Type 2 diabetes mellitus (T2DM) carries a high prevalence in the United States and worldwide. Therefore, the number of medication classes being developed and studied has grown. The individualized management of diabetes is accomplished by evaluating a medication's efficacy, safety, and cost, along with the patient's preference and tolerance to the medication. Sodium glucose co-transporter 2 inhibitors are a new therapeutic class indicated for the treatment of diabetes and have a unique mechanism of action, independent of beta-cell function. The first agent approved by the Food and Drug Administration (FDA) was canagliflozin in March 2013. Two agents - dapagliflozin and empagliflozin - were FDA-approved in January and July 2014, respectively. A clear understanding of the new class is needed to identify its appropriate use in clinical practice. Members of the American College of Clinical Pharmacy Endocrine and Metabolism Practice and Research Network reviewed available literature regarding this therapeutic class. The article addresses the advantages, disadvantages, emerging role, and patient education for sodium glucose co-transporter 2 inhibitors. Key limitations for this article include limited access to clinical trial data not published by the pharmaceutical company and limited data on products produced outside the United States.

Dopamine β-hydroxylase (DBH) and norepinephrine (NE) transporter (NET) are the noradrenergic phenotypes for their functional importance to noradrenergic neurons. It is known that in vivo N-(2-chloroethyl)-N-ethyl-2- bromobenzylamine (DSP4) treatment induces degeneration of noradrenergic terminals by interacting with NET and depleting intracellular NE. However, DSP4's precise mechanism of action remains unclear. In this study various biochemical approaches were employed to test the hypothesis that DSP4 down-regulates the expression of DBH and NET, and to determine molecular mechanisms that may be involved. The results showed that treatment of SH-SY5Y neuroblastoma cells with DSP4 significantly decreased mRNA and protein levels of DBH and NET. DSP4-induced reduction of DBH mRNA and proteins, as well as NET proteins showed a time- and concentration-dependent manner. Flow cytometric analysis demonstrated that DSP4-treated cells were arrested predominantly in the S-phase, which was reversible. The arrest was confirmed by several DNA damage response markers (phosphorylation of H2AX and p53), suggesting that DSP4 causes replication stress which triggers cell cycle arrest via the S-phase checkpoints. Moreover, the comet assay verified that DSP4 induced single-strand DNA breaks. In summary, the present study demonstrated that DSP4 down-regulates the noradrenergic phenotypes, which may be mediated by its actions on DNA replication, leading to replication stress and cell cycle arrest. These action mechanisms of DSP4 may account for its degenerative consequence after systematic administration for animal models.

Chronic stress and dysfunction of the serotonergic system in the brain have been considered two of the major risks for development of depression. In this study, adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD). To mimic stressful conditions, some rats were not exposed to CSD, but instead treated with corticosterone (CORT) in oral solution while maintained in their home cage. Protein levels of the serotonin transporter (SERT) in the dorsal raphe nucleus (DRN), hippocampus, frontal cortex, and amygdala were examined by Western blotting or immunofluorescence staining. The results showed that CSD up-regulated SERT protein levels in the DRN, hippocampus, frontal cortex, and amygdala regions. This up-regulation was abolished or prevented by adrenalectomy, or treatment with antagonists of corticosteroid receptors mifepristone and spironolactone, alone or in combination. Similarly, up-regulated SERT protein levels in these brain regions were also observed in rats treated with oral CORT ingestion, which was analogously prevented by treatment with mifepristone and spironolactone. Furthermore, both CSD- and CORT-induced up-regulation of SERT protein levels in the DRN and three brain regions were attenuated by simultaneous treatment with fluoxetine, an antidepressant that specifically inhibits serotonin reuptake. The results indicate that up-regulation in SERT protein levels in the DRN and forebrain limbic structures caused by CSD regimen was mainly motivated by CORT through corticosteroid receptors. The present findings demonstrate that chronic stress is closely correlated with the serotonergic system by acting on the regulation of the SERT expression in the DRN and its projection regions, which may contribute to the development of depression. Chronic stress and dysfunction of the serotonergic system are etiologically related to depression. In an attempt to explore their interaction, we found that chronic social defeat upregulated expression of serotonin transporter in the DRN and the projection regions, which may induce an alteration of serotonin transformation in the brain. This interaction may account for the development of this disease.

