Human Islet Amyloid Polypeptide Overexpression in INS-1E Cells Influences Amylin Oligomerization under ER Stress and Oxidative Stress.

Human amylin or islet amyloid polypeptide (hIAPP) is synthesized in the pancreatic ß-cells and has been shown to contribute to the pathogenesis of type 2 diabetes (T2D) in vitro and in vivo. This study compared amylin oligomerization/expression and signal transduction under endoplasmic reticulum (ER) stress and oxidative stress. pCMV-hIAPP-overexpressing INS-1E cells presented different patterns of amylin oligomerization/expression under ER stress and oxidative stress. Amylin oligomerization/expression under ER stress showed three amylin oligomers of less than 15 kDa size in pCMV-hIAPP-overexpressing cells, while one band was detected under oxidative stress. Under ER stress conditions, HIF1α, p-ERK, CHOP, Cu/Zn-SOD, and Bax were significantly increased in pCMV-hIAPP-overexpressing cells compared to the pCMV-Entry-expressing cells (control), whereas p-Akt, p-mTOR, Mn-SOD, catalase, and Bcl-2 were significantly decreased. Under oxidative stress conditions, HIF1α, p-ERK, CHOP, Mn-SOD, catalase, and Bcl-2 were significantly reduced in pCMV-hIAPP-overexpressing cells compared to the control, whereas p-mTOR, Cu/Zn-SOD, and Bax were significantly increased. In mitochondrial oxidative phosphorylation (OXPHOS), the mitochondrial complex I and complex IV were significantly decreased under ER stress conditions and significantly increased under oxidative stress conditions in pCMV-hIAPP-overexpressing cells compared to the control. The present study results demonstrate that amylin undergoes oligomerization under ER stress in pCMV-hIAPP-overexpressing cells. In addition, human amylin overexpression under ER stress in the pancreatic ß cells may enhance amylin protein aggregation, resulting in ß-cell dysfunction.

Down-regulation of islet amyloid polypeptide expression induces death of human annulus fibrosus cells via mitochondrial and death receptor pathways.

Islet amyloid polypeptide (IAPP) exerts its biological effects by participating in the regulation of glucose metabolism and cell apoptosis. The main goal of the present study was to investigate the expression of IAPP in degenerated intervertebral disc tissue and IAPP's modulation of extracellular matrix (ECM) catabolic and anabolic genes in human AF cells. We found that the expression of IAPP, the calcitonin receptor, and receptor activity modifying protein decreased considerably in AF cells during the progression of intervertebral disc degeneration (IDD). Meanwhile, transfection with pLV-siIAPP decreased the expression of IAPP and its receptors and reduced glucose uptake and the expression of aggrecan, Col2A1, and BG. Down-regulation of IAPP also induced a significant increase in reactive oxygen species generation in AF cells, along with a decrease in matrix metalloproteinases and an increase in the concentration of cellular Ca2+, ultimately leading to death. Further analysis revealed that siIAPP intervention promoted the release of cytochrome c from mitochondria, resulting in the activation of Caspase-3 and Caspase-9. In contrast, significantly decreased expression of Caspase-3 and Caspase-9 was observed in AF cells transfected with pLV-IAPP. The concentrations of Fas and FasL proteins were significantly decreased in AF cells transfected with PLV-IAPP, while activation of the Fas/FasL system and cell death were induced by siIAPP intervention. Mechanistically, AMPK/Akt-mTOR signaling pathways were involved. In conclusion, down-regulation of IAPP expression induces the death of human AF cells via mitochondrial and death receptor pathways, potentially offering a novel therapeutic target for the treatment of IDD.

Inhibition of amyloid peptide fibril formation by gold-sulfur complexes.

Amyloid-related diseases are characterized by protein conformational change and amyloid fibril deposition. Metal complexes are potential inhibitors of amyloidosis. Nitrogen-coordinated gold complexes have been used to disaggregate prion neuropeptide (PrP106-126) and human islet amyloid polypeptide (hIAPP). However, the roles of metal complexes in peptide fibril formation and related bioactivity require further exploration. In this work, we investigated the interactions of amyloid peptides PrP106-126 and hIAPP with two tetracoordinated gold-sulfur complexes, namely, dichloro diethyl dithiocarbamate gold complex and dichloro pyrrolidine dithiocarbamate gold complex. We also determined the effects of these complexes on peptide-induced cytotoxicity. Thioflavin T assay, morphological characterization, and particle size analysis indicated that the two gold-sulfur complexes effectively inhibited the fibrillation of the amyloid peptides, which led to the formation of nanoscale particles. The complexes reduced the cytotoxicity induced by the amyloid peptides. Intrinsic fluorescence, nuclear magnetic resonance, and mass spectrometry revealed that the complexes interacted with PrP106-126 and hIAPP via metal coordination and hydrophobic interaction, which improved the inhibition and binding of the two gold-sulfur compounds. Our study provided new insights into the use of tetracoordinated gold-sulfur complexes as drug candidates against protein conformational disorders.

