Loss of [C-13]glycerol carbon via the pentose cycle - Implications for gluconeogenesis measurement by mass isotoper distribution analysis

Whereas many reports substantiated the suitability of using [2-C-13]glycero l and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [ C-13]glycerol had been shown to give lower estimates of gluconeogenesis (GN G). The reason for the underestimation has been attributed to asymmetric is otope incorporation during gluconeogenesis as well as zonation of gluconeog enic enzymes and a [C-13]glycerol gradient across the liver. Since the cycl ing of glycerol carbons through the pentose cycle pathways can introduce as ymmetry 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 mo dified Eagle's media containing 1.5 mM [U-C-13]glycerol. Mass isotopomers o f whole glucose from medium or glycogen and those of the C-1-C-4 fragment w ere highly asymmetrical, typical of that resulting from the cycling of gluc ose carbon through the pentose cycle. Substantial exchange of tracer betwee n hexose and pentose intermediates was observed. Our results offer an alter native mechanism for the asymmetrical labeling of glucose carbon from trios e phosphate, The scrambling of C-13 in hexose phosphate via the pentose pho sphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accoun ts for the underestimation of GNG using C-13 tracer methods.