Experimental Physiology

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1 1 Exp Physiol 11.1 (216) pp Reserch Pper Reserch Pper Decresed insulin secretion nd glucose clernce in exocrine pncres-insufficient pigs Liudmyl Lozinsk 1,Björn Weström 1, Olen Prykhodko 1, Andres Lindqvist 2, Nils Wierup 2,BoAhrén 3, Ktrzyn Szwiec 1 nd Stefn G. Pierzynowski 1,4 1 Deprtment of Biology, Lund University, Lund, Sweden 2 Deprtment of Clinicl Sciences in Mlmö, Lund University, Mlmö, Sweden 3 Deprtment of Clinicl Sciences, Lund University, Lund, Sweden 4 Deprtment of Medicl Biology, Institute of Rurl Helth, Lulin, Polnd Experimentl Physiology New Findings Wht is the centrl question of this study? Does the exocrine pncres hve n impct on endocrine pncretic function nd peripherl nutrient utiliztion? Wht is the min finding nd its importnce? In n exocrine pncres-insufficient pig model, the insulin response to glucose lod ws delyed. Orl enzyme supplementtion did not improve the insulin relese ut fcilitted lood glucose clernce. These results suggest n cino-insulr xis communiction ffecting islet function nd n impct of gut pncretic enzymes on lood glucose utiliztion. The effect of exocrine pncretic function on the glucose-medited insulin response nd glucose utiliztion were studied in n exocrine pncres-insufficient (EPI) pig model. Five 1-week-old EPI pigs fter pncretic duct ligtion nd 6 ge-mtched, non-operted control pigs were used in the study. Blood glucose, plsm insulin nd C-peptide concentrtions were monitored during mel (MGTT), orl (OGTT) nd intrvenous (IVGTT) glucose tolernce tests. Upon post-mortem exmintion, the pncretic remnnts of the EPI pigs showed cinr firotic trophy ut norml islets nd β-cell morphology. The EPI pigs displyed incresed fsting glucose concentrtions compred with control nimls (6.4 ±.4 versus 4.8 ±.1 mmol l 1, P <.1) ut unchnged insulin concentrtions (2.4 ±.6 versus 2.1 ±.2 pmol l 1 ). During the OGTT nd IVGTT, the EPI pigs showed slower, impired glucose utiliztion, with the disruption of well-timed insulin response. Plsm C-peptide concentrtions confirmed the delyed insulin response during the IVGTT in EPI pigs. Orl pncretic enzyme supplementtion (PES) of EPI pigs improved glucose clernce during IVGTT [AUC glucose 1295 ± 7 mmol l 1 (12 min) in EPI versus 144 ± 32 mmol l 1 (12 min) in EPI + PES, P <.1] without reinforcing the relese of insulin [AUC C-peptide 14.4 ± 3.8 nmol l 1 (12 min) in EPI versus 6.4 ± 1.3 nmol l 1 (12 min) in EPI + PES, P <.2]. The results suggest the existence of n cino-insulr xis regultory communiction. The presence of pncretic enzymes in the gut fcilittes glucose utiliztion in n insulin-independent mnner, indicting the existence of gut-derived pncretic enzyme-dependent mechnism involved in peripherl glucose utiliztion. (Received 15 July 215; ccepted fter revision 3 Octoer 215) Corresponding uthor S. G. Pierzynowski: Deprtment of Biology, Lund University, Sölvegtn 35, SE , Lund, Sweden nd Deprtment of Medicl Biology, Institute of Rurl Helth, Jczewskiego 2, 2 9 Lulin, Polnd. Emil: stefn.pierzynowski@iol.lu.se DOI: /EP85431 C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

2 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs 11 Introduction The pncres secretes oth digestive enzymes nd islet hormones tht re involved in digestion, sorption nd utiliztion of nutrients (Bilous & Donnelly, 21). Although the exocrine (cini) nd endocrine (islets) portions of the pncres re co-loclized ntomiclly, possile functionl links re not completely understood. Experimentl nd clinicl conditions ffecting one prt ofthepncreshveeenshowntohveeffectsonother prts of the pncres s well (Amromovge et l. 1973; Berkhoff et l. 1987). The effect of the endocrine pncres on exocrine pncretic function, the so-clled insulo-cinr xis, hs een studied profoundly (Willims & Goldfine, 1985). For instnce, insulin hs een shown to pss through the exocrine cinr res of the pncres vi locl portl circultion, in concentrtions higher thn those in the peripherl lood (Lifson et l. 1985), nd in doing so ffects enzyme production, which is known s the hlo phenomenon (Willims & Goldfine, 1985; Schönfeld et l. 1994). Moreover, impired β-cell function my result in impired function of the exocrine pncres (Pierzynowski & Brej, 1984; Yokoym et l. 1988; Hrdt et l. 23; Ewld et l. 27; Czkó et l. 29). In contrst, there is reltively less informtion concerning the possile reverse reltionship, i.e. the impct of the exocrine pncres on islet function nd insulin secretion (Amromovge et l. 1973; Vilsøll et l. 23; Knop et l. 27; Rnkinet l. 213). It hs een shown tht exocrine pncretic insufficiency (EPI) in humns my e ssocited with insulin deficiency nd dietes mellitus (type 3c; Ewld et l. 27; Hrdt et l. 28; Cui & Andersen, 211, 212; Ewld & Bretzel, 213). Cui & Andersen (211) concluded tht in the Western popultion, type 3c dietes ccounts for 5 1% of ll dietic individuls, with 75% of it ttriutle to chronic pncretitis (Cui & Andersen, 211; Ewld & Bretzel, 213). This my suggest tht islet hormone secretion depends on the function of the exocrine pncres, lthough more detiled study of such n cino-insulr communiction xis remins to e done. The effect of the exocrine pncres on endocrine pncretic function hs een studied in pig model of EPI (Imondi et l. 1972; Berkhoff et l. 1987; Boerm et l. 23; Rengmn et l. 29). Pigs re omnivorous nimls with gstrointestinl ntomy nd function similr to those of humns. Owing to the presence of seprte ile nd pncretic ducts, it is reltively simple to perform pncretic duct ligtion (PDL) surgery on pigs without ffecting ile flow, thus creting n EPI pig model. Berkhoff et l. (1987) nd Boerm et l. (23) hve shown tht PDL in pigs ffects β-cell numer or size. We hve previously shown tht following PDL, young pigs experience totl growth