S. L. Howell

Effects of glucose and amino acids on the biosynthesis of DNA and insulin in fetal rat islets maintained in tissue culture.

Islet growth and insulin biosynthesis in fetal rat islets have been studied in a recently developed in vitro culture system. Collagenase-digested pancreases of 22-day-old rat fetuses were maintained for 5 days in culture medium RPMI 1640 (11.1 mM glucose) to allow degeneration of acinar tissue. Intact pure islets, composed of more than 90% B-cells, were then collected and subsequently treated for 3 days in culture with different concentrations of glucose and amino acids. Islets were then labeled for 24 h with 3H-thymidine (DNA synthesis) or for 2 h with 3H-phenylalanine (insulin/protein biosynthesis). The specificity of the incorporation of 3H-thymidine into B-cell DNA has been investigated and characterized by both Chromatographie and autoradiographic studies. Moreover, using a colchicine metaphase arrest technique, a relationship was demonstrated between incorporation of labeled thymidine and mitotic incidence in the B-cells. Incorporation of 3H-thymidine was significantly higher in the fetal than in the adult islets following treatment in all the different concentrations of glucose and amino acids studied. In the presence of 10% fetal calf serum, however, there was no increase in DNA synthesis in the fetal islets in response to high concentrations of glucose or amino acids. In media containing 2.5% fetal calf serum, there was a significant increase in the rates of DNA synthesis after addition of high concentrations of either glucose or amino acids. Fetal islets showed a marked insulin biosynthetic response to an acute glucose challenge. Both the basal and stimulated rates of insulin biosynthesis were increased after treatment in high concentrations of glucose but not after treatment with amino acids. The results suggest an important role of nutrients in the regulation of B-cell growth and insulin biosynthesis and also a dissociation between the regulatory mechanisms of these two processes.

Role of zinc and calcium in the formation and storage of insulin in the pancreatic β-cell

Summary1.The effects of culture of isolated mouse islets of Langerhans for up to 9 days in media which had been depleted of zinc electrochemically or with the chelating agent Tris-(2-aminoethyl) amine, or of calcium, have been compared.2.An 83% reduction of extracellular zinc concentration did not adversely affect proinsulin biosynthesis, conversion of proinsulin to insulin, or the ability of cells to store newly formed insulin in granules. When incubation media were depleted of both zinc and calcium the β-cells produced abnormally large electron-lucent granules, consistent with the failure of insulin to crystallise within the granule sac.3.Very similar results, with formation of large electron lucent granules, were obtained after culture of islets in the absence of calcium but in the presence of normal concentrations of zinc.4.It is suggested that zinc may play a less critical role in the biosynthesis of proinsulin and its conversion to insulin, while calcium may have a more important function in insulin storage, than has sometimes previously been supposed.

Regulation of guanylate cyclase in guinea-pig islets of Langerhans

1. Guanylate cyclase activity was determined in homogenates of guinea-pig islets of Langerhans by measurement of the conversion of [alpha-(32)P]GTP into cyclic [(32)P]GMP, the reaction products being separated on columns of neutral alumina. 2. The pH optimum of the enzyme was 7.3; it showed a requirement for bivalent cations, the effectiveness of the cations tested being Mn(2+)>>Ca(2+)>Mg(2+). 3. About 70% of enzyme activity was sedimented by centrifugation at 105000g for 60min; activity was increased 2.3-fold by treatment of homogenates with 0.1% Triton X-100. 4. Guanylate cyclase activity of homogenates was increased by acetylcholine, secretin or pancreozymin, but was inhibited by adrenaline, noradrenaline or ATP. Insulin, glucagon, prostaglandins E(1) or E(2), glucose, F(-), diazoxide or glibenclamide were ineffective. 5. Determination of cyclic GMP amounts in islets by radioimmunoassay showed a basal concentration of 2.0pmol/mg of protein, which was increased by incubation of the islets in the presence of acetylcholine or the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, but was unaffected by glucose. 6. Dibutyryl cyclic GMP had significant stimulatory effects on rates of insulin biosynthesis in isolated rat islets of Langerhans. 7. These results suggest a possible role for cyclic GMP in the regulation of insulin biosynthesis and secretion.

Direct effects of progesterone on rat islets of Langerhans in vivo and in tissue culture.

SummaryProgesterone and oestradiol did not alter rates of insulin secretion from isolated rat islets of Langerhans during a 60 min period of incubation in vitro. However, islets isolated from rats which had been injected daily for 15 days with progesterone (5 mg) and oestradiol (5 μg) showed enhanced rates of insulin secretion in response to stimulation by 20 mmol/l glucose or 6 and 20 mmol/l glucose plus 5 mmol/l theophylline. Islets from rats which had been injected with the slow-releasing depot progesterone derivative, hydroxyprogesterone hexanoate, 3 times in 15 days, also showed enhanced rates of insulin release in the absence of any alteration in adenylate cyclase activity. In neither experiment could increased food intake, blood glucose levels or islet insulin content account for the observed changes. The possibility of a direct effect of progesterone on the secretory process was investigated in islets which had been cultured for 20 h with progesterone and oestradiol; these islets were then subjected to a variety of stimuli for secretion. They responded significantly more to glucose (6 or 20 mmol/l) in the presence of theophylline (5 mmol/l), while their insulin content was not significantly different from control islets cultured for a similar period. Islets cultured for 20 h in the presence of progesterone and oestradiol did not show any change in their adenylate cyclase activities. Similarly, direct addition of progesterone and oestradiol to islet homogenates did not alter the adenylate cyclase activity during a 30 minute incubation. These results suggest that progesterone and oestradiol affect insulin secretion directly, by a mechanism which does not involve activation of adenylate cyclase.

