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The rumen has several important physiological functions: absorption, transport, metabolic activity, and protection. To clarify the molecular basis underlying the physiological function of the rumen, reticulum, omasum, and abomasum, we used mRNA differential display to isolate and identify differentially expressed genes in these tissues. We isolated 18 transcripts that coexpressed in the rumen, reticulum, and omasum. Five genes, ribosomal protein 19 (RPS19), basic helix-loop-helix domain containing class B2 (BHLHB2), NADH dehydrogenase flavoprotein 2 (NDUFV2), exosome component 9 (EXOSC9), and ribosomal protein 23 (RPS23), were highly expressed in the rumen of adult Holstein and Japanese Black cattle. Significant differences of expression were observed in the abomasum compared with the rumen, reticulum, and omasum. To investigate the expression pattern of these genes during the neonatal growth stage, the relative levels of gene expression were analyzed in the rumen and abomasum of 3-wk-, 13-wk-, and 18- to 20-mo-old Holstein cattle. The expression level of RPS19 did not change with age in the rumen and abomasum. The levels of BHLHB2, NDUFV2, and EXOSC9 mRNA in the abomasum decreased (P < 0.05) after weaning and declined (P < 0.05) further in adults; in contrast, expression in the rumen was not altered. Interestingly, the levels of RPS23 mRNA in the rumen increased (P < 0.05) after weaning and further increased in the adult; however, the level of expression of this gene decreased (P < 0.05) in the abomasum with weaning and age. This indicates that the 4 tissues, especially the rumen and abomasum, have different developmental pathways after birth and subsequent onset of rumination. Key words: abomasum, cattle, differential display reverse transcriptase polymerase chain reaction, rumen

Exposure of testes to high-temperature environment results in defective spermatogenesis. Zebrafish exposed to high temperature exhibited apoptosis not only in germ cells but also in Leydig cells, as expected from studies using mice or salmon. However, the role of testicular somatic cells in spermatogenesis defects remains unclear. We found that in Leydig cells the Trpv4 gene encoding the temperature sensitive ion channel was specifically upregulated in high temperature. High temperature also reduced hormone synthesis in Leydig cells and led to a prompt downregulation of sperm motility. In the Trpv4 null mutant, neither Leydig cell-specific apoptosis nor decreased sperm motility was observed under high temperature. These results indicate that Leydig cell specific-apoptosis is induced via Trpv4 by high temperature. Notably this Trpv4-dependent mechanism was specific to Leydig cells and did not operate in germ cells. Because sperm exposed to high temperature exhibited compromised genome stability, we propose that temperature sensing leading to apoptosis in Leydig cells evolved to actively suppress generation of offspring with unstable genome., (© 2024. The Author(s).)

Background and Aims: Chronic liver congestion reflecting right-sided heart failure (RHF), Budd-Chiari syndrome, or Fontan-associated liver disease (FALD) is involved in liver fibrosis and HCC. However, molecular mechanisms of fibrosis and HCC in chronic liver congestion remain poorly understood., Approach and Results: Here, we first demonstrated that chronic liver congestion promoted HCC and metastatic liver tumor growth using murine model of chronic liver congestion by partial inferior vena cava ligation (pIVCL). As the initial step triggering HCC promotion and fibrosis, gut-derived lipopolysaccharide (LPS) appeared to induce LSECs capillarization in mice and in vitro. LSEC capillarization was also confirmed in patients with FALD. Mitogenic factor, sphingosine-1-phosphate (S1P), was increased in congestive liver and expression of sphingosine kinase 1, a major synthetase of S1P, was increased in capillarized LSECs after pIVCL. Inhibition of S1P receptor (S1PR) 1 (Ex26) and S1PR2 (JTE013) mitigated HCC development and liver fibrosis, respectively. Antimicrobial treatment lowered portal blood LPS concentration, LSEC capillarization, and liver S1P concentration accompanied by reduction of HCC development and fibrosis in the congestive liver., Conclusions: In conclusion, chronic liver congestion promotes HCC development and liver fibrosis by S1P production from LPS-induced capillarized LSECs. Careful treatment of both RHF and liver cancer might be necessary for patients with RHF with primary or metastatic liver cancer., (© 2021 American Association for the Study of Liver Diseases.)

