Mark A. Rizzo, Ph.D. - US grants

DESCRIPTION (provided by applicant): The glucose phosphorylating enzyme, glucokinase (GK),acts as the glucose sensor in glucose stimulated insulin release from the pancreatic B-islet cells. Although the enzymatic characteristics of GK have been characterized extensively, the mechanism of GK activation has remained elusive. Recently, electron micrographs of GK distribution in pancreatic islets have revealed that GK is located on insulin secretory granules. Given that GK activity in other tissues is regulated through changes in localization, it is proposed that GK association with insulin secretory granules may be directly related to GK activity. In order to test this hypothesis, the dynamics of GK association with insulin granules will be assessed in cells expressing a fluorescently tagged GK chimera. Quantitative bleaching studies will be used to characterize the association of GK with insulin granules and specifically examine the role of glucose-GK interactions in triggering GK redistribution to the cytoplasm. The protein domains in GK that participate in targeting OK to insulin granules will be identified by deletion mutagenesis. Identification of the GK targeting domain will allow specific disruption of GK targeting by site-directed mutagenesis and examination of the importance of GK translocation to glucose metabolism in the B-cell. These studies will further our understanding of GK regulation in the pancreatic islet and the role of GK compartmentalization in glucose stimulated insulin secretion.

[unreadable] DESCRIPTION (provided by applicant): Insulin is secreted from the pancreatic islet beta cells in response to elevated blood glucose levels and functions to control systemic metabolism and growth. Dysfunction of glucose-stimulated insulin secretion (GSIS) is a hallmark of both Type I and Type II diabetes. Thus, an understanding of the molecular mechanisms that participate in regulating GSIS is essential to understanding both systemic control of metabolic homeostasis as well as the origin and treatment of diabetic disease states. Our recent work has provided evidence supporting a positive regulatory effect of secreted insulin on GSIS. Insulin treatment of cultured beta cells results in activation of the glucose-sensing enzyme, glucokinase. This process is mediated by production of nitric oxide on secretory granules by nitric oxide synthase. However, the signaling pathway leading to the activation of this enzyme is not understood. In addition, it is unclear whether insulin stimulates a similar auto feedback pathway in the crowd of beta cells and other cell types as occurs in an islet and confers a similar regulatory potential on GSIS through the activation of nitric oxide synthase and glucokinase. Based on our preliminary studies, we hypothesize that insulin activates nitric oxide synthase by stimulating the release of calcium ions from intracellular stores and that this process occurs in pancreatic islets. Therefore, the Specific Aims of this study are to 1) determine the molecular signaling events leading from activated insulin receptors to activation of NOS on secretory granules and [unreadable] 2) determine whether insulin treatment activates nitric oxide synthase and glucokinase in living pancreatic islets. To accomplish these goals, we have developed genetically-encoded biosensors targeted to specific cellular compartments in order to measure insulin signal transduction in living cells. This will allow direct examination of nitric oxide production on individual granules in living beta cells by fluorescence imaging techniques. In addition, a lentiviral vector system will be developed in order to specifically deliver the biosensor constructs to beta cells in isolated pancreatic islets. These studies will further understanding of the physiological regulation of GSIS and may identify novel targets for pharmacological intervention of Type I and Type II diabetes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Progression of type 2 diabetes mellitus tracks with the failure of pancreatic beta cells to compensate for peripheral insulin resistance. Loss of glucose sensitivity, particularly during the sharp rise in blood glucose that follows a meal, is strongly associated with peripheral tissue damage during diabetes. Yet the molecular mechanisms underlying defects in beta-cell glucose sensing during type 2 diabetes are not well understood. This proposal seeks critical information regarding the regulation of glucokinase, which is the glucose sensing protein in insulin-secreting beta cells. Work in the previous funding period focused on hormonal activation of glucokinase through chemical reaction with nitric oxide. These studies revealed important new connections between defects in glucokinase regulation and a genetic form of human diabetes. Even so, major questions remain concerning the mechanism of glucokinase activation and the impact of diabetes-related cell stress on glucokinase function. Three aims are proposed. Aim 1 will utilize a newly developed glucokinase biosensor to reveal the connection between cellular activation by nitric oxide and the underlying biochemical states suggested by its molecular structure. Aim 2 will focus on understanding the molecular mechanism that leads to glucokinase association with nitric oxide synthase, which is a critical interaction required for cellular activation of glucokinase. Experiments in this aim wil also reveal whether defects in glucokinase regulation may explain the association of NOS1AP gene mutations with human diabetes. Aim 3 will focus on the impact of diabetes-related cell stress on glucokinase activation. Our preliminary data show that impaired endoplasmic reticulum function disrupts glucokinase regulation. The planned studies seek to identify the mechanism behind this disruption, and test whether diet-related obesity can similarly disrupt glucokinase regulation. If so, these studies will provide a molecular explanation for inhibited glucose sensing during type 2 diabetes. In summary, these studies have the potential to unify cellular and biochemical models of glucokinase function, identify new molecular regulators of glucokinase activity, and will lead to new ideas about the molecular causes underlying the deficit in ß-cell glucose sensing observed in type 2 diabetes mellitus. Understanding the molecular events that worsen type 2 diabetes mellitus is vital for designing new therapies that target beta-cell glucose sensing.

