Effects of Soy-derived diets on plasma and liver lipids, glucose tolerance, and longevity in normal, long-lived and short-lived mice.

We examined the effects of diets based on a low isoflavone or a high isoflavone soy protein isolates in normal, growth-hormone receptor knockout and Ames dwarf, and Prop 1 (df) mice that are hypoinsulinemic, insulin-sensitive, and exceptionally long-lived, as well as in growth hormone transgenic mice that are hyperinsulinemic, insulin-resistant, dyslipidemic, and short-lived. Soybean diets tended to normalize plasma cholesterol levels in dwarf and transgenic mice, while low isoflavone diet reduced plasma triglycerides in most of the examined genotypes. The effects of low isoflavone and high isoflavone diets on the levels of free and esterified cholesterol in the liver were strongly genotype-dependent. Fasting blood glucose levels were reduced and glucose tolerance improved by both low isoflavone and high isoflavone diets in growth hormone-transgenic mice and in their normal siblings. Glucose tolerance was also improved by high-isoflavone diet in growth hormone receptor knockout mice. Lifespan was increased by low isoflavone diet in normal mice from two of the examined stocks. High isoflavone diet increased lifespan in normal animals from one line, but reduced lifespan of normal mice from a different line. We conclude that dietary soy protein intake can improve plasma and hepatic lipid profiles, reduce fasting glucose, enhance capacity for glucose tolerance, and prolong life, but all of these effects are strongly genotype-dependent.

A cooperative interaction between soy protein and its isoflavone-enriched fraction lowers hepatic lipids in male obese Zucker rats and reduces blood platelet sensitivity in male Sprague-Dawley rats.

Soy protein diets lower plasma cholesterol in hyperlipoproteinemic human subjects, as well as in animal models. We fed 7-wk-old male obese (fa/fa) and lean Zucker rats a modified AIN-76 diet (20 g protein/kg diet) containing casein (C), low isoflavone soy protein (38 mg isoflavones/kg diet; LI), or high isoflavone soy protein (578 mg isoflavones/kg diet; HI) for 70 d. In obese rats, plasma total cholesterol was 21 and 29% lower in the LI and HI groups, respectively, than in the C group (P:

Polyphosphoinositide inclusion in artificial lipid bilayer vesicles promotes divalent cation-dependent membrane fusion.

Recent studies suggest that phosphoinositide kinases may participate in intracellular trafficking or exocytotic events. Because both of these events ultimately require fusion of biological membranes, the susceptibility of membranes containing polyphosphoinositides (PPIs) to divalent cation-induced fusion was investigated. Results of these investigations indicated that artificial liposomes containing PPI or phosphatidic acid required lower Ca2+ concentrations for induction of membrane fusion than similar vesicles containing phosphatidylserine, phosphatidylinositol, or phosphatidylcholine. This trend was first observed in liposomes composed solely of one type of phospholipid. In addition, however, liposomes designed to mimic the phospholipid composition of the endofacial leaflet of plasma membranes (i.e., liposomes composed of combinations of PPI, phosphatidylethanolamine, and phosphatidylcholine) also required lower Ca2+ concentrations for induction of aggregation and fusion. Liposomes containing PPI and phosphatidic acid also had increased sensitivity to Mg(2+)-induced fusion, an observation that is particularly intriguing given the intracellular concentration of Mg2+ ions. Moreover, the fusogenic effects of Ca2+ and Mg2+ were additive in vesicles containing phosphatidylinositol bisphosphate. These data suggest that enzymatic modification of the PPI content of intracellular membranes could be an important mechanism of fusion regulation.

Glucose transport activity and ligand binding (cytochalasin B, IAPS-forskolin) of chimeric constructs of GLUT2 and GLUT4 expressed in COS-7-cells.

