Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Escherichia coli phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxylase (transphorsphorylating) EC 4.1.1.49) catalyzes the decarboxylation and subsequent phosphorylation of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) in the presence of Mg2+ATP and synergistic catalysis has been observed in the presence of Ca2+ or Mn2+. Structural analyses have shown that active site residues Arg333, Ser250 and Tyr207 are coordinated differently in E. coli PCK structures complexed with Mg2+ATP-oxalate, Mg2+ATP-Mn2+-pyruvate and Mg2+ATP-Ca2+-pyruvate; hence, we hypothesize that the function of Arg333, Ser250 and Tyr207, depends on the absence or presence of Ca2+ or Mn2+ during catalysis by E. coli phosphoenolpyruvate carboxykinase (PCK). In order to verify this hypothesis, site directed mutagenesis of the pckA gene was used to convert Arg333 to Gln, Ser250 to Ala and Tyr207 to Phe, while 14CO2 exchange assay and x-ray crystallography were used to determine the effects of these mutations on catalysis by E. coli PCK in the presence of OAA and Mg2+ATP with Ca2+ or Mn2+ metal ions. Kinetic analysis showed that the Tyr207Phe mutation decrease kcat by 1.7 fold, while Ser250Ala and Arg333Gln reduced kcat by 10.8 and 4,555 fold respectively in the presence of Mg2+ATP and OAA. In the presence of Mg2+ATP, OAA and Ca2+, Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 11,688, 44 and 2 fold respectively. In the presence of Mg2+ATP, OAA and Mn2+ Arg333Gln, Ser250Ala and Tyr207Phe mutations reduced kcat by 2,880, 4 and 5.5 fold respectively. The crystal structure of Ser250Ala complexed with Mg2+ATP-Mn2+-pyruvate, showed that in the presence of Mn2+, Ser250Ala mutation reduced the angle between the γ-phosphate of ATP and residue 250 by 6.2 Å and increased the distance between the hydroxyl group of Tyr207 and the CH2 group of pyruvate by 0.5 Å. As a result we conclude that Arg333 is important for oxaloacetate decarboxylation and phosphorylation. During catalysis in the presence of Mg2+ATP with or without Ca2+ or Mn2+, Ser250 functions to maintain one γ-phosphate oxygen of ATP in an eclipsed conformation, while Tyr207 functions to drive oxaloacetate decarboxylation during catalysis in the presence of Mn2+ ion. Kinetic and structural studies of E. coli PCK have previously been used to show that Asp269 is involved in metal coordination, while Lys254 and Arg65 are important for Mg2+ATP and OAA binding to E. coli PCK respectively. In this study the E. coli PCK Asp269Asn-Mg2+ATP-Ca2+-pyruvate crystal structure showed that the Asp269Asn mutation reduced the number of ligands coordinating Ca2+ from seven to three, while no electron density was observed for Mg2+ATP and OAA in Lys254Ser and Arg65Gln crystal structures respectively.

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Phosphoenolpyruvate carboxykinase (Pck) catalyzes the first step of gluconeogenesis, and the gene which encodes this enzyme (pckA) is transcriptionally regulated. High pckA expression is observed in succinate-grown cells, while little expression is observed in glucose-grown cells. pckA regulatory mutants have previously been isolated (Osteras et al. 1997) and pckR, a gene encoding a Lacl-GaIR DNA-binding transcriptional regulator, has been implicated in the regulation of pckA transcription. Here we shew that pckR insertion mutations result in a dramatic decrease in pckA expression even in succinate-grown cells. We demonstrate that the previously identified rpk-9 mutation is tightly linked to pckR. The rpk-9 mutation results in constitutive pckA expression, and we show that plasmids carrying the pckR gene complement the rpk-9 mutation in glucose-grown cells. A putative Lacl-GaIR operator binding site has been identified in the pckA promoter, however no evidence of an interaction between this site and the pckR gene product could be found. / Thesis / Master of Science (MS)

