Isotopomer studies of gluconeogenesis and the Krebs cycle with 13C-labeled lactate

Fasted rats were intragastrically infused with either [2,3-13C]lactate or [1,2,3-13C]lactate. The infusate also contained 14C-labeled lactate and [3-3H]glucose. Glucose, alanine, glutamate, and glutamine were isolated from liver and blood. There was near complete equilibration of lactate and alanine, and the relative specific activities and relative enrichments were the same in blood and liver. Glucose was cleaved enzymatically to lactate. The compounds were examined by gas chromatography-mass spectroscopy. From the mass isotopomer spectra of the lactate, glutamate, and glutamine and their cleavage fragments the positional isotopomer composition of these compounds was obtained. The enrichment and isotopomer pattern in the lactate from cleaved glucose represents that in phosphoenolpyruvate (PEP). When [1,2,3-13C]lactate was infused the mass isotopomer spectrum of glutamates consisted only of compounds containing either one, two, or three 13C carbons per molecule (m1, m2, and m3). There was little 13C in C-4 and C-5 of glutamate. The rate of pyruvate decarboxylation is low, and 3-4% of the acetyl-CoA flux in the Krebs cycle is contributed by lactate carbon. The major isotopomers in lactate, alanine, and PEP were m3 and m2 with 13C in C-2 and C-3. The predominant isotopomer in PEP from [2,3-13C]lactate was m2 with 13C in C-2 and C-3. There was much more of m1 isotopomer with 13C in C-3 and C-2 than the m1 isotopomer with 13C in C-1. There was very little m3, the isotopomer with 13C in all three carbons. Most of the 13C in C-3 and C-4 of glucose and C-1 of glutamate was introduced via 13CO2 fixation. From the isotopomer distribution and the rate of glucose turnover we deduced, applying the analysis described in the "Appendix," the absolute rates of gluconeogenic pathways, recycling of PEP and the Cori cycle, and flux in the Krebs cycle. The flux from oxaloacetate (OAA)-->PEP was seven times that of OAA-->citrate, and about half of PEP was recycled to pyruvate via pyruvate kinase. The mass isotopomer patterns in glutamate and glutamine were similar but differed from those of lactate and glucose. It appears that the glutamates are derived from alpha-ketoglutarate from a different Krebs cycle pool than PEP. The flux from OAA to PEP in this pool was two to three times that of OAA to citrate.(ABSTRACT TRUNCATED AT 400 WORDS)