The bloodstream forms of Trypanosoma brucei (BSF), the parasite protist causing sleeping sickness, primarily proliferate in the blood of their mammalian hosts. The skin and adipose tissues were recently identified as additional major sites for parasite development. Glucose was the only carbon source known to be used by bloodstream trypanosomes to feed their central carbon metabolism, however, the metabolic behaviour of extravascular tissue-adapted parasites has not been addressed yet. Since the production of glycerol is an important primary function of adipocytes, we have adapted BSF trypanosomes to a glucose-depleted but glycerol-rich culture medium (CMM_Glyc/GlcNAc) and compared their metabolism and proteome to those of parasites grown in standard glucose-rich conditions (CMM_Glc). BSF were shown to consume 2-folds more oxygen per consumed carbon unit in CMM_Glyc/GlcNAc and were 11.5-times more sensitive to SHAM, a specific inhibitor of the plant-like alternative oxidase (TAO), which is the only mitochondrial terminal oxidase expressed in BSF. This is consistent with (i) the absolute requirement of the mitochondrial respiratory activity to convert glycerol into dihydroxyacetone phosphate, as deduced from the updated metabolic scheme and (ii) with the 1.8-fold increase of the TAO expression level compared to the presence of glucose. Proton NMR analysis of excreted end products from glycerol and glucose metabolism showed that these two carbon sources are metabolised through the same pathways, although the contributions of the acetate and succinate branches are more important in the presence of glycerol than glucose (10.2% versus 3.4% of the excreted end products, respectively). In addition, metabolomic analyses by mass spectrometry showed that, in the absence of glucose, 13C-labelled glycerol was incorporated into hexose phosphates through gluconeogenesis. As expected, RNAi-mediated down-regulation of glycerol kinase expression abolished glycerol metabolism and was lethal for BSF grown in CMM_Glyc/GlcNAc. Interestingly, BSF have adapted their metabolism to grow in CMM_Glyc/GlcNAc by concomitantly increasing their rate of glycerol consumption and decreasing that of glucose. However, the glycerol kinase activity was 7.8-fold lower in CMM_Glyc/GlcNAc, as confirmed by both western blotting and proteomic analyses. This suggests that the huge excess in glycerol kinase that is not absolutely required for glycerol metabolism, might be used for another yet undetermined non-essential function in glucose rich-conditions. Altogether, these data demonstrate that BSF trypanosomes are well-adapted to glycerol-rich conditions that could be encountered by the parasite in extravascular niches, such as the skin and adipose tissues.
A fast 3D NMR method is described (see picture), which gives access to site-specific 13C isotopic enrichments in complex biological mixtures in a few minutes. It relies on a hybrid strategy combining ultrafast and conventional 2D NMR acquisition techniques.
We have developed a robust workflow to measure high-resolution fluxotypes (metabolic flux phenotypes) for large strain libraries under fully controlled growth conditions. This was achieved by optimizing and automating the whole high-throughput fluxomics process and integrating all relevant software tools. This workflow allowed us to obtain highly detailed maps of carbon fluxes in the central carbon metabolism in a fully automated manner. It was applied to investigate the glucose fluxotypes of 180 Escherichia coli strains deleted for y-genes. Since the products of these y-genes potentially play a role in a variety of metabolic processes, the experiments were designed to be agnostic as to their potential metabolic impact. The obtained data highlight the robustness of E. coli’s central metabolism to y-gene deletion. For two y-genes, deletion resulted in significant changes in carbon and energy fluxes, demonstrating the involvement of the corresponding y-gene products in metabolic function or regulation. This work also introduces novel metrics to measure the actual scope and quality of high-throughput fluxomics investigations.
Microorganisms must make the right choice for nutrient consumption to adapt to their changing environment. As a consequence, bacteria and yeasts have developed regulatory mechanisms involving nutrient sensing and signaling, known as “catabolite repression,” allowing redirection of cell metabolism to maximize the consumption of an energy-efficient carbon source. Here, we report a new mechanism named “metabolic contest” for regulating the use of carbon sources without nutrient sensing and signaling. Trypanosoma brucei is a unicellular eukaryote transmitted by tsetse flies and causing human African trypanosomiasis, or sleeping sickness. We showed that, in contrast to most microorganisms, the insect stages of this parasite developed a preference for glycerol over glucose, with glucose consumption beginning after the depletion of glycerol present in the medium. This “metabolic contest” depends on the combination of 3 conditions: (i) the sequestration of both metabolic pathways in the same subcellular compartment, here in the peroxisomal-related organelles named glycosomes; (ii) the competition for the same substrate, here ATP, with the first enzymatic step of the glycerol and glucose metabolic pathways both being ATP-dependent (glycerol kinase and hexokinase, respectively); and (iii) an unbalanced activity between the competing enzymes, here the glycerol kinase activity being approximately 80-fold higher than the hexokinase activity. As predicted by our model, an approximately 50-fold down-regulation of the GK expression abolished the preference for glycerol over glucose, with glucose and glycerol being metabolized concomitantly. In theory, a metabolic contest could be found in any organism provided that the 3 conditions listed above are met.
