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Hyperlipidemic Mice Present Enhanced Catabolism and Higher Mitochondrial ATP-Sensitive K+ Channel Activity
2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.07.021
ISSN1528-0012
AutoresLuciane C. Alberici, Helena C.F. Oliveira, P.R. Patrício, Alicia J. Kowaltowski, Anı́bal E. Vercesi,
Tópico(s)Diet and metabolism studies
ResumoBackground & Aims: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. Methods: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoKATP) agonists and antagonists were measured in isolated mitochondria, livers, and mice. Results: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoKATP) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoKATP agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoKATP. The higher content and activity of liver mitoKATP resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body co2 release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. Conclusions: These results show that primary hyperlipidemia leads to an elevation in liver mitoKATP activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoKATP, in addition to UCPs, may be involved in the control of energy metabolism and body weight. Background & Aims: Changes in mitochondrial energy metabolism promoted by uncoupling proteins (UCPs) are often found in metabolic disorders. We have recently shown that hypertriglyceridemic (HTG) mice present higher mitochondrial resting respiration unrelated to UCPs. Here, we disclose the underlying mechanism and consequences, in tissue and whole body metabolism, of this mitochondrial response to hyperlipidemia. Methods: Oxidative metabolism and its response to mitochondrial adenosine triphosphate (ATP)-sensitive K+ channel (mitoKATP) agonists and antagonists were measured in isolated mitochondria, livers, and mice. Results: Mitochondria isolated from the livers of HTG mice presented enhanced respiratory rates compared with those from wild-type mice. Changes in oxygen consumption were sensitive to adenosine triphosphate (ATP), diazoxide, and 5-hydroxydecanoate, indicating they are attributable to mitochondrial ATP-sensitive K+ channel (mitoKATP) activity. Indeed, mitochondria from HTG mice presented enhanced swelling in the presence of K+ ions, sensitive to mitoKATP agonists and antagonists. Furthermore, mitochondrial binding to fluorescent glibenclamide indicates that HTG mice expressed higher quantities of mitoKATP. The higher content and activity of liver mitoKATP resulted in a faster metabolic state, as evidenced by increased liver oxygen consumption and higher body co2 release and temperature in these mice. In agreement with higher metabolic rates, food ingestion was significantly larger in HTG mice, without enhanced weight gain. Conclusions: These results show that primary hyperlipidemia leads to an elevation in liver mitoKATP activity, which may represent a regulated adaptation to oxidize excess fatty acids in HTG mice. Furthermore, our data indicate that mitoKATP, in addition to UCPs, may be involved in the control of energy metabolism and body weight. High plasma levels of triglycerides and free fatty acids can occur due to primary inherited disorders or secondarily to other metabolic diseases such as diabetes and the metabolic syndrome, which also includes insulin resistance, obesity, and hypertension.1Grundy S.M. Brewer Jr, H.B. Cleeman J.I. Smith Jr, S.C. Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition.Circulation. 2004; 109: 433-438Crossref PubMed Scopus (4247) Google Scholar In conjunction with these disorders, or even individually, hypertriglyceridemia is a risk factor for coronary heart disease, stroke, and nonalcoholic fatty liver disease.1Grundy S.M. Brewer Jr, H.B. Cleeman J.I. Smith Jr, S.C. Lenfant C. Definition of metabolic syndrome: report of the National Heart, Lung, and Blood Institute/American Heart Association conference on scientific issues related to definition.Circulation. 