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Comparative Study
. 2014 Dec;55(12):2501-8.
doi: 10.1194/jlr.M050955. Epub 2014 Oct 15.

Inflammation stimulates niacin receptor (GPR109A/HCA2) expression in adipose tissue and macrophages

Kenneth R Feingold  1 , Arthur Moser  1 , Judy K Shigenaga  1 , Carl Grunfeld  1
Affiliations
Comparative Study

Inflammation stimulates niacin receptor (GPR109A/HCA2) expression in adipose tissue and macrophages

Kenneth R Feingold et al. J Lipid Res. 2014 Dec.

Abstract

Many of the beneficial and adverse effects of niacin are mediated via a G protein receptor, G protein-coupled receptor 109A/hydroxycarboxylic acid 2 receptor (GPR109A/HCA2), which is highly expressed in adipose tissue and macrophages. Here we demonstrate that immune activation increases GPR109A/HCA2 expression. Lipopolysaccharide (LPS), TNF, and interleukin (IL) 1 increase GPR109A/HCA2 expression 3- to 5-fold in adipose tissue. LPS also increased GPR109A/HCA2 mRNA levels 5.6-fold in spleen, a tissue rich in macrophages. In peritoneal macrophages and RAW cells, LPS increased GPR109A/HCA2 mRNA levels 20- to 80-fold. Zymosan, lipoteichoic acid, and polyinosine-polycytidylic acid, other Toll-like receptor activators, and TNF and IL-1 also increased GPR109A/HCA2 in macrophages. Inhibition of the myeloid differentiation factor 88 or TIR-domain-containing adaptor protein inducing IFNβ pathways both resulted in partial inhibition of LPS stimulation of GPR109A/HCA2, suggesting that LPS signals an increase in GPR109A/HCA2 expression by both pathways. Additionally, inhibition of NF-κB reduced the ability of LPS to increase GPR109A/HCA2 expression by ∼50% suggesting that both NF-κB and non-NF-κB pathways mediate the LPS effect. Finally, preventing the LPS-induced increase in GPR109A/HCA2 resulted in an increase in TG accumulation and the expression of enzymes that catalyze TG synthesis. These studies demonstrate that inflammation stimulates GPR109A/HCA2 and there are multiple intracellular signaling pathways that mediate this effect. The increase in GPR109A/HCA2 that accompanies macrophage activation inhibits the TG accumulation stimulated by macrophage activation.

Keywords: G protein-coupled receptor 109A; G proteins; cytokines; diacylglycerol transferase; hydroxycarboxylic acid 2 receptor; lipopolysaccharide; triglycerides.

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Figures

Fig. 1.
Fig. 1.
Effect of LPS or cytokines on GPR109A/HCA2 mRNA levels in adipose. A: Mice were injected intraperitoneally with LPS (5 mg/kg), and the animals were euthanized at 16 h after LPS administration. Total RNA was isolated from adipose tissue. B: 3T3-L1 adipocytes were treated for 24 h with LPS (100 ng/ml), TNFα (10 ng/ml), or IL-1β (10 ng/ml), and total RNA was isolated. GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3–4 per group. ** P < 0.01, *** P < 0.001.
Fig. 2.
Fig. 2.
Effect of LPS on GPR109A/HCA2 mRNA levels in immune tissue and cells. A: Mice were injected intraperitoneally with LPS (5 mg/kg), and the animals were euthanized at 16 h after LPS administration. Total RNA was isolated from spleen. B: Peritoneal macrophages were harvested from C57BL/6 mice, treated for 16 h with LPS (100 ng/ml), and total RNA was isolated. C: RAW cells were treated for 16 h with LPS (100 ng/ml), and total RNA was isolated. GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3–4 per group. *** P < 0.001.
Fig. 3.
Fig. 3.
Immunofluorescence analysis for GPR109A/HCA2 in RAW cells. Cells were treated with LPS at 100 ng/ml in serum-free medium for 16 h. Immunostaining was performed as described in Materials and Methods. Fluorescent GPR109A/HCA2 staining was visualized by confocal microscopy with a 40× oil immersion objective lens. All images were acquired with identical settings.
Fig. 4.
Fig. 4.
LPS induction of GPR109A/HCA2 mRNA levels in RAW cells. A: RAW cells were treated with LPS (100 ng/ml) for indicated times. B: RAW cells were treated for 16 h with indicated concentrations of LPS. Total RNA was isolated, and GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3 per group. ** P < 0.01, *** P < 0.001.
Fig. 5.
Fig. 5.
Effect of immune stimulators on GPR109A/HCA2 mRNA levels in RAW cells. A: RAW cells were treated with zymosan (500 μg/ml), LTA (1 μg/ml), or poly I:C (50 μg/ml) for 16 h. B: RAW cells were treated with TNFα, IL-1β, or IL-6 at10 ng/ml for 16 h. Total RNA was isolated, and GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3 per group. ** P < 0.01, *** P < 0.001.
Fig. 6.
Fig. 6.
Effect of LPS on GPR109A/HCA2 mRNA in AcLDL-loaded RAW cells. RAW cells were coincubated with AcLDL (100 μg/ml) and LPS (100 ng/ml) for 16 h. Total RNA was isolated, and GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3 per group. *** P < 0.001.
Fig. 7.
Fig. 7.
Effect of inhibitors on GPR109A/HCA2 mRNA levels in RAW cells. Cells were preincubated for 1 h with thalidomide (500 μg/ml) (A), BX795 (10 μM) (B), or PTN (20 μM) (C) and LPS (100 ng/ml) for 16 h. Total RNA was isolated, and GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3 per group. * P < 0.05, ** P < 0.01, *** P < 0.001.
Fig. 8.
Fig. 8.
Effect of inhibition of GPR109A/HCA2 on TG accumulation in RAW cells. Cells were transiently transfected with murine GPR109A/HCA2 siRNA or negative control siRNA, followed by treatment with LPS. A: Cells were treated with LPS for 16 h, total RNA was isolated, and GPR109A/HCA2 mRNA levels were quantified by qPCR performed as described in Materials and Methods. Data are expressed as fold increase over controls. B: Cells were treated with LPS for 24 h, and TG levels were measured as described in Materials and Methods. Data are presented as μg TG per mg protein. C: Cells were coincubated with Intralipid and LPS for 24 h, and TG levels were measured. Data are presented as μg TG per mg protein. D: Cells were coincubated with MPN and LPS for 24 h, and TG levels were measured. Data are presented as μg TG per mg protein. The data are presented as the mean ± SEM. N = 3 per group. ** P < 0.01, *** P < 0.001.
Fig. 9.
Fig. 9.
Effect of inhibition of GPR109A/HCA2 on gene expression in RAW cells. Cells were transiently transfected with murine GPR109A/HCA2 siRNA or negative control siRNA, followed by treatment with LPS. A: Cells were treated with LPS for 16 h, total RNA was isolated, and GPAT3 mRNA levels were quantified by qPCR performed as described in Materials and Methods. B: Cells were treated with LPS for 16 h, total RNA was isolated, and DGAT2 mRNA levels were quantified by qPCR. Cells were coincubated with MPN and LPS for 16 h, total RNA was isolated, and GPAT3 mRNA levels (C) or DGAT2 mRNA levels (D) were quantified by qPCR performed as described in Materials and Methods. Data are expressed as fold increase over controls. The data are presented as the mean ± SEM. Data are expressed as fold increase over controls. N = 3 per group. ** P < 0.01, *** P < 0.001.
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