Role of 20-HETE in Diet-Induced Obesity and Insulin Resistance: Interference with Insulin Signaling

Studies in humans and animal models have shown that levels of 20-Hydroxyeicosatetraenoic acid (20-HETE), the omega-hydroxylation product of arachidonic acid, positively correlate with body mass index, hyperglycemia and plasma insulin levels. In the present study, we seek to identify a causal relationship between 20-HETE and obesity-driven insulin resistance using two distinct transgenic mouse models with overexpression of Cyp4a12-20-HETE-synthase. These include the Cyp4a14 knockout (Cyp4a14-/-) mice which is an androgen-driven model of 20-HETE overproduction and the Cyp4a12 transgenic mice (Cyp4a12tg) which is an androgen-independent doxycycline (DOX)-inducible model of 20-HETE overproduction. Cyp4a14-/- male mice were fed a regular control (CD) or high-fat diet (HFD) for 15 weeks. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl20-hydroxyeicosa-6(Z),15(Z)-dienoate], a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed Cyp4a14-/- male mice gained significant weight (16.7 ± 3.2 vs. 3.8 ± 0.35 g, P \u3c 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P \u3c 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P \u3c 0.05) compared with corresponding CD-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P \u3c 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P \u3c 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P \u3c 0.05). The Cyp4a12tg mice in which 20-HETE overproduction occurs in response to DOX were fed either i) control diet (No Dox + CD); ii) high-fat diet (No Dox + HFD); iii) control diet with doxycycline (Dox + CD); or iv) high-fat diet with doxycycline (Dox + HFD) for 15 weeks. 20-SOLA was administered in drinking water from week 0 of HFD (Dox or No Dox). Dox + HFD-fed mice gained significant body weight compared to regular diet-fed mice (20.63 ± 2.9 vs. 3.75 ± 0.6 g, P \u3c 0.05) or mice receiving HFD alone (20.63 ± 2.9 vs. 13.51 ± 0.94 g, P \u3c 0.05). Mice fed HFD with doxycycline developed hyperglycemia (144 ± 3.53 vs. 101.2 ± 6.1 mg/dl, P \u3c 0.05) and hyperinsulinemia (2.52 ± 0.58 vs. 0.55 ± 0.10 ng/ml, P \u3c 0.05) compared to CD-fed mice. However, this effect was not seen in mice fed HFD without doxycycline. 20-SOLA attenuated HFD-induced weight gain (11.37 ± 1.78 vs. 20.63 ± 2.9 g, P \u3c 0.05) and normalized the hyperglycemia (104.2 ± 9.13 vs 144.4 ± 3.53 mg/dl, P \u3c 0.05) and hyperinsulinemia (0.71 ± 0.09 vs 2.52 ± 0.58 ng/ml, P \u3c 0.05) in animals receiving doxycycline. 20-SOLA did not change body-weight gain, plasma glucose levels or plasma insulin levels in animals fed a HFD without doxycycline. Both transgenic mice overexpressing Cyp4a12-20-HETE synthase, viz. the Cyp4a14-/- and the Cyp4a12tg mice, displayed HFD-induced impairment of glucose homeostasis and insulin resistance evident by reduced insulin and glucose tolerance which was ameliorated by 20-SOLA treatment. HFD feeding to transgenic mice overexpressing Cyp4a12-20-HETE synthase resulted in a significant increase in circulatory and adipose tissue 20-HETE levels correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, hyperglycemia and insulin resistance, possibly by interfering with insulin signaling and glucose homeostasis

MicroRNAs as Mediators of Adipose Thermogenesis and Potential Therapeutic Targets for Obesity

Obesity is a growing health problem worldwide, associated with an increased risk of multiple chronic diseases. The thermogenic activity of brown adipose tissue (BAT) correlates with leanness in adults. Understanding the mechanisms behind BAT activation and the process of white fat “browning” has important implications for developing new treatments to combat obesity. MicroRNAs (miRNAs) are small transcriptional regulators that control gene expression in various tissues, including adipose tissue. Recent studies show that miRNAs are involved in adipogenesis and adipose tissue thermogenesis. In this review, we discuss recent advances in the role of miRNAs in adipocyte thermogenesis and obesity. The potential for miRNA-based therapies for obesity and recommendations for future research are highlighted, which may help provide new targets for treating obesity and obesity-related diseases

