A growing body of literature has linked atypical antipsychotics (AAPs) to an increased propensity for weight gain and metabolic disturbances, including type 2 diabetes. While weight gain is a leading risk factor for diabetes, evidence suggests that AAPs may influence glucose homeostasis independently of changes in adiposity. These ‘direct’ drug effects have been consistently supported by animal models, where following even a single dose of certain AAPs immediate effects are observed with noted perturbations on insulin sensitivity, and insulin secretion. However, the mechanisms underlying these effects remain poorly understood. Also, the translational value of the acute dosing rodent model has not been established in humans. As such, we set out to first elucidate mechanisms of these ‘direct’ effects by deconstructing antipsychotic receptor binding profiles using selective antagonists and gold standard clamping techniques to examine effects on glucose metabolism. We also investigated antipsychotic administration directly into the brain in rodents to tease out central vs. peripheral effects on glucose metabolism. Finally, we examined whether the effects of a single dose of olanzapine on glucose metabolism could be replicated in healthy humans, independently of adiposity or the confounding effects of the illness of schizophrenia. Our findings suggest that cholinergic, serotonergic, and dopaminergic pathways may be involved in antipsychotic-induced glucose dysregulation. We also suggest that such effects may be mediated in part through the central nervous system. Our results in humans suggest that acute drug effects may be less pronounced than in rodents, failing to note an effect on insulin sensitivity or secretion, but observing other early perturbations in lipid and glucose metabolism. Taken together, the work here begins to elucidate mechanisms underlying the diabetogenic risk associated with AAPs, findings which have important implications given the widespread use of these drugs, as well as the increased mortality attributable to cardiovascular disease that defines those with schizophrenia.

Novel Regulatory Mechanisms of D1 Dopamine Receptor Maturation and Internalization Michael Ming Chuen Kong Degree of Doctor of Philosophy, 2008 Department of Pharmacology University of Toronto ABSTRACT Dopamine is the most abundant catecholamine neurotransmitter in the mammalian brain and controls various physiological processes. The D1 dopamine receptor (D1DR) is the predominant dopamine receptor in the brain and traditionally couples to stimulatory G proteins, such as Gs, to activate adenylyl cyclase and generate cAMP. Although the trafficking itinerary of ER/Golgi maturation, agonist-induced internalization, and recycling/degradation are features common to many G protein-coupled receptors (GPCRs), the molecular regulation of these individual processes for the D1DR is not fully elucidated. Many GPCRs have been shown to form homo-oligomers; the work presented in this thesis explores how multimerization of D1DR has a role in regulating how these receptors are trafficked to the plasma membrane. In addition, the regulation of D1DR internalization is investigated in the context of emerging evidence highlighting the importance of lipid rafts. Using strategically designed point mutations of the D1DR, specific receptor mutants were found to intracellularly sequester the wild-type receptor by oligomerization. This level of scrutiny by the quality control machinery in the cell could be circumvented by treatment with cell permeable dopaminergic agonists, but not antagonists or inverse agonists. This finding suggests that specific conformational requirements must be achieved before full maturation and anterograde trafficking of the D1DR can proceed. Furthermore, it was determined that cell surface bound D1DRs could internalize through a novel clathrin independent pathway that required binding to the scaffolding protein, caveolin-1. This interaction with caveolin-1 was identified in whole rat brain and was found to require a putative caveolin binding motif in transmembrane domain 7. Palmitoylation of D1DR was found to regulate the rate of agonist-induced caveolae mediated internalization. Finally, we determined that the integrity of caveolae was important in regulating cAMP signaling through D1DR. These findings provide novel insight into the trafficking requirements of newly synthesized D1DRs as well as alternative mechanisms of regulation of receptors after agonist activation. The oligomerization of GPCRs and the localization of GPCRs in lipid rafts represent two emerging concepts important to many aspects of GPCR function. Future work aimed at integrating these overlapping processes will further our understanding of this important group of cell surface receptors.

Dopamine receptors are members of the G protein-coupled receptor superfamily and play important roles in neuronal transmission. A D1-D2 receptor heterooligomer generating a G-protein linked PLC-dependent intracellular calcium signal was previously identified. The discovery of this dopamine mediated calcium signal implicated a direct link between dopamine receptors and calcium generation, but its regulation remained to be elucidated. By measuring calcium signaling with Fluo-4 fluorescence or cameleon FRET, rapid desensitization of the calcium signal in heterologous cells and striatal neurons was demonstrated by pre-treatment with SKF 83959, which selectively activates D1-D2 receptor heteromers, or SKF 83822 which only activates D1 receptor homooligomers. Although SKF 83822 was unable to activate D1-D2 receptor heteromers, it still permitted desensitization of the calcium signal. This suggested that occupancy of the D1 receptor binding pocket by SKF 83822 resulted in conformational changes sufficient for desensitization without activation of the heteromer. BRET and co-immunoprecipitation studies indicated an agonist induced interaction between the D1-D2 receptor heteromer and GRK2. Increased expression of GRK2 led to a decrease in the calcium signal and decreased expression of GRK2 led to an increased calcium signal. Expression of the catalytically inactive and RGS mutated GRK2 constructs each led to a partial recovery of the GRK2-attenuated calcium signal. These results indicated that desensitization of the D1-D2 receptor heteromer mediated calcium signal can occur by agonist occupancy even without activation and is regulated by two distinct functions of GRK2. Immunocytochemistry and calcium assays demonstrated that recycling of internalized D1 and D2 receptors and resensitization of the desensitized calcium signal occurred after dopamine pre-treatment but not SKF 83959, suggesting that the trafficking and resensitization response associated with the D1-D2 receptor heteromer is differentially regulated by specific ligands. Overall, these results suggest that D1-D2 receptor heterooligomers are uniquely regulated from their constituent receptors which are not coupled to Gq.

A growing body of literature has linked atypical antipsychotics (AAPs) to an increased propensity for weight gain and metabolic disturbances, including type 2 diabetes. While weight gain is a leading risk factor for diabetes, evidence suggests that AAPs may influence glucose homeostasis independently of changes in adiposity. These ‘direct’ drug effects have been consistently supported by animal models, where following even a single dose of certain AAPs immediate effects are observed with noted perturbations on insulin sensitivity, and insulin secretion. However, the mechanisms underlying these effects remain poorly understood. Also, the translational value of the acute dosing rodent model has not been established in humans. As such, we set out to first elucidate mechanisms of these ‘direct’ effects by deconstructing antipsychotic receptor binding profiles using selective antagonists and gold standard clamping techniques to examine effects on glucose metabolism. We also investigated antipsychotic administration directly into the brain in rodents to tease out central vs. peripheral effects on glucose metabolism. Finally, we examined whether the effects of a single dose of olanzapine on glucose metabolism could be replicated in healthy humans, independently of adiposity or the confounding effects of the illness of schizophrenia. Our findings suggest that cholinergic, serotonergic, and dopaminergic pathways may be involved in antipsychotic-induced glucose dysregulation. We also suggest that such effects may be mediated in part through the central nervous system. Our results in humans suggest that acute drug effects may be less pronounced than in rodents, failing to note an effect on insulin sensitivity or secretion, but observing other early perturbations in lipid and glucose metabolism. Taken together, the work here begins to elucidate mechanisms underlying the diabetogenic risk associated with AAPs, findings which have important implications given the widespread use of these drugs, as well as the increased mortality attributable to cardiovascular disease that defines those with schizophrenia.

