A STAT3 palmitoylation cycle promotes TH17 differentiation and colitis.

Cysteine palmitoylation (S-palmitoylation) is a reversible post-translational modification that is installed by the DHHC family of palmitoyltransferases and is reversed by several acyl protein thioesterases1,2. Although thousands of human proteins are known to undergo S-palmitoylation, how this modification is regulated to modulate specific biological functions is poorly understood. Here we report that the key T helper 17 (TH17) cell differentiation stimulator, STAT33,4, is subject to reversible S-palmitoylation on cysteine 108. DHHC7 palmitoylates STAT3 and promotes its membrane recruitment and phosphorylation. Acyl protein thioesterase 2 (APT2, also known as LYPLA2) depalmitoylates phosphorylated STAT3 (p-STAT3) and enables it to translocate to the nucleus. This palmitoylation-depalmitoylation cycle enhances STAT3 activation and promotes TH17 cell differentiation; perturbation of either palmitoylation or depalmitoylation negatively affects TH17 cell differentiation. Overactivation of TH17 cells is associated with several inflammatory diseases, including inflammatory bowel disease (IBD). In a mouse model, pharmacological inhibition of APT2 or knockout of Zdhhc7-which encodes DHHC7-relieves the symptoms of IBD. Our study reveals not only a potential therapeutic strategy for the treatment of IBD but also a model through which S-palmitoylation regulates cell signalling, which might be broadly applicable for understanding the signalling functions of numerous S-palmitoylation events.

Shati/Nat8l deficiency disrupts adult neurogenesis and causes attentional impairment through dopaminergic neuronal dysfunction in the dentate gyrus.

Successful completion of daily activities relies on the ability to select the relevant features of the environment for memory and recall. Disruption to these processes can lead to various disorders, such as attention-deficit hyperactivity disorder (ADHD). Dopamine is a neurotransmitter implicated in the regulation of several processes, including attention. In addition to the higher-order brain function, dopamine is implicated in the regulation of adult neurogenesis. Previously, we generated mice lacking Shati, an N-acetyltransferase-8-like protein on a C57BL/6J genetic background (Shati/Nat8l-/- ). These mice showed a series of changes in the dopamine system and ADHD-like behavioral phenotypes. Therefore, we hypothesized that deficiency of Shati/Nat8l would affect neurogenesis and attentional behavior in mice. We found aberrant morphology of neurons and impaired neurogenesis in the dentate gyrus of Shati/Nat8l-/- mice. Additionally, research has suggested that impaired neurogenesis might be because of the reduction of dopamine in the hippocampus. Galantamine (GAL) attenuated the attentional impairment observed in the object-based attention test via increasing the dopamine release in the hippocampus of Shati/Nat8l-/- mice. The α7 nicotinic acetylcholine receptor antagonist, methyllycaconitine, and dopamine D1 receptor antagonist, SCH23390, blocked the ameliorating effect of GAL on attentional impairment in Shati/Nat8l-/- mice. These results suggest that the ameliorating effect of GAL on Shati/Nat8l-/- attentional impairment is associated with activation of D1 receptors following increased dopamine release in the hippocampus via α7 nicotinic acetylcholine receptor. In summary, Shati/Nat8l is important in both morphogenesis and neurogenesis in the dentate gyrus and attention, possible via modulation of dopaminergic transmission. Cover Image for this issue: https://doi.org/10.1111/jnc.15061.

N-terminal acetylation of actin by NAA80 is essential for structural integrity of the Golgi apparatus.

