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Frontiers in Immunology 2022Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen...
INTRODUCTION
Activation of T cell receptor (TCR) signaling is critical for clonal expansion of CD8+ T cells. However, the effects of augmenting TCR signaling during chronic antigen exposure is less understood. Here, we investigated the role of diacylglycerol (DAG)-mediated signaling downstream of the TCR during chronic lymphocytic choriomeningitis virus clone 13 (LCMV CL13) infection by blocking DAG kinase zeta (DGKζ), a negative regulator of DAG.
METHODS
We examined the activation, survival, expansion, and phenotype of virus-specific T cell in the acute and chronic phases of LCMV CL13-infected in mice after DGKζ blockade or selective activation of ERK.
RESULTS
Upon LCMV CL13 infection, DGKζ deficiency promoted early short-lived effector cell (SLEC) differentiation of LCMV-specific CD8+ T cells, but this was followed by abrupt cell death. Short-term inhibition of DGKζ with ASP1570, a DGKζ-selective pharmacological inhibitor, augmented CD8+ T cell activation without causing cell death, which reduced virus titers both in the acute and chronic phases of LCMV CL13 infection. Unexpectedly, the selective enhancement of ERK, one key signaling pathway downstream of DAG, lowered viral titers and promoted expansion, survival, and a memory phenotype of LCMV-specific CD8+ T cells in the acute phase with fewer exhausted T cells in the chronic phase. The difference seen between DGKζ deficiency and selective ERK enhancement could be potentially explained by the activation of the AKT/mTOR pathway by DGKζ deficiency, since the mTOR inhibitor rapamycin rescued the abrupt cell death seen in virus-specific DGKζ KO CD8+ T cells.
DISCUSSION
Thus, while ERK is downstream of DAG signaling, the two pathways lead to distinct outcomes in the context of chronic CD8+ T cell activation, whereby DAG promotes SLEC differentiation and ERK promotes a memory phenotype.
Topics: Animals; Mice; CD8-Positive T-Lymphocytes; Diglycerides; Lymphocytic Choriomeningitis; Lymphocytic choriomeningitis virus; Receptors, Antigen, T-Cell; Signal Transduction; TOR Serine-Threonine Kinases; MAP Kinase Signaling System
PubMed: 36846018
DOI: 10.3389/fimmu.2022.1032113 -
Arteriosclerosis, Thrombosis, and... Mar 2024Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into...
BACKGROUND
Oxidized phospholipids play a key role in the atherogenic potential of lipoprotein(a) (Lp[a]); however, Lp(a) is a complex particle that warrants research into additional proinflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a).
METHODS
Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNA sequencing, ELISA, Western blot, and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions.
RESULTS
Compared with healthy individuals with low/normal Lp(a) levels (median, 7 mg/dL [18 nmol/L]; n=13), individuals with elevated Lp(a) levels (median, 87 mg/dL [218 nmol/L]; n=12) demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in proinflammatory pathways related to leukocyte migration, chemotaxis, NF-κB (nuclear factor kappa B) signaling, and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL (interleukin)-6, IL-8, and IL-1β after DG(40:6) and DG(38:4) stimulation, which was, in part, mediated via the NLRP3 (NOD [nucleotide-binding oligomerization domain]-like receptor family pyrin domain containing 3) inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated colocalization of Lp(a) with M1 macrophages, and an enrichment of CD68IL-18TLR4 (toll-like receptor) TREM2 (triggering receptor expressed on myeloid cells) resident macrophages and CD68CASP1 (caspase) IL-1BSELL (selectin L) inflammatory macrophages compared with patients with low Lp(a). Finally, potent Lp(a)-lowering treatment (pelacarsen) resulted in a reduction in specific circulating DG lipid subspecies in patients with cardiovascular disease with elevated Lp(a) levels (median, 82 mg/dL [205 nmol/L]).
CONCLUSIONS
Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is, in part, NLRP3 inflammasome dependent.
