Identification of 9-cis-retinoic acid as a pancreas-specific autacoid that attenuates glucose-stimulated insulin secretion

Abstract

The all-trans-retinoic acid (atRA) isomer, 9-cis-retinoic acid (9cRA), activates retinoic acid receptors (RARs) and retinoid X receptors (RXRs) in vitro. RARs control multiple genes, whereas RXRs serve as partners for RARs and other nuclear receptors that regulate metabolism. Physiological function has not been determined for 9cRA, because it has not been detected in serum or multiple tissues with analytically validated assays. Here, we identify 9cRA in mouse pancreas by liquid chromatography/tandem mass spectrometry (LC/MS/MS), and show that 9cRA decreases with feeding and after glucose dosing and varies inversely with serum insulin. 9cRA reduces glucose-stimulated insulin secretion (GSIS) in mouse islets and in the rat β-cell line 832/13 within 15 min by reducing glucose transporter type 2 (Glut2) and glucokinase (GK) activities. 9cRA also reduces Pdx-1 and HNF4α mRNA expression, ∼8- and 80-fold, respectively: defects in Pdx-1 or HNF4α cause maturity onset diabetes of the young (MODY4 and 1, respectively), as does a defective GK gene (MODY2). Pancreas β-cells generate 9cRA, and mouse models of reduced β-cell number, heterozygous Akita mice, and streptozotocin-treated mice have reduced 9cRA. 9cRA is abnormally high in glucose-intolerant mice, which have β-cell hypertropy, including mice with diet-induced obesity (DIO) and ob/ob and db/db mice. These data establish 9cRA as a pancreas-specific autacoid with multiple mechanisms of action and provide unique insight into GSIS.

Fig. 1.

9cRA occurs in pancreas. (A) Representative LC/MS/MS chromatograms of RA isomers from analyses of mouse pancreas, liver, and serum. (B) Representative LC/MS/MS chromatograms of pancreatic RA isomers before (solid lines) and after addition (dashed lines) of retinoids before homogenization, extraction, and analysis. Each chromatogram shows one of triplicate analyses. (C) Quantification of RA isomers in mouse liver, serum, and pancreas: ND, not detected; eight mice per group (SEM).

Fig. 2.

9cRA reflects fasting vs. feeding. (A) Blood glucose, serum insulin, and pancreas RA isomers in fed or 12-h fasted mice. Data are means of three experiments with 6–10 mice per group per experiment; *P ≅ 0.03, **P < 0.004, ***P < 0.003 vs. fed values. (B) 9cRA and atRA responses to a glucose challenge (2 g/kg glucose). Values are means of two to five experiments with 5–10 mice per point per experiment, except 9,13dcRA (one experiment, 10 mice per time); *P < 0.05 from 0 time. The three glucose values after 0 min differ from control; *P < 0.05. (C) Inverse relationship between pancreas 9cRA and serum insulin after a glucose challenge: the slope differs significantly from 0, P = 0.02; 10 mice per point. (D) 9cRA hinders insulin secretion: 9cRA (0.5 mg/kg in 60 μL DMSO) or vehicle alone were injected i.p. in mice 15 min before an i.p dose of glucose (0.5 g/kg). Data are means of six to seven mice; *P < 0.05. All data ± SEM.

Fig. 3.

Exogenous 9cRA induces glucose intolerance. (A) Increases in total pancreas 9cRA after dosing with 9-cis retinol or 9cRA (0.5 mg/kg in 100 μL DMSO). 9-cis retinol and 9cRA were injected 60 and 15 min before glucose, respectively. Glucose (2 g/kg) was injected at 0 min: five to eight mice per group; *P ≅ 0.01 and **P < 0.003 vs. vehicle control; ^&P < 0.005 vs. 0 min. (B) GTT in mice dosed with 9-cis retinol or 9cRA: five to seven mice per group; *P < 0.04, **P < 0.002, ***P < 0.005 vs. control. Mice were dosed with retinoids as described in A. All data are ±SEM.

Fig. 4.

9cRA attenuates pancreas glucose sensing. Pancreas 832/13 β-cells and islets were preincubated 2 h with 3 mM glucose. At 0 min, the medium was exchanged for medium containing 23 mM and agents indicated for the duration of experiments. (A) 9cRA reduces Glut2 activity in 832/13 cells after 15 min incubation: 4–8 replicates per group; *P ≤ 0.0003 vs. no addition. (B) 9cRA reduces GK activity in 832/13 cells: 3–7 replicates per group, *P < 0.02, **P < 0.002 vs. control. (C) 9cRA reduces ATP content in 832/13 cells: 2–4 replicates per group, *P < 0.008 vs. control. (D) 9cRA decreases Ca²⁺ influx into 832/13 cells: 2 replicates per group; *P < 0.02. (E) 9cRA decreases GSIS by 832/13 cells: 3–11 replicates per group; *P < 0.01 vs. control. (F) 9cRA decreases GSIS by pancreatic islets. The graph shows baseline insulin secretion during 3 mM glucose and the effect of 9cRA on stimulation of insulin secretion by 23 mM glucose: 8–9 replicates per group; *P < 0.02, **P < 0.002 vs. control. (G) 9cRA does not affect KCl-stimulated insulin secretion from islets; *P < 0.02 vs. 0 time. (H) 9cRA reduces Pdx-1 and HNF4α mRNA after 2 h in 832/13 cells: 3 replicates per group. One hundred nanomolar 9cRA was used in all experiments, unless noted otherwise. All data are ±SEM.

Fig. 5.

Pancreas β-cells produce 9cRA. (A) Representative LC/MS/MS chromatograms and quantification of RA isomers in pancreas of WT and Akita mice: 8 mice per group; *P < 0.05 vs. WT. (B) Immunohistochemistry showing loss of insulin in pancreas with time after a Stz dose. (Scale bars, 100 μm.) (C) Effect of Stz on β-cell numbers (3–6 islets) and pancreas 9cRA (9–18 mice per group). (D) Biosynthesis of RA isomers from retinol and retinal isomers by the pancreas β-cell line 832/13: 3 replicates/substrate. (E) HPLC of retinol standards and representative analyses of pancreas microsomes. Arrows denote elution positions of 13-cis-, 9-cis-, and all-trans-retinol, respectively. All data are ±SEM.

Fig. 6.

Increased pancreas 9cRA accompanies glucose intolerance. (A) Representative LC/MS/MS chromatograms and RA isomers in pancreas of fed WT and ob/ob mice: 8 mice per group; *P < 0.008 vs. WT. (B) Increased 9cRA in pancreas of mice with DIO: 8–10 mice per group: *P < 0.001 vs. lean. (C) RA isomers in pancreas of fed WT and db/db mice: 8 mice per group; **P = 0.033. All data are ±SEM.