2004; 109: 433-438Crossref PubMed Scopus (4247) Google Scholar, 2Marchesini G. Brizi M. Morselli-Labate A.M. Bianchi G. Bugianesi E. McCullough A.J. Forlani G. Melchionda N. Association of nonalcoholic fatty liver disease with insulin resistance.Am J Med. 1999; 107: 450-455Abstract Full Text Full Text PDF PubMed Scopus (1283) Google Scholar, 3Reid A.E. Nonalcoholic steatohepatitis.Gastroenterology. 2001; 121: 710-723Abstract Full Text Full Text PDF PubMed Scopus (477) Google ScholarA group of proteins that plays a role in the pathogenesis or consequences of metabolic diseases is the uncoupling proteins (UCPs).4Jezek P. Garlid K.D. Mammalian mitochondrial uncoupling proteins.Int J Biochem Cell Biol. 1998; 30: 1163-1168Crossref PubMed Scopus (82) Google Scholar, 5Boss O. Muzzin P. Giacobino J.P. The uncoupling proteins, a review.Eur J Endocrinol. 1998; 139: 1-9Crossref PubMed Scopus (226) Google Scholar, 6Brand M.D. Esteves T.C. Physiological functions of the mitochondrial uncoupling proteins UCP2 and UCP3.Cell Metab. 2005; 2: 85-93Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar These proteins act as mitochondrial inner membrane fatty acid anion transporters and are widely distributed in many mammalian organs.7Garlid K.D. Jaburek M. Jezek P. Varecha M. How do uncoupling proteins uncouple?.Biochim Biophys Acta. 2000; 1459: 383-389Crossref PubMed Scopus (106) Google Scholar Because of the proton gradient and free permeability of protonated fatty acids across the inner membrane, a result of UCP activity is mitochondrial uncoupling, including increased resting respiration and decreased membrane potentials and oxidative phosphorylation efficiency.7Garlid K.D. Jaburek M. Jezek P. Varecha M. How do uncoupling proteins uncouple?.Biochim Biophys Acta. 2000; 1459: 383-389Crossref PubMed Scopus (106) Google Scholar Interestingly, UCP expression is altered by obesity and diabetes8Boss O. Bobbioni-Harsch E. Assimacopoulos-Jeannet F. Muzzin P. Munger R. Giacobino J.P. Golay A. Uncoupling protein-3 expression in skeletal muscle and free fatty acids in obesity.Lancet. 1998; 351: 1933Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 9Bao S. Kennedy A. Wojciechowski B. Wallace P. Ganaway E. Garvey W.T. Expression of mRNAs encoding uncoupling proteins in human skeletal muscle: effects of obesity and diabetes.Diabetes. 1998; 47: 1935-1940Crossref PubMed Scopus (74) Google Scholar, 10Hidaka S. Yoshimatsu H. Kakuma T. Sakino H. Kondou S. Hanada R. Oka K. Teshima Y. Kurokawa M. Sakata T. Tissue-specific expression of the uncoupling protein family in streptozotocin-induced diabetic rats.Proc Soc Exp Biol Med. 2000; 224: 172-177Crossref PubMed Scopus (23) Google Scholar and in some conditions where circulating lipid levels are modified by hormones,11Gong D.W. He Y. Karas M. Reitman M. Uncoupling protein-3 is a mediator of thermogenesis regulated by thyroid hormone, beta3-adrenergic agonists, and leptin.J Biol Chem. 1997; 272: 24129-24132Crossref PubMed Scopus (738) Google Scholar dietary fat,12Samec S. Seydoux J. Dulloo A.G. Post-starvation gene expression of skeletal muscle uncoupling protein 2 and uncoupling protein 3 in response to dietary fat levels and fatty acid composition: a link with insulin resistance.Diabetes. 1999; 48: 436-441Crossref PubMed Scopus (97) Google Scholar and intravenous heparin plus lipid infusion.13Nisoli E. Carruba M.O. Tonello C. Macor C. Federspil G. Vettor R. Induction of fatty acid translocase/CD36, peroxisome proliferator-activated receptor-gamma2, leptin, uncoupling proteins 2 and 3, and tumor necrosis factor-alpha gene expression in human subcutaneous fat by lipid infusion.Diabetes. 