The effect of insulin treatment and of islet transplantation on the resistance artery function in the STZ-induced diabetic rat

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1 Bridsh Joumal of Phamaology (1996) 119, B 1996 Stokton Press All rights reserved /96 $12. The effet of insulin treatment and of islet transplantation on the resistane artery funtion in the STZ-indued diabeti rat tkatherine M. Heygate, *Joanne Davies, *Mihael Holmes, *Roger F.L. James & 1'tHerbert Thurston tdepartment of Mediine/*Surgery, Clinial Sienes Building, Leiester Royal nfirmary, PO Bo 65, Leiester L2 7LX. 1 This study was designed to investigate the influene of insulin treatment and islet transplantation on the smooth musle ontratility and endothelium-dependent and independent relaation of resistane arteries in the hemially indued streptozotoin (STZ) diabeti rat after 6-8 weeks, and weeks of diabetes, ompared to non-diabeti age-mathed ontrols. 2 The morphology, and ontratile responses to high potassium physiologial salt solution (KPSS), KPSS ontaining 1- M noradrenaline (NAK), and onentration-response urves to noradrenaline (NA) of mesenteri resistane arteries were reorded, along with the endothelium-dependent relaation responses to aetylholine (ACh) and bradykinin (BK), and endothelium-independent relaation to sodium nitroprusside (SNP). Conentration-response urves were then repeated in the presene of a nitri oide synthase inhibitor, NG-nitro-L-arginine (L-NOARG). 3 nsulin-treated diabeti rats in the 12 week study demonstrated enhaned vasular ontratility to KPSS, NAK and NA, ompared to age-mathed non-diabeti ontrols. 4 nubation with L-NOARG resulted in both a signifiant inrease in maimum ontratile response, and sensitivity (pd2) to NA in the untreated diabeti group (6 weeks). A signifiant shift in sensitivity was also seen in the insulin-treated diabeti group. n the 12 week study, inubation with L-NOARG resulted in an inreased maimum ontratile response and sensitivity to NA in the insulin-treated diabetis. An inrease in sensitivity was also observed in the untreated diabeti group. 5 ndothelium-dependent relaation to ACh was signifiantly augmented in the untreated diabetis (6- weeks), ompared to the ontrol group. n the 12-week study, relaation to both ACh and BK was not signifiantly different in any of the eperimental groups when ompared to the sham-operated nondiabeti ontrols. 6 nubation with L-NOARG resulted in a signifiant attenuation of the maimum relaation response to ACh and BK in all of the eperimental groups, in the 6- and the 12-week study. 7 There was no signifiant differene in the maimum relaation response or sensitivity to sodium nitroprusside between the diabeti groups and their age-mathed ontrols in either the 6-week or the 12- week study. 8 The results of this study suggest an enhaned release of nitri oide in the early stages of diabetes, whih is more evident in the untreated diabeti rats than the insulin treated, and appears to normalize as the duration of diabetes progresses. This study also shows that the alteration in vasular reativity of the resistane arteries an be restored to within normal limits by the transplantation of islets of Langerhans, and that islet transplantation is an effetive strategy in the orretion of the metaboli abnormalities assoiated with insulin-dependent diabetes. Keywords: Mesenteri resistane arteries; endothelium; nitri oide; endothelium derived relaing fator (DRF); streptozotoin; islet transplantation; aetylholine; bradykinin; sodium nitroprusside; N-nitro-L-arginine (L-NOARG) ntrodution nsulin-dependent diabetes mellitus (DDM) haraterized by poor glyaemi ontrol leads to preapillary vasodilatation with inreasing blood flow and pressure (Jaap & Tooke, 1995), and this may be the initiating fator in diabeti miroangiopathy. ndeed inreased regional blood flow and pressure has been shown in the retina (Kohner et al., 1975), kidney (Ditzel & Junker, 1972), and forearm (Halkin, 1991) of diabeti patients. The endothelium plays an important role in modulating the vasular tone and reativity of blood vessels (Furhgott & Zawadaski, 198), by the release of vasoative agents inluding prostaylin, endothelium derived relaing fator (DRF), and the vasoonstritor peptide, endothelin-1 (Yanagisawa et al., 1988); and it has been suggested that abnormal endothelial funtion may be a ontributory fator to large and small vessel disease in diabetes mellitus (Porta et al., 1987). Moreover, vas- Author for orrespondene. ular smooth musle from hemially indued diabeti animals appears to be more sensitive to vasoonstritors and less so to vasodilators (Gebremedhin et al., 1988; Mayhan et al., 1991). Alteration of vasular reativity and impaired endotheliumdependent relaation has been demonstrated in onduit arteries from a number of eperimental animal models of diabetes. Attenuated responses to endothelium-dependent vasodilators have been observed in both the spontaneously diabeti Bio Bred (BB) rat (Durante et al., 1988), and the hemially indued diabetes model (Oyama et al., 1986; Mayhan et al., 1991; Cameron & Cotter, 1992). On the other hand, others have reported no hange in the response to endothelium-dependent vasodilators (White & Carrier, 1986; Head et al., 1987; Mulhern & Doherty, 1989). Studies of the vasular responses to noradrenaline (NA) have also yielded inonsistent results, showing both an inrease in sensitivity to noradrenaline (MaLeod & MNeill, 1985; Cohen et al., 199), and attenuated responses (Pfaffman et al., 1982; Head et al., 1987).

