20 J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 20–23 Research Objective Interleukin (IL)-15 is highly expressed in skeletal muscles, where it exerts anabolic effects, increase protein content in muscle fibres and promotes muscle growth. Alcoholics frequently suffer myopathy. Therefore, we analyse the level of IL-15 [and other myokines, such as tumor necrosis factor-α (TNF-α)] in alcoholics. Follow-up of skeletal muscle cytokines (myokines) such as IL-15 and TNF-α level in alcoholism, in an attempt to reveal if a certain level of myokines can be considered as a risk factor for short-term motility. Methods IL-15 and TNF-α were determined by enzyme-linked immunoassay analytic techniques in blood samples of 70 chronic alcoholics and 70 age- and sex-matched controls, and then the levels of myokines were correlated with liver enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma glutamate transferase (GGT), amount of ethanol consumed, duration and creatine kinase (CK) activity levels. Results All the alcoholic patients were heavy drinkers (217.04 ± 149.93 g/day), who started at an early age (13.97 ± 8.96 years). IL-15, TNF-α levels and liver enzyme activity were significantly higher in these patients than in controls. Significant relationship was found between IL-15, quantity of ethanol consumption, TNF-α, CK, AST/ALT and between TNF-α and daily ethanol consumption (quantity) and GGT. Conclusion A certain level of myokines such as IL-15 and TNF-α can be considered as a risk factor of alcoholics for short-term motility. Keywords IL-15, TNF-α, alcoholic myopathy, liver impairment Myokines in alcoholic myopathy Kammela Hadi Shammam,a Fadhil Jawad Al-Tu’mab & Hedef Dafer El-Yassinc Introduction Interleukin (IL)-15 is a relatively newly described cytokine, highly expressed in skeletal muscle. IL-15 mRNA levels are highly up-regulated in response to strength training, and it seems to exert anabolic effects, increasing protein content in muscle fibres1 and promoting myogenic differentiation and muscle growth. It would exert an opposite effect to that of tumor necrosis factor α (TNF-α), since it is able to antagonize muscle protein break-down in a cancer cachexia model.2 Since its discovery in 1994,3 it has become clear that it is secreted by many tissues, such as muscle, kidney, heart, lung, dendritic cells, monocytes and macrophages and also enterocytes,4 but muscle is the major site of IL-15 mRNA transcription and probably secretion.3 In addition to its actions on muscle, it also exerts many other functions on memory T cells, natural killer (NK) cells, eosinophils, neutrophils, monocytes and mac- rophages5 and shares stimulatory actions on T-cells with IL-2, partly due to the similarity of their respective receptors. Indeed, the heterotrimeric IL-15 receptor is composed of beta and gamma chains identical to those of the IL-2 receptor, together with a specific alpha chain. It is, therefore, not sur- prising that it acts as a potent activator of T and B lympho- cytes. It is also involved in the maintenance of T cell memory and in the activation of other immune cells, such as neutro- phils and NK cells.6 It may be also related to the pathogenesis of autoimmune diseases.7 Regarding its metabolic function, it probably participates in the cross-talk between muscle and fat, leading to a reduction of the latter. In chronic alcoholism, muscle wasting is a prominent fea- ture. Chronic alcoholic myopathy has been found in nearly 50–60% alcoholics,8–10 and it is defined by muscle atrophy, pre- dominantly affecting type IIb fibres11,12 and leading, some- times, to incapacitating weakness. IL-15 increased after exercises and physical activity that occurs in alcoholics, due to encourage aggression or violence by disrupting normal brain function. According to the disinhibition hypothesis, for example, alcohol weakens brain mechanisms that normally restrain impulsive behaviours, including inappropriate aggres- sion.13 