Article
Reference
Modulation of glutamate generation in mitochondria affects hormone secretion in INS-1E beta cells
MAECHLER, Pierre, ANTINOZZI, Peter, WOLLHEIM, Claes
Abstract
The mitochondria play a pivotal role in regulating glucose-induced insulin secretion in the pancreatic beta cell. We have recently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS-1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Glutamate is formed by the mitochondrial enzyme glutamate dehydrogenase from alpha-ketoglutarate.
Transient overexpression of glutamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal release. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.
MAECHLER, Pierre, ANTINOZZI, Peter, WOLLHEIM, Claes. Modulation of glutamate
generation in mitochondria affects hormone secretion in INS-1E beta cells. IUBMB Life , 2000, vol. 50, no. 1, p. 27-31
DOI : 10.1080/15216540050176557 PMID : 11087117
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Original Research Article
Modulation of Glutamate Generation in Mitochondria Affects Hormone Secretion in INS-1E Beta Cells
Pierre Maechler, Peter A. Antinozzi, and Claes B. Wollheim
Division of Clinical Biochemistry, Department of Internal Medicine, University Medical Center, 1211 Geneva 4, Switzerland
Summary
The mitochondria play a pivotal role in regulating glucose- induced insulin secretion in the pancreatic beta cell. We have re- cently demonstrated that glutamate derived from mitochondria participates directly in the stimulation of insulin exocytosis. In the present study, mitochondria isolated from the beta cell line INS- 1E generated glutamate when incubated with the tricarboxylic acid cycle intermediate succinate. The generation of glutamate correlated with stimulated mitochondrial activity monitored as oxygen consumption and was inhibited by the mitochondrial uncoupler carbonyl cyanidep-tri uoromethoxyphenylhydrazone . Glutamate is formed by the mitochondrial enzyme glutamate dehy- drogenase from®-ketoglutarate. Transient overexpression of glu- tamate dehydrogenase in INS-1E cells resulted in potentiation of glucose-stimulated hormone secretion without affecting basal re- lease. These results further point to glutamate as an intracellular messenger playing a key role in the control of insulin exocytosis.
IUBMB
Life
, 50: 27–31, 2000Keywords Exocytosis; glutamate dehydrogenase; insulin; oxygen consumption; succinate.
INTRODUCTION
In the pancreatic beta cell, mitochondrial metabolism plays a pivotal role in the generation of signals that couple glucose recognition to insulin secretion (1–3). ATP is generated by mito- chondrial metabolism, promoting the closure of ATP-sensitive K+ channels and depolarization of the plasma membrane (4).
This leads to an in ux of Ca2+ through voltage-gated Ca2+
Received 25 April 2000; accepted 6 June 2000.
Address correspondence to Dr. Pierre Maechler, Division de Biochimie Clinique, Centre M´edical Universitaire, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. Fax: 41 22 702 55 43. E-mail: pierre.
maechler@medecine.unige.ch
channels and an increase in cytosolic Ca2+ ([Ca2+]c)1 (5). The increase in[Ca2+]cis the main trigger of exocytosis, the process by which the insulin-containing secretory granules fuse with the plasma membrane (4–6). However, the Ca2+ signal alone is not suf cient for sustained secretion; furthermore, glucose elicits secretion even under conditions of clamped, increased[Ca2+]c (7,8). This secretory response requires another messenger gen- erated by mitochondrial metabolism, distinct from ATP (9,10), recently identi ed as glutamate (11).
In the mitochondria, the tricarboxylic acid (TCA) cycle inter- mediate succinate transfers reducing equivalents to the electron transport chain on complex II in the reaction catalyzed by suc- cinate dehydrogenase. In permeabilized INS-1 cells, succinate hyperpolarizes the mitochondrial membrane potential (D W m), consequently rapidly increasing[Ca2+]in the mitochondrial ma- trix (9,10). This mitochondrial activation is associated with a marked stimulation of insulin release, which requires both acti- vation of the mitochondria and a supply of carbon atoms for the TCA cycle (9,11).