2 496 K.M. Heygate et al slet transplantation in STZ rats There have been omparatively few studies of endothelial funtion in resistane arteries whih reflet the true nature of the mirovasulature, and play an important role in the loal ontrol of blood flow and pressure. However, impaired endothelium-dependent relaation has been desribed in resistane arteries from hemially indued diabeti animals (Taylor et al., 1992; 1994a), and also in the diabeti BB rat (Heygate et al., 1995). Similarly, endothelial dysfuntion has been onfirmed in studies of human Type 1 (de Tejada et al., 1989; MNally et al., 1994). These observations partiularly in the MNally study, support the suggestion that alterations in vasular reativity may preede the strutural hanges whih are the hallmark of established diabeti miroangiopathy. The introdution of insulin earlier this entury removed deompensated diabetes from being the ause of death in DDM. Subsequently, marovasular and mirovasular disease have been the main auses of morbidity and mortality. mproved glyaemi ontrol, with intensive insulin regimes have redued the inidene of diabeti miroangiopathy (DCCT, 1993). However, optimal diabeti ontrol remains a pratial impossibility in many diabetis, sine even the most metiulous form of insulin therapy annot aurately mimi the more preise and subtle blood gluose ontrol afforded by a working panreas. At present this an be ahieved only by the transplantation of islets of Langerhans, or whole panreas (Orloff et al., 1987; 1988) to replae the funtion of the damaged beta ells. n the rat, islet ell transplantation has been shown to reverse metaboli abnormalities, and also has benefiial effets on seondary ompliations assoiated with the diabetes (Mauer et al., 1974; Shmidt et al., 1983; Britland et al., 1991). However, to date no studies have investigated whether improved diabeti ontrol following islet transplantation ould ameliorate diabetes-indued endothelial dysfuntion in the miroirulation. Aordingly, we have investigated the effets of hemially indued diabetes on resistane artery ontratile and relaation funtion at si and twelve weeks in rats after the indution of diabetes and observed the response to long-term treatment with either subutaneous insulin injetions, or intra-portal islet transplantation. Methods Female WAG/Leiester rats (8-9 weeks old and weighing approimately 15 g) from an inbred olony maintained at the Biomedial Servies Department of Leiester University, were used for these studies. n the first study of 6 weeks, rats were randomly divided into three eperimental groups: diabeti, ontrol, and insulin-treated diabeti. Diabetes was indued by a singly femoral vein injetion of streptozotoin (STZ) 55 mg kg-' dissolved in 1 ml of itrate buffer (.1 M solution, ph 4.2) under light anaesthesia. Rats alloated to the ontrol group reeived the buffer solution alone. Blood gluose levels were measured on a daily basis with a blood gluose test strip (BM-Test 1-44) and a Reflolu meter (Boehringer Mannheim). Rats with blood gluose readings >2 mmol were onsidered to be diabeti and either reeived insulin treatment or reeived no insulin and therefore remained hyperglyaemi for the duration of the study. The insulintreated group reeived daily subutaneous injetions of.8 u insulin 1 g-' body weight (Ultralente, Novo Nordisk). The dose of insulin was then adjusted in order to maintain euglyaemia aording to the following dosing regime: blood gluose mmol 1-' the previous day's insulin dose was halved; blood gluose 5-9.9mmol 1, no adjustment; blood gluose >1m m -oa',dose was inreased by.15 u insulin. A seond, etended study was performed to investigate the effet of weeks of diabetes on the mirovasulature. This study onsisted of four eperimental groups: group 1 animals were injeted with STZ and subsequently treated with insulin; group 2 were injeted with STZ but did not reeive insulin therapy; group 3 were injeted with STZ and were treated with insulin for days, before reeiving an intraportal isograft of freshly isolated panreati islets (approimately 1-12 per reipient) from syngenei WAG donor rats (3 donors reipient); and group 4 omprised nondiabeti ontrol rats whih underwent a sham islet transplant operation, whereby only the vehile was injeted into the hepati portal vein. Panreati islets were isolated from male WAG/Leis rats whih had been anaesthetized by inhalation of a miture of halothane, nitrous oide and oygen. During this proedure, the panreati dut was anulated, and the panreas distended with ollagenase. slets were then isolated one ollagenase digestion had taken plae (Sutton et al., 1986), and separated by bovine serum albumin gradient entrifugation (Lake et al., 1987). slets were inubated in a ulture medium overnight at 37 C, prior to hand-piking and transplantation. Reversal of diabetes in the islet-transplanted group was