Available online at www.sciencedirect.com BIOCHIMICA ET BIOPHYSICA ACT SCIENCE )DIRECT BBA ELSEVIER Biochimica et Biophysica Acta 1639(2003)113-120 www.bba-direct.com Diabetes induces metabolic adaptations in rat liver mitochondria: role of coenzyme Q and cardiolipin contents Fernanda M.Ferreira",Raquel Seica,Paulo J.Oliveira",Pedro M.Coxito,Antonio J.Moreno", Carlos M.Palmeira,Maria S.Santos3.* Department of Zoology.University of Coimbra,Center for Neuroscience and Cell Biology of Coimbra.3004-517 Coimbra.Portugal Faculty of Medicine.University of Coimbra.Center for Neuroscience and Cell Biology of Coimbra.3004-517 Coimbra.Portugal Received 6 January 2003;received in revised form 29 July 2003;accepted 12 August 2003 Abstract Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats.However,results are sometimes controversial,since experimental conditions diverge,including age and strain of used animals.The purpose of this study was to evaluate the metabolic modifications in liver mitochondria,both in the presence of severe(STZ-treated rats)and mild hyperglycaemia [Goto-Kakizaki (GK)rats],when compared with control animals of similar age.Moreover,metabolic alterations were evaluated also at initial and advanced stages of the disease We observed that both models of diabetes(type I and type 2)presented a decreased susceptibility of liver mitochondria to the induction of permeability transition(MPT).Apparently,there is a positive correlation between the severity of diabetes mellitus(and duration of the disease) and the decline in the susceptibility to MPT induction.We also found that liver mitochondria isolated from diabetic rats presented some metabolic adaptations,such as an increase in coenzyme Q and cardiolipin contents,that can be responsible for the observed decrease in the susceptibility to multiprotein pore(MPTP)opening. 2003 Published by Elsevier B.V. Keywords:Diabetes mellitus;Type 1 diabetes:Type 2 diabetes;Goto-Kakizaki (GK)rat;Streptozotocin-induced diabetic (STZ)rat;Mitochondrial permeability transition (MPT) 1.Introduction Another important role for mitochondria is the calcium buffering capacity,avoiding excessive cytosolic calcium In higher animals,mitochondria play an essential role in accumulation.Mitochondrial calcium uptake also controls cellular energy metabolism since most of the ATP is pro- the activity of Ca2+-sensitive dehydrogenases and other duced by mitochondrial oxidative phosphorylation.There- metabolic processes.In the presence of high extemal Ca2+ fore,it is essential to have mitochondria intimately involved concentrations,isolated mitochondria can easily undergo an in metabolic regulation and maintenance of ATP production increased permeability to solutes with molecular masses up efficiency [1,2]. to 1500 kDa.This event is called the mitochondrial perme- The inner mitochondrial membrane(IMM)possesses an ability transition (MPT)and is caused by the opening of intrinsically low permeability to ions and solutes,allowing multiprotein pore(MPTP)formed by several proteins of the energy conservation in the form of a proton electrochemical outer and IMM,including the voltage-dependent anion potential difference.The IMM also possesses a set of channel (VDAC),the adenine nucleotide translocator channels and transporters that regulate ion fluxes across (ANT)and the matrix protein cyclophilin [3,4].MPTP the membrane since this management is essential for both opening can be induced by increasing amounts of Ca2+ metabolic regulation and energy conservation. and oxidative stress. Previous studies have focused in abnormalities in the MPT during diabetes [5,6],as well as changes in mitochon- Corresponding author.Tel:+351-239-834729;fax:+351-239. drial bioenergetics and antioxidant defences [7-9]. 826798. Diabetes mellitus is a common degenerative disease and E-mail address:mssantos @ci.uc.pt (M.S.Santos) one of the leading causes of morbidity and mortality in 0925-4439/S-see front matter 2003 Published by Elsevier B.V. doi:10.1016 i.bbadis.2003.08.001
Diabetes induces metabolic adaptations in rat liver mitochondria: role of coenzyme Q and cardiolipin contents Fernanda M. Ferreiraa , Raquel Seicab , Paulo J. Oliveiraa , Pedro M. Coxitoa , Anto´nio J. Morenoa , Carlos M. Palmeiraa , Maria S. Santos a,* a Department of Zoology, University of Coimbra, Center for Neuroscience and Cell Biology of Coimbra, 3004-517 Coimbra, Portugal bFaculty of Medicine, University of Coimbra, Center for Neuroscience and Cell Biology of Coimbra, 3004-517 Coimbra, Portugal Received 6 January 2003; received in revised form 29 July 2003; accepted 12 August 2003 Abstract Several studies have been carried out to evaluate the alterations in mitochondrial functions of diabetic rats. However, results are sometimes controversial, since experimental conditions diverge, including age and strain of used animals. The purpose of this study was to evaluate the metabolic modifications in liver mitochondria, both in the presence of severe (STZ-treated rats) and mild hyperglycaemia [Goto –Kakizaki (GK) rats], when compared with control animals of similar age. Moreover, metabolic alterations were evaluated also at initial and advanced stages of the disease. We observed that both models of diabetes (type 1 and type 2) presented a decreased susceptibility of liver mitochondria to the induction of permeability transition (MPT). Apparently, there is a positive correlation between the severity of diabetes mellitus (and duration of the disease) and the decline in the susceptibility to MPT induction. We also found that liver mitochondria isolated from diabetic rats presented some metabolic adaptations, such as an increase in coenzyme Q and cardiolipin contents, that can be responsible for the observed decrease in the susceptibility to multiprotein pore (MPTP) opening. D 2003 Published by Elsevier B.V. Keywords: Diabetes mellitus; Type 1 diabetes; Type 2 diabetes; Goto –Kakizaki (GK) rat; Streptozotocin-induced diabetic (STZ) rat; Mitochondrial permeability transition (MPT) 1. Introduction In higher animals, mitochondria play an essential role in cellular energy metabolism since most of the ATP is produced by mitochondrial oxidative phosphorylation. Therefore, it is essential to have mitochondria intimately involved in metabolic regulation and maintenance of ATP production efficiency [1,2]. The inner mitochondrial membrane (IMM) possesses an intrinsically low permeability to ions and solutes, allowing energy conservation in the form of a proton electrochemical potential difference. The IMM also possesses a set of channels and transporters that regulate ion fluxes across the membrane since this management is essential for both metabolic regulation and energy conservation. Another important role for mitochondria is the calcium buffering capacity, avoiding excessive cytosolic calcium accumulation. Mitochondrial calcium uptake also controls the activity of Ca2 +-sensitive dehydrogenases and other metabolic processes. In the presence of high external Ca2 + concentrations, isolated mitochondria can easily undergo an increased permeability to solutes with molecular masses up to 1500 kDa. This event is called the mitochondrial permeability transition (MPT) and is caused by the opening of multiprotein pore (MPTP) formed by several proteins of the outer and IMM, including the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT) and the matrix protein cyclophilin [3,4]. MPTP opening can be induced by increasing amounts of Ca2 + and oxidative stress. Previous studies have focused in abnormalities in the MPT during diabetes [5,6], as well as changes in mitochondrial bioenergetics and antioxidant defences [7 –9]. Diabetes mellitus is a common degenerative disease and one of the leading causes of morbidity and mortality in 0925-4439/$ - see front matter D 2003 Published by Elsevier B.V. doi:10.1016/j.bbadis.2003.08.001 * Corresponding author. Tel.: +351-239-834729; fax: +351-239- 826798. E-mail address: mssantos@ci.uc.pt (M.S. Santos). www.bba-direct.com Biochimica et Biophysica Acta 1639 (2003) 113 – 120