N-alpha-acetyltransferase 80 (NAA80) was recently demonstrated to acetylate the N-terminus of actin, with NAA80 knockout cells showing actin cytoskeleton-related phenotypes, such as increased formation of membrane protrusions and accelerated migration. Here we report that NAA80 knockout cells additionally display fragmentation of the Golgi apparatus. We further employed rescue assays to demonstrate that this phenotype is connected to the ability of NAA80 to modify actin. Thus, re-expression of NAA80, which leads to re-establishment of actin's N-terminal acetyl group, rescued the Golgi fragmentation, whereas a catalytic dead NAA80 mutant could neither restore actin Nt-acetylation nor Golgi structure. The Golgi phenotype of NAA80 KO cells was shared by both migrating and non-migrating cells and live-cell imaging indicated increased Golgi dynamics in migrating NAA80 KO cells. Finally, we detected a drastic increase in the amount of F-actin in cells lacking NAA80, suggesting a causal relationship between this effect and the observed re-organization of Golgi structure. The findings further underscore the importance of actin Nt-acetylation and provide novel insight into its cellular roles, suggesting a mechanistic link between actin modification state and Golgi organization.

Regulation of mitochondrial morphology and function by stearoylation of TFR1.

Mitochondria are involved in a variety of cellular functions, including ATP production, amino acid and lipid biogenesis and breakdown, signalling and apoptosis. Mitochondrial dysfunction has been linked to neurodegenerative diseases, cancer and ageing. Although transcriptional mechanisms that regulate mitochondrial abundance are known, comparatively little is known about how mitochondrial function is regulated. Here we identify the metabolite stearic acid (C18:0) and human transferrin receptor 1 (TFR1; also known as TFRC) as mitochondrial regulators. We elucidate a signalling pathway whereby C18:0 stearoylates TFR1, thereby inhibiting its activation of JNK signalling. This leads to reduced ubiquitination of mitofusin via HUWE1, thereby promoting mitochondrial fusion and function. We find that animal cells are poised to respond to both increases and decreases in C18:0 levels, with increased C18:0 dietary intake boosting mitochondrial fusion in vivo. Intriguingly, dietary C18:0 supplementation can counteract the mitochondrial dysfunction caused by genetic defects such as loss of the Parkinson's disease genes Pink or Parkin in Drosophila. This work identifies the metabolite C18:0 as a signalling molecule regulating mitochondrial function in response to diet.

Polyunsaturated fatty acids synthesized by freshwater fish: A new insight to the roles of elovl2 and elovl5 in vivo.

At present, fish provide an important supply of long-chain polyunsaturated fatty acids (LC-PUFAs) for human consumption. Previous studies have shown that fatty acyl elongase 2 (elovl2) and elovl5 play important roles in fish LC-PUFA synthesis. Generally, freshwater fish have a stronger ability to synthesize LC-PUFAs than marine fish. However, the roles of elovl2, elovl5 and elovl2 + elovl5 in LC-PUFA synthesis of freshwater fish in vivo are not very clear. In this study, the elovl2 knockout zebrafish (elovl2-/-), elovl5 knockout zebrafish (elovl5-/-) and the double gene knockout zebrafish (DKO) were generated by CRISPR/Cas9 technology for the first time. Compared with wild type zebrafish (WT), elovl5-deletion zebrafish showed a significant increase in C22 PUFA content, which might be due to the up-regulation expressions of elovl4b and elovl2. elovl5 expressed at very low levels in livers of elovl2-/- relative to WT, indicating that elovl5 may be an "assistant attacker" of elovl2 in LC-PUFA synthesis of zebrafish. Moreover, there were no significant differences in levels of C18-C22 PUFAs between DKO and WT, indicating that besides elovl2 + elovl5 path, LC-PUFA synthesis in zebrafish could be performed by other paths. In addition, the hepatic lipidomic analysis results revealed that the contents of C22:6n-3 in phosphatidyl ethanolamine (PE-DHA) and PE-C22 PUFAs were more easily affected by the absence of elovl2 and elovl5. Our results suggest that the elovl2+elovl5 path is not the only path for LC-PUFA synthesis in zebrafish, and provide novel insights into the roles of elovl2 and elovl5 in LC-PUFA synthesis of freshwater fish.

Ablation of polyamine catabolic enzymes provokes Purkinje cell damage, neuroinflammation, and severe ataxia.

