Impact of glucotoxicity induced in vivo and in vitro in Psammomys obesus


Objective: Chronic hyperglycemia characteristic of type diabetes 2 is responsible for the accelerated atherosclerosis with increased cardiovascular risk. In this study, we will propose to analyze the effect of a long-term of glucotoxicity in vivo in Psammomys obesus by addition of sucrose to 30% for 11 months and in vitro study of adventitial fibroblasts in the presence of D-glucose 0.6% for 7 days. Materials and methods: Evaluation of plasma biochemical parameters was carried out at the initial time and at the end of experiment. At autopsy, a morphological study of the aorta was performed after fixation in aqueous Bouin and staining with Masson’s trichrome. The experimental glucotoxicity is induced by incubation of fibroblasts in DMEM enriched with D-glucose at 0.6% for 7 days. The impact of glucotoxicity is assessed in the intracellular compartments through dosage of total nitrite and malondialdehyde, a product of lipid peroxidation, and thanks to a morphological assay after fixation of cells with aqueous bouin and blood staining with May Grünwald Giemsa. The evaluation of cell proliferation is accomplished by cell counting. Collagens I and III of the extracellular compartment are characterized by SDS-PAGE. Results: Animals subjected to sucrose showed hyperglycemia associated with hyperinsulinemia, dyslipidemia, hyperproteinemia, increased CPK and VLDL-LDL and decreased HDL. Histology of aortas revealed endothelial cells hypertrophy, severe disorganization of intima and media. In the presence of glucose, the proliferation of fibroblasts increases very significantly (P = 2.34 × 10-5), the rate of malonaldehyde, nitrite and total density of chains α2 (I) and α1 (I + III) extra-cellular collagens I and III increased significantly. After staining, the cells showed hypertrophy, vacuolation of cytoplasm and chromatin condensation with nuclear fragmentation, indicative of apoptosis. Conclusion: The glucotoxicity induced in vivo and in vitro is responsible for major structural and metabolic alterations leading to the acceleration of the atherosclerotic process.

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Sihem, B. , Leila, S. , Kheira, O. , Samia, N. , Nadjiba, H. , Saliha, B. , Abdelhamid, S. , Ghouti, K. , Mahdi, H. , Yasmina, B. and Souhila, A. (2012) Impact of glucotoxicity induced in vivo and in vitro in Psammomys obesus. Journal of Diabetes Mellitus, 2, 59-71. doi: 10.4236/jdm.2012.21010.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Pandolfi, A. and De Filippis, E.A. (2007) Chronic hyperglicemia and nitric oxide bioavailability play a pivotal role in pro-atherogenic vascular modifications. Genes Nutrition, 2, 195-208. doi:10.1007/s12263-007-0050-5
[2] Chen, S., Evans, T., Mukherjee, K., Karmazyn, M. and Chakrabarti, S. (2000) Diabetes-induced Myocardial structural changes: Role of endothelin-1 and its receptors. The Journal of Molecular and Cardiologie, 32, 1621-1629. doi:10.1006/jmcc.2000.1197
[3] Goel, A., Zhang, Y., Anderson, L. and Rahimian, R. (2007) Gender difference in rat aorta vasodilation after acute exposure to hight glucose: Involvement of protein kinase Cβ an superoxide but not Rho Kinase. Cardiovascular Research, 76, 351-360. doi:10.1016/j.cardiores.2007.06.029
[4] Brunner, Y., Schvartz, D., Priego-Capote, F., Couté, Y. and Jean-Charles Sanchez, J. C. (2009) Glucotoxicity and pancreatic proteomics. Journal of Proteomics, 71, 576-591. doi:10.1016/j.jprot.2008.10.002
[5] Andreea, S. I., Marieta, C. and Anca, D. (2008) AGEs and glucose levels modulate TypeI and III Procollagen mRNA synthesis in dermal fibroblastes cells culture. The Experimental Diabetes Research, 2008, 1-7. doi:10.1155/2008/473603
[6] Daly, M and Daly, S. (1973) On the ecology of Psammomys obesus (Rodentia gerbillidae) in the wadi saoura Algeria. Mammalia, 37, 546-561. doi:10.1515/mamm.1973.37.4.545
[7] Aouichat Bouguerra, S., Benazzoug, Y., Bekkhoucha, F. and Bourdillon, M. C. (2004) Effect of High Glucose Concentration on Collagen Synthesis and Cholesterol Level in the Phenotypic Modulation of Aortic Cultured Smooth Muscle Cells of Sand Rat (Psammomys obesus). Experimental Diabetes Research, 5, 227-235. doi:10.1080/15438600490489793
[8] Martoja, R. (1967) Initiation aux techniques de l’histologie animale. Masson, Paris, 349 pages.