rrest, even if provided with intrvenous totl prenterl nutrition ensuring 1% dietry requirements, until orl supplementtion with pncretic enzymes is initited (Rengmn et l. 29). Also, orl pncretic enzyme supplementtion (PES) therpy improves ppetite nd feed conversion in oth EPI nd norml control pigs (Fedkiv et l. 29). These results suggest reltionship etween the presence of pncretic enzymes in the gut lumen nd the ssimiltion of elementry food components from the lood. But more in-depth investigtion of the insulin response in the EPI condition in the porcine model hs not een performed nd would e useful for understnding how EPI ffects pncretic islet function. Thus, the present study ws designed to investigte the influence of the exocrine pncretic products (enzymes) in the cini nd in the gut on insulin secretion nd glucose tolernce. In order to mesure the levels of insulin regultion, young PDL pigs tht hd developed EPI underwent mel (MGTT), orl (OGTT) nd intrvenous (IVGTT) glucose tolernce tests either efore or during PES. The ove tests were chosen s the est-stndrdized methods to follow direct nd indirect (incretin-dependent) insulin regultion. Methods Animls The study ws pproved y the Mlmö/Lund Ethicl Review Committee on Animl Experiments. The experimentswereperformedoncrossred[(yorkshire Swedish Lndrce) Hmpshire] pigs, otined from the Odrslöv reserch frm, elonging to the Swedish University of Agriculturl Sciences (Alnrp, Sweden). All pigs were housed individully in pens, in the sme stle, t 2 ± 2 C nd with lights on from 7. to 19. h. The pigs hd free ccess to wter vi drinking nipple nd were offered commercil pelleted pig feed (Växtill 32, Lntmännen, Sweden). In totl, the feed ws offered to the pigs in n mount of 4% of their ody weight (.54 MJ kg 1 dy 1 ), which ws divided etween the morning nd fternoon feeds. The pelleted feed ws composed of the following: strch, 6%; wter, 12%; crude protein, 17.5%; crude fire, 4.7%; crude ft, 4%; shes, 5.9%; clcium,.9%; phosphorus,.7%; nitrogen, 2.8%; potssium,.6%; sodium,.15%; lysine, 1.1%; methionine,.38%; cysteine + methionine,.69%; threonine,.66%; nd energy 12.6 MJ kg 1. Surgery At 6 weeks of ge, five pigs (1.9 ±.2 kg) underwent PDL surgery to induce EPI, s previously descried (Gewert et l. 24; Fedkiv et l. 29). After n overnight fsting period, the pigs were pre-edicted with C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

3 12 L. Lozinsk nd others Exp Physiol 11.1 (216) pp zperone (Stresnil; Jnssen Phrmceutic, Belgium; 2.2 mg kg 1, I.M.) nd wshed using surgicl sop. The pigs were then nesthetized vi n inhltion msk with.5 1.5% ir mixture of hlothne (Fluothne; Astr Läkemedel, Södertälje, Sweden) in O 2 s crrier gs t pproximtely.5 1 l min 1 using closed-circuit respirtory flow system (Komesroff Medicl Developments, Melourne, Victori, Austrli). Immeditely fter surgicl nesthesi ws reched (indicted y lck of cornel reflex), n endotrchel tue ws inserted to mintin surgicl/generl nesthesi. The surgery ws performed in septic conditions. For PDL, 14- to 18-cm-long incision ws mde posterior to the sternum long the line l, nd the ccessory pncretic duct (the min duct in pigs) ws isolted nd ligted t 2 nd 3 cm distnce from the duodenl ppill with doule silk sutures nd trnsected etween the ligtures. The domen ws closed with three lyers of sutures. Postopertive pin ws treted y dministrtion of uprenorphine (Temgesic; Schering-Plough AB, Stockholm, Sweden;.1 mg kg 1, I.M.) for 3 dys. Ampicillin (Doktcillin; Astr Läkemedel) ws dministrted I.V. (15mgkg 1 ) nd t the incision site (25 5 mg). At 1 weeks of ge (1.5 ±.4 kg), in order to collect lood smples during the experiments, second surgicl procedure ws performed on the EPI pigs to implnt ctheter in the nterior ven cv, vi the externl jugulr vein, using the sme nesthetic protocol s descried ove.sixnon-operted,controlpigstthegeof1weeks (17.5 ±.4 kg) were fitted with venous ctheter in the sme mnner, 1 week efore strting the experiments. At 13 weeks of ge, t the end of experiments, the EPI pigs (14. ±.6 kg) nd non-operted pigs (22.6 ±.7 kg) were sedted with Stresnil, killed y I.V. injection of n overdose of pentoritl sodium (Allftl Vet. Omnide, Stockholm, Sweden; 1 mg kg 1 ) nd sumitted to post-mortem exmintion. The pncretic re nd the pncretic duct system in EPI pigs were exmined for involution nd pthologicl chnges. Specimens of pncretic tissue/remnnts from helthy nd EPI pigs were hrvested for immunohistochemicl nlyses. All methods were performed ccording to ethicl principles of niml experiments nd comply with the niml ethics checklist (Grundy, 215). Glucose tolernce tests Glucose tolernce tests were performed on ll pigs fter n overnight fsting period of 18 h. The dose of glucose or its precursor (dietry strch) in prticulr tests ws clculted to e similr [1 g of glucose (kg ody weight) 1 ] for ech type of tolernce test. Thus, for the MGTT the pigs were fed 2. g of the commercil feed per kilogrm ody weight. Tht mount of feed (homogenized in tp wter) ws consumed during 1min.FortheOGTTndIVGTT, the pigs were orlly gvged or infused, respectively, with 1. g glucose (kg ody weight) 1, using 2% glucose solution for the orl lod nd 5% glucose solution for the I.V. lod. The glucose solutions in oth instnces were dministered within 1 min using syringe. Four weeks fter PDL, when EPI hd developed, pigs underwent MGTT nd OGTT with one rest dy in etween tests. During 1 week of PES therpy, mel nd orl glucose tolerncetestswererepetedndnivgttwsperformed (dys 3, 5 nd 7). The IVGTT in EPI pigs ws performed week lter, fter the enzyme wshout period ws over. Glucose tolernce tests on the non-operted (control) pigs were performed in prllel to those on the EPI pigs. During PES therpy, the EPI pigs were dministered four cpsules of enteric-coted pncrelipse (Aott Helthcre