Owen McGuinness - US grants

Carbohydrate and lipid metabolism are under tight hormonal control by insulin, glucagon and the autonomic nervous system. Following a chronic stress, such as infection, carbohydrate and lipid metabolism are markedly accelerated with associated changes in insulin, glucagon, the sympathetic nervous system and other hormones. This increase in carbohydrate metabolism is not readily suppressed by exogenous nutritional support. Although in vivo studies suggest that gluconeogenesis is increased in infection, in vitro data suggest that hepatic gluconeogenic potential is decreased. An explanation for this paradox is that the hormonal and substrate changes commonly seen in infection may compensate for the hepatic impairment. Glucagon and insulin are potent regulators of glucose metabolism and they have been implicated in directing the increase in glucose production seen in sepsis. The first goal of this proposal is to determine if sepsis alters hepatic insulin sensitivity and the role of basal insulin in regulating hepatic glucose production. The second goal is to determine what role the hyperglucagonemia seen in sepsis plays in directing the gluconeogenic response to infection. The third goal of this proposal is to determine whether the capacity of the liver to remove and metabolize glucose is altered by infection. Experiments will be carried out in dogs which have received either a sterile or an E-coli-containing fibrinogen clot in the peritoneum 36 hrs prior to the study. Hormone levels will be controlled both by surgical (pancreatectomy) and pharmacological (somatostatin) methods. Hepatic glucose metabolism (glycogenolysis, gluconeogenesis, glucose uptake) will be measured using combined tracer and A-V difference techniques. This proposal is a first step in defining some of the many factors that are responsible for the acceleration of gluconeogenesis in stressful situations, more specifically in the infectious state. In addition, it will determine if infection modifies the ability of the liver to take up glucose or to respond to factors, which normally play an important role in stimulating hepatic glucose uptake. The combination of both tracer and balance techniques will allow assessment of the rates of hepatic glucose uptake, glycogenolysis and gluconeogenesis and the factors that are important in determining their relative rates. A knowledge of the factors that regulate hepatic metabolism in infection and the identification of the processes involved will contribute to our understanding of the role they might play in directing the metabolic response to infection and the metabolic fate of nutritional support.

The metabolism of exogenous nutrients administered is markedly altered by infection. One of the hallmarks of an individual that develops an infection while receiving nutritional support is hyperglycemia. There is also a concomitant increase in the metabolic rate and the inability of exogenous nutrients to suppress the elevated nitrogen excretion, fat oxidation and increased gluconeogenesis. The primary cause of the hyperglycemia is due to the inability of the body to efficiently dispose of the exogenous glucose. The role of specific tissues in contributing to the impairment is unclear. Based upon our studies the liver is a major site of glucose disposal (approximately 50 percent of the exogenous glucose infused) in normal animals receiving total parenteral nutrition and the uptake of glucose by the liver is markedly suppressed by infection. And peripheral tissues dispose of the glucose carbon. It is also known that the route of nutrient support (enteral or parenteral) alters the ability of the liver to take up glucose in the acute setting but this benefit does not persist chronically. The first goal of the proposal is to determine the time course of and the mechanism for the normal adaptation to nutritional support given enterally and parenterally. The second goal is to determine if the route by which glucose is delivered can chronically regulate liver glucose uptake and if it is affected by infection. The third goal to determine how chronic hyperinsulinemia, hyperglucagonemia and hyperglycemia interact to regulate liver glucose uptake during infection. Experiments will be carried out in chronically catheterized conscious dogs receiving continuous nutritional support. Hepatic glucose metabolism (unidirectional hepatic glucose uptake and production, glucose oxidation) will be assessed using a combination of tracer and arterio-venous difference techniques. In addition we will simultaneously assess limb glucose uptake and disposal. While previous work has been examining the response of whole body glucose metabolism to infection, we will be uniquely able to directly examine the role that individual organs (liver muscle) plays in the infection induced modulation of nutrient disposition. And by using Pharmacological techniques (somatostatin, phosphorylase a inhibition) we can not only determine the factors responsible for the impairment but we will be able to determine their mechanism as well.

