Insulin protects against amyloid β-peptide toxicity in brain mitochondria of diabetic rats

www.elsevier.com/locate/ynbdi Neurobiology of Disease 18 (2005) 628 – 637

Insulin protects against amyloid B-peptide toxicity in brain mitochondria of diabetic rats Paula I. Moreira,a Maria S. Santos,a Cristina Sena,b Raquel Seic¸a,b and Catarina R. Oliveirac,* a

Center for Neuroscience of Coimbra, Department of Zoology, Faculty of Sciences and Technology, University of Coimbra, 3004-517 Coimbra, Portugal Center for Neuroscience of Coimbra, Institute of Physiology, Faculty of Medicine, University of Coimbra, 3004-517 Coimbra, Portugal c Center for Neuroscience of Coimbra, Institute of Biochemistry, Faculty of Medicine, University of Coimbra, 3004-517 Coimbra, Portugal b

Received 25 June 2004; revised 13 October 2004; accepted 26 October 2004

This study compared the status of brain mitochondria isolated from 12week streptozotocin (STZ)-diabetic rats versus STZ-diabetic animals treated with insulin during a period of 4 weeks. Brain mitochondria isolated from 12-week citrate (vehicle)-treated rats were used as control. For that purpose, several mitochondrial parameters were evaluated: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (DCm), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, production of H2O2, ATPase activity, and the capacity of mitochondria to accumulate Ca2+. Furthermore, the effect of AB1–40 was also analyzed. We observed that STZ-induced diabetes promoted a significant decrease in mitochondrial CoQ9, ATPase activity, and a lower capacity of mitochondria to accumulate Ca2+ when compared with control and insulin-treated diabetic rats. The presence of 4 AM AB1–40 induced a significant decrease in RCR in the three groups of rats. However, this peptide induced a significant increase in the repolarization lag phase and a significant decrease in the repolarization level in control and diabetic animals without insulin treatment. Furthermore, this peptide exacerbated significantly the production of H2O2 in STZ-diabetic rats, this effect being avoided by insulin treatment. Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid B-peptide, insulin seems to prevent the decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer’s disease. D 2004 Elsevier Inc. All rights reserved. Keywords: Brain; Amyloid h-peptide; Diabetes; Insulin; Mitochondria

Abbreviations: CoQ, coenzyme Q; CsA, cyclosporin A; DCm, mitochondrial transmembrane potential; MPTP, mitochondrial permeability transition pore; RCR, respiratory control ratio; STZ, streptozotocin; TPP+, tetraphenylphosphonium. * Corresponding author. Fax: +351 239 822776. E-mail address: [email protected] (C.R. Oliveira). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2004.10.017

Introduction In type 1 diabetes, pancreatic h-cells are destroyed leading to an, eventually, absolute insulin deficiency. Without insulin to move glucose into cells, blood glucose levels become excessively high, a condition known as hyperglycemia (Ro¨sen et al., 2001). The central significance of glucose as the major nutrient of the brain, its metabolism, and control have been well documented (Hoyer, 2002). Data from the literature indicate that neuronal glucose metabolism is controlled by insulin. Although the blood–brain barrier allows the passage of insulin produced by pancreatic h-cells, it can be partially formed in the hippocampus, prefrontal cortex, entorhinal cortex, and the olfactory bulb (Hoyer, 2003). Insulin receptors are also present in several brain areas such as the olfactory bulb, hypothalamus, cerebral cortex, and hippocampus (Henneberg and Hoyer, 1995). Insulin-sensitive glucose transporters are found not only at the blood–brain barrier, but also in glia expressing partially insulin-sensitive GLUT1 (Ngarmukos et al., 2001; Vannucci et al., 1997) and in some neurons expressing GLUT1 (Vannucci et al., 1997) or GLUT4 (Cheng et al., 2001; Choeiri et al., 2002). Bingham et al. (2002) have shown that insulin has a significant effect on global brain glucose metabolism, mainly in the cerebral cortex. The authors suggest that this effect may either be a direct effect of insulin, stimulating glucose uptake and metabolism, or an indirect effect achieved via insulin-stimulated neuronal activation with secondary increment in cell glucose metabolism. These results raise the hypothesis that insulin can access insulin receptors in the brain and have a metabolic effect in this organ, which may be maximal at basal circulating insulin concentrations. Insulin affects several brain functions including cognition and memory, and several clinical studies have established links between insulin resistance, diabetes mellitus, and Alzheimer’s disease (Gasparini et al., 2002). Recent evidence indicates that insulin regulates the metabolism of amyloid h and tau proteins, which are involved in the formation of two hallmarks of Alzheimer’s disease, amyloid deposits, and neurofibrillary tangles, respectively (Gasparini et al., 2001; Mandelkow et al., 1992; Solano et al., 2000).

