Increasingly, cell-based therapy with autologous progenitor populations, such as endothelial progenitor cells (EPC), are being utilized for treatment of vascular diseases. However, both the number and functional capacity are diminished when cells are derived from patients with established risk factors for coronary artery disease (CAD). Herein, we report that inhibition of glycogen synthase kinase 3 (GSK) can improve both the number and function of endothelial progenitor cells in patients with CAD or diabetes mellitus (DM) leading to greater therapeutic benefit. Specifically, use of various small molecule inhibitors of GSK (GSKi) results in a 4-fold increased number of EPCs. Moreover, GSKi treatment improves the functional profile of EPCs through reductions in apoptosis, improvements in cell adhesion through up-regulation of very-late antigen-4 (VLA-4), and by increasing paracrine efficacy by increasing vascular endothelial growth factor (VEGF)secretion. Therapeutic improvement was confirmed in vivo by increased reendothelialization(RE) and reductions of neointima (NI) formation achieved when GSKi-treated cells were administered following vascular injury to CD-1 nude mice. Because cell-based therapy is technically challenging, we also tested a strategy of local delivery of GSKi at the site of arterial injury through GSKi-eluting stents. In vitro, GSKi elution increased EPC attachment to stent struts. In vivo, GSKi-eluting stents deployed in rabbit carotid arteries resulted in systemic mobilization of EPCs, improved local RE, and important reductions in in-stent NI formation. Finally, we tested the ability of GSKi to improve EPC-mediated arterial repair in patients with DM. As in patients with CAD, GSKi treatment improved EPC yield and diminished in vitro apoptosis. Utilizing a proteomics approach, we identified Cathepsin B (catB) as a differentially regulated protein necessary for reductions in apoptosis. Indeed, antagonism of catB prevented GSKi improvements in GSKi treated EPC mediated arterial repair in a xenotransplant wire injury model. Thus, our data demonstrates that GSKi treatment results in improvements in EPC number and function in vitro and in vivo resulting in enhanced arterial repair following mechanical injury. Accordingly, GSK antagonism is an effective cell enhancement strategy for autologous cell-based therapy with EPCs from high risk patients such as CAD or DM.

Performance and exercise capacity in prolonged continuous exercise has been shown to be enhanced by the ingestion of dilute (-6%) carbohydrate-electrolyte (CHO-E) solutions during exercise. There is a wealth of literature on the effects of CHO-E solutions on prolonged, continuous exercise however this is not the case for highintensity intermittent exercise. One reason for this is the difficulty of replicating the demands of multiple-sprint sports within the laboratory. The development of the Loughborough Intermittent Shuttle Running Test (LIST), a protocol that simulates the physiological demand of multiple-sprint sports, has allowed for further study in this area. Sports drinks contain a variety of different types of carbohydrate, including synthetic polymers of maltodextrins. The rationale for these is two-fold; firstly there is some evidence to suggest that these glucose polymers of low osmolality may empty faster than isoenergetic glucose solutions especially at dilute (-6%) concentrations. Secondly CHO-E solutions formulated from maltodextrins with low dextrose equivalents are less acidogenic and have lower cariogenic potential than simple glucose solutions, thus making them preferable for tooth health. Therefore the purpose of this thesis was to examine the effects of a 6.4% hypotonic synthetic polymer maltodextrin CHO-E solution on muscle metabolism and physiological performance during prolonged high-intensity intermittent running (LIST). This thesis contains four experimental studies. From the results of study 1 it was concluded that the ingestion of a 6.4% hypotonic CHO-E solution provided no ergogenic benefit to running capacity during the LIST despite blood and hormonal data suggesting that the solution was effective at delivering glucose. As isotonic CHO-E solutions have shown to have ergogenic properties during LIST running the purpose of study 2 was to compare isoenergetic hypotonic and isotonic CHO-E solutions during LIST running. The results from this study suggested that performance, as measured by exercise capacity and sprint speed, was not affected by either treatment. In study 3 the protocol was modified to extend the exercise duration so that a greater demand was placed on the subjects' glycogen reserves. The results from this study further confirmed that exercise capacity in LIST running was not influenced by CHO provision during exercise however the habitual CHO intake of the subjects may have masked any ergogenic properties of the CHO-E solution and led to increased performance in the control trials. In the final study the subjects' pre-trial exercise and dietary CHO intake was manipulated to increase endogenous glycogen concentrations. The results showed that in these subjects the ingestion of a 6.4% hypotonic CHO-E solution increased exercise capacity during the LIST by 21%. Muscle biopsy analyses revealed a lower net muscle glycogen utilisation in the CHO trial post-90 min (p=0.07). There is evidence in the literature to suggest that there is an ergogenic effect of CHO ingestion during the LIST. However the results presented in this thesis suggest that when endogenous glycogen stores are moderate the ingestion of a CHO-E solution may suppress lipid oxidation without a concomitant increase in CHO oxidation and thus LIST capacity is not enhanced. In contrast, when endogenous glycogen concentrations are elevated through dietary manipulation there is an ergogenic benefit from the ingestion of hypotonic CHO-E solutions during LIST exercise.

