The current study is the first to demonstrate the distinct effects of Lcarnitine deficiency and supplementation on nuclear receptors in general and on PPARs in adipocytes, liver- and muscle cells in a comparative manner. No detailed studies have been published so far comparing the effects of short-term and extended Lcarnitine substitution on the expression levels and activity of nuclear receptors. Some results regarding the effects of Lcarnitine on the nuclear level have been found already, however all papers only described effects over one time period of supplementation. In vitro substitution of Lcarnitine for 24 hours in 3T3L1 cells led to a upregulation of lipolytic genes, like hormonesensitive lipase, as well as a significant downregulation of PPARγ . Since PPARγ was proven to be involved in adipogenesis, this would suggest a role of Lcarnitine as a stimulator of lipolysis and energy dissipation. A corresponding effect was found in the case of PPARα in rats, where in vivo substitution of Lcarnitine for 28 days led to a significant upregulation of PPARα expression . PPARα is already known to play a crucial role as an enhancer of fatty acid oxidation, therefore substantiating the role of Lcarnitine as an important stimulator of the catabolism.
By reportergene assays, we were able to prove a direct effect of Lcarnitine on nuclear receptor protein activity (Fig. 1–3). The protocol for induction of Lcarnitine deficiency with the use of dialyzed FCS is an established procedure and has been previously used in publications from our research group [8, 10]. Lcarnitine deficiency, achieved via dialysis, in WRL68 cells led to an initial increase of seven examined nuclear receptor pathways (RAR, RXR, VDR, PPAR, HNF4, ER, LXR). No changes could be observed in the AR, PR and GR pathway, where the relative firefly luciferase activity remained at the level of physiological cultivation conditions. Subsequent shortterm Lcarnitine supplementation for 4 hours resulted in a further stimulation of the seven above mentioned pathways. Short term Lcarnitine pulses for 4 hours can only address preformed transcription complexes, because no significant de novo protein synthesis can take place in this limited time period. Therefore, Lcarnitine substitution obviously has the potential to interact with preformed nuclear receptor complexes, thereby increasing pathway signaling activity.
The pathways which were affected the most by 4 hours of Lcarnitine supplementation were the VDR and PPAR pathway. Interestingly, a link between these two pathways has already been revealed, with 1,25dihydroxyvitamin D being able to upregulate the PPAR pathway and thereby stimulating lipid metabolism in diabetic rats . The PPAR signaling pathway is already known to be influenced by numerous metabolites, thereby acting as a key regulator of lipid homeostasis [16, 17]. Since Lcarnitine serves as an essential esterefication partner of fatty acids getting transported across the mitochondrial membrane, alterations in intracellular Lcarnitine levels have a direct consequence for the lipid metabolism. Based on our results, Lcarnitine levels have a direct effect on the PPAR activity, thereby adjusting lipid catabolism depending on how much Lcarnitine is available to import lipids into the mitochondria.
Lcarnitine supplementation for 24 hours only led to an increase of activity in the case of the LXR and ER pathway. Regarding the nuclear receptor vitamin D pathway, a 24 hours lasting Lcarnitine supplementation exerted a diverging timedependent effect. The respective pathway activity was upregulated after 4 hours, but suppressed significantly after 24 hours of L-carnitine supplementation. The activity of the RAR, RXR, HNF4 and PPAR pathway were downregulated to physiologial steady state mRNA levels after 24 hours of supplementation.
In addition to this effect, we were able to reveal a further close connection between the PPARsystem and Lcarnitine on the transcriptional level in different tissues. Therefore, primary human skeletal muscle cells were investigated together with the WRL68 cell line, representative of mature human hepatocytes, as well as differentiated 3T3L1 cells, representative of mature murine adipocytes. The combination of human hepatocyte cell lines and murine adipocyte cell lines has already been successfully used in previous studies [18, 19].
