Autocrine Regulation of Single Pancreatic -Cell Survival Vı´ctor Navarro-Tableros, M. Carmen Sa´nchez-Soto, Santiago Garcı´a, and Marcia Hiriart
Function and survival of cells depend in part on the presence of growth factors. We explored the autocrine regulation of insulin and nerve growth factor (NGF) on single adult rat pancreatic -cell survival and hormone secretion. When NGF or insulin signaling were blocked in culture media, cell survival decreased compared with control cells, with apoptosis being the main mechanism of cell death. To further explore the role of glucose in -cell survival, we cultured the cells for 16 h in 2.6 mmol/l glucose and observed that nearly 17% of the cells developed apoptosis; this effect was partially prevented by NGF and almost completely inhibited by insulin treatment. A high Kⴙ concentration had the same effect, suggesting that insulin and NGF secretion by the cells was responsible for the survival effects and not glucose per se. Blocking NGF signaling with an NGF antibody or with K252a reduced insulin biosynthesis and secretion in the cells that survived the treatment. Moreover, the functional -cell subpopulation with a higher insulin secretion rate is more susceptible to K252a. These results further indicate that NGF and insulin play important autoregulatory roles in pancreatic -cell survival and function and strongly suggest the need to explore new focuses in diabetes treatment. Diabetes 53: 2018 –2023, 2004
nadequate -cell mass is a crucial factor in diabetes. Immune destruction of -cells is the main defect in type 1 diabetes (1), whereas in type 2 diabetes, -cell mass is decreased to some extent compared with a normal pancreas. In the latter, the remaining cells are not capable of secreting as much insulin as normal -cells to maintain euglycemic patients (2,3). Apoptosis is the mechanism of pancreatic -cell death in both types of diabetes (4). Pancreatic -cell function and survival depend on a number of intrinsic and environmental factors. Among them, it is widely accepted that glucose promotes survival and prevents apoptosis (5,6); however, this mechanism is not entirely clear. Glucose also stimulates insulin and nerve growth factor
I
From the Biophysics Department, Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, Mexico City, Mexico. Address correspondence and reprint requests to Dr. Marcia Hiriart, Department of Biophysics, Instituto de Fisiologı´a Celular, Universidad Nacional Auto´noma de Me´xico, Ciudad Universitaria, A.P. 70-253 Coyoaca´n, Me´xico D.F. 04510, Mexico. E-mail:
[email protected]. Received for publication 12 December 2003 and accepted in revised form 22 April 2004. HBSS, Hank’s balanced salt solution; HO 342, Hoechst 33342; LP, large plaque–forming; NGF, nerve growth factor; PI, phosphatidylinositol; TUNEL, Tdt-mediated dUTP nick-end labeling. © 2004 by the American Diabetes Association. 2018
(NGF) secretion (7). Moreover, -cells express functional receptors for these hormones (8). Insulin and NGF receptors have tyrosine kinase activity that triggers intracellular phosphorylation cascades, including the phosphatidylinositol (PI) 3-kinase/Akt survival-signaling pathway (9 –11). It is then possible that glucose regulates an autocrine pathway for -cell survival by increasing insulin and NGF secretion. It has been shown that insulin protects different mammalian cells from apoptosis through the activation of insulin receptors and a PI 3-kinase– dependent pathway (12,13). Moreover, it has been recently reported that an insulin analog and, with a lesser potency, exogenous insulin have anti-apoptotic activity in the rat insulinoma cell line INS-1 (14). Some observations in -cells also suggest an insulin autocrine regulation. For example, when insulin autoregulation is disrupted in mice homozygous for null alleles of insulin receptor substrate-2 (IRS-2⫺/⫺), the animals develop hyperglycemia associated with pancreatic -cell failure and apoptosis (15). It is also well accepted that NGF is important for neuronal survival, and recently it was reported that NGF withdrawal induces apoptosis in cultured human -cells and in the TC6-F7 cell line (10). We have previously shown that single rat -cells cultured at a low density (1,000 cells/cm2) lose their sensitivity to glucose because they secrete the same amount of insulin in different extracellular glucose concentrations, with increasing time in culture (16). This desensitization is not observed in higher-density cultures (10,000 cells/cm2) (17). These observations suggest that autocrine interactions among -cells, which are increased when cell density is high, are important for the correct function of -cells. It is then possible that glucose-stimulated insulin and NGF secretion constitute autocrine/paracrine signals that are required to suppress apoptosis in -cells and that deprivation of these survival signals results in activation of the apoptosis program. We investigated the autocrine regulation of single -cell survival by insulin and NGF. We also explored insulin biosynthesis and secretion by cultured -cells that survived NGF withdrawal and analyzed -cell subpopulations that lasted. RESEARCH DESIGN AND METHODS Reagents were obtained from the following sources: collagenase type IV from Worthington (Freehold, NJ); guinea pig insulin antiserum from Biogenesis (Sandown, NH); rabbit anti-mouse NGF 2.5-s antibody, wortmannin, BSA, Hank’s balanced salt solution (HBSS), chromium chloride, staphylococcal protein A, HEPES, pig insulin, 2.5-s NGF, trypsin, triton, sodium citrate, trypan DIABETES, VOL. 53, AUGUST 2004
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blue, Hoechst 33342 (HO 342), propidium iodide, and poly-L-lysine from Sigma (St. Louis, MO); tissue culture dishes (Corning); K252a from Alomone Labs (Jerusalem, Israel); fetal bovine serum from Equitech-Bio (Ingram, TX); guinea pig complement, RPMI-1640 salts, and penicillin-streptomycin-amphotericin B solution from Life Technologies (Grand Island, NY); and in situ cell death detection kit fluorescein and RNA PCR Core kit from Roche (Mannheim, Germany). Pancreatic -cell culture. Animal care was performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH number 85-23, revised 1985). Young adult male Wistar rats (250 –280 g) were obtained from the local animal facility, maintained in a 14-h light (0600 –2000)/10-h dark cycle, and allowed free access to standard laboratory rat diet and tap water. Animals were anesthetized with sodium pentobarbital (40 mg/kg) and, after pancreas dissection, were killed by cervical dislocation. Pancreatic -cells were obtained with collagenase digestion, Ficoll gradient centrifugation, and mechanical dissociation in calcium-free solution, as previously described (15). Single cells were cultured in RPMI-1640 (11.6 mmol/l glucose) and supplemented with 200 units/ml penicillin G, 200 mg/ml streptomycin, and 0.5 mg/ml amphotericin B, with 1% of fetal bovine serum, for 16 h to recover from the isolation before starting the experimental procedures. Cell viability measurement. After the recovery period, islet cells were cultured in the following conditions: 1) 11 days in different densities of 2.1 ⫻ 103 (low-density cultures), 4.2 ⫻ 103 (medium-density cultures), 8.4 ⫻ 103, and 16.8 ⫻ 103 cells/cm2 (high-density cultures) in tissue culture dishes; and 2) cells were seeded at a low-density for 12, 16, and 48 h on glass coverslips previously treated with poly-L-lysine. Cell viability was measured by trypan blue exclusion by incubating the cells for 10 min with a 0.04% trypan blue in isotonic Krebs-Ringer buffer solution and counting stained cells (dead cells) versus nonstained cells. All the experiments reported were done in duplicate, and at least 300 cells were counted per experimental condition. Results were expressed as the percentage of viable cells. Apoptosis quantification. To determine apoptosis, cells were cultured at a low density for 12, 16, 24, or 48 h with or without K252a (200 nmol/l), insulin antiserum (1:20), or 5 nmol/l wortmannin (in vitro half-maximal inhibitory concentration [IC50] for PI 3-kinase is 5 nmol/l). We also cultured the cells with an anti-NGF monoclonal antibody (15.5 g/ml) for 12 and 24 h. For another set of experiments, apoptosis was determined in cells cultured at a low density in 2.6 mmol/l glucose with or without NGF (50 nmol/l), insulin (10 nmol/l), and/or KCl (40 mmol/l) by two different methods:
