Type 2 diabetes candidate genes, including PAX5, cause impaired insulin secretion in human pancreatic islets.

Type 2 diabetes (T2D) is caused by insufficient insulin secretion from pancreatic ß cells. To identify candidate genes contributing to T2D pathophysiology, we studied human pancreatic islets from approximately 300 individuals. We found 395 differentially expressed genes (DEGs) in islets from individuals with T2D, including, to our knowledge, novel (OPRD1, PAX5, TET1) and previously identified (CHL1, GLRA1, IAPP) candidates. A third of the identified expression changes in islets may predispose to diabetes, as expression of these genes associated with HbA1c in individuals not previously diagnosed with T2D. Most DEGs were expressed in human ß cells, based on single-cell RNA-Seq data. Additionally, DEGs displayed alterations in open chromatin and associated with T2D SNPs. Mouse KO strains demonstrated that the identified T2D-associated candidate genes regulate glucose homeostasis and body composition in vivo. Functional validation showed that mimicking T2D-associated changes for OPRD1, PAX5, and SLC2A2 impaired insulin secretion. Impairments in Pax5-overexpressing ß cells were due to severe mitochondrial dysfunction. Finally, we discovered PAX5 as a potential transcriptional regulator of many T2D-associated DEGs in human islets. Overall, we have identified molecular alterations in human pancreatic islets that contribute to ß cell dysfunction in T2D pathophysiology.

Islet Gene View-a tool to facilitate islet research.

Characterization of gene expression in pancreatic islets and its alteration in type 2 diabetes (T2D) are vital in understanding islet function and T2D pathogenesis. We leveraged RNA sequencing and genome-wide genotyping in islets from 188 donors to create the Islet Gene View (IGW) platform to make this information easily accessible to the scientific community. Expression data were related to islet phenotypes, diabetes status, other islet-expressed genes, islet hormone-encoding genes and for expression in insulin target tissues. The IGW web application produces output graphs for a particular gene of interest. In IGW, 284 differentially expressed genes (DEGs) were identified in T2D donor islets compared with controls. Forty percent of DEGs showed cell-type enrichment and a large proportion significantly co-expressed with islet hormone-encoding genes; glucagon (<i>GCG</i>, 56%), amylin (<i>IAPP</i>, 52%), insulin (<i>INS</i>, 44%), and somatostatin (<i>SST</i>, 24%). Inhibition of two DEGs, <i>UNC5D</i> and <i>SERPINE2</i>, impaired glucose-stimulated insulin secretion and impacted cell survival in a human ß-cell model. The exploratory use of IGW could help designing more comprehensive functional follow-up studies and serve to identify therapeutic targets in T2D.

A critical role of the mechanosensor PIEZO1 in glucose-induced insulin secretion in pancreatic ß-cells.

Glucose-induced insulin secretion depends on ß-cell electrical activity. Inhibition of ATP-regulated potassium (KATP) channels is a key event in this process. However, KATP channel closure alone is not sufficient to induce ß-cell electrical activity; activation of a depolarizing membrane current is also required. Here we examine the role of the mechanosensor ion channel PIEZO1 in this process. Yoda1, a specific PIEZO1 agonist, activates a small membrane current and thereby triggers ß-cell electrical activity with resultant stimulation of Ca2+-influx and insulin secretion. Conversely, the PIEZO1 antagonist GsMTx4 reduces glucose-induced Ca2+-signaling, electrical activity and insulin secretion. Yet, PIEZO1 expression is elevated in islets from human donors with type-2 diabetes (T2D) and a rodent T2D model (db/db mouse), in which insulin secretion is reduced. This paradox is resolved by our finding that PIEZO1 translocates from the plasmalemma into the nucleus (where it cannot influence the membrane potential of the ß-cell) under experimental conditions emulating T2D (high glucose culture). ß-cell-specific Piezo1-knockout mice show impaired glucose tolerance in vivo and reduced glucose-induced insulin secretion, ß-cell electrical activity and Ca2+ elevation in vitro. These results implicate mechanotransduction and activation of PIEZO1, via intracellular accumulation of glucose metabolites, as an important physiological regulator of insulin secretion.