Stress has been reported to activate the locus coeruleus (LC)-noradrenergic system. However, the molecular link between chronic stress and noradrenergic neurons remains to be elucidated. In the present study adult Fischer 344 rats were subjected to a regimen of chronic social defeat (CSD) for 4 weeks. Measurements by in situ hybridization and Western blotting showed that CSD significantly increased mRNA and protein levels of the norepinephrine transporter (NET) in the LC region and NET protein levels in the hippocampus, frontal cortex and amygdala. CSD-induced increases in NET expression were abolished by adrenalectomy or treatment with corticosteroid receptor antagonists, suggesting the involvement of corticosterone and corticosteroid receptors in this upregulation. Furthermore, protein levels of protein kinase A (PKA), protein kinase C (PKC), and phosphorylated cAMP-response element binding (pCREB) protein were significantly reduced in the LC and its terminal regions by the CSD paradigm. Similarly, these reduced protein levels caused by CSD were prevented by adrenalectomy. However, effects of corticosteroid receptor antagonists on CSD-induced down-regulation of PKA, PKC, and pCREB proteins were not consistent. While mifeprestone and spironolactone, either alone or in combination, totally abrogate CSD effects on these protein levels of PKA, PKC and pCREB in the LC and those in the hippocampus, frontal cortex and amygdala, their effects on PKA and PKC in the hippocampus, frontal cortex and amygdala were region-dependent. The present findings indicate a correlation between chronic stress and activation of the noradrenergic system. This correlation and CSD-induced alteration in signal transduction molecules may account for their critical effects on the development of symptoms of major depression.

Previously, we have found glucocorticoids up-regulate norepinephrine (NE) transporter (NET) expression in vitro. However, the underlying transcriptional mechanism is poorly understood. In this study, the role of glucocorticoids on the transcriptional regulation of NET was investigated. Exposure of neuroblastoma SK-N-BE(2)M17 cells to dexamethasone (Dex) significantly increased NET mRNA and protein levels in a time- and dose-dependent manner. This effect was attenuated by glucocorticoid receptor (GR) antagonist mifepristone, suggesting that up-regulation of NET by Dex was mediated by the GR. In reporter gene assays, exposure of cells to Dex resulted in dose-dependent increases of luciferase activity that were also prevented by mifepristone. Serial deletions of the NET promoter delineated Dex-responsiveness to a -301 to -148 bp region containing a CCAAT/enhancer binding protein-β (C/EBP-β) response element. Co-immunoprecipitation experiments demonstrated that Dex treatment caused the interaction of the GR with C/EBP-β. Chromatin immunoprecipitation (ChIP) assay revealed that Dex exposure resulted in binding of both GR and C/EBP-β to the NET promoter. Further experiments showed that mutation of the C/EBP-β response element abrogated C/EBP-β- and GR-mediated transactivation of NET. These findings demonstrate that Dex-induced increase in NET expression is mediated by the GR via a non-conventional transcriptional mechanism involving interaction of C/EBP-β with a C/EBP-β response element.

Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-L-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.

