Transplantation of PC1/3-Expressing α-cells Improves Glucose Handling and Cold Tolerance in Leptin-resistant Mice

Transcription

1 The Amerin Soiety of Gene Therpy originl rtile Trnsplnttion of PC1/3-Expressing α-ells Improves Gluose Hndling nd Cold Tolerne in Leptin-resistnt Mie Rhond D Widemn 1, Srh L Gry 1, Sott D Covey 1, Gene C We 2 nd Timothy J Kieffer 1,3 1 Lortory of Moleulr nd Cellulr Mediine, Deprtment of Cellulr nd Physiologil Sienes, Life Sienes Institute, University of British Columi, Vnouver, British Columi, Cnd; 2 Deprtment of Mediine, University of Chigo, Chigo, Illinois, USA; 3 Deprtment of Surgery, Life Sienes Institute, University of British Columi, Vnouver, British Columi, Cnd Type 2 dietes (T2D) is hrterized y elevted lood gluose levels owing to insuffiient seretion nd/or tivity of the gluose-lowering hormone insulin. Glugonlike peptide-1 (GLP-1) hs reeived muh ttention s new tretment for dietes euse of its multiple lood gluose lowering effets, inluding gluose-dependent enhnement of insulin seretion, inhiition of gstri emptying, nd promotion of the survivl nd growth of insulin-produing β-ells. GLP-1, long with GLP-2 nd oxyntomodulin, is produed in the intestinl L-ell vi proessing of proglugon y prohormone onvertse 1/3 (PC1/3), while in the pnreti α-ell, oexpression of proglugon nd the lternte enzyme PC2 typilly results in differentil proessing of proglugon to yield glugon. We used lginte-enpsulted α-ells s model to evlute ontinuous delivery of PC1/3- or PC2-derived proglugon produts. In high ft fed nd d/d mie, PC1/3-, ut not PC2-expressing α-ells improved gluose hndling nd trnsiently lowered fsting gluose levels, suggesting tht ontinuous delivery of PC1/3-derived proglugon produts vi ell therpy my e useful for dietes tretment. In ddition, we show tht long-term tretment with PC1/3-expressing, ut not PC2-expressing, α-ells improved old-indued thermogenesis in d/d mie, demonstrting previously unppreited effet of one or more PC1/3-derived α-ell produts. Reeived 21 April 28; epted 1 Septemer 28; pulished online 21 Otoer 28. doi:1.1/mt Introdution Proglugon, whih is expressed in pnreti α-ells, intestinl L-ells, nd in speifi neurons in the entrl nervous system, is differentilly proessed to yield numerous peptides elonging to the glugon superfmily of hormones. 1 Although memers of this superfmily shre signifint peptide sequene homology, they hve diverse nd sometimes opposing regultory funtions. In the pnreti α-ells, prohormone onvertse (PC2) is the predominnt proessing enzyme, with the result tht the mjor iotive proglugon-derived peptide (PGDP) rising in these ells is glugon, 2,3 gluose-rising hormone opposing insulin tion. While glugon tivity is ruil for preventing hypoglyemi under norml onditions, hyperglugonemi ompnies nd my exerte type 2 dietes (T2D). 4 In ontrst to the α-ell, in the enteroendorine L-ell nd the rin, PC1/3 is the predominnt proessing enzyme ting on proglugon, lierting glugon-like peptide-1 (GLP-1), GLP-2, nd oxyntomodulin. 5,6 GLP-1 hs grnered signifint therpeuti interest for the tretment of dietes euse of its pleiotropi lood gluose lowering effets, inluding enhnement of gluose- stimulted insulin seretion, inhiition of gstri emptying nd glugon seretion, nd promotion of β-ell prolifertion nd survivl. 1,7 GLP-2 nd oxyntomodulin lso tend to lower lood gluose levels y promoting stiety nd inhiiting gstri emptying. 8,9 Thus proglugon, depending on whether it is proessed y PC2 or PC1/3, gives rise to produts tht tend to hve either gluose-rising or gluose-lowering effets. We hve previously shown tht expression of PC1/3 rther thn PC2 in α-ells indues GLP-1 prodution nd onverts the α-ell from hyperglyemi-promoting ell to one tht lowers lood gluose levels nd promotes islet survivl in rodent models of type 1 dietes. 1,11 Here we hve exmined the metoli effets of trnsplnting enpsulted PC1/3-expressing α-ells in rodent models of T2D, nd demonstrte tht ell therpy with PC1/3- expressing α-ells improves gluose hndling nd improves old thermogenesis in leptin-resistnt mie. Results We nd others hve previously reported tht αtcδpc2 ells express PC1/3, ut no funtionl PC2, nd produe GLP-1. 1,12 We found tht ompred to PC2-expressing αtc-1 ells, αtcδpc2 ells serete higher levels of immunoretive GLP-2 (255.8 ± 2.6 ng/ml versus ± 17. ng/ml; n = 2) nd oxyntomodulin (5. ± 5.7 ng/ml versus 27.7 ± 1.5 ng/ml; P <.1; n = 4; dt not shown). In this study, we used enpsulted αtcδpc2 ells to ssess the effiy of ell therpy with PC1/3-expressing α-ells in mouse models of T2D. We trnsplnted enpsulted Correspondene: Timothy J. Kieffer, Deprtment of Cellulr nd Physiologil Sienes, Life Sienes Institute, University of British Columi, 235 Helth Sienes Mll, Vnouver, British Columi V6T 1Z3, Cnd. E-mil: tim.kieffer@u. Moleulr Therpy vol. 17 no. 1, jn

