Islet Amyloid Polypeptide Gene Research Articles

Protective roles of adiponectin and molecular signatures of HNF4α and PPARα as downstream targets of adiponectin in pancreatic β cells

The disease progression of the metabolic syndrome is associated with prolonged hyperlipidemia and insulin resistance, eventually giving rise to impaired insulin secretion, often concomitant with hypoadiponectinemia. As an adipose tissue derived hormone, adiponectin is beneficial for insulin secretion and β cell health and differentiation. However, the down-stream pathway of adiponectin in the pancreatic islets has not been studied extensively. Here, along with the overall reduction of endocrine pancreatic function in islets from adiponectin KO mice, we examine PPARα and HNF4α as additional down-regulated transcription factors during a prolonged metabolic challenge. To elucidate the function of β cell-specific PPARα and HNF4α expression, we developed doxycycline inducible pancreatic β cell-specific PPARα (β-PPARα) and HNF4α (β-HNF4α) overexpression mice. β-PPARα mice exhibited improved protection from lipotoxicity, but elevated β-oxidative damage in the islets, and also displayed lowered phospholipid levels and impaired glucose-stimulated insulin secretion. β-HNF4α mice showed a more severe phenotype when compared to β-PPARα mice, characterized by lower body weight, small islet mass and impaired insulin secretion. RNA-sequencing of the islets of these models highlights overlapping yet unique roles of β-PPARα and β-HNF4α. Given that β-HNF4α potently induces PPARα expression, we define a novel adiponectin-HNF4α-PPARα cascade. We further analyzed downstream genes consistently regulated by this axis. Among them, the islet amyloid polypeptide (IAPP) gene is an important target and accumulates in adiponectin KO mice. We propose a new mechanism of IAPP aggregation in type 2 diabetes through reduced adiponectin action.

2091-P: Effects of Caveolin-1 on the Synthesis and Decomposition of Islet ß-Cell IAPP and Its Mechanism

Objective: Islet amyloid polypeptide(IAPP) is a major component of islet amyloid deposition in type 2 diabetic patients. Our preliminary study showed that membrane protein Caveolin-1 (Cav1) silencing inhibited β-cell apoptosis and promote insulin secretion. The purpose of this study was to investigate the regulation of Cav1 on islet β-cell IAPP and its mechanism. Materials and Methods: Firstly, IAPP secretion of each group of primary islets was detected by ELISA, then Western blot and real-time quantitative PCR (qPCR) were used to detect the expression levels of IAPP, PAM(Peptidyl-glycine alpha-amidating monooxygenase), PC1(Prohormone Convertase-1), BACE2(Beta-secretase 2), IDE(Insulin-Degrading Enzyme), TXNIP(Thioredoxin interacting protein), and finally, it confirmed that Cav1 interacted with the protein of TXNIP by immunofluorescence confocal and co-immunoprecipitation (Co-IP). Results: The secretion of IAPP was decreased in primary pancreatic islets and Cav1 deficiency significantly down-regulated the expression of IAPP in NIT-1 cells, while over-expression of Cav1 in βTC-6 cells significantly up-regulated the expression of IAPP. This effect correlated with Cav1 silencing leading to down-regulation of PAM expression associated with IAPP synthesis and over-expression of Cav1 leading to up-regulation of PC1 associated with IAPP synthesis. On the other hand,Cav1 deficiency resulted in the up-regulation of the expression of BACE2 and IDE, the enzymes associated with IAPP decomposition. While over-expression of Cav1 leaded to down-regulation of BACE2 and IDE. Further studies indicated that Cav1 co-localized with TXNIP, and it interacted with and regulated the expression of TXNIP, which regulates IAPP gene transcription. Conclusion: Cav-1 can regulate the synthesis and decomposition of IAPP in islet β cell by interacting with TXNIP. This study suggests that Cav-1 may protect diabetic islet beta cells by reducing islet amyloid deposition. Disclosure K. Liu: None. W. Zeng: None. S. Lin: None. C. Lin: None. F. Xu: None. N. Cai: None. L. Zeng: None. Funding National Natural Science Funding of China (51873071); Natural Science Foundation of Guangdong Province (2018B030311012)

Phylogenetic and promoter analysis of islet amyloid polypeptide gene causing type 2 diabetes in mammalian species

Mutation in islet amyloid polypeptide (IAPP) gene results into its protein misfolding and fibril formation. Isolation of various mammalian species shows its conservation from 89- to 93-amino acid sequence sharing homology with cats, rats, mice, and guinea pigs. For the present study, detailed phylogenetic analysis is carried out for the upstream promoter region of IAPP gene, IAPP messenger RNA (mRNA), and proIAPP protein of nine mammalian species. Sequence analysis has shown partial conservation among the rodent species and the canidae group; however, the primate group species has shown maximum conservation of all sequences under considerations with respect to the human IAPP sequences. Our novel approach of analyzing mRNA and protein sequence conservation of proIAPP delineates the importance for developing IAPP models in closely related species sharing a common ancestor with the time divergence. Further, transcription factor binding sites were critically analyzed for the upstream promoter region of the IAPP gene. These probable binding sites were predicted for DNA motifs recognized by transcription factors, which may prove to be helpful in predicting the regulatory mechanism in understanding the regulation of IAPP gene under in vitro or in vivo conditions. Therefore, confirming the co-evolutionary relationship of IAPP among different species will help us guide in studying transcription factor binding site for poorly studied IAPP gene and targeting disease mutation initiating pancreatic amyloidosis in clinical applications. The phylogenetic and gene promoter analysis presented in the paper reveals regulatory elements for beta cell death caused by pancreatic amyloidosis and resulting in type 2 diabetes (T2D) onset.

Inhibition of BACE2 counteracts hIAPP-induced insulin secretory defects in pancreatic β-cells.

BACE2 (β-site APP-cleaving enzyme 2) is a protease localized in the brain, where it appears to play a role in the development of Alzheimer disease (AD). It is also found in the pancreas, although its biologic function is not fully known. Amyloidogenic diseases, including AD and type 2 diabetes mellitus (T2D), share the accumulation of abnormally folded and insoluble proteins that interfere with cell function. Islet amyloid polypeptide (IAPP) deposits are a key pathogenic feature of T2D. Within this context, we found by global gene expression profiling that BACE2 was up-regulated in the rat pancreatic β-cell line INS1E stably transfected with human IAPP gene (hIAPP-INS1E). Glucose-stimulated insulin secretion (GSIS) in hIAPP-INS1E cells was 30% lower than in INS1E cells. Additionally, INS1E cells transfected with a transient overexpression of BACE2 showed a 60% decrease in proliferation, a 3-fold increase in reactive oxygen species production, and a 25% reduction in GSIS compared to control cells. Remarkably, silencing of endogenous BACE2 in hIAPP-INS1E cells resulted in a significant improvement in GSIS (3-fold increase vs. untransfected cells), revealing the significant role of BACE2 expression in β-cell function. Thus, BACE2 inhibition may be useful to recover insulin secretion in hIAPP-INS1E defective cells and may be proposed as a therapeutic target for T2D.

The study of regulatory effects of Pdx-1, MafA and NeuroD1 on the activity of porcine insulin promoter and the expression of human islet amyloid polypeptide.

