Phosphatidylserine synthesis controls oncogenic B cell receptor signaling in B cell lymphoma.

Cancer cells harness lipid metabolism to promote their own survival. We screened 47 cancer cell lines for survival dependency on phosphatidylserine (PS) synthesis using a PS synthase 1 (PTDSS1) inhibitor and found that B cell lymphoma is highly dependent on PS. Inhibition of PTDSS1 in B cell lymphoma cells caused a reduction of PS and phosphatidylethanolamine levels and an increase of phosphoinositide levels. The resulting imbalance of the membrane phospholipidome lowered the activation threshold for B cell receptor (BCR), a B cell-specific survival mechanism. BCR hyperactivation led to aberrant elevation of downstream Ca2+ signaling and subsequent apoptotic cell death. In a mouse xenograft model, PTDSS1 inhibition efficiently suppressed tumor growth and prolonged survival. Our findings suggest that PS synthesis may be a critical vulnerability of malignant B cell lymphomas that can be targeted pharmacologically.

PPP1R12A is a recycling endosomal phosphatase that facilitates YAP activation.

Yes-associated protein (YAP) is a transcriptional coactivator that is essential for the malignancy of various cancers. We have previously shown that YAP activity is positively regulated by phosphatidylserine (PS) in recycling endosomes (REs). However, the mechanism by which YAP is activated by PS in REs remains unknown. In the present study, we examined a group of protein phosphatases (11 phosphatases) that we had identified previously as PS-proximity protein candidates. Knockdown experiments of these phosphatases suggested that PPP1R12A, a regulatory subunit of the myosin phosphatase complex, was essential for YAP-dependent proliferation of triple-negative breast cancer MDA-MB-231 cells. Knockdown of PPP1R12A increased the level of phosphorylated YAP, reduced that of YAP in the nucleus, and suppressed the transcription of CTGF (a YAP-regulated gene), reinforcing the role of PPP1R12A in YAP activation. ATP8A1 is a PS-flippase that concentrates PS in the cytosolic leaflet of the RE membrane and positively regulates YAP signalling. In subcellular fractionation experiments using cell lysates, PPP1R12A in control cells was recovered exclusively in the microsomal fraction. In contrast, a fraction of PPP1R12A in ATP8A1-depleted cells was recovered in the cytosolic fraction. Cohort data available from the Cancer Genome Atlas showed that high expression of PPP1R12A, PP1B encoding the catalytic subunit of the myosin phosphatase complex, or ATP8A1 correlated with poor prognosis in breast cancer patients. These results suggest that the "ATP8A1-PS-YAP phosphatase" axis in REs facilitates YAP activation and thus cell proliferation.

Disease-related PSS1 mutant impedes the formation and function of osteoclasts.

Phosphatidylserine (PS) is an acidic phospholipid that is involved in various cellular events. Heterologous dominant mutations have been identified in the gene encoding PS synthase 1 (PSS1) in patients with a congenital disease called Lenz-Majewski syndrome (LMS). Patients with LMS show various symptoms, including craniofacial/distal-limb bone dysplasia and progressive hyperostosis. The LMS-causing gain-of-function mutants of PSS1 (PSS1LMS) have been shown to synthesize PS without control, but why the uncontrolled synthesis would lead to LMS is unknown. Here we investigated the effect of PSS1LMS on osteoclasts (OCs) to elucidate the causative mechanism of LMS. PSS1LMS did not affect the expression of OC-related genes but inhibited the formation, multinucleation, and activity of OCs. Especially, OCs expressing PSS1LMS showed abnormal patterns and dynamics of actin podosome clusters, which have roles in OC migration and fusion. PSS1LMS did not affect the level of PS but changed the acyl chain compositions of PS and phosphatidylethanolamine, and decreased the level of phosphatidylinositol. The introduction of a catalytically inactive mutation into PSSLMS canceled the changes in phospholipids and the phenotypes observed in OCs expressing PSS1LMS. A gain-of-function mutant of PSS2 (PSS2 R97K) also impaired OC formation and caused changes in phospholipid composition similar to the changes caused by PSS1LMS. Our results suggest that uncontrolled PS synthesis by PSS1LMS causes changes in the quantity or fatty acid composition of certain phospholipid classes, impairing OC formation and function, which might be a cause of osteosclerosis in patients with LMS.

The plasma membrane of focal adhesions has a high content of cholesterol and phosphatidylcholine with saturated acyl chains.

