Inhibition of de novo ceramide synthesis by Sirtuin-1 improves beta-cell function and glucose metabolism in type 2 diabetes.

BACKGROUND: Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular diseases (CVD). Dysregulated pro-apoptotic ceramide synthesis reduces ß-cell insulin secretion, thereby promoting hyperglycemic states which may manifest as T2D. Pro-apoptotic ceramides modulate insulin sensitivity and glucose tolerance while being linked to poor cardiovascular outcomes. Sirtuin-1 (SIRT1) is a NAD + - dependent deacetylase that protects against pancreatic ß-cell dysfunction; however, systemic levels are decreased in obese T2D mice and may promote pro-apoptotic ceramide synthesis and hyperglycemia. Herein, we aimed to assess the effects of restoring circulating SIRT1 levels to prevent metabolic imbalance in obese and diabetic mice. METHODS AND RESULTS: Circulating SIRT1 levels were reduced in obese diabetic mice (db/db) as compared to age-matched non-diabetic db/+ controls. Restoration of SIRT1 plasma levels with recombinant murine SIRT1 for 4-weeks prevented body weight gain, improved glucose tolerance, insulin sensitivity and vascular function in mice models of obesity and T2D. Untargeted lipidomics revealed that SIRT1 restored insulin-secretory function of ß-cells by reducing synthesis and accumulation of pro-apoptotic ceramides. Molecular mechanisms involved direct binding to and deacetylation of Toll-like receptor 4 (TLR4) by SIRT1 in ß-cells thereby decreasing the rate limiting enzymes of sphingolipid synthesis SPTLC1/2 via AKT/NF-κB. Among T2D patients, those with high baseline plasma levels of SIRT1 prior to metabolic surgery displayed restored ß-cell function (HOMA2- ß) and were more likely to have T2D remission during follow-up. CONCLUSION: Acetylation of TLR4 promotes ß-cell dysfunction via ceramide synthesis in T2D, which is blunted by systemic SIRT1 replenishment. Hence, restoration of systemic SIRT1 may provide a novel therapeutic strategy to counteract toxic ceramide synthesis and mitigate cardiovascular complications of T2D.

ROS Induction Targets Persister Cancer Cells with Low Metabolic Activity in NRAS-Mutated Melanoma.

Clinical management of melanomas with NRAS mutations is challenging. Targeting MAPK signaling is only beneficial to a small subset of patients due to resistance that arises through genetic, transcriptional, and metabolic adaptation. Identification of targetable vulnerabilities in NRAS-mutated melanoma could help improve patient treatment. Here, we used multiomics analyses to reveal that NRAS-mutated melanoma cells adopt a mesenchymal phenotype with a quiescent metabolic program to resist cellular stress induced by MEK inhibition. The metabolic alterations elevated baseline reactive oxygen species (ROS) levels, leading these cells to become highly sensitive to ROS induction. In vivo xenograft experiments and single-cell RNA sequencing demonstrated that intratumor heterogeneity necessitates the combination of a ROS inducer and a MEK inhibitor to inhibit both tumor growth and metastasis. Ex vivo pharmacoscopy of 62 human metastatic melanomas confirmed that MEK inhibitor-resistant tumors significantly benefited from the combination therapy. Finally, oxidative stress response and translational suppression corresponded with ROS-inducer sensitivity in 486 cancer cell lines, independent of cancer type. These findings link transcriptional plasticity to a metabolic phenotype that can be inhibited by ROS inducers in melanoma and other cancers. SIGNIFICANCE: Metabolic reprogramming in drug-resistant NRAS-mutated melanoma cells confers sensitivity to ROS induction, which suppresses tumor growth and metastasis in combination with MAPK pathway inhibitors.

Sphingolipid subtypes differentially control proinsulin processing and systemic glucose homeostasis.