Quantitative information on the carbon isotope content of metabolites is essential for flux analysis. Whereas this information is in principle present in proton NMR spectra through both direct and long-range heteronuclear coupling constants, spectral overlap and homonuclear coupling constants both hinder its extraction. We demonstrate here how pure shift 2D J-resolved NMR spectroscopy can simultaneously remove the homonuclear couplings and separate the chemical shift information from the heteronuclear coupling patterns. We demonstrate the power of this method on cell lysates from different bacterial cultures and investigate in detail the branched chain amino acid biosynthesis.
The mitochondrial respiratory chain (RC) enables many metabolic processes by regenerating both mitochondrial and cytosolic NAD+ and ATP. The oxidation by the RC of the NADH metabolically produced in the cytosol involves redox shuttles as the malate-aspartate shuttle (MAS) and is of paramount importance for cell fate. However, the specific metabolic regulations allowing mitochondrial respiration to prioritize NADH oxidation in response to high NADH/NAD+ redox stress have not been elucidated. The recent discovery that complex I (NADH dehydrogenase), and not complex II (Succinate dehydrogenase), can assemble with other respiratory chain complexes to form functional entities called respirasomes, led to the assumption that this supramolecular organization would favour NADH oxidation. Unexpectedly, characterization of heart and liver mitochondria demonstrates that the RC systematically favours electrons provided by the 'respirasome free' complex II. Our results demonstrate that the preferential succinate driven respiration is tightly controlled by OAA levels, and that OAA feedback inhibition of complex II rewires RC fuelling increasing NADH oxidation capacity. This new regulatory mechanism synergistically increases RC's NADH oxidative capacity and rewires MDH2 driven anaplerosis of the TCA, preventing malate production from succinate to favour oxidation of cytosolic malate. This regulatory mechanism synergistically adjusts RC and TCA fuelling in response to extramitochondrial malate produced by the MAS.
Two-dimensional nuclear magnetic resonance (2D NMR) is a promising tool for studying metabolic fluxes by measuring 13C-enrichments in complex mixtures of 13C-labeled metabolites. However, the methods reported so far are hampered by very long acquisition durations limiting the use of 2D NMR as a quantitative tool for fluxomics. In this paper, we propose a new approach for measuring specific 13C-enrichments in a very fast way, by using new experiments based on ultrafast 2D NMR. Two homonuclear 2D experiments (ultrafast COSY and zTOCSY) are proposed to measure 13C-enrichments in a single scan. Their advantages and limitations are discussed, and their high analytical potentialities are highlighted. Both methods are characterized by an accuracy of 1−2%, an average precision of 3%, and an excellent linearity. The analytical performance is equivalent or better than any of the conventional methods previously reported. The two ultrafast experiments are applied to the measurement of 13C-enrichments on a biomass hydrolyzate, showing the first known application of ultrafast 2D NMR to a real biological extract. The experiment duration is divided by 200 compared to the conventional methods, while preserving 80% of the quantitative information. This new approach opens new perspectives of application for fluxomics and metabonomics.
Activation of energy-dissipating brown/beige adipocytes represents an attractive therapeutic strategy against metabolic disorders. While lactate is known to induce beiging through the regulation of Ucp1 gene expression, the role of lactate transporters on beige adipocytes' ongoing metabolic activity remains poorly understood. To explore the function of the lactate-transporting monocarboxylate transporters (MCTs), we used a combination of primary cell culture studies, 13C isotopic tracing, laser microdissection experiments, and in situ immunofluorescence of murine adipose fat pads. Dissecting white adipose tissue heterogeneity revealed that the MCT1 is expressed in inducible beige adipocytes as the emergence of uncoupling protein 1 after cold exposure was restricted to a subpopulation of MCT1-expressing adipocytes suggesting MCT1 as a marker of inducible beige adipocytes. We also observed that MCT1 mediates bidirectional and simultaneous inward and outward lactate fluxes, which were required for efficient utilization of glucose by beige adipocytes activated by the canonical β3-adrenergic signaling pathway. Finally, we demonstrated that significant lactate import through MCT1 occurs even when glucose is not limiting, which feeds the oxidative metabolism of beige adipocytes. These data highlight the key role of lactate fluxes in finely tuning