Peroxisome proliferator-activated receptor beta/delta activation inhibits hypertrophy in neonatal rat cardiomyocytes.

OBJECTIVE: Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is the predominant PPAR subtype in cardiac cells and plays a prominent role in the regulation of cardiac lipid metabolism. However, the role of PPARbeta/delta activators in cardiac hypertrophy is not yet known. METHODS AND RESULTS: In cultured neonatal rat cardiomyocytes, the selective PPARbeta/delta activator L-165041 (10 micromol/L) inhibited phenylephrine (PE)-induced protein synthesis ([(3)H]leucine uptake), induction of the fetal-type gene atrial natriuretic factor (ANF) and cardiac myocyte size. Induction of cardiac hypertrophy by PE stimulation also led to a reduction in the transcript levels of both muscle-type carnitine palmitoyltransferase (50%, P<0.05) and pyruvatedehydrogenase kinase 4 (30%, P<0.05), and these changes were reversed in the presence of the PPARbeta/delta agonist L-165041. Stimulation of neonatal rat cardiomyocytes with PE and embryonic rat heart-derived H9c2 cells with lipopolysaccharide (LPS) enhanced the expression of the nuclear factor (NF)-kappaB-target gene monocyte chemoattractant protein 1 (MCP-1). The induction of MCP-1 was reduced in the presence of L-165041, suggesting that this compound prevented NF-kappaB activation. Electrophoretic mobility shift assay (EMSA) revealed that L-165041 significantly decreased LPS-stimulated NF-kappaB binding activity in H9c2 myotubes. Finally, coimmunoprecipitation studies showed that L-165041 strongly enhanced the physical interaction between PPARbeta/delta and the p65 subunit of NF-kappaB, suggesting that increased association between these two proteins is the mechanism responsible for antagonizing NF-kappaB activation by PPARbeta/delta activators. CONCLUSION: These results suggest that PPARbeta/delta activation inhibits PE-induced cardiac hypertrophy and LPS-induced NF-kappaB activation.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Potential role for peroxisome proliferator activated receptor (PPAR) in preventing colon cancer.

BACKGROUND: Peroxisome proliferator activated receptors (PPARs) are nuclear hormone receptors involved in genetic control of many cellular processes. PPAR and PPAR have been implicated in colonic malignancy. Here we provide three lines of evidence suggesting an inhibitory role for PPAR in colorectal cancer development. METHODS: Levels of PPAR mRNA and protein in human colorectal cancers were compared with matched non-malignant mucosa using RNAse protection and western blotting. APC(Min)/+ mice were randomised to receive the PPAR activator methylclofenapate 25 mg/kg or vehicle for up to 16 weeks, and small and large intestinal polyps were quantified by image analysis. The effect of methylclofenapate on serum stimulated mitogenesis (thymidine incorporation), linear cell growth, and annexin V and propidium iodide staining were assessed in human colonic epithelial cells. RESULTS: PPAR (mRNA and protein) expression levels were significantly depressed in colorectal cancer compared with matched non-malignant tissue. Methylclofenapate reduced polyp area in the small intestine from 18.7 mm(2) (median (interquartile range 11.1, 26.8)) to 9.90 (4.88, 13.21) mm(2) (p=0.003) and in the colon from 9.15 (6.31, 10.5) mm(2) to 3.71 (2.71, 5.99) mm(2) (p=0.009). Methylclofenapate significantly reduced thymidine incorporation and linear cell growth with no effect on annexin V or propidium iodide staining. CONCLUSIONS: PPAR may inhibit colorectal tumour progression, possibly via inhibition of proliferation, and may be an important therapeutic target.

Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity

Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogasterretina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila suppressor of cytokine signalling 36E (SOCS36E) protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. Here, we apply the Förster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrate that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. We propose a mechanistic model in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras-MAPK signal only in the R7 precursor.

Stoichiometry of Heteromeric BAFF and APRIL Cytokines Dictates Their Receptor Binding and Signaling Properties.

The closely related TNF family ligands B cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL) serve in the generation and maintenance of mature B-lymphocytes. Both BAFF and APRIL assemble as homotrimers that bind and activate several receptors that they partially share. However, heteromers of BAFF and APRIL that occur in patients with autoimmune diseases are incompletely characterized. The N and C termini of adjacent BAFF or APRIL monomers are spatially close and can be linked to create single-chain homo- or hetero-ligands of defined stoichiometry. Similar to APRIL, heteromers consisting of one BAFF and two APRILs (BAA) bind to the receptors B cell maturation antigen (BCMA), transmembrane activator and CAML interactor (TACI) but not to the BAFF receptor (BAFFR). Heteromers consisting of one APRIL and two BAFF (ABB) bind to TACI and BCMA and weakly to BAFFR in accordance with the analysis of the receptor interaction sites in the crystallographic structure of ABB. Receptor binding correlated with activity in reporter cell line assays specific for BAFFR, TACI, or BCMA. Single-chain BAFF (BBB) and to a lesser extent single-chain ABB, but not APRIL or single-chain BAA, rescued BAFFR-dependent B cell maturation in BAFF-deficient mice. In conclusion, BAFF-APRIL heteromers of different stoichiometries have distinct receptor-binding properties and activities. Based on the observation that heteromers are less active than BAFF, we speculate that their physiological role might be to down-regulate BAFF activity.

