Epigenetic inheritance of diet-induced and sperm-borne mitochondrial RNAs.

Spermatozoa harbour a complex and environment-sensitive pool of small non-coding RNAs (sncRNAs)1, which influences offspring development and adult phenotypes1-7. Whether spermatozoa in the epididymis are directly susceptible to environmental cues is not fully understood8. Here we used two distinct paradigms of preconception acute high-fat diet to dissect epididymal versus testicular contributions to the sperm sncRNA pool and offspring health. We show that epididymal spermatozoa, but not developing germ cells, are sensitive to the environment and identify mitochondrial tRNAs (mt-tRNAs) and their fragments (mt-tsRNAs) as sperm-borne factors. In humans, mt-tsRNAs in spermatozoa correlate with body mass index, and paternal overweight at conception doubles offspring obesity risk and compromises metabolic health. Sperm sncRNA sequencing of mice mutant for genes involved in mitochondrial function, and metabolic phenotyping of their wild-type offspring, suggest that the upregulation of mt-tsRNAs is downstream of mitochondrial dysfunction. Single-embryo transcriptomics of genetically hybrid two-cell embryos demonstrated sperm-to-oocyte transfer of mt-tRNAs at fertilization and suggested their involvement in the control of early-embryo transcription. Our study supports the importance of paternal health at conception for offspring metabolism, shows that mt-tRNAs are diet-induced and sperm-borne and demonstrates, in a physiological setting, father-to-offspring transfer of sperm mitochondrial RNAs at fertilization.

A paternal methyl donor-rich diet altered cognitive and neural functions in offspring mice.

Dietary intake of methyl donors, such as folic acid and methionine, shows considerable intra-individual variation in human populations. While it is recognized that maternal departures from the optimum of dietary methyl donor intake can increase the risk for mental health issues and neurological disorders in offspring, it has not been explored whether paternal dietary methyl donor intake influences behavioral and cognitive functions in the next generation. Here, we report that elevated paternal dietary methyl donor intake in a mouse model, transiently applied prior to mating, resulted in offspring animals (methyl donor-rich diet (MD) F1 mice) with deficits in hippocampus-dependent learning and memory, impaired hippocampal synaptic plasticity and reduced hippocampal theta oscillations. Gene expression analyses revealed altered expression of the methionine adenosyltransferase Mat2a and BK channel subunit Kcnmb2, which was associated with changes in Kcnmb2 promoter methylation in MD F1 mice. Hippocampal overexpression of Kcnmb2 in MD F1 mice ameliorated altered spatial learning and memory, supporting a role of this BK channel subunit in the MD F1 behavioral phenotype. Behavioral and gene expression changes did not extend into the F2 offspring generation. Together, our data indicate that paternal dietary factors influence cognitive and neural functions in the offspring generation.

A history of obesity leaves an inflammatory fingerprint in liver and adipose tissue.

BACKGROUND/OBJECTIVES: Dieting is a popular yet often ineffective way to lower body weight, as the majority of people regain most of their pre-dieting weights in a relatively short time. The underlying molecular mechanisms driving weight regain and the increased risk for metabolic disease are still incompletely understood. Here we investigate the molecular alterations inherited from a history of obesity. METHODS: In our model, male high-fat diet (HFD)-fed obese C57BL/6J mice were switched to a low caloric chow diet, resulting in a decline of body weight to that of lean mice. We measured body composition, as well as metrics of glucose, insulin and lipid homeostasis. This was accompanied by histological and gene expression analysis of adipose tissue and liver to assess adipose tissue inflammation and hepatosteatosis. Moreover, acute hypothalamic response to (re-) exposure to HFD was assessed by qPCR. RESULTS & CONCLUSIONS: Within 7 weeks after diet switch, most obesity-associated phenotypes, such as body mass, glucose intolerance and blood metabolite levels were reversed. However, hepatic inflammation, hepatic steatosis as well as hypertrophy and inflammation of perigonadal, but not subcutaneous, adipocytes persisted in formerly obese mice. Transcriptional profiling of liver and perigonadal fat revealed an upregulation of pathways associated with immune function and cellularity. Thus, we show that weight reduction leaves signs of inflammation in liver and perigonadal fat, indicating that persisting proinflammatory signals in liver and adipose tissue could contribute to an increased risk of formerly obese subjects to develop the metabolic syndrome upon recurring weight gain.

