Publications by authors named "Lars Bellner"

44 Publications

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

Metab Syndr Relat Disord 2021 Feb 9. Epub 2021 Feb 9.

Department of Pediatrics, Marshall University School of Medicine, Huntington, West Virginia, USA.

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. 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 ( = 130), and Group B, prospective participants, referred between July 1, 2018 and December 31, 2018, followed with laboratory studies pre- and postdietary intervention ( = 8). 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 ( = 0.02). Participants <12 years age were almost twice as likely to complete the diet compared with those 12-18 years (64% vs. 36%,  < 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 ( < 0.001), body mass index (BMI) 2.5 kg/m ( < 0.001), and percentage weight loss 6.9% ( < 0.001). Group B had reductions in weight 9.6 kg ( < 0.01), BMI 4 kg/m ( < 0.01), and percentage weight loss 9% ( < 0.01). In addition, participants had significant reductions of fasting serum insulin ( < 0.01), triglycerides ( < 0.01), and 20-hydroxyeicosatetraenoic acid ( < 0.01). 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.
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http://dx.doi.org/10.1089/met.2020.0078DOI Listing
February 2021

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

J Food Biochem 2020 12 12;44(12):e13522. Epub 2020 Oct 12.

Department of Medicine, New York Medical College, Valhalla, NY, USA.

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.
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http://dx.doi.org/10.1111/jfbc.13522DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7770619PMC
December 2020

Cold-Pressed 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.

Antioxidants (Basel) 2020 Jun 4;9(6). Epub 2020 Jun 4.

Department of Medicine, New York Medical College, Valhalla, NY 10595, USA.

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 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 Ser 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 tyr), insulin receptor beta (IRβ), UCP1, and pIRS1 Ser 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.
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http://dx.doi.org/10.3390/antiox9060489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7346210PMC
June 2020

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

Antioxidants (Basel) 2020 Jan 1;9(1). Epub 2020 Jan 1.

Departments of Medicine and Pharmacology, New York Medical College, Valhalla, NY 10595, USA.

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.
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http://dx.doi.org/10.3390/antiox9010040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7022335PMC
January 2020

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

Antioxid Redox Signal 2020 05 10;32(14):1045-1060. Epub 2020 Feb 10.

Department of Medicine, Marshall University, Joan C. Edwards School of Medicine, Huntington, West Virginia.

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. 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. 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. 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.
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http://dx.doi.org/10.1089/ars.2019.7970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7153645PMC
May 2020

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

Exp Cell Res 2019 07 27;380(2):180-187. Epub 2019 Apr 27.

Department of Drug Science, University of Catania, Catania, Italy. Electronic address:

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.
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http://dx.doi.org/10.1016/j.yexcr.2019.04.029DOI Listing
July 2019

High-fat diet-induced obesity and insulin resistance in CYP4a14 mice is mediated by 20-HETE.

Am J Physiol Regul Integr Comp Physiol 2018 11 8;315(5):R934-R944. Epub 2018 Aug 8.

Departments of Pharmacology, New York Medical College School of Medicine, Valhalla, New York.

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.
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http://dx.doi.org/10.1152/ajpregu.00125.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6295494PMC
November 2018

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

Int J Hepatol 2018 2;2018:3484107. Epub 2018 Jul 2.

Departments of Medicine, Pharmacology and Gastroenterology, New York Medical College, Valhalla, NY 10595, USA.

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.
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http://dx.doi.org/10.1155/2018/3484107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051135PMC
July 2018

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.

Prostaglandins Other Lipid Mediat 2018 09 21;138:1-8. Epub 2018 Jul 21.

Department of Pharmacology, New York Medical College, Valhalla, NY, 10595, USA; Joan Edward School of Medicine, Marshall University, Huntington, WV, 25701, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.prostaglandins.2018.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6314013PMC
September 2018

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.

Prostaglandins Other Lipid Mediat 2018 07 19;137:30-39. Epub 2018 May 19.

