- Email: [email protected]
The role of glycation in diabetic retinopathy
Thursday, Sep 24, 1992 La Palms/B
X ICER Abstracts
561
7 MYO-INOSITOLTRANSFORTINCULTLJRBDL.BNSBpflwELRTM: VITRO MODEL FOR HYPERGLYCEMIC-ASSOCIATED COMPLICATIONS
THE ROLE OF GLYCATION IN DIABETIC t &&mes. H.-P.‘, and Brw. N. *III. Bed. Klinik und Poliklinik,
ANIN DIABETIC
-Universitat, Albert
t.TexasCoIkgeof Texas 76107
osteopathic MedicintRlniversl
fa mye-ino&oI
uptake
Treatment inhibitor 2.6-fold precapillary occurred.
with myo-inositol
Research
Center,
New York
kinoguanidine treatment completely cell proliferation and significantly
and suggest in this
prevented diminished
abnormal pericyte
that
that advanced to the development
aminoguanidine
glycosylation of diabetic
may have future
product retinopathy,
therapeutic
utility
disorder.
depletion.
8
THE ROLE OF DIACYLGLYCEROL AND PROTEIN KINASE C CHANGES IN THE DEVELOPMENT OF DIABETIC RETINOPATHY. K T. ,“,““i. In T.. Bursell. SE, Joslin Diabetes Center, Harvard%i?&f!&o&n,
Simon P Pharmacology Medicine, of Medicine,
In animal studies total DAG aad membrane PKC kvels are increased in multiple
560 HYPOXIA
AND DIABETIC
Washington
University,
RETlNOPATHY St. Louis,
OXIDATIVE
STRESS
AND
DIABETES
Wolff,
Toxicology Laboratory, Clinical and Toxicology Section, Department of University College and Middlesex School 5 ilniversity Street, LONDON WClE 6JJ
Hyperglycaemia is considered to be an important risk Factor for cataract and retinopethy in diabetes mellitus. The hypotheses of non-enzymatic ulycosylation (glycntion) and polyol accumulation (vi*1 II I dose rx.dUC( nsc , have more rccr?nt.ly led elevatrd glycnemia to be considered as a causative fnctov of t hesr oc111ar compl ications. There are, however, dQ!ibt,S that elevated glycaemia is a cnuset1ve factor in the 0CUl.W diabetic complications. There are also separate concerns that the polyol and glycation hypotheses may not be relevant to their aetiology. Nevertheless studies of these hypotheses of glucose toxicity have provided important data concerning development of the diabetic complications and have led to the formulation of a hypothesis involving oxidative stress and long term tissue alterations initiated by abnormalities in transition metal metabolism. The evidence in support of this new hypothesis of transition metal-catalyzed oxidetive stress as a causative factor in the 0CUlS.r diabetic complications will be reviewed.
vascular tissues in the diabetic rat, such as retina, aorta, heart, and renal glomemli, but not in tbe brain or liver. Similarly, in cultured vascular endothelial cells and smooth muscle cells elevation of glucose in the media will also increase total DAG and PKC membranous activity as well. By Western blotting, we have identified that PKC& isoform appears to be activated by glucose or diabetes as compared to the (Y isoform. Physiologically, using digitali& computer assisted fluorescein angiography, we have found that after one week of diabetes there is a prolongation of mean circulation time (MCT) suggesting a decrease in retinal blood flow. This abnormal prolongation in MCT is correlated with increases in DAG and PKC. Inhibitors of PKC such as staumsporin can normalize the MCT in diabetic. In addition PKC activator such as PDBU intravitreously applied will prolong MCT to similar level as observed in diabetes. Tight glycemic control with insulin will normalize both PKC and DAG changes and MCT. These data suggests that the diabetes will increase DAG and activate PKC activity which may play a role in regulating retinal hemodynamics of diabetic animal and humans. The resulting effect of normalizing hemodynamic changes may lead to prevention of pathologies observed in diabetic vascular diseases.
