scholarly journals Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

2021 ◽  
pp. mbc.E20-11-0695
Author(s):  
Santosh Adhikari ◽  
Joe Moscatelli ◽  
Elias M. Puchner
Keyword(s):  
Fatty Acid ◽  
Single Molecule ◽  
Lipid Droplets ◽  
Fold Increase ◽  
Yeast Cells ◽  
Exogenous Fatty Acid ◽  
Localization Microscopy ◽  
Fatty Acid Chain ◽  
Chain Lengths ◽  
Live Yeast

Lipid droplets (LDs) are dynamic organelles for lipid storage and homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs and enzymes required for LD growth and turnover. The small size of LDs precludes the observation of their associated enzyme densities and dynamics with conventional fluorescence microscopy. Here, we employ quantitative photo-activated localization microscopy to study the density of the fatty acid activating enzyme Faa4 on LDs in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where their emergence and expansion is mediated by the highest observed Faa4 densities. During transition to the stationary phase LDs with a ∼2-fold increased surface area translocate to the vacuolar surface and lumen and exhibit a ∼2.5-fold increase in Faa4 density. The increased Faa4 density on LDs further suggests its role in LD expansion, is caused by its ∼5-fold increased expression level and is specific to exogenous fatty acid chain-lengths. When lipolysis is induced by refreshed medium, Faa4 shuttles through ER- and lipophagy to the vacuole, where it may activate fatty acids for membrane expansion and degrade to reset cellular Faa4 abundance to levels in the log phase. [Media: see text] [Media: see text] [Media: see text] [Media: see text]

scholarly journals Quantitative live-cell PALM reveals nanoscopic Faa4 redistributions and dynamics on lipid droplets during metabolic transitions of yeast

2020 ◽  
Author(s):  
Santosh Adhikari ◽  
Joe Moscatelli ◽  
Elias M. Puchner
Keyword(s):  
Stationary Phase ◽  
Single Molecule ◽  
Lipid Droplets ◽  
Fold Increase ◽  
Diffraction Limit ◽  
Optical Diffraction ◽  
Yeast Cells ◽  
Lipid Homeostasis ◽  
Localization Microscopy ◽  
Live Yeast

AbstractLipid droplets (LDs) are dynamic lipid storage organelles needed for lipid homeostasis. Cells respond to metabolic changes by regulating the spatial distribution of LDs, as well as enzymes required for LD growth and turnover. Due to LD size below the optical diffraction limit, bulk fluorescence microscopy cannot observe the density and dynamics of specific LD enzymes. Here, we employ quantitative photo-activated localization microscopy (PALM) to study the density of the fatty acid activating protein Faa4 on LDs during log, stationary and lag phases in live yeast cells with single-molecule sensitivity and 30 nm resolution. During the log phase LDs co-localize with the Endoplasmic Reticulum (ER) where the highest Faa4 densities are measured. During transition to the stationary phase LDs translocate to the vacuolar surface and lumen with a ~2-fold increased surface area and a ~2.5-fold increase in Faa4 density, suggesting its role in LD expansion. The increased Faa4 density on LDs is caused by its ~5-fold increased expression level. When lipolysis is induced in stationary-phase cells by diluting them for 2 hrs in fresh medium, Faa4 shuttles to the vacuole through the two observed routes of ER- and lipophagy. The observed vacuolar localization of Faa4 may help activating fatty acids for membrane expansion and reduces Faa4 expression to levels found in the log phase.

scholarly journals Intracellular localization of AMP deaminase and its novel role in BCAA and lipid metabolism in diabetic cardiomyopathy

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
T Ogawa ◽  
H Kouzu ◽  
A Osanami ◽  
Y Tatekoshi ◽  
H Oshima ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Lipid Metabolism ◽  
Fatty Acid Oxidation ◽  
Lipid Droplets ◽  
Intracellular Localization ◽  
Fold Increase ◽  
Acid Oxidation ◽  
Transcriptional Level ◽  
Amp Deaminase ◽  
Funding Sources

