Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced
TNF-? production in cultured macrophage cell lines
Zheng Shen,1Joanne M. Ajmo,1Christopher Q. Rogers,1Xiaomei Liang,1Lisa Le,1Michel M. Murr,2
Yanhua Peng,2and Min You1
1Departments of Molecular Pharmacology and Physiology and2Department of Surgery, James A. Haley Veterans Affairs
Medical Center, University of South Florida Health Sciences Center, Tampa, Florida
Submitted 13 January 2009; accepted in final form 16 March 2009
Shen Z, Ajmo JM, Rogers CQ, Liang X, Le L, Murr MM, Peng
Y, You M. Role of SIRT1 in regulation of LPS- or two ethanol
metabolites-induced TNF-? production in cultured macrophage cell
lines. Am J Physiol Gastrointest Liver Physiol 296: G1047–G1053, 2009.
First published March 19, 2009; doi:10.1152/ajpgi.00016.2009.—Dys-
regulation of proinflammatory cytokines such as tumor necrosis fac-
tor-? (TNF-?) has been implicated in the pathogenesis of alcoholic
liver injury. Sirtuin 1 (SIRT1) is an NAD?-dependent class III protein
deacetylase that is known to be involved in regulating production of
proinflammatory cytokines including TNF-?. In the present study, we
examined the role of SIRT1 signaling in TNF-? generation stimulated
by either lipopolysaccharide (LPS), acetaldehyde (AcH), or acetate
(two major metabolites of ethanol) in two cultured macrophage cell
lines. In both rat Kupffer cell line 1 (RKC1) and murine RAW 264.7
macrophages, treatment with either LPS, AcH, or acetate caused
significant decreases in SIRT1 transcription, translation, and activa-
tion, which essentially demonstrated an inverse relationship with
TNF-? levels. LPS, AcH, and acetate each provoked the release of
TNF-? from RKC1 cells, whereas coincubation with resveratrol (a
potent SIRT1 agonist) inhibited this effect. Conversely, addition of
sirtinol (a known SIRT1 inhibitor) or knocking down SIRT1 by the
small silencing SIRT1 plasmid (SIRT1shRNA) augmented TNF-?
release, suggesting that impairment of SIRT1 may contribute to
TNF-? secretion. Further mechanistic studies revealed that inhibition
of SIRT1 by LPS, AcH, or acetate was associated with a marked
increase in the acetylation of the RelA/p65 subunit of nuclear tran-
scription factor (NF-?B) and promotion of NF-?B transcriptional
activity. Taken together, our findings suggest that SIRT1-NF-?B
signaling is involved in regulating LPS- and metabolites-of-ethanol-
mediated TNF-? production in rat Kupffer cells and in murine
macrophages. Our study provides new insights into understanding the
molecular mechanisms underlying the development of alcoholic ste-
inflammation; adiponectin; signal transduction
ALCOHOLIC LIVER DISEASE (ALD) is clinically associated with the
development of steatosis, inflammation, and eventually fibrosis
and cirrhosis. The underlying cellular and molecular mecha-
nisms by which alcohol causes liver injury are complex and
Hepatic ethanol metabolism has long been implicated in the
development of ALD (22, 23). Metabolic byproducts of etha-
nol, such as acetaldehyde (AcH) and acetate, have been pre-
sumed agents of such damage. Additionally, lipopolysaccharide
(LPS), one of the components of the outer wall of gram-negative
bacteria, has been thought to participate in the etiology (10, 31).
Chronic ethanol exposure alters gut microflora and permeabil-
ity, resulting in elevated release of LPS, which enters the liver
through the portal vein and activates resident macrophages
(Kupffer cells), which release proinflammatory cytokines such
as tumor necrosis factor-? (TNF-?), which, in turn, promote
liver damage. Meanwhile, a corollary process occurs in
which chronic ethanol feeding sensitizes Kupffer cells to
LPS signaling, enhancing LPS-stimulated TNF-? produc-
tion in rodents (31).
Sirtuin 1 (SIRT1) is an NAD?-dependent class III protein
deacetylase that exerts anti-inflammatory effects by deacetyla-
tion of modified lysine residues on transcriptional regulators,
particularly nuclear transcription factor-?B (NF-?B), a master
transcription factor involved in regulation of proinflammatory
cytokines (29). SIRT1 physically interacts with and deacety-
lates the RelA/p65 subunit of NF-?B at lysine 310 and subse-
quently inhibits NF-?B transcriptional activity (6, 36, 38, 39).
