Uncoupling protein 2 regulates glucagon-like peptide-1 secretion in L-cells

Abstract

Aim: To investigate whether uncoupling protein 2 (UCP2) affects oleic acid-induced secretion of glucagon-like peptide-1 (GLP-1) in L-cells.

Methods: mRNA and protein expression of UCP2 were analyzed in human NCI-H716 cells, which serve as a model for enteroendocrine L-cells, by quantitative reverse transcription-polymerase chain reaction and Western blotting before and after treatment with oleic acid. Localization of UCP2 and GLP-1 in NCI-H716 cells was assessed by immunofluorescence labeling. NCI-H716 cells were transiently transfected with a small interfering RNA (siRNA) that targets UCP2 (siUCP2) or with a non-specific siRNA using Lipofectamine 2000. The concentrations of bioactive GLP-1 in the medium were measured by enzyme linked immunosorbent assay.

Results: Both GLP-1 and UCP2 granules were expressed mainly in the cytoplasm of NCI-H716 cells. NCI-H716 cells that secreted GLP-1 also expressed UCP2. Time-course experiments revealed that release of GLP-1 from NCI-H716 cells into the medium reached a maximum at 120 min and remained stable until at least 180 min after treatment with oleic acid (the level of GLP-1 increased about 2.3-fold as compared with the level of GLP-1 in the control cells, P < 0.05). In an experiment to determine dose dependence, stimulation of NCI-H716 cells with ≤ 8 mmol oleic acid led to a concentration-dependent release of GLP-1 into the medium; 10 mmol oleic acid diminished the release of GLP-1. Furthermore, GLP-1 secretion induced by oleic acid from NCI-H716 cells that were transfected with siUCP2 decreased to 41.8%, as compared with NCI-H716 cells that were transfected with a non-specific siRNA (P < 0.01).

Conclusion: UCP2 affected GLP-1 secretion induced by oleic acid. UCP2 plays an important role in L-cell secretion that is induced by free fatty acids.

Keywords: Glucagon-like peptide-1; L-cell; NCI-H716 cells; Oleic acid; Uncoupling protein 2.

Figure 1

Localization of uncoupling protein-2 and glucagon-like peptide-1 in NCI-H716 cells. Immunofluorescence staining in human NCI-H716 cells which serve as a model for L-cells. A1, A2: Glucagon-like peptide-1 (GLP-1) antibody staining (red); B1, B2: Uncoupling protein-2 (UCP2) antibody staining (green); C1, C2: Merged image shows co-expression of UCP2 and GLP-1. Blue (Hoechst) staining indicates the nuclei. Original magnification, ×400.

Figure 2

Glucagon-like peptide-1 secretion in NCI-H716 cells after treatment with oleic acid. A: Time course of oleic acid-induced release of glucagon-like peptide-1 (GLP-1) from NCI-H716 cells. NCI-H716 cells were incubated with 8 mmol oleic acid, and the culture medium was collected at 0.25, 0.5, 1, 2, and 3 h to detect GLP-1 levels; B: Dose-dependent oleic acid-induced release of GLP-1 from NCI-H716 cells. NCI-H716 cells were incubated with 2, 4, 6, 8, and 10 mmol oleic acid for 2 h. The culture medium was then analyzed for GLP-1 concentration. NCI-H716 cells without oleic acid exposure. ^aP < 0.05, ^bP < 0.01 vs baseline value.

Figure 3

Expression of uncoupling protein-2 in NCI-H716 cells after treatment with oleic acid. A: Total RNA was extracted, and relative mRNA levels of uncoupling protein-2 (UCP2) were determined by quantitative reverse transcriptase polymerase chain reaction with normalization to β-actin (P < 0.05); B: UCP2 protein was detected by western blotting. Cytochrome C was used as the loading control (P < 0.01); Individual data points in this figure represent the mean ± SE of 12 determinants from four independently prepared samples each with three measurements. NCI-H716 cells were incubated with medium alone (blank control), medium containing 1% dimethyl sulfoxide (negative control), medium with 30 mmol glucose (glucose), and medium with 8 mmol oleic acid (oleic acid).

Figure 4

Knockdown of uncoupling protein-2 expression decreased oleic acid-induced secretion of glucagon-like peptide-1 in NCI-H716 cells. Up panel: The knock-down efficiency of small interfering RNA which targets UCP2 (siUCP2) in NCI-H716 cells. NCI-H716 cells were left untransfected (blank), were mock transfected (no siRNA), or were transfected with siUCP2. After 48 h, the mRNA levels of uncoupling protein-2 were determined as in Figure 4. Protein levels were determined as in Figure 4; Down panel: Effects of oleic acid on levels of secreted glucagon-like peptide-1 (GLP-1). NCI-H716 cells were transfected as described above and were then incubated with 8 mmol oleic acid for 2 h. GLP-1 concentrations were determined as in Figure 4. ^bP < 0.01 vs siRNA control.