Elucidation of the molecular mechanism of the breakage-fusion-bridge (BFB) cycle using a CRISPR-dCas9 cellular model.

Chromosome instability (CIN) is frequently observed in many tumors. The breakage-fusion-bridge (BFB) cycle has been proposed to be one of the main drivers of CIN during tumorigenesis and tumor evolution. However, the detailed mechanisms for the individual steps of the BFB cycle warrants further investigation. Here, we demonstrated that a nuclease-dead Cas9 (dCas9) coupled with a telomere-specific single-guide RNA (sgTelo) can be used to model the BFB cycle. First, we showed that targeting dCas9 to telomeres using sgTelo impeded DNA replication at telomeres and induced a pronounced increase of replication stress and DNA damage. Using Single-Molecule Telomere Assay via Optical Mapping (SMTA-OM), we investigated the genome-wide features of telomeres in the dCas9/sgTelo cells and observed a dramatic increase of chromosome end fusions, including fusion/ITS+ and fusion/ITS-.Consistently, we also observed an increase in the formation of dicentric chromosomes, anaphase bridges, and intercellular telomeric chromosome bridges (ITCBs). Utilizing the dCas9/sgTelo system, we uncovered many novel molecular and structural features of the ITCB and demonstrated that multiple DNA repair pathways are implicated in the formation of ITCBs. Our studies shed new light on the molecular mechanisms of the BFB cycle, which will advance our understanding of tumorigenesis, tumor evolution, and drug resistance.

Alcohol and caffeine synergistically induce spontaneous ventricular tachyarrhythmias: ameliorated with dantrolene treatment.

Background: Alcohol and caffeine are the 2 frequently consumed substances in the general population, and the 2 substances are frequently co-consumed. Both substances may increase cardiac arrhythmia risk. However, it is unknown whether alcohol and caffeine co-consumption can synergistically enhance cardiac arrhythmogenesis. Objective: The study sought to investigate whether caffeine and binge drinking synergistically affect cardiac arrhythmogenesis. Methods: A binge drinking rat model (alcohol 2 g/kg, intraperitoneal, every other day for 3 times) was used. Rats (4 months old, both sexes) were randomized into the following 4 groups: binge alcohol-only group (A) (n = 8), nonalcohol, caffeine-only (60 mg/kg, intraperitoneal) group (C) (n = 8), binge alcohol plus caffeine group (A+C) (n = 8), and binge alcohol + caffeine + dantrolene group (A+D) (n = 7, treated with dantrolene 10 mg/kg before each alcohol injection). We also investigated whether alcohol induces Ca2+ sparks and dantrolene treatment attenuates alcohol-induced Ca2+ leak in ventricular myocytes. Results: No arrhythmia was induced with caffeine alone (group C, n = 0 of 8) or alcohol alone (group A, n = 0 of 8). However, alcohol + caffeine induced spontaneous ventricular tachyarrhythmias in all rats (group A+C, n = 8 of 8; P < .001 vs group C or A). Dantrolene prevented ventricular tachyarrhythmia induction in all 7 rats (group A+D, n = 0 of 7; P < .001 vs group A+C). In isolated ventricular myocytes, alcohol significantly increased Ca2+ sparks and dantrolene treatment reduced alcohol-induced Ca2+ sparks. Conclusion: Co-consumption of caffeine and binge drinking synergistically promote spontaneous ventricular tachyarrhythmias in rats. Dantrolene treatment can decrease alcohol-enhanced Ca2+ sparks in vitro and prevented alcohol and caffeine induced ventricular tachyarrhythmias in vivo.

Stabilizing Cardiac Ryanodine Receptor With Dantrolene Treatment Prevents Binge Alcohol-Enhanced Atrial Fibrillation in Rats.

