Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Glucose utilization rates are linked to the internal free glucose gradient in the rat tapeworm

Hymenolepis diminuta is able to acquire plasma-borne glucose 3-O-[14C]methylglucose in vivo. Free glucose concentrations estimated for this helminth in vivo are comparable to that of the host intestine. Both in vivo and in vitro examinations indicate that the scolex-neck regions (first quartile) of this tapeworm have the highest glucose content, and an anterior-posterior gradient along the second, third, and fourth quartiles was observed. Substrate concentration was rate affecting for glucose utilization rates (measured as substrate depletion from the medium in vitro). Glucose utilization per minute exceeds glucose content by a factor of more than 5. The half-life of glucose was about 10 sec, emphasizing that sugar metabolism is a very rapid process. In addition, utilization was highest in the first quartile and decreased in succession in the second, third, and fourth quartiles. It is concluded that while the exogenous glucose concentration remains stable, regional differences in glucose utilization rates are linked (R = 0.98; P less than 0.01) to free glucose content in H. diminuta.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Characterization of the glucose transporter from rat brain synaptosomes

Our goal was to characterize the glucose transporter in synaptosomes and to compare it to the different forms of transporter already identified. Cross-reactivity with antibodies to the human erythrocyte transporter, Km of glucose uptake, reversibility of NEM inhibition of transport, and insulin sensitivity were all examined. Immunoblotting showed a band at Mr 40,000, and the Km of glucose uptake was determined to be about 4 mM. Treatment with NEM caused irreversible inhibition of glucose uptake, while incubation with insulin failed to stimulate uptake. The results suggest that the transporter in synaptosomes resembles the human erythrocyte transporter.     

The role of glucose in the lipid metabolism of the rat tapeworm Hymenolepis diminuta

The composition of the neutral lipids and the phospholipids, and the role of glucose in the lipid metabolism of prepatent (12-day-old) Hymenolepis diminuta has been studied in vitro. Triglyceride was the most abundant lipid present; substantial amounts of sterol and sterol ester, diglyceride, free fatty acids and monoglycerides were also present. The phospholipids, which were qualitatively and quantitatively similar to those of other invertebrates and vertebrates, were, in order of abundance, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphoinositide, lysophosphatidylcholine, cardiclipin, phosphatidic acid, lysophosphatidic acid and phosphatidylglycerol. Small amounts of glucose carbon were incorporated into the lipids, principally the water soluble (glycerol) moiety of the triglycerides; only traces were incorporated into the phospholipids. Small amounts of glucose were converted to inositol and galactose. The principal pathway of triglyceride synthesis is suggested to be via the α-glycerophosphate-phosphatidic acid-diglyceride pathway.     

Stimulation of glucose transport and glucose transporter phosphorylation by okadaic acid in rat adipocytes

Okadaic acid, an inhibitor of Type I and IIa protein phosphatases, was recently found to stimulate 2-deoxyglucose uptake in rat adipocytes (Haystead, T. A. J., Sim, A. T. R., Carling, D., Honnor, R. C., Tsukitani, Y., Cohen, P., and Hardie, D. G. (1989) Nature 337, 78-81). In the present experiments the effect of okadaic acid on the phosphorylation and subcellular distribution of the insulin-regulatable glucose transporter (IRGT) was investigated. At maximally effective concentrations, insulin and okadaic acid increased the amount of IRGT in the plasma membrane by 10- and 4-fold, respectively. Thus, the stimulation of glucose transport by okadaic acid was apparently due to an increase in the surface concentration of the IRGT. However, despite its stimulatory actions, okadaic acid partially inhibited the ability of insulin to enhance glucose transport and translocation of the transporter. When cells were incubated with okadaic acid alone or in combination with insulin, phosphorylation of the IRGT in the plasma membrane was increased by approximately 3-fold relative to the intracellular pool of transporters in control cells. Phosphorylation of the IRGT was confined to the presumed cytoplasmic domain at the COOH terminus of the protein. Glucose transporters were dephosphorylated in vitro by Type I or Type IIa protein phosphatases, indicating that inhibition of one or both of these phosphatases could account for the increased phosphorylation produced by okadaic acid. The observation that okadaic acid stimulated translocation of the IRGT implicated a serine/threonine phosphorylation event in triggering movement of the intracellular IRGT-containing vesicles (GTV) to the cell surface. Immunoadsorption of GTV from 32P-labeled adipocytes revealed that the IRGT was the major phosphoprotein in these vesicles. The phosphorylation of at least three other GTV proteins was increased by okadaic acid, and these species would appear to be candidates for regulators of GTV movement to the plasma membrane. It is unlikely that phosphorylation of the IRGT is the signal for translocation because insulin did not increase phosphorylation of the protein. Rather, the inhibitory effect of okadaic acid on insulin-stimulated translocation is consistent with the hypothesis that phosphorylation of the IRGT promotes its internalization.     

