Type 2 diabetes (T2D) is characterized by hyperglycemia, which arises due to insulin resistance and beta cell failure and/or decreased beta cell mass. Genome-wide association studies have shown that single nucleotide polymorphisms in the SLC30A8 locus confer altered risk of T2D. Additional human genetic data show that SLC30A8 haploinsufficiency is protective against T2D development. Zinc transporter 8 (ZnT8), encoded by SLC30A8, is predominantly expressed in islet beta cells and localizes to insulin secretory vesicles. Studies of Slc30a8 KO mouse models observed a consistent decrease in islet zinc content, but no clear role for ZnT8 in glucose-stimulated insulin secretion (GSIS). We hypothesized that (i) ZnT8 is critical for the regulation of GSIS in mice, but this only becomes apparent in the absence of another zinc transporter, ZnT7; and (ii) Slc30a8 haploinsufficiency has beneficial effects on glucose metabolism in mice as in humans. To address these hypotheses, we generated several mouse models. A double KO (DKO) mouse lacking both ZnT7 and ZnT8 was generated, and the data demonstrated that deletion of ZnT7 alone had complex effects on glucose and insulin levels in vivo but no effect on GSIS in isolated islets. In contrast, GSIS was abolished in DKO islets. We also examined the protective effect of Slc30a8 haploinsufficiency in the context of diet-induced obesity (DIO). The results demonstrated a protection against DIO and improved glucose tolerance in ZnT8 heterozygous and KO mice compared to WT mice. Additionally, we examined the expression of Slc30a8 in Guinea pig pancreatic tissue and discovered that Slc30a8 is a pseudogene in Guinea pigs. Together, these data demonstrate that ZnT8 is not essential for beta cell function. Overall, the results of these studies suggest that altered ZnT8 function may affect T2D susceptibility through actions in other tissues where it is expressed at low levels rather than through effects on pancreatic islet function.

The pancreas is a dual function organ contributing to both blood glucose homeostasis and digestion. These functions are carried out by the endocrine and exocrine compartments of the pancreas, respectively, which derive from common multipotent progenitor cells (MPCs) during embryonic development. The differentiation process for the cells composing both the endocrine and exocrine compartments is highly orchestrated by regulatory transcription factors. Previous work from our lab showed that one such factor, Onecut 1 (Oc1), is essential for initiating endocrine development, proper duct development, and appears necessary for acinar cell development. Using gene expression and physiologic analyses of genetically altered mouse models we have determined that threshold-dependent cooperation between Oc1 and another transcription factor, Pancreatic and duodenal homeobox 1 (Pdx1) in MPCs is necessary for proper endocrine specification, differentiation, maturation, and function. Additionally, we have concluded that Oc1 is not necessary in differentiated acinar cells, however, we have identified novel targets of Oc1 in exocrine pancreas development.

The endothelium is among the most mechanically enriched environments in the body. It is exposed to a range of hemodynamic-induced and extracellular forces. Of these extracellular forces, the migration of leukocytes through the endothelium will contribute both to classic immune response and development of certain pathologies. While the path of migration across the endothelium will depend on leukocyte and vascular bed type, recent evidence has suggested that the intercellular mechanical microenvironment and forces are also equally as important to this process. Therefore, we present here a model that mimics specific physiological states of a stagnant hemodynamic flow in which we hypothesize that leukocytes will demonstrate attachment preferences to particular areas of differing intercellular stresses on the endothelial bed. Using a model such as this one, it may be possible to exploit these intercellular stresses when developing macrophage-targeted therapies.

