Article Figures & Data
Figures
- FIG. 1.
Conditional deletion of Arfrp1 in adipose tissue. (A) Arfrp1 mRNA levels in the indicated tissues of Arfrp1flox/flox and Arfrp1ad−/− mice at the age of 7 days were detected by quantitative RT-PCR as described in Materials and Methods. No white adipose tissue could be collected from Arfrp1ad−/− mice without contamination from adjacent tissues. Values are means + standard errors of the mean (SEM) of 9 to 12 animals per group. Ct, threshold cycle. (B) ARFRP1 protein levels of Arfrp1flox/flox and Arfrp1ad−/− mice at the age of 14 days were detected by Western blotting with an ARFRP1-specific antibody.
- FIG. 2.
Postnatal growth retardation and lack of triglyceride stores in white and brown adipocytes. (A) Photographs of Arfrp1flox/flox (+/+) and Arfrp1ad−/− (−/−) embryos (embryonic day 18.5) and mice at the age of 7 and 14 days. (B) Dorsal view of Arfrp1flox/flox and Arfrp1ad−/− littermates at the age of 14 days after removal of the skin. (C) Reduced fat mass (left) and plasma leptin (middle) levels and no alteration in adiponectin levels (right) in 14-day-old Arfrp1ad−/− mice. HM, high molecular mass; MM, medium molecular mass; LM, low molecular mass. (D) Transverse sections at the level of the neck were obtained from 2-day-old Arfrp1flox/flox and Arfrp1ad−/− littermates and stained with hematoxylin-eosin (HE). C, cutis; M, muscle.
- FIG. 3.
Expression analysis of adipocyte-specific genes in adipose tissues from Arfrp1flox/flox and Arfrp1ad−/− littermates. (A) Immunohistochemical detection of GLUT4 and FATP1 in WAT and BAT of 2-day-old Arfrp1flox/flox and Arfrp1ad−/− mice. (B) Quantitative RT-PCR for the indicated mRNAs in BAT at postnatal day 7. Values are mean ± SEM of 7 to 8 animals per group.
- FIG. 4.
Reduced size and lipid content of BAT and lower body temperature of Arfrp1ad−/− mice. (A) Size, histology, and lipid content as detected by HE and oil Red O staining of BAT of Arfrp1flox/flox and Arfrp1ad−/− mice at the age of 7 days. Bars, 50 μm. (B) Body temperature of 7-day-old mice was measured by thermographic monitoring with a noncontact thermometer (FLIR Systems i 50). Images of mice taken by the integrated camera (left). Quantification of the axillary body temperature of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice (right). Values are mean ± SEM of 7 to 8 animals per group. (C) Lysates of BAT of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice were analyzed by Western blotting with an anti-UCP1 antibody.
- FIG. 5.
Proportion of lipids in BAT of Arfrp1flox/flox and Arfrp1ad−/− mice. Total lipids were extracted from BAT of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice and triacylglycerol (TAG), diacylglycerol (DAG), free cholesterol (FC), and monoacylglcerol (MAG) levels (A), as well as the composition of fatty acids (B), were measured. Values are mean + SEM of 7 to 8 animals per group.
- FIG. 6.
Reduced size and defective fusion of lipid droplets in BAT of Arfrp1ad−/− mice. (A) Ultrastructural analysis of BAT of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice. Electron microscopic overview of ultrathin sections of BAT of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice (left) and quantification and calculation of the number and volume of LDs (right). (B) Sections of BAT analyzed by electron microscopy at higher magnifications (white arrows indicate association of electron-dense droplets with large LD; black arrows indicate membranes which might represent extensions of ER).
- FIG. 7.
Altered localization of SNAP23 in Arfrp1ad−/− mice. (A) Immunohistochemical staining of SNAP23 in BAT sections of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice (left; white arrows depict SNAP23 associated at lipid droplets). Western blots of BAT lysates stained with the anti-SNAP23 antibody. (B) Detection of SNAP23 in total membranes (P) and the cytosol (SN) of homogenized BAT of 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice by Western blotting.
- FIG. 8.
Investigation of LD proteins in Arfrp1flox/flox and Arfrp1ad−/− BAT. (A) Immunohistochemical detection of perilipin, ADRP, TIP47, and Rab18 in Arfrp1flox/flox and Arfrp1ad−/− adipocytes. Paraffin sections of BAT from 7-day-old Arfrp1flox/flox and Arfrp1ad−/− mice were stained with the indicated antibodies in combination with an Alexa-488- or an Alexa-546-conjugated secondary antibody and analyzed by confocal laser scanning microscopy. (B) Expression of LD proteins perilipin, caveolin 1 (Cav1), and caveolin 2 (Cav2) detected in lysates of BAT of 7-day-old mice by Western blotting with indicated antibodies. GAPDH was detected as loading control.
- FIG. 9.
Impaired LD formation in MEFs lacking Arfrp1. MEFs of Arfrp1+/+ and Arfrp1flox/flox embryos were infected with Cre-expressing adenovirus and incubated with oleate for 2 days. Cells were stained with an anti-ARL1 antibody in order to identify cells with deleted Arfrp1. LDs were visualized by staining with Bodipy.
- FIG. 10.
Enhanced lipolysis in Arfrp1ad−/− BAT and in 3T3-L1 adipocytes after downregulation of Arfrp1. (A) Levels of HSL, phosphorylated HSL (pHSL), and ATGL were detected by Western blotting with lysates (left) and by immunostaining with specific antibodies on sections of BAT from 7-day-old Arfrp1flox/flox and Arfrp1ad−/− animals (right). (B) Arfrp1 expression was suppressed in 3T3-L1 adipocytes by siRNA and analyzed by Western blotting with an anti-ARFRP1 specific antibody (left). Immunocytochemical detection of ATGL costained with Bodipy (right). (C) Palmitate uptake (left) was measured under basal and insulin-stimulated conditions. Lipolysis (right) was measured under basal conditions and after stimulation with isoproterenol (iso) and insulin together with isoproterenol in scrambled and si-Arfrp1transfected 3T3-L1 adipocytes (*, P = 0.00117; **, P = 0.00002).
