Loss of function G-protein coupled receptor 75 (GPR75) variants in humans are associated with leanness, and Gpr75 null mice are protected from diet-induced obesity (DIO). However, the mechanisms underlying this protection are largely unknown. Here, we investigated the contribution of adipocyte-derived Gpr75 to DIO. Adipocyte-specific Gpr75 knockout (adipo-Gpr75-/-) male and female mice and their wild-type (WT) littermates were placed on a high-fat diet (HFD) for 14 weeks. Metabolic parameters including body weight, energy intake and expenditure, activity, and glucose metabolism were monitored before and after diet feeding. While WT mice obtained a diabetogenic phenotype on HFD, the adipo-Gpr75-/- counterparts were protected. This protection showed sexual dimorphism. Female adipo-Gpr75-/- mice displayed a 50% (p < 0.001) decrease in weight gain and adiposity compared to WT, whereas male adipo-Gpr75-/- gained weight like WT mice. Interestingly, both male and female adipo-Gpr75-/- mice exhibited improved glucose handling compared to WT, which was correlated to decreased adiposity, abrogated adipose tissue inflammation, and increased insulin sensitivity in skeletal muscle. Importantly, no differences in food intake were observed; however, adipo-Gpr75-/- mice exhibited increased activity and energy expenditure, regardless of sex. Taken together, these findings demonstrate that deletion of GPR75 specifically in adipocytes is sufficient to confer protection against DIO and suggest that adipocyte-derived GPR75 contributes importantly to the pathogenesis of DIO potentially by mechanisms that may include promotion of inflammation, impairment of insulin signaling, and disruption of metabolic homeostasis.

Weight loss remains one of the primary strategies for reducing cardiometabolic risk, particularly with the advent of glucagon-like peptide-1 receptor agonists, which have been demonstrated to induce significant weight loss. Recent evidence suggests, however, that weight loss does not completely normalize the underlying biology of obesity. When weight loss medications are discontinued, patients may regain lost weight along with an increase in cardiometabolic disease risk, indicating that these medications may contribute to transiently altering the phenotype of obesity, but do not produce long-term remission. Adipose tissue is increasingly recognized as an active organ regulating systemic inflammation and metabolic homeostasis. In obesity, adipose tissue becomes inflamed through the activation of innate and adaptive immune pathways. This response has a lasting effect on the immune system. T cells in adipose tissue develop memory-like qualities via epigenetic and transcriptional reprogramming and can persist after weight loss, ready for rapid activation upon renewed metabolic stress. These immunologic memory effects drive repeated weight gain, progressive metabolic dysfunction, and ongoing cardiovascular risk. An immune process is working alongside the endocrine and metabolic adjustments that facilitate energy conservation and fat regain. The repetitive cycles of weight loss and regain further amplify these responses, leading to greater inflammation. Memory T-cell populations are maintained primarily through the CD70-CD27 axis; therefore, targeting this axis may be an effective approach to developing therapies that modify immune memory and achieve long-term cardiometabolic remission when combined with weight loss.

Metabolic syndrome increases the risk of stroke, cardiovascular disease, and diabetes. The morbidity and mortality associated with this constellation of risk factors are equally alarming when considering the economic and global significance that this epidemic has on an institutional and patient level. Despite several current treatments available, there needs to be a continuous effort to explore more specific and effective druggable entities for preventative and therapeutic interventions. Within this context, the G-protein coupled receptor, GPR75, is an attractive pharmacological target. GPR75 and its association with its ligand, 20-hydroxyeicosatetraenoic acid, have been shown to promote hypertension, inflammation, obesity, and insulin resistance. This review will help shed light on this novel signaling pathway and offer a perspective on a promising new direction of targeting different aspects of the metabolic syndrome involving GPR75. Gene targeting of GPR75 is more effective than current pharmacologic therapies without the known side effects.