2000; 49: 319-324Crossref PubMed Scopus (90) Google ScholarAll these conditions present a complex metabolic context where it is difficult to discriminate the role of hyperlipidemia. To study the effects of elevated plasma lipid levels per se, without other metabolic confounding factors, we used mice overexpressing the apolipoprotein CIII, which develop severe hypertriglyceridemia and high plasma levels of free fatty acids14Ito Y. Azrolan N. O’Connell A. Walsh A. Breslow J.L. Hypertriglyceridemia as a result of human apo CIII gene expression in transgenic mice.Science. 1990; 249: 790-793Crossref PubMed Scopus (448) Google Scholar but retain normal glucose homeostasis.15Reaven G.M. Mondon C.E. Chen Y.D. Breslow J.L. Hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic.J Lipid Res. 1994; 35: 820-824PubMed Google Scholar, 16Amaral M.E.C. Oliveira H.C.F. Carneiro E.M. Delghingaro-Augusto V. Vieira E. Berti J.A. Boschero A.C. Plasma glucose regulation and insulin secretion in hypertriglyceridemic mice.Horm Metab Res. 2002; 34: 21-26Crossref PubMed Scopus (21) Google Scholar The increased apolipoprotein CIII content in the surface of triglyceride-rich lipoproteins hampers their recognition by specific liver receptors, thus increasing their half-life and free fatty acid release in the plasma compartment.17Aalto-Setala K. Fisher E.A. Chen X. Chajek-Shaul T. Hayek T. Zechner R. Walsh A. Ramakrishnan R. Ginsberg H.N. Breslow J.L. Mechanism of hypertriglyceridemia in human apolipoprotein (apo) CIII transgenic mice Diminished very low density lipoprotein fractional catabolic rate associated with increased apo CIII and reduced apo E on the particles.J Clin Invest. 1992; 90: 1889-1900Crossref PubMed Scopus (399) Google Scholar We found that liver mitochondria from these mice presented higher resting respiratory rates.18Alberici L.C. Oliveira H.C. Bighetti E.J. de Faria E.C. Degaspari G.R. Souza C.T. Vercesi A.E. Hypertriglyceridemia increases mitochondrial resting respiration and susceptibility to permeability transition.J Bioenerg Biomembr. 2003; 35: 451-457Crossref PubMed Scopus (23) Google Scholar This increase in respiration was not related to the activity of UCPs because (1) the effect was present even in media in which free fatty acids were quenched by bovine serum albumin and (2) uncoupling was not eliminated by the UCP inhibitor GDP (guanosine 5′-diphosphate).18Alberici L.C. Oliveira H.C. Bighetti E.J. de Faria E.C. Degaspari G.R. Souza C.T. Vercesi A.E. Hypertriglyceridemia increases mitochondrial resting respiration and susceptibility to permeability transition.J Bioenerg Biomembr. 2003; 35: 451-457Crossref PubMed Scopus (23) Google ScholarTo uncover the cause of this mitochondrial uncoupling in hypertriglyceridemic mice, we now focus our attention on another recently described mild mitochondrial uncoupling pathway: the activity of adenosine triphosphate–sensitive K+ channels (mitoKATP).19Garlid K.D. Paucek P. Mitochondrial potassium transport: the K+ cycle.Biochim Biophys Acta. 2003; 1606: 23-41Crossref PubMed Scopus (300) Google Scholar, 20Facundo H.T. Fornazari M. Kowaltowski A.J. Tissue protection mediated by mitochondrial K+ channels.Biochim Biophys Acta. 2006; 40: 469-479Google Scholar These inner membrane uniporters promote K+ influx in a manner counteracted by the K+/H+ antiporter (see Figure 1 and Garlid and Paucek19Garlid K.D. Paucek P. Mitochondrial potassium transport: the K+ cycle.Biochim Biophys Acta. 2003; 1606: 23-41Crossref PubMed Scopus (300) Google Scholar for review). The resulting uptake of H+ through the antiporter decreases the efficiency of oxidative phosphorylation. Uncoupling is limited by K+ transport rates of mitoKATP, which are quite slow and only allow for mild uncoupling. In addition to mild uncoupling, these channels also promote low-amplitude mitochondrial swelling when active due to the uptake of K+, the counter-ion phosphate, and water.21Beavis A.D. Lu Y. Garlid K.D. On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.J Biol Chem. 1993; 268: 997-1004Abstract Full Text PDF PubMed Google Scholar, 22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google Scholar We found strong evidence that the activity and quantity of mitoKATP channels