By impairing information processing alcohol can also lead a person to misjudge social cues. TNF-α, a pro-inflammatory cytokine, also increases in acute or chronic alcoholics due to the impairment of liver. Studies have found that alcohol may increase the liver’s sensi- tivity to inflammatory cytokines, such as TNF-α, in two ways. First, alcohol may directly or indirectly stimulate Kupffer cells to produce and release TNF-α into small channels (i.e. sinu- soids) in which the blood flows through the liver. One indirect mechanism is that the alcohol induces an increase in the levels of a bacterial endotoxin in the blood. Second, alcohol may enhance the sensitivity of hepatocytes to TNF-α.14 The processes by which alcohol is broken down in the hepatocytes generate a variety of molecules that can be toxic to the liver, or it may interfere with normal physiological pro- cesses. For example, alcohol breakdown through the enzyme known as cytochrome P450 2E1(CYP2E1) leads to the forma- tion of small oxygen-containing molecules called reactive oxygen species (ROS). Unless they are rapidly eliminated or converted into harmless molecules by antioxidants, it can interact with and damage complex molecules in the cells (e.g. proteins and DNA).15 Both increased and decreased levels of ROS can lead to apoptosis of hepatocytes. Materials and Methods Seventy alcoholic patients were included (patient group), who were admitted to Ibn-Rushd Teaching Hospital, Baghdad, a Institute of Forensic Medicine, Ministry of Health, Baghdad, Iraq. b Department of Biochemistry, College of Medicine, University of Karbala, Karbala, Iraq. c Department of Biochemistry, College of Medicine, University of Baghdad, Baghdad, Iraq. Correspondence to Fadhil Jawad Al-Tu’ma (email: f_altoma_56@yahoo.com). (Submitted: 17 April 2015 – Revised version received: 21 May 2015 – Accepted: 23 May 2015 – Published online: Summer 2015) ISSN 2413-0516 21J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 20–23 Research Myokines in alcoholic myopathyKammela Hadi Shammam et al. Iraq. Their mean [± standard deviation (SD)] age was 40.35 ± 11.01 years. All of them were heavy drinkers of ethanol (>217 g/day) during a prolonged period (>13 years). In the control group (70 subjects), the mean (± SD) age was 33.0 ± 7.83 years, and they were nonalco- holic. Some clinical and biochemical parameters of patients and controls are shown in Table 1. Cytokines and Biochemical Parameters Blood samples were taken at 8.00 am in fasting conditions, and were immedi- ately frozen at −80°C. The following parameters were determined: TNF-α by enzyme linked immune sorbent assay (ELISA; range of detection 32–2000 pg/ml); and IL-15 by ELISA (sensitivity <3 pg/ml; range of detection 15.6–1000 pg/ml). In addition, patients underwent liver enzyme assays, including aspartate and alanine aminotransferases (AST and ALT), alkaline phosphatase ALP, gamma-glutamyl transferase (GGT) and creatine kinase (CK). Biostatistics Spearman’s correlation coefficient was used to compare quantitative parame- ters. When variables did show a normal distribution, Student’s t-test and Pear- son’s test were employed. Results As shown in Table 1, all the cytokines were determined, including IL-15 and TNF-α levels; liver enzyme activity were significantly higher in alcoholic patients than in controls. All the patients were heavy drinkers (217.04 ± 149.93 g/day) with many years of consuption (13.97 ± 8.96 years). Significant relationship was found between IL-15, quantity of eth- anol consumption, TNF-α, CK, AST/ ALT and between TNF-α and daily eth- anol consumption (quantity) and GGT (see Figs. 1–6), respectively. Significant correlations were found between some studied parameters, as presented in Figs 1–6. Discussion The high value of IL-15 levels may be due to muscle contraction and violence associated with convulsion and cramps happened to the alcoholic patients, resulted from many alteration in elec- trolyte, calcium and sodium ions, caused Table 1. Mean ± SD of serum AST, ALT, ALP, CK, GGT activity levels, AST/ALT activity ratio, IL-15 and TNF-α level for studied groups Parameter Patient Group N = 70 Mean ± SD Control Group N = 70 Mean ± SD P value AST (U/L) 20.01 ± 9.80 8.68 ± 3.79 0.08 ALT (U/L) 13.84 ± 6.27 7.51 ± 2.57 0.030 ALP (U/L) 62.98 ± 22.90 50.87 ± 17.81 0.001 AST/ALT* 1.68 ± 0.89 0.83 ± 0.24 0.001 GGT (U/L) 97.45 ± 15.58 28.68 ± 13.55 0.061 CK (U/L) 133.68 ± 44.43 50.40 ± 24.49 0.001 IL-15 (pg/ml) 59.01 ± 11.09 34.92 ± 9.03 0.053 TNF-α (pg/ml) 83.42 ± 10.12 41.68 ± 15.74 0.072 ALT: alanine aminotransferase; AST: aspartate aminotransferase; ALP: alkaline phosphate; CK: creatine kinase; GGT: gamma glutamate transferase; IL-15: interleukin-15; TNF-α: tumor necrosis factor-alpha. Fig. 1 Correlation between IL-15 level and quantity of ethanol consumed per day in alcoholic patients group. Fig. 2 Correlation between IL-15 level and TNF-α in alcoholic patients group. 22 J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 20–23 Myokines in alcoholic myopathy Research Kammela Hadi Shammam et al. by changes in the membrane permea- bility, due to lipid peroxidation which occurred from reaction of acetaldehyde and phospholipids of muscle cells wall.16 IL-15 mRNA levels were up-regulated in human skeletal muscle following strength training, suggesting that IL-15 may accumulate within the muscle as a consequence of regular training or physical activity as happened to alcoholics.17 Oxidative stress produced from alcohol consumption is associated with numerous deleterious consequences for the cell (e.g. lipid peroxidation or even cell apoptosis or necrosis) and leads to different enzyme leakage to outside the cells, causing an increase in the level of these enzymes in the blood. So there were appositive correlation between IL-15, quantity of ethanol consumed, CK and AST/ALT ratio as shown in Figs. 1, 3 and 4, respectively. The study found positive correla- tion between IL-15 and TNF-α (Fig. 2) due to liver impairment that happened in chronic alcohol due to damaging its cells (Kupffer cells) which converted to macrophages when activated by viruses or any foreign substances like alcohol, and endotoxins which were secreted from intestine bacteria due to alcohol effects travel to blood stream, then to Kupffer cells which activate them and secrete numerous cytokines including TNF-α.18 TNF-α was higher in alcoholics than in nonalcoholics (Table 1), this result agree with what was mentioned about the effects of alcohol consump- tion on different body organs. One of the factors that can enhance the production of TNF-α is alcohol, endotoxin is released from the bacteria (gram-negative) normally living in the intestine when those bacteria die from alcohol and acetaldehyde (toxic sub- stance). If endotoxin enters the blood stream and reaches the liver, it interacts with Kupffer cells, activating the cells to produce cytokines. In a healthy person, endotoxin interacts primarily with Kupffer cells, and this interaction is considered cru- cial to secretion of TNF-α, which then interacts with receptors on both Kupffer cells and hepatocytes. Endotoxins seem to be important in the development of early alcoholic liver disease. Thus TNF-α was corre- lated positively with quantity and GGT, Figs. 5 and 6, respectively. Alcohol Fig. 3 Correlation between IL-15 and CK activity level in alcoholic patient group. Fig. 4 Correlation between IL-15 level and AST/ALT ratio in alcoholic patient group. Fig. 5 Correlation between TNF-α and quantity of ethanol consumed per day in alcoholic patient group. 23J Contemp Med Sci | Vol. 1, No. 3, Summer 2015: 20–23 Research Myokines in alcoholic myopathyKammela Hadi Shammam et al. References 1. Furmanczyk PS, Quinn LS. Interleukin 15 increases myosin accretion in human skeletal myogenic cultures. Cell Biol Int. 2003;27:845–51. doi: http:// dx.doi.org/10.1016/s1065-6995(03)00172-0 PMID: 14499665 2. Carbó N, López Soriano J, Costelli P, Alvarez B, Busquets S, Baccino FM, et al. Interleukin 15 mediates reciprocal regulation of adipose and muscle mass: a potential role in body weight control. Br J Cancer 2000;83:526–31. 