Department of Pharmacology, New York Medical College, Valhalla, NY, 10595, United States; Department of Medicine, New York Medical College, Valhalla, NY, 10595, United States; Department of Medicine, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 25701, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.prostaglandins.2018.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6075657PMC
July 2018

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

Horm Mol Biol Clin Investig 2017 Sep 1;31(2). Epub 2017 Sep 1.

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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.
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http://dx.doi.org/10.1515/hmbci-2017-0029DOI Listing
September 2017

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

J Nutr Metab 2016 20;2016:9039754. Epub 2016 Dec 20.

New York Medical College, Departments of Medicine and Pharmacology, Valhalla, NY, USA; The Rockefeller University, New York, NY, USA.

. 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-1 in adipose and hepatic tissue. . Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess the functional relationship among EETs, PGC-1, HO-1, and mitochondrial signaling using an EET-agonist (EET-A) and PGC-1-deficient cells and mice using lentiviral PGC-1(sh). . EET-A is a potent inducer of PGC-1, 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) ( < 0.05), fasting glucose, BW, and blood pressure. These beneficial effects were prevented by administration of lenti-PGC-1(sh). EET-A administration prevented HF diet induced mitochondrial and dysfunction in adipose tissue and restored VO effects that were abrogated in PGC-1-deficient mice. . EET is identified as an upstream positive regulator of PGC-1 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.
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http://dx.doi.org/10.1155/2016/9039754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5206458PMC
December 2016

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

Prostaglandins Other Lipid Mediat 2016 09 11;125:8-18. Epub 2016 Jul 11.

Department of Pharmacology, New York Medical College, Valhalla, NY 10595, United States. Electronic address:

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.
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http://dx.doi.org/10.1016/j.prostaglandins.2016.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536246PMC
September 2016

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

Stem Cells Dev 2016 07 27;25(14):1084-94. Epub 2016 Jun 27.

1 Department of Pharmacology, New York Medical College , Valhalla, New York.

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 β-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.
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http://dx.doi.org/10.1089/scd.2016.0072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4939374PMC
July 2016

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

Am J Physiol Cell Physiol 2016 06 27;310(11):C993-C1000. Epub 2016 Apr 27.

Department of Pharmacology, New York Medical College, Valhalla, New York

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.
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http://dx.doi.org/10.1152/ajpcell.00023.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935204PMC
June 2016

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

FASEB J 2015 Jan 23;29(1):105-15. Epub 2014 Oct 23.

Department of Pharmacology, Department of Ophthalmology, and.

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.
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http://dx.doi.org/10.1096/fj.14-256503DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4285548PMC
January 2015

NFAT5 is protective against ischemic acute kidney injury.

Hypertension 2014 Mar 30;63(3):e46-52. Epub 2013 Dec 30.

Department of Pharmacology, New York Medical College, Valhalla, NY 10595.

NFAT5 is a transcription factor that protects the kidney from hypertonic stress and also is activated by hypoxia. We hypothesized that NFAT5 mitigates the extent of renal damage induced by ischemia-reperfusion injury (IRI). Mice were subjected to IRI by unilateral clamping of the left renal pedicle for 30 minutes followed by reperfusion. After 3 hours of reperfusion, the level of NFAT5 mRNA was similar in contralateral and clamped kidneys. However, after 48 hours, NFAT5 mRNA accumulation increased ≈3-fold in both outer medulla and medullary thick ascending limb tubules. NFAT1 levels were elevated at 3 hours but did not increase further at 48 hours. Mice were then either pretreated for 72 hours with an intrarenal injection of a lentivirus short-hairpin RNA construct to silence NFAT5 (enhanced green fluorescent protein-U6-N5-ex8) or a control vector (enhanced green fluorescent protein-U6) before induction of IRI. Neutrophil gelatinase-associated lipocalin and kidney ischemia molecule-1 mRNA levels increased after IRI and further increased after knockdown of NFAT5, suggesting that silencing of NFAT5 exacerbates renal damage during IRI. In contrast, silencing of NFAT1 had no effect on the levels of neutrophil gelatinase-associated lipocalin or kidney ischemia molecule-1 mRNA. Hematoxylin and eosin staining revealed patchy denudation of renal epithelial cells and tubular dilation when NFAT5 was silenced. The number of TUNEL-positive cells in the outer and inner medulla of the clamped kidney increased nearly 2-fold after knockdown of NFAT5 and was associated with an increase in the number of caspase-3-positive cells. Collectively, the data suggest that NFAT5 is part of a protective mechanism that limits renal damage induced by IRI.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.113.02476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3945409PMC
March 2014

Inhibition of miR-205 impairs the wound-healing process in human corneal epithelial cells by targeting KIR4.1 (KCNJ10).

