Transport and metabolism of D-glucose in human adipocytes. Studies of the dependence on medium glucose and insulin concentrations

Uptake and metabolism of the physiologically labelled D-glucose (D-[U-14C]glucose) has been characterized in human adipocytes at several unlabelled D-glucose concentrations in the absence and presence of insulin. Following a 90 min incubation, about 80% of the intracellular radioactivity was incorporated into total lipids at tracer glucose concentration, as well as at higher glucose concentrations in basal and insulin-stimulated cells, whereas 20% was recovered as hydrophilic metabolites. The only 14C-labelled metabolite escaping the cells in detectable amounts was CO2, which accounted about 4%. At trace glucose concentrations (5 mumol/l), the rate of glucose uptake was linear with time. Comparative studies of initial glucose uptake after 10 s and tracer D-glucose conversion to total lipids after 90 min showed high coefficients of correlation between basal rates (r = 0.87), maximal response above basal level to insulin (r = 0.92) and insulin sensitivity (r = 0.78). Thus, under these conditions glucose transport is rate-limiting for net glucose uptake, and measurements over long time intervals of rates for total cell-associated radioactivity or lipogenesis may serve as reliable estimates of initial glucose influx rates. However, the conversion rate of tracer glucose to metabolites decreased progressively with the glucose concentration and with an apparent Km of about 0.2 mmol/l. The three metabolic pathways exhibited similar percentage decreases in their activities, suggesting that a common enzymatic step is rate-limiting. In comparison, the Km for initial D-glucose uptake rate was about 7 mmol/l. Hence, the capacity for total glucose metabolism comprised only a small fraction of the glucose transport capacity at medium glucose concentrations above tracer concentrations. Both basal, half-maximal and maximal insulin-stimulated rates of adipocyte glucose utilization were dependent on the glucose concentration. Thus, comparing lipogenesis at tracer and at 0.5 mmol/l medium glucose concentration, it was shown that the higher medium glucose concentration was associated with a 60% lowering of the basal rate, a 35% reduction in the percentage response above baseline to maximal insulin stimulation and a 4-fold increase in the insulin sensitivity. Obviously, these findings reflect some intracellular step(s) being rate-limiting at medium glucose levels above tracer values.     

Stimulation of glucose uptake in skeletal muscle using bolus or continuous insulin delivery

We investigated glucose uptake in the non-cyclically perfused rat hindlimb in response to continuous infusion (CI) or bolus injection (BI) of insulin. Ten mM glucose was infused at 3 ml/min, venous glucose was monitored at two minute intervals, and glucose uptake was calculated on the basis of arteriovenous-difference and expressed as micron/min/100 g body wt. Insulin BI given every ten minutes equaled the amount of insulin given by CI for ten minutes. Insulin doses of 1500, 3000, 6000, and 45,000 microU/30 min showed no significant difference between the two modes of delivery in either onset of stimulation or maximal stimulation of glucose uptake. At the lowest insulin dose tested (1500 microU/30 min) neither BI nor CI stimulated glucose uptake above the control of 1.849 micron/min/100 g. A dose response curve for glucose uptake was obtained using insulin boluses ranging from 2000 to 20,000 microU. Insulin uptake by the muscle was always greater when insulin was administered CI. Net disappearance of immunoreactive insulin over the entire 30 minutes of perfusion was 29.4 +/- 2.6% for CI but only 7.1 +/- 1.6% for BI. Thus in the perfused rat hindlimb, stimulation of glucose uptake in skeletal muscle is comparable with BI and CI delivery of insulin but insulin uptake by the muscle is several-fold greater with CI delivery.     

Transport enhancement and reversal: glucose and 3-O-methyl glucose.