Negative Regulation of Receptor Tyrosine Kinases by T-cell Protein Tyrosine Phosphatase

Protein tyrosine phosphorylation controls a wide array of cellular responses such as growth, migration, proliferation, differentiation, metabolism and cytoskeletal organisation. Tyrosine phosphorylation is a dynamic process involving the competing activities of protein tyrosine kinases and protein tyrosine phosphatases. The protein tyrosine kinases are further divided into non-receptor- and receptor tyrosine kinases. The latter are transmembrane glycoproteins activated by the binding of specific ligands, mostly growth factors, to their extracellular domain, transmitting different signals to the cell. Growth factor receptors such as the epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor β, belong to the receptor tyrosine kinases, the signalling of which is often disturbed in various diseases, including cancer. This has led to the development of receptor tyrosine kinase antagonists for use as anti-cancer drugs. As the receptor tyrosine kinases, also the protein tyrosine phosphatases can be divided into receptor- and non-receptor types. The protein tyrosine phosphatases have attained much less attention than the receptor tyrosine kinases partly because they were identified later. However, accumulating evidence shows that the protein tyrosine phosphatases have important roles as specific and active regulators of tyrosine phosphorylation in cells and of physiological processes. Consequently, the protein tyrosine phosphatases are receiving arising interest as novel drug targets. The aim of this work was to elucidate the negative regulation of receptor tyrosine kinases by one non-receptor protein tyrosine phosphatase, T-cell protein tyrosine phosphatase TCPTP. The results show that TCPTP activated by cell adhesion receptor integrin α1 functions as a negative regulator of the epidermal growth factor receptor. It was also found that TCPTP affects vascular endothelial growth factor receptor 2 signalling and angiogenesis. Lastly, a High-throughput screen with 64,280 compounds was performed to identify novel TCPTP activators, resulting in identification of one small molecule compound capable of exerting similar effects on TCPTP signalling as integrin α1. This compound is shown to downregulate signalling of epidermal growth factor receptor and platelet-derived growth factor receptor β, as well as to inhibit cell proliferation and angiogenesis. Our results suggest that a suitable small-molecule TCPTP activator could be utilized in the development of novel anti-cancer drugs.

CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and G(i) proteins

The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with G(i), G(o) and G(q) species. Interaction with Gi leads to G protein activation, whereas G. does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2). Activation of JAK2 is independent of Gi or Gq activation. Gi protein activation was not prevented by inhibition of JAK, showing that heterotrimeric G protein activation and activation of the JAK/signal transducer and activator of transcription (STAT) pathway are independent of each other. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin.

There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.

Interaction between vitamin D receptor gene polymorphisms and 25-hydroxyvitamin D concentrations on metabolic and cardiovascular disease outcomes

AIM: 25-hydroxyvitamin D (25OHD) concentrations have been shown to be associated with major clinical outcomes, with a suggestion that individual risk may vary according to common genetic differences in the vitamin D receptor (VDR) gene. Hence, we tested for the interactions between two previously studied VDR polymorphisms and 25OHD on metabolic and cardiovascular disease-related outcomes in a large population-based study. METHODS: Interactions between two previously studied VDR polymorphisms (rs7968585 and rs2239179) and 25OHD concentrations on metabolic and cardiovascular disease-related outcomes such as obesity- (body mass index, waist circumference, waist-hip ratio (WHR)), cardiovascular- (systolic and diastolic blood pressure), lipid- (high- and low-density lipoprotein, triglycerides, total cholesterol), inflammatory- (C-reactive protein, fibrinogen, insulin growth factor-1, tissue plasminogen activator) and diabetes- (glycated haemoglobin) related markers were examined in the 1958 British Birth cohort (n up to 5160). Interactions between each SNP and 25OHD concentrations were assessed using linear regression and the likelihood ratio test. RESULTS: After Bonferroni correction, none of the interactions reached statistical significance except for the interaction between the VDR SNP rs2239179 and 25OHD concentrations on waist-hip ratio (WHR) (P=0.03). For every 1nmol/L higher 25OHD concentrations, the association with WHR was stronger among those with two major alleles (-4.0%, P=6.26e-24) compared to those with either one or no major alleles (-2.3%, P≤8.201e-07, for both) of the VDR SNP rs2239179. CONCLUSION: We found no evidence for VDR polymorphisms acting as major modifiers of the association between 25OHD concentrations and cardio-metabolic risk. Interaction between VDR SNP rs2239179 and 25OHD on WHR warrants further confirmation.

