The “Free Hormone Hypothesis” posits that only free, unbound hormone is biologically active and available to tissues. Conventional biomedical wisdom proposes that corticosteroid-binding globulin (CBG) normally binds 90-95% of blood glucocorticoid (GC), rendering it unavailable to tissues. Under chronic stress, GC levels greatly exceed binding capacity resulting in impaired bodily function and reduced fitness. However, under normal conditions in northern and southern flying squirrels, less than 10% of GC is bound, presenting a major challenge to the hypothesis. To assess the extent of variation in these properties among vertebrates, I compared all species (88) with known GC and CBG and levels. 92% conform reasonably to known convention. Flying squirrels appear as extreme species, as do New World monkeys, yet both groups evolved from ancestors that followed normal convention. I speculate as to how this state evolved and persisted through time.

Androgens (such as testosterone) have been shown to increase lean body mass and reduce fat body mass in men through activation of androgen receptors (AR). While this suggests a potential clinical use for androgens, attempts at utilization of this class of hormones as a therapeutic are limited by side effects due to indiscriminate AR activation in various tissues. Thus, a greater understanding of the tissues and cells involved in promoting these changes would be beneficial. Here we show that selective overexpression of AR in muscle cells of transgenic (HSA-AR) rodents both increases lean muscle mass and significantly reduces fat mass in males. Similar effects can be induced in HSA-AR females treated with testosterone. Metabolic analyses of HSA-AR males show that these animals demonstrate increased O2 consumption and hypermetabolism. Thus, targeted activation of AR in muscle regulates body composition and metabolism, suggesting a novel target for drug development.

The “Free Hormone Hypothesis” posits that only free, unbound hormone is biologically active and available to tissues. Conventional biomedical wisdom proposes that corticosteroid-binding globulin (CBG) normally binds 90-95% of blood glucocorticoid (GC), rendering it unavailable to tissues. Under chronic stress, GC levels greatly exceed binding capacity resulting in impaired bodily function and reduced fitness. However, under normal conditions in northern and southern flying squirrels, less than 10% of GC is bound, presenting a major challenge to the hypothesis. To assess the extent of variation in these properties among vertebrates, I compared all species (88) with known GC and CBG and levels. 92% conform reasonably to known convention. Flying squirrels appear as extreme species, as do New World monkeys, yet both groups evolved from ancestors that followed normal convention. I speculate as to how this state evolved and persisted through time.

Benzene and toluene are representative volatile organic compounds (VOCs) released during production, storage, and transportation associated with the oil and gas industry. Benzene and toluene are chemicals of concern because they are released in greater and possibly more biologically significant concentrations than other compounds. <p>Most studies of air pollution in high oil and gas activity areas have neglected to consider risks to top-level predators. Birds can be used as highly sensitive monitors of air quality. Since the avian respiratory tract is physiologically different from a rodent respiratory tract, effects of gases cannot be safely extrapolated from rodent studies. I hypothesized that benzene, being haematotoxic and immunotoxic, along with the neurological and possible endocrine disrupting effects of toluene would be more toxic in birds than in mammals. <p>After two summers of experimental exposure of wild and captive American kestrels to high (10ppm and 80ppm) or environmentally relevant (0.1ppm and 0.8ppm) levels of benzene and toluene, respectively, altered immune, haematopoeitic, behavioural, and endocrine responses characteristic in mammals, were evident in the kestrels.<p>There was a decreased cell mediated immune response as measured by delayed type hypersensitivity tests in all exposed birds (p = 0.028, 0.004). An increase in humoral immunity as compared to control individuals (p = 0.041, 0.031) was also apparent in both dose groups. Plasma retinol levels were decreased in 2005 and 2006 high dose individuals (p = 0.008, 0.048). <p>The majority of haematopoeitic effects involved the erythroid lineage in the bone marrow and the polychromatophilic erythrocytes systemically. There were no significantly adverse responses in the bone marrow with regards to the granuloid lineage but systemically there was a prominent eosinophilia (p = 0.045) and basophilia (p = 0.006) in low exposure groups. The loss of communication between polychromatophilic erythrocytes in the post-mitotic pool within the bone marrow and the peripheral blood was present in low and high exposure individuals compared to control birds (p = 0.013, 0.402, 0.974). The number of polychromatophils in the circulation of low dose group individuals was decreased compared to control birds (p = 0.029). This may be a function of toluenes inability to inhibit biotransformation enzymes at low concentrations leading to blood cell targeting by benzenes increased phenolic metabolite production. This theory is corroborated by the possible decreased benzene metabolism and increased toluene distribution manifesting as increased aggressive responses such as wing beating and vocalization time in the high dose group (p = 0.025, 0.086). <p>The work here has shown American kestrels are sensitive to the air contaminants, benzene and toluene. The present study illustrates the need for reference concentrations for airborne pollutants that are calculated based on data measuring sensitive endpoints specific for avian models. Future studies should evaluate immune, haematopoeitic, and behavioural endpoints, as well as develop more sensitive isoform specific enzyme activity assays to further determine the susceptibility of birds to inhaled toxicants.

Graduation date: 1993

<p>Sex differences in drug addiction are mediated in part by effects of the ovarian hormone estradiol (E2) within the ascending dopamine (DA) system from the midbrain to the striatum. Estradiol enhances the effects of psychostimulants, but the exact underlying mechanisms are unknown. Mice could serve as an ideal genetically-tractable model for mechanistic studies into sex and hormone effects within the DA system but have been under-utilized. This study sought to: 1) characterize psychostimulant-induced behavior in mice as an indirect but quantifiable measure of DA neurotransmission, and 2) elucidate the mechanism underlying E2's enhancement of psychostimulant effects in females using surgical, pharmacological, and genetic manipulations. The spontaneous behavior of mice during habituation to a novel environment and after the psychostimulants d-amphetamine (AMPH; 1, 2.5, and/or 5 mg/kg) and cocaine (COC; 5, 15, and/or 30 mg/kg) were assessed in open field chambers using both automated photobeam interruptions and behavioral observations. Behaviors were assessed in the following groups of mice: intact males and females; ovariectomized mice replaced with either E2 for 2 days or 30 minutes or with estrogen receptor-selective agonists; and female mice lacking either ER&alpha; (&alpha;ERKO) or ER&beta; (&beta;ERKO) versus wildtype (WT) littermates. Brain psychostimulant concentrations and tissue content of DA and its metabolites were determined at the time of maximum behavioral stimulation. Psychostimulants induced behavioral activation in mice including both increased locomotion as detected with an automated system and a sequence of behaviors progressing from stereotyped sniffing at low doses to patterned locomotion and rearing at high doses. Intact female mice exhibited more patterned locomotion and a shift towards higher behavior scores after psychostimulants despite having lower AMPH and equivalent COC brain levels as males. Actively ovariectomized mice exhibited fewer ambulations and lower behavior scores during habituation and after psychostimulants than Sham females. Two days but not 30 minutes of E2 replacement restored COC-induced behavioral responses to Sham levels. ER&alpha;-selective PPT replacement in ovariectomized mice and genetic ablation of ER&alpha; in &alpha;ERKO mice altered COC-stimulated behavior. Immunohistochemistry revealed that midbrain DA neurons in mice express ER&beta; but not ER&alpha;, and that non-DA cells in the midbrain and the striatum express ER&alpha;. These results indicate that E2 enhances COC-stimulated locomotion in mice through an indirect effect of ER&alpha;. ER&alpha; may alter behavior through presynaptic effects on DA neuron activity and/or through postsynaptic effects on transcription and signal transduction pathways within striatal neurons.</p> / Dissertation

