A lipidome landscape of aging in mice.

Understanding the molecular mechanisms of aging is crucial for enhancing healthy longevity. We conducted untargeted lipidomics across 13 biological samples from mice at various life stages (2, 12, 19 and 24 months) to explore the potential link between aging and lipid metabolism, considering sex (male or female) and microbiome (specific pathogen-free or germ-free) dependencies. By analyzing 2,704 molecules from 109 lipid subclasses, we characterized common and tissue-specific lipidome alterations associated with aging. For example, the levels of bis(monoacylglycero)phosphate containing polyunsaturated fatty acids increased in various organs during aging, whereas the levels of other phospholipids containing saturated and monounsaturated fatty acids decreased. In addition, we discovered age-dependent sulfonolipid accumulation, absent in germ-free mice, correlating with Alistipes abundance determined by 16S ribosomal RNA gene amplicon sequencing. In the male kidney, glycolipids such as galactosylceramides, galabiosylceramides (Gal2Cer), trihexosylceramides (Hex3Cer), and mono- and digalactosyldiacylglycerols were detected, with two lipid classes-Gal2Cer and Hex3Cer-being significantly enriched in aged mice. Integrated analysis of the kidney transcriptome revealed uridine diphosphate galactosyltransferase 8A (UGT8a), alkylglycerone phosphate synthase and fatty acyl-coenzyme A reductase 1 as potential enzymes responsible for the male-specific glycolipid biosynthesis in vivo, which would be relevant to sex dependency in kidney diseases. Inhibiting UGT8 reduced the levels of these glycolipids and the expression of inflammatory cytokines in the kidney. Our study provides a valuable resource for clarifying potential links between lipid metabolism and aging.

SOX9-mediated UGT8 expression promotes glycolysis and maintains the malignancy of non-small cell lung cancer.

UDP-glycosyltransferases (UGTs) catalyze the covalent addition of sugars to small lipophilic chemicals and are associated with a wide range of diseases including cancer. The human genome contains 22 UGT genes which could be classified into four families: UGT1, UGT2, UGT3, and UGT8. The UGT8 family contains only one member which utilizes UDP galactose to galactosidate ceramide. Although higher UGT8 mRNA was observed in some types of cancer, its pathological significances remain elusive. Here, by integrating the Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO), and the Genotype-Tissue Expression (GTEx) databases, we showed that UGT8 was selectively highly expressed in non-small cell lung cancer (NSCLC) and associated with worse prognosis. The transcription factor SOX9 promoted UGT8 expression in NSCLC by recognizing two putative response elements localized on the promoter region of UGT8. Silencing UGT8 impaired glycolysis and reduced the malignancy of NSCLC cells both in vitro and in vivo. On the contrary, inhibition of glycolysis by 2-deoxy-d-glucose (2-DG) significantly impaired the pro-proliferation function of UGT8 in NSCLC cells. In conclusion, our results suggest that UGT8 maintains the malignancy of NSCLC mainly via enhanced glycolysis and provides a promising therapeutic target for NSCLC.

Substrate reduction therapy for Krabbe disease and metachromatic leukodystrophy using a novel ceramide galactosyltransferase inhibitor.

Krabbe disease (KD) and metachromatic leukodystrophy (MLD) are caused by accumulation of the glycolipids galactosylceramide (GalCer) and sulfatide and their toxic metabolites psychosine and lysosulfatide, respectively. We discovered a potent and selective small molecule inhibitor (S202) of ceramide galactosyltransferase (CGT), the key enzyme for GalCer biosynthesis, and characterized its use as substrate reduction therapy (SRT). Treating a KD mouse model with S202 dose-dependently reduced GalCer and psychosine in the central (CNS) and peripheral (PNS) nervous systems and significantly increased lifespan. Similarly, treating an MLD mouse model decreased sulfatides and lysosulfatide levels. Interestingly, lower doses of S202 partially inhibited CGT and selectively reduced synthesis of non-hydroxylated forms of GalCer and sulfatide, which appear to be the primary source of psychosine and lysosulfatide. Higher doses of S202 more completely inhibited CGT and reduced the levels of both non-hydroxylated and hydroxylated forms of GalCer and sulfatide. Despite the significant benefits observed in murine models of KD and MLD, chronic CGT inhibition negatively impacted both the CNS and PNS of wild-type mice. Therefore, further studies are necessary to elucidate the full therapeutic potential of CGT inhibition.