Products Ltd, Southmpton, UK), orlly (vi pill-pusher), 1 h efore the glucose tolernce tests. A totl of eight cpsules of pncrelipse were dministered per dy together with the morning (four cpsules) nd fternoon (four cpsules) mels. One cpsule of pncrelipse contins 1 units of lipse, 8 units of mylse nd 6 units of protese (Europen Phrmcopei). Blood smpling Fsting lood smples were tken vi the ctheter in the morning, 1 h efore the glucose tolernce tests nd PES dministrtion. Blood smples for glucose tolernce tests were drwn efore ( 5 min)nd t 5, 15, 3, 6 nd 12 min fter feeding or infusion of glucose. Blood ws collected vi the venous ctheter into 5 ml syringes contining EDTA (.2 mg) nd protese inhiitor, protinin (Trsylol, 1 kiu; Byer, Leverkusen, Germny), s previously descried (Rntzer et l. 1995). The lood smples were immeditely chilled on ice nd then centrifuged t 3g for 15 min t 4 C. Plsm ws collected nd stored t 7 C for further nlyses. Anlysis of lood glucose nd plsm insulin nd C-peptide Glucose ws mesured in the fresh lood smples using glucose-meter with test strips (Accu-Chek Aviv; Roche Dignostics, Mnnheim, Germny). Plsm insulin nd C-peptide concentrtions were mesured using porcine insulin or C-peptide enzyme-linked immunossy kits,respectively(mercodi,uppsl,sweden),minly ccording to mnufcturer s protocol, ut with n incresed smple volume (5 μl) nd n dditionl stndrd point t the lower end of the stndrd curve, to otin lower detection limit of.2 pmol l 1 for insulin nd 2 pmol l 1 for C-peptide. C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

4 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs 13 Clcultions of insulin sensitivity nd β-cell function To evlute insulin sensitivity, the quntittive insulin sensitivity check index (QUICKI) ws ssessed s 1/[log fsting glucose (in millimoles per litre) + log fsting insulin (in picomoles per litre) (Ktz et l. 2). The QUICKI hs een shown to provide resonle nd relile pproximtions of insulin efficiency when pplied to humns (Coelli et l. 27) nd pigs (Jönsson et l. 26; Christoffersen et l. 29), without species-specific component incorportion or djustment. Theilityofthepncreticβ-cells to relese insulin ws ssessed using the insulinogenic index, clculted s the rtio of insulin 3min / glucose 3min,where indictes the reltive differences from initil concentrtions mesured during the OGTT (Christoffersen et l. 29; Blt et l. 212). The totl re under the curve (AUC) ws clculted for post-lod lood glucose (AUC glucose ), insulin (AUC insulin ) nd C-peptide (AUC C-peptide ) concentrtions, using the trpezoidl rule. The reltive mesure of heptic insulin extrction ws clculted s the molr rtio etween totl AUC C-peptide nd AUC insulin during the IVGTT nd represents the mount of insulin present in the circultion per mole of insulin secretion (C-peptide) (Osei et l. 1984). Histology At post-mortem exmintion, the domen ws opened, nd smples from the pncretic remnnt in EPI pigs nd the intct pncres in control pigs were dissected out nd immeditely plced in uffered 1% formlin for fixtion. After 72 h, formlin ws replced y 7% ethnol, nd smples were stored t room temperture until stndrd-procedure prffin emedding. Then, tissue locks were sliced into sections, 5 μm thick, nd deprffinted nd stined y the routine Hemtoxylin nd Eosin method (Histol Products AB, Västr Frölund, Sweden) or y immunohistochemistry, using guine-pig nti-insulin ntiody (# M93, Euro Dignostic AB, Mlmö, Sweden; 1:1) s primry ntiody nd got nti-guine-pig Cy2-lelled secondry ntiody (1:4). The slides were nlysed using n Olympus (PROVIS AX7) microscope. Sttisticl nlyses Sttisticl nlyses were performed using the R (version 3..1) progrmming environment (R Core Tem, 212). We used the repeted-mesures, mixed-effect model to compre tretment, time nd the interction etween tretment nd time, while controlling for non-independence in repeted mesurements of the sme sujects (pigs). Given tht the interction etween tretment nd time ws significnt, we chose to nlyse AUC nd the differences etween time points insted of testing the min tretment effects with post hoc tests. We compred AUC using mixed-effect model with suject (pig) s rndom effect followed y Tukey s post hoc test (which controls for multiple comprisons), compring ech tretment (PES, PES+EPI nd Control) with one nother. The pek ws recognized s the mximl vlue ginst time zero using the repeted-mesures, mixed-effect model. A P vlue of.5 ws considered to e sttisticlly significnt, wheres P vlue of <.1 ws considered s tendency. Results Feed consumption nd growth Almost complete growth rrest ws oserved in the pigs during the 4 weeks fter PDL surgery, with n verge of only.6% ody weight gin per week. At the sme time, EPI pigs fully consumed their dily feed portions (4% of their ody weight per dy given during morning nd fternoon mels). During PES therpy, growth of the EPI pigs recovered, nd 15% ody weight gin per week ws oserved. The feed consumption per kilogrm ody weight ws the sme s efore the therpy. Non-operted, control pigs gined 2% ody weight per week with the sme feed consumption (4% of their ody weight per dy), nd growth ws not distured y glucose loding tests. Fsting glucose nd insulin Fsting glucose nd insulin vlues for ech pig were tken s the verge fsting glucose vlue otined for the 3 dys of the glucose tolernce tests, in order to minimize the effects of pulstile sl insulin secretion (Meier et l. 25). After the development of EPI, the pigs showed higher (P <.1) fsting lood glucose concentrtions compred with non-operted, control pigs (Fig. 1). However, the fsting lood glucose concentrtion in EPI pigs ws normlized towrds the control pigs following PES therpy. Fsting plsm insulin concentrtions in EPI pigs were not significntly different from those of the control pigs, ut PES cused decrese in insulin concentrtions (P =.2) when compred with vlues otined from EPI pigs efore therpy (Fig. 1). Insulin sensitivity (QUICKI vlues) ws not significntly different etween EPI nd control pigs. However, PES tretment in EPI pigs elevted insulin sensitivity, with higher QUICKI vlues thn those clculted efore PES therpy (P <.1; Tle 1). Mel glucose tolernce test After ingestion of the feed, the lood glucose concentrtion of EPI pigs did not significntly increse, nd no cler C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

