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• W2972134410 abstract "•TLQP-21 binds to C3aR1 and potentiates lipolysis via intracellular calcium•A cluster of mutations was identified in C3aR1 and TLQP-21 in Murinae•These mutations result in enhanced binding affinity and pharmacological potency•Mouse TLQP-21 enhances adrenergic-receptor-induced lipolysis in human adipocytes Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs. Structural and functional diversity of peptides and GPCR result from long evolutionary processes. Even small changes in sequence can alter receptor activation, affecting therapeutic efficacy. We conducted a structure-function relationship study on the neuropeptide TLQP-21, a promising target for obesity, and its complement 3a receptor (C3aR1). After having characterized the TLQP-21/C3aR1 lipolytic mechanism, a homology modeling and molecular dynamics simulation identified the TLQP-21 binding motif and C3aR1 binding site for the human (h) and mouse (m) molecules. mTLQP-21 showed enhanced binding affinity and potency for hC3aR1 compared with hTLQP-21. Consistently, mTLQP-21, but not hTLQP-21, potentiates lipolysis in human adipocytes. These findings led us to uncover five mutations in the C3aR1 binding pocket of the rodent Murinae subfamily that are causal for enhanced calculated affinity and measured potency of TLQP-21. Identifying functionally relevant peptide/receptor co-evolution mechanisms can facilitate the development of innovative pharmacotherapies for obesity and other diseases implicating GPCRs. G protein-coupled receptors (GPCR) are one of the largest membrane protein families in eukaryotes, with over 800 different GPCRs found in the human proteome (Shimada et al., 2019Shimada I. Ueda T. Kofuku Y. Eddy M.T. Wüthrich K. GPCR drug discovery: integrating solution NMR data with crystal and cryo-EM structures.Nat. Rev. Drug Discov. 2019; 18: 59-82Crossref PubMed Scopus (130) Google Scholar). Importantly, GPCRs are associated with a large number of human diseases (Tschöp et al., 2016Tschöp M.H. Finan B. Clemmensen C. Gelfanov V. Perez-Tilve D. Müller T.D. DiMarchi R.D. Unimolecular Polypharmacy for Treatment of Diabetes and Obesity.Cell Metab. 2016; 24: 51-62Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, Wacker et al., 2017Wacker D. Stevens R.C. Roth B.L. How Ligands Illuminate GPCR Molecular Pharmacology.Cell. 2017; 170: 414-427Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), a finding that has stimulated, over the last several decades, increased interest in identifying their endogenous ligands and mechanism of activation as well as the development of pharmacological agonists and antagonists with improved therapeutic efficacy (Hauser et al., 2017Hauser A.S. Attwood M.M. Rask-Andersen M. Schiöth H.B. Gloriam D.E. Trends in GPCR drug discovery: new agents, targets and indications.Nat. Rev. Drug Discov. 2017; 16: 829-842Crossref PubMed Scopus (1227) Google Scholar). In this respect, obesity and obesity-associated diseases, such as type 2 diabetes, hypertension, etc., are a therapeutic area in which the study of the biological mechanisms regulated by peptides/GPCRs and the development of drugs based on these mechanisms have flourished (Rodgers et al., 2012Rodgers R.J. Tschöp M.H. Wilding J.P.H. Anti-obesity drugs: past, present and future.Dis. Model. Mech. 2012; 5: 621-626Crossref PubMed Scopus (304) Google Scholar). For example, insulin and glucagon-like peptide 1 analogs are currently in the market or in advanced stages of clinical trials for several common diseases (Clemmensen et al., 2019Clemmensen C. Finan B. Müller T.D. DiMarchi R.D. Tschöp M.H. Hofmann S.M. Emerging hormonal-based combination pharmacotherapies for the treatment of metabolic diseases.Nat. Rev. Endocrinol. 