Monomethyl branched-chain fatty acids are critical for Caenorhabitis elegans survival in elevated glucose conditions

Abstract

The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose. The maintenance of optimal membrane composition under basal and stress conditions is critical for the survival of an organism. High-glucose stress has been shown to perturb membrane properties by decreasing membrane fluidity, and the membrane sensor PAQR-2 is required to restore membrane integrity. However, the mechanisms required to respond to elevated dietary glucose are not fully established. In this study, we used a 13C stable isotope-enriched diet and mass spectrometry to better understand the impact of glucose on fatty acid dynamics in the membrane of Caenorhabditis elegans. We found a novel role for monomethyl branched-chain fatty acids (mmBCFAs) in mediating the ability of the nematodes to survive conditions of elevated dietary glucose. This requirement of mmBCFAs is unique to glucose stress and was not observed when the nematode was fed elevated dietary saturated fatty acid. In addition, when worms deficient in elo-5, the major biosynthesis enzyme of mmBCFAs, were fed Bacillus subtilis (a bacteria strain rich in mmBCFAs) in combination with high glucose, their survival rates were rescued to wild-type levels. Finally, the results suggest that mmBCFAs are part of the PAQR-2 signaling response during glucose stress. Taken together, we have identified a novel role for mmBCFAs in stress response in nematodes and have established these fatty acids as critical for adapting to elevated glucose. In many disease states, including diabetes, cancer, and neurodegenerative diseases, defects in membrane structure and composition have been identified (1Gianfrancesco M.A. Paquot N. Piette J. Legrand-Poels S. Lipid bilayer stress in obesity-linked inflammatory and metabolic disorders.Biochem. Pharmacol. 2018; 153: 168-183Google Scholar, 2Pilon M. Revisiting the membrane-centric view of diabetes.Lipids Health Dis. 2016; 15: 167Google Scholar, 3Samuel V. Shulman G. Mechanisms for insulin resistance: Common threads and missing links.Cell (Cambridge). 2012; 148: 852-871Google Scholar, 4Bandu R. Mok H.J. Kim K.P. Phospholipids as cancer biomarkers: Mass spectrometry-based analysis.Mass Spectrom. Rev. 2018; 37: 107-138Google Scholar, 5Fanning S. Haque A. Imberdis T. Baru V. Barrasa M.I. Nuber S. Termine D. Ramalingam N. Ho G.P.H. Noble T. Sandoe J. Lou Y. Landgraf D. Freyzon Y. Newby G. et al.Lipidomic analysis of α-synuclein neurotoxicity identifies stearoyl CoA desaturase as a target for Parkinson treatment.Mol. Cell. 2019; 73: 1001-1014.e8Google Scholar, 6Fecchio C. Palazzi L. de Laureto P.P. A-synuclein and polyunsaturated fatty acids: Molecular basis of the interaction and implication in neurodegeneration.Molecules. 2018; 23: 1531Google Scholar). Biological membranes are essential barriers between the intracellular and extracellular environment and are also crucial in the compartmentalization of subcellular organelles. Membranes are not limited to establishing passive barriers, but they also influence numerous and diverse cellular bioactivities such as signaling and regulation (7Budin I. de Rond T. Chen Y. Chan L.J.G. Petzold C.J. Keasling J.D. Viscous control of cellular respiration by membrane lipid composition.Science. 2018; 362: 1186-1189Google Scholar, 8Hedger G. Sansom M.S.P. Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations.Biochim. Biophys. Acta. 2016; 1858: 2390-2400Google Scholar, 9Koshy C. Ziegler C. Structural insights into functional lipid–protein interactions in secondary transporters.Biochim. Biophys. Acta. 2015; 1850: 476-487Google Scholar, 10Levental K.R. Malmberg E. Symons J.L. Fan Y. Chapkin R.S. Ernst R. Levental I. Lipidomic and biophysical homeostasis of mammalian membranes counteracts dietary lipid perturbations to maintain cellular fitness.Nat. Commun. 2020; 11: 1339Google Scholar, 11Spector A.A. Yorek M.A. Membrane lipid composition and cellular function.J. Lipid Res. 1985; 26: 1015-1035Google Scholar). Although membranes allow a certain degree of variation in their makeup, it is critical to maintain a membrane composition within an acceptable range for optimal cellular function. Several studies have shown that there are regulatory mechanisms, including membrane sensors, that detect perturbations in the biophysical properties of the membrane and can allow membranes to adapt to variations in environmental cues including temperature and dietary composition (12Siliakus M.F. van der Oost J. Kengen S.W.M. Adaptations of archaeal and bacterial membranes to variations in temperature, pH and pressure.Extremophiles. 