Hyperglycemic Medium Research Articles

Erythrocyte changes in canine diabetes mellitus: in vitro effects of hyperglycaemia and ketoacidosis

Diabetes mellitus (DM) is a common metabolic disorder in dogs, which occurs often in association with some complications, including haematological problems. Some abnormalities of erythrocyte metabolism have been described both in human patients and in dogs affected by DM. The aim of this work was to test in vitro the direct effects of hyperglycaemia and ketoacidosis on canine erythrocytes (RBCs). RBCs from healthy dogs were incubated in normoglycaemic, mild hyperglycaemic, severe hyperglycaemic and severe hyperglycaemic + ketoacidotic media. The following parameters were examined: osmotic fragility (OF), the concentrations of 2,3-diphosphoglycerate (2,3DPG), reduced glutathione (GSH) and adenosine triphosphate (ATP), the activities of glutathione peroxidase (GPX), glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK), sodium (Na+) and potassium (K+) intracellular concentrations, production of lactate. In comparison with t0, incubated cells showed higher OF (P<0.001) and lower 2,3DPG (P<0.001) and ATP (P<0.001) concentrations. In comparison with normoglycaemic conditions, hyperglycaemia induced minimal changes, such as increased OF (P<0.001) and 2,3DPG (P<0.01) concentrations. Major changes were induced by hyperglycaemic + ketoacidotic media, such as increases in mean corpuscular volume (MCV), OF, PK activity and ATP concentration; this suggested that oxidative stress had occurred. In hyperglycaemic media, lactate production was not associated with glucose concentration, but the higher consumption of glucose suggests the activation of metabolic pathways other than glycolysis. These alterations could be partly responsible for some hypoxic complications during DM.

Hyperglycemia and insulin resistance: possible mechanisms.

Sustained hyperglycemia impairs insulin-stimulated glucose utilization and glycogen synthesis in human and rat skeletal muscles, a phenomenon referred to clinically as glucose toxicity. In rat extensor digitorum longus (EDL) muscle preparations preincubated for 2-4 h in a hyperglycemic medium (25 mM vs. 0 mM glucose), we have shown that the ability of insulin to stimulate glucose incorporation into glycogen is impaired. Interestingly, this was associated with a decreased activation of Akt/PKB, but not its upstream regulator, PI3-kinase. A similar pattern of signaling abnormalities has been observed in adipocytes, L6 muscle cells, C2C12 cells, and (as reported here) EDL incubated with C(2)-ceramide. On the other hand, no increase was observed in ceramide mass in EDL incubated with 25 mM glucose. Hyperglycemia-induced insulin resistance also has been described in adipocytes, where it has been linked to activation of novel and conventional protein kinase C isoforms that phosphorylate the insulin receptor and IRS. In addition, we have recently shown that hyperglycemia causes insulin resistance in cultured human umbilical vein endothelial cells (HUVEC). Here, it was associated with an increased propensity to apoptosis and, as in muscle, with an impaired ability of insulin to activate Akt. Interestingly, these effects of hyperglycemia and an increase in diacylglycerol synthesis, which is also caused, were prevented by adding AICAR, an activator of AMP-activated protein kinase (AMPK), to the incubation medium. These results suggest that hyperglycemia causes insulin resistance in cells other than those in classic insulin target tissues. Whether AMPK activation can reverse or prevent insulin resistance in all of these cells remains to be determined.

The effect of hyperglycaemia on expression and re‐distribution of VE‐cadherin in human endothelial cells

