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Microvascular metabolism in diabetes
Microvascular Timothy
Metabolism
in Diabetes
S. Kern and Ronald L. Engerman
Hyperglycemia has been implicated in the development of retinal vascular disease. Consequently, the effects of excessive hexose concentration on cells of the vascular wall are receiving increasing attention. Techniques for isolating metabolically active microvessels from tissues such as those from the retina and cerebral cortex are providing new opportunities for the study of the uptake and metabolism of hexose by microvessels. Such studies indicate that hexose uptake by microvessels is not insulin dependent and that microvessels are capable of metabolizing hexose by pathways common to many diverse tissues, including anaerobic and aerobic glycolysis, pentose phosphate shunt, and glycogenogenesis. Microvessels isolated from diabetic animals metabolize glucose at a subnormal rate. Hexitol production and accumulation has been implicated in the pathogenesis of diabetic complications in a variety of tissues and might also play a role in the development of diabetic microvascular disease. We have quantitated hexitol-producing metabolic activity of retinal and cerebral microvessels isolated from dogs, a species known to develop a retinopathy similar to that seen in diabetic patients. Erythrocytes were removed by perfusion prior to microvessel isolation because they are known to have hexitol-producing activity. Both retinal and cerebral microvessels produce galactitol from galactose. and this activity is inhibited in the presence of the aldose reductase inhibitor sorbinil. The presence of hexitol-producing activity within microvessels is consistent with a possible role of polyol production in the etiology of diabetic microvascular disease. Nevertheless, it is not even clear that the primary defect leading to vascular disease in diabetes resides in the vasculature itself: nonvascular cells such as erythrocytes, platelets, and olial cells have been postulated to plav. a role in the development of the vascular lesions. The steps linking diabetic retinopathy to hyperglycemia await clarification. @ 1966 by Grune & Stratton, Inc.
M
disease accounts for much of the ICROVASCULAR morbidity and mortality of diabetes mellitus. Surprisingly little is known about metabolism in microvessels in diabetes, or the role of vascular metabolism in the etiology of diabetic microvascular disease. The metabolism of microvessels has been explored using a variety of methodologies. Histochemic and immunochemic techniques have provided information on the tissue distribution of enzymes and have allowed semiquantitative estimation of enzymatic activity.le5 More recently, it has become possible to isolate metabolically active microvessels from a variety of tissues including retina, cerebral cortex, adrenal, spinal cord, and adipose. &lo These isolated microvessels have been studied biochemically and have been used to establish cell cultures of pure microvessel cell types, such as endothelial cells and pericytes. (Renal glomeruli also can be isolated by such techniques, but glomerular metabolic activity cannot be equated with microvascular metabolism due to the presence of nonvascular as well as vascular cell types of glomeruli.) The availability of purified microvessels and their cells for study offers new opportunities for examining the effects of diabetes, insulin deficiency, and hyperglycemia on vascular metabolism.
From the Department of Ophthalmology, University of Wisconsin, Madison. Presented at the Pfizer Sorbinil Symposium-The Eflects of Sorbinil on the Pathophysiology of Diabetic Complications, Dorado, Puerto Rico, May 3kJune 2,1985. Supported in part by a Lions Club International Research and Development Award from the American Diabetes Association, and by Public Health Service grant EY00300 from the National Eye Institute. Address reprint requests to Timothy S. Kern, PhD. Department of Ophthalmology, University of Wisconsin. 1300 University Ave. Madison, WI 53706. D 1986 by Grune & Stratton, Inc. 0026-0495/86/3504-1006$03.00/O
24
INSULIN AND MICROVESSELS
Retinal and cerebral microvessels have been found to possess specific, high-affinity insulin receptors.“-‘5 The hormone can be internalized into vascular cells’6 and reportedly can be transported across endothelial cells in vitro,17 although release of insulin on the abluminal side has not been documented in situ.14 Exposure of microvessels to insulin causes a variety of metabolic responses, stimulating both the conversion of glucose into CO2 and lipid, and CAMP phosphodiesterase and glycogen synthetase activities.“,‘* Both endothelial cells and pericytes respond to physiologic levels of insulin.” Pericytes seem particularly sensitive to the hormone at least with respect to stimulation of thymidine incorporation in vitro. The number of micropinocytotic vesicles in endothelia of muscle capillaries is subnormal in insulin deficiency and tends to be normalized by insulin.‘9 Insulin deficiency in diabetes conceivably could have adverse effects on microvascular cells. Nonetheless, diabetic-like microvascular disease has been shown to develop even in the absence of insulin deficiency.” A retinopathy that seems morphologically indistinguishable from that of diabetic patients and dogs has recently been found to develop in nondiabetic dogs made “hyperglycemic” by the consumption of a galactose-rich diet2’ This microvascular pathology develops in the apparent absence of many metabolic disorders typical of diabetes mellitus such as subnormal blood concentrations of insulin and supranormal concentrations of glucose, lipids, and branched-chain amino acids. The evidence strongly suggests that hyperglycemia and its sequelae play a key role in the development of diabetic microvascular disease, at least in the retina. HEXOSE UPTAKE AND METABOLISM
BY MICROVESSELS
of other sugars are transported into retinal and cerebral microvessels by a stereo-specific, carrier-mediated transport system22-25 similar to that described in vivo.26*27This transport is saturable Glucose,
galactose,
and a variety
Metabolism, Vol 35,
No 4, Suppl 1 (April), 1986: pp 24-27
