This study was conducted to test the hypothesis that dietary arginine supplementation reduces fat mass in diet-induced obese rats. Male Sprague-Dawley rats were fed either low- or high-fat diets for 15 wks (16 rats/diet). Thereafter, lean or obese rats continued to be fed their same respective diets and received drinking water containing either 1.51% L-arginine-HCl or 2.55% alanine (isonitrogenous control) (n=8/treatment group). Twelve weeks after the initiation of the arginine treatment, rats were euthanized to obtain tissues for biochemical analyses. Results were statistically analyzed as a 2x2 factorial experimental design using ANOVA. High-fat diet increased the mass of white adipose tissues at different anatomical locations by 49-96% compared to the low-fat diet. Concentrations of serum cholesterol as well as lipids in skeletal muscle and liver were higher in obese rats than in lean rats. L-Arginine supplementation reduced white adipose tissue mass by 20-40% while increasing brown adipose tissue mass by 15-20%. In addition, arginine treatment decreased adipocyte size, serum concentrations of glucose, triglycerides and leptin, improved glucose tolerance, and enhanced glucose and oleic acid oxidation in skeletal muscles. The mRNA levels for hepatic fatty acid synthase and stearoyl-CoA desaturase were reduced, but mRNA levels for hepatic AMP-activated protein kinase (AMPK), PPAR coactivator-1 and carnitine palmitoyltransferase I (CPT-I) as well as muscle CPT-I were increased in response to the arginine treatment. Subsequent experiments were conducted with cell models to define the direct effects of arginine on energy-substrate metabolism in insulin-sensitive cells. In BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes and 3T3-L1 mouse adipocytes, increasing extracellular concentrations of arginine from 0 to 400 µM increased AMPK expression as well as glucose and oleic acid oxidation. Inhibition of nitric oxide synthesis moderately attenuated the arginine-stimulated increases of substrate oxidation as well as AMPK and ACC phosphorylation in BNL CL.2 cells, but had no effect in C2C12 and 3T3-L1 cells. Collectively, these results suggest that arginine increases AMPK expression and energy-substrate oxidation in a cell-specific manner, thereby reducing fat mass in diet-induced obese rats. The findings have important implications for treating obesity in humans and companion animals as well as decreasing fat deposition in livestock species.

The existence and molecular basis of an amino acid sensitive cell cycle control mechanism in human cells is described for the first time. Withdrawal of a single amino acid (arginine) from normal human fibroblast cultures caused a rapid cessation of proliferation characterised with a loss of accumulation of cells with G1 DNA content, consistent with a loss of cyclin D1-associated kinase activity and the predominance of hypo-phosphorylated pRb. Restoration of amino acid caused a synchronous reentry to cycle after a delay in excess of that for M to S transit in freely cycling populations, indicating exit from a quiescent-like state. The cellular response was thus consistent with Pardee's concept of a pivotal cell cycle control mechanism in G1, sensitive to extracellular conditions (<I>ie</I> the R-point) (Pardee, 1974). Inhibition of expression of the pRb phosphorylating kinase, cdk4, was identified as the key regulatory element in the response. A hypothetical cellular communication pathway coupling amino acid shortage to translational suppression of cdk4 (the '-Arg/cdk4 response pathway') has been synthesised from the known biochemical effects of deprivation and the recognised determinants of this suppression, including a 5'UTR mediated wild-type p53 dependency. A strategy for analysis and interrogation of this translational control mechanism, based upon the synthesis of epitope-tagged protein from full length or 5'URT truncated cdk4 cDNAs, and attempts to confirm the primacy of cdk4 suppression to the antiproliferative response by its enforced expression, are described. A highly deranged human tumour cell line (HeLa) was found to be deficient in amino acid sensitive cell cycle control. These cells continued in cycle after withdrawal but this was accompanied by a rapid loss of viability and cell disintegration. Simultaneous cell cycle blocks conferred partial protection from arginine deprivation induced cell death. The possibility that inappropriate cell cycle progression was the cause of cell death is discussed. Not all human tumour lines were vulnerable to arginine deprivation. This responsivity was found to be predicted by the status of the functional determinants identified or inferred (<I>ie</I> wild-type pRb, with cdk4 as the predominant phosphorylating kinase, intact'-Arg'cdk4 response pathway). This work describes a novel cellular response mechanism, complementing recent similar findings from elsewhere, to connect cellular biosynthetic capacity with control of cell cycle progression, with significance to the maintenance of normal cell growth regulation and suppression of the malignant phenotype, and providing a broader understanding of 'physiological' cell cycle control.

