Type 2 diabetes mellitus (T2DM) is characterized by an ∼60% deficit in β-cell mass, increased β-cell apoptosis, and islet amyloid derived from islet amyloid polypeptide (IAPP). Human IAPP (hIAPP) forms oligomers, leading to either amyloid fibrils or toxic oligomers in an aqueous solution in vitro. Either application of hIAPP on or overexpression of hIAPP in cells induces apoptosis. It remains controversial whether the fibrils or smaller toxic oligomers induce β-cell apoptosis. Rifampicin prevents hIAPP amyloid fibril formation and has been proposed as a potential target for prevention of T2DM. We examined the actions of rifampicin on hIAPP amyloid fibril and toxic oligomer formation as well as its ability to protect β-cells from either application of hIAPP or endogenous overexpression of hIAPP (transgenic rats and adenovirus-transduced β-cells). We report that rifampicin (Acocella G. Clin Pharmacokinet 3: 108–127, 1978) prevents hIAPP fibril formation, but not formation of toxic hIAPP oligomers (Bates G. Lancet 361: 1642–1644, 2003), and does not protect β-cells from apoptosis induced by either overexpression or application of hIAPP. These data emphasize that toxic hIAPP oligomers, rather than hIAPP fibrils, initiate β-cell apoptosis and that screening tools to identify inhibitors of amyloid fibril formation are likely to be less useful than those that identify inhibitors of toxic oligomer formation. Finally, rifampicin and related molecules do not appear to be useful as candidates for prevention of T2DM.
- islet amyloid polypeptide
- human islet amyloid polypeptide
type 2 diabetes mellitus (T2DM) is characterized by an ∼60% reduction in β-cell mass and the intraislet formation of amyloid (5, 7). Islet amyloid is derived from islet amyloid polypeptide (IAPP), a 37-amino acid protein that is cosecreted along with insulin from pancreatic β-cells (8, 35). Although an association between islet amyloid and diabetes was recognized at the beginning of the past century (28), there is now increasing evidence for a link between the formation of islet amyloid and the β-cell loss in T2DM (11, 26), even though it is unclear whether this is due to the actions of mature human IAPP (hIAPP) fibrils (11, 15, 34) or their oligomeric precursors (13, 14). Thus exposure of isolated human islets or β-cells in culture to hIAPP peptide results in increased apoptosis (19, 21, 29), and overexpression of hIAPP induces cell death (25). Furthermore, mice and rats transgenic for hIAPP show a progressive β-cell loss due to increased β-cell apoptosis, and this process has been linked to the formation rather than the mere presence of islet amyloid (4, 6). Therefore, inhibition of hIAPP fibril formation is a theoretical approach to prevent β-cell loss and delay or prevent type 2 diabetes in predisposed subjects.
The inhibition of amyloid formation has been proposed as a strategy to prevent or delay several neurodegenerative diseases characterized by local aggregates of amyloidogenic proteins and neuronal cell death. For example, Alzheimer β-protein 1–42 (AβP1–42) derived aggregates in Alzheimer's disease (10), α-synuclein derived aggregates in the form of Lewy bodies in Parkinson's disease (12), and Huntington derived aggregates in Huntington's disease (2).
It has been reported (23) that there was an apparent decreased frequency of neurofibrillary tangles and senile plaques in the brains of patients chronically treated with antibiotic drugs for leprosy. This observation prompted investigators to study whether drugs commonly used in leprosy have any effects on AβP1–42 fibril formation. It was reported that rifampicin inhibited AβP1–42 fibril formation in vitro (30), and similar results were subsequently obtained with hIAPP (31) and α-synuclein (18, 27). However, it has not been examined whether inhibition of hIAPP fibril formation by rifampicin prevents hIAPP-induced apoptosis; a prerequisite before this approach is explored therapeutically.
Also, there is accumulating evidence that toxicity by hIAPP and other amyloidogenic proteins is mediated through toxic oligomers that are distinct from the insoluble amyloid fibrils that make up the extracellular amyloid deposits (16). Recently, it has become possible to detect the toxic form of hIAPP oligomers by antibodies specific to toxic oligomers that do not cross-react with IAPP monomers or amyloid fibrils (9, 16). Taking advantage of this tool, we sought to address the following questions in the present study. 1) Does rifampicin inhibit the formation of hIAPP oligomers and/or fibrils, and 2) does rifampicin prevent hIAPP-induced β-cell apoptosis? Through these studies, we planned to establish whether toxic oligomers or amyloid fibrils are the form in which hIAPP induces β-cell apoptosis and thus shed light on the most appropriate readout of screening studies for candidate compounds for the prevention of hIAPP toxicity. Finally, we sought to establish the potential of rifampicin as a candidate starting point for molecules that can prevent hIAPP toxicity.