In chick embryo fibroblast cultures the 15- to 30-fold enhancement of D-glucose uptake observed when cells are starved of glucose for 24 hours is not duplicated for derivatives of glucose that compete effectively for uptake and have generally been considered to use the same carrier. 2-deoxy-D-glucose, D-mannose, D-galactose and D-glucosamine are derepressed progressively less sharply in that order with glucosamine uptake never more than doubled by starvation. D-glucose at a concentration of 5.5 mM in the 24-hour conditioning medium is a strong "repressor" resulting in low "transport" behavior for each of the five sugars cited. D-glucosamine is equally effective at the same concentration. A 10-fold reduction in the concentration of glucosamine (0.55 mM) allows for the escape from repression of mannose, glucose, and deoxyglucose uptake while the others remain repressed. Mannose uptake escapes as well when the glucose concentration in the "conditioning" medium is similarly reduced. Under certain conditions of starvation and cell density dramatic effects of supplemental stimulation by insulin can be achieved. Insulin withdrawal interrupts the supplemental stimulation process. Cycloheximide, actinomycin D and cordycepin block both non-insulin and insulin-induced derepression. Short exposure (15-30 minutes) of 24-hour starved cells to glucose (5.5 mM) reduces glucose sharply but does not affect 3-O-methyl glucose uptake. If the exposure is to 2-deoxyglucose (5.5 mM) further derepression of glucose uptake results.     

Insulin stimulation of glucose uptake fails to decrease palmitate oxidation in muscle if AMPK is activated.

Fatty acid oxidation in muscle has been reported to be diminished when insulin and glucose levels are elevated. This study was designed to determine whether activation of AMP-activated protein kinase (AMPK) will prevent inhibitory effects of insulin and glucose on the rate of fatty acid oxidation. Rat hindlimbs were perfused with medium containing 0, 0.3, or 60 nM insulin with or without 2 mM 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR). Glucose uptake was stimulated four- to fivefold by inclusion of insulin in the medium. Insulin attenuated the increase in AMPK caused by AICAR both in perfused hindlimbs and in isolated epitrochlearis muscles. The activation constant for citrate activation of acetyl-CoA carboxylase (ACC) was significantly increased in response to AICAR, and the increase was slightly attenuated if insulin was present in the perfusion medium. Insulin stimulated an increase in malonyl-CoA content of the muscles in the absence of AICAR. Malonyl-CoA was decreased to approximately the same value in AICAR-perfused muscle, regardless of insulin concentration. Muscle glucose 6-phosphate and citrate were significantly increased in response to AICAR and insulin. The rate of palmitate oxidation tended to decrease in response to insulin and in the absence of AICAR. AICAR increased palmitate oxidation to approximately the same level regardless of the insulin concentration or the rate of glucose uptake into the muscle. The rate of palmitate oxidation showed a curvilinear relationship as a function of muscle malonyl-CoA content, with half-maximal inhibition at approximately 0.6 nmol/g. We conclude that AMPK activation can prevent high rates of glucose uptake and glycolytic flux from inhibiting palmitate oxidation in predominantly fast-twitch muscle under these conditions.     

Interval between insulin injection and eating in relation to blood glucose control in adult diabetics.

In a survey of 225 diabetics treated with insulin 24 (10.6%) claimed never to have received advice concerning the interval between insulin injection and eating. Of the remainder, 67 (33%) admitted disregarding advice and using shorter intervals. There was a significant (p less than 0.01) difference between the reported frequencies of clinical hypoglycaemia in patients using different intervals. The effects on glucose control of intervals between insulin injection and breakfast of zero, 15, 30, and 45 minutes were studied for periods of one week in 11 patients with type I diabetes who were receiving twice daily injections of monocomponent porcine insulins and high fibre, high carbohydrate diets, using standard home blood glucose monitoring techniques to measure blood glucose concentrations each morning. The delay of 45 minutes resulted in the lowest frequency of hypoglycaemia and the most acceptable pattern of glucose concentrations measured one and two hours after breakfast and before lunch. Combining results obtained at these three times, the mean increment in blood glucose concentration was smaller after allowing a delay of 45 minutes than after delays of zero (p less than 0.001), 15 (p less than 0.03), and 30 (NS) minutes. A delay of 30 minutes resulted in smaller mean increments in blood glucose concentration than did delays of zero (p less than 0.001) and 15 (NS) minutes. These results suggest that this aspect of diabetic management may be neglected, with important consequences for blood glucose control. An increase in delay between insulin injection and eating to 45 minutes would be a simple and safe way of improving blood glucose control in at least the 37% of the diabetic population surveyed in this study who currently allow less than 15 minutes.     