MICROVASCULAR
METABOLISM
25
IN DIABETES
and is not energy dependent or Na’ dependent. Microvessels do not require insulin for glucose uptake22s24; the vascular cells presumably are exposed to ambient blood glucose concentrations in diabetes, unlike insulin-dependent cells such as muscle and adipose cells. Since intracellular concentra.ions of hexose in microvessels tend not to exceed extracellular concentrations, hexose transport is equilibrative rather than accumulative. Similar transport characteristics have been found in endothelial cells cultured from bovine retinas .28 Microvessels incubated in physiologic concentrations of glucose contain more free glucose than phosphorylated glucost, suggesting that hexose uptake is not rate limiting for meFabolism.2’.24 I The majority of glucose transported into vascular cells is believed to be available for release on the abluminal side, presumably in response to a concentration gradienl created by glucose oxidation by perivascular cells.29 Anaerobic glycolysis accounts for much of the glucose me:abolism by microvessels with the production of lactate exceeding that of CO, by severalfold.30.3’ Oxidation of glucose occurs via both the glycolytic and the pentose phosphate pathways, but estimates of each pathway’s role in such oxijlation are conRicting.3’,32 Nonmammalian microvessels (re.e mirabile of the eel) likewise metabolize glucose predorlinantly to lactate.33 Glucose is not the only fuel used by mammalian microvessels; microvessels also have an apprecia’>le capacity for energy production from fatty acids.34l35
MICROVASCULAR EXCESSIVE
METABOLISM, HEXOSE
DIABETES,
AND
CONCENTRATION
The effects of diabetes or hyperglycemia per se on metabo11sn by microvessels have not been extensively studied. Cerebral microvessels from diabetic rats reportedly metabolize glucose3’ and palmitate’j at subnormal rates, but these conclusions are based on in vitro assays at single concentrations of substrate (2 mmol/L and 0.75 mmol/L, respectively). In view of the elevated concentrations of glucose and fatly acids in diabetic blood, it is not clear that microvessels would have a similar depression of metabolism in vivo. Transport of glucose across the blood-retinal and blood-brain barriers also seems to be subnormal in diabetic rats,36-38 but again it is not clear that this would be of metabolic consequence in view of the persistant hyperglycemia. It has been postulated that these observed alterations in glucose uptake and metabolism by cerebral microvessels in diabetes are secondary to hyperglycemia and not due to insulin deficiency per se; starvation of insulin-deficient rats tends to normalize blood glucose concentrations, glucose metabolism by isolated microvessels, and hexose uptake in vivo without any effect on insulin concentration. Supranormal activities of hydoxyacylCo,I-dehydrogenase, aspartate aminotransferase, adenyl kinaseq3’ and normal levels of fatty acid?’ have been observed in retinal microvessels from obese hyperglycemic rats and diabetic patients, respectively, but the method used to solate the microvessels (formalin fixation followed by trypsin digestion) makes the validity of these claims unclear. Pericytes cultured from retinal capillaries reportedly show
signs of cell degeneration.4’ multiply at a subnormal rate.42.43 synthesize supranormal amounts of collagen and protein43 when incubated in hyperglycemic media, and accumulate appreciably more glycogen than do endothelia in diabetic rats.44 These studies are of considerable interest inasmuch as retinal pericytes are selectively lost in diabetes, but the specificity of the above physiologic alterations for pericytes remains to be demonstrated. Inositol deficiency, which recently has been found to be involved in neural complications of diabetes,45 has not been observed in retinal pericytes cultured for several days under hyperglycemic conditions4’ Excessive production of polyol has been implicated in the development of diabetic complications in several tissues, including lens and nerve, and recently has also become identified with microvascular pathology. Considerable interest is developing in the potential role of polyol production in the etiology of diabetic microvascular disease. POLYOL
PRODUCTION
BY MICROVESSELS
The ability of microvessels to produce and accumulate polyol has been studied using microvessels isolated from dogs, a species known to develop significant retinopathy in diabetes and experimental galactosemia.4h The microvessels were carefully isolated from the retina and cerebral cortex using a sieving method, and the production of galactitol from galactose was measured in vitro. The amount of purified microvessels isolated per dog retina was extremely small, usually about 30 to 50 FLg protein per retina. Therefore, retinas from many perfused dogs were pooled for each experiment. Erythrocytes, known to have hexitol-producing activity,47 were removed from the vasculature by perfusion prior to microvessel isolation. The majority of isolated vessels were less than 20 pm in diameter, and the microvessel preparations seemed remarkably free of nonvascular contaminants. The results in Table 1 indicate that galactitol is produced from galactose by retinal and cerebral microvessels but that the activity in microvessels is severalfold less than that seen in freshly isolated lens epithelium. The aldose reductase inhibitor sorbinil was found to inhibit the hexitolproducing activity in all three sites. Bovine retinal and cerebral microvessels,3’ rat glomeruli,48 and the rete mirable of eels49 also are claimed to have polyol-producing activity, but the evidence is open to question. In none of these studies were erythrocytes, which are known to have polyol-producing activity, removed, and thus the contribution of vascular cells to the observed activity is
Table 1. Polyol
Production by Erythrocyte-Free
Retinal Vessels
In Vitro
pmol Galactitollg G&lCtOSe
Swbinil
(30 mmol/L)
Protein* (2.5
x 10m5 mol/L;
+ Galactose (30 mmol/L)
Microvessels retinal cerebral
(n = 5) (n = 6)
24.5
k 4.1
13.1
t
2.7
1 1 .O i 3.4
1.3
(54%
inhibition)
t
1.6
(79%
inhibition)
i
10.2
(70%
inhibition)
Lens epithelium
‘5-hour
(n = 20)
167.9
incubation:
mean
+ 36.8 f
SEM.
