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Autocrine Regulation of Single Pancreatic ß-Cell Survival

From the Biophysics Department, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico

	ABSTRACT

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
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 ß-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 ß-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 K⁺ 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 ß-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 ß-cell survival and function and strongly suggest the need to explore new focuses in diabetes treatment.

Pancreatic ß-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 (NGF) secretion (7). Moreover, ß-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 ß-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 ß-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 ß-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 ß-cells and in the ßTC6-F7 cell line (10).

We have previously shown that single rat ß-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 ß-cells, which are increased when cell density is high, are important for the correct function of ß-cells.

It is then possible that glucose-stimulated insulin and NGF secretion constitute autocrine/paracrine signals that are required to suppress apoptosis in ß-cells and that deprivation of these survival signals results in activation of the apoptosis program.

We investigated the autocrine regulation of single ß-cell survival by insulin and NGF. We also explored insulin biosynthesis and secretion by cultured ß-cells that survived NGF withdrawal and analyzed ß-cell subpopulations that lasted.

	RESEARCH DESIGN AND METHODS

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
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 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 ß-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 ß-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 x 10³ (low-density cultures), 4.2 x 10³ (medium-density cultures), 8.4 x 10³, and 16.8 x 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 µg/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:

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 µg/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 insulin-secreting cells. All experiments were performed by duplicate, and at least 100 cells were counted per experimental condition.

The overall secretory activity of ß-cells under a given experimental 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 ß-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 ß-cell. We could clearly distinguish a subpopulation of cells that formed small plaques, with a plaque diameter of 2,000 µm², and another large plaque–forming (LP) subpopulation, with a plaque diameter of >2,000 µm².

Semiquantitative RT-PCR.
Total RNA was extracted from medium-density cultures treated with or without a monoclonal NGF antibody (15.5 µg/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 x 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

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

 
Trophic autocrine effects of insulin and NGF on ß-cell survival.
We explored the effects of inhibiting autocrine insulin and NGF regulation on ß-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 ß-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).

View larger version (18K): [in this window] [in a new window]   FIG. 1. Inhibition of autocrine regulation of ß-cells increases cell death. To assess the role of endogenous insulin and NGF on survival, 2.1 x 103 cells/cm2 were cultured during 12, 16, 24, and 48 h in control conditions (•) or with the following treatments: 1) K252a (), 2) insulin antiserum (), 3) wortmannin (), and 4) NGF antibody (). A: Viability percentage with trypan blue exclusion. B: Percentage of apoptotic cells measured with TUNEL. Data are means ± 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.

We also cultured ß-cells with wortmannin to explore whether autocrine modulation of ß-cell survival is mediated by the PI 3-kinase pathway. Figure 1 shows that both 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.

View larger version (13K): [in this window] [in a new window]   FIG. 2. Cellular density is important for preservation of ß-cell viability. Single ß-cells were cultured during 11 days at different densities in control conditions (•) or treated with NGF (). Data are means ± SE for four different experiments. Symbol denotes statistically significant difference: *P < 0.01 between control and NGF.

View larger version (23K): [in this window] [in a new window]   FIG. 3. Insulin and NGF are survival factors for ß-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. C+, 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 ± 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.

View larger version (44K): [in this window] [in a new window]   FIG. 4. Neutralizing endogenous NGF decreases insulin mRNA in ß-cells. A: RT-PCR products from rat pancreatic ß-cells cultured in control conditions (C) and with NGF antibody (N, 15.5 µg/ml) for 2 or 5 days. B: Normalized insulin to GAPDH mRNA levels. Data are means ± SE for four different experiments. *P < 0.01.

Functional ß-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 that corresponds to insulin secretion of functional subpopulations of pancreatic ß-cells in 15.6 mmol/l glucose (19). In contrast, in ß-cells that survived K252a treatment, no LP cells can be observed.

View larger version (16K): [in this window] [in a new window]   FIG. 5. Behavior of functional subpopulations of ß-cells that survived the NGF signaling blockade. Cells were cultured in control conditions (•) or in the presence of K252a (200 nmol/l) () 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.

	DISCUSSION

TOP ABSTRACT RESEARCH DESIGN AND METHODS RESULTS DISCUSSION REFERENCES

It has been observed that insulin secreted by ß-cells can bind to membranal autoreceptors, activating intracellular signaling cascades and promoting insulin gene transcription (23) and secretion (24). Pancreatic ß-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 ß-cells cultured for 5 days with NGF (17,27). We have demonstrated that adult rat pancreatic ß-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 ß-cell survival. Among the explanations for this observation are that glucose suppresses a constitutive apoptotic program in ß-cells (5) through a PI 3-kinase/Akt signaling pathway (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 ß-cells is not enough to maintain them. In fact, it has been shown that NGF increases ß-cell survival through inhibition of apoptosis (10). It is then possible that a critical ß-cell mass is required to reach an optimal concentration of insulin and NGF, which exert a positive feedback for ß-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 ß-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 ß-cells (19,29,30). When heterogeneity is studied with the reverse hemolytic plaque assay, we observe that under the same stimulus, one subpopulation of ß-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 ß-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 hyperglycemia, ß-cells with the highest secretion rate become exhausted and type 2 diabetes develops.

The autocrine regulation of ß-cell survival has important consequences for understanding ß-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 insulin-enriched media.

	ACKNOWLEDGMENTS

 
This work was supported by the following grants: IN211800 from Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México, and D39822-Q from the Consejo Nacional de Ciencia y Tecnología.

We thank Dr. J.A. García Saina for valuable discussion; C. Aguayo, D. Castañares, E. Arellanes, and A. Caso for proofreading and discussion; Alejandro Sánchez for participating in some experiments; and A. Escalante and F. Pérez for computing assistance.

Address correspondence and reprint requests to Dr. Marcia Hiriart, Department of Biophysics, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Ciudad Universitaria, A.P. 70-253 Coyoacán, México D.F. 04510, Mexico. E-mail: mhiriart{at}ifc.unam.mx

Received for publication December 12, 2003 and accepted in revised form April 22, 2004

Abbreviations: 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

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