ABSTRACT: Binge drinking is a risk factor for cardiac arrhythmias, known as the holiday heart syndrome. Atrial fibrillation (AF) is the most frequently diagnosed arrhythmia in this condition. Recent reports indicated that cardiac ryanodine receptor (RyR2) dysfunction and Ca 2+ leak contribute to alcohol-enhanced AF. In this study, we investigated whether stabilizing RyR2 with dantrolene treatment can prevent alcohol-enhanced AF in rats. A binge drinking rat model was established with alcohol (2 g /kg, IP) delivered once every other day for 4 times. The study consisted of following 3 groups: control group (n = 9), binge alcohol group (n = 10), and binge alcohol + dantrolene (A+D) group (dantrolene, 10 mg/kg, IP before each alcohol injection, n = 9). Echocardiography, left ventricular hemodynamics, in vivo atrial electrophysiology and AF inducibility test, RyR2 phosphorylation level, and blood norepinephrine level were studied 24 hours after the last injection. Ca 2+ leak in isolated atrial myocytes from control and binge alcohol rats was examined. Binge alcohol significantly increased AF inducibility (1/9 in control vs. 8/9 in binge alcohol group, P < 0.05) and AF duration. Dantrolene treatment significantly reduced both AF inducibility (2/9 in dantrolene group, P < 0.05) and AF duration. Binge alcohol significantly increased Ca 2+ leak in isolated atrial myocytes, which was reduced by dantrolene treatment. Blood norepinephrine,7 RyR2 phosphorylation level, cardiac echocardiography, and left ventricular hemodynamics were not significantly affected 24 hours after binge drinking. In conclusion, stabilizing RyR2 with dantrolene treatment significantly attenuated binge drinking-enhanced AF, suggesting that therapeutic strategies stabilizing RyR2 could be a preventive measure to blunt binge drinking-enhanced AF arrhythmogenesis.

Carbohydrate-Restricted Diet: A Successful Strategy for Short-Term Management in Youth with Severe Obesity-An Observational Study.

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.

Milk thistle seed cold press oil attenuates markers of the metabolic syndrome in a mouse model of dietary-induced obesity.

Milk thistle cold press oil (MTO) is an herbal remedy derived from Silybum marianum which contains a low level of silymarin and mixture of polyphenols and flavonoids. The effect of MTO on the cardiovascular and metabolic complications of obesity was studied in mice that were fed a high-fat diet (HFD) for 20 weeks and treated with MTO for the final 8 weeks of the diet. MTO treatment attenuated HFD-induced obesity, fasting hyperglycemia, hypertension, and induced markers of mitochondrial fusion and browning of white adipose. Markers of inflammation were also attenuated in both adipose and the liver of MTO-treated mice. In addition, MTO resulted in the improvement of liver fibrosis. These results demonstrate that MTO has beneficial actions to attenuate dietary obesity-induced weight gain, hyperglycemia, hypertension, inflammation, and suggest that MTO supplementation may prove beneficial to patients exhibiting symptoms of metabolic syndrome. PRACTICAL APPLICATIONS: Natural supplements are increasingly being considered as potential therapies for many chronic cardiovascular and metabolic diseases. Milk thistle cold press oil (MTO) is derived from Silybum marianum which is used as a dietary supplement in different parts of the world. The results of the present study demonstrate that MTO supplementation normalizes several metabolic and cardiovascular complications arising from dietary-induced obesity. MTO supplementation also had anti-inflammatory actions in the adipose as well as the liver. These results suggest that supplementation of MTO into the diet of obese individuals may afford protection against the worsening of cardiovascular and metabolic disease and improve inflammation and liver fibrosis.

Cold-Pressed Nigella Sativa Oil Standardized to 3% Thymoquinone Potentiates Omega-3 Protection against Obesity-Induced Oxidative Stress, Inflammation, and Markers of Insulin Resistance Accompanied with Conversion of White to Beige Fat in Mice.