45.3
26
KERN
not apparent. In addition, the method used to assess the polyol-producing activity in the bovine retinal and cerebral microvessels recently has been claimed to yield invalid results.50 Rat glomeruli have been found immunohistochemitally to possess aldose reductase, but the enzyme seems localized in glomerular epithelial cells and not in vascular cells.” Other workers have reported that rat glomeruli do not accumulate sorbitol in diabetes or when cultured in hyperglycemic media.‘* Aldose reductase, a key enzyme in the reduction of hexoses to hexitols, has been identified immunohistochemitally in human retinal capillaries by Akagi and coworkers,53 who isolated the vasculature with trypsin prior to immunostaining. The observed distribution of immunostain led them to conclude that retinal vessels have aldose reductase only in pericytes, and they speculated that polyol accumulation within the pericytes might account for the loss of pericytes in diabetes. Others have failed to detect the enzyme in vessels of paraffin-sectioned retinas,5’*54 perhaps due to the thinness of capillary cells in cross-section. Ultrastructural localization of aldose reductase within capillaries in tissue sections is lack-
AND ENGERMAN
ing but is needed to define precisely the enzyme’s distribution and to confirm that the reported absence of immunostain from the endothelia is not an artifact perhaps of endothelial damage during trypsin digestion. Endothelial cells cultured from retinal microvessels reportedly do have aldose reductase-like (hexitol-producing) activity.32 Although it is not yet clear what cells or enzymes are responsible for the hexitolproducing activity in microvessels, it seems clear that this activity can be inhibited by sorbinil. Inhibitors of hexitol production also have been found to inhibit basement membrane thickening of retinal capillaries in both experimental diabetes and galactosemia.55-57 The presence of aldose reductase in retinal microvessels is consistent with a possibility that excessive polyol production within vascular cells causes the basement membrane thickening. It remains conceivable, however, that the vascular lesions might be secondary to abnormalities of cells other than those of the vasculature. Whether the polyol pathway is involved in the etiology of the clinically significant microvascular disease remains to be elucidated.
REFERENCES 1. Levene R, Horton G, Einaugler R: Retinal vessel dehydrogenase enzymes. Arch Ophthal 72:99-103, 1964 2. Landers JW, Chason JL, Gonzalez JE, et al: Morphology and enzymatic activity of rat cerebral capillaries. Lab Invest 11:12531259, 1962 3. Kuwabara, T, Cogan DG: Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch Ophthal 69:492-502, 1963 4. Fried GH, Zweifach BW: Neotetrazolium studies of blood vessels. Anatomical Record 121:97-107, 1955 5. Smith WL, Gutekunst DI, Lyons RH, Jr: Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex. Prostaglandins 19:61-69, 1980 6. Meezan E, Brendel K. Carlson EC: Isolation of a purified preparation of metabolically active retinal blood vessels. Nature 251~65567,1974 7. Brendel K, Meezan E, Carlson EC: Isolated brain microvessels: A purified, metabolically active preparation from bovine cerebral cortex. Science 185:953-955, 1974 8. MrSulja BB, MrSulja BJ, Fujimoto T, et al: Isolation of brain capillaries: A simplified technique. Brain Res 110:361-365, 1976 9. Goldstein GW, Wolinsky JS, Csejtey J, et al: Isolation of metabolically active capillaries from rat brain. J Neurochem 25:715-717, 1975 10. Wagner RC, Matthews MA: The isolation and culture of capillary endothelium from epididymal fat. Microvasc Res 10:286291, 1975 11. Pillion DJ, Haskell JF, Meezan E: Cerebral cortical microvessels: An insulin-sensitive tissue. Biochem Biophy Res Comm 104:686-692, 1982 12. Frank HJL, Pardridge WM: A direct in vitro demonstration of insulin binding to isolated brain microvessels. Diabetes 30:757761,198l 13. King GL, Buzney SM. Kahn CR, et al: Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels. J Clin Invest 71:974-979, 1983 14. James CRH, Cotlier E: Fate of insulin in the retina: An autoradiographic study. Brit J Ophthal67:80-88, 1983
15. Van Houten M, Posner BI: Insulin binds to brain blood vessels in vivo. Nature 282:623-625, 1979 16. Jialal I, King GL, Buchwald S, et al: Processing of insulin by bovine endothelial cells in culture. Internalization without degradation. Diabetes 33:794-800, 1984 17. King GL, Johnson SM: Receptor-mediated transport insulin across endothelial cells. Science 227: 1583-l 586, 1985
of
18. Nemecek GM, Chamberlain RH: Insulin alters cyclic nucleotide metabolism and glycogen synthase activity in microvessels from hamster adipose tissue. Microvasc Res 26:339-343, 1983 19. &.terby R, Gundersen HJG, Christensen NJ: The acute effect of insulin on capillary endothelial cells. Diabetes 27:745-749, 1978 20. Engerman duces diabetic-like
RL, Kern TS: Experimental galactosemia retinopathy. Diabetes 33:97-99, 1984
pro-
21. Engerman RL, Kern TS: Diabetic retinopathy: Is it a consequence of hyperglycaemia? Diabetic Medicine 2:200-203, 1985 22. Betz AL. Goldstein GW: Transport of hexoses, potassium and neutral amino acids into capillaries isolated from bovine retina. Exptl Eye Res 30:593-605,198O 23. Stefanovich V, Gojowczyk G: Transport of 2-deoxy-D-[H] glucose in microvessels isolated from bovine cerebral cortex. Neurothem Res 6:43 l-440, 198 1 24. Betz AL, Csejtey J, Goldstein GW: Hexose transport and phosphorylation by capillaries isolated from rat brain. Am J Physiol 236:C96-C102, 1979 25. Kolber AR, Bagnell CR, Krigman MR. et al: Transport of sugars into microvessels isolated from rat brain: A model for the blood-brain barrier. J Neurochem 33:419-432, 1979 26. Betz AL, Gilboe DD, Drewes LR: The characteristics of glucose transport across the blood-brain barrier and its relation to cerebral glucose metabolism, in Levi G, Battistin L, Lajtha A (eds): Transport Phenomena in the Nervous System. New York, Plenum Press, 1976, pp 1333149 27. Lund-Andersen H: Transport Physiol Rev 59:305-352, 1979 28. Betz AL, Bowman
of glucose from blood to brain.
