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1.
We used-escin-permeabilized pig cerebral microvessels (PCMV) to study theorganization of carbohydrate metabolism in the cytoplasm of vascularsmooth muscle (VSM) cells. We have previously demonstrated (Lloyd PGand Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262,1999) that intact PCMV metabolize the glycolytic intermediate[1-13C]fructose 1,6-bisphosphate (FBP) to[1-13C]glucose with negligible production of[3-13C]lactate, while simultaneouslymetabolizing [2-13C]glucose to[2-13C]lactate. Thus gluconeogenic andglycolytic intermediates do not mix freely in intact VSM cells(compartmentation). Permeabilized PCMV retained the ability tometabolize [2-13C]glucose to[2-13C]lactate and to metabolize[1-13C]FBP to[1-13C]glucose. The continued existence ofglycolytic and gluconeogenic activity in permeabilized cells suggeststhat the intermediates of these pathways are channeled (directlytransferred) between enzymes. Both glycolytic and gluconeogenic flux inpermeabilized PCMV were sensitive to the presence of exogenous ATP andNAD. It was most interesting that a major product of[1-13C]FBP metabolism in permeabilized PCMV was[3-13C]lactate, in direct contrast to ourprevious findings in intact PCMV. Thus disruption of the plasmamembrane altered the distribution of substrates between the glycolyticand gluconeogenic pathways. These data suggest that organization of theplasma membrane into distinct microdomains plays an important role insorting intermediates between the glycolytic and gluconeogenic pathwaysin intact cells.

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2.
To determine whether channeling of glycolytic intermediates canoccur in vascular smooth muscle (VSM), we permeabilized freshly isolated VSM cells from hog carotid arteries with dextran sulfate. Thedextran sulfate-treated cells did not exclude trypan blue, a dye withmolecular weight of ~1,000. If glycolytic intermediates freelydiffuse, plasmalemmal permeabilization would allow intermediates toexit the cell and glycolytic flux should cease. We incubated permeabilized and nonpermeabilized cells with 5 mM[1-13C]glucose at37°C for 3 h. 13C nuclearmagnetic resonance (NMR) was used to determine relative [3-13C]lactateproduction and to identify any13C-labeled glycolyticintermediates that exited from the permeabilized cells.[3-13C]lactateproduction from[1-13C]glucose wasdecreased by an average of 32% (n = 6) in permeabilized cells compared with intact cells. No13C-labeled glycolyticintermediates were observed in the bathing solution of permeabilizedcells. We conclude that channeling of glycolytic intermediates canoccur in VSM cells.

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3.
We have previously found that glycolysis and gluconeogenesis occur in separate "compartments" of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [1999] Am J Physiol Cell Physiol. 277:C1250-C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [2000] Am J Physiol Cell Physiol. 278:C803-C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane-associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non-caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with beta methyl-cyclodextrin (CD) and examined the metabolism of [2-(13)C]glucose (a glycolytic substrate) and [1-(13)C]fructose 1,6-bisphosphate (FBP, a gluconeogenic substrate in PCMV) using (13)C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2-(13)C]glucose to [2-(13)C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1-(13)C]FBP to [3-(13)C]lactate was increased by CD treatment. Alternatively, the increased [3-(13)C]lactate production may reflect changes in FBP uptake, since conversion of [1-(13)C]FBP to [3-(13)C]glucose was also elevated in CD-treated cells. Thus, a link between caveolar organization and metabolic organization may exist.  相似文献   

4.
Péronnet, F., Y. Burelle, D. Massicotte, C. Lavoie,and C. Hillaire-Marcel. Respective oxidation of13C-labeled lactate and glucoseingested simultaneously during exercise. J. Appl.Physiol. 82(2): 440-446, 1997.The purpose ofthis experiment was to measure, by using13C labeling, the oxidation rateof exogenous lactate (25 g, as Na+,K+,Ca2+, andMg2+ salts) and glucose (75 g)ingested simultaneously (in 1,000 ml of water) during prolongedexercise (120 min, 65 ± 3% maximum oxygen uptake in 6 male subjects). The percentage of exogenous glucose and lactateoxidized were similar (48 ± 3 vs. 45 ± 5%, respectively). However, because of the small amount of oral lactate that could be tolerated without gastrointestinal discomfort, the amountof exogenous lactate oxidized was much smaller than that of exogenousglucose (11.1 ± 0.5 vs. 36.3 ± 1.3 g, respectively) andcontributed to only 2.6 ± 0.4% of the energy yield(vs. 8.4 ± 1.9% for exogenous glucose). The cumulative amount ofexogenous glucose and lactate oxidized was similar to that observedwhen 100 g of[13C]glucose wereingested (47.3 ± 1.8 vs. 50.9 ± 1.2 g, respectively). When[13C]glucose wasingested, changes in the plasma glucose13C/12Cratio indicated that between 39 and 61% of plasma glucose derived fromexogenous glucose. On the other hand, the plasma glucose 13C/12Cratio remained unchanged when[13C]lactate wasingested, suggesting no prior conversion into glucose before oxidation.