Excessive lipid accumulation in white adipose tissue (WAT) results in adipocyte hypertrophy and chronic low-grade inflammation, which is the major cause of obesity-associated insulin resistance and consequent metabolic disease. The development of beige adipocytes in WAT (browning of WAT) increases energy expenditure and has been considered as a novel strategy to counteract obesity. Thymoquinone (TQ) is the main bioactive quinone derived from the plant Nigella Sativa and has antioxidative and anti-inflammatory capacities. Fish oil omega 3 (ω3) enhances both insulin sensitivity and glucose homeostasis in obesity, but the involved mechanisms remain unclear. The aim of this study is to explore the effects of TQ and ω3 PUFAs (polyunsaturated fatty acids) on obesity-associated inflammation, markers of insulin resistance, and the metabolic effects of adipose tissue browning. 3T3-L1 cells were cultured to investigate the effects of TQ and ω3 on the browning of WAT. C57BL/6J mice were fed a high-fat diet (HFD), supplemented with 0.75% TQ, and 2% ω3 in combination for eight weeks. In 3T3-L1 cells, TQ and ω3 reduced lipid droplet size and increased hallmarks of beige adipocytes such as uncoupling protein-1 (UCP1), PR domain containing 16 (PRDM16), fibroblast growth factor 21 (FGF21), Sirtuin 1 (Sirt1), Mitofusion 2 (Mfn2), and heme oxygenase 1 (HO-1) protein expression, as well as increased the phosphorylation of Protein Kinase B (AKT) and insulin receptors. In the adipose tissue of HFD mice, TQ and ω3 treatment attenuated levels of inflammatory adipokines, Nephroblastoma Overexpressed (NOV/CCN3) and Twist related protein 2 (TWIST2), and diminished adipocyte hypoxia by decreasing HIF1α expression and hallmarks of beige adipocytes such as UCP1, PRDM16, FGF21, and mitochondrial biogenesis markers Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), Sirt1, and Mfn2. Increased 5' adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) phosphorylation and HO-1 expression were observed in adipose with TQ and ω3 treatment, which led to increased pAKT and pIRS1 Ser307 expression. In addition to the adipose, TQ and ω3 also increased inflammation and markers of insulin sensitivity in the liver, as demonstrated by increased phosphorylated insulin receptor (pIR tyr972), insulin receptor beta (IRß), UCP1, and pIRS1 Ser307 and reduced NOV/CCN3 expression. Our data demonstrate the enhanced browning of WAT from TQ treatment in combination with ω3, which may play an important role in decreasing obesity-associated insulin resistance and in reducing the chronic inflammatory state of obesity.

Heme Oxygenase-1 Upregulation: A Novel Approach in the Treatment of Cardiovascular Disease.

Significance: Heme oxygenase (HO) plays a pivotal role in both vascular and metabolic functions and is involved in many physiological and pathophysiological processes in vascular endothelial cells (ECs) and adipocytes. Recent Advances: From the regulation of adipogenesis in adipose tissue to the adaptive response of vascular tissue in the ECs, HO plays a critical role in the capability of the vascular system to respond and adjust to insults in homeostasis. Recent studies show that HO-1 through regulation of adipocyte and adipose tissue functions ultimately aid not only in local but also in systemic maintenance of homeostasis. Critical Issues: Recent advances have revealed the existence of a cross talk between vascular ECs and adipocytes in adipose tissue. In the pathological state of obesity, this cross talk contributes to the condition's adverse chronic effects, and we propose that specific targeting of the HO-1 gene can restore signaling pathways and improve both vascular and adipose functions. Future Directions: A complete understanding of the role of HO-1 in regulation of cardiovascular homeostasis is important to comprehend the homeostatic regulation as well as in cardiovascular disease. Efforts are required to highlight the effects and the ability to target the HO-1 gene in models of obesity with an emphasis on the role of pericardial fat on cardiovascular health.

Adipocyte Specific HO-1 Gene Therapy is Effective in Antioxidant Treatment of Insulin Resistance and Vascular Function in an Obese Mice Model.