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Increased oxidative stress has been implicated in the pathology of several diseases including diabetes and Alzheimer’s disease (Eckert et al., 2003; Yorek, 2003). The fact that mitochondria are the major generators and direct targets of ROS has led several investigators to foster the idea that oxidative stress and damage in mitochondria are contributory factors to several disorders. Since the brain possesses high energetic requirements, any decline in brain mitochondria electron chain could have a severe impact on brain function and particularly on the etiology of neurodegenerative diseases. Recently, we reported that brain mitochondria isolated from GK rats (a model of type 2 diabetes) are highly susceptible to the presence of amyloid h-peptide (Moreira et al., 2003), suggesting that type 2 diabetes is a risk factor for the development of Alzheimer’s disease. The present study was aimed to evaluate the role of insulin in diabetic brain mitochondrial function at basal conditions and when exposed to the amyloid h-peptide. For this purpose we used, as an experimental model, brain mitochondria isolated from streptozotocin (STZ)-diabetic rats, diabetic rats treated with insulin, and control animals. Several mitochondrial parameters were analyzed: respiratory indexes (respiratory control ratio (RCR) and ADP/O ratio), transmembrane potential (DCm), repolarization lag phase, repolarization level, ATP, glutathione and coenzyme Q (CoQ) contents, ATPase activity, production of H2O2, and the capacity of mitochondria to accumulate Ca2+.

Experimental procedures Materials Streptozotocin (STZ) and protease (Subtilisin, Carlsberg) type VII were obtained from Sigma (Germany). Ah1–40 and Ah40–1 were obtained from Bachem AG (Bubendorf, Germany). Insulin (Mixtard 30 Novolet) was obtained from Novonardisk A/S (Dinamark). Digitonin was obtained from Calbiochem. All the other chemicals were of the highest grade of purity commercially available.

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the massive destruction of h-cells and release of intracellular insulin associated with STZ treatment (Rodrigues et al., 1999). After 8 weeks of STZ-induced diabetes, diabetic animals were divided into two groups and one group was injected daily with insulin (5–20 UN/kg weight). The other group of diabetic rats remained without treatment. Animals were sacrificed after for 4 weeks of insulin treatment. During that period, their weight was measured and blood glucose concentration was determined from the tail vein using a commercial glucometer (Glucometer-Elite, Bayer, Portugal). Values were taken in fasting and in nonfasting conditions just before or after STZ or insulin administration, respectively. If feeding blood glucose in the tail vein exceeded 250 mg/dl, animals were used as diabetic. Hemoglobin A1C (HbA1c) levels were determined by ion exchange chromatography (Abbott Imx Glicohemoglobin, Abbott Laboratories, Portugal). Isolation of brain mitochondria Rat brain mitochondria were isolated by the method of Rosenthal et al. (1987), with slight modifications, using 0.02% digitonin to free mitochondria from the synaptosomal fraction. In brief, one rat was decapitated, and the whole brain minus the cerebellum was rapidly removed, washed, minced, and homogenized at 48C in 10 ml of isolation medium (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, 1 mM EGTA, 1 mg/ml bovine serum albumin, pH 7.4) containing 5 mg of the bacterial protease. Single brain homogenates were brought to 30 ml and then centrifuged at 2500 rpm for 3 min. The pellet, including the fluffy synaptosomal layer, was resuspended in 10 ml of the isolation medium containing 0.02% digitonin and centrifuged at 10,000 rpm for 8 min. The brown mitochondrial pellet without the synaptosomal layer was then resuspended in 10 ml of medium and recentrifuged at 10,000 rpm for 10 min. The mitochondrial pellet was resuspended in 300 Al of resupension medium (225 mM mannitol, 75 mM sucrose, 5 mM Hepes, pH 7.4). Mitochondrial protein was determined by the biuret method calibrated with bovine serum albumin (Gornall et al., 1949). Respiration measurements

Animals Male Wistar and STZ rats (10 weeks) were housed in our animal colony (Laboratory Research Center, University Hospital, Coimbra, Portugal). They were maintained under controlled light (12-h day/night cycle) and humidity with free access to water (except in the fasting period) and powdered rodent chow (AO4 Panlab). Glucose tolerance tests were used to select diabetic rats for study. Adhering to procedures approved by the Institutional Animal Care and Use Committee, the animals were sacrificed by cervical displacement and decapitation. Induction, characterization of STZ-induced diabetes, and insulin treatment Rats (10 weeks) were divided randomly into two groups of animals each. Diabetes was induced in one group of animals after a 16-h fasting period with a single intraperitoneal injection of STZ, 50 mg/kg, freshly dissolved in citrate 100 mM, pH 4.5. The volume used was always 0.5 ml/200 g animal weight. Control animals were injected with the same volume of citrate (vehicle) solution. In the following 24 h, animals were orally fed with glycosylated serum in order to avoid hypoglycemia resulting from

Oxygen consumption of isolated mitochondria was monitored polarographically with a Clark oxygen electrode (Estabrook, 1967) connected to a suitable recorder in a 1-ml thermostated waterjacketed closed chamber with magnetic stirring. The reactions were carried out at 308C in 1 ml of reaction medium with 0.6 mg protein. The isolates were incubated with 4 AM Ah1–40 or Ah40–1 for 5 min before mitochondria energization with succinate. Membrane potential (DWm), repolarization level, and repolarization lag phase measurements The mitochondrial transmembrane potential was monitored by evaluating the transmembrane distribution of tetraphenylphosphonium ion (TPP+) with a TPP+-selective electrode prepared according to Kamo et al. (1979) using a Ag/AgCl2 electrode as reference. Reactions were carried out in a chamber with magnetic stirring in 1 ml of reaction medium (100 mM sucrose, 100 mM KCl, 2 mM KH2PO4, 5 mM Hepes, 10 AM EGTA, pH 7.4) supplemented with 3 AM TPP+. The experiments were started by adding 5 mM succinate to mitochondria in suspension at 0.6 mg protein/ ml. After a steady-state distribution of TPP+ had been reached

