Effect of insulin on ascorbic acid uptake by heart endothelial cells: Possible relationship to retinal atherogenesis

Increasing concentrations of insulin were found to increase transport of radioactive ascorbic acid into fetal bovine heart endothelial cells (FBHE). A linear relationship was found between the log of the insulin concentration (range 0 μU/ml to 400 μU/ml) and the uptake of ascorbic acid quantified as dpm/μg protein. Evidence has accrued which relates ascorbic acid to atherogenesis by its possible effect on preventing the breakdown of the glycosaminoglycan matrix of the intimal layer of the artery. Since insulin was found to increase ascorbic acid uptake, any compound, like glucose, that competes for the carrier mechanism may, if present in high enough concentrations, competitively inhibit ascorbic acid transport into the cell. The hyperglycemia and inadequate insulin production associated with diabetes mellitus may cause an ascorbic acid deficiency within the cell. This deficiency would lead to intimal matrix breakdown with subsequent increase in atherogenesis. The microangiopathies associated with diabetes and with the aging process itself may be related to this mechanism.     

Nitric oxide-induced oxidant stress in endothelial cells: amelioration by ascorbic acid

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.     

Short-term fatty acid effects on adipocyte glucose uptake: Mechanistic insights

The modulation of insulin sensitivity in visceral fat tissue could be important in the treatment of Type 2 diabetes mellitus. Selected fatty acids may impact on insulin-stimulated and basal glucose uptake in adipocytes, thus isolated rat epididymal adipocytes were exposed to 100 μM oleic, arachidonic, eicosapentaenoic, docosahexaenoic or stearic acids and insulin (15 nM) or vehicle for 30 min. Glucose uptake was quantified by measuring uptake of ³H-deoxyglucose/mg adipocyte protein/min. Where appropriate, inhibitors were included to elucidate the mechanisms involved.In this model, insulin stimulated glucose uptake with 62±7%. All fatty acids tested, except for stearic acid, depressed insulin-stimulated glucose uptake by an average of 33±4.2%. On the other hand, all fatty acids tested except stearic and arachidonic acids, stimulated basal glucose uptake with an average of 34±8.1%. Inhibitor studies showed the involvement of prostaglandins, lipoxins, protein kinase C and tyrosine kinase in these processes.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

Oxidized LDL up-regulates the ascorbic acid transporter SVCT2 in endothelial cells

Endothelial dysfunction is an early manifestation of atherosclerosis caused in part by oxidized LDL (oxLDL). Since vitamin C, or ascorbic acid, prevents several aspects of endothelial dysfunction, the effects of oxLDL on oxidative stress and regulation of the ascorbate transporter, SVCT2, were studied in cultured EA.hy926 endothelial cells. Cells cultured for 18 h with 0.2 mg/ml oxLDL showed increased lipid peroxidation that was prevented by a single addition of 0.25 mM ascorbate at the beginning of the incubation. This protection caused a decrease in intracellular ascorbate, but no change in the cell content of GSH. In the absence of ascorbate, oxLDL increased SVCT2 protein and function during 18 h in culture. Although culture of the cells with ascorbate did not affect SVCT2 protein expression, the oxLDL-induced increase in SVCT2 protein expression was prevented by ascorbate. These results suggest that up-regulation of endothelial cell SVCT2 expression and function may help to maintain intracellular ascorbate during oxLDL-induced oxidative stress, and that ascorbate in turn can prevent this effect.     

The effect of elevated extracellular glucose on adherens junction proteins in cultured rat heart endothelial cells

Vascular permeability is a proof of vascular endothelial cell dysfunction induced by diabetes. Vascular permeability is directly related to the width of intercellular endothelial cells junctions, which may become permeable to macromolecules as a result of a change in endothelial cell shape. To determine the role of hyperglycemia in endothelial cell shape, the study examined the effect of high concentrations of glucose on the shape of cultured rat heart endothelial cells. This result indicated that the high-glucose-induced changes in the morphology of endothelial cells, via the glucose-mediated reorganization of F-actin. In endothelial cells, the actin cytoskeleton is tethered to the zonula adherens and focal adhesions, which mediate cell-cell and cell-matrix interactions respectively. The present study demonstrated that the high-glucose-induced changes in the actin-binding protein such as filamin, zonula adherens proteins such as alpha-, beta-, and gamma-catenin, focal adhesions proteins such as focal adhesion kinase, paxillin, and tyrosine phosphorylation of paxillin. It appears that differences in expression of adherens junctions molecules on rat heart endothelial cells in response to high glucose reflect endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.     

