Robert C McEvoy

Central and peripheral localization of somatostatin. Immunoenzyme immunocytochemical studies.

Early in 1973, Brazeau et al. (5), using monolayer cultures of rat pituitary as a bioassay system (34), reported their findings on the chemical structure and physiology of a tetradecapeptide extracted from sheep hypothalami. The new neunohonmone was named somatostatin (SRIF) because it blocked the secretion of growth hormone. Synthetic SRIF proved to be an effective hapten in conjunction with immunogenic molecules, and anti-SRIF antisera were raised in rabbits (3, 7) and guinea pigs (10). Thus, very soon after the initial description of SRIF (5), specific antibodies became available for use in quantitative immunochemical and morphologic cytoendoncninologic studies. Accordingly, Animura et al. (2) and Patel et al. (23) synthesized a tyrosine-containing analogue of SRIF that could be readily iodinated and developed radioimmunoassays (RIA). Subsequent RIA of extracts of brains and peripheral tissues of experimental animals showed that SRIF was not localized exclusively within the hypothalamus. Immunoassayable SRIF is present in significant amounts within extnahypothalamic neural tissue (6, 23, 28) and also within tissues of the gastroentenopancreatic (GEP; see Fujita and Kobayashi (13)) system of endocrine cells (3, 28). Thus SRIF may play a physiologically significant role in these tissues as well as in the hypothalamus. This prospect has become more intriguing in light of numerous reports which have demonstrated that synthetic SRIF, in addition to its inhibitory actions upon adenohypophysial function, blocks pancreatic insulin and glucagon release (see Genich et aL (14), Reichlin et at. (28) and Vale et at. (33)).

Changes in the Volumes of the A-, B-, and D-Cell Populations in the Pancreatic Islets During the Postnatal Development of the Rat

The changes in the volumes of three of the principal islet cell types in the developing rat pancreas were quantitated from day 10 to day 210 of life. The insulin-, glucagon-, and somatostatin-positive cell populations of the islets were identified by immunocytochemical staining, and their volumes were determined by linear scanning. The insulin and glucagon content of the pancreata, and the concentrations of these hormones in plasma, were also determined at each age by radioimmunoassays. The volumes of the A- and D-cells reached their maximum values by day 50 and day 35, respectively. The content of glucagon within the pancreas reached adult levels by day 50. In contrast, the volume of B-cells and the insulin content were highest on day 210. The concentrations of both insulin and glucagon in the plasma reached adult levels by approximately day 50. There were no differences in the pancreatic parameters between male and female rats until day 25, when the wet weight of the male gland became significantly greater. The concentrations of the islet hormones and the percentages of the islet cell types within the pancreas did not differ between the sexes at any age. However, the greater weight of the pancreas resulted in a greater total content of islet cells and hormones in the male gland after day 25. The data suggest that, in the rat, the B-cell volume may continue to increase with age, while the A- and D-cells apparently do not. The physiologic consequences of these changes remain to be determined.

Morphometric Quantitation of the Pancreatic Insulin-, Glucagon-, and Somatostatin-positive Cell Populations in Normal and Alloxan-diabetic Rats

The pancreatic insulin-, glucagon-, and somatostatin-positive cell populations were quantitated in normal and alloxan-diabetic rats. The method of quantitation (linear scanning) allowed an estimation of absolute changes in these cell populations through 14 months of diabetes. The changes in cell masses were correlated with changes in plasma and pancreatic immunoreactive insulin and glucagon. A marked reduction in the insulin-positive beta cells was demonstrated within seven days after alloxan treatment. No significant change in the glucagon-positive alpha cell population was noted in the diabetic rats when compared with normoglycemic controls. A statistically significant increase in the pancreatic somatostatin-positive delta cell population was demonstrable only after 14 months of alloxan diabetes. The results would suggest that the hyperglucagonemia of insulin-deficient diabetes is not a consequence of an increased pancreatic alpha cell population. In addition, since the increase in the pancreatic delta cell mass was found only late in the course of alloxan diabetes in the rat, the increase in delta cells is probably not of significance in the pathophysiology of diabetes in this experimental model.

Fetal Rat Pancreas: Differentiation of the Acinar Cell Component in Vivo and in Vitro

Fetal rat pancreases, Day 16 through Day 22 postcoitum, were analyzed biochemically for amylase and chymotrypsinogen to assess acinar cell development in vivo. The interval between -eighteen and twenty days proved to be a critical period in the differentiation of the acinar component. Eighty- and thirtyfold increases in, respectively, amylase and chymotrypsinogen concentration were accompanied by a large increase in the acinar cell percentage of the pancreas. Explantation of pancreas either before or after this critical period resulted in a developmental pattern which varied from that observed in vivo. The present organ culture system neither supported the differentiated acinar cell nor favored the further development of new acinar cells. Using the present organ culture system, explants can be produced which are devoid of acinar cells and their exocrine enzymes.

Effect of Ingested Interferon-α on β-Cell Function in Children With New-Onset Type 1 Diabetes

OBJECTIVE To evaluate the safety and efficacy of ingested human recombinant interferon-α (hrIFN-α) for preservation of β-cell function in young patients with recent-onset type 1 diabetes. RESEARCH DESIGN AND METHODS Subjects aged 3–25 years in whom type 1 diabetes was diagnosed within 6 weeks of enrollment were randomly assigned to receive ingested hrIFN-α at 5,000 or 30,000 units or placebo once daily for 1 year. The primary outcome was change in C-peptide secretion after a mixed meal. RESULTS Individuals in the placebo group (n = 30) lost 56 ± 29% of their C-peptide secretion from 0 to 12 months, expressed as area under the curve (AUC) in response to a mixed meal. In contrast, children treated with hrIFN-α lost 29 ± 54 and 48 ± 35% (for 5,000 [n = 27] and 30,000 units [n = 31], respectively, P = 0.028, ANOVA adjusted for age, baseline C-peptide AUC, and study site). Bonferroni post hoc analyses for placebo versus 5,000 units and placebo versus 30,000 units demonstrated that the overall trend was determined by the 5,000-unit treatment group. Adverse events occurred at similar rates in all treatment groups. CONCLUSIONS Ingested hrIFN-α was safe at the doses used. Patients in the 5,000-unit hrIFN-α treatment group maintained more β-cell function 1 year after study enrollment than individuals in the placebo group, whereas this effect was not observed in patients who received 30,000 units hrIFN-α. Further studies of low-dose ingested hrIFN-α in new-onset type 1 diabetes are needed to confirm this effect.

Effect of MtTW15 Mammosomatotropic Tumors on Pancreatic Islet Hormones

The effects of hypersecretion of growth hormone and prolactin on islet endocrine cells have been studied by radioimmunoassays, immunocytochemistry, and morphometry in randomized samples of pancreata from MtTW15 mammosomatotropic tumor-bearing and control rats. The randomized sampling procedure, validated by immunoassays, allowed evaluation of both hormone content (immunoassay) and endocrine cell population (immunocytochemistry) on samples derived from the same origin. Hyperinsulinemia (2×) and non-fasting hypoglycemia in 10-wk-tumor rats were normalized 3 wk after tumor removal. Pancreatic weight was doubled, but proportional to body weight increases. Islet/pancreas ratio was constant (1.29 ± 0.05%) and the same in tumor, tumor-removed, and control animals, but average islet dimensions were increased by 30% and average area doubled in tumor animals. Frequency analysis showed fewer small (< 70 μm) and more large (> 140 μm) islets in tumor animals, but no change in average islet shape shown by average axis ratios of 1.4 in all groups. Pancreatic content of insulin and glucagon was doubled, while that of somatostatin was constant. These changes were not completely reversed in tumor-removed animals. Similarly, a significant doubling in islet-derived mass was mainly due to a doubling of the B-cell mass as the average proportion of endocrine cells per islet shifted from 66%, 26%, and 18% to 81%, 18%, and 3% for B-, A-, and D-cells of control and tumor-bearing rats, respectively. Immunocytochemically detectable insulin was found in duct cells of tumor animals, but not controls. Whether such cells represent a functional reserve remains to be determined.