The purpose of the present study was to determine the activation of porcine insulin promoter (PIP) by three transcription factors: pancreatic and duodenal homeobox 1 (Pdx-1), v-maf musculoaponeurotic fibrosarcoma oncogene (MafA) and neurogenic differentiation 1 (NeuroD1) in non-beta islet cells cultured in vitro. In addition, the expression of the exogenous human islet amyloid polypeptide (hIAPP) gene driving by PIP in porcine kidney 15 (PK15) cells co-transfected with these transcription factors was also examined. In the present study, a series of vectors for gene overexpression were constructed, including pGL3-Pdx-1, pGL3-MafA, pGL3-NeuroD1, pGL3-PIP-LUC and pcDNA3.1-PIP-hIAPP. The dual-luciferase reporter assay showed that the PIP activity was increased in PK15 cells when overexpressing the exogenous transcription factors Pdx-1, MafA and NeuroD1. Introducing the PIP-hIAPP expression vector into PK15 cells combined with exogenous Pdx-1, MafA and NeuroD1 resulted in the efficient expression of hIAPP at the gene level, but not the protein. The current systematic porcine insulin promoter analysis provided the basic information for future utilization of porcine insulin.

Protective role of human insulin against the cytotoxicity associated with human mutant S20G islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP) is a main component of islet amyloid in type 2 diabetes and cosecreted from β-cell with insulin. Clinical evidence from the patients with S20G mutation of the IAPP gene, as well as experimental evidence that insulin could inhibit amyloid formation of IAPP, suggests that a gradual reduction of insulin could be related to the cytotoxicity associated with S20G-IAPP through long-term deterioration of β-cells in type 2 diabetes. Our objective was to show an effect of human insulin on S20G-IAPP associated cytotoxicity. We analyzed the cytotoxicity associated with S20G-IAPP by controlling human insulin expression using adenovirus vectors with micro ribonucleic acid specifically against human insulin in endocrine AtT-20ins cells, which express human insulin permanently. Additionally, we carried out a follow-up study of circulating IAPP and insulin in type 2 diabetic patients. S20G-IAPP expression was associated with a decrease in viability and an increase in terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling-positive cells in AtT-20ins cells. Furthermore, downregulation of human insulin enhanced the cytotoxicity associated with S20G-IAPP, and induced the cytotoxicity associated with wild-type (WT)-IAPP. Reduction of ubiquitin carboxy-terminal hydrolase L1 activity enhanced cytotoxicity under the downregulation of human insulin expression in both S20G- and WT-IAPP transduced cells. A 5-year follow up of type 2 diabetic patients showed a disproportionate increase of serum fasting IAPP-to-insulin ratio from baseline. Human insulin plays a protective role against the cytotoxicity associated with S20G-IAPP, as well as WT-IAPP. The findings could suggest long-term deterioration of insulin secretion associates with IAPP linked cytotoxicity in type 2 diabetes.

Involvement of ATP-sensitive Potassium (KATP) Channels in the Loss of Beta-cell Function Induced by Human Islet Amyloid Polypeptide

Islet amyloid polypeptide (IAPP) is a major component of amyloid deposition in pancreatic islets of patients with type 2 diabetes. It is known that IAPP can inhibit glucose-stimulated insulin secretion; however, the mechanisms of action have not yet been established. In the present work, using a rat pancreatic beta-cell line, INS1E, we have created an in vitro model that stably expressed human IAPP gene (hIAPP cells). These cells showed intracellular oligomers and a strong alteration of glucose-stimulated insulin and IAPP secretion. Taking advantage of this model, we investigated the mechanism by which IAPP altered beta-cell secretory response and contributed to the development of type 2 diabetes. We have measured the intracellular Ca(2+) mobilization in response to different secretagogues as well as mitochondrial metabolism. The study of calcium signals in hIAPP cells demonstrated an absence of response to glucose and also to tolbutamide, indicating a defect in ATP-sensitive potassium (K(ATP)) channels. Interestingly, hIAPP showed a greater maximal respiratory capacity than control cells. These data were confirmed by an increased mitochondrial membrane potential in hIAPP cells under glucose stimulation, leading to an elevated reactive oxygen species level as compared with control cells. We concluded that the hIAPP overexpression inhibits insulin and IAPP secretion in response to glucose affecting the activity of K(ATP) channels and that the increased mitochondrial metabolism is a compensatory response to counteract the secretory defect of beta-cells.