is augmented in hypertriglyceridemic (HTG) mice, providing a mechanistic explanation for the observed uncoupling. In addition, we show evidence that this uncoupling leads to increases in overall respiratory rates and catabolism in HTG livers in mice.Materials and MethodsAnimalsHuman apolipoprotein CIII transgenic (line 3707)23Walsh A. Azrolan N. Wang K. Marcigliano A. O’Connell A. Breslow J.L. Intestinal expression of the human apoA-I gene in transgenic mice is controlled by a DNA region 3′ to the gene in the promoter of the adjacent convergently transcribed apoC-III gene.J Lipid Res. 1993; 34: 617-623Abstract Full Text PDF PubMed Google Scholar founders were donated by Dr Alan R. Tall (Columbia University, New York, NY) and crossbred with wild-type (WT) C57Bl6 mice. The apolipoprotein CIII transgenic colony has been kept for 10 years at the animal facilities of the Department of Physiology and Biophysics at the State University of Campinas (Campinas, Brazil). The experiments were approved by the university’s ethics committee and are in accordance with the Guidelines for Handling and Training of Laboratory Animals published by the University’s Federation for Animal Welfare. Mice had access to standard laboratory rodent chow (CR1; Nuvital, Colombo, Paraná, Brazil) and water ad libitum and were housed at 22°C ± 2°C on a 12-hour light-dark cycle. Male and female heterozygous apolipoprotein CIII transgenic (HTG) and nontransgenic (WT) littermates, aged 4–6 months, were used in this study. Total cholesterol and triglyceride (Chod-Pap; Roche Diagnostic GmbH, Mannheim, Germany) and plasma free fatty acid (Wako Chemical, Neuss, Germany) levels were determined by enzymatic-colorimetric methods according to the manufacturers’ instructions. Transgenic mice presented fasting plasma triglyceride levels >300 mg/dL and WT mice levels <100 mg/dL. Four groups (n = 12) of male and female transgenic and WT mice had their body weight gain and food ingestion followed up from weaning (30 days of age) to 6 months of age. Mice and ingested food were weighed 3 times a week. Body weights were taken individually, whereas food ingestion was measured as the average consumed by 4 mice per cage per day. Two groups of transgenic mice (n = 6) were treated with insulin or saline as previously described.24Berti J.A. Casquero A.C. Patricio P.R. Bighetti E.J.B. Carneiro E.M. Boschero A.C. Oliveira H.C.F. Cholesteryl ester transfer protein expression is down-regulated in hyperinsulinemic transgenic mice.J Lipid Res. 2003; 44: 1870-1876Crossref PubMed Scopus (11) Google Scholar Briefly, mice received daily subcutaneous injections of increasing doses of NPH insulin (0.14–1.63 U/30 g body wt, Iolin; Eli Lilly, Indianapolis, IN) or the same volume of saline solution for 7 days. Two thirds of the dose was given at 8 pm and one third at 8 am. To prevent hypoglycemia, these mice had free access to sugar cubes in addition to the chow diet, and a 5% glucose solution was the only drinking solution offered.Isolation of Mouse Liver MitochondriaMitochondria were isolated by conventional differential centrifugation25Kaplan R.S. Pedersen P.L. Characterization of phosphate efflux pathways in rat liver mitochondria.Biochem J. 1983; 212: 279-288Crossref PubMed Scopus (69) Google Scholar at 4°C. No differences between sexes of the animals were noted in isolated mitochondrial studies, so analyzed samples from male and female animals were pooled. A liver homogenate was prepared in 250 mmol/L sucrose, 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 10 mmol/L HEPES buffer (pH 7.2), and 0.01% bovine serum albumin and centrifuged at 600g for 10 minutes. The supernatant was recentrifuged at 7000g for 10 minutes. The pellet was washed in the same medium devoid of bovine serum albumin and containing 0.1 mmol/L EGTA. The final mitochondrial pellet was diluted in 250 mmol/L sucrose to a protein concentration of 60–80 mg/mL, measured using the Biuret method