Origin and Evolution of Two Independently Duplicated Genes Encoding UDP- Glucose: Glycoprotein Glucosyltransferases in Caenorhabditis and Vertebrates.

UDP- glucose: glycoprotein glucosyltransferase (UGGT) is a protein that operates as the gatekeeper for the endoplasmic reticulum (ER) quality control mechanism of glycoprotein folding. It is known that vertebrates and Caenorhabditis genomes harbor two uggt gene copies that exhibit differences in their properties.Bayesian phylogenetic inference based on 195 UGGT and UGGT-like protein sequences of an ample spectrum of eukaryotic species showed that uggt genes went through independent duplications in Caenorhabditis and vertebrates. In both lineages, the catalytic domain of the duplicated genes was subjected to a strong purifying selective pressure, while the recognition domain was subjected to episodic positive diversifying selection. Selective relaxation in the recognition domain was more pronounced in Caenorhabditis uggt-b than in vertebrates uggt-2 Structural bioinformatics analysis revealed that Caenorhabditis UGGT-b protein lacks essential sequences proposed to be involved in the recognition of unfolded proteins. When we assayed glucosyltrasferase activity of a chimeric protein composed by Caenorhabditis uggt-b recognition domain fused to S. pombe catalytic domain expressed in yeast, no activity was detected.The present results support the conservation of the UGGT activity in the catalytic domain and a putative divergent function of the recognition domain for the UGGT2 protein in vertebrates, which would have gone through a specialization process. In Caenorhabditis, uggt-b evolved under different constraints compared to uggt-a which, by means of a putative neofunctionalization process, resulted in a non-redundant paralog. The non-canonical function of uggt-b in the worm lineage highlights the need to take precautions before generalizing gene functions in model organisms.

High FA2H and UGT8 transcript levels predict hydroxylated hexosylceramide accumulation in lung adenocarcinoma.

Lung cancer causes more deaths than any other cancer. Sphingolipids encompass metabolically interconnected species whose balance has pivotal effects on proliferation, migration, and apoptosis. In this study, we paralleled quantification of sphingolipid species with quantitative (q)PCR analyses of metabolic enzymes in order to identify dysregulated routes of sphingolipid metabolism in different subtypes of lung cancers. Lung samples were submitted to histopathological reexamination in order to confirm cancer type/subtype, which included adenocarcinoma histological subtypes and squamous cell and neuroendocrine carcinomas. Compared with benign lesions and tumor-free parenchyma, all cancers featured decreased sphingosine-1-phosphate and SMs. qPCR analyses evidenced differential mechanisms leading to these alterations between cancer types, with neuroendocrine carcinomas upregulating SGPL1, but CERT1 being downregulated in adenocarcinomas and squamous cell carcinomas. 2-Hydroxyhexosylceramides (2-hydroxyHexCers) were specifically increased in adenocarcinomas. While UDP-glycosyltransferase 8 (UGT8) transcript levels were increased in all cancer subtypes, fatty acid 2-hydroxylase (FA2H) levels were higher in adenocarcinomas than in squamous and neuroendocrine carcinomas. As a whole, we report differing mechanisms through which all forms of lung cancer achieve low SM and lysosphingolipids. Our results also demonstrate that FA2H upregulation is required for the accumulation of 2-hydroxyHexCers in lung cancers featuring high levels of UGT8.

Microbubble-based enhancement of radiation effect: Role of cell membrane ceramide metabolism.

