©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Glucose Modulates -Aminobutyric Acid Release from the Pancreatic TC6 Cell Line (*)

(Received for publication, August 23, 1995)

H. Rex Gaskins (1)(§) Manuel E. Baldeón (1)(¶) Leelie Selassie (1)(**) J. Lee Beverly (2)

From the  (1)Division of Nutritional Sciences and Department of Animal Sciences, University of Illinois, Urbana, Illinois 61801 and the (2)Department of Nutrition, Texas Tech University, Lubbock, Texas 79409

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To determine if endogenous -aminobutyric acid (GABA) is secreted by a pancreatic beta-cell-derived cell line and to determine the effects of glucose on GABA release, betaTC6 cultures were incubated in the presence of 1 or 10 mmol/l glucose for 12 h and then subjected to a 2-h secretion test in Krebs-Ringer buffer containing 1 or 10 mmol/l glucose. betaTC6-conditioned medium was collected at 15, 30, 60, and 120 min after glucose stimulation for GABA analysis by high pressure liquid chromatography-electrochemical detection. After 30 min, medium GABA concentrations were significantly higher (p < 0.05) in cultures that were exposed to high glucose during both the 12-h incubation period and the 2-h secretion test than in the remaining three glucose combinations. To address possible roles of beta-cell-derived GABA, the effect of GABA on glucagon secretion from pancreatic alphaTC6 cells was tested at concentrations released from betaTC6 cells. Inhibition of glucagon secretion by alphaTC6 cells was observed in the presence of GABA at concentrations equivalent to concentrations secreted by betaTC6 cells. The inhibitory effects of GABA on glucagon secretion by alphaTC6 cells were blocked by the GABA(A) receptor antagonist bicuculline and were dissociated from the inhibitory effects of glucose. Together, these results provide the first documentation that endogenous GABA is released from a highly differentiated beta-cell line and that glucose and GABA independently attenuate glucagon secretion by a pancreatic alpha-cell line.


INTRODUCTION

Glutamic acid decarboxylase (L-glutamate 1-carboxylase; EC 4.1.1.15) has been identified as an early target antigen of the T-lymphocyte-mediated destruction of pancreatic beta-cells causing insulin-dependent diabetes mellitus(1, 2) . Development of insulitis and diabetes was prevented in NOD mice by inactivating glutamic acid decarboxylase-reactive T-cells(1, 2) , and serum antibodies to glutamic acid decarboxylase are predictive of prediabetes in humans(3) . Recent focus on glutamic acid decarboxylase's role in insulin-dependent diabetes mellitus has exposed a striking lack of knowledge as to the functional relevance of glutamic acid decarboxylase and its synthetic product, -aminobutyric acid (GABA), (^1)in the endocrine pancreas.

Briel and associates (4) and Okada and associates (5) were among the first to report that GABA was present in the endocrine pancreas at concentrations comparable with those found in the central nervous system. There is limited GABAergic innervation of the acinar pancreas, and GABA-containing neuronal processes are located adjacent to islets, but few neuronal processes are found within an islet(5) . The highest concentrations of GABA in the pancreas are found within endocrine islets, with up to 10-fold higher concentrations than in the exocrine acinar pancreas(6) . Within pancreatic islets, GABA is confined to beta-cells as are the two enzymes responsible for GABA synthesis and metabolism, glutamic acid decarboxylase and GABA transaminase (GABA-alpha-ketoglutarate transaminase, EC 2.6.1.19 (reviewed in Refs. 7 and 8). Within isolated beta-cells and betaTC3 cells, GABA is found both in the cytoplasm and in distinct synaptic-like microvesicles(9, 10, 11) , which is suggestive of regulated release; however, neither constitutive nor regulated release of GABA from purified beta-cells has been demonstrated.

The role of GABA in islet function is also unclear. GABA receptors have been demonstrated on both pancreatic alpha-cells and -cells but not on beta-cells(12) . In vitro studies with isolated islets and with perfused pancreas of mice, rats, and dogs (8, 12, 13, 14) support the idea that beta-cell GABA may modulate alpha-cell function in a paracrine manner, although data from purified cells have not been reported. In the present study, differentiated pancreatic alpha- and beta-cell lines derived from transgenic mice (15, 16) were used to determine (i) if GABA is released from transformed beta-cells, (ii) if GABA secretion is influenced by extracellular glucose concentrations, and (iii) to test the effects of GABA on glucagon secretion from a purified alpha-cell line.


