Diabetes mellitus secondary to pancreatic diseases (type 3c): The effect of smoking on the exocrine endocrine interactions of the pancreas

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1 764062DVR / Dibetes & Vsculr Disese ReserchŚliwińsk-Mossoń et l. reserch-rticle2018 Originl Article Dibetes mellitus secondry to pncretic diseses (type 3c): The effect of smoking on the exocrine endocrine interctions of the pncres Dibetes & Vsculr Disese Reserch 2018, Vol. 15(3) The Author(s) 2018 Reprints nd permissions: sgepub.co.uk/journlspermissions.nv DOI: journls.sgepub.com/home/dvr Mriol Śliwińsk-Mossoń 1, Stnisłw Milnerowicz 2 nd Hlin Milnerowicz 1 Abstrct The present study ws conducted to scertin how cigrette smoke ffects the exocrine endocrine interctions of the humn pncres with dibetes mellitus secondry to pncretic diseses (type 3c). Blood hs been collected from smoking nd non-smoking helthy individuls s well s from ptients with dignosed chronic pncretitis nd dibetes type 3c. The concentrtions of interleukin-6, endothelin-1 nd insulin in the plsm were determined by enzyme-linked immunosorbent ssy (ELISA) tests. The ctivities of mylse nd lipse in the serum, s well s the lipid profile, cretinine, uric cid nd ure concentrtions, were mesured using colorimetric methods. Smples of norml pncretic tissue nd chronic pncretitis were verified histopthologiclly nd then interleukin-6, endothelin-1, insulin nd glucgon were loclized by immunohistochemicl stining using monoclonl nti-humn ntibody. The highest levels of interleukin-6 nd endothelin-1 nd the lowest levels of insulin nd glucgon intensity from the immunostining were observed in smoking ptients with dibetes. In ll smoking ptients with pncretitis nd dibetes, there ws significnt elevtion in interleukin-6 nd endothelin-1 concentrtion nd mylse nd lipse ctivities, hyperlipidemi nd lower vlue of estimted glomerulr filtrtion rte nd blood ure nitrogen when compred to non-smokers. Our study confirmed tht smoking exerts pro-inflmmtory effect nd disturbs the exocrine endocrine interctions of the pncres. Keywords Cigrette smoking, dibetes type 3c, complictions, chronic pncretitis Introduction Dibetes mellitus is defined s regultory dysfunction of metbolism minly chrcterized by chronic hyperglycemi. The underlying cuses my be impired insulin secretion or insulin resistnce, or both. Dibetes cn lso develop s direct consequence of other diseses, including diseses of the exocrine pncres. Becuse of this heterogeneity in underlying cuses, dibetes mellitus is currently clssified into different types, comprising types The wreness of type 1 nd type 2 dibetes mellitus mong physicins nd in the generl popultion is rther brod. Dibetes mellitus secondry to pncretic diseses (type 3c), however, is condition rrely considered in everydy prctice. Yet, recent dt on type 3c dibetes mellitus (T3cDM) show tht it might be more common thn previously thought. Studies propose tht this cliniclly importnt condition might be consistently underdignosed nd misdignosed. 1,4 T3cDM is frequent comorbidity of chronic pncretitis (CP), with prevlence estimtes rnging from 25% to 80%. 3 7 Pncretic cncer (8% of ll T3cDM) seems to be the second most common cuse ccording to recent study. 4 The risk of dibetes in ptients with CP increses the longer the durtion of the disese, with worsening pncretic dmge, especilly erly-onset pncretic clcifiction. It lso rises with prior distl pncretectomy. 5 Multiple clinicl studies hve shown tht using tobcco, prticulrly vi smoking cigrettes, elevtes the risk of 1 Deprtment of Biomedicl nd Environmentl Anlyses, Fculty of Phrmcy with Division of Lbortory Dignostics, Wrocłw Medicl University, Wrocłw, Polnd 2 Deprtment nd Clinic of Gstrointestinl nd Generl Surgery, Fculty of Postgrdute Medicl Trining, Wrocłw Medicl University, Wrocłw, Polnd Corresponding uthor: Mriol Śliwińsk-Mossoń, Deprtment of Biomedicl nd Environmentl Anlyses, Fculty of Phrmcy with Division of Lbortory Dignostics, Wrocłw Medicl University, Borowsk 211, Wrocłw , Polnd. Emil: mriol.sliwinsk-mosson@umed.wroc.pl

2 244 Dibetes & Vsculr Disese Reserch 15(3) developing pncretic diseses such s pncretitis nd cncer Furthermore, the risk increses s function of the mount of tobcco consumed. Smoking tobcco hs often been linked s co-fctor with lcohol buse in predisposition to pncretic disorders. However, the inclusion of smokers who do not drink lcohol in some of these studies hs highlighted the fct tht cigrette smoking cn lso be considered n independent risk fctor. 11 Despite significnt clinicl dvncements in this field, scientific dt exploring how tobcco toxins ffect the pncres t the cellulr level re scrce. 9 Smoking hs n influence on chnges in hormonl secretion in the pncres Endocrine dysfunction of pncretic islet bet (β) cells is common compliction of pncretic inflmmtion resulting from long-term smoking, nd s consequence, dibetes occurs. Substnces present in tobcco smoke undoubtedly trigger free rdicl processes, interfere with vsculr homeostsis nd the proper functioning of the vsculr endothelium, 16,17 nd lso increse inflmmtion nd oxidtive stress. 18 Becuse higher levels of inflmmtory mrkers [C-rective protein (CRP) nd interleukin-6 (IL- 6)] herld the development of dibetes, 19 smoking my lso contribute to the development of dibetes by incresing levels of inflmmtory mrkers. 18 The min chrcteristic of T3cDM is the grdul loss of β-cell function leding to impirment in or lck of insulin secretion. 3 7 However, dipose tissue, gstro-intestinl trct, pncretic β-cell ctivity nd kidney function my lso be involved in disese development. 20 In prllel to impirment in endocrine pncretic function, reduction in exocrine function hs lso been observed in ll types of dibetes. 21 A decrese in mylse nd lipse ctivity hs been mentioned by Ydv et l., 22 lthough cses with elevted mylse hve lso been reported. And studies evluting the pncretic exocrine function in smoking ptients with CP showed significnt impirment in the ctivity of pncretic enzymes. 10,23 27 Another study found tht cigrette smoking my be n importnt fctor in potentil chnges in the lipid profile in helthy young people. 26 Most recently, trend of positive correltions between high-density lipoprotein (HDL) cholesterol nd mylse in dibetes type 2 ptients hs been discovered. 