(Pro)renin Receptor Inhibition Reprograms Hepatic Lipid Metabolism and Protects Mice From Diet-Induced Obesity and Hepatosteatosis

Abstract

Rationale: An elevated level of plasma LDL (low-density lipoprotein) is an established risk factor for cardiovascular disease. Recently, we reported that the (pro)renin receptor ([P]RR) regulates LDL metabolism in vitro via the LDLR (LDL receptor) and SORT1 (sortilin-1), independently of the renin-angiotensin system.

Objectives: To investigate the physiological role of (P)RR in lipid metabolism in vivo.

Methods and results: We used N-acetylgalactosamine modified antisense oligonucleotides to specifically inhibit hepatic (P)RR expression in C57BL/6 mice and studied the consequences this has on lipid metabolism. In line with our earlier report, hepatic (P)RR silencing increased plasma LDL-C (LDL cholesterol). Unexpectedly, this also resulted in markedly reduced plasma triglycerides in a SORT1-independent manner in C57BL/6 mice fed a normal- or high-fat diet. In LDLR-deficient mice, hepatic (P)RR inhibition reduced both plasma cholesterol and triglycerides, in a diet-independent manner. Mechanistically, we found that (P)RR inhibition decreased protein abundance of ACC (acetyl-CoA carboxylase) and PDH (pyruvate dehydrogenase). This alteration reprograms hepatic metabolism, leading to reduced lipid synthesis and increased fatty acid oxidation. As a result, hepatic (P)RR inhibition attenuated diet-induced obesity and hepatosteatosis.

Conclusions: Collectively, our study suggests that (P)RR plays a key role in energy homeostasis and regulation of plasma lipids by integrating hepatic glucose and lipid metabolism.

Keywords: dyslipidemia; hypercholesterolemia; hypertriglyceridemia; liver; renin–angiotensin system; vacuolar H+-ATPase.

Figure 1.. Inhibiting hepatic (pro)renin receptor ([P]RR) induces hypercholesterolemia by reducing hepatic LDL (low-density lipoprotein) clearance in normal diet (ND)–fed C57BL/6 mice.

Eight-week-old male C57BL/6 mice were injected with either saline (blue), G-control (magenta), or G-(P)RR (green) intraperitoneally. Mice were euthanized after 7 days, and blood samples were collected for (A) determining circulating levels of cholesterol (n=12–18 per group). Each bar and error represent the mean±SEM; ***P<0.001, or (B) pooled plasma samples were loaded on FPLC (fast protein liquid chromatography) for lipoprotein fractionation analysis, and cholesterol content in each fraction was determined. C, Seven days after injection, mice (n=6 per group) were injected with 50 μg Dil-labeled human LDL. Blood samples were drawn retro-orbitally at the indicated time points, and the Dil-LDL was determined. Each point represents the mean±SEM, and the area under curve (AUC) was constructed for each group and used to compare the difference in LDL clearance. ***P<0.001. D, Blood was collected as in (A) and used to determine plasma triglyceride levels. n=12–18 per group; Each bar and error represent the mean±SEM. ***P<0.001. E, Seven days after injection, mice (n=6 per group) were fasted for 6 hours and injected with Pluronic F127 to inhibit lipoprotein lipase. Blood samples were drawn retro-orbitally at the indicated time points, and the concentration of triglycerides was determined. The mean VLDL (very-low-density lipoprotein) secretion for saline-injected, G-control–injected, or G-(P) RR–injected mice is 474±16, 460±14, and 342±10 mg/dL·h, respectively. The AUC was calculated for individual mice and used to compare the differences in the rate of VLDL secretion. ***P<0.001; G-control vs G-(P)RR. HDL indicates high-density lipoprotein; and IDL, intermediate-density lipoprotein.

Figure 2.. Inhibiting hepatic (pro)renin receptor ([(P]RR) does not result in hypercholesterolemia in high-fat diet (HFD)–fed C57BL/6 mice.

