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Lutein effect on retina and hippocampus of diabetic mice
Free Radical Biology & Medicine 41 (2006) 979 – 984 www.elsevier.com/locate/freeradbiomed
Original Contribution
Lutein effect on retina and hippocampus of diabetic mice María Muriach a , Francisco Bosch-Morell a,b , George Alexander c , Rune Blomhoff c , Jorge Barcia a , Emma Arnal a , Inma Almansa a , Francisco J. Romero a,b,⁎, María Miranda a a
Departamento de Fisiología, Farmacología & Toxicología, Universidad CEU Cardenal Herrera, Valencia, Spain b Fundación Oftalmológica del Mediterráneo, Valencia, Spain c Institute for Nutrition Research, University Oslo, Oslo, Norway Received 24 March 2006; revised 7 June 2006; accepted 14 June 2006 Available online 4 July 2006
Abstract Oxidative stress markers and functional tests were studied to confirm early biochemical and functional changes in retina and hippocampus of diabetic mice. The effects of lutein treatment were also tested. Mice were induced diabetic by alloxan injection and divided into subgroups: control, control + lutein, diabetic, diabetic + lutein, diabetic + insulin, and diabetic + insulin + lutein. Treatments started on Day 4 after alloxan injection and animals were sacrificed on Day 14. Malondialdehyde and glutathione concentrations and glutathione peroxidase activity were measured as oxidative stress markers. The following functional tests for retina and hippocampus were performed: electroretinogram and Morris water maze test. NFκB activity was also measured. Oxidative stress and NFκB activity increase in the retina and hippocampus after 15 days of diabetes. Impairment of the electroretinogram and a correlation between latencies of the water maze test and glycated hemoglobin (HbA1c) levels were observed. Lutein prevented all these changes even under hyperglycemic conditions. Retina appears to be affected earlier than hippocampus by diabetes-induced oxidative stress. Although a proper glycemic control is desirable in preventing the development of diabetic complications, it is not sufficient to prevent them completely. Lutein could be an appropriate coadjuvant treatment for the changes observed in this study. © 2006 Elsevier Inc. All rights reserved. Keywords: Diabetes; Oxidative stress; Retina; Hippocampus; Lutein
Introduction Although strict glycemic control is desirable for preventing diabetes complications, this is seldom achievable and sustained high plasma glucose levels may lead to microangiopathy. Clearly, adjuvant therapies are needed to help in preventing or delaying the onset of diabetes complications. It has been repeatedly suggested that oxidative stress is involved in the pathogenesis of late diabetes complications [1], though it is not definitely demonstrated if this is the cause or the consequence of these complications [2]. It is clear that the Abbreviations: DM, diabetes mellitus; ROS, reactive oxygen species; NFκB, transcription factor-kappaB; GPx, glutathione peroxidase; MDA, malondialdehyde; HbA1c, glycated hemoglobin; ERG, electroretinogram. ⁎ Corresponding author. Department of Physiology, Pharmacology & Toxicology, Universidad CEU Cardenal Herrera, Av. Seminario s/n, 46113Moncada, Valencia, Spain. Fax: +34 961395272. E-mail address: [email protected] (F.J. Romero). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.023
elevated glucose levels present in diabetes and the existence of oxidative stress are inseparable [3]. Hyperglycemia reduces antioxidant levels and concomitantly increases the production of free radicals. These effects contribute to tissue damage in diabetes mellitus (DM), leading to alterations in the redox potential of the cell with subsequent activation of redox-sensitive genes [4]. The production of reactive oxygen species (ROS) leads to the activation of the transcription factor-kappaB (NFκB), which is a redox-sensitive transcription factor involved in immune and inflammatory responses. The DM-induced production of ROS activates NFκB, which, in turn, translocates from the cytosol into the nucleus and subsequently activates a variety of target genes which are linked to the development of diabetic complications. In retina, for example, it was demonstrated [5] that the activation of retinal NFκB in DM is an early event in the development of retinopathy [5]. These elevated levels of NFκB last until retinal capillary cell death occurs.
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DM has long been considered a risk factor for the development of vascular pathologies. Furthermore, epidemiological evidence suggests that DM significantly increases risk for the development of Alzheimer's disease, in a way independent of vascular risk factors [6]. Decreased peripheral glucose regulation has been shown to be associated with decreased general cognitive performance, memory impairment, and atrophy of the hippocampus in humans, a brain area that is key for learning and memory [7]. However, one of the mechanisms by which hyperglycemia is known to cause neural degeneration is via the increased oxidative stress that accompanies diabetes. This is further evidenced by the observation that metabolic and oxidative insults associated to DM often cause rapid changes in glial cells [8]. Other experimental studies [9] suggested that neuronal apoptosis, which is related to NFκB activation, may play an important role in neuronal loss and impaired cognitive function. Additionally, in the hippocampi of streptozotocin-treated rats, not only a strong increase in oxygen reactive species is observed but also a persistent activation of NFκB [10]. Diabetic retinopathy is the first cause of adult blindness in industrialized countries [11]. Strict glycemic control is desirable for preventing diabetic complications, but it is not always achievable. Moreover, it has been observed that in humans resistant to insulin, this impaired glycemic control is associated with decreased general cognitive performance and atrophy of the hippocampus [7]. These make adjuvant therapies necessary to help in preventing or delaying the onset of diabetic complications. The biochemical and functional changes in the retina and hippocampus of diabetic mice, and the ability of lutein, one of the two major carotenoids in the human macula and retina [12] to reverse these effects, have been studied, compared to the effect of insulin therapy.