The incidences of obesity and type 2 diabetes are reaching epidemic proportions worldwide. A cardinal feature of these conditions is resistance to the effects of the hormone insulin and a resulting hepatic overproduction of glucose. Insulin resistance is also implicated in a range of liver diseases including non-alcoholic fatty liver disease (NAFLD) and hepatitis C infection. Insulin is released after a meal and acts on liver, skeletal muscle and adipose tissue to reduce blood glucose concentration. In the liver, insulin inhibits the production and release of glucose into the circulation and stimulates its storage as glycogen. Glucagon, on the other hand, is present in the fasting state and causes breakdown of hepatic glycogen along with production of new glucose. This glucose is released from hepatocytes into the circulation. For the studies in this thesis, functional assays to measure various aspects of hepatic glucose metabolism in vitro were developed. This included measuring glucose output into culture medium, hepatocyte uptake of radiolabelled glucose and incorporation into glycogen, and total cellular glycogen content. These assays were used to investigate glucose metabolism in primary rat hepatocytes and FaO rat hepatoma cells. Both cell types responded to physiological concentrations of insulin, showing decreased glucose output and increased glycogen synthesis. Glucagon increased glucose output and reduced glycogen synthesis in primary cells but had no effect on FaO cells. Factors that have been identified that may inhibit or potentiate insulin action were investigated. Increased body iron stores have been linked with insulin resistance. De-ironing patients improves insulin sensitivity, suggesting a causal relationship between iron and insulin resistance. Hepatocytes store the majority of the body’s excess iron. This project investigated the effects of increasing hepatocyte iron stores, through addition of ferric ammonium citrate (FAC), or depleting iron stores by chelation with dipyridyl. Small increases or decreases of iron in primary cells had negative effects on cell viability, resulting in significantly reduced glucose output and glycogen synthesis. Dipyridyl treatment had similar effects on FaO cells as on primary cells but FAC treatment increased FaO glucose output, although significant iron loading was not achieved. With concentrations of FAC and dipyridyl low enough to not significantly influence cell viability, insulin sensitivity was not affected. Adiponectin is an insulin sensitiser and appears to exert this effect primarily through the liver. Adiponectin can also reduce hepatic glucose output (HGO) independent of insulin. It is believed adiponectin mediates its effects in liver, skeletal muscle and adipose tissue through activation of AMP-activated protein kinase (AMPK). In muscle, p38 mitogen-activated protein kinase (p38 MAPK) has been implicated as a downstream component of adiponectin signalling. In this study, recombinant human adiponectin was produced and collected in culture medium which was then concentrated. Despite the presence of both high molecular weight (HMW) and low molecular weight (LMW) adiponectin multimers, the concentrated medium had no effect on HGO in the presence or absence of insulin. Concentrated adiponectin medium did not affect AMPK or p38 MAPK phosphorylation in hepatocytes or other cell types previously shown to respond to adiponectin. However, commercially-sourced purified recombinant adiponectin also failed to elicit any observable responses. AICAR and metformin are pharmacological activators of AMPK and were used to treat primary rat hepatocytes and FaO cells. These treatments reduced HGO independent of insulin in both cell types. In primary cells, these reductions were partially inhibited with Compound C, an AMPK inhibitor, suggesting that both AICAR and metformin action is at least partly AMPK dependent. In FaO cells, Compound C only inhibited the AICAR-mediated reduction of glucose output, indicating that metformin may act independently of AMPK in these cells. Compound C significantly inhibited AICAR and metformin-mediated increases in AMPK phosphorylation in primary hepatocytes and FaO cells. There was a trend towards inhibition of AICAR-mediated p38 MAPK phosphorylation with Compound C treatment, suggesting that p38 MAPK may lie downstream of AMPK in hepatocytes. Adenoviral expression of constitutively active (CA) and dominant negative (DN) AMPK in primary rat hepatocytes was used to further study the role of AMPK in hepatic glucose metabolism. Despite significant expression of CA AMPK, phosphorylation of downstream acetyl-CoA carboxylase (ACC) was not affected nor was HGO. CA AMPK did, however, increase phosphorylation of p38 MAPK. DN AMPK completely inhibited AICAR-mediated AMPK phosphorylation and partially inhibited phosphorylation of ACC. In addition, AICAR-mediated phosphorylation of p38 MAPK was inhibited by DN AMPK. Taken together, these results suggest that p38 MAPK is downstream of AMPK in hepatocytes. The implication that p38 MAPK is involved in hepatic AMPK signalling is a novel finding. A greater understanding of this pathway in the liver may identify novel therapeutic targets, leading to improved treatment strategies for metabolic disorders linked to obesity and type 2 diabetes.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2004. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2009. / Title from first page of PDF file (viewed on June 8, 2009). Includes bibliographical references.

Thesis (Ph.D.)--Georgetown University, 2008. / Includes bibliographical references.

Thesis (Ph.D.)--University of Melbourne, Dept. , 200. / Typescript. Includes bibliographical references.

Thesis (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 118-133.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 75-84).