In cultured rat hepatocytes the key gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PCK) is known to be induced by glucagon via an elevation of cAMP. Prostaglandin E₂ has been shown to antagonize the glucagon-activated cAMP formation, glycogen phosphorylase activity and glucose output in hepatocytes. It was the purpose of the current investigation to study the potential of PGE₂ to inhibit the glucagon-induced expression of PCK on the level of mRNA and enzyme activity. PCK mRNA and enzyme activity were increased by 0.1 nM glucagon to a maximum after 2 h and 4 h, respectively. This increase was completely inhibited if 10 μM PGE2 was added concomitantly with glucagon. This inhibition by PGE₂ of glucagon-induced PCK activity was abolished by pertussis toxin treatment. When added at the maximum of PCK mRNA at 2 h, PGE₂ accelerated the decay of mRNA and reduced enzyme activity. This effect was not reversed by pertussis toxin treatment. Since in liver PGE₂ is derived from Kupffer cells, which play a key role in the local inflammatory response, the present data imply that during inflammation PGE₂ may reduce the hepatic gluconeogenic capacity via a Gᵢ-linked signal chain.

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The study was to investigate the changes in (1) triacylglycerol (TAG) contents and its relationship to (2) lipid synthesis- and metabolism-related enzyme activity and (3) their gene expression in cobia (Rachycentron canadum) during the fast growth period (from October 2006 to April 2007) in ventral muscle and liver in Hsiao-Lu-Chiao island in southwestern Taiwan. The crude lipid was 12% for fed diet, 30-40% for liver while 13% in February and 11% to 9% in other month for muscle. The TAG content of crude lipid was 36 % for fed diets, and from 22% (December) to 40% (February) for muscle, and from 63% (October to February) to 47% (March) for liver. Oil red-O (ORO) staining showed that TAG accumulated in muscle in February but in December in liver. Muscle TAG contents and enzyme activities and mRNA levels of GPDH and FAS increased in February. A decrease in GPDH enzyme activity and mRNA levels but an increase in PEPCK enzyme activity and mRNA levels indicate the increased supply of acetyl-CoA for fatty acid synthesis is in muscle. An increase in FATP2 mRNA levels suggest the influx of fatty acid also contributes to increased fatty acid accumulation in muscle.In liver, TAG and fatty acid contents decreased in March April but increased FAS and PEPCK enzyme activity and mRNA levels. It is possible that fatty acid synthesis is enhanced in March, but a fast transport to other organs results in a net decline in liver fatty acid contents and subsequently a decrease in TAG contents. FATP contents decreased in March-April mRNA, indicating that the influx of fatty acid in decreasing in liver in adult fish. GPDH and GAPDH were not related to lipid metabolism in liver. These data from enzyme activity and mRNA level, demonstrated that a potentially increase in acetyl-CoA via PEPCK contributes to fatty acid synthesis and GPDH-mediated synthesis of G-3-P provide the C skeleton for TAG synthesis.

