Quantitation of gluconeogenesis in humans

Abstract

Several methods have been introduced for estimating gluconeogenesis, but none provides a direct measurement without assumptions that condition the accuracy of the estimate. The aim of this work has been to develop a new method for quantitative studies of gluconeogenesis, applicable in humans. We postulated that by using (U-13C3) glyceroland application of mass isotopomer distribution analysis (MIDA) to the enrichments of 13C incorporated into blood glucose the contribution of gluconeogenesis to wholebody glucose production could be determined. However, when applied in healthy subjects fasted for 60 h, the estimated contribution was only 50-60% when close to 100% of the glucose production was expected to derive from gluconeogenesis. The values obtained were incompatible with glucose being formed from a single pool of triose phosphate in the liver, which was a condition for the validity of the method. Thus, we concluded that the MIDA technique based on administration of 13C-labeled glycerol does not provide a valid quantitation of gluconeogenesis in vivo. Gluconeogenesis can be estimated a few hours after ingestion of a dose of deuterated water that can be safely administered to humans (body water is enriched to 0.5%). The method is based on the incorporation of deuterium into the hydrogen bound to carbon 5 of glucose (C-5) formed by gluconeogenesis and to carbon 2 (C-2) of all newly formed glucose molecules (i.e formed after the ingestion of deuterated water). The contribution of gluconeogenesis to whole body glucose production thus equals the ratio of enrichments at C-5 to C-2. Simultaneously, glucose production can be estimated by isotope dilution technique using (6,6-2H2) glucose infusion. Together, the methods provide a quantitative measurement of gluconeogenesis. The applicability of the techniques rests on the conversion of the tracer hydrogens and their carbon into formaldehyde and its further polymerization to hexamethylenetetramine (HMT). Thereby, an accurate assessment of the enrichments is facilitated although body water has only been enriched to 0.5%. Moreover, the glucose rate of appearance can be determined without influence from the ingested deuterated water. Applied in healthy subjects in the overnight fasted state, approximately equal proportions of glucose production derived from gluconeogenesis and glycogenolysis. During continued fasting the contribution from gluconeogenesis increased gradually (54% at 14 h and 62% at 22 h), but in absolute terms it stayed constant between 14h and 22 h of fasting (5.5 µmol/kg/min). Thus, the fall in glucose production observed between 14 and 22 h of fasting, from 10.2 to 8.6 lµmol/kg/min, could be attributed to a decrease in the rate of glycogenolysis After 42 h of fasting almost all glucose produced (94%) derived from gluconeogenesls. In the overnight fasted state, gluconeogenesis, as reported above, may be equated with hepatic gluconeogenesis. This was evident from the application of catheterization technique in healthy subjects together with labeled glucose infusion for estimation across both splanchnic and renal tissues: in the 12 h fasted state, hepatic glucose production (the sum of net splanchnic output and glucose uptake to the splanchnic bed) accounted for 100% of whole body glucose production as measured by the isotope dilution technique. No net renal production of glucose and no or only a minimal fractional extraction of labeled glucose were found. In the 60 h fasted state, hepatic glucose production accounted for 80% of whole body glucose production. No renal extraction of glucose but a significant net renal glucose production were found. Thus the renal contribution to glucose production may represent 10-25% after 60 h of fasting.