G. Eric Bauer

Isolation of human and porcine islets of langerhans and islet transplantation in pigs

Abstract Using a modification of the collagenase digestion, Ficoll gradient separation technique, we isolated the islets of Langerhans from fresh human pancreata removed from braindead cadavers. Comparison of the tissue insulin/amylase ratios between whole pancreas and isolated material revealed a significant purification of islet from acinar tissue. Also, islets isolated by this technique, when incubated in vitro , incorporated radiolabeled amino acid precursors into acid-alcohol-soluble islet proteins, thus indicating their viability and protein hormonal synthesis capabilities. Pig pancreata were processed by a similar technique, and the isolated material was transplanted to pigs with diabetes induced by total pancreatectomy. The mean survival time of the 11 pancreatectomized pigs that received an islet transplant was 15.3 days, while 13 pancreatectomized control pigs had a mean survival time of 6.0 days. Control pigs had no detectable circulating insulin 2 days after pancreatectomy. The pancreatectomized pigs that received an islet transplant, although remaining hyperglycemic, had insulin levels ranging from 5 to 14 μU/ml for up to 25 days after transplantation. The present studies demonstrate the feasibility of an approach designed for our ultimate goal—the large scale isolation and transplantation of islets in man.

Immunoassayable insulin in carcinoma of the cervix associated with hypoglycemia

Immunoassayable insulin was elevated in the plasma and in the primary and metastatic lesions of a patient with carcinoma of the cervix. The release of insulin from the tumor tissue could have been the important factor in precipitating acute episodes of hypoglycemia in this patient.

Characterization of the Conversion of a Somatostatin Precursor to Somatostatin by Islet Secretory Granules

SUMMARY The existence of a biosynthetic precursor for somatostatin has been previously demonstrated in angler-fish pancreatic islet tissue. In the present study, the subcellular conversion of this precursor to somatostatin was examined. When subcellular fractions were assayed for immunoreactive somatostatin, 86% of the immunoreactivity was found in the microsome and secretory granule fractions. Based on this finding and results from previous studies on proglucagon and proinsulin conversion, only these two fractions were examined for converting activity. After incubating islet tissue with [3H]tryptophan and [35S]cystine or [14C]iso-leucine for varying time periods, the tissue was fractionated and the microsome and granule fractions were subjected to postincubation. Conversion was monitored by the calculation of percent accumulation of somatostatin in eluates after Bio-Gel P-10 filtration of fraction extracts. Both secretory granules and microsomes converted endogenously labeled 8000–15,000-dalton precursor peptides to somatostatin. Disruption of the granules by repeated freeze-thawing or osmotic shock did not affect converting activity. Conversion of exogenously labeled precursors was also observed in both fractions. When granules were subjected to repeated freeze-thawing and high-speed centrifugation to separate membranes from sol, the membranes were found to have 29% more converting activity than the resulting supernates. Both membranous and soluble converting activities were significantly inhibited in the presence of leupeptin but not phenylmethyl sulfonyl fluoride. Significant inhibition of conversion was observed when lysed granules were postincubated in the presence of antipain, leupeptin, and p-chloromercuribenzoate but not chloroquine, di-isopropyl fluorophosphate, EDTA, or N-p-tosyl-L-lysine-chloromethyl ketone HCI. These results indicate that the prosomatostatin converting enzyme is a unique membrane-associated thiol proteinase with a possible specificity for arginine residues. The findings also indicate that the intracellular transport and processing of prosomatostatin is similar to that of other peptide hormone precursors.

Studies on Rats with Islet Beta Cell Tumors Induced by Nicotinamide and Streptozotocin