DESCRIPTION (provided by applicant): Tissue specific defects in insulin action or in metabolic pathways thought to be essential to maintaining glucose homeostasis do not always produce the expected outcome. For example, complete removal of either GLUT4 or insulin receptor in muscle does not exhibit a significant phenotype unless challenged by aging or high fat feeding. The failure to manifest marked hyperglycemia despite significant impairments in muscle glucose uptake suggests that other tissues compensate for the defect. The pancreas compensates by increasing insulin secretion and adipose tissue compensates by enhancing glucose uptake and lipid deposition or secreting a factors that modifies glucose metabolism in other tissues. Little or no work has addressed the role of the liver. The primary variables measured in vivo to detect changes in hepatic metabolism especially in mice are tracer determined glucose turnover and the ability of insulin to inhibit hepatic glucose production during a euglycemic hyperinsulinemic clamp. Interestingly in most cases it has been difficult to detect underlying defects in hepatic insulin action except when diabetes and the associated hyperglycemia are present. Our data indicate that the liver has the unique ability to adapt to a sustained increase in glucose availability by enhancing its capacity to take up glucose. Moreover, while the liver initially stores the glucose as glycogen, as the duration of high glucose exposure increases a large fraction of the glucose is released as lactate which is subsequently removed by peripheral tissues. The euglycemic hyperinsulinemic clamp, while effective in detecting alterations in peripheral glucose uptake, cannot be used to discriminate between changes in peripheral and liver glucose uptake. To directly assess liver glucose uptake requires arterio-venous difference techniques that, while readily available in large animal models, cannot be implanted in conscious mice. Consequently very little work in mouse models has examined how liver glucose uptake is regulated and how the liver adapts to impairments in peripheral insulin action and glucose disposal. The Mouse Metabolic Phenotyping Center (MMPC) here at Vanderbilt has developed surgical approaches that will allow us for the first time to study the adaptive response of the liver in a conscious mouse. We will combine novel tracer methodology with a chronically catheterized conscious mouse model to quantify the adaptive response of the liver and then test this adaptive response in mouse models of insulin resistance.

DESCRIPTION (provided by applicant): The liver has a unique adaptive response to continuous nutritional support (NS). The liver switches from a site of modest glucose uptake and glycogen storage to an organ that efficiently converts glucose to lactate (i.e. enhanced glycolytic capacity). As a consequence of this shift in hepatic metabolic flux we hypothesize the normal regulators of liver glucose uptake and disposition in the acute setting (glucose and insulin levels and route of glucose delivery) no longer exert the same degree of control in the adapted setting. We believe the fall in glucagon during NS is essential to allowing the adaptation to occur. The enhanced hepatic glucose disposition observed in the adapted setting decreases the fraction of the exogenous glucose removed by peripheral tissues, while still preserving total carbohydrate uptake (glucose + lactate) by peripheral tissues. Following chronic NS greater than 1/3 of the peripheral carbohydrate uptake is as lactate. Since lactate is more efficiently cleared than glucose and less dependent upon insulin for its removal and the absolute rate of glucose uptake by peripheral tissues is diminished, the insulin and glucose concentrations are decreased as well. Infection impairs this adaptive response despite compensatory hyperinsulinemia with the liver reverting back to a state similar to the un-adapted (i.e. acute) state. The failure to adapt shifts the responsibility of glucose removal to peripheral tissues. When combined with the infection induced peripheral insulin resistance the risk of developing hyperglycemia increases. We believe the infection induced rise in glucagon contributes to this impaired adaptive response and aggravates the peripheral insulin resistance. Moreover, while the presence of compensatory hyperinsulinemia and/or hyperglycemia or enteral (or portal vein) glucose delivery can acutely correct the impairment in liver glucose uptake, we hypothesize this can not be sustained because they can not overcome the deficit in hepatic glycolytic capacity. The questions we will address are. Does the suppression of glucagon secretion play an essential role in facilitating the metabolic response to NS and does its failure to suppress during infection contribute to the abnormal hepatic and peripheral metabolism? Are the compensatory hyperinsulinemia and hyperglycemia during infection able to sustain the adaptive response to NS? Do cytokines released during infection modify the adaptive response to NS? Does infection impair the ability of portal and enteral glucose delivery to facilitate liver glucose uptake and inhibit peripheral glucose uptake during NS? Experiments will be carried out in chronically catheterized conscious dogs receiving continuous nutritional support. Hepatic glucose metabolism (unidirectional hepatic glucose uptake and production, glucose oxidation) will be assessed using a combination of tracer and arterio-venous difference techniques. In addition we will simultaneously assess limb glucose uptake and disposal. While previous work has examined the response of whole body glucose metabolism to infection, our model provides the unique ability to directly examine the role that individual organs (liver and muscle) play in the infection-induced modulation of nutrient disposition and the factors responsible for the impairment and the mechanisms by which they occur.