Progressive deterioration of insulin secretion in Japanese type 2 diabetic patients in comparison with those who carry the S20G mutation of the islet amyloid polypeptide gene: A long-term follow-up study.

Aims/Introduction: In order to clarify the enhanced β-cell dysfunction in type 2 diabetic patients carrying the S20G mutation of the islet amyloid polypeptide gene (S20G-patients), we first estimated the decline of insulin secretion in Japanese type 2 diabetic patients without the S20G mutation (non-S20G-T2D-patients) by long-term observation, and then compared it with that of the S20G-patients. We followed 70 non-S20G-T2D-patients (body mass index <30 kg/m(2)) for more than 10 years and six S20G-patients for more than 5 years. We measured fasting C-peptide (F-CP) every 1-2 years and carried out a glucagon test at least once during the follow-up period. F-CP and a 5-min value of C-peptide after glucagon injection (5'-CP) were used as the indices of insulin secretion. We excluded patients who had renal dysfunction and/or anti-insulin antibodies in the insulin-treated patients. The individual annual declines were calculated from the slopes of the regression lines between C-peptide levels and duration (years after diagnosis). The mean individual annual declines of both F-CP and 5'-CP were significantly greater in the S20G-patients than the non-S20G-T2D-patients (F-CP; 0.047 ± 0.026 vs 0.011 ± 0.037 nmol/L/year, P = 0.025, 5'-CP; 0.139 ± 0.055 vs 0.022 ± 0.012 nmol/L/year, P = 0.008). We established the annual decline of insulin secretion in the Japanese type 2 diabetic patients by the long-term observation. The results show that the decline of insulin secretion is more rapid in the S20G-patients than the non-S20G-T2D-patients. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2011.00102.x, 2011).

Islet amyloid polypeptide gene variation ( IAPP) and the risk of incident type 2 diabetes mellitus: The women's genome health study

Background Islet amyloid polypeptide ( IAPP) gene variation has recently been implicated in type 2 diabetes mellitus (T2D). However, to date, no prospective epidemiological data are available. Methods The association between 10 IAPP tag-single nucleotide polymorphisms (tSNPs) and incident T2D was investigated in 22,715 Caucasian participants of the prospective Women's Genome Health Study. All were free of known cardiovascular disease, cancer, and diabetes at baseline. During a 13-year follow-up period, 1445 participants developed an incident T2D. Multivariable Cox regression analysis was performed to investigate the relationship between genotypes and T2D risk. Haplotype-based analysis was also performed. Results No evidence for an association of any of the tSNPs tested or haplotypes thereof with T2D risk. Conclusions If corroborated in other large, prospective studies, the present findings further suggest that the IAPP gene locus may not be useful predictor for T2D risk assessment.

Arrest of Amyloid Fibril Formation Associated to Type II Diabetes: Structural and Functional Links to the Mechanism of Alzheimers &amp;#946;- Amyloid Fibrillization

Pancreatic amyloid deposits composed of the islet amyloid polypeptide (IAPP) are found in nearly all type II diabetes patients. These fibrillar deposits are cytotoxic and their production is clearly associated with the progression of pancreatic βcells death. Such progressive formation of cytotoxic amyloid fibrils by IAPP in Type II diabetes is parallel in many ways to the process of cerebral amyloid fibril formation by the βamyloid polypeptide in the case of Alzheimers disease. Another line of parallelism between Alzheimers disease and Type II diabetes is the occurrence of a mutation within the IAPP gene that is correlated with an early onset of Type II diabetes in the Asian population. This finding resembles the occurrence familial mutations within the βamyloid polypeptide that are correlated with early onset of Alzheimers disease. Biochemical and biophysical analysis demonstrated an increased amyloidogenic potential as well as cytotoxicity for the altered IAPP protein. Arrest of amyloid formation by IAPP therefore seems to be significant to halt the deterioration in βcell function of Type II diabetes patients and maybe used as a treatment to delay onset for parts of the population with predisposition for this disease. Extensive studies by several academic and industrial groups already resulted in lead molecules that can inhibit amyloid fibril formation by IAPP. Some of these lead molecules are based of the same methodologies that were successfully used for the development of potential drugs that inhibit formation of amyloid fibrils by the βamyloid polypeptide. Keywords: alzheimers disease, amyloid formation, islet amyloid polypeptide, type II diabetes, prion disease, protein misfolding, p-stacking

