Identification of genetic elements in metabolism by high-throughput mouse phenotyping.

Metabolic diseases are a worldwide problem but the underlying genetic factors and their relevance to metabolic disease remain incompletely understood. Genome-wide research is needed to characterize so-far unannotated mammalian metabolic genes. Here, we generate and analyze metabolic phenotypic data of 2016 knockout mouse strains under the aegis of the International Mouse Phenotyping Consortium (IMPC) and find 974 gene knockouts with strong metabolic phenotypes. 429 of those had no previous link to metabolism and 51 genes remain functionally completely unannotated. We compared human orthologues of these uncharacterized genes in five GWAS consortia and indeed 23 candidate genes are associated with metabolic disease. We further identify common regulatory elements in promoters of candidate genes. As each regulatory element is composed of several transcription factor binding sites, our data reveal an extensive metabolic phenotype-associated network of co-regulated genes. Our systematic mouse phenotype analysis thus paves the way for full functional annotation of the genome

A dual Ucp1 reporter mouse model for imaging and quantitation of brown and brite fat recruitment

Objectives: Brown adipose tissue (BAT) dissipates nutritional energy as heat through uncoupling protein 1 (UCP1). The discovery of functional BAT in healthy adult humans has promoted the search for pharmacological interventions to recruit and activate brown fat as a treatment of obesity and diabetes type II. These efforts require in vivo models to compare the efficacy of novel compounds in a relevant physiological context. Methods: We generated a knock-in mouse line expressing firefly luciferase and near-infrared red florescent protein (iRFP713) driven by the regulatory elements of the endogenous Ucp1 gene. Results: Our detailed characterization revealed that firefly luciferase activity faithfully reports endogenous Ucp1 gene expression in response to physiological and pharmacological stimuli. The iRFP713 fluorescence signal was detected in the interscapular BAT region of cold-exposed reporter mice in an allele-dosage dependent manner. Using this reporter mouse model, we detected a higher browning capacity in female peri-ovarian white adipose tissue compared to male epididymal WAT, which we further corroborated by molecular and morphological features. In situ imaging detected a strong luciferase activity signal in a previously unappreciated adipose tissue depot adjunct to the femoral muscle, now adopted as femoral brown adipose tissue. In addition, screening cultured adipocytes by bioluminescence imaging identified the selective Salt-Inducible Kinase inhibitor, HG-9-91-01, to increase Ucp1 gene expression and mitochondrial respiration in brown and brite adipocytes. Conclusions: In our mouse model, firefly luciferase activity serves as a bona fide reporter for dynamic regulation of Ucp1. In addition, by means of iRFP713 we are able to monitor Ucp1 expression in a non-invasive fashion. Keywords: BAT, WAT, Firefly luciferase, iRFP713, UCP1, Thermogenesis, Brownin

Adaptive thermogenesis and thermal conductance in wild-type and UCP1-KO mice

We compared maximal cold-induced heat production (HPmax) and cold limits between warm (WA; 27°C), moderate cold (MCA; 18°C), or cold acclimated (CA; 5°C) wild-type and uncoupling-protein 1 knockout (UCP1-KO) mice. In wild-type mice, HPmax was successively increased after MCA and CA, and the cold limit was lowered to −8.3°C and −18.0°C, respectively. UCP1-KO mice also increased HPmax in response to MCA and CA, although to a lesser extent. Direct comparison revealed a maximal cold-induced recruitment of heat production by +473 mW and +227 mW in wild-type and UCP1-KO mice, respectively. The increase in cold tolerance of UCP1-KO mice from −0.9°C in MCA to −10.1°C in CA could not be directly related to changes in HPmax, indicating that UCP1-KO mice used the dissipated heat more efficiently than wild-type mice. As judged from respiratory quotients, acutely cold-challenged UCP1-KO mice showed a delayed transition toward lipid oxidation, and 5-h cold exposure revealed diminished physical activity and less variability in the control of metabolic rate. We conclude that BAT is required for maximal adaptive thermogenesis but also allows metabolic flexibility and a rapid switch toward sustained lipid-fuelled thermogenesis as an acute response to cold. In both CA groups, expression of contractile proteins (myosin heavy-chain isoforms) showed minor training effects in skeletal muscles, while cardiac muscle of UCP1-KO mice had novel expression of beta cardiac isoform. Neither respiration nor basal proton conductance of skeletal muscle mitochondria were different between genotypes. In subcutaneous white adipose tissue of UCP1-KO mice, cold exposure increased cytochrome-c oxidase activity and expression of the cell death-inducing DFFA-like effector A by 3.6-fold and 15-fold, respectively, indicating the recruitment of mitochondria-rich brown adipocyte-like cells. Absence of functional BAT leads to remodeling of white adipose tissue, which may significantly contribute to adaptive thermogenesis during cold acclimation

Neuronal expression of glucosylceramide synthase in central nervous system regulates body weight and energy homeostasis.

Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis

POMC and NPY neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to leptin.

<p>(A) Leptin engages POMC neurons in the Arc of control (<i>Ugcg</i>^f/f) mice and <i>Ugcg</i>^{f/f//CamKCreERT2} mice 1–2 wk p.i., as indicated by elevated c-Fos. This response was decreased in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (B) Elevated leptin-induced PStat3 levels in POMC neurons of <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This response was blunted in Ugcg^{f/f//CamKCreERT2} mice 6 wk p.i. (C) Leptin slightly decreased the activity of NPY neurons in <i>Ugcg</i>^f/f mice and <i>Ugcg</i>^{f/f///CamKCreERT2} mice 1–2 wk p.i. This was not detected in <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. (D) Unlike 1–2 wk p.i., leptin did not elevate PStat3 in NPY neurons of <i>Ugcg</i>^{f/f///CamKCreERT2} mice 6 wk p.i. Datasets for each time point were acquired individually, and quantification contains normalized data from two (1–2 wk p.i.; <i>n</i> = 4–11) or three (6 wk p.i.; <i>n</i> = 18–27) independent animal groups. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 20 µm; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

rAAV-mediated <i>Ugcg</i> gene delivery to the hypothalamic Arc ameliorates obesity and hyperleptinemia in <i>Ugcg</i>^{f/f//CamKCreERT2} mice.

<p>(A) Double immunofluorescence showed that Cre activity, indicated by beta galactosidase staining (b-gal), was targeted to Arc neurons expressing the long form of the ObR, as indicated by PStat3 staining in leptin-injected <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} mice (5 mg/kg leptin, 120 min). (B) Stereotactic delivery of rAA viruses encoding <i>Ugcg</i> and <i>lacZ</i> to the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice resulted in a significant amelioration in body weight increase compared to rAAV-Empty/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 6–8). (C) Serum leptin tended to be lower in rAAV-Ugcg/lacZ-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice 8 wk p.i. (<i>n</i> = 6–8). (D–F) Targeting of rAAV Ugcg/lacZ- and rAAV Empty/lacZ-injected animals that were included in the analyses. At the end of the experiments, brains were removed and stained for X-Gal to indicate vector delivery. Red marks depict exemplarily areas of strong X-Gal staining in animals considered as Arc targeted. Depicted are areas between bregma −1.9 (D), bregma −2.1 (E), and bregma −2.3 (F). (G) Restored ganglioside biosynthesis in the Arc of rAAV-Ugcg-injected <i>Ugcg</i>^{f/f//CamKCreERT2} mice, as shown by GD1a immunofluorescence 8 wk p.i. Scale bar: 18 µm. *<i>p</i>≤0.05. Means ± SEM.</p

Normal ultrastructure in ganglioside-depleted neurons.

<p>(A) Major pathway for biosynthesis of GSL including gangliosides in the brain. (B) X-Gal staining in brains of <i>R26R/Ugcg</i>^{f/+//CamKCreERT2} reporter mice revealed strong Cre activity in the hypothalamic Arc. GD1a immunofluorescence visualized ganglioside depletion in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. Scale bar: 75 µm. (C) Ceramide levels were not significantly altered in hippocampus of <i>Ugcg</i>^{f/f//CamKCreERT2} mice. Quantification from densitometry analysis of TLC results is depicted (<i>n</i> = 3). (D) Neurons in the Arc of <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed normal ultrastructural morphology of plasma membrane (pm), nucleus (N), mitochondria (M), endoplasmic reticulum (ER), golgi (G), projections (P), and myelin sheaths (my) 6 and 12 wk p.i. Scale bar: 2 µm. 3^rdv, third ventricle.</p

<i>Ugcg</i>^{f/f//CamKCreERT2} mice develop progressive obesity.

<p>Both female (A) and male (B) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed a progressive increase in body weight after tamoxifen induction (<i>n</i> = 6–9). (C) <i>Ugcg</i>^{f/f//CamKCreERT2} mice were larger than <i>Ugcg</i>^f/f littermates (16 wk p.i.), and body fat mass was prominently elevated. (D) Enlarged adipocytes in <i>Ugcg</i>^{f/f//CamKCreERT2} mice 9 wk p.i. (E) Increased weight of epigonadal WAT 9 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 4–5). (F) NMR analysis revealed significant and progressive accumulation of body fat mass in <i>Ugcg</i>^{f/f//CamKCreERT2} mice (<i>n</i> = 9–10). *<i>p</i>≤0.05; **<i>p</i>≤0.01;***<i>p</i>≤0.001. Means ± SEM.</p

Hypothalamic neurons of <i>Ugcg</i>^{f/f//CamKCreERT2} mice are less responsive to peripheral leptin.