2019; 15: 90-104Crossref PubMed Scopus (70) Google Scholar, Müller et al., 2018Müller T.D. Clemmensen C. Finan B. DiMarchi R.D. Tschöp M.H. Anti-Obesity Therapy: from Rainbow Pills to Polyagonists.Pharmacol. Rev. 2018; 70: 712-746Crossref PubMed Scopus (94) Google Scholar). Pharmacotherapy for obesity aimed at increasing lipolysis and free fatty acid utilization would be ideal because it would directly target the excess in fat stores. Lipolysis is a complex and finely regulated multistep biological process in which norepinephrine (NE), released by the postganglionic sympathetic neurons innervating adipose fat depots, activates the GPCR β-adrenergic receptors (β-ARs) on adipocyte membranes and the downstream signaling cascade, including cyclic AMP (cAMP)-dependent activation of protein kinase A (PKA), and phosphorylation and translocation of hormone-sensitive lipase (HSL) to lipid droplets (Arner, 1999Arner P. Catecholamine-induced lipolysis in obesity.Int. J. Obes. Relat. Metab. Disord. 1999; 23: 10-13Crossref PubMed Scopus (138) Google Scholar, Granneman and Moore, 2008Granneman J.G. Moore H.P.H. Location, location: protein trafficking and lipolysis in adipocytes.Trends Endocrinol. Metab. 2008; 19: 3-9Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, Lafontan and Langin, 2009Lafontan M. Langin D. Lipolysis and lipid mobilization in human adipose tissue.Prog. Lipid Res. 2009; 48: 275-297Crossref PubMed Scopus (545) Google Scholar, Lönnqvist et al., 1992Lönnqvist F. Wahrenberg H. Hellström L. Reynisdottir S. Arner P. Lipolytic catecholamine resistance due to decreased beta 2-adrenoceptor expression in fat cells.J. Clin. Invest. 1992; 90: 2175-2186Crossref PubMed Scopus (73) Google Scholar, Reilly and Saltiel, 2017Reilly S.M. Saltiel A.R. Adapting to obesity with adipose tissue inflammation.Nat. Rev. Endocrinol. 2017; 13: 633-643Crossref PubMed Scopus (630) Google Scholar). As an end result of lipolysis, free fatty acids and glycerol are released from adipocytes and delivered to other metabolic organs and undergo β-oxidation. Obesity perturbs the physiological regulation of lipolysis, leading to lipolytic catecholamine resistance, characterized by, among other molecular changes, downregulation of β-ARs and HSL (Arner, 1999Arner P. Catecholamine-induced lipolysis in obesity.Int. J. Obes. Relat. Metab. Disord. 1999; 23: 10-13Crossref PubMed Scopus (138) Google Scholar, Lafontan and Langin, 2009Lafontan M. Langin D. Lipolysis and lipid mobilization in human adipose tissue.Prog. Lipid Res. 2009; 48: 275-297Crossref PubMed Scopus (545) Google Scholar, Lönnqvist et al., 1992Lönnqvist F. Wahrenberg H. Hellström L. Reynisdottir S. Arner P. Lipolytic catecholamine resistance due to decreased beta 2-adrenoceptor expression in fat cells.J. Clin. Invest. 1992; 90: 2175-2186Crossref PubMed Scopus (73) Google Scholar). Identifying new mechanisms that can overcome lipolytic catecholamine resistance and safely enhance lipolysis would be extremely helpful for obese patients because it would directly target excessive fat mass (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Reilly and Saltiel, 2017Reilly S.M. Saltiel A.R. Adapting to obesity with adipose tissue inflammation.Nat. Rev. Endocrinol. 2017; 13: 633-643Crossref PubMed Scopus (630) Google Scholar). However, previous attempts largely failed because most potent lipolytic sympathomimetic drugs (such as β-AR agonists) induced adverse cardiovascular effects or aggravated metabolic syndrome (Clapham and Arch, 2011Clapham J.C. Arch J.R.S. Targeting thermogenesis and related pathways in anti-obesity drug discovery.Pharmacol. Ther. 2011; 131: 295-308Crossref PubMed Scopus (23) Google Scholar, James et al., 2010James W.P.T. Caterson I.D. Coutinho W. Finer N. Van Gaal L.F. Maggioni A.P. Torp-Pedersen C. Sharma A.M. Shepherd G.M. Rode R.A. Renz C.L. SCOUT InvestigatorsEffect of sibutramine on cardiovascular outcomes in overweight and obese subjects.N. Engl. J. Med. 2010; 363: 905-917Crossref PubMed Scopus (658) Google Scholar, Rodgers et al., 2012Rodgers R.J. Tschöp M.H. Wilding J.P.H. Anti-obesity drugs: past, present and future.Dis. Model. Mech. 2012; 5: 621-626Crossref PubMed Scopus (304) Google Scholar). Several neurotransmitters and neuropeptides, often co-secreted with NE by sympathetic nerves, endocrine glands, or macrophages/adipocytes and targeting GPCRs on adipocyte membranes play a key role as sensitizers of adrenergic-induced lipolysis (Bandyopadhyay et al., 2012Bandyopadhyay G.K. Vu C.U. Gentile S. Lee H. Biswas N. Chi N.W. O’Connor D.T. Mahata S.K. Catestatin (chromogranin A(352-372)) and novel effects on mobilization of fat from adipose tissue through regulation of adrenergic and leptin signaling.J. Biol. Chem. 2012; 287: 23141-23151Crossref PubMed Scopus (48) Google Scholar, Nguyen et al., 2011Nguyen K.D. Qiu Y. Cui X. Goh Y.P.S. Mwangi J. David T. Mukundan L. Brombacher F. Locksley R.M. Chawla A. Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis.Nature. 2011; 480: 104-108Crossref PubMed Scopus (771) Google Scholar, Pellegrinelli et al., 2018Pellegrinelli V. Peirce V.J. Howard L. Virtue S. Türei D. Senzacqua M. Frontini A. Dalley J.W. Horton A.R. Bidault G. et al.Adipocyte-secreted BMP8b mediates adrenergic-induced remodeling of the neuro-vascular network in adipose tissue.Nat. Commun. 2018; 9: 4974Crossref PubMed Scopus (78) Google Scholar, Pirzgalska et al., 2017Pirzgalska R.M. Seixas E. Seidman J.S. Link V.M. Sánchez N.M. Mahú I. Mendes R. Gres V. Kubasova N. Morris I. et al.Sympathetic neuron-associated macrophages contribute to obesity by importing and metabolizing norepinephrine.Nat. Med. 2017; 23: 1309-1318Crossref PubMed Scopus (232) Google Scholar, Villarroya and Vidal-Puig, 2013Villarroya F. Vidal-Puig A. Beyond the sympathetic tone: the new brown fat activators.Cell Metab. 2013; 17: 638-643Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, Whittle et al., 2012Whittle A.J. Carobbio S. Martins L. Slawik M. Hondares E. Vázquez M.J. Morgan D. Csikasz R.I. Gallego R. Rodriguez-Cuenca S. et al.BMP8B increases brown adipose tissue thermogenesis through both central and peripheral actions.Cell. 2012; 149: 871-885Abstract Full Text Full Text PDF PubMed Scopus (424) Google Scholar). These sensitizer mechanisms have the potential to serve as targets alternative to or synergistic with β-ARs agonists for pharmacotherapy of obesity, directly targeting excess adiposity, but so far, this approach remains poorly understood and investigated. In the present study, we focused the complement 3a receptor (C3aR1) (Hollmann et al., 1998Hollmann T.J. Haviland D.L. Kildsgaard J. Watts K. Wetsel R.A. Cloning, expression, sequence determination, and chromosome localization of the mouse complement C3a anaphylatoxin receptor gene.Mol. Immunol. 1998; 35: 137-148Crossref PubMed Scopus (23) Google Scholar), a receptor expressed in adipocytes (Mamane et al., 2009Mamane Y. Chung Chan C. Lavallee G. Morin N. Xu L.J. Huang J. Gordon R. Thomas W. Lamb J. Schadt E.E. et al.The C3a anaphylatoxin receptor is a key mediator of insulin resistance and functions by modulating adipose tissue macrophage infiltration and activation.Diabetes. 2009; 58: 2006-2017Crossref PubMed Scopus (126) Google Scholar, Quell et al., 2017Quell K.M. Karsten C.M. Kordowski A. Almeida L.N. Briukhovetska D. Wiese A.V. Sun J. Ender F. Antoniou K. Schröder T. et al.Monitoring C3aR Expression Using a Floxed tdTomato-C3aR Reporter Knock-in Mouse.J. Immunol. 