5 14 L. Lozinsk nd others Exp Physiol 11.1 (216) pp pek ws oserved (mximl vlue t 5 min, with P =.9; Fig. 2). However, hyperglycemic conditions were mintined compred with control nimls within 12 min fter mel ingestion (P =.1). Orl PES cused significnt pek in the elevtion of lood glucose t 3 min fter mel ingestion (P <.1), which ws similr to tht oserved in control pigs. Also, t 12 min following mel ingestion during PES in EPI pigs, lood glucose concentrtions decresed to level similr to tht oserved in control pigs. The insulin relese in response to the MGTT in EPI pigs ws much lower compred with tht of the control group, with slight, non-significnt pek t 15 min, compred with the initil, sl vlue (P =.8; Fig. 2). The impired insulin relese in the EPI pigs ws reflected y significntly lower AUC insulin compred with the control group (P <.1). The PES therpy ugmented the insulin response (AUC insulin ), with lte pek (mximl vlue) in plsm insulin concentrtions t 6 min fter mel ingestion (P =.2). However, plsm insulin concentrtions in EPI pigs during PES never reched the control vlues. Glucose (mmol l 1 ) Insulin (pmol l 1 ) Control EPI EPI + PES Figure 1. Fsting lood glucose nd plsm insulin concentrtions (mens ± SEM) in exocrine pncres-insufficient (EPI) pigs (n = 5) efore nd during therpeutic enzyme tretment with pncrelipse (EPI + PES), compred with control pigs (n = 6) Different letters given with result rs indicte significnt differences (P.5). Orl glucose tolernce test Following orl glucose dministrtion, control pigs demonstrted significntly incresed lood glucose concentrtions t 3 min, which returned to initil (fsting) concentrtions within 6 min (Fig. 3). The EPI pigs lso displyed significntly incresed lood glucose concentrtions, which were mintined even t 6 nd 12 min fter glucose dministrtion (P.2) compred with control vlues. The PES therpy in EPI pigs did not ffect lood glucose t 6 min (P <.1), nd the AUC glucose during PES ws still significntly higher (P =.5) compred with tht oserved in control pigs. However, PES normlized lood glucose vlues in EPI pigs t 12 min fter glucose dministrtion. The insulin response to orl glucose loding in EPI pigs ws lso reduced nd delyed (pek t 6 min, P =.8) compred with tht oserved in the control group (Fig. 3). The totl AUC insulin in EPI pigs hd tendency to e lower thn control vlues (P =.6). However, PES therpy significntly decresed the totl insulin relese in EPI pigs, resulting in lower AUC insulin compred with the non-operted, control pigs (P <.1). In ddition, EPI pigs displyed significntly lower (P =.1) insulinogenic index in response to orl glucose dministrtion compred with the control pigs. The PES therpy did not ffect the insulinogenic index in EPI pigs (Tle 1). Intrvenous glucose tolernce test In EPI pigs, glucose elimintion from the lood ws delyed, following the I.V. glucose lod, compred with control pigs (Fig. 4). Blood glucose concentrtions t 15, 3 nd 6 min fter the glucose lod were significntly higher in EPI pigs compred with control nimls (P <.1). The PES therpy significntly lowered lood glucose concentrtions in EPI pigs etween 15 nd 6 min fter glucose dministrtion (P.1), nd this incresed glucose elimintion ws reflected in significntly lower AUC glucose during PES in EPI pigs (P <.1). The insulin response during the IVGTT in EPI pigs ws delyed, in tht plsm insulin concentrtions reched pek t 6 min following glucose loding (P =.4), wheres in the control pigs the insulin pek ws oserved 15 min fter the glucose lod (P <.1; Fig. 4). No significnt differences in AUC insulin etween EPI nd control pigs were oserved. The PES ltered the pttern of insulin response during EPI, with significntly lower AUC insulin compred with EPI pigs efore therpy (P =.4) nd control pigs (P =.5). The plsm C-peptide concentrtions oserved during the IVGTT were nlysed to clrify the true rnge C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

6 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs 15 Tle 1. The quntittive insulin sensitivity check index (QUICKI), insulinogenic index from orl glucose tolernce test nd C-peptide-to-insulin molr rtios from intrvenous glucose tolernce test (mens ± SEM) for exocrine pncres-insufficient pigs (n = 5) efore nd during therpeutic tretment with pncrelipse (EPI + PES), compred with control pigs (n = 6) Prmeter QUICKI 1.1 ± ± ±.2 Insulinogenic index (pmol mmol 1 ) 7.5 ± ±.7.9 ±.3 C-Peptide-to-insulin molr rtio 6.3 ± ±.7 1. ±.7 Different letters given with results in row indicte significnt differences (P <.5). of insulin secretion. Thus, delyed C-peptide pek (6 min, P =.3) ws oserved in EPI pigs, with totl relese of C-peptide (AUC C-peptide ) similr to tht of control pigs. The PES tretment significntly decresed C-peptide production (P =.2) in EPI pigs. The plsm C-peptide-to-insulin molr rtio ws clculted s n indictor of heptic insulin removl; higher rtio ws oserved in EPI pigs compred with control pigs (P <.1; Tle 1), nd PES hd no effect on this rtio Glucose (mmol l 1 ) AUC glucose [mmol l 1 (12 min)] Insulin (pmol l 1 ) AUC insulin [pmol l 1 (12 min)] c Figure 2. Blood glucose nd plsm insulin concentrtions (mens ± SEM) during mel glucose tolernce test in EPI pigs (n = 5) efore (open dimonds, dshed line) nd during therpeutic enzyme tretment with pncrelipse (EPI + PES, filled squres, continuous line), compred with control pigs (n = 6, open circles, continuous line) The res under curves (AUC glucose nd AUC insulin, mens ± SEM), clculted using the trpezoidl rule, re shown eside the corresponding curves. Different letters given with result rs indicte significnt differences (P <.5). C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

