Abstract
Background:
Leptin is one of the possible mediators of ethanol intake. On the other hand, the concentration of total plasma homocysteine (Hcy) is a well-established indicator for the risk of cardiovascular disease and seems to be related to ethanol consumption. So, the aim of the present study was to investigate the effect of acute (70%) and chronic (10%) exposure to evaporated ethanol on: 1-brain leptin and Hcy concentration on the 15th day of embryonic development of chick. 2- brain leptin and Hcy concentration immediately after hatch of chick and 3- serum leptin concentration immediately after hatch of chick.Materials and Methods:
In this experimental study 60 fertilized eggs were used. Eggs were divided into control; acute exposure to ethanol and chronic exposure to ethanol. Hcy was measured by using enzyme-linked assay and leptin was measured with the chick leptin radioimmunoassay kit.Results:
Data showed brain Hcy concentration on the 15th day of embryonic stage of chicken that acute and chronic exposure to ethanol significantly (p<0.05) decreased, but did not have any effect on brain Hcy concentration immediately after hatch in chicken that acute and chronic exposure to ethanol during embryonic stages. Acute and chronic exposure to ethanol during embryonic stages significantly (p<0.05) increased brain leptin on the 15th day of embryonic stage, brain leptin immediately after hatch of chicken and plasma leptin immediately after hatch of chicken.Conclusion:
Present results indicated that acute and chronic exposure to ethanol by evaporation in embryonic stage of chicken can change the brain Hcy, brain leptin and serum leptin.Keywords
Introduction
Leptin is a 16 K-Da polypeptide hormone (167 amino acids). The OB gene codes for hormones are secreted mainly by adipose tissue, placenta, fetal tissue and membranes, and stomach. Leptin reaches the brain, crossing the blood-brain barrier or the chroroid-cerebrospinal fluid barrier and informs the brain about the size of the fat stores [1]. Leptin has a wide variety of central and peripheral actions such as reproduction, food intake, energy expenditure, lipid metabolism, immune system, blood pressure and angiogenesis [2, 3]. Recent evidence has shown leptin as a risk factor for vascular disease. It may be an important link between cardiovascular disease and obesity. Leptin has a procoagulnat, atherosclerotic role and platelet aggregation effect [4].
Homocysteine (Hcy) is a sulfur-containing amino acid produced from food methionine metabolism in the body. It can be converted to methionine or cysteine by remethylation or trans-sulforation cycles with some enzymes (MS, MTHFR, SAM) and cofactors (B6, B12). Folate as a methyl group donor is essential in the remethylation cycle, too. Hcy is transported from blood brain barrier into the CSF and brain, although human neural cells are capable of producing Hcy [5]. Insufficient folic acid, B6, B12 and impairment in enzymes functions cause hyperhomocysteinemia. Hcy>12 µM/dL has been shown as a risk factor for vascular disease, brain athrophy and neurodegenrative disease [6]. Hyper-homocysteinemia has been linked to atherosclerosis and thrombosis [7].
Ethanol has weakly charged molecules that move easily through the cell membrane, rapidly equilibrating between blood and tissue. Alcohol, at low doses can have some beneficial effects such as decreased rates of myocardial infarction, stroke, gallstone, and possibly vascular or Alzheimer’s dementia, but the consumption of more than 2 standard drinks per day increases the risk for health problems in many organ systems and hormonal changes [8]. Ethanol intake by changes in adipose tissue and BMI can affect leptin concentration [9]. Some studies have indicated an inverse relation between alcohol use and leptin level [10]. In other investigations ethanol is shown as a powerful inducer of hyperleptinemia in both animals and humans. Alcohol intake has the potential to alter body weight as it is energy-dense and may also alter eating behavior at higher levels of consumption. Each of these lifestyle factors, therefore, has the ability to alter adipose tissue mass, possibly via leptin [9]. On the other hand, short-term and chronic ethanol intake influences Hcy concentration by changes in the methylation pathway, folate and cofactors concentration [11]. Ethanol-induced increase in serum Hcy levels has been observed in active alcoholics. Exogenous ethanol caused elevated endogenous brain Hcy level, reduced S-adenosyl methionine (SAM) levels, and increased S- adenosyl Hcy (SAH) levels, which correlated to increased brain caspase-3 activities [12].