Invest Ophthalmol Vis Sci 2013 Sep 11;54(9):6167-78. Epub 2013 Sep 11.

Department of Pharmacology, New York Medical College, Valhalla, New York.

Purpose: The aim of the study was to test the hypotheses that injury stimulates the expression of miR-205, which in turn inhibits KCNJ10 channels by targeting its 3' UTR, thereby facilitating the wound-healing process in human corneal epithelial cells (HCECs).

Methods: A stem-loop qRT-PCR was used to examine the miR-205 expression. BrdU cell proliferation assay and wound scratch assay were applied to measure the effect of miR-205 mimic or antagomer in HCECs. The patch-clamp technique, dual luciferase reporter assay, and Western blot analysis were employed to test whether miR-205 regulates KCNJ10, one of the target genes of miR-205. Both of the primary human and mouse corneal epithelial cells (pH/MCECs) were employed to further confirm the observations obtained in HCECs.

Results: The scratch injury in pH/MCECs increased the expression of miR-205 and decreased the expression of KCNJ10 within 24 hours. The notion that miR-205 may target KCNJ10 was supported by dual luciferase reporter assay showing an inhibition effect of miR-205 on 3' UTR of KCNJ10. Application of miR-205 antagomer significantly delayed the regrowth in wounded HCECs. However, inhibition of KCNJ10 partially abolished the effect from miR-205 antagomer and restored the healing process. Moreover, overexpression miR-205 antagomer enhanced the protein expression of KCNJ10 but not KCNJ16. In addition, patch-clamp demonstrated that inhibition of endogenous miR-205 expression increased Ba²⁺-sensitive inwardly rectifying K⁺ channels. In addition, an electrophysiological study of pHCECs showed the presence of KCNJ10-like 20 pS K⁺ channels and scratch injury significantly decreased the Ba²⁺-sensitive inwardly rectifying K⁺ currents.

Conclusions: miR-205 stimulates wound healing by inhibiting its target gene KCNJ10.
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http://dx.doi.org/10.1167/iovs.12-11577DOI Listing
September 2013

Dysregulated heme oxygenase-ferritin system in pterygium pathogenesis.

Cornea 2013 Sep;32(9):1276-82

Department of Pharmacology, New York Medical College, Valhalla, NY, USA.

Purpose: Cyclooxygenase (COX)-, lipoxygenase (LOX)-, and cytochrome P450 monooxygenase (CYP)-derived eicosanoids have been implicated in ocular surface inflammation and neovascularization. These eicosanoids are subjected to regulation by enzymes, such as heme oxygenases (HOs) and ferritin.

Methods: Quantitative polymerase chain reaction and lipidomics based on liquid chromatography-tandem mass spectrometry were performed on pterygia from patients undergoing surgical pterygium excision. Control tissues consisted of donor corneas. In addition, lipidomics based on liquid chromatography-tandem mass spectrometry was performed on tears collected from patients before the surgery.

Results: Messenger RNA (mRNA) expression of HO-2, the constitutive HO isoform, was upregulated by 40% in pterygia compared with control tissue, whereas the mRNA level of the inducible form, HO-1, was downregulated by more than 50%. Levels of CYP4B1 mRNA showed an approximate 2-fold increase in pterygia compared with control. Lipidomic analysis of tissues indicated a moderate elevation in Prostaglandin E2 and thromboxane B2 levels in pterygia compared with control. Among the LOX-derived metabolites, the antiinflammatory-hydroxyeicosatetraenoic acid (15-HETE) levels were significantly reduced in pterygia (79.3 ± 48.11 pg/mg protein) compared with control (586.2 ± 213.5 pg/mg protein), whereas the proinflammatory LOX- and CYP4B1-derived 12-HETE levels were 10-fold higher in pterygia (2768 ± 832.3 pg/mg protein) compared with control (231.4 ± 87.35 pg/mg protein). Prostaglandin E2 and HETEs were also present in tears from patients with pterygium but were not detected in tears from healthy volunteers. The mRNA expression levels of both light and heavy chain ferritin were 60% and 30% lower, respectively, in pterygia compared with control.