114 F.M.Ferreira et al.Biochimica et Biophysica Acta 1639 (2003)113-120 developed countries.Clinically,diabetes mellitus is a het- Animals were kept under controlled light(12-h day/night erogeneous disease,with a common phenotype of impaired cycle),temperature (22-24 C)and humidity (50-60%) glucose tolerance and,depending on the basis of the man- conditions and with free access to powdered rodent chow agement required to control glucose homeostasis,it can be (diet URF1,Charles Rivers,France)and water (pH 5.5), divided into type 1 and type 2 diabetes [10-12]. except during the fasting periods.In this study,the "Princi- With the purpose of understanding the physiological and ples of Laboratory Animal Care"(NIH publication 83-25, pathological changes in this complex disease,animal models revised 1985)were followed.During this period glycaemia for diabetes are important research tools since they allow for was determined from the tail vein using a commercial a tight control over experimental conditions,almost impos- glucometer(Glucometer-Elite,Bayer). sible to achieve in human populations [13,14].In the present study,two animal models for diabetes were used in order to 2.3.Induction and characterization of STZ-induced study abnormalities in liver MPT.STZ-induced diabetic rats, diabetes commonly used as an animal model for type 1 diabetes mellitus,were obtained after selective destruction of B-cells Male Wistar rats weighing about 200 g (10 weeks)were by streptozotocin (STZ),a broad-spectrum antibiotic with randomly divided into two groups of 10 animals each.In diabetogenic effects.STZ-injected rats present many char- order to induce diabetes,one group was injected intraper- acteristics seen in insulin-dependent diabetic human patients: itoneally with a single injection of streptozotocin (STZ,50 hypoinsulinemia,hyperglycaemia,ketonuria and hyperlipi- mg/kg),after a 16-h fasting period.The volume used was daemia [13,15].Goto-Kakizaki(GK)rats are currently used always 0.5 ml/200 g body weight.Control animals were as a non-obese animal model of type 2 diabetes mellitus [16], injected with the same volume of citrate solution.In the obtained by selective breeding of normal Wistar rats,using following 24 h,animals were orally feed with glycosilated glucose intolerance as selection index [17-191. serum in order to avoid hypoglycaemia resulting from In the present study we investigated the impact of diabetes massive destruction of B-cells and release of intracellular mellitus on mitochondrial calcium fluxes and on the MPT insulin associated with STZ treatment [13].Animals were susceptibility in the presence of Ca2/P both in STZ and GK kept 3 or 9 weeks before the experiments.In this study,in rats.The study was conducted during the progression of order to avoid another variability factor than diabetes,we diabetes,with the purpose of assessing the mitochondrial used two groups of animals with similar age:13 weeks ofage metabolic modifications in liver mitochondria.As much as in one group (STZ-treated rats with 3 weeks after treatment, we know,no reports were available concerning mitochon- GK rats 13 weeks of age and control rats with similar age, drial alterations in calcium handling during the progression Wistar rats with 3 weeks after injection with vehicle and/or of the disease in the two described models.Our results 13 weeks of age)and 19-20 weeks of age in the other group showed that in liver mitochondria diabetes decreased the (STZ-treated rats with 9 weeks after treatment,GK rats 20 susceptibility to the induction of MPT. weeks of age and control group with similar age,Wistar with 9 week after injection with vehicle and/or 20 weeks of age. Our results show that intravenous administration of citrate 2.Materials and methods does not modify the parameters studied. During this period,weight was measured and glycaemia 2.1.Materials was determined from the tail vein as described before.Values were taken in fasting conditions just before STZ administra- Streptozotocin [2-deoxy-2-(3-methyl-3-nitrosurea)1-D- tion and in non-fasting conditions in the weeks after.If glucopyranose]was obtained from Sigma Chemical Co. feeding blood glucose in the tail vein exceeded 250 mg/dl. (St.Louis,MO,USA),and prepared prior to use in 100 animals were used as diabetic. mM citrate,pH 4.5.Calcium Green 5-N was obtained from Molecular Probes (Eugene,OR,USA).All other reagents 2.4.Glycaemia and HbAic evaluation and chemicals used were of the highest grade of purity commercially available. Blood glucose concentration was determined immediate- ly after animal sacrifice (Glucometer-Elite,Bayer).The 2.2.Animals glycated hemoglobin (HbAic)values were determined in blood collected at the time of animals'death through ionic Spontaneously diabetic male GK rats,13 or 20 weeks of exchange chromatography (Abbott Imx Glicohemoglobin, age,were obtained from our local breeding colony (Animal Abbott Laboratories,Portugal). Research Center Laboratory,University Hospitals,Coim- bra),established in 1995 with breeding couples from the 2.5.Preparation of liver mitochondria colony at the Tohoku University School of Medicine(Sen- dai,Japan;courtesy of Dr.K.Susuki).Control animals were Mitochondria were isolated from the livers of normal and non-diabetic male Wistar rats of similar age. diabetic rats (maintained ad libitum for at least 12 h before
developed countries. Clinically, diabetes mellitus is a heterogeneous disease, with a common phenotype of impaired glucose tolerance and, depending on the basis of the management required to control glucose homeostasis, it can be divided into type 1 and type 2 diabetes [10 –12]. With the purpose of understanding the physiological and pathological changes in this complex disease, animal models for diabetes are important research tools since they allow for a tight control over experimental conditions, almost impossible to achieve in human populations [13,14]. In the present study, two animal models for diabetes were used in order to study abnormalities in liver MPT. STZ-induced diabetic rats, commonly used as an animal model for type 1 diabetes mellitus, were obtained after selective destruction of h-cells by streptozotocin (STZ), a broad-spectrum antibiotic with diabetogenic effects. STZ-injected rats present many characteristics seen in insulin-dependent diabetic human patients: hypoinsulinemia, hyperglycaemia, ketonuria and hyperlipidaemia [13,15]. Goto –Kakizaki (GK) rats are currently used as a non-obese animal model of type 2 diabetes mellitus [16], obtained by selective breeding of normal Wistar rats, using glucose intolerance as selection index [17 – 19]. In the present study we investigated the impact of diabetes mellitus on mitochondrial calcium fluxes and on the MPT susceptibility in the presence of Ca2 +/Pi both in STZ and GK rats. The study was conducted during the progression of diabetes, with the purpose of assessing the mitochondrial metabolic modifications in liver mitochondria. As much as we know, no reports were available concerning mitochondrial alterations in calcium handling during the progression of the disease in the two described models. Our results showed that in liver mitochondria diabetes decreased the susceptibility to the induction of MPT. 2. Materials and methods 2.1. Materials Streptozotocin [2-deoxy-2-(3-methyl-3-nitrosurea) 1-Dglucopyranose] was obtained from Sigma Chemical Co. (St. Louis, MO, USA), and prepared prior to use in 100 mM citrate, pH 4.5. Calcium Green 5-N was obtained from Molecular Probes (Eugene, OR, USA). All other reagents and chemicals used were of the highest grade of purity commercially available. 