The mitochondrial selenoenzyme thioredoxin reductase 2 (TXNRD2) plays a critical role in redox homeostasis and reactive oxygen species (ROS) scavenging. While heart-specific deletion of Txnrd2 in mice resulted in cardiac dysfunction, TXNRD2 function in skeletal muscle, the major component of lean body mass, remains unclear. In human GWAS the TXNRD2 locus is associated with total lean mass. Here, we show that Txnrd2 muscle-specific knockout (mTKO) induces a lean phenotype characterized by muscle atrophy and diminished adipose tissues. mTKO mice were resistant to weight gain on standard and high-fat diet. Whole body glucose clearance was increased, and ATP levels in muscle were decreased, suggesting impaired mitochondrial energy production. Transcriptomic and metabolomic analyses revealed alterations in one-carbon metabolism and related pathways. Despite elevated glutathione levels, changes in key factors of cellular detoxification were consistent with compromised antioxidant defence system. In sum, we unravel that Txnrd2 deficiency in skeletal muscle rewires whole-body energy metabolism through mitochondrial dysfunction and impaired redox capacity.

BACKGROUND & AIMS:Oxidative stress and antioxidant defense mechanisms have long been implicated in the pathogenesis of acute pancreatitis (AP). However, there is a notable lack of in vivo experimental evidence clarifying their precise role. METHODS:We generated and analyzed mice with a pancreas-specific deletion of Txnrd1 (Txnrd1Δpanc). AP was induced in these mice using cerulein injections. Pancreatic tissue was subsequently analyzed using immunoblotting, histology, immunohistochemistry, RNA sequencing, and biochemical assays. RESULTS:Txnrd1Δpanc mice exhibited normal growth, pancreatic weight, histology, and pancreatic function comparable to controls, although they experienced a slightly more severe course of AP. An increase in glutathione levels and upregulation of components within the glutathione system were observed in these mice. However, depletion of the glutathione pool led to pancreatic necrosis, followed by regeneration. When glutathione depletion was combined with AP, Txnrd1Δpanc mice suffered a profound and permanent loss of acinar tissue. CONCLUSIONS:These findings indicate that the response to AP is closely linked to alterations in antioxidant systems. The thioredoxin and the glutathione systems appear to perform overlapping protective roles in safeguarding acinar cells during AP. A simultaneous disruption of both systems proves detrimental to pancreatic integrity during acute pancreatitis.

Objectives: We here assessed whether typical pathogens of laboratory mice affect the development of diet-induced obesity and glucose intolerance, and whether colonization affects the efficacy of the GLP-1R agonist liraglutide and of the GLP-1/GIP co-agonist MAR709 to treat obesity and diabetes. Methods: Male C57BL/6J mice were experimentally infected with Helicobacter hepaticus, Rodentibacter pneumotropicus and Staphylococcus aureus and compared to a group of uninfected specific and opportunistic pathogen free (SOPF) mice. The development of diet-induced obesity and glucose intolerance was monitored over a period of 26 weeks. To study the influence of pathogens on drug treatment, mice were then subjected for 6 days daily treatment with either the GLP-1 receptor agonist liraglutide or the GLP-1/GIP co-agonist MAR709. Results: Colonized mice did not differ from SOPF controls regarding HFD-induced body weight gain, food intake, body composition, glycemic control, or responsiveness to treatment with liraglutide or the GLP-1/GIP co-agonist MAR709. Conclusions: We conclude that the occurrence of H. hepaticus, R. pneumotropicus and S. aureus does neither affect the development of dietinduced obesity or type 2 diabetes, nor the efficacy of GLP-1-based drugs to decrease body weight and to improve glucose control in mice.

Teleost fish such as Danio rerio (zebrafish) have been successfully used in biomedical research since decades. Genetically altered fish lines obtained by state-of-the-art genetic technologies are serving as well-known model organisms. In Europe, following Directive 2010/63/EU, generation, breeding, and husbandry of new genetically altered lines of laboratory animals require governmental state approval in case pain, suffering, distress, or long-lasting harm to the offspring derived by breeding of these lines cannot be excluded. The identification and assessment of pain, distress, or harm, according to a severity classification of mild, moderate, severe, or humane endpoint, became a new challenging task for all scientists, animal technicians, and veterinarians for daily work with laboratory zebrafish. In this study, we describe the performance of the assessment of welfare parameters of selected pathologic phenotypes and abnormalities frequently found in laboratory fish facilities based on veterinary, biological, and physiological aspects by using a dedicated score sheet. In a colony of zebrafish, we evaluated the frequency of genotype-independent abnormalities observed within 3 years. We give examples for severity classification and measures once an abnormality has been identified according to the 3Rs (Replacement, Reduction and Refinement).