BACKGROUND: Polyamine catabolism plays a key role in maintaining intracellular polyamine pools, yet its physiological significance is largely unexplored. Here, we report that the disruption of polyamine catabolism leads to severe cerebellar damage and ataxia, demonstrating the fundamental role of polyamine catabolism in the maintenance of cerebellar function and integrity. METHODS: Mice with simultaneous deletion of the two principal polyamine catabolic enzymes, spermine oxidase and spermidine/spermine N1-acetyltransferase (Smox/Sat1-dKO), were generated by the crossbreeding of Smox-KO (Smox-/-) and Sat1-KO (Sat1-/-) animals. Development and progression of tissue injury was monitored using imaging, behavioral, and molecular analyses. RESULTS: Smox/Sat1-dKO mice are normal at birth, but develop progressive cerebellar damage and ataxia. The cerebellar injury in Smox/Sat1-dKO mice is associated with Purkinje cell loss and gliosis, leading to neuroinflammation and white matter demyelination during the latter stages of the injury. The onset of tissue damage in Smox/Sat1-dKO mice is not solely dependent on changes in polyamine levels as cerebellar injury was highly selective. RNA-seq analysis and confirmatory studies revealed clear decreases in the expression of Purkinje cell-associated proteins and significant increases in the expression of transglutaminases and markers of neurodegenerative microgliosis and astrocytosis. Further, the α-Synuclein expression, aggregation, and polyamination levels were significantly increased in the cerebellum of Smox/Sat1-dKO mice. Finally, there were clear roles of transglutaminase-2 (TGM2) in the cerebellar pathologies manifest in Smox/Sat1-dKO mice, as pharmacological inhibition of transglutaminases reduced the severity of ataxia and cerebellar injury in Smox/Sat1-dKO mice. CONCLUSIONS: These results indicate that the disruption of polyamine catabolism, via coordinated alterations in tissue polyamine levels, elevated transglutaminase activity and increased expression, polyamination, and aggregation of α-Synuclein, leads to severe cerebellar damage and ataxia. These studies indicate that polyamine catabolism is necessary to Purkinje cell survival, and for sustaining the functional integrity of the cerebellum.

Deletions of the Idh1, Eco1, Rom2, and Taf10 Genes Differently Control the Hyphal Growth, Drug Tolerance, and Virulence of Candida albicans.

The most recent genome-editing system called CRISPR-Cas9 (clustered regularly interspaced short palindromic repeat system with associated protein 9-nuclease) was employed to delete four non-essential genes (i.e., Caeco1, Caidh1, Carom2, and Cataf10) individually to establish their gene functionality annotations in pathogen Candida albicans. The biological roles of these genes were investigated with respect to the cell wall integrity and biogenesis, calcium/calcineurin pathways, susceptibility of mutants towards temperature, drugs and salts. All the mutants showed increased vulnerability compared to the wild-type background strain towards the cell wall-perturbing agents, (antifungal) drugs and salts. All the mutants also exhibited repressed and defective hyphal growth and smaller colony size than control CA14. The cell cycle of all the mutants decreased enormously except for those with Carom2 deletion. The budding index and budding size also increased for all mutants with altered bud shape. The disposition of the mutants towards cell wall-perturbing enzymes disclosed lower survival and more rapid cell wall lysis events than in wild types. The pathogenicity and virulence of the mutants was checked by adhesion assay, and strains lacking rom2 and eco1 were found to possess the least adhesion capacity, which is synonymous to their decreased pathogenicity and virulence.

α-Tubulin Acetylation Restricts Axon Overbranching by Dampening Microtubule Plus-End Dynamics in Neurons.