Binding of3H-progesterone by isolated rat islets of Langerhans

SummaryIslets of Langerhans isolated from normal female rats have been used in studies of3H-progesterone uptake by intact islet cells. The intracellular sites which are involved in binding of the hormones have been determined by subcellular fractionation and autoradiography. Uptake of3H-progesterone into islets occurred in a temperature and concentration dependent manner. The uptake increased rapidly in the first 30 min, and could be partially displaced by addition of excess unlabelled progesterone.3H-progesterone uptake was lowered by incubation of the islets in the absence of Ca++, at 0° compared to 37° C, or to a much lesser extent when islet cycilic AMP levels were raised by addition of 3-isobutyl-l-methylxanthine. However, uptake was unaffected by prior treatment of the islets with neuraminidase orp-hydroxymercuribenzoate. Differential centrifugation of islets which had previously been incubated with3H-progesterone showed the highest specific activity of binding in the nuclei and debris fraction. Isolation of a purified nuclear fraction by sedimentation through sucrose solutions confirmed that binding was present in the nuclear component of this heterogeneous fraction, while autoradiographic studies suggested both nuclear and cytosolic localisation of3H-progesterone. In a separate series of experiments, evidence was obtained for the existence of saturable cytosolic binding of progesterone, and for movement of labelled hormone from the cytosol to the nuclear fraction.It is suggested that, in the islets of Langerhans as in other target tissues, the action of progesterone involves penetration into the cells, binding to a cytosolic receptor protein, and subsequent transfer to the nucleus. The nuclear events which lead to subsequent alteration of rates of insulin secretion remain to be determined.

Role of microtubules in the intracellular transport of growth hormone

SummaryPulse-chase experiments utilising(3H)leucine have been used to study the effects of colchicine and vinblastine on intracellular transport and secretion of newly synthesised growth hormone from rat anterior pituitary fragments. Growth hormone was isolated from medium and fragments by polyacrylamide gel electrophoresis. When colchicine or vinblastine, which disrupt microtubules, were added immediately after pulse labelling, inhibition of the subsequent secretion of newly synthesised growth hormone was detected throughout the succeeding 5 h. Similar inhibition was seen if the drugs were added after a 1 h delay. However, if colchicine or vinblastine were added only after a 2 h chase incubation, then no significant effect on subsequent release of labelled growth hormone was seen. The results suggest that these agents may inhibit the transport of newly formed growth hormone storage granules from the Golgi complex to the cytoplasmic pool. Microtubules do not appear to be involved in the mechanism of the final secretion of newly synthesised hormone by exocytosis.

Biochemical and ultrastructural changes in A and B cells of the islets of Langerhans of mice infected with EMC virus

SummaryInfection of DBA2 mice with the M strain of EMC virus was used to study the effects of virusinduced diabetes on the A and B cells of the islets of Langerhans. A transient hypoglycaemia was seen in 48% of mice 2–3 days after infection and probably resulted from increased serum insulin concentrations together with inhibition of glucagon secretion at that time. Islets from hypoglycaemic mice showed no significant alterations from control level in basal or fluoride-stimulated adenylate cyclase activity. Overall, 70% of infected mice became hyperglycaemic with a maximum incidence 6 days after infection. Hyperglycaemia was accompanied by a dramatic reduction in the total pancreatic insulin content and in insulin secretory responses to glucose and theophylline, while A-cell structure and function appeared relatively unaffected in diabetic animals. Basal adenylate cyclase activity was increased in hyperglycaemic mice at 7 days after infection, while fluoride-stimulated adenylate cyclase activity was normal throughout the course of infection. Ultrastructural alterations were observed in a small proportion of B cells from two days after infection and included abnormalities of mitochondrial structure and increased electron opacity of the cytoplasm of affected cells, which subsequently led to complete necrosis. The results suggest that EMC virus specifically affects the B cells of the islets and that disturbances of A cell function may be secondary to B cell damage.

INSULIN SECRETION - THE ROLE AND MODE OF ACTION OF CYCLIC AMP

There is much evidence to suggest that intracellular levels of cyclic AMP in endocrine cells may play an important role in determining rates of hormone release, and that many secretagogues influence cyclic AMP levels, and hence rates of secretion, by interaction with receptors which are related to adenylate cyclase, or by inhibition of phosphodiesterase. A mechanism of this type seems to operate during activation of secretion by physiological agents in, for instance, the anterior pituitary (1) and thyroid (2). A widely accepted model for the regulation of hormone secretion is shown in Fig. 1, and i t is the purpose of this paper to determine how far this model is applicable to the regulation of insulin secretion by the B cells of mammalian islets of Langerhans.

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.