Alternative splicing encodes functional intracellular CD59 isoforms that mediate insulin secretion and are down-regulated in diabetic islets.

Human pancreatic islets highly express CD59, which is a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein and is required for insulin secretion. How cell-surface CD59 could interact with intracellular exocytotic machinery has so far not been described. We now demonstrate the existence of CD59 splice variants in human pancreatic islets, which have unique C-terminal domains replacing the GPI-anchoring signal sequence. These isoforms are found in the cytosol of ß-cells, interact with SNARE proteins VAMP2 and SNAP25, colocalize with insulin granules, and rescue insulin secretion in CD59-knockout (KO) cells. We therefore named these isoforms IRIS-1 and IRIS-2 (Isoforms Rescuing Insulin Secretion 1 and 2). Antibodies raised against each isoform revealed that expression of both IRIS-1 and IRIS-2 is significantly lower in islets isolated from human type 2 diabetes (T2D) patients, as compared to healthy controls. Further, glucotoxicity induced in primary, healthy human islets led to a significant decrease of IRIS-1 expression, suggesting that hyperglycemia (raised glucose levels) and subsequent decreased IRIS-1 expression may contribute to relative insulin deficiency in T2D patients. Similar isoforms were also identified in the mouse CD59B gene, and targeted CRISPR/Cas9-mediated knockout showed that these intracellular isoforms, but not canonical CD59B, are involved in insulin secretion from mouse ß-cells. Mouse IRIS-2 is also down-regulated in diabetic db/db mouse islets. These findings establish the endogenous existence of previously undescribed non­GPI-anchored intracellular isoforms of human CD59 and mouse CD59B, which are required for normal insulin secretion.

Genetic variant effects on gene expression in human pancreatic islets and their implications for T2D.

Most signals detected by genome-wide association studies map to non-coding sequence and their tissue-specific effects influence transcriptional regulation. However, key tissues and cell-types required for functional inference are absent from large-scale resources. Here we explore the relationship between genetic variants influencing predisposition to type 2 diabetes (T2D) and related glycemic traits, and human pancreatic islet transcription using data from 420 donors. We find: (a) 7741 cis-eQTLs in islets with a replication rate across 44 GTEx tissues between 40% and 73%; (b) marked overlap between islet cis-eQTL signals and active regulatory sequences in islets, with reduced eQTL effect size observed in the stretch enhancers most strongly implicated in GWAS signal location; (c) enrichment of islet cis-eQTL signals with T2D risk variants identified in genome-wide association studies; and (d) colocalization between 47 islet cis-eQTLs and variants influencing T2D or glycemic traits, including DGKB and TCF7L2. Our findings illustrate the advantages of performing functional and regulatory studies in disease relevant tissues.

Loss of ZnT8 function protects against diabetes by enhanced insulin secretion.

A rare loss-of-function allele p.Arg138* in SLC30A8 encoding the zinc transporter 8 (ZnT8), which is enriched in Western Finland, protects against type 2 diabetes (T2D). We recruited relatives of the identified carriers and showed that protection was associated with better insulin secretion due to enhanced glucose responsiveness and proinsulin conversion, particularly when compared with individuals matched for the genotype of a common T2D-risk allele in SLC30A8, p.Arg325. In genome-edited human induced pluripotent stem cell (iPSC)-derived ß-like cells, we establish that the p.Arg138* allele results in reduced SLC30A8 expression due to haploinsufficiency. In human ß cells, loss of SLC30A8 leads to increased glucose responsiveness and reduced KATP channel function similar to isolated islets from carriers of the T2D-protective allele p.Trp325. These data position ZnT8 as an appealing target for treatment aimed at maintaining insulin secretion capacity in T2D.

Decreased Expression of Cilia Genes in Pancreatic Islets as a Risk Factor for Type 2 Diabetes in Mice and Humans.

