Effect of cytochalasin B on the uptake of ascorbic acid and glucose by 3T3 fibroblasts: mechanism of impaired ascorbate transport in diabetes

Hyperglycemia and/or hypoinsulinemia have been found to inhibit L-ascorbic acid cellular transport. The resultant decrease in intracellular ascorbic acid may de-inhibit aryl sulfatase B and increase degradation of sulfated glycosaminoglycans (sGAG). This could lead to a degeneration of the extracellular matrix and result in increased intimal permeability, the initiating event in atherosclerosis. The present studies show that the glucose transport inhibitor cytochalasin B blocked the uptake of 3H-2-deoxy-D-glucose (2.5 mg%) by mouse 3T3 fibroblasts. Cytochalasin B also blocked the uptake of 14C-L-ascorbic acid (1.25 mg%). The results of these studies further support the hypothesis that glucose and ascorbate share a common transport system. This may have important implications concerning the vascular pathology associated with diabetes mellitus.     

The effects of glucose on ascorbic acid uptake in heart endothelial cells: Possible pathogenesis of diabetic angiopathies

Glucose in concentrations of 20 mg% (or greater) significantly inhibited ¹⁴C-labelled ascorbic acid (1.25 mg%) uptake in endothelial cells in the presence of insulin (1600 ωU/ml). The absence of insulin also significantly reduced ascorbic acid uptake. Furthermore, this reduction could be exacerbated by glucose (40, 160 mg%) but not equimolar concentrations of fructose. Increased ascorbic acid concentrations (two-fold) in the absence of insulin (1) significantly enhanced uptake, and (2) reversed the inhibition by glucose. These findings support earlier reports that ascorbic acid uptake into the cell may be compromised by decreased insulin and/or increased extracellular glucose levels. Since previous animal studies have correlated experimental ascorbic acid deficiencies with atherogenic processes (presumably by altering glycosaminoglycan metabolism), the postulation that the “diabetic condition” (low insulin, hyperglycemia) accelerates the cellular changes leading to atherosclerosis by impairing ascorbic acid uptake into the vascular endothelium, may now be supported.     

Insulin binding to erythroblastic leukemic cells is decreased by insulin and increased by amino acids

Erythroblastic leukemic cells incubated in media supplemented with high amino acid concentrations subsequently bound 55.5% more [¹²⁵I]insulin than cells incubated in low amino acid media. Cycloheximide (1 μg/ml) abolished this effect. Incubation of cells with physiological (100 μU/ml) doses of insulin in low amino acid media decreased insulin binding by 34.7%. In high amino acid media the same dose of insulin decreased insulin binding by 33% compared to supplemented media without insulin but increased binding by 22.5% compared to basal media without insulin. The data suggest that amino acids or specific amino acids may exert a regulatory influence on insulin receptor homeostasis.     

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.     

Glucose inhibits cellular ascorbic acid uptake by fibroblasts in vitro

It has been suggested earlier that the local deficiency of ascorbic acid in tissues could be responsible for development of various angiopathies in diabetes. Hyperglycemia is one of the factors which could contribute considerably to the development of local ascorbic acid deficiency. Therefore, the effect of glucose on uptake of L-[1-14C] ascorbic acid by fibroblasts was studied in vitro. The data clearly show that ascorbic acid uptake is inhibited instantly by glucose in a concentration dependent fashion. The results support the contention that local ascorbic acid deficiency in tissues could be a natural consequence of hyperglycemia of whatever cause. The rate of ascorbic acid uptake under various conditions suggests that additional supplements of ascorbic acid might be helpful to individuals in averting deleterious effects of hyperglycemia on tissue ascorbic acid supply.     

Insulin-induced insulin resistance of alpha-aminoisobutyric acid transport in cultured human skin fibroblasts.