Due to their high energy demands and characteristic morphology, retinal photoreceptor cells require a specialized lipid metabolism for survival and function. Accordingly, dysregulation of lipid metabolism leads to the photoreceptor cell death and retinal degeneration. Mice bearing a frameshift mutation in the gene encoding lysophosphatidylcholine acyltransferase 1 (Lpcat1), which produces saturated phosphatidylcholine (PC) composed of two saturated fatty acids, has been reported to cause spontaneous retinal degeneration in mice; however, the mechanism by which this mutation affects degeneration is unclear. In this study, we performed a detailed characterization of LPCAT1 in the retina and found that genetic deletion of Lpcat1 induces light-independent and photoreceptor-specific apoptosis in mice. Lipidomic analyses of the retina and isolated photoreceptor outer segment (OS) suggested that loss of Lpcat1 not only decreased saturated PC production but also affected membrane lipid composition, presumably by altering saturated fatty acyl-CoA availability. Furthermore, we demonstrated that Lpcat1 deletion led to increased mitochondrial reactive oxygen species levels in photoreceptor cells, but not in other retinal cells, and did not affect the OS structure or trafficking of OS-localized proteins. These results suggest that the LPCAT1-dependent production of saturated PC plays critical roles in photoreceptor maturation. Our findings highlight the therapeutic potential of saturated fatty acid metabolism in photoreceptor cell degeneration-related retinal diseases., Competing Interests: Conflict of interest The Department of Lipid Signaling, National Center for Global Health and Medicine, is financially supported by ONO Pharmaceutical Co, Ltd, Japan., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Platelet-activating factor (PAF) is a potent proinflammatory phospholipid mediator that elicits various cellular functions and promotes several pathological events, including anaphylaxis and neuropathic pain. PAF is biosynthesized by two types of lyso-PAF acetyltransferases: lysophosphatidylcholine acyltransferase 1 (LPCAT1) and LPCAT2, which are constitutive and inducible forms of lyso-PAF acetyltransferase, respectively. Because LPCAT2 mainly produces PAF under inflammatory stimuli, understanding the structure of LPCAT2 is important for developing specific drugs against PAF-related inflammatory diseases. Although the structure of LPCAT2 has not been determined, the crystal structure was reported for Thermotoga maritima PlsC, an enzyme in the same gene family as LPCAT2. Here, we identified residues in mouse LPCAT2 essential for its enzymatic activity and a potential acyl-coenzyme A (CoA)-binding pocket, based on homology modeling of mouse LPCAT2 with PlsC. We also found that Ala115 of mouse LPCAT2 was important for acyl-CoA selectivity. In conclusion, these results predict the three-dimensional (3D) structure of mouse LPCAT2. Our findings have implications for the future development of new drugs against PAF-related diseases., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA) and arachidonic acid (ARA), play fundamental roles in mammalian physiology. Although PUFA imbalance causes various disorders, mechanisms of the regulation of their systemic levels are poorly understood. Here, we report that hepatic DHA-containing phospholipids (DHA-PLs) determine the systemic levels of PUFAs through the SREBP1-mediated transcriptional program. We demonstrated that liver-specific deletion of Agpat3 leads to a decrease of DHA-PLs and a compensatory increase of ARA-PLs not only in the liver but also in other tissues including the brain. Together with recent findings that plasma lysophosphatidylcholine (lysoPC) is the major source of brain DHA, our results indicate that hepatic AGPAT3 contributes to brain DHA accumulation by supplying DHA-PLs as precursors of DHA-lysoPC. Furthermore, dietary fish oil-mediated suppression of hepatic PUFA biosynthetic program was blunted in liver-specific Agpat3 deletion. Our findings highlight the central role of hepatic DHA-PLs as the molecular rheostat for systemic homeostasis of PUFAs., Competing Interests: Declaration of Interests The Department of Lipid Signaling, National Center for Global Health and Medicine, is financially supported by Ono Pharmaceutical Co., Ltd., Japan. The Department of Lipidomics, Graduate School of Medicine, The University of Tokyo, is funded by Shimadzu Corp., Japan., (Copyright © 2020. Published by Elsevier Inc.)

