Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 4: FAIM deficiency suppressed the effects of PACAP on alleviating hepatic lipid accumulation in HFD-fed mice. (A) FAIM protein level of mice liver was subjected to western blot. Statistical analysis of FAIM/b-actin was shown in a bar chart (right) (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CD group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. HFD group). (B) Schematic diagram of HFD mouse model with PACAP treatment (0.4 mg/kg) or control saline solution and lentivirus mediated Faim knockdown. (C) Hepatic Faim mRNA level was determined by qRT-PCR, n ¼ 3. (DeH) Modulation of Faim level in the liver influenced HFD-induced hepatic steatosis, (D) bodyweight, liver weight, and WAT weight; (E) liver TG and TC contents; (F) plasma TG, TC, HDL-c, and LDL-c levels; (G) fasting blood glucose level; blood glucose concentrations were measured on day 12 after lentivirus injection; (H) representative photomicrographs of Oil Red O and H&E staining of liver tissues in different groups (scale bars: 50 or 200 mM). (I and J) Ablation of Faim impaired glucose tolerance and insulin sensitivity in mice. IPGTT (I) and IPITT (J), blood glucose was measured at different time points after glucose or insulin injection (left), and quantification of the AUC (right). Data are presented as mean SEM, n ¼ 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Western Blot, Control, Saline, Knockdown, Quantitative RT-PCR, Clinical Proteomics, Injection, Staining

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 5: PACAP activates the FAIM-AMPK-IRb axis to govern the SREBP and lipid synthetic gene program. (A and B) PACAP activated AMPK-IRb signaling pathway and down- regulated the expression of lipogenesis genes in HFD-fed mice, such effects were suppressed following knockdown of Faim. Western blot analysis were used to detect the protein level of indicated antibodies. (C and D) The overexpression of FAIM activated AMPK-IRb signaling pathway, the protein levels of FAIM, p-AMPK, AMPK, p-IRb, IRb, and lipid synthesis genes SREBP1, SREBP2, FAS, SCD1, HMGCR were determined by western blot with quantifications. Data are presented as mean SEM, n ¼ 3 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Knockdown, Western Blot, Over Expression

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 6: Knockdown of Faim inhibited the effects of PACAP on decreasing lipid accumulation and activating AMPK-IRb signaling pathway. AML12 cells were transiently transfected with Faim siRNA or scramble siRNA in cell culture medium containing 200 mM PA or 10% BSA (as control of PA solution) for 24 h, then the cells were treated with PACAP (1 mM) and Max.D.4 (10 mM) for another 24 h as indicated. (A) Representative for Oil Red O staining of AML12 (Scale bars, 50 mM). (B and C) Impacts of Faim knockdown on cellular TG and TC level (B), glucose uptake and glycogen content (C) of AML12. (D and E) Faim knockdown impaired the activation of AMPK-IRb pathway in AML12 treated with PACAP, the protein levels were analyzed by western blot and quantitative measurements (right). (F and G) AML12 were exposed to PA for 24 h, then treated with PACAP (1 mM), Max.D.4 (10 mM) and Compound C (10 mM) (F) or AICAR (0.5 mM) (G) for another 24 h. The protein level of FAIM was detected by western blot analysis and quantitative measurements (down). Data are presented as mean SEM. n ¼ 3 (&P < 0.05, &&P < 0.01, &&&P < 0.001 vs. control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PA þ PACAP group, OP < 0.05, OOP < 0.01, OOOP < 0.001 vs. PA þ PACAP þ Max.D.4 group).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Knockdown, Transfection, Cell Culture, Control, Staining, Activation Assay, Western Blot

Journal: Molecular metabolism

Article Title: PACAP attenuates hepatic lipid accumulation through the FAIM/AMPK/IRβ axis during overnutrition.

doi: 10.1016/j.molmet.2022.101584

Figure Lengend Snippet: Figure 7: PACAP activates the PAC1-PKA-CREB signaling pathway to stimulate FAIM expression. (A) mRNA level of Faim in PA-induced AML12 treated with PACAP, determined by qRT-PCR (n ¼ 3). (B) The effect of PACAP on the activation of PKA and CREB in PA induced AML12 cells, the protein levels of PKA, CREB and FAIM, and phosphorylation of PKA and CREB were determined by western blot. (*P < 0.05, **P < 0.01, ***P < 0.001 vs. PA group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. PACAP (1 mM) group). (C) Schematic of the sequence of two CRE binding sites that CREB targets the WT or mutated promoter region of Faim mRNA. (DeF) Luciferase activities in AML12 transiently transfected with either wild type (WT), truncated, or mutant Faim promoter-luciferase reporter constructs. Then the cells were treated with PACAP (1 mM), or co- transfected with CREB cDNA or empty vectors. (G) Luciferase activities of FAIM WT reporter plasmids in AML12 cells with the treatment of KG-501 (a specific CREB inhibitor) and PACAP (1 mM) as indicated. Data are shown as mean SEM, n ¼ 3 or 5 (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: In CREB inhibitor experiment, AML12 were transfected with FAIM reporter plasmids for 24 h and treated with 1 mM PACAP for an additional 24 h in the presence or absence of the CREB inhibitor KG-501 (S8409, 18228-17- 6, Selleck Chemicals, USA).

Techniques: Expressing, Quantitative RT-PCR, Activation Assay, Phospho-proteomics, Western Blot, Sequencing, Binding Assay, Luciferase, Transfection, Mutagenesis, Construct