These authors contributed equally to this work.
Article first published online: 22 APR 2013
Copyright © 2012 American Association for the Study of Liver Diseases
Volume 57, Issue 5, pages 2037–2048, May 2013
How to Cite
Martin, J., Maurhofer, O., Bellance, N., Benard, G., Graber, F., Hahn, D., Galinier, A., Hora, C., Gupta, A., Ferrand, G., Hoppeler, H., Rossignol, R., Dufour, J.-F. and St-Pierre, M. V. (2013), Disruption of the histidine triad nucleotide-binding hint2 gene in mice affects glycemic control and mitochondrial function. Hepatology, 57: 2037–2048. doi: 10.1002/hep.26060
Potential conflict of interest: Nothing to report.
This work was supported by Swiss National Foundation Grant 31003A-140930 (to J.-F. D.). J.-F. D. received funding from the European Community's Seventh Framework Programme under grant agreement HEALTH-F2-2009-241762 for the project FLIP. N. B. received a grant from INSERM-Région Aquitaine. J. M. received a fellowship from the European Association for the Study of the Liver.
- Issue published online: 22 APR 2013
- Article first published online: 22 APR 2013
- Accepted manuscript online: 7 SEP 2012 09:03AM EST
- Manuscript Accepted: 30 JUL 2012
- Manuscript Received: 5 NOV 2011
Additional Supporting Information may be found in the online version of this article.
|HEP_26060_sm_SuppFig1.tif||78K||Supporting 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.tif||5135K||Supporting 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.tif||276K||Supporting 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.tif||141K||Supporting 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.tif||100K||Supporting 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.tif||6152K||Supporting 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.tif||100K||Supporting 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_SuppTab1.doc||50K||Supporting Information Table 1. List of primary antibodies.|
|HEP_26060_sm_SuppTab2.doc||21K||Supporting Information Table 2. List of secondary antibodies for Western Blot.|
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