Leptin mediates its effects by the binding with the its specific LepR, a member of the class I cytokine receptor family (which includes receptors for IL-6, IL-12, OSM, and prolactin) and the pleiotropic biological effects of leptin can be partly explained by the wide distribution of LepRs on different types of cells, including those in extraneural tissues. Alternative splicing of LepR results in six receptor isoforms with different length of cytoplasmic domains, known as LepRa, LepRb, LepRc, LepRd, LepRe, and LepRf (49). Among all the LepR isoforms, only full-length isoform (LepRb) is able to fully transduce activation signals into the cell, as its cytoplasmic region contains several motifs required for signal transduction. The other LepR isoforms lack some or all of these motifs and their function is still unclear, even though several data suggest that they could be involved in the transport of leptin across the blood-brain barrier or in its degradation. Intracellularly, the LepR does not have an intrinsic tyrosine kinase domain, therefore it binds cytoplasmic kinases – mainly Janus tyrosine kinase 2 (Jak2) (50). LepR contains a highly conserved, proline-rich box 1 (51) and two putative, less conserved, box2 motifs (52, 53). Box 1 and box 2 motifs are considered important in recruiting and binding Jaks (54, 55) for full Jak activation (56). Recent studies indicate that, under physiological conditions, only Jak2 is activated during LepR signaling (53). Once activated, Jaks proteins trans-phosphorylate each other, as well as other tyrosine residues (Tyr985, Tyr1138, and Tyr 1077) of the LepR (57, 58), providing docking sites for downstream molecules such as signal transducer and activation of transcriptions (STATs). These proteins dissociate from the receptor and form homo- or hetero-dimers, which translocate into the nucleus and act as transcription factors by binding specific response elements in the promoter region of their target genes, such as sis-inducible-element (SIE), acute-phase-response-element (APRE), and GAS-like elements (59, 60) (Figure 1).

In response to leptin, STAT3 binds to phospho-Tyr1138, allowing Jak2 to phosphorylate and activate STAT3. Confirming the importance of this site of phosphorylation, mutation of Tyr1138 abolishes the ability of leptin to activate STAT3, resulting in severe hyperphagia and morbid obesity (61–63). Leptin stimulates also phosphorylation of LepRb on Tyr1077, which binds to STAT5 and subsequently mediates STAT5 phosphorylation (64, 65).

The Jak/STAT pathway is under the negative-feedback control of suppressors of cytokine signaling (SOCS) proteins, which are induced upon cytokine stimulation and act as negative regulators of signaling by binding to phosphorylated Jak proteins or by direct interaction with tyrosine-phosphorylated receptors (66, 67). Structurally, SOCS proteins are characterized by a central SH-2 domain, an N-terminal preSH-2 domain, in some cases a kinase inhibitory region (KIR) domain, which abolishes the kinase activity of the Jaks, and a more conserved C-terminal SOCS-box – which represents a key mediator of proteasomal degradation (by linking ubiquitin to the substrate) (66). Only SOCS1 and 3 carry a KIR domain in their N-terminal region and it is involved in the inhibition of the Jak activity and thus leptin signaling. Recent data showed that SOCS3 inhibits kinase activity through its KIR domain after the binding through its SH-2 domain with phosphotyrosine motifs in the receptor in the proximity of the Jaks. Interestingly, leptin can induce SOCS3 expression (68–71) and the Tyr985 of LepRb is a high-affinity binding site for SOCS3 (57, 70). In this context, the participation of SOCS3 in the negative-feedback mechanism of leptin signaling has been proposed to underlie the development of leptin resistance in relation to the hyperleptinemia observed during obesity (69).

Another negative regulator of leptin signaling is represented by the SH-2 domain-containing phosphatase-2 (SHP-2), which is a constitutively expressed tyrosine phosphatase involved in the dephosphorylation of Jak2 (72). SHP-2 carries two tandem SH-2 domains followed by a tyrosine phosphatase catalytic domain. When one SH-2 domain interacts with a tyrosine-phosphorylated ligand, a conformational change occurs and brings this phosphatase to activation of LepR at position Y985 (73). This specific site has an important role in leptin-induced extracellular signal-regulated kinases (ERK) activation (57). More specifically, as a result of leptin administration, Tyr985 becomes phosphorylated by recruited Jaks (mainly Jak2 and Jak1), and provides a docking site for SHP-2. After binding to that specific tyrosine residue, SHP-2 is phosphorylated at the C-terminus and together with its adapter molecule Grb2, it activates downstream signaling, leading to the activation of the p21Ras/ERK signaling cascade (57), with the final induction of specific target genes expression, such as c-fos or egr-1, a zinc-finger transcription factor that influences the initiation of growth and differentiation (74) (Figure 1).