2 PC1/3-expressing α-ell Therpy in Dieti Mie The Amerin Soiety of Gene Therpy ells to the intrperitonel (IP) vity of 16-week-old C57BL/6 mie tht hd een fed 6% kl from ft diet from 4 weeks of ge. Cell trnsplnttion hd no effet on ody weight (Figure 1), nd we ould not detet ny hnges in food intke (dt not shown). However, αtcδpc2 reipients hd lower fsted lood gluose levels thn shm-operted ontrols t two posttrnsplnt time points (Figure 1; 8.8 ±.3 mmol/l versus 1.4 ±.5 mmol/l t dy 4, P <.5; 9.8 ±.3 mmol/l versus 11. ±.3 mmol/l t dy 13, P <.5). An IP gluose tolerne test performed 7 dys fter trnsplnt reveled tht αtcδpc2 reipients hd etter gluose tolerne thn ontrols, s indited y more rpid lowering of lood gluose levels (Figure 1; P <.1 t 6 minutes nd P <.5 t 12 minutes) nd n overll derese in glyemi exursion (Figure 1 inset; P <.1). Plsm insulin levels were not signifintly different efore gluose injetion (1.1 ±.12 μg/l for ell trnsplnt reipients versus.52 ±.1 μg/l for shm-operted ontrols t time ; P =.12; n = 5 6) ut ell trnsplnt reipients tended to hve higher plsm insulin levels 7 minutes fter gluose injetion (1.47 ±. μg/l versus.78 ±.13 μg/l for shm; P =.8; n = 5 6). As αtcδpc2 ells improved glyemi in the high ft fed model of gluose intolerne, we hypothesized tht trnsplnttion of αtcδpc2, ut not αtc-1 ells might dely the onset of dietes in the d/d mouse model of T2D. We did not detet ny impt of ell trnsplnt on either ody weight or food intke (dt Body weight (g) Dys fter trnsplnt Dys fter trnsplnt AUC Figure 1 Trnsplnttion of enpsulted prohormone onvertse 1/3 expressing α-ells improves gluose homeostsis in mouse model of diet-indued oesity nd hyperglyemi. Sixteen-week-old mle mie fed 6% ft diet from 4 weeks of ge reeived shm surgery or ell trnsplnt on dy (n = 8 per ondition). () Body weight nd () lood gluose were monitored fter 4-hour morning fst. () An intrperitonel gluose tolerne test (2 g/kg) ws performed 7 dys fter trnsplnt. AUC, re under the urve. P <.5, P <.1, P <.1 ompred to shm. not shown). Although αtcδpc2 ell trnsplnt initilly lowered fsting lood gluose levels fter trnsplnt (6.6 ±.2 mmol/l versus 7.7 ±.3 mmol/l t 2 dys fter trnsplnt; P <.1), lood gluose levels were not different therefter nd oth groups of nimls developed dietes t similr rte (Figure 2). In ontrst, αtc-1 reipients hd elevted lood gluose levels ompred to shm-operted ontrols t severl time points (Figure 2; 18.7 ± 1.8 mmol/l versus 12.7 ± 1.9 mmol/l t dy 2, P <.5; 27.1 ±.9 mmol/l versus 2.1 ± 2.5 mmol/l t dy 2, P <.5). To determine whether αtcδpc2 ell trnsplnt influened ody omposition in d/d mie, we performed nuler mgneti resonne imging 17 dys fter trnsplnt. αtcδpc2 reipients were found to hve slight ut sttistilly signifint inrese in the rtio of len:lipid ody mss (Figure 2; P <.5) without ny hnge in totl ody mss (dt not shown), inditing tht produt from the αtcδpc2 ell trnsplnts deresed ft mss nd/or inresed len mss in these mie. Despite their only trnsient lowering of fsted gluose levels, αtcδpc2 ells profoundly improved gluose tolerne 7 dys fter trnsplnt, with ell-treted mie hving lower lood gluose levels t eh time point (Figure 3; P <.5 t minutes, P <.1 t 7, 6, nd 12 minutes, nd P <.1 t 15 minutes) nd 4% redution in totl glyemi exursion ompred to shm-operted ontrols (Figure 3 inset; P <.1). In ontrst, t 4 dys fter trnsplnt, αtc-1 reipients hd no improvement in gluose tolerne ompred to shm-operted ontrols (Figure 3). Indeed lood gluose levels were tully inresed t severl time points (11.5 ± 2.2 mmol/l versus 5.8 ± 1. mmol/l t Len:lipid mss Dys fter trnsplnt αtc Dys fter trnsplnt 2 Figure 2 Metoli prmeters in d/d mie reeiving prohormone onvertse 1/3 (PC1/3)-expressing or PC2-expressing α-ell therpy. Blood gluose ws mesured followed 4-hour morning fst in mie reeiving () αtcδpc2 or () αtc-1 ell trnsplnts or shm surgery. () Len-to-lipid ody mss rtio ws determined y nuler mgneti resonne imging 17 dys fter trnsplnt. P <.5, P <.1 ompred to shm; n = 5 6 per group vol. 17 no. 1 jn. 29