Non-insulin dependent diabetic patients have increased glucose uptake in red blood cells

The kinetic characteristics of 3-O-methyl glucose (3-OMG) uptake were examined in red blood cells (RBC) from seven normal individuals (controls) and nine patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet and oral hypoglycemic medication. Comparison of rates of 3-OMG uptake at 5 different substrate concentrations revealed significantly higher overall 3-OMG uptake in the diabetic group (P less than 0.0001). Kinetic parameters obtained for individual subjects showed there was not a significant difference in the Km between the diabetic (3.17 +/- 0.45 mM; mean +/- SE) and the control (2.46 +/- 0.25 mM) groups. However, Vmax was significantly increased (61%; P less than 0.025) in the diabetics (217.8 +/- 28.9 pmol/2 sec per 10(6) cells) compared to controls (135.2 +/- 15.6 pmol/2 sec per 10(6) cells). There was no correlation between HbA1C levels in the diabetic patients and Vmax values for 3-OMG uptake, suggesting that the increased hexose uptake was not accounted for simply by increased glycosylation in these cells. Glucose transport in RBC in hyperglycemic states may be a useful model for delineating the regulation of the non-insulin-mediated disposal of glucose in diabetes.     

De novo emergence of insulin-stimulated glucose uptake in human aortic endothelial cells incubated with high glucose

Elevated glucose concentrations have profound effects on cell function. We hypothesized that incubation of human aortic endothelial cells (HAEC) with high glucose increases insulin signaling and develops the appearance of insulin-stimulated glucose uptake by the cells. Compared with 5 mM glucose, incubation of HAEC with 30 mM glucose for up to 48 h increased in a time-dependent manner expression of insulin receptor, insulin receptor substrate (IRS)-1, IRS-2, and GLUT1 proteins. High glucose also increased the specific binding of (125)I-labeled insulin in HAEC accompanied by accelerated production of interleukin (IL)-6 and IL-8. Short-term stimulation by 50 microU/ml insulin did not activate [(14)C]glucose uptake by HAEC incubated in 5 mM glucose. However, an addition of insulin to high glucose-exposed endothelial cells led to a significant increase in [(14)C]glucose uptake in a glucose concentration- and time-dependent fashion, reaching a plateau at 48 h of incubation. Furthermore, incubation of HAEC with 30 mM glucose resulted in a new insulin-stimulated extracellular signal-regulated kinase-1/2 mitogen-activated protein kinase phosphorylation and increased lipid peroxidation and production of reactive oxygen species. These studies show for the first time that high glucose increases expression of insulin receptors and downstream elements of the insulin-signaling pathway and transforms "insulin-resistant" aortic endothelial cells into "insulin-sensitive" tissue regarding glucose uptake.     

Effect of hyperoxia on glutathione levels and glutamic acid uptake in endothelial cells

Intracellular glutathione was increased by 80% after exposure of bovine pulmonary arterial endothelial cells to 80% O2 (hyperoxia) for 24 h. No change in glutathione occurred in cells exposed to hypoxia (3% O2) for a corresponding period of time. The rate of uptake of [3H]glutamic acid also increased by 35-55% after 24 h of exposure of cells to hyperoxia, whereas exposure to hypoxia had no effect on the [3H]glutamic acid uptake. The increase in glutamic acid uptake reflected a specific effect on amino acid transport systems rather than a change in cell membrane permeability. The major portion of the increased uptake was inhibited by the elimination of sodium and the addition of the competitive inhibitor, cystine, to the incubation medium. Thus increases in glutamic acid uptake parallel increases in cellular glutathione, and glutamic acid may be a regulating factor in the increase in glutathione after exposure to hyperoxia.     

Impaired insulin signaling in endothelial cells reduces insulin-induced glucose uptake by skeletal muscle

In obese patients with type 2 diabetes, insulin delivery to and insulin-dependent glucose uptake by skeletal muscle are delayed and impaired. The mechanisms underlying the delay and impairment are unclear. We demonstrate that impaired insulin signaling in endothelial cells, due to reduced Irs2 expression and insulin-induced eNOS phosphorylation, causes attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduces glucose uptake by skeletal muscle. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reverses the reduction in capillary recruitment and insulin delivery in tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle is restored in these mice. Taken together, our results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Furthermore, improving endothelial insulin signaling may serve as a therapeutic strategy for ameliorating skeletal muscle insulin resistance.     

