Gordon C. Weir

Induced pluripotent stem cells generated without viral integration.

Pluripotent stem cells have been generated from mouse and human somatic cells by viral expression of the transcription factors Oct4, Sox2, Klf4, and c-Myc. A major limitation of this technology is the use of potentially harmful genome-integrating viruses. We generated mouse induced pluripotent stem (iPS) cells from fibroblasts and liver cells by using nonintegrating adenoviruses transiently expressing Oct4, Sox2, Klf4, and c-Myc. These adenoviral iPS (adeno-iPS) cells show DNA demethylation characteristic of reprogrammed cells, express endogenous pluripotency genes, form teratomas, and contribute to multiple tissues, including the germ line, in chimeric mice. Our results provide strong evidence that insertional mutagenesis is not required for in vitro reprogramming. Adenoviral reprogramming may provide an improved method for generating and studying patient-specific stem cells and for comparing embryonic stem cells and iPS cells.

Insulinotropin: glucagon-like peptide I (7-37) co-encoded in the glucagon gene is a potent stimulator of insulin release in the perfused rat pancreas.

Insulin secretion is controlled by a complex set of factors that include not only glucose but amino acids, catecholamines, and intestinal hormones. We report that a novel glucagon-like peptide, co-encoded with glucagon in the glucagon gene is a potent insulinotropic factor. The glucagon gene encodes a proglucagon that contains in its sequence glucagon and additional glucagon-like peptides (GLPs). These GLPs are liberated from proglucagon in both the pancreas and intestines. GLP-I exists in at least two forms: 37 amino acids GLP-I(1-37), and 31 amino acids, GLP-I(7-37). We studied the effects of synthetic GLP-Is on insulin secretion in the isolated perfused rat pancreas. In the presence of 6.6 mM glucose, GLP-I(7-37) is a potent stimulator of insulin secretion at concentrations as low as 5 X 10(-11) M (3- to 10-fold increases over basal). GLP-I(1-37) had no effect on insulin secretion even at concentrations as high as 5 X 10(-7) M. The earlier demonstration of specific liberation of GLP-I(7-37) in the intestine and pancreas, and the magnitude of the insulinotropic effect at such low concentrations, suggest that GLP-I(7-37) participates in the physiological regulation of insulin secretion.

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.

New sources of pancreatic |[beta]|-cells

Two major initiatives are under way to correct the β-cell deficit of diabetes: one would generate β-cells ex vivo that are suitable for transplantation, and the second would stimulate regeneration of β-cells in the pancreas. Studies of ex vivo expansion suggest that β-cells have a potential for dedifferentiation, expansion, and redifferentiation. Work with mouse and human embryonic stem (ES) cells has not yet produced cells with the phenotype of true β-cells, but there has been recent progress in directing ES cells to endoderm. Putative islet stem/progenitor cells have been identified in mouse pancreas, and formation of new β-cells from duct, acinar and liver cells is an active area of investigation. Peptides, including glucagon-like peptide-1/exendin-4 and the combination of epidermal growth factor and gastrin, can stimulate regeneration of β-cells in vivo. Recent progress in the search for new sources of β-cells has opened promising new opportunities and spawned clinical trials.

β-Cell Dysfunction Induced by Chronic Hyperglycemia: Current Ideas on Mechanism of Impaired Glucose-Induced Insulin Secretion

