Thesis (Ph.D.)--Case Western Reserve University, 2009 / Abstract Department of Biomedical Engineering Title from OhioLINK (viewed on 20 April 2009) Available online via the OhioLINK ETD Center

Thesis (M.D.)—Boston University

Choline is an essential nutrient, in the liver it is a precursor necessary for the synthesis of phosphatidylcholine (PC) and is also required as a methyl donor towards the synthesis of betaine and later regeneration of S-adenomethionine (SAM). Choline deficiency is known to trigger the development of non-alcoholic fatty liver disease and affect mitochondrial homeostasis along with a myriad of methylation related regulatory mechanisms. Because of its importance in maintaining liver lipid and mitochondrial homeostasis, choline metabolism has been well characterized with the exception of its transport. The identification of choline transporters has only been recently discovered and because of this, relatively little is known about their expression and regulation. This study has established that choline transporter like proteins 1-5 (CTL1-5) is an intermediate affinity transport system responsible for ~80% of hepatic choline uptake with a smaller percentage accomplished through the low affinity organic cation transporter 1-3/N1-2 (OCT1-3/N1-2) transporters. SLC44A1 expression and choline incorporation have been shown to follow a 24 hour rhythmic trend suggesting the presence of a circadian regulatory mechanism. This finding is supported by the significant decrease in choline expression and aberrant pattern of choline incorporation discovered among rhythmic deficient BMAL-/- mice and through a bioinformatics analysis which revealed the existence of four REV-ERBα consensus sequences. Hepatic SLC44A1 expression and choline incorporation have also been shown to decrease with the onset of obesity. Choline uptake was also shown to decrease following treatment with the free fatty acid oleate. This work increases our knowledge of hepatic choline transport and demonstrates a link between the circadian rhythm and obesity with the hepatic CTL1 transporter.

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The essential nutrient choline is known to serve as a precursor for phospholipids and the neurotransmitter acetylcholine, and to feed into methylation pathways. The role and fate of choline in immune cells, however, is not yet fully elucidated. To act as a methyl group donor, choline must first undergo oxidation in mitochondria, a process which has long been thought to occur exclusively in the liver and kidney. The recent identification of choline transporters on the mitochondrial membrane has highlighted the possibility of mitochondrial choline oxidation in other cell types. Here, I show that choline transporters are present on the mitochondrial membrane of primary and immortalized mouse macrophages. The interaction of CTL2 with mitochondria is further augmented following pro-inflammatory polarization with the bacterial endotoxin lipopolysaccharide. I show that mitochondrial choline uptake occurs in macrophages using radiolabelled choline assays; however, it remains unclear whether this process is conducted through the identified transporters. Preliminary data suggest that mitochondrial choline oxidation to betaine was increased in LPS-stimulated macrophages, revealing a potential additional input into one-carbon metabolism in polarized macrophages. This project broadens the existing paradigm that choline oxidation occurs strictly in hepatic and renal tissue and suggests that choline oxidation may be a regulated process in macrophage polarization.

Choline is universal among plants as phosphatidylcholine and in many plants it serves as a precursor for the compatible osmolytes glycine betaine and choline-𝘖-sulphate. In spinach, choline is synthesized by the sequential 𝘕-methylation of phosphoethanolamine (PEA) → phosphomethylethanolamine (PMEA) → phosphodimethylethanolamine (PDEA) → phosphocholine (PCho) and PCho is hydrolyzed to choline. The objective of this biochemical survey was to determine whether the activities of enzyme(s) converting PEA to PCho could be found in leaves of diverse plants and, if so, if common regulatory properties could be identified. Leaf tissue was harvested from 14 diverse dicot plants. Enzyme activities for the three sequential 𝘕-methylations were quantified in vitro and for representative assays using PEA as substrate, reaction products were identified by thin layer chromatography. Extracts of all plants tested could metabolize PEA to PMEA, the rate of conversion varied from 0.04 to 25 nmol · min⁻¹ · g⁻¹ Fresh wt for soybean and cotton, respectively. In vitro PMEA → PDEA and PDEA → PCho rates varied between the plant species tested. Both steps were highest in sugar beet (22 and 24 nmol · min⁻¹ · g⁻¹ Fresh wt for · PMEA → PDEA and PDEA → PCho, respectively) to below detection limits for soybean (<0.03 nmol· min⁻¹ · g⁻¹ Fresh wt). Upon dark exposure, PEA → PMEA reaction rates were reduced from the light period levels but not in all cases was the activity reduced beyond the level that could be attributed to a general loss in plant vigour. The reduction in 𝘕-methylation capacity was most pronounced in plants with the highest levels of PEA → PMEA activity, with reduction greatest in sugar beet > amaranth > spinach. Thus PEA → PMEA is catalyzed by a light-regulated enzyme in many but apparently not all dicot plants. Datko and Mudd (1988a) have proposed that PEA → PMEA conversion is ubiquitous among plants and the enzyme responsible catalyzes a committing step for PCho synthesis. PEAMeT activity was found in leaf extracts of 10 additional dicot species. The level of in vitro activity and light regulation is not equivalent among all plants examined. If in vitro rates faithfully reflect the in vivo capacity to synthesize choline, why the variability among plants with respect to their capacity to synthesize a universal metabolite? Alternate routes of PCho/phosphatidyl choline synthesis may explain these differences (Datko and Mudd, 1988a,b; Hanson and Rhodes, 1983; Hitz 𝘦𝘵 𝘢𝘭., 1981; Weretilnyk and Summers, 1992). Interestingly, the plants with the highest rates of PEA → PMEA activity, (cotton, sugar beet, amaranth, sunflower, spinach and statice) are all documented glycine betaine accumulating species. These plants may have higher rates of PEA methylation in order to meet requirements for osmolyte synthesis. Thus, it would be interesting to see if PEA metabolism to PCho is up-regulated in response to osmotic stress. / Thesis / Master of Science (MS)

