Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.     

Improvement of insulin sensitivity by antagonism of the renin-angiotensin system

The reduced capacity of insulin to stimulate glucose transport into skeletal muscle, termed insulin resistance, is a primary defect leading to the development of prediabetes and overt type 2 diabetes. Although the etiology of this skeletal muscle insulin resistance is multifactorial, there is accumulating evidence that one contributor is overactivity of the renin-angiotensin system (RAS). Angiotensin II (ANG II) produced from this system can act on ANG II type 1 receptors both in the vascular endothelium and in myocytes, with an enhancement of the intracellular production of reactive oxygen species (ROS). Evidence from animal model and cultured skeletal muscle cell line studies indicates ANG II can induce insulin resistance. Chronic ANG II infusion into an insulin-sensitive rat produces a markedly insulin-resistant state that is associated with a negative impact of ROS on the skeletal muscle glucose transport system. ANG II treatment of L6 myocytes causes impaired insulin receptor substrate (IRS)-1-dependent insulin signaling that is accompanied by augmentation of NADPH oxidase-mediated ROS production. Further critical evidence has been obtained from the TG(mREN2)27 rat, a model of RAS overactivity and insulin resistance. The TG(mREN2)27 rat displays whole body and skeletal muscle insulin resistance that is associated with local oxidative stress and a significant reduction in the functionality of the insulin receptor (IR)/IRS-1-dependent insulin signaling. Treatment with a selective ANG II type 1 receptor antagonist leads to improvements in whole body insulin sensitivity, enhanced insulin-stimulated glucose transport in muscle, and reduced local oxidative stress. In addition, exercise training of TG(mREN2)27 rats enhances whole body and skeletal muscle insulin action. However, these metabolic improvements elicited by antagonism of ANG II action or exercise training are independent of upregulation of IR/IRS-1-dependent signaling. Collectively, these findings support targeting the RAS in the design of interventions to improve metabolic and cardiovascular function in conditions of insulin resistance associated with prediabetes and type 2 diabetes.     

Participation of protein kinases in the stimulant action of GLP-1 on 2-deoxy-D-glucose uptake by normal rat skeletal muscle.

Several protein kinases were recently proposed for involvement in GLP-1-stimulated D-glucose transport in skeletal muscle from both normal subjects and type 2 diabetic patients. This study was mainly aimed at investigating the effect of potential inhibitors of distinct protein kinases and protein phosphatase-1 upon insulin- and GLP-1-stimulated 2-deoxy-D-glucose net uptake by normal rat skeletal muscle. The basal uptake of the D-glucose analog was decreased by wortmannin--a phosphatidylinositol-3-kinase inhibitor--, PD98059--a mitogen-activated protein kinases inhibitor--, and TNFalpha--a protein phosphatase-1 inhibitor--, but not by either rapamycin--a p70s6 kinase inhibitor--, or H-7--, a protein kinase C inhibitor--. The enhancing action of both insulin and GLP-1 upon 2-deoxy-D-glucose transport was abolished by PD98059 and H-7, but largely unaffected by TNFalpha. Wortmannin and rapamycin preferentially affected the response to GLP-1 and insulin, respectively. These findings thus document both analogies and dissimilarities in the participation of the concerned enzymes in the stimulant action of insulin versus GLP-1 upon D-glucose transport in normal rat skeletal muscle.     

The D-glucose transporter is tissue-specific. Skeletal muscle and adipose tissue have a unique form of glucose transporter

Using isotopic equilibration with [3H]D-glucose and measurement of D-glucose inhibitable cytochalasin B binding, I show that the erythrocytes of embryonic and newborn rats contain D-glucose transporters. On the basis of cytochalasin B binding and the time course of isotopic exchange, the number of transporters in rat embryonic erythrocytes is only 5% of that in human erythrocytes. Antibodies raised against the human erythrocyte glucose transporter were used as a probe to investigate the structural similarity between transporters. On this basis, the polypeptides of the glucose transporter of human erythrocytes and of embryonic rat erythrocytes are similar but not identical; in addition, certain antibodies showed similar reactivity toward the transporter of rat embryonic erythrocytes and that of rat brain. These antibodies, however, react with brain transporters 5 to 10 times better than with those of skeletal muscle and adipocytes suggesting that insulin responsive tissues may have a different type of glucose transporter. The cellular location of glucose transporters in skeletal muscle, determined by immunofluorescence, is on the plasma membrane or very close to the plasma membrane.     