Objective: Polypharmacotherapy shows superior efficacy compared to monotherapy in correcting obesity and its co-morbidities in preclinical studies and clinical trials. Female organisms have been traditionally neglected in this research potentially contributing to an increased rate of adverse advents in women. To address this disparity we herein determined the efficacy of our monomeric peptide with a balanced agonism at the receptors for glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) to correct obesity, glucose metabolism, nonalcoholic fatty liver disease (NAFLD) and dyslipidemia in both sexes of a mouse model for diet-induced obesity (DIO) by combining physiological treatment endpoints with plasma proteomic profiling (PPP), a new unbiased diagnostic tool for the efficacy and optimization of pharmacological interventions. Methods: We performed metabolic phenotyping along with PPP in body weight matched male and female DIO mice treated for 21 days with either PBS, the single GIP and GLP-1 monoagonists, or our GLP-1/GIP coagonist. Results: GLP-1R/GIPR coagonism improved obesity, glucose intolerance, NAFLD and dyslipidemia with superior efficacy in both male and female mice compared to monoagonist treatments. PPP revealed in both sexes broader changes of plasma proteins after GLP-1/GIP coagonist compared to monoagonist treatments, including established and novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP. Conclusions: We herein report enhanced efficacy of our GLP-1/GIP coagonist in both sexes relative to monoagonists for the treatment of metabolic disease. Wider sex-specific reductions of circulating proteins after GLP-1/GIP coagonist treatment may reflect additional metabolic benefits that are currently achieved exclusively after bariatric surgery. Disclosure S.M. Hofmann: Advisory Panel; Spouse/Partner; Novo Nordisk A/S. N.J. Wewer Albrechtsen: Research Support; Self; Mercodia, Novo Nordisk A/S, Novo Nordisk Foundation. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. M.H. Tschöp: Advisory Panel; Self; ERX Pharmaceuticals, Novo Nordisk Foundation. A. Feuchtinger: None. M. Brielmeier: None. B. Finan: Employee; Self; Novo Nordisk A/S. R. DiMarchi: Employee; Self; Novo Nordisk Inc. M. Kleinert: None. S. Sachs: None. T.D. Müller: Research Support; Self; Novo Nordisk Inc., Sanofi-Aventis. K. Stemmer: None. M. Mann: None.

Aims Unimolecular peptides targeting the receptors for glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) (GLP-1/GIP co-agonist) have been shown to outperform each single peptide in the treatment of obesity and cardiometabolic disease in preclinical and clinical trials. By combining physiological treatment endpoints with plasma proteomic profiling (PPP), we aimed to identify biomarkers to advance non-invasive metabolic monitoring of compound treatment success and exploration of ulterior treatment effects on an individual basis. Materials and methods We performed metabolic phenotyping along with PPP in body weight-matched male and female diet-induced obese (DIO) mice treated for 21 days with phosphate-buffered saline, single GIP and GLP-1 mono-agonists, or a GLP-1/GIP co-agonist. Results GLP-1R/GIPR co-agonism improved obesity, glucose intolerance, non-alcoholic fatty liver disease (NAFLD) and dyslipidaemia with superior efficacy in both male and female mice compared with mono-agonist treatments. PPP revealed broader changes of plasma proteins after GLP-1/GIP co-agonist compared with mono-agonist treatments in both sexes, including established and potential novel biomarkers for systemic inflammation, NAFLD and atherosclerosis. Subtle sex-specific differences have been observed in metabolic phenotyping and PPP. Conclusions We herein show that a recently developed unimolecular GLP-1/GIP co-agonist is more efficient in improving metabolic disease than either mono-agonist in both sexes. PPP led to the identification of a sex-independent protein panel with the potential to monitor non-invasively the treatment efficacies on metabolic function of this clinically advancing GLP-1/GIP co-agonist.