Islet amyloid polypeptide gene promoter polymorphisms are not associated with Type 2 diabetes or with the severity of islet amyloidosis

The over-expression of the islet amyloid polypeptide (IAPP) gene could be a causal factor for islet amyloidosis and beta-cell destruction in Type 2 diabetes (T2DM). An IAPP gene promoter polymorphism, IAPP-132G to A, has been associated with T2DM in Spain. To investigate this polymorphism in other cohorts and in relation to therapy, DNA from 425 T2DM and 279 unrelated, non-diabetic UK subjects (ND) and 102 T2DM and 80 ND Finnish subjects was examined. The relationship of amyloid severity (percent amyloid/islet) to prevalence (number of islets affected) and the association of IAPP-132G/A with amyloid was determined in post-mortem pancreas from 38 T2DM subjects. The -132G/A was not associated with T2DM in the UK cohorts (4.5% T2DM; 3.2% ND) or associated with requirement for insulin therapy by 6 years. The mutation was and undetected in the Finnish samples but a new variant, -166T/C, was identified in 2 Finnish T2DM subjects. -132G/A was found in 2/38 diabetic, amyloid-containing and 3/19 ND, amyloid-free subjects. The islet amyloid severity was linearly correlated with the prevalence in T2DM. The IAPP-132G/A promoter polymorphism is not associated with T2DM, a requirement for insulin therapy or with the degree of islet amyloidosis in cohorts from the UK or Finland.

Mutation at position -132 in the islet amyloid polypeptide ( IAPP) gene promoter enhances basal transcriptional activity through a new CRE-like binding site.

Mutations in the islet amyloid polypeptide ( IAPP) gene may play a potential role in the abnormal regulation or expression of the peptide. The aim of this study was to determine the functional role of the -132 G/A mutation reported in the promoter region of the IAPP gene in a population of Spanish Type 2 diabetic patients. We investigated the transcriptional activity using MIN6 cells and luciferase reporter plasmids in several culture conditions. Key regulatory elements of the IAPP promoter region were also analysed by electrophoretic mobility shift assays (EMSA). The mutant construct doubled IAPP transcriptional activity ( p<0.001). Both constructs showed severely reduced promoter activity (four-fold decrease) in the presence of verapamil and diazoxide. In contrast, IAPP promoter activity was doubled after incubation with forskolin or dexamethasone, regardless of the glucose concentrations in the culture media. EMSA revealed that the -132 G/A mutation increased the binding affinity through two DNA-protein complexes. In addition, a cAMP-responsive element binding protein (CREB) was identified by super-shift EMSA. Our studies show that the wild-type and the mutant constructs are regulated in a similar pattern under all conditions, strongly indicating that the -132 G/A mutation increases basal but not inducible transcription. These results may be explained by new binding to the mutant region through CREB and other transcription factors not yet identified.

Islet amyloid: a complication of islet dysfunction or an aetiological factor in Type 2 diabetes?