Hematopoietic stem cells (HSCs) maintain lifelong hematopoiesis by remaining quiescent in the bone marrow niche. Recapitulation of a quiescent state in culture has not been achieved, as cells rapidly proliferate and differentiate in vitro. After exhaustive analysis of different environmental factor combinations and concentrations as a way to mimic physiological conditions, we were able to maintain engraftable quiescent HSCs for 1 month in culture under very low cytokine concentrations, hypoxia, and very high fatty acid levels. Exogenous fatty acids were required likely due to suppression of intrinsic fatty acid synthesis by hypoxia and low cytokine conditions. By contrast, high cytokine concentrations or normoxia induced HSC proliferation and differentiation. Our culture system provides a means to evaluate properties of steady-state HSCs and test effects of defined factors in vitro under near-physiological conditions., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Adaption of skeletal muscle to endurance exercise includes PPARδ- and AMP-activated protein kinase (AMPK)/PPARγ coactivator 1α-mediated transcriptional responses that result in increased oxidative capacity and conversion of glycolytic to more oxidative fiber types. These changes are associated with whole-body metabolic alterations including improved glucose handling and resistance to obesity. Increased DHA (22:6n-3) content in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is also reported in endurance exercise-trained glycolytic muscle; however, the DHA-metabolizing enzymes involved and the biological significance of the enhanced DHA content are unknown. In the present study, we identified lysophosphatidic acid acyltransferase (LPAAT)3 as an enzyme that was upregulated in myoblasts during in vitro differentiation and selectively incorporated DHA into PC and PE. LPAAT3 expression was increased by pharmacological activators of PPARδ or AMPK, and combination treatment led to further increased LPAAT3 expression and enhanced incorporation of DHA into PC and PE. Our results indicate that LPAAT3 was upregulated by exercise-induced signaling pathways and suggest that LPAAT3 may also contribute to the enhanced phospholipid-DHA content of endurance-trained muscles. Identification of DHA-metabolizing enzymes in the skeletal muscle will help to elucidate broad metabolic effects of DHA., (Copyright © 2018 by the American Society for Biochemistry and Molecular Biology, Inc.)

