Browsing by Subject "metabolism"

Glutamine, the conditionally essential amino acid, is a major carbon and nitrogen carrier required for a range of cell functions, such as protein synthesis and maintaining redox balance. While healthy cells adjust their activities in response to glutamine availability, tumor cells display deregulated glutamine uptake and metabolism allowing quick proliferation and survival in cellular stress conditions. Hence, further knowledge of the glutamine sensing network is of interest. Utilizing Drosophila melanogaster, the roles of formerly identified glutamine sensing regulator candidates, Forkhead box O (FoxO), Super sex combs (Sxc), Spalt major (Salm) and Spalt-related (Salr), were explored. Drosophila is an efficient model organism for analyzing gene regulatory mechanisms, with its simple genome but conserved genes and metabolic pathways. Loss-of function and gain-of-function mutants of the candidates were cultured with/without glutamine, and their physiological response and gene expression changes were analyzed. The results show the glutamine intolerant phenotype of FoxO and Sxc deficiency, not dependent on altered food intake levels of larvae. However, glutamine intolerance of Salr and Salm deficiency was not observed. Moreover, we aimed to gain further insight to the roles of FoxO and Sxc in glutamine metabolism. Since amino acid catabolism produces ammonia, and glutamine metabolism plays a vital role in ammonia detoxification, we performed a pH-based measurement of foxo and sxc mutant larvae hemolymph on food with/without glutamine. However, we could not associate FoxO or Sxc with regulation of glutamine-derived ammonia clearance. In addition, we explored FoxO downstream regulator candidates. Putative promoter areas of Paics, Uro, Sesn, salr, Prat2 and Gdh were cloned into reporter vectors and the luciferase activity was analyzed under the expression of foxo. The results indicate that FoxO is a regulator of all of the 6 genes. Next we could utilize the here constructed plasmids to see whether the FoxO-mediated regulation is affected by altered glutamine levels in cell culture.

Over the past years sugar consumption has seen great increases worldwide, together with a rise in the prevalence of metabolic diseases. There is a growing need for a comprehensive characterisation of the genes involved in sugar metabolism, yet the mechanisms by which cells sense and respond to sugars in vivo have remained incompletely understood. Here, I analyse members of a protein family best known for their regulation of differentiation during development with regards to their role in sugar metabolism. The Hairy and Enhancer of Split (HES) protein family are a group of basic helix-loop-helix (bHLH) transcription factors that function as major downstream effectors of the Notch signalling pathway. In mammals, the HES proteins have mostly been studied for their role in cell differentiation, but HES1 has been implicated in metabolic control. Drosophila has several transcription factors belonging to the HES family, including Hairy and seven bHLH transcription factors located in the Enhancer of split complex (E(spl)-C). The E(spl)-C bHLH transcription factors display high homology and are considered to be genetically redundant, and therefore little is known about their individual functions. The other HES family members in Drosophila have not previously been linked to metabolic regulation, but Hairy has been shown to repress the tricarboxylic acid cycle. In light of the findings implicating HES1 and Hairy in the regulation of metabolism, I systematically investigated the role of the HES transcription factors in sugar metabolism. By using the GAL4/UAS system in Drosophila melanogaster, I knocked down gene expression of each of the family members, and raised the flies on diets varying in sugar content to identify possible sugar intolerance phenotypes. Here, I show that knockdown of one of the E(spl)-C bHLH genes led to severe sugar intolerance that affected both survival and organismal growth, but did not alter the levels of circulating carbohydrates and storage lipids as measured with colorimetric assays and lipid staining. Furthermore, I identify the tissues in which this transcription factor functions to provide sugar tolerance. Using analysis of publically available chromatin-immunoprecipitation sequencing data coupled with quantitative RT-PCR, I uncover mTOR target Thor/4E-BP as a putative target gene. Additionally, I show that Hairy is similarly required for complete sugar tolerance, but that the mechanism differs from the E(spl)-C bHLH transcription factor. Hairy binds to and positively regulates expression of genes involved in glycolysis and the pentose phosphate pathway, suggestive of a cooperation with earlier known regulators of sugar sensing. In conclusion, I have shown that only two HES family members are involved in the regulation of sugar metabolism and that their regulatory mechanisms are distinct, implying that the HES family members have more diverse roles than previously assumed.

