The peptides encoded by the VGF gene are gaining biomedical interest and are increasingly being scrutinized as biomarkers for human disease. An endocrine/neuromodulatory role for VGF peptides has been suggested but never demonstrated. Furthermore, no study has demonstrated so far the existence of a receptor-mediated mechanism for any VGF peptide. In the present study, we provide a comprehensive in vitro, ex vivo and in vivo identification of a novel pro-lipolytic pathway mediated by the TLQP-21 peptide. We show for the first time that VGF-immunoreactivity is present within sympathetic fibres in the WAT (white adipose tissue) but not in the adipocytes. Furthermore, we identified a saturable receptor-binding activity for the TLQP-21 peptide. The maximum binding capacity for TLQP-21 was higher in the WAT as compared with other tissues, and selectively up-regulated in the adipose tissue of obese mice. TLQP-21 increases lipolysis in murine adipocytes via a mechanism encompassing the activation of noradrenaline/β-adrenergic receptors pathways and dose-dependently decreases adipocytes diameters in two models of obesity. In conclusion, we demonstrated a novel and previously uncharacterized peripheral lipolytic pathway encompassing the VGF peptide TLQP-21. Targeting the sympathetic nerve–adipocytes interaction might prove to be a novel approach for the treatment of obesity-associated metabolic complications.

INTRODUCTION

Obesity-associated pathologies are increasing their incidence worldwide to pandemic levels [1]. The obesity pandemic has stimulated the identification of molecular mechanisms regulating metabolic functions and controlling BW (body weight) with the final goal to generate new pharmacotherapy to control obesity and/or its deleterious consequences [1,2]. The WAT (white adipose tissue) is a complex organ with multiple topographical locations (e.g. visceral and subcutaneous), specific functional characteristics, with direct sympathetic and sensory innervations and capable of influencing global metabolic response through their repertoire of secreted adipokines [3,4]. Lipolysis, the process ultimately required to degrade TAGs (triacylglycerols) and release non-esterified fatty acids, is a very complex process with multiple pathways involved [57]. A key player in lipolysis is the activation of the sympathetic nervous system which sends efferent projections to the adipose tissue and activates β-ARs (β-adrenergic receptors) in adipocyte membranes [4,5]. In agreement, mice deficient in the three known β-ARs show obesity due to a failure of diet-induced thermogenesis [8]. β-AR-less mice also show a transdifferentiation of brown into white-like adipocytes [8]. Unfortunately, sympathomimetic drugs and β-agonists are often associated with mild-to-severe side effects or with contrasting clinical outcome, which has limited their development or use in clinical practice [2,7]. Peptidergic candidates are increasingly considered in drug discovery programmes [2,9,10]. In this context, peptides encoded by the granin protein family member VGF are gaining increasing biomedical interest (e.g. [1113]). A potential link between VGF and obesity is demonstrated by the observation that VGF peptides are up-regulated in rat hypothalamus after a high-fat diet or cold exposure [14]. VGF-deficient mice are hypermetabolic, lean and resist obesity [15,16], a complex phenotype probably resulting from the deletion of several encoded bioactive peptides as demonstrated by the observation that VGF-derived peptides TLQP-21 and NERP-2 centrally modulate metabolic functions and adiposity in opposite directions, with the former being catabolic and the latter being anabolic [17,18]. The C-terminal internal peptide termed TLQP-21 has emerged as a new promising anti-obesity target [17]. Chronic intracerebroventricular injection of TLQP-21 in mice increased energy expenditure and rectal temperature and prevented obesity [17,19]. Besides having a central role in nerve terminal- and in neuron-derived cell cultures secretory granules, VGF co-localizes with molecules as diverse as substance P, proSAAS, noradrenaline (norepinephrine), adrenaline (epinephrine) and insulin [14,2022]. However, no results supporting a peripheral role for VGF peptides have been published so far. In the present paper, we report for the first time that VGF is present in sympathetic fibres in WAT, binds with high affinity to adipocytes membranes and increases lipolysis via a mechanism that requires the activation of noradrenaline/β-AR pathways.

EXPERIMENTAL PROCEDURE

Animals and diet

Male CD1 mice (Charles River) were individually housed on arrival in a fully controlled environment (light on at 07:00 h, light off at 19:00 h, T=21±1°C). Standard chow (STD) was the 4RF21 (Mucedola; 10.6% kcal from fat and 3.9 kcal/g; 1 kcal=4.184 kJ); the high-fat diet (HFD) was a special pelleted diet derived from the standard 4RF21 (Mucedola; 45% kcal from fat and 5.2 kcal/g). All animal experimentation was conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/EEC), and was approved by local ethical committee and the Italian Institute of Health.

Identification of VGF in WAT

VGF immunohistochemistry

Perigonadal rat WAT was fixed by immersion or transcardial perfusion using modified Zamboni's fixative (4% paraformaldehyde and 0.2% picric acid). The tissue was cryoprotected in 10% sucrose for a minimum of 24 h before freezing and sectioning. Slide-mounted cryostat sections (30 μm) were incubated in blocking buffer (PBS containing 0.03% Triton X-100, 1% BSA, 1% normal donkey serum and 0.01% sodium azide) for 1 h at room temperature (20–22°C), followed by overnight incubation at 4°C in primary antisera, which were guinea pig anti-TLQP-21, 1:1000; rabbit anti-TLQP-21, 1:3000; guinea pig anti-AQEE-30, 1:3000 [23]; mouse anti-TH (tyrosine hydroxylase, 1:1000; Immunostar); rabbit anti-TH, 1:1000 (Millipore). TLQP-21 antisera were generated using previously described methods [24]. Briefly, the peptides TLQP-21 and SSRR-14 (corresponding to the last 14 amino acids of TLQP-21) were conjugated to bovine thyroglobulin (Sigma) using glutaraldehyde. The peptide conjugates (1 mg/ml) were emulsified with an equal volume of Freund's adjuvant (Difco). TLQP-21 was injected into female New Zealand rabbits (n=3, Harlan) at 2-week intervals (1 mg of peptide for initial and 0.5 mg of peptide for subsequent immunizations). SSRR-14 was injected in female guinea pigs (n=4, Harlan) at 2-week intervals (0.5 mg of peptide for initial and 0.25 mg of peptide for subsequent immunizations). No cross-reactivity was observed with the C-terminal peptide AQEE-30 which is adjacent to TLQP-21 (Supplementary Figure S1 available at http://www.BiochemJ.org/bj/441/bj4410511add.htm). The specificity of the antisera was ascertained by dot blot analysis, in which the antisera bound preferentially to TLQP-21 rather than the extended fragment TLQP-62 (Supplementary Figure S1). The slides were rinsed in PBS, incubated in secondary antisera [Cy2 (carbocyanine)- and Cy3 (indocarbocyanine)-conjugated donkey anti-guinea pig or anti-rabbit respectively obtained from Jackson Immunoresearch Laboratories] for 1 h at room temperature and overlaid with a coverslip. Images were collected using an Olympus FluoView 1000 BX2 Upright Confocal microscope and processed using ImageJ and Adobe Photoshop.

