Final Examination for the Degree of PhD - NAWAZ AHMED

Examining Committee

Dr. Loong-Tak Lim, Chair

Dr. Paul Spagnuolo, Advisor

Dr. Douglas Goff, Advisory Committee Member 

Dr. Donald Mercer, Department Member

Dr. Chris Perry, York University, External Examiner  
 
TITLE: DEVELOPMENT OF AVOCADO DERIVED POLYHYDROXYLATED FATTY ALCOHOLS AS METABOLIC MODULATORS 
 
ABSTRACT:  Obesity and associated disorders like cardiovascular disease and Type 2 Diabetes share a common pathophysiological process in which adipose tissue dysfunction causes fatty acid spill-over and accumulation in non-adipose tissues like skeletal muscle, heart, pancreas and liver. Recent evidence strongly points towards the inhibition of fatty acid oxidation (FAO) in metabolically active tissue as an effective treatment strategy for obesity and associated disorders, however, safe and efficacious FAO inhibitors are currently not clinically available. Avocado derived polyhydroxylated fatty alcohols (PFAs), avocadene and avocadyne, have been previously shown to selectively induce cell death in leukemia stem cells by inhibiting FAO. This thesis is an investigation on the potential of avocado PFAs to be developed as FAO inhibitors or metabolic modulators for the treatment of obesity and insulin resistance. First, a sensitive and reliable analytical method for the quantitation of avocadene and avocadyne in an array of biological matrices (avocado seed and pulp lipid extracts, mouse whole blood and key metabolic tissues) was developed. Second, two in vitro digestion models (i.e. static and dynamic (i.e. TIM-1)) were applied to avocado pulp powder to determine the bioaccessibility of PFAs from avocado pulp digestion. Additionally, a self-emulsifying oil-in-water emulsion-based delivery system was developed for a 1:1 mixture of pure avocadene and avocadyne (AVO) that showed enhanced bioaccesibility in vitro (TIM-1) and significant bioavailability in a pilot pharmacokinetic in vivo study. The effects of AVO were then evaluated in treatment and prevention mouse models of diet-induced obesity (DIO). In the treatment DIO study, five-week oral administration of AVO (twice weekly) slowed weight gain, improved whole body glucose tolerance, reversed insulin resistance, and increased postprandial skeletal muscle glucose utilization. In contrast, AVO’s effects in the prevention DIO study, where AVO was administered at the onset of high fat diet feeding for 13 weeks, were not as pronounced. AVO’s mechanism of action was further explored in vitro where C2C12 myotubes (mouse skeletal muscle cells) and INS-1 (832/13) cells (rat pancreatic β-cell line) were exposed to excessive amounts of palmitate for 24 h. AVO inhibited excessive FAO and restored glucose oxidation in vitro which restored insulin action in C2C12 myotubes and increased glucose stimulated insulin secretion (GSIS) in INS-1 cells, respectively. Finally, in collaboration with an industry partner (Advanced Orthomolecular Research), lyophilized avocado pulp powder was utilized to manufacture a nutritional supplement containing 50 or 200 mg AVO for evaluation in a Phase I, double-blind, placebo-controlled human clinical trial. AVO consumption (once daily for 60 days) showed no dose limiting toxicities in healthy human participants. In summary, this work addresses significant controversy in obesity and metabolism research where enhancing or inhibiting FAO are highly debated as therapeutic strategies. In the context of chronic overnutrition and low energy expenditure, the use of a safe and potent FAO inhibitor like AVO was shown to ameliorate obesity-associated pathologies. Furthermore, this thesis lays the groundwork for future large scale preclinical and clinical studies for AVO that can now be associated with the health benefits of avocado pulp consumption.