Naji N. Abumrad - US grants

The long range objective of our research is to elucidate the complex regulatory mechanisms involved in protein homeostasis in normal man. Several hormones (i.e. insulin, glucagon, cortisol, catecholamines and thyroid hormones) and the overall nutritional status can directly or indirectly exert their effects on peripheral tissues and the liver in the th modulation of these processes. Our laboratory has been extensively involved over the past few years in assessing these regulatory effects on the liver in normal man; we are presently planning to extend these studies to the peripheral tissues. To meaningfully assess the direct effects and the complex interaction of these hormones in regulating muscle homeostasis in man, it is necessary to measure 1) peripheral substrate release, 2) total body amino acid kinetics and 3) the efficiency of transamination and oxidation of these amino acids. To accomplish this, we utilize a heated superficial hand vein (a suitable substitute for the arter), and a depp forearm vein, combined with he constant infusion of L-1-14C-leucine and the measurement of forearm blood flow. Utilizing these techniques we wish to accomplish the following: (1) to determine in normal man the metabolic changes that occur with early starvation (12h, 36h and 60h) with special reference to the kinetics of leucine and its keto-acid, Alpha-keto-isocarproate, and to forearm metabolism during those periods; (2) to evaluate the influence of residual insulin secretion on in situ metabolism by (a) producing insulinopenia via infusion of somatostatin and (b) by replacing basal insulin levels during somatostatin infusion. Finally we will, (3) evaluate the influence of hypercortisolism of differing duration perse and its interaction with progressive stages of fasting on all these metabolic processes. These studies will provide considerable insight into the mechanisms of enhanced amino acid fluxes and protein breakdown in such disease states as trauma, sepsis, infection, diabetes mellitus etc.., information which may ultimately be translated into improved health care for such patients.

The objective is to continue our studies already begun fiv years ago to understand the way in which the body regulates protein and amino acid metabolism in vivo. We will continue to examine the interaction of various hormones in modulating the metabolism of the branched chain amino acids (BCAA) and their corresponding keto acids (BCKA). Over the past few years, our laboratory has been extensively involved in assessing these regulatory effects on the liver and forearm tissues in man, and the dog. We are presently planning to continue these studies in both man and the dog, and to examine the tissue sensitivities to these amino and keto acids during periods of fasting. We will also examine the modulatory role of two specific hormones, insulin and cortisol on these tissues. To adequately assess the direct interactions in vivo, it is necessary to measure 1) the total body flux of these amino and keto acids 2) and to examine the relative contributions of various organs to these fluxes. These are easily accomplished in the conscious dog model with catheters surgically implanted in an artery, and in the portal, hepatic, renal, and splenic veins; and when needed, in the sagital and coronary sinuses. Since these amino and ketoacids are presumed to primarily metabolized in skeletal muscle, we will examine the metabolism of these substances across the human forearm, and estimate the contribution of human skeletal muscle to their metabolism under vraious fasting, and hormonal preturbations. Utilizing these techniques we will (a) investigate the interorgan flow of these amino and keto acids in vivo. (b) Examine, whether these substrates can regulate their own metabolism. (c) Examine the sensitivities and specificities of the various tissues in the body to the metabolism of the BCAA and BCKA and, (d) Examine how the changes in these parameters with fasting. (d) Finally, we will examine how insulin glucocorticoids modulate their metabolism. These studies will provide insight into the mechanism of enhanced amino acid fluxes and protein breakdown during situations of excessive stress, or during periods of malnutrition. The ultimate hope is to be able to translate this information into improved health care for patients who are subjected to periods of excessive stress, such as trauma, injury, sepsis, diabetes mellitus, etc.

Strict glycemic control reduces the occurrence of microvascular complications of diabetes at the cost of a three-fold increase in the frequency of severe hypoglycemia. Euglycemic levels are restored by hormonal and neural counter-regulatory mechanisms at the expense of enhance rates of substrate mobilization and rates of glucose production. Our studies have demonstrates that insulin-induced hypoglycemia is also associated with enhanced rates of whole body proteolysis and amino acid oxidation, as well as a significant enhancement in gut protein breakdown. This response, in its majority, is controlled by CNS glucopenia. Our studies suggest that sites responsible for controlling protein and amino acid metabolism during hypoglycemia are most likely located in the hind brain. The general hypothesis of the present proposal is that the "CNS-Gut" axis plays a pivotal role in mobilization of amino acids and proteins during the catabolic response to stress. The studies designed will be performed in the chronically catheterized conscious dog and will investigate the contribution of forebrain and hindbrain neuroglucopenia to triggering the proteolytic responses. In addition, the studies proposed will use surgical and pharmacological interventions to investigate the involvement of direct extrinsic innervation of the "gut" as well as sympathetic, parasympathetic and serotoninergic contribution to modulation the proteolytic responses. The associated hormonal and glucoregulatory responses as well as the contribution of the gut-derived amino acids to enhanced rates of hepatic gluconeogenic amino acid utilization will be assessed using a combination of isotopic and AV difference techniques. Complimentary studies in a rodent model of hypoglycemia to be performed in the final stages of this proposal will be aimed at defining the contribution of specific brain region glucopenia to the proteolytic responses to hypoglycemia. Furthermore, they will determine the specific neurotransmitter alterations that are associated with hypoglycemia. The results from these studies responses to hypoglycemia. These findings will contribute to the understanding of CNS modulation of the peripheral protein and amino acid alterations that are an integral part of the metabolic response to stress.