Chimeric constructs of glucose transporters GLUT2 and GLUT4 were transiently expressed in COS-7 cells in order to determine regions of the proteins responsible for their differences in activity and ligand binding. Exchange of the C-terminal tail (aa 479-509) of GLUT4 failed to affect glucose transport activity assayed at 1 mM glucose or ligand binding (cytochalasin B, IAPS-forskolin). In contrast, exchange of the C-terminal half of GLUT4 (aa 222-509) for that of GLUT2 markedly reduced ligand binding (Kd of cytochalasin B binding 1.88 +/- 0.2 microM vs. 0.21 +/- 0.06 in the wild-type GLUT4), and moderately (25%) reduced glucose transport activity. These data support the conclusion that the domains determining differences in ligand binding between GLUT4 and GLUT2 are located in the C-terminal half of the glucose transporters.

Mutation of two conserved arginine residues in the glucose transporter GLUT4 supresses transport activity, but not glucose-inhibitable binding of inhibitory ligands.

Two arginine residues (RR333/334) in the conserved GRR motif located in the endofacial loop between helix 8 and 9 of the glucose transporter GLUT4 were substituted for leucine and alanine, respectively. Reconstituted glucose transport activity of the construct (GLUT4-RR333/4LA) expressed in COS-7 or LM(TK-) cells was less than 10% of that of the wild-type GLUT4. In contrast, binding of the inhibitory ligand cytochalasin B and glucose-inhibitable photolabeling with IAPS-forskolin were not significantly affected. Exchange of a histidine residue (H337Q) previously believed to be involved in the binding of inhibitory ligands failed to affect any of the investigated parameters. These data suggest that positive charges in the GRR motif at the cytoplasmic surface of the transporter participate in the conformational changes of the carrier protein during the process of facilitated diffusion.

Forskolin specifically inhibits the bacterial galactose-H+ transport protein, GalP.

Forskolin is a potent inhibitor of mammalian passive glucose transporters. Here we show that forskolin is a remarkably specific inhibitor of energized D-galactose transport by the GalP sugar-H+ symport protein of Escherichia coli. Surprisingly, it does not inhibit transport of L-arabinose or D-xylose by the related E. coli AraE and XylE transporters, even though the amino acid sequences of their proteins are 30-64% identical to GalP and to the mammalian GLUT family. However, unlike GLUT1, photoactivation of the [3H]forskolin-GalP complex fails to incorporate radioactivity covalently into the protein, in contrast to the effective incorporation of radioactivity from [3H]cytochalasin B into both proteins. However, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldesacetylforskol in ([125I]APS-forskolin), which labels GLUT1, is a potent labeling reagent for GalP and, to a lesser extent, for AraE. The appropriate sugar substrates of each transporter protect it against the [125I]APS-forskolin. Equilibrium binding studies using membranes from an E. coli strain that overexpresses GalP reveal a single set of high affinity binding sites for [3H]forskolin with a Kd of 1.3-1.4 microM, probably forming a 1:1 complex, compared with a value of 7.5 microM for GLUT1. Sugar substrates of GalP and cytochalasin B displace forskolin from the protein. The nonhomologous sugar-H+ symporters for L-rhamnose (RhaT), L-fucose (FucP) and lactose (LacY) in E. coli are insensitive to forskolin. Forskolin and [125I]APS-forskolin, therefore, constitute novel probes for exploring the structure-activity relationship of the bacterial GalP protein. GalP will provide an excellent model for the human glucose transporters and for elucidating the molecular basis of subtle differences in substrate and inhibitor recognition by individual members of this widespread family of transport proteins.

Substitution of conserved tyrosine residues in helix 4 (Y143) and 7 (Y293) affects the activity, but not IAPS-forskolin binding, of the glucose transporter GLUT4.

