Figure 6

The ER stress-induced UPR and autophagy signaling during fasting are impaired in the liver of TXNIP-KO mice. (a) PDI activity was measured by the ProteoStat(R) PDI assay kit. PDI activity decreased during the fasted state in WT mice, but not in TXNIP-KO mice. (b) Differences in the expression of ER stress signaling-related genes between WT and TXNIP-KO mice were examined by RT-qPCR. In WT mice, expression of these genes increased in the fasted state, but this change was not observed in TXNIP-KO mice, and the expression of IRE1 mRNA, which is important in the induction of autophagy, was significantly lower in TXNIP-KO mice in the fasted state. (c) The expression of sXBP and uXBP1 mRNA was evaluated by PCR after RNA extraction and cDNA synthesis. Although upregulation of sXBP1 mRNA was found in WT mice during the fasted state, this upregulation was not found in TXNIP-KO mice. (d) Phosphorylation of IRE1 and the levels of uXBP1 and sXBP1 were examined by western blotting. Decreased phosphorylation of IRE1 and decreased level of sXBP1 were observed in TXNIP-KO mice, which implies decreased ER stress signaling for the induction of autophagy during the fasted state in TXNIP-KO mice. (e) Other proteins important for ER stress signaling, BiP, cATF6, and p-EIF2a, were also examined. Although the expression of these protein tended to be upregulated during the fasted state in WT mice, this tendency was not found in TXNIP-KO mice. Statistically significant differences were found in the expression of cATF6 between the fed and fasted states in WT mice, and WT and TXNIP-KO mice during fasted state. (f) Protein expression of LC3-II was restored in the fasted state when bacitracin was injected to the TXNIP-KO mice before 24-h fasting. *p < 0.05, **p < 0.01 (in each group, six mice were used for PDI analysis, RT-qPCR or PCR, and ten were used for western blotting). UPR unfolded protein response, ER endoplasmic reticulum, TXNIP thioredoxin-interacting protein, PDI protein disulfide isomerase, BiP binding immunoglobulin protein, PERK PKR-like endoplasmic reticulum kinase, ATF6 activating transcription factor 6, IRE1 inositol-requiring enzyme 1, p-IRE1 phosphorylated IRE1, sXBP1 X-box binding protein 1 sliced form, uXBP1 XBP1 unsliced form, cATF6 cleaved ATF6, p-EIF2a phosphorylated eukariotic initiation factor 2a, WT wild type mice, KO knockout mice, NS normal saline, Bac bacitracin.