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Blocking pyrimidine de novo synthesis by inhibiting dihydroorotate dehydrogenase is used to treat autoimmunity and prevent expansion of rapidly dividing cell populations including activated T cells. Here we show memory T cell precursors are resistant to pyrimidine starvation. Although the treatment effectively blocked effector T cells, the number, function and transcriptional profile of memory T cells and their precursors were unaffected. This effect occurred in a narrow time window in the early T cell expansion phase when developing effector, but not memory precursor, T cells are vulnerable to pyrimidine starvation. This vulnerability stems from a higher proliferative rate of early effector T cells as well as lower pyrimidine synthesis capacity when compared with memory precursors. This differential sensitivity is a drug-targetable checkpoint that efficiently diminishes effector T cells without affecting the memory compartment. This cell fate checkpoint might therefore lead to new methods to safely manipulate effector T cell responses.

Purpose Secretin activates brown adipose tissue (BAT) and induces satiation in both mice and humans. However, the exact brain mechanism of this satiety inducing, secretin-mediated gut-BAT-brain axis is largely unknown. Methods and results In this placebo-controlled, single-blinded neuroimaging study, firstly using [18F]-fluorodeoxyglucose (FDG) PET measures (n = 15), we established that secretin modulated brain glucose consumption through the BAT-brain axis. Predominantly, we found that BAT and caudate glucose uptake levels were negatively correlated (r = -0.54, p = 0.037) during secretin but not placebo condition. Then, using functional magnetic resonance imaging (fMRI; n = 14), we found that secretin improved inhibitory control and downregulated the brain response to appetizing food images. Finally, in a PET-fMRI fusion analysis (n = 10), we disclosed the patterned correspondence between caudate glucose uptake and neuroactivity to reward and inhibition, showing that the secretin-induced neurometabolic coupling patterns promoted satiation. Conclusion These findings suggest that secretin may modulate the BAT-brain metabolic crosstalk and subsequently the neurometabolic coupling to induce satiation. The study advances our understanding of the secretin signaling in motivated eating behavior and highlights the potential role of secretin in treating eating disorders and obesity. Trial registration EudraCT no. 2016-002373-35, registered 2 June 2016; Clinical Trials no. NCT03290846, registered 25 September 2017.

Brown adipose tissue (BAT) thermogenesis is activated by feeding. Recently, we revealed a secretin-mediated gut-BAT-brain axis, which stimulates satiation in mice, but the purpose of meal-induced BAT activation in humans has been unclear. In this placebo-controlled, randomized crossover study, we investigated the effects of intravenous secretin on BAT metabolism (measured with [18F]FDG and [15O]H2O positron emission tomography) and appetite (measured with functional magnetic resonance imaging) in healthy, normal weight men (GUTBAT trial no. NCT03290846). Participants were blinded to the intervention. Secretin increased BAT glucose uptake (primary endpoint) compared to placebo by 57% (median (interquartile range, IQR), 0.82 (0.77) versus 0.59 (0.53) μmol per 100 g per min, 95% confidence interval (CI) (0.09, 0.89), P = 0.002, effect size r = 0.570), while BAT perfusion remained unchanged (mean (s.d.) 4.73 (1.82) versus 6.14 (3.05) ml per 100 g per min, 95%CI (-2.91, 0.07), P = 0.063, effect size d = -0.549) (n = 15). Whole body energy expenditure increased by 2% (P = 0.011) (n = 15). Secretin attenuated blood-oxygen level-dependent activity (primary endpoint) in brain reward circuits during food cue tasks (significance level false discovery rate corrected at P = 0.05) (n = 14). Caloric intake did not significantly change, but motivation to refeed after a meal was delayed by 39 min (P = 0.039) (n = 14). No adverse effects were detected. Here we show in humans that secretin activates BAT, reduces central responses to appetizing food and delays the motivation to refeed after a meal. This suggests that meal-induced, secretin-mediated BAT activation is relevant in the control of food intake in humans. As obesity is increasing worldwide, this appetite regulating axis offers new possibilities for clinical research in treating obesity.

How to adjust metabolic rate (MR) in mice that differ in body mass and composition continues to lead to controversies. Here, the challenges that reside in the analysis of mouse MR are highlighted to spur consensus on the unequivocal use of regression-based analysis to maximize reliability and relevance of conclusions.

