Loss of <i>flcn-1</i> increases glycogen content, which mediates resistance to hyperosmotic stress.

<p>(A) Representative electron micrographs from longitudinal sections of the hypodermis in indicated nematodes strains exposed or not to 400mM NaCl for 16 hours. Arrows represent glycogen stores. Scale bars: 2μm. (B, C) Iodine staining (B) and quantification of staining intensities (C) of indicated worm strains treated or not with 400mM NaCl for 16 hours. Data represent mean ± SEM, n≥ 3. (D, E) Percent survival to 400mM NaCl of indicated worm strains treated with indicated RNAi. (F) Relative mRNA levels of indicated target genes in indicated strains with or without 400mM NaCl treatment for 2 hours. Data represent the mean ± SEM, n≥ 3.</p

Graphical representation of FLCN-1/AMPK hyperosmotic stress resistance pathway.

<p>Loss of <i>flcn-1</i> chronically activates AMPK and leads to glycogen accumulation under normal conditions. Upon exposure to hyperosmotic stress, glycogen is rapidly degraded leading to the production of glycerol and animal survival.</p

The increased survival to hyperosmotic stress and the accumulation of glycogen in <i>flcn-1</i> mutant worms require AMPK.

<p>(A-D) Percent survival of indicated worm strains exposed to 400mM NaCl. (A) <i>aak-2(ok524)</i>, (B) <i>aak-2(gt33)</i>, (C) <i>aak-1(tm1944)</i>, (D) <i>aak-1(tm1944)</i>; <i>aak-2(ok524)</i>. (E, F) Iodine staining (E) and quantification of staining intensities (F) of indicated worm strains. Scale bar:100μm. Data represent the mean ± SEM of at least 3 independent experiments.</p

Glycogen degradation heightens glycerol levels and protects animals from hyperosmotic stress.

<p>(A) Representative scheme of glycogen metabolism and osmolyte production in worms. (B-D) Relative mRNA levels of <i>gpdh-1</i> and <i>gpdh-2</i> (B, C) and glycerol content (D) in wt and <i>flcn-1(ok975)</i> L4/young adult animals treated with or without 400mM NaCl for 2 hours. Data represent mean ± SEM, n ≥3. (E) Percent survival of indicated worm strains exposed to 400mM NaCl.</p

Folliculin Regulates Ampk-Dependent Autophagy and Metabolic Stress Survival

<div><p>Dysregulation of AMPK signaling has been implicated in many human diseases, which emphasizes the importance of characterizing AMPK regulators. The tumor suppressor <i>FLCN</i>, responsible for the Birt-Hogg Dubé renal neoplasia syndrome (BHD), is an AMPK-binding partner but the genetic and functional links between FLCN and AMPK have not been established. Strikingly, the majority of naturally occurring <i>FLCN</i> mutations predisposing to BHD are predicted to produce truncated proteins unable to bind AMPK, pointing to the critical role of this interaction in the tumor suppression mechanism. Here, we demonstrate that FLCN is an evolutionarily conserved negative regulator of AMPK. Using <i>Caenorhabditis elegans</i> and mammalian cells, we show that loss of FLCN results in constitutive activation of AMPK which induces autophagy, inhibits apoptosis, improves cellular bioenergetics, and confers resistance to energy-depleting stresses including oxidative stress, heat, anoxia, and serum deprivation. We further show that AMPK activation conferred by FLCN loss is independent of the cellular energy state suggesting that FLCN controls the AMPK energy sensing ability. Together, our data suggest that FLCN is an evolutionarily conserved regulator of AMPK signaling that may act as a tumor suppressor by negatively regulating AMPK function.</p></div

The FLCN-dependent glycogen accumulation is conserved from <i>C</i>. <i>elegans</i> to humans.

<p>(A-B) PAS staining of kidney sections from wt and <i>Flcn</i> kidney-specific KO mice (A) and human BHD kidney tumor in comparison with an adjacent region from the same individual (B). Scale bars:100μm. (C) Table indicating the upregulated glycogen metabolism genes in kidney tumors (KIRC, KIRP, and KICH) as compared to normal kidney. The sign (+) indicates genes that are upregulated in these tumors. The values are indicated in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005520#pgen.1005520.s011" target="_blank">S5 Table</a>. (D) Heat map indicating correlation of expression between glycogen metabolism genes and <i>FLCN</i> in KIRC, KIRP, and KICH tumors. Green and red colors indicate genes that are negatively or positively correlated with FLCN expression, respectively.</p

Loss of <i>flcn-1</i> confers resistance to hyperosmotic stress.

<p>(A-C, F) Percent survival (A, B, F) and mean survival (C) of indicated worm strains exposed to 400mM and 500mM NaCl. (D) Percent recovery from paralysis of wt and <i>flcn-1(ok975)</i> animals after 2 hours from exposure to 400mM NaCl. Data represent mean ± SEM, n≥ 3. (E) Representative images of wt and <i>flcn-1(ok975)</i> animals treated with 400mM NaCl for 48 hours.</p

Loss of FLCN stimulates cellular energy production and resistance to energy stress.

<p>(A) Relative ATP levels measured in the indicated worm strains treated with or without PQ. (B) Percent survival of wild-type and <i>flcn-1(ok975)</i> nematodes upon heat stress (35°C). (C) Recovery rate of wild-type and <i>flcn-1(ok975)</i> strains after 26 hours anoxic injury. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s013" target="_blank">Tables S4</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s014" target="_blank">S5</a>. Data represent the mean ± SEM, n≥3.</p

Loss of <i>flcn-1</i> confers resistance to oxidative stress in <i>C. elegans</i>.

<p>(A) Amino acid alignment of the human and <i>C. elegans</i> Folliculin sequences (accession numbers: human AF517523, <i>C. elegans</i> HE963850). Identical (black) and similar (grey) amino acids are highlighted. (B) Genomic structure of <i>flcn-1</i>. The <i>ok975</i> mutation and the genomic region targeted by RNAi are indicated. (C) Western blot analysis of FLCN-1 protein levels in wild-type and <i>flcn-1(ok975)</i> worm protein lysates. (D) Lifespan of wild-type and <i>flcn-1(ok975)</i> nematodes at 20°C (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s010" target="_blank">Table S1</a>). (E-H) Percent survival of indicated worm strains treated with 4 mM or 100 mM PQ (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s011" target="_blank">Tables S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s012" target="_blank">S3</a>). (I) Percent survival of indicated worm strains treated with 7.5 mM H₂O₂.</p

The FLCN-dependent regulation of AMPK, autophagy, and metabolic stress survival is evolutionarily conserved.

<p>(A) Percent survival of wild-type, <i>Flcn</i>^−/− and FLCN-rescued MEFs (resc.) upon serum starvation (-FBS). (B) Western blot analysis of pAMPK (Thr172) and AMPK protein levels in indicated MEFs lines. (C) Percent survival of the indicated MEF cell lines upon serum starvation. Data represent the means ± SEM, n≥3. (D and E) Representative immunofluorescence pictures (D) and quantification (E) of LC3 positive GFP puncta (arrows) in wild-type or <i>Flcn</i>^−/− MEFs under basal or 24 hours serum starvation conditions (-FBS). When indicated, cells were pretreated with chloroquine (CQ) 12 hours prior to serum starvation, N>200 cells for every trial. (F) Percent survival of indicated cell lines upon serum starvation, treated with or without 10 µM CQ. Data represent the mean ± SEM, n≥3. (G) Relative ATP levels measured in the indicated MEFs lines, pre-treated with or without 10 µM CQ prior to serum starvation. (H) Graphical model that summarizes findings of this study.</p