Biomedical subjects

M Beylot

Publications and source records attributed to M Beylot.

At least 37 records · Page 2Linked to original sources

PubMed1997

Use of labeling pattern of liver glutamate to calculate rates of citric acid cycle and gluconeogenesis.

The use of the labeling pattern of hepatic glutamate during infusion of L-[3-13C]- or [3-14C]lactate to calculate rates of citric acid cycle activity and gluconeogenesis has been proposed. We tested the validity of this approach by perfusing isolated rat livers (48 h starved) with pyruvate and lactate (10% enriched with [3-13C]lactate) without (control) or with infusion of glucagon (to inhibit pyruvate kinase), mercaptopicolinate (to inhibit phosphoenolpyruvate carboxykinase), or dichloroacetate (to stimulate pyruvate dehydrogenase). Compared with control experiments, glucagon increased glucose output (P < 0.05) and decreased the calculated flux through pyruvate kinase (P < 0.05). Mercaptopicolinate almost totally suppressed glucose production and dramatically reduced the calculated gluconeogenic rate and flux through phosphoenolpyruvate carboxykinase (P < 0.001). Dichloroacetate moderately increased the calculated flux through pyruvate dehydrogenase (P < 0.05). In experiments with perfused livers from fed rats, the calculated gluconeogenic rate and flux through phosphoenolpyruvate carboxykinase were very low compared with control experiments (P < 0.001), whereas the pyruvate dehydrogenase flux was increased (P < 0.05). Therefore, the expected modifications of the citric acid cycle activity and gluconeogenic rate were clearly detected using the labeling pattern of glutamate to calculate these metabolic rates. Except for the perfusions with mercaptopicolinate, the dilution by isotopic exchange in the oxaloacetate pool calculated from the model agreed with the actual dilution of enrichment between liver pyruvate and phosphoenolpyruvate. The present results support the validity of this approach to trace liver metabolism.

Animals↗

PubMed1997

In vivo studies of intrahepatic metabolic pathways.

In vivo studies of liver metabolism have long been limited to measurement by the balance technique or isotope dilution method of the amounts of substrates taken up or produced by the liver. New methods, based mainly on the use of stable isotopes, now allow important data to be obtained on intrahepatic metabolic pathways. Nuclear magnetic resonance and chemical biopsy of glucuronic acid by acetaminophen facilitate the study of glycogen metabolism. Chemical biopsies of liver glutamine by phenylacetate and of cytosolic acetylCoA by sulfamethoxazole provide important data respectively on Krebs cycle activity and gluconeogenesis and on lipogenesis and cholesterol synthesis. Mass isotopomer distribution analysis of molecules synthesised during infusion of 13C-labelled precursors allows an estimation of in vivo gluconeogenesis as well as cholesterol synthesis and lipogenesis. Finally, these metabolic pathways can be studied through the incorporation of deuterium from deuterated water in glucose, fatty acids and cholesterol. All these non-invasive techniques allow investigations to be undertaken in human beings to study the nutritional and hormonal regulation of liver metabolism in normal subjects and in pathological situations.

Biopsy↗

PubMed1996

Assay of the deuterium enrichment of water via acetylene.

A technique is presented for measuring the 2H enrichment of water in biological samples when this enrichment is greater than 0.2%. The sample is reacted with calcium carbide to form acetylene gas, which is determined by gas chromatography electron impact ionization mass spectrometry. Ion-molecule reactions, resulting in proton abstraction, are minimized by lowering the electron ionization energy from the usual 70 eV to 45 eV. This technique is much more rapid and economical than the classical isotope ratio mass spectrometric assay of the enrichment of hydrogen gas derived from reduction of water.

Acetylene↗

PubMed1996

Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH4, enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. Therefore, to assay the enrichment of [2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [U-13C3]lactate or [U-13C3]glycerol. There is a 78% isotopic equilibration of 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.

Animals↗

PubMed1996

In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.

