The role of PNPLA1 in ω-O-acylceramide synthesis and skin barrier function

The human genome encodes nine enzymes belonging to the patatin-like phospholipase domain-containing lipase (PNPLA)/Ca²⁺-independent phospholipase A₂ (iPLA₂) family. Although most PNPLA/iPLA₂ enzymes are widely distributed and act on phospholipids or neutral lipids as (phospho)lipases to play homeostatic roles in lipid metabolism, the function of PNPLA1 remained a mystery until a few years ago. However, the recent finding that mutations in the human PNPLA1 gene are linked to autosomal recessive congenital ichthyosis (ARCI), as well as evidence obtained from biochemical and gene knockout studies, has shed light on the function of this enzyme in skin-specific sphingolipid metabolism rather than glycerophospholipid metabolism. PNPLA1 is specifically expressed in differentiated keratinocytes and plays a crucial role in the biosynthesis of ω-O-acylceramide, a particular class of sphingolipids that is essential for skin barrier function. PNPLA1 acts as a unique transacylase that specifically transfers linoleic acid from triglyceride to ω-hydroxy fatty acid in ceramide, thus giving rise to ω-O-acylceramide. In this review, we overview the biosynthetic route and biological role of epidermal ω-O-acylceramide, highlight the function of PNPLA1 as a bona fide acylceramide synthase required for proper skin barrier function and keratinocyte differentiation, and summarize the mutations of PNPLA1 currently identified in ARCI patients.This article is part of a Special Issue entitled Novel functions of phospholipase A₂ Guest Editors: Makoto Murakami and Gerard Lambeau     

δ-Aminolevulinate synthase is required for apical transcellular barrier formation in the skin of the Drosophila larva

Animals construct a layered skin to prevent dehydration and pathogen entrance. The barrier function of the skin relies on the extensive cross-linking of specialised components. In insects, for instance, epidermal cells produce an apical extracellular cuticle that consists of a network of proteins, chitin and lipids. We have identified mutations in the Drosophila gene coding for the δ-aminolevulinate synthase (Alas) that cause massive water loss. The cuticle of alas mutant larvae detaches from the epidermis and its basal region is frayed suggesting that an Alas dependent pathway is needed to organise the contact between the cuticle and the epidermis and anchor the cuticle to the apical surface of epidermal cells. Concomitantly, reduction of Alas function results in weakening of the extracellular dityrosines network in the cuticle, whereas glutamyl-lysine isopeptide bonds are not affected. The lateral septate junctions of epidermal cells that serve as a paracellular plug are intact, as well. Taken together, we hypothesise that Alas activity, which initiates heme biosynthesis in the mitochondrion, is needed for the formation of a dityrosine-based barrier that confers resistance to the internal hydrostatic pressure protecting both the cuticle from transcellular infiltration of body fluid and the animal from dehydration. We conclude that at least two modules--an apical protein-chitin lattice and the lateral septate junctions, act in parallel to ensure Drosophila skin impermeability.     

Effect of fatty acids of ω6 series on the biosynthesis of arachidonic acid in HTC cells

Summary The influence of the preincubation of HTC cells with fatty acids of 6 series and columbinic acid (St, 9c, 12c 18:3) on the biosynthesis of arachidonic acid was studied. The cells were incubated on a chemically defined medium with or without the addition of unlabeled linoleic, -linolenic, eicosatrienoic, arachidonic, docosatetraenoic, docosapentaenoic and columbinic acids. After 24 hr of preincubation in the presence of the aforementioned fatty acids, [1-¹⁴C]eicosa-8,11,14-trienoic acid was added to the culture medium as the only lipidic source. Twenty-four hours later the synthesis of arachidonic acid and the fatty acid composition of the cells were determined. At 20 MM concentration the 6 fatty acids studied except docosapentaenoic acid produced an increase on the biosynthesis of arachidonic acid compared to the cells incubated in the absence of unlabeled fatty acids in the medium. The fatty acids added to the culture medium were incorporated into the cells and modified their fatty acid composition. Columbinic acid, with a similar structure to linoleic acid, also produced a significant increase on the conversion of eicosatrienoic acid to arachidonic acid. These results would suggest that the effect of both, linoleic and columbinic acids, may be adscribed to their configuration and not necessarily to their transformation in higher homologs, since columbinic acid is unable to be desaturated.All authors are members of the Carrera del Investigador Cientifico of the Consejo Nacional de Investigaciones Cientifícas y Técnicas, Argentina.     