EXPERIMENTAL PROCEDURES

Cell Culture

The beta-cell line used in these experiments was derived from transgenic mice expressing the large T-antigen of SV40 under control of the rat insulin promoter(15) . The betaTC6 cells are one of several beta-cell lines that were developed with normal glucose-regulated insulin secretion from insulinomas obtained by breeding of the hybrid transgene from the original C57BL/6J mouse strain onto the C3HeB/FeJ genetic background(16) . Low passages of betaTC6 cells maintain a high insulin content and exhibit a normal glucose concentration dependence for glycolysis and insulin secretion, thus representing an accurate model of beta-cell function(16) . betaTC6 cells were kindly provided by Dr. Shimon Efrat, Albert Einstein College of Medicine.

The alphaTC6 cell line was derived by a similar approach from a glucagonoma created in transgenic mice expressing the SV40 large T-antigen oncogene under the control of the rat preproglucagon promoter (15) . Glucagon-secreting alphaTC6 cells were selectively cloned from the original alphaTC1 cell line by the limiting dilution method(17) . These cells maintain many of the differentiated characteristics of alpha-cells in situ including the synthesis and secretion of glucagon and not insulin(17, 18) . alphaTC6 cells were kindly provided by Dr. Edward H. Leiter, The Jackson Laboratory.

The cell lines were routinely maintained in Dulbecco's minimal essential medium (DMEM) supplemented to a final glucose concentration of 25 mmol/l with Eagle's minimal essential medium nonessential amino acids, 44 mmol/l sodium bicarbonate, 15 mmol/l HEPES, 50 mg/l gentamicin sulfate, 15% horse serum, and 2.5% FetalClone II (HyClone Laboratories, Inc., Logan, UT). Unless otherwise specified, cell culture reagents were purchased from Life Technologies, Inc. Cells were seeded at a density of 5 times 10^4 cells/well into 24-well plates (Corning, Corning, NY). Cultures were maintained in a humidified atmosphere of 95% air, 5% CO(2) at 37 °C. Experiments were performed when cells were approximately 70% confluent (approximately 4 times 10^5 cells/well). Following the culture period, cells were harvested and sonicated in 0.5 ml of phosphate-buffered saline, 0.1% Triton X-100 (Fisher Biotech, Fair Lawn, NJ). Samples were stored at -20°C for later protein determination by the Bradford microassay method (Bio-Rad).

Cell Secretion Experiments

To determine if endogenous GABA is secreted from beta-cells and if glucose influences release, betaTC6 cultures (passage 17) were established in 24-well tissue culture plates according to standard culture protocol (see above) and used in a 2 times 2 factorial design study. At approximately 70% confluence, cells were washed twice with DMEM (no glucose) and then exposed to DMEM containing 0.5% FetalClone II and supplemented with either 1 or 10 mmol/l glucose for a 12-h incubation period. The amount of GABA released by betaTC6 cells in response to 1 or 10 mmol/l glucose in HEPES-buffered Krebs-Ringer buffer (KRB; 119 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl(2), 1.2 mmol/l MgSO(4), 1.2 mmol/l KH(2)PO(4), 25 mmol/l NaHCO(3), 10 mmol/l HEPES at pH 7.4 and 0.1% bovine serum albumin) was then determined during a subsequent 2-h secretion test. During the secretion test, 50-µl aliquots of betaTC6 cell-conditioned KRB were collected at intervals of 15, 30, 60, and 120 min. Samples were stored at -80 °C until assayed for GABA content by HPLC analysis. Insulin concentrations were measured in the 120-min samples by radioimmunoassay (RIA). Cells were seeded equally among wells, and protein values per well were consistently similar across glucose treatments at the end of the culture period.

