Neuroinflammation is a component of age-related neurodegenerative diseases and cognitive decline. Saturated (SFA) and monounsaturated (MUFA) fatty acids are bioactive molecules that may play different extrinsic and intrinsic roles in neuroinflammation, serving as exogenous ligands for cellular receptors, or endogenous components of cell structural, energetic and signaling pathways. We determined the fatty acyl profile of BV2 microglial cells before and after acute activation with lipopolysaccharide (LPS). We also investigated the effect of SFA and MUFA pretreatment on the production of an invasive, neurotoxic phenotype in BV2 cells. Acute activation of BV2 microglia resulted in an increase in the relative content of SFA (12:0, 16:0, 18:0, 20:0, 22:0, and 24:0 increased significantly), and a relative decrease in the content of MUFA (16:1n7, 18:1n7, 18:1n9, 20:1n9, 24:1n9 decreased significantly). In agreement, the major stearoyl-CoA desaturase (SCD) isoform in BV2 cells, SCD2, was significantly down-regulated by LPS. We next treated cells with SFA (16:0 or 18:0) or MUFA (16:1n7 or 18:1n9), and found that levels of secreted IL6 were increased, as was secreted MMP9-mediated proteolytic activity. To test the functional significance, we treated SH-SY5Y neuronal cells with conditioned medium from BV2 cells pretreated with fatty acids, and found a small but significant induction of cell death. Our findings suggest differential intrinsic roles for SFA and MUFA in activated microglial cells, but similar extrinsic roles for these fatty acid species in inducing activation. Expansion of SFA is important during microglial cell activation, but either supplemental SFA or MUFA may contribute to chronic low-grade neuroinflammation.
Tissue-specific cardiolipin fatty acyl profiles are achieved by remodeling of de novo synthesized cardiolipin, and four remodeling enzymes have thus far been identified. We studied the enzyme phospholipase A and acyltransferase 1 (PLAAT1), and we report the discovery that it has phosphatidylcholine (PC):monolysocardiolipin (MLCL) transacylase activity. Subcellular localization was analyzed by differential centrifugation and immunoblotting. Total levels of major phospholipids, and the fatty acyl profile of cardiolipin, were analyzed in HEK293 cells expressing murine PLAAT1 using gas chromatography. Apparent enzyme kinetics of affinity-purified PLAAT1 were calculated using radiochemical enzyme assays. This enzyme was found to localize predominantly to the endoplasmic reticulum (ER) but was detected at low levels in the mitochondria-associated ER matrix. Cells expressing PLAAT1 had higher levels of total cardiolipin, but not other phospholipids, and it was primarily enriched in the saturated fatty acids myristate, palmitate, and stearate, with quantitatively smaller increases in the n-3 polyunsaturated fatty acids linolenate, eicosatrienoate, and eicosapentanoate and the monounsaturated fatty acid erucate. Affinity-purified PLAAT1 did not catalyze the transacylation of MLCL using 1-palmitoyl-2-[14C]-linoleoyl-PC as an acyl donor. However, PLAAT1 had an apparent Vmax of 1.61 μmol/min/mg protein and Km of 126 μM using [9,10-3H]-distearoyl-PC as an acyl donor, and 0.61 μmol/min/mg protein and Km of 16 μM using [9,10-3H]-dioleoyl-PC. PLAAT1 is therefore a novel PC:MLCL transacylase.