Axon growth is tightly controlled to establish functional neural circuits during brain development. Despite the belief that cytoskeletal dynamics is critical for cell morphology, how microtubule acetylation regulates axon development in the mammalian central nervous system remains unclear. Here, we report that loss of α-tubulin acetylation by ablation of MEC-17 in mice predisposes neurons to axon overbranching and overgrowth. Introduction of MEC-17F183A lacking α-tubulin acetyltransferase activity into MEC-17-deficient neurons failed to rescue axon defects. Moreover, loss of α-tubulin acetylation led to increases in microtubule debundling, microtubule invasion into filopodia and growth cones, and microtubule plus-end dynamics along the axon. Taxol application dampened microtubule hyperdynamics and suppressed axon overbranching and overgrowth in MEC-17-deficient neurons. Thus, our study reveals that α-tubulin acetylation acts as a brake for axon overbranching and overgrowth by dampening microtubule dynamics, providing insight into the role of microtubule post-translational modifications in regulating neural development.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

Ketoacylsynthase Domains of a Polyunsaturated Fatty Acid Synthase in Thraustochytrium sp. Strain ATCC 26185 Can Effectively Function as Stand-Alone Enzymes in Escherichia coli.

Thraustochytrium sp. strain ATCC 26185 accumulates a high level of docosahexaenoic acid (DHA), a nutritionally important ω-3 very-long-chain polyunsaturated fatty acid (VLCPUFA) synthesized primarily by polyunsaturated fatty acid (PUFA) synthase, a type I polyketide synthase-like megaenzyme. The PUFA synthase in this species comprises three large subunits, each with multiple catalytic domains. It was hypothesized that among these domains, ketoacylsynthase (KS) domains might be critical for catalyzing the condensation of specific unsaturated acyl-acyl carrier proteins (ACPs) with malonyl-ACP, thereby retaining double bonds in an extended acyl chain. To investigate the functions of these putative KS domains, two segment sequences from subunit A (KS-A) and subunit B (KS-B) of the PUFA synthase were dissected and then expressed as stand-alone enzymes in Escherichia coli The results showed that both KS-A and KS-B domains could complement the defective phenotypes of both E. colifabB and fabF mutants. Overexpression of these domains in wild-type E. coli led to increases in total fatty acid production. KS-B produced a higher ratio of unsaturated fatty acids (UFAs) to saturated fatty acids (SFAs), while KS-A could improve the overall production of fatty acids more effectively, particularly for the production of SFAs, implying that KS-A is more comparable to FabF, while KS-B is more similar to FabB in catalytic functions. Successful complementation and functional expression of the embedded KS domains in E. coli are the first step forward in studying the molecular mechanism of the PUFA synthase for the biosynthesis of VLCPUFAs in ThraustochytriumIMPORTANCE Very-long-chain polyunsaturated fatty acids (VLCPUFAs) are important for human health. They can be biosynthesized in either an aerobic pathway or an anaerobic pathway in nature. However, abundant VLCPUFAs in marine microorganisms are primarily synthesized by polyunsaturated fatty acid (PUFA) synthase, a megaenzyme with multiple subunits, each with multiple catalytic domains. Furthermore, the fundamental mechanism for this enzyme to synthesize these fatty acids still remains unknown. This report started with dissecting the embedded KS domains of the PUFA synthase from marine protist Thraustochytrium sp. strain ATCC 26185 and then expressing them in wild-type E. coli and mutants defective in condensation of acyl-ACP with malonyl-ACP. Successful complementation of the mutants and improved fatty acid production in the overexpression experiments indicate that these KS domains can effectively function as stand-alone enzymes in E. coli This result has paved the way for further studying of molecular mechanisms of the PUFA synthase for the biosynthesis of VLCPUFAs.

Inactivating mutations of acetyltransferase genes in B-cell lymphoma.

B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.

Neuroinflammation, mitochondrial defects and neurodegeneration in mucopolysaccharidosis III type C mouse model.

