A nuclear-receptor-dependent phosphatidylcholine pathway with antidiabetic effects

Abstract

Nuclear hormone receptors regulate diverse metabolic pathways and the orphan nuclear receptor LRH-1 (also known as NR5A2) regulates bile acid biosynthesis. Structural studies have identified phospholipids as potential LRH-1 ligands, but their functional relevance is unclear. Here we show that an unusual phosphatidylcholine species with two saturated 12 carbon fatty acid acyl side chains (dilauroyl phosphatidylcholine (DLPC)) is an LRH-1 agonist ligand in vitro. DLPC treatment induces bile acid biosynthetic enzymes in mouse liver, increases bile acid levels, and lowers hepatic triglycerides and serum glucose. DLPC treatment also decreases hepatic steatosis and improves glucose homeostasis in two mouse models of insulin resistance. Both the antidiabetic and lipotropic effects are lost in liver-specific Lrh-1 knockouts. These findings identify an LRH-1 dependent phosphatidylcholine signalling pathway that regulates bile acid metabolism and glucose homeostasis.

Figure 1. DLPC activates and binds hLRH-1

a, , HeLa cells were transfected with a hLRH-1 expression vector and a luciferase reporter and treated with 100 μM of indicated PCs. Error bars represent mean ± s.e.m. b. The hLRH-1 ligand binding domain (LBD) was expressed and purified as described previously and was incubated at molar ratios of 1:1 and 1:5 (hLRH-1 LBD:PC) with DLPC, DPPC or vehicle for two hours at 37°C, and then repurified by size exclusion chromatography to remove unbound phospholipids. Bound lipids were analyzed using electrospray mass injection-MS in the negative-ion mode. Results with DLPC (1:1), DPPC (1:5) and vehicle are shown, along with analysis of re-extracted DLPC; DLPC (1:5) and DPPC (1:1) incubations were very similar to those shown. The re-extracted DPPC peak is at 768.5, and is not detectable in any of the DPPC incubations.

Figure 2. DLPC and DUPC modulate expression of LRH-1 target genes in liver

a, , 8-week-old male C57BL/6 mice were challenged orally with vehicle, CA, DPPC, DUPC, and DLPC for 3 days. Total liver RNA was isolated and prepared for the cDNA. Hepatic gene expression was determined using qPCR. mRNA levels are relative to 36B4. b, Total BA pool and serum BAs, glucose, TG, NEFA, and cholesterol were measured in the same animals. c, Hepatic TG, NEFA, and cholesterol were measured in the same animals. Error bars represent mean ± s.e.m. (*P<0.05, **P<0.01 vs veh; n=5 animals/group).

Figure 3. DLPC improves glucose homeostasis in mouse models of insulin resistance

a, , Glucose and insulin tolerance were assessed in Lrh-1^f/f and LKO DIO mice 2–3 weeks after vehicle or DLPC treatment. b, Fasting serum glucose and insulin levels were measured in the same animals shown in a. HOMA-IR was calculated from fasting serum glucose and insulin levels. c, The high dose (10 mU/kg/min) hyperinsulinemic-euglycemic clamp (insulin dose (10 mU/kg/min) was used to assess glucose homeostasis in Lrh-1^f/f DIO mice following 3 weeks of vehicle or DLPC treatment. d, Hepatic insulin signaling was examined in Lrh-1^f/f and LKO DIO mice 2 weeks after vehicle or DLPC treatment. Liver tissue homogenates from 3 mice per group were pooled and immunoprecipitation and immunoblotting was as indicated. Results are representative of 3 independent experiments. Error bars represent mean ± s.e.m. (*P<0.05, **P<0.01 vs Lrh-1^f/f DIO mice treated with veh; n=4 animals/group).

Figure 4. DLPC reduces liver fat accumulation by suppressing lipogenesis

a, , Liver sections from Lrh-1^f/f and LKO DIO mice treated for 3 weeks with vehicle or DLPC were stained with Hematoxylin/Eosin (H & E) for general morphology or Oil Red O (ORO) for lipid accumulation. Original magnification, X10. b, Hepatic and serum BA, TG, and NEFA levels were measured in the same mice described in a. c, Lipogenic gene expression in the liver was determined using qPCR. mRNA levels are relative to 36B4. Error bars represent mean ± s.e.m. (*P<0.05, **P<0.01 vs Lrh-1^f/f DIO mice treated with veh; n=4 animals/group).