Switch to Anaerobic Glucose Metabolism with NADH Accumulation in the β-Cell Model of Mitochondrial Diabetes
2002; Elsevier BV; Volume: 277; Issue: 44 Linguagem: Inglês
10.1074/jbc.m207690200
ISSN1083-351X
AutoresMitsuhiko Noda, Shigeo Yamashita, Noriko Takahashi, Kazuhiro Eto, Lin-Ming Shen, Kazuo Izumi, Samira Daniel, Yoshiharu Tsubamoto, Tomomi Nemoto, Masamitsu Iino, Haruo Kasai, Geoffrey W.G. Sharp, Takashi Kadowaki,
Tópico(s)Adipose Tissue and Metabolism
ResumoTo elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 μg/ml ethidium bromide (EtBr) to the murine pancreatic β cell line βHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998)J. Biol. Chem. 273, 20300–20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in βHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p< 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in β cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca2+]i increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion. To elucidate the mechanism underlying diabetes caused by mitochondrial gene mutations, we created a model by applying 0.4 μg/ml ethidium bromide (EtBr) to the murine pancreatic β cell line βHC9; in this model, transcription of mitochondrial DNA, but not that of nuclear DNA, was suppressed in association with impairment of glucose-stimulated insulin release (Hayakawa, T., Noda, M., Yasuda, K., Yorifuji, H., Taniguchi, S., Miwa, I., Sakura, H., Terauchi, Y., Hayashi, J.-I., Sharp, G. W. G., Kanazawa, Y., Akanuma, Y., Yazaki, Y., and Kadowaki, T. (1998)J. Biol. Chem. 273, 20300–20307). To elucidate fully the metabolism-secretion coupling in these cells, we measured glucose oxidation, utilization, and lactate production. We also evaluated NADH autofluorescence in βHC9 cells using two-photon excitation laser microscopy. In addition, we recorded the membrane potential and determined the ATP and ADP contents of the cells. The results indicated 22.2 mm glucose oxidation to be severely decreased by EtBr treatment compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01). By contrast, glucose utilization was only marginally decreased. Lactate production under 22.2 mm glucose was increased by 2.9- and 3.5-fold by EtBr treatment on days 4 and 6, respectively (both p< 0.01). Cellular NADH at 2.8 mm glucose was increased by 35 and 43% by EtBr on days 4 and 6 (both p < 0.01). These data suggest that reduced expression of the mitochondrial electron transport system causes NADH accumulation in β cells, thereby halting the tricarboxylic acid cycle on one hand, and on the other hand facilitating anaerobic glucose metabolism. Glucose-induced insulin secretion was lost rapidly along with the EtBr treatment with concomitant losses of membrane potential depolarization and the [Ca2+]i increase, whereas glibenclamide-induced changes persisted. This is the first report to demonstrate the connection between metabolic alteration of electron transport system and that of tricarboxylic acid cycle and its impact on insulin secretion. Krebs-Ringer bicarbonate intracellular free Ca2+ concentration lactate dehydrogenase The mitochondrion has long been one of the main areas of interest for investigators in the field of insulin secretion because it is the primary organelle that metabolizes glucose and other nutrients eliciting insulin secretion. The notion that mitochondria play an important role in insulin secretion has become more feasible with cloning of the ATP-sensitive K+ channel/sulfonylurea receptor (1Inagaki N. Gonoi T. Clement J.P., IV Namba N. Inazawa J. Gonzalez G. Aguilar-Bryan L. Seino S. Bryan J. Science. 1995; 270: 1166-1170Crossref PubMed Scopus (1618) Google Scholar). This is because it is now clear that these channels important for insulin secretion are, in fact, governed by ATP produced mostly by mitochondria. This importance of mitochondria for insulin secretion is further confirmed by the fact that mutations in mitochondrial DNA are associated with some types of diabetes mellitus (2Ballinger S.W. Schoffner J.M. Hedaya E.V. Trounce I. Polak M.A. Koontz D.A. Wallace D.C. Nat. Genet. 