In vitro studies using animal vascular endothelial cells have shown that hyperglycaemia increases monolayer permeability. The mechanism underlying this increase is thought to involve disruption of adherens junctions by phosphorylation of the transmembrane molecule vascular‐endothelium cadherin (VE‐cadherin) and loss of its perijunctional actin anchorage. Recently an alternate mechanism of disruption, i.e. inflammation induced loss of actin independent VE‐cadherin, has been proposed (Lim et al. Microvas. Res. 2001 on IDEAL). The purpose of this study was to expose human endothelial cells to high glucose and follow the fate of VE‐cadherin. Human vein umbilical endothelial cells (HUVEC) were grown to confluence before being exposed to media containing different concentrations of glucose: 5 mm d‐glucose as euglycaemic control, 25 mm d‐glucose as hyperglycaemic medium, and 25 mm l‐glucose as osmotic control. VE‐cadherin localisation was studied using immunocytochemistry and systemic randomised count of junctional profiles which allowed classification of the VE‐cadherin immunoreactivity at cell‐cell borders as continuous or discontinuous. Immunoblot studies and selective protein extraction into Triton‐soluble and Triton‐insoluble fractions were performed in order to measure total protein levels of expression and actin association of VE‐cadherin under the different experimental conditions. Immunoprecipitation studies were used to investigate tyrosine phosphorylation status of VE‐cadherin. Hyperglycaemic exposure of HUVEC for 2 h resulted in a statistically significant (one‐way Anova, P &lt; 0.01) increase in the percentage of discontinuous VE‐cadherin at cell‐cell contacts of HUVEC monolayers, this significance was lost after longer durations (4 d). After 2 h of glucose insult, the total VE‐cadherin expression was not altered, whilst the selective extraction studies showed VE‐cadherin had a greater association with the actin‐cytoskeleton. The tyrosine phosphorylation level of VE‐cadherin was not altered. Our studies reveal that hyperglycaemia does cause redistribution of VE‐cadherin. However the pattern of VE‐cadherin redistribution favours the hypothesis of a loss of VE‐cadherin from actin‐independent membrane microdomains. Furthermore we find this loss is not mediated by the tyrosine kinase signalling pathway.Funded by the European Social Fund.

Region-specific pathophysiological alterations occurring in calf lenses in vitro during hyperglycemia.

The early onset of cataract during diabetes may come about via a variety of pathogenic pathways, but an uncertainty about the significance of each of them exists. Calf lenses cultured in a high glucose medium were investigated for regional variations in sorbitol accumulation, changes in lactate dehydrogenase activity, and formation of carbonyl groups in proteins. The results obtained were used to evaluate the contributions of various pathways to the alterations in the lens during hyperglycemia and to relate these findings to morphologically diverse lens substructures. The highest sorbitol accumulation was found in both the anterior and posterior cortex of lenses incubated in hyperglycemic medium. Lactate dehydrogenase activity was strongly affected by high sugar concentration, but the alterations in the equatorial part of lenses were more moderate relative to other substructures. After incubation with glucose, the concentration of Amadori products did not increase significantly compared to non-incubated and incubated controls. Nuclear proteins exhibited the highest level of oxidation. The process of sorbitol accumulation is more evident than glycation in the initial stage of hyperglycemia. Lens cortex is affected faster by elevated glucose, while the nucleus is more susceptible to prolonged effects of oxidation, glycation, and glycoxidation.

Hyperglycemia-Induced Apoptosis in Human Umbilical Vein Endothelial Cells

Apoptosis has been observed in vascular cells, nerve, and myocardium of diabetic humans and experimental animals, although whether it contributes to or is a marker of complications in these tissues is unclear. Previous studies have shown that incubation of human umbilical vein endothelial cells (HUVECs) with 30 vs. 5 mmol/l glucose for 72 h causes a significant increase in apoptosis, possibly related to an increase in oxidative stress. We report here that this increase in apoptosis (assessed morphologically by TdT-mediated dUTP nick- end labeling staining) is preceded (24 h of incubation) by inhibition of fatty acid oxidation, by increases in diacylglycerol synthesis, the concentration of malonyl CoA, and caspase-3 activity, and by decreases in mitochondrial membrane potential and cellular ATP content. In addition, the phosphorylation of Akt in the presence of 150 microU/ml insulin was impaired. No increases in ceramide content or its de novo synthesis were observed. AMP-activated protein kinase (AMPK) activity was not diminished; however, incubation with the AMPK activator 5-aminoimidazole-4-carboxamide-riboside increased AMPK activity twofold and completely prevented all of these changes. Likewise, expression of a constitutively active AMPK in HUVEC prevented the increase in caspase-3 activity. The results indicate that alterations in fatty-acid metabolism, impaired Akt activation by insulin, and increased caspase-3 activity precede visible evidence of apoptosis in HUVEC incubated in a hyperglycemic medium. They also suggest that AMPK could play an important role in protecting the endothelial cell against the adverse effects of sustained hyperglycemia.

