Susan Bonner-Weir

Disruption of IRS-2 causes type 2 diabetes in mice

Human type 2 diabetes is characterized by defects in both insulin action and insulin secretion. It has been difficult to identify a single molecular abnormality underlying these features. Insulin-receptor substrates (IRS proteins) may be involved in type 2 diabetes: they mediate pleiotropic signals initiated by receptors for insulin and other cytokines. Disruption of IRS-1 in mice retards growth, but diabetes does not develop because insulin secretion increases to compensate for the mild resistance to insulin,. Here we show that disruption of IRS-2 impairs both peripheral insulin signalling and pancreatic β-cell function. IRS-2-deficient mice show progressive deterioration of glucose homeostasis because of insulin resistance in the liver and skeletal muscle and a lack of β-cell compensation for this insulin resistance. Our results indicate that dysfunction of IRS-2 may contribute to the pathophysiology of human type 2 diabetes.

Translational control is required for the unfolded protein response and in vivo glucose homeostasis.

The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF2alpha) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2alpha phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER stress. Mice with a homozygous mutation at the eIF2alpha phosphorylation site (Ser51Ala) died within 18 hr after birth due to hypoglycemia associated with defective gluconeogenesis. In addition, homozygous mutant embryos and neonates displayed a deficiency in pancreatic beta cells. The results demonstrate that regulation of translation through eIF2alpha phosphorylation is essential for the ER stress response and in vivo glucose homeostasis.

p16INK4a induces an age-dependent decline in islet regenerative potential.

The p16INK4a tumour suppressor accumulates in many tissues as a function of advancing age. p16INK4a is an effector of senescence and a potent inhibitor of the proliferative kinase Cdk4 (ref. 6), which is essential for pancreatic β-cell proliferation in adult mammals. Here we show that p16INK4a constrains islet proliferation and regeneration in an age-dependent manner. Expression of the p16INK4a transcript is enriched in purified islets compared with the exocrine pancreas, and islet-specific expression of p16INK4a, but not other cyclin-dependent kinase inhibitors, increases markedly with ageing. To determine the physiological significance of p16INK4a accumulation on islet function, we assessed the impact of p16INK4a deficiency and overexpression with increasing age and in the regenerative response after exposure to a specific β-cell toxin. Transgenic mice that overexpress p16INK4a to a degree seen with ageing demonstrated decreased islet proliferation. Similarly, islet proliferation was unaffected by p16INK4a deficiency in young mice, but was relatively increased in p16INK4a-deficient old mice. Survival after toxin-mediated ablation of β-cells, which requires islet proliferation, declined with advancing age; however, mice lacking p16INK4a demonstrated enhanced islet proliferation and survival after β-cell ablation. These genetic data support the view that an age-induced increase of p16INK4a expression limits the regenerative capacity of β-cells with ageing.

A Second Pathway for Regeneration of Adult Exocrine and Endocrine Pancreas: A Possible Recapitulation of Embryonic Development

Substantial regeneration of both the endocrine and exocrine pancreas occurs after a 90% partial pancreatectomy in the young adult rat. We have reported previously that replication of preexisting islet and exocrine cells is enhanced 3- to 4-fold. Here, we report a second pathway of regeneration, that of proliferation and differentiation of precursor cells in the ductal epithelium. As shown with in vivo pulse labeling using 5-bromo-2′-deoxyuridine, an expansion of the ductal epithelium occurs. Proliferation is seen first in the common pancreatic duct and sequentially in smaller ducts of the ductal tree as focal areas of proliferation small ductules form. By 60 h after pancreatectomy, only these focal areas show heavy 5-bromo-2′-deoxyuridine staining. These proliferating ductules comprise 12.8% of the pancreatic volume at 3 days after pancreatectomy but are uncommon at 7 days after pancreatectomy. Coincident with the appearance and disappearance of these regions was a 3.5-fold increased growth of the pancreatic remnant compared with its equivalent of sham animals. These small ductules differentiate into new pancreatic islets and exocrine tissue, forming new lobules of pancreas that are indistinguishable from the preexisting ones. This second pathway of rapid regeneration recapitulates embryonic development in its pattern of ductal proliferation and subsequent differentiation. Furthermore, these studies provide evidence of the presence of precursor/stem cells in the adult pancreas.

