Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.

Human amylin (hA) is a small protein cosecreted with insulin from pancreatic islet b-cells upon stimulation by glucose or other chemical signals [1,2]. Wild-type hA, also known as insulinoma amyloid peptide, insulinoma amyloid polypeptide (IAPP) or islet amyloid polypeptide [3], demonstrates a strong in vitro tendency to aggregate into fibrils [4,5], which is dependent on specific residues in different regions of the molecule, notably, the amyloidogenic region of amino acids 20-29 [6]. It is the main protein in the amyloid aggregates that are frequently present in the islets of human subjects with type 2 diabetes mellitus (T2DM) [7,8], as well as in diabetic cats and primates [9]. It has been reported that aggregated hA might contribute to the loss of insulin-producing pancreatic b-cells cells in T2DM [10,11]. The following lines of evidence have associated hA with the pancreatic pathology of T2DM: (a) amyloid is often concentrated near areas of islet b-cell degeneration in humans or primates with T2DM [10,[12][13][14]; (b) synthetic hA is toxic to pancreatic b-cells in vitro [11,[15][16][17]; (c) spontaneous loss of b-cells has been reported in a line of hA-transgenic mice [18]; and (d) toxicity of hA towards cultured cells correlates with its ability to form fibrils -for example, rat amylin does not form fibrils and nor does it evoke b-cell apoptosis in vitro [11,17,19]. 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 b-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 b-cells. Highly toxic amylin contained few preformed fibrils and initially showed little b-sheet content, but underwent marked time-dependent aggregation and b-conformer formation following dissolution. By contrast, low-toxicity amylin contained abundant preformed fibrils, and demonstrated high initial b-sheet content but little propensity to aggregate further once dissolved. Thus, mature amylin fibrils are not toxic to b-cells, and aggregates of fibrils such as occur in pancreatic islet amyloid in vivo are unlikely to contribute to b-cell loss. Rather, the toxic molecular species is likely to comprise soluble oligomers with significant b-sheet content. Attempts to find ways of protecting b-cells from amylin-mediated death might profitably focus on preventing the conformational change from random coil to b-sheet.Abbreviations AM, acetomethoxyl ester; EthD, ethidium homodimer; hA, human amylin; HFIP, hexafluoroisopropanol; IAPP, insulinoma amyloid polypeptide; JNK1, Jun NH 2 -terminal kinase 1; KRB, Krebs-Ringer bicarbonate buffer; RINm5F, rat insulinoma m5F; T2DM, type 2 diabetes mellitus; TEM, transmission electron microscopy; ThT, thioflav...

No abstract

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.

OBJECTIVEAggregation 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 METHODSWe chronically treated hemizygous hA/hIAPP transgenic mice with oral tetracycline to determine its effects on rates of diabetes initiation, progression, and survival.RESULTSHomozygous 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.CONCLUSIONSThis 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.

Human amylin (hA) is a small protein that is cosecreted with insulin from pancreatic islet b-cells upon stimulation by glucose or other chemical signals [1,2]. It is the main proteinaceous constituent of the amyloid deposits commonly found in the islets of human subjects with type-2 diabetes mellitus (T2Dm) [3,4], as well as in diabetic cats and primates [5,6], and has also been referred to as islet amyloid polypeptide (IAPP) [5]. The toxicity of hA towards cultured pancreatic b-cells is a well-documented phenomenon [7][8][9][10]. Post mortem examination of pancreatic tissue from T2Dm patients has shown a correlation between the presence of amyloid deposits in pancreatic islets and b-cell loss [11][12][13]. Evidence from longitudinal studies in spontaneously diabetic primates has also indicated an inverse relationship between b-cell number and the extent of islet amyloidosis [6]. Studies of hA transgenic animals have shown that the occurrence of islet amyloid is Keywords amylin; b-cell apoptosis; diabetes; oxidative stress; reactive oxygen species; N-acetyl- 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 b-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.Abbreviations BSO, buthionine-(S,R)-sulfoximine; carboxy-H 2 DCFDA, 5-(and-6)-carboxy-2¢,7¢-dichlorodihydrofluorescein diacetate; EthD-1, ethidium homodimer-1; hA, human amylin; JNK, c-Jun NH 2 -terminal kinase; KRH, Hepes-balanced Krebs-Ringer bicarbonate buffer; NAC, N-acetyl-L-cysteine; n-PG, n-propyl gallate; rA, rat amylin; RIN, rat insulinom...

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.

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 ) 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/␣CGRP 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 ␣CGRP differ from one another. Our chimeric approach enabled the identification of two nonselective highaffinity 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, ␣CGRP, and AM2 and how AM interacts with CGRP and AM 2 receptors.Human adrenomedullin (AM) is a 52-amino acid peptide belonging to the calcitonin family of peptides, which also includes calcitonin gene-related peptide (CGRP) and AM2 (also known as intermedin). The calcitonin peptide family shows weak homology at the level of the primary sequence, but there are stronger relationships at the secondary structure level. Each member of the family has an N-terminal ring structure and an amidated carboxyl terminus. Both of these structures are critical for receptor binding and subsequent signaling (Eguchi et al., 1994;Conner et al., 2002). Truncation of the N-terminal ring structure produces antagonist peptides in all family members. The ring structure of AM is created by the formation of a disulfide bond between two cysteine residues at positions 16 and 21 of the peptide (Eguchi et al., 1994). Truncation of AM yields the AM antagonist AM . Interfering with the C-terminal amidated tyrosine residue of AM results in a peptide with reduced receptor affinity (Eguchi et al., 1994). AM 13-52 and AM 15-52 are both agonists with similar affinity to full-length AM, however, little is known structurally about AM.The combination of the calcitonin receptor-like receptor (CL) with a receptor activity-modifying protein (RAMP) constitutes receptors that bind AM . CL is unusual for a G protein-coupled receptor in that it cannot f...