Commercially available LA supplements are being used as alternative and/or complementary therapeutics for diseases such as Alzheimer’s disease, diabetic polyneuropathy and atherosclerosis. The therapeutic benefit of LA is assumed to be related to its antioxidant properties, when in fact, little is known about the biochemical and cellular mechanisms that mediate the effects of LA in vivo
. Toward this end, we first reported the novel finding that LA stimulates the production of cAMP [7
], a signaling molecule with potent anti-inflammatory properties. In this study, we expand our initial findings. We discovered that only LA, not its reduced form, DHLA or the derivatives DMLA or LPM, are able to stimulate cAMP production in NK cells (). This is consistent with our data in T cells suggesting that this is not a cell type specific event [8
]. The inability of DHLA to stimulate cAMP supports the hypothesis that stimulation of cAMP production is occurring independent of the conversion of LA to DHLA. Furthermore, this notion is strengthen by data obtained from DMLA, whereby the molecule cannot be reduced due to the presence of two methyl groups attached to the sulfur molecule. Taken together with data showing no elevation in cAMP levels after LPM treatment, the results indicate that the closed disulfide pentane ring and carboxy group are necessary for LA to stimulate cAMP production. Given the importance of cAMP as a second messenger involved in the regulation of a large number of genes, we believe the ability of LA to stimulate cAMP may be critical to its therapeutic effects.
We also discovered that there is no significant difference between the R and S isomers of LA or between R-LA and racemic LA in increasing cAMP levels (). There is no consensus on whether or not R-LA is biologically more active than S-LA. A study on the stereoselectivity and specificity of LA for the pyruvate dehydrogenase complex and its component enzymes demonstrated more selectivity for R-LA than S-LA, with R-LA reacting 24 times faster in binding reactions while S-LA exhibited inhibitory effects on R-LA, reducing its biological activity [48
]. Similarly, treatment of adipocytes in culture showed higher glucose uptake with R-LA than either S-LA or the racemic mixture [49
]. R-LA was more effective at enhancement of aortic flow in rat heart during hypoxia than S-LA [37
]. Smith et al
. reported that racemic and S-LA were less effective than R-LA in protecting against tertiary butyl hydroperoxide damaged C6 glioma cells [50
]. However, the authors reported that all forms of LA protected against hydrogen peroxide toxicity. Investigating lipid peroxidation in both nerve and brain, Nickander et al. found that R and S-LA both reduced peroxidation, and that there was no difference between the two enantiomers [51
]. Both R and S-LA protected brain tissue against ischemic damage with similar potency [39
]. Other investigators have also reported similar potencies between R and S-LA [52
]. As discussed here, the evidence for different or similar functional behavior for R and S-LA are equally compelling. It is unclear what the reason is for these differences, but studies by Smith et al
. suggest that whether or not R and S-LA behave in a similar fashion may be dependent on the cell type and/or treatment variables under investigation [50
The generation of cAMP is historically believed to be due to activation of adenylyl cyclases by ligand-receptor binding of GPCRs, subsequent dissociation of G proteins and activation of tmACs. Our previous data indicate that LA activates tmACs after binding to the prostanoid EP2/EP4 receptors [7
]. Here, we determined that LA is a weak competitor of 3
for binding of these receptors in transfected HEK 293 EBNA cells (). Although LA and PGE2
are both hydrophobic and can exist in multiple conformers, which likely allow them to bind the same receptors, they are structurally very different. These differences may explain the different binding affinities observed. As discussed by Gether et al
., the inactive conformation of GPCRs are stabilized by constraining intramolecular interactions that have been evolutionarily conserved to maintain the receptor preferentially in an inactive conformation in the absence of agonists [54
]. Receptor activation requires disruption of these intramolecular interactions by ligands. Ligand-receptor binding occurs by intermolecular forces, such as ionic bonds, hydrogen bonds and Van der Waals forces, which lead to conformational changes in the tertiary structure of the receptor, allowing the receptor to more readily convert from the inactive to active state. The type of interaction is determined by the specific amino acid residues involved in binding [55
has two hydroxyl groups in addition to the carboxylic tail. LA, on the other hand, only has a carboxylic tail. Thus, PGE2
is able to form more interactions with the residues located inside the binding pockets of the EP2/EP4 receptors than LA, which may contribute to more efficient disruption of the native intramolecular interactions.