Cultured fibroblasts derived from skin biopsies were used to develop a system for studying insulin resistance in human tissue in vitro. Uptake of alpha-aminoisobutyric acid by cultured human skin fibroblasts was found to occur by a combination of saturable and nonsaturable processes. Insulin stimulated uptake by decreasing the Km of the saturable transport system from 0.58 mM to 0.26 mM. The maximal velocity of saturable uptake was 16.6 nmol/10(7) cells/min in both the presence and absence of insulin. Uptake of alpha-aminoisobutyric acid at 0.2 mM was studied in human skin fibroblasts with and without chronic exposure to insulin for 4 days at an initial concentration of 10 micrograms/ml. Unstimulated uptake was increased from 17 to 20 nmol/10(8) cells/min, and the increase in uptake due to maximal stimulation by insulin was unchanged at 16 nmol/10(8) cells/min in the cells exposed chronically to insulin. The apparent Km for insulin was increased from 80 microunits/ml to 2400 microunits/ml in the insulin-exposed cells. Thus, chronic exposure to insulin induces resistance of alpha-aminoisobutyric acid uptake by decreasing the apparent affinity for insulin.     

Effect of insulin on human intestinal smooth muscle.

The action of insulin (0.1 U/ml) on the metabolism of human intestinal smooth muscle was studied in vitro. The experiments were performed on the muscle layer of human jujunum obtained from patients undergoing intestinal shunt operations because of obesity. Insulin significantly increased glucose uptake, glycogen content, the membrane transport of alpha-amino-isobutyric acid (AIB), the incorporation of leucine into protein and tended to increase the membrane transport of the nonutilizable model monosaccharide 3-0-methylglucose. The effects of insulin were moderate and appeared after incubation times of 120 to 180 min.     

Inhibition of intracellular proteolysis in muscle cultures by multiplication-stimulating activity. Comparison of effects of multiplication-stimulating activity and insulin on proteolysis, protein synthesis, amino acid uptake, and sugar transport

The effects of the insulin-like growth factor, multiplication-stimulating activity (MSA), on chick myotube cultures were investigated. In serum-free media, MSA at levels reported to be present in fetal serum (5 ng/ml) significantly inhibited overall rates of protein degradation and stimulated protein synthesis and amino acid uptake. Half-maximal effects on protein degradation (-30%), synthesis (+25%), and amino acid uptake (+50%) occurred at approximately 0.05 micrograms/ml. In contrast, 10(2)-10(3)-fold higher concentrations (5 micrograms/ml) were required to stimulate transport of the glucose analog 2-deoxyglucose. The results indicate that MSA is an effective anabolic agent regulating protein metabolism and amino acid uptake, but not sugar transport in these cells. Parallel studies conducted with insulin demonstrated similar size effects on protein metabolism and amino acid uptake in serum-free media. However, unlike MSA, insulin levels (10(-2) units/ml) well in excess of its normal physiological range were required to produce significant effects. In addition, the relative sensitivity of sugar transport with respect to protein metabolic effects differed for insulin and MSA. Thus, 2-deoxyglucose transport was approximately 10 times more sensitive to insulin than protein synthesis, proteolysis, or amino acid uptake in contrast to MSA where the reverse was true. However, despite the relatively higher sensitivity of sugar transport to insulin, supraphysiological levels (10(-3) units/ml) of this hormone were still required for significant stimulation. These results suggest a generally low insulin sensitivity in cultured chick myotubes relative to adult tissues. In contrast, the effects of MSA are consistent with a possible role of this or similar factors in regulating growth and development of embryonic muscle.     