Impaired proinsulin-to-insulin processing in pancreatic ß-cells is a key defective step in both type 1 diabetes and type 2 diabetes (T2D) (refs. 1,2), but the mechanisms involved remain to be defined. Altered metabolism of sphingolipids (SLs) has been linked to development of obesity, type 1 diabetes and T2D (refs. 3-8); nonetheless, the role of specific SL species in ß-cell function and demise is unclear. Here we define the lipid signature of T2D-associated ß-cell failure, including an imbalance of specific very-long-chain SLs and long-chain SLs. ß-cell-specific ablation of CerS2, the enzyme necessary for generation of very-long-chain SLs, selectively reduces insulin content, impairs insulin secretion and disturbs systemic glucose tolerance in multiple complementary models. In contrast, ablation of long-chain-SL-synthesizing enzymes has no effect on insulin content. By quantitatively defining the SL-protein interactome, we reveal that CerS2 ablation affects SL binding to several endoplasmic reticulum-Golgi transport proteins, including Tmed2, which we define as an endogenous regulator of the essential proinsulin processing enzyme Pcsk1. Our study uncovers roles for specific SL subtypes and SL-binding proteins in ß-cell function and T2D-associated ß-cell failure.

Discovery and Mechanism of Action of Small Molecule Inhibitors of Ceramidases.

Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro-apoptotic lipid catabolized by ceramidase enzymes to produce pro-proliferative sphingosine-1-phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug-like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non-lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.

The BET Protein Inhibitor Apabetalone Rescues Diabetes-Induced Impairment of Angiogenic Response by Epigenetic Regulation of Thrombospondin-1.

Aims: Therapeutic modulation of blood vessel growth holds promise for the prevention of limb ischemia in diabetic (DM) patients with peripheral artery disease (PAD). Epigenetic changes, namely, posttranslational histone modifications, participate in angiogenic response suggesting that chromatin-modifying drugs could be beneficial in this setting. Apabetalone (APA), a selective inhibitor of bromodomain (BRD) and bromodomain and extraterminal containing protein family (BET) proteins, prevents bromodomain-containing protein 4 (BRD4) interactions with chromatin thus modulating transcriptional programs in different organs. We sought to investigate whether APA affects angiogenic response in diabetes. Results: Compared with vehicle, APA restored tube formation and migration in human aortic endothelial cells (HAECs) exposed to high-glucose (HG) levels. Expression profiling of angiogenesis genes showed that APA prevents HG-induced upregulation of the antiangiogenic molecule thrombospondin-1 (THBS1). ChIP-seq and chromatin immunoprecipitation (ChIP) assays in HG-treated HAECs showed the enrichment of both BRD4 and active marks (H3K27ac) on THBS1 promoter, whereas BRD4 inhibition by APA prevented chromatin accessibility and THBS1 transcription. Mechanistically, we show that THBS1 inhibits angiogenesis by suppressing vascular endothelial growth factor A (VEGFA) signaling, while APA prevents these detrimental changes. In diabetic mice with hind limb ischemia, epigenetic editing by APA restored the THBS1/VEGFA axis, thus improving limb vascularization and perfusion, compared with vehicle-treated animals. Finally, epigenetic regulation of THBS1 by BRD4/H3K27ac was also reported in DM patients with PAD compared with nondiabetic controls. Innovation: This is the first study showing that BET protein inhibition by APA restores angiogenic response in experimental diabetes. Conclusions: Our findings set the stage for preclinical studies and exploratory clinical trials testing APA in diabetic PAD. Antioxid. Redox Signal. 36, 667-684.

Metabolism of HSAN1- and T2DM-associated 1-deoxy-sphingolipids inhibits the migration of fibroblasts.

