TLQP-21, a VGF-derived , increases energy expenditure and prevents the early phase of diet-induced obesity

A. Bartolomuccia,b,c, G. La Corteb,d, R. Possentid,e, V. Locatellif, A. E. Rigamontig, A. Torsellof, E. Brescianif, I. Bulgarellif, R. Rizzie, F. Pavonea, F. R. D’Amatoa, C. Severinid, G. Mignognah, A. Giorgih, M. E. Schinina` h, G. Eliai, C. Branciaj, G.-L. Ferrij, R. Contik, B. Cianik, T. Pascuccil, G. Dell’Omom, E. E. Mullerg, A. Levic,d,n, and A. Molesa,c,n

aInstitute of Neuroscience, Consiglio Nazionale delle Ricerche, 00143 Rome, Italy; dInstitute of Neurobiology and Molecular Medicine, Consiglio Nazionale delle Ricerche, 00143 Rome, Italy; eDepartment of Neuroscience, University of Roma II–Tor Vergata, 00161 Rome, Italy; fDepartment of Experimental and Environmental Medicine and Biotechnology and Interdepartmental Center for Bioinformatics and Proteomics, University of Milan–Bicocca, 20052 Monza, Italy; gDepartment of Pharmacology, Chemotherapy, and Medical Toxicology, University of Milan, 20129 Milan, Italy; hDepartment of Biochemical Science, University ‘‘La Sapienza,’’ 00185 Rome, Italy; iDepartment of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Eidgeno¨ssische Technische Hochschule Zu¨rich, CH-8093 Zu¨rich, Switzerland; jNEF Laboratory, Department of Cytomorphology, University of Cagliari, 09042 Monserrato, Italy; kDepartment of Endocrinology and , Sigma-Tau Pharmaceuticals Industries S.p.A., 00040 Rome, Italy; lFoundation Santa Lucia, 00143 Rome, Italy; and mInstitute of Anatomy and Center for Neuroscience, University of Zu¨rich, CH-8057 Zu¨rich, Switzerland

Communicated by Rita Levi-Montalcini, European Brain Research Institute, Rome, Italy, July 20, 2006 (received for review May 15, 2006) The vgf gene has been identified as an energy homeostasis arcuate nucleus of fasted rats (14) and hamsters that are exposed regulator. Vgf encodes a 617-aa precursor protein that is processed to a short or long day’s length (16). However, up until now, it was to yield an incompletely characterized panel of . still unproved that VGF-derived are metabolic neuro- Until now, it was an unproved assumption that VGF-derived modulators (13). Vgf encodes a 617-aa protein in rodents that, upon peptides could regulate metabolism. Here, a VGF peptide desig- processing by prohormone convertase (PC) 1͞3 and PC2, yields nated TLQP-21 was identified in rat brain extracts by means of several peptides that are stored in dense core granules and secreted immunoprecipitation, microcapillary liquid chromatography–tan- through the regulated pathway (13, 17). Based on the phenotype of dem MS, and database searching algorithms. Chronic intracerebro- VGFϪ/Ϫ mice (14, 15), we predicted that VGF-derived peptides are ventricular (i.c.v.) injection of TLQP-21 (15 ␮g͞day for 14 days) anabolic neuropeptides. Here, we identified in rat brain extracts a increased resting energy expenditure (EE) and rectal temperature previously uncharacterized VGF-derived peptide, designated as in mice. These effects were paralleled by increased epinephrine TLQP-21, which spans from residue 556 to residue 576 of the and up-regulation of brown adipose tissue ␤2-AR (␤2 adrenergic precursor sequence. Contrary to our prediction, TLQP-21 increases receptor) and white adipose tissue (WAT) PPAR-␦ (peroxisome energy expenditure (EE) and blocks the early phase of high-fat proliferator-activated receptor ␦), ␤3-AR, and UCP1 (uncoupling diet-induced obesity. protein 1) mRNAs and were independent of locomotor activity and thyroid hormones. Hypothalamic gene expression of orexigenic Results and anorexigenic neuropeptides was unchanged. Furthermore, in Identification of TLQP-21. VGF peptides were immunopurified and mice that were fed a high-fat diet for 14 days, TLQP-21 prevented characterized from rat brain extracts by means of an antiserum the increase in body and WAT weight as well as hormonal changes raised against the N terminus of TLQP-62 (residues 556–617), a that are associated with a high-fat regimen. Biochemical and major VGF form in the CNS (18). MALDI-TOF MS analysis of molecular analyses suggest that TLQP-21 exerts its effects by immunoprecipitates revealed one pseudomolecular ion (MHϩ)at stimulating autonomic activation of adrenal medulla and adipose a mass-to-charge ratio (m͞z) of 2,433.53 (Fig. 1a) corresponding to tissues. In conclusion, we present here the identification in the CNS the peptide at position 556–576 of the VGF sequence, designated of a previously uncharacterized VGF-derived peptide and prove TLQP-21. In addition, the singly charged as well as the doubly that its chronic i.c.v. infusion effected an increase in EE and limited charged ions of TLQP-62 were detected at m͞z ϭ 7,403.19 and the early phase of diet-induced obesity. 3,701.79, respectively (Fig. 1a). TLQP-21 was unambiguously iden- tified by microcapillary liquid chromatography–tandem MS (MS͞ autonomic nervous system ͉ ␤ adrenergic receptor ͉ MALDI-TOF ͉ MS) analysis of the immunoprecipitates and sequence database ͉ peroxisome proliferator-activated receptor ␦ searching algorithms. TLQP-21 was separated and detected as a doubly charged ion at m͞z ϭ 1,217.27 (Fig. 1b). On the basis of the nergy homeostasis is a complex physiological function that is MS͞MS spectra resulting from low-energy collision-induced disso- Ecoordinated at multiple levels. Stimulated by the discovery of ciation of the precursor ion (Fig. 1c), an automated SEQUEST and the pandemic diffusion of obesity and type-2 diabetes, search against the National Center for Biotechnology Information the regulation of energy homeostasis has received increasing at- nonredundant database indicated a cross-correlation value (Xcorr) tention (1–4). New players are being continuously identified and of 1.75 and a ⌬ correlation value (dCn) of 0.09 (Table 1, which is screened as molecular candidates to counteract obesity (5–10). Vgf, initially identified as a -responsive gene, is also robustly induced by BDNF and neurotrophin 3 and marginally Conflict of interest statement: No conflicts declared. induced by epidermal and fibroblast growth factors, IL-6, and Abbreviations: BAT, brown adipose tissue; ␤-AR, ␤ adrenergic receptor; UCP1, uncoupling protein 1; WAT, white adipose tissue; i.c.v., intracerebroventricular; PPAR-␦, peroxisome (11–13). Vgf received great attention after the observation proliferator-activated receptor ␦;MS͞MS, tandem MS; EE, energy expenditure; NE, nor- that VGF-deficient mice are lean, hypermetabolic, and resistant to epinephrine; E, epinephrine; WAT͞bw, WAT͞body weight; TG, triglyceride; FFA, free fatty various types of obesity (14, 15). In the rat brain, VGF is abundant acid; aCSF, artificial cerebrospinal fluid. in the cortex, hypothalamus, , and olfactory system bA.B. and G.L.C. contributed equally to this work. and in a number of thalamic, septal, amygdaloid, and brainstem nA.L. and A.M. contributed equally to this work. nuclei, with the local availability of neurotrophins for receptor cTo whom correspondence may be addressed. E-mail: [email protected], a.levi@ occupation being the critical parameter in determining its selective inmm.cnr.it, or [email protected]. expression (12, 13). Changes in vgf expression also increase in the © 2006 by The National Academy of Sciences of the USA

