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We previously reported that substrates of semicarbazide-sensitive amine oxidase in combination with low concentrations of vanadate exert potent insulin-like effects. Here we performed homology modeling of the catalytic domain of mouse SSAO/VAP-1 and searched through chemical databases to identify novel SSAO substrates. The modeling of the catalytic domain revealed that aromatic residues Tyr384, Phe389, and Tyr394 define a pocket of stable size that may participate in the binding of apolar substrates. We identified a number of amines as substrates of human, rat, and mouse SSAO. The compounds PD0119035, 2,3-dimethoxy-benzylamine, and C-naphthalen-1-yl-methylamine showed high affinity as substrates of rat SSAO. C-Naphthalen-1-yl-methylamine was the only substrate that showed high affinity for human SSAO. C-Naphthalen-1-yl-methylamine and 4-aminomethyl-benzenesulfonamide showed the highest capacity to stimulate glucose transport in isolated rat adipocytes. The impact of these findings on the development of new treatments for diabetes is discussed.

Glucose constitutes a major fuel for the heart, and high glucose uptake during fetal development is coincident with the highest level of expression of the glucose transporter GLUT-1 during life. We have previously reported that GLUT-1 is repressed perinatally in rat heart, and GLUT-4, which shows a low level of expression in the fetal stage, becomes the main glucose transporter in the adult. Here, we show that the perinatal expression of GLUT-1 and GLUT-4 glucose transporters in heart is controlled directly at the level of gene transcription. Transient transfection assays show that the -99/-33 fragment of the GLUT-1 gene is sufficient to drive transcriptional activity in rat neonatal cardiomyocytes. Electrophoretic mobility shift assays demonstrate that the transcription factor Sp1, a trans-activator of GLUT-1 promoter, binds to the -102/-82 region of GLUT-1 promoter during the fetal state but not during adulthood. Mutation of the Sp1 site in this region demonstrates that Sp1 is essential for maintaining a high transcriptional activity in cardiac myocytes. Sp1 is markedly down-regulated both in heart and in skeletal muscle during neonatal life, suggesting an active role for Sp1 in the regulation of GLUT-1 transcription. In all, these results indicate that the expression of GLUT-1 and GLUT-4 in heart during perinatal development is largely controlled at a transcriptional level by mechanisms that might be related to hyperplasia and that are independent from the signals that trigger cell hypertrophy in the developing heart. Furthermore, our results provide the first functional insight into the mechanisms regulating muscle GLUT-1 gene expression in a live animal.

Insulin-like growth factors (IGFs) are potent inducers of skeletal muscle differentiation and phosphatidylinositol (PI) 3-kinase activity is essential for this process. Here we show that IGF-II induces nuclear factor-kappaB (NF-kappaB) and nitric-oxide synthase (NOS) activities downstream from PI 3-kinase and that these events are critical for myogenesis. Differentiation of rat L6E9 myoblasts with IGF-II transiently induced NF-kappaB DNA binding activity, inducible nitric-oxide synthase (iNOS) expression, and nitric oxide (NO) production. IGF-II-induced iNOS expression and NO production were blocked by NF-kappaB inhibition. Both NF-kappaB and NOS activities were essential for IGF-II-induced terminal differentiation (myotube formation and expression of skeletal muscle proteins: myosin heavy chain, GLUT 4, and caveolin 3), which was totally blocked by NF-kappaB or NOS inhibitors in rat and human myoblasts. Moreover, the NOS substrate L-Arg induced myogenesis in the absence of IGFs in both rat and human myoblasts, and this effect was blocked by NOS inhibition. Regarding the mechanisms involved in IGF-II activation of NF-kappaB, PI 3-kinase inhibition prevented NF-kappaB activation, iNOS expression, and NO production. Moreover, IGF-II induced, through a PI 3-kinase-dependent pathway, a decrease in IkappaB-alpha protein content that correlated with a decrease in the amount of IkappaB-alpha associated with p65 NF-kappaB.

The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.

Skeletal muscle differentiation involves myoblast alignment, elongation, and fusion into multinucleate myotubes, together with the induction of regulatory and structural muscle-specific genes. Here we show that two phosphatidylinositol 3-kinase inhibitors, LY294002 and wortmannin, blocked an essential step in the differentiation of two skeletal muscle cell models. Both inhibitors abolished the capacity of L6E9 myoblasts to form myotubes, without affecting myoblast proliferation, elongation, or alignment. Myogenic events like the induction of myogenin and of glucose carrier GLUT4 were also blocked and myoblasts could not exit the cell cycle, as measured by the lack of mRNA induction of p21 cyclin-dependent kinase inhibitor. Overexpresssion of MyoD in 10T1/2 cells was not sufficient to bypass the myogenic differentiation blockade by LY294002. Upon serum withdrawal, 10T1/2-MyoD cells formed myotubes and showed increased levels of myogenin and p21. In contrast, LY294002-treated cells exhibited none of these myogenic characteristics and maintained high levels of Id, a negative regulator of myogenesis. These data indicate that whereas phosphatidylinositol 3-kinase is not indispensable for cell proliferation or in the initial events of myoblast differentiation, i.e. elongation and alignment, it appears to be essential for terminal differentiation of muscle cells.