The effect of insulin on the uptake and metabolic fate of glucose in isolated perfused hearts of dyslipemic rats

Male Wistar rats chronically (15 weeks) fed a sucrose-rich diet (SRD; 63% w/w) developed hypertriglyceridemia and impaired glucose homeostasis. Hearts from these animals were isolated and perfused using the Langendorff recirculating method. Glucose at levels similar to those found in the animal in vivo was used as the only exogenous substrate. The hearts were perfused for 30 minutes in the presence or absence of insulin (30 mU/mL) in the perfusion medium. In the absence of the hormone, glucose uptake was impaired and the glucose utilization was reduced, with a significant increase of lactate release. Glucose oxidation, which was estimated from the activation state of the enzyme pyruvate dehydrogenase complex (PDHc), was depressed mainly due to both an increase of PDH kinase and a decrease of PDHa (active form of PDHc) activities. Although the addition of insulin in the perfusion medium improved the above parameters, it was unable to normalize them. The present results suggest that at least two different mechanisms might contribute to insulin resistance and to the impaired glucose metabolism in the perfused hearts of the dyslipemic SRD-fed animals: (1) reduced basal and insulin-stimulated glucose uptake and its utilization or (2) increased availability and oxidation of lipids (low PDHa and high PDH kinase activities), which in turn decrease glucose uptake and utilization. Thus, this nutritional experimental model may be useful to study how impaired glucose homeostasis, increases plasma free fatty acid levels and hypertriglyceridemia could contribute to heart tissue malfunction.     

Alanine Uptake and Release by Sympathetic Ganglia of Chicken Embryos

Uptake and release of alanine were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14-15 days old, and incubated in a modified Eagle's minimum essential medium. In the presence of [U-14C]glucose, glucose carbon accumulated in alanine in the medium at a rate that increased when unlabeled alanine was added and sometimes exceeded the rate of appearance in lactate. When combined with uptake data, the increase in appearance of labeled alanine in the medium could be accounted for quantitatively by interference with its reuptake, without assuming a change in the unidirectional output of labeled alanine, provided allowance was made for the measured properties of exchange between the extracellular space and the surrounding medium. According to this model, the constant unidirectional outflux of labeled alanine was about 50 mumol/g dry weight/h. When [U-14C]alanine was added to medium containing unlabeled glucose, the alanine was consumed at a rate that increased as the concentration of alanine in the medium was elevated. The uptake rate was found to fit a modified Michaelis-Menten equation with a Umax of about 120 mumol/g dry weight/h, a Km of 0.5-1.0 mM, and a Kd of 0.75 ml/g dry weight/h. By chemical measurement of changes in alanine concentration in the medium during incubation, the uptake rate was shown to equal the output rate when about 0.2 mM alanine was present. Much of the alanine consumed in the presence of glucose was metabolized to CO2, raising the total CO2 output above the rate obtained with glucose alone. When alanine was present at a concentration of 10-20 mM, it contributed almost as much carbon to CO2 as did the glucose. A higher percentage of the carbon from alanine was incorporated into tissue constituents than was carbon from either glucose or lactate. It is concluded that alanine can be significant both as a product and as a substrate, but that its role as substrate would not be great at typical concentrations of alanine in blood.     

