Paradoxical prosteatotic effect of hedgehog signaling pathway inhibition under conditions of steatosis


  • Potential conflict of interest: Nothing to report.

To the Editor:

We read with great interest the article recently published in this journal by Dr. Guy et al.1 The authors show a direct correlation between liver damage and deregulated Hedgehog (HH)-pathway in liver biopsies from a cohort of 90 nonalcoholic fatty liver disease (NAFLD) patients. They demonstrate the association between HH-producing/responsive target cells and fibrosis stage. Shh and Gli2-expressing cells have been positively correlated with portal inflammation, ballooning, and fibrosis stage. Furthermore, they reported a pivotal role of the HH-pathway in both hepatic and extrahepatic tissue, highlighted by the colocalization of Gli2 with vimentin or α-smooth muscle actin.

Guy et al. hypothesize the possibility to control the HH signaling pathway through specific inhibitors as a useful tool to hamper the progression of NAFLD.

In this regard we wish to report our preliminary data. We treated Huh7.5.1 cells with a combination of fatty acids (FAs), palmitic and oleic acid (1 mM), for 14 hours to mimic the intrahepatic fat accumulation typical of NAFLD. Cells were concomitantly treated with cyclopamine (5 μM), a well-known HH-pathway antagonist that inhibits Smoothened, for three different timepoints (2, 7, and 12 hours) during the 14-hour treatment with FAs.2

By a fluorescence-based AdipoRed assay we observed a discrete lipid accumulation in the FA-treated cells compared to controls. Strikingly, in contrast to the therapeutic approach suggested by Guy et al., addition of cyclopamine within the 14 hours to the FA-containing medium did not counteract the intracellular lipid accumulation as expected, but rather, increased the lipid content (Fig. 1A). Moreover, in our experimental conditions, real-time polymerase chain reaction (RT-PCR) analysis showed that cyclopamine did not decrease the expression levels of the HH-target genes (Shh and Gli1) in FA-treated cells. Conversely, in line with the evidence reported in the literature, by administering only cyclopamine to the control cultures, with the timing described above, we observed its known inhibitory effect on the expression of the HH-target genes (Fig. 1B).3

Figure 1.

Prosteatotic effect of cyclopamine in FA-treated Huh7.5.1 cells. (A) Cyclopamine increases intracellular lipid accumulation in cells treated with FAs. Experiments were conducted in triplicate. (B) Cyclopamine and cyclopamine plus FA effect on relative expression of Shh and Gli1 mRNAs measured by RT-PCR. Experiments were conducted in triplicate.

Therefore, even though HH-antagonists could be useful to correct liver damage occurring in nonalcoholic steatohepatitis (NASH) (i.e., inflammation, ballooning, and fibrosis), cyclopamine does not work as HH-inhibitor when used under conditions of FA excess and even exacerbates simple steatosis with still unknown consequences. Based on these findings, we want to point out the fact that pharmacologic administration of HH-pathway antagonists in NAFLD should be carefully evaluated.

Manuele Gori Ph.D.*, Barbara Barbaro Ph.D.* †, Mario Arciello Ph.D.*, Clara Balsano M.D.* †, * Laboratory of Molecular Virology and Oncology, Fondazione A. Cesalpino, Rome, Italy, † Department of Internal Medicine (M.I.S.P), University of L'Aquila, L'Aquila, Italy.