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Improvement of dyslipidemia in OLETF rats by the prostaglandin I2 analog beraprost sodium
Prostaglandins & other Lipid Mediators 93 (2010) 14–19
Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators
Improvement of dyslipidemia in OLETF rats by the prostaglandin I2 analog beraprost sodium Maho Watanabe, Hitoshi Nakashima ∗ , Kenji Ito, Katsuhisa Miyake, Takao Saito Division of Nephrology and Rheumatology, Department of Internal Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonann-ku, Fukuoka 814-0180, Japan
a r t i c l e
i n f o
Article history: Received 21 December 2009 Received in revised form 12 April 2010 Accepted 27 April 2010 Available online 5 May 2010 Keywords: Prostaglandin I2 Beraprost sodium (BPS) Dyslipidemia OLETF rat LETO rat
a b s t r a c t The Otsuka Long-Evans Tokushima Fatty (OLETF) rat was established as an animal model of human type 2 diabetes. Improvement of dyslipidemia by BPS has been confirmed in OLETF rats. The aim of this report is to clarify the mechanisms associated with improvement of dyslipidemia by BPS in OLETF rats. We divided male OLETF rats into three groups; 400 g/kg BPS treated (Group H), 200 g/kg BPS treated (Group L), and untreated control (Group C). After sacrifice, using the quantitative real-time PCR, we assayed the transcription levels of the HMG-CoA reductase (Hmgcr) for cholesterol biosynthesis, monoacylglycerol O-acyltransferase 1 (Mogat1) as TG synthetase, hepatic triglyceride lipase (Lipc) and lipoprotein lipase (Lpl) as triglycerides (TG) reductase in the liver. The mRNA expression of transketolase (Tkt) for pentose phosphate pathway (PPP) enzyme was also evaluated in the liver and kidney. Hmgcr and Mogat1 RNA expression levels were reduced in the livers and those of Tkt were increased in the kidney of BPS treated rats compared with those in untreated rats. The protein expressions of transketolase (Tkt) of BPS treated rats were similarly increased both in the kidney and liver. These results suggest that dyslipidemia was not improved by the acceleration of TG metabolism but by the suppression of activated cholesterol and TG biosyntheses in OLETF rats treated with BPS. High activity of Tkt induced by BPS may be involved in the suppression of such synthetic mechanisms. © 2010 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
We have previously reported that the Prostaglandin I2 (PGI2 ) analog, beraprost sodium (sodium-2,3,3a,8b-tetrahydro-2hydroxy-1-1-[(E)-(3S)-3-hydroxy-4-methyl-1-octen-6-ynyl]1H-cyclopentabenzofuran-5-butyrate) (BPS), ameliorated diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats [1]. The OLETF rat was established as an animal model of human type 2 diabetes that exhibits obesity, hyperinsulinemia, hypertriglyceridemia, and hyperglycemia [2]. Long-Evans Tokushima Otsuka (LETO) rats are the non-diabetic control model of OLETF rats. We demonstrated histologically that BPS attenuated the severity of diabetic nephropathy in OLETF rats and improved the abnormalities in lipid profile [1]. In the current study we used a totally new groups of rats to evaluate the transcription levels of various enzymes that influence lipid and glucose metabolism and tried to clarify the mechanisms for improvement of dyslipidemia by BPS in OLETF rats.
2.1. Experimental protocols
∗ Corresponding author. Tel.: +81 92 801 1011; fax: +81 92 873 8008. E-mail address: [email protected] (H. Nakashima). 1098-8823/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2010.04.003
Experimental protocols were previously described [1]. Briefly, 50-week-old male OLETF and LETO rats were divided into three groups (Groups H, L and C) according to the treatment type of BPS. Using osmotic pumps (ALZA Co., Palo Alto, CA, USA), 400 g/kg body weight (BW) and 200 g/kg BW of BPS were continuously injected into rats of Groups H and L respectively (Group H: OLETF n = 6, LETO n = 5; Group L: OLETF n = 7, LETO n = 7). An equivalent volume of 0.9% saline was injected into rats of Group C (OLETF n = 9, LETO n = 5). Every 4 weeks, osmotic pumps were exchanged. Body weight, serum albumin, serum total cholesterol (TC), triglycerides (TG), and urinary protein were assessed every 4 weeks. This study was carried out in accordance with the Guideline for Animal Experiments at Fukuoka University, Japanese Government Animal Protection and Management Law (No. 105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6). Rats were bred and maintained in specific pathogen free (SPF) facility of Fukuoka University. Rats were maintained in a temperature-controlled room (22 ◦ C) with a 12-h light/dark cycle. Rats were allowed free access to standard food (CRF-1 food, containing normal 5.7% fat and 0% cholesterol, Japan Chares River Ltd., Yokohama, Japan) and water.
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2.2. Histopathology of the kidney and liver
2.6. Statistical analysis
Rats were sacrificed at 16 weeks after the beginning of administration. The kidneys and livers were fixed in 10% formaldehyde or in cold 95% ethanol for 24 h. Formaldehyde-fixed portions were processed according to the method for lipid stain [3] and embedded in paraffin. Sections (2–3 m) were stained with Sudan-IV.