condition was expressed as a secretion index, calculated by multiplying the average plaque area by the percentage of plaque-forming cells (19). To identify functional subpopulations of -cells and to determine if the NGF signaling blockage differentially affected these subpopulations, we constructed a frequency distribution of plaque areas with data pooled from three different experiments by duplicate. We modeled a new way of calculating frequency distribution of plaque areas, by measuring the size of the ring of lysed erythrocytes around the -cell. We could clearly distinguish a subpopulation of cells that formed small plaques, with a plaque diameter of ⱕ2,000 m2, and another large plaque–forming (LP) subpopulation, with a plaque diameter of ⬎2,000 m2. Semiquantitative RT-PCR. Total RNA was extracted from medium-density cultures treated with or without a monoclonal NGF antibody (15.5 g/ml) for 2 or 5 days or with K252a (200 nmol/l) for 5 days, using the TRIzol reagent (Gibco) as instructed by a technical bulletin. Cells were lysed in the culture dishes by adding 1 ml TRIzol per 1 ⫻ 106 cells. RT-PCR was performed according to the protocol recommended by the supplier. cDNA synthesis was performed by oligo-dT–primed RT of 200 ng total RNA. Amplification was carried out during 16 cycles to avoid reaching a plateau in the reaction. For the semiquantitative determinations, parallel amplifications of the housekeeping gene GAPDH were performed. The oligonucleotides used to prime the amplification of the cDNA template were synthesized in the local facility and were designed based on the published sequences of rat insulin gene and rat GAPDH. For insulin detection, we chose the 5⬘-AAGAGCCATCAGCAAGC-3⬘ sequence for the sense (5⬘) primer and the 5⬘-GAGCAGATGCTGGTGCAGC-3⬘ sequence for the antisense (3⬘) primer. For GAPDH mRNA, we used the 5⬘-GCCCCCATGTTTGTGAT-3⬘ sequence for the sense (5⬘) primer and 5⬘-GCCCCAGCATCAAAGGT-3⬘ for the antisense (3⬘) primer. Amplifications were performed with an annealing temperature of 56°C. Reaction products were sequenced and proved to have 100% identity with the sequence reported for the insulin and GAPDH gene. The amplified material was visualized by ethidium bromide staining on a 1% agarose gel electrophoresis. Quantification of the RT-PCR products was determined by densitometry and analyzed with Scion Image analysis software. Statistical analysis. All data are reported as means ⫾ SE; n denotes the number of the experiments performed. The statistical significance was obtained with one-way ANOVA, followed by Fisher’s multiple range test using the Number Cruncher statistical system (NCSS 4.2; Dr. Jerry L. Hintze, Kaysville, UT), and Bonferroni analysis (Statview 4.57; Abacus Concepts, Cary, NC).
RESULTS 1.
Apoptotic cells were detected using the Tdt-mediated dUTP nick-end labeling (TUNEL) method, following the manufacturer’s instructions. Briefly, the cells were fixed for 30 min in 0.4% paraformaldehyde in PBS (0.1 mol/l, pH 7.4). Then cells were perforated with 0.1% Triton in sodium citrate PBS solution during 2 min on ice, washed three times with PBS, and incubated during 90 min with the TUNEL reaction mixture at 37°C in a humid chamber protected from light. Positive cells were counted under a Nikon Axiophot inverted microscope connected to a fluorescence lamp. 2. To detect apoptosis, cells treated for 12 and 24 h were also incubated with the DNA binding dye HO 342 (20 g/ml for 10 min at room temperature) and propidium iodide (2 nmol/l). Morphological changes were detected with excitation filters 340 –380 nm and emission filter ⬎430 nm for HO 342 and 536 and 620 nm, respectively, for propidium iodide.