PREVENTION OF PERICYTE LOSS BY TROLOX IN DIABETIC RAT RETINA

Prolonged hyperglycemia results in a number of diabetic complications, including retinopathy. Pericyte degeneration is one of the earliest histological changes observed in the development of diabetic retinopathy. Increased free radicals generated under hyperglycemia could damage the retina, which abounds in polyunsaturated fatty acids. In the current study, a severalfold increase in thiobarbituric acid-reactive substances was found in rat retina cultured in hyperglycemic medium, which decreased significantly when trolox, an amphipathic antioxidant, was included in the medium. To examine the contribution of oxidative stress in vivo, diabetic rats were fed trolox ( 0.4% in the diet) during the course of the experiments. After 5 mo of hyperglycemia, whole mounts of retinal vessels were prepared and endothelial cells ( E) and pericytes (P) were counted. The ratio of E/P in the retinas obtained from normal rats, diabetic rats, and diabetic rats fed trolox were 1.74 +/- 0.186, 3.78 +/- 0.47, and 2.32 +/- 0.24, respectively. A significant restoration of pericytes by trolox suggests the involvement of oxidative injury during pericyte loss in diabetic retinopathy.

The defence against free radicals protects endothelial cells from hyperglycaemia-induced plasminogen activator inhibitor 1 over-production.

It has been shown that hyperglycaemia may stimulate plasminogen activator inhibitor 1 (PAI-1) over-production in human endothelial cells in culture. At the same time, it has been shown that glucose may enolize, producing free radicals. In this study, the possibility that hyperglycaemia stimulates PAI-1 over-production in human endothelial cells in culture by generating free radicals has been evaluated. For this purpose two experimental models were used: human endothelial cells transfected to express high glutathione peroxidase levels cultured in hyperglycaemic media, and human endothelial cells cultured in hyperglycaemic media with the antioxidant GSH. Cells grown in 20 mM glucose produced higher values of PAI-1 with respect to controls. The production of PAI-1 was not influenced by hyperglycaemia in transfected cells. GSH in the medium reduced hyperglycaemia-induced PAI-1 over-production, but also reduces the basal production of PAI-1 in the cells grown in normal glucose concentration. These data show that antioxidant defences may reduce hyperglycaemia-induced PAI-1 over-production in human endothelial cells in culture. The hypothesis that oxidative stress may play an important role in the pathogenesis of diabetic complications is then supported by this study.

Platelet serotonin transport is altered in streptozotocin-induced diabetic rats

The present work was conducted to examine whether experimental diabetes (streptozotocin-induced) promotes changes in mean platelet volume, and platelet serotonin (5HT) uptake and content. These variables were measured in from four experimental groups: control, diabetic, diabetic+insulin, and non-diabetic+insulin. Animals treated fifteen days before with streptozotocin had platelets with higher 5HT uptake affinity, 5HT content, and volume. The insulin therapy reestablished the control values of all of these three variables. Non-diabetic animals treated one week with insulin did not show any variations. The effects of in vitro application of insulin, hyperglycaemic incubation medium, and streptozotocin on platelet amine uptake and release were also examined. Only those platelets incubated with streptozotocin showed an altered platelet 5HT uptake. No changes were observed for spontaneous 5HT release. The results are consistent with: a) an increase of platelet uptake capacity, as a consequence of an increase in platelet turnover, for explaining alterations of intraplatelet 5HT contents in experimental diabetes; b) a non-direct effect of insulin and glucose levels on platelet 5HT uptake -for explaining its dysfunctions in experimental diabetes-; c) the contribution of alterations in platelet 5HT transport for explaining the higher incidence of vascular complications in diabetic patients; d) the suitability of platelet as a model for investigating neuronal 5HT reuptake.

Effect of glucose on corneal endothelial ATPase assays

Background: previous biochemical methods of determining corneal endothelial ATPase (adenosine triphosphatase) activity have excluded glucose from incubation media. As glucose is an important metabolite for the formation of adenosine triphosphate (ATP) it is possible that glucose may be required in incubations investigating ATPase activity. Methods: standard ATP hydrolysis assays for ATPase activity were performed on bovine corneal endothelial homogenates using incubation media glucose levels of zero, 10, 20 and 30 mM. Results: corneal endothelial Na,K ATPase activity was not significantly different at the four glucose levels tested. Corneal endothelial Mg++ ATPase activity was significantly reduced (13.8 per cent) in the 10, 20 and 30 mM glucose conditions when compared to the zero glucose condition. Conclusions: corneal endothelial Na,K ATPase activity is unaffected by short-term exposure to hyperglycaemic media. While the data of this study are not conclusive, it is possible that short-term exposure of corneal endothelia to glucose may be associated with a transient decrease in Mg++ ATPase activity. Interactions between cellular ATP levels and Mg++ ATPase activity may have a role in helping regulate Na,K ATPase activity. (Clin Exp Optom 1994; 78: 1: 21–23)

Cellular-tissue localization and regulation of the GLUT-1 protein in both the embryo and the visceral yolk sac from normal and experimental diabetic rats during the early postimplantation period.