until the end of the experiment. Mice were killed by cervical dislocation on Day 14, and hippocampus and retina were removed and homogenized in prechilled 0.2 M potassium phosphate buffer, pH 7.0. These homogenates were used to assay glutathione peroxidase (GPx) activity and protein, malondialdehyde (MDA), and glutathione (GSH) concentrations. Samples were kept frozen (- 80° C) until biochemical assays were performed. Blood was taken from the tail vein daily to assay blood glucose levels. Glycated hemoglobin (HbA1c) was determined in blood samples obtained by heart puncture immediately before the animals were killed.
Materials and methods
Transgenic mice containing the NFκB-luciferase reporter gene were used for this assay. The NFκB-reporting transgenic mice were heterozygous 3x-kb-luc mice with a (C57BL/6J) genetic background. These mice were housed as described above. After 15 days of diabetic condition, mice were killed, and retina and hippocampus were obtained. Tissue homogenates luminescence was assayed with a luminometer according to Alexander et al. [19], and expressed as arbitrary units per microgram of tissue protein.
Experimental design Male albino mice (5-6 weeks of age) housed in an environmentally regulated room on a dark-light cycle of 12 h with free access to food and water were used throughout the study. Principles of laboratory animal care (NIH Publication No. 85-23, revised 1985) were followed, as well as specific Spanish regulations. Animals were made diabetic with a single subcutaneous injection of 200 mg alloxan/kg body weight (66 mg/ml) in 0.1 M citrate buffer, pH 4.5, at Day 0 of the experiment. Control mice were injected with the same volume of vehicle also at Day 0 of the experiment. Mice were identified as diabetic on the basis of blood glucose levels (higher than 16 mM at least 4 days after alloxan treatment). Animals were divided into subgroups as required by the experiment (control, control + lutein, diabetic, diabetic + lutein, diabetic + insulin, and diabetic + insulin + lutein). Lutein (Sigma Química, Alcobendas, Madrid) (70% purity, 0.2 mg/kg body weight, administered by stomach tube) and insulin (500 mU/g body weight, injected intraperitoneally) daily treatments started on Day 4 after alloxan injection and lasted
Biochemical assays MDA concentration was measured by liquid chromatography according to a modification of the method of Richard et al. [13], as previously described [14]. GPx activity was assayed as reported by Lawrence et al. [15] toward hydrogen peroxide; the GSH content of the retina was quantified by the method of Reed et al. [16]; and protein content was measured by means of the Lowry method [17]. Electroretinogram (ERG) ERG was carried out under scotopic conditions and registered in McLab software. B-wave amplitude was measured. Morris water maze test Spatial learning and memory were tested using the Morris water maze test [18]. Latency times were measured in minutes. NF-kappa B
Statistical analysis The results are presented as mean values ± SE. Statistical significance was assessed by ANOVA followed by the Student t test. The level of significance was set at P < 0.05. Differences between groups in the water maze data were established by means of the ANOVA test for the different measurements. Results The diabetes model in mice, 14 days after alloxan injection, was used to achieve hyperglycemia, which was confirmed by body weight changes (Fig. 1A), blood glucose levels (Fig. 1B),
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Fig. 1. Diabetes-induced changes of weight and metabolic parameters in control and mice with different treatments. Body weight changes (A), blood glucose levels (B), glycated hemoglobin (C), and serum MDA concentrations (D) from control (c), control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs control group, **P < 0.05 vs diabetic group.
and percentage of glycated hemoglobin (Fig. 1C). Lutein treatment did not alter the hyperglycemic status of alloxan diabetic mice. MDA concentration in blood was elevated in diabetic animals, confirming the overall existence of an oxidative burden (Fig. 1D). Lutein and/or insulin treatments restored MDA concentrations in serum. Retina Ocular MDA concentration (Fig. 2A) was higher than controls, whereas GSH concentration (Fig. 2B) and GPx
activity (Fig. 2C) decreased in the diabetic retina. ERG Bwave amplitude decreased in diabetic animals respect to controls (Fig. 2D). Lutein treatment restored MDA and GSH levels as well as GPx activity and ERG B-wave amplitude to control values (Figs. 2A to D). Insulin alone was only able to recover GPx activity and ERG B-wave amplitude to control data. No major changes were observed when using insulin and lutein together on any of these parameters in this tissue. NFκB activity was increased in the retina of diabetic mice and all treatments used were able to prevent this increase (Fig. 5A).
