Tumoren weisen oftmals einen veränderten Energiestoffwechsel auf, der durch den Begriff „Warburg-Effekt“ bzw. „aerobe Glykolyse“ charakterisiert ist. Hierunter wird die Stoffwechselsituation verstanden, auch in Gegenwart von Sauerstoff aus Glukose Laktat zu bilden. Benigne Zellen zeigen diesen Effekt in aller Regel nicht so stark wie Tumorzellen. In dieser Arbeit wurden neun Tumorzelllinien, die von unterschiedlichen humanen Tumoren etabliert wurden, hinsichtlich ihres Energiestoffwechsels untersucht. Da der Warburg-Effekt auf einem erhöhten Glukosemetabolismus beruht, wurde die Stärke der Glukoseaufnahme und Laktatbildung für die neun Tumorzelllinien bestimmt. Wesentliches Ziel dieser Arbeit war, einen Zusammenhang zwischen dem Warburg-Effekt und weiteren Eigenschaften der Tumorzellen wie ihrem Oberflächenprofil bzw. ihrer Malignität nachzuweisen. Zur Phänotypisierung wurden die Oberflächenmoleküle CD44 und CD24 ausgewählt, da sie Zellinteraktionen, Zelladhäsion und Zellmigration von Tumorzellen vermitteln. Die Malignität wurde im Xenograft-Modell überprüft. Hierzu wurden Tumorzellen unter die Haut immuninkompetenter Mäuse injiziert und anschließend die Wachstumsgeschwindigkeit der entstehenden, soliden Tumoren gemessen. Die Menge an produziertem Laktat durch Tumorzellen war eindeutig von der Anwesenheit von Glukose im Nährmedium abhängig. Insgesamt waren fünf der neun Tumorzelllinien starke Laktatbildner, was einer Laktatbildung von über 2,5 mmol/L innerhalb von 48 Stunden entspricht. Hierzu gehört u.a. die Mammakarzinomzelllinie MDAMB-231, wohingegen MCF-7, eine andere Mammakarzinomzelllinie, nur wenig Laktat bildete. Im Gegensatz zu MDAMB-231 wuchs MCF-7 zudem auch nicht im Xenograft-Modell. Dies scheint ein Hinweis darauf zu sein, dass starke Laktatbildner aggressiver, d.h. schneller wachsen, als schwache Laktatbildner. Darüber hinaus wurde in dieser Arbeit eine temperaturabhängige Aufnahme des Glukoseanalogons 2-NBDG beo-bachtet. Auch hier fiel die Tumorzelllinie MCF-7 im Vergleich zur Tumorzelllinie MDAMB-231 durch eine geringere Aufnahme von 2-NBDG auf. Dies wird als Hinweis darauf gedeutet, dass MCF-7 Glukose in erster Linie in den Mitochondrien veratmet, anstatt sie zu Laktat zu vergären. Die beiden Glukosetransporter GLUT-1 und GLUT-3 wurden hingegen auf den Zellen aller neun Tumorzelllinien gleichermaßen nachgewiesen. Lediglich auf den Zellen der Tumorzelllinien HT-29 und MDAMB-468 war GLUT-3 schwach exprimiert. Sechs der neun Tumorzelllinien weisen den so genannten „Phänotyp 1“ auf. Hierunter wird in dieser Arbeit die gemeinsame Expression von CD44 und CD24 auf der Zelloberfläche verstanden. Zwei Zelllinien waren ausschließlich positiv für CD44 („Phänotyp 2“), eine Zelllinie ausschließlich positiv für CD24 („Phänotyp 3“). Keiner dieser Phänotypen wies eine charakteristische Glukoseaufnahme bzw. Laktatbildung auf, auch wenn drei der fünf Zelllinien mit besonders starker Laktatbildung zum Phänotyp 2 bzw. 3 gehörten. Die im menschlichen Blut vorzufindende nicht-essentielle Aminosäure Glutamin ist auch in Kulturmedien unerlässlich. Deshalb wurde die Laktatbildung aus Glukose in neun Tumorzelllinien sowohl in An- als auch Abwesenheit von Glutamin gemessen. Dabei wurde festgestellt, dass sich die Menge an gebildetem Laktat nach Inkubation in Glukose-haltigem, aber Glutamin-freiem Medium (Glc+ Gln-) besonders stark verringerte. Dieses Phänomen wurde für die Tumorzelllinien HT-29, 23132/87, BXPC-3 und HRT-18 beobachtet und deutet darauf hin, dass Glutamin in diesen Tumorzelllinien in irgendeiner Form auf die Laktatbildung aus Glukose Einfluss