Regulation of amino acid transport in L6 muscle cells: I. Stimulation of transport system a by amino acid deprivation

The regulation of amino acid transport in L6 muscle cells by amino acid deprivation was investigated. Proline uptake was Na⁺-dependent, saturable and concentrative, and was predominantly through system A. Proline uptake was inhibited by alanine, α-amino isobutyric acid (AIB), and by α-methylamino isobutyric acid, but not by lysine or valine. At 25°C, Km of proline uptake was 0.5 mM. Amino acid-deprivation resulted in a progressive increase in the rate of proline uptake, reaching up to 6-fold stimulation after 6 hours. The basal and stimulated transport were equally Na⁺-dependent, and both were inhibited by competition with the same amino acids. Kinetic analysis showed that Km decreased by a factor of 2.4 and Vmax increased 1.9-fold in deprived cells. Amino acid-deprivation did not stimulate amino acid uptake through systems other than system A. This suggests that the higher Km in proline-supplemented cells is not due to release of intracellular amino acids into unstirred layers surrounding the cells. The presence of amino acids which are substrates of system A (including AIB) during proline-deprivation, prevented stimulation of proline uptake, whereas those transported by systems Ly⁺ or L exclusively were ineffective. The stimulation of the transport-rate in deprived cells could be reversed by subsequent exposure to proline or other substrates of system A. L6 cells, deprived of proline for 6 hours, retained the stimulation of transport after detachment from the monolayers with trypsin. Uptake rates were comparable in suspended and attached cells in monolayer culture. Thus, amino acid-depreivation of L6 cells results in an adaptive increase in proline uptake, which is not due to unstirred layers but appears to be mediated by other mechanisms of selective transport regulation.     

Characteristics of proline transport into R3230AC mammary tumor cells

Cells separated by enzyme treatment of the R3230AC mammary carcinoma were used to characterize the entry of proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+. Proline was acitvely transported by these tumor cells, reaching a distribution ratio ([proline] intracellular/[proline] extracellular) of 20 after 2 h. Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-isobutyric acid (Ki = 0.34 mM). Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of proline uptake in the absence of Na+ increases linearly with increasing proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-isobutyric acid, 50 mM glycine +20 mM phenylalanine, or 50 mM serine +20 mM phenylalanine when proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that proline transport appears to be restricted to the A (alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the amino acid transport process.     

The importance of amino acids as carbon sources for Micromonospora echinospora (ATCC 15837)

AIMS: This study set out to investigate the effect of amino acids on the uptake of glucose by Micromonospora eichinospora (ATCC 15837). METHODS AND RESULTS: The specific rate of glucose uptake was found to be reduced when organic nitrogen components were present in the medium. Radioactive uptake studies revealed that the Km for glucose in this organism was 53 mm, indicating a low affinity for uptake compared with other actinomycete sugar transport systems. Individual amino acids negatively influenced the rate of glucose transport, suggesting a relationship between amino acid metabolism and glucose uptake in this organism. The sugar transport system was found to be an active process being inhibited by ionophores and KCN. CONCLUSIONS: The data suggest a direct link between amino acid metabolism and glucose uptake at the level of sugar transport. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that the uptake of glucose, a major carbon source for many antibiotic fermentations, is significantly reduced in the presence of amino acids. This fact should inform the medium design and feeding regimes of fermentations involving similar actinomycetes.     

Glucose transport and its inhibition by short-chain n-alkanes in Cladosporium resinae.