Quantitative data were given as the mean value ± SE. ANOVA, followed by Fisher’s method (StatView version 5.0), was performed to analyze the differences among groups. A p value of less than 0.05 was considered statistically significant. 3. Results
2.3. Immunopathological studies of the kidneys After de-paraffinization in xylene and ethanol, and washing in phosphate-buffered saline (PBS), the paraffin-embedded sections were incubated with mouse anti-rat advanced glycation end product (AGEs) Ab (Trans Genic Inc., Hyogo, Japan), anti-macrophage scavenger receptor A (MSR-A) Ab (Trans Genic Inc., Hyogo, Japan), at concentrations of 1 g IgG/ml PBS, including 1% bovine serum albumin (BSA-PBS). Staining was performed using anti-mouse IgG-HRP labeled polymer (DakoCytomation Inc, Carpinteria, CA, USA). 2.4. RNA expression assay by real-time quantitative polymerase chain reaction (PCR) We assessed the transcription levels of HMG-CoA reductase (Hmgcr), hepatic triglyceride lipase (Lipc), Lipoprotein lipase (Lpl), and monoacylglycerol O-acyltransferase 1 (Mogat1) gene relative to that of -actin (Actb) in the liver. That of proliferator activated receptor␦ (Ppard) gene was assessed in the kidney section. Macrophage type-I and type-II class-A scavenger receptors (MSRA) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins [4–9]. We assessed the transcription levels of Fc receptor-like S, scavenger receptor (Fcrls) gene in kidneys and livers. Transketolase (Tkt) gene in kidney and liver was also evaluated, and the transcription levels were quantified as relative amounts to those of Actb in respective organs. Reverse transcription reactions and TaqMan PCR were performed in accordance with the manufacturer’s instructions (Applied Biosystems Japan, Tokyo, Japan). Sequence-specific amplification was detected with an increased fluorescence signal of FAM during the amplification cycles, using an ABI Prism 7500 sequence detection system (PerkinElmer Japan, Yokohama, Japan). Oligonucleotide primers and probes were designed using the Primer Express Program (Applied Biosystems, Japan) and synthesized; Rn00565598-m1 for Hmgcr, Rn0056147-m1 for Lipc, Rn00561482m1 for Lpl, Rn01400743-m1 for Mogat1, Rn00565707-m1 for Ppard, Rn01453465-g1 for Tkt, and Rn01455191-m1 for Fcrls. 2.5. Protein expression assay by Western blotting The protein extractions from kidneys and livers were performed in the accordance with the protocol in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific K.K, Yokohama, Japan). The extracted proteins were separated on a 12.5% SDSpolyacrylamide gel electrophoresis (5 g/lane) and transferred onto a membrane. The membrane was incubated with polyclonal goat anti-transketolase antibody (1:1000 dilution) (Santa Cruz Biotechnology Inc, Santa Cruz, CA) in the blocking buffer at 4 ◦ C for 16 h. The membrane was subsequently incubated with horseradish peroxidase conjugate rabbit anti-goat IgG (1:100,000 dilution) (Millipore Co., Billerica, MA) at room temperature for 1 h and analyzed using ECL Plus Western Blotting Detection Reagent (GE Healthcare UK Ltd., Buckinghamshire, UK). Hyperfilm ECL (GE Healthcare, UK) with cassette closure times of 1 min resulted in adequate exposure to visualize the bands.
3.1. Body weight, serum TC and TG concentrations were improved by BPS The body weight of OLETF rats had been decreasing during study period. The body weight ratio to original weight of Group C at 8 and 16 weeks were 1.00 ± 0.03 and 0.98 ± 0.05, respectively. Those of Group L were 0.91 ± 0.01 and 0.90 ± 0.01. In Group H those were 0.93 ± 0.01 and 0.90 ± 0.01. There was no significant difference in decreasing ratio among three groups. Concerning food intake significant difference was not observed among three groups. While in the levels of serum TC and TG between BPS treated and control groups there were dose dependent significant differences. At 16 weeks after the beginning of administration, the TC values of Group C, Group L, and Group H were 273.2 ± 20.3, 219.7 ± 15.5, and 199.7 ± 11.2 mg/dl, respectively (Group C vs H; p < 0.01, Group C vs L; p < 0.05) (Fig. 1A). The TG values of Group C, Group L, and Group H were 461.2 ± 45.2, 377.0 ± 64.5, and 195.3 ± 30.1 mg/dl, respectively (Group C vs H; p < 0.01, Group C vs L; p < 0.05) (Fig. 1B). On the other hand, in LETO rats, no significant differences in TC levels were seen (Fig. 1C). However, the TG values of Group C, Group L, and Group H in LETO rats at 16 weeks after the beginning of administration were 64.6 ± 10.6, 72.4 ± 5.2, and 37.4 ± 3.6 mg/dl, respectively, and there was a significant difference in the TG value between Group C and H (Group C vs H; p < 0. 05) (Fig. 1D). 3.2. Reduction of Hmgcr and Mogat1 mRNA expression in the liver of BPS treated rats RNA expression levels of Hmgcr and Mogat1 were reduced in the livers of BPS treated rats compared to the untreated rats; Relative abundance of Hmgcr mRNA, normalized by Actb mRNA, in Group C, Group L, and Group H were 2.08 ± 1.40, 0.49 ± 0.09, and 0.04 ± 0.38, respectively (Group C vs H; p < 0.05) (Fig. 2A). Relative abundance of Mogat1 mRNA were 1.79 ± 0.57, 0.77 ± 0.12, and 0.69 ± 0.43 (Fig. 2D), respectively (Group C vs H; p < 0.05) (Fig. 2D). Similarly, in LETO rats, there was a significant difference in the levels of Mogat1 mRNA between Group C and Group H; Group C 1.78 ± 0.25, Group L 1.01 ± 0.37, and Group H 0.81 ± 0.12, respectively (Group C vs H; p < 0.05) (Fig. 2D). However, significant differences in the levels of Lipc and Lpl mRNA were not seen when comparing OLETF and LETO rats. Relative abundance of Lipc mRNA were 0.72 ± 0.19, 0.68 ± 0.12, and 0.51 ± 0.24, respectively (Fig. 2B), and Lpl mRNA relative abundance values were 0.91 ± 0.60, 1.18 ± 0.62, and 1.16 ± 1.65 in the liver of BPS treated OLETF rats, respectively (Fig. 2C). 3.3. Abnormal lipid depositions in liver and renal glomeruli were attenuated by BPS Lipid was observed as russet deposits with Sudan IV staining, and abundant deposits were detected in the glomerulus of the kidneys in Group C. The degree of lipid deposition in the glomerulus of the kidney from BPS treated groups was attenuated in a dose dependent manner. Deposition was hardly seen in the glomerulus of Group H. Similarly, apparent lipid depositions around the central vein were revealed in liver specimens of group C, but not in liver of Group H (Fig. 2E).
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Fig. 1. BPS improved dyslipidemia in OLETF rats. The serum levels of TC and TG were determined in Group C, Group L, and Group H every 4 weeks. (A) The levels of TC in OLETF and LETO rats. (B) The levels of TG in OLETF and LETO rats. (C) The levels of urinary protein in OLETF and LETO rats. (D) The levels of serum albumin in OLETF and LETO rats. Data shown are mean ± SE of each group. White circle; Group C, triangle: Group L and black circle; Group H, solid line; OLETF rat, dotted line; LETO rats.
