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Immune rejection presents a significant challenge in xenogenic meniscal transplantation. Pigs are widely regarded as an advantageous tissue source for such transplants, with porcine GGTA1, CMAH, and B4GALNT2 being among the most common xenoreactive antigen (Ag) genes. While some studies have suggested that allogeneic meniscus (AM) transplants may exhibit immunoprivileged properties, our study observed slight immunological rejection has been observed following contact between human meniscal cells (HMCs) and human peripheral blood mononuclear cells (PBMCs). Given the limited systematic research on immune responses following xenograft meniscus transplantation, we established porcine meniscus transplantation (PMT) models to comprehensively assess the immunogenicity of porcine meniscus (PM) from both innate and adaptive immune perspectives. Our investigations confirmed that PMT beneath the epidermis led to innate cell infiltration into the xenografts and T-cell activation in local lymph nodes. T-cell activation upregulated the interleukin (IL)-17 signaling pathway, disrupting collagen organization and metabolic processes, thereby hindering PM regeneration. Using freeze-thaw treatment on PM alleviated T-cell activation post-transplantation by eliminating xenogenic DNA. In vitro findings demonstrated that gene editing in porcine meniscal cells (PMCs) suppressed human T-cell activation by downregulating the expression of xenoreactive Ag genes. These results suggest that GGTA1/CMAH/B4GALNT2 knockout (KO) pigs hold significant promise for advancing the field of meniscal transplantation.

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ß-1,4-N-acetylgalactosamine transferase 2 (B4GALNT2) is a vital candidate gene that affects the growth traits in sheep. However, whether it has the same function in goats remains to be investigated further. This study selected 348 Nanjiang Yellow goats, screened all exons, and conserved non-coding regions of the B4GALNT2 gene for single-nucleotide polymorphisms (SNPs). Our results revealed the presence of a synonymous mutation, rs672215506, within the exon of the B4GALNT2 gene in the Nanjiang Yellow goat population. The mutation resulted in a decrease in the mRNA stability of the B4GALNT2 gene. The results of SNP detection of the conserved non-coding region of the B4GALNT2 gene showed five potential regulatory SNPs in the Nanjiang Yellow goat population. Except for rs66095343, the ~500 bp fragments of the other four SNPs (rs649127714, rs649573228, rs652899012, and rs639183528) significantly increased the luciferase activity both in goat skeletal muscle satellite cells (MuSCs) and 293T cells. The genetic diversity indexes indicated low or intermediate levels for all six SNPs analyzed, and the genotype frequencies were in Hardy-Weinberg equilibrium. Association analysis showed that rs660965343, rs649127714, and rs649573228 significantly correlate with growth traits in the later stage of growth and development of Nanjiang Yellow goats. The haplotype combinations of H2H3 and H2H2 had higher body weight and greater body size. Moreover, H2H2 haplotype combinations significantly correlated with the litter size of the Nanjiang Yellow goats. The results of our study demonstrate the potential role of the B4GALNT2 gene as a functional genetic marker in the breeding programs of Nanjiang Yellow goats.

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Introduction: Infectious viruses in poultry, such as avian influenza virus (AIV) and Newcastle disease virus (NDV), are one of the most major threats to the poultry industry, resulting in enormous economic losses. AIVs and NDVs preferentially recognize α-2,3-linked sialic acid to bind to target cells. The human beta-1,4-N-acetyl-galactosaminyltransferase 2 (B4GALNT2) modifies α-2,3-linked sialic acid-containing glycan by transferring N-acetylgalactosamine to the sub-terminal galactose of the glycan, thus playing a pivotal role in preventing viruses from binding to cell surfaces. However, chickens lack a homolog of the B4GALNT2 gene. Methods: Here, we precisely tagged the human B4GALNT2 gene downstream of the chicken GAPDH so that the engineered cells constitutively express the human B4GALNT2. We performed a lectin binding assay to analyze the modification of α-2,3-linked sialic acid-containing glycan by human B4GALNT2. Additionally, we infected the cells with AIV and NDV and compared cell survivability, viral gene transcription, and viral titer using the WST-1 assay, RT-qPCR and TCID50 assay, respectively. Results: We validated human B4GALNT2 successfully modified α-2,3-linked sialic acid-containing glycan in chicken DF-1 cells. Following viral infection, we showed that human B4GALNT2 reduced infection of two AIV subtypes and NDV at 12-, 24-, and 36-hours post-infection. Moreover, cells expressing human B4GALNT2 showed significantly higher cell survivability compared to wild-type DF-1 cells, and viral gene expression was significantly reduced in the cells expressing human B4GALNT2. Discussion: Collectively, these results suggest that artificially expressing human B4GALNT2 in chicken is a promising strategy to acquire broad resistance against infectious viruses with a preference for α-2,3-linked sialic acids such as AIV and NDV.

