Alexandra Nichols

pNaKtide Attenuates Steatohepatitis and Atherosclerosis by Blocking Na/K-ATPase/ROS Amplification in C57Bl6 and ApoE Knockout Mice Fed a Western Diet

We have previously reported that the α1 subunit of sodium potassium adenosine triphosphatase (Na/K-ATPase), acts as a receptor and an amplifier for reactive oxygen species, in addition to its distinct pumping function. On this background, we speculated that blockade of Na/K-ATPase-induced ROS amplification with a specific peptide, pNaKtide, might attenuate the development of steatohepatitis. To test this hypothesis, pNaKtide was administered to a murine model of NASH: the C57Bl6 mouse fed a “western” diet containing high amounts of fat and fructose. The administration of pNaKtide reduced obesity as well as hepatic steatosis, inflammation and fibrosis. Of interest, we also noted marked improvement in mitochondrial fatty acid oxidation, insulin sensitivity, dyslipidemia and aortic streaking in this mouse model. To further elucidate the effects of pNaKtide on atherosclerosis, similar studies were performed in ApoE knockout mice also exposed to the western diet. In these mice, pNaKtide not only improved steatohepatitis, dyslipidemia, and insulin sensitivity, but also ameliorated significant aortic atherosclerosis. Collectively, this study demonstrates that the Na/K-ATPase/ROS amplification loop contributes significantly to the development and progression of steatohepatitis and atherosclerosis. And furthermore, this study presents a potential treatment, the pNaKtide, for the metabolic syndrome phenotype.

Existence of a Strong Correlation of Biomarkers and miRNA in Females with Metabolic Syndrome and Obesity in a Population of West Virginia

Objectives: Metabolic syndrome causes complications like cardiovascular disease and type 2 diabetes mellitus (T2DM). As metabolic syndrome develops, altered levels of cytokines and microRNAs (miRNA) are measurable in the circulation. We aimed to construct a panel detecting abnormal levels of cytokines and miRNAs in patients at risk for metabolic syndrome. Methods: Participants included 54 patients from a Family Medicine Clinic at Marshall University School of Medicine, in groups of: Control, Obese, and Metabolic Syndrome (MetS). Results: Serum levels of leptin, adiponectin, leptin: adiponectin ratio, IL-6, six miRNAs (320a, 197-3p, 23-3p, 221-3p, 27a-3p, and 130a-3p), were measured. Among the three groups, leptin, and leptin: adiponectin ratio, and IL-6 levels were highest in MetS, and levels in Obese were greater than Control (p>0.05). Adiponectin levels were lower in Obese compared to Control, but lowest in MetS (p<0.05). MiRNAs levels were lowest in MetS, and levels in Obese were lower than Control (p>0.05). Conclusion: Our results support the clinical application of biomarkers in diagnosing early stage MetS, which will enable attenuation of disease progression before onset of irreversible complications. Since West Virginians are high-risk for developing MetS, our biomarker panel could reduce the disease burden on our population.

Role of Serum Biomarkers in Early Detection of Diabetic Cardiomyopathy in the West Virginian Population.

Objectives: Diabetic cardiomyopathy (DCM) is an established complication of diabetes mellitus. In West Virginia, the especially high incidence of diabetes and heart failure validate the necessity of developing new strategies for earlier detection of DCM. Since most DCM patients remain asymptomatic until the later stages of the disease when the fibrotic complications become irreversible, we aimed to explore biomarkers that can identify early-stage DCM. Methods: The patients were grouped into 4 categories based on clinical diabetic and cardiac parameters: Control, Diabetes (DM), Diastolic dysfunction (DD), and Diabetes with diastolic dysfunction (DM+DD), the last group being the preclinical DCM group. Results: Echocardiography images indicated severe diastolic dysfunction in patients with DD+DM and DD compared to DM or control patients. In the DM and DM+DD groups, TNFα, isoprostane, and leptin were elevated compared to control (p<0.05), as were clinical markers HDL, glucose and hemoglobin A1C. Fibrotic markers IGFBP7 and TGF-β followed the same trend. The Control group showed higher beneficial levels of adiponectin and bilirubin, which were reduced in the DM and DM+DD groups (p<0.05). Conclusion: The results from our study support the clinical application of biomarkers in diagnosing early stage DCM, which will enable attenuation of disease progression prior to the onset of irreversible complications.

