Animal models of diabetes II

POS-004-040 ROLE OF L3T4 + AND LYT 2 + T CELLS AND MACROPHAGES IN 8 CELL DESTRUCTION IN SPONTANEOUSLY DIABETIC (NOD) MICE.

B CHARLTON, A BACELJ, RM SLA'I-I'ERY, TE MANDEL Walter and Eliza Hall Institute of Medical Research, Victoria, Australia

Non-obese diabetic (NOD) mice are a rodent homologue of human Type I diabetes. They develop a mononuclear cell infiltration of pancreatic islets by 50 days age which may later progress to 8 cell destruction and insulin dependent diabetes. Administration of cyclophosphamide (CY) promotes the rapid onset of diabetes (within 2 wks). MAbs H129.19 (anti- L3T4 + T cells) or 53-6.7 (anti-Lyt 2 + T cells), or silica particles (anti-macrophage) were administered to groups of female NOD mice following CY administration to determine which mononuclear cell populations were involved in 8 cell destruction. Random blood glucose (RBG) measurements were made on days 0 (CY administration) and 18, and histological, immunocytochemical and flow cytometric studies also performed where appropriate. Incidence of hvoeralvcaemia. Treatment Qontrol Treated anti-L3T4 8/15 0/15 (p<0.01) anti-Lyt 2 10/26 1/21 (p<0.01) silica 8/19 1/19 (p<0.01) Histological assessment showed that lymphocytic infiltration and 8 cell destruction was also significantly reduced (p<0.001) in anti-Lyt2 treated mice and splenic Lyt 2 + cells were >95% depleted. Insulitis was less markedly reduced by anti-L3T4 treatment or silica treatment. We therefore conclude that L3T4 + and Lyt 2 + T cells, and macrophages are all involved in 8 cell destruction in NOD mice. We propose that macrophages present 8 cell antigen to L3T4 + cells which are able to provide 'help' to cytotoxic Lyt 2 + T cells which then destroy 8 cells.

POS-004-041 SPONTANEOUS INSULIN DEPENDENT DIABETES MELLITUS IN A CHIMPANZEE (PAN TROGLODYTES)

S K SINHA, G REDDACLIFF Sydney, Australia.

Symptomatic insulin requiring diabetes with persisting insulin dependence has not been previously described in chimpanzees. This report presents the clinical data from one such chimpanzee.

A 34 year old female African chimpanzee who had been in captivity for at least 29 years, was found to have glycosuria (200-S00mg%) in early June 1982. There was no polyuria or polydypsia. Examination under general anaesthesia showed mild hepatomegaly and possible periodontal infection. Weight 47Kg, serum glucose 23mmol/l. Re animal remained unwell and lethargic with persisting glycosuria. Six weeks later she was comatose. 100units of Isophane insulin were given. Within 2 hours the animal appeared much brighter. Urinary ketones and glucose both fell from 3+ to i+. Daily insulin was continued according to the degree of glycosuria and the clinical condition showed steady improvement. Eight days later discontinuation of insulin was attempted over 3 days, but was unsuccessful because of marked increase in glycosuria and ketonuria, and clinical deterioration with pronounced weakness and diminished mobility. The arms were particularly affected and she used her feet to raise her arms. Within 2 weeks of restarting insulin there was evidence of weight gain and some improvement in arm mobility. Physical examination after 8 weeks of insulin therapy revealed a weight of 37.5Kg and marked asymmetrical wasting of the biceps and interossei. Laboratory studies showed an elevated serum glucose (22.3mmols) and HbAlc 15.9%. Values for control animal were in the human range. C peptide measurements on 2 occasions yielded values of 0.2 and less than 0.03mcg - (control 1.3mcg). Electrophysiologic studies carried out on the upper limbs revealed low conduction velocities in the left upper arm. By November 1982 the upper limbs were in full use. By July 1983 the weight had risen to 42.8Kg and there was no muscle wasting evident on clinical examination. She remains well on daily insulin injections - some 6~ years after presentation.