Omega-3 (ω-3) fatty acids (FAs) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are known to have important roles in human health and disease. Besides being utilized as fuel, ω-3 FAs have specific functions based on their structural characteristics. These functions include serving as ligands for several receptors, precursors of lipid mediators, and components of membrane glycerophospholipids (GPLs). Since ω-3 FAs (especially DHA) are highly flexible, the levels of DHA in GPLs may affect membrane biophysical properties such as fluidity, flexibility, and thickness. Here, we summarize some of the cellular mechanisms for incorporating DHA into membrane GPLs and propose biological effects and functions of DHA-containing membranes of several cell and tissue types., (© 2017 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Docosahexaenoic acid (DHA) has essential roles in photoreceptor cells in the retina and is therefore crucial to healthy vision. Although the influence of dietary DHA on visual acuity is well known and the retina has an abundance of DHA-containing phospholipids (PL-DHA), the mechanisms associated with DHA's effects on visual function are unknown. We previously identified lysophosphatidic acid acyltransferase 3 (LPAAT3) as a PL-DHA biosynthetic enzyme. Here, using comprehensive phospholipid analyses and imaging mass spectroscopy, we found that LPAAT3 is expressed in the inner segment of photoreceptor cells and that PL-DHA disappears from the outer segment in the LPAAT3-knock-out mice. Dynamic light-scattering analysis of liposomes and molecular dynamics simulations revealed that the physical characteristics of DHA reduced membrane-bending rigidity. Following loss of PL-DHA, LPAAT3-knock-out mice exhibited abnormalities in the retinal layers, such as incomplete elongation of the outer segment and decreased thickness of the outer nuclear layers and impaired visual function, as well as disordered disc morphology in photoreceptor cells. Our results indicate that PL-DHA contributes to visual function by maintaining the disc shape in photoreceptor cells and that this is a function of DHA in the retina. This study thus provides the reason why DHA is required for visual acuity and may help inform approaches for overcoming retinal disorders associated with DHA deficiency or dysfunction., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Docosahexaenoic acid (DHA) is one of the essential ω-3 polyunsaturated fatty acids with a wide range of physiological roles important for human health. For example, DHA renders cell membranes more flexible and is therefore important for cellular function, but information on the mechanisms that control DHA levels in membranes is limited. Specifically, it is unclear which factors determine DHA incorporation into cell membranes and how DHA exerts biological effects. We found that lysophosphatidic acid acyltransferase 3 (LPAAT3) is required for producing DHA-containing phospholipids in various tissues, such as the testes and retina. In this study, we report that LPAAT3-KO mice display severe male infertility with abnormal sperm morphology. During germ cell differentiation, the expression of LPAAT3 was induced, and germ cells obtained more DHA-containing phospholipids. Loss of LPAAT3 caused drastic reduction of DHA-containing phospholipids in spermatids that led to excess cytoplasm around its head, which is normally removed by surrounding Sertoli cells via endocytosis at the final stage of spermatogenesis. In vitro liposome filtration assay raised the possibility that DHA in phospholipids promotes membrane deformation that is required for the rapid endocytosis. These data suggest that decreased membrane flexibility in LPAAT3-KO sperm impaired the efficient removal of sperm content through endocytosis. We conclude that LPAAT3-mediated enrichment of cell membranes with DHA-containing phospholipids endows these membranes with physicochemical properties needed for normal cellular processes, as exemplified by spermatogenesis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Polyunsaturated fatty acids (PUFAs) in phospholipids affect the physical properties of membranes, but it is unclear which biological processes are influenced by their regulation. For example, the functions of membrane arachidonate that are independent of a precursor role for eicosanoid synthesis remain largely unknown. Here, we show that the lack of lysophosphatidylcholine acyltransferase 3 (LPCAT3) leads to drastic reductions in membrane arachidonate levels, and that LPCAT3-deficient mice are neonatally lethal due to an extensive triacylglycerol (TG) accumulation and dysfunction in enterocytes. We found that high levels of PUFAs in membranes enable TGs to locally cluster in high density, and that this clustering promotes efficient TG transfer. We propose a model of local arachidonate enrichment by LPCAT3 to generate a distinct pool of TG in membranes, which is required for normal directionality of TG transfer and lipoprotein assembly in the liver and enterocytes.

The acyl-chain composition of the major mammalian phospholipid phosphatidylcholine (PC) is distinct in various tissues. Although it was classically suggested that PC diversity is acquired through acyl-chain remodeling, the mechanisms and biological relevance of acyl-chain diversity remain unclear. Here, we show that differences in the substrate selectivity of lysophospholipid acyltransferases regulate tissue PC acyl-chain composition through contribution of both the de novo and remodeling pathways, depending on the fatty acid species. Unexpectedly, while dipalmitoyl-PC (DPPC) is enriched through the remodeling pathway, several polyunsaturated PC molecules accumulate during the de novo pathway. We confirmed this concept for DPPC in pulmonary surfactant and showed that the biophysical properties of this lipid are important to prevent the early onset of acute lung injury. We propose a model of harmonized processes for phospholipid diversification to satisfy in vivo requirements, with an example of its biological relevance., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Cellular membranes are composed of numerous kinds of glycerophospholipids with different combinations of polar heads at the sn-3 position and acyl moieties at the sn-1 and sn-2 positions, respectively. The glycerophospholipid compositions of different cell types, organelles, and inner/outer plasma membrane leaflets are quite diverse. The acyl moieties of glycerophospholipids synthesized in the de novo pathway are subsequently remodeled by the action of phospholipases and lysophospholipid acyltransferases. This remodeling cycle contributes to the generation of membrane glycerophospholipid diversity and the production of lipid mediators such as fatty acid derivatives and lysophospholipids. Furthermore, specific glycerophospholipid transporters are also important to organize a unique glycerophospholipid composition in each organelle. Recent progress in this field contributes to understanding how and why membrane glycerophospholipid diversity is organized and maintained.