Severe progressive neurological paediatric disease mucopolysaccharidosis III type C is caused by mutations in the HGSNAT gene leading to deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase involved in the lysosomal catabolism of heparan sulphate. To understand the pathophysiology of the disease we generated a mouse model of mucopolysaccharidosis III type C by germline inactivation of the Hgsnat gene. At 6-8 months mice showed hyperactivity, and reduced anxiety. Cognitive memory decline was detected at 10 months and at 12-13 months mice showed signs of unbalanced hesitant walk and urinary retention. Lysosomal accumulation of heparan sulphate was observed in hepatocytes, splenic sinus endothelium, cerebral microglia, liver Kupffer cells, fibroblasts and pericytes. Starting from 5 months, brain neurons showed enlarged, structurally abnormal mitochondria, impaired mitochondrial energy metabolism, and storage of densely packed autofluorescent material, gangliosides, lysozyme, phosphorylated tau, and amyloid-ß. Taken together, our data demonstrate for the first time that deficiency of acetyl-CoA: α-glucosaminide N-acetyltransferase causes lysosomal accumulation of heparan sulphate in microglial cells followed by their activation and cytokine release. They also show mitochondrial dysfunction in the neurons and neuronal loss explaining why mucopolysaccharidosis III type C manifests primarily as a neurodegenerative disease.

Cohesin acetylation speeds the replication fork.

Cohesin not only links sister chromatids but also inhibits the transcriptional machinery's interaction with and movement along chromatin. In contrast, replication forks must traverse such cohesin-associated obstructions to duplicate the entire genome in S phase. How this occurs is unknown. Through single-molecule analysis, we demonstrate that the replication factor C (RFC)-CTF18 clamp loader (RFC(CTF18)) controls the velocity, spacing and restart activity of replication forks in human cells and is required for robust acetylation of cohesin's SMC3 subunit and sister chromatid cohesion. Unexpectedly, we discovered that cohesin acetylation itself is a central determinant of fork processivity, as slow-moving replication forks were found in cells lacking the Eco1-related acetyltransferases ESCO1 or ESCO2 (refs 8-10) (including those derived from Roberts' syndrome patients, in whom ESCO2 is biallelically mutated) and in cells expressing a form of SMC3 that cannot be acetylated. This defect was a consequence of cohesin's hyperstable interaction with two regulatory cofactors, WAPL and PDS5A (refs 12, 13); removal of either cofactor allowed forks to progress rapidly without ESCO1, ESCO2, or RFC(CTF18). Our results show a novel mechanism for clamp-loader-dependent fork progression, mediated by the post-translational modification and structural remodelling of the cohesin ring. Loss of this regulatory mechanism leads to the spontaneous accrual of DNA damage and may contribute to the abnormalities of the Roberts' syndrome cohesinopathy.

NAT8L (N-acetyltransferase 8-like) accelerates lipid turnover and increases energy expenditure in brown adipocytes.

NAT8L (N-acetyltransferase 8-like) catalyzes the formation of N-acetylaspartate (NAA) from acetyl-CoA and aspartate. In the brain, NAA delivers the acetate moiety for synthesis of acetyl-CoA that is further used for fatty acid generation. However, its function in other tissues remained elusive. Here, we show for the first time that Nat8l is highly expressed in adipose tissues and murine and human adipogenic cell lines and is localized in the mitochondria of brown adipocytes. Stable overexpression of Nat8l in immortalized brown adipogenic cells strongly increases glucose incorporation into neutral lipids, accompanied by increased lipolysis, indicating an accelerated lipid turnover. Additionally, mitochondrial mass and number as well as oxygen consumption are elevated upon Nat8l overexpression. Concordantly, expression levels of brown marker genes, such as Prdm16, Cidea, Pgc1α, Pparα, and particularly UCP1, are markedly elevated in these cells. Treatment with a PPARα antagonist indicates that the increase in UCP1 expression and oxygen consumption is PPARα-dependent. Nat8l knockdown in brown adipocytes has no impact on cellular triglyceride content, lipogenesis, or oxygen consumption, but lipolysis and brown marker gene expression are increased; the latter is also observed in BAT of Nat8l-KO mice. Interestingly, the expression of ATP-citrate lyase is increased in Nat8l-silenced adipocytes and BAT of Nat8l-KO mice, indicating a compensatory mechanism to sustain the acetyl-CoA pool once Nat8l levels are reduced. Taken together, our data show that Nat8l impacts on the brown adipogenic phenotype and suggests the existence of the NAT8L-driven NAA metabolism as a novel pathway to provide cytosolic acetyl-CoA for lipid synthesis in adipocytes.