The reported sequences of leptin from human, cow, pig, sheep, mouse, rat, dog, and chicken showed a high degree of sequence conservation. This similarity suggests a common function or mechanism of hormone across species [13]. On the other hand, in previous studies exogenous ethanol caused a 1.6 fold increase in chick brain Hcy level at 11 days of development [12].
The present study was to investigate the effect of acute (70%) and chronic (10%) exposure to evaporated ethanol on brain leptin and Hcy concentration on the 15th day of embryonic development of chick, brain and serum leptin, and Hcy concentration immediately after hatch of chick. Fifteenth day that we used in present study was according to our previous study [14].
Materials and Methods
In this experimental study all procedures were approved by the animal care committee of the Iran academy of veterinary medicine. This study was fundamental experimental. Sixty fertile, pathogen-free, cub eggs were purchased from Fars Poultry center. More than 20 eggs (natural fertility is rarely 100%) of each group (acute, chronic exposure, control) were incubated under standard conditions (75% humidity at 37ºC). Humidity and temperature were checked by thermometer and a hygrometer. Incubator turning (3 times a day) was considered essential in the early stages. For the last 3 days of incubation when the chick was preparing to hatch, turning was stopped.
Test groups: eggs were divided into three groups 1- control group (eggs incubated in normal condition, N=20), 2- chronic group (eggs incubated while the water in incubator was replaced by 10 % ethanol, N=20), and 3- acute group (eggs incubated while the water in incubator was replaced by 70 % ethanol at the 6th, 13th and 20th days). In all groups half of the eggs were examined on the 15th day of developmental stage and the other half were examined immediately after hatching of chick.
For the extraction of brain, single whole brain was mixed with a phosphate buffer solution after homogenization, the mixture was centrifuged, and the brain extract was prepared for measurement of brain HCY and leptin. HCY were assayed by liquid stable 2-part HCY reagent cobas mira plus and leptin was assayed by chicken leptin ELISA kit from Cosabio Inc. The data were analyzed by SPSS-18 program and using one way ANOVA and Tukey as post hoc test. Significant level was considered to be p<0.05.
Results
Our data showed that brain Hcy concentration significantly (p=0.01) decreased on the 15th day of embryonic stage of chicken in acute and chronic groups relative to control group (Fig. 1). Figure 2 shows that acute and chronic exposure to ethanol had no significant effect on Hcy concentration on the first day of hatch.
Brain Hcy concentration on the 15th day of embryonic stage and the first day of hatch had not significant difference between acute and chronic groups.
Present data showed that following acute and chronic exposure to ethanol, brain leptin significantly (p<0.001) increased on the 15th day of embryonic stage of chick and on the first day of chick hatching (Fig. 3, 4). Also, serum leptin significantly (p<0.01) increased following acute and chronic exposure to ethanol on the first day of chick hatching (Fig. 5).
Brain leptin concentration on the 15th day of embryonic stage and the first day of hatch, significantly was lower in acute than chronic exposure to ethanol. But serum leptin concentration on the first day of hatch was higher in acute than chronic exposure to ethanol.
Effect of acute and chronic exposure to ethanol on brain Hcy on the 15th day of embryonic stage. *Significant difference relative to control (p<0.05)
Effect of chronic and acute exposure to ethanol on brain Hcy on the first day of chick hatching
Effect of acute and chronic exposure to ethanol on brain leptin on the 15th day of embryonic stage *Significant difference relative to control (p<0.05) **Significant difference relative to chronic (p<0.05)
Effect of acute and chronic exposure to ethanol on brain leptin on the first day of chick hatching *Significant difference relative to control (p<0.05) ** Significant difference relative to chronic (p<0.05)
Effect of acute and chronic exposure to ethanol on serum leptin on the first day of chick hatching * Significant difference relative to control (p<0.05) ** Significant difference relative to chronic (p<0.05)
Acknowledgements
References
-
1.