Glucose transport in Cladosporium resinae was studies with the aid of the non-metabolizable glucose analogue 3-O-methyl-D-glucose (3-O-MG). 3-O-MG, transported as a free sugar without phosphorylation, was found to inhibit glucose uptake competitively. Conversely, glucose was a competitive inhibitor of 3-O-MG uptake. Moreover, both glucose and 3-O-MG were able to bring about rapid counterflow intracellular 3-O-MG. Thus, glucose and 3-O-MG share the same entry and exit systems. The transport of 3-O-MG is carrier mediated and energy dependent as shown by saturation kinetics, strong temperature dependence, accumulation of unaltered 3-O-MG against a concentration gradient, and inhibition of uptake by NaN3, NaCN, and 2,4-dinitrophenol. The glucose transport system appeared to be constitutive for glucose transport in cells grown on fructose, galactose, mannose, xylose, or glucose. There was no derepressible low-Km glucose transport system in C. resinae. n-Hexane and n-heptane were found to inhibit 3-O-MG uptake rapidly at temperatures above 20 C. Over 50% inhibition of the uptake rate occurred after only 10 min of incubation with n-hexane at 30 C. The percentage of inhibition in the presence of n-hexane, compared to controls in the absence of n-hexane, was found to increase with increasing temperature. Longer-chain n-alkanes (C8 to C18) had no significant effect on uptake. The efflux of intracellular 3-O-MG, which appeared to occur by facilitated diffusion, was not affected by any of the n-alkanes tested including n-hexane.     

Proline Uptake and Utilization by Chlorella pyrenoidosa

Conditions for proline uptake and utilization by Chlorella pyrenoidosa Chick are described. Proline is taken up by growing cultures during late log phase growth after depletion of glucose from the medium. However, proline uptake by stationary phase cultures requires the presence of glucose in the medium. The results are consistent with the interpretation that some carbohydrate is required for proline uptake but proline uptake is inhibited by the accumulation of intracellular carbohydrates.     

Proline porter II is activated by a hyperosmotic shift in both whole cells and membrane vesicles of Escherichia coli K12

Proline porter II is rapidly activated when nongrowing bacteria are subjected to a hyperosmotic shift (Grothe, S., Krogsrud, R. L., McClellan, D. J., Milner, J. L., and Wood, J. M. (1986) J. Bacteriol. 166, 253-259). Proline porter II was active in membrane vesicles prepared from bacteria grown under optimal conditions, nutritional stress, or osmotic stress. That activity was: (i) dependent on the presence of the energy sources phenazine methosulphate plus ascorbate or D-lactate; (ii) observed only when a hyperosmotic shift accompanied the transport measurement; (iii) inhibited by glycine betaine in a manner analogous to that observed in whole cells; and (iv) eliminated by lesions in proP. Membrane vesicles were able to transport serine but not glutamine and serine transport was reduced by the hyperosmotic shift. In whole cells, proline porter II activity was supported by glucose and by D-lactate in a strain defective for proline porters I and III and the F1F0-ATPase. Glucose energized proline uptake was eliminated by carbonyl cyanide m-chlorophenylhydrazone and KCN as was serine uptake. These results suggested that proline porter II was respiration-dependent and probably ion-linked. Activation of proline porter II in whole cells by sucrose or NaCl was sustained over 30 min, whereas activation by glycerol was transient. Proline porter II was activated by NaCl and sucrose with a half-time of approximately 1 min in both whole cells and membrane vesicles. Thus, activation of proline porter II was reversible. It occurred at a rate comparable to that of K+ influx and much more rapid than the genetic regulatory responses that follow a hyperosmotic shift.     

Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose

Proline metabolism has been studied in procyclic form Trypanosoma brucei. These parasites consume six times more proline from the medium when glucose is in limiting supply than when this carbohydrate is present as an abundant energy source. The sensitivity of procyclic T. brucei to oligomycin increases by three orders of magnitude when the parasites are obliged to catabolize proline in medium depleted in glucose. This indicates that oxidative phosphorylation is far more important to energy metabolism in this latter case than when glucose is available and the energy needs of the parasite can be fulfilled by substrate level phosphorylation alone. A gene encoding proline dehydrogenase, the first enzyme of the proline catabolic pathway, was cloned. RNA interference studies revealed the loss of this activity to be conditionally lethal. Proline dehydrogenase defective parasites grew as wild-type when glucose was available, but, unlike wild-type cells, they failed to proliferate using proline. In parasites grown in the presence of glucose, proline dehydrogenase activity was markedly lower than when glucose was absent from the medium. Proline uptake too was shown to be diminished when glucose was abundant in the growth medium. Wild-type cells were sensitive to 2-deoxy-D-glucose if grown using proline as the principal carbon source, but not in glucose-rich medium, indicating that this non-catabolizable glucose analogue might also stimulate repression of proline utilization. These results indicate that the ability of trypanosomes to use proline as an energy source can be regulated depending upon the availability of glucose.     

