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Fabrice Rebeille - One of the best experts on this subject based on the ideXlab platform.
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lc ms ms versus tlc plus gc methods consistency of Glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation
PLOS ONE, 2017Co-Authors: Juliette Jouhet, Eric Maréchal, Josselin Lupette, Olivier Clerc, Leonardo Magneschi, Mariette Bedhomme, Severine Collin, Fabrice RebeilleAbstract:Methods to analyze lipidomes have considerably evolved, more and more based on mass spectrometry technics (LC-MS/MS). However, accurate quantifications using these methods require 13C-labeled standards for each lipid, which is not feasible because of the very large number of molecules. Thus, quantifications rely on standard molecules representative of a whole class of lipids, which might lead to false estimations of some molecular species. Here, we determined and compared Glycerolipid distributions from three different types of cells, two microalgae (Phaeodactylum tricornutum, Nannochloropsis gaditana) and one higher plant (Arabidopsis thaliana), using either LC-MS/MS or Thin Layer Chromatography coupled with Gas Chromatography (TLC-GC), this last approach relying on the precise quantification of the fatty acids present in each Glycerolipid class. Our results showed that the Glycerolipid distribution was significantly different depending on the method used. How can one reconcile these two analytical methods? Here we propose that the possible bias with MS data can be circumvented by systematically running in tandem with the sample to be analyzed a lipid extract from a qualified control (QC) of each type of cells, previously analyzed by TLC-GC, and used as an external standard to quantify the MS results. As a case study, we applied this method to compare the impact of a nitrogen deficiency on the three types of cells.
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membrane Glycerolipid remodeling triggered by nitrogen and phosphorus starvation in phaeodactylum tricornutum
Plant Physiology, 2015Co-Authors: Heni Abida, Maryse A. Block, Olivier Bastien, Linajuana Dolch, Valeria Villanova, Melissa Conte, Giovanni Finazzi, Leila Tirichine, Chris Bowler, Fabrice RebeilleAbstract:Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane Glycerolipids have not yet been studied. Our goal was first to define a reference for the Glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the Glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the Glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.
Juliette Jouhet - One of the best experts on this subject based on the ideXlab platform.
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interplay between jasmonic acid phosphate signaling and the regulation of Glycerolipid homeostasis in arabidopsis
Plant and Cell Physiology, 2019Co-Authors: Florian Chevalier, Juliette Jouhet, Maryse A. Block, Laura Cuyas, Vali Rie Gros, Serge Chiarenza, David Secco, James Whelan, Khawla Seddiki, Laurent NussaumeAbstract:: Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane Glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of Glycerolipid remodeling in response to Pi deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and Glycerolipid remodeling in wild-type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine, phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi deprivation, JA seems rather associated with a specific MGDG increase. Following Pi resupply, whereas the expression of Glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, that is, downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.
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lc ms ms versus tlc plus gc methods consistency of Glycerolipid and fatty acid profiles in microalgae and higher plant cells and effect of a nitrogen starvation
PLOS ONE, 2017Co-Authors: Juliette Jouhet, Eric Maréchal, Josselin Lupette, Olivier Clerc, Leonardo Magneschi, Mariette Bedhomme, Severine Collin, Fabrice RebeilleAbstract:Methods to analyze lipidomes have considerably evolved, more and more based on mass spectrometry technics (LC-MS/MS). However, accurate quantifications using these methods require 13C-labeled standards for each lipid, which is not feasible because of the very large number of molecules. Thus, quantifications rely on standard molecules representative of a whole class of lipids, which might lead to false estimations of some molecular species. Here, we determined and compared Glycerolipid distributions from three different types of cells, two microalgae (Phaeodactylum tricornutum, Nannochloropsis gaditana) and one higher plant (Arabidopsis thaliana), using either LC-MS/MS or Thin Layer Chromatography coupled with Gas Chromatography (TLC-GC), this last approach relying on the precise quantification of the fatty acids present in each Glycerolipid class. Our results showed that the Glycerolipid distribution was significantly different depending on the method used. How can one reconcile these two analytical methods? Here we propose that the possible bias with MS data can be circumvented by systematically running in tandem with the sample to be analyzed a lipid extract from a qualified control (QC) of each type of cells, previously analyzed by TLC-GC, and used as an external standard to quantify the MS results. As a case study, we applied this method to compare the impact of a nitrogen deficiency on the three types of cells.