: Obesity is a risk factor for vascular dysfunction and insulin resistance. The study aim was to demonstrate that adipocyte-specific HO-1 (heme oxygenase-1) gene therapy is a therapeutic approach for preventing the development of obesity-induced metabolic disease in an obese-mice model. Specific expression of HO-1 in adipose tissue was achieved by using a lentiviral vector expressing HO-1 under the control of the adiponectin vector (Lnv-adipo-HO-1). Mice fed a high-fat diet (HFD) developed adipocyte hypertrophy, fibrosis, decreased mitochondrial respiration, increased levels of inflammatory adipokines, insulin resistance, vascular dysfunction, and impaired heart mitochondrial signaling. These detrimental effects were prevented by the selective expression of HO-1 in adipocytes. Lnv-adipo-HO-1-transfected mice on a HFD display increased cellular respiration, increased oxygen consumption, increased mitochondrial function, and decreased adipocyte size. Moreover, RNA arrays confirmed that targeting adipocytes with HO-1 overrides the genetic susceptibility of adiposopathy and correlated with restoration of the expression of anti-inflammatory, thermogenic, and mitochondrial genes. Our data demonstrate that HO-1 gene therapy improved adipose tissue function and had positive impact on distal organs, suggesting that specific targeting of HO-1 gene therapy is an attractive therapeutic approach for improving insulin sensitivity, metabolic activity, and vascular function in obesity.

Epoxyeicosatrienoic intervention improves NAFLD in leptin receptor deficient mice by an increase in PGC1α-HO-1-PGC1α-mitochondrial signaling.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is associated with obesity and is considered to be an inflammatory disorder characterized by fatty acid accumulation, oxidative stress, and lipotoxicity. We have previously reported that epoxyeicosatrienoic acid-agonist (EET-A) has multiple beneficial effects on cardiac, renal and adipose tissue function while exhibiting both anti-inflammatory and anti-oxidant activities. We hypothesized that EET-A intervention would play a central role in attenuation of obesity-induced steatosis and hepatic fibrosis that leads to NAFLD. METHODS: We studied the effect of EET-A on fatty liver using db/db mice as a model of obesity. Mice were fed a high fat diet (HFD) for 16 weeks and administered EET-A twice weekly for the final 8 weeks. RESULTS: db/db mice fed HFD significantly increased hepatic lipid accumulation as manifested by increases in NAS scores, hepatic fibrosis, insulin resistance, and inflammation, and decreases in mitochondrial mitofusin proteins (Mfn 1/2) and anti-obesity genes Fibroblast growth factor 21 (FGF21) and Cellular Repressor of E1A-Stimulated Genes 1 (CREG1). EET-A administration reversed the decrease in these genes and reduced liver fibrosis. Knockout of Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) in EET-A treated mice resulted in a reversal of the beneficial effects of EET-A administration. CONCLUSIONS: EET-A intervention diminishes fatty acid accumulation, fibrosis, and NFALD associated with an increase in HO-1-PGC1α and increased insulin receptor phosphorylation. A pharmacological strategy involving EETs may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NAFLD.

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 wk. 20-SOLA [2,5,8,11,14,17-hexaoxanonadecan-19-yl 20-hydroxyeicosa-6( Z),15( Z)-dienoate], 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 < 0.05) and developed hyperglycemia (157 ± 3 vs. 121 ± 7 mg/dl, P < 0.05) and hyperinsulinemia (2.3 ± 0.4 vs. 0.5 ± 0.1 ng/ml, P < 0.05) compared with regular diet-fed mice. 20-SOLA attenuated HFD-induced weight gain (9.4 ± 1 vs. 16.7 ± 3 g, P < 0.05) and normalized the hyperglycemia (157 ± 7 vs. 102 ± 5 mg/dl, P < 0.05) and hyperinsulinemia (1.1 ± 0.1 vs. 2.3 ± 0.4 ng/ml, P < 0.05). 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.

Development of NASH in Obese Mice is Confounded by Adipose Tissue Increase in Inflammatory NOV and Oxidative Stress.