More than one-third of the worldwide population is overweight or obese and therefore at risk of developing type 2 diabetes mellitus. In order to mitigate this pandemic, safer and more potent therapeutics are urgently required. This necessitates the continued use of animal models to discover, validate and optimize novel therapeutics for their safe use in humans. In order to improve the transition from bench to bedside, researchers must not only carefully select the appropriate model but also draw the right conclusions. In this Review, we consolidate the key information on the currently available animal models of obesity and diabetes and highlight the advantages, limitations and important caveats of each of these models.

Detailed protocols and recommendations for the assessment of energy balance have been provided to address the problems associated with different body mass and body composition as apparent for mouse models in obesity research. Here, we applied these guidelines to investigate energy balance in two inbred mouse strains with contrasting susceptibilities for diet-induced obesity (DIO). Mice of the AKR/J strain are highly susceptible, whereas the SWR/J mice are almost completely resistant. The proximate mechanisms responsible for this striking phenotypic difference are only partially understood. Body mass and body composition, metabolizable energy, energy expenditure (EE), body temperature and spontaneous physical activity behavior were first assessed in a cohort of male AKR/J (N=29) and SWR/J (N=30) mice fed on a low-fat control diet (CD) to identify metabolic adaptations determining resistance to DIO. Thereafter, the immediate metabolic responses to high-fat diet (HFD) feeding for 3 days were investigated. Groups of weight-matched AKR/J (N=8) and SWR/J (N=8) mice were selected from the initial cohort for this intervention. Strain differences in body mass, fat mass and lean mass were adjusted by body mass as this was the only covariate significantly correlated with metabolizable energy and EE. On the CD, EE and fat oxidation was higher in SWR/J than in AKR/J mice, whereas no difference was found for metabolizable energy. In response to HFD feeding, both strains increased metabolizable energy intake, but also increased EE, body temperature, and fat oxidation. The catabolic adaptations to HFD feeding opposed the development of positive energy balance. Increased EE was not due to increased spontaneous physical activity. A significant strain difference was found when balancing metabolizable energy and daily energy expenditure (DEE). The guidelines were applicable with some limitations related to the adjustment of differences in body composition. Metabolic phenotyping revealed that metabolizable energy, DEE and metabolic fuel selection all contribute to the development of DIO. Therefore, assessing both sides of the energy balance equation is essential to identify the proximate mechanisms.

Animal models resembling human mutations are valuable tools to research the features of complex human craniofacial syndromes. This is the first report on a viable dominant mouse model carrying a non-synonymous sequence variation within the endothelin receptor type A gene (Ednra c.386A>T, p.Tyr129Phe) derived by an ENU mutagenesis program. The identical amino acid substitution was reported recently as disease causing in three individuals with the mandibulofacial dysostosis with alopecia (MFDA, OMIM 616367) syndrome. We performed standardized phenotyping of wild-type, heterozygous, and homozygous Ednra Y129F mice within the German Mouse Clinic. Mutant mice mimic the craniofacial phenotypes of jaw dysplasia, micrognathia, dysplastic temporomandibular joints, auricular dysmorphism, and missing of the squamosal zygomatic process as described for MFDA-affected individuals. As observed in MFDA-affected individuals, mutant Ednra Y129F mice exhibit hearing impairment in line with strong abnormalities of the ossicles and further, reduction of some lung volumetric parameters. In general, heterozygous and homozygous mice demonstrated inter-individual diversity of expression of the craniofacial phenotypes as observed in MFDA patients but without showing any cleft palates, eyelid defects, or alopecia. Mutant Ednra Y129F mice represent a valuable viable model for complex human syndromes of the first and second pharyngeal arches and for further studies and analysis of impaired endothelin 1 (EDN1)–endothelin receptor type A (EDNRA) signaling. Above all, Ednra Y129F mice model the recently published human MFDA syndrome and may be helpful for further disease understanding and development of therapeutic interventions. Electronic supplementary material The online version of this article (doi:10.1007/s00335-016-9664-5) contains supplementary material, which is available to authorized users.

The number of brown adipocytes residing within murine white fat depots (brite adipocytes) varies a lot by depot, strain and physiological condition. Several endocrine fibroblast growth factors are implicated in the regulation of brite adipocyte abundance. The family of fibroblast growth factors can be categorized by their site of action into endocrine, paracrine and intracellular peptides. We here screened paracrine fibroblast growth factors for their potential to drive brite adipogenesis in differentiating epididymal white adipocytes and identified fibroblast growth factor 8b to induce uncoupling protein 1 expression, but at the same time to interfere in adipogenesis. In an in vivo trial, fibroblast growth factor 8b released into the epididymal fat depot failed to robustly increase the number of brite adipocytes. The specific action of fibroblast growth factor 8b on the uncoupling protein 1 promoter in cultured epididymal adipocytes provides a model system to dissect specific gene regulatory networks.

Increased levels of blood plasma urea were used as phenotypic parameter for establishing novel mouse models for kidney diseases on the genetic background of C3H inbred mice in the phenotype-driven Munich ENU mouse mutagenesis project. The phenotypically dominant mutant line HST014 was established and further analyzed. Analysis of the causative mutation as well as the standardized, systemic phenotypic analysis of the mutant line was carried out. The causative mutation was detected in the potassium channel tetramerization domain containing 1 (Kctd1) gene which leads to the amino acid exchange Kctd1 I27N thereby affecting the functional BTB domain of the protein. This line is the first mouse model harboring a Kctd1 mutation. Kctd1 I27N homozygous mutant mice die perinatally. Standardized, systemic phenotypic analysis of Kctd1 I27N heterozygous mutants was carried out in the German Mouse Clinic (GMC). Systematic morphological investigation of the external physical appearance did not detect the specific alterations that are described in KCTD1 mutant human patients affected by the scalp-ear-nipple (SEN) syndrome. The main pathological phenotype of the Kctd1 I27N heterozygous mutant mice consists of kidney dysfunction and secondary effects thereof, without gross additional primary alterations in the other phenotypic parameters analyzed. Genome-wide transcriptome profiling analysis at the age of 4 months revealed about 100 differentially expressed genes (DEGs) in kidneys of Kctd1 I27N heterozygous mutants as compared to wild-type controls. In summary, the main alteration of the Kctd1 I27N heterozygous mutants consists in kidney dysfunction. Additional analyses in 9–21 week-old heterozygous mutants revealed only few minor effects.

Aging and neurodegeneration are often accompanied by a functionally impaired ubiquitin–proteasome system (UPS). In tauopathies and polyglutamine diseases, a mutant form of ubiquitin B (UBB+1) accumulates in disease-specific aggregates. UBB+1 mRNA is generated at low levels in vivo during transcription from the ubiquitin B locus by molecular misreading. The resulting mutant protein has been shown to inhibit proteasome function. To elucidate causative effects and neuropathological consequences of UBB+1 accumulation, we used a UBB+1 expressing transgenic mouse line that models UPS inhibition in neurons and exhibits behavioral phenotypes reminiscent of Alzheimer’s disease (AD). In order to reveal affected organs and functions, young and aged UBB+1 transgenic mice were comprehensively phenotyped for more than 240 parameters. This revealed unexpected changes in spontaneous breathing patterns and an altered response to hypoxic conditions. Our findings point to a central dysfunction of respiratory regulation in transgenic mice in comparison to wild-type littermate mice. Accordingly, UBB+1 was strongly expressed in brainstem regions of transgenic mice controlling respiration. These regions included, e.g., the medial part of the nucleus of the tractus solitarius and the lateral subdivisions of the parabrachial nucleus. In addition, UBB+1 was also strongly expressed in these anatomical structures of AD patients (Braak stage #6) and was not expressed in non-demented controls. We conclude that long-term UPS inhibition due to UBB+1 expression causes central breathing dysfunction in a transgenic mouse model of AD. The UBB+1 expression pattern in humans is consistent with the contribution of bronchopneumonia as a cause of death in AD patients. Electronic supplementary material The online version of this article (doi:10.1007/s00401-012-1003-7) contains supplementary material, which is available to authorized users.

Mammalian uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis in brown adipose tissue. We previously reported on the presence of a UCP1 orthologue in ectothermic fish and observed downregulation of UCP1 gene expression in the liver of the common carp. Neither the function of UCP1, nor the mode of UCP1 activation is known in carp liver mitochondria. Here, we compared the proton conductance at 25 degrees C of liver mitochondria isolated from carp either maintained at 20 degrees C (warm-acclimated, WA) or exposed to 8 degrees C (cold-acclimated, CA) water temperature for 7-10 days. Liver mitochondria from WA carp had higher state four rates of oxygen consumption and greater proton conductance at high membrane potential. Liver mitochondria from WA, but not from CA, carp showed a strong increase in proton conductance when palmitate (or 4-hydroxy-trans-2-nonenal, HNE) was added, and this inducible proton conductance was prevented by addition of GDP. This fatty acid sensitive proton leak is likely due to the expression of UCP1 in the liver of WA carp. The observed biochemical properties of proton leak strongly suggest that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as mammalian UCP1. Significant UCP1 expression was also detected in our previous study in whole brain of the carp. We here observed a twofold increase of UCP1 mRNA in carp brain following cold exposure, suggesting a role of UCP1 in the thermal adaptation of brain metabolism. In situ hybridization located the UCP1 gene expression to the optic tectum responsible for visual system control, the descending trigeminal tract and the solitary tract. Taken together, this study characterises uncoupling protein activity in an ectotherm for the first time.