Hereditary sensory neuropathy type 1 (HSAN1) is a rare axonopathy, characterized by a progressive loss of sensation (pain, temperature, and vibration), neuropathic pain, and wound healing defects. HSAN1 is caused by several missense mutations in the serine palmitoyltransferase long-chain base subunit 1 and serine palmitoyltransferase long-chain base subunit 2 of the enzyme serine palmitoyltransferase-the key enzyme for the synthesis of sphingolipids. The mutations change the substrate specificity of serine palmitoyltransferase, which then forms an atypical class of 1-deoxy-sphinglipids (1-deoxySLs). Similarly, patients with type 2 diabetes mellitus also present with elevated 1-deoxySLs and a comparable clinical phenotype. The effect of 1-deoxySLs on neuronal cells was investigated in detail, but their impact on other cell types remains elusive. Here, we investigated the consequences of externally added 1-deoxySLs on the migration of fibroblasts in a scratch assay as a simplified cellular wound-healing model. We showed that 1-deoxy-sphinganine (1-deoxySA) inhibits the migration of NIH-3T3 fibroblasts in a dose- and time-dependent manner. This was not seen for a non-native, L-threo stereoisomer. Supplemented 1-deoxySA was metabolized to 1-deoxy-(dihydro)ceramide and downstream to 1-deoxy-sphingosine. Inhibiting downstream metabolism by blocking N-acylation rescued the migration phenotype. In contrast, adding 1-deoxy-sphingosine had a lesser effect on cell migration but caused the massive formation of intracellular vacuoles. Further experiments showed that the effect on cell migration was primarily mediated by 1-deoxy-dihydroceramides rather than by the free base or 1-deoxyceramides. Based on these findings, we suggest that limiting the N-acylation of 1-deoxySA could be a therapeutic approach to improve cell migration and wound healing in patients with HSAN1 and type 2 diabetes mellitus.

Subunit composition of the mammalian serine-palmitoyltransferase defines the spectrum of straight and methyl-branched long-chain bases.

Sphingolipids (SLs) are chemically diverse lipids that have important structural and signaling functions within mammalian cells. SLs are commonly defined by the presence of a long-chain base (LCB) that is normally formed by the conjugation of l-serine and palmitoyl-CoA. This pyridoxal 5-phosphate (PLP)-dependent reaction is mediated by the enzyme serine-palmitoyltransferase (SPT). However, SPT can also metabolize other acyl-CoAs, in the range of C14 to C18, forming a variety of LCBs that differ by structure and function. Mammalian SPT consists of three core subunits: SPTLC1, SPTLC2, and SPTLC3. Whereas SPTLC1 and SPTLC2 are ubiquitously expressed, SPTLC3 expression is restricted to certain tissues only. The influence of the individual subunits on enzyme activity is not clear. Using cell models deficient in SPTLC1, SPTLC2, and SPTLC3, we investigated the role of each subunit on enzyme activity and the LCB product spectrum. We showed that SPTLC1 is essential for activity, whereas SPTLC2 and SPTLC3 are partly redundant but differ in their enzymatic properties. SPTLC1 in combination with SPTLC2 specifically formed C18, C19, and C20 LCBs while the combination of SPTLC1 and SPTLC3 yielded a broader product spectrum. We identified anteiso-branched-C18 SO (meC18SO) as the primary product of the SPTLC3 reaction. The meC18SO was synthesized from anteiso-methyl-palmitate, in turn synthesized from a precursor metabolite generated in the isoleucine catabolic pathway. The meC18SO is metabolized to ceramides and complex SLs and is a constituent of human low- and high-density lipoproteins.

Structure-function relationships of HDL in diabetes and coronary heart disease.

High-density lipoproteins (HDL) contain hundreds of lipid species and proteins and exert many potentially vasoprotective and antidiabetogenic activities on cells. To resolve structure-function-disease relationships of HDL, we characterized HDL of 51 healthy subjects and 98 patients with diabetes (T2DM), coronary heart disease (CHD), or both for protein and lipid composition, as well as functionality in 5 cell types. The integration of 40 clinical characteristics, 34 nuclear magnetic resonance (NMR) features, 182 proteins, 227 lipid species, and 12 functional read-outs by high-dimensional statistical modeling revealed, first, that CHD and T2DM are associated with different changes of HDL in size distribution, protein and lipid composition, and function. Second, different cellular functions of HDL are weakly correlated with each other and determined by different structural components. Cholesterol efflux capacity (CEC) was no proxy of other functions. Third, 3 potentially novel determinants of HDL function were identified and validated by the use of artificially reconstituted HDL, namely the sphingadienine-based sphingomyelin SM 42:3 and glycosylphosphatidylinositol-phospholipase D1 for the ability of HDL to inhibit starvation-induced apoptosis of human aortic endothelial cells and apolipoprotein F for the ability of HDL to promote maximal respiration of brown adipocytes.

FADS3 is a Δ14Z sphingoid base desaturase that contributes to gender differences in the human plasma sphingolipidome.