Using VAAST to identify an X-linked disorder resulting in lethality in male infants due to N-terminal acetyltransferase deficiency.

We have identified two families with a previously undescribed lethal X-linked disorder of infancy; the disorder comprises a distinct combination of an aged appearance, craniofacial anomalies, hypotonia, global developmental delays, cryptorchidism, and cardiac arrhythmias. Using X chromosome exon sequencing and a recently developed probabilistic algorithm aimed at discovering disease-causing variants, we identified in one family a c.109T>C (p.Ser37Pro) variant in NAA10, a gene encoding the catalytic subunit of the major human N-terminal acetyltransferase (NAT). A parallel effort on a second unrelated family converged on the same variant. The absence of this variant in controls, the amino acid conservation of this region of the protein, the predicted disruptive change, and the co-occurrence in two unrelated families with the same rare disorder suggest that this is the pathogenic mutation. We confirmed this by demonstrating a significantly impaired biochemical activity of the mutant hNaa10p, and from this we conclude that a reduction in acetylation by hNaa10p causes this disease. Here we provide evidence of a human genetic disorder resulting from direct impairment of N-terminal acetylation, one of the most common protein modifications in humans.

Deletion of SHATI/NAT8L increases dopamine D1 receptor on the cell surface in the nucleus accumbens, accelerating methamphetamine dependence.

In a previous report, we identified a novel molecule, SHATI/NAT8L, having an inhibitory effect on methamphetamine (METH)-induced hyperlocomotion, sensitization, and conditioned place preference (CPP). SHATI/NAT8L attenuates the METH-induced increase in dopamine overflow in the nucleus accumbens (NAc) by promoting plasmalemmal and vesicular dopamine uptake. However, the biological functions of the protein remain unclear. In this study, we explored NAT8L-binding proteins using pull-down assays and identified a number of components of the adaptor protein (AP)-2 complex, which is a multimeric protein localized to the plasma membrane that functions to internalize cargo during clathrin-mediated endocytosis. To investigate whether NAT8L regulates the receptor localization to the cell surface, cell-surface dopamine D1 receptor in the NAc of Nat8l knockout (KO) mice was quantified. We found that dopamine D1 receptor on the cell surface was increased in the NAc of Nat8l KO mice compared with the wild type (WT) animals. Consistent with this finding, Nat8l KO mice showed higher basal locomotor activity and heightened sensitivity to D1 agonist compared with WT mice. In addition, METH-induced sensitization and CPP were enhanced in Nat8l KO mice. These results suggest that NAT8L might regulate the localization of cell-surface dopamine D1 receptor, thereby controlling basal behaviour and sensitivity to METH. Furthermore, we observed a single nucleotide polymorphism (SNP) in the human NAT8L gene related to reward dependence, a personality trait, and grey matter volume in the caudate nucleus in healthy subjects, suggesting that NAT8L might also affect human personality.

Crucial role of a long-chain fatty acid elongase, Elovl6, in obesity-induced insulin resistance.

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C epsilon activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity.

A shift in sphingolipid composition from C24 to C16 increases susceptibility to apoptosis in HeLa cells.