While LA binds with lower affinity to the EP receptors, LA routinely induces greater cAMP production than PGE2
. We also determined that LA and PGE2
have synergistic effects on cAMP production in PBMCs and NK cells. These data suggest two possibilities: 1) LA and PGE2
in combination exhibit cooperative binding behaviors to enhance cAMP production or 2) LA stimulates other pathways in addition to activation of the EP receptors. We will first address the former possibility. Cooperative interactions could occur through binding of identical sites or between multiple independent sites. Binding studies and information obtained from the crystallized structures of rhodopsin, β-adrenergic and adenosine receptors indicate that GPCRs contain a single binding pocket for endogenous ligands [54
]. However, these studies do not exclude the possibility that other allosteric sites exist on these receptors. While our binding data did not fit to a two binding site competition model, further studies would be necessary to confirm if LA is allosterically binding to a site unique from the PGE2
binding site on the EP receptors. Alternatively, LA may be stimulating additional pathways. The synergistic effects of LA and PGE2
are likely attributable to LA activating other pathways in addition to the EP receptors. The following sections will discuss the evidence to support this theory.
Recent identification of a second pool of ACs (sACs) presented a possible explanation for the effects of LA on cAMP production in vivo
. By blocking sAC activation using the specific inhibitor KH7, we discovered that sACs mediate LA stimulation of cAMP production (). This is the first data showing that sAC mediates cAMP production in PBMCs and NK cells (data not shown) and that sAC is activated by LA. Our data is consistent with reports by Stessin et al.
showing reduction in cAMP synthesis in PC12 cells treated with nerve growth factor [25
]. Similar results were found in human neutrophils treated with ionomycin [60
]. sAC was initially characterized in mammalian sperm, but is now known to be expressed in a variety of tissues including brain, liver, heart, spleen, epithelial cells and neutrophils [21
]. In addition to regulating sperm function, sAC is implicated in mediating neuronal differentiation and fast migration [25
], tumor necrosis factor (TNF) signal transduction to inhibit TNF induced hydrogen peroxide release [60
], and regulation of ciliary beat frequency in the airway [62
]. sAC is localized at multiple, subcellular compartments throughout the cell [22
] suggesting that there may be independently modulated cAMP signaling microdomains, which contribute to the specificity of cAMP signaling [63
]. It will be interesting to see if the cAMP signaling microdomains determine the specificity of cellular responses to LA treatment.
We next identified other GPCRs that may be activated by LA. We narrowed our research by focusing on some of the most studied receptors, including histamine, adenosine and β-adrenergic receptors, specifically the subtypes that are coupled to stimulatory G-proteins and cAMP signaling. Using pharmacological inhibitors to prevent receptor binding, we discovered that LA activates histamine and adenosine, but not β-adrenergic receptors (). The histamine receptors play important roles in regulating inflammation. Activation of H2 receptors on peripheral monocytes potently suppresses interleukin (IL)-12 and stimulates IL-10 production [64
], which may shift from Th1 to Th2 immune response. In autoimmune disorders such as arthritis and multiple sclerosis (MS), activation of Th1 is thought to be pathogenic while Th2 response is protective. This may explain why LA treatment is effective in the animal model of arthritis and MS. However, the shift to a Th2 response is implicated in promoting allergic reactions and tumorigenesis [64
]. Whether LA promotes a shift to Th2 immune response, and whether or not this has detrimental consequences remain to be elucidated.
Activation of the adenosine receptors have also been shown to be important in inflammation. Takahashi et al.
showed that treatment with adenosine resulted in inhibition of IL-18 induced intercellular adhesion molecule (ICAM)-1 expression in monocytes and production of the proinflammatory cytokines IL-12, TNF-α and interferon (IFN)-γ by PBMC [67
]. Adenosine has also been shown to inhibit cytotoxic activity and cytokine production in NK cells [68
]. Similarly, LA treatment has been shown to reduce ICAM-1 and vascular cell adhesion molecule (VCAM)-1 expression in spinal cords and stimulated murine brain endothelial cells [4
], inhibit VCAM-1 and endothelial adhesion of human monocytes [5
], down modulate CD4 expression from human T cells [6
] and inhibit IFN-γ production and cytotoxic activity in human NK cells [7
]. Our data suggest that activation of the adenosine receptors may mediate the anti-inflammatory effects of LA. It will be exciting to test this hypothesis in the future.
In summary, we provide novel evidence that LA, not its derivatives, is responsible for stimulating cAMP production, weakly competes for EP2/EP4 binding, and activates sAC, histamine and adenosine receptors. These data indicate that LA utilizes many mechanisms to generate cAMP. Activation of the histamine receptor suggests that LA supplementation may not be completely without long term side effects and that some cautionary approaches may be necessary to prevent potential problems such as the development of allergies and tumors. These data provide a foundation for future studies to determine the mechanisms by which LA can be beneficial in human health and identify any potential long term issues.