Ascorbic acid uptake in guinea pig intestinal mucosa

Cellular accumulation of ascorbic acid was investigated in vitro in distal intestinal mucosa of guinea pig. With ¹⁴C-ascorbic acid present at 8 μM/L in the bathing media, tissue/media (T/M) concentration ratios of at least 5 were routinely achieved. Recently absorbed ascorbic acid appeared to be free in solution in the cellular fluid in that it diffused from tissue exposed to poisons with a disappearance half-time of approximately 10 minutes. Ascorbic acid uptake was highly dependent on the presence of sodium in the bathing media; total Tris substitution resulted in a 97% decrease in uptake. Also, metabolically depleted tissue did not accumulate ascorbic acid against a concentration gradient. Uptake of ¹⁴C-ascorbic acid from a bathing solution concentration of 8 μM/L was reduced 67% in the presence of 0.8 mM/L nonlabeled ascorbic acid. Recently absorbed ¹⁴C-ascorbic acid moved more rapidly back into the lumen when the luminal solution contained nonlabeled ascorbic acid (5 mM) than when it contained mannitol (5mM). This demonstration of counter transport substantiates a carrier mechanism in the brush border.     

Effect of Escherichia coli endotoxin on ascorbic acid transport in isolated adrenocortical cells

The active uptake of ascorbic acid by isolated rat adrenocortical cells increases with ascorbic acid concentration, depends on time and calcium, and is inhibited by ACTH concentrations required for maximal steroidogenesis. Lipopolysaccharide of Escherichia coli 0111:B4 modifies the ascorbic acid uptake in a calcium-dependent manner. At low calcium concentrations, lipopolysaccharide exerts a stimulatory effect on ascorbic acid transport and at high concentrations lipopolysaccharide produces a dose-dependent inhibitory effect. This inhibition of the ascorbic acid transport by the endotoxin can alter the ascorbic acid accumulation in the adrenal gland during endotoxin shock.     

Stimulation of Na+-Ca2+ exchange in heart sarcolemma by insulin

Insulin was found to stimulate Na+-dependent Ca2+ uptake in dog heart sarcolemma in a concentration dependent manner (0.001 to 1 milliunits/ml). Maximal stimulation (160 to 170%) was seen at 0.1 to 1 milliunits/ml of insulin. Unlike Na+-dependent Ca2+ uptake, ATP-dependent Ca2+ uptake was unaltered by 1 microunit/ml of insulin. However, high concentrations of insulin (0.01 to 1 milliunits/ml) significantly increased the ATP-dependent Ca2+ uptake activity of heart sarcolemma; maximal increase (60%) was observed at 1 milliunit/ml of insulin. The Na+ K+-ATPase activity did not change upon incubating sarcolemma with insulin. The membrane preparation exhibited specific insulin binding characteristics. The Scatchard plot analysis of the data indicated two binding sites for insulin; the association constants for the high and low affinity sites were 2 X 10(9) M-1 and 4.4 X 10(8) M-1, respectively. These results support the view regarding the presence of insulin receptors in the heart cell membrane and indicate a dramatic effect of insulin on the sarcolemmal Ca2+ transport systems.     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Chromium and chronic ascorbic acid depletion effects on tissue ascorbate,manganese, and14C retention from14C-ascorbate in guinea pigs

Chromium (Cr) potentiates the effects of insulin and a role for insulin in ascorbic acid transport has been reported. Therefore, the effects of Cr and ascorbate depletion on tissue ascorbic acid and¹⁴C distribution and excretion after a¹⁴C ascorbate dose were investigated in guinea pigs. As utilization of dietary Cr is affected by interaction with other minerals, tissue manganese (Mn), zinc (Zn), copper (Cu), and iron (Fe) were examined. For 20 wk, 40 weanling animals were fed either a Cr-deficient (<0.06 μg Cr/g diet, ?Cr) or a Cr-adequate (2 μg Cr from CrCl₃/g diet, +Cr) casein-based diet and were given 1 mg ascorbate/d (?C) or 10 mg ascorbate/d (+C) for 20 wk. Animals fed the Cr-depleted diet had decreased weight at 20 wk (p<0.01). Six hours before necropsy, animals were dosed by micropipette with 1.8 μCi ofl-[carboxyl-¹⁴C] ascorbic acid and placed in metabolic cages. Ascorbate supplementation increased Fe concentrations in most analyzed tissues, hepatic¹⁴C, tissue ascorbate and Mn concentration in the adrenal and testes, but decreased the concentrations of Cu in the kidney and Mn in the spleen. Liver Mn concentration was higher and kidney Mn concentration was lower in +Cr animals. Interactions between Cr and ascorbic acid affected Mn concentrations in bone and brain. These results indicate that ascorbate and Cr may affect Mn distribution. Chromium supplementation decreased plasma cortisol, brain¹⁴C and the amount of¹⁴C expired as carbon dioxide. These findings suggest that dietary Cr may affect ascorbic acid metabolism and the metabolic response to stress.     

