Enhanced Glucose Transport Research Articles

'Insulin-like' effects of lithium ion on isolated rat adipocytes. I. Stimulation of glycogenesis beyond glucose transport.

Both insulin and lithium ion stimulated cytochalasin B-sensitive glucose transport in isolated rat adipocytes. As a result of enhanced glucose transport, the incorporation of [14C]glucose into CO2, glycogen and lipid were increased by both agents. However, the action of these two agents was distinguished. Cytochalasin B decreased insulin-stimulated glucose oxidation, glycogenesis and lipogenesis. In contrast, lithium-stimulated glycogenesis was decreased but lithium-stimulated glucose oxidation and lipogenesis were completely blocked. These results indicate that lithium ion in rat adipocytes has a specific insulin-like effect on glycogenesis without affecting glucose oxidation and lipogenesis.

The effect of graded doses of insulin on total glucose uptake, glucose oxidation, and glucose storage in man.

The dose-response relationship between plasma insulin concentration and total glucose uptake, glucose oxidation, and glucose storage was examined in 22 healthy young volunteers by employing the euglycemic insulin clamp technique in combination with indirect calorimetry. Insulin was infused at five rates to achieve steady-state hyperinsulinemic plateaus of 62 ± 4, 103 ± 5, 170 ± 10, 423 ± 16, and 1132 ± 47 μU/ml. With increasing plasma insulin concentrations within the physiologic range, there was a linear increase in glucose uptake with a half maximally effective insulin concentration of 72 μU/ml. Glucose uptake by all tissues of the body reached 80% of its maximum value (12.6 mg/kg · min) at a plasma insulin concentration of ∼200 μU/ml. In contrast to total glucose uptake, glucose oxidation plateaued more quickly, achieved a maximum rate of only 4.0 mg/kg · min, and displayed a lower half maximally effective insulin concentration of 40 μU/ml. The increase in glucose uptake with progressively increasing plasma insulin levels was primarily the result of an increase in glucose storage, with a half maximally effective insulin concentration of 105 μU/ml and maximum rate of 8.7 mg/kg · min. Glucose storage represented over 60–70% of total glucose uptake at all insulin concentrations. After achieving maximum rates of insulin-mediated glucose uptake (plasma insulin concentration = 1132 μU/ml), hyperglycemia (+125 mg/dl) was superimposed on hyperinsulinemia to further enhance glucose transport. Under these conditions, total glucose uptake (32.5 mg/kg · min, P < 0.001) was markedly augmented but no significant increase in glucose oxidation was observed. These results indicate a true saturation of the glucose oxidation pathway. With pro-gressively increasing doses of insulin, the glucose storage represents the major route of glucose disposal.

Intestinal maturation in the rat: the effect of glucocorticoids on sodium, potassium, water and glucose absorption.

The effect of glucocorticoids on the intestinal transport of water, electrolytes, and glucose in the infancy period is not known. Therefore, we studied the effect of parenteral glucocorticoids on intestinal transport of water, electrolytes and glucose in suckling rats with an in vivo single pass perfusion technique. Glucocorticoids enhanced significantly net sodium transport only in the colon segments compared to controls. Net sodium transport in the small intestinal segments was similar in glucocorticoid and control groups. Similar to adult animals, glucocorticoids enhanced glucose transport in the ileum, but not in jejunum segments. Net potassium absorption was decreased five-fold in jejunum segments and reversed to net secretion in the ileum and colon segments by glucocorticoids. These results are the first to extend the known effects of glucocorticoids into the infancy period.

Glucose transport in adipocytes and its control by growth hormone in vivo.

Previous results showed maximally enhanced basal glucose transport in adipocytes of hypophysectomized rats and restoration to normal after human growth hormone (hGH) administration. The data suggested a hGH-dependent "limiting factor" for glucose transport in the adipocyte membrane, which is acutely inhibited by insulin resulting in enhanced glucose transport. In this study the effect of hGH was investigated with respect to dose and time dependence. hGH was administered by continuous infusion from subcutaneously implanted Alzet minipumps. A significant decrease of basal glucose transport was obtained at the lowest hGH dose of 50 mU/day for 6 days. This effect of hGH was strictly correlated to the effects on growth (tibial epiphyseal width, DNA synthesis, body weight, serum level of insulin-like growth factor). The effect of hGH on basal glucose transport was already observed after 12 h of infusion, and it increased to a maximum after 3 days. The data support the concept that GH regulates the glucose transport system in adipose tissue in vivo.