Fig. 2. Diabetes-induced changes of biochemical and functional parameters in control mouse retina and treated animals. Percentage of control values of: (A) MDA concentration (control = 0.59 nmol/mg protein), (B) GSH concentration (control = 23.7 nmol/mg protein), (C) GPx activity (control = 383 nmol/mg protein × min), and (D) B-wave amplitude of the ERG (control = 46.2 mv), in the retina from control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs control group, **P < 0.05 vs diabetic group.
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Hippocampus MDA (Fig. 3A) and GSH (Fig. 3B) contents, as well as GPx (Fig. 3C) and NFκB (Fig. 5B) activities, were assayed in hippocampus homogenate. MDA concentration and NFκB activity increased, whereas GSH content and GPx activity decreased under diabetic conditions. Lutein and/or insulin treatment restored all markers to their control values. Animals were examined for spatial learning ability, and no statistical significant differences were observed in the task acquisition between diabetic and control mice (Fig. 3D). A positive correlation, statistically significant (P < 0.01), was established between the HbA1c values and the accumulated latencies to find the hidden platform of all animals without lutein (Fig. 4). Discussion Because of high levels of polyunsaturated lipids, nervous tissue is markedly sensitive to oxygen free radical damage. Our results show that after 2 weeks of diabetes, MDA levels in retina and hippocampus of diabetic mice are increased when compared to controls and that lutein and insulin are able to prevent these effects. Moreover, it is accepted that MDA determination by HPLC is a good marker of oxidative stress implication in a pathological process [20]. GPx activity and glutathione content were decreased after 2 weeks under diabetic conditions, not only in retina but also in hippocampus. Likewise, ERG B-wave amplitude was decreased in diabetic mice when compared to control, indicating a retinal functional impairment in diabetic mice. Within the central nervous system, the hippocampus is considered a special target for alterations associated with
Fig. 4. Correlation between HbA1c values and accumulated latency to find the hidden platform, without lutein-treated mice, R = 0.388, P < 0.01.
diabetes [21]. Latency time to find a hidden platform in the Morris water maze is widely used to test spatial memory, a hippocampus-dependent task. There are studies showing that this test resulted in significantly prolonged latencies in 8-month diabetic rats, but not after 2 months of diabetes, and that this cognitive impairment in type 1 diabetes is associated with a duration-related apoptosis-induced neuronal loss [22]. Our results are in agreement with these studies, suggesting that 2 weeks of diabetic condition are not enough time to cause spatial memory loss, and that the oxidative stress situation is only starting in this tissue, in contrast to what happens in the retina after 2 weeks (Fig. 2D). The statistically significant correlation between HbA1c values (i.e., prolonged high glucose levels) and accumulated latencies to find the hidden platform (Fig. 4) suggests an initial status of hippocampal impairment. A
Fig. 3. Diabetes-induced changes of biochemical and functional parameters in control mouse hippocampus and treated animals. Percentage of control values in: (A) MDA concentration (control = 0.77 nmol/mg protein), (B) GSH concentration (control = 22.6 nmol/mg protein), (C) GPx activity (control = 103 nmol/mg protein × min), and (D) accumulated latency to find the hidden platform (control = 22.3 min), in the hippocampus from control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs all groups.
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the hippocampus of type 1 diabetic rats [22]. Peroxynitrite S (ONOO-), the product of the reaction between nitric oxid ( NO) and superoxide, has been suggested to be involved in apoptotic S cell death; therefore, cells that constitutively express NO synthase, such as neurons, may be more vulnerable to ONOO–induced cell death in conditions favoring the production of superoxides [28]. Although a proper glycemic control is desirable in reducing the development of diabetic complications, it is not sufficient to prevent them completely; therefore, lutein, and antioxidant that has been suggested to regulate NFκB activity [29], and with peroxynitrite scavenging properties [30], seems to be an appropriate coadjuvant treatment for the impairments observed in this study. The fact that insulin and lutein both prevent biochemical and functional changes, whereas insulin but not lutein normalizes glycemia, further supports a key role for NFκB, that under diabetic conditions may be chronically activated through an oxidative/nitrosative mechanism. Further studies are needed to prove this hypothesis. Acknowledgments
Fig. 5. NF-κB activity in control and diabetic mice. Percentage of NF-κB activity from control values in: (A) retina (control = 6.4 arbitrary units/μg protein) and (B) hippocampus (control = 2.1 arbitrary units/μg protein). *P < 0.05 vs all groups.
