Biomedical subjects
L C MacGregor
Publications and source records attributed to L C MacGregor.
Effect of myo-inositol on renal Na-K-ATPase in experimental diabetes.
The activity of Na-K-ATPase in the kidney is increased by experimental diabetes. Because the kidney is rich in myo-inositol and abnormal inositol metabolism has been implicated in early neural complications of diabetes, we studied the effect of myo-inositol supplementation on Na-K-ATPase activity in renal medullary and cortical homogenates of Sprague-Dawley rats made diabetic with streptozotocin. Myo-inositol (650 mg/kg) was administered by gavage daily for 1 and 2 weeks after induction of diabetes. Medullary Na-K-ATPase (mumol/mg protein/h) was increased at 1 week by approximately 60% in diabetic rats versus control (25.9 +/- 0.07 vs 16.3 +/- 0.7; P less than .01). This increase was completely prevented by myo-inositol supplementation, despite persistent hyperglycemia. At 2 weeks, similar results were seen; medullary Na-K-ATPase activity was increased by 50% in diabetic rats compared with control, and once again myo-inositol prevented this increase. Sorbinil, the aldose reductase inhibitor, was also administered by gavage (20 mg/kg) for 2 weeks and partially prevented the increase in medullary Na-K-ATPase activity (20.0 +/- 0.9; P less than .05). At both 7 and 14 days, Na-K-ATPase activity in the cortex of untreated diabetic rats was also significantly increased compared with nondiabetic control rats and the increase was prevented by myo-inositol or Sorbinil. Myo-inositol or Sorbinil did not reduce Na-K-ATPase activity of nondiabetic control rats, nor did they prevent the increase in medullary Na-K-ATPase in compensatory hypertrophy following uninephrectomy. Myo-inositol content of outer medulla was about five to six times that of cortex, but was unaltered by the diabetic state.(ABSTRACT TRUNCATED AT 250 WORDS)
Diabetes and the myo-inositol paradox.
To test the general applicability of the hypothesis that diabetes mellitus causes increased polyol pathway activity, decreased tissue free myo-inositol, and resultant pathological changes in tissues susceptible to the ravages of diabetes, we measured glucose, sorbitol, and myo-inositol with quantitative histochemical techniques in layers of the cornea, the aortic myointima, the cardiac left ventricle and atrioventricular node (AVN), and retina and kidney after 19 days or 2 mo (mildly diabetic non-insulin-treated [MD] and severely diabetic insulin-treated [SD] groups) in the alloxan-induced diabetes model. In the aqueous humor, glucose rose linearly with increased serum glucose, sorbitol was markedly increased in the MD and SD groups, and myo-inositol did not change in any diabetic group. There was no change in glucose or sorbitol in aortic myointima in any group, but myoinositol was decreased in 19-day diabetic rabbits by 26%, unchanged in MD rabbits but paradoxically increased by 60% in SD rabbits. Glucose, sorbitol, and myo-inositol increased in all three corneal layers in SD rabbits but only in epithelium and stroma in 19-day and MD rabbits. AVN glucose and sorbitol did not change in 19-day diabetic, MD, or SD diabetic rabbits. AVN myo-inositol was three times higher than ventricular myo-inositol and did not appear to change in SD rabbits. Retinal pigmented epithelium myo-inositol was decreased 30% in SD rabbits. Glomerular myo-inositol was also decreased, but not significantly, in SD rabbits. We conclude that the paradoxical increase in corneal and aortal myo-inositol raises fundamental questions about the general applicability of the myo-inositol-depletion hypothesis.
Differential regulation of protein kinase C and (Na,K)-adenosine triphosphatase activities by elevated glucose levels in retinal capillary endothelial cells.