22 To our knowledge, no study correlting cholesterol levels nd mylse in T3cDM ptients hs been conducted. Incresed hyperlipidemi combined with oxidtive stress (resulting from hyperglycemi nd cigrette smoking) re mong the min fctors inducing theromtosis, dibetic ngiopthy, thrombotic incidents nd crdiovsculr problems, ll of which re commonly observed dibetic complictions. 27 In ddition, CP is disese with poor clinicl prognosis. Almost hlf of the ptients with lcoholic pncretitis die fter yers of disese durtion. 28 The development of secondry dibetes in the course of CP is n independent risk fctor for deth in this group of ptients. 29 As it is chronic, it my lso ffect other orgns in the body. So fr, little is known bout the long-term consequences of ongoing CP nd dibetes in smoking ptients. In smoking ptients, episodes of cute pncretitis (AP) occur often enough, 18 resulting in orgn ischemi tht my cuse complictions typicl of the disese (shock, cute respirtory filure, cute renl filure nd others). 30 After stbiliztion of the cute stte in the course of CP, exocrine nd endocrine functions of the pncres re primrily monitored; distnt orgns re rrely dignosed. In our erlier studies, we hve shown tht tobcco smoke disorders pro-oxidnt nd ntioxidnt prmeters in pncretic fluids. 18 However, there re no reports on the locliztion of IL-6, endothelin-1 (ET-1) or the mjor pncretic hormones insulin nd glucgon in the pncretic tissue of non-smoking nd smoking ptients with CP nd dibetes. The concentrtions of IL-6 nd ET-1, mylse nd lipse ctivity, chnges in the lipid profile nd chnges in the prmeters responsible for renl function in the blood of non-smoking nd smoking ptients with CP nd dibetes re lso unknown. Therefore, the objective of this study ws to summrize clinicl nd scientific knowledge of the effects tobcco hs upon the pncres, nd how tobcco my contribute to CP, the development of dibetes nd the complictions ssocited with it. Mterils nd methods Mterils The study ws conducted on 24 ptients with CP nd dibetes (18 men, 6 women; ged yers; men ge 44 yers) dmitted to the Deprtment of Gstrointestinl nd Generl Surgery, Wrocłw Medicl University, Polnd, for evlution of their disese (Tble 1). This work ws crried out in ccordnce with the Declrtion of Helsinki (2000) of the World Medicl Assocition. The study ws pproved by the Bioethics Commission t Wrocłw Medicl University (No. KB: 4353/2011). All helthy individuls nd ptients provided their informed consent in writing. The ptient clssifiction nd ssessment of pncretitis were bsed on the following: routine clinicl exmintion nd lbortory tests (medicl history: pin ppernce bckground, disese durtion; physicl exmintions); ultrsonogrphy (US) nd computed tomogrphy (CT) of the bdomen nd, in specil cses, endoscopic retrogrde cholngiopncretogrphy (ERCP) nd mgnetic resonnce cholngiopncretogrphy (MRCP) of the pncres nd bile ducts. The physicl exmintion, supplemented by the forementioned dignostic procedures, permitted the identifiction of the pncretitis etiology in most cses. The dignosis of CP ws combined with history of lcohol buse, where the ptient consumed more thn 40 g

3 Śliwińsk-Mossoń et l. 245 Tble 1. Chrcteristics of non-smoking nd smoking CP ptients with dibetes. Non-smoking Smoking The number of ptients 9 15 Femle/mle 2/7 4/11 The ge rnge (yers) The durtion of the CP from the onset (yers) Episodes of cute pncretitis 10.0 ± ± ± ± Alcohol/no lcohol 3/6 11/4 The number of cigrettes (n/24 h) 23.0 ± Cotinine concentrtion (ng/ml) CP: chronic pncretitis. Sttisticlly significnt differences: p < ± ± of lcohol per dy for more thn 5 yers. The obstructive pncretitis etiology ws defined on the bsis of ERCP nd US tests. The fmily type of pncretitis ws not dignosed. Cses which could not be clssified ccording to the bove criteri were defined s cses of idiopthic pncretitis. Dt on smoking nd lcohol consumption were obtined during direct personl interviews conducted with ech ptient. Bsed on the interview bout smoking hbits nd on the elevted levels of cotinine nicotine metbolite in the plsm CP ptients were divided into two groups: non-smokers nd smokers. Among the study group of ptients, 9 CP non-smokers nd 15 CP smokers were dignosed with dibetes. The dignosis ws mde bsed on the dignostic criteri for T3cDM proposed by Ewld nd Bretzel 1 (Tble 1). A control group ws selected bsed on interviews by employees of the Medicl University nd crried out during regulr medicl exmintions for the Polish socil security system. The control group comprised 53 helthy individuls, ged yers (men ge 41 yers). Among the control subjects, no cses of dibetes mellitus nor individuls with history or symptoms of pncretitis were observed. All hospitlized ptients nd the control group of volunteers gve informed consent to prticipte in the study. Blood nd tissues served s the study mteril. Venous blood ws collected from fsting ptients during the first 24 h of hospitliztion. Serum ws obtined ccording to the stndrd procedure by collecting venous blood in disposble trce element-free tubes (No. Ct , Srstedt, Germny) with serum clotting ctivtor, left t 25 C to complete thrombosis nd centrifuged (1.200g/20 min). In order to obtin plsm, blood ws collected into trce element-free tubes contining EDTA-K 2 (No. Ct , Srstedt, Germny) nd lithium heprin (No. Ct , Srstedt, Germny), immeditely gently mixed nd centrifuged (2.500g/15 min). The obtined serum nd plsm were portioned nd stored in seled tubes (No. Ct , Eppendorf, Germny) t 80 C until used. Fourteen CP ptients with dibetes (non-smoking, n = 5; smoking, n = 9) underwent surgery during which tissue mteril ws obtined. Four utopsy cses, free from CP nd dibetes mellitus, served s controls. All norml pncres smples were obtined from non-smoking subjects who hd died in cr ccidents. All tissue sections were routinely fixed in phosphtebuffered 10% formldehyde solution nd embedded in prffin. Seril 4-µm-thick sections were plced on histologicl slides (Super Frost Plus, Ct No.: , Menzel- Gloser, Germny). Preprtions stined with hemtoxylin nd eosin were verified histologiclly in the Deprtment of Pthomorphology, Wrocłw Medicl University. Methods Method for the determintion of concentrtion nd ctivity prmeters. The cotinine determintion ws crried out by method described erlier. 