Leptin can activate also another member of the MAP kinase family, p38 MAPK (75) and stress-activated protein kinase c-Jun N-terminal kinase (JNK). Among the possible downstream targets of leptin-induced activation of p38 and JNK MAPK pathways, the regulation of the transcription factor nuclear factor-κB (NF-κB) appears to be crucial for the transcriptional regulation of pro-inflammatory cytokines such as TNFα and IL-1β.

In addition, leptin is able to regulate phosphoinositide 3-kinase (PI3K) activity, indeed the binding of PI3K regulatory subunit to tyrosine-phosphorylated proteins induces a conformational change allowing the activation of its catalytic subunit and consequent full activation of PI3K, whose products typically stimulate protein kinases such as Akt, also called protein kinase B (PKB), protein kinase C (PKC) (76), and Forkhead box O1 (FOXO1), a transcriptional factor that is phosphorylated and inactivated by Akt (77–80). Leptin inhibits both the activity and expression of hypothalamic FOXO1 through the PI 3-kinase pathway (77). Indeed, overexpression of a constitutively active FOXO1 mutant decreases leptin sensitivity in mice with consequent increase in food intake and body weight, whereas small interfering RNA-mediated knockdown of FOXO1 increases leptin sensitivity and decreases food intake and body weight (77, 78).

Finally, leptin stimulates phosphorylation of ribosomal S6 kinase (S6K), a major physiological substrate of the mammalian target of rapamycin (mTOR) kinase in the hypothalamus. Indeed, rapamycin, a specific inhibitor of mTOR attenuates leptin’s anorexigenic effects (81), conversely, activation of S6K enhances leptin sensitivity (82) (Figure 1). mTOR binds to raptor and GβL to form the mTOR complex 1 (mTORC1), which directly phosphorylates and activates S6K (83). mTORC1 is inhibited by the TSC1/TSC2 complex (84–86). Akt phosphorylates TSC2 and inactivates the TSC1/TSC2 complex (85). Therefore, the mTOR/S6K pathway is likely to be a downstream target of the PI 3-kinase/Akt pathway in leptin-stimulated neurons.

Leptin stimulates and promotes the proliferation of human peripheral blood mononuclear cells (PBMC) (40, 48), as the presence of LepR on monocytes and lymphocytes has been shown in mice (46, 87) and confirmed in human peripheral blood T-lymphocytes (both CD4 and CD8) (88).

In PBMCs, leptin stimulation induces tyrosine phosphorylation and translocation of STAT3 molecules to the nucleus (89–91) and the phosphorylation of the STAT3-associated RNA binding protein Sam68 (a tyrosine-phosphorylated adaptor protein in TCR activation, which is associated with the SH2 and SH3 domains of Src and other signaling molecules, such as Grb2, PLC-γ-1, and PI3K) (92–95).

Recent evidence has shown that leptin induces tyrosine phosphorylation of Sam68 and Insulin receptor substrate 1 (IRS-1), which associate with p85 (96, 97), the regulatory subunit of PI3K via the SH-2 domain, recruiting and leading to stimulation of PI3K activity (98). In this context, leptin has been shown to inhibit apoptosis of thymocytes through an IRS-1/PI3K-dependent pathway since this effect was inhibited by the PI3K inhibitor LY294002 (99). Moreover, Martín-Romero et al. have shown that both ERK-1 and ERK-2 were found phosphorylated in a dose-dependent fashion in PBMC after incubation with human leptin (98).

It was also found that leptin could induce sustained phosphorylation of p38 MAPK in human PBMCs and the phosphorylation of the ribosomal protein S6 – the only protein in the large 40S subunit that has been shown to be phosphorylated in response to growth factors and mitogens (100). One route of leptin-induced S6 phosphorylation in human PBMCs is via MEK and p42/p44 MAPK (101–103), which activate MAPK-dependent S6 Kinase p90 RSK and S6. The other way seems to be mediated via activation of p70 S6 kinase, since it has been shown that leptin phosphorylates p70 S6 kinase at Thr389 (104). Accordingly, pre-treatment of cells with rapamycin abolished this phosphorylation (104). Strikingly, the MEK inhibitor PD98059 has been shown to inhibit not only p90 RSK phosphorylation, as expected, but also p70 S6 Kinase and S6 phosphorylation, thus suggesting an essential role of MEK activation in a full induction of p70 S6 kinase activity in human PBMC (105, 106).

In CD4⁺CD25⁻ effector T cells (Teff), De Rosa et al. have shown that leptin-induced strong STAT3 phosphorylation, while stimulation of CD4⁺CD25⁺ Treg cells was not associated with a marked increase of phosphorylated STAT3 (107). SOCS3, a negative regulator of cytokine signaling, was activated by leptin blockade in Treg cells, in which the stimulation with anti-CD3/28 induced phosphorylation of ERK1/2 and subsequent cell proliferation (107). In the same subset of cells, the cyclin-dependent kinase inhibitor p27 (p27kip1, a molecule involved in the control of cell cycle and T cell anergy) was elevated before and after anti-CD3/28 stimulation, and leptin neutralization induced degradation of this molecule, partly explaining the reversal of the anergic state and proliferation of these cells.

