Diet-Microbiota Interactions Mediate Global Epigenetic Programming in Multiple Host Tissues.

Histone-modifying enzymes regulate transcription and are sensitive to availability of endogenous small-molecule metabolites, allowing chromatin to respond to changes in environment. The gut microbiota produces a myriad of metabolites that affect host physiology and susceptibility to disease; however, the underlying molecular events remain largely unknown. Here we demonstrate that microbial colonization regulates global histone acetylation and methylation in multiple host tissues in a diet-dependent manner: consumption of a "Western-type" diet prevents many of the microbiota-dependent chromatin changes that occur in a polysaccharide-rich diet. Finally, we demonstrate that supplementation of germ-free mice with short-chain fatty acids, major products of gut bacterial fermentation, is sufficient to recapitulate chromatin modification states and transcriptional responses associated with colonization. These findings have profound implications for understanding the complex functional interactions between diet, gut microbiota, and host health.

Genetic determinants of gut microbiota composition and bile acid profiles in mice.

The microbial communities that inhabit the distal gut of humans and other mammals exhibit large inter-individual variation. While host genetics is a known factor that influences gut microbiota composition, the mechanisms underlying this variation remain largely unknown. Bile acids (BAs) are hormones that are produced by the host and chemically modified by gut bacteria. BAs serve as environmental cues and nutrients to microbes, but they can also have antibacterial effects. We hypothesized that host genetic variation in BA metabolism and homeostasis influence gut microbiota composition. To address this, we used the Diversity Outbred (DO) stock, a population of genetically distinct mice derived from eight founder strains. We characterized the fecal microbiota composition and plasma and cecal BA profiles from 400 DO mice maintained on a high-fat high-sucrose diet for ~22 weeks. Using quantitative trait locus (QTL) analysis, we identified several genomic regions associated with variations in both bacterial and BA profiles. Notably, we found overlapping QTL for Turicibacter sp. and plasma cholic acid, which mapped to a locus containing the gene for the ileal bile acid transporter, Slc10a2. Mediation analysis and subsequent follow-up validation experiments suggest that differences in Slc10a2 gene expression associated with the different strains influences levels of both traits and revealed novel interactions between Turicibacter and BAs. This work illustrates how systems genetics can be utilized to generate testable hypotheses and provide insight into host-microbe interactions.

Islet proteomics reveals genetic variation in dopamine production resulting in altered insulin secretion.

The mouse is a critical model in diabetes research, but most research in mice has been limited to a small number of mouse strains and limited genetic variation. Using the eight founder strains and both sexes of the Collaborative Cross (C57BL/6J (B6), A/J, 129S1/SvImJ (129), NOD/ShiLtJ (NOD), NZO/HILtJ (NZO), PWK/PhJ (PWK), WSB/EiJ (WSB), and CAST/EiJ (CAST)), we investigated the genetic dependence of diabetes-related metabolic phenotypes and insulin secretion. We found that strain background is associated with an extraordinary range in body weight, plasma glucose, insulin, triglycerides, and insulin secretion. Our whole-islet proteomic analysis of the eight mouse strains demonstrates that genetic background exerts a strong influence on the islet proteome that can be linked to the differences in diabetes-related metabolic phenotypes and insulin secretion. We computed protein modules consisting of highly correlated proteins that enrich for biological pathways and provide a searchable database of the islet protein expression profiles. To validate the data resource, we identified tyrosine hydroxylase (Th), a key enzyme in catecholamine synthesis, as a protein that is highly expressed in ß-cells of PWK and CAST islets. We show that CAST islets synthesize elevated levels of dopamine, which suppresses insulin secretion. Prior studies, using only the B6 strain, concluded that adult mouse islets do not synthesize l-3,4-dihydroxyphenylalanine (l-DOPA), the product of Th and precursor of dopamine. Thus, the choice of the CAST strain, guided by our islet proteomic survey, was crucial for these discoveries. In summary, we provide a valuable data resource to the research community, and show that proteomic analysis identified a strain-specific pathway by which dopamine synthesized in ß-cells inhibits insulin secretion.

The Transcription Factor Nfatc2 Regulates ß-Cell Proliferation and Genes Associated with Type 2 Diabetes in Mouse and Human Islets.