Facilitated Transport of Glucose from Blood into Peripheral Nerve

D-Glucose is the major substrate for energy metabolism in peripheral nerve. The mechanism of transfer of glucose across the blood-nerve barrier is unclarified. In this study an in situ perfusion technique was utilized, in anesthetized rats, to examine monosaccharide transport from blood into peripheral nerve. Unidirectional influxes of D-[14C]glucose, L-[14C]glucose, and [14C]3-O-methyl-D-glucose across capillaries of the tibial nerve were measured at different perfusate concentrations of unlabelled D-glucose. The permeability-surface area product (PA) for D-[14C]glucose and [14C]3-O-methyl-D-glucose decreased, whereas the PA for L-[14C]glucose remained constant, as the perfusate concentration of D-glucose was increased. In the presence of no added unlabelled D-glucose in the perfusate, the PA for L-[14C]glucose equaled one-fifth the PA for D-[14C]glucose. These results demonstrate self-saturation, competitive inhibition, and stereospecificity of glucose transfer, and for the first time show a unidirectional facilitated transport mechanism for D-monosaccharides at capillaries of mammalian peripheral nerve. The data were fit to a model for facilitated transport and passive diffusion. The half-saturation constant and maximal rate of transport for the saturable component of D-glucose influx equaled 23 +/- 11 mumol X ml-1 and 6.6 +/- 3.2 X 10(-3) mumol X s-1 X g-1, respectively. The constant of nonsaturable glucose influx equaled 0.5 +/- 0.1 X 10(-4) s-1. At normal plasma glucose concentrations, the saturable component comprises about 80% of total D-glucose influx into nerve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Distribution of glucose transporters and insulin receptors in the plasma membrane and transverse tubules of skeletal muscle

The distribution of glucose transporters and of insulin receptors on the surface membranes of skeletal muscle was studied, using isolated plasma membranes and transverse tubule preparations. (i) Plasma membranes from rabbit skeletal muscle were prepared according to Seiler and Fleischer (1982, J. Biol. Chem. 257, 13862-13871), and transverse tubules from rabbit skeletal muscle were prepared according to Rosemblatt et al. (1981, J. Biol. Chem. 256, 8140-8148) as modified by Hidalgo et al. (1983, J. Biol. Chem. 258, 13937-13945). The membranes were identified by the abundance of nitrendipine receptors in the transverse tubules, and their relative absence from the plasma membranes. (ii) Plasma membranes and transverse tubules were also isolated from rat skeletal muscle, according to a novel procedure that isolates both fractions from the same common homogenate. (iii) Glucose transporters were detected by D-glucose protectable binding of the specific inhibitor [3H]cytochalasin B, and insulin receptors were detected by saturable binding of 125I-insulin. The concentration of glucose transporters was about threefold (rabbit) or fivefold (rat) higher in the transverse tubule membrane compared to the plasma membrane, whereas the insulin receptor concentration was about the same in both membranes. These results indicate that the glucose transporters on the surface of the muscle are preferentially segregated to the transverse tubules, and this poses interesting consequences on the functional response of glucose transport to insulin in skeletal muscle.     