onfirmed by three onseutive blood gluose readings being onsistently below 1 mmol 1-'. Transplanted animals were ulled if blood gluose readings were > 2 mmol l- l. n both the si and twelve week studies rats were fed standard rat how, allowed aess to water ad libitum, and were weighed daily. Systoli blood pressure was measured by the tail uff method using plethymography and a physiograph reorder (TT system), one week before the animal was due to be killed. On the day they were killed insulin-treated diabeti rats did not reeive their usual morning injetion of insulin. Rats were ulled by stunning followed by ervial disloation. The mesentery and small intestine were removed and plaed in a small beaker ontaining old physiologial salt solution (PSS). The hind limbs were removed at the pelvi joint for tibia length measurement, and the heart eised from the thorai avity, blotted dry, and weighed. Rats that had reeived transplanted islets also had their liver removed and fied in formalin prior to histologial eamination and staining for the presene of islets. Preparation of vessels Third order vessels (internal diameter <35 gm) were disseted from the mesenteri bed at a point approimately 5 m from the posterior end of the olon. Two vessel segments of 2 mm in length were mounted in a Mulvany myograph on two 4 gm steel wires. One wire was attahed to a fore transduer, and the other to a mirometer; this permitted wall tension measurements to be taken at a predetermined internal diameter. Dissetion and mounting of the vessels was arried out in old (4 C) PSS. perimental protool and solutions After mounting, the vessels were left to equilibrate for 3 min in PSS at 37 C, gassed with 5% CO2 and 95%2 to maintain a ph of 7.4 (PSS omposition (mm): NaCl 118, NaHCO3 25, KCl 4.5, KH2PO4 1., CaCl2 2.5, MgSO4 1. and gluose 6). A high potassium physiologial salt solution (KPSS) was made by replaing NaCl with KCl. Morphologial measurements of media thikness and internal diameter were then made through glass windows in the myograph bath using a water immersion light mirosope and a pre-alibrated filar mirometer eyepiee with a resolution of 1 gm. The passive tension and internal irumferene of the vessels were determined by a proess known as normalisation. ah vessel underwent a series of strethes by adjusting the mirometer. The arteries were then set to.9 L(1) where L(loo) is the internal irumferene the artery would have in vivo when relaed under a transmural pressure of 1 mghg (Mulvany & Halpern, 1976). After ompleting the normalisation proedure, the vessels were maintained in PSS for 1 h before starting the eperimental protool. During this time the bathing medium was hanged at 2 min intervals. The eperimental protool was initiated with three stimulations of KPSS, followed by one stimulation of KPSS on-

3 K.M. Heygate et al taining i' M NA. Contrations were maintained for 2 min before rinsing with PSS to base line. After ompleting the ativation proedure, the vessels were rinsed three times and left to reover at baseline for 15 min. Then a umulative onentration-response urve to NA (1-8-3 i-' M) was performed in the presene of 1-6 M oaine, whih was added 2 min prior to the ontration urve to blok neuronal reuptake of noradrenaline. The vessels were then rinsed to baseline and allowed to reover for 15 min. The arteries were then maimally ontrated with a bolus dose of NA-5 M and one a plateau had been reahed, the relaation response to aetyholine (ACh l-'- i-' M) observed. Fifteen minutes after washout, arteries were maimally ontrated again with NA (1'- M), and were subsequently relaed with umulative doses of bradykinin (BK, 1-9- O- M). The vessels were brought to base line with three rinses of PSS, and inubated with PSS ontaining i' M NG-nitro- L-arginine (L-NOARG) for 1 h. The NA, ACh, and BK onentration-response urves were repeated in the presene of L- NOARG. Finally, the vessels were maimally ontrated with NA (l-' M) and relaed with umulative onentrations of sodium nitroprusside (SNP M). At the end of eah study, all vessels were rinsed in alium-free PSS before fiing overnight in 1% formalin for histologial eamination. Drugs All drugs were freshly prepared on the day of study and dissolved in distilled water, with the eeption of the streptozotoin whih was made up in itrate buffer. The STZ was dissolved immediately prior to injetion in saline aidified to ph 4.2 with itri aid (.1 M). Streptozotoin, noradrenaline hydrohloride, oaine hydrohloride, aetylholine hloride, bradykinin, sodium nitroprusside, and L-NOARG were obtained from Sigma Chemial Company, Poole, Dorset, UK. Data and statistial analysis Results are epressed as the mean + standard error of the mean (s.e.mean). Contratile responses to NA are epressed as ative tension (mn mm-'), whih is alulated from the measured fore divided by twie the vessel