diabetic complications/basic mechanisms i

11
Diabetic complications/Basic mechanisms I POS-003-115 IN SEARCH OF A DIABETIC CARDIOMYOPATHY - & STUDY OF LEFT VENTRICULAR FUNCTION IN 25 ASXNIVtX)MATIC DIABETICS USING M MODE ECHOPat~nlOCRAPBfl[ - A PILOT STUDY S BALASINGAM, PK MAN, CH LEE Department of Medicine, Alexandra Hospital, Singapore. 13 Insulin dependant (IDDM) and 12 non insulin dependant (NIDDM) diabetics and 15 age and sex matched controls without clinical cardiac disease, hypertension, anaemia, or renal failure and with normal electrocardiographs and chest roentgenograms were studied. The fractional shortening (FS) and ejections fractions (EF) and the left atrium to aortic root index were measured by echocardiography. HbAlc (glycosylated haemoglobin) was used as an index of glycaemic control in our patients. The results were analysed using student's t statistic. The FS and EF were reduced in 64% of patients. The FS in the entire group appeared to decrease as the HbAlc increased, but the correlation was not statistically significant. The FS in the IDDM group was significantly lower than in the NIDDM group p = 0.006. The HbAlc in the NIDDM group was significantly lower than the HbAlc in the IDDM group. The FS and EF did not correlate with age, lipids or duration of diabetes mellitus. Conclusions (i) A significant proportion of diabetics in this study had asymptomatic left ventricular dysfunction in the absence of clinical coronary artery disease. (2) In the patients we studied, the IDDM group had significantly ~.~.;:.3L~;~;F,?";;,"-'-"~,,~,: .......... poorer glycaemic control and more severe left ventricular :[~:'~'L?,,:':£-'.t::,.~..~ ..... dysfunction when compared to the glycaemic control and left ~.~.:::;:~:,::.';~"-:~::~" ventricular function of the NIDDM group. ~ ',~:;:".;:;" ........ ,.r., ,., I~": :;:~:I."~:,;'.. "'I'' I i ',I ........ 'I ....... i .6 , ~ r Ii !i' !1 !il iliii' ...... _=' i = = : ' ' i ......... i . i n i ,, i ,~ i ,.. i , t ,~ i . t., t ,, t o.. t ~ I I I ~" ":T,'.:;:"' POS-003-116 STUDIES ON DIABETIC MICROANGIOPATHY IN STREPTOZOTOCIN-DIABETIC RIIESUS MONKEYS. R.J.DASH, A MAZUMDAR AND R.N. CHAKRAVARTI* Department of Endocrinology and Experimental Medicine*, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012 (India) This study was designed to define the significance of persistent hyperglycemia and Hb-Alc in the development of diabetic microangiopathy. Twentyone healthy, tuberculosis-free rhesus monkeys of 3.5-4 Kg body weight were employed. Diabetes was induced by intravenous administration of streptozotocin I00 mg/Kg body weight. Six of them died early because of diabetic ketocidosis and infection. Remainder were grouped 5 each into (a) well controlled (FBG 100-120 mg/dl) (b) poorly controlled (FBG 200-300 mg/dl) and uncontrolled (FBG 300-400 mg/dl or higher). Five healthy sham treated monkeys served as controls. All monkeys had BG, llb-Alc, serum cholesterol,triglycerides, phospholipids, free fatty acid and ECG at monthly intervals. At 24 months of diabetes, the monkeys were sacrificed and representative tissues from muscles, skin, heart, kidney and retina were studied in H & E and PAS stained sections. Additionally, Jones silver methenamine stain was used to examine glomerular pathology. Employing a randomised scoring system for overall glomerular microangiopathy as : frequency of glomerular basement membrane thickening x frequency of mesangial widening x frequency of exudate localisation below the Bowman's capsule/duration of diabetes, a positive correlation was recorded between the extent and degree of glomemlopathy and the elevated levels of FBG, Hb-Alc, cholesterol and triglyceride and decreased renal Beta -NAG activity. Microangiopathy in other organs including mild retinal changes also positively correlated with the biochemical parameters. The study thus indicated the role of persistance hyperglycemia in development and progress of microangiopathy in diabetes. $415

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Page 1: Diabetic complications/Basic mechanisms I

Diabetic complications/Basic mechanisms I

POS-003-115 IN SEARCH OF A DIABETIC CARDIOMYOPATHY - & STUDY OF LEFT VENTRICULAR FUNCTION IN 25 ASXNIVtX)MATIC

DIABETICS USING M MODE ECHOPat~nlOCRAPBfl[ - A PILOT STUDY

S BALASINGAM, PK MAN, CH LEE Department of Medicine, Alexandra Hospital, Singapore.

13 Insulin dependant (IDDM) and 12 non insulin dependant (NIDDM) diabetics and 15 age and sex matched controls without clinical cardiac disease, hypertension, anaemia, or renal failure and with normal electrocardiographs and chest roentgenograms were studied. The fractional shortening (FS) and ejections fractions (EF) and the left atrium to aortic root index were measured by echocardiography. HbAlc (glycosylated haemoglobin) was used as an index of glycaemic control in our patients. The results were analysed using student's t statistic. The FS and EF were reduced in 64% of patients. The FS in the entire group appeared to decrease as the HbAlc increased, but the correlation was not statistically significant. The FS in the IDDM group was significantly lower than in the NIDDM group p = 0.006. The HbAlc in the NIDDM group was significantly lower than the HbAlc in the IDDM group. The FS and EF did not correlate with age, lipids or duration of diabetes mellitus.

Conclusions (i) A significant proportion of diabetics in this study had

asymptomatic left ventricular dysfunction in the absence of clinical coronary artery disease.

(2) In the patients we studied, the IDDM group had significantly ~.~.;:.3L~;~;F,?";;,"-'-"~,,~,: .......... poorer glycaemic control and more severe left ventricular :[~:'~'L?,,:':£-'.t::,.~..~ ..... dysfunction when compared to the glycaemic control and left ~.~.:::;:~:,::.';~"-:~::~" ventricular function of the NIDDM group. ~ ' ,~:;:". ; : ;" . . . . . . . . , .r., ,.,

I~" : :;:~:I."~:,;'.. "'I''

I i ',I . . . . . . . . 'I . . . . . . .

i . 6 , ~ r

Ii

!i' !1 !il i l i i i ' ...... _=' i = = : ' ' i . . . . . . . . .

i . i n i , , i , ~ i , . . i ,

t , ~ i . t . , t , , t o . . t ~ I I I

~ " " :T , ' . : ; : " '

POS-003-116 STUDIES ON DIABETIC MICROANGIOPATHY IN STREPTOZOTOCIN-DIABETIC RIIESUS MONKEYS.

R.J.DASH, A MAZUMDAR AND R.N. CHAKRAVARTI* Department of Endocrinology and Experimental Medicine*, Postgraduate Institute of Medical Education and Research, Chandigarh-160 012 (India)

This study was designed to define the significance of persistent hyperglycemia and Hb-Alc in the development of diabetic microangiopathy.

Twentyone healthy, tuberculosis-free rhesus monkeys of 3.5-4 Kg body weight were employed. Diabetes was induced by intravenous administration of streptozotocin I00 mg/Kg body weight. Six of them died early because of diabetic ketocidosis and infection. Remainder were grouped 5 each into (a) well controlled (FBG 100-120 mg/dl) (b) poorly controlled (FBG 200-300 mg/dl) and uncontrolled (FBG 300-400 mg/dl or higher). Five healthy sham treated monkeys served as controls. All monkeys had BG, llb-Alc, serum cholesterol,triglycerides, phospholipids, free fatty acid and ECG at monthly intervals.

At 24 months of diabetes, the monkeys were sacrificed and representative tissues from muscles, skin, heart, kidney and retina were studied in H & E and PAS stained sections. Additionally, Jones silver methenamine stain was used to examine glomerular pathology. Employing a randomised scoring system for overall glomerular microangiopathy as : frequency of glomerular basement membrane thickening x frequency of mesangial widening x frequency of exudate localisation below the Bowman's capsule/duration of diabetes, a positive correlation was recorded between the extent and degree of glomemlopathy and the elevated levels of FBG, Hb-Alc, cholesterol and triglyceride and decreased renal Beta -NAG activity. Microangiopathy in other organs including mild retinal changes also positively correlated with the biochemical parameters.

The study thus indicated the role of persistance hyperglycemia in development and progress of microangiopathy in diabetes.

$415

Page 2: Diabetic complications/Basic mechanisms I

POS-003-117 DEFECTS IN THEMYOCARDIAL CONDUCTING SYSTEM IN STREPTOZOTOCIN-DIABETIC RATS.

ROSEN P., TSCHOPE D., DHEIN S.*, R6sen R.* Diabetes Research Institute, D~sseldorf, and * Pharmacological Institute of the University, Cologne, F.R.G.

Recently we demonstrated that cardiac function in vivo -under physiological work load- was maximally impaired 6 to 8 weeks after induction of diabetes by streptozotocin (60 mg/kg b.w.). Additionally, several abnormalities in the ECG occurred: the QRS-complex was broadened, the R-wave was partly splitted, the PQ-interval seemed to be prolonged. These findings cannot be explained by changes in the electrolytical status, which was not significantly altered by dlabetes. To get a deeper insight the myocardial conducting system was studied in more detail using a 20 electrode matrix mounted on the epicardial surface of isolated perfused hearts of control and diabetic rats. In diabetes, the atrio- ventricular delay was prolonged from 47 ms to 58 ms, the delay of epicardial activation was slowed from 5.6 ms in controls to 10 ms in diabetes. The epicar- dial impuls duration was increased from 53 ms in controls to 72 ms in diabetic hearts. Total global ischemia caused a prolongation of epicardial impuls dura- tion in both controls and diabetic hearts, however, the prolongation was inten- sivied and accelerated in diabetes. Treatment of the animals with insulin did not only prevent, but also reversed the observed disturbances. The significance of an treatment of diabetic rats with an aldose-reductase inhibitor will be dis- cussed. These data suggest that disturbances in the conducting system, in regional per- fusion, and ventricuiar performance occur nearly simultaneously already after a short period of diabetes. These findings might explain the diminished tole- rance of the diabetic heart for ichemic episodes and the higher incidence for arrhythmias.

POS-003-118 ESPONTANEOUS CORRECTION OF BLOOD HYPERVISCOSITY IN DIABETIC SUBJECTS WITH MICROANGIOPATHY BY MEANS OF HAEMATOCRIT REDUCTION

I.SALAMA,A~R~DE SALAMA,C.BREGNI Department of Diabetology0P.Lagleyze National Hospital of Ophthalmology Buenos Aires° Argentina

The haematocrit,the whole blood viscosity,and the viscosity at constant haema- tocrit of 45%,were investigated in 271 diabetic subjects with retinopathy and 20 healthYlcontrol subjects.It was used a rotational viscometer cone-plate (230 Sego- ) at constant temperature.In the whole blood hyperviscosity wa~ de- tecte~ i~ 16% of the diabetic patients;but in blood at haematocrit of 45%, hyperviscosity was found in 40,21% of the patients.When the viscosity measures are performed at constant haematocrit,the parameter which has more influence on viscosity is the red cells rigidityoA very significant correlationbetween the haematocrit and the whole blood viscosity values was found out (r:0,82) On the other hand,a satisfactory inverse correlation was found out between'@~c. (haematocrit of 45%)and the real haematocrit (r:O,69)oIn the mayority o~ the diabetic patients with high red cell rigidity are observed haemodilutiona anemia and consequently normal blood viscosityoit is a compensation mechanism T~is observation suggests the existence of viscoreceptors,according to the theory presented by Dintenfass,that detect the red cell rigidity and release compensate mechanisms in order to improve the blood flowoThis mechanism is not present in diabetic with poor metabolic control during a long period of time° Particular emphasis is placed on blood viscosity control in diabetic patients°

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Page 3: Diabetic complications/Basic mechanisms I

POS-003-119 PROSTAGLANDIN E 2 PRODUCTION BY MONOCYTES FROM DIABETIC PATIENTS

N AOKI, T SAIKA, T YAMAMOTO Department of Medicine, Kinki University School of Medicine, Osakasayama, Osaka 589, Japan.