During β-adrenergic stimulation of brown adipose tissue (BAT), p38 phosphorylates the activating transcription factor 2 (ATF2) which then translocates to the nucleus to activate the expression of Ucp1 and Pgc-1α . The mechanisms underlying ATF2 target activation are unknown. Here we demonstrate that p62 (Sqstm1) binds to ATF2 to orchestrate activation of the Ucp1 enhancer and Pgc-1α promoter. P62 Δ69-251 mice show reduced expression of Ucp1 and Pgc-1α with impaired ATF2 genomic binding. Modulation of Ucp1 and Pgc-1α expression through p62 regulation of ATF2 signaling is demonstrated in vitro and in vivo in p62 Δ69-251 mice, global p62 −/− and Ucp1-Cre p62 flx/flx mice. BAT dysfunction resulting from p62 deficiency is manifest after birth and obesity subsequently develops despite normal food intake, intestinal nutrient absorption and locomotor activity. In summary, our data identify p62 as a master regulator of BAT function in that it controls the Ucp1 pathway through regulation of ATF2 genomic binding.

Precise knowledge of the health status of experimental fish is crucial to obtain high scientific and ethical standards in biomedical research. In addition to the use of sentinel fish, the examination of diseased fish is a fundamental part of all health monitoring concepts. PCR assays offer excellent sensitivity and the ability to test a broad variety of pathogenic agents in different sample types. Recently, it was shown that analysis of environmental samples such as water, sludge or detritus from static tanks can complement PCR analysis of fish and is actually more reliable for certain pathogens. In our study, we investigated whether the analysis of filtered water mixed with detritus of tanks including fish showing clinical signs of illness is suitable to complement health monitoring programs in recirculating systems. The obtained data indicate that pathogens such as Pseudoloma neurophilia or Myxidium streisingeri were exclusively or mainly found in fish, while mycobacteria were predominantly present in environmental samples. A combination of both sample types seems to be required for the detection of a broad range of infectious agents in zebrafish colonies using real-time PCR technology.

Murine Astrovirus is one of the most prevalent viral agents in laboratory rodent facilities worldwide, but its influence on biomedical research results is poorly examined. Due to possible influence on research results and high seroprevalence rates in mice, it appears useful to include this virus into routine health monitoring programs. In order to establish exhaust air particle PCR as a reliable detection method for Murine Astrovirus infections in mice kept in individually ventilated cages (IVC) and compare the method to sentinel mice monitoring regarding reproducibility and detection limit, we conducted a study with defined Murine Astrovirus cage prevalence. In parallel, the efficacy of both detection strategies (soiled-bedding sentinel (SBS) and exhaust air dust (EAD) analysis) was tested for Myocoptes musculinus. The fur mite was used as a reference organism during the whole study period to ensure the validity of this method. Because some publications already demonstrated successful detection of several pathogens, including murine fur mite species, via EAP-PCR. Detection of Murine Astrovirus infections at low prevalence is possible with both methods tested. Detection by exhaust air particles (EAP) is faster, more sensitive and more reliable compared to soiled bedding sentinels (SBS). Exhaust air particle PCR also detected the reference organism Myocoptes musculinus, which was not detected at all by sentinel mice, not even by high sensitivity fur swab qPCR. In conclusion, Murine Astrovirus can be detected by both exhaust air particle PCR and soiled bedding sentinels. We recommend exhaust air particle PCR as the better detection technique for Murine Astrovirus, because it is more reliable. Environmental samples are the method of choice for detection of Myocoptes musculinus because relying on soiled bedding sentinels harbors a big risk of missing existing infestations.

Accurate knowledge of the health status of experimental animals is pivotal to high scientific and ethical standards in biomedical research. Individually ventilated cages (IVCs) are becoming the predominant system for housing laboratory mice, as they prevent cage-to-cage infections. However, this feature constitutes a major drawback for hygienic monitoring of mouse colonies, as traditional screening programs build on reliable transmission of infectious agents from experimental animals to sentinel mice commonly tested as representatives for the mouse colonies. In recent years, the laboratory animal community has realized that sentinels are ineffectual for screening mouse colonies in IVC systems because infections are often not transmitted to sentinels and therefore remain undetected. Furthermore, sentinel monitoring results in high numbers of used animals. In contrast, environmental monitoring provides a more reliable approach to identify and exclude pathogens in rodent colonies. In recent studies we provided evidence that polymerase chain reaction analysis of exhaust air particles is superior to soiled bedding sentinels for different agents. In this study, we show that testing pooled environmental samples generates more meaningful information compared to soiled bedding sentinels during routine hygienic monitoring in different barriers.