the metabolic activity of beige adipocytes according to extracellular metabolic conditions and reinforce the emerging role of lactate metabolism in the control of energy homeostasis. Activation of energy-dissipating brown/beige adipocytes represents an attractive therapeutic strategy against metabolic disorders. While lactate is known to induce beiging through the regulation of Ucp1 gene expression, the role of lactate transporters on beige adipocytes' ongoing metabolic activity remains poorly understood. To explore the function of the lactate-transporting monocarboxylate transporters (MCTs), we used a combination of primary cell culture studies, 13C isotopic tracing, laser microdissection experiments, and in situ immunofluorescence of murine adipose fat pads. Dissecting white adipose tissue heterogeneity revealed that the MCT1 is expressed in inducible beige adipocytes as the emergence of uncoupling protein 1 after cold exposure was restricted to a subpopulation of MCT1-expressing adipocytes suggesting MCT1 as a marker of inducible beige adipocytes. We also observed that MCT1 mediates bidirectional and simultaneous inward and outward lactate fluxes, which were required for efficient utilization of glucose by beige adipocytes activated by the canonical β3-adrenergic signaling pathway. Finally, we demonstrated that significant lactate import through MCT1 occurs even when glucose is not limiting, which feeds the oxidative metabolism of beige adipocytes. These data highlight the key role of lactate fluxes in finely tuning the metabolic activity of beige adipocytes according to extracellular metabolic conditions and reinforce the emerging role of lactate metabolism in the control of energy homeostasis. Thermogenic brown and beige adipose tissues increase systemic energy expenditure and represent putative targets to cure obesity and related metabolic diseases including type II diabetes (1Betz M.J. Enerback S. Targeting thermogenesis in brown fat and muscle to treat obesity and metabolic disease.Nat. Rev. Endocrinol. 2018; 14: 77-87Crossref PubMed Scopus (175) Google Scholar, 2Sidossis L. Kajimura S. Brown and beige fat in humans: thermogenic adipocytes that control energy and glucose homeostasis.J. Clin. Invest. 2015; 125: 478-486Crossref PubMed Scopus (453) Google Scholar). The energy-dissipating capacity of brown adipocytes is due to a high mitochondrial content and the expression of uncoupling protein 1 (UCP1) inside the mitochondrial inner membrane, which enables heat production through acceleration of the mitochondrial electron transport chain. Although sharing phenotypic, metabolic, and functional similarities with brown adipocytes, multilocular adipocytes expressing UCP1 interspaced within white adipose tissue (3Cousin B. Cinti S. Morroni M. Raimbault S. Ricquier D. Penicaud L. Casteilla L. Occurrence of brown adipocytes in rat white adipose tissue: molecular and morphological characterization.J. Cell Sci. 1992; 103: 931-942Crossref PubMed Google Scholar, 4Loncar D. Convertible adipose tissue in mice.Cell Tissue Res. 1991; 266: 149-161Crossref PubMed Scopus (150) Google Scholar, 5Young P. Arch J.R. Ashwell M. Brown adipose tissue in the parametrial fat pad of the mouse.FEBS Lett. 1984; 167: 10-14Crossref PubMed Scopus (282) Google Scholar) are distinct cells, with specific molecular expression profiles and different developmental origins. The number of these so-called beige adipocytes sharply increases during cold exposure through a process known as beiging, particularly in the murine inguinal fat pad, while the perigonadal depot is refractory to beiging (6Giordano A. Frontini A. Cinti S. Convertible visceral fat as a therapeutic target to curb obesity.Nat. Rev. Drug Discov. 2016; 15: 405-424Crossref PubMed Scopus (149) Google Scholar). We recently highlighted the structural heterogeneity of the inguinal fat pad and localized cold-induced beige adipocytes in the core of the depot, a region defined by the tissue autofluorescence signal (7Barreau C. Labit E. Guissard C. Rouquette J. Boizeau M.L. Gani Koumassi S. Carriere A. Jeanson Y. Berger-Muller S. Dromard C. Plouraboue F. Casteilla L. Lorsignol A. Regionalization of browning revealed by whole subcutaneous adipose tissue imaging.Obesity (Silver Spring). 2016; 24: 1081-1089Crossref PubMed Scopus (38) Google Scholar) and constituted by interconnected and complex 3D polylobular entities (8Dichamp J. Barreau C. Guissard C. Carriere A. Martinez Y. Descombes X. Penicaud L. Rouquette J. Casteilla L. Plouraboue F. Lorsignol A. 3D analysis of the whole subcutaneous adipose tissue reveals a complex spatial network of interconnected lobules with heterogeneous browning ability.Sci. Rep. 2019; 9: 6684Crossref PubMed Scopus (17) Google Scholar). Different studies suggest that, depending on the nature or the length of the stimulation of the fat pad, cold/β-adrenergic signaling promotes de novo beige fat differentiation and/or induction of UCP1 in mature adipocytes (9Barbatelli G. Murano I. Madsen L. Hao Q. Jimenez M. Kristiansen K. Giacobino J.P. De Matteis R. Cinti S. The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E1244-E1253Crossref PubMed Scopus (556) Google Scholar, 10Lee Y.H. Petkova A.P. Mottillo E.P. Granneman J.G. In vivo identification of bipotential adipocyte progenitors recruited by beta3-adrenoceptor activation and high-fat feeding.Cell Metab. 