Several studies have shown that the cRNA of human, rabbit, or rat rBAT induces in Xenopus oocytes sodium-independent, high affinity uptake of L-cystine via a system b0,(+)-like amino acid exchanger. We have shown that mutations in rBAT cause type I cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-425; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). Apart from oocytes, no other expression system has been used for transfection of functional rBAT activity. Furthermore, the b0,(+)-like transport activity has not been clearly described in the kidney or intestine. Here, we report that a "proximal tubular-like" cell line derived from opossum kidney (OK cells) expresses an rBAT transcript. Poly(A)+ RNA from OK cells induced by system b0,(+)-like transport activity in oocytes. This was hybrid-depleted by human rBAT antisense oligonucleotides. A polymerase chain reaction-amplified cDNA fragment (approximately 700 base pairs) from OK cell RNA corresponds to an rBAT protein fragment 65-69% identical to those from human, rabbit and rat kidneys. We have also examined transport of l-cystine in OK cells and found characteristics very similar to the amino acid exchanger activity induced by rBAT cRNA in oocytes. Uptake of L-cystine was of high affinity, sodium-independent and shared with L-arginine and L-leucine. It was trans-stimulated by amino acids with the same specificity as rBAT-induced transport activity in oocytes. Furthermore, it was localized to the apical pole of confluent OK cells. To demonstrate that the rBAT protein is functionally related to this transport activity, we have transfected OK cells with human rBAT antisense and sense sequences. Transfection with rBAT antisense, but not with rBAT sense, resulted in the specific reduction of rBAT mRNA expression and b0,(+)-like transport activity. These results demonstrate that rBAT is functionally related to the L-cystine uptake via system b0,(+)-like in the apical pole of the renal OK cell line.

Cystinuria is an autosomal recessive amino-aciduria where three urinary phenotypes have been described (I, II, and III). An amino acid transporter gene, SLC3A1 (formerly rBAT), was found to be responsible for this disorder. To assess whether mutations in SLC3A1 are involved in different cystinuria phenotypes, linkage with this gene and its nearest marker (D2S119) was analyzed in 22 families with type I and/or type III cystinuria. Linkage with heterogeneity was proved (alpha = 0.45; P < 0.008). Type I/I families showed homogeneous linkage to SLC3A1 (Zmax > 3.0 at theta = 0.00; alpha = 1), whereas types I/III and III/III were not linked. Our data suggest that type I cystinuria is due to mutations in the SLC3A1 gene, whereas another locus is responsible for type III. This result establishes genetic heterogeneity for cystinuria, classically considered as a multiallelic monogenic disease.

In this study we have used wortmannin, a highly specific inhibitor of phosphatidylinositol (PI) 3-kinase, to assess the role of this enzyme on GLUT1 glucose carrier distribution and glucose transport activity in myoblasts from two skeletal-muscle cell lines, L6E9 and Sol8. As detected in L6E9 cells, myoblasts exhibited basal and insulin-stimulated PI 3-kinase activities. Incubation of intact myoblasts with wortmannin resulted in a marked inhibition of both basal and insulin-stimulated PI 3-kinase activities. L6E9 and Sol8 myoblasts showed basal and insulin-stimulated glucose transport activities, both of them inhibited by wortmannin in a dose-dependent manner (IC50 approximately 10-20 nM). Concomitantly, immunofluorescence analysis revealed that 1 h treatment with wortmannin led to a dramatic intracellular accumulation of GLUT1 carriers (the main glucose transporter expressed in L6E9 and Sol8 myoblasts) in both cell systems. The effect of wortmannin on GLUT1 cellular redistribution was independent of the presence of insulin. The cellular distribution of two structural plasma-membrane components such as beta 1-integrin or the alpha 1 subunit of the Na(+)-K(+)-ATPase were unaffected by wortmannin in both the absence and the presence of insulin. As a whole, our results indicate that PI 3-kinase is necessary to basal and insulin-stimulated glucose transport in L6E9 and Sol8 myoblasts. Moreover, immunofluorescence assays suggest that in both cellular models there is a constitutive GLUT 1 trafficking pathway (independent of insulin) that involves PI 3-kinase and which, when blocked, locks GLUT1 in a perinuclear compartment.

(1) In this study we have determined the distribution of various membrane proteins involved in insulin-activated glucose transport in T-tubules and in sarcolemma from rat skeletal muscle. Two independent experimental approaches were used to determine the presence of membrane proteins in T-tubules: (i) the purification of T-tubules free from sarcolemmal membranes by lectin agglutination, and (ii) T-tubule vesicle immunoadsorption. These methods confirmed that T-tubules from rat skeletal muscle were enriched with dihydropyridine receptors and tt28 protein and did not contain the sarcolemmal markers dystrophin or beta 1-integrin. Both types of experiments revealed an abundant content of GLUT4 glucose carriers, insulin receptors and SCAMPs (secretory carrier membrane proteins) in T-tubule membranes. (2) Acute administration in vivo of insulin caused an increased abundance of GLUT4 in T-tubules and sarcolemma. On the contrary, insulin led to a 50% reduction in insulin receptors present in T-tubules and in sarcolemma, demonstrating that insulin-induced insulin receptor internalization affects T-tubules in the muscle fibre. The alteration in the content of GLUT4 and insulin receptors in T-tubules was a consequence of insulin-induced redistribution of these proteins. SCAMPs also redistributed in muscle membranes in response to insulin. They were recruited by insulin from intracellular high-density fractions to intracellular lighter-density fractions and to the cell surface, showing a pattern of insulin-induced cellular redistribution distinct from those of GLUT4 and the insulin receptor. (3) In conclusion, the T-tubule is a cell-surface target for membrane proteins involved in recycling such as SCAMPs or for membrane proteins that acutely redistribute in response to insulin such as GLUT4 or insulin receptors.