Knockdown of NYGGF4 increases glucose transport in C2C12 mice skeletal myocytes by activation IRS-1/PI3K/AKT insulin pathway

NYGGF4, an obesity-related gene, is proposed to be involved in the development of insulin resistance. Skeletal muscle is a primary target organ for insulin and NYGGF4 showed a relatively high expression level in skeletal muscle. Therefore, this study aimed to explore the effect of NYGGF4 on insulin sensitivity of skeletal muscle cells. RNA interference (RNAi) was adopted to silence NYGGF4 expression in mice C2C12 skeletal myocytes. A remarkably increased insulin-stimulated glucose uptake and GLUT4 translocation was observed in NYGGF4 silencing C2C12 cells. Importantly, the enhanced glucose uptake induced by NYGGF4 silencing could be abrogated by the PI3K inhibitor LY294002. In addition, the crucial molecules involved in PI3K insulin signaling pathway were detected by western blotting. The results showed that NYGGF4 knockdown dramatically activate the insulin-stimulated phosphorylation of IRS-1 and AKT. Taken together, these data demonstrate that NYGGF4 knockdown increases glucose transport in myocytes by activation of the IRS-1/PI3K/AKT insulin pathway.     

The influence of insulin on glucose and fatty acid metabolism in the isolated perfused rat hind quarter.

Glucose and fatty acid metabolism of resting skeletal muscle were studied by perfusion of the isolated rat hind leg with a hemoglobin-free medium. Tissue integrity was demonstrated by normal ATP, ADP and creatine phosphate levels, by a sufficient oxygen supply, and by a normal appearance of perfused muscle specimens under the electron microscope. The rates of glucose and fatty acid uptake, and of lactate, alanine, glycerol and fatty acid release were constant over a perfusion period of 60 min. Insulin (1 unit/l) caused a more than threefold increase in glucose uptake, a stimulation of lactate production, and a 20% increase in the muscular glycogen levels. Fatty acids and alanine release were significantly diminished by insulin, but glycerol release did not change. The uptake of oleate by the rat hind leg was dependent on the medium concentration in a range of 0.7-1.9mM oleate, and was stimulated by insulin. Glucose uptake was not influenced by oleate, whether sodium was present or not. When the leg was perfused with [1-14C]oleate, 75% of the incorporated fatty acids were found in muscle lipids, 10% were oxidized to CO2, and 5% were recovered in bone lipids. The absolute amount of oleate oxidation was not altered by insulin. In all experiments with and without glucose in the medium, 70-80% of the 14C label incorporated into muscle lipids was found in the triglyceride fraction. In the presence of glucose, insulin significantly increased the incorporation of [1-14C]oleate into muscle triglycerides, whereas no insulin effect, either on fatty acid uptake or on triglyceride formation, could be observed when glucose was omitted from the perfusate. The present results indicate that a "glucose-fatty acid cycle" as found in rat heart muscle does not operate in resting peripheral skeletal muscle tissue. They also demonstrate that the stimulating effect of insulin on muscular fatty acid uptake and triglyceride synthesis is dependent on glucose supply. This finding can be intrepreted as a stimulation of fatty acid esterification by sn-glycerol 3-phosphate derived from an increased glucose turnover, which is in turn due to insulin.     

Muscle atrophy by limb immobilization is not caused by insulin resistance

Hindlimbs of adult female rats were immobilized for 1 day. The hindquarter was then perfused either without or with 200 microunits of insulin/ml perfusate. The percentage increase in muscle protein synthesis rates by the inclusion of insulin in the perfusate was similar between control and hindlimb immobilized groups. Thus, the previously reported inability of insulin to stimulate any increase in glucose metabolism in skeletal muscle after 1 day of immobilization ( Seider , Nicholson and Booth 1982) does not also extend to an inability of insulin to stimulate an increase in protein synthesis in muscles of immobilized limbs.     