2012; 15: 480-491Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 11Rosenwald M. Perdikari A. Rulicke T. Wolfrum C. Bi-directional interconversion of brite and white adipocytes.Nat. Cell Biol. 2013; 15: 659-667Crossref PubMed Scopus (558) Google Scholar, 12Wang Q.A. Tao C. Gupta R.K. Scherer P.E. Tracking adipogenesis during white adipose tissue development, expansion and regeneration.Nat. Med. 2013; 19: 1338-1344Crossref PubMed Scopus (824) Google Scholar). Besides the therapeutic perspectives associated with the beiging-dependent remodeling of adipose tissues, the mechanisms regulating beige adipocyte metabolic activity still remain incompletely understood. It has been recently demonstrated that beige adipocytes appear in different physiopathological conditions, including cancer-associated cachexia (13Kir S. White J.P. Kleiner S. Kazak L. Cohen P. Baracos V.E. Spiegelman B.M. Tumour-derived PTH-related protein triggers adipose tissue browning and cancer cachexia.Nature. 2014; 513: 100-104Crossref PubMed Scopus (405) Google Scholar, 14Petruzzelli M. Schweiger M. Schreiber R. Campos-Olivas R. Tsoli M. Allen J. Swarbrick M. Rose-John S. Rincon M. Robertson G. Zechner R. Wagner E.F. A switch from white to brown fat increases energy expenditure in cancer-associated cachexia.Cell Metab. 2014; 20: 433-447Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar), intermittent fasting (15Li G. Xie C. Lu S. Nichols R.G. Tian Y. Li L. Patel D. Ma Y. Brocker C.N. Yan T. Krausz K.W. Xiang R. Gavrilova O. Patterson A.D. Gonzalez F.J. Intermittent fasting promotes white adipose browning and decreases obesity by shaping the gut microbiota.Cell Metab. 2017; 26: 672-685.e4Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar), or physical exercise (16Bostrom P. Wu J. Jedrychowski M.P. Korde A. Ye L. Lo J.C. Rasbach K.A. Bostrom E.A. Choi J.H. Long J.Z. Kajimura S. Zingaretti M.C. Vind B.F. Tu H. Cinti S. et al.A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis.Nature. 2012; 481: 463-468Crossref PubMed Scopus (3107) Google Scholar), suggesting function(s) that are distinct from thermogenesis (17Jeanson Y. Carriere A. Casteilla L. A new role for browning as a redox and stress adaptive mechanism?.Front. Endocrinol. 2015; 6: 158Crossref PubMed Scopus (31) Google Scholar). We recently described that lactate, a metabolite produced when the glycolytic production of pyruvate exceeds mitochondrial oxidative capacities and acting as a redox substrate and signaling metabolite (18Brooks G.A. The science and translation of lactate shuttle theory.Cell Metab. 2018; 27: 757-785Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 19Ferguson B.S. Rogatzki M.J. Goodwin M.L. Kane D.A. Rightmire Z. Gladden L.B. Lactate metabolism: historical context, prior misinterpretations, and current understanding.Eur. J. Appl. Physiol. 2018; 118: 691-728Crossref PubMed Scopus (152) Google Scholar), is a strong inducer of UCP1 expression in adipocytes (20Carriere A. Jeanson Y. Berger-Muller S. Andre M. Chenouard V. Arnaud E. Barreau C. Walther R. Galinier A. Wdziekonski B. Villageois P. Louche K. Collas P. Moro C. Dani C. et al.Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure.Diabetes. 2014; 63: 3253-3265Crossref PubMed Scopus (186) Google Scholar). This occurs through intracellular redox modifications subsequent to its transport. The regulation of UCP1 expression by lactate has been confirmed by others (21Bai Y. Shang Q. Zhao H. Pan Z. Guo C. Zhang L. Wang Q. Pdcd4 restrains the self-renewal and white-to-beige transdifferentiation of adipose-derived stem cells.Cell Death Dis. 2016; 7e2169Crossref PubMed Scopus (16) Google Scholar, 22Kim N. Nam M. Kang M.S. Lee J.O. Lee Y.W. Hwang G.S. Kim H.S. Piperine regulates UCP1 through the AMPK pathway by generating intracellular lactate production in muscle cells.Sci. Rep. 2017; 7: 41066Crossref PubMed Scopus (40) Google Scholar). This mechanism might be part of a redox regulatory and adaptive loop (17Jeanson Y. Carriere A. Casteilla L. A new role for browning as a redox and stress adaptive mechanism?.Front. Endocrinol. 2015; 6: 158Crossref PubMed Scopus (31) Google Scholar, 23Carriere A. Lagarde D. Jeanson Y. Portais J.C. Galinier A. Ader I. Casteilla L. The emerging roles of lactate as a redox substrate and signaling molecule in adipose tissues.J. Physiol. Biochem. 