length. Relaation responses to ACh, BK, and SNP are epressed as a perentage deline of the maimum ontratile response. The sensitivity (C5) i.e. the onentration of the drug required to produe 5% of the maimum response, was alulated by omputer-aided urve fitting programme for NA, ACh, BK and SNP onentrationresponse urves. The C5 values were then onverted to -log C5s or pd2s, and parametri statistial tests were applied. Multiple omparisons were firstly made between means of the eperimental groups by a one-way analysis of variane (ANOVA). f P<.5, individual pairs of means were then ompared by the Bonferroni multiple omparisons test. Comparisons within groups were determined by the Student's paired t test. slet transplantation in STZ rats Results Morphology There were no signifiant differenes in media lumen, media volume, media/lumen ratio or vessel diameter between the eperimental groups in the si week or the twelve week study (Table 1) Physial harateristis of diabeti animals (Table 2) Si week study nsulin-treated rats were signifiantly heavier than the untreated diabeti rats (194 g±6 versus 174 g±4, P<.5, n=8), although they were not signifiantly heavier than the ontrol non-diabeti rats ( , n = 5), and the body weight of the untreated diabeti rats was not signifiantly different from that of the ontrols. Heart weight/tibia ratio was signifiantly higher in diabeti rats treated with insulin ompared to non-diabeti ontrols ( versus , P<.5). Blood gluose levels were signifiantly greater in the untreated diabeti group ( mmol 1'-, n = 8) ompared to the ontrol group ( mmol -', n = 9 P<.1), or those reeiving insulin ( mmol, n = 7 P<.1). The diabeti animals reeiving insulin also had signifiantly higher blood gluose levels than the ontrol animals (P<.1). There was not signifiant differene between blood pressure of the eperimental groups. Table 2 Physial harateristis of STZ-indued diabeti rats, insulin-treated STZ rats, islet-transplanted rats and their age-mathed ontrols 6-wk Control (n = 5) 6-wk Diabeti 174+4** (n=8) 6-wk Diabeti insulin 12-wk Control (n= 1) 12-wk Diabeti Heart wt. Blood pressure Weight (g) /tibia (mmhg) *tt (n = 9) 12-wk Diabeti insulin slet-transplanted * (n =7) * (n = 6) (n= 1) ' (n =8) (n = 7) (n = 6) (n = 6) (n = 4) (n = 7) *P<.5 (unpaired t test versus ontrol); **P<.1 (unpaired t test diabeti versus diabeti + insulin); tp <.5 (unpaired t test versus ontrol, diabeti + insulin, and transplanted animals); ttp <.1 (unpaired t test diabeti versus transplant) 497 Table 1 Morphologial measurements of vessels of STZ-indued diabeti rats, insulin-treated diabeti rats, islet-transplanted rats and age-mathed ontrols 6-wk Control (n = 9) 6-wk Diabeti 6-wk Diabeti + insulin 12-wk Control (n = 12) 12-wk Diabeti (n = 9) 12-wk Diabeti + insulin slet-transplanted Diameter (gm) Media thikness Media Vol. Medial lumen

4 498 K.M. Heygate et al Twelve week study The body weight of the untreated diabeti animals was signifiantly lower than that of the remaining three eperimental groups (152 g + 7 versus ontrol 214 g + 4, P<O.OO1; insulin-treated diabetis 21 g± 14, P<.1; and transplanted animals 187 g ± 7, P <.1). The transplanted animals were also signifiantly lighter than the ontrol group (187 g + 7 versus 214 g ± 4, P <.1). The untreated diabeti animals developed atarats in both eyes by the final week of the twelve week study. Heart weight/tibia ratio was signifiantly different in the untreated diabeti animals ompared to the other eperimental groups ( versus ontrol , P<.5; insulin-treated diabetis , P<.1; and the transplanted group , P<.5). At the end of the study, blood gluose levels were signifiantly greater in the untreated diabeti group ( mmol 1-', n = 8, P<.1) ompared to those reeiving insulin ( mmol -l1, n=8), and the ontrol animals ( mmol -', n = 6). Blood gluose was signifiantly lowered from hyperglyaemi levels to the normal range approimately ten days after islet transplantation (pretransplant: mmol ' versus post-transplant: mmol -', n=8, P<.1). There was no signifiant differene between the blood pressure of the eperimental groups. Contration studies Si week study n the si week study, there was no signifiant differene in maimum ontratile response to KPSS (ontrol; mn mm-', n = 9; untreated diabeti: mn mm-', n =8; insulin-treated diabeti: mn mm-', n =8), KPSS ontaining NA -5 M (NAK) (ontrol: z 1)._ H ~ o- lo io-6 * Figure 1 Contratile response to noradrenaline of rat mesenteri resistane vessels (12 week study) from sham-operated non-diabeti rats (), insulin-treated diabeti rats (Ol), untreated diabeti rats (A), and islet-transplanted rats (V). Points show mean+s.e.mean. slet transplantation in STZ rats mn mm-', n=9; untreated diabeti mn mm-', n =8; insulin-treated diabeti: mn mm 1, n = 8), or to NA between the groups (ontrol: mn mm-l; untreated diabeti: mn mm'; and insulin-treated