25 Immunoenzymtic methods n IL-6 DuoSet ELISA Development System (Ct. No. DY, R&D Systems; 206) nd Humn ET-1 Immunossy (Code: BBE5; R&D Systems, Minnepolis, MN, USA) were used to determine plsm IL-6 nd ET-1 levels with specific ntibodies ginst humn IL-6 nd ET-1. The IL-6 nd ET-1 vlues were expressed in picogrms per millilitre. Fsting glucose levels were mesured in plsm using n oxidtion method with regent for the quntittive determintion of glucose (Ct. No , BIOLABO, Frnce). Glucose is oxidized by glucose oxidse to gluconic cid nd hydrogen peroxide. Hydrogen peroxide involving peroxidse rects with 4-chlorophenol nd 4-minontipyrine to form red quinone-imine. The bsorbnce of the coloured complex proportionl to the concentrtion glucose in the smple is mesured t 500 nm. Another immunoenzymtic method n Insulin ELISA Kit (Ct. No. 6219, DAKO, UK) ws used to determine plsm insulin levels with specific ntibodies ginst humn insulin. The insulin vlues were expressed in units per millilitre. The enzyme ctivity ws determined using colorimetric method with substrtes for mylse: ethylidene-g7-pnp (4,6-ethylidene-p-nitrophenyl-lph,D-mlthoheptozyde) nd for lipse: 1,2-o-diluryl-rc-glycero-3-glutric cid- (6-methylresorufin) ester. Totl cholesterol ws mesured in the serum using regent (Ct. No: ; BioMxim, Polnd). Cholesterol ws determined using n enzymtic method. One of the

4 246 Dibetes & Vsculr Disese Reserch 15(3) rection by-products, H 2 O 2, ws mesured quntittively in peroxidse-ctlysed rection tht produces colour, with intensity proportionl to cholesterol concentrtion. Absorbnce ws mesured t λ = 500 nm t 25 C. Triglycerides (TGs) were determined in the serum using regent (Ct. No: ; BioMxim, Polnd). TGs were hydrolysed to produce glycerol, then glycerol ws oxidized using glycerol oxidse, nd H 2 O 2, one of the rection products, ws determined s described bove for cholesterol. Absorbnce ws mesured t λ = 500 nm t 25 C. HDL concentrtion ws determined in the serum using direct method (Ct. No: , BioMxim, Polnd). In this test, ll cholesterol frctions except HDL were blocked. HDL ws subjected to n enzymtic rection tht involved the formtion of colour compounds. The mount of colour product ws proportionl to the HDL concentrtion in the smple. The bsorbnce ws mesured t λ = 600 nm t 25 C. Low-density lipoprotein (LDL) concentrtion ws clculted using the Friedewld formul. 31 The vlue for the Cstelli risk index I [totl cholesterol (TC)/HDL rtio] ws clculted ccording to Asre et l. 32 The mrker of lipid peroxidtion, thiobrbituric cid rective substnces (TBARS), ws mesured in the plsm using thiobrbituric cid, ccording to the method described erlier. 33 Serum cretinine concentrtion ws determined by the Jffe colorimetric method (Ct. No.: , BioMxim, Polnd). Uric cid levels were determined by colorimetric ssy of urokinse (Ct. No.: , BioMxim, Polnd). Ure concentrtion ws determined by the Urese Glutmte dehydrogense (GLDH) method. For dignostic purposes nd nlysis of results, ure concentrtion ws converted to ure nitrogen concentrtion (blood ure nitrogen, BUN). The estimted glomerulr filtrtion rte (egfr) ws clculted using the CKD-EPI Cretinine Eqution (s recommended by the Ntionl Kidney Foundtion). Immunohistochemicl nlysis. The tissue sections were deprffinized nd rehydrted in n lcohol series. All sections were then incubted with 3% hydrogen peroxide to block the intrcellulr ctivity of peroxidse. The non specific binding ws blocked with 0.05 M Tris HCl buffer, ph 7.4 with 1% bovine serum lbumin (Ct. No.: S0809, Antibody Diluent, DkoCytomtion, Denmrk). The tissue sections were subsequently incubted for 30 min t room temperture with specific monoclonl mouse ntibodies ginst IL-6 (Ct. No.: MAB206, R&D Systems, USA), ET-1 (Ct. No.: ABIN120978, Abcm, Cmbridge, UK), insulin (Ct. No.: N1542; DkoCytomtion, Denmrk) nd glucgon (Ct. No.: N1541; DkoCytomtion, Denmrk). After wshing in 0.05 M Tris HCL sline (Ct. No.: , Bio-Rd Lbortories; Hercules, USA) with 0.1% Tween 20 (Ct. No.: P1379, Sigm Chemicl, St. Louis, MO, USA), the IL-6, ET-1, insulin nd glucgon complexes were visulized using the LSAB2-HRP test (Ct. No.: K0673, DkoCytomtion, Crpinteri, CA, USA). Peroxidse ctivity ws loclized ginst 3,3 -diminobenzidine in n imidzole HCl buffer, ph 7.5 (Ct. No.: K0637, DAB, DkoCytomtion, Denmrk). Consequently, the sections were wshed in distilled wter, contrsted with hemtoxylin (Ct. No.: S2020, Chem Mte, DkoCytomtion, Denmrk) nd finlly closed in glycerine gel nd left until dry. A negtive control ws performed for ech tissue section, replcing the primry ntibody with the nti-rbbit immunoglobulin control IgG ntibody (Ct. No.: X0903, Negtive Control DkoCytomtion, Crpinteri, CA, USA). The incubtion time of individul stges of the test were mtched experimentlly in lbortory conditions. The stined tissue sections were viewed under high power using n Olympus BX41 light microscope (Olympus Opticl Co. Ltd, Jpn) tht interfced with n Olympus DP70 digitl cmer (Olympus Opticl Co. Ltd, Jpn) in order to digitize the light microscopic imge. Evlution of the immunohistochemicl stining results. The intensity of the prmeters IL-6, ET-1, insulin nd glucgon immunorections within the cells ws grded semi-quntittively in the following mnner: negtive (no immunohistochemicl rection: 0), wekly positive (lightly stined but clerly differentited from negtive bckground: +/ ), modertely positive (between wek nd strong: +), strongly positive (drk brown with high contrst: + +) nd very strongly positive (+ + +). Digitl imging methodology. In order to determine the prmeters content in the immunohistochemiclly stined sections, digitl imging method for DAB quntifiction on slides with hemtoxylin counterstin ws used. The imge nlysis llows for quntittive numericl comprisons of stining intensity bsed on the vlue of signls red from the DAB stining, which is possible due to the existence of liner reltionship between stining intensity nd ntigen concentrtion. Stined tissue sections were viewed under light microscope tht interfced with cmer for scientific pplictions. Both units were controlled by personl computer. The Fiji mesurement system (opertes in the Windows environment) ws used to perform the quntittive nlysis of the digitized imges. The system quntified the verge drkness of the imge due to the DAB signl from ntigen stining. Then, it converted the signl stining into men gry vlue, nd the results were quntified in DAB signl intensity units. The rection intensity ws evluted in rbitrry unit (AU) of opticl density (OD). A totl of 10 res were cquired from ech tissue section of the control ptients s well s the smoking nd nonsmoking CP ptients with dibetes.