The Enrichment, Training, and Outreach Program of the DRTC orchestrates a broad range of essential DRTC activities that greatly enhance and enrich the research and training environment at Vanderbilt, and in combination with the Administrative Core, that also facilitates cooperative activities and interactions of the Vanderbilt DRTC with other academic medical centers. The target audience of DRTC efforts to enhance the research and training environment is not only DRTC-affiliated investigators, but also their students and postdoctoral fellows, and other members of the Vanderbilt scientific community. The DRTC has multiple mechanisms for achieving these goals, including a weekly seminar series, an annual Diabetes Research Day, and symposia on specific research areas. Furthermore, as part of the Vanderbilt DRTC's mission to promote the education and training of scientists and health professionals, the DRTC is affiliated with three diabetes-related training grants and assists in additional training venues. Plus, the laboratories of DRTCaffiliated investigators provide the scientific home for 83 graduate students and 101 post-doctoral fellows. Substantial resources provided by Vanderbilt augment the enrichment funds supplied by the DRTC grant.

Section V. Metabolic Pathophysiology Core (MFC) The MFC is divided into three subcores: Metabolic Regulation, Islet Isolation, and Tissue and In Vivo Imaging. The Metabolic Regulation Subcore uses state-of-the-art techniques to assess metabolism and energy balance in the conscious mouse or perfused organ. The Islet Isolation Subcore provides high quality islets to investigators and characterizes pancreatic islet function in vitro. The Tissue and In vivo Imaging Subcore provides sensitive optical imaging techniques to monitor in vivo real time kinetics of a metabolic process or to monitor the kinetics of gene expression in vivo. Details of animal procedures can be found in "Vertebrate Animals" located at the end of the core description.

