The accumulation of lipid droplets (LDs) and ceramides (Cer) is linked to non-alcoholic fatty liver disease (NAFLD), regularly co-existing with type 2 diabetes and decreased immune function. Chronic inflammation and increased disease severity in viral infections are the hallmarks of the obesity-related immunopathology. The upregulation of neutral sphingomyelinase-2 (NSM2) has shown to be associated with the pathology of obesity in tissues. Nevertheless, the role of sphingolipids and specifically of NSM2 in the regulation of immune cell response to a fatty acid (FA) rich environment is poorly studied. Here, we identified the presence of the LD marker protein perilipin 3 (PLIN3) in the intracellular nano-environment of NSM2 using the ascorbate peroxidase APEX2-catalyzed proximity-dependent biotin labeling method. In line with this, super-resolution structured illumination microscopy (SIM) shows NSM2 and PLIN3 co-localization in LD organelles in the presence of increased extracellular concentrations of oleic acid (OA). Furthermore, the association of enzymatically active NSM2 with isolated LDs correlates with increased Cer levels in these lipid storage organelles. NSM2 enzymatic activity is not required for NSM2 association with LDs, but negatively affects the LD numbers and cellular accumulation of long-chain unsaturated triacylglycerol (TAG) species. Concurrently, NSM2 expression promotes mitochondrial respiration and fatty acid oxidation (FAO) in response to increased OA levels, thereby shifting cells to a high energetic state. Importantly, endogenous NSM2 activity is crucial for primary human CD4+ T cell survival and proliferation in a FA rich environment. To conclude, our study shows a novel NSM2 intracellular localization to LDs and the role of enzymatically active NSM2 in metabolic response to enhanced FA concentrations in T cells.

Physiological activation by light of the Drosophila TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP2) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells. Unlike previous studies, we added OAG into the cytosol via a patch-clamp pipette and observed robust activation of the expressed TRPL channels. However, TRPL channel activation was much slower than the physiologically activated TRPL by light. Therefore, we used a picosecond-fast optically activated DAG analogue, OptoDArG. Inactive OptoDArG was added into the intracellular solution with the patch-clamp pipette, and it slowly accumulated on the surface membrane of the recorded HEK cell in the dark. A fast application of intense UV light to the recorded cell resulted in a robust and relatively fast TRPL-dependent current that was greatly accelerated by the constitutively active TRPLF557I pore-region mutation. However, this current of the mutant channel was still considerably slower than the native light-induced TRPL current, suggesting that DAG alone is not sufficient for TRPL channel activation under physiological conditions.

Transient Receptor Potential (TRP) channels constitute a large superfamily of polymodal channel proteins with diverse roles in many physiological and sensory systems that function both as ionotropic and metabotropic receptors. From the early days of TRP channel discovery, membrane lipids were suggested to play a fundamental role in channel activation and regulation. A prominent example is the Drosophila TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of Drosophila. Light activation of the TRP and TRPL channels, the founding members of the TRP channel superfamily, requires activation of phospholipase Cβ (PLC), which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into Diacylglycerol (DAG) and Inositol 1, 4,5-trisphosphate (IP3). However, the events required for channel gating downstream of PLC activation are still under debate and led to several hypotheses regarding the mechanisms by which lipids gate the channels. Despite many efforts, compelling evidence of the involvement of DAG accumulation, PIP2 depletion or IP3-mediated Ca2+ release in light activation of the TRP/TRPL channels are still lacking. Exogeneous application of poly unsaturated fatty acids (PUFAs), a product of DAG hydrolysis was demonstrated as an efficient way to activate the Drosophila TRP/TRPL channels. However, compelling evidence for the involvement of PUFAs in physiological light-activation of the TRP/TRPL channels is still lacking. Light-induced mechanical force generation was measured in photoreceptor cells prior to channel opening. This mechanical force depends on PLC activity, suggesting that the enzymatic activity of PLC converting PIP2 into DAG generates membrane tension, leading to mechanical gating of the channels. In this review, we will present the roles of membrane lipids in light activation of Drosophila TRP channels and present the many advantages of this model system in the exploration of TRP channel activation under physiological conditions.

Lifestyle interventions with weight loss can improve insulin sensitivity in type 2 diabetes (T2D), but mechanisms are unclear. We explored circulating and skeletal muscle metabolite signatures of altered peripheral (pIS) and hepatic insulin sensitivity (hIS) in overweight and obese T2D individuals that were randomly assigned a 12-week Paleolithic-type diet with (diet-ex, n = 13) or without (diet, n = 13) supervised exercise. Baseline and post-intervention measures included: mass spectrometry-based metabolomics and lipidomics of skeletal muscle and plasma; pIS and hIS; ectopic lipid deposits in the liver and skeletal muscle; and skeletal muscle fat oxidation rate. Both groups lowered BMI and total % fat mass and increased their pIS. Only the diet-group improved hIS and reduced ectopic lipids in the liver and muscle. The combined improvement in pIS and hIS in the diet-group were associated with decreases in muscle and circulating branched-chain amino acid (BCAA) metabolites, specifically valine. Improved pIS with diet-ex was instead linked to increased diacylglycerol (34:2) and triacylglycerol (56:0) and decreased phosphatidylcholine (34:3) in muscle coupled with improved muscle fat oxidation rate. This suggests a tissue crosstalk involving BCAA-metabolites after diet intervention with improved pIS and hIS, reflecting reduced lipid influx. Increased skeletal muscle lipid utilization with exercise may prevent specific lipid accumulation at sites that perturb insulin signaling.

