The corticotropin-releasing hormone (CRH) and its type 1 receptor (CRHR1) play a central role in coordinating the endocrine, autonomic, and behavioral responses to stress. A prerequisite to functionally dissect the complexity of the CRH/CRHR1 system is to unravel the identity of CRHR1-expressing neurons and their connectivities. Therefore, we used a knockin approach to genetically label CRHR1-expressing cells with a tau-lacZ (tZ) reporter gene. The distribution of neurons expressing β-galactosidase in the brain and the relative intensity of labeling is in full accordance with previously described Crhr1 mRNA expression. Combining the microtubule-binding properties of TAU with the Cre-loxP system allowed to direct the β-galactosidase to proximal dendrites, and in particular to axons. Thereby, we were able to visualize projections of CRHR1 neurons such as glutamatergic and dopaminergic afferent connections of the striatum and GABAergic CRHR1-expressing neurons located within its patch compartment. In addition, the tZ reporter gene revealed novel details of CRHR1 expression in the spinal cord, skin, and eye. CRHR1 expression in the retina prompted the identification of a new physiological role of CRHR1 related to the visual system. Besides its reporter properties, this novel CRHR1 allele comprises the possibility to conditionally restore or delete CRHR1 via Flp and Cre recombinase, respectively. Finally, the allele is suitable for further manipulations of the CRHR1 locus by recombinase-mediated cassette exchange. Taken together, this novel mouse allele will significantly facilitate the neuroanatomical analysis of CRHR1 circuits and opens up new avenues to address CRHR1 function in more detail.

Accumulation of visceral fat is associated with metabolic risk whereas excessive amounts of peripheral fat are considered less problematic. At the same time, altered white adipocyte mitochondrial bioenergetics has been implicated in the pathogenesis of insulin resistance and type 2 diabetes. We therefore investigated whether the metabolic risk of visceral vs peripheral fat coincides with a difference in mitochondrial capacity of white adipocytes. We assessed bioenergetic parameters of subcutaneous inguinal and visceral epididymal white adipocytes from male C57BL/6N mice employing a comprehensive respirometry setup of intact and permeabilized adipocytes as well as isolated mitochondria. Inguinal adipocytes clearly featured a higher respiratory capacity attributable to increased mitochondrial respiratory chain content compared with epididymal adipocytes. The lower capacity of mitochondria from epididymal adipocytes was accompanied by an increased generation of reactive oxygen species per oxygen consumed. Feeding a high-fat diet (HFD) for 1 week reduced white adipocyte mitochondrial capacity, with stronger effects in epididymal when compared with inguinal adipocytes. This was accompanied by impaired body glucose homeostasis. Therefore, the limited bioenergetic performance combined with the proportionally higher generation of reactive oxygen species of visceral adipocytes could be seen as a candidate mechanism mediating the elevated metabolic risk associated with this fat depot.

Brown fat is a potential target in the treatment of metabolic disorders as recruitment and activation of this thermogenic organ increases energy expenditure and promotes satiation. A large variety of G-protein coupled receptors, known as classical drug targets in pharmacotherapy, is expressed in brown adipocytes. In the present study, we analyzed transcriptome data for the expression of these receptors to identify potential pathways for the recruitment and activation of thermogenic capacity in brown fat. Our analysis revealed 12 Gs-coupled receptors abundantly expressed in murine brown fat. We screened ligands for these receptors in brown adipocytes for their ability to stimulate UCP1-mediated respiration and Ucp1 gene expression. Adrenocorticotropic hormone (ACTH), a ligand for the melanocortin 2 receptor (MC2R), turned out to be the most potent activator of UCP1 whereas its capability to stimulate Ucp1 gene expression was comparably low. Adrenocorticotropic hormone is the glandotropic hormone of the endocrine hypothalamus-pituitary-adrenal-axis stimulating the release of glucocorticoids in response to stress. In primary brown adipocytes ACTH acutely increased the cellular respiration rate similar to isoproterenol, a β-adrenergic receptor agonist. The effect of ACTH on brown adipocyte respiration was mediated via the MC2R as confirmed by using an antagonist. Inhibitor-based studies revealed that ACTH-induced respiration was dependent on protein kinase A and lipolysis, compatible with a rise of intracellular cAMP in response to ACTH. Furthermore, it is dependent on UCP1, as cells from UCP1-knockout mice did not respond. Taken together, ACTH is a non-adrenergic activator of murine brown adipocytes, initiating the canonical adenylyl cyclase-cAMP-protein kinase A-lipolysis-UCP1 pathway, and thus a potential target for the recruitment and activation of thermogenic capacity. Based on these findings in primary cell culture, the physiological significance might be that cold-induced ACTH in concert with norepinephrine released from sympathetic nerves contributes to BAT thermogenesis. Notably, dexamethasone attenuated isoproterenol-induced respiration. This effect increased gradually with the duration of pretreatment. In vivo, glucocorticoid release triggered by ACTH might oppose beta-adrenergic stimulation of metabolic fuel combustion in BAT and limit stress-induced hyperthermia.