Animals regulate their metabolism dynamically as a response to changes in nutritional landscape. Intestine is emerging as a key regulator of systemic metabolism. It possesses secretory enteroendocrine cells (EECs), which have a central role in intestinal nutrient sensing and signaling. However, how the number and function of EECs is regulated in response to nutrients remains poorly understood. Previous work in Hietakangas lab has shown that a transcriptional cofactor, C-terminal binding protein (CtBP), regulates the number of EECs in response to sugar feeding and loss of CtBP function in EECs causes sugar intolerance in Drosophila. CtBP’s transcriptional activity is modulated through homodimerization, which is controlled by redox coenzyme NAD+/NADH, whose levels are dependent on sugar metabolism. Therefore, I hypothesise that CtBP is a sugar- and redox-responsive regulator of EEC function. In this thesis, I aimed to understand how CtBP is regulated and what are its downstream effectors. My results show that the formation of CtBP homodimers is responsive to dietary sugars and cellular redox state. In addition, I observed that CtBP heterodimerizes with EEC fate determining transcription factor Prospero. Functional analysis of CtBP downstream effector genes shows significant overlap with those of Prospero. In conclusion, CtBP is a sugar- and redox-responsive cellular regulator of EEC function, which acts in cooperation with Prospero.

A natural sweetener from Stevia rebaudiana has the potential to reduce calorie intake. Recent data suggest that glycosides from Stevia display favorable pharmacological properties for the metabolic syndrome. Skeletal muscle constitutes a major extrahepatic tissue responsible for glucose utilization. We studied whether steviol can modulate glucose uptake in L6 myocytes. Signaling targets that are central in regulating cellular glucose metabolism were assessed with western blotting. Our data show that the highest concentration of steviol tested decreased slightly and statistically significantly the glucose uptake rate in insulin-stimulated muscle cells. This was supported by western blotting data from targets Akt and GSK-3β. Therefore, our data suggest that direct exposure of myocytes to steviol causes insulin resistance. The results can aid in planning further experiments to understand the pharmacological effects of Stevia-derived products. This would ultimately enhance our understanding of the mechanism behind insulin resistance and be used in future drug development.

Autophagy is an essential pathway that evolved to sustain cellular integrity by removing damaged and aged organelles. During this process, our cells sense, encapsulate and deliver defective cellular components to the lysosome for destruction. Over the past decade, many laboratories have demonstrated that damaged mitochondria can be selectively eliminated, during a process known as "mitophagy". Mitophagy senses, targets, and engulfs defective mitochondria for elimination via lysosomal hydrolysis. The identification of factors that promote or prevent mitophagy has high therapeutic relevance, particularly those that alter PINK1/Parkin-independent mitophagy. Recent research in the McWilliams lab uncovered a novel role for lipid metabolism in the regulation of PINK1/Parkin-independent mitophagy. Briefly, the team discovered that DGAT1-dependent lipid droplet (LD) biosynthesis occurred several hours upstream of mitochondrial clearance, with LDs accumulation upon iron chelation. LDs accumulate in a DGAT1-dependent fashion as mitochondria are eliminated. Pharmacological or genetic inhibition of DGAT1, restricts mitophagy levels in vitro and in vivo. However, the mechanism that linked defective lipid metabolism to reduced mitophagy remained mysterious. We hypothesized that defective lipid signalling may compromise lysosomal activity leading to reduced levels of mitophagy. Accordingly, my project examined the functional contribution of DGAT-dependent LD biogenesis to lysosomal homeostasis in the context of PINK1/Parkin-independent mitophagy. After first verifying the DGAT1-dependent nature of LD accumulation in human cells, I established assays to investigate lysosomal homeostasis in the context of iron chelation-induced mitophagy. Using a variety of labelling approaches, live cell imaging experiments revealed a significant displacement of endolysosomes upon DGAT1/2 inhibition, in addition to possible alterations in lysosomal dynamics. My data suggest that loss of DGAT1 activity impairs lysosomal homeostasis when iron levels are low. This likely explains the mitophagy impairments and might account for additional phenotypes of impaired cell viability upon DGAT1 inhibition. Changes in lysosomal acidity were inconclusive, indicating further timepoints may need to be analysed to detect transient impairments in hydrolysis. My results emphasize the importance of organelle crosstalk in mitophagy and the emerging role of LDs in cellular integrity. These data further highlight that targeting lipid metabolism may provide a means to sustain efficient mitochondrial turnover.

