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J. Biol. Chem., Vol. 281, Issue 6, 3261-3267, February 10, 2006

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Glucose-stimulated DNA Synthesis through Mammalian Target of Rapamycin (mTOR) Is Regulated by K_ATP Channels

EFFECTS ON CELL CYCLE PROGRESSION IN RODENT ISLETS^*

Guim Kwon, Connie A. Marshall, Hui Liu, Kirk L. Pappan, Maria S. Remedi^¶, and Michael L. McDaniel¹

From the Departments of Pathology and Immunology and ^¶Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the School of Pharmacy, Southern Illinois University, Edwardsville, Illinois 62026

Received for publication, August 10, 2005 , and in revised form, December 8, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 K_ATP channel and changes in cytosolic Ca²⁺, we determined whether glyburide, an inhibitor of K_ATP 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 K_ATP 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Both types 1 and 2 diabetes result from the inability of pancreatic -cells to secrete sufficient amounts of insulin to maintain normal glucose homeostasis due to an acquired secretory defect and/or inadequate -cell mass. Increased metabolic demands or stress responses that exert a positive effect on -cell mass include obesity, pregnancy, partial pancreatectomy, or chronic glucose exposure. -Cell mass is regulated by cellular mechanisms that include replication, neogenesis, hypertrophy, and apoptosis (1, 2). Recent studies have emphasized the importance of the proliferative capacity of existing adult -cells as a major source of new -cells during adult life that may significantly contribute to the maintenance of -cell mass (3).

Mammalian target of rapamycin (mTOR)² 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 (4, 5). 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 (6–9). Insulin secreted by the -cell and growth factors also provide input to mTOR through the insulin signaling cascade to Akt (6). 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 (10). Apparently, this negative feedback to IRS2 does not reduce nutrient-stimulated mTOR activation in the -cell as S6K1 remains fully activated during a 4- or 6-day exposure to elevated glucose (7), 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 (7). 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 K_ATP channel and changes in cytosolic Ca²⁺ in rodent islets (7). Agents that have been used to modulate the K_ATP channel in -cells include: 1) glyburide, a sulfonylurea type agent, which directly inhibits the K_ATP channel, causes depolarization, increases in Ca²⁺ influx and insulin secretion and 2) diazoxide, an activator of K_ATP channels, that causes hyperpolarization, inhibition of Ca²⁺ influx, and a blockage of insulin secretion.

At basal glucose (3 mM), glyburide-induced closure of K_ATP 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 K_ATP channel partially inhibited S6K1 phosphorylation by glucose. These results suggested that diazoxide might attenuate glucose-stimulated DNA synthesis.

Guiot et al. (11) reported in 1994 that glibenclamide (glyburide) stimulated -cell replication and increased -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 1D subunit is required for proper -cell development in the postnatal pancreas (12). In 1D gene knock-out mice, -cell proliferation was decreased, suggesting that a defect in Ca²⁺ influx may be partly responsible for a reduction in -cell mass. In addition, recent findings indicated that S6K1 requires an initial Ca²⁺-dependent priming event for activation (13, 14).

Diazoxide has been used extensively in vitro to characterize the regulation of stimulus-secretion coupling mechanisms of -cells that are independent of plasma membrane K_ATP channels. Although diazoxide has provided important mechanistic insights into the role of plasma membrane K_ATP channels in -cells, it is also reported to exert significant effects on mitochondrial energetics (15, 16). A direct effect of K_ATP channel openers including diazoxide on mitochondrial function has been shown to be critical in the preservation of heart function against injury (17). 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 (18) reported that diazoxide at concentrations used routinely to open plasma membrane K_ATP channels in -cells also exerted direct effects on -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 -cells that are not limited to plasma membrane K_ATP channels. Studies by Grill and co-workers (19–21) 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 -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 -cells was not due to a lasting effect of diazoxide on plasma membrane K_ATP channels and may involve other targets possibly including the -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 K_ATP 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 PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—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 (22). 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 x 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 (23).

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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In 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.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Glyburide dose-dependently enhances DNA synthesis at a basal glucose concentration. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mM glucose ± glyburide as indicated at 37 °C, under 95% air/5% CO2. The medium was changed after 2 days. During the final 24 h of the 96-h incubation, [3H]thymidine was added to each dish and [3H]thymidine incorporation determined as described under "Experimental Procedures." Data are the means ± S.E. of n = 3 experiments with triplicate samples in each experiment.

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 (7). 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.

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.

View larger version (29K): [in this window] [in a new window]   FIGURE 2. Stimulatory 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.

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.

View larger version (23K): [in this window] [in a new window]   FIGURE 3. Diazoxide 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.

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 K_ATP 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).

Rapamycin-mediated Inhibition of Glucose-stimulated DNA Synthesis Is Irreversible in Vitro—An important question raised in these in vitro studies is whether the ability of rapamycin to inhibit basal and glucose-stimulated DNA synthesis in primary rodent islets is reversible. This concern is also relevant to the current use of rapamycin as an immunosuppressant in islet transplantation protocols in type 1 diabetes (24). As shown in Fig. 7, the inhibitory effects of rapamycin (1 nM) present for 4 days on glucose-stimulated DNA synthesis (lane 3 versus lane 2) is nearly identical to the inhibition observed when rapamycin is present for only 1 day and absent during the remaining 3 days of the incubation period (lane 6). As shown in the inset, S6K1 phosphorylation, based on gel shift analysis, correlates well with the effects of rapamycin on DNA synthesis. These results indicate that inhibition of DNA synthesis by rapamycin (1 nM) is essentially irreversible during this 3-day exposure period under these in vitro conditions with primary adult islets. Furthermore, these findings suggest that the synthesis of new mTOR protein may be required to restore mTOR activity following the removal of the inhibitor, rapamycin.

View larger version (19K): [in this window] [in a new window]   FIGURE 4. Nifedipine 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 larger version (20K): [in this window] [in a new window]   FIGURE 5. Nifedipine 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 larger version (19K): [in this window] [in a new window]   FIGURE 6. Glyburide 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 larger version (36K): [in this window] [in a new window]   FIGURE 7. Inhibition of DNA synthesis by rapamycin is irreversible. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mM glucose, or rapamycin as indicated. The medium was changed each day with rapamycin being washed away as indicated. During the final 24 h of incubation [3H]thymidine was added to each dish. [3H]Thymidine incorporation was determined. Data are the means ± S.E. of n = 3 experiments. 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 using 1 nM rapamycin and n = 1 using 25 nM rapamycin.

View larger version (20K): [in this window] [in a new window]   FIGURE 8. Cell cycle analysis of glucose- and glyburide-stimulated rat islets. Islets were cultured for 4 days in 1 ml of cCMRL, 3 or 20 mM glucose, or 3 mM glucose + 10 nM glyburide as indicated at 37 °C, under 95% air/5% CO2. The medium was changed after 2 days. At the end of 4 days, islets were stained with propidium iodide and processed for flow cytometry to determine cell cycle progression as described under "Experimental Procedures." Data are representative of n = 3 experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In rodents, chronic exposure of islets to elevated glucose has been shown to increase hypertrophy, proliferation, and/or the neogenesis of -cells. Steil et al. (25) have shown that glucose infusions in rats for 1–2 days resulted in an increase in -cell mass that paralleled increased rates of -cell proliferation. Paris et al. (26) also reported that chronic exposure of rodent islets to glucose in vivo for 48 h provoked a rapid compensatory increase in -cell mass that was independent of plasma insulin levels. Lipsett et al. (27) reported that -cell neogenesis contributed significantly to glucose-stimulated expansion of -cell mass during prolonged hyperglycemia in rats infused with elevated glucose. In in vitro models, the mitogenic effects produced by elevated glucose were shown to be long lasting in rat islets and more transitory in human islets (28). In addition, prolonged exposure of rodent islets to elevated glucose resulted in impaired insulin secretion and increased -cell apoptosis that is attributed, in part, to glucose toxicity.