MATERIALS AND METHODS
hIAPP (lot no. 0504-011) was purchased from PolyPeptide (Wolfenbuettel, Germany). Lyophilized hIAPP was dissolved in 0.5% acetic acid to prepare a 2 mM stock solution. The stock solution was then diluted with the culture medium to obtain the desired final hIAPP concentrations. Acetic acid concentrations in the culture medium applied to cells were always <0.003%. Rifampicin was purchased from Sigma (St. Louis, MO) and dissolved in DMSO at stock concentrations of 50 mM. The antibody for the toxic oligomers of hIAPP (rabbit I-11) was prepared in the laboratory of Charles Glabe, University of Irvine, Irvine, CA, in an identical fashion to that previously prepared against AβP1–42 oligomers, as previously described (16). This antibody binds specifically to cytotoxic oligomers formed by hIAPP but not to its monomeric or fibrillar form. To determine the specificity of the I-11 antibody, increasing amounts of hIAPP monomers, oligomers, and fibrils were plated on an ELISA plate, as previously described (16), and detected with 1 μg/ml affinity-purified I-11 antibody (Fig. 1). These experiments showed specific binding to hIAPP oligomers but minimal cross-reactivity with monomers and fibrils of hIAPP. In addition, dot blot experiments carried out in analogy with those previously described for the A-11 antibody (16) revealed specific binding of the I-11 antibody with Aβ42 oligomers, α-synuclein oligomers, and IAPP oligomers but not with Aβ42 monomers or Aβ42 fibrils (Fig. 1).
Thioflavin T fluorescence assay.
hIAPP fibril formation in the presence or absence of rifampicin was monitored using thioflavin T (ThT) fluorescence, a dye known to preferentially bind amyloid fibrils as decribed (19). ThT fluorescence increases in a solution of freshly reconstituted hIAPP as amyloid fibrils grow. Each fibril formation reaction was performed at 5 μM ThT in buffer (10 mM sodium phosphate, 100 mM NaCl), and real-time emission intensities were measured at 482 nm with excitation at 450 nm. Measurements were performed at room temperature with excitation and emission slit widths of 1 and 10 nm, respectively. Fluorescence measurements were taken using a Jasco FP-6500 spectrofluorometer. Plots of ThT emission intensity as a function of time were fitted to a sigmoidal curve (nonlinear regression analysis).
Detection of hIAPP oligomers by dot blot.
hIAPP peptide was freshly dissolved in 0.5% acetic acid at a final concentration of 100 μM in combination with rifampicin at concentrations of 3, 10, and 100 μM or vehicle. Aliquots of rifampicin at 3, 10, and 100 μM or vehicle were prepared as controls. The samples were kept at room temperature in light-protected vials. Single dots (∼2 μl each) of each sample were spotted on nitrocellulose membranes (Schleicher and Schuell, Keene, NH) immediately after preparation of the solutions and 1, 2, 4, 6, 8, 24, 48, 72, and 96 h after incubation at room temperature. Subsequently, the membranes were washed five times in TBS-0.001% Tween 20 (TBST) and blocked in 10% milk in TBST for 1 h at room temperature. The membranes were then incubated at 4°C overnight with rabbit anti-oligomer antibody (I-11, 1:3,000 dilution in 3% BSA in TBST). After being washed repeatedly (5 times) in TBST, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:3,000 dilution; Zymed Laboratories, San Francisco, CA) for 1 h. After five washes with TBST, proteins were visualized using enhanced chemiluminescence (ECL; Bio-Rad, Hercules, CA).
The reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) was used to assess cell viability as described previously (16). To test the effects of rifampicin or exogenous hIAPP, rat insulinoma (RIN) cells were seeded on a 96-well plate at 2.0 × 104 cells/well. The next day, the medium was replaced with fresh medium containing freshly dissolved hIAPP, rifampicin, or vehicle at the respective desired concentrations. Twenty-four hours later, the medium was changed to 1 mg/ml MTT (Sigma) containing medium according to the manufacturer's instructions. Colorimetric measurements were made 4 h after addition of the MTT reagent at 570 nm with an ELISA plate reader (Spectramax 250; Molecular Devices, Sunnyvale, CA). The background wavelength at 690 nm was subtracted from the 570-nm measurement.