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5.
The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measumed CO2 and lactate production from [6-14C]glucose, [1-14C]glucose, and [U-14C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO2 production by glycolysis, but increased CO2 production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO2. Lactate production from [1-, and 6-14C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-14C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism.  相似文献   

6.
Glycogenolysis and gluconeogenesis are sensitive to nutritional state, and the net direction of flux is controlled by multiple enzymatic steps. This delicate balance in the liver is disrupted by a variety of pathological states including cancer and diabetes mellitus. Hyperpolarized carbon-13 magnetic resonance is a new metabolic imaging technique that can probe intermediary metabolism nondestructively. There are currently no methods to rapidly distinguish livers in a gluconeogenic from glycogenolytic state. Here we use the gluconeogenic precursor dihydroxyacetone (DHA) to deliver hyperpolarized carbon-13 to the perfused mouse liver. DHA enters gluconeogenesis at the level of the trioses. Perfusion conditions were designed to establish either a gluconeogenic or a glycogenolytic state. Unexpectedly, we found that [2-13C]DHA was metabolized within a few seconds to the common intermediates and end products of both glycolysis and gluconeogenesis under both conditions, including [2,5-13C]glucose, [2-13C]glycerol 3-phosphate, [2-13C]phosphoenolpyruvate (PEP), [2-13C]pyruvate, [2-13C]alanine, and [2-13C]lactate. [2-13C]Phosphoenolpyruvate, a key branch point in gluconeogenesis and glycolysis, was monitored in functioning tissue for the first time. Observation of [2-13C]PEP was not anticipated as the free energy difference between PEP and pyruvate is large. Pyruvate kinase is the only regulatory step of the common glycolytic-gluconeogenic pathway that appears to exert significant control over the kinetics of any metabolites of DHA. A ratio of glycolytic to gluconeogenic products distinguished the gluconeogenic from glycogenolytic state in these functioning livers.  相似文献   

7.
Intramuscular fatty acid metabolism evaluated with stable isotopic tracers   总被引:1,自引:0,他引:1  
We evaluated the applicability of stableisotopic tracers to the study of intramuscular fatty acid metabolism byinfusing both[U-13C]palmitateand [1-13C]oleateintravenously for 4 h into fasted conscious rats. Skeletal muscles weresequentially biopsied, and the concentration and 13C enrichment of fatty acids weremeasured by gas chromatography/combustion/isotope ratio massspectrometry. Throughout the study, the13C enrichment of plasma palmitateand oleate remained substantially greater than intramuscularnonesterified palmitate and oleate enrichment, which in turn wasgreater than intramuscular triglyceride palmitate and oleateenrichment. Fractional synthesis rates of intramuscular triglyceridesin gastrocnemius and soleus were 0.267 ± 0.075 and 0.100 ± 0.030/h (P = 0.04), respectively, asdetermined by using[U-13C]palmitate, andwere 0.278 ± 0.049 and 0.075 ± 0.013/h(P = 0.02), respectively, by using[1-13C]oleate. Weconclude that plasma free fatty acids are a source for intramusculartriglycerides and nonesterified fatty acids; the latter are likely thesynthetic precursors of the former. Uniformly and singly labeled[13C]fatty acidtracers will provide an important tool to study intramuscular fattyacid and triglyceride metabolism.