Conclusions: We believe that a dysfunctional HO-ferritin system leads to increased levels of proinflammatory mediators, thus contributing to the inflammation characteristic of pterygia.
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http://dx.doi.org/10.1097/ICO.0b013e3182936915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3740074PMC
September 2013

Increased heme-oxygenase 1 expression in mesenchymal stem cell-derived adipocytes decreases differentiation and lipid accumulation via upregulation of the canonical Wnt signaling cascade.

Stem Cell Res Ther 2013 Mar 12;4(2):28. Epub 2013 Mar 12.

Introduction: Heme oxygenase (HO), a major cytoprotective enzyme, attenuates oxidative stress and obesity. The canonical Wnt signaling cascade plays a pivotal role in the regulation of adipogenesis. The present study examined the interplay between HO-1and the Wnt canonical pathway in the modulation of adipogenesis in mesenchymal stem cell (MSC)-derived adipocytes.

Methods: To verify the role of HO-1 in generating small healthy adipocytes, cobalt protoporphyrin (CoPP), inducer of HO-1, was used during adipocyte differentiation. Lipid accumulation was measured by Oil red O staining and lipid droplet size was measured by BODIPY staining.

Results: During adipogenesis in vitro, differentiating pre-adipocytes display transient increases in the expression of genes involved in canonical Wnt signaling cascade. Increased levels of HO-1 expression and HO activity resulted in elevated levels of β-catenin, pGSK3β, Wnt10b, Pref-1, and shh along with increased levels of adiponectin (P < 0.05). In addition, induction of HO-1 resulted in a reduction in C/EBPα, PPARγ, Peg-1/Mest, aP2, CD36 expression and lipid accumulation (P < 0.05). Suppression of HO-1 gene by siRNA decreased Wnt10b, pGSK3β and β-catenin expression, and increased lipid accumulation. The canonical Wnt responsive genes, IL-8 and SFRP1, were upregulated by CoPP and their expression was decreased by the concurrent administration of tin mesoporphyrin (SnMP), an inhibitor of HO activity. Furthermore, knockdown of Wnt10b gene expression by using siRNA showed increased lipid accumulation, and this effect was not decreased by concurrent treatment with CoPP. Also our results show that blocking the Wnt 10b antagonist, Dickkopf 1 (Dkk-1), by siRNA decreased lipid accumulation and this effect was further enhanced by concurrent administration of CoPP.

Conclusions: This is the first study to demonstrate that HO-1 acts upstream of canonical Wnt signaling cascade and decreases lipogenesis and adipocyte differentiation suggesting that the HO-1 mediated increase in Wnt10b can modulate the adipocyte phenotype by regulating the transcriptional factors that play a role in adipogenesis. This is evidenced by a decrease in lipid accumulation and inflammatory cytokine levels, increased adiponectin levels and elevation of the expression of genes of the canonical Wnt signaling cascade.
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http://dx.doi.org/10.1186/scrt176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706794PMC
March 2013

NKCC2A and NFAT5 regulate renal TNF production induced by hypertonic NaCl intake.

Am J Physiol Renal Physiol 2013 Mar 26;304(5):F533-42. Epub 2012 Dec 26.

Dept. of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.