and bovine serum albumin as the protein standard. Isolated mitochondria were kept over ice and used within 90 minutes of preparation to ensure mitoKATP activity. Mitochondria isolated in this manner lose matrix K+ and contract due to low levels of this ion in the isolation buffer and recover K+ when suspended in K+-rich buffers.21Beavis A.D. Lu Y. Garlid K.D. On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.J Biol Chem. 1993; 268: 997-1004Abstract Full Text PDF PubMed Google Scholar, 22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google ScholarMitochondrial and Respiratory RatesOxygen consumption was measured using a temperature-controlled computer-interfaced Clark-type oxygen electrode from Hansatech Instruments Ltd. (King’s Lynn, Norfolk, England) equipped with magnetic stirring at 28°C.Mitochondrial SwellingMitochondrial swelling was estimated from the decrease in absorbance of the mitochondrial suspension measured at 520 nm using a temperature-controlled SLM Aminco DW 2000 spectrophotometer (SLM Instruments, Inc., Urbana, IL) equipped with continuous stirring at 37°C. Swelling rates of freshly isolated mitochondria were measured soon after their addition of K+-rich, hyposmotic buffers. This procedure allows for a magnified measurement of K+ uptake rates due to prior K+ depletion during the mitochondrial isolation procedure.22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google ScholarLiver Respiratory RatesMouse livers were rapidly dissected and chopped into 1-mm cubes using a tissue chopper. Approximately 50-mg liver samples were incubated in 1 mL Krebs–Henseleit solution (37°C) containing 10 mmol/L glucose. Oxygen consumption was measured using a Clark-type electrode as described previously. The exact protein content of each homogenized tissue sample was then determined using the Biuret method, and respiratory rates were calculated.co2 Production Rates In Vivoco2 production in vivo was measured in a temperature-monitored respirometer described by Calegario et al.26Calegario F.F. Cosso R.G. Fagian M.M. Almeida F.V. Jardim W.F. Jezek P. Arruda P. Vercesi A.E. Stimulation of potato tuber respiration by cold stress is associated with an increased capacity of both plant uncoupling mitochondrial protein (PUMP) and alternative oxidase.J Bioenerg Biomembr. 2003; 35: 211-220Crossref PubMed Scopus (45) Google Scholar Fed mice weighing between 24 and 28 g were adapted to the respirometer chamber twice a day for 5 minutes for 5 days. After the adaptation period, co2 expiration of each mouse was monitored for 5 minutes once a day, between 9 am and 11 am, for 5 consecutive days. co2 production rates were calculated as averages of 5 measurements for each mouse.Body TemperatureRectal temperatures were measured using a digital thermometer (BD Basic; Becton Dickinson and Company, São Paulo, Brazil). Mice were adapted to rapid comfortable immobilization and rectal temperature measurements for 5 days, between 2 pm and 3 pm. Measurements were then conducted for 5 days, during three 20-second periods. Average rectal temperatures for each animal during these measurements were then determined and compared.Data AnalysisData shown as traces are representative of at least 3 repetitions using different preparations. Other data are averages ± SEM. Statistical analysis was performed using one-way analysis of variance comparisons (Figure 2, Figure 3, Figure 4) and analysis of variance and Student t tests (with similar results; Figure 5, Figure 6) conducted using Origin 7.0 software (OringinLab Corp., Northampton, MA). P ≤ .05 was considered significant.Figure 2Enhanced resting respiration in HTG mitochondria is due to ATP-sensitive K+ uptake. Typical traces are shown in A, and averages ± SEM are depicted in B. WT and HTG mitochondria (0.5 mg/mL) were added to 28°C, pH 7.2 (KOH) medium containing 125 mmol/L sucrose, 65 mmol/L KCl, 10 mmol/L HEPES, 2 mmol/L Pi, 1 mmol/L Mg2+, 0.4 mmol/L EGTA, 4 mmol/L