Pancreatic Islets Of Transgenic Mice Research Articles

Ectopic serotonin production in β-cell specific transgenic mice

Genetically modified mice have been widely used in the field of β-cell research. However, analysis of results gathered using genetically modified organisms should be interpreted carefully as the results may be confounded by several factors. Here, we showed the ectopic serotonin (5-HT) production in β-cells of RIP-CreMgn, MIP-GFP, and MIP-Cre/ERT mice. These mice contained a human growth hormone (hGH) cassette to enhance transgene expression and hGH expression and Stat5 phosphorylation were detected in pancreatic islets of these mice. The expression level of tryptophan hydroxylase 1 (Tph1) was upregulated in pancreatic islets of transgenic mice with an hGH cassette but not in transgenic mice without an hGH cassette. Ectopic 5-HT production was not observed in β-cell-specific prolactin receptor (Prlr) knockout mice or Stat5 knockout mice crossed with RIP-CreMgn. We further confirmed that 5-HT production in β-cells of several transgenic mice was induced by hGH expression followed by the activation of the Prlr-Stat5-Tph1 pathway. These findings indicate that results obtained using transgenic mice containing the hGH cassette should be interpreted with care.

The anti-inflammatory protein NUPR1 protects pancreatic islets from diabetogenic lipotoxic injury ex vivo

Background and aim: According to the so-called “nutrition overflow hypothesis”, type 2 diabetes results from nutrition-induced local tissue inflammation leading to insulin resistance, insulin secretory dysfunction and finally pancreatic β-cell loss. Free fatty acids (FFA) acting through toll-like receptors are key mediators of this lipotoxic inflammatory response. We previously demonstrated that the intracellular protein NUPR1 (p8) exerts potent anti-inflammatory and anti-diabetogenic effects in β-cells in vivo. Here we investigated the anti-lipotoxic potential of NUPR1 ex vivo in pancreatic islets of transgenic mice with β-cell-specific Nupr1 overexpression under the control of the rat insulin 1 promoter (Nupr1-Tg).

The anti-inflammatory protein p8 protects pancreatic islets from diabetogenic lipotoxic injury ex vivo

Background and aim: According to the so-called “nutrition overflow hypothesis”, type 2 diabetes results from nutrition-induced local tissue inflammation leading to insulin resistance, insulin secretory dysfunction and finally pancreatic beta cell loss. Free fatty acids acting through toll-like receptors are key mediators of this lipotoxic inflammatory response. We previously demonstrated that the intracellular protein p8 exerts potent anti-inflammatory and anti-diabetogenic effects in pancreatic beta cells in vivo. Here we investigated the anti-lipotoxic potential of p8 ex vivo in pancreatic islets of transgenic mice with beta cell-specific p8 overexpression.

Inhibition of human insulin gene transcription by peroxisome proliferator‐activated receptor γ and thiazolidinedione oral antidiabetic drugs

The transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is essential for glucose homeostasis. PPARgamma ligands reducing insulin levels in vivo are used as drugs to treat type 2 diabetes mellitus. Genes regulated by PPARgamma have been found in several tissues including insulin-producing pancreatic islet beta-cells. However, the role of PPARgamma at the insulin gene was unknown. Therefore, the effect of PPARgamma and PPARgamma ligands like rosiglitazone on insulin gene transcription was investigated. Reporter gene assays were used in the beta-cell line HIT and in primary mature pancreatic islets of transgenic mice. Mapping studies and internal mutations were carried out to locate PPARgamma-responsive promoter regions. Rosiglitazone caused a PPARgamma-dependent inhibition of insulin gene transcription in a beta-cell line. This inhibition was concentration-dependent and had an EC(50) similar to that for the activation of a reporter gene under the control of multimerized PPAR binding sites. Also in normal primary pancreatic islets of transgenic mice, known to express high levels of PPARgamma, rosiglitazone inhibited glucose-stimulated insulin gene transcription. Transactivation and mapping experiments suggest that, in contrast to the rat glucagon gene, the inhibition of the human insulin gene promoter by PPARgamma/rosiglitazone does not depend on promoter-bound Pax6 and is attributable to the proximal insulin gene promoter region around the transcription start site from -56 to +18. The human insulin gene represents a novel PPARgamma target that may contribute to the action of thiazolidinediones in type 2 diabetes mellitus.