Receptor-binding assay and lipolysis

TLQP-21-binding assay

Binding of TLQP-21 to crude membranes (30000 g pellet) isolated from pWAT (perigonadal WAT), scWAT (subcutaneous inguinal WAT), rpWAT (retroperitoneal WAT), BAT (brown adipose tissue), adrenals, cerebellum, skeletal muscle and liver was carried out using 125I-YA-TLQP-21 as a ligand (New England Biolabs). The addition of tyrosine-alanine (YA) amino acidic residues to the TLQP-21 molecule made possible the radio-iodination of the peptide and to obtain a highly specific radioligand with the same in vitro biological activity as TLQP-21 (results not shown). For the single-point binding assay, cell membranes (corresponding to 100 μg of membrane protein) were incubated in triplicate at 23°C for 4 h under constant shaking with 0.5 nM 125I-YA-TLQP-21 in a final volume of 0.5 ml of assay buffer [25]. Parallel incubations, in which 1 μM unlabelled YA-TLQP-21 was also present, were used to determine non-specific binding, which was subtracted from total binding to yield specific binding values. The binding reaction was terminated by the addition of ice-cold assay buffer, followed by rapid filtration through Whatman GF/B filters and the radioactivity bound to membranes was measured by a Packard auto-γ-counter. Specific binding was expressed as a percentage of the total radioactivity added. For saturation-binding studies, cell membranes were incubated with increasing concentrations of the radioligand (0.03–4 nM). Competition studies were performed by incubating cell membranes (150 μg/tube) with 1 nM 125I-YA-TLQP-21 with or without increasing concentrations (10 pM–0.1 μM) of unlabelled YA-TLQP-21 or TLQP-21. Binding specificity was also tested with a scrambled peptide made using the same amino acid residues of TLQP-21. Results were plotted and curves fit using the GraphPad Prism 4 software assuming that the binding was due to a single class of binding sites, thus allowing determination of the maximum binding capacity (Bmax), dissociation constant (Kd), Hill slope and concentration of the competitor causing 50% inhibition (IC50) of specific radioligand binding.

Glycerol assay

Approx. 2–3 g of the epididymal fat was surgically removed and adipose tissue suspension was incubated as described by Muccioli et al. [26] and Student et al. [27]. Fat cells were incubated in the presence of TLQP-21 (0.1–100 nM) with or without isoprenaline (15 nM, also called isoproterenol). Glycerol levels were measured in triplicate using the Free Glycerol Reagent (Sigma) and expressed relative to the cellular protein content. EC50 for inhibition of isoprenaline-induced lipolysis was determined for each compound under study with GraphPad Prism 4 software.

Signal transduction pathway in 3T3-L1 adipocytes

Cells and differentiation

3T3-L1 cell line was originally obtained from the ATCC (Manassas, VA, U.S.A.). Cells were maintained according to standard procedures. The cells were differentiated according to a well-established protocol described previously [28].

Western blotting 3T3-L1 cells

Equivalent amounts of cell extracts (~5×105 cells) were mixed with SDS-reducing sample buffer according to NuPAGE® (Invitrogen). For detailed procedures, see [29]. Incubation with primary antibodies was performed overnight at 4°C: rabbit polyclonal antibodies anti-phospho-p44/p42 MAPK (mitogen-activated protein kinase; Thr202/Tyr204), anti-phospho-AMPK (AMP-activated protein kinase; Thr172) anti-phospho-PKA (protein kinase A) substrate (serine/threonine) and anti-phospho-HSL (hormone-sensitive lipase; Ser660) (Cell Signaling Technology). Anti-tubulin was a mAb (monoclonal antibody) from Sigma. Secondary antibodies were horseradish peroxidase-coupled donkey anti-rabbit or anti-mouse (GE Healthcare). A semi-quantitative analysis of a Western blotting densitometry scan was performed using Scan Analysis software ImageJ. Five–six different experiments run in duplicate taking the time point 15 min were analysed with non-parametric Kruskall–Wallis test followed by Mann–Whitney U test for pair-wise comparisons.

Physiological and biochemical effects of chronic TLQP-21 delivery in vivo in animal model of obesity

TLQP-21 peptide and surgery

TLQP-21 peptide (Primm) 40 and 400 μg/day (referred to as the LOW and HIGH dose respectively) was suspended in sterile saline and delivered (subcutaneously) via Alzet osmotic mini-pumps (model 1002) for 14 days. Controls received saline only. Prefilled osmotic mini-pumps were surgically implanted subcutaneously in the dorsal area of the animals. Surgery was performed in sterile conditions in mice anaesthetized with a mixture of ketamin/xilazine (100–5 mg/kg).

Animal models of obesity: DIO (diet-induced obesity)

Male mice initially weighing 32–35 g were fed for 12 weeks on an HFD. DIO mice had a final BW being at least higher than the average mean plus 2 S.D. of the BW of mice fed with STD for the same amount of time. DIO mice were randomly allocated to saline or TLQP-21 LOW or HIGH dose avoiding any pre-experimental difference in BW or food intake. An additional group of mice fed with STD and treated with saline was used as lean control. BW and food intake were determined every other day. Perigonadal fat pads were manually dissected and weighed. Fat pads were split into two parts and one-half was snap-frozen in liquid nitrogen and stored at −80°C for later measurement of TH enzymatic activity, noradrenaline (norepinephrine) and TAG/DNA ratio and TNFα (tumour necrosis factor α) mRNA (see below). The second half was immersed in an ice-cold solution of 4% paraformaldehyde and embedded in paraffin for morphometric analyses and immunohistochemistry (see below). Plasma was analysed for adrenaline concentration. BW, food intake, fat pad weight, TH activity, noradrenaline, adipocyte diameter and TNFα were analysed with a two-way ANOVA for repeated measures. All other parameters were analysed with a one-way between factor ANOVA. Mean comparisons were performed with Tukey's HSD (Honestly Significant Difference) post-hoc after ANOVA or Student's unpaired t test. Correlations were evaluated with Pearson's test.