<p>(A–C) Brains of leptin-stimulated mice were analyzed for neuronal activity indicated by c-Fos immunofluorescence. Detailed pictures in the upper lane indicate regions of the Arc that are outlined in overview pictures (frames). Arrowheads mark c-Fos-positive neurons located in the VMH. Axis indicators were included indicating the medial (m) and ventral (v) axes. (A) <i>Ugcg</i>^{f/f//CamKCreERT2} mice showed leptin-induced neuronal activation comparable to <i>Ugcg</i>^f/f mice in the Arc 1–2 wk p.i. (B) Leptin response in the Arc was decreased in nonobese <i>Ugcg</i>^{f/f//CamKCreERT2} mice weight-matched to controls 3–4 weeks p.i. (C) Decreased c-Fos staining in the Arc was also observed in obese leptin-induced <i>Ugcg</i>^{f/f//CamKCreERT2} mice 6 wk p.i. The percentage of c-Fos-positive neurons per Arc section was depicted as values normalized to saline-injected <i>Ugcg</i>^f/f mice (<i>n</i> = 14–22 sections). Depicted sections are located between bregma levels −1.5 to −1.8. Quantification contains data from bregma levels −1.4 to −2.3. (D–F) <i>Ugcg</i>^{f/f//CamKCreERT2} mice retained leptin responsiveness in the VMH, as elevated c-Fos after leptin stimulation indicated (<i>n</i> = 8–20 sections). Quantification contains data from bregma levels −1.4 to −2.0. Datasets for each time point were acquired individually. Two (1–2 and 3–4 wk) or three (6 wk) independent animal groups were analyzed. Immunofluorescence and image acquisition for each dataset (treated and untreated controls and knockouts) were performed simultaneously. Scale bar: 75 µm; 3^rdv, 3^rd ventricle; *<i>p</i>≤0.05; **<i>p</i>≤0.01; ***<i>p</i>≤0.001. Means ± SEM.</p

GCS in hypothalamic neurons regulates neuronal leptin signaling at the plasma membrane.

<p>(A) Stat3 phosphorylation was markedly decreased in Arc sections of <i>Ugcg</i>^{f/f//CamKCreERT2} mice in response to peripheral leptin (5 mg/kg; 45 min) 6 wk p.i. (<i>n</i> = 16–33). Three independent animal groups were analyzed. (B) Serum leptin levels were unchanged 3 wk p.i. and increased prominently 7 wk p.i. in <i>Ugcg</i>^{f/f//CamKCreERT2} mice, reflecting increased body fat mass (<i>n</i> = 12–14). (C) mRNA expression analysis for suppressor of cytokine signaling 3 (SOCS-3) expression in Arc-enriched hypothalamic tissue was carried out 2, 6, and 9 wk p.i. <i>Socs-3</i> expression normalized to the housekeeping gene tubulin was unaltered (<i>n</i> = 3–5). (D) mRNA expression analysis for the long form of the leptin receptor, <i>Leprb</i>, in mediobasal hypothalamus was carried out 6 wk p.i. <i>Leprb</i> expression normalized to the housekeeping gene tubulin was unaltered at that time point (<i>n</i> = 4–5). (E) Immortalized mouse hypothalamic cells (N-41 cells) were analyzed for cell surface expression of ObR. Non-detergent-treated cells were fixed and simultaneously stained with two ObR antibodies. A proximity ligation assay (PLA) indicated quantifiable and unchanged ObR expression on the surface of controls and cells treated with the specific GCS inhibitor NB-DNJ (<i>n</i> = 41–47 cells). PLA principle is depicted on the right side. (F–G) N-41 cells were incubated with either saline or 100 ng/ml leptin (10 min). Close interactions between GCS-derived neuronal gangliosides GD1a/ObR (F) and GM1/ObR (G) were detected by PLA. Leptin treatment dynamically increased the GD1a/ObR and GM1/ObR PLA spots per cell (<i>n</i> = 48–67 cells). (H) Extracts from saline- and leptin-treated N-41 cells were immunoprecipitated with an ObR antibody, lipids were extracted, and GD1a and GM1 were visualized by immune overlay TLC. GD1a and GM1 co-immunoprecipitated (Co-IP) with ObR, which tended to be stronger in leptin-treated cells. Addition of a blocking peptide almost totally abolished ganglioside signals. Gangliosides GD1b and GT1b, expressed in mouse brain tissue, were not co-precipitated with ObR from hypothalamic tissue of <i>Ugcg</i>^f/f mice (5 mg/kg leptin, 45 min). (I) Co-IP showed significantly decreased leptin-induced complex formation between ObR and Jak in NB-DNJ-treated N-41 cells (<i>n</i> = 4). (J) Sustainable Jak phosphorylation could be induced in N-41 cells after 15 min of leptin treatment (0.5 µg/ml). NB-DNJ-treated cells showed a markedly delayed response to leptin. (K) Thirty minutes after leptin treatment, Jak phosphorylation was decreased in NB-DNJ-treated cells (<i>n</i> = 4). *<i>p</i>≤0.05; ***<i>p</i>≤0.001. Means ± SEM.</p