Acute bioenergetic insulin sensitivity of skeletal muscle cells: ATP-demand-provoked glycolysis contributes to stimulation of ATP supply

Skeletal muscle takes up glucose in an insulin-sensitive manner and is thus important for the maintenance of blood glucose homeostasis. Insulin resistance during development of type 2 diabetes is associated with decreased ATP synthesis, but the causality of this association is controversial. In this paper, we report real-time oxygen uptake and medium acidification data that we use to quantify acute insulin effects on intracellular ATP supply and ATP demand in rat and human skeletal muscle cells. We demonstrate that insulin increases overall cellular ATP supply by stimulating the rate of glycolytic ATP synthesis. Stimulation is immediate and achieved directly by increased glycolytic capacity, and indirectly by elevated ATP demand from protein synthesis. Raised glycolytic capacity does not result from augmented glucose uptake. Notably, insulin-sensitive glucose uptake is increased synergistically by nitrite. While nitrite has a similar stimulatory effect on glycolytic ATP supply as insulin, it does not amplify insulin stimulation. These data highlight the multifarious nature of acute bioenergetic insulin sensitivity of skeletal muscle cells, and are thus important for the interpretation of changes in energy metabolism that are seen in insulin-resistant muscle.     

Time-dependent Mechanisms in Beta-cell Glucose Sensing

The relation between plasma glucose and insulin release from pancreatic beta-cells is not stationary in the sense that a given glucose concentration leads to a specific rate of insulin secretion. A number of time-dependent mechanisms appear to exist that modify insulin release both on a short and a longer time scale. Typically, two phases are described. The first phase, lasting up to 10 min, is a pulse of insulin release in response to fast changes in glucose concentration. The second phase is a more steady increase of insulin release over minutes to hours, if the elevated glucose concentration is sustained. The paper describes the glucose sensing mechanism via the complex dynamics of the key enzyme glucokinase, which controls the first step in glucose metabolism: phosphorylation of glucose to glucose-6-phosphate. Three time-dependent phenomena (mechanisms) are described. The fastest, corresponding to the first phase, is a delayed negative feedback regulating the glucokinase activity. Due to the delay, a rapid glucose increase will cause a burst of activity in the glucose sensing system, before the glucokinase is down-regulated. The second mechanism corresponds to the translocation of glucokinase from an inactive to an active form. As the translocation is controlled by the product(s) of the glucokinase reaction rather than by the substrate glucose, this mechanism gives a positive, but saturable, feedback. Finally, the release of the insulin granules is assumed to be enhanced by previous glucose exposure, giving a so-called glucose memory to the beta-cells. The effect depends on the insulin release of the cells, and this mechanism constitutes a second positive, saturable feedback system. Taken together, the three phenomena describe most of the glucose sensing behaviour of the beta-cells. The results indicate that the insulin release is not a precise function of the plasma glucose concentration. It rather looks as if the beta-cells just increase the insulin production, until the plasma glucose has returned to normal. This type of integral control has the advantage that the precise glucose sensitivity of the beta-cells is not important for normal glucose homeostasis.     

Influence of the carnitine palmitoyltransferase inhibitor POCA on myocardial performance and metabolism of insulin resistant rats

The specific carnitine palmitoyltransferase I (CPT I)-inhibitor POCA - sodium-2(5-(4-chlorphenyl)pentyl-oxirane carboxylate - was used in isolated perfused hearts of acutely diabetic, ketotic (AD, 100 mg streptozotocin/kg body weight), chronically diabetic (CD, 60 mg streptozotocin/kg body weight), and obese ZUCKER rats (fa/fa) to study different forms of insulin resistance. In hearts of AD rats an absolute insulin resistance was observed which could be attenuated by perfusion of the hearts with POCA (10 microM). The insulin sensitivity could be fully restored and was not any longer significantly different from control hearts. In hearts of CD rats, which show a relative insulin resistance, POCA only slightly stimulated glucose oxidation and uptake, but the total rate of uptake and conversion of glucose as well as the responsiveness of these hearts to insulin remained low. In hearts of obese ZUCKER rats, the rate of glucose oxidation was accelerated to control levels by perfusion with POCA, however, the rate of glycolysis and glucose uptake remained reduced as compared to controls. Thus, POCA shifted the glucose metabolism by stimulating oxidation without normalizing the reduced glucose uptake. It follows that in hearts of AD rats the insulin resistance is due to the accelerated lipid metabolism described and is, therefore, fully reversible if the oxidation of fatty acids is inhibited. In hearts of ZUCKER rats a form of insulin resistance mediated by lipid metabolism seems to be responsible for the reduced glucose oxidation and the lowered rate of glycolysis. The insulin resistance can be eliminated and has to be distinguished from a defect in the glucose uptake system not affected by POCA. In hearts of CD rats insulin resistance is not dependent on disturbances in lipid metabolism and is practically not influenced by POCA. Thus, a CPI I-inhibitor might be useful to differentiate various forms of insulin resistance and therapeutically beneficial in forms mediated by lipid metabolic defects.     