Sphingolipids (SLs) are structurally diverse lipids that are defined by the presence of a long-chain base (LCB) backbone. Typically, LCBs contain a single Δ4E double bond (DB) (mostly d18:1), whereas the dienic LCB sphingadienine (d18:2) contains a second DB at the Δ14Z position. The enzyme introducing the Δ14Z DB is unknown. We analyzed the LCB plasma profile in a gender-, age-, and BMI-matched subgroup of the CoLaus cohort (n = 658). Sphingadienine levels showed a significant association with gender, being on average ∼30% higher in females. A genome-wide association study (GWAS) revealed variants in the fatty acid desaturase 3 (FADS3) gene to be significantly associated with the plasma d18:2/d18:1 ratio (p = -log 7.9). Metabolic labeling assays, FADS3 overexpression and knockdown approaches, and plasma LCB profiling in FADS3-deficient mice confirmed that FADS3 is a bona fide LCB desaturase and required for the introduction of the Δ14Z double bond. Moreover, we showed that FADS3 is required for the conversion of the atypical cytotoxic 1-deoxysphinganine (1-deoxySA, m18:0) to 1-deoxysphingosine (1-deoxySO, m18:1). HEK293 cells overexpressing FADS3 were more resistant to m18:0 toxicity than WT cells. In summary, using a combination of metabolic profiling and GWAS, we identified FADS3 to be essential for forming Δ14Z DB containing LCBs, such as d18:2 and m18:1. Our results unravel FADS3 as a Δ14Z LCB desaturase, thereby disclosing the last missing enzyme of the SL de novo synthesis pathway.

DEGS1-associated aberrant sphingolipid metabolism impairs nervous system function in humans.

BACKGROUND: Sphingolipids are important components of cellular membranes and functionally associated with fundamental processes such as cell differentiation, neuronal signaling, and myelin sheath formation. Defects in the synthesis or degradation of sphingolipids leads to various neurological pathologies; however, the entire spectrum of sphingolipid metabolism disorders remains elusive. METHODS: A combined approach of genomics and lipidomics was applied to identify and characterize a human sphingolipid metabolism disorder. RESULTS: By whole-exome sequencing in a patient with a multisystem neurological disorder of both the central and peripheral nervous systems, we identified a homozygous p.Ala280Val variant in DEGS1, which catalyzes the last step in the ceramide synthesis pathway. The blood sphingolipid profile in the patient showed a significant increase in dihydro sphingolipid species that was further recapitulated in patient-derived fibroblasts, in CRISPR/Cas9-derived DEGS1-knockout cells, and by pharmacological inhibition of DEGS1. The enzymatic activity in patient fibroblasts was reduced by 80% compared with wild-type cells, which was in line with a reduced expression of mutant DEGS1 protein. Moreover, an atypical and potentially neurotoxic sphingosine isomer was identified in patient plasma and in cells expressing mutant DEGS1. CONCLUSION: We report DEGS1 dysfunction as the cause of a sphingolipid disorder with hypomyelination and degeneration of both the central and peripheral nervous systems. TRIAL REGISTRATION: Not applicable. FUNDING: Seventh Framework Program of the European Commission, Swiss National Foundation, Rare Disease Initiative Zurich.

Metabolic Syndrome, Neurotoxic 1-Deoxysphingolipids and Nervous Tissue Inflammation in Chronic Idiopathic Axonal Polyneuropathy (CIAP).