State of metabolic control determines role of epinephrine-glucagon interaction in glucoregulation in diabetes.

Epinephrine (0.1 micrograms.kg-1.min-1) was infused with or without somatostatin (0.1 microgram.kg-1.min-1) in six depancreatized dogs, studied under normo- and hypoinsulinemia to determine whether the participation of glucagon in epinephrine-induced hepatic glucose overproduction is governed by the degree of metabolic control. When normoglycemia was achieved by basal intraportal insulin replacement, insulin levels remained constant during the epinephrine infusion, and there was a twofold increase in extrapancreatic immunoreactive glucagon (eIRG) and glucose production (Ra). Although eIRG increments were prevented by somatostatin, the increase in Ra was undiminished, indicating that epinephrine can act independently of glucagon as in normal animals. During subbasal intraportal insulin infusion in the depancreatized dogs, insulin levels remained 35% lower than with basal replacement, and the animals were hyperglycemic. Epinephrine induced a similar twofold increase in eIRG as during normoglycemia, and again this rise was prevented by somatostatin. There was a significantly greater, threefold increase in Ra with epinephrine when the animals were hyperglycemic. This exaggerated response to epinephrine was not seen during eIRG suppression by somatostatin, suggesting that glucagon participated in the epinephrine-induced hepatic glucose overproduction when the depancreatized dogs were in poor metabolic control, as seen previously in alloxan-diabetic dogs. However, in the depancreatized, unlike in the alloxan-diabetic dogs, epinephrine-induced glucagon release was small. Thus, hypoinsulinemia appears to sensitize the liver to eIRG during epinephrine infusion. The fact that epinephrine induces hyperglycemia both in physiology and diabetes could indicate an important role in enhancing glucose transport in insulin-insensitive tissues.

Transport of 6-deoxy-D-glucose and D-xylose by untransformed and SV40-transformed 3T3 cells.

Transport rates of the nonphosphorylated D-glucose analogs 6-deoxy-D-glucose and D-xylose were measured in quiescent and serum-stimulated cultures of mouse 3T3 cells, in SV40-transformed 3T3 cells (SV101), and in a density revertant cell line derived from SV101 (FI-SV101). Initial rates of both entry and exit of 6-deoxy-D-glucose and D-xylose were more than threefold higher in serum-stimulated 3T3 and in SV101 cells than they were in quiescent 3T3 cells, but transport rates were not higher in the transformed cells (SV101) than they were in serum-stimulated 3T3. Confluent cultures of FI-SV101 showed lower rates of transport than serum-stimulated FI-SV101, but not as low as quiescent 3T3 cells enter the quiescent G0 state, but emphasize that SV40-transformed 3T3 cells do not show higher activity of the D-glucose carrier than do actively growing 3T3 cells. Thus, enhanced glucose transport appears not to be a specific consequence of transformation, but a reflection of the active growth state of the cell.

In vivo control of insulin-sensitive phosphodiesterase in rat adipocytes by growth hormone and its parallelism to glucose transport.

As shown previously in adipocytes of hypophysectomized (hypox) rats, 3-O-methyl-glucose transport is already maximal in the basal state and insensitive to insulin. It is normalized by prolonged administration of GH to hypox rats. This study shows glucose transport in the presence and absence of phosphodiesterase (PDE) inhibitors in the fat cells of normal and hypox rats. Enhanced glucose transport in insulin-stimulated normal fat cells as well as enhanced glucose transport in adipocytes of hypox rats is inhibited by PDE inhibitors. The low Km phosphodiesterase activity, which is known to be acutely stimulated by insulin in normal adipocytes, is found to be increased in the fat cells of hypox rats, and further stimulation by insulin is not possible. Normalization occurs after GH administration for 4 days. Then, the low Km PDE activity is again low and stimulated in the presence of insulin. The similarity between the behavior of the activity of the glucose carrier system and that of the low Km PDE suggests that both may be dependent on a GH-induced membrane factor which would be acutely inhibited by insulin.