The hygienic status of laboratory animals is pivotal for animal health as well as for the reliability and reproducibility of experimental results in biomedical research. Common health monitoring strategies of individually ventilated cage (IVC) reared rodent colonies involve sentinel animals exposed to soiled bedding of the colony to monitor. After an adequate exposure time these sentinel mice representing all animals housed in the respective racks are euthanized and examined for unwanted organisms which results in higher numbers of animals used for the sole purpose of health monitoring. Sentinel monitoring is subject to limitations: IVCs prevent transmission of airborne pathogens; transmission of unwanted organisms to sentinels by used bedding is uncertain due to dilution and susceptibility of sentinels to some pathogens is low. Screening of exhaust air dust of IVC racks for nucleic acids of unwanted pathogens is a relatively new and promising approach to improve health monitoring in IVC systems whilst avoiding the additional use of animals for this purpose. This review article summarizes current studies on the new health monitoring technology. Exhaust Air Particle (EAP) PCR outperformed sentinel monitoring by far. Infections have been detected newly or at lower prevalence. Hence, laboratory animals can be replaced in biomedical research and knowledge on the hygienic status of rodent colonies can be improved. Since the technology has a broad applicability the possible impact as replacement according to the 3Rs is remarkable.

Animal welfare requires the adequate housing of animals to ensure health and well-being. The application of environmental enrichment is a way to improve the well-being of laboratory animals. However, it is important to know whether these enrichment items can be incorporated in experimental mouse husbandry without creating a divide between past and future experimental results. Previous small-scale studies have been inconsistent throughout the literature, and it is not yet completely understood whether and how enrichment might endanger comparability of results of scientific experiments. Here, we measured the effect on means and variability of 164 physiological parameters in 3 conditions: with nesting material with or without a shelter, comparing these 2 conditions to a “barren” regime without any enrichments. We studied a total of 360 mice from each of 2 mouse strains (C57BL/6NTac and DBA/2NCrl) and both sexes for each of the 3 conditions. Our study indicates that enrichment affects the mean values of some of the 164 parameters with no consistent effects on variability. However, the influence of enrichment appears negligible compared to the effects of other influencing factors. Therefore, nesting material and shelters may be used to improve animal welfare without impairment of experimental outcome or loss of comparability to previous data collected under barren housing conditions.

Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.

One limitation to housing rodents in individually ventilated cages (IVCs) is the ineffectiveness of traditional health monitoring programs that test soiled bedding sentinels every quarter. Aerogen transmission does not occur with this method. Moreover, the transmission of numerous pathogens in bedding is uncertain, and sentinel susceptibility to various pathogens varies. A novel method using particle collection from samples of exhaust air was developed in this study which was also systematically compared with routine health monitoring using soiled bedding sentinels. We used our method to screen these samples for the presence of murine norovirus (MNV), a mouse pathogen highly prevalent in laboratory animal facilities. Exhaust air particles from prefilters of IVC racks with known MNV prevalence were tested by quantitative reverse transcription polymerase chain reaction (RT–qPCR). MNV was detected in exhaust air as early as one week with one MNV-positive cage per rack, while sentinels discharged MNV RNA without seroconverting. MNV was reliably and repeatedly detected in particles collected from samples of exhaust air in all seven of the three-month sampling rounds, with increasing MNV prevalence, while sentinels only seroconverted in one round. Under field conditions, routine soiled bedding sentinel health monitoring in our animal facility failed to identify 67% ( n = 85) of positive samples by RT–qPCR of exhaust air particles. Thus, this method proved to be highly sensitive and superior to soiled bedding sentinels in the reliable detection of MNV. These results represent a major breakthrough in hygiene monitoring of rodent IVC systems and contribute to the 3R principles by reducing the number of animals used and by improving experimental conditions.

Reliable detection of unwanted organisms is essential for meaningful health monitoring in experimental animal facilities. Currently, most rodents are housed in IVC systems, which prevent the aerogenic transmission of pathogens between cages. Typically soiled-bedding sentinels (SBS) exposed to soiled bedding collected from a population of animals within an IVC rack are tested as representatives, but infectious agents often go undetected due to inefficient transmission. Pasteurellaceae are among the most prevalent bacterial pathogens isolated from experimental mice, and the failure of SBS to detect these bacteria is well established. In this study, we investigated whether analysis of exhaust air dust (EAD) samples by using a sensitive and specific real-time PCR assay is superior to conventional SBS monitoring for the detection of Pasteurella pneumotropica (Pp) infections. In a rack with a known prevalence of Pp-positive mice, weekly EAD sampling was compared with the classic SBS method over 3 mo. In 6 rounds of testing, with a prevalence of 5 infected mice in each of 7 cages in a rack of 63 cages, EAD PCR detected Pp at every weekly time point; SBS failed to detect Pp in all cases. The minimal prevalence of Pp-infected mice required to obtain a reliable positive result by EAD PCR testing was determined to be 1 in 63 cages. Reliable detection of Pp was achieved after only 1 wk of exposure. Analysis of EAD samples by real-time PCR assay provides a sensitive, simple, and reliable approach for Pp identification in laboratory mice.

