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Department of Animal and Avian Sciences, University of Maryland College Park, MD 20742
* To whom correspondence should be addressed. E-mail: bdhumphr{at}calpoly.edu.
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ABSTRACT |
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 TOP ABSTRACT IntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Introduction |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Developing T cell glucose metabolism and differentiation are closely linked by cytokines and developmental ligands that are part of the thymic microenvironment (6). For example, interleukin-7 increases glucose transport and metabolism in developing T cells to promote their differentiation from the DP to CD8+ developmental stage (7). Notch signals promote viability of pre-T cells via maintenance of glucose metabolism (1). However, our understanding of thymocyte metabolism has in large part accumulated from studies conducted in single cell suspensions or reaggregate cell culture models (8) that lack aforementioned and other hematopoietic cytokines and developmental ligands known to regulate developing T cell metabolism (9). The embryonic thymic organ culture (ETOC)4 system supports thymocyte development in vitro (10,11) and maintains the thymic microenvironment and its associated hematopoietic cytokines and developmental ligands that regulate developing T cell metabolism. Therefore, the ETOC system is an alternative model system for the study of the effect of glucose availability on developing T cell metabolism.
In chickens, T cell development is initiated during embryogenesis and continues for several weeks posthatch. We previously showed that developing T cells increase their ability to acquire and metabolize glucose during the embryonic to posthatch transition and that the plasma glucose concentration is low during embryogenesis and increases 3-fold by 1 wk posthatch (12). The objective of these studies was to determine the effect of glucose availability on thymocyte glucose metabolism in chickens. Here, we measure mRNA abundance of glycolytic genes, glucose transport, hexokinase (HK-1) enzyme activity, mitochondria membrane potential (
), viability, and apoptosis in thymocytes to answer whether or not glucose availability affects developing T cell metabolism, energy status, and survival.
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Materials and Methods |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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ETOC. To maintain the thymic microenvironment, thymic architecture was maintained by culturing whole thymic lobes in vitro. Thymic lobes were cultured on transwell inserts as previously described (13) with modifications. Thymic lobes were harvested on embryonic d (e)15 and were placed on a polycarbonate membrane (Corning, no. 3401). Replicates consisted of 4 thymic lobes per embryo per transwell insert. In all experiments, RPMI 1640 with glutamine (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin (ETOC media) was used to achieve final glucose concentrations ranging from 0 to 20 mmol/L. Either glucose-deplete RPMI 1640 (0 mmol/L glucose; no. 11879–020), glucose-replete RPMI 1640 (10 mmol/L glucose; no. 11875–085), or their combination was used to achieve 0, 5, and 10 mmol/L final glucose concentrations and D-glucose (Sigma; no. G8270) was added to glucose-replete RPMI 1640 to achieve 15 and 20 mmol/L final glucose concentrations. ETOC media was added to each well (900 µL) and ETOC were maintained at 37°C with 5% CO2.
Lymphocyte isolation. Thymocytes were isolated from thymic lobes by mincing and straining as previously described (12). We determined lymphocyte viability by trypan blue exclusion using a hemocytometer or propidium iodide staining using FACSCalibur flow cytometer (BD Biosciences). For total RNA and protein isolation, thymocyte pellets were immediately snap-frozen in liquid nitrogen and stored at –80°C until further analysis. For all other assays, isolated thymocytes were immediately used at the indicated cell number.
Quantification of mRNA abundance by real-time PCR. Thymocyte glucose transporter-1 (Glut-1), Glut-3, and HK-1 mRNA abundance was determined after 12 and 24 h of culture in ETOC media containing 0, 10, or 20 mmol/L glucose (n = 6). Total RNA was isolated from 2.5 x 106 thymocytes using the Trizol reagent (Invitrogen, no. 15596–026) and OD at 260 nm was used to determine total RNA concentrations. RT, real-time PCR primers, cycling conditions, and analysis were performed as previously described (12) with minor modifications. Target gene mRNA abundance was normalized by geometric averaging of hypoxanthine phosphoribosyltransferase-1, glyceraldehyde 3-phosphate dehydrogenase, TATA box-binding protein, and β2-microglobulin raw nonnormalized values using the geNorm software (14). Data are presented as the normalized fold-change in mRNA abundance of a gene relative to its mRNA abundance at 0 h.