To test the effects of adenoviral overexpression of hIAPP, cells were transduced with adenovirus expressing Ad-hIAPP-EGFP at a multiplicity of infection (MOI) of 1,000. At the same time, rifampicin or vehicle was added to the medium. After 48 h, cells were washed, MTT containing medium was added, and measurements were carried out as described above.
Measurement of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining.
RIN cells were cultured in chamber slides coated with a human tumor bladder-9 matrix, as previously described (3, 29). Twenty-four hours after treatment, cells were fixed and permeabilized, followed by the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining, which was performed according to the manufacturer's instructions (in situ Cell Death Detection Kit, TMR red; Roche Diagnostics, Mannheim, Germany) with the nuclear marker 4,6-diamidino-2-phenylindole. Fluorescent slides were viewed using a Leica DM6000 microscope (Leica Microsystems, Bannockburn, IL), and images were acquired using Openlab software (Improvision, Lexington, MA). The percentage of cells positive for apoptosis was quantified in each chamber and expressed as a percentage of the total cell number.
The complementary cDNA encoding the full-length human preproIAPP was used as a template to replace the stop codon with TCG and introduce an EcoRI restriction site using PCR mutagenesis. The amplified products were digested with HindIII and EcoRI and ligated into pEGFP-N2 vector (Clontech, Palo Alto, CA) in frame with an enhanced green fluorescent protein (EGFP). The plasmid pEGFP-N2/human preproIAPP-EGFP was subsequently digested with HindIII and XbaI and inserted into pShuttle-CMV adenovirus vector (Stratagene, La Jolla, CA). The recombinant adenovirus human preproIAPP-EGFP (Ad-hIAPP-EGFP) was generated and purified as described previously (20).
RIN cells were lysed in 2× Laemmli sample buffer, and protein concentrations were determined using Bio-Rad protein assay reagents. To establish the expression of IAPP in Ad-IAPP-EGFP-transduced RIN cells, 10 μg of protein were separated on a 4–12% Bis-Tris NuPAGE gel in MES buffer (Invitrogen) and transferred to polyvinylidene difluoride membranes. As a control, 5 ng of human amylin peptide (Bachem, Torrance, CA) were loaded. Membranes were blocked with 1% gelatin in 0.25% Triton X-100-PBS (PBS-T) for 1 h at room temperature and incubated overnight at room temperature in 1% gelatin PBS-T containing anti-amylin 25–37 amide (human) IgG (1:1,000; Peninsula Laboratories, San Carlos, CA) or anti-GFP (1:1,000; Zymed Laboratories) antibodies. Membranes were washed with PBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized using ECL (Bio-Rad).
Islet isolation and pancreatic tissue processing.
Pancreatic islets from 3-mo-old rats transgenic for human islet amyloid polypeptide (HIP rats; n = 3) and control Sprague-Dawley rats (n = 3) were isolated as described previously (17). Rats were bred and housed at the animal housing facility of the University of California Los Angeles. To obtain islets, rats were killed by intraperitoneal injection of pentobarbital sodium (50 mg/kg). The bile duct was cannulated, and Hanks' solution (Flow Labs, Irvine, UK) containing 7.5 mM calcium chloride, 20 mM HEPES buffer, and collagenase (1 mg/ml of type II; Sigma) was injected to uniformly distend the pancreas. The pancreas was then removed and incubated for 20 min in Hanks' solution at 37°C, followed by transfer into Hanks' solution containing 5 g/l BSA and 20 mM HEPES buffer at 4°C. The pancreas was dispersed by gentle shaking and washed four times in HBSS to remove collagenase and small debris of digested tissue. Subsequently, islets were transferred into RPMI medium (containing 11 mM glucose) and handpicked three times to exclude exocrine debris from the culture. Islets were then incubated in RPMI medium (containing 11 mM glucose) for 24 h to recover from the isolation procedure.
Detection of islet cell death using propidium iodide staining.
