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Department of Genetic Medicine and Development (E.Z., P.A.H.), University Medical Centre and Division of Surgical Research (E.Z., T.B., D.B.), Department of Surgery, University Hospital, Geneva CH-1211, Switzerland; and Division of Endocrinology and Diabetes (K.M., M.Y.D.), University Hospital, Zurich CH-8091, Switzerland
Address all correspondence and requests for reprints to: Philippe A. Halban, Department of Genetic Medicine and Development, University Medical Center, 1 rue Michel-Servet, 1211 Geneva 4, Switzerland. E-mail: philippe.halban{at}medecine.unige.ch.
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Abstract |
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B (NFB)/Fas pathway. The aim of this study was to determine whether two drugs believed to block NFB activation, the thiazolidinedione (glitazone) pioglitazone and the nonsteroidal antiinflammatory drug sodium salicylate, can protect human ß-cells against the toxic effects of IL-1ß and high glucose in vitro. Human islets were maintained in culture 2–4 d at 100 mg/dl (5.5 mM) glucose with or without (control) IL-1ß or at 600 mg/dl (33.3 mM) glucose. IL-1ß and 600 mg/dl glucose increased ß-cell apoptosis and abolished short-term glucose-stimulated insulin secretion. Both drugs protected partially against loss of glucose-stimulated insulin secretion and prevented completely increased apoptosis caused by IL-1ß or 600 mg/dl glucose. IL-1ß secretion from islets was increased by 4-d culture at 600 mg/dl, and this was blocked by pioglitazone. Both drugs prevented activation of ß-cell NFB by high glucose. Pioglitazone and sodium salicylate thus protect human islets against the detrimental effects of IL-1ß and high glucose by blocking NFB activation and may therefore be useful in retarding the manifestation and progression of diabetes. |
Introduction |
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B (NFB) and the Fas-pathway (4, 5, 6). A central role of NFB in proapoptotic signaling is unusual, but it follows that preventing its activation should improve human ß-cell survival and function, and this has indeed been demonstrated by others (7). However, the approaches used to inhibit NFB, for example by overexpression of its natural inhibitor IB (7) or by down-regulation of IB kinase (IKKß) (8), may not be readily applicable in the clinical setting and would most probably not be suited to treatment of patients (8). In the present study, we have evaluated the antiapoptotic effect of two drugs (pioglitazone, a thiazolidinedione; and the nonsteroidal antiinflammatory agent, sodium salicylate) that have strong antiinflammatory effects via inhibition of NFB.
Thiazolidinediones (glitazones) are a class of oral agents used for treatment of type 2 diabetes and acting as ligands of the peroxisome proliferator-activated receptor-
(PPAR) (9, 10). It is commonly understood that the major clinical impact of this class of hypoglycemic drugs is to improve insulin sensitivity of target tissues, thereby augmenting glucose uptake and decreasing hepatic glucose output. However, PPAR is also expressed in human islet cells (11, 12). Indeed, and possibly as a consequence of direct action on ß-cells, troglitazone has been shown to improve insulin secretion in patients with impaired glucose tolerance (13) or type 2 diabetes (14), and this drug restored insulin secretion in the Zucker fatty diabetic rat (15). Pioglitazone, one of the later glitazones in current clinical use, similarly improves insulin secretion in patients with diabetes (16, 17) and in various murine models of diabetes (18, 19). Most recently, rosiglitazone was shown to preserve insulin secretion from human islets exposed to fatty acids in vitro (20).
Although originally described as a modulator of metabolism and adipocyte differentiation (21, 22), PPAR has now been shown to be expressed in immune-competent cells, leading to suggested use of its ligands, such as glitazones, in the treatment of inflammatory diseases, including atherosclerosis (23). Reports that activation of PPAR represses NFB activity in endothelial cells (24) and macrophages (25) are of particular relevance to the present study, even if an opposite (activation) effect has been reported in pre-B-cells (26), suggesting that there may be a complex dose-response curve and/or peculiar tissue-specificity. Troglitazone (27, 28) and rosiglitazone (29) decrease (lipotoxic) apoptosis in Zucker rat islets. However, in vitro, troglitazone did not protect sorted primary rat ß-cells against lipotoxicity (30). It remains unclear whether glitazones can exert a direct effect on human ß-cell survival in vitro.