In the beta cell, glutamate is generated by the mitochon- dria during glucose stimulation (11,12). Under conditions of permissive, clamped[Ca2+]cin permeabilized cells, exogenous glutamate directly stimulates insulin exocytosis independently of mitochondrial function, suggesting that glutamate acts as an intracellular messenger for coupling glucose metabolism to in- sulin secretion (11). Glutamate is formed in the mitochondria from the TCA cycle intermediatea -ketoglutarate by glutamate dehydrogenase (13).
In the present study, we measured glutamate generation from activated mitochondria isolated from rat insulinoma cells INS- 1E. Moreover, glutamate dehydrogenase was overexpressed in
1Abbreviations:[Ca2+]c, cytosolic[Ca2+];D W m, mitochondrial membrane potential; TCA, tricarboxylic acid; GDH, glutamate dehydrogenase ; hGH, human growth hormone; FCCP, carbonyl cyanide p-tri uoromethoxyphenyl - hydrazone.
27
28 MAECHLER ET AL.
INS-1E cells to increase the cellular concentrations of glutamate in response to glucose stimulation.
MATERIALS AND METHODS
Cell Culture and Transient Transfection. INS-1E cells, cloned from the INS-1 cell line (14), were cultured in RPMI 1640 medium with 5% fetal calf serum (11). Cells were seeded at 105 cells/2 ml of RPMI 1640 medium in polyornithine-treated 24- well plates. Three days later, the cells were transiently transfected as previously described (15), using for each well 6l l of the poly- cationic lipid LipofectAMINE (Gibco BRL, Basel, Switzerland) combined with the cDNA mix diluted in RPMI 1640 medium (25 mM HEPES). The cDNA mix was composed of 1l g of full-length GLUD1, the cDNA encoding human glutamate de- hydrogenase (GDH) in pcDNA3, along with 1 l g of human growth hormone (hGH) plasmid as a secretion indicator of ef-
ciently transfected cells (16). Control cells were cotransfected with hGH and an inert mitochondrial protein (mitochondrially targeted aequorin without its prosthetic group) (17). The lipo- some and plasmid solutions were then mixed and after incuba- tion for 30 min at room temperature, this transfection mixture was added to the cells and further incubated for 5 h at 37±C in air enriched with 5% CO2. Two days after transfection, cells were assayed for glucose-stimulated hormone secretion.
Isolation of Mitochondria. Attached INS-1E cells were cul- tured in 10-cm-diameter Petri dishes for 3–4 days. Mitochon- dria were then isolated by Potter-Elvehjem homogenization, fol- lowed by serial centrifugation as described previously (18). The mitochondrial pellet was resuspended in a buffer adjusted to
» 500 nM free Ca2+ (140 mM KCl, 5 mM NaCl, 7 mM MgSO4, 1 mM ATP, 20 mM HEPES, pH 7.0, 10.2 mM EGTA, and 6.67 mM CaCl2) before protein determination by Bradford’s assay.
Mitochondrial Glutamate Determination. Mitochondria were incubated in 0.25 ml of the mitochondrial buffer at 37±C for the times indicated. The reactions were stopped by adding 2l M of the mitochondrial poison carbonyl cyanide p-tri uorome- thoxyphenylhydrazone (FCCP) and placing the samples on ice.
Mitochondria were then lysed by adding an equal volume of lysis buffer (20 mM Tris-HCl pH 8.0, 2 mM cyclohexanediaminetet- raacetate, 0.2% Tween-20) and further sonication. Glutamate concentrations were measured by monitoring the increase in NADH uorescence during oxidation of glutamate in mitochon- dria extracts in the presence of an excess of GDH (Boehringer Mannheim, Mannheim, Germany) as described previously (11).
Oxygen Consumption. Rates of oxygen consumption were measured in a 0.5-ml reaction chamber at 37±C with a Clark- type electrode (Rank Brothers Ltd., Cambridge, UK). Measure- ments were made with 35l g of mitochondrial protein in the mitochondrial buffer.