Human studies suggest that a meal elevates glucose uptake in brown adipose tissue (BAT). However, in postprandial state the thermogenic activity and the metabolism of non-esterified fatty acids (NEFAs) in BAT remain unclear. Using indirect calorimetry combined with positron emission tomography and computed tomography (PET/CT), we showed that whole-body and BAT thermogenesis (oxygen consumption) increases after the ingestion of a mixed carbohydrate-rich meal, to the same extent as in cold stress. Postprandial NEFA uptake into BAT is minimal, possibly due to elevated plasma insulin inhibiting lipolysis. However, the variation in postprandial NEFA uptake is linked to BAT thermogenesis. We identified several genes participating in lipid metabolism to be expressed at higher levels in BAT compared with white fat in postprandial state, and to be positively correlated with BAT UCP1 expression. These findings suggest that substrates preferred by BAT in postprandial state are glucose or LPL-released NEFAs due to insulin stimulation.

βB2-crystallin (gene symbol: Crybb2/CRYBB2) was first described as a structural protein of the ocular lens. This gene, however, is also expressed in several regions of the mammalian brain, although its function in this organ remains entirely unknown. To unravel some aspects of its function in the brain, we combined behavioral, neuroanatomical, and physiological analyses in a novel Crybb2 mouse mutant, O377. Behavioral tests with male O377 mutants revealed altered sensorimotor gating, suggesting modified neuronal functions. Since these mouse mutants also displayed reduced hippocampal size, we concentrated further investigations on the hippocampus. Free intracellular Ca(2+) levels were increased and apoptosis was enhanced in the hippocampus of O377 mutants. Moreover, the expression of the gene encoding calpain 3 (gene symbol Capn3) was elevated and the expression of genes coding for the NMDA receptor subunits was downregulated. Additionally, the number of parvalbumin-positive interneurons was decreased in the hippocampus but not in the cortex of the mutants. High-speed voltage-sensitive dye imaging demonstrated an increased translation of input-to-output neuronal activity in the dentate gyrus of this Crybb2 mutant. These results point to an important function of βB2-crystallin in the hippocampal network. They indicate pleiotropic effects of mutations in the Crybb2 gene, which previously had been considered to be specific to the ocular lens. Moreover, our results are the first to demonstrate that βB2-crystallin has a role in hippocampal function and behavioral phenotypes. This model can now be further explored by future experiments.

Metformin, the first line drug for treatment of type 2 diabetes, suppresses hepatic gluconeogenesis and reduces body weight in patients, the latter by an unknown mechanism.

The SH2B1 gene (Src-homology 2B adaptor protein 1 gene) is a solid candidate gene for obesity. Large scale GWAS studies depicted markers in the vicinity of the gene; animal models suggest a potential relevance for human body weight regulation.

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.

Within a mutagenesis screen, we identified the new mouse mutant Aey80 with small eyes; homozygous mutants were not obtained. The aim of the study was its molecular characterization.

