3,171 research outputs found

    Adipose Tissue Protects against Hepatic Steatosis in Male Rats Fed a High-Fat Diet plus Liquid Fructose: Sex-Related Differences

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    Non-alcoholic fatty liver disease is a sexual dimorphic disease, with adipose tissue playing an essential role. Our previous work showed that female rats fed a high-fat high-fructose diet devoid of cholesterol (HFHFr) developed simple hepatic steatosis dissociated from obesity. This study assessed the impact of the HFHFr diet on the male rat metabolism compared with data obtained for female rats. A total of 16 Sprague Dawley (SD) male rats were fed either a control (standard rodent chow and water) or HFHFr (high-fat diet devoid of cholesterol, plus 10% fructose in drinking water) diet for 3 months. Unlike female rats, and despite similar increases in energy consumption, HFHFr males showed increased adiposity and hyperleptinemia. The expression of hormone-sensitive lipase in the subcutaneous white adipose tissue was enhanced, leading to high free fatty acid and glycerol serum levels. HFHFr males presented hypertriglyceridemia, but not hepatic steatosis, partially due to enhanced liver PPARα-related fatty acid β-oxidation and the VLDL-promoting effect of leptin. In conclusion, the SD rats showed a sex-related dimorphic response to the HFHFr diet. Contrary to previous results for HFHFr female rats, the male rats were able to expand the adipose tissue, increase fatty acid catabolism, or export it as VLDL, avoiding liver lipid deposition. Keywords: adipose tissue; fructose; high-fat diet; leptin; non-esterified fatty acids

    Marine fungus Aspergillus c1. sp metabolite activates the HSF1/PGC-1α axis, inducing a thermogenic program for treating obesity

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    Background and aims: Obesity is one of the most prevalent diseases worldwide with less ideal approved agents in clinic. Activating the HSF1/PGC-1α axis in adipose tissues has been reported to induce thermogenesis in mice, which presents a promising therapeutic avenue for obesity treatment. The present study aimed to identified novel natural HSF1 activator and evaluated the therapeutic effects of the newly discovered compound on obesity-associated metabolic disorders and the molecular mechanisms of these effects.Methods: Our previous reported HSF1/PGC-1α activator screening system was used to identify novel natural HSF1 activator. The PGC-1α luciferase activity, immunoblot, protein nuclear-translocation, immunofluorescence, chromatin immunoprecipitation assays were used to evaluate the activity of compound HN-001 in activating HSF1. The experiments of mitochondrial number measurement, TG assay and imaging, cellular metabolic assay, gene assays, and CRISPR/Cas 9 were applied for investigating the metabolic effect of HN-001 in C3H10-T1/2 adipocytes. The in vivo anti-obesity efficacies and beneficial metabolic effects of HN-001 were evaluated by performing body and fat mass quantification, plasma chemical analysis, GTT, ITT, cold tolerance test, thermogenesis analysis.Results: HN-001 dose- and time-dependently activated HSF1 and induced HSF1 nuclear translocation, resulting in an enhancement in binding with the gene Pgc-1α. This improvement induced activation of adipose thermogenesis and enhancement of mitochondrial oxidation capacity, thus inhibiting adipocyte maturation. Deletion of HSF1 in adipocytes impaired mitochondrial oxidation and abolished the above beneficial metabolic effects of HN-001, including adipocyte browning induction, improvements in mitogenesis and oxidation capacity, and lipid-lowering ability. In mice, HN-001 treatment efficiently alleviated diet-induced obesity and metabolic disorders. These changes were associated with increased body temperature in mice and activation of the HSF1/PGC-1α axis in adipose tissues. UCP1 expression and mitochondrial biogenesis were increased in both white and brown adipose tissues of HN-001-treated mice.Conclusion: These data indicate that HN-001 may have therapeutic potential for obesity-related metabolic diseases by increasing the capacity of energy expenditure in adipose tissues through a mechanism involving the HSF1/PGC-1α axis, which shed new light on the development of novel anti-obesity agents derived from marine sources

    Autophagy in spinal muscular atrophy: from pathogenic mechanisms to therapeutic approaches