Involvement of membrane sulfhydryls in the activation and maintenance of nutrient transport in chick embryo fibroblasts

At 5 μg/ml, insulin stimulates hexose, A-system amino acid, and nucleoside transport by serum-starved chick embryo fibroblasts (CEF). This stimulation, although variable, is comparable to that induced by 4% serum. The sulfhydryl oxidants diamide (1–20 μM). hydrogen peroxide (500 μM), and methylene blue (50 μM) mimic the effect of insulin in CEF. PCMB-S,¹ a sulfhydryl-reacting compound which penetrates the membrane slowly, has a complex effect on nutrient transport in serum- and glucose-starved CEF. Hexose uptake is inhibited by 0.1–1 mM PCMB-S in a time- and concentration-dependent manner, whereas A-system amino acid transport is inhibited maximally within 10 min of incubation and approaches control rates after 60 min. A differential sensitivity of CEF transport systems is also seen in cells exposed to membrane-impermeant glutathione-maleimide I, designated GS-Mal. At 2 mM GS-Mal reduces the rate of hexose uptake 80–100% in serum- and glucose-starved CEF; in contrast A-system amino acid uptake is unaffected. D-glucose, but not L-glucose or cytochalasin B, protects against GS-Mal inhibition. These results are consistent with the hypothesis that sulfhydryl groups are involved in nutrient transport and that those sulfhydryls associated with the hexose transport system and essential for its function are located near the exofacial surface of the membrane in CEF.     

Ascorbic acid modulates collagen type I gene expression by cells from an eye tissue--trabecular meshwork.

The trabecular meshwork, a specialized tissue in the anterior chamber of the eye, plays a major role in the regulation of aqueous humor outflow. We studied the effects of ascorbic acid, a significant component in the aqueous humor, on gene expression of type I collagen in cultures of bovine trabecular meshwork cells. These cells were plated for 6 days, exposed to ascorbic acid in concentrations of 100, 250 and 500 micrograms/ml for 3 days and labeled with (3H)proline for the last 24 hrs. Cultures that did not receive ascorbic acid served as controls. Bacterial collagenase assays showed enhanced incorporation of (3H)proline into collagenous proteins in cultures treated with 100 and 250 micrograms/ml of ascorbic acid. Gel electrophoresis and fluorography revealed that ascorbic acid caused a 2.6- to 4.9-fold increase in production of alpha 1 (I) and alpha 2(I) collagen chains by trabecular meshwork cells. Such an increase was found, using a cDNA probe specific for pro alpha 1(I) chains, to be accompanied by an increase in steady-state mRNA levels. Similar findings were also yielded from in situ hybridization experiments. These results, coupled with previously demonstrated ascorbate-induced effects on glycosaminoglycan, fibronectin and laminin synthesis, suggest that ascorbic acid is a key mediator of the extracellular matrix production by trabecular meshwork cells. Fluctuations in its concentration may lead to alterations in the makeup and assembly of matrices underlying the cells.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Induction of tyrosine aminotransferase and amino acid transport in rat hepatoma cells by insulin and the insulin-like growth factor,multiplication-stimulating activity: Mediation by insulin and multiplication-stimulating activity receptors

