Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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/+.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Western Blot, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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 differ- ent preparations of hepatocytes.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Over Expression, Cell Culture, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Phospho-proteomics, Western Blot

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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 ani- mal. Results are representative of 3 independent experiments.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Expressing, Injection, Quantitative RT-PCR, Western Blot

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Injection, Isolation, Western Blot, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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 respec- tive 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.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Targeted Gene Expression, Staining, Injection, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Targeted Gene Expression, Injection, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: Figure 10 Consequences of GRP78 overexpression on insulin sensitivity in awake ob/+ and ob/ob mice. (A–C) Hepatic glucose production (A), glucose utili- zation (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.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Over Expression

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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 rela- tive 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.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: Gene Expression, Injection, Western Blot, Expressing, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

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

doi: 10.1172/jci37007

Figure Lengend Snippet: 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 liv- ers. (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, cul- tured 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 precur- sor 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 irrel- evant antibody, Glut1, was made on microsomal proteins from a pooled preparation of ob/+ mouse livers. The quanti- fied ratio of GRP78 to SREBP-1 precursor is shown below. ***P < 0.001 versus ob/+.

Article Snippet: The membranes were hybridized with the following antibodies: SREBP-1 (clone 2A4; Thermo Scientific), ATF6 (Imgenex), ChREBP (Novus Biological), XBP-1 (Prosci), and GRP78 and Insig-1 (Santa Cruz Biotechnology Inc.).

Techniques: In Vitro, Over Expression, Western Blot, Infection, Isolation, Immunoprecipitation, Control