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Rat pancreatic beta and alpha 2 cells were purified by autofluorescence-activated cell sorting and used for electrophysiological patch clamp studies and measurements of the initial uptake of 45Ca. Both beta and alpha 2 cells were electrically active, the action potentials of the latter cells also were detected in the absence of glucose. Furthermore, alpha 2 cells differed from beta cells in lacking a glucose-sensitive K+ channel with a single conductance of 50-60 pS (in symmetric 140 mM K+ solutions). The rate of Ca2+ entry into the alpha 2 cells was slower than that into the beta cells, being equivalent to 0.2 mmol, kg-1 dry wt min-1. Whereas raising the glucose concentration to 20 mM significantly increased the amount of Ca2+ entering the beta cells, the sugar was without effect on Ca2+ entry into the alpha 2 cells.

Surface major histocompatibility complex (MHC) class I and class II expression by pancreatic islet cells is considered a local initiator or regulator of immune processes that can lead to diabetes. Locally released cytokines, in particular interferon-gamma, are known to stimulate MHC antigen expression by islet cells. The present study quantifies MHC expression in cultured pancreatic beta- and non-beta-cells from both rat and human organs. Interferon-gamma increased MHC class I expression in endocrine beta- and non-beta-cells as well as in pancreatic ductal cells. The cytokine induced a 6-fold increase in the MHC class I messenger ribonucleic acid levels in pancreatic beta-cells; this effect was 2-fold amplified in the presence of elevated glucose levels (20 mmol/L instead of 6 mmol/L). No MHC class II expression was observed in endocrine beta- or non-beta-cells; human, but not rat, ductal cells exhibited MHC class II expression that increased in the presence of interferon-gamma. These data indicate that the increase in beta-cell MHC class I expression described in the pancreata of diabetic patients may result from stimulated transcription after exposure to locally released interferon-gamma and/or to a hyperglycemic state. The association of human islets with ductal cells in which MHC class II expression is stimulated by interferon-gamma makes these cells potential participants in the autoimmune process in diabetes.

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.

Similar to other endocrine glands, the endocrine pancreas displays a characteristic topography of its constituent cells. The functional significance of this structural organization was examined by measuring the secretory activity of the B cell in rat islet cell preparations of different composition. Glucose released 30-fold more insulin from B cells lodged within intact islets as from purified single B cells; structurally coupled B cells and single B cells isolated with A cells or incubated with glucagon responded 4- and 2-fold, respectively, more effectively to glucose than single B cells alone. Glucose homeostasis is thus dependent not only on the number and integrity of the insulin-containing B cells but also on their interactions with neighboring B and non-B cells. This study provides direct support for the concept that the microanatomy of the islet creates the anatomical basis for functional cooperation between islet cells and hence for an appropriate glucose-induced insulin release.

Insulin-dependent diabetes develops when more than 90% of the insulin containing B cells are destroyed. The present study investigates whether the target B cells can counteract the damaging effects of cytotoxic substances. Purified islet cells were first exposed for 3-10 min to t-butylhydroperoxide, alloxan, streptozotocin, or B-cell surface antibodies plus complement, then cultured for 20 hr before the percent of dead cells was counted. t-Butylhydroperoxide destroyed all islet cell types whereas the three other agents exerted a dose-dependent toxicity upon islet B cells only. The survival of drug- and complement-treated cells varied with the culture conditions present between the initial cellular attack and the moment of cell death. For the four B-cell toxic agents tested, an increase in medium glucose following any of these treatments reduced the percent of dead cells. This protective effect was not observed with galactose or fructose, nor could it be induced in islet non-B cells; it was additive to the protective action glucose induced during preincubation of the cells prior to their exposure to certain cytotoxic agents such as alloxan. Nicotinamide also enhanced the survival of drug-treated B cells, irrespective of the damaging compound. The vitamin was most effective when applied immediately after the initial drug or complement treatment; it also protected islet non-B cells--in contrast to glucose. The present in vitro study has led to the recognition of defense mechanisms in pancreatic B cells. Physiologic compounds such as glucose and nicotinamide were found to stimulate islet B cells to counteract the damaging effects of B-cell toxic conditions. It is conceivable that the events involved in this protection are implicated in the pathogenesis and/or prevention of insulin-dependent diabetes.

