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. 2016 Jun 29;36(14):1961-76.
doi: 10.1128/MCB.00244-16. Print 2016 Jul 15.

AMPK Phosphorylates Desnutrin/ATGL and Hormone-Sensitive Lipase To Regulate Lipolysis and Fatty Acid Oxidation Within Adipose Tissue

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AMPK Phosphorylates Desnutrin/ATGL and Hormone-Sensitive Lipase To Regulate Lipolysis and Fatty Acid Oxidation Within Adipose Tissue

Sun-Joong Kim et al. Mol Cell Biol. .
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Abstract

The role of AMP-activated protein kinase (AMPK) in promoting fatty acid (FA) oxidation in various tissues, such as liver and muscle, has been well understood. However, the role of AMPK in lipolysis and FA metabolism in adipose tissue has been controversial. To investigate the role of AMPK in the regulation of adipose lipolysis in vivo, we generated mice with adipose-tissue-specific knockout of both the α1 and α2 catalytic subunits of AMPK (AMPK-ASKO mice) by using aP2-Cre and adiponectin-Cre. Both models of AMPK-ASKO ablation show no changes in desnutrin/ATGL levels but have defective phosphorylation of desnutrin/ATGL at S406 to decrease its triacylglycerol (TAG) hydrolase activity, lowering basal lipolysis in adipose tissue. These mice also show defective phosphorylation of hormone-sensitive lipase (HSL) at S565, with higher phosphorylation at protein kinase A sites S563 and S660, increasing its hydrolase activity and isoproterenol-stimulated lipolysis. With higher overall adipose lipolysis, both models of AMPK-ASKO mice are lean, having smaller adipocytes with lower TAG and higher intracellular free-FA levels. Moreover, FAs from higher lipolysis activate peroxisome proliferator-activated receptor delta to induce FA oxidative genes and increase FA oxidation and energy expenditure. Overall, for the first time, we provide in vivo evidence of the role of AMPK in the phosphorylation and regulation of desnutrin/ATGL and HSL and thus adipose lipolysis.

