A sphingolipid rheostat controls apoptosis versus apical cell extrusion as alternative tumour-suppressive mechanisms.

Evasion of cell death is a hallmark of cancer, and consequently the induction of cell death is a common strategy in cancer treatment. However, the molecular mechanisms regulating different types of cell death are poorly understood. We have formerly shown that in the epidermis of hypomorphic zebrafish hai1a mutant embryos, pre-neoplastic transformations of keratinocytes caused by unrestrained activity of the type II transmembrane serine protease Matriptase-1 heal spontaneously. This healing is driven by Matriptase-dependent increased sphingosine kinase (SphK) activity and sphingosine-1-phosphate (S1P)-mediated keratinocyte loss via apical cell extrusion. In contrast, amorphic hai1afr26 mutants with even higher Matriptase-1 and SphK activity die within a few days. Here we show that this lethality is not due to epidermal carcinogenesis, but to aberrant tp53-independent apoptosis of keratinocytes caused by increased levels of pro-apoptotic C16 ceramides, sphingolipid counterparts to S1P within the sphingolipid rheostat, which severely compromises the epidermal barrier. Mathematical modelling of sphingolipid rheostat homeostasis, combined with in vivo manipulations of components of the rheostat or the ceramide de novo synthesis pathway, indicate that this unexpected overproduction of ceramides is caused by a negative feedback loop sensing ceramide levels and controlling ceramide replenishment via de novo synthesis. Therefore, despite their initial decrease due to increased conversion to S1P, ceramides eventually reach cell death-inducing levels, making transformed pre-neoplastic keratinocytes die even before they are extruded, thereby abrogating the normally barrier-preserving mode of apical live cell extrusion. Our results offer an in vivo perspective of the dynamics of sphingolipid homeostasis and its relevance for epithelial cell survival versus cell death, linking apical cell extrusion and apoptosis. Implications for human carcinomas and their treatments are discussed.

Drosophila HNF4 acts in distinct tissues to direct a switch between lipid storage and export in the gut.

Nutrient digestion, absorption, and export must be coordinated in the gut to meet the nutritional needs of the organism. We used the Drosophila intestine to characterize the mechanisms that coordinate the fate of dietary lipids. We identified enterocytes specialized in absorbing and exporting lipids to peripheral organs. Distinct hepatocyte-like cells, called oenocytes, communicate with these enterocytes to adjust intestinal lipid storage and export. A single transcription factor, Drosophila hepatocyte nuclear factor 4 (dHNF4), supports this gut-liver axis. In enterocytes, dHNF4 maximizes dietary lipid export by preventing their sequestration in cytoplasmic lipid droplets. In oenocytes, dHNF4 promotes the expression of the insulin antagonist ImpL2 to activate Foxo and suppress lipid retention in enterocytes. Disruption of this switch between lipid storage and export is associated with intestinal inflammation, suggesting a lipidic origin for inflammatory bowel diseases. These studies establish dHNF4 as a central regulator of intestinal metabolism and inter-organ lipid trafficking.

High-fat feeding drives the intestinal production and assembly of C16:0 ceramides in chylomicrons.

Consumption of a diet rich in saturated fat increases lipid absorption from the intestine, assembly into chylomicrons, and delivery to metabolic tissues via the lymphatic and circulatory systems. Accumulation of ceramide lipids, composed of sphingosine and a fatty acid, in metabolic tissues contributes to the pathogenesis of cardiovascular diseases, type 2 diabetes mellitus and cancer. Using a mesenteric lymph duct cannulated rat model, we showed that ceramides are generated by the intestine and assembled into chylomicrons, which are transported via the mesenteric lymphatic system. A lipidomic screen of intestinal-derived chylomicrons identified a diverse range of fatty acid, sphingolipid, and glycerolipid species that have not been previously detected in chylomicrons, including the metabolically deleterious C16:0 ceramide that increased in response to high-fat feeding in rats and human high-lipid meal replacement enteral feeding. In conclusion, high-fat feeding increases the export of intestinal-derived C16:0 ceramide in chylomicrons, identifying a potentially unknown mechanism through which ceramides are transported systemically to contribute to metabolic dysfunction.