An insufficient adaptive beta-cell compensation is a hallmark of type 2 diabetes (T2D). Primary cilia function as versatile sensory antennae regulating various cellular processes, but their role on compensatory beta-cell replication has not been examined. Here, we identify a significant enrichment of downregulated, cilia-annotated genes in pancreatic islets of diabetes-prone NZO mice as compared with diabetes-resistant B6-ob/ob mice. Among 327 differentially expressed mouse cilia genes, 81 human orthologs are also affected in islets of diabetic donors. Islets of nondiabetic mice and humans show a substantial overlap of upregulated cilia genes that are linked to cell-cycle progression. The shRNA-mediated suppression of KIF3A, essential for ciliogenesis, impairs division of MIN6 beta cells as well as in dispersed primary mouse and human islet cells, as shown by decreased BrdU incorporation. These findings demonstrate the substantial role of cilia-gene regulation on islet function and T2D risk.

Complement Component C3 Is Highly Expressed in Human Pancreatic Islets and Prevents ß Cell Death via ATG16L1 Interaction and Autophagy Regulation.

We show here that human pancreatic islets highly express C3, which is both secreted and present in the cytosol. Within isolated human islets, C3 expression correlates with type 2 diabetes (T2D) donor status, HbA1c, and inflammation. Islet C3 expression is also upregulated in several rodent diabetes models. C3 interacts with ATG16L1, which is essential for autophagy. Autophagy relieves cellular stresses faced by ß cells during T2D and maintains cellular homeostasis. C3 knockout in clonal ß cells impaired autophagy and led to increased apoptosis after exposure of cells to palmitic acid and IAPP. In the absence of C3, autophagosomes do not undergo fusion with lysosomes. Thus, C3 may be upregulated in islets during T2D as a cytoprotective factor against ß cell dysfunction caused by impaired autophagy. Therefore, we revealed a previously undescribed intracellular function for C3, connecting the complement system directly to autophagy, with a broad potential importance in other diseases and cell types.

Genetic determinants of circulating GIP and GLP-1 concentrations.

The secretion of insulin and glucagon from the pancreas and the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) from the gastrointestinal tract is essential for glucose homeostasis. Several novel treatment strategies for type 2 diabetes (T2D) mimic GLP-1 actions or inhibit incretin degradation (DPP4 inhibitors), but none is thus far aimed at increasing the secretion of endogenous incretins. In order to identify new potential therapeutic targets for treatment of T2D, we performed a meta-analysis of a GWAS and an exome-wide association study of circulating insulin, glucagon, GIP, and GLP-1 concentrations measured during an oral glucose tolerance test in up to 7,828 individuals. We identified 6 genome-wide significant functional loci associated with plasma incretin concentrations in or near the SLC5A1 (encoding SGLT1), GIPR, ABO, GLP2R, F13A1, and HOXD1 genes and studied the effect of these variants on mRNA expression in pancreatic islet and on metabolic phenotypes. Immunohistochemistry showed expression of GIPR, ABO, and HOXD1 in human enteroendocrine cells and expression of ABO in pancreatic islets, supporting a role in hormone secretion. This study thus provides candidate genes and insight into mechanisms by which secretion and breakdown of GIP and GLP-1 are regulated.

Glucose-Induced Changes in Gene Expression in Human Pancreatic Islets: Causes or Consequences of Chronic Hyperglycemia.

Dysregulation of gene expression in islets from patients with type 2 diabetes (T2D) might be causally involved in the development of hyperglycemia, or it could develop as a consequence of hyperglycemia (i.e., glucotoxicity). To separate the genes that could be causally involved in pathogenesis from those likely to be secondary to hyperglycemia, we exposed islets from human donors to normal or high glucose concentrations for 24 h and analyzed gene expression. We compared these findings with gene expression in islets from donors with normal glucose tolerance and hyperglycemia (including T2D). The genes whose expression changed in the same direction after short-term glucose exposure, as in T2D, were considered most likely to be a consequence of hyperglycemia. Genes whose expression changed in hyperglycemia but not after short-term glucose exposure, particularly those that also correlated with insulin secretion, were considered the strongest candidates for causal involvement in T2D. For example, ERO1LB, DOCK10, IGSF11, and PRR14L were downregulated in donors with hyperglycemia and correlated positively with insulin secretion, suggesting a protective role, whereas TMEM132C was upregulated in hyperglycemia and correlated negatively with insulin secretion, suggesting a potential pathogenic role. This study provides a catalog of gene expression changes in human pancreatic islets after exposure to glucose.