1. The heterologous expression of glucose transporters GLUT4 and GLUT1 in Xenopus oocytes has been shown to cause a differential targeting of these glucose-carrier isoforms to cellular membranes and a distinct induction of glucose transport activity. In this study we have evaluated the effect of insulin and insulin-like growth factor I (IGF-I) on glucose uptake and glucose transporter distribution in Xenopus oocytes expressing mammalian GLUT4 and GLUT1 glucose carriers. 2. Insulin and IGF-I stimulated 2-deoxyglucose uptake in GLUT4-expressing oocytes, but not in GLUT1-expressing oocytes or in water-injected oocytes. The stimulatory effect of insulin and IGF-I on 2-deoxyglucose uptake in GLUT4-expressing oocytes occurred via activation of the IGF-I receptor. 3. Subcellular-fractionation studies indicated that insulin and IGF-I stimulated translocation of GLUT4 to the cell surface of the oocyte. 4. Incubation of intact oocytes with insulin stimulated phosphatidylinositol 3-kinase activity, an effect that was blocked by the additional presence of wortmannin. Furthermore, wortmannin totally abolished the insulin-induced stimulation of 2-deoxyglucose uptake in GLUT4-expressing oocytes. 5. In this study, both the insulin-induced GLUT4 carrier translocation and GLUT4-dependent insulin-stimulated glucose transport have been reconstituted in the Xenopus oocyte. These observations, together with the fact that wortmannin, as found in adipocytes, inhibits insulin-stimulated glucose transport in oocytes, suggest that the heterologous expression of GLUT4 in oocytes is a useful experimental model by which to study the cell biology of insulin-induced GLUT4 translocation.

1. Several cell-surface domains of sarcolemma and T-tubule from skeletal-muscle fibre were isolated and characterized. 2. A protocol of subcellular fractionation was set up that involved the sequential low- and high-speed homogenization of rat skeletal muscle followed by KCl washing, Ca2+ loading and sucrose-density-gradient centrifugation. This protocol led to the separation of cell-surface membranes from membranes enriched in sarcoplasmic reticulum and intracellular GLUT4-containing vesicles. 3. Agglutination of cell-surface membranes using wheat-germ agglutinin allowed the isolation of three distinct cell-surface membrane domains: sarcolemmal fraction 1 (SM1), sarcolemmal fraction 2 (SM2) and a T-tubule fraction enriched in protein tt28 and the alpha 2-component of dihydropyridine receptor. 4. Fractions SM1 and SM2 represented distinct sarcolemmal subcompartments based on different compositions of biochemical markers: SM2 was characterized by high levels of beta 1-integrin and dystrophin, and SM1 was enriched in beta 1-integrin but lacked dystrophin. 5. The caveolae-associated molecule caveolin was very abundant in SM1, SM2 and T-tubules, suggesting the presence of caveolae or caveolin-rich domains in these cell-surface membrane domains. In contrast, clathrin heavy chain was abundant in SM1 and T-tubules, but only trace levels were detected in SM2. 6. Immunoadsorption of T-tubule vesicles with antibodies against protein tt28 and against GLUT4 revealed the presence of GLUT4 in T-tubules under basal conditions and it also allowed the identification of two distinct pools of T-tubules showing different contents of tt28 and dihydropyridine receptors. 7. Our data on distribution of clathrin and dystrophin reveal the existence of subcompartments in sarcolemma from muscle fibre, featuring selective mutually exclusive components. T-tubules contain caveolin and clathrin suggesting that they contain caveolin- and clathrin-rich domains. Furthermore, evidence for the heterogeneous distribution of membrane proteins in T-tubules is also presented.

The inhibition of insulin-stimulated glucose transport by isoprenaline, a mixed beta-adrenergic-receptor (AR) agonist, is well documented in rat adipocytes. Since it has been described that rat adipocytes possess not only beta 1- and beta 2- but also beta 3-ARs, the influence of various subtype-selective beta-AR agonists and antagonists on 2-deoxyglucose (2-DG) transport was assessed in order to characterize the beta-AR subtype involved in the adrenergic counter-regulation of the insulin effect. The stimulation of 2-DG transport by insulin was counteracted, in a dose-dependent manner, by all the beta-AR agonists tested, and the magnitude of the inhibition followed the rank order: BRL 37344 > isoprenaline = noradrenaline >> dobutamine = procaterol. The same rank order of potency was obtained for lipolysis activation. This is not in accordance with the pharmacological definition of a beta 1- or a beta 2-adrenergic effect, but agrees with the pharmacological pattern of a beta 3-adrenergic effect. The inhibitory effect of the beta 3-agonist BRL 37344 on insulin-stimulated 2-DG transport was not reversed by either the selective beta 1-antagonist ICI 89406 or the beta 2-antagonist ICI 118551. In addition, neither of these beta-antagonists was able to block the isoprenaline and noradrenaline effects, supporting major beta 3-adrenoceptor-subtype involvement in the adrenergic inhibition of insulin-stimulated 2-DG transport. Like isoprenaline, BRL 37344 inhibited (60% inhibition) insulin-stimulated glucose transport only when adenosine deaminase was present in the assay. Furthermore, the maximal inhibitory effects of isoprenaline and BRL 37344 were not additive, and were both dependent on albumin concentration in the incubation medium: they increased when the albumin concentration decreased in the medium from 3.5 to 1%. To conclude, the similarities between isoprenaline and BRL 37344 action on insulin-stimulated 2-DG transport, the poor efficacy of the beta 1-/beta 2-agonists and the lack of effect of selective beta 1- and beta 2-antagonists are compelling arguments to support the important role of beta 3-adrenoceptors in the adrenergic inhibition of glucose transport in rat adipocytes.