The Na/K-ATPase Oxidant Amplification Loop Regulates Aging

As aging involves oxidant injury, we examined the role of the recently described Na/K-ATPase oxidant amplification loop (NKAL). First, C57Bl6 old mice were given a western diet to stimulate oxidant injury or pNaKtide to antagonize the NKAL. The western diet accelerated functional and morphological evidence for aging whereas pNaKtide attenuated these changes. Next, human dermal fibroblasts (HDFs) were exposed to different types of oxidant stress in vitro each of which increased expression of senescence markers, cell-injury, and apoptosis as well as stimulated the NKAL. Further stimulation of the NKAL with ouabain augmented cellular senescence whereas treatment with pNaKtide attenuated it. Although N-Acetyl Cysteine and Vitamin E also ameliorated overall oxidant stress to a similar degree as pNaKtide, the pNaKtide produced protection against senescence that was substantially greater than that seen with either antioxidant. In particular, pNaKtide appeared to specifically ameliorate nuclear oxidant stress to a greater degree. These data demonstrate that the NKAL is intimately involved in the aging process and may serve as a target for anti-aging interventions.

Role of Serum Biomarkers in Early Detection of Non-Alcoholic Steatohepatitis and Fibrosis in West Virginian Children

Background Obesity, an epidemic among West Virginia children, as well as insulin resistance (IR), is well-established contributors to nonalcoholic steatohepatitis (NASH). Progression of NASH can lead to hepatic fibrosis and cirrhosis, making early detection imperative. The standard for diagnosing NASH is histologically via liver biopsy, which is highly invasive and generally contraindicated in children. By studying serum biomarkers associated with NASH, we aim to identify high risk children who can benefit from a less invasive, alternative approach to the early detection of NASH. Methods Seventy one children were prospectively recruited and divided into 3 groups: normal weight without IR (control), obese without IR, and obese with IR. Serum samples were drawn for each patient and biomarker levels were assessed via ELISA kits. Results Obese without IR and obese with IR patients had significantly elevated levels of lipid metabolism and accumulation markers (FGF-21, NEFA, FATP5, ApoB), oxidative stress markers (dysfunctional HDL, 8-Isoprostane), inflammatory markers(dysfunctional HDL, CK-18) and apoptosis markers (CK-18) compared to control patients (p<0.02). Bilirubin (an antioxidant) was significantly decreased in the obese without IR and obese with IR patients compared to control (p<0.02). Conclusion This study showed a correlation between obesity, IR, and biomarkers associated with NASH in pediatrics patients from West Virginia, with obese with IR patients showing the strongest correlation. These findings support the clinical application of these serum biomarkers as a less invasive method for early detection of NASH and hepatic fibrosis.

Spin Trapping: A Review for the Study of Obesity Related Oxidative Stress and Na + /K + -ATPase

Reactive oxygen species (ROS) have gained attention with mounting evidence of their importance in cell signaling and various disease states. ROS is produced continuously as a natural by-product of normal oxygen metabolism. However, high levels ROS causes oxidative stress and damage to biomolecules. This results in loss of protein function, DNA cleavage, lipid peroxidation, or ultimately cell injury or death. Obesity has become a worldwide epidemic; studies show fat accumulation is associated with increased ROS and oxidative stress. Evidence exists supporting oxidative stress as a factor driving forward insulin resistance (IR), potentially resulting in diabetes. Na+/K+-ATPase signaling is also a potential source of ROS promoting oxidative stress. The best way to observe radical species in biological systems is electron paramagnetic resonance spectroscopy with spin trapping. EPR spin trapping is an important technique to study the mechanisms driving disease states attributed to ROS.

Heme Oxygenase Induction Suppresses Hepatic Hepcidin and Rescues Ferroportin and Ferritin Expression in Obese Mice

Hepcidin, a phase II reactant secreted by hepatocytes, regulates cellular iron levels by increasing internalization of ferroportin-a transmembrane protein facilitating egress of cellular iron. Chronic low-grade inflammatory states, such as obesity, have been shown to increase oxidative stress and enhance hepcidin secretion from hepatocytes and macrophages. Heme-heme oxygenase (HO) is a stress response system which reduces oxidative stress. We investigated the effects of HO-1 induction on hepatic hepcidin levels and on iron homeostasis in hepatic tissues from lean and obese mice. Obese mice exhibited hyperglycemia (p < 0.05); increased levels of proinflammatory cytokines (MCP-1, IL-6, p < 0.05); oxidative stress (p < 0.05); and increased hepatic hepcidin levels (p < 0.05). Enhancement of hepcidin was reflected in the reduced expression of ferroportin in obese mice (p < 0.05). However, this effect is accompanied by a significant decline in ferritin expression. Additionally, there are reduced insulin receptor phosphorylation and attenuation of metabolic regulators pAMPK, pAKT, and pLKB1. Cobalt protoporphyrin- (CoPP-) induced HO-1 upregulation in obese mice reversed these alterations (p < 0.05), while attenuating hepatic hepcidin levels. These effects of CoPP were prevented in obese mice concurrently exposed to an inhibitor of HO (SnMP) (p < 0.05). Our results highlight a modulatory effect of HO on iron homeostasis mediated through the suppression of hepatic hepcidin.

