Gluconeogenesis in hepatocytes determined with [2-13C] acetate and quantitative 13C NMR spectroscopy |
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Institution: | 1. Biomedical Research Division, Diabetes and Obesity Research Institute, Department of Biomedical Science, Cedars-Sinai Medical Center, Los Angeles, CA, United States;2. Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX, United States |
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Abstract: | - 1.1. In the present study the major metabolic pathways of glucose metabolism were determined in isolated liver cells using 2-13C]acetate and 13C magnetic resonance spectroscopy.
- 2.2. The relative reaction rates of glucose synthesis to the TCA cycle were determined from the 13C distribution in glucose where the overall 13C enrichment of glucose was 6.41 ± 1.94% (mean ± SD; n = 6) and the mean 13C enrichment of C1, C2, C5, C6 to C3, C4 was 2.63 ± 0.30.
- 3.3. Since the distribution of tracer in glucose is a function of the relative entry rates of pyruvate to acetyl-CoA into the oxaloacetate pool this was calculated to be 0.32 ± 0.15 and the factor for carbon exchange (1/P) between the gluconeogenic pathway and the TCA cycle was calculated to be 1.03 ± 0.20.
- 4.4. With this carbon exchange factor and the approximated 13C enrichment of acetyl-CoA the intramitochondrial 13C enrichment of phosphoenolpyruvate was calculated and the “true” rate of hepatic gluconeogenesis from phosphoenolpyruvate estimated.
- 5.5. Since acetate was metabolized solely in liver cells the 13C enrichment of acetyl-CoA could be approximated from that of 3-hydroxybutyrate.
- 6.6. The carbon 13 enrichment of 3-hydroxybutyrate and phosphoenolpyruvate was 5.89 ± 0.90% and 5.96 ± 1.67%, respectively.
- 7.7. The per cent gluconeogenesis from phosphoenolpyruvate calculated as the ratio of the 13C enrichment of glucose to that of 3-hydroxybutyrate times 1/P was 107 ± 8%.
- 8.8. In this study the validity of assessing isotopic exchange at oxaloacetate as suggested by Katz Katz J. (1985) Am. J. Physiol.248, R391–R399] when interpretation of the data are not obscured by pseudoketogenesis.
- 9.9. Magnetic resonance spectroscopy provides direct information about intramolecular tracer distribution by which flux rates in major metabolic pathways are derived.
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