Substrate regulation of the glucose transport system in rat skeletal muscle. Characterization and kinetic analysis in isolated soleus muscle and skeletal muscle cells in culture

A self-regulatory mechanism of the glucose transport in rat skeletal muscle cells is described. In isolated rat soleus muscles and rat skeletal myocytes and myotubes in culture, pre-exposure to varying glucose concentrations modulated the rate of 2-deoxyglucose uptake. Maximal uptake was observed at glucose concentrations below 3 mM. Between 2.5 and 4.0 mM glucose it was reduced by 25-35%; further elevation of the glucose concentration resulted in a gradual decrease of the transport rate by approximately 2% for each millimolar glucose. The effect of glucose was time-dependent and fully reversible. Insulin rapidly increased the 2-deoxyglucose uptake in the soleus muscle; however, the insulin effect depended on the glucose concentration of the preincubation. Insulin was totally ineffective in muscles pre-exposed to 1.0-3.0 mM glucose, whereas its stimulatory action increased with increasing glucose concentrations above 4 mM. The effect of low glucose and insulin were not additive, and the maximal 2-deoxyglucose uptake rates induced by both conditions were of identical magnitude. It is postulated that glucose may "up- and down-regulate" its transport by affecting the number of active glucose transporters in the plasma membrane, and that insulin exerts its stimulatory effect only when the extracellular glucose reaches a threshold concentration.     

Comparison of insulin and insulin-like growth factor I receptors from rat skeletal muscle and L-6 myocytes

Insulin and IGF-I receptors were solubilized from fused L-6 myocytes, a rat skeletal muscle derived cell line, and compared to rat skeletal muscle receptors. In skeletal muscle, 125I-insulin binding was competed by insulin greater than IGF-I greater than MSA, whereas in L-6 cells IGF-I greater than insulin greater than MSA. 125I-IGF-I binding was competed by IGF-I greater than insulin = MSA in both tissues. On electrophoresis, differences in Mr were observed between skeletal muscle and L-6 derived receptors both in the alpha- and beta-subunits. Six antibodies directed against the human insulin receptor beta-subunit recognized the rat skeletal muscle insulin receptor, while only two reacted strongly with L-6 derived receptors. Skeletal muscle has receptors with relative specificity for insulin and IGF-I respectively; L-6 cells also have two classes of receptors, one is kinetically similar to the IGF-I receptor from skeletal muscle; the other, which binds insulin with relatively high affinity has even greater affinity for IGF-I. This unusual receptor may represent a developmental stage in muscle or the transformed nature of L-6 cells.     

Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.     

The use of a cell-free perfusate in the perfused rat hindquarter: methodological concerns

The viability of using a cell-free perfusate in a rat hindlimb preparation to assess skeletal muscle glycogenesis was investigated. A perfusate containing 10 mM glucose and 10 microCi (1 Ci = 37 GBq) of D-[5-3H]glucose was recycled for a 60-min period. In agreement with other studies using more complex media, oxygen uptake of the preparation indicated adequate tissue oxygenation (8 mumol.min-1.g-1). Skeletal muscle fiber type heterogeneity in basal glycogen synthesis from glucose was shown (slow oxidative greater than fast oxidative glycolytic greater than fast glycolytic fibres). Insulin (4.2 mU/mL) markedly stimulated glycogenesis from D-[5-3H]glucose in the soleus (slow oxidative fiber), red gastrocnemius (fast oxidative glycolytic fiber), and white gastrocnemius muscles (p less than 0.05). A recent report indicates that tissue edema in this preparation did not affect insulin responsiveness of the tissue. In contrast, our observations indicate that glucos uptake was enhanced by insulin when edema was absent (p less than 0.05), but not when edema was present (p less than 0.05). In addition, the presence of tissue edema negated insulin-mediated glycogenesis in slow oxidative and fast oxidative glycolytic muscle (p less than 0.05 compared with control) but not in fast glycolytic muscle (p less than 0.05). These data warrant caution when using a cell-free media in the perfused rat hindquarter; however, in the absence of edema, normal responses of glucose metabolism are observed.     

Glucose-induced insulin resistance of skeletal-muscle glucose transport and uptake.