Ultrasound (US) stimulated microbubbles (MB) is a new treatment approach that sensitizes cancer cells to radiation (XRT). The molecular pathways in this response remain unelucidated, however, previous data has supported a role for cell membrane-metabolism related pathways including an up regulation of UDP glycosyltransferase 8 (UGT8), which catalyzes the transfer of galactose to ceramide, a lipid that is associated with the induction of apoptotic signalling. In this study, the role of UGT8 in responses of prostate tumours to ultrasound-stimulated microbubble radiation enhancement therapy is investigated. Experiments were carried out with cells in vitro and tumours in vivo in which UGT8 levels had been up regulated or down regulated. Genetically modified PC3 cells were treated with XRT, US+MB, or a combination of XRT+US+MB. An increase in the immunolabelling of ceramide was observed in cells where UGT8 was down-regulated as opposed to cells where UGT8 was either not regulated or was up-regulated. Clonogenic assays have revealed a decreased level of cellular survival with the down-regulation of UGT8. Xenograft tumours generated from stably transfected PC3 cells were also treated with US+MB, XRT or US+MB+XRT. Histology demonstrated more cellular damage in tumours with down-regulated UGT8 in comparison with control tumours. In contrast, tumours with up-regulated UGT8 had less damage than control tumours. Power Doppler imaging indicated a reduction in the vascular index with UGT8 down-regulation and photoacoustic imaging revealed a reduction in oxygen saturation. This was contrary to when UGT8 was up regulated. The down regulation of UGT8 led to the accumulation of ceramide resulting in more cell death signalling and therefore, a greater enhancement of radiation effect when vascular disruption takes place through the use of ultrasound-stimulated microbubbles.

Chronic ethanol consumption decreases serum sulfatide levels by suppressing hepatic cerebroside sulfotransferase expression in mice.

Epidemiological studies demonstrate a possible relationship between chronic ethanol drinking and thrombotic diseases, such as myocardial infarction and stroke. However, the precise mechanism for this association remains unclear. Sulfatides are endogenous glycosphingolipids composed of ceramide, galactose, and sulfate, known to have anti-thrombotic properties. Low (0.5 g/kg/day), middle (1.5 g/kg/day), and high (3.0 g/kg/day) doses of ethanol were administered for 21 days intraperitoneally to female wild-type mice, and serum/liver sulfatide levels were measured. No significant changes in cholesterol and triglycerides were seen in serum and liver by ethanol treatment. However, serum/liver sulfatide levels were significantly decreased by middle- and high-dose ethanol treatment, likely due to downregulation of hepatic cerebroside sulfotransferase (CST) levels. Marked decreases in the expression of catalase and superoxide dismutases and ensuing increases in lipid peroxides were also observed in the livers of mice with middle- and high-dose ethanol treatment, suggesting the association between the suppression of hepatic CST expression and enhancement of oxidative stress. Furthermore, serum levels of tissue factor, a typical pro-coagulant molecule, were significantly increased in the mice with middle- and high-dose ethanol treatment showing decreases in serum sulfatide levels. Collectively, these results demonstrate that chronic ethanol consumption reduces serum sulfatide levels by increasing oxidative stress and decreasing the expression of CST in the liver. These findings could provide a mechanism by which chronic ethanol drinking increases thrombotic events.

Evidence of intracellular stages in Trypanosoma (Megatrypanum) theileri in non-phagocytic mammalian cells.

Trypanosoma (subgenus Megatrypanum) theileri was first identified over one hundred years ago, and is a widespread parasite in cattle. Its life cycle within the mammalian host has rarely been reported. Whether there is an intracellular stage in tissues is unknown and such a stage has not been demonstrated experimentally. Intriguingly, using Giemsa staining with light microscopy and transmission electron microscopy examination, we found that the parasite was able not only to attach to cells but also to invade several phagocytic and non-phagocytic mammalian cells. Based on these findings, we conducted further investigations using a special antibody in immunofluorescence confocal images. Moreover, we examined a series of possible events of cell invasion in T. theileri. The results revealed that GM1, a marker of membrane rafts, was implicated in the mechanism of entry by this parasite. After incubation with tissue culture trypomastigotes, the gelatinolytic activity was significantly increased and accumulated at the attachment sites. Using ultrastructural localization detection by CytoTracker live imaging and confocal immunofluorescence microscopy, we found that lysosome fusion and the autophagy pathway were engaged in invaginating processes. T. theileri amastigotes also invaded cells and were enclosed by the lysosomes. Furthermore, tissue-cultured trypomastigotes were found to be capable of triggering intracellular free Ca(2+) transients and TGF-ß-signaling. Our findings that intracellular amastigote stages exist in mammalian cells infected with T. theileri and that the invasion processes involved various host cell components and cell signalings were extremely surprising and warrant further investigation.