diabeti: mn mm The NA sensitivity (pd2) was also unhanged between the groups (ontrol: ; untreated diabeti: 5.74±.1; and insulin-treated diabeti: ). Twelve week study n the twelve week study, both the ontratile responses to KPSS, and NAK+ were signifiantly inreased in the insulin-treated diabeti group (KPSS: mn mm-', P <.5; and NAK: mn mm-', P<.5, n=8) ompared to the ontrols (KPSS: mn mm-'; NAK: , n= 12), untreated diabetis (KPSS: mn mm'; NAK: mn mm 1, n = 8), and the transplanted animals (KPSS: mNmm-'; NAK mnmm-', n=8). Also, the maimum ontratile response to NA was signifiantly greater in the insulin-treated group ( mn mm-', P<.5, n=7) ompared to the other three eperimental groups (ontrols: mn mm-'; untreated diabeti: mn mm-1; transplants: mn mm-1), although there was no hange in the NA sensitivity (insulin-treated: ; ontrol: ; untreated diabeti: ; transplants: ) (Figure 1, Table 3). ffets of L-NOARG on the NA ontratile response and sensitivity Si week study nubation with the nitri oide synthase inhibitor, L-NOARG resulted in a signifiant inrease in maimum NA ontratile response in the untreated diabeti group (P<.5), along with a signifiant shift in the sensitivity to NA (pd2) (Figure 2a). There was no signifiant inrease in either the maimum ontratility or sensitivity to NA in the ontrol group (Figure 2b). The maimum ontratile response also remained unaltered in the insulin-treated diabeti group, although there was evidene of a signifiant shift in sensitivity (P<.5) (Figure 2, see Table 3). The perentage shift after the addition of L-NOARG was also alulated within eah group by the following formula: (pd2 )- (pd2 + L - NOARG) 1 pd2 An analysis of variane (ANOVA) was then arried out between the eperimental groups. As a result, there was no signifiant shift evident between the groups (ontrol: %, n = 9; untreated diabeti: %, n = 8; insulin-treated diabeti: %, n = 8). Twelve week study nubation with L-NOARG resulted in an inrease in both maimum ontratile response and sensitivity to NA in the insulin-treated diabetis (P <.5) (Figure 3a). n ontrast to the findings of the si week study, the untreated diabetis showed no signifiant inrease in ontratility, although an inrease in sensitivity (pd2) was evident (P<.5) Contratile responses and sensitivity to noradrenaline before and after inubation with NG-nitro-L-arginine (L-NOARG) in Table 3 si and twelve week STZ study 6-week Control (n = 9) 6-week Diabeti 6-week Diabeti + insulin 12-week Control (n = 12) 12-week Diabeti 12-week Diabeti + insulin (n = 7) Transplants (n = 7) *P<.5 (paired t test before and after L-NOARG). NA Ma * * L-NOARG pd * * * * L-NOARG

5 K.M. Heygate et al z._4 8) z C. a b / e /f /e /* 4 i ## 4 / slet transplantation in STZ rats (Figure 3b). However, the ontratility and sensitivity in both the ontrol and the islet-transplanted group were not signifiantly different after inubation with L-NOARG (Table 3). Similarly, the shift in sensitivity was alulated for eah group using the aforementioned formula, and an ANOVA was arried out between all of the eperimental groups. This showed that there was no signifiant differene in the perentage shift evident between the eperimental groups (ontrol: %, n=1; untreated diabeti: %, n=8; insulin-treated diabeti: %, n = 7; transplants: %, n = 8). Aetylholine relaation responses Si week study Maimum relaation to the endothelium-dependent vasodilator ACh was signifiantly greater in the un- -; ;;treated diabeti group ompared to the ontrol group (92+1%, n=7 versus 64+1%, n=8, P<.5), although there was no signifiant differene in sensitivity between the groups (ontrol: ; untreated diabeti: ; insulin-treated diabeti: ). Relaation in the insulin treated-diabeti group was not signifiantly greater than that seen in the ontrol group (79+7%, n=8 versus 64+1%) (Figure 4a). nubation with the nitri oide synthase inhibitor L- NOARG aused signifiant attenuation of the maimum relaation response to ACh in all three eperimental groups (P<.5) with the untreated diabeti group displaying the least inhibition with L-NOARG. However, there was no i hange in ACh sensitivity (pd2) in any of the three eperi mental groups (Table 4). z C C a 4 i # * i,... i C b io-4 z._ C n i, * / z _8 o o-5 o Figure 2 Contratile response to noradrenaline of rat mesenteri resistane vessels (6 week study) before and after inubation with NGnitro-L-arginine (L-NOARG) taken from (a) untreated diabeti rats (A); + L-NOARG (A); (b) ontrols (); + L-NOARG (); and () insulin-treated rats ([1); and + L-NOARG (L). Points show means + s.e.mean o_ io-4 Figure 3 Contratile response to noradrenaline of rat mesenteri resistane vessels (12 week study) before and after inubation with NG-nitro-L-arginine (L-NOARG) taken from (a) insulin treated rats (O); + L-NOARG (O); and (b) untreated diabeti rats (A); and + L-NOARG (A). Points show means+s.e.mean.