5 Śliwińsk-Mossoń et l. 247 Tble 2. Concentrtions IL-6 nd ET-1 in plsm of smoking nd non-smoking control groups nd CP ptients with dibetes. Vrible Control group CP ptients with dibetes Non-smoking Smoking Non-smoking Smoking IL-6 (pg/ml) Men ± SD 0.96 ± ± ± ± 11.4 Medin ,b 15.6,b,c Min. Mx ET-1 (pg/ml) Men ± SD 1.23 ± ± ± ± 2.43 Medin ,b Min. Mx IL-6: interleukin-6; ET-1: endothelin-1; CP: chronic pncretitis; SD: stndrd devition. p < 0.05, when compred to non-smoking control group. b p < 0.05, when compred to smoking control group. c p < 0.05, when compred to non-smoking ptients with dibetes. Sttisticl nlysis. Prticipnts were ctegorized into four groups: non-smoking nd smoking control groups, nd nonsmoking nd smoking CP ptients with dibetes. The normlity of the vribles ws tested by the Shpiro Wilk test. The differences between nlysed groups were tested using the Mnn Whitney U test. Spermn rnk correltion nlysis ws used to evlute the correltions. In ll instnces, p < 0.05 ws considered sttisticlly significnt. Sttisticl clcultions were done using the Sttistic Softwre Pckge, version 13.0 (Polish version; SttSoft, Krkow, Polnd). Results Concentrtion of IL-6, ET-1 nd insulin The concentrtion of IL-6, n estblished meditor of inflmmtion, ws found to be higher in the plsm of both the control group nd CP ptients with dibetes who hd been exposed to cigrette smoke. The study confirmed significnt differences in the concentrtion of plsm IL-6 between the non-smoking nd smoking control groups, nd non-smoking nd smoking ptients with dibetes (Tble 2). The concentrtion of serum IL-6 ws found to be four times higher in smoking thn in non-smoking CP ptients with dibetes. Interestingly, the IL-6 levels were found to be only twice higher in the plsm of the smoking control thn in tht of non-smoking control. The highest level of IL-6 ws found in smokers with dibetes. The men vlues of plsm ET-1 concentrtion long with stndrd devitions in the non-smoking nd smoking control groups nd ptients with CP re summrized in Tble 2. The highest concentrtion of ET-1 ws observed in the plsm of smoking ptients with dibetes followed by non-smoking ptients with dibetes. The differences in ET-1 concentrtion between the non-smoking control group, the smoking control group nd dibetic ptients were sttisticlly significnt (p < 0.05). Sttisticlly higher levels of ET-1 in smoking CP ptients with dibetes were observed compred to the control group of smokers. No significnt difference ws found in the concentrtion of ET-1 in the plsm of non-smoking nd smoking ptients with dibetes, which suggests tht dibetes hs very strong effect on endothelil dysfunction nd the secretion of ET-1, with cigrette smoking incresing this secretion. The men vlues of plsm insulin concentrtion long with stndrd devitions in non-smoking nd smoking CP ptients with dibetes nd in the control groups re summrized in Tble 2. The highest level of insulin ws found in the non-smoking control, the lowest in smoking CP ptients with dibetes. The differences in insulin concentrtion between non-smoking nd smoking controls nd non-smoking nd smoking ptients with dibetes were sttisticlly significnt (Tble 3). A lower, similr vlue of insulin concentrtion ws observed in non-smoking nd smoking CP ptients with dibetes compred with the non-smoking control. Amylse nd lipse ctivity The highest mylse nd lipse ctivity ws found in smoking CP ptients with dibetes. It ws noted tht the serum enzyme ctivity ws significntly higher in the smoking control thn in the non-smoking control. A sttisticlly significnt increse in mylse nd lipse ctivity in non-smoking nd smoking CP ptients with dibetes ws observed compred to the non-smoking control. Higher enzyme ctivity ws found in the serum of smoking ptients thn in non-smoking ptients (Tble 3). The lipid profile, TBARS, cretinine nd egfr Chnges in lipid peroxidtion nd the lipid profile were observed in the serum of non-smoking nd smoking CP ptients with dibetes. The concentrtion of HDL ws sttisticlly lower in dibetic CP ptients, non-smoking or smoking, compred to the control group, while the concentrtions of TC, TG nd LDL were sttisticlly highest in non-smoking nd smoking dibetics, compred to control

6 248 Dibetes & Vsculr Disese Reserch 15(3) Tble 3. Concentrtions nd ctivities of nlysed prmeters in the blood of smoking nd non-smoking control groups nd CP ptients with dibetes. Vrible Control group CP ptients with dibetes Non-smoking Smoking Non-smoking Smoking Glucose (mg/dl) Men ± SD 88.9 ± ± ± ± 52.6 Medin Min. Mx Insulin (μu/ml) Men ± SD 10.4 ± ± ± ± 0.7 Medin ,b 1.0,b Min. Mx Amylse (U/L) Men ± SD 47.4 ± ± ± ± 51.3 Medin ,b,c Min. Mx Lipse (U/L) Men ± SD 28.4 ± ± ± ± 30.4 Medin 25.0 b ,b 82.0,b Min. Mx TC (mg/dl) Men ± SD ± ± ± ± 15.9 Medin ,b Min. Mx LDL (mg/dl) Men ± SD ± ± ± ± 19.5 Medin Min. Mx HDL (mg/dl) Men ± SD 51.7 ± ± ± ± 5.7 Medin ,b 37.4,b Min. Mx TG (mg/dl) Men ± SD 97.7 ± ± ± ± 23.2 Medin ,b 204.0,b,c Min. Mx Cstelli risk index I (TC/HDL) Men ± SD 3.6 ± ± ± ± 0.5 Medin ,b 5.8,b Min. Mx TBARS (μmol/l) Men ± SD 3.4 ± ± ± ± 3.6 Medin ,b,c Min. Mx CP: chronic pncretitis; SD: stndrd devition; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TC: totl cholesterol; TG: triglyceride; TBARS: thiobrbituric cid rective substnces. Reference rnge/vlue for TC is <200 mg/dl, TG mg/dl (detection limit 10 mg/dl) nd HDL >35 mg/dl. Sttisticlly significnt differences: p < 0.05, when compred to non-smoking control group. b Sttisticlly significnt differences: p < 0.05, when compred to smoking control group. c Sttisticlly significnt differences: p < 0.05, when compred to non-smoking ptients with dibetes. groups (Tble 3). When we nlysed the vlue of the Cstelli risk index I (TC/HDL), we found higher vlue in CP ptients with dibetes when compred to the control group (non-smoking nd smoking) (Tble 3). It ws lso observed tht the concentrtion of TBARS ws sttisticlly significntly higher in the smoking control nd smoking CP ptients with dibetes (4.0 ± 2.0 nd 6.3 ± 3.6 μmol/l), compred to the non-smoking control group (3.4 ± 1.9 μmol/l). The highest TBARS concentrtion ws found in smoking CP ptients with dibetes (Tble 3). The highest serum cretinine level ws found in the smoking group of dibetic ptients: 1.3 ± 0.2 mg/dl. A sttisticlly significnt increse in cretinine nd uric cid concentrtions in smoking CP ptients with dibetes ws present when compred to the non-smoking control. A significnt decrese in the vlues of BUN nd the BUN/cretinine rtio ws observed in non-smoking nd smoking ptients with dibetes s compred to non-smoking nd smoking controls. The lowest vlue of egfr ws observed in smoking ptients with dibetes. A sttisticl decrese in the vlue of egfr in smoking dibetics compred to the non-smoking control ws lso observed (Tble 4). Correltions between prmeters Significnt correltions in the non-smoking group of ptients with dibetes. The correltion coefficients of specific biochemicl prmeters s determined in non-smoking CP