Dopamine (DA) is central to movement, reward, learning, sleep, and anxiety. The dopamine transporter (DAT) spatially and temporally controls extracellular dopamine levels by taking DA back up into the presynaptic neuron. Multiple lines of evidence from studies using pharmacological DAT blockade or genetic DAT deletion demonstrate that DAT availability at the plasma membrane is required for maintenance of homeostatic DA levels and DA tone. Therefore, intrinsic mechanisms that regulate the transporter’s availability at the plasma membrane may directly impact downstream DA signaling cascades and DA-dependent behavior. Acute, regulated DAT internalization in response to protein kinase C (PKC) activation has been well documented, however the physiological importance of this mechanism remains untested. Due to DAT’s critical role in regulating DA levels, It is essential to understand mechanisms that acutely regulate DAT function and surface expression, and further, how these mechanisms contribute to DA related behaviors. DAT has intracellular amino and carboxy termini, which contain domains for transporter phosphorylation, recruitment to and from the plasma membrane, and sites for protein-protein interactions. To test whether these domains work synergistically for DAT function and regulated endocytosis I made DAT/SERT chimeras, in which I switched DAT’s amino, carboxy, or both termini with that of SERT, a homologous transporter with highly divergent intracellular domains. I demonstrated that DAT’s amino and carboxy termini synergistically contribute to substrate and select competitive inhibitor affinities. Additionally, I demonstrated that the amino terminus is required for PKC-stimulated DAT endocytosis, and that both N- and C-termini are required for downstream Ack1-dependent regulation of DAT endocytosis. To test the physiological importance of PKC-stimulated DAT endocytosis in vivo, I knocked down Rin, a GTPase required for PKC-stimulated DAT trafficking, in mouse DA neurons. This study was the first to achieve AAV-mediated, conditional, and inducible gene silencing in neurons. Using this AAV approach, I demonstrated a critical role for Rin GTPase signaling and DAT trafficking in both anxiety and locomotor response to cocaine. Taken together, this thesis 1) adds to the understanding of DAT functional and endocytic mechanisms and 2) is the first to report the physiological impact of Rin signaling and DAT endocytosis in DA behavior.

Proton-coupled and sodium-dependent monocarboxylate transporters are encoded by the SLC16A and SLC5A gene family of solute carriers, and are responsible for the transport of essential nutrients such as L-lactate, pyruvate, and ketone bodies. Basigin, or CD147, acts as an ancillary protein for MCT1 and MCT4, and is involved in membrane surface expression of transporters. MCT's are also involved in the shuttling of monocarboxylic xenobiotics across cell membranes, including the drugs valproate and gamma hydroxybutyrate. MCT’s are also important for normal mammalian development, particularly during embryogenesis and early neonatal life. Previous studies have shown that ketogenic diets increase MCT expression in the brain, and the obesity biomarker leptin increases MCT1 and CD147 expression and colocalization in colonocytes. Clinical studies in post-mortem tissue demonstrated that hepatic MCT1 expression changes nonlinearly from birth to adulthood. We hypothesize that age and high fat dietary intake regulate monocarboxylate transporter and ancillary protein expression in the liver, and other organs of drug disposition during childhood obesity. The purpose of this study was to elucidate just how diet and ontogeny regulate MCT1, MCT4, CD147, and SMCT1 mRNA and protein expression in the liver, kidney, and ileum. Timed-pregnant rats were fed either normal or high fat diet, and tissue was harvested from the progeny of both cohorts at predetermined postnatal timepoints. Serum leptin levels were measured, and MCT1, MCT4, CD147, and SMCT1 transcripts were evaluated using real time quantitative PCR. Whole cell and total membrane proteins were extracted and transporter expression was analyzed via western blot. In summary, we have demonstrated age, diet, and sex dependent regulation of MCT1, MCT4, CD147, and SMCT1 expression in the liver, kidneys, and intestine, and that these effects are tissue specific. Pediatric drug-dosing is both a pressing and understudied clinical field, with the possibility of altered pharmacokinetics in obese children. Changes in hepatic, renal, and intestinal monocarboxylate transporter expressions during mammalian development may affect functional activity of these transporters and lead to altered metabolism and drug disposition. Further studies of this animal model can shine new light on the dynamic and highly-variable nature of drug pharmacokinetics in pediatric obesity.

