Untersuchungen zur Dynamik des Glukosestoffwechsels in Wistar und Zucker Diabetic Fatty Ratten : in vivo / ex vivo Studien zur Regulation des Pyruvatdehydrogenase-Komplexes

Gegenstand dieser Arbeit ist das Studium der Blutglukosehomöostase und die Regulation des PDH-Komplexes in der Ratte. Als Tiermodelle wurden die Wistar Ratte und die ZDF-Ratte, ein genetisches Tiermodell für die spontane Entwicklung des Typ 2 Diabetes, gewählt. Die Glukoseoxidation spielt eine entscheidende Rolle im Energiestoffwechsel. Der PDH-Komplex verbindet die Glykolyse mit dem Zitronensäure-Zyklus und fungiert als Schlüsselenzym im oxidativen Glukosemetabolismus. Hungerzustand und Diabetes verursachen einen Anstieg der PDK4-Expression in mehreren Geweben. Der Anstieg der PDK4-Proteinmenge ist nach einer Nahrungskarenz sehr wichtig, um die PDH-Aktivität zu senken und damit eine Hypoglykämie zu vermeiden. Im Diabetes jedoch ist diese Regulation nachteilig, da die Erhaltung glukoneogenetischer Substrate zur Hyperglykämie beiträgt. Anhand eines oGTT (2 g/kg KM) wurden bei insulinsensitiven, insulinresistenten und diabetischen Tieren die Anteile des oxidativen und nicht-oxidativen Glukosestoffwechsels bilanziert. Zur Untersuchung der Glukoseoxidation wurde als Methode die indirekte Kalorimetrie gewählt. Der prozentuale Anteil der KHO an der applizierten Glukosemenge betrug bei den 8 Wochen alten ZDF-Ratten 35% (45% obese) und bei dem 16 Wochen alten Kollektiv jeweils 34%. Der nicht-oxidative Fluss der Glukose in Leber und Muskel wurde über die Bestimmung von Glykogen und freier Glukose quantifiziert. Die Studien demonstrierten, dass die (Patho)physiologie im Leberstoffwechsel sowohl der schlanken als auch der obesen ZDF-Ratten nicht auf die Situation beim Mensch projiziert werden kann. Während bei schlanken Tieren die hepatische Glukoseaufnahme bei einer oralen Glukosebelastung eine minore Rolle spielte, speicherte die Leber der diabetischen Tiere paradoxerweise beträchtliche Mengen an Glykogen. In der Leber der 8 und 16 Wochen alten, obesen ZDF-Ratten wurden von der applizierten Glukosemenge 18% bzw. 25% als Glykogen gespeichert; bei den schlanken Tieren betrug dieser Anteil nur 1% bzw. 2%. Der als Muskelglykogen gespeicherte Anteil ergab bei den 8 Wochen alten Tieren 21% (schlank) bzw. 18% (obese) und bei den 16 Wochen alten Ratten 16% (schlank) bzw. 26% (obese). Zur Untersuchung der Regulation des PDH-Komplexes im Leber- und Muskelgewebe wurde ex vivo die PDK4, PDK2 und PDH-E1&#945;/&#946; Proteinmenge mittels Western-Blot-Analyse bestimmt und die PDH-Aktivität über die Produktion von 14CO2 aus [1-14C]-Pyruvat gemessen. Die Analysen der biochemischen Parameter sollten die Gemeinsamkeiten und Unterschiede in der Kurz- und Langzeitregulation des PDH-Komplexes bei der ZDF-Ratte, sowohl in Abhängigkeit der diabetischen Stoffwechsellage als auch nach akut angebotener Glukose aufdecken. Durch die Versuchsdaten konnte gezeigt werden, dass durch eine orale Glukosebelastung eine Reaktivierung der PDH-Aktivität binnen weniger Minuten möglich ist und dabei kurzfristig die Aktivitätswerte gefütterter Tiere überschritten werden können. Eine Stunde nach der Glukosebelastung war die spezifische PDH-Aktivität bei den 16 Wochen alten Tieren 3,6 (Leber) bzw. 4,6-fach (Muskel) höher als zu Beginn des Versuchs. Während des oGTT war die Änderung der PDH-Aktivität im Leber- und Muskelgewebe zwischen schlanken und obesen ZDF-Ratten nicht signifikant unterschiedlich. Aus den Ergebnissen der in vitro und in vivo Studien