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(2 min of recording), Ca2+ was added and DCm recorded. Membrane potential was estimated from the decrease of TPP+ concentration in the reaction medium as described elsewhere (Moreno and Madeira, 1991). The isolates were incubated with 4 AM Ah1–40 for 5 min before succinate addition, while 0.85 AM cyclosporin A (CsA) and 2 Ag/ml oligomycin plus 1 mM ADP were added 2 min prior to Ah preincubation or mitochondria energization. In mitochondrial permeability transition pore (MPTP) experiments, after a steady-state distribution of TPP+ had been reached (2 min of recording), Ca2+ was added and DCm recorded.

a stream of N2 and suspended in ethanol for HPLC analysis. Liquid chromatography was performed using a Gilson high-performance liquid chromatography using a reverse phase column (RP18; Spherisorb; S5 ODS2) as earlier described (Chung et al., 1997). Samples were eluted from the column with methanol/heptane (10:2), by volume) at a flow of 2 ml/min. Detection was performed with a UV detector at 269 nm. Identification and quantification were based in pure standards by their retention times and peak areas, respectively. CoQ levels (CoQ9 and CoQ10) in mitochondrial membranes were expressed in pmol/mg protein. Measurement of ATPase activity

Analysis of ATP content At the end of the DCm experiments, an aliquot of 250 Al of each mitochondrial suspension was rapidly centrifuged at 14,000 rpm for 6 min with 0.3 M perchloric acid. The supernatants were neutralized with 10 M KOH in 5 M Tris and centrifuged at 14,000 rpm for 5 min. The resulting supernatants were assayed for ATP by separation in a reverse-phase high-performance liquid chromatography. The chromatography apparatus was a BeckmanSystem Gold, consisting of a 126 Binary Pump Model and 166 Variable UV detector controlled by a computer. The detection wavelength was 254 nm, and the column was a Lichrospher 100 RP-18 (5 Am) from Merck. An isocratic elution with 100 mM phosphate buffer (KH2PO4) pH 6.5 and 1.0% methanol was performed with a flow rate of 1 ml/min. The required time for each analysis was 6 min. Measurement of glutathione content Reduced (GSH) and oxidized (GSSG) glutathione were determined with fluorescence detection after reaction of the supernatant of the H3PO4/EDTA–NaH2PO4 or H3PO4/NaOH deproteinized mitochondria solution, respectively, with o-phthalaldehyde (OPT), pH 8.0, according to Hissin and Hilf (1976). In brief, at the end of the DCm experiments, 500 Al of each mitochondrial suspension was rapidly centrifuged at 50,000 rpm (Beckman, TL-100 Ultracentrifuge) for 30 min with 1.5 ml phosphate buffer (100 mM NaH2PO4, 5 mM EDTA, pH 8.0) and 500 Al H3PO4 2.5%. For GSH determination, 100 Al of the supernatant was added to 1.8 ml phosphate buffer and 100 Al OPT. After mixing and incubation at room temperature for 15 min, the solution was transferred to a quartz cuvette and the fluorescence was measured at 420 nm. For GSSG determination, 250 Al of the supernatant was added to 100 Al of N-ethylmaleymide and incubated at room temperature for 30 min. After incubation, 140 Al of the mixture was added to 1.76 ml NaOH (100 mM) buffer and 100 Al OPT. After mixing and incubation at room temperature for 15 min, the solution was transferred to a quartz cuvette and the fluorescence was measured at 420 and 350 nm emission and excitation wavelength, respectively (slits 5, 5). The GSH and GSSG contents were determined from comparisons with a linear reduced or oxidized glutathione standard curve, respectively. Measurement of CoQ content CoQs were extracted from aliquots of mitochondria containing 0.5 mg protein/ml according to the previously described method (Takada et al., 1984). The extract was evaporated to dryness under

ATPase activity was measured by the colorimetric determination of Pi hydrolyzed from ATP by the method of Taussky and Shorr (1953). The reaction was carried out at 308C in 1 ml of reaction medium (130 mM sucrose, 50 m KCl, 5 mM MgCl2, 5 mM Hepes, pH 7.2) containing 0.25 mg protein. The reaction was started by the addition of 1 mM ATP-Mg, allowed to proceed for 15 min, and stopped by the addition of 500 Al of 40% trichloroacetic acid. Samples were chilled on ice for 10 min, centrifuged, and 1-ml samples taken for assay of released Pi. The mitochondrial (F1) ATPase is expressed in nmol Pi liberated/mg protein/15 min and represents the difference between the activity in the absence and presence of oligomycin (1 Ag/mg protein). Measurement of H2O2 production The rate of hydrogen peroxide (H2O2) production was measured fluorometrically using a modification of the method described by Barja (1999). Briefly, mitochondria were incubated at 378C with 10 mM succinate in 1.5 ml of phosphate buffer, pH 7.4, containing 0.1 mM EGTA, 5 mM KH2PO4, 3 mM MgCl2, 145 mM KCl, 30 mM Hepes, 0.1 mM homovalinic acid, and 6 U/ml horseradish peroxidase in the presence or absence of 4 AM Ah1–40. The incubation was stopped at 15 min with 0.5 ml of cold 2 M glycine buffer containing 25 mM EDTA and NaOH, pH 12. The fluorescence of supernatants was measured at 312 nm as excitation wavelength and 420 nm as emission wavelength. The rate of peroxide production was calculated using a standard curve of H2O2. Statistical analysis Results are presented as mean F SEM of the indicated number of experiments. Statistical significance was determined using Student’s t test and one-way ANOVA test for multiple comparisons, followed by the post hoc Tukey–Kramer test.

Results Insulin reverses the increase in glycemia levels and the decrease in body weight promoted by STZ-induced diabetes STZ rats presented a significant increase in blood glucose levels and HbA1C, and a significant decrease in body weight when compared with Wistar control rats (Table 1). These data confirm the state of diabetes mellitus in STZ-treated rats. However, insulin treatment attenuated the alterations promoted by STZ-induced diabetes. Diabetic animals treated with insulin presented a significant decrease in glycemia and HbA1C levels and a significant

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increase in body weight when compared with diabetic animals without insulin treatment (Table 1). Insulin reverses the decrease in the respiratory control ratio promoted by Ab 1–40 Respiratory control ratio (RCR) is the ratio between mitochondrial respiration states 3 (consumption of oxygen in the presence of substrate and ADP) and 4 (consumption of oxygen after ADP has been consumed). ADP/O ratio, an indicator of oxidative phosphorylation efficiency, is expressed by the ratio between the amount of ADP added and the oxygen consumed during the state 3 of respiration. At basal conditions, neither diabetes nor insulin treatment induced any significant alteration in both parameters (Figs. 1A and B). However, the presence of 4 AM Ah1–40 induced a significant decrease in RCR in the three groups of rats while ADP/ O ratio remained statistically unchanged (Figs. 1A and B). Incubation of mitochondria with the reverse sequence peptide (4 AM Ah40–1) did not induce any significant effect (data not shown). Insulin prevents the decrease in oxidative phosphorylation efficiency induced by diabetes or Ab 1–40 Mitochondrial transmembrane potential (DCm) is fundamental for the phenomenon of oxidative phosphorylation, the conversion of ADP to ATP via ATP synthase. Mitochondrial respiratory chain pumps H+ out of the mitochondrial matrix across the inner mitochondrial membrane. The H+ gradient originates an electrochemical potential (Dp) resulting in a pH (DpH) and a voltage gradient (DCm) across the mitochondrial inner membrane. As shown in Table 2, neither diabetes nor insulin treatment induced any significant change on DCm, repolarization level (time necessary for mitochondria to reestablish the DCm, after ADP phosphorylation), and repolarization lag phase (corresponding to ADP phosphorylation) (Table 2). However, STZ-diabetic animals presented a significant decrease in ATPase activity (Table 3) and ATP content (Table 2) when compared with control and diabetic rats treated with insulin. The presence of 4 AM Ah1–40 induced a significant decrease in repolarization level and a significant increase in repolarization lag phase of mitochondria isolated from control and STZ-diabetic animals being these effects prevented by insulin treatment (Table 2). Insulin prevents the depletion of antioxidant defenses in diabetic animals Endogenous antioxidants such as reduced glutathione (GSH) and CoQ belong to the first line of defense and act by scavenging potentially damaging free radical moieties. In our study, we did not

Fig. 1. Effect of STZ-induced diabetes and insulin treatment on mitochondrial respiratory indexes: role of Ah1–40. (A) Respiratory control ratio (RCR). (B) ADP/O ratio. Freshly isolated brain mitochondria (0.6 mg) in 1 ml of standard medium supplemented with 2 AM rotenone were energized with 5 mM succinate. Isolates were incubated with 4 AM Ah1–40 for 5 min, at 308C, before mitochondria energization. **P b 0.01; *P b 0.05, statistically significant when compared with Wistar control rats. #P b 0.05, statistically significant when compared with STZ-diabetic rats. &P b 0.05, statistically significant when compared with STZ + INS rats. Data shown represent the mean F SEM from six animals for each condition studied.

observe any significant alteration promoted by STZ-induced diabetes, insulin treatment (Figs. 2A and B), and Ah1–40 peptide (Fig. 2B) on glutathione content. The levels of GSH in Fig. 2A are slightly higher than those in Fig. 2B because in the first case we prepared the samples immediately after mitochondria isolation. In Fig. 2B, the samples were incubated during 5 min, at 308C, resulting in the loss of some mitochondrial GSH. However, mitochondrial CoQ content was significantly decreased in STZ-diabetic animals being this effect prevented by insulin treatment (Fig. 3).