Dynamics of beta-cell mass in the growing rat pancreas. Estimation with a simple mathematical model.

The growth and development of the endocrine pancreas has been studied for many years, but questions remain concerning the regulation of the mass of insulin-producing β-cells both in the normal growing pancreas and during the pathogenesis of diabetes. The homeostatic control of β-cell mass in both normal and pathophysiological conditions is based on the balance of cell proliferation, cell growth, and cell death. To gain insight into the relative contribution of each of these dynamic processes, we first mathematically analyzed the data available on the components involved in the maintenance of β-cell mass, including rates of replication, β-cell volume, and the β-cell mass itself, at various ages in normal Sprague-Dawley rats. Then these data were combined in a simple mass balance equation to construct a mathematical model of the dynamics of the β-cell mass in the normal growing rat pancreas. Such a model has allowed us to infer the contributions of fluxes that cannot be measured, i.e., neogenesis and cell death, to the known mass of β-cells. Another important contribution of this model is to raise unanswered questions concerning the control of the balance of cell death and cell renewal in the endocrine pancreas.

Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles.

NIDDM is a polygenic disease characterized by insulin resistance in muscle, fat, and liver, followed by a failure of pancreatic beta cells to adequately compensate for this resistance despite increased insulin secretion. Mice double heterozygous for null alleles in the insulin receptor and insulin receptor substrate-1 genes exhibit the expected approximately 50% reduction in expression of these two proteins, but a synergism at a level of insulin resistance with 5- to 50-fold elevated plasma insulin levels and comparable levels of beta cell hyperplasia. At 4-6 months of age, 40% of these double heterozygotes become overtly diabetic. This NIDDM mouse model in which diabetes arises in an age-dependent manner from the interaction between two genetically determined, subclinical defects in the insulin signaling cascade demonstrates the role of epistatic interactions in the pathogenesis of common diseases with non-Mendelian genetics.

Chronic hyperglycemia triggers loss of pancreatic beta cell differentiation in an animal model of diabetes.

Differentiated pancreatic β cells are unique in their ability to secrete insulin in response to a rise in plasma glucose. We have proposed that the unique constellation of genes they express may be lost in diabetes due to the deleterious effect of chronic hyperglycemia. To test this hypothesis, Sprague-Dawley rats were submitted to a 85–95% pancreatectomy or sham pancreatectomy. One week later, the animals developed mild to severe chronic hyperglycemia that was stable for the next 3 weeks, without significant alteration of plasma nonesterified fatty acid levels. Expression of many genes important for glucose-induced insulin release decreased progressively with increasing hyperglycemia, in parallel with a reduction of several islet transcription factors involved in β cell development and differentiation. In contrast, genes barely expressed in sham islets (lactate dehydrogenase A and hexokinase I) were markedly increased, in parallel with an increase in the transcription factor c-Myc, a potent stimulator of cell growth. These abnormalities were accompanied by β cell hypertrophy. Changes in gene expression were fully developed 2 weeks after pancreatectomy. Correction of blood glucose by phlorizin for the next 2 weeks normalized islet gene expression and β cell volume without affecting plasma nonesterified fatty acid levels, strongly suggesting that hyperglycemia triggers these abnormalities. In conclusion, chronic hyperglycemia leads to β cell hypertrophy and loss of β cell differentiation that is correlated with changes in c-Myc and other key transcription factors. A similar change in β cell differentiation could contribute to the profound derangement of insulin secretion in human diabetes.