Pathways that contribute to TNF production by the kidney are not well defined. Mice given 1% NaCl in the drinking water for 3 days exhibited a 2.5-fold increase in urinary, but not plasma, TNF levels compared with mice given tap water. Since furosemide attenuated the increase in TNF levels, we hypothesized that hypertonic NaCl intake increases renal TNF production by a pathway involving the Na(+)-K(+)-2Cl(-) cotransporter (NKCC2). A 2.5-fold increase in NKCC2A mRNA accumulation was observed in medullary thick ascending limb (mTAL) tubules from mice given 1% NaCl; a concomitant 2-fold increase in nuclear factor of activated T cells 5 (NFAT5) mRNA and protein expression was observed in the outer medulla. Urinary TNF levels were reduced in mice given 1% NaCl after an intrarenal injection of a lentivirus construct designed to specifically knockdown NKCC2A (EGFP-N2A-ex4); plasma levels of TNF did not change after injection of EGFP-N2A-ex4. Intrarenal injection of EGFP-N2A-ex4 also inhibited the increase of NFAT5 mRNA abundance in the outer medulla of mice given 1% NaCl. TNF production by primary cultures of mTAL cells increased approximately sixfold in response to an increase in osmolality to 400 mosmol/kgH2O produced with NaCl and was inhibited in cells transiently transfected with a dnNFAT5 construct. Transduction of cells with EGFP-N2A-ex4 also prevented increases in TNF mRNA and protein production in response to high NaCl concentration and reduced transcriptional activity of a NFAT5 promoter construct. Since NKCC2A expression is restricted to the TAL, NKCC2A-dependent activation of NFAT5 is part of a pathway by which the TAL produces TNF in response to hypertonic NaCl intake.
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http://dx.doi.org/10.1152/ajprenal.00243.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3602716PMC
March 2013

Antioxidants condition pleiotropic vascular responses to exogenous H(2)O(2): role of modulation of vascular TP receptors and the heme oxygenase system.

Antioxid Redox Signal 2013 Feb 28;18(5):471-80. Epub 2012 Sep 28.

Department of Pharmacology, New York Medical College, Valhalla, New York, USA.

Aims: Hydrogen peroxide (H(2)O(2)), a nonradical oxidant, is employed to ascertain the role of redox mechanisms in regulation of vascular tone. Where both dilation and constriction have been reported, we examined the hypothesis that the ability of H(2)O(2) to effect vasoconstriction or dilation is conditioned by redox mechanisms and may be modulated by antioxidants.

Results: Exogenous H(2)O(2) (0.1-10.0 μM), dose-dependently reduced the internal diameter of rat renal interlobular and 3rd-order mesenteric arteries (p<0.05). This response was obliterated in arteries pretreated with antioxidants, including tempol, pegylated superoxide dismutase (PEG-SOD), butylated hydroxytoluene (BHT), and biliverdin (BV). However, as opposed to tempol or PEG-SOD, BHT & BV, antioxidants targeting radicals downstream of H(2)O(2), also uncovered vasodilation.

Innovations: Redox-dependent vasoconstriction to H(2)O(2) was blocked by inhibitors of cyclooxygenase (COX) (indomethacin-10 μM), thromboxane (TP) synthase (CGS13080-10 μM), and TP receptor antagonist (SQ29548-1 μM). However, H(2)O(2) did not increase vascular thromboxane B(2) release; instead, it sensitized the vasculature to a TP agonist, U46619, an effect reversed by PEG-SOD. Antioxidant-conditioned dilatory response to H(2)O(2) was accompanied by enhanced vascular heme oxygenase (HO)-dependent carbon monoxide generation and was abolished by HO inhibitors or by HO-1 & 2 antisense oligodeoxynucleotides treatment of SD rats.

Conclusion: These results demonstrate that H(2)O(2) has antioxidant-modifiable pleiotropic vascular effects, where constriction and dilation are brought about in the same vascular segment. H(2)O(2)-induced oxidative stress increases vascular TP sensitivity and predisposes these arterial segments to constrictor prostanoids. Conversely, vasodilation is reliant upon HO-derived products whose synthesis is stimulated only in the presence of antioxidants targeting radicals downstream of H(2)O(2).
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http://dx.doi.org/10.1089/ars.2012.4587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3545357PMC
February 2013

Apo A1 Mimetic Rescues the Diabetic Phenotype of HO-2 Knockout Mice via an Increase in HO-1 Adiponectin and LKBI Signaling Pathway.