After recovery from the islet isolation procedure, aliquots of ∼30 islets each were hand picked in culture dishes with RPMI medium (11 mM glucose) containing rifampicin at concentrations of 3, 10, 100, or 1,000 μM, or vehicle, and incubated at 37°C for 48 h. Subsequently, islets were washed in RPMI medium and stained with 0.5 μM propidium iodide (PI; Molecular Probes, Eugene, OR) for 20 min at 37° C. Immediately after PI staining, the culture dishes were transferred into the incubation chamber of a Leica AS MDW confocal microscope, which allows imaging of the islets under regular culture conditions (37°C, constant inflow of humidified air containing 5% CO2). Images of 10 islets each were acquired in bright field and fluorescence at 568 nm (Cy3) at ×20 objective magnification. The fractional area of the islets positive for PI was digitally quantified using Image-Pro Plus software (Media Cybernetics, Silver Spring, MD).
Data are presented as the means ± SE. Statistical analyses were carried out by ANOVA followed by Duncan's post hoc test using Statistica, version 6, (Statsoft, Tulsa, OK). A P value of <0.05 was taken as evidence of statistical significance.
Formation of hIAPP fibrils.
Formation of hIAPP fibrils from monomeric hIAPP was monitored using ThT, a dye known to avidly bind amyloid fibrils. The emission intensity at 482 nm was measured over a time course of 1,000–1,500 s until a plateau for the respective intensity levels was reached. With 25 μM hIAPP, a maximum intensity in ThT emission was observed within ∼1,000 s. In contrast, both the time course and the maximal intensity of ThT emission for hIAPP were dose-dependently delayed in the presence of rifampicin, indicating inhibition of fibril formation (Fig. 2). Of note, a reduction in hIAPP fibril formation was found at rifampicin concentrations as low as 3 μM.
Detection of hIAPP oligomers by dot blot.
Anti-toxic oligomer staining using the I-11 antibody revealed the presence of hIAPP oligomers in the freshly dissolved hIAPP preparation (Fig. 3). There was no detectable difference in the amount of hIAPP oligomers between the samples with hIAPP plus vehicle or with rifampicin at 3 and 10 μM, but the overall staining intensity appeared to be lower with hIAPP plus 100 μM rifampicin. However, inspection of the control samples with rifampicin treatment in the absence of hIAPP revealed that this was likely due to bleaching of the nitrocellulose membrane by high rifampicin levels rather than to a true rifampicin effect on hIAPP oligomerisation. Oligomers of hIAPP were detectable at similar amounts during all time points with a trend toward lower oligomer concentrations immediately after preparation of the hIAPP solution.
RIN cell viability assays.
Treatment of RIN cells with rifampicin for 24 h at concentrations from 0 to 3 μM did not influence cell viability, whereas a dose-dependent reduction in cell viability was found at concentrations from 12.5 to 200 μM (P < 0.0001; Fig. 4).
Application of exogenous hIAPP at concentrations from 20 to 80 μM led to a dose-dependent reduction in cell viability (P < 0.0001; Fig. 5). Since rifampicin treatment was found to be independently cytotoxic at higher concentrations, the highest nontoxic concentration (3 μM) that had been shown to inhibit hIAPP fibril formation was used to examine the possible protective effects of rifampicin on hIAPP-induced cell death. However, no changes in cell viability were found when rifampicin was added alone or together with hIAPP at concentrations from 20 to 40 μM (Fig. 5).
To assess the effect of rifampicin on cell death induced by endogenous expression of hIAPP, RIN cells were transduced with Ad-hIAPP-EGFP at an MOI of 1,000. A high proportion of RIN cells (∼90%) was fluorescent 24 h after preproIAPP-EGFP transduction (Fig. 6A). Human preproIAPP-EGFP was processed to hIAPP in RIN cells, as shown in Fig. 6B. Transduction of RIN cells for 48 h led to a reduction in cell viability to 76 ± 3% of controls (P < 0.001). Addition of rifampicin at concentrations from 3 to 200 μM did not protect from this deleterious effect of hIAPP overexpression. In contrast, the reduction in cell viability was even aggravated at increasing concentrations of rifampicin (P < 0.0001; Fig. 7).
Measurement of apoptosis in RIN cells.