2020; 76: 241-250Crossref PubMed Scopus (15) Google Scholar) where a high redox (NADH/NAD+) pressure drives UCP1 expression, just as mitochondrial reactive oxygen species positively control UCP1 protein activity (24Chouchani E.T. Kazak L. Jedrychowski M.P. Lu G.Z. Erickson B.K. Szpyt J. Pierce K.A. Laznik-Bogoslavski D. Vetrivelan R. Clish C.B. Robinson A.J. Gygi S.P. Spiegelman B.M. Mitochondrial ROS regulate thermogenic energy expenditure and sulfenylation of UCP1.Nature. 2016; 532: 112-116Crossref PubMed Scopus (279) Google Scholar). Lactate also stimulates fibroblast growth factor-21 expression and release by beige adipocytes (25Jeanson Y. Ribas F. Galinier A. Arnaud E. Ducos M. Andre M. Chenouard V. Villarroya F. Casteilla L. Carriere A. Lactate induces FGF21 expression in adipocytes through a p38-MAPK pathway.Biochem. J. 2016; 473: 685-692Crossref PubMed Scopus (34) Google Scholar), independently of the redox state, highlighting the diversity of signaling mechanisms responsive to lactate in these cells (23Carriere A. Lagarde D. Jeanson Y. Portais J.C. Galinier A. Ader I. Casteilla L. The emerging roles of lactate as a redox substrate and signaling molecule in adipose tissues.J. Physiol. Biochem. 2020; 76: 241-250Crossref PubMed Scopus (15) Google Scholar). Among several members of the proton-linked monocarboxylate transporters (MCTs) family that transport lactate, pyruvate, and ketone bodies (26Felmlee M.A. Jones R.S. Rodriguez-Cruz V. Follman K.E. Morris M.E. Monocarboxylate transporters (SLC16): function, regulation, and role in health and disease.Pharmacol. Rev. 2020; 72: 466-485Crossref PubMed Scopus (109) Google Scholar, 27Perez-Escuredo J. Van Hee V.F. Sboarina M. Falces J. Payen V.L. Pellerin L. Sonveaux P. Monocarboxylate transporters in the brain and in cancer.Biochim. Biophys. Acta. 2016; 1863: 2481-2497Crossref PubMed Scopus (228) Google Scholar), the MCT1 isoform is known to be expressed by several oxidative tissues, including heart, muscle, and brown adipose tissue (BAT) (28Bonen A. The expression of lactate transporters (MCT1 and MCT4) in heart and muscle.Eur. J. Appl. Physiol. 2001; 86: 6-11Crossref PubMed Scopus (200) Google Scholar, 29De Matteis R. Lucertini F. Guescini M. Polidori E. Zeppa S. Stocchi V. Cinti S. Cuppini R. Exercise as a new physiological stimulus for brown adipose tissue activity.Nutr. Metab. Cardiovasc. Dis. 2013; 23: 582-590Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 30Fukano K. Okamatsu-Ogura Y. Tsubota A. Nio-Kobayashi J. Kimura K. Cold exposure induces proliferation of mature brown adipocyte in a ss3-adrenergic receptor-mediated pathway.PLoS One. 2016; 11e0166579Crossref PubMed Scopus (22) Google Scholar, 31Iwanaga T. Kuchiiwa T. Saito M. Histochemical demonstration of monocarboxylate transporters in mouse brown adipose tissue.Biomed. Res. 2009; 30: 217-225Crossref PubMed Scopus (27) Google Scholar, 32Okamatsu-Ogura Y. Nio-Kobayashi J. Nagaya K. Tsubota A. Kimura K. Brown adipocytes postnatally arise through both differentiation from progenitors and conversion from white adipocytes in Syrian hamster.J. Appl. Physiol. 2018; 124: 99-108Crossref PubMed Scopus (11) Google Scholar). However, the expression of MCTs by beige adipocytes and their role in their metabolic activity remain to be elucidated to efficiently recruit and/or activate them. Herein, we report that the MCT1 protein is a marker of inducible beige adipocytes. MCT1 is expressed at the plasma membrane of a subset of adipocytes present in the core region of the inguinal fat pad. These fat cells possess a beige adipocyte gene signature at 21 °C and express the UCP1 protein after short-term cold exposure. Using isotopic labeling experiments, we identified MCT1 as a key transporter mediating simultaneous outward and inward lactate fluxes in beige adipocytes and as a critical target and mediator of the β3 adrenergic signaling. MCT1-dependent lactate fluxes are critical for both glycolysis and oxidative metabolism of beige adipocytes, finely tuning their metabolism according to the extracellular metabolic conditions. Because of the heterogeneity of the beiging-sensitive inguinal fat pad (7Barreau C. Labit E. Guissard C. Rouquette J. Boizeau M.L. Gani Koumassi S. Carriere A. Jeanson Y. Berger-Muller S. Dromard C. Plouraboue F. Casteilla L. Lorsignol A. Regionalization of browning revealed by whole subcutaneous adipose tissue imaging.Obesity (Silver Spring). 