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SUMMARY
Tissue and plasma insulin concentrations were measured in foetal and postnatal rabbits. Insulin was detected in 18-day foetuses and pancreatic insulin concentrations from the 22nd day of foetal life onwards were similar to one another. Insulin was detected in foetal plasma from the earliest time that foetal blood could be collected: 20 days. Foetal plasma insulin levels did not change significantly with the length of gestation but maternal levels were lower on day 30 than on day 20. There was a positive maternofoetal plasma insulin gradient on day 20 and a negative gradient on day 30.
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SUMMARY
Pieces of pancreas from 24-day and 30-day rabbit foetuses, 1-day-old rabbits and rabbits aged approximately 8 weeks were incubated in vitro and insulin secretion into the incubation medium was measured in response to a variety of stimuli. Glucagon, leucine, ouabain and potassium were effective stimuli at all ages studied. By the criteria of response chosen for these experiments, glucose did not stimulate insulin secretion from 24-day foetal pancreas but did so when pancreas from older animals was studied. It was concluded that the foetal β cell of the rabbit on the 24th day of gestation, although morphologically immature, shows evidence of functional competence.
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Pieces of rabbit pancreas were incubated in vitro in an incubation medium containing no glucose or 1·5 mg. glucose/ml. In each of these conditions the effect on insulin release of each of the essential amino acids at 5 mm concentration was studied. Leucine was the only essential amino acid that stimulated insulin release to a level which reached statistical significance in an incubation medium containing no glucose. In medium containing 1·5 mg. glucose/ml., arginine, isoleucine, leucine and lysine stimulated insulin release and phenylalanine inhibited insulin release. Glucagon, theophylline or dibutyryl cyclic adenosine monophosphate stimulated insulin release significantly in the presence of leucine but not in the presence of arginine. Arginine stimulated insulin release in the presence of leucine. The results of these experiments characterize further the difference in the mechanism of action of leucine and arginine on the pancreatic β-cell and indicate possible explanations for results obtained in other species in vivo.
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ABSTRACT
The actions of partially purified porcine platelet-derived growth factor (PDGF) and highly purified multiplication-stimulating activity (MSA) II and MSA III-2, which are somatomedins, were investigated on the incorporation of [3H]thymidine and [35S]sulphate by fetal rat costal cartilage in vitro. This was compared with their effects in the presence of 1% fetal calf serum (FCS) on the uptake of thymidine by growth-arrested fetal rat fibroblasts. Platelet-derived growth factor at concentrations of 0·21–21 μg/l enhanced the incorporation of both isotopes by fetal cartilage in the presence of 1% FCS, but had an inconsistent action on thymidine uptake and no significant action on sulphate uptake in serum-free medium. Platelet-derived growth factor promoted thymidine uptake by growth-arrested, isolated fetal rat fibroblasts. Multiplication-stimulating activity II (10–100 μg/l) stimulated the uptake of thymidine and sulphate by fetal cartilage in medium containing 1% FCS but had no consistent action in serum-free medium, although MSA II and PDGF had a synergistic effect on thymidine uptake in the absence of serum. Multiplication-stimulating activity III-2 had no consistent action on thymidine or sulphate incorporation by fetal cartilage in either serum-free or serum-supplemented medium. However, the same preparation of MSA III-2 stimulated the uptake of [3H]thymidine into fetal rat fibroblasts with a half-maximal response at a concentration of 5–10 μg/l. The results identify PDGF as a possible mitogenic agent for fetal rat connective tissues in vitro and show a differential sensitivity of fetal cartilage to MSA peptides.
J. Endocr. (1984) 103, 195–203
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SUMMARY
Foetal rat pancreatic rudiments explanted on day 14 of gestation were grown for 6 days in organ culture in medium containing glucose (5·5 or 16·5 mmol/l) and amino acids at the 'physiological' or seven times the 'physiological' concentration. At the end of the period of culture, the rudiments were compared with normal 20-day foetal pancreas for DNA content, insulin concentration and quantitative morphology. The secretion of insulin from the explants was tested during 2 h incubations in medium containing glucose (5·5 or 16·5 mmol/l).
