Untersuchungen des Leptin-Systems und des Stoffwechsels juveniler Ratten : Zwei Adipositas-Modelle

Endogene, funktionelle Teratogene wie auch chemische Noxen können in kritischen Entwicklungsperioden zur Zerstörung zentraler Regler und damit zu lebenslang anhaltenden Fehlfunktionen endokriner Regelungsprozesse führen. In der vorliegenden Studie wurden die akuten Änderungen in der Energiebilanz während der Säugephase an zwei unterschiedlichen Modellen untersucht, die durch Eingriffe in den ersten Lebenstagen Adipositas im adulten Tier bewirken. Im ersten Adipositasmodell wurde eine postnatale Überernährung (PNO) 21 Tage alter Rattenwelpen durch die Aufzucht in einem Nest mit nur vier Tieren, gegenüber zwölf Tieren in normal großen Nestern, induziert, wobei neben Wistarratten entweder Zuckerratten, die heterozygot für einen Defekt des Leptinrezeptors sind (+/fa), oder deren Wildtyp-Wurfgeschwister (+/+) untersucht wurden. Bei allen PNO-Tieren ergab sich gegenüber den im normalen Nest aufgezogenen Kontrolltieren sowohl eine größere fettfreie Trockenmasse und Fettmasse, wobei die normalerweise phänotypisch kaum ausgeprägte genetische Prädisposition der +/fa-Tiere zu vermehrter Fettakkumulation bei den PNO-Tieren erheblich verstärkt wurde. Neben Zunahmen der Körperfettmasse entwickelten die +/fa-PNO-Tiere, aber auch die PNO-Wistarratten, eine relative Hyperleptinämie. Diese war mit einer Leptinresistenz assoziiert, wie sich in einer nahezu vollständigen Insensitivität insbesondere der PNO-Wistarratten und der +/fa-PNO-Tiere gegenüber exogen appliziertem Leptin zeigte. Eine Bestimmung der hypothalamischen Leptinrezeptorbindung an Hypothalamus-Homogenaten verdeutlichte, dass diese weder bei den PNO-Tieren noch durch Leptinbehandlung im Vergleich zu den Kontrolltieren verändert wurde. Unabhängig davon wiesen zum einen +/fa- und zum anderen weibliche Tiere eine verminderte Leptinrezeptorbindung auf. Diese Befunde am Untersuchungsmodell I zeigen, dass eine erhöhte Energiezufuhr im Säuglingsalter nicht nur zu einem verstärktem Wachstum, sondern auch zu einer exzessiven Fettdeposition führt. Zusätzlich konnte eine auffallende Interaktion zwischen einem definierten, normalerweise rezessiv eingestuften Merkmal und frühen postnatalen Unwelteinflüssen gezeigt werden, die modellhaft für die Programmierung der phänotypischen Ausprägung genetischer Störungen sein könnte. Unabhängig vom genetischen Hintergrund tritt dabei eine Leptinresistenz als Leitsymptom auf, die jedoch nicht auf einer Änderung der Rezeptorendichte beruht. Eine analoge Beziehung zwischen genetischem Hintergrund undÜberernährung wäre auch bei der Wistarratte zu vermuten, ist aber noch nicht identifiziert worden. Grundlage des zweiten Adipositasmodelles ist die chemische Läsionierung von Nervenzellkörpern durch Verabreichung von Monosodiumglutamat (MSG), das in hypothalamischen Gebieten mit durchlässiger Blut-Hirnschranke wirksam wird. Die postnatale MSG-Behandlung führte bei den MSG-behandelten Tiere zu einer geringeren Fettdeposition gegenüber den NaCl-behandelten Kontrolltieren. Bei identischer Nahrungszufuhr künstlich über intraösophageale Katheter aufgezogener Tiere kam es zu einem nahezu vollständigen Verschwinden der Stoffwechselabsenkung im Minimum der Tiere und einer erhöhten Energiedissipation der MSG-behandelten Tiere. Auch die künstlich aufgezogenen Tiere zeigten eine starke Abnahme besonders des Körperfettgehaltes, was die Änderungen in der Energiebilanz der Tiere als Ursache bestätigt. Mit dem verminderten Körperfettgehalt ging auch eine Abnahme der Plasmaleptinspiegel einher. Um die Auswirkungen der MSG-Behandlung auf die für die Energiehomöostase wichtigen hypothalamischen Kerngebiete insbesondere des Nucleus Arcuatus (ARC) zu untersuchen wurden kresylviolett-gefärbte coronale Gehirnschnitte mikroskopisch untersucht. Während andere Kerngebiete nicht von der chemischen Läsionierung durch MSG betroffen waren, trat eine weitgehende Zerstörung hypothalamischer Neurone im ARC ein. Zudem wurde zur Funktionskontrolle am zehn Tage alten Rattenhirn die Reagibilität auf Leptin anhand des Nachweises eines \u27immediate-early-gene\u27 Produkts, des Fos Proteins, als Marker für neuronale Aktivierung nachgewiesen und die Verteilung des durch das Neuropeptid Y (NPY) als Transmitter gekennzeichneten, hirn-intrinsischen Neuronensystems, dessen Beitrag zur Stimulation der Nahrungsaufnahme bekannt ist, trotz der neuronalen Unreife der juvenilen Tiere nachgewiesen. Die Befunde des Adipositasmodells II zeigen, dass der MSG-induzierten Adipositas im Erwachsenenalter eine Phase erhöhten Energieumsatzes vorausgeht, die in ihrer Wirkung auf den circadianen Rhythmus der einer Leptinapplikation bei der juvenilen Ratte vergleichbar ist und vor allem zu einer Verringerung des Körperfettgehaltes führt. Bei der Deutung dieses Befundes sind die histochemischen Hinweise zu beachten, dass normalerweise bereits im Alter von zehn Tagen eine Fortleitung des Leptinsignals zum Nucleus paraventricularis als integrative Struktur für die Kontrolle der Energiebilanz existiert und dass insbesondere das an dieser Verbindung beteiligte NPY-System entwickelt ist. Der beobachtete Zelluntergang im ARC wirkt sich akut wie eine durch Leptin hervorgerufene Enthemmung der normalerweise wirksamen Maßnahmen der juvenilen Energieeinsparung im Tagesminimum aus. Zusammenfassend läßt sich