The role of islet amyloidosis in the onset and progression of Type 2 diabetes remains obscure. Islet amyloid polypeptide is a 37 amino-acid, beta-cell peptide which is co-stored and co-released with insulin. Human islet amyloid polypeptide refolds to a beta-conformation and oligomerises to form insoluble fibrils; proline substitutions in rodent islet amyloid polypeptide prevent this molecular transition. Pro-islet amyloid polypeptide (67 amino acids in man) is processed in secretory granules. Refolding of islet amyloid polypeptide may be prevented by intragranular heterodimer formation with insulin (but not proinsulin). Diabetes-associated abnormal proinsulin processing could contribute to de-stabilisation of granular islet amyloid polypeptide. Increased pro-islet amyloid polypeptide secretion as a consequence of islet dysfunction could promote fibrillogenesis; the propeptide forms fibrils and binds to basement membrane glycosamino-glycans. Islet amyloid polypeptide gene polymorphisms are not universally associated with Type 2 diabetes. Transgenic mice expressing human islet amyloid polypeptide gene have increased islet amyloid polypeptide concentrations but develop islet amyloid only against a background of obesity and/or high fat diet. In transgenic mice, obese monkeys and cats, initially small perivascular deposits progressively increase to occupy 80% islet mass; the severity of amyloidosis in animal models is related to the onset of hyperglycaemia, suggesting that islet amyloid and the associated destruction of islet cells cause diabetes. In human diabetes, islet amyloid can affect less than 1% or up to 80% of islets indicating that islet amyloidosis largely results from diabetes-related pathologies and is not an aetiological factor for hyperglycaemia. However, the associated progressive beta-cell destruction leads to severe islet dysfunction and insulin requirement.

Studies of variability in the islet amyloid polypeptide gene in relation to Type 2 diabetes.

To explore whether the coding region of the islet amyloid polypeptide (IAPP) gene contains genetic variants associated with Type 2 diabetes and whether a previously reported association of the promoter variant -132g-->a with Type 2 diabetes could be reproduced in Danish Caucasians. The coding region was analyzed using single strand conformation polymorphism (SSCP) and heteroduplex analysis in 192 Type 2 diabetic patients. Restriction fragment length polymorphism (RFLP) was employed to screen for the promoter variant in 414 Type 2 diabetic patients and 182 glucose-tolerant control subjects. The SSCP analysis identified an IVS+75a-->g variant in two patients. The frequency of heterozygous carriers of the promoter variant in the case-control study was 4.1% (17/414) and 7.1% (13/182), respectively. Odds ratio of the prevalence of Type 2 diabetes in carriers compared with non-carriers was estimated to be 0.47 (95% confidence interval 0.19, 1.15). Neither variability in the coding region of the IAPP gene nor the -132g-->a promoter variant was associated with Type 2 diabetes among the studied Danish Caucasians.

Transcription Factor Occupancy of the Insulin Gene in Vivo: EVIDENCE FOR DIRECT REGULATION BY Nkx2.2

Consensus-binding sites for many transcription factors are relatively non-selective and found at high frequency within the genome. This raises the possibility that factors that are capable of binding to a cis-acting element in vitro and regulating transcription from a transiently transfected plasmid, which would not have higher order chromatin structure, may not occupy this site within the endogenous gene. Closed chromatin structure and competition from another DNA-binding protein with similar nucleotide specificity are two possible mechanisms by which a transcription factor may be excluded from a potential binding site in vivo. Multiple transcription factors, including Pdx-1, BETA-2, and Pax6, have been implicated in expression of the insulin gene in pancreatic beta cells. In this study, the chromatin immunoprecipitation assay has been used to show that these factors do, in fact, bind to insulin control region sequences in intact beta cells. In addition, another key islet-enriched transcription factor, Nkx2.2, was found to occupy this region using the chromatin immunoprecipitation assay. In vitro DNA-binding and transient transfection assays defined how Nkx2.2 affected insulin gene expression. Pdx-1 was also shown to bind within a region of the endogenous islet amyloid polypeptide, pax-4, and glucokinase genes that were associated with control in vitro. Because Pdx-1 does not regulate gene transcription in isolation, these sequences were examined for occupancy by the other insulin transcriptional regulators. BETA-2, Pax6, and Nkx2.2 were also found to bind to amyloid polypeptide, glucokinase, and pax-4 control sequences in vivo. These studies reveal the broad application of the Pdx-1, BETA-2, Pax6, and Nkx2.2 transcription factors in regulating expression of genes selectively expressed in islet beta cells.

