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. 2010 Sep 8;12(3):224-36.
doi: 10.1016/j.cmet.2010.06.009.

Hepatic-specific Disruption of SIRT6 in Mice Results in Fatty Liver Formation Due to Enhanced Glycolysis and Triglyceride Synthesis

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Free PMC article

Hepatic-specific Disruption of SIRT6 in Mice Results in Fatty Liver Formation Due to Enhanced Glycolysis and Triglyceride Synthesis

Hyun-Seok Kim et al. Cell Metab. .
Free PMC article

Abstract

Under various conditions, mammals have the ability to maintain serum glucose concentration within a narrow range. SIRT1 plays an important role in regulating gluconeogenesis and fat metabolism; however, the underlying mechanisms remain elusive. Here, we show that SIRT1 forms a complex with FOXO3a and NRF1 on the SIRT6 promoter and positively regulates expression of SIRT6, which, in turn, negatively regulates glycolysis, triglyceride synthesis, and fat metabolism by deacetylating histone H3 lysine 9 in the promoter of many genes involved in these processes. Liver-specific deletion of SIRT6 in mice causes profound alterations in gene expression, leading to increased glycolysis, triglyceride synthesis, reduced beta oxidation, and fatty liver formation. Human fatty liver samples exhibited significantly lower levels of SIRT6 than did normal controls. Thus, SIRT6 plays a critical role in fat metabolism and may serve as a therapeutic target for treating fatty liver disease, the most common cause of liver dysfunction in humans.