Addition of hIAPP at concentrations of 20 and 40 μM led to a dose-dependent increase in the percentage of apoptotic RIN cells, as detected by TUNEL staining (Fig. 8). Addition of rifampicin alone at 3 μM did not have any effect on the frequency of apoptosis (P = 0.85 vs. controls). Likewise, rifampicin did not prevent the deleterious effects of 20 or 40 μM hIAPP (P = 0.52 and P = 0.44 vs. controls, respectively).
Rifampicin effects on isolated rat islets.
In control rats, the extent of cell death estimated by the fractional islet area positive for PI staining was relatively low (0.40 ± 0.13%; Fig. 9), assuring sufficient recovery of the islets after the isolation procedure. Although treatment with rifampicin at 3 and 10 μM did not have a measurable effect on islet cell viability, there was a dose-dependent increase in fractional PI staining at higher rifampicin concentrations, with ∼20% of the islet being PI positive after 1,000 μM rifampicin (P < 0.0001). In islets isolated from HIP rats, the PI-positive area was already ∼15-fold higher during vehicle treatment compared with control rats (P < 0.001; Fig. 9). There was no detectable protection from cell death induced by the hIAPP transgene at any concentration of rifampicin. To the contrary, rifampicin treatment led to an increase in the extent of cell death in the HIP rat islets (P < 0.01).
The present studies confirm that rifampicin does prevent IAPP fibril formation. However, using new specific antibodies, our experiments for the first time demonstrate that rifampicin does not prevent the formation of toxic hIAPP oligomers. Therefore, not surprisingly, we also report that rifampicin does not protect cells from the toxic actions of either exogenously applied hIAPP or high endogenous expression rates of hIAPP and is therefore not a useful target for diabetes prevention. These data imply that screening compounds for their capacity to prevent fibril formation may not be a rational approach to establish novel candidates for therapies for type 2 diabetes (or amyloid-associated neurodegenerative diseases).
To examine the consequences arising from the rifampicin-mediated inhibition of fibril formation for the cytotoxic effect of hIAPP, we measured the viability of RIN cells using the MTT assay. As expected, addition of freshly dissolved hIAPP led to a dose-dependent reduction in the viability of RIN cells, but this process was not prevented by the addition of rifampicin. However, under physiological conditions, hIAPP is produced by and not applied to β-cells (24). Therefore, to more closely recapitulate in vivo conditions, we transduced a β-cell line (RIN cells) with Ad-hIAPP-EGFP to create a model of intracellular hIAPP expression, a strategy that did indeed increase β-cell apoptosis. However, even under these conditions, rifampicin did not protect β-cells from the toxic effects of hIAPP. Finally, since β-cell lines have an increased vulnerability to apoptosis due to increased replication (22, 29), we studied the effects of rifampicin on isolated pancreatic rat islets. For these purposes, we employed a hIAPP transgenic rat model characterized by progressive β-cell loss due to increased β-cell apoptosis (HIP-rats) (4), features that closely resemble the islet pathology in humans with T2DM (5). Consistent with previous studies of this rat model (4), there was a substantial increase in the frequency of cell death compared with islets from control rats. Rifampicin did not alleviate the high frequency of β-cell destruction conferred by the hIAPP transgene in these islets in culture. Taken together, these data therefore demonstrate that, despite its effects on hIAPP fibril formation, rifampicin does not protect against the deleterious effects of hIAPP on β-cells.
Interestingly, the sensitivity of isolated rat islets to rifampicin-induced toxicity was less than that of RIN cells. Most likely, this is due to the higher rate of replication in cell lines compared with primary β-cells in culture, given the increased vulnerability of replicating β-cells to cell death (22, 29).
One limitation of this as well as of previous studies that examined the effects of rifampicin on fibril formation of hIAPP or amyloid β-peptide (18, 30–32) is that, for the detection of ThT binding, the respective peptides had to be diluted in buffer to prevent interference with the spectrofluorometric measurements. Therefore, it is theoretically possible that the actual effects of rifampicin on hIAPP fibril formation in vivo differ from those obtained in the present in vitro experiments. However, since the effects of rifampicin on hIAPP fibril formation are mediated through a direct chemical interaction between both molecules (Fig. 2), there is little reason to expect different results under in vivo conditions.