2.2. Animals Spontaneously diabetic male GK rats, 13 or 20 weeks of age, were obtained from our local breeding colony (Animal Research Center Laboratory, University Hospitals, Coimbra), established in 1995 with breeding couples from the colony at the Tohoku University School of Medicine (Sendai, Japan; courtesy of Dr. K. Susuki). Control animals were non-diabetic male Wistar rats of similar age. Animals were kept under controlled light (12-h day/night cycle), temperature (22 – 24 jC) and humidity (50 –60%) conditions and with free access to powdered rodent chow (diet URF1, Charles Rivers, France) and water (pH 5.5), except during the fasting periods. In this study, the ‘‘Principles of Laboratory Animal Care’’ (NIH publication 83-25, revised 1985) were followed. During this period glycaemia was determined from the tail vein using a commercial glucometer (Glucometer-Elite, Bayer). 2.3. Induction and characterization of STZ-induced diabetes Male Wistar rats weighing about 200 g (10 weeks) were randomly divided into two groups of 10 animals each. In order to induce diabetes, one group was injected intraperitoneally with a single injection of streptozotocin (STZ, 50 mg/kg), after a 16-h fasting period. The volume used was always 0.5 ml/200 g body weight. Control animals were injected with the same volume of citrate solution. In the following 24 h, animals were orally feed with glycosilated serum in order to avoid hypoglycaemia resulting from massive destruction of ß-cells and release of intracellular insulin associated with STZ treatment [13]. Animals were kept 3 or 9 weeks before the experiments. In this study, in order to avoid another variability factor than diabetes, we used two groups of animals with similar age: 13 weeks of age in one group (STZ-treated rats with 3 weeks after treatment, GK rats 13 weeks of age and control rats with similar age, Wistar rats with 3 weeks after injection with vehicle and/or 13 weeks of age) and 19– 20 weeks of age in the other group (STZ-treated rats with 9 weeks after treatment, GK rats 20 weeks of age and control group with similar age, Wistar with 9 week after injection with vehicle and/or 20 weeks of age. Our results show that intravenous administration of citrate does not modify the parameters studied. During this period, weight was measured and glycaemia was determined from the tail vein as described before. Values were taken in fasting conditions just before STZ administration and in non-fasting conditions in the weeks after. If feeding blood glucose in the tail vein exceeded 250 mg/dl, animals were used as diabetic. 2.4. Glycaemia and HbA1C evaluation Blood glucose concentration was determined immediately after animal sacrifice (Glucometer-Elite, Bayer). The glycated hemoglobin (HbA1C) values were determined in blood collected at the time of animals’ death through ionic exchange chromatography (Abbott Imx Glicohemoglobin, Abbott Laboratories, Portugal). 2.5. Preparation of liver mitochondria Mitochondria were isolated from the livers of normal and diabetic rats (maintained ad libitum for at least 12 h before 114 F.M. Ferreira et al. / Biochimica et Biophysica Acta 1639 (2003) 113–120
FM.Ferreira et al.Biochimica et Biophysica Acta 1639 (2003)113-120 115 being sacrificed),according to a previously established units of fluorescence (AFU),determined 100 and 210 s method [201,with slight modifications. after the addition of Ca2+. Homogenization medium contained 210 mM mannitol. 70 mM sucrose,5 mM HEPES (pH 7.4),0.2 mM EGTA,0.1 2.8.Extraction and quantification of coenzyme O mM EDTA and 0.1%defatted bovine serum albumin(BSA). EDTA,EGTA and defatted BSA were omitted from the final Aliquots of mitochondria containing 2 mg protein/ml washing medium and adjusted to pH 7.2.The mitochondrial were extracted according to the method described previously pellet was washed twice,suspended in the washing medium [24].The extract was evaporated to dryness under a stream of and immediately used.Protein was determined by the biuret N2,and stored at -80 C,for HPLC analysis.Liquid method,using BSA as a standard [21]. chromatography was performed using a Gilson HPLC appa- ratus with a reverse phase column(RP18;Spherisorb;S5 2.6.Membrane potential(☑Ψ)measurements ODS2),as described by Chung et al.[25].Samples were eluted from the column with methanol/heptane(10:2 v/v)at a The mitochondrial transmembrane potential was estimat- flow rate of 2 ml/min.Detection was performed using a UV ed by calculating transmembrane distribution of tetraphe- detector at 269 nm.Identification and quantification were nylphosphonium ion (TPP)with a TPP*-selective based on pure standards by their retention times and peak electrode,prepared as previously reported [22],using a areas,respectively.The levels of coenzyme Q(CoQo and calomel electrode as a reference.TPP*uptake was mea- CoQio)were expressed in pmol/mg protein. sured from the decrease in TPP*concentration in the medium.The potential difference between the selective 2.9.Quantification of cardiolipin content and the reference electrodes was measured with an elec- trometer and continuously recorded.A matrix mitochondri- Mitochondrial cardiolipin content was quantified using al volume of 1.1 ul/mg was assumed and valinomycin was an established spectrophotometric assay using 10-nonyl- used to calibrate the baseline.Reactions were carried out at acridine orange (NAO)[26].Briefly,rat liver mitochondria 25 C in 1 ml of the reaction media(200 mM sucrose,10 (0.25 mg protein/ml)were suspended in 210 mM mannitol, mM Tris-CI,10 uM EGTA,1 mM KH2PO4,pH 7.2), 70 mM sucrose,5 mM HEPES (pH 7.2).Aliquots of this supplemented with 3 uM TPP,2 uM rotenone and 0.1 ug suspension (150 ul)were added to increasing amounts of oligomycin,1 mg mitochondria and 5 mM succinate.The NAO(1-25 uM)and the final volume adjusted to 1.5 ml calcium accumulation capacity was determined by adding with the suspension buffer.The samples were incubated for small pulses of CaCl2 (10 nmol/mg protein each);these 5 min at room temperature and then centrifuged at small amounts of calcium were added until the opening of 35,000 xg for 5 min,using a Beckman ultracentrifuge MPTP was observed (as an irreversible drop in A). (model TL-100 and TL-100 rotor).The pellets were dis- carded and the amount of unbound NAO in the supernatant 2.7.Measurement of mitochondrial Ca fluxes was measured spectrophotometrically at 495 nm.A standard curve was generated using NAO(1-25 uM)in the absence The uptake and following release of Ca2 by isolated of mitochondria.The number of moles of NAO per milli- mitochondria was evaluated with the hexapotassium salt of gram of protein was calculated by subtracting the sample the calcium-sensitive fluorescent probe Calcium Green 5-N, absorbance (unbound NAO)from the value of absorbance according to Oliveira et al.