PD, Goldstein
GW: Hexose transport
in
MICROVASCULAR
METABOLISM
27
IN DIABETES
microvascular endothelial cells cultured from bovine retina. Exptl Eye Res 361269%217, 1983 2’). Goldstein GW, Betz AL, Recent advances in understanding brain capillary function. Ann Neurol 14:389-395. 1983 30. McCall AL. Gould JB. Ruderman NB: Diabetes-induced site-ations of glucose metabolism in rat cerebral microvessels. Am J Physiol 247:E462-E467, 1984 31. Chan CT, Brecher P, Haudenschild C, et al: The effect of cholesterol feeding on the metabolism of rat cerebral microvessels. Microvasc Res 18:3533369. 1979 32. Kennedy A, Frank RN, Varma SD: Aldose reductase activity in retinal and cerebral microvessels and cultured vascular cells. Invest Ophthalmol Vis Sci 24:1250-1258, 1983 33. Rasio EA: Glucose metabolism in an isolated blood capillary preparation. Canad J Biochem 51:701-708, 1973 34. Goldstein GW: Relation of potassium transport to oxidative metabolism in isolated brain capillaries. J Physiol 286:185-195. 1973 35. Morisaki N. Saito Y, Kumagai A: Fatty acid oxidation in rat bra n microvessels in hypertension, aging and experimental diabetes. Atherosclerosis 42:221-227, 1982 36. Ennis SR. Johnson JE, Pautler EL: In situ kinetics of glucose trarsport across the blood-retinal barrier in normal rats and rats wit11 streptozotocin-induced diabetes. Invest Ophthalmol Vis Sci :3:447-456. 1982 37. McCall AL, Millington WR, Wurtman RJ: Metabolic fuel and amino acid transport into the brain in experimental diabetes mellitus. Proc Nat Acad Sci 79:5406-5410, 1982 38. Gjedde A, Crone C: Blood-brain glucose transfer: Repression in chronic hyperglycemia. Science 214:456-457, 1981 39. Naeser P. Agren A: Morphology and enzyme activities of the retinal capillaries in mice with the obese-hyperglycemic syndrome. Acta Ophthalmol 56:607-615, 1978 40. Futterman S. Kupfer C: The fatty acid composition of the retinal vasculature of normal and diabetic human eyes. Invest Op ithalmol 7:105-10X, 1968 4 I. Buzney SM. Frank RN, Varma SD, et al: Aldose reductase in retinal mural cells. Invest Ophthalmol Vis Sci 16:392-396, 1977 42. King GL, Buzney SM. Goodman AD: Modulation of growth c,f .etinal capillary cells by insulin and hyperglycemia. Diabetes ?2:125. 1983 (Suppl 1) 13. Li W. Shen S. Khatami M, et al: Stimulation of retinal capillary pericyte protein and collagen synthesis in culture by big-i-glucose concentration. Diabetes 33:785-789, 1984
44. Sosula L, Beaumont P. Hollows FC, et al: Dilatation and endothelial proliferation of retinal capillaries in streptozotocindiabetic rats: quantitative electron microscopy. Invest Ophthalmol 11:926-935. 1972 45. Mayer transport and myo-inositol diabetic rats.
JH. Tomlinson DR: Prevention of defects of axonal nerve conduction velocity by oral administration of or an aldose reductase inhibitor in streptozotocinDiabetologia 25:4333438, 1983
46. Kern TS, Engerman RL: Hexitol production by canine retinal microvessels. Invest Ophthalmol Vis Sci 26:382-.384, 1985 47. Malone JI, Knox G, Benford S, et al: Red cell sorbitol. indicator of diabetic control. Diabetes 29:861-864, 1980
An
48. Beyer-Mears A, Ku L, Cohen MP: Glomerular polyol accumulation in diabetes and its prevention by oral Sorbinil. Diabetes 33:604-607. 1984 49. Rasio EA. Morrison AD: Glucose-induced metabolism of an isolated capillary preparation. 113, 1978
alterations of the Diabetes 27:108-
50. Wolff SP, Crabbe MJC: Low apparent aldose reductase activity produced by monosaccharide autoxidation. Biochem J 226:625-630. I985 5 1. Ludvigson MA, Sorenson RL: Immunohistochemical localization of aldose reductase. II. Rat eye and kidney. Diabetes 29:45&459, 1980 52. Hutton JC, Schofield PJ, Williams JF. et al: The localization of sorbitol pathway activity in the rat renal cortex and its relationship to the pathogenesis of the renal complications of diabetes mellitus. Aust J Exp Biol Med 53:49-57, 1975 53. Akagi Y, Kador PF, Kuwabara T, et al: Aldose reductase localization in human retinal mural cells. Invest Ophthalmol Vis Sci 24:1516~1519, 1983 54. Kern TS, Engerman RL: Distribution of aldose reductase ocular tissues. Exp Eye Res 33: 175- 182.198 1
in
55. Frank RN, Keirn RJ, Kennedy A, et al: Galactose-induced retinal capillary basement membrane thickening: Prevention by sorbinil. Invest Ophthalmol Vis Sci 24: I5 19- 1 524, I983 56. Robison WG, Jr. Kador PF, Kinoshita JH: Retinal capillaries: Basement membrane thickening by galactosemia prevented with aldose reductase inhibitor. Science 221: 1177 I 179, I983 57. Chandler retinal capillary aldose reductase (suPPI)
ML, Shannon WA. DeSantis L: Prevention of basement membrane thickening in diabetic rats by inhibitors. Invest Ophthalmol Vis Sci 25: 159, 1984
Metabolism
in Diabetes
S. Kern and Ronald L. Engerman
Hyperglycemia has been implicated in the development of retinal vascular disease. Consequently, the effects of excessive hexose concentration on cells of the vascular wall are receiving increasing attention. Techniques for isolating metabolically active microvessels from tissues such as those from the retina and cerebral cortex are providing new opportunities for the study of the uptake and metabolism of hexose by microvessels. Such studies indicate that hexose uptake by microvessels is not insulin dependent and that microvessels are capable of metabolizing hexose by pathways common to many diverse tissues, including anaerobic and aerobic glycolysis, pentose phosphate shunt, and glycogenogenesis. Microvessels isolated from diabetic animals metabolize glucose at a subnormal rate. Hexitol production and accumulation has been implicated in the pathogenesis of diabetic complications in a variety of tissues and might also play a role in the development of diabetic microvascular disease. We have quantitated hexitol-producing metabolic activity of retinal and cerebral microvessels isolated from dogs, a species known to develop a retinopathy similar to that seen in diabetic patients. Erythrocytes were removed by perfusion prior to microvessel isolation because they are known to have hexitol-producing activity. Both retinal and cerebral microvessels produce galactitol from galactose. and this activity is inhibited in the presence of the aldose reductase inhibitor sorbinil. The presence of hexitol-producing activity within microvessels is consistent with a possible role of polyol production in the etiology of diabetic microvascular disease. Nevertheless, it is not even clear that the primary defect leading to vascular disease in diabetes resides in the vasculature itself: nonvascular cells such as erythrocytes, platelets, and olial cells have been postulated to plav. a role in the development of the vascular lesions. The steps linking diabetic retinopathy to hyperglycemia await clarification. @ 1966 by Grune & Stratton, Inc.
