Effects of Egg White Derived Peptides on Metabolic Syndrome Complications: Hypertension, Inflammation, and Insulin resistance

• Author / Creator

  Jahandideh, Forough

• Metabolic syndrome (MetS), a cluster of several abnormalities of hypertension, inflammation, glucose intolerance and dyslipidemia, enhances a person’s risk for cardiovascular disease and type 2 diabetes. The number of people with MetS is increasing largely worldwide. The multi-faceted nature of MetS makes patients take several medications to target different aspects of this disease. In addition to significant side effects associated with synthetic drugs and possible drugs interactions, adherence to life-long therapies is usually poor. Therefore, there is increasing interest in developing functional foods or natural health products as an alternative for the prevention and management of the complications of this disease. Bioactive peptides may potentially alleviate several complications of MetS namely hypertension and insulin resistance, the key features of the disease. Several peptides with antioxidant and antihypertensive activity were previously identified from egg white protein ovotransferrin hydrolysate. Egg white contains several proteins including ovotransferrin, therefore, we hypothesized that egg white hydrolysate would have beneficial effects on hypertension, oxidative stress, and inflammation. Moreover, due to the link between hypertension and insulin resistance, egg white hydrolysate may positively affect glucose tolerance and insulin resistance. The main purpose of the current research was to investigate the potential of egg white-derived peptides on hypertension, inflammation, glucose intolerance, and insulin resistance. The specific objectives of this thesis were to 1) evaluate the anti-inflammatory and antioxidant activities of ovotransferrin derived antioxidant peptides on endothelial cells and their gastrointestinal stability, 2) test the in vivo antihypertensive effect of egg white hydrolysate (EWH) prepared with thermolysin and pepsin with in vitro ACE-inhibitory effect using spontaneously hypertensive rats (SHRs), 3) explore the possible effects of the EWH on adipocyte differentiation and insulin signaling in vitro, 4) evaluate the potential effects of EWH on glucose and insulin tolerance in insulin resistant rats, and 5) identify bioactive peptides in EWH responsible for the observed adipogenic differentiating effects in adipocytes. We assessed the anti-inflammatory and antioxidant capacity of sixteen antioxidant peptides-previously identified from the egg protein ovotransferrin by in vitro oxygen radical absorbance capacity (ORAC) method-using human umbilical vein endothelial cells (HUVECs). Surprisingly, none of the peptides showed anti-inflammatory effects in endothelial cells. While several digested peptides significantly reduced expression of pro-inflammatory adhesion molecules (ICAM-1 and VCAM-1) in response to tumor necrosis factor-α (TNF-α) stimulation, only GWNI reduced TNF-α activated superoxide generation (71.0 ± 12.9%) after GID when tested with Dihydroethidium (DHE) assay. Mass spectrometer analysis identified two new peptides, GWN and GW, in the GWNI digest; however, only GW reduced TNF-α induced VCAM-1 expression (64.3 ± 20.6%) significantly compared to the TNF-α treated cells. Our study suggested that the in vitro ORAC test lacked biological relevance in assessing bioactive peptides. In vivo antihypertensive activity was studied in SHRs using the telemetric method. The effect of oral administration of EWH at a low dose (250 mg/kg BW) and a high dose (1000 mg/kg BW) over a period of 12 days was studied on blood pressure (BP). BP showed a significant reduction in the EWH high dose group compared to untreated controls. BP reduction was associated with enhanced ex vivo vasodilation, reduced oxidative/nitrosative stress, reduced angiotensin converting enzyme and angiotensin II type 1 receptor expression, while enhanced angiotensin II type 2 receptor expression. Circulating level of angiotensin II was unaffected. Thus, EWH exerted antihypertensive effects in SHRs through multiple mechanisms of vascular relaxation and RAS modulation. Egg white ovotransferrin derived bioactive peptides have shown beneficial effects against hypertension, oxidative stress and inflammation in vitro and in vivo. We also observed that EWH exerted antihypertensive effects in SHRs. Given the interplay among hypertension, inflammation, oxidative stress and metabolic syndrome, we aimed to test the effects of EWH on differentiation, insulin signaling and inflammatory responses in 3T3-F442A pre-adipocytes. Our study suggested that EWH could promote adipocyte differentiation as shown by increased lipid accumulation, increased the release of adiponectin and upregulation of peroxisome proliferator associated receptor gamma (PPARγ) and CCAAT/ enhancer binding protein alpha (C/EBP-α). In addition to enhanced insulin effects on the upregulation of protein kinase B/Akt phosphorylation, EWH treatment increased extracellular signal regulated kinase 1/2 (ERK1/2) phosphorylation to a level similar to that of insulin, indicating insulin sensitizing and mimetic properties. EWH further attenuated cytokine-induced inflammatory marker; cyclooxygenase-2 (COX-2) was reduced by 48.8%, possibly through the AP-1 pathway by down regulating c-Jun phosphorylation in adipocytes. Given the critical role of adipose in the pathogenesis of insulin resistance and MetS, EWH may have potential applications in the prevention and management of MetS and its complications. Next, we evaluated the effectiveness of EWH on glucose and insulin tolerance in diet-induced insulin resistant rats. Supplementing a high-fat diet (HFD) with 4% EWH (equivalent to 1.2 g/Kg BW) improved glucose tolerance, muscle and adipose tissue insulin sensitivity, and inflammatory profile in insulin resistant rats. We also observed some evidence for the enhanced adipocyte differentiation or reduced hepatic glucose production in EWH-treated rats, which needs further experiments. Since EWH is a combination of multiple peptides, we identified the bioactive peptides in EWH with beneficial effects on adipocyte differentiation in 3T3-F442A adipocytes. Our data on the purification and characterization of peptides from EWH with PPARγ stimulatory activity in 3T3-F442A cells identified 42 potent peptides from the major egg white proteins ovalbumin and ovotransferrin. We successfully validated the stimulatory effects of several novel peptide sequences including WEKAFKDED, QAMPFRVTEQE, and VFKGL. ERYPIL was another potent peptide with stimulatory effects on PPARγ expression. For the first time, adipogenic differentiating peptides have been characterized from the hydrolysate of a food-derived protein. Thus, the present study confirmed the effectiveness of EWH on several complications of MetS, including hypertension, inflammation, oxidative stress, glucose tolerance and insulin resistance in vitro and in vivo. We could also identify the bioactive peptides in EWH with beneficial effects on pre-adipocyte differentiation. Findings from this study support the potential of egg protein-derived peptides for uses in the prevention and management of MetS complications.

• Subjects / Keywords

  • Egg white derived peptides
  • Inflammation
  • Hypertension
  • Metabolic syndrome
  • Insulin resistance

• Graduation date

  Spring 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3WS8J20D

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.