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POS-004-042 ISLET B CELL CONTENT AND INSULIN SECRETION IN ONE DAY DIABETIC BB RATS.

R.A. PEDERSON, A.M.J. BUCHAN, J.C. BROWN MRC Regulatory Peptide Group, Department of Physiology, University of British Columbia, Vancouver, Canada.

Onset of diabetes in the BB rat is accompanied by rapid and selective destruction of islet B cells (insulitis). We compared islet B cell numbers and insulin secretion in one day (24 hours post-onset) diabetic BB (n=4) and control rats (n=4). Insulin secretion from the isolated vascularly perfused pancreas was stimulated for 45 minutes with 300 mg/dl glucose and 1 ng/ml Glucose-Dependent Insulinotropic Polypeptide (GIP). Pancreatic tissue was taken for immunostaining (insulin) and morphometric determination of % of islet area occupied by insulin-containing cells. In 120 diabetic islets, B cells occupied 44% of the islet area compared to 67% for controls. Although BB islet insulin cell content was 65% of control islets, total insulin output from BB pancreas perfusions was only 12% of controls (i0,000 vs 85,000 p U). It was concluded that the initial dramatic reduction in insulin secretion following onset of diabetes in the BB rat is not totally due to depletion of islet insulin.

POS-004-043 NATURAL COURSE OF PREDIABETIC ISLET INFLAMMATION IN THE BB-RAT : QUANTITATIVE ANALYSIS

H. Kolb, H. HANENBERG, G. KANTWERK-FUNKE, U. KIESEL and V. KOLB- BACHOFEN. Diabetes Research Institute and Institute for Biophysics and Electron Microscopy, University of DHsseldorf, DHsseldorf, FRG

Serial cryostat sections of pancreata from untreated BB rats of different ages were analysed for islet infiltrates using a panel of Ii monoclonal antibodies. A second group of rats received macrophage toxic silica particles i.p. . Islet inflammation started with increased MHC class I expression at one pole of the islet with concomitant infiltration of activated macrophages (EDI +, CD4 +, 13 per islet section). At the same time electron microscopy reveals macrophage phagocyte activity and vascular damage. The exocrine tissue is also affected. Stage 2 shows enhanced MHC class I expression also in exocrine tissue and now lymphocytes infiltratinq (OXI9+: 8/islet, + + OX8:8/islet, W3/25 :>20/islet, including W3/25 -macrophages). Stage 3 shows more intense T-Tell infiltration (OXl9÷:~20/islet) and in addition massive B-cell infiltrates (O~12:>20/islet). The order of infiltrates thus is: macrophages -- CD4+-,--CD8+-lymphocytes -- B- lymphocytes. Silica treatment prevented diabetes development. Immunocytochemistry showed suppression of macrophage infiltration and a complete lack of lymphocytic infiltration (stages 2 and 3). Thus macrophage infiltration not only precedes but seems to be a precondition for lymphocytic infiltration.

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POS-004-044 ISLET CELL ANTIBODIES AND INSULIN AUTOANTIBODIES AS PREDICTIVE MARKERS IN THE NON-OBESE DIABETIC MOUSE.

S. REDDY, N J BIBBY, R B ELLIOTT The University of Auckland, School of Medicine, Dept. of Paediatrics, Auckland New Zealand

The expression of i s le t cell cytoplasmic antibodies (ICA) and insulin autoantibodies (IAA) and their degree of association were studied longitudinally in the non-obese diabetic (NOD) mouse in order to assess their predictive value for insulin-dependent diabetes. Sera from 14 NOD mice (diabetes rate : 30%) were examined for ICA and IAA fortnight ly from day 40 until the onset of diabetes (n=9) or unti l day 250 in non-diabetic mice (n=5). BoUin's fixed normal Swiss albino mouse pancreas was used as substrate for ICA while IAA were measured by elisa with porcine insulin as the primary ligand. The IAA values for al l test sera were expressed after subtraction of the corresponding optical densities for sera assayed in parallel but without the insulin ligand. Our results show that both ICA and IAA were present in al l animals from day 40 irrespective of diabetes. Most animals showed peak IAA values between day 80-160 and in 5/9 diabetic animals the onset of the disease was preceded by declining levels. Four animals with diabetes were ICA-negative at diagnosis. Although sera from majority of the animals showed a strong association for both the markers, some had the single marker only. We conclude that both ICA and IAA precede the onset of diabetes but their expression may be independent. Either marker on i ts own or in association do not appear to be specific predictors of the disease in the NOD mouse.

