Genetic Analysis of Obesity-Induced Diabetic Nephropathy in BTBR Mice.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the U.S. and has a significant impact on human suffering. Leptin-deficient BTBR (BTBRob/ob) mice develop hallmark features of obesity-induced DN, whereas leptin-deficient C57BL/6J (B6ob/ob) mice do not. To identify genetic loci that underlie this strain difference, we constructed an F2 intercross between BTBRob/ob and B6ob/ob mice. We isolated kidneys from 460 F2 mice and histologically scored them for percent mesangial matrix and glomerular volume (∼50 glomeruli per mouse), yielding ∼45,000 distinct measures in total. The same histological measurements were made in kidneys from B6 and BTBR mice, either lean or obese (Lepob/ob), at 4 and 10 weeks of age, allowing us to assess the contribution of strain, age, and obesity to glomerular pathology. All F2 mice were genotyped for ∼5,000 single nucleotide polymorphisms (SNPs), ∼2,000 of which were polymorphic between B6 and BTBR, enabling us to identify a quantitative trait locus (QTL) on chromosome 7, with a peak at ∼30 Mbp, for percent mesangial matrix, glomerular volume, and mesangial volume. The podocyte-specific gene nephrin (Nphs1) is physically located at the QTL and contains high-impact SNPs in BTBR, including several missense variants within the extracellular immunoglobulin-like domains.

Missense variants in SORT1 are associated with LDL-C in an Amish population.

Common noncoding variants at the human 1p13.3 locus associated with SORT1 expression are among those most strongly associated with low-density lipoprotein cholesterol (LDL-C) in human genome-wide association studies. However, validation studies in mice and cell lines have produced variable results regarding the directionality of the effect of SORT1 on LDL-C. This, together with the fact that the 1p13.3 variants are associated with expression of several genes, has raised the question of whether SORT1 is the causal gene at this locus. Using whole exome sequencing in members of an Amish population, we identified coding variants in SORT1 that are associated with increased (rs141749679, K302E) and decreased (rs149456022, Q225H) LDL-C. Further, analysis of plasma lipoprotein particle subclasses by ion mobility in a subset of rs141749679 (K302E) carriers revealed higher levels of large LDL particles compared to noncarriers. In contrast to the effect of these variants in the Amish, the sortilin K302E mutation introduced into a C57BL/6J mouse via CRISPR/Cas9 resulted in decreased non-high-density lipoprotein cholesterol, and the sortilin Q225H mutation did not alter cholesterol levels in mice. This is indicative of different effects of these mutations on cholesterol metabolism in the two species. To our knowledge, this is the first evidence that naturally occurring coding variants in SORT1 are associated with LDL-C, thus supporting SORT1 as the gene responsible for the association of the 1p13.3 locus with LDL-C.

Identification of genetic drivers of plasma lipoprotein size in the Diversity Outbred mouse population.

Despite great progress in understanding lipoprotein physiology, there is still much to be learned about the genetic drivers of lipoprotein abundance, composition, and function. We used ion mobility spectrometry to survey 16 plasma lipoprotein subfractions in 500 Diversity Outbred mice maintained on a Western-style diet. We identified 21 quantitative trait loci (QTL) affecting lipoprotein abundance. To refine the QTL and link them to disease risk in humans, we asked if the human homologs of genes located at each QTL were associated with lipid traits in human genome-wide association studies. Integration of mouse QTL with human genome-wide association studies yielded candidate gene drivers for 18 of the 21 QTL. This approach enabled us to nominate the gene encoding the neutral ceramidase, Asah2, as a novel candidate driver at a QTL on chromosome 19 for large HDL particles (HDL-2b). To experimentally validate Asah2, we surveyed lipoproteins in Asah2-/- mice. Compared to wild-type mice, female Asah2-/- mice showed an increase in several lipoproteins, including HDL. Our results provide insights into the genetic regulation of circulating lipoproteins, as well as mechanisms by which lipoprotein subfractions may affect cardiovascular disease risk in humans.

Novel regulators of islet function identified from genetic variation in mouse islet Ca2+ oscillations.

Insufficient insulin secretion to meet metabolic demand results in diabetes. The intracellular flux of Ca2+ into ß-cells triggers insulin release. Since genetics strongly influences variation in islet secretory responses, we surveyed islet Ca2+ dynamics in eight genetically diverse mouse strains. We found high strain variation in response to four conditions: (1) 8 mM glucose; (2) 8 mM glucose plus amino acids; (3) 8 mM glucose, amino acids, plus 10 nM glucose-dependent insulinotropic polypeptide (GIP); and (4) 2 mM glucose. These stimuli interrogate ß-cell function, α- to ß-cell signaling, and incretin responses. We then correlated components of the Ca2+ waveforms to islet protein abundances in the same strains used for the Ca2+ measurements. To focus on proteins relevant to human islet function, we identified human orthologues of correlated mouse proteins that are proximal to glycemic-associated single-nucleotide polymorphisms in human genome-wide association studies. Several orthologues have previously been shown to regulate insulin secretion (e.g. ABCC8, PCSK1, and GCK), supporting our mouse-to-human integration as a discovery platform. By integrating these data, we nominate novel regulators of islet Ca2+ oscillations and insulin secretion with potential relevance for human islet function. We also provide a resource for identifying appropriate mouse strains in which to study these regulators.

PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy.

PPTC7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass and metabolic capacity with elevated hepatic triglyceride accumulation. Pptc7 knockout animals exhibit increased expression of the mitophagy receptors BNIP3 and NIX, and Pptc7-/- mouse embryonic fibroblasts (MEFs) display a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs, including multiple sites on BNIP3 and NIX, and our molecular studies demonstrate that PPTC7 can directly interact with and dephosphorylate these proteins. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that PPTC7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for PPTC7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

An Enhancer Within Abcb11 Regulates G6pc2 in C57BL/6 Mouse Pancreatic Islets.

G6PC2 is predominantly expressed in pancreatic islet ß-cells where it encodes a glucose-6-phosphatase catalytic subunit that modulates the sensitivity of insulin secretion to glucose by opposing the action of glucokinase, thereby regulating fasting blood glucose (FBG). Prior studies have shown that the G6pc2 promoter alone is unable to confer sustained islet-specific gene expression in mice, suggesting the existence of distal enhancers that regulate G6pc2 expression. Using information from both mice and humans and knowledge that single nucleotide polymorphisms (SNPs) both within and near G6PC2 are associated with variations in FBG in humans, we identified several putative enhancers 3' of G6pc2. One region, herein referred to as enhancer I, resides in the 25th intron of Abcb11 and binds multiple islet-enriched transcription factors. CRISPR-mediated deletion of enhancer I in C57BL/6 mice had selective effects on the expression of genes near the G6pc2 locus. In isolated islets, G6pc2 and Spc25 expression were reduced ∼50%, and Gm13613 expression was abolished, whereas Cers6 and nostrin expression were unaffected. This partial reduction in G6pc2 expression enhanced islet insulin secretion at basal glucose concentrations but did not affect FBG or glucose tolerance in vivo, consistent with the absence of a phenotype in G6pc2 heterozygous C57BL/6 mice.

Genetic ablation of synaptotagmin-9 alters tomosyn-1 function to increase insulin secretion from pancreatic ß-cells improving glucose clearance.

Stimulus-coupled insulin secretion from the pancreatic islet ß-cells involves the fusion of insulin granules to the plasma membrane (PM) via SNARE complex formation-a cellular process key for maintaining whole-body glucose homeostasis. Less is known about the role of endogenous inhibitors of SNARE complexes in insulin secretion. We show that an insulin granule protein synaptotagmin-9 (Syt9) deletion in mice increased glucose clearance and plasma insulin levels without affecting insulin action compared to the control mice. Upon glucose stimulation, increased biphasic and static insulin secretion were observed from ex vivo islets due to Syt9 loss. Syt9 colocalizes and binds with tomosyn-1 and the PM syntaxin-1A (Stx1A); Stx1A is required for forming SNARE complexes. Syt9 knockdown reduced tomosyn-1 protein abundance via proteasomal degradation and binding of tomosyn-1 to Stx1A. Furthermore, Stx1A-SNARE complex formation was increased, implicating Syt9-tomosyn-1-Stx1A complex is inhibitory in insulin secretion. Rescuing tomosyn-1 blocked the Syt9-knockdown-mediated increases in insulin secretion. This shows that the inhibitory effects of Syt9 on insulin secretion are mediated by tomosyn-1. We report a molecular mechanism by which ß-cells modulate their secretory capacity rendering insulin granules nonfusogenic by forming the Syt9-tomosyn-1-Stx1A complex. Altogether, Syt9 loss in ß-cells decreases tomosyn-1 protein abundance, increasing the formation of Stx1A-SNARE complexes, insulin secretion, and glucose clearance. These outcomes differ from the previously published work that identified Syt9 has either a positive or no effect of Syt9 on insulin secretion. Future work using ß-cell-specific deletion of Syt9 mice is key for establishing the role of Syt9 in insulin secretion.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 (Abhd2) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 (Abhd2), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2. The Abhd2KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

Pptc7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy.

Pptc7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass concomitant with elevation of the mitophagy receptors Bnip3 and Nix. Consistently, Pptc7-/- mouse embryonic fibroblasts (MEFs) exhibit a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs-including multiple sites on Bnip3 and Nix. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that Pptc7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for Pptc7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

Lipidomic QTL in Diversity Outbred mice identifies a novel function for α/ß hydrolase domain 2 ( Abhd2 ) as an enzyme that metabolizes phosphatidylcholine and cardiolipin.

We and others have previously shown that genetic association can be used to make causal connections between gene loci and small molecules measured by mass spectrometry in the bloodstream and in tissues. We identified a locus on mouse chromosome 7 where several phospholipids in liver showed strong genetic association to distinct gene loci. In this study, we integrated gene expression data with genetic association data to identify a single gene at the chromosome 7 locus as the driver of the phospholipid phenotypes. The gene encodes α/ß-hydrolase domain 2 ( Abhd2 ), one of 23 members of the ABHD gene family. We validated this observation by measuring lipids in a mouse with a whole-body deletion of Abhd2 . The Abhd2 KO mice had a significant increase in liver levels of phosphatidylcholine and phosphatidylethanolamine. Unexpectedly, we also found a decrease in two key mitochondrial lipids, cardiolipin and phosphatidylglycerol, in male Abhd2 KO mice. These data suggest that Abhd2 plays a role in the synthesis, turnover, or remodeling of liver phospholipids.

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.

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.

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.

INFIMA leverages multi-omics model organism data to identify effector genes of human GWAS variants.

Genome-wide association studies reveal many non-coding variants associated with complex traits. However, model organism studies largely remain as an untapped resource for unveiling the effector genes of non-coding variants. We develop INFIMA, Integrative Fine-Mapping, to pinpoint causal SNPs for diversity outbred (DO) mice eQTL by integrating founder mice multi-omics data including ATAC-seq, RNA-seq, footprinting, and in silico mutation analysis. We demonstrate INFIMA's superior performance compared to alternatives with human and mouse chromatin conformation capture datasets. We apply INFIMA to identify novel effector genes for GWAS variants associated with diabetes. The results of the application are available at http://www.statlab.wisc.edu/shiny/INFIMA/ .

Application of 2D IR Bioimaging: Hyperspectral Images of Formalin-Fixed Pancreatic Tissues and Observation of Slow Protein Degradation.

We used two-dimensional IR bioimaging to study the structural heterogeneity of formalin-fixed mouse pancreas. Images were generated from the hyperspectral data sets by plotting quantities associated with the amide I vibrational mode, which is created by the backbone carbonyl stretch. Images that measure the fundamental vibrational frequencies, cross peaks, and anharmonic shifts are presented. Histograms are generated for each quantity, providing averaged values and distributions around the mean that serve as metrics for protein structures. Images were generated from tissue that had been stored in a formalin fixation for 3, 8, and 48 weeks. Over this period, all three metrics show that that the ß-sheet content of the samples increased, consistent with protein aggregation. Our results indicate that formalin fixation does not entirely arrest the degradation of a protein structure in pancreas tissue.

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.

Secretion of Recombinant Interleukin-22 by Engineered Lactobacillus reuteri Reduces Fatty Liver Disease in a Mouse Model of Diet-Induced Obesity.

The incidence of metabolic syndrome continues to rise globally. In mice, intravenous administration of interleukin-22 (IL-22) ameliorates various disease phenotypes associated with diet-induced metabolic syndrome. In patients, oral treatment is favored over intravenous treatment, but methodologies to deliver IL-22 via the oral route are nonexistent. The goal of this study was to assess to what extent engineered Lactobacillus reuteri secreting IL-22 could ameliorate nonalcoholic fatty liver disease. We used a mouse model of diet-induced obesity and assessed various markers of metabolic syndrome following treatment with L. reuteri and a recombinant derivative. Mice that received an 8-week treatment of wild-type probiotic gained less weight and had a smaller fat pad than the control group, but these phenotypes were not further enhanced by recombinant L. reuteri However, L. reuteri secreting IL-22 significantly reduced liver weight and triglycerides at levels that exceeded those of the probiotic wild-type treatment group. Our findings are interesting in light of the observed phenotypes associated with reduced nonalcoholic liver disease, in humans the most prevalent chronic liver disease, following treatment of a next-generation probiotic that is administered orally. Once biological and environmental containment strategies are in place, therapeutic applications of recombinant Lactobacillus reuteri are on the horizon.IMPORTANCE In humans, nonalcoholic fatty liver disease (NAFLD) is the most prevalent liver disease due to the increased prevalence of obesity. While treatment of NAFLD is often geared toward lifestyle changes, such as diet and exercise, the use of dietary supplements such as probiotics is underinvestigated. Here, we report that probiotic Lactobacillus reuteri reduces fatty liver in a mouse model of diet-induced obesity. This phenotype was further enhanced upon delivery of recombinant interleukin-22 by engineered Lactobacillus reuteri These observations pave the road to a better understanding of probiotic mechanisms driving the reduction of diet-induced steatosis and to development of next-generation probiotics for use in the clinic. Ultimately, these studies may lead to rational selection of (engineered) probiotics to ameliorate fatty liver disease.

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.

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.