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Prolificacy is a crucial characteristic of livestock, particularly for species such as sheep that have many births. The objectives of this study were as follows: (1) to investigate the genetic diversity of the 13 new and 7 known variants in the BMPRIB, GDF9, BMP15, LEPR, and B4GALNT2 genes in Ujimqin (UM), the F1 population of Dorper × Ujimqin crossbred (DPU), the F1 population of Suffolk × Ujimqin crossbred (SFKU), Sonid sheep (SN), Tan sheep (Tan), Hu sheep (Hu), and Small-tailed Han sheep (STH) sheep breeds/populations; (2) to perform an association analysis of the above 20 variants with litter size in 325 UM, 304 DPU, and 66 SFKU sheep populations; (3) to compare the frequencies of the litter-size-related alleles of these 20 variants among 8 sheep breeds/populations (the above seven sheep breeds + Mongolia sheep breed). With the use of the Sequenom MassARRAY®SNP assay technology, these 20 mutations were genotyped. The association analysis results showed that the c.746A>G (FecB) mutation in BMPR1B was significantly associated with the litter size of UM and DPU, the c.994A>G (FecGA) in GDF9 was significantly associated with the litter size of SFKU, and the c.31_33CTTinsdel (B1) in BMP15 was significantly associated with the litter size of UM. Our findings might provide valuable genetic markers for expanding sheep litter sizes.

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The Sda carbohydrate epitope and its biosynthetic B4GALNT2 enzyme are expressed in the healthy colon and down-regulated to variable extents in colon cancer. The human B4GALNT2 gene drives the expression of a long and a short protein isoform (LF-B4GALNT2 and SF-B4GALNT2) sharing identical transmembrane and luminal domains. Both isoforms are trans-Golgi proteins and the LF-B4GALNT2 also localizes to post-Golgi vesicles thanks to its extended cytoplasmic tail. Control mechanisms underpinning Sda and B4GALNT2 expression in the gastrointestinal tract are complex and not fully understood. This study reveals the existence of two unusual N-glycosylation sites in B4GALNT2 luminal domain. The first atypical N-X-C site is evolutionarily conserved and occupied by a complex-type N-glycan. We explored the influence of this N-glycan using site-directed mutagenesis and showed that each mutant had a slightly decreased expression level, impaired stability, and reduced enzyme activity. Furthermore, we observed that the mutant SF-B4GALNT2 was partially mislocalized in the endoplasmic reticulum, whereas the mutant LF-B4GALNT2 was still localized in the Golgi and post-Golgi vesicles. Lastly, we showed that the formation of homodimers was drastically impaired in the two mutated isoforms. An AlphaFold2 model of the LF-B4GALNT2 dimer with an N-glycan on each monomer corroborated these findings and suggested that N-glycosylation of each B4GALNT2 isoform controlled their biological activity.

