Browsing by Author "Day, Richard"

Now showing 1 - 8 of 8
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    Endoplasmic reticulum calcium dynamics and insulin secretion in pancreatic β cells
    (2017-08-15) Yamamoto, Wataru; Evans-Molina, Carmella; Day, Richard; Sturek, Michael; Obukhov, Alexander; Wek, Ronald
    Under normal conditions, ER Ca2+ levels are estimated to be at least three orders of magnitude higher than intracellular Ca2+. This steep Ca2+ concentration gradient is maintained by the balance of Ca2+ uptake into the ER via the sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) pump and ER Ca2+ release through Ryanodine receptors (RyR) and Inositol 1,4,5-triphosphate (IP3) receptors (IP3R). Emerging data suggest that alterations in β cell ER Ca2+ levels lead to diminished insulin secretion and reduced β cell survival in both type 1 and type 2 diabetes. However, the mechanisms leading to β cell ER Ca2+ loss remain incompletely understood, and a specific role for either RyR or IP3R dysfunction in diabetes has been largely untested. To this end, we applied intracellular and ER-Ca2+ imaging techniques in INS-1 β cells and isolated mouse and human islets to define whether RyR or IP3R activity were altered under diabetogenic conditions. Results revealed preferential alterations in RyR function in response to ER stress, while pro-inflammatory cytokine stress primarily impacted IP3R activity. Consistent with this, pharmacological inhibition of RyR and IP3Rs prevented ER Ca2+ loss under ER and pro-inflammatory stress, respectively. However, RyR inhibition was unique in its ability to prevent β cell death, delayed initiation of the unfolded protein response (UPR), and dysfunctional glucose-induced Ca2+ oscillations in tunicamycin treated INS-1 β cells and islets from Akita mice. Monitoring at the single cell level revealed that ER stress acutely increased intracellular Ca2+ transients and this was dependent on both ER Ca2+ leak from the RyR and plasma membrane depolarization, suggesting ER Ca2+ dynamics regulate cellular excitability. Collectively, our findings suggest that ER-stress induced RyR dysfunction regulates β cell ER Ca2+ dynamics, propagation of the UPR, insulin secretion, and cell survival. These data indicate that RyR-mediated loss of ER Ca2+ and β cell hyperexcitability may be early pathogenic events in diabetes.
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    IUPUI Imaging Research Initiative
    (Office of the Vice Chancellor for Research, 2013-04-05) Holland, Mark; Barnett, William; Burr, David B.; Day, Richard; Du, Eliza Y.; Gattone, Vincent, III; Fletcher, James; Johnson, Daniel P.; Long, Eric; Molitoris, Bruce A.; Palakal, Mathew; Salama, Paul; Sturek, Michael; Hutchins, Gary D.
    Imaging has become an essential research tool in a majority of scientific disciplines. The IUPUI Imaging Research Initiative (IRI) has been established to bring together researcher investigators who develop novel imaging technologies with those who utilize imaging tools to advance their research with the primary objective of building a large scale imaging research infrastructure at IUPUI. An Imaging Research Council has been created to establish priorities for the IRI and help guide the development of an IUPUI research imaging infrastructure and sustainable research funding base. The specific goals of the council include: • To encourage and coordinate collaboration among IUPUI researchers from different disciplines • To provide advice and guidance in the realization of highly competitive large grant proposals that will support and grow the IUPUI imaging efforts into major nationally and internationally recognized programs • To develop a strategic plan that will enable IUPUI to become nationally and internationally known as the place for imaging research and its applications • To determine strategic areas of strength and growth • To determine available and needed resources • To determine strategic external partnerships
