Leptin activates AMP-activated protein kinase in hepatic cells via a JAK2-dependent pathway

AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis within the individual cell. Recent reports have suggested that leptin, an adipocyte-secreted hormone, phosphorylates AMPK in skeletal muscle directly. However, little is known about the interaction between leptin signaling and AMPK activation. Here, we report that the leptin-induced phosphorylation of AMPK was detected in Huh7 cells expressing long form leptin receptor (OBRb) as well as short form leptin receptor (OBRa). In addition, we demonstrate that AMPK activation does not require the phosphorylation of either Tyr-985 or Tyr-1138 within the OBRb and may occur via a STAT3-independent signaling pathway. We also show that Huh7 cells expressing OBRb and SOCS3 (inhibitor of JAK2) resulted in a marked reduction of AMPK activation in response to leptin. These findings suggest that the activation of JAK2, but not STAT3, may play a critical role in leptin-induced AMPK activation in Huh7 cells.     

Chronic leptin treatment stimulates lipid oxidation in immortalized and primary mouse skeletal muscle cells

Leptin administration enhances lipid oxidation in skeletal muscle. Nevertheless, direct and chronic effect of leptin has not been well characterized. Here, we measured the effect of leptin on skeletal muscles and their signaling pathways using differentiated C₂C₁₂ myotubes and primary myotube cultures. Differentiated myotubes expressed both the short and long forms of leptin receptors. Leptin increased lipid oxidation in myotubes in a concentration- and time-dependent manner, with significant induction of lipid oxidation occurring after 6 h. Actinomycin D completely blocked leptin-induced lipid oxidation. Leptin significantly increased phosphorylation of JAK2 and STAT3 in myotubes, and leptin-induced lipid oxidation was abolished by treatment with a JAK2 inhibitor or STAT3 siRNA. We then used mouse myotubes to measure these effects under physiological conditions. Leptin increased lipid oxidation, which again was blocked by a JAK2 inhibitor and STAT3 siRNA. These results suggest that the JAK2/STAT3 signaling pathway may underlie the chronic effects of leptin on lipid oxidation in skeletal muscles.     

Leptin activates STAT and ERK2 pathways and induces gastric cancer cell proliferation

Although leptin is known to induce proliferative response in gastric cancer cells, the mechanism(s) underlying this action remains poorly understood. Here, we provide evidence that leptin-induced gastric cancer cell proliferation involves activation of STAT and ERK2 signaling pathways. Leptin-induced STAT3 phosphorylation is independent of ERK2 activation. Leptin increases SHP2 phosphorylation and enhances binding of Grb2 to SHP2. Inhibition of SHP2 expression with siRNA but not SHP2 phosphatase activity abolished leptin-induced ERK2 activation. While JAK inhibition with AG490 significantly reduced leptin-induced ERK2, STAT3 phosphorylation, and cell proliferation, SHP2 inhibition only partially reduced cancer cell proliferation. Immunostaining of gastric cancer tissues displayed local overexpression of leptin and its receptor indicating that leptin might be produced and act locally in a paracrine or autocrine manner. These findings indicate that leptin promotes cancer growth by activating multiple signaling pathways and therefore blocking its action at the receptor level could be a rational therapeutic strategy.     

Glucose enhances leptin signaling through modulation of AMPK activity

Leptin exerts its action by binding to and activating the long form of leptin receptors (LEPRb). LEPRb activates JAK2 that subsequently phosphorylates and activates STAT3. The JAK2/STAT3 pathway is required for leptin control of energy balance and body weight. Defects in leptin signaling lead to leptin resistance, a primary risk factor for obesity. Body weight is also regulated by nutrients, including glucose. Defects in glucose sensing also contribute to obesity. Here we report crosstalk between leptin and glucose. Glucose starvation blocked the ability of leptin to stimulate tyrosyl phosphorylation and activation of JAK2 and STAT3 in a variety of cell types. Glucose dose-dependently enhanced leptin signaling. In contrast, glucose did not enhance growth hormone-stimulated phosphorylation of JAK2 and STAT5. Glucose starvation or 2-deoxyglucose-induced inhibition of glycolysis activated AMPK and inhibited leptin signaling; pharmacological inhibition of AMPK restored the ability of leptin to stimulate STAT3 phosphorylation. Conversely, pharmacological activation of AMPK was sufficient to inhibit leptin signaling and to block the ability of glucose to enhance leptin signaling. These results suggest that glucose and/or its metabolites play a permissive role in leptin signaling, and that glucose enhances leptin sensitivity at least in part by attenuating the ability of AMPK to inhibit leptin signaling.     

