Jacqueline F. Aitken

The aggregation potential of human amylin determines its cytotoxicity towards islet β-cells

Human amylin is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type 2 diabetes. There is evidence that it can elicit in vitro apoptosis in islet β‐cells, but the physical properties that underpin its cytotoxicity have not been clearly elucidated. Here we employed electron microscopy, thioflavin T fluorescence and CD spectroscopy to analyze amylin preparations whose cytotoxic potential was established by live–dead assay in cultured β‐cells. Highly toxic amylin contained few preformed fibrils and initially showed little β‐sheet content, but underwent marked time‐dependent aggregation and β‐conformer formation following dissolution. By contrast, low‐toxicity amylin contained abundant preformed fibrils, and demonstrated high initial β‐sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to β‐cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to β‐cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant β‐sheet content. Attempts to find ways of protecting β‐cells from amylin‐mediated death might profitably focus on preventing the conformational change from random coil to β‐sheet.

Suppression by polycyclic compounds of the conversion of human amylin into insoluble amyloid.

There is a significant correlation between the occurrence of pancreatic islet amyloid and beta-cell failure in advanced type II diabetes mellitus. Islet amyloid is composed primarily of the fibrillar form of the pancreatic hormone, amylin. Using thioflavin-T fluorescence binding and radioprecipitation assays, we investigated whether or not a series of small tricyclic compounds, tetracycline or Congo Red could interfere with the conversion of synthetic human amylin into its insoluble amyloid form. Of the compounds investigated, incubation of human amylin with a 20-fold molar excess of either Congo Red or Acridine Orange resulted in significant inhibition in the rate of amyloid formation. With Congo Red, maximal inhibition effectively occurred at a 1:1 molar ratio or greater over human amylin, whereas inhibition by Acridine Orange was dose-dependent. A 20-fold molar excess of the compound tetracycline also decreased insoluble amyloid content after extended incubation periods of approx. 20 h. Amyloid fibril morphology in the presence of tetracycline, as measured by transmission electron microscopy, was characterized by short fragmented fibrils compared with the longer and denser appearance of fibrils formed by amylin alone. These findings show that polycyclic compounds can suppress the formation of amyloid by human amylin, providing support for an alternative approach to peptide-based strategies by which islet amyloid formation could be modulated.

Thiol reducing compounds prevent human amylin-evoked cytotoxicity

Human amylin (hA) is a small fibrillogenic protein that is the major constituent of pancreatic islet amyloid, which occurs in most subjects with type‐2 diabetes mellitus (T2Dm). There is growing evidence that hA toxicity towards islet β‐cells is responsible for their gradual loss of function in T2Dm. Preventing hA‐mediated cytotoxicity has been proposed as a route to halt the progression of this disease, although this has not yet been demonstrated in vivo. The aim of our studies, in which we show that a small number of hA‐treated cells exhibit intracellular accumulation of reactive oxygen species (ROS), was to evaluate the role of oxidative stress in the mechanism of hA‐mediated cytotoxicity. Here we report that catalase and n‐propyl gallate, antioxidants that are thought to act mainly as free radical scavengers, afford RINm5F cells only limited protection against hA‐mediated toxicity. By contrast, the thiol antioxidants, N‐acetyl‐L‐cysteine (NAC), GSH and dithiothreitol, which not only react with ROS, but also modulate the cellular redox potential by increasing intracellular levels of GSH and/or by acting as thiol reducing agents, afford almost complete protection and inhibit the progression of hA‐evoked apoptosis. We also show that hA treatment is not associated with changes in intracellular GSH levels and that inhibition of GSH biosynthesis has no effect on either hA‐mediated cytotoxicity or NAC‐mediated protection. These results indicate that, in addition to the induction of oxidative stress, hA appears to mediate cytotoxicity through signalling pathways that are sensitive to the actions of thiol antioxidants.