Coupling of insulin receptors to glucose transport: a temperature-dependent time lag in activation of glucose transport.

We have studied the ability of occupied insulin receptors to activate (or couple to) the glucose transport system in isolated rat adipocytes. Maximal insulin action is seen when only a small proportion (<10%) of the receptors is occupied, and this fraction can be rapidly filled (<5 s) at an insulin concentration of 100 ng/ml. Additionally, control studies show that when the extracellular glucose concentration is tripled, the rate of transport triples within 10 s, indicating that changes in transport activity can be observed nearly instantaneously. Therefore, when cells are exposed to a high insulin concentration (100 ng/ml), any delay in the onset of insulin action beyond this time must be due to the time required for coupling of occupied insulin receptors to the glucose transport system. At 24 °C there is a lag of at least 200 s after insulin addition before a significant stimulation of 2-deoxyglucose transport is seen. The length of this lag phase is temperature dependent, decreasing to 45 s at 37 °C. An Arrhenius plot of the coupling lag is linear, with an activation energy of 25 kcal/mol. After the delay in the onset of initial transport activation the full response appears in a gradual manner, requiring 20 min at 24 °C to attain maximal stimulation. The time required for the full insulin response to appear is also temperature dependent, decreasing to 5 min at 37 °C. Similar results were obtained for the kinetics of insulin activation of 3-O-methyl glucose transport. Thus, the coupling of insulin receptors to the glucose transport system can be divided into two components: an initial absolute time lag followed by a gradual incremental process before the maximal, or full, effect of insulin is achieved. In conclusion, (1) there is an absolute delay in the onset of the insulin's initial action on glucose transport, (2) after an initial delay, activation of transport proceeds in a gradual manner, and (3) the coupling process between insulin receptors and the glucose transport system is temperature dependent and can be described by a linear Arrhenius plot. This suggests that the rate of activation is not limited by membrane fluidity.     

Sensitivity of glucose uptake and lipolysis of white adipocytes of the rat to insulin and effects of some metabolites.

1. Insulin increased glucose uptake and inhibited lipolysis in white adipocytes of the rat over the same concentration range of the hormone: the half-maximal effects were observed at approx. 10 microunits of insulin/ml. Thus, contrary to previous reports, no difference in sensitivity of the two processes to insulin could be found, which suggests that both these effects of insulin are important in increasing the rate of glucose utilization after a meal. 2. Adenosine deaminase, which lowers the concentration of adenosine in the incubation medium, decreased the sensitivity of both processes (lipolysis and glucose uptake) to insulin: this suggests that adenosine increases the sensitivity of both processes. Similarly, lactate and 3-hydroxybutyrate increased the sensitivity of both processes (to the same extent) to insulin. It is suggested that this increased sensitivity will improve the response (of adipose tissue) to insulin on refeeding after a prolonged period of starvation (when the hydroxybutyrate concentration is high), and after a short burst of exercise, when the blood lactate concentration is high and when large amounts of glucose are produced from lactate via gluconeogenesis in the liver.     