POS-004-045 ALTERATION OF INSULIN AND GLUCAGON SECRETION FROM THE PERFUSED BB RAT PANCREAS

BEFORE AbD AFTER THE ONSET OF DIABETES

M KANAZAWA, S SHIRABE, J IKEDA, Y NOTOYA, H ITO, Y ~ W A + 3rd Department of Internal Medicine, Tokyo Medical College, Tokyo, Japan. 3rd Department of Internal Medicine, Tokyo 5hiversity School of Medicine, Tokyo, Japan.*

~he spontaneous diabetic BB rat was established in Ottawa in 1974. ~he infiltration of mononuclear cells in the pancreatic islets danages the islet B cells and the animals finally develop insullin dependent diabetes mellitus. We examined insulin and glucagon secretion at two concentrations of glucose in the perfused isolated pancreas of BB rat, just before the initiation of cell infiltration to the islet and just after the onset of diabetes mellitus, to elucidate changes In responsiveness of A and B cells to glucose during these periods. (Method) BB rats, before the onset (5 weeks old, n=6) and 2 weeks after the onset (13 weeks old, n=5) of overt diabetes mellitus were used in the study. Age-matched Wistar rats served as control. The isolated rat pancreas was perfused by the method of Grodsky et al. Flow rate of the meditTn was 2 ml/min. After 30 minutes of preperi%asion with KRBB containIng 2.8 mM glucose, sample collection was started for 5 minutes in the seine glucose concentration as control. The glucose concentration of the perfUsate was then elevated to 16.5 mM and collection was continued for another 30 minutes. (Result) In 5-week old BB rats, glucagon secretion was higher at low glucose concentration and insulin secretion lower at high glucose concentration than the corresponding values in age- matched control rats. In 13-week-old BB rats, insulin secretion was undetectable irrespective of glucose concentration. Glucagon secretion was only moderately suppressed by the perfUsion of high glucose and was significantly higher than that of age-matched controls. (Conclusion) The reduction in insulin secretion observed in 5-week-old rats without Insulitis indicated that pancreatic B cells of BB rats of this age are somehow injured by humoral factors rather than cell infilltration.

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POS-004-046 THE CHARACTERISTICS OF PANCREATIC LESION IN A NEW DIABETIC STAR/iN OF RAT (WBN/Kob)

Y MORI, J YOKOYAMA, A SASAKI, M OHNO, H KURATA, Y IEEDA 3rdDepartment of Internal Medicine, Jikei University School of Medicine, Tokyo, Japan.

We previously reported a new diabetic strain of rat (WBN/Kob) with endo-exocrine pancreatic insufficiency ( Diabetes 36 suppl. I, 23A ). In the present study, to investigate the patho- physiology of this rat strain, pathological examination of the pancreas and measurements of pancreatic amylase, insulin and glucagon contents were performed.

