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The non-phosphorylated oxidative pathway of glucose dissimilation has been established in Pseudomonas aeruginosa and evidence for phosphorylated pathways, other than the Embden-Meyerhof scheme, has been obtained. In the present study the non-phosphorylated and phosphorylated pathways of glucose degradation have been investigated with cell-free extracts of this organism. Gluconolactone was shown to be an intermediate in the oxidation of glucose to gluconic acid. The enzymatic hydrolysis of the lactone ring has an absolute magnesium ion, or divalent cation requirement. In the presence of phosphate buffer magnesium was chelated and effectively removed from participation in the enzymatic reaction. As has been reported in the literature, the product of glucose and gluconic acid oxidation was identified as 2-ketogluconate. In the presence of adenosine triphosphate (ATP), glucose and gluconate are phosphorylated and the kinases involved, therefore, link the non-phosphorylated with the phosphorylated pathways. The demonstration of triphosphopyridine nucleotide (TPN) linked dehydrogenases for glucose-6-phosphate and 6-phosphogluconate, as well as the production of glucose-6-phosphate and 3-phosphoglyceraldehyde from cell-free extracts with gluconate or ribose plus ATP, illustrated the presence of a functional pentose phosphate cycle in this organism. An active 6-phosphogluconate dehydrase and a 2-keto-3-deoxy-6-phosphogluconate aldolase were demonstrated by the production of pyruvic acid from 6-phosphogluconate and indicated the presence of the Entner-Doudoroff pathway. The oxidation of 3-phosphoglyceraldehyde to 3-phosphoglyceric acid initiated by a TPN specific 3-phosphoglyceraldehyde dehydrogenase, and the conversion of phospho-enol-pyruvate to pyruvic acid was shown. It is suggested that the trioses are immediately concerned in the observed CO₂ fixation by this organism. Fructose-1,6-diphosphate aldolase, fructose-1,6-diphosphate phosphatase and phosphohexoisomerase may be involved in the formation of glucose-6-phosphate from triose phosphates. A direct link between 2-ketogluconate and the phosphorylated pathways could not be shown but the reduction of the phosphate ester of the compound was demonstrated. The feasibility of 2-ketogluconate undergoing a 3:3 split is presented. No attempt has been made to estimate the relative importance of the various pathways of glucose dissimilation as it is felt that this is determined by the conditions and stages of growth of the organism. / Land and Food Systems, Faculty of / Graduate

An effort has been made to demonstrate that the major pathway for glucose oxidation in Pseudomonas aeruginosa (ATCC 9027) involves the sequence of reactions: glucose →gluconate → 2-ketogluconate → 2-keto-6- phosphogluconate → 6-phosphogluconate. It appears however, that extracts of this organism are capable of phosphorylating glucose directly, that is, to yield glucose-6-phosphate and subsequently 6-phosphogluconate. A study of this latter pathway was felt to be necessary in order to evaluate the likelihood of it being a major alternative to the established non-phosphorylated pathway. Since it is known that glucose-6-phosphate dehydrogenase from P. aeruginosa and other microorganisms as well as from certain animal tissues exhibits a marked sensitivity to various nucleotides particularly to adenosine triphosphate. A study of this inhibition was made in order to assess the possible role that this sensitivity may play in determining the importance of this pathway as the major route of glucose oxidation. Enzyme fractionation studies revealed that hexokinase and glucose-6-phosphate dehydrogenase could be separated either by an ethanol fractionation or by an alkaline ammonium sulfate fractionation. The best separation of dehydrogenase was obtained using ethanol although hexokinase could only be isolated using the alkaline ammonium sulfate method. Cell free extracts of P. aeruginosa oxidize glucose to 2-keto-gluconate but carry the reaction no further. This represents a consumption of l µM of oxygen per µM glucose. In the presence of ATP the amount of oxygen consumed was reduced to a maximum of 0.5 µM per µM glucose, indicating the accumulation of a compound less oxidized than 2-ketogluconic acid. 6-phosphogluconate appeared to conform to the requirements of such a compound. Chromatographic analysis of reaction mixtures containing ATP revealed the accumulation of a phosphorylated compound which could not be identified. Under in vitro conditions both pathways appear to be operable but the non-phosphorylated pathway accounts for most of the glucose in the metabalizing organism. / Land and Food Systems, Faculty of / Graduate