Reverse hemolytic plaque assay. To identify insulin-secreting cells and measure insulin secretion by single cells, we used the reverse hemolytic plaque assay (18) as described previously (19). Briefly, medium-density cultures were exposed to K252a (200 nmol/l) for 5 days. After this period, cells were challenged for 1 h in HBSS containing 5.6 or 15.6 mmol/l glucose, in the presence of an insulin antiserum (1:20 in HBSS), and further incubated for 30 min with guinea pig complement. Insulin released during the incubation time was revealed by the presence of hemolytic plaques around secretory cells. The size of the plaques was measured by projecting the image on a monitor attached to a video camera and Nikon Axiophot inverted microscope, with the aid of the JAVA video analysis software (Version 1.40; Jandel Scientific, Corte Madera, CA). The plaque size was expressed as area; cells that formed plaques were counted, and the results were expressed as the percentage of insulinsecreting cells. All experiments were performed by duplicate, and at least 100 cells were counted per experimental condition. The overall secretory activity of -cells under a given experimental DIABETES, VOL. 53, AUGUST 2004
Trophic autocrine effects of insulin and NGF on -cell survival. We explored the effects of inhibiting autocrine insulin and NGF regulation on -cell survival and apoptosis in 11.6 mmol/l glucose. As shown in Fig. 1A, single cell viability in control cells was around 67% and tended to decrease with time in culture. When autocrine NGF signaling was disrupted with K252a, the viability of cells decreased by nearly 27% at 12 h and was further reduced during the next hours, compared with control cells. The most significant effect was observed when insulin was blocked, where viability decreased to half of the control value during the first 12 h and nearly 90% of the cells died after 48 h in culture. We investigated if, under these conditions, cell death was apoptotic (Fig. 1B). At 12 h, apoptosis is the main mechanism of death in response to NGF or insulin deprivation in -cells. We did not find differences between results obtained treating cells with the inhibitor of TrkA phosphorylation, K252a, or a neutralizing NGF antibody (Table 1). Interestingly, the percentage of apoptosis did not increase within the 48 h in culture in all experimental groups; the additional reduction of viability observed in Fig. 1A corresponds to necrotic death. We also cultured -cells with wortmannin to explore whether autocrine modulation of -cell survival is mediated by the PI 3-kinase pathway. Figure 1 shows that both 2019
AUTOCRINE REGULATION OF -CELL SURVIVAL
FIG. 2. Cellular density is important for preservation of -cell viability. Single -cells were cultured during 11 days at different densities in control conditions (F) or treated with NGF (E). Data are means ⴞ SE for four different experiments. Symbol denotes statistically significant difference: *P < 0.01 between control and NGF.
FIG. 1. Inhibition of autocrine regulation of -cells increases cell death. To assess the role of endogenous insulin and NGF on survival, 2.1 ⴛ 103 cells/cm2 were cultured during 12, 16, 24, and 48 h in control conditions (F) or with the following treatments: 1) K252a (E), 2) insulin antiserum (Œ), 3) wortmannin (‚), and 4) NGF antibody (f). A: Viability percentage with trypan blue exclusion. B: Percentage of apoptotic cells measured with TUNEL. Data are means ⴞ SE for four different experiments in duplicate. Symbols denote statistically significant differences: *P < 0.01, **P < 0.05, in all treatment groups with respect to control conditions.
Insulin and NGF prevent low glucose–induced apoptosis. We explored the possibility that glucose itself would be a survival factor by culturing islet cells for 12 h in RPMI-1640 with 2.6 mmol/l glucose. Figure 3 shows that in this condition, nearly 17% of control cells experimented apoptotic death. When NGF or insulin was added to the culture media, apoptosis decreased by 46 and 80%, respectively, compared with control cells. Interestingly, apoptotic death in cells depolarized with potassium decreased by nearly 90%, indicating that endogenous insulin and NGF secreted in this condition is enough to prevent apoptosis. Blocking autocrine NGF signals decreases insulin biosynthesis and secretion. We analyzed the effect of neutralizing NGF with a monoclonal NGF antibody on insulin biosynthesis in single -cells cultured for 2 or 5