Benzene and toluene are representative volatile organic compounds (VOCs) released during production, storage, and transportation associated with the oil and gas industry. Benzene and toluene are chemicals of concern because they are released in greater and possibly more biologically significant concentrations than other compounds. <p>Most studies of air pollution in high oil and gas activity areas have neglected to consider risks to top-level predators. Birds can be used as highly sensitive monitors of air quality. Since the avian respiratory tract is physiologically different from a rodent respiratory tract, effects of gases cannot be safely extrapolated from rodent studies. I hypothesized that benzene, being haematotoxic and immunotoxic, along with the neurological and possible endocrine disrupting effects of toluene would be more toxic in birds than in mammals. <p>After two summers of experimental exposure of wild and captive American kestrels to high (10ppm and 80ppm) or environmentally relevant (0.1ppm and 0.8ppm) levels of benzene and toluene, respectively, altered immune, haematopoeitic, behavioural, and endocrine responses characteristic in mammals, were evident in the kestrels.<p>There was a decreased cell mediated immune response as measured by delayed type hypersensitivity tests in all exposed birds (p = 0.028, 0.004). An increase in humoral immunity as compared to control individuals (p = 0.041, 0.031) was also apparent in both dose groups. Plasma retinol levels were decreased in 2005 and 2006 high dose individuals (p = 0.008, 0.048). <p>The majority of haematopoeitic effects involved the erythroid lineage in the bone marrow and the polychromatophilic erythrocytes systemically. There were no significantly adverse responses in the bone marrow with regards to the granuloid lineage but systemically there was a prominent eosinophilia (p = 0.045) and basophilia (p = 0.006) in low exposure groups. The loss of communication between polychromatophilic erythrocytes in the post-mitotic pool within the bone marrow and the peripheral blood was present in low and high exposure individuals compared to control birds (p = 0.013, 0.402, 0.974). The number of polychromatophils in the circulation of low dose group individuals was decreased compared to control birds (p = 0.029). This may be a function of toluenes inability to inhibit biotransformation enzymes at low concentrations leading to blood cell targeting by benzenes increased phenolic metabolite production. This theory is corroborated by the possible decreased benzene metabolism and increased toluene distribution manifesting as increased aggressive responses such as wing beating and vocalization time in the high dose group (p = 0.025, 0.086). <p>The work here has shown American kestrels are sensitive to the air contaminants, benzene and toluene. The present study illustrates the need for reference concentrations for airborne pollutants that are calculated based on data measuring sensitive endpoints specific for avian models. Future studies should evaluate immune, haematopoeitic, and behavioural endpoints, as well as develop more sensitive isoform specific enzyme activity assays to further determine the susceptibility of birds to inhaled toxicants.

Individual day-to-day management and effective control of type 1 diabetes (T1D) is ultimately driven by decisions made by the individual. Individuals with attention deficit hyperactivity disorder (ADHD) have a higher tendency to be inattentive, impulsive, and hyperactive. Attention deficits and impulsivity among adolescents and adults with T1D could result in poor diabetes management through infrequent self-monitoring of blood glucose and inadequate insulin dosing – key components of achieving optimal glycemic control. This study included 7,380 adolescents and young adults, ages 13-25 years, participating in the T1D Exchange clinic registry (median age 17.4 years, duration 7.7 yrs, 50% female, 81% white). Participants were classified into 3 groups for the primary analyses using participant reported diagnosis and medication use: 1) No ADHD; 2) ADHD with current medication; 3) ADHD without current medication. Groups were compared in logistic and linear regression models for: self-monitoring of blood glucose (SMBG) /day, missed insulin dose ≥1 time/week, HbA1c, and at least 1 diabetic ketoacidosis (DKA) and severe hypoglycemic (SH) event in the past 3 months. Overall, 774 (10%) participants reported a diagnosis of ADHD of whom 371 currently took medication. Mean SMBG/day was 4.7, 5.0, and 4.9 in the ADHD w/o meds, ADHD with meds, and no ADHD groups, respectively. ADHD patients w/o meds (36%, P=0.02) or with meds (39%, P=0.003) were more likely to report missing insulin doses compared with no ADHD pts (30%). Mean HbA1c was higher in ADHD w/o meds (9.0%, P<0.001) and ADHD with meds (8.9%, P=0.002) compared with no ADHD pts (8.6%). The odds of having at least one DKA event in the past 3 months was 1.8 and 1.5 times higher in the ADHD w/o meds (P<0.001) and ADHD with meds (P=0.01) group compared with no ADHD. The ADHD w/o meds group was significantly more likely to have had a SH event (OR 1.7 95% CI 1.2-2.3; P<0.001) compared with the no ADHD group but the occurrence of SH in the ADHD with meds group was similar to the no ADHD. Results of this study supported the working hypothesis that ADHD without treatment with medication has a negative impact on aspects of diabetes management and glycemic control. Participants with ADHD with and without medication were more likely to miss insulin doses, less likely to use an insulin pump, more likely to have high HbA1c levels, and had a higher frequency of DKA and SH. These results have important public health implications for adolescents and young adults with T1D who are already at risk for poor glycemic control. Since ADHD has a meaningful impact on glycemic outcomes it is important for providers of adolescents and young adults with T1D to review history and signs of an ADHD diagnosis along with diagnosis of other psychosocial disorders with their patients and consider recommending psychosocial services.

