Islet Amyloid Polypeptide Gene Expression Research Articles

Glucose and forskolin regulate IAPP gene expression through different signal transduction pathways.

Molecular mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene expression remain unclear. In the present study, we investigated the effects of glucose and forskolin on IAPP gene regulation in the INS-1 islet beta-cell line. Both glucose and forskolin increased the level of expression of this gene, as measured by Northern blot analysis, and increased IAPP gene transcription in a time- and concentration-dependent manner, as demonstrated in a reporter gene assay. Although inhibition of protein kinase A activity with H-89 eliminated the effect of forskolin on this gene, the glucose effect was unaffected. This supported the predominant use of a protein kinase A-independent signaling pathway for glucose regulation of the IAPP gene. Electrophoretic mobility shift assay further indicated that glucose and forskolin regulated expression of this gene by targeting different elements of the promoter. Mutation of the cAMP regulatory element flanking the IAPP coding region resulted in the loss of most of the forskolin-stimulated IAPP gene promoter activity, whereas glucose-enhanced IAPP gene transcription was unaffected. These results demonstrate parallel and distinct regulatory pathways involved in glucose- and forskolin-induced IAPP gene expression in this model beta-cell system.

Glucose Regulates Islet Amyloid Polypeptide Gene Transcription in a PDX1- and Calcium-dependent Manner

Islet amyloid polypeptide (IAPP) and insulin are expressed in the beta-cells of the islets of Langerhans. They are co-secreted in response to changes in glucose concentration, and their mRNA levels are also regulated by glucose. The promoters of both genes share similar cis-acting sequence elements, and both bind the homeodomain transcription factor PDX1, which plays an important role in the regulation of the insulin promoter and insulin mRNA levels by glucose. Here we examine the role of PDX1 in the regulation of the human IAPP promoter by glucose. The experiments were facilitated by the availability of a human beta-cell line (NES2Y) that lacks PDX1. NES2Y cells also lack operational K(ATP) channels, resulting in a loss of control of calcium signaling. We have previously used these cells to show that glucose regulation of the insulin gene is dependent on PDX1, but not calcium. In the mouse beta-cell line Min6, glucose (16 mm) stimulated a 3.5-4-fold increase in the activity of a -222 to +450 IAPP promoter construct compared with values observed in 0.5 mm glucose. In NES2Y cells, glucose failed to stimulate transcriptional activation of the IAPP promoter. Overexpression of PDX1 in NES2Y cells failed to reinstate glucose-responsive control of the IAPP promoter. Glucose effects on the IAPP promoter were observed only in the presence of PDX1 when normal calcium signaling was restored by overexpression of the two K(ATP) channel subunits SUR1 and Kir6.2. The importance of calcium was further emphasized by an experiment in which glucose-stimulated IAPP promoter activity was inhibited by the calcium channel blocker verapamil (50 microm). Verapamil was further shown to inhibit the stimulatory effect of glucose on IAPP mRNA levels. These results demonstrate that like the insulin promoter, glucose regulation of the IAPP promoter is dependent on the activity of PDX1, but unlike the insulin promoter, it additionally requires the activity of another, as yet uncharacterized factor(s), the activity of which is calcium-dependent.

Identification of cis- and trans-Active Factors Regulating Human Islet Amyloid Polypeptide Gene Expression in Pancreatic β-Cells

Islet amyloid polypeptide is expressed almost exclusively in pancreatic beta- and delta-cells. Here we report that beta cell-specific expression of the human islet amyloid polypeptide gene is principally regulated by promoter proximal sequences. The sequences that control tissue-specific expression were mapped between nucleotides -2798 and +450 of the human islet amyloid polypeptide (IAPP) gene using transgenic mice. To localize the cis-acting elements involved in this response, we examined the effects of mutations within these sequences using transfected islet amyloid polypeptide promoter expression constructs in pancreatic beta cell lines. The sequences between -222 and +450 bp were found to be necessary for beta cell-specific expression. Linker-scanning mutations of the 5'-promoter proximal region defined several key distinct control elements, including a negative-acting element at -111/-102 base pairs (bp), positive-acting elements like the basic helix-loop-helix-like binding site at -138/-131 bp, and the three A/T-rich, homeobox-like sites at -172/-163, -154/-142, and -91/-84 bp. Mutations within any one of these elements eliminated transcriptional expression by the promoter. Gel mobility shift assays revealed that the PDX-1 homeobox factor, which is required for insulin gene transcription in beta cells, interacted specifically at the -154/-142- and -91/-84-bp sites. Since PDX-1 is highly enriched in beta and delta cells, these results suggest that this factor plays a principal role in defining islet beta cell- and delta cell-specific expression of the IAPP gene.