Topics: Humans; Diglycerides; Inflammasomes; Inflammation; Interleukin-1beta; Interleukin-6; Lipoprotein(a); Lysophospholipids; Monocytes; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein
PubMed: 38269588
DOI: 10.1161/ATVBAHA.123.319937 -
Cell Research Nov 2019The response of endothelial cells to signaling stimulation is critical for vascular morphogenesis, homeostasis and function. Vascular endothelial growth factor-a (VEGFA)...
The response of endothelial cells to signaling stimulation is critical for vascular morphogenesis, homeostasis and function. Vascular endothelial growth factor-a (VEGFA) has been commonly recognized as a pro-angiogenic factor in vertebrate developmental, physiological and pathological conditions for decades. Here we report a novel finding that genetic ablation of CDP-diacylglycerol synthetase-2 (CDS2), a metabolic enzyme that controls phosphoinositide recycling, switches the output of VEGFA signaling from promoting angiogenesis to unexpectedly inducing vessel regression. Live imaging analysis uncovered the presence of reverse migration of the angiogenic endothelium in cds2 mutant zebrafish upon VEGFA stimulation, and endothelium regression also occurred in postnatal retina and implanted tumor models in mice. In tumor models, CDS2 deficiency enhanced the level of tumor-secreted VEGFA, which in-turn trapped tumors into a VEGFA-induced vessel regression situation, leading to suppression of tumor growth. Mechanistically, VEGFA stimulation reduced phosphatidylinositol (4,5)-bisphosphate (PIP2) availability in the absence of CDS2-controlled-phosphoinositide metabolism, subsequently causing phosphatidylinositol (3,4,5)-triphosphate (PIP3) deficiency and FOXO1 activation to trigger regression of CDS2-null endothelium. Thus, our data indicate that the effect of VEGFA on vasculature is context-dependent and can be converted from angiogenesis to vascular regression.
Topics: Animals; Cell Line, Tumor; Diacylglycerol Cholinephosphotransferase; Endothelial Cells; Humans; Melanoma, Experimental; Mice; Mice, Knockout; Neoplasms; Neovascularization, Pathologic; Neovascularization, Physiologic; Nucleotidyltransferases; Vascular Endothelial Growth Factor A; Zebrafish; Zebrafish Proteins
PubMed: 31501519
DOI: 10.1038/s41422-019-0229-5 -
Nature May 2020Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans. DGAT1 belongs to the...
Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.
Topics: Acyl Coenzyme A; Binding Sites; Cryoelectron Microscopy; Diacylglycerol O-Acyltransferase; Diglycerides; Humans; Models, Molecular; Protein Multimerization; Structure-Activity Relationship; Triglycerides
PubMed: 32433610
DOI: 10.1038/s41586-020-2280-2 -
International Journal of Molecular... Sep 2020A large number of studies reported an association between elevated circulating and tissue lipid content and metabolic disorders in obesity, type 2 diabetes (T2D) and... (Review)
Review
A large number of studies reported an association between elevated circulating and tissue lipid content and metabolic disorders in obesity, type 2 diabetes (T2D) and aging. This state of uncontrolled tissue lipid accumulation has been called lipotoxicity. It was later shown that excess lipid flux is mainly neutralized within lipid droplets as triglycerides, while several bioactive lipid species such as diacylglycerols (DAGs), ceramides and their derivatives have been mechanistically linked to the pathogenesis of insulin resistance (IR) by antagonizing insulin signaling and action in metabolic organs such as the liver and skeletal muscle. Skeletal muscle and the liver are the main sites of glucose disposal in the body and IR in these tissues plays a pivotal role in the development of T2D. In this review, we critically examine recent literature supporting a causal role of DAGs and ceramides in the development of IR. A particular emphasis is placed on transgenic mouse models with modulation of total DAG and ceramide pools, as well as on modulation of specific subspecies, in relation to insulin sensitivity. Collectively, although a wide number of studies converge towards the conclusion that both DAGs and ceramides cause IR in metabolic organs, there are still some uncertainties on their mechanisms of action. Recent studies reveal that subcellular localization and acyl chain composition are determinants in the biological activity of these lipotoxic lipids and should be further examined.