Effect of thiamin deficiency and acute ethanol ingestion on jejunal glucose transport in rats

Chronic alcoholism is often accompanied by malnutrition and thiamin deficiency. To determine the influence of thiamin deficiency on the absorption of an essential nutrient, the transport of '4C-D-glucose was measured using rat everted jejunal sacs and 3H-dextran as a marker of adherent mucosal volume in diet-induced thiamin-deficient (TD) rats and pair-fed controls (PFC). In addition, ethanol (2.5 g/kg) was given by gavage to these rats to assess the combined effect of thiamin deficiency and ethanol. At 1, 2, 3, 5, and 10 mM glucose, tissue uptake of glucose was higher (p <0.05) in TD than in PFC (n = 8 to 14) by 73, 35, 46, 28, 21, and 17%, respecrtively; but was unchanged at 15 and 20 mM glucose. Thus, TD lowered *i(m to 2.77 ± 0.43 from the PFC value of 5.00 ± 0.39 mM, while the maximal transport velocity (Jmax) was unchanged. TD similarly affected the transmural transfer of glucose to the serosal compartment. Treatment of TD rats with thiamin HCI 500 �g IP daily x 2 reversed these changes. Ethanol given to TD rats (n = 8) decreased the uptake of 1, 2, 3, 5 mM glucose by 34.9,44.19,44.46 and 34.15% (p <0.001) to rates even lower than those of untreated rats (p <0.001). Similarly ethanol reduced glucose uptake in PFC. In both TD and PFC rats ethanol decreased *Jmbut did not change *Km. These changes were achieved at TD and PFC plasma ethanol concentrations of 243 ±34 and 240±46 (SEM) mg/dl, respectively. These studies indicate that TD enhances glucose transport by lowering *Km. This finding extends our previous observation that in TD the *Km for thiamin uptake is reduced along with a decrease in the unstirred water layer thickness, suggesting that this change may be a general phenomenon. In contrast, ethanol decreases *Jmand obviates the enhancement of glucose transport in thiamin deficiency. These findings contribute to the understanding of malabsorption and malnutrition in alcoholism. Am. I Clin. Nutr. 34: 14-19, 1981.

The insulin-like effect of hydrogen peroxide on pathways of lipid synthesis in rat adipocytes.

In addition to the well known insulin-like effects of certain concentrations of H2O2 on glucose transport and oxidation in isolated rat adipocytes, the present work demonstrates that lipid synthesis from glucose is also enhanced over a narrow range of H2O2 concentrations (0.15 to 0.5 mM) added to the incubation medium. As in the case of insulin, H2O2 was found to stimulate greater glucose incorporation into glyceride-fatty acid than incorporation into glyceride-glycerol. As part of a multifaceted regulation of lipogenesis, H2O2, like insulin, increased the amount of pyruvate dehydrogenase in the active form without increasing the total amount of pyruvate dehydrogenase. Pyruvate dehydrogenase activity increased within 5 min of H2O2 incubation, reached a maximum at 15 min and declined thereafter as the H2O2 disappeared from the incubation medium. While medium glucose per se was found to activate the enzyme, it is unlikely that the effect of H2O2 was mediated by the known enhancement of glucose transport since the effects on the enzyme were maximal in the absence of glucose in the incubation medium. These findings add to the growing list of insulin effects that are reproduced by H2O2, and strengthen the hypothesis that assigns H2O2 the role of "second messenger" of insulin.

Insulin-like effects of polyamines in fat cells. Mediation by H2O2 formation.

Significant amounts of H2O2 were produced when the polyamines, spermine, or spermidine were incubated with a Krebs-Ringer phosphate buffer that contained bovine serum albumin (Fraction V). This effect was specific for certain amines and could be prevented by treatment of the albumin fraction with isoniazid or aminoguanidine. These features suggest that H2O2 is formed during oxidative deamination of the polyamines catalized by spermine oxidase, a known contaminant of Fraction V bovine serum albumin. The insulin-like effects elicited by polyamines in fat cells (e.g. enhancement of glucose transport and inhibition of cAMP-mediated lipolysis) were dependent on H2O2 production. Incubation of cells with catalase or treatment of the albumin fraction with isoniazid abolished the stimulation of glucose uptake by polyamines but did not alter the stimulatory effects of insulin or vitamin K5. The H2O2 generating activity was partially separated from the albumin by gel filtration; only those fractions which formed H2O2 provided support for the activation of glucose transport by polyamines. Also, the time needed to activate glucose uptake was markedly shortened by incubation of the albumin buffer with the polyamines before addition of the cells. These findings indicate that the polyamines do not themselves mimic the actions of insulin but that the insulin-like effects result from the formation of H2O2 which has been shown to stimulate glucose transport.

