RESUMEN
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.
Asunto(s)
Amiloide/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Polipéptido Amiloide de los Islotes Pancreáticos/metabolismo , Pliegue de Proteína/efectos de los fármacos , Rutina/uso terapéutico , Amiloide/genética , Animales , Glucemia/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Hipoglucemiantes/farmacología , Polipéptido Amiloide de los Islotes Pancreáticos/genética , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Masculino , Ratones Transgénicos , Agregación Patológica de Proteínas/tratamiento farmacológico , Agregación Patológica de Proteínas/genética , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , Deficiencias en la Proteostasis/tratamiento farmacológico , Deficiencias en la Proteostasis/genética , Deficiencias en la Proteostasis/metabolismo , Deficiencias en la Proteostasis/patología , Rutina/farmacologíaRESUMEN
The recent discovery of two unique manuka marker fluorescence wavelengths (MM1 and MM2) potentially offers a rapid and cost-effective approach for manuka honey authentication using spectroscopy. The fluorophore responsible for the MM1 marker has been identified as leptosperin. We investigated whether lepteridine may be responsible for the MM2 fluorescence. We quantified the lepteridine in manuka honey and manuka nectar, which ranged between 5-52mg/kg and 80-205mg/kg, respectively. Notably, the fluorescent spectrum of synthetic lepteridine matched the MM2 fluorescence signature. Fluorescence quenching was observed in the honey matrix but otherwise, lepteridine was stable over prolonged storage at 37°C. Lepteridine was also found in Australian Leptospermum honeys and nectars. Lepteridine concentration was positively correlated with concentrations of the MM1 fluorescence marker leptosperin in honeys. These findings identify lepteridine as the principle compound responsible for MM2 fluorescence, and support the utility as a marker compound for manuka honey authentication.
Asunto(s)
Miel/análisis , Pteridinas/química , Espectrometría de Fluorescencia/métodos , BiomarcadoresRESUMEN
New Zealand manuka (Leptospermum scoparium) honey exhibits two unique fluorescence signatures that distinguish it from other honey types. One of these is the MM1 fluorescence marker (270-365nm excitation-emission) which we show is due to a Leptospermum nectar-derived compound, leptosperin. Synthetic or honey-purified leptosperin not only displayed an identical fluorescence spectrum, but supplementation of leptosperin into clover or artificial honeys generated the MM1 fluorescence signature. There was a quenching effect of the honey matrix on leptosperin fluorescence but otherwise leptosperin was chemically stable over prolonged storage at 37°C. Leptosperin was also present in the woody-fruited Australian Leptospermum species at elevated concentrations but virtually absent in Leptospermum subtenue suggesting its elevated expression developed following the mid-Miocene separation of the genus. These findings suggest that fluorescence spectroscopy could offer a rapid and high-throughput screening method for identification of Leptospermum honeys using the MM1 fluorescence marker.
Asunto(s)
Miel/análisis , Leptospermum/química , Néctar de las Plantas/química , Australia , Fluorescencia , Colorantes Fluorescentes , Ensayos Analíticos de Alto Rendimiento , Espectrometría de FluorescenciaRESUMEN
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].
RESUMEN
MaÌ nuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine maÌ nuka honey are increasingly in demand due to the presence of counterfeit maÌ nuka honey. This work reports the identification of a compound previously unreported in maÌ nuka honey by HPLC, and determination of the structure of the as 3,6,7-trimethyllumazine using NMR, MS, IR, and UV/vis spectroscopy. This assignment was confirmed by total synthesis. The natural product, renamed lepteridine, was only observed in maÌ nuka honeys and could potentially serve as a biomarker for genuine maÌ nuka honey.
Asunto(s)
Biomarcadores/química , Miel/análisis , Pteridinas/química , Cromatografía Líquida de Alta Presión , Leptospermum/química , Espectroscopía de Resonancia Magnética , Néctar de las Plantas/química , Pteridinas/aislamiento & purificaciónRESUMEN
We report here a unique amyloidoma of the radial nerve which could not be subtyped by available techniques, including immunohistochemistry and standard clinical and laboratory evaluation. In order to identify the amyloid monomer, we developed a novel preparative procedure designed to optimize conditions for liquid chromatography tandem mass spectrometry analysis of formalin-fixed/paraffin-embedded (FFPE) tissue. Subsequent mass spectrometric analysis clearly identified kappa light chain as the monomer, with no evidence of lambda light chain. Manual interpretation of the matched spectra revealed no evidence of polyclonality. This study also enabled detailed characterisation of twelve likely amyloid matrix components. Finally, our analysis revealed extensive hydroxylation of collagen type I but, unexpectedly, an almost complete lack of hydroxylated residues in the normally heavily-hydroxylated collagen type VI chains, pointing to structural/functional alterations of collagen VI in this matrix that could have contributed to the pathogenesis of this very unusual tumour. Given the high quality of the data here acquired using a standard quadrupole-time of flight tandem mass spectrometer of modest performance, the robust and straightforward preparative method described constitutes a competitive alternative to more involved approaches using state-of-the-art equipment.
Asunto(s)
Amiloidosis/patología , Placa Amiloide/patología , Nervio Radial/patología , Espectrometría de Masas en Tándem/métodos , Secuencia de Aminoácidos , Amiloidosis/metabolismo , Cromatografía Líquida de Alta Presión , Colágeno Tipo VI/química , Colágeno Tipo VI/metabolismo , Femenino , Fijadores , Secciones por Congelación , Humanos , Hidroxilación , Cadenas kappa de Inmunoglobulina/química , Cadenas kappa de Inmunoglobulina/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Adhesión en Parafina , Placa Amiloide/metabolismo , Nervio Radial/metabolismo , Fijación del TejidoRESUMEN
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.