AIM: Nonalcoholic steatohepatitis (NASH) is the consequence of insulin resistance, fatty acid accumulation, oxidative stress, and lipotoxicity. We hypothesize that an increase in the inflammatory adipokine NOV decreases antioxidant Heme Oxygenase 1 (HO-1) levels in adipose and hepatic tissue, resulting in the development of NASH in obese mice. METHODS: Mice were fed a high fat diet (HFD) and obese animals were administered an HO-1 inducer with or without an inhibitor of HO activity to examine levels of adipose-derived NOV and possible links between increased synthesis of inflammatory adipokines and hepatic pathology. RESULTS: NASH mice displayed decreased HO-1 levels and HO activity, increased levels of hepatic heme, NOV, MMP2, hepcidin, and increased NAS scores and hepatic fibrosis. Increased HO-1 levels are associated with a decrease in NOV, improved hepatic NAS score, ameliorated fibrosis, and increases in mitochondrial integrity and insulin receptor phosphorylation. Adipose tissue function is disrupted in obesity as evidenced by an increase in proinflammatory molecules such as NOV and a decrease in adiponectin. Importantly, increased HO-1 levels are associated with a decrease of NOV, increased adiponectin levels, and increased levels of thermogenic and mitochondrial signaling associated genes in adipose tissue. CONCLUSIONS: These results suggest that the metabolic abnormalities in NASH are driven by decreased levels of hepatic HO-1 that is associated with an increase in the adipose-derived proinflammatory adipokine NOV in our obese mouse model of NASH. Concurrently, induction of HO-1 provides protection against insulin resistance as seen by increased insulin receptor phosphorylation. Pharmacological increases in HO-1 associated with decreases in NOV may offer a potential therapeutic approach in preventing fibrosis, mitochondrial dysfunction, and the development of NASH.

Ablation of soluble epoxide hydrolase reprogram white fat to beige-like fat through an increase in mitochondrial integrity, HO-1-adiponectin in vitro and in vivo.

We have shown that epoxyeicosatrienoic acids (EETs), specifically 11,12- and 14,15-EETs, reduce adipogenesis in human mesenchymal stem cells and mouse preadipocytes (3T-3L1). In this study, we explore the effects of soluble epoxide hydrolase (sEH) deletion on various aspects of adipocyte-function, including programing for white vs. beige-like fat, and mitochondrial and thermogenic gene-expressions. We further hypothesize that EETs and heme-oxygenase 1 (HO-1) form a synergistic, functional module whose effects on adipocyte and vascular function is greater than the effects of sEH deletion alone. In in vitro studies, we examined the effect of sEH inhibitors on MSC-derived adipocytes. MSC-derived adipocytes exposed to AUDA, an inhibitor of sEH, exhibit an increased number of small and healthy adipocytes, an effect reproduced by siRNA for sEH. in vivo studies indicate that sEH deletion results in a significant decrease in adipocyte size, inflammatory adipokines NOV, TNFα, while increasing adiponectin (p < 0.05). These findings are associated with a decrease in body weight (p < 0.05), and visceral fat (p < 0.05). Importantly, sEH deletion was associated with a significant increase in Mfn1, COX 1, UCP1 and adiponectin (p < 0.03). sEH deletion was manifested by a significant increase in EETs isomers 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET and an increased EETs/DHETEs ratio. Notably, activation of HO-1 gene expression further increased the levels of EETs, suggesting that the antioxidant HO-1 system protects EETs from degradation by ROS. These results are novel in that sEH deletion, while increasing EET levels, resulted in reprograming of white fat to express mitochondrial and thermogenic genes, a phenotype characteristic of beige-fat. Thus, EETs agonist(s) and sEH inhibitors may have therapeutic potential in the treatment of metabolic syndrome and obesity.

EET enhances renal function in obese mice resulting in restoration of HO-1-Mfn1/2 signaling, and decrease in hypertension through inhibition of sodium chloride co-transporter.