7 Śliwińsk-Mossoń et l. 249 Tble 4. Cretinine nd uric cid concentrtions, nd vlues of egfr in serum non-smoking nd smoking helthy persons nd ptients with chronic pncretitis. Vrible Control group CP ptients with dibetes Non-smoking Smoking Non-smoking Smoking Cretinine (mg/dl) 0.9 ± ± ± ± Uric cid (µmol/l) ± ± ± ± b BUN (mg/dl) 10.4 ± ± ± ± b,c 3.0 b,c egfr (ml/min/1.73 m 2 ) ± ± ± b egfr: estimted glomerulr filtrtion; CP: chronic pncretitis; BUN: blood ure nitrogen. Reference rnge/vlue for cretinine is mg/dl, uric cid mol/l, BUN mg/dl nd egfr 90 ml/min/1.73 m 2. Sttisticlly significnt differences: p < 0.05, when compred to non-smoking ptients with dibetes. b Sttisticlly significnt differences: p < 0.05, when compred to non-smoking control group. c Sttisticlly significnt differences: p < 0.05, when compred to smoking control group. Tble 5. Correltions of the biochemicl prmeters in the non-smoking group of ptients with dibetes. Vrible Insulin Glucose Amylse Lipse ET-1 IL b 0.88 b 0.82 Insulin b ET IL-6: interleukin-6; ET-1: endothelin-1. p < b p < Tble 6. Correltions of the lipid profile concentrtions with the blood glucose nd insulin concentrtions nd the enzymes ctivities of the non-smoking ptients with dibetes. Vrible Insulin Glucose Amylse Lipse TBARS TC b 0.54 b 0.79 c 0.56 LDL 0.42 b 0.14 b NS 0.62 b 0.45 HDL 0.48 b 0.81 b NS 0.61 NS TG 0.72 b b 0.65 c NS Cstelli risk index I b 0.59 b b TBARS: thiobrbituric cid rective substnces; TC: totl cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglyceride; NS: not significnt. p < b p < c p < ptients with dibetes re summrized in Tbles 5 nd 6. There were strong positive correltions between the enzyme ctivity of lipse nd mylse, between these enzymes nd IL-6 nd between ET-1 nd IL-6 nd glucose. The results of the study confirmed the existence of correltion between decresed insulin levels nd incresed pncretic enzyme ctivity nd between elevted ET-1 nd glucose levels in ptients with dibetes (negtive correltion). In ddition, there ws significnt positive correltion between lipse ctivity nd mylse ctivity, which confirms the reltionship between the prmeters (r = 0.96; p < 0.01).

8 250 Dibetes & Vsculr Disese Reserch 15(3) Tble 7. Correltions of the biochemicl prmeters in the smoking group of ptients with dibetes. Vrible Insulin Glucose Amylse Lipse ET-1 IL-6 Cotinine b 0.94 c 0.81 c 0.89 c 0.90 c IL c 0.94 c 0.95 c 0.81 c 0.90 c ET c 0.69 c NS 0.90 c Insulin c 0.90 c IL-6: interleukin-6; ET-1: endothelin-1; NS: not significnt. p < b p < c p < Tble 8. Correltions of the lipid profile concentrtions with the blood glucose nd insulin concentrtions nd the enzymes ctivities of the smoking ptients with dibetes. Vrible Cotinine Insulin Glucose Amylse Lipse TBARS TC b c 0.87 b LDL b 0.54 NS b HDL 0.72 b NS 0.91 b NS 0.61 b NS TG c NS Cstelli risk index I b b 0.63 b TBARS: thiobrbituric cid rective substnces; TC: totl cholesterol; LDL: low-density lipoprotein; HDL: high-density lipoprotein; TG: triglyceride; NS: not significnt. p < b p < c p < A negtive correltion between insulin nd the TC, LDL nd TG levels s well s the vlue of the Cstelli risk index I ws found. A lower insulin level correlted with lower HDL level. A negtive correltion between HDL level nd glucose concentrtion nd lipse ctivity ws detected. A higher glucose concentrtion ws ccompnied by higher TC, LDL nd TG levels nd higher vlues of the Cstelli risk index I. Higher mylse ctivity correlted with higher TC nd TG levels nd higher vlues of the Cstelli risk index I. Positive correltion coefficients between lipse ctivity nd the nlysed prmeters were noted in Tble 6. Positive correltion coefficients were observed between TBARS concentrtion nd TC nd LDL levels nd the vlue of the Cstelli risk index I. Significnt correltions in the smoking group of ptients with dibetes. The correltion coefficients of specific biochemicl prmeters determined in smoking CP ptients with dibetes re summrized in Tbles 7 nd 8. Cigrette smoking in dibetic ptients resulted in incresed secretion of the cytokine of inflmmtory IL-6, with simultneous pncretic blood flow disorder, incresed lipse nd mylse enzyme ctivity nd decresed insulin secretion. Evidence of this is found in the positive correltions between cotinine nd IL-6, pncretic enzymes nd ET-1, nd the negtive correltion between cotinine nd insulin. A negtive correltion ws lso found between the cytokine level nd insulin (the increse in IL-6 correlted with decrese in insulin concentrtion). In ddition, the reduction in insulin levels correlted with the intensity of lipse nd mylse ctivity. In contrst, pncretic circultion disorders in smoking dibetic ptients were confirmed by n increse in endothelin concentrtion, which positively correlted with n increse in lipse ctivity nd glucose concentrtion, nd negtively correlted with insulin secretion from pncretic B cells. There ws positive significnt correltion between lipse ctivity nd mylse ctivity, which confirms the reltionship between the prmeters (r = 0.94; p < 0.05). A positive correltion between cotinine nd the TC, LDL nd TG levels s well s the vlue of the Cstelli risk index I ws found. A higher cotinine level correlted with lower HDL level. A negtive correltion between insulin concentrtion nd TC, LDL nd TG levels nd the vlue of the Cstelli risk index I ws found. Higher TC nd TG levels were correlted with higher glucose levels, higher TBARS levels nd higher mylse nd lipse ctivity. A negtive correltion between HDL level nd glucose level nd lipse ctivity ws detected. A higher vlue of the Cstelli risk index I ws ccompnied by higher levels of the nlysed prmeters (except insulin). Positive correltion coefficients between LDL levels nd glucose levels, TBARS levels nd