Fig. 2. BPS reduced the mRNA expression of Hmgcr in the liver and Mogat1 in the kidney of OLETF rats. Enzyme mRNA expression levels in the liver or kidney cortex in Group C, Group L, and Group H of OLETF and LETO rats were evaluated (N = 5 each group). (A–C) mRNA expression levels in the liver of Hmgcr, Lipc and Lpl, respectively. (D) Mgat1 mRNA expression levels in the kidney cortex. (E) BPS attenuated abnormal lipid deposition in the kidney and liver. Representative results of specimens stained with Sudan IV.
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Fig. 3. BPS inhibited AGE formation and MSR-labeled macrophage influx in the kidneys of OLETF rats. (A) The evaluations of MSR-A expression and AGE formation in the glomeruli of the OLETF rats were performed by immunohistological method using anti-MSR-A Ab and anti-AGE Ab (stained with H.E.; original magnification 400×). (B) Real-time RT-PCR quantifications of Fcrls mRNA expression and (C) those of Tkt mRNA expression in the kidneys and livers of rats in Group C, Group L, and Group H (N = 5 each). Shown in the relative abundance of mRNAs normalized by Actb. (D) Representative results of Western blot analysis of Tkt in the kidney and liver of OLETF and LETO rats. C, L, and H indicate Group C, L, and H, respectively.
3.4. Immunohistological studies of the kidneys in OLETF rats Intraglomerular macrophages were detected by anti-MSR-A Ab. Macrophage influx is found in the some glomeruli of untreated rats, while in BPS treated rats a few macrophage influx was observed. The area of AGEs in the glomerulus of BPS treated rats was apparently reduced compared to untreated rats; The proportions of AGEs area for glomerulus in Group C, Group L and Group H were 13.34 ± 2.05%, 8.56 ± 1.56%, 3.02 ± 0.17%, respectively (Group H vs C; p < 0.001, Group L vs C; p < 0.01) (Fig. 3A). 3.5. Increased Tkt mRNA expression and decreased Fcrls mRNA expression in the kidney of BPS treated rats Fcrls and Tkt mRNA expression levels were increased in both kidney and liver of BPS treated OLETF rats compared with the untreated OLETF rats; Fcrls relative mRNA expression levels were 0.85 ± 0.20, 0.68 ± 0.08, and 0.07 ± 0.05, respectively, in OLETF rats (Group C vs H; p < 0.05). Relative abundance of Tkt mRNA normalized by ACTB in Group C, Group L, and Group H were 0.61 ± 0.20, 1.00 ± 0.34, and 1.94 ± 0.98, respectively in OLETF rats (Group C vs H; p < 0.05) (Fig. 3C and B). In LETO rats, there were no significant differences in the RNA expression levels of Fcrls and Tkt between Group H and Group C. Ppard expression levels were no significant differences between BPS treated and untreated rats in both of OLETF and LETO rats (data not shown). 3.6. Increased Tkt protein expression in the kidney and liver of BPS treated rats The protein expression levels of Tkt were evaluated by Western blotting analysis (Fig. 3D). The bands for the kidney were much
weaker than those for the liver. The depth of the bands of Group H in both OLETF and LETO rats were more concentrated than those of Group C. 4. Discussion In this study we demonstrated that BPS improved dyslipidemia in OLETF rats. There were significant differences in the levels of total cholesterol between BPS treated and control groups in a dose dependent manner in OLETF, not in LETO rats (Fig. 1A). Although sufficient proteinuria has persisted, the level of serum albumin in OLETF rat has been maintained more than 4.0 g/dl (Fig. 1C and D). This fact indicated that dyslipidemia in OLETF rat may not result from nephrotic syndrome. The transcription level of Hmgcr was significantly suppressed by BPS administration in OLETF rats, but the level in LETO rats was not influenced by BPS (Fig. 2A). HMG-CoA reductase is the rate-limiting enzyme for cholesterol biosynthesis, and heavy proteinuria leads to up-regulation of HMGCoA reductase [10]. In OLETF rats, urine protein excretions in BPS treated groups were significantly decreased compared to those of the control group [1], and the decline of urine protein by BPS administration may induce the suppression of Hmgcr transcription. Intriguingly, BPS reduced the level of serum TG in not only OLETF, but also in LETO rats (Fig. 1B). A cyclic AMP elevating agent such as BPS might have an anti-oxidative property, and might increase lipid metabolism through increased glycolysis [11]. Therein we examined the transcription levels of Lipc and Lpl to assess TG reductase, and Mogat1 to assess TG synthetase. Contrary to our expectation, the transcription levels of TG reductases were not increased, and those of Mogat1 were significantly suppressed by BPS administration in both OLETF and LETO rats (Fig. 2B–D). Although the serum TG values in LETO rats were significantly lower
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Fig. 4. Shunting of glycolytic intermediates. Shunting of glycolytic intermediates from the Embden–Meyerhof pathway (dotted enclosure) to the reductive PPP (broken line enclosure) in anaerobic glycolysis. G-6-P, glucose-6-phosphate; F6P, fructose-6-phosphate; F-1, 6-bis-P, fructose-1,6-bisphosphate; DHAP, dihydroxyacetonephosphate; GA3P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehydes phosphate dehydrogenase; 1,3-bis-PG, 1,3-bisphosphoglycerate; 3PG, glycerate 3-phosphate; 2PG, glycerate 2-phosphate; PEP, phosphoenolpyruvic acid; ATP, adenosine triphosphate; ADP, adenosine diphosphate NAD+, nicotinamide adenine dinucleotide; NADH, the reduced form of NAD+; TCA cycle; tricarboxylic acid cycle.
than those in OLETF rats (Fig. 1B), the transcription levels of Mogat1 in control LETO rats were comparable to those in OLETF rats, and these values were lowered by BPS administration in a dose dependent manner (Fig. 2D). On the other hand, the transcription levels of Lipc and Lpl in LETO rats were significantly higher than those in OLETF rats, and these values were not influenced by BPS administration (Fig. 2B and C). These results suggest that normalization of TG in LETO rats was maintained with high TG reductase activity, and that BPS administration may not activate TG reduction, but induces suppression of TG synthesis in both rats. Therefore excessive lipid deposits in the kidney and liver of OLETF rats were significantly reduced after BPS administration (Fig. 2E). In the current study, immunohistological findings revealed that the formation of AGEs in the glomerulus of BPS treated OLETF rats were apparently reduced in a dose dependent manner (Fig. 3A). Hyperglycemia induces overproduction of superoxide by the mitochondrial electron transport chain, and this superoxide partially inhibits the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) [12,13]. Therefore upstream
metabolites of glycolysis are diverted into glucose-driven signaling pathways of glucose overuse [13,14] (Fig. 4). Reducing sugars, including glucose, fructose and trioses, can react nonenzymatically with the amino groups of proteins to form reversible Schiff bases and, subsequently, Amadori products which undergo further complex reactions to become AGEs [15,16]. The accumulation of glyceraldehyde-3-phosphate (GA3P) especially accelerates intracellular AGE formation [17]. Tkt is a thiamine-dependent pentose phosphate pathway (PPP) enzyme, and converts excess fructose-6-phosphate and GA3P to pentose-5-phosphates and erythrose-4-phosphate and activates reductive PPP shunt (Fig. 4). Therefore, increasing reductive PPP shunt through Tkt activation may reduce the accumulation of GA3P and prevent the formation of AGE [18,19]. Therefore we examined Tkt expression to clarify whether reductive PPP shunt was increased by BPS administration in OLETF rats. In fact, the transcription level of Tkt in OLEFT rats was increased in both kidney and liver by BPS administration in a dose dependent manner (Fig. 3C). The protein expression levels was also increased, and it was revealed that Tkt mRNA expression
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pattern reflect protein expression of Tkt in the kidney and liver (Fig. 3D). These results indicate that administration of BPS induced increased Tkt and reduced the formation of AGEs. Accordingly, it has been reported that stimulation of the reductive PPP by high-dose therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphate in experimental diabetes and inhibited the development of incipient nephropathy [20]. Furthermore it was shown that benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy through PPP shunt by induction of Tkt activation [17], and that high dose thiamine also corrected dyslipidemia in experimental diabetes [21–23]. Although the detailed mechanism of the decline in serum levels of TG by BPS was not clarified, these reports suggested that reductive PPP shunt activation may cause practical improvement in dyslipidemia, and BPS may also improve dyslipidemia in OLETF rats by the same mechanism. Improvement of dyslipidemia and reduction of AGE formation might cause amelioration of macrophage scavenger receptor A (MSRA) positive macrophage infiltration in glomeruli (Fig. 3A). MSR-A is a multi-ligand and multifunctional receptor expressed mainly on macrophages [5,24]. MSR-A is involved in foam cell formation, activation of macrophages, and adhesion of macrophages to atherosclerotic lesions [25–28]. It was shown that MSR-A is expressed on the macrophage positive AGE in glomerular lesions of patients with diabetic nephropathy [29]. The transcription level of Fcrls in OLEFT rats was decreased in both kidney and liver by BPS administration in a dose dependent manner (Fig. 3B), and this decline was apparently related to reduced AGE formation by activation of Tkt. The data reported here indicate the ability of BPS to improve dyslipidemia and to inhibit AGE formation, and BPS might be clinically useful in preventing the development and progression of diabetic complications. Acknowledgements This study was supported in part by a grant for the Progressive Renal Diseases Research Projects from the Ministry of Health, Labor and Welfare, Japan. References [1] Watanabe M, Nakashima H, Mochizuki S, et al. Amelioration of diabetic nephropathy in oletf rats by prostaglandin i(2) analog, beraprost sodium. Am J Nephrol 2009;30:1–11. [2] Kawano K, Hirashima T, Mori S, et al. Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (oletf) strain. Diabetes 1992;41:1422–8. [3] Mochizuki S, Moriya T, Naganuma H, et al. Significance of fat stains in serial sections from epon-embedded tissue samples for electron microscopy in renal diseases. Clin Exp Nephrol 2001;4:240–5. [4] Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: Implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223–61.
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[5] Kodama T, Freeman M, Rohrer L, et al. Type i macrophage scavenger receptor contains alpha-helical and collagen-like coiled coils. Nature 1990;343: 531–5. [6] Naito M, Suzuki H, Mori T, et al. Coexpression of type i and type ii human macrophage scavenger receptors in macrophages of various organs and foam cells in atherosclerotic lesions. Am J Pathol 1992;141:591–9. [7] Steinberg D, Parthasarathy S, Carew TE, et al. Beyond cholesterol. Modifications of low-density lipoprotein that increase its atherogenicity. N Engl J Med 1989;320:915–24. [8] Krieger M, Herz J. Structures and functions of multiligand lipoprotein receptors: macrophage scavenger receptors and ldl receptor-related protein (lrp). Annu Rev Biochem 1994;63:601–37. [9] Rohrer L, Freeman M, Kodama T, et al. Coiled-coil fibrous domains mediate ligand binding by macrophage scavenger receptor type ii. Nature 1990;343:570–2. [10] Vaziri ND, Liang KH. Hepatic hmg-coa reductase gene expression during the course of puromycin-induced nephrosis. Kidney Int 1995;48:1979–85. [11] Mishima K, Baba A, Matsuo M, et al. Protective effect of cyclic amp against cisplatin-induced nephrotoxicity. Free Radic Biol Med 2006;40:1564–77. [12] Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature 2000;404:787–90. [13] Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:813–20. [14] Du XL, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing sp1 glycosylation. Proc Natl Acad Sci USA 2000;97:12222–6. [15] Brownlee M, Cerami A, Vlassara H. Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 1988;318:1315–21. [16] Makita Z, Vlassara H, Rayfield E, et al. Hemoglobin-age: a circulating marker of advanced glycosylation. Science 1992;258:651–3. [17] Hammes HP, Du X, Edelstein D, et al. Benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy. Nat Med 2003;9:294–9. [18] Stracke H, Hammes HP, Werkmann D, et al. Efficacy of benfotiamine versus thiamine on function and glycation products of peripheral nerves in diabetic rats. Exp Clin Endocrinol Diabetes 2001;109:330–6. [19] La Selva M, Beltramo E, Pagnozzi F, et al. Thiamine corrects delayed replication and decreases production of lactate and advanced glycation end-products in bovine retinal and human umbilical vein endothelial cells cultured under high glucose conditions. Diabetologia 1996;39:1263–8. [20] Babaei-Jadidi R, Karachalias N, Ahmed N, et al. Prevention of incipient diabetic nephropathy by high-dose thiamine and benfotiamine. Diabetes 2003;52:2110–20. [21] Babaei-Jadidi R, Karachalias N, Kupich C, et al. High-dose thiamine therapy counters dyslipidaemia in streptozotocin-induced diabetic rats. Diabetologia 2004;47:2235–46. [22] Thornalley PJ. The potential role of thiamine (vitamin b1) in diabetic complications. Curr Diabetes Rev 2005;1:287–98. [23] Naveed AK, Qamar T, Ahmad I, et al. Effect of thiamine on lipid profile in diabetic rats. J Coll Physicians Surg Pak 2009;19:165–8. [24] Platt N, Gordon S. Is the class a macrophage scavenger receptor (sr-a) multifunctional?—the mouse’s tale. J Clin Invest 2001;108:649–54. [25] Santiago-Garcia J, Kodama T, Pitas RE. The class a scavenger receptor binds to proteoglycans and mediates adhesion of macrophages to the extracellular matrix. J Biol Chem 2003;278:6942–6. [26] Coller SP, Paulnock DM. Signaling pathways initiated in macrophages after engagement of type a scavenger receptors. J Leukoc Biol 2001;70:142–8. [27] Suzuki H, Kurihara Y, Takeya M, et al. A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection. Nature 1997;386:292–6. [28] de Winther MP, van Dijk KW, Havekes LM, et al. Macrophage scavenger receptor class a: a multifunctional receptor in atherosclerosis. Arterioscler Thromb Vasc Biol 2000;20:290–7. [29] Uesugi N, Sakata N, Horiuchi S, et al. Glycoxidation-modified macrophages and lipid peroxidation products are associated with the progression of human diabetic nephropathy. Am J Kidney Dis 2001;38:1016–25.
Contents lists available at ScienceDirect
Prostaglandins and Other Lipid Mediators
Improvement of dyslipidemia in OLETF rats by the prostaglandin I2 analog beraprost sodium Maho Watanabe, Hitoshi Nakashima ∗ , Kenji Ito, Katsuhisa Miyake, Takao Saito Division of Nephrology and Rheumatology, Department of Internal Medicine, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonann-ku, Fukuoka 814-0180, Japan
a r t i c l e
i n f o
Article history: Received 21 December 2009 Received in revised form 12 April 2010 Accepted 27 April 2010 Available online 5 May 2010 Keywords: Prostaglandin I2 Beraprost sodium (BPS) Dyslipidemia OLETF rat LETO rat
a b s t r a c t The Otsuka Long-Evans Tokushima Fatty (OLETF) rat was established as an animal model of human type 2 diabetes. Improvement of dyslipidemia by BPS has been confirmed in OLETF rats. The aim of this report is to clarify the mechanisms associated with improvement of dyslipidemia by BPS in OLETF rats. We divided male OLETF rats into three groups; 400 g/kg BPS treated (Group H), 200 g/kg BPS treated (Group L), and untreated control (Group C). After sacrifice, using the quantitative real-time PCR, we assayed the transcription levels of the HMG-CoA reductase (Hmgcr) for cholesterol biosynthesis, monoacylglycerol O-acyltransferase 1 (Mogat1) as TG synthetase, hepatic triglyceride lipase (Lipc) and lipoprotein lipase (Lpl) as triglycerides (TG) reductase in the liver. The mRNA expression of transketolase (Tkt) for pentose phosphate pathway (PPP) enzyme was also evaluated in the liver and kidney. Hmgcr and Mogat1 RNA expression levels were reduced in the livers and those of Tkt were increased in the kidney of BPS treated rats compared with those in untreated rats. The protein expressions of transketolase (Tkt) of BPS treated rats were similarly increased both in the kidney and liver. These results suggest that dyslipidemia was not improved by the acceleration of TG metabolism but by the suppression of activated cholesterol and TG biosyntheses in OLETF rats treated with BPS. High activity of Tkt induced by BPS may be involved in the suppression of such synthetic mechanisms. © 2010 Elsevier Inc. All rights reserved.
1. Introduction
2. Materials and methods
We have previously reported that the Prostaglandin I2 (PGI2 ) analog, beraprost sodium (sodium-2,3,3a,8b-tetrahydro-2hydroxy-1-1-[(E)-(3S)-3-hydroxy-4-methyl-1-octen-6-ynyl]1H-cyclopentabenzofuran-5-butyrate) (BPS), ameliorated diabetic nephropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats [1]. The OLETF rat was established as an animal model of human type 2 diabetes that exhibits obesity, hyperinsulinemia, hypertriglyceridemia, and hyperglycemia [2]. Long-Evans Tokushima Otsuka (LETO) rats are the non-diabetic control model of OLETF rats. We demonstrated histologically that BPS attenuated the severity of diabetic nephropathy in OLETF rats and improved the abnormalities in lipid profile [1]. In the current study we used a totally new groups of rats to evaluate the transcription levels of various enzymes that influence lipid and glucose metabolism and tried to clarify the mechanisms for improvement of dyslipidemia by BPS in OLETF rats.
2.1. Experimental protocols
∗ Corresponding author. Tel.: +81 92 801 1011; fax: +81 92 873 8008. E-mail address: [email protected] (H. Nakashima). 1098-8823/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.prostaglandins.2010.04.003
Experimental protocols were previously described [1]. Briefly, 50-week-old male OLETF and LETO rats were divided into three groups (Groups H, L and C) according to the treatment type of BPS. Using osmotic pumps (ALZA Co., Palo Alto, CA, USA), 400 g/kg body weight (BW) and 200 g/kg BW of BPS were continuously injected into rats of Groups H and L respectively (Group H: OLETF n = 6, LETO n = 5; Group L: OLETF n = 7, LETO n = 7). An equivalent volume of 0.9% saline was injected into rats of Group C (OLETF n = 9, LETO n = 5). Every 4 weeks, osmotic pumps were exchanged. Body weight, serum albumin, serum total cholesterol (TC), triglycerides (TG), and urinary protein were assessed every 4 weeks. This study was carried out in accordance with the Guideline for Animal Experiments at Fukuoka University, Japanese Government Animal Protection and Management Law (No. 105) and Japanese Government Notification on Feeding and Safekeeping of Animals (No. 6). Rats were bred and maintained in specific pathogen free (SPF) facility of Fukuoka University. Rats were maintained in a temperature-controlled room (22 ◦ C) with a 12-h light/dark cycle. Rats were allowed free access to standard food (CRF-1 food, containing normal 5.7% fat and 0% cholesterol, Japan Chares River Ltd., Yokohama, Japan) and water.
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2.2. Histopathology of the kidney and liver
2.6. Statistical analysis
Rats were sacrificed at 16 weeks after the beginning of administration. The kidneys and livers were fixed in 10% formaldehyde or in cold 95% ethanol for 24 h. Formaldehyde-fixed portions were processed according to the method for lipid stain [3] and embedded in paraffin. Sections (2–3 m) were stained with Sudan-IV.
Quantitative data were given as the mean value ± SE. ANOVA, followed by Fisher’s method (StatView version 5.0), was performed to analyze the differences among groups. A p value of less than 0.05 was considered statistically significant. 3. Results
2.3. Immunopathological studies of the kidneys After de-paraffinization in xylene and ethanol, and washing in phosphate-buffered saline (PBS), the paraffin-embedded sections were incubated with mouse anti-rat advanced glycation end product (AGEs) Ab (Trans Genic Inc., Hyogo, Japan), anti-macrophage scavenger receptor A (MSR-A) Ab (Trans Genic Inc., Hyogo, Japan), at concentrations of 1 g IgG/ml PBS, including 1% bovine serum albumin (BSA-PBS). Staining was performed using anti-mouse IgG-HRP labeled polymer (DakoCytomation Inc, Carpinteria, CA, USA). 2.4. RNA expression assay by real-time quantitative polymerase chain reaction (PCR) We assessed the transcription levels of HMG-CoA reductase (Hmgcr), hepatic triglyceride lipase (Lipc), Lipoprotein lipase (Lpl), and monoacylglycerol O-acyltransferase 1 (Mogat1) gene relative to that of -actin (Actb) in the liver. That of proliferator activated receptor␦ (Ppard) gene was assessed in the kidney section. Macrophage type-I and type-II class-A scavenger receptors (MSRA) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins [4–9]. We assessed the transcription levels of Fc receptor-like S, scavenger receptor (Fcrls) gene in kidneys and livers. Transketolase (Tkt) gene in kidney and liver was also evaluated, and the transcription levels were quantified as relative amounts to those of Actb in respective organs. Reverse transcription reactions and TaqMan PCR were performed in accordance with the manufacturer’s instructions (Applied Biosystems Japan, Tokyo, Japan). Sequence-specific amplification was detected with an increased fluorescence signal of FAM during the amplification cycles, using an ABI Prism 7500 sequence detection system (PerkinElmer Japan, Yokohama, Japan). Oligonucleotide primers and probes were designed using the Primer Express Program (Applied Biosystems, Japan) and synthesized; Rn00565598-m1 for Hmgcr, Rn0056147-m1 for Lipc, Rn00561482m1 for Lpl, Rn01400743-m1 for Mogat1, Rn00565707-m1 for Ppard, Rn01453465-g1 for Tkt, and Rn01455191-m1 for Fcrls. 2.5. Protein expression assay by Western blotting The protein extractions from kidneys and livers were performed in the accordance with the protocol in T-PER Tissue Protein Extraction Reagent (Thermo Fisher Scientific K.K, Yokohama, Japan). The extracted proteins were separated on a 12.5% SDSpolyacrylamide gel electrophoresis (5 g/lane) and transferred onto a membrane. The membrane was incubated with polyclonal goat anti-transketolase antibody (1:1000 dilution) (Santa Cruz Biotechnology Inc, Santa Cruz, CA) in the blocking buffer at 4 ◦ C for 16 h. The membrane was subsequently incubated with horseradish peroxidase conjugate rabbit anti-goat IgG (1:100,000 dilution) (Millipore Co., Billerica, MA) at room temperature for 1 h and analyzed using ECL Plus Western Blotting Detection Reagent (GE Healthcare UK Ltd., Buckinghamshire, UK). Hyperfilm ECL (GE Healthcare, UK) with cassette closure times of 1 min resulted in adequate exposure to visualize the bands.
3.1. Body weight, serum TC and TG concentrations were improved by BPS The body weight of OLETF rats had been decreasing during study period. The body weight ratio to original weight of Group C at 8 and 16 weeks were 1.00 ± 0.03 and 0.98 ± 0.05, respectively. Those of Group L were 0.91 ± 0.01 and 0.90 ± 0.01. In Group H those were 0.93 ± 0.01 and 0.90 ± 0.01. There was no significant difference in decreasing ratio among three groups. Concerning food intake significant difference was not observed among three groups. While in the levels of serum TC and TG between BPS treated and control groups there were dose dependent significant differences. At 16 weeks after the beginning of administration, the TC values of Group C, Group L, and Group H were 273.2 ± 20.3, 219.7 ± 15.5, and 199.7 ± 11.2 mg/dl, respectively (Group C vs H; p < 0.01, Group C vs L; p < 0.05) (Fig. 1A). The TG values of Group C, Group L, and Group H were 461.2 ± 45.2, 377.0 ± 64.5, and 195.3 ± 30.1 mg/dl, respectively (Group C vs H; p < 0.01, Group C vs L; p < 0.05) (Fig. 1B). On the other hand, in LETO rats, no significant differences in TC levels were seen (Fig. 1C). However, the TG values of Group C, Group L, and Group H in LETO rats at 16 weeks after the beginning of administration were 64.6 ± 10.6, 72.4 ± 5.2, and 37.4 ± 3.6 mg/dl, respectively, and there was a significant difference in the TG value between Group C and H (Group C vs H; p < 0. 05) (Fig. 1D). 3.2. Reduction of Hmgcr and Mogat1 mRNA expression in the liver of BPS treated rats RNA expression levels of Hmgcr and Mogat1 were reduced in the livers of BPS treated rats compared to the untreated rats; Relative abundance of Hmgcr mRNA, normalized by Actb mRNA, in Group C, Group L, and Group H were 2.08 ± 1.40, 0.49 ± 0.09, and 0.04 ± 0.38, respectively (Group C vs H; p < 0.05) (Fig. 2A). Relative abundance of Mogat1 mRNA were 1.79 ± 0.57, 0.77 ± 0.12, and 0.69 ± 0.43 (Fig. 2D), respectively (Group C vs H; p < 0.05) (Fig. 2D). Similarly, in LETO rats, there was a significant difference in the levels of Mogat1 mRNA between Group C and Group H; Group C 1.78 ± 0.25, Group L 1.01 ± 0.37, and Group H 0.81 ± 0.12, respectively (Group C vs H; p < 0.05) (Fig. 2D). However, significant differences in the levels of Lipc and Lpl mRNA were not seen when comparing OLETF and LETO rats. Relative abundance of Lipc mRNA were 0.72 ± 0.19, 0.68 ± 0.12, and 0.51 ± 0.24, respectively (Fig. 2B), and Lpl mRNA relative abundance values were 0.91 ± 0.60, 1.18 ± 0.62, and 1.16 ± 1.65 in the liver of BPS treated OLETF rats, respectively (Fig. 2C). 3.3. Abnormal lipid depositions in liver and renal glomeruli were attenuated by BPS Lipid was observed as russet deposits with Sudan IV staining, and abundant deposits were detected in the glomerulus of the kidneys in Group C. The degree of lipid deposition in the glomerulus of the kidney from BPS treated groups was attenuated in a dose dependent manner. Deposition was hardly seen in the glomerulus of Group H. Similarly, apparent lipid depositions around the central vein were revealed in liver specimens of group C, but not in liver of Group H (Fig. 2E).