figure representing all data, including lutein groups, gives a worse correlation than that in Fig. 4 (data not shown), confirming that there is already an initial correlation between high glucose levels (i.e., HbA1c percentage) and hippocampal function, and that lutein exerts a positive effect on these changes. Our group has previously described that after 1 week under diabetic conditions there is a modification in retinal oxidative stress markers, as well as impairment in the electroretinogram [23,24]. In these reports, no statistical differences between the hippocampal oxidative stress markers or functional tests of diabetic and control mice were observed (data not shown). NFκB is a redox-sensitive nuclear factor, which is involved in the control of a large number of normal cellular and organ processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. In fact, it can induce antiapoptotic or proapoptotic genes, depending on the size and duration of the insult [25]. The results herein show that NFκB activity is increased in both retina and hippocampus of diabetic mice (Figs. 5A and B), and that this could be an indicator of inflammatory response. However, the NFκB increases observed after 2 weeks could be considered to be evidence of a chronic NFκB elevation. Chronic elevated NFκB is almost always associated with a negative disease outcome. Apoptosis occurs in diabetes as well as in its chronic complications, like retinopathy [26], and more recently apoptosis has been demonstrated in neuropathy [27] and in
M. Muriach was a research fellow from the Fundación Universitaria San Pablo-CEU. This work was supported by projects PI03/1710 from the Fondo de Investigación Sanitaria to F.B.-M., and PRUCH04/30 to F.J.R. References [1] Baynes, J. W.; Thorpe, S. R. The role of oxidative stress in diabetic complications. Curr. Opin. Endocrinol. 3:277–284; 1996. [2] Baynes, J. W.; Thorpe, S. R. Role of oxidatives stress in diabetic complications. A new perspective on an old paradigm. Diabetes 48:1–9; 1999. [3] Packer, L.; Kraemer, K.; Rimbach, G. Molecular aspects of lipoic acid in the prevention of diabetic complications. Nutrition 17:888–895; 2001. [4] Bonnefont-Rousselot, D. Glucose and reactive oxygen species. Curr. Opin. Clin. Nutr. Metab. Care 5:561–568; 2002. [5] Kowluru, R. A.; Koppolu, P.; Chakrabarti, S.; Chen, S. Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants. Free Radical Res. 37:1169–1180; 2003. [6] Grossman, H. Does diabetes protect or provoke Alzheimer's disease? Insights into the pathobiology and future treatment of Alzheimer's disease. CNS Spectr. 8:815–823; 2003. [7] Convit, A.; Wolf, O. T.; Tarshish, C.; de Leon, M. J. Reduced glucose tolerance is associated with poor memory performance and hippocampal atrophy among normal elderly. Proc. Natl. Acad. Sci. U. S. A. 100: 2019–2022; 2003. [8] Baydas, G.; Reiter, R. J.; Yasar, A.; Tuzcu, M.; Akdemir, I.; Nedzvetskii, V. S. Melatonin reduces glial reactivity in the hippocampus, cortex, and cerebellum of streptozotocin-induced diabetic rats. Free Radical Biol. Med. 35:797–804; 2003. [9] Li, Z. G.; Sima, A. A. C-peptide and central nervous system complications in diabetes. Exp. Diab. Res. 5:79–90; 2004. [10] Aragno, M.; Mastrocola, R.; Brignardello, E.; Catalano, M.; Robino, G.; Manti, R.; Parola, M.; Danni, O.; Boccuzzi, G. Dehydroepiandrosterone modulates nuclear factor-kappaB activation in hippocampus of diabetic rats. Endocrinology 143:3250–3258; 2002. [11] Trautner, C.; Haastert, B.; Giani, G.; Berger, M. Incidence of blindness in southern Germany between 1990 and 1998. Diabetologia 44:147–150; 2001. [12] Handelman, G. J.; Dratz, E. A.; Reay, C. C.; Van Kuijk, J. G. Carotenoids in the human macula and whole retina. Invest. Ophthalmol. Vis. Sci. 29:850–855; 1988.
