Recovery of viable endocrine-specific cells and transcriptomes from human pancreatic islet-engrafted mice.

Human pancreatic islets engrafted into immunodeficient mice serve as an important model for in vivo human diabetes studies. Following engraftment, islet function can be monitored in vivo by measuring circulating glucose and human insulin; however, it will be important to recover viable cells for more complex graft analyses. Moreover, RNA analyses of dissected grafts have not distinguished which hormone-specific cell types contribute to gene expression. We developed a method for recovering live cells suitable for fluorescence-activated cell sorting from human islets engrafted in mice. Although yields of recovered islet cells were relatively low, the ratios of bulk-sorted ß, α, and δ cells and their respective hormone-specific RNA-Seq transcriptomes are comparable pretransplant and posttransplant, suggesting that the cellular characteristics of islet grafts posttransplant closely mirror the original donor islets. Single-cell RNA-Seq transcriptome analysis confirms the presence of appropriate ß, α, and δ cell subsets. In addition, ex vivo perifusion of recovered human islet grafts demonstrated glucose-stimulated insulin secretion. Viable cells suitable for patch-clamp analysis were recovered from transplanted human embryonic stem cell-derived ß cells. Together, our functional and hormone-specific transcriptome analyses document the broad applicability of this system for longitudinal examination of human islet cells undergoing developmental/metabolic/pharmacogenetic manipulation in vivo and may facilitate the discovery of treatments for diabetes.

HLA Class II Antigen Processing and Presentation Pathway Components Demonstrated by Transcriptome and Protein Analyses of Islet ß-Cells From Donors With Type 1 Diabetes.

Type 1 diabetes studies consistently generate data showing islet ß-cell dysfunction and T cell-mediated anti-ß-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the ß-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single ß-cells. Consistent with immunohistological studies, ß-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These ß-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin+CD68- ß-cells, specifically found in islets with lymphocytic infiltrates. ß-Cell surface expression of HLA Class II was detected on a portion of CD45-insulin+ ß-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic ß-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. ß-Cell expression of Class II molecules suggests that ß-cells may interact directly with islet-infiltrating CD4+ T cells and may play an immunopathogenic role.

α Cell Function and Gene Expression Are Compromised in Type 1 Diabetes.

Many patients with type 1 diabetes (T1D) have residual ß 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 ß cells and α 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 ß cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and ß 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 α-to-ß cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes.

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood-derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2-DQ8 heterodimer-restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Surprising Heterogeneity of Pancreatic Islet Cell Subsets.

Two studies clearly demonstrate that pancreatic islets and, more specifically, their cellular constituents, display a much greater complexity than previously appreciated.

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'-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 ß-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.

Novel Observations From Next-Generation RNA Sequencing of Highly Purified Human Adult and Fetal Islet Cell Subsets.

Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and ß-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase ß-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, ß-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and ß-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal ß-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and ß-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

The pancreatic ß-cell transcriptome and integrated-omics.

PURPOSE OF REVIEW: ß Cells represent one of many cell types in heterogeneous pancreatic islets and play the central role in maintaining glucose homeostasis, such that disrupting ß-cell function leads to diabetes. This review summarizes the methods for isolating and characterizing ß cells, and describes integrated 'omics' approaches used to define the ß cell by its transcriptome and proteome. RECENT FINDINGS: RNA sequencing and mass spectrometry-based protein identification have now identified RNA and protein profiles for mouse and human pancreatic islets and ß cells, and for ß-cell lines. Recent publications have outlined these profiles and, more importantly, have begun to assign the presence or absence of specific genes and regulatory molecules to ß-cell function and dysfunction. Overall, researchers have focused on understanding the pathophysiology of diabetes by connecting genome, transcriptome, proteome, and regulatory RNA profiles with findings from genome-wide association studies. SUMMARY: Studies employing these relatively new techniques promise to identify specific genes or regulatory RNAs with altered expression as ß-cell function begins to deteriorate in the spiral toward the development of diabetes. The ultimate goal is to identify the potential therapeutic targets to prevent ß-cell dysfunction and thereby better treat the individual with diabetes. VIDEO ABSTRACT: http://links.lww.com/COE/A5.

Disease allele-dependent small-molecule sensitivities in blood cells from monogenic diabetes.

Even as genetic studies identify alleles that influence human disease susceptibility, it remains challenging to understand their functional significance and how they contribute to disease phenotypes. Here, we describe an approach to translate discoveries from human genetics into functional and therapeutic hypotheses by relating human genetic variation to small-molecule sensitivities. We use small-molecule probes modulating a breadth of targets and processes to reveal disease allele-dependent sensitivities, using cells from multiple individuals with an extreme form of diabetes (maturity onset diabetes of the young type 1, caused by mutation in the orphan nuclear receptor HNF4α). This approach enabled the discovery of small molecules that show mechanistically revealing and therapeutically relevant interactions with HNF4α in both lymphoblasts and pancreatic ß-cells, including compounds that physically interact with HNF4α. Compounds including US Food and Drug Administration-approved drugs were identified that favorably modulate a critical disease phenotype, insulin secretion from ß-cells. This method may suggest therapeutic hypotheses for other nonblood disorders.

