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With age, metabolic perturbations accumulate to elevate our obesity burden. While age-onset obesity is mostly driven by a sedentary lifestyle and high calorie intake, genetic and epigenetic factors also play a role. Among these, members of the large histone deacetylase (HDAC) family are of particular importance as key metabolic determinants for healthy ageing, or metabolic dysfunction. Here, we aimed to interrogate the role of class 2 family member HDAC5 in controlling systemic metabolism and age-related obesity under non-obesogenic conditions. Starting at 6 months of age, we observed adult-onset obesity in chow-fed male global HDAC5-KO mice, that was accompanied by marked reductions in adrenergic-stimulated ATP-consuming futile cycles, including BAT activity and UCP1 levels, WAT-lipolysis, skeletal muscle, WAT and liver futile creatine and calcium cycles, and ultimately energy expenditure. Female mice did not differ between genotypes. The lower peripheral sympathetic nervous system (SNS) activity in mature male KO mice was linked to higher dopaminergic neuronal activity within the dorsomedial arcuate nucleus (dmARC) and elevated hypothalamic dopamine levels. Mechanistically, we reveal that hypothalamic HDAC5 acts as co-repressor of STAT5b over the control of Tyrosine hydroxylase (TH) gene transactivation, which ultimately orchestrates the activity of dmARH dopaminergic neurons and energy metabolism in male mice under non-obesogenic conditions., Competing Interests: Declaration of competing interest The author is an Editorial Board Member/Editor-in-Chief/Associate Editor/Guest Editor for Molecular Metabolism and was not involved in the editorial review or the decision to publish this article. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: PTP received speaker honoraria by Novo Nordisk. As a scientist, MHT participated in a scientific advisory board meeting of ERX Pharmaceuticals, Inc., Cambridge, MA, in 2019. He was a member of the Research Cluster Advisory Panel (ReCAP) of the Novo Nordisk Foundation between 2017 and 2019. He received funding for his research projects by Novo Nordisk (2016–2020) and Sanofi-Aventis (2012–2019). He consulted twice for Böhringer Ingelheim Pharma GmbH & Co. KG (2020 & 2021) and delivered a scientific lecture for Sanofi-Aventis Deutschland GmbH (2020) and for AstraZeneca GmbH (2024). As CEO and CSO of Helmholtz Munich, he is co-responsible for countless collaborations of the employees with a multitude of companies and institutions, worldwide. In this capacity, he discusses potential projects with and has signed/signs contracts for the centers institute(s) related to research collaborations worldwide, including but not limited to pharmaceutical corporations like Boehringer Ingelheim, Novo Nordisk, Roche Diagnostics, Arbormed, Eli Lilly, SCG Cell Therapy and others. As the CEO of Helmholtz Munich, he was/is further overall responsible for commercial technology transfer activities. MHT confirms that to the best of his knowledge none of the above funding sources or collaborations were involved in or had an influence on the preparation of this manuscript. All other authors declare that they have no conflict of interest related to this study., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons regulate fertility and integrate hormonal status with environmental cues to ensure reproductive success. Here we show that GnRH neurons in the olfactory bulb (GnRH OB ) of adult mice can mediate social recognition. Specifically, we show that GnRH OB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRH OB neurons in males express vomeronasal and olfactory receptors, are activated by female odors and mediate gonadotropin release in response to female urine. Male preference for female odors required the presence and activation of GnRH OB neurons, was impaired after genetic inhibition or ablation of these cells and relied on GnRH signaling in the posterodorsal medial amygdala. GnRH receptor expression in amygdala kisspeptin neurons appear to be required for GnRH OB neurons' actions on male mounting behavior. Taken together, these results establish GnRH OB neurons as regulating fertility, sex recognition and mating in male mice., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Background: The development of obesity-associated comorbidities such as type 2 diabetes (T2D) and hepatic steatosis has been linked to selected microRNAs in individual studies; however, an unbiased genome-wide approach to map T2D induced changes in the miRNAs landscape in human liver samples, and a subsequent robust identification and validation of target genes are still missing., Methods: Liver biopsies from age- and gender-matched obese individuals with (n=20) or without (n=20) T2D were used for microRNA microarray analysis. The candidate microRNA and target genes were validated in 85 human liver samples, and subsequently mechanistically characterized in hepatic cells as well as by dietary interventions and hepatic overexpression in mice., Results: Here, we present the human hepatic microRNA transcriptome of type 2 diabetes in liver biopsies and use a novel seed prediction tool to robustly identify microRNA target genes, which were then validated in a unique cohort of 85 human livers. Subsequent mouse studies identified a distinct signature of T2D-associated miRNAs, partly conserved in both species. Of those, human-murine miR-182-5 p was the most associated with whole-body glucose homeostasis and hepatic lipid metabolism. Its target gene LRP6 was consistently lower expressed in livers of obese T2D humans and mice as well as under conditions of miR-182-5 p overexpression. Weight loss in obese mice decreased hepatic miR-182-5 p and restored Lrp6 expression and other miR-182-5 p target genes. Hepatic overexpression of miR-182-5 p in mice rapidly decreased LRP6 protein levels and increased liver triglycerides and fasting insulin under obesogenic conditions after only seven days., Conclusions: By mapping the hepatic miRNA-transcriptome of type 2 diabetic obese subjects, validating conserved miRNAs in diet-induced mice, and establishing a novel miRNA prediction tool, we provide a robust and unique resource that will pave the way for future studies in the field. As proof of concept, we revealed that the repression of LRP6 by miR-182-5 p, which promotes lipogenesis and impairs glucose homeostasis, provides a novel mechanistic link between T2D and non-alcoholic fatty liver disease, and demonstrate in vivo that miR-182-5 p can serve as a future drug target for the treatment of obesity-driven hepatic steatosis., Funding: This work was supported by research funding from the Deutsche Forschungsgemeinschaft (KI 1887/2-1, KI 1887/2-2, KI 1887/3-1 and CRC-TR296), the European Research Council (ERC, CoG Yoyo LepReSens no. 101002247; PTP), the Helmholtz Association (Initiative and Networking Fund International Helmholtz Research School for Diabetes; MB) and the German Center for Diabetes Research (DZD Next Grant 82DZD09D1G)., Competing Interests: CK, JB, MB, AM, NT, NL, CG, MK, KI, AJ, JW, SW, OM, PP, IC, HL, KS, SS, HK No competing interests declared, (© 2023, Krause et al.)

