1. Volume 146, Issue 2, Pages 539-549.e7 (February 2014)
Hepatic SIRT1 Attenuates Hepatic Steatosis and Controls Energy Balance in Mice by Inducing Fibroblast Growth Factor 21 
Yu Li, Kimberly Wong, Amber Giles, Jianwei Jiang, Jong Woo Lee, Andrew C. Adams, Alexei Kharitonenkov, Qin Yang, Bin Gao, Leonard Guarente, Mengwei Zang 
Gastroenterology 
Volume 146, Issue 2, Pages e7 (February 2014)
DOI: /j.gastro
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2. Figure 1
Hepatocyte-specific deletion of SIRT1 in mice increases susceptibility to developing fasting-induced fatty liver. (A) Representative immunoblots for SIRT1 in 2 mouse livers from each group are shown. The expected deletion mutant protein in SIRT1 LKO mice, which migrates slightly faster than WT SIRT1, was visualized by immunoblotting. (B) Blood glucose and plasma insulin levels in WT and SIRT1 LKO mice at 6 and 7 months of age that were fed a normal diet (Fed) or fasted for 24 hours (Fasted) (n = 10–18). (C) Plasma triglyceride and cholesterol levels in mice (n = 4–6). (D and E) Hepatic steatosis was assessed by Oil Red O staining and quantified by measuring Oil Red O–stained areas. (F) Hepatic triglyceride and cholesterol levels in WT and SIRT1 LKO mice (n = 4–6). Results are presented as the mean ± SEM. *P < .05 vs the fed WT mice; #P < .05 vs the fasted WT mice.
Gastroenterology  , e7DOI: ( /j.gastro )
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3. Figure 2
Production and secretion of hepatic FGF21 are reduced in SIRT1 LKO mice in response to nutrient deprivation. SIRT1 LKO mice and their WT littermates at 6 and 7 months of age (n = 4–7) were subjected to feeding (Fed), fasting for 24 hours (Fasted), or refeeding for 6 hours after a 24-hour fast (Refed). (A) Hepatic SIRT1 expression was increased by fasting and decreased by refeeding in WT mice, and the induction was eliminated in SIRT1 LKO mice (n = 4–7). (B and C) Fasting-induced gene expression and protein production of hepatic FGF21 were diminished in SIRT1 LKO mice (n = 4–7), as determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assays (ELISA). (D) The circulating levels of FGF21 were increased robustly by fasting in WT mice, and the induction was impaired in SIRT1 LKO mice. *P < .05 vs the fed WT mice; #P < .05 vs the fasted WT mice.
Gastroenterology  , e7DOI: ( /j.gastro )
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4. Figure 3
Expression of metabolic genes involving fatty acid oxidation, ketogenesis, and lipogenesis is altered in livers of SIRT1 LKO mice. (A) Hepatic expression of key genes of fatty acid oxidation such as CPT1α and MCAD was induced by fasting in WT mice and inhibited in SIRT1 LKO mice (n = 4–7). (B and C) Fasting-induced expression of ketogenic enzymes including acetyl-CoA acetyltransferase 1 (ACAT1), 3-hydroxy-3-methylglutaryl–CoA synthase 2 (HMGCS2), 3-hydroxy-3-methylglutaryl-CoA lyase (HMGCL), and β-hydroxybutyrate dehydrogenase 1 (BDH1) was suppressed in livers of SIRT1 LKO mice. (D) Effects of fasting and refeeding on circulating ketone bodies in mice (n = 5–10). (E) mRNA amounts of SREBP-1c and key lipogenic enzymes including FAS and stearoyl-CoA desaturase (SCD)1 were up-regulated in livers of SIRT1 LKO mice (n = 4–7). *P < .05 vs the fed WT mice; #P < .05 vs the fasted WT mice.
Gastroenterology  , e7DOI: ( /j.gastro )
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5. Figure 4
SIRT1 stimulates mitochondrial fatty acid oxidation in an FGF21-dependent manner in HepG2 cells. (A) Increased expression of SIRT1 progressively stimulated transcriptional activity of the FGF21 (-2090/+117) promoter-driven reporter and increased FGF21 gene expression. Overexpression of FLAG-tagged mouse SIRT1 (FLAG-mSIRT1) in HEK293T cells was confirmed by immunoblots. (B) Protein production of FGF21 and mRNA amounts of CPT1α were stimulated by SIRT1 activators resveratrol (10 μmol/L) and SRT1720 (5 μmol/L). (C) Oxygen consumption rates were determined by analyzing a bioenergetics profile of SIRT1+/+ and SIRT1-/- MEFs using the Seahorse Bioscience (North Billerica, MA). The oxygen consumption rates of basal respiration, adenosine triphosphate (ATP) turnover, and respiratory capacity were measured and calculated as averages for each phase. (D) The mitochondrial oxygen consumption rate was increased by SIRT1 activators. (E) The ability of resveratrol (10 μmol/L) to induce CPT1α was abrogated by small interfering RNA (siRNA)-mediated FGF21 knockdown in HepG2 cells. (F) Resveratrol-induced CPT1α expression was diminished by a competitive inhibitor of FGF21 (FGF21ΔN17, 100 nmol/L) in HepG2 cells (n = 3–4). *P < .05 vs control cells; #P < .05 vs treatment cells. pcDNA, an empty pcDNA plasmid as a negative control.