The ability of glucose and insulin to modify insulin-stimulated glucose transport and uptake was investigated in perfused skeletal muscle. Here we report that perfusion of isolated rat hindlimbs for 5 h with 12 mM-glucose and 20,000 microunits of insulin/ml leads to marked, rapidly developing, impairment of insulin action on muscle glucose transport and uptake. Thus maximal insulin-stimulated glucose uptake at 12 mM-glucose decreased from 34.8 +/- 1.9 to 11.5 +/- 1.1 mumol/h per g (mean +/- S.E.M., n = 10) during 5 h perfusion. This decrease in glucose uptake was accompanied by a similar change in muscle glucose transport as measured by uptake of 3-O-[14C]-methylglucose. Simultaneously, muscle glycogen stores increased to 2-3.5 times initial values, depending on fibre type. Perfusion for 5 h in the presence of glucose but in the absence of insulin decreased subsequent insulin action on glucose uptake by 80% of the effect of glucose with insulin, but without an increase in muscle glycogen concentration. Perfusion for 5 h with insulin but without glucose, and with subsequent addition of glucose back to the perfusate, revealed glucose uptake and transport similar to initial values obtained in the presence of glucose and insulin. The data indicate that exposure to a moderately increased glucose concentration (12 mM) leads to rapidly developing resistance of skeletal-muscle glucose transport and uptake to maximal insulin stimulation. The effect of glucose is enhanced by simultaneous insulin exposure, whereas exposure for 5 h to insulin itself does not cause measurable resistance to maximal insulin stimulation.     

Sarcolemmal glucose transport in Ca2+-tolerant myocytes from adult rat heart. Calcium dependence of insulin action

Cardiac myocytes were isolated from adult rat ventricles by a method which preserves their functional integrity, including long survival in physiological concentrations of Ca2+. Sarcolemmal glucose transport was assessed by measuring linear initial uptake rates of the nonmetabolized glucose analog 3-O-methyl-D-glucose. Transport was saturable and showed competition by D-glucose and other features of chemical and stereo-selectivity. Transport was stimulated by insulin in a dose-dependent manner, resulting in an almost 5-fold increase in Vmax, with little change in Km. Stimulation of 3-methylglucose transport by insulin was largely Ca2+-dependent. Omission of Ca2+ from the incubation medium caused a minor rise in basal 3-methylglucose uptake but the insulin-stimulated rise in Vmax was only 30%. The Ca2+ antagonist D600 also antagonized stimulation of hexose transport by insulin. In all the above respects, 3-methylglucose transport in myocytes is identical to that in intact heart muscle. In addition, the decrease in insulin response by Ca2+ omission was partially reversed by subsequent return to a Ca2+-containing medium. ATP levels remained stable in the absence of Ca2+, showing that the Ca2+ dependence did not reflect nonspecific cell damage.     

Vanadate stimulates D-glucose transport into sarcolemmal vesicles from rat skeletal muscles.

Vanadate is known to have various insulin-like actions including activation of D-glucose uptake into the skeletal muscle and adipose tissue. In this study, we examined the effect of orthovanadate on D-glucose uptake into sarcolemmal vesicles prepared from rat hind limb skeletal muscles. In the presence of 10 mM vanadate, the initial rate of D-glucose uptake into sarcolemmal vesicles was enhanced 4-5 times above the basal value. Half-maximal concentration for this effect of vanadate was 3 mM. The D-glucose uptake was also stimulated by metavanadate, but not by selenite, selenate, or molybdate. When vanadate was removed from the vesicles by dilution and centrifugation, D-glucose uptake into the vesicles returned to the basal level, indicating that the effect of vanadate was reversible. Saturation curves showed that the Vmax value for the D-glucose uptake was enhanced more than 4-fold by 10 mM vanadate. Therefore, the activation of D-glucose uptake was due, at least in part, to a large increase in the Vmax value. These results suggest that vanadate increases the intrinsic activity (turnover number) of skeletal muscle glucose transporters in a reversible manner.     

Glucose metabolism in perfused skeletal muscle. Interaction of insulin and exercise on glucose uptake.