Six tyrosine residues (Y28, Y143, Y292, Y293, Y308, Y432(1)) which are conserved in all mammalian glucose transporters were substituted for phenylalanine by site-directed mutagenesis, and mutant glucose transporters were transiently expressed in COS-7 cells. Glucose transport activity as assessed by reconstitution of the solubilized transporters into lecithin liposomes was reduced by 70% in the mutant Y143F and appeared to be abolished in Y293F, but was not affected by substitution of Y28, Y292, Y308 and Y432. In contrast, covalent binding of the photolabel 125IAPS-forskolin was normal in all mutants. Stable expression of the mutants Y143F, Y293F, and Y292F in LTK cells yielded identical results. These data indicate that only two of the 6 conserved helical tyrosine residues, located in helices 4 and 7, are essential for full activity, but not for IAPS-forskolin binding of the GLUT4.

Role of tryptophan-388 of GLUT1 glucose transporter in glucose-transport activity and photoaffinity-labelling with forskolin.

GLUT1 glucose-transporter cDNA was modified to substitute leucine for Trp-388 and transfected into Chinese hamster ovary cells using the expression vector termed pMTHneo. This tryptophan residue is conserved among most of the facilitative glucose-transporter isoforms and has been proposed to be the photolabelling site of forskolin, a competitive inhibitor of glucose transport. In addition, this residue is located on membrane-spanning helix 10 which is suggested to contain the dynamic segment of the transporter. The mutated glucose transporter was expressed and inserted into the plasma membrane in a fashion similar to the wild-type. Unexpectedly, this mutation did not abolish photolabelling with forskolin. However, the mutation induced a marked decrease in 2-deoxyglucose uptake with a 4-fold decrease in turnover number and a 1.25-fold increase in Km compared with the wild-type GLUT1. A similar decrease in zero-trans influx activity was also observed for 3-O-methylglucose. In contrast, no apparent decrease was observed in zero trans efflux activity for 3-O-methylglucose. The mutation decreased the turnover number of the glucose transporter in equilibrium exchange influx for 3-O-methylglucose by 33% without any change in Km. These results indicate that (1) Trp-388 is not the photolabelling site for forskolin, if we assume that the labelling occurs at a single site and (2) Trp-388 is more likely to be involved in interconversion between the inward-facing and outward-facing conformers of GLUT1 than binding of glucose, and thus, substitution of leucine for Trp-388 in this dynamic segment would decrease the rate of alternating conformation, which would preferentially affect the influx activity.

Glucose transport activity and photolabelling with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (IAPS)-forskolin of two mutants at tryptophan-388 and -412 of the glucose transporter GLUT1: dissociation of the binding domains of forskolin and glucose.

The tryptophan residues 388 and 412 in the glucose transporter GLUT1 were altered to leucine (L) by site-directed mutagenesis and were transiently expressed in COS-7 cells. As assessed by immunoblotting, comparable numbers of glucose transporters were present in plasma membranes from cells transfected with wild-type GLUT1, GLUT1-L388 or GLUT1-L412. Transfection of the wild-type GLUT1 gave rise to a 3-fold increase in the reconstituted glucose transport activity recovered from plasma membranes. In contrast, transfection of GLUT1-L412 failed to increase the reconstituted transport activity, whereas transfection of GLUT1-L388 produced only a 70% increase. Photolabelling of GLUT1-L412 with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl (125IAPS)-forskolin was not different from that of the wild-type GLUT1, whereas the GLUT1-L388 incorporated 70% less photolabel than did the wild-type GLUT1. These data suggest a dissociation of the binding sites of forskolin and glucose in GLUT1. Whereas both tryptophan-388 and tryptophan-412 appear indispensable for the function of the transporter, only tryptophan-388 is involved in the binding of the inhibitory ligand forskolin.

Localization of the binding domain of the inhibitory ligand forskolin in the glucose transporter GLUT-4 by photolabeling, proteolytic cleavage and a site-specific antiserum.

The binding domain of forskolin in the adipocyte/muscle-type glucose transporter (GLUT-4) was localized with the aid of the photoreactive derivative, [125I]IAPS-forskolin (3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin). Plasma membranes from insulin-treated rat adipocytes containing predominantly the GLUT-4 isoform were irradiated with UV light in the presence of [125I]IAPS-forskolin. The covalently labeled glucose transporters were isolated by immunoprecipitation with specific antiserum and partially digested with trypsin and elastase. The fragments were separated by gel electrophoresis, transferred on to nitrocellulose membranes, and identified by direct autoradiography and by immunoassay with antiserum against a peptide sequence corresponding to the C-terminus of GLUT-4. Digestion with a high-purity grade trypsin generated two photolabeled fragments with apparent molecular weights of 21 and 16 kDa. Since the antiserum detected two fragments with identical electrophoretic mobility, both labeled fragments appeared to contain the intact C-terminus of GLUT-4. In contrast, digestion with elastase generated only one photolabeled fragment with intact C-terminus at 21 kDa, and a smaller unlabeled fragment with intact C-terminus at 15 kDa. A less pure trypsin preparation generated two labeled (21 and 17 kDa) and one unlabeled (15 kDa) fragment with intact C-terminus. These data suggest that the site of covalent binding of IAPS-forskolin in the GLUT-4 is located within a region of 1-6 kDa defined by the difference between the unlabeled C-terminal fragment (15 kDa) and the labeled fragments (21, 17 and 16 kDa). Based on a tentative allocation of the fragments to the sequence of the GLUT-4, it is suggested that the covalent binding site of IAPS-forskolin is located between the membrane spanning helices 7-9, possibly in the proximity of helix 9.

Localization of the forskolin photolabelling site within the monosaccharide transporter of human erythrocytes.

Chemical and proteolytic digestion of intact erythrocyte glucose transporter as well as purified transporter protein has been used to localize the derivatization site for the photoaffinity agent 3-[125I]iodo-4-azido-phenethylamino-7-O-succinyldeacetylforskol in [( 125I]IAPS-forskolin). Comparison of the partial amino acid sequence of the labelled 18 kDa tryptic fragment with the known amino acid sequence for the HepG2 glucose transporter confirmed that the binding site for IAPS-forskolin is between the amino acid residues Glu254 and Tyr456. Digestion of intact glucose transporter with Pronase suggests that this site is within the membrane bilayer. Digestion of labelled transporter with CNBr generated a major radiolabelled fragment of Mr approximately 5800 putatively identified as residues 365-420. Isoelectric focusing of Staphylococcus aureus V8 proteinase-treated purified labelled tryptic fragment identified two peptides which likely correspond to amino acid residues 360-380 and 381-393. The common region for these radiolabelled peptides is the tenth putative transmembrane helix of the erythrocyte glucose transporter, comprising amino acid residues 369-389. Additional support for this conclusion comes from studies in which [125I]APS-forskolin was photoincorporated into the L-arabinose/H(+)-transport protein of Escherichia coli. Labelling of this transport protein was protected by both cytochalasin B and D-glucose. The region of the erythrocyte glucose transporter thought to be derivatized with IAPS-forskolin contains a tryptophan residue (Trp388) that is conserved in the sequence of the E. coli arabinose-transport protein.

Stimulation of glucose transport in rat cardiac myocytes by guanosine 3',5'-monophosphate.

Glucose transport in isolated rat cardiomyocytes is stimulated by insulin, catecholamines, and anoxia approximately 2- to 3-fold over basal rates. The molecular mechanisms controlling these responses are unknown. In our search for possible cellular mediators of glucose transport stimulation, we examined the effects of a number of nucleotides on 3-O-methylglucose transport in heart cells. The nucleotides and/or permeable analogs (monosuccinyl, 8-bromo, and dibutyryl derivatives) included cUMP, cIMP, cCMP, cAMP, and cGMP at concentrations ranging from 10 nM to 1 mM. Of all the nucleotides tested only cGMP analogs induced a significant stimulation of transport at concentrations as low as 100 nM. This effect was observed in both the 8-bromo- and dibutyryl derivatives and with 1 mM cGMP itself. The effect was concentration dependent for both analogs and produced a maximal response equivalent to that of 100 nM insulin. This insulinomimetic effect of cGMP was examined in more detail in order to evaluate its role as a potential mediator of this response. Agents that are known to stimulate guanylate cyclase in the heart produced a clear stimulation of transport when added to cardiomyocytes. These include insulin, aminophylline, histamine, beta-estradiol, and biotin-nitrophenyl ester. Methylene blue, an inhibitor of guanylate cyclase, blocked the insulin response when added to cells before insulin, but was ineffective when added after insulin. In addition, agents that raise intracellular cGMP levels by inhibiting cyclic nucleotide phosphodiesterases were also examined for effects on glucose transport. Out of several phosphodiesterase inhibitors tested, only Zaprinast (which selectively increases cGMP in heart) stimulated transport in a concentration-dependent manner to within 80% of the maximal insulin effect. These results are consistent with the notion that cGMP may be involved in glucose transport stimulation.