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8.
A novel inhibitor of lactate transport, AR-C122982, was used to study the effect of inhibiting the monocarboxylate transporters MCT1 and MCT2 on cortical brain slice metabolism. We studied metabolism of l-[3-13C]lactate, and d-[1-13C]glucose under a range of conditions. Experiments using l-[3-13C]lactate showed that the inhibitor AR-C122982 altered exchange of lactate. Under depolarizing conditions, net flux of label from d-[1-13C]glucose was barely altered by 10 or 100 nM AR-C122982. In the presence of AMPA or glutamate there were increases in net flux of label and metabolic pool sizes. These data suggest lactate may supply compartments in the brain not usually directly accessed by glucose. In general, it would appear that movement of lactate between cell types is not essential for metabolic activity, with the heavy metabolic workloads imposed being unaffected by inhibition of MCT1 and MCT2. Further experiments investigating the mechanism of operation of AR-C122982 are necessary to corroborate this finding.  相似文献   

9.
The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-13C]glucose and [2-13C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the 13C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-13C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.  相似文献   

10.
Abstract: Cerebral pentose phosphate pathway (PPP) activity has been linked to NADPH-dependent anabolic pathways, turnover of neurotransmitters, and protection from oxidative stress. Research on this potentially important pathway has been hampered, however, because measurement of regional cerebral PPP activity in vivo has not been possible. Our efforts to address this need focused on the use of a novel isotopically substituted glucose molecule, [1,6-13C2,6,6-2H2]glucose, in conjunction with microdialysis techniques, to measure cerebral PPP activity in vivo, in freely moving rats. Metabolism of [1,6-13C2,6,6-2H2]glucose through glycolysis produces [3-13C]lactate and [3-13C,3,3-2H2]lactate, whereas metabolism through the PPP produces [3-13C,3,3-2H2]lactate and unlabeled lactate. The ratios of these lactate isotopomers can be quantified using gas chromatography/mass spectrometry (GC/MS) for calculation of PPP activity, which is reported as the percentage of glucose metabolized to lactate that passed through the PPP. Following addition of [1,6-13C2,6,6-2H2]glucose to the perfusate, labeled lactate was easily detectable in dialysate using GC/MS. Basal forebrain and intracerebral 9L glioma PPP values (mean ± SD) were 3.5 ± 0.4 (n = 4) and 6.2 ± 0.9% (n = 4), respectively. Furthermore, PPP activity could be stimulated in vivo by addition of phenazine methosulfate, an artificial electron acceptor for NADPH, to the perfusion stream. These results show that the activity of the PPP can now be measured dynamically and regionally in the brains of conscious animals in vivo.  相似文献   

11.
Bertocci, Loren A., John G. Jones, Craig R. Malloy, RonaldG. Victor, and Gail D. Thomas. Oxidation of lactateand acetate in rat skeletal muscle: analysis by13C-nuclear magnetic resonancespectroscopy. J. Appl. Physiol. 83(1): 32-39, 1997.The balance between carbohydrate and fatty acidutilization in skeletal muscle previously has been studied in vivo byusing a variety of methods such as arteriovenous concentrationdifferences and radioactive isotope tracer techniques. However, thesemethodologies provide only indirect estimates of substrate oxidation.We used 13C-nuclear magneticresonance (NMR) spectroscopy and non-steady-state isotopomer analysisto directly quantify the relative oxidation of two competing exogenoussubstrates in rat skeletal muscles. We infused[1,2-13C]acetate and[3-13C]lactateintravenously in anesthetized rats during the final 30 min of 35 (n = 10) or 95 (n = 10) min of intense, unilateral, rhythmic hindlimb contractions.13C-NMR spectroscopy andisotopomer analysis were performed on extracts of gastrocnemius andsoleus muscles from both the contracting and contralateralresting hindlimbs. We found that1)[13C]lactate and[13C]acetate were taken up and oxidized by both restingand contracting skeletal muscles; and2) high-intensity musclecontractions altered the pattern of substrate utilization such that therelative oxidation of acetate decreased while that of lactate remainedunchanged or increased. Based on these findings, we propose that13C-NMR spectroscopy incombination with isotopomer analysis can be used to study the generaldynamics of substrate competition between carbohydrates and fats in ratskeletal muscle.