Gastroenterology  , e7DOI: ( /j.gastro )
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6. Figure 5
Hepatic overexpression of FGF21 rescues hepatic steatosis and metabolic defects in fasted SIRT1 LKO mice. (A) An adenovirus vector encoding FGF21 (Ad-FGF21) was overexpressed in HEK293A cells. (B) Adenoviral overexpression of FGF21 in SIRT1 LKO mice (n = 4–7) was shown by increased hepatic gene expression, protein production, and circulating levels of FGF21. (C and D) Fasting-induced hepatic steatosis in SIRT1 LKO mice was ameliorated by hepatic overexpression of FGF21, as indicated by decreased Oil Red O–stained areas and reduced hepatic triglyceride levels. (E and F) Suppression of hepatic fatty acid oxidation and ketogenesis in SIRT1 LKO mice was prevented by hepatic overexpression of FGF21. *P < .05 vs Ad-GFP–injected WT mice; #P < .05 vs Ad-GFP–injected SIRT1 LKO mice. HMGCS2, 3-hydroxy-3-methylglutaryl–CoA synthase 2; HMGCL, 3-hydroxy-3-methylglutaryl–CoA lyase; BDH1, β-hydroxybutyrate dehydrogenase 1.
Gastroenterology  , e7DOI: ( /j.gastro )
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7. Figure 6
SIRT1 LKO mice are susceptible to developing obesity with increased adiposity and decreased total oxygen consumption and energy expenditure. (A) Chow-fed SIRT1 LKO mice at 6 and 7 months of age (n = 8) gained more body weight. Body weight curves of WT and SIRT1 LKO mice over a 15-month period (n = 8–16). (B) A representative photograph of increased adiposity and adipocyte size in WAT of SIRT1 LKO mice. (C) Body composition analysis of WT and SIRT1 LKO mice. (D) Food intake (n = 10–12) and body temperature (n = 6) were similar between WT and SIRT1 LKO mice. (E) The rates of Vo2, Vco2, and energy expenditure were measured by comprehensive metabolic monitoring in mice (n = 4) over a 24-hour period with food and over a 24-hour fast and normalized to lean body mass. Upper panels: circadian changes in metabolic parameters in mice in the fed state during light and dark cycles, as indicated by the white and gray areas. Lower panels: metabolic rates in mice in fed and fasted states. (F) Respiratory quotient and locomotor activity were determined in metabolic cages. The physical activity is expressed as total counts measured by summing X and Y beam breaks, and the bar graph represents average locomotor activity over a 12-hour block of light and dark phases. *P < .05 vs WT mice.
Gastroenterology  , e7DOI: ( /j.gastro )
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8. Figure 7
Hepatic overexpression of FGF21 rescues the deregulation of whole-body energy metabolism and promotes browning of WAT in SIRT1 LKO mice. (A) Circadian changes in Vo2 were measured by indirect calorimetry over 48 hours in mice in fed and fasted states. (B) The rates of Vo2 and energy expenditure were increased by hepatic overexpression of FGF21 in SIRT1 LKO mice in fed and fasted states through light and dark cycles (n = 4). Respiratory quotient (RQ) and locomotor activity were similar between the 2 genotypes of mice. (C) Body weight and body composition in mice (n = 4–7). (D and E) Adenoviral delivery of FGF21 increased the transcription of brown-fat-like genes and adiponectin in (D) WAT and (E) BAT of SIRT1 LKO mice. *P < .05 vs Ad-GFP–injected SIRT1 LKO mice. (F) The proposed model for nutrient regulation of FGF21 through SIRT1. Prolonged fasting increases the NAD/ form of NADH ratio and stimulates hepatic SIRT1, which in turn promotes expression, production, and release of hepatic FGF21. Induction of FGF21 by hepatic SIRT1 stimulates fatty acid oxidation and ketogenesis, enhances the metabolic adaptation to fasting, and improves fatty liver disease. Activation of the SIRT1-FGF21 cascade may represent a liver-adipose tissue axis that contributes to reduced adiposity and body weight by promoting white fat browning and increasing energy expenditure.
Gastroenterology  , e7DOI: ( /j.gastro )
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9. Supplementary Figure 1
Hepatic expression of SREBP-1c and FAS in fasted SIRT1 LKO mice is not affected by hepatic overexpression of FGF21. Adenoviruses (5 × 109 pfu) encoding either FGF21 or control green fluorescent protein (GFP) were delivered into 8- to 10-month-old mice (n = 4–7) via tail vein injection. The animals were killed in a 24-hour fasted state after 2 weeks of injection.
Gastroenterology  , e7DOI: ( /j.gastro )
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