Pulmonary surfactant, a complex composed primarily of lipids and associated proteins, is synthesized in alveolar type II (ATII) cells and secreted into alveoli to prevent collapse during respiration. Although numerous studies have clarified the fundamental roles of pulmonary surfactant, the molecular mechanisms of transport and secretion of pulmonary surfactant remain totally unknown. Thus, we screened candidate genes by comparing genes with the expressed sequence tag (EST) libraries of embryonic and adult lungs by using the digital differential display method in the National Center for Biotechnology Information (NCBI) database. We identified Sec14-like 3 (Sec14L3) as a new class of lipid-associated proteins highly expressed in ATII cells. We found that Sec14L3 expression is >100-fold increased during the perinatal period in the lung. Furthermore, Sec14L3 bound to small-sized liposomes (30 nm in diameter), but not to large-sized liposomes (100 nm diameter), through its Sec14 domain. Because of the increased curvature, lipid-packing defects are more likely to occur in small-sized liposomes than in large-sized liposomes. Based on these results, we conclude that Sec14L3 is a new class of lipid-packing sensor. Sec14L3 may play important roles in the lung, such as intracellular lipid transport, surfactant maturation, and endo/exocytosis.

Glycerophospholipids are main components of cellular membranes and have numerous structural and functional roles to regulate cellular functions. Polyunsaturated fatty acids, such as arachidonic acid and eicosapentaenoic acid, are mainly located at the sn-2, but not the sn-1 position of glycerophospholipids in an asymmetrical manner and the fatty acid compositions at both the sn-1 and sn-2 positions differ in various cell types and tissues. Asymmetry and diversity of membrane glycerophospholipids are generated in the remodelling pathway (Lands' cycle), which are conducted by the concerted actions of phospholipases A2 (PLA2s) and lysophospholipid acyltransferases (LPLATs). The Lands' cycle was first reported in the 1950s. While PLA2s have been well characterized, little is known about the LPLATs. Recently, several laboratories, including ours, isolated LPLATs that function in the Lands' cycle from the 1-acylglycerol-3-phosphate O-acyltransferase family and the membrane bound O-acyltransferases family. In this review, we summarize recent studies on cloning and characterization of LPLATs that contribute to membrane asymmetry and diversity.

Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and beta-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis.

Cells of all organisms are enclosed by a plasma membrane containing bipolar lipids, cholesterol, and proteins. Cellular membranes contain several classes of glycerophospholipids, which have numerous structural and functional roles in cells. Polyunsaturated fatty acids including arachidonic acid and eicosapentaenoic acid are usually located at the sn-2 position, but not the sn-1 position, of glycerophospholipids in an asymmetrical manner. Glycerophospholipids are first formed by the de novo pathway (Kennedy pathway) using acyl-CoAs as donors. Subsequently, in the remodeling pathway (Lands' cycle), cycles of deacylation and reacylation of glycerophospholipids modify the fatty acid composition to generate mature membrane with asymmetry and diversity. Both pathways were proposed in the 1950s. Whereas the enzymes involved in the Kennedy pathway have been well characterized, little is known about the enzymes involved in the Lands' cycle. Recently, several laboratories, including ours, have identified enzymes working in the Lands' cycle from the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family, and also from the membrane bound O-acyltransferases (MBOAT) family. These discoveries have prompted a robust surge of research in this field. In this review, we focus on the cloning and characterization of lysophospholipid acyltransferases (LPLATs), which contribute to membrane asymmetry and diversity.

Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) plays a critical role in inflammatory disorders including experimental allergic encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). Although PAF accumulation in the spinal cord (SC) of EAE mice and cerebrospinal fluid of MS patients has been reported, little is known about the metabolic processing of PAF in these diseases. In this study, we demonstrate that the activities of phospholipase A(2) (PLA(2)) and acetyl-CoA:lyso-PAF acetyltransferase (LysoPAFAT) are elevated in the SC of EAE mice on a C57BL/6 genetic background compared with those of naive mice and correlate with disease severity. Correspondingly, levels of groups IVA, IVB, and IVF cytosolic PLA(2)s, group V secretory PLA(2), and LysoPAFAT transcripts are up-regulated in the SC of EAE mice. PAF acetylhydrolase activity is unchanged during the disease course. In addition, we show that LysoPAFAT mRNA and protein are predominantly expressed in microglia. Considering the substrate specificity and involvement of PAF production, group IVA cytosolic PLA(2) is likely to be responsible for the increased PLA(2) activity. These data suggest that PAF accumulation in the SC of EAE mice is profoundly dependent on the group IVA cytosolic PLA(2)/LysoPAFAT axis present in the infiltrating macrophages and activated microglia.

All organisms consist of cells that are enclosed by a cell membrane containing bipolar lipids and proteins. Glycerophospholipids are important not only as structural and functional components of cellular membrane but also as precursors of various lipid mediators. Polyunsaturated fatty acids comprising arachidonic acid or eicosapentaenoic acid are located at sn-2 position, but not at sn-1 position of glycerophospholipids in an asymmetrical manner. In addition to the asymmetry, the membrane diversity is important for membrane fluidity and curvature. To explain the asymmetrical distribution of fatty acids, the rapid turnover of sn-2 position was proposed in 1958 by Lands [Lands WE (1958) Metabolism of glycerolipides: A comparison of lecithin and triglyceride synthesis. J Biol Chem 231:883-888]. However, the molecular mechanisms and biological significance of the asymmetry remained unknown. Here, we describe a putative enzyme superfamily consisting mainly of three gene families, which catalyzes the transfer of acyl-CoAs to lysophospholipids to produce different classes of phospholipids. Among them, we characterized three important enzymes with different substrate specificities and tissue distributions; one, termed lysophosphatidylcholine acyltransferase-3 (a mammalian homologue of Drosophila nessy critical for embryogenesis), prefers arachidonoyl-CoA, and the other two enzymes incorporate oleoyl-CoAs to lysophosphatidylethanolamine and lysophosphatidylserine. Thus, we propose that the membrane diversity is produced by the concerted and overlapped reactions with multiple enzymes that recognize both the polar head group of glycerophospholipids and various acyl-CoAs. Our findings constitute a critical milestone for our understanding about how membrane diversity and asymmetry are established and their biological significance.

Recently, it has been found that long-chain fatty acids activate the G protein-coupled receptors (GPRs), GPR120 and GPR40. However, there have been no reports to date on the possible physiological roles of these GPRs in adipose tissue development and adipocyte differentiation. GPR120 mRNA was highly expressed in the four different adipose tissues, and the amount of mRNA was elevated in adipose tissues of mice fed a high fat diet. However, GPR40 mRNA was not detected in any of the adipose tissues. The expression of GPR120 mRNA was higher in adipocytes compared to stromal-vascular (S-V) cells. The level of GPR120 mRNA increased during adipocyte differentiation in 3T3-L1 cells. Similar results were observed in human adipose tissue, human preadipocytes, and cultured adipocytes. Moreover, use of a small interference RNA (siRNA) to down-regulate GPR120 expression resulted in inhibition of adipocyte differentiation. Our results suggest that GPR120 regulates adipogenic processes such as adipocyte development and differentiation.

Platelet-activating factor (PAF) is a potent proinflammatory lipid mediator eliciting a variety of cellular functions. Lipid mediators, including PAF are produced from membrane phospholipids by enzymatic cascades. Although a G protein-coupled PAF receptor and degradation enzymes have been cloned and characterized, the PAF biosynthetic enzyme, aceyl-CoA:lyso-PAF acetyltransferase, has not been identified. Here, we cloned lyso-PAF acetyltransferase, which is critical in stimulus-dependent formation of PAF. The enzyme is a 60-kDa microsomal protein with three putative membrane-spanning domains. The enzyme was induced by bacterial endotoxin (lipopolysaccharide), which was suppressed by dexamethasone treatment. Surprisingly, the enzyme catalyzed not only biosynthesis of PAF from lyso-PAF but also incorporation of arachidonoyl-CoA to produce PAF precursor membrane glycerophospholipids (lysophosphatidylcholine acyltransferase activity). Under resting conditions, the enzyme prefers arachidonoyl-CoA and contributes to membrane biogenesis. Upon acute inflammatory stimulation with lipopolysaccharide, the activated enzyme utilizes acetyl-CoA more efficiently and produces PAF. Thus, our findings provide a novel concept that a single enzyme catalyzes membrane biogenesis of inflammatory cells while producing a prophlogistic mediator in response to external stimuli.

Phosphatidylcholine (1,2-diacyl-sn-glycero-3-phosphocholine, PC), is an important constituent of biological membranes. It is also the major component of serum lipoproteins and pulmonary surfactant. In the remodeling pathway of PC biosynthesis, 1-acyl-sn-glycero-3-phosphocholine (LPC) is converted to PC by acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT, EC 2.3.1.23). Whereas LPCAT activity has been detected in several tissues, the structure and detailed biochemical information on the enzyme have not yet been reported. Here, we present the cloning and characterization of a cDNA for mouse lung-type LPCAT (LPCAT1). The cDNA encodes an enzyme of 60 kDa, with three putative transmembrane domains. When expressed in Chinese hamster ovary cells, mouse LPCAT1 exhibited Ca(2+)-independent activity with a pH optimum between 7.4 and 10. LPCAT1 demonstrated a clear preference for saturated fatty acyl-CoAs, and 1-myristoyl- or 1-palmitoyl-LPC as acyl donors and acceptors, respectively. Furthermore, the enzyme was predominantly expressed in the lung, in particular in alveolar type II cells. Thus, the enzyme might synthesize phosphatidylcholine in pulmonary surfactant and play a pivotal role in respiratory physiology.

It has recently been discovered that G protein-coupled receptors (GPCR) 41 and 43 are characterized by having the short chain fatty acids acetate and propionate as their ligands. The objective of this study was to investigate the involvement of GPCR41, GPCR43, and their ligands in the process of adipogenesis. We measured the levels of GPCR41 and GPCR43 mRNA in both adipose and other tissues of the mouse. GRP43 mRNA expression was higher in four types of adipose tissue than in other tissues, whereas GPCR41 mRNA was not detected in any adipose tissues. A high level of GPCR43 expression was found in isolated adipocytes, but expression level was very low in stromal-vascular cells. Expression of GPCR43 was up-regulated in adipose tissues of mice fed a high-fat diet compared with those fed a normal-fat diet. GPCR43 mRNA could not be detected in confluent and undifferentiated 3T3-L1 adipocytes; however, the levels rose with time after the initiation of differentiation. GPCR41 expression was not detected in confluent and differentiated adipocytes. Acetate and propionate treatments increased lipids present as multiple droplets in 3T3-L1 adipocytes. Propionate significantly elevated the level of GPCR43 expression during adipose differentiation, with up-regulation of PPAR-gamma2. Small interfering RNA mediated a reduction of GPCR43 mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. In addition, both acetate and propionate inhibited isoproterenol-induced lipolysis in a dose-dependent manner. We conclude that acetate and propionate short chain fatty acids may have important physiological roles in adipogenesis through GPCR43, but not through GPCR41.

To investigate the role of claudin-6 in adipogenesis, claudin-6 mRNA was examined in adipose tissues and adipocyte differentiation. Claudin-6 mRNA was found to be differentially expressed in four different adipose tissues, and up-regulated in each fat depot of mice fed a high-fat diet as compared to a normal-fat diet. Levels of claudin-6 transcripts were increased during differentiation of 3T3-L1 cells in vitro. Moreover, small interfering RNA (siRNA)-mediated reduction of claudin-6 mRNA inhibited differentiation of 3T3-L1 cells. These results suggest that claudin-6 is another important regulator in adipogenesis and fat deposition.

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through 'Search Expression' on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor gamma2 (PPAR-gamma2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development., ((Mol Cell Biochem 276: 133-141, 2005))

The factors that control fat deposition in adipose tissues are poorly understood. It is known that visceral adipose tissues display a range of biochemical properties that distinguish them from adipose tissues of subcutaneous origin. However, we have little information on gene expression, either in relation to fat deposition or on interspecies variation in fat deposition. The first step in this study was to identify genes expressed in fat depot of cattle using the differential display RT-PCR method. Among the transcripts identified as having differential expression in the two adipose tissues were cell division cycle 42 homolog (CDC42), prefoldin-5, decorin, phosphate carrier, 12S ribosomal RNA gene, and kelch repeat and BTB domain containing 2 (Kbtbd2). In subsequent experiments, we determined the expression levels of these latter genes in the pig and in mice fed either a control or high-fat diet to compare the regulation of fat accumulation in other animal species. The levels of CDC42 and decorin mRNA were found to be higher in visceral adipose tissue than in subcutaneous adipose tissue in cattle, pig, and mice. However, the other genes studied did not show consistent expression patterns between the two tissues in cattle, pigs, and mice. Interestingly, all genes were upregulated in subcutaneous and/or visceral adipose tissues of mice fed the high-fat diet compared with the control diet. The data presented here extend our understanding of gene expression in fat depots and provide further proof that the mechanisms of fat accumulation differ significantly between animal species.

Visceral adipocytes differ in various biochemical properties from adipocytes of subcutaneous origin. However, information on differences in gene expression between visceral and subcutaneous fat depots is limited. Expression of the genes for the nonmuscle and muscle isoforms of the actin-binding protein cofilin was examined in subcutaneous and visceral fat depots of mice, pigs, and cattle by semiquantative reverse transcription and polymerase chain reaction analysis. The abundance of nonmuscle-type cofilin mRNA was markedly higher in visceral adipose tissue than in subcutaneous adipose tissue of mouse and pig. This difference was more pronounced in mice fed a high-fat diet than in those fed a standard diet. In cattle, however, the amount of nonmuscle-type cofilin mRNA was greater in subcutaneous fat than in visceral fat. Muscle-type cofilin mRNA was not detected in either adipose tissue of any of the three species. These results suggest that the nonmuscle isoform of cofilin, and therefore the cytoskeleton, may play a role in lipid accumulation in visceral adipose tissue.

Recent research progress indicates a close link between ghrelin, a natural ligand of GH secretagogues receptor (GHS-R), and both the metabolic balance and body composition. To clarify the involvement of ghrelin and GHS-R in the process of adipogenesis, we measured the expression of GHS-R and peroxisome proliferator-activated receptor gamma 2 (PPAR-gamma 2) mRNA in rat adipocytes using semiquantitative RT-PCR methods. The levels of GHS-R mRNA increased by up to 4-fold in adipose tissue from epididymal and parametrial regions as the rat aged from 4-20 wk and were significantly elevated during the differentiation of preadipocytes in vitro. Ghrelin (10(-8) M for 10 d) stimulated the activity of glycerol-3-phosphate dehydrogenase and the differentiation of rat preadipocytes in vitro. Ghrelin treatment also significantly increased the levels of PPAR-gamma 2 mRNA in primary cultured rat differentiated adipocytes. In addition, isoproterenol (10(-8) M, 40 min)-stimulated lipolysis was significantly reduced by simultaneous ghrelin treatment in a dose-dependent manner in vitro. In conclusion, the expression of GHS-R in rat adipocytes increases with the age and during adipogenesis. Ghrelin in vitro stimulates the differentiation of preadipocytes and antagonizes lipolysis. Ghrelin may therefore play an important role in the process of adipogenesis in rats.