Date of Award

7-1-1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Phosphoenolpyruvate carboxykinase (PEPCK) is a key gluconeogenic enzyme. In gluconeogenic tissues, cytosolic PEPCK is essential for glucose formation whereas no metabolic role is defined for mitochondrial PEPCK. Large amounts of PEPCK activity are present in the mucosa of small intestine from rabbits although this tissue is not known to be a significant site for gluconeogenesis in adult animals. Thus the intent of the project was to investigate the intracellular distribution and role of PEPCK in rabbit enterocytes from the small intestine.Initially, a procedure was developed to isolate viable enterocytes from the small intestine of rabbits for use in enzyme and metabolic studies. Subcellular fractions of enterocytes were assayed for PEPCK, glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (FBPase), glycerol kinase (GlyK), and pyruvate carboxylase (PC) activities to evaluate the feasibility of gluconeogenesis in enterocytes. Enterocytes were also incubated with various gluconeogenic precursors and assayed for glucose. Enterocytes from fasted rabbits metabolize fructose and dihydroxyacetone to glucose, which is consistent with their content of G6Pase and FBPase. However, the lack of significant activities of PC, GlyK and cytosolic PEPCK, and of glucose production from lactate and amino acids suggests that rabbit small intestine is not a gluconeogenic tissue.Since PEPCK activity in enterocytes is $>$90% mitochondrial, viable mitochondria were isolated and metabolic studies conducted to characterize further the role of the mitochondrial isozyme. The activities of various lipogenic enzymes in cytosol of enterocytes were also determined. Mitochondria incubated with exogenous PEP, acetyl carnitine, and HCO$\sbsp{3}{-}$ produce citrate, and citrate production is stimulated by guanine nucleotides. Furthermore, inhibition of citrate production by 3-mercaptopicolinate and benzenetricarboxylate indicates that the route of citrate production is via mitochondrial PEPCK. Significant amounts of four lipogenic enzymes, ATP-citrate lyase, malic enzyme, NADP$\sp+$:isocitrate dehydrogenase, and NADP$\sp+$:glucose 6-phosphate dehydrogenase, are present in cytosol from enterocytes. In conclusion, the PEPCK activity in rabbit enterocytes could contribute to the formation of citrate from PEP thereby playing a direct and/or indirect role in lipogenesis.

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