Isoleucine, a potent plasma glucose-lowering amino acid, stimulates glucose uptake in C2C12 myotubes

To examine which branched-chain amino acids affect the plasma glucose levels, we investigated the effects of leucine, isoleucine, and valine (0.3 g/kg body weight p.o.) in normal rats using the oral glucose tolerance test (OGTT, 2 g/kg). A single oral administration of isoleucine significantly reduced plasma glucose levels 30 and 60 min after the glucose bolus, whereas administration of leucine and valine did not produce a significant decrease. Oral administration of valine significantly enhanced the plasma glucose level at 30 min after the glucose administration and leucine had a similar effect at 120 min. At each measurement timepoint, the insulin levels of the treated groups were lower than that of the control group. We then investigated the effects of leucine, isoleucine or valine at the same concentration (1 mM) on glucose metabolism in C(2)C(12) myotubes in the absence of insulin. Glucose consumption was elevated by 16.8% in the presence of 1 mM isoleucine compared with the control. Conversely, 1 mM leucine or valine caused no significant changes in glucose consumption in the C(2)C(12) myotubes. The 2-deoxyglucose uptake of C(2)C(12) myotubes significantly increased upon exposure to 1-10 mM isoleucine and 5-10 mM leucine. However, isoleucine caused no significant difference in glycogen synthesis in C(2)C(12) myotubes, although leucine and valine caused a significant increase in intracellular glycogen compared with the control. The isoleucine effect on glucose uptake was mediated by phosphatidylinositol 3-kinase (PI3K), but was independent of mammalian target of rapamycin (mTOR). These results suggest that isoleucine stimulates the insulin-independent glucose uptake in skeletal muscle cells, which may contribute to the plasma glucose-lowering effect of isoleucine in normal rats.     

Formation of a complex between valine and intestinal mucosal lipid; its possible role in valine absorption

During intestinal absorption amino acids must traverse the lipid-rich epithelial cell membrane, possibly in a lipid-soluble form. In a search for such a form, we have determined the ability of lipid extracted from intestinal mucosa to bind valine. After incubation in a valine-containing medium this lipid (defined as the heptane-soluble fraction) contained, on the average, 3.63 micromoles of valine per 100 mg of lipid. Cyanide (0.002 m), 2,4-dinitrophenol (0.0002 m), and anaerobic conditions had little effect on this process. Valine uptake into the lipid fraction of mucosa was complete after 2.5 min. Of a number of sugars and amino acids tested, isoleucine, methionine, and leucine were the most potent inhibitors of valine uptake into lipid. The inhibition by leucine appeared to be competitive. A similar uptake of glucose into the mucosal lipid was not inhibited by leucine, methionine, or isoleucine but was inhibited by galactose. Various phosphoglycerides (but not sphingomyelin) from other sources, used in place of mucosal lipid, were able to carry 20-150 times as much valine into heptane-soluble fraction as were other lipid classes. Some characteristics of the complex are similar to those of the valine transport system.     