Summary Islet beta cell adenomata were induced in rats by combined treatment with nicotinamide and streptozotocin. Three weeks after treatment marked alterations in glucose tolerance were noted in animals which later exhibited large beta cell tumors. Eight months after treatment, the rats known to have beta cell tumors on the basis of marked hypoglycemia and later confirmed by autopsy showed variable response to a glucose load. Some tumor-bearing rats showed fast response to glucose load, their blood sugar levels were elevated moderately and returned to normal or below normal levels rapidly; these animals are described as having “fast-acting tumors.” Rats with “slow-acting tumors” responded sluggishly to a glucose load; their blood glucose pattern was similar to that of subdiabetic animals. Animals with beta cell tumors exhibited elevated serum insulin levels 30 min after glucose administration. Insulin biosynthesis by beta cell adenomata was demonstrated by in vitro incorporation of [14C]leucine into proinsulin and insulin. In the small number of tumor samples studied, a stimulatory effect of glucose on insulin biosynthesis was observed. Our grateful thanks to Dr. William E. Dulin, The Upjohn Co., Kalamazoo, Michigan, for the gift of streptozotocin, and to Mr. Steven Koziol of Pharmacia Lab, Piscataway, New Jersey, for Phadebas Insulin Test Kits. The Minnesota Medical Foundation aided this research. This paper is dedicated to the memory of the late Dr. Arnold Lazarow.

STUDIES ON THE ISOLATED ISLET TISSUE OF FISH. IV. IN VITRO INCORPORATION OF C14- AND H8-LABELED AMINO ACIDS INTO GOOSEFISH ISLET TISSUE PROTEINS

1. Goosefish islet tissue, incubated in vitro with C14- or H3-labeled amino acids showed significant labeling of the protein fractions.2. The purified alcohol-soluble fraction is presumed to contain the insulin which is synthesized in vitro.3. The rate of amino acid incorporation into the alcohol-soluble fraction increases progressively with increasing time of incubation; it is decreased in the absence of oxygen.4. The addition of glucose decreases the number of counts incorporated into the alcohol-soluble fraction.5. In the C14-amino acid incorporation experiments, the specific activity of the purified alcohol-soluble fraction (128,000 cpm/mg. protein) is three times greater than that of the trichlor-precipitable protein residue.6. These studies Support the thesis that amino acids, added to the islet tissue in vitro, are incorporated into insulin.

[125I]-Insulin metabolism by the rat liver in vivo: Evidence that a neutral thiol-protease mediates rapid intracellular insulin degradation

The subcellular site where insulin is degraded by rat hepatocytes in vivo is controversial. While several potential insulin-degrading enzyme systems, each with its own characteristic cellular location, are known to exist in the liver, questions remain about which of them participates in the degradation of physiologic doses of insulin. These studies examine the proteases that degrade physiologic doses of [125I]-insulin in vivo to determine (1) when and where initial degradation occurs, and (2) which of the potential degradative enzymes is active. Following injection into the mesenteric veins of male rats, intact [125I]-insulin and its labeled degradation products were analysed by reverse-phase high-performance liquid chromatography (RP-HPLC) of biopsy homogenates. [125I]-insulin was rapidly degraded in vivo; the t 1/2 of degradation was approximately 2.7 minutes. To test for extracellular protease activity, an isolated perfused liver system was employed. [125I]-insulin (or [125I]-glucagon) uptake was controlled by changing the temperature of the perfusion medium. Five minutes after [125I]-insulin injection, surface-bound label was recovered in an acidic (pH 3.5) wash. In perfusion at 15 degrees C, both the internalization and degradation of [125I]-insulin were inhibited; 7.2% of unbound hormone was degraded and 5.1% of surface-bound insulin was degraded. Only 11.4% of unbound insulin and 17.4% of surface-bound insulin were degraded at 35 degrees C. In contrast, 95.5% of unbound glucagon and 89.9% of surface-bound glucagon were degraded at 35 degrees C. Thus, although glucagon degradation occurs at the sinusoidal plasmalemma of perfused livers, the same membrane does not mediate the rapid degradation of insulin observed in vivo. Analysis of the RP-HPLC [125I]-insulin elution profiles from liver biopsy homogenates, and comparison of them to profiles produced by purified proteases, suggested that insulin protease is responsible for most hepatic degradation of physiologic doses of insulin. Some cathepsin D-like activity was also observed in vivo, confirming that two pathways exist for insulin metabolism. The time course over which insulin was degraded was more rapid than previous studies in vitro would have predicted. This suggests that more insulin was receptor-bound at the time of its initial degradation, and that the active protease was soluble and was introduced into endocytic peripheral endosomes within seconds after their formation.