Polymorphism in intron 2 of islet amyloid polypeptide gene is associated with lower low-density lipoprotein cholesterol in nondiabetic subjects and in type 2 diabetic patients.

The aim of this study was to investigate the presence of mutations in the islet amyloid polypeptide (IAPP) gene in a Spanish population with type 2 diabetes and gestational diabetes mellitus (GDM). Using polymerase chain reaction single-stranded conformation polymorphism, we examined the coding region and the 5'-untranslated region (UTR) of the IAPP gene in 177 unrelated type 2 diabetic patients, 110 healthy control subjects, 38 women with GDM, and 38 gestational control subjects. Mutations were confirmed by DNA sequencing. A heterozygous C-to-A nucleotide substitution at +79 bp in intron 2 of the IAPP gene was detected. The frequencies of the +79-bp polymorphism (A allele) were 6.8% in type 2 diabetic patients, 7.7% in nondiabetic control subjects, 11.8% in women with GDM, and 9.2% in gestational control subjects. No AA genotypes were detected. Nondiabetic subjects and patients with type 2 diabetes bearing the CA genotype had lower low-density lipoprotein (LDL) cholesterol levels than subjects bearing wild genotype. Multivariate logistic regression analysis showed an independent association (p < 0.001; odds ratio: 0.33; 95% confidence interval: 0.17-0.63). We did not detect any sequence variant within exons 1 or 2. One diabetic patient was heterozygous for a silent mutation at codon 31 of exon 3 (Asn31 AAC --> AAT). Our findings indicate that the presence of the +79-bp polymorphism of the IAPP gene in nondiabetic subjects and in patients with type 2 diabetes is associated with lower levels of LDL cholesterol. Furthermore, abnormalities of the coding regions or the 5'-UTR of the IAPP gene are not associated with type 2 diabetes or GDM in the Spanish population.

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.

A novel mutation in islet amyloid polypeptide (IAPP) gene promoter is associated with Type II diabetes mellitus.

A novel mutation in islet amyloid polypeptide (IAPP) gene promoter is associated with Type II diabetes mellitus.

Islet amyloid polypeptide in the islets of Langerhans: friend or foe?

Islet amyloid polypeptide (IAPP), or amylin, was originally discovered as the constituent peptide in amyloid occurring in human insulinomas and in pancreatic islets in human subjects with Type II (non-insulin-dependent) diabetes mellitus. Its normal expression in beta cells and its co-secretion with insulin in response to nutrient stimuli, suggest a metabolic function for the peptide. Specifically, IAPP has most frequently been shown to inhibit insulin secretion, implying that IAPP has a role in the regulation of islet hormone homeostasis. The physiological significance of IAPP in islets has been difficult to assess; very high IAPP concentrations are required to alter insulin secretion. Moreover, until recently, IAPP receptors have not been characterised at the molecular level, thus leaving the actual target cells for IAPP unidentified. Furthermore, in experimental diabetes in rodents, the ratio of IAPP expression to that of insulin invariably is increased. In view of the pleiotropic effects attributed to IAPP, such regulation could be both adverse and beneficial in diabetes. Metabolic characterisation of mice carrying a null mutation in the IAPP gene or which overexpress IAPP in beta cells have recently confirmed that IAPP is a physiological inhibitor of insulin secretion. Based on experiments in which IAPP-deficient mice develop a more severe form of alloxan-induced diabetes, we argue that the action of IAPP in the islets normally is beneficial for beta-cell function and survival; thus, the established up regulation of IAPP expression compared with that of insulin in experimental rodent diabetes could serve to protect islets under metabolically challenging circumstances.

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.