Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/β hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/β-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

(A) Despite significant reductions in several cardiolipin species in male mice, total hepatic cardiolipin levels in male and female mice did not differ by genotype. However, total phosphatidylglycerol concentrations were decreased Abhd2 KO mice compared to WT males (p<0.01). (B) Mitochondrial gene expression, measured as a proxy for mitochondrial number, was not different by sex or genotype. Neither genotype nor sex affected fatty acyl composition of PC, PG or CL in the livers of HF/HS-fed mice. (C) The hepatic phosphatidylcholine landscape was diverse and were primarily comprised of acyl chains of C16 or C18 in length and were saturated or monounsaturated. (D) Phosphatidylglycerols were equally represented by fatty acyl lengths of C16 and C18 and contained 0 or 1 double bond. (E) Cardiolipins were highly represented by linoleate, with C18 being 95% of acyl lengths and 98% of CLs containing 1 or more double bonds. Figure S2. Whole-body deletion of Abhd2 did not alter growth rates nor fasting blood profiles in C57Bl6/J mice. Abhd2 KO female (A) and male (E) mice showed similar growth curves to WT mice. Fasting glucose (B, F), insulin (C, G), and triglycerides (D, H) did not differ by genotype.
. CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. Figure S3. Loss of Abhd2 altered body compositions of female mice by increasing fat mass as measured by DEXA. Body compositions of mice were measured at ~24 weeks of age by DEXA. (A) Body mass of female mice were not significantly different. Fat mass, both as total weight (B) and %body weight (C) increased in Abhd2 KO female mice. Lean mass weight (D) did not change with genotype in females, but lean mass as %body weight (E) was reduced in Abhd2 KO female mice. Male mice were not different in total body weight, fat, nor lean mass (F-J). *p<0.05 . CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ; Figure S4. Assessment of insulin action by oral glucose tolerance test (oGTT) elicited similar responses between genotypes of the same sex (A) Female Abhd2 KO mice showed a trend for increased plasma glucose at 15 and 30minute timepoints during the oGTT. Male Abhd2 KO mice were not different. (B) Area under the curve (AUC) for plasma glucose during the oGTT did not differ by genotype. (C) Plasma insulin response to glucose stimulation were the same for genotypes of each sex, with all mice returning to baseline within two hours of receiving the glucose bolus. (D) Insulin curve AUCs were not different. (E) C-peptide, a marker of insulin secretion, was the same for genotypes of each sex during the oGTT, with no difference in AUC (F). (G) The C-peptide/insulin ratio, used as a surrogate for insulin clearance, were not different at 0, 15, and 30 minutes. (H) AUCs for C-peptide/insulin ratio were similar between genotypes of the same sex.
. CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ; https://doi.org/10.1101/2023.03.23.533902 doi: bioRxiv preprint Figure S5. β3-adregeneric receptor agonist stimulation failed to produce a physiologic response in Abhd2 KO mice Plasma glucose concentrations at various time points (A) and total AUC for glucose (B) during β3-adregeneric receptor agonist stimulation was not different in male or female Abhd2 KO mice. Plasma insulin concentrations (C) and total AUC for insulin (D) during the B3TT were the same for genotypes of each sex. Non-esterified fatty acid (NEFA) concentration (E) and total AUC for NEFA (F), and glycerol concentration (G), and AUC for glycerol (H) during the β3TT did not different for Abhd2 KO female or male mice.
. CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ; Figure S6. Loss of Abhd2 does not alter the physiological response to prolonged fasting or refeeding. Following a 24-hr fast, female mice averaged a 1.7 ± 0.9 gm weight loss and an average 1.1 ± 0.1 gm weight gain following the 6-hour refeed period and were not different for Abhd2 KO versus WT mice (A). Plasma NEFAs, measured before and after prolonged fast, were similar between genotypes (B). Male mice lost 2.5 ± 0.2 gm with prolonged fasting and regained 0.6 ± 0.1 gm following refeeding, and were not different between genotypes (C). Plasma NEFAs of male mice during the fast/refeed protocol did not differ by genotype (D).
. CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ; Figure S7. Loss of Abhd2 did not alter insulin secretion in response to glucose or monoacylglycerol Insulin secretion in response to varying glucose concentration, or two different monoacylglycerols (2-AG or 1-PG) (A) or total islet insulin content (B) remained unchanged in cultured islets from female and male Abhd2 KO versus WT mice.
. CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made   CC-BY 4.0 International license available under a hich was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted March 24, 2023. ;https://doi.org/10.1101https://doi.org/10. /2023