Vesiculin derived from IGF-II drives increased islet cell mass in a mouse model of pre-diabetes.

Pancreatic islet-cell function and volume are both key determinants of the maintenance of metabolic health. Insulin resistance and islet-cell dysfunction often occur in the earlier stages of type 2 diabetes (T2D) progression. The ability of the islet cells to respond to insulin resistance by increasing hormone output accompanied by increased islet-cell volume is key to maintaining blood glucose control and preventing further disease progression. Eventual ß-cell loss is the main driver of full-blown T2D and insulin-dependency. Researchers are targeting T2D with approaches that include those aimed at enhancing the function of the patient's existing ß-cell population, or replacing islet ß-cells. Another approach is to look for agents that enhance the natural capacity of the ß-cell population to expand. Here we aimed to study the effects of a new putative ß-cell growth factor on a mouse model of pre-diabetes. We asked whether: 1) 4-week's treatment with vesiculin, a two-chain peptide derived by processing from IGF-II, had any measurable effect on pre-diabetic mice vs vehicle; and 2) whether the effects were the same in non-diabetic littermate controls. Although treatment with vesiculin did not alter blood glucose levels over this time period, there was a doubling of the Proliferating Cell Nuclear Antigen (PCNA) detectable in the islets of treated pre-diabetic but not control mice and this was accompanied by increased insulin- and glucagon-positive stained areas in the pancreatic islets.

Glucoregulatory activity of vesiculin in insulin sensitive and resistant mice.

Pancreatic islet-derived peptide hormones play key roles in the maintenance of systemic energy homeostasis and glucose balance and defects in their regulation are strongly implicated in the pathogenesis of obesity and diabetes. Peptides have also been used as lead compounds for therapeutics targeting metabolic disease. It is therefore important to understand the activity and function of islet hormones in both their target tissues and the whole organism. Insulin-like growth factor II (IGF-II) is an insulin homolog secreted by the islet ß-cells. Vesiculin is a newly discovered peptide hormone, processed from IGF-II and secreted from islet ß-cells in response to glucose. We postulated that vesiculin might act to regulate systemic glucose metabolism. Here we report our original investigations of vesiculin's activity in relation to glucoregulation. Vesiculin and IGF-II displayed similar dose-response relationships for lowering blood glucose in insulin-responsive FVB/n mice. By contrast, the ability of IGF-II to lower blood glucose was blunted in insulin-resistant triprolyl human-amylin transgenic mice, whereas vesiculin's ability to lower blood glucose remained unaffected. We also confirmed the ability of vesiculin to bypass insulin resistance in a second mouse model. In vitro analysis of signalling by vesiculin and IGF-II indicates that, like IGF-II, vesiculin signals through the IR/ IGF1R. Overall, we show that removal of only four amino acids from IGF-II has generated a peptide hormone with different bioactivity relevant to blood-glucose regulation. Investigating the differences among vesiculin, IGF-II and insulin signalling and activity may provide new insights into insulin resistance and potentially inform the design of novel therapeutics.

Quantitative data describing the impact of the flavonol rutin on in-vivo blood-glucose and fluid-intake profiles, and survival of human-amylin transgenic mice.

Here we provide data describing the time-course of blood-glucose and fluid-intake profiles of diabetic hemizygous human-amylin (hA) transgenic mice orally treated with rutin, and matched control mice treated with water. We employed "parametric change-point regression analysis" for investigation of differences in time-course profiles between the control and rutin-treatment groups to extract, for each animal, baseline levels of blood glucose and fluid-intake, the change-point time at which blood glucose (diabetes-onset) and fluid-intake (polydipsia-onset) accelerated away from baseline, and the rate of this acceleration. The parametric change-point regression approach applied here allowed a much more accurate determination of the exact time of onset of diabetes than do the standard diagnostic criteria. These data are related to the article entitled "Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice" (J.F. Aitken, K.M. Loomes, I. Riba-Garcia, R.D. Unwin, G. Prijic, A.S. Phillips, A.R.J. Phillips, D. Wu, S.D. Poppitt, K. Ding, P.E. Barran, A.W. Dowsey, G.J.S. Cooper. 2016) [1].

Rutin suppresses human-amylin/hIAPP misfolding and oligomer formation in-vitro, and ameliorates diabetes and its impacts in human-amylin/hIAPP transgenic mice.