Insulin-stimulated plasma membranes from rat adipocytes: their physiological and physicochemical properties

Using methods previously described, we have isolated plasma membranes from insulin-treated adipocytes of the rat epididymal fat pad. Such membranes showed an accelerated uptake and release of d-glucose when compared with similar preparations from cells not exposed to the hormone. The only change observed was in the rate of d-glucose uptake; at equilibrium, d-glucose space was identical in both preparations. Vigorous alkaline hydrolysis of the insulin resulted in loss of its typical effect on both intact cells and derived plasma membranes. Anti-insulin antiserum, potent enough to inhibit the effect of the hormone when both were added to intact cells, did not prevent the insulin effect when added to plasma membranes prepared from insulin-treated cells. Addition of insulin directly to plasma membranes was without effect; exposure of cells prior to rupture was required. Studies of infrared spectra, native membrane protein fluorescence, and fluorescence of 8-anilino-1-naphthalene sulfonate added to the membranes, showed no differences between control plasma membranes and those prepared from insulin-treated cells. We conclude that: (1) plasma membranes can be prepared from insulin-treated fat cells which retain an enhanced glucose transport; (2) the effect of insulin on glucose transport does not involve large scale changes in the structure of the plasma membrane; (3) the insulin unresponsiveness of isolated plasma membranes, as well as the resistance to anti-insulin serum of membranes prepared from insulin-treated cells, appears to result from an uncoupling of insulin binding from glucose transport, the basis of which requires further exploration.

Direct action of insulin on plasma membrane ATPase activity in human lymphocytes.

LITTLE is known about the effects of insulin on lymphocytes. Helmreich and Eisen1 concluded that it has insignificant effects, but others2–5 have made a case for a role in inflammatory and immunological responses. We6,7 have demonstrated that noradrenaline enhances the uptake of both glucose and potassium by lymphocytes, as does insulin in several tissues. We have associated this action of noradrenaline with a direct effect on membrane adenosine triphosphatase (ATPase) activity7. The observation8 that insulin bound to ‘Sepharose’ polymers enhances glucose transport while in contact only with the plasma membrane indicated that insulin might have a direct action similar to that of noradrenaline on membrane ATPase. The observations reported here show that insulin stimulates ATPase activity and glucose uptake in the lymphocyte and suggest a relationship between membrane ATPase activity and glucose transport.

Membrane Sialic Acid and the Mechanism of Insulin Action in Adipose Tissue Cells: EFFECTS OF DIGESTION WITH NEURAMINIDASE

Mild digestion of isolated adipose tissue cells with Clostridium perfringens neuraminidase (10 to 20 mµg of purified enzyme per ml, 15 min at 37°) results in enhanced glucose transport by these cells. This effect appears to mimic closely the physiological effect of insulin on glucose transport by normal cells, and there is evidence to suggest that these two apparently different stimuli may affect similar membrane structures. Digestion of fat cells with higher concentrations of neuraminidase (about 1 µg of purified enzyme per ml, 15 min, 37°) causes a fall in the enhanced basal rate of glucose transport described above and abolishes the effects of insulin on glucose transport and on lipolysis. Such enzymatic digestion, however, has no effect on the total quantity of specific cell membrane receptor for insulin or in the affinity of the insulin-receptor complex. The biological and the receptor-binding properties of insulin are thus dissociated. The loss of insulin-stimulated glucose transport caused by neuraminidase digestion does not reverse spontaneously by prolonged incubation of the cells after digestion. Neuraminidases from C. perfringens, Vibrio cholerae and influenza virus were purified by affinity chromatography using selective agarose adsorbents containing an oxamic acid derivative. The three enzymes, at low concentration, can destroy the glucose transport effects of insulin on fat cells. However, the specific patterns of digestion observed with the three different enzymes are not identical, probably as a result of the differences in substrate specificity of the enzymes. Differently linked sialic acid residues at the cell surface may participate differently in various membrane functions. The effects of digesting fat cells with various combinations of C. perfringens neuraminidase and trypsin on glucose oxidation and the patterns of release of sialic acid and sialyl peptides suggest that the biological effects resulting from digesting cells with these enzymes are a consequence of cleavage of similar portions of the same or closely related membrane structures. Sialic acid at the cell surface is intimately involved in the processes concerned with transmitting the effects of insulin on membrane glucose transport and lipolysis, but it is not involved in the recognition of insulin by the receptor. Despite the unessential role of sialic acid in the function of insulinrecognition by the receptor, this moiety may nonetheless be a constituent of a glycoprotein insulin receptor.