Glucose uptake. ChT1+ gated thymocyte 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (Invitrogen, no. N13195) uptake after 24 h of culture in ETOC media containing 0, 10, or 20 mmol/L glucose (n = 6) or after 6 and 12 h of culture in 5 or 15 mmol/L glucose (n = 5) was measured as previously described (12). Data are presented as background-corrected MFI.
HK-1 activity. Thymocyte HK-1 activity after 24 h of culture in ETOC media containing 0, 10, or 20 mmol/L glucose (n = 6) or after 6 and 12 h of culture in 5 or 15 mmol/L glucose (n = 5) was measured as previously described (12). HK-1 activity is presented as nmol of NADPH produced/(mg protein· min).
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Thymocyte mitochondria after 24 h of culture in ETOC media containing 5, 10, or 15 mmol/L glucose (n = 4) was determined by staining with tetramethylrhodamine ethyl ester perchlorate (TMRE) (Invitrogen, no. T-669). Thymocytes (106) were incubated in their respective ETOC media containing 100 nmol/L TMRE for 30 min at 37°C with 5% CO2. To determine whether TMRE staining is sensitive to , the was depolarized by adding 50 µmol/L carbonyl cyanide m-chlorophenylhydrazone (CCCP). To determine thymocyte mitochondrial biomass, thymocytes were stained with 50 nmol/L 10-nonyl acridine orange (NAO; Invitrogen, no. A-1372). Fluorescence was measured for 10,000 ChT1+ gated events for each replicate using a FACSCalibur flow cytometer (BD Biosciences). Nonspecific fluorescence in TMRE- and CCCP-treated samples was subtracted from fluorescence of TMRE-stained samples. Fluorescence in NAO and CCCP samples did not differ from NAO-stained samples (data not shown); therefore, fluorescence due to NAO was not corrected for nonspecific fluorescence. Data on are presented as a fold-change in TMRE fluorescence relative to 5 mmol/L glucose.
Apoptosis. Thymocyte apoptosis after 24 h of culture in ETOC media containing 5, 10, or 15 mmol/L glucose (n = 4) or after 6 and 12 h culture in 5 or 15 mmol/L glucose (n = 5) was determined by staining with fluorescein isothiocyanate-conjugated annexin V (Invitrogen, no. PHN1008). Thymocytes (106) were incubated in 0.1 L 5x annexin binding buffer containing 5 µL FITC-annexin V and 1 mg/L PI for 15 min at 37°C with 5% CO2. Fluorescence was measured for 10,000 ChT1+ gated events for each replicate using a FACSCalibur flow cytometer (BD Biosciences). Thymocytes were gated into apoptotic or dead populations based on annexin V and PI fluorescence, respectively.
Statistical analysis.
Dependent variables were analyzed by general linear model procedure (JMP). A 1-way ANOVA was used to determine the main effect of glucose on thymocyte mRNA abundance, glucose uptake, , viability, and apoptosis and means were compared by Tukey's means comparison. Prior to analysis, data were assessed for homogeneity of variance by Levene's test and when significant (P < 0.05) were log-transformed to attain homogeneity of variance. Data are reported as nontransformed means and pooled standard errors. A 2-way ANOVA was used to determine the main effect of glucose, time, and their interaction on thymocyte HK-1 activity, glucose uptake, viability, and apoptosis and main effects were compared by Tukey's means comparison. Differences between means or interactions were considered significant at P < 0.05.
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Results |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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Effect of glucose concentration on thymocyte glucose transporter mRNA abundance, glucose uptake, and HK-1 activity. Glut-1, Glut-3, and HK-1 mRNA abundance was measured to determine the effect of glucose on the transcription of genes involved in glucose uptake and metabolism. Thymocytes incubated with 20 mmol/L glucose had 3-fold greater Glut-1 mRNA abundance than in those incubated with 0 mmol/L glucose at 12 h (P < 0.05; Table 1). Glut-3 mRNA abundance was at least 2-fold greater in thymocytes incubated with 10 and 20 mmol/L glucose than in those incubated with 0 mmol/L glucose at 12 h (P < 0.05; Table 1). Thymocyte HK-1 mRNA abundance did not differ between glucose concentrations at either 12 or 24 h (data not shown). Glucose uptake was greater in thymocytes incubated with 20 mmol/L glucose than in those incubated with 0 or 10 mmol/L (P < 0.05; Table 2). Thymocyte HK-1 enzyme activity was measured to determine the effect of glucose concentration on entry into glycolysis. HK-1 activity was 185% higher in thymocytes incubated with 0 and 20 mmol/L glucose than in those incubated with 10 mmol/L at 24 h (P < 0.05; Table 2).