GABA dose-response studies were conducted to determine the effects of GABA on glucagon secretion by alphaTC6 cells (passage 28). The GABA concentrations tested were in the same molar range as those secreted by betaTC6 cells. On the day of the experiment, medium was removed and alphaTC6 cells were washed twice with KRB (10 mmol/l glucose). Cells were then preincubated at 37 °C for 1 h in KRB containing 10 mmol/l glucose, followed by incubation in fresh KRB containing 1 mmol/l glucose alone or with increasing GABA concentrations (50-200 nmol/l GABA) or with 10 mmol/l glucose plus 100 nmol/l GABA for 2 h. Cells exposed to 1 or 10 mmol/l glucose plus 100 nmol/l GABA were also treated with bicuculline (100 nmol/l), a specific competitive antagonist of the GABA(A) receptor(19) . At the end of secretion test, alphaTC6-conditioned medium was collected for later glucagon determination by RIA. Cells were seeded equally among wells; protein values per well were similar across glucose, GABA, and bicuculline treatments at the end of the culture period. Data are expressed as percent of control (1 mmol/l glucose).

GABA HPLC Determinations

Bioanalytical Systems HPLC (West Lafayette, IN) was used to analyze GABA in betaTC6-conditioned KRB by a modified isocratic procedure with electrochemical detection(20, 21) . Ten microliters of sample was mixed with 10 µl of KRB, 4 µl of internal standard (D-aminovaleric acid), and 4 µl of derivatization reagent (27 mg of o-phthaldialdehyde in 10 ml of 50% 0.1 M carbonate buffer (pH 9.6), 50% methanol, and 22 ml of t-butylthiol) 3 min before injection onto a 100 times 4.6-mm C18 (3-µm) reverse-phase column (Microsorb-MV, Ranin Instruments, Woburn, MA) and 35 times 4-mm C18 (5-µm) guard column (Alltech Associates, Inc., Deerfield, IL). Mobile phase (pH 5.4) was 0.15 M sodium acetate buffer containing 1 mmol/l EDTA and 50% (v/v) acetonitrile delivered at a flow rate of 1.0 ml/min. Quantitation was by electrochemical detection, using a glass carbon electrode set at +0.70 V. Limit of detection for the GABA assay was 50-100 fmol of GABA with a coefficient of variation for 1 pmol of GABA standards consistently less than 0.08(22) . A five-point GABA standard curve between 0.5 and 10 pmol is used routinely with correlations consistently greater than 0.95.

Insulin and Glucagon RIA

Insulin concentrations in betaTC6-conditioned medium were determined by double antibody RIA using an assay validated in our laboratory following a protocol described previously(23) . Rat insulin was used as a standard. Standards, antibodies, and I-Insulin were obtained from Linco Research, Inc., St. Louis, MO. The inter- and intraassay coefficients of variation were 9 and 3%, respectively.

Glucagon concentrations in alphaTC6-conditioned medium were determined by double antibody RIA using a published assay that has been validated in our laboratory(24) . Porcine/bovine glucagon was used as a standard. Standards, antibodies, and I-glucagon were obtained from Linco Research, Inc., St. Louis, MO. The inter- and intraassay coefficients of variation were both less than 5%.

Statistical Analysis

Data were analyzed by two-way repeated measures analysis of variance (ANOVA; Experiment 1) and one-way ANOVA (Experiments 2 and 3). Means were compared by Scheffe's multiple comparison test when an ANOVA was significant. Data are presented as the mean ± S.E.


RESULTS

Effect of Extracellular Glucose Concentrations on GABA Secretion by betaTC6 Cells