Insulin stimulates a 2-fold increase in the amount of tyrosine aminotransferase and a 5–10-fold increase in the rate of amino acid transport in dexamethasone-treated rat hepatoma cells. In order to determine whether these effects are mediated by insulin receptors or receptors for insulin-like growth factors, we have examined the binding of ¹²⁵I-labeled insulin and ¹²⁵I-labeled multiplication-stimulating activity, a prototype insulin-like growth factor, and compared the biological effects of these polypeptides. Insulin and multiplication-stimulating activity cause an identical increase in transaminase activity and transport velocity; half-maximal biological effects were observed at 35 ng/ml (5.5 nM) insulin and 140 ng/ml multiplication-stimulating activity. The hepatoma cells display typical insulin receptors of appropriate specificity; half-maximal displacement of tracer insulin binding occured at 33 ng/ml unlabeled insulin, but only at 2500 ng/ml unlabeled multiplication-stimulating activity. Specific multiplication-stimulating activity receptors also were demonstrated with which insulin did not interact even at 10 μg/ml. Half-maximal displacement of tracer multiplication-stimulating activity occured at 200 ng/ml unlabeled multiplication-stimulating activity. We conclude that insulin cannot act via the multiplication-stimulating activity receptor and presumably acts via typical insulin receptors. The effects of multiplication-stimulating activity on enzyme induction and amino acid transport are probably mediated primarily via the multiplication-stimulating activity receptor.     

Inhibition of ascorbic acid transport in human neutrophils by glucose.

Because of the structural similarity between glucose and ascorbic acid, we investigated the effect of glucose on uptake and accumulation of ascorbic acid in isolated normal human neutrophils. Ascorbic acid accumulation was determined using high-performance liquid chromatography with coulometric electrochemical detection, in conjunction with liquid scintillation spectrometry. Ascorbic acid accumulation in neutrophils is mediated by a high and a low affinity transport activity. In neutrophils from different volunteers, glucose inhibited uptake and accumulation of ascorbic acid by both transport activities 3-9-fold. The mechanism of inhibition was different for each transport activity: inhibition of the high affinity transport activity was noncompetitive, while inhibition of the low affinity activity was competitive. Glucose-induced inhibition of both ascorbic acid transport activities occurred in neutrophils of all donors tested and was fully reversible. Although the mechanism of ascorbic acid accumulation appeared to be different than that for glucose transport, other monosaccharides and glucose transport inhibitors also inhibited ascorbic acid accumulation. These are the first data to suggest that ascorbic acid accumulation in neutrophils can be regulated by compounds of similar structure.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Divergent effects of rosiglitazone on protein-mediated fatty acid uptake in adipose and in muscle tissues of Zucker rats

Thiazolidinediones (TZDs) increase tissue insulin sensitivity in diabetes. Here, we hypothesize that, in adipose tissue, skeletal muscle, and heart, alterations in protein-mediated FA uptake are involved in the effect of TZDs. As a model, we used obese Zucker rats, orally treated for 16 days with 5 mg rosiglitazone (Rgz)/kg body mass/day. In adipose tissue from Rgz-treated rats, FA uptake capacity increased by 2.0-fold, coinciding with increased total contents of fatty acid translocase (FAT/CD36; 2.3-fold) and fatty acid transport protein 1 (1.7-fold) but not of plasmalemmal fatty acid binding protein, whereas only the plasmalemmal content of FAT/CD36 was changed (increase of 1.7-fold). The increase in FA uptake capacity of adipose tissue was associated with a decline in plasma FA and triacylglycerols (TAGs), suggesting that Rgz treatment enhanced plasma FA extraction by adipocytes. In obese hearts, Rgz treatment had no effect on the FA transport system, yet the total TAG content decreased, suggesting enhanced insulin sensitivity. Also, in skeletal muscle, the FA transport system was not changed. However, the TAG content remained unaltered in skeletal muscle, which coincided with increased cytoplasmic adipose-type FABP content, suggesting that increased extramyocellular TAGs mask the decline of intracellular TAG in muscle. In conclusion, our study implicates FAT/CD36 in the mechanism by which Rgz increases tissue insulin sensitivity.