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• W1978463531 abstract "The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the KATP channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of KATP channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10–100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 μm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2–4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an ∼62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation. The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the KATP channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of KATP channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10–100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 μm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2–4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an ∼62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation. Both types 1 and 2 diabetes result from the inability of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate β-cell mass. Increased metabolic demands or stress responses that exert a positive effect on β-cell mass include obesity, pregnancy, partial pancreatectomy, or chronic glucose exposure. β-Cell mass is regulated by cellular mechanisms that include replication, neogenesis, hypertrophy, and apoptosis (1Rhodes C.J. Science. 2005; 307: 380-384Crossref PubMed Scopus (750) Google Scholar, 2Montanya E. Nacher V. Biarnes M. Soler J. Diabetes. 2000; 49: 1341-1346Crossref PubMed Scopus (177) Google Scholar). Recent studies have emphasized the importance of the proliferative capacity of existing adult β-cells as a major source of new β-cells during adult life that may significantly contribute to the maintenance of β-cell mass (3Dor Y. Brown J. Martinez O.I. Melton D.A. Nature. 2004; 429: 41-46Crossref PubMed Scopus (1884) Google Scholar).Mammalian target of rapamycin (mTOR) 2The abbreviations used are: mTOR, mammalian target of rapamycin; Akt (also known as PKB), protein kinase B; eIF-4E, eukaryotic initiation factor 4E; 4EBP1, eukaryotic initiation factor 4E-binding protein 1; KATP channel, ATP-sensitive potassium channel; IRS1 and -2, insulin receptor substrates 1 and 2; PI3K, phosphoinositide 3-kinase; S6K1, 70-kDa ribosomal protein S6 kinase; TSC1 (also known as hamartin) and TSC2 (also known as tuberin), respective protein products of mutated tuberous sclerosis genes TSC1 and TSC2; PI, propidium iodide; FBS, fetal bovine serum; PBS, phosphate-buffered saline. 2The abbreviations used are: mTOR, mammalian target of rapamycin; Akt (also known as PKB), protein kinase B; eIF-4E, eukaryotic initiation factor 4E; 4EBP1, eukaryotic initiation factor 4E-binding protein 1; KATP channel, ATP-sensitive potassium channel; IRS1 and -2, insulin receptor substrates 1 and 2; PI3K, phosphoinositide 3-kinase; S6K1, 70-kDa ribosomal protein S6 kinase; TSC1 (also known as hamartin) and TSC2 (also known as tuberin), respective protein products of mutated tuberous sclerosis genes TSC1 and TSC2; PI, propidium iodide; FBS, fetal bovine serum; PBS, phosphate-buffered saline. is a serine/threonine protein kinase that integrates signals derived from growth factors and nutrients to regulate cell growth and proliferation through the regulatory proteins 70-kDa ribosomal protein S6 kinase (S6K1) and the eukaryotic initiation factor 4E-binding protein-1 (4EBP1). This signaling cascade stimulates protein translation and increases the capacity of the ribosomal protein machinery necessary for the onset of DNA synthesis (4Harris T.E. Lawrence Jr., J.C. Sci. STKE. 2003; 212: re15Google Scholar, 5Hay N. Sonenberg N. Genes Dev. 2004; 18: 1926-1945Crossref PubMed Scopus (3398) Google Scholar). Our previous studies have demonstrated that glucose robustly activates mTOR/S6K1/4EBP1 in an amino acid-dependent manner via its metabolism in both rodent and human islets. Glucose and amino acids, especially leucine and glutamate, are the most prominent activators of mTOR in islets, possibly through ATP production mediated by mitochondrial metabolism (6McDaniel M.L. Marshall C.A. Pappan K.L. Kwon G. Diabetes. 2002; 51: 2877-2885Crossref PubMed Scopus (103) Google Scholar, 7Kwon G. Marshall C.A. Pappan K.L. Remedi M.S. McDaniel M.L. Diabetes. 