cell viability and apoptosis percentage were similar to those observed with K252a. We investigated autocrine modulation on in vitro cell survival by calculating cell viability in cells cultured in different densities for 11 days in 11.6 mmol/l glucose. Figure 2 shows that only 20% of cells cultured in a low density survived; this percentage increased by threefold in cells cultured with NGF. This difference was not observed in cells cultured at higher densities, suggesting that autocrine survival modulation by NGF is saturated in a later condition. TABLE 1 Percentage of apoptotic cells measured with two different techniques
Technique TUNEL HO 342
Control
NGF antibody
K252a
14.8 ⫾ 3 10.9 ⫾ 5
30.2 ⫾ 5 27.1 ⫾ 9
27.9 ⫾ 3 25.9 ⫾ 8
Data are means ⫾ SE. 2020
FIG. 3. Insulin and NGF are survival factors for -cells in low glucose concentrations. Single islet cells were cultured at low density for 12 h in 2.6 mmol/l glucose medium. The apoptosis percentage was determined with TUNEL, and data were normalized with respect to control conditions. Cⴙ, control cells, 2.6 mmol/l glucose; I, cells treated with insulin (10 nmol/l); K, cells exposed to KCl (40 mmol/l); N, cells exposed to NGF; NI, cells incubated with NGF and insulin; NIK, cells treated with NGF, insulin, and KCl. Data are means ⴞ SE for four different experiments. Symbols denote statistically significant differences: *P < 0.01 between all treatment groups and 2.6 mmol/l glucose alone; **P < 0.01 between 2.6 and 11.6 mmol/l glucose. DIABETES, VOL. 53, AUGUST 2004
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FIG. 5. Behavior of functional subpopulations of -cells that survived the NGF signaling blockade. Cells were cultured in control conditions (F) or in the presence of K252a (200 nmol/l) (E) for 5 days. Interestingly, in cells that survived the treatment, the LP subpopulation (see RESEARCH DESIGN AND METHODS) disappeared. Data are the mean of six different experiments.
FIG. 4. Neutralizing endogenous NGF decreases insulin mRNA in -cells. A: RT-PCR products from rat pancreatic -cells cultured in control conditions (C) and with NGF antibody (␣N, 15.5 g/ml) for 2 or 5 days. B: Normalized insulin to GAPDH mRNA levels. Data are means ⴞ SE for four different experiments. *P < 0.01.
days in a medium density. Figure 4 shows that insulin mRNA decreased by nearly 40% in cells treated with NGF antibody, and a similar effect was observed in cells treated with K252a for 5 days (data not shown). Table 2 shows that the insulin secretion index, in response to a 1-h challenge in 5.6 and 15.6 mmol/l glucose, decreased by 50 and 76%, respectively, in -cells cultured with K252a, compared with control cells. This effect resulted from a decrease of both the percentage of insulin secretors and the amount of insulin secreted by single cells cultured with K252a in both glucose concentrations. It is interesting to note that blocking NGF signaling not only reduced the capability of the cells to respond to glucose but also insulin basal secretion in 5.6 mmol/l glucose. Functional -cell subpopulations. We have previously described that NGF increases the percentage of LP cells by nearly twofold in 15.6 mmol/l glucose (20). Figure 5 shows the multimodal distribution of plaque areas of control cells TABLE 2 Insulin secretion by single -cells treated for 4 days with K252a
Treatment Control K252a
Glucose (mmol/l)
% of plaqueforming cells
Plaque area (m2)
Secretion index
5.6 15.6 5.6 15.6
40 ⫾ 2 56 ⫾ 1* 33 ⫾ 3* 36 ⫾ 3†
2,536 ⫾ 447 4,441 ⫾ 214* 1,528 ⫾ 234* 1,653 ⫾ 238†
1,014 ⫾ 161 2,487 ⫾ 130* 504 ⫾ 82* 595 ⫾ 146†
Data are means ⫾ SE. *P ⬍ 0.01 with respect to 5.6 mmol/l glucose; †P ⬍ 0.01 with respect to 15.6 mmol/l glucose. DIABETES, VOL. 53, AUGUST 2004
that corresponds to insulin secretion of functional subpopulations of pancreatic -cells in 15.6 mmol/l glucose (19). In contrast, in -cells that survived K252a treatment, no LP cells can be observed. DISCUSSION