feststellen, dass zwei völlig unterschiedliche Einflüsse, nämlich eine chemische Noxe, genauso wie eine postnatale Überernährung in einer kritischen juvenilen Entwicklungsphase, obwohl sie zunächst gegenläufige Veränderung der Fettdeposition auslösen, schließlich zu Adipositas und deren assoziierten Begleitstörungen im adulten Tier führen können.Endogenous as well as chemical functional teratogens, if administered in critical periods of neuronal development, may destroy central regulatory mechanisms and therefore lead to lifelong-lasting dysfunctions of endocrine regulatory loops. In this study acute changes in energy balance during suckling period has been examined in two rat obesity models, which are known to induce an obese phenotype in the adult animal. In the first obesity model a postnatal overnutrition (PNO) has been induced by rearing pups in small litters of only 4 animals (small litter size) compared to pups reared under control conditions with 12 pups (normal litter size). Besides Wistar rats either Zucker rats, which are heterozygous for a leptin receptor defect \u27fatty\u27 (+/fa), or wildtyp Zucker rats have been used in this study. All PNO-animal showed an enhanced fat-free dry-mass (FFDM) as well as an increased body-fat mass compared to their control littermates, which had been reared in litters of normal size. The enlarged fat deposition of heterozygous animals which is normally hardly detectable by phenotype is massively enhanced by the postnatal overnutrition. Besides the increase in body fat mass the +/fa-PNO as well as the PNO-Wistar rats developed a relative hyperleptinaemia. This was associated with a leptin resistance, which was expressed in a nearly total insensitivity exspecially of the PNO-Wistar- and PNO-+/fa-Zucker rats against peripheral leptin treatment. The investigation of hypothalamic leptin receptor binding in Zucker rats revealed that neither PNO- nor leptin-treated animals had a different binding compared to the controll animals. Independent of these findings, a diminished leptin receptor binding of heterozygous and female animals could be detected. These results of the first obesity model show that a higher energy supply in postnatal life not only contributes to a higher body weight but exspecially supports an exaggerated fat deposition. Additionally, this study demonstrated an obvious interaction between an usually inconspicous genetic trait and early postnatal enviromental influences, which could be exemplary for the programming of phenotypical expression of genetic disorders. Independant of genetic background, a leptin resistance as a lead-symptom develops, which is not caused by an altered expression of the leptin receptor. In the second obesity model monosodiumglutamate (MSG) in high doses has been used to lesion neuronal cell bodies in specific hypothalamic areas without a blood-brain-barrier. Postnatal MSG-treatment causes a diminshed fat-deposition of the treated suckling-age animals compared to the control (NaCl-treated) animals. Artificial rearing which ensures asupply with identical amounts of milk via intraosophageal catheters revealed a nearly complete suppression of the circadian torpor-like decreases of Tc and MR in the daily minimum and therefore lead to a higher energy dissipation of the MSG-treated animals. The artificially reared animals showed also a diminished fat mass compared to the control animals in accordance with the observed changes in energy metabolism which thus were causal for the reduction of body weight. To study the effect of MSG treatment on hypothalamic nuclei, which are known to be involved in regulation of energy homeostasis, cresyl-violet stained brain sections have been examined. A marked loss of cells in the arcuate nucleus (ARC) could be detected while other brain regions were not affected by the treatment. Additionally, to test whether those brain regions are already functional at an age of ten days, the expression of the Fos protein, an immediate early-gene-product, which is known as a marker for neuronal activity, could be demonstrated. Despite the neuronal immaturity of ten day old rat pups the NPY-ergic projections, which have been shown to be essential for signal transmission in the control of food intake, already exist. The results show that MSG-induced obesity, which develops in adulthood, is preceded by a period of expanded energy expenditure in juvenile life. With respect to their effects on energy metabolism leptin treatment and MSG lesioning of juvenile pups are comparable and lead mainly to a diminished fat deposition. For the interpretation of these findings one has to keep in mind the histological results, which revealed that even at this early age the transmission of the leptin signal to the nucleus paraventricularis is already possible, because the NPY-ergic connectivities are already existent. The neuronal cell death in the ARC leads to an leptin-like disinhibition of normal energy saving mechanisms in the minimum of the daily circadian rhythm in 10 day old pups. Summarizing the results, this study shows that two totally different influences - postnatal overnutrition or a chemical teratogene - if administered in periods that are critical for developing organisms, can induce changes in energy metabolism and nutritional state of animals which may lead to a higher obesity susceptibility in adulthood

