Differential distribution of VGF-derived peptides in the adrenal medulla and evidence for their selective modulation Filomena D’Amato 1 , Barbara Noli 1 , Carla Brancia 1 , Cristina Cocco 1 , Giovanna Flore 2 , Maria Collu 2 , Paola Nicolussi 3 and Gian-Luca Ferri 1 1 NEF-Laboratory, Department of Cytomorphology, 2 Department of Neuroscience, University of Cagliari, I-09042 Monserrato (Cagliari), Italy 3 Istituto Zooprofilattico Sperimentale, I-07100 Sassari (SS), Italy (Correspondence should be addressed to G-L Ferri; Email: ferri@unica.it) Abstract While vg f gene knockout mice are hyperactive and hypermetabolic, surprisingly the TLQP-21 brain VGF peptide increased energy consumption, suggesting that opposing regulatory effects could be exerted by peptides alternatively cleaved from the VGF precursor. Using antisera to the VGF precursor C-terminus and three cleavage products, we revealed a distinct differential distribution in adrenal, certain peptides (VGF 422–430 : PGH peptides) being found throughout bovine and swine medulla, while C-terminus and TLQP peptides were confined to adrenaline cells in the above species and in rat and C-terminally shortened forms (VGF 604–612 : HVLL peptides) to nor-adrenaline cells. Ran- dom abattoir samples of bovine and swine adrenal contained 520G40 and 450G60 pmol/g (meanGS.E.M. respectively) of C-terminus peptides and similar or lower amounts of others. Upon gel chromatography, bona fide VGF precursor, ~7 . 5 and ~3 . 5 kDa forms were revealed by C-terminus assays, HVLL peptides being limited to small fragments. TLQP peptides included ~7 . 5 kDa form and peaks accounting for TLQP-21 and predicted TLQP-30 and TLQP-42. Low molecular weight (MW) PGH peptides were revealed, together with a high MW form possibly encompassing the VGF precursor N-terminus. In acutely stressed swine, a striking increase was seen for C-terminus and TLQP peptides, with no significant differences for PGH peptides. A similar response was found in rat TLQP peptides showing a major increase upon an acute swimming stress and 30 min thereafter. A differential processing of the VGF precursor encompassing many areas of its primary sequence and selective modulations of its derived peptides occur in adrenal medullary cells, possibly relevant to adaptive homeostatic responses. Journal of Endocrinology (2008) 197, 359–369 Introduction The vg f (non-acronymic) gene was discovered because of its distinct, selective up-regulation by nerve growth factor in the PC12 rat pheochromocytoma cell line (Levi et al. 1985) and proved to be selectively expressed in a range of neuro- endocrine cell types (Ferri et al. 1992, Salton et al. 2000, Levi et al. 2004). The primary gene product, VGF protein or VGF precursor, is composed of 617 or 615 amino acids (in rat/ mouse and man respectively, O85% identity, ~65 kDa calculated molecular weight (MW)) and migrates as ~90 kDa MW band in western blot (Salton et al. 2000). The primary sequence of VGF shows at least ten short stretches of basic amino acid residues, highly conserved across species, which could be target sites for endoprotease activity, with cleavage to smaller VGF peptides (Salton et al. 2000). The 30 amino acid ‘peptide V’ was isolated from bovine posterior pituitary (Liu et al. 1994), and, upon sequencing of the relevant region of human (Canu et al. 1997, Salton et al. 2000) and bovine VGF (accession No. XP_875466.2), proved to correspond to the 30 amino acid portion of VGF comprised between the Arg 584 -Arg 585 (numbering according to human VGF) putative cleavage site and the C-terminus. In rat brain, lower MW peptides (10, 18 and 20 kDa) were detected by an antiserum against the C-terminal VGF nonapeptide (Trani et al. 1995, 2002). Processing at the rat VGF Arg 553 -Pro 554 -Arg 555 cleavage site resulted in further VGF peptides isolated from rat brain and called TLQP peptides from their N-terminal sequence (Trani et al. 2002). When VGF peptide release in response to secretory stimuli was studied in vitro, low MW C-terminal peptides were released preferentially, as opposed to high MW forms (Possenti et al. 1989, 1999, Trani et al. 1995). VGF-deficient (knockout) mice are hyperactive and hypermetabolic, with altered energy homeostasis, decreased body weight and especially body fat, and a deranged hypothalamic response to feeding (Hahm et al. 1999, 2002). Nonetheless, intracerebroventricular injection of the VGF precursor-derived TLQP-21 brain peptide increased resting energy expenditure, body temperature and circulating adrenaline levels (Bartolomucci et al. 2006), indicative of a stimulatory action on the autonomic nervous system and 359 Journal of Endocrinology (2008) 197, 359–369 DOI: 10.1677/JOE-07-0346 0022–0795/08/0197–359 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org