3 The Amerin Soiety of Gene Therpy PC1/3-expressing α-ell Therpy in Dieti Mie minutes; 17.8 ± 2.3 mmol/l versus 1.8 ± 2. mmol/l t 12 minutes; P <.5; n = 6) nd there ws no hnge in overll glyemi exursion (Figure 3 inset). As hs een reported previously, 13 d/d mie were hyperinsulinemi even fter n overnight fst nd hd defetive gluose-stimulted insulin seretion in the IP gluose tolerne test. However, αtcδpc2 ell reipients displyed lower fsting insulin levels (2.13 ±.28 μg/l versus 3.47 ±.43 μg/l for shm-operted ontrols; P <.5) nd re-estlishment of insulin seretion in response to gluose injetion (Figure 3; P <.5 t 7 nd 15 minutes, P <.1 t 6 minutes). By 28 dys fter trnsplnt the enefiil impt of αtcδpc2 ells on gluose tolerne ws no longer evident, exept for slight lowering of lood gluose levels t the 15-minute time point (Figure 3d; P <.5 t 7 minutes). Plsm GLP-1 levels were ssessed in lood smple tken 14 dys fter trnsplnt, nd lthough GLP-1 ws elow the level of detetion (~2 pmol/l) in ll ut one shm-operted mouse, αtcδpc2 reipients hd plsm GLP-1 levels of 71.3 ± 9.2 pmol/l (n = 6) AUC 2 d Plsm insulin (%) AUC AUC2 1 αtc-1 αtc Figure 3 Gluose homeostsis in d/d mie reeiving prohormone onvertse 1/3 (PC1/3)-expressing or PC2-expressing α-ell therpy. Intrperitonel gluose tolerne tests (IPGTTs) (2 g/kg) were performed following n overnight fst 7 dys (, ), 4 dys (), or 28 dys (d) fter trnsplnttion of αtcδpc2 (,, d) or αtc-1 () ells. AUC, re under the urve. For the dy 7 IPGTT, plsm ws olleted t the indited time points nd ssyed for insulin (), expressed s % inrese ompred to sl. P <.5, P <.1, P <.1 ompred to shm; n = 5 6 per group. Mie with geneti defets in the leptin reeptor signling pthwy (e.g., d/d mie) re known to hve lower ody temperture in oth the light nd drk periods, s well s defetive old-indued thermogenesis, ompred to littermte ontrols. 14 To ssess whether α-ell produts ould improve ody temperture regultion in this model, we implnted suutneous temperture sensors in d/d mie tht hd reeived αtcδpc2 or αtc-1 ell trnsplnt, or shm surgery. At 29 dys fter trnsplnt, αtcδpc2 reipients hd remrkle improvement in their ility to mintin their ody temperture during ute housing in 4 C environment, with higher ody temperture t lmost every time point (Figure 4; P <.5 t 2, 75, 9, nd 15 minutes; P <.1 t 45, 6, nd 12 minutes). In ontrst, αtc-1 reipients hd no improvement in their ility to mintin their ody temperture ompred to ontrol nimls t dys fter trnsplnt (Figure 4). To further hrterize the enefiil effets of αtcδpc2 ell trnsplnt on ody temperture regultion, dditionl ell trnsplnts were performed in d/d mie nd shm-operted wild-type C57BLKS/J ontrols. As in our previous study, trnsplnttion of αtcδpc2 ells did not dely dietes onset in d/d mie, lthough trnsplnt reipients displyed improved gluose tolerne 7 dys fter trnsplnt, with lower lood gluose levels t severl time points nd 45% derese in re under the urve ompred to shm-operted d/d mie (dt not shown). In this seond ohort of mie, we performed n initil old tolerne test 5 dys fter trnsplnt, nd found no differene etween shm-operted nd αtcδpc2-trnsplnted d/d mie, though oth groups hd n impired ility to mintin their ody temperture ompred to wild-type ontrols (Figure 5). However, y 26 dys fter trnsplnt, d/d αtcδpc2 ell reipients were etter le to mintin their ody temperture during old exposure Wild type d/d d/d αtc d/d GLP-1 d/d OXM d/d GLP Figure 4 Cold-indued thermogenesis in d/d mie reeiving prohormone onvertse 1/3 (PC1/3)-expressing or PC2-expressing α-ell therpy or ontinuous infusion of proglugon-derived peptide. Cold tolerne tests were performed 29 dys fter trnsplnttion of αtδpc2 ells () or dys fter trnsplnttion of αtc-1 ells or implnttion of miniosmoti pump loded with glugon-like peptide-1 (GLP-1), GLP-2, or oxyntomodulin (OXM; ). Mie were housed t 4 C nd ody temperture ws monitored using hnd-held trnsponder (n = 5 6 per group). P <.5, P <.1 ompred to shm. Moleulr Therpy vol. 17 no. 1 jn