7 16 L. Lozinsk nd others Exp Physiol 11.1 (216) pp Histology Histologicl nd immunohistologicl exmintion of the pncretic remnnts from the EPI pigs showed decresed nd dmged cini, with infiltrted immune cells nd firosis ut with pprently unchnged morphology of the pncretic islets nd insulin-producing cells (Fig. 5). Discussion The porcine EPI model Pncretic duct ligtion in young pigs leds to the immedite elimintion of pncretic enzymes from the gut lumen without ffecting ile flow (Prykhodko et l. 214). The min consequence of PDL is the development of EPI. Exocrine pncretic insufficiency in young pigs tht would normlly e growing rpidly is mnifested y poor nutrient ssimiltion nd growth retrdtion (Fedkiv et l. 29; Rengmn et l. 29; Prykhodko et l. 214). Pncretic duct ligtion results in the dilttion of the pncretic duct, trophy of the cinr cells, nd their replcement with firous tissue in the pncretic remnnt (Imondi et l. 1972). This is similr to wht is seen in ostructive chronic pncretitis in humns (Amromovge et l. 1973; Berkhoff et l. 1987; Boerm et l. 23; Klöppel, 27). Our dt prove tht PDL surgery in pigs lso leds to trophy of the exocrine tissue, inflmmtion nd firosis, ending with the development of EPI. After PDL surgery, the reduction in exocrine pncres function is reflected in the lood plsm y low concentrtions, fter n initil urst, of ctionic trypsinogen, the min pncretic enzyme, (Berkhoff et l. 1987; Lozinsk et l. 213). At the sme time, lekge of Glucose (mmol l 1 ) AUC glucose [mmol l 1 (12 min)] c Insulin (pmol l 1 ) AUC insulin [pmol l 1 (12 min)] Figure 3. Blood glucose nd plsm insulin concentrtions (mens ± SEM) during orl glucose tolernce test in EPI pigs (n = 5) efore (open dimonds, dshed line) nd during therpeutic enzyme tretment with pncrelipse (EPI + PES, filled squres, continuous line), compred with control pigs (n = 6, open circles, continuous line) The res under the curves (AUC glucose nd AUC insulin, mens ± SEM), clculted using the trpezoidl rule, re shown eside the corresponding curves. Different letters given with result rs indicte significnt differences (P <.5). C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

8 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs Glucose (mmol l 1 ) AUC glucose [mmol l 1 (12 min)] c Insulin (pmol l 1 ) AUC insulin [pmol l 1 (12 min)] C-peptide (pmol l 1 ) AUC C-peptide [mmol l 1 (12 min)] Figure 4. Blood glucose, plsm insulin nd C-peptide concentrtions (mens ± SEM) during intrvenous glucose tolernce test in EPI pigs (n = 5) efore (open dimonds, dshed line) nd during therpeutic enzyme tretment with pncrelipse (EPI + PES, filled squres, continuous line), compred with control pigs (n = 6, open circles, continuous line) The res under the curves (AUC glucose, AUC insulin nd AUC C-peptide, mens ± SEM), clculted using the trpezoidl rule, re shown eside the corresponding curves. Different letters given with result rs indicte significnt differences (P <.5). C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

9 18 L. Lozinsk nd others Exp Physiol 11.1 (216) pp pncretic enzymes (high plsm mylse) resulting from PDL in the porcine model reflects n exggerted insulin relese during n IVGTT compred with preopertive vlues (Berkhoff et l. 1987). In contrst to erlier studies (Berkhoff et l. 1987; Boerm et l. 23), we did not find tht EPI in pigs led to ny pprent chnges of the endocrine islets nd insulin-producing cells. In fct, the ove-mentioned studies reported unchnged pncretic endocrine function long with miguous dt out the mount of insulin-positive cells. Conversely, Imondi et l. (1972), working with pigs fter PDL, lso showed firotic chnges in the pncres glnd ut with little effect on the islet cells, which ws ccompnied y lower glucose tolernce, similr to our oservtions. The role of exocrine pncretic function in the glucose-induced insulin response In the present study, we found tht fsting lood glucose concentrtions were higher in EPI pigs compred with the non-operted control pigs, similr to erlier oservtions of Imondi et l. (1972). Together with the unltered sl plsm insulin concentrtions, this my indicte ineffective regultion of lood glucose concentrtions y insulin during EPI. Moreover, peripherl insulin sensitivity did not pper to chnge in the EPI pigs, in tht the QUICKI vlues were similr to those otined in the control pigs. We performed more thorough investigtion of lood glucose control during EPI y mking use of different glucose tolernce tests (MGTT, OGTT nd IVGTT). It hs een shown previously tht EPI pigs retin some cpcity to digest dietry strch, proly owing to the ction of slivry mylse (Kmmlott et l. 25). During the MGTT, however, the EPI pigs displyed smll, insignificnt increse in lood glucose compred with the fsting concentrtions, which explins the wek insulin response oserved during the test. This suggests tht the MGTT should e used with cution in sujects with reduced exocrine pncretic Figure 5. Representtive photomicrogrphs of porcine pncres stined y Hemtoxylin nd Eosin (A nd C) or immunohistochemistry for insulin-producing cells (B nd D) Islets of Lngerhns re indicted y dshed lines. The control non-operted pig shows no pthologicl chnges in pncretic tissue, A, nd insulin-producing cells, B wheres the EPI pig shows inflmmtion (lck rrow) nd firosis, C, with unchnged insulin-producing cells, D. Scle rs: 1 μm. C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