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Role of the Envelope Membranes in Chloroplast Glycerolipid Biosynthesis
Photosynthesis, 2011Co-Authors: Maryse A. Block, Juliette Jouhet, Eric Maréchal, Olivier Bastien, Jacques JoyardAbstract:All plastid membranes are characterized by the presence of large amounts of galactolipids. The galactolipid content of thylakoids is especially high since galactolipids represent up to 80% of the membrane Glycerolipids, out of which monogalactosyldiacylglycerol (MGDG) constitutes the main part (50%). In its first part, this chapter describes the structure of the main Glycerolipids present in plastids, particularly of galactolipids and details their subcellular localization. Functional analysis of mutants deleted of specific lipids and structural data obtained from Photosystem I and Photosystem II crystallization are presented to characterize the specific role of each Glycerolipid. In the second part, this chapter summarizes our current understanding of the role of the chloroplast envelope in Glycerolipid biogenesis and points out the emerging lipid trafficking mechanisms that envelope membranes take part in. The building up of eukaryotic and prokaryotic lipid structures proceeds from at least two distinct pathways requiring different trafficking of lipids. Whereas synthesis of prokaryotic Glycerolipids is entirely realized in the envelope, synthesis of eukaryotic Glycerolipids relies on the transfer of precursors from the endoplasmic reticulum to the envelope. The final assembly of Glycerolipids is made in the envelope membranes prior to transfer to thylakoids membranes. A special focus is given to current results concerning MGDG and digalactosyldiacylglycerol (DGDG) synthesis in plants grown in phosphate-deficient conditions.
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Glycerolipid transfer for the building of membranes in plant cells.
Progress in Lipid Research, 2007Co-Authors: Juliette Jouhet, Eric Maréchal, Maryse BlockAbstract:Membranes of plant organelles have specific Glycerolipid compositions. Selective distribution of lipids at the levels of subcellular organelles, membrane leaflets and membrane domains reflects a complex and finely tuned lipid homeostasis. Glycerolipid neosynthesis occurs mainly in plastid envelope and endoplasmic reticulum membranes. Since most lipids are not only present in the membranes where they are synthesized, one cannot explain membrane specific lipid distribution by metabolic processes confined in each membrane compartment. In this review, we present our current understanding of Glycerolipid trafficking in plant cells. We examine the potential mechanisms involved in lipid transport inside bilayers and from one membrane to another. We survey lipid transfers going through vesicular membrane flow and those dependent on lipid transfer proteins at membrane contact sites. By introducing recently described membrane lipid reorganization during phosphate deprivation and recent developments issued from mutant analyses, we detail the specific lipid transfers towards or outwards the chloroplast envelope.
Robert E. Anderson - One of the best experts on this subject based on the ideXlab platform.
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Biosynthesis of docosahexaenoate-containing Glycerolipid molecular species in the retina.
Journal of Molecular Neuroscience, 2001Co-Authors: Feng Li, Huiming Chen, Robert E. AndersonAbstract:Vertebrate retinas are highly enriched in long-chain polyunsaturated fatty acids (PUFA), especially docosahexaenoic acid (22:6n-3, DHA). In the present study, we investigated the role of de novo synthesis in the enrichment of 22:6n-3 in characteristic molecular species of retinal Glycerolipids. Following the incubation of fresh dark-adapted retinas with [2-3H]-glycerol, individual Glycerolipids were isolated and converted into either diacylglycerol acetates (DGAC) or diacylglycerol benzoates (DGBZ), followed by high-performance liquid chromatography (HPLC) and flow-through radioactivity detection. Total lipids from rat retinas incubated with [3H]-glycerol were analyzed. Unlike what was observed with frog retinas, relative larger of amounts of di-22:6 molecular species were synthesized de novo. In both rat and frog retinas, there was synthesis of Glycerolipid molecular species containing two PUFA (one of which was 22:6) in larger amounts than predicted by their steady-state mass levels. These results demonstrate that the unique molecular species of retinal Glycerolipids are derived only in part through de novo synthesis, but that molecular rearrangement (remodeling) and differential turnover must also play a role in maintaining the high levels of 22:6 found in rod phohtoreceptor outer segments (ROS) membranes.