4 PC1/3-expressing α-ell Therpy in Dieti Mie The Amerin Soiety of Gene Therpy d Wild type d/d d/d : 18: 2: 22: Time of dy thn d/d ontrols (Figure 5; P <.5 t,, 6, 75, nd 9 minutes; P <.1 t 45 minutes), though they still hd lower ody tempertures thn wild-type ontrols towrd the end of the old exposure period. We monitored hnges in ody temperture over 24-hour period in ohort of singly housed mie, nd found tht t ll time points, oth groups of d/d mie hd lower ody temperture thn wild-type ontrols. However, αtcδpc2 reipients tended to hve higher ody tempertures thn shm- operted d/d mie, prtiulrly during the drk phse (Figure 5; P <.5 t 24: hours; P =.11 for entire 24-hour period using pired Student s t-test). Our oservtion tht αtcδpc2 ells improved ody temperture regultion in d/d mie led us to hypothesize tht one or more of the PC1/3-derived proglugon produts might hve role in ody temperture regultion. We resoned tht if GLP-1 were involved in mintenne of ody temperture, then mie with ltion of the GLP-1R might hve impired old tolerne. 24: GLP-1R / GLP-1R +/+ o/o 6: 8: 1: 12: 14: Figure 5 Cold tolerne nd diurnl ody temperture in d/d mie reeiving prohormone onvertse 1/3 (PC1/3)-expressing α-ell therpy. Mie were individully housed for 2 hours t 4 C 5 dys () nd 26 dys () fter trnsplnt. () Diurnl ody temperture ws mesured in mie housed individully t room temperture. (d) Cold tolerne ws ssessed in GLP-1R +/+ nd GLP-1R / mie. P <.5, P <.1 for d/d shm versus d/d αtcδpc2 trnsplnt; n = 7 8 ( ) or n = 3 5 (d) per group. GLP-1, glugon-like peptide-1. However, 2-hour old tolerne test reveled no differene in ody temperture etween GLP-1R / nd GLP-1R +/+ mie (ged 1 12 weeks), nd oth groups were etter le to thermoregulte thn o/o ontrol mie (Figure 5d). We lso implnted IP miniosmoti pumps infusing GLP-1 7-NH2, GLP-2, or oxyntomodulin into d/d mie, nd tested old thermogenesis dys fter implnttion. We deteted no differene in the old thermogeni ility of mie reeiving ny of the peptides ompred to ontrol mie (Figure 4). Wild-type mie nd αtcδpc2-trnsplnted or shm-operted d/d mie were killed dys fter trnsplnt nd tissues were olleted for nlysis. As expeted, d/d mie hd greter epididyml white dipose tissue (WAT) mss (Figure 6; P <.1 for oth groups versus wild type) nd intrspulr rown dipose tissue (BAT) mss (Figure 6; P <.1 for shm nd P <.5 for αtcδpc2 reipients versus wild type) thn did wildtype ontrols, though the mss of these ft depots did not differ etween shm-operted nd ell-trnsplnted d/d mie. Although oth groups of d/d mie hd lrger dipoytes in the WAT depot nd lrger lipid droplets in the dipoytes in the BAT depot ompred to wild-type ontrols, we were not le to detet ny differene etween the groups of d/d mie (Figure 6 nd dt not shown). Using western lot nlysis, we ould not detet ny differene in unoupling protein 1 (UCP-1) protein levels in the BAT depot of shm- versus αtcδpc2-treted d/d mie (Figure 6d,e). Disussion In this study, we sought to use ell therpy to evlute the onsequenes of ontinuous delivery of either PC1/3- or PC2- derived α-ell produts in mouse models of T2D. Unlike prototypil α-ells whih express PC2, αtcδpc2 ells re derived from mie lking iotive PC2 nd therefore the pity for glugon prodution, ut express high levels of PC1/3. 1,12 We hve previously shown tht αtcδpc2 ells improve gluose hndling in norml mie, nd prevent streptozotoin-indued hyperglyemi, effets tht ppered to e primrily ttriutle to the seretion of GLP-1 from the trnsplnted ells. 1 Here we seleted αtcδpc2 ells nd αtc-1 ells to evlute ontinuous ell therpy with either PC1/3-expressing or PC2-expressing α-ells in T2D. Alginte enpsultion protets trnsplnted ells from immune ttk nd hs een used for long-term mintenne of trnsplnted islets in models of dietes. 15,16 We used enpsultion to evlute long-term therpy with PC1/3- or PC2-expressing α-ells in mie without the need for immunosuppression or use of immunoompromised reipients. Cpsules remined interspersed throughout the IP vity of reipients, though eyond ~3 4 weeks fter trnsplnt psules osionlly hrdened nd lumped together. However, it hs een previously demonstrted tht plsm GLP-1 levels remin elevted in C57BL6 mie dys fter trnsplnttion of enpsulted αtcδpc2 ells, 1 nd in the urrent study plsm glugon levels remined elevted dys fter trnsplnttion of enpsulted αtc-1 ells in d/d mie (dt not shown). Tken together these dt suggest tht the enpsulted ells remined funtionl throughout the durtion of our studies vol. 17 no. 1 jn. 29

