Subpopulation Of Beta-cells Research Articles

Dynamic pacing of cell metabolism by intracellular Ca2+ transients.

During cell activation, Ca2+, by stimulating the NADH-producing mitochondrial dehydrogenases, triggers the generation of reducing equivalents whereby ATP production is sustained. In cell populations, [Ca2+] changes in the mitochondrial matrix were demonstrated to parallel rapidly those in the cytosol ([Ca2+]i). There is still no indication as to whether metabolic activation follows oscillatory patterns similar to those of [Ca2+]i. Therefore, changes in NAD(P)H were monitored in single pancreatic beta-cells, adrenal glomerulosa cells, and liver cells during oscillatory [Ca2+]i transients. Rapid NAD(P)H and [Ca2+]i oscillations with similar frequency and sensitive both to changes in glucose concentration and to extracellular Ca2+ removal were identified in a subpopulation of pancreatic beta-cells in primary culture. Furthermore, Ca(2+)-dependent oscillatory NAD(P)H formation could be evoked by the pulsatile application of depolarizing [K+], demonstrating the pacing effect of increased [Ca2+]i on beta-cell metabolism. In adrenal glomerulosa cells, angiotensin II, a physiological stimulator of aldosterone production, could be shown to elicit the oscillatory formation of mitochondrial NAD(P)H through frequency modulation of [Ca2+]i transients. In contrast to the two former endocrine cell types, in hepatocytes, [Arg8]vasopressin and epinephrine caused the amplitude modulation of NAD(P)H formation. Taken together, these results provide unprecedented evidence for a cell-specific pacing of metabolism by [Ca2+]i transients coordinated with cell activation and function.

Amino-Acid Responsiveness in Beta Cell Subpopulations with Different Sensitivity to Glucose

Isolated rat β-cells differ in their individual responsiveness to glucose. The present study examines whether two β-cell subpopulations with different thresholds for glucose stimulation also differ in their responsiveness to amino acids that are known to stimulate insulin release. The subpopulations were separated by autofluorescence-activated cell sorting using their metabolic responsiveness to 7.5 mM glucose as discriminating parameter. The 7.5 mM glucose responsive and unresponsive subpopulations were perifused in parallel in order to compare their secretory responses to leucine (10 mM) or to arginine (5 mM); responses to glucose were taken as control. Under maximal glucose stimulation (20 mM), the responsive subpopulation released two-fold more insulin than the unresponsive one whereas maximal stimulation with leucine (10 mM) elicited similar first and second phase responses in the two subpopulations. On the other hand, a maximal arginine stimulus (5 mM) amplified release only from glucose-activated β-cells; neither did it correct the differences in glucose-induced insulin release between both subpopulations. These results indicate that rat β-cells exhibit a heterogeneity in secretory responsiveness to glucose but not to leucine, a metabolized secretagogue which can induce release in the absence of glucose. A heterogeneity is also observed in the cellular responsiveness to arginine, and its secretory effect consists of an amplification of glucose-activated cells.

Preservation of glucose-responsive islet beta-cells during serum-free culture.

This study describes a serum-free medium in which adult rat islet beta-cells can be cultured in suspension for at least 9 days without a detectable loss in cell number or function. The medium is composed of Ham's F-10 with 10 mM glucose, 1% BSA, and 50 microM isobutylmethylxanthine. After 9 days of culture, beta-cell aggregates had preserved their initial DNA content, with more than 80% ultrastructurally intact cells. Their rates of glucose-inducible insulin synthesis (64 +/- 13 fmol/10(3) cells.2 h) and release (173 +/- 44 fmol/10(3) cells.2 h) were comparable to those previously determined in overnight cultured beta-cells. Their secretory response to 20 mM glucose plus 10(-8) M glucagon was biphasic and 10-fold elevated above the basal level. Their secretory and biosynthetic activities at basal (1.25 mM) glucose levels were significantly higher than after culture with serum. These elevated basal activities are attributed to a rise in the proportion of beta-cells with high content in pale secretory granules. Supplementing the serum-free medium with GH (1 micrograms/ml) plus glucagon (10(-8) M) further increased basal activities, leading to cellular degranulation and reduced hormone release after stimulation. Control cultures in Ham's F-10 with 10 mM glucose and 10% fetal calf serum reduced the initial DNA content by 40% and, consequently, the total amount of hormone synthesis and release. Surviving cells exhibited a lower secretory responsiveness than those recovered from serum-free medium; their lower basal activities coincided with an absence of cells with high content in pale granules. It is concluded that preservation of glucose-responsive beta-cells during suspension culture requires conditions that keep the cells recruited into glucose-dependent functions. Such a condition is achieved by the presently defined serum-free medium. It is characterized by the presence of a subpopulation of beta-cells with a high proportion of pale secretory granules.