The early metabolic responses to injury are characterized by alterations in the neuroendocrine responses and in abnormalities in carbohydrate and amino acid metabolism. The abnormalities in glucose metabolism occur early and are characterized by sustained hyperglycemia resulting from enhanced hepatic glucose production, decreased glucose utilization and increased rates of hepatic glycogenolysis, gluconeogenesis and ureagenesis. The metabolic abnormalities in protein metabolism occur later and are characterized by relative increases in protein breakdown over those of protein synthesis, which if they persist, will ultimately lead to nitrogen wasting and muscle loss. The exact mechanisms for such abnormalities remains obscure. Several hypotheses have been put forth to explain the metabolic events, with some attributing the changes to the associated hormonal alterations, others relating the changes to enhanced biosynthesis and release of cytokines, prostaglandins, leukotrienes, etc. Based on preliminary data from our laboratory, the present application proposes a unifying hypothesis explaining many of the hormonal and metabolic events occurring with trauma and injury. Our data indicate that injury is associated with immediate increases in plasma and CSF levels of beta-endorphin, a derivative of proopiomelanocortin. We also presented evidence to suggest that injury is also associated with delayed increases in the formation of endogenous alkaloids (non-peptide opiates), namely recent independent work by Spector et al. nd later confirmed by collaborative work with Dr. Spector in our laboratory identified the presence of these endogenous non-peptide alkaloids in high quantities in plasma, CSF and in brain. Interestingly, the temporal changes in the beta-endorphin corresponded with the changes in carbohydrate metabolism while the changes in endogenous alkaloids corresponded with those of amino acid metabolism. We also presented evidence showing that many of the catabolic events seen with trauma are mediated by CNS activation of mu-receptors. ICV administration of morphine or beta-endorphin or i.v. administration of morphine resulted in significant alterations in carbohydrate metabolism identical to those seen after injury. The General Hypothesis of this proposal is that beta-endorphin and the opioid alkaloids act synergistically as neurotransmitters integrating most of the endocrine, autonomic and metabolic responses to stressful stimuli. The rises in CNS beta-endorphin precede those of morphine, and that this temporal relationship is responsible for the time-dependent changes in carbohydrate and in amino acid metabolism. The Specific Aims of this proposal are: a) to define the temporal changes in the endogenous responses of beta-endorphin, and non-peptide opiate alkaloids (morphine, codeine, thebaine) during the stress of surgery. b) Examine the central mechanisms involved in the metabolic changes in glucose and amino acid metabolism following surgical intervention. We will isolate the actions of the hypothalamic-pituitary-adrenal axis, the autonomic nervous system, and the endocrine pancreas to determine their relative contributions to the observed changes. c) Characterize the central opiate receptors involved in modulating the catabolic response to operative trauma. Localize the specific actions of peripheral versus central receptors and define the interactions between peptide and non-peptide opiate systems in eliciting those responses.

