Kerry M. Loomes

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.

Crystal structure of a substrate complex of myo-inositol oxygenase, a di-iron oxygenase with a key role in inositol metabolism.

Altered metabolism of the inositol sugars myo-inositol (MI) and d-chiro-inositol is implicated in diabetic complications. In animals, catabolism of MI and d-chiro-inositol depends on the enzyme MI oxygenase (MIOX), which catalyzes the first committed step of the glucuronate–xylulose pathway, and is found almost exclusively in the kidneys. The crystal structure of MIOX, in complex with MI, has been determined by multiwavelength anomalous diffraction methods and refined at 2.0-Å resolution (R = 0.206, Rfree = 0.253). The structure reveals a monomeric, single-domain protein with a mostly helical fold that is distantly related to the diverse HD domain superfamily. Five helices form the structural core and provide six ligands (four His and two Asp) for the di-iron center, in which the two iron atoms are bridged by a putative hydroxide ion and one of the Asp ligands, Asp-124. A key loop forms a lid over the MI substrate, which is coordinated in bidentate mode to one iron atom. It is proposed that this mode of iron coordination, and interaction with a key Lys residue, activate MI for bond cleavage. The structure also reveals the basis of substrate specificity and suggests routes for the development of specific MIOX inhibitors.

Pharmacological characterization of rat amylin receptors: implications for the identification of amylin receptor subtypes

BACKGROUND AND PURPOSE Amylin (Amy) is an important glucoregulatory peptide and AMY receptors are clinical targets for diabetes and obesity. Human (h) AMY receptor subtypes are complexes of the calcitonin (CT) receptor with receptor activity‐modifying proteins (RAMPs); their rodent counterparts have not been characterized. To allow identification of the most clinically relevant receptor subtype, the elucidation of rat (r) AMY receptor pharmacology is necessary.

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.

Mice Lacking the Neuropeptide α-Calcitonin Gene-Related Peptide Are Protected Against Diet-Induced Obesity

Alpha-calcitonin gene-related peptide (alphaCGRP) is a neuropeptide that is expressed in motor and sensory neurons. It is a powerful vasodilator and has been implicated in diverse metabolic roles. However, its precise physiological function remains unclear. In this study, we investigated the role of alphaCGRP in lipid metabolism by chronically challenging alphaCGRP-specific knockout (alphaCGRP(-/-)) and control mice with high-fat diet regimens. At the start of the study, both animal groups displayed similar body weights, serum lipid markers, and insulin sensitivity. However, alphaCGRP(-/-) mice displayed higher core temperatures, increased energy expenditures, and a relative daytime (nonactive) depression in respiratory quotients, which indicated increased beta-oxidation. In response to fat feeding, alphaCGRP(-/-) mice were comparatively protected against diet-induced obesity with an attenuated body weight gain and an overall reduction in adiposity across all the three diets examined. AlphaCGRP(-/-) mice also displayed improved glucose handling and insulin sensitivity, lower im and hepatic lipid accumulation, and improved overall metabolic health. These findings define a new role for alphaCGRP as a mediator of energy metabolism and opens up therapeutic opportunities to target CGRP action in obesity.

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.

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.

Fluorescence markers in some New Zealand honeys.

The fluorescence characteristics of various New Zealand honeys were investigated to establish if this technique might detect signatures unique to manuka (Leptospermum scoparium) and kanuka (Kunzea ericoides) honeys. We found unique fluorescence profiles for these honeys which distinguished them from other New Zealand honey floral types. Two excitation-emission (ex-em) marker wavelengths each for manuka and kanuka honeys were identified; manuka honey at 270-365 (MM1) and 330-470 (MM2) nm and kanuka honey at 275-305 (KM1) and 445-525 (KM2) nm. Dilution of manuka and kanuka honeys with other honey types that did not possess these fluorescence profiles resulted in a proportional reduction in fluorescence signal of the honeys at the marker wavelengths. By comparison, rewarewa (Knightia excelsa), kamahi (Weinmannia racemosa), and clover (Trifolium spp.) honeys did not exhibit unique fluorescence patterns. These findings suggests that a fluorescence-based screening approach has potential utility for determining the monoflorality status of manuka and kanuka honeys.

Purification, crystallization and preliminary crystallographic analysis of mouse myo‐inositol oxygenase

Myo-inositol oxygenase (MIOX) catalyzes the novel oxidative cleavage of myo-inositol (MI) and its epimer D-chiro inositol (DCI) to D-glucuronate. MIOX utilizes an Fe(II)/Fe(III) binuclear iron centre for the dioxygen-dependent cleavage of the C1-C6 bond in MI. Despite its key role in inositol metabolism, the structural basis of its unique four-electron oxidation mechanism and its substrate specificity remain unknown. In order to answer these questions and to facilitate the use of this key enzyme for the development of new therapeutic strategies for diabetes, the mouse enzyme has been cloned, expressed in Escherichia coli, purified and crystallized from 4.4 M sodium formate. The crystals belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 44.87, b = 77.26, c = 84.84 angstroms, and diffract to 2.8 angstroms resolution.

Isolation, Structural Elucidation, and Synthesis of Lepteridine From Ma̅nuka (Leptospermum scoparium) Honey

Ma̅nuka honey, made from the nectar of Leptospermum scoparium, has garnered scientific and economical interest due to its nonperoxide antibacterial activity. Biomarkers for genuine ma̅nuka honey are increasingly in demand due to the presence of counterfeit ma̅nuka honey. This work reports the identification of a compound previously unreported in ma̅nuka 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 ma̅nuka honeys and could potentially serve as a biomarker for genuine ma̅nuka honey.