Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 May 1;30(9):1034-46.
doi: 10.1101/gad.281410.116.

Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα

Ming Yan  1 , Étienne Audet-Walsh  1 , Sanaz Manteghi  1 , Catherine Rosa Dufour  2 , Benjamin Walker  1 , Masaya Baba  3 , Julie St-Pierre  1 , Vincent Giguère  4 , Arnim Pause  1
Affiliations

Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1α/ERRα

Ming Yan et al. Genes Dev. .

Abstract

The tumor suppressor folliculin (FLCN) forms a repressor complex with AMP-activated protein kinase (AMPK). Given that AMPK is a master regulator of cellular energy homeostasis, we generated an adipose-specific Flcn (Adipoq-FLCN) knockout mouse model to investigate the role of FLCN in energy metabolism. We show that loss of FLCN results in a complete metabolic reprogramming of adipose tissues, resulting in enhanced oxidative metabolism. Adipoq-FLCN knockout mice exhibit increased energy expenditure and are protected from high-fat diet (HFD)-induced obesity. Importantly, FLCN ablation leads to chronic hyperactivation of AMPK, which in turns induces and activates two key transcriptional regulators of cellular metabolism, proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα). Together, the AMPK/PGC-1α/ERRα molecular axis positively modulates the expression of metabolic genes to promote mitochondrial biogenesis and activity. In addition, mitochondrial uncoupling proteins as well as other markers of brown fat are up-regulated in both white and brown FLCN-null adipose tissues, underlying the increased resistance of Adipoq-FLCN knockout mice to cold exposure. These findings identify a key role of FLCN as a negative regulator of mitochondrial function and identify a novel molecular pathway involved in the browning of white adipocytes and the activity of brown fat.