2017; 199: 688-706Crossref PubMed Scopus (43) Google Scholar), and its ligand, the VGF-derived neuropeptide TLQP-21, a sensitizer of β-AR-induced lipolysis shown previously to oppose obesity and normalize molecular markers of catecholamine resistance without causing cardiovascular side effects, a chronic increase in circulating fatty acids, or ectopic fat deposition in mice, overall suggesting a concomitant increase in tissue β-oxidation (Bartolomucci et al., 2006Bartolomucci A. La Corte G. Possenti R. Locatelli V. Rigamonti A.E. Torsello A. Bresciani E. Bulgarelli I. Rizzi R. Pavone F. et al.TLQP-21, a VGF-derived peptide, increases energy expenditure and prevents the early phase of diet-induced obesity.Proc. Natl. Acad. Sci. USA. 2006; 103: 14584-14589Crossref PubMed Scopus (127) Google Scholar, Cero et al., 2014Cero C. Vostrikov V.V. Verardi R. Severini C. Gopinath T. Braun P.D. Sassano M.F. Gurney A. Roth B.L. Vulchanova L. et al.The TLQP-21 peptide activates the G-protein-coupled receptor C3aR1 via a folding-upon-binding mechanism.Structure. 2014; 22: 1744-1753Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Fargali et al., 2014Fargali S. Garcia A.L. Sadahiro M. Jiang C. Janssen W.G. Lin W.J. Cogliani V. Elste A. Mortillo S. Cero C. et al.The granin VGF promotes genesis of secretory vesicles, and regulates circulating catecholamine levels and blood pressure.FASEB J. 2014; 28: 2120-2133Crossref PubMed Scopus (35) Google Scholar, Guo et al., 2018Guo Z. Sahu B.S. He R. Finan B. Cero C. Verardi R. Razzoli M. Veglia G. Di Marchi R.D. Miles J.M. Bartolomucci A. Clearance kinetics of the VGF-derived neuropeptide TLQP-21.Neuropeptides. 2018; 71: 97-103Crossref PubMed Scopus (10) Google Scholar, Jethwa et al., 2007Jethwa P.H. Warner A. Nilaweera K.N. Brameld J.M. Keyte J.W. Carter W.G. Bolton N. Bruggraber M. Morgan P.J. Barrett P. Ebling F.J. VGF-derived peptide, TLQP-21, regulates food intake and body weight in Siberian hamsters.Endocrinology. 2007; 148: 4044-4055Crossref PubMed Scopus (79) Google Scholar, Possenti et al., 2012Possenti R. Muccioli G. Petrocchi P. Cero C. Cabassi A. Vulchanova L. Riedl M.S. Manieri M. Frontini A. Giordano A. et al.Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.Biochem. J. 2012; 441: 511-522Crossref PubMed Scopus (50) Google Scholar). Here, combining pharmacology, homology modeling, and evolutionary analysis, we first characterized the mechanism of TLQP-21 potentiation of β-AR-induced lipolysis in adipocytes and subsequently identified a unique peptide/receptor co-evolution in the Murinae subfamily of rodents, which explains its pharmacological efficacy by virtue of enhanced hydrophobicity representing a gain of function for a neuropeptide that becomes a potent agonist at C3aR1. Because we also demonstrated that changes in gene expression of critical nodes in TLQP-21/C3aR1-mediated lipolysis are conserved in obesity in mice and humans, and because the rodent peptide retains its biological activity in humans (potentiation of adrenergic-induced lipolysis), our discovery can pave the way for investigation of this molecular target to develop innovative pharmacotherapies for obesity and associated diseases. To test the hypothesis that C3aR1 is required for TLQP-21-mediated lipolysis and to understand the molecular mechanism of action of TLQP-21, we generated C3aR1 knockdown (KD) 3T3-L1 cell lines. Cells were infected with 3 different lentiviruses harboring short hairpin RNA (shRNA) against C3aR1, or a non-targeting shRNA was used as a control. qPCR showed strong depletion of C3aR1 in KD cells (Figure 1A). We selected one of the KD lines for the experiments and replicated critical findings in a second cell line (see below). Adipogenesis and expression of key genes involved in adipogenesis and lipolysis, such as PPAR-γ, leptin, βARs, HSL, etc., were normal, except for a modest