10 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs 19 function, owing to diminished digestive function during EPI. A etter understnding of glycemic control during EPI ws expected from the OGTT nd IVGTT, where the effect of digestion is voided. The EPI pigs showed grdully elevted lood glucose concentrtions during the OGTT, which indicted sustined glucose sorption from the gut, ut this glucose ingestion did not stimulte n dequte insulin response compred with tht oserved in the control pigs. The slow relese of insulin, with delyed insulin pek, in EPI pigs cused divergent ptterns in the glucose utiliztion from the lood, with the totl AUC glucose eing higher compred with tht of the control pigs. The inpproprite insulin response, together with the mrkedly lower clculted insulinogenic index, my suggest n impired β-cell function nd the lck of well-timed insulin relese in EPI conditions. The higher totl AUC insulin otined during the MGTT in control pigs, when compred with the OGTT (with indistinguishle vlues for AUC glucose ), perhps indictes postprndil stimultion of insulin relese, owing to the presence of insulinogenic food components in the gut fter digestion (e.g. lysine, methionine). Incretins my lso e responsile for this insulinotrophic effect, which in ny event ws not oserved in EPI conditions, even during PES therpy. A low incretin stimultion in EPI conditions hs previously een reported (Rogers et l. 1983; Knop et l. 27,), ut t the sme time should not e considered s the only possile reson for the wek insulin response. Previous studies hve lso shown tht the insulinotrophic stimultion of β-cells is diminished in EPI in humns, even fter injection of incretin (glucose-dependent insulinotrophic polypeptide) (Vilsøll et l. 23; Knop et l. 27). Interestingly, delyed insulin (nd C-peptide) relese during MGTT hs lso een reported for ptients with dietes secondry to chronic pncretitis (Knop et l. 27). As further test of β-cell function nd n evlution of the glucose-medited insulin secretion, while voiding possile incretin effects, we lso performed the IVGTT. Direct stimultion of the β-cells y glucose still resulted in delyed insulin relese in EPI pigs compred with control pigs, which ws similr to tht oserved during the OGTT. Additionlly, the similr ptterns of plsm insulin nd C-peptide concentrtions within EPI nd control pigs supports the conclusion of n indequte glucose-medited stimultion of the β-cells in the EPI pigs. C-Peptide in plsm, owing to its slower degrdtion rte, cn e ssumed to indicte the rel β-cell secretion rte (Coelli & Pcini, 1988). The reltionship etween plsm C-peptide nd insulin concentrtions cn e used s model-sed, non-invsive pproch to estimte heptic insulin extrction. Therefore, ccording to the C-peptide-to-insulin molr rtio, EPI increses heptic insulin removl. However, high heptic insulin extrction tht is ccompnied y higher peripherl insulin sensitivity is feture of cloric restriction, which is evident in EPI conditions (Brzili et l. 1998). The delyed insulin response in EPI conditions strengthens the evidence tht dysfunction of the exocrine pncres leds to diminished β-cell function, nd orl PES therpy does not improve the insulin relese. This my suggest n intrpncretic exocrine endocrine xis communiction, with n impct of properly functionl cinr cells on the insulin response. The existence of such n intrpncretic cino-insulr xis hs een previously investigted to some extent, ut owing to miguous results, is still unproved. For exmple, it hs een shown tht in EPI dogs during n OGTT, sunorml insulin response occurs in ssocition with dietic type glucose tolernce (Amromovge et l. 1973). Additionlly, Isksson nd collegues (1983) investigted the connection etween the exocrine nd endocrine pncretic function fter PDL in dult rts. Despite the fct tht PDL in the rt model does not cuse totl pncretic insufficiency, evidence of incresed insulin resistnce ttriutle to inefficient insulin production ws oserved, thus confirming the influence of the pncretic cini on β-cell function (Isksson et l. 1983). Finlly, cystic firosis ptients with EPI hve limited insulin relese nd show higher sl (fsting) sensitivity to insulin, s n dptive mechnism to the diminished mount of insulin (Morn et l. 1994). In contrst, unchnged β-cellmssndinsulincontent were oserved in the pncres of PDL mice (Rnkin et l. 213). Boerm et l. (23) oserved reduced size, ut not numer, of Lngerhns islets in n EPI pig model. They suggested tht the secretion of insulin in EPI conditions ws dequte, sed on the unchnged glucose tolernce during n IVGTT, without the estimtion of insulin response (Boerm et l. 23). Severl clinicl oservtionl studies mention tht ptients with chronic pncretitis developed glucose intolernce nd lower β-cell responsiveness, resulting in lower insulin relese during glucose tolernce tests (Vilsøll et l. 23; Knop et l. 27, 21). The physiologicl conditions tht cuse chnges in glycemic control, insufficient insulin production nd dietes secondry to pncretic diseses in humns re not fully understood (Cui & Andersen, 212; Ewld & Bretzel, 213). In fct, pncretic cncer nd chronic pncretitis, when cini cells re injured, re reported s the most common cuses of this type of dietes. Tken together, dt from previous studies nd the dt presented in the present study indicte impired β-cell function in EPI conditions nd suggest n intrpncretic influence of the exocrine pncres on the endocrine pncres. The interruption of the cino-insulr xis my explin the presence of dietic symptoms, recognized s pncretogenic type 3c dietes mellitus. C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

