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The neutral lipids diacylglycerols (DAGs) are involved in a plethora of metabolic pathways. They function as components of cellular membranes, as building blocks for glycero(phospho)lipids, and as lipid second messengers. Considering their central role in multiple metabolic processes and signaling pathways, cellular DAG levels require a tight regulation to ensure a constant and controlled availability. Interestingly, DAG species are versatile in their chemical structure. Besides the different fatty acid species esterified to the glycerol backbone, DAGs can occur in three different stereo/regioisoforms, each with unique biological properties. Recent scientific advances have revealed that DAG metabolizing enzymes generate and distinguish different DAG isoforms, and that only one DAG isoform holds signaling properties. Herein, we review the current knowledge of DAG stereochemistry and their impact on cellular metabolism and signaling. Further, we describe intracellular DAG turnover and its stereochemistry in a 3-pool model to illustrate the spatial and stereochemical separation and hereby the diversity of cellular DAG metabolism.

Plants accumulate the lipids phosphatidic acid (PA), diacylglycerol (DAG), and triacylglycerol (TAG) during cold stress, but how plants balance the levels of these lipids to mediate cold responses remains unknown. The enzymes ACYL-COENZYME A:DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) and DIACYLGLYCEROL KINASE (DGK) catalyze the conversion of DAG to TAG and PA, respectively. Here, we show that DGAT1, DGK2, DGK3, and DGK5 contribute to the response to cold in Arabidopsis (Arabidopsis thaliana). With or without cold acclimation, the dgat1 mutants exhibited higher sensitivity upon freezing exposure compared with the wild type. Under cold conditions, the dgat1 mutants showed reduced expression of C-REPEAT/DRE BINDING FACTOR2 and its regulons, which are essential for the acquisition of cold tolerance. Lipid profiling revealed that freezing significantly increased the levels of PA and DAG while decreasing TAG in the rosettes of dgat1 mutant plants. During freezing stress, the accumulation of PA in dgat1 plants stimulated NADPH oxidase activity and enhanced RbohD-dependent hydrogen peroxide production compared with the wild type. Moreover, the cold-inducible transcripts of DGK2, DGK3, and DGK5 were significantly more up-regulated in the dgat1 mutants than in the wild type during cold stress. Consistent with this observation, dgk2, dgk3, and dgk5 knockout mutants showed improved tolerance and attenuated PA production in response to freezing temperatures. Our findings demonstrate that the conversion of DAG to TAG by DGAT1 is critical for plant freezing tolerance, acting by balancing TAG and PA production in Arabidopsis.

Adiponectin has emerged as a potential therapy for type 2 diabetes mellitus, but the molecular mechanism by which adiponectin reverses insulin resistance remains unclear. Two weeks of globular adiponectin (gAcrp30) treatment reduced fasting plasma glucose, triglyceride (TAG), and insulin concentrations and reversed whole-body insulin resistance, which could be attributed to both improved insulin-mediated suppression of endogenous glucose production and increased insulin-stimulated glucose uptake in muscle and adipose tissues. These improvements in liver and muscle sensitivity were associated with ∼50% reductions in liver and muscle TAG and plasma membrane (PM)-associated diacylglycerol (DAG) content and occurred independent of reductions in total ceramide content. Reductions of PM DAG content in liver and skeletal muscle were associated with reduced PKCε translocation in liver and reduced PKCθ and PKCε translocation in skeletal muscle resulting in increased insulin-stimulated insulin receptor tyrosine1162 phosphorylation, IRS-1/IRS-2–associated PI3-kinase activity, and Akt-serine phosphorylation. Both gAcrp30 and full-length adiponectin (Acrp30) treatment increased eNOS/AMPK activation in muscle and muscle fatty acid oxidation. gAcrp30 and Acrp30 infusions also increased TAG uptake in epididymal white adipose tissue (eWAT), which could be attributed to increased lipoprotein lipase (LPL) activity. These data suggest that adiponectin and adiponectin-related molecules reverse lipid-induced liver and muscle insulin resistance by reducing ectopic lipid storage in these organs, resulting in decreased plasma membrane sn-1,2-DAG–induced nPKC activity and increased insulin signaling. Adiponectin mediates these effects by both promoting the storage of TAG in eWAT likely through stimulation of LPL as well as by stimulation of AMPK in muscle resulting in increased muscle fat oxidation.