Much of our understanding of physiology and metabolism is derived from investigating mouse mutants and transgenic mice, and open-access platforms for standardized mouse phenotyping such as the German Mouse Clinic (GMC) are currently viewed as one powerful tool for identifying novel gene-function relationships. Phenotyping or phenotypic screening involves the comparison of wild-type control mice with their mutant or transgenic littermates. In our study, we explored the extent to which standardized phenotyping will succeed in detecting biologically relevant phenotypic differences in mice generated and provided by different collaborators. We analyzed quantitative metabolic data (body mass, energy intake, and energy metabolized) collected at the GMC under the current workflow, and used them for statistical power considerations. Our results demonstrate that there is substantial variability in these parameters among lines of wild-type C57BL/6 (B6) mice from different sources. Given this variable background noise in mice that serve as controls, subtle phenotypes in mutant or transgenic littermates may be overlooked. Furthermore, a phenotype observed in one cohort of a mutant line may not be reproducible (to the same extent) in mice coming from a different environment or supplier. In the light of these constraints, we encourage researchers to incorporate information on intrastrain variability into future study planning, or to perform advanced hierarchical analyses. Both will ultimately improve the detectability of novel phenotypes by phenotypic screening.

During seasonal acclimation, Djungarian hamsters spontaneously exhibit a reduction in food intake, body mass and body fat stores, which is externally cued by shortening of day length in autumn and controlled by a sliding set-point. We investigated the function of the leptin adipostatic feedback system in the photoperiodic control of seasonal acclimation. In response to mouse recombinant leptin injections for 10 days, long day photoperiod (LD) and short day photoperiod (SD)-acclimated hamsters decreased food intake and body mass. The reduction of body mass was due to the depletion of body fat, as revealed by carcass composition analysis. In SD hamsters, leptin caused a larger reduction of body fat mass than observed under LD conditions, whereas the anorectic effect was similar in both photoperiods. The serum leptin concentration was 9.3 +/- 1.2 ng/ml in LD-acclimated hamsters and decreased significantly to 4.2 +/- 0.8 ng/ml and 2.1 +/- 0.6 ng/ml in hamsters exposed to SD for 66 days and 116 days, respectively (P < 0.001). A strong positive correlation between total body fat mass and serum leptin concentration was found (rS = 0.935, P < 0.0001, n = 70). Despite the anorectic action of exogenous leptin, higher endogenous leptin levels in LD hamsters were paralleled by higher food intake in LD hamsters as compared to SD hamsters. This paradoxical finding further supports the increased leptin sensitivity in SD hamsters as judged from leptin treatment experiments. We tested the functional significance of leptin for the controlled down-regulation of food intake and body mass induced by short photoperiod. Food restriction for 10 days during the transition phase decreased body mass below the desired sliding set-point, which was recovered in control hamsters following ad libitum refeeding. Treatment with mouse recombinant leptin during ad libitum refeeding inhibited the recovery of body mass and blunted the increase of food intake observed in controls, indicating that the sliding set-point utilizes leptin as a signal for the adjustment of the appropriate body mass level.

The bilateral lobe of interscapular brown adipose tissue of the Djungarian hamster was unilaterally denervated in order to study the role of the sympathetic innervation for maintenance and cold-induced increase of non-shivering thermogenesis. Denervation decreased the noradrenaline content of brown adipose tissue to less than 9% of the intact contralateral pad. This low noradrenaline level was maintained for 1-14 days after denervation. First, to study the role of the sympathetic innervation of brown adipose tissue in the maintenance of the high thermogenic capacity characteristic of the cold acclimated state, brown adipose tissue was denervated in hamsters either kept at thermoneutrality or acclimated to 5 degrees C ambient temperature for 4 weeks. Cold-acclimated hamsters had elevated levels of uncoupling protein messenger ribonucleic acid (8.1-fold) and cytochrom-c oxidase-activity (3-fold). Denervation of brown adipose tissue decreased uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity as compared to the intact pad in thermoneutral and in cold-acclimated hamsters. However, in cold-acclimated hamsters uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity in denervated brown adipose tissue both were maintained on an elevated 6-fold higher level as compared to thermoneutral controls. Second, to study the role of the sympathetic innervation of brown adipose tissue in the cold-induced increase in thermogenic capacity, hamsters were denervated prior to cold acclimation and responses were measured after 3 and 14 days of cold exposure. Uncoupling protein-messenger ribonucleic acid level and cytochrom-c-oxidase-activity of intact brown adipose tissue increased after 14 days cold acclimation.(ABSTRACT TRUNCATED AT 250 WORDS)

