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Nucleotide coenzymes dot the map of metabolic pathways providing energy to drive the reactions of the pathway and play an important role in regulating and controlling energy metabolism through their shared potential energy, which is widely unobserved due to the paradox that the energy in the coenzyme pools cannot be determined from the concentration of the coenzyme couples. The potential energy of the nucleotide couples in the mitochondria or the cytoplasm is expressed in the enzyme reactions in which they take part. The energy in these couples, [NAD+]/[NADH], [NADP+]/[NADPH], [acetyl CoA]/[CoA], and [ATP]/[ADP]x[Pi], regulates energy metabolism. The energy contained in the couples can be altered by suppling energy equivalents in the form of ketones, such as, D-β-hydroxybutyrate to overcome insulin resistance, to restore antioxidants capacity, to form potential treatments for Alzheimer's and Parkinson's diseases, to enhance life span, and to increase physiological performance. © 2019 IUBMB Life, 71(5):565–579, 2019.

Cardiovascular disease (CVD) is the leading cause of death in the United States. Age is a primary risk factor for the development of CVD and middle-age is a vulnerable period where risk factors for the disease begin to exceed diagnostic thresholds. Interest has increased for the use of low carbohydrate high fat (LCHF) ketogenic diets due to their reported improvements for cardiometabolic health. Supplementation with exogenous ketone esters (KE) has been shown to increase plasma β-hydroxybutyrate (BHB) and mimic the metabolic effects of LCHF ketogenic diets. Evidence suggests elevated concentrations of plasma BHB may lower blood pressure, improve vascular function, attenuate hyperglycemic responses, and enhance cognitive function. The majority of research has been conducted in preclinical models, and whether exogenous KE supplementation has similar improvements in humans of any ages remains relatively unanswered. To address this we conducted a randomized, placebo controlled, crossover design study in healthy, sedentary, middle to older aged adults who received the exogenous KE (or placebo), and consumed the supplement for 2-weeks (3x/day, 15 minutes prior to each meal; breakfast, lunch, and dinner). Our first hypothesis was to test that KE supplementation would improve vascular function by increasing flow-mediated dilation, reducing arterial stiffness, and lowering blood pressure. Secondly, we hypothesized that KE supplementation would attenuate the glycemic response to an oral glucose tolerance test, improve glycemic variability, and show reductions in postprandial glucose levels. Thirdly, we tested the hypothesis that KE supplementation would improve cognitive performance by showing improvements in processing speed, memory, attention control, and executive functions. In support of our first hypotheses, KE supplementation increased flow-mediated dilation (8.1 ± 1.3 vs. 7.7 ± 1.2%, p = 0.023), but it did not show any difference in arterial stiffness or blood pressure. In contrast to our second hypotheses, following the KE supplementation intervention there were no significant difference from the placebo in terms of glycemic response, variability or mean 2-hour post-meal glucose. In support of our third hypotheses, we found a significant improvement in measures of working memory (7.55 ± 0.93 vs. 7.27 ± 0.29, p = 0.026) and inhibitory control (80 ± 38 vs. 87 ± 32ms, p = 0.035) following the 14-day KE supplementation. More research is needed to elucidate the effects of KE on cardiometabolic health and cognition. / Doctor of Philosophy / Recently there has been an increase in the popularity of low carbohydrate high fat (LCHF) ketogenic diets, with advocates for the diet claiming increased benefits in weight loss and blood glucose control, therefore leading to an increased interest for its use in the treatment for cardiovascular disease, obesity, and diabetes. As more evidence has accumulated much of the impact LCHF ketogenic diets are said to have, has been attributed to a state known as nutritional ketosis, which occurs in response to the restriction of carbohydrates from the diet. Ketone esters (KE) have been shown to effectively elevate ketone bodies (alternative energy produced by the body during times when glucose stores are low) without the need of altering one's own diet, however, this method of inducing ketosis is still lacking evidence for its impact on cardiometabolic health in humans. The purpose of these studies is to determine the effect of having sustained elevations of ketone bodies on our health and cognition in humans. Study 1 included healthy, sedentary middle to older aged adults who consumed a KE for 2-weeks (3x/day, prior to each meal) and a placebo. Following supplementation participants completed test to assess our vascular health and blood sugar control. Study 2 included healthy, sedentary middle to older aged adults who consumed a KE for the same 2-weeks (3x/day, prior to each meal) and a placebo. Participants underwent a series of tests to assess cognitive performance. Overall, after a 2-week supplementation period we found significant improvements in our blood vessel function and with cognitive performance where we saw improvements in working memory, and inhibitory control.