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12.
Cellular pyruvate is an essential metabolite at the crossroads of glycolysis and oxidative phosphorylation, capable of supporting fermentative glycolysis by reduction to lactate mediated by lactate dehydrogenase (LDH) among other functions. Several inherited diseases of mitochondrial metabolism impact extracellular (plasma) pyruvate concentrations, and [1-13C]pyruvate infusion is used in isotope-labeled metabolic tracing studies, including hyperpolarized magnetic resonance spectroscopic imaging. However, how these extracellular pyruvate sources impact intracellular metabolism is not clear. Herein, we examined the effects of excess exogenous pyruvate on intracellular LDH activity, extracellular acidification rates (ECARs) as a measure of lactate production, and hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates across a panel of tumor and normal cells. Combined LDH activity and LDHB/LDHA expression analysis intimated various heterotetrameric isoforms comprising LDHA and LDHB in tumor cells, not only canonical LDHA. Millimolar concentrations of exogenous pyruvate induced substrate inhibition of LDH activity in both enzymatic assays ex vivo and in live cells, abrogated glycolytic ECAR, and inhibited hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in cellulo. Of importance, the extent of exogenous pyruvate-induced inhibition of LDH and glycolytic ECAR in live cells was highly dependent on pyruvate influx, functionally mediated by monocarboxylate transporter-1 localized to the plasma membrane. These data provided evidence that highly concentrated bolus injections of pyruvate in vivo may transiently inhibit LDH activity in a tissue type- and monocarboxylate transporter-1–dependent manner. Maintaining plasma pyruvate at submillimolar concentrations could potentially minimize transient metabolic perturbations, improve pyruvate therapy, and enhance quantification of metabolic studies, including hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging and stable isotope tracer experiments.  相似文献   

13.
The proplastid fraction containing no cytosol and mitochondrionwas isolated from developing castor bean endosperm by stepwisesucrose density centrifugation. This fraction possesses thecapacity to synthesize LFAs from [u-14C]sucrose, [u-14C]-glucose,[u-14C]G-1-P, [u-14C]G-6-P, [2-14C]pyruvate and [1-14C]acetate.Little was incorporated from [1-14C]pyruvate into LFAs, butmuch into 14COa. Addition of cytosol to the proplastid fractiondid not enhance the LFA synthesis. From these data, the wholepath from sucrose to LFAs through glycolytic path and pyruvatedecarboxylation seems to be located within the proplastid indeveloping castor bean endosperm. The difference in utilizationof substrates indicates that the rate of LFA synthesis in castorbean proplastids is limited at a step between sucrose and hexosephosphate. In addition, experiments with CO2 output and LFAsynthesis from [1-14C]glucose, [6-14C]glucose and [u-14C]G-6-Pstrongly suggest that the path flow branches actively throughG-6-P to the pentose phosphate path and little through acetylCoAto the TCA cycle. (Received May 12, 1975; )  相似文献   

14.
The Metabolism of Abscisic Acid   总被引:7,自引:2,他引:5  
The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-14C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-14D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-14C]trans-abscisic acid in an equimolar mixturewith (±)-[2-14C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-14C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-14C]-, and (–)-[2-14C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-l4C]ABA.  相似文献   

15.
The purpose of this study was to compare the oxidation of[13C]glucose (100 g)ingested at rest or during exercise in six trained (TS) and sixsedentary (SS) male subjects. The oxidation of plasma glucose was alsocomputed from the volume of13CO2and13C/12Cin plasma glucose to compute the oxidation rate of glucose released from the liver and from glycogen stores in periphery (mainly muscle glycogen stores during exercise). At rest, oxidative disposal of bothexogenous (8.3 ± 0.3 vs. 6.6 ± 0.8 g/h) and liver glucose (4.4 ± 0.5 vs. 2.6 ± 0.4 g/h) was higher in TS than in SS.This could contribute to the better glucose tolerance observed at rest in TS. During exercise, for the same absolute workload [140 ± 5 W: TS = 47 ± 2.5; SS = 68 ± 3 %maximal oxygen uptake(O2 max)], [13C]glucose oxidationwas higher in TS than in SS (39.0 ± 2.6 vs. 33.6 ± 1.2 g/h),whereas both liver glucose (16.8 ± 2.4 vs. 24.0 ± 1.8 g/h) and muscle glycogen oxidation (36.0 ± 3.0 vs. 51.0 ± 5.4 g/h) were lower. For the same relative workload (68 ± 3% O2 max:TS = 3.13 ± 0.96; SS = 2.34 ± 0.60 lO2/min), exogenous glucose(44.4 ± 1.8 vs. 33.6 ± 1.2 g/h) and muscle glycogen oxidation (73.8 ± 7.2 vs. 51.0 ± 5.4 g/h) were higher in TS. However,despite a higher energy expenditure in TS, liver glucose oxidation was similar in both groups (22.2 ± 3.0 vs. 24.0 ± 1.8 g/h). Thus exogenous glucose oxidation was selectively favored in TSduring exercise, reducing both liver glucose and muscle glycogen oxidation.