Lipid unsaturation promotes BAX and BAK pore activity during apoptosis.

BAX and BAK are proapoptotic members of the BCL2 family that directly mediate mitochondrial outer membrane permeabilition (MOMP), a central step in apoptosis execution. However, the molecular architecture of the mitochondrial apoptotic pore remains a key open question and especially little is known about the contribution of lipids to MOMP. By performing a comparative lipidomics analysis of the proximal membrane environment of BAK isolated in lipid nanodiscs, we find a significant enrichment of unsaturated species nearby BAK and BAX in apoptotic conditions. We then demonstrate that unsaturated lipids promote BAX pore activity in model membranes, isolated mitochondria and cellular systems, which is further supported by molecular dynamics simulations. Accordingly, the fatty acid desaturase FADS2 not only enhances apoptosis sensitivity, but also the activation of the cGAS/STING pathway downstream mtDNA release. The correlation of FADS2 levels with the sensitization to apoptosis of different lung and kidney cancer cell lines by co-treatment with unsaturated fatty acids supports the relevance of our findings. Altogether, our work provides an insight on how local lipid environment affects BAX and BAK function during apoptosis.

ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway.

Complexes of ERLIN1 and ERLIN2 (ER lipid raft-associated 1 and 2) form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3 ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 (transmembrane and ubiquitin-like domain-containing 1) and RNF170 (RING finger protein 170). We identify a luminal N-terminal conserved region in TMUB1 and RNF170, which is required for this interaction. Three-dimensional modelling shows that this conserved motif binds the stomatin/prohibitin/flotillin/HflKC domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia. Using omics-based approaches in combination with phenotypic characterization of HeLa cells lacking both ERLINs, we demonstrate a role of ERLIN scaffolds in limiting cholesterol esterification, thereby favouring cholesterol transport from the ER to the Golgi apparatus and regulating Golgi morphology and the secretory pathway.

Glutamine promotes human CD8+ T cells and counteracts imiquimod-induced T cell hyporesponsiveness.

T cells protect tissues from cancer. Although investigations in mice showed that amino acids (AA) critically regulate T cell immunity, this remains poorly understood in humans. Here, we describe the AA composition of interstitial fluids in keratinocyte-derived skin cancers (KDSCs) and study the effect of AA on T cells using models of primary human cells and tissues. Gln contributed to ∼15% of interstitial AAs and promoted interferon gamma (IFN-γ), but not granzyme B (GzB) expression, in CD8+ T cells. Furthermore, the Toll-like receptor 7 agonist imiquimod (IMQ), a common treatment for KDSCs, down-regulated the metabolic gatekeepers c-MYC and mTORC1, as well as the AA transporter ASCT2 and intracellular Gln, Asn, Ala, and Asp in T cells. Reduced proliferation and IFN-γ expression, yet increased GzB, paralleled IMQ effects on AA. Finally, Gln was sufficient to promote IFN-γ-production in IMQ-treated T cells. Our findings indicate that Gln metabolism can be harnessed for treating KDSCs.

Identification of TFG- and Autophagy-Regulated Proteins and Glycerophospholipids in B Cells.

Autophagy supervises the proteostasis and survival of B lymphocytic cells. Trk-fused gene (TFG) promotes autophagosome-lysosome flux in murine CH12 B cells, as well as their survival. Hence, quantitative proteomics of CH12tfgKO and WT B cells in combination with lysosomal inhibition should identify proteins that are prone to lysosomal degradation and contribute to autophagy and B cell survival. Lysosome inhibition via NH4Cl unexpectedly reduced a number of proteins but increased a large cluster of translational, ribosomal, and mitochondrial proteins, independent of TFG. Hence, we propose a role for lysosomes in ribophagy in B cells. TFG-regulated proteins include CD74, BCL10, or the immunoglobulin JCHAIN. Gene ontology (GO) analysis reveals that proteins regulated by TFG alone, or in concert with lysosomes, localize to mitochondria and membrane-bound organelles. Likewise, TFG regulates the abundance of metabolic enzymes, such as ALDOC and the fatty acid-activating enzyme ACOT9. To test consequently for a function of TFG in lipid metabolism, we performed shotgun lipidomics of glycerophospholipids. Total phosphatidylglycerol is more abundant in CH12tfgKO B cells. Several glycerophospholipid species with similar acyl side chains, such as 36:2 phosphatidylethanolamine and 36:2 phosphatidylinositol, show a dysequilibrium. We suggest a role for TFG in lipid homeostasis, mitochondrial functions, translation, and metabolism in B cells.