Fractional lipid synthesis can be measured using the incorporation of deuterium from deuterated water. The calculations require knowledge of the maximum incorporation number (N) of deuterium atoms in the molecules synthesized. For both tissue palmitate and cholesterol, N values have been found to be higher during in vivo versus in vitro experiments. We determined the N values to be used for measuring the fractional synthesis of plasma cholesterol and of palmitate triglycerides (TG). Rats were given drinking water enriched (7% to 10%) with deuterated water, and N was determined from the mass isotopomer distributions of plasma cholesterol and plasma TG palmitate and the deuterium enrichment of plasma water. We found N to be 21 for palmitate and 27 for cholesterol. These values agree with those reported for tissue palmitate and cholesterol in vivo, and are higher than values found in vitro. We also found large deuterium enrichments in plasma glucose and in liver lactate and pyruvate. We suggest that, compared with in vitro studies, in vivo metabolism of these compounds leads to an additional pathway of incorporation of deuterium into lipids through deuterium-labeled acetyl coenzyme A (CoA). This could explain why N values are higher in vivo than in vitro.

Acetyl Coenzyme A↗

PubMed1996

Measuring glycerol turnover, gluconeogenesis from glycerol, and total gluconeogenesis with [2-13C] glycerol: role of the infusion-sampling mode.

Mass isotopomer distribution analysis (MIDA) of glucose during infusion of [2-13C]glycerol is a new method for measuring total gluconeogenesis (GNG). Since this method relies on calculation of the isotopic enrichment (IE) of hepatic triose phosphates (TP), the results should be independent of the sites of tracer infusion and blood sampling. Postabsorptive and starved rats were infused with [2-13C]glycerol and sampled either in the arterial-venous (A-V) or venous-arterial (V-A) modes. Blood was also sampled from the portal vein. In both postabsorptive and starved rats, glycerol turnover rate (Rt) and the percent contribution of glycerol to total glucose production were higher in the A-V mode than in the V-A mode (P < .05). Glycerol IE in portal venous blood was intermediate between IE values observed in peripheral arterial and venous blood. Its use for calculating the contribution of glycerol to glucose production reconciled the results obtained with the two infusion-sampling modes in both postabsorptive and starved rats; this contribution was increased by starvation (P < .01). In postabsorptive rats, total GNG calculated from MIDA of glucose accounted for approximately 50% of glucose production whatever the infusion-sampling mode (A-V, 48.8% +/- 4.7%; V-A, 52.2% +/- 3.9%). This contribution increased to 90% in starved rats, again, with no difference between A-V (95.2% +/- 1.8%) and V-A (89.2% +/- 1.3%) modes. In conclusion, during infusion of [2-13C]glycerol, total GNG measured from MIDA of glucose is independent of the infusion-sampling mode, contrary to calculations of Rt and GNG from glycerol. Measurement of glycerol IE in portal venous blood reconciles the results obtained with the two modes with respect to the contribution of glycerol to GNG.

Animals↗

PubMed1996

Measurement of post-absorptive glucose kinetics in non-insulin-dependent diabetic patients: methodological aspects.

Post-absorptive glucose metabolism was studied in non-insulin-dependent diabetes mellitus (NIDDM) patients and normal subjects using dideuterated glucose as tracer. From the progressive fall in blood glucose levels and the increase in isotopic enrichment, the post-absorptive situation could not be regarded as a steady state for glucose metabolism, and non-steady-state approximations had therefore to be applied. However, this did not alter significantly the results in the 10 NIDDM patients studied. Significantly higher values of endogenous glucose production (EGP) were obtained (178 center dot 1 +/- 24 center dot 0 mg m-2 min-1 vs. 80 center dot 2 +/- 14 center dot 4; P < 0 center dot 01) if the tracer priming dose was not adapted to the degree of hyperglycaemia. Valid measurements could be made after only 1 h isotopic equilibration time if an appropriately matched priming dose was employed. Methodologically acceptable values for EGP in the 10 NIDDM patients did not differ significantly from those of 10 normal control subjects (80 center dot 2 +/- 14 center dot 4 mg m-2 min-1 vs. 85 center dot 6 +/- 3 center dot 9; not significant). The post-absorptive hyperglycaemia in these patients was assumed to stem essentially from a defect in peripheral glucose uptake.

