Modulation of HDL metabolism by the niacin receptor GPR109A in mouse hepatocytes

Abstract

The niacin receptor GPR109A is a G(i)-protein-coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.

Figure 1

Treatment of mice with a diet containing 1% niacin decreases plasma HDL-C in wild-type mice but not in GPR109A knockout mice. GPR109A knockout and wild-type control mice (n=8) were fed a 1% niacin diet for three weeks. (A) Plasma HDL-C levels. Data are expressed as mean ± SEM, **P<0.01, compared with control diet; ##P<0.01 compared with wild type. Total cholesterol FPLC profile in GPR109A knockout (C) and wild-type mice(B). (D) Intracellular cAMP levels in the primary hepatocyte. Primary hepatocytes from GPR109A deficient or wild-type C57BL/6 mice were isolated and plated in 12-well plates. After pretreatment with or without 0, 10, 30 or 100 μM niacin for 1 hour, hepatocytes were treated with 1 mM IBMX and 30 μM forskolin for 15 min at 37° C. The amount of cAMP present was calculated from a standard curve prepared using nonradioactive cAMP and was expressed as picomoles per 1 × 10⁶ cells. Data are mean ± SEM, n = 3, **P<0.01, compared with no niacin treatment.

Figure 2

Effects of liver specific expression of GPR109A or GPR109B on plasma lipids in vivo. C57BL/6 mice (n=7) were injected intraperitoneally with 1x10¹² genome copies (GC) AAV2/8 GPR109A or control virus. (A) Western blot of GPR109A and ABCA1 in liver tissue from GPR109A overexpression and control mice. C57BL/6 mice were injected with 1x10¹² GC AAV2/8 GPR109A or control virus IP, and 8 weeks later livers were harvested for total protein extraction. Experiments were repeated 3 times. (B) Plasma levels of total cholesterol and HDL-C after 8 weeks of transduction. Data are expressed as mean ± SEM. n=7, **P <0.01. (C) Total cholesterol FPLC Profile. C57BL/6 mice (n=8) were injected intraperitoneally with 1x10¹² GC AAV2/8 GPR109B or control virus. (D) Plasma levels of total cholesterol and HDL-C after 8 weeks of transduction. Data are expressed as mean ± SEM. (E) Total cholesterol FPLC Profile.

Figure 3

Effects of 24 hour fasting on both control and liver-specific expression of GPR109A. C57BL/6 mice were injected intraperitoneally with 1x10¹² genome copies AAV2/8 GPR109A or control AAV2/8 Null. After 11 weeks of virus injection, chow-fed mice were subjected to 24 hour fasting; plasma levels of β– hydroxybutyrate (A), total cholesterol (B) and HDL-C (C) were measured. Data are expressed as mean ± SEM, n=7. *P<0.05 compared with 4 hours fasting, #P <0.05, ##P<0.01 compared with AAV-Null.

Figure 4

Plasma kinetics of ³H-cholesteryl ether and ¹²⁵I-labeled tyramine-cellobiose labeled human HDL₃. Blood samples were drawn at the time points indicated and analyzed for radioactivity. (A) ³H-HDL-cholesteryl ether clearance from plasma. (B) ¹²⁵I-Tyramine cellobiose HDL clearance from plasma. (C) Plasma HDL-cholesteryl ether selective uptake. Data are expressed as mean ± SEM, n=6. (D) Plasma HDL-cholesteryl ester transport rate. Data are expressed as mean ± SEM, n = 6, **P<0.01.

Figure 5

GPR109A overexpression inversely related to free cholesterol efflux to apoA-I from hepatocytes. C57BL/6 mice were injected with 1x10¹² GC AAV2/8 GPR109A or control virus IP, and 8 weeks later liver and hepatocytes were isolated. Primary hepatocytes from mice injected with either AAV-GPR109A or AAV-Null were cultured in 12-well plates. (A) GPR109A overexpression decreased intracellular cAMP release in response to forskolin. The amount of cAMP present was calculated from a standard curve prepared using nonradioactive cAMP and was expressed as picomoles per 1 × 10⁶ cells. Data are mean ± SEM, n = 3, **P<0.01, compared with AAV-Null. Primary hepatocytes were labeled with 2 μCi/ml ³H-cholesterol in medium for 24 h; cells were then equilibrated for 18 h at 37°C with DMEM/BSA (B) or containing the indicated reagents 30 μM forskolin plus 1 mM IBMX (C), separately. For cholesterol efflux, medium containing 10 μg/ml free apoA-I was added to cells. 9 hours cholesterol efflux levels were measured. Values represent the average of triplicate determinations normalized to AAV-Null group (mean ± SD), **P<0.01 compared with control vector. (D) Western blot of ABCA1 in liver tissue from GPR109A knockout and wild-type control mice. Experiments were repeated 3 times. (E) Cholesterol efflux to apoA-I from forskolin treated GPR109A knockout and wild-type control primary hepatocytes. Primary hepatocytes from GPR109A deficient or wild type C57BL/6 mice were isolated, labeled, equilibrated with DMEM/BSA containing 30 μM forskolin plus 1 mM IBMX, then performed 9 hours efflux study to human apoA-I. Values represent the average of triplicate determinations normalized to wild type group (mean ± SD), **P<0.01 compared with wild-type.

Figure 6

Overexpression of GPR109A reduces ABCA1 expression and activity in HepG2 cells. (A) Western Blot of GPR109A in HepG2 cells. (B) Intracellular cAMP assay. HepG2 cells transfected with GPR109A or GPR109B or control HepG2 cells were treated with 30 μM forskolin and 1 mM 3-isobutyl-1-methylxanthine for 15 min at 37 °C. Data from 3 independent experiments are expressed as mean ± SD, n =6, **P<0.01, compared with mock group, ##P<0.01, compared with GPR109B group. (C) Western Blot of cell lysate of ABCA1. Experiments were repeated 3 times. (D) Cholesterol efflux to apoA-I from HepG2 cells. Efflux of free cholesterol into the media is expressed as percentage of the total radioactivity in the media and cells (³H-cholesterol in the medium × 100)/(³H-cholesterol in medium + ³H-cholesterol in the cells). Values represent the average of triplicate determinations (mean ± SD), **P<0.01 compared with control vector, ##P<0.01, compared with GPR109B group.