A homoeostatic switch causing glycerol-3-phosphate and phosphoethanolamine accumulation triggers senescence by rewiring lipid metabolism.

Cellular senescence affects many physiological and pathological processes and is characterized by durable cell cycle arrest, an inflammatory secretory phenotype and metabolic reprogramming. Here, by using dynamic transcriptome and metabolome profiling in human fibroblasts with different subtypes of senescence, we show that a homoeostatic switch that results in glycerol-3-phosphate (G3P) and phosphoethanolamine (pEtN) accumulation links lipid metabolism to the senescence gene expression programme. Mechanistically, p53-dependent glycerol kinase activation and post-translational inactivation of phosphate cytidylyltransferase 2, ethanolamine regulate this metabolic switch, which promotes triglyceride accumulation in lipid droplets and induces the senescence gene expression programme. Conversely, G3P phosphatase and ethanolamine-phosphate phospho-lyase-based scavenging of G3P and pEtN acts in a senomorphic way by reducing G3P and pEtN accumulation. Collectively, our study ties G3P and pEtN accumulation to controlling lipid droplet biogenesis and phospholipid flux in senescent cells, providing a potential therapeutic avenue for targeting senescence and related pathophysiology.

Mitochondrial dysfunction abrogates dietary lipid processing in enterocytes.

Digested dietary fats are taken up by enterocytes where they are assembled into pre-chylomicrons in the endoplasmic reticulum followed by transport to the Golgi for maturation and subsequent secretion to the circulation1. The role of mitochondria in dietary lipid processing is unclear. Here we show that mitochondrial dysfunction in enterocytes inhibits chylomicron production and the transport of dietary lipids to peripheral organs. Mice with specific ablation of the mitochondrial aspartyl-tRNA synthetase DARS2 (ref. 2), the respiratory chain subunit SDHA3 or the assembly factor COX10 (ref. 4) in intestinal epithelial cells showed accumulation of large lipid droplets (LDs) in enterocytes of the proximal small intestine and failed to thrive. Feeding a fat-free diet suppressed the build-up of LDs in DARS2-deficient enterocytes, which shows that the accumulating lipids derive mostly from digested fat. Furthermore, metabolic tracing studies revealed an impaired transport of dietary lipids to peripheral organs in mice lacking DARS2 in intestinal epithelial cells. DARS2 deficiency caused a distinct lack of mature chylomicrons concomitant with a progressive dispersal of the Golgi apparatus in proximal enterocytes. This finding suggests that mitochondrial dysfunction results in impaired trafficking of chylomicrons from the endoplasmic reticulum to the Golgi, which in turn leads to storage of dietary lipids in large cytoplasmic LDs. Taken together, these results reveal a role for mitochondria in dietary lipid transport in enterocytes, which might be relevant for understanding the intestinal defects observed in patients with mitochondrial disorders5.

CerS6-dependent ceramide synthesis in hypothalamic neurons promotes ER/mitochondrial stress and impairs glucose homeostasis in obese mice.

Dysregulation of hypothalamic ceramides has been associated with disrupted neuronal pathways in control of energy and glucose homeostasis. However, the specific ceramide species promoting neuronal lipotoxicity in obesity have remained obscure. Here, we find increased expression of the C16:0 ceramide-producing ceramide synthase (CerS)6 in cultured hypothalamic neurons exposed to palmitate in vitro and in the hypothalamus of obese mice. Conditional deletion of CerS6 in hypothalamic neurons attenuates high-fat diet (HFD)-dependent weight gain and improves glucose metabolism. Specifically, CerS6 deficiency in neurons expressing pro-opiomelanocortin (POMC) or steroidogenic factor 1 (SF-1) alters feeding behavior and alleviates the adverse metabolic effects of HFD feeding on insulin sensitivity and glucose tolerance. POMC-expressing cell-selective deletion of CerS6 prevents the diet-induced alterations of mitochondrial morphology and improves cellular leptin sensitivity. Our experiments reveal functions of CerS6-derived ceramides in hypothalamic lipotoxicity, altered mitochondrial dynamics, and ER/mitochondrial stress in the deregulation of food intake and glucose metabolism in obesity.