Amino acid, but not glucose enrichment of the culture medium stimulated cellular growth of the rudiment which remained, nevertheless, smaller than that occurring in vivo. All culture conditions produced a smaller proportion of exocrine cells and a greater proportion of β and duct cells than were found in normal 20-day foetal pancreas. Enrichment with amino acids favoured the development of exocrine cells at the expense of duct cells. Glucose had no effect on the development of any type of cell. Enrichment with amino acids resulted in a higher concentration of insulin per β cell but the highest value observed in vitro was only one sixth of that occurring in vivo. The absolute number of β cells cultured in amino-acid-enriched medium was twice that occurring in vivo. Culture in a glucose-enriched medium had no effect on the ability of the explant to respond to an acute glucose challenge during a short incubation, but the basal and glucose-stimulated release of insulin from cultures grown in amino-acid-enriched medium were significantly greater.
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Fetal rat pancreatic rudiments explanted on day 14 of gestation were grown for 6 days in organ culture in medium containing glucose (5·5 or 16·5 mmol/l) and essential amino acids (3·5 or 13·1 mmol/l). Non-essential amino acids (alanine, aspartic acid, asparagine, glycine, proline and serine) were added to the culture medium in a number of combinations and at a maximum total concentration of 4·0 mmol/l. At the end of the period of culture rudiments were compared for DNA content, insulin concentration and quantitative morphology. The release of insulin from the rudiments was tested during 6-h incubations on day 6 of culture.
Enrichment of the culture medium with any combination of non-essential amino acids had a slight or no effect on the growth or cellular composition of the rudiment or insulin release from it. Addition of all the non-essential amino acids to medium containing 16·5 mmol glucose and 13·1 mmol essential amino acids/l caused a dramatic reduction in the net insulin accumulation by the cultured rudiment. Combinations of non-essential amino acids in which one or more were omitted did not have the same effect. These findings suggest that fetal rat pancreas grown in vitro may require both essential and non-essential amino acids for the full expression of insulin biosynthesis and secretion.
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Foetal rat pancreatic rudiments, explanted on day 14 of gestation and grown for 7 days in organ culture, were highly sensitive to the stimulatory effect of glucose on insulin secretion. The autonomic innervation of the cultivated explant was investigated as a possible explanation of this phenomenon. No sign of sympathetic innervation was detected in the pancreas explanted on day 14; the uptake of alpha-methyl-noradrenaline was not detectable by fluorescence microscopy, the concentration of noradrenaline/pancreatic rudiment was less than 7 pg and no nerve-endings containing granular vesicles were visible by electron microscopy. It was concluded that sympathetic innervation of the pancreas occurs after day 14 of gestation. Sympathetic receptors developed independently of innervation since phentolamine, and, to a lesser extent, phenoxybenzamine, potentiated the glucose-induced insulin release from cultured explants. The beta-agonists isoproterenol or salbutamol were inactive alone, but in combination with phentolamine, they potentiated the effect of glucose. Alpha-antagonists reversed the inhibitory effect of adrenaline in a dose-dependent manner. Intrapancreatic neurones were demonstrated microscopically as cholinesterase-positive cells; parasympathetic receptors were shown to be present after potentiation of glucose-stimulated insulin release by carbamylcholine and its reversal by atropine. The preservation of parasympathetic innervation and the absence of sympathetic innervation in the cultured pancreatic rudiment may explain in part the high sensitivity of the tissue to the stimulatory effect of glucose on the release of insulin.
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SUMMARY
Foetal rat pancreatic rudiments explanted on day 14 of gestation were grown for 6 days in organ culture in medium containing glucose (5·5 (1G) or 16·5 (3G) mmol/l) and amino acids at the 'physiological' (1AA) or seven times the 'physiological' (7AA) concentration. Cultures were also performed in medium to which zinc sulphate had been added at 10−7 to 10−5 mol/l concentration. At the end of the period of culture the diameters of insulin, glucagon and zymogen granule profiles in the rudiments were compared with those in normal 20-day foetal pancreas by quantitative morphology. The β cell volume, the number of granules per β cell, the insulin granular volume fraction and the area of insulin granule core and halo were also measured under selected experimental conditions.
Zymogen granule profiles were largest in vivo, intermediate in diameter when grown in 1G × 7AA medium and smallest in 1G × 1AA medium. The mean diameter of glucagon granule profiles remained constant for growth in vivo, in 1G × 1AA medium or in 1G × 7AA medium. Insulin granule profiles were largest in 1G ×7AA medium, smallest in 3G × 1AA medium and of intermediate diameter in vivo. Amino-acid enrichment increased the diameter of insulin granules and glucose enrichment decreased it. The addition of zinc to the culture medium had no effect on insulin granule diameter. In 1G × 7AA cultures the β cells were of similar size to those in vivo, but there were 29% fewer insulin granules per cell. The increased size of the insulin granules in 1G × 7AA cultures resulted in the insulin granule volume fraction in 1G × 7AA being 17·6 compared with 10·8% in vivo. Insulin granule cores were made larger by amino-acid enrichment of the culture medium but they were unaffected by glucose. The haloes were larger in 7AA medium and smaller in 3G medium. Glucose and amino-acid enrichment had a significant interaction on halo area, the mean area in 3G × 7AA medium being less than would have been expected from the summation of the effects of the two conditions.
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ABSTRACT
Insulin has been implicated in the regulation of fetal growth. The aim of these studies was to determine if insulin has a direct mitogenic effect on fetal and neonatal rat cells in vitro. Myoblasts and fibroblasts were isolated from skeletal muscle and grown until myotube formation began or until fibroblasts were confluent. The cultures were then incubated in the presence of insulin (10−5–10−1 units/ml) and its effects were measured by the cellular incorporation of [3H]-thymidine. Myoblasts from fetuses of 21 days of gestation showed a marked, linear dose–response to insulin, significant increases over control values being observed at 2 × 10−5 units/ml or 2 × 10−4 units/ml in five out of seven experiments. Neither myoblasts from 19-day fetuses or neonates nor fibroblasts from animals of any of the three ages showed a significant thymidine uptake response to insulin. Myoblasts released immunoreactive somatomedin-C-like activity into the culture medium, but this was not related to fetal age nor to the presence of insulin in the culture medium. The results suggest that insulin may have a direct role in fetal muscle growth.
J. Endocr. (1985) 104, 63–68
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One rabbit foetus within each litter was decapitated in utero on day 24 of gestation. Plasma somatomedin activity and costal cartilage metabolism were studied 5 days later in the experimental foetuses and control litter-mates. Somatomedin was assayed by the uptake of [35S]sulphate in vitro into costal cartilage from intact foetuses. Uptake was proportional to logarithmic increases in the concentration of both foetal and maternal rabbit plasma. The mean (± 1 s.d.) somatomedin activity of four plasma pools, each pool being derived from the intact foetuses within each of four litters, was 1·3 ± 0·3 compared with a potency of unity for the reference pool of maternal plasma. The plasma somatomedin activity of decapitated foetuses did not differ significantly from that of control litter-mates when analysed by rank test, but the costal cartilage of decapitated foetuses took up less [35S]-sulphate in basal medium when compared with that of intact litter-mates. The headless body weight of the decapitated foetuses did not rank in a position significantly different from the one expected. The concentration of plasma growth hormone in the decapitated foetuses was less than 5 ng/ml and that of the intact foetuses was more than 157 ng/ml. It is concluded that plasma somatomedin activity in the rabbit foetus is not dependent on foetal growth hormone.