DESCRIPTION (provided by the applicant): Obesity is often associated with insulin resistance and abnormal production of inflammatory cytokines. Central obesity represents a major risk for the development of type 2 diabetes mellitus (T2DM) and cardiovascular complications. Adipose tissue and especially omentum (adipocytes and resident macrophages) release several cytokines. Bariatric surgery and specifically Roux-en-Y gastric bypass (RYGB) is the only modality that results in sustained weight loss. Our studies and those of others demonstrate that RYGB is effective in reversing T2DM in a high proportion of patients. The mechanisms remain unknown. We have evidence showing that weight loss after surgery is not the sole mechanism behind the metabolic improvements. The improvements occur very early (within 10 days) post-op and precede any significant weight loss; they are related to visceral fat distribution and are racially biased, with African Americans showing blunted and more delayed responses than Caucasians. The central hypotheses of this application is that improvements in insulin sensitivity after bariatric surgery are racially biased and begin early in the postoperative period (10-30 days) and are mediated by changes in the secretion of energy-related peptides, while the long-term effects (greater than 1 month) are mediated by down-regulation of inflammatory factors. Additionally, we hypothesize that the removal of the omentum in combination with bariatric surgery enhances the reversal of the insulin resistance and will diminish the racial differences in response to bariatric surgery. We propose a randomized study in Caucasian and African American morbidly obese patients to evaluate changes in glucose and fatty acid metabolism. Patients will be randomized to two groups, one with RYGB alone and the second with RYGB with omentectomy. Three specific aims are proposed. In Specific Aim 1, we will determine the mechanism for the metabolic improvements after RYGB. Specifically, we will examine alterations in the secretion and action of energy related peptides and inflammatory responses. Specific Aim 2 explores the mechanism for the blunted/delayed metabolic improvement after RYGB in African American patients. We will examine the genetic basis for differences in the two races using microarray analysis of muscle and visceral and peripheral adipose tissues. We will explore the role of resident macrophages in mediating associated inflammatory responses. Specific Aim 3 will determine if combining omentectomy with RYGB accelerates and sustains improvements especially in the African American population. The studies include determination of regional fat stores, adipocyte size, tissue macrophage content and macrophage gene expression, diurnal and food-induced secretion of adipokines (leptin, resistin and adiponectin) and of energy regulating-peptides such as ghrelin and PYY. Parameters will be correlated with time dependent changes in inflammatory markers (e.g. CRP, IL-6, TNF-a2R, etc.) and with tissue insulin responsiveness using hyperinsulinemic euglycemic clamps.

CRISP; Computer Retrieval of Information on Scientific Projects Database; Energy Expenditure; Energy Metabolism; Funding; Grant; Human; Human, General; Individual; Institution; Intestinal; Intestines; Investigators; Man (Taxonomy); Man, Modern; NIH; National Institutes of Health; National Institutes of Health (U.S.); Obesity; Research; Research Personnel; Research Resources; Researchers; Resources; Rest; Source; Testing; United States National Institutes of Health; adiposity; bowel; corpulence; corpulency; corpulentia; defined contribution; experiment; experimental research; experimental study; obese; obese people; obese person; obese population; research study; volunteer

African American; Afro American; Afroamerican; Black Populations; Black or African American; CRISP; Computer Retrieval of Information on Scientific Projects Database; Down-Regulation; Down-Regulation (Physiology); Downregulation; Effects, Longterm; Funding; Grant; Inflammatory; Institution; Investigators; Long-Term Effects; Mediating; Metabolic; Morbid Obesity; NIH; National Institutes of Health; National Institutes of Health (U.S.); Obesity, Morbid; Omental Fat; Omentum; Peptides; Population; Post-Operative; Postoperative; Postoperative Period; Refractory; Research; Research Personnel; Research Resources; Researchers; Resources; Severe obesity; Source; United States National Institutes of Health; bariatric surgery; black American; day; gastric banding; gastric bypass surgery; implantable gastric stimulation banding; insulin sensitivity; obesity surgery; obesity, extreme; stomach stapling; weight loss surgery

DESCRIPTION (provided by applicant): Obesity is a major public health problem; nearly 60% of the US population is considered obese or overweight. More alarming is the increase in prevalence of super-obesity (Class III obese) reaching in some localities 7% of the population, as in Tennessee where the PI resides. This condition is associated with severe co- morbidities, such as cardiovascular disease and type 2 diabetes, many of which are attributed to chronic inflammation, oxidative stress and insulin resistance. Roux-en-Y gastric bypass (RYGB) surgery is the most effective and sustainable weight loss procedure. Data of ours and others have shown that many of the metabolic benefits of RYGB occur in the first week postoperatively, prior to significant weight loss. These improvements are preceded by significant reductions in the circulating levels of gastric-derived ghrelin and leptin, occurring as early as 15 minutes after the surgical interruption of stomach during the RYGB procedure. These changes associate with significant reductions in oxidative stress in adipose tissue. The general hypothesis is that RYGB results in interruption of a gastric-adipose tissue axis leading to immediate (within the first week) improvements in oxidative stress and insulin sensitivity. In specific aim 1 we will examine the cellular, tissue-specific and whole-body metabolic alterations 7 days following RYGB. Two cohorts of matched controls will be studied before and 7 days following caloric restriction (to match the post-RYGB diet) without stomach interruption: one with LAGB (laparoscopic adjusted gastric banding) and the other without any surgical procedure. In specific aim 2 we will examine whether ghrelin replacement (restoration of unacylated to acylated ghrelin ratio) in the first week following RYGB reverses improvements in oxidative stress in adipose tissue and in insulin sensitivity. We will utilize three complimentary and comprehensive approaches: (i) In vivo studies to determine insulin sensitivity in liver and skeletal muscle and microdialysis of subcutaneous adipose tissue to assess tissue-specific oxidative stress, cytokine production and lipolysis. We will correlate metabolic improvements to intra-hepatic triglyceride content using magnetic resonance spectroscopy (MRS), and visceral adipose tissue mass using MRI and dual-energy x-ray absorptiometry (DXA). (ii) Ex vivo studies will assess mechanistic aspects of stomach-derived peptides on markers of oxidative stress and inflammation in adipose tissue explants. (iii) In vitro studies will examine changes in: (a) cellular factors of ROS production and pro- and anti-oxidative stress enzymes in adipose tissue biopsies (b) adipose tissue macrophage content via flow cytometry, RT-qPCR and immunohistochemistry (c) cellular factors involved in insulin signaling in adipose tissue and skeletal muscle. The information derived could lead to the combination of less invasive surgical procedures with pharmacologic manipulation of the levels of acylated ghrelin and/or leptin for the treatment of morbid obesity.