2004; 53: S225-S232Crossref PubMed Scopus (125) Google Scholar, 8Xu G. Kwon G. Cruz W.S. Marshall C.A. McDaniel M.L. Diabetes. 2001; 50: 353-360Crossref PubMed Scopus (190) Google Scholar, 9Xu G. Kwon G. Marshall C.A. Lin T.-A. Lawrence Jr., J.C. McDaniel M.L. J. Biol. Chem. 1998; 273: 28178-28184Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Insulin secreted by the β-cell and growth factors also provide input to mTOR through the insulin signaling cascade to Akt (6McDaniel M.L. Marshall C.A. Pappan K.L. Kwon G. Diabetes. 2002; 51: 2877-2885Crossref PubMed Scopus (103) Google Scholar). Akt may directly activate mTOR but also has been shown to inhibit the tumor suppressor proteins TSC1/2. These proteins are activated by AMP-dependent protein kinase that is regulated by the ATP/AMP ratio. Rapamycin specifically inhibits mTOR activation and signaling to 4EBP1 and S6K1. Recent reports have demonstrated a negative feedback pathway from chronically stimulated mTOR to IRS2 that inhibits the insulin signaling pathway (10Briaud I. Dickson L.M. Lingohr M.K. McCuaig J.F. Lawrence J.C. Rhodes C.J. J. Biol. Chem. 2005; 280: 2282-2293Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Apparently, this negative feedback to IRS2 does not reduce nutrient-stimulated mTOR activation in the β-cell as S6K1 remains fully activated during a 4- or 6-day exposure to elevated glucose (7Kwon G. Marshall C.A. Pappan K.L. Remedi M.S. McDaniel M.L. Diabetes. 2004; 53: S225-S232Crossref PubMed Scopus (125) Google Scholar), although other IRS2-dependent pathways may be inhibited.Our previous studies demonstrated that the majority of glucose-stimulated [3H]thymidine incorporation by rodent islets is inhibited by rapamycin, thus mediated through mTOR (7Kwon G. Marshall C.A. Pappan K.L. Remedi M.S. McDaniel M.L. Diabetes. 2004; 53: S225-S232Crossref PubMed Scopus (125) Google Scholar). Studies have further indicated that initial signaling events responsible for glucose-stimulated insulin secretion are also shared by the glucose-stimulated mTOR pathway. Thus, mTOR/S6K1 activation by glucose is mediated, in part, through modulation of the KATP channel and changes in cytosolic Ca2+ in rodent islets (7Kwon G. Marshall C.A. Pappan K.L. Remedi M.S. McDaniel M.L. Diabetes. 2004; 53: S225-S232Crossref PubMed Scopus (125) Google Scholar). Agents that have been used to modulate the KATP channel in β-cells include: 1) glyburide, a sulfonylurea type agent, which directly inhibits the KATP channel, causes depolarization, increases in Ca2+ influx and insulin secretion and 2) diazoxide, an activator of KATP channels, that causes hyperpolarization, inhibition of Ca2+ influx, and a blockage of insulin secretion.At basal glucose (3 mm), glyburide-induced closure of KATP channels caused a partial phosphorylation of S6K1. Since mTOR is a regulator of DNA synthesis, these results suggested that glyburide might enhance DNA synthesis at basal glucose. In addition, diazoxide-induced activation of the KATP channel partially inhibited S6K1 phosphorylation by glucose. These results suggested that diazoxide might attenuate glucose-stimulated DNA synthesis.Guiot et al. (11Guiot Y. Henquin J.-C. Rahier J. Eur. J. Pharmacol. 1994; 261: 157-161Crossref PubMed Scopus (18) Google Scholar) reported in 1994 that glibenclamide (glyburide) stimulated β-cell replication and increased β-cell mass in normal young mice. However, the cellular mechanisms responsible for the effects of glyburide were not addressed. More recent findings have indicated that the L-type Ca2+ channel α1D subunit is required for proper β-cell development in the postnatal pancreas (12Namkung Y. Skrypnyk N. Jeong M.-J. Lee T. Lee M.-S. Kim H.-L. Chin H. Suh P.-G. Kim S.-S. Shin H.-S. J. Clin. Invest. 2001; 108: 1015-1022Crossref PubMed Scopus (113) Google Scholar). In α1D gene knock-out mice, β-cell proliferation was decreased, suggesting that a defect in Ca2+ influx may be partly responsible for a reduction in β-cell mass. In addition, recent findings indicated that S6K1 requires an initial Ca2+-dependent priming event for activation (13Hannan K.M. Thomas G. Pearson R.B. Biochem. J. 2003; 370: 469-477Crossref PubMed Scopus (50) Google Scholar, 14Conus N.M. Hemmings B.A. Pearson R.B. J. Biol. Chem. 1998; 273: 4776-4782Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar).Diazoxide has been used extensively in vitro to characterize the regulation of stimulus-secretion coupling mechanisms of β-cells that are independent of plasma membrane KATP channels. Although diazoxide has provided important mechanistic insights into the role of plasma membrane KATP channels in β-cells, it is also reported to exert significant effects on mitochondrial energetics (15Ozcan C. Holmuhamedov E.L. Jahangir A. Terzic A. J. Thorac. Cardiovasc. Surg. 2001; 121: 298-306Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 16Holmuhamedov E.L. Wang L. Terzic A. J. Physiol. (Lond.). 1999; 519.2: 347-360Crossref Scopus (310) Google Scholar). A direct effect of KATP channel openers including diazoxide on mitochondrial function has been shown to be critical in the preservation of heart function against injury (17Holmuhamedov E. Lewis L. Bienengraeber M. Holmuhamedova M. Jahangir A. Terzic A. FASEB J. 2002; 16: 1010-1016Crossref PubMed Scopus (64) Google Scholar). The cardioprotective effects of diazoxide include decreased Ca2+ uptake due to depolarization of the mitochondrial membrane potential and an activated Ca2+ release that maintains and/or preserves mitochondrial function under conditions of increased metabolic stress. Grimmsmann and Rustenbeck (18Grimmsmann T. Rustenbeck I. Brit. J. Pharmacol. 1998; 123: 781-788Crossref PubMed Scopus (108) Google Scholar) reported that diazoxide at concentrations used routinely to open plasma membrane KATP channels in β-cells also exerted direct effects on β-cell mitochondrial function, resulting in a decrease in mitochondrial membrane potential, an efflux of Ca2+ from the mitochondria, and a decrease in ATP concentration. These findings demonstrated that diazoxide exerts multiple effects on β-cells that are not limited to plasma membrane KATP channels. Studies by Grill and co-workers (19Grill V. Bjorklund A. Diabetes. 2001; 50: S122-S124Crossref PubMed Google Scholar, 20Yoshikawa H. Ma Z. Bjorklund A. Grill V. Am. J. Physiol. 2004; 287: E1202-E1208Crossref Scopus (15) Google Scholar, 21Bjorklund A. Lansner A. Grill V.E. Diabetes. 2000; 49: 1840-1848Crossref PubMed Scopus (62) Google Scholar) have further demonstrated that chronic glucose exposure of pancreatic islets for 48 h under both in vitro and in vivo conditions produced nonresponsiveness to a subsequent glucose challenge. However, if the chronic exposure of islets to glucose was performed in the presence of diazoxide, the ability of β-cells to respond to glucose was maintained. A conclusion drawn from these studies was that the protective effect of diazoxide against glucose-induced desensitization of β-cells was not due to a lasting effect of diazoxide on plasma membrane KATP channels and may involve other targets possibly including the β-cell mitochondria.Our data indicate that [3H]thymidine incorporation in adult rodent islets chronically exposed to glucose or glyburide is largely mediated through mTOR via the KATP channel and changes in intracellular Ca2+. Based on [3H]thymidine incorporation and cell cycle analysis, we determined that chronic exposure of adult islets to basal glucose (3 mm) results in DNA synthesis and cell cycle progression consistent with a low level of cell proliferation. In contrast, chronic exposure to elevated glucose or glyburide results in cell cycle progression from G0/G1 to an accumulation of newly synthesized DNA in S phase and a reduction in G2/M. Rapamycin (100 nm) resulted in an ∼62% reduction of S phase accumulation. The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation.EXPERIMENTAL PROCEDURESMaterials—Male Sprague-Dawley rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN). Collagenase type XI, Ficoll type 400-DL, diazoxide, glyburide, RNase A, and propidium iodide were obtained from Sigma. CMRL-1066 tissue culture media was from Invitrogen. Defined fetal bovine serum (FBS) was from Hyclone (Logan, UT). Penicillin, streptomycin, Hanks' balanced salt solution, and l-glutamine were obtained from the Washington University Tissue Culture Support Center. Dispase and Versene were from Roche Applied Science. Rapamycin was from Biomol (Plymouth Meeting, PA). [methyl-3H]Thymidine-aqueous, 2.0 Ci/mmol, 1.0 mCi/ml was from PerkinElmer Life Sciences. Nifedipine was from Calbiochem. The primary antibody for S6K1 was from R&D Systems (Minneapolis, MN). The secondary antibody, peroxidase-conjugated donkey anti-rabbit IgG, was obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). All other chemicals were from commercially available sources.Islet Isolation—Islets were isolated from male Sprague-Dawley rats (200–250 g) by collagenase digestion as described previously (22McDaniel M.L. Colca J.R. Kotagal N. Lacy P.E. Methods Enzymol. 