increase in β3AR (Figure 1A; Figure S1). C3aR1 KD and shRNA control cells were differentiated in adipocytes and incubated with TLQP-21 (using previously validated doses; Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar) in the presence or absence of the nonselective β-AR agonist isoproterenol (ISO), essentially as established previously (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar) and as described in the STAR Methods. As expected (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Possenti et al., 2012Possenti R. Muccioli G. Petrocchi P. Cero C. Cabassi A. Vulchanova L. Riedl M.S. Manieri M. Frontini A. Giordano A. et al.Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.Biochem. J. 2012; 441: 511-522Crossref PubMed Scopus (50) Google Scholar), TLQP-21 enhanced ISO-induced lipolysis in control cells without being pro-lipolytic per se (Figure 1B). Conversely, TLQP-21 failed to potentiate ISO-induced lipolysis in C3aR1 KD cells (Figure 1B). Essentially the same phenotype was obtained in a second independent cell line harboring a different shRNA targeting C3aR1 (Figure S1F), ruling out nonspecific targeting of the shRNA. Next we carried out immunoblots and densitometry analysis for phosphorylated hormone sensitive lipase (pHSL) and phosphorylated extracellular signal–regulated kinase (pERK), the key signaling nodes in the TLQP-21 pro-lipolytic pathway (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Possenti et al., 2012Possenti R. Muccioli G. Petrocchi P. Cero C. Cabassi A. Vulchanova L. Riedl M.S. Manieri M. Frontini A. Giordano A. et al.Characterization of a novel peripheral pro-lipolytic mechanism in mice: role of VGF-derived peptide TLQP-21.Biochem. J. 2012; 441: 511-522Crossref PubMed Scopus (50) Google Scholar). Consistent with previous findings, incubating 3T3-L1 adipocytes with TLQP-21 enhanced pERK and potentiated ISO-induced phosphorylation of ERK and HSL (Figure 1C). Importantly, experiments conducted in C3aR1 KD cells established that C3aR1 expression is necessary for TLQP-21-induced phosphorylation of ERK and potentiation of ISO-induced pERK and pHSL (Figure 1C). Overall, our data demonstrate that C3aR1 is necessary for TLQP-21-mediated lipolysis via ERK activation of HSL in murine adipocytes. However, the upstream signals remained unknown. To answer this question, we focused on the mechanism of peptide-mediated calcium uptake. TLQP-21-mediated lipolysis can be prevented by pretreatment with the calcium chelant EGTA, suggesting calcium influx from the extracellular compartment (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar). However, the mechanism is not well understood. We carried out PCR-based microarray experiments and found a number of calcium channels expressed in both 3T3-L1 fibroblasts and adipocytes (Table S2), including transient receptor potential channel 1 (TRPC1), TRPC5, and TRPC6, channels that have been identified previously in this cell type (Sukumar et al., 2012Sukumar P. Sedo A. Li J. Wilson L.A. O’Regan D. Lippiat J.D. Porter K.E. Kearney M.T. Ainscough J.F.X. Beech D.J. Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin.Circ. Res. 2012; 111: 191-200Crossref PubMed Scopus (72) Google Scholar). Based on the TLQP-21 pharmacological profile, we tested the hypothesis that C3aR1 is functionally coupled to a putative TRPC channel upon TLQP-21 activation, leading to calcium influx from the extracellular compartment. To test this hypothesis, we used two methods, the non-ratiometric Fluo-4 Ca2+ indicator and radiometric Ca452+ assays. Fluo-4, unlike ratiometric indicators such as Fura-2-acetoxymethyl ester (FURA-2AM), is less prone to quenching of the signal by lipid droplets in adipocytes and more sensitive to transient calcium influx, which would be undetectable by