We investigated the tissue-specific developmental expression and localization of GLUT-1 protein in the rat embryo and visceral yolk sac (VYS) during the organogenic periods of normal rats. The expression of GLUT-1 protein was then compared to that of experimental diabetic rats to test whether the diabetic state would affect the regulation of the glucose transporter during the early postimplantation periods (9.5-14.5 days), as we have previously demonstrated that GLUT-1 protein in embryo and VYS was down-regulated in culture with hyperglycemic medium. In the embryo, GLUT-1 protein was highly expressed during the early stages of organogenesis (between 9.5-12.5 days) and declined thereafter, whereas in the VYS, its strong expression was observed at the later stages (from 12.5-14.5 days). Immunohistochemical localization of the GLUT-1 protein in the embryo during the main periods of neurulation (9.5-11.5 days) showed that GLUT-1 immunoreactivity was principally observed in the neuroepithelial cells of the neural tube and also noted in the primitive heart, primitive gut, otic, and optic vesicles. At 12.5 days, GLUT-1 protein started to be expressed in the microvessels at the cranial portions of the neural tube, although its expression in the neuroepithelial cells still remained at the caudal (tail) portions of the neural tube. In the later stages (13.5-14.5 days) after completion of neural tube formation, GLUT-1 protein immunoreactivity substantially decreased in the neuroepithelial cells and was found mainly in the microvessels of the brain vesicles and spinal cord, whereas it continued to be expressed in the heart and eyes. In the VYS, its immunoreactivity was noticeably confined to the endodermal layer, which started as a simple layer and developed wave-like folds in the later stages. The levels of GLUT-1 protein in embryo and VYS from diabetic rats, determined by Western blot analysis, were not down-regulated compared to those in control rats at the different gestational days. Likewise, comparison of GLUT-1 protein immunoreactivity of various tissues in embryo and VYS, focusing on the neural tube, also revealed no significant differences between the two groups. We demonstrated that GLUT-1 protein is abundantly expressed in embryonic tissues and VYS during the early periods of organogenesis. The lack of down-regulation and the continuous abundant expression of the GLUT-1 protein despite the diabetic state in embryo and VYS during the early postimplantation periods may increase delivery of glucose from the VYS into various differentiating embryonic cells, leading to diabetes-induced congenital malformations.

Glucose transporter gene expression in rat conceptus during high glucose culture.

We investigated the expression of glucose transporter genes and protein in embryo and yolk sac during organogenesis and the regulation of glucose transporters during culture in hyperglycaemic media. Erythrocyte-type glucose transporter (GLUT 1) and brain-type glucose transporter (GLUT 3) mRNA were expressed in embryo and yolk sac. The expression of GLUT-1 and GLUT-3 mRNA was abundant on day 9-11 and day 9-10 in the embryo, respectively, and day 9-14 and day 10-11 in the yolk sac, respectively. The levels of GLUT-1 protein in the embryo increased in parallel with the expression of GLUT-1 mRNA during the corresponding period. Immunohistochemical staining of GLUT-1 protein was found principally in the neuroepithelial cells surrounding the neural tube in the embryo on day 10 and appeared in the microvessels surrounding the neural tube after day 12. To test whether the expression of glucose transporter genes and protein was suppressed during hyperglycaemia, conceptuses were cultured in high glucose medium. The abundant expression of GLUT-1 protein was not decreased during culture in high glucose media for 24 h (day 9-10) and was only down-regulated by prolonged exposure to this media for 48 h (day 9-11). We have demonstrated the predominant expression of the high affinity glucose transporter (GLUT 1 and GLUT 3) genes and (GLUT 1) protein in embryo during the early period of organogenesis. The persistently abundant expression of glucose transporter during the critical period of neural tube formation (day 9-10) even in the presence of hyperglycaemia may explain one of the mechanism of increased glucose flux into the neuroepithelium, which may lead to neural tube defects.

The role of glycogenin in glycogen synthesis and non-insulin dependent diabetes mellitus.

