Figure 5.

Macrovesicular steatosis in HLCs results in truncation of the TCA cycle, inhibiting conversion of succinate to fumarate.

Paradoxically, this is associated with increased accumulation of fumarate.

(A) Schematic outlining the TCA cycle and possible ¹³C labeled isotopomers from a single cycle.

(B) Steady state measurements of TCA cycle-associated metabolites that could be measured by GC-MS.

(C) PC-derived αKG is unchanged in the presence of steatosis, and PDH-derived αKG is reduced.

(D–E) Labeling patterns of αKG are not due to increased cataplerosis through glutamate, as both PC- and PDH-derived glutamate are reduced in response to steatosis.

(F) Furthermore, reduced ¹³C labeling of succinate indicates inefficient conversion from αKG.

(G–H) Despite reduced labeling of succinate, both fumarate and malate show increased PC-derived label incorporation.

(I) Proposed model of preferential anaplerosis of pyruvate into the TCA cycle, with concomitant truncation, preventing conversion of succinate to fumarate. For NMR data, labels are: 1 = ¹³C labeling; 0 = no ¹³C labeling. All GC-MS data consisted of 10 biological replicates and 2 technical replicates. Isotopomer data were calculated by multiplying MID (multiple ion detection) by normalized total ion count. For NMR data (E) n = 4 biological replicates/group. Data were analyzed by two-way ANOVA with Sidak post-hoc testing and are expressed as mean ± SD.