6
562
HYPERGLYCAEMIA,
Hyperglycemia has been shown to be a major causal factor for the development of diabetic retinopathy. Recently we have found that diabetes and elevation of glucose will increase the level of diacylglycerol (DAG) and activate protein kinase C (PKC) activity. Since these two biochemical parameters have been shown to alter many hemodynamic functions which are abnormal in diabetes, we postulate that these changes in DAG aad PKC may pIay a roIe in the development of diabetic retinopathy.
of Pathology,
Bronx,
rats for 26 weeks with aminoguanidine, an glycosylation product formation, prevented a of these products at branching sites of where abnormal PAS positive deposits also
These findings indicate accumulation contributes
559
Dept.
of Medicine,
Justus-Liebig and ‘Diabetes
dropout. After 75 weeks, untreated animals developed an lB.6-fold increase in the number of acellular capillaries and forned capillary microaneurysms. In contrast, aminoguanidine-treated diabetic animals had only a 3.6.fold increase in acellular capillaries, and no microaneurysms.
on tbc high- &initytransportsitewasuncovend
5
HYPERGLYCEMlC
College
of diabetic of advanced accumulation arterioles
endothelial
Dixon plot analysis revealed that glucose was a competitive inhibitor of the highaffinity transport site. Exposure of cultured cells to exogenous sorbitol also diminished myo-inositol accum&tian. Dixon plot analysis established that the effect of sorbitol was due to axupuitive inhibition of the low-affinity myoinositol trans site. *se data su port the contention that myc+inositol transport in B c? Cs is maintained by at PeBst two processes: a glucose-sensitive, sodiumdependent, high-affinity my&msitol transport system; and a sorbitolsensitive, sodium-dependent, low-affinity myo-inositol transport system. Moreover, these results suggest a plausible mechanism whereby hyperglycemia is associated
Germany,
Increased retinal vascular permeability and progressive retinal vessel closure are the principal pathophysiologic abnormalities underlying diabetic retinopathy. Non-enzymatic modification of extracellular and intracellular components by hyperglycemia may contribute to both these processes by a variety of mechanisms.
The underlying biochemical deftits which contribute to the pathogenesis of diabetic ccmplitzatiom IO tbe kns Md other tissues mmains c~~~tmvc.rsiaI. Despite thidentiRcatimtof*&ndnt~manifesaiionsscellcultureandanimal models of the disuse, explanations ot the me&a&m leading to c&ilar damage are primwily specptuive. The stahanism by which hyperglycemia &its dte depletion of inteac&kr myo-htositol remains unexplained. Myo-inositol uptake in cultured lmvine leas epithelial crlls (BLECs) is linear and greater than 95% sodiumdependa. High- and low-affmity uans sites have been identified. Glucose attenuuca myo-inositol uptake in a r ose-depeudent fashion. The concomitant addition of the akbse ttductare inhibitor, sorbii, partially prevents the. dezrcase in m o-inositol uptake by ghtcose. While sorbmil prevented the inhibiting effect cl glucose on the low-aft3 aansport site, a glucose-sensitive process
Einstein
Giessen,
RETINOPATHV
MO. U.S.A.
Recent investigations in our laboratory suggest that many of the metabolic imbalances and mechanisms of glucotoxicty linked to early glucose/diabetes-induced structural and functional vascular changes either contribute to or are the consequence of an increase in the ratio of cytosolic NADHINAD+. In vitro incubation of retinas (removed from normal rats) in 30 versus 5 mM glucose for only 2 h causes a marked increase in the ratio of lactatelpyruvate (a reliable parameter of the cytosolic ratio of free NADH/NAD+) which is completely prevented in retinas from rats treated with an inhibitor of akiose reductase. Since an increase in the ratio of NADH/NAD+ is an immediate and characteristic response to hypoxia (i.e. a decrease in tissue pop) or to cyanide poisoning (both of which inhibit oxidation of NADH to NAD+) and is associated with vasodilation and increased blood flow, we hypothesize that glucose-induced, sorbitol pathway-linked vasodilation and increased blood flow may be the consequence of an increase in NADH/NAD+, i.e. hyperglycemic hypoxia, resulting from increased oxidation of sorbitol to fructose which is coupled to reduction of NAD+ to NADH.
