Ryan M. Bradley

Microglial Cell Activation Increases Saturated and Decreases Monounsaturated Fatty Acid Content, but Both Lipid Species are Proinflammatory

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.

Acylglycerophosphate acyltransferase 4 (AGPAT4) is a mitochondrial lysophosphatidic acid acyltransferase that regulates brain phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol levels

The acylglycerophosphate acyltransferase/lysophosphatidic acid acyltransferase (AGPAT/LPAAT) family is a group of homologous acyl-CoA-dependent lysophospholipid acyltransferases. We performed studies to better understand the subcellular localization, activity, and in vivo function of AGPAT4/LPAATδ, which we found is expressed in multiple mouse brain regions. Endogenous brain AGPAT4 and AGPAT4 overexpressed in HEK293 or Sf9 insect cells localizes to mitochondria and is resident on the outer mitochondrial membrane. Further fractionation showed that AGPAT4 is present specifically in the mitochondria and not in the mitochondria-associated endoplasmic reticulum membrane (i.e. MAM). Lysates from Sf9 cells infected with baculoviral Agpat4 were tested with eight lysophospholipid species but showed an increased activity only with lysophosphatidic acid as an acyl acceptor. Analysis of Sf9 phospholipid species, however, indicated a significant 72% increase in phosphatidylinositol (PI) content. We examined the content of major phospholipid species in brains of Agpat4(-/-) mice and found also a >50% decrease in total levels of PI relative to wildtype mice, as well as significant decreases in phosphatidylcholine (PC) and phosphatidylethanolamine (PE), but no significant differences in phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid (PA). A compensatory upregulation of Agpats 1, 2, 3, 5, and 9 may help to explain the lack of difference in PA. Our findings indicate that AGPAT4 is a mitochondrial AGPAT/LPAAT that specifically supports synthesis of brain PI, PC, and PE. This understanding may help to explain apparent redundancies in the AGPAT/LPAAT family.

Influence of tissue, diet, and enzymatic remodeling on cardiolipin fatty acyl profile

Cardiolipin is a specialized phospholipid found primarily in the inner mitochondrial membrane. Because of its unique dimeric structure, cardiolipin plays an important role in mitochondrial function, stability, and membrane fluidity. As such, cardiolipin is subject to a high degree of remodeling by phospholipases, acyltransferases, and transacylases that create a fatty acyl profile that tends to be highly tissue-specific. Despite this overarching regulation, the molecular species of cardiolipin produced are also influenced by dietary lipid composition. A number of studies have characterized the tissue-specific profile of cardiolipin species and have investigated the specific nature of cardiolipin remodeling, including the role of both enzymes and diet. The aim of this review is to highlight tissue specific differences in cardiolipin composition and, collectively, the enzymatic and dietary factors that contribute to these differences. Consequences of aberrant cardiolipin fatty acyl remodeling are also discussed.

Fasting upregulates adipose triglyceride lipase and hormone-sensitive lipase levels and phosphorylation in mouse kidney.

Circulating non-esterified fatty acids (NEFA) rise during fasting and are taken up by the kidneys, either directly from the plasma or during re-uptake of albumin from glomerular filtrate, and are stored as triacylglycerol (TAG). Subsequent utilization of stored fatty acids requires their hydrolytic release from cellular lipid droplets, but relatively little is known about renal lipolysis. We found that total [(3)H]triolein hydrolase activity of kidney lysates was significantly increased by 15% in the fasted state. Adipose triglyceride lipase (Atgl) and hormone-sensitive lipase (Hsl) mRNA expression was time-dependently increased by fasting, along with other fatty acid metabolism genes (Pparα, Cd36, and Aox). ATGL and HSL protein levels were also significantly induced (by 239 ± 7% and 322 ± 8%, respectively). Concomitant with changes in total protein levels, there was an increase in ATGL phosphorylation at the AMPK-regulated serine 406 site in the 14-3-3 binding motif, and an increase in HSL phosphorylation at serines 565 and 660 that are regulated by AMPK and PKA, respectively. Using immunofluorescence, we further demonstrate nearly ubiquitous expression of ATGL in the renal cortex with a concentration on the apical/lumenal surface of some cortical tubules. Our findings suggest a role for ATGL and HSL in kidney lipolysis.

The HRASLS (PLA/AT) subfamily of enzymes

The H-RAS-like suppressor (HRASLS) subfamily consists of five enzymes (1–5) in humans and three (1, 3, and 5) in mice and rats that share sequence homology with lecithin:retinol acyltransferase (LRAT). All HRASLS family members possess in vitro phospholipid metabolizing abilities including phospholipase A1/2 (PLA1/2) activities and O-acyltransferase activities for the remodeling of glycerophospholipid acyl chains, as well as N-acyltransferase activities for the production of N-acylphosphatidylethanolamines. The in vivo biological activities of the HRASLS enzymes have not yet been fully investigated. Research to date indicates involvement of this subfamily in a wide array of biological processes and, as a consequence, these five enzymes have undergone extensive rediscovery and renaming within different fields of research. This review briefly describes the discovery of each of the HRASLS enzymes and their role in cancer, and discusses the biochemical function of each enzyme, as well as the biological role, if known. Gaps in current understanding are highlighted and suggestions for future research directions are discussed.

Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia

Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1].

Data on oxygen consumption rate, respiratory exchange ratio, and movement in C57BL/6J female mice on the third day of consuming a high-fat diet

Whole animal physiological measures were assessed following three days of either standard diet or high fat diet, in either the fasted or non-fasted states. Our data shows that acute 3-day high fat feeding increases whole body lipid oxidation. When this feeding protocol is followed by an overnight fast, oxygen consumption (VO2) in the light phase is reduced in both dietary groups, but oxygen consumption in the dark phase is only reduced in mice fed the high-fat diet. Furthermore, the fasting-induced rise in dark cycle activity level observed in mice maintained on a standard diet is abolished when mice are fed a high-fat diet.

Agpat4/Lpaatδ deficiency highlights the molecular heterogeneity of epididymal and perirenal white adipose depots

Acylglycerophosphate acyltransferase 4 (AGPAT4)/lysophosphatidic acid acyltransferase delta catalyzes the formation of phosphatidic acid (PA), a precursor of triacylglycerol (TAG). We investigated the effect of Agpat4 gene ablation on white adipose tissue (WAT) after finding consistent expression across depots. Epididymal WAT mass was 40% larger in male Agpat4−/− mice than wild-type littermates, but unchanged in perirenal, retroperitoneal, and inguinal WAT and subscapular brown adipose tissue. Metabolic changes were identified in epididymal WAT that were not evident in perirenal WAT, which was analyzed for comparison. The total epididymal TAG content doubled, increasing adipocyte cell size without changing markers of differentiation. Enzymes involved in de novo lipogenesis and complex lipid synthesis downstream of phosphatidic acid production were also unchanged. However, total epididymal TAG hydrolase activity was reduced, and there were significant decreases in total ATGL and reduced phosphorylation of hormone-sensitive lipase at the S563 and S660 PKA-activation sites. Analysis of Agpats 1, 2, 3, and 5, as well as Gpats 1, 2, 3, and 4, demonstrated compensatory upregulation in perirenal WAT that did not occur in epididymal WAT. Our findings therefore indicate depot-specific differences in the redundancy of Agpat4 and highlight the molecular and metabolic heterogeneity of individual visceral depots.

Mice Deficient in lysophosphatidic acid acyltransferase delta (Lpaatδ)/acylglycerophosphate acyltransferase 4 (Agpat4) Have Impaired Learning and Memory

ABSTRACT We previously characterized LPAATδ/AGPAT4 as a mitochondrial lysophosphatidic acid acyltransferase that regulates brain levels of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Here, we report that Lpaatδ−/− mice display impaired spatial learning and memory compared to wild-type littermates in the Morris water maze and our investigation of potential mechanisms associated with brain phospholipid changes. Marker protein immunoblotting suggested that the relative brain content of neurons, glia, and oligodendrocytes was unchanged. Relative abundance of the important brain fatty acid docosahexaenoic acid was also unchanged in phosphatidylserine, phosphatidylglycerol, and cardiolipin, in agreement with prior data on PC, PE and PI. In phosphatidic acid, it was increased. Specific decreases in ethanolamine-containing phospholipids were detected in mitochondrial lipids, but the function of brain mitochondria in Lpaatδ−/− mice was unchanged. Importantly, we found that Lpaatδ−/− mice have a significantly and drastically lower brain content of the N-methyl-d-asparate (NMDA) receptor subunits NR1, NR2A, and NR2B, as well as the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1, compared to wild-type mice. However, general dysregulation of PI-mediated signaling is not likely responsible, since phospho-AKT and phospho-mTOR pathway regulation was unaffected. Our findings indicate that Lpaatδ deficiency causes deficits in learning and memory associated with reduced NMDA and AMPA receptors.

Data on hepatic lipolysis, adipose triglyceride lipase, and hormone-sensitive lipase in fasted and non-fasted C57BL/6J female mice.

Liver homogenates produced from fasted and non-fasted C57BL/6J female mice were assayed for total lipolytic activity measured as hydrolysis of [9,10-3H(N)]-triolein into [3H] free fatty acids (FFA). Liver homogenates were also used for immunoblotting to determine levels of the lipolytic enzymes adipose-triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), as well as site specific phosphorylation at the 14-3-3 binding site of ATGL and the serine 565 and serine 660 sites of HSL. Significantly higher triolein hydrolysis activity was observed in fasted liver samples, as well as a significant increase in total ATGL and a significant decrease in HSL phosphorylation at the S565 site.