[9] Kinugawa, K., Shimizu, T., Yao, A., Kohmoto. O., Serizawa. T. and Takahashi, T. (1997) Transcriptional regulation of inducible nitric oxide synthase in cultured neonatal rat cardiac myocytes. Circulation Research, 81, 911-921.
[10] Heath, R.L. and Packer, L. (1968) Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Archives in Biochemistry and Biophysics, 125, 189-198. doi:10.1016/0003-9861(68)90654-1
[11] Grand, F., Guitton, J. and Goudable, J. (2001) Optimisation des parametres du dosage des nitrites et nitrates seriques par la technique de Griess. Annales de Biologie Clinique, 59, 559-565.
[12] Bradford, M. (1976) A rapid sensitive method for quantification of microgram quantities of protein utilizing the principe of protein dye binding. Anall of Biochemestry, 72, 248-254. doi:10.1016/0003-2697(76)90527-3
[13] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0
[14] Marquie, G., Duhault, J. and Jacotot, B. (1984) Diabetes mellitus in sand rats (Psammomys obesus). Metabolic pattern during development of the diabetic syndrome. Diabetes, 33, 438-443. doi:10.2337/diabetes.33.5.438
[15] Shafrir, E. (2001) Albert Renold Memorial Lecture: Molecular Background of Nutritionally Induced Insulin Resistance leading to Type 2 Diabetes-From Animal Models to humans. The International Journal of Experimental Diabetes Research, 2, 299-319. doi:10.1155/EDR.2001.299
[16] Ikeda, Y., Olsen, G. S., Ziv, E., Hansen, L.L., Busch, A.K., Hansen Bo, F., Shafrir, E. and Mosthaf-Seedorf, L. (2001) Cellular Mechanism of Nutritionally Induced Insulin Resistance in Psammomys Obesus. Overexpression of PKC ξ in Skeleletal Muscle Precedes the Onset of Hyperinsulinemia and Hyperglycemia. Diabetes, 50, 584-592. doi:10.2337/diabetes.50.3.584
[17] Stentz, F.B. and Kitabchi, A.E. (2005) Hyperglycemia-induced activation of human T-lymphocytes with de novo emergence of insulin receptors and generation of reactive oxygen species. Biochemical and Biophysical Research Communication, 335, 491-495. doi:10.1016/j.bbrc.2005.07.109
[18] Clay, F.S. (2006) Insulin resistance and atherosclerosis. The Journal of Clinical Investigation, 116, 1813-1822. doi:10.1172/JCI29024
[19] Goel, A., Zhang, Y., Anderson, L. and Rahimian, R. (2007) Gender difference in rat aorta vasodilation after acute exposure to hight glucose: Involvement of protein kinase Cβ an superoxide but not Rho Kinase. Cardiovascular Research, 76, 351-360. doi:10.1016/j.cardiores.2007.06.029
[20] Dagre, A.G., Lekakis, J.P., Protogerou, A.D., Douridas, G.N., Papaioannou, T.G., Tryfonopoulos, D.J., Papamichael, C.M. and Alevizaki, M. (2007) Abnormal endothelial function in female patients with hypothyroidism and borderline thyroid function. The International Journal of Cardiology, 114, 332-338. doi:10.1016/j.ijcard.2005.12.013
[21] Schaffer, J.E. (2003) Lipotoxicity: When tissues overeat. Current Opinion in Lipidoogy, 14, 281-287. doi:10.1097/00041433-200306000-00008
[22] Schmiz-Peiffer, C., Browne, C.L., Oakes, N.D., Watkinson, A., Chisholm, D.J., Kraegen, E.W. and Biden, T.J. (1997) Alteration in the expression and cellulaire localization of protein kinase C isozymes ξ and θ are associated with insulin résistance in skeletal muscle of the hight-fat-fed rat. Diabetes, 46, 169-178.