6 5 K.M. Heygate et at Twelve week study There was no signifiant differene in ACh maimum relaation between the eperimental groups and the sham-operated non diabeti ontrols (untreated diabetis: %, n = 8; insulin-treated diabetis %, n =8; and transplants: %; versus ontrols: %, n = 1) (Figure a slet transplantation in STZ rats 4b). The ACh sensitivity remained unaltered with no signifiant differene between the eperimental groups (ontrol: ; untreated diabeti: ; insulin-treated dia- a L-C 5 - -o b Aetylholine (M) 16 b B1 i 1 7 Bradykinin (M) (a C 4- C) " '" vv l Aetylholine (M) Figure 4 Aetylholine-indued relaation response of rat mesenteri resistane vessels from ontrols (); insulin-treated diabeti rats (O); untreated diabeti rats (A) and islet transplanted rats (V-12 weeks only) in (a) 6 week study and (b) 12 week study. Points show means + s.e.mean Bradykinin (M) Figure 5 Bradykinin-indued relaation response of rat mesenteri resistane vessels from ontrols (); insulin-treated diabeti rats (1); untreated diabeti rats (A) and islet transplanted rats (V-12 weeks only) in (a) 6 week study and (b) 12 week study. Points show means + s.e.mean.

7 K.M. Heygate et al beti: ; transplants: ). nubation with L- NOARG aused signifiant attenuation in the maimum relaation responses in all groups. However, in keeping with the si week study, there was no signifiant hange in sensitivity following the addition of L-NOARG (Table 4). Relaation responses to bradykinin Si week study There was no signifiant differene in maimum relaation response in the diabeti animals ompared to their age-mathed ontrols (ontrol: %, n = 8; untreated diabetis: %, n = 7; insulin-treated diabetis: %, n = 8), although there was a signifiant shift in sensitivity between the insulin-treated diabetis and the ontrols ( versus ) (Figure 5a). A 1 h inubation with L- NOARG, signifiantly inhibited the BK relaation response in all three groups. There was also a shift in sensitivity evident in both the insulin-treated (P =.59), and the untreated dia- -betis (P =.52), although statistial signifiane was not quite reahed (Table 5). Twelve week study There was no signifiant differene between maimum BK relaation response in the eperimental groups ompared to the sham-operated non-diabeti ontrols (untreated: %, n = 8; insulin-treated: %, n = 8; and transplants: , n = 7 versus ontrols: , n = 8). Also there was no hange in the sensitivity between the eperimental groups and the non-diabeti ontrols (ontrols: ; untreated diabetis: ; insulin-treated diabetis: ; transplants: ) (Figure Sb). nubation with the nitri oide synthase inhibitor, L-NOARG, aused signifiant attenuation of the maimum relaation response to bradykinin in all of the eperimental groups, but there was no hange in BK sensitivity after the addition of L-NOARG (Table 5). Relaation responses to sodium nitroprusside Si week study There was not signifiant differene in the maimum relaation response or sensitivity to the endothelium-independent vasodilator, sodium nitroprusside between the diabeti groups and the age-mathed ontrols slet transplantation in STZ rats (ontrols: %, , n = 9; untreated diabetis: 87+3%, , n=8; insulin-treated diabetis: 76+5%, , n = 8), or when the untreated rats were ompared diretly to the insulin treated rats (Figure 6a). Twelve week study The maimum relaation and sensitivity to sodium nitroprusside was similar in all of the eperimental groups (ontrols: 86+3%, , n=8; untreated diabetis: 84 +4%, , n = 8; insulin-treated diabetis: 9+4%, , n = 7; transplants: 87+4%, , n = 8; Figure 6b). Disussion 51 The results of this study support inreased basal and stimulated release of nitri oide in streptozotoin-indued diabeti rats at both si and twelve weeks duration. Thus inubation with the nitri oide synthase inhibitor, NG-nitro-L-arginine (L- NOARG), revealed a signifiant inrease in ontratility and sensitivity in the untreated diabeti vessels at si weeks, with inreased sensitivity also displayed at 12-weeks. nsulin-treated diabeti rats showed inreased sensitivity at both 6 and 12- weeks, with the 12-week insulin-treated diabetis displaying enhaned ontratility to noradrenaline. However, when the ratio of shift in sensitivity to noradrenaline (after inubation with L-NOARG) was alulated, and then ompared between eah of the eperimental groups, no signifiant enhanement of sensitivity to noradrenaline was evident in either the 6 or the 12-week study. Vasular reativity to noradrenaline in the transplanted group remained unaltered, and was not signifiantly different to the ontrols. This is in agreement with the only other transplant study whih has diretly investigated the responses of rat blood vessels to ontratile and relaant agents (Pieper et al., 1995). n ontrast to these findings, some groups who investigated aorti responses to noradrenaline reported that there was no signifiant differene in ontratility in the diabeti vessels (Cameron & Cotter, 1992), espeially when ontratile fore was related to tissue weight (Oyama et al., 1986). However, other studies of rats with hemially indued diabetes have demonstrated an inreased ontratile response to noradrena- Table 