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, viability, and apoptosis.
We measured thymocyte as a marker of thymocyte energy status. The glucose concentrations used were 5, 10, or 15 mmol/L to better reflect in ovo and in vivo serum glucose concentrations during the embryonic to posthatch transition, respectively (12). Thymocytes incubated with 15 mmol/L glucose had 2.5-fold higher than in those incubated with 5 mmol/L glucose at 24 h (P < 0.05; Table 3). Thymocyte mitochondria biomass did not differ between glucose concentrations (data not shown), indicating that changes in were not due to differences in mitochondria number. Thymocyte apoptosis was measured to determine the effect of glucose on cell survival. Thymocytes incubated with 5 mmol/L glucose had lower viability than those incubated with either 10 or 15 mmol/L glucose (P < 0.05; Table 3). The percentage of thymocytes undergoing apoptosis was higher when incubated with 5 mmol/L glucose than with 15 mmol/L glucose (P < 0.05; Table 3).
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Discussion |
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TOPABSTRACTIntroductionMaterials and MethodsResultsDiscussionLITERATURE CITED |
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In response to different glucose concentrations, thymocytes adapted their glucose metabolism by altering Glut-1 and Glut-3 gene expression, glucose transport, and HK-1 activity. Rat thymocytes in single cell suspension increase glucose metabolism, glycolytic gene expression, and enzyme activity in response to high glucose concentrations (16,17). Chicken thymocytes in our studies increased their glucose metabolism in relation to media glucose concentration, which suggests that these cells are able to sense and adapt their metabolism to glucose availability. However, specific glucose-sensing mechanisms and pathways in thymocytes are yet to be elucidated in detail. Eukaryotic cells take cues from glycolytic and pentose phosphate pathway metabolites to sense glucose availability through the action of protein kinases and transcription factors, such as stimulatory protein-1 (Sp1) (18,19) and AMP-activated kinase (AMPK) (20). In thymocytes, high glucose concentrations dephosphorylate Sp1 and increase the binding of this transcription factor to promoter regions of glycolytic genes to increase their transcription (21). AMPK is transcribed in chicken thymocytes (12) and in mammalian T lymphocytes AMPK functions to regulate energy metabolism according to their ATP demand (22). Collectively, AMPK and Sp1 may be important molecular determinants in thymocytes that increase the transcription of genes to facilitate glucose uptake and metabolism in response to elevated glucose availability.
Glucose transport and metabolism in mammalian lymphocytes contributes to their (23). Cell is a measurement of the proton gradient across the inner mitochondria membrane and this serves as a marker of cell energy status, because is directly related to ATP production potential (24). In this study, thymocytes cultured in high glucose concentrations had increased , suggesting greater ATP-producing potential and, consequently, an elevated cell energy status. The increase in in response to high glucose levels could be due to increased substrate level phosphorylation via glycolysis, because lymphocytes metabolize most of their glucose carbon to lactate (3). Consequently, is maintained, because cytosolic ADP:ATP ratios decrease and inhibit further proton dissipation from the inner mitochondria membrane. On the other hand, low in glucose-depleted thymocytes could be due to a combination of low cellular ATP levels associated with glucose deprivation (25) and/or loss of outer mitochondria membrane integrity leading to proton leakage. The latter is consistent with previous reports demonstrating that decreases in glycolysis due to glucose depletion increases outer mitochondria membrane permeability and serves as the first step in the activation of apoptosis (26). Overall, glucose levels regulate the energy status of chicken thymocytes and indicate that changes in glucose availability or glucose metabolism may affect energy-dependent processes, such as lymphocyte proliferation associated with the development of the T cell receptor.
