Additional Supporting Information may be found in the online version of this article.

HEP_26060_sm_SuppFig1.tif78KSupporting Information Figure 1. Western blot of Hint2 in liver homogenate. Liver homogenate from Hint2-/- and Hint2+/+ mice at 10, 20 and 30 weeks was immunoblotted. Expression of Hint2, normalized for actin, was constant throughout the experimental period. Hint2 was absent from Hint2-/- livers (n=9-10 mice per group).
HEP_26060_sm_SuppFig2.tif5135KSupporting Information Figure 2. Immunoblotting and immunohistochemistry of mitochondrial-shaping proteins in livers from Hint2+/+ and Hint2-/- mice. A. Immunoblots of mitochondrial fusion proteins and fission proteins were performed in cytosolic (Cyt) and mitochondrial (Mito) fractions of liver tissue isolated from Hint2+/+ and Hint2-/- mice. The expression of outer membrane mitochondrial fusion proteins, Mfn1 and Mfn2 were not different. The cytosolic dynamin-related protein 1 (Drp1) and the outer mitochondrial membrane fission protein (Fis1) were slightly decreased in Hint2-/- livers. The inner membrane fusion protein, optic atrophy 1 (Opa1), was slightly increased in Hint2-/- livers. Actin and Hsp60 were used to normalize for differences in protein loading (n=7 mice per group). B. Immunohistochemical staining for Opa1 and Fis1 in livers from Hint2+/+ and Hint2-/- mice. The expression of Opa1 was slightly higher and the expression of Fis1 was slightly lower in Hint2-/- than in Hint2+/+ livers (n=7 mice per group).
HEP_26060_sm_SuppFig3.tif276KSupporting Information Figure 3. Immunoblotting of insulin signalling proteins in Hint2-/- and Hint2+/+ mice. A. Quantification of immunoblots shown in Fig. 5B. Expression of insulin signalling proteins were compared in liver, muscle and white adipose tissue (WAT) of fasted Hint2-/- and Hint2+/+ mice before and 5 min after insulin injection (* p < 0.05, *** p<0.001; Mann Whitney U test, Hint2−/− vs. Hint2+/+ same condition, p < 0.05, p < 0.001; Mann Whitney U, + vs. − insulin, same strain) (n=3 − insulin; n=6 + insulin). B. Immunoblot of basal levels of insulin signalling proteins in fed mice. The basal of ratio phosphorylated Akt/Akt was not different in Hint2−/− and Hint2+/+ mice. The basal ratio of phosphorylated FoxO1/FoxO1 was elevated at 30 weeks (* p < 0.05) but the expression of total FoxO1 decreased in both groups at 30 weeks. The basal expression of phosphoenolpyruvate carboxykinase (Pck1) was significantly lower in Hint2−/− livers at 10 and 20 weeks (* p < 0.05) (n=9–10 mice per group).
HEP_26060_sm_SuppFig4.tif141KSupporting Information Figure 4. A. Immunoblots tested the expression levels of respiratory enzymes in isolated mitochondria. Expression was not different in Hint2-/- and Hint2+/+ livers (n=7 mice per group).
HEP_26060_sm_SuppFig5.tif100KSupporting Information Figure 5. Immunoblots of HINT2 expression in HepG2 cells. HepG2 cells were stably transfected with TransFast (Promega) using 5μg of pControl, HINT2-pControl, pSuppressor and siHINT2-pSuppressor constructs and selected with Geneticin. Immunoblotting confirmed the over-expression and silencing of HINT2 and the HINT2 expression in the corresponding control cell lines (n=3 experiments, *** p< 0.001 vs pControl or pSuppressor).
HEP_26060_sm_SuppFig6.tif6152KSupporting Information Figure 6. Comparison of HIF expression level and steady-state level of reactive oxygen species (ROS) in the livers of Hint2-/- and Hint2+/+ mice. A. ROS were measured in isolated hepatocytes. The oxidation of 2'7'-dichlorofluorescin (5 μM) to fluorescent 2'7'-dichlorofluorescein was measured at 485 nm (excitation) and 530 nm (emission). The hepatocytes from Hint2-/- generated a higher level of ROS than did the hepatocytes from Hint2+/+ mice (n=3 experiments, * p < 0.05). B. Immunohistochemical examination of liver sections showed significantly more nuclei positive for HIF-2a in Hint2−/− mice than in Hint2+/+ mice (* p < 0.05) (n=12 mice per group). No change was detected in HIF-1a.
HEP_26060_sm_SuppFig7.tif100KSupporting Information Figure 7. A. Carnitine palmitoyltransferase (CPT) activity in mitochondria from Hint2+/+ and Hint2-/- mice, aged 20 weeks. Palmitoyl-CoA was used as the substrate. No significant difference was detected between Hint2+/+ and Hint2-/- mitochondria (n=9 mice per group). B. Time profile of blood glucose in Hint2+/+ and Hint2-/- mice, aged 20 weeks, in response to fasting. Blood was collected from the tail vein and glucose was measured at 4 h intervals. Hint2+/+ and Hint2-/- mice responded similarly to food deprivation. C. Fasting-induced hepatic steatosis in livers of Hint2+/+ and Hint2-/- mice (aged 20 weeks). After fasting (16 h), triglycerides increased slightly but not significantly in Hint2+/+ and Hint2-/- livers. (ANOVA, * p < 0.05; fed Hint2−/− vs. fed Hint2+/+).
HEP_26060_sm_SuppInfo.doc54KSupporting Information
HEP_26060_sm_SuppTab1.doc50KSupporting Information Table 1. List of primary antibodies.
HEP_26060_sm_SuppTab2.doc21KSupporting Information Table 2. List of secondary antibodies for Western Blot.

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