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Infectious disease is widely considered to be a major driver of evolution. A preponderance of signatures of balancing selection at blood group-related genes is thought to be driven by inherent trade-offs in susceptibility to disease. B4galnt2 is subject to long-term balancing selection in house mice, where two divergent allele classes direct alternative tissue-specific expression of a glycosyltransferase in the intestine versus blood vessels. The blood vessel allele class leads to prolonged bleeding times similar to von Willebrand disease in humans, yet has been maintained for millions of years. Based on in vivo functional studies in inbred lab strains, it is hypothesized that the cost of prolonged bleeding times may be offset by an evolutionary trade-off involving susceptibility to a yet unknown pathogen(s). To identify candidate pathogens for which resistance could be mediated by B4galnt2 genotype, we here employed a novel "pathometagenomic" approach in a wild mouse population, which combines bacterial 16S rRNA gene-based community profiling with histopathology of gut tissue. Through subsequent isolation, genome sequencing and controlled experiments in lab mice, we show that the presence of the blood vessel allele is associated with resistance to a newly identified subspecies of Morganella morganii, a clinically important opportunistic pathogen. Given the increasing importance of zoonotic events, the approach outlined here may find useful application in the detection of emerging diseases in wild animal populations.

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The structure Siaα2,3(GalNAcß1,4)Gal- is the epitope of the Sda antigen, which is expressed on the erythrocytes and secretions of the vast majority of Caucasians, carried by N- and O-linked chains of glycoproteins, as well as by glycolipids. Sda is very similar, but not identical, to ganglioside GM2 [Siaα2,3(GalNAcß1,4)Galß1,4Glc-Cer]. The Sda synthase ß1,4 N-acetylgalactosaminyl transferase 2 (B4GALNT2) exists in a short and a long form, diverging in the aminoterminal domain. The latter has a very long cytoplasmic tail and displays a Golgi- as well as a post-Golgi localization. The biosynthesis of Sda is mutually exclusive with that of the cancer-associated sialyl Lewis antigens, whose structure is Siaα2,3Galß1,3/4(Fucα1,4/3)GlcNAc-. B4GALNT2 is down-regulated in colon cancer but patients with higher expression survive longer. In experimental systems, B4GALNT2 inhibits colon cancer progression,not only through inhibition of sialyl Lewis antigen biosynthesis. By contrast, in breast cancer B4GALNT2 is associated with malignancy. In colon cancer, the B4GALNT2 gene is regulated by multiple mechanisms, which include miRNA and transcription factor expression, as well as CpG methylation. In addition, Sda/B4GALNT2 regulates the susceptibility to infectious agents, the protection from muscle dystrophy, the activity of immune system in pregnancy and the immune rejection in xenotransplantation.

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Histo-blood group antigens in the intestinal mucosa play important roles in host-microbe interactions and modulate the susceptibility to enteric pathogens. The B4galnt2 gene, expressed in the GI tract of most mammals, including humans, encodes a beta-1,4-N-acetylgalactosaminyltransferase enzyme which catalyzes the last step in the biosynthesis of the Sd(a) and Cad blood group antigens by adding an N-acetylgalactosamine (GalNAc) residue to the precursor molecules. In our study, we found that loss of B4galnt2 expression is associated with increased susceptibility to Citrobacter rodentium infection, a murine model pathogen for human enteropathogenic Escherichia coli. We observed increased histopathological changes upon C. rodentium infection in mice lacking B4galnt2 compared to B4galnt2-expressing wild-type mice. In addition, wild-type mice cleared the C. rodentium infection faster than B4galnt2-/- knockout mice. It is known that C. rodentium uses its type 1 fimbriae adhesive subunit to bind specifically to D-mannose residues on mucosal cells. Flow cytometry analysis of intestinal epithelial cells showed the absence of GalNAc-modified glycans but an increase in mannosylated glycans in B4galnt2-deficient mice compared to B4galnt2-sufficient mice. Adhesion assays using intestinal epithelial organoid-derived monolayers revealed higher C. rodentium adherence to cells lacking B4galnt2 expression compared to wild-type cells which in turn was reduced in the absence of type I fimbriae. In summary, we show that B4galnt2 expression modulates the susceptibility to C. rodentium infection, which is partly mediated by fimbriae-mannose interaction.

