Fatty Acid Transport Protein 1 Is Required for Nonshivering Thermogenesis in Brown Adipose Tissue
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Figures
- FIG. 1.
A: The effect of cold treatment on the expression of FATPs in brown fat. Mice were exposed to 4°C for 12 h before their BATs were taken. a, no cold-exposed BAT; b, cold-exposed BAT; c, muscle lysate control; d, liver lysate control; e, lung lysate control; f, heart lysate control. B: Cold-induced gene expression of FATP1 and UCP-1 expression in brown fat tissues. Expression level was analyzed by densitometry and normalized to the expression of β-tublin.
- FIG. 2.
A: Localization of FATP1 in brown fat. Frozen brown fat sections were paraformaldehyde fixed and incubated with anti-FATP1 antibody, followed by Cy5-tagged goat anti-rabbit secondary antibody, and analyzed by immunofluorescence microscopy. B: Three-dimensional surface projects of FATP1 (red), integrin α6 (green), as well as their superimposition (right panels) of 15-μm sections of BAT from wild-type (WT) mice at 4°C. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylinodole) (blue).
- FIG. 3.
Histology of brown fat shows lipid accumulation in adipocytes. A: Interscapular brown fat was removed from 8-week-old FATP1 KO and wild-type (WT) littermates. Paraffin-embedded sections were stained with hematoxylin and eosin. B: Three-dimensional reconstructions of frozen BAT section from FATP1 wild-type and KO mice stained with BODIPY fluorophore 493/503 for neutral lipids (yellow) and DAPI for nuclei (blue). One grid corresponds to 23 μmol/l. C: Uptake of a fluorescently labeled long-chain fatty acid by primary brown adipocytes from FATP1 wild-type and KO animals following cold or 0.5 mg/kg body wt of norepinephrine (NE) treatment. D: Western blot analysis of FATP1 and UCP-1. The expression of the UCP-1 increase after exposure to cold in both wild-type and FATP1-null BAT pads. Level of β-tublin (Tub) in BAT pads were used as a loading control.
- FIG. 4.
FATP-null mice are unable to maintain core body temperature during fasting and cold exposure. A: Colonic temperature was recorded at 1, 3, 6, 12, and 24 h of fasting mice in cold treatment; right panel is the reading of fed mice in cold treatment. B: Levels of serum nonesterified fatty acids in both wild-type (WT) and FATP1-null mice in response to the cold exposure. C: Indirect calorimetry comparing the increases in O2 consumption and CO2 production rates during a 1-h phase, following injection of the β3-AR agonist CL316,243 in FATP1 KO mice (□) and wild-type littermates (▪). *P < 0.05 in paired Student’s t test.
- FIG. 5.
Effect of isoproterenol on long-chain fatty acid uptake. A: Expression of FATP1 and UCP-1 in cultured cells. Fibroblasts 3T3-L1 (black bar), HIB-1B preadipocytes (gray bar), HIB-1B adipocytes (green bar), and HIB-1B adipocytes + isoproterenol are indicated. Isotproterenol (10 μmol/l) was added into the medium 1 h before harvesting the cells for Western blot. Level of expression was analyzed by densitometry and normalized to the expression of β-tublin. B: Comparison of kinetic readings of long-chain fatty acid uptake by HIB adipocytes. □, untreated cells; ♦, 10 μmol/l isoproterenol treated cells. C: Effects of duration and dose of isopreterenol stimulus on initial uptake of long-chain fatty acid in HIB adipocytes. D: Fatty acid uptake 1 h after a 10 μmol/l isoproterenol stimulation and FATP1 expression. /, control; GP, Gs inhibitor (2 nmol/l pertussis toxin); MEK, MEK II inhibitor (10 nmol/l U0126); PKA, PKA inhibitor (10 nmol/l H89); TC, transcription inhibitor (1 μmol/l α-amanitin); TL, translation inhibitor (1 μmol/l NSC119889).
- FIG. 6.
Effects of FATP1 RNA interference (RNAi) on FATP1 expression and fatty acid uptake in HIB-1B cells. Upper panel: Expression of FATP1 was analyzed by Western blot analysis in HIB, HIB-SCR (scrambled RNAi), and HIB-1.5 (FATP1 RNAi) cell lines. These cells were incubated with solvent (basal), 1 nmol/l insulin, 50 nmol/l insulin, 100 nmol/l insulin (ins), 10 μmol/l isoproterenol (iso), and both (iso+ins) for 1 h. Lower panel: Stimulation of adrenergic agonist and insulin induce increased uptake fatty acid in HIB cells, and this effect was suppressed by RNA interference in the stable cell line of FATP1 RNAi. HIB cell lines were incubated in the presence of insulin, isoproterenol, or both at indicated concentration for 1 h and then assayed for fatty acid transport into the cells as described in research design and methods. Data are representative of three independent experiments.
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