Aim: The general aim of this research is development and evaluation of novel methods for predicting active transport over the human blood-brain-barrier (BBB), while this project specifically aims to i) review the literature and select suitable methods and substrates, ii) develop models for determining in vitro kinetic properties of selected compounds, analyze the in vitro data using the developed models and to use Maximum Transport Activity (MTA) approach (Karlgren et al., 2012), iii) perform Physiology Based Pharmacokinetic (PBPK) modelling and compare to in vivo literature data. Background: Drug permeation to the brain through blood circulation is primarily limited by blood-brain barrier (BBB), due to existence of tight junctions in endothelial cells of blood vessels as well as active efflux and influx transporters in the barrier. Toxicity and CNS related side effects can be caused by peripheral targeted drugs crossing BBB. Hence, prediction of BBB permeability and estimation of drug concentration in the brain tissue are challenging in drug discovery. To resolve this, estimating the human BBB permeability using improved in vitro and in silico predictive models can be a facilitator. Methods: In vitro data provided by the Drug Delivery research group was used to develop in vitro predictive models for BBB penetration of Verapamil, Risperidone, and Prazosin using R-studio 1.2.5. The MTA approach was adjusted for extrapolation of BBB in vitro transporter activity to in vivo condition. For PBPK modelling, we took advantage of PK-Sim® to simulate drug disposition and time profile of Risperidone in human and animal species. Results: It was shown that MDR1 is the major transporter for efflux transport of Prazosin and Risperidone in brain while both BCRP and MDR1 have similar impact on transport of Verapamil. Furthermore, it was presented in PBPK models that the predicted brain concentration of Risperidone increases in rat and nonhuman primate (NHP) when MDR1 And BCRP are knocked out while the brain concentration of Risperidone in dog is not affected by expression level of the efflux transporters. Conclusion: Both MDR1 and BCRP are contributing in efflux transport of Verapamil, Risperidone, and Prazosin across the BBB. Additionally, expression of the efflux transporters shown to have an impact on brain exposure of Risperidone in animal PBPK models.

Many omics-level studies have been undertaken on Aiptasia, however, our understanding of the genes and processes associated with symbiosis regulation and maintenance is still limited. To gain deeper insights into the molecular processes underlying this association, we investigated this relationship using multipronged approaches combining next generation sequencing with metabolomics and immunohistochemistry. We identified 731 high-confident symbiosis-associated genes using meta-analysis. Coupled with metabolomic profiling, we exposed that symbiont-derived carbon enables host recycling of ammonium into nonessential amino acids, which may serve as a regulatory mechanism to control symbiont growth through a carbon-dependent negative feedback of nitrogen availability to the symbiont. We then characterized two symbiosis-associated ammonium transporters (AMTs). Both of the proteins exhibit gastrodermis-specific localization in symbiotic anemones. Their tissuespecific localization consistent with the higher ammonium assimilation rate in gastrodermis of symbiotic Aiptasia as shown by 15N labeling and nanoscale secondary ion mass spectrometry (NanoSIMS). Inspired by the tissue-specific localization of AMTs, we investigated spatial expression of genes in Aiptasia. Our results suggested that symbiosis with Symbiodiniaceae is the main driver for transcriptional changes in Aiptasia. We focused on the phagosome-associated genes and identified several key factors involved in phagocytosis and the formation of symbiosome. Our study provided the first insights into the tissue specific complexity of gene expression in Aiptasia. To investigate symbiosis-induced response in symbiont and to find further evidence for the hypotheses generated from our host-focused analyses, we explored the growth and gene expression changes of Symbiodiniaceae in response to the limitations of three essential nutrients: nitrogen, phosphate, and iron, respectively. Comparisons of the expression patterns of in hospite Symbiodiniaceae to these nutrient limiting conditions showed a strong and significant correlation of gene expression profiles to the nitrogen-limited culture condition. This confirmed the nitrogen-limited growing condition of Symbiodiniaceae in hospite, and further supported our hypothesis that the host limits the availability of nitrogen, possibly to regulate symbiont cell density. In summary, we investigated different molecular aspects of symbiosis from both the host’s and symbiont’s perspective. This dissertation provides novel insights into the function of nitrogen, and the potential underlying molecular mechanisms, in the metabolic interactions between Aiptasia and Symbiodiniaceae.