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Terminal carbohydrate structures are particularly relevant in oncology because they can serve as cancer markers and alter the phenotype of cancer cells. The Sda antigen and the sialyl Lewisx and sialyl Lewisa (sLex and sLea) antigens are terminal structures whose biosynthesis is mutually exclusive. In this review, we describe the main features of the Sda antigen in cancer and its relationship with sLex/a antigens. Information was obtained from an extensive literature search and from The Cancer Genome Atlas (TCGA) public database. The Sda biosynthetic enzyme B4GALNT2 undergoes downregulation in colorectal (CRC) and stomach cancer, while it is ectopically expressed by a minority of breast cancer (BRCA) patients. High expression of B4GALNT2 is associated with better prognosis and a less malignant gene expression profile in CRC, while the opposite occurs in BRCA. The regulation of B4GALNT2 expression in CRC is multifactorial, involving gene methylation and miRNA expression. Forced expression of B4GALNT2 inhibited sLea/sLex and reduced malignancy and stemness in cells constitutively expressing sLex/a antigens. However, consistent effects were observed upon B4GALNT2 forced expression and in cells not expressing sLex/a antigens. Thus, B4GALNT2 and the Sda antigen exert a tumor-restraining activity in CRC and probably other gastrointestinal cancers, independently of sLex/a antigens.

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B4GALNT2 gene encodes the enzyme ß1,4-N-acetylgalactosaminyltransferase 2 that biosynthesizes the histo-blood group antigen Sda, which is expressed on the surface of erythrocytes and in body secretions. Analysis of The Cancer Genome Atlas (TCGA) database revealed that this gene was highly expressed in breast cancer tissues in comparison with adjacent healthy ones. In-vitro lentivirus-assisted B4GALNT2 gene knockdown experiments in model triple negative breast cancer (TNBC) cell lines (HCC1937 and MDA-MB-231) showed inhibition in cell proliferation, decrease in cell viability, promotion of cell apoptosis and inhibitions in cell migration and invasiveness abilities in comparison with empty lentivirus transfectant controls. Also, in cell cycle tests, the number of cells in the G1 phase increased, in the S phase decreased and did not change in the G2/M phase (indicative of the presence of a block in the G1 phase). In-vivo tumor formation experiments in mice revealed that knockdown of the B4GALNT2 gene in MDA-MB-231 cells inhibited their proliferation. Using co-immunoprecipitation (Co-IP) mass spectroscopy-assisted analysis, it was found that HLA-B protein [a product of the human leukocyte antigen (HLA) class I gene] interacts with B4GALNT2 protein. In-vitro overexpression of HLA-B in B4GALNT2-knocked down MDA-MB-231 cell lines significantly recovered the cell proliferation, viability and migration ability of B4GALNT2 gene. These indicate that HLA-B is one of the interaction proteins in the downstream pathway of the B4GALNT2 gene.

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BACKGROUND: The Sda antigen and corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in normal colonic mucosa and are down-regulated to a variable degree in colon cancer tissues. Although their expression profile is well studied, little is known about the underlying regulatory mechanisms. METHODS: To clarify the molecular basis of Sda expression in the human gastrointestinal tract, we investigated the transcriptional regulation of the human B4GALNT2 gene. The proximal promoter region was delineated using luciferase assays and essential trans-acting factors were identified through transient overexpression and silencing of several transcription factors. RESULTS: A short cis-regulatory region restricted to the -72 to +12 area upstream of the B4GALNT2 short-type transcript variant contained the essential promoter activity that drives the expression of the human B4GALNT2 regardless of the cell type. We further showed that B4GALNT2 transcriptional activation mostly requires ETS1 and to a lesser extent SP1. CONCLUSIONS: Results presented herein are expected to provide clues to better understand B4GALNT2 regulatory mechanisms.