DESCRIPTION (provided by applicant): Leading causes of death, such as heart disease, stroke, and diabetes, and are all associated with vascular dysfunction. Thus, understanding the physiologic mechanisms that control vascular function is vital for understanding the pathogenesis of these conditions and for developing new treatments. Many important classes of vasomodulators work by binding to G-protein coupled receptors (GPCRs) that initiate signaling cascades that converge on the small GTPase, RhoA. RhoA-GTP activates Rho-associated kinase (ROK), which regulates contraction of smooth muscle through inhibition of myosin light chain phosphatase (MLCP) and is also involved in pathophysiological responses of arteries; vascular remodeling, smooth muscle cell proliferation, and recruitment of inflammatory cells. RhoA can therefore be regarded as an integrative control point that translates diverse GPCR signaling to numerous artery functions. The fraction of RhoA molecules that are bound to GTP constitutes the 'fractional activation' of RhoA, and is a quantitative measure of the potential activation of ROK. In preliminary work we have constructed a high performance FRET-based RhoA activation sensor molecule, RhoA.v2. RhoA.v2 utilizes mCerulean3 and mCitrine to provide outstanding characteristics for quantitative FRET measurements, particularly with two-photon excitation. Two-photon excitation also provides the ability to image RhoA.v2 within cells of intact tissues of the living mouse and even entirely non-invasively, through the skin. The major Aims of this proposal are to 1) develop a novel transgenic mouse model that expresses RhoA.v2 specifically in smooth muscle cells, 2) develop methods, utilizing two- photon imaging, that unlock the full quantitative power inherent to the design of the RhoA.v2, such that the fractional activation of RhoA can be quantified in arteries in vivo, and 3) pursue a preliminary investigation into the role of RhoA in control of contraction of smooth muscle cells in arteries by the sympathetic nervous system (SNS) activity. SNS hyperactivity, which can exist only in living animals, is a key factor in hypertension, metabolic syndrome, heart failure and other conditions. We will test the hypothesis that RhoA is a critical effector of SNS in certain arteries in vivo. Ths work will be accomplished by a team of investigators with complimentary expertise in optical probe development/FRET imaging (Dr. Rizzo) and vascular biology and in vivo imaging (Dr. Wier). In summary, a novel RhoA biosensor mice will be created and methods, utilizing two-photon imaging, will be developed for quantification of RhoA activation in vivo, with subcellular resolution. The model and methods developed by this proposal will be broadly impactful to hypertension, diabetes, many areas of vascular biology (including stroke), and areas of general smooth muscle involvement, such as gastrointestinal and bladder function.