Table 1 Characterization of Wistar control, STZ-diabetic, and STZ-diabetic rats treated with insulin (STZ + INS) Glycemia (mg/ml) HbA1C (percentage of total hemoglobin) Weight (g)

Wistar control

STZ

STZ + INS

92.8 F 3.77 5.58 F 0.45 517.8 F 8.1

527.4 F 24.87*** 11.61 F 0.63*** 331.8 F 21.6***

393.4 F 46.66***,y 8.43 F 0.54**,yy 395.0 F 4.54***,yy

Data are the mean F SEM from six animals for each condition studied. *** P b 0.001, when compared with Wistar control rats. ** P b 0.01, when compared with Wistar control rats. y P b 0.05, when compared with STZ diabetic rats. yy P b 0.01, when compared with STZ diabetic rats.

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Table 2 Effect of STZ-induced diabetes and insulin treatment on DCm, repolarization lag phase, repolarization level, and ATP content: impact of Ah1–40 Control DCm (mV) Repolarization level (mV) Repolarization lag phase (min) ATP content (nmol/mg protein)

177.3 153.7 0.522 153.0

F F F F

STZ 2.20 4.21 0.04 3.04

172.4 144.8 0.545 128.2

F F F F

2.83 3.36 0.02 2.28*

STZ + INS

Control + Ah1–40

STZ + Ah1–40

STZ + INS + Ah1–40

175.1 151.2 0.568 146.5

172.9 138.9 0.734 135.7

171.6 137.1 0.738 126.2

173.7 141.2 0.665 136.8

F 1.40 F 2.50 F 0.02 F 3.96#

F F F F

2.20 3.59* 0.07* 8.62

F F F F

2.63 4.12* 0.07* 7.13**,y

F F F F

1.87 2.93 0.04 8.00

Freshly isolated brain mitochondria (0.6 mg) in 1 ml of reaction medium supplemented with 3 AM TPP+ and 2 AM rotenone were energized with 5 mM succinate. **P b 0.01; *P b 0.05, when compared with Wistar control rats. #P b 0.05, when compared with STZ rats. yP b 0.05, when compared with STZ + INS rats. Data shown represent the mean F SEM from six animals for each condition studied.

Insulin reverses the increase in H2O2 production induced by diabetes The production of H2O2 by mitochondria gives an indication about the propensity of mitochondria to originate or exacerbate oxidative stress. We observed that STZ rats presented a significant higher production of H2O2, this effect being partially reversed by insulin treatment (Fig. 4). Insulin prevents the decrease in the capacity of mitochondria to accumulate Ca2+ promoted by diabetes

effect of 4 AM Ah1–40 was also evaluated. We observed that STZinduced diabetes induced a significant decrease in mitochondrial CoQ9 and ATP contents, ATPase activity, and a lower capacity of mitochondria to accumulate Ca2+. In the presence of Ah1–40, STZinduced diabetic brain mitochondria produced higher levels of H2O2, probably due to the decreased content in CoQ9 that acts as an antioxidant defense. Furthermore, Ah1–40 induced an impairment of the respiratory chain and a decrease in the oxidative phosphorylation efficiency in both control and STZ-diabetic rats. Insulin treatment prevented the mitochondrial alterations induced by Ah1–40 or STZ-induced diabetes (Fig. 6).

Mitochondrial transmembrane potential (DCm) drop is a typical phenomenon that follows the induction of MPTP. In control conditions (Fig. 5A), the addition of 5 mM succinate produced an DCm of c185 mV (negative inside mitochondria), corresponding to respiratory state 4. Then, the first pulse of 10 AM Ca2+ led to a rapid depolarization (decrease of DCm) followed by a small repolarization (recover of DCm). The depolarization was due to the entry of Ca2+ into the electronegative mitochondrial matrix, followed by the efflux of H+ in an attempt to restore DCm. However, a second and third pulse of 10 AM Ca2+ led to total depolarization of mitochondria. Mitochondria can tolerate some amount of Ca2+, but ultimately their capacity to adapt to Ca2+ loads is overwhelmed and mitochondria depolarize completely due to a profound change in the inner membrane permeability. Brain mitochondria isolated from STZ-diabetic animals presented a slight decreased capacity to accumulate this cation (Fig. 5B). However, the behavior of brain mitochondria of diabetic animals treated with insulin was similar to that of control rats (Fig. 5C). The effects of Ca2+ were prevented when mitochondria were preincubated with 0.85 AM CsA (specific inhibitor of the MPTP) (Fig. 5D) or 1 mM ADP plus 1 Ag/ml oligomycin (Fig. 5E).

Discussion This study analyzed the role of insulin on the function of brain mitochondria isolated from STZ-diabetic rats. Furthermore, the

Table 3 Effect of STZ-induced diabetes and insulin treatment on ATPase activity ATPase (nmol Pi/mg/15 min)

Control

STZ

STZ + INS

578.3 F 68.8

293.8 F 69.8*

426.4 F 81.6

Data shown represent the mean F SEM from six animals for each condition studied. * P b 0.05, when compared with Wistar control rats.