1992; 1: 11-15Crossref PubMed Scopus (577) Google Scholar, 3van den Ouweland J.M.W. Lemkes H.H.P.J. Ruitenbeek W. Sandkuijl L.A. de Vijlder M.F. Struyvenberg P.A.A. van de Kamp J.J.P. Maassen J.A. Nat. Genet. 1992; 1: 368-371Crossref PubMed Scopus (1056) Google Scholar, 4Kadowaki T. Kadowaki H. Mori Y. Tobe K. Sakuta R. Suzuki Y. Tanabe Y. Sakura H. Awata T. Goto Y.-I. Hayakawa T. Matsuoko K. Kawamori R. Kamada T. Horai S. Nonaka I. Hagura R. Akanuma Y. Yazaki Y. N. Engl. J. Med. 1994; 330: 962-968Crossref PubMed Scopus (545) Google Scholar) (for review, see Ref. 5Maassen J.A. Kadowaki T. Diabetologia. 1996; 39: 375-382Crossref PubMed Scopus (163) Google Scholar), which is estimated to be the cause of disease in ∼1% of people suffering from diabetes (4Kadowaki T. Kadowaki H. Mori Y. Tobe K. Sakuta R. Suzuki Y. Tanabe Y. Sakura H. Awata T. Goto Y.-I. Hayakawa T. Matsuoko K. Kawamori R. Kamada T. Horai S. Nonaka I. Hagura R. Akanuma Y. Yazaki Y. N. Engl. J. Med. 1994; 330: 962-968Crossref PubMed Scopus (545) Google Scholar, 6Vionnet N. Passa P. Froguel P. Lancet. 1993; 342: 1429-1430Abstract PubMed Scopus (74) Google Scholar). Considering that diabetes mellitus is one of the most common diseases, especially in developed countries (e.g. in both the United States (7King H. Aubert R.E. Herman W.H. Diabetes Care. 1998; 21: 1414-1431Crossref PubMed Scopus (5064) Google Scholar) and Japan (8Akazawa H. Diabetes Res. Clin. Pract. 1994; 24: S23-S27Abstract Full Text PDF PubMed Scopus (23) Google Scholar) ∼10% of people over 40 have this disease), the significance of the percentage must not be overlooked. Recently, we demonstrated (9Hayakawa T. Noda M. Yasuda K. Yorifuji H. Taniguchi S. Miwa I. Sakura H. Terauchi Y. Hayashi J.-I. Sharp G.W.G. Kanazawa Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 20300-20307Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) that ethidium bromide (EtBr), an inhibitor of DNA/RNA synthesis which more specifically affects transcription and replication of extrachromosomal genetic components (10Zylber E. Vesco C. Penman S. J. Mol. Biol. 1969; 44: 195-204Crossref PubMed Scopus (224) Google Scholar, 11Hayakawa T. Tanaka T. Sakaguchi K. Otake N. Yonehara H. J. Gen. Appl. Microbiol. 1979; 25: 255-260Crossref Scopus (70) Google Scholar, 12Desjardins P. Frost E. Morais R. Mol. Cell. Biol. 1985; 5: 1163-1169Crossref PubMed Scopus (168) Google Scholar), i.e. mitochondrial DNA in animal cells, suppresses the transcription of mitochondrial DNA by ∼90% at a low dosage (0.4 μg/ml) without affecting nuclear gene transcription, thereby resulting in deterioration of glucose-induced insulin release from the murine insulin-secreting cell line βHC9 (13Radvanyi F. Christgau S. Baekkeskov S. Jolicoeur C. Hanahan D. Mol. Cell. Biol. 1993; 13: 4223-4233Crossref PubMed Google Scholar, 14Liang Y. Bai G. Doliba N. Buetteger C. Wang L. Berner D.K. Matschinsky F.M. Am. J. Physiol. 1996; 270: E846-E857PubMed Google Scholar, 15Noda M. Komatsu M. Sharp G.W.G. Diabetes. 1996; 45: 1766-1773Crossref PubMed Scopus (23) Google Scholar). Similar results have been obtained with other cell types (16Kennedy E.D. Maechler P. Wollheim C.B. Diabetes. 1998; 47: 374-380Crossref PubMed Scopus (128) Google Scholar, 17Tsuruzoe K. Araki E. Furukawa N. Shirotani T. Matsumoto K. Kaneko K. Motoshima H. Yoshizato K. Shirakami A. Kishikawa H. Miyazaki J.-I. Shichiri M. Diabetes. 1998; 47: 621-631Crossref PubMed Scopus (65) Google Scholar) and with the use of another compound to inhibit nucleic acid synthesis (18Soejima A. Inoue K. Takai D. Kaneko M. Ishihara H. Oka Y. Hayashi J.