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16.
After exposure to [U-13C3]glycerol, the liver produces primarily [1,2,3-13C3]- and [4,5,6-13C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other 13C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex 13C-labeling patterns in exported glucose. Here, we investigated 13C labeling in plasma glucose in rats given [U-13C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a 13C-13C bond within the trioses contributing to glucose, the appearance of [1,2-13C2]-, [2,3-13C2]-, [5,6-13C2]-, and [4,5-13C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-13C2]glucose/[2,3-13C2]glucose was significantly greater than [5,6-13C2]glucose/[4,5-13C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by 13C distribution in blood glucose after [U-13C3]glycerol administration.  相似文献   

17.
Immune cell functions can be evaluated in vivo by measuring their specific protein fractional synthesis rates (FSR). Using stable isotope dilution techniques, we describe a new method allowing simultaneous in vivo assessment of FSR in two leukocyte populations in healthy human subjects, using small blood samples. Peripheral blood mononuclear cell (PBMC) and polymorphonuclear neutrophil (PMN) FSR were measured during primed continuous intravenous infusion of L-[1-13C]leucine. Immune cells from 6 ml of whole blood were isolated by density gradient centrifugation. In a first study, we calculated the FSR using plasma [13C]leucine or -[13C]ketoisocaproate (KIC) enrichments as precursor pools. In a second study, we compared protein FSR in leukocytes, using enrichments of either intracellular or plasma free [13C]leucine as immediate precursor pools. The present approach showed a steady-state enrichment of plasma and circulating immune cell free [13C]leucine precursor pools. The linearity of labeled amino acid incorporation rate within mixed PBMC and PMN proteins also was verified. Postabsorptive protein FSR was 4.09 ± 0.39%/day in PBMC and 1.44 ± 0.08%/day in PMN when plasma [13C]KIC was the precursor pool. The difference between PBMC and PMN FSR was statistically significant, whatever the precursor pool used, suggesting large differences in their synthetic activities and functions. Use of the plasma [13C]KIC pool led to an underestimation of leukocyte FSR compared with the intracellular pool (PBMC: 6.04 ± 0.94%/day; PMN: 2.98 ± 0.30%/day). Hence, the intracellular free amino acid pool must be used as precursor to obtain reliable results. In conclusion, it is possible to assess immune cell metabolism in vivo in humans by using small blood samples to directly indicate their metabolic activity in various clinical situations and in response to regulating factors. peripheral blood mononuclear cells; polymorphonuclear neutrophils; protein metabolism; stable isotopes; leucine  相似文献   

18.
The present study compared the microdialysis ethanoloutflow-inflow technique for estimating blood flow (BF) in skeletalmuscle of humans with measurements by Doppler ultrasound of femoralartery inflow to the limb(BFFA). The microdialysis probeswere inserted in the vastus lateralis muscle and perfused with a Ringeracetate solution containing ethanol,[2-3H]adenosine (Ado),andD-[14C(U)]glucose.BFFA at rest increased from0.16 ± 0.02 to 1.80 ± 0.26 and 4.86 ± 0.53 l/minwith femoral artery infusion of Ado (AdoFA,i) at 125 and 1,000 µg · min1 · l1thigh volume (low dose and high dose, respectively;P < 0.05) and to 3.79 ± 0.37 and6.13 ± 0.65 l/min during one-legged, dynamic, thigh muscle exercisewithout and with high AdoFA,i,respectively (P < 0.05). The ethanoloutflow-to-inflow ratio (38.3 ± 2.3%) and the probe recoveries(PR) for [2-3H]Ado(35.4 ± 1.6%) and forD-[14C(U)]glucose(15.9 ± 1.1%) did not change withAdoFA,i at rest (P = not significant). During exercisewithout and with AdoFA,i, theethanol outflow-to-inflow ratio decreased(P < 0.05) to a similar level of17.5 ± 3.4 and 20.6 ± 3.2%, respectively(P = not significant), respectively,while the PR increased (P < 0.05) toa similar level (P = not significant)of 55.8 ± 2.8 and 61.2 ± 2.5% for[2-3H]Ado and to 42.8 ± 3.9 and 45.2 ± 5.1% forD-[14C(U)]glucose.Whereas the ethanol outflow-to-inflow ratio and PR correlated inverselyand positively, respectively, to the changes in BF during muscularcontractions, neither of the ratio nor PR correlated tothe AdoFA,i-induced BF increase.Thus the ethanol outflow-to-inflow ratio does not represent skeletalmuscle BF but rather contraction-induced changes in molecular transport in the interstitium or over the microdialysis membrane.