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Fig. 1. BPS improved dyslipidemia in OLETF rats. The serum levels of TC and TG were determined in Group C, Group L, and Group H every 4 weeks. (A) The levels of TC in OLETF and LETO rats. (B) The levels of TG in OLETF and LETO rats. (C) The levels of urinary protein in OLETF and LETO rats. (D) The levels of serum albumin in OLETF and LETO rats. Data shown are mean ± SE of each group. White circle; Group C, triangle: Group L and black circle; Group H, solid line; OLETF rat, dotted line; LETO rats.
Fig. 2. BPS reduced the mRNA expression of Hmgcr in the liver and Mogat1 in the kidney of OLETF rats. Enzyme mRNA expression levels in the liver or kidney cortex in Group C, Group L, and Group H of OLETF and LETO rats were evaluated (N = 5 each group). (A–C) mRNA expression levels in the liver of Hmgcr, Lipc and Lpl, respectively. (D) Mgat1 mRNA expression levels in the kidney cortex. (E) BPS attenuated abnormal lipid deposition in the kidney and liver. Representative results of specimens stained with Sudan IV.
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Fig. 3. BPS inhibited AGE formation and MSR-labeled macrophage influx in the kidneys of OLETF rats. (A) The evaluations of MSR-A expression and AGE formation in the glomeruli of the OLETF rats were performed by immunohistological method using anti-MSR-A Ab and anti-AGE Ab (stained with H.E.; original magnification 400×). (B) Real-time RT-PCR quantifications of Fcrls mRNA expression and (C) those of Tkt mRNA expression in the kidneys and livers of rats in Group C, Group L, and Group H (N = 5 each). Shown in the relative abundance of mRNAs normalized by Actb. (D) Representative results of Western blot analysis of Tkt in the kidney and liver of OLETF and LETO rats. C, L, and H indicate Group C, L, and H, respectively.
3.4. Immunohistological studies of the kidneys in OLETF rats Intraglomerular macrophages were detected by anti-MSR-A Ab. Macrophage influx is found in the some glomeruli of untreated rats, while in BPS treated rats a few macrophage influx was observed. The area of AGEs in the glomerulus of BPS treated rats was apparently reduced compared to untreated rats; The proportions of AGEs area for glomerulus in Group C, Group L and Group H were 13.34 ± 2.05%, 8.56 ± 1.56%, 3.02 ± 0.17%, respectively (Group H vs C; p < 0.001, Group L vs C; p < 0.01) (Fig. 3A). 3.5. Increased Tkt mRNA expression and decreased Fcrls mRNA expression in the kidney of BPS treated rats Fcrls and Tkt mRNA expression levels were increased in both kidney and liver of BPS treated OLETF rats compared with the untreated OLETF rats; Fcrls relative mRNA expression levels were 0.85 ± 0.20, 0.68 ± 0.08, and 0.07 ± 0.05, respectively, in OLETF rats (Group C vs H; p < 0.05). Relative abundance of Tkt mRNA normalized by ACTB in Group C, Group L, and Group H were 0.61 ± 0.20, 1.00 ± 0.34, and 1.94 ± 0.98, respectively in OLETF rats (Group C vs H; p < 0.05) (Fig. 3C and B). In LETO rats, there were no significant differences in the RNA expression levels of Fcrls and Tkt between Group H and Group C. Ppard expression levels were no significant differences between BPS treated and untreated rats in both of OLETF and LETO rats (data not shown). 3.6. Increased Tkt protein expression in the kidney and liver of BPS treated rats The protein expression levels of Tkt were evaluated by Western blotting analysis (Fig. 3D). The bands for the kidney were much
weaker than those for the liver. The depth of the bands of Group H in both OLETF and LETO rats were more concentrated than those of Group C. 4. Discussion In this study we demonstrated that BPS improved dyslipidemia in OLETF rats. There were significant differences in the levels of total cholesterol between BPS treated and control groups in a dose dependent manner in OLETF, not in LETO rats (Fig. 1A). Although sufficient proteinuria has persisted, the level of serum albumin in OLETF rat has been maintained more than 4.0 g/dl (Fig. 1C and D). This fact indicated that dyslipidemia in OLETF rat may not result from nephrotic syndrome. The transcription level of Hmgcr was significantly suppressed by BPS administration in OLETF rats, but the level in LETO rats was not influenced by BPS (Fig. 2A). HMG-CoA reductase is the rate-limiting enzyme for cholesterol biosynthesis, and heavy proteinuria leads to up-regulation of HMGCoA reductase [10]. In OLETF rats, urine protein excretions in BPS treated groups were significantly decreased compared to those of the control group [1], and the decline of urine protein by BPS administration may induce the suppression of Hmgcr transcription. Intriguingly, BPS reduced the level of serum TG in not only OLETF, but also in LETO rats (Fig. 1B). A cyclic AMP elevating agent such as BPS might have an anti-oxidative property, and might increase lipid metabolism through increased glycolysis [11]. Therein we examined the transcription levels of Lipc and Lpl to assess TG reductase, and Mogat1 to assess TG synthetase. Contrary to our expectation, the transcription levels of TG reductases were not increased, and those of Mogat1 were significantly suppressed by BPS administration in both OLETF and LETO rats (Fig. 2B–D). Although the serum TG values in LETO rats were significantly lower
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Fig. 4. Shunting of glycolytic intermediates. Shunting of glycolytic intermediates from the Embden–Meyerhof pathway (dotted enclosure) to the reductive PPP (broken line enclosure) in anaerobic glycolysis. G-6-P, glucose-6-phosphate; F6P, fructose-6-phosphate; F-1, 6-bis-P, fructose-1,6-bisphosphate; DHAP, dihydroxyacetonephosphate; GA3P, glyceraldehyde-3-phosphate; GAPDH, glyceraldehydes phosphate dehydrogenase; 1,3-bis-PG, 1,3-bisphosphoglycerate; 3PG, glycerate 3-phosphate; 2PG, glycerate 2-phosphate; PEP, phosphoenolpyruvic acid; ATP, adenosine triphosphate; ADP, adenosine diphosphate NAD+, nicotinamide adenine dinucleotide; NADH, the reduced form of NAD+; TCA cycle; tricarboxylic acid cycle.
