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. 2014 Sep 12;289(37):25925-35.
doi: 10.1074/jbc.M114.567628. Epub 2014 Jul 25.

Liver clock protein BMAL1 promotes de novo lipogenesis through insulin-mTORC2-AKT signaling

Deqiang Zhang  1 , Xin Tong  1 , Blake Arthurs  1 , Anirvan Guha  1 , Liangyou Rui  1 , Avani Kamath  1 , Ken Inoki  1 , Lei Yin  2
Affiliations

Liver clock protein BMAL1 promotes de novo lipogenesis through insulin-mTORC2-AKT signaling

Deqiang Zhang et al. J Biol Chem. .

Abstract

The clock protein BMAL1 (brain and muscle Arnt-like protein 1) participates in circadian regulation of lipid metabolism, but its contribution to insulin AKT-regulated hepatic lipid synthesis is unclear. Here we used both Bmal1(-/-) and acute liver-specific Bmal1-depleted mice to study the role of BMAL1 in refeeding-induced de novo lipogenesis in the liver. Both global deficiency and acute hepatic depletion of Bmal1 reduced lipogenic gene expression in the liver upon refeeding. Conversely, Bmal1 overexpression in mouse liver by adenovirus was sufficient to elevate the levels of mRNA of lipogenic enzymes. Bmal1(-/-) primary mouse hepatocytes displayed decreased levels of de novo lipogenesis and lipogenic enzymes, supporting the notion that BMAL1 regulates lipid synthesis in hepatocytes in a cell-autonomous manner. Both refed mouse liver and insulin-treated primary mouse hepatocytes showed impaired AKT activation in the case of either Bmal1 deficiency or Bmal1 depletion by adenoviral shRNA. Restoring AKT activity by a constitutively active mutant of AKT nearly normalized de novo lipogenesis in Bmal1(-/-) hepatocytes. Finally, Bmal1 deficiency or knockdown decreased the protein abundance of RICTOR, the key component of the mTORC2 complex, without affecting the gene expression of key factors of insulin signaling. Thus, our study uncovered a novel metabolic function of hepatic BMAL1 that promotes de novo lipogenesis via the insulin-mTORC2-AKT signaling during refeeding.

Keywords: Akt; Circadian Clock; Insulin Signaling; Lipid Metabolism; Lipogenesis; Liver; Rictor-mTORC2.