Fluoride is a nucleophilic reagent which has been reported to inhibit a variety of different enzymes such as esterases, asymmetrical hydrolases and phosphatases. In this report, we demonstrate that fluoride inhibits tyrosine kinase activity of insulin receptors partially purified from rat skeletal muscle and human placenta. Fluoride inhibited in a similar dose-dependent manner both beta-subunit autophosphorylation and tyrosine kinase activity for exogenous substrates. This inhibitory effect of fluoride was not due to the formation of complexes with aluminum and took place in the absence of modifications of insulin-binding properties of the insulin receptor. Fluoride did not complete with the binding site for ATP or Mn2+. Fluoride also inhibited the autophosphorylation and tyrosine kinase activity of receptors for insulin-like growth factor I from human placenta. Addition of fluoride to the pre-phosphorylated insulin receptor produced a slow (time range of minutes) inhibition of receptor kinase activity. Furthermore, fluoride inhibited tyrosine kinase activity in the absence of changes in the phosphorylation of prephosphorylated insulin receptors, and the sensitivity to fluoride was similar to the sensitivity of the unphosphorylated insulin receptor. The effect of fluoride-on tyrosine kinase activity was markedly decreased when insulin receptors were preincubated with the copolymer of glutamate/tyrosine. Prior exposure of receptors to free tyrosine or phosphotyrosine also prevented the inhibitory effect of fluoride. However, the protective effect of tyrosine or phosphotyrosine was maximal at low concentrations, suggesting the interaction of these compounds with the receptor itself rather than with fluoride. These data suggest: (i) that fluoride interacts directly and slowly with the insulin receptor, which causes inhibition of its phosphotransferase activity; (ii) that the binding site of fluoride is not structurally modified by receptor phosphorylation; and (iii) based on the fact that fluoride inhibits phosphotransferase activity in the absence of alterations in the binding of ATP, Mn2+ or insulin, we speculate that fluoride binding might affect the transfer of phosphate from ATP to the tyrosine residues of the beta-subunit of the insulin receptor and to the tyrosine residues of exogenous substrates.

1. GLUT-4 glucose-transporter protein and mRNA levels were assessed in heart, red muscle and white muscle, as well as in brown and white adipose tissue from 7-day streptozotocin-induced diabetic and 48 h-fasted rats. 2. In agreement with previous data, white adipose tissue showed a substantial decrease in GLUT-4 mRNA and protein levels in response to both diabetes and fasting. Similarly, GLUT-4 mRNA and protein markedly decreased in brown adipose tissue in both insulinopenic conditions. 3. Under control conditions, the level of expression of GLUT-4 protein content differed substantially in heart, red and white skeletal muscle. Thus GLUT-4 protein was maximal in heart, and red muscle had a greater GLUT-4 content compared with white muscle. In spite of the large differences in GLUT-4 protein content, GLUT-4 mRNA levels were equivalent in heart and red skeletal muscle. 4. In heart, GLUT-4 mRNA decreased to a greater extent than GLUT-4 protein in response to diabetes and fasting. In contrast, red muscle showed a greater decrease in GLUT-4 protein than in mRNA in response to diabetes or fasting, and in fact no decrease in GLUT-4 mRNA content was detectable in fasting. On the other hand, preparations of white skeletal muscle showed a substantial increase in GLUT-4 mRNA under both insulinopenic conditions, and that was concomitant to either a modest decrease in GLUT-4 protein in diabetes or to no change in fasting. 5. These results indicate that (a) the effects of diabetes and fasting are almost identical and lead to changes in GLUT-4 expression that are tissue-specific, (b) white adipose tissue, brown adipose tissue and heart respond similarly to insulin deficiency by decreasing GLUT-4 mRNA to a larger extent than GLUT-4 protein, and (c) red and white skeletal muscle respond to insulinopenic conditions in a heterogeneous manner which is characterized by enhanced GLUT-4 mRNA/protein ratios.

1. In the present study we have examined the sensitivity of A and ASC amino-acid-carrier activities in rat liver plasma-membrane vesicles to the thiol-group modifying reagents N-ethylmaleimide (NEM) and iodoacetamide (IA). To this end, the different Na(+)-dependent entities involved in alanine transport were assessed. 2. NEM inactivated Na(+)-dependent alanine transport as a result of the inhibition of both system A and ASC transport activities. The functional sensitivity of system A to NEM was greater than that of system ASC. 3. The presence of L-alanine (10 mM) during the exposure of vesicles to NEM afforded partial protection to system A, but not to the ASC, carrier. This effect was specific, since the presence of L-phenylalanine (10 mM) did not cause any protection. 4. Na+ did not protect A or ASC carriers against NEM inactivation; however, the presence of Na+ (100 mM-NaCl) and L-alanine (10 mM) during the exposure of the vesicles to NEM protected against inactivation of system A and ASC transport activities. The extent of protection was greater in the case of the system ASC transport activity than in the case of the A carrier. 5. IA also diminished Na(+)-dependent alanine transport by inhibition of A and ASC transport activities. Sodium and L-alanine afforded protection to both A and ASC transport activities from the inhibitory action of IA. The extent of protection induced by substrates was similar for both carriers. 6. It is concluded that there is one, or several, free thiol groups in A and ASC carriers, the integrity of which is essential for transport activity. Sensitivity to thiol-group-specific reagents and the pattern of protection with substrates against inactivation is different in A and ASC carriers. That suggests the existence of topological dissimilarities regarding the thiol-group containing site(s) in A and ASC amino acid carriers.