M
disease accounts for much of the ICROVASCULAR morbidity and mortality of diabetes mellitus. Surprisingly little is known about metabolism in microvessels in diabetes, or the role of vascular metabolism in the etiology of diabetic microvascular disease. The metabolism of microvessels has been explored using a variety of methodologies. Histochemic and immunochemic techniques have provided information on the tissue distribution of enzymes and have allowed semiquantitative estimation of enzymatic activity.le5 More recently, it has become possible to isolate metabolically active microvessels from a variety of tissues including retina, cerebral cortex, adrenal, spinal cord, and adipose. &lo These isolated microvessels have been studied biochemically and have been used to establish cell cultures of pure microvessel cell types, such as endothelial cells and pericytes. (Renal glomeruli also can be isolated by such techniques, but glomerular metabolic activity cannot be equated with microvascular metabolism due to the presence of nonvascular as well as vascular cell types of glomeruli.) The availability of purified microvessels and their cells for study offers new opportunities for examining the effects of diabetes, insulin deficiency, and hyperglycemia on vascular metabolism.
From the Department of Ophthalmology, University of Wisconsin, Madison. Presented at the Pfizer Sorbinil Symposium-The Eflects of Sorbinil on the Pathophysiology of Diabetic Complications, Dorado, Puerto Rico, May 3kJune 2,1985. Supported in part by a Lions Club International Research and Development Award from the American Diabetes Association, and by Public Health Service grant EY00300 from the National Eye Institute. Address reprint requests to Timothy S. Kern, PhD. Department of Ophthalmology, University of Wisconsin. 1300 University Ave. Madison, WI 53706. D 1986 by Grune & Stratton, Inc. 0026-0495/86/3504-1006$03.00/O
24
INSULIN AND MICROVESSELS
Retinal and cerebral microvessels have been found to possess specific, high-affinity insulin receptors.“-‘5 The hormone can be internalized into vascular cells’6 and reportedly can be transported across endothelial cells in vitro,17 although release of insulin on the abluminal side has not been documented in situ.14 Exposure of microvessels to insulin causes a variety of metabolic responses, stimulating both the conversion of glucose into CO2 and lipid, and CAMP phosphodiesterase and glycogen synthetase activities.“,‘* Both endothelial cells and pericytes respond to physiologic levels of insulin.” Pericytes seem particularly sensitive to the hormone at least with respect to stimulation of thymidine incorporation in vitro. The number of micropinocytotic vesicles in endothelia of muscle capillaries is subnormal in insulin deficiency and tends to be normalized by insulin.‘9 Insulin deficiency in diabetes conceivably could have adverse effects on microvascular cells. Nonetheless, diabetic-like microvascular disease has been shown to develop even in the absence of insulin deficiency.” A retinopathy that seems morphologically indistinguishable from that of diabetic patients and dogs has recently been found to develop in nondiabetic dogs made “hyperglycemic” by the consumption of a galactose-rich diet2’ This microvascular pathology develops in the apparent absence of many metabolic disorders typical of diabetes mellitus such as subnormal blood concentrations of insulin and supranormal concentrations of glucose, lipids, and branched-chain amino acids. The evidence strongly suggests that hyperglycemia and its sequelae play a key role in the development of diabetic microvascular disease, at least in the retina. HEXOSE UPTAKE AND METABOLISM
BY MICROVESSELS
of other sugars are transported into retinal and cerebral microvessels by a stereo-specific, carrier-mediated transport system22-25 similar to that described in vivo.26*27This transport is saturable Glucose,
galactose,
and a variety
Metabolism, Vol 35,
No 4, Suppl 1 (April), 1986: pp 24-27
MICROVASCULAR
METABOLISM
25
IN DIABETES
and is not energy dependent or Na’ dependent. Microvessels do not require insulin for glucose uptake22s24; the vascular cells presumably are exposed to ambient blood glucose concentrations in diabetes, unlike insulin-dependent cells such as muscle and adipose cells. Since intracellular concentra.ions of hexose in microvessels tend not to exceed extracellular concentrations, hexose transport is equilibrative rather than accumulative. Similar transport characteristics have been found in endothelial cells cultured from bovine retinas .28 Microvessels incubated in physiologic concentrations of glucose contain more free glucose than phosphorylated glucost, suggesting that hexose uptake is not rate limiting for meFabolism.2’.24 I The majority of glucose transported into vascular cells is believed to be available for release on the abluminal side, presumably in response to a concentration gradienl created by glucose oxidation by perivascular cells.29 Anaerobic glycolysis accounts for much of the glucose me:abolism by microvessels with the production of lactate exceeding that of CO, by severalfold.30.3’ Oxidation of glucose occurs via both the glycolytic and the pentose phosphate pathways, but estimates of each pathway’s role in such oxijlation are conRicting.3’,32 Nonmammalian microvessels (re.e mirabile of the eel) likewise metabolize glucose predorlinantly to lactate.33 Glucose is not the only fuel used by mammalian microvessels; microvessels also have an apprecia’>le capacity for energy production from fatty acids.34l35
