Immune Netw.  2016 Oct;16(5):296-304. 10.4110/in.2016.16.5.296.

Cytokine-like Activity of Liver Type Fatty Acid Binding Protein (L-FABP) Inducing Inflammatory Cytokine Interleukin-6

• Kim H
• Gil G
• Lee S
• Kwak A
• Jo S
• Kim E
• Nguyen TT
• Kim S
• Jhun H
• Kim S
• Kim M
• Lee Y
• Kim S

• KMID: 2355023
• DOI: http://doi.org/10.4110/in.2016.16.5.296

Abstract

It has been reported that fatty acid binding proteins (FABPs) do not act only as intracellular mediators of lipid responses but also have extracellular functions. This study aimed to investigate whether extracellular liver type (L)-FABP has a biological activity and to determined serum L-FABP levels in patients with end-stage renal disease (ESRD). We isolated L-FABP complementary deoxyribonucleic acid (cDNA) from the Huh7 human hepatocarcinoma cell line and expressed the recombinant L-FABP protein in Escherichia coli. A549 lung carcinoma and THP-1 monocytic cells were stimulated with the human recombinant L-FABP. Human whole blood cells were also treated with the human recombinant L-FABP or interleukin (IL)-1α. IL-6 levels were measured in cell culture supernatants using IL-6 enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in A549, THP-1 cells, and whole blood cells. The blood samples of healthy volunteers and patients with ESRD were taken after an overnight fast. The serum levels of L-FABP in healthy volunteers and ESRD patients were quantified with L-FABP ELISA. The values of L-FABP in patients with ESRD were significantly lower than those in the control group. Our results demonstrated the biological activity of L-FABP in human cells suggesting L-FABP can be a mediator of inflammation.

Keyword

Liver type fatty acid binding protein; Whole blood cells; End stage renal disease

MeSH Terms

Blood Cells
Carrier Proteins*
Cell Culture Techniques
Cell Line
DNA
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Fatty Acid-Binding Proteins
Healthy Volunteers
Humans
Inflammation
Interleukin-6*
Interleukins
Kidney Failure, Chronic
Liver*
Lung
Carrier Proteins
DNA
Fatty Acid-Binding Proteins
Interleukin-6
Interleukins

Figure

• 

  Figure 1 Isolation of human recombinant liver-fatty acid binding protein by using high performance liquid chromatography (HPLC). Human recombinant L-FABP was expressed in E. coli and first purified by a Talon metal affinity chromatography (not shown). Then the protein was further purified by HPLC. The peaks corresponding to human recombinant L-FABP were observed right after 51 and 63 min. Units are in milli Absorbance Units (mAU) at 280 nm. The data presents one of five independent experiments.

• 

  Figure 2 Silver staining of human recombinant liver-fatty acid binding protein. The purified human recombinant liver-fatty acid binding protein was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and visualized by silver staining. (A) The eluants from fraction 45 to 55 were visualized by silver stain. A serious of protein bands with molecular weight of 16- (monomer form), 32- (dimer form), and 48 kDa (trimer form) were identified in nearly all fractions examined. (B) These protein bands were detected mainly in fractions 63 and 64 especially. The data represent one of three independent experiments. Molecular weight (Mw) is indicated on the left. BSA; bovine serum albumin, kDa; kilodalton.

• 

  Figure 3 Quantification of human recombinant liver-fatty acid binding protein concentration. The purified human recombinant L-FABP was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, visualized by silver staining, and compared with various known amount of bovine serum albumin (BSA). Twelve µl of the eluants from fraction 47 to 54 was loaded in the second lane. One and 2 µl of the eluants from fraction 63 to 64 was loaded in the third and fourth lanes, respectively. Purified human recombinant L-FABP proteins from fraction 47 to 54 typically migrate as monomer, dimer, and trimer otherwise those from fraction 63 and 64 mainly migrate as monomer and dimer. A standard curve was created with density measurement of BSA standards and used to calculate estimates of human recombinant L-FABP protein concentration. The data represent one of three independent experiments. Molecular weight (Mw) is indicated on the left. BSA; bovine serum albumin, kDa; kilodalton.