Secretion Assay. INS-1E cells were cotransfected and cul- tured in complete RPMI 1640 medium as described above. Be- fore the experiments, cells were maintained for 2 h in glucose- free culture medium, washed, and preincubated in glucose-free Krebs–Ringer bicarbonate HEPES buffer containing (in mM):
135 NaCl, 3.6 KCl, 10 HEPES (pH 7.4), 5 NaHCO3, 0.5 NaH2PO4, 0.5 MgCl2, and 1.5 CaCl2. After 30 min of preincuba- tion, the cells were incubated in the presence of basal (2.8 mM) or stimulatory concentrations of glucose (7.5 and 15.0 mM) for 30 min at 37±C. Insulin secretion was determined by radioim- munoassay, with rat insulin used as a standard (9); hGH secre- tion was measured with an ELISA kit (Boehringer Mannheim).
For all secretion experiments, 0.1% of bovine serum albumin (Sigma, St. Louis, MO) was added to buffers as carrier.
RESULTS
Glutamate Generation from Isolated Mitochondria. Mito- chondria were isolated from the beta cell line INS-1E and in- cubated for various periods at 37 ±C in a KCl buffer contain- ing 500 nM free Ca2+ and 1 mM succinate (added at time 0) before glutamate contents were measured. The time course of succinate stimulation showed that glutamate was increased by 4.6-fold (P < 0.01) after 5 min and by 23.6-fold (P < 0.001) after 30 min of incubation (Fig. 1A). Glutamate generation over a 30-min stimulation period with succinate was inhibited by 87% (P < 0.001) when mitochondria were kept on ice or by 68% (P < 0.01) when treated with 1l M of the mitochondrial uncoupler FCCP, thus demonstrating that glutamate formation requires mitochondrial activation (Fig. 1B). Endogenous amino acids, such as glutamine and aspartate, are the likely donors of ammonia for glutamate synthesis (13,19).
The production of glutamate should also depend on the pro- vision of carbons to the mitochondria. To test this, we incubated mitochondria witha -glycerophosphate, which, like succinate, activates the respiratory chain at complex II but, unlike suc- cinate, does not provide carbons to the TCA cycle. As shown in Fig. 2B,a -glycerophosphate (2 mM) was inef cient in in- creasing glutamate concentrations in isolated mitochondria even though it stimulated oxygen consumption as potently as succi- nate (Fig. 1C). Succinate-stimulated respiration was completely abolished in the presence of 2.5 mM of the succinate dehydro- genase inhibitor malonate. In these INS-1E–derived mitochon- dria, glutamate (1 mM) is not a substrate for the activation of oxidative metabolism, as demonstrated by the lack of effect on oxygen consumption (Fig. 1C). This is in agreement with ex- periments performed in rat liver mitochondria, which showed that the GDH pathway is not permissive to glutamate oxidation (20).
Secretion from INS-1E Cells Overexpressing GDH. Over- expression of GDH was performed in INS-1E cells by transient cotransfection of the GLUD1 cDNA with a hGH plasmid as a secretion reporter of ef ciently transfected cells (16). Two days after transfection, cells were assayed for glucose-stimulated se- cretion in static incubation over a 30-min period. To verify that the cells ordinarily were responsive to glucose, we measured both overall insulin secretion (re ecting the total cell popula- tion) and hGH secretion (re ecting only the subpopulation of transfected cells: 10%). As expected, overall insulin secretion
Figure 1. Glutamate generation and respiration of mitochon- dria isolated from INS-1E cells. (A) Time course of glutamate generation from isolated mitochondria incubated at 37±C in the presence of 1 mM succinate (added at time 0). (B) Glutamate formation by mitochondria during 30 min of incubation at 37±C in the presence of 1 mM succinate, succinate at 4±C, succinate plus the protonophore FCCP (1l M), anda -glycerophosphate (2 mM). (C) Rates of oxygen consumption stimulated by glu- tamate (1 mM), succinate (1 mM), succinate plus 2.5 mM mal- onate (Suc+Mal), anda -glycerophosphate (2 mM). The results are shown as means§SE ofn =3 to 6 independent experiments done in duplicate. *,P < 0.05; **, P < 0.01 versus results for t = 0 in A, versus results for succinate in B, versus results for the control in C.