OBJECTIVE: Mitochondrial thioredoxin reductase is essential for mouse embryogenesis. Its ubiquitous deletion leads to early embryonic lethality1. Whilst several tissue-specific Txnrd2 knockouts have been described2, the significance of Txnrd2 in aging skeletal muscle is yet to be examined. We therefore generated a muscle-specific knockout of the mitochondrial thioredoxin reductase and investigated its function in the aging murine skeletal muscle. METHODOLOGY: Mice expressing cre recombinase under the control of the muscle-specific actin α1 promotor3 were mated to mice carrying floxed Txnrd2 alleles1 to generate the muscle-specific Txnrd2 knockout mice (KO). Mitochondria were isolated from gastrocnemius muscle and mitochondrial respiration was measured using a Clark-type electrode. RESULTS: A significantly lower O2 consumption in isolated mitochondria of old KO compared with control mice was observed. Isolated mitochondria of young KO showed a tendency towards a higher O2 consumption compared to control mice. Mitochondrial membrane integrity was not compromised in KO mitochondria. CONCLUSION: These results show altered mitochondrial function in skeletal muscle due to a loss of Txnrd2 and indicate Txnrd2 as a key player in sustaining muscle function during aging.

BACKGROUND:Ubiquitous deletion of thioredoxin reductase 2 (Txnrd2) in mice is embryonically lethal and associated with abnormal heart development, while constitutive, heart-specific Txnrd2 inactivation leads to dilated cardiomyopathy and perinatal death. The significance of Txnrd2 in aging cardiomyocytes, however, has not yet been examined.METHODS AND RESULTS:The tamoxifen-inducible heart-specific αMHC-MerCreMer transgene was used to inactivate loxP-flanked Txnrd2 alleles in adult mice. Hearts and isolated mitochondria from aged knockout mice were morphologically and functionally analyzed. Echocardiography revealed a significant increase in left ventricular end-systolic diameters in knockouts. Fractional shortening and ejection fraction were decreased compared with controls. Ultrastructural analysis of cardiomyocytes of aged mice showed mitochondrial degeneration and accumulation of autophagic bodies. A dysregulated autophagic activity was supported by higher levels of lysosome-associated membrane protein 1 (LAMP1), microtubule-associated protein 1A/1B-light chain 3-I (LC3-I), and p62 in knockout hearts. Isolated Txnrd2-deficient mitochondria used less oxygen and tended to produce more reactive oxygen species. Chronic hypoxia inducible factor 1, α subunit stabilization and altered transcriptional and metabolic signatures indicated that energy metabolism is deregulated.CONCLUSIONS:These results imply a novel role of Txnrd2 in sustaining heart function during aging and suggest that Txnrd2 may be a modifier of heart failure.

Pasteurellaceae are among the most prevalent bacterial pathogens isolated from mice housed in experimental animal facilities. Reliable detection and differentiation of Pasteurellaceae are essential for high-quality health monitoring. In this study, we combined a real-time PCR assay amplifying a variable region in the 16S rRNA sequence with high-resolution melting curve analysis (HRM) to identify and differentiate among the commonly isolated species Pasteurella pneumotropica biotypes “Jawetz” and “Heyl”, Actinobacillus muris, and Haemophilus influenzaemurium. We used a set of six reference strains for assay development, with the melting profiles of these strains clearly distinguishable due to DNA sequence variations in the amplicon. For evaluation, we used real-time PCR/HRM to test 25 unknown Pasteurellaceae isolates obtained from an external diagnostic laboratory and found the results to be consistent with those of partial 16S rRNA sequencing. The real-time PCR/HRM method provides a sensitive, rapid, and closed-tube approach for Pasteurellaceae species identification for health monitoring of laboratory mice.