Comprehensive genomic analyses associate UGT8 variants with musical ability in a Mongolian population.

BACKGROUND: Musical abilities such as recognising music and singing performance serve as means for communication and are instruments in sexual selection. Specific regions of the brain have been found to be activated by musical stimuli, but these have rarely been extended to the discovery of genes and molecules associated with musical ability. METHODS: A total of 1008 individuals from 73 families were enrolled and a pitch-production accuracy test was applied to determine musical ability. To identify genetic loci and variants that contribute to musical ability, we conducted family-based linkage and association analyses, and incorporated the results with data from exome sequencing and array comparative genomic hybridisation analyses. RESULTS: We found significant evidence of linkage at 4q23 with the nearest marker D4S2986 (LOD=3.1), whose supporting interval overlaps a previous study in Finnish families, and identified an intergenic single nucleotide polymorphism (SNP) (rs1251078, p = 8.4 × 10(-17)) near UGT8, a gene highly expressed in the central nervous system and known to act in brain organisation. In addition, a non-synonymous SNP in UGT8 was revealed to be highly associated with musical ability (rs4148254, p = 8.0 × 10(-17)), and a 6.2 kb copy number loss near UGT8 showed a plausible association with musical ability (p = 2.9 × 10(-6)). CONCLUSIONS: This study provides new insight into the genetics of musical ability, exemplifying a methodology to assign functional significance to synonymous and non-coding alleles by integrating multiple experimental methods.

Expression profiles of podocytes exposed to high glucose reveal new insights into early diabetic glomerulopathy.

Podocyte injury has been suggested to have a pivotal role in the pathogenesis of diabetic glomerulopathy. To glean insights into molecular mechanisms underlying diabetic podocyte injury, we generated temporal global gene transcript profiles of podocytes exposed to high glucose for a time interval of 1 or 2 weeks using microarrays. A number of genes were altered at both 1 and 2 weeks of glucose exposure compared with controls grown under normal glucose. These included extracellular matrix modulators, cell cycle regulators, extracellular transduction signals and membrane transport proteins. Novel genes that were altered at both 1 and 2 weeks of high-glucose exposure included neutrophil gelatinase-associated lipocalin (LCN2 or NGAL, decreased by 3.2-fold at 1 week and by 7.2-fold at 2 weeks), endothelial lipase (EL, increased by 3.6-fold at 1 week and 3.9-fold at 2 week) and UDP-glucuronosyltransferase 8 (UGT8, increased by 3.9-fold at 1 week and 5.0-fold at 2 weeks). To further validate these results, we used real-time PCR from independent podocyte cultures, immunohistochemistry in renal biopsies and immunoblotting on urine specimens from diabetic patients. A more detailed time course revealed changes in LCN2 and EL mRNA levels as early as 6 hours and in UGT8 mRNA level at 12 hours post high-glucose exposure. EL immunohistochemistry on human tissues showed markedly increased expression in glomeruli, and immunoblotting readily detected EL in a subset of urine samples from diabetic nephropathy patients. In addition to previously implicated roles of these genes in ischemic or oxidative stress, our results further support their importance in hyperglycemic podocyte stress and possibly diabetic glomerulopathy pathogenesis and diagnosis in humans.

GM1 / GD1b / GA1 synthase expression results in the reduced cancer phenotypes with modulation of composition and raft-localization of gangliosides in a melanoma cell line.

