Pathological and biochemical investigation of the effects of l-carnitine and gemfibrozil on peroxisome proliferator activated Receptors (PPARS) and lipidosis in rabbits on a high-fat diet

Abstract

Obesity and fatty liver is a widespread growing health problem in human with detrimental consequences that encouraged researchers to find ways to overcome it. In this study, gemfibrozil and L-carnitine were evaluated in prevention of obesity and hepatic lipidosis also the role of L-carnitine in avoiding side effects of gemfibrozil was investigated. The study involved 56 New-Zealand Albino rabbits, divided into 2 main groups and then subdivided into 4 equal groups (n=7). The groups I (normal diet), II (normal diet+gemfibrozil), III (normal diet+L-carnitine) and IV (normal diet+gemfibrozil+L-carnitine) received normal diet and the groups V (fat rich diet), VI (fat rich diet+gemfibrozil), VII (fat rich diet+L-carnitine) and VIII (fat rich diet+gemfibrozil+L-carnitine) received fat rich diet for 8 weeks. Animals were blood sampled and wieght weekly during the experiment and at the end of the experiment for determination of biochemical (HDL, High-density lipoproteins; LDL, Low-density lipoprotein; VLDL, Very low-density lipoprotein; ALT, Alanine amino transferase; AST, Aspartate aminotransferase; GGT, Gamma glutamyltransferase; GLDH, Glutamate lactate dehydrogenase; LDH, Lactate dehydrogenase) and oxidative stress (MDA, Malondialdehyde; GSH, Reduced gluthation; NO, Nitric oxide; SOD, Superoxide dismutase) parameters. All rabbits were euthanised for histopathological examination and for distrubition of Peroxisome proliferator activated receptors (PPARs) in tissues by immunohystochemistry. Liver enzymes increased in fat rich diet group throughout the study. Addition of gemfibrozil and L-carnitin in fat rich diet resulted in statistically significant decreasein lipid profile when compared to those only received fat rich diet. Beta oxidation of fat rich diet group was significantly higher than that of groups additionally received gemfibrozil and L-carnitine. Immunohistochemistry revealed an increase in PPAR PPAR-α and β but not PPAR-γ expression in fat rich diet group. On the contrary L-carnitin administration did have any effect on tissue PPAR expression. PPAR-α expression differed between groups received gemfibrozil and fat rich diet and those did not. Fat rich diet increased MDA level while decreased GSH and catalase. Addition of gemfibrozil and L-carnitine to fat rich diet significantly decreased MDA level and increased antioxidants. The most marked macroscopy finding was abdominal fat increase in fat rich diet group (group V). On the other hand gemfibrozil administration resulted in significant abdominal fat decrease. Furthermore decreased abdominal fat was marked in gemfibrozil and L-carnitine given animals (group VIII) when compared to other groups. In conclusion, gemfibrozil and L-carnitine administration alleviated abdominal and hepatic fattening and improved lipid profile. Gemfibrozil also caused a significant increase in PPAR-α expression in the liver. It may be of use in avoiding abdominal fat (obesity) due to high fat by use of gemfibrozil, a synthetic PPAR-a ligand, and L-carnitine.

Keywords

• Gemfibrozil
• hepatic lipidosis
• L-carnitine
• obesity
• PPARs
• rabbits

References

1. Aebi, H. (1984). Catalase in vitro assay methods. Methods in Enzymology, 105, 121-126. doi: 10.1016/s0076-6879(84)05016-3
2. Akbiyik, F., Cinar, K., Demirpence, E., Ozsullu, T., Tunca, R., Haziroglu, R., Yurdaydın, C., Uzunalimoglu, O. & Bozkaya, H. (2007). Ligand-induced expression of peroxisome proliferator-activated receptor  and activation of fatty acid oxidation enzymes in fatty liver. European Journal of Clinical Investigation, 34, 429-435.
