Faculty Members in Research Theme 2:
Insulin and Counterregulatory Hormone Secretion and Action
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Eugene Barrett * Paula Barrett David Brautigan
David Castle William Evans * Leon Farhi
James Garrison Erik Hewlett Susanna Keller
Joel Linden Chien Li Zhenqi Liu *
John Marshall * Raghu Mirmira Suzanne Moenter
Julie Sando Heidi Scrable Thomas Sturgill
Michael Thorner * Michael Weber
Eugene J. Barrett
Professor of Medicine and Pediatrics
Director DERC
Research focus: Regulation of skeletal muscle perfusion and metabolism in vivo by insulin
(434) 924-1175
Laboratory and clinical Investigations focus on: 1) the direct action of insulin to modulate the nitric oxide dependent recruitment of capillaries in rat and human skeletal muscle; 2) the role of the endothelium in regulating insulin delivery to skeletal muscle; and 3) the effect of insulin resistance and exercise on insulin delivery to muscle.
Paula Q. Barrett
Professor of Pharmacology
Research focus: Molecular mechanisms regulating low-voltage-activated, T-type, calcium channels
(434) 924-5454
Low-voltage-activated T-type calcium channels of the Cav3 family are especially prominent in: the His-purkinje fiber system of the heart, thalamic neurons of the brain, sympathetic neurons of the dorsal root ganglion, and aldosterone producing cells of the adrenal cortex. As such, these calcium channels have been implicated in the pathogenesis of arrhythmias, epilepsy, hyperalgesia, hypertension, and in the progression of congestive heart failure. Our laboratory is interested in understanding how the activity of this channel type is regulated, and how changes in channel activity underlie changes in physiological function.
Regulatory mechanism: Using a combination of whole cell and single channel electrophysiologcial recording techniques, we have demonstrated that Cav3.2 channel activity is increased by calcium calmodulin-dependent protein kinase II (CaMKII) and inhibited by heterotrimeric Gβγ subunits containing Gβ2. CaMKII induces a hyperpolarizing shift in the voltage-dependence of activation (opening) that is the consequence of an increase in the frequency of single channel opening. This selective increase in channel activity at negative potentials can be reconstituted in a heterologous expression system, is selective for the Cav3.2 (α1H) channel subtype and is the direct result of phosphorylation of a single residue on the intracellular linker that connects transmembrane domains II and III that is absent from the unregulated Cav3.1 channel. By contrast, Gβ2 induces a tonic inhibition of Cav3.2 channel activity at all voltages that is not relieved by a strong depolarizing prepulse and is the consequence of a decrease in the number of functional channels. This voltage independent inhibition can also be reconstituted in a heterologous expression system, is also selective for the Cav3.2 channel subtype and is the direct result of an interaction between the II-III intracellular linker and four residues on Gβ2 located on the d strands of blades 2 and 3.
Physiological function: Recently, we have demonstrated that during Ang II stimulation in vivo the first of these mechanisms operates to stimulate Cav3.2 activity. Notably, inhibition of this modulation dramatically blunts Ang II-stimulated aldosterone secretion. Currently, we are working on how Gβ2-elicited inhibition of Cav3.2 channel activity may mediate the tonic inhibition of aldosterone production evoked by dopamine.
David Brautigan
Professor of Microbiology & Medicine
Director, Center for Cell Signaling
Research focus: Hormone Regulation of protein Ser(P)/Thr(P) Phosphatases
(434) 924-5892
Studies in this laboratory focus on understanding the molecular basis of regulation of Ser/Thr phosphatases and phosphatase inhibitor proteins by mitogen and insulin signals. Emphasis is on the major Ser(P)/Thr(P) phosphatases, PP1, PP2A, PP6 and PP2C which are studied using a combination of biochemical and cell biology methods. Insulin second messengers derived from membrane precursors activate oxidative and non-oxidative glucose disposal via PP2C phosphatases and working with Prof. Joe Larner we have shown these messengers act as allosteric activators of the phosphatase. Chromium enhances insulin signaling and glucose uptake, and we have shown this occurs through at least two mechanisms, one involving insulin receptor kinase activation, the other phosphorylation-independent activation of GLUT4. PP6 counteracts inflammatory cytokines such as TNF and Il-1 and we have found this enzyme is anchored in signaling complexes downstream from these receptors. Negative regulation of these phosphatases is an important component to insulin and hormone signaling.
David Castle
Professor of Cell Biology
Research focus: Regulation of endocrine/neuroendocrine secretion and trafficking of cell surface receptors regulating cell growth
(434) 924-1786
The roles of membrane proteins in regulating exocytosis and compensatory endocytosis of dense core vesicles and in organizing receptor down-regulation in endosomes are examined using expression of recombinant cDNAs, siRNA-mediated knockdown, and real-time analysis of secretion and membrane trafficking by amperometry, microscopic analysis of fluorescently-tagged proteins, and total internal reflection fluorescence (TIRF) microscopy. The emphasis is on proteins that couple exo- and endocytic events and that organize endocytic sorting sites.
William Evans
Professor of Medicine and Ob-Gyn
Research focus: Polycystic ovarian disease and hyperinsulinemia
(434) 924-5629
The relationship between the polycystic ovarian syndrome and hyperinsulinemia is being investigated with an emphasis on developing novel strategies for therapy that will improve rates of ovulation and subsequent pregnancy. The relationship of hyperinsulinemia to early pregnancy loss and potential therapeutic strategies to address this issue are also under investigation.
Leon Farhi
Assistant Professor of Research
Research focus: Intraislet network control of glucagon secretion
(434) 924-2496
Studies focus on pancreatic system-level regulation of glucagon secretion and counterregulation. We examine the hypothesis that in the face of beta cell deficiency the dominant factors controlling the release of glucagon unify dose-response interactions between delta cell somatostatin, alpha cell glucagon and blood glucose. Experimental work and advanced mathematical methods are combined to reconstruct the mechanisms by which alpha cell inhibitory "switch-off" signals restore glucagon counterregulation. The ultimate goal is to identify the endocrine intraislet network mechanisms that regulate glucagon secretion and response to hypoglycemia, and understand how they are altered in diabetes.
James Garrison
Professor and Chair of Pharmacology
Research focus: G-proteins in hormonal and chemokine signaling
(434) 924-5618
The overall goal of our research is to understand how the large number of G protein α and βγ isoforms lead to specificity in cell signaling, especially how the multiple isoforms of the βγ dimer selectively regulate signaling. The βγ dimer plays a major role in activating hematopoietic cells and we are attempting to understand the interaction between two major signaling pathways in the cell membrane, one utilized by Gi linked receptors to activate hematopoietic cells which mediate many of the vascular complications of diabetes and another used by Gs linked receptors to inhibit these cells. In neutrophils, macrophages and monocytes, activation of Gi coupled receptors such as those for chemokines, f-Met-Leu-Phe, S-1P or LPA release the G protein βγ dimer. This response activates a number of inflammatory responses including superoxide production, cell shape changes and cell migration. Two importatnt targets for Gβγ dimers are phosphatidylinositol 3-kinase (PI 3-kinase) and the Rac guanine nucleotide exchange factor (GEF), P-Rex1. Activation of PI 3-kinase generates PIP3 in the plasma membrane and Rac is a central regulator of cell shape changes and cell migration. As P-Rex1 activation is modulated by PIP3 and the G protein βγ subunit, the synergistic actions of the Gβγ dimer on PI 3-kinase and P-Rex1 combine to activate cells such as neutrophils, monocytes and macrophages. Importantly, these responses can be markedly inhibited by activation of G protein coupled receptors such as the β-adrenergic and adenosine A2a receptors which raise cyclic AMP levels. The major target of cyclic AMP in cells is the cyclic AMP depended protein kinase (PKA). Thus, phosphorylation of important regulatory sites in hematopoietic cells by PKA must be central to the inhibitory response. We have discovered that both PI 3-kinase and P-Rex1 can be phosphorylated in vitro by PKA with a marked reduction in their activity. We are also using small, inhibitory RNA's delivered to these cells by transfection or stable infection with lentiviruses to determine which isoforms of G proteins regulate PI 3-kinase and P-Rex1 activity in a cellular context.