Properties of Bacillus acidocaldarius mutants deficient in ω-cyclohexyl fatty acid biosynthesis

Mutants of the thermoacidophilic Bacillus acidocaldarius, auxotrophic for shikimate or cyclohyxyl-carboxylate, were isolated and characterized. The cyclohexylcarboxylate auxotrophs could be divided by crossfeeding experiments into two groups according to their genetic block. The cyclohexylcarboxylate auxotrophs were deficient in -cyclohexyl fatty acid biosynthesis. If the mutants were fed with branched-chain amino acids or short branched-chain fatty acids instead of cyclohexylcarboxylate they form a fatty acid pattern consisting of branched-chain fatty acids. In the high temperature/low pH range the growth yield of cells with this fatty acid pattern is lower as compared to wild type cells or mutants fed with cyclohexylcarboxylate. The same cells are also more sensitive to heat shocks and ethanol. The transport systems for lysine, glutamate and glucose are severely altered by the fatty acid pattern. It was also shown that the density of the lipids containing -cyclohexyl fatty acids is higher compared to cells with branched-chain fatty acids. Thus it could be supposed that this alteration influences transport systmes in a direct manner or via energization of the cytoplasmic membrane.     

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function?

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.     

Isomers of conjugated linoleic acid induce insulin resistance through a mechanism involving activation of protein kinase Cε in liver cells

Conjugated linoleic acid (CLA) constitutes a group of isomers derived from linoleic acid. Diverse studies have suggested that these unsaturated fatty acids have beneficial effects on human health. However, it has also been reported that their consumption can generate alterations in hepatic tissue. Thus, in the present study, we evaluated the effect of two of the major isomers of CLA, cis-9, trans-11-CLA and trans-10, cis-12-CLA, in the regulation of insulin signaling in a hepatic cell model, clone 9 (C9). We found that the two isomers decrease insulin-stimulated phosphorylation of the main proteins involved in insulin signaling, such as Akt at Ser⁴⁷³ and Thr³⁰⁸, the insulin receptor at Tyr¹¹⁵⁸, IRS-1 at Tyr⁶³², and GSK-3 at Ser^9/21. Protein expression, however, was unaffected. Interestingly, both isomers of CLA promoted phosphorylation and activation of PKCε. Inhibition of PKCε activity by a dominant-negative form or knockdown of endogenous PKCε prevented the adverse effects of CLA isomers on insulin-induced Akt phosphorylation. Additionally, we also found that both isomers of CLA increase phosphorylation of IRS-1 at Ser⁶¹², a mechanism that probably underlies the inhibition of IRS-1 signaling by PKCε. Using confocal microscopy, we found that both isomers of CLA induced lipid accumulation in C9 cells with the presence of spherical cytosolic vesicles, suggesting their identity as neutral lipid droplets. These findings indicate that cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers could have a significant role in the development of insulin resistance in hepatic C9 cells through IRS-1 serine phosphorylation, PKCε activation, and hepatic lipid accumulation.     

Is there an optimal dose for dietary linoleic acid? Lessons from essential fatty acid deficiency supplementation and adipocyte functions in rats

Differential effects of n-3 and n-6 polyunsaturated fatty acids (PUFAs) have been demonstrated on adipose tissue physiology. Facing to the widely recognized beneficial effects of n-3 PUFAs, the n-6 PUFA effects remain controversial. Thus, we further analyzed the linoleic acid (LA) influence on adipocyte functions. To this aim, we treated by LA supplementation at three distinct doses (1, 2.5, or 5 % of energy intake) rats with essential fatty acids deficiency (EFAD). PUFA composition was determined in blood and white adipose tissue (WAT), while lipolytic and lipogenic activities were measured in isolated adipocytes. EFAD rats exhibited reduced WAT mass and increased EFAD biomarkers. WAT mass was completely recovered after supplementation, irrespective of LA dose. However, neither body mass nor EFAD biomarkers returned to control with 1 % LA, while LA abundance doubled in adipocytes from rats supplemented with 5 % LA. Regarding lipolysis, 2.5 % LA normalized the EFAD-induced alterations. A trend to decrease the maximal stimulation of lipolysis was observed with 1 and 5 % LA. Regarding lipogenesis, the lower and higher LA doses increased basal activity and hampered insulin to further stimulate glucose incorporation into lipids whereas 2.5 % LA normalized the basal or insulin-stimulated levels. Our results show that dietary linoleate at 2.5 % restored anatomical, biochemical, and functional disturbances induced by EFAD. At higher dose, LA tended to reduce triacylglycerol breakdown, to increase triacylglycerol assembly, and to provoke insulin resistance. Therefore, LA influence on adipocyte functions does not appear to follow a typical dose–response relationship, adding further complexity to the definition of its dietary requirement.     

Adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetic mice

Rosiglitazone is a PPARγ agonist commonly used to treat diabetes. In addition to improving insulin sensitivity, rosiglitazone restores normal vascular function by a mechanism that remains poorly understood. Here we show that adiponectin is required to mediate the PPARγ effect on vascular endothelium of diabetic mice. In db/db and diet-induced obese mice, PPARγ activation by rosiglitazone restores endothelium-dependent relaxation of aortae, whereas diabetic mice lacking adiponectin or treated with an anti-adiponectin antibody do not respond. Rosiglitazone stimulates adiponectin release from fat explants, and subcutaneous fat transplantation from rosiglitazone-treated mice recapitulates vasodilatation in untreated db/db recipients. Mechanistically, adiponectin activates AMPK/eNOS and cAMP/PKA signaling pathways in aortae, which increase NO bioavailability and reduce oxidative stress. Taken together, these results demonstrate that adipocyte-derived adiponectin is required for PPARγ-mediated improvement of endothelial function in diabetes. Thus, the adipose tissue represents a promising target for treating diabetic vasculopathy.     

Enzymes for biosynthesis de novo and elongation of fatty acids in mycobacteria grown in host cells: is Mycobacterium leprae competent in fatty acid biosynthesis?

Fatty acid synthetase activity in extracts of Mycobacterium leprae was equivalent to 1.7 pmol malonyl-CoA incorporated into fatty acid min-1 (mg protein)-1. This activity--if representative of living M. leprae organisms--is insufficient to enable them to synthesize their lipid requirements rapidly enough to support growth. The major activity for scavenging fatty acids in extracts of Mycobacterium microti and Mycobacterium avium, as well as in extracts of M. leprae, was acetyl-CoA-dependent fatty acyl-CoA 'elongase'. This activity was about four times higher in M. avium and M. microti grown in a medium which contained lipids, or when grown in mice, than in medium without added lipids. In contrast, the de novo fatty acid synthetase activity was repressed in M. avium and M. microti when grown in medium that contained lipids, or when grown in mice. These results are consistent with the hypothesis that mycobacteria grown in vivo preferentially scavenge lipids from the host cells, and suggest that a source of lipid should be included in media for attempted axenic isolation of M. leprae.     

TRPV1 activation improves exercise endurance and energy metabolism through PGC-1α upregulation in mice

Impaired aerobic exercise capacity and skeletal muscle dysfunction are associated with cardiometabolic diseases. Acute administration of capsaicin enhances exercise endurance in rodents, but the long-term effect of dietary capsaicin is unknown. The capsaicin receptor, the transient receptor potential vanilloid 1 (TRPV1) cation channel has been detected in skeletal muscle, the role of which remains unclear. Here we report the function of TRPV1 in cultured C2C12 myocytes and the effect of TRPV1 activation by dietary capsaicin on energy metabolism and exercise endurance of skeletal muscles in mice. In vitro, capsaicin increased cytosolic free calcium and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) expression in C2C12 myotubes through activating TRPV1. In vivo, PGC-1α in skeletal muscle was upregulated by capsaicin-induced TRPV1 activation or genetic overexpression of TRPV1 in mice. TRPV1 activation increased the expression of genes involved in fatty acid oxidation and mitochondrial respiration, promoted mitochondrial biogenesis, increased oxidative fibers, enhanced exercise endurance and prevented high-fat diet-induced metabolic disorders. Importantly, these effects of capsaicin were absent in TRPV1-deficient mice. We conclude that TRPV1 activation by dietary capsaicin improves energy metabolism and exercise endurance by upregulating PGC-1α in skeletal muscles. The present results indicate a novel therapeutic strategy for managing metabolic diseases and improving exercise endurance.     