3. Ament, Z., West, J.A., Stanley, E., Ashmore, T., Roberts, L.D., Wright, J., Nicholls, A.W. & Griffin, J.L. (2016). PPAR-pan activation induces hepatic oxidative stress and lipidomic remodelling. Free Radical Biology and Medicine, 95, 357-368. doi:10.1016/j.freeradbiomed.2015.11.033
4. Arockia Rani, P.J. & Panneerselvam, C. (2001). Carnitine as a free radical scavenger in aging. Experimental Gerontology, 36, 1713-1726. doi: 10.1016/S0531-5565(01)00116-4
5. Atherton, H.J., Bailey, N.J., Zhang, W., Taylor, J., Major, H., Shockcor, J., Clarke, K. & Griffin, J.L. (2006). A combined 1H-NMR spectroscopy- and mass spectrometry-based metabolomic study of the PPAR-{alpha} null mutant mouse defines profound systemic changes in metabolism linked to the metabolic syndrome. Physiological Genomics, 27, 178-186. doi: 10.1152/physiolgenomics.00060.2006
6. Barter, P.J. & Rye, K.A. (2006). Cardioprotective properties of fibrates. Which fibrate, Which patients, What mechanism?. Circulation, 113, 1553-1555. doi: 10.1161/CIRCULATIONAHA.105.620450
7. Beutler, E., Duran, O. & Kelley, B.M. (1963). Improved method for determination of blood glutathione. Journal of Laboratory and Clinical Medicine, 61, 882-888.
8. Binienda, Z.K. & Ali, S.F. (2001). Neuroprotective role of l-carnitine in the 3-nitropropionic acid induced neurotoxicity. Toxicology Letters, 25, 67-73. DOI: 10.1016/s0378-4274(01)00415-5

9. Brown, J.D. & Plutzky, J. (2007). Peroxisome proliferator-activated receptors as transcriptional nodal points and therapeutic targets. Circulation, 115, 518-533.
10. Brown, G.C. (1999). Nitric oxide and mitochondrial respiration. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1411, 351-369. doi:10.1016/S0005-2728(99)00025-0
11. Costet, P., Legendre, J., More, J., Edgar, A., Galtier, P. & Pineau, T. (1998). Peroxisome proliferator-activated receptor alpha-isoform deficiency leads to progressive dyslipidemia with sexually dimorphic obesity and steatosis. Journal of Biological Chemistry, 273, 29577-29585. doi: 10.1074/jbc.273.45.29577
12. DeFronzo, R.A. (1999). Pharmacologic therapy for type 2 diabetes mellitus. Annals of Internal Medicine, 131, 281-303. doi: 10.7326/0003-4819-131-4-199908170-00008
13. Dokmeci, D., Akpolat, M., Aydogdu, N., Doganay, L. & Turan, F.N. (2005). L-carnitine inhibits ethanol-induced gastric mucosal injury in rats. Pharmacological Reports, 57, 481-488.
14. Dreyer, C., Krey, G., Keller, H., Givel, F., Helftenbein, G. & Wahli, W. (1992). Control of the peroxisomal -oxidation pathway by a novel family of nuclear hormone receptors. Cell, 68, 879-887. doi: 10.1016/0092-8674(92)90031-7
15. Elijah, I.E., Børsheim, E., Maybauer, D.M., Finnerty, C.C., Herndon, D.N. & Maybauer, M.O. (2012). Role of the PPAR-α agonist fenofibrate in severe pediatric burn injury. Burns, 38, 481-86. doi: 10.1016/j.burns.2011.12.004
16. Fritz, I.B. (1955). The effect of muscle extracts on the oxidation of palmitic acid by liver slices and homogenates. Acta Physiologica Scandinavica, 34, 367-385. doi: 10.1111/j.1748-1716.1955.tb01256.x
17. Frei, B, Stocker, R. & Ames, B.N. (1988). Antioxidant defences and lipid peroxidation in human blood plasma. Proceedings of the National Academy of Sciences, 85, 9748-9752. doi: 10.1073/pnas.85.24.9748
18. Guerre-Millo, M., Gervois, P., Raspé, E., Madsen, L., Poulain, P., Derudas, B., Herbert, J.M., Winegar, D.A., Willson, T.M., Fruchart, J.C., Berge, R.K. & Staels, B. (2000). Peroxisome proliferator-activated receptor α activators improve insulin sensitivity and reduce adiposity. Journal of Biological Chemistry, 275, 16638-16642. doi: 10.1074/jbc.275.22.16638