Figures

Fig. 1
Fig. 1. Regulation of SIRT1 and SIRT6 expression by nutrient deprivation
(A-C) Levels of mRNA (A,B) and protein (C) of SIRT1 and SIRT6 in multiple organs of mice under fed ad libitum (AL), starved (S), or re-fed for 24 hours. (D-F) Levels of SIRT1 and SIRT6 protein (D), G6Pase and PEPCK mRNA (E), and GK and LPK mRNA (F) in the liver of wild type mice during a time course of fasting. (G,H) Levels of SIRT6 mRNA revealed by Real-Time RT-PCR (G) and protein (H) in the liver from wild type and Sirt1−/− mice. (I,J) Over-expression of SIRT1 increased endogenous SIRT6 in the presence and absence of glucose (I), while RNAi-mediated knockdown of SIRT1 blocked SIRT6 induction in the absence of glucose (J) revealed by Real-Time PCR. * represents p < 0.05 by Student T-test in all figures.
Fig. 2
Fig. 2. Regulation of SIRT6 by SIRT1, FOXO3a, and NRF1
(A) Absence of glucose in Hepa1-6 cells induced luciferase activity of a SIRT6 promoter reporter, while mutation of NRF1 binding sites abolished the induction. (B,C) Effect of NRF1 ectopic expression (B) and RNAi-mediated knockdown (C) on a SIRT6 promoter reporter. (D,E) Ectopic expression of SIRT1 increased SIRT6 promoter activity in the presence or absence of glucose (D), while mutation of NRF1 binding sites (D), or RNAi-mediated knockdown of SIRT1 (E) abolished the induction. (F,G) Ectopic expression of FOXO3a increased SIRT6 promoter activity in the presence or absence of glucose (F), while RNAi-mediated knockdown of FOXO3a inhibits it (G). (H,I) Absence of glucose induces expression of SIRT6 mRNA, which is further increased by overexpression of FOXO3a (H), while RNAi-mediated knockdown of FOXO3a abolished the induction (I).
Fig. 3
Fig. 3. SIRT1, FOXO3a and NRF1 form a protein complex on the NRF1 binding sites of the SIRT6 promoter
(A) Expression of SIRT1 and FOXO3a synergistically activated a SIRT6 promoter reporter, which is blocked by siRNA specific to NRF1. (B) Binding of NRF1, SIRT1, and FOXO3a to the SIRT6 promoter in the presence or absence of glucose as revealed by ChIP assay. (C) Interaction of FOXO3a, SIRT1, and NRF1 in cultured cells in either the presence or absence of glucose as revealed by immunoprecipitation (IP). (D) Pull-down of 35S-FOXO3a, but not 35S-SIRT1, by GST tagged-NRF1. (E) SIRT1 pulls down both FOXO3a and NRF1 in Hepa1-6 cells. However, SIRT1 could not pull down NRF1 in Hepa1-6 cells carrying a stably transfected shRNA for FOXO3a (SS3-16). (F) Wild-type SIRT1 deacetylates FOXO3a, which is enhanced in the absence of glucose in Hepa1-6 cells, and the deacetylated form of FOXO3a interacts more abundantly with NRF1.
Fig. 4
Fig. 4. Phenotypic analysis of mice carrying a liver specific knockout of SIRT6
(A) Blood glucose level (mg/dL) of 8–9 months old SIRT6 MT and WT mice under fed or 24 hours fasting condition. (B) Glucose tolerance test. Mutant mice had a slightly higher, but not significantly different glucose levels at 15 and 30 minutes than wild type mice. (C) Insulin tolerance test expressed as percentage of basal glucose level. We have also measured glucose value in the Area Under the Curve (AUC) for both GTT and ITT, and no difference is found between wild type and mutant mice. (D) Body weight (gram) of SIRT6 mice at 2 months: WT 17, MT 15; 5–6 months: WT 18, MT 15; and 8–10 months: WT 15, MT 22. (E,F) Percent of liver weight/body weight (E) and TG levels (F) of 8–9 months old SIRT6 MT and WT mice. (G-L) Morphology (G,H), H&E sections (I,J) and Oil Red O staining (K,L) of livers from MT (G,I,K) and WT (H,J,L) mice. Bar in (G,H) is 1 centimeter. At least 6 pairs of mice were analyzed in each experiment. (M) TG secretion rate to plasma (mg/hour/gram of liver). (N,O) TG content in primary hepatocytes measured by 3H-palmitate incorporation (N) and TG level (O) at different times. In panel (O), TG production increases 1.54 fold in wild type cells and 2.45 fold in mutant cells from 0 hour to 12 hours, respectively. This increase is statistically significant with p<0.04.
Fig. 5
Fig. 5. SIRT6 regulates the expression of genes related to glycolysis and lipid metabolism
(A,B) mRNA levels of GK and LPK in the liver of WT and MT mice under fed Ad Libitum (AL) or 24 hours starvation (S) detected by using Real-Time RT-PCR. (C) Protein expression of genes involved in glycolysis and lipid metabolism. (D) GK enzymatic activity in the liver of WT and MT mice. (E-G) mRNA levels of FAT and FATP (E,F) and several genes involved in TG synthesis (G). Mice used in panels (A,F,G) were 8–9 months old, and in panels (B-E) were 2–3 months old. At least 5 pairs of mice were used for each experiment. (H) De Novo lipogenesis activity in the primary hepatocytes under fed or fasted condition. (I,J) Absence of SIRT6 decreased the expression levels of genes involved in β-oxidation revealed by Real-Time RT-PCR (I) and fatty acid β-oxidation revealed by enzymatic activity (J). (K) Gene expression in primary hepatocytes upon acute knockdown of SIRT6 for 36 hours revealed by Real-Time PCR. Abbreviation of genes that is not mentioned in the text: ACC: acetyl-CoA carboxylase; Elovl6: long-chain elongase; FAS: Fatty acid synthase; GPAT: mitochondrial glycerol 3-phosphate acyltransferase; SCD1: stearoyl-CoA desaturase-1.
Fig. 6
Fig. 6. SIRT6 is recruited to and deacetylates H3K9 in the promoter of many genes
(A) Western blot analysis of nuclear extracts prepared from the liver of WT and MT mice under fed (AL) or 24 hours starvation (S). (B) Western blot analysis of nuclear extracts prepared from primary hepatoctyes infected with pCDH-FLAG-SIRT6 or vector control under fed or fasting condition. (C-G) ChIP analysis showing the acetylation pattern of H3K9 (C-G) in the promoter of several genes in samples extracted from the liver of WT and MT mice. Four fragments (two in promoter region, and two in exon/intron) for each gene were analyzed and in all cases the absence of SIRT6 increased acetylation in the promoter, but not in the exon/intron region (some data not shown). (H) ChIP analysis showing the binding of SIRT6 to the promoter of several genes in samples extracted from the liver of WT and MT mice. (I,J) Expression of Flag-tagged SIRT6 in SIRT6 mutant liver mediated by injection of lentivirus carrying Flag-tagged SIRT6 significantly reversed increased levels of AcH3K9 (I) and gene expression (J). At least three pairs of SIRT6 mutant and control mice were used for each experiment.
Fig. 7
Fig. 7
Reduced levels of SIRT1 and SIRT6, and increased levels of GK and LPK in human fatty liver samples. A, Western blot analysis of protein levels of SIRT1 and SIRT6 in fatty liver samples. Quantification of gel intensity of 8 normal livers and 8 nonalcoholic fatty livers was shown in right. B, mRNA levels of GK and LPK revealed by Real-Time RT-PCR in these samples. The fatty livers and controls were provided by the Liver Tissues Procurement and Distribution System (University of Minnesota). C, An integrated model for functions of SIRT6 in inhibiting fatty liver formation through regulation of glycolysis and lipid metabolism. SIRT6 deficiency, consequently, results in fatty liver fomation.

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