The anti-inflammatory action of SIRT1 is further supported by a
recent study demonstrating that activation of SIRT1 protects mice
from high-fat diet-induced hepatic inflammation through decreas-
ing the NF-?B-mediated induction of inflammatory cytokines
including TNF-? (27).
Adiponectin is an adipocyte-derived protein that has po-
tent anti-inflammatory properties. ALD is associated with
reduced circulating adiponectin levels and impaired hepatic
adiponectin signaling (28). The anti-inflammatory proper-
ties of adiponectin have been implicated in the protective
action of adiponectin against development of alcoholic liver
injury (25). However, the underlying molecular mechanisms
by which adiponectin exerts its protective effects are still
not fully understood.
In the present study, we used immortalized rat Kupffer cell
line 1 (RKC1) and murine RAW 264.7 macrophages to test the
hypothesis that SIRT1 signaling is involved in regulating the
TNF-? production that is stimulated by three putative inducers
of ALD [LPS and two major metabolites of ethanol (AcH and
acetate)]. Furthermore, we investigated the corollary hypothe-
sis, that SIRT1 plays a role in adiponectin’s suppression of
these compounds’ abilities to induce TNF-?.
MATERIALS AND METHODS
Materials. Most chemicals and supplies were purchased from
Sigma Chemical, Schleicher and Schuell, GIBCO-BRL, and Dupont
NEN Research Products. Acetate, AcH, LPS (tissue culture-tested,
L-2654), and resveratrol were obtained from Sigma. Sirtinol and
splitomicin were purchased from Calbiochem (Gibbstown, NJ). Re-
combinant human globular adiponectin (gAcrp) was purchased from
PeproTech (Rockyhill, NJ).
Plasmids. NF-?B-responsive reporter-3x?B luciferase plasmid, wild-
type SIRT1 (SIRT1wt) or deacetylase-defective SIRT1(H363Y) mutant
Address for reprint requests and other correspondence: M. You, Dept. of
Molecular Pharmacology & Physiology, School of Basic Biomedical Sciences,
College of Medicine, Box 8, Univ. of South Florida, 12901 Bruce B. Downs
Blvd, Tampa, FL 33612 (e-mail: email@example.com).
Am J Physiol Gastrointest Liver Physiol 296: G1047–G1053, 2009.
First published March 19, 2009; doi:10.1152/ajpgi.00016.2009.
0193-1857/09 $8.00 Copyright © 2009 the American Physiological Societyhttp://www.ajpgi.orgG1047
1. Abdelmohsen K, Pullmann R Jr, Lal A, Kim HH, Galban S, Yang X,
Blethrow JD, Walker M, Shubert J, Gillespie DA, Furneaux H,
Gorospe M. Phosphorylation of HuR by Chk2 regulates SIRT1 expres-
sion. Mol Cell 25: 543–557, 2007.
2. Ajmo JM, Liang X, Rogers CQ, Pennock B, You M. Resveratrol
alleviates alcoholic fatty liver in mice. Am J Physiol Gastrointest Liver
Physiol 295: G833–G842, 2008.
3. Cao Q, Mak KM, Lieber CS. Dilinoleoylphosphatidylcholine decreases
acetaldehyde-induced TNF-alpha generation in Kupffer cells of ethanol-
fed rats. Biochem Biophys Res Commun 299: 459–464, 2002.
4. Cao Q, Mak KM, Lieber CS. Dilinoleoylphosphatidylcholine decreases
LPS-induced TNF-alpha generation in Kupffer cells of ethanol-fed rats:
respective roles of MAPKs and NF-kappaB. Biochem Biophys Res Com-
mun 294: 849–853, 2002.
5. Civitarese AE, Carling S, Heilbronn LK, Hulver MH, Ukropcova B,
Deutsch WA, Smith SR, Ravussin E. Calorie restriction increases
muscle mitochondrial biogenesis in healthy humans. PLoS Med 4: e76,
6. Csiszar A, Labinskyy N, Podlutsky A, Kaminski PM, Wolin MS,
Zhang C, Mukhopadhyay P, Pacher P, Hu F, de Cabo R, Ballabh P,
Ungvari Z. Vasoprotective effects of resveratrol and SIRT1: attenuation
of cigarette smoke-induced oxidative stress and proinflammatory pheno-
typic alterations. Am J Physiol Heart Circ Physiol 294: H2721–H2735,
7. De Minicis S, Brenner DA. Oxidative stress in alcoholic liver disease:
role of NADPH oxidase complex. J Gastroenterol Hepatol 23: S98–S103,
8. Feige JN, Auwerx J. Transcriptional targets of sirtuins in the coordination
of mammalian physiology. Curr Opin Cell Biol 20: 303–309, 2008.