Diss. (sammanfattning) Stockholm : Karol. inst., 2000. / Härtill 6 uppsatser.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1937. / Typescript. Includes abstract and vita. With this is bound: Further studies on the growth factor required by chicks : the essential nature of arginine / By Aaron Arnold, O.L. Kline, C.A. Elvehjem, and E.B. Hart, reprinted from Journal of biological chemistry, vol. 116, no. 2 (Dec. 1936), p. 699-709. Includes bibliographical references (leaves 26-28).

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published_or_final_version / Physiology / Master / Master of Philosophy

The effect of arginine on gastric cancer cell behaviour: Molecular mechanisms of action – Abstract of thesis In gastric cancer patients undergoing surgical resection, the immunosuppression associated with surgery together with the malnutrition, which these patients often have, contribute substantially to a 40% risk of major peri-operative morbidity. Standard nutritional support in these patients has had mixed results. However, the ingestion of key nutrients, which modulate immune, inflammatory and metabolic pathways, also known as immunonutrition, offers a therapeutic modality, by reducing infectious complications by approximately 50%. However, studies have shown that arginine a key nutrient included in immunonutritional regimens not only has immune-enhancing effects but also has the ability to both stimulate and inhibit tumour growth. Therefore concerns remain with regard to the peri-operative use of arginine with regard to tumour growth and dissemination around the time of surgery. The aims of this study were to evaluate the in vitro effects of arginine on gastric cancer cell growth and invasion and the potential molecular mechanisms underlying any changes. A feasibility study was conducted to evaluate the influence of immunonutrition on gastric cancer patients by way of effect on expression of genes involved with tumour growth and invasion. The in vitro data confirmed that both stimulation of apoptosis associated with an increase in caspase 8 expression and cell cycle arrest at G2 phase independent of the effects of both p21 and p53 were associated with inhibition of AGS cell growth. No significant effect on invasion was demonstrated on AGS cells treated with arginine. The feasibility study demonstrated the challenges associated with extracting adequate quantity and quality of RNA from gastric tumour tissue. However, a total of 668 genes demonstrating a two fold change in gene expression were identified in the gastric tumour biopsies following feeding with immunonutrition. In summary, our data confirms inhibition of gastric cancer cell growth with arginine supplementation. However, the peri-operative use of arginine enriched nutritional support in patients with gastric cancer requires further assessment.

E2F1 is a transcription factor which paradoxically has major influence on both apoptosis and cell cycle progression. One of the most important questions in E2F1 biology therefore is the mechanism underlying regulation of these opposing physiological outcomes. Post-translational modifications (PTM) provide proteins with an additional layer of complexity, potentially altering interactions with partner DNA and protein. The importance of arginine methylation has recently been implicated in modulating the activity of the tumour suppression pathway proteins, p53 and E2F1. Previous studies have established that the methyltransferase, PRMT5, is responsible for the symmetrical dimethylation of E2F1, which inhibits its pro-apoptotic activity. In this thesis, E2F1 was found to be a substrate of PRMT1, which catalysed asymmetrical dimethylation of E2F1 at arginine 109. In addition, a positive correlation was found between the percentage of apoptotic cells and levels of PRMT1. Conversely, an increase in cancer cell colony formation was shown when the site of PRMT1 methylation on E2F1 was changed from arginine to lysine at position 109. These findings suggested a growth inhibition effect by PRMT1 methylation on E2F1. At the transcriptional level, depletion of PRMT1 increased E2F1 binding to the promoter region of Cdc6, a cell cycle regulator, and decreased binding to the promoter region of Apaf1, which has a pro- apoptotic role. Genome-wide ChIP-sequencing technology was undertaken and results further clarified that the depletion of PRMT1 preferably enriched E2F1 binding to promoters of positive regulators of cell proliferation and promoters of the cell cycle. Collectively, the findings of this thesis suggested that the opposing roles E2F1 demonstrated in promoting both cell proliferation and apoptosis was due to different types of arginine methylation which trigger E2F1 binding to different promoters. Lastly, arginine methylation was shown to influence protein-protein interactions. PRMT5 induction resulted in the identification by mass spectrometry of &beta;-catenin as an E2F1 interacting partner. As the Wnt/&beta;-catenin signalling pathway is broadly recognised as having pro- cell proliferation activity, this finding is consistent with previous reports that suggest the oncogenic role PRMT5 methylation has on E2F1.