Palmitate- and lipopolysaccharide-activated macrophages evoke contrasting insulin responses in muscle cells

Factors secreted by macrophages contribute to whole body insulin resistance, acting in part on adipose tissue. Muscle is the major tissue for glucose disposal, but how macrophage-derived factors impact skeletal muscle glucose uptake is unknown, or whether the macrophage environment influences this response. We hypothesized that conditioned medium from macrophages pretreated with palmitate or LPS would directly affect insulin action and glucose uptake in muscle cells. L6-GLUT4myc myoblasts were exposed to conditioned medium from RAW 264.7 macrophages pretreated with palmitate or LPS. Conditioned medium from palmitate-treated RAW 264.7 macrophages inhibited myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation while activating JNK p38 MAPK, decreasing IkappaBalpha, and elevating inflammation markers. Surprisingly, and opposite to its effects on adipose cells, conditioned medium from LPS-treated macrophages stimulated myoblast insulin-stimulated glucose uptake, GLUT4 translocation, and Akt phosphorylation without affecting stress kinases or inflammation indexes. This medium had markedly elevated IL-10 levels, and IL-10, alone, potentiated insulin action in myoblasts and partly reversed the insulin resistance imparted by medium from palmitate-treated macrophages. IL-10 neutralizing antibodies blunted the positive influence of LPS macrophage-conditioned medium. We conclude that myoblasts and adipocytes respond differently to cytokines. Furthermore, depending on their environment, macrophages negatively or positively influence muscle cells. Macrophages exposed to palmitate produce a mixture of proinflammatory cytokines that reduce insulin action in muscle cells; conversely, LPS-activated macrophages increase insulin action, likely via IL-10. Macrophages may be an integral element in glucose homeostasis in vivo, relaying effects of circulating factors to skeletal muscle.     

Measurement of flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol and glycerol in rat heart and epididymal adipose tissue: Effects of insulin, adrenaline and alloxan-diabetes

1. Flow of carbon atoms from glucose and glycogen glucose to glyceride glycerol, glyceride fatty acids and glycerol was calculated in the perfused rat heart and incubated epididymal adipose tissue from the incorporation of (14)C from [U-(14)C]-glucose (into glyceride glycerol, glyceride fatty acids and glycerol in the medium), and from measurements of the specific activity of l-glycerol 3-phosphate, and the effects of insulin, adrenaline and alloxan-diabetes were studied. Measurements were also made of the uptake of glucose and the outputs of lactate, pyruvate and glycerol. 2. New methods are described for the measurement of radioactivity in small amounts of metabolites (glycerol, glucose 6-phosphate and fructose 6-phosphate and l-glycerol 3-phosphate) in which use has been made of alterations in charge induced by enzymic conversions to effect resolution by ion-exchange chromatography. 3. In hearts the specific activity of l-glycerol 3-phosphate was less than that of glucose in the medium but similar to that of lactate released during perfusion. Because repeated measurements of the specific activity of l-glycerol 3-phosphate was impracticable, the specific activity of lactate has been used as an indirect measurement of glycerol phosphate specific activity. 4. In fat pads, specific activity of lactate was the same as that of glucose in the medium and thus the specific activity of l-glycerol 3-phosphate was taken to be the same as that of medium glucose. 5. In hearts from alloxan-diabetic rats, despite decreased glucose uptake and l-glycerol 3-phosphate concentration, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased about threefold. 6. In fat pads, flow of carbon atoms through l-glycerol 3-phosphate to glyceride glycerol was increased by insulin (twofold), by adrenaline in the presence of insulin (fivefold) and by diabetes in pads incubated with insulin (1.5-fold). These increases could not be correlated either with increases in glucose uptake, which was unchanged by adrenaline and decreased in diabetes, or with the concentration of l-glycerol 3-phosphate, which was decreased by adrenaline and unchanged in diabetes. 7. These results are discussed in relation to the control of glyceride synthesis in heart and adipose tissue and to the regulation of glyceride fatty acid oxidation in the perfused rat heart.     