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    Mechanisms of HIV-Nef Induced Endothelial Cell Stress: Implications of HIV-Nef Protein Persistence in Aviremic HIV Patients
    (2019-05) Chelvanambi, Sarvesh; Clauss, Matthias; Basile, David; Day, Richard; Yu, Andy
    HIV-associated cardio-pulmonary vascular pathologies such as coronary artery disease, pulmonary hypertension and emphysema remain a major issue in the HIVinfected population even in the era of antiretroviral therapy (ART). The continued production of HIV encoded pro-apoptotic protein, such as Nef in latently HIV-infected cells is a possible mechanism for vascular dysfunction underlying these diseases. HIVNef persists in two compartments in these patients: (i) extracellular vesicles (EV) of plasma and bronchoalveolar lavage (BAL) fluid and (ii) PBMC and BAL derived cells. Here I demonstrate that the presence of HIV-Nef protein in cells and EV is capable of stressing endothelial cells by inducing ROS production leading to endothelial cell apoptosis. HIV-Nef protein hijacks host cell signaling by interacting with small GTP binding protein Rac1 which activates PAK2 to promote the release of pro-apoptotic cargo containing EV and surface expression of pro-apoptotic protein Endothelial Monocyte Activating Polypeptide II (EMAPII). Using this mechanism, Nef protein robustly induces apoptosis in Human Coronary Artery Endothelial Cells and Human Lung microvascular endothelial cells. Endothelial specific expression of HIV-Nef protein in transgenic mice was sufficient to induce vascular pathologies as evidenced by impaired endothelium mediated vasodilation of the aorta and vascular remodeling and emphysema like alveolar rarefaction in the lung. Furthermore, EV isolated from HIV patients on ART was capable of inducing endothelial apoptosis in a Nef dependent fashion. Of therapeutic interest, EMAPII neutralizing antibodies to block EMAPII mediated apoptosis and statin treatment to ameliorate Nef induced Rac1 signaling was capable of blocking Nef induced endothelial stress in both in vivo and in vitro. In conclusion, HIV-Nef protein uses a Rac1-Pak2 signaling axis to promote its dissemination in EV, which in turn induces endothelial cell stress after its uptake.
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    Quantitative Kinetic Models from Intravital Microscopy: A Case Study Using Hepatic Transport
    (ACS, 2019-08-29) Tsekouras, Konstantinos; Day, Richard; Dunn, Kenneth W.; Pressé, Steve; Physics, School of Science
    The liver performs critical physiological functions, including metabolizing and removing substances, such as toxins and drugs, from the bloodstream. Hepatotoxicity itself is intimately linked to abnormal hepatic transport, and hepatotoxicity remains the primary reason drugs in development fail and approved drugs are withdrawn from the market. For this reason, we propose to analyze, across liver compartments, the transport kinetics of fluorescein-a fluorescent marker used as a proxy for drug molecules-using intravital microscopy data. To resolve the transport kinetics quantitatively from fluorescence data, we account for the effect that different liver compartments (with different chemical properties) have on fluorescein's emission rate. To do so, we develop ordinary differential equation transport models from the data where the kinetics is related to the observable fluorescence levels by "measurement parameters" that vary across different liver compartments. On account of the steep non-linearities in the kinetics and stochasticity inherent to the model, we infer kinetic and measurement parameters by generalizing the method of parameter cascades. For this application, the method of parameter cascades ensures fast and precise parameter estimates from noisy time traces.