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats

Background

There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.

Methodology/Principal Findings

Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally) once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin), plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium.

Conclusions/Significance

Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.     

Characterization of glutamic acid uptake by bovine pulmonary arterial endothelial cells

L-Glutamic acid uptake by bovine pulmonary arterial endothelial cells in culture increased linearly with time up to 30 min and did not show saturation with increased substrate concentration up to 6 X 10(-3) M. The uptake per cell decreased as cell density increased and was lowest when the cells became fully confluent. Most of the uptake was sodium dependent, although the relative contribution of sodium-independent uptake increased with an increase in cell density. Cysteic and aspartic acid strongly inhibited L-glutamic acid uptake, but at higher cell densities this effect was less pronounced than at low densities. Other amino acids, including leucine, glutamine, and serine, exerted a modest inhibitory effect at both high and low cell densities. Thus pulmonary arterial endothelial cells contain similar membrane transport systems for L-glutamic acid as those previously described for fibroblasts, hepatocytes, and nerve cells. However, quantitative properties of the transport systems differ depending on the state of cellular density in monolayers.     

Reduced viability of vascular endothelial cells by high concentration of ascorbic acid in vitreous humor

Normal mammalian vitreous humor maintains its avascularity after regression of hyaloid vessels. Neovascularization in adults is only detected under pathological conditions which suggests that antiangiogenic factors are present in the vitreous humor. To elucidate the mechanism of vitreal angiogenic inhibition, we investigated the effect of vitreous humor on cultured vascular endothelial cells. When bovine aortic endothelial cells were cultured in the presence of bovine vitreous humor in medium, a decrease in cell viability was observed within 24 h. Ascorbic acid from vitreous humor has been identified as a cell death inducing factor with high performance liquid chromatography (HPLC) and molecular mass analysis. Ascorbic acid reduced endothelial cell viability at concentrations normally present in vitreous humor. This effect was completely inhibited by antioxidants, N-acetylcysteine and catalase. Amongst the ascorbic acid derivatives tested, ascorbic acid 2-phosphate did not induce cell death, suggesting that the production of ascorbyl radical is required for induction of cell death. Furthermore, capillary formation in three-dimensional collagen gel cultures characteristic of vascular endothelial cells were disrupted in the presence of ascorbic acid. Since ascorbic acid is highly concentrated in ocular tissues, especially in vitreous humor, it may function as a neovascularization inhibitor.     

Inhibition of sugar uptake by ascorbic acid in Escherichia coli

The uptake of glucose by the glucose phosphotransferase system in Escherichia coli was inhibited greater than 90% by ascorbate. The uptake of the nonmetabolizable analog of glucose, methyl-alpha-glucoside, was also inhibited to the same extent, confirming that it was the transport process that was sensitive to ascorbate. Similarly, it was the transport function of mannose phosphotransferase for which mannose and nonmetabolizable 2-deoxyglucose were substrates that was partially inhibited by ascorbate. Other phosphotransferase systems, including those for the uptake of sorbitol, fructose and N-acetylglucosamine, but not mannitol, were also inhibited to varying degrees by ascorbate. The inhibitory effect on the phosphotransferase systems was reversible, required the active oxidation of ascorbate, was sensitive to the presence of free-radical scavengers, and was insensitive to uncouplers. Because ascorbate was not taken up by E. coli, it was concluded that the active inhibitory species was the ascorbate free radical and that it was interacting reversibly with a membrane component, possibly the different enzyme IIB components of the phosphotransferase systems. Ascorbate also inhibited other transport systems causing a slight reduction in the passive diffusion of glycerol, a 50% inhibition of the shock-sensitive uptake of maltose, and a complete inhibition of the proton-symport uptake of lactose. Radical scavengers had little or no effect on the inhibition of these systems.     

