20-Hydroxyeicosatetraenoic acid (HETE), the cytochrome P-450 (CYP) 4A ω-hydroxylation product of arachidonic acid, has potent biological effects on renal tubular and vascular functions and on the control of arterial pressure. We have expressed high levels of the rat CYP4A1, -4A2, -4A3, and -4A8 cDNAs, using baculovirus and Sf 9 insect cells. Arachidonic acid ω- and ω-1-hydroxylations were catalyzed by three of the CYP4A isoforms; the highest catalytic efficiency of 947 nM−1 ⋅ min−1for CYP4A1 was followed by 72 and 22 nM−1 ⋅ min−1for CYP4A2 and CYP4A3, respectively. CYP4A2 and CYP4A3 exhibited an additional arachidonate 11,12-epoxidation activity, whereas CYP4A1 operated solely as an ω-hydroxylase. CYP4A8 did not catalyze arachidonic or linoleic acid but did have a detectable lauric acid ω-hydroxylation activity. The inhibitory activity of various acetylenic and olefinic fatty acid analogs revealed differences and indicated isoform-specific inhibition. These studies suggest that CYP4A1, despite its low expression in extrahepatic tissues, may constitute the major source of 20-HETE synthesis. Moreover, the ability of CYP4A2 and -4A3 to catalyze the formation of two opposing biologically active metabolites, 20-HETE and 11,12-epoxyeicosatrienoic acid, may be of great significance to the regulation of vascular tone.

Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of endogenous inflammatory and antiapoptotic mediators. However, the roles of sEH in diabetes and the pancreas are unknown. Our aims were to determine whether sEH is involved in the regulation of hyperglycemia in diabetic mice and to investigate the reasons for the regulation of insulin secretion by sEH deletion or inhibition in islets. We used two separate approaches, targeted disruption of Ephx2 gene [sEH knockout (KO)] and a selective inhibitor of sEH [trans-4-[4-(3-adamantan-1-ylureido)-cyclohexyloxy]-benzoic acid (t-AUCB)], to assess the role of sEH in glucose and insulin homeostasis in streptozotocin (STZ) mice. We also examined the effects of sEH KO or t-AUCB on glucose-stimulated insulin secretion (GSIS) and intracellular calcium levels in islets. Hyperglycemia in STZ mice was prevented by both sEH KO and t-AUCB. In addition, STZ mice with sEH KO had improved glucose tolerance. More important, when insulin levels were assessed by hyperglycemic clamp study, sEH KO was found to promote insulin secretion. In addition, sEH KO and t-AUCB treatment augmented islet GSIS. Islets with sEH KO had a greater intracellular calcium influx when challenged with high glucose or KCl in the presence of diazoxide. Moreover, sEH KO reduced islet cell apoptosis in STZ mice. These results show not only that sEH KO and its inhibition prevent hyperglycemia in diabetes, but also that sEH KO enhances islet GSIS through the amplifying pathway and decreases islet cell apoptosis in diabetes.The prevalence of diabetes continues to increase. It is estimated that 225 million people are affected worldwide (Mazzone, 2009). Moreover, the diabetic population is subject to a high incidence of cardiovascular and renal diseases (Breyer et al., 2005;Mazzone, 2009). Diabetes is characterized by hyperglycemia related to abnormalities in the function of pancreatic ␤ cells. Because ␤-cell destruction and dysfunction are the central events in the development and progression of diabetes, the prevention of ␤-cell destruction and the improvement of ␤-cell function could be important strategies for controlling the advance of diabetes (Kahn et al., 2006;Donath et al., 2008).In pancreatic ␤ cells, glucose stimulates insulin secretion by activating the triggering and amplifying pathways (Henquin, 2000). In the triggering pathway, products of glucose metabolism enter the mitochondrial respiratory chain, which uses them to generate ATP. Increased ATP levels close the K ATP -sensitive channels, followed by membrane depolarization and opening of the voltage-sensitive Ca 2ϩ channels, which in turn increase intracellular Ca 2ϩ concentration and

Aims/hypothesis Animal models of diabetic nephropathy show increased levels of glomerular vascular endothelial growth factor (VEGF)-A, and several studies have shown that inhibiting VEGF-A in animal models of diabetes can prevent albuminuria and glomerular hypertrophy. However, in those studies, treatment was initiated before the onset of kidney damage. Therefore, the aim of this study was to investigate whether transfecting mice with the VEGF-A inhibitor sFlt-1 (encoding soluble fms-related tyrosine kinase 1) can reverse pre-existing kidney damage in a mouse model of type 1 diabetes. In addition, we investigated whether transfection with sFlt-1 can reduce endothelial activation and inflammation in these mice. Methods Subgroups of untreated 8-week-old female C57BL/ 6J control (n = 5) and diabetic mice (n = 7) were euthanised 5 weeks after the start of the experiment in order to determine the degree of kidney damage prior to treatment with sFLT-1. Diabetes was induced with three i.p. injections of streptozotocin (75 mg/kg) administered at 2 day intervals. Diabetic nephropathy was then investigated in diabetic mice transfected with sFlt-1 (n = 6); non-diabetic, non-transfected control mice (n = 5); non-diabetic control mice transfected with sFlt-1(n = 10); and non-transfected diabetic mice (n = 6). These mice were euthanised at the end of week 15. Transfection with sFlt-1 was performed in week 6. Results We found that transfection with sFlt-1 significantly reduced kidney damage by normalising albuminuria, glomer-ular hypertrophy and mesangial matrix content (i.e. glomeru-lar collagen type IV protein levels) (p < 0.001). We also found that transfection with sFlt-1 reduced endothelial activation (p < 0.001), glomerular macrophage infiltration (p < 0.001) and glomerular TNF-α protein levels (p < 0.001). Finally, sFLT-1 decreased VEGF-A-induced endothelial activation in vitro (p < 0.001). Conclusions/interpretation These results suggest that sFLT-1 might be beneficial in treating diabetic nephropathy by inhibiting VEGF-A, thereby reducing endothelial activation and glomerular inflammation, and ultimately reversing kidney damage.

