Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / Typescript. Vita. Includes bibliographical references (leaves 141-150). Also available on the Internet.

published_or_final_version / Anaesthesiology / Master / Master of Philosophy

Pyrithiamine-induced thiamine deficiency in rats is a well-established animal model of Wernicke's Encephalopathy (WE). This thesis project, submitted as four articles, presents an examination of metabolic events that contribute to the selective neuronal lesions observed in the medial thalamus (MT) of thiamine-deficient (TD) rat. In addition, the phenomenon of glucose-precipitated worsening of neurological status in WE patients (Wallis et al., 1978; Watson et al., 1981) is explored. / Lactate accumulation is known to occur selectively in regions of the TD brain, which ultimately express neuronal cell death (McCandless, 1982; Munujos et al., 1996). In Article 1, the metabolic origin and cellular localization of region-selective lactate accumulation in the MT of TD rats was studied using combined 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Parallel studies were performed to examine the effects of glucose loading on regional brain lactate synthesis in TD animals. Thiamine deficiency caused focal increases in the de novo synthesis of lactate via elevated glycolytic flux in the MT, while contribution via pyruvate recycling and the periphery remained nominal. Lactate levels remained unaltered in the frontal cortex (FC), a brain region that is spared in thiamine deficiency. Administration of a glucose load intensified the selective increases in lactate de novo synthesis and accumulation in the MT of TD rats, positing a role for lactic acidosis in the glucose-precipitated worsening of neurological status in TD patients. Accordingly, Article 2 addresses the effect of glucose loading on local cerebral pH in the vulnerable MT, compared to the FC, of TD rats. Administration of a glucose load resulted in detrimental decreases in regional pH selectively in the MT, implying that alterations of brain pH contribute to the pathogenesis of thalamic neuronal damage and consequent cerebral dysfunction in WE. / Region-specific alterations in the steady state levels of cerebral amino acid neurotransmitters have been well-documented in experimental animal models of thiamine deficiency (Butterworth et al., 1979; Butterworth & Heroux, 1989; Gaitonde et al., 1975; Plaitakis et al., 1979); however, the dynamics of these changes have never been systematically explored. In Article 3, we examined the metabolic fluxes through thiamine-dependent pyruvate dehydrogenase (PDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) using multinuclear NMR spectroscopy. Furthermore the cellular localization of the metabolic changes in relation to regional vulnerability to thiamine deficiency was addressed. Our studies clearly demonstrate that early decreases m metabolic flux through alpha-KGDH result in commensurate declines in aspartate concentrations in the MT of TD rats. Impairments to PDH flux manifest secondarily to the metabolic block at alpha-KGDH, likely due to depleted oxaloacetate pools. As a result of impaired pyruvate oxidation, declines in the de novo synthesis of glutamate and GABA ensue. The present findings also suggest that inhibition of flux through alpha-KGDH in TD brain occurs primarily in the neurons, while astrocytes possess compensatory mechanisms, i.e. the anaplerotic pathway, to replenish oxaloacetate concentrations via metabolic pathways that do not involve thiamine-dependent enzymes. / In Article 4, we investigated the regional effects of thiamine deficiency on the activity of thiamine-dependent branched-chain alpha-ketoacid dehydrogenase (BCKDH) and the resultant effects on regional cerebral branched-chain amino acid (BCAA) oxidation. Thiamine deficiency resulted in significant impairments in BCKDH activity; while parallel studies on enzyme distribution confirmed a lower oxidative capacity for BCAAs in the MT compared with the Fe. / The data presented in these four articles confirm and extend findings for the region-selective impairments in thiamine-dependent metabolic processes as the foundation of vulnerability of the MT to thiamine deficiency. In addition, glucose loading of TD rats exacerbates both lactic acidosis and impaired pyruvate oxidation in this vulnerable brain region, positing a role for these processes in the glucose-precipitated worsening of neurological status in TD patients. Impaired oxidative metabolism of glucose and BCAAs in the MT leads to the accumulation of potentially harmful metabolic intermediates, contributing to the mitochondrial dysfunction, cellular energy failure and ultimately neuronal cell death observed in thiamine deficiency.