[23].The reactions were carried correspondent in standard curve (full amount of NAO)[27]. out in 2 ml of reaction medium(200 mM sucrose,10 mM Cardiolipin content was calculated as the half of this value Tris-CI,10 uM EGTA,1 mM KH2PO4;pH 7.2),supple- due to the 2:1 stoichiometric relationship between NAO and mented with 0.6 mg liver mitochondria,2 uM rotenone,0.1 cardiolipin [27]. ug oligomycin,5 mM succinate and 100 nM Calcium Green 5-N,and stirred continuously in a water-jacketed 2.10.Citrate synthase assay cuvette holder at 25 C.Fluorescence was continuously monitored with a Perkin-Elmer LS-50B spectrofluorometer Citrate synthase was determined according to Coore et al. (excitation 506 nm and emission 531 nm),for 50 s,prior to [28].Briefly,freeze-thawed liver mitochondria(100 ug) the addition of Ca2+(33 nmol/mg protein).Excess EGTA were incubated with 1 ml of Tris-based media [100 mM was used to stop the reaction and to obtain the baseline.As Tris.200 uM Acetyl-CoA and 200 uM DTNB(5.5'-dithio- the addition of Ca2+produced a different peak in control bis-2-nitrobenzoic acid)].The absorbance of the suspension and diabetic rats (probably,due to different cardiolipin was continuously measured at 412 nm under stirring and at contents in mitochondrial inner membrane).the data were 25 C.After a basal line setting,100 uM oxaloacetate was 'corrected'based on the ratio of these peaks [(1/mean value added.In this protocol,we measured the formation rate of a of Wistar peaks)x 100];this correction was performed to coloured product resulting from the condensation of DTNB each different pair of mitochondrial preparations (diabetic and coenzyme A.Our results did not show any significant and control).Calcium fluxes were expressed as arbitrary difference between citrate synthase contents,thus pointing
being sacrificed), according to a previously established method [20], with slight modifications. Homogenization medium contained 210 mM mannitol, 70 mM sucrose, 5 mM HEPES (pH 7.4), 0.2 mM EGTA, 0.1 mM EDTA and 0.1% defatted bovine serum albumin (BSA). EDTA, EGTA and defatted BSA were omitted from the final washing medium and adjusted to pH 7.2. The mitochondrial pellet was washed twice, suspended in the washing medium and immediately used. Protein was determined by the biuret method, using BSA as a standard [21]. 2.6. Membrane potential (DW) measurements The mitochondrial transmembrane potential was estimated by calculating transmembrane distribution of tetraphenylphosphonium ion (TPP+ ) with a TPP+ -selective electrode, prepared as previously reported [22], using a calomel electrode as a reference. TPP+ uptake was measured from the decrease in TPP+ concentration in the medium. The potential difference between the selective and the reference electrodes was measured with an electrometer and continuously recorded. A matrix mitochondrial volume of 1.1 Al/mg was assumed and valinomycin was used to calibrate the baseline. Reactions were carried out at 25 jC in 1 ml of the reaction media (200 mM sucrose, 10 mM Tris –Cl, 10 AM EGTA, 1 mM KH2PO4, pH 7.2), supplemented with 3 AM TPP+ , 2 AM rotenone and 0.1 Ag oligomycin, 1 mg mitochondria and 5 mM succinate. The calcium accumulation capacity was determined by adding small pulses of CaCl2 (10 nmol/mg protein each); these small amounts of calcium were added until the opening of MPTP was observed (as an irreversible drop in DWm). 2.7. Measurement of mitochondrial Ca2+ fluxes The uptake and following release of Ca2 + by isolated mitochondria was evaluated with the hexapotassium salt of the calcium-sensitive fluorescent probe Calcium Green 5-N, according to Oliveira et al. [23]. The reactions were carried out in 2 ml of reaction medium (200 mM sucrose, 10 mM Tris –Cl, 10 AM EGTA, 1 mM KH2PO4; pH 7.2), supplemented with 0.6 mg liver mitochondria, 2 AM rotenone, 0.1 Ag oligomycin, 5 mM succinate and 100 nM Calcium Green 5-N, and stirred continuously in a water-jacketed cuvette holder at 25 jC. Fluorescence was continuously monitored with a Perkin-Elmer LS-50B spectrofluorometer (excitation 506 nm and emission 531 nm), for 50 s, prior to the addition of Ca2 + (33 nmol/mg protein). Excess EGTA was used to stop the reaction and to obtain the baseline. As the addition of Ca2 + produced a different peak in control and diabetic rats (probably, due to different cardiolipin contents in mitochondrial inner membrane), the data were ‘corrected’ based on the ratio of these peaks [(1/mean value of Wistar peaks) 100]; this correction was performed to each different pair of mitochondrial preparations (diabetic and control). Calcium fluxes were expressed as arbitrary units of fluorescence (AFU), determined 100 and 210 s after the addition of Ca2 +. 2.8. Extraction and quantification of coenzyme Q Aliquots of mitochondria containing 2 mg protein/ml were extracted according to the method described previously [24]. The extract was evaporated to dryness under a stream of N2, and stored at 80 jC, for HPLC analysis. Liquid chromatography was performed using a Gilson HPLC apparatus with a reverse phase column (RP18; Spherisorb; S5 ODS2), as described by Chung et al. [25]. Samples were eluted from the column with methanol/heptane (10:2 v/v) at a flow rate of 2 ml/min. Detection was performed using a UV detector at 269 nm. Identification and quantification were based on pure standards by their retention times and peak areas, respectively. The levels of coenzyme Q (CoQ9 and CoQ10) were expressed in pmol/mg protein. 2.9. Quantification of cardiolipin content Mitochondrial cardiolipin content was quantified using an established spectrophotometric assay using 10-nonylacridine orange (NAO) [26]. Briefly, rat liver mitochondria (0.25 mg protein/ml) were suspended in 210 mM mannitol, 70 mM sucrose, 5 mM HEPES (pH 7.2). Aliquots of this suspension (150 Al) were added to increasing amounts of NAO (1– 25 AM) and the final volume adjusted to 1.5 ml with the suspension buffer. The samples were incubated for 5 min at room temperature and then centrifuged at 35,000 g for 5 min, using a Beckman ultracentrifuge (model TL-100 and TL-100 rotor). The pellets were discarded and the amount of unbound NAO in the supernatant was measured spectrophotometrically at 495 nm. A standard curve was generated using NAO (1– 25 AM) in the absence of mitochondria. The number of moles of NAO per milligram of protein was calculated by subtracting the sample absorbance (unbound NAO) from the value of absorbance correspondent in standard curve (full amount of NAO) [27]. Cardiolipin content was calculated as the half of this value due to the 2:1 stoichiometric relationship between NAO and cardiolipin [27]. 2.10. Citrate synthase assay Citrate synthase was determined according to Coore et al. [28]. Briefly, freeze-thawed liver mitochondria (100 Ag) were incubated with 1 ml of Tris-based media [100 mM Tris, 200 AM Acetyl-CoA and 200 AM DTNB (5,5V-dithiobis-2-nitrobenzoic acid)]. The absorbance of the suspension was continuously measured at 412 nm under stirring and at 25 jC. After a basal line setting, 100 AM oxaloacetate was added. In this protocol, we measured the formation rate of a coloured product resulting from the condensation of DTNB and coenzyme A. Our results did not show any significant difference between citrate synthase contents, thus pointing F.M. Ferreira et al. / Biochimica et Biophysica Acta 1639 (2003) 113–120 115