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Unique molecular species composition of Glycerolipids of frog rod outer segments
Experimental Eye Research, 1990Co-Authors: Hong-gwan Choe, Robert E. AndersonAbstract:Abstract The composition and metabolism of molecular species of Glycerolipids, including phosphatidic acid (PA), phosphatidylinositol (PI) and diacylglycerol (DG), were studied in four frog retinal fractions prepared by discontinuous sucrose gradient centrifugation. Six Glycerolipid classes were isolated from the lipid extracts of each fraction and converted to their corresponding 1,2-diacylglycerol acetates by acetolysis for quantitation of their molecular species by HPLC. Rod outer segments (ROS) showed a distinctive molecular species composition in all Glycerolipid classes except phosphatidylcholine (PC). The relative amounts of dipolyunsaturated species in ROS were higher in phosphatidylethanolamine (PE), phosphatidylserine (PS), and PA, compared to the other retinal fractions. PI and DG of ROS had a similar molecular species composition and contained only small amounts of dipolyunsaturated species. A unique feature of the molecular species of ROS PI and DG was that they had high amounts of species containing docosahexaenoic acid (22:6ω3), while PI and DG from the other retinal membranes consisted mostly of species containing arachidonic acid (20:4ω6). Following in vitro incubation of frog retinas with [2- 3 H] glycerol, the mass and radioactivity distributions among molecular species were determined following HPLC fractionation. The unique species composition of PS in ROS is determined mainly by selective translocation from the inner segments to ROS, since the dpm %, representative of newly synthesized species, was similar to the steady state mole %. In contrast, the dpm % of PE and PA in ROS did not resemble their steady state composition (mole %). Instead, these were similar to newly synthesized species composition of the same Glycerolipid classes in the other membrane fractions. This suggests that the distinctive species composition of PE and PA in ROS is determined not by selective translocation from the inner segments, but by remodeling processes taking place in the ROS. However, both the selective translocation and the remodeling processes seem to be responsible for the molecular species compositions of PC, PI, and DG in ROS. The dpm %s of these in ROS were not similar to those of the same Glycerolipid classes in the other membrane fractions indicating the selective translocation of the newly synthesized species to the ROS. In addition, the remodeling processes on these Glycerolipids were suggested because the dpm %s were not similar to the steady state species compositions of PC, PI, and DG in ROS.
Maryse A. Block - One of the best experts on this subject based on the ideXlab platform.
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interplay between jasmonic acid phosphate signaling and the regulation of Glycerolipid homeostasis in arabidopsis
Plant and Cell Physiology, 2019Co-Authors: Florian Chevalier, Juliette Jouhet, Maryse A. Block, Laura Cuyas, Vali Rie Gros, Serge Chiarenza, David Secco, James Whelan, Khawla Seddiki, Laurent NussaumeAbstract:: Jasmonic acid (JA) biosynthesis and signaling are activated in Arabidopsis cultivated in phosphate (Pi) deprived conditions. This activation occurs mainly in photosynthetic tissues and is less important in roots. In leaves, the enhanced biosynthesis of JA coincides with membrane Glycerolipid remodeling triggered by the lack of Pi. We addressed the possible role of JA on the dynamics and magnitude of Glycerolipid remodeling in response to Pi deprivation and resupply. Based on combined analyses of gene expression, JA biosynthesis and Glycerolipid remodeling in wild-type Arabidopsis and in the coi1-16 mutant, JA signaling seems important in the determination of the basal levels of phosphatidylcholine, phosphatidic acid (PA), monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol. JA impact on MGDG steady state level and fluctuations seem contradictory. In the coi1-16 mutant, the steady state level of MGDG is higher, possibly due to a higher level of PA in the mutant, activating MGD1, and to an increased expression of MGD3. These results support a possible impact of JA in limiting the overall content of this lipid. Concerning lipid variations, upon Pi deprivation, JA seems rather associated with a specific MGDG increase. Following Pi resupply, whereas the expression of Glycerolipid remodeling genes returns to basal level, JA biosynthesis and signaling genes are still upregulated, likely due to a JA-induced positive feedback remaining active. Distinct impacts on enzymes synthesizing MGDG, that is, downregulating MGD3, possibly activating MGD1 expression and limiting the activation of MGD1 via PA, might allow JA playing a role in a sophisticated fine tuning of galactolipid variations.