succinate, and 1 μg/mL oligomycin in the presence of 0.1 mmol/L ATP, 12 μmol/L DZX, and 60 μmol/L 5-HD, as shown. No K+, experiments conducted in media in which all K+ salts were substituted by Li-positive salts. *P < .01 vs WT under control conditions; #P < .01 vs HTG under control conditions; ×P = .05 vs HTG in the presence of ATP; ϕP < 05 vs HTG in the presence of ATP plus DZX.View Large Image Figure ViewerDownload (PPT)Figure 3HTG mitochondria present enhanced ATP-sensitive K+ uptake. Typical traces are shown in A, and averages ± SEM of swelling rates during the first 15 seconds are depicted in B Mitochondria (0.5 mg/mL) were added to 28°C, pH 7.2 (KOH) medium containing 100 mmol/L KCl, 5 mmol/L HEPES, 2 mmol/L Pi, 1 mmol/L Mg2+, 0.1 mmol/L EGTA, 2 mmol/L succinate, and 1 μg/mL oligomycin in the presence of 0.2 mmol/L ATP, 30 μmol/L DZX, 10 μmol/L GLY, and 60 μmol/L 5-HD as indicated. No K+, experiments conducted in media in which all K+ salts were substituted by Li-positive salts. *P < .05 vs WT under control conditions; #P < .05 vs HTG under control conditions; ×P < 05 vs HTG in the presence of ATP; ϕP ≤ .05 vs HTG in the presence of ATP plus DZX.View Large Image Figure ViewerDownload (PPT)Figure 4MitoKATP activity increases respiratory rates in HTG livers. Data shown are average ± SEM respiratory rates of liver fragments incubated at 37°C in Krebs–Henseleit solution, as described in Materials and Methods. Mice received intraperitoneal saline (control) or 5-HD (10 mg/kg body wt) 1 hour before liver excision. *P < .05 vs WT under control conditions; #P < .05 vs HTG under control conditions.View Large Image Figure ViewerDownload (PPT)Figure 5Body temperature and CO2 production is enhanced in HTG mice. (A) Rectal body temperatures and (B) CO2 release rates were compared in WT and HTG mice, as described in Materials and Methods. *P < .05 vs WT.View Large Image Figure ViewerDownload (PPT)Figure 6Food ingestion is enhanced in HTG mice. (A) Body weight, (B) food ingestion, and (C) the efficiency of the conversion of ingested food in HTG and WT mice were measured from weaning (1 month) to 6 months of age, as described in Materials and Methods. Data represent means ± SEM (in grams). *P < .05 vs WT.View Large Image Figure ViewerDownload (PPT)ResultsConfirming previous results,18Alberici L.C. Oliveira H.C. Bighetti E.J. de Faria E.C. Degaspari G.R. Souza C.T. Vercesi A.E. Hypertriglyceridemia increases mitochondrial resting respiration and susceptibility to permeability transition.J Bioenerg Biomembr. 2003; 35: 451-457Crossref PubMed Scopus (23) Google Scholar we found that mitochondria isolated from livers of HTG mice present higher o2 consumption rates than WT mitochondria when incubated under basal conditions in which no oxidative phosphorylation occurs (see representative traces in Figure 2A and averages in Figure 2B). In our prior study,18Alberici L.C. Oliveira H.C. Bighetti E.J. de Faria E.C. Degaspari G.R. Souza C.T. Vercesi A.E. Hypertriglyceridemia increases mitochondrial resting respiration and susceptibility to permeability transition.J Bioenerg Biomembr. 2003; 35: 451-457Crossref PubMed Scopus (23) Google Scholar no increases in respiratory rates under state III conditions, in which oxidative phosphorylation is stimulated, were observed, indicating these changes are not due to higher maximal respiratory capacity. Instead, we found here that enhanced respiratory rates of HTG mitochondria were significantly prevented by the presence of ATP (Figure 2). Furthermore, the effects of ATP were reversed by the mitoKATP agonist diazoxide (DZX),27Garlid K.D. Paucek P. Yarov-Yarovoy V. Sun X. Schindler P.A. The mitochondrial KATP channel as a receptor for potassium channel openers.J Biol Chem. 1996; 271: 8796-8799Crossref PubMed Scopus (385) Google Scholar strongly suggesting that the increase in respiration is due to K+ cycling stimulated by the activity of this channel. Indeed, the DZX effect was completely abrogated by the mitoKATP antagonist 5-hydroxydecanoate (5-HD),28Jaburek M. Yarov-Yarovoy V. Paucek P. Garlid K.D. State-dependent inhibition of the mitochondrial KATP channel by glyburide and 5-hydroxydecanoate.J Biol Chem. 1998; 273: 13578-13582Abstract Full Text Full Text PDF PubMed Google Scholar and no differences in respiratory rates could be observed in media devoid of K+ salts. These results indicate that the increased respiratory rates observed in HTG mitochondria are due to the activity of mitoKATP.To directly assess if ATP-sensitive K+ transport was enhanced in HTG compared to WT mitochondria, we measured light scattering changes in these suspensions. Mitochondria lose K+ during isolation, and the uptake of this ion during the first few seconds of incubation in K+-rich media is enhanced by mitoKATP activity. Because K+ uptake is accompanied by phosphate (as a counter-ion) and water, mitochondrial matrix swelling occurs, with concomitant decreases in light scattering of the suspension.21Beavis A.D. Lu Y. Garlid K.D. On the regulation of K+ uniport in intact mitochondria by adenine nucleotides and nucleotide analogs.J Biol Chem. 1993; 268: 997-1004Abstract Full Text PDF PubMed Google Scholar, 22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google Scholar Swelling experiments are an important complement to the respiratory rate measurements conducted earlier, because DZX is known to have protonophoric effects and leads to respiratory inhibition in mitochondria when used at toxic concentrations.22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google Scholar Because the inner membrane potential is a driving force for K+ uptake,19Garlid K.D. Paucek P. Mitochondrial potassium transport: the K+ cycle.Biochim Biophys Acta. 2003; 1606: 23-41Crossref PubMed Scopus (300) Google Scholar both respiratory inhibition and protonophoric activity decrease mitochondrial swelling, while mitoKATP activation promoted by low doses of DZX enhances swelling.22Kowaltowski A.J. Seetharaman S. Paucek P. Garlid K.D. Bioenergetic consequences of opening the ATP-sensitive K+ channel of heart mitochondria.Am J Physiol Heart Circ Physiol. 2001; 280: H649-H657PubMed Google ScholarWe found that swelling in WT mitochondria was poorly inhibited by ATP, indicating low levels of mitoKATP activity (see Figure 3A for typical traces and Figure 3B for average swelling rates). On the other hand, swelling rates in HTG mitochondria were significantly larger and prevented by ATP. This difference in swelling rates was only noted in media containing K+ ions. Indeed, as expected for mitoKATP-mediated swelling, DZX reversed the ATP effect in a manner prevented by mitoKATP antagonists glibenclamide (glyburide [GLY]) and 5-HD. Based on these findings, we conclude that mitoKATP activity is enhanced in the livers of HTG mice.Next, we assessed if larger quantities of mitoKATP were present in HTG mitochondria compared with WT. Because the molecular identity of mitoKATP is a matter of debate, we compared levels of bound fluorescent GLY (BODIPY FL GLY; Molecular Probes, Eugene, OR) in isolated mitochondrial preparations. This measurement estimates the content of mitochondrial sulfonylurea receptors, a component of mitoKATP.29Bajgar R. Seetharaman S. Kowaltowski A.J. Garlid K.D. Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain.J Biol Chem. 2001; 276: 33369-33374Crossref PubMed Scopus (257) Google Scholar We found that the fluorescent GLY binding to HTG mitochondria was significantly enhanced (110.4% ± 2.4% of WT; P < .05), confirming that liver content of mitochondrial sulfonylurea receptors is higher in HTG mice.The activation of mitoKATP in HTG mice could be a consequence of higher circulating or intracellular lipids. We showed previously18Alberici L.C. Oliveira H.C. Bighetti E.J. de Faria E.C. Degaspari G.R. Souza C.T. Vercesi A.E. Hypertriglyceridemia increases mitochond
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