Determination of in vivo kinetics of the starvation-induced Hxt5 glucose transporter of Saccharomyces cerevisiae

We have investigated the role and the kinetic properties of the Hxt5 glucose transporter of Saccharomyces cerevisiae. The HXT5 gene was not expressed during growth of the yeast cells in rich medium with glucose or raffinose. However, it became strongly induced during nitrogen or carbon starvation. We have constructed yeast strains constitutively expressing only Hxt5, Hxt1 (low affinity) or Hxt7 (high affinity), but no other glucose transporters. Aerobic fed-batch cultures at quasi steady-state conditions, and aerobic and anaerobic chemostat cultures at steady-state conditions of these strains were used for estimation of the kinetic properties of the individual transporters under in vivo conditions, by investigating the dynamic responses of the strains to changes in extracellular glucose concentration. The K(m) value and the growth properties of the HXT5 single expression strain indicate that Hxt5 is a transporter with intermediate affinity.     

Rapid kinetics of the glucose transporter from human erythrocytes. Detection and measurement of a half-turnover of the purified transporter

The stopped flow method combined with fluorescence detection has been employed to study the rapid kinetics of the glucose transporter from human erythrocytes. Upon mixing the purified transporter reconstituted into unsealed membranes of erythrocyte lipids with 4,6-ethylidene D-glucose, a derivative that binds preferentially to the substrate site on the outer domain of the transporter, there was a rapid, first-order decrease in the intrinsic fluorescence of the protein. Three properties of this transient indicate that it represents a half-turnover of the transporter from a conformation with the substrate site facing inward to one with this site facing outward. The first-order rate constant decreased as the concentration of ethylidene glucose was increased; the value of the rate constant for the process is similar to that expected from steady-state kinetic studies of transport in the erythrocyte; and D-glucose at low concentration increased the rate of reaction. This study is the first determination of the kinetics of a half-turnover for a transport system of the facilitated diffusion type. The identification of this step provides direct evidence for the alternating conformation mechanism of transport.     

Effects of medium-chain triglyceride feeding or glucose infusion on glucose kinetics in the newborn rat

Hypoglycaemia which develops in starved newborn rats (0.15 +/- 0.01 mg/ml) is reversed by feeding medium-chain triglycerides (0.66 +/- 0.05 mg/ml). Despite similar glycaemia (0.71 +/- 0.07 mg/ml) starved newborns infused with glucose (10.7 mg/min/kg) show a 30% higher glucose turnover rate than medium-chain triglyceride fed animals (14.1 +/- 0.6 versus 10.6 +/- 0.3 mg/min/kg, p less than 0.01). For a comparable [6-3H]glucose turnover rate (10.5 +/- 0.3 mg/min/kg), glucose-infused (5.25 mg/min/kg) newborns have a 30% lower glycaemia (0.50 +/- 0.03 mg/ml, p less than 0.01) than medium-chain triglyceride-fed newborns. Thus, medium chain triglyceride feeding leads to a 30% decreased capacity of the tissues to utilize glucose. For a similar glucose turnover rate, medium-chain triglyceride-fed newborns have a higher blood lactate concentration than glucose-infused newborns (0.26 +/- 0.03 versus 0.15 +/- 0.02 mg/ml). However, in medium-chain triglyceride-fed newborns, the increase of blood lactate is not only due to the Cori cycle, as glucose recycling is less increased than glucose production. Thus medium-chain triglyceride increases the release of gluconeogenic precursors which are not derived from blood glucose. In presence of a glucose infusion (15.25 mg/min/kg) producing hyperglycaemia (1.35 +/- 0.05 mg/ml), endogenous glucose production is suppressed by only 37%. If 3-mercaptopicolinate, an inhibitor or gluconeogenesis, is given concomitantly, hyperglycaemia is prevented (0.72 +/- 0.08 mg/ml) and endogenous glucose production is suppressed. Glucose infusion in the hypoglycaemic newborn rat might thus lead to a precarious glucose homeostasis.     

Immunohistochemical localization of Na+-dependent glucose transporter in rat jejunum

Summary Glucose is actively absorbed via a Na⁺-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564–575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.     

Expression of facilitative glucose transporter in rat liver and choroid plexus

Summary Two isoforms of facilitative glucose transporters (GLUT), namely the erythroid/brain-type GLUT 1 and the liver-type GLUT 2, were demonstrated in native cryostat sections of normal rat liver and brain by immunofluorescence and a very sensitive immunoalkaline phosphatase reaction. Fixation with 0.1% alcoholic periodic acid resulted in an excellent localization of GLUT 2 in liver and GLUT 1 in brain. GLUT 1 in liver, however, could successfully be demonstrated after fixation with 1% alcoholic formaldehyde. GLUT 2 occurred in all hepatocytes as a basolateral membrane protein with a gradient of high expression in the periportal area and a lower one in the perivenous part. The first layer of hepatocytes adjacent to the hepatic vein coexpressed GLUT 1. In addition, GLUT 1 could be detected in the smooth muscle layer of the portal vein and in the apical and lateral plasma membrane of the bile duct epithelium. In brain, GLUT 1 showed a high expression in the microvessels, the ependym and in the basal plasma membrane of choroid plexus epithelial cells. The blood capillaries associated with the choroidal epithelium were, however, negative for GLUT 1. The importance of the new findings in this study for the physiological role of the respective facilitative glucose transport proteins is discussed.     

