Date of Award
6-1-2021
Degree Name
Doctor of Philosophy
Department
Molecular, Cellular, and Systemic Physiology
First Advisor
Hales, Dale
Abstract
We are the first lab to report the occurrence of a diet-induced transsulfuration (TS) blockade associating with elevated S-adenosylmethionine (i.e. SAM) synthesis, enhanced lifespan and enhanced reproductive capacity, in a vertebrate animal model. In this paradoxical study, we used LC-MS/MS-derived metabolomics data to report the effects of flaxseed (Linum Usitatissimum) on one-carbon metabolism in White Leghorn laying hens (Gallus gallus). Flaxseed contains a vitamin B6-antagonizing molecule called ‘linatine’ that is particularly effective at reducing vitamin B6 levels in small rodents and poultry. Linatine reduces TS flux through its inhibition of the vitamin B6-dependent enzymes cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE). In this study, our flaxseed-fed hens displayed decreased 4-pyridoxic acid and decreased pyridoxamine, concomitant with 15-fold elevated cystathionine. Homocysteine levels were stable in flaxseed-fed hens despite such highly elevated cystathionine. This is an astonishing finding, because mammalian models would predict the induction of hyperhomocysteinemia (i.e. elevated homocysteine) when cystathionine is so highly elevated (1). We are therefore reporting a phenomenon that might be unique to birds. Our metabolomics data indicate increased consumption of one-carbon donor molecules (e.g. choline, betaine, dimethylglycine, serine, etc) in flaxseed-fed hens, probably as a means of fueling the betaine homocysteine methyltransferase (BHMT) and methionine synthase-B12 (MS-B12) reactions. This modeling approach provides a rationale that flaxseed-fed hens increase their rate of homocysteine remethylation via BHMT and MS-B12, and in turn this would maintain stable homocysteine levels in the animal. We observed that the culminating outcome is elevated synthesis of SAM and an elevated SAM:SAH ratio. The associated biological outcomes are extended lifespan and increased reproductive capacity (i.e. increased daily egg laying) in flaxseed-fed hens. Our data further indicate that flaxseed tremendously stimulates a glucagon-like phenotype in hens. Specifically, flaxseed-fed hens exhibit 3-fold elevated glycated hemoglobin (HbA1c), 2-fold elevated serum free fatty acids (FFAs), 10 to 14% reduced body weight, and slightly reduced plasma pyruvate. These phenotypic markers suggest that glucagon might also play a large role in extending lifespan and enhancing reproductive capacity in flaxseed-fed hens. We predict that the anti-vitamin B6 effects of flaxseed are responsible for stimulating this glucagon-like phenotype in hens. Therefore, we conclude that the vitamin B6-antagonizing effects of linatine (via flaxseed dieting) improve liver function, body leanness, egg laying and survival, in a manner associated with increased SAM synthesis, increased blood FFA levels and increased blood glucose levels, in laying hens. The second part of this dissertation is used to test the hypothesis that polyunsaturated fatty acids (PUFAs) regulate lipid metabolism, increase mitochondrial respiration and decrease E-cadherin expression, in laying hen ovarian tumors. Researchers are unaware of the mitochondrial effects of PUFAs within laying hen ovarian tumors, and only a few publications exist regarding the analysis of E-cadherin in laying hen ovarian cancer. The results of this present work suggest that dietary PUFAs accumulate within laying hen ovarian tumors, and these same tumors exhibit decreased gene transcripts that govern de novo lipogenesis (i.e. FASN). Simultaneously, these tumors exhibit elevated transcripts for oxidative phosphorylation (OXPHOS) and decreased transcripts for phase 2 antioxidant enzymes. These patterns associated with decreased transcript levels of CDH1 (the gene for E-cadherin) in ovarian tumors, but no effect on full length 120kDa protein was observed by PUFAs versus our control diet. Interestingly, the effect of PUFAs on E-cadherin occurred at the level of the cleaved 37kDa and 80kDa E-cadherin fragments, such that PUFAs reduced their levels in hen ovarian tumors. We therefore developed a system for depleting the 80kDa E-cadherin fragment from hen ascites fluid (i.e. fluid from a hen that had ovarian cancer), so that we could test our hypothesis that the 80kDa E-cadherin fragment is important for supporting mitochondrial respiration in normal ovarian surface epithelial cells (IOSE80s). Mitochondrial membrane potential was decreased in IOSE80s when the 80kDa fragment was removed from the ascites; however, there was no effect on basal oxygen consumption in subsequent tests using extracellular flux analysis with Seahorse XFp. During our analysis of microRNA-200a-3p (miR-200a) we did not observe any effect of diet on miR-200a within hen ovarian tumors; however, we did observe that miR-200a levels increased within the tumor when going from stage 2 to stage 4 disease. Overall, we observed reduced risk of ovarian cancer, stage 4 ovarian cancer, multiple peritoneal tumor involvement and cancer-associated mortality, in laying hens that consumed a diet that was supplemented with PUFAs.
Access
This dissertation is Open Access and may be downloaded by anyone.