14584–14589 ͉ PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606102103 Downloaded by guest on September 25, 2021 Fig. 2. Physiological effects in mice receiving TLQP-21. (a)EE(n ϭ 6 per group). (b) Rectal temperature (n ϭ 8 per group). (c) E levels. (d) NE levels. (e) TG levels. (f) FFA͞TG ratio. Unless otherwise stated, n ϭ 16–18 per group. *, P Ͻ 0.05.

weight and food intake were unaffected, whereas WAT͞bw and circulating leptin were slightly reduced (Fig. 7 and Table 2). TLQP-21 treatment also lowered circulating triglycerides (TGs) but not free fatty acids (FFAs) or glucose; therefore, an increased FFA͞TG ratio was observed (Fig. 2 e and f; TG, U(17,18) ϭ 66, P Ͻ 0.01; FFA͞TG, U(17,18) ϭ 72, P Ͻ 0.01). TG also was positively Fig. 1. TLQP-21 indentification. (a) MALDI-TOF MS spectrum-immuno- ͞ r ϭ P Ͻ selected VGF peptides. (b) MicroLC MS͞MS full mass spectrum recorded at a correlated with WAT bw ( 0.35; 0.05). retention time of 21 min and 41 sec, showing the doubly charged ion corre- In the glucose tolerance test, TLQP-21 treatment reduced TG sponding to TLQP-21 at an m͞z of 1,217.3. (c) Fragment ion spectrum. levels (F(1,9) ϭ 6.1, P Ͻ 0.05; post hoc, P Ͻ 0.05) and, to a lesser extent, FFA levels (F(3,27) ϭ 6.4, P Ͻ 0.01; post hoc, P ϭ 0.079) 30 min after glucose load (Fig. 3 c and d), leading to a transient ͞ ϭ Ͻ published as supporting information on the PNAS web site). normalization in the FFA TG ratio (Fig. 3e; F(3,27) 3.1, P Manual interpretation of the MS͞MS analysis by a MASCOT 0.05; control vs. TLQP-21 at baseline, 60 min, and 120 min, Ͻ search in the Sequence Query mode led to the identification of the P 0.05; TLQP-21 at 30 min vs. baseline, 60 min, and 120 Ͻ TLQPPASSRRRHFHHALPPAR peptide with a high confidence min, P 0.05). (ion score ϭ 62; P ϭ 0.0083). Furthermore, TLQP-21 was identified TLQP-21 Up-Regulates ␤ Adrenergic Receptor (␤-AR), Peroxisome as a triply charged ion by SEQUEST in a second independent ␦ ␦ experiment (Xcorr ϭ 2.98; dCn ϭ 0.44; data not shown). Proliferator-Activated Receptor (PPAR- ), and Uncoupling Protein 1 (UCP1) Gene Expression in the Adipose Tissue. Hypothalamic expres- sion of agouti-related peptide, , melanocyte- TLQP-21 Upsets Energy Balance. Fourteen-day i.c.v. TLQP-21 treat- concentrating hormone, , and corticotropin- ment increased EE at rest (ϩ12%; P Ͻ 0.05) but less so when releasing hormone was unaffected by TLQP-21 (Table 3, which is animals showed moderate (ϩ7%) or high (ϩ4%) activity level published as supporting information on the PNAS web site). (F ϭ 5.6; P Ͻ 0.05; Fig. 2a). TLQP-21 also increased rectal (1,10) TLQP-21 determined major changes in the adipose tissues. temperature (T) (Fig. 2b; U(8,8) ϭ 10; P Ͻ 0.05). Increased EE and ␤2-AR mRNA increased in the BAT (Fig. 4a; U(5,6) ϭ 3; P Ͻ 0.05) T were paralleled by increased serum epinephrine (E) and de- ϭ Ͻ ϭ and was positively correlated with serum E (r 0.68; P 0.05) and creased norepinephrine (NE) levels (Fig. 2 c and d;E,U(16,18) FFA͞TG (r ϭ 0.7; P Ͻ 0.05). In the WAT, TLQP-21 increased Ͻ ϭ Ͻ 75.5, P 0.05; NE, U(17,18) 93, P 0.05) independent of PPAR-␦ and UCP1 mRNAs, whereas ␤3-AR expression slightly locomotor activity and free triiodothyronine and free thyroxine ␦ ϭ Ͻ ϭ