Pancreatic islet ß-cells secrete the hormones insulin and amylin, and defective ß-cell function plays a central role in the pathogenesis of type-2 diabetes (T2D). Human amylin (hA, also termed hIAPP) misfolds and forms amyloid aggregates whereas orthologous mouse amylin does neither. Furthermore, hA elicits apoptosis in cultured ß-cells and ß-cell death in ex-vivo islets. In addition, hA-transgenic mice that selectively express hA in their ß-cells, manifest ß-cell apoptosis and progressive islet damage that leads to diabetes closely resembling that in patients with T2D. Aggregation of hA is thus linked to the causation of diabetes. We employed time-dependent thioflavin-T spectroscopy and ion-mobility mass spectrometry to screen potential suppressors of hA misfolding for anti-diabetic activity. We identified the dietary flavonol rutin as an inhibitor of hA-misfolding and measured its anti-diabetic efficacy in hA-transgenic mice. In vitro, rutin bound hA, suppressed misfolding, disaggregated oligomers and reverted hA-conformation towards the physiological. In hA-transgenic mice, measurements of glucose, fluid-intake, and body-weight showed that rutin-treatment slowed diabetes-progression by lowering of rates of elevation in blood glucose (P = 0.030), retarding deterioration from symptomatic diabetes to death (P = 0.014) and stabilizing body-weight (P < 0.0001). In conclusion, rutin treatment suppressed hA-aggregation in vitro and doubled the lifespan of diabetic mice (P = 0.011) by a median of 69 days compared with vehicle-treated control-diabetic hA-transgenic mice.

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 ß 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 ß 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.

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

Misfolding of the islet ß-cell peptide hA (human amylin) into ß-sheet-containing oligomers is linked to ß-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 ß-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 ß-cell chaperone biology could contribute to hA misfolding and initiation of apoptosis in T2DM.

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 beta-sheet-containing oligomers is linked to islet beta-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 beta-cell loss. Hemizygous transgenic animals also developed spontaneous diabetes, although severity was less and progression rates slower. Pathogenesis was characterized by initial islet beta-cell dysfunction followed by progressive beta-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 beta-cell dysfunction followed by beta-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.

Novel peptide antagonists of adrenomedullin and calcitonin gene-related peptide receptors: identification, pharmacological characterization, and interactions with position 74 in receptor activity-modifying protein 1/3.

Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin peptide family, which also includes calcitonin gene-related peptide (CGRP) and AM2. The two AM receptors, AM(1) and AM(2), are calcitonin receptor-like receptor (CL)/receptor activity-modifying protein (RAMP) (RAMP2 and RAMP3, respectively) heterodimers. CGRP receptors comprise CL/RAMP1. The only human AM receptor antagonist (AM(22-52)) is a truncated form of AM; it has low affinity and is only weakly selective for AM(1) over AM(2) receptors. To develop novel AM receptor antagonists, we explored the importance of different regions of AM in interactions with AM(1), AM(2), and CGRP receptors. AM(22-52) was the framework for generating further AM fragments (AM(26-52) and AM(30-52)), novel AM/alphaCGRP chimeras (C1-C5 and C9), and AM/AM(2) chimeras (C6-C8). cAMP assays were used to screen the antagonists at all receptors to determine their affinity and selectivity. Circular dichroism spectroscopy was used to investigate the secondary structures of AM and its related peptides. The data indicate that the structures of AM, AM2, and alphaCGRP differ from one another. Our chimeric approach enabled the identification of two nonselective high-affinity antagonists of AM(1), AM(2), and CGRP receptors (C2 and C6), one high-affinity antagonist of AM(2) receptors (C7), and a weak antagonist selective for the CGRP receptor (C5). By use of receptor mutagenesis, we also determined that the C-terminal nine amino acids of AM seem to be responsible for its interaction with Glu74 of RAMP3. We provide new information on the structure-activity relationship of AM, alphaCGRP, and AM2 and how AM interacts with CGRP and AM(2) receptors.

The aggregation potential of human amylin determines its cytotoxicity towards islet beta-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 beta-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 beta-cells. Highly toxic amylin contained few preformed fibrils and initially showed little beta-sheet content, but underwent marked time-dependent aggregation and beta-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial beta-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to beta-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to beta-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant beta-sheet content. Attempts to find ways of protecting beta-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to beta-sheet.

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 beta-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.

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.