M. Van De Winkel

Differences in glucose recognition by individual rat pancreatic B cells are associated with intercellular differences in glucose-induced biosynthetic activity

In vitro incubated rat islet B cells differ in their individual rates of protein synthesis. The number of cells in biosynthetic activity increases with the glucose concentration. Flow cytometric monitoring of the cellular redox states indicated that islet B cells differ in their individual metabolic responsiveness to glucose. A shift from basal to increased NAD(P)H fluorescence occurred for 18% of the cells at 1 mM glucose, for 43% at 5 mM, and for 70% at 20 mM. The functional significance of this metabolic heterogeneity was assessed by comparing protein synthesis in metabolically responsive and unresponsive subpopulations, shortly after their separation by autofluorescence-activated cell sorting. The glucose-sensitive subpopulation exhibited four- to fivefold higher rates of insulin synthesis during 60-min incubations at 2.5-10 mM glucose. Its higher biosynthetic activity was mainly caused by recruitment of cells into active synthesis and, to a lesser extent, by higher biosynthetic activity per recruited cell. Cells from the glucose-sensitive subpopulation were larger, and presented a threefold higher density of a pale secretory vesicle subtype, which is thought to contain unprocessed proinsulin. It is concluded that intercellular differences in metabolic responsiveness result in functional heterogeneity of the pancreatic B cell population.

Autofluorescence-activated cell sorting of pancreatic islet cells: Purification of insulin-containing B-cells according to glucose-induced changes in cellular redox state

Autofluorescence-activated cell sorting can be employed for the subfractionation of insulin-containing islet B-cells according to their responsiveness to their physiologic stimulus, glucose. The method utilizes a flow cytometric detection of the rapid variations in endogenous NAD (P) H - and FAD - fluorescence after exposure to 20 mM glucose. Under these conditions, a two-fold increase in NAD (P) H and a 40% decrease in FAD was observed in more than 75% of B-cells isolated from fed normal rats. The technique makes it possible to study the metabolic behaviour of the B-cell population in (physio)pathological conditions of impaired glucose-induced insulin release; the availability of functionally homogenous B-cell preparations facilitates studies on stimulus-secretion coupling. In view of the universal role of the cellular metabolic redox state in cell regulation, it is suggested that similar techniques can be developed for the metabolic analysis of other cell types and for their purification according to their responsiveness to specific stimuli.

Presence of islet amyloid polypeptide in rat islet B and D cells determines parallelism and dissociation between rat pancreatic islet amyloid polypeptide and insulin content

The islet amyloid polypeptide (IAPP) immunoreactivity of the adult rat pancreas is located in insulin-containing B cells as well as in somatostatin-containing D cells. In both cell types, the IAPP immunoreactivity is identical to rat synthetic IAPP in terms of its elution position after reversed phase HPLC and its binding to IAPP antibodies. The IAPP content per 10(6) B-cells is more than 100 fold lower than the corresponding insulin content, but comparable to the IAPP content of D cells. After induction of diabetes by streptozotocin, pancreatic IAPP seems predominantly located in somatostatin-containing cells. In normal rats, pancreatic insulin and IAPP content increase 20 fold from birth to 12 weeks of age; beyond week 12, the further rise in pancreatic insulin was not paralleled by an increase in IAPP content.

Cell surface antibodies in Type 1 (insulin-dependent) diabetic patients

SummaryA standardized method has been developed for the assay of cell surface antibodies in IgM- and IgG-fractions from human serum. Suspensions of adult rat islet B cells, islet non-B cells, and anterior pituitary cells were used as antigen source and a cell sorter as analyser of the immunoglobulin binding to individual cells. Assay conditions were selected wherein no surface antibodies were detected in 33 control subjects younger than 20 years. In 30% of Type 1 (insulin-dependent) diabetic patients, surface antibodies were measured with rat anterior pituitary cells as well as with rat islet B cells. Binding to pituitary cells occurred with IgM- and IgG-fractions and correlated positively with IgG binding to islet B cells. At onset of the disease, the prevalence of IgM-rat anterior pituitary cell surface antibodies was higher than that of IgG-rat anterior pituitary cell surface antibodies. Cell surface antibodies were also detected in first-degree relatives of Type 1 diabetic patients, but corresponded primarily to IgM-rat anterior pituitary cell surface antibodies. It is concluded that the development of Type 1 diabetes in subjects younger than 20 years is associated with the generation of both IgM and IgG cell surface antibodies. The IgM surface antibodies may result from stimulated production of polyreactive natural autoantibodies and could precede the switch to the formation of monoreactive IgG autoantibodies. The assay of IgM cell surface antibodies can be useful in studies on the sequence of immune events in diabetes and other autoimmune disease.