MICROVASCULAR EXCESSIVE
METABOLISM, HEXOSE
DIABETES,
AND
CONCENTRATION
The effects of diabetes or hyperglycemia per se on metabo11sn by microvessels have not been extensively studied. Cerebral microvessels from diabetic rats reportedly metabolize glucose3’ and palmitate’j at subnormal rates, but these conclusions are based on in vitro assays at single concentrations of substrate (2 mmol/L and 0.75 mmol/L, respectively). In view of the elevated concentrations of glucose and fatly acids in diabetic blood, it is not clear that microvessels would have a similar depression of metabolism in vivo. Transport of glucose across the blood-retinal and blood-brain barriers also seems to be subnormal in diabetic rats,36-38 but again it is not clear that this would be of metabolic consequence in view of the persistant hyperglycemia. It has been postulated that these observed alterations in glucose uptake and metabolism by cerebral microvessels in diabetes are secondary to hyperglycemia and not due to insulin deficiency per se; starvation of insulin-deficient rats tends to normalize blood glucose concentrations, glucose metabolism by isolated microvessels, and hexose uptake in vivo without any effect on insulin concentration. Supranormal activities of hydoxyacylCo,I-dehydrogenase, aspartate aminotransferase, adenyl kinaseq3’ and normal levels of fatty acid?’ have been observed in retinal microvessels from obese hyperglycemic rats and diabetic patients, respectively, but the method used to solate the microvessels (formalin fixation followed by trypsin digestion) makes the validity of these claims unclear. Pericytes cultured from retinal capillaries reportedly show
signs of cell degeneration.4’ multiply at a subnormal rate.42.43 synthesize supranormal amounts of collagen and protein43 when incubated in hyperglycemic media, and accumulate appreciably more glycogen than do endothelia in diabetic rats.44 These studies are of considerable interest inasmuch as retinal pericytes are selectively lost in diabetes, but the specificity of the above physiologic alterations for pericytes remains to be demonstrated. Inositol deficiency, which recently has been found to be involved in neural complications of diabetes,45 has not been observed in retinal pericytes cultured for several days under hyperglycemic conditions4’ Excessive production of polyol has been implicated in the development of diabetic complications in several tissues, including lens and nerve, and recently has also become identified with microvascular pathology. Considerable interest is developing in the potential role of polyol production in the etiology of diabetic microvascular disease. POLYOL
PRODUCTION
BY MICROVESSELS
The ability of microvessels to produce and accumulate polyol has been studied using microvessels isolated from dogs, a species known to develop significant retinopathy in diabetes and experimental galactosemia.4h The microvessels were carefully isolated from the retina and cerebral cortex using a sieving method, and the production of galactitol from galactose was measured in vitro. The amount of purified microvessels isolated per dog retina was extremely small, usually about 30 to 50 FLg protein per retina. Therefore, retinas from many perfused dogs were pooled for each experiment. Erythrocytes, known to have hexitol-producing activity,47 were removed from the vasculature by perfusion prior to microvessel isolation. The majority of isolated vessels were less than 20 pm in diameter, and the microvessel preparations seemed remarkably free of nonvascular contaminants. The results in Table 1 indicate that galactitol is produced from galactose by retinal and cerebral microvessels but that the activity in microvessels is severalfold less than that seen in freshly isolated lens epithelium. The aldose reductase inhibitor sorbinil was found to inhibit the hexitolproducing activity in all three sites. Bovine retinal and cerebral microvessels,3’ rat glomeruli,48 and the rete mirable of eels49 also are claimed to have polyol-producing activity, but the evidence is open to question. In none of these studies were erythrocytes, which are known to have polyol-producing activity, removed, and thus the contribution of vascular cells to the observed activity is
Table 1. Polyol
Production by Erythrocyte-Free
Retinal Vessels
In Vitro
pmol Galactitollg G&lCtOSe
Swbinil
(30 mmol/L)
Protein* (2.5
x 10m5 mol/L;
+ Galactose (30 mmol/L)
Microvessels retinal cerebral
(n = 5) (n = 6)
24.5
k 4.1
13.1
t
2.7
1 1 .O i 3.4
1.3
(54%
inhibition)
t
1.6
(79%
inhibition)
i
10.2
(70%
inhibition)
Lens epithelium
‘5-hour
(n = 20)
167.9
incubation:
mean
+ 36.8 f
SEM.