Thymocytes developing in low glucose concentrations (5 mmol/L) had elevated rates of apoptosis and thymocyte HK-1 activity tended to decrease before the activation of apoptosis. This suggests that altered glycolytic metabolism may be an upstream stress factor that leads to apoptosis. Apoptosis in T lymphocytes can be initiated via the death ligand-receptor–regulated extrinsic pathway or the Bcl-2 family protein-regulated intrinsic pathway (27). Inhibiting glycolysis by depleting media glucose concentrations activates proapoptotic factors, such as activation of the proapoptotic protein Noxa in human T cells deprived of glucose (28). Furthermore, intermediary metabolites of glucose metabolism regulate apoptosis by altering the activity of caspase enzymes, which cleave the key cytosolic and nuclear proteins to activate apoptosis (29). Glucose-6-phosphate, a glycolysis intermediate, and/or NADPH, a product of the pentose phosphate pathway, inhibit caspase-2 activation and promote cell survival (30). Because thymocyte glucose metabolism was related to glucose concentration, it is likely that either one or both of these survival signaling pathways may mediate the initiation of apoptosis in chicken thymocytes in response to glucose availability.
Glucose-deprived (0 mmol/L) thymocytes increased glucose transporter (Glut-1) gene expression between 12 and 24 h and HK-1 activity was similar to 20 mmol/L glucose after 24 h of culture. This could be a part of a salvage response, because increased thymocyte Glut-1 gene expression and metabolism are generally associated with high glucose concentrations (16,17). Nutrient depletion induces salvage responses in an attempt to increase the expression and activity of proteins required for nutrient acquisition and metabolism (31). Glucose deprivation activates AMPK, which induces glucose transport and glycolytic enzyme activity to improve cellular energy status (20). However, long-term expression of AMPK leads to the breakdown of salvage efforts and the activation of apoptosis (32). It is possible that glucose-deprived thymocytes may increase their ability to acquire and metabolize glucose between 12 and 24 h to prevent the activation of apoptosis.
Despite lacking glucose, the glucose-depleted media contained other energy substrates, such as glutamine and fatty acids. Glutamine has long been recognized as an important metabolic fuel for leukocytes and is conditionally essential for lymphocyte proliferation (33). Lymphocyte activation results in increased fatty acid uptake but not oxidation (8), suggesting that lipid may be partitioned more toward membrane synthesis rather than energy generation in these cell types. Despite the availability of other energy substrates, thymocyte energy status was reduced and rates of apoptosis increased when thymic lobes were cultured in low (5 mmol/L) or glucose-deplete media. This indicates that in addition to being an important metabolic fuel for lymphocytes, glucose may also serve as an important metabolic signal involved in cell fate decisions. Indeed, different T cell maturation stages, from the double negative (CD4–CD8–) to the single positive stage (CD4+ or CD8+), differ in their ability to acquire glucose (2,7). Additionally, glucose metabolism in developing T cells is elevated during T cell antigen receptors-β rearrangement (1) and a subset of double positive (CD4+CD8+) T cells in early post-β selection have high rates of glucose transport and glycolysis (7).
Because apoptosis plays a key role in thymocyte selection and differentiation (27) and low glucose concentrations used in these studies were similar to embryonic serum glucose concentrations, it is possible that glucose availability may play an important role in their development during embryonic to posthatch transition. Based upon the results obtained in these studies, the low glucose concentrations present during embryogenesis may alter the susceptibility of developing T cells to apoptosis. Therefore, increasing the in ovo availability of glucose or increasing the synthesis of trophic factors that promote T cell glucose metabolism may help promote the development of the chick's cell mediated immune system in early life.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Present address: Animal Science Department, California Polytechnic State University, 1 Grand Ave, San Luis Obispo, CA 93407.
3 Present address: Fels Cancer Institute and Department of Biochemistry, Temple University School of Medicine, 3307 N. Broad Street, Philadelphia, PA 19140.
4 Abbreviations used: AMPK, AMP-activated kinase; , mitochondria membrane potential; CCCP, carbonyl cyanide m-chlorophenylhydrazone; ETOC, embryonic thymic organ culture; Glut-1, glucose transporter 1; HK-1, hexokinase-1; NAO, 10-nonyl acridine orange; SP, single positive; TMRE, tetramethylrhodamine ethyl ester perchlorate.
Manuscript received 18 December 2007. Initial review completed 4 January 2008. Revision accepted 21 March 2008.
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