Activation of mitogen-activated protein kinases (p38-MAPKs, SAPKs/JNKs and ERKs) by the G-protein-coupled receptor agonist phenylephrine in the perfused rat heart.

We investigated the ability of phenylephrine (PE), an alpha-adrenergic agonist and promoter of hypertrophic growth in the ventricular myocyte, to activate the three best-characterized mitogen-activated protein kinase (MAPK) subfamilies, namely p38-MAPKs, SAPKs/JNKs (i.e. stress-activated protein kinases/c-Jun N-terminal kinases) and ERKs (extracellularly responsive kinases), in perfused contracting rat hearts. Perfusion of hearts with 100 microM PE caused a rapid (maximal at 10 min) 12-fold activation of two p38-MAPK isoforms, as measured by subsequent phosphorylation of a p38-MAPK substrate, recombinant MAPK-activated protein kinase 2 (MAPKAPK2). This activation coincided with phosphorylation of p38-MAPK. Endogenous MAPKAPK2 was activated 4-5-fold in these perfusions and this was inhibited completely by the p38-MAPK inhibitor, SB203580 (10 microM). Activation of p38-MAPK and MAPKAPK2 was also detected in non-contracting hearts perfused with PE, indicating that the effects were not dependent on the positive inotropic/chronotropic properties of the agonist. Although SAPKs/JNKs were also rapidly activated, the activation (2-3-fold) was less than that of p38-MAPK. The ERKs were activated by perfusion with PE and the activation was at least 50% of that seen with 1 microM PMA, the most powerful activator of the ERKs yet identified in cardiac myocytes. These results indicate that, in addition to the ERKs, two MAPK subfamilies, whose activation is more usually associated with cellular stresses, are activated by the Gq/11-protein-coupled receptor (Gq/11PCR) agonist, PE, in whole hearts. These data indicate that Gq/11PCR agonists activate multiple MAPK signalling pathways in the heart, all of which may contribute to the overall response (e.g. the development of the hypertrophic phenotype).

The plasmodium receptor for activated C kinase protein inhibits Ca(2+) signaling in mammalian cells

Plasmodium falciparum, the most lethal malarial parasite, expresses an ortholog for the protein kinase C (PKC) activator RACK1. However, PKC has not been identified in this parasite, and the mammalian RACK1 can interact with the inositol 1,4,5-trisphosphate receptor (InsP3R). Therefore we investigated whether the Plasmodium ortholog PfRACK also can affect InsP3R-mediated Ca(2+) signaling in mammalian cells. GFP-tagged PfRACK and endogenous RACK1 were expressed in a similar distribution within cells. PfRACK inhibited agonist-induced Ca(2+) signals in cells expressing each isoform of the InsP3R, and this effect persisted when expression of endogenous RACK1 was reduced by siRNA. PfRACK also inhibited Ca(2+) signals induced by photorelease of caged InsP3. These findings provide evidence that PfRACK directly inhibits InsP3-mediated Ca(2+) signaling in mammalian cells. Interference with host cell signaling pathways to subvert the host intracellular milieu may be an important mechanism for parasite survival. (C) 2009 Elsevier Inc. All rights reserved.

Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells

Carraro-Lacroix LR, Malnic G, Girardi AC. Regulation of Na(+)/H(+) exchanger NHE3 by glucagon-like peptide 1 receptor agonist exendin-4 in renal proximal tubule cells. Am J Physiol Renal Physiol 297: F1647-F1655, 2009. First published September 23, 2009; doi:10.1152/ajprenal.00082.2009.-The gut incretin hormone glucagon-like peptide 1 (GLP-1) is released in response to ingested nutrients and enhances insulin secretion. In addition to its insulinotropic properties, GLP-1 has been shown to have natriuretic actions paralleled by a diminished proton secretion. We therefore studied the role of the GLP-1 receptor agonist exendin-4 in modulating the activity of Na(+)/H(+) exchanger NHE3 in LLC-PK(1) cells. We found that NHE3-mediated Na(+)-dependent intracellular pH (pH(i)) recovery decreased similar to 50% after 30-min treatment with 1 nM exendin-4. Pharmacological inhibitors and cAMP analogs that selectively activate protein kinase A (PKA) or the exchange protein directly activated by cAMP (EPAC) demonstrated that regulation of NHE3 activity by exendin-4 requires activation of both cAMP downstream effectors. This conclusion was based on the following observations: 1) the PKA antagonist H-89 completely prevented the effect of the PKA activator but only partially blocked the exendin-4-induced NHE3 inhibition; 2) the MEK1/2 inhibitor U-0126 abolished the effect of the EPAC activator but only diminished the exendin-4-induced NHE3 inhibition; 3) combination of H-89 and U-0126 fully prevented the effect of exendin-4 on NHE3; 4) no additive effect in the inhibition of NHE3 activity was observed when exendin-4, PKA, and EPAC activators were used together. Mechanistically, the inhibitory effect of exendin-4 on pHi recovery was associated with an increase of NHE3 phosphorylation. Conversely, this inhibition took place without changes in the surface expression of the transporter. We conclude that GLP-1 receptor agonists modulate sodium homeostasis in the kidney, most likely by affecting NHE3 activity.