Treatment of diabetes mellitus is mainly focused on glycaemic control regulated by insulin and takes place in insulin sensitive tissues like skeletal muscle which accounts for 75% of glucose metabolism. Plant derived compounds that have anti-diabetic potential are currently being investigated for diabetes treatment as they are cheap and non-toxic. Oleanolic acid (OA), a triterpene found in a wide variety of plants has been shown to have anti-diabetic effects but its mechanism of action, especially on the insulin signalling cascade has not been fully elucidated. The aim of the present study was to investigate the effects of OA on the PI3K/Akt insulin signalling cascade in skeletal muscle and skin of streptozotocin induced diabetic male Sprague-Dawley rats. Male Sprague-Dawley rats (non-diabetic and diabetic) were treated with insulin (4IU/ kg bw), OA (80 mg/kg bw) and a combination of OA + insulin in an acute and sub-chronic study. The study showed that OA does not reduce blood glucose levels in type 1 diabetic rats but enhances insulin stimulated hypoglycaemic effects. In the acute study OA was shown to activate Akt and dephosphorylate GS in skeletal muscle of streptozotocin induced diabetic rats. In the sub-chronic study OA and OA + insulin increased expression of GS in skeletal muscle of diabetic rats. GP expression was decreased by OA and OA + insulin treatments in skeletal muscle whilst in skin it was increased by both treatments. OA increased both GS and GP in skeletal muscle whilst in skin they were decreased. OA + insulin treatment increased GS and decreased GP activities in skeletal muscle and increased activity of both enzymes in skin of diabetic rats. OA increased the amount of glycogen in both muscle and skin whilst OA + insulin reduced the amount of glycogen. OA and OA + insulin treatment showed some protective effects against liver and muscle damage as there were reductions in serum LDH, ALT and AST levels. In conclusion, oleanolic acid in synergy with insulin can enhance activation of the insulin signalling pathway and there was evidence of OA activation of insulin signaling enzymes independent of insulin. / Thesis (M.Sc.)-University of KwaZulu-Natal, Durban, 2013.

Human milk contains ~ 25 µg/mL of soluble cluster of differentiation 14 (sCD14) protein, a pattern recognition receptor (PRR) that triggers the innate immune system to respond to bacterial lipopolysaccharide (LPS). To date, the role of CD14 in the digestive tract of breast fed infants has not been well characterized and is the subject of this thesis. To investigate the biodistribution of proteins such as CD14 in vivo, a novel method for 14C radiolabeling of proteins to high specific radioactivity was developed using in vacuo methylation. Bovine serum albumin (BSA) and casein were used as test proteins to determine the following: 1) The efficacy of the in vacuo radiolabeling procedure; 2) The extent of incorporation of the 14C-label into the organs of oro-gastric gavaged 10 day old Sprague Dawley rats. [14C]BSA, [14C]casein and [14C]CD14 were prepared with specific radioactivities of 10 400, 10 800 and 163 000 dpm/µg, respectively. After feeding 6.25 µg of 14C-labeled proteins, quantifiable levels of 14C were found in the stomach, jejunum, duodenum, ileum, large intestine, intestinal luminal flushes, blood, liver, spleen and kidneys of rats. The accumulation of radiolabel in the organs of [14C]CD14 fed rats was temporally and spatially distinct from [14C]BSA and [14C]casein. Most notably, the label persisted in the stomach 480 min post-gavage. To design a neonate animal model for biodistribution, the segmental and total gastrointestinal transit times (GItt) were measured in two litters of 10 and 15 day old Sprague Dawley rat pups using barium sulfate. Ten day old rat pups that remained with and without the dam had a total gastrointestinal transit time of 13.8 ± 0.9 hr and 9.3 ± 0.7 hr, respectively. This decrease (p<0.05) in total gastrointestinal transit time in the absence of the dam was age dependent, as it was not observed (p>0.05) in the 15 day old rat pup litter. The immunological impact of an exogenous sCD14 source was examined in human peripheral blood mononuclear cells (PBMC). Pre-treatment of CD14+ monocytes with sCD14 had a protective effect, one of reducing the production of proinflammatory cytokines (TNF-α, IL-6, IL-8, IL-1β) when challenged with LPS. 14C was absorbed by neonate rats upon ingestion of [14C]CD14 and exposure to relatively high concentrations of rCD14 led to a reduction in inflammation. This may be beneficial to initial gut colonization in breast-fed newborns. / Alexander Graham Bell NSERC CGS M scholarship. Japan Society for the Promotion of Sciences, Summer in Japan Fellowship. Funded by the Canadian Institutes of Health Research, Institute of Nutrition Metabolism and Diabetes Grant #82816 “Fate and function of breast milk and recombinant human CD14 at mammary and newborn gastrointestinal mucosal epithelia”.