116 FM.Ferreira et al.Biochimica et Biophysica Acta 1639 (2003)113-120 toward no significant differences in the number of mitochon- -220 10 nmol Ca2 10 nmol Ca+ dria per milligram of protein in control and diabetic (GK and -215 STZ☑rats. 210 ■AΨ 2.11.Statistic analysis of data E -200 after 3 ulses of The results are presented as mean+S.E.of the number a2 of experiments indicated and statistical significance between diabetic rats and their control group was determined using .175■ Number of unpaired Student's t test.Multiple comparison was per- pulses of Ca2+ formed using one-way ANOVA,with the Student-New- man-Keuls as a post-test.P380 mitochondrial preparations. mg/dl).In order to estimate the severity of diabetes,glycated hemoglobin (HbAic)was also evaluated since HbAie is a the effect of known amounts of calcium ('pulses'of 10 very useful parameter to evaluate the severity of diabetes, nmol CaCl2 each)in the mitochondrial A(Fig.I and indicating the average blood glucose levels presented 2-3 Table 2).Mitochondria possess a finite capacity for accu- months prior to the analysis.The determined levels of HbAie mulating calcium before undergoing the calcium-dependent (Table 1)confirmed that blood glucose levels were signifi- MPT.Our results showed that liver mitochondria from cantly increased in diabetic rats and these levels in STZ- diabetic rats were able to accumulate a higher amount of treated rats were also significantly augmented as compared added Ca2.Therefore,liver mitochondria isolated from to GK rats. diabetic rats could handle a larger number of pulses of calcium (5 to 11 pulses of 10 nmol Ca2/mg protein) 3.2.Calcium accumulation capacity compared to Wistar rats(about 4 pulses of 10 nmol Ca2+/ mg protein;see Fig.1 for typical experiment)before the Calcium accumulation capacity of liver mitochondria irreversible drop in Ay,compatible with the MPTP open- from diabetic and control rats was determined observing ing.This decline in Ay was abolished in the presence of Table 1 Characterization of animals Condition 3 weeks after treatment 9 weeks after treatment 13 weeks of age 20 weeks of age Wistar STZ Wistar STZ Wistar GK Wistar GK Glycaemia 115.1±4.5 409.3±17.9 97.3±5.5 468.6±15.6 101.3±5.0 141.2±9.4 85.7±5.0 162.0±28.6 (mg/dl) (n=6 (初=6**本，战&& (n=6 (n=6***,&& (n=8) (行=8)*，#，& (n=9) (仿=9)*，w HbAIc 5.23+0.21 10.25±0.43 5.84±0.47 11.71±0.39 5.04±0.12 7.32±0.67 5.43±0.33 8.46±0.89 (n=6 (1=6)**,机&& (n=6) (1=6**,是& (n=6) (H=6)*, (n=6) (行=6)*率，#特 Data are mean+S.E.obtained from the number of samples indicated,each obtained from a different animal.Glycaemia and HbAic were determined as described in Materials and methods. P<0.05 as compared to controls. **P<0.01 as compared to controls. *P<0.001 as compared to controls. P<0.05 compared with STZ (9 weeks after treatment) P<0.01 compared with STZ (9 weeks after treatment). P<0001 compared with STZ(9 weeks after treatment) p<0.05 compared with GK(20 weeks of age). P<0.01 compared with GK (20 weeks of age)
toward no significant differences in the number of mitochondria per milligram of protein in control and diabetic (GK and STZ) rats. 2.11. Statistic analysis of data The results are presented as mean F S.E. of the number of experiments indicated and statistical significance between diabetic rats and their control group was determined using unpaired Student’s t test. Multiple comparison was performed using one-way ANOVA, with the Student –Newman –Keuls as a post-test. P 380 mg/dl). In order to estimate the severity of diabetes, glycated hemoglobin (HbA1c) was also evaluated since HbA1c is a very useful parameter to evaluate the severity of diabetes, indicating the average blood glucose levels presented 2 – 3 months prior to the analysis. The determined levels of HbA1c (Table 1) confirmed that blood glucose levels were significantly increased in diabetic rats and these levels in STZtreated rats were also significantly augmented as compared to GK rats. 3.2. Calcium accumulation capacity Calcium accumulation capacity of liver mitochondria from diabetic and control rats was determined observing the effect of known amounts of calcium (‘pulses’ of 10 nmol CaCl2 each) in the mitochondrial DW (Fig. 1 and Table 2). Mitochondria possess a finite capacity for accumulating calcium before undergoing the calcium-dependent MPT. Our results showed that liver mitochondria from diabetic rats were able to accumulate a higher amount of added Ca2 +. Therefore, liver mitochondria isolated from diabetic rats could handle a larger number of pulses of calcium (5 to 11 pulses of 10 nmol Ca2 +/mg protein) compared to Wistar rats (about 4 pulses of 10 nmol Ca2 +/ mg protein; see Fig. 1 for typical experiment) before the irreversible drop in DW, compatible with the MPTP opening. This decline in DW was abolished in the presence of Table 1 Characterization of animals Condition 3 weeks after treatment 9 weeks after treatment 13 weeks of age 20 weeks of age Wistar STZ Wistar STZ Wistar GK Wistar GK Glycaemia (mg/dl) 115.1 F 4.5 (n = 6) 409.3 F 17.9 (n = 6)***, #,&& 97.3 F 5.5 (n = 6) 468.6 F 15.6 (n = 6) ***, && 101.3 F 5.0 (n = 8) 141.2 F 9.4 (n = 8) *, ###, & 85.7 F 5.0 (n = 9) 162.0 F 28.6 (n = 9)*, ## HbA1C 5.23 F 0.21 (n = 6) 10.25 F 0.43 (n = 6)***, #, && 5.84 F 0.47 (n = 6) 11.71 F 0.39 (n = 6) ***, && 5.04 F 0.12 (n = 6) 7.32 F 0.67 (n = 6)*, ## 5.43 F 0.33 (n = 6) 8.46 F 0.89 (n = 6)**, ## Data are mean F S.E. obtained from the number of samples indicated, each obtained from a different animal. Glycaemia and HbA1C were determined as described in Materials and methods. * P < 0.05 as compared to controls. ** P < 0.01 as compared to controls. *** P < 0.001 as compared to controls. # P < 0.05 compared with STZ (9 weeks after treatment). ## P < 0.01 compared with STZ (9 weeks after treatment). ### P < 0.001 compared with STZ (9 weeks after treatment). & P < 0.05 compared with GK (20 weeks of age). && P < 0.01 compared with GK (20 weeks of age). Fig. 1. Calcium accumulation capacity of liver mitochondria isolated from Wistar control and diabetic rats: The Ca2 + accumulation capacity, measured with a TPP+ selective electrode, was determined by adding several pulses of CaCl2 (10 nmol/mg protein each), in order to induce MPT. The MPTP opening was assessed as a drop in membrane potential. In the presence of cyclosporin A, a specific inhibitor of MPT induction, the number of pulses of calcium needed to induce a similar decrease membrane potential is much higher (both for diabetic and control mitochondrial preparations, not shown). The traces are representative of experiments performed with different mitochondrial preparations. 116 F.M. Ferreira et al. / Biochimica et Biophysica Acta 1639 (2003) 113–120
EM.Ferreira et al./Biochimica et Biophysica Acta 1639 (2003)113-120 117 Table 2 with 13 weeks of age)compared to controls (Fig.2C). Effect of diabetes on Caaccumulation capacity Therefore,our results indicated that mitochondrial calcium Condition △里m(-mV)△里m(-mV) Pulses of Ca2+ (after 3 pulses of (mean value) 10 nmol Ca2*) Wistar Wistar 225.0±1.20 219.9±3.13 4.70±0.62 Diabetic (3 weeks (n=6 (m=6 (1=6# (STZ 9 weeks) after treatment) STZ 228.2±1.61 2233±0.65 100AF 5.50±0.29 (3 weeks (n=6 (n=6) (H=6)*,#脚 after treatment) Wistar 223.1±2.96 218.2±1.37 4.59±0.22 Ca (9 weeks (n=6) (n=6) (n=6) after treatment) Cyclosporin A STZ 230.6±2.46 227.3±2.74 (Wistar and STZ) 11.20±0.70 100 210 (9 weeks (H=6)* (n=6)* (切=6)** Time(s) after treatment) Wistar 350 220.6±2.00 215.8±2.95 3.62±0.19 (13 weeks (n=7) (n=8) (切=7) 300 of age) GK 225.0±1.12 222.2±1.37 5.56±0.45 (13 weeks (n=7)&d (们=7)*，特 250 (n=7) of age) 200 Wistar 222.6±2.45 212.5±2.55 4.00±0.22 (20 weeks (n=8) (n=8) (n=8)"# 150 of age) GK 235.8±1.99 228.0±1.86 5.62±0.28 twatein (20 weeks (1=8)** (n=8)*** (1=8)*,# 100 of age) ★★女 50 The mitochondrial electric potential measurements (AV)were performed with a TPP*selective electrode.AP was evaluated as described in Materials 0 and methods.Data are mean+S.E.of the number of independent Wistar GK Wistar GK Wistar STZ Wistar STZ 13 weeks 20 weeks 3 weeks 9 weeks experiments indicated,performed with at least three different mitochondrial preparations. C 400 *P<0.05 compared to controls. ***P<0.001 compared to controls. 