CSS (chronic subordination stress)-induced obesity

The CSS-induced obesity procedure has been previously described in detail [29]. Briefly, subordinate male mice were exposed to chronic sensory contact with a dominant male and received a brief aggressive encounter daily. The stress phase feeding regimen was scheduled as follows (see Figure 6A): STD during baseline and first 3 weeks of stress; HFD during the fourth and fifth week of stress. Subordinate mice were surgically implanted mini-pumps delivering 40 μg/day TLQP-21 or saline. For the subsequent 2 weeks mice were fed on the HFD. The following experimental groups were used: CSS/TLQP-21 (n=7), CSS/saline (n=7). BW and food intake were determined every other day. pWAT and interscapular (i)BAT were manually dissected and weighed. BW and food intake were analysed with a two-way ANOVA for repeated measures followed by Tukey's HSD post-hoc. Fat pad weight, TH-activity, noradrenaline and adipocyte diameter were analysed with Student's unpaired t test.

Home cage phenotyping

Body temperature was recorded using temperature-sensing subcutaneous transponders (Bio Medic Data Systems). Sensors were implanted at least 15 days before the beginning of the experiment. The assessment of individual locomotor activity in the home cage was carried out by means of an automated system that uses small passive infrared sensors positioned on the top of each cage (TechnoSmart), as described previously [29]. Activity and body temperature results were analysed with ANOVA for repeated measures.

Tissue and plasma biochemical assays

Detailed methods of tissue and plasma biochemical assays can be found in [30]. Briefly, tissue biopsies homogenate was incubated with L-tyrosine and TH activity was determined by calculating the amount of L-dopa (L-3,4-dihydroxyphenylalanine) generated from L-tyrosine per minute per mg of tissue measuring with HPLC–ECD (electron-capture-induced dissociation) system. Noradrenaline was measured in fat biopsies by HPLC using electrochemical detection, as previously described [30]. TAG/DNA content was analysed in tissue biopsies by measuring TAG content using a Sigma TAG assay kit and by normalizing to the DNA content (DNeasy tissue kit; Qiagen). Adrenaline was determined in plasma by HPLC using electrochemical detection, as previously described [17].

Real-time RT–PCR (reverse transcription–PCR)

pWAT TNFα mRNA was analysed in triplicate by real-time RT–PCR. Briefly, 400 ng of total digested RNA of each sample was subjected to RT with revertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). QPCR (quantitative PCR) for TNFα was performed with Assay-on-Demand technology (Applied Biosystems, Warrington, U.K.), with a Brilliant QPCR Master Mix (Stratagene, La Jolla, CA, U.S.A.). All QPCRs were performed on the ABI 7900 HT system (Applied Biosystems). Samples were normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase; Assay-on-Demand, Applied Biosystems) and 2ΔΔCt analysis applied.

WAT morphometric analysis

Paraffin-embedded specimens of perigonadal adipose tissues from different mice (DIO and CSS) were processed for morphometric analysis. Sections 5 μm thick were stained with haematoxylin and eosin. Optical microscopy images (Nikon Microscope Eclipse 80i) were digitally captured with NIS-Elements imaging software F 2.20, and the diameter of 200 adipocytes for each mouse was measured with ImageJ software (NIH).

WAT TH immunohistochemistry

Paraffin sections 3 μm thick of pWAT DIO mice were used for immunohistochemical localization of TH protein and MAC-2 according to the ABC (avidin–biotin-peroxidase) method [30]. The primary antibodies were polyclonal anti-rabbit TH (Chemicon; 1:600 dilution) and monoclonal anti-MAC-2 (Cedarlane Laboratories; 1:1000 dilution).

Heart histomorphology and morphometric analysis

The abdominal cavity was opened and the aorta was cannulated with a PE-50 catheter connected to a perfusion apparatus. The chest was opened and the heart was stopped in diastole by LV (left ventricular) intracavitary injection of cadmium chloride (100 nmol). The right atrium was then cut and the myocardial vasculature was shortly perfused at a physiological pressure with a heparinized PBS solution, followed by 10 min of perfusion with 10% formalin. The heart was separated by surgical excision of the major thoracic vessels and fixated in 4% formalin. After 24 h, the free walls of the RV (right ventricle) and the LV inclusive of interventricular S were separated and their weights were recorded. The major cavity axis of the LV, from the aortic valve to the apex, was measured under a stereomicroscope with a ruler calibrated exactly to 0.1 μm (2Biological Instruments). LV transversal diameters and wall thickness were analysed with a stereomicroscope connected to a digital camera (Kodak, DC290 ZOOM). Captured microphotographs were analysed by the use of Image-Pro Plus 4.0 software (Media Cybernetics). The cavity volume was calculated using the Dodge equation [31]. The volume of the LV myocardium was computed by dividing ventricular weight by the specific gravity of the tissue (1.06 g/ml). The following parameters were determined: HW (heart weight) to BW ratio, LV to BW, RV to BW, LV free wall thickness, and LV transversal diameters and volume, LV thickness to chamber radius and mass to chamber volume [32]. One section, stained with Masson's trichrome, was analysed by optical microscopy (magnification ×250) in order to evaluate the volume fraction of myocytes, and perivascular and interstitial fibrosis in the LV myocardium. In accordance with a procedure previously described [33], for each section a quantitative evaluation of the volume fraction of myocytes and fibrotic tissue was performed in 60 adjacent fields with the aid of a grid defining a tissue area of 0.160 mm2.