Intracellular acidosis is a putative agent of skeletal muscle fatigue, in part, because acidosis depresses the calcium (Ca2+) sensitivity and force production of muscle (18, 50). However, the molecular mechanisms behind this depression in Ca2+ sensitivity and force production are unknown. This gap in knowledge poses a significant challenge in generating a complete understanding of the fatigue process. To close this gap, the ability of myosin to bind to a single actin filament was measured under acidic conditions, in a laser trap assay, with and without regulatory proteins. Decreasing pH from 7.4 to 6.5 reduced the frequency of single actomyosin binding events at submaximal (pCa 8 – pCa 6), but not at maximal (pCa 5 - 4) concentrations. To delineate whether this was due to a direct effect on myosin versus an indirect effect on the regulatory proteins, troponin (Tn) and tropomyosin (Tm), binding frequency was also quantified in the absence of Tn and Tm. Acidosis did not alter the frequency of actomyosin binding events in the absence of regulatory proteins (1.4 ± 0.05 vs 1.4 ± 0.13 events/sec for pH 7.4 and 6.5). Additionally, acidosis did not significantly affect the size of myosin’s powerstroke, or the duration of binding events in the presence of regulatory proteins, at every pCa. These data suggest that acidosis impedes activation of the thin filament by competitively inhibiting Ca2+ binding to TnC. This slows the rate at which myosin initially attaches to actin, therefore less cross-bridges will be bound and generating force at any given sub-maximal pCa.

<p>Adhesion of the blastocyst to the uterine wall is a highly sensitive phenomenon referred to as implantation. Novel-males are capable of disrupting the success of this process (the Bruce effect). A leading hypothesis invokes the transfer of estradiol from the male to the female via urine. This estradiol has direct effect on the uterus which may include morphology and molecular dynamics. Estradiol has been related to closure of the uterus around the blastocyst during implantation, which may assist in bringing the blastocyst close to the uterine wall for strong adhesion. E-cadherin, a cellular adhesion molecule, is found on both blastocyst and uterine surfaces and has been suggested to be involved in their interaction during implantation. Estradiol has been observed to reduce e-cadherin expression in hormonally sensitive tissues like the mammary glands, ovaries and uteri. Here, male-induced disruption of implantation was examined across days 2-8 of gestation. Luminal area was quantified in isolated and male-exposed females as a measure of extent of luminal closure. This area was larger in male-exposed animals. E-cadherin was found to have reduced expression on luminal epithelial cells. I suggest that the reduction in e-cadherin may lead to weaker attachment of the blastocyst to the uterine wall as well as reduced adhesion between opposing uterine walls leading to the “opening” of the uterus observed in male exposed animals. Together, these data may in part explain the blastocyst implantation failure observed in male-exposed animals during the Bruce effect.</p> / Master of Science (MSc)

Transient receptor potential vanilloid 2 channel (TRPV2) is a Ca2+-permeable ion channel that is highly expressed in leukocytes but is also present in skeletal and cardiac muscle and endocrine cells. The TRPV2 function is implicated in a number of physiological processes, including bacterial phagocytosis, pro-inflammatory cytokine production, cardiac hypertrophy, and cancer development. TRPV2 knockout mice exhibit a high incidence of perinatal mortality, arguing that the channel plays essential roles in physiology. Despite the importance of TRPV2 for normal homeostasis, the mechanisms that control TRPV2 gating in response to pharmacological agonists, heating, membrane stretch, bioactive lipids and reactive oxygen species (ROS) remain poorly understood. Here we demonstrate that TRPV2 is functionally expressed in microglia (i.e., ‘brain macrophages’) and the microglia-like BV-2 cell line, and demonstrate that the gating of an endogenous TRPV2-like conductance is positively modulated by the bacterial toxin lipopolysaccharide (LPS), which is known to cause pro-inflammatory (M1) activation and increase ROS production by NADPH oxidase. To determine how TRPV2 gating is modulated by ROS, we recorded single channel activity in inside-out patches excised from HEK-293 cells expressing GFP-rTRPV2. Unitary currents elicited by the TRPV2 agonist 2-aminophenyl borinate (2-APB) or cannabidiol (CBD) are linear in monovalent recording solutions and give rise to an estimated unitary conductance of ~100pS, which is similar to TRPV1 but significantly smaller than TRPV3. Intriguingly, we find that although TRPV2 is insensitive to ROS (in the form of exogenously applied H2O2) alone, apparent open probability is synergistically enhanced when H2O2 is applied together with CBD. We identify two intracellular Cys residues that are necessary for TRPV2 responses to H2O2 sensitivity and find that these residues are located close to one another, albeit in different subunits, in the TRPV2 structure, suggesting that ROS promote the formation of an inter-subunit disulfide bond that alters sensitivity to pharmacological agonists. We hypothesize that ROS-dependent modulation of TRPV2 activity may be an important contributor to pro-inflammatory activation of microglia underline central nervous system diseases and that TRPV2 antagonism could be a useful therapeutic strategy in the treatment of neuroinflammation.