Eight-week-old male C57BL/6 mice were injected with either saline (blue), G-control (magenta), or G-(P)RR (green), and fed anHFD for 4 weeks. A, Plasma cholesterol concentrations were determined weekly, and each point represents the mean±SEM. n=10 per group; ***P<0.001; G-control vs G-(P)RR. B, C, Pooled plasma samples were collected after the (B) first week of diet, or (C) after 4 weeks of diet, and the lipoprotein distribution was determined. The cholesterol content in each fraction was determined and is plotted. D, Two weeks after start of HFD diet, mice (n=6 per group) were injected with 50 μg Dil-labeled human LDL, and LDL clearance was assessed. Each point represents the mean±SEM, and the area under curve (AUC) was constructed for each treatment and used to compare the differences in LDL (low-density lipoprotein) clearance. *P<0.05. E, Two weeks after (P)RR inhibition, mice were fasted for 6 hours, and VLDL (very-low-density lipoprotein) secretion was assessed (n=6 per group) by injecting mice with Pluronic F127 to inhibit lipoprotein lipase. Blood samples were drawn retro-orbitally at the indicated time points, and the concentration of triglycerides was determined, and the AUC was calculated and used to compare the differences in the rate of VLDL secretion. **P<0.01; G-control vs G-(P)RR. F, G, Plasma triglyceride levels were analyzed in samples collected after 4 weeks of HFD. Each bar represents the mean±SEM, n=10 per group. ***P<0.001, or (G) pooled plasma samples were analyzed by FPLC (fast protein liquid chromatography). H, Plasma LPL (lipoprotein lipase) activity was determined for mice were injected with G-control or G-(P)RR and fed HFD for 4 weeks. n=9 per group. HDL indicates high-density lipoprotein; IDL, intermediate-density lipoprotein; and N.S., not significant.

Figure 3.. SORT1 (sortilin-1) overexpression prevents PRR-dependent hypercholesterolemia, but does not affect reduction in plasma triglycerides.

Eight-week-old male C57BL/6 mice were injected with G-control or G-(pro)renin receptor ([P] RR) intraperitoneally and subsequently injected with either adenovirus carrying GFP (green fluorescent protein; Ad-GFP) or adenovirus carrying human SORT1 (Ad-SORT1) via the tail vein. Mice were fed with normal diet (ND) or high-fat diet (HFD) for 1 week and (A) plasma cholesterol levels were determined. Each bar represents the mean±SEM (n=6 per group). **P<0.01; ***P<0.001. Alternatively, (B) lipoprotein composition in pooled plasma samples was analyzed by fractionation. C, Plasma was collected as in (A) and analyzed for triglyceride content. Each bar and error represent the mean±SEM (n=6 per group). **P<0.01. HDL indicates high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; and VLDL, very-low-density lipoprotein.

Figure 4.. Hepatic (pro)renin receptor ([P]RR) inhibition in the absence of LDLR (low-density lipoprotein receptor) reduces plasma lipid levels and hepatic lipid deposition.

Eight-week-old male C57BL/6 mice were injected intraperitoneally with 10×10¹⁰ genomic copies of mouse PCSK9 (proprotein convertase subtilisin/kexin type 9) D377Y adeno-associated virus (AAV), and fed with normal diet (ND) for 4 weeks. Subsequently, mice were injected with either saline (blue), G-control (magenta), or G-(P)RR (green) and fed with ND or high-fat diet (HFD) for an additional 4 weeks. A–D, Plasma cholesterol levels and lipoprotein profiles at the end of study were determined for ND-fed (A, B) and HFD-fed (C, D) mice. E–H, Plasma triglycerides and lipoprotein distribution were determined for ND-fed (E, G)and HFD-fed (F, H) mice. (n=6 per group.) **P<0.01; ***P<0.001; G-control vs G-(P)RR. I, Representative images of Oil Red O–stained liver samples from above-indicated mice fed with ND or HFD for 4 weeks. Scale bar=100 μm. J, K, Lipids were extracted from liver samples and analyzed for triglycerides and cholesterol levels. *P<0.05; G-control vs G-(P)RR. HDL indicates high-density lipoprotein; IDL, intermediate-density lipoprotein; LDL, low-density lipoprotein; and VLDL, very-low-density lipoprotein.