We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid transport in muscle.

Muscle cell differentiation caused a reduction of glucose transport, GLUT1 glucose transporter expression, and GLUT1 mRNA levels. A fragment of 2.1 kilobases of the rat GLUT1 gene linked to chloramphenicol acetyltransferase drove transcriptional activity in myoblasts, and differentiation caused a decrease in transcription. Transient transfection of 5' and 3' deletion constructs showed that the fragment -99/-33 of the GLUT1 gene drives transcriptional activity of the GLUT1 gene and participates in the reduced transcription after muscle differentiation. Electrophoretic mobility shift assays showed the binding of Sp1 protein to the fragment -102/-37 in the myoblast state but not in myotubes, and Sp1 was found to transactivate the GLUT1 promoter. Western blot analysis indicated that Sp1 was drastically down-regulated during myogenesis. Furthermore, the forced over-expression of MyoD in C3H10T1/2 cells mimicked the effects observed during myogenesis, Sp1 down-regulation and reduced transcriptional activity of the GLUT1 gene promoter. In all, these data suggest a regulatory model in which MyoD activation during myogenesis causes the down-regulation of Sp1, which contributes to the repression of GLUT1 gene transcription and, therefore, leads to the reduction in GLUT1 expression and glucose transport.

Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.

1. GLUT5 gene expression was studied in small intestine under a variety of conditions characterized by altered intestinal absorption of monosaccharides. 2. RNA-blotting studies showed that GLUT5 mRNA was abundantly expressed in rat and rabbit intestine and kidney, but it was not detected in heart or brown adipose tissue. GLUT5 mRNA levels were higher in the upper segments of the small intestine (duodenum and proximal jejunum) than in the lower segments (distal jejunum and ileum). 3. The intestinal expression of GLUT5 mRNA in rat proximal jejunum showed circadian rhythm. A 12-fold increase in GLUT5 mRNA levels was detected at the end of the light cycle and at the beginning of the dark cycle when compared with the early light period. In keeping with this, GLUT5 protein content in brush-border membranes was also increased at the beginning of the dark cycle compared with that in the light period. 4. In streptozotocin-induced diabetes an 80% increase in GLUT5 mRNA levels in mucosa from the proximal jejunum was detected under conditions in which enhanced intestinal absorption of monosaccharides has been reported. 5. The intestinal expression of GLUT5 mRNA showed regulation during perinatal development. Levels of GLUT5 mRNA were low during fetal life, increased progressively during the postnatal period and reached levels comparable with the adult state after weaning. Weaning on to a high-fat diet partially prevented the induction of GLUT5 gene expression. 6. Our results indicate that GLUT5 gene expression is tightly regulated in small intestine. Regulation involves maximal expression in the upper part of the small intestine, circadian rhythm, developmental regulation dependent on the fat and carbohydrate content in the diet at weaning and enhanced expression in streptozotocin-induced diabetes. Furthermore, changes observed in intestinal GLUT5 expression correlate with reported alterations in intestinal absorption of fructose. This suggests a regulatory role for GLUT5 in fructose uptake by absorptive enterocytes.

In this study, the relationship between the concentration of extracellular insulin, insulin binding and insulin action was evaluated in skeletal muscle. Initially we investigated the dose-response relationship of insulin action using three different experimental models that are responsive to insulin, i.e. the isolated perfused rat hindquarter, incubated strips of soleus muscle, and insulin receptors partially affinity-purified from skeletal muscle. We selected as insulin-sensitive parameters glucose uptake in the perfused hindquarter, lactate production in the incubated muscle preparation, and tyrosine receptor kinase activity in the purified receptor preparation. Our results showed that the dose-response curves obtained in the perfused hindquarter and in the incubated muscle were superimposable. In contrast, the dose-response curve for insulin-stimulated receptor tyrosine kinase activity in partially purified receptors was displaced to the left compared with the curves obtained in the perfused hindquarter and in the incubated muscle. The differences between the dose-response curve for receptor tyrosine kinase and those for glucose uptake and lactate production were not explained by a substantial insulin concentration gradient between medium and interstitial space. Thus the medium/interstitial insulin concentration ratio, when assayed in the incubated intact muscle at 5 degrees C, was close to 1. We also compared the dose-response curve of insulin-stimulated receptor tyrosine kinase with the pattern of insulin-binding-site occupancy. The curve of insulin-stimulated receptor kinase activity fitted closely with the occupancy of high-affinity binding sites. In summary, assuming that the estimation of the medium/interstitial insulin concentration ratio obtained at 5 degrees C reflects the actual ratio under more physiological conditions, our results suggest that maximal insulin action is obtained in skeletal muscle at insulin concentrations which do allow full occupancy of high-affinity binding sites. Therefore our data provide evidence for a lack of spare high-affinity insulin receptors in skeletal muscle.