Syngeneic Transplantation of Fetal Rat Pancreas: II. Effect of Insulin Treatment on the Growth and Differentiation of Pancreatic Implants Fifteen Days After Transplantation

Eight 18-days-postcoltum fetal pancreases were transplanted to isogenic alloxan-diabetic male rats. Some recipients were treated with insulin for seven days immediately after transplantation. Eight animals in both the insulin-treated group and control group were killed 15 days after transplantation for morphologic and hormonal studies of the transplanted tissue. Using the morphometric technique of linear scanning, the insulin, glucagon, and somatostatin immunocytochemically positive, cell masses of the fetal pancreatic implants were quantitated. The beta cell mass of the implants from the control animals increased roughly eightfold from the time of transplant; insulin treatment resulted in a further two- to threefold increase. The insulin content of the implants increased more than did the beta cell mass, resulting in the fivefold increase in insulin per beta cell. The alpha cell and delta cell masses did not change during the transplant site, the mass of functional beta cells, and the cell-to-cell content of the implanted tissue. These results are discussed in relation to previous quantitative studies of pancreatic islet cell growth. The relationships of the transplant site, the mass of functional beta cells, and the cell-to-cell interaction within the islet to the maintenance of glucose homeostasis are also discussed.

Syngeneic Transplantation of Fetal Rat Pancreas: III. Effect of Insulin Treatment on the Growth and Differentiation of the Pancreatic Implants After Reversal of Diabetes

Eight 18-day fetal pancreases were transplanted to syngeneic alloxan diabetic male rats. Some of the recipients were treated with insulin for a 7-day period immediately after transplant. By previously published clinical criteria, three groups of recipients could be identified after reversal of diabetes by the transplanted tissue: insulin-treated rapid reversal; insulin-treated slow reversal; and control (not treated with insulin). Five animals in each group were sacrificed after glucose tolerance testing for morphologic and hormonal analysis of the transplanted tissue. The insulin-, glucagon-, and somatostatin-positive islet cell masses of the fetal pancreatic implants were quantitated. There was a correlation between the beta cell mass of of the implants and the glucose tolerance exhibited by the host animals. The rapid response insulin-treated recipients had significantly greater implant beta cell mass and insulin content compared with the other groups. There was no difference in implant alpha cell mass among the groups, but the insulin-treated implants had a significantly greater glucagon content. The delta cell mass of insulin-treated rapid response was less than that of the other two groups. The results are discussed in relation to previously reported morphometric analysis 15 days after transplantation. The relationships of transplanted beta cell mass, beta cell differentiation, transplant site, and cell-to-cell interactions within the transplanted islet to the control of glucose homeostasis are also discussed.

Syngeneic transplantation of fetal rat pancreas. I. Effect of insulin treatment of the reversal of alloxan diabetes.

Sixty-nine alloxan-diabetic male Fischer rats received syngeneic transplants of eight 18-days-postcoitum fetal pancreases at the renal subcapsular site. One half of the recipients were given 2 to 4 U. protamine-zinc insulin for seven days immediately after transplantation. This insulin-treatment regimen effectively normalized blood glucose rapidly. Forty-seven transplant recipients survived, and diabetes was reversed in all. Insulin treatment had no effect on recovery time or glucose tolerance. Those animals requiring longer periods to reach normoglycemia had impaired glucose tolerance. Some insulin-treated recipients returned to normoglycemia rapidly while others required an extended period. Those animals that showed rapid reversal exhibited elevated concentrations of plasma insulin both in the fasting state and during glucose tolerance tests. No pretransplant parameters could be identified as predictors of rapid reversal.

Syngeneic transplantation of the fetal rat pancreas IV. Dissociated versus whole organ implantation

Reversal of insulinopenia, hyperglycemia, glycosuria, and polyuria associated with severe alloxan diabetes in the rat was accomplished by syngeneic transplantation of whole late-gestation fetal rat pancreata. Intravenous glucose tolerance test (GTT) revealed an improved yet still abnormal glucose and insulin response in reversed recipients reconstituted with as few as two pancreata from fetal donors. Eight fetal donors were sufficient to return glucose and insulin response following GTT to normal. Seventy to eighty percent fewer donors were required when the pancreata were transplanted in their entirely as opposed to transplantation of pancreata subjected to prior enzymatic and mechanical dissociation. The facility and simplicity of the whole fetal pancreas implantation technique makes it an appealing model for further study of islet growth and differentiation at the transplant site and of its effect on the metabolic state of the recipient.