Journal: Nature Communications

Article Title: Hepatic Crtc2 controls whole body energy metabolism via a miR-34a-Fgf21 axis

doi: 10.1038/s41467-017-01878-6

Figure Lengend Snippet: Hepatic miR-34a is critical in reducing Sirt1-Pparα-mediated expression of Fgf21 in a Creb/Crtc2-dependent manner. a Effects of hepatic Crtc2 knockout on protein levels of Atf4, Foxo1, and Pparα in the livers of 16 h-fasted, 9-week HFD-fed mice. b Effects of hepatic Crtc2 knockout on mature microRNA expression in the livers of 16 h-fasted, 9-week HFD-fed mice (left) (Q-PCR, n = 4 mice per group). Levels of AGO2-associated miR-34a were also shown (right) (Q-PCR, n = 4 mice per group). c Effects of hepatic Crtc2 knockout on protein levels in the livers of 16 h fasted, 9-week HFD-fed mice. Quantitation of protein levels of each condition was shown at right. d , e Effects of expression of constitutively active form of Crtc2 (S171A) on hepatic genes. 8-week old C57BL/6 mice were injected with adenovirus for 5 days before being killed after 24 h fasting. Hepatic miR-34a levels ( d , left), hepatic expression of Pparα target genes ( d , right), hepatic protein levels ( e , left), and quantitation of protein levels ( e , right) from C57BL/6 mice that were infected with Ad-GFP or Ad-Crtc2 S171A adenovirus ( n = 5 mice per group). f 5′- and 3′-deletion analysis was performed to map the putative Creb/Crtc2 response element on the promoter of mouse miR-34a (top). Luciferase reporter assay was performed in 293T cells to determine the effects of Creb/Crtc2 on miR-34a promoter activity (bottom). N = 3 independent experiments in triplicate. g Location of putative CREs on the mouse miR-34a promoter (top), the effects of CRE mutations on the mouse miR-34a promoter activity (left), and the chromatin immunoprecipitation assay showing the occupancy of Creb/Crtc2 over mouse miR-34a promoter (right) were shown. N = 3 independent experiments in triplicate. Note that the HFD was initiated at 4 weeks of age and maintained throughout the experimental period ( a – c ). Data in b – g represent mean ± s.d. (* P < 0.05, ** P < 0.01, t -test)

Article Snippet: Human miR-34a promoter was excised from the pGL3-PMT-miR-34a plasmid (a gift from Judy Lieberman (Addgene plasmid #25799) , and was subcloned into the pGL4 vector to generate the human full-length miR-34a luciferase plasmid.

Techniques: Expressing, Knock-Out, Quantitation Assay, Injection, Infection, Luciferase, Reporter Assay, Activity Assay, Chromatin Immunoprecipitation

Journal: Nature Communications

Article Title: Hepatic Crtc2 controls whole body energy metabolism via a miR-34a-Fgf21 axis

doi: 10.1038/s41467-017-01878-6

Figure Lengend Snippet: Effect of miR-34a on adiposity is reversed by co-expression of Fgf21 in Crtc2 liver-specific knockout mice. a – d Effects of miR-34a and/or Fgf21 on Crtc2 LKO mice under 9-week HFD ( n = 5 mice per group). Schematics of AAV injection ( a , top) and the effect of miR-34a and Fgf21 on fat size ( a , bottom) were shown. H&E staining of the liver, scWAT, visWAT, BAT of each group of mice were shown ( b ). c Western blot analysis showing hepatic protein levels, as well as Ucp1 protein levels in scWAT and BAT of each genotype. Data represent four independent experiments ( n = 4 mice per group). Note that the HFD was initiated at 4 weeks of age and maintained throughout the experimental period. d Schematic diagram showing the effects of Creb/Crtc2-driven expression of miR-34a on hepatic fatty acid metabolism and whole-body energy homeostasis under diet-induced obesity (DIO) and insulin-resistant conditions. Depletion of Crtc2 promotes the activation of Sirt1/Pparα pathway under DIO conditions, leading to the increased hepatic fatty acid beta oxidation and the amelioration of fatty liver symptoms. In addition, enhanced hepatic Fgf21 expression is also essential in improving lipid metabolism in the peripheral tissues including the liver and adipose tissues, resulting in the increased energy expenditure and the improved insulin sensitivity

Article Snippet: Human miR-34a promoter was excised from the pGL3-PMT-miR-34a plasmid (a gift from Judy Lieberman (Addgene plasmid #25799) , and was subcloned into the pGL4 vector to generate the human full-length miR-34a luciferase plasmid.

Techniques: Expressing, Knock-Out, Injection, Staining, Western Blot, Activation Assay