Fig. 2. Effect of STZ-induced diabetes and insulin treatment on mitochondrial-reduced (GSH) and oxidized (GSSG) glutathione content (A) and the role of Ah1–40 on reduced glutathione (GSH) content (B). Freshly isolated brain mitochondria (0.6 mg) in 1 ml of standard medium were immediately frozen (A) or were supplemented with 2 AM rotenone and energized with 5 mM succinate (B). Isolates were incubated with 4 AM Ah1–40 for 5 min, at 308C, before mitochondria energization. Data shown represent the mean F SEM from six animals for each condition studied.

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Fig. 3. Effect of STZ-induced diabetes and insulin treatment on mitochondrial CoQ content. Freshly isolated brain mitochondria (0.5 mg) were handled under standard conditions as described in Experimental procedures. ***P b 0.001, statistically significant when compared to Wistar control rats. Data shown represent the mean F SEM from six animals for each condition studied.

For the characterization of the animals, blood glucose levels and glycated hemoglobin (HbA1C) were determined. We observed that STZ-diabetic animals presented a significant increase in glycemia and glycated hemoglobin levels as well as a significant decrease in body weight (Table 1). These data demonstrate that diabetes mellitus was successfully induced in these rats injected with STZ. When we compared diabetic animals treated with insulin with those without treatment, we observed that insulin induced a significant increase in body weight and a significant decrease in the levels of blood glucose and HbA1C (Table 1). These results are in accordance with human-based studies (Alvarsson et al., 2003; Park and Choi, 2002), indicating that insulin treatment improves metabolic control. Diabetes mellitus is associated with damage to the central nervous system and cognitive deficits (Gispen and Biessels, 2000; Knopman et al., 2001). Impairment of learning and memory has been documented in both type 1 and type 2 diabetes. Central nervous system deficits range from moderate to severe, depending on the quality of glycemic control (Gasparini et al., 2002). Population-based studies (Leibson et al., 1997; Ott et al., 1999) indicate a link between insulin resistance, diabetes mellitus, and Alzheimer’s disease (AD). Evidence from the literature indicates that there is an increase in oxidative stress in human type 1 diabetes (Desco et al., 2002) and experimental diabetes (Baynes, 1991; Reagan et al., 2000) and a decrease in the antioxidant capacity (Evans et al., 2002; Maritim et al., 2003). Oxidative damage in rat brain is increased by experimentally induced hyperglycemia (Aragno et al., 1997). Schmeichel et al. (2003) suggested that oxidative stress leads to oxidative injury of dorsal root ganglion neurons, the mitochondria being a specific target. Our results show that STZ-diabetic mitochondria possess a lower content on CoQ9 (Fig. 3), indicating a deficit in antioxidant defenses in diabetic animals and, consequently, an increased probability of oxidative stress. The reduced form of CoQ may function as an antioxidant, protecting membrane phospholipids and serum low-density lipoprotein from lipid peroxidation by quenching lipid radicals or lipid peroxidation-initiating species and, as recent data indicate, also protects mitochondrial membrane proteins and DNA from free radical-induced oxidative damage (Beyer, 1990; Ernster and Dallner, 1995; Lenaz, 1998). Accordingly, the higher production

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of H2O2 resulting from preincubation of mitochondria isolated from STZ-diabetic rats with the neurotoxic agent Ah1–40 may be due to the decrease in mitochondrial CoQ9 content (Fig. 3). In vitro studies have shown that Ah-mediated cell death in both neuronal and non-neuronal cells is mediated in part by the increase in cellular H2O2 (Behl et al., 1994) and that catalase has a protective role as an H2O2-degrading enzyme (Milton, 2001). In this study, the ATP synthase activity was assayed under substrate saturation in the direction of ATP hydrolysis (ATPase) that is proportional to ATP synthesis (Gresser et al., 1979). According to the chemiosmotic theory (Mitchell, 1972), the flux of electrons along the mitochondrial respiratory chain leads to an electrochemical gradient. The electron flux results in pumping out H+ from the mitochondrial matrix space that generates a pH and voltage gradient across the inner mitochondrial membrane. This decrease in ATPase/synthase activity can be a protective mechanism developed by diabetic rats to preserve the energetic levels. Previous reports indicate that under ischemic or anoxic conditions the cardiac mitochondrial ATP synthase works in reverse hydrolyzing ATP (Das, 2003; Das and Harris, 1999). However, downregulation of the ATP synthase under these conditions thus conserves ATP levels. As the activity of the enzyme is not downregulated to zero, the electrochemical gradient across the inner mitochondrial membrane can be maintained or is only slightly reduced by hydrolysis of a small amount of ATP, which could presumably preserve intracellular homeostasis. The membrane potential prevents uncontrolled influx of ATP into the mitochondrial matrix space via the electrogenic ATP/ADP translocase thus limiting ATP hydrolysis (Das, 2003; Das and Harris, 1999). Our results are in accordance with these reports because although a decrease in ATPase/synthase (Table 3) and ATP contents (Table 2) was observed, the other oxidative phosphorylation parameters were maintained (Table 2). Furthermore, the decrease in the enzyme activity can also be related with a decreased number of F0F1-ATPase units. Jorda et al. (1988) reported that the

Fig. 4. Effect of STZ-induced diabetes and insulin treatment on mitochondrial H2O2 production. Freshly isolated brain mitochondria were incubated at 0.2 mg protein/ml under standard conditions as described in Experimental procedures. ##P b 0.01, statistically significant when compared to STZ-diabetic rats. &P b 0.01, statistically significant when compared to STZ + INS rats. $P b 0.05, statistically significant when compared to Wistar control rats in the presence of 4 AM Ah1–40. $P b 0.05, statistically significant when compared to Wistar rats in the presence of 4 AM Ah1–40. +P b 0.05, statistically significant when compared to STZdiabetic rats in the presence of 4 AM Ah1–40. Data shown represent the mean F SEM from six animals for each condition studied.