Absorption↗

PubMed1996

New methods for in vivo studies of hepatic metabolism.

In vivo studies of liver metabolism have been limited for a long time to measurements, by the balance technique or the isotope dilution method, of the amounts of substrates taken up or produced by liver. New methods have been developed that now permit us to obtain important information on intrahepatic metabolic pathways. Nuclear magnetic resonance permits noninvasive studies of liver glycogen synthesis and breakdown. Chemical biopsy of glucuronic acid by acetaminophen also permits the study of glycogen synthesis whereas chemical biopsies of liver glutamine by phenylacetate and of cytosolic acetyl-CoA by sulfamethoxazole give important information concerning, respectively, Krebs cycle activity and glconeogenesis and on lipogenesis and cholesterol synthesis. Mass isotopomer distribution analysis of molecules synthesized during the infusion of a deuterium of 13C-labeled precursor permits the estimation of in vivo gluconeogenesis as well as cholesterol synthesis and lipogenesis. Finally, these metabolic pathways can be studied through the incorporation of deuterium from deuterated water in glucose, fatty acids and cholesterol. All these noninvasive techniques will allow investigations to be undertaken in humans, addressing the nutritional and hormonal regulation of liver metabolism in normal subjects and in pathological situations.

Biopsy↗

PubMed1996

Metabolic fate of an oral long-chain triglyceride load in humans.

To determine the steps involved in the metabolism of ingested triglycerides (TG), 10 healthy women were studied during 6 h after ingestion of 30 g olive oil labeled with [1,1,1-13C3] triolein. The appearance of 13C was followed in chylomicron-TG (CM-TG), nonesterified fatty acid (NEFA), very low-density lipoprotein (VLDL)-TG, and in expired gas. Indirect calorimetry was used to determine total lipid oxidation. After 90 min, labeling was higher in CM-TG than in NEFA or VLDL. At 180 min, a plateau of enrichment was obtained for CM-TG and NEFA, demonstrating the entry of exogenous lipids in the NEFA pool. After 300 min, a plateau was observed for VLDL-TG with levels of enrichment (0.38 +/- 0.04%) similar to those observed for NEFA (0.36 +/- 0.03%), suggesting a precursor-product relationship. Only 19 +/- 2% of the load was oxidized. From 300 to 360 min, 70% of total lipid oxidation was from exogenous TG. We conclude that, after ingestion of a lipid load, a cycle of fatty acids-TG occurs from CM to NEFA and from NEFA to VLDL. Furthermore, this lipid load has a sparing effect on endogenous lipid stores.

Adult↗

PubMed1996

Noninvasive probing of citric acid cycle intermediates in primate liver with phenylacetylglutamine.

In human and primate liver, phenylacetate and glutamine form phenylacetylglutamine, which is excreted in urine. Probing noninvasively the labeling pattern of liver citric acid cycle intermediates with phenylacetylglutamine assumes that the labeling pattern of its glutamine moiety reflects that of liver alpha-ketoglutarate. To validate this probe, we infused monkeys with [U-13C3]lactate, [3-13C]lactate, [1, 2-13C2]acetate, [2-13C]acetate, [U-13C3]glycerol, or 2-[3-13C]ketoisocaproate and compared the labeling patterns of urinary phenylacetyl-glutamine with those of glutamate and glutamine in liver, plasma, muscle, and kidney and liver alpha-ketoglutarate. Only with [U-13C3]lactate or [3-13C]lactate does the labeling pattern of phenylacetylglutamine reflect patterns of liver alpha-ketoglutarate and glutamate. With [13C]acetate, muscle and kidney glutamate are more labeled than liver metabolites. This confirms that with [13C]acetate, the labeling pattern of liver metabolites is influenced by 13CO2 and [13C]glutamine made in peripheral tissues. Our data validate the use of phenylacetylglutamine labeled from [3-13C]lactate or [3-13C]pyruvate to probe noninvasively the pyruvate carboxylase-to-pyruvate dehydrogenase flux ratio in human subjects.