5 The Amerin Soiety of Gene Therpy PC1/3-expressing α-ell Therpy in Dieti Mie BAT mss (% of BW) WAT mss (% of BW) WT WT ### # Wild type WAT H&E BAT H&E e d β-atin UCP-1 UCP-1 Expression Wild type WT Figure 6 Brown dipose tissue (BAT) nd white dipose tissue (WAT) morphology in d/d mie reeiving prohormone onvertse 1/3 (PC1/3)- expressing α-ell therpy. Intrspulr BAT nd epididyml WAT were olleted dys fter trnsplnt. (,) WAT nd BAT mss s % of ody weight. () Representtive hemtoxylin nd eosin stined imges of WAT nd BAT (sle r = 12 μm). (d,e) Western lot showing unoupling protein 1 (UCP-1) nd β-tin levels. Blots re representtive of three to five experiments. UCP-1 expression is expressed s pixel intensity nd is normlized for lot kground nd for β-tin expression.,### P <.1 versus wild type; # P <.5 versus wild type; n 4 per group. In oth high ft fed mie nd d/d mie, αtcδpc2 ells improved gluose tolerne nd modestly, if trnsiently, redued fsting lood gluose, while tretment of d/d mie with αtc-1 ells inresed fsted gluose levels nd worsened gluose tolerne. In high ft fed mie, αtcδpc2 ells inresed insulin output in response to gluose, onsistent with the known insulinotropi effet of GLP Moreover, in d/d mie αtcδpc2 ells redued fsting insulin levels nd restored gluose-stimulted insulin seretion. However, αtcδpc2 ell trnsplnt ws unle to dely the onset of overt dietes in d/d mie, nd hd miniml impt on gluose tolerne y 1 month fter trnsplnt. We initilly suspeted tht hronilly elevted lood gluose levels in d/d mie might hve limited seretion from the α-ell trnsplnts. However, in ontrst to the ntive α-ell, whih inreses seretion of glugon in response to low irulting gluose onentrtions, 18 in vitro experiments showed tht αtcδpc2 ells were unresponsive to hnges in medi gluose onentrtion (dt not shown). This suggests tht hyperglyemi itself proly did not limit the relese of GLP-1 nd other PC1/3-derived proglugon produts from the ell trnsplnts. In rodents, however, hroni hyperglyemi my indue downregultion of the GLP-1R. 19 It is therefore possile tht s lood gluose ontrol worsened, d/d mie grdully eme insensitive to the effets of trnsplnt-derived GLP-1. Interestingly, despite the known noreti effets of GLP-1 2,21 nd oxyntomodulin, 22 we were unle to detet ny hnges in food intke or ody weight in d/d mie reeiving αtcδpc2 ell trnsplnt ompred to shm surgery. It is possile tht the sensitivity of our methods ws insuffiient to oserve hnges in food intke. An lterntive possiility is tht the pthwys mediting these effets were desensitized euse of hroni reeptor stimultion, lthough there is little evidene to support the notion tht hroni GLP-1R gonism indues desensitiztion in rodents 23 or humns. 24 Leptin normlly ts vi the long form of the leptin reeptor (OR) to indite the sttus of dipose tissue stores to the entrl nervous system. In d/d mie, the ltion of OR nd loss of this signling xis renders these mie in pereived stte of negtive energy lne, resulting in mssive hyperphgi nd oesity, deresed energy expenditure, nd suppression of energy-intensive tivities suh s reprodution nd growth. 25,26 It is likely tht αtcδpc2 ell trnsplnt ws simply unle to overome this energy onservtion phenotype in d/d mie for more thn short period. Loss of leptin reeptor signling in mie leds to norml thermoregultion nd deresed pity for dptive thermogenesis. 14 This is thought to relte to deresed tivtion of leptin-sensitive symptheti nerves innervting BAT. 27,28 In ordne with previous oservtions, 14 in our study d/d mie displyed lower ody tempertures thn wild-type mie, lthough the mplitude nd phse of their diurnl ody temperture flututions mimiked those of wild-type mie. Unexpetedly, we oserved tht shortterm exposure to 4 C unmsked n improvement in the thermogeni ility of d/d mie ering n αtcδpc2 ell trnsplnt, lthough this ws evident only fter severl weeks of exposure to trnsplnted αtcδpc2 ells. This is onsistent with the ide tht inreses in dptive thermogeni pity develop over the ourse of weeks, rther thn dys. 27,29 While ute exposure to suthermoneutrl temperture minly tivtes shivering s mehnism for mintining ody temperture, longer-term exposure tivtes nonshivering (or dptive) thermogenesis. The key meditor of this proess is thought to e UCP-1, whih unouples ellulr respirtion from denosine triphosphte synthesis suh tht het is generted. 