• 

  Figure 4 Biological activities of human recombinant liver-fatty acid binding protein. Human recombinant L-FABP was examined with A549 lung carcinoma and THP-1 monocytic and human whole blood cells. A549 cells (A) and THP-1 cells (B) were treated with various concentrations of human recombinant L-FABP as indicated under the horizontal axis for 24 h, and interleukin IL-6 levels were assessed by enzyme-linked immunosorbent assay (ELISA). Human recombinant L-FABP induced IL-6 in a dose-dependent manner in these cells. (C) Whole bloods from healthy volunteers were collected with heparin- or ethylenediamine tetraacetic acid (EDTA)-coated tube and IL-6 supernatant levels were measured with ELISA after 24 h of incubation with human recombinant L-FABP or IL-1α at concentrations indicated under the horizontal axis. Both the human recombinant L-FABP and IL-1α induced IL-6 in human whole blood cells compared with non-treated control cells. These effects were stronger in the blood cells collected with heparin-coated tube than in those collected with EDTA-coated tube. The data represent one of three independent experiments. cont, control; hr, human recombinant; L-FABP, liver-fatty acid binding protein.

• 

  Figure 5 Liver-fatty acid binding protein concentration in human serum. The serum L-FABP levels of the healthy control group (n=63), renal diseases of the ESRD dialysis (n=75), and diabetes patients (n=87). Blood samples of healthy volunteers were taken after an overnight fast and those of ESRD patients were drawn before the start of a routine hemodialysis treatment. Levels were measured using our ELISA assay. The values of L-FABP in patients with ESRD were significantly lower than those in the control group (2.7 ng/ml vs. 0.23 ng/ml, p<0.001). ESRD, end-stage renal disease; L-FABP, liver-fatty acid binding protein.

Reference

1. Furuhashi M, Hotamisligil GS. Fatty acid-binding proteins: role in metabolic diseases and potential as drug targets. Nat Rev Drug Discov. 2008; 7:489–503.

2. Atshaves BP, Martin GG, Hostetler HA, McIntosh AL, Kier AB, Schroeder F. Liver fatty acid-binding protein and obesity. J Nutr Biochem. 2010; 21:1015–1032.

3. Kanda T, Fujii H, Tani T, Murakami H, Suda T, Sakai Y, Ono T, Hatakeyama K. Intestinal fatty acid-binding protein is a useful diagnostic marker for mesenteric infarction in humans. Gastroenterology. 1996; 110:339–343.

4. Piumngam P, Schachtrup C, Owada Y, Kondo H, Promptmas C, Spener F. Expression of liver-type fatty acid-binding protein in murine lung and its release into serum upon challenge of lung with lipopolysaccharide. Eur J Lipid Sci Technol. 2005; 107:145–152.

5. Pelsers MM, Morovat A, Alexander GJ, Hermens WT, Trull AK, Glatz JF. Liver fatty acid-binding protein as a sensitive serum marker of acute hepatocellular damage in liver transplant recipients. Clin Chem. 2002; 48:2055–2057.

6. Monbaliu D, de VB, Crabbe T, van HE, Verwaest C, Roskams T, Fevery J, Pirenne J, Buurman WA. Liver fatty acid-binding protein: an early and sensitive plasma marker of hepatocellular damage and a reliable predictor of graft viability after liver transplantation from non-heart-beating donors. Transplant Proc. 2005; 37:413–416.

7. Akbal E, Koklu S, Kocak E, Cakal B, Gunes F, Basar O, Tuna Y, Senes M. Liver fatty acid-binding protein is a diagnostic marker to detect liver injury due to chronic hepatitis C infection. Arch Med Res. 2013; 44:34–38.

8. Morrissey PE, Gollin G, Marks WH. Small bowel allograft rejection detected by serum intestinal fatty acid-binding protein is reversible. Transplantation. 1996; 61:1451–1455.