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    Regulation of endoplasmic reticulum calcium homeostasis in pancreatic β cells
    (2016-06-21) Tong, Xin; Evans-Molina, Carmella; Day, Richard; Tune, Johnathan; Fueger, Patrick T.; Dong, X. Charlie
    Diabetes mellitus is a group of metabolic diseases characterized by disordered insulin secretion from the pancreatic β cell and chronic hyperglycemia. In order to maintain adequate levels of insulin secretion, the β cell relies on a highly developed and active endoplasmic reticulum (ER). Calcium localized in this compartment serves as a cofactor for key proteins and enzymes involved in insulin production and maturation and is critical for ER health and function. The ER Ca2+ pool is maintained largely through activity of the sarco-endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) pump, which pumps two Ca2+ ions into the ER during each catalytic cycle. The goal of our research is to understand the molecular mechanisms through which SERCA2 maintains β cell function and whole body glucose metabolism. Our previous work has revealed marked dysregulation of β cell SERCA2 expression and activity under diabetic conditions. Using a mixture of pro-inflammatory cytokines to model the diabetic milieu, we found that SERCA2 activity and protein stability were decreased through nitric oxide and AMP-activated protein kinase (AMPK)mediated signaling pathways. Moreover, SERCA2 expression, intracellular Ca2+ storage, and β cell death under diabetic conditions were rescued by pharmacologic or genetic inhibition of AMPK. These findings provided novel insight into pathways leading to altered β cell Ca2+ homeostasis and reduced β cell survival in diabetes. To next define the role of SERCA2 in the regulation of whole body glucose homeostasis, SERCA2 heterozygous mice (S2HET) were challenged with high fat diet (HFD). Compare to wild-type controls, S2HET mice had lower serum insulin and significantly reduced glucose tolerance with similar adiposity and systemic and tissue specific insulin sensitivity, suggesting an impairment in insulin secretion rather than insulin action. Consistent with this, S2HET mice exhibited reduced β cell mass, decreased β cell proliferation, increased ER stress, and impaired insulin production and processing. Furthermore, S2HET islets displayed impaired cytosolic Ca2+ oscillations and reduced glucose-stimulated insulin secretion, while a small molecule SERCA2 activator was able to rescue these defects. In aggregate, these data suggest a critical role for SERCA2 and the maintenance of ER Ca2+ stores in the β cell compensatory response to diet induced obesity.
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    Resurgent sodicum current modulation by auxiliary subunits in dorsal root ganglia neurons and potential implications in pain pathologies
    (2016-04-11) Barbosa Nuñez, Cindy Marie; Cummins, Theodore R.; Fehrenbacher, Jill C.; Hudmon, Andy; Nicol, Grant D.; Day, Richard
    Increased electrical activity in peripheral sensory neurons contributes to pain. A unique type of sodium current, fast resurgent current, is proposed to increase nerve activity and has been associated with pain pathologies. While sodium channel isoform Nav1.6 has been identified as the main carrier of fast resurgent currents, our understanding of how resurgent currents are modulated in sensory neurons is fairly limited. Thus the goal of this dissertation was to identify resurgent current modulators. In particular, we focused on sodium channel beta subunits (Navβs) and fibroblast growth factor homologous factors (FHFs) in dorsal root ganglion (DRG) neurons. We hypothesized that Navβ4 and FHF2B act as positive regulators by mediating resurgent currents and modulating Nav1.6 inactivation, respectively. In contrast, we hypothesized FHF2A negatively regulates resurgent current by increasing the probability of channels in inactivated states. Thus, the aims of this dissertation were to 1) determine if Navβ4 regulates fast resurgent currents in DRG neurons, 2) examine the effects of Navβ4 knockdown on resurgent currents, firing frequency and pain associated behavior in an inflammatory pain model and 3) determine if FHF2A and FHF2B functionally regulate Nav1.6 currents, including resurgent currents in DRG neurons. To examine the aims, we used biochemical, electrophysiological and behavioral assays. Our results suggest that Navβ4 is a positive regulator of resurgent currents: in particular, the C-terminus likely mediates these currents. Localized knockdown of Navβ4 decreased inflammation-induced enhancement of resurgent currents and neuronal excitability, and prevented the development of persistent pain associated behavior in an inflammatory pain model. FHF2B increased resurgent currents and delayed inactivation. In contrast, FHF2A limited resurgent currents; an effect that is mainly contributed by FHF2A's N-terminus activity that increased accumulation of channels in inactivated states. Interestingly, in an inflammatory pain model FHF2B was upregulated and FHFA isoforms were downregulated. Together these results suggest that FHF2A/B modulation might contribute to enhanced resurgent currents and increased neuronal excitability observed in the inflammatory pain model. Overall, our work has identified three resurgent current modulators FHF2A, FHF2B and Navβ4. Manipulation of these proteins or their activity might result in novel strategies for the study and treatment of pain.
