IMPORTANCE OF THE LEPTIN/GRELIN RATIO AS A BIOMARKER IN DIETARY INDUCED HYPERLIPIDEMIA IN FEMALE C57Black/6 MICE

Visceral obesity, dyslipidemia and insulin resistance are considered the main causes of metabolic disorders in metabolic syndrome. Leptin and ghrelin are the most important factors involved in regulation of the metabolic processes. The purpose of this study was to evaluate the significance of leptin-to-ghrelin ratio (L/Gh) and cytokine profiles as biomarkers of metabolic and immune disorders in an in vivo model of a dietary induced dyslipidemia in mice.

The studies were carried out on 48 female C57Black/6 mice, which were divided into 6 groups of 8 animals. Group 1 (control) received the AIN93 diet; group 2, excess fat administration (30% dry weight); the mice from group 3 were supplied with 20% fructose in drinking water added to the main diet; group 4 got fats and fructose excess, group 5, cholesterol excess (0.5% dry weight); group 6 was fed with cholesterol and fructose in excess. Duration of the experiment was 63 days. In all animals, the relative mass of internal organs was determined. The levels of cytokines, leptin and ghrelin in plasma were determined by means of Luminex 200 analyzer using Bio-Plex kits.

There were no significant differences for plasma leptin and ghrelin concentrations between the control and most of experimental groups, except of the 6th group (combined diet with excess fructose and cholesterol) which a significantly lower leptin levels as compared to the controls (group 6: 2.12 pg/ml, min 1.57 – max 3.83 vs group 1: 3.92 pg/ml, min 2.45 – max 27.88, p < 0.05). The changes in plasma ghrelin contents, depending on the diet, showed a generally opposite trend when compared to leptin levels.

The value of L/Gh ratio in mice fed with excess fat (group 2) and cholesterol (group 5) showed a statistically unsignificant trend for increase. Fructose added to a diet with fat or cholesterol excess caused a significant decrease in L/Gh ratio (p < 0.05). In animals of the 6th group (fructose + cholesterol) with minimal L/Gh values, the lowest total body fat deposition was found. The relationship between the L/Gh and the fat mass was confirmed by the linear regression relationship between the indicators considered. We have also found a correlation between L/Gh and animal fat weight (r = 0.424, α = 0.004), relative mass of adipose tissue (r = 0.663, α = 0.000), liver (r = -0.315, α = 0.035) and spleen (r = -0.585; α = 0.000), statistically significant correlations between L/Gh, organ and tissue weight and concentrations of IL-12(p40), IL-2, IL-9, IL-13, G-CSF and RANTES. In addition, significant differences were found between the control and test groups for plasma concentrations of G-CSF, IL-12(p40), IL-2, IL-3 and IL-9. The IL-12(p40) concentration in plasma from the group 2 mice was the lowest against controls. Meanwhile, the 6th group exhibited highest IL-12(p40) levels against the lowest L/Gh ratios and total fat levels.

Thus, a significant relationship was found between L/Gh ratio and changing mass of organs and tissues, as well as with the levels of cytokines involved into the regulation of inflammation. The most significant relationship was found between the relative mass of body fat, L/Gh ratio and the IL-12(p40) concentration. L/ Gh ratio and IL-12p(40) showed both content Not only a correlation dependence, but also significant changes were noted between the experimental groups in the in vivo model of alimentary dyslipidemia in mice.

1. Apryatin S.A., Mzhelskaya K.V., Trusov N.V., Balakina A.S., Kulakova S.N., Soto S.Kh., Makarenko M.A., Riger N.A., Tutelyan V.A. Comparative characteristics of in vivo models of hyperlipidemia in Wistar rats and C57Bl/6 mice. Voprosy pitaniya = Problems of Nutrition, 2016, Vol. 85, no. 6, pp. 14-23. (In Russ.)

2. Aroor A.R., McKarns S., Demarco V.G., Jia G., Sowers J.R. Maladaptive immune and inflammatory pathways lead to cardiovascular insulin resistance. Metabolism, 2013, Vol. 62, no. 11, pp. 1543-1552.

3. Baumann H., Morella K.K., White D.W., Dembski M., Bailon P.S., Kim H., Lai C.F., Tartaglia L.A. The fulllength leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc. Natl. Acad. Sci. USA, 1996, Vol. 93, no. 16, pp. 8374-8378.

4. Choi K.M., Ryu O.H., Lee K.W., Kim H.Y., Seo J.A., Kim S.G., Choi D.S., Baik S.H. Serum adiponectin, interleukin-10 levels and inflammatory markers in the metabolic syndrome. Diabetes Research and Clinical Practice, 2007, Vol. 75, no. 2, pp. 235-240.