Viable rat islet cells were used to detect islet cell surface antibodies (ICSA) in the sera of diabetic and control patients. ICSA were present in almost all recent-onset insulin-dependent diabetics younger than 30 yr (15/16); their incidence in other diabetics (6/22) was also higher than in normal controls (1/18) or in patients with autoimmune thyroiditis (1/12). The varying specificity of the ICSA for the different islet cell types led to the recognition of class I sera, whose ICSA bind exclusively to B cells, class II sera, binding only to A and pancreatic polypeptide (PP) cells and class III sera, reacting with the three islet cell types but not with D cells. Most recent-onset insulin-dependent diabetics younger than 30 contained class I-ICSA, which is consistent with an autoimmune basis of their disease and with an involvement of surface antibodies in the B cell destruction. The presence of class II ICSA in three older diabetics and in one normal control raises the question whether autoimmune reactions against A and PP cells exist and are associated with a distinct entity in islet disease. It is concluded that the autoimmune form of diabetes mellitus represents a heterogeneous group, in which ICSA-positive patients can be distinguished on the basis of their ICSA-binding to one or more islet cell types. Three techniques can be used for the further identification of circulating ICSA, namely binding experiments with purified A or B cells, electron microscopical analysis of ICSA-binding islet cells purified by fluorescence-activated cell sorting, and the immunocytochemical characterization of ICSA-positive cells.

A 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 beta cells, islet non-beta cells, and anterior pituitary cels 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 isle beta cells. Binding to pituitary cells occured with IgM- and IgG-fractions and correlated positively with IgG binding to islet beta 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 surfaca 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.

A standardized cell surface antibody assay was used to measure binding of circulating human immunoglobulins to rat or piglet splenocytes. In 100-fold diluted serum fractions, lymphocyte surface antibodies were detected in 30% of type 1 (insulin-dependent) diabetic patients under 20 years of age but in none of 33 control subjects. Binding occurred with T and B lymphocytes, appeared unrelated to Fc receptors or protein glycosylation and was not attributable to insulin or albumin antibodies. At clinical onset of the disease, the lymphocyte surface antibodies belonged primarily to the IgM-class. Their presence was positively correlated to that of IgM-pituitary cell surface antibodies and their absorption by anterior pituitary cells occurred as well as by splenocytes. Lymphocyte surface antibodies remained present during the first years of insulin, treatment. They were also detected in first degree relatives of lymphocyte surface antibody-positivie patients. It is unlikely that IgM-lymphocyte surface antibodies mark the destructive process in the pancreatic B cell population. They may, instead, express a stat of immune reactivity which precedes the formation of IgG-autantibodies and therefore be associated with an event in the development of diseases such as type 1 (insulin-dependent) diabetes.

Glucose caused a sustained and dose-related increase in the fructose 2,6-bisphosphate content of isolated pancreatic islets, as well as of purified pancreatic B-cells. With isolated B-cells, the glucose saturation curve was sigmoidal and superimposable on that obtained with hepatocytes isolated from unfed rats. However, the response to glucose was notably faster in purified B-cells than in isolated hepatocytes. In contrast again with the situation prevailing in the liver, glucagon failed to decrease significantly the concentration of fructose 2,6-bisphosphate in either islets or purified B-cells. It is proposed that, in the process of glucose-stimulated insulin secretion, an early increase in fructose 2,6-bisphosphate formation may, by causing activation of 6-phosphofructo-1-kinase, allow glycolysis to keep pace with the rate of glucose phosphorylation.

Islet cell surface antibodies (ICSA) have been detected in insulin-dependent diabetes (IDD). Current techniques for the detection of ICSA often suffer from a lack of standardization and from a poorly defined specificity. We have developed a standardized method to quantify binding of circulating IgM and IgG fractions with the surface of purified rat pancreatic B cells; parallel experiments on islet non-B cells serve as control for islet cell specificity. Using this procedure no abnormalities were detected in the binding of circulating IgM from 30 recent onset IDD to islet B or non-B cells, comparison being made with 30 healthy age-matched controls. On the other hand, the IgG fraction from IDD patients was found to bind selectively to islet B cells (p 0.01) while no binding was observed with IgG from the normal controls. It is concluded that the developed procedure allows the recognition, quantification and specification of autoimmune reactions against surface components of islet cells.