGH2, little or no secretion by either cell type with TXA2, TXB2, or PGI2, and a unique selective stimulatory action of PGD2 upon the alpha-cell.     

Insulin releasing activity of gastrointestinal glucagon-like immunoreactive materials in perfused rat pancreas

Insulin-releasing activity of porcine gastrointestinal glucagon-like immunoreactive materials purified by affinity chromatography was examined in the perfused rat pancreas. When glucose concentration of the perfusate was raised from 60 to 100 mg/dl, augmented insulin release was observed. The mean incremental area of immunoreactive insulin (sigma delta IRI) during the first 10 min thus observed was 19.07 +/- 3.76 ng/10 min. Pancreatic glucagon and the extract from the gastric fundus showed the enhancement of insulin release in this system when they were added to the perfusate at the rate of 100 ng/min for 5 min; delta IRI were 41.92 +/- 8.47 and 71.70 +/- 18.09 ng/10 min, respectively, which were significantly higher than that of 100 mg/dl of glucose alone. However, no significant difference in the insulinogenic activity was noticed between the extracts from the small intestine and the control. These results suggest that the extract from the gastric fundus has insulinogenic activity similar to that of pancreatic glucagon.