Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice

Obesity: <i>In vivo</i> expression studies about the obesity factor <i>DOR</i> and studies of translational control in early adipogenesis

In der vorliegenden Arbeit wurden Grundlagen zur Entstehung einer Adipositas erforscht. Dies erfolgte über Expressionsstudien des Adipositasfaktors DORim Tiermodell sowie durch eine Studie zur Translationskontrolle in der frühen Adipogenese im Zellmodell. Im ersten Teil der Arbeit wurden in vivo Expressionsstudien des Adipositas Faktors DOR mittels q-PCR durchgeführt. Dafür wurden Fett- und Muskelgewebe von fettreich gefütterten Tieren oder genetisch dicken Mäusen sowie entsprechender Kontrolltiere verwendet. Um einen Einfluss von fettreicher Nahrung auf die DOR-Expression zu untersuchen wurden die Tiere für eine Woche mit einer Diät gefüttert, welche einen Fettanteil von 18% oder 80% enthielt. Bei den Mäusen mit einer genetisch bedingten Adipositas wurde die DOR-Expression in zwei verschiedenen Altersstufen, 45 und 100 Tage nach der Geburt, gemessen. Veränderungen in der DOR-Expression zwischen fettreich gefütterten Mäusen und Kontrolltieren wurden durch die Höhe des Fettgehaltes der Diät, den untersuchten Gewebetyp, sowie das Geschlecht der Tiere beeinflusst. Bei den Mäusen mit genetisch bedingter Adipositas wurde ebenfalls ein Einfluss des Geschlechtes der untersuchten Tiere auf die DOR expression beobachtet. Zwischen den zwei untersuchten Altersgruppen konnten keine Unterschiede in der Expression von DORfestgestellt werden. Möglicherweise ist DORTeil eines Schutzmechanismus gegen eine vermehrte Einlagerung von Körperfett bei fettreichen Diäten. Für den zweiten Teil der Arbeit wurden Veränderungen in der Translationsrate in 3T3-L1 Zellen in der frühen Adipogenese, 6 Stunden nach hormoneller Induktion, bestimmt. mRNAs, die nicht mit Ribosomen besetzt sind (freie RNAs) werden nicht translatiert und haben eine geringere Dichte als mRNAs, welche mit Ribosomen besetzt sind (polysomale mRNAs) und folglich translatiert werden. Die Auftrennung der mRNAs aus 3T3-L1 Zell-Lysat erfolgte über eine Dichtegradientenzentrifugation. Durch eine anschließende Microarray-Analyse wurde die Identifikation polysomaler mRNAs ermöglicht. Es wurden 43 Gene identifiziert, deren Translationsrate in der frühen Adipogenese heraufreguliert wurde, während sie bei zwei Genen herabreguliert war. Diese alternativ regulierten mRNAs sind involviert in die Kontrolle von Zellzyklus, Transkription und Translation. Zu den heraufregulierten Faktoren gehören der Translations-Initiationsfaktor eIF4B und eine große Anzahl an ribosomalen Proteinen. Diese ermöglichen eine Modifikation der Translationsrate durch eine Veränderung der Translationsaktivität. Das erlaubt die Schlussfolgerung, dass eine moderat

Comparison of hemostatic dressings for superficial wounds using a new spectrophotometric coagulation assay

Background: Due to demographical changes the number of elderly patients depending on oral anticoagulation is expected to rise. Prolonged bleeding times in case of traumatic injuries represent the drawback of these medications, not only in major trauma, but also in superficial wounds. Therefore, dressings capable of accelerating coagulation onset and shortening bleeding times are desirable for these patients. Methods: The hemostatic potential and physical properties of different types of superficial wound dressings (standard wound pad, two alginates, chitosan, collagen (Lyostypt (R)), oxidized cellulose, and QuikClot (R)) were assessed in vitro. For this purpose the clotting times of blood under the influence of the named hemostatics from healthy volunteers were compared with Marcumar (R) or ASS (R) treated patients. For that, a newly developed coagulation assay based on spectrophotometric extinction measurements of thrombin activity was used. Results: The fastest coagulation onset was observed for oxidized cellulos (empty set 2.47 min), Lantor alginate-L (empty set 2.50 min) and QuikClot (R) (empty set 3.01 min). Chitosan (empty set 5.32 min) and the collagen Lyostypt (R) (empty set 7.59 min) induced clotting comparatively late. Regarding physical parameters, QuikClot (R) showed the lowest absorption capacity and speed while chitosan and both alginates achieved the highest. While oxidized cellulose displayed the best clotting times, unfortunately it also revealed low absorption capacity. Conclusions: All tested specimens seem to induce clotting independently from the administered type of oral anticoagulant, providing the possibility to neglect the disadvantage in clotting times arising from anticoagulation on a local basis. QuikClot (R), oxidized cellulose and unexpectedly alginate-L were superior to chitosan and Lyostypt (R). Due to its additional well-known positive effect on wound healing alginate-L should be considered for further investigations

Extrinsic and intrinsic regulation of DOR/TP53INP2 expression in mice: effects of dietary fat content, tissue type and sex in adipose and muscle tissues

BACKGROUND: DOR/TP53INP2 acts both at the chromosomal level as a nuclear co-factor e.g. for the thyroid hormone receptor and at the extrachromosomal level as an organizing factor of the autophagosome. In a previous study, DOR was shown to be down-regulated in skeletal muscle of obese diabetic Zucker fa/fa rats. METHODS: To identify sites of differential DOR expression in metabolically active tissues, we measured differences in DOR expression in white adipose tissue (WAT), brown adipose tissue (BAT), skeletal muscle (SM) and heart muscle (HM) by qPCR. To assess whether DOR expression is influenced in the short term by nutritional factors, NMRI mice were fed different fat rich diets (fat diet, FD: 18% or high fat diet, HFD: 80% fat) for one week and DOR expression was compared to NMRI mice fed a control diet (normal diet, ND: 3.3% fat). Additionally, DOR expression was measured in young (45 days old) and adult (100 days old) genetically obese (DU6/DU6i) mice and compared to control (DUKs/DUKsi) animals. RESULTS: ANOVA results demonstrate a significant influence of diet, tissue type and sex on DOR expression in adipose and muscle tissues of FD and HFD mice. In SM, DOR expression was higher in HFD than in FD male mice. In WAT, DOR expression was increased compared to BAT in male FD and HFD mice. In contrast, expression levels in female mice were higher in BAT for both dietary conditions. DOR expression levels in all tissues of 100 days old genetically obese animals were mainly influenced by sex. In HM, DOR expression was higher in male than female animals. CONCLUSIONS: DOR expression varies under the influence of dietary fat content, tissue type and sex. We identified target tissues for further studies to analyze the specific function of DOR in obesity. DOR might be part of a defense mechanism against fat storage in high fat diets or obesity