increased (Fig. 4 b–d; PPAR- , U(5,5) 3, P 0.05; UCP1, U(4,6) NEUROSCIENCE serum levels (Fig. 7 and Table 2, which are published as supporting 3, P ϭ 0.05; ␤3-AR, U(5,6) ϭ 5, P ϭ 0.06). ␤3-AR positively information on the PNAS web site). Brown adipose tissue correlated with PPAR-␦ (r ϭ 0.77; P Ͻ 0.05) and FFA͞TG ratio (BAT)-NE was slightly increased in TLQP-21-treated mice (con- (r ϭ 0.61; P Ͻ 0.05) but negatively correlated with serum NE (r ϭ trol, 13.2 Ϯ 1.2 ng͞mg; TLQP-21, 15.1 Ϯ 1.2 ng͞mg), and white Ϫ0.62; P Ͻ 0.05), WAT͞bw (r ϭϪ0.66; P Ͻ 0.05), and TG levels adipose tissue (WAT)-NE was inversely correlated with relative (r ϭϪ0.8; P Ͻ 0.01). WAT͞body weight (WAT͞bw) (r ϭϪ0.6; P Ͻ 0.01). T and E values were positively correlated (r ϭ 0.56; P Ͻ 0.05). TLQP-21 also TLQP-21 Confers Resistance to the Early Phase of Diet-Induced Obe- increased adrenal gland weight, whereas corticosterone level re- sity. We investigated whether TLQP-21 could prevent the early mained unaffected (U(17,18) ϭ 89; P Ͻ 0.05; Table 2). Finally, body phase of obesity induced by 14 days of a high-fat diet (20% lard

Bartolomucci et al. PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 ͉ 14585 Downloaded by guest on September 25, 2021 Fig. 5. Physiological and behavioral parameters in mice receiving a high-fat (HF) diet. Shown are body weight changes from baseline (a), food intake (b), caloric efficiency (c), and WAT͞bw (d). Standard, standard chow; Con, mice treated with aCSF. n ϭ 11–12 per group; *, P Ͻ 0.05.