RESULTS

VGF co-localizes with TH in pWAT

VGF immunoreactivity (-ir) has been detected in the dorsal root and sympathetic ganglia and in a variety of neuroendocrine and endocrine cells [12] and in sympathetic neural pathways innervating iBAT [34]. Our result shows that VGF is expressed in perigonadal (p)WAT as protein but not as mRNA (Supplementary Figure S2 available at http://www.BiochemJ.org/bj/441/bj4410511add.htm), thus suggesting that peptides are sorted into the regulated secretory pathway granules [35,36]. To unambiguously identify VGF within sympathetic nerve fibres, we examined the co-localization of VGF with TH, a marker of catecholaminergic nerves and terminals [37]. We used antisera generated against TLQP-21 (anti-TLQP-21) that recognized preferentially the processed peptide rather than the unprocessed precursor TLQP-62 (Supplementary Figure S1), as well as antisera generated against a region located C-terminally to TLQP-21 (anti-AQEE-30) that recognized VGF C-terminal fragments of various lengths (Supplementary Figure S1). No cross-reactivity was observed with the C-terminal peptide AQEE-30 which is adjacent to TLQP-21 (Supplementary Figure S1). Both anti-TLQP-21 and anti-AQEE-30 labelled fibres within pWAT that were also TH-positive (Figure 1). Staining with the two TLQP-21 antisera was blocked by pre-absorption of the antisera with the cognate peptides (10 μg/ml). Moreover, TLQP-21 and AQEE-30 co-localized extensively in nerve fibres, consistent with the notion that the antisera recognize fragments of the same neuropeptide precursor. We observed some fibres that were anti-AQEE-30-positive and anti-TLQP-21-negative (but not vice versa) (Figures 1E and 1I).

Localization of VGF-peptides in the adipose tissue

Figure 1
Localization of VGF-peptides in the adipose tissue

(AE) Co-localization of TH-ir (red) and TLQP-21-ir (green) in mouse pWAT. (A and B) Volume of 3 μm (stack of four optical sections, 1 μm apart). (CE) A single optical section of a portion of the nerve fibre seen in (A and B). Yellow puncta in (E) represent the co-localization of TH-ir (C, red) and TLQP-21-ir (D, green). TH-ir was visualized using rabbit anti-TH. TLQP-ir was visualized using guinea pig anti-TLQP-21. (FI) Co-localization of TH-ir (red), TLQP-21-ir (green) and AQEE-30-ir (blue) in rat pWAT. The images represent a volume of 6 μm (stack of seven optical sections, 1 μm apart). In (I), the merging of (F) and (G) shows the overlap of TH-ir, TLQP-21-ir and AQEE-30-ir. TH-ir was visualized using mouse anti-TH. TLQP-ir was visualized using rabbit anti-TLQP-21. AQEE-30-ir was visualized using guinea pig anti-AQEE-30.

Figure 1
Localization of VGF-peptides in the adipose tissue

(AE) Co-localization of TH-ir (red) and TLQP-21-ir (green) in mouse pWAT. (A and B) Volume of 3 μm (stack of four optical sections, 1 μm apart). (CE) A single optical section of a portion of the nerve fibre seen in (A and B). Yellow puncta in (E) represent the co-localization of TH-ir (C, red) and TLQP-21-ir (D, green). TH-ir was visualized using rabbit anti-TH. TLQP-ir was visualized using guinea pig anti-TLQP-21. (FI) Co-localization of TH-ir (red), TLQP-21-ir (green) and AQEE-30-ir (blue) in rat pWAT. The images represent a volume of 6 μm (stack of seven optical sections, 1 μm apart). In (I), the merging of (F) and (G) shows the overlap of TH-ir, TLQP-21-ir and AQEE-30-ir. TH-ir was visualized using mouse anti-TH. TLQP-ir was visualized using rabbit anti-TLQP-21. AQEE-30-ir was visualized using guinea pig anti-AQEE-30.

A saturable receptor binding site for TLQP-21 in adipocyte membranes

Having established that VGF is present in sympathetic fibres in WAT we aimed to characterize binding sites for TLQP-21 [26]. We used 125I-YA-TLQP-21 (an analogue of TLQP-21 with similar biological activity, results not shown) to show that the binding to cell membranes was saturable, with high Bmax in different WAT fat pads as well as in the adrenals and BAT, whereas low-binding activity occurred in CNS (central nervous system), liver and skeletal muscle (Figure 2A). Representative saturation isotherms and Scatchard plots of 125I-YA-TLQP-21 binding to membranes from WAT and iBAT are shown in Figure 2(B). Experiments using increasing concentrations of radiotracer revealed evidence of saturable specific binding in the scWAT, pWAT and rpWAT pads as well as in the iBAT. Scatchard analysis (Figure 2C) showed the existence in all four tissues of a single class of high-affinity site with Kd values ranging from 0.54 to 0.65 nM. The specificity of 125I-YA-TLQP-21 binding to tissue membranes was assessed by competitive binding studies with a scrambled TLQP-21 peptide (LRSP-21) and isoprenaline (Figure 2D). Unlabelled TLQP-21 and YA-TLQP-21 completely displaced radiolabelled 125I-YA-TLQP-21 from binding sites. In contrast, scrambled peptide and isoprenaline were capable of displacing only 10–25% of the specifically bound radiolabelled 125I-YA-TLQP-21. Finally, obese mice (fed on an HFD for 4 weeks which induced a BW gain of ~25% when compared with baseline and controls; results not shown) showed increased density of 125I-YA-TLQP-21 binding site in WAT and BAT fat pads only, whereas Bmax in the other tissues remained unaffected (Figure 2A).

Pharmacological characterization and tissue distribution of TLQP-21-binding sites

Figure 2
Pharmacological characterization and tissue distribution of TLQP-21-binding sites

(A) The highest 125I-YA-TLQP-21-binding activity was observed in WAT as well as in the adrenals, whereas much lower binding activity occurred in the BAT, CNS, liver and skeletal muscle. Binding activity was significantly increased by exposure to 4 weeks of HFD in the adipose tissue only (P<0.001), whereas no change occurred in the other tissues. (B and C) Representative saturation isotherms and Scatchard plots of 125I-YA-TLQP-21 binding to membranes from pWAT, rpWAT, scWAT and iBAT. (D) The specificity of TLQP-21 peptide to adipocyte membranes was assessed by competitive binding studies with 125I-YA-TLQP-21. Scrambled peptide or isoprenaline was unable to significantly displace 125I-YA-TLQP-21 from tissue membranes. Pooled results are represented as means+S.E.M.

Figure 2
Pharmacological characterization and tissue distribution of TLQP-21-binding sites

(A) The highest 125I-YA-TLQP-21-binding activity was observed in WAT as well as in the adrenals, whereas much lower binding activity occurred in the BAT, CNS, liver and skeletal muscle. Binding activity was significantly increased by exposure to 4 weeks of HFD in the adipose tissue only (P<0.001), whereas no change occurred in the other tissues. (B and C) Representative saturation isotherms and Scatchard plots of 125I-YA-TLQP-21 binding to membranes from pWAT, rpWAT, scWAT and iBAT. (D) The specificity of TLQP-21 peptide to adipocyte membranes was assessed by competitive binding studies with 125I-YA-TLQP-21. Scrambled peptide or isoprenaline was unable to significantly displace 125I-YA-TLQP-21 from tissue membranes. Pooled results are represented as means+S.E.M.

