Human Erythrocyte Glucose Transporter (GLUT1) Structure, Function, and Regulation: A Dissertation

The structure-function relationship explains how the human erythrocyte glucose transport protein (GLUT1) catalyzes sugar transport across the plasma membrane. This work investigates the glucose transport mechanism, the structural arrangement and dynamics of GLUT1 membrane-spanning α-helices, the molecular basis for glucose transport regulation by ATP, and how cysteine accessibility contributes to GLUT1 structure. A rapid kinetics approach was applied to examine the conformational changes GLUT1 undergoes during the transport cycle. To transition from a global to molecular focus, a novel mass spectrometry technique was developed to resolve GLUT1 sequence that is associated either with membrane embedded GLUT1 subdomains or with water exposed domains. By studying accessibility changes of specific amino acids to covalent modification by a Sulfo-NHS-LC-Biotin probe, specific protein regions associated with glucose transport modulation by ATP were identified. Finally, mass spectrometry was applied to examine cysteine residue accessibility under native and reducing conditions. This thesis presents data supporting the isolation of an intermediate, occluded GLUT1 conformational state that temporally bridges import and export configurations during glucose translocation. Our results confirm that amphipathic α-helices line the translocation pathway and promote interactions with the aqueous environment and substrate. In addition, we show that GLUT1 is conformationally dynamic, undergoes reorganization in the cytoplasmic region in response to ATP modulation, and that GLUT1 contains differentially exposed cysteine residues that affect its folding

Gene Expression and Profiling of Human Islet Cell Subtypes: A Master’s Thesis

Background: The endocrine pancreas contains multiple cell types co-localized into clusters called the Islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential for normal metabolism and to prevent diabetes and complications from the disease. Given the heterogeneous nature of islet composition and absence of unique surface markers, many previous studies have focused on the whole islet. Sorting islet cells by intracellular hormone expression overcomes this limitation and provides pure populations of individual islet cell subsets, specifically alpha and beta cells. This technique provides the framework for characterizing human islet composition and will work towards identifying the genetic changes alpha and beta cells undergo during development, growth, and proliferation. Methods: Human islets obtained from cadaveric donors are dissociated into a single cell suspension, fixed, permeabilized, and labeled with antibodies specific to glucagon, insulin, and somatostatin. Individual alpha, beta, and delta cell populations are simultaneously isolated using fluorescence activated cell sorting. Candidate gene expression and microRNA profiles have been obtained for alpha and beta cell populations using a quantitative nuclease protection assay. Thus far, RNA has been extracted from whole islets and beta cells and subjected to next generation sequencing analysis. Results: The ratio of beta to alpha cells significantly increases with donor age and trends higher in female donors; BMI does not appear to significantly alter the ratio. Further, we have begun to investigate the unique gene expression profiles of alpha and beta cells versus whole islets, and have characterized the microRNA profiles of the two cell subsets. Conclusions: By establishing methods to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein expression patterns change under environmental conditions that lead to beta cell failure or promote beta cell development, growth, and proliferation

Gene Expression and Profiling of Human Islet Cell Subtypes

The endocrine pancreas contains multiple cell types co-localized into clusters called the islets of Langerhans. The predominant cell types include alpha and beta cells, which produce glucagon and insulin, respectively. The regulated release of these hormones maintains whole body glucose homeostasis, essential to prevent complications from diabetes (e.g. blindness, kidney failure, and cardiovascular disease). In type 1 diabetes, an autoimmune reaction destroys the beta cells and patients must monitor their blood sugar levels and inject insulin in order to maintain euglycemia. In type 2 diabetes, the beta cells fail to produce sufficient insulin to overcome the individual’s decreased insulin sensitivity. Most studies to date have focused on whole islets, which are very heterogeneous. Recent focus has shifted to studying the individual islet cell subsets (i.e. alpha, beta, delta, PP, and other cell types). Unlike immunological cells, surface molecule reagents do not yet exist to specifically distinguish beta from alpha cells. We have successfully isolated pure populations of insulin producing beta cells and glucagon producing alpha cells by using intracellular hormone staining and fluorescence activated cell sorting. We present data that describe the ratio of beta cells to alpha cells across gender, age, and BMI. Further, we have characterized the miRNA profiles of alpha and beta cells and have begun to investigate the unique gene expression patterns of the two cell types. By developing the ability to profile multiple characteristics of alpha and beta cells, we hope to determine how gene, miRNA, and protein profiles change under environmental conditions that lead to beta cell failure, and others that may promote beta cell health or stimulate beta cell growth and proliferation