Objective: The glucose-dependent insulinotropic polypeptide (GIP) decreases body weight via central GIP receptor (GIPR) signaling, but the underlying mechanisms remain largely unknown. Here, we assessed whether GIP regulates body weight and glucose control via GIPR signaling in cells that express the leptin receptor (Lepr)., Methods: Hypothalamic, hindbrain, and pancreatic co-expression of Gipr and Lepr was assessed using single cell RNAseq analysis. Mice with deletion of Gipr in Lepr cells were generated and metabolically characterized for alterations in diet-induced obesity (DIO), glucose control and leptin sensitivity. Long-acting single- and dual-agonists at GIPR and GLP-1R were further used to assess drug effects on energy and glucose metabolism in DIO wildtype (WT) and Lepr-Gipr knock-out (KO) mice., Results: Gipr and Lepr show strong co-expression in the pancreas, but not in the hypothalamus and hindbrain. DIO Lepr-Gipr KO mice are indistinguishable from WT controls related to body weight, food intake and diet-induced leptin resistance. Acyl-GIP and the GIPR:GLP-1R co-agonist MAR709 remain fully efficacious to decrease body weight and food intake in DIO Lepr-Gipr KO mice. Consistent with the demonstration that Gipr and Lepr highly co-localize in the endocrine pancreas, including the β-cells, we find the superior glycemic effect of GIPR:GLP-1R co-agonism over single GLP-1R agonism to vanish in Lepr-Gipr KO mice., Conclusions: GIPR signaling in cells/neurons that express the leptin receptor is not implicated in the control of body weight or food intake, but is of crucial importance for the superior glycemic effects of GIPR:GLP-1R co-agonism relative to single GLP-1R agonism., Competing Interests: Declaration of competing interest MHT is a member of the scientific advisory board of ERX Pharmaceuticals, Cambridge, Mass. He was a member of the Research Cluster Advisory Panel (ReCAP) of the Novo Nordisk Foundation between 2017 and 2019. He attended a scientific advisory board meeting of the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, in 2016. He received funding for his research projects by Novo Nordisk (2016–2020) and Sanofi-Aventis (2012–2019). He was a consultant for Bionorica SE (2013–2017), Menarini Ricerche S.p.A. (2016), and Bayer Pharma AG Berlin (2016). As former Director of the Helmholtz Diabetes Center and the Institute for Diabetes and Obesity at Helmholtz Zentrum München (2011–2018), and since 2018, as CEO of Helmholtz Zentrum München, he has been responsible for collaborations with a multitude of companies and institutions, worldwide. In this capacity, he discussed potential projects with and has signed/signs contracts for his institute(s) and for the staff for research funding and/or collaborations with industry and academia, worldwide, including but not limited to pharmaceutical corporations like Boehringer Ingelheim, Eli Lilly, Novo Nordisk, Medigene, Arbormed, BioSyngen, and others. In this role, he was/is further responsible for commercial technology transfer activities of his institute(s), including diabetes related patent portfolios of Helmholtz Zentrum München as, e.g., WO/2016/188932 A2 or WO/2017/194499 A1. MHT confirms that to the best of his knowledge none of the above funding sources were involved in the preparation of this paper. TDM and K.S. receive research funding by Novo Nordisk but these funds are unrelated the here described work. DJD has received speaking and consulting fees from Merck and Novo Nordisk Inc and consulting fees from Forkhead Biopharmaceuticals and Kallyope Inc. R.D.D is a co-inventor on intellectual property owned by Indiana University and licensed to Novo Nordisk. He was previously employed by Novo Nordisk. P.J.K, S.M., and B.F. are current employees of Novo Nordisk. TDM receives funding from Novo Nordisk and received speaking fees within the last 3 years from Novo Nordisk, Eli Lilly, AstraZeneca, Merck, Berlin Chemie AG, and Mercodia., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Prader-Willi Syndrome (PWS) is a rare neurodevelopmental disorder of genetic etiology, characterized by paternal deletion of genes located at chromosome 15 in 70% of cases. Two distinct genetic subtypes of PWS deletions are characterized, where type I (PWS T1) carries four extra haploinsufficient genes compared to type II (PWS T2). PWS T1 individuals display more pronounced physiological and cognitive abnormalities than PWS T2, yet the exact neuropathological mechanisms behind these differences remain unclear. Our study employed postmortem hypothalamic tissues from PWS T1 and T2 individuals, conducting transcriptomic analyses and cell-specific protein profiling in white matter, neurons, and glial cells to unravel the cellular and molecular basis of phenotypic severity in PWS sub-genotypes. In PWS T1, key pathways for cell structure, integrity, and neuronal communication are notably diminished, while glymphatic system activity is heightened compared to PWS T2. The microglial defect in PWS T1 appears to stem from gene haploinsufficiency, as global and myeloid-specific Cyfip1 haploinsufficiency in murine models demonstrated. Our findings emphasize microglial phagolysosome dysfunction and altered neural communication as crucial contributors to the severity of PWS T1's phenotype., (© 2024. The Author(s).)