Plasma glucose and insulin responses to oral and intravenous glucose in cold-exposed humans

Although glucose tolerance and skeletal muscle glucose uptake are markedly improved by cold exposure in animals, little is known about such responses in humans. This study used two variations of a glucose tolerance test (GTT) to investigate changes in carbohydrate metabolism in healthy males during nude exposure to cold. In experiment 1, an oral GTT was performed in the cold and in the warm (3 h at 10 or 29 degrees C). To bypass the gastrointestinal tract, and to suppress hepatic glucose output, a second experiment was carried out as described above, using an intravenous GTT. Even though cold exposure raised metabolic rate greater than 2.5 times, plasma glucose and insulin responses to an oral GTT remained unaltered. In contrast, cold exposure reduced the entire plasma glucose profile as a function of time during the intravenous GTT (P less than 0.05), as plasma glucose was returned to basal levels within 1 h in comparison to a full 2 h in the warm, despite low insulin levels. The results of the intravenous GTT demonstrate that even with low insulin levels, carbohydrate metabolism is increased in cold-exposed males. This effect could be masked in the oral GTT by gastrointestinal factors and a high hepatic glucose output. Cold exposure may enhance insulin sensitivity and/or responsiveness for glucose uptake, mainly in shivering skeletal muscles.     

In vitro glucose and 2-aminoisobutyric acid uptake by rat interscapular brown adipose tissue

The dependence upon substrate and insulin concentrations, as well as on sodium and potassium concentrations in the medium of the uptake of glucose and 2-aminoisobutyric acid, was determined for fragments of brown and white adipose tissues incubated in vitro. Brown adipose tissue showed a high capacity for glucose uptake at high glucose concentrations, this uptake being dependent on both glucose and insulin concentration. White adipose tissue showed much more limited uptake capabilities. The presence of Na+ and K+ had little effect on the uptake. The uptake of 2-aminoisobutyric acid was similar in both adipose tissues, being enhanced by physiological levels of insulin and depressed by ouabain. This amino acid transport was dependent on Na+ and K+ concentrations, and the overall transporting capability was two to three orders of magnitude lower than that for glucose. It was concluded that amino acids could not play a significant role as bulk thermogenic substrates for brown adipose tissue, as their transporters lack the plasticity of response to high substrate and insulin concentrations which characterize brown adipose tissue uptake of glucose.     

Fluid-phase endocytosis by isolated rat adipocytes

We have developed an assay, which uses radiolabeled sucrose as the marker, to measure the rate of fluid-phase endocytosis in isolated rat adipocytes. In addition, the assay was adapted to allow measurement of the release of sucrose from previously loaded cells (fluid-phase exocytosis). Adipocytes take up sucrose at an approximately linear rate for at least 1.5 hours. A portion of the pinocytosed sucrose is rapidly (half-time about 20 minutes) returned to the medium. The minimal value for fluid uptake by endocytosis is 57 nl/10(6) cells-h at 37 degrees C; this value corresponds to the formation of 110,000 endocytic vesicles of 100-nm diameter per cell per hour and the internalization of about 20% of the plasma membrane per hour. Insulin caused a small and variable increase in the rate of sucrose uptake. The average increase of 31% from 11 experiments is statistically significant at the level of P less than 0.01. A small insulin effect upon the uptake of the calcium complex of [14C]EDTA was also observed. Since this complex was taken up at 2.5 times the rate of sucrose, it probably entered by a combination of fluid-phase and adsorptive pinocytosis. Insulin did not elicit a significant change in the rate of sucrose release from preloaded cells.     

Regulation of glucose transporters by insulin and extracellular glucose in C2C12 myotubes