C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Mechanistic Contributions of Biological Cofactors in Islet Amyloid Polypeptide Amyloidogenesis.

Type II diabetes mellitus is associated with the deposition of fibrillar aggregates in pancreatic islets. The major protein component of islet amyloids is the glucomodulatory hormone islet amyloid polypeptide (IAPP). Islet amyloid fibrils are virtually always associated with several biomolecules, including apolipoprotein E, metals, glycosaminoglycans, and various lipids. IAPP amyloidogenesis has been originally perceived as a self-assembly homogeneous process in which the inherent aggregation propensity of the peptide and its local concentration constitute the major driving forces to fibrillization. However, over the last two decades, numerous studies have shown a prominent role of amyloid cofactors in IAPP fibrillogenesis associated with the etiology of type II diabetes. It is increasingly evident that the biochemical microenvironment in which IAPP amyloid formation occurs and the interactions of the polypeptide with various biomolecules not only modulate the rate and extent of aggregation, but could also remodel the amyloidogenesis process as well as the structure, toxicity, and stability of the resulting fibrils.

Effect of Diabetes Mellitus on Adipocyte-Derived Stem Cells in Rat.

Diabetes mellitus affects the adipose tissue and mesenchymal stem cells derived from the adipose stroma and other tissues. Previous reports suggest that bone morphogenetic protein 4 (BMP4) is involved in diabetic complications, at the same time playing an important role in the maintenance of stem cells. In this study, we used rats transgenic for human islet amyloid polypeptide (HIP rats), a model of type 2 diabetes, to study the effect of diabetes on adipocyte-derived stem cells, referred to as dedifferentiated fat (DFAT) cells. Our results show that BMP4 expression in inguinal adipose tissue is significantly increased in HIP rats compared to controls, whereas matrix Gla protein (MGP), an inhibitor of BMP4 is decreased as determined by quantitative PCR, and immunofluorescence. In addition, adipose vascularity and expression of multiple endothelial cell markers was increased in the diabetic tissue, visualized by immunofluorescence for endothelial markers. The endothelial markers co-localized with the enhanced BMP4 expression, suggesting that vascular cells play a role BMP4 induction. The DFAT cells are multipotent stem cells derived from white mature adipocytes that undergo endothelial and adipogenic differentiation. DFAT cells prepared from the inguinal adipose tissue in HIP rats exhibited enhanced proliferative capacity compared to wild type. In addition, their ability to undergo both endothelial cell and adipogenic lineage differentiation was enhanced, as well as their response to BMP4, as assessed by lineage marker expression. We conclude that the DFAT cells are affected by diabetic changes and may contribute to the adipose dysfunction in diabetes.

Combinatorial gene construct and non-viral delivery for anti-obesity in diet-induced obese mice.

The combinatorial peptidergic therapy of islet amyloid polypeptide (IAPP) and leptin (LEP) analogues was once an optimistic option in treating obese animals and patients. However, the need for frequent administrations and its negative side effects prevent it from being a viable choice. Here, we developed a combinatorial gene therapy of IAPP and LEP, where two genes are inserted into a single plasmid with self-cleaving furin and 2A sites to treat diet-induced obese (DIO) mice. The developed plasmid DNA (pDNA) individually produced both IAPP and LEP peptides in vitro and in vivo. The pDNA was delivered with a non-viral polymeric carrier, and its once-a-week administrations demonstrated a synergistic loss of body weight and significant reductions of fat mass, blood glucose, and lipid levels in DIO mice. The results suggest that the combinatorial gene therapy would have higher potential than the peptidergic approach for future translation due to its improved practicability.

Engineered aggregation inhibitor fusion for production of highly amyloidogenic human islet amyloid polypeptide.