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[13] Richard, M. J.; Guiraud, P.; Meo, J.; Favier, A. High performance liquid chromatography separation of malondialdehyde thiobarbituric acid adduct in biological materials (plasma and human cell) using a commercially available reagent. J. Chromatogr. 577:9–18; 1992. [14] Romero, M. J.; Bosch-Morell, F.; Romero, B.; Rodrigo, J. M.; Serra, M. A.; Romero, F. J. Serum malondialdehyde: possible use for the clinical management of chronic hepatitis C patients. Free Radical Biol. Med. 25: 993–997; 1998. [15] Lawrence, R. A.; Parkhill, L. K.; Burk, R. F. Hepatic cytosolic nonselenium dependent glutathione peroxidase activity: its nature and the effect of selenium deficiency. J. Nutr. 108:981–987; 1978. [16] Reed, D. J.; Babson, J. R.; Beatty, P. W.; Brodie, A. E.; Ellis, W. W.; Potter, D. W. High-performance liquid chromatography analysis of nanomole levels of glutathione, glutathione disulfide, and related thiols and disulfides. Anal. Biochem. 106:55–62; 1980. [17] Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265–275; 1951. [18] Morris, R. G.; Garrud, P.; Rawlins, J. N.; O'Keefe, J. Place navigation impaired in rats with hippocampal lesions. Nature 24:681–683; 1982. [19] Alexander, G.; Carlsen, H.; Blomhoff, R. Strong in vivo activation of NFkappaB in mouse lenses by classic stressors. Invest. Ophthalmol. Vis. Sci. 44:2683–2688; 2003. [20] Halliwell, B. Oxidative stress markers in human disease: application to diabetes and to evaluation of the effects of antioxidants. In: Packer, L., Rösen, P., Tritschler, H.J., King, G.L., Azzi, A. (Eds.), Antioxidants in Diabetes Management. Dekker, New York,pp. 33–62; 2000. [21] Biessels, G. J.; Van der Heide, L. P.; Kamal, A.; Bleys, R. L.; Gispen, W. H. Ageing and diabetes: implications for brain function. Eur. J. Pharmacol. 441:1–14; 2002.
[22] Li, Z. G.; Zhang, W.; Grunberger, G.; Sima, A. A. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res. 946:221–231; 2002. [23] Miranda, M.; Muriach, M.; Johnsen, S.; Bosch-Morell, F.; Araiz, J.; Roma, J.; Romero, F. J. Oxidative stress in a model for experimental diabetic retinopathy: treatment with antioxidants. Arch. Soc. Esp. Oftalmol. 79: 289–294; 2004. [24] Miranda, M.; Muriach, M.; Romá, J.; Bosch-Morell, F.; Genovés, J. M.; Barcia, J.; Araiz, J.; Diaz-Llopis, M.; Romero, F. J. Oxidative stress in a model of experimental diabetic retinopathy. II. Peroxynitrites scavengers utility. Arch. Soc. Esp. Oftalmol. 80:27–32; 2006. [25] Clemens, J. A.; Stephenson, D. T.; Smalstig, E. B.; Dixon, E. P.; Little, S. P. Global ischemia activates nuclear factor-kappa B in forebrain neurons of rats. Stroke 28:1073–1080; 1997. [26] Barber, A. J.; Lieth, E.; Khin, S. A.; Antonetti, D. A.; Buchanan, A. G.; Gardner, T. W. The Penn State Research Group. Neural apoptosis in the retina during experimental and human diabetes. J. Clin. Invest. 102: 783–791; 1998. [27] Cellek, S.; Qu, W.; Schmidt, A. M.; Moncada, S. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes. Diabetologia 47:331–339; 2004. [28] Cowell, R. M.; Russell, J. W. Nitrosative injury and antioxidant therapy in the management of diabetic neuropathy. J. Invest. Med. 52:33–44; 2004. [29] Chew, B. P.; Brown, C. M.; Park, J. S.; Mixter, P. F. Dietary lutein inhibits mouse mammary tumor growth by regulating angiogenesis and apoptosis. Anticancer Res. 23:3333–3339; 2003. [30] Panasenko, O. M.; Sharov, V. S.; Briviba, K.; Sies, H. Interaction of peroxynitrite with carotenoids in human low density lipoproteins. Arch. Biochem. Biophys. 373:302–305; 2000.