The modulation of the electron transport during ischemia-reperfusion has been shown to be protective. We hypothesized that metformin, a Complex I inhibitor, may exhibit characteristics of a pharmacological agent that could achieve long-term therapeutic intervention against ischemia-reperfusion injury. Mitochondria were harvested from adult male mice and incubated with or without metformin at 30oC for 15 minutes, while being shaken at 300 rpm. Metformin decreased Complex I oxidative phosphorylation and Complex I activity. However, metformin also increased injury and decreased the maximum membrane potential. Even though there was a decrease in maximum membrane potential, the proton motive force (PMF) was still intact as the ADP/O ratio was not affected. In conclusion, metformin does exhibit some characteristics of a drug that could achieve long-term therapeutic benefit against ischemia-reperfusion.

Cathinones are psychostimulant compounds heavily implicated as drugs of abuse. They exert their physiological actions at the monoamine transporters, which are responsible for maintaining synaptic neurotransmitter homeostasis. Monoamine transporters produce currents during transport and have been shown to depolarize cell membranes and activate voltage-gated calcium channels in mammalian expression systems. This phenomenon is harnessed in an assay which measures these induced calcium transients, allowing for quantification of pharmacodynamic effects of compounds at monoamine transporters. It is unknown if this electrical coupling occurs in neurons, but the implications if it does are significant. In the current work, fluorescent resonance energy transfer studies of HEK cells expressing hDAT suggest that a subpopulation of monoamine transporters and calcium channels may be interacting directly. Additionally, this work presents calcium assay data comparing several novel methcathinone analogs. Of the compounds tested, a single α-methyl substituent at the α-carbon yields the greatest potency at hDAT. The implications of these results shed light on future psychostimulant studies and further define the physiological relationship of the components of a system used to study these compounds.