9. Pelsers MM, Namiot Z, Kisielewski W, Namiot A, Januszkiewicz M, Hermens WT, Glatz JF. Intestinal-type and liver-type fatty acid-binding protein in the intestine. Tissue distribution and clinical utility. Clin Biochem. 2003; 36:529–535.

10. Okamoto F, Sohmiya K, Ohkaru Y, Kawamura K, Asayama K, Kimura H, Nishimura S, Ishii H, Sunahara N, Tanaka T. Human heart-type cytoplasmic fatty acid-binding protein (H-FABP) for the diagnosis of acute myocardial infarction. Clinical evaluation of H-FABP in comparison with myoglobin and creatine kinase isoenzyme MB. Clin Chem Lab Med. 2000; 38:231–238.

11. Setsuta K, Seino Y, Ogawa T, Arao M, Miyatake Y, Takano T. Use of cytosolic and myofibril markers in the detection of ongoing myocardial damage in patients with chronic heart failure. Am J Med. 2002; 113:717–722.

12. Pelsers MM, Hanhoff T, d Van V, Arts B, Peters M, Ponds R, Honig A, Rudzinski W, Spener F, de K Jr, Twijnstra A, Hermens WT, Menheere PP, Glatz JF. Brain- and heart-type fatty acid-binding proteins in the brain: tissue distribution and clinical utility. Clin Chem. 2004; 50:1568–1575.

13. Foucaud L, Grillasca J, Niot I, Domingo N, Lafont H, Planells R, Besnard P. Output of liver fatty acidbinding protein (L-FABP) in bile. Biochim Biophys Acta. 1999; 1436:593–599.

14. Specht B, Bartetzko N, Hohoff C, Kuhl H, Franke R, Borchers T, Spener F. Mammary derived growth inhibitor is not a distinct protein but a mix of heart-type and adipocyte-type fatty acid-binding protein. J Biol Chem. 1996; 271:19943–19949.

15. Bronsky J, Karpisek M, Bronska E, Pechova M, Jancikova B, Kotolova H, Stejskal D, Prusa R, Nevoral J. Adiponectin, adipocyte fatty acid binding protein, and epidermal fatty acid binding protein: proteins newly identified in human breast milk. Clin Chem. 2006; 52:1763–1770.

16. Xu A, Wang Y, Xu JY, Stejskal D, Tam S, Zhang J, Wat NM, Wong WK, Lam KS. Adipocyte fatty acid-binding protein is a plasma biomarker closely associated with obesity and metabolic syndrome. Clin Chem. 2006; 52:405–413.

17. Mita T, Furuhashi M, Hiramitsu S, Ishii J, Hoshina K, Ishimura S, Fuseya T, Watanabe Y, Tanaka M, Ohno K, Akasaka H, Ohnishi H, Yoshida H, Saitoh S, Shimamoto K, Miura T. FABP4 is secreted from adipocytes by adenyl cyclase-PKA- and guanylyl cyclase-PKG-dependent lipolytic mechanisms. Obesity (Silver Spring). 2015; 23:359–367.

18. Lamounier-Zepter V, Look C, Alvarez J, Christ T, Ravens U, Schunck WH, Ehrhart-Bornstein M, Bornstein SR, Morano I. Adipocyte fatty acid-binding protein suppresses cardiomyocyte contraction: a new link between obesity and heart disease. Circ Res. 2009; 105:326–334.

19. Cao H, Sekiya M, Ertunc ME, Burak MF, Mayers JR, White A, Inouye K, Rickey LM, Ercal BC, Furuhashi M, Tuncman G, Hotamisligil GS. Adipocyte lipid chaperone AP2 is a secreted adipokine regulating hepatic glucose production. Cell Metab. 2013; 17:768–778.

20. Kralisch S, Kloting N, Ebert T, Kern M, Hoffmann A, Krause K, Jessnitzer B, Lossner U, Sommerer I, Stumvoll M, Fasshauer M. Circulating adipocyte fatty acid-binding protein induces insulin resistance in mice in vivo. Obesity (Silver Spring). 2015; 23:1007–1013.