Alternative bone repair strategies are frequently sought after in orthopaedic surgery to address the growing need for improved morbidity and healing rates. This thesis sought to initiate and validate such an alternative, harnessing the flexible nature of a biomaterial substrate and the unique potential of glycosaminoglycan sugars. A novel, biodegradable biomaterial polymer, PHBV, has previously been identified to have the potential to mimic the characteristics of bone necessary for tissue repair and in this study, it was hypothesized that PHBV would be able to support bone formation. When tested in vitro, PHBV was found to support osteoblast cell attachment, proliferation and differentiation, despite its rougher, more hydrophobic surface characteristics compared to tissue culture plastic (TCP). However, unlike the progression of cells on TCP, PHBV caused a developmental delay at each stage of osteogenesis, suggesting a sub-optimal cell-substrate interaction. The expression profiles of genes involved in the maintenance of the extracellular matrix were monitored to investigate this phenomenon further. The results suggested that cells cultured on PHBV appeared to preference 7 against a collagen-based ECM and, instead, trigger an increase in the expression of other factors, such as osteopontin, presumably to modify the biomaterial microenvironment to optimise continued growth and differentiation. This finding led to the next hypothesis that functionalisation of PHBV with suitable compounds could optimise and enhance the osteogenic development at the implant site by facilitating the desired and appropriate cell-substrate interactions. Non-protein factors are often preferred for functionalisation to material scaffolds over proteins, as they are relatively robust and can survive many of the processes used in the manufacture of biomaterials. Glycosaminoglycan (GAG) sugars were appropriate candidates for this purpose, as they are not only abundantly expressed in bone, but more importantly, they are capable of binding and facilitating the activity of growth factors. Furthermore, they are resistant to several environmental influences including changes in pH, heat and desiccation. To identify a GAG that could be integrated with PHBV or any other biomaterial substrate, GAGs were extracted from phenotypically-distinct stages of MG-63 osteosarcoma cells. These GAGs were identified to display gross structural differences, as well as differences in the enzymes synthesising them, between immature and mature osteoblastic cells, with the increased production of a larger GAG species observed as the cells differentiated. Unexpectedly, however, when these GAGs were subsequently dosed back into the media of growing MG-63 cells, their bioactivity did not match the stage at which they had been harvested: all GAG species were able to influence cell survival and growth to varying degrees but were not capable of affecting cell differentiation. However, if these same GAGs were exposed to cells by first being attached to the growth substrate, they induced varying degrees of aggregation in human mesenchymal stem cells (hMSCs), with more mature GAGs producing the most profound effects. Interestingly, a similar phenomenon was not observed when MG-63 cells where cultured in a similar manner. A direct correlation between the GAGs expressed by osteoblasts and the specific cellular processes they functionally influence has yet to be identified. While the experiments presented here demonstrate an effect of GAGs in osteoblastic cell survival, a role for GAGs in the progression of bone formation was not revealed. Loss-of-function studies were therefore necessary to determine the role of GAGs in bone, but this was hampered by the limited availability of procedures that allow the alteration of GAGs and the subsequent detection of these effects. Therefore, a tool to screen the efficacy of a loss of GAG function was developed. TAT-EGFP, a purpose-designed fluorescent GAG-binding peptide, was able to confirm that treatment with sodium chlorate was an effective 8 strategy to hinder GAG expression in MG-63 cells with minimal cytotoxicity to the cells. Following more extensive studies with chlorate treatment, it was found that a recoverable disruption to both proliferation and mineralisation could be induced in MG-63 cells. This suggested a role for GAGs in osteogenesis. A series of experiments then carried out following gene expression microarray analysis indicated that GAG de-sulfation by chlorate gives rise to an S-phase block in the cell cycle and a disruption to the actin cytoskeleton, which appeared to be associated with a change in the activity of cell-surface proteoglycans, most likely syndecan 4. It was also found that cells up-regulated plasma membrane ALP activity and cholesterol synthesis, presumably in an attempt to recover from a chlorate-induced loss in GAG function. Cholesterol is known to be important in establishing connections between membrane elements and the actin cytoskeleton, and its up-regulation here may reflect dysfunctions in these units and a dysfunction in syndecan 4 activity. With further confirmation, this would suggest that syndecan 4 plays a pivotal role in maintaining osteogenesis, in at least MG-63 cells, and that sulfated GAGs function principally to facilitate this role. The effective use of GAGs in bone repair strategies will require further understanding of GAG/syndecan 4/osteogenesis relationship.

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