Human genome-wide association studies (GWAS) have shown that genetic variation at >130 gene loci is associated with type 2 diabetes (T2D). We asked if the expression of the candidate T2D-associated genes within these loci is regulated by a common locus in pancreatic islets. Using an obese F2 mouse intercross segregating for T2D, we show that the expression of ~40% of the T2D-associated genes is linked to a broad region on mouse chromosome (Chr) 2. As all but 9 of these genes are not physically located on Chr 2, linkage to Chr 2 suggests a genomic factor(s) located on Chr 2 regulates their expression in trans. The transcription factor Nfatc2 is physically located on Chr 2 and its expression demonstrates cis linkage; i.e., its expression maps to itself. When conditioned on the expression of Nfatc2, linkage for the T2D-associated genes was greatly diminished, supporting Nfatc2 as a driver of their expression. Plasma insulin also showed linkage to the same broad region on Chr 2. Overexpression of a constitutively active (ca) form of Nfatc2 induced ß-cell proliferation in mouse and human islets, and transcriptionally regulated more than half of the T2D-associated genes. Overexpression of either ca-Nfatc2 or ca-Nfatc1 in mouse islets enhanced insulin secretion, whereas only ca-Nfatc2 was able to promote ß-cell proliferation, suggesting distinct molecular pathways mediating insulin secretion vs. ß-cell proliferation are regulated by NFAT. Our results suggest that many of the T2D-associated genes are downstream transcriptional targets of NFAT, and may act coordinately in a pathway through which NFAT regulates ß-cell proliferation in both mouse and human islets.

Phosphorylation and degradation of tomosyn-2 de-represses insulin secretion.

The abundance and functional activity of proteins involved in the formation of the SNARE complex are tightly regulated for efficient exocytosis. Tomosyn proteins are negative regulators of exocytosis. Tomosyn causes an attenuation of insulin secretion by limiting the formation of the SNARE complex. We hypothesized that glucose-dependent stimulation of insulin secretion from ß-cells must involve reversing the inhibitory action of tomosyn. Here, we show that glucose increases tomosyn protein turnover. Within 1 h of exposure to 15 mM glucose, ~50% of tomosyn was degraded. The degradation of tomosyn in response to high glucose was blocked by inhibitors of the proteasomal pathway. Using (32)P labeling and mass spectrometry, we showed that tomosyn-2 is phosphorylated in response to high glucose, phorbol esters, and analogs of cAMP, all key insulin secretagogues. We identified 11 phosphorylation sites in tomosyn-2. Site-directed mutagenesis was used to generate phosphomimetic (Ser → Asp) and loss-of-function (Ser → Ala) mutants. The Ser → Asp mutant had enhanced protein turnover compared with the Ser → Ala mutant and wild type tomosyn-2. Additionally, the Ser → Asp tomosyn-2 mutant was ineffective at inhibiting insulin secretion. Using a proteomic screen for tomosyn-2-binding proteins, we identified Hrd-1, an E3-ubiquitin ligase. We showed that tomosyn-2 ubiquitination is increased by Hrd-1, and knockdown of Hrd-1 by short hairpin RNA resulted in increased abundance in tomosyn-2 protein levels. Taken together, our results reveal a mechanism by which enhanced phosphorylation of a negative regulator of secretion, tomosyn-2, in response to insulin secretagogues targets it to degradation by the Hrd-1 E3-ubiquitin ligase.

Integrative analysis of a cross-loci regulation network identifies App as a gene regulating insulin secretion from pancreatic islets.

