*Purpose: Glycogen synthase kinase 3 (Gsk3) has α and β isoforms. They are constitutively active in cells and inhibited upon stimulations by N-terminal serine phosphorylation. Although roles of active Gsk3 in ischemia reperfusion injury (IRI) have been well appreciated, the significance of Gsk3 inhibitory phosphorylation has not been fully understood. The potential functional difference of Gsk3 isoforms in the disease processes has never been properly addressed.

*Methods: In a murine liver partial warm ischemia model (90min. ischemia), we compared Gsk3 wild-type (WT), α and β double or single phosphorylation-resistant (Gsk3 αS21A and/or βS9A) mutant knock-in (KI) mice to study whether and how Gsk3 inhibitory phosphorylation regulated liver IRI.

*Results: Liver Gsk3 phosphorylation was transient downregulated by ischemia in both its isoforms and gradually increased by reperfusion up to 6h. Gsk3 α but not β phosphorylation-resistant mutation protected mice from liver IRI, as evidenced by lower sALT levels and better preserved liver histological architectures at 6h and 24h post reperfusion in Gsk3 α single and αβ double mutant KI mice, compared with β single mutant KI and WT counterparts. To determine the regulatory mechanism of Gsk3 inhibitory phosphorylation in the disease process, we studied macrophage activation and hepatocyte death in vitro. Gsk3 mutant KI macrophages, both Kupffer cells and bone-marrow derived macrophages, produced more TNF-a and IL-6, but less IL-10, upon TLR4 stimulation. Hepatocytes of the same genotype, however, were indeed protected from TNF-a and stress-induced cell death. To confirm that Gsk3 phosphorylation-resistant mutation plays distinctive roles in liver parenchymal vs. non-parenchymal cells, we studied Gsk3 mutant KI bone marrow (BM) chimeras. Only mutant KI recipients of WT BM were protected from liver IRI, while WT recipients of KI mutant BM suffered more severe liver injuries. Liver autophagy was enhanced in Gsk3 mutant KI mice upon IR. Inhibition of autophagy abrogated liver protection from IRI in these mice. Gsk3 mutant KI resulted in increased activation of HIV-1 TAT-interactive protein 60 (TIP60) and AMP-activated protein kinase, but inhibition of mammalian target of rapamycin complex 1, leading to enhanced autophagy induction and increased resistance to inflammatory cell death in hepatocytes.

*Conclusions: Gsk3 α but not β phosphorylation promoted liver IRI by the inhibition of hepatocyte autophagy.

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