Adipose tissue expands via differentiation of preadipocytes into adipocytes (adipogenesis) and/or hypertrophy of existing adipocytes. A low adipogenic capacity promotes adipocyte hypertrophy, causing inflammatory macrophage accumulation and insulin resistance. Macrophage-conditioned medium (MacCM) inhibits adipogenesis and promotes adipocyte inflammation, but it is unknown if these effects are altered by high glucose (HG) versus normal glucose (NG) concentrations. The effect of HG on adipogenesis was assessed. Human subcutaneous abdominal preadipocytes were induced to differentiate in HG or NG conditions. HG did not affect adipogenesis. HG increased ChREBP-β mRNA and protein levels, and increased GLUT4 mRNA, in differentiated adipocytes. It did not change mRNA levels of ACC, SCD, and FAS. The increase in ChREBP-β mRNA was positively correlated with HG-induced increase in GLUT4 mRNA. The effect of HG-MacCM versus NG-MacCM on human adipogenesis and adipocyte inflammation was compared. Human monocyte-derived macrophages (MDM) were placed in NG or HG glucose for 24 hours to generate MacCM. HG-MacCM, but not NG-MacCM inhibited triacylglycerol accumulation and protein expression of PPARγ during human adipogenesis. Preadipocytes differentiated in HG-MacCM displayed a more pro-inflammatory phenotype, as assessed by increased MCP-1 and IL-6 and reduced adiponectin mRNA expression. HG increased phosphorylation of IKK-β and decreased protein expression of IκBα in MDMs. In addition, HG reduced protein expression of PPARγ in MDMs. The pro-inflammatory effect of HG-MacCM on MCP-1 expression in adipocytes was partially inhibited when MDMs were treated with sc-514 (IKKβ inhibitor). My data demonstrate that HG-induced expression of ChREBP-β in adipocytes may be associated with increased GLUT4 mRNA. The anti-adipogenic and pro-inflammatory effects of HG-MacCM are more potent than NG-MacCM. This suggests the possibility that adipose tissue cellular remodeling in vivo may be altered with hyperglycemia.

Elasmobranchs (sharks, skates, and rays) are a primarily carnivorous group of vertebrates that consume very few carbohydrates and have little reliance on glucose as an oxidative fuel, the one exception being the rectal gland. This has led to a dearth of information on glucose transport and metabolism in these fish, as well as the presumption of glucose intolerance. Given their location on the evolutionary tree however, understanding these aspects of their physiology could provide valuable insights into the evolution of glucose homeostasis in vertebrates. In this thesis, the presence of glucose transporters in an elasmobranch was determined and factors regulating their expression were investigated in the North Pacific spiny dogfish (Squalus suckleyi). In particular, the presence of a putative GLUT4 transporter, which was previously thought to have been lost in these fish, was established and its mRNA levels were shown to be upregulated by feeding (intestine, liver, and muscle), glucose injections (liver and muscle), and insulin injections (muscle). These findings, along with that of increases in muscle glycogen synthase mRNA levels and muscle and liver glycogen content, indicate a potentially conserved mechanism for glucose homeostasis in vertebrates, and argue against glucose intolerance in elasmobranchs. In contrast to the other tissues examined, there was a decrease in glut4 mRNA levels within the rectal gland in response to natural feeding, a factor known to activate the gland, suggesting mRNA storage for rapid protein synthesis upon activation. A similar trend was also shown for sglt1 in the rectal gland, and the ability of GLUT and SGLT inhibitors to prevent chloride secretion solidified the importance of glucose uptake for gland function. The exogenous factor of salinity was also investigated and high levels of glut mRNA were observed within the rectal glands of low salinity-acclimated fish relative to control and high salinity fish, reiterating the idea of mRNA storage when the gland is expected to be inactive. Taken together, the results of this thesis demonstrate that glucose is an important fuel in the dogfish (and likely other elasmobranchs) and that the dogfish is fully capable of regulating its storage and circulation, contrary to prior beliefs.

The non-phosphorylated oxidative pathway of glucose dissimilation has been confirmed in Pseudomonas aeruginosa using whole cells and cell-free extracts. The oxidation of glucose to 2-ketogluconate was complete and stoichiometric in cell-free extracts and cell-free extracts of glucose grown cells were shown to be incapable of metabolizing 2-ketogluconate. It was shown that whole cells completely degraded 2-ketogluconate and quantitatively accumulated pyruvic acid in the presence of specific inhibitors. The initial step involved in 2-ketogluconate dissimilation was found to be exceptionally labile to the effects of a variety of metabolic inhibitors. The metabolism of 2-ketogluconate was demonstrated to involve the initial phosphorylation with adenosine triphosphate (ATP) as the phosphate donor. The resultant intermediate, 2-keto-6-phosphogluconate, was identified and was shown to undergo reduction by a nicotinamide adenine dinucleotide phosphate linked reductase to 6-phosphogluconate which, in turn, was metabolized to pyruvate by enzymes of the Entner-Doudoroff pathway. Radioactivity from 2-ketogluconate-C¹⁴ was rapidly incorporated into cellular constituents, primarily protein, by washed cell suspensions of P. aeruginosa, but oxidation of 2-ketogluconate did not involve the accumulation of keto-acid intermediates. The role of 2-ketogluconic acid as a key intermediate for the conservation of excess carbon under conditions where nitrogen is limiting was discussed. / Land and Food Systems, Faculty of / Graduate

The graduate studies of the writer began at Brigham Young University in the Autunm of 1951, at which time he filed his intention of majoring in Zoology and minoring in Chemistry. During the first three quarters he received instruction under his committee chairman in a sequence of courses entitled: "Body Fluids", "Neurology", and "Endocrinology". During the first of these three courses the role of the kidneys in the maintenance of a relatively constant internal environment was discussed. During the course in Endocrinology the following statement was found in the text which suggested that the thyroid hormone might have an influence on the part of the kidney function involved in the maintenance of a rather constant blood clucose concentration: The metabolism of carbohydrates .... is influenced by the functional state of the thyroid gland .... the rate of absorption of sugar from the intestinal tract is low in thyroidectomized animals and high in hyperthyroid individuals. However, no direct reference we.s found in this text about the thyroid hormone and kidney function.

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