45.3
26
KERN
not apparent. In addition, the method used to assess the polyol-producing activity in the bovine retinal and cerebral microvessels recently has been claimed to yield invalid results.50 Rat glomeruli have been found immunohistochemitally to possess aldose reductase, but the enzyme seems localized in glomerular epithelial cells and not in vascular cells.” Other workers have reported that rat glomeruli do not accumulate sorbitol in diabetes or when cultured in hyperglycemic media.‘* Aldose reductase, a key enzyme in the reduction of hexoses to hexitols, has been identified immunohistochemitally in human retinal capillaries by Akagi and coworkers,53 who isolated the vasculature with trypsin prior to immunostaining. The observed distribution of immunostain led them to conclude that retinal vessels have aldose reductase only in pericytes, and they speculated that polyol accumulation within the pericytes might account for the loss of pericytes in diabetes. Others have failed to detect the enzyme in vessels of paraffin-sectioned retinas,5’*54 perhaps due to the thinness of capillary cells in cross-section. Ultrastructural localization of aldose reductase within capillaries in tissue sections is lack-
AND ENGERMAN
ing but is needed to define precisely the enzyme’s distribution and to confirm that the reported absence of immunostain from the endothelia is not an artifact perhaps of endothelial damage during trypsin digestion. Endothelial cells cultured from retinal microvessels reportedly do have aldose reductase-like (hexitol-producing) activity.32 Although it is not yet clear what cells or enzymes are responsible for the hexitolproducing activity in microvessels, it seems clear that this activity can be inhibited by sorbinil. Inhibitors of hexitol production also have been found to inhibit basement membrane thickening of retinal capillaries in both experimental diabetes and galactosemia.55-57 The presence of aldose reductase in retinal microvessels is consistent with a possibility that excessive polyol production within vascular cells causes the basement membrane thickening. It remains conceivable, however, that the vascular lesions might be secondary to abnormalities of cells other than those of the vasculature. Whether the polyol pathway is involved in the etiology of the clinically significant microvascular disease remains to be elucidated.
REFERENCES 1. Levene R, Horton G, Einaugler R: Retinal vessel dehydrogenase enzymes. Arch Ophthal 72:99-103, 1964 2. Landers JW, Chason JL, Gonzalez JE, et al: Morphology and enzymatic activity of rat cerebral capillaries. Lab Invest 11:12531259, 1962 3. Kuwabara, T, Cogan DG: Retinal vascular patterns. VI. Mural cells of the retinal capillaries. Arch Ophthal 69:492-502, 1963 4. Fried GH, Zweifach BW: Neotetrazolium studies of blood vessels. Anatomical Record 121:97-107, 1955 5. Smith WL, Gutekunst DI, Lyons RH, Jr: Immunocytochemical localization of the prostaglandin-forming cyclooxygenase in the cerebellar cortex. Prostaglandins 19:61-69, 1980 6. Meezan E, Brendel K. Carlson EC: Isolation of a purified preparation of metabolically active retinal blood vessels. Nature 251~65567,1974 7. Brendel K, Meezan E, Carlson EC: Isolated brain microvessels: A purified, metabolically active preparation from bovine cerebral cortex. Science 185:953-955, 1974 8. MrSulja BB, MrSulja BJ, Fujimoto T, et al: Isolation of brain capillaries: A simplified technique. Brain Res 110:361-365, 1976 9. Goldstein GW, Wolinsky JS, Csejtey J, et al: Isolation of metabolically active capillaries from rat brain. J Neurochem 25:715-717, 1975 10. Wagner RC, Matthews MA: The isolation and culture of capillary endothelium from epididymal fat. Microvasc Res 10:286291, 1975 11. Pillion DJ, Haskell JF, Meezan E: Cerebral cortical microvessels: An insulin-sensitive tissue. Biochem Biophy Res Comm 104:686-692, 1982 12. Frank HJL, Pardridge WM: A direct in vitro demonstration of insulin binding to isolated brain microvessels. Diabetes 30:757761,198l 13. King GL, Buzney SM. Kahn CR, et al: Differential responsiveness to insulin of endothelial and support cells from micro- and macrovessels. J Clin Invest 71:974-979, 1983 14. James CRH, Cotlier E: Fate of insulin in the retina: An autoradiographic study. Brit J Ophthal67:80-88, 1983
15. Van Houten M, Posner BI: Insulin binds to brain blood vessels in vivo. Nature 282:623-625, 1979 16. Jialal I, King GL, Buchwald S, et al: Processing of insulin by bovine endothelial cells in culture. Internalization without degradation. Diabetes 33:794-800, 1984 17. King GL, Johnson SM: Receptor-mediated transport insulin across endothelial cells. Science 227: 1583-l 586, 1985
of
18. Nemecek GM, Chamberlain RH: Insulin alters cyclic nucleotide metabolism and glycogen synthase activity in microvessels from hamster adipose tissue. Microvasc Res 26:339-343, 1983 19. &.terby R, Gundersen HJG, Christensen NJ: The acute effect of insulin on capillary endothelial cells. Diabetes 27:745-749, 1978 20. Engerman duces diabetic-like
RL, Kern TS: Experimental galactosemia retinopathy. Diabetes 33:97-99, 1984
pro-
21. Engerman RL, Kern TS: Diabetic retinopathy: Is it a consequence of hyperglycaemia? Diabetic Medicine 2:200-203, 1985 22. Betz AL. Goldstein GW: Transport of hexoses, potassium and neutral amino acids into capillaries isolated from bovine retina. Exptl Eye Res 30:593-605,198O 23. Stefanovich V, Gojowczyk G: Transport of 2-deoxy-D-[H] glucose in microvessels isolated from bovine cerebral cortex. Neurothem Res 6:43 l-440, 198 1 24. Betz AL, Csejtey J, Goldstein GW: Hexose transport and phosphorylation by capillaries isolated from rat brain. Am J Physiol 236:C96-C102, 1979 25. Kolber AR, Bagnell CR, Krigman MR. et al: Transport of sugars into microvessels isolated from rat brain: A model for the blood-brain barrier. J Neurochem 33:419-432, 1979 26. Betz AL, Gilboe DD, Drewes LR: The characteristics of glucose transport across the blood-brain barrier and its relation to cerebral glucose metabolism, in Levi G, Battistin L, Lajtha A (eds): Transport Phenomena in the Nervous System. New York, Plenum Press, 1976, pp 1333149 27. Lund-Andersen H: Transport Physiol Rev 59:305-352, 1979 28. Betz AL, Bowman
of glucose from blood to brain.