Until menopause, women have a lower propensity to develop metabolic diseases than men, suggestive of a protective role for sex hormones. Although a functional synergy between central actions of estrogens and leptin has been demonstrated to protect against metabolic disturbances, the underlying cellular and molecular mechanisms mediating this crosstalk have remained elusive. By using a series of embryonic, adult-onset, and tissue/cell-specific loss-of-function mouse models, we document an unprecedented role of hypothalamic Cbp/P300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 1 (Cited1) in mediating estradiol (E2)-dependent leptin actions that control feeding specifically in pro-opiomelanocortin (Pomc) neurons. We reveal that within arcuate Pomc neurons, Cited1 drives leptin's anorectic effects by acting as a co-factor converging E2 and leptin signaling via direct Cited1-ERα-Stat3 interactions. Together, these results provide new insights on how melanocortin neurons integrate endocrine inputs from gonadal and adipose axes via Cited1, thereby contributing to the sexual dimorphism in diet-induced obesity., Competing Interests: Declaration of interests M.H.T. is a member of the scientific advisory board of ERX Pharmaceuticals, Inc., Cambridge, MA. He was a member of the Research Cluster Advisory Panel (ReCAP) of the Novo Nordisk Foundation between 2017 and 2019. He attended a scientific advisory board meeting of the Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, in 2016. He received funding for his research projects by Novo Nordisk (2016–2020) and Sanofi-Aventis (2012–2019). He consulted for Böhringer Ingelheim Pharma GmbH & Co. KG (2020). He delivered a scientific lecture for Sanofi-Aventis Deutschland GmbH in 2020. He is an editorial board member of Cell Metabolism. He confirms that to the best of his knowledge none of the above were involved in the preparation of this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The Allan-Herndon Dudley Syndrome (AHDS) is a rare disease caused by the progressive loss of monocarboxylate transporter 8 (MCT8). In patients with AHDS, the absence of MCT8 impairs transport of thyroid hormones (TH) through the blood brain barrier, leading to a central state of TH deficiency. In mice, the AHDS is mimicked by simultaneous deletion of the TH transporters MCT8 and the solute carrier organic anion transporter family member 1c1 (OATP1C1). To support preclinical mouse studies, an analytical methodology was developed and successfully applied for quantifying selected thyroid hormones in mouse whole brain and in specific regions using liquid chromatography tandem mass-spectrometry (LC-MS/MS). An important requirement for the methodology was its high sensitivity since a very low concentration of THs was expected in MCT8/OATP1C1 double-knockout (dko) mouse brain. Seven THs were targeted: L-thyroxine (T4), 3,3´,5-triiodo-L-thyronine-thyronine (T3), 3,3´,5´-triiodo-L-thyronine-thyronine (rT3), 3,3´-diiodo-L-thyronine (3,3´-T2, T2), 3,5-diiodo-L-thyronine (rT2, 3,5-T2), 3-iodo-L-thyronine (T1), 3-iodothyronamine (T1AM). Isotope dilution liquid chromatography triple-quadrupole mass spectrometry methodology was applied for detection and quantification. The method was validated in wild-type animals for mouse whole brain and for five different brain regions (hypothalamus, hippocampus, prefrontal cortex, brainstem and cortex). Instrumental calibration curves ranged from 0.35 to 150 pg/µL with good linearity (r 2 >0.996). The limit of quantification was from 0.08 to 0.6 pg/mg, with an intra- and inter-day precision of 4.2-14.02% and 0.4-17.9% respectively, and accuracies between 84.9% and 114.8% when the methodology was validated for the whole brain. In smaller, distinct brain regions, intra- and inter-day precision were 0.6-20.7% and 2.5-15.6% respectively, and accuracies were 80.2-128.6%. The new methodology was highly sensitive and allowed for the following quantification in wild-type mice: (i) for the first time, four distinct thyroid hormones (T4, T3, rT3 and 3,3´-T2) in only approximately 100 mg of mouse brain were detected; (ii) the quantification of T4 and T3 for the first time in distinct mouse brain regions were reported. Further, application of our method to MCT8/OATP1C1 dko mice revealed the expected, relative lack of T3 and T4 uptake into the brain, and confirmed the utility of our analytical method to study TH transport across the blood brain barrier in a preclinical model of central TH deficiency., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Morphine can be synthesized endogenously by mammals from dopamine via the intermediate norlaudanosoline. Previously, both compounds have been detected separately in whole brains of mice and brain regions of rats, and in urine of humans. Here, we report a novel method for the analysis of both compounds in single murine brain regions. Initially, a variant of dispersive liquid-liquid microextraction was established by using methanol as an extractant, cyclohexane as solvent, and tributylphosphate as disperser. The extraction method was applied to murine brain regions homogenized with perchloric acid while the subsequent detection was carried out by HPLC with electrochemical detection. In the thalamus of C57Bl/6J mice (n = 3, male, age 4-8 months), morphine and norlaudanosoline could be detected at levels of 19 ± 3.9 and 7.2 ± 2.3 pg/mg, respectively. Overall, we provide a novel method for the simultaneous extraction and detection of both morphine and norlaudanosoline in single murine brain regions., (Copyright © 2021. Published by Elsevier Ltd.)

Objective: Technological advances have brought a steady increase in the availability of various types of omics data, from genomics to metabolomics. Integrating these multi-omics data is a chance and challenge for systems biology; yet, tools to fully tap their potential remain scarce., Methods: We present here a fully unsupervised and versatile correlation-based method - termed Correlation guided Network Integration (CoNI) - to integrate multi-omics data into a hypergraph structure that allows for the identification of effective modulators of metabolism. Our approach yields single transcripts of potential relevance that map to specific, densely connected, metabolic subgraphs or pathways., Results: By applying our method on transcriptomics and metabolomics data from murine livers under standard Chow or high-fat diet, we identified eleven genes with potential regulatory effects on hepatic metabolism. Five candidates, including the hepatokine INHBE, were validated in human liver biopsies to correlate with diabetes-related traits such as overweight, hepatic fat content, and insulin resistance (HOMA-IR)., Conclusion: Our method's successful application to an independent omics dataset confirmed that the novel CoNI framework is a transferable, entirely data-driven, flexible, and versatile tool for multiple omics data integration and interpretation., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