Genome Wide Conditional Mouse Knockout Resources.

The International Knockout Mouse Consortium (IKMC) developed high throughput gene trapping and gene targeting pipelines that produced mostly conditional mutations of more than 18,500 genes in C57BL/6N mouse embryonic stem (ES) cells which have been archived and are freely available to the research community as a frozen resource. From this unprecedented resource more than 6,000 mutant mouse strains have been produced by the IKMC and mostly the International Mouse Phenotyping Consortium (IMPC). In addition, a cre-driver resource was established including 250 inducible cre-driver mouse strains in a C57BL/6 background. Complementing the cre-driver resource, a collection of comprising 27 cre-driver rAAVs has also been produced. The resources can be easily accessed at the IKMC/IMPC web portal (www.mousephenotype.org). The IKMC/IMPC resource is a standardized reference library of mouse models with defined genetic backgrounds that enables the analysis of gene-disease associations in mice of different genetic makeup and should therefore have a major impact on biomedical research.

Peri-conceptional obesogenic exposure induces sex-specific programming of disease susceptibilities in adult mouse offspring.

Vulnerability of the fetus upon maternal obesity can potentially occur during all developmental phases. We aimed at elaborating longer-term health outcomes of fetal overnutrition during the earliest stages of development. We utilized Naval Medical Research Institute (NMRI) mice to induce pre-conceptional and gestational obesity and followed offspring outcomes in the absence of any postnatal obesogenic influences. Male adult offspring developed overweight, insulin resistance, hyperleptinemia, hyperuricemia and hepatic steatosis; all these features were not observed in females. Instead, they showed impaired fasting glucose and a reduced fat mass and adipocyte size. Influences of the interaction of maternal diet∗sex concerned offspring genes involved in fatty liver disease, lipid droplet size regulation and fat mass expansion. These data suggest that a peri-conceptional obesogenic exposure is sufficient to shape offspring gene expression patterns and health outcomes in a sex- and organ-specific manner, indicating varying developmental vulnerabilities between sexes towards metabolic disease in response to maternal overnutrition.

Identification of biomarkers for apoptosis in cancer cell lines using metabolomics: tools for individualized medicine.

BACKGROUND: Metabolomics is a versatile unbiased method to search for biomarkers of human disease. In particular, one approach in cancer therapy is to promote apoptosis in tumour cells; this could be improved with specific biomarkers of apoptosis for monitoring treatment. We recently observed specific metabolic patterns in apoptotic cell lines; however, in that study, apoptosis was only induced with one pro-apoptotic agent, staurosporine. OBJECTIVE: The aim of this study was to find novel biomarkers of apoptosis by verifying our previous findings using two further pro-apoptotic agents, 5-fluorouracil and etoposide, that are commonly used in anticancer treatment. METHODS: Metabolic parameters were assessed in HepG2 and HEK293 cells using the newborn screening assay adapted for cell culture approaches, quantifying the levels of amino acids and acylcarnitines with mass spectrometry. RESULTS: We were able to identify apoptosis-specific changes in the metabolite profile. Moreover, the amino acids alanine and glutamate were both significantly up-regulated in apoptotic HepG2 and HEK293 cells irrespective of the apoptosis inducer. CONCLUSION: Our observations clearly indicate the potential of metabolomics in detecting metabolic biomarkers applicable in theranostics and for monitoring drug efficacy.

Genome-wide, large-scale production of mutant mice by ENU mutagenesis.

In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).

Inflammation and mitochondrial fatty acid beta-oxidation link obesity to early tumor promotion.