11 11 L. Lozinsk nd others Exp Physiol 11.1 (216) pp The effect of pncretic enzymes on glucose ssimiltion Pncretic enzyme supplementtion therpy with porcine pncretic enzyme preprtions sufficiently restores the digestive cpcity in oth EPI humns nd nimls (Kmmlott et l. 25; Knop et l. 27; Fedkiv et l. 29). When the EPI pigs were treted with PES, the hyperglycemi oserved in fsting conditions ws improved nd lowered to vlues similr to those oserved in control pigs. In ddition, PES incresed the insulin sensitivity, ccording to the QUICKI vlues, in the EPI pigs, indicting improved responsiveness to insulin of the peripherl trget cells. In contrst, the β-cell sensitivity, ccording to the insulinogenic index, ws low nd unchnged in EPI pigs receiving PES compred with the untreted EPI pigs, indicting tht PES therpy did not lter β-cell function. Decresed fsting lood glucose concentrtions nd decresed insulin concentrtions following orl PES therpy my lso indicte tht enzymes presentintheguthvenimpctonperipherlglucose utiliztion in the EPI pigs, in n insulin-independent mnner. This notion ws further strengthened y the results otined in the glucose tolernce tests performed on the EPI pigs receiving PES. During oth the OGTT nd the IVGTT, PES ccelerted glucose clernce from the lood towrds vlues oserved in the control pigs. As is cler from the OGTT nd IVGTT results, the PES-dependent improvement in glucose clernce ws chieved without n enhnced insulin response, ut with decreses in totl insulin response (AUC insulin,epiversus EPI + PES), with lower plsm C-peptide concentrtion (IVGTT). Moreover, PES in EPI pigs did not increse the C-peptide-to-insulin rtio, suggesting no chnge in the heptic insulin extrction s result of the tretment. Therefore, the results otined from the glucose tolernce tests performed on EPI pigs receiving PES therpy, together with dt otined from the fsting glucose nd insulin vlues, i.e. the incresed insulin sensitivity nd depressed insulin secretion, showed tht the orl PES resulted in improved peripherl glucose utiliztion. This my suggest tht the components of pncretic juice, or more likely some pncretic enzymes, y interctions with intestinl cells cn stimulte the relese of n lterntive gut-derived fctor(s), which contriutes to the regultion of glucose utiliztion in n insulin-independent mnner. Similr to our oservtions, Mohn et l. (1998) showed tht pncrelipse supplementtion leds to improvement in control of dietes, with significnt decreses in postprndil plsm glucose nd glycosylted hemogloin concentrtions. Besides, there were no differences in the fsting C-peptide concentrtions efore nd fter pncretic enzyme therpy, which suggests tht there ws no impct on insulin production (Mohn et l. 1998). These uthors proposed tht the mechnism of improved dietes control ws proly secondry to the regultion of crohydrte sorption. Conclusion In conclusion, the elimintion of exocrine function in the porcine PDL model revels n cino-insulr xis communiction, which ppers to e importnt for well-timed insulin secretion. Nonetheless, we cnnot exclude the effects of mldigestion on enterl hormonl relese tht might hve n impct on insulin secretion in EPI pigs, ecuse incretin secretion depends on the sorption of mino cids nd glucose (Eert & Creutzfeldt, 198). Additionlly, orl enzyme supplementtion in the young EPI pig model suggests the existence of n unknown gut-derived mechnism/fctor (ssimilin), which might e involved in insulin-independent peripherl glucose utiliztion. Our findings my explin the glucose metolic normlities seen in cystic firosis ptients nd in ptients with type 3c dietes nd e of ssistnce in understnding the growth retrdtion oserved in EPI conditions in young individuls. References Amromovge AM, Pirent FW & Howrd JM (1973). Pncretic exocrine insufficiency. V. The effects of long-term pncretic duct ligtion on serum insulin levels nd glucose metolism in the dog. Ann Surg 177, Brzili N, Bnerjee S, Hwkins M, Chen W & Rossetti L (1998). Cloric restriction reverses heptic insulin resistnce in ging rts y decresing viscerl ft. J Clinicl Invest 11, Berkhoff M, Grossner D, Klpdor R, KlöppelG& vonkrogeh (1987). [Intrvenously stimulted insulin reserve of the pncres following experimentl pncretic duct ligtion in the swine studies 2, 4 nd out 6 dys following ligtion]. Lngenecks Archiv fur Chirurgie 373, Bilous R & Donnelly R (21). Hndook of Dietes.4th Edition. John Wiley & Sons Ltd., Chichester, pp Blt S, Morise A, Suret A, Louveu I, McéK,Le Huërou-Luron I & Sève B (212). The protein level of isoenergetic formule does not modulte postprndil insulin secretion in piglets nd hs no consequences on lter glucose tolernce. 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12 Exp Physiol 11.1 (216) pp Insulin secretion in exocrine pncres-insufficient pigs 111 Coelli C, Toffolo GM, Dll Mn C, Cmpioni M, Denti P, Cumo A, Butler P & Rizz R (27). Assessment of β-cell function in humns, simultneously with insulin sensitivity nd heptic extrction, from intrvenous nd orl glucose tests. Am J Physiol Endocrinol Met 293, E1 E15. Cui Y & Andersen DK (211). Pncretogenic dietes: specil considertions for mngement. Pncretology 11, Cui Y & Andersen DK (212). Dietes nd pncretic cncer. Endocr Relt Cncer 19, F9 F26. Czkó L, Hegyi P, Rkonczy Z Jr, Wittmnn T & Otsuki M (29). Interctions etween the endocrine nd exocrine pncres nd their clinicl relevnce. Pncretology 9, Eert R & Creutzfeldt W (198). Reversl of impired GIP nd insulin secretion in ptients with pncretogenic stetorrhe following enzyme sustitution. Dietologi 19, EwldN&BretzelRG(213). Dietes mellitus secondry to pncretic diseses (Type 3c) re we neglecting n importnt disese? 