PPARdelta is a fatty acid sensor that enhances mitochondrial oxidation in insulin-secreting cells and protects against fatty acid-induced dysfunction

Abstract

The peroxisome proliferator-activated receptor delta (PPARdelta) is implicated in regulation of mitochondrial processes in a number of tissues, and PPARdelta activation is associated with decreased susceptibility to ectopic lipid deposition and metabolic disease. Here, we show that PPARdelta is the PPAR subtype expressed at the highest level in insulinoma cells and rat pancreatic islets. Furthermore, PPARdelta displays high transcriptional activity and acts in pronounced synergy with retinoid-X-receptor (RXR). Interestingly, unsaturated fatty acids mimic the effects of synthetic PPARdelta agonists. Using short hairpin RNA-mediated knockdown, we demonstrate that the ability of unsaturated fatty acids to stimulate fatty acid metabolism is dependent on PPARdelta. Activation of PPARdelta increases the fatty acid oxidation capacity in INS-1E beta-cells, enhances glucose-stimulated insulin secretion (GSIS) from islets, and protects GSIS against adverse effects of prolonged fatty acid exposure. The presented results indicate that the nuclear receptor PPARdelta is a fatty acid sensor that adapts beta-cell mitochondrial function to long-term changes in unsaturated fatty acid levels. As maintenance of mitochondrial metabolism is essential to preserve beta-cell function, these data indicate that dietary or pharmacological activation of PPARdelta and RXR may be beneficial in the prevention of beta-cell dysfunction.

Fig. 1.

PPARδ is the most abundant PPAR subtype in pancreatic β-cells. Total RNA was extracted from INS-1E cells (A) and Wistar rat islets (B) cultured in RPMI with 11 mM glucose and supplemented as described. Expression of the PPAR subtypes α, γ, and δ was quantified by quantitative PCR and normalized to TFIIB expression. RNA was harvested in duplicates and range is indicated. The presented results are representative of at least three independent experiments. C: Nuclear proteins were extracted from INS-1E cells (NE) and separated by SDS-PAGE, and PPAR levels were visualized by immunoblotting. Protein extracts from NIH-3T3 cells ectopically expressing murine PPARα (Pα), PPARγ (Pγ), PPARδ (Pδ), or control adenoviral vector (E) were run in parallel as control for protein migration. Arrows indicate the PPAR subtypes (NS, nonspecific band).

Fig. 2.

A PPARδ-specific agonist and unsaturated fatty acids activate a PPAR-responsive reporter in synergy with RXRα in INS-1E cells. INS-1E cells were transfected with the 3xACO-PPRE luciferase reporter construct and incubated in RPMI (11 mM glucose) for 24 h with DMSO, PPAR subtype-specific agonists, or fatty acids in the presence or absence of the RXRα agonist LG100268 (0.2 μM). A: The specific PPAR agonists used were for PPARα, WY14.643 (30 μM); for PPARγ, BRL49653 (1 μM); and for PPARδ, L165041 (1 μM). B: The fatty acids used were oleic acid (100 μM), γ-linolenic acid (100 μM), or palmitic acid (100 μM). Luciferase activities were measured and presented relative to DMSO control. Transfections were performed in triplicate, and standard deviations are indicated. Results are representative of three independent experiments, and P values were calculated using two-way ANOVA. Addition of LG100268 differs from corresponding DMSO control (*P < 0.05).

Fig. 3.

PPARδ activation and fatty acids stimulate expression of fatty acid handling genes in synergy with RXRα in INS-1E cells and rat islets. INS-1E cells were incubated for 24 h in RPMI (11 mM glucose) with DMSO, PPAR subtype-specific agonists, or fatty acids in the presence or absence of the RXRα agonist LG100268 (0.2 μM). A: The specific PPAR agonists used were for PPARα, WY14.643 (WY, 30 μM); for PPARγ, BRL49653 (BRL, 1 μM); and for PPARδ, L165041 (L, 1 μM). B: The fatty acids used were oleic acid (OA, 100 μM) or γ-linolenic acid (LA, 100 μM). Total RNA was extracted, and expression levels of CD36, muscle CPT-1, and UCP2 were quantified using quantitative PCR and normalized to TFIIB expression. Expression levels are presented relative to DMSO control and are representative of three independent experiments. For each experiment, RNA was harvested in triplicates. C: Rat islets were isolated and incubated for 24 h in RPMI (11 mM glucose) with the indicated ligands. Results are means of three independent experiments, and P values are calculated using two-way ANOVA. Addition of LG100268 differs from corresponding DMSO control (*P < 0.05).

Fig. 4.