4 ndothelium-dependent relaation and sensitivity to aetylholine before and after inubation with NG-nitro-L-arginine (L- NOARG) in si and twelve week STZ study 6-week Control 6-week Diabeti (n = 7) 6-week Diabeti + insulin 12-week Control (n = 1) 12-week Diabeti 12-week Diabeti + insulin Transplants (n = 7) ACh Ma %* 92+1%* 79+7%* 86+3%* 84+ 3%* %* 72+7%* + L-NOARG 3+9% 71+6% 35+11% 34+8% 48+9% 48+8% % pd *P<.5 (paired t test before and after L-NOARG); P <.5 (unpaired t test, untreated diabeti versus ontrol). + L-NOARG Table 5 ndothelium-dependent relaation and sensitivity to bradykinin before and after inubation with NG-nitro-L-arginine (L- NOARG) in si and twelve weeks STZ study 6-week Control 6-week Diabeti (n = 7) 6-week Diabeti + insulin 12-week Control 12-week Diabeti 12-week Diabeti + insulin Transplants (n = 7) BK Ma. 54+8%** 71+6%* 63+8%* 68+7%** 63+7%** 56+9%* 49+11%* +L-NOARG 18+5% 49+4% % 2+7% 25+7% % 19±7% pd t *P<.5, **P<.1 (paired t test before and after L-NOARG); tp<.5 (diabeti+insulin versus ontrol). + L-NOARG

8 b ' Cu o io io-6 Sodium nitroprusside (M) -o '% 1-7 lo-6 Sodium nitroprusside (M) K.M. Heygate et al slet transplantation in STZ rats line in aortae (Mulhern & Doherty, 1989), mestenteri arteries (Bhardwaj & Moore, 1988). Another investigation of vasular (Agrawal & MNeill, 1987; White & Carrier, 1988), isolated reativity of both aortae and mesenteri arteries in hroni perfused hindquarters (Friedman, 1989), and pe: rfused kidneys eperimental diabetes showed that the inreased ontratile response to noradrenaline was evident only in the untreated diabetis, whereas treatment with insulin ompletely prevented the inrease (Maleod, 1985). Similar findings were reported in a a long-term study of diabetes where the aortae from untreated diabeti rats showed an inreased sensitivity to the agonist, phenylephrine, whih was partially orreted in the insulintreated group (Chang & Stevens, 1992). n one study of resistane artery funtion, of similar eperimental design to our study, evidene of enhaned ontratility to noradrenaline was revealed in both untreated and - insulin treated diabeti rats, and similarly, inubation with a nitri oide synthase inhibitor (L-NAM) resulted in an inrease in sensitivity to noradrenaline in the diabeti vessels (Taylor et al., 1994a). Likewise, the maimum ontratile response to depolarizing K+PSS also was signifiantly inreased 25- in the insulin-treated diabeti group. nterestingly, in the ativation proedure of the 12-week study, ontratile responses to the depolarizing K+PSS, and 4- NAK+ were signifiantly augmented in the insulin-treated diabeti group alone, suggesting a hroni effet of raised a) 5- eogenous insulin on the ontratile mehanism of the vasular 1-o~ smooth musle. n this study, the insulin-treated rats reeived their last injetion 24 h previously and so plasma insulin levels in the freshly ulled rat should be negligible. t is however, possible that the insulin-treated rats were hyperinsulinaemi 75- and that this effet was arried over into the myograph bath. ndeed, other workers have also demonstrated a similar effet after in vivo treatment of diabeti rats with insulin (Taylor et Figure 6 ndothelium-indued relaation respo nitroprusside of rat mesenteri resistane vessels fro insulin-treated diabeti rats ([l); untreated diabeti islet-transplanted rats (V-12 weeks only) in (a) 6 we 12 week study. Points show means+s.e.mean. al., 1994a). There was no evidene of impaired endothelium-dependent relaation to aetylholine or bradykinin in either the untreated or the insulin-treated diabetis of the si or twelve week 1o 1 study; this is in agreement with other studies whih investigated the vasodilator response of the mesenteri vasular bed (Kiff et al., 1991a), and the aortae (Head et al., 1987; Taylor et al., 1994b) or hemially-indued diabeti rats. n our 6-week study, maimum relaation to ACh was signifiantly augmented in the untreated diabeti group. This inreased vasodilator response again suggests an inreased release of nitri oide, whih appears to be absent in more established diabetes of longer duration (i.e weeks). ndeed, further evidene to support this is again provided by the inubation of the vessels with L-NOARG. n the presene of the nitri oide synthase inhibitor, the untreated diabeti vessels from the 6-week study failed to display suh a marked redution in endothelium-dependent relaation as that of the insulin-treated diabeti and ontrol vessels. An alternative eplanation for the inreased relaation response to ACh is the release of an additional relaing fator, namely endothelium-derived hyperpolarizing fator (DHF). This has been shown to rela vasular smooth musle ells by opening potassium hannels, and hyperpolarizing the ell membrane (Van de Voorde et al., 1992). t is possible that the simultaneous release of vasodilator eiosanoids ould also be responsible for altering endothelium-mediated relaation to aetylholine in the diabeti blood vessels. Several studies have demonstrated augmented release of prostaylin from diabeti arteries, and this inreased prodution, oupled with its synergisti ation on DRF may play an important role in mediating endothelium-dependent relaation in ertain diabeti vasular beds (White & Carrier, 1986). n keeping with ontratile agonists, the literature 1-5 io-4 responses to endothelium-dependent vasodilators is onsistent; with inreased relaation being reported in one study (White & on the Carrier, 1986), and a derease in sensitivity, but not maimum,nse to sodium relaation, being reported in another (Kamata et al., 1989). n m ontrols (Q); ontrast, other investigations of endothelial-dependent rei rats (A\) and sponses in hemial indued diabetes, have demonstrated atek study and (b) tenuation of endothelium-dependent relaation to ACh in both mesenteri arteries (Taylor et al., 1994b) and aortae (Oyama et

