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ABSTRACT

Melanin-concentrating hormone (MCH) is a neurohypophysial peptide that induces pigmentary pallor in teleosts and which is released when the fish are placed on a white background. An additional effect of the peptide is the depression of ACTH and hence cortisol secretion during moderate stress. The present work on rainbow trout shows that plasma MCH concentrations, while unaffected by a single stress, are raised by repeated stress (1 ml saline injected i.p. without anaesthesia) and remain high for several hours thereafter. The response to stress is observed only in white-adapted fish and not in fish kept in black-coloured tanks, when MCH release is normally low. Plasma concentrations of MCH vary diurnally but stress induces an equivalent incremental rise in plasma MCH, whether administered in the middle or towards the end of the photophase. The stress-induced rise in MCH concentrations is prevented by treatment with dexamethasone. The results support the suggestion that the modulatory effect of MCH on the hypothalamopituitary-interrenal axis of fish might be enhanced under conditions of stress.

Journal of Endocrinology (1991) 128, 261–266

SUMMARY

The secretory response of rat islets of Langerhans was examined during pregnancy and compared with insulin release in normal rat islets. The threshold for a secretory response to glucose was lowered for islets from pregnant rats by comparison with non-pregnant controls. In addition, such islets showed a greatly increased sensitivity to glucose concentrations over the range 3·5–20 mmol/1. Significantly lower fasting blood glucose levels were found in pregnant rats in vivo, compared with controls.

Insulin secretagogues other than glucose were tested for their effects on islets during pregnancy. Despite the high baseline of insulin secretion in response to glucose in pregnancy, there was an additional increased secretory response to arginine and theophylline. In contrast to their response to glucose, pregnant rat islets did not display an increased sensitivity to leucine. Glucagon, while it increased the insulin response of normal islets, had no significant effect on increasing the insulin response from pregnant rat islets suggesting that adenyl cyclase activity is already highly stimulated in pregnancy.

In addition, the insulin, DNA and protein content of islets during pregnancy were increased significantly above normal values.

The results suggested that rat islets are not only larger in pregnancy, but that they possess a more sensitive mechanism for detecting and responding to glucose and other secretagogues.

SUMMARY

The effects of diet on the altered insulin secretory responses of islets of Langerhans of pregnant rats have been investigated. The daily food intake of pregnant rats was found to exceed that of control non-pregnant rats by 20% on average. Depriving pregnant rats of this additional food resulted in an alteration in the pattern of insulin secretion seen in pregnancy, such that the sensitivity to stimulation by low glucose concentrations was abolished. The contribution made by different components of the diet to the secretory response in pregnancy was investigated. When additional carbohydrate, though not protein, was fed to pregnant rats on a restricted food intake, the sensitivity of the islets to glucose stimulation was restored. It was concluded that the quantity and in particular the carbohydrate content of food eaten by pregnant rats exerts an important influence on the changes in insulin secretion in pregnancy.

Ovariectomized rats showed a 30% increase in body weight and food intake over a 9 day period after operation. When pair-fed with normal controls, they had comparatively higher fasting serum levels of glucose and lower levels of insulin and progesterone than the normal rats. Treatment with oestradiol reversed these results. Insulin release from islets isolated from ovariectomized rats was significantly lower than from those of normal controls; the secretory responses were improved after administration of oestradiol to ovariectomized rats.

Cytosol receptors for progesterone and oestradiol were measured in a 105 000g supernatant fraction of islets from normal, ovariectomized and oestrogen-treated ovariectomized rats. Progesterone-receptor binding was dramatically reduced after ovariectomy but was restored to normal levels by oestradiol treatment of the rats. Oestradiol-receptor binding was not significantly affected by ovariectomy but was increased several fold by oestrogen treatment of ovariectomized rats.

These results suggest that islets of Langerhans contain receptors for both progesterone and oestradiol, and that this receptor population is subject to change. Progesterone—but not oestradiol—receptor measurements could be correlated with alterations in the rates of secretion of insulin from islets. Oestrogen administration in vivo had profound effects on subsequent insulin release from islets, though this may have been mediated by way of an increase in the quantity of the progesterone receptor.