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.

? DESCRIPTION (provided by applicant): Bariatric surgery, specifically Roux-en-Y gastric bypass (RYGB), is the most effective and durable treatment for morbid obesity and potentially a viable treatment for type 2 diabetes (T2D). The resolution rate of T2D following RYGB approaches 80% and far surpasses that achieved by medical management alone. The molecular basis for this improvement is not entirely understood. We hypothesize that the altered enterohepatic circulation of bile acids (BA) after RYGB mediates the metabolic improvements that underlie the resolution of T2D after the procedure. In this proposal we will determine how BA contribute to improved lipid and glucose homeostasis, insulin sensitivity and energy expenditure after RYGB. We will apply a novel surgical procedure to diet-induced obese (DIO) mice where bile is diverted from the gallbladder (GB) to the duodenum (GB-D, a sham procedure), the jejunum (GB-J), or ileum (GB-IL). The biliary diversion (BD) procedure permits examination of altered bile flow, similar to that observed after RYGB, independent of alterations in gastrointestinal tract anatomy. Remarkably, the GB-IL procedure, but not GB-D or GB-J, recapitulates many of the beneficial metabolic and physiologic outcomes observed after RYBG. Specific Aim 1 will elucidate the contribution of altered fat absorption, chylomicron generation and clearance in the efficacy of GB-IL or RYGB. Studies in Specific Aim 2 are designed to define the role of membrane-bound BA receptor, TGR5, on glucose homeostasis after RYGB and GB-IL. In Specific Aim 3 we extend our investigations to the nuclear BA receptor, farnesoid x receptor (FXR), investigating the efficacy of RYGB and BD in mice genetically-engineered for intestine- and liver-specific FXR deficiency. The long-term goal of this work is to understand the mechanisms of metabolic improvement after bariatric procedures and to develop technically simple procedures and pharmacologic strategies to treat obesity and its sequelae.

ABSTRACT: The scavenger receptor CD36 has lipid and non-lipid ligands and versatile functions in metabolism and immunity. An important function of CD36 is its ability to transduce intracellular signals triggered by its ligands. CD36 signaling contributes to the regulation of several aspects of fatty acid (FA) utilization such as fat taste perception, chylomicron production, enteroendocrine secretion, FA oxidation etc. We recently showed that CD36 interacts with the AMPK and insulin signaling pathways and we propose that this mediates an important part of its actions on nutrient utilization. We document presence of CD36 in a molecular complex with insulin receptor beta (IR?) and CD36-mediated recruitment to IR? of Fyn and the catalytic p85 subunit of PI3-kinase. We also find that palmitic acid binding to CD36 interferes with Fyn and p85 recruitment and blunts Akt phosphorylation. In this project, which involves a multidisciplinary collaboration between basic and clinical scientists, we will test the novel hypothesis that the membrane protein CD36 via its interaction with the PI3K pathway influences endothelial cell function, insulin?s action on microvasculature recruitment and consequently nutrient flux and the effect of high fat feeding to cause endothelial dysfunction. Our preliminary data document diminished vascular compliance in CD36-/- mice and more importantly in African Americans carrying coding SNP rs3211938 that reduces CD36 level by 50%. We will examine the functional implications of CD36 signaling in endothelial cells and the consequences of endothelial cell CD36 deletion in mice on vascular function and insulin regulation of tissue perfusion and energetics. We will determine in these mice the effect of high fat feeding to induce endothelial dysfunction and whether this is reversed by treatment with phosphodiesterase 5 (PDE5) inhibition which blocks cGMP degradation. In a parallel approach we will examine influence of partial CD36 deficiency in African American carriers of rs3211938 on endothelial dysfunction, microvascular recruitment by insulin and the efficacy of PDE5 inhibition treatment. Obesity and endothelial dysfunction are highly prevalent especially in African Americans and associate with negative cardiovascular and metabolic outcomes. The proposed work will provide fundamental information that is currently unavailable and that could influence treatment of endothelial dysfunction and insulin resistance in humans.