FURA-2AM (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Sukumar et al., 2012Sukumar P. Sedo A. Li J. Wilson L.A. O’Regan D. Lippiat J.D. Porter K.E. Kearney M.T. Ainscough J.F.X. Beech D.J. Constitutively active TRPC channels of adipocytes confer a mechanism for sensing dietary fatty acids and regulating adiponectin.Circ. Res. 2012; 111: 191-200Crossref PubMed Scopus (72) Google Scholar). Using the Fluo-4 assay, we established that TLQP-21 rapidly and concentration-dependently increased intracellular calcium ([Ca2+]i) in 3T3-L1 cells (Figures S2A and S2B). This effect is specific to TLQP-21/C3aR1 activation, as demonstrated by the following: (1) the TLQP-21 mutant R21A (Cero et al., 2014Cero C. Vostrikov V.V. Verardi R. Severini C. Gopinath T. Braun P.D. Sassano M.F. Gurney A. Roth B.L. Vulchanova L. et al.The TLQP-21 peptide activates the G-protein-coupled receptor C3aR1 via a folding-upon-binding mechanism.Structure. 2014; 22: 1744-1753Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) did not increase [Ca2+]i (Figure S2A); (2) the C3aR1 antagonist SB290157 antagonized TLQP-21-mediated calcium influx (Figure 2A); (3) C3aR1 KD prevented the TLQP-21-mediated increase in [Ca2+]i (Figure 2B; Figure S2C). Additionally, multiple pharmacological experiments allowed us to exclude a role of the C5a receptor in TLQP-21-induced increases in [Ca2+]i (Figures S3A and S3B). Next, to unambiguously establish that TLQP-21 causes an increase in calcium from the extracellular compartment, we used a high-sensitivity radiometric Ca452+ assay. TLQP-21 caused significant Ca452+ uptake compared with control treatment, whereas the R21A mutant consistently failed to elicit calcium influx (Figure 2C; Figure S4A). Additionally, ISO per se did not increase Ca452+ uptake under our experimental conditions and did not potentiate TLQP-21-mediated effects (Figure 2D), further supporting the hypothesis that the first messengers downstream of NE- and TLQP-21-mediated signaling pathways are independent in our cellular model. Next we directly tested the hypothesis that TLQP-21 mediates lipolysis via activation of a TRPC channel. We first measured TLQP-21-mediated calcium influx in 3T3-L1 cells in the presence or absence of the pan-specific TRPC inhibitor SKF-96365 (Singh et al., 2010Singh A. Hildebrand M.E. Garcia E. Snutch T.P. The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels.Br. J. Pharmacol. 2010; 160: 1464-1475Crossref PubMed Scopus (135) Google Scholar). SKF-96365 blocked the TLQP-21-induced increase in [Ca2+]i, whereas the response to the purinergic agonist uridine triphosphate (UTP) was maintained even in the presence of a mild SKF-96365-induced elevation of basal fluorescence (Figure 2E; Figure S4B). Next we incubated 3T3-L1 adipocytes with TLQP-21 in the presence or absence of ISO and in the presence or absence of SKF-96365. SKF-96365 concentration-dependently blocked TLQP-21-mediated enhancement of ISO-induced lipolysis without affecting basal or ISO-induced lipolysis (Figure 2F; Figures S4C and S4D). Because SKF-96365 can potentially inhibit store-operated calcium release (Sabourin et al., 2016Sabourin J. Bartoli F. Antigny F. Gomez A.M. Benitah J.P. Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES.J. Biol. Chem. 2016; 291: 13394-13409Crossref PubMed Scopus (49) Google Scholar), we also carried out a second set of lipolysis experiments, incubating adipocytes with AncoA4, a selective inhibitor of store-operated calcium release (Sadaghiani et al., 2014Sadaghiani A.M. Lee S.M. Odegaard J.I. Leveson-Gower D.B. McPherson O.M. Novick P. Kim M.R. Koehler A.N. Negrin R. Dolmetsch R.E. Park C.Y. Identification of Orai1 channel inhibitors by using minimal functional domains to screen small molecule microarrays.Chem. Biol. 2014; 21: 