21. Kamijo A, Sugaya T, Hikawa A, Yamanouchi M, Hirata Y, Ishimitsu T, Numabe A, Takagi M, Hayakawa H, Tabei F, Sugimoto T, Mise N, Omata M, Kimura K. Urinary liver-type fatty acid binding protein as a useful biomarker in chronic kidney disease. Mol Cell Biochem. 2006; 284:175–182.

22. Yamamoto T, Noiri E, Ono Y, Doi K, Negishi K, Kamijo A, Kimura K, Fujita T, Kinukawa T, Taniguchi H, Nakamura K, Goto M, Shinozaki N, Ohshima S, Sugaya T. Renal L-type fatty acid--binding protein in acute ischemic injury. J Am Soc Nephrol. 2007; 18:2894–2902.

23. Matsui K, Kamijo-Ikemori A, Imai N, Sugaya T, Yasuda T, Tatsunami S, Toyama T, Shimizu M, Furuichi K, Wada T, Shibagaki Y, Kimura K. Clinical significance of urinary liver-type fatty acid-binding protein as a predictor of ESRD and CVD in patients with CKD. Clin Exp Nephrol. 2016; 20:195–203.

24. Lachmann RA, Werchan S, Schachtrup C, Haitsma JJ, Spener F, Lachmann B. Liver-type fatty acid binding protein in serum and broncho-alveolar lavage in a model of acute respiratory failure because of surfactant depletion--a possible marker for lung damage? Clin Physiol Funct Imaging. 2006; 26:371–375.

25. Kawai A, Kusaka M, Kitagawa F, Ishii J, Fukami N, Maruyama T, Sasaki H, Shiroki R, Kurahashi H, Hoshinaga K. Serum liver-type fatty acid-binding protein predicts recovery of graft function after kidney transplantation from donors after cardiac death. Clin Transplant. 2014; 28:749–754.

26. Akbal E, Kocak E, Akyurek O, Koklu S, Batgi H, Senes M. Liver fatty acid-binding protein as a diagnostic marker for non-alcoholic fatty liver disease. Wien Klin Wochenschr. 2016; 128:48–52.

27. Shi J, Zhang Y, Gu W, Cui B, Xu M, Yan Q, Wang W, Ning G, Hong J. Serum liver fatty acid binding protein levels correlate positively with obesity and insulin resistance in Chinese young adults. PLoS One. 2012; 7:e48777.

28. Ishimura S, Furuhashi M, Watanabe Y, Hoshina K, Fuseya T, Mita T, Okazaki Y, Koyama M, Tanaka M, Akasaka H, Ohnishi H, Yoshida H, Saitoh S, Miura T. Circulating levels of fatty acid-binding protein family and metabolic phenotype in the general population. PLoS One. 2013; 8:e81318.

29. Kwan BC, Kronenberg F, Beddhu S, Cheung AK. Lipoprotein metabolism and lipid management in chronic kidney disease. J Am Soc Nephrol. 2007; 18:1246–1261.

30. Jin K, Norris K, Vaziri ND. Dysregulation of hepatic fatty acid metabolism in chronic kidney disease. Nephrol Dial Transplant. 2013; 28:313–320.

31. Fuseya T, Furuhashi M, Yuda S, Muranaka A, Kawamukai M, Mita T, Ishimura S, Watanabe Y, Hoshina K, Tanaka M, Ohno K, Akasaka H, Ohnishi H, Yoshida H, Saitoh S, Shimamoto K, Miura T. Elevation of circulating fatty acid-binding protein 4 is independently associated with left ventricular diastolic dysfunction in a general population. Cardiovasc Diabetol. 2014; 13:126.

32. Yeung DC, Xu A, Tso AW, Chow WS, Wat NM, Fong CH, Tam S, Sham PC, Lam KS. Circulating levels of adipocyte and epidermal fatty acid-binding proteins in relation to nephropathy staging and macrovascular complications in type 2 diabetic patients. Diabetes Care. 2009; 32:132–134.