Identification of the glucose transporter in mammalian cell membranes with a 125I-forskolin photoaffinity label.

The glucose transporter has been identified in a variety of mammalian cell membranes using a photoactivatable carrier-free radioiodinated derivative of forskolin, 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetylforskoli n ([125I]IAPS-forskolin) at 1-3 nM. The membranes that were photolabelled with [125I]IAPS-forskolin were human placental membranes, rat cortical and cerebellar synaptic membranes, rat cardiac sarcolemmal membranes, rat adipocyte plasma membranes, smooth-muscle membranes, and S49 wild-type (WT) lymphoma-cell membranes. The glucose transporter in plasma membranes prepared from the insulin-responsive rat cardiac sarcolemmal cells, rat adipocytes and smooth-muscle cells were determined to be approx. 45 kDa by SDS/polyacrylamide-gel electrophoresis (PAGE). Photolysis of human placental membranes, rat cortical and cerebellar synaptic membranes, and WT lymphoma membranes with [125I]-IAPS-forskolin, followed by SDS/PAGE, indicated specific derivatization of a broad band (43-55 kDa) in placental membranes and a narrower band (approx. 45 kDa) in synaptic membranes and WT lymphoma membranes. Digestion of the [125I]IAPS-forskolin-labelled placental and WT lymphoma membranes with endo-beta-galactosidase showed a reduction in the apparent molecular mass of the radiolabelled band to approx. 40 kDa. The membranes that were photolabelled with [125I]IAPS-forskolin and trypsin-treated produced a radiolabelled proteolytic fragment with an apparent molecular mass of 18 kDa. [125I]IAPS-forskolin is a highly effective probe for identifying low levels of glucose transporters in mammalian tissues.

Derivatization of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label.

An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-O-succinyldeacetyl-forskolin (IAPS-forskolin), has been synthesized, purified, and characterized. The I50 for inhibition of 3-O-methylglucose transport in red blood cells by IAPS-forskolin was found to be 0.05 microM. The carrier free radioiodinated label is a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes (ghosts) and purified glucose transporter preparations with 1-2 nM [125I]IAPS-forskolin and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed specific derivatization of a broad band with an apparent molecular mass of 40-70 kDa. Photoincorporation into erythrocyte membranes using 2 nM [125I]IAPS-forskolin was protected with D-glucose (I50 400 mM), cytochalasin B (I50 0.5 microM), and forskolin (I50 10 microM). No protection was observed with L-glucose (600 mM). Endo-beta-galactosidase digestion of [125I] IAPS-forskolin-labeled ghosts and purified transporter resulted in a dramatic sharpening of the specifically radiolabeled transporter to 40 kDa. Trypsinization of [125I]IAPS-forskolin-labeled ghosts and purified transporter reduced the specifically radiolabeled transporter to a sharp peak at 18 kDa. [125I]IAPS-forskolin will be a useful tool to study the structural aspects of the glucose transporter.

[3H]forskolin. Direct photoaffinity labeling of the erythrocyte D-glucose transporter.