Poly(A)+ mRNA was isolated from rabbit kidney cortex and injected into Xenopus laevis oocytes. Injection of mRNA resulted in a time- and dose-dependent increase in Na(+)-independent uptake of L-[3H]alanine and L-[3H]arginine. L-Alanine uptake was stimulated about 3-fold and L-arginine uptake was stimulated about 8-fold after injection of mRNA (25-50 ng, after 3-6 days) as compared with water-injected oocytes. T.I.C. of oocyte extracts suggested that the increased uptake actually represented an increase in the oocyte content of labelled L-alanine and L-arginine. The expressed L-alanine uptake, obtained by subtracting the uptake in water-injected oocytes from that in mRNA-injected oocytes, showed saturability and was inhibited completely by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) and L-arginine. The expressed L-arginine uptake in mRNA-injected oocytes also showed saturability, being completely inhibited by L-dibasic amino acids) and partially inhibited by BCH. Expression of both L-alanine and L-arginine uptake showed clear cis-inhibition by cationic (e.g. L-arginine) and neutral (e.g. L-leucine) amino acids. In all, this points to the expression of a Na(+)-independent transport system with broad specificity (i.e. b degree, (+)-like). In addition, part of the expressed uptake of L-arginine could be due to a system y(+)-like transporter. After size fractionation through a sucrose density gradient, the mRNA species encoding these increased transport activities (Na(+)-independent transport of L-alanine and of L-arginine) were found in fractions of an average mRNA chain-length of 1.8-2.4 kb. On the basis of these results, we conclude that Na(+)-independent transport system(s) for L-alanine and L-arginine from rabbit renal cortical tissues, most likely proximal tubules, are expressed in Xenopus laevis oocytes. These observations may represent the first steps towards expression and cloning of these transport pathways.

1. Insulin and adaptive regulation are known to stimulate system A amino acid transport activity in skeletal muscle. The present study was designed to investigate whether activation of system A in muscle is a consequence of processes which rely on microtubule or microfilament function. To that end, extensor digitorum longus (EDL) muscles were incubated in the presence of colchicine and cytochalasin D, well-known inhibitors of microtubule and microfilament activity respectively. 2. Basal alpha-(methyl)aminoisobutyric acid (MeAIB) uptake decreased after incubation with 5 microM-colchicine in a time-dependent manner. In keeping with this, adaptive regulation of MeAIB uptake caused by prolonged incubation in the absence of amino acids was substantially decreased in the presence of colchicine. 3. Under these conditions, stimulation of MeAIB uptake by insulin was unaltered in muscle in the presence of colchicine. This contrasted with the insulin-induced stimulation of MeAIB uptake by isolated rat hepatocytes, which was markedly decreased by colchicine. 4. Cytochalasin D, an agent that disrupts microfilaments, did not inhibit basal or insulin-stimulated MeAIB uptake by the incubated muscle. 5. Neither colchicine nor cytochalasin D modified the stimulatory effect of insulin on 3-O-methylglucose uptake by EDL muscle. 6. We conclude that up-regulation of system A by synthesis of new carriers depends on the integrity of microtubular function both in skeletal muscle and in hepatocytes. Microtubules might play a role in the movement of system A-containing vesicles from the Golgi network to the plasma membrane.

1. The present study was designed to explore the mechanisms by which insulin stimulates system A of amino acid transport in extensor digitorum longus (EDL) muscles, by using a system A analogue, alpha-(methyl)aminoisobutyric acid (MeAIB). 2. Insulin stimulation of MeAIB uptake was noted after only 30 min of incubation and was maximal at 60 min. Kinetics of the insulin effect on MeAIB uptake were characterized by an increased Vmax. without modification of Km for MeAIB. 3. Incubation of EDL muscles with cycloheximide for 90 min did not modify MeAIB uptake in either the presence or the absence of insulin, indicating the independence of insulin action from protein synthesis de novo. Incubations for 180 min with cycloheximide caused a decrease in basal MeAIB uptake; however, the percentage stimulation of amino acid transport by insulin was unaltered. Basal MeAIB uptake was increased by incubation for 180 min, but under these conditions no change in the percentage effect of insulin was found. 4. Ouabain, gramicidin D, or both, markedly decreased basal MeAIB uptake by EDL muscle, but the percentage effect of insulin was unaltered. 5. We conclude that insulin action on amino acid transport through system A in muscle is rapid, is characterized by an increased Vmax., and is independent of protein synthesis de novo and the Na+ electrochemical gradient. Our data are compatible with insulin acting directly on the system A transporter.

The cDNAs of mammalian amino acid transporters already identified could be grouped into four families. One of these protein families is composed of the protein rBAT and the heavy chain of the cell surface antigen 4F2 (4F2hc). The cRNAs of rBAT and 4F2hc induce amino acid transport activity via systems b0,+ -like and y+L -like inXenopus oocytes respectively. Surprisingly, neither rBAT nor 4F2hc is very hydrophobic, and they seem to be unable to form a pore in the plasma membrane. This prompted the hypothesis that rBAT and 4F2hc are subunits or modulators of the corresponding amino acid transporters. The association of rBAT with a light subunit of ~40kDa has been suggested, and such an association has been demonstrated for 4F2hc.

The previous characterization of an abundant population of non-adrenergic imidazoline-I2 binding sites in adipocytes and the recent demonstration of the interplay between these binding sites and amine oxidases led us to analyze the amine oxidase activity in membranes from isolated rat adipocytes. Adipocyte membranes had substantial levels of semicarbazide-sensitive amine oxidase (SSAO). SSAO activity and immunoreactive SSAO protein were maximal in plasma membranes, and they were also detectable in intracellular membranes. Vesicle immunoisolation analysis indicated that GLUT4-containing vesicles from rat adipocytes contain substantial levels of SSAO activity and immunoreactive SSAO protein. Immunotitration of intracellular GLUT4 vesicles indicated that GLUT4 and SSAO colocalize in an endosomal compartment in rat adipocytes. SSAO activity was also found in GLUT4 vesicles from 3T3-L1 adipocytes and rat skeletal muscle. Benzylamine, a substrate of SSAO activity, caused a marked stimulation of glucose transport in isolated rat adipocytes in the presence of very low vanadate concentrations that by themselves were ineffective in exerting insulin-like effects. This synergistic effect of benzylamine and vanadate on glucose transport was totally abolished in the presence of semicarbazide, a specific inhibitor of SSAO. Subcellular membrane fractionation revealed that the combination of benzylamine and vanadate caused a recruitment of GLUT4 to the plasma membrane of adipose cells. The stimulatory effects of benzylamine and vanadate on glucose transport were blocked by catalase, suggesting that hydrogen peroxide production coupled to SSAO activity plays a crucial regulatory role. Based on these results we propose that SSAO activity might contribute through hydrogen peroxide production to the in vivo regulation of GLUT4 trafficking in adipose cells.