-I. J. Biol. Chem. 1996; 271: 26194-26199Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). However, the overall mechanism that elicits such alteration is still poorly understood, especially from a viewpoint of cellular metabolism. Herein, we investigated the changes in substrate metabolism and stimulus-secretion coupling caused by suppression of the transcription of mitochondrial DNA by EtBr, which exclusively encodes components of the electron transport system (complexes I, III, IV, and V) and mitochondrial transfer RNA molecules that are used for the synthesis of these components. We have found metabolic derangements, such as accelerated anaerobic metabolism with increased lactate production, indirectly caused by suppression of mitochondrial DNA transcription. Our observations provide insight not only into the physiological importance of mitochondrial function in insulin release from islet β cells, but also into the pathophysiology of mitochondrial diseases, especially diabetes mellitus, resulting from dysfunctional mitochondria, and may further suggest clinical approaches to managing this disease. EtBr, glibenclamide, tetrabutylammonium hydrogen sulfate, fura-2 acetoxymethyl ester, and sulfinpyrazone were purchased from Sigma. Nitrendipine was obtained from Research Biochemicals, norepinephrine from Fluka. Glibenclamide and nitrendipine were dissolved in dimethyl sulfoxide (with final concentrations up to 0.1%), and the same final concentration of dimethyl sulfoxide was added to the controls when they were used. βHC9 cells were cultured in Dulbecco's modified Eagle's medium containing 25 mmol/liter glucose, 1 mmol/liter pyruvate, 15% horse serum, 2.5% fetal bovine serum, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C in a 95% air plus 5% CO2 atmosphere. Supplemental pyruvate was added to ensure cell growth, as with ρ0 cells (cells lacking mitochondrial DNA) (19Hayashi J.-I. Ohta S. Kikuchi A. Takemitsu M. Goto Y.-I. Nonaka I. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10614-10618Crossref PubMed Scopus (513) Google Scholar). Cells were plated at a density of 0.1–0.15 × 106/cm2. For the treated cells, 0.4 μg/ml EtBr was added to the culture medium 22–26 h after plating. This EtBr concentration was chosen based on the observation that culturing βHC9 cells with 0.4 μg/ml EtBr for 4–6 days resulted in an ∼90% decrease in the level of mitochondrial DNA transcription without changing its copy number (9Hayakawa T. Noda M. Yasuda K. Yorifuji H. Taniguchi S. Miwa I. Sakura H. Terauchi Y. Hayashi J.-I. Sharp G.W.G. Kanazawa Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 20300-20307Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cells at passages from 25 to 38 were used for the experiments. Insulin secretion experiments were performed as described previously (15Noda M. Komatsu M. Sharp G.W.G. Diabetes. 1996; 45: 1766-1773Crossref PubMed Scopus (23) Google Scholar). Briefly, four (occasionally three) 16-mm-diameter wells containing cells were used for one test situation. Cells at a concentration of 0.2–0.3 × 106/well (0.1–0.15 × 106/cm2) were plated in each 16-mm-diameter well in a 24-well plate. Four (or occasionally 3) of the wells treated with or without EtBr were used for one test situation (usual cell concentrations for control wells at the time of the experiments were 0.5–1.0 × 106/well). In all experiments, cells were preincubated for 30 min at 37 °C in Krebs-Ringer bicarbonate (KRB)1 buffer solution (pH 7.4) composed of (in mmol/liter) 129 NaCl, 5 NaHCO3, 4.8 KCl, 1.2 KH2PO4, 2.0 CaCl2, 1.2 MgSO4, and 10 HEPES (pH 7.4) containing 0.1 glucose and 0.2% bovine serum albumin before incubation. Then the medium was removed, and 1 ml of fresh KRB buffer (0.1 mm glucose) containing test substances was introduced. Incubation under the test conditions was then carried out for the indicated times (usually 60 min) at 37 °C. At the end of each incubation, media were sampled and centrifuged, and the supernatants were used for insulin radioimmunoassay. Rat insulin was used as the standard. For determination of insulin content in the cells, each well was suspended in a solution containing 77% (v/v) ethanol and 1% (v/v) HCl, and kept at −20 °C overnight. All samples were stored at −20 °C until the insulin radioimmunoassay. Insulin release was expressed as fractional release during the incubation time. The temporal profiles of insulin secretion from βHC9 cells were investigated using a perifusion system (20Kikuchi M. Rabinovitch A. Blackard W.G. Renold A.E. Diabetes. 