Complex diseases result from molecular changes induced by multiple genetic factors and the environment. To derive a systems view of how genetic loci interact in the context of tissue-specific molecular networks, we constructed an F2 intercross comprised of >500 mice from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mouse strains made genetically obese by the Leptin(ob/ob) mutation (Lep(ob)). High-density genotypes, diabetes-related clinical traits, and whole-transcriptome expression profiling in five tissues (white adipose, liver, pancreatic islets, hypothalamus, and gastrocnemius muscle) were determined for all mice. We performed an integrative analysis to investigate the inter-relationship among genetic factors, expression traits, and plasma insulin, a hallmark diabetes trait. Among five tissues under study, there are extensive protein-protein interactions between genes responding to different loci in adipose and pancreatic islets that potentially jointly participated in the regulation of plasma insulin. We developed a novel ranking scheme based on cross-loci protein-protein network topology and gene expression to assess each gene's potential to regulate plasma insulin. Unique candidate genes were identified in adipose tissue and islets. In islets, the Alzheimer's gene App was identified as a top candidate regulator. Islets from 17-week-old, but not 10-week-old, App knockout mice showed increased insulin secretion in response to glucose or a membrane-permeant cAMP analog, in agreement with the predictions of the network model. Our result provides a novel hypothesis on the mechanism for the connection between two aging-related diseases: Alzheimer's disease and type 2 diabetes.

Positional cloning of Sorcs1, a type 2 diabetes quantitative trait locus.

We previously mapped the type 2 diabetes mellitus-2 locus (T2dm2), which affects fasting insulin levels, to distal chromosome 19 in a leptin-deficient obese F2 intercross derived from C57BL/6 (B6) and BTBR T+ tf/J (BTBR) mice. Introgression of a 7-Mb segment of the B6 chromosome 19 into the BTBR background (strain 1339A) replicated the reduced insulin linked to T2dm2. The 1339A mice have markedly impaired insulin secretion in vivo and disrupted islet morphology. We used subcongenic strains derived from 1339A to localize the T2dm2 quantitative trait locus (QTL) to a 242-kb segment comprising the promoter, first exon and most of the first intron of the Sorcs1 gene. This was the only gene in the 1339A strain for which we detected amino acid substitutions and expression level differences between mice carrying B6 and BTBR alleles of this insert, thereby identifying variation within the Sorcs1 gene as underlying the phenotype associated with the T2dm2 locus. SorCS1 binds platelet-derived growth factor, a growth factor crucial for pericyte recruitment to the microvasculature, and may thus have a role in expanding or maintaining the islet vasculature. Our identification of the Sorcs1 gene provides insight into the pathway underlying the pathophysiology of obesity-induced type 2 diabetes mellitus.

A potent α/ß-peptide analogue of GLP-1 with prolonged action in vivo.

Glucagon-like peptide-1 (GLP-1) is a natural agonist for GLP-1R, a G protein-coupled receptor (GPCR) on the surface of pancreatic ß cells. GLP-1R agoinsts are attractive for treatment of type 2 diabetes, but GLP-1 itself is rapidly degraded by peptidases in vivo. We describe a design strategy for retaining GLP-1-like activity while engendering prolonged activity in vivo, based on strategic replacement of native α residues with conformationally constrained ß-amino acid residues. This backbone-modification approach may be useful for developing stabilized analogues of other peptide hormones.

Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion.

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic ß-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes.

Tsc2, a positional candidate gene underlying a quantitative trait locus for hepatic steatosis.

Nonalchoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction and is associated with metabolic diseases, including obesity, insulin resistance, and type 2 diabetes. We mapped a quantitative trait locus (QTL) for NAFLD to chromosome 17 in a cross between C57BL/6 (B6) and BTBR mouse strains made genetically obese with the Lep(ob/ob) mutation. We identified Tsc2 as a gene underlying the chromosome 17 NAFLD QTL. Tsc2 functions as an inhibitor of mammalian target of rapamycin, which is involved in many physiological processes, including cell growth, proliferation, and metabolism. We found that Tsc2(+/-) mice have increased lipogenic gene expression in the liver in an insulin-dependent manner. The coding single nucleotide polymorphism between the B6 and BTBR strains leads to a change in the ability to inhibit the expression of lipogenic genes and de novo lipogenesis in AML12 cells and to promote the proliferation of Ins1 cells. This difference is due to a different affinity of binding to Tsc1, which affects the stability of Tsc2.

ß Cell-specific deletion of Zfp148 improves nutrient-stimulated ß cell Ca2+ responses.