350 P<0.01 compared to STZ(9 weeks after treatment). P<0.001 compared to STZ(9 weeks after treatment). 300 P<0.001 compared to GK (20 weeks of age). (000 250 200 cyclosporine A,a specific inhibitor of MPT (data not 150 shown). Previous results,showing the higher calcium capacity of 100 diabetic mitochondria.were confirmed by determinations of 50 extramitochondrial calcium movements,using the fluores- cent calcium-sensitive probe Calcium Green 5-N.The addi- 09 Wistar GK Wistar GK Wistar STZ Wistar STZ tion of Ca2+leads to an increase of the Ca2+content in the 13 weeks 20 weeks 3 weeks 9 weeks external medium,which is associated to the rapid increase in fluorescence (the first peak in Fig.2A).As the calcium is Fig.2.Mitochondrial Cafuxes in liver mitochondria isolated from diabetic and Wistar control rats:(A)Typical measurements of calcium accumulated by mitochondria,the fluorescence decreases. movements using the fluorescent calcium sensitive probe Calcium Green 5- The MPTP opening leads to the release of the previous N,as described in Materials and methods.The reactions were carried out in accumulated calcium and consequently to an increase in 2 ml of reaction medium supplemented with 0.6 mg protein,2 uM fluorescence.The release of calcium was completely abol- rotenone,0.1 uM oligomycin,5 mM succinate and 100 nM Calcium Green ished(both in Wistar and diabetic rat liver mitochondria)by 5-N.Fluorescence was monitored continuously for 50s,prior to the addition of 33 nmol Ca2/mg protein,and stopped with excess EGTA to obtain the cyclosporine A,indicating that the release was due to MPTP baseline.The calcium release was inhibited by 0.8 uM cyclosporin A, opening(Fig.2A).We observed that 100 s after the addition indicating that the observed release was due to MPTP opening.Calcium of calcium (33 nmol CaCl2/mg protein),the Ca2*loss by fluxes are expressed as arbitrary units of fluorescence (AFU),determined diabetic liver mitochondria was significantly lower when 100s(B)and 210s(C)after the addition of 33 nmol Ca2*/mg protein,as described in Materials and methods.Data are mean+S.E.of four compared to control Wistar rats(Fig.2B).Moreover,210 s independent experiments performed with different mitochondrial prepara- after the addition of Ca2,the release of calcium was tions.*P<0.05.*+P<0.01,***P<0.001.as compared to Wistar control significantly decreased in diabetic rats (except for GK rats rats
cyclosporine A, a specific inhibitor of MPT (data not shown). Previous results, showing the higher calcium capacity of diabetic mitochondria, were confirmed by determinations of extramitochondrial calcium movements, using the fluorescent calcium-sensitive probe Calcium Green 5-N. The addition of Ca2 + leads to an increase of the Ca2 + content in the external medium, which is associated to the rapid increase in fluorescence (the first peak in Fig. 2A). As the calcium is accumulated by mitochondria, the fluorescence decreases. The MPTP opening leads to the release of the previous accumulated calcium and consequently to an increase in fluorescence. The release of calcium was completely abolished (both in Wistar and diabetic rat liver mitochondria) by cyclosporine A, indicating that the release was due to MPTP opening (Fig. 2A). We observed that 100 s after the addition of calcium (33 nmol CaCl2/mg protein), the Ca2 + loss by diabetic liver mitochondria was significantly lower when compared to control Wistar rats (Fig. 2B). Moreover, 210 s after the addition of Ca2 +, the release of calcium was significantly decreased in diabetic rats (except for GK rats with 13 weeks of age) compared to controls (Fig. 2C). Therefore, our results indicated that mitochondrial calcium Table 2 Effect of diabetes on Ca2 + accumulation capacity Condition DWm ( mV) DWm ( mV) (after 3 pulses of 10 nmol Ca2 +) Pulses of Ca2 + (mean value) Wistar (3 weeks after treatment) 225.0 F 1.20 (n = 6) 219.9 F 3.13 (n = 6) 4.70 F 0.62 (n = 6)### STZ (3 weeks after treatment) 228.2 F 1.61 (n = 6) 223.3 F 0.65 (n = 6) 5.50 F 0.29 (n = 6)*, ### Wistar (9 weeks after treatment) 223.1 F 2.96 (n = 6) 218.2 F 1.37 (n = 6) 4.59 F 0.22 (n = 6) STZ (9 weeks after treatment) 230.6 F 2.46 (n = 6)* 227.3 F 2.74 (n = 6)* 11.20 F 0.70 (n = 6)*** Wistar (13 weeks of age) 220.6 F 2.00 (n = 7) 215.8 F 2.95 (n = 8) 3.62 F 0.19 (n = 7)### GK (13 weeks of age) 225.0 F 1.12 (n = 7)&&& 222.2 F 1.37 (n = 7) 5.56 F 0.45 (n = 7)*, ## Wistar (20 weeks of age) 222.6 F 2.45 (n = 8) 212.5 F 2.55 (n = 8) 4.00 F 0.22 (n = 8)### GK (20 weeks of age) 235.8 F 1.99 (n = 8)*** 228.0 F 1.86 (n = 8)*** 5.62 F 0.28 (n = 8)*, ### The mitochondrial electric potential measurements (DW) were performed with a TPP+ selective electrode. DW was evaluated as described in Materials and methods. Data are mean F S.E. of the number of independent experiments indicated, performed with at least three different mitochondrial preparations. * P < 0.05 compared to controls. *** P < 0.001 compared to controls. ## P < 0.01 compared to STZ (9 weeks after treatment). ### P < 0.001 compared to STZ (9 weeks after treatment). &&& P < 0.001 compared to GK (20 weeks of age). Fig. 2. Mitochondrial Ca2 + fluxes in liver mitochondria isolated from diabetic and Wistar control rats: (A) Typical measurements of calcium movements using the fluorescent calcium sensitive probe Calcium Green 5- N, as described in Materials and methods. The reactions were carried out in 2 ml of reaction medium supplemented with 0.6 mg protein, 2 AM rotenone, 0.1 AM oligomycin, 5 mM succinate and 100 nM Calcium Green 5-N. Fluorescence was monitored continuously for 50s, prior to the addition of 33 nmol Ca2 +/mg protein, and stopped with excess EGTA to obtain the baseline. The calcium release was inhibited by 0.8 AM cyclosporin A, indicating that the observed release was due to MPTP opening. Calcium fluxes are expressed as arbitrary units of fluorescence (AFU), determined 100s (B) and 210s (C) after the addition of 33 nmol Ca2 +/mg protein, as described in Materials and methods. Data are mean F S.E. of four independent experiments performed with different mitochondrial preparations. *P < 0.05, **P < 0.01, ***P < 0.001, as compared to Wistar control rats. F.M. Ferreira et al. / Biochimica et Biophysica Acta 1639 (2003) 113–120 117
118 F.M.Ferreira et al.Biochimica et Biophysica Acta 1639 (2003)113-120 6.00 ★★★ mitochondrial preparations was very low.Therefore,the ★★★ contents of CoQ determined can be attributed to the IMM. 5.00 3.4.Mitochondrial cardiolipin content 4.00 In order to help in the understanding of the observed 3.00 resistance of diabetic mitochondria to MPT induction.car- diolipin (diphosphatidylglycerol)contents were evaluated 2.00 [26].Our results showed that liver mitochondria isolated from diabetic rats presented a significantly higher content of 1.00 cardiolipin,except for the STZ-treated rats,3 weeks after STZ treatment(Fig.4). 