Background: Tracers should be maximally descriptive with minimal interference to the metabolic system of interest. Divergent concepts of in vivo hepatic metabolism (e.g., anaplerosis) may have emerged, in part, from invalid assumptions and/or perturbations associated with common tracers.
Aim: We evaluated three common substrate tracers for intrahepatic metabolism vis-à-vis a novel independent [1,2-13C2]-L-glutamine strategy.
Methods: Overnight fasted Sprague-Dawley rats underwent primed, continuous infusions of reported tracer rates for lactate, propionate, acetate or glutamine. Plasma/freeze-clamped livers were obtained at 0, 5, 15, 30, 60, and 120 min. Plasma/tissue isotopomers were subject to steady state and kinetic Mass Isotopomer MultiOrdinate Spectral Analysis (MIMOSA) and reported relative to citrate synthase (CS).
Results: In contrast to glutamine infusion, plasma insulin ([i]) glucose concentration ([g]), and/or plasma glucose turnover (Ra) were increased by acetate ([i] = 1.5 fold, p = 0.015; [g] = 129 mg/dl, p=0.021), lactate ([i]= 2.03 fold, p = 0.0001; [g] = 156mg/dl, p = 0.0001, Ra = 7.2 mg/kg/min, p=0.006), and propionate ([i]= 1.7 fold, p = 0.0013; [g] = 165 mg/dl, p = 0.0001, Ra = 7.8 mg/kg/min, p = 0.0017). [1,2-13C2]-L-glutamine quickly reached isotopic equilibrium without impacting mitochondrial metabolite concentration or glutamate anaplerosis. Transaminase exchange (Vx) was >>CS. PEPCK was 1.6 x CS with pyruvate only accounting for 76% of anaplerosis (remainder from propionate and glutamate anaplerosis). PK was less than CS while ATP citrate lyase (ACLy) was 18% of CS.
Conclusion: Direct in vivo measurements of pyruvate cycling rates were low relative to CS while ACLy, IDH exchange, and non-pyruvate anaplerosis contributed substantially to hepatic metabolism. [1,2-13C2]-L-glutamine MIMOSA delivered an unencumbered, direct assessment of hepatic mitochondrial metabolism.
S. Siebel: None. R.L. Cardone: None. A. Abulizi: None. R. Raaisa: None. R.M. Williams: None. R. Sehgal: None. G. Butrico: None. G. Cline: None. D.L. Rothman: None. G.F. Mason: None. R. Kibbey: None.
National Institutes of Health (2K12DK094714-06, R01DK108283)