Pathological examination of thepancreaswasperformedat 2 to 3month intervals. Serial sections of thepancreaswere stained withHEand immunostained for insulin and glucagonby ABC peroxidase method. At I month of age, no abnormal change was observed in the islet or acinar cells. At 3 months of age, infl~torychanges and fibrous exudation ~_re observed butthe islet cells were intact. A gradual increase in fibrous tissue was observed at 6 and 9 months. At 12 months of age, fibrous tissue replaced an extensive area of the pancreas and involved the islets. The amylase content of the pancreas in WBN/Kob rats was markedly decreased in cc~ison with control Wistar rats at the same age. The islets were remarkably decreased in number and size, and only small islets composed few endocrine cells were detected. ~ohistoc~hemical study of the pancreatic islets using anti-insulin and anti-glucagon antibodies by ABC peroxidase method showed the decreased numbers of not only B cells but also A cells. For insulin and glucagon assay, the pancreas was homogenized in acid alcohol and extracted overnight at 4°C. Insulin and glucagon contents weremeasuredbyradio~oassayusing double anti/xm~ymethod against rat insulin and bovine glucagon standard respectively. Both of the pancreatic insulin and glucagon contents were markedly decreased in WBN/Kob rat in comparison with control Wistar rats.

In conclusion, tNis new diabetic strain of rat (WBN/Kob) is characterized by the destruction of not only B cell but also A cell, accompanied with fibrous changes of exocrine pancreas. The pathophysiology of this diabetes is quite different from that of BB rats or NOD mice.

POS-004-047 ANALYSIS OF THE PASSIVE TRANSFER OF INSULIN DIABETES MELLITUS IN NOD MICE.

P. ~ G S , L. O'REILLY, K. JONES, T. IA/ND,*E. SIMPSON,+H. ~ILDMANN & A. COOKE. Dept. of Irmmlnology, University College & Middlesex School of Medicine, London WI U.K. ~CRC, Northwick Park, Harrow, Middlesex U.K. Dept. Pathology, Cambridge University Cambridge, U.K.

Diabetes can be transferred by splenic T cel ls from diabetic NOD mice to irradiated non diabetic sxngeneic recipients. The effects of T cell depletion of donors and reciplents using subset specific antibodies in vivo on disease transfer has been investigated. We have furthermore found that diabetes can be transferred from these recipients of diabetic spleen to a new group of i r radiated recipients (a double transfer). This shows the presence of expanded diabetogenic T cells in the spleens of the f i rs t recipients.

The time course of development of diabetes in the recipients has been investigated. The phenotypes of the cel ls i n f i l t r a t i n g the pancreas and causing selective ~ cell destruction in this transfer situation have been identi f ied histologically.

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POS-004-048 ANALYSIS OF THE MAJOR HISTOCOMPATIBILITY COMPLEX CLASS II REGION IN THE NOD MOUSE

T. LUND, K. JONES, A-M VAREY, L. SIMPSON* & A. COOKE Dept. of ~ology, University College & Middlesex School of Medicine, London W1 U.K.

*CRC, Northwick Park, Harrow, Middlesex U.K.

One o f the a t l e a s t t h ree r ecess i ve genes c o n t r o l l i n g the deve lopment o f d i abe tes in the non-obese d i a b e t i c mouse Is a s s o c i a t e d w i t h the H-K to H-I r eg ion of the ma jor h i s t o ¢ o m p a t £ b i l i t y complex. The hap lo t ype o f the NOD mouse has been ass igned K ° I -A n°a I-E ° D b. To be ab le to i d e n t i f y the H-2 l inked diabetes s u s c e p t i b l l i t y gene Idd-L we have i n i t i a t e d a cha rac te r i za t i on of the c lass I I r eg ion a t the chromosomal and m o l e c u l a r l e v e l . We have i d e n t i f i e d a RFLP w i t h the r e s t r i c t i o n enzyme Taql which a ] l o w s us tO d i s t i n g u i s h the A~ o f the NOD mouse from t h a t o f the b, d and k h a p l o t y p e , and a RFLP w i th Pst l which a l lows us to d i sc r im ina te between the E ~ o f the NOD haplotype and the E~ gene of the other three haplotypes. We have so fa r not been ab le to d i s t i n g u i s h the A~ of the NOD mouse and the A~ of the d- haplotype. For a more de ta i l ed cha rac te r i za t i on of the MHC Class I I region a cosmid genomic l i b r a r y has been constructed and is being screened wi th Class I I spec i f i c probes. So fa r clones cross hyb r id i z ing wl th AF , A~ and E~ have been i d e n t i f i e d . These clones are being character ized.

