Itaconate Links Inhibition of Succinate Dehydrogenase with Macrophage Metabolic Remodeling and Regulation of Inflammation

Remodeling of the tricarboxylic acid (TCA) cycle is a metabolic adaptation accompanying inflammatory macrophage activation. During this process, endogenous metabolites can adopt regulatory roles that govern specific aspects of inflammatory response, as recently shown for succinate, which regulates the pro-inflammatory IL-1β-HIF-1α axis. Itaconate is one of the most highly induced metabolites in activated macrophages, yet its functional significance remains unknown. Here, we show that itaconate modulates macrophage metabolism and effector functions by inhibiting succinate dehydrogenase-mediated oxidation of succinate. Through this action, itaconate exerts anti-inflammatory effects when administered in vitro and in vivo during macrophage activation and ischemia-reperfusion injury. Using newly generated Irg1(−/−) mice, which lack the ability to produce itaconate, we show that endogenous itaconate regulates succinate levels and function, mitochondrial respiration, and inflammatory cytokine production during macrophage activation. These studies highlight itaconate as a major physiological regulator of the global metabolic rewiring and effector functions of inflammatory macrophages

Metformin Antagonizes Cancer Cell Proliferation by Suppressing Mitochondrial-Dependent Biosynthesis.

Metformin is a biguanide widely prescribed to treat Type II diabetes that has gained interest as an antineoplastic agent. Recent work suggests that metformin directly antagonizes cancer cell growth through its actions on complex I of the mitochondrial electron transport chain (ETC). However, the mechanisms by which metformin arrests cancer cell proliferation remain poorly defined. Here we demonstrate that the metabolic checkpoint kinases AMP-activated protein kinase (AMPK) and LKB1 are not required for the antiproliferative effects of metformin. Rather, metformin inhibits cancer cell proliferation by suppressing mitochondrial-dependent biosynthetic activity. We show that in vitro metformin decreases the flow of glucose- and glutamine-derived metabolic intermediates into the Tricarboxylic Acid (TCA) cycle, leading to reduced citrate production and de novo lipid biosynthesis. Tumor cells lacking functional mitochondria maintain lipid biosynthesis in the presence of metformin via glutamine-dependent reductive carboxylation, and display reduced sensitivity to metformin-induced proliferative arrest. Our data indicate that metformin inhibits cancer cell proliferation by suppressing the production of mitochondrial-dependent metabolic intermediates required for cell growth, and that metabolic adaptations that bypass mitochondrial-dependent biosynthesis may provide a mechanism of tumor cell resistance to biguanide activity

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

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 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

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