Leptin-induced STAT3 Activation Research Articles

Synthetic adiponectin-receptor agonist, AdipoRon, induces glycolytic dependence in pancreatic cancer cells

Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.

Pharmacological Inhibition of c-Jun N-terminal Kinase Reduces Food Intake and Sensitizes Leptin\u2019s Anorectic Signaling Actions

The role for c-Jun N-terminal Kinase (JNK) in the control of feeding and energy balance is not well understood. Here, by use of novel and highly selective JNK inhibitors, we investigated the actions of JNK in the control of feeding and body weight homeostasis. In lean mice, intraperitoneal (i.p.) or intracerebroventricular (i.c.v.) administration of SR-3306, a brain-penetrant and selective pan-JNK (JNK1/2/3) inhibitor, reduced food intake and body weight. Moreover, i.p. and i.c.v. administrations of SR11935, a brain-penetrant and JNK2/3 isoform-selective inhibitor, exerted similar anorectic effects as SR3306, which suggests JNK2 or JNK3 mediates aspect of the anorectic effect by pan-JNK inhibition. Furthermore, daily i.p. injection of SR3306 (7 days) prevented the increases in food intake and weight gain in lean mice upon high-fat diet feeding, and this injection paradigm reduced high-fat intake and obesity in diet-induced obese (DIO) mice. In the DIO mice, JNK inhibition sensitized leptin’s anorectic effect, and enhanced leptin-induced STAT3 activation in the hypothalamus. The underlying mechanisms likely involve the downregulation of SOCS3 by JNK inhibition. Collectively, our data suggest that JNK activity promotes positive energy balance, and the therapeutic intervention inhibiting JNK activities represents a promising approach to ameliorate diet-induced obesity and leptin resistance.

Clusterin/ApoJ enhances central leptin signaling through Lrp2-mediated endocytosis.

Hypothalamic leptin signaling plays a central role in maintaining body weight homeostasis. Here, we show that clusterin/ApoJ, recently identified as an anorexigenic neuropeptide, is an important regulator in the hypothalamic leptin signaling pathway. Coadministration of clusterin potentiates the anorexigenic effect of leptin and boosts leptin-induced hypothalamic Stat3 activation. In cultured neurons, clusterin enhances receptor binding and subsequent endocytosis of leptin. These effects are mainly mediated through the LDL receptor-related protein-2 (Lrp2). Notably, inhibition of hypothalamic clusterin, Lrp2 or endocytosis abrogates anorexia and hypothalamic Stat3 activation caused by leptin. These findings propose a novel regulatory mechanism in central leptin signaling pathways.

Stat3 pathway correlates with the roles of leptin in mouse liver fibrosis and sterol regulatory element binding protein-1c expression of rat hepatic stellate cells

Leptin, the adipocyte-derived hormone, plays an unique role in promoting liver fibrosis. Hepatic stellate cell (HSC) activation is the key step in liver fibrogenesis and sterol regulatory element binding protein-1c (SREBP-1c, a pivotal transcription factor for adipocyte differentiation) exerts a critical function in inhibition of HSC activation. Stat3 pathway is the main pathway induced by leptin and its role in liver fibrogenesis is controversial. Our previous results demonstrated the inhibitory effect of leptin on SREBP-1c expression in HSCs. The present study aimed to explore the role of Stat3 pathway in leptin-induced liver fibrogenesis in mouse model, focusing on examining the effect of leptin-induced Stat3 pathway on SREBP-1c expression in HSCs in vitro and in vivo. Results suggested that Stat3 pathway mediated the promotional role of leptin in liver fibrosis in mouse and was involved in leptin inhibition of SREBP-1c expression in HSCs. Leptin-induced Stat3 activation was, at least partially, ERK pathway-dependent in cultured HSCs and was correlated positively with β-catenin activity and negatively with liver X receptor α expression and activity which influenced SREBP-1c expression in HSCs. The decrease in SREBP-1c expression by leptin-induced Stat3 pathway led to the increase in the marker for HSC activation and in α1(I) collagen expression in HSCs. In summary, the effect of leptin-induced Stat3 pathway on SREBP-1c expression in HSCs might contribute to the role of leptin in liver fibrosis in mouse, thus advancing understanding of the mechanisms of liver fibrogenesis associated with leptin.