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Figures

FIGURE 1.
FIGURE 1.
Induction of hepatic lipogenic and circadian clock gene upon refeeding and insulin. C57BL6 WT mice (n = 3–4) were fasted for 16 h before refeeding for 2 or 8 h. Mice were then sacrificed for assessment of the mRNA levels of lipogenic genes (Fasn, Gck, and L-pk) (A) and circadian genes (Dbp and Per2) (B). C, adenoviral shRNA for Bmal1 reduces the protein level of BMAL1 in the mouse liver. WT mice were administrated with recombinant adenoviral control (Ad-shLacZ) (n = 3) or Bmal1-specific shRNA (Ad-shBmal1) (n = 3) via tail-vein injection. 2 weeks post-injection, mice were fasted for 16 h and then refed for 8 h before sacrifice for measurement of liver BMAL1 protein by immunoblotting. The loading was determined by the level of Lamin A/C. The protein expression was quantified and normalized by the levels of lamin A/C. D, adenoviral Bmal1 knockdown blocks clock gene induction by refeeding. Two weeks after Ad-shBmal1 or Ad-shLacZ control injection, WT mice (n = 3) were subjected to fasting for 16 h followed by refeeding for 8 h. The hepatic mRNA levels of Dbp, Nr1d1, and Per2 were determined by RT-QPCR. The data are plotted as the mean ± S.D. E, loss of Bmal1 impairs induction of circadian genes by insulin in PMHs. PMHs were isolated from WT and Bmal1−/− mice and treated with insulin at 10 nm for 6 h. RT-QPCR was performed to assess the induction of clock gene Dbp and Per2. Each condition was done in triplicate. *, p < 0.05. **, p < 0.01. Data are representative of 3–4 independent experiments.
FIGURE 2.
FIGURE 2.
Loss of Bmal1 impairs refeeding-induced lipogenic program in the liver. Both WT and Bmal1−/− (8-week male mice, n = 5) were fasted overnight and refed for 8 h. Mice were then sacrificed and harvested for analysis of levels of mRNA and protein in the liver. A, the liver mRNA levels of Bmal1 and Dbp determined by RT-QPCR. B and C, the liver mRNA and protein levels of major lipogenic enzymes or regulators in WT and Bmal1−/− mice determined by RT-QPCR and immunoblotting. The data are plotted as the mean ± S.D. (n = 5). * p < 0.05. **p < 0.01.
FIGURE 3.
FIGURE 3.
Acute Bmal1 depletion in the liver impairs refeeding-induced expression of lipogenic enzymes. Three weeks after tail vein injection of Ad-shLacz or Ad-shBmal1, mice (n = 3) were fasted overnight and sacrificed either right after overnight fasting or after 8-h refeeding. Liver tissues were harvested for mRNA and protein analysis. A, protein levels of BMAL1 and the lipogenic enzyme, FASN, in the liver. Quantified protein band intensity is shown in the right panel. B, hepatic mRNA levels of lipogenic genes Acc1, Fasn, and Gck. The data were plotted as the mean ± S.D. (n = 3). *, p < 0.05.
FIGURE 4.
FIGURE 4.
Liver-specific overexpression of Bmal1 elevates the expression level of lipogenic genes. WT mice (n = 3) were injected with Ad-GFP (control virus) or Ad-Bmal1 through tail vein. One week after injection mouse livers were collected for confirmation of BMAL1 protein expression (A) and for measurement of mRNA levels by RT-QPCR for clock genes (B) and lipogenic enzymes (C). The data are plotted as the mean ± S.D. (n = 3). *, p < 0.05; **, p < 0.01.
FIGURE 5.
FIGURE 5.
BMAL1 regulates de novo lipogenesis in a cell-autonomous manner. PMHs were isolated from either Bmal1−/− or WT mice and then used for measuring the levels of mRNA and protein of lipogenic enzymes and the rate of de novo lipogenesis. A, mRNA levels of Dbp in both WT and Bmal1−/− hepatocytes (n = 3). B, mRNA levels of lipogenic enzymes and regulators (n = 3). C, Bmal1−/− PMHs show decreased rate of de novo lipogenesis. PMHs from both WT and Bmal1−/− mice were treated with high glucose and insulin in six replicates before incubating with 3H-labeled acetate for 4 h. Radiolabeled lipids were extracted in petroleum, and the 3H radioactivity was normalized by protein amount for each sample. The data are plotted as the mean ± S.D. (n = 6). *, p < 0.05. **, p < 0.01. Data are representative of 3–4 independent experiments.
FIGURE 6.
FIGURE 6.