The principal defect in the majority of non-insulin dependent diabetes mellitus (NIDDM) patients is impaired glycogenesis in skeletal muscle in response to insulin. Glycogen synthesis is initiated by transfer of glucosyl residues to the tyrosine OH at position 194 of a 37KD primer protein named glycogenin (1,2). While the enzymatic details of the first glucose addition have not yet been determined, addition of the next 8 or 9 glucose residues is autocatalytic. Subsequent elongation of this product by an enzyme named proglycogen synthase, yields an intermediate (proglycogen, M.W. 400,000) that is subsequently enlarged some 25-fold to mature glycogen of M.W. lo7 by glycogen synthase (3). Cultured quail skeletal muscle cells, starved of glucose by incubation in glucose-free medium overnight, contained very little glycogen, (Table 1) that was principally in the form of proglycogen. Following transfer to hyperglycemic medium (25 mM glucose), the glycogen content increased several hundred-fold within 4 hrs. This increase was stimulated by insulin (Table 1). The insulin effect was not observed when the cells were pretreated for 2 hrs with dexamethasone (1 pM). Glucocorticoids also block insulin stimulation in skeletal muscle in vivQ (4).

Phosphoinositide metabolism in the developing conceptus. Effects of hyperglycemia and scyllo-inositol in rat embryo culture.

Culture of the postimplantation rat conceptus in hyperglycemic medium causes developmental abnormalities and is associated with a diminished water-soluble myo-inositol content. We investigated the effect myo-inositol depletion has on lipid-soluble phosphoinositides, precursors, and water-soluble inositol phosphates. Rat conceptuses were cultured from gestational day 9.5 (presomite, early head fold) to day 10.5 (7-15 somites) in 6.7-73.3 mM D-glucose. Significant decreases in the phosphoinositides of the embryo were observed with increased culture D-glucose concentrations. PI was reduced 15-34%, PIP 18-46%, and PIP2 26-46%. Yolk sac phosphoinositides also were reduced but to a lesser degree. Culture in hyperglycemic media also mediated significant reductions of conceptus inositol phosphates. To investigate whether effects similar to those induced by D-glucose could be mediated by another agent capable of decreasing myo-inositol content, we used scyllo-inositol, a transported but nonmetabolized isomer of myo-inositol. Conceptuses cultured in medium containing scyllo-inositol (0.06-16.7 mM) had dose-dependent decreases of myo-[3H]inositol in water-soluble and lipid-soluble fractions. Incorporation of myo-[3H]inositol into phosphoinositides and inositol phosphates was decreased concomitantly. Developmental effects of D-glucose and scyllo-inositol were assessed in rat conceptuses cultured from day 9.5 (presomite, early head fold) to day 11.5 (22-28 somites). Culture in 40.0-73.3 mM glucose and 0.06-33.3 mM scyllo-inositol impaired growth while increasing dysmorphogenesis in a dose-dependent manner. The results suggest that decreases in conceptus myo-inositol and associated diminution of phosphoinositides, which are the inositol/lipid cycle precursors, are dysmorphogenic and may contribute to the etiology of diabetic embryopathy.

Delayed Maturation of Fetal Lung in Yellow KK Mice

Yellow KK mice have the Ay allele and spontaneously develop non-insulin dependent diabetes mellitus (NIDDM). In the present study, female yellow KK mice were mated at 7 or 13 weeks of age (the average blood glucose levels were ca. 260 and 500 mg/dl, respectively), and their fetuses were examined on days 18 or 19 of gestation. The fetuses from 13-wk-old dams were smaller than those from 7-wk-old dams. The average lung weight on day 19 of gestation was slightly greater in the fetuses from 13-wk-old dams than in those from 7-wk-old dams (23.3 vs 21.2 mg). Histologically, alveolar spaces were smaller and alveolar walls were thicker in the fetuses from 13-wk-old dams than in those from 7-wk-old dams. Morphometric analysis revealed that the percentage area of the fetal lung occupied by alveolar spaces was significantly smaller in the former fetuses than in the latter. Day-15 fetal lungs of KK mice, which do not have the Ay allele, were cultured in hyperglycemic media (glucose levels: 400 and 700 mg/dl) for 48 hr. The development of alveoli was inhibited in hyperglycemic media as compared to the development of the lungs grown in the control medium. The inhibitory effect was dependent on the glucose concentration in the media. Thus, it seems that maternal hyperglycemia itself is a major cause of the delayed maturation of the fetal lung in NIDDM mice.

Effects of brief exposure to insulin-induced hypoglycemic serum during organogenesis in rat embryo culture.