BACKGROUND: We have previously reported that epoxyeicosatrienoic acid (EET) has multiple beneficial effects on renal and adipose tissue function, in addition to its vasodilatory action; it increases insulin sensitivity and inhibits inflammation. In an examination of the signaling mechanisms by which EET reduces renal and peri-renal fat function, we hypothesized that EET ameliorates obesity-induced renal dysfunction by improving sodium excretion, reducing the sodium-chloride cotransporter NCC, lowering blood pressure, and enhancing mitochondrial and thermogenic gene levels in PGC-1α dependent mice. METHODS: EET-agonist treatment normalized glucose metabolism, renal ENaC and NCC protein expression, urinary sodium excretion and blood pressure in obese (db/db) mice. A marked improvement in mitochondrial integrity, thermogenic genes, and PGC-1α-HO-1-adiponectin signaling occurred. Knockout of PGC-1α in EET-treated mice resulted in a reversal of these beneficial effects including a decrease in sodium excretion, elevation of blood pressure and an increase in the pro-inflammatory adipokine nephroblastoma overexpressed gene (NOV). In the elucidation of the effects of EET on peri-renal adipose tissue, EET increased adiponectin, mitochondrial integrity, thermogenic genes and decreased NOV, i.e. "Browning' peri-renal adipose phenotype that occurs under high fat diets. Taken together, these data demonstrate a critical role of an EET agonist in the restoration of healthy adipose tissue with reduced release of inflammatory molecules, such as AngII and NOV, thereby preventing their detrimental impact on sodium absorption and NCC levels and the development of obesity-induced renal dysfunction.

The association of NOV/CCN3 with obstructive sleep apnea (OSA): preliminary evidence of a novel biomarker in OSA.

Obstructive sleep apnea (OSA) has a strong association with cardiovascular and metabolic abnormalities, although the mechanism driving this association is not well established. NOV/CCN3, a multifunctional extracellular matrix protein, may play a mechanistic and/or prognostic role in these associations. We hypothesized that patients with OSA, which primarily affects obese individuals, will have increased levels of NOV, and that NOV can serve as a biomarker in patients to predict OSA as well as metabolic and cardiac risk. Ten morbidly obese and 10 healthy lean subjects underwent overnight polysomnography (PSG) and clinical evaluation. Blood samples were analyzed for NOV levels, adiponectin and IL-6. OSA was found in nine obese subjects and three lean subjects. NOV levels were significantly higher in the OSA vs. no OSA group (2.1 ± 0.9 vs. 1.3 ± 0.8, p < 0.03). NOV levels were significantly higher in the obese vs. lean group (2.2 ± 0.3 vs. 1.4 ± 0.2-fold change, p < 0.03). Among lean subjects, NOV levels were significantly higher in the OSA vs. no OSA group (2.1 ± 0.9 vs. 1.0 ± 0.4, p < 0.05). NOV and AHI were positively correlated (ρ = 0.49, p = 0.033). IL-6 and adiponectin differences in obese vs. lean and OSA vs. no OSA were consistent with an inflammatory phenotype in obese subjects and OSA subjects. NOV is a novel biomarker of the presence and severity of OSA and a potential marker of future cardiovascular and metabolic disease in OSA patients.

PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid.

BACKGROUND/OBJECTIVES: Obesity is a risk factor in the development of type 2 diabetes mellitus (DM2), which is associated with increased morbidity and mortality, predominantly as a result of cardiovascular complications. Increased adiposity is a systemic condition characterized by increased oxidative stress (ROS), increased inflammation, inhibition of anti-oxidant genes such as HO-1 and increased degradation of epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs attenuate mitochondrial ROS. We postulate that EETs increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which controls mitochondrial function, oxidative metabolism and induction of HO-1. METHODS: Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess functional relationship between EETs, HO-1 and (PGC-1α) using an EET analogue (EET-A) and lentivirus to knock down the PPARGC1A gene. RESULTS: EET-A increased PGC-1α and HO-1 in cultured adipocytes and increased the expression of genes involved in thermogenesis and adipocyte browning (UCP1 and PRDM16, respectively). PGC-1α knockdown prevented EET-A-induced HO-1expression, suggesting that PGC-1α is upstream of HO-1. MRI data obtained from fat tissues showed that EET-A administration to mice on a HF diet significantly reduced total body fat content, subcutaneous and visceral fat deposits and reduced the VAT: SAT ratio. Moreover EET-A normalized the VO2 and RQ (VCO2/VO2) in mice fed a HF diet, an effect that was completely prevented in PGC-1α deficient mice. In addition, EET-A increased mitochondrial biogenesis and function as measured by OPA1, MnSOD, Mfn1, Mfn2, and SIRT3, an effect that was inhibited by knockdown of PGC-1α. CONCLUSION: Taken together, our findings show that EET-A increased PGC-1α thereby increasing mitochondrial viability, increased fusion potential thereby providing metabolic protection and increased VO2 consumption in HF-induced obesity in mice, thus demonstrating that the EET-mediated increase in HO-1 levels require PGC-1α expression.