Abstract-P450-dependent arachidonic acid (AA) metabolites regulate arterial tone by modulating calcium-activated (BK) potassium channels in vascular smooth muscle cells (VSMC). Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent epoxygenation of EPA produces alternative vasoactive compounds. We synthesized the 5 regioisomeric epoxyeicosattrienoic acids (EETeTr) and examined them for effects on K ϩ currents in rat cerebral artery VSMCs with the patch-clamp technique. 11(R),12(S)-epoxyeicosatrienoic acid (50 nmol/L) was used for comparison and stimulated K ϩ currents 6-fold at ϩ60 mV. However, 17(R),18(S)-EETeTr elicited a more than 14-fold increase. 17(S),18(R)-EET and the remaining four regioisomers were inactive. The effect of 17(R),18(S)-EETeTr was blocked by tetraethylammonium but not by 4-aminopyridine. VSMCs expressed P450s 4A1 and 4A3. Recombinant P450 4A1 hydroxylated EPA at C-19 and C-20 and epoxygenated the 17,18-double bond, yielding the R, S-and S, R-enantiomers in a ratio of 64:36. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK potassium channels. Furthermore, this metabolite can be directly produced in VSMCs. We suggest that 17(R),18(S)-EETeTr may function as an important hyperpolarizing factor, particularly with EPA-rich diets. Key Words: vascular smooth muscle cells Ⅲ endothelium-derived factors Ⅲ potassium channels Ⅲ cytochrome P450 D ietary fish oil or purified (n-3) long-chain polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) exert a wide range of beneficial effects on vascular function. 1,2 Endothelium-dependent relaxation is enhanced and the vasoconstrictor response to angiotensin II and norepinephrine is reduced because of mechanisms that are incompletely understood. [3][4][5][6] Possibly, EPA and other (n-3) PUFA compete with arachidonic acid (AA) for enzymatic conversion by P450 enzymes. This competition may lead to a reduced formation of vasoactive AA metabolites while alternative metabolites originating from EPA are increased. The P450-dependent AA metabolites result from epoxygenation and hydroxylation and include the epoxyeicosatrienoic acids (EET) 5,6-, 8,9-, 11,12 to 14,15-EET, and the /(-1)-hydroxyeicosatetraenoic acids . 7 EETs are produced in the endothelium by the P450 subfamilies 2C and 2J. 8,9 EETs activate large-conductance, calcium-activated (BK) K ϩ channels in vascular smooth muscle cells (VSMC) and are considered as leading candidates for the endothelium-derived hyperpolarizing factor (EDHF). 8,10,11 20-HETE is produced by P450 4A enzymes in VSMC and acts as endogenous vasoconstrictor that inhibits BK channels. 20-HETE is important for the autoregulation of renal and cerebral blood flow. [12][13][14][15] How AA metabolite production is influenced by EPA competition for P450 enzymes, and whether or not P450-dependent EPA metabolites modulate BK channels, is unknown. We synthesized the five possible regioisomeric epoxyeicosatetraenoic acids: 5,6...

Arachidonic acid (AA) is metabolized by cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes into eicosanoids, which are involved in diverse diseases, including type 1 and type 2 diabetes. During the last thirty years, evidence has been accumulated that suggests important functions for eicosanoids in the control of pancreatic β-cell function and destruction. AA metabolites of the COX pathway, especially prostaglandin E2 (PGE2), appear to be significant factors to β-cell dysfunction and destruction, participating in the pathogenesis of diabetes and its complications. Several elegant studies have contributed to the sorting out of the importance of 12-LOX eicosanoids in cytokine-mediated inflammation in pancreatic β cells. The role of CYP eicosanoids in diabetes is yet to be explored. A recent publication has demonstrated that stabilizing the levels of epoxyeicosatrienoic acids (EETs), CYP eicosanoids, by inhibiting or deleting soluble epoxide hydrolase (sEH) improves β-cell function and reduces β-cell apoptosis in diabetes. In this review we summarize recent findings implicating these eicosanoid pathways in diabetes and its complications. We also discuss the development of animal models with targeted gene deletion and specific enzymatic inhibitors in each pathway to identify potential targets for the treatment of diabetes and its complications.