Oleic acid activation of fatty acid handling genes is dependent on PPARδ in insulin secreting cells. INS-1E cells were transduced with adenoviral vectors (∼20 pfu/cell) expressing shRNA targeting PPARα [AdshPPARα (shPα)] or PPARδ [AdshPPARδ (shPδ)] or with empty control virus [AdshEmpty (E)] and cultured for 48 h in RPMI (11 mM glucose) before harvest of total RNA and nuclear protein extracts. A: Expression levels of PPARα and PPARδ were quantified using quantitative PCR, normalized to TFIIB expression, and presented relative to AdshEmpty control transduced cells. B: Nuclear proteins (NE) were separated by SDS-PAGE, and PPAR levels were visualized by immunoblotting. Protein extracts from NIH-3T3 cells ectopically expressing murine PPARα (Pα), PPARγ2 (Pγ), PPARδ (Pδ), or control adenoviral vector (E) were run in parallel as control for protein migration. Arrows indicate migration of the subtypes. C: INS-1E cells were transduced with the indicated adenoviral vectors and cultured for 24 h in RPMI (11 mM glucose) before incubation with DMSO or the fatty acid oleic acid (OA, 100 μM) in the presence or absence of the RXRα agonist LG100268 (0.2 μM) for additional 24 h. Total RNA was extracted, and expression levels of CD36, muscle CPT-1, and UCP2 were quantified using quantitative PCR and normalized to TFIIB expression. Expression levels are presented relative to DMSO control. Results are means of three independent experiments, and P values were calculated by two-way ANOVA. Addition of LG100268 differs from corresponding DMSO control (*P < 0.05).

Fig. 5.

Oleic acid activation of PPARδ increases fatty acid oxidation capacity in synergy with RXRα in insulin-secreting cells. A: INS-1E cells were incubated for 24 h in RPMI (11 mM glucose) with DMSO, the specific PPARα agonist WY14.643 (30 μM), or the specific PPARδ agonist L165041 (1 μM) in the presence or absence of the RXRα agonist LG100268 (0.2 μM). Cells were incubated with [1-14C]oleic acid for 4 h in serum-free RPMI1640 containing 5 mM glucose, and oxidation was measured in triplicate, background was subtracted, and values were normalized to total cellular protein. B: INS-1E cells were transduced with adenoviral vectors (∼20 pfu/cell) expressing shRNA against PPARδ (AdshPPARδ) or a control shRNA vector (AdshEmpty). After 24 h in RPMI (11 mM glucose), DMSO or oleic acid (OA; 100 μM) together with LG100268 (LG; 0.2 μM) was added to the media. Cells were incubated for another 24 h, and fatty acid oxidation was measured as described above. The experiments were performed in triplicate, and standard deviations are indicated. Results are representative of three independent experiments, and P values were calculated using two-way ANOVA. Addition of LG100268 differs from corresponding DMSO control (*P < 0.05).

Fig. 6.

PPARδ activity potentiates GSIS and protects against fatty acid-induced insulin secretion defects. A: INS-1E cells were incubated for 24 h in RPMI (11 mM glucose) with DMSO or the specific PPARδ agonist L165041 (1 μM). Insulin secretion over 30 min at 2.5 and 15 mM glucose was determined and normalized to total insulin content. GSIS is significantly different from basal insulin secretion (*P < 0.01). GSIS in cells preincubated with L165041 is elevated compared with DMSO-treated cells (P < 0.02). B: Rat islets were incubated for 24 h in RPMI (11 mM glucose) with DMSO or the specific PPARδ agonist L165041 (1 μM). Insulin secretion over 30 min at 2.8 and 11.2 mM glucose was determined and normalized to islet number. GSIS is significantly different from basal insulin secretion (*P < 0.01). GSIS in cells preincubated with L165041 is elevated compared with DMSO-treated cells (P < 0.05). C: INS-1E cells were transduced with adenoviral vectors (∼20 pfu/cell) expressing shRNA against PPARδ (shPδ) or control shRNA (shE). After 24 h of incubation in RPMI (11 mM glucose) with DMSO or oleic acid (OA; 400 μM), insulin secretion over 30 min at 2.5 and 15 mM glucose was determined and normalized to total insulin content. Where indicated, GSIS is significantly different from basal insulin secretion (*P < 0.05) as assessed by two-way ANOVA. ** Significantly different from control transduced, DMSO-treated cells at 2.5 mM glucose (P < 0.001). Insulin secretion was significantly reduced at 15 mM glucose (P < 0.025) and increased at 2.5 mM glucose (P < 0.01) for shPδ cells compared with wild-type cells treated with oleic acid. The results are means of three independent experiments.