There are at least two possible explanations for the lack of rifampicin effect on hIAPP toxicity. It is conceivable that the cytotoxicity induced by rifampicin itself outweighed protection from the deleterious effects of hIAPP. However, cytotoxic effects of rifampicin required concentrations of >12.5 μM, whereas hIAPP fibril formation was inhibited at concentrations of 3 and 10 μM rifampicin, at least in vitro. An alternative explanation is that the cytotoxic effects of hIAPP are primarily mediated by toxic oligomers that are unaffected by rifampicin. This explanation seems the more plausible since we were able to measure toxic hIAPP oligomers directly, and in contrast to hIAPP fibrils, these were not decreased by rifampicin. In fact, if production of hIAPP fibrils is blocked by rifampicin, this might even lead to an accumulation of toxic hIAPP oligomers and therefore increase rather than inhibit the toxicity of hIAPP. However, although this mechanism cannot be fully excluded from the present data, the dot blot experiments showing no effects of rifampicin on hIAPP oligomer formation do not support such reasoning (Fig. 3). Therefore, the present studies support the concept that formation of toxic oligomers of amyloidogenic proteins is a distinct pathway from that of amyloid fibril formation. Also, these data support the emerging concept that it is these toxic oligomers that induce cell death rather than intermediates of amyloid fibril formation (protofibrils; Fig. 10).
Several lines of evidence are supportive of these concepts in relation to hIAPP. First, it has previously been noted that the formation of islet amyloid from hIAPP in transgenic rodent models is readily dissociated from β-cell apoptosis. Thus, in a homozygous hIAPP transgenic mouse model, almost all β-cells had undergone apoptosis before the first extracellular islet amyloid was present, whereas small intracellular, nonfibrillar toxic hIAPP oligomers were present at the time of maximal β-cell loss (14). In contrast, in an alternative hIAPP transgenic mouse model on high-fat feeding, extensive islet amyloid developed without the mice developing diabetes (33), consistent with formation of inert IAPP fibrils in the absence of toxic oligomers. In line with these findings, studies of both rats and mice transgenic for hIAPP revealed no relationship between the extent of islet amyloid (i.e., amyloid fibrils) and the frequency of β-cell death (4, 6), and the location of apoptotic cells bears no physical relation to the site of the extracellular amyloid deposits. Freshly dissolved hIAPP is cytotoxic to β-cells and causes membrane disruption to membrane bilayers, whereas addition of preformed hIAPP fibrils has no toxic effects (21). Consistent with this, an antibody that binds specifically to the toxic oligomers, but not the amyloid fibrils, inhibited hIAPP-induced apoptosis in a human neuroblastoma cell line (16).
The failure of rifampicin to prevent hIAPP-induced β-cell death despite its potent inhibition of hIAPP fibril formation also demonstrates that this property alone is insufficient to identify agents with the potential to inhibit the toxicity associated with amyloidogenic peptides. The screening of strategies that seek targets that inhibit formation of toxic oligomers of amyloidogenic proteins, rather than fibrils, appears to be a more rational approach.
The β-cytotoxic effects of rifampicin reported here may, at first sight, raise concerns as to whether rifampicin treatment in patients with leprosy or tuberculosis might cause β-cell destruction. However, β-cytotoxicity was observed only at rifampicin concentrations >12.5 μM, levels higher than the typical plasma concentrations achieved during therapeutic administration of rifampicin (∼5–10 μM) and presumably substantially higher than typical tissue levels (1). Moreover, it is likely that the susceptibility of β-cell lines in culture to rifampicin toxicity is greater than that of adult human β-cells in vivo, given the increased vulnerability of replicating β-cells to cell death (22, 29).
In conclusion, the present studies confirm that rifampicin dose-dependently inhibits fibril formation of hIAPP. However, this action of rifampicin was not accompanied by any attenuation of the toxic effects of hIAPP either applied to β-cells or expressed endogenously by β-cells. We conclude that the toxic actions of amyloidogenic proteins are not directed through amyloid fibrils but rather through toxic oligomers. We also conclude that screening candidate compounds for the property to prevent amyloidogenic proteins from inducing apoptosis is not best served by quantifying the impact on fibril formation. Finally, these studies do not support the potential of rifampicin or related structures as promising candidates for the prevention of β-cell death induced by hIAPP in patients with T2DM.
These studies were funded by grants from the National Institutes of Health (DK-59579 to P. C. Butler and NS-31230 to C. G. Glabe), the Larry Hillblom Foundation, and the Deutsche Forschungsgemeinschaft (ME 2096/2-1).
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- Copyright © 2006 by American Physiological Society