High attrition rates of novel anti-cancer drugs highlight the need for improved models to predict toxicity. Although polo-like kinase 1 (Plk1) inhibitors are attractive candidates for drug development, the role of Plk1 in primary cells remains widely unexplored. Therefore, we evaluated the utility of an RNA interference-based model to assess responses to an inducible knockdown (iKD) of Plk1 in adult mice. Here we show that Plk1 silencing can be achieved in several organs, although adverse events are rare. We compared responses in Plk1-iKD mice with those in primary cells kept under controlled culture conditions. In contrast to the addiction of many cancer cell lines to the non-oncogene Plk1, the primary cells' proliferation, spindle assembly and apoptosis exhibit only a low dependency on Plk1. Responses to Plk1-depletion, both in cultured primary cells and in our iKD-mouse model, correspond well and thus provide the basis for using validated iKD mice in predicting responses to therapeutic interventions., Polo-like kinase 1 is a key regulator of mitosis and is a candidate for drug development to treat cancer. Here, reduced expression of polo-like kinase 1 in adult mice has a minor impact on animal physiology, suggesting that polo-like kinase 1 inhibitors may be useful in the killing of tumour cells while sparing normal cells.

In Djungarian hamsters,Phodopus sungorus, daily torpor occurs spontaneously in winter in the presence of abundant food, but individuals show different tendencies to enter torpor. The results show that in hamsters fed rodent chow ad libitum individual torpor frequencies were negatively correlated with both food consumption and the amount of nocturnal locomotor activity. Provision of cafeteria diet at ambient temperatures below thermoneutrality significantly lowered torpor frequencies and induced body weight gains. However, in hamsters fed seeds with a high fat or carbohydrate content (i.e., sunflower seeds or wheat, respectively) neither a decrease of torpor frequencies nor an increase of body weights was observed. The results suggest that in Djungarian hamsters, daily torpor is an intrinsic component of energy balance control and is functionally linked to individual physiological adjustments of food consumption and foraging activity. In addition, the employment of daily torpor can be affected by social interactions, since the long-term pattern of alternations between torpor and normothermia was found to be synchronized in breeding pairs caged together.

Ucp3 is an integral protein of the inner mitochondrial membrane with a role in lipid metabolism preventing deleterious effects of fatty acids in states of high lipid oxidation. Ucp3 is expressed in brown adipose tissue and skeletal muscle and controlled by a transcription factor complex including PPARalpha, MyoD and the histone acetyltransferase p300. Several studies have demonstrated interaction of these factors with chicken ovalbumin upstream promoter transcription factor II (Coup-TFII). This nuclear receptor is involved in organogenesis and other developmental processes including skeletal muscle development, but also co-regulates a number of metabolic genes. In this study we in silico analyzed the upstream region of Ucp3 of the Djungarian hamster Phodopus sungorus and identified several putative response elements for Coup-TFII. We therefore investigated whether Coup-TFII is a further player in the transcriptional control of the Ucp3 gene in rodents. By quantitative PCR we demonstrated a positive correlation of Coup-TFII and Ucp3 mRNA expression in skeletal muscle and brown adipose tissue in response to food deprivation and cold exposure, respectively. In reporter gene assays Coup-TFII enhanced transactivation of the Ucp3 promoter conveyed by MyoD, PPARalpha, RXRalpha and/or p300. Using deletions and mutated constructs, we identified a Coup-TFII enhancer element 816–840 bp upstream of the transcriptional start site. Binding of Coup-TFII to this upstream enhancer was confirmed in electrophoretic mobility shift and supershift assays. Transcriptional regulation of the Coup-TFII gene in response to starvation and cold exposure seems to be the regulatory mechanism of Ucp3 mRNA expression in brown adipose and skeletal muscle tissue determining the final appropriate rate of transcript synthesis. These findings add a crucial component to the complex transcriptional machinery controlling expression of Ucp3. Given the substantial evidence for a function of Ucp3 in lipid metabolism, Coup-TFII may not only be a negative regulator of glucose responsive genes but also transactivate genes involved in lipid metabolism.