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SUMMARY
Pieces of human foetal pancreas were incubated under control conditions and in media containing different stimuli of insulin release. Insulin secretion was stimulated from the pancreases of foetuses (83–625 g body weight) which were of 16–24 weeks gestational age. Potassium (60 mmol/l), barium (2·54 mmol/l) and ouabain (10−5 mol/l) were effective stimuli in all experiments. Glucagon (5 μg/ml), theophylline (1 mmol/l) and dibutyryl 3′,5′-cyclic adenosine monophosphate (1 mmol/l) stimulated insulin secretion in media containing 0, 0·6 or 3·0 mg glucose/ml. Theophylline and dibutyryl 3′,5′-cyclic adenosine monophosphate were effective in all experients and glucagon stimulated insulin release in four out of six experiments. At all ages studied, histological examination of the pancreas after each experiment revealed islets of Langerhans containing β cells. In most cases the islets were of the mantle type but occasionally bipolar islets were seen. Cellular normality, as judged by light microscopy, was preserved after periods of incubation for up to 5½ h. Glycogen was demonstrable in the pancreatic acinar tissue but not in the islets.
The results of these experiments indicate that, between the 16th and 24th week of foetal life, the human β cell is capable of releasing insulin in vitro when stimulated appropriately.
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SUMMARY
Pieces of pancreas from human foetuses of 14–24 weeks gestational age and weighing between 50 and 625 g were incubated in vitro. Insulin release was studied under control conditions and in media supplemented with glucose (3 mg/ml), leucine (5 mmol/1) or arginine (5 mmol/1). Glucose never caused a significant rise in insulin release from the pancreas. The failure of mannoheptulose (3 mg/ml) and 2-deoxyglucose (3 mg/ml) to suppress basal insulin release in a glucose-free medium indicated that basal insulin release was not governed by the liberation of glucose from glycogen in the exocrine pancreas. Arginine stimulated insulin release in four experiments using pancreas from foetuses weighing more than 200 g, but failed to cause insulin release from the pancreas of foetuses weighing less than 200 g in three experiments. Leucine consistently stimulated insulin release from the pancreas of foetuses of less than 200 g body weight but was only variably effective in causing insulin release from pancreas of foetuses weighing more than 200 g. The experimental results illustrate the development of different mechanisms for the release of insulin from the human foetal β cell.
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SUMMARY
Various methods of tissue culture were studied in an attempt to grow human foetal pancreas under conditions favourable for insulin release. Simple dicing of pancreas was superior to plasma clot adhesion or collagenase digestion for the preparation of tissue for culture. The culture medium described by Kahri (1966) (containing 25% heated postnatal calf serum) was the most suitable of four tested for the study of insulin release from the tissue culture. In this medium insulin could be measured quantitatively by radioimmunoassay and insulin degradation occurred at the rate of 20–25%/24 h. In other media the pancreas grew less well or insulin degradation was much greater.
Human foetal pancreas grown under optimal conditions released insulin for up to 34 days. Insulin released into the culture medium did not appear to inhibit the further release of insulin. In some experiments the total amount of insulin released into the culture medium was several-fold greater than that in the pancreas originally seeded. In acute incubation experiments barium and theophylline stimulated insulin release from pancreas cultures. It proved impossible to identify the cells from which insulin was released but they did not appear to be in the monolayer which was composed of fibroblasts.
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ABSTRACT
A new TRH-like peptide pyroglutamylglutamylprolineamide (pGlu-Glu-ProNH2) has recently been purified and characterized from both the rabbit prostate complex and human semen. In this study, TRH-immunoreactive peptides were extracted from anterior pituitary, posterior pituitary and hypothalamus and subjected to gel exclusion chromatography. For each tissue, TRH was resolved from pGlu-Glu-ProNH2 by anion-exchange chromatography at pH 7.6 In the anterior pituitary, 63% of the TRH immunoreactivity was chromatographically identical to pGlu-Glu-ProNH2 whereas in the posterior pituitary the new peptide represented less than 5% of the total TRH immunoreactivity. Only trace levels of pGlu-Glu-ProNH2 were observed in hypothalamus, suggesting that the acidic TRH-related peptide found in the anterior pituitary may not be of hypothalamic origin. The new TRH-like peptide was purified from whole pituitaries by gel exclusion and ion-exchange chromatography, followed by high power liquid chromatography and was shown to have chromatographic properties identical to pGlu-Glu-ProNH2. Amino acid analysis of the purified peptide revealed glutamic acid and proline residues in the ratio Glx:2 Pro:1, which is the expected composition of pGlu-Glu-ProNH2 after acid hydrolysis.
Search for other papers by R. D. G. MILNER in
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Search for other papers by P. M. B. JACK in
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SUMMARY
Insulin release was studied in vitro using pieces of pancreas from rabbits of between 24 days gestational age and 6 weeks postnatal age. When allowance was made for the fraction of pancreas which was endocrine, 16·5 mm-glucose caused increasing stimulation of insulin release as development advanced and 3·3 mm-glucose caused a similar rate of secretion at all ages. Secretion was not significantly influenced by insulin destruction in the incubation medium. Glucagon (5 μg/ml) did not stimulate insulin secretion from 24-day foetal pancreas but did so postnatally. Theophylline (1 mmol/l) stimulated insulin release at all ages and was equipotent on 24-day foetal pancreas in 3·3 or 16·5 mm-glucose. The stimulation of insulin release from 24-day foetal pancreas by 1 mm-theophylline occurred in the absence of extracellular glucose, pyruvate, fumarate and glutamate and in the presence of mannoheptulose and 2-deoxyglucose (each 3 mg/ml). Adrenaline (1 μmol/l) and diazoxide (250 μg/ml) abolished or attenuated the stimulation of insulin release by glucose, leucine plus arginine or theophylline from 24-day foetal, 1 day and 6 weeks postnatal pancreas. The stimulation of insulin release from 6-week-old pancreas by 1 mm-barium was blocked by adrenaline and diazoxide but the effect became less with increasing immaturity.
The experimental results illustrate some of the ways in which insulin secretion by the rabbit β cell changes as a function of development and draw attention to the importance of glucose and cyclic adenosine monophosphate in this process.