Characterization of the biotin transport system in Saccharomyces cerevisiae

The characteristics of the biotin transport mechanism of Saccharomyces cerevisiae were investigated in nonproliferating cells. Microbiological and radioisotope assays were employed to measure biotin uptake. The vitamin existed intracellularly in both free and bound forms. Free biotin was extracted by boiling water. Chromatography of the free extract showed it to consist entirely of d-biotin. Cellular bound biotin was released by treating cells with 6 n H(2)SO(4). The rate of biotin uptake was linear with time for 10 min, reaching a maximum at about 20 min followed by a gradual loss of accumulated free vitamin from the cells. Biotin was not degraded or converted to vitamers during uptake. Transport was temperature- and pH-dependent, optimum conditions for uptake being 30 C and pH 4.0. Glucose markedly stimulated biotin transport. In its presence, large intracellular free-biotin concentration gradients were established. Iodoacetate inhibited the glucose stimulation of biotin uptake. The rate of vitamin transport increased in a linear fashion with increasing cell mass. The transport system was saturated with increasing concentrations of the vitamin. The apparent K(m) for uptake was 3.23 x 10(-7)m. Uptake of radioactive biotin was inhibited by unlabeled biotin and a number of analogues including homobiotin, desthiobiotin, oxybiotin, norbiotin, and biotin sulfone. Proline, hydroxyproline, and 7,8-diaminopelargonic acid did not inhibit uptake. Unlabeled biotin and desthiobiotin exchanged with accumulated intracellular (14)C-biotin, whereas hydroxyproline did not.     

Mechanism of regulation of glucose transport in Rhizobium leguminosarum.

Multiple glucose transport systems were distinguished in Rhizobium leguminosarum. We found nonlinear Lineweaver-Burk plots for the uptake of glucose, 2-deoxy-D-glucose, and alpha-methyl-D-glucoside, and this implied the existence of at least two uptake mechanisms. Different patterns of inhibition of 2-deoxy-D-glucose uptake and alpha-methyl-D-glucoside uptake at 0.1 mM by various carbohydrates revealed differences in the stereospecificities of the transport systems. Osmotic shock treatment abolished transport activities, and two independent glucose-binding activities were detected in the supernatants. Induction of glucose transport was repressed strongly by L-malate, even in the presence of excess D-glucose. Rhizobium bacteroids showed no significant glucose uptake activity at different oxygen concentrations. These results suggested that glucose transport is repressed by dicarboxylic acids during R. leguminosarum symbiosis.     

Proline uptake through the major transport system of Salmonella typhimurium is coupled to sodium ions.

Strains of Salmonella typhimurium deficient in one or more of the proline transport systems have been constructed and used to study the mechanism of energy coupling to transport. Proline uptake through the major proline permease (PP-I, putP) is shown to be absolutely coupled to Na+ ions and not to H+ ions as has previously been assumed. Transport through the minor proline permease (PP-II, proP), however, is unaffected by the presence or absence of Na+. The effect of Na+ on the kinetics of proline uptake shows that external Na+ increases the Vmax for transport. It seems probable that proline transport through PP-I is also coupled to Na+ ions in Escherichia coli.     

Characterization of sulfate, proline, and glucose transport systems in anterior cruciate and medial collateral ligament cells

The present study was undertaken to define the nature of key transport processes for sodium, glucose, proline, and sulfate in primary culture of canine anterior cruciate ligament (ACL) and medial collateral ligament (MCL) cells. Uptake studies using radiolabeled isotopes were performed and Na,K-ATPase activity was determined in cell lysates. At 25 degrees C both ACL and MCL cells showed a significant uptake of 86Rb. Ouabain inhibited Rb uptake by 55% in ACL cells and by 60% in MCL cells. The transport activity of Na,K-ATPase in intact cells was calculated to be 57 and 71 nmol.(mg protein)-1.(15 min)-1, respectively. The enzymatic activity of Na,K-ATPase in cell lysates was observed to be 104 for ACL cells and 121 nmol.(mg protein)-1.(15 min)-1 for MCL cells. Cytochalasin B, a known inhibitor of sodium-independent D-glucose transport, completely inhibited D-glucose uptake in ACL and MCL cells. Removal of Na+ or addition of 10-5 mol/L phlorizin, a potent inhibitor of the sodium-D-glucose cotransporter, did not alter D-glucose uptake, suggesting that glucose entered the cells using a sodium-independent pathway. Both ACL and MCL cells exhibited high sulfate uptake that was not altered by replacement of Na+ by N-methyl-D-glucamine, whereas DIDS, an inhibitor of sulfate/anion exchange abolished sulfate uptake in both cell types. Thus, neither cell type seems to possess a sodium-sulfate cotransport system. Rather, sulfate uptake appeared to be mediated by sulfate/anion exchange. Proline was rapidly taken up by ACL and MCL cells and its uptake was reduced by 85% when Na+ was replaced by N-methyl-D-glucamine, indicating that proline entered the cells via sodium-dependent cotransport systems. The data demonstrate that both ACL and MCL cells possess a highly active sodium pump, a secondary active sodium-proline cotransport system, and sodium-independent transport systems for D-glucose and sulfate.     