Glucose regulation of islet amyloid polypeptide gene expression in rat pancreatic islets

Intracellular pathways by which glucose regulates islet amyloid polypeptide (IAPP) gene expression in pancreatic islets were studied. IAPP mRNA levels were threefold higher in islets cultured with 16.7 mM glucose compared with control (5.5 mM glucose). Mannose and amino acids but not 2-deoxyglucose or 6-deoxyglucose mimicked the effect of glucose. Mannoheptulose (a glycolysis inhibitor) and verapamil and diazoxide (which affect calcium signaling pathway) abolished the difference in islet IAPP mRNA content between high and low glucose. At low glucose, IAPP mRNA levels were increased 1.9-fold in islets treated with forskolin or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) but not with 12-O-tetradecanoylphorbol 13-acetate. Insulin mRNA levels were 1.6-fold higher in islets cultured at high glucose than controls; glucose metabolism was required, whereas no effects of cAMP or diazoxide were observed. IAPP and insulin were cosecreted into the media. We conclude that glucose regulation of IAPP mRNA abundance requires intracellular metabolism of the hexose and that calcium may serve as a mediator of this effect; cAMP but not protein kinase C possibly participates in this regulation.

Signals related to glucose metabolism regulate islet amyloid polypeptide (IAPP) gene expression in human pancreatic islets

Intracellular pathways involved in glucose stimulation of IAPP gene expression were studied in human pancreatic islets. Glucose (16.7 mM), but not mannose, caused a 2.3-fold increase in IAPP mRNA levels; this effect was inhibited by actinomycin D. In the presence of the non-metabolizable 6-deoxyglucose (16.7 mM) IAPP mRNA levels were markedly depleted. Both mannoheptulose and verapamil blocked glucose-induced stimulation of the IAPP gene. The magnitude of the insulin gene response to glucose was smaller (1.3-fold); none of the above-mentioned agents had significant effects on insulin mRNA content. Tunicamycin elicited a 2.4- and 2.7-fold increase in IAPP mRNA levels in the low and high glucose media, respectively; however, it did not change insulin mRNA. It had no effect on rat IAPP or insulin mRNAs, either. We conclude that IAPP gene expression is regulated by signals derived from glucose metabolism and that intracellular calcium may be involved in this response. IAPP and insulin genes are not co-regulated in cultured human pancreatic islets.

Islet amyloid polypeptide and insulin gene expression are regulated in parallel by glucose in vivo in rats.

Islet amyloid polypeptide (IAPP) is a novel amyloid-forming beta-cell hormone with putative roles in glucose metabolism and non-insulin-dependent diabetes mellitus (NIDDM) pathogenesis. To study how IAPP and insulin expression are regulated by glucose, rats were fasted for 48h followed by administration of glucose at repeated 4-h intervals; IAPP and insulin mRNA levels were determined by quantitative in situ hybridization. Fasting markedly reduced IAPP and insulin mRNA levels. Two (6 h) and four (14 h) glucose injections dose dependently increased both mRNA levels; the effects were matched by similar changes in plasma glucose levels. Actinomycin D blocked the glucose-induced increase in IAPP expression. IAPP and insulin mRNA levels were significantly correlated over the range of glucose levels. The parallel regulation of IAPP and insulin gene expression by glucose is consistent with a role for IAPP in glucose homeostasis. Thus, under hyperglycemic conditions such as NIDDM, IAPP gene expression is likely to increase. Hence, IAPP could, by elevated local concentrations, contribute to amyloid formation and/or affect metabolism unfavorably by inhibition of insulin release and action.

Islet amyloid polypeptide gene expression in the endocrine pancreas of the rat: a combined in situ hybridization and immunocytochemical study.

The expression of the islet amyloid polypeptide (IAPP) gene within the endocrine pancreas and its correlation with insular neuroendocrine peptide localization were investigated in the rat. In situ hybridization with a 35S-labelled IAPP-mRNA specific oligonucleotide probe was combined with immunocytochemistry. In situ hybridization alone showed strong autoradiographic labelling of the pancreatic islets. In situ hybridization combined with immunocytochemistry for IAPP, revealed labelling of the IAPP-immunoreactive cells. However, when in situ hybridization was combined with immunocytochemistry for proinsulin, we noted a lack of proinsulin immunoreactivity in some peripherally located autoradiographically labelled islet cells. Furthermore, combination of in situ hybridization and immunocytochemistry for somatostatin showed autoradiographic labelling of somatostatin cells to a varying degree. This was further confirmed by showing cellular co-localization of IAPP and somatostatin by immunocytochemical double staining. We conclude that IAPP is mainly synthesized in insulin cells. Additionally, a subpopulation of the somatostatin cells is capable of IAPP synthesis. This may account for the relatively small reduction in the content of IAPP-mRNA in islets compared to the marked reduction of insulin mRNA after streptozotocin-induced diabetes in rats as previously reported.

The dissociation of tumor-induced weight loss from hypoglycemia in a transplantable pluripotent rat islet tumor results in the segregation of stable alpha- and beta-cell tumor phenotypes.