Topics: Animals; Diabetes Mellitus, Type 2; Glucose Intolerance; Humans; Insulin Resistance; Lipids
PubMed: 32887221
DOI: 10.3390/ijms21176358 -
Cancer Communications (London, England) Feb 2024Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and lethal hepatobiliary tumor with few therapeutic strategies. The metabolic reprogramming of tumor...
BACKGROUND
Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and lethal hepatobiliary tumor with few therapeutic strategies. The metabolic reprogramming of tumor cells plays an essential role in the development of tumors, while the metabolic molecular classification of iCCA is largely unknown. Here, we performed an integrated multiomics analysis and metabolic classification to depict differences in metabolic characteristics of iCCA patients, hoping to provide a novel perspective to understand and treat iCCA.
METHODS
We performed integrated multiomics analysis in 116 iCCA samples, including whole-exome sequencing, bulk RNA-sequencing and proteome analysis. Based on the non-negative matrix factorization method and the protein abundance of metabolic genes in human genome-scale metabolic models, the metabolic subtype of iCCA was determined. Survival and prognostic gene analyses were used to compare overall survival (OS) differences between metabolic subtypes. Cell proliferation analysis, 5-ethynyl-2'-deoxyuridine (EdU) assay, colony formation assay, RNA-sequencing and Western blotting were performed to investigate the molecular mechanisms of diacylglycerol kinase α (DGKA) in iCCA cells.
RESULTS
Three metabolic subtypes (S1-S3) with subtype-specific biomarkers of iCCA were identified. These metabolic subtypes presented with distinct prognoses, metabolic features, immune microenvironments, and genetic alterations. The S2 subtype with the worst survival showed the activation of some special metabolic processes, immune-suppressed microenvironment and Kirsten rat sarcoma viral oncogene homolog (KRAS)/AT-rich interactive domain 1A (ARID1A) mutations. Among the S2 subtype-specific upregulated proteins, DGKA was further identified as a potential drug target for iCCA, which promoted cell proliferation by enhancing phosphatidic acid (PA) metabolism and activating mitogen-activated protein kinase (MAPK) signaling.
CONCLUSION
Via multiomics analyses, we identified three metabolic subtypes of iCCA, revealing that the S2 subtype exhibited the poorest survival outcomes. We further identified DGKA as a potential target for the S2 subtype.
Topics: Humans; Diacylglycerol Kinase; Multiomics; Cholangiocarcinoma; Bile Ducts, Intrahepatic; Bile Duct Neoplasms; RNA; Tumor Microenvironment
PubMed: 38143235
DOI: 10.1002/cac2.12513 -
Respiratory Research Jun 2023Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that...
BACKGROUND
Diacylglycerol kinase (DGK) regulates intracellular signaling and functions by converting diacylglycerol (DAG) into phosphatidic acid. We previously demonstrated that DGK inhibition attenuates airway smooth muscle (ASM) cell proliferation, however, the mechanisms mediating this effect are not well established. Given the capacity of protein kinase A (PKA) to effect inhibition of ASM cells growth in response to mitogens, we employed multiple molecular and pharmacological approaches to examine the putative role of PKA in the inhibition of mitogen-induced ASM cell proliferation by the small molecular DGK inhibitor I (DGK I).
METHODS
We assayed cell proliferation using CyQUANT™ NF assay, protein expression and phosphorylation using immunoblotting, and prostaglandin E (PGE) secretion by ELISA. ASM cells stably expressing GFP or PKI-GFP (PKA inhibitory peptide-GFP chimera) were stimulated with platelet-derived growth factor (PDGF), or PDGF + DGK I, and cell proliferation was assessed.
RESULTS
DGK inhibition reduced ASM cell proliferation in cells expressing GFP, but not in cells expressing PKI-GFP. DGK inhibition increased cyclooxygenase II (COXII) expression and PGE secretion over time to promote PKA activation as demonstrated by increased phosphorylation of (PKA substrates) VASP and CREB. COXII expression and PKA activation were significantly decreased in cells pre-treated with pan-PKC (Bis I), MEK (U0126), or ERK2 (Vx11e) inhibitors suggesting a role for PKC and ERK in the COXII-PGE-mediated activation of PKA signaling by DGK inhibition.