Lentiviral vector-mediated knockdown of SOCS3 in the hypothalamus protects against the development of diet-induced obesity in rats

Leptin resistance is a feature of most cases of obesity in both humans and rodents. The suppressor of cytokine signalling 3 (SOCS3) is a negative-feedback regulator of leptin signalling involved in leptin resistance; therefore, the suppression of SOCS3 is a potential therapy for leptin resistance in obesity. In the studies, we investigated whether hypothalamic silencing of SOCS3 would attenuate diet-induced obesity in rats. First we established hypothalamic SOCS3-deficient rats through lentiviral vector (LV)-mediated RNA interference (RNAi) technique, then provided a high-fat diet or a chow diet to the rats. After 8 weeks of the diet, the serum leptin and insulin concentrations were measured by RIA, and the gene expressions of SOCS3 and the long form of leptin receptor in hypothalamus were detected by a real time RT-PCR. The leptin-induced Stat3 activation was examined by Western blot. The RNAi protocol specifically knocked down the expression of SOCS3 mRNA by 50% approximately. The rats treated with LV-SOCS3-shRNA exhibited enhanced leptin-induced Stat3 activation, decreased body weight gain and improved metabolic parameters when exposed to a high-fat diet. Our results provide evidence that the rats treated with hypothalamic SOCS3 silencing are significantly protected against the development of diet-induced obesity and SOCS3 is a potential target molecule for therapeutic intervention of obesity.

Ablation of the Leptin Receptor in the Hypothalamic Arcuate Nucleus Abrogates Leptin-Induced Sympathetic Activation

The hypothalamic arcuate nucleus (ARC) is considered a major site for leptin signaling that regulates several physiological processes. To test the hypothesis that leptin receptor in the ARC is required to mediate leptin-induced sympathetic activation. First, we used the ROSA Cre-reporter mice to establish the feasibility of driving Cre expression in the ARC in a controlled manner with bilateral microinjection of adenovirus-expressing Cre-recombinase (Ad-Cre). Ad-Cre microinjection into the ARC of ObR(flox/flox) mice robustly reduced ObR expression and leptin-induced Stat3 activation in the ARC but not in the adjacent nuclei, confirming the efficacy and selectivity of the ARC deletion of ObR. Critically, deletion of ObR in the ARC attenuated brown adipose tissue and renal sympathetic nerve responses to leptin. We also examined whether ObR in the ARC is required for the preserved leptin-induced increase in renal sympathetic activity in dietary obesity. We found that deletion of ARC ObR abrogated leptin-induced increases in renal sympathetic discharge and resolved arterial pressure elevation in diet-induced obese ObR(flox/flox) mice. These data demonstrate a critical role for ObR in the ARC in mediating the sympathetic nerve responses to leptin and in the adverse sympathoexcitatory effects of leptin in obesity.

Disruption of Leptin Receptor–STAT3 Signaling Enhances Leukotriene Production and Pulmonary Host Defense against Pneumococcal Pneumonia