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    © 2024 Rashid and Dimitriadi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). https://creativecommons.org/licenses/by/4.0/Spinal muscular atrophy (SMA) is a devastating neuromuscular disorder caused by the depletion of the ubiquitously expressed survival motor neuron (SMN) protein. While the genetic cause of SMA has been well documented, the exact mechanism(s) by which SMN depletion results in disease progression remain elusive. A wide body of evidence has highlighted the involvement and dysregulation of autophagy in SMA. Autophagy is a highly conserved lysosomal degradation process which is necessary for cellular homeostasis; defects in the autophagic machinery have been linked with a wide range of neurodegenerative disorders, including amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease. The pathway is particularly known to prevent neurodegeneration and has been suggested to act as a neuroprotective factor, thus presenting an attractive target for novel therapies for SMA patients. In this review, (a) we provide for the first time a comprehensive summary of the perturbations in the autophagic networks that characterize SMA development, (b) highlight the autophagic regulators which may play a key role in SMA pathogenesis and (c) propose decreased autophagic flux as the causative agent underlying the autophagic dysregulation observed in these patients.Peer reviewe

    The KINGS mouse as a model of beta cell endoplasmic reticulum (ER) stress and sex differences in diabetes.

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    Background: The KINGS mouse is a novel model of beta cell endoplasmic reticulum (ER) stress which shows stark sex differences in diabetes, with males developing overt and progressive hyperglycaemia whilst females are protected. Beta cell ER stress has been implicated in many types of diabetes and underpins numerous factors known to drive beta cell failure. Sex differences also exist in diabetes in humans with premenopausal women having a lower diabetes incidence compared to men. Further characterisation of the KINGS mice may provide valuable insight into these phenomena. Aims: The objectives of this thesis were to 1) further characterise beta cell ER stress and associated cellular response in the KINGS mice, 2) investigate the influence of sex hormones and beta cell ER stress manipulation on glycaemic control in the KINGS mice and 3) investigate whether diabetes development can be prevented in the male KINGS mice. Methods: Western blotting and immunofluorescent staining were used to investigate the expression of ER stress and unfolded protein response (UPR) markers in KINGS islets, as well as beta cell turnover and mass. To determine the influence of oestradiol on the KINGS phenotype, endogenous oestradiol was removed from female mice via ovariectomy, and exogenous oestradiol was delivered to male KINGS mice through implantation of oestradiol- containing capsules. A western diet was used to exacerbate beta cell ER stress in female KINGS mice, whilst liraglutide administration, TUDCA administration and removal of endogenous testosterone (via orchidectomy) was used in an attempt to reduce ER stress and prevent diabetes in the male KINGS mice. For all in vivo studies, glycaemic control was assessed through blood glucose concentration monitoring, glucose tolerance testing and insulin tolerance testing. Results: Male KINGS mice developed diabetes by 5-6 weeks of age whereas female KINGS mice were protected, in line with previous studies. Protein markers of ER stress and the UPR were observed in KINGS islets from 4 weeks of age and a sex difference was observed in expression profiles with males largely showing an increased expression of markers. Despite this, we did not observe a loss of beta cell mass in either male or female KINGS mice. However, subtle changes in beta cell proliferation and apoptosis in the male KINGS mice are suggestive of mild changes to beta cell turnover which may contribute to diabetes development. A western diet exacerbated beta cell ER stress in female KINGS mice, however this only led to a mild impairment in glycaemic control which was not as severe as that seen in male KINGS mice. This may suggest that even under conditions of further ER stress, female mice are still able to respond adaptively. Removal of endogenous oestradiol also exacerbated beta cell ER stress, however again this was only associated with a subtle impairment in glycaemic control. On the contrary, exogenous oestradiol delivery in the male KINGS mice prevented the development of overt diabetes. Treatment with liraglutide was used in an attempt to alleviate ER stress in the male KINGS mice. Although liraglutide prevented the development of diabetes and reduced blood glucose concentrations once diabetes was established, this protection only lasted during the treatment window and cessation of treatment was associated with increases in blood glucose concentrations. In addition, liraglutide had no effect on beta cell ER stress levels. Treatment with TUDCA, a chemical chaperone previously found to reduce beta cell ER stress, had no impact on blood glucose concentrations in the KINGS mice. However, removal of endogenous testosterone through orchidectomy prevented the development of overt diabetes. Conclusion: In this study we have confirmed that the KINGS mutation drives beta cell ER stress and that sex differences exist in beta cell response to this. Interestingly, an adaptive response to beta cell ER stress was still maintained in female KINGS mice when ER stress was exacerbated through a western diet. We also found that whilst oestradiol likely contributes in-part to sex differences in diabetes, it cannot be the sole mediator and other factors must be involved. Indeed, we found that endogenous testosterone removal prevented the development of diabetes in male mice. Liraglutide treatment also prevented diabetes development in male mice, however this was likely to be mediated through mechanisms unrelated to beta cell ER stress. Further study is required to investigate how testosterone removal and liraglutide protect male mice.</div