9. Feng YH, Zou JP, Li XY. Effects of resveratrol and ethanol on produc-
tion of pro-inflammatory factors from endotoxin activated murine macro-
phages. Acta Pharmacol Sin 23: 1002–1006, 2002.
10. Fukui H. Relation of endotoxin, endotoxin binding proteins and macro-
phages to severe alcoholic liver injury and multiple organ failure. Alcohol
Clin Exp Res 29: 172S–179S, 2005.
11. Gao Z, Ye J. Inhibition of transcriptional activity of c-JUN by SIRT1.
Biochem Biophys Res Commun 376: 793–796, 2008.
12. Gobejishvili L, Barve S, Joshi-Barve S, McClain C. Enhanced PDE4B
expression augments LPS-inducible TNF expression in ethanol-primed
monocytes: relevance to alcoholic liver disease. Am J Physiol Gastrointest
Liver Physiol 295: G718–G724, 2008.
13. Gustot T, Lemmers A, Moreno C, Nagy N, Quertinmont E, Nicaise C,
Franchimont D, Louis H, Deviere J, Le Moine O. Differential liver
sensitization to toll-like receptor pathways in mice with alcoholic fatty
liver. Hepatology 43: 989–1000, 2006.
14. Hou X, Xu S, Maitland-Toolan KA, Sato K, Jiang B, Ido Y, Lan F,
Walsh K, Wierzbicki M, Verbeuren TJ, Cohen RA, Zang M. SIRT1
regulates hepatocyte lipid metabolism through activating AMP-activated
protein kinase. J Biol Chem 283: 20015–20026, 2008.
15. Kasdallah-Grissa A, Mornagui B, Aouani E, Hammami M, El May M,
Gharbi N, Kamoun A, El-Fazaa ˆ S. Resveratrol, a red wine polyphenol,
attenuates ethanol-induced oxidative stress in rat liver. Life Sci 80: 1033–
16. Kasdallah-Grissa A, Mornagui B, Aouani E, Hammami M, Gharbi N,
Kamoun A, El-Fazaa S. Protective effect of resveratrol on ethanol-
induced lipid peroxidation in rats. Alcohol Alcohol 41: 236–239, 2006.
17. Kishore R, Hill JR, McMullen MR, Frenkel J, Nagy LE. ERK1/2 and
Egr-1 contribute to increased TNF-? production in rat Kupffer cells after
chronic ethanol feeding. Am J Physiol Gastrointest Liver Physiol 282:
18. Kowalski J, Samojedny A, Paul M, Pietsz G, Wilczok T. Effect of
apigenin, kaempferol and resveratrol on the expression of interleukin-
1beta and tumor necrosis factor-alpha genes in J774.2 macrophages.
Pharmacol Rep 57: 390–394, 2005.
19. Lieber CS, Leo MA, Wang X, Decarli LM. Alcohol alters hepatic
FoxO1, p53, and mitochondrial SIRT5 deacetylation function. Biochem
Biophys Res Commun 373: 246–252, 2008.
20. Lieber CS, Leo MA, Wang X, Decarli LM. Effect of chronic alcohol
consumption on hepatic SIRT1 and PGC-1alpha in rats. Biochem Biophys
Res Commun 370: 44–48, 2008.
21. Ma ZH, Ma QY, Wang LC, Sha HC, Wu SL, Zhang M. Effect of
resveratrol on NF-kappaB activity in rat peritoneal macrophages. Am J
Chin Med 34: 623–630, 2006.
22. Meier P, Seitz HK. Age, alcohol metabolism and liver disease. Curr Opin
Clin Nutr Metab Care 11: 21–26, 2008.
23. Mello T, Ceni E, Surrenti C, Galli A. Alcohol induced hepatic fibrosis:
role of acetaldehyde. Mol Aspects Med 29: 17–21, 2008.
24. Park PH, Huang H, McMullen MR, Mandal P, Sun L, Nagy LE.
Suppression of lipopolysaccharide-stimulated tumor necrosis factor-alpha
production by adiponectin is mediated by transcriptional and post-tran-
scriptional mechanisms. J Biol Chem 283: 26850–26858, 2008.
25. Park PH, Thakur V, Pritchard MT, McMullen MR, Nagy LE. Regu-
lation of Kupffer cell activity during chronic ethanol exposure: role of
adiponectin. J Gastroenterol Hepatol 21: S30–S3, 2006.