9 Śliwińsk-Mossoń et l. 251 Figure 1. Immunohistochemicl locliztion of IL-6 (, b, c) nd ET-1 (d, e, f) in norml pncres nd pncres from non-smoking nd smoking CP ptients with dibetes. IL-6: () (210 ): No immunohistochemicl rection in the cinr cells. (b) (210 ): A strong expression in cinr, pncretic duct nd islet cells. (c) (210 ): A very strong rection in cinr nd moderte rection in pncretic duct cells. ET-1: (d) (210 ): No nd wek immunohistochemicl rection in the cinr cells. (e) (175 ): A strong immunohistochemicl rection in islet nd cinr cells. (f) (137 ): A strong nd very strong rection in cinr nd pncretic duct cells (rrows red: pncretic islets; blue: cinr cells; green: pncretic duct cells; blck: fibrous lesions in the sublyer). lipse ctivity were noted (Tble 8). A strongly positive correltion between cotinine nd cretinine nd BUN concentrtions (r = 0.83; r = 0.86, p < 0.05) nd negtive correltion between cotinine nd the vlue of egfr (r = 0.73, p < 0.05) were found in smoking CP ptients with dibetes. Histologicl dignosis of pncres Norml tissue. The histopthologic exmintion confirmed the regulrity of the pncretic exocrine nd endocrine cells. Pncretic islets were clerly seprted. There were no chnges indicting the existence of chronic inflmmtion, tht is, no clcifiction or fibrosis. In the four smples collected from helthy subjects, lck of or wek immunohistochemicl rection to IL-6 nd ET-1 ws observed in the pncretic islet nd exocrine cells (Tble 9). The nlysis of IL-6- nd ET-1-immunostined sections of the norml, semiquntittive digitl imge ws positive for the pncretic islet nd cinr cells (81.5 ± 13.5 nd ± 23.5 AU, respectively) (Figure 1() nd (d)). Isolted islet cells clerly

10 252 Dibetes & Vsculr Disese Reserch 15(3) Tble 9. Prmeters immunostining of the pncretic tissues control groups nd non-smoking nd smoking CP ptients with dibetes. Vrible Control group CP ptients with dibetes Non-smoking Non-smoking Smoking IL-6 Men ± SD 81.5 ± ± ± Medin ,b Min. Mx ET-1 Men ± SD ± ± ± Medin ,b Min. Mx Insulin Men ± SD ± ± ± 17.2 Medin ,b Min. Mx Glucgon Men ± SD ± ± ± 45.2 Medin ,b Min. Mx IL-6: interleukin-6; ET-1: endothelin-1; CP: chronic pncretitis; SD: stndrd devition. p < 0.05, when compred to control group. b p < 0.05, when compred to non-smoking ptients with dibetes. showed strong nd very strong expression of insulin in the tissue of the pncres on their whole surfce (Figure 2()). The nlysis of the insulin-immunostined sections of the control tissues semi-quntittive digitl imge resulted in very intense positive identifiction of bet cells (282.5 ± 53.3 AU). In the control pncres, specific response to glucgon ws observed in cells loclized minly t the periphery of the pncretic islets with single cells in the centrl prt of the islet (Figure 2(d)). In the control group, the intensity of the immunohistochemicl rection rnged from 87.4 to AU. The glucgon expression ws bout two times lower thn tht of insulin (Tble 9). Non-smoking nd smoking CP ptients with dibetes. The histopthologic dignosis confirmed the occurrence of chrcteristics of the inflmmtory process in ll the tissues of CP ptients. In the exocrine nd endocrine prts of the pncres, fibrous connective tissues were observed. In ll sections tken from non-smoking nd smoking ptients with CP nd dibetes, much stronger immunohistochemicl rection to IL-6 nd ET-1 in the cinr, islet nd ductl cells ws found when compred to the control sections. Significnt differences were detected between nonsmoking nd smoking ptients with dibetes regrding the IL-6 nd ET-1 stining intensity in the pncretic tissue (Tble 9). In smoking CP ptients with dibetes, fibrosis nd progressive trophy of cells nd functionl defect of these cells ws observed, chrcterized by the much stronger immunohistochemicl rection of IL-6 (diffused nd focl) reltive to control nd non-smoking ptients with CP. Interestingly, the highest IL-6 concentrtions were detected in the plsm of smoking ptients with dibetes. A less diffuse nd less intense stining pttern ws seen in the bet cells of specimens retrieved from non-smoking nd smoking CP ptients with dibetes compred to the control. Finlly, in specimens of smoking CP ptients, stining in the bet cells ws mrkedly decresed when compred with both the norml pncres from the control nd the pncres from non-smoking CP ptients with dibetes. There ws sttisticl decrese in the immunohistochemicl rection for insulin in specimens from non-smoking nd smoking ptients with dibetes compred with the control. There ws mrked decrese in the number of bet cells nd the intensity of insulin in tissues from smoking CP specimens with dibetes compred to non-smoking CP specimens with dibetes nd the controls (Tble 9). Compred to helthy subjects, the wekest rection for glucgon occurred in the pncres of the group of smoking CP ptients with dibetes (Figure 2(f)). A comprtive nlysis between the intensity of the immunohistochemicl response to glucgon in non-smoking nd smoking CP ptients with dibetes not showed significnt differences. On the other hnd, significnt decrese in the intensity of the immunohistochemicl rection for glucgon in smoking dibetic ptients ws observed compred to non-smoking dibetic ptients (Tble 9). Discussion Dibetes is one of the most common metbolic disorders nd the resons underlying its development re genetic, environmentl nd lifestyle-relted. Mny epidemiologicl studies indicte tht cigrette smoking is n independent risk fctor for type 2 dibetes. 30,34 36