Animals↗

PubMed1996

Regulation of in vivo ketogenesis: role of free fatty acids and control by epinephrine, thyroid hormones, insulin and glucagon.

The production of ketone bodies (KB) is dependent on the amount of free fatty acids (FFA) supplied to the liver and on the hepatic metabolic fate of fatty acids and their orientation towards oxidation and ketogenesis or reesterification. In vivo ketogenesis can thus be regulated at the pre-hepatic (lipolysis) or hepatic level. We first investigated the role of FFA availability on the rate of KB production and then the effects of epinephrine, thyroid hormones, insulin and glucagon on the relationship between FFA availability and KB production. An increase in FFA availability augmented KB production not only by a mass effect but also by a diversion of hepatic fatty acid metabolism towards ketogenesis. The ketogenic effect of epinephrine and thyroid hormones depended only on their stimulatory action on lipolysis and FFA availability. An excess of thyroid hormones had no direct effect on hepatic ketogenesis, whereas the direct action of epinephrine on liver was rather anti-ketogenic. Glucagon stimulated hepatic ketogenesis, whereas a short-term increase in insulinemia within the physiological range appeared to have no restrictive action.

Animals↗

PubMed1995

Tracing hepatic gluconeogenesis relative to citric acid cycle activity in vitro and in vivo. Comparisons in the use of [3-13C]lactate, [2-13C]acetate, and alpha-keto[3-13C]isocaproate.

The validity of the use of a carbon tracer for investigating liver intermediary metabolism in vivo requires that the labeling pattern of liver metabolites not be influenced by metabolism of the tracer in other tissues. To identify such specific tracer, livers from 48-h starved rats were perfused with recirculating buffer containing [3-13C]lactate, [2-13C]acetate, or alpha-keto[3-13C]isocaproate. Conscious 48-h starved rats were infused with the same tracers for 5 h. The labeling patterns of liver glutamate and extracellular glucose were assayed by gas chromatography-mass spectrometry. In vivo data were corrected for 13CO2 reincorporation into C-1 of glutamate and C-3 and C-4 of glucose, using data from control rats infused with NaH13CO3. With [3-13C]lactate the labeling pattern of liver glutamate was the same in perfused organs and in vivo. In contrast, with [2-13C]acetate and alpha-keto[3-13C]isocaproate the labeling pattern of liver glutamate in vivo was clearly influenced by the expected labeling pattern of citric acid cycle intermediates formed in non-gluconeogenic organs, presumably glutamine made in muscle. Indeed, the labeling pattern of plasma glutamine and liver glutamate were similar in experiments with [3-13C]lactate but different in experiments with [2-13C]acetate and alpha-keto[3-13C]isocaproate. Similar conclusions were drawn from the labeling patterns of glucose. Therefore, labeled lactate appears as the best tracer for studies of liver intermediary metabolism in vivo. Our data also show that a substantial fraction of alpha-ketoisocaproate metabolism occurs in peripheral tissues.

Acetates↗

PubMed1995

Use of gas chromatography/isotope ratio-mass spectrometry to study triglyceride metabolism in humans.

