Human Fetal Islet-like Cell Clusters Research Articles

Expression of glucokinase in glucose-unresponsive human fetal pancreatic islet-like cell clusters.

Glucokinase (GK) is the glucose sensor in the adult beta-cell, resulting in fuel for insulin synthesis and secretion. Defects in this enzyme in the beta-cell are responsible for the genetic disorder maturity-onset diabetes of the young, with the beta-cell being unable to secrete insulin appropriately when challenged with glucose. The human fetal beta-cell is also unable to secrete insulin when exposed to glucose, but whether GK is present and functional in this developing cell is unknown. To determine the expression of GK in human fetal pancreatic tissue, cytosolic protein was extracted from human fetal islet-like cell clusters (ICCs) at 17-19 weeks gestation and examined for protein content and enzyme activity. On Western blots, a single band corresponding to GK was seen at 52 kDa, and this was similar to that obtained from human adult islets. The maximal velocity (Vmax) of GK was less in fetal ICCs than that in adult islets (8.7 vs. 20.7 nmol/mg protein x h); similar K(m) values were found in both ICCs and islets. No attempt was made to determine which cells in an ICC contained GK. Glucose utilization was determined radiometrically; the Vmax of the high K(m) component was less in ICCs than in islets (31.3 pmol/ICC x h vs. 101.4 pmol/islet.h). Culture of ICCs for 3-7 days in medium containing 11.2 mmol/L glucose resulted in a 3.7-fold increase in the Vmax of GK and a 1.8-fold increase in glucose utilization. These enhanced activities of glucose phosphorylation and glycolysis, however, did not lead to the beta-cell being able to secrete insulin when exposed to glucose. In conclusion, glucokinase is present and functional in human fetal ICCs, but the inability of the human fetal beta-cell to secrete insulin in response to an acute glucose challenge is not due to immaturity of this enzyme.

Constitution of a biphasic insulin response to glucose in human fetal pancreatic beta-cells with glucagon-like peptide 1.

Insulin release from fetal beta-cells responds only minimally to acute glucose stimulation. Glucagon-like peptide-1 (GLP-1) is able to correct glucose sensitivity in some models of glucose-resistant beta-cells. We have now tested whether GLP-1 can induce glucose-responsive insulin release in human fetal islet-like cell clusters (ICCs). For this purpose we used perifusion and static incubation of ICCs and isolated adult human islets. In perifusion, the fetal ICCs responded with only minimal insulin release to 16.7 mmol/L glucose or 10 nmol/L GLP-1 combined with 1.67 mmol/L glucose. However, in the presence of GLP-1, the insulin response to glucose was markedly potentiated and appeared in a biphasic manner (10-fold first phase and 3-fold second phase). The first phase response was equal to that of adult human islets in similar experiments, whereas the second phase of the fetal cells was significantly lower. In static incubations, the relative insulin release responses to 16.7 mmol/L glucose plus 10 nmol/L GLP-1 were equal in the fetal and adult cells. The cAMP content of the cells was increased by glucose only in the presence of GLP-1. Our studies indicate that the glucoincretin hormone GLP-1 is able to constitute biphasic insulin release in the immature beta-cell, possibly as the result of cAMP-mediated priming of the glucose sensor mechanism.

Effect of Alloxan on the Endocrine Function of Human Fetal Islet-like Cell Clusters—An In Vitro Study

We studied the effects of alloxan on insulin and glucagon secretion, islet insulin content, and morphology of human fetal islet-like cell clusters (ICCs). ICCs were derived after collagenase digestion and culture of pancreata from two fetuses. Culture medium (RPMI 1640) containing either 2.0 (low) or 11.1 (high) mM glucose was used during the alloxan exposure. Alloxan exposure lasted for 5 min at room temperature, with final concentrations of 0.3, 1, 3, 10, 30 and 100 mM. Medium samples were collected for hormone assays on days 0, 1, 2, 3, 6, and 10 and islet insulin contents were measured on day 10 after alloxan treatment. Electron microscopy of ICCs was done 24 h after the drug exposure. Control ICCs steadily increased their insulin secretion during the whole study period. Alloxan concentrations above 0.3 mM significantly (p < 0.01) decreased insulin secretion at the low glucose concentration. High glucose protected beta cells from alloxan toxicity. There was no difference in islet insulin contents between alloxan-treated and control cultures. Glucagon secretion by glucose media was not affected by alloxan exposure. All islet cells including beta cells remained intact in electron microscopy. The results suggest a block in insulin secretion by alloxan, but beta cells appear to recover at least partly in their insulin-secreting capacity.