0.00 Wistar STZ Wistar STZ Wistar GK Wistar GK 3 weeks g weeks 13 weeks 20 weeks 4.Discussion Fig.3.Coenzyme Q content in liver mitochondria isolated from Wistar control and diabetic rats.The levels of coenzyme Q(CoQo and CoQo)were Mitochondria are the major ATP producer in eukaryotic measured by HPLC as described in Materials and methods and expressed in mammalian cells and also the main intracellular sources and pmol/mg protein.Data are mean+S.E.of five different mitochondrial preparations.001as compared to Wistar control rats. target of reactive oxygen species (ROS).Moreover,mito- chondria play an important role in the regulation of intracel- lular Ca2 homeostasis.Previous reports describe some accumulation capacity increased with the increase in diabe- abnormalities in MPTP associated with diabetes [5,6].Since tes severity. the opening of MPTP can be induced by increasing amounts of Ca2+and by oxidative stress,and considering that 3.3.Mitochondrial Coo content diabetes leads to increased oxidative stress,due to constant hyperglycaemia [29-31],it should be expected an increase CoQ in rat mitochondria consists of two main homo- in Ca-dependent MPT induction during diabetes [32]. logues,CoQ and CoQ1o In Wistar non-diabetic rats,CoQo However,mitochondria isolated from STZ-treated rats liver represented 10-16%of the total amount of mitochondrial [5]and from GK rats heart [6]presented a lower suscepti- CoQ.while in diabetic rats this ratio varied between 8%and bility to MPT induction in the presence of Ca2+.These 12%.However,the amount of CoQ,particularly CoQo,was observations were on the basis of the present study,designed significantly increased in liver mitochondria isolated from to evaluate differences in MPT susceptibility due to diabetes diabetic rats (up to 2.5-fold)(Fig.3).Results of citrate in liver mitochondria.Our results showed that mitochondria synthase quantification and analysis by electron microscopy from diabetic rats were less susceptible to the induction of (data not shown)showed that the contamination level of MPTP assessed by Ca2/Pi.Surprisingly,we noted that the STZ 9w Group I (20 weeks of age) GK Wistar STZ 3w Group ll (13 weeks of age) GK Wistar 0.00 5.00 10.00 15.00 20.00 25.00 Cardiolipin(nmol/mg protein) Fig.4.Cardiolipin content in liver mitochondria isolated from diabetic and Wistar control rats.Mitochondrial cardiolipin content was quantified using an established spectrophotometric assay using 10-NAO,as described in Materials and methods.Data are meanS.E.of five different mitochondrial preparations. .,compared to STZ3 weeks after treatment:P<05.P<01 as compared to Wistar 13 weeks of age (3 weeks after treatment with STZ);"P<0.05 as compared to Wistar 20 weeks of age(9 weeks after treatment with STZ)
accumulation capacity increased with the increase in diabetes severity. 3.3. Mitochondrial CoQ content CoQ in rat mitochondria consists of two main homologues, CoQ9 and CoQ10. In Wistar non-diabetic rats, CoQ10 represented 10– 16% of the total amount of mitochondrial CoQ, while in diabetic rats this ratio varied between 8% and 12%. However, the amount of CoQ, particularly CoQ9, was significantly increased in liver mitochondria isolated from diabetic rats (up to 2.5-fold) (Fig. 3). Results of citrate synthase quantification and analysis by electron microscopy (data not shown) showed that the contamination level of mitochondrial preparations was very low. Therefore, the contents of CoQ determined can be attributed to the IMM. 3.4. Mitochondrial cardiolipin content In order to help in the understanding of the observed resistance of diabetic mitochondria to MPT induction, cardiolipin (diphosphatidylglycerol) contents were evaluated [26]. Our results showed that liver mitochondria isolated from diabetic rats presented a significantly higher content of cardiolipin, except for the STZ-treated rats, 3 weeks after STZ treatment (Fig. 4). 4. Discussion Mitochondria are the major ATP producer in eukaryotic mammalian cells and also the main intracellular sources and target of reactive oxygen species (ROS). Moreover, mitochondria play an important role in the regulation of intracellular Ca2 + homeostasis. Previous reports describe some abnormalities in MPTP associated with diabetes [5,6]. Since the opening of MPTP can be induced by increasing amounts of Ca2 + and by oxidative stress, and considering that diabetes leads to increased oxidative stress, due to constant hyperglycaemia [29 – 31], it should be expected an increase in Ca2 +-dependent MPT induction during diabetes [32]. However, mitochondria isolated from STZ-treated rats liver [5] and from GK rats heart [6] presented a lower susceptibility to MPT induction in the presence of Ca2 +. These observations were on the basis of the present study, designed to evaluate differences in MPT susceptibility due to diabetes in liver mitochondria. Our results showed that mitochondria from diabetic rats were less susceptible to the induction of MPTP assessed by Ca2 +/Pi. Surprisingly, we noted that the Fig. 3. Coenzyme Q content in liver mitochondria isolated from Wistar control and diabetic rats. The levels of coenzyme Q (CoQ9 and CoQ10) were measured by HPLC as described in Materials and methods and expressed in pmol/mg protein. Data are mean F S.E. of five different mitochondrial preparations. ***P < 0.001 as compared to Wistar control rats. Fig. 4. Cardiolipin content in liver mitochondria isolated from diabetic and Wistar control rats. Mitochondrial cardiolipin content was quantified using an established spectrophotometric assay using 10-NAO, as described in Materials and methods. Data are mean F S.E. of five different mitochondrial preparations. $$P < 0.01,$$$P < 0.001, compared to STZ 3 weeks after treatment; *P < 0.05. **P < 0.01 as compared to Wistar 13 weeks of age (3 weeks after treatment with STZ); # P < 0.05 as compared to Wistar 20 weeks of age (9 weeks after treatment with STZ). 118 F.M. Ferreira et al. / Biochimica et Biophysica Acta 1639 (2003) 113–120
FM.Ferreira et al.Biochimica et Biophysica Acta 1639 (2003)113-120 119 Ca2+accumulation capacity increased,with increased dia- Previous reports from our group [6,47,48]on the sus- betes time length and severity,as observed in STZ-treated ceptibility on heart and brain mitochondrial preparations to rats after 9 weeks of treatment and GK rats 20 weeks of age. MPTP induction showed that GK mitochondrial prepara- These observations were even more surprising since the tions from heart had reduced susceptibility to MPT induc- levels of endogenous oxidation products in these mitochon- tion [6],in agreement with the liver mitochondria results dria and in liver mitochondria from STZ-treated rats 3 weeks reported in this study,while heart mitochondria isolated after treatment were lower when compared to controls from STZ-treated rats after 3 weeks of treatment had an [evaluated as thiobarbituric acid reactive substances increase susceptibility to the induction of MPT [471.Brain (TBARS)formation [33];data not shown]. mitochondria isolated from STZ rats after 9 weeks of In order to find some explanations for the observed treatment were less susceptible to MPT induction. results,we evaluated the CoQ content in liver mitochondria. In conclusion,our results suggest that liver mitochon- It is well known that oxidative stress can trigger the MPT dria isolated from diabetic rats presented some metabolic pore opening [32,34].Coenzyme Q acts as an electron carrier adjustments,namely increase in CoQ and membrane car- from mitochondrial respiratory complexes I and II to com- diolipin contents that can be responsible for the observed plex III;also,CoQ in its fully reduced form (ubiquinol)is a decrease in the susceptibility to MPT induction in the potent antioxidant [35-38],preventing lipid peroxidation. presence of Ca2.These adjustments seem to vary between Furthermore,as ubiquinone-binding site regulates the MPT the tissues studied in our lab (liver,brain and heart).We pore 39,an increase in CoQ contents (CoQo and,probably, cannot exclude the possibility that liver mitochondria from also CoQo and CoQo)may enhance the calcium retention control rats present a higher