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Fig. 5. Effect of STZ-induced diabetes and insulin treatment on the mitochondrial capacity of mitochondria to accumulate Ca2+. Freshly isolated brain mitochondria (0.6 mg) in 1 ml of standard medium supplemented with 3 AM TPP+ and 2 AM rotenone were energized with 5 mM succinate. (A) Wistar control rats; (B) STZ-diabetic rats; (C) STZ + INS rats. (D) Mitochondria isolated from STZ-diabetic rats were pre-incubated with CsA. (E) Mitochondria isolated from STZ-diabetic rats were pre-incubated with ADP plus oligomycin. CsA and ADP plus oligomycin were added 2 min prior to mitochondria energization. Ca2+ was added 1.5 min after mitochondria energization with succinate. The traces are typical of three experiments.

half-life of ATPase of STZ-diabetic rat liver mitochondria decreased by approximately 20%. They hypothesized that diabetes produces alterations in lysosomes leading to an increase in the degradation of ATPase. Furthermore, we observed that insulin treatment partially prevents the effect of diabetes in ATPase/ synthase activity (Table 3). AD is associated with mitochondrial impairment (Mattson et al., 2001; Mecocci et al., 1994). Specific defects in energy metabolism in AD brains have been reported in a number of PET studies (Azari et al., 1993; Pettegrew et al., 1994). Postmortem studies revealed a

decline in the activities of pyruvate dehydrogenase and aketoglutarate dehydrogenase (Blass et al., 2000), key enzymes in energy metabolism that are localized in the mitochondria. Furthermore, defects on cytochrome oxidase activity have also been described (Kish et al., 1992). Consistently, our data show that Ah1–40 induced a significant decrease in RCR (Fig. 1A), indicating an impairment of the respiratory chain. Furthermore, we observed that this neurotoxic peptide decreases oxidative phosphorylation efficiency in control and STZ-diabetic rats by delaying the process of ATP production (Table 2). However, mitochondria maintained the

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Fig. 6. Insulin prevents mitochondrial dysfunction induced by diabetes or the presence of the amyloid h-peptide. STZ-induced diabetes promotes a significant decrease in mitochondrial CoQ9 and ATP contents, ATPase activity, and a lower capacity of mitochondria to accumulate Ca2+. In the presence of Ah1–40, STZinduced diabetic brain mitochondria produce higher levels of H2O2. Furthermore, Ah1–40 induces an impairment of the respiratory chain and a decrease in the oxidative phosphorylation efficiency. Insulin treatment prevents the mitochondrial alterations induced by diabetes or Ah1–40. : negative impact on mitochondria. : positive impact on mitochondria.

capacity to produce ATP, which may be related to the unchanged DCm (Table 2). The maintenance of oxidative phosphorylation capacity is extremely important in the brain since about 90% of the ATP required for the normal functioning of neurons is provided by the mitochondria. DCm, which normally accounts for 80% of the protomotive force, contributes for the high degree of reduction of the matrix NADPH/NADP+ pool and, in turn, this pool helps to maintain the matrix glutathione pool in the reduced state. So, the maintenance of DCm can also be correlated with the unchanged content of GSH (Figs. 2A and B). GSH is abundant in the mitochondria and is a first-line defense in the cellular antioxidant system. Baydas et al. (2003) observed that although STZ-diabetic rats presented higher levels of lipid peroxidation in hippocampus, cortex, and cerebellum as compared to control rats, no significant alterations were found in GSH levels in the same brain regions. Mitochondria are important cytoplasmic Ca2+ buffers since they avoid the increase of Ca2+ above a critical value termed bset pointQ. In oxidative stress conditions, a sustained increase in intracellular Ca2+ concentration occurs (Biessels et al., 2002). Changes in the cytosolic Ca2+ levels play a role in the modulation of several intracellular signaling pathways, including protein kinase C-a and calmodulin-dependent signaling (Clapham, 1995), which have also been implicated in apoptotic processes. The cytosolic Ca2+ level can be increased by ROS in various cell types through mobilization of intracellular Ca2+ stores or through the influx of extracellular Ca2+ (Dro¨gue, 2002). We observed that diabetes decreases the capacity of mitochondria to accumulate Ca2+ (Fig. 5), a favorable intracellular environment for mitochondrial permeability transition pore (MPTP)

