Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR

Abstract

A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply and hormonal control, but the effects of these parameters on the flux through biochemical pathways which integrate to control glucose production are less clear. A combination of 13C and [2H]water tracers and NMR isotopomer analysis were used to investigate metabolic fluxes in response to altered cytosolic redox state and insulin. In livers isolated from fed mice and perfused with a mixture of substrates including lactate/pyruvate (10:1, w/w), hepatic glucose production had substantial contributions from glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising decrease in the contribution from glycogen and an increase in that from PEP to glucose production. A change in the lactate/pyruvate ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption and overall glucose output unchanged. When lactate and pyruvate were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower. Insulin lowered glucose production by inhibiting glycogenolysis at both low and high doses, but only at high levels of insulin did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards lower values (P<0.1). Our data demonstrate that, in the isolated mouse liver, substrate availability and cellular redox state have a dramatic impact on liver metabolism in both the tricarboxylic acid cycle and gluconeogenesis. The tight correlation of these two pathways under multiple conditions suggest that interventions which increase or decrease hepatic tricarboxylic-acid-cycle flux will have a concomitant effect on gluconeogenesis and vice versa.

Scheme 1. Metabolic pathways probed by the combination of ²H and ¹³C tracers

Hepatic glucose production (v1) has contributions from v2 (glycogenolysis, GLY), v3 (GNG from glycerol, GNG_glycerol), v4 (GNG from phosphoenolpyruvate, GNG_pep) as determined by ²H NMR of perfusate glucose. PEPCK flux is presumed to be the major constituent of total efflux from the hepatic tricarboxylic acid cycle, represented by v6. Pyruvate cycling, v5, denotes pathways such as PK or the malic enzyme which regenerate pyruvate rather than contributing to GNG. Tricarboxylic-acid-cycle (TCA Cycle) flux is shown as v7.

Figure 1. Typical ²H spectra of MAG from isolated perfused mouse livers under varying redox conditions in the presence and absence of insulin

Proton-decoupled ²H-NMR spectra of MAG derived from glucose are shown. Glucose was produced by mouse livers supplied with 0.2 mM octanoate, 0.25 mM glycerol, 0.5 mM sodium [U-¹³C₃]propionate, and 3% ²H₂O plus (A) 0.15 mM lactate/1.5 mM pyruvate, (B) 1.5 mM lactate/0.15 mM pyruvate, or (C) 1.5 mM lactate/0.15 mM pyruvate plus 0.81 ng/ml insulin, and (D) no lactate, pyruvate or insulin.

Figure 2. Typical ¹³C-NMR spectra of MAG derived from glucose

The insets show expanded views of the C2 resonance of MAG under the three conditions. (A) 1:10 lactate/pyruvate; (B) 10:1 lactate/pyruvate; (C) no lactate or pyruvate. The areas of the multiplet components (D12, D23 and Q) labelled in (B) were used to evaluate the relative fluxes summarized in the bottom panel of Table 1 and the absolute fluxes summarized in Table 2.

Figure 3. Ketone production with and without lactate/pyruvate (Lac/Pyr) by livers isolated from fed mice and perfused with 0.2 mM octanoate, 0.25 mM glycerol and 0.5 mM [U-¹³C]propionate

Ketone production represents the sum of ACAC and BHB (n=3).