CONCLUSIONS
Our study provides insight into the molecular pathway (DAG-PKC/ERK-COXII-PGE-PKA) regulated by DGK in ASM cells and identifies DGK as a potential therapeutic target for mitigating ASM cell proliferation that contributes to airway remodeling in asthma.
Topics: Diacylglycerol Kinase; Cyclic AMP-Dependent Protein Kinases; Cells, Cultured; Cell Proliferation; Myocytes, Smooth Muscle
PubMed: 37301818
DOI: 10.1186/s12931-023-02465-8 -
Biochimica Et Biophysica Acta.... Oct 2019Lipins are phosphatidic acid phosphatase enzymes whose cellular function in regulating lipid metabolism has been known for decades, particularly in metabolically active... (Review)
Review
Lipins are phosphatidic acid phosphatase enzymes whose cellular function in regulating lipid metabolism has been known for decades, particularly in metabolically active tissues such as adipose tissue or liver. In recent years evidence is accumulating for key regulatory roles of the lipin family in innate immune cells. Lipins may help regulate signaling through relevant immune receptors such as Toll-like receptors, and are also integral part of the cellular machinery for lipid storage in these cells, thereby modulating certain inflammatory processes. Mutations in genes that encode for members of this family produce autoinflammatory hereditary diseases or diseases with an important inflammatory component in humans. In this review we summarize recent findings on the role of lipins in cells of the innate immune system and in the onset and progress of inflammatory processes.
Topics: Animals; Diglycerides; Humans; Immunity, Innate; Inflammation; Macrophages; Phosphatidate Phosphatase; Phosphatidic Acids
PubMed: 31220616
DOI: 10.1016/j.bbalip.2019.06.003 -
Frontiers in Immunology 2022Brain lipid dysregulation is a hallmark of depression and Alzheimer's disease, also marked by chronic inflammation. Early-life stress (ELS) and dietary intake of...
Brain lipid dysregulation is a hallmark of depression and Alzheimer's disease, also marked by chronic inflammation. Early-life stress (ELS) and dietary intake of polyunsaturated fatty acids (PUFAs) are risk factors for these pathologies and are known to impact inflammatory processes. However, if these early-life factors alter brain lipid homeostasis on the long-term and thereby contribute to this risk remains to be elucidated. We have recently shown that an early diet enriched in omega(ω)-3 PUFAs protected against the long-term negative effects of ELS on cognition and neuroinflammation. Here, we aim to understand if modulation of brain lipid and oxylipin profiles contributes to the detrimental effects of ELS and the protective ones of the diet. We therefore studied if and how ELS and early dietary PUFAs modulate the brain lipid and oxylipin profile, basally as well as in response to an inflammatory challenge, to unmask possible latent effects. Male mice were exposed to ELS the limited bedding and nesting paradigm, received an early diet with high or low ω6/ω3 ratio (HRD and LRD) and were injected with saline or lipopolysaccharide (LPS) in adulthood. Twenty-four hours later plasma cytokines (Multiplex) and hypothalamic lipids and oxylipins (liquid chromatography tandem mass spectrometry) were measured. ELS exacerbated the LPS-induced increase in IL-6, CXCL1 and CCL2. Both ELS and diet affected the lipid/oxylipin profile long-term. For example, ELS increased diacylglycerol and LRD reduced triacylglycerol, free fatty acids and ceramides. Importantly, the ELS-induced alterations were strongly influenced by the early diet. For example, the ELS-induced decrease in eicosapentaenoic acid was reversed when fed LRD. Similarly, the majority of the LPS-induced alterations were distinct for control and ELS exposed mice and unique for mice fed with LRD or HRD. LPS decreased ceramides and lysophosphotidylcholine, increased hexosylceramides and prostaglandin E, reduced triacylglycerol species and ω6-derived oxylipins only in mice fed LRD and ELS reduced the LPS-induced increase in phosphatidylcholine. These data give further insights into the alterations in brain lipids and oxylipins that might contribute to the detrimental effects of ELS, to the protective ones of LRD and the possible early-origin of brain lipid dyshomeostasis characterizing ELS-related psychopathologies.
