Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms.     

Cloning and characterization of ATRAP, a novel protein that interacts with the angiotensin II type 1 receptor.

The carboxyl-terminal cytoplasmic domain of the angiotensin II type 1 (AT1) receptor has recently been shown to interact with several classes of cytoplasmic proteins that regulate different aspects of AT1 receptor physiology. Employing yeast two-hybrid screening of a mouse kidney cDNA library with the carboxyl-terminal cytoplasmic domain of the murine AT1a receptor as a bait, we have isolated a novel protein with a predicted molecular mass of 18 kDa, which we have named ATRAP (for AT1 receptor-associated protein). ATRAP interacts specifically with the carboxyl-terminal domain of the AT1a receptor but not with those of angiotensin II type 2 (AT2), m3 muscarinic acetylcholine, bradykinin B2, endothelin B, and beta2-adrenergic receptors. The mRNA of ATRAP was abundantly expressed in kidney, heart, and testis but was poorly expressed in lung, liver, spleen, and brain. The ATRAP-AT1a receptor association was confirmed by affinity chromatography, by specific co-immunoprecipitation of the two proteins, and by fluorescence microscopy, showing co-localization of these proteins in intact cells. Overexpression of ATRAP in COS-7 cells caused a marked inhibition of AT1a receptor-mediated activation of phospholipase C without affecting m3 receptor-mediated activation. In conclusion, we have isolated a novel protein that interacts specifically with the carboxyl-terminal cytoplasmic domain of the AT1a receptor and affects AT1a receptor signaling.     

beta-Adrenergic stimulated lipolysis in pony adipocytes is exclusively via a beta2-subtype and is not affected by lactation

Catecholamines are important lipolytic agents in horses and ponies but the nature of the adrenergic receptor subtype distribution in their adipocytes is uncertain. A first objective was to identify the beta-adrenergic receptor subtype(s) present in adipocytes from horses and ponies. A second objective was to evaluate if the lipolytic responsiveness of isolated adipocytes to beta-adrenergic agonists is altered during lactation, a condition known to affect markedly maternal fat metabolism. Isoproterenol and salbutamol elicited strong lipolytic responses in adipocytes isolated from horse and pony subcutaneous adipose tissue. There were weak lipolytic responses to norepinephrine, dobutamine and BRL37344. The weak lipolytic response to NE compared to isoproterenol or salbutamol suggests an antilipolytic action from alpha2-adrenergic receptors. The relative order of potency for the beta-adrenergic agonists was isoproterenol>/=salbutamol>dobutamine=BRL37344. There was expression of beta2-adrenergic receptor mRNA in pony and horse adipose tissues, as estimated by relative RT-PCR, but no expression of mRNAs for beta1- or beta3-adrenergic receptors. Early lactation did not alter the lipolytic responses to beta-adrenergic agonists, nor the expression of beta2-adrenergic receptor mRNA. Thus, these results indicate a dominant if not exclusive presence of beta2-adrenergic receptors in pony and horse adipocytes that is not affected by lactation.     

Inhibition of JAK2/STAT3 signalling induces colorectal cancer cell apoptosis via mitochondrial pathway

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.     

Hyperglycemia and angiotensin II cooperate to enhance collagen I deposition by cardiac fibroblasts through a ROS-STAT3-dependent mechanism

Cardiac fibroblasts significantly contribute to diabetes-induced structural and functional changes in the myocardium. The objective of the present study was to determine the effects of high glucose (alone or supplemented with angiotensin II) in the activation of the JAK2/STAT3 pathway and its involvement in collagen I production by cardiac fibroblasts. We observed that the diabetic environment 1) enhanced tyrosine phosphorylation of JAK2 and STAT3; 2) induced nuclear localization of tyrosine phosphorylated STAT3 through a reactive oxygen species-mediated mechanism, with angiotensin II stimulation further enhancing STAT3 nuclear accumulation; and 3) stimulated collagen I production. The effects were inhibited by depletion of reactive oxygen species or silencing of STAT3 in high glucose alone or supplemented with exogenous angiotensin II. Combined, our data demonstrate that increased collagen I deposition in the setting of high glucose occurred through a reactive oxygen species- and STAT3-dependent mechanism. Our results reveal a novel role for STAT3 as a key signaling molecule of collagen I production in cardiac fibroblasts exposed to a diabetic environment.     

Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development

It is commonly accepted that activation of most signalling pathways is induced by ligand receptor dimerisation. This belief has been challenged for some vertebrate cytokine receptors of the JAK/STAT pathway. Here we study whether DOME, the Drosophila receptor of the JAK/STAT pathway, can dimerise and if the dimerisation is ligand-dependent. To analyse DOME homo-dimerisation, we have applied a beta-gal complementation technique that allows the detection of protein interactions in situ. This technique has been used previously in cell culture but this is the first time that it has been applied to whole embryos. We show that this technique, which we rename betalue-betalau technique, can be used to detect DOME homo-dimerisation in Drosophila developing embryos. Despite DOME being ubiquitously expressed, dimerisation is developmentally regulated. We investigate the state of DOME dimerisation in the presence or absence of ligand and show that DOME dimerisation is not ligand-induced, indicating that ligand independent cytokine receptor dimerisation is a conserved feature across phyla. We have further analysed the functional significance of ligand-independent receptor dimerisation by comparing the effects of ectopic ligand expression in cells in which the receptor is, or is not, dimerised. We show that ligand expression can only activate STAT downstream targets or affect embryo development in cells in which the receptor is dimerised. These results suggest a model in which ligand-independent dimerisation of the JAK/STAT receptor confers cells with competence to activate the pathway prior to ligand reception. Thus, competence to induce the JAK/STAT signalling pathway in Drosophila can be regulated by controlling receptor dimerisation prior to ligand binding. These results reveal a novel level of JAK/STAT signalling regulation that could also apply to vertebrates.     

Differential activities of the Drosophila JAK/STAT pathway ligands Upd, Upd2 and Upd3

JAK/STAT signalling in vertebrates is activated by multiple cytokines and growth factors. By contrast, the Drosophila genome encodes for only three related JAK/STAT ligands, Upd, Upd2 and Upd3. Identifying the differences between these three ligands will ultimately lead to a greater understanding of this disease-related signalling pathway and its roles in development. Here, we describe the analysis of the least well characterised of the Upd-like ligands, Upd3. We show that in tissue culture-based assays Upd3-GFP is secreted from cells and appears to interact with the extracellular matrix (ECM) in a similar manner to Upd, while still non-autonomously activating JAK/STAT signalling. Quantification of each of the Upd-like ligands in conditioned media has allowed us to determine the activity of equal amounts of each ligand on JAK/STAT ex vivo and reveals that Upd is the most potent ligand in this system. Finally, investigations into the effects of ectopic expression of Upd3 in vivo have confirmed its ability to activate pathway signalling at long-distance.     

Soy hydrolysate enhances the isoproterenol-stimulated lipolytic pathway through an increase in β-adrenergic receptor expression in adipocytes

ABSTRACT

Activation of the adipose lipolytic pathway during lipid metabolism is mediated by protein kinase A (PKA), which responds to β-adrenergic stimulation, leading to increased lipolysis. Soy is well known as a functional food and it is able to affect lipolysis in adipocytes. However, the mechanism by which soy components contribute to the lipolytic pathway remains to be fully elucidated. Here, we show that hydrolyzed soy enhances isoproterenol-stimulated lipolysis and activation of PKA in 3T3-L1 adipocytes. We also found that the expression of β-adrenergic receptors, which coordinate the activation of PKA, is elevated in adipocytes differentiated in the presence of soy hydrolysate. The activity of the soy hydrolysate towards β-adrenergic receptor expression was detected in its hydrophilic fraction. Our results suggest that the soy hydrolysate enhances the PKA pathway through the upregulation of β-adrenergic receptor expression and thereby, increase lipolysis in adipocytes.     

Oncostatin M decreases adiponectin expression and induces dedifferentiation of adipocytes by JAK3- and MEK-dependent pathways