TLQP-21 potentiates β-AR-induced lipolysis and phosphorylation of HSL in vitro

Having established that TLQP-21 binds to adipocytes, we asked whether TLQP-21 might affect spontaneous or β-AR-mediated lipolysis in mouse mature adipocytes. Cultured mouse adipocytes were incubated with TLQP-21, with and without the non-selective β-AR agonist isoprenaline. TLQP-21 did not affect glycerol release (Figure 3A), but dose-dependently increased isoprenaline-induced glycerol release (Figure 3B). The maximal dose of TLQP-21 tested (100 nM) reduced lipolytic EC50 of isoprenaline from 16 to 7.5 nM (Figure 3C). TLQP-21 did not show any binding affinity for β-AR, thus ruling out a primary agonist effect (Figure 3D). Noradrenaline promotes lipolysis via β-AR induction of cAMP-dependent PKA-mediated phosphorylation of HSL [6,38]. Based on TLQP-21-induced potentiation of isoprenaline-induced lipolysis (Figure 3B), we asked whether TLQP-21 might potentiate isoprenaline-induced phosphorylation of HSL and upstream protein kinases in 3T3-L1 adipocytes as a model. As expected [39,40], isoprenaline increased phosphorylation of AMPK, ERK (extracellular-signal-regulated kinase), PKA and HSL (Figure 4). In the absence of isoprenaline, equimolar TLQP-21 increased phosphorylation of AMPK and ERK but not PKA or HSL (Figure 4). TLQP-21 also failed to phosphorylate Akt (Ser473), PKC (protein kinase C; pan Ser660), p38 (Thr180/Tyr182) and JNK (c-Jun N-terminal kinase; Thr183/Tyr185; results not shown), suggesting that TLQP-21 does not increases intracellular cAMP. In contrast, in the presence of isoprenaline, TLQP-21 potentiated and prolonged phosphorylation of AMPK and HSL (Figure 4), thus providing a mechanistic support to its potentiation of isoprenaline-induced lipolysis (Figure 3). Overall, in vitro cellular models support a mechanism whereby TLQP-21 potentiates β-AR-induced lipolysis by up-regulating HSL activity.

Effects of TLQP-21 on lipolysis in mouse adipocytes

Figure 3
Effects of TLQP-21 on lipolysis in mouse adipocytes

(AC) TLQP-21 did not affect glycerol release in mouse adipocytes, whereas the same ineffective dose, up-regulated isoprenaline (ISO)-induced glycerol release. (D) ISO binding to β-AR is not displaced by increasing concentrations of TLQP-21. *P<0.05, **P<0.01. Scrambled peptide (LRPS-21). Pooled results are represented as means+S.E.M.

Figure 3
Effects of TLQP-21 on lipolysis in mouse adipocytes

(AC) TLQP-21 did not affect glycerol release in mouse adipocytes, whereas the same ineffective dose, up-regulated isoprenaline (ISO)-induced glycerol release. (D) ISO binding to β-AR is not displaced by increasing concentrations of TLQP-21. *P<0.05, **P<0.01. Scrambled peptide (LRPS-21). Pooled results are represented as means+S.E.M.

Effect of TLQP-21 on phosphorylation cascade in 3T3-L1 adipocytes

Figure 4
Effect of TLQP-21 on phosphorylation cascade in 3T3-L1 adipocytes

(A) An equimolar concentration (10 μM) of TLQP-21 in the presence or absence of isoprenaline induced a time- and substrate-dependent phosphorylation of AMPK, ERK and HSL which reached a maximum between 15 and 30 min which were used for quantitative experiments in (CE). (B) cAMP-activated PKA substrates are not affected by TLQP-21. (C) TLQP-21 increased phosphorylation of AMPK and potentiated the ISO-induced effect (Mann–Whitney U test, **P<0.01, *P<0.05). (D) TLQP-21 increased phosphorylation of ERK but failed to further up-regulate ISO-induced effects (Mann–Whitney U test, **P<0.01). (E) TLQP-21 did not increase phosphorylation of HSL in the absence of ISO, but potentiated the ISO-induced effect (Mann–Whitney U test, **P<0.01, *P<0.05). Pooled results are represented as means+S.E.M.

Figure 4
Effect of TLQP-21 on phosphorylation cascade in 3T3-L1 adipocytes

(A) An equimolar concentration (10 μM) of TLQP-21 in the presence or absence of isoprenaline induced a time- and substrate-dependent phosphorylation of AMPK, ERK and HSL which reached a maximum between 15 and 30 min which were used for quantitative experiments in (CE). (B) cAMP-activated PKA substrates are not affected by TLQP-21. (C) TLQP-21 increased phosphorylation of AMPK and potentiated the ISO-induced effect (Mann–Whitney U test, **P<0.01, *P<0.05). (D) TLQP-21 increased phosphorylation of ERK but failed to further up-regulate ISO-induced effects (Mann–Whitney U test, **P<0.01). (E) TLQP-21 did not increase phosphorylation of HSL in the absence of ISO, but potentiated the ISO-induced effect (Mann–Whitney U test, **P<0.01, *P<0.05). Pooled results are represented as means+S.E.M.

Chronic subcutaneous infusion of TLQP-21 decreases adipocyte diameter in two mouse models of obesity

Having established that TLQP-21 is present in nerve terminals, binds to adipocytes and potentiates lipolysis in vitro, we set out to investigate whether chronic peripheral in vivo treatment with TLQP-21 might affect adipocyte function in two models of obesity. In DIO mice, TLQP-21 dose-dependently decreased the diameter of pWAT adipocytes [F(2,18)=49.5, P<0.0001; Figure 5A]. TLQP-21 also dose-dependently increased enzymatic activity of TH in both pWAT [F(2,24)=25.1, P<0.0001] and scWAT [F(2,24)=34.5, P<0.0001] (Figure 5B) as well as the concentration of noradrenaline in pWAT [F(2,24)=24.3, P<0.0001] and scWAT [F(2,24)=14.2, P<0.0001] (Figure 5C). As expected, TH enzymatic activity and tissue noradrenaline were positively correlated in both pWAT (r=0.67, P<0.001) and scWAT (r=0.65, P<0.001). In addition, the density of TH-positive nerve fibres was increased in pWAT of mice receiving the HIGH dose of TLQP-21 (Figure 5D). Increased sympathetic-related parameters and reduced adipocytes diameter suggest increased sympathetic-driven lipolysis in the adipose tissue [37]. Two lines of evidence suggest that the reduction in diameter and the increased sympathetic tone could be functionally related. First, the high dose of TLQP-21 increased lipolysis in the pWAT expressed as TAG/DNA ratio [41] when compared with the HFD-saline group (Figure 5E; F(2,18)=4.5. P<0.05). Secondly, the diameter of pWAT adipocytes was negatively correlated with both TH (r=−0.69, P<0.001) and noradrenaline content (r=−0.65, P<0.001), but positively correlated with TAG/DNA ratio (r=0.54, P<0.05). The TAG/DNA ratio also negatively correlates with tissue noradrenaline (r=−0.52, P<0.05).