BMAL1 is required for AKT activation by insulin in the liver and hepatocytes. A, Bmal1−/− mice show reduced AKT activity in the liver. Both WT and Bmal1−/− mice (n = 5) were fasted overnight and refed for 8 h. Liver tissues were used to examine the AKT activity (phosphorylation of AKTSer(P)-473) and the total AKT by immunoblotting. The levels of AKTSer(P)-473 were quantified and normalized to total AKT level. B, acute Bmal1 depletion in the liver reduces AKT phosphorylation induced by refeeding. Two weeks after tail vein injection of Ad-shLacZ or Ad-shBmal1, WT mice (n = 3) were fasted overnight and then refed for 8 h. After sacrifice, mouse liver lysates were prepared for Western blot analysis of AKTSer(P)-473 and total AKT. The levels of AKTSer(P)-473 were quantified and normalized to total AKT expression. C, loss of Bmal1 blocks insulin-induced AKT activation in PMHs. PMHs from WT or Bmal1−/− mice were treated with 0.2 nm insulin for the indicated time points before assessment of AKTSer(P)-473 by Western blot. D, acute depletion of Bmal1 expression attenuates insulin-induced AKT phosphorylation in Hepa1 cell. Hepa1 cells were transduced with Ad-shLacZ or Ad-shBmal1 for 36 h and then treated with 0.2 nm insulin for indicated time points. AKTSer(P)-473 levels were determined by immunoblotting. E, adenoviral overexpression of Bmal1 restores insulin-induced AKT phosphorylation in Bmal1-deleted PMHs. Bmal1flox/flox PMHs were transduced by AdGFP, or AdCre plus AdGFP, or AdCre plus AdBmal1 for 48 h before insulin treatment (0.2 nm) for the indicated time points. AKTSer(P)-473 and BMAL1 levels were measured by immunoblotting along with HSP90 as the loading control. The levels of AKT-P were quantified and normalized to total AKT expression.
FIGURE 7.
FIGURE 7.
Overexpression of constitutively active AKT2 restores de novo lipogenesis in primary hepatocytes with Bmal1 deficiency. Bmal1−/− mice (n = 3) were injected with Ad-Akt-CA or Ad-GFP adenovirus through tail vein. One week later PMHs were isolated for measuring the mRNA and protein levels of lipogenic enzymes (A and B) and the rate of de novo lipogenesis (C). The data are plotted as the mean ± S.D. (n = 3). *, p < 0.05. Data are representative of three independent experiments.
FIGURE 8.
FIGURE 8.
BMAL1 regulates RICTOR protein expression in the mouse liver and cultured hepatocytes. A–C, loss of Bmal1 shows no effect on the gene expression of key factors of insulin signaling. After overnight fasting and refeeding for 8 h, the liver mRNA levels of key components of the insulin-signaling pathway were determined in both WT and Bmal1−/− mice (n = 5). The data are plotted as the mean ± S.D. (n = 5). D, liver-specific deletion of Bmal1 lowers the RICTOR protein levels in the liver of Bmal1-LKO mice. RICTOR, mTOR, BMAL1, and β-tubulin protein expression in the liver of Bmal1-LKO mice was detected by immunoblotting and quantified. E, acute deletion of Bmal1 reduces the abundance of RICTOR in PMHs. Bmal1 deletion was achieved by transducing Bmal1flox/flox PMHs with Ad-Cre versus Ad-GFP. 36 h later cells in triplicate were harvested for examining the protein expression of RICTOR and BMAL1. F, acute depletion of Bmal1 accelerates RICTOR protein degradation. Hepa1 or U2OS cells were transfected with either shLacZ control or shBmal1 construct for 48 h before treatment with protein synthesis inhibitor cycloheximide for 0, 2, 4, or 6 h. Cells were then harvested for examining the protein expression of RICTOR, BMAL1, and loading control β-tubulin. G, proteasome inhibitor MG-312 blocks RICTOR protein degradation after acute Bmal1 depletion. Hepa1 cells were transduced with either Ad-shLacZ control or Ad-shBmal1 virus for 48 h before treatment with MG-132 (10 μm) for 0, 4, and 8 h. Cells were then harvested to examine the protein abundance of RICTOR, BMAL1, and loading control β-tubulin. The protein level of RICTOR was quantified from three independent experiments and normalized to the expression of β-tubulin. **, p value <0.01 (n = 3). H, the working model of BMAL1 regulation of de novo lipogenesis in the liver in response to nutritional cues. Upon refeeding, insulin or other nutrients activate BMAL1-dependent transcription activity. BMAL1 is required for maintaining RICTOR protein stability and promoting AKT activation and subsequent de novo lipogenesis. In the case of Bmal1 deletion or depletion, destabilization of RICTOR is likely to attenuate AKT activation and suppress lipogenic pathway.
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