ABSTRACT

Rainbow trout were reared in black or off-white coloured tanks for up to 18 months of age to achieve maximum differences in the synthesis of the neuropeptide, melanin-concentrating hormone (MCH). White-reared fish had greatly increased MCH concentrations in their pituitary glands, in their MCH perikarya and in the presumptive neuromodulatory fibres of the dorsal hypothalamus/thalamus when compared with black-reared and commercially reared trout. Following transfer to brighter white tanks, white-reared fish showed a significant increase in plasma MCH concentration and a reduction of MCH in the pituitary and MCH perikarya. The additional challenge of repeated stress further increased plasma MCH concentration in these fish and also reduced MCH in the dorsal hypothalamus/thalamus. In black-reared fish transferred to white tanks, plasma MCH concentrations were significantly raised after transfer, although they were lower after 11 days than in white-reared counterparts. Transfer from black to white background caused a fall in the MCH concentration in all regions— pituitary gland, perikarya and dorsal hypothalamus/thalamus; if transfer was accompanied by repeated stress, the hormone in the pituitary gland and MCH perikarya became so depleted that plasma MCH concentrations declined. Within each experimental situation (control, background transfer and transfer with stress) there was in inverse correlation between plasma MCH concentrations of black- and white-reared fish and the cortisol concentration. MCH had no direct effect on the secretion of cortisol by interrenal tissue but incubated hypothalami, in which endogenous MCH had been immunoabsorbed, provided evidence that MCH can depress the release of corticotrophin-releasing bioactivity.

Journal of Endocrinology (1991) 128, 267–274

ABSTRACT

Pancreatic islets, isolated from rats starved for 48 h, secreted significantly less insulin in the presence of 2 mmol glucose/l than islets of fed controls. In contrast, the insulin secretory response of islets from fed and starved rats to a challenge of 20 mmol glucose/l was similar. Concentrations of prostaglandin E₂ (PGE₂) in islets from starved rats incubated with 2 mmol glucose/l were significantly lower compared with those in control islets obtained from fed animals. Although glucose (20 mmol/l) stimulated PGE₂ production in islets from starved and fed rats by 2·7- and 1·6-fold respectively, the concentrations achieved were the same as a consequence of the different prestimulated concentrations. Incubation with [14C]arachidonic acid of sonicated islet preparations from fed rats and separation of metabolites generated by high-pressure liquid chromatography, indicated the biosynthesis of a number of cyclo-oxygenase- and lipoxygenase-derived compounds, including 6-keto-PGF_1α, PGF_2α, PGE₂ and 12-hydroxyeicosatetraenoic acid. Metabolism of arachidonic acid to cyclo-oxygenase-derived compounds occurred with the same efficiency, but production of lipoxygenase-derived compounds was reduced by 50% in sonicated islets from starved compared with fed rats. Activity of phospholipase A₂ of islets from starved rats was significantly less than that measured in islets from fed rats, although the degree of stimulation by 20 mmol glucose/l was the same in both types of islet. These alterations in the phospholipase A₂/arachidonic acid cascade may contribute to the diminished insulin secretory response of islets from starved rats to relatively low concentrations of glucose.

Journal of Endocrinology (1990) 124, 455–461

ABSTRACT

Male hypophysectomized rats treated with bovine (b)GH–monoclonal antibody complexes showed enhanced weight gain compared with animals treated with bGH alone over a 12-day treatment period. Liver microsomes prepared from animals showing enhanced weight gain exhibited increased specific binding of human (h)GH. Studies on the specificity of these binding sites showed that they were lactogenic, ¹²⁵I-labelled hGH being displaced by ovine prolactin, but not by non-mammalian growth hormones. In this respect they were similar to lactogenic binding sites in the liver of pregnant rats. Monoclonal antibodies to hGH blocked binding to lactogenic receptors to different extents. The pattern of such inhibition was similar, but not identical, for the receptors induced in hypophysectomized rats and those from pregnant rat liver. The evidence available suggests that the lactogenic receptors induced by bGH–monoclonal antibody complexes are not directly involved in the enhancement of growth.