Folate deficiency (FD) has been found to cause number of medical conditions varying from megaloblastic anemia to fetal neural tube defects but the molecular basis behind these has remained poorly understood. We studied the metabolic consequences of FD in mouse liver and brain, concentrating on transsulfuration pathway and cysteine-dependent pathways. Data was acquired with mass spectrometry based metabolomics and western blotting. We found that FD induces lack of cysteine in liver and brain, causing further imbalances in cysteine-derived metabolites such as glutathione and taurine. Changes in enzyme expression show that hepatic cells prioritize glutathione synthesis over taurine synthesis, while the brain does vice versa. We then supplemented FD mice with N-acetylcysteine (NAC), precursor of cysteine. NAC supplementation restored hepatic bile acid and blood glutathione levels of FD tissues. These results improved our understanding of FD induced metabolic imbalances and proved that NAC significantly rescues some of these changes.

Cardiovascular diseases are the most common causes of mortality worldwide. More adequate human-based models would be needed for the purposes of disease modeling and drug development. One of the most promising fields of in vitro modeling is the use of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). A central problem of hPSC-CMs is their immature or fetal-like phenotype compared to adult human cardiomyocytes regarding many structural, functional, and metabolic properties. The development of metabolic properties is considered to be a central driver of cardiomyocyte maturation. One practicable way to promote the metabolic maturation of hPSC-CMs in vitro is the use of various biochemical cues in the cell culturing media. The topic of this study was the metabolic maturation of hPSC-CMs. The research questions were: What biochemical cues have been suggested to be involved in the hPSC-CM maturation in vitro? What signaling pathways connected to the biochemical cues have been explored in the context of the maturation of hPSC-CM? What experimental results have been achieved on the effects of the biochemical cues and the involvement of the signaling pathways? The study was conducted as a systematic review with the database Scopus (Elsevier). The final set of materials consisted of 46 original research articles published in peer-reviewed journals in English in the years 2013–2022. Out of the materials, 11 articles (24%) were characteristically longitudinal studies. They indicated that the pathways leading to metabolic changes such as PPARs (peroxisome proliferator-activated receptors) and PGC-1α (peroxisome proliferator-activated receptor γ coactivator 1α) are activated already in early stages. In 12 articles (26%), pharmacological agents were used to target the metabolic pathways, and in 8 articles (17%) techniques affecting the gene expression were utilized. The most recent studies involved ever more frequently combinations of different techniques. Considering the use of biochemical cues, the trend has been to favor fatty acids, thyroid hormone and dexamethasone over glucose, insulin and insulin-like growth factor. Some cues such as retinoic acid and neuregulin 1 have been tested only in single experiments. In addition to the nuclear receptor mediated pathways, the energy sensors AMPK (AMP-activated protein kinase) and mTOR (mechanistic target of rapamycin), the oxygen sensor HIF-1α (hypoxia-inducible factor 1α), and the microRNAs turned out to be central.