27,29, We were unle to detet ny differenes in UCP-1 protein levels etween shm-operted nd ell-treted d/d mie; however, it remins possile tht ell trnsplnts ltered the degree of tivtion of UCP-1, whih ontriutes to funtionl UCP-1 levels. 29 Alterntively, ell trnsplnttion my hve inresed shivering thermogenesis, possiility tht seems plusile given our oservtion tht αtcδpc2 reipients hd slight inrese in len:lipid ody mss rtio ompred to shm-operted ontrols. The mehnism driving the αtcδpc2 ell indued inreses in ody temperture of d/d mie therefore remins unler, s does the speifi PGDP mediting this effet. Moleulr Therpy vol. 17 no. 1 jn

6 PC1/3-expressing α-ell Therpy in Dieti Mie The Amerin Soiety of Gene Therpy It is intriguing to note tht for some time, glugon hs een proposed to hve thermogeni properties, lthough only when dministered t ~1, irulting levels. 31 A more reent report found no thermogeni effet of purified glugon nd suggested tht the erlier oservtions my hve risen from ontmintion of the glugon preprtion with nother PGDP. Indeed we lso oserved no impt of glugon-produing αtc-1 ell trnsplnts on old thermogenesis in d/d mie. There is evidene tht GLP-1 n modulte ore ody temperture, ut results from vrious studies hve een somewht ontrditory. Severl studies in rts hve reported tht GLP-1 dministered ICV or intrvenously inreses ody temperture, 33, wheres in the Jpnese quil GLP-1 hs een reported to derese ody temperture when dministered ICV or intrvenously. 35 However, role for GLP-1 in old-indued thermogenesis hs not, to our knowledge, een desried. Nevertheless, the notion tht GLP-1 rising from trnsplnted αtcδpc2 ells my hve improved thermogenesis in d/d mie is onsistent with reent studies showing tht peripherlly delivered GLP-1 n tivte utonomi ontrol enters in the entrl nervous system nd therey influene peripherl symptheti funtions suh s lood pressure nd hert rte.,37 We found tht GLP-1R / mie do not hve defetive ility to thermoregulte in the old, suggesting tht GLP-1R signling is not essentil for old thermogenesis. This does not, however, prelude ny involvement of GLP-1R signling in ody temperture regultion, euse ny deleterious effet of GLP-1R ltion on temperture regultion my simply e msked y other redundnt mehnisms ontrolling this key metoli prmeter. Whether other PC1/3-derived PGDPs (i.e., GLP-2, oxyntomodulin) might ontriute to the oserved effets lso remins possile. However, t the doses tested we were unle to detet n improvement in old thermogenesis in response to longterm ontinuous infusion of GLP-1, GLP-2, or oxyntomodulin in isoltion. It is lso possile tht nother s yet unidentified non- PGDP sereted from αtcδpc2 ells my medite this effet. In summry, this study provides model for evluting longterm delivery of PC1/3-derived PGDPs vi ell therpy. Our studies demonstrte tht trnsplnttion of αtcδpc2 ells improves gluose hndling in mouse models of T2D, nd indite tht one or more produts of αtcδpc2, ut not αtc-1 ells, my hve previously unppreited effet on thermogenesis nd ody temperture regultion. This work provides dditionl evidene to support the notion tht mnipultion of the proessing enzyme profile of the α-ell n lter its overll hrteristis y hnging the lne of peptides rising from it. Mterils And Methods Tissue ulture medi, ntiiotis, nd fetl ovine serum were otined from Invitrogen Cnd (Burlington, ON, Cnd). αtc-1 (lone 9) ells were otined from the Amerin Type Culture Colletion. Totl GLP-1 RIA nd GLP-2 ELISA kits were from Lino Reserh (St. Chrles, MO); ultrsensitive insulin ELISA kits were from Alpo Dignostis (Slem, NH); nd oxyntomodulin RIA kits were from Phoenix Phrmeutils (Burlingme, CA). Western lotting regents were from GE Helthre (Bukinghmshire, UK), nd the BCA kits for protein quntifition were from Piere (Rokford, IL). Imging nd quntifition ws performed using n Axiovert 2 mirosope (Crl Zeiss, Toronto, ON, Cnd) onneted to digitl mer (Retig 2R; QImging, Burny, BC, Cnd) ontrolled with Openl 5. softwre (Improvision, Lexington, MA). Animls. All experiments were pproved y the University of British Columi Animl Cre Committee. d/d mie nd wild-type C57BLKS/J mie (BKS.Cg-m +/+ Leprd/J, stok #642 nd stok #662, respetively; reeived t 4-week ge), nd diet-indued oese C57BL/6 mie (fed 6% kl from ft diet from 6-week ge nd otined t 16-week ge) were otined from Jkson Lortories (Br Hror, ME). GLP-1R / mie, provided y Dr D. Druker (University of Toronto), were mintined on C57BL/6 kground nd genotyped s desried elsewhere. 