Human islet amyloid polypeptide (IAPP) is the major component of pancreatic amyloid deposits in type 2 diabetes. The structural conversion of IAPP from a monomeric state into amyloid assemblies is the subject of intense research. Recombinant production of IAPP is, however, difficult due to its extreme aggregation propensity. Here we describe a novel strategy for expression of IAPP in Escherichia coli, based on an engineered protein tag, which sequesters IAPP monomers and prevents IAPP aggregation. The IAPP-binding protein HI18 was selected by phage display from a ß-wrapin library. Fusion of HI18 to IAPP enabled the soluble expression of the construct. IAPP was cleaved from the fusion construct and purified to homogeneity with a yield of 3mg of isotopically labeled peptide per liter of culture. In the monomeric state, IAPP was largely disordered as evidenced by far-UV CD and liquid-state NMR spectroscopy but competent to form amyloid fibrils according to atomic force microscopy. These results demonstrate the ability of the engineered ß-wrapin HI18 for shielding the hydrophobic sequence of IAPP during expression and purification. Fusion of aggregation-inhibiting ß-wrapins is a suitable approach for the recombinant production of aggregation-prone proteins.

Inhibition of human amylin fibril formation by insulin-mimetic vanadium complexes.

The toxicity of amyloid-forming proteins can be linked to many degenerative and systemic diseases. Human islet amyloid polypeptide (hIAPP, amylin) has been associated with type II diabetes. Methods for efficient inhibition of amyloid fibril formation are highly clinically important. This study demonstrated the significant inhibitory effects of six vanadium complexes on hIAPP aggregation. Vanadium complexes, such as bis(maltolato)-oxovanadium (BMOV), have been used as insulin-mimetic agents for the treatment of diabetes for many years. Different biophysical methods were applied to investigate the interaction between V complexes and hIAPP. The results indicated that the selected compounds affected the peptide aggregation by different action modes and protected the cells from the cytotoxicity induced by hIAPP. Both the high binding affinity and the ligand spatial effect on inhibiting hIAPP aggregation are significant. Although some of these compounds undergo biotransformation under the conditions of the experiments, and the active species are not identified, it is understood that the effect results from a particular compound and its conversion products. Importantly, our work provided information on the effects of the selected V complexes on hIAPP and demonstrated multiple levels of effects of V complexes against amyloid-related diseases.

In silico cross seeding of Aß and amylin fibril-like oligomers.

Recent epidemiological data have shown that patients suffering from Type 2 Diabetes Mellitus have an increased risk to develop Alzheimer's disease and vice versa. A possible explanation is the cross-sequence interaction between Aß and amylin. Because the resulting amyloid oligomers are difficult to probe in experiments, we investigate stability and conformational changes of Aß-amylin heteroassemblies through molecular dynamics simulations. We find that Aß is a good template for the growth of amylin and vice versa. We see water molecules permeate the ß-strand-turn-ß-strand motif pore of the oligomers, supporting a commonly accepted mechanism for toxicity of ß-rich amyloid oligomers. Aiming for a better understanding of the physical mechanisms of cross-seeding and cell toxicity of amylin and Aß aggregates, our simulations also allow us to identify targets for the rational design of inhibitors against toxic fibril-like oligomers of Aß and amylin oligomers.

One year of sitagliptin treatment protects against islet amyloid-associated ß-cell loss and does not induce pancreatitis or pancreatic neoplasia in mice.

The dipeptidyl peptidase-4 (DPP-4) inhibitor sitagliptin is an attractive therapy for diabetes, as it increases insulin release and may preserve ß-cell mass. However, sitagliptin also increases ß-cell release of human islet amyloid polypeptide (hIAPP), the peptide component of islet amyloid, which is cosecreted with insulin. Thus, sitagliptin treatment may promote islet amyloid formation and its associated ß-cell toxicity. Conversely, metformin treatment decreases islet amyloid formation by decreasing ß-cell secretory demand and could therefore offset sitagliptin's potential proamyloidogenic effects. Sitagliptin treatment has also been reported to be detrimental to the exocrine pancreas. We investigated whether long-term sitagliptin treatment, alone or with metformin, increased islet amyloid deposition and ß-cell toxicity and induced pancreatic ductal proliferation, pancreatitis, and/or pancreatic metaplasia/neoplasia. hIAPP transgenic and nontransgenic littermates were followed for 1 yr on no treatment, sitagliptin, metformin, or the combination. Islet amyloid deposition, ß-cell mass, insulin release, and measures of exocrine pancreas pathology were determined. Relative to untreated mice, sitagliptin treatment did not increase amyloid deposition, despite increasing hIAPP release, and prevented amyloid-induced ß-cell loss. Metformin treatment alone or with sitagliptin decreased islet amyloid deposition to a similar extent vs untreated mice. Ductal proliferation was not altered among treatment groups, and no evidence of pancreatitis, ductal metaplasia, or neoplasia were observed. Therefore, long-term sitagliptin treatment stimulates ß-cell secretion without increasing amyloid formation and protects against amyloid-induced ß-cell loss. This suggests a novel effect of sitagliptin to protect the ß-cell in type 2 diabetes that appears to occur without adverse effects on the exocrine pancreas.

