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. 2012 May;122(5):1677-87.
doi: 10.1172/JCI61248. Epub 2012 Apr 2.

Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice

Ding Ai  1 , Juan M Baez, Hongfeng Jiang, Donna M Conlon, Antonio Hernandez-Ono, Maria Frank-Kamenetsky, Stuart Milstein, Kevin Fitzgerald, Andrew J Murphy, Connie W Woo, Alanna Strong, Henry N Ginsberg, Ira Tabas, Daniel J Rader, Alan R Tall
Affiliations

Activation of ER stress and mTORC1 suppresses hepatic sortilin-1 levels in obese mice

Ding Ai et al. J Clin Invest. 2012 May.

Abstract

Recent GWAS have identified SNPs at a human chromosom1 locus associated with coronary artery disease risk and LDL cholesterol levels. The SNPs are also associated with altered expression of hepatic sortilin-1 (SORT1), which encodes a protein thought to be involved in apoB trafficking and degradation. Here, we investigated the regulation of Sort1 expression in mouse models of obesity. Sort1 expression was markedly repressed in both genetic (ob/ob) and high-fat diet models of obesity; restoration of hepatic sortilin-1 levels resulted in reduced triglyceride and apoB secretion. Mouse models of obesity also exhibit increased hepatic activity of mammalian target of rapamycin complex 1 (mTORC1) and ER stress, and we found that administration of the mTOR inhibitor rapamycin to ob/ob mice reduced ER stress and increased hepatic sortilin-1 levels. Conversely, genetically increased hepatic mTORC1 activity was associated with repressed Sort1 and increased apoB secretion. Treating WT mice with the ER stressor tunicamycin led to marked repression of hepatic sortilin-1 expression, while administration of the chemical chaperone PBA to ob/ob mice led to amelioration of ER stress, increased sortilin-1 expression, and reduced apoB and triglyceride secretion. Moreover, the ER stress target Atf3 acted at the SORT1 promoter region as a transcriptional repressor, whereas knockdown of Atf3 mRNA in ob/ob mice led to increased hepatic sortilin-1 levels and decreased apoB and triglyceride secretion. Thus, in mouse models of obesity, induction of mTORC1 and ER stress led to repression of hepatic Sort1 and increased VLDL secretion via Atf3. This pathway may contribute to dyslipidemia in metabolic disease.