Neurobeachin (Nbea) regulates neuronal membrane protein trafficking and is required for the development and functioning of central and neuromuscular synapses. In homozygous knockout (KO) mice, Nbea deficiency causes perinatal death. Here, we report that heterozygous KO mice haploinsufficient for Nbea have higher body weight due to increased adipose tissue mass. In several feeding paradigms, heterozygous KO mice consumed more food than wild-type (WT) controls, and this consumption was primarily driven by calories rather than palatability. Expression analysis of feeding-related genes in the hypothalamus and brainstem with real-time PCR showed differential expression of a subset of neuropeptide or neuropeptide receptor mRNAs between WT and Nbea+/- mice in the sated state and in response to food deprivation, but not to feeding reward. In humans, we identified two intronic NBEA single-nucleotide polymorphisms (SNPs) that are significantly associated with body-mass index (BMI) in adult and juvenile cohorts. Overall, data obtained in mice and humans suggest that variation of Nbea abundance or activity critically affects body weight, presumably by influencing the activity of feeding-related neural circuits. Our study emphasizes the importance of neural mechanisms in body weight control and points out NBEA as a potential risk gene in human obesity.

Mitochondria are functionally specialized in different tissues, and a detailed understanding of this specialization is important to elucidate mitochondrial involvement in normal physiology and disease. In adaptive thermogenesis, brown fat converts mitochondrial energy to heat, whereas tissue-specific functions of mitochondria in white fat are less characterized. Here we apply high-resolution quantitative mass spectrometry to directly and accurately compare the in vivo mouse mitochondrial proteomes of brown and white adipocytes. Their proteomes are substantially different qualitatively and quantitatively and are furthermore characterized by tissue-specific protein isoforms, which are modulated by cold exposure. At transcript and proteome levels, brown fat mitochondria are more similar to their counterparts in muscle. Conversely, white fat mitochondria not only selectively express proteins that support anabolic functions but also degrade xenobiotics, revealing a protective function of this tissue. In vivo comparison of organellar proteomes can thus directly address functional questions in metabolism.

Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.

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.

Several human diseases are associated with a lack of caveolae. Yet, the functions of caveolae and the molecular mechanisms critical for shaping them still are debated. We show that muscle cells of syndapin III KO mice show severe reductions of caveolae reminiscent of human caveolinopathies. Yet, different from other mouse models, the levels of the plasma membrane-associated caveolar coat proteins caveolin3 and cavin1 were both not reduced upon syndapin III KO. This allowed for dissecting bona fide caveolar functions from those supported by mere caveolin presence and also demonstrated that neither caveolin3 nor caveolin3 and cavin1 are sufficient to form caveolae. The membrane-shaping protein syndapin III is crucial for caveolar invagination and KO rendered the cells sensitive to membrane tensions. Consistent with this physiological role of caveolae in counterpoising membrane tensions, syndapin III KO skeletal muscles showed pathological parameters upon physical exercise that are also found in CAVEOLIN3 mutation-associated muscle diseases.

The vertebrate Scube (Signal peptide, CUB, and EGF-like domain-containing protein) family consists of three independent members, Scube1-3, which encode secreted cell surface-associated membrane glycoproteins. Limited information about the general function of this gene family is available, and their roles during adulthood. Here, we present the first Scube3 mutant mouse line (Scube3N294K/N294K), which clearly shows phenotypic alterations by carrying a missense mutation in exon 8, and thus contributes to our understanding of SCUBE3 functions. We performed a detailed phenotypic characterization in the German Mouse Clinic (GMC). Scube3N294K/N294K mutants showed morphological abnormalities of the skeleton, alterations of parameters relevant for bone metabolism, changes in renal function, and hearing impairments. These findings correlate with characteristics of the rare metabolic bone disorder Paget disease of bone (PDB), associated with the chromosomal region of human SCUBE3 In addition, alterations in energy metabolism, behavior, and neurological functions were detected in Scube3N294K/N294K mice. The Scube3N294K/N294K mutant mouse line may serve as a new model for further studying the effect of impaired SCUBE3 gene function.