DESCRIPTION (provided by applicant): This application is in response to RFA GM-08-010 (Short Courses in Integrative and Organ Systems Pharmacology). A two-week short course entitled, an organ systems approach to experimental targeting of the Metabolic Syndrome is proposed. The Metabolic Syndrome is a cluster of metabolic risk factors that when they occur together increase the risk of heart disease, stroke and diabetes. These risk factors include insulin resistance, central obesity, dyslipidemia and hypertension. The Metabolic Syndrome is an epidemic and its prevalence is still on the rise. The demands on the US Health Care System resulting from its pathology are debilitating to the economy. Clearly, there is a need to understand the pathogenesis of the Metabolic Syndrome and learn ways to treat it. This is challenging because it is a condition that involves organ system cross talk and understanding it requires knowledge of integrated physiology. Over the last 30 years physiology and pharmacology graduate programs have shifted their emphasis to cell biology. Consequently, there is a generation of physiologists and pharmacologists that lack experience in organ system physiology. Thus, there is a great need to restore and contemporize the toolbox for the study of integrated physiology. The objective of the course will be to give students the tools needed to assess whether an experimental intervention (pharmacologic, genetic, dietary, or environmental) alters macronutrient metabolism, energy balance, cardiovascular homeostasis or animal behavior. Moreover students will learn how to measure whole body and tissue specific kinetics, the principles of which can be applied to the kinetics of drugs, substrates and hormones. To accomplish this we will use a combination of lectures, hands on laboratories, demonstrations and data problem sessions. Three guiding principles thread through the course components. 1) organ systems do not function in isolation; 2) primary mechanisms can best be identified by disrupting compensatory feedback loops using tools such as a glucose clamp. 3) proper animal care is critical to good outcomes. With regard to the last the privilege of animal research is accompanied by the responsibility of treating animals humanely. Students will learn that the quality of data obtained in animal models is directly related to the health and well-being of the animals. All procedures involving animals will follow USDA and AAALAC guidelines. Vanderbilt is the ideal place to conduct such a course. Vanderbilt has a strong history in the physiologic regulation of metabolic, hemodynamic, neurobehavioral processes, and pharmacological testing. Vanderbilt is internationally recognized for its excellence in translational research that uses bench to bedside research to improve drug therapy for human disease. The Vanderbilt Metabolic Physiology Shared Resource (VMPSR) is comprised of core resources of the Vanderbilt Diabetes Research and Training Center (VDRTC) and the Vanderbilt Mouse Metabolic Phenotyping Center (VMMPC) and will provide infrastructure for the course. VMPSR personnel have extensive experience with experimental surgery and procedures for studying metabolism and cardiovascular function in vivo. Organ system approaches will be used to study metabolic flux, endocrine function, animal behavior and cardiovascular function in conscious animals and these will be correlated with results in vitro systems. The VMMPC gives a course annually in which it teaches the skills necessary to assess insulin action in mice. This course has attracted investigators from all over the world. We will use this experience in developing An organ systems approach to experimental targeting of the Metabolic Syndrome. Vanderbilt also has strong Murine Neurobehavioral Core Laboratories and a state of the art imaging institute that actively support both basic research and drug discovery programs. We will combine the strengths of the Vanderbilt community with faculty from other institutions in the national MMPC program to provide a comprehensive course for students to learn how to study the Metabolic Syndrome and its components in animals. PUBLIC HEALTH RELEVANCE: The purpose of this course is to train investigators to do studies in animals so they can effectively develop and evaluate therapeutic targets to treat diabetes and obesity before they enter the clinic. Most graduate students in the basic biomedical sciences receive limited training in physiology and integrative pharmacology. The benefit to human disease is that these trainees will now be equipped with the tools necessary to fill a void in our training programs.

DESCRIPTION (provided by applicant): Hyperglycemia is very common in patients with sepsis even if there is no history of diabetes. Insulin resistance of skeletal muscle glucose uptake (MGU) is a major cause of this hyperglycemia. Control of MGU is distributed between delivery of glucose to muscle, glucose transport into muscle, and glucose phosphorylation within muscle;insulin resistance is due to defects in one or more of them. The impact of obesity on MGU has been studied by a number of groups, but the impact of inflammation on the distribution of control of MGU is unknown. The experiments described in this proposal will examine the extent to which inflammation induced by lipopolysaccharide (LPS) redistributes the control of MGU. In this proposal the roles transport and phosphorylation play in controlling MGU will be assessed by using germline manipulation (partial knockout or over expression) of transport and phosphorylation capacity (e.g. hexokinase) to modulate a single step or multiple steps and measure the impact on MGU. We hypothesize that defects in glucose phosphorylation capacity play a central role in the inflammation induced insulin resistance. Experiments will be performed in chronically catheterized, conscious mice. This approach allows for comprehensive metabolic assessment of MGU in vivo in the absence of stress. The experimental strategy is to perturb proteins or processes involved in control of MGU and measure the effect of the perturbation on glucose influx. Whole body glucose uptake and MGU will be measured using [3-3H] glucose and [14C] 2- deoxyglucose, respectively, in combination with methods for sampling blood and tissues and measuring muscle blood flow. The relationship of MGU to long chain fatty acid (LCFA) uptake will simultaneously be measured using a radiolabeled fatty acid analog. Muscle ATP flux will be assessed using 31NMR spectroscopy. Tissues will be analyzed for glycogen synthesis, insulin signaling, oxidative stress and GLUT4 translocation. Our specific aims are to determine: 1. The impact of LPS on the relative control transport and glucose phosphorylation have in determining MGU 2. If LPS amplifies the impact NEFA and glucose availability have in modulating MGU 3. If modulating oxidative stress (NO availability and NF-:B activation) following LPS will improve MGU by augmenting glucose phosphorylation and mitochondrial ATP flux Our long term goal is to identify the steps controlling MGU that are impacted by inflammation and assess which of those steps are more responsive to changes in oxidative stress. Future therapies can then have a more targeted approach in correcting MGU during an inflammatory stress such as sepsis. PUBLIC HEALTH RELEVANCE: Hyperglycemia is very common in hospitalized patients and clinical trials suggest if the hyperglycemia can be minimized morbidity and mortality are improved. A major cause of the hyperglycemia is insulin resistance of skeletal muscle glucose uptake This proposal will determine where the defect is and address the questions is it due simply to a failure of insulin to activate it signaling pathway or are underlying defects in the mitochondria caused by the inflammation aggravating and limiting the ability of insulin to exert is beneficial effects.

