Cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids contribute to insulin sensitivity in mice and in humans

Abstract

Aims/hypothesis: Insulin resistance is frequently associated with hypertension and type 2 diabetes. The cytochrome P450 (CYP) arachidonic acid epoxygenases (CYP2C, CYP2J) and their epoxyeicosatrienoic acid (EET) products lower blood pressure and may also improve glucose homeostasis. However, the direct contribution of endogenous EET production on insulin sensitivity has not been previously investigated. In this study, we tested the hypothesis that endogenous CYP2C-derived EETs alter insulin sensitivity by analysing mice lacking CYP2C44, a major EET producing enzyme, and by testing the association of plasma EETs with insulin sensitivity in humans.

Methods: We assessed insulin sensitivity in wild-type (WT) and Cyp2c44 -/- mice using hyperinsulinaemic-euglycaemic clamps and isolated skeletal muscle. Insulin secretory function was assessed using hyperglycaemic clamps and isolated islets. Vascular function was tested in isolated perfused mesenteric vessels. Insulin sensitivity and secretion were assessed in humans using frequently sampled intravenous glucose tolerance tests and plasma EETs were measured by mass spectrometry.

Results: Cyp2c44 -/- mice showed decreased glucose tolerance (639 ± 39.5 vs 808 ± 37.7 mmol/l × min for glucose tolerance tests, p = 0.004) and insulin sensitivity compared with WT controls (hyperinsulinaemic clamp glucose infusion rate average during terminal 30 min 0.22 ± 0.02 vs 0.33 ± 0.01 mmol kg-1 min-1 in WT and Cyp2c44 -/- mice respectively, p = 0.003). Although glucose uptake was diminished in Cyp2c44 -/- mice in vivo (gastrocnemius Rg 16.4 ± 2.0 vs 6.2 ± 1.7 μmol 100 g-1 min-1, p < 0.01) insulin-stimulated glucose uptake was unchanged ex vivo in isolated skeletal muscle. Capillary density was similar but vascular KATP-induced relaxation was impaired in isolated Cyp2c44 -/- vessels (maximal response 39.3 ± 6.5% of control, p < 0.001), suggesting that impaired vascular reactivity produces impaired insulin sensitivity in vivo. Similarly, plasma EETs positively correlated with insulin sensitivity in human participants.

Conclusions/interpretation: CYP2C-derived EETs contribute to insulin sensitivity in mice and in humans. Interventions to increase circulating EETs in humans could provide a novel approach to improve insulin sensitivity and treat hypertension.

Keywords: Arachidonic acid; Epoxygenases; Hypertension; Insulin secretion in vitro and in vivo; Insulin sensitivity.

Conflict of interest statement

Duality of interest

The authors declare that there is no duality of interest associated with this manuscript.

Figures

Fig. 1

Cyp2c44 disruption impairs glucose tolerance and insulin sensitivity. (a, b) Glucose tolerance was impaired in Cyp2c44−/− mice (black circles, n=15) vs WT controls (white circles, n=15) after glucose administration (a) and as quantified by glucose AUC (b). (c–f) WT (white circles) and Cyp2c44−/− mice (black circles) on a regular chow diet were studied during hyperinsulinaemic–euglycaemic clamps. Glucose was similarly maintained in both groups during the hyperinsulinaemic studies (c). The glucose infusion rate (GIR, d) and rate of glucose disappearance (Rd, e) were significantly reduced in Cyp2c44−/− mice compared with WT mice. The EndoRa was incompletely suppressed in Cyp2c44−/− mice at the end of the clamp (f). (g, h) Liver tissues were collected 10–15 min after i.p. injection of insulin (10 mU) or saline, and western blots for pAkt and Akt (g) showed significantly impaired phosphorylation of Akt after insulin treatment in Cyp2c44−/− mice (h). *p<0.05, **p<0.01 and ***p<0.001, Cyp2c44−/− vs WT

