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.

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

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.270.5.e882 ◽

1996 ◽

Vol 270 (5) ◽

pp. E882-E889 ◽

Cited By ~ 2

Author(s):

D. Yang ◽

S. F. Previs ◽

C. A. Fernandez ◽

S. Dugelay ◽

M. V. Soloviev ◽

...

Keyword(s):

Citric Acid ◽

Pyruvate Dehydrogenase ◽

Pyruvate Carboxylase ◽

Citric Acid Cycle ◽

Human Subjects ◽

Flux Ratio ◽

Peripheral Tissues ◽

Acid Cycle ◽

Labeling Pattern ◽

Made In

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.

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Validation of the Use of Urinary Phenylacetyl-Glutamine to Assess the Labeling Pattern of Liver Citric Acid Cycle Intermediates in Monkeys

IFAC Proceedings Volumes ◽

10.1016/s1474-6670(17)46288-4 ◽

1994 ◽

Vol 27 (1) ◽

pp. 417-418

Author(s):

D. Yang ◽

S.F. Previs ◽

C.A. Fernandez ◽

M.V. Soloviev ◽

F. David ◽

...

Keyword(s):

Citric Acid ◽

Citric Acid Cycle ◽

Acid Cycle ◽

Labeling Pattern

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Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes

Biochemical Journal ◽

10.1042/bj2890821 ◽

1993 ◽

Vol 289 (3) ◽

pp. 821-828 ◽

Cited By ~ 20

Author(s):

C G Jones ◽

S K Hothi ◽

M A Titheradge

Keyword(s):

Pyruvate Kinase ◽

Pyruvate Dehydrogenase ◽

Pyruvate Carboxylase ◽

Phosphoenolpyruvate Carboxykinase ◽

Tricarboxylic Acid Cycle ◽

Acute Effect ◽

Isolated Hepatocytes ◽

Pyruvate Oxidation ◽

Parallel Increase ◽

Glucose Output

Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both 1.0 mM pyruvate and 1.0 mM lactate plus 0.2 mM pyruvate as the substrate. The magnitude of stimulation was comparable with both substrates. The increase in glucose output was accompanied by an increased flux through pyruvate carboxylase, although the absolute flux and magnitude were considerably less in the presence of the more reduced substrate. The effect of the steroid on the flux through pyruvate dehydrogenase was substrate-dependent, an inhibition occurring with the more oxidized substrate. There was no effect of steroid treatment on [1-14C]lactate or pyruvate oxidation or on tricarboxylic-acid-cycle flux as measured by [3-14C]pyruvate oxidation. Dexamethasone treatment resulted in a parallel increase in both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvate between pyruvate and lactate formation and gluconeogenesis. Similarly there was no effect of the steroid on either the activity ratio or the total pyruvate kinase activity in the cells. It is suggested that the acute effect of the dexamethasone to increase gluconeogenesis resides at the level of phosphoenolpyruvate formation, i.e. pyruvate carboxylase and possibly phosphoenolpyruvate carboxykinase.

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Influence of Liver Triglycerides on Suppression of Glucose Production by Insulin in Men

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2014-2404 ◽

2015 ◽

Vol 100 (1) ◽

pp. 235-243 ◽

Cited By ~ 18

Author(s):

Eunsook S. Jin ◽

Magdalene Szuszkiewicz-Garcia ◽

Jeffrey D. Browning ◽

Jeannie D. Baxter ◽

Nicola Abate ◽

...

Keyword(s):

Citric Acid ◽

Fatty Liver ◽

Citric Acid Cycle ◽

Hepatic Glucose Production ◽

Glucose Production ◽

Hepatic Insulin Resistance ◽

Resonance Spectroscopy ◽

Acid Cycle ◽

Liver Triglycerides ◽

Total Glucose

Abstract Context: The ability of insulin to suppress hepatic glucose production is impaired among subjects with increased intrahepatic triglycerides (IHTG). However, little is known about the roles of insulin on the supporting fluxes of glucose production among patients with fatty liver. Objective: To evaluate the effects of insulin on fluxes through the three potential sources of plasma glucose (glycerol, the citric acid cycle, and glycogen) among patients with fatty liver. Design, Settings, Participants, and Intervention: Nineteen men with a range of IHTG (∼0.5% to 23%) were studied after an overnight fast and during hyperinsulinemia using magnetic resonance spectroscopy and stable isotope tracers. Main Outcome Measures: IHTG, gluconeogenesis from glycerol, gluconeogenesis from the citric acid cycle, glycogenolysis, and 13C-labeled glucose produced from the citric acid cycle during hyperinsulinemia were measured. Results: Men with high IHTG had higher fluxes through all pathways contributing to glucose production during hyperinsulinemia, compared to men with low IHTG, but they had similar fluxes after the fast. Consequently, men with fatty liver had impaired insulin efficiency in suppressing total glucose production as well as fluxes through all three biochemical pathways contributing to glucose. The detection of glucose isotopomers with 13C arising from [U-13C3]propionate ingested during hyperinsulinemia demonstrated continuous gluconeogenesis from the citric acid cycle in all subjects. Conclusions: These findings challenge the concept that individual glucose production pathways are selectively dysregulated during hepatic insulin resistance. Overproduction of glucose during hyperinsulinemia in men with fatty liver results from inadequate suppression of all the supporting fluxes of glucose production in response to insulin.

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