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.

Evidence for different in vitro oligomerization behaviors of synthetic hIAPP obtained from different sources.

Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24 h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process. Graphical abstract.

Human islet amyloid polypeptide (hIAPP) aggregation in type 2 diabetes: Correlation between intrinsic physicochemical properties of hIAPP aggregates and their cytotoxicity.

A large number of pathological diseases are known now to be associated with the misfolding and the aberrant oligomerization and deposition of peptides and proteins into various aggregates. One of these peptides is islet amyloid polypeptide (IAPP), which is responsible for amyloid formation in type 2 diabetes. The mechanism of IAPP amyloid formation in vivo and in vitro is not well understood and the factors behind the peptide aggregates toxicity are not fully defined. Therefore, the precise nature of toxic agents still remains to be elucidated. In this context, first we used a complementary biophysical approach to undertake a systematic study of the hIAPP aggregation process with focus on the lag phase, followed by the study of their degrees of toxicity when added to the extracellular medium of pancreatic cells. The structural properties of hIAPP aggregates are characterized by evaluating their size with DLS, their surface hydrophobicity with ANS, and the interactions between monomers through the intrinsic fluorescence of aromatic residues or by the quenching of these residues mainly the tyrosine in position 37. Our results indicate that despite the method used to study hIAPP aggregation, the obtained curve is easily well fitted in a sigmoidal curve but with some differences. In fact, the analysis of the kinetic parameters gives different information about the hIAPP aggregation process such as lag time and growth rate. Moreover, a high surface hydrophobicity and small size of the aggregates, mainly for the species formed during the lag time, shows strong correlation with the cytotoxicity. These findings provide new insights into the structural changes during hIAPP aggregation and are consistent with a model in which the exposure of hydrophobic surfaces and the small size of aggregates formed during the early stage of the process are crucial for their cytotoxicity.

Human Amylin: From Pathology to Physiology and Pharmacology.

The histopathological hallmark of type 2 diabetes is islet amyloid implicated in the developing treatment options. The major component of human islet amyloid is 37 amino acid peptide known as amylin or islet amyloid polypeptide (IAPP). Amylin is an important hormone that is co-localized, copackaged, and co-secreted with insulin from islet ß cells. Physiologically, amylin regulates glucose homeostasis by inhibiting insulin and glucagon secretion. Furthermore, amylin modulates satiety and inhibits gastric emptying via the central nervous system. Normally, human IAPP is soluble and natively unfolded in its monomeric state. Pathologically, human IAPP has a propensity to form oligomers and aggregate. The oligomers show misfolded α-helix conformation and can further convert themselves to ß-sheet-rich fibrils as amyloid deposits. The pathological findings and physiological functions of amylin have led to the introduction of pramlintide, an amylin analog, for the treatment of diabetes. The history of amylin's discovery is a representative example of how a pathological finding can translate into physiological exploration and lead to pharmacological intervention. Understanding the importance of transitioning from pathology to physiology and pharmacology can provide novel insight into diabetes mellitus and Alzheimer's disease.

Protein aggregates and proteostasis in aging: Amylin and ß-cell function.

The ubiquitin-proteasomal-system (UPS) and the autophagy-lysosomal-system (ALS) are both highly susceptible for disturbances leading to the accumulation of cellular damage. A decline of protein degradation during aging results in the formation of oxidatively damaged and aggregated proteins finally resulting in failure of cellular functionality. Besides protein aggregation in response to oxidative damage, amyloids are a different type of protein aggregates able to distract proteostasis and interfere with cellular functionality. Amyloids are clearly linked to the pathogenesis of age-related degenerative diseases such as Alzheimer's disease. Human amylin is one of the peptides forming fibrils in ß-sheet conformation finally leading to amyloid formation. In contrast to rodent amylin, human amylin is prone to form amyloidogenic aggregates, proposed to play a role in the pathogenesis of Type 2 Diabetes by impairing ß-cell functionality. Since aggregates such as lipofuscin and ß-amyloid are known to impair proteostasis, it is likely to assume similar effects for human amylin. In this review, we focus on the effects of IAPP on UPS and ALS and their role in amylin degradation, since both systems play a crucial role in maintaining proteome balance thereby influencing, at least in part, cellular fate and aging.