Irradiation of erythrocyte ghosts in the presence of [3H]forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into several of the major membrane protein bands. Most of the incorporation occurred in four regions of the gel. Peak 1 (216 kDa) was a sharp peak near the top of the gel in the region corresponding to spectrin. Peak 2 appeared to be associated with band 3 (89 kDa), while a third peak occurred around the position of band 4.2 (76 kDa). The fourth region of labeling was a broad area between 43-75 kDa which corresponds to the region of the glucose transporter. Forskolin labeling of this region was inhibited by cytochalasin B and D-glucose, but not L-glucose. Extraction of extrinsic membrane proteins resulted in a loss of radiolabeled protein from the 216- and 76-kDa regions. Treatment of membranes labeled with either cytochalasin B or forskolin with endo-beta-galactosidase resulted in identical shifts of the 43 to 75-kDa peaks to 42 kDa. Similarly, trypsinization of membranes photolabeled with either cytochalasin B or forskolin resulted in the generation of a 17-kDa radiolabeled fragment in both cases. Photoincorporation of [3H]cytochalasin B into the glucose transporter was blocked in a concentration-dependent manner by unlabeled forskolin.

Interactions of insulin, catecholamines and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes.

The regulation of the glucose transport system by catecholamines and insulin has been studied in isolated rat cardiomyocytes. In the basal state, 1-isoproterenol exhibited a biphasic concentration-dependent regulation of 3-O-methylglucose transport. At low concentrations (less than 10 nM), isoproterenol induced a maximal inhibition of 65-70% of the basal rates, while at higher concentrations (greater than 10 nM) a 25-70% stimulation of transport was observed. In the presence of adenosine deaminase, the inhibition of isoproterenol at low doses was attenuated. No effect of adenosine deaminase was observed on the stimulation of transport at high doses of isoproterenol. The inhibitory effect of isoproterenol returned when N6-phenylisopropyladenosine (a non-metabolizable analog of adenosine) was included along with adenosine deaminase. Dibutyryl cAMP and forskolin both inhibited basal transport rates. In the presence of maximally stimulating concentrations of insulin, cardiomyocyte 3-O-methylglucose transport was generally elevated 200-300% above basal levels. In the presence of isoproterenol, insulin stimulation was inhibited at both high and low concentrations of catecholamine, with maximum inhibition occurring at the lowest concentrations tested. When cells were incubated with both adenosine deaminase and isoproterenol, the inhibition of the insulin response was greater at all concentrations of catecholamine and was almost completely blocked at isoproterenol concentrations of 10 nM or less. Dibutyryl cAMP inhibited the insulin response to within 10% of basal transport levels, while forskolin completely inhibited all transport activity in the presence of insulin. These results suggest that catecholamines regulate basal and insulin-stimulated glucose transport via both cAMP-dependent and cAMP-independent mechanisms and that this regulation is modulated in the presence of extracellular adenosine.

Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose.

A photoreactive, radioiodinated derivative of glucose, N-(4-iodoazidosalicyl)-6-amido-6-deoxyglucopyranose (IASA-glc), has been synthesized and used as a photoaffinity label for the human erythrocyte monosaccharide transporter. Photoinactivation and photoinsertion are both light-dependent and result in a marked decrease in the absorption spectra of the compound. When [125I]IASA-glc was photolyzed with erythrocyte ghost membranes, photoinsertion of radiolabel was observed in three major regions, spectrin, band 3, and a protein of 58,000 daltons located in the zone 4.5 region. Of the three regions which were photolabeled, only labeling of polypeptides in the zone 4.5 region was partially blocked by D-glucose. In the non-iodinated form, N-(4-azidosalicyl)-6-amido-6-deoxy-glucopyranose inhibited the labeling of the transporter by [125I]IASA-glc more effectively than D-glucose. The ability to synthesize this [125I]containing photoprobe for the monosaccharide transporter at carrier-free levels offers several new advantages for investigating the structure of this transport protein in the erythrocyte.

Fetal insulin and placental 3-O-methyl glucose clearance in near-term sheep.

