George G. Holz, Ph.D. - US grants

The specific aim of this proposal is to characterize the surface lipids of WHO-reference stocks of species of the protozoan parasite Leishmania, responsible for cutaneous, mucocutaneous and visceral disease of man in many temperature and tropical regions of the world, and to determine the roles of those lipids in the host-parasite relationships of the life-cycle stages. This aim is to be realized by (1) in vivo propagation of the amastigote stage in laboratory animals and in vitro in cultures of macrophage-like cells, (2) in vitro propagation of the promastigote stage (characteristic of the vector sand fly) in culture under a variety of environmental stresses chosen to mimic those to which the parasite is likely to be exposed (Delta T, Delta pH, Delta osmolarity, Delta 02 and CO2), (3) isolation, collection and purification of amastigotes from macrophages by density gradient centrifugation and lectin agglutination techniques, (4) isolation, collection and purification of plasma membranes of leishmanias and macrophages by fractionation (N2 cavitation, vesiculation, polyacrylamide bead complexing) and centrifugation methods, (5) analysis of the lipid constituents of parasites and their surface membranes, in detail, by chromatographic, chemical degradative and spectrometric means (GC-MS, PNMR, IR, UV), and (6) assessment of the antigenicity of surface glycolipids by attempts to raise antibodies to them in rabbits.

The long term objective of this project is to identify and to study novel chemotherapeutic approaches to the control of leishmanial and trypanosmal infections of substantial medical importance, by exploiting new knowledge that aspects of the lipid biochemistry of the responsible trypanosomatid flagellates are characteristic of fungi and not of vertebrates. The immediate specific aims to realize this objective are to investigate: a) the normal sterol metabolism of the life-cycle stages of representative species of Leishmania and Trypanosoma, b) the perturbations of that metabolism, and impairment of growth, caused by hypercholesterolemic and other cytotoxic drugs, and c) the mechanisms of action of the most effective of those drugs. Life-cycle stages are to be cultured in vitro, exposed to drugs, and the consequences to growth and to lipid metabolism assessed by population enumeration, and by lipid analysis employing chromatographic (CC, TLC, HPLC, GLC), spectrometric (GC/MS, 1H and 13C NMR, UV/VIS, FTIR), and radio- and stable isotopic tracer methodologies.

It is well recognized that treatment of non-insulin-dependent diabetes mellitus (NIDDM) is complicated by the risks of hypoglycemia, as well as large swings in the blood glucose level, inherent to insulin and sulfonylurea therapy. Recent clinical studies of NIDDM indicate the usefulness of incretin hormone glucagon-like peptide-1-(7-37) (INSULINOTROPIN, GLP-1) as the alternative treatment for this disorder. When administered during a meal to patients with NIDDM, GLP-1 restores the missing first phase component of insulin secretion and delays the post-prandial hyperglycemic excursion. Since the insulinotropic action of GLP-1 is self-terminating as blood glucose levels begin to fall, hypoglycemia is not a significant complicating factor. Although it is generally accepted that NIDDM is characterized by ineffective coupling of beta-cell glucose uptake to insulin secretion, it is not yet understood how short-term administration of GLP-1 corrects this defective secretory response. Here we report preliminary findings indicating rapid conversion of glucose-insensitive beta-cells to fully responsive cells by GLP-1, a phenomenon we term glucose competence. The objective of this study is to determine the mechanism by which GLP-1 renders pancreatic beta-cells glucose-competent, as assessed by patch clamp electrophysiological analysis. Perforated patch, cell-attached patch, and excised patch recordings will be used to study modulation of Ik GLP, an inwardly-rectifying potassium current that is inhibited by GLP-1, and which mediates the depolarizing action of this hormone on beta-cells. The biophysical and pharmacologic properties of Ik GLP will be characterized and an assessment made as to whether it represents a previously unrecognized beta-cell potassium current. since inhibition of Ik GLP by GLP-1 is contingent on the simultaneous application of glucose, the synergistic interaction of these two agents to depolarize beta-cells will be quantified by dose-response analysis. This will allow a direct test of the hypothesis that induction of glucose competence by GLP-1 results from a shift in the dose-dependence of the glucose response so that concentrations of glucose normally considered to be substimulatory become fully effective. Since cross-talk between the GLP- 1 and glucose signalling systems is a requirement for inhibition of Ik GLP, experiments will be directed at identifying the cytosolic second messenger that mediates the action of GLP-1. Understanding the molecular events that underlie the response to GLP-1 will provide insight into why this hormone is of therapeutic value in the treatment of NIDDM, as well as further our understanding of the glucose-sensing mechanism of beta- cells.