Elevated cellular sorbitol levels resulting from conversion of increased glucose by aldose reductase might deplete cellular myoinositol content, which could then lower inositol phosphates (InsPs) and diacylglycerol levels, key regulators of protein kinase C (PKC). Secondary to altered PKC activity, other cellular enzymes such as (Na,K)-ATPase could be affected. To test this hypothesis we examined the association between PKC activity, (Na,K)-ATPase activity, and sorbitol, myoinositol, and InsP levels in cultured bovine retinal capillary endothelial cells, a cell type prominently involved in diabetic retinopathy. Elevating glucose concentration in culture media from 100 to 400 mg/dl led to a 100% increase in sorbitol levels, which could be inhibited completely by sorbinil, an aldose reductase inhibitor. In contrast, no changes were observed in myoinositol or InsP levels. Subfractionated PKC activities showed a 100% increase in the membranous pool with a parallel decrease in the cytosolic fraction. Adding sorbinil did not affect PKC activity, whereas the PKC agonist, phorbol myristate acetate (PMA), stimulated translocation of PKC. Ouabain-inhibitable (Na,K)-ATPase activity was decreased 70% by elevated glucose levels. This decrease could be prevented by adding either PMA or sorbinil. Thus, in retinal capillary endothelial cells elevated glucose concentration can affect PKC and (Na,K)-ATPase activities, probably via different mechanisms.
Quantitative histochemical determination of Na+ and K+ in microscopic samples using carbon furnace atomic absorption spectrometry.
Carbon furnace atomic absorption spectrometry was used to measure the Na and K content of freeze-dried microscopic tissue samples. This method was sufficiently sensitive to measure pmol amounts of Na and K from tissue weighing 10-60 ng. Within the spatial resolution of the microdissection procedure, ion diffusion that might occur during cryosectioning, freeze-drying, and dissection of the tissue did not seem to be a problem. Data obtained with this methodology were in agreement with previously reported values of the Na and K content of various tissues, thus supporting the usefulness of this quantitative histochemical technique.
Altered retinal metabolism in diabetes. II. Measurement of sodium-potassium ATPase and total sodium and potassium in individual retinal layers.
Pathological changes in retinas of diabetics include specific morphological, biochemical, and functional abnormalities. As biochemical manifestations of the disease, increased sorbitol and decreased myo-inositol were found in retinas of experimentally diabetic animals. Similar alterations in polyol metabolism have been associated in nerves of diabetics with a reduction of Na+-K+-ATPase activity. To determine whether this association extends to the retinas of diabetic animals, we applied quantitative histochemical techniques to measure ATPase activities and the amounts of sodium and potassium in samples from nine individual layers of cryostat sections of rabbit retina. ATPase activities were determined fluorimetrically, and the ions were measured by atomic absorption with a carbon rod atomizer. The activity of Na+-K+-ATPase was reduced in the retinal pigmented epithelium (retinal pigment epithelium) and in selected layers of the neural retina, and total sodium in the retinal pigment epithelium layer was elevated in diabetes. The retinal pigment epithelium forms the outer component of the blood-retinal barrier and partly determines the composition of the retinal interstitial fluid. Changes in retinal pigment epithelium biochemistry and function might alter the intraretinal environment, predisposing neural retina or retinal blood vessels to disease. The morphologically and functionally well defined retinal pigment epithelium may provide a useful model for studying the pathogenesis of diabetic complications.
Altered retinal metabolism in diabetes. I. Microanalysis of lipid, glucose, sorbitol, and myo-inositol in the choroid and in the individual layers of the rabbit retina.