Morphometric analysis of the endocrine cell composition of rat pancreas following treatment with streptozotocin and nicotinamide

Using immunohistochemistry and linear scanning, a morphometric analysis was made of the composition of the rat endocrine pancreas at sequential intervals after combined injections of streptozotocin (SZ) and nicotinamide (NA). One week after treatment, the volume of islet tissue was significantly higher than that of the corresponding, saline-injected controls, probably as the result of acute hyperplasia of insulin- and somatostatin-positive cells. However, at all time periods thereafter (6, 20, and 36 weeks), the drug-treated rats showed decreased islet volumes compared to controls. Analysis of aggregate (total) volumes of hormone producing cells at various time periods after drug treatment indicated that decreases in insulin (B-cell) volumes only partially accounted for the observed changes in total islet volume. There were, in addition, early decreases in glucagon (A-cell) and increases in somatostatin (D-cell) volumes. The results suggest that SZ/NA treatment caused limited islet B-cell destruction and transient changes in the proportions of islet A and D cells. Microscopic endocrine tumors were observed at 20 weeks, and both gross and microscopic tumors were observed 36 weeks after SZ/NA treatment. When islet and tumor tissues were included in computation, aggregate volumes of insulin and somatostatin-positive cells were markedly increased, with no significant changes in glucagon-positive cell volumes compared to controls, indicating that the tumors were rich in B and D cells, but poor in A cells. These results are discussed in relation to changes in glucose tolerance and serum insulin levels, and to islet cell volumes following treatment with a diabetogenic dose of streptozotocin alone.

The Biosynthesis of C14- and H3-Labeled Insulin

In recent years, there has been much interest in the biosynthesis of insulin because of its role in diabetes. In addition, the exact amino acid sequence of the two peptide chains is known [1] and, therefore, the insulin molecule has become a model protein for research in synthetic mechanisms. Many techniques for the purification and characterization of insulin have been developed [2,3,4]. In the study of insulin biosynthesis, several investigators have incubated mammalian pancreas slices in the presence of radioactive amino acids in vitro, and then isolated the labeled insulin [3,5,6] This approach is limited because the beta cells of the islets of Langerhans (which manufacture and store insulin) comprise only a small fraction of the total pancreas. During the development of the pancreas in certain teleost fish, however, the islet tissue becomes separated from the exocrine pancreas and it appears in the adult as a macroscopic aggregate of endocrine tissue called the principal islet or Brockmann body. The principal islet contains cells which are functionally and anatomically analogous to the B-cells of mammalian islets; fish insulin is similar to mammalian insulin in structure and in biological activity [7,8]. Our studies on insulin biosynthesis using fish islets were begun in 1959; this report is a summary of these investigations.

Biosynthesis of somatostatin in pancreatic islets.

Abstract In order to determine whether the somatostatin (SRIF) that can be localized by immunocytochemical means in pancreatic islets is produced in situ, we have investigated this tissue for evidence of SRIF biosynthesis. We have employed the islets of anglerfish, which are free of pancreatic exocrine tissue and consist of nearly 30% SRIF-containing D cells. 1 Results from our initial studies demonstrate that SRIF is synthesized in pancreatic islet tissue and suggest that a larger precursor form may play a role in SRIF biosynthesis.

Immunochemical identification of endocrine cell types in the streptozotocin nicotinamide-induced rat islet adenoma

Abstract The streptozotocin nicotinamide-induced rat islet adenoma is a benign encapsulated tumor that exhibits considerable histologic and endocrine variability. Individual tumors differ in their general pattern of cellular arrangement and often contain necrotic regions, ductlike structures and large amounts of interspersed connective tissue. The majority of tumors are polyhormonal. All contain insulin by radioimmunoassay and immunohistochemistry, although the amounts vary greatly. Glucagon and/or somatostatin immunoreactive cells are present in 62% of tumors by immunohistochemistry and in 86% by radioimmunoassay. Again, the amounts vary greatly. Pancreatic polypeptide immunoreactivity was localized in a small number of scattered cells in 31% of tumors studied. Tumors demonstrated considerable hormonal intra- and intertumor heterogeneity both by immunohistochemistry and by radioimmunoassay. From these data it is indicated that the tumor can neither be considered an “insulinoma” nor “pure” in endocrine composition. This histologic and endocrine variability must be taken into account when using the tumor as a source of islet mRNA or as a source of tissue in insulin biosynthetic work.