Original Contribution
Lutein effect on retina and hippocampus of diabetic mice María Muriach a , Francisco Bosch-Morell a,b , George Alexander c , Rune Blomhoff c , Jorge Barcia a , Emma Arnal a , Inma Almansa a , Francisco J. Romero a,b,⁎, María Miranda a a
Departamento de Fisiología, Farmacología & Toxicología, Universidad CEU Cardenal Herrera, Valencia, Spain b Fundación Oftalmológica del Mediterráneo, Valencia, Spain c Institute for Nutrition Research, University Oslo, Oslo, Norway Received 24 March 2006; revised 7 June 2006; accepted 14 June 2006 Available online 4 July 2006
Abstract Oxidative stress markers and functional tests were studied to confirm early biochemical and functional changes in retina and hippocampus of diabetic mice. The effects of lutein treatment were also tested. Mice were induced diabetic by alloxan injection and divided into subgroups: control, control + lutein, diabetic, diabetic + lutein, diabetic + insulin, and diabetic + insulin + lutein. Treatments started on Day 4 after alloxan injection and animals were sacrificed on Day 14. Malondialdehyde and glutathione concentrations and glutathione peroxidase activity were measured as oxidative stress markers. The following functional tests for retina and hippocampus were performed: electroretinogram and Morris water maze test. NFκB activity was also measured. Oxidative stress and NFκB activity increase in the retina and hippocampus after 15 days of diabetes. Impairment of the electroretinogram and a correlation between latencies of the water maze test and glycated hemoglobin (HbA1c) levels were observed. Lutein prevented all these changes even under hyperglycemic conditions. Retina appears to be affected earlier than hippocampus by diabetes-induced oxidative stress. Although a proper glycemic control is desirable in preventing the development of diabetic complications, it is not sufficient to prevent them completely. Lutein could be an appropriate coadjuvant treatment for the changes observed in this study. © 2006 Elsevier Inc. All rights reserved. Keywords: Diabetes; Oxidative stress; Retina; Hippocampus; Lutein
Introduction Although strict glycemic control is desirable for preventing diabetes complications, this is seldom achievable and sustained high plasma glucose levels may lead to microangiopathy. Clearly, adjuvant therapies are needed to help in preventing or delaying the onset of diabetes complications. It has been repeatedly suggested that oxidative stress is involved in the pathogenesis of late diabetes complications [1], though it is not definitely demonstrated if this is the cause or the consequence of these complications [2]. It is clear that the Abbreviations: DM, diabetes mellitus; ROS, reactive oxygen species; NFκB, transcription factor-kappaB; GPx, glutathione peroxidase; MDA, malondialdehyde; HbA1c, glycated hemoglobin; ERG, electroretinogram. ⁎ Corresponding author. Department of Physiology, Pharmacology & Toxicology, Universidad CEU Cardenal Herrera, Av. Seminario s/n, 46113Moncada, Valencia, Spain. Fax: +34 961395272. E-mail address: [email protected] (F.J. Romero). 0891-5849/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2006.06.023
elevated glucose levels present in diabetes and the existence of oxidative stress are inseparable [3]. Hyperglycemia reduces antioxidant levels and concomitantly increases the production of free radicals. These effects contribute to tissue damage in diabetes mellitus (DM), leading to alterations in the redox potential of the cell with subsequent activation of redox-sensitive genes [4]. The production of reactive oxygen species (ROS) leads to the activation of the transcription factor-kappaB (NFκB), which is a redox-sensitive transcription factor involved in immune and inflammatory responses. The DM-induced production of ROS activates NFκB, which, in turn, translocates from the cytosol into the nucleus and subsequently activates a variety of target genes which are linked to the development of diabetic complications. In retina, for example, it was demonstrated [5] that the activation of retinal NFκB in DM is an early event in the development of retinopathy [5]. These elevated levels of NFκB last until retinal capillary cell death occurs.
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DM has long been considered a risk factor for the development of vascular pathologies. Furthermore, epidemiological evidence suggests that DM significantly increases risk for the development of Alzheimer's disease, in a way independent of vascular risk factors [6]. Decreased peripheral glucose regulation has been shown to be associated with decreased general cognitive performance, memory impairment, and atrophy of the hippocampus in humans, a brain area that is key for learning and memory [7]. However, one of the mechanisms by which hyperglycemia is known to cause neural degeneration is via the increased oxidative stress that accompanies diabetes. This is further evidenced by the observation that metabolic and oxidative insults associated to DM often cause rapid changes in glial cells [8]. Other experimental studies [9] suggested that neuronal apoptosis, which is related to NFκB activation, may play an important role in neuronal loss and impaired cognitive function. Additionally, in the hippocampi of streptozotocin-treated rats, not only a strong increase in oxygen reactive species is observed but also a persistent activation of NFκB [10]. Diabetic retinopathy is the first cause of adult blindness in industrialized countries [11]. Strict glycemic control is desirable for preventing diabetic complications, but it is not always achievable. Moreover, it has been observed that in humans resistant to insulin, this impaired glycemic control is associated with decreased general cognitive performance and atrophy of the hippocampus [7]. These make adjuvant therapies necessary to help in preventing or delaying the onset of diabetic complications. The biochemical and functional changes in the retina and hippocampus of diabetic mice, and the ability of lutein, one of the two major carotenoids in the human macula and retina [12] to reverse these effects, have been studied, compared to the effect of insulin therapy.
until the end of the experiment. Mice were killed by cervical dislocation on Day 14, and hippocampus and retina were removed and homogenized in prechilled 0.2 M potassium phosphate buffer, pH 7.0. These homogenates were used to assay glutathione peroxidase (GPx) activity and protein, malondialdehyde (MDA), and glutathione (GSH) concentrations. Samples were kept frozen (- 80° C) until biochemical assays were performed. Blood was taken from the tail vein daily to assay blood glucose levels. Glycated hemoglobin (HbA1c) was determined in blood samples obtained by heart puncture immediately before the animals were killed.