1278-1292Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Unlike SKF-96365, AncoA4 did not prevent TLQP-21-mediated lipolysis (Figure S4E), further strengthening the conclusion that TLQP-21 activates calcium influx from the extracellular compartment (Figure 2). Finally, Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated by increased [Ca2+]i and causes phosphorylation of the ERK/HSL pathways (Rapold et al., 2013Rapold R.A. Wueest S. Knoepfel A. Schoenle E.J. Konrad D. Fas activates lipolysis in a Ca2+-CaMKII-dependent manner in 3T3-L1 adipocytes.J. Lipid Res. 2013; 54: 63-70Crossref PubMed Scopus (21) Google Scholar), which are critical nodes in TLQP-21 signaling (Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar). Consistently, the CaMKII inhibitor KN-62 prevented TLQP-21-induced potentiation of ISO-induced lipolysis without affecting basal or ISO-induced lipolysis per se (Figure S4F). Overall, our results demonstrate that the TLQP-21 lipolytic mechanism is mediated by C3aR1 activation of a TRPC channel that is responsible for calcium uptake from the extracellular compartment and activation of the CaMKII/ERK signaling cascade, resulting in potentiation of adrenergic-induced HSL phosphorylation. Having identified the TLQP-21 mechanism of action in vitro, we aimed to establish whether the identified signaling pathway is conserved in mouse and human obesity. The current knowledge of TLQP-21/C3aR1-mediated biological effects is limited to rodent models. Therefore, we first aimed to determine whether the critical molecular nodes in the TLQP-21/C3aR1 lipolytic pathway identified in this study and elsewhere are conserved in adipose tissue in humans (Figure 3A). We quantified the expression of several genes in 208 well-characterized and non-diabetic humans (see STAR Methods for a description of the study population). We observed a negative correlation between BMI and/or percent fat mass with β2ARs, TRPC1, and HSL and a positive association of the same biometrical parameters with C3aR1 (Figure 3B). Consistently, 9 weeks of a high-fat diet (HFD) induced very similar molecular changes in obese mice, with a significant increase in C3aR1 expression and downregulation of βARs (particularly β3AR), HSL, and TRPC1 (Figures 3C and 3D). Conversely, the expression of C5aR1 and C5aR2, receptors closely related to C3aR1, is not significantly associated with obesity in humans or in mice (Figure S3C). These results suggest that the molecular pathway critical for TLQP-21-mediated lipolysis is conserved in obese mice and humans. Specifically, although C3aR1 is upregulated in adipose tissue in obesity in both species, the critical nodes downstream of C3aR1 (i.e., TRPC1 and HSL) are downregulated in obesity. This finding is in line with and adds to the existing literature on molecular changes observed in lipolytic catecholamine resistance (Arner, 1999Arner P. Catecholamine-induced lipolysis in obesity.Int. J. Obes. Relat. Metab. Disord. 1999; 23: 10-13Crossref PubMed Scopus (138) Google Scholar, Cero et al., 2016Cero C. Razzoli M. Han R. Sahu B.S. Patricelli J. Guo Z. Zaidman N.A. Miles J.M. O’Grady S.M. Bartolomucci A. The neuropeptide TLQP-21 opposes obesity via C3aR1-mediated enhancement of adrenergic-induced lipolysis.Mol. Metab. 2016; 6: 148-158Crossref PubMed Scopus (23) Google Scholar, Reilly and Saltiel, 2017Reilly S.M. Saltiel A.R. Adapting to obesity with adipose tissue inflammation.Nat. Rev. Endoc" @default.
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• W2972134410 date "2019-09-01" @default.
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• W2972134410 title "Peptide/Receptor Co-evolution Explains the Lipolytic Function of the Neuropeptide TLQP-21" @default.
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