Gene Expression Profiling of Islet Cell Subtypes

Abstract Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and proliferation. We have shown that we can purify adult human islets into individual cellular populations. This is the first step in understanding the genetic and environmental components that regulate increased beta cell proliferation and beta cell mass. In the absence of full-length mRNA for RT-PCR or next generation sequencing, the qNPA technique provides candidate gene expression profiles for these cells

alpha Cell Function and Gene Expression Are Compromised in Type 1 Diabetes

Many patients with type 1 diabetes (T1D) have residual beta cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual beta cells and alpha cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant beta cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D alpha cells was markedly reduced, and these cells had alterations in transcription factors constituting alpha and beta cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of alpha-to-beta cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia

Structural Basis of GLUT1 Inhibition by Cytoplasmic ATP

Cytoplasmic ATP inhibits human erythrocyte glucose transport protein (GLUT1)–mediated glucose transport in human red blood cells by reducing net glucose transport but not exchange glucose transport (Cloherty, E.K., D.L. Diamond, K.S. Heard, and A. Carruthers. 1996. Biochemistry. 35:13231–13239). We investigated the mechanism of ATP regulation of GLUT1 by identifying GLUT1 domains that undergo significant conformational change upon GLUT1–ATP interaction. ATP (but not GTP) protects GLUT1 against tryptic digestion. Immunoblot analysis indicates that ATP protection extends across multiple GLUT1 domains. Peptide-directed antibody binding to full-length GLUT1 is reduced by ATP at two specific locations: exofacial loop 7–8 and the cytoplasmic C terminus. C-terminal antibody binding to wild-type GLUT1 expressed in HEK cells is inhibited by ATP but binding of the same antibody to a GLUT1–GLUT4 chimera in which loop 6–7 of GLUT1 is substituted with loop 6–7 of GLUT4 is unaffected. ATP reduces GLUT1 lysine covalent modification by sulfo-NHS-LC-biotin by 40%. AMP is without effect on lysine accessibility but antagonizes ATP inhibition of lysine modification. Tandem electrospray ionization mass spectrometry analysis indicates that ATP reduces covalent modification of lysine residues 245, 255, 256, and 477, whereas labeling at lysine residues 225, 229, and 230 is unchanged. Exogenous, intracellular GLUT1 C-terminal peptide mimics ATP modulation of transport whereas C-terminal peptide-directed IgGs inhibit ATP modulation of glucose transport. These findings suggest that transport regulation involves ATP-dependent conformational changes in (or interactions between) the GLUT1 C terminus and the C-terminal half of GLUT1 cytoplasmic loop 6–7

Strophatrypa, a new genus of Brachiopoda (Atrypidae), from upper Silurian strata of the Alexander terrane, Southeast Alaska

The atrypid brachiopod Strophatrypa skaflestadi gen. et sp. nov. occurs in unnamed upper Silurian strata exposed near the town of Hoonah on northeast Chichagof Island, Southeast Alaska. The new genus represents the first strophic atrypoid with many characters otherwise similar to those of the Atrypidae. Although this genus is known from only a single bed at one Alaskan locality, its unique morphology is worth noting in anticipation that similar forms may be discovered elsewhere in the Alexander terrane, or especially somewhere in Siberia where other taxa restricted to the Silurian of the Alexander terrane are shared. This paper represents a further contribution to our limited knowledge of the Silurian brachiopods of the Alexander terrane of Southeast Alaska.Keywords: new genus, Southeast Alaska, U.S.A., palaeogeography, Alexander terrane, late Silurian, Brachiopoda, palaeoecology, slope facie

The Application of the Haddon Matrix to Public Health Readiness and Response Planning

State and local health departments continue to face unprecedented challenges in preparing for, recognizing, and responding to threats to the public’s health. The attacks of 11 September 2001 and the ensuing anthrax mailings of 2001 highlighted the public health readiness and response hurdles posed by intentionally caused injury and illness. At the same time, recent natural disasters have highlighted the need for comparable public health readiness and response capabilities. Public health readiness and response activities can be conceptualized similarly for intentional attacks, natural disasters, and human-caused accidents. Consistent with this view, the federal government has adopted the all-hazards response model as its fundamental paradigm. Adoption of this paradigm provides powerful improvements in efficiency and efficacy, because it reduces the need to create a complex family of situation-specific preparedness and response activities. However, in practice, public health preparedness requires additional models and tools to provide a framework to better understand and prioritize emergency readiness and response needs, as well as to facilitate solutions; this is particularly true at the local health department level. Here, we propose to extend the use of the Haddon matrix—a conceptual model used for more than two decades in injury prevention and response strategies—for this purpose

End Sequence Analysis Toolkit (ESAT) expands the extractable information from single-cell RNA-seq data

RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3\u27-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct beta-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing

Engaging the Dynamics of Pastoral Imagination for Field Education

The importance and the process of engaging pastoral imagination in field education