The adipocyte-derived hormone leptin regulates energy homeostasis and the innate immune response. We previously reported that leptin plays a protective role in bacterial pneumonia, but the mechanisms by which leptin regulates host defense remain poorly understood. Leptin binding to its receptor, LepRb, activates multiple intracellular signaling pathways, including ERK1/2, STAT5, and STAT3. In this study, we compared the responses of wild-type and s/s mice, which possess a mutant LepRb that prevents leptin-induced STAT3 activation, to determine the role of this signaling pathway in pneumococcal pneumonia. Compared with wild-type animals, s/s mice exhibited greater survival and enhanced pulmonary bacterial clearance after an intratracheal challenge with Streptococcus pneumoniae. We also observed enhanced phagocytosis and killing of S. pneumoniae in vitro in alveolar macrophages (AMs) obtained from s/s mice. Notably, the improved host defense and AM antibacterial effector functions in s/s mice were associated with increased cysteinyl-leukotriene production in vivo and in AMs in vitro. Augmentation of phagocytosis in AMs from s/s mice could be blocked using a pharmacologic cysteinyl-leukotriene receptor antagonist. Phosphorylation of ERK1/2 and cytosolic phospholipase A(2) α, known to enhance the release of arachidonic acid for subsequent conversion to leukotrienes, was also increased in AMs from s/s mice stimulated with S. pneumoniae in vitro. These data indicate that ablation of LepRb-mediated STAT3 signaling and the associated augmentation of ERK1/2, cytosolic phospholipase A(2) α, and cysteinyl-leukotriene synthesis confers resistance to s/s mice during pneumococcal pneumonia. These data provide novel insights into the intracellular signaling events by which leptin contributes to host defense against bacterial pneumonia.

MyD88 Signaling in the CNS Is Required for Development of Fatty Acid-Induced Leptin Resistance and Diet-Induced Obesity

Obesity-associated activation of inflammatory pathways represents a key step in the development of insulin resistance in peripheral organs, partially via activation of TLR4 signaling by fatty acids. Here, we demonstrate that palmitate acting in the central nervous system (CNS) inhibits leptin-induced anorexia and Stat3 activation. To determine the functional significance of TLR signaling in the CNS in the development of leptin resistance and diet-induced obesity in vivo, we have characterized mice deficient for the TLR adaptor molecule MyD88 in the CNS (MyD88(DeltaCNS)). Compared to control mice, MyD88(DeltaCNS) mice are protected from high-fat diet (HFD)-induced weight gain, from the development of HFD-induced leptin resistance, and from the induction of leptin resistance by acute central application of palmitate. Moreover, CNS-restricted MyD88 deletion protects from HFD- and icv palmitate-induced impairment of peripheral glucose metabolism. Thus, we define neuronal MyD88-dependent signaling as a key regulator of diet-induced leptin and insulin resistance in vivo.

FoxO1 Inhibits Leptin Regulation of Pro-opiomelanocortin Promoter Activity by Blocking STAT3 Interaction with Specificity Protein 1

Leptin controls food intake and energy expenditure by regulating hypothalamic neuron activities. Leptin exerts its actions through complex signaling pathways including STAT3 phosphorylation, nuclear translocation, and binding to target gene promoter/cofactor complexes. Deficient or defective leptin signaling leads to obesity, which may be caused by insufficient leptin levels and/or resistance to leptin signaling. To understand the molecular mechanisms of leptin resistance, we studied the regulation of pro-opiomelanocortin (POMC) gene expression by leptin. We show that phospho-STAT3 activates POMC promoter in response to leptin signaling through a mechanism that requires an SP1-binding site in the POMC promoter. Furthermore, FoxO1 binds to STAT3 and prevents STAT3 from interacting with the SP1.POMC promoter complex, and consequently, inhibits STAT3-mediated leptin action. Our study suggests that leptin action could be inhibited at a step downstream of STAT3 phosphorylation and nuclear translocation, and provides a potential mechanism of leptin resistance in which an increased FoxO1 antagonizes STAT3-mediated leptin signaling.

Mapping of Binding Site III in the Leptin Receptor and Modeling of a Hexameric Leptin·Leptin Receptor Complex

The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.

Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators of this signalling pathway. Specifically, recent evidence has suggested that PTP1B might be a key regulator of leptin signalling, based on the resistance to diet-induced obesity and increased leptin signalling observed in PTP1B-deficient mice. The present study was undertaken to investigate the mechanism by which PTP1B mediates the cessation of the leptin signal transduction. Leptin-induced activation of a STAT3 responsive reporter was dose-dependently inhibited by co-transfection with PTP1B. No inhibition was observed when a catalytically inactive mutant of PTP1B was used or when other PTPs were co-transfected. PTP1B was able to dephosphorylate activated JAK2 and STAT3 in vitro, whereas either no or a minimal effect was observed with cluster of differentiation 45 (CD45), PTPalpha and leukocyte antigen-related (LAR). By utilisation of a selective PTP1B inhibitor, the leptin-induced STAT3 activation was enhanced in cells. In conclusion, these results suggested that the negative regulatory role of PTP1B on leptin signalling is mediated through a direct and selective dephosphorylation of the two signalling molecules, JAK2 and STAT3.

Alteration of Leptin-Induced STAT3 Activation in the Brain of Senescence-Accelerated Mouse (SAM) P8

We used senescence-accelerated mouse prone 8 (SAMP8), a useful model of accelerated aging, to investigate the responsiveness to leptin with aging. The state of leptin-induced STAT3 phosphorylation in the hypothalamus was found to be higher in SAMP8 than in SAMR1, a control mouse showing normal aging, at 14-18 months of age but not at 2 months of age. Moreover, leptin receptor Ob-Rb expression in the hypothalamus was up-regulated in SAMP8. The results indicate that leptin sensitivity increases with aging in the SAM mouse brain.

Insulin attenuates leptin-induced STAT3 tyrosine-phosphorylation in a hepatoma cell line

Leptin, the 16 kDa protein product of the ob gene, is secreted by adipocytes. The long form leptin receptor (ObRb) is expressed at high levels in the hypothalamus, and regulates appetite and energy expenditure. The fact that serum concentration of leptin is correlated with body mass index (BMI) suggests reduced sensitivity to leptin. Even though hyperinsulinemia and hyperleptinemia could coexist in obese humans, little is known about the interaction of insulin and leptin. In this study, we examined the effect of insulin on leptin signaling using Huh 7 cells transiently transfected with ObRb cDNA. Insulin inhibits leptin-induced STAT3 phosphorylation in a time- and dose-dependent manner without affecting Janus tyrosine kinases (JAKs) JAK2 phosphorylation. Okadaic acid prevents the inhibitory effect of insulin on leptin-induced STAT3 activation.

Ethanol inhibits leptin-induced STAT3 activation in Huh7 cells

Leptin, an adipocyte-derived hormone, regulates food intake and energy expenditure in the hypothalamus via its receptor, member of the class I cytokine receptor family. Leptin resistance has been observed in rodents and in humans. However, the mechanisms could not be explained in most cases of human obesity, except for rare cases with mutations in the leptin receptor. Recent reports demonstrated that ethanol inhibited the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway activated by some members of the class I cytokine receptor family. In this study, we examined the effects of ethanol on the leptin-induced JAK/STAT signaling pathway using human hepatoma cell lines transiently expressing long form of the leptin receptor. A 30 min pretreatment with ethanol dose-dependently inhibited the leptin-induced STAT3 phosphorylation. Furthermore, to determine the time course of ethanol inhibitory effects, the cells were incubated in 10 mM ethanol for various times. Partial inhibition of leptin-induced STAT3 activation was seen after 1 min of treatment with ethanol and completely inhibited after 30 min pretreatment. SB 202190, a p38 mitogen-activated protein kinase (MAPK) inhibitor, partly prevented this inhibition by ethanol of leptin-induced STAT3 activation. These findings suggest that ethanol time- and dose-dependently inhibits the leptin action, in part via p38 MAPK.