Death of the Pancreatic B-Cell

Insulin-dependent diabetes is the clinical definition for a condition of absolute insulin deficiency which leads to ketosis when no exogenous insulin is administered (Lefebvre, this volume). The syndrome was attributed to an insufficient number of pancreatic B-cells after it became clear that newly diagnosed cases contained at least a five-fold smaller B-cell mass than normal controls [1-5]. Little is known about the events which lead to such quantitative deficit. Clarification of the underlying mechanisms has been hindered by two major obstacles. The first is the inability to measure the pancreatic B-cell mass in vivo, which prevents the detection of the disease at an earlier stage and hence its follow-up in the light of extrapancreatic events. Secondly, it has been impossible so far to screen for agents which impair the growth or survival of the pancreatic B-cells or their precursor cells. During recent years, both issues have been investigated in man and in rodents with a genetic predisposition for insulin-dependent diabetes. It was shown that abnormal glucose tolerance curves can precede the onset of clinical diabetes and may thus serve as a more sensitive parameter for a reduced B-cell mass [6-8]. It remains nevertheless questionable whether this functional test can be employed as a reliable index for the actual number of pancreatic B-cells. Observations in recent-onset diabetic patients also suggested a heterogeneous origin of the disease, but succeeded only in exceptional cases in identifying an agent which was primarily responsible for the killing of B-cells [9-11].

Cellular Endogenous Fluorescence: A Basis for Preparing Subpopulations of Functionally Homogeneous Cells

Publisher Summary This chapter reviews the technique of autofluorescence-activated cell sorting and discusses its usefulness in recognizing and isolating cell preparations with different functional properties. Earlier studies demonstrated how the measurement of the cellular FAD and NAD(P)H fluorescent intensities allows the distinction and isolation of various types of pancreatic endocrine cells. Autofluorescence-activated cell sorting permitted the preparation of pure and single insulin-containing B cells, which were used to investigate the functional properties of this cell type without interference from other cell types. In the course of these studies, it was noticed that this endocrine cell population does not represent a homogeneous group of identical cells, but rather is composed of functionally diverse subpopulations. Several of these subpopulations have been recognized during flow cytometric analysis, whereby the cellular autofluorescent compounds FAD and NAD(P)H appeared again to be particularly powerful discriminators. The observation that a pure pancreatic B-cell population is composed of functionally diverse subpopulations underlines the need to purify cells according to their functional properties rather than according to their structural characteristics.

Separation of Pancreatic Islet Cells according to Functional Characteristics

Publisher Summary This chapter discusses the methods for the separation of pancreatic islet cells according to functional characteristics. The physical properties of cells, such as their size and density, have served as a basis for their isolation. The ability to isolate cells according to the functional characteristics would permit a more precise cellular analysis of the tissue function, and it could also provide the appropriate in vitro models for investigating the physiopathological basis of certain diseases. In the study of diabetes mellitus, it is not clear whether and how the various pancreatic islet cells cooperate in the generation of the in vivo insulin response. A separation of the various islet cell types and their further distribution into the functionally homogeneous subpopulations may provide an experimental approach to the questions. The autofluorescence-activated cell sorting permits the isolation of islet cells with similar size and density, but with a different metabolic responsiveness to their physiological regulator glucose. The chapter reviews the work on islet cells and addresses investigations involved in diabetes research. It also intends for cell biologists in search of ways to isolate cells according to functional criteria.