Figure 1.

The livers of liver-specific Crif1-deficient mice exhibit altered glucose metabolism and impaired insulin signaling

(A) Gross morphology of Ctrl and LKO mice at 8 weeks of age.

(B) Western blot analysis showing lower levels of CRIF1 and subunits of the OxPhos complex in the livers of Ctrl and LKO mice. The results of one representative experiment of the two conducted are shown.

(C) BN-PAGE analysis of the assembled OxPhos complex in the livers of Ctrl and LKO mice (∗: abnormal sub-complexes).

(D) Representative transmission electron microscopic images of livers from Ctrl and LKO mice (n = 4). The white arrows indicate hepatic glycogen granules.

(E and F) Quantitative PCR analysis of UPR^mt mediators (E) and UPR^er and transcription factors involved in the mitochondrial stress response (F) in the livers of Ctrl and LKO mice (n = 4–5 biological replicates from three independent experiments).

(G) Western blot analysis of UPR^mt and UPR^er mediators in the livers of Ctrl and LKO mice. The results of one representative experiment out of the three conducted are shown. Data are mean ± SEM and were analyzed using Student's t test (∗p < 0.05 versus Ctrl).

(H) OCR (left panel) and individual parameters (right panel) in primary hepatocytes isolated from Ctrl and LKO mice (n = 10 biological replicates from two independent experiments) treated with oligomycin (2 μg/mL), CCCP (10 μM), or rotenone (1 μM). Basal respiration, ATP production, and proton leakage were calculated after oligomycin treatment, and the maximal and non-mitochondrial respiration were calculated after CCCP and rotenone treatment, respectively.

(I) Glycolysis assay (left panel) and glycolytic parameters (right panel) in primary hepatocytes isolated from the livers of Ctrl and LKO mice (n = 6 biological replicates from two independent experiments). Non-glycolytic acidification was calculated after the addition of 2-DG (50 mM). Glycolysis, glycolytic capacity, and glycolytic reserve were calculated after the addition of glucose (10 mM) and oligomycin (1 μM), respectively.

(J) Quantitative PCR analysis of Glut1 and Glut2 mRNA expression in livers from 8-week-old Ctrl and LKO mice (n = 6 biological replicates from three independent experiments).

(K) Glucose uptake by primary hepatocytes from Ctrl and LKO mice (n = 6 biological replicates from two independent experiments). Insulin (1 μM) was added 20 min before the measurements.

(L) Western blot analysis of insulin signaling after the addition of insulin (200 nM) for 15 min to primary hepatocytes isolated from Ctrl and LKO mice. The results of one representative experiment of the two conducted are shown. Data are mean ± SEM and were analyzed using two-way ANOVA followed by Scheff's post-hoc test in (K) and Student's t test in (E and F) and (H–J) (∗p < 0.05 versus Ctrl or Ctrl-Vehicle). See also Figure S1.