Analysis of glucose transporter topology and structural dynamics.

Homology modeling and scanning cysteine mutagenesis studies suggest that the human glucose transport protein GLUT1 and its distant bacterial homologs LacY and GlpT share similar structures. We tested this hypothesis by mapping the accessibility of purified, reconstituted human erythrocyte GLUT1 to aqueous probes. GLUT1 contains 35 potential tryptic cleavage sites. Fourteen of 16 lysine residues and 18 of 19 arginine residues were accessible to trypsin. GLUT1 lysine residues were modified by isothiocyanates and N-hydroxysuccinimide (NHS) esters in a substrate-dependent manner. Twelve lysine residues were accessible to sulfo-NHS-LC-biotin. GLUT1 trypsinization released full-length transmembrane helix 1, cytoplasmic loop 6-7, and the long cytoplasmic C terminus from membranes. Trypsin-digested GLUT1 retained cytochalasin B and d-glucose binding capacity and released full-length transmembrane helix 8 upon cytochalasin B (but not D-glucose) binding. Transmembrane helix 8 release did not abrogate cytochalasin B binding. GLUT1 was extensively proteolyzed by alpha-chymotrypsin, which cuts putative pore-forming amphipathic alpha-helices 1, 2, 4, 7, 8, 10, and 11 at multiple sites to release transmembrane peptide fragments into the aqueous solvent. Putative scaffolding membrane helices 3, 6, 9, and 12 are strongly hydrophobic, resistant to alpha-chymotrypsin, and retained by the membrane bilayer. These observations provide experimental support for the proposed GLUT1 architecture; indicate that the proposed topology of membrane helices 5, 6, and 12 requires adjustment; and suggest that the metastable conformations of transmembrane helices 1 and 8 within the GLUT1 scaffold destabilize a sugar translocation intermediate.

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.

Quench-flow analysis reveals multiple phases of GluT1-mediated sugar transport.

Standard models for carrier-mediated nonelectrolyte transport across cell membranes do not explain sugar uptake by human red blood cells. This means that either (1) the models for sugar transport are incorrect or (2) measurements of sugar transport are flawed. Most measurements of red cell sugar transport have been made over intervals of 10 s or greater, a range which may be too long to measure transport accurately. In the present study, we examine the time course of sugar uptake over intervals as short as 5 ms to periods as long as 8 h. Using conditions where transport by a uniform population of cells is expected to be monophasic (use of subsaturating concentrations of a nonmetabolizable but transported sugar, 3-O-methylglucose), our studies demonstrate that red cell sugar uptake is comprised of three sequential, protein-mediated events (rapid, fast, and slow). The rapid phase is more strongly temperature-dependent than the fast and slow phases. All three phases are inhibited by extracellular (maltose or phloretin) or intracellular (cytochalasin B) sugar-transport inhibitors. The rate constant for the rapid phase of uptake is independent of the 3-O-methylglucose concentration. The magnitude (moles of sugar associated with cells) of the rapid phase increases in a saturable manner with [3-O-methylglucose] and is similar to (1) the amount of sugar that is retained by red cell membrane proteins upon addition of cytochalasin B and phloretin and (2) the d-glucose inhibitable cytochalasin B binding capacity of red cell membranes. These results are consistent with the hypothesis that previous studies have both under- and overestimated the rate of erythrocyte sugar transport. These data support a transport mechanism in which newly bound sugars are transiently sequestered within the translocation pathway where they become inaccessible to extra- and intracellular water.

Conventional transport assays underestimate sugar transport rates in human red cells.

The time course of protein-mediated 3-O-methylglucose uptake by human red cells and by red cell ghosts containing or lacking 4 mM MgATP was measured at ice temperature and sub-saturating sugar levels by conventional sampling procedures and at 20 degrees C by use of a quench-flow apparatus. The temporal resolution of the quench-flow apparatus (as fast as 5-ms sample times) was confirmed by analysis of alkaline hydrolysis of dinitrophenolacetate. Red cell sugar uptake at 4 degrees C is consistent with two processes [fast (tau = 120 s) and slow (tau = 1100 s)] that occur in series. Intracellular ATP increases the size and the rate of equilibration of the fast compartment and slows the rate of filling of the slow compartment. Red cell ghost volume and protein content are unaffected by lysis/resealing in the presence of ATP. Uptake at 20 degrees C is also consistent with two processes [fast (tau = 10 ms) and slow (tau = 15 s)] that occur in series. ATP increases the size of both compartments and the rate of filling of the small compartment at 20 degrees C. Preliminary estimates indicate that the sugar uptake capacity of human red cells at 20 degrees C is underestimated by as much as 8-fold by measuring sugar uptake over 2 s vs. 26 ms. We discuss the implications of multiphasic sugar uptake in the context of models for protein-mediated sugar transport.