Prostaglandin E~ (PGE~) has been known to be a product of a variety of cells including tumor-cells-and macrophages. It is now clear that there are several immunoregulatory molecules such as inleterleukins, interferon y and PGEp in immune responses. PGEp seems to be of special importance from an immunological point of view and with regard to pathophysiology of diabetes mellitus, since diabetes closely links to immunity and PGE^ is an important product of monocytes

. z . .

whlch produces IL-I as well. The purpose of thls study is to assess the PGE 2 production in monocytes from diabetic patients which are stimulated wit~ lipopolysaccharide (LPS). Monocytes, collected from the peripheral blood of healthy subjects and di~etic patients, were cultured in RPMI 1640 with 5 % fetal calf serum (2.5xi0 cells/ml) in the presence of LPS (25 ~g/ml) at 37°C for 24h. PGE 2 in the culture supernatants was measured by radioimmunoassay. Diabetic patients (15 IDDM and 33 NIDDM patients) were subgrouped with the control level, the morbid duration, the presence of microangiopathy, and the type of treatment. As a whole, PGE^ production was 15.3 ± 8.4 ng/ml (mean ± SD) and 13.1 ± 8.0 ng/ml for IDDM a~d NIDDM respectively, whcih did not differ significantly from the data obtained from healthy subjects (12.3 ± 7.0 ng/ml). In IDDM patients, the subgroup with the disease duration of 10 or more years, or with microangiopathy showed significantly high PGE~ production in comparison

• . z

with healthy sub3ects. In any group of NIDDM patxents, however, no difference of PGE 2 prodcution was found as compared with healthy subjects.

POS-003-120 ACTION OF THE PENTOXIFYLLINE ON THE BLOOD VISCOSITY IN THE DIABETIC PATIENTS WITH RETINOPATHY

A.R.DE SALAMA,I.SAL~MA,C.BREGNI Department of Diabetology,P.Lagleyz~ National Hospital of Ophthalmology Buenos Aires. Argentina

50 patients with hyperviscosity in their blood (at haematocrit of 45%) have bee~ studied in a double blind trial° 28 randomized patients had received 12OO mg.of pentoxifylline daily,the rest had placebo. After 3 months of treatment the viscosity of the two groups was studied again in the same way° In the pentoxifylline treated group a statistically significant fall (P<O,OI) in blood viscosity was observed. The conclution was that this drug is able to reduce the blood viscosity (at haematocrit of 45%). Theorically this effect id due to an improvement in red cell deformability taking into account that our viscosity values were performe~ at constant haematocrit

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Page 4: Diabetic complications/Basic mechanisms I

POS-003-121

EFFECT OF a2-ADRENOCEPTOR ANTAGONIST ON PLATELET ACTIVATION DURING INSULIN-INDUCED HYPOGLYCEMIA IN TYPE 2 DIABETES MELLITUS

M SHINOHARA, H TAKEDA, H KISHIKAWA, T MIYATA, H FUKUSHIMA, M. SHICHIRI Department of Metabolic Medicine, Kumamoto University Medical School, Kumamoto, Japan

The role of epinephrine on platelet activation and the effect of an a2-adrenoceptor antagonist, midaglizole, during insulin-induced hypoglycemia in Type 2 diabetes mellitus were examined. The action of midaglizole as a platelet a2-antagonist was confirmed by in vitro studies using platelet-rich plasma obtained from 8 normal subjects. This agent (2.5 ~M) strongly suppressed the platelet aggregation induced not only by the near-threshold concentrations of epinephrine alone (0.31±0.07 ~M), but also by the synergistic action of epinephrine (0.i ~M) with ADP (0.88t0.20 ~M), collagen (0.44±0.13 ~g/ml), or arginine vasopressin (23.3±?.? nM). Hypoglycemia were induced by a bolus injection of short-acting insulin (0.1-0.2 u/kg) in 18 diabetic subjects, and the blood levels of glucose, C- peptide, counter-regulatory hormones, arginine vasopressin, 8-thromboglobulin, and thromboxane B 2 before and after insulin injection were compared. They were divided into two groups, control group (n=9) and a2-grou p (n=9), and midaglizole (300 mg) were administered per os 60 min before insulin injection in the latter. Blood glucose and plasma C-peptide levels were significantly decreased by insulin injection in both groups. Counter-regulatory hormones, including epinephrine, and arginine vasopressin were significantly increased at the hypoglycemic nadir compared with the 0 min in both groups. Plasma 8-thromboglobulin was increased at the hypoglycemic nadir (157.0±18.0 ng/ml) compared with the 0 min (122.7±16.4, p<O.05) in the control group, whereas no significant increment was demonstrated in the e2-grou p (i00.7t11.4 v s 108.7±17.0). On the other hand, plasma thromboxane B 2 levels demonstrated no significant increase in both groups. These results suggest that plasma epinephrine has an important role on platelet activation during hypoglycemia in Type 2 diabetes mellitus, and that the platelet abnormality is prevented by e2-adrenoceptor antagonist.

POS-003-122 DOES GLYCEMIC CONTROL INCREASE IN BINDING CAPACITY OF FIBRONECTIN FOR FIBRIN(OGEN) IN PLASMA SOLUBLE FIBRIN MONOMER COMPLEXES IN DIABETICS ?

S ISOGAI, S TAKEUCHI, K NAKAMURA, M KAMEYA~, T INOKUCHI The Second Department of Internal Medicine, Toho University School of Medicine, Tokyo, Japan.