Human model of primary carnitine deficiency cardiomyopathy reveals ferroptosis as a novel mechanism.

Primary carnitine deficiency (PCD) is an autosomal recessive monogenic disorder caused by mutations in SLC22A5. This gene encodes for OCTN2, which transports the essential metabolite carnitine into the cell. PCD patients suffer from muscular weakness and dilated cardiomyopathy. Two OCTN2-defective human induced pluripotent stem cell lines were generated, carrying a full OCTN2 knockout and a homozygous OCTN2 (N32S) loss-of-function mutation. OCTN2-defective genotypes showed lower force development and resting length in engineered heart tissue format compared with isogenic control. Force was sensitive to fatty acid-based media and associated with lipid accumulation, mitochondrial alteration, higher glucose uptake, and metabolic remodeling, replicating findings in animal models. The concordant results of OCTN2 (N32S) and -knockout emphasizes the relevance of OCTN2 for these findings. Importantly, genome-wide analysis and pharmacological inhibitor experiments identified ferroptosis, an iron- and lipid-dependent cell death pathway associated with fibroblast activation as a novel PCD cardiomyopathy disease mechanism.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

Organ Protection by Caloric Restriction Depends on Activation of the De Novo NAD+ Synthesis Pathway.

SIGNIFICANCE STATEMENT: AKI is a major clinical complication leading to high mortality, but intensive research over the past decades has not led to targeted preventive or therapeutic measures. In rodent models, caloric restriction (CR) and transient hypoxia significantly prevent AKI and a recent comparative transcriptome analysis of murine kidneys identified kynureninase (KYNU) as a shared downstream target. The present work shows that KYNU strongly contributes to CR-mediated protection as a key player in the de novo nicotinamide adenine dinucleotide biosynthesis pathway. Importantly, the link between CR and NAD+ biosynthesis could be recapitulated in a human cohort. BACKGROUND: Clinical practice lacks strategies to treat AKI. Interestingly, preconditioning by hypoxia and caloric restriction (CR) is highly protective in rodent AKI models. However, the underlying molecular mechanisms of this process are unknown. METHODS: Kynureninase (KYNU) knockout mice were generated by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and comparative transcriptome, proteome and metabolite analyses of murine kidneys pre- and post-ischemia-reperfusion injury in the context of CR or ad libitum diet were performed. In addition, acetyl-lysin enrichment and mass spectrometry were used to assess protein acetylation. RESULTS: We identified KYNU as a downstream target of CR and show that KYNU strongly contributes to the protective effect of CR. The KYNU-dependent de novo nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway is necessary for CR-associated maintenance of NAD+ levels. This finding is associated with reduced protein acetylation in CR-treated animals, specifically affecting enzymes in energy metabolism. Importantly, the effect of CR on de novo NAD+ biosynthesis pathway metabolites can be recapitulated in humans. CONCLUSIONS: CR induces the de novo NAD+ synthesis pathway in the context of IRI and is essential for its full nephroprotective potential. Differential protein acetylation may be the molecular mechanism underlying the relationship of NAD+, CR, and nephroprotection.

Caloric restriction reduces the pro-inflammatory eicosanoid 20-hydroxyeicosatetraenoic acid to protect from acute kidney injury.