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membrane Glycerolipid remodeling triggered by nitrogen and phosphorus starvation in phaeodactylum tricornutum
Plant Physiology, 2015Co-Authors: Heni Abida, Maryse A. Block, Olivier Bastien, Linajuana Dolch, Valeria Villanova, Melissa Conte, Giovanni Finazzi, Leila Tirichine, Chris Bowler, Fabrice RebeilleAbstract:Diatoms constitute a major phylum of phytoplankton biodiversity in ocean water and freshwater ecosystems. They are known to respond to some chemical variations of the environment by the accumulation of triacylglycerol, but the relative changes occurring in membrane Glycerolipids have not yet been studied. Our goal was first to define a reference for the Glycerolipidome of the marine model diatom Phaeodactylum tricornutum, a necessary prerequisite to characterize and dissect the lipid metabolic routes that are orchestrated and regulated to build up each subcellular membrane compartment. By combining multiple analytical techniques, we determined the Glycerolipid profile of P. tricornutum grown with various levels of nitrogen or phosphorus supplies. In different P. tricornutum accessions collected worldwide, a deprivation of either nutrient triggered an accumulation of triacylglycerol, but with different time scales and magnitudes. We investigated in depth the effect of nutrient starvation on the Pt1 strain (Culture Collection of Algae and Protozoa no. 1055/3). Nitrogen deprivation was the more severe stress, triggering thylakoid senescence and growth arrest. By contrast, phosphorus deprivation induced a stepwise adaptive response. The time scale of the Glycerolipidome changes and the comparison with large-scale transcriptome studies were consistent with an exhaustion of unknown primary phosphorus-storage molecules (possibly polyphosphate) and a transcriptional control of some genes coding for specific lipid synthesis enzymes. We propose that phospholipids are secondary phosphorus-storage molecules broken down upon phosphorus deprivation, while nonphosphorus lipids are synthesized consistently with a phosphatidylglycerol-to-sulfolipid and a phosphatidycholine-to-betaine lipid replacement followed by a late accumulation of triacylglycerol.
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Glycerolipid Biosynthesis and Chloroplast Biogenesis
Plastid Development in Leaves during Growth and Senescence, 2013Co-Authors: Maryse A. Block, Emmanuelle Dubots, Eric MaréchalAbstract:Chloroplast membranes are enriched with galactoGlycerolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG). These galactolipids do not contain phosphorus and chloroplast membranes are therefore very poor in phospholipids, primarily represented as a special trans Δ3-hexadecenoic acid-containing phosphatidylglycerol (PG), a finely compartmentalized amount of phosphatidylcholine (PC) and a very low and transitory level of phosphatidic acid (PA). The biogenesis of chloroplasts requires a highly efficient Glycerolipid-synthesis system for the development and functioning of both the chloroplast envelope and the thylakoids. Photosynthesis notably relies on the presence of galactolipids and PG. In this chapter, we review the properties of these Glycerolipids, their role in photosynthesis and the characteristics of their synthetic pathways. We focus on the role of MGDG synthase in chloroplast biogenesis, the enzyme functioning in the chloroplast envelope membrane, and different mechanisms involved in its regulation. The regulation of MGDG synthase by the phospholipids, PG and PA, is discussed in terms of membrane homeostasis and plant cell biology.
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Role of the Envelope Membranes in Chloroplast Glycerolipid Biosynthesis
Photosynthesis, 2011Co-Authors: Maryse A. Block, Juliette Jouhet, Eric Maréchal, Olivier Bastien, Jacques JoyardAbstract:All plastid membranes are characterized by the presence of large amounts of galactolipids. The galactolipid content of thylakoids is especially high since galactolipids represent up to 80% of the membrane Glycerolipids, out of which monogalactosyldiacylglycerol (MGDG) constitutes the main part (50%). In its first part, this chapter describes the structure of the main Glycerolipids present in plastids, particularly of galactolipids and details their subcellular localization. Functional analysis of mutants deleted of specific lipids and structural data obtained from Photosystem I and Photosystem II crystallization are presented to characterize the specific role of each Glycerolipid. In the second part, this chapter summarizes our current understanding of the role of the chloroplast envelope in Glycerolipid biogenesis and points out the emerging lipid trafficking mechanisms that envelope membranes take part in. The building up of eukaryotic and prokaryotic lipid structures proceeds from at least two distinct pathways requiring different trafficking of lipids. Whereas synthesis of prokaryotic Glycerolipids is entirely realized in the envelope, synthesis of eukaryotic Glycerolipids relies on the transfer of precursors from the endoplasmic reticulum to the envelope. The final assembly of Glycerolipids is made in the envelope membranes prior to transfer to thylakoids membranes. A special focus is given to current results concerning MGDG and digalactosyldiacylglycerol (DGDG) synthesis in plants grown in phosphate-deficient conditions.