The study of triglyceride (TG) metabolism using stable isotope tracers would be facilitated by being able to detect low 13C enrichment. To meet this goal, we developed a gas chromatography/isotope ratio-mass spectrometry technique to measure the enrichment of palmitate in nonesterified fatty acids (NEFA) and TG as its methyl derivative. This method allows accurate and reproducible measurements of enrichment as low as 0.009 mole percent excess (MPE), in a range between 0-0.65 MPE. The usefulness of this method is shown by two studies of lipid metabolism in human beings. First, we studied the metabolic fate of an oral TG load labeled with [1,1,1-13C3]tripalmitin. Labeled palmitate appeared concurrently in plasma NEFA and TG, and four hours after the load, the labeling was higher in NEFA than in TG (MPE NEFA: 1.53 +/- 0.31 vs. MPE TG: 0.78 +/- 0.06, P < 0.05). In a second study, the hepatic reesterification of NEFA was estimated by measuring the appearance of infused [1-13C]palmitate in circulating TG. The estimated contribution of plasma NEFA to circulating TG increased to a maximum of 22%. Thus, gas chromatography/isotope ratio-mass spectrometry appears to be a useful tool for future studies of lipid metabolism in humans.

Fatty Acids↗

PubMed1995

Respective role of plasma nonesterified fatty acid oxidation and total lipid oxidation in lipid-induced insulin resistance.

To investigate the respective role of nonesterified fatty acids (NEFA) oxidation and total lipid oxidation in lipid-induced insulin resistance, we measured the response of glucose metabolism to insulin in normal subjects without (control study) or with either heparin (heparin study) or triglycerides (TG) emulsion (Ivelip study) infusion. Three-step euglycemic-mild-hyperinsulinemic clamp studies were performed. Lipid and glucose metabolism were studied using indirect calorimetry and [6,6-2H2]glucose and [1-13C]palmitate infusions. NEFA concentration and turnover and oxidation rates were decreased by insulin in the control study, but were maintained during the heparin and Ivelip infusion studies. Total lipid oxidation was decreased similarly in the control and heparin studies, but was increased during the Ivelip infusion. Stimulation of glucose oxidation and utilization by insulin was reduced in the Ivelip study, but not in the heparin study. Thus, peripheral insulin resistance was observed in the presence of a combined increase in total lipid and NEFA oxidation, but not during an isolated increase in NEFA oxidation. On the other hand, insulin-induced inhibition of glucose production was impaired in both the heparin and Ivelip studies. We conclude that total lipid oxidation is a major determinant of peripheral insulin resistance, whereas hepatic insulin resistance could be induced even by a moderate increase in NEFA availability.

Adult↗

PubMed1995

Lactate and pyruvate isotopic enrichments in plasma and tissues of postabsorptive and starved rats.

It has been proposed that plasma pyruvate isotopic enrichment (IE) during infusion of labeled lactate could be used to estimate the intracellular IE of lactate and pyruvate and thus to calculate their turnover rate. We determined the relations of plasma and tissue IE of lactate and pyruvate in anesthetized rats infused with [3-13C]lactate in an artery and sampled from a vein (A-V mode) or infused in a vein and sampled from an artery (V-A mode). In both groups of rats, the ratio of tissue to plasma lactate IE was < 1 with large differences between tissues: the highest ratio was observed in heart and the lowest in soleus. With the exception of liver, this ratio was higher in the A-V than in the V-A mode. Pyruvate IE was lower than lactate IE in tissues, with a few exceptions, and in plasma. This ratio of pyruvate to lactate IE was approximately 0.70 in plasma in A-V and V-A modes. Moreover pyruvate IE was also always higher in plasma than in tissues. This seemingly surprising result could be explained by the production of labeled pyruvate from labeled lactate inside the circulation by erythrocytes, because we observed a rapid isotopic equilibrium between lactate and pyruvate in blood "in vitro." Apparent lactate turnover was higher in the A-V than in the V-A mode when it was calculated using lactate as well as pyruvate IE. Therefore plasma pyruvate IE cannot be used in rats to estimate tissue IE and did not reconcile turnover rates measured using the A-V or V-A mode.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Measuring gluconeogenesis with [2-13C]glycerol and mass isotopomer distribution analysis of glucose.