Many studies have reported that metabolic dysfunction is closely involved in the complex mechanism underlying the development of non-alcoholic fatty liver disease (NAFLD), which has prompted a movement to consider renaming NAFLD as metabolic dysfunction-associated fatty liver disease (MAFLD). Metabolic dysfunction in this context encompasses obesity, type 2 diabetes mellitus, hypertension, dyslipidemia, and metabolic syndrome, with insulin resistance as the common underlying pathophysiology. Imbalance between energy intake and expenditure results in insulin resistance in various tissues and alteration of the gut microbiota, resulting in fat accumulation in the liver. The role of genetics has also been revealed in hepatic fat accumulation and fibrosis. In the process of fat accumulation in the liver, intracellular damage as well as hepatic insulin resistance further potentiates inflammation, fibrosis, and carcinogenesis. Increased lipogenic substrate supply from other tissues, hepatic zonation of Irs1, and other factors, including ER stress, play crucial roles in increased hepatic de novo lipogenesis in MAFLD with hepatic insulin resistance. Herein, we provide an overview of the factors contributing to and the role of systemic and local insulin resistance in the development and progression of MAFLD.

Phospholipase C (PLC) β and ε enzymes hydrolyze phosphatidylinositol (PI) lipids in response to direct interactions with heterotrimeric G protein subunits and small GTPases, which are activated downstream of G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). PI hydrolysis generates second messengers that increase the intracellular Ca2+ concentration and activate protein kinase C (PKC), thereby regulating numerous physiological processes. PLCβ and PLCε share a highly conserved core required for lipase activity, but use different strategies and structural elements to autoinhibit basal activity, bind membranes, and engage G protein activators. In this review, we discuss recent structural insights into these enzymes and the implications for how they engage membranes alone or in complex with their G protein regulators.

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.

Phosphoinositides, together with the phospholipids phosphatidylserine and phosphatidic acid, are important components of the plasma membrane acting as second messengers that, with diacylglycerol, regulate a diverse range of signaling events converting extracellular changes into cellular responses. Local changes in their distribution and membrane charge on the inner leaflet of the plasma membrane play important roles in immune cell function. Here we discuss their distribution and regulators highlighting the importance of membrane changes across the immune synapse on the cytoskeleton and the impact on the function of cytotoxic T lymphocytes.

Hydration⁻dehydration cycles can frequently cause stress to seeds, but can also be used to improve germination. However, the molecular basis of the stress caused is poorly understood. Herein, we examine the effects of hydration⁻dehydration cycles on seed viability and profile the membrane glycerolipid molecular species. We find that seed viability was not affected during the first two cycles, but significantly decreased as further cycles were applied, until all viability was lost. The abundances of seven glycerolipid classes increased and decreased through hydration and dehydration, respectively, but the phosphatidic acid and diacylglycerol abundances changed in the opposite sense, while total glycerolipid contents remained constant. This suggests that during hydration⁻dehydration cycles, turnover of glycerolipid metabolite pools take place, while no significant lipid synthesis or degradation is involved. As further hydration⁻dehydration cycles occurred, lipid unsaturation increased, plastidic lipids decreased, and phosphatidylserine acyl chains lengthened. The latter two could be lethal for seeds. Our findings reveal a novel model of membrane lipid changes, and provide new insights into the responses of seeds to hydration⁻dehydration cycles.

Non-alcoholic fatty liver disease (NAFLD) is in parallel with the obesity epidemic and it is the most common cause of liver diseases. The development of hepatic steatosis in majority of patients is linked to dietary fat ingestion. NAFLD is characterized by excess accumulation of triglyceride in the hepatocyte due to both increased inflow of free fatty acids and de novo hepatic lipogenesis. Insulin resistance with the deficiency of insulin receptor substrate-2 (IRS-2)-associated phosphatidylinositol 3-kinase (PI3K) activity causes an increase in intracellular fatty acid-derived metabolites such as diacylglycerol, fatty acyl CoA or ceramides. Lipotoxicity-related mechanism of NAFLD could be explained still best by the \"double-hit\" hypothesis. Insulin resistance is the major mechanism in the development and progression of NAFLD/Non-alcoholic steatohepatitis (NASH). Metabolic oxidative stress, autophagy, and inflammation induce NASH progression. In the \"first hit\" the hepatic concentrations of diacylglycerol increase with rising saturated liver fat content in human NAFLD. Activities of mitochondrial respiratory chain complexes are decreased in liver tissue of patients with NASH. Furthermore, hepatocyte lipoapoptosis is a critical feature of NASH. In \"second hit\" reduced glutathione levels due to oxidative stress lead to overactivation of c-Jun N-terminal kinase (JNK)/c-Jun signaling that induces cell death in the steatotic liver. Accumulation of toxic levels of reactive oxygen species (ROS) is caused by the ineffectual cycling of the endoplasmic reticulum (ER) oxidoreductin (Ero1)-protein disulfide isomerase oxidation cycle through the downstream of the inner membrane mitochondrial oxidative metabolism and Kelch like-ECH-associated protein 1 (Keap1)- Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway.