Supplementary Material for: Effects of a DPP4 Inhibitor on Progression of NASH-related HCC and the p62/ Keap1/Nrf2-Pentose Phosphate Pathway in a Mouse Model

<b><i>Background and Aims:</i></b> Diabetes mellitus is a risk factor for hepatocellular carcinoma (HCC) in patients with nonalcoholic steatohepatitis (NASH). Dipeptidyl peptidase-4 inhibitor (DPP4i), an antidiabetic agent, is reported to affect cell proliferation. We aimed to investigate the effects of DPP4i on the progression of NASH-related HCC and its metabolic pathway in a mouse model. <b><i>Methods:</i></b> A mouse model of NASH-related HCC was used in this study. Eight-week-old mice were administered either DPP4i (sitagliptin 30 mg/kg/day; DPP4i group; <i>n</i> = 8) or distilled water (control group; <i>n</i> = 8) for 10 weeks. Then, HCC progression was evaluated by computed tomography. Changes in metabolites of HCC tissue were analyzed by metabolomic analysis. The localization and expression of p62, Keap1, Nrf2, and MCM7 were evaluated by immunostaining and immunoblotting, respectively. <b><i>Results:</i></b> The number and volume of HCC were significantly lower in the DPP4i group than in the control group (1.8 ± 1.2 vs. 4.5 ± 1.7/liver, <i>p</i> < 0.01; 11.2 ± 20.8 vs. 37.5 ± 72.5 mm<sup>3</sup>/tumor, <i>p</i> < 0.05). Metabolome analysis revealed that DPP4i significantly increased 6-phosphogluconic acid and ribose 5-phosphate levels and decreased the AMP-to-adenine and GMP-to-guanine ratios (AMP-to-adenine ratio 0.7 ± 0.2 vs. 2.0 ± 1.2, <i>p</i> < 0.01; GMP-to-guanine ratio 0.6 ± 0.3 vs. 1.5 ± 0.7, <i>p</i> < 0.01). Immunostaining showed that p62 was localized in the cytoplasm of HCC in the DPP4i group, while p62 was localized in the nucleus of HCC in the control group. Keap1, Nrf2, and MCM7 expression decreased significantly in the DPP4i group compared to that in the control group. <b><i>Conclusions:</i></b> We demonstrated that DDP4i prevented the progression of NASH-related HCC in a mouse model. Furthermore, metabolome analysis revealed that DDP4i downregulated the pentose phosphate pathway with suppression of the p62/Keap1/Nrf2 pathway. Thus, DDP4i may prevent tumor progression through inhibition of metabolic reprogramming in NASH-related HCC.