Impact of Transgenic Overexpression of SH2-Containing Inositol 5′-Phosphatase 2 on Glucose Metabolism and Insulin Signaling in Mice

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a 5'-lipid phosphatase hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI(3,4,5)P(3) to PI(3,4)P(2) in the regulation of insulin signaling, and is shown to be increased in peripheral tissues of diabetic C57BL/KSJ-db/db mice. To clarify the impact of SHIP2 in the pathogenesis of insulin resistance with type 2 diabetes, we generated transgenic mice overexpressing SHIP2. The body weight of transgenic mice increased by 5.0% (P < 0.05) compared with control wild-type littermates on a normal chow diet, but not on a high-fat diet. Glucose tolerance and insulin sensitivity were mildly but significantly impaired in the transgenic mice only when maintained on the normal chow diet, as shown by 1.2-fold increase in glucose area under the curve over control levels at 9 months old. Insulin-induced phosphorylation of Akt was decreased in the SHIP2-overexpressing fat, skeletal muscle, and liver. In addition, the expression of hepatic mRNAs for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was increased, that for sterol regulatory element-binding protein 1 was unchanged, and that for glucokinase was decreased. Consistently, hepatic glycogen content was reduced in the 9-month-old transgenic mice. Structure and insulin content were histologically normal in the pancreatic islets of transgenic mice. These results indicate that increased abundance of SHIP2 in vivo contributes, at least in part, to the impairment of glucose metabolism and insulin sensitivity on a normal chow diet, possibly by attenuating peripheral insulin signaling and by altering hepatic gene expression for glucose homeostasis.

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

CD8 T cell ignorance or tolerance to islet antigens depends on antigen dose.

There are two major mechanisms reported to prevent the autoreactivity of islet-specific CD8(+) T cells: ignorance and tolerance. When ignorance is operative, naïve autoreactive CD8(+) T cells ignore islet antigens and recirculate without causing damage, unless activated by an external stimulus. In the case of tolerance, CD8(+) T cells are deleted. Which factor(s) contributes to each particular outcome was previously unknown. Here, we demonstrate that the concentration of self antigen determines which mechanism operates. When ovalbumin (OVA) was expressed at a relatively low concentration in the pancreatic islets of transgenic mice, there was no detectable cross-presentation, and the CD8(+) T cell compartment remained ignorant of OVA. In mice expressing higher doses of OVA, cross-presentation was detectable and led to peripheral deletion of OVA-specific CD8(+) T cells. When cross-presentation was prevented by reconstituting the bone marrow compartment with cells incapable of presenting OVA, deletional tolerance was converted to ignorance. Thus, the immune system uses two strategies to avoid CD8(+) T cell-mediated autoimmunity: for high dose antigens, it deletes autoreactive T cells, whereas for lower dose antigens, it relies on ignorance.

The effect of local production of cytokines in the pathogenesis of insulin-dependent diabetes mellitus.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease mediated by self-reactive T cells that induce inflammation and destruction of pancreatic islet beta cells. A widely held belief is that T helper lymphocytes carrying a type 1 inflammatory phenotype are the major players in generating IDDM. However, recent evidence shows that cytokines belonging to the Th2 pathway can also induce autoimmune diabetes. The expression of cytokines directly within the pancreatic islets of transgenic mice helped to characterize the modulatory effects that Th1 or Th2 cytokines play on T cell-mediated autoimmune responses and diabetogenesis. This review describes the new information that these transgenic models have provided in understanding the exceedingly complex cytokine network and its role in the pathogenesis of IDDM.

Expression of human islet amyloid polypeptide/amylin impairs insulin secretion in mouse pancreatic β cells

Non—insulin-dependent diabetes mellitus (NIDDM) is associated histopathologically with islet amyloid deposits of which a major component is islet amyloid polypeptide (IAPP)/amylin. We examined whether endogenous IAPP controls insulin secretion via a local effect within pancreatic islets and whether overexpression of this peptide contributes to pancreatic β-cell dysfunction in this disease. Transgenic mice expressing human IAPP in pancreatic β cells were used in this study. Human IAPP expression did not influence the mouse proinsulin mRNA level and insulin content. Glucose-induced insulin secretion was decreased in the isolated pancreatic islets of transgenic mice. MIN6, a glucose-responsive pancreatic β-cell line, was transfected with human IAPP cDNA by a lipofectin method. Human IAPP expression was confirmed by RNA blot and immunohistochemical analysis. In two transfectants expressing the largest amount of human IAPP, insulin secretion was increased in response to glucose stimulation; however, the magnitude of the insulin response in cells transfected with human IAPP was smaller than in control clones. Insulin content was not influenced by the expression. We conclude that endogenous IAPP inhibits insulin secretion via an autocrine effect within pancreatic islets, and that the impaired insulin secretion in this disease may be partly caused by overexpression of IAPP.

New perspectives in clinical oncology from angiogenesis research.