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Figures

Figure 1
Figure 1. Regulation of sortilin-1 in obesity.
Shown are results for (A and B) ob/ob mice (8–10 weeks old) and (C and D) DIO mice (C57BL/6 fed high-fat diet for 20 weeks). (A and C) Western blot analysis of hepatic protein levels after a 6-hour fast. β-Actin was used as internal control. P-, phosphorylated; T-, total. (B and D) Quantification of sortilin-1 protein level and measurement of hepatic Sort1 mRNA by QPCR. *P < 0.05, unpaired t test.
Figure 2
Figure 2. Restoration of sortilin-1 abolished apoB overproduction in ob/ob mice.
(A) Hepatic protein expression of sortilin-1 in WT and ob/ob mice. Animals were fed WTD for 1 week before injection with control AAV8 or AAV8-Sort1, fed WTD for an additional 2 weeks, then sacrificed after a 6-hour fast. (B) TG production was determined at the indicated times after P407 injection. ANOVA revealed significant differences between treatment (F2,14 = 8.753; P = 0.0034) and time point (*P < 0.05 at 2.5 hours, Bonferroni post-test). (C) apoB levels in plasma collected at the 2.5-hour time point. (D) Quantification and ANOVA revealed significant differences between treatments for apoB100 (F2,11 = 27.91) and apoB48 (F2,11 = 5.554) from C. *P < 0.05, AAV8-Sort1 vs. control AAV8, Bonferroni post-test.
Figure 3
Figure 3. Downregulation of sortilin-1 in Li-Tsc1KO mice.
(A) Western blot analysis of hepatic S6K, sortilin-1, and β-actin protein levels in Li-Tsc1KO and control mice sacrificed 7 days after injection (Cre and control adenovirus, respectively) via tail vein. (B) Hepatic Sort1, Mttp, Apob, and Atf3 mRNA levels were measured by QPCR. (C) apoB levels in plasma collected at 2.5 hours; quantification is also shown. (D) TG production over time after P407 injection. ANOVA revealed significant differences only for time point (F2, 12 = 134.3; P < 0.0001), not for treatment. (E) Liver weight/body weight ratio (LW/BW). (F) Hepatic Srebp1c and Fas mRNA levels were measured by QPCR. *P < 0.05, unpaired t test.
Figure 4
Figure 4. Tunicamycin decreased sortilin-1 expression in vivo.
(A) Western blot analysis and quantification of hepatic sortilin-1 and Atf3 in WT mice injected with DMSO or tunicamycin (Tu; 1 mg/kg) for 6, 12, or 24 hours. (B) Hepatic Sort1 and Atf3 mRNA was determined by QPCR. (C) Western blot analysis of hepatic sortilin-1 in WT or Chop–/– mice injected with DMSO or 1 mg/kg tunicamycin for 24 hours. *P < 0.05 vs. control at the respective time point, unpaired t test.
Figure 5
Figure 5. Expression of sortilin-1 was increased by suppression of ER stress.
8- to 10-week-old ob/ob mice were orally administered PBA (0.5 g/kg body weight, twice daily) or PBS control, fed WTD for 21 days, then fasted for 6 hours and subsequently killed. (A) Western blot analysis of hepatic p-eIF2α, total eIF2α, Atf3, Grp78, and sortilin-1 protein levels. (B) Hepatic Sort1, Mttp, and Apob mRNA levels were measured by QPCR. (C) apoB production was determined 2.5 hours after P407 injection; quantification is also shown. (D) TG secretion was determined. ANOVA revealed significant differences for treatment (F2,46 = 4.385; P = 0.0181) and time point (#P < 0.05 at 2.5 hours, Bonferroni post-test). (E) Basal levels of plasma glucose, insulin, and cholesterol were determined. *P < 0.05, unpaired t test.
Figure 6
Figure 6. Regulation of sortilin-1 by ER stress.
(A) Western blot analysis of sortilin-1 and Atf3 in cells of the McArdleH7777 line treated with DMSO or 5 μg/ml tunicamycin at the indicated time points. (B) Sort1 mRNA was determined by QPCR. McArdle cells were treated with DMSO control or 5 μg/ml tunicamycin or were preincubated with 5 μg/ml actinomycin D (ActD) for 30 minutes with or without 5 μg/ml tunicamycin for the indicated times. (C) Western blot analysis of sortilin-1 and Atf3 protein levels in McArdle cells treated with 1 μM thapsigargin (Tg) for the indicated times. (D) Grp78, Atf3, and Sort1 mRNA was measured by QPCR in McArdle cells treated with 1 μM thapsigargin for the indicated times. 3 separated experiments were performed. *P < 0.05 vs. control for the respective time point, unpaired t test.
Figure 7
Figure 7. Mechanism of sortilin-1 regulation by ATF3.
(A) 2 putative ATF3 binding sites were predicted in the SORT1 promoter region. SNPs (asterisk) in CELSR2-PSRC1-SORT1 locis, which can regulate the expression of SORT1, identified a high association with CVD by GWAs studies. Two putative ATF3 binding sites were predicted in SORT1 promoter region. (B) Human SORT1 promoter fragments SORT1–2.66 kb, SORT1–674 bp, and SORT1–458 bp (see Methods) were inserted into the reporter system and cotransfected with or without ATF3 plasmid in the 293 cell line. (C) SORT1–674 bp was used as template to make a mutation construct (674M), and both were cotransfected with or without ATF3 in 293 cells. (D) Sortilin-1 expression was reduced by overexpressing adeno-Atf3 in mouse primary hepatocytes. Quantification is also shown. Empty adenoviral vector served as control. (E) EMSA was performed in the nuclear extract (NE) protein (5 μg) from 293 cells with biotin-labeled probes containing the ATF3 or mutant binding site in the SORT1 promoter at –538 bp. Cells were treated directly with DMSO control or thapsigargin and preincubated with cold probe or ATF3 antibody. (F and G) ChIP analysis in Li-Tsc1KO mice and Li-Tsc1fl/fl controls (F) and in ob/ob and control lean mice (G) using IgG or ATF3 antibody. The approximate –310 to –440 region in the mouse promoter is homologous to a region in the human promoter containing a proximal ATF3 binding site. ANOVA revealed significant differences for treatment with –310 to –440 primers (F, F3,12 = 58.40, P < 0.0001; G, F3,15 = 523.1, P < 0.0001) and IL-6 primers (F, F3,12 = 127.0, P < 0.0001; G, F3,15 = 269.9, P < 0.0001). *P < 0.05, unpaired t test. #P < 0.05 as shown by brackets, Bonferroni post-test.
Figure 8
Figure 8. Atf3 deficiency rescued sortilin-1 expression and reduced apoB production in ob/ob mice.
(A) WT and ob/ob mice were injected with control or Atf3 siRNA, and expression of Atf3 and sortilin-1 was measured by Western blot. (B) Mice were fasted for 5 hours before TritonWR1339 and 35S-methionine injection. TG secretion was measured at the indicated times after injection. ANOVA revealed significant differences for both treatment (F6,34 = 3.103, P = 0.0156) and time point (#P < 0.05, control vs. Atf3 siRNA in ob/ob animals at 1.5 hours, Bonferroni post-test). (C) Representative autoradiogram showing plasma apoB at the 2-hour time point. (D) Quantification of relative apoB levels of WT and ob/ob mice was performed by cutting out apoB100 and apoB48 bands for each sample and counting. *P < 0.05, unpaired t test.
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