Tetracycline Treatment Retards the Onset and Slows the Progression of Diabetes in Human Amylin/Islet Amyloid Polypeptide Transgenic Mice

OBJECTIVE Aggregation of human amylin/islet amyloid polypeptide (hA/hIAPP) into small soluble β-sheet–containing oligomers is linked to islet β-cell degeneration and the pathogenesis of type 2 diabetes. Here, we used tetracycline, which modifies hA/hIAPP oligomerization, to probe mechanisms whereby hA/hIAPP causes diabetes in hemizygous hA/hIAPP-transgenic mice. RESEARCH DESIGN AND METHODS We chronically treated hemizygous hA/hIAPP transgenic mice with oral tetracycline to determine its effects on rates of diabetes initiation, progression, and survival. RESULTS Homozygous mice developed severe spontaneous diabetes due to islet β-cell loss. Hemizygous transgenic animals also developed spontaneous diabetes, although severity was less and progression rates slower. Pathogenesis was characterized by initial islet β-cell dysfunction followed by progressive β-cell loss. Islet amyloid was absent from hemizygous animals with early-onset diabetes and correlated positively with longevity. Some long-lived nondiabetic hemizygous animals also had large islet-amyloid areas, showing that amyloid itself was not intrinsically cytotoxic. Administration of tetracycline dose-dependently ameliorated hyperglycemia and polydipsia, delayed rates of diabetes initiation and progression, and increased longevity compared with water-treated controls. CONCLUSIONS This is the first report to show that treating hA/hIAPP transgenic mice with a modifier of hA/hIAPP misfolding can ameliorate their diabetic phenotype. Fibrillar amyloid was neither necessary nor sufficient to cause diabetes and indeed was positively correlated with longevity therein, whereas early- to mid-stage diabetes was associated with islet β-cell dysfunction followed by β-cell loss. Interventions capable of suppressing misfolding in soluble hA/hIAPP oligomers rather than mature fibrils may have potential for treating or preventing type 2 diabetes.

The chaperone proteins HSP70, HSP40/DnaJ and GRP78/BiP suppress misfolding and formation of β-sheet-containing aggregates by human amylin: A potential role for defective chaperone biology in Type 2 diabetes

Misfolding of the islet β-cell peptide hA (human amylin) into β-sheet-containing oligomers is linked to β-cell apoptosis and the pathogenesis of T2DM (Type 2 diabetes mellitus). In the present study, we have investigated the possible effects on hA misfolding of the chaperones HSP (heat-shock protein) 70, GRP78/BiP (glucose-regulated protein of 78 kDa/immunoglobulin heavy-chain-binding protein) and HSP40/DnaJ. We demonstrate that hA underwent spontaneous time-dependent β-sheet formation and aggregation by thioflavin-T fluorescence in solution, whereas rA (rat amylin) did not. HSP70, GRP78/BiP and HSP40/DnaJ each independently suppressed hA misfolding. Maximal molar protein/hA ratios at which chaperone activity was detected were 1:200 (HSP70, HSP40/DnaJ and GRP78/BiP). By contrast, none of the chaperones modified the secondary structure of rA. hA, but not rA, was co-precipitated independently with HSP70 and GRP78/BiP by anti-amylin antibodies. As these effects occur at molar ratios consistent with chaperone binding to relatively rare misfolded hA species, we conclude that HSP70 and GRP78/BiP can detect and bind misfolded hA oligomers, thereby effectively protecting hA against bulk misfolding and irreversible aggregation. Defective β-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

Is type 2 diabetes an amyloidosis and does it really matter (to patients)

Degeneration of pancreatic islet beta cells is increasingly ranked as a key disease mechanism in type 2 diabetes [1, 2], but it is not entirely clear what the underlying molecular processes might be and how they attack insulin production, ultimately causing type 2 diabetes [3, 4]. However, recent studies based on quantitative measurements in post mortem pancreatic tissue from humans with type 2 diabetes have reinforced earlier observations concerning the probable role of lowered beta cell numbers [5, 6] and pointed to a linkage between beta cell disappearance and beta cell apoptosis [7, 8]. ‘Glucotoxicity’ and ‘lipotoxicity’ are widely discussed possible causes of beta cell failure, but both are thought to exert their effects only after significant metabolic deterioration and functional islet impairment are already well underway [9] and so cannot be the prime movers. So what might the initiating insults be? Recent data from genome-wide association studies have highlighted the impact of particular alleles of certain susceptibility genes, giving further support to the idea that beta cell abnormalities are fundamental in the pathogenesis of this disease [4]. Some of these alleles encode variants of certain beta cell proteins linked to insulin production and/or secretion, and can hasten the development of functional islet deterioration and diabetes onset in carriers [1, 2]. However, currently identified alleles do not provide an explanation for most cases of ‘common or garden’ type 2 diabetes [10]. Against this background, Zraika et al. in this issue of Diabetologia have reviewed aspects of another, underappreciated phenomenon, which could well initiate beta cell degeneration in type 2 diabetes [11]. As they point out, it has long been known that many [8, 12–14] or perhaps most [15, 16] type 2 diabetic patients are found to have islet amyloid deposits adjacent to their beta cells [17] when post mortem pancreatic tissue is examined histologically. Although islet amyloid has also been reported to occur in numbers of humans not known to have diabetes, the author of a major study cautioned that ‘nondiabetic persons with hyaline islets may represent individuals with undiagnosed or potential diabetes’ [18]. Varying sensitivities of different histological methods may also have contributed to betweenstudy differences in prevalence estimates [11]. Islet amyloid, or hyaline as it was once known [12], has the beta cell hormone human amylin or human islet G. J. S. Cooper (*) : J. F. Aitken : S. Zhang School of Biological Sciences, and Maurice Wilkins Centre of Excellence for Molecular Biodiscovery, Faculty of Science, University of Auckland, Auckland, New Zealand e-mail: g.cooper@auckland.ac.nz