opening. The MPTP is a phenomenon that is characterized by the opening of pores in the inner mitochondrial membrane and by its sensitivity to a very low concentration of cyclosporin A (CsA). Ca2+ and oxidative stress have long been known to favor the mitochondrial permeability transition (Zoratti and Szabo, 1995). Our results are in agreement with the bCalcium hypothesisQ that first proposes that among the many biochemical and histological changes involved in brain aging and in age-related diseases, Ca2+ alteration is a central defect (Khachaturian, 1994; Kostyuk et al., 2001). Recent findings (Stump et al., 2003) indicate that insulin is a major regulating factor of mitochondrial oxidative phosphorylation in human skeletal muscle. Previously, it was shown (Boirie et al., 2001) that insulin selectively stimulates mitochondrial protein synthesis in skeletal muscle and activates mitochondrial enzyme activity. However, a direct stimulatory action on ATP production was not shown. Our results are in accordance with these data because although we did not observe any significant change on ATP content, insulin treatment increased mitochondrial oxidative phosphorylation efficiency by decreasing the repolarization lag phase and increasing the repolarization level (Table 2). Furthermore, insulin was capable to increase mitochondrial antioxidant defenses (CoQ9 content) that had been reduced by diabetes (Fig. 3). Growing evidence suggests the importance of insulin and insulin growth factors (IGFs) in intracellular antioxidant status by playing a pivotal role in protein kinase B-mediated expression of Bcl2 protein, which prevents the escape of ROS by opposing the oxidative-stressinduced pro-apoptotic action of Bax (Hong et al., 2001). Another study showed that pretreatment of cells with IGF-1 suppresses

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H 2 O 2 -induced apoptosis by subsequent inhibition of Bax expression (Hong et al., 2001; Napier et al., 1999). Furthermore, Ko et al. (2001) demonstrated that insulin treatment induces an increase in myocardial vitamin C and a decrease in vitamin E. Our data also indicate that insulin is capable to increase the capacity of mitochondria to accumulate Ca2+ (Fig. 5), suggesting a role of insulin in Ca2+ homeostasis. Moreover, it has been shown that insulin modulates the cellular clearance of amyloid h (Gasparini et al., 2001) and IGF-1 protects neurons against its neurotoxic effects (Niikura et al., 2001). Recently, Rensink et al. (2004) reported that insulin inhibits amyloid h-induced cell death in cultured human brain pericytes. In accordance, our data indicate that insulin treatment also protects against mitochondrial injury induced by Ah1–40 (Fig. 4). Our data show that although diabetes induces some alterations in brain mitochondrial activity, those alterations do not interfere significantly with mitochondria functional efficiency. Similarly, insulin does not affect basal mitochondria function. However, in the presence of amyloid h-peptide, insulin prevents a drastic decline in mitochondrial oxidative phosphorylation efficiency and avoids an increase in the oxidative stress, improving or preserving the function of neurons under adverse conditions, such as Alzheimer’s disease (Fig. 6). In this line, insulin therapy, besides its well-known importance in the maintenance of glycemic control, may play an important role against brain mitochondrial dysfunction associated to several age-related disorders such as diabetes and Alzheimer’s disease. Acknowledgments We are grateful to Dr. Anto´nio Moreno for his helpful discussions. This work was supported by FCT (Portuguese Research Council). Paula Moreira is the recipient of a grant SFRH/BD/5320/2001. References Alvarsson, M., Sundkvist, G., Lager, I., Hemricsson, M., Berntorp, K., ¨ rn, Fernqvist-Forbes, E., Steen, L., Westermark, G., Weestremark, P., O T., Grill, V., 2003. Beneficial effects of insulin versus sulphonylurea on insulin secretion and metabolic control in recently diagnosed type 2 diabetic patients. Diabetes Care 26, 2231 – 2237. Aragno, M., Brignardello, E., Tamagno, E., Gatto, V., Danni, O., Boccuzzi, G., 1997. Dehydroepiandrosterone administration prevents the oxidative damage induced by acute hyperglycemia in rats. J. Endocrinol. 155, 233 – 240. Azari, N.P., Pettigrew, K.D., Schapiro, M.B., Haxby, J.V., Grady, C.L., Pietrini, P., Salerno, J.A., Heston, L.L., Rapoport, S.I., Horwitz, B.J., 1993. Early detection of Alzheimer’s disease: a statistical approach using positron emission tomographic data. Cereb. Blood Flow Metab. 13, 438 – 447. Barja, J., 1999. Mitochondrial oxygen radical generation and leak: sites of production in states 4 and 3, organ specificity, and relation to aging and longevity. J. Bioenerg. Biomembr. 31, 347 – 366. Baydas, G., Nedzvetskii, V.S., Tuzcu, M., Yasar, A., Kirichenko, S.V., 2003. Increase of glial fibrillary acidic protein and S-100B in hippocampus and cortex of diabetic rats: effects of vitamin E. Eur. J. Pharmacol. 462, 67 – 71. Baynes, J.W., 1991. Role of oxidative stress in development of complications in diabetes. Diabetes 40, 405 – 412. Behl, C., Davies, J.B., Lesley, R., et al., 1994. Hydrogen peroxide mediates amyloid beta protein toxicity. Cell 77, 817 – 827.

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