Metformin suppresses glucose- and glutamine-dependent TCA cycle activity.

<p><b>A.</b> Relative abundance of TCA metabolites in metformin-treated H1299 cells. Cells were treated with or without metformin (5 mM) for 8 h, and TCA cycle metabolites determined by gas chromatography-mass spectrometry (GC-MS). Data are expressed as the ratio of metabolite levels in metformin-treated cells relative to cells cultured without metformin. Data shown is normalized to cell number. The data represent the mean ± SEM for triplicate samples. <b>B.</b> Heat map of relative metabolite abundances in metformin-treated H1299 cells. H1299 cells were treated with (+) or without (−) 5 mM metformin for 6 h, followed by culture with U-[¹³C]-glucose or U-[¹³C]-glutamine for an additional 2 h. Shown is the relative abundance of U-[¹³C]-glucose-derived (left panel) or U-[¹³C]-glutamine-derived (right panel) TCA cycle metabolites under each culture condition. Data are expressed relative to the ¹³C metabolite abundance in H1299 cells cultured under control conditions (no metformin). <b>C.</b> Relative abundance of glucose-derived citrate in metformin-treated H1299 cells. Cells were treated for 24 h with the indicated doses of metformin followed by incubation with U-[¹³C]-glucose for 2 h. The abundance of unlabeled (¹²C, white) and U-[¹³C]-glucose-labeled (¹³C, black) citrate was determined by GC-MS. Data are normalized to cell number. <b>D.</b> Schematic of U-[¹³C]-glucose labeling in the TCA cycle. Input of fully-labeled Ac-CoA (m + 2) results in the generation of m + 2-labeled metabolites on the first turn of the TCA cycle, and m + 4-labeled metabolites on the second turn. <b>E.</b> Distribution of U-[¹³C]-glucose-derived isotopomers of citrate in H1299 cells cultured with or without metformin as in (<b>B</b>). The data represent the mean ± SEM for triplicate samples. <b>F.</b> Relative abundance of glutamine-derived citrate in metformin-treated H1299 cells. Cells were treated as in (<b>B</b>), and the abundance of unlabeled (¹²C, white) and U-[¹³C]-glutamine-labeled (¹³C, black) citrate was determined by GC-MS. Data are normalized to cell number. <b>G.</b> Schematic of U-[¹³C]-glutamine labeling in the TCA cycle. Anaplerotic U-[¹³C]-glutamine flux into the TCA cycle follows clockwise flow resulting in m + 4 labeling during the first round of the TCA cycle. Reductive carboxylation of α-KG results in m + 5 labeling in citrate. <b>H.</b> Distribution of U-[¹³C]-glutamine-derived isotopomers of citrate in H1299 cells cultured with or without metformin as in (<b>B</b>). The data represent the mean ± SEM for triplicate samples. <b>I.</b> Relative abundance of citrate produced via oxidative (m + 4) and reductive (m + 5) pathways in H1299 cells treated with (+) or without (−) metformin. Cells were treated as in (<b>B</b>), and the abundance of U-[¹³C]-glutamine-labeled m+4 and m+5 citrate was determined by GC-MS. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01; ***, <i>p</i> < 0.001. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s003" target="_blank">S3 Data</a>.</p

Cancer cells with defective ETC activity display resistance to the antiproliferative effects of metformin.

<p><b>A.</b> Proliferation of 143B<i>wt</i> and 143B<i>cytb</i> cells cultured in medium containing the indicated doses of metformin. Cell numbers were determined after 72 h of treatment, and are expressed relative to cell counts in control conditions (0 mM metformin). Each data point represents the mean ± SEM for each condition (<i>n</i> = 10). <b>B.</b> Proliferation of 143B<i>wt</i> and 143B<i>cytb</i> cells treated with control siRNA (CTL) or ACL-specific siRNA. Cell counts were determined after 48 h of culture in medium containing 10 mM metformin and expressed relative to cell counts in control conditions. The data represent the mean ± SEM for each condition (<i>n</i> = 10) and are representative of three independent experiments. <b>C.</b> U-[¹³C]-acetate-derived lipogenic acetyl-CoA in 143B<i>wt</i> cells with or without 5 mM metformin treatment for 72 h. <b>D.</b> Proliferation of 143B<i>wt</i> cells cultured in the absence or presence of 5 mM metformin under control (white) or acetate supplementation (black, 5 mM) conditions. Growth curves over time are shown. Each data point represents the mean ± SD for each condition (<i>n</i> = 10), and is representative of three independent experiments. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s006" target="_blank">S6 Data</a>.</p

Metformin exerts AMPK-independent effects on cancer cell metabolism.