[23] Avignon, A., Yamada, K., Zhou, X., Spencer, B., Cardona, O., Saba-Siddique, S., Galloway, L., Galloway, L., Standaert, M.L. and Farese, R.V. (1996) Chronic activation of protein kinase C in soleus muscle and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats: A mechanism for inhibiting glycogene synthesis. Diabetes, 45, 1396-1404. doi:10.2337/diabetes.45.10.1396
[24] Navab, M., Hama, S.Y., Reddy, S.T., Ng, C.J., Van Lenten, B.J., Laks, H. and Fogelman, A.M. (2002) Oxidized lipids as mediators of coronary heart disease. Current Opinion in Lipidology, 13, 363-372. doi:10.1097/00041433-200208000-00003
[25] Brewer, H.B. (2004) High density lipoprotein: a new potential therapeutic target for the prevention of cardiovascular disease. Arteriosclerosis Thrombosis and Vascular Biology, 24, 387-391. doi:10.1161/01.ATV.0000121505.88326.d2
[26] Oldegren, J., Wallentin, L., Grip, L., Linder, R., Norgaard, B.L. and Siegbahn, A. (2003) Miocardial damage, inflamemation and thrombin inhibition in unstable coronary artery disease. Eureupean Heart Journal, 24, 21-25.
[27] Baudouy, P.Y. and Beaufils, P. (1998) Diagnostic de l’infarctus du myocarde aigu. Encyclopédie Médico-chirurgicale Cardiologie, Elsevier, Paris, Tome 2.1
[28] Kim, P.K.M., Zamora, R., Petrosko, P. and Billiar, T.R. (2001) The regulatory role of nitric oxide in apoptosis. International Immunopharmacology, 1, 1421-1441. doi:10.1016/S1567-5769(01)00088-1
[29] Orbe, J., Rodriguez, J.A., Ardies, R., Belzunce, M., Nespereira, B., Pérez, I., Zarbe. M., Rancal, C. and Paramo, J.A. (2003) Antioxidant vitamins increase the collagen content and reduce MMP-1 in a porcine model of atherosclerosis: Implication for plaque stabilization. Atherosclerosis, 167, 45-53. doi:10.1016/S0021-9150(02)00392-1
[30] Lusis, A.J. (2000) Atherosclerosis. Nature, 407, 233-241. doi:10.1038/35025203
[31] Zwijsen, R.M., Japenga, S.C., Heijen, A.M., Van den Bros, R.C. and Koeman, J.H. (1992) Induction of platelet-derived growth factor chain a gene expression in human smooth muscle cells by oxidized low density lipoproteins. Biochemical and Biophysical Research. Communication, 186, 1410-1416.