Int J Hypertens 2012 4;2012:628147. Epub 2012 Apr 4.

Department of Geriatric Cardiology, Chinese PLA General Hospital, Beijing 100853, China.

Insulin resistance, with adipose tissue dysfunction, is one of the hallmarks of metabolic syndrome. We have reported a metabolic syndrome-like phenotype in heme oxygenase (HO)-2 knockout mice, which presented with concurrent HO-1 deficiency and were amenable to rescue by an EET analog. Apo A-I mimetic peptides, such as L-4F, have been shown to induce HO-1 expression and decrease oxidative stress and adiposity. In this study we aimed to characterize alleviatory effects of HO-1 induction (if any) on metabolic imbalance observed in HO-2 KO mice. In this regard, HO-2((-/-)) mice were injected with 2 mg/kg/day L-4F, or vehicle, i.p., for 6 weeks. As before, compared to WT animals, the HO-2 null mice were obese, displayed insulin resistance, and had elevated blood pressure. These changes were accompanied by enhanced tissue (hepatic) oxidative stress along with attenuation of HO-1 expression and activity and reduced adiponectin, pAMPK, and LKB1 expression. Treatment with L-4F restored HO-1 expression and activity and increased adiponectin, LKB1, and pAMPK in the HO-2((-/-)) mice. These alterations resulted in a decrease in blood pressure, insulin resistance, blood glucose, and adiposity. Taken together, our results show that a deficient HO-1 response, in a state with reduced HO-2 basal levels, is accompanied by disruption of metabolic homeostasis which is successfully restored by an HO-1 inducer.
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http://dx.doi.org/10.1155/2012/628147DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3335301PMC
August 2012

Heme oxygenase (HO-1) rescue of adipocyte dysfunction in HO-2 deficient mice via recruitment of epoxyeicosatrienoic acids (EETs) and adiponectin.

Cell Physiol Biochem 2012 1;29(1-2):99-110. Epub 2012 Mar 1.

Department of Physiology and Pharmacology, University of Toledo, Toledo, USA.

Background/aims: HO-1 and EETs are functionally linked and their interactions influence body weight, insulin sensitivity, and serum levels of inflammatory cytokines in metabolic syndrome phenotype of HO-2 null mice. The HO-2 isozyme is essential for regulating physiological levels of ROS. Recent studies have suggested a potential role of EET in modifying adipocyte differentiation through up-regulation of HO-1-adiponectin-AkT signaling in human mesenchymal stem cells (MSCs). Our aim was to examine the consequences of HO deficiency on MSC-derived adipogenesis in vitro using MSC derived from HO-2 null and WT mice in vivo.

Methods: Four-month-old HO-2 null (HO-2(-/-)) and B6/129SF2/J (WT) mice were divided into three groups (four mice/group): WT, HO-2(-/-), and HO-2(-/-) +CoPP. Adipogenesis was performed on purified MSC-derived adipocytes cultured in adipogenic differentiation media and an EET-agonist was added every 3 days.

Results: HO-2 depletion of MSC adipocytes resulted in increased adipogenesis (p<0.01) and increased levels of inflammatory cytokines including (TNF)-alpha (p<0.05), (MCP)-1 (p<0.05), and (IL-1)-beta (p<0.05). These results were accompanied by decreases in HO-1 (p<0.05) and subsequently EET and HO activity (p<0.05). Up-regulation of HO-1 resulted in decreased MSC-derived adipocyte differentiation, decreased production of TNF-alpha and MCP-1 and increased levels of adiponectin (p<0.05). Cyp2J5 (p<0.05), HO-1 (p<0.05), and adiponectin mRNA levels (p<0.05) were also decreased in visceral adipose tissue isolated from HO-2 null compared to WT mice. EET agonist stimulation of MSC adipocytes derived from HO-2 null mice yielded similar results.