Problem: Gestational obesity and insulin resistance pose a significant threat to the future health of our population. Mothers and children of these metabolically maladaptive pregnancies experience extensive morbidity and mortality. This study characterized postprandial insulin sensitivity in female Sprague Dawley rats at 5-days and 15-days gestation. Based on these findings, a model of gestational obesity was developed using 35% sucrose supplementation (SS). The efficacy of a preventative and a therapeutic intervention at modulating sucrose-induced gestational insulin resistance in Sprague Dawley rats was elucidated. Methods: Insulin sensitivity in the post-prandial state includes insulin-dependent and Hepatic Insulin Sensitizing Substance (HISS)-dependent components, and can be characterized with the Rapid Insulin Sensitivity Test (RIST). HISS is a putative hepatic factor released in the fed-state that selectively increases glucose uptake in skeletal muscle, kidney and heart. In the first phase of this study, the effects of insulin were assessed in pregnant animals (5 and 15-days gestation) and their virgin controls. Groups of 15-day gestation and virgin animals had SS for 8-weeks (with a 2-week recovery), 10-weeks or 22-weeks. Half of all of the 10-week SS animals were treated with either SAMEC (given chronically, containing S-adenosyl-methionine, vitamin C and vitamin E) or BENAC (given once the night before the acute study, containing bethanechol chloride and n-acetyl-l-cysteine). Body weight, weight gained over the gestational period, fat pad mass, post-prandial glycemia, plasma insulin and triglyceride concentrations were measured in all groups. Results: 5-days gestation was associated with preserved direct insulin action and increased HISS-dependent insulin action. 15-days gestation was associated with a mixed insulin resistance: both direct and HISS-dependent insulin action were reduced. SS in these pregnant and virgin rats eliminated HISS-dependent insulin action, associated with hyperinsulinemia, hypertriglyceridemia and obesity. In the SS group given 8-weeks of sucrose (then a 2-week recovery), virgins spontaneously partially recovered HISSdependent insulin action. At 15-days gestation, recovery was complete with reductions in plasma insulin and triglyceride concentrations, and normalization of body weight and fat pad mass. 10-week SS resulted in complete absence of HISS-dependent insulin action, and produced a model of gestational obesity. Prolonged (22-week) SS did not result in hyperglycemia or elevation of plasma insulin concentration above 10-week SS. SAMEC in 10-week SS 15-day pregnant and virgin rats prevented the loss of HISS-dependent insulin action, and normalized plasma insulin and triglyceride concentrations. BENAC given to 10-week SS virgin and 15-day pregnant rats normalized overall insulin responses secondary to restoration of HISS-dependent insulin action. This was accompanied by a reduction (for virgins) and normalization (at 15-days gestation) of plasma insulin and triglyceride concentrations. In 15-day pregnant controls (no sucrose), BENAC increased the HISS-dependent insulin action significantly above baseline and reduced plasma triglycerides and insulin below control levels. Conclusions: These results suggest an explanation for the insulin resistance occurring in pregnancy, whereby HISS may facilitate metabolic adaptation. HISS may represent a pathophysiological missing link in the insulin resistant disorders of pregnancy. These findings substantiate a series of unexplored treatments (including BENAC and SAMEC) for the epidemic of gestational obesity and diabetes in mothers-to-be and the deleterious metabolic programming occurring in the next generation.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references (leaves 180-204). Also available online.

Thesis (Ph.D.). / Written for the Dept. of Pharmacology and Therapeutics. Title from title page of PDF (viewed 2009/06/05). Includes bibliographical references.

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Pharmacology and Toxicology. Title from resource description page. Includes bibliographical references.

Thesis (Ph.D.)--Uniformed Services University of the Health Sciences, 2006 / Typescript (photocopy)