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Structure, Distribution and Biosynthesis of Glycerolipids from Higher Plant Chloroplasts
Lipids in Photosynthesis: Structure Function and Genetics, 1998Co-Authors: Jacques Joyard, Eric Maréchal, Maryse A. Block, Christine Miège, Albert-jean Dorne, Roland DouceAbstract:Galactolipids (MGDG and DGDG), sulfolipid and phosphatidylglycerol are the main constituents of plastid membranes. Glycerolipid biosynthesis requires first the assembly of glycerol and esterification by fatty acids at the sn-1 and sn-2 positions of the glycerol backbone. Then, the sn-3 position of phosphatidic acid or diacylglycerol is modified, for instance by addition of a third fatty acid for triacylglycerol, of a galactose for galactolipids, of a sulfoquinovose for sulfolipid, and phosphorylglycerol for phosphatidylglycerol. Directly or indirectly, the compounds used for the biosynthesis of Glycerolipids derive from photosynthesis, i.e. from endogenous CO2 fixation by chloroplasts or from photosynthates produced in leaves. The two main MGDG molecular species found in chloroplasts have (a) 18:3 at both the sn-1 and sn-2 positions of the glycerol backbone, and (b) 18:3 and 16:3 respectively at the sn-1 and sn-2 positions of the glycerol backbone. The occurrence of such structures within plastid membranes reflects the existence of different pathways for the biosynthesis of these two types of molecules. Sulfolipid and phosphatidylglycerol molecular species also contain the typical structure of prokaryotic lipids with C16 fatty acids at the sn-2 position of glycerol. In contrast, a wide variety of diacylglycerol molecular species (i.e. with different acyl chain length and saturation levels at both sn positions) can be found in extremely variable amounts in envelope membranes where the synthesis of all typical plastid lipids takes place. Several enzymes, such as the inner envelope phosphatidate phosphatase and the outer envelope galactolipid:galactolipid galactosyltransferase, are involved in diacylglycerol formation, others, like the MGDG synthase or the sulfolipid synthase, use diacylglycerol within the inner envelope membrane as a substrate for the biosynthesis of membrane Glycerolipids. A puzzling question is how such enzymes could be involved in the formation of the characteristic structural features of chloroplast Glycerolipids and their final distribution within membranes. Although little molecular data are presently available on enzymes such as the phosphatidate phosphatase, the galactolipid:galactolipid galactosyltransferase or the MGDG synthase, detailed analysis of the biochemical properties of these key enzymes in galactolipid biosynthesis recently provided some clues to the problem. These observations suggest that the biochemical properties of the envelope MGDG synthase are highly responsible for the final MGDG molecular species found in plastid membranes.
Charles F Burant - One of the best experts on this subject based on the ideXlab platform.
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increased glucose metabolism and Glycerolipid formation by fatty acids and gpr40 receptor signaling underlies the fatty acid potentiation of insulin secretion
Journal of Biological Chemistry, 2014Co-Authors: Mahmoud Elazzouny, Charles R Evans, Mary K Treutelaar, Robert T Kennedy, Charles F BurantAbstract:Acute fatty acid (FA) exposure potentiates glucose-stimulated insulin secretion in β cells through metabolic and receptor-mediated effects. We assessed the effect of fatty acids on the dynamics of the metabolome in INS-1 cells following exposure to [U-13C]glucose to assess flux through metabolic pathways. Metabolite profiling showed a fatty acid-induced increase in long chain acyl-CoAs that were rapidly esterified with glucose-derived glycerol-3-phosphate to form lysophosphatidic acid, mono- and diacylglycerols, and other Glycerolipids, some implicated in augmenting insulin secretion. Glucose utilization and glycolytic flux increased, along with a reduction in the NADH/NAD+ ratio, presumably by an increase in conversion of dihydroxyacetone phosphate to glycerol-3-phosphate. The fatty acid-induced increase in glycolysis also resulted in increases in tricarboxylic cycle flux and oxygen consumption. Inhibition of fatty acid activation of FFAR1/GPR40 by an antagonist decreased Glycerolipid formation, attenuated fatty acid increases in glucose oxidation, and increased mitochondrial FA flux, as evidenced by increased acylcarnitine levels. Conversely, FFAR1/GPR40 activation in the presence of low FA increased flux into Glycerolipids and enhanced glucose oxidation. These results suggest that, by remodeling glucose and lipid metabolism, fatty acid significantly increases the formation of both lipid- and TCA cycle-derived intermediates that augment insulin secretion, increasing our understanding of mechanisms underlying β cell insulin secretion.