It is well established that insulin stimulation of glucose uptake in skeletal muscle cells is mediated through translocation of GLUT4 from intracellular storage sites to the cell surface. However, the established skeletal muscle cell lines, with the exception of L6 myocytes, reportedly show minimal insulin-dependent glucose uptake and GLUT4 translocation. Using C(2)C(12) myocytes expressing exofacial-Myc-GLUT4-enhanced cyan fluorescent protein, we herein show that differentiated C(2)C(12) myotubes are equipped with basic GLUT4 translocation machinery that can be activated by insulin stimulation ( approximately 3-fold increase as assessed by anti-Myc antibody uptake and immunostaining assay). However, this insulin stimulation of GLUT4 translocation was difficult to demonstrate with a conventional 2-deoxyglucose uptake assay because of markedly elevated basal glucose uptake via other glucose transporter(s). Intriguingly, the basal glucose transport activity in C(2)C(12) myotubes appeared to be acutely suppressed within 5 min by preincubation with a pathophysiologically high level of extracellular glucose (25 mM). In contrast, this activity was augmented by acute glucose deprivation via an unidentified mechanism that is independent of GLUT4 translocation but is dependent on phosphatidylinositol 3-kinase activity. Taken together, these findings indicate that regulation of the facilitative glucose transport system in differentiated C(2)C(12) myotubes can be achieved through surprisingly acute glucose-dependent modulation of the activity of glucose transporter(s), which apparently contributes to obscuring the insulin augmentation of glucose uptake elicited by GLUT4 translocation. We herein also describe several methods of monitoring insulin-dependent glucose uptake in C(2)C(12) myotubes and propose this cell line to be a useful model for analyzing GLUT4 translocation in skeletal muscle.     

The effect of insulin on glucose uptake in soleus muscle during hemorrhagic shock.

Hemorrhagic shock was produced by bleeding conscious rats to a mean arterial pressure of 40 mm Hg, which was maintained for 2 h. Basal glucose uptake by isolated soleus muscle from normal rats and rats subjected to hemorrhagic shock ('shock' muscles) increased with the increase ib medium glucose concentration. Uptake values were similar in both groups of muscles. This indicates that there were no alterations in the basal glucose carrier mechanism during shock. Whereas insulin (0.1 U/ml) stimulated glucose uptake in control muscles under aerobic as well as under anaerobic conditions, it had no stimulatory effect in 'shock' muscles under either environment. Maximal stimulation of glucose uptake in 'shock' muscles was observed at an insulin concentration of 0.2 U/ml. The ability of muscle to bind insulin was not altered during shock. The present experiments indicate that insulin responsiveness to tissues is altered in shock. This could be due to alterations in the insulin sensitivity of the glucose carrier mechanism during shock.     

Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-L1 adipocytes

Fluid phase endocytosis by monolayers of 3T3-L1 adipocytes has been followed by measuring [14C]sucrose uptake, a well characterized pinocytic marker. Insulin, at a maximal stimulatory concentration, increased the pinocytic rate by 2-fold within 5 min of its addition; this activation persisted for at least 2 h. The dose-response curve for the enhancement of fluid-phase endocytosis by insulin was identical with that for the stimulation of hexose transport, as measured by the uptake of 2-deoxyglucose. The concentration of insulin eliciting half-maximal effects was 6 nM. These results suggest that activation of endocytosis and hexose uptake by insulin are triggered by the same signalling event. Insulin-activated pinocytosis was not dependent upon the increased metabolism of D-glucose that occurs in response to the hormone, since the stimulation of fluid-phase endocytosis occurred in the absence of 5 nM glucose. Fluid-phase exocytosis was examined by loading cells with [14C]sucrose for various times and then measuring tracer efflux. The rate of sucrose release was biphasic; a portion of the internalized sucrose was rapidly released from the cell (t1/2 approximately 5 min), whereas the remainder was released slowly (t1/2 approximately to 5 h). These results are consistent with a sequential two-compartment model in which the [14C] sucrose first enters a compartment from which about 70% of the sucrose is rapidly released back into the medium and the remaining 30% is transferred to a second compartment. Therefore, the true rate of endocytosis is much greater than the observed accumulation rates, except after short uptake times. Insulin increases the rate of sucrose efflux from both compartments as well as the rate of transfer from the first compartment to the second compartment by about 2-fold. Furthermore, insulin increased the apparent size of the first and second compartments by 1.6- and 3-fold, respectively. The lysosomotropic agent chloroquine (200 muM) had only a small effect on fluid movements in these cells. The rapid and prolonged stimulation of fluid-phase endocytosis and exocytosis by insulin are hitherto unrecognized effects of this hormone.