Epoxyeicosatrienoic Acids Regulate Adipocyte Differentiation of Mouse 3T3 Cells, Via PGC-1α Activation, Which Is Required for HO-1 Expression and Increased Mitochondrial Function.

Epoxyeicosatrienoic acid (EET) contributes to browning of white adipose stem cells to ameliorate obesity/diabetes and insulin resistance. In the current study, we show that EET altered preadipocyte function, enhanced peroxisome proliferation-activated receptor γ coactivator α (PGC-1α) expression, and increased mitochondrial function in the 3T3-L1 preadipocyte subjected to adipogenesis. Cells treated with EET resulted in an increase, P < 0.05, in PGC-1α and a decrease in mitochondria-derived ROS (MitoSox), P < 0.05. The EET increase in heme oxygenase-1 (HO-1) levels is dependent on activation of PGC-1α as cells deficient in PGC-1α (PGC-1α knockout adipocyte cell) have an impaired ability to express HO-1, P < 0.02. Additionally, adipocytes treated with EET exhibited an increase in mitochondrial superoxide dismutase (SOD) in a PGC-1α-dependent manner, P < 0.05. The increase in PGC-1α was associated with an increase in ß-catenin, P < 0.05, adiponectin expression, P < 0.05, and lipid accumulation, P < 0.02. EET decreased heme levels and mitochondria-derived ROS (MitoSox), P < 0.05, compared to adipocytes that were untreated. EET also decreased mesoderm-specific transcript (MEST) mRNA and protein levels (P < 0.05). Adipocyte secretion of EET act in an autocrine/paracrine manner to increase PGC-1α is required for activation of HO-1 expression. This is the first study to dissect the mechanism by which the antiadipogenic and anti-inflammatory lipid, EET, induces the PGC-1α signaling cascade and reprograms the adipocyte phenotype by regulating mitochondrial function and HO-1 expression, leading to an increase in healthy, that is, small, adipocytes and a decrease in adipocyte enlargement and terminal differentiation. This is manifested by an increase in mitochondrial function and an increase in the canonical Wnt signaling cascade during adipocyte proliferation and terminal differentiation.

Caveolin-1 regulates corneal wound healing by modulating Kir4.1 activity.

The expression of caveolin-1 (Cav1) in corneal epithelium is associated with regeneration potency. We used Cav1(-/-) mice to study the role of Cav1 in modulating corneal wound healing. Western blot and whole cell patch clamp were employed to study the effect of Cav1 deletion on Kir4.1 current density in corneas. We found that Ba(2+)-sensitive K(+) currents in primary cultured murine corneal epithelial cells (pMCE) from Cav1(-/-) were dramatically reduced (602 pA) compared with those from wild type (WT; 1,300 pA). As a consequence, membrane potential was elevated in pMCE from Cav1(-/-) compared with that from WT (-43 ± 7.5 vs. -58 ± 4.0 mV, respectively). Western blot showed that either inhibition of Cav1 expression or Ba(2+) incubation stimulated phosphorylation of the EGFR. The transwell migration assay showed that Cav1 genetic inactivation accelerated cell migration. The regrowth efficiency of human corneal epithelial cells (HCE) transfected with siRNA-Cav1 or negative control was evaluated by scrape injury assay. With the presence of mitomycin C (10 µg/ml) to avoid the influence of cell proliferation, Cav1 inhibition with siRNA significantly increased migration compared with control siRNA in HCE. This promoting effect by siRNA-Cav1 could not be further enhanced by cotransfection with siRNA-Kcnj10. By using corneal debridement, we found that wound healing was significantly accelerated in Cav1(-/-) compared with WT mice (70 ± 10 vs. 36 ± 3%, P < 0.01). Our findings imply that the mechanism by which Cav-1 knockout promotes corneal regrowth is, at least partially, due to the inhibition of Kir4.1 which stimulates EGFR signaling.