TUMOUR BURDEN AFFECTS TUMOUR ANGIOGENESIS SINCE THE early 197Os, the conventional wisdom in angiogenesis research was that the neovascularisation in a given tumour bed was an isolated event, unrelated to other turnours. In the past 2 years, it has been recognised that total tumour burden can affect angiogenesis in remote metastases. This recognition came about because of the demonstration by O’Reilly and associates [l] that a primary tumour could suppress angiogenesis in its metastases and that this would iead to inhibition of growth of the metastases. This process was found to be mediated by a novel endogenous angiogenesis inhibitor, angiostatin, generated by the primary tumour [ 11. Angiostatin is a 38 kD protein with homology to the first four kringle structures of plasminogen. It is a specific inhibitor of endothehal cell proliferation. Three converging lines of experimental work led up to O’Reilly’s discovery of angiostatin. The first was that, during the 198Os, my laboratory had been trying to understand how certain primary tumours could suppress the growth of their metastases. Since the turn of the century, clinicians and laboratory investigators have recognised that certain human and animal primary tumours could suppress the growth of their metastases [2-81. Furthermore, Prehn argued with compelling data that the rate of tumour growth was inversely proportional to total tumour burden, regardless of how the tumour was distributed, e.g. single tumour versus multiple implants or metastases [9, lo]. Oncologists have observed that very large tumours may grow very slowly and that ‘debulking’ of a large tumour mass may render the residual tumour more susceptible to cytotoxic chemotherapy. Many explanations have been offered to explain these phenomena, including mechanisms based on immunity (concomitant immunity), hormonal interactions and metabolic changes (accumulation of catabolites). The second line of experiments was a collaboration with Douglas Hanahan (of the University of California, San Francisco). We were studying the mechanism of the switch to the angiogenic phenotype by beta cell carcinomas arising de nowo in the pancreatic islets of transgenic mice [ 11, 121. Two angiogenic proteins, aFGF (acidic fibroblast growth factor) and VEGF (vascular endothelial growth factor) revealed similarly high levels of expression by these islets, before and after the angiogenic switch [13, 141. This provocative result forced us to think that the angiogenic switch could not be explained entirely by positive regulators of angiogenesis, but could also involve some role by negative angiogenic regulators. The third line of experiments was reported in 1989, from Noel Bouck’s laboratory at Northwestern University Medical School, Chicago, Illinois, U.S.A. 1151. Thrombospondin was identified as a negative regulator of angiogenesis that was downregulated during the switch to the angiogenic phenotype in transformed fibroblasts. Subsequently, thrombospondin was shown to be upregulated in human tumour cells by the restoration of ~53 tumour suppressor function, thus negating the angiogenesis inducers that the cells themselves produced as well as added bFGF, leading to blocked angiogenesis [ 161. It immediately occurred to us that a primary tumour elaborating both angiogenic stimulators and ‘left-over’ inhibitor(s) could generate accumulating levels of the putative inhibitor in the circulation, if such an inhibitor had a longer half-life in the circulation than the stimulator. Thus, when O’Reilly found that angiostatin accumulated in the serum and urine of tumour-bearing mice, but disappeared from serum and urine after removal of the tumour, this suggested a role for angiostatin as a mediator of the suppression of metastases by the primary tumour. It became clear that a primary tumour could influence the growth of a metastasis by inhibiting angiogenesis in the metastasis, without communicating directly with its tumour cells.

Overexpression of Parathyroid Hormone-related Protein in the Pancreatic Islets of Transgenic Mice Causes Islet Hyperplasia, Hyperinsulinemia, and Hypoglycemia

Overexpression of Parathyroid Hormone-related Protein in the Pancreatic Islets of Transgenic Mice Causes Islet Hyperplasia, Hyperinsulinemia, and Hypoglycemia

Overexpression of Parathyroid Hormone-related Protein in the Pancreatic Islets of Transgenic Mice Causes Islet Hyperplasia, Hyperinsulinemia, and Hypoglycemia

Parathyroid hormone-related protein (PTHrP) is produced by the pancreatic islet. It also has receptors on islet cells, suggesting that it may serve a paracrine or autocrine role within the islet. We have developed transgenic mice, which overexpress PTHrP in the islet through the use of the rat insulin II promoter (RIP). Glucose homeostasis in these mice is markedly abnormal; RIP-PTHrP mice are hypoglycemic in the postprandial and fasting states and display inappropriate hyperinsulinemia. At the end of a 24-hour fast, blood glucose values are 49 mg/dl in RIP-PTHrP mice, as compared to 77 mg/dl in normal littermates; insulin concentrations at this time are 6.3 and 3.9 ng/ml, respectively. Islet perifusion studies failed to demonstrate abnormalities in insulin secretion. In contrast, quantitative islet histomorphometry demonstrates that the total islet number and total islet mass are 2-fold higher in RIP-PTHrP mice than in their normal littermates. PTHrP very likely plays a normal physiologic role within the pancreatic islet. This role is most likely paracrine or autocrine. PTHrP appears to regulate insulin secretion either directly or indirectly, through developmental or growth effects on islet mass. PTHrP may have a role as an agent that enhances islet mass and/or enhances insulin secretion.

A 338-bp proximal fragment of the glucose transporter type 2 (GLUT2) promoter drives reporter gene expression in the pancreatic islets of transgenic mice

The high K m glucose transporter GLUT2 is a membrane protein expressed in tissues involved in maintaining glucose homeostasis, and in cells where glucose-sensing is necessary. In many experimental models of diabetes, GLUT2 gene expression is decreased in pancreatic β-cells, which could lead to a loss of glucose-induced insulin secretion. In order to identify factors involved in pancreatic β-cell specific expression of GLUT2, we have recently cloned the murine GLUT2 promoter and identified cis-elements within the 338-bp of the proximal promoter capable of binding islet-specific trans-acting factors. Furthermore, in transient transfection studies, this 338-bp fragment could efficiently drive the expression of the chloramphenicol acetyl transferase (CAT) gene in cell lines derived from the endocrine pancreas, but displayed no promoter activity in non-pancreatic cells. In this report, we tested the cell-specific expression of a CAT reporter gene driven by a short (338 bp) and a larger (1311 bp) fragment of the GLUT2 promoter in transgenic mice. We generated ten transgenic lines that integrated one of the constructs. CAT mRNA expression in transgenic tissues was assessed using the RNAse protection assay and the quantitative reverse transcribed polymerase chain reaction (RT-PCR). Overall CAT mRNA expression for both constructs was low compared to endogenous GLUT2 mRNA levels but the reporter transcript could be detected in all animals in the pancreatic islets and the liver, and in a few transgenic lines in the kidney and the small intestine. The CAT protein was also present in Langerhans islets and in the liver for both constructs by immunocytochemistry. These findings suggest that the proximal 338 bp of the murine GLUT2 promoter contain cis-elements required for the islet-specific expression of GLUT2.

Intra- and extracellular amyloid fibrils are formed in cultured pancreatic islets of transgenic mice expressing human islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is the constituent peptide of amyloid deposits found in the islets of non-insulin-dependent diabetic patients. Formation of islet amyloid is associated with a progressive destruction of insulin-producing beta cells. Factors responsible for the conversion of IAPP into insoluble amyloid fibrils are unknown. Both the amino acid sequence of human IAPP (hIAPP) and hypersecretion of hIAPP have been implicated as factors for amyloid fibril formation in man. We have generated transgenic mice using rat insulin promoter-hIAPP or rat IAPP (rIAPP) gene constructs. No fibrillar islet amyloid was detectable in vivo in these normoglycemic mice, although small amorphous perivascular accumulations of IAPP were observed in hIAPP mice only. To determine the effects of glucose on IAPP secretion and fibrillogenesis, pancreatic islets from transgenic and control mice were examined in vitro. Islet IAPP secretion and content were increased in transgenic islets compared with control islets. IAPP-immunoreactive fibrils were formed at both intra- and extracellular sites in isolated hIAPP islets cultured with glucose at 11.1 and 28 mM for only 7 days. At 28 mM glucose, fibrils were present in deep invaginations of beta cells as observed in non-insulin-dependent diabetic patients. No fibrils were present at low glucose concentrations in hIAPP islets or at any glucose concentration in rIAPP or control islets. Thus, glucose-induced expression and secretion of hIAPP in transgenic mouse islets can lead to formation of amyloid fibrils similar to that found in non-insulin-dependent diabetes mellitus.

Localisation of islet amyloid polypeptide (IAPP) in pancreatic islets of transgenic mice expressing the human or rat IAPP gene.

Localisation of islet amyloid polypeptide (IAPP) in pancreatic islets of transgenic mice expressing the human or rat IAPP gene.

Tolerance induced by physiological levels of secreted proteins in transgenic mice expressing human insulin.

We have used transgenic mice to study immune tolerance to autologous, non-MHC encoded proteins that are expressed at physiological levels in the circulation. The transgenic mice used in these studies express the human preproinsulin gene and synthesize human proinsulin. Human and mouse insulin are secreted from the pancreatic islets of transgenic mice in response to normal physiological stimuli, such as glucose. Our data demonstrate that the transgenic mice have acquired tolerance to human insulin. The repertoire of T cells specific for exogenous antigens is shaped by the acquired tolerance to autologous proteins since pork but not beef or sheep insulin is also nonimmunogenic in the transgenic mice. We also found that the transgenic mice were tolerant to human proinsulin, the intracellular precursor of insulin. Unresponsiveness to human proinsulin most likely results from tolerance of insulin-specific and proinsulin-specific T cells that recognize the secreted enzymatic cleavage products of proinsulin, insulin and C-peptide.