1974; 23: 550-559Crossref PubMed Scopus (43) Google Scholar), as described previously (15Noda M. Komatsu M. Sharp G.W.G. Diabetes. 1996; 45: 1766-1773Crossref PubMed Scopus (23) Google Scholar). Briefly, glass coverslips with attached cells were placed in each 0.6-ml perifusion chamber and perifused with KRB buffer containing 2.8 mm glucose at a rate of 1 ml/min at 37 °C. Experiments were started after 60-min perifusion equilibration periods with KRB buffer containing 2.8 mm glucose. At the end of the experiments, each coverslip was suspended in a solution containing 77% ethanol and 1% HCl and kept at −20 °C overnight for determination of the residual insulin content. Perifusate samples were collected every 1 or 2 min. Insulin release rates were expressed in terms of fractional secretion/content at each time point. The [Ca2+]i of the cells was measured fluorometrically using fura-2 as described previously (15Noda M. Komatsu M. Sharp G.W.G. Diabetes. 1996; 45: 1766-1773Crossref PubMed Scopus (23) Google Scholar). Cells dispersed by trypsin and EDTA were suspended in KRB buffer (0.1 mm glucose) containing 1 μm fura-2 acetoxymethyl ester and 0.25 mm sulfinpyrazone, then loaded with fura-2 by incubation at 37 °C for 30 min with continuous shaking. The fura-2-loaded cells thus obtained were washed and resuspended in KRB buffer (containing 0.1 mm glucose and 0.25 mm sulfinpyrazone), and 3 ml of the suspension was introduced into individual quartz cuvettes held in a spectrofluorometer (PerkinElmer Life Sciences LS-5). The cell suspension in each cuvette was stirred continuously with a small magnetic bar within the cuvette during each experiment. The temperature of the cell suspension was maintained at 37 °C by circulating warm water through the cuvette holder. An excitation wavelength of 340 nm and an emission wavelength of 510 nm were used for the measurement. In some cases, an excitation wavelength of 380 nm was used for the reciprocal monitor and, to confirm the Ca2+ dependence of the fluorescence, the [Ca2+]i was calculated as described previously (15Noda M. Komatsu M. Sharp G.W.G. Diabetes. 1996; 45: 1766-1773Crossref PubMed Scopus (23) Google Scholar) employing values of auto- and extracellular fura-2-emitted fluorescence, which were determined using two of the four cuvettes applied for each experiment. For stimulation, a 100-fold concentration of the stimulus was added, gently, as an injection. Membrane potential was measured by the patch clamp method in a conventional whole cell mode. Pipettes were pulled from glass 1740 (Garner Glass Co.) using a two-stage puller, fire polished, tip filled with pipette solution (containing 70 mmK2SO4, 10 mm KCl, 10 mmNaCl, 2 mm MgCl2, and 5 mm HEPES, pH adjusted to 7.4), and then back filled with the same solution containing 200 μg/ml nystatin. Before each experiment, cells were transferred into a bath solution. A gigaohm seal was obtained within 10 min after transfer of the cells into an experimental chamber. A whole cell configuration was considered to be successfully established when the conductance observed was more than 30 microsiemens. Membrane potential was measured by patch clamps in the current clamp mode. Experiments were carried out at 37 °C. Nucleotide extraction was performed as described previously (21Deitemary P. Van den Gerghe G. Henquin J.-C. J. Biol. Chem. 1996; 271: 20559-20565Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) with minor modifications. Cells at a concentration of 0.2–0.3 × 106/well (0.1–0.15 × 106/cm2) were plated onto each 16-mm-diameter well in a 24-well plate. Three of the wells treated with or without EtBr were used for one test condition. After the indicated culture periods, cells were preincubated for 30 min at 37 °C in fresh KRB buffer (containing 0.1 mm glucose) after being washed twice with the same buffer. Then, the medium was removed, and 1 ml of fresh KRB buffer containing 22.2 mm glucose was introduced. Incubation was carried out for 5 or 30 min at 37 °C and stopped by adding trichloroacetic acid (final concentration 5%); for determination of the initial nucleotide content, trichloroacetic