S-167
X ICER Abstracts
561
7 MYO-INOSITOLTRANSFORTINCULTLJRBDL.BNSBpflwELRTM: VITRO MODEL FOR HYPERGLYCEMIC-ASSOCIATED COMPLICATIONS
THE ROLE OF GLYCATION IN DIABETIC t &&mes. H.-P.‘, and Brw. N. *III. Bed. Klinik und Poliklinik,
ANIN DIABETIC
-Universitat, Albert
t.TexasCoIkgeof Texas 76107
osteopathic MedicintRlniversl
fa mye-ino&oI
uptake
Treatment inhibitor 2.6-fold precapillary occurred.
with myo-inositol
Research
Center,
New York
kinoguanidine treatment completely cell proliferation and significantly
and suggest in this
prevented diminished
abnormal pericyte
that
that advanced to the development
aminoguanidine
glycosylation of diabetic
may have future
product retinopathy,
therapeutic
utility
disorder.
depletion.
8
THE ROLE OF DIACYLGLYCEROL AND PROTEIN KINASE C CHANGES IN THE DEVELOPMENT OF DIABETIC RETINOPATHY. K T. ,“,““i. In T.. Bursell. SE, Joslin Diabetes Center, Harvard%i?&f!&o&n,
Simon P Pharmacology Medicine, of Medicine,
In animal studies total DAG aad membrane PKC kvels are increased in multiple
560 HYPOXIA
AND DIABETIC
Washington
University,
RETlNOPATHY St. Louis,
OXIDATIVE
STRESS
AND
DIABETES
Wolff,
Toxicology Laboratory, Clinical and Toxicology Section, Department of University College and Middlesex School 5 ilniversity Street, LONDON WClE 6JJ
Hyperglycaemia is considered to be an important risk Factor for cataract and retinopethy in diabetes mellitus. The hypotheses of non-enzymatic ulycosylation (glycntion) and polyol accumulation (vi*1 II I dose rx.dUC( nsc , have more rccr?nt.ly led elevatrd glycnemia to be considered as a causative fnctov of t hesr oc111ar compl ications. There are, however, dQ!ibt,S that elevated glycaemia is a cnuset1ve factor in the 0CUl.W diabetic complications. There are also separate concerns that the polyol and glycation hypotheses may not be relevant to their aetiology. Nevertheless studies of these hypotheses of glucose toxicity have provided important data concerning development of the diabetic complications and have led to the formulation of a hypothesis involving oxidative stress and long term tissue alterations initiated by abnormalities in transition metal metabolism. The evidence in support of this new hypothesis of transition metal-catalyzed oxidetive stress as a causative factor in the 0CUlS.r diabetic complications will be reviewed.
vascular tissues in the diabetic rat, such as retina, aorta, heart, and renal glomemli, but not in tbe brain or liver. Similarly, in cultured vascular endothelial cells and smooth muscle cells elevation of glucose in the media will also increase total DAG and PKC membranous activity as well. By Western blotting, we have identified that PKC& isoform appears to be activated by glucose or diabetes as compared to the (Y isoform. Physiologically, using digitali& computer assisted fluorescein angiography, we have found that after one week of diabetes there is a prolongation of mean circulation time (MCT) suggesting a decrease in retinal blood flow. This abnormal prolongation in MCT is correlated with increases in DAG and PKC. Inhibitors of PKC such as staumsporin can normalize the MCT in diabetic. In addition PKC activator such as PDBU intravitreously applied will prolong MCT to similar level as observed in diabetes. Tight glycemic control with insulin will normalize both PKC and DAG changes and MCT. These data suggests that the diabetes will increase DAG and activate PKC activity which may play a role in regulating retinal hemodynamics of diabetic animal and humans. The resulting effect of normalizing hemodynamic changes may lead to prevention of pathologies observed in diabetic vascular diseases.