T2R receptors are the classical bitter taste receptors which detect and transduce bitter taste in a subset of taste receptor cells (TRCs). The TRPM5-dependent T2Rs are G-protein coupled receptors (GPCRs) and are linked to G protein, gustducin to initiate an intracellular signaling cascade for the transduction of bitter tastants. Nicotine is bitter. However, at present the transduction mechanisms for the detection of nicotine in are poorly understood. Previous studies from our laboratory using TRPM5 knockout (KO) mice demonstrated that the T2R pathway is insufficient in explaining the taste perception of nicotine. TRPM5 KO mice elicited chorda tympani (CT) taste nerve responses to nicotine, albeit significantly smaller than the wild type (WT) mice and still responded to nicotine as an aversive stimulus. Following addition of mecamylamine (Mec), a non-specific blocker of neuronal nicotinic acetylcholine receptors (nAChRs), CT responses to nicotine were partially inhibited in both WT and TRPM5 KO mice. Mec also decreases the aversive response to nicotine in both WT and TRPM5 KO mice. These studies led to the hypothesis that both a TRPM5-independent and TRPM5-dependent pathways are responsible for the detection and transduction of the bitter taste of nicotine in TRCs. The TRPM5-independent pathway most likely utilizes the nAChRs expressed in TRCs and function as bitter taste receptors for nicotine. We have subsequently demonstrated the expression of nAChRs in a subset of TRPM5-positive TRCs. However, this mechanism is not well understood in other cell types, particularly undifferentiated epithelial cells, such as HEK293T cells. The specific aims of this project were: (i) To identify which components of T2R-dependent taste reception as well as components of nAChRs are expressed in HEK293T cells; (ii) To determine if HEK293T cells co-express these components; (iii) To identify if exposure to nicotine modulates the expression of T2R and nAChR dependent components in HEK293T cells; (iv) To determine if TRCs express functional nAChR ion channels; and (v) To determine if nAChRs are involved in the release of neuropeptides, such as brain-derived neurotrophic factor (BDNF) in HEK293T cells. The data obtained in HEK293T cells was compared with parallel studies on adult cultured human fungiform taste cells (HBO) done independently by Dr. Jie Qian, a postdoctoral fellow in Dr. Vijay Lyall’s lab. The results of combined studies on HBO and HEK293T cells indicates that TRPM5-positive cells also co-express ionotropic nAChRs, comprising a and β subunits. The nAChRs are capable of forming ion pores and when stimulated by nicotine and create a parallel TRPM5-independent pathway for the detection of nicotine. Using molecular and immunocytochemical techniques, our results demonstrate that mRNAs and proteins for bitter taste receptors and downstream intracellular signaling components as well as subunits necessary for the formation of nAChRs are expressed in HBO and HEK293T cells. Results demonstrated that TRPM5-positive HEK293T cells co-expressed nAChR subunits throughout the entire population. Nicotine increased the influx of Ca2+ in a dose dependent manner, which was somewhat reduced by the addition of TRPM5 blocker, triphenylphosphine oxide (TPPO). Both mRNA and protein expression were altered in a biphasic pattern with a maximum increased observed at 0.5 µM nicotine with a decrease in expression at higher concentrations. The synthesis of neurotrophic factor BDNF, required for maturation of taste bud cells and their innervating nerves, increased in HEK293T cells exposed to nicotine, however, nicotine did not trigger the release of BDNF. These results were then compared and contrasted with HBO cells to better understand the comparative effects of nicotine on both undifferentiated and differentiated cells. The data on HBO cells is presented in the Appendix.

Intertidal mussels regularly experience emersion-induced anoxia, in contrast to normoxic conditions experienced during submersion. We therefore hypothesized that acclimation to a tidal rhythm, as opposed to a rhythm of constant submersion, preconditions the proteome of the California mussel, Mytilus californianus, to respond differently to emersion-induced anoxia. Following acclimation, mussels either continued to receive the acclimation conditions (control) or were exposed to 100% nitrogengas (anoxia) during aerial emersion. We collected gill tissue for subsequent analysis of protein abundance with 2D gel electrophoresis and protein identification with tandem mass spectrometry. Relative to subtidally-acclimated mussels, tidally-acclimated mussels showed a greater propensity to respond to distrupted protein homeostasis during emersion through higher levels of several small heat shock protein isoforms, while they showed lower levels of several chaperones involved in redox-sensitive protein maturation in the endoplasmic reticulum during acute anoxia. Several metabolic proteins showed elevated levels in tidally-acclimated mussels, suggesting a compensatory response to reduced feeding times. However, changes in the abundance of several tricarboxylic acid cycle enzymes (e.g. aconitase, succinate dehydrogenase) suggest that tidally-acclimated mussels are also primed to sense reactive oxygen species (ROS) and limit their production, respectively. These findings are further supported by higher abundances of several aldehyde dehydrogenases and thioredoxin peroxidase, which function as scavengers of aldehydes and ROS, common products of lipid peroxidation. Finally, tidally-acclimated mussels are also more responsive to changes in cytoskeletal and vesicular trafficking dynamics in response to acute anoxia. Together, our analysis showed that proteostasis, energy metabolism, oxidative stress and cytoskeletal and trafficking processes are all involved in priming tidally-acclimated mussels to respond more dynamically to acute emersion-induced anoxia in Mytilus gill.