acid was introduced just after preincubation. Then, the cells were scraped, and the buffer containing lysate was moved to a tube and centrifuged for 1 min. A fraction of the supernatant was mixed with 1.5 ml of diethyl ether, and the ether phase containing trichloroacetic acid was discarded. This step was repeated three times to ensure complete elimination of the trichloroacetic acid. The extracts were diluted with a buffer containing 20 mm HEPES, 3 mmMgCl2, and KOH, as required to adjust pH to 7.75, and then frozen at −80 °C until the assay. Another set of three wells of cells, with or without EtBr treatment, was trypsinized, detached, and used to assay protein amounts. ATP and ADP contents of the cells were assayed in duplicate by the luminometric method as described previously (21Deitemary P. Van den Gerghe G. Henquin J.-C. J. Biol. Chem. 1996; 271: 20559-20565Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) using an ATP bioluminescent assay kit (Sigma). For measurement of the sum of ATP + ADP, ADP was first converted into ATP as described previously (21Deitemary P. Van den Gerghe G. Henquin J.-C. J. Biol. Chem. 1996; 271: 20559-20565Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar), and ADP levels were calculated by subtraction. Glucose utilization by βHC9 cells was measured as described previously (22Ashcroft S.J.H. Weeransinghe L.C.C. Bassett J.M. Randle P.J. Biochem. J. 1972; 126: 525-535Crossref PubMed Scopus (247) Google Scholar, 23Miwa I. Murata T. Okuda J. Biochem. Biophys. Res. Commun. 1991; 180: 709-715Crossref PubMed Scopus (14) Google Scholar, 24Aizawa T. Sato Y. Ishihara F. Taguchi N. Komatsu M. Suzuki N. Hashizume K. Yamada T. Am. J. Physiol. 1994; 266: C622-C627Crossref PubMed Google Scholar) with modifications based ond-[5-3H]glucose. In brief, ∼0.5 × 106 cells dispersed by trypsinization were first incubated for 30 min in 1 ml of KRB buffer containing 0.1 mm glucose (preincubation). At the end of preincubation, the cells were briefly centrifuged, supernatants were aspirated, 100 μl of KRB buffer containing 1 μCi of d-[5-3H]glucose (Amersham Biosciences, specific activity 16.8 Ci/mmol) was introduced, and incubation was continued for another 60 min. Then, the reaction was terminated by adding 10 μl of 3 n HCl and 40 μl of ethanol to the incubation mixture. Formed 3H2O was equilibrated with 0.5 ml of H2O in outer vessels, as described previously (23Miwa I. Murata T. Okuda J. Biochem. Biophys. Res. Commun. 1991; 180: 709-715Crossref PubMed Scopus (14) Google Scholar), for 24 h at room temperature. The glucose concentration (radioactive and nonradioactive combined) was set at 0.8 or 22.2 mm during the incubations. Glucose oxidation of βHC9 cells was measured as described previously (22Ashcroft S.J.H. Weeransinghe L.C.C. Bassett J.M. Randle P.J. Biochem. J. 1972; 126: 525-535Crossref PubMed Scopus (247) Google Scholar, 24Aizawa T. Sato Y. Ishihara F. Taguchi N. Komatsu M. Suzuki N. Hashizume K. Yamada T. Am. J. Physiol. 1994; 266: C622-C627Crossref PubMed Google Scholar, 25McDaniel M.L. King S. Anderson S. Fink J. Lacy P.E. Diabetologia. 1974; 10: 303-308PubMed Google Scholar) by using uniformly labeledd-[14C]glucose with modifications. In brief, ∼0.5 × 106 cells dispersed by trypsinization were first incubated for 30 min in 1 ml of KRB buffer containing 0.1 mm glucose (preincubation). At the end of the preincubation, the cells were briefly centrifuged, the supernatants were aspirated, 100 μl of KRB buffer containing 1 μCi ofd-[14C]glucose (PerkinElmer Life Sciences, specific activity 13.0 mCi/mmol) was introduced, and incubation was continued for another 60 min. Then, the reaction was terminated by adding 200 μl of 0.1 n HCl to the incubation mixture. Formed 14CO2 was trapped by 300 μl of 1m benzethonium hydroxide (Sigma) in methanol for 48 h at room temperature. The glucose concentration (radioactive and nonradioactive combined) was set at 0.8 or 22.2 mm during the incubations. Lactate production was measured by the lactate oxidase method using a Determiner LA assay kit (Kyowa Medics, Tokyo, Japan). Five 