POS-OOg-Og9 SCREENING FOR LINKAGE OF VARIOUS GENETIC MARKERS WITH DIABETES IN THE BB RAT

W.KASTERN; I.KRYSPIN-SZRENSEN I Hagedorn Research Laboratory , Gentof te, Denmark I ) Danish National Food Agency, Seborg, Denmark

In an attempt to locate the genes responsible f o r diabetes in the BB r a t , pedigrees of d iabe- t i c BB rats have been examined fo r genet ic l inkage wi th var ious genet ic markers. These markers were e i the r isoenzymes which show polymorphism between var ious ra t s t ra ins and l i n e s , or cloned fragments of random, r e p e t i t i v e DNA iso la ted from the ra t genome and shown to detect polymorphic DNA fragments a f t e r hyb r i d i za t i on to Southern b lo t s . Within the Hagedorn BB colony, there is a s ing le recessive gene separat ing the diabetes-prone l i n e (BB/H) from the d iabe tes - res i s tan t l i n e (der ived from the W-subline). To locate th i s gene, a c ross / in te rc ross breeding has been per- formed where the F 2 generat ion has been fo l lowed both fo r diabetes and the inher i tance of the markers. There was a 25% incidence of diabetes in th is generat ion , and thus f a r , we have seen a 2.3 k i lobase Rsal fragment detected wi th a r e p e t i t i v e DNA probe and which was present in 8/13 of the d iabe t i c F 2 animals. While th is number is s i g n i f i c a n t to show l inkage, i t is a very weak l inkage, i nd ica t ing tha t the marker sequence l i e s some distance from the diabetes gene, but on the same chromosome. To a l low the i d e n t i f i c a t i o n of the other genes involved in diabetes but which were shared between the two BB l ines (and thus unable to be detec ted) , we have performed an outcross of the BB/H to the Long Evans Hooded (LEH) s t r a i n ) . This was then fo l lowed by F I i n t e r - crosses and backcrosses to the BB/H. The Fp of th is cross y ie lded a 6% incidence of diabetes in 200 animals. We have detected a s ing le Mspl r e s t r i c t i o n fragment which was inher i ted by 83% of the d iabe t ic F 2 animals. This ind icates a very close l inkage wi th another of the diabetes genes. In ne i the r of the crosses, have we detected the l inkage of any known isoenzyme markers wi th the disease. More animals are needed to a l low a more precise determinat ion of the actual map d is - tances involved to e i t he r of these new markers, but both of them should at leas t a l low the iden- t i f i c a t i o n of the chromosomes involved fo r each of these two genes which are necessary fo r the development of diabetes in the BB ra t .

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POS-O04-05O EVIDENCE THAT THE MACROPHAGE-MEDIATED T CELL DYSFUNCTION IN THE DIABETIC BB (BBd) RAT OCCURS AT THE LEVEL OF CALCIUM MOBILIZATION.

MD DAYER-METROZ, EB MARLISS, P POUSSIER McGill Nutrition and Food Science Centre, McGill University, Montreal, Canada.

To study the possible step(s I involved in BBd T ce l l dysfunction, we compared the mitogeni responses of puri f ied splenlc T ce l ls to direct act ivat ion of protein kinase C by phorbo~ ~yristate acetate (PMA) and/or calcium mobilization with the ionophore ionomycin. Spontaneous H thymidine incorporation in BBd (8510 cpm) was significantly higher than in the non diabetes-

prone (BBn) (856 cpm, p<0.05), re f lect ing probable in vivo act ivat ion. Ionomycin (not mitogenic alone in BBn, 850 cpm) caused significant proliferation in BBd (25937 cpm, p< 0.05), whereas in BBn addition of interleukin 2 (IL-2) to ionomycin was required for such a response (34783 cpm). This suggests that BBd T cel ls were in a (pre)activated state, ionomycin acting as second signal. In BBn addition of either ionomycin (48341 cpm) or PMA (50842 cpm), together with Concanavalin A (Con A) gave similar responses. In contrast, in BBd, addition of ionomycin with Con A great ly enhanced the response (143991 cpm) as compared to PMA + Con A (58550 cpm). The maximal stimulation was obtained with PMA + ionomycin (227284 cpm in BBd vs 139548 in BBn). In BBn when autologous macrophages were depleted, the T cel l mitogenic responses to a l l the agents tested, i.e. IL-2, Con A, PMA and ionomycin, used alone or in combination, were decreased by 20-80%. In contrast, depletion of macrophages in BBd resulted in responses increased up to 10-fold with these agents, indicating that the BBd T ce l l defect is due at least in part to macrophages. CONCLUSIONS: I. BBd T cell activation has been demonstrated in vitro by increased spontaneous proliferation, consistent with the increased expression of Class I I molecules previously reported in BBd T ce l l s in vivo. 2. Macrophage-mediated T ce l l dysfunction can be explained in BBd at least par t ia l ly by an impaired calcium mobilization.

POS-004-051 INSULIN SENSITIVITY IN BB RATS AT ONSET OF DIABETES

P FERRE, M-A BAUDON, L PENICAUD*, A KTORZA*, L CASTANO**, J GIRAR D . Centre de Recherches sur la Nutrition, CNRS, Meudon : *UA307 CNRS : **U188 INSERM, Paris, France

In the BB rat, 30 to 50 % of animals undergo a autoimmune destruction of the pancreatic B cells leading to a short period of glucose intolerance followed by an abrupt onset of hyperglycemia and glucosuria. We have examined whether the onset of diabetes is associated with changes in insulin sensitivity. Animals were tested each day for glycosuria. On the first day of glycosuria, insulin sensitivity of liver and perioheral tissues was studied using the euglycemic hyperinsulinemic clamp and [3-3H]glucose, and compared to age and sex-matched controls. Glycemia was clamped at i00 mg/dl in all groups. A decreased insulin response was observed at submaximal and maximal insulin concentrations for both inhibition of hepatic glucose production and stimulation of glucose utilization in diabetic rats. No insulin antibodies could be detected in diabetic animals. The plasma concentration of glucagon, catecholamines, ketone bodies and fatty acids were similar in control and diabetic rats during the clamp studies. Glycated hemoglobin was only slightly higher in diabetics (5.6 + 0.3 %, n = i0) than in controls (4.3 + 0.2 %, n = i0). In vitro studies on adipocytes isolated from controls and diabetics (first day of glycosuria) pointed out to a defect in glucose transport and lipogenesis. In conclusion, an insulin resistance is p~eseQt in BB rats at onset of diabetes and could be linked to ~e~ects of intracellular eff~tors of insulin rather than to circulating antagonists.

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POS-004-052 SPONTANEOUS RECOVERY OF EXPERIMENTAL DIABETES IN SOME STRAINS OF RATS.

RR RODRIGUEZ, CF REYES TOSO, RL PEREZ

I n s t i t u t e o f P h y s i o l o g y , School o f M e d i c i n e , U n i v e r s i t y o f Buenos A i r e s , Buenos A i r e s , A r g e n t i n a

Swiss Albino rats of the I n s t i t u t e , Plebald, SHR and WKy were e i t he r In jec ted with s t reptozotoc in (SO mg/kg body weight, i n t r a p e r i t o n e a l l y ) o r p a r t i a l l y depancreatized (removal of around 95 % of the pancreatic mass). The animals were followed fo r one year, with monthly determinations of body weight and fas t ing blood sugar l e v e l . They drank water and eat a