nimmt. Denkbar wäre, dass Glutamin Enzyme der Glykolyse über einen allosterischen Effekt moduliert, wodurch die Laktatbildung ansteigt. Dass diese Zellen eine Glutamin-abhängige Laktatbildung in Form der Glutaminolyse aufweisen, konnte in dieser Arbeit nicht beobachtet werden. Um diese Beobachtung abschließend zu klären, sind jedoch weiterführende Untersuchungen notwendig. / Unlike normal cells, the energy metabolism in tumour cells is characterised by the Warburg effect, which means high aerobic glycolysis even in the presence of oxygen. Most of the glucose is metabolised to lactate, which is not observed to this extent in normal cells. The present thesis examines the energy metabolism of nine cancer cell lines which were all established from different human solid tumours. Because of the high glucose consumption of the Warburg effect, glucose uptake and lactate output were quantified. The objective was to find out whether there is a link between the Warburg effect and other properties of the tumour cells, e.g. the cell-surface phenotype or malignancy. The surface proteins CD44 and CD24, markers for cellular interactions, cellular adhesion and migration, were selected as phenotyping molecules. Tumour malignancy was validated in xenograft experiments in which tumour cells were injected subcutaneously into immunodeficient mice and the subsequent rate of tumour growth determined. The amount of lactate produced in tumours cells was dependent on the presence of glucose in the culture medium. Five out of nine tumour cell lines produced lactate in high concentrations (on average more than 2.5 mmol/L within 48 hours). One of these was MDAMB-231, a breast tumour cell line. In contrast, another breast tumour cell line, MCF-7, showed a low rate of lactate production and no growth in xenograft experiments. This implies that lactate production correlates with malignancy. Moreover, the uptake of the glucose analogon 2-NBDG was dependent on temperature. Again, MCF-7 showed a lower uptake of 2-NBDG than the cells of MDAMB-231, which suggests that MCF-7 metabolizes glucose in the mitochondria rather than via aerobic glycolysis. However, the expression of glucose carriers GLUT-1 and GLUT-3 was nearly equal on all tumour cell lines except for the cells of HT-29 and MDAMB-468 where GLUT-3 was seen only sparsely. Six out of nine tumour cell lines displayed the phenotype CD44+ / CD24+, meaning the concomitant expression of CD44 and CD24. The single expression of CD44 was seen on two tumour cell lines and one tumour cell line showed the single expression of CD24. A correlation between glucose uptake, lactate production and a particular phenotype respectively could not be shown unambiguously, albeit three out of five tumour cell lines with a high lactate production exhibited the single expression of CD44 or CD24. The non-essential amino-acid glutamine is a constituent of human blood and therefore, was added to the culture medium. The tumour cell lines’ lactate production was measured in the presence and absence of glutamine. The lactate production in some tumour cell lines decreased strongly in glutamine-free but glucose-containing culture medium (Glc+ Gln-). The tumour cell lines HT-29, 23132/87, BXPC-3 and HRT-18 showed this phenomenon, indicating an influence of glutamine on the metabolism of glucose to lactate. For example, glutamine may affect glycolysis by an allosteric impact on particular enzymes, resulting in a higher lactate production. Lactate production by glutaminolysis could not be proven within this thesis. Further investigations are required to elucidate this.