Topics: Animals; Male; Mice; Brain; Ceramides; Cytokines; Diglycerides; Eicosapentaenoic Acid; Fatty Acids, Nonesterified; Fatty Acids, Omega-3; Fatty Acids, Unsaturated; Interleukin-6; Lipopolysaccharides; Oxylipins; Phosphatidylcholines; Prostaglandins; Triglycerides; Stress, Psychological
PubMed: 36131915
DOI: 10.3389/fimmu.2022.967437 -
Nutrients Nov 2022Lipid droplet is a dynamic organelle that undergoes periods of biogenesis and degradation under environmental stimuli. The excessive accumulation of lipid droplets is...
Lipid droplet is a dynamic organelle that undergoes periods of biogenesis and degradation under environmental stimuli. The excessive accumulation of lipid droplets is the major characteristic of non-alcoholic fatty liver disease (NAFLD). Moderate aerobic exercise is a powerful intervention protecting against the progress of NAFLD. However, its impact on lipid droplet dynamics remains ambiguous. Mice were fed with 15 weeks of high-fat diet in order to induce NAFLD. Meanwhile, the mice performed 15 weeks of treadmill exercise. Our results showed that 15 weeks of regular moderate treadmill exercise alleviated obesity, insulin intolerance, hyperlipidemia, and hyperglycemia induced by HFD. Importantly, exercise improved histological phenotypes of NAFLD, including hepatic steatosis, inflammation, and locular ballooning, as well as prevented liver fat deposition and liver injury induced by HFD. Exercise reduced hepatic lipid droplet size, and moreover, it reduced PLIN2 protein level and increased PLIN3 protein level in the liver of HFD mice. Interestingly, our results showed that exercise did not significantly affect the gene expressions of , , or , which were involved in TG synthesis. However, it did reduce the expressions of FITM2, , and , which were major involved in lipid droplet growth and budding, and lipid droplet expansion. In addition, exercise reduced ATGL protein level in HFD mice, and regulated lipophagy-related markers, including increasing ATG5, LAMP1, LAMP2, LAL, and CTSD, decreasing LC3II/I and p62, and promoting colocalization of LAMP1 with LDs. In summary, our data suggested that 15 weeks of moderate treadmill exercise was beneficial for regulating liver lipid droplet dynamics in HFD mice by inhibiting abnormal lipid droplets expansion and enhancing clearance of lipid droplets by lysosomes during the lipophagic process, which might provide highly flexible turnover for lipid mobilization and metabolism. Abbreviations: β-actin: actin beta; ATG5: autophagy related 5; LAMP2: lysosomal-associated membrane protein 2; LAMP1: lysosomal-associated membrane protein 1; SQSTM1/p62: sequestosome 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; ATGL: adipose triglyceride lipase; CSTD: cathepsin D; LAL: lysosomal acid lipase; DGAT1: diacylglycerol-o-acyltransferase 1; DGAT2: diacylglycerol-o-acyltransferase 2; CIDEA: cell death inducing dffa-like effector a; CIDEC/FSP27: cell death inducing dffa-like effector c; FITM2: fat storage-inducing transmembrane protein 2; PLIN2: adipose differentiation related protein; PLN3: tail-interacting protein 47; HSP90: heat shock protein 90; SREBP1c: sterol regulatory element binding protein-1c; chREBP: carbohydrate response element binding protein.
Topics: Mice; Animals; Non-alcoholic Fatty Liver Disease; Lipid Droplets; Diglycerides; Autophagy; Perilipin-2; Sterol Regulatory Element Binding Protein 1; Diacylglycerol O-Acyltransferase
PubMed: 36432597
DOI: 10.3390/nu14224910