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.     

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.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

Human leptin activates PI3K and MAPK pathways in human peripheral blood mononuclear cells: possible role of Sam68.

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. Thus, we have recently found that human leptin promotes stimulation and proliferation of human peripheral blood mononuclear cells. In the present work, we sought to study the mechanisms underlying these effects. First, we have assessed the presence of the long isoform of the human leptin receptor by RT-PCR. Next, we have studied tyrosine phosphorylation of cell proteins in response to leptin stimulation. We have found that leptin receptor, IRS-1 and the RNA-binding protein Sam68 are tyrosine phosphorylated upon leptin challenge in a dose-dependent manner. Moreover, tyrosine phosphorylation of IRS-1 and Sam68 promotes their association with p85, the regulatory subunit of PI3K, and this association leads to the stimulation of PI3K activity. On the other hand, the leptin-stimulated tyrosine phosphorylation of Sam68 mediates the dissociation from RNA as assessed by Sepharose-conjugated poly(U) binding. Finally, leptin receptor activation also triggers MAPK signaling pathway. Thus, leptin dose-dependently stimulates tyrosine and threonine phosphorylation of MAPK in mononuclear cells. In summary, the present work demonstrates the presence of the long isoform of the human leptin receptor in peripheral blood mononuclear cells and the activation of two signaling pathways, PI3K and MAPK. The effects on Sam68 phosphorylation may modulate its binding to RNA, although the physiological implications remain to be studied. These signal transduction pathways may mediate the described effects of human leptin on human peripheral blood mononuclear cells.     

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.     

Leptin potentiates antiproliferative action of cAMP elevation via protein kinase A down‐regulation in breast cancer cells

Previously, we have shown that leptin potentiates the antiproliferative action of cAMP elevating agents in breast cancer cells and that the protein kinase A (PKA) inhibitor KT‐5720 prevented the antiproliferative effects induced by the leptin plus cAMP elevation. The present experiments were designed to gain a better understanding about the PKA role in the antitumor interaction between leptin and cAMP elevating agents and on the underlying signaling pathways. Here we show that exposure of MDA‐MB‐231 breast cancer cells to leptin resulted in a strong phosphorylation of both ERK1/2 and STAT3. Interestingly, intracellular cAMP elevation upon forskolin pretreatment completely abrogated both ERK1/2 and STAT3 phosphorylation in response to leptin and was accompanied by a consistent CREB phosphorylation. Notably, leptin plus forskolin cotreatments resulted in a strong decrease of both PKA regulatory RIα and catalytic subunits protein levels. Importantly, pretreatment with the PKA inhibitor KT‐5720 blocked the forskolin‐induced CREB phosphorylation and prevented both the inhibition by forskolin of leptin‐induced ERK1/2 and STAT3 phosphorylation and the PKA subunits down‐regulation induced by the combination of leptin and forskolin. Altogether, our results indicate that leptin‐dependent signaling pathways are influenced by cAMP elevation and identify PKA as relevantly involved in the pharmacological antitumor interaction between leptin and cAMP elevating drugs in MDA‐MB‐231 cells. We propose a molecular model by which PKA confers its effects. Potential therapeutic applications by our data will be discussed. J. Cell. Physiol. 225: 801–809, 2010. © 2010 Wiley‐Liss, Inc.     

Selective insulin and leptin resistance in metabolic disorders

Obesity represents a major risk factor for the development of insulin and leptin resistance, ultimately leading to a pleiotropic spectrum of metabolic alterations. However, resistance to both hormones does not uniformly affect all target cells and intracellular signaling pathways. In contrast, numerous clinical phenotypes arise from selective hormone resistance, leading to inhibition of defined intracellular signaling pathways in some tissues, while in other cell types hormone action is maintained or even overactivated. Here, we review the molecular mechanisms and clinical outcomes resulting from selective insulin and leptin resistance, which should ultimately guide future strategies for the treatment of obesity-associated diseases.     

Glycoxydation promotes vascular damage via MAPK-ERK/JNK pathways

Oxidation and glycation enhance foam cell formation via MAPK/JNK in euglycemic and diabetic subjects. Here, we investigated the effects of glycated and oxidized LDL (glc-oxLDL) on MAPK-ERK and JNK signaling pathways using human coronary smooth muscle cells. Glc-oxLDL induced a broad cascade of MAPK/JNK-dependent signaling transduction pathways and the AP-1 complex. In glc-oxLDL treated coronary arterioles, tumor necrosis factor (TNF) α increased JNK phosphorylation, whereas protein kinase inhibitor dimethylaminopurine (DMAP) prevented the TNF-induced increase in JNK phosphorylation. The role of MKK4 and JNK were then investigated in vivo, using apolipoprotein E knockout (ApoE(-/-)) mice. Peritoneal macrophages, isolated from spontaneously hyperlipidemic but euglycemic mice showed increases in both proteins and phosphorylated proteins. Compared to streptozotocin-treated diabetic C57BL6 and nondiabetic C57BL6 Wt mice, in streptozotocin-diabetic ApoE(-/-) mice, the increment of foam cell formation corresponded to an increment of phosphorylation of JNK1, JNK2, and MMK4. Thus, we provide a first line of evidence that MAPK-ERK/JNK pathways are involved in vascular damage induced by glycoxidation.     