<p><b>A.</b> Proliferation of H1299 NSCLC cells treated with the indicated concentrations of metformin for 72 h. Cell numbers are expressed relative to cell counts in control conditions (0 mM metformin). Each data point represents the mean ± standard error of the mean (SEM) for triplicate samples. <b>B.</b> Immunoblot of AMPK activation in metformin-treated H1299 cells. H1299 cells were treated for 1 h with various doses of metformin (from left to right: 0, 2.5, 5, and 10 mM), and cell lysates analyzed for AMPK (total and pT172), ACCα (total and pS79), and Raptor (total and pS792) levels. <b>C.</b> ATP:ADP ratio of H1299 cells cultured with varying doses of metformin for 14 h. Ratios are expressed relative to cells grown in complete growth medium. The data represents the mean ± standard deviation (SD) for triplicate samples. <b>D–G.</b> Metabolic characterization of metformin-treated mouse embryonic fibroblasts (MEFs). Wild type (WT) or AMPKα-deficient (knockout, KO) MEFs were cultured in the presence or absence of metformin. Shown are the O₂ consumption rate (OCR) (<b>D</b>) and extracellular acidification rate (ECAR) (<b>E</b>) of cells cultured for 24 h in the presence or absence of 10 mM metformin. Glucose consumption (<b>F</b>) and lactate production (<b>G</b>) were assessed after 48 h of culture with metformin (5 mM). All data are normalized to cell number and represent the mean ± SEM for triplicate samples per condition. The data are representative of three independent experiments. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s001" target="_blank">S1 Data</a>.</p

Metformin suppresses cancer cell proliferation independent of metabolic checkpoints.

<p>Cells were cultured with the indicated concentrations of metformin, and cell number was determined by crystal violet incorporation after 72 h of culture. Cell number is expressed relative to control cell number (no metformin) at 72 h. Shown are cell numbers for control (WT) and AMPKα-deficient (KO) MEFs (<b>A</b>), control (WT) and LKB1-deficient (KO) MEFs (<b>B</b>), A549 cells expressing empty vector (A549/Vec) or re-expressing LKB1 (A549/LKB1) (<b>C</b>), and control (WT) or 4EBP1/2-deficient (double knockout, DKO) MEFs (<b>D</b>) in response to metformin treatment. Each data point represents the mean ± SEM for triplicate samples. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s002" target="_blank">S2 Data</a>.</p

Hypoxia results in adaptive resistance to the anti-proliferative effects of metformin.

<p><b>A</b>. Proliferation of 143B<i>wt</i> cells cultured in the absence or presence of 5 mM metformin under normoxic (white) or hypoxic (black, 1% O₂) conditions. Growth curves over time are indicated. <b>B.</b> Proliferation of 143B<i>wt</i> cells cultured under normoxia (white) or hypoxia (black, 1% O₂) in medium containing the indicated doses of metformin. Cell numbers were determined after 72 h of treatment, and are expressed relative to cell counts in control conditions. Each data point represents the mean ± SEM for each condition (<i>n</i> = 10), and is representative of three independent experiments. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s007" target="_blank">S7 Data</a>.</p

Metformin requires functional mitochondrial electron transport to suppress de novo lipogenesis.

<p>A–B. Abundance and distribution of U-[¹³C]-glucose-derived citrate in metformin-treated 143B osteosarcoma cells. 143B<i>wt</i> and 143B<i>cytb</i> cells were cultured with (+) or without (−) metformin (10 mM) for 12 h, and intracellular metabolites extracted and analyzed by GC-MS. U-[¹³C]-glucose was added for the final 6 h of culture. Shown is the isotopomer distribution (<b>A</b>) and relative abundance (<b>B</b>) of U-[¹³C]-glucose-derived citrate in 143B<i>wt</i> and 143B<i>cytb</i> cells treated as indicated. Data are normalized to cell number and are presented as mean ± SEM for each condition (<i>n</i> = 3), and are representative of two independent experiments. <b>C–D.</b> Abundance and distribution of U-[¹³C]-glutamine-derived citrate in metformin-treated 143B cells. 143B Cells were treated as in (<b>A</b>), with U-[¹³C]-glutamine added for the final 6 h of culture. Isotopomer distribution (<b>C</b>) and relative abundance (<b>D</b>) of U-[¹³C]-glutamine-derived citrate in 143B<i>wt</i> and 143B<i>cytb</i> cells is shown. Data are normalized to cell number. <b>E</b>. Relative palmitate abundance in 143B<i>wt</i> and 143B<i>cytb</i> cells cultured with (+) or without (−) metformin (10 mM) for 72 h. Data are normalized to cell number and presented as mean ± SEM for each condition (<i>n</i> = 3). <b>F–G</b>. Relative abundance of U-[¹³C]-glucose-derived (<b>F</b>) and U-[¹³C]-glutamine-derived (<b>G</b>) lipogenic acetyl-CoA and palmitate in 143B<i>wt</i> and 143B<i>cytb</i> cells cultured in the presence (+) or absence (−) of metformin (10 mM) for 72 h. Cells were cultured for 72 h, with U-[¹³C]-glucose or U-[¹³C]-glutamine added for the final 24 h of culture. Data are normalized to cell number, are presented as mean ± SEM for triplicate samples, and are representative of three independent experiments. *, <i>p</i> < 0.05; **, <i>p</i> < 0.01. Raw data for this figure can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002309#pbio.1002309.s005" target="_blank">S5 Data</a>.</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

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 an <i>aak-2</i>-dependent resistance to oxidative stress.

<p>(A, B, C, D) Percent survival of indicated worm strains treated with 4 mM or 100 mM PQ. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s011" target="_blank">Tables S2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004273#pgen.1004273.s012" target="_blank">S3</a>. (E) Western blot analysis of pAAK-2 (Thr234) protein levels in indicated worm strains. Levels were normalized to Tubulin. (F) Fold change in pAAK-2 levels in <i>flcn-1(ok975);par-4(it57)</i> and <i>par-4(it57)</i> animals. Data represent the means ± SEM, n≥3.</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