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bioRxiv · 10.1101/2024.08.02.606015

PNPLA3(148M) Promotes Hepatic Steatosis by Interfering with Triglyceride Hydrolysis Through a Gain-of-Function Mechanism

Wang, Y.·Hong, S.·Hudson, H.·Kory, N.·Kinch, L. N.·Kozlitina, J.·Cohen, J. C.·Hobbs, H. H.
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Abstract

Background & AimsPNPLA3(148M) (patatin-like phospholipase domain-containing protein 3) is the most impactful genetic risk factor for steatotic liver disease (SLD), thus motivating a search for therapeutic modulators of its expression. A key unresolved issue is whether PNPLA3(148M) confers a loss- or gain-of-function. Here we used multiple approaches to further test the hypothesis that PNPLA3 causes steatosis by sequestering ABHD5 (/{beta} hydrolase domain containing protein 5), the cofactor of ATGL (adipose TG lipase), thus limiting mobilization of hepatic triglyceride (TG). MethodsWe quantified the physical interactions between ABHD5 and PNPLA3/ATGL in cultured hepatocytes using NanoBiT complementation assays. Immunocytochemistry was used to compare the relative binding of PNPLA3 and ATGL to ABHD5 and to determine if PNPLA3 must associate with lipid droplets (LDs) to inhibit ATGL. Adenoviruses and adeno-associated viruses were used to express PNPLA3 in liver-specific Atgl-/- mice and ABHD5 in livers of Pnpla3148M/M mice, respectively. We used purified recombinant proteins to compare the TG hydrolytic activities of PNPLA3 and ATGL in the presence and absence of ABHD5. ResultsABHD5 interacted preferentially with PNPLA3 relative to ATGL in cultured hepatocytes and in vitro, with no differences observed between PNPLA3(WT) or PNPLA3(148M). PNPLA3(148M)-associated inhibition of TG hydrolysis required localization of PNPLA3 to LDs and the presence of ATGL. Finally, overexpression of ABHD5 reversed the hepatic steatosis in Pnpla3M/M mice. ConclusionsThese findings support the premise that PNPLA3(148M) promotes hepatic steatosis by accumulating on LDs and inhibiting ATGL-mediated lipolysis in an ABHD5-dependent manner. Our results predict that reducing, rather that increasing PNPLA3 expression will be the best strategy to treat PNPLA3(148M)-associated SLD. Impact and implicationsSteatotic liver disease (SLD) is a common complex disorder associated with both environmental and genetic risk factors. PNPLA3(148M) is the most impactful genetic risk factor for SLD and yet its pathogenic mechanism remains controversial. Here we provide evidence that PNPLA3(148M) promotes triglyceride (TG) accumulation by sequestering ABHD5, thus limiting its availability to activate ATGL. Although the substitution of methionine for isoleucine reduces the TG hydrolytic activity of PNPLA3, the loss-of-function is only indirectly related to the steatotic effect of the variant. Here we provide evidence that PNPLA3(148M) confers a gain-of-function by interfering with ATGL-mediated TG hydrolysis. These findings have implications for the design of potential PNPLA3-based therapies. Reducing, rather than increasing, PNPLA3 levels is predicted to reverse steatosis in susceptible individuals. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/606015v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1dd7acaorg.highwire.dtl.DTLVardef@1a869dborg.highwire.dtl.DTLVardef@1bde743org.highwire.dtl.DTLVardef@99effc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIABHD5 binds preferentially to PNPLA3 relative to ATGL. C_LIO_LIPNPLA3(WT) and PNPLA3(148M) compete similarly for binding and inhibition of ATGL. C_LIO_LIABHD5 activates the triglyceride lipase activity of PNPLA3, as well as ATGL. C_LIO_LIThe steatotic effect of PNPLA3(148M) requires expression of ATGL. C_LIO_LIOverexpression of ABHD5 can rescue the steatosis associated with PNPLA3(148M). C_LI

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Wang, Y., Hong, S., Hudson, H., Kory, N., Kinch, L. N., Kozlitina, J., Cohen, J. C., Hobbs, H. H.. 2024-08-04. PNPLA3(148M) Promotes Hepatic Steatosis by Interfering with Triglyceride Hydrolysis Through a Gain-of-Function Mechanism. https://doi.org/10.1101/2024.08.02.606015

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