Figures

FIG 1
FIG 1
Generation of aP2 and Adn-AMPKα1/α2 ASKO mice. (A) RT-qPCR for AMPKα1 and AMPKα2 expression in the SVFs and adipocytes of wild-type mouse WAT (n = 4). (B) Cre-Lox strategy for removal of AMPKα1 (Prkaa1) and -α2 (Prkaa2) from adipose tissue. Open boxes, untranslated regions; gray boxes, translated regions that do not encode the kinase domain; black boxes, translated regions that encode the respective kinase domain; triangles, loxP sites; arrows, primer sets for genotyping (top). RT-qPCR for AMPKα1 (left) and AMPKα2 (right) expression in various tissues from aP2-ASKO (a) and Adn-ASKO (b) mice and control Flox/Flox mice (n = 4). (C) Immunoblotting and its quantification of lysates from various adipose depots, gonadal (Gon), inguinal (Ing), renal (Reno), and liver, from aP2-ASKO (a) and Adn-ASKO (b) mice with AMPKα, AMPKα1, and AMPKα2 antibodies. GAPDH was used as an internal control (n = 4). Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; NS, no significant change. Experiments were repeated twice, and representative data are shown.
FIG 2
FIG 2
AMPK phosphorylates desnutrin/ATGL and HSL. (A) Immunoblotting and its quantification of WAT lysates with antibodies against phosphorylated desnutrin/ATGL (P-S406) and total desnutrin/ATGL in aP2-ASKO (a) and Adn-ASKO (b) mice. WAT were harvested under fasted conditions (9 a.m.). (B) Immunoblotting and its quantification of WAT lysates with antibodies against HSL and phospho-HSL (P-S565, P-S660, and P-S563) in aP2-ASKO (a) and Adn-ASKO (b) mice (n = 4). (C and D) Immunoblotting and its quantification of desnutrin/ATGL and its phosphorylation (C) and HSL and its phosphorylation (D) in WAT lysates under basal or isoproterenol (Iso)-stimulated conditions (n = 4). Experiments were repeated twice, and a representative blot is shown. *, P < 0.05; **, P < 0.01.
FIG 3
FIG 3
Effect of AMPK on lipase activities. (A) Fluorescent-lipase assay with pyrene-labeled TAG (1,3-dioleolyl-3-(pyren-1-yl) decanoyl-rac-glycerol) (a, left) and lipase activities with [carboxyl-14C]triolein (a, middle) and [1-14C]diolein (1,3-dioleoylglycerol [1-14C]oleoyl) (a, left) as substrates for WAT lysates from aP2-ASKO mice (a) and lipase activity with [14C]triolein as a substrate for WAT lysates from Adn-ASKO mice (b) (n = 4). (B) Lipase activities with a [14C]triolein substrate in the presence of 10 μM HSL inhibitor CAY10499 in aP2-ASKO (a) or Adn-ASKO (b) mouse WAT. (C) Lipase activities with [14C]triolein in the presence of the desnutrin/ATGL inhibitor atglistatin at 50 μM in aP2-ASKO (a) or Adn-ASKO (b) mouse WAT. Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01. Experiments were repeated twice, and representative data are shown.
FIG 4
FIG 4
AMPK induces basal lipolysis and inhibits stimulated lipolysis. (A) FFA release from WAT of aP2-ASKO (a) and Adn-ASKO (b) mice under basal and isoproterenol-stimulated conditions. Cellular AMP/ATP ratios in WAT of Flox/Flox and Adn-ASKO mice under basal conditions (c, left) and immunoblotting for AMPKα and AMPKα phosphorylated at T172 (c, right). (B) Immunoblotting with HSL antibody after immunoprecipitation (IP) of WAT lysates with perilipin antibody. (C) FFA release from WAT of aP2-ASKO (a) and Adn-ASKO (b) mice upon treatment with 2 mM A-769662 or 50 μM compound C under basal or stimulated conditions (n = 5). (D) FFA release from isolated adipocytes of aP2-ASKO mice upon treatment with 2 mM A-769662 or 50 μM compound C under basal and stimulated conditions (n = 4). (E) FFA release from Adn-ASKO mouse WAT explants upon treatment with 50 μM atglistatin or 10 μM CAY10499 under basal and isoproterenol-stimulated conditions. Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; NS, no significant change. Experiments were repeated twice, and representative data are shown.
FIG 5
FIG 5
AMPK-ASKO mice show a lean phenotype. (A) BW gain, food intake, lean mass, and fat mass gain of aP2-ASKO (a) and Adn-ASKO (b) mice (n = 8) fed a standard chow diet. (B) Fat pad and liver weights of aP2-ASKO (a) and Adn-ASKO (b, right side) mice (n = 8). Data from male Adn-ASKO mice on a chow diet at 16 weeks of age (b, left side) and gonadal (Gon), inguinal (Ing), renal (Reno), and liver samples from these mice (b, middle) are also shown. (C) Hematoxylin-and-eosin staining of gonadal fat pads (left) and quantification of cell size (right, n = 3). Scale bar = 20 μm. (D) TAG contents (left), relative levels of DAG (middle), and FFA levels (right). The levels in Flox/Flox mice were defined as 100% (middle and right, n = 4). (E) Serum FFA levels in aP2-ASKO (a) and Adn-ASKO (b) mice (n = 4). (F, a) RT-qPCR for lipogenic gene expression in WAT (n = 4). (F, b) Immunoblotting of WAT lysates with antibodies to ACC, phosphorylated ACC (P-S79ACC), and AMPKα. GAPDH was used as an internal control. (F, c) Lipids generated were measured by counting [14C]TAG after incubation of gonadal WAT fragments (with or without treatment with insulin at 1 μg/ml for 15 min) with d-[6-14C]glucose for 2 h. Lipids were separated by TLC. Experiments were repeated twice, and representative data are shown. Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; NS, no significant change.
FIG 6
FIG 6
AMPKα1/α2 ablation promotes FA oxidation in adipose tissue. (A) O2 consumption rate (VO2) determined by indirect calorimetry in 16-week-old mice on a chow diet (left and middle; n = 6). The RER is shown on the right. (B) FA oxidation was measured by determining captured CO2 and ASMs after the incubation of lysates with [14C]palmitate for aP2-ASKO (a) and Adn-ASKO (b) mouse WAT, Adn-ASKO mouse liver (c), and Adn-ASKO mouse muscle (d). (n = 4). (C) RT-qPCR for FA oxidative genes in aP2-ASKO (a) and Adn-ASKO (b) mouse WAT and aP2-ASKO (c) and Adn-ASKO (d) mouse liver (n = 4). Data are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; NS, no significant change.
FIG 7
FIG 7
AMPKα1/α2 ablation promotes FA oxidation through PPARδ. (A) RT-qPCR for PPARα and PPARδ expression in WAT (a, left). PPARα and PPARδ binding activities in nuclear extracts from aP2-ASKO (a, right) and Adn-ASKO (b) mice are shown. (B) PPARα and PPARδ binding activities in WAT after GSK3787 injection for 3 days in aP2-ASKO (a) and Adn-ASKO (b) mice (n = 5). (C) Expression of FA oxidative genes in GSK3787-treated mice (n = 5) (a and b). (D) PPARδ binding activity (absorbance at 450 nm) and Cox8b (2−ΔΔCT) mRNA levels from Adn-ASKO mouse WAT explants upon treatment with 50 μM atglistatin (n = 4) or 10 μM CAY10499 (n = 4). Data are expressed as means ± SEM. *, P < 0.05; **P < 0.01; NS, no significant change. Experiments were repeated twice. (E) Function of AMPK in adipose lipolysis.

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