We have previously shown that long-term exposure to medium containing insulin-induced hypoglycemic serum during the early phase of organogenesis can adversely affect embryonic development in rat embryo culture and that these effects were mediated through the interruption of glycolytic flux that constituted the principal pathway at this embryonic stage. Further experiments were performed to examine whether brief exposure to the hypoglycemic medium during critical developmental periods would have adverse effects on embryogenesis during embryo culture not only in normal but also in high glucose concentrations. Rat embryos in the early head-fold stage (9.5 days gestation) were grown in vitro for 48 h until neural tube closure occurred; dysmorphogenic lesions were not elicited in either the basal culture medium containing 6.6 mM glucose (control medium) or the hyperglycemic medium supplemented with glucose at a concentration of 33.3 mM. Hypoglycemic mediums (2.2-2.5 mM glucose) were prepared from the serum of rats given insulin intraperitoneally. Postimplantation embryos (in early neural tube formation) were briefly exposed (1 h) to hypoglycemic medium on day 10.3 of gestation during the basal culture. After exposure to the hypoglycemic medium for 1 h during culture in the control medium, embryos showed minor growth retardation and dysmorphogenic lesions (7.1% open neural pores). Exposure to the hypoglycemic medium for 1 h during culture in hyperglycemic medium suplemented with a subteratogenic concentration of glucose (33.3 mM) resulted in greater growth retardation and increased occurrence of dysmorphogenic lesions (17.3% open neural pores).(ABSTRACT TRUNCATED AT 250 WORDS)

Polyol and vacuole formation in cultured canine lens epithelial cells

Polyol accumulation and myo-inositol depletion were accompanied by extensive vacuole formation in cultured canine lens epithelial cells that were incubated for up to 96 hr in growth medium supplemented with 30 m m d-galactose or 30 m m d-glucose. These changes did not occur in cells incubated in a hypergalactosemic or hyperglycemic medium which also contained an aldose reductase inhibitor (20 μ m sorbinil). In addition, these changes were not observed in lens cells incubated in growth medium supplemented with either 30 m m mannitol, which is known to enter cells only slowly, or in 30 m m l-galactose, which is not a substrate for aldose reductase. The vacuoles were visible at the ultrastructural level after 6 hr of incubation in 30 m m >d-galactose and increased in both number and size with time. These vacuoles had a unique fine structure. They did not result from swelling of mitochondria or other cell organelles. As demonstrated cytochemically, they did not represent either lysosomes or Golgi saccules. The proliferation pattern of cells incubated with 30 m m d-galactose was clearly different from that of control cells, but approached normal when an aldose reductase inhibitor was added to the incubation medium. Together these findings suggest that vacuole formation and altered cell proliferation were caused by polyol accumulation and/or myo-inositol loss, both of which result from aldose reductase activity.

Arachidonic acid prevents hyperglycemia-associated yolk sac damage and embryopathy

Light microscopic, electron microscopic, and morphometric studies were performed on rat conceptuses cultured between day 10 and day 12 in normal, hyperglycemic, arachidonic acid-supplemented normal, and arachidonic acid-supplemented hyperglycemic rat serum. The results were compared with those of 12-day-old conceptuses grown in utero. No major differences were observed between in vivo and in vitro control conceptuses. Arachidonic acid supplementation of control culture medium resulted in an improvement of conceptus development. Addition of 20 micrograms/ml of arachidonic acid to an otherwise teratogenic hyperglycemic serum medium (950 mg/dl of D-glucose) prevented the malformations induced by hyperglycemic conditions: open neural tube, advanced neuropil formation in the neuroepithelium, significant reduction of rough endoplasmic reticulum, decreased size and number of lipid droplets, and increased number of lysosome-like structures in the visceral endodermal yolk sac cells.

Heparin Osteoporosis

Light microscopic, electron microscopic, and morphometric studies were performed on rat conceptuses cultured between day 10 and day 12 in normal, hyperglycemic, arachidonic acid-supplemented normal, and arachidonic acid-supplemented hyperglycemic rat serum. The results were compared with those of 12-day-old conceptuses grown in utero. No major differences were observed between in vivo and in vitro control conceptuses. Arachidonic acid supplementation of control culture medium resulted in an improvement of conceptus development. Addition of 20 μg/ml of arachidonic acid to an otherwise teratogenic hyperglycemic serum medium (950 mg/dl of D-glucose) prevented the malformations induced by hyperglycemic conditions: open neural tube, advanced neuropil formation in the neuroepithelium, significant reduction of rough endoplasmic reticulum, decreased size and number of lipid droplets, and increased number of lysosome-like structures in the visceral endodermal yolk sac cells.