Translational control of glucose-induced islet amyloid polypeptide production in pancreatic islets.

Dysfunctional islet amyloid polypeptide (IAPP) biosynthesis and/or processing are thought contribute to formation of islet amyloid in type 2 diabetes. However, it is unclear how normal pro-IAPP biosynthesis and processing are regulated to be able to define such dysfunction. Here, it was found that acute exposure to high glucose concentrations coordinately regulated the biosynthesis of pro-IAPP, proinsulin, and its proprotein convertase PC1/3 in normal isolated rat islets, without affecting their respective mRNA levels. Pro-7B2 biosynthesis, like that of pro-PC2, did not appreciably change, but this was likely due to a much higher expression in pancreatic α-cells masking glucose regulation of their biosynthesis in ß-cells. Biosynthesis of pro-SAAS, the putative PC1/3 chaperone, was unaffected by glucose, consistent with its scarce expression in ß-cells. We conclude that translational control of pro-IAPP biosynthesis, in parallel to the pro-PC1/3, pro-PC2, and pro-7B2 proprotein-processing endopeptidases/chaperones, is the predominate mechanism to produce IAPP in islet ß-cells.

Evaluation of plasma islet amyloid polypeptide and serum glucose and insulin concentrations in nondiabetic cats classified by body condition score and in cats with naturally occurring diabetes mellitus.

OBJECTIVE: To evaluate and compare circulating concentrations of islet amyloid polypeptide (IAPP), insulin, and glucose in nondiabetic cats classified by body condition score (BCS) and in cats with naturally occurring diabetes mellitus. ANIMALS: 109 (82 nondiabetic, 21 nonketoacidotic diabetic, and 6 ketoacidotic diabetic) cats. PROCEDURES: Cats were examined and BCSs were assessed on a scale of 1 to 9. After food was withheld for 12 hours, blood was collected and plasma concentrations of IAPP and serum concentrations of insulin and glucose were measured. Differences in these values were evaluated among nondiabetic cats grouped according to BCS and in diabetic cats grouped as ketoacidotic or nonketoacidotic on the basis of clinicopathologic findings. Correlations were determined among variables. RESULTS: In nondiabetic cats, BCS was significantly and positively correlated with circulating IAPP and insulin concentrations. Mean plasma IAPP concentrations were significantly different between cats with BCSs of 5 and 7, and mean serum insulin concentrations were significantly different between cats with BCSs of 5 and 8. Serum glucose concentrations were not significantly different among nondiabetic cats. Mean IAPP concentrations were similar between nonketoacidotic diabetic cats and nondiabetic cats with BCSs of 8 or 9. Mean IAPP concentrations were significantly reduced in ketoacidotic diabetic cats, compared with those of nondiabetic cats with BCSs of 6 through 8 and of nonketoacidotic diabetic cats. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that increased BCS (a measure of obesity) is associated with increased circulating concentrations of IAPP and insulin in nondiabetic cats.

The role of protein sequence and amino acid composition in amyloid formation: scrambling and backward reading of IAPP amyloid fibrils.

The specific functional structure of natural proteins is determined by the way in which amino acids are sequentially connected in the polypeptide. The tight sequence/structure relationship governing protein folding does not seem to apply to amyloid fibril formation because many proteins without any sequence relationship have been shown to assemble into very similar ß-sheet-enriched structures. Here, we have characterized the aggregation kinetics, seeding ability, morphology, conformation, stability, and toxicity of amyloid fibrils formed by a 20-residue domain of the islet amyloid polypeptide (IAPP), as well as of a backward and scrambled version of this peptide. The three IAPP peptides readily aggregate into ordered, ß-sheet-enriched, amyloid-like fibrils. However, the mechanism of formation and the structural and functional properties of aggregates formed from these three peptides are different in such a way that they do not cross-seed each other despite sharing a common amino acid composition. The results confirm that, as for globular proteins, highly specific polypeptide sequential traits govern the assembly pathway, final fine structure, and cytotoxic properties of amyloid conformations.