Birds and longevity: does flight driven aerobicity provide an oxidative sink?

Birds generally age slower and live longer than similar sized mammals. For birds this occurs despite elevated blood glucose levels that for mammals would in part define them as diabetic. However these data were acquired in respiration states that have little resemblance to conditions in healthy tissues and mitochondrial RS production is probably minimal in healthy animals. Indeed mitochondria probably act as net consumers rather than producers of RS. Here we propose that (1) if mitochondria are antioxidant systems, the greater mitochondrial mass in athletic species, such as birds, is advantageous as it should provide a substantial sink for RS. (2) The intense drive for aerobic performance and decreased body density to facilitate flight may explain the relative insensitivity of birds to insulin, as well as depressed insulin levels and apparent sensitization to glucagon. Glucagon also associates with the sirtuin protein family, most of which are associated with caloric restriction regulated pathways, mitochondrial biogenesis and life span extension. (3) We note that telomeres, which appear to be unusually long in birds, bind Sirtuins 2 and 4 and therefore may stabilize and protect nuclear DNA. Ultimately these flight driven responses may suppress somatic growth and protect DNA from oxidative damage that would otherwise lead to ageing and non-viral cancers.

Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers

A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four β globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a “monohemoglobin” comprised of two β globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.

Antisera to synthetic peptide recognize high molecular weight enkephalin-containing proteins.

Antisera to a synthetic peptide corresponding to the 95-117 sequence of proenkephalin were used to develop a sensitive radioimmunoassay. Gel-filtration of acid extracts of bovine adrenal medulla and purified chromaffin granules revealed that the antisera recognized high molecular weight material (Mr approximately 5,000-30,000). The material in peak I ( Mr 20 ,000-30,000) and peak II (Mr 10,000-20,000) was further purified by immunoaffinity chromatography. Sequential digestion of each of these fractions with trypsin and carboxypeptidase B generated immunoreactive Met-enkephalin. This study demonstrates that antisera against a synthetic peptide cross-react with high molecular weight enkephalin-containing precursors, validating the use of these antisera in studies of enkephalin biosynthesis.

Human Amylin Transgenic Mice Show Elevated Core Body Temperature

Aberberga-Augskalne L PP087 Abou-Raya A PP059 Abou-Raya S PP059 AbuElKheir H PP059 Ackermans M OP21 Adamu V PP068 Aechundia-Cabanez H PP021 Afrashteh B PP001 Agapakis D PP043 Agrawal R OP25, PP010, PP091 Ahmed M PP084 Ahn C PP022, OP30 Aitken J OP02 Aitken J OP15 Aivars J PP087 Akbaba G PP035 Akbarov PP085, PP086 Akbarov Z PP062, PP063 Akbarzadeh M PP029 Akubude D PP068 Al Bannai F PP084 Al Daghri N PP016 Alashek W PP060 Albassas A PP012 Alebouyeh M PP029 Alexe O PP030 Alexia K PP019 Alexiadou K PP029, OP61, PP099 Alfadda A PP012, PP016 Alfieri A OP06 Alhammad A PP016 Alikhanova N PP062, PP063, PP085, PP086 Alnaamy M PP012 Alomaim W PP012 Amashukeli M OP23 Andreucci A OP33 Angelakas I PP079 Angelopoulos P PP031 Arambasic J OP01 Aranza-Doniz C PP020 Archundia-Cabanez H PP070 Argyrakopoulou G OP29 Aronne L OP31 Arribas C OP18 Asatiani K OP23, PP076 Aslanoglou D PP080, PP079, PP078 Avgeri A OP03, OP05