Caveolin is believed to play an important role in sorting processes, vesicular trafficking, transmembrane signaling, and molecular transport across membranes. In this study we have evaluated the expression and distribution of caveolin in skeletal muscle and its interaction with GLUT4 glucose carriers. Caveolin was expressed to substantial levels in muscle and its expression was regulated in muscle; aging and high fat diet enhanced caveolin expression in skeletal muscle and inversely, myogenesis down-regulated caveolin in L6E9 cells. Under fasting conditions, most of caveolin was found in intracellular membranes and the caveolin present in the cell surface was found in both sarcolemma and T-tubules. Insulin administration led to a redistribution of caveolin from intracellular high density membrane fractions to intracellular lighter density fractions and to the cell surface; this pattern of insulin-induced redistribution was different to what was shown by GLUT4. These results suggests that caveolin is a component of an insulin-regulated machinery of vesicular transport in muscle. Quantitative immunoisolation of GLUT4 vesicles obtained from different intracellular GLUT4 populations revealed the absence of caveolin which substantiates the lack of colocalization of intracellular GLUT4 and caveolin. This indicates that caveolin is not involved in intracellular GLUT4 trafficking in skeletal muscle.

Using isolated rat cardiomyocytes we have examined: 1) the effect of insulin on the cellular distribution of glucose transporter 4 (GLUT4) and GLUT1, 2) the total amount of these transporters, and 3) the co-localization of GLUT4, GLUT1, and secretory carrier membrane proteins (SCAMPs) in intracellular membranes. Insulin induced 5.7- and 2.7-fold increases in GLUT4 and GLUT1 at the cell surface, respectively, as determined by the nonpermeant photoaffinity label [3H]2-N-[4(1-azi-2,2,2-trifluoroethyl)benzoyl]-1, 3-bis-(D-mannos-4-yloxy)propyl-2-amine. The total amount of GLUT1, as determined by quantitative Western blot analysis of cell homogenates, was found to represent a substantial fraction ( approximately 30%) of the total glucose transporter content. Intracellular GLUT4-containing vesicles were immunoisolated from low density microsomes by using monoclonal anti-GLUT4 (1F8) or anti-SCAMP antibodies (3F8) coupled to either agarose or acrylamide. With these different immunoisolation conditions two GLUT4 membrane pools were found in nonstimulated cells: one pool with a high proportion of GLUT4 and a low content in GLUT1 and SCAMP 39 (pool 1) and a second GLUT4 pool with a high content of GLUT1 and SCAMP 39 (pool 2). The existence of pool 1 was confirmed by immunotitration of intracellular GLUT4 membranes with 1F8-acrylamide. Acute insulin treatment caused the depletion of GLUT4 in both pools and of GLUT1 and SCAMP 39 in pool 2. In conclusion: 1) GLUT4 is the major glucose transporter to be recruited to the surface of cardiomyocytes in response to insulin; 2) these cells express a high level of GLUT1; and 3) intracellular GLUT4-containing vesicles consist of at least two populations, which is compatible with recently proposed models of GLUT4 trafficking in adipocytes.

Mutations in the rBAT gene cause type I cystinuria, a common inherited aminoaciduria of cystine and dibasic amino acids due to their defective renal and intestinal reabsorption (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Gallucci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426; Calonge, M. J., Volipini, V., Bisceglia, L., Rousaud, F., De Sanctis, L., Beccia, E., Zelante, L., Testar, X., Zorzano, A., Estivill, X., Gasparini, P., Nunes, V., and Palacín, M.(1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9667-9671). One important question that remains to be clarified is how the apparently non-concentrative system bo,+-like, associated with rBAT expression, participates in the active renal reabsorption of these amino acids. Several studies have demonstrated exchange of amino acids induced by rBAT in Xenopus oocytes. Here we offer evidence that system bo,+-like is an obligatory amino acid exchanger in oocytes and in the "renal proximal tubular" cell line OK. System bo, +-like showed a 1:1 stoichiometry of exchange, and the hetero-exchange dibasic (inward) with neutral (outward) amino acids were favored in oocytes. Obligatory exchange of amino acids via system bo,+-like fully explained the amino acid-induced current in rBAT-injected oocytes. Exchange via system bo,+-like is coupled enough to ensure a specific accumulation of substrates until the complete replacement of the internal oocyte substrates. Due to structural and functional analogies of the cell surface antigen 4F2hc to rBAT, we tested for amino acid exchange via system y+L-like. 4F2hc-injected oocytes accumulated substrates to a level higher than CAT1-injected oocytes (i.e. oocytes expressing system y+) and showed exchange of amino acids with the substrate specificity of system y+L and L-leucine-induced outward currents in the absence of extracellular sodium. In contrast to L-arginine, system y+L-like did not mediate measurable L-leucine efflux from the oocyte. We propose a role of systems bo,+-like and y+L-like in the renal reabsorption of cystine and dibasic amino acids that is based on their active tertiary transport mechanism and on the apical and basolateral localization of rBAT and 4F2hc, respectively, in the epithelial cells of the proximal tubule of the nephron.