Non-insulin-dependent diabetes mellitus is characterized by abnormal β-cell function. The characteristic secretory defect is a selective loss of glucose-induced insulin secretion. Substantial data have been generated in animal models to support the concept that chronic hyperglycemia causes the loss of glucorecognition (the so-called glucose toxicity hypothesis). This review summarizes the data supporting the concept of hyperglycemia-induced β-cell dysfunction and then focuses on the ideas for the mechanism of the glucose unresponsiveness. The lack of access to islet tissue in humans means that these studies have all been conducted in animal models. Another major stumbling block continues to be the lack of in vitro systems that faithfully reproduce the secretory abnormalities that occur in vivo. Despite these limitations, many hypotheses are being investigated that span most of the major intracellular steps for glucose-induced insulin secretion, including abnormalities in glucose transport, storage, metabolism/oxidation, and the second messengers. No single hypothesis stands out as being able to explain all of the characteristics of the secretory abnormalities. In the last few years major advances have occurred in our knowledge about the events that normally cause glucose-induced insulin secretion. Similarly, biochemical and molecular tools have become available to probe the different steps. As better in vitro models of the selective glucose unresponsiveness become available, rapid progress can be expected in unraveling the biochemical basis for the loss of glucose responsiveness in diabetic rat models. The long-term hope is that this information will lead to innovative new strategies for the therapy of non-insulin-dependent diabetes mellitus.

Responses of Neonatal Rat Islets to Streptozotocin: Limited B-Cell Regeneration and Hyperglycemia

Streptozotocin (SZ) was given to 2-day-old neonatal rats, and, during their subsequent development, the interrelationships between plasma glucose, plasma insulin, pancreatic islet morphology, and hormone content were examined. At 4 days of age, a peak of hyperglycemia was observed (SZ, 349 ± 8 mg/dl versus control (C), 127 ± 2) that was associated with a marked reduction of B-cell numbers (SZ, 26.5 ± 2.6% B-cell per islet versus C, 72.8 ± 0.8%). By 10 days of age the SZ animals became normoglycemic with partial recovery of the B-cell number (SZ, 39.6 ± 2.1% versus C, 64.0 ± 2.6%). By 6 weeks hyperglycemia returned (SZ, 345 ± 5.2 mg/dl versus C, 171 ± 6.2) with B-cell number of the SZ being 72% of the C (SZ, 48.8 ± 2.4% versus C, 67.5 ± 1.5%). This hyperglycemia and reduced B-cell number persisted to at least 13 wk age. Despite a marked reduction of pancreatic insulin content observed during development, there was little effect upon glucagon or somatostatin content. At 6 wk of age, the plasma insulin concentration was only 30% of C, which suggests an insulin secretory defect beyond that which could be accounted for by the modest B-cell reduction. The present study indicates that even though active regeneration of B-celle occurred after early injury, the capacity for ultimate normalization was limited. The resultant moderate reduction in B-cell number may be associated with a functional defect in glucose-stimulated insulin secretion.

Involvement of c-Jun N-terminal Kinase in Oxidative Stress-mediated Suppression of Insulin Gene Expression

Oxidative stress, which is found in pancreatic β-cells in the diabetic state, suppresses insulin gene transcription and secretion, but the signaling pathways involved in the β-cell dysfunction induced by oxidative stress remain unknown. In this study, subjecting rat islets to oxidative stress activates JNK, p38 MAPK, and protein kinase C, preceding the decrease of insulin gene expression. Adenovirus-mediated overexpression of dominant-negative type (DN) JNK, but not the p38 MAPK inhibitor SB203580 nor the protein kinase C inhibitor GF109203X, protected insulin gene expression and secretion from oxidative stress. Moreover, wild type JNK overexpression suppressed both insulin gene expression and secretion. These results were correlated with changes in the binding of the important transcription factor PDX-1 to the insulin promoter; adenoviral overexpression of DN-JNK preserved PDX-1 DNA binding activity in the face of oxidative stress, whereas wild type JNK overexpression decreased PDX-1 DNA binding activity. Furthermore, to examine whether suppression of the JNK pathway can protect β-cells from the toxic effects of hyperglycemia, rat islets were infected with DN-JNK expressing adenovirus or control adenovirus and transplanted under renal capsules of streptozotocin-induced diabetic nude mice. In mice receiving DN-JNK overexpressing islets, insulin gene expression in islet grafts was preserved, and hyperglycemia was ameliorated compared with control mice. In conclusion, activation of JNK is involved in the reduction of insulin gene expression by oxidative stress, and suppression of the JNK pathway protects β-cells from oxidative stress.