? DESCRIPTION (provided by applicant): Hypertension involves elevated vascular resistance and only exists in a living animal, where the physiologic regulators of vascular tone (i.e. neuronal activity, blood flow, and endocrine factors) are intact. Therefore, hypertension research will greatly benefit from the in vivo exploration of the molecular regulators of arterial smooth muscle cell contraction. Our overall goal is to elucidate mechanisms of increased vascular resistance, for the first time, in conscious animals, during experimental salt-dependent hypertension. The use of conscious animals (as opposed to anesthetized) is key to understanding the putative role of sympathetic nerve activity (SNA), increasingly recognized as a key mechanism of salt-dependent hypertension. This overall goal is to be achieved through the use of non-invasive, fluorescence imaging of molecular signaling in arterioles of conscious optical biosensor mice. Given that phosphorylation of myosin regulatory light chains is the critical determinant of smooth muscle contraction, Specific Aims 1 and 2 are to develop optical biosensor mice that express novel, genetically-encoded activity biosensors for the key molecular regulators of smooth muscle myosin phosphorylation; a) myosin light chain kinase, (MLCK), b) myosin light chain phosphatase, MLCP), and c) a key upstream regulator of MLCP, the small GTPase, RhoA. In Aim 3, we will use these optical biosensor mice to determine, in vivo, 1) the regulation of MLCK, MLCP and RhoA by certain vascular G-protein coupled receptors (GPCR) putatively involved in hypertension, including adrenoceptors (?1-AR), Angiotensin II receptors (AT1-R), endothelin-1 receptors, (ETA), and sphingosine-1-phosphate receptors (S1P1). The regulation of MLCK, MLCP and RhoA by SNA will be determined through the use of complete autonomic ganglionic blockade (hexamethonium) in conscious mice. Mice will be implanted with telemetric arterial blood pressure transducers to allow continuous measurement of arterial blood pressure during imaging (and all other times). In Specific Aim 4, the time course of the activation levels of MLCK, MLCP and RhoA will be measured in ear arterioles of conscious individual mice (i.e. a `longitudinal' study) during 14 days of Angiotensin II/salt hypertension. Mice are infused chronically with Angiotensin II and fed a high-salt (NaCl) diet. Increased vascular resistance in this model of salt-dependent hypertension is believed to involve heightened SNA (`sympathoexcitation) emanating from salt-sensitive CNS cardiovascular control regions, mandating use of conscious mice. These Specific Aims will be performed in this multi-PI project under the direction of two Principal Investigators with the necessary expertise to generate the proposed sensors (Rizzo) and perform the physiologic experiments (Wier). In summary, we expect to achieve dynamic imaging of myosin phosphorylation regulatory processes during the development of salt-sensitive hypertension in a living animal for the first time. These studies will reveal new insights on the molecular basis of increased vascular resistance in hypertension, as it actually occurs in living animals.

Deciphering neural coding will require deconstructing the complex and intertwined signaling mechanisms that drive cellular excitability, synaptic plasticity, and circuit dynamics in the brain. This fundamental objective has been extremely challenging because unraveling the temporal and spatial interactions of multiple signaling pathways requires coordinated observation of multiple networks within individual cells and multiple neurons within intact circuits. Large gaps in knowledge remain because our current tools for tracking the dynamics of molecular activity are poorly suited for investigating more than one reporter at a time. Here, we propose to tackle this constraint through development of a novel methodology for simultaneous optical imaging of multiple quantitative FRET biosensors within single neurons, using FLuorescence Anisotropy Reporters (FLAREs). Numerous FLAREs targeting canonical signaling pathways, including calcium, cAMP, and the MAPK cascade, have been constructed in several colors allowing simultaneous imaging of up to three sensors in a single preparation, either in the same or complimentary pathways. We propose three aims to validate and further develop this technology to tailor it for studying cells and circuitry in acute and cultured slices from the mouse brain during neural coding. We will first adapt an optical sectioning microscopy method that is highly advantageous for fluorescence polarization imaging, known as dual-inverted Selective Plane Illumination Microscopy (diSPIM), for FLARE imaging. We will also expand the FLARE palette to include key regulators of synaptic function (Rac, CaMKII) and membrane excitability (voltage). Construction of the FLARE-SPIM instrument will enable proof of principle studies on two high-value neuronal circuits. First, pushing the limits of subcellular spatial resolution, FLARE-SPIM imaging will be performed on key signaling molecules in single dendritic spines in acute hippocampal brain slices during induction of long-term potentiation. Second, pushing the limits of cellular temporal resolution, we will track the rhythmic fluctuations of voltage, calcium, PKA and ERK activities during circadian oscillations of neuronal activity exhibited in organotypically-cultured suprachiasmatic nucleus brain slices. Together, these studies will lay the foundation for systematic exploration of neuromodulation within cells and neuronal circuitry, providing critical and unprecedented new insights for the spatial and temporal interactions between signaling pathways. Through collaboration with other Brain Initiative groups working on similar problems, this foundational work will be scalable to add suites of sensors that visualize nodes of coordinated cellular activity and reveal and measure the complexity of neural coding within intact brain circuits.