Interaction between leptin and insulin signaling pathways differentially affects JAK-STAT and PI 3-kinase-mediated signaling in rat liver

Chronic leptin treatment markedly enhances the effect of insulin on hepatic glucose production unproportionally with respect to body weight loss and increased insulin sensitivity. In the present study the cross-talk between insulin and leptin was evaluated in rat liver. Upon stimulation of JAK2 tyrosine phosphorylation, leptin induced JAK2 co-immunoprecipitation with STAT3, STAT5b, IRS-1 and IRS-2. This phenomenon parallels the leptin-induced tyrosine phosphorylation of STAT3, STAT5b, IRS-1 and IRS-2. Acutely injected insulin stimulated a mild increase in tyrosine phosphorylation of JAK2, STAT3 and STAT5b. Leptin was less effective than insulin in stimulating IRS phosphorylation and their association with PI 3-kinase. Simultaneous treatment with both hormones yielded no change in maximal phosphorylation of STAT3, IRS-1, IRS-2 and Akt, but led to a marked increase in tyrosine phosphorylation of JAK2 and STAT5b when compared with isolated administration of insulin or leptin. This indicates that there is a positive cross-talk between insulin and leptin signaling pathways at the level of JAK2 and STAT5b in rat liver.     

Direct evidence for leptin-induced lipid oxidation independent of long-form leptin receptor

Leptin administration has been shown to enhance muscle lipid oxidation in relation to the energy expenditure. Both long-form (Ob-R_L) and short-form leptin receptors (Ob-R_S) are expressed in skeletal muscle, but the role of Ob-R_S is unclear. In the present study, the role of Ob-R_S in leptin-induced lipid oxidation in skeletal muscles was investigated using primary murine myotubes from m/m and db/db mice. Primary myotubes were treated with leptin (0.1, 1, 10, 100 nM) for 24 h. Lipid oxidation was determined by ¹⁴CO₂ production rate from [1-¹⁴C] palmitate. Leptin was found to increase lipid oxidation in a dose- and time-dependent manner in db/db myotubes as well as in m/m myotubes. Leptin significantly increased phosphorylation of JAK2 and STAT3 in both types of myotube. Leptin-induced lipid oxidation was abolished by STAT3 siRNA. To investigate the mechanism underlying leptin-induced lipid oxidation, the effects of pharmacological inhibitors were examined. JAK2 or p38 MAPK inhibitor suppressed leptin-induced lipid oxidation and decreased STAT3 phosphorylation in both types of myotube, respectively. Leptin significantly increased phosphorylation of p38 MAPK, and leptin-induced lipid oxidation was abolished by treatment with p38 MAPK siRNA in both types of myotube. These results suggest that leptin induces lipid oxidation in skeletal muscle through the JAK2/p38 MAPK/STAT3 signaling pathway via not only Ob-R_L but also Ob-R_S.     

Suppressor of cytokine signaling 7 inhibits prolactin, growth hormone, and leptin signaling by interacting with STAT5 or STAT3 and attenuating their nuclear translocation

We report here the role of one of the less studied members of the family of suppressors of cytokine signaling (SOCS), namely SOCS-7, in cytokine signaling. We demonstrate that SOCS-7 inhibits prolactin (PRL), growth hormone (GH), or leptin (LEP) signaling mediated through STAT3 and STAT5 in a dose-dependent manner. SOCS-7 also attenuated STAT3 and STAT5 signaling induced by overexpression of JH1, the catalytic subdomain of JAK2. Since SOCS-7 interacted with phosphorylated STAT3 or STAT5, we assumed that SOCS-7 acts at the level of STAT proteins. Indeed, we showed that SOCS-7 inhibits PRL- and leptin-induced STAT5 and STAT3 phosphorylation and prevented the nuclear translocation of activated STAT3. Taken together, our results indicate that SOCS-7 is a physiological dysregulator of PRL, leptin, and probably also GH signaling and that its mode of action is a novel variation of SOCS protein inhibition of cytokine-inducible STAT-mediated signal transduction.     