Acute kidney injury is a frequent complication in the clinical setting and associated with significant morbidity and mortality. Preconditioning with short-term caloric restriction is highly protective against kidney injury in rodent ischemia reperfusion injury models. However, the underlying mechanisms are unknown hampering clinical translation. Here, we examined the molecular basis of caloric restriction-mediated protection to elucidate the principles of kidney stress resistance. Analysis of an RNAseq dataset after caloric restriction identified Cyp4a12a, a cytochrome exclusively expressed in male mice, to be strongly downregulated after caloric restriction. Kidney ischemia reperfusion injury robustly induced acute kidney injury in male mice and this damage could be markedly attenuated by pretreatment with caloric restriction. In females, damage was significantly less pronounced and preconditioning with caloric restriction had only little effect. Tissue concentrations of the metabolic product of Cyp4a12a, 20-hydroxyeicosatetraenoic acid (20-HETE), were found to be significantly reduced by caloric restriction. Conversely, intraperitoneal supplementation of 20-HETE in preconditioned males partly abrogated the protective potential of caloric restriction. Interestingly, this effect was accompanied by a partial reversal of caloric restriction--induced changes in protein but not RNA expression pointing towards inflammation, endoplasmic reticulum stress and lipid metabolism. Thus, our findings provide an insight into the mechanisms underlying kidney protection by caloric restriction. Hence, understanding the mediators of preconditioning is an important prerequisite for moving towards translation to the clinical setting.

Mitochondrial respiration in B lymphocytes is essential for humoral immunity by controlling the flux of the TCA cycle.

To elucidate the function of oxidative phosphorylation (OxPhos) during B cell differentiation, we employ CD23Cre-driven expression of the dominant-negative K320E mutant of the mitochondrial helicase Twinkle (DNT). DNT-expression depletes mitochondrial DNA during B cell maturation, reduces the abundance of respiratory chain protein subunits encoded by mitochondrial DNA, and, consequently, respiratory chain super-complexes in activated B cells. Whereas B cell development in DNT mice is normal, B cell proliferation, germinal centers, class switch to IgG, plasma cell maturation, and T cell-dependent as well as T cell-independent humoral immunity are diminished. DNT expression dampens OxPhos but increases glycolysis in lipopolysaccharide and B cell receptor-activated cells. Lipopolysaccharide-activated DNT-B cells exhibit altered metabolites of glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle and a lower amount of phosphatidic acid. Consequently, mTORC1 activity and BLIMP1 induction are curtailed, whereas HIF1α is stabilized. Hence, mitochondrial DNA controls the metabolism of activated B cells via OxPhos to foster humoral immunity.

Hyperthermia-induced doxorubicin delivery from thermosensitive liposomes via MR-HIFU in a pig model.

PURPOSE: Encapsulation of cytotoxic drugs for a localized release is an effective way to increase the therapeutic window of such agents. In this article we present the localized release of doxorubicin (DOX) from phosphatidyldiglycerol (DPPG2) based thermosensitive liposomes using MR-HIFU mediated hyperthermia in a swine model. MATERIALS AND METHODS: German landrace pigs of weights between 37.5 and 53.5 kg received a 30-min infusion of DOX containing thermosensitive liposomes (50 mg DOX/m2). The pigs' biceps femoris was treated locally in two separate target areas with mild hyperthermia using magnetic resonance guided high intensity focused ultrasound, starting 10 min and 60 min after initiation of the infusion, respectively. The pharmacokinetics and biodistribution of DOX were determined and an analysis of the treatment parameters' influence was performed. RESULTS: Compared to untreated tissue, we found a 15-fold and a 7-fold increase in DOX concentration in the muscle volumes that had undergone hyperthermia starting 10 min and 60 min after the beginning of the infusion, respectively. The pharmacokinetic analysis showed a prolonged circulation time of DOX and a correlation between the AUC of extra-liposomal DOX in the bloodstream and the amount of DOX accumulated in the target tissue. CONCLUSIONS: We have demonstrated a workflow for MR-HIFU hyperthermia drug delivery that can be adapted to a clinical setting, showing that HIFU-hyperthermia is a suitable method for local drug release of DOX using DPPG2 based thermosensitive liposomes in stationary targets. Using the developed pharmacokinetic model, an optimization of the drug quantity deposited in the target via the timing of infusion and hyperthermia should be possible.

A Potential Role for Bile Acid Signaling in Celiac Disease-Associated Fatty Liver.

Celiac disease (CeD) is a chronic autoimmune disorder characterized by an intolerance to storage proteins of many grains. CeD is frequently associated with liver damage and steatosis. Bile acid (BA) signaling has been identified as an important mediator in gut-liver interaction and the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Here, we aimed to analyze BA signaling and liver injury in CeD patients. Therefore, we analyzed data of 20 CeD patients on a gluten-free diet compared to 20 healthy controls (HC). We furthermore analyzed transaminase levels, markers of cell death, BA, and fatty acid metabolism. Hepatic steatosis was determined via transient elastography, by MRI and non-invasive scores. In CeD, we observed an increase of the apoptosis marker M30 and more hepatic steatosis as compared to HC. Fibroblast growth factor 19 (FGF19) was repressed in CeD, while low levels were associated with steatosis, especially in patients with high levels of anti-tissue transglutaminase antibodies (anti-tTG). When comparing anti-tTG-positive CeD patients to individuals without detectable anti-tTG levels, hepatic steatosis was accentuated. CeD patients with significant sonographic steatosis (defined by CAP ≥ 283 db/m) were exclusively anti-tTG-positive. In summary, our results suggest that even in CeD patients in clinical remission under gluten-free diet, alterations in gut-liver axis, especially BA signaling, might contribute to steatotic liver injury and should be further addressed in future studies and clinical practice.

Distribution and daily oscillation of GABA in the circadian system of the cockroach Rhyparobia maderae.

Gamma-aminobutyric acid (GABA) is the prevalent inhibitory neurotransmitter in nervous systems promoting sleep in both mammals and insects. In the Madeira cockroach, sleep-wake cycles are controlled by a circadian clock network in the brain's optic lobes, centered in the accessory medulla (AME) with its innervating pigment-dispersing factor (PDF) expressing clock neurons at the anterior-ventral rim of the medulla. GABA is present in cell clusters that innervate different circuits of the cockroach's AME clock, without colocalizing in PDF clock neurons. Physiological, immunohistochemical, and behavioral assays provided evidence for a role of GABA in light entrainment, possibly via the distal tract that connects the AME's glomeruli to the medulla. Furthermore, GABA was implemented in clock outputs to multiple effector systems in optic lobe and midbrain. Here, GABAergic brain circuits were analyzed further, focusing on the circadian system in search for sleep/wake controlling brain circuits. All GABA-immunoreactive neurons of the cockroach brain were also stained with an antiserum against the GABA-synthesizing enzyme glutamic acid decarboxylase. We found strong overlap of the distribution of GABA-immunoreactive networks with PDF clock networks in optic lobes and midbrain. Neurons in five of the six soma groups that innervate the clock exhibited GABA immunoreactivity. The intensity of GABA immunoreactivity in the distal tract showed daily fluctuations with maximum staining intensity in the middle of the day and weakest staining at the end of the day. Quantification via enzyme-linked immunosorbent assay and quantitative liquid chromatography coupled to electrospray ionization tandem mass spectrometry, likewise, showed higher GABA levels in the optic lobe during the inactivity phase of the cockroach during the day and lower levels during its activity phase at dusk. Our data further support the hypothesis that light- and PDF-dependently the circadian clock network of the cockroach controls GABA levels and thereby promotes sleep during the day.

High fat / high cholesterol diet does not provoke atherosclerosis in the ω3-and ω6-polyunsaturated fatty acid synthesis-inactivated Δ6-fatty acid desaturase-deficient mouse.