19. Hashimoto, T., Fujita, T., Usuda, N., Cook, W., Qi, C., Peters, J.M., Gonzales, F.J., Yeldandi, A.V., Rao, S.M. & Reddy JK (1999). Peroxisomal and mitochondrial fatty acid -oxidation in mice nullizygous for both PPARα and fatty acyl CoA oxidase: Genotype correlation with fatty liver phenotype. Journal of Biological Chemistry, 274, 19228-19236. doi: 10.1074/jbc.274.27.19228
20. Huang, T.H., Teoh, A.W., Lin, B.L., Lin, D.S. & Roufogalis, B. (2009). The role of herbal PPAR modulators in the treatment of cardiometabolic syndrome. Pharmacological Research, 60, 195-206. doi: 10.1016/j.phrs.2009.03.020
21. Jeffrey, M.P., Susanna, S.T.L., Wen, L., Jerrold, M.W., Oksana, G., Carrie, E., Marc, L.R., Lynn, D.H. & Frank, J.G. (2000). Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor-. Molecular and Cellular Biology, 20, 5119-5128. doi: 10.1128/mcb.20.14.5119-5128.2000
22. Kalaiselvi, T. & Panneersalvam, C. (1998). Effect of L-carnitine on the status of lipid peroxidation and antioxidants in aging rats. The Journal of Nutritional Biochemistry, 9, 575-581. doi: 10.1016/S0955-2863(98)00052-7
23. Keene, B.W. (1991). L-carnitine supplementation in the therapy of canine dilated cardiomyopathy. Veterinary Clinics of North America: Small Animal Practice, 21, 1005-1009. doi: 10.1016/S0195-5616(91)50108-X
24. Kim, H., Haluzik, M., Asghar, Z., Yau, D., Joseph, J.W., Fernandez, A.M., Reitman M.L., Yakar, S., Stannard, Bethel., Heron-Milhavet, L., Wheeler, M.B. & LeRoith, D. (2003). Peroxisome proliferator-activated receptor-α agonist treatment in a transgenic model of type 2 diabetes reverses the lipotoxic state and improves glucose homeostasis. Diabetes, 52, 1770-1778. doi: 10.2337/diabetes.52.7.1770
25. Kopec, B. & Fritz, I.B. (1973). Comparison of properties of carnitine palmitoyltransferase I with those of carnitine palmitoyltransferase II, and preparations of antibodies to carnitine palmitoyltransferases. Journal of Biological Chemistry, 248, 4069-4074. doi: 10.1016/S0021-9258(19)43840-4
26. Kraja, A.T., Province, M.A., Straka, R.J., Ordovas, J.M., Borecki, I.B. & Arnett, D.K. (2010). Fenofibrate and metabolic syndrome. Endocrine Metabolic and Immune Disorders Drug Targets, 10, 138-148. doi: 10.2174/187153010791213047
27. Kremser, K., Stangl, H., Pahan, K. & Singh, I. (1995). Nitric oxide regulates peroxisomal enzyme activities. European Journal of Clinical Chemistry and Clinical Biochemistry, 33, 763-774. doi: 10.1515/cclm.1995.33.11.763
28. LaCount, D.W., Drackley, J.K. & Weigel, D.J. (1995). Responses of dairy cows during early lactation to ruminal or abomasal administration of L-carnitine. Journal of Dairy Science, 78, 1824-1836. doi: 10.3168/jds.S0022-0302(95)76807-2
29. Lawrence, J.W., Li, Y., Chen, S., DeLuca, J.G., Berger, J.P., Umbenhauer, D.R., Moller, D.E. & Zhou, G. (2001). Differential gene regulation in human versus rodent hepatocytes by peroxisome proliferator-activated receptor (PPAR) alpha. PPAR alpha fails to induce peroxisome proliferation-associated genes in human cells independently of the level of receptor expresson. Journal of Biological Chemistry, 276, 31521-31527.
30. Lazarow, P.B. (1981). Assay of peroxisomal β-oxidation of fatty acids. Methods in Enzymology, 72, 315-319. doi: 10.1016/S0076-6879(81)72021-4
31. Lehrke, M. & Lazar, M.A. (2005). The many faces of PPARγ. Cell, 123, 993-999. doi: 10.1016/j.cell.2005.11.026
32. Lohninger, A., Kaiser, E., Legenstein, E. & Staniek, H. (1987). Carnitine, metabolism and function. Ed. Kaiser E., Lohninger A., Carnitine-Its Role in Lung and Heart Disorders, Karger München, pp: 1-25.