A unique autophosphorylation site on Tie2/Tek mediates Dok-R phosphotyrosine binding domain binding and function

Tie2/Tek is an endothelial cell receptor tyrosine kinase that induces signal transduction pathways involved in cell migration upon angiopoietin-1 (Ang1) stimulation. To address the importance of the various tyrosine residues of Tie2 in signal transduction, we generated a series of Tie2 mutants and examined their signaling properties. Using this approach in conjunction with a phosphorylation state-specific antibody, we identified tyrosine residue 1106 on Tie2 as an Ang1-dependent autophosphorylation site that mediates binding and phosphorylation of the downstream-of-kinase-related (Dok-R) docking protein. This tyrosine residue is contained within a unique interaction motif for the phosphotyrosine binding domain of Dok-R, and the pleckstrin homology domain of Dok-R further contributes to Tie2 binding in a phosphatidylinositol 3'-kinase-dependent manner. Introduction of a Tie2 mutant lacking tyrosine residue 1106 into endothelial cells interferes with Dok-R phosphorylation in response to Ang1. Furthermore, this mutant is unable to restore the migration potential of endothelial cells derived from mice lacking Tie2. Together, these findings demonstrate that tyrosine residue 1106 on Tie2 is critical for coupling downstream cell migration signal transduction pathways with Ang1 stimulation in endothelial cells.     

leptin-induced growth stimulation of breast cancer cells involves recruitment of histone acetyltransferases and mediator complex to CYCLIN D1 promoter via activation of Stat3

Numerous epidemiological studies documented that obesity is a risk factor for breast cancer development in postmenopausal women. Leptin, the key player in the regulation of energy balance and body weight control also acts as a growth factor on certain organs in both normal and disease state. In this study, we analyzed the role of leptin and the molecular mechanism(s) underlying its action in breast cancer cells that express both short and long isoforms of leptin receptor. Leptin increased MCF7 cell population in the S-phase of the cell cycle along with a robust increase in CYCLIN D1 expression. Also, leptin induced Stat3-phosphorylation-dependent proliferation of MCF7 cells as blocking Stat3 phosphorylation with a specific inhibitor, AG490, abolished leptin-induced proliferation. Using deletion constructs of CYCLIN D1 promoter and chromatin immunoprecipitation assay, we show that leptin induced increase in CYCLIN D1 promoter activity is mediated through binding of activated Stat3 at the Stat binding sites and changes in histone acetylation and methylation. We also show specific involvement of coactivator molecules, histone acetyltransferase SRC1, and mediator complex in leptin-mediated regulation of CYCLIN D1 promoter. Importantly, silencing of SRC1 and Med1 abolished the leptin induced increase in CYCLIN D1 expression and MCF7 cell proliferation. Intriguingly, recruitment of both SRC1 and Med1 was dependent on phosphorylated Stat3 as AG490 treatment inhibited leptin-induced recruitment of these coactivators to CYCLIN D1 promoter. Our data suggest that CYCLIN D1 may be a target gene for leptin mediated growth stimulation of breast cancer cells and molecular mechanisms involve activated Stat3-mediated recruitment of distinct coactivator complexes.     

Integrins stimulate phosphorylation of neurofilament NF-M subunit KSP repeats through activation of extracellular regulated-kinases (Erk1/Erk2) in cultured motoneurons and transfected NIH 3T3 cells

Integrin-mediated interactions of cells with components of the extracellular matrix (ECM) regulate cell survival, cell proliferation, cell differentiation and cell migration through activation of multiple intracellular signal transduction pathways. In this study, we have demonstrated that integrin-matrix interactions promote KSP tail-domain phosphorylation of neurofilament medium molecular weight subunits (NF-M) in cultured rat spinal cord motoneurons and NF-M transfected NIH 3T3 cells. We found that laminin and fibronectin induce NF-M tail-domain phosphorylation in motoneurons and NIH 3T3 cells transfected with NF-M, respectively. This phosphorylation was selectively inhibited by PD98059, a specific MEK1 inhibitor. This suggests that laminin and fibronectin-induced MEK1 activation and the downstream targets Erk1 and Erk2 are involved in NF-M KSP tail-domain phosphorylation. This pathway appears to represent one of the mechanisms whereby integrin-extracellular matrix interactions are involved in phosphorylation of the NF-M KSP tail domain.