Fig. 3. Glucose tolerance test. Basal levels and levels 30, 60, and 120 min after the rise of leptin of HF-CON mice and a normalization of . oral glucose (3 mg͞kg) treatment are shown for the following: insulin (a), glucose TG levels, but not FFA or glucose levels, decreased in both (b), FFAs (c), TGs (d), and FFA͞TG ratio (e). n ϭ 6 per group; *, P Ͻ 0.05 control vs. HF-treated groups compared with ST-CON, whereas HF- TLQP-21; °, TLQP-21 at 30 min vs. TLQP-21 at baseline, 60 min, and 120 min. TLQP-21 did not differ from ST-TLQP-21 (Fig. 6b; TG, diet, F(1,52) ϭ 21.3, P Ͻ 0.001; diet X treatment, F(1,52) ϭ 13.7, P Ͻ 0.001). Accordingly, HF resulted in up-regulation of the FFA͞TG ratio in addition to rodent chow; HF). As expected, control animals (HF- both HF groups when compared with ST-CON. HF-CON, and CON) showed increased body weight gain (Fig. 5a; post hoc, day 13, ϭ Ͻ ϭ Ͻ slightly so HF-TLQP-21 (P 0.06), also differed from ST-TLQP-21 P 0.01), caloric efficiency (Fig. 5c; diet, F(1,51) 4.3, P 0.05; diet ϭ Ͻ ϭ ϭ ͞ mice for the same parameter (Fig. 6b; diet, F(1,52) 28.6, P X treatment, F(1,51) 4.3, P 0.08), and WAT bw (Fig. 5d; F ϭ P Ͻ ϭ Ͻ ϭ Ͻ 0.0001; treatment X diet, (1,52) 2.7, 0.05; see Table 4, which treatment, F(1,54) 5.7, P 0.05; diet, F(1,54) 18.6, P 0.0001) is published as supporting information on the PNAS web site, for when compared with mice receiving a standard diet (ST-CON and further results). ST-TLQP-21). TLQP-21 conferred resistance to obesity: HF- TLQP-21 mice showed almost unaffected body and WAT weight Discussion and caloric efficiency (Fig. 5). These effects occurred despite the TLQP-21 Is a Neuropeptide That Upsets Energy Balance. Ever since the fact that HF- and ST-fed mice ingested a similar number of discovery that targeted deletion of the Vgf gene determined a lean, kilocalories and showed similar locomotor activity (Fig. 5b; see also hypermetabolic, obesity-resistant phenotype (14, 15), it has been Figs. 7 and 8, which are published as supporting information on the crucial to know the molecular basis of Vgf’s activity on energy PNAS web site). balance. The present study shows the immunoprecipitation from rat As expected, HF-CON mice had increased serum leptin (Fig. 6a; brain extracts of a previously uncharacterized VGF peptide desig- diet, F(1,34) ϭ 13.8; P Ͻ 0.0001) and decreased ghrelin (diet, F(1,38) ϭ nated TLQP-21 and its identification by MS͞MS and database 10.9; P Ͻ 0.001). On the contrary, HF-TLQP-21 mice showed half search engines. TLQP-21 appears to derive from further processing of TLQP-62, an abundant VGF C-terminal peptide that is produced inside dense core vesicles by prohormone convertase 1͞3 (18). The lean and hypermetabolic phenotype of VGFϪ/Ϫ mice led us to predict that VGF peptides are anabolic neuropeptides. On the contrary, we demonstrated that chronic i.c.v. treatment of TLQP-21 increased resting EE, rectal temperature, and serum E without affecting body weight or food intake. Increased EE was not paralleled by changes in locomotor activity or thyroid hormones. In addition, it was not paralleled by increased gene expression of ␤3-AR or UCP1 expression in the BAT and was accompanied by only a slight increase of BAT-NE. Therefore, activation of BAT thermogenesis does not appear to be the primary mediator of the effects observed (19, 20). However, increased BAT ␤2-AR expres- sion (42% increase) might contribute to EE and hyperthermia (4, 19–21). On the contrary, increased EE and T may be attributed to the permissive role of increased E (22) and to changes occurring in the WAT. WAT receives sympathetic innervation downstream of Fig. 4. ␤2-AR mRNA in the BAT (a) and PPAR-␦ (b), UCP1 (c), and ␤3-AR (d) the paraventricular nucleus and ventromedial hypothalamus, the mRNA in the WAT. Data are presented as net intensity͞GAPDH expression. n ϭ nucleus of the solitary tract, the intermediolateral cell group, and 4–6 per group; *, P Ͻ 0.05; §, P ϭ 0.06. the central autonomic nucleus of the spinal cord (23–25). Sympa-

14586 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606102103 Bartolomucci et al. Downloaded by guest on September 25, 2021 Fig. 6. Hormones and serological parameters in mice receiving a high-fat (HF) diet. (a) Serum levels of leptin and ghrelin. (b) Levels of TGs, FFAs, and glucose and FFA͞TG ratio. Standard, standard chow; Con, mice treated with aCSF. n ϭ 11–12 per group; **, P Ͻ 0.01.