3. Grabstein KH, Eisenman J, Shanebeck K, Rauch C, Srinivasan S, Fung V, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 1994 May;264(5161):965–8. doi: http:// dx.doi.org/10.1126/science.8178155 PMID: 8178155 4. Van Heel DA. Interleukin 15: its role in intestinal inflammation. Gut. 2006 Apr;55(4):444–5. doi: http://dx.doi.org/10.1136/gut.2005.079335 PMID: 16531523 5. Quinn LS. Interleukin 15: a muscle-derived cytokine regulating fat-to-lean body composition. J Anim Sci. 2008 Apr;86(14 Suppl):E75–E83. doi: http:// dx.doi.org/10.2527/jas.2007-0458 PMID: 17709786 6. Fehniger TA, Caliguri MA. Interleukin 15: biology and relevance to human disease. Blood 2001 Jan 1;97(1):14–32. doi: http://dx.doi.org/10.1182/blood. v97.1.14 PMID: 11133738 7. Waldmann TA. Targeting the interleukin-15/Interleukin 15 receptor system in inflammatory autoimmune diseases. Arthritis Res Ther. 2004;6(4):174–7. PMID: 15225362 8. Peters TJ, Martin F, Ward K. Chronic alcoholic myopathy—common and reversible. Alcohol 1985 May;2(3):485–9. doi: http://dx.doi. org/10.1016/0741-8329(85)90120-x 9. Urbano-Márquez A, Estruch R, Navarro-Lopez F, Grau JM, Mont L, Rubin E. The effects of alcoholism on skeletal and cardiac muscle. N Engl J Med. 1989 Feb 16;320(7):409–15. doi: http://dx.doi.org/10.1056/ nejm198902163200701 PMID: 2913506 also increased TNF-α by another way, it oxidises to acetaldehyde, which reacts with proteins and DNA and to form adducts. These adducts induce certain immune cells (recognize these adducts as foreign bodies) to produce many deafens cells like interleukins, interfer- on’s and pro-inflammatory molecules like TNF-α. Conclusion IL-15 and TNF-α levels were higher in Iraqi alcoholics than in nonalcoholic. Both of them exert dangerous effects on the body and can consider as a risk factor for short-term motility.  Fig. 6 Correlation between TNF-α level and GGT activity levels in alcoholic patient group. 10. Preedy VR, Paice A, Mantle D, Dhillon AS, Palmer TN, Peters TJ. Alcoholic myopathy: biochemical mechanisms. Drug Alcohol Depend. 2001 Aug;63(3):199–205. doi: http://dx.doi.org/10.1016/s0376-8716(00)00219-2 11. Preedy VR, Marway JS, Macpherson AJS, Peters TJ. Ethanol-induced smooth and skeletal muscle myopathy: use of animal studies. Drug Alcohol Depend. 1990 Aug;26:1–8. doi: http://dx.doi.org/10.1016/0376-8716(90)90076-q PMID: 1698602 12. Conde-Martel A, González-Reimers E, González-Hernández T, Santolaria F, Martinez-Riera A, Romero-Perez JC, et al. Relative and combined roles of ethanol and protein malnutrition on skeletal muscle. Alcohol Alcohol. 1992 Mar;27(2):159–63. PMID: 1524607 13. Gustafson R. Alcohol and aggression. J Offender Rehabil. 1994;21(3/4):41–80. 14. Nanj AA, Jokelainen K, Fotouhinia M, Rahemtulla A, Thomas P, Tipoe GL, et al. Increased severity of alcoholic liver injury in female rats: role of oxidative stress, endotoxin, and chemokines. Am J Physiol Gastrointest Liver Physiol. 2001 Dec;281(6):G1348–G1356. PMID: 11705739 15. Wu D, Cederbaum AI. Oxidative stress and alcoholic liver disease. Semin Liver Dis. 2009 Apr 22;29(2):141–54. doi: http://dx.doi. org/10.1055/s-0029-1214370 16. Wickramasinghe SN, Marjot DH, Rosalki SB, Fink RS. Correlations between serum proteins modified by acetaldehyde and biochemical variables in heavy drinkers. J Clin Pathol. 1989 Mar;42(3):295–9. doi: http://dx.doi. org/10.1136/jcp.42.3.295 PMID: 2703546 17. Nielsen AR, Hojman P, Erikstrup C, Fischer CP, Plomgaard P, Mounier R, et al. Association between IL-15 and obesity: Il-15 as a potential regulator of fat mass. J Clin Endocrinol Metab. 2008 Nov;93(11):4486–93. doi: http://dx.doi. org/10.1210/jc.2007-2561 PMID: 18697873 18. Neuman MG. Apoptosis in diseases of the liver. Critic Rev Clin Lab Sci. 2001;38:109–66.