Pyruvate dehydrogenase | EPFL Graph Search

Pyruvate dehydrogenase is an enzyme that catalyzes the reaction of pyruvate and a lipoamide to give the acetylated dihydrolipoamide and carbon dioxide. The conversion requires the coenzyme thiamine pyrophosphate. Pyruvate dehydrogenase is usually encountered as a component, referred to as E1, of the pyruvate dehydrogenase complex (PDC). PDC consists of other enzymes, referred to as E2 and E3. Collectively E1-E3 transform pyruvate, NAD+, coenzyme A into acetyl-CoA, CO2, and NADH. The conversion is crucial because acetyl-CoA may then be used in the citric acid cycle to carry out cellular respiration. To distinguish between this enzyme and the PDC, it is systematically called pyruvate dehydrogenase (acetyl-transferring). Mechanism The thiamine pyrophosphate (TPP) converts to an ylide by deprotonation. The ylide attack the ketone group of pyruvate. The resulting adduct decarboxylates. The resulting 1,3-dipole reductively acetylates lipoamide-E2. In terms

Multidisciplinary Analysis of the Metabolic Shift to Lactate Consumption in CHO Cell Culture

Francesca Zagari

Mammalian cells represent the most widely used host system for the industrial production of recombinant therapeutic proteins. In order to increase productivity, chemically defined media are often used and optimized to provide the cells with the necessary nutrients. However, a deregulated cell metabolism is often observed in these culture conditions. In particular, carbon sources are fast and inefficiently consumed with a consequent accumulation of byproducts, mainly lactate and ammonia. Lactate, in particular, causes a decrease of the medium pH which can be detrimental for cells growth and productivity. Its metabolic profile is normally characterized by a first production phase, which is associated to the cells exponential growth. Afterwards, when the cells enter into the stationary phase, a shift to net lactate consumption can occur under optimal culture conditions. However, such a metabolic shift is not easily controlled, since the mechanisms modulating lactate production in cell culture are still under investigation. This work aims to understand which factors could influence the lactate metabolic shift in CHO cell culture, focusing, in particular, on the mitochondrial role. To this purpose, cell lines with opposite lactate profiles were compared. The initial lactate production phase was common to all the fast growing cultures and was concomitant to a rapid glutamine consumption. After glutamine depletion, two different scenario occurred. In one case, the cells started to consume lactate until complete depletion. Alternatively, lactate continued to be accumulated, causing a decrease of media pH. The mitochondrial oxidative capacity was hypothesized as a possible cause of the different lactate profile. Indeed, mitochondrial membrane potential and oxygen consumption measurements highlighted a correlation between a reduced oxidative metabolism and a state of high lactate production. This correlation was confirmed by evaluating other cell lines or media compositions which resulted in the same divergence of lactate metabolism. Afterwards, the expression of selected genes was analysed in correlation with the observed lactate profile. Among 22 genes, two were identified as significantly downregulated (absolute fold change ≥2) in conditions of high lactate accumulation; namely, the mitochondrial aspartate-glutamate carrier (aralar1) and the translocase of the inner mitochondrial membrane 8 (timm8a). Aralar1, in particular, is an important component of the malate-aspartate shuttle (MAS). This system promotes the recycling of the cytosolic NADH pool, which is necessary for maintaining the glycolytic flux. Alternatively, NADH can be oxidised through pyruvate conversion into lactate, catalysed by the lactate dehydrogenase enzyme. Therefore, an inefficient MAS activity can engender an increased lactate accumulation. Finally, stable cell lines, which overexpressed aralar1 or timm8a, were generated. Clones derived from a lactate-producing cell line showed an improvement of lactate metabolism. In particular, they switched more easily to lactate consumption compared to the untransfected control, while maintaining a similar glucose consumption rate. On the other hand, no impact of the transgene expression was observed in the clones derived from the lactate-consuming cell line, since the switch to lactate consumption was maintained and no other effect on cell growth or glucose metabolism was detected. The data obtained indicate that lactate consumption was most likely promoted by a better NADH oxidation through the malate-aspartate shuttle, which resulted in a more efficient link between glycolysis and the mitochondrial oxidative metabolism. In conclusion, the results presented in this thesis indicate that the mitochondrial oxidative capacity plays a central role in the control of lactate production. Media composition and intrinsic cell characteristics have both an impact on mitochondrial activity. Moreover, the expression of specific genes can also be influenced by the culture media. In particular, aralar1 and timm8a have been identified as promising targets for the generation of a host cell line with an improved lactate metabolism.