Leptin Induces Cyclin D1 Expression and Proliferation of Human Nucleus Pulposus Cells via JAK/STAT,PI3K/Akt and MEK/ERK Pathways

Increasing evidence suggests that obesity and aberrant proliferation of nucleus pulposus (NP) cells are associated with intervertebral disc degeneration. Leptin, a hormone with increased circulating level in obesity, has been shown to stimulate cell proliferation in a tissue-dependent manner. Nevertheless, the effect of leptin on the proliferation of human NP cells has not yet been demonstrated. Here, we show that leptin induced the proliferation of primary cultured human NP cells, which expressed the leptin receptors OBRa and OBRb. Induction of NP cell proliferation was confirmed by CCK8 assay and immunocytochemistry and Real-time PCR for PCNA and Ki-67. Mechanistically, leptin induced the phosphorylation of STAT3, Akt and ERK1/2 accompanied by the upregulation of cyclin D1. Pharmacological inhibition of JAK/STAT3, PI3K/Akt or MEK/ERK signaling by AG490, Wortmannin or U0126, respectively, reduced leptin-induced cyclin D1 expression and NP cell proliferation. These experiments also revealed an intricate crosstalk among these signaling pathways in mediating the action of leptin. Taken together, we show that leptin induces human NP cell cyclin D1 expression and proliferation via activation of JAK/STAT3, PI3K/Akt or MEK/ERK signaling. Our findings may provide a novel molecular mechanism that explains the association between obesity and intervertebral disc degeneration.     

Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.     

APPL1-Mediating Leptin Signaling Contributes to Proliferation and Migration of Cancer Cells

Leptin has been implicated in tumorigenesis and tumor progression, particularly in obese patients. As a multifunctional adaptor protein, APPL1 (containing pleckstrin homology domain, phosphotyrosine binding domain, and a leucine zipper motif 1) plays a critical role in regulating adiponectin and insulin signaling pathways. Currently, high APPL1 level has been suggested to be related to metastases and progression of some types of cancer. However, the intercourse between leptin signaling pathway and APPL1 remains poorly understood. Here, we show that the protein levels and phosphorylation statues of APPL1were highly expressed in tissues from human hepatocellular carcinoma and triple-positive breast cancer. Leptin stimulated APPL1 phosphorylation in a time-dependent manner in both human hepatocellular carcinoma HepG2 cell and breast cancer MCF-7 cell. Overexpression or suppression of APPL1 promoted or attenuated, respectively, leptin-induced phosphorylation of STAT3, ERK1/2, and Akt in the cancer cells, accompanied with enhanced or mitigated cell proliferation and migration. In addition, we identified that APPL1 directly bound to both leptin receptor and STAT3. This interaction was significantly enhanced by leptin stimulation. Our results suggested that APPL1 positively mediated leptin signaling and promoted leptin-induced proliferation and migration of cancer cells. This finding reveals a novel mechanism by which leptin promotes the motility and growth of cancer cells.     

Leptin induces IL-8 expression via leptin receptor, IRS-1, PI3K, Akt cascade and promotion of NF-kappaB/p300 binding in human synovial fibroblasts

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-8 production caused by leptin in both rheumatoid arthritis synovial fibroblasts (RASF) and osteoarthritis synovial fibroblasts (OASF). RASF and OASF expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-8 production. Leptin-mediated IL-8 production was attenuated by OBRl receptor antisense oligonucleotide, JAK2 inhibitor or STAT3 small interference RNA (siRNA). Transfection with insulin receptor substrate (IRS)-1 siRNA or dominant-negative mutant of p85 and Akt or pretreatment with phosphatidylinositol 3-kinase inhibitor (Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (PDTC) and NF-kappaB inhibitor peptide also inhibited the potentiating action of leptin. Stimulation of RASF with leptin activated IkappaB kinase alpha/beta (IKK alpha/beta), p65 phosphorylation at Ser(276), p65 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. Moreover, pretreatment with p300 inhibitor (curcumin) also blocked IL-8 expression. The binding of p65 to the NF-kappaB elements, as well as the recruitment of p300 and the enhancement of histone H3 acetylation on the IL-8 promoter was enhanced by leptin, which was inhibited by wortmannin, Akt inhibitor or IRS-1 siRNA. These results suggest that leptin increased IL-8 production in synovial fibroblast via the OBRl/JAK2/STAT3 pathway, as well as the activation of IRS1/PI3K/Akt/NF-kappaB-dependent pathway and the subsequent recruitment of p300.