Sodium salicylate at high doses was first shown to be beneficial in some cases of diabetes over 100 years ago (31, 32). Recently, interest in the compound has been revived by a study demonstrating that it can reverse obesity and insulin-resistance (33, 34). This action is not attributed to the well-known ability of low-dose aspirin to inhibit the cyclooxygenase enzymes, Cox1 and Cox2. Rather, it has been shown to be due to blocking NFB action (35) and most specifically by inhibiting the regulatory kinase IKKß that normally favors displacement of NFB from its complex with IB and its movement from the cytosol to the nucleus (33, 34). Sodium salicylate has been shown to block activation of NFB by IL-1ß in rat islets and to thereby prevent the loss of islet function normally induced by this cytokine (36). The possible antiapoptotic effect of sodium salicylate on human islets has not been investigated previously.
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Materials and Methods |
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Human islets were isolated from heart-beating organ donors at the Department of Surgery, University of Geneva Medical Centre as described (37, 38, 39, 40). If islet purity was less than 90% as judged by dithizone staining, they were handpicked. After isolation, islets were rinsed with CMRL 1066 culture medium containing 100 mg/dl (5.5 mM) glucose, 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.29 mg/ml (2 mM) glutamine (Invitrogen Ltd., Carlsbad, CA): CMRL. They were maintained in CMRL at 24 C for approximately 24 h.
Some of the islets were then cultured in batches of 20–30 in 2 ml CMRL at 37 C for a further 24 h on 3-cm-diameter plastic dishes coated with extracellular matrix derived from bovine corneal endothelial cells (Novamed Ltd., Jerusalem, Israel), allowing the cells to attach and spread to the dishes and thereby preserving their functional integrity (41, 42). The rest of the islets were cultured on nonadherent plastic Petri dishes in CMRL at 37 C. During the experiment itself, the culture medium contained either 100 or 600 mg/dl glucose. Concentrations of IL-1ß or drugs and time of culture were adapted according to the culture conditions (adherent or nonadherent). When indicated, islets were cultured with recombinant human IL-1ß (R&D Systems Inc., Minneapolis, MN): 2 ng/ml for the adherent islets and 5 ng/ml for the nonadherent islets, and/or with 0.4 µg/ml (1 µM) pioglitazone for adherent islets or 2 µg/ml (5 µM) for nonadherent islets (the generous gift of Takeda Chemical Industry, Ltd., Osaka, Japan) or 0.04 mg/ml sodium salicylate. This culture period lasted 4 d for the adherent islets and 2 d for the nonadherent islets (which is usually the longest period of culture before a graft).
Apoptosis
Adherent islets. The free 3'-OH strand breaks resulting from DNA fragmentation were detected by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL) technique (43). This method has been shown to reflect predominantly apoptosis, and not necrosis, in our conditions (5). After washing with PBS, cultured islets were fixed in 4% paraformaldehyde for 30 min at room temperature, followed by permeabilization with 0.5% Triton X-100 for 4 min at room temperature. The TUNEL assay was performed according to the manufacturer’s instructions (In Situ Cell Death Detection Kit, AP; Roche Molecular Biochemicals, Mannheim, Germany). The preparations were then rinsed with Tris-buffered saline and incubated for 10 min at room temperature with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium liquid substrate system (Sigma-Aldrich, St. Louis, MO). Thereafter, and to identify ß-cells, islets were incubated with a guinea pig antiinsulin antibody, followed by detection using streptavidin-biotin-horseradish peroxidase complex (Zymed Laboratories Inc., South San Francisco, CA). Only islets that had spread completely on the matrix to form a monolayer or had a central zone presenting a bilyaer of cells were scored for apoptosis. Apoptotic cells appeared randomly dispersed, presenting indifferently at the periphery or toward the center of each cluster. A mean of 25 islets/clusters was scored per condition and per experiment (range, 17–45; n = 3 independent experiments).
Nonadherent islets. The islets in suspension were fixed in paraformaldehyde 4% for 20 min. They were rinsed three times in PBS, embedded in agar, dehydrated, and embedded in paraffin. Sections (5 µm) were deparaffinated in ethanol and xylol. They were either stained with hematoxylin-eosin, to assess the quality of the tissue slices, or processed for apoptosis detection. To this end, the TUNEL technique was performed as above except that labeling of DNA strand breaks was with fluorescein. The slides were washed three times with PBS and blocked 20 min in PBS BSA 0.1%. An antiinsulin guinea pig antibody (home made), diluted 1:800, was applied to the sections during 2 h at room temperature. The slides were washed three times in PBS, and a rhodamine-linked anti-guinea pig goat IgG (The Jackson Laboratory, Bar Harbor, ME), diluted 1:400, was applied for 1 h to the sections at room temperature. The slides were washed three times in PBS and incubated with bisbenzimide (10 µg/ml) for 15 min at room temperature to color the nuclei. A mean of 364 (insulin-positive) ß-cells were scored per condition and per experiment (range, 74–898).
Insulin release and content
To determine short-term insulin release in response to glucose stimulation, islets adhering to extracellular matrix were washed in Krebs-Ringer bicarbonate buffer, 0.25% BSA, 2.38 mg/ml (10 mM) HEPES (KRB-HEPES), containing 60 mg/dl glucose, and preincubated for 30 min in the same buffer. The KRB-HEPES was then discarded and replaced with fresh buffer containing 60 mg/dl glucose for 1 h for basal secretion, followed by an additional 1 h incubation in KRB-HEPES containing 300 mg/dl glucose. Supernatants were collected and frozen for insulin assays. Thereafter, islets were washed with PBS and insulin extracted in acid ethanol for 24 h at 4 C. Insulin in KRB-HEPES and acid-ethanol extracts was determined using a human insulin RIA kit (CIS Bio International, Gif-sur-Yvette, France).
IL-1ß release
Adherent islets were cultured 4 d under the conditions described above. The culture medium was collected, and IL-1ß was measured by ELISA (R&D Systems, Inc.).
Determination of nitrite generation
Islets in suspension were cultured for 48 h under the conditions described above. Culture media were analyzed for nitrite levels using the Griess method. In brief, an equal volume of 1% sulfanilamide in 0.1 M HCl and 0.1% N-[-1-naphthyl-ethylenediamine dihydrochloride] was added to culture media. The samples were analyzed by spectrophotometry (570 nm). The results were correlated to a standard curve of NaNO2 (44, 45)
Measurement of NFB activation
Islets were cultured in suspension for 2 d, and activation of NFB complex was quantified with an ELISA-based Kit, using attached oligonucleotides binding to an NFB consensus site and detected by an anti-p65 subunit antibody, according to the manufacturer’s instructions (Trans-AM NFB, Active Motif, Carlsbad, CA). In parallel, islets were cultured on extracellular matrix-coated dishes. After the indicated culture time, islets were fixed in paraformaldehyde and incubated with mouse anti-NFB (p65) (Active Motif) antibody, detected by donkey antimouse Cy3 conjugated antibody and double-stained for insulin with guinea pig antiinsulin, followed by detection with fluorescein-conjugated rabbit anti-guinea pig antibody (Dako, Carpinteria, CA).
Statistical analysis
Comparison between groups was made using Student’s two-tailed t test for unpaired groups. A P value < 0.05 was considered statistically significant.
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Results |
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Human islets adhering to extracellular matrix were exposed to IL-1ß in the presence of 100 mg/dl (5.5 mM) glucose or to 600 mg/dl (33.3 mM) glucose for 4 d. After fixation, apoptosis was assessed using the TUNEL technique, with insulin-containing ß-cells identified concomitantly by immunostaining. The incidence of apoptotic ß-cells was 0.38 ± 0.05 cells/islet under standard (basal) conditions. ß-Cell apoptosis increased approximately 3-fold after exposure to IL-1ß or to high glucose (Fig. 1A
). Both pioglitazone and sodium salicylate prevented increased apoptosis due to either IL-1ß or glucose but had no effect on basal apoptosis. Human islets are not routinely cultured on extracellular matrix, regardless of whether they are used for experimental purposes or for clinical transplantation. Furthermore, the particular matrix used in this study (derived from bovine corneal endothelial cells) cannot be considered representative of the natural matrix encountered by human islet cells in vivo. Because the extracellular matrix may have provided an environment facilitating, in some way, the antiapoptotic effects of these two agents, the effects of pioglitazone were also tested using islets cultured in suspension. The results were quite similar to those seen for adherent islets, with protection against apoptosis induced by either IL-1ß or 600 mg/dl glucose (Fig. 1B). Interestingly, ß-cell apoptosis under basal conditions (culture at 100 mg/dl glucose) was higher in islets cultured in suspension than on matrix (1.29 ± 0.54 vs. 0.38 ± 0.05 cells/islet, respectively).
