Ingemar Swenne

The Role of Glucose in the In Vitro Regulation of Cell Cycle Kinetics and Proliferation of Fetal Pancreatic B-Cells

To study the cell cycle and regulation by glucose of Bcell proliferation, B-cell-rich pancreatic islets of rat fetuses (22 days gestational age) were maintained in tissue culture at various glucose concentrations. The proliferating islet cells were synchronized with hydroxyurea and their rate of progress through the cell cycle studied by pulse labeling with 3H-thymidine and after exposure to colchicine. The time for a full cell cycle was estimated to 14.9 h and could be subdivided into a G1 phase of a duration of 2.5 h, an S phase of 6.4 h, a G2 phase of 5.5 h, and an M phase of 0.5 h. Although glucose significantly stimulated B-cell proliferation, the progression of cells through the cell cycle was similar at different glucose concentrations. The experiments furthermore suggested that only a limited pool of islet cells was able to proliferate. Glucose seemed to stimulate B-cell proliferation by increasing the number of cells entering the cell cycle. From the cell cycle data it was possible to calculate the rate of formation of new B-cells, which ranged from 4.2%/24 h in 2.7 mM glucose to 10.4%/24 h in 16.7 mM. When the accumulation of mitotic figures during colchicine treatment was used as an alternative method for estimation of newly formed B-cells the cell birth rates were found to be 3.1 and 6.0%/24 h at 2.7 and 16.7 mM glucose, respectively. The notion that only a small fraction of B-cells takes part in proliferation would explain the limited regenerative capacity of this cell type. It remains to be established whether such a limitation is of significance in the development of maturity-onset diabetes.

Persistent Impairment of Insulin Secretory Response to Glucose in Adult Rats After Limited Period of Protein-Calorie Malnutrition Early in Life

The effect of a limited period of protein-calorie malnutrition in young rats on glucose tolerance, insulin secretory response to glucose, and tissue composition in the adult was studied. Three-week-old rats were weaned onto semisynthetic diets containing either 5% protein (low protein; LP) or 15% protein (control; C) and maintained for 3 wk on their respective diets. At 6 wk of age all rats were returned to a commercial rat chow diet (18% protein). Glucose tolerance, insulin secretory response to glucose, and the protein/DNA ratio in liver, skeletal muscle, heart, kidney, small intestine, and lung were investigated at 3, 6, and 12 wk of age. Rats receiving LP diet failed to gain weight, but growth resumed immediately when they were transferred to commercial rat chow. They did not, however, catch up with C rats. Glucose tolerance and insulin secretory response to glucose remained similar between 3 and 12 wk in C rats. In 6-wk-old LP rats, glucose tolerance was impaired, and the insulin secretory response to glucose was absent. At 12 wk of age the glucose tolerance of the LP rats had normalized, but the insulin secretory response was still blunted. In 6-wk-old LP rats there was an inhibition of the age-dependent increase in cell size, shown by lowered protein/DNA ratios in all tissues studied. This decrease in cell size persisted at 12 wk in liver, skeletal muscle, heart, and lung. We conclude that protein-calorie malnutrition early in life persistently impairs the insulin secretion. The persistently lowered protein/DNA ratios in many tissues may be related to this lowered capacity for insulin secretion. The individual could have an impaired ability to respond to diabetogenic and nutritional challenges, and it is thus possible that the early malnutrition may predispose for diabetes.

Growth Hormone Regulation of Somatomedin C/Insulin-Like Growth Factor I Production and DNA Replication in Fetal Rat Islets in Tissue Culture