2016; 24: 1081-1089Crossref PubMed Scopus (38) Google Scholar, 8Dichamp J. Barreau C. Guissard C. Carriere A. Martinez Y. Descombes X. Penicaud L. Rouquette J. Casteilla L. Plouraboue F. Lorsignol A. 3D analysis of the whole subcutaneous adipose tissue reveals a complex spatial network of interconnected lobules with heterogeneous browning ability.Sci. Rep. 2019; 9: 6684Crossref PubMed Scopus (17) Google Scholar), we analyzed mRNA levels of the different Mct isoforms described as lactate transporters (26Felmlee M.A. Jones R.S. Rodriguez-Cruz V. Follman K.E. Morris M.E. Monocarboxylate transporters (SLC16): function, regulation, and role in health and disease.Pharmacol. Rev. 2020; 72: 466-485Crossref PubMed Scopus (109) Google Scholar, 27Perez-Escuredo J. Van Hee V.F. Sboarina M. Falces J. Payen V.L. Pellerin L. Sonveaux P. Monocarboxylate transporters in the brain and in cancer.Biochim. Biophys. Acta. 2016; 1863: 2481-2497Crossref PubMed Scopus (228) Google Scholar), i.e., Mct1, Mct2, Mct3, and Mct4, in specific regions with different beiging abilities (Fig. 1A). The spatial heterogeneity of this depot was highlighted by the gradient in Ucp1 expression, with lowest mRNA levels at the periphery (region 1), intermediate levels in the core of the tissue lying the extremity (region 2), and highest levels close to the lymph node (region 3) (Fig. 1B). This gradient was observed in mice housed at 21 °C as well as after 48 h of cold exposure (Fig. 1B). Mct1 expression displayed the same pattern as Ucp1, as Mct1 mRNA levels also increased from regions 1 to 3 and were significantly upregulated following cold exposure, specifically in the region close to the lymph node that exhibited the highest levels of Ucp1 (Fig. 1C). This gene expression profile is specific to Mct1 as no significant difference was observed regarding Mct2 or Mct4 expression, irrespective of the different fat pad regions or in response to cold exposure (Fig. 1, D–E). Mct3 was not detected, in accordance with its exclusive expression in retinal cells and choroid plexus (26Felmlee M.A. Jones R.S. Rodriguez-Cruz V. Follman K.E. Morris M.E. Monocarboxylate transporters (SLC16): function, regulation, and role in health and disease.Pharmacol. Rev. 2020; 72: 466-485Crossref PubMed Scopus (109) Google Scholar, 27Perez-Escuredo J. Van Hee V.F. Sboarina M. Falces J. Payen V.L. Pellerin L. Sonveaux P. Monocarboxylate transporters in the brain and in cancer.Biochim. Biophys. Acta. 2016; 1863: 2481-2497Crossref PubMed Scopus (228) Google Scholar). To analyze gene expression with higher precision in this very heterogeneous tissue, we analyzed Mct1 expression in clusters of cells dissected by laser capture microdissection (LCM; Fig. 1F), in the three regions exhibiting different levels of Mct1 expression, at 21 °C. These experiments revealed a tight and significant positive correlation between Mct1 and Ucp1 mRNA levels (R = 0.881; p < 0.0001; Fig. 1, G–H) and between Mct1 and additional thermogenic markers such as Cidea and Cox8b (R = 0.925 and R = 0.884, respectively; p < 0.0001; Fig. 1, G, I, and J). Conversely, Mct1 mRNA levels were negatively correlated with leptin (Ob) expression (R = 0.425; p < 0.01; Fig. 1, G and K), known to be enriched in white adipocytes (33Cinti S. Frederich R.C. Zingaretti M.C. De Matteis R. Flier J.S. Lowell B.B. Immunohistochemical localization of leptin and uncoupling protein in white and brown adipose tissue.Endocrinology. 1997; 138: 797-804Crossref PubMed Scopus (171) Google Scholar). No significant correlation was found between Mct1 and other adipogenic genes including Ap2 and Pparg2 (Fig. 1, G, L, and M). Thus, the fine dissection of the cellular heterogeneity within the subcutaneous white fat pad highlighted Mct1 expression as tightly and positively correlated with the gene signature of beige adipocytes. In agreement with Mct1 mRNA expression patterns, immunofluorescence experiments performed on the whole inguinal fat pad revealed the existence of a gradient in MCT1 protein levels, from the periphery to the core of the tissue (Fig. 2A), in 21 °C-housed animals. MCT1 was detected at the plasma membrane of adipocytes, primarily in large clusters in the region prone to beiging close to the lymph node (Fig. 2A). In contrast, a very faint signal was detected at the periphery, which is highly refractory to beiging (Fig. 2A). In addition, heterogeneous MCT1 protein expression was also observed within the region prone to beiging itself because MCT1− and MCT1+ adjacent adipocytes (white and yellow arrows, respectively; Fig. 2A) were identified. MCT1+ adipocytes display several small lipid droplets in their cytoplasm and exhibit a paucilocular phenotype (as described (9Barbatelli G. Murano I. Madsen L. Hao Q. Jimenez M. Kristiansen K. Giacobino J.P. De Matteis R. Cinti S. The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.Am. J. Physiol. Endocrinol. Metab. 