Fernandez-Galaz MC, Fernandez-Agullo T, Carrascosa JM, Ros M, Garcia-Segura LM. Leptin accumulation in hypothalamic and dorsal raphe neurons is inversely correlated with brain serotonin content. Brain Res. 2010;1329:194-202. [PubMed ID: 20211152]. https://doi.org/10.1016/j.brainres.2010.02.085.
-
2.
Balasubramaniyan V, Nalini N. The potential beneficial effect of leptin on an experimental model of hyperlipidemia, induced by chronic ethanol treatment. Clin Chim Acta. 2003;337(1-2):85-91. [PubMed ID: 14568184].
-
3.
Beltowski J. Leptin and atherosclerosis. Atherosclerosis. 2006;189(1):47-60. [PubMed ID: 16580676]. https://doi.org/10.1016/j.atherosclerosis.2006.03.003.
-
4.
Wannamethee SG, Tchernova J, Whincup P, Lowe GD, Kelly A, Rumley A, et al. Plasma leptin: associations with metabolic, inflammatory and haemostatic risk factors for cardiovascular disease. Atherosclerosis. 2007;191(2):418-26. [PubMed ID: 16712853]. https://doi.org/10.1016/j.atherosclerosis.2006.04.012.
-
5.
Obeid R, Herrmann W. Mechanisms of homocysteine neurotoxicity in neurodegenerative diseases with special reference to dementia. FEBS Lett. 2006;580(13):2994-3005. [PubMed ID: 16697371]. https://doi.org/10.1016/j.febslet.2006.04.088.
-
6.
Sachdev PS. Homocysteine and brain atrophy. Prog Neuropsychopharmacol Biol Psychiatry. 2005;29(7):1152-61. [PubMed ID: 16102882]. https://doi.org/10.1016/j.pnpbp.2005.06.026.
-
7.
Spencer TA, Chai H, Fu W, Ramaswami G, Cox MW, Conklin BS, et al. Estrogen blocks homocysteine-induced endothelial dysfunction in porcine coronary arteries(1,2). J Surg Res. 2004;118(1):83-90. [PubMed ID: 15093721]. https://doi.org/10.1016/j.jss.2004.01.021.
-
8.
Kasper. D. L, Braunwald. E, Hauser S. Harrison's principles of internal medicine. 18th ed. Philadelphia: McGraw-Hill; 2004.
-
9.
de Silva A, de Courten M, Zimmet P, Nicholson G, Kotowicz M, Pasco J, et al. Lifestyle factors fail to explain the variation in plasma leptin concentrations in women. Nutrition. 1998;14(9):653-7. https://doi.org/10.1016/s0899-9007(98)00065-3.
-
10.
Donahue RP, Zimmet P, Bean JA, Decourten M, Donahue RA, Collier GR, et al. Cigarette Smoking, Alcohol Use, and Physical Activity in Relation to Serum Leptin Levels in a Multiethnic Population. Annals of Epidemiology. 1999;9(2):108-13. https://doi.org/10.1016/s1047-2797(98)00037-4.
-
11.
Carrasco MP, Jiménez-López JM, Segovia JL, Marco C. Comparative study of the effects of short- and long-term ethanol treatment and alcohol withdrawal on phospholipid biosynthesis in rat hepatocytes. Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology. 2002;131(3):491-7. https://doi.org/10.1016/s1096-4959(02)00006-4.
-
12.
Berlin KN, Cameron LM, Gatt M, Miller RJ. Reduced de novo synthesis of 5-methyltetrahydrofolate and reduced taurine levels in ethanol-treated chick brains. Comp Biochem Physiol C Toxicol Pharmacol. 2010;152(3):353-9. [PubMed ID: 20541623]. https://doi.org/10.1016/j.cbpc.2010.06.002.