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1^−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1^−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1^−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1^−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1^+/+ cells, which was not the case in Cav1^−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells.The transforming growth factor-beta (TGF-β) belongs to a family of polypeptide factors, whose cytostatic and apoptotic functions help restrain the growth of mammalian cells. However, paradoxically, TGF-β also modulates processes such as cell invasion, immune regulation and microenvironment modification, which cancer cells may exploit to their advantage.¹ Indeed, a better knowledge about the mechanistic basis and clinical relevance of TGF-β is required for a better understanding of the complexity and therapeutic potential of this pathway. In hepatocytes, TGF-β induces both pro- and anti-apoptotic signals, whose balance decides cell fate.² Those hepatocytes that survive to TGF-β-mediated apoptosis induce an epithelial–mesenchymal transition (EMT) process, which confers migratory properties and apoptosis resistance.³ The anti-apoptotic signals are at least partially mediated by the epidermal growth factor receptor (EGFR) pathway, which is transactivated by TGF-β through a mechanism that involves upregulation of the EGFR ligands and activation of the metalloprotease TACE/ADAM17 responsible for their shedding.^{4, 5}Many efforts have been done in the recent years for a better understanding of spatial requirements on TGF-β signaling, including endocytic TGF-β receptors trafficking. Strong pieces of evidence support that TGF-β receptors can be located both in clathrin-coated pits and caveolin/cholesterol-enriched lipid rafts.^{6, 7} A pioneer study from Di Guglielmo et al.⁸ proposed that Smad2 phosphorylation would require clathrin-dependent endocytosis, whereas TGF-β receptors internalization via caveolae/lipid rafts would inhibit its signaling. From then, different studies have suggested that the endocytic pathways'' role on TGF-β signaling depend on the cell type and a general rule about the role of endocytosis in TGF-β signaling is not well understood yet.⁹ In hepatocytes, Smad activation is in a large extent accomplished on the hepatocyte plasma membrane in an AP-2 complex-dependent manner, being unnecessary the formation of clathrin-coated pits.¹⁰ In contrast, the non-Smad/AKT pathway activation requires caveolin-1 (Cav1)-dependent endocytosis,¹⁰ which is required for counteracting apoptosis.¹¹Cav1 is required for caveolae formation, which regulates not only endocytosis, but also lipid metabolism and energy homeostasis.¹² The localization of membrane receptors in the lipid raft pushed to investigate the role of Cav1 in regulating signaling events. In the case of epidermal growth factor (EGF) signaling, it was proposed that non-caveolae-coated pits are involved in the compartmentalization and internalization of the EGFR, although caveolin-rich domains may be required for signaling.¹³ In this line of evidence, different studies revealed an important role for Cav1 in EGFR-induced effects on cell proliferation and migration.^{14, 15}In this work, we have examined the role of Cav1 in the anti-apoptotic signals induced by TGF-β in hepatocytes, postulating that it may be required for TGF-β-mediated regulation of EGFR signaling. Using different experimental approaches and an immortalized hepatocyte cell line derived from Cav1^−/− mice, we demonstrate that transactivation of the EGFR pathway by TGF-β is impaired in Cav1^−/− hepatocytes. However, Cav1 is not required for the cellular response to EGFR ligands, but is necessary for TGF-β-mediated activation of the metalloprotease TACE/ADAM17, which is responsible for shedding of EGFR ligands and requires an intact lipid raft domain to be activated by TGF-β.     

Importin α7 is essential for zygotic genome activation and early mouse development

Importin α is involved in the nuclear import of proteins. It also contributes to spindle assembly and nuclear membrane formation, however, the underlying mechanisms are poorly understood. Here, we studied the function of importin α7 by gene targeting in mice and show that it is essential for early embryonic development. Embryos lacking importin α7 display a reduced ability for the first cleavage and arrest completely at the two-cell stage. We show that the zygotic genome activation is severely disturbed in these embryos. Our findings indicate that importin α7 is a new member of the small group of maternal effect genes.