Journal of Endocrinology (1990) 124, 469–474

It was possible to vary the replication rate of cells in the islets of Langerhans of adult rats. The rate of incorporation of [³H]thymidine into islet DNA was increased at 12 days of pregnancy to 2·3-fold and at 19 days of pregnancy to 1·3-fold that in control rats. Ovariectomy, which leads to lowered plasma levels of ovarian steroids, induced a significant and unexpected increase in the rate of thymidine incorporation into islets; treatment of ovariectomized rats with 2 μg oestradiol/rat per day for 3 days reversed this upward trend. When islets from normal rats were cultured with certain combinations of steroid hormones including progesterone and oestradiol or with insulin secretagogues, with the exception of glucose, a decreased rate of DNA synthesis was usually found compared with that in control rats.

Since treatment with steroid hormones inhibited incorporation of [³H]thymidine into islets from ovariectomized rats and directly reduced incorporation into tissue-cultured islets from normal rats in vitro, it was concluded that increased levels of steroid hormones were not responsible for the higher rate of regeneration of islet cells in pregnant rats. However, a striking correlation between levels of blood glucose ^{in vivo} and DNA synthesis in islets ^{in vitro} has been observed.

Abstract

Insulin delivery by somatic cell gene therapy was evaluated using murine pituitary AtT20MtIns-1.4 cells. These cells have been stably transfected to release human insulin by the introduction of a recombinant plasmid bearing a human preproinsulin cDNA under the control of a zinc-sensitive metallothionein promoter. 6 × 10⁷ AtT20MtIns-1.4 cells were implanted subcutaneously into streptozotocin-diabetic mice immunosuppressed with cyclosporin A. Release of human insulin was assessed using a specific plasma human C-peptide assay. On days 1 and 2 after implantation human C-peptide concentrations were about 0·02 pmol/ml. Consumption of zinc sulphate solution (500 mg/l) as drinking fluid for days 3–5 increased plasma human C-peptide concentrations to 0·11 ±0·01 pmol/ml (mean±s.e.m.), n=11, P<0·01, and concentrations declined when zinc was discontinued. The extent of hyperglycaemia was slightly lower (P<0·05) than in a group implanted with non-transfected AtT20 cells. The study was terminated after 9 days, and tumour-like aggregations of implanted cells were identified at autopsy. These comprised a large necrotic core with insulin-containing cells at the periphery. The study provides support for the view that somatic cell gene therapy offers a potential approach to insulin delivery in diabetes mellitus.

Journal of Endocrinology (1994) 142, 339–343

ABSTRACT

Since porcine islets are considered a likely tissue source for islet transplantation we have studied the insulin secretory responses to stimuli and some of the cell surface antigen characteristics of porcine islet cells.

In a static incubation system, the threshold level of glucose required for the stimulation of insulin secretion from freshly isolated porcine islets was found to be between 2·8 and 4·2 mmol glucose/l. Arginine (5 mmol/l) and 3-isobutyl-1-methylxanthine (1 mmol/l) potentiated insulin release induced by 8·3 mmol glucose/l. Leucine (5 mmol/l) initiated release in the presence of 2 mmol glucose/l. Neither β-hydroxybutyrate (10 mmol/l) nor octanoate (5 mmol/l) potentiated insulin release induced by 8·3 mmol glucose/l, but β-hydroxybutyrate initiated release in the presence of 2 mmol glucose/l while octanoate did not.

A ¹²⁵I-labelled protein A binding assay and an enzyme-linked immunosorbent assay system were used to detect antibody binding to islet and non-islet cells. Monoclonal antibodies raised against intact rat islets were shown to bind to both porcine and rat islet cells but not to rat hepatoma tissue culture cells or rat insulinoma cells. The serum from recently diagnosed type I diabetics was shown to bind to rat islet cells in a ¹²⁵I-labelled protein A binding assay, while serum from control subjects showed little, if any, binding. Porcine islet cells were unable to distinguish between the sera of recently diagnosed type I diabetics and controls in a similar assay.

In conclusion, porcine islets respond to many of the major insulin secretagogues to which human islets are sensitive. However, they have a lower secretory response to these compounds compared with that reported for human islets. Monoclonal antibody and human sera binding studies show that rat and porcine islets share some cell surface antigens but it remains to be seen whether the inability of porcine islets to discriminate between diabetic and non-diabetic sera is an advantage for transplantation, indicating less likelihood of an islet-specific autoimmune-like humoral attack.

Journal of Endocrinology (1990) 126, 43–4