Effects of endotoxin induced inflammation on glucose metabolism and contractility of the rat heart in vivo and in vitro [Elektronische Ressource] / vorgelegt von Jean-Philippe Tessier

Cristoph Becker

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2003
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Effects of endotoxin-induced inflammation on
glucose metabolism and contractility
of the rat heart
in vivo and in vitro
Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der
Rheinisch-Westfälischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades eines Doktors der
Naturwissenschaften genehmigte Dissertation
vorgelegt von
Diplom-Biologe
Jean-Philippe Tessier
aus
Nantes (Frankreich)
Berichter: Universitätsprofessor Dr. rer. nat. Fritz Kreuzaler
Professor Dr. rer. nat. Yvan Fischer
Tag der mündlichen Prüfung: 28. März 2003
Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online
verfügbar.Part of the work described in this thesis has been submitted for publication
and currently in press:
Cardiovasc. Res. (2003): Impairment of glucose metabolism in hearts from rats
treated with endotoxin Tessier J-P., Thurner B., Jüngling, E., Lückhoff A., and
Fischer Y.Table of contents
I Introduction ........................................................................................................................1
I.1 Endotoxin and inflammation .............................................................................................1
I.2 Cytokines and cardiac contractile function .......................................................................2
I.3 Cytokines and the metabolism of glucose.........................................................................3
I.4 Aims and organisation of the thesis...................................................................................5
II Materials and methods ...........................................................................................................7
II.1 Materials......7
II.1.1 Chemicals and consumables.....................................................................................7
II.1.2 Experimental animals...............................................................................................8
II.1.3 Equipment ...............................................................................................................8
II.2 Methods ........................................................................................................................9
II.2.1 Experiments with isolated hearts .............................................................................9
II.2.1.1 Experimental model .......................................................................................................................... 9
II.2.1.2 Rat blood analyses....................... 10
II.2.1.2.1 Plasma insulin measurements................................................................................................. 10
II.2.1.2.2 Cortisol measurements............ 10
II.2.1.2.3 Lactate.................................................................................................................................... 11
II.2.1.2.4 Glucose...................... 11
II.2.1.2.5 Non esterified fatty acids........................................................................................................ 12
II.2.1.2.6 Partial thromboplastin (PTT) time.......................................................................................... 12
II.2.1.2.7 Blood picture.......................................................................................................................... 13
II.2.1.3 Isolated heart procedure .................................................................................................................. 13
II.2.1.4 Experimental setup and heart perfusion .......................................................................................... 14
II.2.1.5 Assessment of cardiac function ....................................................................................................... 16
II.2.1.6 Experimental design of experiments with isolated hearts................................................................17
II.2.1.7 Cardiac metabolism......................................................................................................................... 18
II.2.1.7.1 Glucose uptake and glycolysis ............................................................................................... 18
II.2.1.7.2 pH and Osmolarity ................................................................................................................. 21
II.2.1.7.3 Glucose consumption and lactate formation .......................................................................... 21
II.2.1.8 Action of isoproterenol in isolated hearts........................................................................................ 21
II.2.1.9 Direct effects of LPS on the contractility of isolated hearts ............................................................ 21
II.2.1.10 Nitrite release in recycling perfusion............................................................................................. 22
II.2.1.11 Myocardial inducible NO synthase (iNOS) expression................................................................. 22
II.2.1.12 Preparation of heart extracts for metabolite measurements........................................................... 25
II.2.1.13 Cardiac content of high energy phosphates....... 26
II.2.1.14 Cardiac glycogen content .............................................................................................................. 26
II.2.1.15 Cardiac citrate content................................................................................................................... 27
II.2.2 Experiments with isolated cardiomyocytes............................................................28
II.2.2.1 Glucose metabolism ........................................................................................................................ 28
II.2.2.1.1 Cell isolation ...................................................................................................... 28
II.2.2.1.2 Protein assay .......................................................................................................................... 30
II.2.2.1.3 Gylcolytic measurement........... 31
II.2.2.1.4 Effect of LPS incubation in vitro on glycolysis ..................................................................... 32
II.2.2.2 Patch-clamp analyses ...................................................................................................................... 32
II.2.2.2.1 Patch clamp setup................................................................................................................... 32
II.2.2.2.2 Cell isolation.................... 35
II.2.2.2.3 Cell treatment................... 37
II.2.2.2.4 Measurements of currents through calcium channels............................................................. 38
II.3 Statistical analysis....40III Results ......................................................................................................................41
III.1 Model validation..............................................................................................................41
III.1.1 Rectal temperature .................................................................................................41
III.1.2 Animal behaviour...................................................................................................42
III.1.3 Blood analyses .......................................................................................................42
III.1.4 Cardiodepression ex vivo.......................................................................................44
III.1.5 pH and osmolarity ..................................................................................................46
III.1.6 Glucose consumption and lactate formation ..........................................................46
III.2 Effect of LPS on cardiac glycolysis and glucose uptake.................................................48
III.2.1 With glucose as sole substrate................................................................................48
III.2.2 In the presence of 2 substrates (glucose + β-hydroxybutyric acid) .......................49
III.2.3 Influence of LPS on cardiac citrate content ...........................................................51
III.2.4 Influence of LPS on cardiac glycogen content ......................................................53
III.2.5 Signals mediating the LPS effects on cardiac glycolysis.......................................54
III.2.5.1Role of nitric oxide....................... 54
III.2.5.1.1 Inhibitors of NOS................ 55
III.2.5.1.2 Cardiac expression of inducible NOS... 56
III.2.5.1.3 Cardiac nitrite production ..................................................................................................... 56
III.2.5.2 Role of sphingomyelin pathway..................................................................................................... 57
III.2.5.3 Role of cyclooxygenase-derived metabolites................................................................................. 58
III.2.5.3.1 Cyclooxygenase-2................................................................................................................. 58
III.2.5.3.2 Thromboxane A2 .................................................................................................................. 59
III.2.6 Metabolic changes induced in myocytes after LPS treatment ...............................60