Erik Hewlett
Professor of Medicine, Senior Associate Dean for Research
Research focus: Mechanism of bacterial toxin action and their use to study homeostasis
(434) 924-5945
Dr. Erik Hewlett’s research program operates at the interface between infectious diseases and cell signaling. During his training in the Clinical Endocrinology Branch of the National Institute of Arthritis, Metabolism and Digestive Diseases (name changed in the interim) Dr. Hewlett began his studies of several bacterial toxins that affect second messenger pathways and, as a result, he is especially interested in the uses of toxins as research tools in endocrinology and related fields. His work on pertussis toxin (PT), also known as “islet-activating protein”, includes investigation of the mechanisms by which PT-mediated ADP-ribosylation of heterotrimeric-G proteins affects insulin secretion and other regulatory processes in endocrine cells. Similarly, his studies of adenylate cyclase (AC) toxins, from Bordetella pertussis and Bacillus anthracis, are addressing their effects on regulation of progression through the cell cycle, as well as the mechanisms by which their intracellular signals are linked to effects, such as insulin release or apoptosis.
Susanna Keller
Assistant Professor of Internal Medicine and Cell Biology
Director Animal Characterization Core
Research focus: Insulin signaling and regulation of the trafficking of membrane proteins
(434) 243-5780
The present focus of this research is to establish the physiological function of the insulin-regulated aminopeptidase (IRAP) and the role it plays in insulin action. Research performed in the laboratory over the last few years has demonstrated that IRAP cleaves vasopressin and that insulin increases vasopressin clearance by IRAP in vivo. As kidney is one of the major targets of vasopressin action and IRAP is expressed in vasopressin target cells in the kidney, the role of IRAP in kidney function is currently evaluated in wild type, IRAP knockout and diabetic mouse models. It is well established that in adipocytes and myocytes cell surface expression of IRAP is regulated by insulin, and consequently cleavage of extracellular IRAP substrates increased. Thus, the regulation of the subcellular localization of IRAP by insulin and the intracellular IRAP-containing compartments are currently also characterized in the kidney. This research may uncover novel insulin actions in the kidney and elucidate molecular defects that lead to kidney complications in insulin-resistant and diabetic individuals.
Chien Li
Assistant Professor of Pharmacology
Research focus: Urocortin 3 regulation of food intake and energy expenditure
(434) 982-6752
The primary focus of the Li lab is to determine the functional importance and the underlying mechanisms of a novel anorectic neuropeptide, Urocortin 3 (Ucn 3), in the brain in regulating food intake and energy expenditure. In addition, they are also investigating the functional role of Ucn 3 in pancreatic β cells in modulating insulin secretion. These studies will make significant contribution in understanding the regulation of energy balance and provide valuable information for pharmacological interventions to treat obesity and diabetes.