The optimisation of strength and power through resistance training has been the source of debate amongst health professionals and researchers for many years. As resistance training involves the repeated activation and contraction of skeletal muscles, continuous training will ultimately result in a failure to sustain the training intensity especially when performing multiple sets and / or repetitions. Therefore the prescription of rest periods within the training session becomes an important consideration. Relatively short rest periods (60-90 seconds) have been traditionally used for the maximal strength adaptation involving increases in the cross-sectional area of the muscle, whereas longer rest periods (180-300 seconds) have traditionally been used for the maximal strength adaptation involving enhancement of neural function and maximal power adaptation. However, there is very little scientific evidence to support these current practices. In fact, the effect of different rest periods on maximal strength and power development has received very little research attention. Additionally, research that has been conducted in this area has been typified by a number of methodological inconsistencies, within and between studies, which confound scientific understanding. Although traditionally resistance training has employed continuous training schemes with inter-set rest periods, intra-set rest training methods which distribute rest intervals between groups of repetitions have also been investigated. It has been theorised that the short rest periods within the training set allow partial resynthesis of the intramuscular phosphocreatine stores, potentially allowing an athlete to increase their training volume by training at high intensities for longer durations, or performing additional repetitions (Berg, 2003). This is thought to lead to an increased exposure of the muscle to the kinematic and kinetic stimuli thought important for strength and power adaptation whilst minimising performance-inhibiting metabolic accumulation and substrate depletion. However, research into intra-set rest training schemes is still in its infancy, and many of the theories surrounding intra-set rest training are currently unsubstantiated. It is thought that examinations of the acute kinematic, kinetic and blood lactate profiles of continuous and intra-set rest training schemes may enhance scientific understanding regarding the efficacy of intra-set rest training. The purpose of this study was to investigate and compare the acute kinematic, kinetic and blood lactate responses to continuous and intra-set rest loading schemes. Nine male subjects performed an isoinertial Smith machine bench press task (6RM load) with a continuous loading scheme (CONT), an intra-set rest loading scheme equated by total rest time, volume and load (ISRV) and an intra-set rest loading scheme equated by total rest time and load (ISRR). The order of the loading schemes was assigned in a block randomised order with a minimum of 48 hours recovery between each testing session. Attached to the bar of the Smith machine was a linear position transducer that measured vertical displacement with an accuracy of 0.01cm. Displacement data was sampled at 1000Hz and collected by a laptop computer running custom built data acquisition software. Finger prick blood lactate samples were taken from the non-dominant hand using sterile techniques at the following time points: pre-exercise (Pre), immediately post-exercise (P0), five (P5), fifteen (P15) and thirty minutes (P30) post exercise. Blood glucose samples were taken pre-exercise only. It was observed that manipulating the rest period, by increasing the frequency but decreasing the length of each rest period, did not significantly influence the kinematics and kinetics associated with resistance training, but did have an effect on the post-exercise blood lactate response when the load, rest duration and training volume was equated (ISRV). This finding may be of practical significance if fatigue is important in strength development or conversely if power training needs to be performed with minimal fatigue. It was also observed that increasing the frequency of the rest period enabled the subjects to perform a greater number of repetitions (ISRR), resulting in significantly greater kinematics, kinetics and blood lactate accumulation. It may be speculated, therefore, that ISRR training may offer a superior training stimulus for the development of maximal strength and hypertrophy than CONT training methods, as ISRR loading increased the exposure of the muscle to the kinematic, kinetic and metabolic stimuli thought important for the development of these qualities.