Conclusion: Increased levels of EET and HO-1 are essential for protection against the adverse effects of adipocyte hypertrophy and the ensuing metabolic syndrome. These results offer a portal into therapeutic approaches for the prevention of the metabolic syndrome.
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http://dx.doi.org/10.1159/000337591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3711769PMC
July 2012

ApoA1: mimetic peptide reverses adipocyte dysfunction in vivo and in vitro via an increase in heme oxygenase (HO-1) and Wnt10b.

Cell Cycle 2012 Feb 15;11(4):706-14. Epub 2012 Feb 15.

Department of Physiology & Pharmacology, University of Toledo College of Medicine, Toledo, OH, USA.

Insulin resistance is a risk factor in the development of type 2 diabetes and is a major cause of atherosclerosis. Reduction in heme oxygenase (HO-1) has been shown to exacerbate vascular dysfunction and insulin resistance in obese mice and involves a decrease in adiponectin levels. Adiponectin is released from mesenchymal stem cell (MSC)-derived adipocytes, its levels are decreased in type 2 diabetes. We hypothesized that the apoA1 mimetic peptide, L-4F, will target the expression of the HO-1-adiponectin axis and reverse adipocyte dysfunction both in vivo and in vitro. The administration of L-4F [2 mg/Kg/daily (i.p.) for 4-week to 8-week-old obese (ob) mice restored adipocyte function, increased adiponectin release (p < 0.05) and decreased the levels of IL-1 and IL-6 (p < 0.05)]. These perturbations were associated with an increase in insulin sensitivity (p < 0.01 vs. untreated ob mice) and decreased glucose levels (309 + 42 vs. 201 + 8 mg/d after L-4F treatment). Treatment of both mesenchymal stem cell (MSC)-derived adipocytes with L-4F (50 μg/ml) increased adiponectin (p < 0.05), decreased IL-1 and IL-6 (p < 0.05) levels and increased MSC-derived adipocyte cell numbers by 50% in S phase (p < 0.05). MSC-derived adipocytes treated with L-4F increased WNT10b and decreased Peg 1/Mest. Inhibition of HO activity reversed the decrease in the adipogenic response gene, Peg 1/Mest. An increase of HO-1 expression by L-4F increased insulin-receptor phosphorylation. These findings support the hypothesis that L-4F increases early adipocyte markers, HO-1-adiponectin, WNT10b and decreases Peg1/Mest, negative regulators of adipocyte differentiation.
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http://dx.doi.org/10.4161/cc.11.4.19125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3318105PMC
February 2012

Reciprocal Effects of Oxidative Stress on Heme Oxygenase Expression and Activity Contributes to Reno-Vascular Abnormalities in EC-SOD Knockout Mice.

Int J Hypertens 2012 12;2012:740203. Epub 2012 Jan 12.

Department of Anesthesiology and Resuscitology, Okayama University Medical School, Okayama, Japan.

Heme oxygenase (HO) system is one of the key regulators of cellular redox homeostasis which responds to oxidative stress (ROS) via HO-1 induction. However, recent reports have suggested an inhibitory effect of ROS on HO activity. In light of these conflicting reports, this study was designed to evaluate effects of chronic oxidative stress on HO system and its role in contributing towards patho-physiological abnormalities observed in extracellular superoxide dismutase (EC-SOD, SOD3) KO animals. Experiments were performed in WT and EC-SOD((-/-)) mice treated with and without HO inducer, cobalt protoporphyrin (CoPP). EC-SOD((-/-)) mice exhibited oxidative stress, renal histopathological abnormalities, elevated blood pressure, impaired endothelial function, reduced p-eNOS, p-AKT and increased HO-1 expression; although, HO activity was significantly (P < 0.05) attenuated along with attenuation of serum adiponectin and vascular epoxide levels (P < 0.05). CoPP, in EC-SOD((-/-)) mice, enhanced HO activity (P < 0.05) and reversed aforementioned pathophysiological abnormalities along with restoration of vascular EET, p-eNOS, p-AKT and serum adiponectin levels in these animals. Taken together our results implicate a causative role of insufficient activation of heme-HO-adiponectin system in pathophysiological abnormalities observed in animal models of chronic oxidative stress such as EC-SOD((-/-)) mice.
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http://dx.doi.org/10.1155/2012/740203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3265091PMC
August 2012

Epoxyeicosatrienoic acids and heme oxygenase-1 interaction attenuates diabetes and metabolic syndrome complications.