Keywords: brown fat; high-fat diet; metabolic reprogramming; nuclear receptors; obesity; thermogenesis.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Adipose-specific Flcn deletion protects from HFD-induced obesity. (A) Western blot analysis of FLCN expression levels in iWAT (left) and BAT (right) of 5-mo-old mice. Tubulin was used as loading control. (B–I) Six-week-old wild-type or Adipoq-FLCN knockout mice were fed with either chow or a HFD. (B) Body weight gain of mice during a 14-wk period measured weekly. (C) Representative mouse images of 5-mo-old wild-type or Adipoq-FLCN knockout mice fed a HFD for 13 wk. (D) Blood glucose during a GTT in HFD-fed wild-type or Adipoq-FLCN knockout mice following a 16-h fast and intraperitoneal glucose administration of 2 g per kilogram of body weight. (E) Blood glucose during an ITT in HFD-fed wild-type or Adipoq-FLCN knockout mice following a 16-h fast and intraperitoneal insulin administration of 0.75 U per kilogram of body weight. (F–I) Metabolic cage analyses of 4-mo-old mice fed with chow or HFD and housed individually for 3 d. The VO2 consumption during a 12-h light:12-h dark cycle (F,G), mean VO2 consumption levels (H), and the respiratory exchange ratio (RER; VCO2/VO2) (I) were measured simultaneously. Data in B, D, E, H, and I are presented as mean ± SEM. n = 8–10 mice per genotype per diet. (*) P < 0.05.
Figure 2.
Figure 2.
Loss of FLCN decreases lipid accumulation and adipocyte cell size. (A) Fat index (percentage of fat pad weight relative to the whole-body weight) of iWAT, gWAT, and BAT. (B) Mean adipocyte area of adipose tissues quantified from hematoxylin and eosin (H&E)-stained tissue sections, five fields per mouse, using Metaxpress software. (C) Representative images of H&E-stained adipose tissue sections from 5-mo-old mice fed either chow or a HFD. Magnification, 20×. Bar, 100 µm. Lipid droplet production as demonstrated by Oil-Red-O staining (D) and relative units of cell size (E) measured by FACS analysis in differentiated 3T3-L1 adipocytes at day 6 after induction of differentiation. (Undiff.) Undifferentiated 3T3-L1 cells as a control. Bar, 20 µm. (F) Determination of FLCN protein levels following FLCN knockdown in 3T3-L1 adipocytes using shRNA (shFlcn-A and shFlcn-B) or control shRNA (shEV). (G) Relative mRNA levels of genes involved in lipid oxidation (Acadm and Ppara) as well as adipocyte differentiation markers (Pparg, Plin1, and AP2). Data in A and B are presented as mean ± SEM (n = 8–10 mice per genotype per diet), and the data in E and G are presented as mean ± SEM of four independent experiments performed in triplicate. (*) P < 0.05.
Figure 3.
Figure 3.
FLCN repression up-regulates an AMPK/PGC-1α/ERRα molecular axis, promoting metabolic reprogramming and WAT browning. (A,B) Western blot analysis performed with the indicated antibodies on iWAT (A) and BAT (B) of 4-mo-old mice fed either chow or a HFD. Relative expression of mRNA transcripts encoding PGC-1α (Ppargc1a) and ERRα (Esrra) (C) as well as mRNA levels of mitochondrial associated genes (D) in iWAT and BAT of wild-type and Adipoq-FLCN knockout mice are shown. (E) Adipose tissue mitochondrial content was determined by the ratio of mtDNA:nDNA. Quantitative RT–PCR (qRT–PCR) analysis of genes related to lipid metabolism (F) and thermogenesis (G) in iWAT and BAT of wild-type and Adipoq-FLCN knockout mice. (H) UCP1 protein levels in BAT of wild-type and Adipoq-FLCN-null mice fed chow or a HFD. Tubulin levels are shown as a loading control. Data in C–G are presented as mean ± SEM. n = 8–10 mice per group. (*) P < 0.05.
Figure 4.
Figure 4.
PGC-1α/ERRα expression/activity and mitochondrial respiration are induced upon loss of FLCN in an AMPK-dependent manner. (A) Western blot analysis of the indicated proteins is shown in wild-type and FLCN-null MEFs. (B) Transcript levels of Ppargc1a, Esrra, and genes related to mitochondrial activity in FLCN wild-type or knockout MEFs are shown. (C) PGC-1/ERR activity assessed using an ERRE luciferase reporter assay in MEFs expressing FLCN or not. (D) Relative fold enrichments of ERRα binding to metabolic target genes by ChIP-qPCR in wild-type or FLCN knockout MEFs. n = 3. (E) Extracellular OCR profiles following addition of the indicated mitochondrial perturbing drugs in FLCN wild-type or knockout MEFs. (Oligo) Oligomycin. (F, left) Etomoxir, an inhibitor of FAO, abolished the induced OCR levels in FLCN-null MEFs. (Right) Western blot analysis of FLCN protein levels in wild-type and FLCN knockout MEFs. β-Actin levels are shown as a loading control. (G) Extracellular OCR profiles following addition of the indicated mitochondrial perturbing drugs in FLCN wild-type or knockout MEFs treated with siRNAs against ERRα (siERRα) or control (siC). (Oligo) Oligomycin. (H, left) The increased OCR in FLCN knockout MEFs was lost when ERRα expression was inhibited. (Right) Western blot analysis of FLCN and ERRα protein levels in the wild-type or FLCN MEFs treated with or without siERRα or siC. β-Actin levels are shown as a loading control. (I) Western blot analysis of the indicated proteins are shown in wild-type and AMPKα-null MEFs treated with shRNAs against FLCN (shFlcn) or control (shEV). (J) Transcript levels of Ppargc1a, Esrra, and genes related to FAO and thermogenesis in AMPKα wild-type and knockout MEFs treated with or without shFlcn. (K) Loss of AMPKα in MEFs abolished the increased recruitment of ERRα to metabolic target genes following FLCN repression as determined by ChIP-qPCR analyses. n = 3. Data in B, C, E–H, and J are presented as mean ± SEM taken from at least three independent experiments. (*) P < 0.05.
Figure 5.
Figure 5.
FLCN ablation in adipose tissues promotes cold resistance and browning of iWAT. Three-month-old wild-type or Adipoq-FLCN knockout mice were housed for 24 h at 30°C (thermoneutrality) and then transferred to 4°C in individual precooled cages. (A) Rectal temperature recordings taken hourly during 5 h of cold exposure using a rectal digital probe (Physitemp Instruments, Inc.). (B) Relative mRNA expression of thermogenic markers determined by qRT–PCR in BAT and iWAT of FLCN wild-type or knockout mice isolated following 5 h of cold exposure. (C) Western blot analysis of UCP1 proteins levels in BAT of wild-type or FLCN knockout mice after 5 h of cold exposure. FLCN protein levels are shown, and tubulin was used as loading control. (D) Immunohistochemistry (IHC) staining with a UCP1-specific antibody (left) and H&E staining (right) in iWAT sections of cold-exposed FLCN wild-type or knockout mice. Bar, 100 µm. Data in A and B are presented as mean ± SEM. n = 6 animals pre genotype. (*) P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Audet-Walsh E, Papadopoli DJ, Gravel SP, Yee T, Bridon G, Caron M, Bourque G, Giguère V, St-Pierre J. 2016. The PGC-1α/ERRα axis represses one-carbon metabolism and promotes sensitivity to anti-folate therapy in breast cancer. Cell Rep 14: 920–931. - PubMed
    1. Baba M, Hong SB, Sharma N, Warren MB, Nickerson ML, Iwamatsu A, Esposito D, Gillette WK, Hopkins RF III, Hartley JL, et al. 2006. Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling. Proc Natl Acad Sci 103: 15552–15557. - PMC - PubMed
    1. Baba M, Furihata M, Hong SB, Tessarollo L, Haines DC, Southon E, Patel V, Igarashi P, Alvord WG, Leighty R, et al. 2008. Kidney-targeted Birt-Hogg-Dube gene inactivation in a mouse model: Erk1/2 and Akt–mTOR activation, cell hyperproliferation, and polycystic kidneys. J Natl Cancer Inst 100: 140–154. - PMC - PubMed
    1. Behrends C, Sowa ME, Gygi SP, Harper JW. 2010. Network organization of the human autophagy system. Nature 466: 68–76. - PMC - PubMed
    1. Carrier JC, Deblois G, Champigny C, Levy E, Giguère V. 2004. Estrogen-related receptor α (ERRα) is a transcriptional regulator of apolipoprotein A-IV and controls lipid handling in the intestine. J Biol Chem 279: 52052–52058. - PubMed

MeSH terms

Substances

LinkOut - more resources