C4b-binding Protein Protects ß-Cells from Islet Amyloid Polypeptide-induced Cytotoxicity.

C4BP (C4b-binding protein) is a polymer of seven identical α chains and one unique ß chain synthesized in liver and pancreas. We showed previously that C4BP enhances islet amyloid polypeptide (IAPP) fibril formation in vitro Now we report that polymeric C4BP strongly inhibited lysis of human erythrocytes incubated with monomeric IAPP, whereas no lysis was observed after incubation with preformed IAPP fibrils. In contrast, incubation with the monomeric α-chain of C4BP was less effective. These data indicate that polymeric C4BP with multiple binding sites for IAPP neutralizes lytic activity of IAPP. Furthermore, addition of monomeric IAPP to a rat insulinoma cell line (INS-1) resulted in decreased cell viability, which was restored in the presence of physiological concentrations of C4BP. Treatment of INS-1 cells and primary rat islets with IAPP also diminished their ability to secrete insulin upon stimulation with glucose, which was reversed in the presence of C4BP. Further, C4BP was internalized together with IAPP into INS-1 cells. Pathway analyses of mRNA expression microarray data indicated that cells exposed to C4BP and IAPP in comparison with IAPP alone increased expression of genes involved in cholesterol synthesis. Depletion of cholesterol through methyl-ß-cyclodextrin or cholesterol oxidase abolished the protective effect of C4BP on IAPP cytotoxicity of INS-1 cells. Also, inhibition of phosphoinositide 3-kinase but not NF-κB had a similar effect. Taken together, C4BP protects ß-cells from IAPP cytotoxicity by modulating IAPP fibril formation extracellularly and also, after uptake by the cells, by enhancing cholesterol synthesis.

Global genomic and transcriptomic analysis of human pancreatic islets reveals novel genes influencing glucose metabolism.

Genetic variation can modulate gene expression, and thereby phenotypic variation and susceptibility to complex diseases such as type 2 diabetes (T2D). Here we harnessed the potential of DNA and RNA sequencing in human pancreatic islets from 89 deceased donors to identify genes of potential importance in the pathogenesis of T2D. We present a catalog of genetic variants regulating gene expression (eQTL) and exon use (sQTL), including many long noncoding RNAs, which are enriched in known T2D-associated loci. Of 35 eQTL genes, whose expression differed between normoglycemic and hyperglycemic individuals, siRNA of tetraspanin 33 (TSPAN33), 5'-nucleotidase, ecto (NT5E), transmembrane emp24 protein transport domain containing 6 (TMED6), and p21 protein activated kinase 7 (PAK7) in INS1 cells resulted in reduced glucose-stimulated insulin secretion. In addition, we provide a genome-wide catalog of allelic expression imbalance, which is also enriched in known T2D-associated loci. Notably, allelic imbalance in paternally expressed gene 3 (PEG3) was associated with its promoter methylation and T2D status. Finally, RNA editing events were less common in islets than previously suggested in other tissues. Taken together, this study provides new insights into the complexity of gene regulation in human pancreatic islets and better understanding of how genetic variation can influence glucose metabolism.

The complement inhibitor CD59 regulates insulin secretion by modulating exocytotic events.

Type 2 diabetes is triggered by reduced insulin production, caused by genetic and environmental factors such as inflammation originating from the innate immune system. Complement proteins are a component of innate immunity and kill non-self cells by perforating the plasma membrane, a reaction prevented by CD59. Human pancreatic islets express CD59 at very high levels. CD59 is primarily known as a plasma membrane protein in membrane rafts, but most CD59 protein in pancreatic ß cells is intracellular. Removing extracellular CD59 disrupts membrane rafts and moderately stimulates insulin secretion, whereas silencing intracellular CD59 markedly suppresses regulated secretion by exocytosis, as demonstrated by TIRF imaging. CD59 interacts with the exocytotic proteins VAMP2 and Syntaxin-1. CD59 expression is reduced by glucose and in rodent diabetes models but upregulated in human diabetic islets, potentially reflecting compensatory reactions. This unconventional action of CD59 broadens the established view of innate immunity in type 2 diabetes.