PPARγ, PPARα and PPARδ steady state mRNA levels in WRL68 cells were upregulated in the absence of Lcarnitine via the use of dialyzed FCS, according to the results of the reporter gene assays (Fig. 4). In contrast, in SKMC and 3T3L1 cells, dialysis led to a downregulation of the whole PPAR system. In addition, transcript amounts of all PPAR members were downregulated in all three observed cell lines in the course of shortterm Lcarnitine supplementation for 4 hours, whilst the reporter gene assay showed an increase of PPAR activity after short-term supplementation. This observation further substantiates the hypothesis that Lcarnitine is able to rapidly interact with preformed protein complexes on the nuclear level.
In contrast to the short time pulse, extended Lcarnitine supplementation for 24 and 48 hours led to a slight increase of PPARγ transcription levels in WRL68 cells. PPARδ and PPAR-α mRNA amounts in WRL68 were markedly increased 6.7- and 7.7-fold, respectively. In contrast, in the reporter gene assay for PPAR protein signaling, activity decreased down to physiological levels in the course of 24 hours of Lcarnitine supplementation. A possible reason for this disparity could be the transcriptional downregulation, which was observed after 4 hours and 15 hours of supplementation. Steady state mRNA levels were significantly downregulated after short-term supplementation and did not increase again up until 24 hours of supplementation. The most pronounced upregulation of transcription was observed in the case of 48 hours of supplementation. Thus, possible effects of extended Lcarnitine supplementation on nuclear pathway protein activity could probably be observed via protein activity measurements after more than 24 hours. Therefore, effects of L-carnitine supplementation for longer time periods of supplementation on the protein activity of nuclear receptors would be worthwhile being investigated in future studies.
Whereas, extended supplementation in 3T3L1 cells led to a return of the PPARγ steady state mRNA levels to normal degrees and to a downregulation of PPARγ in SKMC. An upregulation of PPAR-γ was only observed in the case of WRL68. These divergent effects can be directly explained by the different functions of PPAR-γ in the investigated cell lines, as described by Dubois et al. . Few studies on the effects of Lcarnitine supplementation in skeletal muscles have been published so far. Interestingly, Lcarnitine substitution for 6 weeks in rats led to an increased expression of PPARγ in skeletal muscles . Since our experiments only measured the expression levels after supplementation for 4 hours to 48 hours, Lcarnitine supplementation over the course of weeks may lead to a different expression pattern as part of a response to changing metabolic conditions. Thus, possible effects of Lcarnitine supplementation on nuclear receptor expression for longer time periods are worthwhile being investigated in the future.
Different to PPARγ, the transcript levels of PPARα were significantly increased in all the cell lines after extended L-carnitine supplementation, concordant with previous studies . PPAR-δ mRNA amounts remained relatively lowered in SKMC and 3T3-L1 cells after prolonged L-carnitine supplementation but were as well significantly increased 6.7-fold in WRL68 cells. PPARγ upregulation is known to lead to an increase of lipogenesis and a decrease of lipolysis in adipose tissue . In contrast, PPARα and PPARδ promote an increase of βoxidation in skeletal muscles and hepatocytes as well as a decrease of de novo lipogenesis in the liver . Therefore, based on our results, extended Lcarnitine supplementation seems to be able to potently promote catabolic pathways, by inducing expression of catabolic PPARs and thus enhancing βoxidation and reducing lipogenesis.
The additional promoter active factors LXRα and RARβ were downregulated following the absence of Lcarnitine in all cell lines. In SKMC and WRL68 cells, mRNA levels remained significantly lowered after both short-term and extended supplementation. In WRL68 cells, steady state expression levels even dropped down to 2% under dialysis and remained at 15% even after extended supplementation, compared to normal growth conditions. These results show a clear disparity between decreasing mRNA abundance and the increase of LXR-α and RAR-β protein activity in WRL68 cells observed in the reporter gene assays (Fig. 1–2). This phenomenon could be due to several reasons. Whilst transcription is downregulated, the pool of already synthesized mRNA might however be more efficiently translated. Another reason could be an increase of the proteins’ halflife due to a reduced rate of degradation via posttranslational modifications. Lcarnitine administration has already been shown to modulate posttranslational modifications, leading to an increase of phosphorylation of AMPactivated protein kinase (AMPK), PI3K, Akt and mTOR [22–24]. Additionally, Lcarnitine supplementation has been proven to exert a decrease in proteasome activity and downregulate genes of the ubiquitin proteasome system [25, 26]. A similar effect has been described for the nutrigenomic metabolite Niacin, where supplementation reduced ubiquitination of hepatic ACOX1 and CYP4A1, thereby increasing protein levels without affecting mRNA transcription .