thetic stimulation determines lipolysis and EE primarily by means infusion of the peptide are physiological or pharmacological is only of ␤-AR stimulation (26). In agreement with our proposal, speculative. However, the concentration used seems compatible TLQP-21 treatment mildly decreased WAT weight, whereas with a physiological range based on independent studies (37–39). ␤3-AR expression was slightly (15% increase) but significantly Another relevant issue is the identification of the brain structure(s) up-regulated within the same tissue; the two parameters were mediating TLQP-21 action. Anabolic (agouti-related peptide, neu- inversely correlated. WAT weight was also negatively correlated ropeptide Y, and melanocyte-concentrating hormone) and cata- with tissue NE level. TLQP-21 treatment also increased PPAR-␦ bolic (proopiomelanocortin and corticotropin-releasing hormone) mRNA (74% increase) in the WAT. Overall, this effect may hypothalamic peptides were not grossly affected by TLQP-21. contribute to the observed increase in EE by determining fatty acid Although whole hypothalamic semiquantitative RT-PCR is not oxidation and energy uncoupling (2, 8, 21, 25). informative of regional differences in gene expression, it is often TLQP-21 effected increased serum E and BAT-NE levels, which used to make a preliminary evaluation of changes in mRNA are consistent with other physiological effects, whereas the reduced (40–42). Even considering this limitation, our results would rule out serum NE level seems at odds. Circulating NE mainly derives from a primary role of the hypothalamus in mediating the effects of synaptic leakage after exocytotic release from nerve terminals, and TLQP-21 while being compatible with the activation of brainstem sympathetic-stimulated catabolism results in increased tissue re- nuclei downstream of the hypothalamus. This conclusion agrees uptake of NE (27, 28). Therefore, decreased serum NE might result with the proposal that VGF would function in the outflow pathways from increased tissue reuptake. Alternatively, TLQP-21 may dif- downstream of hypothalamic 4 receptors (Mc4Rs) ferentially regulate functionally specific autonomic outputs inner- that project by means of the autonomic nervous system to periph- vating different tissues (28, 29). eral metabolic tissues and regulate energy homeostasis (13, 15). Ϫ Ϫ TLQP-21 also increased UCP1 in the WAT (84% increase). Fat Recent evidence (43) in Mc4R / mice suggests a divergent pads are a mix of brown (expressing UCP1) and white (not melanocortin pathway in the control of food intake (hypothalamic͞ expressing UCP1) adipocytes (19, 30). The epididymal WAT is amygdaloid nuclei) and EE [possibly brainstem͞spinal cord neu- mainly composed of white adipocytes. Increased UCP1 mRNA rons where Mc4R colocalizes with pseudorabies virus injected in would then imply transdifferentiation of brown adipocytes (3, 31). the inguinal WAT (44)]. TLQP-21, affecting EE but not affecting Interestingly, increased expression of catabolic mediators in the feeding or hypothalamic peptides, may modulate one such Mc4R- WAT was paralleled by a reduction of serum TG levels and high sensitive extrahypothalamic site controlling EE. FFA͞TG ratio. A reappraisal of published data has revealed a high FFA͞TG ratio after treatments (e.g., ␤3-AR agonists) or gene TLQP-21 Prevents the Effects of High-Fat Diet. In mice that were fed overexpression increasing lipolysis and blocking obesity (e.g., refs. a standard diet, TLQP-21 upset energy balance but did not deter- 5–7 and 32). Therefore, TLQP-21 effect mimics ␤-agonist activity mine an overall shift in energy homeostasis.o We hypothesized that and further points to sustained sympathetic stimulation of adipose energy homeostasis could have been positively boosted by chal- tissue. We have also observed that TLQP-21 induces contractions lenging mice with a high-fat diet. TLQP-21 almost completely of gastric fundus strips (G.L.C., C.S., R.P., and A.L., unpublished blocked the early phases of diet-induced obesity. Despite TLQP- work). This effect is inhibited by the cyclooxygenase inhibitors 21-treated mice ingesting a similar number of kilocalories with indomethacine and naproxen, and, accordingly, an increased respect to controls, both body weight and visceral WAT showed only a modest increase. Consistent with these effects, HF-TLQP-21 production of prostaglandin E2 (PGE2) follows TLQP-21 admin- mice showed half the rise in leptin that was observed in HF-CON istration. Central induction or treatment with PGE2 induces hyperthermia and increases EE (33–35). Therefore, chronic TLQP- mice and a normalization of ghrelin. Fig. 5a shows that body weight 21-induced upset of energy balance is compatible with a mechanism of HF-CON and HF-TLQP-21 mice at the end of the 2-week treatment phase (time constrained by the limited capacity of the of action involving central PGE2 production. TLQP-21 also determined a stronger decline of FFA and TG in osmotic pumps) substantially diverged. We can predict that, by increasing HF exposure and peptide delivery, the curves will spread