konnte somit gezeigt werden, dass weder die prädiabetische noch die manifest diabetische ZDF-Ratte einen Defekt im oxidativen Glukosestoffwechsel besitzt. Die totale PDH-Aktivität war bei den diabetischen ZDF-Ratten im Vergleich zu den schlanken Tieren signifikant erhöht. Die annähernd doppelt so hohe Proteinexpression von E1&#945;/&#946; in Leber und Muskel der obesen Tiere deutet darauf hin, dass die gesteigerte totale PDH-Aktivität obeser Tiere auf die erhöhten Proteinspiegel dieser Untereinheiten zurückzuführen ist. Durch die Western-Blot-Analyse konnte demonstriert werden, dass während des oGTT ein schneller Proteinabbau der PDK4 erfolgte. Die PDK4-Proteinspiegel waren, mit Ausnahme der Leber der obesen Tiere, ab der 2. Stunde nach der Glukosebelastung im Vergleich zu den Werten zu Versuchsbeginn signifikant erniedrigt. Daher muss die Degradation der PDK4 als neuer Mechanismus in der Kurzzeitkontrolle des PDH-Enzymkomplexes diskutiert werden. Sowohl die Proteinspiegel der PDK4 als auch der PDK2 waren bei den diabetischen ZDF-Ratten im Vergleich zu den schlanken Tieren signifikant erhöht. Die Ergebnisse der Proteinanalyse sprechen für eine additive Funktion der PDK2 zur PDK4, die in der Langzeitkontrolle des PDH-Komplexes als Hauptregulator angesehen wird. In pharmakologischen Experimenten wurde verdeutlicht, dass die Senkung der Blutglukose auf den Wirkmechanismus einer gesteigerten PDH-Aktivität zurückzuführen ist. Bei den diabetischen ZDF-Ratten bewirkte eine einmalige Applikation von DCA (500 mg/kg KM) eine Verdopplung der spezifischen PDH-Aktivität in Leber und Muskel. Bei Untersuchungen zur Hemmung der PDK am Tiermodell der ZDF-Ratte ist der Unterschied im Leberstoffwechsel zwischen schlanken und obesen Tieren zu beachten. Trotz einer Nahrungskarenz von 18 Stunden konnten die obesen Tiere über eine Mobilisierung der hohen Leberglykogenreserven (962 ± 121 mg/Leber) einen Blutglukoseabfall bis zu etwa sechs Stunden kompensieren, während bei insulinsensitiven Ratten ein gesteigerter Substratumsatz über die PDH unmittelbar in sinkenden Blutglukosespiegeln resultierte. Da PDK-Inhibitoren unter hungerähnlichen Stoffwechselbedingungen die Glukoseoxidation erhöhen, könnten diese Substanzen in Zukunft zu einer interessanten Perspektive werden, um die diabetische Blutglukosehomöostase zu verbessern.This study investigates the blood glucose homeostasis and the regulation of the PDC in the rat. The Wistar rat and the ZDF-rat, a genetic model for the spontaneous development of type 2 diabetes, were investigated. Glucose oxidation plays a major role in energy metabolism. The pyruvate dehydrogenase complex (PDC) links the glycolysis with the citric acid cycle and functions as a key enzyme in the oxidative glucose pathway. Starvation and diabetes cause an increase in PDK4 expression in various tissues. The increase in PDK4 expression is critically important in the starved state to prevent hypoglycemia. However, it is detrimental in diabetes because conservation of gluconeogenic substrates contributes to hyperglycemia. By means of an oGTT, the proportions of the oxidative and non-oxidative glucose metabolism were calculated in rats with a physiological responsiveness to insulin, in insulin resistant rats and in diabetic ones. Indirect calorimetry was chosen as a method to analyze glucose oxidation. The percentage of CHO in the 8 weeks old ZDF-rats was 35% (45% obese) of the administered glucose and in the 16 weeks old collective 34% in each case. The non-oxidative flux into