Recently, the contribution of leptin to mTOR activation in human Teffs has been well defined. Indeed, it has been shown that leptin treatment had little effect on mTOR phosphorylation, but it induced a significant increase in p70S6K and S6 phosphorylation, concomitant with a consistent increase in AKT phosphorylation. The induction of mTOR, as well as AKT phosphorylation induced by TCR engagement, was significantly reduced by leptin blockade and this inhibition was partially reversed by the addition of recombinant leptin to cultures, thus suggesting suggest a link between autocrine secretion of leptin and mTOR activation in Teffs through an AKT-dependent mechanism (108). A recent study by Galgani et al. shows that nutritional status, through leptin, directly affects survival and proliferation of autoreactive T cells, modulating the activity of the survival protein Bcl-2, the Th1/Th17 cytokines, and the nutrient/energy-sensing AKT-mTOR pathway (109). Moreover, a paper by the same group has shown that leptin activates the mTOR pathway to control also Treg cells responsiveness (110, 111). More specifically leptin inhibited rapamycin-induced proliferation of Tregs, by increasing activation of the mTOR pathway. In addition, under normal conditions, Tregs secreted leptin, which activated mTOR in an autocrine manner to maintain their state of hyporesponsiveness. Finally, Tregs from db/db mice exhibited a decreased mTOR activity and increased proliferation compared with that of wild-type cells (110, 111). Together, these data suggest that the leptin-mTOR axis sets the threshold for the responsiveness of Tregs and that this pathway might integrate cellular energy status with metabolic-related signaling in Treg cells that use this information to control immune tolerance.

Little is know about the effect of adiponectin on T cell function. Several data suggest that adiponectin is a negative regulator of T cell activity. In particular, although a small percentage of T cells express ADIPOR on their surface, a great amount of T cells store ADIPORs within clathrin-coated vesicles and these receptors colocalized with Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) molecules. After stimulation of T cells, the expression of both ADIPORs and CTLA-4 has been shown to be upregulated. Interestingly, it has observed that the addition of adiponectin results in a significant decrease of antigen-specific T cell proliferation and cytokines production, through the enhancement of T cells apoptosis. Confirming these findings in vivo, adiponectin-deficient mice had higher frequencies of CD137⁺ T cells upon Coxsackie B virus infection, thus suggesting that adiponectin is a novel negative T-cell regulator (136).

Adiponectin has been shown to inhibit allograft rejection in murine cardiac transplantation, indeed Okamoto et al. have shown that allografts transplanted to APN^−/− mice showed severe acute rejection to transplants in APN^+/+ hosts accompanied by increased accumulation of CD4⁺ and CD8⁺ T cells and macrophages (137). A recent paper by Tsang et al. suggests that the immunomodulatory effect of adiponectin on immune response could be at least in part mediated by its ability to alter dendritic cell functions (138). Indeed, adiponectin-treated dendritic cells show a lower production of IL-12p40 and a lower expression of CD80, CD86, and histocompatibility complex class II (MHCII). Moreover, in co-culture experiments of T cells and adiponectin-treated dendritic cells, a reduction in T cells proliferation and IL-2 production and an higher percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells was observed (138) suggesting that adiponectin could also control regulatory T cell homeostasis. Moreover adiponectin inhibits the production of CXC receptor 3 chemokine ligands in macrophages and consequently reduces T-lymphocyte recruitment and accumulation during atherogenesis (139).

On the contrary, Cheng et al. have recently shown that addition of adiponectin to polyclonally activated CD4⁺ T cells induced secretion of IFN-γ and IL-6, increased phosphorylation of p38 MAPK and STAT4 and augmented T-bet expression, indicating that adiponectin enhances Th1 differentiation (140). In the same direction, the paper by Jung et al. has shown that adiponectin-induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, co-stimulatory molecules in both mouse and human DCs, and it significantly enhanced production of pro-inflammatory cytokines. moreover, adiponectin-treated DCs significantly induced both Th1 and Th17 responses in allogeneic T cells, leading to enhanced pro-inflammatory responses (141).

A small number of studies have been performed to address the role of resistin in T cell functions, but recent evidence has showed that resistin strongly up-regulates the expression of TNF and IL-6 by human PBMCs and induces arthritis after injection into the joints of healthy mice (152). These pro-inflammatory properties of resistin were abrogated by an NF-κB inhibitor, thus showing the key role of NF-κB in resistin-induced modulation of inflammatory reactions. Moreover Son et al. have recently shown that resistin induces expansion of functional Tregs, as testified by increased protein and mRNA expression of FoxP3, only when CD4⁺ T cells are co-cultured with DCs (153).