The Senescence-associated Secretome of Hedgehog-deficient Hepatocytes Drives MASLD Progression

The burden of senescent hepatocytes correlates with MASLD severity but mechanisms driving senescence, and how it exacerbates MASLD are poorly understood. Hepatocytes become senescent when Smoothened (Smo) is deleted to disrupt Hedgehog signaling. We aimed to determine if the secretomes of Smo-deficient hepatocytes perpetuate senescence to drive MASLD progression. RNA seq analysis confirmed that hepatocyte populations of MASLD livers are depleted of Smo(+) cells and enriched with senescent cells. When fed CDA-HFD, Smo(-) mice had lower antioxidant markers and developed worse DNA damage, senescence, MASH and liver fibrosis than Smo(+) mice. Sera and hepatocyte-conditioned medium from Smo(-) mice were depleted of thymidine phosphorylase (TP), a protein that maintains mitochondrial fitness. Treating Smo(-) hepatocytes with TP reduced senescence and lipotoxicity; inhibiting TP in Smo(+) hepatocytes had the opposite effects and exacerbated hepatocyte senescence, MASH, and fibrosis in CDA-HFD-fed mice.Therefore, we found that inhibiting Hedgehog signaling in hepatocytes promotes MASLD by suppressing hepatocyte production of proteins that prevent lipotoxicity and senescence. Overall design: Adult male Smo tm2Amc /J (Smoflox/flox) mice on a C57Bl6/J background (JAX stock# 004526; The Jackson Laboratory, Bar Harbor, ME) were used. To examine the impact of Smo on MASLD susceptibility, Smo flox/flox mice were fed with a CDA-HFD diet (A06071302, choline-deficient, L-amino acid defined diet with 60 kcal% fat; Research Diets, Inc, New Brunswick, NJ) for 6 weeks. 1 week before sacrifice, mice were injected by tail vein with 5 × 10e11 genome equivalents of AAV8-TBG-Luciferase (control) or AAV8-TBG-Cre Recombinase (Smo KO) to selectively delete Smo in hepatocytes. RNA was isolated from liver tissue and sent for RNA-seq analyses.