-
13.
Ashwell CM, Czerwinski SM, Brocht DM, McMurtry JP. Hormonal regulation of leptin expression in broiler chickens. Am J Physiol. 1999;276(1 Pt 2):R226-32. [PubMed ID: 9887199].
-
14.
Taherianfard M, Davazdahemamy M, Shojaeifard M, Sharifi M. Acute and chronic exposure of chick embryo to ethanol alters brain neurosteroid levels. J Physiol Biochem. 2013;69(1):141-5. [PubMed ID: 22826196]. https://doi.org/10.1007/s13105-012-0198-3.
-
15.
Ozguven I, Ersoy B, Ozguven A, Ozkol M, Onur E. Factors affecting carotid intima media thickness predicts early atherosclerosis in overweight and obese adolescents. Obes Res Clin Pract. 2010;4(1):e1-e82. [PubMed ID: 24345625]. https://doi.org/10.1016/j.orcp.2009.06.003.
-
16.
Bleich S, Degner D, Sperling W, Bonsch D, Thurauf N, Kornhuber J. Homocysteine as a neurotoxin in chronic alcoholism. Prog Neuropsychopharmacol Biol Psychiatry. 2004;28(3):453-64. [PubMed ID: 15093951]. https://doi.org/10.1016/j.pnpbp.2003.11.019.
-
17.
Sakuta H, Suzuki T. Alcohol consumption and plasma homocysteine. Alcohol. 2005;37(2):73-7. [PubMed ID: 16584970]. https://doi.org/10.1016/j.alcohol.2005.12.005.
-
18.
Sakuta H, Suzuki T, Ito T, Yasuda H. Beer ethanol consumption and plasma homocysteine among patients with type 2 diabetes. Diabetes Res Clin Pract. 2007;78(2):202-7. [PubMed ID: 17521771]. https://doi.org/10.1016/j.diabres.2007.03.016.
-
19.
Lakshman R, Garige M, Gong M, Leckey L, Varatharajalu R, Zakhari S. Is alcohol beneficial or harmful for cardioprotection? Genes Nutr. 2010;5(2):111-20. [PubMed ID: 20012900]. https://doi.org/10.1007/s12263-009-0161-2.
-
20.
Bleich S, Bandelow B, Javaheripour K, Müller A, Degner D, Wilhelm J, et al. Hyperhomocysteinemia as a new risk factor for brain shrinkage in patients with alcoholism. Neurosci Lett. 2003;335(3):179-82. https://doi.org/10.1016/s0304-3940(02)01194-1.
-
21.
de Bree A, Verschuren WM, Blom HJ, Kromhout D. Association between B vitamin intake and plasma homocysteine concentration in the general Dutch population aged 20-65 y. Am J Clin Nutr. 2001;73(6):1027-33. [PubMed ID: 11382655].
-
22.
Shinohara M, Ji C, Kaplowitz N. Differences in betaine-homocysteine methyltransferase expression, endoplasmic reticulum stress response, and liver injury between alcohol-fed mice and rats. Hepatology. 2010;51(3):796-805. [PubMed ID: 20069651]. https://doi.org/10.1002/hep.23391.
-
23.
Balasubramaniyan V, Nalini N. Intraperitoneal leptin regulates lipid metabolism in ethanol supplemented Mus musculas heart. Life Sci. 2006;78(8):831-7. [PubMed ID: 16137712]. https://doi.org/10.1016/j.lfs.2005.05.079.
-
24.
Patterson-Buckendahl P, Pohorecky LA, Kvetnansky R. Differing effects of acute and chronic stressors on plasma osteocalcin and leptin in rats. Stress. 2007;10(2):163-72. [PubMed ID: 17514585]. https://doi.org/10.1080/10253890701317601.