Molecular Mechanisms of Amylin Turnover, Misfolding and Toxicity in the Pancreas

Abstract

Amyloidosis is a common pathological event in which proteins self-assemble into misfolded soluble and insoluble molecular forms, oligomers and fibrils that are often toxic to cells. Notably, aggregation-prone human islet amyloid polypeptide (hIAPP), or amylin, is a pancreatic hormone linked to islet β-cells demise in diabetics. The unifying mechanism by which amyloid proteins, including hIAPP, aggregate and kill cells is still matter of debate. The pathology of type-2 diabetes mellitus (T2DM) is characterized by extracellular and intracellular accumulation of toxic hIAPP species, soluble oligomers and insoluble fibrils in pancreatic human islets, eventually leading to loss of β-cell mass. This review focuses on molecular, biochemical and cell-biology studies exploring molecular mechanisms of hIAPP synthesis, trafficking and degradation in the pancreas. In addition to hIAPP turnover, the dynamics and the mechanisms of IAPP-membrane interactions; hIAPP aggregation and toxicity in vitro and in situ; and the regulatory role of diabetic factors, such as lipids and cholesterol, in these processes are also discussed.

Keywords: aggregation; cholesterol; human islet amyloid polypeptide; islet amyloidosis; lipids; pancreas; proteotoxicity; secretion; transcription; type-2 diabetes mellitus.

Figure 1

Molecular determinants of hIAPP synthesis, aggregation and toxicity in pancreatic islets. (A) Diagram depicts main transcriptional regulatory sites and factors from hIAPP and insulin promoters. Primary sequence of fully processed mature hIAPP form is shown on the right. Amyloidogenic region in hIAPP amino acids sequence is underlined. AFM micrograph of a single fibril self-assembled from mature synthetic hIAPP monomers is shown below. Adapted from Reference [46]. (B) Diagram depicts main steps in hIAPP synthesis in glucose-challenged or ER-stressed pancreatic β-cells, including activation of a central TXNIP/FOXA2-mediated signaling pathway. Following processing, hIAPP is stored together with insulin in secretory vesicles. Disproportionate production and/or processing of hIAPP in human islets may initiate its aggregation and consequently β-cell stress and islet amyloidosis. (C) Excessive intracellular and/or extracellular accumulation of protein aggregates in hIAPP transgenic mice induces a loss in β-cell mass and hyperglycemia, which are main pathological attributes of T2DM. Note a decrease in insulin levels (green) with simultaneous accumulation of hIAPP (red) and thioflavin T (ThT)-positive protein aggregates (white) in hIAPP transgenic mouse islets as compared to wild-type mice islets which are hIAPP- and aggregate-free. Additionally, note severe islet cells atrophy and distortion of hIAPP transgenic mouse islets as compared to morphologically and functionally preserved islets from non-diabetic wild-type mice. Confocal micrographs adapted from Reference [33].

Figure 2

Regulatory mechanisms driving IAPP transcription in normal and stressed pancreatic β-cells. (1) High glucose induces expression of the major glucose regulated gene TXNIP, which, in turn, upregulates IAPP transcription by increasing the expression and promoter binding of IAPP specific transcription factor FoxA2 in the β-cell. (2) Similarly, ER stress upregulates IAPP transcription in β-cells by increasing IAPP promoter’s occupancy for transcription factor FoxA2, for which the binding of could be TXNIP-dependent or independent. (3) Severe inhibition of proteasome function and associated protein stress downregulate IAPP transcription by attenuating FoxA2 binding at the IAPP promoter in the β-cell. Green arrows depict positive regulatory pathways, and red arrow depicts an inhibitory pathway. Different signaling branches are numbered. Dashed arrows depict hypothetical signaling branches.

Figure 3

Proteolytic pathways involved in IAPP degradation. (1) ALS is involved in degradation of misfolded IAPP localized outside nucleus. (2) UPS regulates degradation of misfolded IAPP localized in cytosol and nucleus. Diabetogenic stresses downregulate both (1) and (2). UCHL1 probably regulates both (1–3) hIAPP aggregate inhibits both (1) and (2). (4) IDE degrades cytosolic hIAPP aggregates. (5) MMP-9 and neprilysin degrade secreted form of hIAPP and/or inhibits fibril formation, thereby preventing its aggregation on the beta-cell.

Figure 4

Polymerization pathways and polymorphic structures of hIAPP on different surfaces. (A) Time-lapse 3D-AFM analysis of hIAPP aggregation on soft negatively charged lipid/sterol planar membranes. Freshly dissolved monomeric hIAPP was added to preformed planar lipidic membranes of different composition, and the modulatory effect of lipids and cholesterol on the extent, size, organization and morphology of amylin aggregates was analyzed by AFM. Upper micrographs, 5 × 5 μm. At higher magnification (2.5 × 2.5 μm), single highly ordered hIAPP tetrameric and pentameric oligomeric assemblies are resolved (lower insets; scale bar, 100 nm), two of which feature a central pore (hIAPP/Lipids, 10 min, top inset). Tetrameric (lower left micrograph) and pentameric (lower right micrograph) subunits of individual hIAPP supramolecular complexes are outlined for clarity (scale bar, 50 nm). (B) Time-lapse 3D-AFM analysis of hIAPP aggregation on stiff mica surface. In contrast to planar membranes, self-assembled hIAPP oligomers (black arrowheads, AFM micrograph) bi-directionally extended into a mature fibril. hIAPP polymerization on mica was visualized and quantified with time-lapse AFM and 3D-section analysis, which revealed the width, length and height of full-grown hIAPP fibrils and their intermediates. (C) Structural diversity of hIAPP polymorphic forms on different surfaces. AFM micrographs of a single fibril, a pore and cluster self-assembled from hIAPP monomers on different surfaces are presented for clarity. AFM micrographs and fibril growth curve were adapted from References [46,84].

Figure 5

Cholesterol controls hIAPP trafficking, aggregation and toxicity in pancreatic islets. (A) Time-lapse laser scanning confocal microscopy (LSCM) analysis of hIAPP oligomerization and internalization in cultured human islets. Freshly dissolved hIAPP was incubated with cholera toxin (CTX) in acute human islets for indicated periods of time, in the presence or absence of cholesterol depleting agents, and the subcellular distribution, size and accumulation of A₁₁-positive hIAPP oligomeric clusters was quantified by confocal microscopy and a conformation specific anti-oligomer A₁₁ antibody. Bar, 5 μm. Organization and plasma membrane distribution of hIAPP oligomers (clusters) prior to and following depletion of PM cholesterol are shown on the right (boxes). LSCM micrographs adapted from Reference [66]. (B) Intact cholesterol organization on PM is required for internalization of hIAPP soluble oligomeric assemblies. Following internalization, hIAPP monomeric and oligomeric structures are targeted for degradation by 20S proteasome complex and intracellular proteolytic enzymes, such as insulin-degrading enzyme (IDE) [71,79]. Cholesterol depleting agents, betacyclodextrin (BCD) and lovostatin (Lov), disturb cholesterol homeostasis, leading to less hIAPP clearance and, consequently, its enhanced oligomerization and aggregation in solution and on the cell surface. Graph depicts inverse relationship between PM cholesterol content and hIAPP toxicity in human islets. Graph adapted from Reference [66]. (C) Disruption of cholesterol efflux in hIAPP-transgenic rodent islets stimulate hIAPP aggregation, islet amyloidosis and β-cell dysfunction. Β-cell-specific downregulation of cholesterol-specific ATP-binding cassette transporter 1 (ABCA1) was achieved by using knockout and RNAi-silencing approaches [109].