20-HETE Interferes with Insulin Signaling and Contributes to Obesity-Driven Insulin Resistance

20-HETE, a metabolite of arachidonic acid produced by Cytochrome P450 (CYP) 4A/4 F, has been implicated in the development of obesity-associated complications such as diabetes and insulin resistance. In this study, we examined whether the acute elevation of 20-HETE levels contributes to the development of diet-driven hyperglycemia and insulin resistance. We employed a conditional transgenic mouse model to overexpress Cyp4a12 (Cyp4a12tg), a murine 20-HETE synthase, together with high fat diet (HFD) feeding. Mice in which Cyp4a12 was induced by doxycycline (DOX) at the onset of HFD feeding gained weight at a greater rate and extent than corresponding DOX-untreated Cyp4a12 mice. Cyp4a12tg mice fed HFD + DOX displayed hyperglycemia and impaired glucose metabolism while corresponding HFD-fed Cyp4a12tg mice (no DOX) did not. Importantly, administration of a 20-HETE antagonist, 20-SOLA, to Cyp4a12tg mice fed HFD + DOX significantly attenuated weight gain and prevented the development of hyperglycemia and impaired glucose metabolism. Levels of insulin receptor (IR) phosphorylation at Tyrosine 972 and insulin receptor substrate-1 (IRS1) phosphorylation at serine 307 were markedly decreased and increased, respectively, in liver, skeletal muscle and adipose tissues from Cyp4a12tg mice fed HFD + DOX; 20-SOLA prevented the IR and IRS1 inactivation, suggesting that 20-HETE interferes with insulin signaling. Additional studies in 3T3-1 differentiated adipocytes confirmed that 20-HETE impairs insulin signaling and that its effect may require activation of its receptor GPR75. Taken together, these results provide strong evidence that 20-HETE interferes with insulin function and contributed to diet-driven insulin resistance

Proximal Tubular-Targeted Overexpression of the Cyp4a12-20-HETE Synthase Promotes Salt-Sensitive Hypertension in Male Mice

20-Hydroxyeicosatetraenoic acid (20-HETE) has been linked to blood pressure (BP) regulation via actions on the renal microvasculature and tubules. We assessed tubular 20-HETE contribution to hypertension by generating transgenic mice overexpressing the CYP4A12-20-HETE synthase (PT-4a12 mice) under the control of the proximal tubule (PT)-specific promoter, phosphoenolpyruvate carboxykinase (PEPCK). 20-HETE levels in the kidney cortex of male (967±210 vs. 249±69 pg/mg protein), but not female (121±15 vs. 92±11 pg/mg protein) PT-4a12 mice, showed a 2.5-fold increase compared to WT. Renal cortical Cyp4a12 mRNA and CYP4A12 protein in male, but not female PT-4a12 mice increased by 2-3-fold compared to WT. Male PT-4a12 mice displayed elevated BP (142±1 vs. 111±4 mmHg,

The Blood Pressure Lowering Effect of 20-HETE Blockade in Cyp4a14(-/-) Mice is associated With Natriuresis

20-Hydroxy-5,8,11,14-eicosatetraenoic acid (20-HETE) has been linked to pro- and anti-hypertensive actions through its ability to promote vasoconstriction and inhibit Na transport in the ascending limb of the loop of Henle, respectively. In this study, we assessed the effects of 20-HETE blockade on blood pressure, renal hemodynamics and urinary sodium excretion in Cyp4a14(-/-) male mice, which display androgen-driven 20-HETE-dependent hypertension. Administration of 20-SOLA, a water-soluble 20-HETE antagonist, in the drinking water normalized blood pressure of male Cyp4a14(-/-) hypertensive mice (124+/- vs 153+/- mmHg) while having no effect on age-matched normotensive wild-type male mice. Hypertension in Cyp4a14(-/-) male mice was accompanied by decreased renal perfusion and reduced glomerular filtration rates which were corrected by treatment with 20-SOLA. Interestingly, Cyp4a14(-/-) male mice treated with 20-SOLA displayed increased urinary sodium excretion that paralleled by the reduction of blood pressure suggestive of an anti-natriuretic activity of endogenous 20-HETE in the hypertensive mice. This interpretation is in line with the observation that the natriuretic response to acute isotonic saline loading in hypertensive Cyp4a14(-/-) male mice was significantly impaired relative to that in WT mice; this impairment was corrected by 20-SOLA treatment. Hence, endogenous 20-HETE appears to promote sodium conservation in hypertensive Cyp4a14(-/-) male mice, presumably, as a result of associated changes in renal hemodynamics and/or direct stimulatory action on tubular sodium reabsorption