The results of the present study represent, to our knowledge, the first description of an autocrine regulation of normal single rat -cell survival directly caused by insulin. NGF secreted by -cells is also an autocrine regulator of survival and preserves insulin biosynthesis and secretion. Moreover, analysis of the functional subpopulations of -cells that survived NGF withdrawal showed that the subpopulation of high insulin secretors disappears when the NGF pathway is blocked. These observations may contribute toward a better understanding of the physiopathology of diabetes, where serum insulin and NGF levels are diminished (21,22). It has been observed that insulin secreted by -cells can bind to membranal autoreceptors, activating intracellular signaling cascades and promoting insulin gene transcription (23) and secretion (24). Pancreatic -cells also express the high-affinity NGF receptor TrkA (8,20,25). Among other effects, we have previously observed that exogenous NGF increases glucose-stimulated insulin secretion and content (8,26). Moreover, an increase in Na⫹ and Ca2⫹ current densities is observed in -cells cultured for 5 days with NGF (17,27). We have demonstrated that adult rat pancreatic -cells synthesize and secrete NGF in response to increasing extracellular glucose concentrations and to potassium-induced depolarization (7). Endogenous NGF modulates glucose-induced insulin secretion because the acute blockage of NGF signaling with K252a, or with a monoclonal NGF antibody, decreases insulin secretion stimulated by glucose (20). It has been described that glucose promotes -cell survival. Among the explanations for this observation are that glucose suppresses a constitutive apoptotic program in -cells (5) through a PI 3-kinase/Akt signaling pathway 2021
AUTOCRINE REGULATION OF -CELL SURVIVAL
(6). We observed that when insulin is neutralized with a polyclonal antibody or the NGF pathway is disturbed, in the presence of 11.6 mmol/l glucose, the viability of cells decreases with time in culture and that most of the cells die by an apoptotic mechanism. Moreover, when cells are cultured in 2.6 mmol/l glucose, apoptotic death can be partially prevented with NGF and almost completely with insulin. Interestingly, high K⫹ depolarization, which stimulates insulin and NGF secretion, almost completely prevented apoptosis. Based on these observations, we consider that the effects of glucose on cell survival could be mainly mediated by an autocrine loop of insulin and secondarily by NGF secretion. This consideration is also supported by the observation that NGF increases cell survival in low-density cultures, probably because, in this condition, the amount of NGF secreted by -cells is not enough to maintain them. In fact, it has been shown that NGF increases -cell survival through inhibition of apoptosis (10). It is then possible that a critical -cell mass is required to reach an optimal concentration of insulin and NGF, which exert a positive feedback for -cell function and survival. IGF-I is also considered a survival factor that has a widespread antiapoptotic effect on many death signals (28). TrkA, insulin, and IGF receptors are different proteins that have tyrosine kinase activity. When the ligand binds to the receptor, it autophosphorylates on tyrosine residues and activates, initiating cascades of protein phosphorylation. The intracellular signaling cascade of insulin and NGF converge in downstream-located effector proteins, such as PI 3-kinase/Akt, which are associated with the antiapoptotic systems in different cell types (9 –11). We observed that treatment of cells with the PI 3-kinase blocker wortmannin decreases cell viability and increases the percentage of apoptotic cells, compared with control in approximately the same extent of K252a, which suggests that the trophic effect of NGF on -cell survival is mediated by the activation of PI 3-kinase; however, we cannot discard the possibility of activation of other survival pathways. The insulin secretion index in cells that survived NGF withdrawal decreased in both glucose concentrations (5.6 and 15.6 mmol/l). This result can partially be explained because insulin mRNA declined by nearly 40% in cells treated with K252a. It has been shown that there are functional subpopulations of adult rat -cells (19,29,30). When heterogeneity is studied with the reverse hemolytic plaque assay, we observe that under the same stimulus, one subpopulation of -cells secretes more insulin (LP cells) than the other one (small plaque cells). It is important to note that LP cells are responsible for nearly 75% of the insulin secreted (18). We have previously observed that LP cells are preferentially modulable by NGF (20). In this study, we demonstrate that the LP subpopulation is more sensitive to NGF withdrawal than the low-rate secretors (small plaque). Moreover, increasing glucose concentrations result in recruitment of -cells into the secretory pool (19,30). This indicates that the gland has a large reserve of secretory capacity that can be recruited when glucose remains high, for example, in insulin-resistant conditions. It could then be possible that after a prolonged period of 2022
hyperglycemia, -cells with the highest secretion rate become exhausted and type 2 diabetes develops. The autocrine regulation of -cell survival has important consequences for understanding -cell dysfunctions in diabetes and may suggest new means of therapeutic intervention by trying to preserve plasmatic insulin near to normal values in the first stages of type 1 diabetes. Moreover, transplantation of pancreatic islets is a potential treatment for patients with type 1 diabetes; however, a limiting factor for success is insufficient insulin secretion from grafted islets. It has been observed that treatment with NGF and vascular endothelial growth factor increases survival of grafted islets, as well as their reinnervation (31). It is also possible that transplanted islets would exhibit better survival and physiology in an insulinenriched media. ACKNOWLEDGMENTS
This work was supported by the following grants: IN211800 from Direccio´ n General de Asuntos del Personal Acade´ mico, Universidad Nacional Auto´ noma de Me´ xico, and D39822-Q from the Consejo Nacional de Ciencia y Tecnologı´a. We thank Dr. J.A. Garcı´a Saina for valuable discussion; C. Aguayo, D. Castan˜ ares, E. Arellanes, and A. Caso for proofreading and discussion; Alejandro Sa´ nchez for participating in some experiments; and A. Escalante and F. Pe´ rez for computing assistance. REFERENCES 1. Pipeleers D, Hoorens A, Marichal-Pipeleers M, Van de Casteele M, Bouwens L, Ling Z: Role of pancreatic beta-cells in the process of beta-cell death. Diabetes 50 (Suppl. 1):S52–S57, 2001 2. Federici M, Hribal M, Perego L, Ranalli M, Caradonna Z, Perego C, Usellini L, Nano R, Bonini P, Bertuzzi F, Marlier LN, Davalli AM, Carandente O, Pontiroli AE, Melino G, Marchetti P, Lauro R, Sesti G, Folli F: High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes 50:1290 –1301, 2001 3. Butler A, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC: -Cell deficit and increased -cell apoptosis in humans with type 2 diabetes. Diabetes 52:102–110, 2003 4. Sesti G: Apoptosis in the beta cells: cause or consequence of insulin secretion defect in diabetes? Ann Med 34:444 – 450, 2002 5. Hoorens A, Van de Castelee M, Ko¨ ppel G, Pipeleers D: Glucose promotes survival of rat pancreatic  cells by activating synthesis of proteins which suppress a constitutive apoptotic program. J Clin Invest 98:1568 –1574, 1996 6. Srinivasan S, Bernal-Mizrachi E, Ohsugi M, Permutt MA: Glucose promotes pancreatic islet -cell survival through a PI 3-kinase/Akt-signaling pathway. Am J Physiol Endocrinol Metab 283:E784 –E793, 2002 7. Rosenbaum T, Vidaltamayo R, Sa´ nchez-Soto C, Zentella A, Hiriart M: Pancreatic  cells synthesize and secrete nerve growth factor. Proc Natl Acad Sci U S A 95:7784 –7788, 1998 8. Kanaka-Gantenbein C, Dicou E, Czernichow P, Scharfmann R: Presence of nerve growth factor and its receptors in an in vitro model of islet cell development. Endocrinology 136:3154 –3162, 1995 9. Hetman M, Xia Z: Signaling pathways mediating anti-apoptotic action of neurotrophins. Acta Neurobiol Exp 60:531–545, 2000 10. Pierucci D, Cicconi S, Bonini P, Ferrelli F, Pastore D, Matteucci C, Marselli L, Marchetti P, Ris F, Halban P, Oberholzer J, Federici M, Cozzolino F, Lauro R, Borboni P, Marlier LN: NGF-withdrawal induces apoptosis in pancreatic beta cells in vitro. Diabetologia 44:1281–1295, 2001 11. Leibiger IB, Leibiger B, Berggren PO: Insulin feedback action on pancreatic -cell function. FEBS Lett 532:1– 6, 2002 12. Bertrand F, Atfi A, Cadoret A, L’Allemain G, Robin H, Lascols O, Capeau J, le Cherquii G: A role for nuclear factor kB in the antiapoptotic function of insulin. J Biol Chem 273:2931–2938, 1998 13. Kang S, Song J, Kang H, Kim S, Lee Y, Park D: Insulin can block apoptosis DIABETES, VOL. 53, AUGUST 2004
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