DESCRIPTION (provided by applicant): Bariatric surgery is currently the only effective treatment for severe obesity, and the only effective cure for type II diabetes. Research on the mechanism of action of the different bariatric surgical procedures in humans and model systems including pigs, dogs, rats, and mice supports the hypothesis that the beneficial effects result from more than the restrictive or malabsorptive effects of the procedures on food intake. Indeed, data argue that neuroendocrine changes in gut-brain signaling resulting from the Roux-en-Y and gastric sleeve procedures alter satiety, hunger, food preferences, and glucose homeostasis prior to the achievement of significant weight loss. Understanding the cellular and molecular basis of these changes induced by bariatric surgery might lead to the development of pharmaceutical interventions, or improved surgical procedures for the treatment of obesity and diabetes. While several animal models can be used for research on the physiology of bariatric surgery, the mouse provides the best model for studies of cellular and molecular mechanisms because transgenesis can be used to alter individual genes, and to label specific cell types. We show results here demonstrating successful creation of murine bariatric surgery models at Vanderbilt, and the use of the models to identify the first gene that plays an essential role in th efficacy of RYGB for long term maintenance of significant weight loss. The unique hypothesis to be tested is that the efficacy of bariatric surgery results not solely from a collection of changesto Gl signaling, but rather that essential changes in both Gl signaling AND in the plasticity and responsiveness of CNS homeostatic and hedonic circuits act synergistically to restore glucose homeostasis, and create a new weight set point. In this interdisciplinary team grant application, we bring together leading experts in human and murine bariatric surgery, murine pathology, Gl anatomy and function, obesity and diabetes, and quantitative human genetics to jointly study surgical preparations from humans and mice in order to identify the genes and cell types mediating the efficacy of bariatric surgery.

The Metabolic Regulation Core (MRC) provides services that are distinct in that they permit complex study of mouse models without handling or stress. The MRC has continually refined its methodology to accurately assess metabolism in healthy, conscious mice. The Core has improved the accuracy of methods to measure metabolic flux using isotopes and developed blood replacement methods to prevent anemia due to blood withdrawal. Besides the unique skills of the MRC in chronically implanting catheters into the carotid artery and jugular vein for sampling and infusions, it has developed techniques to catheterize the portal vein and stomach to deliver hormones and nutrients by their physiologic routes. The MRC has developed experimental designs to apply stable isotopes to measure hepatic metabolic flux in vivo and in vitro. The ability of the MRC to gain vascular access in the absence of stress permits flexibility as well as sophistication. For example the intracerebroventricular delivery of a compound on whole body metabolic flux or hormone secretion has been assessed. Additional services include in vitro perfusion techniques (liver, pancreas, skeletal muscle). It partners with the DRTC Islet Procurement and Analysis Core to provide high quality islets to investigators as well as tools to characterize islet function. MRC has developed novel imaging technology to study metabolic processes in real time at the physiological and molecular levels. Imaging Resources include: a multi-photon excitation confocal microscope to visualize real time kinetics of calcium, NAD(P)H, pH the movement of biomolecules (e.g. insulin) across the vascular endothelium and tissue blood flow using fluorescent probes in whole organ preparations and in vivo; The MRC provides state-of-the-art technology to comprehensively characterize metabolic regulation in mouse models of metabolic disease.