2010; 298: E1244-E1253Crossref PubMed Scopus (556) Google Scholar)), in contrast to unilocular MCT1− adipocytes (yellow and white arrows, respectively; Fig. 2, B–C). The same heterogeneous MCT1 staining was observed in the inguinal fat pad of mice acclimated at 28 °C (Fig. 2D). Analysis of the outer mitochondrial membrane protein TOM20 indicated that MCT1+ adipocytes exhibited a high abundance of mitochondria (Fig. 2E; yellow arrows), in contrast to MCT1− adipocytes, which very weakly express TOM20 (Fig. 2E; white arrows). TOM20 is almost undetectable in MCT1− adipocytes of the region of the inguinal fat pad, which is refractory to beiging (Fig. 2E; top panels). Together, these results reveal a high MCT1 expression in a subset of mature adipocytes gathered in large clusters in the core region of the inguinal pad of mice housed at 28 °C and 21 °C, which may exhibit distinct oxidative capacities. We next analyzed the expression of the UCP1 protein, at 21 °C and after cold exposure, and quantified in both MCT1+ and MCT1− adipocyte subpopulations the percentage of UCP1+ and UCP1− cells. These experiments revealed that UCP1 was detected exclusively in the subpopulation of adipocytes expressing MCT1 (Fig. 2, F–H). Interestingly, while only 29 ± 11% of MCT1+ adipocytes expressed the UCP1 protein at 21 °C, almost all of them turn UCP1+ after cold exposure (90 ± 4% and 92 ± 3% of MCT1+ adipocytes after 24 and 48 h of cold exposure, respectively, Fig. 2G, top panel), suggesting that MCT1 is expressed by cold-inducible beige adipocytes. This is reinforced by the fact that the percentage of UCP1+ adipocytes in the MCT1− fraction was negligible and did not significantly increase after cold exposure (0.5 ± 0.3 to 4 ± 2%; Fig. 2G, bottom panel). In conclusion, in the inguinal fat pad, all the paucilocular/multilocular mitochondrial-enriched adipocytes do express MCT1, and the majority of MCT1+ adipocytes express UCP1 after cold exposure. In agreement with MCT1 as a bona fide marker of inducible beige adipocytes, we could not detect any MCT1 protein in adipocytes from the perigonadal depot at 21 °C or after 4 °C exposure (Fig. 3, A and C), which is refractory to cold-induced beiging (6Giordano A. Frontini A. Cinti S. Convertible visceral fat as a therapeutic target to curb obesity.Nat. Rev. Drug Discov. 2016; 15: 405-424Crossref PubMed Scopus (149) Google Scholar). As previously reported (29De Matteis R. Lucertini F. Guescini M. Polidori E. Zeppa S. Stocchi V. Cinti S. Cuppini R. Exercise as a new physiological stimulus for brown adipose tissue activity.Nutr. Metab. Cardiovasc. Dis. 2013; 23: 582-590Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 30Fukano K. Okamatsu-Ogura Y. Tsubota A. Nio-Kobayashi J. Kimura K. Cold exposure induces proliferation of mature brown adipocyte in a ss3-adrenergic receptor-mediated pathway.PLoS One. 2016; 11e0166579Crossref PubMed Scopus (22) Google Scholar, 31Iwanaga T. Kuchiiwa T. Saito M. Histochemical demonstration of monocarboxylate transporters in mouse brown adipose tissue.Biomed. Res. 2009; 30: 217-225Crossref PubMed Scopus (27) Google Scholar, 32Okamatsu-Ogura Y. Nio-Kobayashi J. Nagaya K. Tsubota A. Kimura K. Brown adipocytes postnatally arise through both differentiation from progenitors and conversion from white adipocytes in Syrian hamster.J. Appl. Physiol. 2018; 124: 99-108Crossref PubMed Scopus (11) Google Scholar), we detected high expression of MCT1 in BAT (Fig. 3B [yellow arrows], Fig. 3D). Indeed, all multilocular brown adipocytes expressing UCP1 are MCT1+ (Fig. 3D). Note that the MCT1 protein was not detected in white adipocytes surrounding BAT (Fig. 3B; white arrows). Together, these findings reveal that MCT1 is expressed in classical brown adipocytes and white adipocytes susceptible to beiging remodeling but not in white adipocytes refractory to this process. To determine whether the cold-induced Mct1 expression observed in vivo (Fig. 1C) could be due to the β3-adrenergic signaling pathway, we treated primary differentiated adipocytes with the β3-adrenergic receptor agonist CL316.243 (CL). We found that CL, which upregulated Ucp1 expression as expected (Fig. 4A), also increased Mct1 expression in primary differentiated adipocytes (Fig. 4B). As the same effect was observed with the cAMP-rising agent forskolin (Fig. 4, A–B), we concluded that Mct1 expression was regulated by a cAMP-dependent signaling, further suggesting its functional role in cold-induced beige adipocytes. To study the involvement of MCT1 in β3-adrenergic regulation of Ucp1 expression, we tested the effect of AZD3965 (AZD), an established MCT1 inhibitor (34Bola B.M. Chadwick A.L. Michopoulos F. Blount K.G. Telfer B.A. Williams K.J. Smith P.D. Critchlow S.E. Stratford I.J. Inhibition of monocarboxylate transporter-1 (MCT1) by AZD3965 enhances radiosensitivity by reducing lactate transport.Mol. Cancer Ther. 2014; 13: 2805-2816Crossref PubMed Scopus (109) Google Scholar, 35Curtis N.J. Mooney L. Hopcroft L. Michopoulos F. Whalley N. Zhong H. Murray C. Logie A. Revill M. Byth K.F. Benjamin A.D. 