Adiponectin, an adipokine secreted from adipocytes, plays a crucial role in the regulation of glucose and lipid metabolism. In the present study, we examine the role of the IL-6 family of cytokines in the expression of adiponectin in human adipocytes derived from human adipose tissue-derived stromal cells. Oncostatin M (OSM), but not IL-6, attenuated the expression level of adiponectin dose- and time-dependently, and the inhibitory effect of OSM on adiponectin expression was as potent as that of TNF-alpha. The OSM-induced down-regulation of adiponectin expression was correlated with the down-regulation of PPARgamma2 and lipoprotein lipase, markers for adipogenic differentiation, and depletion of intracellular lipid droplets, suggesting dedifferentiation of adipocytes in response to OSM. OSM induced phosphorylation of STAT1, and treatment of adipocytes with JAK3 inhibitor WHI-P131 or MEK inhibitor U0126, but not with JAK2 inhibitor AG490, prevented the activation of STAT1. Furthermore, the OSM-induced suppression of adiponectin expression and dedifferentiation of adipocytes were ameliorated by WHI-P131 or U0126, but not by AG490. These results suggest that OSM inhibits adiponectin expression by inducing dedifferentiation of adipocytes through signaling pathways involving JAK3 and MEK, but not JAK2.     

Pharmacological characterization of alpha1- and beta-adrenergic synergism of 5'DII activity in rat brown adipocytes

The role of adrenoceptor subtypes was studied in rat brown adipose tissue (BAT). The type II 5'-deiodinase (5'DII) was activated in response to simultaneous stimulation by beta3- and alpha1-adrenergic agonists, BRL 37344 or CGP 12177, and cirazoline, in brown adipocytes. Inhibition of the alpha1- and beta-adrenergic phenylephrine-stimulated 5'DII activity was obtained by the alpha1-adrenergic antagonists in the order of prazosin >/= wb 4101 > 5-methylurapidil. In comparison, the binding of [3H]prazosin to rat BAT plasma membranes was inhibited by alpha1-adrenergic antagonists in the order of prazosin > WB 4101 = benoxathian > 5-methylurapidil. Although the order of the alpha1-adrenergic competition seemed to be rather typical for the alpha1B-adrenergic receptors, a molecular analysis on adrenoceptor mRNAs should be made to confirm the exact alpha1-adrenergic subtypes at the level of brown adipocytes, since the possibility of a mixture of different receptor subtypes in brown fat cells and/or tissue may interact with the pharmacological characterization. Thus, specific alpha1- and beta-adrenoceptor subtypes participate in the regulation of 5'DII activity in the rat brown adipocytes, and therefore, an impaired alpha1- and beta-adrenergic co-work may be involved in a defective BAT function, e.g., in obese Zucker rats, too. An interesting possibility is that the decreased number of alpha1-adrenoceptors in the BAT of obese Zucker rats is due to the decrease in the alpha1B-adrenoceptor subtype which would further be involved especially in the regulation of BAT 5'DII activity.     

Receptor interacting protein 140 regulates expression of uncoupling protein 1 in adipocytes through specific peroxisome proliferator activated receptor isoforms and estrogen-related receptor alpha

Expression of uncoupling protein 1 (Ucp1) mRNA is elevated in differentiated adipocytes derived from brown or white adipose tissue devoid of the nuclear receptor corepressor receptor interacting protein 140 (RIP140). Increased expression is mediated in part by the recruitment of peroxisome proliferator activated receptors alpha and gamma, together with estrogen-related receptor alpha, which functions through a novel binding site on the Ucp1 enhancer. This demonstrates that regulation of Ucp1 expression in the absence of RIP140 involves derepression of at least three different nuclear receptors. The ability to increase expression of Ucp1 by beta-adrenergic signaling is independent of RIP140, as shown by the action of the beta(3)-adrenergic agonist CL 316,243 to stimulate expression in both brown and white adipocytes in the presence and absence of the corepressor. Therefore, the expression of this metabolic uncoupling protein in adipose cells is regulated by inhibition as well as activation of distinct signaling pathways.     

C-X-C motif chemokine receptor 4 aggravates renal fibrosis through activating JAK/STAT/GSK3β/β-catenin pathway