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The Sda carbohydrate antigen and the corresponding biosynthetic enzyme B4GALNT2 are primarily expressed in human normal colonic mucosa and are down-regulated to variable degrees in colon cancer. On the other hand, the tumor associated antigen SLex is not detected in the healthy colon and is upregulated in colon cancer. High level of B4GALNT2 gene expression appears to be a good marker of prognosis in colon cancer; however, the molecular mechanisms regulating these carbohydrate antigens' expression are still poorly understood. We review here the most recent progress made towards understanding this balanced expression of blood group carbohydrate epitopes Sda and SLex . In particular in recent years, we have attained a better understanding of genetic and epigenetic regulation of the B4GALNT2 gene and of the subcellular fate of B4GALNT2 isoforms.

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Xenoantigens cause hyperacute rejection and limit the success of interspecific xenografts. Therefore, genes involved in xenoantigen biosynthesis, such as GGTA1, CMAH, and B4GALNT2, are key targets to improve the outcomes of xenotransplantation. In this study, we introduced a CRISPR/Cas9 system simultaneously targeting GGTA1, CMAH, and B4GALNT2 into in vitro-fertilized zygotes using electroporation for the one-step generation of multiple gene-edited pigs without xenoantigens. First, we optimized the combination of guide RNAs (gRNAs) targeting GGTA1 and CMAH with respect to gene editing efficiency in zygotes, and transferred electroporated embryos with the optimized gRNAs and Cas9 into recipient gilts. Next, we optimized the Cas9 protein concentration with respect to the gene editing efficiency when GGTA1, CMAH, and B4GALNT2 were targeted simultaneously, and generated gene-edited pigs using the optimized conditions. We achieved the one-step generation of GGTA1/CMAH double-edited pigs and GGTA1/CMAH/B4GALNT2 triple-edited pigs. Immunohistological analyses demonstrated the downregulation of xenoantigens; however, these multiple gene-edited pigs were genetic mosaics that failed to knock out some xenoantigens. Although mosaicism should be resolved, the electroporation technique could become a primary method for the one-step generation of multiple gene modifications in pigs aimed at improving pig-to-human xenotransplantation.

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OBJECTIVE: Cytoplasmic microinjection and electroporation of the CRISPR/Cas9 system into zygotes are used for generating genetically modified pigs. However, these methods create mosaic mutations in embryos. In this study, we evaluated whether the gene editing method and embryonic stage for gene editing affect the gene editing efficiency of porcine embryos. RESULTS: First, we designed five guide RNAs (gRNAs) targeting the B4GALNT2 gene and evaluated mutation efficiency by introducing each gRNA with Cas9 protein into zygotes by electroporation. Next, the optimized gRNA with Cas9 protein was introduced into 1-cell and 2-cell stage embryos by either microinjection or electroporation. The sequence of gRNA affected the bi-allelic mutation rate and mutation efficiency of blastocysts derived from electroporated embryos. Microinjection significantly decreased the cleavage rates in each embryonic stage and blastocyst formation rates in 2-cell stage embryos compared with electroporation (p < 0.05). However, the bi-allelic mutation rate and mutation efficiency of blastocysts from the 1-cell stage embryos edited using microinjection were significantly higher (p < 0.05) than those of blastocysts from the 2-cell stage embryos edited by both methods. These results indicate that the gene editing method and embryonic stage for gene editing may affect the genotype and mutation efficiency of the resulting embryos.

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BACKGROUND: Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. METHODS: Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. RESULTS: Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. CONCLUSION: We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

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Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.