Fibronectin(Fn) inhibits fibrin polymerization and fibrin exists as soluble fibrin monomer complexes(SFMC) in circulating plasma. In addition, the in- creased Fn to fibrin(ogen) molar ratio has been proposed to raise plasma solubility, followed by less coagulability. In order to elucidate the effect of glycemic control on the binding capacity in SFMC consisting of Fn and fibrin- (ogen) in the plasma, SFMC was evaluated in 13 diabetics and 14 healthy controls. Eight patients were controlled with insulin and the remaining 5 with sulfonyl- urea over a period of 4 weeks. The constituents of SFMC in each subject were analyzed densitometrically with SDS-polyacrylamide gel electrophoresis. Diabetic patients initially showed more elevated diurnal mean plasma glucose, HbAI and SFMC levels than controls. In the diabetics, diurnal mean plasma glucose de- creased significantly(p<0.01) from 337 + 84(mean + SD) to 152 + 44 mg/dl, HbAI from 13.5 + 4.5 to 10.6 + 2.6 %(p<0.01)-and SFMC from 10.2 + 7?2 to 6.1 + 1.6 mg/dl(p<0.01), before and after the treatment, respectively? In contrast~ the ratio of Fn to fibrin(ogen) in SFMC was significantly(p40.01) elevated from 0.I0 + 0.08 up to 0.37 + 0.31. On the other hand, no changes in these parameters, including the Fn to fibrin(ogen) ratio (before and after the treatment, 0.09 + 0.05 vs 0.ii +_ 0.06), were found in controls throughout the observation period. These results suggest that improvement in hyperglycemia brings about an enhanced binding capacity of Fn for fibrin(ogen). Although the precise mechanism is not clear, it appears to be a useful mechanism for the solubilization and prevention of the silting of fibrin fibers in diabetics.

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Page 5: Diabetic complications/Basic mechanisms I

POS-003-123 TISSUE PLASMINOGEN ACTIVATOR (t-PA) AND INHIBITOR (PAl) IN PATIENTS WITH DIABETES MELLITUS

S KADOWAKI, T MATSUO, S OKADA AND 0 MATSUO Department of In ternal Medicine, Hyogo Prefectura l Awaji Hospi tal , Sumoto Department of Physiology, Kink1 Univers i ty School of Medicine, Osaka, sayama, Japan

t-PA and PAl are known to be important fac tors [o control local f ibr i i~olys is , and may

cooperate each o ther to regula te f i b r i n o l y t i c system iu r ive . This study was udertaken to

c l a r i f y the role of t-PA and PAl in the development of d iabet ic vascular complicat ions.

t-PA and PAl levels were measured in 82 d iabet ic pa t i en t s and these concent ra t ions were

compared with age and sex mached normal con t ro l s . Corelat ion between these two fac tors

and glycosylated hemoglobin which is not only a useful indica tor of d iabet ic control

s t a t e , but one of the markers of the prote in g lycosy la t ion , was a lso assessed.

[-PA level was s i g n i f i c a n t l y decreased in the d iabe t ics complicated with maeroangiopathy

including hypertension and ishemic heart d i sease , though both of t-PA and PAl were not

s i g n i f i c a n t l y changed in the d iabe t ics with microangiopathy. Although there was a reverse

eore la t ion between t-PA and glyeosylated hemoglobin level , no s i g n i f i c a n t r e l a t i onsh ip

between PAl and glycesyla ted hemoglobin was found.

I t is speculated that the decreased [-PA a c t i v i t y , probably induced by non enzymatic glycosy

la t ion in t-PA molecular s t r u c t u r e , might cont r ibute to the i~paired f i b r i n o l y t i c system

responsible for the development of the vascular complication.

POS-003-124 EFFECT OF AN ALDOSE REDUCTASE INHIBITOR ON AXON REFLEX VASODILATATION IN STREPTOZOTOCIN DIABETIC RATS

A LOW, W KOZAK*, R WESTERMAN Physiology Dept, Monash University, Clayton, Vic, Australia Engineering Program, Carnegie-Mellon University, Pittsburg, PA, USA.

and *Biomedical

The efficacy of sorbinil (Pfizer) in ameliorating the neurovascular function in inflammation ~as examined in streptozotocin (Upjohn Co., 70mg/kg) diabetic rats using non-invasive tests (Westerman et al., 1987). Untreated diabetic rats monitored at 20-day intervals up to I00 days, showed a progressive reduction in their vasodilator responses to trains of 1,2,4,8,16 and 32 pulses of electrical stimulation (100V, 2Hz, ims). The cutaneous axon reflex vasodilatation was measured by the laser Doppler flowmeter (Periflux Pfld). In the first 60 days of sorbinil treatment (4.38g/kg/24h), diabetic rats showed a steeper decline in their inflammatory responses compared to untreated diabetic rats. The size of the reduction was dependent on the strength of the stimulus. Improvement of the vasodilator responses was observed after the 60-day testings. These responses were partially restored with time. A single dose of subcutaneously administered insulin (3 U) restored the reduced responses fully (not significantly different from pre-diabetic responses). Results of this study suggest that the polyol pathway is in part responsible for the reduced vasodilator responses. Furthermore, restoration of responses by insulin suggests that the presence of insulin and/or normoglyeemic condition are/is necessary for maintaining normal axon reflex vasodilatation.

Westerman et al. (1987) Clin. Exp. Neurol. 23: 81-89.

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Page 6: Diabetic complications/Basic mechanisms I

POS-003-125 BLOOD VISCOSITY IS ELEVATED IN DIABETIC DOGS AND GALACTOSEMIC DOGS DEVELOPING RETINOPATHY.

T.S. KERN, D. SOBKOWICZ, R.L. ENGERMAN and C.M. PETERSON University of Wisconsin, Madison, Wisconsin and Sansum Medical Research Foundation, Santa Barbara, California

Supranormal blood viscosity is associated with retinopathy in diabetic patients, and may play a role in its development. We have compared blood viscosity in alloxan diabetic dogs and experimentally galactosemic dogs, both models known to develop a retinopathy similar to that seen in patients. Viscosity of whole blood, plasma and washed erythrocytes was measured with a narrow gap concentric cylinder viscosimeter (Silenus Instruments) at low (0.1 sec -1) and high (100 sec -1) shear rates. Viscosity of whole blood from insulin-deficient diabetic dogs was significantly greater than normal at either shear rate (P<0.05). In galactosemic dogs, whole blood viscosity was found to be significantly greater than normal at low shear rate (not at high shear rate). Plasma viscosity showed significant correlation with plasma fibrinogen concentrations. No abnormality in the viscosity of washed erythrocytes (tested at 40% v/v) was observed in any group. The presence of supranormal blood viscosity in both animal models of diabetic retinopathy is consistent with a potential role of hyperviscosity in the etiology of the retinopathy.

POS-003-126 A METHOD FOR COMPARING ERYTHROCYTE AND PLASMA PERFUSION OF THE RETINAL MICROVASCUIATURE, IN NORMAL AND DIABETIC RETINAE.

V.A.ALDER, J.B~-NUN & I.J.CONSTABLE. Lions Eye Institute, University of Western Australia, Nedlands W.A. Australia.

Microvascular changes in diabetic retinopathy involve pericyte loss, loss of endothelial and epithelial cells, acellular capillaries and finally complete capillary closure. Currently only the last stage in this process is detectable by angiography. Our hypothesis is that in an earlier stage there is plasma perfusion but reduced and finally no erythrocyte perfusion of the capillaries, resulting in retinal hypoxia. We have devised a histological method for demonstrating erythrocyte and very small particle perfusion in the same capillaries, at all retinal locations. The method involves injecting fluorescent microspheres, diameter 0.2 micron via a continous slow intravenous infusion. The spheres do not disturb normal perfusion by erythrocytes. When the spheres are distributed throughout the body both eyes are enucleated and fixed in 10% paraformaldehyde. The retina is whole mounted and stained with Martius yellow which stains erythrocytes. The whole mount is then observed and photographed under light and fluorescent microscopy : the former showing the positions of the erythrocytes in both capillary beds of the retina and the latter shewing the location of the microspheres. Any vessel patent to microspheres will be patent to plasma. With this technique it is therefore possible to demonstrate that normal retinae have uniform perfusion of erythrocyte and pla~na. We now plan to use the technique in a rat induced model of the early vascular changes in diabetes to test our hypothesis that in early stages of diabetic retinopathy there will be capillaries perfused by plasma only.

$420

Page 7: Diabetic complications/Basic mechanisms I

POS-003-127 ALDOSE REDUCTASE INHIBITION EFFECTS ON MICROALBUMINURIA, PLATELET AGGREGATION AND MUSCLE CAPILLARY BASEMENT MEMBRANE THICKENING IN PATIENTS WITH PERIPHERAL NEUROPATHY.

P E JENNINGS, S N NIGHTINGALE, N LAWSON, C LeGUEN, J H WILLIAMSON~ A H BARNETT Department of Medicine, East Birmingham Hospital, Birmingham and Washington School of + Medicine, St. Louis, Missouri, U.S.A.

Animal s t u d i e s show t h a t a l d o s e r e d u c t a s e i n h i b i t i o n (ARI) p r e v e n t s p r o t e i n u r i a and b a s e m e n t membrane t h i c k e n i n g (CBMT). We i n v e s t i g a t e d ARI on t h e s e p a r a m e t e r s and p l a t e l e t a g g r e g a t i o n d u r i n g a p l a c e b o c o n t r o l l e d d o u b l e b l i n d n e u r o p a t h y t r i a l . S e v e n t e e n p a t i e n t s r a n d o m l y a l l o c a t e d t o p l a c e b o o r S o r b i n i l (250 mg od) c o m p l e t e d t h e 2 y e a r s t u d y . E i g h t t o o k S o r b i n i l and w e r e m a t c h e d f o r a g e and d u r a t i o n o f d i a b e t e s t o t h e p l a c e b o g r o u p . Albumin e x c r e t i o n r a t e s (AER) w e r e m e a s u r e d f r o m 2 h o u r s u p i n e u r i n e c o l l e c t i o n s , p l a t e l e t a g g r e g a t i o n was e s t i m a t e d f r o m p e a k r e s p o n s e s t o a d r e n a l i n e , ADF and c o l l a g e n and CBMT f r o m f i x e d g a s t r o c n e m i u s m u s c l e b i o p s i e s . M e a s u r e m e n t s w e r e p e r f o r m e d a t b a s e l i n e and 6 m o n t h l y i n t e r v a l s d u r i n g wh ich HbAI% and Bp r e m a i n e d c o n s t a n t . Both g r o u p s had s i m i l a r AER a t b a s e l i n e . In t h e place~oo g r o u p AER r o s e s i g n i f i c a n t l y f r o m 3 7 . 7 ± 58 u g / m i n t o 7 0 . 5 ± 102 u g / m i n ( p < 0 . 0 2 5 ) r e m a i n i n g u n c h a n g e d a f t e r two y e a r s i n t h e t r e a t m e n t g r o u p ( 3 4 . 5 ± 51 u g / m i n ) . B a s e l i n e p l a t e l e t r e s p o n s e s w e r e i n c r e a s e d s i m i l a r l y in b o t h g r o u p s b u t f e l l i n t h e t r e a t m e n t g r o u p o n l y , t o b o t h c o l l a g e n and ADP f r o m 84 ± 8% and 85 ± 5% t o 71 ± 10% (p<O.02 ) and 73 ± 20% ( p < 0 . 0 2 5 ~ r e s p e c t i v e l y a f t e r 2 y e a r s , o CBMT was n o t i n f l u e n c e d by t r e a t m e n t b e i n g 1622 ± 530 A a t b a s e l i n e and 2137 ± 741 A a f t e r 2 y e a r s , s h o w i n g a s i m i l a r p e r c e n t a g e i n c r e a s e a s s e e n in t h e p l a c e b o g r o u p . ARI may, t h e r e f o r e , p r e v e n t t h e p r o g r e s s i o n o f m i c r o p r o t e i n u r i a and i m u r o v e p l a t e l e t r e a c t i v i t y .