Cellular functions, such as differentiation and migration, are regulated by the extracellular microenvironment, including the extracellular matrix (ECM). Cells adhere to ECM through focal adhesions (FAs) and sense the surrounding microenvironments. Although FA proteins have been actively investigated, little is known about the lipids in the plasma membrane at FAs. In this study, we examine the lipid composition at FAs with imaging and biochemical approaches. Using the cholesterol-specific probe D4 with total internal reflection fluorescence microscopy and super-resolution microscopy, we show an enrichment of cholesterol at FAs simultaneously with FA assembly. Furthermore, we establish a method to isolate the lipid from FA-rich fractions, and biochemical quantification of the lipids reveals that there is a higher content of cholesterol and phosphatidylcholine with saturated fatty acid chains in the lipids of the FA-rich fraction than in either the plasma membrane fraction or the whole-cell membrane. These results demonstrate that plasma membrane at FAs has a locally distinct lipid composition compared to the bulk plasma membrane.

Cell density-dependent membrane distribution of ganglioside GM3 in melanoma cells.

Monosialoganglioside GM3 is the simplest ganglioside involved in various cellular signaling. Cell surface distribution of GM3 is thought to be crucial for the function of GM3, but little is known about the cell surface GM3 distribution. It was shown that anti-GM3 monoclonal antibody binds to GM3 in sparse but not in confluent melanoma cells. Our model membrane study evidenced that monoclonal anti-GM3 antibodies showed stronger binding when GM3 was in less fluid membrane environment. Studies using fluorescent GM3 analogs suggested that GM3 was clustered in less fluid membrane. Moreover, fluorescent lifetime measurement showed that cell surface of high density melanoma cells is more fluid than that of low density cells. Lipidomics and fatty acid supplementation experiment suggested that monounsaturated fatty acid-containing phosphatidylcholine contributed to the cell density-dependent membrane fluidity. Our results indicate that anti-GM3 antibody senses GM3 clustering and the number and/or size of GM3 cluster differ between sparse and confluent melanoma cells.

Hypercholesterolemic Dysregulation of Calpain in Lymphatic Endothelial Cells Interferes With Regulatory T-Cell Stability and Trafficking.

BACKGROUND: Although hypercholesterolemia reportedly counteracts lymphocyte trafficking across lymphatic vessels, the roles of lymphatic endothelial cells (LECs) in the lymphocyte regulations remain unclear. Previous studies showed that calpain-an intracellular modulatory protease-interferes with leukocyte dynamics in the blood microcirculation and is associated with hypercholesterolemic dysfunction in vascular endothelial cells. METHODS: This study investigated whether the calpain systems in LECs associate with the LEC-lymphocyte interaction under hypercholesterolemia using gene-targeted mice. RESULTS: Lipidomic analysis in hypercholesterolemic mice showed that several lysophospholipids, including lysophosphatidic acid, accumulated in the lymphatic environment. Lysophosphatidic acid enables the potentiation of calpain systems in cultured LECs, which limits their ability to stabilize regulatory T cells (Treg) without altering Th1/Th2 (T helper type1/2) subsets. This occurs via the proteolytic degradation of MEKK1 (mitogen-activated protein kinase kinase kinase 1) and the subsequent inhibition of TGF (transforming growth factor)-ß1 production in LECs. Targeting calpain systems in LECs expanded Tregs in the blood circulation and reduced aortic atherosclerosis in hypercholesterolemic mice, concomitant with the reduction of proinflammatory macrophages in the lesions. Treg expansion in the blood circulation and atheroprotection in calpain-targeted mice was prevented by the administration of TGF-ß type-I receptor inhibitor. Moreover, lysophosphatidic acid-induced calpain overactivation potentiated the IL (interleukin)-18/NF-κB (nuclear factor κB)/VCAM1 (vascular cell adhesion molecule 1) axis in LECs, thereby inhibiting lymphocyte mobility on the cells. Indeed, VCAM1 in LECs was upregulated in hypercholesterolemic mice and human cases of coronary artery disease. Neutralization of VCAM1 or targeting LEC calpain systems recovered afferent Treg transportation via lymphatic vessels in mice. CONCLUSIONS: Calpain systems in LECs have a key role in controlling Treg stability and trafficking under hypercholesterolemia.

TLCD1 and TLCD2 regulate cellular phosphatidylethanolamine composition and promote the progression of non-alcoholic steatohepatitis.