1 Mie were mintined on 12-hour light/drk yle nd reeived stndrd how diet (#515; LDiet, St. Louis, MO; ontins 19.8% kl from protein, 54.9% kl from rohydrtes, nd 25.3% kl from ft, 6% of whih is soy- or leithin-derived nd 4.% of whih is from lrd) exept for dietindued oese mie, whih were mintined on diet D12492 from Reserh Diets (New Brunswik, NJ; ontins 2.% kl from protein, 2.% kl from rohydrte, nd 6% kl from ft, omprised of 9.3% soyen oil nd 9.7% lrd). Blood gluose nd ody weight were monitored two to three times weekly following 4-hour morning fst. Blood gluose monitoring nd lood smpling were rried out on restrined, unnesthetized mie vi the sphenous vein using heprinized miropillry tues. Plsm ws stored t 2 C until ssy. Gluose tolerne tests were performed y IP delivery of gluose (2 g/kg) to reipient mie following n overnight fst. Blood gluose ws monitored for 2 hours fter gluose delivery nd insulin ws mesured using n ultrsensitive mouse insulin ELISA. Food intke ws mesured y providing known mss of food nd weighing food remining in the hopper 24 hours lter for 4 dys. Cell ulture nd trnsplnttion of enpsulted ells. αtc-1 nd αtcδpc2 ells were ultured in high-gluose Duleo s modified Egle s medium ontining 1% fetl ovine serum, 1 U/ml peniillin, nd 1 μg/ml streptomyin. Cells were mintined t 37 C/5% CO 2 nd pssged s neessry using.25% trypsin/1 mmol/l EDTA with medi refreshment every 2 4 dys. Medium ws olleted fter 24-hour stti inution nd ssyed for immunoretive GLP-2 nd oxyntomodulin. Cells were grown to ~8% onfluene efore enpsultion. For ll trnsplnts, ml of psules (~ ells/mouse) were trnsplnted in <3 ml totl volume. Experiments were performed using pssges 2 35 for αtcδpc2 nd p8-12 for αtc-1. After trypsiniztion, pelleted ells were resuspended in phosphte-uffered sline without CCl 2 nd ell ount ws performed using hemtoytometer. Cells were resuspended in mixture of 1.5% sodium lginte (IE-11; Inoteh Biosystems Interntionl; Rokville, MD; moleulr weight 475 kd; G/M rtio 65 75%/25 35%) nd morpholinepropnesulfoni id nd trnsferred to sterile enpsultor (Inoteh Biosystems Interntionl). Enpsultion ws performed ording to the mnufturer s instrutions using 25-μm nozzle nd enpsultion settings s follows: eletril hrge ~1. kv, virtion frequeny ~1,2 Hz, pump speed ~6. Cpsules (5 7 μm) were wshed in phosphte-uffered sline without CCl 2 nd loded to sterile syringes tthed to 18-G theters. Reipient mie were nesthetized using isoflurne, nd psules were injeted to the IP vity s desried elsewhere. 1 -operted mie reeived n equl volume of sterile sline under identil onditions. Implnttion of miniosmoti pumps. Rt GLP-2, humn GLP-1 7-NH2, nd oxyntomodulin were from Amerin Peptide (Sunnyvle, CA) nd were reonstituted in sterile sline. Miniosmoti pumps (28 dys infusion, #14; Alzet, Cupertino, CA) were loded with peptide (7.5 pmol/kg min) or sline nd preequilirted overnight t 37 C. The following dy pumps were implnted to the peritonel vity of isoflurne-nesthetized d/d mie (ge ~6 weeks). Cold tolerne testing nd ody temperture nlysis. Mie were implnted suutneously with sterile temperture trnsponders (IPTT- ; Bio Medi Dt Systems, Seford, DE) through ~3-mm inision in the intrspulr region t the sme time of ell trnsplnt/pump implnt vol. 17 no. 1 jn. 29