Chronic infusion of TLQP-21 in DIO mice

Figure 5
Chronic infusion of TLQP-21 in DIO mice

TLQP-21 determined a dose-dependent (40–400 μg/day subcutaneously for 14 days) decrease in perigonadal adipocyte diameter (A). TLQP-21 also dose-dependently increased pWAT and scWAT TH enzymatic activity (B) and noradrenaline content (C). (D) Density of TH-ir showed a trend to be increased by the HIGH dose of TLQP-21 in pWAT parenchyma. (E) TLQP-21 increased lipolysis in pWAT as shown by a decrease in the TAGs to DNA content ratio in HIGH dose group. The broken line indicates the mean value for control mice fed with STD. Different letters indicate statistical difference (P<0.01) between groups within each fat pad. *P<0.05. Pooled results are represented as means+S.E.M.

Figure 5
Chronic infusion of TLQP-21 in DIO mice

TLQP-21 determined a dose-dependent (40–400 μg/day subcutaneously for 14 days) decrease in perigonadal adipocyte diameter (A). TLQP-21 also dose-dependently increased pWAT and scWAT TH enzymatic activity (B) and noradrenaline content (C). (D) Density of TH-ir showed a trend to be increased by the HIGH dose of TLQP-21 in pWAT parenchyma. (E) TLQP-21 increased lipolysis in pWAT as shown by a decrease in the TAGs to DNA content ratio in HIGH dose group. The broken line indicates the mean value for control mice fed with STD. Different letters indicate statistical difference (P<0.01) between groups within each fat pad. *P<0.05. Pooled results are represented as means+S.E.M.

Peripheral TLQP-21 delivery did not affect plasma adrenaline levels (STD-saline, 1819.8±138.1; HFD-saline, 2419.2±210.9; HFD-TLQP-21-LOW, 1885.3±163.4; HFD-TLQP-21-HIGH, 2016.3±182.7 pg/ml), suggesting that the peripheral action of this peptide is distinct from its central effects [17].

Obesity has been associated with tissue inflammation [42,43]. However, this is unlikely to be the mechanism of TLQP-21 for two reasons. First, TLQP-21 did not affect density of crown-like structures, a histological evidence of inflamed WAT ([44]; Supplementary Figure S3 available at http://www.BiochemJ.org/bj/441/bj4410511add.htm). Secondly, as expected, TNFα mRNA was increased in obese mice but was not affected by TLQP-21 treatment (STD-saline=0.04±0.007; HFD-saline=0.08±0.008; HFD-TLQP-21-LOW=0.07±0.008; HFD-TLQP-21-HIGH=0.08±0.009). Finally, TLQP-21 did not affect BW, adipose fat pad weight which is likely to be due to the limited infusion time, and the absence of increased food intake and energy expenditure (body temperature and locomotor activity) (Supplementary Table S1 available at http://www.BiochemJ.org/bj/441/bj4410511add.htm).

We also provided an independent validation of the in vivo effects of TLQP-21 in a different model of obesity, CSS (chronic subordination stress) [29]. In agreement with the results obtained in DIO mice, chronic subcutaneous treatment of TLQP-21 (40 mg/day for 14 days) in CSS mice significantly decreased the diameter of pWAT adipocytes (t12=2.912, P<0.05), but increased TH-enzymatic activity (t12=2.2, P<0.05) and noradrenaline content (t12=2.7, P<0.05; Figure 6). BW gain, food intake, adipose fat pad weight and locomotor activity were not affected (Supplementary Table S2 available at http://www.BiochemJ.org/bj/441/bj4410511add.htm).

Chronic infusion of TLQP-21 in CSS-induced obesity in mice

Figure 6
Chronic infusion of TLQP-21 in CSS-induced obesity in mice

(A) Schematic diagram of experimental procedure. (B) TLQP-21 (40 μg/day subcutaneously for 14 days) determined a decrease in perigonadal adipocyte diameter, an increase in pWAT TH enzymatic activity (C) and noradrenaline content (D). *P<0.05. Pooled results are represented as means+S.E.M.

Figure 6
Chronic infusion of TLQP-21 in CSS-induced obesity in mice

(A) Schematic diagram of experimental procedure. (B) TLQP-21 (40 μg/day subcutaneously for 14 days) determined a decrease in perigonadal adipocyte diameter, an increase in pWAT TH enzymatic activity (C) and noradrenaline content (D). *P<0.05. Pooled results are represented as means+S.E.M.

Finally, TLQP-21 did not affect cardiac anatomy and structure as indicated by a preserved mass-to-chamber volume ratio and the absence of myocardial fibrosis (Table S1) [32] in DIO mice (Table S1), either iBAT TH activity or noradrenaline concentration in CSS mice (Supplementary Table S2), thus ruling out a generalized increase in sympathetic tone to peripheral organs.

In conclusion, results from two independent models of obesity show that chronic peripheral TLQP-21 delivery decreased adipocyte size in obese mice and up-regulated sympathetic functions within the WAT.

DISCUSSION

In the present paper we report the original identification of a novel peripheral lipolytic sensitizer mechanism mediated by the VGF-derived peptide TLQP-21. Our results shows that TLQP-21-ir is present in sympathetic nerve terminals in the WAT, that TLQP-21 binds to an as yet unidentified membrane receptor in adipocyte membranes and that ex vivo and in vivo it exerts a pro-lipolytic effect. Chronic peripheral TLQP-21 does not determine a generalized up-regulation of the sympathetic tone as shown by the normal plasma adrenaline, BAT noradrenaline content and the absence of ventricular remodelling and myocardial damage [45,46].