than those in OLETF rats (Fig. 1B), the transcription levels of Mogat1 in control LETO rats were comparable to those in OLETF rats, and these values were lowered by BPS administration in a dose dependent manner (Fig. 2D). On the other hand, the transcription levels of Lipc and Lpl in LETO rats were significantly higher than those in OLETF rats, and these values were not influenced by BPS administration (Fig. 2B and C). These results suggest that normalization of TG in LETO rats was maintained with high TG reductase activity, and that BPS administration may not activate TG reduction, but induces suppression of TG synthesis in both rats. Therefore excessive lipid deposits in the kidney and liver of OLETF rats were significantly reduced after BPS administration (Fig. 2E). In the current study, immunohistological findings revealed that the formation of AGEs in the glomerulus of BPS treated OLETF rats were apparently reduced in a dose dependent manner (Fig. 3A). Hyperglycemia induces overproduction of superoxide by the mitochondrial electron transport chain, and this superoxide partially inhibits the glycolytic enzyme glyceraldehyde phosphate dehydrogenase (GAPDH) [12,13]. Therefore upstream
metabolites of glycolysis are diverted into glucose-driven signaling pathways of glucose overuse [13,14] (Fig. 4). Reducing sugars, including glucose, fructose and trioses, can react nonenzymatically with the amino groups of proteins to form reversible Schiff bases and, subsequently, Amadori products which undergo further complex reactions to become AGEs [15,16]. The accumulation of glyceraldehyde-3-phosphate (GA3P) especially accelerates intracellular AGE formation [17]. Tkt is a thiamine-dependent pentose phosphate pathway (PPP) enzyme, and converts excess fructose-6-phosphate and GA3P to pentose-5-phosphates and erythrose-4-phosphate and activates reductive PPP shunt (Fig. 4). Therefore, increasing reductive PPP shunt through Tkt activation may reduce the accumulation of GA3P and prevent the formation of AGE [18,19]. Therefore we examined Tkt expression to clarify whether reductive PPP shunt was increased by BPS administration in OLETF rats. In fact, the transcription level of Tkt in OLEFT rats was increased in both kidney and liver by BPS administration in a dose dependent manner (Fig. 3C). The protein expression levels was also increased, and it was revealed that Tkt mRNA expression
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pattern reflect protein expression of Tkt in the kidney and liver (Fig. 3D). These results indicate that administration of BPS induced increased Tkt and reduced the formation of AGEs. Accordingly, it has been reported that stimulation of the reductive PPP by high-dose therapy with thiamine and the thiamine monophosphate derivative benfotiamine countered the accumulation of triosephosphate in experimental diabetes and inhibited the development of incipient nephropathy [20]. Furthermore it was shown that benfotiamine blocks three major pathways of hyperglycemic damage and prevents experimental diabetic retinopathy through PPP shunt by induction of Tkt activation [17], and that high dose thiamine also corrected dyslipidemia in experimental diabetes [21–23]. Although the detailed mechanism of the decline in serum levels of TG by BPS was not clarified, these reports suggested that reductive PPP shunt activation may cause practical improvement in dyslipidemia, and BPS may also improve dyslipidemia in OLETF rats by the same mechanism. Improvement of dyslipidemia and reduction of AGE formation might cause amelioration of macrophage scavenger receptor A (MSRA) positive macrophage infiltration in glomeruli (Fig. 3A). MSR-A is a multi-ligand and multifunctional receptor expressed mainly on macrophages [5,24]. MSR-A is involved in foam cell formation, activation of macrophages, and adhesion of macrophages to atherosclerotic lesions [25–28]. It was shown that MSR-A is expressed on the macrophage positive AGE in glomerular lesions of patients with diabetic nephropathy [29]. The transcription level of Fcrls in OLEFT rats was decreased in both kidney and liver by BPS administration in a dose dependent manner (Fig. 3B), and this decline was apparently related to reduced AGE formation by activation of Tkt. The data reported here indicate the ability of BPS to improve dyslipidemia and to inhibit AGE formation, and BPS might be clinically useful in preventing the development and progression of diabetic complications. Acknowledgements This study was supported in part by a grant for the Progressive Renal Diseases Research Projects from the Ministry of Health, Labor and Welfare, Japan. References [1] Watanabe M, Nakashima H, Mochizuki S, et al. Amelioration of diabetic nephropathy in oletf rats by prostaglandin i(2) analog, beraprost sodium. Am J Nephrol 2009;30:1–11. [2] Kawano K, Hirashima T, Mori S, et al. Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (oletf) strain. Diabetes 1992;41:1422–8. [3] Mochizuki S, Moriya T, Naganuma H, et al. Significance of fat stains in serial sections from epon-embedded tissue samples for electron microscopy in renal diseases. Clin Exp Nephrol 2001;4:240–5. [4] Brown MS, Goldstein JL. Lipoprotein metabolism in the macrophage: Implications for cholesterol deposition in atherosclerosis. Annu Rev Biochem 1983;52:223–61.
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