Key insights into the evolutionary origins of human social behavior can be gained via study of our closest living relatives, bonobos (Pan paniscus) and chimpanzees (Pan troglodytes). Despite being equally related to humans, these two species differ importantly in aspects of their morphology, physiology, behavior, and cognition. Morphological comparisons reveal numerous traits in bonobos that can be viewed as paedomorphic, or juvenile, relative to chimpanzees. Meanwhile, comparisons of endocrinology in the two species suggest that aspects of steroid physiology have changed significantly in bonobos in line with their reductions in male mating competition. Based on this evidence, I tested the hypothesis that behavioral and cognitive differences between bonobos and chimpanzees derive from changes in their 1) developmental trajectories of behavioral and cognitive traits and 2) neuroendocrine influences on behavior and cognition. I tested this hypothesis by studying semi free-ranging populations of bonobos and chimpanzees. First, I found that bonobos retained juvenile levels of food sharing and social inhibition into adulthood, leading them to differ from chimpanzees in these traits as adults. Second, I found that bonobos showed muted elevations in their levels of testosterone from infancy to adulthood in comparison to chimpanzees, suggesting that numerous aspects of development differ between these two species. Third, I found that male bonobos and chimpanzees differ in their immediate neuroendocrine shifts surrounding competition, implicating changes in proximate mechanisms influencing social behavior between the two species. Fourth, I found that patterns of cognitive development in these two apes differed significantly from those of human children. These results provide substantial support for my hypothesis that phenotypic differences between bonobos and chimpanzees evolved via shifts in bonobo development and neuroendocrine physiology. More broadly, they illustrate how behavioral and cognitive evolution can occur through changes in ontogenetic trajectories and neuroendocrine mechanisms. These findings thus show the merits of integrating ultimate and proximate levels of analysis in studies of the evolution of human behavior and cognition. / Human Evolutionary Biology

The prevalence of obesity and type 2 diabetes has increased at alarming rates in recent decades. These diseases are prominent components of the metabolic syndrome, which is characterized by marked dyslipidemia. Adipose tissue contributes to the development of obesity-related diabetes through increased release of hormones and non-esterified fatty acids. The development of sensitive analytical tools for the broad detection of lipid biomolecules, such as liquid chromatographymass spectrometry (LC-MS), has spurred interest in the molecular determinants of the metabolic syndrome. The development of mature adipocytes from precursor fibroblasts—adipogenesis—plays a crucial role in the expansion of adipose tissue in obesity. We profiled differentiating 3T3-L1 pre-adipocytes by LC-MS and found that a class of monoglyceride lipids, monoalkylglycerol ethers (MAGEs), was transiently elevated early in adipogenesis. Upon addition to differentiating cells, MAGE specifically promoted adipocyte maturation and expression of adipogenic gene markers, indicating that MAGEs may be signaling molecules during adipogenesis. The insulin-sensitive glucose transporter, GLUT4, is downregulated during obesity and diabetes. In collaboration with Prof. Barbara Kahn, we studied a transgenic mouse model that overexpressed GLUT4 specifically in adipose tissue (AG4OX) and was protected from developing diabetes. We used LC-MS-based metabolomics to discover a previously undescribed class of bioactive lipids that was highly upregulated in AG4OX adipose tissue. We structurally characterized these lipids as fatty acyl hydroxy fatty acids (FAHFAs) and several positional isomers were chemically synthesized to confirm structural assignments via coelution studies. We discovered that individual FAHFAs, such as 5-palmitoyl-hydroxystearic acid (5-PAHSA), were differentially regulated by the transcription factor ChREBP. Circulating 5-PAHSA levels in mice and humans correlated with ChREBP expression and insulin resistance. In order to explore the biochemical regulation of FAHFAs, we developed an LCMS-based assay to measure FAHFA hydrolysis activity. We identified one enzyme, carboxyl ester lipase (CEL), as the major FAHFA hydrolase in pancreas, where the activity was highest. We confirmed its relevance in vivo by feeding labeled FAHFA to CEL inhibitor-treated mice. In this work we used LC-MS-based metabolomics to discover two lipids, MAGE and FAHFA, along with the CEL pathway, that may help us to better understand the pathogenesis of obesity and diabetes. / Chemistry and Chemical Biology