7 The Amerin Soiety of Gene Therpy PC1/3-expressing α-ell Therpy in Dieti Mie Trnsponders were implnted longitudinlly in the suutneous spe, prllel ut to one side of the spine so s not to interfere with the niml s movement. The inision ws losed with wound lip efore the niml ws llowed to reover from nesthesi. GLP-1R / nd o/o mie were implnted with trnsponders 2 dys efore old tolerne testing. Twenty-four-hour ody temperture profiles were ssessed using hnd-held Poket Snner (DAS-57; Bio Medi Dt Systems, Seford, DE) to red the temperture trnsponders in singly housed mie. For old tolerne testing, mie were singly housed in ges without edding for the durtion of the experiment. Mie were housed t 4 C nd ody temperture ws mesured every 15 minutes for 1 2 hours using the Poket Snner. Any mouse tht lost 7 C from sl ody temperture ws returned to mient temperture, wrmed using heting pd, nd exluded from the study. Body omposition nlysis. Mesurements were performed using Bruker Biospe 7/ 7 Tesl MRI snner (Bruker Biospin, Ettlingen, Germny). Nuler mgneti resonne signl from the ody ws quired using qudrture volume RF oil tuned to MHz. The free wter omponent orresponding to ody fluids (e.g., urine nd CSF) ws typilly <5% of the totl signl. The rtio of len/ft tissue (weight/weight) ws lulted s desried elsewhere. 39 Plsm nd tissue olletion. At the indited time, mie were nesthetized with isoflurne, nd rdi punture ws performed to ollet plsm efore ervil dislotion. Pnres, intrspulr BAT, nd epididyml WAT were removed, rinsed in phosphte-uffered sline, fixed in 4% prformldehyde nd prffin setioned. A portion of intrspulr BAT ws olleted, rinsed in phosphte-uffered sline, nd flsh frozen in liquid nitrogen. Plsm GLP-1 ws mesured using totl GLP-1 RIA kit. BAT nd WAT nlysis. BAT nd epididyml WAT setions were stined with hemtoxylin nd eosin nd imged using rightfield mirosopy. Lipid droplet size ws mesured using n inuilt lgorithm. Other setions were stined with rit UCP-1 ntiody (1983; 1:1,; Am, Cmridge, MA) nd horserdish peroxidse onjugted seondry ntiser. Intrspulr BAT ws ground using prehilled mortr nd pestle nd trnsferred to miroentrifuge tue with 1 μl of lysis uffer (5 mmol/l HEPES, 15 mmol/l NCl, 1 mmol/l EDTA, 1 mmol/l N 4 P 2 O 7, 1 mmol/l NF, with 1 mmol/l phenylmethnesulphonylfluoride, 2 mmol/l N 3 VO 4,.4% Triton-X, nd 1% protese inhiitor oktil dded immeditely efore use). Smples were vortexed nd inuted on ie for minutes. The protein-ontining superntnt ws removed to fresh set of tues. Equl mounts of protein were eletrophoresed on 1% rylmide gel using sodium dodeyl sulfte polyrylmide gel eletrophoresis nd trnsferred to.2-μm polyvinylidene fluoride memrne (Bio-Rd, Herules, CA). After loking, the memrne ws inuted with rit nti-ucp-1 ntiody (1:4,; 4 ) overnight t 4 C. The memrne ws inuted with lkline phosphtse linked polylonl seondry ntiody (1:5,) nd developed using n enhned hemiluminesene kit (GE Helthre; Bukinghmshire, UK). The memrne ws wshed, reloked, nd immunolotted with n lkline phosphtse linked β-tin ntiody, nd UCP-1 expression ws normlized for β-tin levels. Dt nlysis. Dt nlysis ws rried out using Prism 4. (GrphPd, Sn Diego, CA). Dt re presented s men ± SEM nd were nlyzed using two-tiled Student s t-test exept where noted. Sttistil signifine ws set t 5% nd the numer of experiments performed re shown in figure legends. P <.5, P <.1, P <.1. Aknowledgments We grtefully knowledge the expert tehnil ssistne provided for these studies y Christine Donld. We re grteful for the provision of αtcδpc2 ells, UCP-1 ntiser, nd GLP-1R / mie y Donld F. Steiner (University of Chigo, Chigo, IL), Brr Cnnon ( Wenner-Gren Institute, Stokholm University, Sweden), nd Dniel Druker (University of Toronto, ON, Cnd), respetively. This reserh ws funded y grnts from the Cndin Dietes Assoition nd the Juvenile Dietes Reserh Foundtion. T.J.K. reeived sholrship support from the Mihel Smith Foundtion for Helth Reserh (MSFHR) nd R.D.W. reeived sholrship support from MSFHR nd the Nturl Sienes nd Engineering Reserh Counil of Cnd. Referenes 1. Kieffer, TJ nd Hener, JF (1999). The glugon-like peptides. Endor Rev 2: Furut, M, Zhou, A, We, G, Crroll, R, Rvzzol, M, Ori, L et l. (21). Severe defet in proglugon proessing in islet A-ells of prohormone onvertse 2 null mie. J Biol Chem 276: Rouille, Y, Binhi, M, Irminger, JC nd Hln, PA (1997). Role of the prohormone onvertse PC2 in the proessing of proglugon to glugon. FEBS Lett 413: Unger, RH (1975). Letter: glugon in pthogenesis of dietes. Lnet 1: Rouille, Y, Kntengw, S, Irminger, JC nd Hln, PA (1997). Role of the prohormone onvertse PC3 in the proessing of proglugon to glugon-like peptide 1. J Biol Chem 272: Zhu, X, Zhou, A, Dey, A, Norrom, C, Crroll, R, Zhng, C et l. (22). 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