Joel Linden
Professor of Medicine
Research focus: Role of A2B Adenosine Receptors in the Regulation of Glucose Metabolism
(434) 924-5600
There are four subtypes of G protein coupled adenosine receptors, A1, A2A, A2B, and A3. We have developed radioligand binding assays to all four receptor subtypes in multiple species (human, rat, mouse and dog). We have synthesized and characterized novel and receptor subtype selective antagonists of the A2B- receptor. Importantly, some of these new compounds are orally active and non-toxic in animals. These antagonists are insulin sensitizers. In insulin-resistant Zuker rats and KKAy mice they improve glucose tolerance, increase the rate of insulin-stimulated glucose uptake into skeletal muscle, and decrease hepatic gluconeogenesis. They also activate insulin-dependent signaling pathways in skeletal muscle, including membrane association of PKC-theta and AKT-phosphorylation. Studies using mice from with each adenosine receptor subtype has been individually deleted indicate confirm that these effects are mediated by blockade of the A2B receptor. We have used marker assisted genetic selection to move the A2B receptor KO locus onto the KKa background. These mice will be used for careful metabolic studies to investigate the effects of A2B blockers on long-term food inake and metabolism. A2B receptors blockers have potential as a new class of therapeutics for the treatment of type II diabetes.
Zhenqi Liu
Assistant Professor of Medicine
Research focus: Cardiovascular Actions of Insulin
(434) 982-0395
Insulin regulates vascular tone and tissue perfusion and is also a pro-survival hormone. Insulin deficiency and/or insulin resistance predisposes patients with diabetes to increased risk of microvascular and macrovascular complications. Dr. Liu’s research focuses on the regulation of insulin signal transduction in the vascular endothelium and myocardium and the mechanisms underlying insulin-mediated microvascular perfusion and protection against ischemia-reperfusion injury in the myocardium.
John C. Marshall
Professor of Medicine
Director- Center for Research in Reproduction
(434) 924-2431
Present research focuses on the etiology of polycystic ovarian syndrome (PCOS). Earlier work had demonstrated an association of hyperandrogenemia with insulin resistance and documented that insulin can act as a co-gonadotropin with LH to increase androgen production by the ovary. In addition, however, numerous studies had documented elevated levels of LH and a rapid frequency of LH (GnRH) secretion. The etiology of this remained unexplained and work by Dr. Marshall’s group provided the first demonstration that the ability of progesterone to slow the GnRH pulse generator was impaired by testosterone, and could be corrected with pre-treatment with the androgen receptor blocker flutamide. Other work demonstrated that a similar rapid frequency GnRH pulse secretion was present in early pubertal girls prior to menarche, indicating that the LH secretory abnormality preceded the onset of menses. Thus abnormal LH secretion is present in girls with hyperandrogenemia and present research focuses on the mechanisms causing hyperandrogenemia and its effects on regulation of GnRH secretion. Studies in progress have demonstrated that adolescent girls with hyperandrogenemia show similar impaired ability of progesterone to slow GnRH pulse frequency. Moreover, in search for the origins of androgen excess, note was made of the marked increase in prevalence of obesity in pre and early pubertal girls (NHANES data 2002), and ongoing work has shown that obese prepubertal girls (before clinical evidence of puberty) all demonstrate marked hyperandrogenemia. This is associated with elevated fasting insulin as expected from insulin resistance and associated abnormalities of Cytokines and Adipokinins such as elevated leptin, decreased adiponectin and PAI-I. Additional research has demonstrated that both progesterone and testosterone are secreted to excess in obese girls and show a diurnal pattern that resembles that of cortisol. This suggests an adrenal contribution and the mechanisms and role of the adrenal in producing hyperandrogenemia in adolescents are being investigated.
In summary, current research focuses on the role of elevated plasma testosterone, before or during pubertal maturation, in modifying hypothalamic regulation of GnRH pulse secretion, leading to elevated levels of LH and maintaining the androgen excess in these young women.