Adiponectin, Leptin, IGF-1, and Tumor Necrosis Factor Alpha As Potential Serum Biomarkers for Non-Invasive Diagnosis of Colorectal Adenoma in African Americans

The potential role of adiponectin, leptin, IGF-1, and tumor necrosis factor alpha (TNF-α) as biomarkers in colorectal adenoma is not clear. Therefore, we aimed to investigate the blood serum levels of these biomarkers in colorectal adenoma. The case–control study consisted of serum from 180 African American patients with colon adenoma (cases) and 198 healthy African Americans (controls) at Howard University Hospital. We used ELISA for adiponectin, leptin, IGF-1, and TNF-α detection and quantification. Statistical analysis was performed by t-test and multivariate logistic regression. The respective differences in median leptin, adiponectin, IGF-1, and TNF-α levels between control and case groups (13.9 vs. 16.4), (11.3 vs. 46.0), (4.5 vs. 12.9), and (71.4 vs. 130.8) were statistically significant (P < 0.05). In a multivariate model, the odds ratio for adiponectin, TNF-α, and IGF-1 were 2.0 (95% CI = 1.6–2.5; P < 0.001), 1.5 (95% CI = 1.5(1.1–2.0); P = 0.004), and 1.6 (95% CI = 1.3–2.0; P < 0.001), respectively. There was a positive correlation between serum adiponectin and IGF-1 concentrations with age (r = 0.17, P < 0.001 and r = 0.13, P = 0.009), TNF-α, IGF-1, and leptin concentration with body mass index (BMI) (r = 0.44, P < 0.001; r = 0.11, P = 0.03; and r = 0.48, P < 0.001), respectively. Also, there was a negative correlation between adiponectin and leptin concentrations with BMI (r = −0.40, P < 0.001), respectively. These data support the hypothesis that adiponectin, IGF-1, and TNF-α high levels correlate with higher risk of colon adenoma and can thus be used for colorectal adenomas risk assessment.

Abstract 3130: Adiponectin, Leptin, IGF1 and TNFα serum biomarker as noninvasive diagnosis of colon adenoma

Background and Aim: The potential role of Adiponectin, Leptin, IGF1 and TNFα as biomarker in colon adenoma has not been studied. Therefore, we investigated the blood serum levels of these biomarkers in colorectal adenoma. Method: The case-control study consisted of 198 African American patients with colon adenoma (cases) and 198 healthy individuals (controls) at Howard University Hospital. We used Elisa for biomarkers detection. Statistical analysis was performed by t-test and multivariate logistic regression. Results: The differences in median leptin, Adiponectin, IGF1 and TNFα levels between control and case groups (6.7 vs.16.4), (11.3 vs.46.0), (4.5 vs.12.9) and (71.4 vs. 130.8) were statistically significant (p Conclusion: These data support the hypothesis that serum Adiponectin, IGF1 and TNFα are risk biomarkers for noninvasive detection of colorectal adenomas. Citation Format: Hassan Ashktorab, Akbar Soleimani, Alexandra Nichols, Komal Sodhi, Lakshmi Kannan, Laiyemo Adeyinka, Mehdi Nouraie, Hassan Brim. Adiponectin, Leptin, IGF1 and TNFα serum biomarker as noninvasive diagnosis of colon adenoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3130.

Su1885 Insulin Resistance Is a Key Factor for Metabolic and Inflammatory Biomarkers in Children Obesity

protein integrity. Hypothesis: Epithelial cell Hsp70 overexpression maintains tight junction protein levels in spite of high fat diet. Materials & Methods: Five week old male mice, [non-transgenic (NTG; n=19) and Hsp70 transgenic littermates (TG; n=14; villin promoter driven epithelial cell Hsp70 expression)] were randomly divided into low fat (LF; 10 kcal% fat) or high fat (HF; 60 kcal% fat) diet groups (NTG/LF, NTG/HF, TG/LF, TG/HF). All mice continued on the protocol for 12 weeks. At week 13, colonic scrapings were collected and homogenized for determining Hsp70 levels (ELISA, R&D Systems) and tight junction protein expression (Western blot). Western blots probed for occludin (Life Technologies) and constitutive heat shock protein 70 (HSC70, Santa Cruz Biotech) were run using pooled samples of each group (n= 6-10 per group). Densitometry readings were completed using a ChemiDoc MP Imager (Bio-Rad) and data normalized using HSC70 as a loading control with NTG/LF as the reference sample. Hsp70 data was analyzed by 2-way ANOVA and presented as a mean ± SEM. Results: Measurement of colonic Hsp70 levels showed a significant (p < 0.05, Fig. 1), 100 fold, increase in the TG group compared to its NTG littermates. Western blot for occludin using pooled samples representative of each group suggested TG mice expressed an increased amount of this protein compared to their NTG littermates regardless of diet (Fig. 2). Discussion: Hsp70 TG mice demonstrated increased levels of occludin, suggesting that it may enhance gut barrier function by increasing tight junction proteins independent of diet. Further examination of Hsp70s affect on colonic tight junction proteins may provide a possible target for the prevention and treatment of obesity