CD9 Counteracts Liver Steatosis and Mediates GCGR Agonist Hepatic Effects

Abstract Glucagon receptor (GCGR) agonism offers potentially greater effects on the mitigation of hepatic steatosis. However, its underlying mechanism is not fully understood. Here, it screened tetraspanin CD9 might medicate hepatic effects of GCGR agonist. CD9 is decreased in the fatty livers of patients and upregulated upon GCGR activation. Deficiency of CD9 in the liver exacerbated diet‐induced hepatic steatosis via complement factor D (CFD) regulated fatty acid metabolism. Specifically, CD9 modulated hepatic fatty acid synthesis and oxidation genes through regulating CFD expression via the ubiquitination‐proteasomal degradation of FLI1. In addition, CD9 influenced body weight by modulating lipogenesis and thermogenesis of adipose tissue through CFD. Moreover, CD9 reinforcement in the liver alleviated hepatic steatosis, and blockage of CD9 abolished the remission of hepatic steatosis induced by cotadutide treatment. Thus, CD9 medicates the hepatic beneficial effects of GCGR signaling, and may server as a promising therapeutic target for hepatic steatosis.


CD9 counteracts liver steatosis and mediates GCGR agonist hepatic effects
Table S1.RNA sequencing analysis of mice treated with a highly selective GCGR agonist (quoted from GEO#GSE135881).
Table S3.Top 5 upregulated and downregulated genes in livers upon CD9 deletion through RNA sequencing analysis.
Table S4.List of primers.AAV-shCtr and AAV-shCD9 mice were fed on HFD for 10 weeks, and received equimolar dosing (30 nmol per kg (body weight)) subcutaneously, once daily for 4 weeks at week 6 of HFD feeding.The protein levels of CD9 and genes related to lipid metabolism in livers.n = 6 mice per group.

Figure S2 .
Figure S2.AAV-shCD9 mice show similar body weight and tissue morphology under NCD.

Figure S3 .
Figure S3.Blood glucose, liver TC, serum lipid contents and lipid metabolism in AAV-shCD9 mice under HFD conditions.

Figure S4 .
Figure S4.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-shCD9 mice after CFD knockdown.

Figure S5 .
Figure S5.Body weight and liver phenotype are comparable in AAV-CFD mice as compared with AAV-Ctr mice under NCD.

Figure S6 .
Figure S6.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-CFD mice.

Figure S7 .
Figure S7.Sequence information of CFD promoter and chromatin immunoprecipitation assay showing the binding of FLI1 on CFD promoter.

Figure S9 .
Figure S9.Hepatic CD9 deficiency in livers does not affect physical activity, respiratory exchange ratio and adipose tissue of CFD expression under HFD.

Figure S10 .
Figure S10.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-CD9 mice.

Figure S11 .
Figure S11.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-shCD9 mice after treated with cotadutide.

Figure S1 .
Figure S1.CD9 and NOX4 expressions responding to cotadutide treatment.(A and B) The mRNA levels (A) and their indicated log-transformed fold change (B) of CD9 and NOX4 in livers of HFD mice treated with cotadutide acetate (Cot).(C) Huh7 cells were treated by free fatty acid (FFA) for 24h, the mRNA and protein levels of CD9 were assessed after Cot treatment.n = 6 mice per group for A and B, n = 3 for C. Data are presented as the mean ± S.E.M. ***, P < 0.001.

Figure S2 .
Figure S2.AAV-shCD9 mice show similar body weight and tissue morphology under NCD.(A-H) AAV-shCtr and AAV-shCD9 mice were fed on NCD for 12 weeks.(A) AAV-shCD9 leads to CD9 attenuation exclusively in the liver.(B) Immunofluorescence analyses validates the suppression of CD9 in mouse liver with AAV-shCD9 treatment.(C) Body weight, liver weight and liver/body weight were determined.(D and E) At week 12 of NCD feeding, fasting blood glucose (D) and glucose tolerance test (GTT, E) were performed after overnight fasting.(F) Liver TG and TC were determined enzymatically.(G) Serum levels of TG, TC, LDL and HDL were

Figure S3 .
Figure S3.Blood glucose, liver TC, serum lipid contents and lipid metabolism in AAV-shCD9 mice under HFD conditions.

Figure S4 .
Figure S4.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-shCD9 mice after CFD knockdown.AAV-shCtr and AAV-shCD9 mice were injected with AAV8-TBG-shCFD and fed HFD for 10 weeks before sacrificed.The protein levels of CD9, CFD, and genes related to fatty acid synthesis and oxidation in livers were assessed.n = 5 mice per group.

Figure S5 .
Figure S5.Body weight and liver phenotype are comparable in AAV-CFD mice as compared with AAV-Ctr mice under NCD.(A-E) AAV-Ctr and AAV-CFD mice were fed on NCD for 12 weeks.(A) AAV-CFD leads to CFD overexpression exclusively in the liver.(B) Immunofluorescence analysis validates the overexpression of CFD in mouse liver with AAV-CFD treatment.(C) Body weight, liver weight and liver/body weight were determined.(D) Liver TG was determined enzymatically.(E) H&E staining and NASH activity score of livers.Scale bar = 20 µm for B, 100 µm for E. n = 6 mice per group for A-E.Data are presented as the mean ± S.E.M. ***, P < 0.001.

Figure S6 .
Figure S6.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-CFD mice.AAV-Ctr and AAV-CFD mice were fed on HFD for 12 weeks before sacrificed.The protein levels of CFD and genes related to fatty acid synthesis and oxidation in livers were assessed.n = 6 mice per group.

Figure S7 .
Figure S7.Sequence information of CFD promoter and chromatin immunoprecipitation assay showing the binding of FLI1 on CFD promoter.(A and B) Sequence information of CFD promoter (A) and chromatin immunoprecipitation assay using different lengths of CFD promoter fragments (B) were shown.

Figure S8 .
Figure S8.CD9 regulates hepatic fatty acid synthesis and oxidation through ubiquitinationproteasomal degradation of FLI1.(A)Huh7 cells were co-transfected with siRNA of CD9 and plasmid of FLI1, and then treated with FFA for 24 hours, the protein levels of genes related to lipid metabolism were determined.(B) Huh7 cells were co-transfected with plasmid of CD9 and siRNA of FLI1, and then treated with FFA for 24 hours, the protein levels of genes related to lipid metabolism were determined.(C) FFA treated Huh7 cells were labeled by 13 C isotope, de novo lipogenesis and fatty acid oxidation were assessed under CFD or FLI1 manipulated conditions.(D) FFA-treated Huh7 cells

Figure S9 .
Figure S9.Hepatic CD9 deficiency in livers does not affect physical activity, respiratory exchange ratio and adipose tissue of CFD expression under HFD.AAV-shCtr and AAV-shCD9 mice were fed on HFD for 12 weeks.(A) Food intake was determined.(B and C) Physical activity (B) and respiratory exchange ratio (RER, C) for a 24 h recording period

Figure S10 .
Figure S10.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-CD9 mice.AAV-Ctr and AAV-CD9 mice were fed on HFD for 12 weeks.The protein levels of genes related to lipid metabolism in livers were shown.n = 6 mice per group.

Figure S11 .
Figure S11.The protein levels of fatty acid synthesis and oxidation genes in HFD-fed AAV-shCD9 mice after treated with cotadutide.