No description available.

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

Thermodynamic and kinetic experiments have been performed at ionic strength 0.30 to elucidate the relationship between the structure of pig heart H₄-LDH (lactate dehydrogenase) and its catalytic function. Calorimetry and fluorescence were used to determine all the thermodynamic parameters for binary and ternary complex formation. TFL (trifluorolactate) and oxamate were employed as nonreactive analogs of the substrates lactate and pyruvate, respectively, to examine ternary complex formation in the absence of the ensuing redox step. At pH 6 where there is no apparent change in the protonation state of LDH upon binary complex formation, LDH binds NADH more tightly than NAD due to an entropy effect, i.e., only 1.1 out of the 3.1 kcal/mole difference in free energy changes is enthalpic in origin. The heat capacities of LDH·NAD (-150 ± 30 cal/K-mole) and LDH·NADH (-220 ± 40 cal/K-mole) formation at pH 6 and 25°C are relatively small and similar. These results suggest the importance of charge interactions in coenzyme binding. Structural information indicates that Arg-106, a positively charged residue of a loop of polypeptide in LDH which at equilibrium alternates between two conformations, open (extended into solvent) and closed (part of the active site), interacts unfavorably with the positively charged nicotinamide ring of NAD when the loop is in the closed conformation. Thermodynamic experiments demonstrate the suitability of TFL as an analog of lactate. TFL displays the correct specificity by binding to LDH·NAD more tightly (Kₐ = 400 M⁻¹) than to LDH·NADH (Kₐ = 34 M⁻¹) at pH 8 and 25°C. This binding requires that an enzymic residue with a pK = 6.7 not be protonated in accordance with the role of His-193 in analog binding in crystalline ternary complexes. Since the free energy change of the redox step is small, the difference in the free energy changes of formation of LDH·NAD·TFL and LDH·NADH·oxamate from LDH+NAD+TFL and LDH+NADH+oxamate, respectively, should approximate the free energy change of the actual enzymic reaction. The free energy and enthalpy changes of this model reaction are within 10% of the values of the actual reaction. Steady-state kinetic experiments further support the use of TFL as an analog of lactate. At pH 8 and 25°C TFL acts mainly as competitive inhibitor of lactate during lactate oxidation. The difference between the TFL dissociation constant (2.5 mM) and its inhibition constant (8.0 mM) means that TFL is not a simple dead-end inhibitor, i.e., LDH·NAD·TFL must be connected to the productive pathway of the reaction at more than one point. This is consistent with the existence of two conformational states of LDH·NAD. Additional support for the existence of two conformational states of LDH comes from analysis of the heat capacity changes of ternary complex formation. The large negative heat capacity changes at 25°C of TFL binding to LDH·NAD at pH 8 (-150 cal/K-mole) and of oxamate binding to LDH·NADH at pH 8 (-330 cal/K-mole) and pH 6 (-420 cal/K-mole) are partly attributed to a reaction heat effect arising from a shift in the conformational equilibrium of LDH to one in which the loop is in the closed position. As shown by calorimetry and fluorescence, phosphate binds to a single class of sites of LDH. The thermodynamic parameters of this process at pH 6 and 25°C are ΔG = -30 kcal/mole, ΔH = -5.1 kcal/mole, and ΔS = -7.0 cal/K-mole. Binding is not at the active site.

No description available.

Ten trained male track athletes (VO2max = 64.7 ml&middot;kg&middot;min -1) performed three workouts (conditions) with repeated 400-m runs. The intensity and number of repetitions varied among conditions. Condition 1 consisted of two all-out 400-m runs. Condition 2 was 4 x 400-m runs with the first three reps performed 4 s slower than condition 1 and the 4 th rep was all-out. Condition 3 consisted of 8 x 400-m runs with the first seven reps performed 8 s slower than condition 1 and the 8th rep was all-out. Dependent variables were HR, blood lactate and run time for the final rep in each condition. Peak HRs for the last run were 201, 194, 189 beats&middot;min-1 for conditions 1, 2 & 3 respectively, and were not significantly different. Blood lactate values measured 4 min after the last run were 16.6, 17.8 and 17.1 mmol&middot;L -1 in conditions 1, 2 and 3 respectively, and were not significantly different. Run times for conditions 1 (55.2 s), 2 (56.9 s) and 3 (61.5 s) were significantly different (P < 0.05). The decline in performance was greatest in condition 3. The three conditions challenged the anaerobic system with similar peak values for lactate and heart rate during the final run.