POS-003-128 THE ROLE OF GLOMERULAR CHARGE BARRIER FOR THE MICROALBUMINURIA IN EARLY DIABETIC RATS

H TSUCHIDA~ K HIROSE? M NOZAWA~, I OTSU *~4~ ~Sakura National Mospital,~Univ. of Library and Information Science,

~Dept. of Surg. Josai Dental University, Japan

Charge barrier defect (CBD) in glomerular basement membrane (GBM) of kidneys is proposed as a major mechanism for the microalbuminuria (MA) in diabetes. This study by electronmicroscopy was carried out in order to explore the contribution of CBD to MA in very early diabetes. The density of polyethyleneimine (PEI) stained anionic sites in GBM was investigated at I, 3, 5, 7 and 14 days after induction of diabetes in the untreated streptozotocin diabetic rats (D), and 24 hour urinary albumin excretion (UAE) was examined at 3, 7 and 14 days. Normal control rats (C) were also subjected to these morphological and functional kidney exami- nations. PEI treated renal biopsy materials were obtained following the methods of Schurer et al and urinary albumin was measured by double anti- body radioimmunoassay. Compared with the normal values in C, 24 hour UAE was abnormally high at the 3rd day and was followed by further increase. However, there was no reduction in the number of PEI stained sites per unit length of GBM at each time point studied. It is concluded that CBD in GBM is unlikely to be responsible for MA in very early diabetes.

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POS-003-129 ASCORBIC ACID DEFICIENCY AND THE POLYOL PATHWAY IN DIABETIC RATS.

S McLENNAN, S HEFFERNAN, C CAPOGRECO, E FISHER, G ROSS, DK YUE, JR TURTLE. Department of Medicine, University of Sydney and the Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia.

Ascorbic acid (AA) is required for collagen synthesis and its deficiency my be important in the pathogenesis of some diabetic complications. We reported previously that plasma AA is decreased in diabetic rats and can be normalized by the aldose reductase inhibitor Tolrestat (T, Ayerst). To further study the mechanism of this phenomenon, plasma and urinary AA was measured by HPLC in normal(N), diabetic (D, 65mE STZ/kg) and D rats treated (DRx) for 4 weeks with T (5mglkg), myoinositol (M, 3Z in diet) or AA (lgm/L in water). Excretion of AA was also measured after iv injection of C14 labelled AA (7.5uCi).

The plasma AA was decreased and urinary AA increased in D. These abnormalities were substantially normalized by T or M. By contrast AA normalized plasma AA but further increase its urinary excretion.

GROUP TOLRESTAT Put MYOINOSITOL Rx AA Rx Plasma Urine Plasma Urine Plasma Urine

N . 7 0 . 5 + 1 4 . 4 1 8 . 0 + 1 . 8 6 3 . 8 + 13.7 7.1 +3 .8 48.9 + 7.4 7 . 0 + 3.4 D 30.8 + 11.5 59.9 + 3.3 19.2 + ii.0 27.7 +--0.5 14.6 + 8.2 25.1 + 1.4 DRx 75.9 + 37.9 36.4 + 2.6 49.5 + 4.9 13.9 +-0.3 109.0 + 4.5 57.8 + 2.5

* Different from N and DRx, n=5-10, ANOVA, p<0.05, Mean~ SD In the ~ix hours after injectiog of C14 AA (41group), tracer excretion was N-l.8+_0.7xl0

D-3.6+_0.Yx10-, DRxwith M=2.4+_l.2x10- DPM. These results confirmed the relationship between AA abnormalities and polyol metabolism

in diabetes. The excretion of AA is increased in diabetes, possibly due to defective Na-K ATPase activity which is important for renal AA reabsorption. M normalized plasma AA in diabetes partly, but not completely, by correcting this excessive urinary AA loss. Supported by NHMRC, Hoechst and Kellion Foundations of Australia.

POS-003-130 EXCHANGEABLE SODIUM, VOLUME STATUS AND GLOMERULAR FILTRATION RATE IN EXPERIMENTAL DIABE'I'F_~ - THE ROLE OF GLYCAEMIC CONTROL.

TJ ALLEN, ME COOPER, RC O'BRIEN, L FRANZE, B JACKSON, G JERUMS Endocrine Unit and Department of Medicine, Austin Hospital, Heidelberg, Australia, 3084.

It has been suggested that altered sodium and water balance may play an important role in the development of hyperfiltration in diabetes (1). This study has investigated the effect of changes in glycaemic control on exchangeable sodium (Ex Na), plasma volume (PV) and glomerular filtration rate (GFR) in 2 strains of rats, Wistar Kyoto (WKY) and Sprague-Dawley (SD) with streptozotocin-diabetes. Within each strain, rats were randomised to four groups: (i) DIAB - no insulin. (ii) 3xINS - 2.4 units of Ultralente Insulin 3 times a week (iii) DAILY - insulin dose titrated daily to maintain euglycaemia (mean dose - 8 units per day) and (iv) CON - Control rats. Plasma glucose (mM), Whole body Ex Na (mmol/kg) by equilibrium 22Na counting, PV (ml/100g) using iodinated rat albumin and GFR (ml/min) by 99mTc DTPA clearance (2) were determined after 4 weeks.

Diabetes increased ExNa in both strains (WKY, DIAB 53.3 + 1.2 vs. CON 43.8 + 0.7; SD, DIAB 56.5 + 1.4 vs CON 42.8 + 0.5 mmol/kg, both p<0.001, mean + SEM). PV paralleled changes in ExNa (SD, DIAB 5.4 + 0.2 vs CON 3.8 + 1.0 ml/100g, p<0.001). 3xlNS reduced plasma glucose in WKY (DIAB 36.5 5:1.0 vs. 3xlNS 29.1 :t: 1.2 mM, p<0.01) with an associated normalisation of ExNa (42.2 5: 0.7, p<0.001) and PV. However, 3xlNS in SD failed to alter plasma glucose levels (DIAB 37.2 5:1.6 vs 3xlNS 37.0 + 2.0 raM) and did not normalise ExNa (51.2 5: 1.4) or PV (5.4 5: 0.3). In WKY rats, a slight decrease in plasma glucose was associated with an increase in GFR when compared to controls (3xlNS 3.7 5:0.2 vs. CON 3.0 5: 0.1ml/min, p<0.01) whereas in untreated rats with more severe hyperglycaemia there was no increase in GFR (DIAB 3.0 5:0.3 vs.CON 3.3 + 0.3 ml/min). Daily insulin therapy normalised all parameters in both strains.