Barth syndrome (BTHS) is caused by mutations in the TAZ gene encoding the cardiolipin remodeling enzyme, Tafazzin. The study objective was to quantitatively examine growth characteristics and mitochondrial morphology of transformed lymphoblast cell lines derived from five patients with BTHS relative to five healthy controls, as well as the therapeutic potential of oleoylethanolamide (OEA) and linoleoylethanolamide (LEA). These bioactive lipids both activate PPARα, which may be therapeutic. BTHS lymphoblasts grew more slowly than controls, suggesting lymphopenia merits clinical investigation. Treatment of BTHS lymphoblasts with OEA, but not LEA, significantly restored mitochondrial membrane potential, as well as colony growth in all BTHS lymphoblast lines, although a full growth rescue was not achieved. Quantification analysis of electron micrographs from three BTHS and healthy lymphoblast donors indicated similar numbers of mitochondria per cell, but lower average cristae length per mitochondrion, and higher mitochondrial density. Additionally, BTHS lymphoblasts had larger mitochondria, and a higher percentage of abnormally large mitochondria (> 1 μm2) than healthy controls. Notably, OEA treatment significantly restored mitochondrial size, without affecting density or cristae lengths. Cardiolipin total content, relative linoleic acid content and monolysocardiolipin:cardiolipin ratios were not improved by OEA, indicating that effects on growth, and mitochondrial morphology and function, occurred without resolving this deficit. However, immunoblotting showed higher levels of OPA1, a biomarker for mitochondrial fusion, in BTHS lymphoblasts, which was attenuated by OEA treatment, implicating altered mitochondrial dynamics in the pathology and treatment of BTHS.
Introduction: Cardiolipin is produced de novo in an immature form, and must be remodeled to contain a fatty acyl profile that is functionally appropriate for the mitochondria in a specific tissue. We have recently discovered that phospholipase A and acyltransferase 1 (PLAAT1) has transacylase activity using monolysocardiolipin as an acyl acceptor, and phosphatidylcholine as an acyl donor, and that cells overexpressing PLAAT1 have higher levels of total cardiolipin, supporting a role for this enzyme in cardiolipin remodeling in vivo. We have generated Plaat1 global knockout mice ( Plaat1-/-) to study the role of this enzyme in whole body physiology. Objectives and methods: The objective of this study was to perform an initial physiological characterization of Plaat1-/- mice, with an emphasis on measures related to mitochondrial function. Activity levels, oxygen utilization, carbon dioxide production, respiratory exchange ratio (RER), and exercise capacity were measured and compared between Plaat1-/- and wildtype mice within sexes. Hypotheses:: We anticipate that altered cardiolipin remodeling due to Plaat1 deficiency will adversely affect mitochondrial function, resulting in changes in bioenergetic metabolism and exercise capacity. As compared to controls, we hypothesize that Plaat1-/- mice will have reduced activity levels and impaired exercise tolerance, but increased oxygen consumption rates and carbon dioxide production, with an elevated RER due to dependance on glycolysis. Increased heat production is also predicted due to an expected increased dissipation of the proton gradient. Results: Male Plaat1-/- mice had 42.1% ± 17.2% lower rearing activity over 24 hours compared to wildtype mice (n=6-8, P<0.05), but no differences in X- or Y-directional locomotor activity, while female Plaat1-/- mice did not differ from their wildtype ( Wt) littermates in these measures. However, Female Plaat1-/- mice had 31.9% ± 14.4% lower oxygen utilization (P<0.05), 34.3% ± 11.5% lower carbon dioxide production (P<0.05), and 45.8% ± 13.9% lower heat production (P<0.01) over 24 h when compared to Wt controls. Both male and female Plaat1-/- mice ran significantly shorter distances in a treadmill time-to-exhaustion test (P<0.05). Male Plaat1-/- mice had a mean run time of 32.4±2.3 minutes (min) before exhaustion compared to Wt littermates (35.8±2.3 min), while female Plaat1-/- mice had mean run times of 34.6±2.1 min compared to Wt littermates (38.3±1.9 min). Interestingly, however, when mice were subjected to two treadmill tests, 1-hour apart, male Wt mice displayed significantly decreased performance from their initial test (-6.7% ± 2.3%), but Plaat1-/- mice did not. Conclusions: Our initial physiological characterization demonstrates differences in behaviour, respiration, and exercise tolerance in both males and females deficient in Plaat1, supporting a role for this enzyme in whole body bioenergetic metabolism and exercise tolerance. This work is supported by the Barth Syndrome Foundation, and the Natural Sciences and Engineering Research Council of Canada (NSERC). R.E.D. was a recipient of an Early Investigator Award from the Canadian Lipoprotein Conference. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The sarco(endo)plasmic reticulum calcium (Ca2+) ATPase (SERCA) pump is a major regulator of cytosolic Ca2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
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