The regulation of DNA replication by growth hormone and the production of somatomedin C/insulin-like growth factor I (SM-C/IGF-I) and insulin by fetal rat islets in culture has been studied. Islets were cultured for 3 days in medium containing 2.7 or 16.7 mM glucose, various concentrations of fetal calf serum (FCS), and 100-1000 ng/ml human growth hormone (GH). DNA replication was determined by incorporation of [3H]thymidine into islet DNA; SM-C/IGF-I and insulin secreted into the medium were measured by specific radioimmunoassays. Glucose caused a twofold stimulation of islet DNA replication in medium containing ≥ 1% FCS but failed to stimulate DNA replication at lower serum concentrations. In the presence of 16.7 mM glucose, GH (100–1000 ng/ml) stimulated DNA replication at all serum concentrations. In medium containing 2.7 mM glucose, GH was stimulatory only in the presence of 1% FCS. Somatomedin C/IGF-I release into the culture medium could be detected in all experimental groups. Glucose alone did not affect SM-C/IGF-I release, and in serum concentrations <0.1% FCS, GH also failed to increase the release of the peptide. In medium containing 1% FCS and 16.7 mM glucose, 100–1000 ng/ml GH caused a 50-100% increase in SM-C/IGF-I release into the medium. Addition of 100 ng/ml exogenous SM-C/IGF-I to medium containing 16.7 mM glucose and 0.1–1.0% FCS caused a twofold stimulation of the islet DNA replication. This effect could be abolished by the addition of an antibody to SM-C/IGF-I. This antibody also significantly reduced GH-stimulated DNA replication, although some stimulation remained in its presence. Glucose stimulated insulin secretion at all serum concentrations. However, with < 1% FCS in the medium, insulin secretion was markedly reduced and could not be stimulated by the addition of GH. In medium containing 1% FCS and 16.7 mM glucose, 1000 ng/ml GH augmented insulin release almost twofold. We conclude that GH, but not glucose, can stimulate islet cell DNA replication by inducing the local production of SM-C/IGF-I. Apparently, however, part of the stimulation of DNA replication by GH is independent of SM-C/IGF-I and may be related to a direct effect of GH on the islets. Whether the insulin production is augmented via the mediation of somatomedins or by a direct effect of GH remains to be resolved.

Effects of Aging on the Regenerative Capacity of the Pancreatic B-Cell of the Rat

Since an increased insulin resistance could be expected to lead to a compensatory B-cell hyperplasia, it may be speculated that human type II diabetes becomes manifest only in those individuals who are unable to respond to an increased insulin demand with a higher rate of B-cell proliferation. To test this hypothesis experimentally, the cell cycle and growth regulation of the pancreatic B cell has been studied in vitro. Islets of fetal, 1-wk, 3-wk, and 3-mo-old rats were isolated and maintained in tissue culture at different glucose concentrations. The proliferating islet cells were synchronized with hydroxyurea and their progression through the cell cycle studied by pulse labeling with 3H-thymidine. From the cell cycle data it was possible to calculate the rate of formation of new B-cells. The cell cycle of the B-cells was similar in all donor age groups. When the glucose concentration of the culture medium was raised from 2.7 to 16.7 mM, the rate of B-cell proliferation increased 2.5-fold in all age groups, but there was no further increase in proliferation at 33.3 mM. At each of the glucose concentrations tested the rate of B-cell formation decreased with increasing age of the donor. Results indicate that only a fraction of the islet cells are capable of entering the cell cycle and undergoing mitosis. This fraction composed about 10% of the fetal islet cells but was less than 3% in the adult islets. The small pool of proliferating cells in adult islets could explain why B-cell multiplication, although present in the aging rat, is insufficient to increase the insulin output to levels at which normal glucose tolerance is maintained. This forms an interesting parallel to the development of type II diabetes in man, in which an inherited low capacity for B-cell regeneration may predispose to the disease.

Effect of genetic background on the capacity for islet cell replication in mice

SummaryProliferation of islet cells may compensate for both an increased peripheral insulin resistance and islet cell destruction but the capacity for regeneration may be genetically determined. For the latter reason, glucose-stimulated islet cell replication was estimated in both inbred C57BL/6J (BL/6) and C57BL/KsJ (BL/Ks) mice. Islets isolated from both strains were exposed to high concentrations of glucose in vitro or in vivo for a prolonged time period. This was achieved either by culturing the islets free-floating in a high glucose concentration medium for 3 days or implanting the islets intrasplenically in insufficient numbers to cure alloxan-diabetic syngeneic recipients. In both strains high glucose concentration culture was found to increase the autoradiographic labelling index of the islets but the replicatory activity decreased with age. The proliferative rate of the islet cells of the BL/6 mice was about twice as high as that of the BL/Ks mice irrespective of age and glucose concentration. Likewise, the labelling index of intrasplenic BL/6 islets implanted into alloxan-diabetic mice was twice as high as that of the islets implanted into alloxan-diabetic BL/Ks mice. The replicatory activity of the latter islets did not differ statistically from that of islets implanted into non-diabetic control BL/Ks mice. No differences in the rates of proinsulin and total protein biosynthetic rates were observed between high glucose concentration-cultured islets of the two mouse strains. The present results indicate that the proliferative response of pancreatic islets to a prolonged glucose stimulation may be genetically determined. This may play a significant role in the development of different diabetic syndromes both in laboratory animals and man.

Effects of glucose and amino acids on the biosynthesis of DNA and insulin in fetal rat islets maintained in tissue culture.

Islet growth and insulin biosynthesis in fetal rat islets have been studied in a recently developed in vitro culture system. Collagenase-digested pancreases of 22-day-old rat fetuses were maintained for 5 days in culture medium RPMI 1640 (11.1 mM glucose) to allow degeneration of acinar tissue. Intact pure islets, composed of more than 90% B-cells, were then collected and subsequently treated for 3 days in culture with different concentrations of glucose and amino acids. Islets were then labeled for 24 h with 3H-thymidine (DNA synthesis) or for 2 h with 3H-phenylalanine (insulin/protein biosynthesis). The specificity of the incorporation of 3H-thymidine into B-cell DNA has been investigated and characterized by both Chromatographie and autoradiographic studies. Moreover, using a colchicine metaphase arrest technique, a relationship was demonstrated between incorporation of labeled thymidine and mitotic incidence in the B-cells. Incorporation of 3H-thymidine was significantly higher in the fetal than in the adult islets following treatment in all the different concentrations of glucose and amino acids studied. In the presence of 10% fetal calf serum, however, there was no increase in DNA synthesis in the fetal islets in response to high concentrations of glucose or amino acids. In media containing 2.5% fetal calf serum, there was a significant increase in the rates of DNA synthesis after addition of high concentrations of either glucose or amino acids. Fetal islets showed a marked insulin biosynthetic response to an acute glucose challenge. Both the basal and stimulated rates of insulin biosynthesis were increased after treatment in high concentrations of glucose but not after treatment with amino acids. The results suggest an important role of nutrients in the regulation of B-cell growth and insulin biosynthesis and also a dissociation between the regulatory mechanisms of these two processes.

Growth Pattern of Pancreatic Islets in Animals

Since the first edition of The Diabetic Pancreas, new facts and observations relevant to islet growth have accumulated rapidly in the literature. Most of this concerns the B cell, but much new information has also been obtained on the other types of islet cells.* This progress, no doubt, reflects the more general use of immunocytochemical staining methods for identification of different islet cell types in combination with morphometric methods for quantitation of islet cell volume and weight. In addition, the application of techniques for cell cycle analyses has made possible a more detailed characterization of the various phases of B-cell life cycle. A review of these latter techniques will be given below. Animal experiments have furthermore expanded the knowledge on the effects of nutrients, hormones, and drugs on B-cell replication and they have contributed significant information on changes in islet cell proliferation with age. New animal models for both type I and type II diabetes have evolved and the role of a possible deficiency of B-cell proliferation for the manifestation of these diabetic syndromes has been a matter of much concern. It is the purpose of the present chapter to review and discuss the literature within this broad frame with particular emphasis on data that have appeared since the publication of the first edition in 1977.1

Persistent reduction of pancreatic Beta-cell mass after a limited period of protein-energy malnutrition in the young rat

SummaryKwashiorkor, the human disease of protein-energy malnutrition, has been implicated in the aetiology of malnutrition-related diabetes mellitus, a form of diabetes not uncommon in developing countries. We have previously demonstrated that temporary protein-energy malnutrition in young rats causes a persisting impairment of insulin secretion. The present study investigates whether this secretory deficiency is accompanied by structural alterations of the endocrine pancreas. Three-week-old rats were weaned onto semi-synthetic diets containing either 15% or 5% protein and these diets were maintained for 3 weeks. From 6 weeks of age all rats were fed a commercial chow containing 18% protein. The endocrine pancreas was investigated by light and electron microscopic morphometry at 3, 6 and 12 weeks of age. In rats not subjected to protein-energy malnutrition there was a progressive increase, with age, of total pancreatic Beta-cell weight and individual Beta-cell size. In 6-week-old rats fed the low protein diet total pancreatic Beta-cell weight and individual Beta-cell size were diminished. In 12-week-old rats previously fed the low protein diet total Beta-cell weight remained lower compared to control rats. It is concluded that protein-energy malnutrition early in life may result in a diminished reserve for insulin production. This may predispose to glucose intolerance or even diabetes in situations with an increased insulin demand.

