Dysregulation of the unfolded protein response in db/db mice with diet-induced steatohepatitis

Abstract

In humans with nonalcoholic fatty liver, diabetes is associated with more advanced disease. We have previously shown that diabetic db/db mice are highly susceptible to methionine choline-deficient diet (MCD)-induced hepatic injury. Because activation of the unfolded protein response (UPR) is an important adaptive cellular mechanism in diabetes, obesity, and fatty liver, we hypothesized that dysregulation of the UPR may partially explain how diabetes could promote liver injury. Db/db and db/m mice were fed the MCD or control diet for 4 weeks to characterize differences in UPR activation and downstream injury. Wildtype mice (C57BLKS/J) fed the MCD or control diet were treated with SP600125; a c-Jun N-terminal kinase (JNK) inhibitor and its effect on liver injury and UPR activation was measured. The MCD diet resulted in global up-regulation of the UPR in both diabetic db/db and nondiabetic db/m mice. db/db mice had an inadequate activation of recovery pathways (GADD34, XBP-1(s)) and accentuated activation of injury pathways related to persistent eif2-α phosphorylation (activating transcription factor 4 [ATF-4], C/EBP homologous transcription factor [CHOP], oxireductase endoplasmic reticulum oxidoreductin-1 [ERO-1α], JNK, nuclear factor kappaB [NF-κB]) compared to db/m mice. This led to increased expression of inflammatory mediators such as tumor necrosis factor alpha (TNF-α), ICAM-1, and MCP-1 compared to db/m mice. Interestingly, whereas pharmacologic JNK inhibition did not prevent the development of MCD diet-induced steatohepatitis, it did attenuate UPR and downstream inflammatory signaling.

Conclusion: MCD-fed db/db mice develop a more proinflammatory milieu than db/m mice associated with an impaired ability to dephosphorylate eif2-α through GADD34, impairing cellular recovery. These data may enhance our understanding of why diabetics with nonalcoholic steatohepatitis are prone to develop more severe liver injury than nondiabetic patients.

Figure 1. The MCD diet increases activation of the PERK pathway

Western immunoblotting of eif2-α, CHOP and GADD34 individual samples with loading controls (n=3) of db/db and db/m mice fed either the control (C) or MCD (M) diet for 4 weeks. (A.) Hepatic real time quantitative PCR of ATF4, ERO-1α and CHOP, in db/m and db/db mice treated with the control or MCD diet for 4 weeks. (B.) Data represent relative values +/− SEM, (N=5–12). ** p≤0.01, *** p≤0.001 for the effect of diet within a strain, ¥ p<0.05 for the effect of the MCD diet between mouse strains.

Figure 2. The MCD diet induces activation of the ATF-6 and IRE-1 pathways

Western immunoblotting of hepatic ATF-6, Bip and XBP-1(s) with loading controls in db/m and db/db mice. Data represent pooled samples (n=4–5) of db/m and db/db mice fed either the control (C) or MCD (M) diet for 4 weeks. (A) Hepatic real time quantitative PCR of XBP-1 and EDEM in db/m and db/db mice treated with the control or MCD diet for 4 weeks. (B.) Data represent relative values +/− SEM, (N=5–12). ** p≤0.01,*** p≤0.001, ¥ p<0.05, for the effect of the MCD diet on mouse strain.

Figure 3. JNK activation in db/m and db/db mice treated with the MCD diet

Western immunoblotting depicting activation of JNK and c-Jun in livers of db/m and db/db mice treated with the control or MCD diet for 4 weeks. For both JNK and c-Jun total (T) and phosphorylated (P) forms are shown. Data represent pooled samples (n=4–5) of db/m and db/db mice fed either the control (C) or MCD (M) diet for 4 weeks.

Figure 4. Decreased translation of IKKβ resulting from ER stress leads to increased nuclear translocation of NFκB

Western immunoblotting of hepatic IKKβ in db/m and db/db mice treated with the control (C) or MCD (M) diet for 4 weeks. Groups represent pooled samples (n=4–5). (A) NFκB activity of subunits p50 (B) and p65 (C) from hepatic nuclear extracts from db/m and db/db mice treated with the control or MCD diet for 4 weeks. Data represent relative values +/− SEM. * p<0.05, ** p<0.01, *** p<0.001

Figure 5. db/db mice on the MCD diet develop more pronounced downstream inflammatory signaling

Real time quantitative PCR of TNF-α (A.) ICAM-1 (B.) and MCP-1 (C.) in hepatic tissue of db/m and db/db mice treated with the control or MCD diet for 4 weeks. Differences represent relative changes in hepatic mRNA expression in cohorts of db/m and db/db mice treated with the control or MCD diet. Data represent relative values +/− SEM. * p<0.05, ** p<0.01,*** p<0.001.

Figure 6. The effect of pharmacologic JNK inhibition with SP600125 on JNK activation and inflammatory signaling

Western immunoblotting of pooled samples (n=5) of mice fed either the control (C) or MCD (M) and either vehicle (V) or SP600125 (SP) intra-peritoneally for 2 weeks. For JNK, total (T) and phosphorylated (P) forms are shown in addition to ICAM-1 and CHOP.

Figure 7. Summary of proposed mechanism of increased liver injury in db/db mice fed the MCD diet

Panel A depicts activation of the UPR with cellular recovery. Phosphorylation of eIF2α activates ATF-4, which then induces CHOP and GADD34 as well as activates NF-κB through IKKβ. Induction of GADD34 acts as a negative feedback mechanism to de-phosphorylate and hence deactivate eIF2a, preventing cell injury from persistent activation of inflammatory mediators. In this compensatory model, CHOP induction is attenuated as is the activation of inflammatory pathways, restoring cellular homeostasis. Panel B depicts an inadequate compensatory response and consequently persistent propagation of the inflammatory cascade. Although the UPR is activated by the MCD diet, recovery from ER stress is impaired in db/db mice. We propose that db/db mice fed the MCD diet have persistent phosphorylation of eif2-α, due to a loss of feedback inhibition from GADD34. This then results in persistent CHOP activation and more robust inflammatory signaling (via JNK, NK-κB and other factors).