PD, Goldstein
GW: Hexose transport
in
MICROVASCULAR
METABOLISM
27
IN DIABETES
microvascular endothelial cells cultured from bovine retina. Exptl Eye Res 361269%217, 1983 2’). Goldstein GW, Betz AL, Recent advances in understanding brain capillary function. Ann Neurol 14:389-395. 1983 30. McCall AL. Gould JB. Ruderman NB: Diabetes-induced site-ations of glucose metabolism in rat cerebral microvessels. Am J Physiol 247:E462-E467, 1984 31. Chan CT, Brecher P, Haudenschild C, et al: The effect of cholesterol feeding on the metabolism of rat cerebral microvessels. Microvasc Res 18:3533369. 1979 32. Kennedy A, Frank RN, Varma SD: Aldose reductase activity in retinal and cerebral microvessels and cultured vascular cells. Invest Ophthalmol Vis Sci 24:1250-1258, 1983 33. Rasio EA: Glucose metabolism in an isolated blood capillary preparation. Canad J Biochem 51:701-708, 1973 34. Goldstein GW: Relation of potassium transport to oxidative metabolism in isolated brain capillaries. J Physiol 286:185-195. 1973 35. Morisaki N. Saito Y, Kumagai A: Fatty acid oxidation in rat bra n microvessels in hypertension, aging and experimental diabetes. Atherosclerosis 42:221-227, 1982 36. Ennis SR. Johnson JE, Pautler EL: In situ kinetics of glucose trarsport across the blood-retinal barrier in normal rats and rats wit11 streptozotocin-induced diabetes. Invest Ophthalmol Vis Sci :3:447-456. 1982 37. McCall AL, Millington WR, Wurtman RJ: Metabolic fuel and amino acid transport into the brain in experimental diabetes mellitus. Proc Nat Acad Sci 79:5406-5410, 1982 38. Gjedde A, Crone C: Blood-brain glucose transfer: Repression in chronic hyperglycemia. Science 214:456-457, 1981 39. Naeser P. Agren A: Morphology and enzyme activities of the retinal capillaries in mice with the obese-hyperglycemic syndrome. Acta Ophthalmol 56:607-615, 1978 40. Futterman S. Kupfer C: The fatty acid composition of the retinal vasculature of normal and diabetic human eyes. Invest Op ithalmol 7:105-10X, 1968 4 I. Buzney SM. Frank RN, Varma SD, et al: Aldose reductase in retinal mural cells. Invest Ophthalmol Vis Sci 16:392-396, 1977 42. King GL, Buzney SM. Goodman AD: Modulation of growth c,f .etinal capillary cells by insulin and hyperglycemia. Diabetes ?2:125. 1983 (Suppl 1) 13. Li W. Shen S. Khatami M, et al: Stimulation of retinal capillary pericyte protein and collagen synthesis in culture by big-i-glucose concentration. Diabetes 33:785-789, 1984
44. Sosula L, Beaumont P. Hollows FC, et al: Dilatation and endothelial proliferation of retinal capillaries in streptozotocindiabetic rats: quantitative electron microscopy. Invest Ophthalmol 11:926-935. 1972 45. Mayer transport and myo-inositol diabetic rats.
JH. Tomlinson DR: Prevention of defects of axonal nerve conduction velocity by oral administration of or an aldose reductase inhibitor in streptozotocinDiabetologia 25:4333438, 1983
46. Kern TS, Engerman RL: Hexitol production by canine retinal microvessels. Invest Ophthalmol Vis Sci 26:382-.384, 1985 47. Malone JI, Knox G, Benford S, et al: Red cell sorbitol. indicator of diabetic control. Diabetes 29:861-864, 1980
An
48. Beyer-Mears A, Ku L, Cohen MP: Glomerular polyol accumulation in diabetes and its prevention by oral Sorbinil. Diabetes 33:604-607. 1984 49. Rasio EA. Morrison AD: Glucose-induced metabolism of an isolated capillary preparation. 113, 1978
alterations of the Diabetes 27:108-
50. Wolff SP, Crabbe MJC: Low apparent aldose reductase activity produced by monosaccharide autoxidation. Biochem J 226:625-630. I985 5 1. Ludvigson MA, Sorenson RL: Immunohistochemical localization of aldose reductase. II. Rat eye and kidney. Diabetes 29:45&459, 1980 52. Hutton JC, Schofield PJ, Williams JF. et al: The localization of sorbitol pathway activity in the rat renal cortex and its relationship to the pathogenesis of the renal complications of diabetes mellitus. Aust J Exp Biol Med 53:49-57, 1975 53. Akagi Y, Kador PF, Kuwabara T, et al: Aldose reductase localization in human retinal mural cells. Invest Ophthalmol Vis Sci 24:1516~1519, 1983 54. Kern TS, Engerman RL: Distribution of aldose reductase ocular tissues. Exp Eye Res 33: 175- 182.198 1
in
55. Frank RN, Keirn RJ, Kennedy A, et al: Galactose-induced retinal capillary basement membrane thickening: Prevention by sorbinil. Invest Ophthalmol Vis Sci 24: I5 19- 1 524, I983 56. Robison WG, Jr. Kador PF, Kinoshita JH: Retinal capillaries: Basement membrane thickening by galactosemia prevented with aldose reductase inhibitor. Science 221: 1177 I 179, I983 57. Chandler retinal capillary aldose reductase (suPPI)
ML, Shannon WA. DeSantis L: Prevention of basement membrane thickening in diabetic rats by inhibitors. Invest Ophthalmol Vis Sci 25: 159, 1984