5. Cicero A.F., Magni P., More M., Ruscica M., Borghi C., Strollo F. Brisighella heart study staff. Metabolic syndrome, adipokines and hormonal factors in pharmacologically untreated adult elderly subjects from the Brisighella Heart Study historical cohort. Obesity Facts, 2012, Vol. 5, no. 3, pp. 319-326.

6. Claycombe K., King L.E., Fraker P.J. A role for leptin in sustaining lymphopoiesis and myelopoiesis. Proc. Natl. Acad. Sci. USA, 2008, Vol. 105, no. 6, pp. 2017-2021.

7. Cong W.N., Golden E., Pantaleo N., White C.M., Maudsley S., Martin B. Ghrelin receptor signaling: a promising therapeutic target for metabolic syndrome and cognitive dysfunction. CNS and Neurological Disorders Drug Targets, 2010, Vol. 9, no. 5, pp. 557-563.

8. Cui H., López M., Rahmouni K. The cellular and molecular bases of leptin and ghrelin resistance in obesity. Nat. Rev. Endocrinol., 2017, Vol. 13, no. 6, pp. 338-351.

9. Deng Y., Scherer P.E. Adipokines as novel biomarkers and regulators of the metabolic syndrome. Annals of the New York Academy of Sciences, 2011, Vol. 1226, no. 1, pp. 50-69.

10. Dong M., Ren J. What fans the fire: insights into mechanisms of leptin in metabolic syndrome-associated heart diseases. Current Pharmaceutical Design, 2014, Vol. 20, no. 4, pp. 652-658.

11. Falahi E., Khalkhali Rad A.H., Roosta S. What is the best biomarker for metabolic syndrome diagnosis? Diabetes Metab. Syndr., 2015, Vol. 9, no. 4, pp. 366-372.

12. Fantuzzi G., Faggioni R. Leptin in the regulation of immunity, inflammation, and hematopoiesis. J. Leukoc. Biol., 2000, Vol. 68, no. 4, pp. 437-446.

13. Finucane F.M., Luan J., Wareham N.J., Sharp S.J., O’Rahilly S., Balkau B., Flyvbjerg A., Walker M., Højlund K., Nolan J., Savage DB. Correlation of the leptin:adiponectin ratio with measures of insulin resistance in non-diabetic individuals. Diabetologia, 2009, Vol. 52, no. 11, pp. 2345-2349.

14. Guide for the care and use of laboratory animals. Committee for the Update of the Guide for the Care and Use of Laboratory Animals; Institute for Laboratory Animal Research (ILAR); Division on Earth and Life Studies (DELS); National Research Council of the national academies. 8th Ed. Washington: The National Academies Press, 2011, 248 p.

15. Howard J.K., Lord G.M., Matarese G., Vendetti S., Ghatei M.A., Ritter M.A., Lechler R.I., Bloom S.R. Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice. J. Clin. Invest., 1999, Vol. 104, no. 8, pp. 1051-1059.

16. Kashani A., Salehi B., Anghesom D., Kawayeh A.M., Rouse G.A., Runyon B.A. Spleen size in cirrhosis of different etiologies. J. Ultrasound. Med., 2015, Vol. 34, no. 2, pp. 233-238.

17. Katare R., Rawal S., Munasinghe P.E., Tsuchimochi H., Inagaki T., Fujii Y., Dixit P., Umetani K., Kangawa K., Shirai M., Schwenke D.O. Ghrelin promotes functional angiogenesis in a mouse model of critical limb ischemia through activation of proangiogenic microRNAs. Endocrinology, 2016, Vol. 157, no. 2, pp. 432-445.

18. Kelly A.S., Jacobs D.R. Jr., Sinaiko A.R., Moran A., Steffen L.M., Steinberger J. Relation of circulating oxidized LDL to obesity and insulin resistance in children. Pediatric. Diabetes, 2010, Vol. 11, no. 8, pp. 552-555.

19. Khazaei M., Tahergorabi Z. Systemic ghrelin administration alters serum biomarkers of angiogenesis in diet-induced obese mice. Int. J. Pept., 2013, ID 249565, 5 p.

20. Kohno D., Nakata M., Maekawa F., Fujiwara K., Maejima Y., Kuramochi M., Shimazaki T., Okano H., Onaka T., Yada T. Leptin suppresses ghrelin-induced activation of neuropeptide Y neurons in the arcuate nucleus via phosphatidylinositol 3-kinase- and phosphodiesterase 3-mediated pathway. Endocrinology, 2007, Vol. 148, no. 5, pp. 2251-2263.

21. Kotani K., Sakane N. Leptin:adiponectin ratio and metabolic syndrome in the general Japanese population. Korean J. Lab. Med., 2011, Vol. 31, no. 3, pp. 162-166.

22. Kraja A.T., Province M.A., Arnett D., Wagenknecht L., Tang W., Hopkins P.N. Do inflammation and procoagulation biomarkers contribute to the metabolic syndrome cluster? Nutrition and metabolism, 2007, Vol. 4, no. 1, pp. 28-40.

