Regulation of energy metabolism in hepatocytes with induced steatosis

Mitochondria are cellular organelles involved in variety of metabolic pathways and important for ATP production. Based on our new findings the Hedgehog (Hh) signaling pathway is an active pathway in healthy mature hepatocytes with relevant impact on endocrine liver function and is involved in lipid metabolism [1,2]. In this study we explored the effects of nutritive induced steatosis in combination with hepatocyte-specific deletion of Hh signaling transcription factor Gli3 on the regulation of energy metabolism in mitochondria. For this analysis we used a transgenic mouse model with deletion of Gli3 (KO) and fed mice for four and ten weeks with control (CD) and high fat diet (HFD). To explore the endogenous respiration a Clark type oxygen electrode was used and the oxygen consumption was monitored. The ATP-level was determined by the CellTiter-Glo® Luminescent Cell Viability Assay. Furthermore, the activity of mitochondrial enzymes of respiratory chain complexes was assessed by photometric assays. The mRNA expression levels of selected complex subunits and genes encoded mitochondrial proteins were analysed by qrt-PCR. In general we could observe that hepatocytes from control mice fed with HFD show higher oxygen consumption than control mice fed with CD after ten weeks. Compared with wild type mice, hepatocytes from Gli3-KO mice fed with CD showed increased oxygen consumption after four and ten weeks, which is also true for Gli3-KO mice fed with HFD. Considering the ATP-level the results show that hepatocytes from control mice fed with HFD have decreased amount compared to control mice fed with CD. Referring Gli3-KO mice fed with CD we measured reduced ATP-amount compared to wild type mice after four and ten weeks. The same was true for Gli3-KO mice fed with HFD for four weeks. Interestingly, ATP-level in Gli3-KO mice fed with HFD for ten weeks was slightly increased compared to control mice. We could show that mice fed with HFD have altered mitochondrial functions characterized by higher oxygen consumption and lower ATP-level. The same was true for Gli3-KO mice fed with CD and become more pronounced when feeding Gli3-KO mice with HFD. These results suggest that interrupting the transcriptional output of Hh signaling in the liver at the level of the transcription factor Gli3 causes changes in mitochondrial function which resemble those seen after feeding HFD. However, it remains to be explored whether the low ATP-levels (in spite of an increased oxygen consumption) are due to a partial uncoupling of the mitochondria or whether they reflect simply a low steady state level resulting from an unusually high energy demand.

[1] Matz-Soja et al., Hepatic Hedgehog signaling contributes to the regulation of IGF1 and IGFBP1 serum levels. Cell Commun Signal. 2014, 18;12:11.

[2] Pospisilik JA et al., Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate. Cell. 2010, 8;140(1):148 – 60

Corresponding author: Schönefeld, Kristin

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