3-iodothyronamine (3-T 1 AM) has been suggested as a novel chemical messenger and potent trace amine-associated receptor 1 ligand in the CNS that occurs naturally as endogenous metabolite of the thyroid hormones. Discrepancies and variations in 3-T 1 AM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-T 1 AM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-T 1 AM and the widely used deuterated quantification standard D 4 -T 1 AM. Here, we report a novel LC-MS/MS method for the quantification of 3-T 1 AM in biological specimens using stable isotope dilution with 13 C 6 -T 1 AM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-T 1 AM. The method detection limit (MDL) and method quantification limit (MQL) of 3-T 1 AM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-T 1 AM were between 85.4% and 94.3%, with a coefficient of variation of 3.7-5.8%. The intra-day and inter-day variations of 3-T 1 AM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-T 1 AM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-T 1 AM values were found when C57BL/6J mice were treated with N-acetyl-3-iodothyronamine or O-acetyl-3-iodothyronamine. Overall, our new stable isotope dilution LC-MS/MS method improves both the sensitivity and selectivity compared with existing methods. The concomitant possibility to quantify additional thyroid hormones such as thyroxine, 3,5,3'-triiodo- L -thyronine, 3,3',5'-triiodo- L -thyronine, 3,3'-diiodo- L -thyronine, and 3,5-diiodo- L -thyronine further adds to the value of our novel method in exploring the natural occurrence and fate of 3-T 1 AM in biological tissues and fluids., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Dusp8 is the first GWAS-identified gene that is predominantly expressed in the brain and has previously been linked with the development of diabetes type 2 in humans. In this study, we unravel how Dusp8 is involved in the regulation of sucrose reward behavior., Methods: Female, chow-fed global Dusp8 WT and KO mice were tested in an observer-independent IntelliCage setup for self-administrative sucrose consumption and preference followed by a progressive ratio task with restricted sucrose access to monitor seeking and motivation behavior. Sixty-three human carriers of the major C and minor T allele of DUSP8 SNP rs2334499 were tested for their perception of food cues by collecting a rating score for sweet versus savory high caloric food., Results: Dusp8 KO mice showed a comparable preference for sucrose, but consumed more sucrose compared to WT mice. In a progressive ratio task, Dusp8 KO females switched to a "trial and error" strategy to find sucrose while control Dusp8 WT mice kept their previously established seeking pattern. Nonetheless, the overall motivation to consume sucrose, and the levels of dopaminergic neurons in the brain areas NAcc and VTA were comparable between genotypes. Diabetes-risk allele carriers of DUSP8 SNP rs2334499 preferred sweet high caloric food compared to the major allele carriers, rating scores for savory food remained comparable between groups., Conclusion: Our data suggest a novel role for Dusp8 in the perception of sweet high caloric food as well as in the control of sucrose consumption and foraging in mice and humans., (© 2020 The Authors. Brain and Behavior published by Wiley Periodicals LLC.)

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.

Light sheet fluorescence microscopy (LSFM) of optically cleared biological samples represents a powerful tool to analyze the 3-dimensional morphology of tissues and organs. Multimodal combinations of LSFM with additional analyses of the identical sample help to limit the consumption of restricted specimen and reduce inter-sample variation. Here, we demonstrate the proof-of-concept that LSFM of cleared brain tissue samples can be combined with Matrix Assisted Laser Desorption/Ionization-Mass Spectrometry Imaging (MALDI-MSI) for detection and quantification of proteins. Samples of freshly dissected murine brain and of archived formalin-fixed paraffin-embedded (FFPE) human brain tissue were cleared (3DISCO). Tissue regions of interest were defined by LSFM and excised, (re)-embedded in paraffin, and sectioned. Mouse sections were coated with sinapinic acid matrix. Human brain sections were pre-digested with trypsin and coated with α-cyano-4-hydroxycinnamic acid matrix. Subsequently, sections were subjected to MALDI-time-of-flight (TOF)-MSI in mass ranges between 0.8 to 4 kDa (human tissue sections), or 2.5-25 kDa (mouse tissue sections) with a lateral resolution of 50 µm. Protein- and peptide-identities corresponding to acquired MALDI-MSI spectra were confirmed by parallel liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. The spatial abundance- and intensity-patterns of established marker proteins detected by MALDI-MSI were also confirmed by immunohistochemistry.

The study aimed to examine the sex specific association of obesity with cortisol metabolism in a sample of older community dwelling people. The cross-sectional analysis included 394 men and 375 women (aged 65-90 years) of the population-based KORA-Age study. Multivariable regression analyses were employed to examine the association between cortisol samples (serum and salivary samples of morning after awakening (M1), 30 min later (M2) and at late night (LNSC)). Obesity was calculated as waist-to-hip ratio (WHR) and body mass index (BMI). Cortisol levels were not significantly different between obesity measures except for elevated serum cortisol (P = 0.02) levels in individuals with a low WHR. Higher M1 levels were especially apparent in women with normal BMI. Serum cortisol levels were inversely related to WHR (P = 0.004) and CAR AUC was inversely associated with BMI (P = 0.007). Sex-stratified analytic models revealed that both obesity measures showed a non-linear association with cortisol diurnal pattern (M1/LNSC) in men. Impaired cortisol patterns emerged at both very ends of the body weight distribution. These findings do not support a cortisol driven obesity etiology in an older population and even point to an inverse association of body weight with cortisol levels. Differences of cortisol secretion patterns in generalized and abdominal fat distribution were marginal.

Celastrol is a natural pentacyclic triterpene extracted from the roots of Tripterygium wilfordi (thunder god vine). Celastrol was reported as a powerful anti-obesity drug with leptin sensitizing properties that decreases food consumption and mediates body weight loss when administered to diet-induced obese mice at 100 μg/kg body weight. The weight lowering properties of celastrol are likely mediated by the CNS, in particular, by the hypothalamus, but the final proof for the accumulation of celastrol in the brain and hypothalamus remains to be established. Here, we aimed to demonstrate that intraperitoneal celastrol administration at 100 μg/kg can rapidly reach the brain and, in particular, the hypothalamus of mice. We developed and validated a sensitive liquid chromatography mass spectrometry method for the quantitative determination of celastrol in murine tissues, namely liver, brain and hypothalamus. Chow-fed lean mice were randomly assigned to the vehicle vs. celastrol groups, injected with saline or 100 μg/kg body weight of celastrol, and sacrificed 30 min or 120 min post injection. Celastrol was extracted from homogenized tissue using ethyl acetate as organic solvent, and quantified using a matrix-matched calibration curve with glycyrrhetinic acid as internal standard. Liver celastrol concentrations were 32.60 ± 8.21 pg/mg and 40.52 ± 15.6 pg/mg, 30 and 120 min after injection, respectively. We found 4.70 ± 0.31 pg/mg celastrol after 30 min, and 16.22 ± 3.33 pg/mg after 120 min in whole brain lysates, and detectable amounts in the hypothalamus. These results corroborate the validity of our methodology, demonstrate the accumulation of celastrol in the brain of mice injected intraperitoneally with a dose of 100 μg/kg, and confirm the CNS as possible site of action for the weight lowering properties of celastrol., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8 WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network.