Total lipid, sucrose, glucose, sorbitol and myo-inositol contents in individual layers from normal and alloxan-diabetic rabbit retinas were measured using gravimetric and enzymatic microtechniques. Pure samples of nine retinal layers were microdissected from freeze-dried retinal cryosections. The lipid content was measured by loss of weight after ethanol and hexane extraction. Retinal lipid varied 3-fold across the retina and was not influenced by diabetes. Sucrose, glucose, sorbitol, and myo-inositol were measured with fluorimetric microassays. Sucrose infused intravenously prior to sampling the tissue did not traverse the outer blood retinal barrier of the normal or the diabetic retina. In both normals and diabetics, glucose followed a diffusional curve, with outer and inner retinal concentrations, respectively, equal to choroidal blood and vitreal glucose concentrations. Sorbitol was elevated in all retinal layers of diabetic animals. The peak sorbitol concentrations, of 2 mmol/kg defatted dry weight in diabetics, were not high enough to be osmotically significant. Retinal myo-inositol, of the order found in brain tissue (10-30 mmol/kg defatted dry tissue), was decreased by 22-40% in all retinal layers of the diabetics. The results indicate that diabetes affects the metabolism of retinal structures independently of the small blood vessel disease that is the hallmark of diabetes of long duration. It is conceivable that primary intraretinal metabolic alterations cause, aggravate, or perpetuate the well known degenerative processes that occur in retinal blood vessels in diabetes.
Experimental diabetes mellitus impairs the function of the retinal pigmented epithelium.
The retinal pigmented epithelium (RPE), which influences the composition of the retinal extracellular fluid, is significantly affected in diabetes. Changes in RPE morphology, permeability, and electrophysiology in experimentally diabetic animals have been described. To facilitate the study of diabetes-related changes in RPE metabolism, we applied the techniques of quantitative histochemistry to pure samples of RPE and individual retinal layers from eyes of normal and alloxan-diabetic rabbits. Glucose within the RPE approximated serum levels in both normal and diabetic animals. Other changes in diabetics included increased sorbitol, decreased myo-inositol, elevated total Na, and loss of measurable Na+-K+-ATPase activity within the RPE. The altered ion metabolism was associated with a progressive decrease in the amplitude of the RPE-generated c-wave of the electroretinogram. The deterioration of the c-wave was arrested by treatment of the diabetic animals with either myo-inositol supplementation or with sorbinil, an inhibitor of aldose reduction. Diabetic alterations in the RPE might impair the ability of the tissue to maintain normal transport functions. The subsequently altered composition of the extracellular environment of the retina may play an important role in the pathogenesis of diabetic retinopathy.
In vitro exhaustion of pancreatic beta-cells.
To learn more about possible limited beta-cell secretory capacity and factors essential for insulin release, a perifusion system was applied that allowed the in vitro study of insulin secretion from isolated pancreatic islets for more than 6 h. Islets isolated from rats were stimulated with various glucose concentrations (7.5, 16.7, and 30 mM), alpha-ketoisocaproate (30 mM), and 30 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine for several hours in Krebs-Ringer-bicarbonate buffer (KRB) or RPMI 1640. Islets showed "exhaustion" with all stimulatory conditions used when KRB was the perifusion medium. This was not prevented by addition of amino acids, phosphate, myo-inositol or arachidonic acid. With RPMI 1640 as the basal medium, exhaustion was not seen at 7.5 mM but was readily approached at higher glucose concentrations. It is possible that the exhaustion phenomenon observed here is due to a depletion of a readily releasable insulin pool.
Treatment with aldose reductase inhibitor or with myo-inositol arrests deterioration of the electroretinogram of diabetic rats.
Biochemical abnormalities in the retinal pigment epithelium of experimentally diabetic animals include increased sorbitol and decreased myo-inositol. Diabetes also causes a progressive reduction in the amplitude of the c-wave of the electroretinogram of the pigmented rat. The c-wave is generated by the retinal pigmented epithelium. Myo-inositol administration or treatment with sorbinil, an inhibitor of aldose reductase, arrested the decline in the c-wave. Therefore, hyperglycemia-associated defects in myo-inositol or sorbitol metabolism may be involved in the pathogenesis of the electrophysiological deficit of the diabetic retina. The homogeneous cell layer of the pigment epithelium may be a useful tissue model for studying the pathogenesis of the complications of diabetes.