EPFL2012

In vivo metabolic studies in skeletal and cardiac muscle using ¹³C magnetic resonance spectroscopy

Josefina Adriana Maria Bastiaansen

Cardiac and skeletal muscle function relies on a continuous energy production via fatty acid metabolism and mitochondrial oxidation of pyruvate, with a fine balance between substrate delivery and utilization. Changes in metabolism are increasingly being implicated as playing an intrinsic role in many diseases, such as diabetes, cancer, and heart failure. Investigating and identifying fundamental metabolic processes are paramount to understanding pathologies. Beyond morphology and functional information, magnetic resonance can provide insights at a metabolic level using spectroscopic techniques such as 13C NMR. The low natural abundance and sensitivity of the 13C nucleus makes 13C NMR in biological systems challenging. In addressing this issue, hyperpolarized methods have emerged as a very promising tool, obtaining signal enhancements up to 10,000 fold. Spectra of hyperpolarized 13C labeled substrates and their downstream metabolic products offer insight into metabolic processes occurring in vivo within seconds after the injection. This thesis focused on the development of MR hyperpolarization methods and applications to study energy metabolism in cardiac and skeletal muscle in vivo. This ranged from development of the experimental frame work to mathematical tools to characterize the observed metabolic processes. Methods were developed to visualize 13C labeling kinetics of acetylcarnitine in vivo in resting skeletal muscle following the administration of hyperpolarized [1-13C]acetate. Two different, novel mathematical models were constructed to quantify the kinetic rate constants. Although separated by two enzymatic reactions, the conversion of acetate to acetylcarnitine was uniquely defined by the enzymatic activity of acetylCoA synthetase (ACS). A 13C MRS protocol was developed and implemented for hyperpolarized studies in the heart, which included selecting appropriate cardiac triggers to align the measurements with the cardiac phase. The 13C label propagation into acetylcarnitine and citrate could be measured in real time in the beating rat heart following the infusion of hyperpolarized [1-13C]acetate, using a newly constructed 13C RF coil which improved the detection sensitivity. The substantial spectral resolution at 9.4T and a triggered shimming and MR acquisition protocol allowed for the detection of citrate for the first time in vivo after injection of hyperpolarized [1-13C]acetate. Mathematical models were successfully extended to include mitochondrial oxidation and analytical expressions were derived to interpret the dynamic 13C labeling of citrate. Cardiac dysfunction is often associated with a shift in substrate preference, while diagnostic methods such as PET provide only information on substrate uptake. The potential of hyperpolarized 13C MRS to measure simultaneously lipid and carbohydrate oxidation was demonstrated in vivo, and the sensitivity of the method to a metabolic perturbation was assessed. Hyperpolarized [1-13C]butyrate and [1-13C]pyruvate were used as representatives of carbohydrate and lipid oxidation. Fasting led to significant changes in preference for the injected substrates. The appearance of a cohort of downstream metabolites (bicarbonate, lactate, alanine, glutamate, citrate, acetylcarnitine, β-hydroxybutyrate and acetoacetate) allowed the independent and simultaneous monitoring of myocardial oxidation of both fatty acid and carbohydrates in vivo and is a sensitive indicator of metabolic shift. Lactate is an important metabolic intermediate for mitochondrial oxidation. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1-13C]lactate, which can be injected at physiological concentrations and leaves other oxidative processes undisturbed. A significant decrease in [1-13C]alanine and 13C bicarbonate were observed comparing both groups, attributed to a change in cellular alanine concentration and pyruvate dehydrogenase (PDH) flux. It was shown that lactate can be used to study carbohydrate oxidation in skeletal muscle at physiological levels and that the downstream metabolite signals are sensitive markers to probe metabolic changes. Since the detection of [5-13C]citrate and [5-13C]glutamate in the heart is hindered by the close proximity of the [1-13C]acetate resonance, acetylcarnitine could be an interesting substrate for hyperpolarized MR. Acetylcarnitine crosses the mitochondrial membrane easily, while skipping a few metabolic steps needed for acetate to cross the membrane. Moreover, it does not interfere with the detection of [5-13C]glutamate and [5-13C]citrate. [1-13C]Acetylcarnitine was successfully hyperpolarized and despite its short T1 it was possible to detect the formation of [5-13C]glutamate in the heart in vivo. Due to the absence of the [5-13C]citrate resonance, we hypothesized the existence of an intricate relationship between reaction, transport and relaxation rates in the choice of hyperpolarized substrates. Acetylcarnitine has relatively small poolsizes and has only been observed using hyperpolarized 13C MRS techniques. Therefore, it has not been possible to obtain reliable quantification of the metabolic turnover of acetylcarnitine, which is an essential intermediate in the metabolism of acetate. Methods were implemented to measure the oxidation of [2-13C]acetate locally with increased sensitivity at high field (14.1T), using a 1H-13C polarization transfer sequence. This allowed the observation of [2-13C]acetylcarnitine in vivo at 21.5 ppm, with a metabolic turnover time τ of 0.34 μmol/g/hr. The acetylcarnitine resonance assignment was confirmed by experiments infusing 13C labeled glucose. The measurement of the time courses of Glu C4 and C3 were also clearly observed, without lipid contamination. To conclude, this work presents novel metabolic information about skeletal and cardiac energy metabolism using developed methods in hyperpolarized 13C MRS. The use of hyperpolarized 13C substrates to measure absolute flux through metabolic pathways in vivo would not only revolutionize clinical diagnostic imaging but also serve as an important tool to better understand diseased metabolism and the metabolic effects of new drugs aimed to combat diseases.

EPFL2013