Figure 2. Glucose-stimulated hormone secretion in INS-1E cells overexpressing GDH. (A)Control insulin secretion (re ect- ing untransfected cells; see text) from INS-1E cells stimulated for 30 min by different glucose concentrations. (B) Secretion of the transfection marker, hGH, from cells transfected with an inert protein (control) or with GDH. The results are shown as means§SE ofn = 3 independent experiments done in dupli- cate. *, P < 0.05; **, P < 0.005 versus results at 2.8 mM glucose; ,P < 0.005 versus results for control cells.
was ef ciently stimulated by 7.5 and 15.0 mM glucose (Fig. 2A).
In transfected cells (Fig. 2B), the basal hGH secretion at 2.8 mM glucose was not signi cantly different between control and GDH-overexpressing cells (741 § 61 and 827§ 167 pg/well, respectively). Compared with basal hGH release, 7.5 mM glu- cose stimulated hGH secretion 3.8-fold in control cells versus 6.3-fold in GDH-transfected cells (P < 0.005). At 15.0 mM glucose, hGH secretion was enhanced 3.3-fold in control cells versus 7.6-fold in cells overexpressing GDH (P < 0.005).
DISCUSSION
In the consensus model of glucose-stimulated insulin secre- tion, ATP is generated by mitochondrial metabolism, promoting
30 MAECHLER ET AL.
an increase in[Ca2+]cthat constitutes the main trigger initiating insulin exocytosis (4–6). In addition, glucose elicits a secretory response under conditions of clamped, increased[Ca2+]c(7,8), which requires a mitochondrial messenger distinct from ATP (9, 10), recently identi ed as glutamate (11). Cellular gluta- mate concentrations are indeed increased during glucose stim- ulation (11,12). In permeabilized beta cells, direct activation of the mitochondria with the TCA cycle intermediate succi- nate markedly stimulates insulin secretion (9,11). Succinate transfers reducing equivalents to the electron transport chain on complex II at succinate dehydrogenase (21), resulting in hyper- polarization ofD W m and an increase in mitochondrial[Ca2+] (9,10). Activation of complex II can also be achieved bya - glycerophosphate (22), which transfers reducing equivalent to the FAD-linked glycerophosphate dehydrogenase without pro- viding carbons to the TCA cycle (23). Here we have shown that both succinate anda -glycerophosphate increased mitochon- drial respiration, but only succinate was ef cient in promoting the generation of glutamate in isolated mitochondria. These re- sults are in accordance with the previous observation that in permeabilized cellsa -glycerophosphate fails to trigger insulin secretion, although it hyperpolarizesD W mand increases mito- chondrial[Ca2+]to the same extent as succinate (9). This dis- crepancy can be explained by the lack of anaplerotic effect of a -glycerophosphate on the TCA cycle, that is, failure to increase a -ketoglutarate, the substrate of GDH in the generation of gluta- mate (13). Taken together, our results suggest that in the beta cell, GDH preferentially operates in the directiona -ketoglutarate! glutamate at stimulatory concentrations of glucose. The direc- tion of the enzyme reaction may vary from tissue to tissue, however. For instance, in the liver the reaction moves mainly froma -ketoglutarate to form glutamate, whereas in astrocytes, oxidative deamination of glutamate to providea -ketoglutarate dominates (19).
The overexpression of GDH in INS-1E cells and transient cotransfection with a secretion marker further substantiated the role of glutamate in coupling metabolism and secretion. The basal rate of secretion at a nonstimulatory glucose concentra- tion was not affected. In contrast, at stimulatory glucose con- centrations, the secretion was potentiated in cells that overex- pressed GDH. This approach also mimics the hyperinsulinism syndrome caused by mutations in the GDH gene that result in an increase in enzyme activity (24,25). These ndings demon- strate that glutamate is generated by mitochondria and that the GDH plays an essential role in the control of glucose-stimulated secretion.
ACKNOWLEDGEMENTS
We thank G. Chaffard and C. Bartley for expert technical as- sistance, Drs. C. A. Stanley and B. Y. Hsu (Philadelphia, PA) for kindly supplying GDH cDNA, and Dr. H. Ishihara for stimulat- ing discussions. This study was supported by the Swiss National Science Foundation (grant no. 32-49755.96 to C.B.W.) and by
a European Union Network Grant (through the Swiss Federal Of ce for Education and Science).
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