Indiana University-Purdue University Indianapolis (IUPUI) / High choline uptake and increased choline kinase activity have been reported in many cancers. This has motivated the use of choline as a biomarker for tumor imaging. Tumors in general are heterogeneous in nature with respect to oxygen tension. There are regions of hypoxia and normoxia that are expected to have different metabolism but regulation of choline metabolism under hypoxia is poorly understood. It is important to clarify the status of choline metabolism in hypoxic microenvironment as it will have an impact on potential of choline as a cancer biomarker. The primary goal was to determine the status of choline phosphorylation in hypoxic cancer cells and its effect on uptake of choline. This was examined by tracer studies in cancer cells exposed to hypoxia. It was observed that hypoxia universally inhibits choline uptake /phosphorylation in cancer cells. Decreased choline phosphorylation resulted in transient uptake of choline radiotracers in cultured cancer cells and 9L tumors suggesting potential problem in using choline as a biomarker for cancers in hypoxic microenvironment. To investigate the mechanism behind decrease in choline phosphorylation, steady state levels of choline metabolites were measured and choline kinase catalyzed choline phosphorylation step was found to be rate-limiting in PC-3 cells. This suggested that modulation in choline kinase levels can alter choline metabolism in hypoxic cancer cells. Expression and activity assays for choline kinase revealed that choline kinase expression is down-regulated in hypoxia. This regulation involved transcriptional level mediation by HIF1 at the conserved HRE7 site in choline kinase promoter. To further understand the importance of down-regulation of choline kinase in hypoxia, stable prostate cancer cell lines over-expressing choline kinase were generated. Effect of over-expression of choline kinase in hypoxia was evaluated in terms of malignant phenotypes like proliferation rate, anchorage independent growth and invasion potential. Both over-expression of choline kinase and hypoxia had a pronounced effect on malignant phenotypes of prostate cancer cells. Further study showed that increased choline kinase activity and hypoxic tumor microenvironment are important for progression of early-stage, androgen-dependent LNCaP prostate cancer cells but confer little survival advantage in undifferentiated, androgen-independent PC-3 prostate cancer cells.

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Plasma choline levels have been reported to be significantly reduced in athletes at the endof prolonged, exhaustive exercise (marathon running), and this may affect the release of acetylcholine at the neuromuscular junction (16). The purpose of the present investigation was to examine the influence of two levels of choline (from soy lecithin) ingestion on plasma choline levels and exercise performance. Ten endurance-trained male cyclists exercised on an electrically resisted ergometer for 105 minutes at a load equal to 70% V02 max, followed by an all-out, self-paced 15 minute performance ride on an isokinetic cycle. Three randomly ordered trials were performed four hours after the feedings of (A) 0.0 g, (B) 1.1 g, or (C) 1.8 g choline. Dietary intake of choline was controlled. Before and after the trials, blood samples were drawn and analyzed for serum choline (umol/liter) using an HPLC method. Feedings of 1.1 g and 1.8 g of choline significantly (p < 0.05) elevated serum choline values compared to control (A = 34.16 [± 0.63], B 38.17 [+ 0.96], and C = 42.32 [+ 0.59]).Post-exercise serum choline values, however, were not significantly different (p > 0.05) from pre-exercise levels. In addition, there were no significant differences (p > 0.05) in performance between placebo, 1.1 g, and 1.8 choline trials (2.11 x 105, 2.07 x 105, 2.07 x 105 Newton-meters, respectively). This study indicates a dose response to choline ingestion, with no effect on performance. / School of Physical Education

This study sought to determine whether caffeine combined with choline could improve short-term memory in healthy adults. The study tested the effect of choline (2 gm) alone and in combination with several concentrations of caffeine (25 mg, 50mg and 100mg) on short-term verbal and visual memory and attention. The Wide Range Assessment of Memory and Learning-2 was utilized. Choline 2 gm + caffeine 25 mg group showed significantly (p&lt;0.05) higher overall memory performance whereas memory performance in the choline 2 gm + caffeine 50 mg group was significantly impaired compared to placebo. The data suggest that specific combinations of caffeine and choline can either facilitate or impair short-term memory in adults with normal cognitive function. Future studies of caffeine and choline combinations will test memory performance in subjects with memory impairment. / Mylan School of Pharmacy and the Graduate School of Pharmaceutical Sciences / Pharmacology / MS / Thesis