International Journal of Obesity (2010) 34, 1599–1607 & 2010 Macmillan Publishers Limited All rights reserved 0307-0565/10 www.nature.com/ijo ORIGINAL ARTICLE Genetic deficiency for proprotein convertase subtilisin/kexin type 2 in mice is associated with decreased adiposity and protection from dietary fat-induced body weight gain YAnini1,2,JMayne3,JGagnon1,2,JSherbafi3,AChen3,NKaefer3,MChre´tien3,4,5 and M Mbikay3,4,5 1Department of Physiology and Biophysics, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; 2Department of Obstetrics and Gynecology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; 3Chronic Disease Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada; 4Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontario, Canada and 5Division of Endocrinology and Metabolism, Ottawa Hospital, Ottawa, Ontario, Canada Background: Proprotein convertase subtilisin/xexin type 2 (PCSK2) is an endoproteinase responsible for proteolytic activation of a number of precursors to active neuropeptides and peptide hormones, known to influence glucose homeostasis, food intake and ultimately body mass. In this study, we examined the consequences of PCSK2 deficiency on these phenotypic traits. Study Design: Weight gain with age under diets of different fat contents was monitored. White adipose tissue (WAT) and muscle masses were evaluated. Plasma levels of triglycerides, leptin, ghrelin, insulin and proglucagon-derived peptides were measured as well as leptin and acetyl coenzyme-a carboxylase (ACCa) mRNA levels in adipose tissue. Results: Compared with their Pcsk2 þ / þ littermates, Pcsk2À/À mice weighed significantly less as weanlings and as adults. As adults, they carried noticeably less fat mass, with similar lean muscle mass: their plasma leptin level and adipose tissue leptin mRNA level were accordingly lower. PCSK2 deficiency did not affect food intake or the level of the orexigenic hormone ghrelin. However, PCSK2 deficiency resulted in decreased plasma triglycerides and reduced ACCa mRNA levels in WAT. Interestingly, unlike their Pcsk2 þ / þ littermates, Pcsk2À/À were resistant to enhanced body weight gain when fed a high-fat diet. Consistent with a role of PCSK2 in body mass gain, diet-induced or genetically obese mice were found to contain significantly higher levels of PCSK2 mRNA in their brain and stomach than their lean counterparts. Conclusion: Collectively, these results suggest that PCSK2 contributes to increase in body mass through the various regulatory peptides generated through its action. It represents a potential target in the prevention and treatment of obesity. International Journal of Obesity (2010) 34, 1599–1607; doi:10.1038/ijo.2010.90; published online 25 May 2010 Keywords: proprotein convertase 2; adiposity; body mass; dietary fat; diet-induced obesity; proglucagon Introduction PCSK1 and carboxypeptidase E, it mediates the proteolytic activation of precursors to a variety of neuropeptides and Proprotein convertase subtilisin/kexin type 2 (PCSK2), also hormones.1,2 known as proprotein convertase 2, is a member of the Kexin- Generation of a PCSK2-null (Pcsk2À/À) mouse by Furuta like subfamily of eukaryotic endoproteinases implicated in et al.3 has offered the opportunity to delineate the enzymatic the activation of secretory precursor proteins by cleavage link between this convertase and many neuroendocrine after selected basic residues. PCSK2 is primarily expressed precursors, and to explore the physiological importance of in neuronal and endocrine cells in which, together with its processing of these precursors. The original Pcsk2À/À mouse,3 which was a 129Sv:C57BL/6 (B6) mixed genetic Correspondence: Dr Y Anini, Departments of Physiology and Biophysics and background, seemed normal at birth, but grew at a slightly Obstetrics and Gynecology, Dalhousie University, 5850 College Street, Room reduced rate. They showed chronic fasting hypoglycemia 4C1, Halifax, Nova Scotia B3H 1X5, Canada. and enhanced glucose tolerance in an intraperitoneal E-mail: [email protected] Received 18 June 2009; revised 16 March 2010; accepted 17 March 2010; glucose tolerance test. They were normoinsulinemic published online 25 May 2010 but severely hypoglucagonemic. Proteolytic activation of Reduced body mass and adiposity in PCSK2-null mice Y Anini et al 1600 pancreatic islet prohormones was variably impaired: the genders were weighed and killed by decapitation; their impairment was more severe for a-cell proglucagon and gonadal fat and gastroc muscle were immediately collected d-cell prosomatostatin than for b-cell proinsulin.3,4 At 3 and weighed. months of age, the islets of mutant mice showed marked To assess food consumption, 4-month-old male mice were