Rationale: Pulmonary hypertension (PH) is a multifactorial disease with a poor prognosis and inadequate treatment options. We found two-fold higher expression of the orphan G-Protein Coupled Receptor 75 (GPR75) in leukocytes and pulmonary arterial smooth muscle cells from idiopathic PH patients and from lungs of C57BL/6 mice exposed to hypoxia. We therefore postulated that GPR75 signaling is critical to the pathogenesis of PH.Methods: To test this hypothesis, we exposed global (Gpr75-/-) and endothelial cell (EC) GPR75 knockout (EC- Gpr75- /-)mice and wild-type (control) mice to hypoxia (10% oxygen) or normal atmospheric oxygen for 5 weeks. We then recorded echocardiograms and performed right heart catheterizations.Results: Chronic hypoxia increased right ventricular systolic and diastolic pressures in wild-type mice but not Gpr75-/-or EC-Gpr75-/-mice. In situ hybridization and qPCR results revealed that Gpr75 expression was increased in the alveoli, airways and pulmonary arteries of mice exposed to hypoxia. In addition, levels of chemokine (C-C motif) ligand 5 (CCL5), a low affinity ligand of GPR75, were increased in the lungs of wild-type, but not Gpr75-/-, mice exposed to hypoxia, and CCL5 enhanced hypoxia-induced contraction of intra-lobar pulmonary arteries in a GPR75-dependent manner. Gpr75 knockout also increased pulmonary cAMP levels and decreased contraction of intra-lobar pulmonary arteries evoked by endothelin-1 or U46619 in cAMP-protein kinase A-dependent manner.Conclusion: These results suggest GPR75 has a significant role in the development of hypoxia-induced PH.

Vascular function is dynamically regulated and dependent on a bevy of cell types and factors that work in concert across the vasculature. The vasoactive eicosanoid, 20-Hydroxyeicosatetraenoic acid (20-HETE) is a key player in this system influencing the sensitivity of the vasculature to constrictor stimuli, regulating endothelial function, and influencing the renin angiotensin system (RAS), as well as being a driver of vascular remodeling independent of blood pressure elevations. Several of these bioactions are accomplished through the ligand-receptor pairing between 20-HETE and its high-affinity receptor, GPR75. This 20-HETE axis is at the root of various vascular pathologies and processes including ischemia induced angiogenesis, arteriogenesis, septic shock, hypertension, atherosclerosis, myocardial infarction and cardiometabolic diseases including diabetes and insulin resistance. Pharmacologically, several preclinical tools have been developed to disrupt the 20-HETE axis including 20-HETE synthesis inhibitors (DDMS and HET0016), synthetic 20-HETE agonist analogues (20-5,14-HEDE and 20-5,14-HEDGE) and 20-HETE receptor blockers (AAA and 20-SOLA). Systemic or cell-specific therapeutic targeting of the 20-HETE-GPR75 axis continues to be an invaluable approach as studies examine the molecular underpinnings activated by 20-HETE under various physiological settings. In particular, the development and characterization of 20-HETE receptor blockers look to be a promising new class of compounds that can provide a considerable benefit to patients suffering from these cardiovascular pathologies.

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.

OBJECTIVE:20-Hydroxyeicosatetraenoic acid (20-HETE) is a vasoactive eicosanoid exhibiting effects on vascular smooth muscle cell (VSMC) via G-protein coupled receptor 75 (GPR75) and include stimulation of contractility, migration, and growth. We examined whether VSMC-targeted overexpression of CYP4A12, the primary 20-HETE-producing enzyme in mice, is sufficient to promote hypertension.METHODS:Mice with VSM-specific Cyp4a12 overexpression (Myh11-4a12) and their littermate controls (WT) were generated by crossbreeding Cyp4a12-floxed with Myh11-Cre mice. The 20-HETE receptor blocker, N-disodium succinate-20-hydroxyeicosa-6(Z),15(Z)-diencarboxamide (AAA), was administered in the drinking water. Experiments were carried out for 12 days. SBP was measured by tail cuff. Renal interlobar and mesenteric arteries were harvested for assessment of gene expression, 20-HETE levels, vascular contractility, vasodilation, and remodeling.RESULTS:Vascular and circulatory levels of 20-HETE were several folds higher in Myh11-4a12 mice compared with WT. The Myh11-4a12 mice compared with WT were hypertensive (145 ± 2 vs. 127 ± 2 mmHg; P < 0.05) and their vasculature displayed a contractile phenotype exemplified by increased contractility, reduced vasodilatory capacity, and increased media to lumen ratio. All these features were reversed by the administration of AAA. The mechanism of increased contractility includes, at least in part, Rho-kinase activation followed by increased myosin light chain phosphorylation and activation of the contractile apparatus.CONCLUSION:VSM-specific Cyp4a12 overexpression is sufficient to alter VSM cell phenotype through changes in contractile markers and enhancement in contractility that promote hypertension and vascular dysfunction in a 20-HETE-dependent manner. The 20-HETE receptor GPR75 may represent a novel target for the treatment of hypertension and associated vascular conditions.