Compensatory Growth of Pancreatic β-Cells in Adult Rats After Short-Term Glucose Infusion

The extent to which adult pancreatic β-cells can respond in vivo to a sustained glucose stimulus by increasing their mass through either hyperplasia or hypertrophy has remained unanswered. Therefore, we studied the in vivo effect of short-term (96-h) hyperglycemia on the growth of β-cells by infusing adult rats with 35 or 50% glucose or 0.45% saline. After 96 h of glucose infusion, the β-cell mass, quantified by point-counting morphometrics of immunoperoxidase-stained paraffin sections, showed a 50% increase (9.57 ± 0.87 mg, n = 5, 50% glucose infused; 9.50 ± 1.23, n = 7, 35% glucose infused; 6.15 ± 0.55, n = 6, 0.45% saline infused). This growth was selective for β-cells; the non-β-cell mass was unchanged. The mitotic index, measured by accumulated mitotic frequency after a 4-h colchicine treatment, increased fivefold in glucose-infused animals compared to saline-infused animals. This enhanced replication of β-cells provides evidence for increase in cell number or hyperplasia. In addition, hypertrophy of the β-cells was also quantified. Mean cell volume, determined from the mean cell cross-sectional area measured planimetrically from low-magnification electron micrographs, increased to 150% of control values after 96 h of 50% glucose infusion. Seven days after the 96-h infusion, in reversal experiments, the β-cell mass had not returned to saline-infused levels. In addition, the non-β-cell mass of glucose-infused animals had increased. The mitotic index of the β-cells of glucose-infused rats was, however, significantly lower than that of the saline controls, but the mean cell volume of the β-cells remained elevated. Thus, with a short-term in vivo stimulus, adultβ-cells have a far greater capacity to respond with compensatory growth by hyperplasia and hypertrophy than has been appreciated before. Even 7 days after discontinuation of the stimulus, β-cell mass remains elevated.

Vulnerability of Islets in the Immediate Posttransplantation Period: Dynamic Changes in Structure and Function

To learn more about islet vulnerability in the immediate posttransplant period, 400 syngeneic islets were transplanted under the kidney capsule of B6AF1 mice. Three groups of recipients were used: normal mice (normal), streptozotocin (STZ)-diabetic (diabetic), and STZ-diabetic kept hypo- or normoglycemic with insulin pellets (diabetic-normalized). Normoglycemia was achieved in all three groups 14 days after transplantation; however, in the diabetic and diabetic-normalized groups, blood glucose levels throughout the posttransplantation period were respectively higher and lower than in the normal group. Grafts were harvested 1, 3, 7, and 14 days after transplantation and analyzed for morphology, β-cell death, β-cell mass, insulin content, and insulin mRNA expression. In all groups, substantial damage in islet grafts was found on days 1 and 3 with apoptotic nuclei and necrotic cores; on day 3, β-cell death was significantly higher in the diabetic group than in the other groups. Tissue remodeling occurred in all groups with stable graft appearance on day 14; the actual β-cell mass of the grafts was lowest in the diabetic group. Graft insulin content decreased in all groups on day 1 and fell even further on days 3 and 7. Insulin mRNA levels of grafts retrieved from both the diabetic and diabetic-normalized group were lower than those from the normal group already by day 1 and remained lower on day 14. In conclusion, the first few days of islet transplantation, even under the most advantageous circumstances of excellent metabolic control, are characterized by dynamic changes, with substantial islet cell dysfunction and death followed by tissue remodeling and then stable engraftment.

Partial pancreatectomy in the rat and subsequent defect in glucose-induced insulin release.

To define the consequences of a known reduction of B cell mass in rats, 90% partial pancreatectomies were performed. For the 6 wk following surgery moderate hyperglycemia was maintained in the fed state but there were no differences in body weight nor plasma insulin concentrations compared with sham-pancreatectomized controls. 8-10 wk following surgery regeneration of the remnant was evident with remnant weight being 26%, B cell mass being 42%, and non-B cell mass being 47% of values found for control whole pancreas. There were comparable increases in the remnant content of insulin, glucagon, and somatostatin. Following meal challenges, intraperitoneal and intravenous glucose tolerance tests and intravenous arginine challenge given 6-7 wk after surgery, the insulin responses to glucose were blunted or absent but the responses following the meals or arginine were intact. Similarly, when the pancreatic remnant was perfused in vitro, insulin release after challenge with 300 mg/dl glucose was markedly reduced whereas intact responsiveness to 10 mM arginine was retained. These data suggest that the chronic stimulation of a reduced B cell mass can lead to a selective loss of glucose-induced insulin secretion.