26. Peng Y, Murr MM. Establishment of immortalized rat Kupffer cell lines.
Cytokine 37: 185–191, 2007.
27. Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M, Tscho ¨p MH.
Sirt1 protects against high-fat diet-induced metabolic damage. Proc Natl
Acad Sci USA 105: 9793–9798, 2008.
28. Rogers CQ, Ajmo JM, You M. Adiponectin and alcoholic fatty liver
disease. IUBMB Life 60: 790–797, 2008.
29. Salminen A, Kauppinen A, Suuronen T, Kaarniranta K. SIRT1 lon-
gevity factor suppresses NF-kappaB-driven immune responses: regulation
of aging via NF-kappaB acetylation? Bioessays 30: 939–942, 2008.
30. Shi L, Kishore R, McMullen MR, Nagy LE. Chronic ethanol increases
lipopolysaccharide-stimulated Egr-1 expression in RAW 264.7 macro-
phages: contribution to enhanced tumor necrosis factor alpha production.
J Biol Chem 277: 14777–14785, 2002.
31. Thakur V, McMullen MR, Pritchard MT, Nagy LE. Regulation of
macrophage activation in alcoholic liver disease. J Gastroenterol Hepatol
22: S53–S56, 2007.
32. Thakur V, Pritchard MT, McMullen MR, Wang Q, Nagy LE. Chronic
ethanol feeding increases activation of NADPH oxidase by lipopolysac-
charide in rat Kupffer cells: role of increased reactive oxygen in LPS-
stimulated ERK1/2 activation and TNF-alpha production. J Leukoc Biol
79: 1348–1356, 2006.
33. Uchikura K, Wada T, Hoshino S, Nagakawa Y, Aiko T, Bulkley GB,
Klein AS, Sun Z. Lipopolysaccharides induced increases in Fas ligand
expression by Kupffer cells via mechanisms dependent on reactive oxygen
species. Am J Physiol Gastrointest Liver Physiol 287: G620–G626, 2004.
34. Uesugi T, Froh M, Arteel GE, Bradford BU, Thurman RG. Toll-like
receptor 4 is involved in the mechanism of early alcohol-induced liver
injury in mice. Hepatology 34: 101–108, 2001.
35. Wu A, Ying Z, Gomez-Pinilla F. Oxidative stress modulates Sir2alpha in
rat hippocampus and cerebral cortex. Eur J Neurosci 23: 2573–2580,
36. Yang SR, Wright J, Bauter M, Seweryniak K, Kode A, Rahman I.
Sirtuin regulates cigarette smoke-induced proinflammatory mediator re-
lease via RelA/p65 NF-?B in macrophages in vitro and in rat lungs in
vivo: implications for chronic inflammation and aging. Am J Physiol Lung
Cell Mol Physiol 292: L567–L576, 2007.
37. Yao J, Mackman N, Edgington TS, Fan ST. Lipopolysaccharide induc-
tion of the tumor necrosis factor-alpha promoter in human monocytic
cells. Regulation by Egr-1, c-Jun, and NF-kappaB transcription factors.
J Biol Chem 272: 17795–17801, 1997.
38. Yeung F, Hoberg JE, Ramsey CS, Keller MD, Jones DR, Frye RA,
Mayo MW. Modulation of NF-kappaB-dependent transcription and cell
survival by the SIRT1 deacetylase. EMBO J 23: 2369–2380, 2004.
39. Yoshizaki T, Milne JC, Imamura T, Schenk S, Sonoda N, Babendure
JL, Lu JC, Smith JJ, Jirousek MR, Olefsky JM. SIRT1 exerts anti-
inflammatory effects and improves insulin sensitivity in adipocytes. Mol
Cell Biol 29: 1363–1374, 2008.
40. You M, Matsumoto M, Pacold CM, Cho WK, Crabb DW. The role of
AMP-activated protein kinase in the action of ethanol in the liver.
Gastroenterology 127: 1798–1808, 2004.
41. You M, Cao Q, Liang X, Ajmo JM, Ness GC. Mammalian sirtuin 1 is
involved in the protective action of dietary saturated fat against alcoholic
fatty liver in mice. J Nutr 138: 497–501, 2008.
42. You M, Liang X, Ajmo JM, Ness GC. Involvement of mammalian
sirtuin 1 in the action of ethanol in the liver. Am J Physiol Gastrointest
Liver Physiol 294: G892–G898, 2008.
SIRT1 AND ALCOHOLIC LIVER INJURY
AJP-Gastrointest Liver Physiol • VOL 296 • MAY 2009 • www.ajpgi.org