A kidney cortex cDNA clone (rBAT) has recently been isolated, which upon in vitro transcription and capping complementary RNA (cRNA) and injection into Xenopus laevis oocytes induces a system b0,(+)-like amino acid transport activity. This cDNA encodes a type II membrane glycoprotein that shows significant homology to another type II membrane glycoprotein, the heavy chain of the human and mouse 4F2 surface antigen (4F2hc). Here we demonstrate that injection of human 4F2hc cRNA into oocytes results in the activation of a cation-preferring amino acid transport system that appears to be identical to the y(+)-like transport already present in the oocyte. This is based on the following results: (i) Injection of in vitro transcripts from 4F2hc cDNA (4F2hc cRNA) into oocytes stimulates up to 10-fold the sodium-independent uptake of L-arginine and up to 4.1-fold the sodium-dependent uptake of L-leucine. In contrast, 4F2hc cRNA does not increase the basal sodium-independent uptake of L-leucine. (ii) Basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine is completely inhibited by L-leucine in the presence of sodium. Similarly, the basal and 4F2hc cRNA-stimulated sodium-dependent uptake of L-leucine is entirely inhibited by L-arginine. (iii) The stimulation of sodium-independent uptake of L-arginine and the stimulation of sodium-dependent uptake of L-leucine induced by injection of 4F2hc cRNA are both completely inhibited by dibasic L amino acids and to a lesser extent by D-ornithine. (iv) Both basal and 4F2hc cRNA-stimulated sodium-independent uptake of L-arginine show two additional characteristics of the system y+ transport activity: inhibition of L-arginine uptake by L-homoserine only in the presence of sodium and an increase in the inhibition exerted by L-histidine as the extracellular pH decreased. Our results allow us to propose that an additional family of type II membrane glycoproteins (composed by rBAT and 4F2hc) is involved in amino acid transport, either as specific activators or as components of amino acid transport systems.

We have isolated a cDNA clone by screening a rabbit kidney cortex cDNA library for expression of sodium-independent transport of L-arginine and L-alanine in Xenopus laevis oocytes. Expressed uptake relates to a single component of sodium-independent transport for dibasic and neutral amino acids. This transport activity resembles the functionally defined system b0,+ and carries cystine and dibasic amino acids with high affinity. The rBAT (b0,+ amino acid transporter-related) mRNA is found mainly in kidney and intestinal mucosa. It encodes a predicted 77.8-kDa protein with only one putative transmembrane domain and seven potential N-glycosylation sites. This protein could either be a constitutive element or a specific activator of system b0,+.

We have recently isolated a renal cDNA clone (rBAT) that induces amino acid transport in oocytes either as a component or as a specific activator of a system bo,(+)-like transporter. (Bertran, J., Werner, A., Moore, M. L., Stange, G., Markovich, D., Biber, J., Testar, X., Zorzano, A., Palacín, M., and Murer, H. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5601-5605. In order to obtain additional information on the rBAT protein, we have investigated the cellular localization of rBAT and its expression during development in rat kidney. A polyclonal antibody raised against rBAT recognizes a specific protein band of approximately 90 kDa, which is highly enriched in rat renal brush border membranes. Immunofluorescence and immunoelectron microscopy studies demonstrated that rBAT is expressed in the microvilli domain of S3 epithelial cells (i.e. straight tubules). The onset of rBAT mRNA expression in kidney was detected during late fetal life. In keeping with this, induction in oocytes of L-cystine uptake due to fetal rBAT-related mRNA was approximately 10% of the induction obtained with rBAT-related mRNA from adult kidneys. rBAT mRNA levels were low in early postnatal life, and only at the end of lactation did they increase steeply, attaining approximately 50% of adult values after weaning. rBAT protein was undetectable in total membrane preparations of kidneys from fetuses and early neonates, weakly detected during lactation, and represented < 15% of adult values after weaning. The postnatal expression of rBAT and its specific location in the microvilli of epithelial cells from the S3 segment of the proximal tubule coincide with postnatal maturation of cystine resorption and with the site of high affinity resorption of cysteine in kidney. This is consistent with the involvement of rBAT in a b(o,+)-like high-affinity resorption system for cysteine in the proximal straight tubule of the nephron.

There is controversy concerning the possible modulation of glucose transfer to the fetus by insulin acting on the maternal side of the placenta. To study this question, 20.5 day pregnant rats were infused simultaneously with (U-14C)-D-glucose (via the jugular vein) and with different doses of insulin (via the left uterine artery) so that placentas from the left uterine horn were exposed to a higher insulin concentration than those from the right uterine horn. Placentas and fetuses from each uterine side were processed separately. No differences were detected in total blood radioactivity, plasma-14C-glucose,-14-C-lactate, -glucose, or -radioimmunoassayable insulin in fetuses from the left versus the right uterine horn. Total placenta radioactivity and 14C-glycogen were also similar in the left and right uterine sides at all insulin doses studied. Infusion of insulin (66 mU/min) to the pregnant rat caused hyperinsulinaemia and hypoglycaemia, decreased blood total radioactivity and plasma 14C-glucose, and increased plasma 14C-lactate in the mother. The level of fetal plasma 14C-glucose paralleled that of the mother. It is concluded that in the rat, placental glucose uptake, its transfer to the fetus, and fetal glucose utilization are not directly affected by maternal circulating insulin. Metabolic changes occurring in fetuses of hyperinsulinaemic mothers are secondary to the decreased availability of glucose.