Obesity is associated with increased risk for developing pancreatic cancer, and it is suggested that insulin resistance provides the missing link. Here we demonstrate that under the context of genetic susceptibility, a high fat diet (HFD) predisposes mice with oncogenic K-ras activation to accelerated pancreatic intraepithelial neoplasm (PanIN) development. Tumor promotion is closely associated with increased inflammation and abrogation of TNFR1 signaling significantly blocks this process underlining a central role for TNFalpha in obesity-mediated enhancement of PanIN lesions. Interestingly, however, despite increased TNFalpha levels, mice remain insulin sensitive. We show that, while aggravating tumor promotion, a HFD exerts dramatic changes in energy metabolism through enhancement of pancreatic exocrine insufficiency, metabolic rates, and expression of genes involved in mitochondrial fatty acid (FA) beta-oxidation that collectively contribute to improved glucose tolerance in these mice. While on one hand these findings provide significant evidence that obesity is linked to tumor promotion in the pancreas, on the other it suggests alterations in inflammatory responses and bioenergetic pathways as the potential underlying cause.

Osteopontin deficiency protects against obesity-induced hepatic steatosis and attenuates glucose production in mice.

AIMS/HYPOTHESIS: Obesity is strongly associated with the development of non-alcoholic fatty liver disease (NAFLD). The cytokine osteopontin (OPN) was recently shown to be involved in obesity-induced adipose tissue inflammation and reduced insulin response. Accumulating evidence links OPN to the pathogenesis of NAFLD. Here we aimed to identify the role of OPN in obesity-associated hepatic steatosis and impaired hepatic glucose metabolism. METHODS: Wild-type (WT) and Opn (also known as Spp1) knockout (Opn (-/-)) mice were fed a high-fat or low-fat diet to study OPN effects in obesity-driven hepatic alterations. RESULTS: We show that genetic OPN deficiency protected from obesity-induced hepatic steatosis, at least in part, by downregulating hepatic triacylglycerol synthesis. Conversely, absence of OPN promoted fat storage in adipose tissue thereby preventing the obesity-induced shift to ectopic fat accumulation in the liver. Euglycaemic-hyperinsulinaemic clamp studies revealed that insulin resistance and excess hepatic glucose production in obesity were significantly attenuated in Opn (-/-) mice. OPN deficiency markedly improved hepatic insulin signalling as shown by enhanced insulin receptor substrate-2 phosphorylation and prevented upregulation of the major hepatic transcription factor Forkhead box O1 and its gluconeogenic target genes. In addition, obesity-driven hepatic inflammation and macrophage accumulation was blocked by OPN deficiency. CONCLUSIONS/INTERPRETATION: Our data strongly emphasise OPN as mediator of obesity-associated hepatic alterations including steatosis, inflammation, insulin resistance and excess gluconeogenesis. Targeting OPN action could therefore provide a novel therapeutic strategy to prevent obesity-related complications such as NAFLD and type 2 diabetes.

Analysis of the sex-specific variability of blood parameters in C3H inbred mice by using data from a long-term, high-throughput project.

Mice are important models for biomedical research by providing the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Use of both sexes in experiments is strongly recommended because of possible differences in the outcome. However, sex-specific phenotypic variability is discussed with regard to putative consequences on the group size which is necessary for achieving valid and reproducible results. Here, we retrospectively analyzed the sex-specific variability of 25 blood parameters of C3H inbred mice in two different mouse facilities withinthe long-term, high-throughput Munich ENU mouse mutagenesis project. Using the 95 % data range, data of4,780-20,706 mice per parameter were analyzed and resulted in ratios of the coefficient of variation (= female CV / (female CV + male CV)) from 0.44 to 0.58 for the 25 parameters, with an overall mean of 0.51 in both facilities. Together with data analyses of three additional, smaller studies with 72-247 animals per parameter examined and various genetic backgrounds (inbred strains, F1 hybrids) included, hints for reproducible sex-specific variability were observed for particular parameters. Thus, the overall analysis comprising all 25 clinical chemical and hematological parameters of the standardized, long-term analysis of a high number of group housed, young adult, twelve-week-old C3H inbred mice showed no evidence for substantial sex-specific variability. The results may provide a basis for the examination of sex-specific variability in particular blood parameters.

Phenotypic and pathomorphological characteristics of a novel mutant mouse model for maturity-onset diabetes of the young type 2 (MODY 2).

Several mutant mouse models for human diseases such as diabetes mellitus have been generated in the large-scale Munich ENU (N-ethyl-N-nitrosourea) mouse mutagenesis project. The aim of this study was to identify the causal mutation of one of these strains and to characterize the resulting diabetic phenotype. Mutants exhibit a T to G transversion mutation at nt 629 in the glucokinase (Gck) gene, leading to an amino acid exchange from methionine to arginine at position 210. Adult Munich Gck(M210R) mutant mice demonstrated a significant reduction of hepatic glucokinase enzyme activity but equal glucokinase mRNA and protein abundances. While homozygous mutant mice exhibited growth retardation and died soon after birth in consequence of severe hyperglycemia, heterozygous mutant mice displayed only slightly elevated blood glucose levels, present from birth, with development of disturbed glucose tolerance and glucose-induced insulin secretion. Additionally, insulin sensitivity and fasting serum insulin levels were slightly reduced in male mutant mice from an age of 90 days onward. While beta-cell mass was unaltered in neonate heterozygous and homozygous mutant mice, the total islet and beta-cell volumes and the total volume of isolated beta-cells were significantly decreased in 210-day-old male, but not female heterozygous mutant mice despite undetectable apoptosis. These findings indicate that reduced total islet and beta-cell volumes of male mutants might emerge from disturbed postnatal islet neogenesis. Considering the lack of knowledge about the pathomorphology of maturity-onset diabetes of the young type 2 (MODY 2), this glucokinase mutant model of reduced total islet and total beta-cell volume provides the opportunity to elucidate the impact of a defective glucokinase on development and maintenance of beta-cell mass and its relevance in MODY 2 patients.

[Characterization of ENU-mutant mice. Animal models for human diseases using morphological and molecular methods]. / Charakterisierung von ENU-Mausmutanten. Tiermodelle für menschliche Erkrankungen mittels morphologischer und molekularer Methoden.

Following sequencing of the human genome there are new challenges to decipher the knowledge concerning gene function and the role of gene interactions and pathways leading to disease. Mouse models have proven to be an ideal tool for this purpose. Point mutations induced by chemical mutagenesis by N-ethyl-N-nitrosourea (ENU) offer possibilities for the analysis of the phenotypic outcome of a single base pair exchange on the entire organism. The Munich ENU mouse mutagenesis project is part of the worldwide efforts to obtain mutations for each gene. The generation of new alleles or allelic series offers relevant insights into the relevance of single gene sections. Various mouse models for human diseases have been generated by a systematic large-scale genome-wide phenotyping screen in the last decade. This work illustrates how the implementation of the ENU mouse mutagenesis project with gene identification and parallel high-throughput screening is taking advantage of local cooperation with experienced phenotyping groups at the Helmholtz Zentrum München, leading to major advances in the functional analysis of the mammalian genome.

Overexpressed vs mutated Kras in murine fibroblasts: a molecular phenotyping study.

Ras acts in signalling pathways regulating the activity of multiple cellular functions including cell proliferation, differentiation, and apoptosis. Amino-acid exchanges at position 12, 13, or 61 of the Kras gene convert the proto-oncogene into an activated oncogene. Until now, a direct comparison of genome-wide expression profiling studies of Kras overexpression and different Kras mutant forms in a single assay system has not been carried out. In our study, we focused on the direct comparison of global gene expression effects caused by mutations in codon 12 or 13 of the Kras gene and Kras overexpression in murine fibroblasts. We determined Kras cellular mRNA, Ras protein and activated Ras protein levels. Further, we compared our data to the proteome analysis of the same transfected cell lines. Both overexpression and mutations of Kras lead to common altered gene expression patterns. Only two genes, Lox and Col1a1, were reversely regulated in the Kras transfectants. They may contribute to the higher aggressiveness of the Kras codon 12 mutation in tumour progression. The functional annotation of differentially expressed genes revealed a high frequency of proteins involved in tumour growth and angiogenesis. These data further support the important role of these genes in tumour-associated angiogenesis.

Novel lymphocyte-independent mechanisms to initiate inflammatory arthritis via bone marrow-derived cells of Ali18 mutant mice.

OBJECTIVE: In a large-scale ENU (N-ethyl-N-nitrosourea) mouse mutagenesis programme, we previously have identified and characterized a novel mutation Ali18 that causes inflammatory arthritis like lesions in peripheral joints. In this study, we analysed the immune system of Ali18 mice to understand mechanisms underlying the spontaneous inflammation. METHODS: Humoral and cellular components of the immune system were phenotyped by ELISA and flow cytometry. The contribution of the immune system for phenotype expression was analysed in disease transfer experiments. The involvement of the adaptive immune system was investigated in Ali18;Rag1 double mutants and the influence of environmental factors was analysed in Ali18 mice reared under germ-free conditions. RESULTS: Bone marrow cells from Ali18 mice were able to transfer the disease phenotype to naïve wild-type recipients suggesting that cellular components of the reconstituted immune system were sufficient to induce arthritis. Ali18 mice revealed abnormal leucocyte populations including lymphocytes and granulocytes, as well as increased plasma IL-5 and IgE levels. Ali18;Rag1 double homozygous mutants, which lack mature lymphocytes, still developed arthritis, suggesting that the phenotype is independent of the adaptive immune system. In addition, the arthritis phenotype appeared to be independent from environmental conditions as demonstrated in mice reared under germ-free conditions. CONCLUSIONS: The Ali18 mutation induces inflammatory arthritis through bone marrow-derived cells. However, non-pro-inflammatory cytokine cascades and mature lymphocyte independent-mechanisms are crucial for initiation and progression of the phenotype. Ali18 mice may thus represent a model to study mechanisms involved in seronegative arthritis induced by cells of the innate immune system.

Glucose tolerance and insulin sensitivity define adipocyte transcriptional programs in human obesity.

OBJECTIVE: Although debated, metabolic health characterizes 10-25% of obese individuals and reduces risk of developing life-threatening co-morbidities. Adipose tissue is a recognized endocrine organ important for the maintenance of whole-body metabolic health. Adipocyte transcriptional signatures of healthy and unhealthy obesity are largely unknown. METHODS: Here, we used a small cohort of highly characterized obese individuals discordant for metabolic health, characterized their adipocytes transcriptional signatures, and cross-referenced them to mouse phenotypic and human GWAs databases. RESULTS AND CONCLUSIONS: Our study showed that glucose intolerance and insulin resistance co-operate to remodel adipocyte transcriptome. We also identified the Nuclear Export Mediator Factor (NEMF) and the Ectoderm-Neural Cortex 1 (ENC1) as novel potential targets in the management of metabolic health in human obesity.

Interaction between Notch signalling and Lunatic fringe during somite boundary formation in the mouse.

BACKGROUND: The process of somitogenesis can be divided into three major events: the prepatterning of the mesoderm; the formation of boundaries between the prospective somites; and the cellular differentiation of the somites. Expression and functional studies have demonstrated the involvement of the murine Notch pathway in somitogenesis, although its precise role in this process is not yet well understood. We examined the effect of mutations in the Notch pathway elements Delta like 1 (Dll1), Notch1 and RBPJkappa on genes expressed in the presomitic mesoderm (PSM) and have defined the spatial relationships of Notch pathway gene expression in this region. RESULTS: We have shown that expression of Notch pathway genes in the PSM overlaps in the region where the boundary between the posterior and anterior halves of two consecutive somites will form. The Dll1, Notch1 and RBPJkappa mutations disrupt the expression of Lunatic fringe (L-fng), Jagged1, Mesp1, Mesp2 and Hes5 in the PSM. Furthermore, expression of EphA4, mCer 1 and uncx4.1, markers for the anterior-posterior subdivisions of the somites, is down-regulated to different extents in Notch pathway mutants, indicating a global alteration of pattern in the PSM. CONCLUSIONS: We propose a model for the mechanism of somite border formation in which the activity of Notch in the PSM is restricted by L-fng to a boundary-forming territory in the posterior half of the prospective somite. In this region, Notch function activates a set of genes that are involved in boundary formation and anterior-posterior somite identity.

Cryopreservation of mouse spermatozoa: double your mouse space.

Cryopreservation of mouse germplasm is an important tool to secure, archive and distribute mouse lines of different origin. While embryo freezing has been standard procedure for many years, major advantages in freezing spermatozoa have been made recently that open new opportunities for management of mouse colonies.

Functional genome analysis indicates loss of 17beta-hydroxysteroid dehydrogenase type 2 enzyme in the zebrafish.

Among the family of 17beta-hydroxysteroid dehydrogenases, the type 2 (17beta-HSD 2) is the main enzyme responsible for inactivation of estrogens and androgens, catalyzing the oxidation of the C17 hydroxyl group. 17beta-HSD 2 has been studied only in mammals, its occurrence and function in other vertebrates hardly known. We investigated the presence of homologs in non-mammalian species and found sequences of 17beta-HSD 2 and its closest homolog 11beta-HSD 2 in zebrafish (Danio rerio), Takifugu rubripes, Tetraodon nigroviridis, Xenopus tropicalis and chicken databases. Furthermore, we cloned zebrafish 17beta-HSD 2 from ovarian tissue and found high expression also in the testis of adult fish and throughout embryogenesis. The enzyme, though, is inactive likely due to a non-sense N-terminal region including a dysfunctional cofactor binding motif. Replacement of the affected part by the corresponding human 17beta-HSD 2 sequence fully restored enzymatic activity. Comparison of all retrieved 17beta-HSD 2 sequences indicates that this functional loss may have occurred only in zebrafish, where steroid inactivation at position C17 seems to pursue without the protein studied. The closely related 11beta-HSD 2 is unlikely to substitute for 17beta-HSD 2 since in our hands it did not catalyze the respective oxidation of testosterone or estradiol.

A mutation in the enamelin gene in a mouse model.

Amelogenesis imperfecta is an inherited disorder affecting tooth enamel formation. We previously isolated a mouse strain with an amelogenesis imperfecta phenotype (ATE1 mice) from a dominant ethylnitrosourea screen and mapped the disease-causing defect to a 9-cM region of mouse chromosome 5. In the current study, we tested the hypothesis that there is a mutation in enamelin (ENAM) or ameloblastin (AMBN), both of which are located within the linkage region, by sequencing these two candidate genes. Analysis of our data shows that the amelogenesis imperfecta phenotype is linked to a C > T transition in exon 8 of the enamelin gene. The mutation predicts a C826T transition, which is present in the enamelin transcript and changes the glutamine (Gln) codon at position 176 into a premature stop codon (Gln176X). Conversely, no mutation could be detected in the ameloblastin gene. These results define the ATE1 mice as a model for local hypoplastic autosomal-dominant amelogenesis imperfecta (AIH2), which is caused by enamelin truncation mutations in humans.

Linking disease-associated genes to regulatory networks via promoter organization.

Pathway- or disease-associated genes may participate in more than one transcriptional co-regulation network. Such gene groups can be readily obtained by literature analysis or by high-throughput techniques such as microarrays or protein-interaction mapping. We developed a strategy that defines regulatory networks by in silico promoter analysis, finding potentially co-regulated subgroups without a priori knowledge. Pairs of transcription factor binding sites conserved in orthologous genes (vertically) as well as in promoter sequences of co-regulated genes (horizontally) were used as seeds for the development of promoter models representing potential co-regulation. This approach was applied to a Maturity Onset Diabetes of the Young (MODY)-associated gene list, which yielded two models connecting functionally interacting genes within MODY-related insulin/glucose signaling pathways. Additional genes functionally connected to our initial gene list were identified by database searches with these promoter models. Thus, data-driven in silico promoter analysis allowed integrating molecular mechanisms with biological functions of the cell.