liver and muscle was quantified by determination of free glucose and glycogen. The studies showed that the (patho)physiology in liver metabolism, both in lean and in obese ZDF-rats, cannot be transmitted to human subjects. In the diabetic animals the liver paradoxically stored large amounts of glycogen whereas in lean animals hepatic glucose uptake played obviously a minor role when an oral glucose load of 2 g per kg body weight was given. In the livers from the 8 and 16 weeks old obese ZDF-rats 18% and 25% were stored as glycogen, respectively; in the lean animals this share was only 1% and 2%, respectively. In liver and muscle tissue PDK4, PDK2 and PDC-E1&#945;/&#946; protein levels were determined by ex vivo western-blot-analysis. PDC activity was measured by production of 14CO2 from [1-14C]-pyruvate. The analyses of biochemical parameters should reveal the similarities and the differences in short- and long-term regulation of the PDC in the ZDF-rat, both the dependence on the diabetic condition and after an acute supply of glucose. The data show that PDC activity can be reactivated within few minutes by an oral glucose load even beyond the values of fed animals. One hour after the glucose load, the specific PDC-activity in 16 weeks old rats was 3.6 (liver) and 4.6-times (muscle) higher than before the test. During oGTT the changes in PDC activity in liver and muscle were not significantly different between lean and obese animals. Neither in the prediabetic nor in the overt diabetic ZDF-rat there was a defect in oxidative glucose metabolism indicated by the in vitro and in vivo studies results. The total PDC activity was significantly higher in the diabetic rats than in lean ones. Both, E1&#945; and &#946; protein levels in liver and muscle were elevated approximately twofold in obese animals. These data indicate that the increase of total activity in obese ZDF-rats is due to elevation of PDC E1&#945;/&#946; protein levels. The Western blot analysis demonstrates that during the oGTT a rapid protein breakdown happens. With the exception of the liver of the obese animals, PDK4 protein levels were significantly decreased in comparison to the levels at the beginning of the test two hours after glucose administration. Thus, this degradation has to be discussed as a new mechanism in short-term regulation of the PDC. Both PDK4 and PDK2 protein levels were significantly increased in diabetic rats compared to lean animals. The results from the protein analysis argue for an additive function of PDK2 for PDK4, which is considered to be the main regulator in long-term control of PDC in diabetic rats. In the pharmacological experiments it was shown that the lowering of blood glucose was originated by an increase in PDC activity. In diabetic ZDF-rats a single application of DCA (500 mg/kg) doubled the specific PDC activity in liver and muscle. In experiments of PDK inhibition in ZDF-rats, the difference in liver metabolism between lean and obese animals must be considered. Even after a fasting period of 18 hours, obese animals were able to compensate a decline in blood glucose for about six hours by mobilising its sizeable liver glycogen reserves (962 ± 121 mg/liver), whereas in insulin sensitive rats the outcome of an increased substrate turnover through PDC was an immediate decrease in blood glucose levels. Because PDK-inhibitors enhance glucose oxidation in starvation-like conditions, they might be an useful perspective to improve diabetic blood glucose homeostasis in future.<BR