The fatty acid composition of phosphatidylethanolamine (PE) determines cellular metabolism, oxidative stress, and inflammation. However, our understanding of how cells regulate PE composition is limited. Here, we identify a genetic locus on mouse chromosome 11, containing two poorly characterized genes Tlcd1 and Tlcd2, that strongly influences PE composition. We generated Tlcd1/2 double-knockout (DKO) mice and found that they have reduced levels of hepatic monounsaturated fatty acid (MUFA)-containing PE species. Mechanistically, TLCD1/2 proteins act cell intrinsically to promote the incorporation of MUFAs into PEs. Furthermore, TLCD1/2 interact with the mitochondria in an evolutionarily conserved manner and regulate mitochondrial PE composition. Lastly, we demonstrate the biological relevance of our findings in dietary models of metabolic disease, where Tlcd1/2 DKO mice display attenuated development of non-alcoholic steatohepatitis compared to controls. Overall, we identify TLCD1/2 proteins as key regulators of cellular PE composition, with our findings having broad implications in understanding and treating disease.

Identification and characterization of LPLAT7 as an sn-1-specific lysophospholipid acyltransferase.

The main fatty acids at the sn-1 position of phospholipids (PLs) are saturated or monounsaturated fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), and oleic acid (C18:1) and are constantly replaced, like unsaturated fatty acids at the sn-2 position. However, little is known about the molecular mechanism underlying the replacement of fatty acids at the sn-1 position, i.e., the sn-1 remodeling. Previously, we established a method to evaluate the incorporation of fatty acids into the sn-1 position of lysophospholipids (lyso-PLs). Here, we used this method to identify the enzymes capable of incorporating fatty acids into the sn-1 position of lyso-PLs (sn-1 lysophospholipid acyltransferase [LPLAT]). Screenings using siRNA knockdown and recombinant proteins for 14 LPLATs identified LPLAT7/lysophosphatidylglycerol acyltransferase 1 (LPGAT1) as a candidate. In vitro, we found LPLAT7 mainly incorporated several fatty acids into the sn-1 position of lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), with weak activities toward other lyso-PLs. Interestingly, however, only C18:0-containing phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were specifically reduced in the LPLAT7-mutant cells and tissues from knockout mice, with a concomitant increase in the level of C16:0- and C18:1-containing PC and PE. Consistent with this, the incorporation of deuterium-labeled C18:0 into PLs dramatically decreased in the mutant cells, while deuterium-labeled C16:0 and C18:1 showed the opposite dynamic. Identifying LPLAT7 as an sn-1 LPLAT facilitates understanding the biological significance of sn-1 fatty acid remodeling of PLs. We also propose to use the new nomenclature, LPLAT7, for LPGAT1 since the newly assigned enzymatic activities are quite different from the LPGAT1s previously reported.

Structure of SARS-CoV-2 membrane protein essential for virus assembly.

The coronavirus membrane protein (M) is the most abundant viral structural protein and plays a central role in virus assembly and morphogenesis. However, the process of M protein-driven virus assembly are largely unknown. Here, we report the cryo-electron microscopy structure of the SARS-CoV-2 M protein in two different conformations. M protein forms a mushroom-shaped dimer, composed of two transmembrane domain-swapped three-helix bundles and two intravirion domains. M protein further assembles into higher-order oligomers. A highly conserved hinge region is key for conformational changes. The M protein dimer is unexpectedly similar to SARS-CoV-2 ORF3a, a viral ion channel. Moreover, the interaction analyses of M protein with nucleocapsid protein (N) and RNA suggest that the M protein mediates the concerted recruitment of these components through the positively charged intravirion domain. Our data shed light on the M protein-driven virus assembly mechanism and provide a structural basis for therapeutic intervention targeting M protein.

Omega-3 fatty acid epoxides produced by PAF-AH2 in mast cells regulate pulmonary vascular remodeling.

Pulmonary hypertension is a fatal rare disease that causes right heart failure by elevated pulmonary arterial resistance. There is an unmet medical need for the development of therapeutics focusing on the pulmonary vascular remodeling. Bioactive lipids produced by perivascular inflammatory cells might modulate the vascular remodeling. Here, we show that ω-3 fatty acid-derived epoxides (ω-3 epoxides) released from mast cells by PAF-AH2, an oxidized phospholipid-selective phospholipase A2, negatively regulate pulmonary hypertension. Genetic deletion of Pafah2 in mice accelerate vascular remodeling, resulting in exacerbation of hypoxic pulmonary hypertension. Treatment with ω-3 epoxides suppresses the lung fibroblast activation by inhibiting TGF-ß signaling. In vivo ω-3 epoxides supplementation attenuates the progression of pulmonary hypertension in several animal models. Furthermore, whole-exome sequencing for patients with pulmonary arterial hypertension identifies two candidate pathogenic variants of Pafah2. Our findings support that the PAF-AH2-ω-3 epoxide production axis could be a promising therapeutic target for pulmonary hypertension.