number of spontaneous (but capacity in mitochondria [3].Our results showing that the reversible)MPTP openings,compared to diabetic liver amount of CoQ was significantly increased in liver mito- mitochondria. chondria from diabetic rats suggest that this increase in CoQ contents can,in part,be responsible for the decreased susceptibility for MPTP opening,observed in diabetic mi- Acknowledgements tochondrial preparations(in addition to the increase in the activity of Complexes II and IV of the respiratory chain [401). This work was supported by Fundacao para a Ciencia e a Additionally,the increase in CoQ contents in diabetic rats Tecnologia (FCT:Portuguese Research Council).Fernanda can be responsible for the higher membrane potential devel- M.Ferreira and Paulo J.Oliveira are recipients of PhD grants oped upon energization with succinate;we also found an from FCT (SFRH/BD/3247/2000 and PRAXIS XXI/BD/ increase in Complex II (and IV)specific activity with 21494/99,respectively). increase in diabetes severity [40]. It has also been found that the membrane lipid composi- tion is able to modulate the MPTP opening [26].Cardiolipin References (diphosphatidylglycerol),the only phospholipid synthesized by the mitochondria [26],has been proposed to regulate the [1]T.E.Gunter,K.K.Gunter,S.S.Sheu,C.E.Gavin,Mitochondrial cal- induction of MPT due to its ability to bind calcium [41-43]. cium transport:physiological and pathological relevance,Am.J. owing to its strongly negatively charged headgroups.The Physiol.267(1994)C313-C339. increased pool of negatively charges is thought to nonspe- [2]P.Bernardi,V.Petronilli,F.Di Lisa,M.Forte,A mitochondrial per- cifically bind Ca2,preventing its action on protein sites that spective on cell death,Trends Biochem.Sci.26(2001)112-117. [3]L.Walter,V.Nogueira,X.Leverve,M.P.Heitz,P.Berardi,E.Fon- play a role in MPTP opening.Our data indicated that the taine,Three classes of ubiquinone analogs regulate the mitochondrial content in cardiolipin was significantly increased in diabetic permeability transition pore through a common site,J.Biol.Chem. mitochondrial preparations (except for STZ-treated rats 3 275(2000)29521-29527. weeks after treatment),when compared to Wistar mitochon- [4]M.Crompton,The mitochondrial permeability transition pore and its role in cell death,Biochem.J.341 (1999)233-249. dria,suggesting that mitochondria have the capacity to [5]B.S.Kristal,M.Matsuda,B.P.Yu,Abnormalities in the mitochondrial enhance the synthesis of cardiolipin or adjust phospholipid permeability transition in diabetic rats,Biochem.Biophys.Res.Com- metabolism,in order to decrease the susceptibility to the mun.222(1996519-523. induction of MPT.The higher content in cardiolipin of the [6]PJ.Oliveira,A.P.Rolo,R.Seiga,C.M.Palmeira,M.S.Santos,A.J. IMM of diabetic rats makes it more impermeable to protons Moreno,Decreased susceptibility of heart mitochondria from diabetic (and other ions),which could explain the higher values of GK rats to mitochondrial permeability transition induced by calcium phosphate,Biosci.Rep.21 (2001)45-53. △Ψobserved. [7]Y.Ihara,Y.Yamada,S.Toyokuni,K.Miyawaki,N.Ban,T.Adachi The higher calcium loading capacity in diabetic rats may A.Kuroe,T.Iwakura,A.Kubota,H.Hiai,Y.Seino,Antioxidant a- interfere with the normal calcium pathways of the cell. tocopherol ameliorates glycemic control of GK rats,a model of type 2 Inside mitochondria,calcium can stimulate some dehydro- diabetes,FEBS Lett.473 (2000)24-26. genases(pyruvate dehydrogenase,isocitrate dehydrogenase [8]E.C.Opara,E.Abdel-Rahman,S.Soliman,W.A.Kamel,S.Souka J.E.Lowe,S.Abdel-Aleem,Depletion of total antioxidant capacity in and 2-oxoglutarate dehydrogenase [44,45])and have a type 2 diabetes,Metabolism 48 (1999)1414-1417. regulatory effect on ATP synthase [46]. [9]R.W.Rinehart,J.Roberson.D.S.Beattie.The effect of diabetes on
Ca2 + accumulation capacity increased, with increased diabetes time length and severity, as observed in STZ-treated rats after 9 weeks of treatment and GK rats 20 weeks of age. These observations were even more surprising since the levels of endogenous oxidation products in these mitochondria and in liver mitochondria from STZ-treated rats 3 weeks after treatment were lower when compared to controls [evaluated as thiobarbituric acid reactive substances (TBARS) formation [33]; data not shown]. In order to find some explanations for the observed results, we evaluated the CoQ content in liver mitochondria. It is well known that oxidative stress can trigger the MPT pore opening [32,34]. Coenzyme Q acts as an electron carrier from mitochondrial respiratory complexes I and II to complex III; also, CoQ in its fully reduced form (ubiquinol) is a potent antioxidant [35 – 38], preventing lipid peroxidation. Furthermore, as ubiquinone-binding site regulates the MPT pore [39], an increase in CoQ contents (CoQ0 and, probably, also CoQ9 and CoQ10) may enhance the calcium retention capacity in mitochondria [3]. Our results showing that the amount of CoQ was significantly increased in liver mitochondria from diabetic rats suggest that this increase in CoQ contents can, in part, be responsible for the decreased susceptibility for MPTP opening, observed in diabetic mitochondrial preparations (in addition to the increase in the activity of Complexes II and IVof the respiratory chain [40]). Additionally, the increase in CoQ contents in diabetic rats can be responsible for the higher membrane potential developed upon energization with succinate; we also found an increase in Complex II (and IV) specific activity with increase in diabetes severity [40]. It has also been found that the membrane lipid composition is able to modulate the MPTP opening [26]. Cardiolipin (diphosphatidylglycerol), the only phospholipid synthesized by the mitochondria [26], has been proposed to regulate the induction of MPT due to its ability to bind calcium [41 – 43], owing to its strongly negatively charged headgroups. The increased pool of negatively charges is thought to nonspecifically bind Ca2 +, preventing its action on protein sites that play a role in MPTP opening. Our data indicated that the content in cardiolipin was significantly increased in diabetic mitochondrial preparations (except for STZ-treated rats 3 weeks after treatment), when compared to Wistar mitochondria, suggesting that mitochondria have the capacity to enhance the synthesis of cardiolipin or adjust phospholipid metabolism, in order to decrease the susceptibility to the induction of MPT. The higher content in cardiolipin of the IMM of diabetic rats makes it more impermeable to protons (and other ions), which could explain the higher values of DW observed. The higher calcium loading capacity in diabetic rats may interfere with the normal calcium pathways of the cell. Inside mitochondria, calcium can stimulate some dehydrogenases (pyruvate dehydrogenase, isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase [44,45]) and have a regulatory effect on ATP synthase [46]. Previous reports from our group [6,47,48] on the susceptibility on heart and brain mitochondrial preparations to MPTP induction showed that GK mitochondrial preparations from heart had reduced susceptibility to MPT induction [6], in agreement with the liver mitochondria results reported in this study, while heart mitochondria isolated from STZ-treated rats after 3 weeks of treatment had an increase susceptibility to the induction of MPT [47]. Brain mitochondria isolated from STZ rats after 9 weeks of treatment were less susceptible to MPT induction. 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