Reference number : GC-2017-Q1-02188, amendment number : , agreement type : G, recipient legal name : Lehner, Richard (University of Alberta), recipient country : CA, recipient province : nan, recipient city : Alberta, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Lipid droplets (LDs) are organelles present in essentially all organisms. LDs are composed of a hydrophobic core of lipids, mainly triacylglycerol (TG) and some steryl esters (SE), which is surrounded by a monolayer of amphipathic phospholipids and decorated with LD-associated proteins. LDs serve as energy (fuel) storage but also play an important role in cell signaling, viral infection and protein degradation. LD biogenesis in eukaryotes initiates from the endoplasmic reticulum (ER) where TG and SE are synthesized. However, little is known about the mechanism by which LDs accrue TG and grow in size. The liver is a central organ for energy metabolism because TG stored in LDs are either oxidized to generate energy (ATP and ketone bodies) in mitochondria or are assembled into very-low density lipoproteins (VLDL) that are then secreted into the blood. Ketone bodies and VLDL-TG serve as an important source of energy for extra-hepatic tissues. The liver contains two enzymes that are responsible TG synthesis: diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2). We have shown that TG synthesized by DGAT1 in hepatocytes is deposited in small LDs that are preferentially used for oxidation, while TG synthesized by DGAT2 is incorporated into large LDs that are preferentially used for VLDL assembly. Therefore, hepatic LD dynamics appear to be very complex. 
 
We hypothesize that hepatic LDs acquire distinct set of proteins and enzymes according to the nutritional needs of the animal (during fasting or refeeding). We also hypothesize that LDs containing TG synthesized by DGAT1 have a distinct set of proteins and possibly lipid composition from those synthesized by DGAT2. 
 
Objectives: 
1) Determine protein and lipid composition of hepatic LDs during various stages of LD synthesis. Methods: Primary mouse hepatocytes will be incubated with fatty acids together with fluorescently tagged fatty acids (tracer) in the presence or absence of DGAT1 or DGAT2 inhibitors. Incubations will be stopped at various time points, LDs will be isolated by centrifugation, sorted by flow cytometry and analyzed for protein and lipid composition. 
2) Characterize mouse liver LD protein and lipid composition in fasted and refed states. Methods: Mice will be either fasted overnight or fasted overnight followed by 6 hours refeeding. Livers will be harvested, lipid droplets isolated and subjected to detailed proteomic (mass spectrometry) and lipidomics (liquid chromatography-mass spectrometry) analyses. 
3) Mechanism of targeting of LD-associated proteins to LDs containing TG synthesized by either DGAT1 or DGAT2 (using specific DGAT1 and DGAT2 inhibitors) will be analyzed. 
 
Expected results: We expect to significantly advance knowledge about hepatic LD synthesis and dynamics. Gaining this knowledge is of fundamental importance because hepatic LDs play a central role in the regulation of fuel supply to other tissues., program purpose french : Lipid droplets (LDs) are organelles present in essentially all organisms. LDs are composed of a hydrophobic core of lipids, mainly triacylglycerol (TG) and some steryl esters (SE), which is surrounded by a monolayer of amphipathic phospholipids and decorated with LD-associated proteins. LDs serve as energy (fuel) storage but also play an important role in cell signaling, viral infection and protein degradation. LD biogenesis in eukaryotes initiates from the endoplasmic reticulum (ER) where TG and SE are synthesized. However, little is known about the mechanism by which LDs accrue TG and grow in size. The liver is a central organ for energy metabolism because TG stored in LDs are either oxidized to generate energy (ATP and ketone bodies) in mitochondria or are assembled into very-low density lipoproteins (VLDL) that are then secreted into the blood. Ketone bodies and VLDL-TG serve as an important source of energy for extra-hepatic tissues. The liver contains two enzymes that are responsible TG synthesis: diacylglycerol acyltransferases 1 and 2 (DGAT1 and DGAT2). We have shown that TG synthesized by DGAT1 in hepatocytes is deposited in small LDs that are preferentially used for oxidation, while TG synthesized by DGAT2 is incorporated into large LDs that are preferentially used for VLDL assembly. Therefore, hepatic LD dynamics appear to be very complex. 
 
We hypothesize that hepatic LDs acquire distinct set of proteins and enzymes according to the nutritional needs of the animal (during fasting or refeeding). We also hypothesize that LDs containing TG synthesized by DGAT1 have a distinct set of proteins and possibly lipid composition from those synthesized by DGAT2. 
 
Objectives: 
1) Determine protein and lipid composition of hepatic LDs during various stages of LD synthesis. Methods: Primary mouse hepatocytes will be incubated with fatty acids together with fluorescently tagged fatty acids (tracer) in the presence or absence of DGAT1 or DGAT2 inhibitors. Incubations will be stopped at various time points, LDs will be isolated by centrifugation, sorted by flow cytometry and analyzed for protein and lipid composition. 
2) Characterize mouse liver LD protein and lipid composition in fasted and refed states. Methods: Mice will be either fasted overnight or fasted overnight followed by 6 hours refeeding. Livers will be harvested, lipid droplets isolated and subjected to detailed proteomic (mass spectrometry) and lipidomics (liquid chromatography-mass spectrometry) analyses. 
3) Mechanism of targeting of LD-associated proteins to LDs containing TG synthesized by either DGAT1 or DGAT2 (using specific DGAT1 and DGAT2 inhibitors) will be analyzed. 
 
Expected results: We expect to significantly advance knowledge about hepatic LD synthesis and dynamics. Gaining this knowledge is of fundamental importance because hepatic LDs play a central role in the regulation of fuel supply to other tissues., agreement title english : Dynamics of liver lipid droplets, agreement title french : Dynamics of liver lipid droplets, agreement number : RGPIN, agreement value : 125000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada