Abstract
Physiological concentrations of leptin stimulate the activity of the endocannabinoid-degrading enzyme anandamide hydrolase (fatty acid amide hydrolase, FAAH) in human T lymphocytes up to approximately 300% over the untreated controls. Stimulation of FAAH occurred through up-regulation of gene expression at transcriptional and translational levels and involved binding of leptin to its receptor with an apparent dissociation constant (K(d)) of 1.95 +/- 0.14 nm and maximum binding (B(max)) of 392 +/- 8 fmol x mg protein(-1). Leptin binding to the receptor triggered activation of STAT3 but not STAT1 or STAT5 or the mitogen-activated protein kinases p38, p42, and p44. Peripheral lymphocytes of leptin knock-out (ob/ob) mice showed decreased FAAH activity and expression (approximately 25% of the wild-type littermates), which were reversed to control levels by exogenous leptin. Analysis of the FAAH promoter showed a cAMP-response element-like site, which is a transcriptional target of STAT3. Consistently, mutation of this site prevented FAAH activation by leptin in transient expression assays. Electrophoretic mobility shift and supershift assays further corroborated the promoter activity data. Taken together, these results suggest that leptin, by up-regulating the FAAH promoter through STAT3, enhances FAAH expression, thus tuning the immunomodulatory effects of anandamide. These findings might also have critical implications for human fertility.
Highlights
Leptin (L)1 is the 16-kDa non-glycosylated product of the obese gene, which is secreted by adipose cells, is released into the circulation, and transported across the blood-brain barrier into the central nervous system where it regulates energy homeostasis (1)
1 The abbreviations used are: L, leptin; LR, leptin receptor; AEA, anandamide; AMT, AEA membrane transporter; Chloramphenicol Acetyltransferase (CAT), chloramphenicol acetyltransferase; CBR, cannabinoid receptors; CP55.940, 5-(1,1Ј-dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; cAMP responsive element (CRE), cAMP-response element; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FAAH, fatty acid amide hydrolase; GAR/M-AP, goat antirabbit/mouse antibodies conjugated with alkaline phosphatase; IGF-IR, insulin-like growth factor I receptor; sLR, soluble leptin receptor; MAPK, mitogen-activated protein kinase; rabbit anti-goat antibodies conjugated to alkaline phosphatase (RAG-AP), rabbit anti-goat antibodies conjugated with alkaline phosphatase; Reverse transcriptase (RT), reverse transcriptase; STAT, signal transducer and activator of transcription
The same anti-FAAH antibodies were used to further quantify FAAH content by ELISA, which showed that L increased FAAH protein in human T lymphocytes in parallel to the increase of enzymic activity (Fig. 1, A and B)
Summary
L, leptin; LR, leptin receptor; AEA, anandamide (arachidonoylethanolamide); AMT, AEA membrane transporter; CAT, chloramphenicol acetyltransferase; CBR, cannabinoid receptors; CP55.940, 5-(1,1Ј-dimethylheptyl)-2-[1R,5R-hydroxy-2R-(3-hydroxypropyl) cyclohexyl]-phenol; CRE, cAMP-response element; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; FAAH, fatty acid amide hydrolase; GAR/M-AP, goat antirabbit/mouse antibodies conjugated with alkaline phosphatase; IGF-IR, insulin-like growth factor I receptor; sLR, soluble leptin receptor; MAPK, mitogen-activated protein kinase; RAG-AP, rabbit anti-goat antibodies conjugated with alkaline phosphatase; RT, reverse transcriptase; STAT, signal transducer and activator of transcription. From those related to food intake and energy expenditure in mammals, including regulation of fertility (2) and modulation of immune response (3) These two actions might be interconnected in humans because leptin alters the production from T lymphocytes of T helper 1 and 2 cytokines (4), which are critical in regulating embryo implantation and materno-fetal exchanges (5, 6). Lymphocyte FAAH has been shown to control the levels of blood AEA in pregnant women, where low FAAH activity implies high AEA levels, leading to spontaneous abortion (21–23) Taken together, these data have suggested a cross-talk between steroid hormones, cytokines, and the peripheral endocannabinoid system in lymphocytes, which is implicated in regulating immunity and fertility in humans (24). Expression by leptin, triggered through binding to LR and subsequent STAT3-dependent up-regulation of promoter activity
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