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To assess whether pioglitazone and sodium salicylate had any impact on insulin secretion, human islets were maintained in culture for 4 d with IL-1ß or 600 mg/dl glucose, and then short-term insulin secretion (1-h) was evaluated at low (60 mg/dl) or high (300 mg/dl) glucose. Under control conditions, high glucose elicited a 2- to 3-fold stimulation of insulin secretion (Fig. 2). Whereas treatment for 4 d with sodium salicylate decreased subsequent short-term insulin secretion under basal conditions, there was a no such effect of pioglitazone. By contrast, short-term basal secretion was decreased after 4 d with IL-1ß in combination with pioglitazone but not sodium salicylate. There was essentially complete suppression of such short-term stimulation of insulin secretion by glucose after 4 d of culture with either IL-1ß or 600 mg/dl glucose, and both pioglitazone and sodium salicylate prevented, in part, this suppression but failed to restore the short-term insulin response to glucose to control levels. As for apoptosis, similar experiments were performed to test the effects of pioglitazone on islets cultured in suspension. Although the drug was able to prevent the decreased secretion seen after a 2-d period with IL-1ß, there was no protection against the toxic effects of high glucose (data not shown).
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Islets on matrix were cultured for 4 d at 100 or 600 mg/dl glucose, with or without pioglitazone, and the secretion of IL-1ß measured by ELISA. IL-1ß release from islets was increased 4.4-fold by high glucose, and this increase was prevented completely by pioglitazone (Fig. 3). No significant effect of sodium salicylate on IL-1ß secretion was observed at either low or high glucose (data not shown).
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Pioglitazone and sodium salicylate protect human ß-cells against activation of NFB by glucose
The ability of both pioglitazone and sodium salicylate to protect against apoptosis induced by glucose or exogenous IL-1ß suggested a possible blockade of NFB activation. To test this directly, human islets were incubated in suspension for 2 d and NFB activation quantified by ELISA. High glucose (600 mg/dl) increased NFB activity to approximately 150% of control (100 mg/dl glucose), and both agents prevented this increase (Fig. 4A). To confirm whether the increase in NFB activity and its prevention by pioglitazone pertained specifically to ß-cells, rather than other islet cell types possibly contributing to the measurement of NFB activity by ELISA, and whether the drug was active in this regard on islet cells in monolayer as well as in suspension, islets adhering to extracellular matrix were immunostained for the presence in the nucleus (nuclear translocation) of the active p65 subunit of NFB. ß-Cells were identified by colabeling using antiinsulin. Culture of human islets at 600 mg/dl glucose increased nuclear staining for p65 in ß-cells, reflecting NFB activation in these cells. Such positive staining for NFB activation in ß-cells at high glucose was not apparent when islets were cultured in the presence of pioglitazone (Fig. 4B).
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Discussion |
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B activation as a major signaling event leading to ß-cell apoptosis. The drugs were selected on the basis of their known or postulated ability to block NFB activation and for their possible beneficial impact on islet function and/or insulin sensitivity.
The impact of NFB signaling on the survival of cells is enigmatic. In many cell types, activation of this transcription factor protects against apoptosis, and indeed there is one recent report of such an activity in insulin-secreting cells (46). However, in this study, aside from use of transformed (MIN6) mouse cells rather than primary human islet cells, attention was focused on apoptosis induced by TNF
. Based on studies on apoptosis induced in primary ß-cells by IL-1ß or glucose, the general understanding is that NFB activation is a major proapoptotic pathway in primary ß-cells and, most importantly, in human ß-cells. In keeping with this, numerous studies have shown that blocking NFB activation or signaling protects ß-cells against apoptosis (4, 7, 45, 47, 48, 49, 50, 51, 52, 53). The results from our previous studies have allowed us to formulate a unifying hypothesis explaining the molecular basis for induction of apoptosis in human ß-cells and centered on NFB activation. It was thus shown that glucose stimulates IL-1ß secretion from ß-cells themselves and that the cytokine, in turn, is responsible for activating NFB, leading to cell death (4). The downstream endpoint of NFB signaling is furthermore governed by intracellular levels of the caspase 8 inhibitor FLIP (FLICE-inhibitory protein), providing a switching mechanism between mitogenic or apoptotic effects (6).