1. The interaction of insulin and isometric exercise on glucose uptake by skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin, 10 m-i.u./ml, added to the perfusate, increased glucose uptake more than 10-fold, from 0.3-0.5 to 5.2-5.4 mumol/min per 30g of muscle in hindquarters of fed and 48h-starved rats respectively. In contrast, it did not stimulate glucose uptake in hindquarters from rats in diabetic ketoacidosis. 3. In the absence of added insulin, isometric exercise, induced by sciatic-nerve stimulation, increased glucose uptake to 4 and 3.4 mumol/min per 30g of muscle in fed and starved rats respectively. It had a similar effect in rats with moderately severe diabetes, but it did not increase glucose uptake in rats with diabetic ketoacidosis or in hindquarters of fed rats that had been "washed out" with an insulin-free perfusate. Insulin, at concentrations which did not stimulate glucose uptake in resting muscle, restored the stimulatory effect of exercise in these situations. 4. The stimulation of glucose uptake by exercise was independent of blood flow and the degree of tissue hypoxia; also it could not be reproduced by perfusing resting muscle with a medium previously used in an exercise experiment. 5. At rest glucose was not detectable in muscle cell water of fed and starved rats even when perfused with insulin. In the presence of insulin, a small accumulation of glucose, 0.25 mM, was noted in the muscle of ketoacidotic diabetic rats, suggesting inhibition of glucose phosphorylation, as well as of transport. 6. During exercise, the calculated intracellular concentration of glucose in the contracting muscle increased to 1.1-1.6mM in the fed, starved and moderately diabetic groups. Insulin significantly increased the already high rates of glucose uptake by the hindquarters of these animals but it did not alter the elevated intracellular concentration of glucose. 7. In severely diabetic rats, exercise did not cause glucose to accumulate in the cell in the absence of insulin. In the presence of insulin, it increased glucose uptake to 6.1 mumol/min per 30g of muscle and intracellular glucose to 0.72 mM. 8. The data indicate that the stimulatory effect of exercise on glucose uptake requires the presence of insulin. They suggest that in the absence of insulin, glucose uptake is not enhanced by exercise owing to inhibition of glucose transport into the cell.     

Polarity of transport of 2-deoxy-D-glucose and D-glucose by cultured renal epithelia (LLC-PK1).

At least two types of glucose transporter exist in cultured renal epithelial cells, a Na(+)-glucose cotransporter (SGLT), capable of interacting with D-glucose but not 2-deoxy-D-glucose (2dglc) and a facilitated transporter (GLUT) capable of interacting with both D-glucose and 2dglc. In order to examine the polarity of transport in cultured renal epithelia, 2dglc and D-glucose uptakes were measured in confluent cultures of LLC-PK1 cells grown on collagen-coated filters that permitted access of medium to both sides of the monolayer. The rates of basolateral uptake of both 1 mM glucose (Km 3.6 mM) and 1 mM 2dglc (Km 1.5 mM) were greater than apical uptake rates and the (apical-to-basolateral)/(basolateral-to-apical) flux ratio was high for glucose (9.4) and low for 2dglc (0.8), thus, confirming the lack of interaction of 2dglc with the apical SGLT. Specific glucose transport inhibitor studies using phlorizin, phloretin and cytochalasin B confirmed the polarised distribution of SGLT and GLUT in LLC-PK1 cells. Basolateral sugar uptake could be altered by addition of insulin (1 mU/ml) which increased 2dglc uptake by 72% and glucose uptake by 50% and by addition of 20 mM glucose to the medium during cell culture which decreased 2dglc uptake capacity at confluence by 30%. During growth to confluence, 2dglc uptake increased to a maximum, then decreased at the time of confluence, coincident with a rise in uptake capacity for alpha-methyl-D-glucoside, a hexose that interacts only with the apical SGLT. It was concluded that the non-metabolisable sugar 2dglc was a useful, specific probe for GLUT in LLC-PK1 cells and that GLUT was localised at the basolateral membrane after confluence.