Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes

Abstract

The contribution of gluconeogenesis to glucose production is estimated from the enrichment of the H bound to C-5 of glucose relative to either that bound to C-2 of glucose or the enrichment in body water on ingesting 2H2O in the fasted state. Contributions of all gluconeogenic substrates are included in the estimate and the limitation of an uncertain precursor enrichment removed. The half-life of 2H2O in body water precludes a repeat study for many weeks. Glycogen cycling could result in underestimation, but there is evidence that glycogen cycling does not occur in liver in the fasted state. Gluconeogenesis has been estimated by mass-isotopomer-distribution analyses, usually by administering 13C-labelled glycerol. Underestimates emphasize the major limitation of the method, i.e. the need to assume a single enrichment of the precursor pool. Estimates of gluconeogenesis from isotopomer distribution in arterial-blood glucose and lactate on infusing [U-13C6] glucose are unreliable, as a proportion of the glucose is formed from glycerol and from amino acids not converted to glucose via pyruvate. Loss of label in the Krebs cycle and relying on enrichment of arterial-blood lactate as a measure of hepatic pyruvate further add to the uncertainty. Estimates of the rate of gluconeogenesis by NMR are obtained by subtraction of the rate of glycogenolysis determined by NMR from the rate of glucose production. Estimates are then the mean rate for the period over which glycogen contents are measured. Technical considerations can limit the accuracy of analyses and result in overestimates.

Changes in hepatic glycogen cycling during a glucose load in healthy humans

“…Glycogen cycling has limited the interpretation of estimates of gluconeogenesis [2,8], since its contribution cannot be excluded by current methods, e.g. using 2 H 2 O [9,10] and magnetic resonance spectroscopy (MRS) [11].…”

Measurement of Fractional Whole-Body Gluconeogenesis in Humans From Blood Samples Using 2H Nuclear Magnetic Resonance Spectroscopy

“…Previous methods for measuring gluconeogenesis yielded a wide range of estimates in humans, which may relate to biochemical limitations of those methods (2)(3)(4)(5). Gluconeogenesis can be also calculated from the difference between the rates of EGP determined with [6-3 H]glucose and of liver glycogen breakdown determined by in vivo 13 C-nuclear magnetic resonance spectroscopy (MRS) (1,6).…”

“…In humans, gluconeogenesis can be calculated from 2 H enrichments in hexamethylenetetramine (HMT) derived from blood glucose measured with gas chromatographymass spectrometry (9,10). Briefly, 2 H is incorporated into position 3 of phosphoenolpyruvate (PEP) during the equilibration of pyruvate with alanine and via conversion of the pyruvate to oxaloacetate to the extent it equilibrates with fumarate. The carbon at position 3 of PEP becomes carbon 6 of glucose.…”

“…2 H is incorporated into position 2 of glyceraldehyde-3-P via its isomerization with dihydroxyacetone-3-P and via hydration of PEP to 2-phosphoglycerate. During glycerol conversion, 2 H is introduced at position 2 of glyceraldehyde-3-P. Carbon two of glyceraldehyde-3-P becomes carbon 5 of glucose.…”

“…Biochemical short-comings of previous approaches and invasive or time-consuming procedures can be avoided by this method. In the present study, 5:2 ratios were obtained by an alternative approach applying in vitro 2 H-NMR on a glucopyranosyl bromide derivative. The estimates of gluconeogenesis were compared with both in vivo 13 C-MRS and the HMT method.…”