Apoptosis signal regulating kinase-1 and NADPH oxidase mediate human amylin evoked redox stress and apoptosis in pancreatic beta-cells.

Misfolded toxic human islet amyloid polypeptide or amylin (hA) and plasma membrane-associated redox complex, NADPH oxidase (NOX), have been implicated in the islet ß-cell demise associated with type-2 diabetes mellitus (T2DM). Studies show that hA accumulation is stressful to ß-cells and that misfolding of human amylin evokes redox stress and activates mitogen activated protein (MAP) kinases, p38 MAPK and c-Jun N-terminal (JNK) kinase. However, the molecular link and causality between hA-evoked redox stress, NOX activity and MAP kinases signaling in pancreatic ß-cells is incompletely understood. Here, we show that in the process of activating JNK, aggregation prone hA also activates an upstream apoptosis signal regulating kinase-1 (ASK1) with concomitant decrease in intracellular levels of reduced glutathione. Inhibition of ASK1 kinase activity, either by specific ASK1 inhibitor, NQDI1 or by thiol antioxidants reduces human amylin-evoked ASK1 and JNK activation and consequently human amylin toxicity in rat insulinoma Rin-m5F cells and human islets. ß-cell specific overexpression of human amylin in mouse islets elicited ASK1 phosphorylation and activation in ß-cells but not in other rodent's islet or exocrine cells. This ASK1 activation strongly correlated with islet amyloidosis and diabetes progression. Cytotoxic human amylin additionally stimulated pro-oxidative activity and expressions of plasma membrane bound NADPH oxidase (NOX) and its regulatory subunits. siRNA mediated NOX1 knockdown and selective NOX inhibitors, ML171 and apocynin, significantly reduced hA-induced mitochondrial stress in insulinoma beta-cells. However, NOX inhibitors were largely ineffective against hA-evoked redox stress and activation of cytotoxic ASK1/JNK signaling complex. Thus, our studies suggest that NOX1 and ASK1 autonomously mediate human amylin-evoked redox and mitochondrial stress in pancreatic ß-cells.

Human amylin induces CD4+Foxp3+ regulatory T cells in the protection from autoimmune diabetes.

Autoimmune diabetes is a disorder of immune homeostasis that leads to targeted insulin-secreting islet ß cell destruction characterized by insulitis. Human amylin (hA) is an important neuroendocrine hormone co-secreted with insulin by pancreatic ß cells. Here, we report hA immune-modulatory action through inducing regulatory T cells. We ex vivo-treated human peripheral blood mononuclear cells (hPBMCs) with hA for 24 h and counted CD4+Foxp3+ regulatory T cells (Treg) using flow cytometry. Diabetic status was monitored and splenic Treg were measured in non-obese diabetic (NOD) male mice. NOD mice were intraperitoneally injected once daily with hA (n = 25) or solvent for control (n = 25) for 7 months continuously. Spleen tissues were collected at the end of intervention and processed for flow cytometry and Western blot. We found a 2.9-fold (p < 0.05) increase of CD4+Foxp3+ Treg in hPBMCs treated with 10 nmol/L hA compared with negative control. Incidence of diabetes in hA-treated NOD mice decreased 44% (p = 0.045) in the 6th month and 57% (p = 0.0002) in the 7th month. Meanwhile, the hA treatment induced a 1.5-fold increase of CD4+Foxp3+ Treg from mouse splenocytes (p = 0.0013). Expression of transforming growth factor-ß (TGF-ß) and toll-like receptor-4 (TLR-4) were upregulated in hA-treated mice. Human amylin might protect against autoimmune diabetes via the induction of CD4+Foxp3+ Treg, which suggests a novel approach to improve autoimmune conditions.