Hormone Assay & Analytical Services Core (HAASC): Project Summary/Abstract The purpose of the Hormone Assay & Analytical Services Core (HAASC) is to provide to investigators assays to measure hormones, cytokines, amino acids (Specific Activity & concentrations), lipids/lipoproteins, nucleotides and markers of oxidant stress in biologic fluids and tissue samples, and more recently glucose (enrichment and isotopomer distribution) in plasma and tissue samples. The services provided by the HAASC and its two subcores (Oxidative Stress Subcore and Lipid Subcore) require unique instrumentation and involve rigorous standardization of procedures. It is neither practical nor economically reasonable to establish these assays in individual laboratories. The consolidation of services into one Core laboratory provides investigators with efficient, high quality, low cost analyses. All services are tailored to meet the needs of the specific investigator in that the exact protocol followed may differ depending on the analysis desired by the investigator and the sample volume to be analyzed. Through training and education, investigators become more aware of the various services offered and how these services might address specific areas of interest. Finally, an important function of the Core is method development, which not only enhances the spectrum of services offered but ultimately also increases efficiency by providing researchers with the opportunity to expand their research programs. The core provides space, equipment, and personnel for sample analysis. Investigators pay a fee-for-service that covers the cost of reagents, supplies, pro-rated service contracts for equipment, and a percentage of personnel salary. The Core also serves as a valuable resource for all investigators without ready access to wet-lab research space and personnel. Core services utilize modern instrumentation run by experienced research assistants in a consistent, quality-controlled environment. This Core works closely with another VDRTC Core, the Metabolic Physiology Shared Resource, to develop standard operating procedures to facilitate translation of this technology to VDRTC investigators performing in vivo and in vitro studies. Those investigators rely on the Core and Subcore directors and their staff for advice on how to collect and process samples collected during in vivo studies. The Hormone Assay and Analytical Services Core provides essential services to VDRTC investigators that allow them to perform important and innovative research. The HAASC has demonstrated its ability to develop and modify assays to meet the evolving needs of VDRTC investigators.

Project Summary This application is in response to Funding Opportunity Announcement PAR-15-139 a five day course entitled? Isotope Tracers in Metabolic Research: Principles and Practice in Kinetic Analysis?. The course faculty will present state of the art methods for using both radioactive and stable isotopes to investigate whole body and organ metabolism in vivo and intracellular flux rates and pathway regulation in vivo and in vitro. The basic aspects of modeling will be considered, as well as specific applications to the study of carbohydrate, fat, protein metabolism and energy balance. Theoretical and practical matters related to sample analysis by mass spectrometry and NMR will be discussed, including detailed numerical examples of calculations involved in determining isotopic enrichment and basic kinetic parameters. Advanced lectures will discuss in more detail the use of positional and mass isotopomer analysis for intracellular flux rates and various aspects of protein and amino acid metabolism. Specific applications (hyperinsulinemic euglycemic clamp) for use in humans and animals will be presented. The course uses a lecture format combined with problem discussions. In addition a popular aspect of the course is that trainees meet one on one with course faculty to discuss their specific research project and can present their project in an evening session. A new addition is detailed theory and experimental protocols to monitor specific pathways in a webinar style format that will be available on the course website to the entire research community. Typically, 75-85 trainees attended the course each year. Feedback from attendees has been very laudatory. A number of trainees have developed new research projects using isotope technologies. Thus this popular course builds on and reinforces fundamental skills needed to study metabolic processes that are relevant to the mission of NIDDK.