Characterization of valine transport in sea urchin eggs

In unfertilized eggs, the mechanism of valine uptake can be summarized as follows. It is saturable over the external concentration of valine and insensitive to the presence of external sodium, depletion of cellular energy supplies and intracellular acidosis. The activation energy for the transport reaction (16.3 kcal/mol) is within the range of values reported for active transport of small molecules. In fertilized eggs, the total rate of valine uptake can be divided into two components: (i) a Na⁺-insensitive uptake which accounts for about 7% of total absorption as shown by studies in Na⁺-free medium seems to possess the same characteristics as in unfertilized eggs, (ii) a Na⁺-dependent transport of valine which constitutes the main entry is formed about 5 min after fertilization. It follows Michaelis-Menten kinetics characterized by 15-fold increase in $\text{V}{}_{\max }$ with no change in $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$. These two mechanisms have characteristics in common, such as their insensitivity to metabolic energy supply, their energy of activation and their ability to concentrate valine. The relationship between the establishment of the Na⁺-dependent valine uptake and the ionic events triggered by fertilization is discussed.     

Facilitated glucose and dehydroascorbate transport in plant mitochondria

Ascorbate, dehydroascorbate, and glucose transport was investigated in plant mitochondria and mitoplasts prepared from cultured BY2 tobacco cells. Using a rapid filtration method with radiolabeled ligands, we observed a specific glucose and dehydroascorbate transport, which was temperature and time dependent and saturable. Inhibition of mitochondrial respiration by KCN and the uncoupler 2,4-dinitrophenol did not influence the transport of the investigated compounds. Dehydroascorbate transport was inhibited by glucose and genistein, while glucose uptake was decreased upon 3-O-methyl-glucose, D-mannose, cytochalasin B or genistein addition. On the other hand, a low affinity low capacity ascorbate transport was found. Oxidizing agents (potassium ferricyanide or ascorbate oxidase) increased ascorbate uptake. The results demonstrate the presence of dehydroascorbate and glucose transport in plant mitochondria and suggest that it is mediated by the same or closely related transporter(s).     

Use of a genetic variant to study the hexose transport properties of human skin fibroblasts.

Human skin fibroblasts from 'normal' subjects were found to possess at least two hexose transport systems. One system was responsible for the uptake of 2-deoxy-D-glucose (dGlc), D-glucose and D-galactose, whereas the other was responsible primarily for the uptake of 3-O-methyl-D-glucose (MeGlc). The transport of dGlc was the rate-limiting step in the uptake process; over 97% of the internalized dGlc was phosphorylated and the specific activity of hexokinase was several times higher than that for dGlc transport. The dGlc transport system was activated by glucose starvation, and was very sensitive to inhibition by cytochalasin B and energy uncouplers. Fibroblasts isolated from a patient with symptoms of hypoglycaemia were found to differ from their normal counterparts in the dGlc transport system. They exhibited a much higher transport affinity for dGlc, D-glucose and D-galactose, with no change in the respective transport capacity. Transport was not the rate-limiting step in dGlc uptake by these cells. Moreover, the patient's dGlc transport system was no longer sensitive to inhibition by cytochalasin B and energy uncouplers. This suggested that the intrinsic properties of the patient's dGlc transport system were altered. It should be noted that the patient's dGlc transport system could still be activated by glucose starvation. Despite the changes in the dGlc transport system, the MeGlc transport system in the patient's fibroblasts remained unaltered. The observed difference in the properties of the two hexose transport systems in the 'normal' and the patient's fibroblasts strongly suggests that the two transport systems may be coded or regulated by different genes. The present finding provides the first genetic evidence from naturally occurring fibroblasts indicating the presence of two different hexose transport systems.     