Abnormal male reproduction and embryonic development induced by downregulation of a phospholipid fatty acid-introducing enzyme Lpgat1 in zebrafish.

Phospholipids in the membrane consist of diverse pairs of fatty acids bound to a glycerol backbone. The biological significance of the diversity, however, remains mostly unclear. Part of this diversity is due to lysophospholipid acyltransferases (LPLATs), which introduce a fatty acid into lysophospholipids. The human genome has 14 LPLATs and most of them are highly conserved in vertebrates. Here, we analyzed the function of one of these enzymes, lysophosphatidylglycerol acyltransferase 1 (Lpgat1), in zebrafish. We found that the reproduction of heterozygous (lpgat1+/-) male mutants was abnormal. Crosses between heterozygous males and wild-type females produced many eggs with no obvious cleavage, whereas eggs produced by crosses between heterozygous females and wild-type males cleaved normally. Consistent with this, spermatozoa from heterozygous males had reduced motility and abnormal morphology. We also found that the occurrence of lpgat1 homozygous (lpgat1-/-) mutants was far lower than expected. In addition, downregulation of lpgat1 by morpholino antisense oligonucleotides resulted in severe developmental defects. Lipidomic analysis revealed that selective phospholipid species with stearic acid and docosahexaenoic acid were reduced in homozygous larvae and spermatozoa from heterozygotes. These results suggest that the specific phospholipid molecular species produced by Lpgat1 have an essential role in sperm fertilization and in embryonic development.

Tricalbin proteins regulate plasma membrane phospholipid homeostasis.

The evolutionarily conserved extended synaptotagmin (E-Syt) proteins are calcium-activated lipid transfer proteins that function at contacts between the ER and plasma membrane (ER-PM contacts). However, roles of the E-Syt family members in PM lipid organisation remain incomplete. Among the E-Syt family, the yeast tricalbin (Tcb) proteins are essential for PM integrity upon heat stress, but it is not known how they contribute to PM maintenance. Using quantitative lipidomics and microscopy, we find that the Tcb proteins regulate phosphatidylserine homeostasis at the PM. Moreover, upon heat-induced membrane stress, Tcb3 co-localises with the PM protein Sfk1 that is implicated in PM phospholipid asymmetry and integrity. The Tcb proteins also control the PM targeting of the known phosphatidylserine effector Pkc1 upon heat-induced stress. Phosphatidylserine has evolutionarily conserved roles in PM organisation, integrity, and repair. We propose that phospholipid regulation is an ancient essential function of E-Syt family members required for PM integrity.

Update and nomenclature proposal for mammalian lysophospholipid acyltransferases, which create membrane phospholipid diversity.

The diversity of glycerophospholipid species in cellular membranes is immense and affects various biological functions. Glycerol-3-phosphate acyltransferases (GPATs) and lysophospholipid acyltransferases (LPLATs), in concert with phospholipase A1/2s enzymes, contribute to this diversity via selective esterification of fatty acyl chains at the sn-1 or sn-2 positions of membrane phospholipids. These enzymes are conserved across all kingdoms, and in mammals four GPATs of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and at least 14 LPLATs, either of the AGPAT or the membrane-bound O-acyltransferase (MBOAT) families, have been identified. Here we provide an overview of the biochemical and biological activities of these mammalian enzymes, including their predicted structures, involvements in human diseases, and essential physiological roles as revealed by gene-deficient mice. Recently, the nomenclature used to refer to these enzymes has generated some confusion due to the use of multiple names to refer to the same enzyme and instances of the same name being used to refer to completely different enzymes. Thus, this review proposes a more uniform LPLAT enzyme nomenclature, as well as providing an update of recent advances made in the study of LPLATs, continuing from our JBC mini review in 2009.

Supercritical fluid chromatography-mass spectrometry enables simultaneous measurement of all phosphoinositide regioisomers.