Gangliosides are expressed in neuroectoderm-derived tumors, and seemed to play roles in the regulation of cancer properties. To examine the behavior and roles of individual gangliosides, GM1/GD1b/GA1 synthase cDNA was introduced into the melanoma cell line SK-MEL-37, and changes in tumor phenotypes were analyzed. The transfectant cells showed neo-expression of GD1b, GT1b, and GM1, and reduced expression of GM3, GM2, GD2, and GD3. Function analyses revealed that the transfectant cells had definite reduction in cell growth and invasion. Tyrosine-phosphorylation levels of proteins such as p130Cas and paxillin were also reduced in the transfectants. These results suggested that the expression of GM1/GD1b/GA1 synthase resulted in the suppression of tumor properties. In the analyses of the floating patterns of gangliosides using fractions from sucrose density gradient ultracentrifugation of TritonX-100 extracts, the majority of gangliosides were found in glycolipid-enriched microdomain (GEM)/raft fractions, while GD3, GD1b, and GT1b in the transfectant cells tended to disperse to non-GEM/raft fractions. Furthermore, GD3, GD1b, and GT1b in non-GEM/raft dominantly had unsaturated fatty acids, while those in GEM/rafts contained more saturated forms than in non-GEM/rafts. This might be a mechanism for the decreased tumor properties in the transfectants of GM1/GD1b/GA1 synthase cDNA.

Ceramide galactosyltransferase (UGT8) is a molecular marker of breast cancer malignancy and lung metastases.

BACKGROUND: It was shown recently on the level of gene expression that UGT8, coding UDP-galactose:ceramide galactosyltransferase, is one of six genes whose elevated expression correlated with a significantly increased the risk of lung metastases in breast cancer patients. In this study primary tumours and their lung metastases as well as breast cancer cell lines were analysed for UGT8 expression at the protein level. METHODS: Expression of UGT8 in breast cancer tissue specimens and breast cancer cell lines was analysed using IHC, real-time PCR and Western blotting. RESULTS: Comparison of the average values of the reaction intensities (IRS scale) showed a significant difference in UGT8 expression between (1) primary and metastatic tumours (Mann-Whitney U, P<0.05), (2) tumours of malignancy grades G3 and G2 (Mann-Whitney U, P<0.01) as well as G3 and G1 (Mann-Whitney U, P<0.001) and (3) node-positive and node-negative tumours (Mann-Whitney U, P<0.001). The predictive ability of increased expression of UGT8 was validated at the mRNA level in three independent cohorts of breast cancer patients (721). Similarly, breast cancer cell lines with the 'luminal epithelial-like' phenotype did not express or weakly expressed UGT8, in contrast to malignant, 'mesenchymal-like,' cells forming metastases in nude mice. CONCLUSION: Our data suggest that UGT8 is a significant index of tumour aggressiveness and a potential marker for the prognostic evaluation of lung metastases in breast cancer.

Absence of oligodendroglial glucosylceramide synthesis does not result in CNS myelin abnormalities or alter the dysmyelinating phenotype of CGT-deficient mice.

To examine the function of glycosphingolipids (GSLs) in oligodendrocytes, the myelinating cells of the central nervous system (CNS), mice were generated that lack oligodendroglial expression of UDP-glucose ceramide glucosyltransferase (encoded by Ugcg). These mice (Ugcg(flox/flox);Cnp/Cre) did not show any apparent clinical phenotype, their total brain and myelin extracts had normal GSL content, including ganglioside composition, and myelin abnormalities were not detected in their CNS. These data indicate that the elimination of gangliosides from oligodendrocytes is not detrimental to myelination. These mice were also used to asses the potential compensatory effect of hydroxyl fatty acid glucosylceramide (HFA-GlcCer) accumulation in UDP-galactose:ceramide galactosyltransferase (encoded by Cgt, also known as Ugt8a) deficient mice. At postnatal day 18, the phenotypic characteristics of the Ugcg(flox/flox);Cnp/Cre;Cgt(-/-) mutants, including the degree of hypomyelination, were surprisingly similar to that of Cgt(-/-) mice, suggesting that the accumulation of HFA-GlcCer in Cgt(-/-) mice does not modify their phenotype. These studies demonstrate that abundant, structurally intact myelin can form in the absence of glycolipids, which normally represent over 20% of the dry weight of myelin.