Nano on reflection

A number of experts from different areas of nanotechnology describe how the field has evolved in the last ten years

Strategic Behavior in the German Balancing Energy Mechanism: Incentives, Evidence, Costs and Solutions

This paper investigates the incentives market participants have in the German electricity balancing mechanism. Strategic over- and undersupply positions are the result of existing stochastic arbitrage opportunities between the spot market and the balancing mechanism. This strategic behavior can be clearly identified in aggregate market data. These structural imbalances increase the need for reserve capacity, raise system security concerns, and thus burden significant cost on the customers. More effective market designs include changes in the balancing mechanism, the reserve capacity and the intraday spot markets

Temporal changes in clinical and radiographic variables in dogs with preclinical myxomatous mitral valve disease: The EPIC study

The Evaluation of pimobendan in dogs with cardiomegaly caused by preclinical myxomatous mitral valve disease (EPIC) study monitored dogs with myxomatous mitral valve disease (MMVD) as they developed congestive heart failure (CHF)

Structure elucidation, biosynthesis, total synthesis and antibacterial in-vivo efficacy of myxobacterial Corramycin

Herein, we describe the myxobacterial natural product Corramycin (1) isolated from Corallococcus coralloides (C. coralloides). Corramycin is a linear peptide containing an unprecedented (2R,3S) N-methyl-β-hydroxy histidine moiety and exhibiting anti-Gram-negative activity against Escherichia coli. Moreover, we describe the Corramycin biosynthetic gene cluster (BGC) and propose a biosynthesis model involving a 12-modular non-ribosomal peptide synthetase (NRPS)/polyketide synthase (PKS). Bioinformatic analysis of the BGC combined with the development of a total synthesis route allowed for the elucidation of the molecule’s absolute configuration. Furthermore, we show that the uptake of Corramycin in E. coli depends on two transporter systems, SbmA and YejABEF. Importantly, intravenous administration of 30 mg kg 1 of Corramycin in an E. coli mouse infection model resulted in significantly reduced colony forming units (CFU) and in 60 % survival of animals, with no toxic effects observed in vitro or in vivo. Corramycin is thus an intriguing innovative starting point to develop a potent antibacterial drug against hospital acquired infections

Temporal changes in clinical and radiographic variables in dogs with preclinical myxomatous mitral valve disease: The EPIC study

Background: The Evaluation of pimobendan in dogs with cardiomegaly caused by preclinical myxomatous mitral valve disease (EPIC) study monitored dogs with myxomatous mitral valve disease (MMVD) as they developed congestive heart failure (CHF). Objectives: To describe the changes in clinical and radiographic variables occurring as dogs with MMVD and cardiomegaly develop CHF, compared to similar dogs that do not develop CHF. Animals: One hundred and thirty-five, and 73 dogs that did or did not develop CHF, respectively. Materials and methods: The following variables were evaluated in 2 groups of dogs (dogs that did or did not develop CHF): Heart rate (HR), clinic respiratory rate (RR), home-measured resting respiratory rate (RRR), rectal temperature (RT), body weight (BW), and vertebral heart sum (VHS). Absolute value and rate of change of each variable were calculated for each day a dog was in study. Daily means were calculated and plotted against time. The onset of CHF or last visit before leaving the study were set as reference time points. Results: The most extreme values and rate of change occurred in variables immediately before onset of CHF. Vertebral heart sum increased earliest. Heart rate, RR, and RRR also increased. Rectal temperature and BW decreased. Increases in RR and RRR were most extreme and occurred immediately before CHF. Conclusions and Clinical Importance: Dogs with MMVD and cardiomegaly experience increases in HR, RR, RRR, and VHS, and decreases in BW and RT as they develop CHF. The variables with highest absolute change and rate of change were RR and RRR. These findings reinforce the value of RR and RRR as indicators of impending or incipient CHF

Temporal changes in clinical and radiographic variables in dogs with preclinical myxomatous mitral valve disease: The EPIC study

Background: The Evaluation of pimobendan in dogs with cardiomegaly caused by preclinical myxomatous mitral valve disease (EPIC) study monitored dogs with myxomatous mitral valve disease (MMVD) as they developed congestive heart failure (CHF). Objectives: To describe the changes in clinical and radiographic variables occurring as dogs with MMVD and cardiomegaly develop CHF, compared to similar dogs that do not develop CHF. Animals: One hundred and thirty-five, and 73 dogs that did or did not develop CHF, respectively. Materials and methods: The following variables were evaluated in 2 groups of dogs (dogs that did or did not develop CHF): Heart rate (HR), clinic respiratory rate (RR), home-measured resting respiratory rate (RRR), rectal temperature (RT), body weight (BW), and vertebral heart sum (VHS). Absolute value and rate of change of each variable were calculated for each day a dog was in study. Daily means were calculated and plotted against time. The onset of CHF or last visit before leaving the study were set as reference time points. Results: The most extreme values and rate of change occurred in variables immediately before onset of CHF. Vertebral heart sum increased earliest. Heart rate, RR, and RRR also increased. Rectal temperature and BW decreased. Increases in RR and RRR were most extreme and occurred immediately before CHF. Conclusions and Clinical Importance: Dogs with MMVD and cardiomegaly experience increases in HR, RR, RRR, and VHS, and decreases in BW and RT as they develop CHF. The variables with highest absolute change and rate of change were RR and RRR. These findings reinforce the value of RR and RRR as indicators of impending or incipient CHF