6
562
HYPERGLYCAEMIA,
Hyperglycemia has been shown to be a major causal factor for the development of diabetic retinopathy. Recently we have found that diabetes and elevation of glucose will increase the level of diacylglycerol (DAG) and activate protein kinase C (PKC) activity. Since these two biochemical parameters have been shown to alter many hemodynamic functions which are abnormal in diabetes, we postulate that these changes in DAG aad PKC may pIay a roIe in the development of diabetic retinopathy.
of Pathology,
Bronx,
rats for 26 weeks with aminoguanidine, an glycosylation product formation, prevented a of these products at branching sites of where abnormal PAS positive deposits also
These findings indicate accumulation contributes
559
Dept.
of Medicine,
Justus-Liebig and ‘Diabetes
dropout. After 75 weeks, untreated animals developed an lB.6-fold increase in the number of acellular capillaries and forned capillary microaneurysms. In contrast, aminoguanidine-treated diabetic animals had only a 3.6.fold increase in acellular capillaries, and no microaneurysms.
on tbc high- &initytransportsitewasuncovend
5
HYPERGLYCEMlC
College
of diabetic of advanced accumulation arterioles
endothelial
Dixon plot analysis revealed that glucose was a competitive inhibitor of the highaffinity transport site. Exposure of cultured cells to exogenous sorbitol also diminished myo-inositol accum&tian. Dixon plot analysis established that the effect of sorbitol was due to axupuitive inhibition of the low-affinity myoinositol trans site. *se data su port the contention that myc+inositol transport in B c? Cs is maintained by at PeBst two processes: a glucose-sensitive, sodiumdependent, high-affinity my&msitol transport system; and a sorbitolsensitive, sodium-dependent, low-affinity myo-inositol transport system. Moreover, these results suggest a plausible mechanism whereby hyperglycemia is associated
Germany,
Increased retinal vascular permeability and progressive retinal vessel closure are the principal pathophysiologic abnormalities underlying diabetic retinopathy. Non-enzymatic modification of extracellular and intracellular components by hyperglycemia may contribute to both these processes by a variety of mechanisms.
The underlying biochemical deftits which contribute to the pathogenesis of diabetic ccmplitzatiom IO tbe kns Md other tissues mmains c~~~tmvc.rsiaI. Despite thidentiRcatimtof*&ndnt~manifesaiionsscellcultureandanimal models of the disuse, explanations ot the me&a&m leading to c&ilar damage are primwily specptuive. The stahanism by which hyperglycemia &its dte depletion of inteac&kr myo-htositol remains unexplained. Myo-inositol uptake in cultured lmvine leas epithelial crlls (BLECs) is linear and greater than 95% sodiumdependa. High- and low-affmity uans sites have been identified. Glucose attenuuca myo-inositol uptake in a r ose-depeudent fashion. The concomitant addition of the akbse ttductare inhibitor, sorbii, partially prevents the. dezrcase in m o-inositol uptake by ghtcose. While sorbmil prevented the inhibiting effect cl glucose on the low-aft3 aansport site, a glucose-sensitive process
Einstein
Giessen,
RETINOPATHV
MO. U.S.A.
Recent investigations in our laboratory suggest that many of the metabolic imbalances and mechanisms of glucotoxicty linked to early glucose/diabetes-induced structural and functional vascular changes either contribute to or are the consequence of an increase in the ratio of cytosolic NADHINAD+. In vitro incubation of retinas (removed from normal rats) in 30 versus 5 mM glucose for only 2 h causes a marked increase in the ratio of lactatelpyruvate (a reliable parameter of the cytosolic ratio of free NADH/NAD+) which is completely prevented in retinas from rats treated with an inhibitor of akiose reductase. Since an increase in the ratio of NADH/NAD+ is an immediate and characteristic response to hypoxia (i.e. a decrease in tissue pop) or to cyanide poisoning (both of which inhibit oxidation of NADH to NAD+) and is associated with vasodilation and increased blood flow, we hypothesize that glucose-induced, sorbitol pathway-linked vasodilation and increased blood flow may be the consequence of an increase in NADH/NAD+, i.e. hyperglycemic hypoxia, resulting from increased oxidation of sorbitol to fructose which is coupled to reduction of NAD+ to NADH.
S-167