11 Śliwińsk-Mossoń et l. 253 Figure 2. Immunohistochemicl locliztion of insulin (, b, c) nd glucgon (d, e, f) in norml pncres nd pncres from nonsmoking nd smoking CP ptients with dibetes. Insulin: () (210 ) A strong nd very strong rection in islet B cells. (b) (137 ) A strong rection in B cells of dmge islet. (c) (210 ) A wek immunohistochemicl rection in the B cells in dmge islet. Glucgon: (d) (210 ) A strong rection in islet A cells. (e) (265 ) A diffuse strong nd moderte rection in A cells of dmge islet. (f) (210 ) A diffuse nd moderte immunohistochemicl rection of glucgon in of dmge islet (rrows red: pncretic islets; blue: cinr cells; green: pncretic duct cells; blck: fibrous lesions in the sublyer). It ws found tht cigrette smoking cn lso be n independent risk fctor for CP, mjor cuse of T3cDM. 37 Ech cigrette smoked leds to n inflmmtory response in the body nd if continued, CP develops. Becuse higher levels of inflmmtory mrkers (CRP nd IL-6) herld the development of dibetes 19 nd CP, 38 smoking my lso contribute to the development of dibetes by incresing levels of inflmmtory mrkers. 18 The presented studies showed tht IL-6 concentrtion in plsm ws significntly higher in non-smoking nd smoking CP ptients with dibetes thn in the non-smoking control (Tble 2). There ws lso sttisticlly higher IL-6 concentrtion in smoking ptients with dibetes compred to non-smoking ptients with dibetes. But the highest level of IL-6 ws found in smokers with dibetes. In smoking CP ptients with dibetes, fibrosis, progressive trophy of cells nd functionl defect of these cells chrcterized by much stronger immunohistochemicl rection of IL-6 (diffused nd focl) were observed reltive to the controls nd to non-smoking ptients with dibetes. The highest intensity

12 254 Dibetes & Vsculr Disese Reserch 15(3) of immunostining for IL-6 in smoking CP with dibetes coincided with the highest mrked cytokine concentrtions in the blood of these ptients. These findings re consistent with erlier studies. 18 A drstic increse in the level of the investigted cytokine in smoking ptients points to proinflmmtory effect of cigrette smoke. Experimentl rts showed significntly incresed levels of serum IL-6 s consequence of oxidtive stress following 12-week exposure to cigrette smoke. 37 Cigrette smoking is fctor tht triggers inflmmtion in the pncres nd induces fibrosis of the orgn, s evidenced by the increse in plsm IL-6 concentrtion, the increse in IL-6 expression in the dmged tissue of smoking ptients (Figure 1(c)) nd the strongly positive correltion between the concentrtion of cotinine ( nicotine metbolite) nd the IL-6 concentrtion (Tble 7). According to recent hypothesis, n inititing event is required for the development of CP; it is first AP until ctivtion of pncretic stellte cells (PSCs). The cigrette smoke components ctivte the nti-inflmmtory response (with the relese of pro-inflmmtory cytokines IL-6) nd stimulte PSCs to produce n extrcellulr mtrix, minly collgen, fibronectin nd proteoglycns. Orgn fibrosis comes s result of continuous inflmmtion nd continuous ctivtion of PSCs. PSCs trnsformtion into myofibroblsts, tht is, ctive forms cpble of producing n extrcellulr mtrix nd vrious dhesion molecules, lso occurs in response to vrious inflmmtory cytokines nd chemokines such s tumor necrosis fctor lph (TNFα), IL-1β, IL-6, IL-8; the growth fctors trnsforming growth fctor bet 1 (TGF-β1) nd pltelet-derived growth fctor (PDGF) relesed by both pncretic cells (dmged prenchym cells) nd by inflmmtory cells Potentil prcrine ctivtors of PSCs cting in vivo lso include rective oxygen species nd ET-1. Active PSCs cn produce utocrine gents such s growth fctors (PDGF, TGF-β1), cytokines (IL-1, IL-6, tumor necrosis fctor-relted poptosis-inducing lignd (TRAIL)), ctivin-a (which increses collgen production nd increses TGF-β1 expression nd secretion) nd ET-1 (which cn stimulte the migrtion nd contrction of PSCs). 40 ET-1 is hormone with strong vsoconstrictor properties, secreted in excessive mounts by dmged endothelil cells. 16,42 Borissov et l. 42 demonstrted tht smoking increses the ET-1 level in plsm. ET-1 reduces peripherl glucose utiliztion 42,43 probbly by inducing vsoconstriction nd subsequent tissue hypoxi. Tobcco smoke hs long been recognized s fctor tht is n impediment to norml endothelil functioning of vessels, including smll vessels in the pncres. The lst study hs demonstrted tht the significntly higher concentrtion of ET-1 in the plsm of smoking helthy individuls nd in ptients with pncretitis, when compred with non-smokers, is consistent with the strong degree of ET-1 expression in pncretic tissue. 16,42 The current study confirmed the previous results. Significnt differences were found between ET-1 levels in the plsm of the non-smoking nd smoking control groups. An increse in the level of this peptide in the smoking control group nd in CP ptients is evidence of the impct of cigrette smoking on the functioning of the blood vessel endothelium. This thesis is confirmed by the strongly positive correltion between the cotinine concentrtion nd the ET-1 concentrtion (Tble 7). The highest plsm concentrtions of ET-1 were observed in non-smoking nd smoking ptients with dibetes mellitus (Tble 2). Hrris et l. 44 lso demonstrted significnt correltion between plsm ET-1 levels nd dibetic complictions. The high level of ET-1 in ptients with dibetes cn be the result of n imblnce between the fctors secreted by the endothelium responsible for the mintennce of norml vsculr wll tension (reduction of NO synthesis in fvour of ET-1). This phenomenon is prticulrly evident in the group of smokers, where in ddition to the developing stte of insulin resistnce, the substnces contined in the cigrette smoke cuse the decomposition of nitric oxide, leding to excessive production of ET The immunohistochemicl ET-1 rection ws negtive or wekly positive in histologic specimens from the control group, wheres in smoking ptients with dibetes, it ws strong or very strong in cinr, ductl nd islet cells. The difference in the intensity of immunostining ws confirmed by densitometric nlysis. Sttisticlly higher ET-1 immunorectivity ws found in non-smoking nd smoking ptients with dibetes compred to the control group (Tble 9). In ddition, the degree of immunorectivity of ET-1 is lso dependent on the severity of fibrogenesis of the study tissues (Figure 1(e) nd (f)). ET-1 is n ctivtor of PSCs for collgen synthesis, suggesting how importnt this hormone is in the development of CP. ET-1 lso strongly stimultes expression of IL-6, which, in ddition to stimulting PSCs, is primrily pro-inflmmtory cytokine. In ddition, strong correltion ws observed between ET-1 nd IL-6 in smokers, which confirms the reltionship between these two fctors. It follows from this tht ET-1 enhnces the locl inflmmtory response in the pncres. Due to the strong vsoconstrictor ET-1, high level of the peptide contributes to locl tissue ischemi, leding to dmge in pncretic tissue. Ongoing inflmmtion, which trnsltes into some degree of tissue destruction, leds to permnent histologicl chnges tht prevent proper functioning of the orgn, mong other things, nd ffect the development of dibetes. It ws lso observed tht s the development of pncretic fibrosis occurs in ptients with CP, the rtio of bet cells to lph cells decreses. 12 In the present study, lower number of bet nd lph cells in the islets of Lngerhns were found in smoking ptients with dibetes compred to the controls (Figure 2(c) nd (f)). The lowest