Modulation of glucose uptake in animal cells. Studies using plasma membrane vesicles isolated from nontransformed and simian virus 40-transformed mouse fibroblast cultures.

Plasma membrane vesicles isolated from nontransformed and Simian virus 40-transformed mouse fibroblast cultures catalyzed carrier-mediated D-glucose transport without detectable metabolic conversion to glucose 6-phosphate. Glucose transport activity was stereospecific, temperature-dependent, sensitive to inactivation by p-chloromercuriphenylsulfonate, and accompanied plasma membrane material during subcellular fractionation. D-Glucose efflux from vesicles was inhibited by phloretin, an inhibitor of glucose uptake in intact cells. Cytochalasin B, a potent inhibitor of glucose uptake when tested with the intact cells used for vesicle isolation did not inhibit glucose transport in vesicles despite the presence of high affinity cytochalasin binding sites in isolated membranes. The enhanced glucose uptake observed in intact cells after viral transformation was not expressed in vesicles: no significant differences in glucose transport specific activity could be detected in vesicle preparations from nontransformed and transformed mouse fibroblast cultures. These findings indicate that cellular components distinct from glucose carriers can mediate changes in glucose uptake in mouse fibroblast cultures in at least two cases: sensitivity to inhibition by cytochalasin B and the enhanced cellular sugar uptake observed after viral transformation.     

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

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

myo-Inositol Transport in Mouse Astroglia-Rich Primary Cultures

Uptake of radiolabeled myo-inositol was studied in astroglia-rich primary cultures derived from neonatal mouse brains. The uptake was saturable in the presence of Na+ with a Km of 25 microM and a Vmax of 60 pmol.min-1.(mg protein)-1, suggesting a high-affinity transport system for myo-inositol in astroglial cells. In addition, a Na(+)-independent, nonsaturable component was found. Carrier-mediated uptake was not inhibited by cytochalasin B (50 microM), but was reduced by depolarizing concentrations of K+ and, to different extents, in the presence of phloretin, ouabain, or amiloride (1 mM each). scyllo-Inositol, glucose, and galactose also reduced myo-inositol uptake; inhibition by the two hexoses was not reversed in the presence of 0.4 mM sorbinil. On the other hand, uptake of 2-deoxyglucose was not inhibited by high concentrations of myo-inositol. Preincubation of the cells with glucose-free or inositol-free medium stimulated uptake of myo-inositol and preincubation with 25 mM glucose in the presence of 0.4 mM sorbinil had no effect on the rate of uptake. The results suggest that myo-inositol is taken up into the astroglial cells by a transport mechanism that is distinct from that of glucose and probably is an active one. Sorbitol pathway activity does not interfere with myo-inositol uptake.     

C2C12 细胞胰岛素增敏的机制

目的:观察口服葡萄糖负荷对小鼠小肠组织网膜素基因表达的影响及网膜素对C2C12肌管细胞胰岛素敏感性的影响,并进一步探讨其机制。方法:半定量逆转录聚合酶链反应(RT-PCR)技术检测小鼠小肠组织网膜素mRNA的表达;葡萄糖转运实验观察网膜素对C2C12肌管细胞胰岛素敏感性的影响;Western blot检测Akt(Ser473)的磷酸化水平。结果:口服葡萄糖负荷30min后小鼠小肠组织网膜素基因表达显著减低(P<0.05),而60min后恢复至负荷前水平;葡萄糖转运实验发现:网膜素作用10min对C2C12肌管细胞基础葡萄糖转运无影响,但显著增加了胰岛素刺激的葡萄糖转运(P<0.05);Western Blot发现:网膜素和AICAR均显著增加了C2C12肌管细胞Ak(tSer473)的磷酸化水平(P<0.05)。结论:网膜素作为一种胃肠激素还受到葡萄糖负荷的调节,在C2C12肌管细胞,网膜素可通过增加Akt的磷酸化发挥胰岛素增敏作用。