Materials and methods
Transgenic mice containing the NFκB-luciferase reporter gene were used for this assay. The NFκB-reporting transgenic mice were heterozygous 3x-kb-luc mice with a (C57BL/6J) genetic background. These mice were housed as described above. After 15 days of diabetic condition, mice were killed, and retina and hippocampus were obtained. Tissue homogenates luminescence was assayed with a luminometer according to Alexander et al. [19], and expressed as arbitrary units per microgram of tissue protein.
Experimental design Male albino mice (5-6 weeks of age) housed in an environmentally regulated room on a dark-light cycle of 12 h with free access to food and water were used throughout the study. Principles of laboratory animal care (NIH Publication No. 85-23, revised 1985) were followed, as well as specific Spanish regulations. Animals were made diabetic with a single subcutaneous injection of 200 mg alloxan/kg body weight (66 mg/ml) in 0.1 M citrate buffer, pH 4.5, at Day 0 of the experiment. Control mice were injected with the same volume of vehicle also at Day 0 of the experiment. Mice were identified as diabetic on the basis of blood glucose levels (higher than 16 mM at least 4 days after alloxan treatment). Animals were divided into subgroups as required by the experiment (control, control + lutein, diabetic, diabetic + lutein, diabetic + insulin, and diabetic + insulin + lutein). Lutein (Sigma Química, Alcobendas, Madrid) (70% purity, 0.2 mg/kg body weight, administered by stomach tube) and insulin (500 mU/g body weight, injected intraperitoneally) daily treatments started on Day 4 after alloxan injection and lasted
Biochemical assays MDA concentration was measured by liquid chromatography according to a modification of the method of Richard et al. [13], as previously described [14]. GPx activity was assayed as reported by Lawrence et al. [15] toward hydrogen peroxide; the GSH content of the retina was quantified by the method of Reed et al. [16]; and protein content was measured by means of the Lowry method [17]. Electroretinogram (ERG) ERG was carried out under scotopic conditions and registered in McLab software. B-wave amplitude was measured. Morris water maze test Spatial learning and memory were tested using the Morris water maze test [18]. Latency times were measured in minutes. NF-kappa B
Statistical analysis The results are presented as mean values ± SE. Statistical significance was assessed by ANOVA followed by the Student t test. The level of significance was set at P < 0.05. Differences between groups in the water maze data were established by means of the ANOVA test for the different measurements. Results The diabetes model in mice, 14 days after alloxan injection, was used to achieve hyperglycemia, which was confirmed by body weight changes (Fig. 1A), blood glucose levels (Fig. 1B),
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Fig. 1. Diabetes-induced changes of weight and metabolic parameters in control and mice with different treatments. Body weight changes (A), blood glucose levels (B), glycated hemoglobin (C), and serum MDA concentrations (D) from control (c), control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs control group, **P < 0.05 vs diabetic group.
and percentage of glycated hemoglobin (Fig. 1C). Lutein treatment did not alter the hyperglycemic status of alloxan diabetic mice. MDA concentration in blood was elevated in diabetic animals, confirming the overall existence of an oxidative burden (Fig. 1D). Lutein and/or insulin treatments restored MDA concentrations in serum. Retina Ocular MDA concentration (Fig. 2A) was higher than controls, whereas GSH concentration (Fig. 2B) and GPx
activity (Fig. 2C) decreased in the diabetic retina. ERG Bwave amplitude decreased in diabetic animals respect to controls (Fig. 2D). Lutein treatment restored MDA and GSH levels as well as GPx activity and ERG B-wave amplitude to control values (Figs. 2A to D). Insulin alone was only able to recover GPx activity and ERG B-wave amplitude to control data. No major changes were observed when using insulin and lutein together on any of these parameters in this tissue. NFκB activity was increased in the retina of diabetic mice and all treatments used were able to prevent this increase (Fig. 5A).
Fig. 2. Diabetes-induced changes of biochemical and functional parameters in control mouse retina and treated animals. Percentage of control values of: (A) MDA concentration (control = 0.59 nmol/mg protein), (B) GSH concentration (control = 23.7 nmol/mg protein), (C) GPx activity (control = 383 nmol/mg protein × min), and (D) B-wave amplitude of the ERG (control = 46.2 mv), in the retina from control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs control group, **P < 0.05 vs diabetic group.