An emerging theme in experimental therapeutics concerns the use of glucagon-like peptide-1-(7-36)- amide (GLP-1) and its synthetic peptide analogs (the "incretin mimetics") to lower levels of blood glucose in Type 2 diabetic subjects. This action of GLP-1 results, at least in part, from its ability to stimulate the secretion of insulin from pancreatic beta cells located in the islets of Langerhans. Given the established importance of GLP-1 for the treatment of diabetes, our laboratory is interested in defining the signal transduction properties of the beta cell GLP-1 receptor (GLP-1-R). To this end, we have focused on a newly-discovered signaling mechanism that uses the second messenger cAMP to activate cAMP- regulated guanine nucleotide exchange factors designated as Epad and Epac2 (the Exchange Proteins directly Activated by Cyclic AMP). Our studies lead us to Hypothesize that GLP-1, a cAMP-elevating hormone, stimulates Ca2+-dependent insulin secretion, and that this insulinotropic action is mediated not simply by protein kinase A (PKA), but also by Epac. To test our Hypothesis concerning the putative role of Epac in GLP-1-R-mediated signal transduction, the Specific Aims of this project are to: 1) determine if GLP-1 uses Epad and/or Epac2 to mobilize intracellular Ca2+ via a process of Ca2+-induced Ca2+ release (CICR) that originates at the endoplasmic reticulum (ER) and which may involve IP3 receptors or ryanodine receptors, 2) assess what role Rap family GTPases play in the process of ER Ca2"1" mobilization, with special emphasis on the potential role of Rap1 as an intermediary linking activation of Epac to the stimulation of phospholipase C-epsilon, and 3) determine the nature of a novel signaling mechanism by which beta cell growth factors and receptor tyrosine kinases utilize the Ras GTPases to recruit Epac2 to the plasma membrane where an interaction of Epac2 with its putative effector molecule the sulfonylurea receptor-1 (SUR1) occurs. The Relevance of this line of investigation is fully apparent. We wish to establish the molecular basis for "antidiabetogenic" properties of a new class of blood glucose-lowering agents that activate the GLP-1-R and which stimulate pancreatic insulin secretion.

Glucagon-like peptide-1 (GLP-1; Insulinotropin) is an intestinally-derived blood glucose-lowering hormone that stimulates pancreatic insulin secretion and which is now under investigation for use as a therapeutic agent in treatment of type-2 diabetes mellitus. The Central Hypothesis presented here is that the beneficial insulinotropic action of GLP-1 at the islets of Langerhans results, in part, from an ability of GLP-1 to stimulate metabolism of D-glucose by the pancreatic beta- cells. Studies are presented demonstrating that GLP-1 augments the glucose-dependent production of ATP in beta-cells, as imaged by single photon counting of mitochondrially-targeted luciferase reporters. The action of GLP-1 is preceded by an increase of [Ca2+]i that reflects mobilization of Ca2+ from endoplasmic reticulum Ca2+ stores, and which is proposed to facilitate the enzymatic activity of mitochondrial dehydrogenases, thereby increasing [ATP]i. Subsequent inhibition of ATP-sensitive K+ channels (K-ATP) produces depolarization, oscillatory Ca2+ influx, and pulsatile exocytosis of insulin. To test our hypothesis, and to validate this model of GLP-1 signal transduction in human beta-cells, luminescence-based measurements of [ATP]i will be combined with fura-2 determinations of [Ca2+]i while monitoring K-ATP using the patch clamp technique. It will be determined: 1) if GLP-1 increases the potency and efficacy of glucose to stimulate production of ATP, 2) if stimulatory effects of GLP-1 on [Ca2+]i and [ATP]i explain how this hormone inhibits K-ATP, and 3) which second messengers and protein kinases mediate stimulatory effects of GLP-1 on beta-cell glucose metabolism. Our goal is to elucidate the complex cellular signal transduction properties of GLP-1 that explain its effectiveness for use in treatment of diabetes mellitus.

hormones; hormone regulation /control mechanism; blood glucose; biomedical resource;

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The overall goal of this project is to establish the systems physiology of glucoregulation in which alpha 7 nicotinic acetylcholine receptors (?7nAChR) regulate the incretin hormone action of Glucagon-Like Peptide-1 (GLP-1). This project may reveal novel features of glucoregulation that are of relevance to our understanding of type 2 diabetes mellitus (T2DM) since we find that the ?7nAChR agonist GTS-21 stimulates intestinal GLP-1 secretion, raises circulating levels of GLP-1, and improves glucose tolerance in mice. Thus, we propose a novel and important role for the ?7nAChR in the control of glucose homeostasis by virtue of its ability to exert inter-organ control over GLP-1 secretion and GLP-1 action. To test this Hypothesis, our aims are as follows: Aim 1: We will use Cre/lox technology in combination with gene knockout or knock-in technology to determine how ?7nAChR agonists lower levels of blood glucose in healthy mice or db/db and ob/ob mouse models of diabetes. A first goal is to evaluate baseline alterations of glucoregulation in tissue-specific ?7nAChR knockout mice, while also assessing how ?7nAChR agonist action is modified. A second goal is to identify which cell types express the GLP-1 receptor (GLP-1R) that mediates glucoregulatory actions of ?7nAChR agonists. Four possibilities exist: 1) when mice are administered GTS-21 in combination with a DPP-4 inhibitor, the concentration of GLP-1 in the blood will reach high levels so that GLP-1 will act at the pancreatic ?-cell GLP-1R to enhance insulin secretion, or 2) GLP-1 released from L-cells in response to GTS-21 might exert a local effect in the intestinal wall to initiate neural reflexes that stimulate insulin secretion, or 3) GTS-21 might act in the hindbrain nucleus tractus solitarius to stimulate GLP-1 release so that glucose homeostasis is improved, or 4) GTS-21 might stimulate GLP-1 release from pancreatic ?-cells so that intra-islet GLP-1 will exert a paracrine hormone action at the ?-cell GLP-1R. Aim 2: We will perform in vitro studies to test if GTS-21 acts exclusively at L-cells, or if it also acts at other types of enteroendocrine cells that may express the ?7nAChR. For example, GTS-21 might lower levels of blood glucose by stimulating the release of GIP from K-cells so that GIP will then act at the ?-cell GIP receptor to stimulate insulin secretion. Potentially, this action of GIP will be enhanced by a DPP-4 inhibitor. Since Peptide YY (PYY) is co-secreted with GLP-1 from L-cells, we may find that its release is also stimulated by GTS-21. This would be significant because PYY is important to the suppression of food intake in obesity-related T2DM. Finally, we will test for actions of GTS-21 to stimulate GLP-1 biosynthesis in L-cells and pancreatic ?-cells. This possibility exists because we find that GTS-21 upregulates expression of a prohormone convertase (PC1/3) that liberates GLP-1 from proglucagon, while also upregulating expression of GPR119, a GPCR that stimulates glucagon gene transcription. It will be especially interesting to determine if the ?7nAChR in pancreatic islets regulates coordinate expression of PC1/3 and GPR119 so that ?-cells acquire the ability to secret GLP-1 under conditions of T2DM. Summary: Our long-term goal is to establish the systems physiology of glucoregulation under ?7nAChR control.