Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

Modification of aquaporin expression in response to fenretinide-induced transdifferentiation of ARPE-19 cells into neuronal-like cells.

PURPOSE: The goal of this study was to investigate the modifications of aquaporin (AQP) expression in ARPE-19 cells in response to fenretinide-induced transdifferentiation into neuronal-like cells METHODS: ARPE-19 cells were treated daily for 7 days with 3 µm fenretinide or dimethyl sulphoxide as control. mRNA and protein expression were evaluated by real-time quantitative PCR, Western blot analysis and immunofluorescence. RESULTS: Control ARPE-19 cells expressed AQP1, AQP4, AQP6 and AQP11 at the mRNA level, but only AQP4, AQP6 and AQP11 at the protein level. Fenretinide induced the transdifferentiation of ARPE-19 cells into neuronal-like cells. Indeed, fenretinide induced morphological changes similar to neurons characterized by elongated cell body and the formation of neurite branching. Moreover, ARPE-19 cells transdifferentiated to neuron-like cells were characterized by significant decrease in retinal pigmented epithelium markers, for example cytokeratin 8 and cellular retinaldehyde-binding protein, as well as an increase in neuronal markers such as synaptophysin and calretinin. AQP4 expression, at both mRNA and protein levels, and AQP6 expression, only at protein level, were significantly decreased in ARPE-19 cells transdifferentiated into neuronal-like cells. CONCLUSIONS: The expression of AQP4 and AQP6 is downregulated during fenretinide-induced transdifferentiation.

Aquaporin-1 expression in proliferative vitreoretinopathy and in epiretinal membranes.

PURPOSE: Aquaporin-1 (AQP1) is involved in cell migration and proliferation; therefore, the purpose of the study was to investigate its expression in proliferative vitreoretinopathy (PVR) and epiretinal membranes (ERM). METHODS: 19 membranes from PVR and ERM were collected following eye surgery. AQP1 mRNA and protein expressions were determined by RT-qPCR and immunofluorescence in the membranes from PVR and ERM. RESULTS: AQP1 mRNA and protein were expressed in both PVR and ERM as shown by RT-qPCR and immunofluorescence. AQP1 protein expression was heterogeneous among and between PVR and ERM and colocalized with alpha-smooth muscle actin ( α SMA) and with glial fibrillary acidic protein (GFAP). There were a higher percentage of cells coexpressing AQP1 and α SMA than AQP1 and GFAP. GFAP and α SMA did not colocalize. CONCLUSION: Our data show for the first time AQP1 expression in both PVR and ERM. AQP1 is expressed mostly by the α SMA-positive cells, presumably myofibroblasts, but also by GFAP-positive cells, assumed to be glial cells. These original findings warrant further functional investigations aiming at studying the potential role of AQP1 in cell migration and proliferation occurring during the development of PVR and ERM.

Pituitary adenylate cyclase activating peptide (PACAP) participates in adipogenesis by activating ERK signaling pathway.

Pituitary adenylate cyclase activating peptide (PACAP) belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2), strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein ß (C/EBPß).

Murine 3T3-L1 adipocyte cell differentiation model: validated reference genes for qPCR gene expression analysis.

BACKGROUND: Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). METHODOLOGY/PRINCIPAL FINDINGS: Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. CONCLUSION: Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Ghrelin interacts with human plasma lipoproteins.

Ghrelin, a peptide hormone produced predominantly by the stomach, stimulates food intake and GH secretion. The Ser(3) residue of ghrelin is mainly modified by a n-octanoic acid. In the human bloodstream, ghrelin circulates in two forms: octanoylated and desacylated. We previously demonstrated that ghrelin is desoctanoylated in human serum by butyrylcholinesterase (EC 3.1.1.8) and other esterase(s), whereas in rat serum, only carboxylesterase (EC 3.1.1.1) is involved. The aims of this study were to determine the role of lipoprotein-associated enzymes in ghrelin desoctanoylation and the role of lipoproteins in the transport of circulating ghrelin. Our results show that ghrelin desoctanoylation mostly occurred in contact with low-density lipoproteins (LDLs) and lipoprotein-poor plasma subfractions. Butyrylcholinesterase and platelet-activating factor acetylhydrolase (EC 3.1.1.47) were responsible for the ghrelin hydrolytic activity of the lipoprotein-poor plasma and LDL subfractions, respectively. Moreover, we observed that ghrelin is associated with triglyceride-rich lipoproteins (TRLs), high-density lipoproteins (HDLs), very high-density lipoproteins (VHDLs), and to some extent LDLs. In conclusion, we report that the presence of the acyl group is necessary for ghrelin interaction with TRLs and LDLs but not HDLs and VHDLs. Ghrelin interacts via its N- and C-terminal parts with HDLs and VHDLs. This suggests that, whereas TRLs mostly transport acylated ghrelin, HDLs and VHDLs transport both ghrelin and des-acyl ghrelin.

Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation.

Ghrelin, a ligand of the GH secretagogue receptor (GHS-R 1a), is a 28-amino acid peptide with an unusual octanoyl group on Ser3, crucial for its biological activity. For the first time, ghrelin and GHS-R 1b, a truncated variant of the receptor resulting from alternative splicing, but not GHS-R 1a, mRNAs were detected in the human erythroleukemic cell line HEL. Two antibodies, used for RIA, were directed against octanoylated and total (octanoylated and desoctanoylated) ghrelin, and the recognized epitopes were characterized. Using reverse phase HPLC analysis followed by RIA, we demonstrated that octanoylated and desoctanoylated ghrelins were present in HEL cells and their culture medium, of which more than 90% was octanoylated. The ghrelin levels were not affected after 24 h treatment with sodium butyrate, phorbol 12-myristate 13-acetate, or forskolin, but a significant 3-fold increase in desoctanoylated ghrelin was detected in the culture medium after 48 h treatment with sodium butyrate. The antighrelin SB801 and SB969 antisera inhibited HEL cell proliferation by 24% and 39%, respectively, after 72 h. Taken together, these data suggested that endogenous ghrelin stimulated HEL cell proliferation by an autocrine pathway involving an unidentified receptor, distinct from GHS-R1a, and that the HEL cell line represents a unique model to study the octanoylation of ghrelin.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2.

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein-coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases through the G(i) class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

Ghrelin degradation by serum and tissue homogenates: identification of the cleavage sites.

The endogenous ligand for the GH secretagogue receptor is ghrelin, a peptide recently purified from the stomach. Ghrelin is n-octanoylated on the Ser(3) residue, and this modification is essential for its interaction with the receptor. The degradation of ghrelin by rat and human serum, purified commercial enzymes, and tissues homogenates was analyzed by combining HPLC and mass spectrometry. In serum, ghrelin was desoctanoylated, without proteolysis. The desoctanoylation was significantly reduced by phenylmethylsulfonyl fluoride, a serine proteases and esterases inhibitor. In rat serum, the carboxylesterase inhibitor bis-p-nitrophenyl-phosphate totally inhibited ghrelin desoctanoylation, and a correlation was found between ghrelin desoctanoylation and carboxylesterase activity. Moreover, purified carboxylesterase degraded ghrelin. Thus, carboxylesterase could be responsible for ghrelin desoctanoylation in that species. In human serum, ghrelin desoctanoylation was partially inhibited by eserine salicylate and sodium fluoride, two butyrylcholinesterase inhibitors, but not by bis-p-nitrophenyl-phosphate and EDTA. Purified butyrylcholinesterase was able to degrade ghrelin, and there was a correlation between the butyrylcholinesterase and ghrelin desoctanoylation activities in human sera. This suggested that several esterases, including butyrylcholinesterase, contributed to ghrelin desoctanoylation in human serum. In contact with tissues homogenates, ghrelin was degraded by both desoctanoylation and N-terminal proteolysis. We identified five cleavage sites in ghrelin between residues -Ser(2)-(acyl)Ser(3)- (stomach and liver), -(acyl?)Ser(3)-Phe(4)- (stomach, liver, and kidney), -Phe(4)-Leu(5)- (stomach and kidney), -Leu(5)-Ser(6)- and -Pro(7)-Glu(8)- (kidney). In all cases, the resulting fragments were biologically inactive.

Ala-scan of ghrelin (1-14): interaction with the recombinant human ghrelin receptor.

Ghrelin, a 28 residues acylated peptide, is the natural ligand of the growth-hormone secretagogue receptor (GHS-R), which also interacts with small synthetic peptides. We investigated the importance of each of the first 14 N-terminal residues by Ala replacement (Ala-scan) and also of the N-terminal positive charge, on the recombinant GHS-R expressed in HEK293 or CHO cells by binding, IP and Ca(2+) assays. Nearly all of the replacements had no significant effect on the ligand binding or IP(3)/Ca(2+) stimulation. Exceptions were the modification of the N-terminal residue to [A(1)]- or N(alpha)-acetyl-ghrelin (1-14), confirming the requirement for the positive charge at the amino-terminus. Mutation of [F(4)]- to [A(4)]- or [Y(4)]-ghrelin (1-14), were detrimental suggesting direct interaction with the GHS-R. [A(8)] and [Y(8)] were more potent than ghrelin (1-14), implying that the naturally occurring Glu(8) residue may not be the optimal.

Hexanoylation of a VPAC2 receptor-preferring ligand markedly increased its selectivity and potency.

We synthesized a VIP analog that combines mutations that decrease the affinity for the VPAC1 receptor but maintain a high affinity for the VPAC2 receptor with an amino-terminal hexanoylation that increases the affinity for the VPAC2 receptor with a limited decrease in the affinity of the VPAC1 receptor. The resulting Hexanoyl[A19,K(27,28)]VIP had the expected properties of a high affinity for the VPAC2 receptor and a low affinity for the VPAC1 receptor and also a low affinity for the PAC1 and secretin receptors. With a 1000-fold preference for the VPAC2 receptor and a IC50 value of binding of 1 nM, this compound is the most potent and the most selective agonist presently described.

The C-terminal nonapeptide of mature chemerin activates the chemerin receptor with low nanomolar potency.

Chemerin is a novel protein identified as the natural ligand of ChemR23 (chemerinR), a previously orphan G protein-coupled receptor expressed in immature dendritic cells and macrophages. Chemerin is synthesized as a secreted precursor, prochemerin, which is poorly active, but converted into a full agonist of chemerinR by proteolytic removal of the last six amino acids. In the present work, we have synthesized a number of peptides derived from the C-terminal domain of human prochemerin and have investigated their functional properties as agonists or antagonists of human chemerinR. We found that the nonapeptide (149)YFPGQFAFS(157) (chemerin-9), corresponding to the C terminus of processed chemerin, retained most of the activity of the full-size protein, with regard to agonism toward the chemerinR. Extension of this peptide at its N terminus did not increase the activity, whereas further truncations rapidly resulted in inactive compounds. The C-terminal end of the peptide appeared crucial for its activity, as addition of a single amino acid or removal of two amino acids modified the potency by four orders of magnitude. Alanine-scanning mutagenesis identified residues Tyr(149), Phe(150), Gly(152), Phe(154), and Phe(156) as the key positions for chemerinR activation. A modified peptide (YHSFFFPGQFAFS) was synthesized and iodinated, and a radioligand binding assay was established. It was found that the ability of the various peptides to activate the chemerin receptor was strictly correlated with their affinity in the binding assay. These results confirm that a precise C-terminal processing is required for the generation of a chemerinR agonist. The possibility to restrict a medium sized protein to a nonapeptide, while keeping a low nanomolar affinity for its receptor is unusual among G protein-coupled receptors ligands. The identification of these short bioactive peptides will considerably accelerate the pharmacological analysis of chemerin-chemerinR interactions.

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells.

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.

Mutational analysis of the glucagon receptor: similarities with the vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP)/secretin receptors for recognition of the ligand's third residue.

Receptor recognition by the Asp(3) residues of vasoactive intestinal peptide and secretin requires the presence of a lysine residue close to the second transmembrane helix (TM2)/first extracellular loop junction and an ionic bond with an arginine residue in TM2. We tested whether the glucagon Gln(3) residue recognizes the equivalent positions in its receptor. Our data revealed that the binding and functional properties of the wild-type glucagon receptor and the K188R mutant were not significantly different, whereas all agonists had markedly lower potencies and affinities at the I195K mutated receptor. In contrast, glucagon was less potent and the Asp(3)-, Asn(3)- and Glu(3)-glucagon mutants were more potent and efficient at the double-mutated K188R/I195K receptor. Furthermore, these alterations were selective for position 3 of glucagon, as shown by the functional properties of the mutant Glu(9)- and Lys(15)-glucagon. Our results suggest that although the Gln(3) residue of glucagon did not interact with the equivalent binding pocket as the Asp(3) residue of vasoactive intestinal peptide or secretin, the Asp(3)-glucagon analogue was able to interact with position 188 of the K188R/I195K glucagon receptor. Nevertheless, the Gln(3) side chain of glucagon probably binds very close to this region in the wild-type receptor.