23. Lam Q.L., Lu L. Role of leptin in immunity. Cell. Mol. Immunol., 2007, Vol. 4, no. 1, pp. 1-13.

24. Lee B.C., Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesityinduced insulin resistance. Biochim. Biophys. Acta, 2014, Vol. 1842, no. 3, pp. 446-462.

25. Li L., Duan M., Chen W., Jiang A., Li X., Yang J., Li Z. The spleen in liver cirrhosis: revisiting an old enemy with novel targets. J. Transl. Med., 2017, Vol. 15, no. 1, pp. 111-120.

26. López-Jaramillo P., Gómez-Arbeláez D., López-López J., López-López C., Martínez-Ortega J., GómezRodríguez A., Triana-Cubillos S. The role of leptin/adiponectin ratio in metabolic syndrome and diabetes. Horm. Mol. Biol. Clin. Invest., 2014, Vol. 18, no. 1, pp. 37-45.

27. Martinelli N., Micaglio R., Consoli L., Guarini P., Grison E., Pizzolo F., Friso S., Trabetti E., Pignatti P.F., Corrocher R., Olivieri O., Girelli D. Low levels of serum paraoxonase activities are characteristic of metabolic syndrome and may influence the metabolic-syndrome-related risk of coronary artery disease. Exp. Diabetes Res., 2012, Vol. 2012, ID 231502, 9 p.

28. Musialik K. The influence of chosen adipocytokines on blood pressure values in patients with metabolic syndrome. Kardiol Pol., 2012, Vol. 70, no. 12, pp. 1237-1242.

29. Myers M.G. Jr. Leptin receptor signaling and the regulation of mammalian physiology. Recent Prog. Horm. Res., 2004, Vol. 59, pp. 287-304.

30. Ouchi N., Parker J.L., Lugus J.J., Walsh K. Adipokines in inflammation and metabolic disease. Nat. Rev. Immunol., 2011, Vol. 11, no. 2, pp. 85-97.

31. Perello M., Scott M.M., Sakata I., Lee C.E., Chuang J.C., Osborne-Lawrence S., Rovinsky S.A., Elmquist J.K., Zigman J.M. Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain. J. Comp. Neurol., 2012, Vol. 520, pp. 281-294.

32. Pérez-Pérez A., Vilariño-García T., Fernández-Riejos P., Martín-González J., Segura-Egea J.J., SánchezMargalet V. Role of leptin as a link between metabolism and the immune system. Cytokine Growth Factor Rev., 2017, Vol. 35, pp. 71-84.

33. Perna V., Perez-Perez A., Fernandez-Riejos P., Polo-Padillo J., Batista N., Dominguez-Castellano A., Sanchez-Margalet V. Effective treatment of pulmonary tuberculosis restores plasma leptin levels. Eur. Cytokine Netw., 2013, Vol. 24, no. 4, pp. 157-161.

34. Reeves P.C. AIN-93 purified diets for the study of trace elements metabolism in rodents. Trace elements in laboratory rodents. Ed. Watson R.R., CRC Press. Ink., 1997, pp. 3-34.

35. Ryo M., Nakamura T., Kihara S., Kumada M., Shibazaki S., Takahashi M., Nagai M., Matsuzawa Y., Funahashi T. Adiponectin as a biomarker of the metabolic syndrome. Circ. J., 2004, Vol. 68, no. 11, pp. 975-981.

36. Silva H.A., Carraro J.C., Bressan J., Hermsdorff H.H. Relation between uric acid and metabolic syndrome in subjects with cardiometabolic risk. Einstein, 2015, Vol. 13, no. 2, pp. 202-208.

37. Srikanthan K., Feyh A., Visweshwar H., Sodhi K. Systematic review of metabolic syndrome biomarkers: a panel for early detection, management, and risk stratification in the West Virginian population. Int. J. Med. Sci., 2016, Vol. 13, no. 1, pp. 25-38.

38. Tartaglia L.A. The leptin receptor. J. Biol. Chem., 1997, Vol. 272, no. 10, pp. 6093-6096.

39. Trottier M.D., Naaz A., Li Y., Fraker P.J. Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice. Proc. Natl. Acad. Sci. USA, 2012, Vol. 109, no. 20, pp. 7622-7629.

40. Varela L., Vazquez M.J., Cordido F., Nogueiras R., Vidal-Puig A., Dieguez C., López M. Ghrelin and lipid metabolism: key partners in energy balance. J. Mol. Endocrinol., 2011, Vol. 46, no 2, pp. 43-63.

41. Williams R.L., Wood L.G., Collins C.E., Morgan P.J., Callister R. Energy homeostasis and appetite regulating hormones as predictors of weight loss in men and women. Appetite, 2016, Vol. 101, pp. 1-7.