OBJECTIVE: An increased ω6/ω3-polyunsaturated fatty acid ratio in the current Western diet is regarded as a critical epigenetic nutritional factor in the pathogenesis of several human lifestyle diseases, metabolic syndrome, cardiovascular disease, the central nervous system and the female and male reproductive systems. The impact of nutrient ω3-and ω6-PUFAs in the pathogenesis of dyslipoproteinemia and atherosclerosis has been a topic of intense efforts for several decades. Cellular homeostasis of the ω3-and ω6- PUFA pool is maintained by the synthesis of ω3-and ω6-PUFAs from essential fatty acids (EFA) (linoleic and α-linolenic acid) and their dietary supply. In this study, we used the auxotrophic Δ6-fatty acid desaturase- (FADS2) deficient mouse (fads2-/-), an unbiased model congenial for stringent feeding experiments, to investigate the molecular basis of the proposed protective role of dietary ω3-and ω6-PUFAs (Western diet) in the pathogenesis of multifactorial dyslipoproteinemia and atherosclerosis. We focused on the metabolic axis-liver endoplasmic reticulum (ER), serum lipoprotein system (Lp) and aorta vessel wall. Furthermore, we addressed the impact of the inactivated fads2-locus with inactivated PUFA synthesis on the development and progression of extended atherosclerosis in two different mouse mutants with disrupted cholesterol homeostasis, using the apoe-/- and ldlr-/- mutants and the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double mutants. METHODS: Cohorts of +/+ and fads2-/- mice underwent two long-term dietary regimens: a) a PUFA-free standard chow diet containing only EFAs, essential for viability, and b) a high fat/high cholesterol (HFHC) diet, a mimicry of the human atherogenic "Western" diet. c) To study the molecular impact of PUFA synthesis deficiency on the development and progression of atherosclerosis in the hypercholesterolemic apoe-/- and ldlr-/- mouse models fed PUFA-free regular and sustained HFHC diets, we generated the fads2-/- x apoe-/- and the fads2-/- x ldlr-/- double knockout mutants. We assessed essential molecular, biochemical and cell biological links between the diet-induced modified lipidomes of the membrane systems of the endoplasmic reticulum/Golgi complex, the site of lipid synthesis, the PL monolayer and neutral lipid core of LD and serum-Lp profiles and cellular reactions in the aortic wall. RESULTS: ω3-and ω6-PUFA synthesis deficiency in the fads2-/- mouse causes a) hypocholesterolemia and hypotriglyceridemia, b) dyslipoproteinemia with a shift of high-density lipoprotein (HDL) to very low-density lipoprotein (VLDL)-enriched Lp-pattern and c) altered liver lipid droplet structures. d) Long-term HFHC diet does not trigger atherosclerotic plaque formation in the aortic arc, the thoracic and abdominal aorta of PUFA-deficient fads2-/- mice. Inactivation of the fads2-/- locus, abolishing systemic PUFA synthesis in the fads2-/- x apoe-/- and fads2-/- x ldlr-/- double knockout mouse lines. CONCLUSIONS: Deficiency of ω3-and ω6-PUFA in the fads2-/- mutant perturbs liver lipid metabolism, causes hypocholesterolemia and hypotriglyceridemia and renders the fads2-/- mutant resistant to sustained atherogenic HFHC diet. Neither PUFA-free regular nor long-term HFHC-diet impacts the apoe- and LDL-receptor deficiency-provoked hypercholesterolemia and atherosclerotic plaque formation, size and distribution in the aorta. Our study strongly suggests that the absence of PUFAs as highly vulnerable chemical targets of autoxidation attenuates inflammatory responses and the formation of atherosclerotic lesions. The cumulative data and insight into the molecular basis of the pleiotropic functions of PUFAs challenge a differentiated view of PUFAs as culprits or benefactors during a lifespan, pivotal for legitimate dietary recommendations.

Modulation of Endocannabinoids by Caloric Restriction Is Conserved in Mice but Is Not Required for Protection from Acute Kidney Injury.

Acute kidney injury (AKI) is a frequent and critical complication in the clinical setting. In rodents, AKI can be effectively prevented through caloric restriction (CR), which has also been shown to increase lifespan in many species. In Caenorhabditis elegans (C. elegans), longevity studies revealed that a marked CR-induced reduction of endocannabinoids may be a key mechanism. Thus, we hypothesized that regulation of endocannabinoids, particularly arachidonoyl ethanolamide (AEA), might also play a role in CR-mediated protection from renal ischemia-reperfusion injury (IRI) in mammals including humans. In male C57Bl6J mice, CR significantly reduced renal IRI and led to a significant decrease of AEA. Supplementation of AEA to near-normal serum concentrations by repetitive intraperitoneal administration in CR mice, however, did not abrogate the protective effect of CR. We also analyzed serum samples taken before and after CR from patients of three different pilot trials of dietary interventions. In contrast to mice and C. elegans, we detected an increase of AEA. We conclude that endocannabinoid levels in mice are modulated by CR, but CR-mediated renal protection does not depend on this effect. Moreover, our results indicate that modulation of endocannabinoids by CR in humans may differ fundamentally from the effects in animal models.