    Divergent roles of the regulatory subunits of class IA PI3K

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    The regulatory subunit of phosphatidylinositol 3-kinase (PI3K), known as p85, is a critical component in the insulin signaling pathway. Extensive research has shed light on the diverse roles played by the two isoforms of p85, namely p85α and p85β. The gene pik3r1 encodes p85α and its variants, p55α and p50α, while pik3r2 encodes p85β. These isoforms exhibit various activities depending on tissue types, nutrient availability, and cellular stoichiometry. Whole-body or liver-specific deletion of pik3r1 have shown to display increased insulin sensitivity and improved glucose homeostasis; however, skeletal muscle-specific deletion of p85α does not exhibit any significant effects on glucose homeostasis. On the other hand, whole-body deletion of pik3r2 shows improved insulin sensitivity with no significant impact on glucose tolerance. Meanwhile, liver-specific double knockout of pik3r1 and pik3r2 leads to reduced insulin sensitivity and glucose tolerance. In the context of obesity, upregulation of hepatic p85α or p85β has been shown to improve glucose homeostasis. However, hepatic overexpression of p85α in the absence of p50α and p55α results in increased insulin resistance in obese mice. p85α and p85β have distinctive roles in cancer development. p85α acts as a tumor suppressor, but p85β promotes tumor progression. In the immune system, p85α facilitates B cell development, while p85β regulates T cell differentiation and maturation. This review provides a comprehensive overview of the distinct functions attributed to p85α and p85β, highlighting their significance in various physiological processes, including insulin signaling, cancer development, and immune system regulation

    Antitumor T‐cell function requires CPEB4‐mediated adaptation to chronic endoplasmic reticulum stress

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    Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license

