Microglial phagolysosome dysfunction and altered neural communication amplify phenotypic severity in Prader-Willi Syndrome with larger deletion.

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.

Fluorescent blood-brain barrier tracing shows intact leptin transport in obese mice.

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.

Calcineurin A beta deficiency ameliorates HFD-induced hypothalamic astrocytosis in mice.

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 Ca2+ homeostasis and activation of Ca2+/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.

Comprehensive analysis of nine monoamines and metabolites in small amounts of peripheral murine (C57Bl/6 J) tissues.

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.

Hormones and diet, but not body weight, control hypothalamic microglial activity.

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.

Digital image analysis approach for lipid droplet size quantitation of Oil Red O-stained cultured cells.

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.

HDAC5 controls a hypothalamic STAT5b-TH axis, the sympathetic activation of ATP-consuming futile cycles and adult-onset obesity in male mice.

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.

Liver microRNA transcriptome reveals miR-182 as link between type 2 diabetes and fatty liver disease in obesity.

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).

A GnRH neuronal population in the olfactory bulb translates socially relevant odors into reproductive behavior in male mice.

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 (GnRHOB) of adult mice can mediate social recognition. Specifically, we show that GnRHOB neurons extend neurites into the vomeronasal organ and olfactory epithelium and project to the median eminence. GnRHOB 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 GnRHOB 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 GnRHOB neurons' actions on male mounting behavior. Taken together, these results establish GnRHOB neurons as regulating fertility, sex recognition and mating in male mice.

Loss of GIPR in LEPR cells impairs glucose control by GIP and GIP:GLP-1 co-agonism without affecting body weight and food intake in mice.

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 ß-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.

Estradiol regulates leptin sensitivity to control feeding via hypothalamic Cited1.

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.

In situ assay of fatty acid ß-oxidation by metabolite profiling following permeabilization of cell membranes.

Quantitative analysis of mitochondrial FA ß-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 ß-oxidation function. The aim was to develop a new assay to quantify ß-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 ß-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.

Development and validation of an LC-MS/MS methodology for the quantification of thyroid hormones in dko MCT8/OATP1C1 mouse brain.

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 (r2 >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.

Determination of morphine and norlaudanosoline in murine brain regions by dispersive liquid-liquid micro-extraction and liquid chromatograpy-electrochemical detection.

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.

Determination of 3-iodothyronamine (3-T1AM) in mouse liver using liquid chromatography-tandem mass spectrometry.

3-iodothyronamine (3-T1AM) 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-T1AM plasma and tissue concentrations have nonetheless caused controversy regarding the existence and biological role of 3-T1AM. These discussions are at least partially based on potential analytical artefacts caused by differential decay kinetics of 3-T1AM and the widely used deuterated quantification standard D4-T1AM. Here, we report a novel LC-MS/MS method for the quantification of 3-T1AM in biological specimens using stable isotope dilution with 13C6-T1AM, a new internal standard that showed pharmacodynamic properties comparable to endogenous 3-T1AM. The method detection limit (MDL) and method quantification limit (MQL) of 3-T1AM were 0.04 and 0.09 ng/g, respectively. The spike-recoveries of 3-T1AM 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-T1AM were 8.45-11.2% and 3.58-5.73%, respectively. Endogenous 3-T1AM liver values in C57BL/6J mice were 2.20 ± 0.49 pmol/g with a detection frequency of 50%. Higher liver 3-T1AM 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-T1AM in biological tissues and fluids.

Diabetes type 2 risk gene Dusp8 is associated with altered sucrose reward behavior in mice and humans.

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.

Correlation guided Network Integration (CoNI) reveals novel genes affecting hepatic metabolism.

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.

Detection and quantification of the anti-obesity drug celastrol in murine liver and brain.

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.

Light sheet fluorescence microscopy guided MALDI-imaging mass spectrometry of cleared tissue samples.

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.

Type 2 diabetes risk gene Dusp8 regulates hypothalamic Jnk signaling and insulin sensitivity.

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.