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. 2009 May;119(5):1201-15.
doi: 10.1172/JCI37007. Epub 2009 Apr 13.

GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice

Hélène L Kammoun  1 , Hervé Chabanon, Isabelle Hainault, Serge Luquet, Christophe Magnan, Tatsuro Koike, Pascal Ferré, Fabienne Foufelle
Affiliations

GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice

Hélène L Kammoun et al. J Clin Invest. 2009 May.

Abstract

Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis. Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c. Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents. Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice. To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector. GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes. Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice. These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation. Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes. These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

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Figures

Figure 1
Figure 1. Expression of SREBP-1c and Insig proteins, SREBP-1c target genes, and ER stress markers in ob/+ and ob/ob mouse livers.
Livers from 8-wk-old mice were used to prepare nuclear extracts, microsomes, and total RNA. (A) Immunoblot analysis of the precursor form (p-) and nuclear (n-) SREBP-1c as well as lamin A/C from ob/+ and ob/ob mice. Quantification by qRT-PCR of SREBP-1c and FAS mRNA in the liver is shown at right. (B) Immunoblot analysis of Insig-1, Insig-2, and SCAP proteins. Quantification of Insig-1 and Insig-2a (liver specific) mRNA levels by qRT-PCR is shown at right. (C) Immunoblot analysis of nuclear XBP-1 in nuclear extracts. Quantification of GRP78, ATF4, TRB3, and EDEM mRNA levels by qRT-PCR is shown at right. Results are mean ± SEM (n = 4–5 per group). **P < 0.01, ***P < 0.001 versus ob/+.
Figure 2
Figure 2. Effect of ER stress on SREBP-1c proteolytic cleavage and SREBP-1c target genes in cultured rat hepatocytes.
(A and B) Hepatocytes were incubated for 6 h in M199 medium supplemented with the LXR agonist TO-901317 (TO; 10 μM) and then treated for 1 h with 100 nM insulin (Ins), 300 nM thapsigargin (Thp), 1 μg/ml tunicamycin (Tu), 500 μM DTT, or 5 mM homocysteine (Hom). (A) Unspliced (u-) and spliced (s-) forms of XBP-1 mRNA, measured by RT-PCR. (B) Immunoblot analysis of SREBP-1c precursor and nuclear SREBP-1c. Quantification of SREBP-1c precursor is shown at right. Results are mean ± SEM of 3 independent cultures of hepatocytes in which 3 plates were collected for each condition. #P < 0.05, ##P < 0.01 versus TO-901317 alone. (C and D) Hepatocytes were treated for 1, 3, 6, and 9 h with 100 nM insulin or 300 nM thapsigargin in medium containing 25 mM glucose. (C) Total RNA from triplicate plates of hepatocytes was analyzed for the expression of GK. For the measurement of nuclear ChREBP, hepatocytes were cultured in the presence of 5 or 25 mM glucose and treated for 3 h in the presence of 100 nM insulin or 300 nM thapsigargin. (D) Total RNA from triplicate plates of hepatocytes was analyzed for expression of SCD1 and FAS mRNA. *P < 0.05, **P < 0.01, ***P < 0.001 versus basal value.
Figure 3
Figure 3. Effect of GRP78 overexpression on thapsigargin-induced ER stress and SREBP-1c proteolytic cleavage in cultured rat hepatocytes.
After a 24-h period of infection with Ad GRP78 or Ad β-gal, cultured hepatocytes were changed to a fresh M199 medium containing 10 μM TO-901317 for 6 h, then treated for 1 h with 300 nM thapsigargin or control DMSO. (A) Total RNA from triplicate plates of hepatocytes were extracted and analyzed for unspliced and spliced forms of XBP-1 mRNA by RT-PCR. The phosphorylation state of PERK in total lysates of hepatocytes was analyzed by Western blot. (B) Immunoblot of SREBP-1c in hepatic microsomal membranes and nuclear extracts and immunoblot of GRP78 measured in the microsomal fraction. Results are representative of 3 independent experiments with different preparations of hepatocytes.
Figure 4
Figure 4. Expression of cytokines and ER stress markers in the livers of ob/+ and ob/ob mice injected with Ad GRP78 or Ad β-gal.
Mice were injected with the indicated adenoviruses and sacrificed in the fed state 72 h later. (A) Total RNA was extracted and analyzed for IL-6 and TNF-α mRNA by qRT-PCR. *P < 0.05, **P < 0.01 versus respective ob/+ value; #P < 0.05 versus respective Ad β-gal value. (B) Microsomal membranes and nuclear extracts were prepared and analyzed by Western blot for the expression of precursor and nuclear ATF6, nuclear XBP-1, and microsomal GRP78. Each lane represents a different animal. Results are representative of 3 independent experiments.
Figure 5
Figure 5. SREBP-1c and ChREBP protein levels and mRNA in the livers of ob/+ and ob/ob mice overexpressing GRP78.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later for the preparation of microsomal membranes, nuclear extracts, and isolation of total RNA. (A) Analysis of SREBP-1c precursor, nuclear SREBP-1c, nuclear ChREBP, and lamin A/C by Western blot. (B) Relative levels of SREBP-1c and ChREBP mRNA measured by qRT-PCR. Results are mean ± SEM (n = 5–6 per group). **P < 0.01 versus respective ob/+ value; ##P < 0.01, ###P < 0.001 versus respective Ad β-gal value.
Figure 6
Figure 6. SREBP-1c target gene expression and Oil Red O staining in the livers of ob/+ and ob/ob mice overexpressing GRP78.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later for the preparation of total RNA. (A) Relative levels of GK, FAS, SCD1, and malic enzyme mRNA measured by qRT-PCR. Results are mean ± SEM (n = 5–6 per group). ***P < 0.001 versus respective ob/+ value; ###P < 0.001 versus respective Ad β-gal value. (B) Liver sections were stained with H&E (top row) or Oil red O (bottom row). Original magnification, ×20.
Figure 7
Figure 7. SREBP-2 and SREBP-2 target gene expression in the livers of ob/+ and ob/ob mice overexpressing GRP78.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later for the preparation of total RNA. Shown are relative levels of SREBP-2, hydroxymethylglutaryl-CoA reductase (HMG CoAR), LDL receptor (LDL-R), hydroxymethylglutaryl-CoA synthase (HMG CoAS), squalene synthase, and farnesyl 1,6 diphosphatase (Farnesyl 1,6 diP) mRNA measured by qRT-PCR. Results are mean ± SEM (n = 5–6 per group). ***P < 0.001 versus respective ob/+ value; #P < 0.05, ##P < 0.01, ###P < 0.001 versus respective Ad β-gal value.
Figure 8
Figure 8. Consequences of GRP78 overexpression on insulin signaling in the livers of ob/+ and ob/ob mice.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later. Total liver extracts and total RNA were prepared and analyzed by Western blotting and qRT-PCR, respectively. (A) Total IRS-1 protein content and relative expression of IRS-1 mRNA. (B) IRS-2 total protein content and relative mRNA expression. (C and D) Tyrosine phosphorylation of IRS-1 (C) and IRS-2 (D). Shown are a representative blot and quantification of 3 independent injection series (n = 4–5 per group). (E) Serine phosphorylation of IRS-1. Shown are a representative blot and quantification of 3 independent injection series (n = 4–5 per group). (F) Total liver extracts were analyzed for phosphorylated and total JNK. (G and H) Association of the PI3K p85 subunit with IRS-1 (G) and IRS-2 (H). Shown are a representative blot and quantification of 3 independent injection series (n = 4–5 per group). *P < 0.05, **P < 0.01, ***P < 0.001 versus respective ob/+ value; #P < 0.05, ##P < 0.01, ###P < 0.001 versus respective Ad β-gal value.
Figure 9
Figure 9. Consequences of GRP78 overexpression on PKB and FoxO phosphorylation and on gluconeogenic enzyme expression in livers of ob/+ and ob/ob mice.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later. Total liver extracts and mRNA were prepared and analyzed by Western blotting or qRT-PCR. (A) Phosphorylation of PKB on Thr308 and Ser473. Shown are representative blots and quantification of 3 independent injection series (n = 4–5 per group). (B) Total liver extracts were analyzed by Western blotting for phosphorylated and total FoxO1. Also shown is relative expression of PEPCK and G6Pase mRNA. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective ob/+ value; #P < 0.05, ##P < 0.01, ###P < 0.001 versus respective Ad β-gal value.
Figure 10
Figure 10. Consequences of GRP78 overexpression on insulin sensitivity in awake ob/+ and ob/ob mice.
(A–C) Hepatic glucose production (A), glucose utilization (B), and glucose infusion rate (GIR; C) were measured in ob/+ and ob/ob mice treated with Ad β-gal or Ad GRP78 during hyperinsulinemic-euglycemic clamps. *P < 0.05, **P < 0.01, ***P < 0.001 versus respective ob/+ value; #P < 0.05, ##P < 0.01 versus respective Ad β-gal value.
Figure 11
Figure 11. Insig-1 and Insig-2 gene expression and protein content in the livers of ob/+ and ob/ob mice overexpressing GRP78.
Mice were injected with Ad β-gal or Ad GRP78, and livers were collected in the fed state 72 h later. Total liver extracts were prepared and analyzed by Western blotting for Insig-1 and Insig-2 protein content. The graph shows relative expression of Insig-1 and Insig-2 mRNA quantified by qRT-PCR. **P < 0.01, ***P < 0.001 versus respective ob/+ value; ##P < 0.01, ###P < 0.001 versus respective Ad β-gal value.
Figure 12
Figure 12. In vitro effects of GRP78 overexpression on insulin-induced SREBP-1c cleavage in rat hepatocytes and analysis of SREBP-1c complex and GRP78 interaction in mouse livers.
(A) Immunoblot of the IR precursor and IR-β in total lysates of hepatocytes infected for 24 h with Ad GRP78 or Ad β-gal. The blot was hybridized with a GRP78 antibody to verify the overexpression of the transgene. (B) After a 24-h period of infection with Ad GRP78 or Ad β-gal, cultured hepatocytes were changed to a fresh M199 medium containing 10 μM TO-901317 for 6 h, then treated for 1 h with 300 nM thapsigargin or 100 nM insulin. Shown is immunoblot analysis of microsomal SREBP-1c precursor and nuclear SREBP-1c. (C) Microsomal membranes were isolated from the livers of fed ob/+ and ob/ob mice. Detergent-solubilized membranes were subjected to immunoprecipitation with polyclonal H160 anti–SREBP-1 antibody. Immunoprecipitated proteins were probed by Western blot with anti-GRP78 and anti–SREBP-1 or anti-calnexin as a nonrelevant microsomal antibody. An aliquot of microsomal proteins before immunoprecipitation from a pooled preparation of ob/+ mice livers was run on the gel in parallel (Input). Control immunoprecipitation with an irrelevant antibody, Glut1, was made on microsomal proteins from a pooled preparation of ob/+ mouse livers. The quantified ratio of GRP78 to SREBP-1 precursor is shown below. ***P < 0.001 versus ob/+.
Figure 13
Figure 13. Model illustrating how an initial ER stress linked to a high triglyceride synthesis rate in the liver could induce SREBP-1c cleavage and insulin resistance and initiate a vicious circle leading to steatosis and dyslipidemia.
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