33. Girona J, Rosales R, Plana N, Saavedra P, Masana L. FABP4 induces vascular smooth muscle cell proliferation and migration through a MAPK-dependent pathway. PLoS One. 2013; 8:e81914.

34. Yeung DC, Wang Y, Xu A, Cheung SC, Wat NM, Fong DY, Fong CH, Chau MT, Sham PC, Lam KS. Epidermal fatty-acid-binding protein: a new circulating biomarker associated with cardio-metabolic risk factors and carotid atherosclerosis. Eur Heart J. 2008; 29:2156–2163.

35. Bagheri R, Qasim AN, Mehta NN, Terembula K, Kapoor S, Braunstein S, Schutta M, Iqbal N, Lehrke M, Reilly MP. Relation of plasma fatty acid binding proteins 4 and 5 with the metabolic syndrome, inflammation and coronary calcium in patients with type-2 diabetes mellitus. Am J Cardiol. 2010; 106:1118–1123.

36. Bowen RA, Remaley AT. Interferences from blood collection tube components on clinical chemistry assays. Biochem Med (Zagreb). 2014; 24:31–44.

37. Brunialti MK, Kallas EG, Freudenberg M, Galanos C, Salomao R. Influence of EDTA and heparin on lipopolysaccharide binding and cell activation, evaluated at single-cell level in whole blood. Cytometry. 2002; 50:14–18.

38. Duvigneau JC, Sipos W, Hartl RT, Bayer M, Moldzio R, Stevenson L, Adair B, Gemeiner M. Heparin and EDTA as anticoagulant differentially affect cytokine mRNA level of cultured porcine blood cells. J Immunol Methods. 2007; 324:38–47.

39. Freitas M, Porto G, Lima JL, Fernandes E. Isolation and activation of human neutrophils in vitro The importance of the anticoagulant used during blood collection. Clin Biochem. 2008; 41:570–575.

40. Iwamoto M, Miyoshi T, Doi M, Takeda K, Kajiya M, Nosaka K, Nakayama R, Hirohata S, Usui S, Kusachi S, Sakane K, Nakamura K, Ito H. Elevated serum adipocyte fatty acid-binding protein concentrations are independently associated with renal dysfunction in patients with stable angina pectoris. Cardiovasc Diabetol. 2012; 11:26.

41. Furuhashi M, Ura N, Hasegawa K, Yoshida H, Tsuchihashi K, Nakata T, Shimamoto K. Serum ratio of heart-type fatty acid-binding protein to myoglobin A novel marker of cardiac damage and volume overload in hemodialysis patients. Nephron Clin Pract. 2003; 93:C69–C74.

42. Furuhashi M, Ura N, Hasegawa K, Tsuchihashi K, Nakata T, Shimamoto K. Utility of serum ratio of heart-type fatty acid-binding protein to myoglobin for cardiac damage regardless of renal dysfunction. Circ J. 2004; 68:656–659.

43. Sommer G, Ziegelmeier M, Bachmann A, Kralisch S, Lossner U, Kratzsch J, Bluher M, Stumvoll M, Fasshauer M. Serum levels of adipocyte fatty acid-binding protein (AFABP) are increased in chronic haemodialysis (CD). Clin Endocrinol (Oxf). 2008; 69:901–905.

44. Furuhashi M, Ishimura S, Ota H, Hayashi M, Nishitani T, Tanaka M, Yoshida H, Shimamoto K, Hotamisligil GS, Miura T. Serum fatty acid-binding protein 4 is a predictor of cardiovascular events in end-stage renal disease. PLoS One. 2011; 6:e27356.

45. Mori Y, Hirano T, Nagashima M, Shiraishi Y, Fukui T, Adachi M. Decreased peroxisome proliferator-activated receptor alpha gene expression is associated with dyslipidemia in a rat model of chronic renal failure. Metabolism. 2007; 56:1714–1718.

46. Staels B, Dallongeville J, Auwerx J, Schoonjans K, Leitersdorf E, Fruchart JC. Mechanism of action of fibrates on lipid and lipoprotein metabolism. Circulation. 1998; 98:2088–2093.