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Treatment with 50 nM AZD did not hamper CL-induced Ucp1 expression (even at higher doses, data not shown) suggesting that MCT1 was not involved in the signaling cascade linking the β3-adrenergic receptor to the regulation of Ucp1 expression (Fig. 4C). We then investigated the effect of AZD on lactate-induced Ucp1 expression, using sodium-L-lactate (and not lactic acid to avoid pH changes) at a concentration of 25 mM, known to give rise to the maximal induction of Ucp1 (as shown by the dose-dependent effects reported (20Carriere A. Jeanson Y. Berger-Muller S. Andre M. Chenouard V. Arnaud E. Barreau C. Walther R. Galinier A. Wdziekonski B. Villageois P. Louche K. Collas P. Moro C. Dani C. et al.Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure.Diabetes. 2014; 63: 3253-3265Crossref PubMed Scopus (186) Google Scholar)). We found that AZD abrogated lactate-induced Ucp1 expression (Fig. 4C), confirming previous data obtained with other MCT inhibitors (20Carriere A. Jeanson Y. Berger-Muller S. Andre M. Chenouard V. Arnaud E. Barreau C. Walther R. Galinier A. Wdziekonski B. Villageois P. Louche K. Collas P. Moro C. Dani C. et al.Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure.Diabetes. 2014; 63: 3253-3265Crossref PubMed Scopus (186) Google Scholar), and further highlighting the role of intracellular lactate on the regulation of Ucp1 expression. These data clearly indicate the existence of two independent signaling pathways regulating Ucp1 expression, one mediated by MCT1/lactate and the other mediated by β3 adrenergic receptor. Altogether these data reveal that MCT1 is a transcriptional target of the β3 adrenergic pathway and is not involved in the regulation of Ucp1 expression by β3 agonists. We next investigated the role of MCT1 in the metabolism of beige adipocytes. The effect of AZD on lactate metabolism was first studied by monitoring lactate content in the supernatant of primary adipocytes. Time-course experiments highlighted a biphasic curve where adipocytes exhibited a first phase of net lactate release and a second phase of net lactate consumption after 32 h in culture (Fig. 5A). Lactate release was significantly reduced by AZD (Fig. 5, A–B), showing the primary role of MCT1 in lactate export. The residual export is probably because of other isoforms such as MCT4 (20Carriere A. Jeanson Y. Berger-Muller S. Andre M. Chenouard V. Arnaud E. Barreau C. Walther R. Galinier A. Wdziekonski B. Villageois P. Louche K. Collas P. Moro C. Dani C. et al.Browning of white adipose cells by intermediate metabolites: an adaptive mechanism to alleviate redox pressure.Diabetes. 2014; 63: 3253-3265Crossref PubMed Scopus (186) Google Scholar, 38Petersen C. Nielsen M.D. Andersen E.S. Basse A.L. Isidor M.S. Markussen L.K. Viuff B.M. Lambert I.H. Hansen J.B. Pedersen S.F. MCT1 and MCT4 expression and lactate flux activity increase during white and brown adipogenesis and impact adipocyte metabolism.Sci. Rep. 2017; 7: 13101Crossref PubMed Scopu
Cyanobacteria receive huge interest as green catalysts. While exploiting energy from sunlight, they co-utilize sugar and CO2. This photomixotrophic mode enables fast growth and high cell densities, opening perspectives for sustainable biomanufacturing. The model cyanobacterium Synechocystis sp. PCC 6803 possesses a complex architecture of glycolytic routes for glucose breakdown that are intertwined with the CO2-fixing Calvin-Benson-Bassham (CBB) cycle. To date, the contribution of these pathways to photomixotrophic metabolism has remained unclear.Here, we developed a comprehensive approach for 13C metabolic flux analysis of Synechocystis sp. PCC 6803 during steady state photomixotrophic growth. Under these conditions, the Entner-Doudoroff (ED) and phosphoketolase (PK) pathways were found inactive but the microbe used the phosphoglucoisomerase (PGI) (63.1%) and the oxidative pentose phosphate pathway (OPP) shunts (9.3%) to fuel the CBB cycle. Mutants that lacked the ED pathway, the PK pathway, or phosphofructokinases were not affected in growth under metabolic steady-state. An ED pathway-deficient mutant (Δeda) exhibited an enhanced CBB cycle flux and increased glycogen formation, while the OPP shunt was almost inactive (1.3%). Under fluctuating light, ∆eda showed a growth defect, different to wild type and the other deletion strains.The developed approach, based on parallel 13C tracer studies with GC-MS analysis of amino acids, sugars, and sugar derivatives, optionally adding NMR data from amino acids, is valuable to study fluxes in photomixotrophic microbes to detail. In photomixotrophic cells, PGI and OPP form glycolytic shunts that merge at switch points and result in synergistic fueling of the CBB cycle for maximized CO2 fixation. However, redirected fluxes in an ED shunt-deficient mutant and the impossibility to delete this shunt in a GAPDH2 knockout mutant, indicate that either minor fluxes (below the resolution limit of 13C flux analysis) might exist that could provide catalytic amounts of regulatory intermediates or alternatively, that EDA possesses additional so far unknown functions. These ideas require further experiments.