Phosphoinositide species, differing in phosphorylation at hydroxyls of the inositol head group, play roles in various cellular events. Despite the importance of phosphoinositides, simultaneous quantification of individual phosphoinositide species is difficult using conventional methods. Here we developed a supercritical fluid chromatography-mass spectrometry method that can quantify the molecular species of all seven phosphoinositide regioisomers. We used this method to analyze (1) profiles of phosphoinositide species in mouse tissues, (2) the effect of lysophosphatidylinositol acyltransferase 1-depletion on phosphoinositide acyl-chain composition in cultured cells, and (3) the molecular species of phosphatidylinositol-3-phosphate produced during the induction of autophagy. Although further improvement is needed for the absolute quantification of minor phosphoinositide regioisomers in biological samples, our method should clarify the physiological and pathological roles of phosphoinositide regioisomers at the molecular species level.

α-Tocopherol transfer protein (α-TTP).

α-Tocopherol transfer protein (α-TTP) is so far the only known protein that specifically recognizes α-tocopherol (α-Toc), the most abundant and most biologically active form of vitamin E, in higher animals. α-TTP is highly expressed in the liver where α-TTP selects α-Toc among vitamin E forms taken up via plasma lipoproteins and promotes its secretion to circulating lipoproteins. Thus, α-TTP is a major determinant of plasma α-Toc concentrations. Familial vitamin E deficiency, also called Ataxia with vitamin E deficiency, is caused by mutations in the α-TTP gene. More than 20 different mutations have been found in the α-TTP gene worldwide, among which some missense mutations provided valuable clues to elucidate the molecular mechanisms underlying intracellular α-Toc transport. In hepatocytes, α-TTP catalyzes the vectorial transport of α-Toc from the endocytotic compartment to the plasma membrane (PM) by targeting phosphatidylinositol phosphates (PIPs) such as PI(4,5)P2. By binding PIPs at the PM, α-TTP opens the lid covering the hydrophobic pocket, thus facilitating the release of bound α-Toc to the PM.

Suppressing postcollection lysophosphatidic acid metabolism improves the precision of plasma LPA quantification.

Lysophosphatidic acid (LPA) is a potent signaling lipid, and state-dependent alterations in plasma LPA make it a promising diagnostic marker for various diseases. However, plasma LPA concentrations vary widely among reports, even under normal conditions. These variations can be attributed, at least in part, to the artificial metabolism of LPA after blood collection. Here, we aimed to develop an optimized plasma preparation method that reflects the concentration of LPA in the circulating blood. The main features of the devised method were suppression of both LPA production and degradation after blood collection by keeping whole blood samples at low temperature followed by the addition of an autotaxin inhibitor to plasma samples. Using this devised method, the LPA level did not change for 30 min after blood collection. Also, human and mouse LPA levels were found to be much lower than those previously reported, ranging from 40 to 50 nM with minimal variation across the individual. Finally, the increased accuracy made it possible to detect circadian rhythms in the levels of certain LPA species in mouse plasma. These results demonstrate the usefulness of the devised plasma preparation method to determine accurate plasma LPA concentrations.

LPIAT1/MBOAT7 depletion increases triglyceride synthesis fueled by high phosphatidylinositol turnover.

OBJECTIVE: Non-alcoholic fatty liver disease (NAFLD) is a common prelude to cirrhosis and hepatocellular carcinoma. The genetic rs641738 C>T variant in the lysophosphatidylinositol acyltransferase 1 (LPIAT1)/membrane bound O-acyltransferase domain-containing 7, which incorporates arachidonic acid into phosphatidylinositol (PI), is associated with the entire spectrum of NAFLD. In this study, we investigated the mechanism underlying this association in mice and cultured human hepatocytes. DESIGN: We generated the hepatocyte-specific Lpiat1 knockout mice to investigate the function of Lpiat1 in vivo. We also depleted LPIAT1 in cultured human hepatic cells using CRISPR-Cas9 systems or siRNA. The effect of LPIAT1-depletion on liver fibrosis was examined in mice fed high fat diet and in liver spheroids. Lipid species were measured using liquid chromatography-electrospray ionisation mass spectrometry. Lipid metabolism was analysed using radiolabeled glycerol or fatty acids. RESULTS: The hepatocyte-specific Lpiat1 knockout mice developed hepatic steatosis spontaneously, and hepatic fibrosis on high fat diet feeding. Depletion of LPIAT1 in cultured hepatic cells and in spheroids caused triglyceride accumulation and collagen deposition. The increase in hepatocyte fat content was due to a higher triglyceride synthesis fueled by a non-canonical pathway. Indeed, reduction in the PI acyl chain remodelling caused a high PI turnover, by stimulating at the same time PI synthesis and breakdown. The degradation of PI was mediated by a phospholipase C, which produces diacylglycerol, a precursor of triglyceride. CONCLUSION: We found a novel pathway fueling triglyceride synthesis in hepatocytes, by a direct metabolic flow of PI into triglycerides. Our findings provide an insight into the pathogenesis and therapeutics of NAFLD.