    Signaling Mechanisms Behind the Benefits of Sleep

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    Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende Würmer nach einer Verletzung häufiger. Insgesamt deutet dies darauf hin, dass AMP als Signalmoleküle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. Für diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlüsseln, durch den verschiedene Reize der angeborenen Immunität, das heißt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche Domänen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-Domäne als aktive Proteasedomäne von NAS-38 angesehen wird, erwartete ich eine Schlüsselrolle dieser Domäne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitätsinduziertem Schlaf? Da gezeigt wurde, dass AMP während des NAS-38 Schlafes und auch nach Verwundung hochreguliert sind, erwartete ich, dass AMP an der Signalisierung von Schlaf von der Epidermis zum Nervensystem beteiligt sind. In einem zweiten Schritt untersuchte ich die molekularen Mechanismen, die den Vorteilen von Schlaf für das Überleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: Verändert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf für viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeinträchtigt. Zweitens, ist Schlaf wichtig für die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? Während der Larvenentwicklung fällt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeinträchtigt. Methoden: Zur Analyse der Signalmechanismen, durch die sowohl NAS-38 als auch Verletzungen Schlaf induzieren, filmte ich das Schlafverhalten von C. elegans mittels Langzeit-Bildgebung in Agarose-Mikrokammern. So führte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 Domäne deletiert war. Darüber hinaus testete ich verschiedene Suppressoren für immunvermittelten Schlaf, der durch NAS 38 oder Verletzungen induziert war. Die Redundanz des Suppressionseffektes der verschiedenen Mitglieder der AMP-Familie auf immunvermittelten Schlaf testete ich, indem ich den Suppressionsphänotyp einer CRISPR/Cas9-editierten Multi-Knockout-Mutante analysierte, in der insgesamt 19 AMP deletiert waren. Um Effektoren zu identifizieren, die den AMP nachgeschaltet sind, induzierte ich Schlaf durch Überexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschäftigte ich mich mit der Frage, wie genau Schlaf das Überleben nach Verletzungen unterstützt. Ich verglich die Expression von literaturbekannten Reportern für verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell für chronisch-genetischen Schlafmangel. Darüber hinaus habe ich die Transkriptome zwischen jeweils adulten verwundeten und unverwundeten Wildtypen und schlaflosen Mutanten verglichen. Um die Struktur der Cuticula des Wildtyps und der schlaflosen Mutante zu vergleichen, analysierte ich außerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-Domäne verlängert. Dieser Prozess wird moderiert durch die TSP-1-Domäne. Weiterhin konnte ich zeigen, dass viele AMP redundant wirken um immunvermittelten Schlaf, verursacht durch NAS-38 oder Verletzungen, zu signalisieren. Ich konnte zeigen, dass das AMP NLP-29 über den Neuropeptidrezeptor NPR-12 wirkt. Dieser kann NLP-29-induzierten Schlaf vermitteln, wenn er in einem neuronalen Netzwerk exprimiert wird, welches nachweislich RIS aktiviert. Interessanterweise fand ich außerdem heraus, dass für NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verändert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verändert. Dies betraf unter anderem eine Gruppe oszillierender Gene, die Cuticula-assoziierte Proteine codieren, und deren Expression normalerweise ihren Höhepunkt gegen Ende des Lethargus erreicht. Da angenommen wird, dass der Zeitpunkt der Kollagenexpression entscheidend für eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsächlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die Strapazierfähigkeit der Cuticula gegenüber bestimmten Belastungen verringern. Daher könnte Schlaf erforderlich sein, Robustheit in Form einer strukturierten Cuticula zu fördern. Schlussfolgerungen: In diesem Dissertationsprojekt vollendete ich die Charakterisierung eines neuentdeckten Mechanismus in C. elegans, durch den Verwundungen Schlaf als Teil der Immunantwort aus der Peripherie zum Nervensystem signalisieren. Ich konnte zeigen, dass AMP gewebeübergreifend Signale von der Epidermis an ein neuronales Netz vermitteln, welches wiederum RIS aktiviert und dadurch Schlaf induziert. Da Komponenten dieses Signalweges konserviert sind, könnten AMP auch in anderen Tieren, einschließlich des Menschen, Schlaf zur Genesung fördern. Darüber hinaus habe ich die Grundlagen für die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes für Heilung und Überleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verändern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfältigere Rolle bei der zeitlichen Regulierung der Genexpression hin.:Summary I Zusammenfassung IV Contents VII List of Figures XII List of Tables XIV Abbreviations XV 1. Introduction 1 1.1. Sleep is fascinating 1 1.1.1. The origin and basic features of sleep 1 1.1.2. Regulation of sleep in higher animals 3 1.1.2.1. Neuronal control of sleep 3 1.1.2.2. Molecular control of sleep 5 1.1.3. The functions of sleep 6 1.2. The immune system and its relationship to sleep 7 1.3. Wound healing and its relationship to sleep 10 1.4. Caenorhabditis elegans is a well-studied model organism 12 1.4.1. Sleep in C. elegans 15 1.4.2. The C. elegans cuticle 18 1.4.3. Immunity in C. elegans 19 1.4.4. Wound healing response in C. elegans 22 2. Previous results 25 2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25 2.2. NAS-38 increases sleep mostly through the RIS neuron 25 2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25 2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27 2.5. Overexpression of AMPs induces RIS dependent quiescence 30 2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31 3. Thesis Aims 34 3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34 3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35 4. Materials and Methods 36 4.1. C. elegans maintenance 36 4.2. C. elegans crossing and genotyping 41 4.3. Creation of transgenic animals 45 4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45 4.3.2. Microparticle bombardment 45 4.3.3. CRISPR/Cas9 system 46 4.4. Synchronizing worm cultures by hypochlorite treatment 48 4.5. Imaging 49 4.5.1. Imaging setups 49 4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50 4.5.2.1. Imaging of heterozygous mutants 50 4.5.3. DIC imaging in the temperature control device 51 4.5.4. Fluorescent imaging experiments 51 4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51 4.5.4.2. nlp-29p::GFP in L4 larvae 52 4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52 4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52 4.5.4.5. Functional Ca2+ imaging in young adults 52 4.5.4.6. Fluorescence imaging across the whole developmental time 54 4.5.4.7. Nuclear decompaction assays 55 4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55 4.5.4.9. Imaging DPY-13::mKate2 in young adults 56 4.6. Image analysis 56 4.6.1. Assessment of developmental time and lethargus detection 56 4.6.2. Sleep detection in DIC mode 56 4.6.3. Analyzing functional Ca2+ images 57 4.6.4. Fluorescent reporter analysis during long-term imaging 57 4.7. RNAi-by-feeding 58 4.8. Transcriptome analysis 59 4.8.1. Analysis of the nas-38(ok3407) transcriptome 59 4.8.2. Analysis of the wounding transcriptome 59 4.9. Epidermal wounding 62 4.9.1. Laser wounding 62 4.9.2. Needle wounding 62 4.9.3. Survival assay 63 4.10. Scanning Electron Microscopy (SEM) 63 4.11. Histamine-inducible hyperpolarization of RIS 64 4.12. Cuticle integrity test with Sodium hypochlorite 64 4.13. NPR-12 receptor modeling 64 4.14. Quantification and statistical analysis 65 5. Results 66 5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66 5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66 5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66 5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69 5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72 5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75 5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77 5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80 5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81 5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83 5.1.10. AMPs contribute to the survival after wounding 85 5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87 5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87 5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87 5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89 5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95 5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101 5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106 5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109 5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114 5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120 5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122 6. Discussion and Outlook 128 6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128 6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130 6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131 6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132 6.5. Genetic sleep deprivation alters cuticle formation 135 6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137 6.7. Conclusion 139 7. References 140 8. Acknowledgements 163 9. Appendix 166 9.1. Standard reagents 166 9.2. Sequence summary of PHX3754 167 9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171 9.4. Permissions to reprint figures 174 9.5. Experimental author contributions 175 9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176 9.7. Overlap of the adult wounding transcriptome with other data sets 179 9.8. Curriculum Vitae – Marina Patricia Sinner 181Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a protective response. This hypothesis, however, was hitherto lacking final evidence and pathway components. Research questions and hypotheses: I aimed to characterize the molecular mechanism by which separate triggers of innate immunity, i. e. NAS-38 and wounding, induce sleep. I specifically addressed two questions: Firstly, which domains of the NAS-38 protein are involved in sleep regulation? As the astacin domain is predicted to be the active protease domain of NAS-38, I expected a role for it also in sleep induction by NAS-38. Secondly, what is the role of AMPs in signaling immunity-induced sleep? As they have been shown to be upregulated during times of increased sleep in the nas-38 mutant and after wounding, I expected AMPs to be involved in signaling sleep from the epidermis to the nervous system. In a second step, I investigated the molecular mechanisms underlying the benefits of sleep for surviving injury. Again, I addressed two questions: Firstly, does genetic sleep deprivation alter the transcriptional wounding response? As sleep has a role in many fundamental processes and sleeplessness increases mortality upon wounding, I hypothesized that genetic sleep deprivation impairs wounding-induced changes of transcriptional activity. Secondly, does sleep help building robustness before encountering injury? During larval development the synthesis of a new cuticle coincides with sleep. Thus, I hypothesized that genetic sleep deprivation impairs proper cuticle formation. Methods: To dissect the signaling mechanisms by which NAS-38 and wounding induced sleep, I followed sleep behavior of C. elegans by long-term imaging in agarose microchambers. I performed a structure-function analysis with different nas-38 mutants, each carrying a deletion of a different domain. Moreover, I screened for suppressors of sleep induced by NAS 38 or wounding. To test for redundancy of the AMP family, I investigated the suppression-phenotype of a CRISPR/Cas9 edited multi-knockout mutant lacking 19 AMPs. To identify downstream effectors of the AMP NLP 29, I induced sleep by overexpressing NLP 29 from a heat-shock promoter and analyzed the suppression-phenotype of different knockout mutants. For the second project, I addressed the question how sleep aids recovery from injury. I followed fluorescent reporters of previously described wounding response pathways by fluorescent long-term imaging in wild-type and genetically sleep-deprived animals. Moreover, I compared the transcriptomes of adult wild-type and genetically sleep-deprived worms both wounded and unwounded. To investigate the structure of the cuticle, I analyzed scanning electron microscopy images. Results: In the first project, I could show that NAS-38 indeed increases sleep via its astacin domain in a process that is modulated by the TSP-1 domain. Moreover, I could show that many AMPs act redundantly in mediating immunity-induced sleep downstream of NAS-38 and after wounding. I demonstrated that the AMP NLP-29 signals sleep via the neuropeptide receptor NPR 12. This receptor can mediate sleep when it is specifically expressed in command interneurons of a circuit that has been shown to activate RIS. Interestingly, I also found that EGFR signaling is required to mediate NLP-29-induced sleep. In the second project, I found that sleeplessness does not dramatically alter the transcriptional wounding response. However, I could show that transcription is altered already in the unwounded non-sleeping mutant. This affects, among others, a specific subset of oscillating collagen-coding genes, whose expression usually peaks around the end of lethargus. As the timing of expression of collagens is thought to be highly important for proper cuticle formation, I characterized the cuticle of the aptf-1(gk794) mutant. I could show that young adult aptf 1(gk794) worms indeed have a structural defect affecting cuticular furrows in the region adjacent to the alae, which could potentially decrease specific aspects of resilience of the cuticle. Thus, sleep might be required to build robustness in the form of a properly structured cuticle. Conclusion: In this PhD project, I completed the characterization of a novel mechanism by which wounding signals sleep from the periphery to the nervous system as part of the immune response in C. elegans. I could show that AMPs act as cross-tissue signals from the epidermis to a neuronal RIS-controlling circuit that ultimately leads to sleep induction. As components of this molecular pathway are highly conserved, AMPs might also induce sleep to promote recovery from injury in other organisms, including humans. Moreover, I laid the foundations for dissecting the molecular mechanisms behind the functions of sleep for healing and survival. Even though the disability to sleep did not seem to drastically change the transcriptional response to wounding, my results indicate a role for

