Korean J Physiol Pharmacol.  2015 Sep;19(5):451-460. 10.4196/kjpp.2015.19.5.451.

Myeloid-specific SIRT1 Deletion Aggravates Hepatic Inflammation and Steatosis in High-fat Diet-fed Mice

• Kim KE
• Kim H
• Heo RW
• Shin HJ
• Yi CO
• Lee DH
• Kim HJ
• Kang SS
• Cho GJ
• Choi WS
• Roh GS

• KMID: 2285590
• DOI: http://doi.org/10.4196/kjpp.2015.19.5.451

Abstract

Sirtuin 1 (SIRT1) is a mammalian NAD+-dependent protein deacetylase that regulates cellular metabolism and inflammatory response. The organ-specific deletion of SIRT1 induces local inflammation and insulin resistance in dietary and genetic obesity. Macrophage-mediated inflammation contributes to insulin resistance and metabolic syndrome, however, the macrophage-specific SIRT1 function in the context of obesity is largely unknown. C57/BL6 wild type (WT) or myeloid-specific SIRT1 knockout (KO) mice were fed a high-fat diet (HFD) or normal diet (ND) for 12 weeks. Metabolic parameters and markers of hepatic steatosis and inflammation in liver were compared in WT and KO mice. SIRT1 deletion enhanced HFD-induced changes on body and liver weight gain, and increased glucose and insulin resistance. In liver, SIRT1 deletion increased the acetylation, and enhanced HFD-induced nuclear translocation of nuclear factor kappa B (NF-kappaB), hepatic inflammation and macrophage infiltration. HFD-fed KO mice showed severe hepatic steatosis by activating lipogenic pathway through sterol regulatory element-binding protein 1 (SREBP-1), and hepatic fibrogenesis, as indicated by induction of connective tissue growth factor (CTGF), alpha-smooth muscle actin (alpha-SMA), and collagen secretion. Myeloid-specific deletion of SIRT1 stimulates obesity-induced inflammation and increases the risk of hepatic fibrosis. Targeted induction of macrophage SIRT1 may be a good therapy for alleviating inflammation-associated metabolic syndrome.

Keyword

Hepatic steatosis; High-fat diet; Nuclear factor kappa B; Sirtuin 1

MeSH Terms

Acetylation
Actins
Animals
Collagen
Connective Tissue Growth Factor
Diet
Diet, High-Fat
Fibrosis
Glucose
Inflammation*
Insulin Resistance
Liver
Macrophages
Metabolism
Mice*
NF-kappa B
Obesity
Sirtuin 1
Sterol Regulatory Element Binding Protein 1
Weight Gain
Actins
Collagen
Connective Tissue Growth Factor
Glucose
NF-kappa B
Sirtuin 1
Sterol Regulatory Element Binding Protein 1

Figure

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  Fig. 1 SIRT1 deletion aggravates HFD-induced weight gain and insulin resistance. (A) Body weights, (B) liver/body weight ratio, (C) food intake, (D) fasting blood glucose levels, (E) GTT, (F) ITT, (G) area under curve (AUC) for GTT and (H) AUC for ITT of WT and KO mice fed ND or HFD. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice.

• 

  Fig. 2 SIRT1 deletion increases NF-κB acetylation and induces NF-κB nuclear translocation in HFD-fed mice. Western blots of SIRT1 (A) and acetylated NF-κB p65 (B) in liver homogenates from WT and KO mice fed ND or HFD. (C, D) Western blots of cytosolic and nuclear NF-κB and the nuclear/cytosolic (N/C) ratio of NF-κB levels. Band intensity was normalized to β-actin or α-tubulin. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice.

• 

  Fig. 3 SIRT1 deletion aggravates HFD-induced hepatic inflammation and JNK activation. (A, B) Western blots of HMGB1, RAGE, TNF-α, and TNFR1 in liver homogenates from WT and KO mice fed an ND or HFD. (C) Immunohistochemistry using a CD68 antibody for detection of activated macrophages in liver sections. The areas in black squares in the top panels were magnified on the bottom. The CD68-positive cells were quantified. Scale bar=100 µm (high magnification, 50 µm). (D) Western blots of p-JNK/JNK in liver homogenates from WT and KO mice fed ND or HFD. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice.

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  Fig. 4 SIRT1 deletion aggravates hepatic steatosis in HFD-fed mice. (A) Histological analysis of hepatic fat accumulation by H&E and Oil Red O staining. Scale bar=100 µm. (B) Western blots of nuclear SREBP1 in liver homogenates from WT and KO mice fed an ND or HFD. Band intensity was normalized to nuclear p84 protein. (C) Hepatic TG levels in supernatant fractions of liver homogenates. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice.

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  Fig. 5 SIRT1 deletion increases hepatic CTGF and α-SMA expression in HFD-fed mice. (A) Western blots of CTGF in liver homogenates from WT and KO mice fed ND or HFD. (B) Immunohistochemistry for CTGF detection in liver sections. (C) Western blots of α-SMA in liver homogenates from WT and KO mice fed ND or HFD. (D) Immunohistochemistry for α-SMA detection in liver sections. Band intensity was normalized to β-actin. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice. Scale bar=100 µm.

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  Fig. 6 SIRT1 deletion induces hepatic fibrogenesis in HFD-fed mice. Histological analysis of hepatic collagen deposition by Masson trichrome (A) and Sirius red staining (B). Scale bar=100 µm. (C) Hepatic collagen levels measured in liver extracts using a Sircol collagen assay kit. (D) Western blots of 4-HNE in liver homogenates from WT and KO mice fed ND or HFD. Band intensity was normalized to β-actin. Data are presented as mean±SEM. *p<0.05 for WH mice versus WN mice. †p<0.05 for KH mice versus KN mice. #p<0.05 for KH mice versus WH mice. WN, ND-fed WT mice; WH, HFD-fed WT mice; KN, ND-fed KO mice; KH, HFD-fed KO mice.

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