Nonenzymic in vitro isolation of perinatal islets of Langerhans

Hegre, Orion D.; Marshall, Sue; Schulte, Bradley A.; Hickey, Gregg E.; Williams, Frank G.; Sorenson, Robert L.; Serie, Janet R.

doi:10.1007/bf02619574

Cited by 39 publications

(23 citation statements)

References 45 publications

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“…This effect on p-cell replication on the addition of exogenous insulin to neonatal rat pancreatic monolayer cultures has recently been reported. 19 Our studies also confirm the data of Hegre et al 20 on islet yield from the perinatal pancreas. These authors utilized a nonenzymatic method to isolate the islets and demonstrated that, compared with amounts from the fetal or mature rat pancreas, islet yield was higher in young postnatal pancreatic tissue and that day 5 of postnatal life yielded the most islets.…”

Section: Discussionsupporting

confidence: 92%

Hormone Release, Islet Yield, and Transplantation of Fetal and Neonatal Rat Dorsal and Ventral Pancreatic Islets

Hayek

Guardian

1986

Diabetes

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Fetal (20-21 day gestation) and neonatal (less than 5-day-old) rat islets were isolated from the glucagon-rich (dorsal) and glucagon-poor (ventral) pancreatic regions. After 1 or 2 wk in culture, groups of islets from each region were transferred to culture dishes containing Krebs-Ringer bicarbonate buffer with low (2.4 mM) and high (16.7 mM) glucose plus aminophylline. After 2 wk in culture, insulin released into medium was higher than after 1 wk, and more so if the islets originated from dorsal tissue (P less than .01). Glucagon release in response to alanine (10 mM) stimulation was also significantly higher in dorsal than in ventral islets (6.38 +/- 1.98 vs. 1.49 +/- 0.89 pg X islet-1 X h-1, respectively; P less than .02). Coincident with higher insulin and glucagon release, islet yield was greater in tissue from the dorsal neonatal pancreas than from that obtained in the fetal and neonatal ventral pancreas [range: dorsal, 131-180 (median, 153), vs. ventral, 53-127 (median, 84), islets obtained on day 5 of culture]. Neonatal islets of dorsal origin were transplanted intrasplenically (500-3000 islets) to streptozocin-induced diabetic inbred Lewis rats. Only rats receiving greater than 2500 islets were cured by transplantation. These experiments show that dorsal fetal islets secrete more insulin than do ventral islets and that islet yield is higher when islets are isolated from dorsal rather than from ventral perinatal pancreatic tissue. Despite their in vitro behavior, more neonatal dorsal islets are required to cure experimental diabetes than are reported with adult islets.

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Section: Discussionsupporting

confidence: 92%

Hormone Release, Islet Yield, and Transplantation of Fetal and Neonatal Rat Dorsal and Ventral Pancreatic Islets

Hayek

Guardian

1986

Diabetes

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“…Islet Isolation and Culture-Islets were isolated and cultured by a modification of the method described (9). Pancreata were harvested from 5 to 10, 4-or 5-day-old neonatal rat pups and placed in 4 ml of Ham's F-12 medium containing 5% (v/v) fetal bovine serum, 100 IU/ml penicillin, and 100 g/ml streptomycin (culture medium).…”

Section: Methodsmentioning

confidence: 99%

A High Affinity Glutamate/Aspartate Transport System in Pancreatic Islets of Langerhans Modulates Glucose-stimulated Insulin Secretion