Sphingolipids, major lipid components of the eukaryotic plasma membrane, have a variety of physiological functions and have been associated with many diseases. They have also been implicated in apoptosis. Sphingolipids are heterogeneous in their acyl chain length, with long-chain (C16) and very long-chain (C24) sphingolipids being predominant in most mammalian tissues. We demonstrate that knockdown of ELOVL1 or CERS2, which catalyze synthesis of C24 acyl-CoAs and C24 ceramide, respectively, drastically reduced C24 sphingolipid levels with a complementary increase in C16 sphingolipids. Under ELOVL1 or CERS2 knockdown conditions, cisplatin-induced apoptosis significantly increased. Enhanced sensitivity to cisplatin-induced apoptosis exhibited close correlation with increases in caspase-3/7 activity. No significant alterations in sphingolipid metabolism such as ceramide generation were apparent with the cisplatin-induced apoptosis, and inhibitors of ceramide generation had no effect on the apoptosis. Apoptosis induced by UV radiation or C6 ceramides also increased in ELOVL1 or CERS2 knockdown cells. Changes in the composition of sphingolipid chain length may affect susceptibility to stimuli-induced apoptosis by affecting the properties of cell membranes, such as lipid microdomain/raft formation.

Loss of Gcn5l2 leads to increased apoptosis and mesodermal defects during mouse development.

Histone acetyltransferases regulate transcription, but little is known about the role of these enzymes in developmental processes. Gcn5 (encoded by Gcn5l2) and Pcaf, mouse histone acetyltransferases, share similar sequences and enzymatic activities. Both interact with p300 and CBP (encoded by Ep300 and Crebbp, respectively), two other histone acetyltransferases that integrate multiple signalling pathways. Pcaf is thought to participate in many of the cellular processes regulated by p300/CBP (refs 2-8), but the functions of Gcn5 are unknown in mammalian cells. Here we show that the gene Pcaf is dispensable in mice. In contrast, Gcn5l2-null embryos die during embryogenesis. These embryos develop normally to 7.5 days post coitum (d.p.c.), but their growth is severely retarded by 8.5 d.p.c. and they fail to form dorsal mesoderm lineages, including chordamesoderm and paraxial mesoderm. Differentiation of extra-embryonic and cardiac mesoderm seems to be unaffected. Loss of the dorsal mesoderm lineages is due to a high incidence of apoptosis in the Gcn5l2 mutants that begins before the onset of morphological abnormality. Embryos null for both Gcn5l2 and Pcaf show even more severe defects, indicating that these histone acetyltransferases have overlapping functions during embryogenesis. Our studies are the first to demonstrate that specific acetyltransferases are required for cell survival and mesoderm formation during mammalian development.

Metabolic and growth inhibitory effects of conjugated fatty acids in the cell line HT-29 with special regard to the conversion of t11,t13-CLA.

Conjugated fatty acids (CFAs) exhibit growth inhibitory effects on colon cancer in vitro and in vivo. To investigate whether the anticancerogenic potency depends on number or configuration of the conjugated double bonds, the effect of conjugated linoleic acid (CLA; C18:2) isomers and conjugated linolenic acid (CLnA; C18:3) isomers on viability and growth of HT-29 cells were compared. Low concentrations of CLnAs (<10µM) yielded a higher degree of inhibitory effects compared to CLAs (40µM). All trans-CFAs were more effective compared to cis/trans-CFAs as follows: t9,t11,t13-CLnA≥c9,t11,t13-CLnA>t11,t13-CLA≥t9,t11-CLA>c9,t11-CLA. The mRNA expression analysis of important genes associated with fatty acid metabolism showed an absence of ∆5-/∆6-desaturases and elongases in HT-29 cells, which was confirmed by fatty acid analysis. Using time- and dose-dependent stimulation experiments several metabolites were determined. Low concentrations of all trans-CFAs (5-20µM) led to dose-dependent increase of conjugated t/t-C16:2 formed by ß-oxidation of C18 CFAs, ranging from 1-5% of total FAME. Importantly, it was found that CLnA is converted to CLA and that CLA is inter-converted (t11,t13-CLA is metabolized to c9,t11-CLA) by HT-29 cells. In summary, our study shows that growth inhibition of human cancer cells is associated with a specific cellular transcriptomic and metabolic profile of fatty acid metabolism, which might contribute to the diversified ability of CFAs as anti-cancer compounds.