We previously established pluripotent transformed rat islet cell lines, MSL-cells, of which certain clones have been used to study processes of islet beta-cell maturation, including the transcriptional activation of the insulin gene induced by in vivo passage. Thus, successive sc transplantation in NEDH rats resulted in stable hypoglycemic insulinoma tumor lines, such as MSL-G2-IN. Occasionally, hypoglycemia as well as severe weight loss were observed in the early tumor passages of MSL-G and the subclone, NHI-5B, which carry the transfected neomycin and human insulin genes as unique clonal markers. By selective transplantation, it was possible to segregate stable anorectic normoglycemic tumor lines, MSL-G-AN and NHI-5B-AN, from both clones. These tumors cause an abrupt onset of anorexia when they reach a size of 400-500 mg (< 0.3% of total body weight), and the observed weight loss parallels that of starved rats until death results from cachexia. After tumor resection, animals immediately resume normal feeding behavior. Comparative studies of hormone release and mRNA content in anorectic lines, MSL-G-AN and NHI-5B-AN, vs. those in the insulinoma line, MSL-G2-IN, revealed selective glucagon gene expression in both of the anorectic tumors, whereas insulin and islet amyloid polypeptide gene expression were confined to the insulinoma. Both tumor phenotypes produced cholecystokinin and gastrin in variable small amounts, making it unlikely that these hormones contribute to the anorectic phenotype. Tumor necrosis factor (cachectin) was not produced by any of the tumors. Proglucagon was processed as in the fetal islet to products representative of both pancreatic alpha-cell and intestinal L-cell phenotypes, with glucagon and Glp-1 (7-36)amide as the major extractable products. In contrast to the administration of cholecystokinin, neither glucagon, Glp-1 (7-36)amide, nor their combination, affected feeding behavior in fasted mice, suggesting the presence of a hitherto unidentified anorectic substance released from the glucagonoma. We conclude 1) that glucagonomas and insulinomas can be derived from a common clonal origin of pluripotent MSL cells, thus supporting the existence of a cell lineage relationship between islet alpha- and beta-cell during ontogeny; and 2) that our glucagonomas release an anorexigenic substance(s) of unknown nature that causes a severe weight loss comparable to that reported in animals carrying tumor necrosis factor-producing experimental tumors.

The dissociation of tumor-induced weight loss from hypoglycemia in a transplantable pluripotent rat islet tumor results in the segregation of stable alpha- and beta-cell tumor phenotypes

We previously established pluripotent transformed rat islet cell lines, MSL-cells, of which certain clones have been used to study processes of islet beta-cell maturation, including the transcriptional activation of the insulin gene induced by in vivo passage. Thus, successive sc transplantation in NEDH rats resulted in stable hypoglycemic insulinoma tumor lines, such as MSL-G2-IN. Occasionally, hypoglycemia as well as severe weight loss were observed in the early tumor passages of MSL-G and the subclone, NHI-5B, which carry the transfected neomycin and human insulin genes as unique clonal markers. By selective transplantation, it was possible to segregate stable anorectic normoglycemic tumor lines, MSL-G-AN and NHI-5B-AN, from both clones. These tumors cause an abrupt onset of anorexia when they reach a size of 400-500 mg (< 0.3% of total body weight), and the observed weight loss parallels that of starved rats until death results from cachexia. After tumor resection, animals immediately resume normal feeding behavior. Comparative studies of hormone release and mRNA content in anorectic lines, MSL-G-AN and NHI-5B-AN, vs. those in the insulinoma line, MSL-G2-IN, revealed selective glucagon gene expression in both of the anorectic tumors, whereas insulin and islet amyloid polypeptide gene expression were confined to the insulinoma. Both tumor phenotypes produced cholecystokinin and gastrin in variable small amounts, making it unlikely that these hormones contribute to the anorectic phenotype. Tumor necrosis factor (cachectin) was not produced by any of the tumors. Proglucagon was processed as in the fetal islet to products representative of both pancreatic alpha-cell and intestinal L-cell phenotypes, with glucagon and Glp-1 (7-36)amide as the major extractable products. In contrast to the administration of cholecystokinin, neither glucagon, Glp-1 (7-36)amide, nor their combination, affected feeding behavior in fasted mice, suggesting the presence of a hitherto unidentified anorectic substance released from the glucagonoma. We conclude 1) that glucagonomas and insulinomas can be derived from a common clonal origin of pluripotent MSL cells, thus supporting the existence of a cell lineage relationship between islet alpha- and beta-cell during ontogeny; and 2) that our glucagonomas release an anorexigenic substance(s) of unknown nature that causes a severe weight loss comparable to that reported in animals carrying tumor necrosis factor-producing experimental tumors.

Strong Promoter Activity of Human and Rat Islet Amyloid Polypeptide / Amylin Gene Constructs in Mouse Beta Cells (βTC 3)

Islet amyloid polypeptide (IAPP)(′amylin′) is co-produced with insulin in pancreatic β cells and is the formative polypeptide of pancreatic amyloid in patients with type 2 (non-insulin-dependent) diabetes mellitus. Islet amyloid and type 2 diabetes occur in man, but not in rat. To study transcription regulation of IAPP gene expression in man and rat, luciferase reporter constructs containing different portions of the upstream region of both IAPP genes were expressed in transfected cells. Both the human and the rat IAPP gene constructs revealed higher promoter activity in β cells (particularly in βTC3 cells) than in non-β cells. In both IAPP genes potential transcription elements, with homology to insulin gene transcription elements, were identified. βTC3 cells provide a good model system in which to study regulation of human and rat IAPP gene expression.