synthet ic d ie t "ad l i b i t u m " . A f te r s t reptozotoc in we had 25 SHR rats with severe diabetes ( fas t i ng glycemia over L6.5 mmol/1) and 12 with moderate diabetes (blood sugar leve l between 11.2 and 16.5 mmol/l). One year l a t e r 9 animals, o r i g i n a l l y with moderate dlabetes completel ly recovered. Only two with severe diabetes surv ived. Simi lar resu l ts were obtained wlth rats of the WKy and Piebald s t ra lns : 6 out of 13 d iabet ic WKy animals had normal fas t ing glycemla (6.7 mmol/l or less) a f t e r the one year period and 3 out of 9 Piebald rats recovered.On the other hand a l l of the 7 d iabet ic Swlss Albino rats remained d iabet ic at the end of the observation period. In the p a r t l a l l y depancretized ra ts , previous resu l ts obtained by us were confirmed. The Swiss Albino anlmals shown e sex d i f fe rence on the onset of diabetes, since a f t e r IZ months of the operat ion, 86 % of 28 males have fas t ing hyperglycemia and only 40 % of 30 females were d iabe t i c . When SHR of Piebald rats were studied, the resu l ts were d i f f e r e n t . Af ter large subtotal pancreatectomy only moderate diabetes was observed, a f t e r 3 to 4 months of the operation in only 40 % of IZ l SHR and 55 % of 136 Piebald animals. The sex d i f fe rence was not c lear in these s t ra ins and at the end of the experiments only Z % of the SHR rats and 15 % of the Piebald had hyperglycemia. In the i s l e t s of Langerhans of the non d iabet ic p a r t l a l l y depancreatized SHR and Piebald rats increased of the size and number of ce l l s were observed. This hypertrophy and hyperplasla insu la r was accompanied by numerous mitosis and, in some cases, adenomatous type hyperplasla.

POS-004-053 THE GK RATS. A MODEL OF NON-OBESE NIDDM. RESULTS OF THE SELECTIVE BREEDING OVER 36 GENERATIONS.

Y GOTO,K SUZUKI,M SASAKI,Y ONO,S ABE Third Department of Internal Medicine,Tohoku University School of Medicine, Sendal,Japan.

Production of diabetic state was carried out by repeating the selective breeding using glucose intolerance as a selection indicator. Two hundred and eleven Wistar rats were obtained from ~he Experimental Animal Farm (Nihon Clea Co.) and 9 male and 9 female rats were selected by OGTT (2g/kg body weight) and bred. This procedure was performed repeatedly. Since the 8th generation, brother-and-sister mating has been carried out until now. After the 10th generation (F9), all offsprings had abnormal GTTs.

In F28 rats, treatment for SPF (specific pathogene free) conditions was performed. Thereafter, improvement of fertility was obtained. Mean levels of FBS and 2 hr. were 168 mg/dl and 235 mg/dl in OGTT, respectively. Glucose intolerance of GK rats were admitted since 8 weeks after birth.

Pancreatic contents of both insulin and glucagon were 67% and 42% of normal Wistar rats, respectively. Experiments on insulin secretion from the isolated pancreas (Grodsky technique) revealed a significant decrease or lack of the first phase response to glucose but normal response to arginine stimulation. Star-fish-like deformity of pancreatic islets and significant reduction of B cells were observed histologically. Islet deformity in GK rats were far more remarkable than that of normal Wistar rats.

Thickening of basement membranes, decreased motor nerve conduction velocity and shrinkage of myelinated nerve fibers were significantly observed in GK rats as compared with those of normal Wistar rats.

Glycolytic and gluconeogenetic liver enzymes were found to be abnormal in GK rats as compared to those of normal Wistar rats.