13 Śliwińsk-Mossoń et l. 255 intensity of immunostining for insulin nd glucgon ws observed in smoking CP ptients with dibetes (Tble 9). Tissues of these ptients were chrcterized by significnt reduction in the strom. In ddition, severe inflmmtion nd multifocl intr- nd inter-lobulr fibrosis nd severely dmged islets were observed in non-smoking nd smoking ptients with dibetes (Figure 2(b), (c), (e) nd (f)). The pthogenesis of T3cDM is ultimtely due to decresed insulin secretion cused by both reduction in the number of islets nd their functionl cpcity s consequence of extensive fibrosis nd sclerosis. This theory of T3cDM ws lso described by other reserchers. 46,47 The mechnism by which the scrring of pncretic tissue induces insulin deficiency cn be explined by the ntomicl nd functionl interply between the pncretic islets nd cinr tissue. In erlier studies, it ws found tht prolonged smoking destroys the exocrine prt of the pncres (i.e. the pncres decreses morphologiclly), while the pncretic islets become restricted nd then destroyed. 12,15 18 The studies conducted by Srles et l. 48 showed tht ddicted smokers re more predisposed to pncretic clcifiction thn non-smokers. In the present study, the significntly lower levels of insulin nd the higher blood glucose levels in smoking ptients with CP in comprison to the controls coincided with the progression of chnges in the structure nd dysfunction of the endocrine orgn. The suppressive effect of cigrette smoking on insulin secretion hs been reveled in hevy mle smokers. 49 In recent yers, it hs lso been shown tht smoking cigrettes ffects the production nd secretion of other pncretic hormones such s glucgon, somtosttin nd pncretic polypeptide. Although the precise role of cigrette smoking in the pthogenesis of the decresed excretion of hormones in CP still remins uncler, the presented dt provide evidence tht tobcco smoking my be regrded s mjor custive fctor of endocrine disorders in CP. There is lso morphologicl evidence tht indictes tht pncretic exocrine functioning my be influenced by the pncretic endocrine hormones. Insulin hs trophic effect on the exocrine pncres, especilly on the peri-insulr cini. Progressive dmge to the pncretic cinr cells is seen in insulin deficiency. The pncretic exocrine tissue in dibetic ptient becomes fibrosed nd shows reduced response to the hormonl stimultion. 50 This mechnism hs been confirmed in the present study. In the histopthologicl exmintion of pncretic tissues from smoking dibetic ptients, the exocrine nd endocrine prts of the pncres were observed to hve been severely destroyed (Figure 2(c) nd (f)). Low serum mylse nd lipse ctivity observed in dibetes type 2 my reflect the impired exocrine endocrine interctions of the pncres. 22,50 However, only few clinicl studies hve ddressed this complex reltionship. In our study, we found significntly higher mylse nd lipse ctivity in dibetic ptients (especilly in smokers) compred to those in the non-smoking control. Another study hs reveled tht the differences in mylse ctivity in both the serum nd urine of smoking ptients compred to non-smoking ptients with pncretitis my prove the significnt influence of tobcco smoking on the exocrine functioning of the pncres. 51 Also, severl niml nd cellulr studies hve tried to find out the pthogenic fetures nd the underlying mechnisms which link the islet cells nd the cinr cells. 50 But the estimtion of the serum mylse ctivity, long with the blood glucose to ssess this reltionship, consolidtes its clinicl relevnce in humns. There is evidence tht the pncretic hormones insulin nd glucgon influence enzyme synthesis nd relese in the exocrine pncres. Insulin hs trophic effect on the cinr cells, wheres glucgon hs been found to hve n inhibitory influence on the exocrine secretions; moreover, there is decrese in the sensitivity of the dibetic pncretic cini to secretgogues. The insulin deficiency nd the glucgon excess in dibetes ffect the norml tmosphere of the pncres, thereby decresing the totl volume, the mylse secretions nd the bicrbonte content of its exocrine secretions. 50 In our work, we showed simultneous decrese in insulin nd glucgon secretion from the pncretic cells of the islets due to their destruction. Our results suggest tht the low level of glucgon did not inhibit the secretion of pncretic enzymes in smoking ptients with dibetes. On the other hnd, the inflmmtory process stimulted by the components of cigrette smoke destroys the cinr cells, which results in the incresed secretion of enzymes. Confirmtion of this cn be found in the strongly positive correltion between cotinine concentrtion nd mylse nd lipse ctivity in smoking ptients with dibetes. Severl studies evluting the pncretic exocrine function in smoking ptients with CP showed significnt increse in the ctivity of the pncretic enzymes. 10,23 26,51 In ptients with type 1 dibetes, n increse in serum mylse levels ws found. In type 1 dibetes, the clinicl nd the pthologicl pictures re different, with the presence of insulitis nd the diffuse lymphocytic infiltrtion of the exocrine pncres. Also, the difference in serum mylse ctivity compred to tht in the controls ws more pronounced in ptients with type 1 dibetes who presented with cute fulminnt ketocidosis In ptients with T3cDM, the chnge in glucose metbolism rnges from only mild impirment to severe form which is chrcterized by frequent episodes of hypoglycemi, commonly referred to s brittle dibetes. 1 It is generlly reported tht T3cDM is difficult to control, lthough there re only few older studies on this topic. 55,56 The hypotheses put forwrd for the difficulty in chieving good glycemic control in ptients with T3cDM