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19.
The hypothesisof this investigation was that insulin and muscle contraction, byincreasing the rate of skeletal muscle glucose transport, would biascontrol so that glucose delivery to the sarcolemma (and t tubule) andphosphorylation of glucose intracellularly would exert more influenceover glucose uptake. Because of the substantial increases in blood flow(and hence glucose delivery) that accompany exercise, we predicted thatglucose phosphorylation would become more rate determining duringexercise. The transsarcolemmal glucose gradient (TSGG; the glucoseconcentration difference across the membrane) is inversely related tothe degree to which glucose transport determines the rate of glucoseuptake. The TSGG was determined by using isotopic methods in consciousrats during euglycemic hyperinsulinemia [Ins; 20 mU/(kg · min); n = 7], during treadmill exercise (Ex,n = 6), and in sedentary,saline-infused rats (Bas, n = 13).Rats received primed, constant intravenous infusions of trace3-O-[3H]methyl-D-glucoseand [U-14C]mannitol.Then2-deoxy-[3H]glucosewas infused for the calculation of a glucose metabolic index(Rg). At the end of experiments,rats were anesthetized, and soleus muscles were excised. Total soleusglucose concentration and the steady-state ratio of intracellular toextracellular3-O-[3H]methyl-D-glucose(which distributes on the basis of the TSGG) were used to calculateranges of possible glucose concentrations ([G]) at theinner and outer sarcolemmal surfaces([G]im and[G]om, respectively).Soleus Rg was increased in Ins andfurther increased in Ex. In Ins, total soleus glucose,[G]om, and the TSGGwere decreased compared with Bas, while[G]im remained near 0. In Ex, total soleus glucose and[G]im were increasedcompared with Bas, and there was not a decrease in[G]om as was observedin Ins. In addition, accumulation of intracellular free2-deoxy-[3H]glucoseoccurred in soleus in both Ex and Ins. Taken together, these dataindicate that, in Ex, glucose phosphorylation becomes an importantlimitation to soleus glucose uptake. In Ins, both glucose delivery andglucose phosphorylation influence the rate of soleus glucose uptakemore than under basal conditions.

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20.
To study effects of the portal-arterial glucose difference on the hepatic glycogenesis, the liver was isolated from fasted rats and was bivascularly perfused. Thirty-five milliliters of Krebs-Ringer buffer (pH 7.4) with 2 mM glucose, 3 mM lactate, 20 ng/ml insulin, and [1-14C]glucose or [U-14C]lactate was recirculated at flow rates of 14 ml/min via the portal vein and 7 ml/min via the hepatic artery. Glucose was continuously infused at a rate of 27.75 μmol/min into the portal (P experiment) and the arterial cannula (A experiment), and the portal-arterial glucose gradients were + 1.98 and −3.96 mM. Perfusate glucose concentration was not different between the P and A experiments within 20 min. Perfusate lactate level was higher in the P experiment than in the A experiment at 20 min. Incorporation of radioactivity from [14C]glucosc into glycogen was higher in the P experiment than in the A experiment (0.245 ± 0.014%/20 min vs 0.175 ± 0.022%/20 min, P < 0.01), and not influenced by the addition of insulin. Incorporation of 14C from [14C]lactate into glycogen was not different between the P and A experiments, and was significantly increased with the addition of insulin. This activity, in the presence of insulin, was higher in the P experiment than in the A experiment (0.490 ± 0,028%/20 min vs 0.406 ± 0.025%/20 min, P < 0.05). These results suggest that the portal-arterial glucose difference has an important role in the regulation of hepatic glycogenesis from exogenous glucose and gluconeogenesis.  相似文献   

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