Prostaglandins Other Lipid Mediat 2012 Jan 15;97(1-2):1-16. Epub 2011 Nov 15.

Department of Physiology and Pharmacology, University of Toledo College of Medicine, Toledo, OH 43614, USA.

MSCs are considered to be the natural precursors to adipocyte development through the process of adipogenesis. A link has been established between decreased protective effects of EETs or HO-1 and their interaction in metabolic syndrome. Decreases in HO-1 or EET were associated with an increase in adipocyte stem cell differentiation and increased levels of inflammatory cytokines. EET agonist (AKR-I-27-28) inhibited MSC-derived adipocytes and decreased the levels of inflammatory cytokines. We further describe the role of CYP-epoxygenase expression, HO expression, and circulating cytokine levels in an obese mouse, ob/ob(-/-) mouse model. Ex vivo measurements of EET expression within MSCs derived from ob/ob(-/-) showed decreased levels of EETs that were increased by HO induction. This review demonstrates that suppression of HO and EET systems exist in MSCs prior to the development of adipocyte dysfunction. Further, adipocyte dysfunction can be ameliorated by induction of HO-1 and CYP-epoxygenase, i.e. EET.
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http://dx.doi.org/10.1016/j.prostaglandins.2011.10.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3261364PMC
January 2012

The role of neutrophils in corneal wound healing in HO-2 null mice.

PLoS One 2011 17;6(6):e21180. Epub 2011 Jun 17.

Department of Pharmacology and Ophthalmology, New York Medical College, Valhalla, New York, United States of America.

Our studies demonstrated that Heme oxygenase (HO), in particular, the constitutive HO-2, is critical for a self-resolving inflammatory and repair response in the cornea. Epithelial injury in HO-2 null mice leads to impaired wound closure and chronic inflammation in the cornea. This study was undertaken to examine the possible relationship between HO-2 and the recruitment of neutrophils following a corneal surface injury in wild type (WT) and HO-2 knockout (HO-2(-/-)) mice treated with Gr-1 monoclonal antibody to deplete peripheral neutrophils. Epithelial injury was performed by removing the entire corneal epithelium. Infiltration of inflammatory cell into the cornea in response to injury was higher in HO-2(-/-) than in WT. However, the rate of corneal wound closure following neutrophil depletion was markedly inhibited in both WT and HO-2(-/-) mice by 60% and 85%, respectively. Neutropenia induced HO-1 expression in WT but not in HO-2(-/-) mice. Moreover, endothelial cells lacking HO-2 expressed higher levels of the Midkine and VE-cadherin and displayed strong adhesion to neutrophils suggesting that perturbation in endothelial cell function caused by HO-2 depletion underlies the increased infiltration of neutrophils into the HO-2(-/-) cornea. Moreover, the fact that neutropenia worsened epithelial healing of the injured cornea in both WT and HO-2(-/-) mice suggest that cells other than neutrophils contribute to the exaggerated inflammation and impaired wound healing seen in the HO-2 null cornea.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0021180PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3117875PMC
October 2011

Targeted suppression of HO-2 gene expression impairs the innate anti-inflammatory and repair responses of the cornea to injury.

Mol Vis 2011 Apr 29;17:1144-52. Epub 2011 Apr 29.

Department of Pharmacology, New York Medical College, Valhalla, NY 10595, USA.

Purpose: Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. In this study, we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea.

Methods: Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily, thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT-PCR.

Results: Local injection of HO-2-specific shRNA led to a 50% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 14%, 20%, and 12% at days 3, 4, and 7 after injury, respectively (n=18-24). The observed delay in the wound repair process in HO-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further, local knockdown of HO-2 lead to an aberrant chronic inflammatory response, as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury; 1.04±0.45×10(6) in HO-2 knockdown mice versus 0.14±0.03×10(6) inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes.

Conclusions: Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization, increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087447PMC
April 2011