Nicotine is the primary substance in tobacco causing addiction. In humans the majority (70-80%) of nicotine is inactivated to cotinine in a reaction predominantly catalyzed by CYP2A6 (80-90%), with a minor possible role for CYP2B6. Substantial interindividual variability is observed in the rate of nicotine’s inactivation to cotinine and this variation contributes to differences in smoking behaviors (e.g. cigarette consumption). Twin studies suggest an important genetic contribution to the variability in nicotine metabolism. However in 2004, genetic variation in CYP2A6 and CYP2B6 accounted for only a small portion of the variability suggesting gaps in our knowledge. Our objective was to identify additional genetic and non-genetic sources of variability in CYP2A6 expression/activity, CYP2B6 expression, and nicotine to cotinine metabolism in vivo and/or in vitro. Participants included individuals from different world populations phenotyped for CYP2A6 activity either following oral nicotine administration or using metabolite ratios derived from baseline smoking. Genotyping and sequencing were utilized to identify and characterize multiple new CYP2A6 alleles. In total 17 novel CYP2A6 alleles were identified, many of which were found predominantly among individuals of black African descent and exhibited lower CYP2A6 activity. In addition, human livers were assessed for CYP2A6 and CYP2B6 expression and nicotine to cotinine metabolism. The mechanisms underlying the lower CYP2A6 activity associated with some of the variant CYP2A6 alleles included either a reduction in hepatic CYP2A6 protein expression, an alteration of CYP2A6’s structural property, or a combination of both. DNA methylation was not associated with altered hepatic CYP2A6 expression/activity. Livers from female donors were associated with higher CYP2A6 and CYP2B6 protein expression compared to male livers, while age did not influence the expression of either CYP. Finally, CYP2B6 and its prevalent altered function genetic variant (CYP2B6*6) did not influence nicotine to cotinine metabolism. Identification of factors that contribute to the variability in CYP2A6 and nicotine metabolism is important to improve future association studies between CYP2A6 genotype, nicotine metabolism, and smoking behaviors. In addition, this information could provide the potential to personalize therapy in order to improve the clinical efficacy of nicotine, particularly as a smoking cessation aid.

Objective: To conduct an open label clinical pilot trial using Ginkgo biloba in the treatment of vitiligo in Toronto to test the feasibility of recruitment, patient retention, variability of outcome measures, identify safety concerns, and magnitude of treatment effect ahead of a full randomized clinical trial. Methods: 12 participants 12 to 35 years old were recruited to a prospective nonrandomized open-label pilot trial and treated with 60 mg of standardized G. biloba BID for 12 weeks. The primary outcome was the validated Vitiligo European Task Force (VETF). Secondary outcomes included the Vitiligo Area Scoring Index (VASI), photographs, and adverse reactions. Results: Ingestion of Ginkgo biloba was associated with a trend towards improvement on VETF measures of vitiligo lesion area and staging, and significant improvement in VETF spread and total VASI vitiligo measures. Conclusions: By achieving full recruitment, showing benefit, and indicating no adverse reactions the pilot study shows that a future RCT is feasible.

Varenicline is used to treat tobacco dependence. While varenicline decreases craving during a quit attempt, no studies have investigated its effect on cue-induced craving. Varenicline has also been shown to decrease alcohol consumption in animal and humans. This double-blind, randomized, placebo-controlled trial investigated the effect of varenicline on tobacco and alcohol cue-induced craving and alcohol consumption in dependent smokers/light drinkers. Tobacco and alcohol craving were assessed at baseline and after 2-weeks of drug administration using the QSU and ACQ. Significant decreases in cigarette and alcohol craving were observed between the pre- and post-drug session in the varenicline group on QSU Factor 1(87.5811.66 vs.70.5820.79, p=0.008) and ACQ Total (3.371.16 vs. 2.661.15, p=0.004) scores. This effect remained significant after correction for craving during neutral cues in the alcohol but not tobacco condition. No significant decreases in alcohol consumption were seen. These results suggest varenicline decreases overall craving, but not cue-induced craving specifically.

Objective: To conduct an open label clinical pilot trial using Ginkgo biloba in the treatment of vitiligo in Toronto to test the feasibility of recruitment, patient retention, variability of outcome measures, identify safety concerns, and magnitude of treatment effect ahead of a full randomized clinical trial. Methods: 12 participants 12 to 35 years old were recruited to a prospective nonrandomized open-label pilot trial and treated with 60 mg of standardized G. biloba BID for 12 weeks. The primary outcome was the validated Vitiligo European Task Force (VETF). Secondary outcomes included the Vitiligo Area Scoring Index (VASI), photographs, and adverse reactions. Results: Ingestion of Ginkgo biloba was associated with a trend towards improvement on VETF measures of vitiligo lesion area and staging, and significant improvement in VETF spread and total VASI vitiligo measures. Conclusions: By achieving full recruitment, showing benefit, and indicating no adverse reactions the pilot study shows that a future RCT is feasible.

Varenicline is used to treat tobacco dependence. While varenicline decreases craving during a quit attempt, no studies have investigated its effect on cue-induced craving. Varenicline has also been shown to decrease alcohol consumption in animal and humans. This double-blind, randomized, placebo-controlled trial investigated the effect of varenicline on tobacco and alcohol cue-induced craving and alcohol consumption in dependent smokers/light drinkers. Tobacco and alcohol craving were assessed at baseline and after 2-weeks of drug administration using the QSU and ACQ. Significant decreases in cigarette and alcohol craving were observed between the pre- and post-drug session in the varenicline group on QSU Factor 1(87.5811.66 vs.70.5820.79, p=0.008) and ACQ Total (3.371.16 vs. 2.661.15, p=0.004) scores. This effect remained significant after correction for craving during neutral cues in the alcohol but not tobacco condition. No significant decreases in alcohol consumption were seen. These results suggest varenicline decreases overall craving, but not cue-induced craving specifically.

Nicotine is the primary substance in tobacco causing addiction. In humans the majority (70-80%) of nicotine is inactivated to cotinine in a reaction predominantly catalyzed by CYP2A6 (80-90%), with a minor possible role for CYP2B6. Substantial interindividual variability is observed in the rate of nicotine’s inactivation to cotinine and this variation contributes to differences in smoking behaviors (e.g. cigarette consumption). Twin studies suggest an important genetic contribution to the variability in nicotine metabolism. However in 2004, genetic variation in CYP2A6 and CYP2B6 accounted for only a small portion of the variability suggesting gaps in our knowledge. Our objective was to identify additional genetic and non-genetic sources of variability in CYP2A6 expression/activity, CYP2B6 expression, and nicotine to cotinine metabolism in vivo and/or in vitro. Participants included individuals from different world populations phenotyped for CYP2A6 activity either following oral nicotine administration or using metabolite ratios derived from baseline smoking. Genotyping and sequencing were utilized to identify and characterize multiple new CYP2A6 alleles. In total 17 novel CYP2A6 alleles were identified, many of which were found predominantly among individuals of black African descent and exhibited lower CYP2A6 activity. In addition, human livers were assessed for CYP2A6 and CYP2B6 expression and nicotine to cotinine metabolism. The mechanisms underlying the lower CYP2A6 activity associated with some of the variant CYP2A6 alleles included either a reduction in hepatic CYP2A6 protein expression, an alteration of CYP2A6’s structural property, or a combination of both. DNA methylation was not associated with altered hepatic CYP2A6 expression/activity. Livers from female donors were associated with higher CYP2A6 and CYP2B6 protein expression compared to male livers, while age did not influence the expression of either CYP. Finally, CYP2B6 and its prevalent altered function genetic variant (CYP2B6*6) did not influence nicotine to cotinine metabolism. Identification of factors that contribute to the variability in CYP2A6 and nicotine metabolism is important to improve future association studies between CYP2A6 genotype, nicotine metabolism, and smoking behaviors. In addition, this information could provide the potential to personalize therapy in order to improve the clinical efficacy of nicotine, particularly as a smoking cessation aid.