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POS-004-054 MODEL ANIMALS OF NON-INSULIN DEPENDENT DIABETES MELLITUS INDUCED WITH VIRUSES. -CHARACTERIZATION OF THE PREDIABETIC PHASE-

M FUKUMA, K DAN, Y SETO D iv i s ion of Chemotherapy, Pharmaceutical Keio Un i ve r s i t y , Tokyo, Japan

I n s t i t u t e , School of Medicine,

Diabetes induced by v i ruses e n t i r e l y belong to insu l in -dependent one. A p u r i f i e d pancreat rop ic v a r i a n t (CP-44) of Venezuelan encepha l i t i s v i rus (VE) t ha t we i so la ted causes moderate hyperglycemia and hypoinsul inemia in weanling male hamsters with an incidence of 60% by 3 • 5 weeks a f t e r the i n f e c t i o n . However, t h i s d i a b e t e s - l i k e syndrome could not c l a s s i f y i n to an IDDM-type, because any c h a r a c t e r i s t i c changes inc lud ing in - s u l i t i s fo r IDDM in the in fec ted pancreas were not observed. CP-44 also induces p red iabe t i c s ta te in adu l t hamsters, in which IGT and impaired g lucose-s t imu la ted i n s u l i n release ( I IR) are observed. To date we showed the consequence of i n i t i a l phase (24-48 hrs of p o s t - i n f e c t i o n : PI) in v i r a l r e p l i c a t i o n fo r the onset of d iabetes, so tha t the f i r s t phase was delayed in higher ages of r e c i p i e n t , in an insuscep t ib le host, and with non-diabetogenic va r i an ts fo r 72 hrs of PI. To determine what fac tors or events would be key in to the development of d iabetes, the mechanism of i n s u l i n de f i c i ency of adu l t hamsters in p red iabe t i c phase ( IGT/ I IR) was examined in a pe r i f us i on system using i so la ted i s l e t s , in which var ious secretagogues such as D-glucose, a rg in ine , glucagon, somatostat ine, su l - fonylureas and a new type of hypoglycemic agent, A-4166, tha t we d is - covered were used to ana lyze the i n t e r a c t i o n between IIR and the c o u n t e r - r e g u l a t o r y hormones. From the study, i t was shown tha t IIR was not only caused by low response of i s l e t s on glucose, but on glucagon. On the other hand, t h i s fIR was p a r t l y recovered only by A-4166 tha t has the a c t i v i t y to enhance g lucose-s t imu la ted i n s u l i n secre t ion .

POS-004-055 ANALYSIS OF INSULIN BY REVERSE-PHASE HPLC, AND RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) OF INSULIN GENE IN "NON MICE", AN ANIMAL MODEL FOR NON- OBESE NIDDM S. OHGAKU, H. MORIOKA, T. SAWA, S. YANO, Y. TOCHINO* The First Dept. of Medicine, Toyama Medical and Pharmaceutical University, Toyama, *Dept. of Clinical Physiology, Setsunan University, Faculty of Pharmaceutical Science, Hirakata, Japan

NON and NOD mice are derived from the same JCL-ICR mice. NON mice are characterized by non-obesity, remarkable glucose intolerance, hypoinsulinemia, lower insulin contents, decreased insulin mRNA and no insulitis in the pancreas. We regard NON mice as an animal model suitable for non-obese NIDDM, and have further studied this animal for the elucidation of the etiology of NIDDM.

Some people with a structurally abnormal insulin are reported to have developed NIDDM and to present mutant insulin syndrome, and we extracted insulins from the pancreas of NON and ICR and analysed them by the reverse- phase HPLC to make it sure that NON has a normal insulin. Insulin from NON eluted as two peaks (insulin I and II) with the same retention time as control ICR and rat standard insulin.

In the next study, we compared RFLP of insulin gene in NON, NOD, and ICR to examine the possibility of some changes on the insulin gene of NON mice. Genomic DNAs were prepared from the livers of these animals, and digested with BamHI, EcoRI, HindIII, KpnI, and TaqI. After BamHI digestion and gel electrophoresis, DNA blot hybridization followed by autoradiography revealed the presence of a 1.7 kbp extra band in NON, but not in both ICR and NOD mice.

In conclusion: NON mice, an animal model for NIDDM produce normal insulin. Reduced insulin mRNA in the pancreas in combination of an extra band of 1.7 kbp in RFLP of insulin gene suggests that IGT in NON is J nduced by the insufficient synthesis of insulin resulting from its reduced expression due to the probable alteration of the insulin gene in NON mice.

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