Angiotensin II AT(1) receptor mutants expressed in CHO cells caused morphological change and inhibition of cell growth

To assess the importance of the leucine residues in positions 262 and 265 of the angiotensin AT, receptor for signaling pathways and receptor expression and regulation, we compared the properties of CHO cells transfected with the wild type or the L262D or L265D receptor point mutants. It was found that the two mutants significantly increased the basal intracellular cyclic AMP (cAMP) formation in an agonist-independent mode. The morphology transformation of CHO cells was correlated with the increased cAMP formation, since forskolin, a direct activator of adenylate cyclase mimicked this effect on WT-expressing CHO cells. DNA synthesis was found to be inhibited in these cell lines, indicating that cAMP may also have determined the inhibitory effect on cell growth, in addition to the cell transformation from a tumorigenic to a non-tumorigenic phenotype. However a role for an increased Ca2(+) influx induced by the mutants in non-stimulated cells cannot be ruled out since this ion also was shown to cause transformed cells to regain the morphology and growth regulation. (c) 2005 Elsevier B.V. All rights reserved.

Gastrin-Releasing Peptide Receptor Antagonism Induces Protection from Lethal Sepsis: Involvement of Toll-like Receptor 4 Signaling

In sepsis, toll-like receptor (TLR)-4 modulates the migration of neutrophils to infectious foci, favoring bacteremia and mortality. In experimental sepsis, organ dysfunction and cytokines released by activated macrophages can be reduced by gastrin-releasing peptide (GRP) receptor (GRPR) antagonist RC-3095. Here we report a link between GRPR and TLR-4 in experimental models and in sepsis patients. RAW 264.7 culture cells were exposed to lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-alpha and RC-3095 (10 ng/mL), Male Wistar rats were subjected to cecal ligation and puncture (CLP), and RC-3095 was administered (3 mg/kg, subcutaneously); after 6 h, we removed the blood, bronchoalveolar lavage, peritoneal lavage and lung. Human patients with a clinical diagnosis of sepsis received a continuous infusion with RC-3095 (3 mg/kg, intravenous) over a period of 12 h, and plasma was collected before and after RC-3095 administration and, in a different set of patients with systemic inflammatory response syndrome (SIRS) or sepsis. GRP plasma levels were determined. RC-3095 inhibited TLR-4, extracellular-signal-related kinase (ERK)-1/2, Jun NH2-terminal kinase (JNK) and Akt and decreased activation of activator protein 1 (AP-1), nuclear factor (NF)-kappa B and interleukin (IL)-6 in macrophages stimulated by LPS. It also decreased IL-6 release from macrophages stimulated by TNF-alpha. RC-3095 treatment in CLP rats decreased lung TLR-4, reduced the migration of cells to the lung and reduced systemic cytokines and bacterial dissemination. Patients with sepsis and systemic inflammatory response syndrome have elevated plasma levels of GRP which associates with clinical outcome in the sepsis patients. These findings highlight the role of GRPR signaling in sepsis outcome and the beneficial action of GRPR antagonists in controlling the inflammatory response in sepsis through a mechanism involving at least inhibition of TLR-4 signaling. Online address: http://www.molmed.org doi: 10.2119/molmed.2012.00083

Leptin receptor 170 kDa (OB-R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

[EN] To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean +/- s.d. age, 31 +/- 5 years; height, 184 +/- 9 cm; weight, 91 +/- 13 kg; and percentage body fat, 24.8 +/- 5.8%) and 10 obese (age, 30 +/- 7 years; height, 184 +/- 8 cm; weight, 115 +/- 8 kg; and percentage body fat, 34.9 +/- 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKalpha and ACCbeta phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKalpha and ACCbeta phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles.