Carnitine: A Nutritional Modulator of Glucococorticoid Receptor Function

S. Alesci

Clinical Neuroendocrinology Branch, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, U.S.A.

Background: L-Carnitine (3-hydroxy-4-N, N, N-trimethylaminobutyrate) is a nutrient that plays a major role in energy metabolism. Human plasma level of L-carnitine (LCAR) is approximately 0.05 mM, as opposed to tissue concentrations of 1 to 100 mM, depending on the active uptake of this compound from the extracellular space. Currently, the only indication to the use of pharmacologic doses of LCAR (up to 600 mg/kg body weight/day) is the treatment of primary and secondary LCAR deficiencies. However, evidence from animal and human studies suggests that, at pharmacologic doses, LCAR may mimic some of the biologic actions of glucocorticoids (GCs), including their well known immunosuppressive effect. We hypothesized that these effects could be mediated through activation of the glucocorticoid receptor (GR) by LCAR. Therefore, we investigated the influence of LCAR on the various activities of this steroid receptor.

Major findings: At millimolar concentrations, which were not cytotoxic in vitro, LCAR significantly reduced the specific whole cell binding (SWCB) of ³H-dexamethasone (³H-Dex) to GR in HeLa cells in a dose dependent fashion (Fig. [1]). The reduced binding was accompanied by a significant increase in the K_d of GR for Dex, with no change in the B_max. Moreover, at the same doses, LCAR triggered the nuclear translocation of green fluorescent protein (GFP)-fused human GR alpha and transactivated the GC-responsive MMTV and TAT3 promoters in transiently transfected HeLa (Fig. [2] a, b) and CV-1 cells (Fig. [2] c). Finally, similarly to GCs, LCAR suppressed TNF alpha and IL-12 release from human primary monocytes stimulated with lipopolysaccharide and primed with IFN gamma ex vivo (Fig. [3]). Both the transcriptional and the cytokine suppressive effect of LCAR were abolished by the well known GR-antagonist mifepristone (RU 486), further supporting the GR dependence of these actions.

Fig. 1 Inhibition of ³H-Dex SWCB by LCAR.

Fig. 2 Transcriptional activation of the GC-responsive MMTV (a and c) and TAT3 (b) promoters by LCAR. LCAR was transcriptionally inactive in HeLa cells transfected with the GRE-devoid pODLO2 vector (b) and in CV-1 cells lacking functional GR alpha (GRα^-) (c). In CV-1 cells, LCAR became able to transactivate MMTV after exogenous human GR alpha was expressed by cotransfection (GRα⁺) (c).

Fig. 3 GC-like suppression of proinflammatory cytokine ex vivo release from human primary monocytes after treatment with LCAR.

Discussion: LCAR is one of a few compounds that are sold in the United States both as a prescription drug and as an over-the-counter nutritional supplement. In the present study we found that, similarly to the synthetic GC Dex, LCAR at millimolar concentrations, is able to: i) trigger nuclear translocation of human GR; ii) stimulate GR-mediated transactivation of known GC-responsive promoters in vitro; iii) suppress in a GR-dependent fashion the ex vivo release of proinflammatory cytokines from human monocytes. In addition, LCAR can compete with Dex for binding to the GR. Taken together, these findings suggest that LCAR, a nutrient structurally diverse from the classic GC Dex, may stimulate GR-mediated transactivation by functioning as an allosteric regulator of this steroid receptor. Indeed, the decreased affinity of GR for Dex in the presence of LCAR might be explained by the ability of this nutrient to interact with a portion of the receptor outside the GC binding pocket and modify the allosteric structure of the latter. This structural modification could, at the same time, reduce the affinity of the binding pocket for Dex, and create conformational changes similar to those induced by the native ligand, ultimately resulting in GR activation (Fig. [4]). The ability of LCAR to reduce GR affinity for Dex, combined with its weaker transactivating effect compared to Dex, brings also up the possibility that this compound may act as a partial GC agonist/antagonist, able to both transactivate GR in the absence of the native ligand, and antagonize GR activation in its presence. The agonist/antagonist effect may depend on the responsive promoter and be cell-specific.

Fig. 4 Hypothetical model of GR function modulation by LCAR: a classic mechanism of GR activation by the native ligand; b alternative mechanism of GR activation by pharmacologic doses of LCAR (see text for details). RAPs = receptor associated proteins; TD = transactivation domain.

Conclusions: In summary, we provide novel evidence that pharmacologic doses of LCAR, a popular widely available nutritional supplement, can activate GR and modulate the transcription of GC-responsive genes in vitro, potentially sharing and/or influencing some of the biologic and pharmacologic actions of these hormones. It was recently reported that, at high concentrations, LCAR has also a positive effect on the bone. Indeed, this invites the speculation that pharmacologic doses of LCAR might share the beneficial immunomodulatory properties of GCs, but not their deleterious effects on the bone. More generally, the modulatory effects of LCAR on GR functions may be tissue- and/or gene-specific, being influenced by receptor abundance and distribution, and/or by transcription regulatory or co-regulatory molecules, such as transcription factors, co-activators and/or co-repressors. The clinical and therapeutic implications of these findings should be evaluated in controlled trials.

Reference

• 1 Alesci S, De Martino M U, Mirani M, Benvenga S, Trimarchi F, Kino T, Chrousos G P. L-Carnitine: a nutritional modulator of glucorticoid receptor functions.  FASEB J. 2003;  17 1553-1555