Autocrine Regulation of Single Pancreatic β-Cell Survival

Vctor Navarro-Tableros 0 M. Carmen S anchez-Soto 0 Santiago Garca 0 Marcia Hiriart 0 0 From the Biophysics Department, Instituto de Fisiolog a Celular, Universidad Nacional Auto noma de Me xico , Mexico City , Mexico. ment of Biophysics, Instituto de Fisiolog a Celular, Universidad Nacional Auto noma de Me xico , Ciudad Universitaria, A.P. 70-253 Coyoaca n, Me xico D.F. 04510 , Mexico 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 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 -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 - Itype 1 diabetes (1), whereas in type 2 diabetes, -cell nadequate -cell mass is a crucial factor in diabetes. Immune destruction of -cells is the main defect in 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 (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, Hanks 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 -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-lysi (...truncated)