It is difficult, if not impossible, to measure the placental transfer of glucose directly because of placental glucose consumption and the low A-V glucose difference across the sheep placenta. We have approached the problem of quantifying placental hexose transfer by using a nonmetabolized glucose analogue (3-O-methyl glucose) which shares the glucose transport system. We have measured the clearance by using a multisample technique permitting least squares linear computing to avoid the errors implicit in the Fick principle. The placental clearance of 3-O-methyl glucose was measured in the control condition and after the administration of insulin to the fetal circulation. A glucose clamp technique was used to maintain constant transplacental glucose concentrations throughout the duration of the experiment. A control series was performed in which the only intervention was the infusion of normal saline. In these experiments the maternal and fetal glucose concentrations remained constant as did the volume of distribution of 3-O-methyl glucose in the fetus. The maternal insulin concentration remained constant and fetal insulin concentration changed from 11 +/- 2 microU/ml to 355 +/- 51 microU/ml (P less than 0.01). In the face of this large increase in fetal plasma insulin, there was no change in the placental clearance of 3-O-methyl glucose. In the control condition the clearance was 14.1 +/- 1.0 ml/min per kg and this was 13.8 +/- 1.0 ml/min per kg in the high insulin condition. Fetal insulin may change placental glucose flux by decreasing fetal plasma glucose concentrations but does not do so by changing the activity of the glucose transport system.

Modulation of hexose transport in cultured skeletal muscle.

The modulation of hexose transport due to insulin and glucose starvation was investigated in cultures derived from the breast musculature of embryonic quail. Fused myotubes at 37 degrees C exhibited a saturable, stereospecific basal uptake of both D-glucose and 3-O-methylglucose which was markedly inhibited by cytochalasin B, a potent inhibitor of hexose transport in other cell systems. In the presence of insulin, 3-O-methylglucose uptake was stimulated relative to untreated controls. Kinetic analysis indicated that insulin increased the Vmax of transport with no significant increase in the apparent Km. Incubation of myotubes in glucose-free medium for 24 h resulted in an increase in D-glucose and 3-O-methylglucose transport activity. Cycloheximide abolished this stimulation effect when it was included during the starvation period, but had no effect on transport in glucose-fed cells. Insulin binding studies on these myotubes indicate that high-affinity insulin receptors are present and continue to increase throughout the life of the culture. This high-affinity binding as well as the capacity to degrade insulin in these cells is characteristically similar to effects observed in other insulin-sensitive cell systems.

Orientation of the glucose transporter in the human erythrocyte membrane. Investigation by in situ proteolytic dissection.

Glucose transporter proteins (zone 4.5) which had been photoaffinity labeled with [3H]cytochalasin B in human erythrocyte ghosts were subjected to enzymatic dissection in order to study the transmembrane disposition of the protein in situ. Proteolytic enzymes as well as glycosidases were used to treat unsealed and resealed ghosts in order to explore the various membrane domains of the transporter in a topographically defined manner. Limited digestion of sealed ghosts with trypsin had no effect on the apparent Mr of the transporter (55,000). Similar treatment in unsealed ghosts, however, resulted in the generation of a major fragment of 21.5 kDa, along with several minor fragments. Thermolysin also had no effect on sealed ghosts but caused a complete loss of radiolabel from the zone 4.5 region with no lower-molecular-weight fragments being retained on the gel. Chymotrypsin treatment resulted in the generation of a single peak, Mr = 18,400, in both sealed and unsealed ghosts indicating its action occurs at the outer surface. Digestion with carboxypeptidase and aminopeptidase indicate the C-terminal end of the transporter is located exterior to the membrane with the N terminus located at the cytoplasmic surface. Treatment with endoglycosidase resulted in a shift of mobility of the transporter to a lower Mr of 49,000. The results obtained indicate that the carbohydrate is located near the C-terminal end and that the cytochalasin B-binding site is located near the cytoplasmic N-terminal end.