Increase in liver glucose transporter mRNA levels during rat liver regeneration

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.     

Hymenolepis diminuta: glucose and glycogen gradients in the adult tapeworm

Adult (20-day-old) Hymenolepis diminuta were cut into 12 pieces of equal length, and the individual pieces of the tapeworm's strobila were analyzed. There was a continuous gradient of decreasing concentrations of glucose (mM) and decreasing levels of glycogen (microgram/mg wt) in the strobila. The ethanol extracts of the individual pieces of strobila contained four compounds tentatively identified as disaccharides; the distributions of these compounds were different from those of glucose and glycogen. The distributions of glucose, glycogen, and the "disaccharides" changed when tapeworms were incubated for 1 h in saline or glucose. When compared, on a per-weight basis, to the most posterior sections of the strobila, the anterior sections absorbed more glucose and incorporated more glucose into glycogen. There was a continuous gradient of decreasing values along the tapeworm's strobila of the Vmax for glucose uptake, while the Kt values for glucose uptake changed only slightly. The data indicate that glucose and glycogen metabolism are most active in the anterior part of the tapeworm's strobila where new proglottids are produced and the initial stages of organogenesis occur.     

Colchicine binding in the rat tapeworm,Hymenolepis diminuta

A fraction with enriched colchicine-binding properties prepared from Hymenolepis diminuta was found to possess many of the properties of tubulin isolated from other sources. Colchicine was bound by a simple saturable process with a dissociation constant of 13.2 μM. The binding capability decayed with a half-life of about 5 h. Binding was unaffected by lumicolchicine, was competitively inhibited by podophyllotoxin (inhibition constant of 4.8 μM) and showed an apparent stimulation by vinblastine sulphate. Sodium chloride also appeared to stimulate the binding process. The ligand/receptor complex had a molecular weight of approx. 112 000 as determined by gel filtration.On the basis of this biochemical and pharmacological evidence it was concluded that the colchicine receptor in the supernatant fraction of the H. diminuta homogenate was almost certainly tubulin. Refinement of the preparation should facilitate further studies on the mode of action of certain types of anthelmintic compound.     

Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat

Antibodies specific for the insulin-regulatable glucose transporter (GLUT 4) were used to immunolocalize this protein in brown adipose tissue from basal- and insulin-treated rats. Cryosections of fixed tissue were incubated with antibodies, which were subsequently labeled with Protein A/gold and examined by EM. Antibodies against albumin and cathepsin D were also used with gold particles of different sizes to identify early and late endosomes, respectively. Under basal conditions 99% of the GLUT 4 labeling was located within the cell. Labeling was predominantly in the trans-Golgi reticulum and tubulo-vesicular structures elsewhere in the cytoplasm. In insulin-stimulated cells approximately 40% of the GLUT 4 labeling was at the cell surface, where it was randomly distributed, except for occasional clustering in coated pits. Moreover, after insulin treatment, GLUT 4 was also enriched in early endosomes. We conclude that translocation of GLUT 4 to the cell surface is the major mechanism by which insulin increases glucose transport. In addition, these results suggest that in the presence of insulin GLUT 4 recycles from the cell surface, probably via the coated pit-endosome pathway that has been characterized for cell surface receptors, and also that insulin causes the redistribution of GLUT 4 by stimulating exocytosis from GLUT 4-containing tubulo-vesicular structures, rather than by slowing endocytosis of GLUT 4.     

Effects of chronic coffee consumption on glucose kinetics in the conscious rat

Epidemiological studies indicate that regular coffee consumption reduces the risk of developing type 2 diabetes. Despite these findings, the biological mechanisms by which coffee consumption exerts these effects are unknown. The aim of this study was twofold: to develop a rat model that would further delineate the effects of regular coffee consumption on glucose kinetics, and to determine whether coffee, with or without caffeine, alters the actions of insulin on glucose kinetics in vivo. Male Sprague-Dawley rats were fed a high-fat diet for 4 weeks in combination with one of the following: (i) drinking water as placebo (PL), (ii) decaffeinated coffee (2 g/100 mL) (DC), or (iii) alkaloid caffeine (20 mg/100 mL) added to decaffeinated coffee (2 g/100 mL) (CAF). Catheters were chronically implanted in a carotid artery and jugular vein for sampling and infusions, respectively. Recovered animals (5 days postoperative) were fasted for 5 h before hyperinsulinemic-euglycemic clamps (2 mU x kg(-1) x min(-1)). Glucose was clamped at 6 mmol/L and isotopes (2-deoxy-[(14)C]glucose and [3-(3)H]glucose) were administered to obtain indices of whole-body and tissue-specific glucose kinetics. Glucose infusion rates and measures of whole-body metabolic clearance were greater in DC than in PL or CAF, indicating increased whole-body insulin sensitivity. As the only difference between DC and CAF was the addition of alkaloid caffeine, it can be concluded that caffeine antagonizes the beneficial effects of DC. Given these findings, decaffeinated coffee may represent a nutritional means of combating insulin resistance.