Interestingly, 3T3-L1 was the only cell line, where an increase of LXR-α and RAR-β transcription was observed after extended L-carnitine supplementation (Fig. 5). After 48 hours of supplementation, mRNA levels increased up to 2-fold compared to the normal untreated cells. LXR activation in both murine and human adipocytes has already been shown to shift substrate oxidation towards utilization of lipids and upregulate mitochondrial β-oxidation . Concordant with these findings, RAR activation in adipocytes seems to suppress adipogenesis and to down-regulate mRNA expression of PPAR-γ, a key regulator of adipocyte differentiation [29, 30]. These results further substantiate our hypothesis, that L-carnitine acts as potent promoter of catabolic pathways via increase of lipolysis and decrease of lipid storage in multiple tissues.
Interestingly, the expression levels of RXR-α increased as well only in 3T3-L1 cells after extended supplementation. In contrast, in SKMC and WRL68 cells, RXR-α mRNA amounts declined under Lcarnitine deficiency but remained relatively unchanged in all states of supplementation, averaging at two-thirds of the expression levels observed in normal growth conditions. A possible reason for this could be the fact that RXRα acts as an obligate hetero-dimerization partner for a number of other nuclear receptors . Since a strong decrease in RXRα levels would also impair the effectiveness of those other receptors, it could be hypothesized that cells must maintain relatively high amounts of RXRα in order to preserve their capacity to modulate transcription of essential target genes.
In addition, the effect of Lcarnitine on the expression levels of key effector genes in WRL68 cells was measured (Fig. 6). ALDH1A1 is a major gene of the oxidative pathway of alcohol metabolism and has been shown to act as a promoter of adipogenesis [29, 30]. Lcarnitine supplementation led to a significant decrease of ALDH1A1 expression, substantiating the hypothesis that Lcarnitine substitution effectively decreases adipogenesis. Olinked βNacetylglucosamine transferase (OGT) catalyzes the addition of Nacetylglucosamine in Oglycosidic linkage to serine or threonine residues . Additionally, hepatic OGT overexpression has been shown to impair the expression of insulinresponsive genes and contribute to insulin resistance and dyslipidemia . Extended Lcarnitine supplementation for 48 hours led to a 0.7-fold reduction of OGT expression, which further elucidates
the beneficial role of Lcarnitine on metabolic pathways. Additionally, extended Lcarnitine supplementation led to a strong increase of key genes of the VDR pathway CYP27A1 and CYP2R1. In the case of CYP2R1, supplementation for 48 hours even led to a dramatic 50fold upregulation of mRNA transcription. HMGCR, a key gene of the LXR pathway, and HSD11B2, a key gene of the GR pathway, were as well upregulated 7.2- and noteworthy 35-fold, respectively. These findings provide additional support for our observations, which indicate that Lcarnitine directly modulates both nuclear receptor gene transcription as well as transcription of key effector genes. In our current study, changes in expression levels of effector genes were measured in WRL68 hepatocytes, because physiologically Lcarnitine is mainly metabolized in liver tissue. In order to assess if the expression of those key genes diverges as well between different tissues after L-carnitine supplementation, changes of effector gene expression levels in other cell lines would be as well worthwhile being investigated in future studies.
Beyond that, in a chipscreen study performed by our lab, we observed that several hundreds of genes throughout the whole genome showed an increased or decreased transcription due to Lcarnitine supplementation, underlining the importance of this nutrigenomic metabolite . Similar intense effects on the transcriptome, which resulted in altered expression of hundreds of genes, were already observed for the nutrigenomic metabolites niacin, vitamin D and leucine [55–57]. In the case of niacin, similar to our results, a tissuespecific pattern of effects has been observed as well. Niacin specifically altered expression of a group of genes in adipose tissue, but not in the liver, heart or skeletal muscle . For a more detailed representation, Table 2 shows a comparison of Lcarnitine with other important nutrigenomic metabolites exerting similar effects.