20‐Hydroxyeicosatetraenoic acid (20‐HETE) and its receptor (20HR), GPR75 (Gq), exhibit diverse bioactions that promote the activation of pro‐hypertensive, ‐diabetic and ‐obesity signals. The pharmacological properties associated with analogues that target GPR75 remain unclear. The screening of 20‐HETE and synthetic 20‐HETE analogues using changes of intracellular calcium (iCa2+) in the endothelial cell line, EA.hy926, as a measure of 20HR activation, revealed that the compounds 20‐HETE (10 nM), sodium 20‐hydroxyeicosa‐5Z,14Z‐dienoate (20‐5,14‐HEDE) (10 nM) and sodium 14‐((6‐hydroxyhexyl)oxy)tetradec‐5(Z)‐enoate (5Z‐HOTE) (10 nM) promote significant and comparable elevations in iCa2+. In EA.hy926 cells, 20‐HETE elicited a half‐maximal effective concentrations (EC50) with respect to iCa2+ of 1.228 e‐9 M while 20‐5,14‐HEDE’s EC50 was 6.908 e‐10 M. The water‐soluble derivative of 20‐5,14‐HEDE, SOLAGO, exhibited a marked and leftward shift in the dose‐response with an EC50 of 2.702 e‐10 M. Interestingly, sodium 19(R)‐ and 19(S)‐hydroxyeicosa‐5(Z),14(Z)‐dienoate, analogues of 19(R)‐HETE, an endogenous 20HR blocker (20HRB), demonstrated partial agonist activity; elevating iCa2+to 1.6‐ and 2‐fold over baseline vehicle treatment, respectively. With respect to 20HR blockers, the water‐soluble 20HRBs 2,5,8,11,14,17‐hexaoxanonadecan‐19‐yl 20‐hydroxyeicosa‐6(Z),15(Z)‐dienoate (20‐SOLA) and N‐disodium succinate‐20‐hydroxyeicosa‐6(Z),15(Z)‐diencarboxamide (AAA) displayed potent half‐maximal inhibitory concentrations (IC50s) of 8.059 e‐10 and 5.356 e‐10 M, respectively. The 19(R)‐HETE analogue sodium (19(R)‐hydroxyeicosa‐5(Z),14(Z)‐dienoyl)glycinate (19(R)‐HEDGE) also demonstrated a strong yet less potent IC50 response of 6.715 e‐9 M. Further studies are necessary to better understand the structure‐function relationships between 20HR agonists, partial agonists, and receptor blockers. These data would allow for the development of novel 20HRBs for the treatment of various pathologies associated with elevations in 20‐HETE including hypertension, cancer, diabetes, and obesity.

The CDC estimates the prevalence of obesity in the United States to be over 42.5% in 2021. With obesity related conditions including hypertension, heart disease, and type 2 diabetes, therapeutic approaches to treat obesity are necessary to prevent premature death. Recent studies have identified the orphan G-protein coupled receptor, GPR75, as a possible target. For example, Akbari et. al., (Science, 2021) found that truncated loss of function variants of GPR75 were associated with 5.3 kg lower bodyweight and 54% lower odds of obesity in heterozygous individuals. Based on these findings, we hypothesized that global deletion of Gpr75 protects against diet-induced obesity (DIO) and insulin resistance. Wild-type and Gpr75null mice were subjected to 14 weeks of regular chow or high-fat diet (HFD) feeding. Body composition, intraperitoneal glucose and insulin tolerance tests, and oxygen consumption were measured initially and at week 14. There were no significant differences in body composition, glucose homeostasis, and oxygen consumption between wild-type (WT), Gpr75 (KO) and Gpr75 (HET) mice initially. However, WT mice obtained a diabetogenic phenotype after HFD-feeding while KO and HET counterparts were robustly protected as indicated by reduced body weight, (45.32 ± 1.387 grams, 28.29 ± 1.47 grams, and 36.60 ± 1.8 grams respectively) and increased sensitivity to insulin (blood glucose 30 min after insulin injection as percent change, -28.5 ± 4.41%, -50.68 ± 4.15% and -39.07 ± 1.79% respectively, p<0.0001). Weekly energy intake calculated from food consumption showed no significant differences between all three groups. However, oxygen consumption measured over 60 minutes indicated an increase in energy expenditure in KO compared to WT mice (73.76 ml/min/kg vs. 44.61 ml/min/kg respectively, p<0.0001). This correlated with a 2.63-fold increase in brown adipose tissue (BAT) UCP1 mRNA expression in KO compared to WT mice. While the abundance of BAT is low in humans compared to mice, our findings suggest that improved glucose homeostasis as a result of GPR75 deficiency is linked to increased BAT thermogenesis. This may provide a potential novel route for the control of metabolism to combat obesity-driven metabolic disorders.