The specific localization of seminolipid molecular species on mouse testis during testicular maturation revealed by imaging mass spectrometry.

More than 90% of the glycolipid in mammalian testis consists of a unique sulfated glyceroglycolipid called seminolipid. The galactosylation of the molecule is catalyzed by UDP-galactose:ceramide galactosyltransferase (CGT). Disruption of the CGT gene in mice results in male infertility due to the arrest of spermatogenesis, indicating that seminolipid plays an important role in reproductive function. Seminolipid molecules can be assigned to different molecular species based on the fatty acid composition. In this report, we investigated the localizations of the molecular species of seminolipid by imaging mass spectrometry and demonstrated that major molecule (C16:0-alkyl-C16:0-acyl) was expressed throughout the tubules: some (C16:0-alkyl-C14:0-acyl and C14:0-alkyl-C16:0-acyl) were predominantly expressed in spermatocytes and the other (C17:0-alkyl-C16:0-acyl) was specifically expressed in spermatids and spermatozoa. This is the first report to show the cell-specific localization of each molecular species of seminolipid during testicular maturation.

Gene expression of ceramide kinase, galactosyl ceramide synthase and ganglioside GD3 synthase is associated with prognosis in breast cancer.

PURPOSE: Sphingolipids are bioactive lipids implicated in apoptosis, cell survival and proliferation. We analyzed the prognostic value of enzymes from sphingolipid metabolism in breast cancer. METHODS: Differences in expression of ceramide galactosyl transferase (UGT8), ceramide kinase (CERK), and Ganglioside GD3-Synthase (ST8SIA1) in breast cancer cells were investigated by using microarray data of 1,581 tumor samples. RESULTS: UGT8, CERK, and ST8SIA1 were associated with poor pathohistological grading (P < 0.001). High CERK expression was correlated with ErbB2 status (P = 0.006). Among ER positive breast cancers a significant worse prognosis for patients with tumors showing low ST8SIA1 and UGT8 expression was observed. In the ER negative subgroup those samples with high CERK expression displayed a worse prognosis. In a multivariate analysis only ST8SIA1 and tumor size remained significant. CONCLUSIONS: Our experiments reveal that expression of enzymes from the sphingolipid metabolism has prognostic implications in breast cancer.

Differential partitioning and trafficking of GM gangliosides and cholesterol-rich lipid rafts in thymic and splenic CD4 T cells.

The GM gangliosides and cholesterol components of plasma membrane lipid rafts play an important role in the recruitment and signaling of protein receptors in eukaryotic cells. Herein, we have analyzed at the single-cell level the partitioning and intracellular trafficking of GM gangliosides and cholesterol in quiescent (CD4+CD69-) and CD3-activated (CD4+CD69+) thymic and splenic T cells. First, regardless the gender and the quiescent or activated status of T cells, the GM and cholesterol content in cytosol and plasma membrane as well as the expression levels of GM synthase, Sphingomyelin phosphodiestarase 2 and HMG Co-A reductase genes involved in GM and cholesterol synthesis were constantly lower in CD4 thymocytes than in CD4 splenocytes. Second, we detected variations in the balance between GM and cholesterol in plasma membrane depending on aging, and found that deprivation of cellular cholesterol does not necessarily affect the GM content in both quiescent CD4 thymocytes and splenocytes. Third, CD3 stimulation up-regulated the GM and little if any the cholesterol content in both thymic and splenic CD4 T cells, suggesting a cross talk between the CD3 signaling and GM but not cholesterol biosynthesis pathway. Fourth, partitioning and trafficking of GM to the plasma membrane depended on the transport of ceramide precursors from endoplasmic reticulum to Golgi network, as well as on the synthesis, glycosylation and vesicular assembly in trans-Golgi, and less on the cytoskeleton architecture in both quiescent and activated CD4 thymic and splenic T cells. Together, these findings suggest that the differential partitioning and intracellular trafficking of GM and cholesterol in thymic and splenic CD4 T cells may account for the stage of functional maturation.