Severe hyperglycaemia increased ExNa and PV in both strains without a rise in GFR. Slight reduction of plasma glucose normalised ExNa and PV but GFR increased paradoxically. This suggests that the hypertiltration of experimental diabetes is not directly related to sodium balance or volume status. 1. Ortola FV et al, J Clin Invest 80, 670 (1987). 2. O'Brien RC et al. J Diab Comp (in press, 1988).

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POS-003-131 EFFECTS OF GLUCOSE AND INSULIN ON MYO-INOSITOL UPTAKE BY CULTURED RAT MESANGIAL CELLS.

S ROGERS, M DUNLOP, M HILL, R LARKINS University of Melbourne Department of Medicine, Royal Melbourne Hospital, Parkville, Australia.

Diabetic complications in kidney, peripheral nerve, retina and aorta have been attributed to compromised cellular Na+-K+-ATPase activity and metabolism, due to increased polyol pathway activity and decreased myo-inositol (MI) uptake. Sodium-dependent uptake of (2-3H)-MI by mesangial cells obtained by cloning glomerular explants was examined under conditions designed to simulate the diabetic environment. Mesangial cells were cultured in DMEM with 10% fetal bovine serum (FBS). The long-term effect of insulin on these cells was examined in a group of cells which were cultured through all passages with 40ug/ml insulin. For 16 to 22 hr prior to measurement of (2-3H)-MI uptake, both groups of cells were incubated without added insulin in medium containing 5.6 mmol/l glucose and i0% FBS. Uptake of (2-~)-MI (lOpCi, final inositol concentration 4xlO s mol/l) was measured over 20 min at 5.6 and 25 mmol/l glucose, and related to cellular protein

content. The results presented as the mean ±SEM are from a representative experiment in which MI uptake was determined in three independent cell incubations. Similar results were obtained in four other experiments. Compared with uptake of (2-~)-MI at 5.6 mmol/l glucose, uptake by cells not exposed to insulin was decreased by acute exposure to 25 mmol/l glucose (89±I Vs 69±2 dpm/Bg protein/20 min, p<O.O01). This effect was also seen in cells cultured long-term in insulin (147±4 Vs 121±9 dpm/ug protein/20 min, P<O.05). Consistent with previous observations of the inhibitory effect of glucose on MI uptake in other cell types, the Ki value for these mesangial cells was 21 mmol/l. Long-term exposure of mesangial cells to insulin in culture increased MI uptake at 5.6 mmol/l glucose (P<O.O01) and 25 mmol/l glucose (P<O.OI). At 5.6 mmol/l glucose, MI uptake by cells not previously exposed to insulin was increased by acute exposure to 4ng/ml insulin (89±1Vs 104±I dpm/~g protein/20 min, P<O.O01). From these observations, a decrease in MI uptake may be expected in mesangial cells in poorly controlled diabetes, and may be responsible for early functional changes in diabetic glomeruli.

POS-003-132 GLOMERULOPATHY IN VACOR-INDUCED DIABETIC MONGOLIAN GERBIL

T.N. LEE, M.Y. CHUNG, S.H. KIM, J.R. YOON Cho~nam University, Kwangju, Korea

It is well known that Vacor, rat poison, developes hyperglycemia in human

and animals, and the patients have also various complications such as ketosis,

cataract, neuropathy and nephropathy even in the early stage of the disease.

This study is the electron-microscopic observation of the kidney of Vacor-

induced diabetic Mongolian Gerbil. The doses of oral administration of Vacor

were 100mg(Vl) and 200mg(V2 group) per kg of body weight. The fine glomerular

structures were observed in acute stage of Vacor poisoning, and the thickness

of basal laminae was measured using the orthogonal intercept method.

MesangJal alterations were noted in Vl and V2 groups. The mean epithelial

basal lamina thickness was 334.0 ! 41.73 nm (~ SD) in control group, and 381.5

90.40 nm in Vl group. The thickness of basal lamina in V2 group was increas-

ed significantly (656.8 ~ 112.70, p <0.005 ) as compared with that of control

and Vl groups.

This study suggests that Vacor with large dose can develope glomerulopathy

in the early stage of disease.

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POS-003-133 STRICT CONTROL OF BLOOD GLUCOSE IMPROVES THE HEMORHEOLOGICAL ABNORMALITIES IN DIABETES

Y ISOGAI, S IKEMOTO, M AKIYAMA, K ITOH, J OGAWA, K KUCHIBA, T MAEDA, Y YOKOSE Department of Internal Medicine, The Jikei University School of Medicine, Tokyo, Japan.

The aims of the study are firstly to elucidate the pathophysiological role of several hemorheological parameters in the development of diabetic microangiopathy and secondly to test the hypotheses that the strict control of blood glucose can prevent the aggravation of microangiopathy through the improvement of hemorheological abnormalities. Seventy-four diabetics(13 IDDM and 61 NIDDM) were investigated to observe the hemorheological changes. Blood viscosity, plasma viscosity, blood viscoelasticity and plasma fibrinogen concentration were determined before and after the strict control of blood glucose with artificial endocrine pancreas (Biostator ). Several hemorheological abnormalities already appeared in the diabetics without microangiopathy and most of all hemorheological parameters showed abnormal values in advanced stage of microangiopathy. These abnormalities were improved significantly after the near normal control of blood glucose. But this improvement of hemorheological parameters was difficult to be attained with the conventional insulin therapy. After the treatment of artificial pancreas the intensive insulin therapy was continued, but the improvement of hemorheology could not be necessarily maintained with this method. These results suggest that the microcirculatory disturbances reflected by the abnormalities of rheological parameters can be improved by the strict control of blood glucose and that hemorheological improvement may ameliorate the course of the microangiopathy, although it would be difficult to maintain good control state both in metabolism and in hemorheology.

POS-003-134 ANTIPLATELET EFFECTS OF K-MAP

T KARIYAI),T SA~AII),s SUZUKI~),S KUME3),H OKA h) , Saga Med.Coll.11,Showa Univ. 2 ,Yamanashi Med. Coll.3;,Univ.of Tokyo ~)

Enhanced platelet aggregation is thought to be involved in the pathogenesis of diabetic angiopathy. Prostagladin(PG) modulates platelet aggregation and K-MAP, which is p-aminobenzoic acid-N-D- mannoside sodium salt, is known to affects PG's metabolism in some cells.