Protein kinase C (PKC) and Protein kinase D (PKD) isoforms can sense diacylglycerol (DAG) generated in the different cellular compartments in various physiological processes. DAG accumulates in multiple organs of the obese subjects, which leads to the disruption of metabolic homeostasis and the development of diabetes as well as associated diseases. Multiple studies proved that aberrant activation of PKCs and PKDs contributes to the development of metabolic diseases. DAG-sensing PKC and PKD isoforms play a crucial role in the regulation of metabolic homeostasis and therefore might serve as targets for the treatment of metabolic disorders such as obesity and diabetes.

Non-alcoholic fatty liver disease and its downstream sequelae, hepatic insulin resistance and type 2 diabetes, are rapidly growing epidemics, which lead to increased morbidity and mortality rates, and soaring health-care costs. Developing interventions requires a comprehensive understanding of the mechanisms by which excess hepatic lipid develops and causes hepatic insulin resistance and type 2 diabetes. Proposed mechanisms implicate various lipid species, inflammatory signalling and other cellular modifications. Studies in mice and humans have elucidated a key role for hepatic diacylglycerol activation of protein kinase Cε in triggering hepatic insulin resistance. Therapeutic approaches based on this mechanism could alleviate the related epidemics of non-alcoholic fatty liver disease and type 2 diabetes.

Muscle insulin resistance is a key feature of obesity and type 2 diabetes and is strongly associated with increased intramyocellular lipid content and inflammation. However, the cellular and molecular mechanisms responsible for causing muscle insulin resistance in humans are still unclear. To address this question, we performed serial muscle biopsies in healthy, lean subjects before and during a lipid infusion to induce acute muscle insulin resistance and assessed lipid and inflammatory parameters that have been previously implicated in causing muscle insulin resistance. We found that acute induction of muscle insulin resistance was associated with a transient increase in total and cytosolic diacylglycerol (DAG) content that was temporally associated with protein kinase (PKC)θ activation, increased insulin receptor substrate (IRS)-1 serine 1101 phosphorylation, and inhibition of insulin-stimulated IRS-1 tyrosine phosphorylation and AKT2 phosphorylation. In contrast, there were no associations between insulin resistance and alterations in muscle ceramide, acylcarnitine content, or adipocytokines (interleukin-6, adiponectin, retinol-binding protein 4) or soluble intercellular adhesion molecule-1. Similar associations between muscle DAG content, PKCθ activation, and muscle insulin resistance were observed in healthy insulin-resistant obese subjects and obese type 2 diabetic subjects. Taken together, these data support a key role for DAG activation of PKCθ in the pathogenesis of lipid-induced muscle insulin resistance in obese and type 2 diabetic individuals.

Diacylglycerol O -acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans 1 . 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 2 , 3 . 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. The structure of human diacylglycerol O -acyltransferase 1, a membrane protein that synthesizes triacylglycerides, is solved with cryo-electron microscopy, providing insight into its function and mechanism of enzymatic activity.

Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding. Perilipin 3 binds to emerging lipid droplets from the endoplasmic reticulum. Choi and Ajjaji et al characterize the molecular mechanisms of Perilipin 3 recruitment to membranes and uncover a function for the PAT domain in diacylglycerol sensing.

Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5′-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) that are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphotransferases (CDP-APs). Here, the authors present the cryo-EM structure of yeast CPT1, a critical enzyme in phospholipid synthesis, identifying residues crucial for substrate preference. This enable a reaction mechanism for the family of CDP-alcohol phosphotransferases to be proposed.

A photoswitchable diacylglycerol enables spatiotemporal control of membrane translocation of C1-domain-containing proteins and protein kinase C activation to modulate calcium oscillations and vesicle release for synaptic transmission. Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans . As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.