Obesity and being overweight have become a worldwide epidemic affecting more than 1.9 billion adults and 340 million children. Efforts to curb this global health burden by developing effective long-term non-surgical weight loss interventions continue to fail due to weight regain after weight loss. Weight cycling, often referred to as Yoyo dieting, is driven by physiological counter-regulatory mechanisms that aim at preserving energy, i.e. decreased energy expenditure, increased energy intake, and impaired brain-periphery communication. Models based on genetically determined set points explained some of the weight control mechanisms, but exact molecular underpinnings remained elusive. Today, gene-environment interactions begin to emerge as likely drivers for the obesogenic memory effect associated with weight cycling. Here, epigenetic mechanisms, including histone modifications and DNA methylation, appear as likely factors that underpin long-lasting deleterious adaptations or an imprinted obesogenic memory to prevent weight loss maintenance. The first part summarizes our current knowledge on the physiology of weight cycling by discussing human and murine studies on the Yoyo-dieting phenomenon and physiological adaptations associated with weight loss and weight re-gain. The second part provides an overview on known associations between obesity and epigenetic modifications. We further interrogate the roles of epigenetic mechanisms in the CNS control of cognitive functions as well as reward and addictive behaviors, and subsequently discuss whether such mechanisms play a role in weight control. The final two parts describe major opportunities and challenges associated with studying epigenetic mechanisms in the CNS with its highly heterogenous cell populations, and provide a summary of recent technological advances that will help to delineate whether an obese memory is based upon epigenetic mechanisms., (Copyright © 2019 Contreras, Schriever and Pfluger.)

Background/objectives: Individuals carrying loss-of-function gene mutations for the adipocyte hormone leptin are morbidly obese, but respond favorably to replacement therapy. Recombinant leptin is however largely ineffective for the vast majority of obese individuals due to leptin resistance. One theory underlying leptin resistance is impaired leptin transport across the blood-brain-barrier (BBB). Here, we aim to gain new insights into the mechanisms of leptin BBB transport, and its role in leptin resistance., Methods: We developed a novel tool for visualizing leptin transport using infrared fluorescently labeled leptin, combined with tissue clearing and light-sheet fluorescence microscopy. We corroborated these data using western blotting., Results: Using 3D whole brain imaging, we display comparable leptin accumulation in circumventricular organs of lean and obese mice, predominantly in the choroid plexus (CP). Protein quantification revealed comparable leptin levels in microdissected mediobasal hypothalami (MBH) of lean and obese mice (p = 0.99). We further found increased leptin receptor expression in the CP (p = 0.025, p = 0.0002) and a trend toward elevated leptin protein levels in the MBH (p = 0.17, p = 0.078) of obese mice undergoing weight loss interventions by calorie restriction or exendin-4 treatment., Conclusions: Overall, our findings suggest a crucial role for the CP in controlling the transport of leptin into the cerebrospinal fluid and from there to target areas such as the MBH, potentially mediated via the leptin receptor. Similar leptin levels in circumventricular organs and the MBH of lean and obese mice further suggest intact leptin BBB transport in leptin resistant mice.

Objective: Obesity has been linked to an inflammation like state in the hypothalamus, mainly characterized by reactive gliosis (RG) of astrocytes and microglia. Here, using two diet models or pharmacological treatment, we assessed the effects of mild and drastic weight loss on RG, in the context of high-fat diet (HFD) induced obesity., Methods: We subjected HFD-induced obese (DIO) male C57BL/6J mice to a weight loss intervention with a switch to standard chow, calorie restriction (CR), or treatment with the Glp1 receptor agonist Exendin-4 (EX4). The severity of RG was estimated by an ordinal scoring system based on fluorescence intensities of glial fibrillary acidic protein, ionized calcium-binding adapter molecule 1 positive (Iba1), cell numbers, and morphological characteristics., Results: In contrast to previous reports, DIO mice fed chronically with HFD showed no differences in microglial or astrocytic RG, compared to chow controls. Moreover, mild or profound weight loss had no impact on microglial RG. However, astrocyte RG was increased in CR and EX4 groups compared to chow fed animals and strongly correlated to body weight loss. Profound weight loss by either CR or EX4 was further linked to increased levels of circulating non-esterified free fatty acids., Conclusions: Overall, our data demonstrate that in a chronically obese state, astrocyte and microglial RG is indifferent from that observed in age-matched chow controls. Nonetheless, profound acute weight loss can induce astrocyte RG in the hypothalamic arcuate nucleus, possibly due to increased circulating NEFAs. This suggests that astrocytes may sense acute changes to both the dietary environment and body weight., (Copyright © 2019 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Celastrol is a natural pentacyclic triterpene used in traditional Chinese medicine with significant weight-lowering effects. Celastrol-administered mice at 100 μg/kg decrease food consumption and body weight via a leptin-dependent mechanism, yet its molecular targets in this pathway remain elusive. Here, we demonstrate in vivo that celastrol-induced weight loss is largely mediated by the inhibition of leptin negative regulators protein tyrosine phosphatase (PTP) 1B (PTP1B) and T-cell PTP (TCPTP) in the arcuate nucleus (ARC) of the hypothalamus. We show in vitro that celastrol binds reversibly and inhibits noncompetitively PTP1B and TCPTP. NMR data map the binding site to an allosteric site in the catalytic domain that is in proximity of the active site. By using a panel of PTPs implicated in hypothalamic leptin signaling, we show that celastrol additionally inhibited PTEN and SHP2 but had no activity toward other phosphatases of the PTP family. These results suggest that PTP1B and TCPTP in the ARC are essential for celastrol's weight lowering effects in adult obese mice.

Celastrol, a plant-derived constituent of traditional Chinese medicine, has been proposed to offer significant potential as an antiobesity drug. However, the molecular mechanism for this activity is unknown. We show that the weight-lowering effects of celastrol are driven by decreased food consumption. Although young Lep ob mice respond with a decrease in food intake and body weight, adult Lep db and Lep ob mice are unresponsive to celastrol, suggesting that functional leptin signaling in adult mice is required to elicit celastrol's catabolic actions. Protein tyrosine phosphatase 1 (PTP1B), a leptin negative-feedback regulator, has been previously reported to be one of celastrol's targets. However, we found that global PTP1B knockout (KO) and wild-type (WT) mice have comparable weight loss and hypophagia when treated with celastrol. Increased levels of uncoupling protein 1 (UCP1) in subcutaneous white and brown adipose tissue suggest celastrol-induced thermogenesis as a further mechanism. However, diet-induced obese UCP1 WT and KO mice have comparable weight loss upon celastrol treatment, and celastrol treatment has no effect on energy expenditure under ambient housing or thermoneutral conditions. Overall, our results suggest that celastrol-induced weight loss is hypophagia driven and age-dependently mediated by functional leptin signaling. Our data encourage reconsideration of therapeutic antiobesity strategies built on leptin sensitization., (© 2018 by the American Diabetes Association.)

Monoamines, acting as hormones and neurotransmitters, play a critical role in multiple physiological processes ranging from cognitive function and mood to sympathetic nervous system activity, fight-or-flight response and glucose homeostasis. In addition to brain and blood, monoamines are abundant in several tissues, and dysfunction in their synthesis or signaling is associated with various pathological conditions. It was our goal to develop a method to detect these compounds in peripheral murine tissues. In this study, we employed a high-performance liquid chromatography method using electrochemical detection that allows not only detection of catecholamines but also a detailed analysis of nine monoamines and metabolites in murine tissues. Simple tissue extraction procedures were optimized for muscle (gastrocnemius, extensor digitorum longus and soleus), liver, pancreas and white adipose tissue in the range of weight 10-200 mg. The system allowed a limit of detection between 0.625 and 2.5 pg μL -1 for monoamine analytes and their metabolites, including dopamine, 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, epinephrine, 3-methoxy-4-hydroxyphenylglycol, serotonin and 5-hydroxyindoleacetic acid. Typical concentrations for different monoamines and their metabolization products in these tissues are presented for C57Bl/6 J mice fed a high-fat diet., (Copyright © 2017 John Wiley & Sons, Ltd.)

ᅟ: Astrocytosis is a reactive process involving cellular, molecular, and functional changes to facilitate neuronal survival, myelin preservation, blood brain barrier function and protective glial scar formation upon brain insult. The overall pro- or anti-inflammatory impact of reactive astrocytes appears to be driven in a context- and disease-driven manner by modulation of astrocytic Ca 2+ homeostasis and activation of Ca 2+ /calmodulin-activated serine/threonine phosphatase calcineurin. Here, we aimed to assess whether calcineurin is dispensable for astrocytosis in the hypothalamus driven by prolonged high fat diet (HFD) feeding. Global deletion of calcineurin A beta (gene name: Ppp3cb) led to a decrease of glial fibrillary acidic protein (GFAP)-positive cells in the ventromedial hypothalamus (VMH), dorsomedial hypothalamus (DMH), and arcuate nucleus (ARC) of mice exposed chronically to HFD. The concomitant decrease in Iba1-positive microglia in the VMH further suggests a modest impact of Ppp3cb deletion on microgliosis. Pharmacological inhibition of calcineurin activity by Fk506 had no impact on IBA1-positive microglia in hypothalami of mice acutely exposed to HFD for 1 week. However, Fk506-treated mice displayed a decrease in GFAP levels in the ARC. In vivo effects could not be replicated in cell culture, where calcineurin inhibition by Fk506 had no effect on astrocytic morphology, astrocytic cell death, GFAP, and vimentin protein levels or microglia numbers in primary hypothalamic astrocytes and microglia co-cultures. Further, adenoviral overexpression of calcineurin subunit Ppp3r1 in primary glia culture did not lead to an increase in GFAP fluorescence intensity. Overall, our results point to a prominent role of calcineurin in mediating hypothalamic astrocytosis as response to acute and chronic HFD exposure. Moreover, discrepant findings in vivo and in cell culture indicate the necessity of studying astrocytes in their "natural" environment, i.e., preserving an intact hypothalamic microenvironment with neurons and non-neuronal cells in close proximity.

Here, we aimed to investigate the potential role of DUSP6, a dual specificity phosphatase, that specifically inactivates extracellular signal-regulated kinase (ERK), for the regulation of body weight and glucose homeostasis. We further assessed whether metabolic challenges affect Dusp6 expression in selected brain areas or white adipose tissue. Hypothalamic Dusp6 mRNA levels remained unchanged in chow-fed lean vs. high fat diet (HFD) fed obese C57Bl/6J mice, and in C57Bl/6J mice undergoing prolonged fasting or refeeding with fat free diet (FFD) or HFD. Similarly, Dusp6 expression levels were unchanged in selected brain regions of Lepob mice treated with 1 mg/kg of leptin for 6 days, compared to pair-fed or saline-treated Lepob controls. Dusp6 expression levels remained unaltered in vitro in primary adipocytes undergoing differentiation, but were increased in eWAT of HFD-fed obese C57Bl/6J mice, compared to chow-fed lean controls. Global chow-fed DUSP6 KO mice displayed reduced body weight and lean mass and slightly increased fat mass at a young age, which is indicative for early-age weight retardation. Subsequent exposure to HFD led to a significant increase in lean mass and body weight in DUSP6 deficient mice, compared to WT controls. Nevertheless, after 26 weeks of high-fat diet exposure, we observed comparable body weight, fat and lean mass in DUSP6 WT and KO mice, suggesting overall normal susceptibility to develop obesity. In line with the increased weight gain to compensate for early-age weight retardation, HFD-fed DUSP6 KO displayed increased expression levels of anabolic genes involved in lipid and cholesterol metabolism in the epididymal white adipose tissue (eWAT), compared to WT controls. Glucose tolerance was perturbed in both chow-fed lean or HFD-fed obese DUSP6 KO, compared to their respective WT controls. Overall, our data indicate that DUSP6 deficiency has limited impact on the regulation of energy metabolism, but impairs systemic glucose tolerance. Our data are in conflict to earlier reports that propose protection from diet-induced obesity and glucose intolerance in DUSP6 deficient mice. Reasons for the discrepancies remain elusive, but may entail differential genetic backgrounds, environmental factors such as the type and source of HFD, or alterations in the gut microbiome between facilities.