We tested the validity of the use of [2-13C]glycerol and of the mass isotopomer distribution analysis of glucose for measuring gluconeogenesis in vitro and in vivo. When isolated rat livers (starved for 48 h) were infused with labeled glycerol without or with lactate+pyruvate, gluconeogenesis accounted for > 90% of glucose production. When glucose was added to the infusate so that glucose produced by the liver represented only 80 or 45% of total glucose output, this dilution could be calculated from the mass isotopomer distribution of glucose. When postabsorptive and starved rats were infused with [2-13C]glycerol, gluconeogenesis accounted for 54 +/- 2 and 89 +/- 1%, respectively, of glucose production. However, accurate measures could be obtained, particularly in postabsorptive rats, only with high tracer infusion rates (representing > or = 50% of endogenous glycerol production rate). In both groups of rats, these infusion rates resulted in an increase in total glycerol turnover rate and gluconeogenesis from glycerol. In addition, hepatic concentration of glycerol 3-phosphate was increased. In conclusion, [2-13C]glycerol infusion and mass isotopomer distribution analysis of glucose appear to be useful methods for studies of gluconeogenesis in vitro and in vivo; however, accurate measurements in vivo can be obtained only at the expense of some perturbation of the metabolic pathway studied.

Animals↗

PubMed1995

Regulation of insulin receptor mRNA splicing in rat tissues. Effect of fasting, aging, and diabetes.

Recent findings suggested that alterations in insulin receptor isoform expression might be involved in the molecular mechanism of insulin resistance. Using reverse transcription reaction followed by competitive polymerase chain reaction, we measured the level of the receptor mRNA variants in rat insulin-sensitive tissues, under conditions of decreased insulin effectiveness (fasting, aging, and diabetes). The liver expressed the mRNA variant with exon 11 predominantly, and the hind limb skeletal muscles expressed the mRNA without exon 11. The heart and epididymal adipose tissue expressed both variants. Fasting and streptozocin-induced diabetes increased the level of receptor mRNAs in the liver but did not modify the repartition between the two variants. The modification of the expression ratio, in favor of the form with exon 11, found by some authors in the skeletal muscle of insulin-resistant patients was not observed in rat muscles that expressed > 99% of the form without exon 11 under all the conditions tested. In adipose tissue, the proportion of both mRNA variants was never altered (45% of exon 11-positive [Ex11+]), while the total receptor mRNA concentration changed markedly during fasting or aging. The only modification observed in the isoform distribution was a significant decrease in Ex11+ mRNA concentration in the liver, muscle, and heart of old rats. We conclude that alternative splicing of insulin receptor mRNA is not involved in the impairment of insulin action during fasting or diabetes. Its potential role in the insulin resistance of old animals remains to be defined.

Adipose Tissue↗

PubMed1994

Nonhomogeneous labeling of liver extra-mitochondrial acetyl-CoA. Implications for the probing of lipogenic acetyl-CoA via drug acetylation and for the production of acetate by the liver.

The labeling of liver extra-mitochondrial acetyl-CoA was investigated in isolated rat livers perfused with [2-(13)C]acetate, [1-(13)C]octanoate, or [1,2,3,4-(13)C4]docosanoate and with drugs that undergo acetylation (phenylaminobutyrate, paraaminobenzoate, and sulfamethoxazole; singly or in combination). The 13C enrichment of mitochondrial acetyl-CoA was probed by the enrichment of R-beta-hydroxybutyrate. The latter was not enriched from [1,2,3,4-(13)C4]docosanoate, thus excluding mitochondrial beta-oxidation of docosanoate. The 13C enrichment of extra-mitochondrial acetyl-CoA was probed by the enrichments of acetylated drugs and of free acetate. In most cases, the four probes yielded different enrichments. Thus, extra-mitochondrial acetyl-CoA appears nonhomogeneous. Competition between drugs alters the labeling of individual acetyl-CoA sub-pools. The labeling pattern of acetylated drugs suggests the existence of more than the two N-acetyltransferases identified so far by others. Our data question the possibility of probing the pool of lipogenic acetyl-CoA via drug acetylation.

4-Aminobenzoic Acid↗