9 K.M. Heygate et al slet transplantation in STZ rats 53 al., 1986; Cameron & Cotter, 1992) from diabeti rats. Also, we have shown impaired endothelium-dependent relaation in the resistane arteries from the spontaneously diabeti Bio Bred (BB) rat (Heygate et al., 1995). However, aetylholine is not an important physiologial mediator of endothelium-dependent relaation in vivo, but the influene of shear stress on the subsequent release of NO has been diretly linked to the same G-protein as ACh (Ohno et al., 1993). t is therefore possible to etrapolate from these findings, and suggest that flow-indued NO release ould be abnormal in diabetes, and may have important impliations in the determination of peripheral tone. ndeed, inreased blood flow has been shown in the early stages of insulin-dependent diabetes mellitus in man, resulting in preapillary vasodilatation (Jaap & Tooke, 1995). One in vivo animal study whih ompared regional blood flow in different vasular beds in rats, whih had been diabeti for four weeks, reported a signifiant inrease in blood flow to the mesenteri bed, whereas, blood flow to other abdominal and thorai organs was unhanged, and those to skin and musle was redued (Hill & Larkins, 1989). Moreover, inreased renal and mesenteri blood flow has been demonstrated in onsious, hronially instrumented rats following streptozotoin treatment (Kiff et al., 1991a,b). The relaation response to the endothelium-independent vasodilator, sodium nitroprusside in this study, was similar in all the eperimental groups irrespetive of the duration of diabetes. The literature reveals onfliting reports about alterations in the SNP response in diabetes. The majority of studies show that the SNP response is not impaired in diabetis versus ontrols (Oyama et al., 1986; Pieper et al., 1995; Taylor et al., 1995), but one study showed a derease in relaation to SNP in diabeti arteries (Kiff et al., 1991a). Similarly, in man some studies in insulin-dependent diabeti subjets, have shown normal responses to SNP (lliott et al., 1993; MNally et al., 1994), whilst another has demonstrated an impaired response (Calver et al., 1992). The transient hanges in reativity observed in these diabeti animal and human studies, suggest that omple alterations in vasular funtion develop in early stages of diabetes, and that these hanges ontinue to be modulated by other intrinsi or etrinsi fators that ome into play as the disease progresses (Katz & MNeill, 1987; Kabbah et al., 1988). Sine the arterioles are the major site of vasular resistane, small hanges in arteriolar sensitivity ould have a signifiant effet on blood pressure, and the regulation of tissue blood flow (Morff, 199). Several studies of islet transplantation in diabeti rats have been performed demonstrating the potential to reverse the diabeti state and assoiated mirovasular ompliations (Mauer et al., 1975; Orloff et al., 1987; Shmidt 1983). One suh study reported that the development of nerve fibre abnormalities ould be prevented if islets were transplanted soon after the onset of diabetes. However, where islet transplantation was delayed, abnormal fibre morphology ourred, and omplete reversal of the diabeti neuropathy was not ahieved despite onsistent euglyaemia (Britland et al., 1991). More reently, a study has shown that islets transplanted either beneath the kidney apsule, or intraportally to the liver, and the subsequent long-term reversal of hyperglyaemia, an prevent the development of glomerular basement membrane thikening found in untreated diabeti animals (Leow et al., 1995). nterestingly, another reent study demonstrated that panreati islet transplantation not only restored euglyaemia, but also reversed the endothelial dysfuntion in rat aortae (Pieper et al., 1995). Moreover, this group showed islet transplantation ompletely normalized indies of oidative stress in various blood vessels even after eight weeks of diabetes (Pieper et al., 1995); and sine oidizing free radials are known to have a detrimental effet on endothelial funtion, this improvement must be onsidered to be an additional benefit of islet transplantation. n onlusion, we an suggest that in the early stages of diabetes there is an enhaned release of nitri oide, whih was more evident in the untreated diabeti rats, than the insulintreated, and appeared to normalize as the duration of diabetes progressed. 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