Ethanol Influences Class I and Class II MHC Antigen Expression on Human Fetal Islet‐Like Cell Clusters

Several autoimmune diseases have been linked to an aberrant expression of major histocompatibility complex (MHC) products Ethanol enhances Class I and Class II products on a variety of cell types, and there is evidence for an autoimmune etiology in numerous pathologies associated with alcoholism. We examined whether ethanol alters the expression of Class I and Class II MHC products on human fetal islet-like cell clusters. Incubation of islet-like clusters for 48 hr in ethanol at a starting concentration of 1.5% increased the percentage of single cells expressing Class I. The percentage of cells expressing Class II did not change, but their relative mean fluorescence increased significantly. These findings suggest that alcohol ingestion could alter MHC expression on pancreatic islet cells in vivo perhaps affecting the development of diabetes in genetically predisposed individuals. These findings also support the hypothesis that the rising incidence of type 1 diabetes seen in areas of the world where the per capita consumption of alcohol is also increasing may be a consequence of the immunological effects of alcohol intake.

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture.

The effects of recombinant human growth hormone (GH, 1 micrograms/ml) and insulin-like growth factor I (IGF-I, 200 ng/ml) on the production of insulin and glucagon by human fetal islet-like cell clusters (ICCs) were studied in tissue culture. ICCs were derived after collagenase digestion and culture of pancreases from 16 fetuses (mean gestational age 15.6 wk). The ICCs were cultured with or without GH or IGF-I for 7 or 31 days. Basal rates of insulin and glucagon production were not altered by GH during the first 17 days of culture, but the release of both hormones was increasingly augmented by GH during the last 2 wk of culture (131% increase in insulin and 85% in glucagon compared with controls). ICCs cultured for 7 days in the presence of GH secreted more insulin when incubated for 120 min in 20 mM than in 2 mM glucose (2.1-fold response, P less than .05), whereas ICCs maintained in basal medium did not respond to glucose. GH had no effect on DNA and insulin content or insulin biosynthesis. Exogenous IGF-I caused a 28% suppression of insulin release (P less than .05) between days 4 and 10 of culture but induced a 49% increase in the mean secretion rate during the last week (days 25-31, P less than .01). Glucagon release was not affected by exogenous IGF-I. In contrast to GH, exogenous IGF-I induced a twofold increase in the DNA content of the 7-day--cultured ICCs. However, insulin biosynthesis and release were markedly suppressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture

Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during long-term tissue culture

Insulin and glucagon secretory responses to arginine, glucagon, and theophylline during perifusion of human fetal islet-like cell clusters.

The secretory responsiveness of human fetal pancreatic endocrine cells was studied by perifusion of cultured islet-like cell clusters (ICC). ICC were obtained from 7 fetuses at 13-15 weeks gestation and 21 fetuses at 17-22 weeks gestation. The ICC were challenged with glucose (20 mmol/L), arginine (10 mmol/L), glucagon (1.4 mumol/L), and theophylline (10 mmol/L) combined with zero, low (2 mmol/L), or high (20 mmol/L) glucose. At 13-15 weeks, glucose and arginine enhanced insulin release in some experiments, whereas glucagon and theophylline were always potent stimuli (mean response, 4-fold regardless of the glucose concentration). At 17-22 weeks, both glucose alone (20 mmol/L) and arginine (10 mmol/L, with 2 mmol/L glucose) induced a small (1.4- to 1.5-fold) increase in insulin release. When arginine was combined with 20 mmol/L glucose, the response was potentiated to become a 2.3-fold increase. In contrast, glucagon was equally effective in 2 and 20 mmol/L glucose (2.9- and 2.6-fold responses, respectively) and produced a half-maximal response even in the absence of glucose. In this age range the most potent stimulus for insulin release was clearly theophylline. The effect of theophylline was also remarkably independent of the glucose concentration of the perifusate (5.6-, 8.1-, and 8.6-fold responses at 0, 2, and 20 mmol/L glucose, respectively). Glucagon release from the ICC of the 17- to 22-week-old fetuses was low (mean basal glucagon release, 2.9; insulin, 24.8 fmol/100 ICC/min). Glucagon release was not affected by 20 mmol/L glucose, but was stimulated by arginine and theophylline. These findings suggest that in the fetal pancreas, in contrast to the adult organ, insulin release results from elevation of intracellular cAMP concentrations (by glucagon or theophylline) relatively independent of the exogenous glucose concentration. Therefore, glucagon may have an important role in regulating insulin release during the early development of human fetal B-cells.