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Sda is a high-frequency carbohydrate histo-blood group antigen, GalNAcß1-4(NeuAcα2-3)Galß, implicated in pathogen invasion, cancer, xenotransplantation and transfusion medicine. Complete lack of this glycan epitope results in the Sd(a-) phenotype observed in 4% of individuals who may produce anti-Sda. A candidate gene (B4GALNT2), encoding a Sda-synthesizing ß-1,4-N-acetylgalactosaminyltransferase (ß4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sda deficiency was never elucidated. Experimental and bioinformatic approaches were used to identify and characterize B4GALNT2 variants in nine Sd(a-) individuals. Homozygosity for rs7224888:T > C dominated the cohort (n = 6) and causes p.Cys466Arg, which targets a highly conserved residue located in the enzymatically active domain and is judged deleterious to ß4GalNAc-T2. Its allele frequency was 0.10-0.12 in different cohorts. A Sd(a-) compound heterozygote combined rs7224888:T > C with a splice-site mutation, rs72835417:G > A, predicted to alter splicing and occurred at a frequency of 0.11-0.12. Another compound heterozygote had two rare nonsynonymous variants, rs148441237:A > G (p.Gln436Arg) and rs61743617:C > T (p.Arg523Trp), in trans. One sample displayed no differences compared to Sd(a+). When investigating linkage disequilibrium between B4GALNT2 variants, we noted a 32-kb block spanning intron 9 to the intergenic region downstream of B4GALNT2. This block includes RP11-708H21.4, a long non-coding RNA recently reported to promote tumorigenesis and poor prognosis in colon cancer. The expression patterns of B4GALNT2 and RP11-708H21.4 correlated extremely well in >1000 cancer cell lines. In summary, we identified a connection between variants of the cancer-associated B4GALNT2 gene and Sda, thereby establishing a new blood group system and opening up for the possibility to predict Sd(a+) and Sd(a‒) phenotypes by genotyping.

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Clustered Regularly Interspaced Short Palindromic Repeat activation-synergistic activation mediator system (CRISPRa-SAM) has been efficiently used to up-regulate the targeted genes in human and mouse. But it is not known whether the CRISPRa-SAM system can be used against porcine disease because its two important transcriptional activation domains (P65 and heat shock transcription factor 1 (HSF1)) are from mouse and human, respectively. Pig is one of the most important meat sources, porcine viral infectious diseases cause massive economic losses to the swine industry and threaten the public health. We aimed to investigate whether the CRISPRa-SAM system could increase porcine antiviral activity by mediating two pig-specific target genes (Mx2 and ß1,4 N-acetylgalactosaminyltransferase (B4galnt2)). First, we constructed PK-15 and IPEC-J2 cell lines expressing nuclease-deficient Cas9 (dCas9)-vp64 and MS2-P65-HSF1 stably. Next, in these two cell models, we activated Mx2 and B4galnt2 expression through CRISPRa-SAM system. Antiviral activity to PRV or H9N2 was improved in PK-15 cells where Mx2 or B4galnt2 was activated. Altogether, our results demonstrated the potential of CRISPRa-SAM system as a powerful tool for activating pig genes and improving porcine antiviral activity.

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While strong evidence from clinical studies suggests beneficial effects of carnitine supplementation on metabolic health, serious safety concerns associated with carnitine supplementation have been raised from studies in mice. Considering that the carnitine doses in these mice studies were up to 100 times higher than those used in clinical studies, the present study aimed to address possible safety concerns associated with long-term supplementation of a carnitine dose used in clinical trials. Two groups of NMRI mice were fed either a control or a carnitine-supplemented diet (1 g/kg diet) from weaning to 19 months of age, and parameters of hepatic lipid metabolism and stress signalling and skeletal muscle gene expression were analysed in the mice at 19 months of age. Concentrations of free carnitine and acetylcarnitine in plasma and tissues were higher in the carnitine than in the control group (P<0·05). Plasma concentrations of free carnitine and acetylcarnitine were higher in mice at adult age (10 and 15 months) than at advanced age (19 months) (P<0·05). Hepatic mRNA and protein levels of genes involved in lipid metabolism and stress signalling and hepatic and plasma lipid concentrations did not differ between the carnitine and the control group. Skeletal muscle transcriptome analysis in 19-month-old mice revealed only a moderate regulation between carnitine and control group. Lifelong carnitine supplementation prevents an age-dependent impairment of plasma carnitine status, but safety concerns associated with long-term supplementation of carnitine at doses used in clinical trials can be considered as unfounded.

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