Elevated levels of oxidative stress and neuronal inflammation in the hypothalamus or ventral midbrain, respectively, represent common denominators for obesity and Parkinson's Disease (PD). However, little is known about defense mechanisms that protect neurons in these regions from oxidative damage. Here, we aimed to assess whether murine Gpx4, a crucial antioxidant enzyme that protects neurons from membrane damage and ferroptosis, is critical for the protection from neuronal inflammation in two distinct pathophysiologic diseases, namely metabolic dysfunction in diet-induced obesity or PD. Gpx4 was deleted from either AgRP or POMC neurons in the hypothalamus, essential for metabolic homeostasis, or from dopaminergic neurons in the ventral midbrain, governing behaviors such as anxiety or voluntary movement. To induce a pro-inflammatory environment, AgRP and POMC neuron-specific Gpx4 knockout mice were subjected to high-fat high-sucrose (HFHS) diet. To exacerbate oxidative stress in dopaminergic neurons of the ventral midbrain, we systemically co-deleted the PD-related gene DJ-1. Gpx4 was dispensable for the maintenance of cellular health and function of POMC neurons, even in mice exposed to obesogenic conditions. In contrast, HFHS-fed mice with Gpx4 deletion from AgRP neurons displayed increased body adiposity. Gpx4 expression and activity were diminished in the hypothalamus of HFHS-fed mice compared to standard diet-fed controls. Gpx4 deletion from dopaminergic neurons induced anxiety behavior, and diminished spontaneous locomotor activity when DJ-1 was co-deleted. Overall, these data suggest a physiological role for Gpx4 in balancing metabolic control signals and inflammation in AgRP but not POMC neurons. Moreover, Gpx4 appears to constitute an important rheostat against neuronal dysfunction and PD-like symptoms in dopaminergic circuitry within the ventral midbrain., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Susceptibility to obesity is linked to genes regulating neurotransmission, pancreatic beta-cell function and energy homeostasis. Genome-wide association studies have identified associations between body mass index and two loci near cell adhesion molecule 1 (CADM1) and cell adhesion molecule 2 (CADM2), which encode membrane proteins that mediate synaptic assembly. We found that these respective risk variants associate with increased CADM1 and CADM2 expression in the hypothalamus of human subjects. Expression of both genes was elevated in obese mice, and induction of Cadm1 in excitatory neurons facilitated weight gain while exacerbating energy expenditure. Loss of Cadm1 protected mice from obesity, and tract-tracing analysis revealed Cadm1-positive innervation of POMC neurons via afferent projections originating from beyond the arcuate nucleus. Reducing Cadm1 expression in the hypothalamus and hippocampus promoted a negative energy balance and weight loss. These data identify essential roles for Cadm1-mediated neuronal input in weight regulation and provide insight into the central pathways contributing to human obesity.

Hypothalamic leptin signalling has a key role in food intake and energy-balance control and is often impaired in obese individuals. Here we identify histone deacetylase 5 (HDAC5) as a regulator of leptin signalling and organismal energy balance. Global HDAC5 KO mice have increased food intake and greater diet-induced obesity when fed high-fat diet. Pharmacological and genetic inhibition of HDAC5 activity in the mediobasal hypothalamus increases food intake and modulates pathways implicated in leptin signalling. We show HDAC5 directly regulates STAT3 localization and transcriptional activity via reciprocal STAT3 deacetylation at Lys685 and phosphorylation at Tyr705. In vivo, leptin sensitivity is substantially impaired in HDAC5 loss-of-function mice. Hypothalamic HDAC5 overexpression improves leptin action and partially protects against HFD-induced leptin resistance and obesity. Overall, our data suggest that hypothalamic HDAC5 activity is a regulator of leptin signalling that adapts food intake and body weight to our dietary environment.

Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We recently reported restoration of leptin responsiveness in diet-induced obese (DIO) mice using a pharmacologically optimized, polyethylene-glycolated (PEG)-leptin analog in combination with exendin-4 or FGF21. However, the return of leptin action required discontinuation of high-fat diet (HFD) exposure. Here we assess whether a single peptide possessing balanced coagonism at the glucagon-like peptide 1 (GLP-1) and glucagon receptors can restore leptin responsiveness in DIO mice maintained on a HFD. DIO mice were treated with PEG-GLP-1/glucagon (30 nmol/kg every fourth day) to induce an ∼15% body weight loss, upon which they were randomized to continue PEG-GLP-1/glucagon therapy or reassigned to receive supplemental daily PEG-leptin (185 nmol/kg/day). The addition of PEG-leptin to PEG-GLP-1/glucagon resulted in an ∼18% greater weight loss as compared with PEG-GLP-1/glucagon alone and was accompanied by further decreases in food intake and improved glucose and lipid metabolism. The beneficial effect of PEG-leptin supplementation occurred after an initial body weight loss similar to what we previously reported following reduced dietary fat along with PEG-leptin and exendin-4 or FGF21 cotreatment. In summary, we report that GLP-1/glucagon coagonism restores leptin responsiveness in mice maintained on a HFD, thus emphasizing the translational value of this polypharmacotherapy for the treatment of obesity and diabetes.

A simple approach was developed for the quantification of lipid droplet size and frequency distribution in images acquired by standard light microscopy. Oil Red O-stained cell images were thresholded for the lipid droplet signal using the freely available imaging software ImageJ. Watershed algorithms allowed analyzing the area of each individual lipid droplet. The method was validated by the decrease in lipid droplet size of 3T3-L1 adipocytes on lowered glucose availability associated with reduced glycerol-3-phosphate dehydrogenase activity and reduced transcription of lipid droplet size markers. This approach can be easily applied using standard laboratory equipment without requiring expensive and complex instrumentation., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The arcuate nucleus (ARC) of the hypothalamus plays a key role in sensing metabolic feedback and regulating energy homeostasis. Recent studies revealed activation of microglia in mice with high-fat diet (HFD)-induced obesity (DIO), suggesting a potential pathophysiological role for inflammatory processes within the hypothalamus. To further investigate the metabolic causes and molecular underpinnings of such glial activation, we analyzed the microglial activity in wild-type (WT), monogenic obese ob/ob (leptin deficient), db/db (leptin-receptor mutation), and Type-4 melanocortin receptor knockout (MC4R KO) mice on either a HFD or on standardized chow (SC) diet. Following HFD exposure, we observed a significant increase in the total number of ARC microglia, immunoreactivity of ionized calcium binding adaptor molecule 1 (iba1-ir), cluster of differentiation 68 (CD68-ir), and ramification of microglial processes. The ob/ob mice had significantly less iba1-ir and ramifications. Leptin replacement rescued these phenomena. The db/db mice had similar iba1-ir comparable with WT mice but had significantly lower CD68-ir and more ramifications than WT mice. After 2 weeks of HFD, ob/ob mice showed an increase of iba1-ir, and db/db mice showed increase of CD68-ir. Obese MC4R KO mice fed a SC diet had comparable iba1-ir and CD68-ir with WT mice but had significantly more ramifications than WT mice. Intriguingly, treatment of DIO mice with glucagon-like peptide-1 receptor agonists reduced microglial activation independent of body weight. Our results show that diet type, adipokines, and gut signals, but not body weight, affect the presence and activity levels of hypothalamic microglia in obesity., (Copyright © 2013 Wiley Periodicals, Inc.)