Background: An important role has been suggested for tissue-specific glucocorticoid metabolism in the development of obesity and its complications. 11ß hydroxysteroid dehydrogenase 1 (11ßHSD1) is an enzyme that catalyzes the interconversion of biologically inactive cortisone to active cortisol, thereby regulating its access to glucocorticoid receptors in target tissues. Indeed, an unfavorable metabolic outcome has been associated with increased 11ßHSD1 gene expression and activity in adipose tissue and liver in humans and rodents. Cortisol is an important regulator of phosphoenolpyruvate carboxykinase (PEPCK) a key enzyme in gluconeogenesis and lipid metabolism. In rodents, overexpression of PEPCK in adipose tissue leads to adiposity and increased fatty acid re-esterification. In human obesity, PEPCK has been positively associated with body fat, total cholesterol levels, and plasma triglycerides. However, few studies have addressed the putative reversibility of peripheral cortisol levels and disturbed fatty acid homeostasis that may accompany weight loss. The aim of this thesis was to investigate alterations in peripheral glucocorticoid metabolism in the context of obesity, and putative modulations of glucocorticoid metabolism in the context of weight changes in humans and rodents. Materials & Methods: 11ßHSD1 expression/activity in different adipose tissue depots and liver, the expression of genes involved in adipogenesis and fatty acid homeostasis, and serum levels of adipose tissue-derived adipokines were investigated in severely obese women before and after surgically induced weight loss. The same parameters were measured in female Sprague-Dawley rats fed on high-fat and control diets. Results: In severely obese women, 11ßHSD1 expression was higher in subcutaneous adipose tissue (SAT), while 11ßHSD1 activity and PEPCK expression were higher in the omental depot. In a multivariate analysis, SAT 11ßHSD1 activity was an independent predictor for central fat accumulation. Hepatic 11ßHSD1 activity and levels of intra-abdominal fat storage correlated negatively, while 11ßHSD1 correlated positively with PEPCK in adipose tissue and liver. Weight loss after gastric bypass surgery was followed by significant and metabolically beneficial reductions in subcutaneous 11ßHSD1 and leptin gene expression, as well as reduced circulating leptin and increased adiponectin levels. In contrast, PEPCK gene expression did not change with weight loss. In rats, a high-fat diet did not affect body weight, but was associated with increased serum leptin and decreased adiponectin levels. Short-term, high-fat diet feeding resulted in the up-regulation of SAT 11ßHSD1 expression, while chronic feeding led to its significant down-regulation (compared with the control diet and short-term, high-fat feeding). Interestingly, hepatic 11ßHSD1 expression was constantly downregulated in rats that were fed a high-fat diet. Conclusions: Severe obesity in women was accompanied by a metabolically adverse increase of 11ßHSD1 in adipose tissue, with a concomitant decrease in the liver. Subcutaneous 11ßHSD1 was an independent predictor for central fat accumulation. As weight loss was followed by significant down-regulation of subcutaneous 11ßHSD1, we suggest that up-regulation of this enzyme was a consequence, rather than a cause of obesity. In rodents, a high-fat diet induced dynamic changes in 11ßHSD1 in SAT and liver, both being down-regulated after chronic high-fat feeding without altered weight. In summary, weight changes and alterations in fat and liver glucocorticoid metabolism are closely linked. Moreover, a high-fat diet significantly influences 11ßHSD1 expression/activity in adipose tissue and liver without affecting body weight.

Rainbow trout are an important salmonid species whose poor utilization of dietary carbohydrates spurred research investigating molecular and physiological components of its glucoregulation. Among the environmental factors described to exert robust changes in glucose metabolism in rainbow trout, nutrition and social stress are among the most studied: Diets exceeding 20% of carbohydrates and chronic social stress induce hyperglycemia in adult and juvenile rainbow trout, respectively. Common to both responses is a contribution of hepatic de novo gluconeogenesis, which has been described to evade repression in response to high dietary carbohydrate content and to be stimulated in subordinate rainbow trout. Compared to previous studies investigating the regulation of hepatic gluconeogenesis at the molecular level, the recent publication of the annotated rainbow trout genome has opened novel possibilities to investigate paralogue-specific and posttranscriptional regulation of gluconeogenesis. In this thesis, I identify and describe the regulation of the novel phosphoenolpyruvate carboxykinase paralogue pck2b in rainbow trout and identify specific miRNA candidates predicted to contribute to gene paralogue-specific regulation of gluconeogenesis in nutritional and social contexts using small RNA next generation sequencing, real-time RT-PCR and in silico target prediction approaches. In nutritional and social status experiments, in silico predicted targets of differentially expressed hepatic miRNAs are enriched for gluconeogenesis regulation, suggesting a posttranscriptional component in regulating gluconeogenic transcript abundance. Differentially expressed hepatic miRNAs in both experiments comprise evolutionarily conserved and teleost-specific miRNAs, and are indicative of both environmental factor-specific and common regulation of gluconeogenesis transcripts in rainbow trout liver. Together this work provides novel comparative insight into hepatic miRNA-dependent glucoregulation and identifies several specific candidate miRNAs for future functional validation in hepatic glucoregulation in rainbow trout.