The Molecular Physiopathogenesis of Islet Amyloidosis.

Human islet amyloid polypeptide or amylin (hA) is a 37-amino acid peptide hormone produced and co-secreted with insulin by pancreatic ß-cells. Under physiological conditions, hA regulates a broad range of biological processes including insulin release and slowing of gastric emptying, thereby maintaining glucose homeostasis. However, under the pathological conditions associated with type 2 diabetes mellitus (T2DM), hA undergoes a conformational transition from soluble random coil monomers to alpha-helical oligomers and insoluble ß-sheet amyloid fibrils or amyloid plaques. There is a positive correlation between hA oligomerization/aggregation, hA toxicity, and diabetes progression. Because the homeostatic balance between hA synthesis, release, and uptake is lost in diabetics and hA aggregation is a hallmark of T2DM, this chapter focuses on the biophysical and cell biology studies investigating molecular mechanisms of hA uptake, trafficking, and degradation in pancreatic cells and its relevance to h's toxicity. We will also discuss the regulatory role of endocytosis and proteolytic pathways in clearance of toxic hA species. Finally, we will discuss potential pharmacological approaches for specific targeting of hA trafficking pathways and toxicity in islet ß-cells as potential new avenues toward treatments of T2DM patients.

Proteasome regulates turnover of toxic human amylin in pancreatic cells.

Toxic human amylin (hA) oligomers and aggregates are implicated in the pathogenesis of type 2 diabetes mellitus (T2DM). Although recent studies demonstrated a causal connection between hA uptake and toxicity in pancreatic cells, the mechanism of amylin's clearance following its internalization and its relationship to toxicity is yet to be determined, and hence was investigated here. Using pancreatic rat insulinoma ß-cells and human islets as model systems, we show that hA, following its internalization, first accumulates in the cytosol followed by its translocation into nucleus, and to a lesser extent lysosomes, keeping the net cytosolic amylin content low. An increase in hA accumulation in the nucleus of pancreatic cells correlated with its cytotoxicity, suggesting that its excessive accumulation in the nucleus is detrimental. hA interacted with 20S core and 19S lid subunits of the ß-cell proteasomal complex, as suggested by immunoprecipitation and confocal microscopy studies, which subsequently resulted in a decrease in the proteasome's proteolytic activity in these cells. In vitro binding and activity assays confirmed an intrinsic and potent ability of amylin to interact with the 20S core complex thereby modulating its proteolytic activity. Interestingly, less toxic and aggregation incapable rat amylin (rA) showed a comparable inhibitory effect on proteasome activity and protein ubiquitination, decoupling amylin aggregation/ toxicity and amylin-induced protein stress. In agreement with these studies, inhibition of proteasomal proteolytic activity significantly increased intracellular amylin content and toxicity. Taken together, our results suggest a pivotal role of proteasomes in amylin's turnover and detoxification in pancreatic cells.

Molecular confinement of human amylin in lipidic nanoparticles.

Amylin is a pancreatic hormone involved in the regulation of glucose metabolism and homeostasis. Restoration of the post-prandial and basal levels of human amylin in diabetic individuals is a key in controlling glycemia, controlling glucagon, reducing the insulin dose and increasing satiety, among other physiologic functions. Human amylin has a high propensity to aggregate. We have addressed this issue by designing a liposomal human amylin formulation. Nanoparticles of multilamellar liposomes comprising human amylin were obtained with 53% encapsulation efficiency. The in vitro kinetic release assay shows a biphasic profile. The stabilization of the lipidic nanoparticle against freeze-drying was achieved by using mannitol as a cryoprotectant, as evidenced by morphological characterization. The effectiveness of the human amylin entrapped in lipidic nanoparticles was tested by the measurement of its pharmacological effect in vivo after subcutaneous administration in mice. Collectively these results demonstrate the compatibility of human amylin with the lipidic interface as an effective pharmaceutical delivery system.