The regulation of cell growth and protein biosynthesis is triggered by the mammalian target of rapamycin complex 1 (mTORC1) responding to amino acids, especially leucine. The molecular mechanisms linking leucine to mTORC1 activation are not well understood. We analyzed whether the free intracellular leucine availability, a metabolite of leucine catabolism or the process of leucine oxidation activates mTORC1 signaling. We further investigated whether mTORC1 signaling is subject to altered regulation in disturbed leucine metabolism. Human fibroblasts with deficiencies in leucine catabolic steps and those from healthy control subjects were utilized. In all cells, leucine-induced mTORC1 signaling was significantly related to leucine pool size and leucine repletion capacity. The leucine/glutamine antiporter SLC7A5/SLC3A2 and the amino acid sensor MAP4K3 were identified as crucial determinants of signaling leucine availability to downstream targets. In cells with defective leucine catabolism, mTORC1 signaling towards phosphorylation of ribosomal protein S6 kinase 1 (S6K1) was significantly increased, whereas transcriptional down-regulation of MAP4K3 upon reduced amino acid supply was abrogated. Remarkably, these effects were observed irrespective of the localization of the enzymatic blockage. Our data provide evidence that mechanisms determining intracellular leucine availability and the amino acid sensor MAP4K3 are key upstream modulators of nutrient-sensitive mTORC1 signaling, whereas specific leucine metabolites or leucine oxidation rates do not play a role. In human fibroblasts deficient in leucine catabolic steps, we observed regulation consistent with sustaining a more efficient MAP4K3 and mTOR-S6K1 signaling. Such regulatory circuit might serve to protect cells against detrimental consequences of reduced nutrient utilization in human conditions associated with disturbed leucine metabolism., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The scaffold protein p62 (sequestosome 1; SQSTM1) is an emerging key molecular link among the metabolic, immune, and proliferative processes of the cell. Here, we report that adipocyte-specific, but not CNS-, liver-, muscle-, or myeloid-specific p62-deficient mice are obese and exhibit a decreased metabolic rate caused by impaired nonshivering thermogenesis. Our results show that p62 regulates energy metabolism via control of mitochondrial function in brown adipose tissue (BAT). Accordingly, adipocyte-specific p62 deficiency led to impaired mitochondrial function, causing BAT to become unresponsive to β-adrenergic stimuli. Ablation of p62 leads to decreased activation of p38 targets, affecting signaling molecules that control mitochondrial function, such as ATF2, CREB, PGC1α, DIO2, NRF1, CYTC, COX2, ATP5β, and UCP1. p62 ablation in HIB1B and BAT primary cells demonstrated that p62 controls thermogenesis in a cell-autonomous manner, independently of brown adipocyte development or differentiation. Together, our data identify p62 as a novel regulator of mitochondrial function and brown fat thermogenesis.

We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1-targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1-estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

The opioid system plays a pivotal role in how our brain regulates hedonic components of ingestive behavior. Duraffourd et al. (2012) add the gut to this opioid landscape, demonstrating direct activation of periportal μ-opioid receptors by food-derived opioid peptides (nutropioids), and a gut-brain feedback spiral that culminates in enhanced satiety., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Quantitative analysis of mitochondrial FA β-oxidation (FAO) has drawn increasing interest for defining lipid-induced metabolic dysfunctions, such as in obesity-induced insulin resistance, and evaluating pharmacologic strategies to improve β-oxidation function. The aim was to develop a new assay to quantify β-oxidation function in intact mitochondria and with a low amount of cell material. Cell membranes of primary human fibroblasts were permeabilized with digitonin prior to a load with FFA substrate. Following 120 min of incubation, the various generated acylcarnitines were extracted from both cells and incubation medium by protein precipitation/desalting and subjected to solid-phase extraction. A panel of 30 acylcarnitines per well was quantified by MS/MS and normalized to citrate synthase activity to analyze mitochondrial metabolite flux. Pretreatment with bezafibrate and etomoxir revealed stimulating and inhibiting regulatory effects on β-oxidation function, respectively. In addition to the advantage of a much shorter assay time due to in situ permeabilization compared with whole-cell incubation systems, the method allows the detection of multiple acylcarnitines from an only limited amount of intact cells, particularly relevant to the use of primary cells. This novel approach facilitates highly sensitive, simple, and fast monitoring of pharmacological effects on FAO.

Selenium (Se) plays a critical role in testis, sperm, and reproduction, and testis Se levels are remarkably maintained in Se deficiency. In most other tissues, Se levels decrease dramatically as do levels of most selenoproteins and levels of a subset of Se-regulated selenoprotein mRNAs. Because of the recent identification of key molecules in the targeted trafficking of Se to the testis, we examined the hierarchy of Se regulation in testis by determining the dietary Se regulation of the full testis selenoproteome in rats fed graded levels of Se (0 to 0.8 microg Se/g) as Na2SeO3 for 28 d. Se status did not significantly affect testis weight or glutathione peroxidase 4 (Gpx4) activity (P>0.05). qRT-PCR analysis of selenoprotein mRNA expression revealed that 21 of the 24 selenoprotein mRNAs and ApoER2 mRNA (the selenoprotein P [Sepp1] receptor) were also not regulated significantly by dietary Se status. In contrast, Gpx1 activity decreased to 28% of Se-adequate levels, and mRNA levels for Gpx1, Sepp1, and Sepw1 (selenoprotein W) decreased significantly in Se-deficient rats to 45, 46, and 55%, respectively, of Se-adequate plateau levels. Overlap of hyperbolic Gpx4 activity and Sepw1 mRNA response curves with testis Se concentration, all with minimum dietary Se requirements<0.016 microg Se/g, showed the priority for synthesis of Gpx4. Higher minimum dietary Se requirements of 0.04 microg Se/g for Gpx1 activity and Sepp1 mRNA, and the even higher minimum dietary Se requirement of 0.08 microg Se/g for Gpx1 mRNA, suggest that the hierarchy of these biomarkers reflects distinct, lower priority pools, cell types, and roles for Se within the testis.