Bibliography: leaves 178-210. / xiii, 230 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis examines the effects of endurance exercise training on whole body blood lactate removal, and the production and removal of blood lactate by skeletal muscle. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1997?

The optimisation of strength and power through resistance training has been the source of debate amongst health professionals and researchers for many years. As resistance training involves the repeated activation and contraction of skeletal muscles, continuous training will ultimately result in a failure to sustain the training intensity especially when performing multiple sets and / or repetitions. Therefore the prescription of rest periods within the training session becomes an important consideration. Relatively short rest periods (60-90 seconds) have been traditionally used for the maximal strength adaptation involving increases in the cross-sectional area of the muscle, whereas longer rest periods (180-300 seconds) have traditionally been used for the maximal strength adaptation involving enhancement of neural function and maximal power adaptation. However, there is very little scientific evidence to support these current practices. In fact, the effect of different rest periods on maximal strength and power development has received very little research attention. Additionally, research that has been conducted in this area has been typified by a number of methodological inconsistencies, within and between studies, which confound scientific understanding. Although traditionally resistance training has employed continuous training schemes with inter-set rest periods, intra-set rest training methods which distribute rest intervals between groups of repetitions have also been investigated. It has been theorised that the short rest periods within the training set allow partial resynthesis of the intramuscular phosphocreatine stores, potentially allowing an athlete to increase their training volume by training at high intensities for longer durations, or performing additional repetitions (Berg, 2003). This is thought to lead to an increased exposure of the muscle to the kinematic and kinetic stimuli thought important for strength and power adaptation whilst minimising performance-inhibiting metabolic accumulation and substrate depletion. However, research into intra-set rest training schemes is still in its infancy, and many of the theories surrounding intra-set rest training are currently unsubstantiated. It is thought that examinations of the acute kinematic, kinetic and blood lactate profiles of continuous and intra-set rest training schemes may enhance scientific understanding regarding the efficacy of intra-set rest training. The purpose of this study was to investigate and compare the acute kinematic, kinetic and blood lactate responses to continuous and intra-set rest loading schemes. Nine male subjects performed an isoinertial Smith machine bench press task (6RM load) with a continuous loading scheme (CONT), an intra-set rest loading scheme equated by total rest time, volume and load (ISRV) and an intra-set rest loading scheme equated by total rest time and load (ISRR). The order of the loading schemes was assigned in a block randomised order with a minimum of 48 hours recovery between each testing session. Attached to the bar of the Smith machine was a linear position transducer that measured vertical displacement with an accuracy of 0.01cm. Displacement data was sampled at 1000Hz and collected by a laptop computer running custom built data acquisition software. Finger prick blood lactate samples were taken from the non-dominant hand using sterile techniques at the following time points: pre-exercise (Pre), immediately post-exercise (P0), five (P5), fifteen (P15) and thirty minutes (P30) post exercise. Blood glucose samples were taken pre-exercise only. It was observed that manipulating the rest period, by increasing the frequency but decreasing the length of each rest period, did not significantly influence the kinematics and kinetics associated with resistance training, but did have an effect on the post-exercise blood lactate response when the load, rest duration and training volume was equated (ISRV). This finding may be of practical significance if fatigue is important in strength development or conversely if power training needs to be performed with minimal fatigue. It was also observed that increasing the frequency of the rest period enabled the subjects to perform a greater number of repetitions (ISRR), resulting in significantly greater kinematics, kinetics and blood lactate accumulation. It may be speculated, therefore, that ISRR training may offer a superior training stimulus for the development of maximal strength and hypertrophy than CONT training methods, as ISRR loading increased the exposure of the muscle to the kinematic, kinetic and metabolic stimuli thought important for the development of these qualities.

Thesis (M.Sc. (Biochemistry)) -- Mahidol University, 1976.