AIM: Chronic idiopathic axonal polyneuropathy (CIAP) is a slowly progressive, predominantly sensory, axonal polyneuropathy, with no aetiology being identified despite extensive investigations. We studied the potential role of the metabolic syndrome, neurotoxic 1-deoxysphingolipids (1-deoxySLs), microangiopathy and inflammation in sural nerve biopsies. METHODS: We included 30 CIAP-patients, 28 with diabetic distal symmetrical polyneuropathy (DSPN) and 31 healthy controls. We assessed standardised scales, tested for the metabolic syndrome, measured 1-deoxySLs in plasma, performed electroneurography and studied 17 sural nerve biopsies (10 CIAP; 7 DSPN). RESULTS: One third of the CIAP-patients had a metabolic syndrome, significantly less frequent than DSPN-patients (89%). Although the metabolic syndrome was not significantly more prevalent in CIAP compared to healthy controls, hypercholesterolemia did occur significantly more frequent. 1-deoxySLs were significantly and equally elevated in both patient groups compared to healthy controls. Mean basal lamina thickness of small endoneurial vessels and the number of CD68- or CD8-positive cells in biopsies of CIAP- and DSPN-patients did not differ significantly. However, the number of leucocyte-common-antigen positive cells was significantly increased in CIAP. CONCLUSIONS: A non-significant trend towards a higher occurrence of the metabolic syndrome in CIAP-patients compared to healthy controls was found. 1-deoxySLs were significantly increased in plasma of CIAP-patients. Microangiopathy and an inflammatory component were present in CIAP-biopsies.

Lack of cyclophilin D protects against the development of acute lung injury in endotoxemia.

Sepsis caused by LPS is characterized by an intense systemic inflammatory response affecting the lungs, causing acute lung injury (ALI). Dysfunction of mitochondria and the role of reactive oxygen (ROS) and nitrogen species produced by mitochondria have already been proposed in the pathogenesis of sepsis; however, the exact molecular mechanism is poorly understood. Oxidative stress induces cyclophilin D (CypD)-dependent mitochondrial permeability transition (mPT), leading to organ failure in sepsis. In previous studies mPT was inhibited by cyclosporine A which, beside CypD, inhibits cyclophilin A, B, C and calcineurin, regulating cell death and inflammatory pathways. The immunomodulatory side effects of cyclosporine A make it unfavorable in inflammatory model systems. To avoid these uncertainties in the molecular mechanism, we studied endotoxemia-induced ALI in CypD(-/-) mice providing unambiguous data for the pathological role of CypD-dependent mPT in ALI. Our key finding is that the loss of this essential protein improves survival rate and it can intensely ameliorate endotoxin-induced lung injury through attenuated proinflammatory cytokine release, down-regulation of redox sensitive cellular pathways such as MAPKs, Akt, and NF-κB and reducing the production of ROS. Functional inhibition of NF-κB was confirmed by decreased expression of NF-κB-mediated proinflammatory genes. We demonstrated that impaired mPT due to the lack of CypD reduces the severity of endotoxemia-induced lung injury suggesting that CypD specific inhibitors might have a great therapeutic potential in sepsis-induced organ failure. Our data highlight a previously unknown regulatory function of mitochondria during inflammatory response.

Diverse in- and output polarities and high complexity of local synaptic and non-synaptic signaling within a chemically defined class of peptidergic Drosophila neurons.

Peptidergic neurons are not easily integrated into current connectomics concepts, since their peptide messages can be distributed via non-synaptic paracrine signaling or volume transmission. Moreover, the polarity of peptidergic interneurons in terms of in- and out-put sites can be hard to predict and is very little explored. We describe in detail the morphology and the subcellular distribution of fluorescent vesicle/dendrite markers in CCAP neurons (NCCAP), a well defined set of peptidergic neurons in the Drosophila larva. NCCAP can be divided into five morphologically distinct subsets. In contrast to other subsets, serial homologous interneurons in the ventral ganglion show a mixed localization of in- and output markers along ventral neurites that defy a classification as dendritic or axonal compartments. Ultrastructurally, these neurites contain both pre- and postsynaptic sites preferably at varicosities. A significant portion of the synaptic events are due to reciprocal synapses. Peptides are mostly non-synaptically or parasynaptically released, and dense-core vesicles and synaptic vesicle pools are typically well separated. The responsiveness of the NCCAP to ecdysis-triggering hormone may be at least partly dependent on a tonic synaptic inhibition, and is independent of ecdysteroids. Our results reveal a remarkable variety and complexity of local synaptic circuitry within a chemically defined set of peptidergic neurons. Synaptic transmitter signaling as well as peptidergic paracrine signaling and volume transmission from varicosities can be main signaling modes of peptidergic interneurons depending on the subcellular region. The possibility of region-specific variable signaling modes should be taken into account in connectomic studies that aim to dissect the circuitry underlying insect behavior and physiology, in which peptidergic neurons act as important regulators.