Our current studies showed that glyburide in the nanomolar range at basal glucose stimulated DNA synthesis to the same magnitude as elevated glucose, and these effects were blocked by rapamycin. Nifedipine prevented glyburide-mediated DNA synthesis indicating that the ability of glyburide to stimulate DNA synthesis was due to an increase in Ca²⁺ influx from the extracellular media. Maedler et al. (29) reported with isolated human islets that 0.1 and 10 nM glyburide in the presence of basal glucose after 4 days of exposure produced small increases (2.2- and 2.5-fold, respectively) in -cell apoptosis. In the -cell line, INS-1, Briaud et al. (30) reported that glyburide (5 µM) increased DNA synthesis at basal glucose (3 mM) but was ineffective at an elevated glucose concentration. Glyburide also failed to stimulate phosphorylation of S6K1 in this -cell line. Their results indicated that glyburide and/or glucose exposure stimulated increases in cytosolic Ca²⁺ that were primarily associated with Erk1/2 phosphorylation, and DNA synthesis likely occurred through a protein kinase A-dependent pathway. The effects of rapamycin on glucose- or glyburide-mediated DNA synthesis were not evaluated in this study. A plausible explanation for the lack of agreement of the effects of glyburide on S6K1 phosphorylation with our findings is that these studies were performed in a rapid proliferating cell line, INS-1, as opposed to primary islets.

In our previous studies, diazoxide treatment resulted in a partial inhibition of S6K1 phosphorylation suggesting that diazoxide might reduce glucose-stimulated DNA synthesis (7). Paradoxically, diazoxide (250 µM) in the presence of glucose (20 mM) resulted in a marked 2–4-fold enhancement of DNA synthesis above that of elevated glucose alone and occurred in a rapamycin-sensitive manner. This potentiating effect also occurred under conditions when diazoxide was absent from the incubation media during the final 24 h of the 96 h total incubation period. These effects of diazoxide were also compared with those of pinacidil, another K_ATP channel opener (18). Pinacidil mimicked the effects produced by diazoxide on glucose-stimulated DNA synthesis but to a significantly lesser degree possibly due to its lower potency in opening K_ATP channels in comparison with diazoxide in pancreatic -cells (data not shown). The ability of diazoxide to attenuate nutrient over-stimulation of mTOR (7) might also be anticipated to down-regulate the negative feedback pathway from mTOR/S6K1 to IRS2. It is possible that attenuating this negative feedback pathway with diazoxide may increase -cell mass by reducing apoptosis through increased growth factor activation of IRS2-, PI3K-, and Akt-regulated pathways other than mTOR in vivo. Functional IRS2/PI3K/Akt signaling is considered essential for -cell survival (1).

Studies were next performed with nifedipine, an inhibitor of L-type Ca²⁺ channels, to determine whether the ability of diazoxide to attenuate Ca²⁺ influx was responsible for the marked potentiation of glucose-stimulated DNA synthesis. Nifedipine failed to mimic the stimulatory effects produced by diazoxide (Fig. 4) even though nifedipine completely blocked insulin secretion in response to glucose (data not shown). Overall, these results suggested that diazoxide might be exerting effects independent of the activation of plasma membrane K_ATP channels that are responsible for the potentiation of glucose-stimulated DNA synthesis. Findings by Kullin et al. (31) also reported that K_ATP channel openers including diazoxide decreased the vulnerability of rat islets to free radicals induced by alloxan, nitroprusside, or IL-1. It was proposed that K_ATP channel openers by their ability to decrease Ca²⁺ influx due to -cell membrane hyperpolarization in combination with their direct effect to decrease the mitochondrial membrane potential reduced ATP production necessary for the activation of apoptotic pathways. In cardiac mitochondria, Holmuhamedov et al. (16) have also proposed that K_ATP channel openers modulate Ca²⁺ homeostasis through dissipation of the mitochondrial membrane potential that leads to reduced mitochondrial Ca²⁺ influx and provides protection against ischemic injury.

Based on cell cycle analysis, our studies suggest that the ability of chronic elevated glucose or glyburide to enhance DNA synthesis in adult islets is linked to an accumulation of cells in S phase. Conditions of chronic hyperglycemia or pregnancy have been shown to cause hypertrophy of islet cells; however, the possibility that hypertrophy is responsible for S phase accumulation of DNA is unlikely, since the synergy of cell growth and proliferation is a G₁ phenomenon regulated by retinoblastoma protein and not by S phase (32). Although the mechanism responsible for this defect in cell cycle progression is unknown, it may be related to excess free radical production which predisposes the mitochondria to Ca²⁺ overload that mediates DNA damage. It has been shown under chronic hyperglycemic conditions that mRNA levels of some stress-related genes are increased in islets cells such as, heme oxygenase-1, glutathione peroxidase, A20, Fas, iNOS, and c-Myc (33, 34). Based on flow cytometry, S phase accumulation represents new DNA synthesis. The arrest of islet cells in S phase may be due to the lack of protein expression, i.e. cyclins, necessary to complete cell cycle progression or possibly a checkpoint in cell cycle progression due to the over expression of stress-related proteins that cause a response to DNA damage.

The ability of diazoxide to significantly enhance [³H]thymidine incorporation above that of elevated glucose alone might be related to the protective effect that diazoxide exerts on the -cell mitochondria. Since the ability of diazoxide to enhance DNA synthesis occurs without any significant alterations in S phase accumulation, this increase in DNA synthesis appears to be linked to an arrest in S phase cell cycle progression and not translated to cell proliferation. In contrast, chronic exposure of islets to basal glucose that only partially activates mTOR results in a typical cell cycle progression pattern that is consistent with a low level of proliferation. Santiago-Walker et al. (35) have recently reported that the ability of PKC to stimulate apoptosis of primary rat thyroid cells is linked to its ability to stimulate G₁ phase cell cycle progression and S phase arrest. Evidence that apoptosis was a direct consequence of replication stress was established by using the PI3K inhibitor, LY294002, that prevented S phase entry and apoptosis. Based on cell cycle analysis by flow cytometry, we have no evidence to indicate that a significant degree of apoptosis occurs under our in vitro conditions with primary rat islets (data not shown). Interestingly, Belanger et al. (36) reported that the anti-apoptotic protein Bcl-2 delays cell proliferation and promotes accumulation of cells in S phase without affecting the rate of apoptosis by human ovarian carcinoma cells. Bcl-2 as a regulator of apoptosis is believed to alter mitochondrial permeability transition through its ion channel activity and inhibits the release of both cytochrome c and apoptosis-inducing factor from the mitochondria, thereby promoting cell survival. Importantly, it has been reported that damage to DNA results in activation of damage response pathways in human epidermal keratinocytes (37). The signal derived from DNA damage to the activation of these response pathways is mediated by mTOR. A similar mechanism may be responsible for enhanced [³H]thymidine incorporation that occurs in a rapamycin-sensitive manner as a consequence of chronic exposure of adult rodent islets to elevated glucose. The ability of rapamycin to partially prevent in a dose-dependent manner the accumulation of DNA in S phase may reflect the necessity of inhibiting mTOR more completely at the higher concentration of 100 nM that maximally inhibits cell cycle progression from G₀/G₁ to S phase. The reason for the differences in sensitivity to rapamycin of [³H]thymidine incorporation versus cell cycle progression are unknown but may be due to different mTOR-dependent pathways involving DNA damage responses and/or cell cycle checkpoints.

Overall, in our model of chronic glucose exposure we observed an accumulation of DNA in S phase that does not translate to an increase in cell proliferation. This is consistent with the well established finding that only a few adult islet cells are progressing through the cell cycle at any one time. Thus, it appears that the enhanced [³H]thymidine incorporation observed under conditions of prolonged elevated glucose alone or in combination with diazoxide or glyburide may be related to a response to DNA damage that is mediated by mTOR. Although an increase in DNA synthesis based on either [³H]thymidine or bromodeoxyuridine incorporation is required for cell division, this essential prerequisite does not always result in completion of the cell cycle. Our studies suggest that the appropriate regulation of mTOR by nutrients and pharmacologic agents that mediate DNA synthesis and cell cycle progression may be a component of an effective strategy to enhance the survival, growth, and proliferative capacity of adult rodent islets.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant DK-06181 (to M. L. M.), an American Diabetes Association Mentor-based Postdoctoral Fellowship (to M. L. M), and an American Diabetes Association/Takeda Pharmaceuticals Mentor-based Minority Postdoctoral Fellowship (to M. L. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Pathology & Immunology, Washington University School of Medicine, Campus Box 8118, 660 South Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7435; Fax: 314-362-4096; E-mail: mmcdaniel{at}wustl.edu.

² The 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; K_ATP 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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