Streptozotocin, but not alloxan, induces DNA repair synthesis in mouse pancreatic islets in vitro

SummaryIn the present investigation, the abilities of streptozotocin and alloxan to induce DNA repair synthesis in isolated mouse pancreatic islets have been compared using an autoradiographic technique. Streptozotocin exposure in vitro induced a dose-dependent DNA repair synthesis, whereas no such effect was observed after alloxan treatment. The hydroxyl radical scavenger dimethyl urea and the poly(ADP-ribose) synthetase inhibitors nicotinamide and theophylline reduced the streptozotocin-induced DNA repair. The results suggest that the initial events in streptozotocin-induced B cell injury are DNA damage and repair and that alloxan exerts its major cytotoxic effect by a different mechanism.

Differential Effects of Age Versus Glycemic Stimulation on the Maturation of Insulin Stimulus-Secretion Coupling During Culture of Fetal Rat Islets

We have cultured islets from 21.5-day-old fetal rats for 1–7 days in RMP11640/10% fetal calf serum containing 2.8 or 11.1 mM glucose to evaluate the differential effects of age vis-à-vis glycemic stimulation on the maturation of selected components of stimulus-secretion coupling. After 1 day of culture in either media, acute stimulation with 3.0 mg/ml glucose during basal perifusion with 0.5 mg/ml glucose elicited only a small first phase of stimulated insulin secretion and no second phase. The acute exposure to 3.0 mg/ml glucose produced no change in the prevailing high rates of oxygen consumption (Q02) and caused only minor increments in phosphate efflux (i.e., peak values for phosphate flush of 126 ± 16% of baseline for islets that had been cultured in 11.1 mM glucose and 162 ± 30% for islets cultured in 2.8 mM glucose). After 7 days of culture in 11.1 mM glucose, acute stimulation with 3.0 mg/ml glucose increased QO2 (as in adult islets) and effected acute increases in the AT32P and GT32P content of prelabeled islets. The 3.0 mg/ml glucose also triggered phosphate flush to 599 ± 45% of baseline and elicited first as well as early second phases of stimulated insulin secretion that replicated the performance of adult islets. By contrast, after 7 days of culture in 2.8 mM glucose, similar acute exposures of fetal islets to 3.0 mg/ml glucose effected only a small first phase and a negligible second phase of stimulated insulin secretion despite the occurrence of the same increments in QO2 as after culture in 11.1 mM glucose, highly significant increases in AT32P and GT32P, and phosphate flushes that peaked at 306 ± 16% of basal values. Thus, the ontogeny of individual components of stimulus-secretion coupling may occur in asynchronous fashion and varyingly require glycemic stimulation in addition to aging per se. The capacities to augment efflux of orthophosphate, enhance respiration, and heighten nucleotide turnover in response to acute stimulation with glucose seem to mature in large measure in time-dependent fashion, whereas some chronic exposure to ambient glucose in excess of basal levels may be required to establish and/or maintain the other coupled components that underlie bi-phasic stimulated insulin release. However, we did not achieve full exocytotic maturation even after 7 days of culture with 11.1 mM glucose. When stimulatory perifusion of such islets was prolonged beyond 30 min, the adult-like second phase of stimulated insulin release began to diminish. The fall-off was not due to limitations in preformed islet stores of immunoreactive insulin and it was not prevented by supplementing the perifusates with secretory-enhancing amounts of theophylline. Our experiences indicate that controlled tissue culture of fetal islets may provide a useful approach for unmasking the relative interdependence and independence of individual steps in insulin stimulus-secretion coupling. They also underscore the multiplicity of potential sites through which arrest of functional maturation could result in faulty insulin release.