Therefore, we evaluated the effect of K-MAP on aggregation and PG metabolism of platelet to clarify if K-MAP is useful for the treat- ment of diabetic angiopathy. K-MAP was added to human platelet-rich plasma(PRP) in vitro and platelet aggregation induced by various agonists was measured. K-MAP inhibited platelet aggregation in a dose-dependent manner and enhanced PG production. In another experi- ment, we examined the effects K-MAP administered for 2 weeks to STZ-induced diabetic rats. STZ diabetic rats showed markedly enhanced platelet aggregation, but K-MAP normalized the aggregation.

Pilot study in diabetic angiopathy was performed. Platelet aggregation was corrected in 65 percent(13/20). 8-TG and PF4 values significantly reduced, and 6Keto-PGFl~ (metabolite of PGI 2) also significantly increased as compared to pre-administer value.

Thus, K-MAP inhibited platelet aggregation not only in vitro but also in vivo. Therefore, K-MAP may be useful for correcting platelet function associated with diabetic angiopathy.

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POS-003-135 EFFECTS OF ALDOSE REDUCTASE INHIBITOR (0N0-2235) ON INCREASED URINARY ALBUMIN EXCRETION IN DIABETIC PATIENTS - A DOUBLE BLIND TRIAL-.

M YAMA~OTO, S FUJII. T SATO, H NORII, N SHIMOJO@ Department of Internal Medicine, Laboratory Medicine~, Osaka City University medical school, Osaka, japan There has been increasing evidence that so rb i to l accumulation in glomeruli may be pathogenet ical ly l inked to the development of d iabet ic nephropath~. 1'o see whether a potent aldose reductase inh ib i t o r (ARI), 0N0-2235 (Ono Pharmaceutical Co., Osaka, Japan), is e f fec t i ve for d iabet ic renal damase, we compared the chan~es in the ur inary albumin measured by RIA, N-acety l -#-D-glucosamini - dase (NAG) and ~ , -m ic rog lobu l i n before and a f te r drug adminis t rat ion. Twenty-£ix d iabet ic pat ients wi thout hypertension were selected for albumin excretion measured in the f i r s t morning ur ine being above the normal l im i t . 27.5mJ8 creat in ine (Cr), and less than 200. The pat ients were put into two groups matched for ur inary albumin, age, d iabet ic durat ion and HbA~c, and given 0N0-2235 (]50mg/day) or a placebo o r a l l y for 8 weeks. Urinary albumin excret ion was low 4 weeks a f te r ARI treatment star ted (52 .6±2 ] . 0 vs. 29.0±]7.0m8/8 Cr, mean±SD, P<O.02) and at 8 weeks (32 .9±20.3 , P<O.02), hut chan~es with placeho adminis t rat ion were not s i gn i f i can t (53 .5±2 ] .7 , 39.8±26.0, and 50.6±3~.6, respect ive ly ) . Patients in the placebo group were given ARI a f te r the p!ac~ho admin is t ra t ion ended, and they had decreased ur inary albumin 8 weeks la ter (34.7±12.4 vs. 26 .0±8.6 , P<0.05) . Urinary NAG levels before. 4 weeks a f t e r , and 8 weeks a f te r ARI treatment were 9 .2± 5.3, 12.8±8.2, and ]O. ]±5 .?L /~ Cr, and a f te r placebo treatment, 9 .6±5 .5 , 11.6±4.2, 9 .3±Q.3, respect ive ly : the di f ferences were not s i gn i f i can t . There was no s ign i f i can t d i f f e r - ence in ur inary (4:-microglobu) in before and a f te r the adminis t rat ion of ARI or the placebo. NF, ifhe¢" HbA:c n{)r t,]ood pressure changed s i g n i f i c a n t l y during the study. These resul ts suggest that a!dose reducta£e i nh ib i t o r may be e f f ec t i ve for mild d iabet ic glomerular disorders.

POS-003-136 EXPERIMENTAL DIABETES AND CENTRAL HYPOXAEMIA CAUSE SIMILAR REDUCTIONS IN OUABAIN- SENSITIVE ATPASE ACTIVITY IN RAT SCIATIC NERVE HOMOGENATES

D R TOMLINSON, S B McDONOUGH, H C MASTERS, G B WILLARS, A M BROWN Department of Physiology and Pharmacology, Medical School, Nottingham NG7 2UH, U.K.

The aim of this work was to determine whether ischaemic hypoxia of the endoneurium in peripheral nerves might part icipate in the development of a biochemical defect character is t ical ly present in short-term experimental diabetes of the rat. The defect studied was impaired ouabain-sensitive ATPase ac t i v i t y in the sciatic nerve homogenates. Possible influences of endoneurial hypoxia were studied by induction of central hypoxaemia. Thus, rats with 4 week streptozotocin-diabetes were compared with non- diabetic rats maintained for 4 weeks in an atmosphere containing 10% oxygen in nitrogen and with a normoxic control group. Motor nerve conduction veloci ty (MNCV) was measured at the end of the 4 week protocol in al l rats as a variable which exhibits a well- characterised de f ic i t in experimental diabetes. A few days la ter the rats were k i l led , sciat ic nerves removed, homogenised and subjected to assay of ATPase assay in the pr~segce and absence of ouabain (5mM) to detemine the ouabain-sensitive (presumptive Na-/K-) ATPase ac t i v i t y .

The diabetic rats showed a s ign i f icant ly reduced MNCV (43.6±3.6 (SD) m/sec; p<O.05) compared to controls (48.9±1.4 m/sec). Hypoxia was without effect on MNCV (47.9±3.6 m/sec). Ouabain-sensitive ATPase ac t i v i t y (nmol ADP formed/h/cm nerve ± 1SD) was reduced in nerves from diabetic rats (93±136) and from hypoxic rats (220±177); changes which were signif icant (p<O.01 and p<O.05 respectively) when compared with the ac t i v i t y of control nerves (617±466), Thus hypoxia of peripheral nerve can cause defective ouabain-sensitive ATPase ac t i v i t y , but over the same time-course does not affect MNCV.

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