[32] Neumann, S., Huse, K., Semrau, R., Diegeler, A., Gebhardt, R., Buniatian, G.H. and Scholz, G.H. (2002) Aldosterone and D-Glucose Stimulate the Proliferation of Human Cardiac Myofibroblasts in Vitro. Hypertenstion, 39, 756-760. doi:10.1161/hy0302.105295
[33] Heo, K.J., Kim, D.U., Kim, L., Nam, M., Baek, S.T., Park, S.K., Park, Y., Myung, C.S., Hwang, S.O. and Hoe, K.L. (2008) Activation of PKCβII and PKCθ is essential for LDL-induced cell proliferation of human aortic smooth muscle cells via Gi-mediated Erk1/2 activation and Egr-1 upregulation. Biochemical and Biophysical Research Communication, 368, 126-131. doi:10.1016/j.bbrc.2008.01.050
[34] McCaffrey, T.A., Fu, C., Du, B., Eksinar, S., Kent, K. C., Bush, J.R., Kreiger, K., Rosengart, T., Cybulsky, M.I., Silverman, E.S. and Collins, T. (2000) High-level expression of Erg-1 inducible gene in mouse and human atherosclerosis. The Journal of Clinical Investigation, 105, 653-662. doi:10.1172/JCI8592
[35] Benazzoug, Y., Borchiellini, C., Labat-Robert, J., Robert, I. and Kern, P. (1998) Effect of high-glucose concentrations on the expression of collagens and fibronectin by fibroblasts in culture. Experimental Gerontology, 33, 445-455. doi:10.1016/S0531-5565(98)00015-1
[36] Hoffman, B.B., Sharma, K., Zhu, Y. and Zyadeh, F.N. (1998) Transcriptional activation of transforming growth factor-β1 in mesangial cell culture by high glucose concentration. Kidney International, 54, 1107-1116. doi:10.1046/j.1523-1755.1998.00119.x
[37] Ryoo, S.W., Kim, D.U., Won, C.M., Chung, K.S., Jang, Y.J., Oh, G.T., Park, S.K., Maeng, P.J., Yoo, H.S. and Hoe, K.L. (2004) Native LDL induces interleukin-8 expression via H2O2, p38 kinase, and activator protein-1 in human aortic smooth muscle cells. Cardiovascular Research, 62, 185-193. doi:10.1016/j.cardiores.2004.01.002
[38] Grimm, D., Jabusch, H.C., Kossmehl, P., Huberb, M., Fredersdorf, S., Griese, D.P., Kramer, B.K. and Kromer, E.P. (2002) Experimental diabetes and left ventricular hypertrophy: Effects of beta-receptor blockade. Cardiovascular Pathology, 11, 229-237. doi:10.1016/S1054-8807(01)00116-8
[39] Liu, X.J., He, A.B., Chang, Y.S. and De Fang, F. (2006) Atypical protein kinase C in glucose metabolism. Cell Signal, 18, 2071-2076. doi:10.1016/j.cellsig.2006.04.007
[40] Babich, H., Zuckerbraun, H.L., Wurzburger, B.J., Rubin, Y.L., Borenfreund, E. and Blau, L. (1996) Benzoyl peroxide cytotoxicity evaluated in vitro with the human keratinocyte cell line, RHEK-1. Toxicology, 106, 187-196. doi:10.1016/0300-483X(95)03189-M
[41] Kolb, J.P. (2001) Role proet anti-apoptotique du monoxide d’azote, NO. C.R. Lifes Sciences, 324, 413-424.
[42] Babich, H., Zuckerbraun, H.L. and Weinerman, S.M. (2007) In vitro cytotoxicity of (?)-catechin gallate, a minor polyphenol in green tea. Toxicology, 71, 171-180.
[43] Boumaza, S., Neggazi, S., Hamlat, N., Sahraoui, H., Berdja, S., Smail, L., Kacimi, G., Gernigon, T., Benazzoug, Y. and Aouichat Bouguerra, S. (2011) Implication of hydrogen peroxyde in biochemical and morphological alterations of cultured adventitiels of Psammomys obesus. Journal of Cell and Animal Biology, 5, 76-88.
[44] Andreea, S. I., Marieta, C. and Anca, D. (2008) AGEs and glucose levels modulate Type I and III Procollagen mRNA synthesis in dermal fibroblastes cells culture. The Experimental Diabetes Research, 2008, 1-7. doi:10.1155/2008/473603
[45] Whiteside, C. I. and Dlugosz, J. A. (2002) Masangial cell protein kinase C isoenzyme activation in the diabetic milieu. American Journal Physiology Renal Physiology, 282, 975-980.
[46] Hayek, T., Kaplan, M., Kerry, R. and Aviram, M. (2007) Macrophage NADPHoxidase activation, impaired cholesterol fluxes, and increased cholesterol biosynthesis in diabetic mice: A stimulatory role for D-glucose. Atherosclorisis, 195, 277-286. doi:10.1016/j.atherosclerosis.2006.12.026
[47] Michel, F., Bonnefont-Rousselot, D., Mas, E., Drai, J. and Thérond, P. (2008) Biomarqueurs de la peroxydation lipidiques: Aspects analytiques. Annales de Biologie Clinique, 66, 605-620.

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