Introduction: Nutritional habits and requirements are changing over the lifespan, but the dynamics of nutritional issues and the diet-health relationship in the major stages of the human life cycle are not sufficiently understood. A human phenotyping research platform for nutrition studies was established to recruit and phenotype selected population groups across different stages of life. The project is the backbone of the highly interdisciplinary enable competence cluster of nutrition research aiming to identify dietary determinants of a healthy life throughout the lifespan and to develop healthier and tasty convenience foods with high consumer acceptance. Methods: The phenotyping program included anthropometry, body composition analysis, assessment of energy metabolism, health and functional status, multisensory perception, metabolic phenotyping, lifestyle, sociodemography, chronobiology, and assessment of dietary intake including food preferences and aversions. Results: In total, 503 healthy volunteers at four defined phases of life including 3-5-year old children (n = 44), young adults aged 18-25 years (n = 94), adults aged 40-65 years ("middle agers," n = 205), and older adults aged 75-85 years (n = 160) were recruited and comprehensively phenotyped. Plasma, serum, buffy coat, urine, feces and saliva samples were collected and stored at -80°C. Significant differences in anthropometric and metabolic parameters between the four groups were found. A major finding was the decrease in fat-free mass and the concomitant increase in % body fat in both sexes across the adult lifespan. Conclusions: The dataset will provide novel information on differences in diet-related parameters over the lifespan and is available for targeted analyses. We expect that this novel platform approach will have implications for the development of innovative food products tailored to promote healthy eating throughout life. Trial registration: DRKS, DRKS00009797. Registered on 20 January 2016, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&_ID=DRKS00009797.

Current concepts regarding the biology of aging are primarily based on studies aimed at identifying factors regulating lifespan. However, lifespan as a sole proxy measure for aging can be of limited value because it may be restricted by specific pathologies. Here, we employ large-scale phenotyping to analyze hundreds of markers in aging male C57BL/6J mice. For each phenotype, we establish lifetime profiles to determine when age-dependent change is first detectable relative to the young adult baseline. We examine key lifespan regulators (putative anti-aging interventions; PAAIs) for a possible countering of aging. Importantly, unlike most previous studies, we include in our study design young treated groups of animals, subjected to PAAIs prior to the onset of detectable age-dependent phenotypic change. Many PAAI effects influence phenotypes long before the onset of detectable age-dependent change, but, importantly, do not alter the rate of phenotypic change. Hence, these PAAIs have limited effects on aging.

Glioblastoma (GBM) is the most common malignant intracranial tumor with a low survival rate. However, only few drugs responsible for GBM therpies, hence new drug development for it is highly required. The natural product Cudraflavone B (CUB) has been reported to potentially kill a variety of tumor cells. Currently, its anit-cancer effect on GBM still remains unknown. Herein, we investigated whether CUB could affect the proliferation and apoptosis of GBM cells to show anti-GBM potential.

Metabolites of omega-6 and omega-3 polyunsaturated fatty acids are important signaling molecules implicated in the control of adipogenesis and energy balance regulation. Some of these metabolites belonging to the group of oxylipins have been associated with non-shivering thermogenesis in mice mediated by brown or brite adipose tissue. We aimed to identify novel molecules with thermogenic potential and to clarify the relevance of these findings in a translational context. Therefore, we characterized and compared the oxylipin profiles of murine and human adipose tissues with different abundance of brown or brite adipocytes. A broad panel of 36 fatty acid metabolites was quantified in brown and white adipose tissues of C57BL/6J mice acclimatized to different ambient temperatures and in biopsies of human supraclavicular brown and white adipose tissue. The oxylipin profile of murine brite adipose tissue was not distinguishable from white adipose tissue, suggesting that adipose tissue browning in vivo is not associated with major changes in the oxylipin metabolism. Human brown and white adipose tissue also exhibited similar metabolite profiles. This is in line with previous studies proposing human brown adipose tissue to resemble the nature of murine brite adipose tissue representing a heterogeneous mixture of brite and white adipocytes. Although the global oxylipin profile served as a marker for the abundance of thermogenic adipocytes in bona fide brown but not white adipose tissue, we identified 5-HETE and 5,6-EET as individual compounds consistently associated with the abundance of brown or brite adipocytes in human BAT and murine brite fat. Further studies need to establish whether these candidates are mere markers or functional effectors of thermogenic capacity.

Classical ATP-independent non-shivering thermogenesis enabled by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) is activated, but not essential for survival, in the cold. It has long been suspected that futile ATP-consuming substrate cycles also contribute to thermogenesis and can partially compensate for the genetic ablation of UCP1 in mouse models. Futile ATP-dependent thermogenesis could thereby enable survival in the cold even when brown fat is less abundant or missing.