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Hippocampus MDA (Fig. 3A) and GSH (Fig. 3B) contents, as well as GPx (Fig. 3C) and NFκB (Fig. 5B) activities, were assayed in hippocampus homogenate. MDA concentration and NFκB activity increased, whereas GSH content and GPx activity decreased under diabetic conditions. Lutein and/or insulin treatment restored all markers to their control values. Animals were examined for spatial learning ability, and no statistical significant differences were observed in the task acquisition between diabetic and control mice (Fig. 3D). A positive correlation, statistically significant (P < 0.01), was established between the HbA1c values and the accumulated latencies to find the hidden platform of all animals without lutein (Fig. 4). Discussion Because of high levels of polyunsaturated lipids, nervous tissue is markedly sensitive to oxygen free radical damage. Our results show that after 2 weeks of diabetes, MDA levels in retina and hippocampus of diabetic mice are increased when compared to controls and that lutein and insulin are able to prevent these effects. Moreover, it is accepted that MDA determination by HPLC is a good marker of oxidative stress implication in a pathological process [20]. GPx activity and glutathione content were decreased after 2 weeks under diabetic conditions, not only in retina but also in hippocampus. Likewise, ERG B-wave amplitude was decreased in diabetic mice when compared to control, indicating a retinal functional impairment in diabetic mice. Within the central nervous system, the hippocampus is considered a special target for alterations associated with
Fig. 4. Correlation between HbA1c values and accumulated latency to find the hidden platform, without lutein-treated mice, R = 0.388, P < 0.01.
diabetes [21]. Latency time to find a hidden platform in the Morris water maze is widely used to test spatial memory, a hippocampus-dependent task. There are studies showing that this test resulted in significantly prolonged latencies in 8-month diabetic rats, but not after 2 months of diabetes, and that this cognitive impairment in type 1 diabetes is associated with a duration-related apoptosis-induced neuronal loss [22]. Our results are in agreement with these studies, suggesting that 2 weeks of diabetic condition are not enough time to cause spatial memory loss, and that the oxidative stress situation is only starting in this tissue, in contrast to what happens in the retina after 2 weeks (Fig. 2D). The statistically significant correlation between HbA1c values (i.e., prolonged high glucose levels) and accumulated latencies to find the hidden platform (Fig. 4) suggests an initial status of hippocampal impairment. A
Fig. 3. Diabetes-induced changes of biochemical and functional parameters in control mouse hippocampus and treated animals. Percentage of control values in: (A) MDA concentration (control = 0.77 nmol/mg protein), (B) GSH concentration (control = 22.6 nmol/mg protein), (C) GPx activity (control = 103 nmol/mg protein × min), and (D) accumulated latency to find the hidden platform (control = 22.3 min), in the hippocampus from control + lutein (c + l), diabetic (d), diabetic + lutein (d + l), diabetic + insulin (d + i), and diabetic + insulin + lutein (d + i + l) mice. *P < 0.05 vs all groups.
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the hippocampus of type 1 diabetic rats [22]. Peroxynitrite S (ONOO-), the product of the reaction between nitric oxid ( NO) and superoxide, has been suggested to be involved in apoptotic S cell death; therefore, cells that constitutively express NO synthase, such as neurons, may be more vulnerable to ONOO–induced cell death in conditions favoring the production of superoxides [28]. Although a proper glycemic control is desirable in reducing the development of diabetic complications, it is not sufficient to prevent them completely; therefore, lutein, and antioxidant that has been suggested to regulate NFκB activity [29], and with peroxynitrite scavenging properties [30], seems to be an appropriate coadjuvant treatment for the impairments observed in this study. The fact that insulin and lutein both prevent biochemical and functional changes, whereas insulin but not lutein normalizes glycemia, further supports a key role for NFκB, that under diabetic conditions may be chronically activated through an oxidative/nitrosative mechanism. Further studies are needed to prove this hypothesis. Acknowledgments
Fig. 5. NF-κB activity in control and diabetic mice. Percentage of NF-κB activity from control values in: (A) retina (control = 6.4 arbitrary units/μg protein) and (B) hippocampus (control = 2.1 arbitrary units/μg protein). *P < 0.05 vs all groups.
figure representing all data, including lutein groups, gives a worse correlation than that in Fig. 4 (data not shown), confirming that there is already an initial correlation between high glucose levels (i.e., HbA1c percentage) and hippocampal function, and that lutein exerts a positive effect on these changes. Our group has previously described that after 1 week under diabetic conditions there is a modification in retinal oxidative stress markers, as well as impairment in the electroretinogram [23,24]. In these reports, no statistical differences between the hippocampal oxidative stress markers or functional tests of diabetic and control mice were observed (data not shown). NFκB is a redox-sensitive nuclear factor, which is involved in the control of a large number of normal cellular and organ processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. In fact, it can induce antiapoptotic or proapoptotic genes, depending on the size and duration of the insult [25]. The results herein show that NFκB activity is increased in both retina and hippocampus of diabetic mice (Figs. 5A and B), and that this could be an indicator of inflammatory response. However, the NFκB increases observed after 2 weeks could be considered to be evidence of a chronic NFκB elevation. Chronic elevated NFκB is almost always associated with a negative disease outcome. Apoptosis occurs in diabetes as well as in its chronic complications, like retinopathy [26], and more recently apoptosis has been demonstrated in neuropathy [27] and in
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