Figure 4. Loss of ALT2 reduces hepatocyte alanine metabolism and alanine-mediated gluconeogenesis.

(A) Representative western blots for ALT2, ALT1, or GAPDH using liver lysates from WT and LS-Gpt2^−/− mice demonstrating loss of ALT2 protein.

(B) Glucose concentrations in the media of hepatocytes isolated from WT or LS-Gpt2^−/− mice stimulated with glucagon in the presence of no substrate, alanine, pyruvate, or glutamine. Some cells were also treated with the transaminase inhibitor β-chloroalanine (β-Cl) (n = 7). *Indicates significant differences (p < 0.05).

(C) Schematic depicting incorporation of ¹³C-alanine into pyruvate and TCA cycle intermediates. Black circles indicate ¹³C. White circles indicate ¹²C. Created with BioRender.com.

(D) Hepatocyte citrate enrichment from ¹³C-alanine is shown. *Significant differences (p < 0.05) between hepatocytes from different genotypes of mice.

(E) Hepatocyte M3 citrate enrichment from ¹³C-alanine is shown. *Significantly different (p < 0.05) from WT hepatocytes treated with vehicle. **Significantly different (p < 0.05) from all other groups.

(F) Media glucose enrichment from ¹³C-alanine is shown. *Significant differences (p < 0.05) between hepatocytes from different genotypes of mice. For (D)–(F), a representative experiment (of 3) performed in triplicate is shown. Data are presented as mean ± SEM.

(G) Blood glucose concentrations during ATT, QTT, and pyruvate tolerance test (PTT) analyses using lean WT or LS-Gpt2^−/− mice.