Effects of common genetic variants associated with type 2 diabetes and glycemic traits on α- and ß-cell function and insulin action in humans.

Although meta-analyses of genome-wide association studies have identified >60 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes and/or glycemic traits, there is little information on whether these variants also affect α-cell function. The aim of the current study was to evaluate the effects of glycemia-associated genetic loci on islet function in vivo and in vitro. We studied 43 SNPs in 4,654 normoglycemic participants from the Finnish population-based Prevalence, Prediction, and Prevention of Diabetes-Botnia (PPP-Botnia) Study. Islet function was assessed, in vivo, by measuring insulin and glucagon concentrations during oral glucose tolerance test, and, in vitro, by measuring glucose-stimulated insulin and glucagon secretion from human pancreatic islets. Carriers of risk variants in BCL11A, HHEX, ZBED3, HNF1A, IGF1, and NOTCH2 showed elevated whereas those in CRY2, IGF2BP2, TSPAN8, and KCNJ11 showed decreased fasting and/or 2-h glucagon concentrations in vivo. Variants in BCL11A, TSPAN8, and NOTCH2 affected glucagon secretion both in vivo and in vitro. The MTNR1B variant was a clear outlier in the relationship analysis between insulin secretion and action, as well as between insulin, glucose, and glucagon. Many of the genetic variants shown to be associated with type 2 diabetes or glycemic traits also exert pleiotropic in vivo and in vitro effects on islet function.

Secreted frizzled-related protein 4 reduces insulin secretion and is overexpressed in type 2 diabetes.

A plethora of candidate genes have been identified for complex polygenic disorders, but the underlying disease mechanisms remain largely unknown. We explored the pathophysiology of type 2 diabetes (T2D) by analyzing global gene expression in human pancreatic islets. A group of coexpressed genes (module), enriched for interleukin-1-related genes, was associated with T2D and reduced insulin secretion. One of the module genes that was highly overexpressed in islets from T2D patients is SFRP4, which encodes secreted frizzled-related protein 4. SFRP4 expression correlated with inflammatory markers, and its release from islets was stimulated by interleukin-1ß. Elevated systemic SFRP4 caused reduced glucose tolerance through decreased islet expression of Ca(2+) channels and suppressed insulin exocytosis. SFRP4 thus provides a link between islet inflammation and impaired insulin secretion. Moreover, the protein was increased in serum from T2D patients several years before the diagnosis, suggesting that SFRP4 could be a potential biomarker for islet dysfunction in T2D.

A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets.

Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion, whereas GPR120 affected apoptosis in islets. Expression variation of the top 20 genes explained 24% of the variance in HbA(1c) with no claim of the direction. The data present a global map of genes associated with islet dysfunction and demonstrate the value of systems genetics for the identification of genes potentially involved in T2D.

Abnormal peripheral chemokine profile in Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disorder characterized by both neurological and systemic abnormalities. Immune activation is a well-established feature of the HD brain and we have previously demonstrated a widespread, progressive innate immune response detectable in plasma throughout the course of HD. In the present work we used multiplex ELISA to quantify levels of chemokines in plasma from controls and subjects at different stages of HD. We found an altered chemokine profile tracking with disease progression, with significant elevations of five chemokines (eotaxin-3, MIP-1ß, eotaxin, MCP-1 and MCP-4) while three (eotaxin-3, MIP-1ß and eotaxin) showed significant linear increases across advancing disease stages. We validated our results in a separate sample cohort including subjects at different stages of HD. Here we saw that chemokine levels (MCP-1 and eotaxin) correlated with clinical scores. We conclude that, like cytokines, chemokines may be linked to the pathogenesis of HD, and that immune molecules may be valuable in tracking and exploring the pathogenesis of HD.

Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells.

Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80%) and protein level (>40%) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P<0.05), 1.6- (P<0.05) and 1.6-fold (P<0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P<0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase 1 expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.