20-Hydroxyeicosatetraenoic acid (20-HETE) is a bioactive lipid generated from the ω-hydroxylation of arachidonic acid (AA) by enzymes of the cytochrome P450 (CYP) family, primarily the CYP4A and CYP4F subfamilies. 20-HETE is most notably identified as a modulator of vascular tone, regulator of renal function, and a contributor to the onset and development of hypertension and cardiovascular disease. 20-HETE-mediated signaling promotes hypertension by sensitizing the vasculature to constrictor stimuli, inducing endothelial dysfunction, and potentiating vascular inflammation. These bioactions are driven by the activation of the G-protein coupled receptor 75 (GPR75), a 20-HETE receptor (20HR). Given the capacity of 20-HETE signaling to drive pro-hypertensive mechanisms, the CYP/20-HETE/GPR75 axis has the potential to be a significant therapeutic target for the treatment of hypertension and cardiovascular diseases associated with increases in blood pressure. In this chapter, we review 20-HETE-mediated cellular mechanisms that promote hypertension, highlight important data in humans such as genetic variants in the CYP genes that potentiate 20-HETE production and describe recent findings in humans with 20HR/GPR75 mutations. Special emphasis is given to the 20HR and respective receptor blockers that have the potential to pave a path to translational and clinical studies for the treatment of 20-HETE-driven hypertension, and obesity/metabolic syndrome.

Pulmonary Hypertension (PH) is a cardiopulmonary disease estimated to affect between 20 million to 70 million individuals globally, with poor prognosis and inadequate treatment. Recent studies showed the orphan G Protein Coupled Receptor 75 (GPR75) is upregulated in the pulmonary vessels of PH patients, especially in females. Therefore, we hypothesized that Gpr75knock out (Gpr75KO) mice will be protected from developing PH. To test our hypothesis, first we determined the vasoreactivity of isolated intra-lobar pulmonary arteries from wild-type (WT) and GPR75KO mice induced by KCl (WT=6; Gpr75KO=8), U46619 (WT=8; Gpr75KO=8), ZD7288 (WT=7; Gpr75KO=7), and Endothelin (WT=7; Gpr75KO=8) to test their vasoreactivity. The Gpr75KO as compared with wild-type pulmonary arteries contracted less to all the contractile agents. Next, we determine if Gpr75KO mice were protected from developing hypoxia-induced PH. We placed 3-month-old female Gpr75KO (n=8) and control wild-type (C57BL/C) mice (n=18) in a hypoxic chamber with 10% Oxygen or at atmospheric oxygen for 5 weeks. At the end of 5 weeks, we performed right heart catheterizations. Gpr75KO mice developed less (P<0.05) right ventricle systolic pressure (RVSP: 24.3±1.2 mmHg) and right ventricle diastolic pressure (RVDP: 2.0±0.0 mmHg) than the wild-type mice under hypoxia (RSVP and RDVP of 56.6±3.5 mmHg and 9.8±0.9 mmHg, respectively). Since GPR75 is a G-protein coupled receptor to determine secondary messengers that potentially contribute to regulating contractile function of pulmonary arteries, we measured cAMP and IP3 in lungs of wild-type and GPR75KO mice. While we found no difference in the IP3 levels between the WT and Gpr75KO groups, cAMP levels were significantly higher in Gpr75KO compared to WT mice. These results suggest GPR75 plays a major role in the development of PH by potentially attenuating cAMP-dependent signaling and concomitantly augmenting pulmonary constriction in response to hypoxia.