We unambiguously identified the presence of VGF peptides in sympathetic nerve fibres within the WAT. Specifically, we observed the presence of both TLQP-21-ir and VGF C-terminal peptide-ir in sympathetic fibres innervating the visceral WAT. Anti-TLQP-21-ir and C-terminal peptide-ir co-localized extensively in nerve fibres. However, we also observed fibres that were anti-AQEE-30-positive and anti-TLQP-21-negative (but not vice versa), which would suggest a discrete processing of C-terminal peptides [20,47,48] and that TLQP-21 can be cleaved from its precursor TLQP-62 within neurite secretory vesicles [17,47]. Furthermore, we have also demonstrated the existence of a high-affinity binding site for TLQP-21 in adipocyte membranes. Taken together, these results suggest that TLQP-21 released locally from synaptic nerves participates in neuromodulation of lipolysis (Figure 7). Diet-induced obesity led to an increase in TLQP-21 maximum binding capacity in the adipose tissue but not in other tissues investigated (adrenals, CNS, muscle and liver). To establish an in vivo functional role for TLQP-21 we chronically delivered subcutaneously the peptide for 2 weeks in obese mice. TLQP-21 dose-dependently decreased adipocyte diameter and increased TAG lipolysis in obese mice. Decreased adipocyte diameter and increased lipolysis were paralleled by increased sympathetic tone in the adipose fat pads as demonstrated by increased enzymatic activity/immunoreactivity of TH, the rate-limiting enzyme for the biosynthesis of catecholamine, as well as the neurotransmitter noradrenaline in both visceral and subcutaneous fat pads. Taken together these observations establish a new and previously unrecognized role for TLQP-21 in obesity and adipocyte biology.

Proposed mechanism of TLQP-21. TLQP-21 is released by nerve terminals, binds a putative receptor (TLQP-21R) in adipocyte membrane, potentiate β-AR-induced phosphorylation of AMPK and HSL and increases lipolysis

Figure 7
Proposed mechanism of TLQP-21. TLQP-21 is released by nerve terminals, binds a putative receptor (TLQP-21R) in adipocyte membrane, potentiate β-AR-induced phosphorylation of AMPK and HSL and increases lipolysis

We hypothesize that intracellular signalling downstream of a putative TLQP-21 receptor activation will encompass activation of a Gq protein and lead to intracellular calcium mobilization. Chronic TLQP-21 treatment in vivo increased WAT TH-activity/immunoreactivity and tissue noradrenaline (NE). We hypothesize two mechanisms of action. Hypothesis 1 (Hyp: 1): a presynaptic TLQP-21 receptor would directly affect nerve functions. Hypothesis 2 (Hyp: 2): TLQP-21-receptor-mediated signalling in adipocytes will activate a retrograde signalling to nerve terminals.

Figure 7
Proposed mechanism of TLQP-21. TLQP-21 is released by nerve terminals, binds a putative receptor (TLQP-21R) in adipocyte membrane, potentiate β-AR-induced phosphorylation of AMPK and HSL and increases lipolysis

We hypothesize that intracellular signalling downstream of a putative TLQP-21 receptor activation will encompass activation of a Gq protein and lead to intracellular calcium mobilization. Chronic TLQP-21 treatment in vivo increased WAT TH-activity/immunoreactivity and tissue noradrenaline (NE). We hypothesize two mechanisms of action. Hypothesis 1 (Hyp: 1): a presynaptic TLQP-21 receptor would directly affect nerve functions. Hypothesis 2 (Hyp: 2): TLQP-21-receptor-mediated signalling in adipocytes will activate a retrograde signalling to nerve terminals.

Three lines of evidence support a mechanism whereby TLQP-21 affects adipocyte diameter via modulation of noradrenaline signalling. First, correlation analysis supports a connection between TLQP-21-dependent changes in TH/noradrenaline, lipolysis and adipocyte diameter in vivo. Secondly, TLQP-21 dose-dependently up-regulated isoprenaline-induced glycerol release in mouse adipocytes. Thirdly, TLQP-21 increases isoprenaline-induced phosphorylation of HSL in 3T3-L1 adipocytes. Lipolysis in adipocytes is facilitated via a series of regulatory phosphorylation events linking receptor-mediated increases in cAMP to the phosphorylation of several key proteins, including perilipin and HSL, resulting in an increase in TAG hydrolysis. Lipolysis is under multidimensional regulation by hormones and neurotransmitters with noradrenaline being considered the main regulator via β-AR activation [6,38]. Several investigators have shown that PKA-dependent phosphorylation of HSL (Ser659 and Ser660) is required for both its translocation to the lipid droplet and increased hydrolytic activity (e.g. [49]). In addition, although the role of AMPK in adipocytes is still controversial [50], AMPK-dependent phosphorylation of HSL (Ser565) also results in activation of the lipase and is necessary for translocation [51]. TLQP-21 did not affect lipolysis in the absence of β-AR activation. However, when TLQP-21 was co-administered with a submaximal lipolytic dose of isoprenaline, glycerol release increased dose-dependently up to ~80%. At the same time TLQP-21 increased phosphorylation of AMPK but not HSL in the absence of isoprenaline, whereas it potentiated the isoprenaline-induced phosphorylation of the same kinase (Figure 7). The TLQP-21 receptor has not been identified so far (see below), but previous results demonstrated that the putative TLQP-21 receptor would increase Ca2+ [52] and not cAMP (e.g. lack of PKA activation). On the other hand, although the physiological relevance and role of AMPK in the regulation of lipolysis in adipocytes remain to be fully addressed and contrasting results have been reported [40,5354], TLQP-21 potentiation of isoprenaline-induced lipolysis might result from convergent isoprenaline-induced cAMP-PKA and TQLP-21-induced Ca2+-AMPK [52,56,57] signalling pathways leading to increased HSL activity (Figure 7). Future studies should clarify this issue.