Nanomolar concentrations of GABA were recovered in betaTC6-conditioned medium at all time points (15 min, 30 min, 1 h, 2 h) throughout a 2-h secretion test for both the low (1.0 mmol/l) and high (10 mmol/l) glucose treatments (Fig. 1). Concentrations of GABA recovered in medium from betaTC6 cells are consistent with reported values for GABA content in islet extracts (190 pmol/mg, wet weight(8) ). Glucose concentrations during the 12-h culture period and during the 2-h secretion test independently influenced GABA concentrations in betaTC6-conditioned medium. At the end of the first 15 min, GABA concentrations did not differ (p > 0.05) between low and high glucose treatments. At all time points thereafter (30, 60, and 120 min), GABA concentrations in the medium were significantly greater (p < 0.05) in cultures that were exposed to high glucose during both the 12-h incubation period and during the 2-h secretion period than in any of the remaining three glucose combinations (1:1, 1:10, and 10:1 mmol/l glucose; Fig. 1). GABA concentrations from cultures incubated for 12 h in high glucose but stimulated with 1 mmol/l glucose during the 2-h secretion test were not different from those cultured in 1 mmol/l glucose and stimulated with 10 mmol/l glucose or from those both cultured and stimulated with 1 mmol/l glucose (Fig. 1). When these results are considered with those observed from cultures exposed to 10 mmol/l glucose during both the incubation period and the secretion test, an effect of glucose on both GABA synthesis and secretion is apparent (Fig. 1). Within a treatment group, the amount of GABA present at the end of the 2-h secretion test was not different (p > 0.05) than the amount collected after 15 min (Fig. 1). Insulin concentrations in betaTC6-conditioned medium at the end of the 2-h secretion test ranged from 370 to 450 pmol/l and did not differ among glucose treatment groups.


Figure 1: Effect of glucose on GABA release from betaTC6 cultures. betaTC6 cells were first exposed to either 1 mmol/l (open symbols) or 10 mmol/l (closed symbols) glucose during a 12-h incubation period. The amount of GABA released by betaTC6 cells in response to 1 mmol/l (diamonds) and 10 mmol/l (squares) glucose in HEPES-buffered KRB was then determined during a subsequent 2-h secretion test. Fifty-microliter aliquots of betaTC6-conditioned KRB, collected at intervals of 15, 30, 60, and 120 min, were assayed for GABA content by HPLC-electrochemical detection. Data are presented as nmol/l and represent the mean ± S.E. from triplicate wells for glucose treatment for each time period. Asterisks indicate significant (p < 0.05) glucose effects within time.



Effect of Extracellular GABA on Glucagon Secretion by alphaTC6 Cells

Initial studies were conducted to define the sensitivity of alphaTC6 cells to glucose at concentrations that influenced GABA secretion by betaTC6 cultures and to GABA at concentrations released by the betaTC6 cells. Glucagon concentrations in alphaTC6-conditioned medium were significantly greater (p < 0.05) in cultures incubated for 2 h in the presence of 1 mmol/l glucose than in the presence of 10 mmol/l glucose ( Fig. 2and Fig. 3). Thus, glucose inversely affected glucagon secretion by alphaTC6 cells and GABA secretion by betaTC6 cells (compare Fig. 1and Fig. 2).


Figure 2: Effect of GABA on glucagon secretion by alphaTC6 cells. alphaTC6 cultures were exposed to KRB supplemented with 1 mmol/l glucose alone or with increasing GABA concentrations (50-200 nmol/l GABA) or with 10 mmol/l glucose plus 100 nmol/l GABA for 2 h. At the end of the secretion test, alphaTC6-conditioned medium was collected for later glucagon determination by RIA. Data represent the mean ± S.E. from triplicate wells from a representative experiment. Glucagon secretion is expressed as a percent of control (1 mmol/l glucose without GABA). Asterisks indicate that the values are significantly (p < 0.05) less than the values obtained from control cultures (1 mmol/l glucose without GABA). Dark shaded bars, -GABA; light shaded bars, +GABA.




Figure 3: Effect of glucose, GABA, and bicuculline, a GABA(A) receptor antagonist, on glucagon secretion by alphaTC6 cells. alphaTC6 cultures were exposed to KRB supplemented with 1 or 10 mmol/l glucose alone or with 100 nmol/l GABA plus or minus 100 nmol/l bicuculline for 2 h. At the end of secretion test, alphaTC6-conditioned medium was collected for later glucagon determination by RIA. Data represent the mean ± S.E. from two experiments. Glucagon secretion is expressed as a percent of control (1 mmol/l glucose without GABA). Asterisks indicate that the values are significantly (p < 0.05) less than the values obtained from control cultures (1 mmol/l glucose without GABA). Dark shaded bars, -GABA; light shaded bars, +GABA (100 nM); striped bars, +GABA (100 nM) + bicuculline (100nM).