Inter-rater reliability, sensitivity to change and responsiveness of the orthopaedic Wolf-Motor-Function-Test as functional capacity measure before and after rehabilitation in patients with proximal humeral fractures

Background: The incidence of proximal humeral fractures (PHF) increased by more than 30% over the last decade, which is accompanied by an increased number of operations. However, the evidence on operative vs. non-operative treatment and post-operative treatments is limited and mostly based on expert opinion. It is mandatory to objectively assess functional capacity to compare different treatments. Clinical tools should be valid, reliable and sensitive to change assessing functional capacity after PHFs. This study aimed to analyse inter-rater reliability of the videotaped Wolf-Motor-Function-Test-Orthopaedic (WMFT-O) and the association between the clinical WMFT-O and the Disability of the Arm, Shoulder and Hand (DASH) and to determine the sensitivity to change of the WMFT-O and the DASH to measure functional capacity before and after rehabilitation in PHF patients. Methods: Fifty-six patients (61.7 ± 14.7 years) after surgical treatment of PHF were assessed using the WMFT-O at two different time points. To determine inter-rater reliability, the videotaped WMFT-O was evaluated through three blinded raters. Inter-rater agreement was determined by Fleiss’ Kappa statistics. Pearson correlation coefficients were calculated to assess the association between the clinical WMFT-O and the video rating as well as the DASH. Sensitivity to change and responsiveness were analysed for the WMFT-O and the DASH in a subsample of forty patients (53.8 ± 1.4 years) who were assessed before and after a three week robotic-assisted training intervention. Results: Inter-rater agreement was indicated by Fleiss’ Kappa values ranging from 0.33–0.66 for functional capacity and from 0.27–0.54 for quality of movement. The correlation between the clinical WMFT-O and the video rating was higher than 0.77. The correlation between the clinical WMFT-O and the DASH was weak. Sensitivity to change was high for the WMFT-O and the DASH and responsiveness was given. In comparison to the DASH, the sensitivity to change of the WMFT-O was higher. Conclusion: The overall results indicate that the WMFT-O is a reliable, sensitive and responsive instrument to measure more objectively functional change over time in rehabilitation after PHF. Furthermore, it has been shown that video assessment is eligible for studies to ensure a full blinding of raters. Trial registration: Clinicaltrials.gov, NCT03100201. Registered on 28 March 2017. The trial was retrospectively registered