1983; 98: 182-200Crossref PubMed Scopus (117) Google Scholar). Briefly, pancreases were inflated with Hanks' balanced salt solution, and the tissue was isolated, minced, and digested with 20 mg of collagenase/pancreas for 6 min at 39 °C. Islets were separated on a Ficoll step density gradient and then placed in CMRL-1066 culture medium supplemented with 10% FBS, 2 mm l-glutamine, 5.6 mm glucose, 100 units/ml penicillin, 100 μg/ml streptomycin (cCMRL) and selected with a stereomicroscope to exclude any contaminating tissues.[3H]Thymidine Incorporation—Islets (100) were counted into Falcon Petri dishes (35 × 10 mm) and were cultured for 4 days in 1 ml of CMRL (3 or 20 mm glucose, 10% FBS, 2 mm l-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin) containing treatment conditions as indicated in the figure legends at 37 °C, under 95% air/5% CO2. The culture media were changed everyday or after 2 days as indicated. During the final 24 h of the 96-h incubation, 10 μCi of [3H]thymidine was added to each dish. [3H]Thymidine incorporation was determined by trichloroacetic acid extraction and scintillation counting (23Bradley L.M. Mishell B.B. Shiigi S.M. “Selected Methods in Cellular Immunology”. W. H. Freeman and Co., N.Y.1980: 156-158Google Scholar).Western Blotting—Islets were treated as described above for [3H]thymidine incorporation. Following incubation, islets were processed for SDS-PAGE electrophoresis and Western blotting for S6K1.Flow Cytometry—Following the 4-day treatment as described above, islets were subjected to cell cycle analysis by flow cytometry. Islets were dispersed into single cells by treatment with Versene, followed by Dispase in Ca2+- and Mg2+-free Hanks' solution at 31 °C. The dispersed cells were washed once with PBS + 1% FBS and pelleted by low speed centrifugation. Cells were resuspended in 500 μl of PBS. Five ml of 100% ethanol was added slowly while vortexing to avoid clumping of cells. Fixed cells were stored at –20 °C until use. Cells were collected by centrifugation, washed once with PBS + 1% FBS, and resuspended in PBS + 1% FBS containing RNase A (250 μg/ml) and propidium iodide (10 μg/ml). PI-stained cells were analyzed for their DNA content using a FACSCALIBUR instrument and data analyzed with CellQuest software (Immunocytometry Systems).Expression of Data and Statistics—Data are presented as mean ± S.E. Statistically significant differences between groups were analyzed using unpaired one-tailed t tests, where p < 0.05 was considered significant.RESULTSIn our experimental design, isolated primary rat islets were chronically exposed to either basal (3 mm) or elevated glucose (20 mm) for 4 days to quantitate the incorporation of [3H]thymidine that was added to the culture media on day 3 as an index of DNA synthesis. Time course studies established that a 4-day incubation period was optimal for [3H]thymidine incorporation under these conditions. Separate studies also demonstrated, based on S6K1 phosphorylation, that mTOR remained fully activated in response to elevated glucose or glyburide and was not down-regulated over this time period. This same 4-day treatment period was used to measure cell cycle progression by flow cytometry. Both methods measure DNA synthesis, although in the case of [3H]thymidine incorporation only the last 24 h of incubation are measured, whereas with the flow cytometry method, total DNA is intercalated with PI.Glyburide Dose Response—Our initial approach was to determine whether glyburide, an inhibitor of KATP channels that enhances Ca2+ influx, modulates DNA synthesis through mTOR. As shown in Fig. 1, chronic exposure of rat islets to elevated glucose for 4 days resulted in an increase in DNA synthesis as assessed by [3H]thymidine incorporation compared with islets exposed to basal glucose (3 mm, lane 2 versus lane 1). Glyburide dose-dependently (1–100 nm) increased DNA synthesis at basal glucose (lanes 4 and 