the glucose tolerance test when compared with controls. This NEUROSCIENCE transient decrease (30 min after glucose intake) determined a out more conspicuously. TLQP-21-induced increase of EE, T, and momentary normalization of the FFA͞TG ratio. Increased liver adipose tissue catabolic mediators is compatible with the block of and adipose tissue sensitivity to the lipostatic action of insulin due weight gain and adiposity (1, 4, 20). These results suggest that to sustained parasympathetic stimulation after glucose overload TLQP-21 can be a promising target for a pharmacological inter- may explain this result (24, 36). vention aimed at limiting adiposity by increasing EE (9, 10). Although we identified TLQP-21 in brain tissue extract, its regional localization and its concentration within the CNS are not oIn a recent meeting abstract, Jethwa et al. (45) confirmed our observations by showing known yet, and the identification of its receptor remains elusive, that subchronic central TLQP-21 treatment may reduce body and adipose tissue weight in too. Presently, whether the effects we observed after 15-␮g͞day the Siberian hamster.

Bartolomucci et al. PNAS ͉ September 26, 2006 ͉ vol. 103 ͉ no. 39 ͉ 14587 Downloaded by guest on September 25, 2021 Does VGF Encode for Several Metabolically Active Peptides? Surpris- spectrum followed by three data-dependent MS͞MS scans on the ingly, the profile of TLQP-21 treated mice closely matches the three most intense peaks of each full MS spectrum. phenotype of VGFϪ/Ϫ mice. Both VGFϪ/Ϫ and TLQP-21-treated mice showed hyperthermia, increased lipolysis, EE and tissue Peptide Identification. MS͞MS results were submitted to SE- sensitivity to insulin (13–15, 46), and even similar molecular changes QUEST 2.0 screening against a nonredundant protein database in BAT and WAT (13). Moreover, both VGF deletion and downloaded from the National Center for Biotechnology Informa- TLQP-21 treatment blunted diet-induced obesity (15). It is not tion home page (www.ncbi.nlm.nih.gov). To confirm the identifi- unusual that results from constitutive gene knockout and pharma- cation of TLQP-21, MASCOT 2.1 Sequence Query was used cological studies result in contrasting findings (47, 48). However, an against the SwissProt Rattus database. Query inputs were adopted intriguing hypothesis would clarify the apparent contradiction of based on the daughter ions list detected by a SEQUEST search (see the present study and studies by Hahm et al. (14, 15): One, or more, Table 1): 217.27 tag (1,495.7, RRH, 1,046.2), tag (553.7, [IL], 440.5); VGF-derived peptide should have an anabolic role that positively enzyme, none; fixed͞variable modifications, none; peptide toler- affects energy homeostasis and opposes TLQP-21 effects. Indeed, ance, Ϯ2 Da; MS͞MS tolerance, Ϯ0.6 Da; peptide charge, ϩ1, ϩ2, vgf encodes for a precursor protein that yields a number of peptides, and ϩ3. Average mass was selected. which have not been fully characterized (13). Three VGF peptides were shown to possess biological activity: TLQP-62 and AQEE-30 Central TLQP-21 Administration. Animals. Male CD1 mice (Charles increase the synaptic charge in hippocampal neurons (18), and River, Calco, Italy) were housed in an environmentally con- AQEE-30 and LQEQ-19 facilitate penile erection (49). In addition, trolled room (temperature of 21–24°C, light on at 0700 and light ͞ we showed that TLQP-21 induces contraction of rat gastric longi- off at 1900), with food (3.4 kcal g Mucedola) and water available tudinal muscle (G.L.C., C.S., R.P., and A.L., unpublished work) and ad libitum. Experiments were carried out in accordance with the ͞ ͞ modulates formalin pain (R.R., A.B., A.M., F.R.D., R.P, A.L., and European Communities Council Directive (86 609 EEC). F.P., unpublished work). If our hypothesis is confirmed, VGF Experimental procedure. All experiments consisted of a 4-day baseline should be considered a polypeptide precursor that encodes for followed by a 14-day treatment phase consisting of continuous i.c.v. different metabolically active neuropeptides in analogy with others injection by means of osmotic pumps of TLQP-21 in artificial (e.g., proopiomelanocortin). Site-specific and͞or time-dependent cerebrospinal fluid (aCSF). For all of the experimental phase, mice regulation of VGF processing would then account for its function, were individually housed, and locomotor activity was monitored. depending on different physiological signals. Body weight and food and water intake were determined (between 0900 and 1000) on the day of osmotic pump positioning (basal), on Conclusion day 1, and every second day. Animals were killed by decapitation This study shows the identification in brain extracts of a previously after brief CO2 exposure between 0900 and 1100. Peptide delivery. Synthetic TLQP-21 peptide (TLQPPASSRRRH- uncharacterized neuropeptide designated TLQP-21. Results dis- FHHALPPAR; Primm, Milan, Italy) at a dose of 1 mM dissolved cussed address a role for this peptide in centrally stimulating the in aCSF or aCSF alone was i.c.v.-delivered through microosmotic autonomic nervous system, possibly by means of central prosta- pumps (model 1002; Alzet, Palo Alto, CA; flow rate of 0.25 ␮l͞h, glandin induction, peripheral adrenomedullary activity, and adi- 15 ␮g͞day) connected to the Alzet brain infusion kit 1. Pumps pose tissue catabolism, to upset energy balance. By virtue of its were filled and incubated overnight at 37°C. TLQP-21 thermo- effect, TLQP-21 also limited weight gain and adiposity associated stability was ascertained by measuring with HPLC (1 mM with a high-fat diet. solution) before and after 14 days at 37°C (data not shown). The Materials and Methods dose was selected based on previous studies in which the acute effect of TLQP-21 on restraint-induced catecholamine release Brain Extracts. Brains of Dark Agouti male rats were dissected, (A.B., T.P., and A.M., unpublished work), hind paw formalin- minced, boiled for 10 min in 10 ml of H2O per gram of tissue, and induced licking behavior (A.B., R.P., and F.P., unpublished subsequently incubated at room temperature for 10 min. The ϫ work), and penile erection (49) was assessed. The dose was also supernatant was cleared at 22,000 g for1hat4°Candkept on consistent with other VGF-derived peptides (37) and neuropep- ice until prompt use to inactivate proteases and enrich extraction of tides infused by means of osmotic minipumps (38, 39). low-molecular-weight peptides. Surgery. Alzet pumps and brain infusion kits were implanted Immunopurification of VGF Peptides. The antiserum used for immu- under anesthesia (100 mg͞kg ketamine i.p. and 5 mg͞kg xylazine noaffinity purification was raised in rabbits against residues 556– i.p.). Cannulae were implanted according to the stereotaxic 576 of rat VGF (C.B. and G.L.F., unpublished work). Extracts were coordinates (anteroposterior, Ϫ0.1 mm; mediolateral, Ϯ1.0 mm; ⅐ adjusted to 20 mM Tris HCl (pH 7.5) (binding buffer) followed by dorsoventral, 3 mm from bregma) (52). Cannulae were fixed to binding onto protein G Sepharose beads for3hatroom temper- the mouse skull with polycarbonate cement (Shofu, Kyoto, ature. Antigen Ig-bound beads were washed three times with Japan). binding buffer and recovered. Elution was performed into 40% ͞ acetonitrile (ACN) 0.2% TFA solution in H2O, and samples were High-Fat Diet. In the baseline phase, mice were fed with powdered stored at 4°C. pellets suspended in water to obtain a dense consistency. In the treatment phase, mice were fed with a high-fat diet consisting of MALDI-TOF MS. Samples were mixed 1:1 with a saturated solution diluted powdered rodent chow plus 20% lard (calculated energy of ␣-cyano-4-hydroxycinnamic acid matrix in 50% ACN͞0.1% content of the dry diet was 4.33 kcal͞g). TFA and deposited onto the stainless-steel target (Perseptive Biosystems, Foster City, CA). Spectra were obtained essentially Oral Glucose Tolerance Test. Mice were fasted for7h(from0730 to as described in ref. 50. 1430) and orally injected with glucose at a dose of 3 g͞kg. Blood samples were obtained from the tail vein before (baseline) and 30, Microcapillary Liquid Chromatography MS͞MS. Analysis was per- 60, and 120 min after glucose load. Insulin, glucose, FFA, and TG formed essentially as described in ref. 51. The following gradient levels were analyzed as described below. was applied: 10% buffer B (80% acetonirile͞0.2% formic acid) at 3min,60%Bat53min,90%bufferBat63min,90%bufferBat Home Cage Locomotor Activity. Activity was scored by means of an 68 min, 0% buffer B at 70 min, and 0% buffer B at 90 min. Spectra automated infrared system (Activiscope; New Behavior, Zu¨rich, were acquired in an automated mode, consisting of a full MS Switzerland).