Increased sympathetic innervation to fat pads has been demonstrated after cold exposure, fasting and chronic angiotensin II treatment [37,58,59]. Local sympathetic denervation of WAT leads to marked increases in WAT mass and fat cell number [60]. Conversely, electrical stimulation of WAT nerves promotes lipid mobilization [61]. In the present study, we demonstrate that chronic TLQP-21 treatment increased TH activity/immunoreactivity and noradrenaline content in fat pads while increasing lipolysis. Therefore we speculate that TLQP-21 might directly (e.g. binding to a presynaptic receptor on nerve terminals, Hypothesis 1 in Figure 7) or indirectly (e.g. increasing neurotrophin signalling in adipocytes [62,63], Hypothesis 2 in Figure 7) increase sympathetic nerve activity, leading to increased noradrenergic signalling, and stimulate nerve growth within the WAT. Following the observation that iBAT noradrenaline, body temperature and heart morphology are not affected by TLQP-21, we conclude that TLQP-21 selectively modulates sympathetic noradrenergic pathways in WAT. Understanding the molecular mechanism of this selective modulation of sympathetic nerves require further studies.

Besides its functional role in the WAT, one of the major findings of the present study is the identification of the first receptor-mediated mechanism for a VGF-derived peptide. Research on granin-derived peptides has so far been constrained by the poor understanding of receptor-mediated mechanisms [64]. In the present study, we identified for the first time a selective and saturable TLQP-21-receptor-binding site and showed that the highest affinity and Bmax is present in white adipocytes. We have also demonstrated that TLQP-21 binds to adrenals and BAT with an affinity comparable with that of WAT. In contrast, binding to the muscle, liver and cerebellum was modest. Previous studies, in a neuronal model, established that TLQP-21 significantly activated ERK1/2 serine/threonine protein kinases as an effect of its anti-apoptotic neurotrophic action [52]. In addition, TLQP-21 significantly increased intracellular calcium [as measured by fura 2/AM (fura 2 acetoxymethyl ester)] in approx. 60% of the recorded neurons [52]. In an attempt to analyse the TLQP-21 signalling in adipocytes we have undertaken a broad approach which took into account different signalling pathways. We confirmed the phosphorylation of ERK and most interestingly the prolonged effect on activation of HSL and AMPK by TLQP-21 with β-AR agonist isoprenaline. On the other hand TLQP-21 failed to activate the cAMP/PKA pathway since PKA substrates were not substantially phosphorylated by TLQP-21 [38]. We can speculate that, similarly to other peptides [65], TLQP-21 will bind a seven transmembrane domain receptor, which leads to the activation of a Gq protein, stimulation of a phospholipase B, increase in DAG and PI3K (phosphoinositide 3-kinase) and activation of PKC and intracellular calcium mobilization (Figure 7). However, lacking a molecular characterization of TLQP-21 receptor, we cannot exclude other types of receptor-mediated effect, such as tyrosine receptor or cytokine receptor, but the mild and weak effect suggests a more neuromodulatory role typical of neuropeptides more than the strong effects typical of grow factors or cytokines (e.g. [66]).

Conclusion

The VGF gene is highly conserved across evolution [13,64]. Studies on VGF in humans are limited (see [11,64] for reviews) and to the best of our knowledge no study directly addressed a potential gene association of VGF with obesity or other metabolic disorders in humans. We report in the present paper the original characterization of a novel peripheral lipolytic mechanism encompassing the VGF peptide TLQP-21. Our findings are remarkable and novel for three different aspects. First, this is the first demonstration of a peripheral biological role for a VGF-derived peptide. Secondly, the present study establishes for the first time a selective receptor-mediated mechanism for a VGF-derived and one of the first for the entire family of granin peptides [64]. Thirdly, the present study identified a completely novel pro-lipolytic mechanism which, in the absence of adverse cardiovascular effects, may open promising drug discovery perspective to counteract obesity. In conclusion, our results provide a mechanistic rationale and translational potential for the development of TLQP-21 analogues for the treatment of obesity.

Abbreviations

     
  • AMPK

    AMP-activated protein kinase

  •  
  • β-AR

    β-adrenergic receptor

  •  
  • BAT

    brown adipose tissue

  •  
  • BW

    body weight

  •  
  • CNS

    central nervous system

  •  
  • CSS

    chronic subordination stress

  •  
  • DIO

    diet-induced obesity

  •  
  • ERK

    extracellular-signal-regulated kinase

  •  
  • HFD

    high-fat diet

  •  
  • HSD

    Honestly Significant Difference

  •  
  • HSL

    hormone-sensitive lipase

  •  
  • iBAT

    interscapular BAT

  •  
  • ir

    immunoreactivity

  •  
  • LV

    left ventricular

  •  
  • PKA

    protein kinase A

  •  
  • PKC

    protein kinase C

  •  
  • QPCR

    quantitative PCR

  •  
  • RT

    reverse transcription

  •  
  • RV

    right ventricle

  •  
  • STD

    standard chow

  •  
  • TAG

    triacylglycerol

  •  
  • TH

    tyrosine hydroxylase

  •  
  • TNFα

    tumour necrosis factor α

  •  
  • WAT

    white adipose tissue

  •  
  • pWAT

    perigonadal WAT

  •  
  • rpWAT

    retroperitoneal WAT

  •  
  • scWAT

    subcutaneous inguinal WAT

AUTHOR CONTRIBUTION

Roberta Possenti and Alessandro Bartolomucci led the design, conceived the study, performed experiments and analysed the results. Roberta Possenti, Bjarne D. Larsen, Jorgen S. Petersen, Anna Moles, Andrea Levi and Alessandro Bartolomucci conceived an initial part of the study. Giampiero Muccioli, Pamela Petrocchi, Cheryl Cero, Aderville Cabassi, Lucy Vulchanova, Maureen S. Riedl, Monia Manieri, Andrea Frontini, Antonio Giordano, Saverio Cinti, Paolo Govoni, Gallia Graiani, Federico Quaini, Corrado Ghè, Elena Bresciani, Ilaria Bulgarelli, Antonio Torsello, Vittorio Locatelli, Valentina Sanghez, Paola Palanza and Stefano Parmigiani performed experiments, and analysed and contributed to the interpretation of results. Alessandro Bartolomucci wrote the paper with input from all other authors.

FUNDING

This work was supported by Zealand Pharma A/S (to A.B. and S.P.), University of Minnesota Medical School (to A.B.), Regione Lazio (to A.L. and R.P.), University of Milano-Bicocca-Fondo di Ateneo per la Ricerca (to A.T. and V.L.), Cariparma-Credit Agricole Foundation (to A.C.) and Fondazione Enrico and Enrica Sovena (to P.P.).

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Author notes

1

Present address: Merck Serono International S.A., Geneva, Switzerland.

2

Jorgen Petersen owns shares in Zealand Pharma A/S.

Supplementary data