Abstract Background A metabolomic study in the human heart suggested a pivotal role of amino acid (AA) metabolism in fatty acid oxidation, which is dysregulated in type 2 diabetes mellitus (T2DM) and heart failure. We previously reported that aberrant up-regulation of AMP deaminase 3 (AMPD3) impairs cardiac energetics in T2DM hearts, and AMPD3 was recently shown to be activated by fasting and to promote AA metabolism and fatty acid oxidation in skeletal muscle. A sodium glucose cotransporter 2 inhibitor (SGLT2i) has been shown to augment systemic AA metabolism, but its effect on cardiac AA metabolism remains unknown. Purpose We hypothesized that AMPD3 has a role in AA and lipid metabolism in cardiomyocytes and that the protective effect of an SGLT2i in diabetic hearts is mediated by modification of AA and lipid metabolism. Methods and results Proteomic analyses of AMPD3 immunoprecipitates in rat hearts revealed that AMPD3 interacted with the E1α and E2 components of the BCKDH complex, a rate-limiting enzyme of branched-chain AA (BCAA) catabolism. Immunoblotting using subcellular fractions revealed that BCKDH localized not only in the mitochondria matrix but also in the cytosol and endoplasmic reticulum (ER) and that AMPD3 interacted with BCKDH in the cytosol and ER. Despite comparable expression of BCKDH components and phosphorylation of E1α at Ser293, significant accumulation of BCAA was observed in T2DM rats (OLETF; 317±30 nmol/g) compared to that in control rats (LETO; 213±16 nmol/g), and the accumulation of BCAA was accompanied by up-regulation of AMPD3 in the cytosol and ER by 98% and 231%, respectively. In cardiomyocytes, disruption of BCAA catabolism by knockdown of BCKDH-E1α resulted in a 5.8-fold increase in AMPD3 at the transcriptional level and blunted lipid droplet biogenesis in response to a long-chain fatty acid challenge. Next, myocardial infarction (MI) was induced in LETO and OLETF pretreated with empagliflozin (10 mg/kg/day, 14 days) or a vehicle. Pathway analysis of cardiac metabolites revealed arginine biosynthesis and BCAA metabolism as the most significantly changed pathways with empagliflozin, with BCAA (791±187 nmol/g), glutamate, glutamine and urea being significantly increased. Empagliflozin restored myocardial ATP and survival after MI in OLETF to levels comparable to those in LETO. Electron microscopy showed a significantly higher prevalence of myocardium lipid droplets in OLETF, which was further increased by empagliflozin. Conclusions The results support the hypotheses that imbalance of extra-mitochondrial AMPD3-BCKDH interaction underlies dysregulated BCAA metabolism in T2DM hearts and that activation of cardiac AA metabolism by an SGLT2i normalizes fatty acid overload through sequestration into intracellular lipid droplets. FUNDunding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Boehringer Ingelheim

scholarly journals Mitochondrial fission governed by Drp1 regulates exogenous fatty acid usage and storage

2020 ◽  
Author(s):  
Jae Eun Song ◽  
Tiago C. Alves ◽  
Bernardo Stutz ◽  
Matija Sestan-Pesa ◽  
Nicole Kilian ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Oxidation ◽  
Lipid Droplets ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Fatty Acid Content ◽  
Acid Oxidation ◽  
Exogenous Fatty Acid ◽  
And Storage

ABSTRACTThe bioenergetic function of mitochondrial fission is associated with uncoupled respiration or elimination of damaged mitochondria to maintain a healthy mitochondrial population. In the presence of a high abundance of exogenous fatty acids, cells can either store fatty acids in lipid droplets or oxidize them in mitochondria. Even though carnitine palmitoyltransferase-1 (CPT1) controls the respiratory capacity of mitochondria in fatty acid oxidation, we observed that it did not dictate the balance of storage and usage of lipids in HeLa cells. On the other hand, inhibition of mitochondrial fission by silencing dynamic-related protein 1 (DRP1) resulted in an increase in fatty acid content of lipid droplets and a decrease in fatty acid oxidation. Mitochondrial fission was not only reflective of the amount of exogenous fatty acid being processed by mitochondria, but also found to be actively involved in the distribution of fatty acids between mitochondria and lipid droplets. Our data reveals a novel function for mitochondrial fission in balancing exogenous fatty acids between usage and storage, assigning a role for mitochondrial dynamics in control of intracellular fuel utilization and partitioning.

scholarly journals Cyanidin-3-glucoside Lipophilic Conjugates for Topical Application: Tuning the Antimicrobial Activities with Fatty Acid Chain Length

Processes ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 340
Author(s):  
Hélder Oliveira ◽  
Patrícia Correia ◽  
Lucinda J. Bessa ◽  
Marta Guimarães ◽  
Paula Gameiro ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
Fatty Acid ◽  
Antioxidant Capacity ◽  
Multidrug Resistant ◽  
Antimicrobial Activities ◽  
Aliphatic Chain ◽  
Fatty Acid Chain ◽  
The Stability ◽  
Application Tuning ◽  
Chain Lengths

