GPR109A (PUMA-G/HM74A) mediates nicotinic acid-induced flushing

Abstract

Nicotinic acid (niacin) has long been used as an antidyslipidemic drug. Its special profile of actions, especially the rise in HDL-cholesterol levels induced by nicotinic acid, is unique among the currently available pharmacological tools to treat lipid disorders. Recently, a G-protein-coupled receptor, termed GPR109A (HM74A in humans, PUMA-G in mice), was described and shown to mediate the nicotinic acid-induced antilipolytic effects in adipocytes. One of the major problems of the pharmacotherapeutical use of nicotinic acid is a strong flushing response. This side effect, although harmless, strongly affects patient compliance. In the present study, we show that mice lacking PUMA-G did not show nicotinic acid-induced flushing. In addition, flushing in response to nicotinic acid was also abrogated in the absence of cyclooxygenase type 1, and mice lacking prostaglandin D(2) (PGD(2)) and prostaglandin E(2) (PGE(2)) receptors had reduced flushing responses. The mouse orthologue of GPR109A, PUMA-G, is highly expressed in macrophages and other immune cells, and transplantation of wild-type bone marrow into irradiated PUMA-G-deficient mice restored the nicotinic acid-induced flushing response. Our data clearly indicate that GPR109A mediates nicotinic acid-induced flushing and that this effect involves release of PGE(2) and PGD(2), most likely from immune cells of the skin.

Figure 1

Nicotinic acid– and acipimox-induced flushing response in the mouse external ear and its desensitization. (A and B) Original recordings of the LDF signal in the ear artery. Mean baseline LDF represents 100% on the vertical scale. Nicotinic acid (NA) or acipimox (APX) were injected i.p. in doses of 200 mg/kg at the time points indicated by arrows. (C) Quantitative analysis of the percentage of LDF increase after administration of NA (white bars, n = 21) and APX (black bars, n = 11) during the first and second peaks of flushing. (D–G) Animals were pretreated with 200 mg/kg (i.p.) of NA (D and E) or APX (F and G) 120 minutes before the administration of NA (D and F) or APX (E and G). Shown are representative recordings of 5–6 experiments per group.

Figure 2

COX-1 but not eNOS is required for NA-induced flushing. (A–C) Original LDF recordings (A and B) and quantitative analysis of the percentage of LDF increase (C) after i.p. administration of 200 mg/kg NA indicate normal first and second peaks of the flushing response in eNOS^–/– mice (B and C, black bars, n = 9) compared with wild-type littermate controls (A and C, white bars, n = 9). (D–F) Original LDF recordings (D and E) and quantitative analysis of the percentage of LDF increase (F) after administration of NA to COX-1^–/– mice (E and F, black bars, n = 7) and wild-type littermates (D and F, white bars, n = 6). **P = 0.002, ***P < 0.001 vs. COX-1^+/+.

Figure 3

NA-induced flushing involves EP₂, EP₄, and DP but not IP. (A–D) Original recordings of the flushing response to nicotinic acid (200 mg/kg i.p.) in DP^–/– (A), EP₂^–/– (B), EP₄^–/– (C), and IP^–/– mice (D). (E) Quantitative analysis of the percentage of LDF increase in response to NA in wild-type control animals (n = 27) and the indicated mutants (n = 6–15). *P = 0.013; ***P < 0.001 vs. control (contr.).

Figure 4

PUMA-G mediates the flushing response to NA. (A–D) Original recordings of the LDF response to i.p. injection of 200 mg/kg NA (A–C) or 2 mg/kg PGD₂ (D) in PUMA-G^+/+ (A), PUMA-G^+/– (B), and PUMA-G^–/– (C and D) mice. (E) Quantitative analysis of the percentage of LDF increase after administration of NA in PUMA-G^+/+ (white bars, n = 8), PUMA-G^+/– (gray bars, n = 10), and PUMA-G^–/– (black bars, n = 10) mice. ***P < 0.001 vs. PUMA-G^+/+ littermate controls.

Figure 5

PUMA-G is expressed in the skin and in various immune cells. (A) Northern blot analysis of PUMA-G mRNA in the ear and brown adipose tissue (BAT) of PUMA-G^+/+ (WT) and PUMA-G^–/– (KO) animals. 18s, ribosomal RNA used as control. (B) RT-PCR of cDNAs from different tissues prepared from PUMA-G^+/+ (WT) and PUMA-G^–/– (KO) mice using PUMA-G– or GAPDH-specific primers. (C) Expression of PUMA-G in dermal MHC class II–positive cells, dendritic cells, and peritoneal macrophages. (D) Effect of NA (100 μM) and ATP (10 μM) on the [Ca²⁺]_i in macrophages prepared from wild-type or PUMA-G–deficient mice. Values on the y axis indicate the measured 340/380-nm fluorescence ratio as an indicator of the intracellular-free [Ca²⁺].

Figure 6

Transplantation of wild-type bone marrow to PUMA-G–deficient recipients restores the flushing response to NA. (A) RT-PCR of cDNAs from BM or MHC class II–positive cells from the outer ear of PUMA-G–deficient mice transplanted with wild-type BM or PUMA-G–deficient bone marrow using PUMA-G– or GAPDH-specific primers. (B) Original LDF recordings of the flushing response induced by 200 mg/kg NA injected i.p. at the time points indicated by arrows. The experiments were performed 20 weeks after transplantation of PUMA-G^+/+ (lower panel) or PUMA-G^–/– (upper panel) BM to PUMA-G^–/– recipients. (C) Statistical evaluation of NA effects on LDF in PUMA-G^–/– mice transplanted with wild-type (n = 8) or PUMA-G^–/– bone marrow (n = 5). *P < 0.05 vs. animals transplanted with PUMA-G^–/– bone marrow.