PPARβ/δ activation prevents the high glucose-induced impairment of insulin stimulated nitric oxide production in human endothelial cells by reducing mitochondrial reactive oxygen species.

AM Quintela1, R Jimenez1, L Piqueras2, M Gomez-Guzman1, P Galindo1, J Haro3, MJ Zarzuelo1, S Boaretto1, A Cogolludo4, MJ Sanz2, F Perez-Vizcaino4, J Duarte1. 1University of Granada, Department of Pharmacology, School of Pharmacy, 18071 Granada, Spain, 2University of Valencia, Department of Pharmacology, School of Medicine, Valencia, Spain, 3Clinic Hospital of Granada, Service of Gynecology, Granada, Spain, 4University Complutense of Madrid and Ciber Enfermedades Respiratorias (CIBERES), Department of Pharmacology, School of Medicine, Madrid, Spain

Activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) improves insulin sensitivity in diabetic db/db mice, ameliorates insulin signaling in interleukin-6-stimulated adipocytes (Serrano-Marco et al., 2011) and prevents fatty acid-induced insulin resistance in skeletal muscle cells (Coll et al., 2010). However, whether PPARβ/δ activation is joined to improvement the insulin signaling in endothelial cells under hyperglycemic conditions is unknown. Thus, the aim of the present study was to examine the possible protective effects of PPARβ/δ agonists, GW0742 and L165041, on impaired insulin signaling induced by high glucose in cultured primary human umbilical vein endothelial cells (HUVEC).

HUVEC were cultured with GW0742 or L165041 (1 or 10 μM) during 24 hours in low (5 mM) or high glucose (30 mM) medium. Cells were then used to measure nitric oxide (NO) production by diaminofluorescein-2 (DAF-2) fluorescence, reactive oxygen species (ROS) production by the fluorescent probe 5-(and-6-)chloromethyl-2’-7’-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) or by test both (Ser-473)-Akt and (Ser-1177)-endothelial NO synthase (eNOS) phosphorylation by western blots, under basal and insulin (100 nM for 30 min) stimulated conditions. In some experiments, HUVECs were transfected with control or PPARβ/δ-specific siRNA for 48 h and then used by insulin-stimulated NO measures. Significant differences were established by one-way ANOVA using the GraphPad Instant program. When significant variations were found, the Tukey-Kramer multiple comparisons test was applied.

HUVECs incubated in high glucose medium showed reduced insulin-dependent NO production (n = 9-15) and (Ser473)-Akt and (Ser1177)-eNOS phosphorylation (n = 5-6) as compared to cells exposed to low glucose. The coincubation with both PPARβ/δ agonists increased the NO production stimulated by insulin in high glucose cultured cells (n = 8), being without effect in cells incubated in low glucose (n = 8). This improvement in endothelial function was accompanied by increased Akt and eNOS phosphorylation (n = 5-6). Both functional and expressional effects induced by both agonists were suppressed when HUVEC were also incubated with the PPAR-β antagonist GSK0660 (1 μM, n = 5-8). Moreover, PPARβ/δ-specific siRNA (n = 10-15) and the piruvate dehydrogenase kinase (PDK)-4 inhibitor dichloroacetate (DCA, 2 mM, n = 8) also abolished the protective effects of PPARβ/δ agonists in insulin-stimulated NO production. Extracellular signal regulated kinase (ERK)1/2 inhibition by PD98059 (10 μM, n = 8), but not c-Jun NH2-terminal kinases (JNK) inhibition by SP600125 (25 μM, n = 8), abolished the impaired insulin-stimulated NO production in high glucose. After 12 of high glucose incubation HUVECs showed increased ERK1/2-Thr183 and Tyr185 phosphorylation, which was suppressed by coincubation with both PPARβ/δ agonists (n = 5-6). ROS production was increased in HUVECs incubated in high glucose as compared to low glucose (2353 ± 113 vs 1744 ± 133 arbitrary units, respectively, n = 5-7, p<0.01). This increase was suppressed by thenoyltrifluoroacetone (TTFA, 10 μM), an inhibitor of complex II mitochondrial electron transport chain (1844 ± 132 arbitrary units, n = 7), and by L165041 (10 μM, 1744 ± 235 arbitrary units, n = 7). PDK-4 inhibition by DCA (2 mM, n = 8) also suppressed the effect of L165041 in ROS production induced by high glucose. Both PPARβ/δ agonists also increased PDK-4 mRNA in HUVECs exposed to low and high glucose (n = 6-8), which was abolished by GSK0660 (1 μM).

Our results suggest that PPAR-β activation improves Akt-eNOS pathway, leading to increase NO production stimulated by insulin in high glucose incubated HUVECs, at least in part, through PDK4 activation. This effect might reduce pyruvate transport into the mitochondria, normalizing mitochondrial ROS production, leading to ERK1/2 inactivation and subsequent improvement of Akt-eNOS signalling.

Coll T et al., (2010). Endocrinology 151:1560-9.

Serrano-Marco L et al., (2011). Diabetes 60:1990-9.

Supported by SAF2010-22066.