Insulin secretion from pancreatic ß cells is essential for glucose homeostasis. An insufficient response to the demand for insulin results in diabetes. We previously showed that ß cell-specific deletion of Zfp148 (ß-Zfp148KO) improves glucose tolerance and insulin secretion in mice. Here, we performed Ca2+ imaging of islets from ß­Zfp148KO and control mice fed both a chow and a Western-style diet. ß-Zfp148KO islets demonstrated improved sensitivity and sustained Ca2+ oscillations in response to elevated glucose levels. ß-Zfp148KO islets also exhibited elevated sensitivity to amino acid-induced Ca2+ influx under low glucose conditions, suggesting enhanced mitochondrial phosphoenolpyruvate-dependent (PEP-dependent), ATP-sensitive K+ channel closure, independent of glycolysis. RNA-Seq and proteomics of ß-Zfp148KO islets revealed altered levels of enzymes involved in amino acid metabolism (specifically, SLC3A2, SLC7A8, GLS, GLS2, PSPH, PHGDH, and PSAT1) and intermediary metabolism (namely, GOT1 and PCK2), consistent with altered PEP cycling. In agreement with this, ß-Zfp148KO islets displayed enhanced insulin secretion in response to l-glutamine and activation of glutamate dehydrogenase. Understanding pathways controlled by ZFP148 may provide promising strategies for improving ß cell function that are robust to the metabolic challenge imposed by a Western diet.

Loss of PDGF-B activity increases hepatic vascular permeability and enhances insulin sensitivity.

Hepatic vasculature is not thought to pose a permeability barrier for diffusion of macromolecules from the bloodstream to hepatocytes. In contrast, in extrahepatic tissues, the microvasculature is critically important for insulin action, because transport of insulin across the endothelial cell layer is rate limiting for insulin-stimulated glucose disposal. However, very little is known concerning the role in this process of pericytes, the mural cells lining the basolateral membrane of endothelial cells. PDGF-B is a growth factor involved in the recruitment and function of pericytes. We studied insulin action in mice expressing PDGF-B lacking the proteoglycan binding domain, producing a protein with a partial loss of function (PDGF-B(ret/ret)). Insulin action was assessed through measurements of insulin signaling and insulin and glucose tolerance tests. PDGF-B deficiency enhanced hepatic vascular transendothelial transport. One outcome of this change was an increase in hepatic insulin signaling. This correlated with enhanced whole body glucose homeostasis and increased insulin clearance from the circulation during an insulin tolerance test. In obese mice, PDGF-B deficiency was associated with an 80% reduction in fasting insulin and drastically reduced insulin secretion. These mice did not have significantly higher glucose levels, reflecting a dramatic increase in insulin action. Our findings show that, despite already having a high permeability, hepatic transendothelial transport can be further enhanced. To the best of our knowledge, this is the first study to connect PDGF-B-induced changes in hepatic sinusoidal transport to changes in insulin action, demonstrating a link between PDGF-B signaling and insulin sensitivity.

BTBR Ob/Ob mutant mice model progressive diabetic nephropathy.

There remains a need for robust mouse models of diabetic nephropathy (DN) that mimic key features of advanced human DN. The recently developed mouse strain BTBR with the ob/ob leptin-deficiency mutation develops severe type 2 diabetes, hypercholesterolemia, elevated triglycerides, and insulin resistance, but the renal phenotype has not been characterized. Here, we show that these obese, diabetic mice rapidly develop morphologic renal lesions characteristic of both early and advanced human DN. BTBR ob/ob mice developed progressive proteinuria beginning at 4 weeks. Glomerular hypertrophy and accumulation of mesangial matrix, characteristic of early DN, were present by 8 weeks, and glomerular lesions similar to those of advanced human DN were present by 20 weeks. By 22 weeks, we observed an approximately 20% increase in basement membrane thickness and a >50% increase in mesangial matrix. Diffuse mesangial sclerosis (focally approaching nodular glomerulosclerosis), focal arteriolar hyalinosis, mesangiolysis, and focal mild interstitial fibrosis were present. Loss of podocytes was present early and persisted. In summary, BTBR ob/ob mice develop a constellation of abnormalities that closely resemble advanced human DN more rapidly than most other murine models, making this strain particularly attractive for testing therapeutic interventions.

Identification of direct transcriptional targets of NFATC2 that promote ß cell proliferation.