Fig. 2

Cyp2c44 disruption impairs peripheral tissue glucose uptake during hyperinsulinaemic–euglycaemic clamps. Tissue glucose uptake (Rg) was decreased in Cyp2c44−/− mice as assessed by 2-DG uptake during hyperinsulinaemic–euglycaemic clamps in vastus lateralis (lat.) (a), gastrocnemius (b) and adipose (c) tissues. **p<0.01, Cyp2c44−/− vs WT

Fig. 3

Glucose-stimulated insulin secretion during hyperglycaemic clamps is unchanged in Cyp2c44−/− mice during regular chow feeding. (a, b) During hyperglycaemic clamp, glucose was increased to 11.1–13.3 mmol/l (200–250 mg/dl) by glucose infusion (a), and plasma insulin was assessed (b). (c) Insulin during the initial 20 min of the study increased to a similar extent in Cyp2c44−/− mice. White circles, WT mice; black circles, Cyp2c44−/− mice. *p<0.05, Cyp2c44−/− vs WT

Fig. 4

Cyp2c44 disruption increases insulin secretion in isolated islets. (a) In isolated islets cultured in normal (5.6 mmol/l) or high glucose (16.7 mmol/l) for 60 min, glucose-stimulated insulin secretion was increased in Cyp2c44−/− (grey bars) compared with WT islets (white bars). IEQ, islet equivalents. (b) Cyp2c44 mRNA expression was detected in isolated WT but not Cyp2c44−/− islets. (c, d) sEH (Ephx2, c) and Acsl4 mRNA expression (d) was increased in Cyp2c44−/− mice. (e) Immunostaining of pancreatic sections for CYP2C (green), insulin (red), and DAPI (blue) demonstrated localisation to insulin-positive cells. Scale bar = 100 μm. *p<0.05 and ***p<0.001 Cyp2c44−/− vs WT

Fig. 4

Cyp2c44 disruption increases insulin secretion in isolated islets. (a) In isolated islets cultured in normal (5.6 mmol/l) or high glucose (16.7 mmol/l) for 60 min, glucose-stimulated insulin secretion was increased in Cyp2c44−/− (grey bars) compared with WT islets (white bars). IEQ, islet equivalents. (b) Cyp2c44 mRNA expression was detected in isolated WT but not Cyp2c44−/− islets. (c, d) sEH (Ephx2, c) and Acsl4 mRNA expression (d) was increased in Cyp2c44−/− mice. (e) Immunostaining of pancreatic sections for CYP2C (green), insulin (red), and DAPI (blue) demonstrated localisation to insulin-positive cells. Scale bar = 100 μm. *p<0.05 and ***p<0.001 Cyp2c44−/− vs WT

Fig. 5

Cyp2c44 disruption impairs KATP-mediated vascular relaxation. (a) Mesenteric resistance artery endothelium-independent vasodilation in response to the ATP-sensitive potassium channel opener pinacidil was impaired in Cyp2c44−/− mice (black circles) compared with WT controls (white circles). (b) After administration of the KATP channel blocker glibenclamide (Glib.), the plasma insulin response was diminished in Cyp2c44−/− mice compared with WT mice. (c, d) Western blots for the KATP channel subunits Kir6.1 (c) and Kir6.2 (d) in skeletal muscle demonstrated similar expression. *p<0.05 and ***p<0.001 Cyp2c44−/− vs WT

Fig. 6

Plasma EETs correlate with insulin sensitivity in humans. Insulin sensitivity assessed during FSIVGTTs correlates with plasma EET isomers (a, 8,9-; b, 11,12-; c, 14,15-; d, total) in mildly hypertensive human participants. Pearson correlation coefficient (r) and p values are presented for each. Each data point represents measurements from an individual participant. Linear regression lines (solid) are displayed with 95% CIs (dashed lines). To convert Si values to SI units multiply by 0.167