    Mechanisms linking hypertriglyceridemia to acute pancreatitis

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    Hypertriglyceridemia (HTG) is a metabolic disorder, defined when serum or plasma triglyceride concentration (seTG) is >1.7 mM. HTG can be categorized as mild to very severe groups based on the seTG value. The risk of acute pancreatitis (AP), a serious disease with high mortality and without specific therapy, increases with the degree of HTG. Furthermore, even mild or moderate HTG aggravates AP initiated by other important etiological factors, including alcohol or bile stone. This review briefly summarizes the pathophysiology of HTG, the epidemiology of HTG-induced AP and the clinically observed effects of HTG on the outcomes of AP. Our main focus is to discuss the pathophysiological mechanisms linking HTG to AP. HTG is accompanied by an increased serum fatty acid (FA) concentration, and experimental results have demonstrated that these FAs have the most prominent role in causing the consequences of HTG during AP. FAs inhibit mitochondrial complexes in pancreatic acinar cells, induce pathological elevation of intracellular Ca2+ concentration, cytokine release and tissue injury, and reduce the function of pancreatic ducts. Furthermore, high FA concentrations can induce respiratory, kidney, and cardiovascular failure in AP. All these effects may contribute to the observed increased AP severity and frequent organ failure in patients. Importantly, experimental results suggest that the reduction of FA production by lipase inhibitors can open up new therapeutic options of AP. Overall, investigating the pathophysiology of HTG-induced AP or AP in the presence of HTG and determining possible treatments are needed
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