Compensatory angiogenesis is an important adaptation for recovery from critical ischemia. We recently identified 20-hydroxyeicosatetraenoic acid (20-HETE) as a novel contributor of ischemia-induced angiogenesis. However, the precise mechanisms by which ischemia promotes 20-HETE increases that drive angiogenesis are unknown. This study aims to address the hypothesis that inflammatory neutrophil-derived myeloperoxidase (MPO) and hypochlorous acid (HOCl) critically contribute to 20-HETE increases leading to ischemic angiogenesis. Using Liquid Chromatography-Mass Spectrometry/Mass Spectrometry, Laser Doppler Perfusion Imaging, and Microvascular Density analysis, we found that neutrophil depletion and MPO knockout mitigate angiogenesis and 20-HETE production in the gracilis muscles of mice subjected to hindlimb ischemia. Furthermore, we found MPO and HOCl to be elevated in these tissues postischemia as assessed by immunofluorescence microscopy and in vivo live imaging of HOCl. Next, we demonstrated that the additions of either HOCl or an enzymatic system for generating HOCl to endothelial cells increase the expression of CYP4A11 and its product, 20-HETE. Finally, pharmacological interference of hypoxia inducible factor (HIF) signaling results in ablation of HOCl-induced CYP4A11 transcript and significant reductions in CYP4A11 protein. Collectively, we conclude that neutrophil-derived MPO and its product HOCl activate HIF-1α and CYP4A11 leading to increased 20-HETE production that drives postischemic compensatory angiogenesis. SIGNIFICANCE STATEMENT Traditionally, neutrophil derived MPO and HOCl are exclusively associated in the innate immunity as potent bactericidal/virucidal factors. The present study establishes a novel paradigm by proposing a unique function for MPO/HOCl as signaling agents that drive critical physiological angiogenesis by activating the CYP4A11-20-HETE signaling axis via a HIF-1α-dependent mechanism. The findings from this study potentially identify novel therapeutic targets for the treatment of ischemia and other diseases associated with abnormal angiogenesis.

Although standard testing guidelines use a species as a representative surrogate, species-specific sensitivity is well-known. The aim of this study was to investigate the species-specific difference in avoidance behaviour among Collembola species exposed to silver (Ag) nanomaterials (NM) (Ag NM300K). The avoidance test was performed with Folsomia candida, an international standard species in laboratory tests, and five widely distributed species with different life history traits, commonly used in small multispecies systems (Folsomia fimetaria, Proisotoma minuta, Mesaphorura macrochaeta, Protaphorura fimata and Ceratophysella denticulata). There was higher avoidance in euedaphic species, such as F. candida and F. fimetaria, compared to the epiedaphic species C. denticulata, which showed the least avoidance behaviour. An explanation may be that euedaphic species (living in deeper soil layers) are more directly exposed within the soil pores and have developed a pronounced avoidance behaviour. In contrast, species living on the surface are likely less directly exposed and hence only avoid at higher total concentrations. Additionally, difference in cuticula between the groups, providing different degrees of protection against exposure, can explain the different behaviours. The present results highlight the importance of biodiversity for the ecosystem and raise awareness on species sensitivity.

Background and Purpose The G-protein-coupled receptor GPR75 (Gq) and its ligand, the cytochrome P450-derived vasoactive eicosanoid 20-hydroxyeicosatetraenoic acid (20-HETE), are involved in the activation of pro-inflammatory and hypertensive signalling cascades contributing to diabetes, obesity, vascular dysfunction/remodelling, hypertension and cardiovascular disease. Little is known as to how, where and with what affinity 20-HETE interacts with GPR75. Experimental Approach To better understand the pairing of 20-HETE and its receptor (GPR75), we used surface plasmon resonance (SPR) to determine binding affinity/kinetics. The PRESTO-Tango receptor-ome methodology for GPR75 overexpression was coupled with FLIPR Calcium 6 assays, homogeneous time-resolved fluorescence (HTRF) IP-1 and beta-arrestin recruitment assays to determine receptor activation and downstream signalling events. Key Results SPR confirmed 20-HETE binding to GPR75 with an estimated K-D of 1.56 x 10(-10) M. In GPR75-transfected HTLA cells, 20-HETE stimulated intracellular Ca2+ levels, IP-1 accumulation and beta-arrestin recruitment, all of which were negated by known 20-HETE functional antagonists. Computational modelling of the putative ligand-binding pocket and mutation of Thr212 within the putative 20-HETE binding site abolished 20-HETE's ability to stimulate GPR75 activation. Knockdown of GPR75 in human endothelial cells nullified 20-HETE-stimulated intracellular Ca2+. The chemokine CCL5, a suggested GPR75 ligand, binds to GPR75 (K-D of 5.85 x 10(-10) M) yet fails to activate GPR75; however, it inhibited 20-HETE's ability to activate GPR75 signalling. Conclusions and Implications We have identified 20-HETE as a high-affinity ligand for GPR75 and CCL5 as a low-affinity negative regulator of GPR75, providing additional evidence for the deorphanization of GPR75 as a 20-HETE receptor.

Aging is associated with a significant deficiency in circulating insulin-like growth factor-1 (IGF-1), which has an important role in the pathogenesis of age-related vascular cognitive impairment (VCI). Impairment of moment-to-moment adjustment of regional cerebral blood flow via neurovascular coupling (NVC) importantly contributes to VCI. Previous studies established a causal link between circulating IGF-1 deficiency and neurovascular dysfunction. Release of vasodilator mediators from activated astrocytes plays a key role in NVC. To determine the impact of impaired IGF-1 signaling on astrocytic function, astrocyte-mediated NVC responses were studied in a novel mouse model of astrocyte-specific knockout of IGF1R ( GFAP-Cre ERT2 /Igf1r f/f ) and accelerated neurovascular aging. We found that mice with disrupted astrocytic IGF1R signaling exhibit impaired NVC responses, decreased stimulated release of the vasodilator gliotransmitter epoxy-eicosatrienoic acids (EETs), and upregulation of soluble epoxy hydrolase (sEH), which metabolizes and inactivates EETs. Collectively, our findings provide additional evidence that IGF-1 promotes astrocyte health and maintains normal NVC, protecting cognitive health.

Background: Obesity affects ∼20% of children in the United States and reports of successful dietary treatment are lacking. This study aimed to determine the change in body weight in severely obese youth after carbohydrate-restricted dietary intervention. Methods: This single-center study of a carbohydrate-restricted diet (≤30 grams per day), with unlimited calories, fat, and protein for 3-4 months, examined two groups of severely obese youth of ages 5-18 years: Group A, retrospectively reviewed charts of severely obese youth referred to the Pediatric Obesity Clinic at Hoops Family Children's Hospital and the Ambulatory Division of Marshall Pediatrics, Marshall University School of Medicine, in Huntington, WV, between July 1, 2014 and June 30, 2017 (n = 130), and Group B, prospective participants, referred between July 1, 2018 and December 31, 2018, followed with laboratory studies pre- and postdietary intervention (n = 8). Results: In Group A, 310 participants began the diet, 130 (42%) returned after 3-4 months. Group B had 14 enrollees who began the diet, and 8 followed up at 3-4 months (57%). Girls compared with boys were more likely to complete the diet (P = 0.02). Participants <12 years age were almost twice as likely to complete the diet compared with those 12-18 years (64% vs. 36%, P < 0.01); however, the older group subjects who completed the diet had the same percentage of weight loss compared with those <12 years (6.9% vs. 6.9%). Group A had reductions in weight of 5.1 kg (P < 0.001), body mass index (BMI) 2.5 kg/m2 (P < 0.001), and percentage weight loss 6.9% (P < 0.001). Group B had reductions in weight 9.6 kg (P < 0.01), BMI 4 kg/m2 (P < 0.01), and percentage weight loss 9% (P < 0.01). In addition, participants had significant reductions of fasting serum insulin (P < 0.01), triglycerides (P < 0.01), and 20-hydroxyeicosatetraenoic acid (P < 0.01). Conclusions: This study demonstrated a carbohydrate-restricted diet, utilized short term, effectively reduced weight in a large percentage of severely obese youth, and can be replicated in a busy primary care office.

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