2017; 21: 651-670Google Scholar, 13Svensk E. Devkota R. Ståhlman M. Ranji P. Rauthan M. Magnusson F. Hammarsten S. Johansson M. Borén J. Pilon M. Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar). Model systems including the nematode, Caenorhabitis elegans, have been established in order to probe the regulation of membrane composition and the response to perturbations. In addition to the genetic tools available, C. elegans has the added advantage of being small enough to allow for high isotope enrichment and subsequent detailed measurements of membrane flux. Our lab has developed stable isotope feeding strategies in C. elegans to track the incorporation of new fatty acid molecules into the membrane (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google Scholar, 15Dancy B.C.R. Chen S. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar). Using this technique, we established that the majority of the membrane lipids are replaced or within a in This is in the in lipids used to in the B.C.R. Chen S. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar, N. Olsen C.P. Using stable isotope to membrane dynamics in C. 2020; Scholar). in addition to to and the phospholipid membrane is a structure with of lipids in and of the membrane under basal is the in membrane but the rates on the of for the maintenance of membrane composition and of that when branched-chain fatty acids (mmBCFAs) are in and and of mmBCFAs in have been to and insulin F. D. E. S. monomethyl branched-chain fatty acids and insulin of and 2015; 23: Scholar). In C. elegans, mmBCFAs are found within the membrane for of the of are from branched-chain acids the of the branched-chain fatty acid and in the of is by and to and M. M. C. M. branched-chain fatty acids an essential role in Caenorhabditis elegans Scholar, M. T. M. A branched-chain fatty acid is in control in to the insulin and biosynthesis is in C. 2008; Scholar). mmBCFAs are critical for in part by signaling to and in nematodes M. M. A and under fed and conditions in C. Cell. 2015; Scholar, M. M. A novel in Caenorhabditis Scholar, M. J.L. J. N. J.D. P. et branched-chain fatty acid in 2018; Scholar). In the of mmBCFAs the of the membrane and membrane R. P. J. role for membrane in of 2012; Scholar, P. of membrane order with of and a of Scholar). In branched-chain fatty acid to as and a the or the and are the fatty acids of membranes of of the The of these fatty acids has a impact on membrane properties such as they properties of saturated and fatty acids The fatty acids is when the temperature is that membrane with D. T. S. of of acids in One. 2017; Scholar, L. in properties between and fatty acids as by incorporation into the membrane lipids of A strain Biophys. Acta. Scholar). However, the of mmBCFAs in the response to stress such as high glucose diet has not been conditions such as temperature that the membrane composition is in order to maintain the membrane in an in the of fatty acids to the membrane to maintain membrane Lipid and in membrane Scholar, J.L. M. Lipid and in Caenorhabditis 2017; Scholar). The of in the or in the the fatty acid and the membrane structure G. Membrane they are and they Rev. Scholar). The of saturated within a membrane with and polyunsaturated fatty acids to the biophysical properties of the membrane L. M. A. P. and biosynthesis by the sites in 2015; Scholar). fatty acids including to membrane and as homeostasis is and have regulatory mechanisms that allow to maintain membrane composition when with glucose. PAQR-2 is a of and to the of saturated within the by PAQR-2 is required for survival in conditions that perturb the membrane properties including dietary glucose R. E. M. Ståhlman M. Borén J. Pilon M. The and Caenorhabditis elegans PAQR-2 membrane by saturated fatty Genet. 2017; Scholar). In the of nematodes fed glucose have a and The role for PAQR-2 is to to the membrane the the into the saturated fatty However, it is PAQR-2 the of metabolic that to survival in elevated glucose Although it is that membrane composition is by glucose the mechanisms in to perturbations in membrane properties have not been In we stable isotope labeling under elevated glucose conditions to the fatty acid of the membrane stress. Our results a novel role for mmBCFAs in elevated glucose We have these to that nematodes not mmBCFAs when with glucose. it has been established that a metabolic in lipid the in the membrane lipids of to glucose have not been the response to dietary glucose, we C. elegans to glucose to as is the are observed in with E. Devkota R. Ståhlman M. Ranji P. Rauthan M. Magnusson F. Hammarsten S. Johansson M. Borén J. Pilon M. Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar). on were fatty acid were from the major lipid and by spectrometry to quantify the of fatty acids (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google Scholar). We the to the fatty acid the of in the fatty and the of for We the in on on the major fatty acid of the we not the fatty acid analysis for the polyunsaturated fatty they and in the mass with the stable isotope studies We found that there were in major fatty acid with the of a in from to and a in from to The in is to the of this in the bacteria to glucose. The of major in the phospholipid has been by studies as E. Devkota R. Ståhlman M. Ranji P. Rauthan M. Magnusson F. Hammarsten S. Johansson M. Borén J. Pilon M. Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar). The the of maintenance mechanisms to control the fatty acid composition of the membrane lipids under elevated glucose maintenance to membrane lipids or lipid in to glucose the within the nematode, we the lipid the and analysis by we found in the fatty acid from there are of fatty acid including a of by by The mmBCFAs, acid and acid were by and the that glucose in lipid and that the lipid are phospholipid the to the membrane composition is that in lipid by the of lipids the of the glucose stress. we a dietary stable isotope to the of lipids to the membrane glucose The nematodes were fed bacteria with by the diet in 13C from B.C.R. Chen S. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar). the in the the stable isotope labeling was in the as studies have observed high of in the of is a in the of to the membrane with of being in worms with in control this is a of saturated fatty we and found in stable isotope we that the of is to and not saturated fatty of the of a stable isotope we can probe the fatty acid in the with the of the that in the mass The dynamics of the and polyunsaturated fatty and were not by the glucose However, we identified a in the of and the to the membrane This is not of the and this de of these the in by and by in membrane lipids are from control but there is an in the labeling of saturated fatty acids and an in mmBCFAs glucose We that these to the elevated glucose levels. In order to there is an in we the of the and and the and glucose In has been shown to with glucose and the analysis a in with glucose feeding This is to as are is a of and by and that these are to in elevated glucose conditions the stable isotope labeling to the membrane glucose we mmBCFAs a role in to the elevated glucose We their with is the essential for the of and A feeding was used to the and with of were on as in the lipid studies or a to glucose glucose there was in the the we the of glucose of the were with of the The fatty acid from fed glucose a in the of but not to the We in survival in we the role of is required for The the glucose to wild-type with that the of by is not to In of this the by the mmBCFAs the role of mmBCFAs in the response to glucose, we fed nematodes a diet Bacillus this bacterial strain high of with results a role for mmBCFAs in to elevated dietary glucose, subtilis feeding the survival rates of on to wild-type The of mmBCFAs in subtilis are and with mmBCFAs are subtilis a high of that in an and not an as in the with results a role for mmBCFAs in to elevated dietary glucose, subtilis feeding the survival rates of on to wild-type that this of nematode with subtilis was not to bacteria we fed wild-type nematodes a to with a bacterial fed the high under glucose subtilis can by the of and we that the incorporation of new mmBCFAs from or from the diet is critical for glucose stress the of in survival with glucose we was on or the of mmBCFAs to glucose We it by during and the on The was with the glucose stress in In the there is survival with with of in as to of in The survival on that there is an role for in to glucose that is of the of mmBCFAs during Finally, we that also on with with in control for the a critical role for mmBCFAs in glucose stress and a novel for these unique fatty acids in studies this PAQR-2 as an membrane sensor that to membrane from glucose. of the high of on we that the by PAQR-2 as part of the metabolic response to glucose stress. probe this we the membrane composition of glucose stress and their mmBCFAs with We found mmBCFAs and in the membranes of on with on control was from to and from to with glucose that PAQR-2 required to maintain under glucose stress. the impact of PAQR-2 in the response to elevated glucose, we the membrane dynamics of with stable isotope labeling as In there is a in the labeling of mmBCFAs with decreasing from to and from to that PAQR-2 required for the of the analysis of the fatty acid in the membrane a in the of in on with the observed in is to that the are on glucose the in a of their E. Devkota R. Ståhlman M. Ranji P. Rauthan M. Magnusson F. Hammarsten S. Johansson M. Borén J. Pilon M. Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar). However, PAQR-2 has been found to and of by results in a of fatty acid to the impact of the fatty acid on the membrane B.C.R. Chen S. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar). PAQR-2 is to impact of metabolic we to the of in these with and glucose. we the requirement of PAQR-2 to with glucose and found that is the of control in PAQR-2 is of wild-type in the of glucose. we found that is of wild-type glucose and of wild-type with glucose the analysis that is by PAQR-2 and that is a major in the PAQR-2 Finally, we used stable isotope labeling to that the in in the not impact the of the fatty acid The control for the studies is can the and has a fatty acid we the elevated on with this diet and found that the wild-type fed high of of on glucose with the mmBCFAs response in In there is also a in on and the is the of is with control the nematode to the of from glucose stress by the of to the membrane but not a that with of that the of the of this to the membrane in control In addition, the is by the was in nematodes these mmBCFAs, the in the impact of this on the properties of the Taken together, results that a of including mmBCFAs is essential to respond to glucose and that is a in the The major established role of PAQR-2 is to the membrane that with dietary glucose or dietary saturated and a within the these fatty acids lipid and within a In order to mmBCFAs a role in membrane fluidity, nematodes were fed and for and we is required for and on with we to as was impact on in fed on in to the in the rates of and were with The incorporation of not impact of or The of impact with that the role of mmBCFAs is not in a response to membrane but to certain conditions such as high glucose. we the of in the phospholipid of the and found a in from to in and from to in We also a of in the bacteria on the and found that this diet In order to the was into the nematode, the fatty acid of lipids was also and there was an from to in wild-type this that is for the but it also mechanisms to protect the membrane from saturated We can also that the role of mmBCFAs is not in membrane suggest that PAQR-2 the response in mmBCFAs, we the subtilis survival of on We found that the subtilis diet the of on that the mmBCFAs are a critical to the metabolic response to elevated glucose we observed that a in the of glucose with a of with on with a of A has been for the was not as as shown to in the dietary fatty acid the we that the addition of the of to Taken together, these mmBCFAs as part of the response to elevated dietary glucose. We to mechanisms that an response to elevated dietary glucose conditions that the optimal membrane composition when Although it is that the PAQR-2 membrane sensor to glucose stress the we a stable isotope labeling to a new of the PAQR-2 response labeling studies identified that under glucose the membrane of acid and of The of is critical for survival on glucose as results in survival on Our results that we have identified mmBCFAs in a novel and essential response to elevated glucose. studies have shown that elevated glucose membrane and have found an incorporation of saturated into the membrane lipids R. E. M. Ståhlman M. Borén J. Pilon M. The and Caenorhabditis elegans PAQR-2 membrane by saturated fatty Genet. 2017; Scholar). has been that the elevated saturated is by the bacteria fed glucose de fatty acid However, we the saturated from the bacteria and found it has a but in that the in in the In addition, have a response and with we found is required for survival on glucose but not is that the in glucose stress to an between the and the diet has of this fatty acid is from diet (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google the nematode a of impact the membrane However, the the that the glucose is the of saturated fatty acid In mmBCFAs have been shown to in M. M. C. M. branched-chain fatty acids an essential role in Caenorhabditis elegans and M. M. A and under fed and conditions in C. Cell. 2015; Scholar). However, mmBCFAs can impact the membrane as mmBCFAs influence membrane the in their structure lipid dietary membrane but the of not this we that the role of mmBCFAs is not in membrane a by et found that the was to the target of M. M. A novel in Caenorhabditis Scholar). The was to metabolic in the with and L. N. F. L. A. A. E. of in mass and glucose in a S. A. 2008; Scholar, M. signaling in control and Scholar). the elevated of mmBCFAs observed in diet to the of signaling to the and of glucose, a of In wild-type there is a of but not as by stable isotope on there are glucose in the but not levels. The of a response by when is not as there are of in We that the role of mmBCFAs is not to of fatty acid but to the of the as there are in in This is by the can rescued by mmBCFAs that the of the not is that PAQR-2 an role to maintain and membrane the membrane composition of PAQR-2 a in the of This that PAQR-2 also to mmBCFAs in response to glucose stress. We were to mmBCFAs were in PAQR-2 isotope labeling analysis a in the lipid of fatty acid The in lipid is with the role of PAQR-2 in we have established is required for rates of lipid However, it is also that PAQR-2 have to Although it was shown that PAQR-2 have a PAQR-2 signaling temperature to C. elegans by Commun. 2019; to a impact with We that the high to in the to the PAQR-2 to by the dietary In addition to the of lipid in we found that a diet rich in mmBCFAs, can not but also glucose This the that is of The of as essential for dietary glucose required the of stable measurements not this Although this the unique of the stable isotope feeding it is to that this has The stable isotope used to detect with it on the of and under stable isotope labeling studies of the membrane in to and it is that the response observed or it has been shown that mmBCFAs are in In the mmBCFAs were in with M. J.L. J. N. J.D. P. et branched-chain fatty acid in 2018; Scholar). The of the role of in the of it critical to the between PAQR-2 and mmBCFAs in and to mmBCFAs impact survival with glucose. In we have identified a novel role for mmBCFAs under elevated dietary glucose stable and mass the were wild-type nematodes from the C. elegans and were from the C. elegans In order to the were to and the were in bacteria on were used to the elo-5, and from the were on R.S. J. in Caenorhabditis Scholar). subtilis was by the lab and was in the and the used with The were to a of glucose or glucose by a glucose to The were with bacteria the worms on were to for the glucose were 13C stable as in the were to with control or the in the of was added to the the bacteria were R. E. M. Ståhlman M. Borén J. Pilon M. The and Caenorhabditis elegans PAQR-2 membrane by saturated fatty Genet. 2017; Scholar). to the bacteria was with and in or and on the bacteria with the control were a of to were a of worms for the nematodes were to for quantify survival on bacteria elo-5, were and worms were for nematodes were to and the of was by with a subtilis in a with bacteria and as with the were with the established in (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google Scholar). and were with to allow the of bacteria for the bacteria were and in in the of A bacteria was to and to from were and stable isotope labeling of bacteria for The of was by worms were and lipid and analysis by The were the In of was the The were and was used for the on a were the and was by a on in a new lipid was and and were for (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google Scholar, 15Dancy B.C.R. Chen S. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar). lipid and were added to and was with for lipids in of were and and were and were and in of in for to fatty acid to on We the of in by the under the of fatty acid in the and as as in (14Perez C.L. Van Gilst M.R. A 13C isotope labeling strategy reveals the influence of insulin signaling on lipogenesis in C. elegans.Cell Metab. 2008; 8: 266-274Google Scholar). with stable for the analysis of the of or of major to the the were and to the incorporation of of or as et of the lipid composition and 13C labeling the of the and were used to between the fatty in this is in the This The that they have of with the of this We to the of the Olsen lab for and on the We also to the Caenorhabditis the of for nematode C. P. A. F. C. V. and C. P. A. F. C. C. and A. J. M. C. P. A. F. C. M. A. C. A. J. M. C. P. C. P. M. A. and C. P. C. P. M. A. and C. P. and This was by the of Health and were by the is by of

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