To test the responsiveness and sensitivity of alphaTC6 cells to GABA, alphaTC6 cultures were incubated for 1 h in the presence of 10 mmol/l glucose and then switched to fresh KRB containing 1 mmol/l glucose and increasing GABA concentrations in a range of concentrations (50-200 nmol/l GABA) determined earlier to be secreted by betaTC6 cells. Glucagon secretion by alphaTC6 cultures exposed to 1 mmol/l glucose was inhibited (p < 0.05) by GABA at concentrations equal to and greater than 100 nmol/l (Fig. 2). These results demonstrate that GABA inhibition of glucagon secretion by alpha-cells occurs at concentrations secreted by beta-cells. GABA (100 nmol/l) did not decrease glucagon secretion by alphaTC6 cells cultured in high (10 mmol/l) glucose during the 2-h secretion test ( Fig. 2and Fig. 3).

To test the specificity of GABA's inhibitory effects on alpha-cell function, glucagon concentrations were compared in glucose-stimulated alphaTC6 cultures incubated in the presence of both GABA (100 nmol/l) and bicuculline (100 nmol/l), a GABA(A) receptor antagonist(19) . The inhibitory effect (p < 0.05) of GABA on glucagon secretion by alphaTC6 cells in response to 1 mmol/l glucose was abolished in the presence of bicuculline (Fig. 3). A slight decrease in glucagon secretion was observed when GABA was added to alphaTC6 cultures exposed to 10 mmol/l glucose ( Fig. 2and Fig. 3). This GABA-mediated decrease was also abolished in response to bicuculline (Fig. 3).


DISCUSSION

The present results provide the first documentation that endogenous GABA is secreted by a pancreatic beta-cell line and that release is influenced by extracellular glucose concentrations. The results further provide evidence that GABA, at concentrations similar to those released from beta-cells, will attenuate glucagon release from pancreatic alpha-cells. While previous studies (reviewed in (8) ) have demonstrated GABA synthesis by isolated islets and GABA release from perfused pancreas, the source of GABA was not identified conclusively in either case. Using a sensitive HPLC procedure similar to the one employed here, Thomas-Reetz and associates (10) did not detect GABA peaks after stimulation of betaTC3 cells with various insulin secretagogues, including glucose. The presence of amino acids in cell culture media limits GABA detection by HPLC, which led us to use KRB routinely in GABA secretion studies. This together with our use of the more differentiated betaTC6 cell line may explain why we were able to measure GABA release from the beta-cell line.

A positive relationship between glucose concentration and GABA secretion by betaTC6 cells was observed in the present study. Few studies investigating glucose modulation of either GABA synthesis or release have been reported in the literature. Gylfe and Hellman (25) reported that GABA concentrations in freeze-dried islet sections and in fresh islet extracts from starved ob/ob mice (background strain not designated) were numerically higher than in preparations from fed mice. The mice were given free access to food or were fasted for 3 days prior to islet isolation. While those results indicate that GABA synthesis was modulated by presumed distinct states of glycemia, neither blood concentrations of glucose nor the islet hormones were provided as conformation. Regarding release, Gerber and Hare (26) reported that external concentrations of GABA doubled within 15 min when pieces of rabbit pancreas were transferred from a buffer containing 2.8 mmol/l glucose to one containing 27.8 mmol/l glucose.

GABA concentrations were highest in betaTC6 cultures exposed to high glucose during both the 12-h incubation period and the subsequent 2-h secretion test in the present study (Fig. 1). Thus, external glucose may influence the amount of GABA released in two ways. First, chronic glucose levels may influence GABA biosynthesis, and second, GABA release may be dependent on a glucose-stimulated membrane depolarization event. That preproinsulin biosynthesis and insulin secretion are stimulated by distinct glucose-activated events provides precedent for more than one glucose-sensitive pathway being operative in pancreatic beta-cells(27, 28) . The observation that GABA concentrations did not differ between betaTC6 treatment groups exposed to low glucose during the 12-h incubation period but differently stimulated with low or high glucose during the 2-h secretion test supports the postulate that glucose availability influences GABA synthesis by beta-cells. A preliminary comparison of GABA concentrations in betaTC6-conditioned medium after 2- or 12-h incubation periods in 1 or 10 mM glucose demonstrated that GABA concentrations were higher after a longer period of incubation for both glucose concentrations (not shown). Moreover, as demonstrated by the present results, the amount of GABA released during a subsequent secretion test was dependent on the glucose concentration present during the incubation period. Notably, Gylfe and Hellman (25) demonstrated that GABA can be synthesized from glucose in islets. In the brain, estimates are that 10-40% of GABA in neurons is derived from glucose. Michalik and Erecinska (8) offered the view that GABA may provide fuel for the generation of ATP, via the GABA shunt, in islets when glucose is deficient. The present demonstration that GABA is synthesized and secreted by betaTC6 cells should facilitate future efforts to define the extent of beta-cell GABA metabolism using these cells as a model system.