14588 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0606102103 Bartolomucci et al. Downloaded by guest on September 25, 2021 Indirect Calorimetry. EE was determined by indirect calorimetry third ventricle) were quickly dissected, collected in RNAlater with an Oxymax system (Columbus Instruments, Columbus, OH) (Sigma-Aldrich, St. Louis, MO), and stored at Ϫ80°C. RT-PCR ͞ with an air flow of 0.75 liter min. O2 and CO2 gas content was was performed as described in ref. 40 (Table 5, which is recorded every 15 min for 21 h, starting at 1000. Mice had food and published as supporting information on the PNAS web site). water ad libitum. The respiratory exchange ratio (VCO2͞VO2) and EE were calculated by using Oxymax 5.9. Calculated data were Statistical Analysis. Data from standard diet-fed groups were ana- analyzed according to mouse activity level that was determined by lyzed with Mann–Whitney’s U test or multivariate ANOVA [fol- infrared sensors as follows: rest, 0 cpm; moderate, 1–80 cpm; high, Ͼ80 cpm. lowed by Tukey’s honestly significant difference (HSD) post hoc tests, which can be used also with nonsignificant F values (53)] Rectal Temperature. Temperature was determined between 0900 where appropriate. Data from high-fat diet groups were analyzed and 1100 (room temperature was 22 Ϯ 1°C) with a rectal probe with two- or three-way ANOVA followed by Tukey’s HSD test. (Physitemp Instruments, Clifton, NJ) connected to a portable Correlations were performed with a nonparametric Spearman’s thermometer (Hanna Instruments, Padua, Italy). test.

Serum Measurements. Ghrelin, fee triiodothyronine, free thyrox- We thank Prof. J. Feldon, C. Ro¨sli, Dr. B. Roschitzki, and the team at the ine, total corticosterone, and insulin were measured with RIA. Functional Genomics Centre Zu¨rich (Zu¨rich, Switzerland). This work was Leptin was measured with ELISA. Glucose was measured by the supported by Fondo per gli Investimenti della Ricerca di Base Grants esokinase method. TG and FFA levels were measured with a RBNE01JKLF࿝004 (to A.M.), RBNE01JKLF࿝006 (to R.P.), colorimetric reaction kit. Serum NE and E levels were deter- RBNE013XSJ࿝004 (to R.P.), RBNE01JKLF࿝008 (to V.L.), and mined with HPLC (for details, see Supporting Materails and RBNE01JKLF࿝01 (to E.E.M.); Instituto Zooprofilattico Sperimentale Methods which is published as supporting information on the Grant SA 002͞02 (to R.P.); the Fondo di Ateneo per la Ricerca of the PNAS web site). University of Milan–Bicocca (A.T. and V.L.); the Fondo Integrativo Speciale per La Ricerca–Neurobiotecnologie and Telethon E0830 (to A.L.); RT-PCR. Interscapular BAT, epididymal WAT, and hypothalami European Molecular Biology Organization Fellowship ASTF 35-2005 (to (landmarks: optic chiasma, lateral sulci, mammillary bodies, and G.L.C.); and Swiss National Science Foundation PIIB 109067͞1 (to G.L.C.).