Figure 5.. Hepatic (pro)renin receptor ([P]RR) inhibition attenuates diet-induced obesity and metabolic dysregulation.

Eight-week-old mice were injected with saline (blue), G-control (magenta), or G-(P)RR (green) and fed a high-fat diet (HFD) for 14 weeks. (n=10 per group.) A, Body weight was monitored during the study period and each point and error represent the mean±SEM. ***P<0.001. Representative picture showing that G-(P)RR–injected mice are leaner than control mice. B, Fat and lean mass were measured by EchoMRI. Each bar and error represent the mean±SEM. ***P<0.001. C, Liver weight, and representative pictures showing G-(P)RR–treated mice have less fatty liver. **P<0.01. D, Weight and representative picture of different adipose tissue depots. Brown fat tissue of saline and G-control–injected mice were surrounded with white fat which was removed to give a correct estimation of the weight of the brown fat. ***P<0.001. E, Representative images of Oil RedO (ORO) and H&E (hematoxylin and eosin) staining of the livers (scale bar=200 μm). F, Hepatic lipids were extracted and measured. *P<0.05; G-control vs G-(P)RR. G, Oxygen consumption and 24-h average respiratory quotient (RQ) of G-control–injected and G-(P)RR–injected mice was monitored with a metabolic monitoring system 4 days before euthanize. n=8 per group. BAT indicates brown adipose tissue; eWAT, epididymal white adipose tissue; iWAT, inguinal white adipose tissue; N.S., not significant; and rWAT, retroperitoneal white adipose tissue.

Figure 6.. Inhibiting the (pro)renin receptor ([P]RR) reduces PDH (pyruvate dehydrogenase) and ACC (acetyl-CoA carboxylase) protein abundance and activity.

A, Representative blot of liver samples from mice injected with saline, G-control, or G-(P)RR, and feda high-fat diet (HFD) for 14 weeks. The protein abundance of PDHA (pyruvate dehyrogenase E1 component subunit α), PDHB (pyruvate dehydrogenase E1 component subunit β), and ACCα/β was quantified and normalized to the level of β-actin in the same lysate. (n=6per group); *P<0.05; ***P<0.001. B, C57BL/6 mice were treated with antisense oligonucleotides (ASOs) and fed with HFD for 14 weeks. Hepatic pyruvate concentrations (B), plasma pyruvate concentrations (C), plasma lactate concentrations (D), hepatic PDH activity (E), and acetyl-CoA concentrations (F) were determined. G, Mouse primary hepatocytes were treated with G-control or G-(P)RR for 36 hours, and cellular Acetyl-CoA concentrations were determined. Three independent experiments in triplicates were performed. ***P<0.001. Oxygen consumption rate (OCR; H) and fuel dependency (I) were measured in mouse primary hepatocytes treated with G-control or G-(P)RR for 36 hours. Arrow 1 to 3 indicates addition of oligomycin, FCCP (carbonyl cyanide-4-[trifluoromethoxy]phenylhydrazone) and the mixture of rotenone and antimycin, respectively. n=6 per group. *P<0.05; ***P<0.001. FA indicates fatty acid; and N.S., not significant.

Figure 7.. Model for reprogrammed hepatic metabolism by (pro)renin receptor ([P]RR) inhibition.

Inhibiting hepatic (P)RR reduces PDH (pyruvate dehydrogenase) activity, impairing pyruvate metabolism and reducing acetyl-CoA supply from pyruvate, which limits fatty acid (FA) biosynthesis. (P)RR inhibition further limits FA biosynthesis by reducing protein abundance of ACC (acetyl-CoA carboxylase), the crucial enzyme in FA biosynthesis. It further signals to increase FA oxidation via reduced malonyl-CoA, an inhibitor of FA oxidation that blocks the transportation of long-chain fatty acylcarnitine by carnitine acyltransferase I (CAT1). TCA indicates tricarboxylic acid.