Gpr75-Deficient Mice Are Protected From High-Fat Diet-Induced Obesity

OBJECTIVE: G-protein coupled receptor 75 (GPR75) has been identified as the high-affinity receptor of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive and proinflammatory lipid, and mice overproducing 20-HETE have been shown to develop insulin resistance when fed a high-fat diet (HFD), which was prevented by a 20-HETE receptor blocker. Simultaneously, a large-scale exome sequencing of 640,000 subjects identified an association between loss-of-function GPR75 variants and protection against obesity. METHODS: Wild-type (WT) and Gpr75-deficient mice were placed on HFD for 14 weeks, and their obesity phenotype was examined. RESULTS: Male and female Gpr75 null (knockout [KO]) and heterozygous mice gained less weight than WT mice when placed on HFD. KO mice maintained the same level of energy expenditure during HFD feeding, whereas WT mice showed a significant reduction in energy expenditure. Diet-driven adiposity and adipocyte hypertrophy were greatly lessened in Gpr75-deficient mice. HFD-fed KO mice did not develop insulin resistance. Adipose tissue from Gpr75-deficient mice had increased expression of thermogenic genes and decreased levels of inflammatory markers. Moreover, insulin signaling, which was impaired in HFD-fed WT mice, was unchanged in KO mice. CONCLUSIONS: These findings suggest that GPR75 is an important player in the control of metabolism and glucose homeostasis and a likely novel therapeutic target to combat obesity-driven metabolic disorders

20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension

RATIONALE: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. OBJECTIVE: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. METHODS AND RESULTS: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Galphaq/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Galphaq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKbeta, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. CONCLUSIONS: This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases

High Fat Diet-Induced Obesity and Insulin Resistance in Cyp4a14-/- Mice is Mediated by 20-HETE

20-hydroxyeicosatetraenoic acid (20-HETE) has been shown to positively correlate with body mass index, hyperglycemia and plasma insulin levels. This study seeks to identify a causal relationship between 20-HETE and obesity-driven insulin resistance. Cyp4a14(-/-) male mice, a model of 20-HETE overproduction, were fed a regular or high-fat diet (HFD) for 15 weeks. 20-SOLA, a 20-HETE antagonist, was administered from week 0 or week 7 of HFD. HFD-fed mice gained significant weight (16.7+/-3.2 vs. 3.8+/-0.35 g, p\u3c0.05) and developed hyperglycemia (157+/-3 vs 121+/-7 mg/dl, p\u3c0.05) and hyperinsulinemia (2.3+/-0.4 vs 0.5+/-0.1 ng/ml, p\u3c0.05) compared to regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4+/-1 vs 16.7+/-3 g, p\u3c0.05), and normalized the hyperglycemia (157+/-7 vs 102+/-5 mg/dl, p\u3c0.05) and hyperinsulinemia (1.1+/-0.1 vs 2.3+/-0.4 ng/ml, p\u3c0.05). The impaired glucose homeostasis and insulin resistance in HFD-fed mice evidenced by reduced insulin and glucose tolerance were also ameliorated by 20-SOLA. Circulatory and adipose tissue 20-HETE levels significantly increased in HFD-fed mice correlating with impaired insulin signaling, including reduction in insulin receptor tyrosine (Y972) phosphorylation and increased serine (S307) phosphorylation of the insulin receptor substrate-1 (IRS-1). 20-SOLA treatments prevented changes in insulin signaling. These findings indicate that 20-HETE contributes to HFD-induced obesity, insulin resistance and impaired insulin signaling