Pioglitazone was seen to protect human ß-cells against apoptosis induced by exogenous IL-1ß or high glucose. The drug was used in vitro at concentrations of 0.4–2 µg/ml, which fall beneath the maximum concentration of approximately 1.5 µg/ml observed in pharmacokinetic studies in individuals with normal renal function after an oral dose of 45 mg (54). There was a concomitant beneficial impact on glucose-stimulated insulin secretion. The antiapoptotic effects of pioglitazone were accompanied by blockade of glucose-induced secretion of IL-1ß from islets and of NFB activation in ß-cells, in keeping with similar blockade by glitazones in other cell types (24, 25, 55, 56, 57). The decrease in IL-1ß may, in turn, impact on ß-cell apoptosis (4). In keeping with our previous study (4) showing that IL-1ß alone does not impact on inducible NO synthase expression by human islets, as discussed in detail in that same study (4) and as shown by others (58), no increase in NO production was seen during 2- to 4-d exposure of human islets to this cytokine or to high glucose. Pioglitazone was, in any event, without effect on this parameter. Use of glitazones in vivo, both in diabetic fatty Zucker rats (15, 59, 60) and in man (13, 14, 16), results in improved ß-cell function (in rats and man) and preserved ß-cell mass or islet morphology (in rats), but it has not been possible from these studies to conclude whether this is due to direct beneficial effects of the drug, or rather secondary to its hypoglycemic action via sensitization of target tissues to insulin and/or possibly inhibition of glucagon gene expression (61). Indeed, the only possibly direct effect of a glitazone demonstrated in vivo was increased protein levels of carboxypeptidase B in islets from both obese and control mice treated with rosiglitazone (62). More recent clinical studies do support a direct effect of glitazones on ß-cell function, albeit without evidence for the underlying molecular mechanism, leading to the hypothesis of ß-cell rejuvenation by this class of drugs (63). Interestingly, troglitazone has been shown to prevent autoimmune diabetes in NOD mice, possibly by direct effects on ß-cells as well as T cells (56). Whether it is valid to extrapolate from this particular model of autoimmune diabetes in the mouse to type 1 diabetes in man remains to be established.
Most of the previous studies examining the direct effects of glitazones on ß-cell function in vitro were performed using troglitazone (30, 64, 65, 66). This particular molecule has been associated with hepatoxicity and may act on ß-cells in ways different from those of the newer glitazones now in use, including pioglitazone used in the present study. Given that fatty acids are natural ligands of PPAR, the focus of earlier studies was furthermore typically on lipotoxicity rather than the possibly more relevant apoptotic effects of IL-1ß/glucose. The interpretation of such earlier studies is made more complicated by the fact that most of those studies used transformed insulin-secreting cell lines rather than primary islets or ß-cells (30). PPAR agonists have been shown to be proapoptotic in a variety of tumor cells or transformed cell lines, and it is possible that survival signaling in ß-cells is altered upon transformation. Finally, it cannot be stressed enough that rodent and human ß-cells differ in many respects and notably in their susceptibility toward cytokine and glucose-induced apoptosis. In contrast to the present study, most previous ones on the effect of glitazones on islet cell survival and function have focused on rodent islets.
In the present study, pioglitazone was without effect on insulin secretion from human islets maintained in culture at 100 mg/dl (5.5 mM) glucose. Others have found similar results using human islets under standard conditions of culture (20). The impact of glitazones on rodent ß-cell secretion is controversial. Whereas rosiglitazone was shown to enhance insulin secretion from the perfused rat pancreas (67) and from isolated mouse islets (68), this effect could not be confirmed using isolated rat islets (69). Whether these contrasting results are the consequence of the different species of ß-cell and/or the experimental conditions used remains to be established. It remains unclear why both pioglitazone and sodium salicylate completely prevented apoptosis induced by culture with IL-1ß or high glucose but only partially the loss of short-term glucose-stimulated insulin secretion induced by these same culture conditions. Presumably, multiple and partially overlapping signaling pathways are triggered, with only those leading to apoptosis being inhibited by the two drugs.
We are aware that the levels of IL-1ß attained in the medium bathing the human islets, and consequent to its secretion from islets, is low compared with the concentration of exogenous IL-1ß needed to elicit ß-cell apoptosis. First, it should be noted that there is degradation of IL-1ß during the culture period. Thus, the amount of IL-1ß produced by the islets is underestimated when measured at the end of this period. Conversely, the bioavailability of exogenous IL-1ß will be compromised by degradation during culture. Regardless, we have shown previously that addition to culture medium of the naturally occurring soluble IL-1ß receptor antagonist IL-1Ra protects human ß-cells against glucose-induced apoptosis, lending support to the hypothesis that increased production by islets of this cytokine does indeed trigger ß-cell apoptosis, at least in this in vitro setting (4). Most recently, we have shown that IL-1Ra is also produced by human islets and that its down-regulation by introduction of small interfering RNA (siRNA) enhanced glucose-induced ß-cell apoptosis (70). The fact that pioglitazone protects islets against apoptosis induced by exogenous IL-1ß at 100 mg/ml glucose, in addition to preventing IL-1ß secretion elicited by culture at high glucose, is intriguing. An explanation will become apparent only when the proapoptotic signaling pathways of glucose and IL-1ß in human islets, and most specifically signals leading to increased IL-1ß secretion, are fully elucidated.