Hypoxia effects on cell volume and ion uptake of cerebral microvascular endothelial cells

Increased transport of Na across an intact blood-brain barrier (BBB) contributes to cerebral edema formation in ischemic stroke. Our previous studies have shown that ischemic factors stimulate activity of a luminal BBB Na-K-Cl cotransporter, and we have hypothesized that during ischemia, the cotransporter together with the abluminal Na/K pump mediates increased transport of Na from blood into the brain. However, it is possible that elevated Na-K-Cl cotransporter activity could also cause cell swelling if it outpaces ion efflux pathways. The present study was conducted to evaluate the effects of hypoxia on intracellular volume of BBB cells. Cerebral microvascular endothelial cell (CMEC) monolayers were exposed to varying levels of hypoxia for 1 to 5 h in an O(2)-controlled glove box, and cell volume was assessed using 3-O-methyl-D-[(3)H]glucose and [(14)C]sucrose as markers of total and extracellular water space, respectively. Cells exposed to either 7.5%, 3%, or 1% O(2) showed gradual increases in volume (compared with 19% O(2) normoxic controls) that became significant after 3 or more hours. By ion chromatography methods, we also found that a 30-min exposure to 7.5% O(2) caused an increase in bumetanide-sensitive net Na uptake by the cells without increasing cell Na content. CMEC Na content was significantly increased, however, following 3 or more hours of exposure to 7.5% O(2). These findings are consistent with the hypothesis that during cerebral ischemia, the BBB Na-K-Cl cotransporter is stimulated to mediate transendothelial uptake of Na into the brain and that increased cotransporter activity also contributes to gradual swelling of the cells.     

Possible inhibitory function of endogenous 15-hydroperoxyeicosatetraenoic acid on prostacyclin formation in bovine aortic endothelial cells

Arachidonic acid is metabolized via the cyclooxygenase pathway to several potent compounds that regulate important physiological functions in the cardiovascular system. The proaggregatory and vasoconstrictive thromboxane A2 produced by platelets is opposed in vivo by the antiaggregatory and vasodilating activity of prostacyclin (prostaglandin I2) synthesized by blood vessels. Furthermore, arachidonic acid is metabolized by lipoxygenase enzymes to different isomeric hydroxyeicosatetraenoic acids (HETE's). This metabolic pathway of arachidonic acid was studied in detail in endothelial cells obtained from bovine aortae. It was found that this tissue produced 6-ketoprostaglandin F1 alpha as a major cyclooxygenase metabolite of arachidonic acid, whereas prostaglandins F2 alpha and E2 were synthesized only in small amounts. The monohydroxy fatty acids formed were identified as 15-HETE, 5-HETE, 11-HETE and 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT). The latter two compounds were produced by cyclooxygenase activity. Nordihydroguaiaretic acid (NDGA), a rather selective lipoxygenase inhibitor and antioxidant blocked the synthesis of 15- and 5-HETE. It also strongly stimulated the cyclooxygenase pathway, and particularly the formation of prostacyclin. This could indicate that NDGA might exert its effect on prostacyclin levels by preventing the synthesis of 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a potent inhibitor of prostacyclin synthetase. 15-HPETE could therefore act as an endogenous inhibitor of prostacyclin production in the vessel wall.     

The effects of ionizing radiation on the pulmonary endothelial cell uptake of alpha-aminoisobutyric acid and synthesis of prostacyclin

The effects of gamma irradiation (150-3000 rad) on prostacyclin synthesis (PGI2) and Na+-dependent amino acid uptake (alpha-aminoisobutyric acid, AIB) were assessed in vitro in bovine pulmonary artery endothelial cells grown in plastic culture dishes. A dose-dependent increase in both PGI2 synthesis and AIB was found 24 h after irradiation at exposure levels greater than 600 rad. The increase in PGI2 synthesis [297% of sham-irradiated values at 3000 rad, P less than 0.01] was due to an increase in release of arachidonic acid from plasma membrane stores as well as stimulation of cyclooxygenase and/or prostacyclin synthetase enzymes. The increase in AIB uptake (75% increase at 3000 rad compared to sham-exposure values) correlated with the increased synthesis of PGI2 (r = 0.94). There was also a dose-dependent increase in the number of cells that became detached from the culture dishes during the 24-h period after irradiation. The changes in PGI2 synthesis and AIB uptake induced by gamma irradiation differed if the endothelial cells were grown on cover slips, indicating that the endothelial response to irradiation may be dependent on the interaction between the endothelial cell and its extracellular basement membrane matrix.