Downregulation of PGC-1α Prevents the Beneficial Effect of EET-Heme Oxygenase-1 on Mitochondrial Integrity and Associated Metabolic Function in Obese Mice.

Background/Objectives. Obesity and metabolic syndrome and associated adiposity are a systemic condition characterized by increased mitochondrial dysfunction, inflammation, and inhibition of antioxidant genes, HO-1, and EETs levels. We postulate that EETs attenuate adiposity by stimulating mitochondrial function and induction of HO-1 via activation of PGC-1α in adipose and hepatic tissue. Methods. Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess the functional relationship among EETs, PGC-1α, HO-1, and mitochondrial signaling using an EET-agonist (EET-A) and PGC-1α-deficient cells and mice using lentiviral PGC-1α(sh). Results. EET-A is a potent inducer of PGC-1α, HO-1, mitochondrial biogenesis (cytochrome oxidase subunits 1 and 4 and SIRT3), fusion proteins (Mfn 1/2 and OPA1) and fission proteins (DRP1 and FIS1) (p < 0.05), fasting glucose, BW, and blood pressure. These beneficial effects were prevented by administration of lenti-PGC-1α(sh). EET-A administration prevented HF diet induced mitochondrial and dysfunction in adipose tissue and restored VO2 effects that were abrogated in PGC-1α-deficient mice. Conclusion. EET is identified as an upstream positive regulator of PGC-1α that leads to increased HO-1, decreased BW and fasting blood glucose and increased insulin receptor phosphorylation, that is, increased insulin sensitivity and mitochondrial integrity, and possible use of EET-agonist for treatment of obesity and metabolic syndrome.

Heme oxygenase-2 deletion impairs macrophage function: implication in wound healing.

Heme oxygenase (HO)-2 deficiency impairs wound healing and exacerbates inflammation following injury. We examine the impact of HO-2 deficiency on macrophage function and the contribution of macrophage HO-2 to inflammatory and repair responses to injury. Corneal epithelial debridement was performed in control and macrophage-depleted HO-2(-/-) and wild-type (WT) mice and in bone marrow chimeras. Peritoneal macrophages were collected for determination of phagocytic activity and classically activated macrophage (M1)-alternatively activated macrophage (M2) polarization. Depletion of macrophages delayed corneal healing (13.2%) and increased neutrophil infiltration (54.1%) by day 4 in WT mice, whereas in HO-2(-/-) mice, it did not worsen the already impaired wound healing and exacerbated inflammation. HO-2(-/-) macrophages displayed an altered M1 phenotype with no significant expression of M2 or M2-like activated cells and a 31.3% reduction in phagocytic capacity that was restored by inducing HO-1 activity or supplementing biliverdin. Macrophage depletion had no effect, whereas adoptive transfer of WT bone marrow improved wound healing (34% on day 4) but did not resolve the exaggerated inflammatory response in HO-2(-/-) mice. These findings indicate that HO-2-deficient macrophages are dysfunctional and that macrophage HO-2 is required for proper macrophage function but is insufficient to correct the impaired healing of the HO-2(-/-) cornea, suggesting that corneal epithelial expression of HO-2 is a key to resolution and repair in wound healing.