hyperplasia of a-cells and d-cells and a relative diminution of fasted for 16 h and then given free access to pre-weighed b-cells.3 Other physiological studies showed that these mice standard chow; the drop in chow weight was measured after are prone to salt-induced hypertension5 and stress-induced 1, 2, 4, 6 and 24 h. analgesia.6 Other substrates whose processing is impaired in the Pcsk2À/À mouse include precursors for pancreatic islet Dietary regimens amyloid peptide,7,8 ileum neuropeptide Y,9 pituitary and To study the effect of dietary fat on weight gain, 3-week-old brain adrenocorticotropin, a-melanocyte-stimulating hor- mice were divided into groups (n ¼ 4–6/gender/genotype) mone10 and b-endorphin,11 brain cholecystokinin (CCK),12 and fed for 7 weeks a Research Diets (North Brunswick, NJ, dynorphin,13 neurotensin,14 Met-enkephalin15 and orpha- USA) low-fat diet (LFD, catalog no. D12450) or a high-fat diet nin FQ/nociceptin.16 Several of these peptides are known to (HFD, catalog no. D12451), containing 10 or 45% kcal as fat, regulate feeding: neuropeptide Y and b-endorphin are respectively. Body weight was recorded weekly. orexigenic, and CCK, neurotensin and a-melanocyte-stimu- To evaluate the effect of diet-induced obesity on the lating hormone are anorexigenic.17 expression of PCSK2, 4-week-old B6 mice were fed either a Using subcongenic mapping of a SPRET/Ei donor DNA on HFD or a LFD for 8 weeks. RNA was then extracted from Chr 2 in a B6 genetic background, Chiu et al.18 have stomach and brain tissues. identified Pcsk2 as a positional candidate gene influencing body weight and adiposity. Mice homozygous for SPRET/Ei donor allele were leaner than their B6 counterparts and Plasma hormone assays expressed less PCSK2 in their brain.18 However, in another Mice were fasted for 16 h, anesthetized under isoflurane mouse study, a negative correlation was observed between (Abbott Laboratories, Montreal, QC, Canada) and bled by body weight and the expression level of PCSK2 and 7B2, its cardiac puncture. Plasma was separated from blood cells by specific chaperone and transient inhibitor.19 In this study, centrifugation. Plasma levels of leptin, insulin, glucagon, we examine how food intake and body mass are affected by glucagon-like peptide 1 (GLP-1) and amylin were measured PCSK2 global deficiency in mice. using the LINCOplex Mouse Endocrine Immunoassay Panel (Linco Research, Inc., St Charles, MI, USA). Total plasma ghrelin was measured using total ghrelin RIA kit (Linco Materials and methods Research, Inc.). Animals The Pcsk2À/À mice and wild-type (WT) control mice were Triglyceride assays littermates obtained from the mating of Pcsk2À/ þ hetero- Plasma was collected from 16-h-fasted mice and assayed for zygotes generated as described in Furuta et al.3 Our mouse triglyceride content using the Triglyceride Quantification Kit colony was initiated from a N4 backcross into the CD-1 from Biovision (Mountain View, CA, USA). genetic background. It was perpetuated by brother–sister heterozygous mating for 45 years. Four-week-old male C57BL/6 (B6) and B6.V-Lep(ob)/j ob/ob Quantitative reverse transcriptase PCR mice were purchased from The Jackson Laboratory (Bar Total RNA was extracted from brain, stomach and adipose Harbor, ME, USA). All mice were housed in temperature- tissue using TRIzol Reagent (Invitrogen, Burlington, ON, controlled rooms with 12-h dark–light cycles and, unless Canada). It was reverse-transcribed into complementary otherwise stated, were provided with food and drink ad DNA using oligo dT-18 primer (100 ng mg–1 of RNA). libitum. They were handled according to the guidelines of the Quantitative PCR reactions were performed in 25 ml using Canadian Council on Animal Care under a protocol approved 2 Â Brilliant SYBR green master mix (Roche, Mississauga, by the institutional animal care committee. Unless otherwise ON, Canada) under the following conditions: initial dena- stated, experimental mice were male and 8–12 weeks of age. turation at 95 1C for 10 min, then 40 cycles of 95 1C for 30 s, 60 1C for 1 min and 72 1C for 30 s, using the following primers for proprotein convertase 2 (forward 50-AACATCT Weight gain, food consumption and adiposity GACTGTGCTCACCTCCA-30 and reverse 50-TTTCACCATGG To measure weight gain with age, 3-week-old male mice were CACCTGCATCAAG -30), leptin (forward 50-TGCTGAAGTTT kept on standard mouse chow and weighed weekly for CAAAGGCCACCAG-30 and reverse 50-ATGCCTTTGGATG 11 weeks and at 17 weeks. To evaluate white adipose tissue GGTGGTCTACA-30) and acetyl coenzyme-a carboxylase (WAT) and muscle mass, 7- and 17-week-old mice of both (ACCa; forward 50-TAACAGAATCGACACTGGCTGGCT-30 International Journal of Obesity Reduced body mass