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    The Role of Mammalian Lipid Transport Protein ORP1 During Coxiella Burnetii Infection
    (2022-05) Schuler, Baleigh Elizabeth; Gilk, Stacey D.; Arrizabalaga, Gustavo; Spinola, Stanley; Harrington, Maureen; Day, Richard
    Coxiella burnetii is an intracellular bacterium that causes the human disease Q fever. C. burnetii is transmitted from infected animals to humans through inhalation of infectious droplets. Acute Q fever is a flu-like illness lasting 10-14 days. Patients often have respiratory symptoms and present with pneumonia. Patients with suppressed immune systems or valvular heart disease can develop chronic Q fever, which causes endocarditis and vasculitis long after initial infection. Chronic Q fever is difficult to treat, and if untreated, is typically fatal. Currently, the United States lacks any vaccine for Q fever. In order to better prevent and treat this disease, it is important to understand how C. burnetii interacts with mammalian cells. Within the host cell, C. burnetii forms a large, acidic Coxiella-containing vacuole (CCV) and uses a Type 4B secretion system (T4SS) to secrete effector proteins into the host cell cytoplasm. While the CCV membrane is rich in sterols, cholesterol accumulation in the CCV is bacteriolytic, suggesting that C. burnetii regulation of lipid transport is critical for infection. The mammalian lipid transport protein ORP1L localizes to the CCV membrane and mediates CCV-ER membrane contact sites. ORP1L functions in lipid transport, including cholesterol efflux from late endosomes/lysosomes. Its sister isoform ORP1S binds cholesterol but localizes to the cytoplasm and nucleus. In ORP1- null cells, we found that CCVs were smaller than in wildtype cells, highlighting the importance of ORP1 in CCV development. CCVs in ORP1-null cells had higher cholesterol content than CCVs in wildtype cells, suggesting ORP1 functions in cholesterol efflux from the CCV. ORP1-null MH-S cells do not accumulate lipid droplets upon C. burnetii infection, supporting our hypothesis that ORP1 promotes cholesterol transfer from the CCV to the ER, as lipid droplets form from neutral lipids in the ER. While the absence of ORP1 led to a C. burnetii growth defect in MH-S cells, there was no growth defect in HeLa cells. Together, our data demonstrate that C. burnetii uses the host sterol transport protein ORP1 to promote CCV development, potentially by using ORP1 to facilitate cholesterol efflux from the CCV to diminish the bacteriolytic effects of cholesterol.
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    Two-Photon Intravital Fluorescence Lifetime Imaging of the Kidney Reveals Cell-Type Specific Metabolic Signatures
    (American Society of Nephrology, 2017-08) Hato, Takashi; Winfree, Seth; Day, Richard; Sandoval, Ruben M.; Molitoris, Bruce A.; Yoder, Mervin C.; Wiggins, Roger C.; Zheng, Yi; Dunn, Kenneth W.; Dagher, Pierre C.; Medicine, School of Medicine
    In the live animal, tissue autofluorescence arises from a number of biologically important metabolites, such as the reduced form of nicotinamide adenine dinucleotide. Because autofluorescence changes with metabolic state, it can be harnessed as a label-free imaging tool with which to study metabolism in vivo Here, we used the combination of intravital two-photon microscopy and frequency-domain fluorescence lifetime imaging microscopy (FLIM) to map cell-specific metabolic signatures in the kidneys of live animals. The FLIM images are analyzed using the phasor approach, which requires no prior knowledge of metabolite species and can provide unbiased metabolic fingerprints for each pixel of the lifetime image. Intravital FLIM revealed the metabolic signatures of S1 and S2 proximal tubules to be distinct and resolvable at the subcellular level. Notably, S1 and distal tubules exhibited similar metabolic profiles despite apparent differences in morphology and autofluorescence emission with traditional two-photon microscopy. Time-lapse imaging revealed dynamic changes in the metabolic profiles of the interstitium, urinary lumen, and glomerulus-areas that are not resolved by traditional intensity-based two-photon microscopy. Finally, using a model of endotoxemia, we present examples of the way in which intravital FLIM can be applied to study kidney diseases and metabolism. In conclusion, intravital FLIM of intrinsic metabolites is a bias-free approach with which to characterize and monitor metabolism in vivo, and offers the unique opportunity to uncover dynamic metabolic changes in living animals with subcellular resolution.