Background: Natural anthocyanins present a low solubility in lipophilic media, which compromises their effective application in lipophilic systems. In this work, cyanidin-3-O-glucoside (Cy3glc) was esterified by the addition of fatty acids with increasing chain-lengths and a structure-activity relationship was performed towards the description of the best analog for skin-care applications. Methods: By enzymatic hemi-synthesis, it was possible to obtain 5 structurally related derivatives of cyanidin-3-O-glucoside with successive C2 increments in the aliphatic chain. The stability in hanks buffer and DMEM with or without FBS was followed by HPLC. The cytotoxicity against keratinocytes was evaluated by MTT assay. The antioxidant capacity was determined by using the fluorescent probe DCF-DA. The effect on enzyme activity was evaluated towards tyrosinase, collagenase, and elastase enzymes by colorimetric assays. MIC and MBC values were obtained against reference strains and against multidrug-resistant isolates. Results: In physiological conditions, cy3glc−fatty acid derivatives are more stable and may be converted to the native anthocyanin. The 5 conjugates showed lower antioxidant capacity and enzymatic inhibitory activities in comparison to the anthocyanin precursor. However, concerning the antibacterial activity, the insertion of a fatty acid chain sprouted the antibacterial activity, showing a clear biphasic effect and a more effective effect on Gram-positive bacteria. Conclusions: Cy3glc-C10 was the most effective compound considering the antimicrobial activity, although a general reduction was observed among the other activities evaluated. This work prompt further assays with a different panoply of derivatives ranging other features including saturation vs. unsaturation, even vs. odd carbon content and linear vs. branched.

scholarly journals Evidence that the Essential Response Regulator YycF in Streptococcus pneumoniae Modulates Expression of Fatty Acid Biosynthesis Genes and Alters Membrane Composition

2005 ◽  
Vol 187 (7) ◽  
pp. 2357-2367 ◽  
Author(s):  
M. Luz Mohedano ◽  
Karin Overweg ◽  
Alicia de la Fuente ◽  
Mark Reuter ◽  
Silvia Altabe ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Streptococcus Pneumoniae ◽  
Fatty Acid Biosynthesis ◽  
Membrane Lipids ◽  
Response Regulator ◽  
Membrane Composition ◽  
Acid Biosynthesis ◽  
Fatty Acid Chain ◽  
Genome Wide ◽  
Chain Lengths

ABSTRACT The YycFG two-component system, originally identified in Bacillus subtilis, is highly conserved among gram-positive bacteria with low G+C contents. In Streptococcus pneumoniae, the YycF response regulator has been reported to be essential for cell growth, but the signal to which it responds and the gene members of the regulon remain unclear. In order to investigate the role of YycFG in S. pneumoniae, we increased the expression of yycF by using a maltose-inducible vector and analyzed the genome-wide effects on transcription and protein expression during the course of yycF expression. The induction of yycF expression increased histidine kinase yycG transcript levels, suggesting an autoregulation of the yycFG operon. Evidence from both proteomic and microarray transcriptome studies as well as analyses of membrane fatty acid composition indicated that YycFG is involved in the regulation of fatty acid biosynthesis pathways and in determining fatty acid chain lengths in membrane lipids. In agreement with recent transcriptome data on pneumococcal cells depleted of YycFG, we also identified several other potential members of the YycFG regulon that are required for virulence and cell wall biosynthesis and metabolism.

The Arabidopsis acyl-CoA oxidase gene family

2000 ◽  
Vol 28 (6) ◽  
pp. 755-757 ◽  
Author(s):  
P. J. Eastmond ◽  
M. Hooks ◽  
I. A. Graham
Keyword(s):  
Fatty Acid ◽  
Full Range ◽  
Biochemical Properties ◽  
Oxidase Gene ◽  
Substrate Specificities ◽  
Fatty Acid Chain ◽  
Acyl Coa Oxidase ◽  
Chain Lengths ◽  
Peroxisomal Fatty Acid

A family of acyl-CoA oxidase isozymes catalyse the first step in the peroxisomal fatty acid β-oxidation spiral. Our group and others have recently characterized four genes from this family in the model oilseed Arabidopsis. These genes encode isozymes with different acyl-CoA substrate specificities, which together encompass the full range of fatty acid chain lengths that exist in vivo. Here we review the biochemical properties and physiological roles of the acyl-CoA oxidase isozymes.

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