Role of Phosphatidylethanolamine Biosynthesis in Herpes Simplex Virus 1-Infected Cells in Progeny Virus Morphogenesis in the Cytoplasm and in Viral Pathogenicity In Vivo.

Glycerophospholipids are major components of cell membranes. Phosphatidylethanolamine (PE) is a glycerophospholipid that is involved in multiple cellular processes, such as membrane fusion, the cell cycle, autophagy, and apoptosis. In this study, we investigated the role of PE biosynthesis in herpes simplex virus 1 (HSV-1) infection by knocking out the host cell gene encoding phosphate cytidylyltransferase 2, ethanolamine (Pcyt2), which is a key rate-limiting enzyme in one of the two major pathways for PE biosynthesis. Pcyt2 knockout reduced HSV-1 replication and caused an accumulation of unenveloped and partially enveloped nucleocapsids in the cytoplasm of an HSV-1-infected cell culture. A similar phenotype was observed when infected cells were treated with meclizine, which is an inhibitor of Pcyt2. In addition, treatment of HSV-1-infected mice with meclizine significantly reduced HSV-1 replication in the mouse brains and improved their survival rates. These results indicated that PE biosynthesis mediated by Pcyt2 was required for efficient HSV-1 envelopment in the cytoplasm of infected cells and for viral replication and pathogenicity in vivo The results also identified the PE biosynthetic pathway as a possible novel target for antiviral therapy of HSV-associated diseases and raised an interesting possibility for meclizine repositioning for treatment of these diseases, since it is an over-the-counter drug that has been used for decades against nausea and vertigo in motion sickness.IMPORTANCE Glycerophospholipids in cell membranes and virus envelopes often affect viral entry and budding. However, the role of glycerophospholipids in membrane-associated events in viral replication in herpesvirus-infected cells has not been reported to date. In this study, we have presented data showing that cellular PE biosynthesis mediated by Pcyt2 is important for HSV-1 envelopment in the cytoplasm, as well as for viral replication and pathogenicity in vivo This is the first report showing the importance of PE biosynthesis in herpesvirus infections. Our results showed that inhibition of Pcyt2, a key cell enzyme for PE synthesis, significantly inhibited HSV-1 replication and pathogenicity in mice. This suggested that the PE biosynthetic pathway, as well as the HSV-1 virion maturation pathway, can be a target for the development of novel anti-HSV drugs.

Palmitate induces cardiomyocyte death via inositol requiring enzyme-1 (IRE1)-mediated signaling independent of X-box binding protein 1 (XBP1).

Overloading of the saturated fatty acid (SFA) palmitate induces cardiomyocyte death. The purpose of this study is to elucidate signaling pathways contributing to palmitate-induced cardiomyocyte death. Palmitate-induced cardiomyocyte death was induced in Toll-like receptor 2/4 double-knockdown cardiomyocytes to a similar extent as wild-type cardiomyocytes, while cardiomyocyte death was canceled out by triacsin C, a long-chain acyl-CoA synthetase inhibitor. These results indicated that palmitate induced cytotoxicity after entry and conversion into palmitoyl-CoA. Palmitoyl-CoA is not only degraded by mitochondrial oxidation but also taken up as a component of membrane phospholipids. Palmitate overloading causes cardiomyocyte membrane fatty acid (FA) saturation, which is associated with the activation of endoplasmic reticulum (ER) unfolded protein response (UPR) signaling. We focused on the ER UPR signaling as a possible mechanism of cell death. Palmitate loading activates the UPR signal via membrane FA saturation, but not via unfolded protein overload in the ER since the chemical chaperone 4-phenylbutyrate failed to suppress palmitate-induced ER UPR. The mammalian UPR relies on three ER stress sensors named inositol requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6). Palmitate loading activated only IRE1 and PERK. Knockdown of PERK did not affect palmitate-induced cardiomyocyte death, while knockdown of IRE1 suppressed palmitate-induced cardiomyocyte death. However, knockdown of X-box binding protein 1 (XBP1), the downstream effector of IRE1, did not affect palmitate-induced cardiomyocyte death. These results were validated by pharmacological inhibitor experiments. In conclusion, we identified that palmitate-induced cardiomyocyte death was triggered by IRE1-mediated signaling independent of XBP1.