5). These concentrations of glyburide are within the physiological range typically used to modulate KATP channels.Role for Ca2+ Influx—Studies were next performed to determine whether glyburide-mediated increases in DNA synthesis were due to cytosolic Ca2+ derived from the extracellular media. In Fig. 2, an increase in glucose concentration enhanced DNA synthesis above basal glucose (lane 2 versus lane 1) and glyburide (10 nm) at basal glucose (lane 4) stimulated DNA synthesis to the same level attained by elevated glucose alone (lane 2). Nifedipine (10 μm), an inhibitor of the L-type Ca2+ channel, blocked glyburide-stimulated DNA synthesis (lane 5) to a level comparable with basal glucose (lane 1), and all of these effects were inhibited by rapamycin (25 nm) as shown in lane 6. The level of DNA synthesis in the presence of rapamycin (lane 6) was substantially lower than basal DNA synthesis (lane 1), supporting our previous findings that mTOR mediates both basal and nutrient stimulated DNA synthesis (7Kwon G. Marshall C.A. Pappan K.L. Remedi M.S. McDaniel M.L. Diabetes. 2004; 53: S225-S232Crossref PubMed Scopus (125) Google Scholar). These results support the conclusion that the ability of glyburide to mediate [3H]thymidine incorporation is due, in part, to an increase in Ca2+ influx through the nifedipine-sensitive L-type Ca2+ channel.FIGURE 2Stimulatory effect of glyburide on DNA synthesis at basal glucose is modulated by Ca2+ influx. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mm glucose, glyburide, nifedipine, or rapamycin as indicated and cultured as described in the legend to Fig. 1. [3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment. Inset, S6K1 Western blot. Islets were treated as described above. Following incubation, islets were processed for SDS-PAGE electrophoresis and Western blotting for S6K1. Blot is representative of n = 2 experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)These results also correlated with S6K1 phosphorylation based on gel shift mobility assays (upper inset) as an indicator of mTOR activation. Elevated glucose robustly enhanced S6K1 phosphorylation as indicated by the intense and more slowly migrating upper band (lane 2) compared with basal glucose (lane 1). An effective concentration of glyburide (10 nm) resulted in a partial activation of S6K1 phosphorylation at basal glucose (lane 4) as indicated by the doublet band in comparison to basal glucose (lane 1) that is sensitive to nifedipine (lane 5), and all of these effects are blocked by rapamycin (lane 6). These studies, however, suggest that the level of S6K1 activation is not linearly related with [3H]thymidine incorporation, since different levels of S6K1 activation (lane 2 versus lane 4) resulted in comparable levels of DNA synthesis. We propose that S6K1 is an excellent indicator of mTOR activation but other mTOR-mediated signals in addition to S6K1 activation are important for the regulation of DNA synthesis.Diazoxide Potentiates Glucose-stimulated DNA Synthesis—Our next approach was to determine whether diazoxide, an opener of KATP channels, would decrease glucose-stimulated DNA synthesis due to its ability to attenuate Ca2+ influx derived from the extracellular media. As shown in Fig. 3, diazoxide (250 μm) paradoxically enhanced the ability of elevated glucose to stimulate DNA synthesis (lane 3 versus lane 2). This enhanced DNA synthesis also occurred in the absence of diazoxide treatment during the final 24 h of the total 96-h incubation period as shown in the washout protocol (lane 4). Rapamycin (25 nm) blocked the ability of diazoxide to significantly enhance glucose-stimulated DNA synthesis (lane 5). These unexpected results suggested that diazoxide might exert multiple effects to mediate the potentiation of glucose-stimulated DNA synthesis in addition to the opening of plasma membrane KATP channels. The observation that diazoxide did not have to be present for the total 4 day incubation period to potentiate glucose-stimulated DNA synthesis was consistent with this conclusion, since the ability of diazoxide to activate plasma membrane KATP channels is rapidly reversible following its removal.FIGURE 3Diazoxide enhances glucose-stimulated DNA synthesis. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mm glucose, diazoxide, or rapamycin as indicated and cultured as described in the legend to Fig. 1 with the exception that during the final 24 h of incubation, diazoxide was washed away from lane 4.[3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Multiple Sites of Action for Diazoxide—Studies were next performed to determine whether the ability of diazoxide to attenuate Ca2+ influx through L-type Ca2+ channels is responsible for the significant potentiation of glucose-stimulated DNA synthesis. If this were the case, an inhibitor of the L-type Ca2+ channel such as nifedipine should mimic the potentiating effects produced by diazoxide on glucose-stimulated DNA synthesis. As shown in Fig. 4, the ability of elevated glucose alone to increase DNA synthesis (lane 2) was significantly enhanced by diazoxide exposure (lane 3). However, nifedipine (0.1–10 μm) failed to mimic the potentiating effect of diazoxide on glucose-stimulated DNA synthesis (lanes 4–6). These results suggest that diazoxide stimulates DNA synthesis by mechanisms other than solely attenuating Ca2+ influx.FIGURE 4Nifedipine does not alter the ability of an elevated glucose concentration to increase DNA synthesis. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mm glucose, diazoxide, or nifedipine as indicated and cultured as described in the legend to Fig. 1. [3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Evidence that the potentiating effect of diazoxide on glucose-stimulated DNA synthesis is associated with a nifedipine-sensitive component of Ca2+ influx is shown in Fig. 5. As shown in lane 3, diazoxide enhanced the ability of glucose (20 mm) to stimulate DNA synthesis compared with glucose alone (lane 2). In lane 4, nifedipine (10 μm) in the presence of diazoxide (250 μm) significantly inhibited glucose-stimulated DNA synthesis to the level produced by glucose alone (lane 2). These results indicate that the significant enhancement of glucose-stimulated DNA synthesis by diazoxide (lane 3) is dependent on a low level of nifedipine-sensitive Ca2+ influx into β-cells. In contrast, chronic exposure of islets to elevated glucose alone as shown in Fig. 4 (lane 2) resulted in a smaller increase in DNA synthesis that is independent of Ca2+ influx due to the lack of inhibition by nifedipine (lanes 4–6).FIGURE 5Nifedipine inhibits the potentiating effects of diazoxide to enhance DNA synthesis. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mm glucose, diazoxide, or nifedipine as indicated and cultured as described in the legend to Fig. 1. [3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment. * indicates a significant difference (p < 0.05) analyzed by an unpaired one-tailed t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The conclusion that the stimulation of Ca2+ influx, under conditions in which primary rat islets are chronically exposed to elevated glucose, will enhance DNA synthesis is further supported by the findings shown in Fig. 6. In this experimental protocol, glyburide due to its ability to directly inhibit KATP channels was used in combination with elevated glucose to enhance Ca2+ influx. Chronic exposure of islets to glucose (20 mm) in combination with glyburide (10 nm) significantly potentiated DNA synthesis compared with an elevated glucose concentration alone (lane 3 versus lane 2) in a nifedipine-sensitive manner (lane 4).FIGURE 6Glyburide enhances DNA synthesis at elevated chronic exposure to glucose through Ca2+ influx. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mm glucose, glyburide, or nifedipine as indicated and cultured as described in the legend to Fig. 1. [3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment. * indicates a significant difference (p < 0.05) analyzed by an unpaired one-tailed t test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Rapamycin-mediated Inhibition of Glucose-stimulated DNA Synthesis Is Irreversible in Vit" @default.
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• W1978463531 date "2006-02-01" @default.
• W1978463531 modified "2023-10-16" @default.
• W1978463531 title "Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by KATP Channels" @default.
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• W1978463531 doi "https://doi.org/10.1074/jbc.m508821200" @default.
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