30-mm-diameter dishes of cells were used for one test condition. Cells at a concentration of 0.7–1.1 × 106/dish (0.1–0.15 × 106/cm2) were plated onto the 30-mm-diameter dishes. After the indicated culture periods, the cells were preincubated for 30 min at 37 °C with 3 ml of a fresh KRB buffer solution (containing 0.1 mm glucose) after being washed twice with the same buffer. Then, the medium was removed, and 3 ml of fresh KRB buffer containing 0.1 or 22.2 mm glucose was introduced. Incubation under the test conditions was carried out for 60 min at 37 °C. At the end of the incubation, media were sampled and centrifuged, and the supernatants were used to assay for lactate released during the incubation period. The cells were trypsinized, detached, and used to assay for protein amount. LDH activity was measured using LDH assay kit (TaKaRa, Ohtsu, Japan). After the indicated culture periods, cells of four of the 32-mm-diameter wells at a concentration of 1.0–5.0 × 106/well, treated with or without EtBr, were used for measurement of one condition after trypsinization and resuspension. Briefly, lysate of 1.0–5.0 × 104 cells in a 100-μl final volume with 1% Triton X-100 were applied for photometric measurement in final volume of 200 μl of assay mixture as indicated by the manufacturer. LDH from rabbit muscle (Wako, Tokyo, Japan) was used for the standard. NADH autofluorescence (400–500 nm) of the monolayer-cultured cells was imaged using two-photon excitation microscopy (26Nemoto T. Kimura R. Ito K. Tachikawa K. Miyashita Y. Iino M. Kasai H. Nat. Cell Biol. 2001; 3: 253-258Crossref PubMed Scopus (149) Google Scholar, 27Xu C. Zipfel W. Shear J.B. Williams R.M. Webb W.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10763-10768Crossref PubMed Scopus (1086) Google Scholar) at an excitation wavelength of 720 nm. Cells were plated at a density of 0.1–0.15 × 106/cm2 and cultured with or without EtBr for the indicated times on 5 mm-in-diameter glass coverslips. Data are expressed as means ± S.E. Statistical significance was evaluated by one-way analysis of variance using Bonferroni's method unless otherwise indicated. For all comparisons in the figures, * and ** denote 0.01 ≤ p < 0.05 andp < 0.01, respectively. In static incubations, during suppression of the electron transport system by EtBr, glucose-induced insulin secretion was decreased by 4-day EtBr treatment and had almost disappeared by day 6, as shown in Fig.1 A. However, glibenclamide-stimulated insulin secretion was not reduced on day 2, 4, or 6 of EtBr treatment (Fig. 1 A). The concentration-secretion relationship curves (Fig. 1 C) showed a decrease in insulin release with glucose stimulation. These results are similar to our previous data (9Hayakawa T. Noda M. Yasuda K. Yorifuji H. Taniguchi S. Miwa I. Sakura H. Terauchi Y. Hayashi J.-I. Sharp G.W.G. Kanazawa Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 20300-20307Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Thus, suppression of the electron transport system ultimately abolished glucose-stimulated insulin secretion (Fig. 1, A and C), whereas insulin secretion elicited by glibenclamide was maintained (Fig. 1 A) because it does not employ metabolism for its effect, as discussed previously (9Hayakawa T. Noda M. Yasuda K. Yorifuji H. Taniguchi S. Miwa I. Sakura H. Terauchi Y. Hayashi J.-I. Sharp G.W.G. Kanazawa Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 20300-20307Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). In addition, the formerly observed increase in basal (nonstimulated) insulin secretion in EtBr-treated cells (9Hayakawa T. Noda M. Yasuda K. Yorifuji H. Taniguchi S. Miwa I. Sakura H. Terauchi Y. Hayashi J.-I. Sharp G.W.G. Kanazawa Y. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 20300-20307Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) was also seen in the current series of experiments on most occasions. However, the degree of the increase was much smaller, possibly because of the more dense initial cell concentration (0.03 × 106/cm2 versus 0.1–0.15 × 106/cm2) with which culture was started in this series of experiments. Perifusion experiments were performed on day 4 (Fig. 1 B), and in these experiments, both the first and the second phase of glucose-elicited insulin secretion were reduced, the latter being nearly abolished by the treatment. The [Ca2+]i measurement was performed on day 4 (Fig. 2). Basal (initial) [Ca2+]i at the start of stimulation did not differ significantly between EtBr-treated (189 nm; n = 13) and control conditions (212 nm; n = 23) by Student's ttest. Both glucose and glibenclamide increased [Ca2+]i under control conditions. However, in EtBr-treated cells, the mean ratio over the initial (start of stimulation) [Ca2+]i was decreased significantly under glucose stimulation at every time point 4 min after stimulation and thereafter. By contrast, the glibenclamide-induced [Ca2+]iincrease did not differ significantly except at 2 min after the start of stimulation. Membrane potential was measured by whole cell mode patch clamp on day 6 (Fig.3). In control cells, both glucose and glibenclamide elicited depolarization of the membrane potential with an action potential burst. Glibenclamide was still able to trigger the potential without being affected by EtBr treatment (Fig.3 D). By contrast, the glucose-induced change in membrane potential was abolished by EtBr treatment, as shown in Fig.3 C. ATP and ADP contents were measured on day 4 and day 6. As shown in Fig.4, the steady-state ATP and ADP contents of the cells (measured after a 30-min incubation in 0.1 mmglucose after continuous culture) were fairly well maintained even with EtBr treatment, i.e. they were equilibrated. The ATP/ADP ratio before stimulation did not differ significantly on either day 4 or 6 between EtBr-treated and control cells. After glucose stimulation, although no significant ATP increase was observed on either day in EtBr-treated cells or even in control cells, the ADP content was decreased significantly in control cells (days 4 and 6) and in EtBr-treated cells (day 4). As a result, the ATP/ADP ratio was increased significantly (at 30 min) in control cells. In EtBr-treated cells, however, this increase in the ATP/ADP ratio was not significant on either day. With 4- and 6-day EtBr treatment, glucose oxidation (Fig.5 B), measured by the rate of14CO2 formation fromd-[14C]glucose, was decreased severely compared with control cells (by 63% on day 4 and by 78% on day 6; both p < 0.01 by Student's t test) under the stimulatory (22.2 mm) condition; under the nonstimulatory (0.8 mm) condition, it was substantially decreased as well (p < 0.05 on day 4 andp < 0.01 on day 6; Student's t test). By contrast, glucose utilization was only marginally affected on both day 4 and day 6 of EtBr treatment, as measured by3H2O formation fromd-[5-3H]glucose, suggesting the glycolytic pathway to be intact (Fig. 5 A). The slight, but significant (Student's t test), difference in glucose utilization could represent a backward effect of suppressed mitochondrial metabolism (see “Discussion”). In control cells, the ratio of glucose oxidation to utilization under the stimulatory (22.2 mm glucose) condition was 10–15%. This percentage was similar to previous data obtained with βHC9 cells (14Liang Y. Bai G. Doliba N. Buetteger C. Wang L. Berner D.K. Matschinsky F.M. Am. J. Physiol. 1996; 270: E846-E857PubMed Google Scholar), which was much smaller than those (∼70% of utilization) with mouse (28Eizirik D.L. Sandler S. Diabetologia. 1989; 32: 769-773Crossref PubMed Scopus (24) Google Scholar) and rat (24Aizawa T. Sato Y. Ishihara F. Taguchi N. Komatsu M. Suzuki N. Hashizume K. Yamada T. Am. J. Physiol. 1994; 266: C622-C627Crossref PubMed Google Scholar) islets at a stimulatory glucose concentration (16.7 mm) measured using the same methods for both oxidation and utilization. Lactate production under basal (0.1 mm glucose) conditions was not different on either day 4 or day 6 (Fig. 5 C). At 22.2 mm glucose, it was increased by 2.9- and 3.5-fold by EtBr on days 4 and 6, respectively (both p < 0.01; Student's t test) (Fig. 5 C). T
Referência(s)