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Is pncretic dietes (type 3c dietes) underdignosed nd misdignosed? Dietes Cre 31, S165 S169. Hrdt PD, Huenschild A, Nlop J, Mrzeion AM, Jeger C, Teichmnn J, Bretzel RG, Hollenhorst M & Kloer HU; S /S Study Group (23). High prevlence of exocrine pncretic insufficiency in dietes mellitus. A multicenter study screening fecl elstse 1 concentrtions in 1,21 dietic ptients. Pncretology 3, Imondi A, Strdley R & Wolgemuth R (1972). Enzyme replcement therpy in the pncretic duct ligted swine. Exp Biol Med 141, Isksson G, Ihse I & Lundquist I (1983). Influence of pncretic duct ligtion on endocrine nd exocrine rt pncres. Act Physiol Scnd 117, Jönsson T, Ahrén B, Pcini G, Sundler F, Wierup N, Steen S, Sjöerg T, Ugnder M, Frostegård J, Görnsson L & Lindeerg S (26). A Pleolithic diet confers higher insulin sensitivity, lower C-rective protein nd lower lood pressure thn cerel-sed diet in domestic pigs. Nutr Met (Lond) 3, 39. Kmmlott E, Krthoff J, Stemme K, Gregory P & Kmphues J (25). Experiments to optimize enzyme sustitution therpy in pncretic duct-ligted pigs. J Anim Physiol Anim Nutr (Berl) 89, Ktz A, Nmi SS, Mther K, Bron AD, Follmnn DA, Sullivn G & Quon MJ (2). Quntittive insulin sensitivity check index: simple, ccurte method for ssessing insulin sensitivity in humns. J Clin Endocrinol Met 85, Klöppel G (27). Chronic pncretitis, pseudotumors nd other tumor-like lesions. Mod Pthol 2, S113 S131. KnopFK,VilsøllT,HøjergPV,LrsenS,MdsdS,HolstJJ & Krrup T (27). The insulinotropic effect of GIP is impired in ptients with chronic pncretitis nd secondry dietes mellitus s compred to ptients with chronic pncretitis nd norml glucose tolernce. Regul Pept 144, Knop FK, Vilsøll T, Lrsen S, Højerg PV, Volund A, Mdsd S, Holst JJ & Krrup T (27). Incresed postprndil responses of GLP-1 nd GIP in ptients with chronic pncretitis nd stetorrhe following pncretic enzyme sustitution. Am J Physiol Endocrinol Met 292, E324 E33. Knop F, Vilsøll T, Lrsen S, Mdsd S, Holst JJ & Krrup T (21). Glucgon suppression during OGTT worsens while suppression during IVGTT sustins longside development of glucose intolernce in ptients with chronic pncretitis. Regul Pept 164, Lifson N, Lss CV & Dixit PK (1985). Reltion etween lood flow nd morphology in islet orgn of rt pncres. Am J Physiol Endocrinol Met 249, E43 E48. Lozinsk L, ArévloSured E,Prykhod ko O,SzwiecK, Pierzynowski S & Weström B (213).Plsm enzyme levels fter the induction of exocrine pncretic insufficiency (EPI) nd pncretic enzyme replcement therpy (PERT) in pig model. Pncretology 13, S3. Meier JJ, Veldhuis JD & Butler PC (25). Pulstile insulin secretion dicttes systemic insulin delivery y regulting heptic insulin extrction in humns. Dietes 54, Mohn V, Poongothi S & Pitchumoni C (1998). Orl pncretic enzyme therpy in the control of dietes mellitus in tropicl clculous pncretitis. Int J Pncretol 24, Morn A, Pyzdrowski KL, Weinre J, Khn BB, Smith SA, Adms KS & Sequist ER (1994). Insulin sensitivity in cystic firosis. Dietes 43, Osei K, Flko JM, O Dorisio TM & Adm DR (1984). Decresed serum C-peptide/insulin molr rtios fter orl glucose ingestion in hyperthyroid ptients. Dietes Cre 7, Pierzynowski S & Brej W (1984). The dependence of exocrine pncretic secretion on insulin in sheep. Exp Physiol 69, PrykhodkoO,FedkivO,Weström BR & Pierzynowski SG (214). Effects on gut properties in exocrine pncretic insufficient (EPI) pigs, eing growth retrded due to pncretic duct ligtion t 7 weeks ut not t 16 weeks of ge. Adv Med Sci 59, Rnkin MM, Wilur CJ, Rk K, Shields EJ, Grnger A & Kushner JA (213). β-cells re not genertedin pncretic duct ligtion-induced injury in dult mice. Dietes 62, C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society

13 112 L. Lozinsk nd others Exp Physiol 11.1 (216) pp Rntzer D, Svendsen J & Weström B (1995). Wening of pigs rised in sow-controlled nd in conventionl housing systems, 1: Description of systems, production nd cteriology. Swed J Agr Res 25, R Core Tem (212). R: Lnguge nd Environment for Sttisticl Computing. R Foundtion for Sttisticl Computing, Vienn, Austri, 212. ISBN Rengmn S, Fedkiv O, Botermns J, Svendsen J, Weström B & Pierzynowski S (29). An elementl diet fed, enterl or prenterl, does not support growth in young pigs with exocrine pncretic insufficiency. Clin Nutr 28, Rogers WA, O Dorisio TM, Johnson SE, Ctlnd S, Strdley RP & Sherding RG (1983). Postprndil relese of gstric inhiitory polypeptide (GIP) nd pncretic polypeptide in dogs with pncretic cinr trophy. Dig Dis Sci 28, Schönfeld JV, Goeell H & Mütter MK (1994). The islet-cinr xis of the pncres. Int J Pncretol 16, Vilsøll T, Knop FK, Krrup T, Johnsen A, Mdsd S, Lrsen S, Hnsen T, Pedersen O & Holst JJ (23). The pthophysiology of dietes involves defective mplifiction of the lte-phse insulin response to glucose y glucose-dependent insulinotropic polypeptide regrdless of etiology nd phenotype. J Clin Endocrinol Met 88, Willims JA & Goldfine ID (1985). The insulin-pncretic cinr xis. Dietes 34, Yokoym J, Mori Y, Iked Y, Nisimur M & Mullen Y (1988). Influence of B-cell impirment on pncretic cini in NOD mice nd streptozotocin-induced dietic rts. Endocrinol Jpn 35, Additionl informtion Competing interests None declred. Author contriutions All the work ws done in the lortories of the Deprtment of Biology nd Deprtment of Clinicl Sciences t Lund University (Sweden). S.G.P., B.W., L.L. nd O.P. conceived nd designed the reserch. L.L., K.S. nd S.G.P. performed experiments. L.L., A.L. nd N.W. nlysed dt. S.G.P., L.L., O.P., B.W., B.A., A.L. nd N.W. interpreted results of experiments. L.L. prepred nd drfted the mnuscript. S.G.P., B.W., L.L., O.P. nd B.A. edited nd revised the mnuscript. All uthors hve pproved the finl version of the mnuscript nd gree to e ccountle for ll spectsofthework.allpersonsdesigntedsuthorsqulify for uthorship, nd ll those who qulify for uthorship re listed. Funding The Swedish Reserch Council for Environment, Agriculturl Sciences nd Sptil Plnning (FORMAS, no , is cknowledged for its finncil support. C 215 The Authors. Experimentl Physiology C 215 The Physiologicl Society