Preservation of glucose-responsive islet beta-cells during serum-free culture

This study describes a serum-free medium in which adult rat islet beta-cells can be cultured in suspension for at least 9 days without a detectable loss in cell number or function. The medium is composed of Ham's F-10 with 10 mM glucose, 1% BSA, and 50 microM isobutylmethylxanthine. After 9 days of culture, beta-cell aggregates had preserved their initial DNA content, with more than 80% ultrastructurally intact cells. Their rates of glucose-inducible insulin synthesis (64 +/- 13 fmol/10(3) cells.2 h) and release (173 +/- 44 fmol/10(3) cells.2 h) were comparable to those previously determined in overnight cultured beta-cells. Their secretory response to 20 mM glucose plus 10(-8) M glucagon was biphasic and 10-fold elevated above the basal level. Their secretory and biosynthetic activities at basal (1.25 mM) glucose levels were significantly higher than after culture with serum. These elevated basal activities are attributed to a rise in the proportion of beta-cells with high content in pale secretory granules. Supplementing the serum-free medium with GH (1 micrograms/ml) plus glucagon (10(-8) M) further increased basal activities, leading to cellular degranulation and reduced hormone release after stimulation. Control cultures in Ham's F-10 with 10 mM glucose and 10% fetal calf serum reduced the initial DNA content by 40% and, consequently, the total amount of hormone synthesis and release. Surviving cells exhibited a lower secretory responsiveness than those recovered from serum-free medium; their lower basal activities coincided with an absence of cells with high content in pale granules. It is concluded that preservation of glucose-responsive beta-cells during suspension culture requires conditions that keep the cells recruited into glucose-dependent functions. Such a condition is achieved by the presently defined serum-free medium. It is characterized by the presence of a subpopulation of beta-cells with a high proportion of pale secretory granules.

Heterogeneity in glucose sensitivity among pancreatic beta-cells is correlated to differences in glucose phosphorylation rather than glucose transport.

Rat beta-cells differ in their individual rates of glucose-induced insulin biosynthesis and release. This functional heterogeneity has been correlated with intercellular differences in metabolic redox responsiveness to glucose. The present study compares glucose metabolism in two beta-cell subpopulations that have been separated on the basis of the presence (high responsive) or absence (low responsive) of a metabolic redox shift at 7.5 mM glucose. Mean rates of glucose utilization and glucose oxidation in high responsive beta-cells were 2- to 4-fold higher than in low responsive beta-cells, whereas their leucine and glutamine oxidation was only 10-50% higher. This heterogeneity in glucose metabolism cannot be attributed to differences in GLUT2 mRNA levels or in glucose transport. In both cell subpopulations, the rates of glucose transport (13-19 pmol/min/10(3) beta-cells) were at least 50-fold higher than corresponding rates of glucose utilization. On the other hand, rates of glucose phosphorylation (0.3-0.7 pmol/min/10(3) beta-cells) ranged within those of total glucose utilization (0.2-0.4 pmol/min/10(3) beta-cells). High responsive beta-cells exhibited a 60% higher glucokinase activity than low responsive beta-cells and their glucokinase mRNA level was 100% higher. Furthermore, glucose phosphorylation via low Km hexokinase was detected only in the high responsive beta-cell subpopulation. Heterogeneity in glucose sensitivity among pancreatic beta-cells can therefore be explained by intercellular differences in glucose phosphorylation rather than in glucose transport.

Fetal antigen 1 (FA1) in the human pancreas: cell type expression, topological and quantitative variations during development.

Monospecific rabbit anti-human fetal antigen 1 (FA1), was used to examine the distribution of FA1 during the development of the human fetal pancreas and liver using an indirect immunoperoxidase technique. FA1 was expressed by 94% of the glandular epithelial cells of the branching ducts in the pancreatic anlage at week 7 of gestation. This pattern changed during the development of the human pancreas, 64% of the glandular cells being FA1 positive at week 17 of gestation, decreasing to 11% in the infant (4 months after birth). In the infant and adults the FA1 expression was restricted to a subpopulation of beta-cells within the islets of Langerhans. Insulin immunoreactive cells were scattered throughout the epithelium of primitive branching pancreatic ducts at week 7 of gestation, well before the formation of islets. From the 7th through to the 17th week of gestation, FA1 was found in the cytoplasm of fetal hepatocytes, whereas no staining was observed in the liver from a 4-month-old infant. No FA1 expression was found in the epithelium of the developing gut. The present findings indicate that the glandular epithelial cells in the developing pancreas may serve as stem cells, which, if appropriately induced, may differentiate into endocrine cells. Fetal antigen 1 (FA1) may take part in or be a result of this differentiation.

Heterogeneity in pancreatic beta-cell population.

All pancreatic beta-cells are identified by specific morphological characteristics. Similarity in microscopic features is not necessarily associated with identity in functional properties. In vitro studies on isolated rat beta-cells have indicated intercellular differences in the threshold for glucose-induced shifts in metabolic redox state. The cellular heterogeneity in glucose sensitivity results in a dose-dependent recruitment of glucose-exposed beta-cells into biosynthetic and secretory activities. The molecular basis of this diversity is not known. Indirect evidence supports the concept that the in situ pancreatic beta-cell population is also composed of functionally diverse subpopulations. The heterogeneity in glucose responsiveness is expected to create subpopulations of beta-cells with either constant, fluctuating, or occasional glucose-dependent functions; whether any subpopulation is preferentially responsive to other regulatory factors and/or committed to other activities is unknown. Morphological markers may help identify beta-cell subpopulations in situ and quantify their size in conditions known to affect total beta-cell mass or function. The concept of a functionally heterogeneous beta-cell population influences views on the role of pancreatic beta-cells in health and disease.