Prodrugs are pharmacologically inactive molecules which undergo metabolic bioactivation in vivo to form pharmaceutically active agents. Prodrugs have been designed to improve so called drug-like properties of active parent compounds (APC) i.e. to increase solubility or absorption and to reduce first-pass metabolism etc. In this master's thesis the goal was to establish non-cell-based in vitro methods to study prodrug bioactivation. Four commercially available prodrugs (bambuterol, olmesartan medoxomil (OM), candesartan cilexetil (CC) and famciclovir) were used as test compounds. The prodrugs were incubated in liver and intestinal S9 fractions and blood plasma to study in vitro bioactivation of these prodrugs. Other metabolism of the prodrug and APC (nonproductive metabolism) was studied by comparing incubation with and without cofactors of metabolizing enzymes. Species differences was studied using human, rat and dog matrices. Prodrug concentrations were quantified from the incubation samples using liquid chromatography- tandem mass spectrometry (LC-MSMS) methods developed for this study. Additionally the effect of promoiety on passive permeability was studied with parallel artificial membrane permeability assay (PAMPA). All of the studied prodrugs produced at least low concentrations of APC in one or more incubations. Terbutaline (APC of bambuterol) formation was observed in human plasma and was concentration dependent which is consisted with the literature. Olmesartan and candesartan were formed in S9 fraction in high rate, but not in buffer: indicating enzyme mediated hydrolysis. However, based on literature CC hydrolysis was not expected to occur in intestinal S9 fractions. Penciclovir (APC of famciclovir) was formed only in presence of human or rat liver S9 fraction which was in line with the pre-existing literature. With the method used the nonproductive metabolism could not be estimated. In PAMPA bambuterol, famciclovir and OM had higher permeability than corresponding APCs whereas CC was only more permeable than candesartan in pH 7.4. The in vitro incubation used in this study can be used for screening prodrugs. However both low and high activation rates were observed thus the clinically relevant in vivo APC formation can be achieved with both high and low bioactivation in vitro. Studying the rate of prodrug formation alone estimations about clinically relevant bioactivation rates cannot be concluded. No clear signs of nonproductive could be seen with the prodrugs studied with current method. For the estimation of nonproductive metabolism, metabolite screening studies would need to be developed and conducted parallel to studies prescribed in this master's thesis.

Respirometry is a polarographic method that provides insights into mitochondrial respiratory capacity – specifically to electron transport chain (ETC) complexes I to V –, mitochondrial integrity and energy metabolism. The limitation of the respiratory measurements has been that it requires freshly isolated mitochondria or tissue sample. Long-term preservation of mitochondrial function in frozen samples has been a considerable challenge, since the membrane integrity of the mitochondria is lost during the freezing process. Thus, samples do not display coupled respiration. However, previous studies have found that despite coupled respiration is impaired the individual ETC complexes and the ability of ETC supercomplexes to consume oxygen are not destroyed due to freezing and thawing. On the basis of this knowledge, recently published article presented a novel protocol that overcomes the damages caused by freeze-thaw cycles. The protocol also enables respiration measurement of ETC complexes I-IV by using Seahorse XF96 Extracellular flux analyzer. In this MSc thesis I modified and optimized the aforementioned protocol for Oroboros O2k high- resolution respirometry using frozen skeletal muscle samples. In addition, this study provides an optimized sample preparation protocol for frozen muscle samples and respiration measurement. The new method broadens the possibilities within mitochondrial respiration studies since Oroboros O2k high-resolution respirometry records results with high sensitivity without limiting the number of substrates used. The possibility to use frozen samples reduces research costs, simplifies logistics and enables retrospective studies with previously stored frozen tissue samples. I also utilized the optimized respiration measurement protocol to study metabolic effects of combined gene therapy in skeletal muscle. This gene therapy mimics the positive effects of exercise by inducing skeletal muscle growth and angiogenesis. The mimicking effect was induced by systemic delivery of adeno-associated viral vectors encoding pro-myostatin and VEGF-B. In previous studies inhibition of myostatin has been connected to compromised oxidative capacity and vascular rarefaction. In contrast, VEGF-B has demonstrated to induce angiogenesis in several tissues. Thus, my hypothesis was that combination gene therapy would result in better mitochondrial function than pro-myostatin alone. Results from this study indicate that moderate inhibition of myostatin signaling by pro-myostatin using rAAV vectors could provide enhancements in ETC function when it is induced independently or combined with rAAV-VEGF-B. This result lays a solid foundation for future research and could provide a new therapeutic option against muscle loss and related metabolic diseases.