33. Longo, N., Frigeni, M. & Pasquali, M. (2016). Carnitine transport and fatty acid oxidation. Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1863, 2422-2435. doi: 10.1016/j.bbamcr.2016.01.023
34. Mandard, S., Muller, M. & Kersten S. (2004). Peroxisome proliferator-activated receptor alpha target genes. Cellular and Molecular Life Sciences, 61, 393-416. doi 10.1007/s00018-003-3216-3
35. Martin, K.R. & Barret, J.C. (2002). Reactive oxygen species as double-edged swords in cellular processes: low-dose cell signaling versus high-dose cell signaling versus high-dose toxicity. Human & Experimental Toxicology, 21, 71-75. doi: 10.1191/0960327102ht213oa
36. McCarty, M. (2001). Hepatothermic therapy of obesity: rationale and invertory of resources. Medical Hypotheses, 57, 324-336.
37. Minnich, A., Tian, N., Byan, L. & Bilder, G. (2001). A potent PPAR alpha agonist stimulates mitochondrial fatty acid beta-oxidation in liver and skeletal muscle. American Journal of Physiology Endocrinology and Metabolism, 280, E270-E279. doi: 10.1152/ajpendo.2001.280.2.E270
38. Mondola, P., Santillo, M., De Mercato, R. & Santangelo, F. (1992). The effect of L-carnitine on cholesterol metabolism in rat (Rattus bubalus) hepatocyte cells. International Journal of Biochemistry, 24, 1047-1050. doi: 10.1016/0020-711x(92)90372-8
39. Moutzouri, E., Kei, A., Elisaf, M.S. & Milionis HJ. (2010). Management of dyslipidemias with fibrates, alone and in combination with statins: Role of delayed-release fenofibric acid. Vascular Health and Risk Management, 6, 525-539. doi: 10.2147/VHRM.S5593
40. Mussoni, L., Mannucci, L., Sirtori, C., Pazzucconi, F., Bonfardeci, G., Cimminiello, C., Notarbartolo, A., Scafidi, V., Bittolo Bon, G., Alessandrini, P., Nenci, G., Parise, P., Colombo, L., Piliego, T. & Tremoli, E. (2000). Effects of gemfibrozil on insulin sensitivity and on haemostatic variables in hypertriglyceridemic patients. Atherosclerosis, 148, 397-406. doi: 10.1016/s0021-9150(99)00283-x
41. Ogawa, S., Takeuchi, K., Sugimura, K., Fukuda, M., Lee, R., Ito, S. & Sato T. (2000). Bezafibrate reduces blood glucose in type 2 diabetes mellitus. Metabolism, 49, 331-334.
42. Owen, K.Q., Nelssen, J.L, Goodband, R.D., Weeden, T.L. & Blum, S.A. (1996). Effect of L-carnitine and soybean oil on growth performance and body composition of early-weaned pigs. Journal of Animal Science, 74, 1612-1619. doi: 10.2527/1996.7471612x
43. Peters, J.M., Hennuyer, N., Staels, B., Fruchart, J.C., Fievet, C., Gonzalez, F.J. & Auwerx, J. (1997). Alterations in lipoprotein metabolism in peroxisome proliferator-activated receptor alpha-deficient mice. Journal of Biological Chemistry, 272, 27307-27312. doi: 10.1074/jbc.272.43.27307
44. Rao, M.S. & Reddy. J.K. (2001). Peroxizomal β-oxidation and steatohepatitis. Seminars in Liver Disease, 21, 43-56. doi: 10.1055/s-2001-12928
45. Ringseis, R., Lüdi, S., Hirche, F. & Eder, K. (2008). Treatment with pharmacological peroxisome proliferator-activated receptor alpha agonist clofibrate increases intestinal carnitine absorption in rats. Pharmacological Research, 58, 58-64. doi: 10.1016/j.phrs.2008.06.006
46. Schoonjans, K., Staels, B. & Auwerx, J. (1996). The peroxisome proliferator activated receptors (PPARs) and their effects on lipid metabolism and adipocyte differentiation. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1302, 93-109. doi: 10.1016/0005-2760(96)00066-5
47. Smith, J.W., Owen, K.Q., Friesen, K.G., Nelssen, J.L., Goodband, R.D. & Tokach, M.D. (1994). The effect of supplemental dietary carnitine, betaine and chromium nicotinate on growth and carcass characteristics in growing-finishing pigs. Kansas State Univ., Swine Day, Agricultural Experiment Station, pp: 154-157.
48. Stalenhoef, A.F., de Graaf, J., Wittekoek, M.E., Bredie, S.J., Demacker, P.N. & Kastelein, J.J. (2000). The effect of concentrated n-3 fatty acids versus gemfibrozil on plasma lipoproteins, low density lipoprotein heterogeneity and oxidizability in patients with hypertriglyceridemia. Atherosclerosis, 153,129-138. doi: 10.1016/s0021-9150(00)00381-6
49. Stienstra, R., Duval, C., Müller, M. & Kersten, S. (2007). PPARs, obesity, and inflammation. PPAR Resesearch, 2007:95974. doi:10.1155/2007/95974
50. Sung, D.J., Kim, S., Kim, J.H.S. & So, W.Y. (2016). Role of l-carnitine in sports performance: Focus on ergogenic aid and antioxidant. Science & Sports, 31, 177-188. doi: 10.1016/j.scispo.2016.02.005
51. Tenenbaum, A., Motro, M. & Fisman, E.Z. (2005). Dual and pan-peroxisome proliferator-activated receptors (PPAR) co-agonism: the bezafibrate lessons. Cardiovascular Diabetology, 4:14. doi: 10.1186/1475-2840-4-14
52. Toda, K., Okoda, T., Miyaura, C. & Saibara, T. (2003). Fenofibrate, a ligant for PPARα, inhibits aromatase cytochrome P450 expression in the ovary of mause. Journal of Lipid Research, 44, 265-270. doi:10.1194/jlr.M200327-JLR200
53. Tunca, R. & Devrim, A.K. (2007). Deneysel olarak obezite olusturulan farelerde ovarioektomi operasyonu ve gemfibrozilin peroksizom proliferatörleri ile aktive olan reseptörler (PPARs) ve obezite üzerine etkilerinin arastirilmasi. TUBITAK TOVAG, proje no: 104 O 297.
54. Uhlenbruck, G. (1996). L-Carnitine and the immune system: from the mode of metabolism to the modulation of membranes. Proc Carnitine Symp Leipzig, pp: 47-60.
55. Wägner, A.M., Jorba, O., Bonet, R., Ordóñez-Llanos, J. & Pérez, A. (2003). Efficacy of atorvastatin and gemfibrozil, alone and in low dose combination, in the treatment of diabetic dyslipidemia. The Journal of Clinical Endocrinology & Metabolism, 88, 3212-3217.
56. Wahli, W. (2002). Peroxisome proliferator-activated receptors (PPARs): from metabolic control to epidermal wound healing. Swiss Medical Weekly, 132, 83-91.
57. Xin, X., Yang, S., Kowalski, J. & Gerritsen, M.E. (1999). Peroxisome proliferator-activated receptor gamma ligands are potent inhibitors of angiogenesis in vitro and in vivo. Journal of Biological Chemistry, 274, 9116-9121. doi: 10.1074/jbc.274.13.9116
58. Xu, H., Barnes, G.T., Yang, Q., Tan, G., Yang, D., Chou, C.J., Sole, J., Nichols, A., Ross, J,S., Tartaglia, L.A. & Chen, H. (2003). Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. Journal of Clinical Investigation, 112, 1821-1830.
59. Yoon, M., Jeong, S., Lee, H., Han, M., Kang, J.H., Kim, E.Y., Kim, M. & Oh, G.T. (2003). Fenofibrate improves lipid metabolism and obesity in ovariectomised LDL receptor-nul mice. Biochemical and Biophysical Research Communications, 302, 29-34. doi: 10.1016/s0006-291x(03)00088-3
60. Yoshoiko, T., Kawada, K. & Shimada, T. (1979). Lipid peroxidation in maternal and cord blood and protective mechanism against activated-oxigen toxicity in the blood. American Journal of Obstetrics and Gynecology, 135, 372-376.
61. Yu, K., Bayona, W., Kallen, C.B., Harding, H.P., Ravera, C.P., McMahon, G., Brown, M. & Lazar, M.A. (1995). Differential activation of peroxisome proliferator-activated receptors by eicosanoids. Journal of Biological Chemistry, 270, 23975-23983. doi: 10.1074/jbc.270.41.23975
62. Zambon, A. & Cusi, K. (2007). The role of fenofibrate in clinical practice. Diabetes and Vascular Disease Research, 4, S15-S20. doi: 10.3132/dvdr.2007.053