Insulin modulates leptin-induced STAT3 activation in rat hypothalamus

Insulin and leptin have overlapping effects in the control of energy homeostasis, but the molecular basis of this synergism is unknown. Insulin signals through a receptor tyrosine kinase that phosphorylates and activates the docking proteins IRSs (insulin receptor substrates), whereas the leptin receptor and its associated protein tyrosine kinase JAK2 (Janus kinase 2) mediate phosphorylation and activation of the transcription factor STAT3 (signal transducer and activator of transcription). Here, we present evidence for the integration of leptin and insulin signals in the hypothalamus. Insulin induced JAK2 tyrosine phosphorylation, leptin receptor phosphorylation which, in the presence of leptin, augmented the interaction between STAT3 and this receptor. Insulin also increased the leptin-induced phosphorylation of STAT3 and its activation. These results indicate that insulin modulates the leptin signal transduction pathway, and may provide a molecular basis for the coordinated effects of insulin and leptin in feeding behavior and weight control.

Hypothalamic leptin resistance is associated with impaired leptin signal transduction in aged obese rats.

Leptin contributes to the regulation of both food intake and energy expenditure. We previously demonstrated that the F344xBN rat, a rodent model for late-onset obesity, is leptin-resistant and that leptin signal transduction following peripheral administration of leptin is impaired in these aged, overweight rats. To determine if leptin signal transduction is impaired in response to central administration of leptin and whether reduced hypothalamic leptin receptors may be contributing to the impaired signal transduction, we examined the in vivo dose-response leptin-induced STAT3 activation (phosphorylation and binding activity to the SIE M67 oligonucleotide) in response to i.c.v. administration of leptin along with the level of hypothalamic leptin receptor protein in young and older, late-onset obese rats. The leptin-induced maximum phosphorylation of STAT3 was 41% greater in young compared with older obese rats, but the dose required for half-maximal phosphorylation of STAT3 was similar in both the young (41 ng) and old-obese (47 ng) rats. There were no changes in total STAT3 protein with leptin or age, and leptin did not increase phosphorylation of STAT1. Leptin increased phosphorylation of STAT3 transcription factor binding eight-fold in the young but only four-fold in the aged-obese rats, and leptin receptor protein was 50% greater in the young compared with aged rats. These data indicate that aged-overweight rats demonstrate reduced signal transduction in response to centrally administered leptin that may be the result of the diminished leptin receptor protein observed in the aged-obese rats. The diminished leptin receptors and impaired leptin signal transduction may explain the diminished physiological responses observed following leptin administration in older rats. This impaired leptin signal transduction may be due either to the elevated obesity with age or to age itself, or to both.

Impaired leptin signal transduction with age-related obesity

Leptin contributes to the regulation of both food intake and energy expenditure. We previously demonstrated that the F-344×BN rat, a rodent model for late-onset obesity, is leptin resistant, suggesting that leptin signal transduction may be impaired in these aged, overweight rats. To test this hypothesis, we examined the in vivo dose–response and time–course response of leptin-induced STAT3 activation (phosphorylation and binding activity to the SIE M67 oligonucleotide) in the hypothalamus of young rats along with the dose–response leptin-induced STAT3 phosphorylation (P-STAT3) and maximum increase in binding activity in young and aged rats. In young rats there was a dose (0–1 mg, iv) and time dependent increase in P-STAT3 and in P-STAT3 binding activity. P-STAT3 paralleled the rise and fall in serum leptin levels with P-STAT3 elevated for at least 4 h with return to basal levels by 14 h after 1 mg leptin. The maximum level of leptin-induced P-STAT3 was unchanged with age, but the dose for half maximal phosphorylation was greater in aged (138 μg) compared with young (26 μg) rats. In addition, the leptin-induced increase in P-STAT3 transcription factor binding was diminished in aged rats. These data suggest that leptin signal transduction, in vivo, demonstrate a time and dose response increase paralleling the rise and fall in serum leptin, suggesting that serum leptin levels are the most important factor in determining leptin-induced phosphorylation of STAT3 in the hypothalamus. In addition, aged, overweight rats demonstrate reduced signal transduction in response to leptin, with reduced sensitivity for STAT3 phosphorylation and diminished leptin-induced P-STAT3 transcription factor binding. This impaired leptin signal transduction may be due to either the elevated obesity with age or due to age itself or both.