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J. Biol. Chem., Vol. 275, Issue 47, 36787-36793, November 24, 2000

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Loss of [¹³C]Glycerol Carbon via the Pentose Cycle
IMPLICATIONS FOR GLUCONEOGENESIS MEASUREMENT BY MASS ISOTOPER DISTRIBUTION ANALYSIS^*

Irwin J. Kurland^§¶, Allison Alcivar^§, Sara Bassilian, and Wai-Nang P. Lee

From the  Molecular Biology Institute and ^§ Department of Medicine, Division of Endocrinology, Diabetes and Metabolism Signaling Laboratory, UCLA, Los Angeles, California 90024 and the  Department of Pediatrics, Harbor-UCLA Medical Center, Torrance, California 90502

Whereas many reports substantiated the suitability of using [2-¹³C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [¹³C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [¹³C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mM [U-¹³C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of ¹³C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using ¹³C tracer methods.

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