1. Schwartz MW, Woods SC, Porte D, Jr, Seeley RJ, Baskin DG (2000) Nature 29. Morrison SF (2001) Am J Physiol 281:R683–R698. 404:661–671. 30. Cinti S (2005) Prostaglandins Leukot Essent Fatty Acids 73:9–15. 2. Barish GD, Narkar VA, Evans RM (2006) J Clin Invest 116:590–597. 31. Tiraby C, Langin D (2003) Trends Endocrinol Metab 14:439–441. 3. Friedman JM (2004) Nat Med 10:563–569. 32. Gomori A, Ishihara A, Ito M, Mashiko S, Matsushita H, Yumoto M, Ito M, 4. Lowell BB, Spiegelman BM (2000) Nature 404:652–660. Tanaka T, Tokita S, Moriya M, et al. (2003) Am J Physiol 284:E583–E588. 5. Cederberg A, Grønning LM, Ahren B, Tasken K, Carlsson P, Enerback S 33. Heleniak EP, Aston B (1989) Med Hypot 28:13–33. (2001) Cell 106:563–573. 34. Amir S, Schiavetto A (1990) Brain Res 528:138–142. 6. Riu E, Ferre T, Hidalgo A, Mas A, Franckhauser S, Otaegui P, Bosch F (2003) 35. Nakamura K, Matsumura K, Hubschle T, Nakamura Y, Hioki H, Fujiyama F, FASEB J 17:1715–1717. Boldogkoi Z, Konig M, Thiel HJ, Gerstberger R, et al. (2004) J Neurosci 7. Solveva V, Graves RA, Rasenick MM, Spiegelman BM, Ross SR (1997) Mol 24:5370–5380. Endocrinol 11:27–38. 36. Uyama N, Geerts A, Reynaert H (2004) Anat Rec A Discov Mol Cell Evol Biol 8. Wang Y, Lee C-H, Tiep S, Yu RT, Ham J, Kang H, Evans RM (2003) Cell 280:808–820. 113:159–170. 37. Pan H, Nanno D, Che F-H, Zhu X, Salton SR, Steiner DF, Fricker LD, Devi 9. Crowley VEF, Yeo GSH, O’Rahilly S (2002) Nat Rev Drug Discov 1:276–286. LA (2005) Biochemistry 44:4939–4948. 10. Jandacek RJ, Woods SC (2004) Drug Discov Today 9:874–880. 38. Baran K, Preston E, Wilks D, Cooney GJ, Kraegen EW, Sainsbury A (2002) 11. Levi A, Eldridge JD, Paterson BM (1985) Science 229:393–395. Diabetes 51:152–158. 12. Salton SR, Ferri GL, Hahm S, Snyder SE, Wilson AJ, Possenti R, Levi A (2000) 39. Overton JM, Williams DT, Chambers JB, Rashotte ME (2001) Hypertension Front Neuroendocrinol 21:199–219. 37:663–669. Cell Mol Neurobiol 13. Levi A, Ferri GL, Watson E, Possenti R, Salton SR (2004) 40. Bresciani E, Nass R, Torsello A, Gaylinn B, Avallone R, Locatelli V, Thorner 24:517–533. MO, Muller EE (2004) Neuroendocrinology 80:52–59. 14. Hahm S, Mizuno TM, Wu TJ, Wisor JP, Priest CA, Kozak CA, Boozer CN, 41. Ziotopulou M, Mantzoros CS, Hileman SM, Flier JS (2000) Am J Physiol Peng B, McEvoy RC, Good P, et al. (1999) Neuron 23:537–548. 279:E838–E845. 15. Hahm S, Fekete C, Mizuno TM, Windsor J, Yan H, Boozer CN, Lee C, Elmquist 42. Sun Y, Wang P, Zheng H, Smith RG (2004) Proc Natl Acad Sci USA JK, Lechan RM, Mobbs CV, Salton SRJ (2002) J Neurosci 22:6929–6938. 101:4679–4684. 16. Ross AW, Bell LM, Littlewood PA, Mercer JG, Barrett P, Morgan PJ (2005) 43. Balthasar N, Dalgaard LT, Lee CE, Yu J, Funahashi H, Williams T, Ferreira Endocrinology 146:1940–1947. M, Tang V, McGovern RA, Kenny CD, et al. (2005) Cell 123:493–505. 17. Trani E, Giorni A, Canu N, Amadoro G, Rinaldi AM, Halban PA, Ferri GL, Am J Possenti R, Schinina ME, Levi A (2002) J Neurochem 81:565–574. 44. Song CK, Jackson RM, Harris RBS, Richard D, Bartness TJ (2005) 18. Alder J, Thakker-Varia S, Bangasser DA, Kuroiwa M, Plummer MR, Shors TJ, Physiol 289:R1467–R1476. Black IB (2003) J Neurosci 23:10800–10808. 45. Jethwa PH, Warner A, Brameld JM, Keyte JW, Nilaweera NK, Morgan PJ, 19. Cannon B, Nedegaard J (2004) Physiol Rev 84:277–359. Barrett P, Francis JP (2006) Front Neuroendocrinol 27:5–6. 20. Lowell BB, Flier JS (1997) Ann Rev Med 48:307–316. 46. Watson E, Hahm S, Mizuno TM, Windsor J, Montgomery C, Scherer PE, 21. Bachman ES, Dhillon H, Zhang CY, Cinti S, Bianco AC, Kobilka BK, Lowell Mobbs CV, Salton SR (2005) Endocrinology 146:5151–5163. BB (2002) Science 297:843–845. 47. Rocha BA, Scearce-Levie K, Lucas JJ, Hiroi N, Castanon N, Crebbe JC, 22. Silva JE (2006) Physiol Rev 86:435–464. Nestler EJ, Hen R (1998) Nature 393:175–178. 23. Rayner DV (2001) Proc Nutr Soc 60:357–364. 48. Yaswen L, Diehl N, Brennan MB, Hochgeschwender U (1999) Nat Med 24. Fliers E, Kreier F, Voshol PJ, Havekes LM, Sauerwein HP, Kalsbeek A, Buijs 5:1066–1070.

RM, Romijn JA (2003) J Neuroendocrinol 15:1005–1010. 49. Succu S, Cocco C, Mascia MS, Melis T, Melis MR, Possenti R, Levi A, Ferri NEUROSCIENCE 25. Bowers RR, Festuccia WT, Song CK, Shi H, Migliorini RH, Bartness TJ (2004) GL, Argiolas A (2004) Eur J Neurosci 20:3035–3040. Am J Physiol 286:R1167–R1175. 50. Scheurer SB, Rybak JN, Roesli C, Brunisholz RA, Potthast F, Schlapbach R, 26. Larsen TM, Toubro S, van Baak MA, Gottesdiener KM, Larson P, Saris WHM, Neri D, Elia G (2005) Proteomics 5:2718–2728. Astrup A (2002) Am J Clin Nutr 76:780–788. 51. Scheurer SB, Roseli C, Neri D, Elia G (2005) Proteomics 5:3035–3039. 27. Garofano MA, Kettelhut IC, Rosolino JE, Migliorini RH (1996) J Auton Nerv 52. Franklin KBJ, Paxinos G (1997) The Mouse Brain in Stereotaxic Coordinates Syst 60:206–208. (Academic, London). 28. Goldstein DS (2001) The Autonomic Nervous System in Health and Disease 53. Wilcox RR (1987) New Statistical Procedures for Social Science (Erlbaum, (Dekker, New York). Hillsdale, NJ).

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