Performance of non-invasive tests and histology for the prediction of clinical outcomes in patients with non-alcoholic fatty liver disease: an individual participant data meta-analysis

BackgroundHistologically assessed liver fibrosis stage has prognostic significance in patients with non-alcoholic fatty liver disease (NAFLD) and is accepted as a surrogate endpoint in clinical trials for non-cirrhotic NAFLD. Our aim was to compare the prognostic performance of non-invasive tests with liver histology in patients with NAFLD.MethodsThis was an individual participant data meta-analysis of the prognostic performance of histologically assessed fibrosis stage (F0–4), liver stiffness measured by vibration-controlled transient elastography (LSM-VCTE), fibrosis-4 index (FIB-4), and NAFLD fibrosis score (NFS) in patients with NAFLD. The literature was searched for a previously published systematic review on the diagnostic accuracy of imaging and simple non-invasive tests and updated to Jan 12, 2022 for this study. Studies were identified through PubMed/MEDLINE, EMBASE, and CENTRAL, and authors were contacted for individual participant data, including outcome data, with a minimum of 12 months of follow-up. The primary outcome was a composite endpoint of all-cause mortality, hepatocellular carcinoma, liver transplantation, or cirrhosis complications (ie, ascites, variceal bleeding, hepatic encephalopathy, or progression to a MELD score ≥15). We calculated aggregated survival curves for trichotomised groups and compared them using stratified log-rank tests (histology: F0–2 vs F3 vs F4; LSM: 2·67; NFS: 0·676), calculated areas under the time-dependent receiver operating characteristic curves (tAUC), and performed Cox proportional-hazards regression to adjust for confounding. This study was registered with PROSPERO, CRD42022312226.FindingsOf 65 eligible studies, we included data on 2518 patients with biopsy-proven NAFLD from 25 studies (1126 [44·7%] were female, median age was 54 years [IQR 44–63), and 1161 [46·1%] had type 2 diabetes). After a median follow-up of 57 months [IQR 33–91], the composite endpoint was observed in 145 (5·8%) patients. Stratified log-rank tests showed significant differences between the trichotomised patient groups (p<0·0001 for all comparisons). The tAUC at 5 years were 0·72 (95% CI 0·62–0·81) for histology, 0·76 (0·70–0·83) for LSM-VCTE, 0·74 (0·64–0·82) for FIB-4, and 0·70 (0·63–0·80) for NFS. All index tests were significant predictors of the primary outcome after adjustment for confounders in the Cox regression.InterpretationSimple non-invasive tests performed as well as histologically assessed fibrosis in predicting clinical outcomes in patients with NAFLD and could be considered as alternatives to liver biopsy in some cases

Machine learning algorithm improves the detection of NASH (NAS-based) and at-risk NASH: A development and validation study

Background and aims: Detecting NASH remains challenging, while at-risk NASH (steatohepatitis and F≥ 2) tends to progress and is of interest for drug development and clinical application. We developed prediction models by supervised machine learning techniques, with clinical data and biomarkers to stage and grade patients with NAFLD. Approach and results: Learning data were collected in the Liver Investigation: Testing Marker Utility in Steatohepatitis metacohort (966 biopsy-proven NAFLD adults), staged and graded according to NASH CRN. Conditions of interest were the clinical trial definition of NASH (NAS ≥ 4;53%), at-risk NASH (NASH with F ≥ 2;35%), significant (F ≥ 2;47%), and advanced fibrosis (F ≥ 3;28%). Thirty-five predictors were included. Missing data were handled by multiple imputations. Data were randomly split into training/validation (75/25) sets. A gradient boosting machine was applied to develop 2 models for each condition: clinical versus extended (clinical and biomarkers). Two variants of the NASH and at-risk NASH models were constructed: direct and composite models.Clinical gradient boosting machine models for steatosis/inflammation/ballooning had AUCs of 0.94/0.79/0.72. There were no improvements when biomarkers were included. The direct NASH model produced AUCs (clinical/extended) of 0.61/0.65. The composite NASH model performed significantly better (0.71) for both variants. The composite at-risk NASH model had an AUC of 0.83 (clinical and extended), an improvement over the direct model. Significant fibrosis models had AUCs (clinical/extended) of 0.76/0.78. The extended advanced fibrosis model (0.86) performed significantly better than the clinical version (0.82). Conclusions: Detection of NASH and at-risk NASH can be improved by constructing independent machine learning models for each component, using only clinical predictors. Adding biomarkers only improved the accuracy of fibrosis