Diabetic rats exhibit decrease in plasma arginine, NO synthesis and tetrahydrobiopterin in endothelial cells (EC). Treatment with L-arginine may be beneficial for enhancing NO synthesis in diseases associated with endothelial dysfunction. However, little is known about the mechanism responsible for the stimulatory effect of arginine on endothelial NO synthesis. We hypothesized that dietary arginine supplementation increases BH4 for NO synthesis in EC of diabetic rats, thereby preventing endothelial dysfunction. In experiment I, streptozotocin (STZ) induced-diabetic male Sprague Dawley (SD) rats (a model of type-I diabetes) were individually pair-fed a casein-based diet on the basis of feed intake (per kg body weight) of non-diabetic SD rats. Addition of arginine-HCl or alanine to drinking water for the rats were adjusted daily to ensure isonitrogenous provision per kg body weight. In non-diabetic rats, arginine supplementation increased plasma arginine (144%), plasma insulin (44%), EC arginine (88%), EC BH4 (106%) and EC NO synthesis (80%), compared with alanine treatment. In diabetic rats, arginine supplementation reduced body weight loss (36%), and plasma glucose (54%), and increased plasma arginine (110%), plasma insulin (209%), EC arginine (173%), EC BH4 (128%) and EC NO synthesis (125%), compared with alanine treatment. In experiment II, male Zucker diabetic fatty (ZDF) rats (a model of type-II diabetes) were individually pair-fed a Purina 5008 diet on the basis of feed intake by alanine-treated diabetic rats (per kg body wt). Addition of arginine-HCl or alanine to drinking water for the rats was adjusted daily to ensure isonitrogenous provision per kg body weight. Arginine supplementation to ZDF rats did not affect plasma glucose and insulin, reduced epidididmal fat (30%), abdominal fat (43%) and body weight gain (18%), and increased plasma arginine (273%), EC arginine (197%), EC BH4 (120%) and EC NO synthesis (122%), compared with alanine-treated ZDF rats. These results show that dietary L-arginine supplementation increases BH4 and NO synthesis in EC of both STZ-diabetic and ZDF rats. Strikingly, arginine treatment prevented hyperglycemia in STZ-diabetic SD rats and reduced obesity in ZDF rats. Collectively, results demonstrate that oral administration of arginine is beneficial for both type-I and type-II diabetic rats.

We hypothesized that subcutaneous (s.c.) adipose tissue would differ in monounsaturated (MUFA) and saturated fatty acid (SFA) composition among different depots throughout a beef carcass. To test this, 50 carcasses from a variety of breed types and backgrounds were sampled. External fat samples were collected from eight different carcass locations: round, sirloin, loin, rib, chuck, brisket, plate and flank. Samples were used to provide information on slip points, fatty acid composition and MUFA:SFA ratios. Lipids were extracted from s.c. adipose tissue by a modified chloroform:methanol procedure, and fatty acid composition and slip points were measured. The brisket was significantly lower in palmitic (16:0) and stearic (18:0) acid than the other seven sampling sites (P = 0.001). The brisket demonstrated the highest values of MUFA (P = 0.001) with the exception of possessing the lowest value of transvaccenic (18:1t11) acid (P = 0.002). There were also significant differences in the amounts of PUFA among the eight sampling sites. The lowest values were from the brisket with a mean of 25.1. The flank had the highest slip point with a mean of 39.0 (P ≤ 0.001). There was a high negative correlation shown between palmitoleic and stearic acid (R2 = 0.827). The brisket displayed the highest values for MUFA:SFA ratios (P = 0.001), whereas the flank was the lowest. Due to the significant differences amongst fat depots within bovine carcasses in their fatty acid composition we conclude that substantial differences exist across fat depots.