Chronic kidney disease (CKD) has a high prevalence worldwide. Renal fibrosis is the common pathological feature in various types of CKD. However, the underlying mechanisms are not determined. Here, we adopted different CKD mouse models and cultured human proximal tubular cell line (HKC-8) to examine the expression of C-X-C motif chemokine receptor 4 (CXCR4) and β-catenin signalling, as well as their relationship in renal fibrosis. In CKD mice and humans with a variety of nephropathies, CXCR4 was dramatically up-regulated in tubules, with a concomitant activation of β-catenin. CXCR4 expression level was positively correlated with the expression of β-catenin target MMP-7. AMD3100, a CXCR4 receptor blocker, and gene knockdown of CXCR4 significantly inhibited the activation of JAK/STAT and β-catenin signalling, protected against tubular injury and renal fibrosis. CXCR4-induced renal fibrosis was inhibited by treatment with ICG-001, an inhibitor of β-catenin signalling. In HKC-8 cells, overexpression of CXCR4 induced activation of β-catenin and deteriorated cell injury. These effects were inhibited by ICG-001. Stromal cell–derived factor (SDF)-1α, the ligand of CXCR4, stimulated the activation of JAK2/STAT3 and JAK3/STAT6 signalling in HKC-8 cells. Overexpression of STAT3 or STAT6 decreased the abundance of GSK3β mRNA. Silencing of STAT3 or STAT6 significantly blocked SDF-1α-induced activation of β-catenin and fibrotic lesions. These results uncover a novel mechanistic linkage between CXCR4 and β-catenin activation in renal fibrosis in association with JAK/STAT/GSK3β pathway. Our studies also suggest that targeted inhibition of CXCR4 may provide better therapeutic effects on renal fibrosis by inhibiting multiple downstream signalling cascades.     

The four-jointed gene is required in the Drosophila eye for ommatidial polarity specification

BACKGROUND: The Drosophila eye is composed of about 800 ommatidia, each of which becomes dorsoventrally polarised in a process requiring signalling through the Notch, JAK/STAT and Wingless pathways. These three pathways are thought to act by setting up a gradient of a signalling molecule (or molecules) often referred to as the 'second signal'. Thus far, no candidate for a second signal has been identified. RESULTS: The four-jointed locus encodes a type II transmembrane protein that is expressed in a dorsoventral gradient in the developing eye disc. We have analysed the function and regulation of four-jointed during eye patterning. Loss-of-function clones or ectopic expression of four-jointed resulted in strong non-autonomous defects in ommatidial polarity on the dorsoventral axis. Ectopic expression experiments indicated that localised four-jointed expression was required at the time during development when ommatidial polarity was being determined. In contrast, complete removal of four-jointed function resulted in only a mild ommatidial polarity defect. Finally, we found that four-jointed expression was regulated by the Notch, JAK/STAT and Wingless pathways, consistent with it mediating their effects on ommatidial polarity. CONCLUSIONS: The clonal phenotypes, time of requirement and regulation of four-jointed are consistent with it acting in ommatidial polarity determination as a second signal downstream of Notch, JAK/STAT and Wingless. Interestingly, it appears to act redundantly with unknown factors in this process, providing an explanation for the previous failure to identify a second signal.     

Prolonged treatment with the β3-adrenergic agonist CL 316243 induces adipose tissue remodeling in rat but not in guinea pig: 1) fat store depletion and desensitization of β-adrenergic responses

Beta3-adrenergic agonists have been considered as potent antiobesity and antidiabetic agents mainly on the basis of their beneficial actions discovered twenty years ago in obese and diabetic rodents. The aim of this work was to verify whether prolonged treatment with a beta3-adrenergic agonist known to stimulate lipid mobilisation, could promote desensitization of beta-adrenergic responses. Wistar rats and guinea pigs were treated during one week with CL 316243 (CL, 1 mg/kg/d) by implanted osmotic minipumps. In control animals, beta3-adrenergic agonists were lipolytic in rat but not in guinea pig adipocytes. CL-treatment did not alter body weight gain in both species, but reduced fat stores in rats. Lipolysis stimulation by forskolin was unmodified but responses to beta1-, beta2- and beta3-agonists were reduced in visceral or subcutaneous white adipose tissues of CL-treated rats. Similarly, the beta3-adrenergic-dependent impairment of insulin action on glucose transport and lipogenesis in rat adipocytes was diminished after CL-treatment. In rat adipocytes, [125I]ICYP binding and beta3-adrenoceptor mRNA levels were reduced after sustained CL administration. These findings show that CL 316243 exerts (beta3-adrenergic lipolytic and antilipogenic effects in rat adipocytes. These actions, which are likely involved in the fat depletion observed in rat, also lead to the desensitization of all beta-adrenergic responses. Therefore this desensitization, together with the lack of slimming action in guinea pig, seriously attenuates the usefulness of beta3-agonists as antiobesity agents, and may explain why such agonists have not been conducted to a widespread clinical use.