GABA secretion from betaTC6 cells reflected extracellular glucose concentrations; however, insulin release did not differ between the low and high glucose treatments. The insulin response noted is consistent with previous reports of diminished glucose responsiveness in the murine beta-cell lines with propagation in culture(16, 29, 30) . The shift in glucose sensitivity in these cell lines has been associated with an increase in hexokinase activity(16) . The present demonstration that GABA secretion, but not insulin secretion, remained responsive to differences in extracellular glucose in passage 17 betaTC6 cells indicates that glucose-regulated pathways modulating GABA secretion may be more sensitive to or otherwise distinct from those that regulate insulin secretion. Clearly, additional studies are required to precisely define the glucose-sensitive pathways that modulate GABA biosynthesis and release by beta-cells.

The role of locally produced GABA is also unresolved. The possibility that GABA released from beta-cells may have a paracrine role in the islet is proposed frequently(7, 8, 31, 32) . Secretion of GABA from betaTC6 cells prompted us to directly test the modulatory effects of GABA on glucagon secretion by alphaTC6 cells, a glucose-sensitive glucagon-producing alpha-cell line(15) . The results provide clear indication that (i) GABA attenuates glucagon release from alphaTC6 cells; (ii) GABA is effective at concentrations that are released from betaTC6 cells; (iii) GABA and glucose independently inhibit glucagon secretion by alphaTC6 cells; and (iv) that GABA's inhibitory effects on glucagon secretion are mediated through GABA(A) receptors. Previous studies demonstrating GABA inhibition of glucagon secretion in islets used supraphysiological concentrations of GABA (µM range(12, 33) ).

Although it is not possible to infer an in vivo role of GABA from these results, the observations do provide clear evidence that GABA and glucose can inhibit glucagon release independently and thereby argue against the proposal that the inhibitory effects of glucose on glucagon are mediated by beta-cell GABA(12) . Having alpha-cells independently responsive to both glucose and GABA may play a key role in the physiological control of blood glucose. Initially the short term rise in blood glucose associated with meal ingestion might inhibit glucagon secretion directly, while indirect inhibition through glucose-stimulated GABA release from neighboring beta-cells may become important for tonic suppression of glucagon as normoglycemia is restored. The present observation that glucose history influences the amount of GABA released by betaTC6 cells agrees with that possibility. A complete understanding of the role of GABA in physiological regulation of glucose will require investigation of strict temporal parameters that can only be addressed in vivo. A clear impetus for such studies is established by the present demonstration that GABA is secreted by a beta-cell line and that the effects of glucose and GABA on glucagon secretion can be dissociated with a defined model system.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grant DK-49192 (to H. R. G.), a Future Leader Award from International Life Sciences Institute (to H. R. G.), and an Extramural Research Promotion Award from Texas Tech University (to J. L. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests may be addressed: University of Illinois, 1207 W. Gregory Dr., Urbana IL 61801. Tel. or Fax: 217-244-3165; hgaskins@ux1.cso.uiuc.edu.

Supported by a graduate fellowship from the Inter-American Foundation.

**
Recipient of a summer scholarship from the Ronald E. McNair Scholars Program (United States Department of Education).

(^1)
The abbreviations used are: GABA, -aminobutyric acid; DMEM, Dulbecco's minimal essential medium; KRB, Krebs-Ringer buffer; HPLC, high performance liquid chromatography; RIA, radioimmunoassay; ANOVA, analysis of variance; l, liter.


ACKNOWLEDGEMENTS

We thank Dr. Shimon Efrat for providing the betaTC6 cell line, Dr. Edward Leiter for providing the alphaTC6 cell line, and Anna Gelfand for technical assistance.


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