Weaver

Gundersen

Verdoorn

1998

Journal of Biological Chemistry

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To examine the role of glutamatergic signaling in the function of pancreatic islets, we have characterized a high affinity glutamate/aspartate uptake system in this tissue. The islet [3 H]glutamate uptake activity was Na ؉ -dependent, and it was blocked by L-trans-pyrrolidine-2,4-dicarboxylic acid, a blocker of neuronal and glial glutamate transporters. Islet glutamate transport activity exhibited a V max of 8.48 ؎ 1.47 fmol/min/islet (n ‫؍‬ 4), which corresponds to 102.2 ؎ 17.7 pmol/min/mg islet protein. The apparent K m of islet glutamate transport activity depended on the glucose concentration used in the assay. In the presence of glucose concentrations that do not stimulate insulin secretion (2.8 mM), the apparent K m was 34.7 ؎ 7.8 M (n ‫؍‬ 3). However, in high glucose (16.7 mM) the apparent K m increased to 112.7 ؎ 16.5 M (n ‫؍‬ 3) with little or no change in V max . Like most known plasma membrane glutamate transporters, islet glutamate transporters also transported D-aspartate. Anti-Daspartate immunoreactivity showed that the islet glutamate/aspartate transport activity was localized to the non-␤ cell islet mantle. In perifusion experiments with isolated islets in the absence of exogenous amino acids, L-trans-pyrrolidine-2,4-dicarboxylic acid in the presence of 8.3 mM glucose potentiated insulin secretion 23.3 ؎ 2.3% (n ‫؍‬ 3) compared with 8.3 mM glucose alone. This effect was abolished in the presence of the ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione. Furthermore, 6-cyano-7-nitroquinoxaline-2,3-dione alone inhibited glucose-stimulated insulin secretion in isolated islets by 15.9 ؎ 5.9% (n ‫؍‬ 3). Taken together these data suggest that a high affinity glutamate transport system exists in pancreatic islets and that this system contributes to a glutamatergic signaling pathway that can modulate glucose-inducible insulin secretion.Although the role of glutamate as a signaling molecule is well established in the central nervous system, a similar role in the periphery has only recently been suggested. We (1) and others (2) have detected functional glutamate receptors in the pancreatic islets of Langerhans. These miniature organs, found dispersed throughout the exocrine pancreas, are composed of four major cell types as follows: the insulin-secreting ␤ cell, the glucagon-secreting ␣ cell, the pancreatic polypeptide-secreting PP cell, and the somatostatin-secreting ␦ cell. The electrically excitable ␤ cells are stimulated to secrete insulin in response to changes in serum glucose concentrations. Secretion of insulin, and the three other major peptide hormones found in islets, is also believed to be affected by other metabolic and neuronal signals (reviewed in Refs. 3 and 4). Bertrand et al. (5,6) have shown that ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1 receptor agonists can potentiate both insulin and glucagon secretion from a perfused pancreas preparation and that oral or intravenous glutamate can increase insulin secretio...

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“…Many procedures exist for the recovery of islets from the adult pancreas [22][23][24][25][26][27]. The scattered distribution of endocrine cells in the human fetal pancreas excludes standard islet isolation procedures [9].…”

Section: Discussionmentioning

confidence: 99%

Enrichment of Beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody

et al. 1992

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The aim of this study was to produce an antibody reactive to the surface of endocrine pancreatic cells and use this antibody for the purification of endocrine cells from the human fetal pancreas by fluorescence activated cell sorting. We describe such an antibody, called N1, reacting with the surface and cytoplasm of endocrine cells in the adult and fetal human pancreas (12 to 18 weeks gestational age). While unreactive to exocrine and mesenchymal cells, it was not specific for endocrine cells, as evidenced by its staining pattern in tissues other than pancreas. Almost 40% of the N1-positive pancreatic cells contained either insulin, glucagon or somatostatin. Conversely, more than 90% of each of the hormone-containing cells was N1 positive. An additional 40% of N1-positive cells, not containing other pancreatic hormones, was shown to contain islet amyloid polypeptide, synaptophysin, chromogranin, tyrosine hydroxylase or CA812. A two-step collagenase digestion protocol yielded 1.29 +/- 0.17 x 10(5) cells per mg pancreatic tissue. After Percoll gradient centrifugation, the suspension contained 15.6 +/- 5.7% (n = 25, mean +/- SD) cells reactive with N1. By fluorescence activated cell sorting using the antibody N1, the single-cell suspension was enriched from 3.0 +/- 1.4% to 16.2 +/- 4.8% (n = 10, p less than 0.01) Beta cells. Alpha and Delta cells were also enriched significantly by this procedure. The percentage of N1-positive cells increased from 17 +/- 4% to 83 +/- 6%. This preparation enriched for endocrine cells allows future studies on possible endocrine precursor cells.

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Nonenzymic in vitro isolation of perinatal islets of Langerhans

Cited by 39 publications

References 45 publications

Hormone Release, Islet Yield, and Transplantation of Fetal and Neonatal Rat Dorsal and Ventral Pancreatic Islets

Hormone Release, Islet Yield, and Transplantation of Fetal and Neonatal Rat Dorsal and Ventral Pancreatic Islets

A High Affinity Glutamate/Aspartate Transport System in Pancreatic Islets of Langerhans Modulates Glucose-stimulated Insulin Secretion

Enrichment of Beta cells from the human fetal pancreas by fluorescence activated cell sorting with a new monoclonal antibody

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