Several kinases have been implicated in the metabolic response of human and rat myocytes to glucagon-like peptide-1 (GLP-1), exendin-4 (Ex-4) and exendin-9 (Ex-9). We have investigated, in isolated rat adipocytes, the changes caused by GLP-1, Ex-4 and Ex-9 compared with those provoked by insulin or glucagon, upon the activity of phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB), p42/44 MAP kinases (MAPKs) and p70s6 kinase (p70s6k), and the participation of these kinases and protein kinase C (PKC) in their action upon 2-deoxy-d-glucose uptake, lipolysis and lipogenesis. The study was conducted in normal rats, and extended to a streptozotocin-induced type-2 diabetic model (STZ-rats). The participation of distinct kinases was estimated by using potential kinase inhibitors, including wortmannin, PD98059, rapamycin, H-7 and RO31–8220. In normal rat adipocytes, GLP-1 and both exendins share with insulin an increasing action upon the activity of all kinases studied (except PKB), PI3K, p44 and p42 MAPKs and possibly PKC, all being required for their stimulating effect upon glucose uptake. Ex-4 and Ex-9, like GLP-1 and insulin, have lipogenic action, while only Ex-4 shares with GLP-1 its lipolytic effect which is antagonized by Ex-9. MAP kinases and PKC seem to have an essential role in the GLP-1 and Ex-4 lipolytic action, as does PI3K in that of Ex-4. An increase in PI3K and MAPKs activity for the lipogenic effect of Ex-4, Ex-9 and GLP-1 are required, and in the case of Ex-4 and Ex-9, a stimulation of p70s6k activity is also needed. In cells from STZ-rats the magnitude of the above parameters was, in general, comparable to that in normal animals, with some exceptions: basal PI3K activity and lipogenesis were higher, GLP-1, Ex-4 and Ex-9 failed to modify basal lipogenesis but increased PKB activity, insulin failed to affect the activity of MAPKs and the insulin-induced glucose uptake was impaired. The impaired insulin effects upon some of the variables in the STZ-rat, distinct from those of GLP-1 and exendins, adds knowledge to the mechanism of the beneficial action of GLP-1 and Ex-4 in diabetic states.
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- Abstract: Adipose x
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Verónica Sancho, María V Trigo, Nieves González, Isabel Valverde, Willy J Malaisse, and María L Villanueva-Peñacarrillo
H Roger Lijnen and Ilse Scroyen
Development and maintenance of fat depots require angiogenesis, in which vascular endothelial growth factor (VEGF) and its receptors play a crucial role. We have evaluated the effect of blocking VEGF receptor 2 (VEGF-R2) with a MAB (DC101) on adipose tissue of mice with established obesity. Therefore, obese male wild-type C57B1/6 mice were treated with i.p. injection of DC101 (40 mg/kg body weight, twice weekly during 13 weeks) or of the control antibody 1C8. Treatment with DC101 resulted in a slightly lower body weight but had no effect on subcutaneous (SC) or gonadal (GON) white adipose tissue mass, as monitored by MRI. Histochemical analysis of isolated SC and GON fat pads did not reveal significant effects of DC101 treatment on adipocyte or blood vessel size or density. Plasma levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase as well as liver triglyceride levels were significantly decreased following DC101 treatment. Plasma glucose levels were markedly lower upon DC101 treatment, whereas insulin and adiponectin levels were not affected. Furthermore, Akt phosphorylation in adipose tissues was not affected. Thus, in vivo VEGF-R2 blockade in mice with established nutritionally induced obesity did not significantly affect insulin signaling in adipose tissue or adiposity.
M-B Debril, L Dubuquoy, J-N Feige, W Wahli, B Desvergne, J Auwerx, and L Gelman
Transcriptional activity relies on coregulators that modify the chromatin structure and serve as bridging factors between transcription factors and the basal transcription machinery. Using the DE domain of human peroxisome proliferator-activated receptor gamma (PPARγ) as bait in a yeast two-hybrid screen of a human adipose tissue library, we isolated the scaffold attachment factor B1 (SAFB1/HET/HAP), which was previously shown to be a corepressor of estrogen receptor α. We show here that SAFB1 has a very broad tissue expression profile in human and is also expressed all along mouse embryogenesis. SAFB1 interacts in pull-down assays not only with PPARγ but also with all nuclear receptors tested so far, albeit with different affinities. The association of SAFB1 and PPARγ in vivo is further demonstrated by fluorescence resonance energy transfer (FRET) experiments in living cells. We finally show that SAFB1 is a rather general corepressor for nuclear receptors. Its change in expression during the early phases of adipocyte and enterocyte differentiation suggests that SAFB1 potentially influences cell proliferation and differentiation decisions.