The transcription factor NFATC2 induces ß cell proliferation in mouse and human islets. However, the genomic targets that mediate these effects have not been identified. We expressed active forms of Nfatc2 and Nfatc1 in human islets. By integrating changes in gene expression with genomic binding sites for NFATC2, we identified approximately 2200 transcriptional targets of NFATC2. Genes induced by NFATC2 were enriched for transcripts that regulate the cell cycle and for DNA motifs associated with the transcription factor FOXP. Islets from an endocrine-specific Foxp1, Foxp2, and Foxp4 triple-knockout mouse were less responsive to NFATC2-induced ß cell proliferation, suggesting the FOXP family works to regulate ß cell proliferation in concert with NFATC2. NFATC2 induced ß cell proliferation in both mouse and human islets, whereas NFATC1 did so only in human islets. Exploiting this species difference, we identified approximately 250 direct transcriptional targets of NFAT in human islets. This gene set enriches for cell cycle-associated transcripts and includes Nr4a1. Deletion of Nr4a1 reduced the capacity of NFATC2 to induce ß cell proliferation, suggesting that much of the effect of NFATC2 occurs through its induction of Nr4a1. Integration of noncoding RNA expression, chromatin accessibility, and NFATC2 binding sites enabled us to identify NFATC2-dependent enhancer loci that mediate ß cell proliferation.

A large-scale genome-lipid association map guides lipid identification.

Despite the crucial roles of lipids in metabolism, we are still at the early stages of comprehensively annotating lipid species and their genetic basis. Mass spectrometry-based discovery lipidomics offers the potential to globally survey lipids and their relative abundances in various biological samples. To discover the genetics of lipid features obtained through high-resolution liquid chromatography-tandem mass spectrometry, we analysed liver and plasma from 384 diversity outbred mice, and quantified 3,283 molecular features. These features were mapped to 5,622 lipid quantitative trait loci and compiled into a public web resource termed LipidGenie. The data are cross-referenced to the human genome and offer a bridge between genetic associations in humans and mice. Harnessing this resource, we used genome-lipid association data as an additional aid to identify a number of lipids, for example gangliosides through their association with B4galnt1, and found evidence for a group of sex-specific phosphatidylcholines through their shared locus. Finally, LipidGenie's ability to query either mass or gene-centric terms suggests acyl-chain-specific functions for proteins of the ABHD family.

Obesity and genetics regulate microRNAs in islets, liver, and adipose of diabetic mice.

Type 2 diabetes results from severe insulin resistance coupled with a failure of b cells to compensate by secreting sufficient insulin. Multiple genetic loci are involved in the development of diabetes, although the effect of each gene on diabetes susceptibility is thought to be small. MicroRNAs (miRNAs) are noncoding 19-22-nucleotide RNA molecules that potentially regulate the expression of thousands of genes. To understand the relationship between miRNA regulation and obesity-induced diabetes, we quantitatively profiled approximately 220 miRNAs in pancreatic islets, adipose tissue, and liver from diabetes-resistant (B6) and diabetes-susceptible (BTBR) mice. More than half of the miRNAs profiled were expressed in all three tissues, with many miRNAs in each tissue showing significant changes in response to genetic obesity. Furthermore, several miRNAs in each tissue were differentially responsive to obesity in B6 versus BTBR mice, suggesting that they may be involved in the pathogenesis of diabetes. In liver there were approximately 40 miRNAs that were downregulated in response to obesity in B6 but not BTBR mice, indicating that genetic differences between the mouse strains play a critical role in miRNA regulation. In order to elucidate the genetic architecture of hepatic miRNA expression, we measured the expression of miRNAs in genetically obese F2 mice. Approximately 10% of the miRNAs measured showed significant linkage (miR-eQTLs), identifying loci that control miRNA abundance. Understanding the influence that obesity and genetics exert on the regulation of miRNA expression will reveal the role miRNAs play in the context of obesity-induced type 2 diabetes.

Overexpression of pre-pro-cholecystokinin stimulates beta-cell proliferation in mouse and human islets with retention of islet function.

Type 1 and type 2 diabetes result from a deficit in insulin production and beta-cell mass. Methods to expand beta-cell mass are under intensive investigation for the treatment of type 1 and type 2 diabetes. We tested the hypothesis that cholecystokinin (CCK) can promote beta-cell proliferation. We treated isolated mouse and human islets with an adenovirus containing the CCK cDNA (AdCMV-CCK). We measured [(3)H]thymidine and BrdU incorporation into DNA and additionally, performed flow cytometry analysis to determine whether CCK overexpression stimulates beta-cell proliferation. We studied islet function by measuring glucose-stimulated insulin secretion and investigated the cell cycle regulation of proliferating beta-cells by quantitative RT-PCR and Western blot analysis. Overexpression of CCK stimulated [(3)H]thymidine incorporation into DNA 5.0-fold and 15.8-fold in mouse and human islets, respectively. AdCMV-CCK treatment also stimulated BrdU incorporation into DNA 10-fold and 21-fold in mouse and human beta-cells, respectively. Glucose-stimulated insulin secretion was unaffected by CCK expression. Analysis of cyclin and cdk mRNA and protein abundance revealed that CCK overexpression increased cyclin A, cyclin B, cyclin E, cdk1, and cdk2 with no change in cyclin D1, cyclin D2, cyclin D3, cdk4, or cdk6 in mouse and human islets. Additionally, AdCMV-CCK treatment of CCK receptor knockout and wild-type mice resulted in equal [(3)H]thymidine incorporation. CCK is a beta-cell proliferative factor that is effective in both mouse and human islets. CCK triggers beta-cell proliferation without disrupting islet function, up-regulates a distinct set of cell cycle regulators in islets, and signals independently of the CCK receptors.

Gene loci associated with insulin secretion in islets from non-diabetic mice.

Genetic susceptibility to type 2 diabetes is primarily due to ß-cell dysfunction. However, a genetic study to directly interrogate ß-cell function ex vivo has never been previously performed. We isolated 233,447 islets from 483 Diversity Outbred (DO) mice maintained on a Western-style diet, and measured insulin secretion in response to a variety of secretagogues. Insulin secretion from DO islets ranged >1,000-fold even though none of the mice were diabetic. The insulin secretory response to each secretagogue had a unique genetic architecture; some of the loci were specific for one condition, whereas others overlapped. Human loci that are syntenic to many of the insulin secretion QTL from mouse are associated with diabetes-related SNPs in human genome-wide association studies. We report on three genes, Ptpn18, Hunk and Zfp148, where the phenotype predictions from the genetic screen were fulfilled in our studies of transgenic mouse models. These three genes encode a non-receptor type protein tyrosine phosphatase, a serine/threonine protein kinase, and a Krϋppel-type zinc-finger transcription factor, respectively. Our results demonstrate that genetic variation in insulin secretion that can lead to type 2 diabetes is discoverable in non-diabetic individuals.

Genetic Drivers of Pancreatic Islet Function.

The majority of gene loci that have been associated with type 2 diabetes play a role in pancreatic islet function. To evaluate the role of islet gene expression in the etiology of diabetes, we sensitized a genetically diverse mouse population with a Western diet high in fat (45% kcal) and sucrose (34%) and carried out genome-wide association mapping of diabetes-related phenotypes. We quantified mRNA abundance in the islets and identified 18,820 expression QTL. We applied mediation analysis to identify candidate causal driver genes at loci that affect the abundance of numerous transcripts. These include two genes previously associated with monogenic diabetes (PDX1 and HNF4A), as well as three genes with nominal association with diabetes-related traits in humans (FAM83E, IL6ST, and SAT2). We grouped transcripts into gene modules and mapped regulatory loci for modules enriched with transcripts specific for α-cells, and another specific for δ-cells. However, no single module enriched for ß-cell-specific transcripts, suggesting heterogeneity of gene expression patterns within the ß-cell population. A module enriched in transcripts associated with branched-chain amino acid metabolism was the most strongly correlated with physiological traits that reflect insulin resistance. Although the mice in this study were not overtly diabetic, the analysis of pancreatic islet gene expression under dietary-induced stress enabled us to identify correlated variation in groups of genes that are functionally linked to diabetes-associated physiological traits. Our analysis suggests an expected degree of concordance between diabetes-associated loci in the mouse and those found in human populations, and demonstrates how the mouse can provide evidence to support nominal associations found in human genome-wide association mapping.