Raghu Mirmira
Associate Professor of Medicine and Pharmacology
Research focus: Transcriptional regulation of beta-cell development
(434) 982-0966
Dr. Mirmira’s laboratory focuses on the regulation of gene transcription in the developing and mature islet. The projects in his laboratory fall into two broad categories: (1) the mechanisms of transcriptional regulation by the factors Pdx-1 and Nkx6.1, and (2) the interrelationships between chromatin structure and gene transcription in the islet. Pdx-1 is considered an important “master” regulator of pancreas development, and also a crucial regulator of β cell-specific genes (insulin, glut2, glucokinase) in the mature β cell. Nkx6.1, on the other hand, is dispensible for pancreas formation, but necessary for the formation and function of the β cell. His group has shown that Pdx-1 directly activates both the Nkx6.1 gene and the insulin gene. The mechanisms of gene activation by Pdx-1 involve direct binding to key elements in the promoter region of the respective genes, activation of basal transcripitonal machinery (through the phosphorylation of Ser2 of RNA polymerase II), and the recruitment of chromatin modifying enzymes that favorably alter the structure of the transcriptional template. Nkx6.1, by contrast, appears to function largely as a repressor of transcription by coordinating the assembly of a “repressor” complex involving mammalian Groucho and histone deacetylases; the targets of repression include the glucagon gene, among others. Together, these factors coordinately maintain the phenotype of the β cell and its ability to appropriately secrete insulin in response to glucose. Ongoing work in the lab focuses on how transcriptional complexes are assembled and how these complexes relate to the basal transcriptional machinery.
A second focus of the laboratory has been on the relationship of gene expression in the β cell to chromatin structure. This area of research as been largely unexplored and Dr. Mirmira is considered one of the leaders in this field. His lab has shown that chromatin exists in a unique “open” state at key genetic loci within the β cell (insulin, glut2, glucokinase); this state of “open” chromatin is marked by hyperacetylation of histones H3 and H4, and a hypermethylation of histone H3 at position Lys4. This latter modification appears to be mediated by a complex that includes Pdx-1 and the histone methyltransferase Set9. Interestingly, Set9 expression appears highly specific for the islet in the pancreas. Ongoing work in his lab now focuses on how histone modifications are specifically achieved and maintained in the β cell, with a particular focus on the role of Set9. His lab is using both biochemical and cell biological techniques to address these questions, and is also using genetically-modified mice in which histone-modifying enzymes are specifically deleted in islet cells.
Suzanne Moenter
Associate Professor of Medicine and Cell Biology
Research focus: Reproductive/metabolic interactions in polycystic ovary syndrome (PCOS)
(434) 982-0076
This lab uses an electrophysiological approach to study gonadotropin-releasing hormone (GnRH) neurons and much of the work focuses on murine models of PCOS. Prenatal androgenization (PNA) produces a reproductive phenotype resembling this disorder and this laboratory has now demonstrated PNA mice are also glucose intolerant. Ongoing studies are defining the mechanisms of this metabolic phenotype by examining islet and adipocyte function in these mice and how these interact with the elevated circulating androgens of this disorder to alter central neural drive to reproduction via GnRH release.
John Marshall
Professor of Medicine
Research focus: Polycystic Ovarian Disease
(434) 924-2431
Present research focuses on the etiology of polycystic ovarian syndrome (PCOS). Earlier work had demonstrated an association of hyperandrogenemia with insulin resistance and documented that insulin can act as a co-gonadotropin with LH to increase androgen production by the ovary. Moreover, in search for the origins of androgen excess, note was made of the marked increase in prevalence of obesity in pre and early pubertal girls (NHANES data 2002), and ongoing work has shown that obese prepubertal girls (before clinical evidence of puberty) all demonstrate marked hyperandrogenemia. This is associated with elevated fasting insulin as expected from insulin resistance and associated abnormalities of Cytokines and Adipokinins such as elevated leptin, decreased adiponectin and PAI-I. Additional research has demonstrated that both progesterone and testosterone are secreted to excess in obese girls and show a diurnal pattern that resembles that of cortisol. This suggests an adrenal contribution and the mechanisms and role of the adrenal in producing hyperandrogenemia in adolescents are being investigated.