As for PPAR agonists, there were data in the literature suggesting that sodium salicylate might present dual action compatible with the requirements of the present investigation: blockade of NFB activation and insulin sensitization. It was thus rewarding to discover that this nonsteroidal antiinflammatory drug does indeed protect human islets against glucose-induced apoptosis. The impact on insulin secretion was predicted by earlier studies on rat islets and has been suggested to reflect blockade by sodium salicylate of IL-1ß activation of NFB, thus preventing up-regulation of expression of cyclooxygenase-2 (COX-2) and prostaglandin E receptor subtype 3 (EP3) (36) as well as to decreased cAMP levels (71). However, extrapolation of such previous studies on rat islets or transformed cell lines to human islets is not straightforward, given the striking differences in response to glucose and cytokines. For example, IL-1ß alone stimulates NO production by rat islets and RIN cells; this is prevented by low levels of sodium salicylate but without concomitant effects on NFB activity (72). Interestingly, it had been observed many years ago that aspirin can improve insulin secretion in individuals with type 2 diabetes (73). Just as for the beneficial impact of glitazones on insulin secretion, whether this is attributable to direct effects on the ß-cell or indirect effects secondary to improved insulin-sensitivity remains to be established. In the present study, sodium salicylate, when present at 0.04 mg/ml over 4 d, was antiapoptotic and preserved, in part, glucose-stimulated insulin secretion from human islets exposed to IL-1ß or high glucose. This corresponds to 0.25 mM, at the lower end of the antiinflammatory therapeutic range. Interestingly, when added at five times this concentration (0.2 mg/ml or 1.25 mM), sodium salicylate was proapoptotic and impaired insulin secretion when added to medium for 4 d under basal conditions (data not shown). Similar dose-dependent opposing effects of sodium salicylate are observed in terms of its impact on insulin sensitivity (74) and have been observed previously using rat islets (72), presumably reflecting its multifactorial mode of action. These results indicate that, if ever sodium salicylate is advocated for preservation of ß-cell function and/or improvement of insulin sensitivity in man, particular attention will have to be paid to possible toxic effects associated with higher concentrations of the drug. It should finally be mentioned that aspirin may have additional beneficial effects in vivo that could favor ß-cell survival, including the intriguing observation of inhibition of amylin aggregation (75) that may underlie the amyloidogenic process, possibly contributing to ß-cell death in type 2 diabetes (76).
In conclusion, both pioglitazone and sodium salicylate can protect human ß-cells against apoptosis and loss of function after exposure to IL-1ß or high glucose in vitro. The ability of both these agents to prevent activation of NFB in ß-cells may underlie such protection. This opens the way to their possible use as a means for improving islet survival and preserving function in type 1 and 2 diabetes and immediately after islet transplantation. Any such beneficial effects of these drugs on ß-cell function and mass would synergize with the known positive impact of both drugs on insulin sensitivity.
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Acknowledgments |
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Footnotes |
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E.Z. and K.M. contributed equally to this study.
Abbreviations: NFB, Nuclear factor B; NO, nitric oxide; PPAR, peroxisome proliferator-activated receptor; TUNEL, transferase-mediated deoxy-UTP nick-end labeling.
Received March 4, 2004.
Accepted June 22, 2004.
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M. Y. Donath, J. A. Ehses, K. Maedler, D. M. Schumann, H. Ellingsgaard, E. Eppler, and M. Reinecke Mechanisms of {beta}-Cell Death in Type 2 Diabetes Diabetes, December 1, 2005; 54(suppl_2): S108 - S113. [Abstract] [Full Text] [PDF]
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 E. B. Hammar, J.-C. Irminger, K. Rickenbach, G. Parnaud, P. Ribaux, D. Bosco, D. G. Rouiller, and P. A. Halban Activation of NF-{kappa}B by Extracellular Matrix Is Involved in Spreading and Glucose-stimulated Insulin Secretion of Pancreatic Beta Cells J. Biol. Chem., August 26, 2005; 280(34): 30630 - 30637. [Abstract] [Full Text] [PDF]
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, P < 0.05 compared with 100 mg/dl glucose alone; #, P < 0.01 compared with IL-1ß alone; **, P < 0.01 compared with 600 mg/dl glucose alone.