Irina G Bogdarina, Peter J King, and Adrian J L Clark
Angiotensin II acts through two pharmacologically distinct receptors known as AT1 and AT2. Duplication of the AT1 receptor in rodents into At1a and b subtypes allows tissue-specific expression of the AT1b in adrenal and pituitary tissue. Adrenal expression of this receptor is increased in the offspring of rat mothers exposed to a low-protein diet and this is associated with the undermethylation of its promoter. This phenomenon is blocked by the inhibition of maternal glucocorticoid synthesis by metyrapone. We have mapped the transcriptional start site of the promoter and demonstrated that a 1.2 kbp fragment upsteam of this site is effective in driving luciferase expression in mouse Y1 cells. A combination of bioinformatic analysis, electrophoretic mobility shift analysis (EMSA), and mutagenesis studies demonstrates: i) the presence of a putative TATA box and CAAT box; ii) the presence of three Sp1 response elements, capable of binding SP1; mutation of any pair of these sites effectively disables this promoter; iii) the presence of four potential glucocorticoid response elements which each bind glucocorticoid receptor in EMSA, although only two confer dexamethasone inhibition on the promoter; iv) the presence of two AP1 sites. Mutagenesis of the distal AP1 site greatly diminishes promoter function but this is also associated with the loss of dexamethasone inhibition. These studies will facilitate an understanding of the mechanisms by which fetal programming leads to long term alterations in gene expression and the development of adult disease.
Anthony L Albiston, Mauricio Cacador, Puspha Sinnayah, Peta Burns, and Siew Yeen Chai
Insulin-regulated aminopeptidase (IRAP) co-localizes with the glucose transporter 4 (GLUT4) in GLUT4 storage vesicles (GSV) in insulin-responsive cells. In response to insulin, IRAP is the only transmembrane enzyme known to translocate together with GLUT4 to the plasma membrane in adipocytes and muscle cells. Although the intracellular region of IRAP is associated with GLUT4 vesicle trafficking, the role of the aminopeptidase activity in insulin-responsive cells has not been elucidated. The aim of this study was to investigate whether the inhibition of the aminopeptidase activity of IRAP facilitates glucose uptake in insulin-responsive cells. In both in vitro and in vivo studies, inhibition of IRAP aminopeptidase activity with the specific inhibitor, HFI-419, did not modulate glucose uptake. IRAP inhibition in the L6GLUT4myc cell line did not alter glucose uptake in both basal and insulin-stimulated state. In keeping with these results, HFI419 did not affect peripheral, whole-body glucose handling after an oral glucose challenge, neither in normal rats nor in the streptozotocin (STZ)-induced experimental rat model of diabetes mellitus (DM). Therefore, acute inhibition of IRAP aminopeptidase activity does not affect glucose homeostasis.
Kun Chen, Ji-Dan Zhou, Feng Zhang, Fang Zhang, Rui-Rui Zhang, Meng-Si Zhan, Xiao-Yin Tang, Bing Deng, Ming-Gang Lei, and Yuan-Zhu Xiong
G protein-coupled receptor 120 (GPR120), an adipogenic receptor critical for the differentiation and maturation of adipocytes, plays an important role in controlling obesity in both humans and rodents and, thus, is an attractive target of obesity treatment studies. However, the mechanisms that regulate the expression of porcine GPR1 20 remain unclear. In this study, electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) techniques were used to analyze and identify the binding of C/EBPβ (transcription factor CCAAT/enhancer binding protein beta) to the GPR120 promoter. C/EBPβ overexpression and RNA interference studies showed that C/EBPβ regulated GPR120 promoter activity and endogenous GPR120 expression. The binding site of C/EBPβ in the GPR120 promoter region from −101 to −87 was identified by promoter deletion analysis and site-directed mutagenesis. Overexpression of C/EBPβ increased endogenous GPR120 expression in pig kidney cells (PK). Furthermore, when endogenous C/EBPβ was knocked down, GPR120 mRNA and protein levels were decreased. The stimulatory effect of C/EBPβ on GPR120 transcription and its ability to bind the transcription factor-binding site were confirmed by luciferase, ChIP, and EMSA. Moreover, the mRNA and protein expression levels of C/EBPβ were induced by high fat diet feeding. Taken together, it can be concluded that C/EBPβ plays a vital role in regulating GPR120 transcription and suggests HFD-feeding induces GPR120 transcription by influencing C/EBPβ expression.
WJ Meadus, R MacInnis, and ME Dugan
Peroxisome proliferator activated receptors (PPARs) represent a family of DNA binding proteins that are activated by a variety of dietary and endogenous fatty acids. The PPAR proteins are expressed throughout the body and are the target of a variety of lipidaemic and insulin sensitizing drugs. Conjugated linoleic acid (CLA) is a collective name for octadecadienoic acid isomers with conjugated double bonds, which can also act as ligands for some of the PPAR family. To gain better understanding of the long-term effects of PPAR activation, CLA was fed at 11 g/kg of feed for 45 days to castrated male pigs (barrows). These barrows had a significant repartitioning of subcutaneous fat to lean tissue in the carcass: fat was reduced by 9 x 2% and lean muscle was increased by 3 x 5%, but intramuscular fat content was also increased by 14% (P<0 x 05). PPARgamma, glutamine-fructose aminotransferase (GFAT), adipocyte fatty acid binding protein (AFABP), but not PPARalpha mRNA levels were significantly increased (P<0 x 05) in the CLA-fed pigs. The increased expression of PPARgamma and AFABP indicates that CLA induced the development of preadipocytes from stromal-vascular (s-v) stem cells to promote intramuscular fat content. The increase in the expression of GFAT mRNA indicates that the glucose supply of the muscle cells had been increased with the CLA diet, possibly sparing intramuscular fatty acid reserves.
Pitchai Balakumar and Gowraganahalli Jagadeesh
The renin–angiotensin system (RAS) plays an important role in the pathophysiology of cardiovascular disorders. Pharmacologic interventions targeting the RAS cascade have led to the discovery of renin inhibitors, angiotensin-converting enzyme inhibitors, and AT1 receptor blockers (ARBs) to treat hypertension and some cardiovascular and renal disorders. Mutagenesis and modeling studies have revealed that differential functional outcomes are the results of multiple active states conformed by the AT1 receptor upon interaction with angiotensin II (Ang II). The binding of agonist is dependent on both extracellular and intramembrane regions of the receptor molecule, and as a consequence occupies more extensive area of the receptor than a non-peptide antagonist. Both agonist and antagonist bind to the same intramembrane regions to interfere with each other's binding to exhibit competitive, surmountable interaction. The nature of interactions with the amino acids in the receptor is different for each of the ARBs given the small differences in the molecular structure between drugs. AT1 receptors attain different conformation states after binding various Ang II analogues, resulting in variable responses through activation of multiple signaling pathways. These include both classical and non-classical pathways mediated through growth factor receptor transactivations, and provide cross-communication between downstream signaling molecules. The structural requirements for AT1 receptors to activate extracellular signal-regulated kinases 1 and 2 through G proteins, or G protein-independently through β-arrestin, are different. We review the structural and functional characteristics of Ang II and its analogs and antagonists, and their interaction with amino acid residues in the AT1 receptor.
Russell Snyder and Thomas Thekkumkara
Recently, we have demonstrated that 13-cis retinoic acid (13cRA) downregulates rat angiotensin type 1A receptor (Agtr1a) gene transcription through a MAP kinase (ERK1/2)-dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA-mediated Agtr1a downregulation. Rat Agtr1a chloramphenicol acetyltransferase (CAT) promoter construct containing a sequence -2541 and -1836 bp upstream of the start site demonstrated reduced CAT activity; this region possesses a specificity protein 1 (SP1) consensus sequence (5′-TGGGGCGGGGCGGGG-3′). Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggests that the trans-acting factor binding to this cis-acting element is SP1. 13cRA significantly reduced specific binding without any change in SP1 protein expression. Studies showed that 13cRA treatment maximally phosphorylates ERK1/2 within 5–10 min, which translocates to the nucleus, activating early growth response protein 1 (Egr1) mRNA expression at 20 min followed by de novo protein synthesis, leading to an EGR1/SP1 interaction. siRNA silencing of Egr1 restored Agtr1a mRNA and protein expression in 13cRA-treated cells, and Sp1 silencing results in complete loss of Agtr1a expression. Our study suggests that 13cRA-mediated activation of ERK1/2, through EGR1, is capable of disrupting SP1, the requisite trans-activator for Agtr1a expression, providing a novel paradigm in Agtr1a gene transcription.
M Montiel and E Jimenez
In this study we showed, for the first time, the existence of a moderate density of specific angiotensin II (Ang II) binding sites (Kd=3.9+/-1.7 nM and Bmax=467.2 130.0 fmol/mg protein) in plasma membrane preparations from rat thyroid gland. Reverse transcriptase/polymerase chain reactions, using primers based on the cloned AT1 and AT2 receptor subtypes, and pharmacological characterization, using the Ang II receptor subtype antagonists Losartan and PD 123319, revealed that these Ang II binding sites match with the AT1 receptor subtypes. To obtain more information on the molecular structure of this Ang II receptor, immunoblotting analyses were carried out using a polyclonal rabbit anti-AT1 antiserum. Western analysis of fresh plasma membrane preparations from thyroid tissue showed three prominent bands of approximately 60, 45 and 40 kDa which appear to be related to different degrees of glycosylation of the receptor molecule. The functional significance of the Ang II receptors in thyroid gland is currently not known. Nevertheless, since Ang II receptors play a pivotal role in the co-ordinated actions of the renin-angiotensin system (RAS), our findings support a reciprocal regulation of thyroid function by the RAS.