Julie Sando
Professor of Anesthesiology
Research focus: PKC Structure and function
(434) 924-5020
The major interest of this laboratory is in activation of Protein Kinase C (PKC) isozymes and in roles of these enzymes in cellular signaling networks. PKC family members are activated by association with cell membranes and help to transduce a variety of extracellular signals. Most cells have several PKC isozymes. Alterations in isozyme expression or activation have been found in a number of diseases including some cancers and some endocrine, neurological, and cardiovascular diseases. Properties of membrane lipids that activate PKC are studied using biochemical and biophysical techniques. Structural analysis of PKC is conducted in collaboration with Dr. Kretsinger (Biology) via generation of 2-dimensional crystals on defined lipid monolayers and with Drs. Grisham and Cafiso (Chemistry) using NMR and EPR. The goal of this work is to understand the structure and activation of the enzymes and the role of individual isozymes in specific cellular processes. This understanding should facilitate design of more specific PKC activators and inhibitors for clinical use. A commercial PKCβ inhibitor is useful in diabetic retinopathy.
Heidi Scrable
Associate Professor of Neuroscience
Research focus: The role of p53 in metabolic signaling pathways
(434) 982-1416
p53 is a transcription factor that regulates cell proliferation, playing a major role as a suppressor of tumorigenesis. We have found that the molecular mechanisms by which p53 controls cell proliferation include pathways mediated by insulin and the insulin-like growth factors. When p53 activity is experimentally enhanced in mice, the insulin and IGF-1 receptors are constitutively activated and cells undergo growth arrest. We would like to understand how enhanced activity of the tumor suppressor p53 leads to loss of pancreatic beta-cells, hyperglycemia, and diabetes in young adult mice.
Thomas Sturgill
Professor of Pharmacology & Medicine
Research focus: Protein kinases in intracellular signaling cascades
(434) 924-8659
His laboratory is presently working in three major areas, all of which are relevant to basic understanding of diabetes and diabetic complications. (i) Classic Map kinases and MAPKAP kinases. (ii) novel TDY motif kinases related to CDKs and MAPKs. The work on the protein kinase MAK-related protein kinase (MRK) has led to a proposal submitted to the Juvenile Diabetes Association and the American Diabetes Association. (iii) the TOR signaling pathways in yeast. Dr. Sturgill will be on sabbatical during 2007 at the Biozentrum of the University of Basel to continue his work in this area.
Michael Thorner
Professor of Medicine
Research focus: GH regulation, GHRH and aging
(434) 982-3297
The regulation of growth hormone secretion and action both in vivo and in vitro has been a major focus of the laboratory. This laboratory first described and isolated GHRH using a human pancreatic tumor as a source. They also cloned the GHRH receptor, a 423 amino acid peptide with seven membrane-spanning units of the B Family of G protein coupled receptors. The focus of the laboratory has been to evaluate the effect of an oral GH secretagogue on increasing pulsatile GH secretion in elderly subjects and evaluating the effects on metabolism and body composition, in addition to its effects on quality of life and function.
In short one year of treatment was able to increase fat free mass by 1.5 kg compared to placebo, and this was associated with an increase in pulsatile GH secretion and serum IGF-I into the normal range for that of 20-30 year old subjects. This is probably the most promising approach to combating sarcopenia of aging. The current focus of the laboratory is elucidation of the interaction of GHRH with its receptor and the lessons learned appear to be applicable to other members of the B family of receptors including GLP-1 and glucagon. Working together with H. Mario Geysen in the Department of Chemistry we have made over 300 analogs of GHRH which have unique structures and properties. These developments and intellectual property are now being translated into the creation of a start up company to develop novel therapeutic agents.
Michael J. Weber
Professor of Microbiology
Director UVA Cancer Center
Research focus: Kinase cascades in cell regulation
(434) 924-5022
The MAP Kinase cascade is an evolutionary conserved regulatory module involved in a wide variety of cellular activities. It is activated in response to many extracellular signals, including insulin, and generates a diverse portfolio of biological responses. Our laboratory focuses on ways that scaffold proteins can regulate the activation, timing, and intracellular location of the components of this module so that specific outcomes can be generated from this ubiquitous system.