Author
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FERNANDEZ-FIGARES, I. - GRANADA, SPAIN |
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Shannon, Amy |
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WRAY-CAHEN, DIANE - FDA |
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Caperna, Thomas |
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Submitted to: Domestic Animal Endocrinology
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 1/31/2004 Publication Date: 1/31/2004 Citation: Fernandez-Figares, I., Shannon, A.E., Wray-Cahen, D., Caperna, T.J. 2004. The role of insulin glucagons, dexamethasone and leptin in the regulation ketogenesis and glycogen storage in primary cultures of porcine hepatocytes prepared from 60 kg pigs. Domestic Animal Endocrinology. 27(2):125-140. Interpretive Summary: Energy utilization and storage is primarily controlled by key hormones (insulin and glucagon) produced in the pancreas. New studies have emerged which suggest that leptin, a peptide hormone produced by fat cells is involved in regulation of feed intake and also plays a role in energy metabolism in liver tissue. Therefore, a study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from 54-68 kg pigs. Monolayer cultures were established and cells were maintained in a serum-free medium for three days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1 or 100 ng/ml. Recombinant human leptin was added during the final 24 hours. Concentrations of ketone bodies in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio. High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of porcine hepatocytes with leptin, over the range of insulin, glucagon and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are very sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine. Technical Abstract: A study was conducted to elucidate hormonal control of ketogenesis and glycogen deposition in primary cultures of porcine hepatocytes. Hepatocytes were isolated from 54-68 kg pigs by collagenase perfusion and seeded into collagen-coated T-25 flasks. Monolayers were established in medium containing fetal bovine serum for one day and switched to a serum-free medium for the remainder of the culture period. Hepatocytes were maintained in DMEM/M199 containing 1% DMSO, dexamethasone (10-6 or 10-7 M), linoleic acid (3.4 x 10-5 M) and carnitine (10-3 M) for three days. On the first day of serum-free culture, insulin was added at 1 or 100 ng/ml and glucagon was added at 0, 1 or 100 ng/ml. Recombinant human leptin (200 ng/ml) was added during the final 24 hr; medium and all cells were harvested on the third day. Concentrations of acetoacetate and b-hydroxybutyrate (ketone bodies) in media and glycogen deposition in the cellular compartment were determined. Ketogenesis was highly stimulated by glucagon (1 and 100 ng/ml) and inhibited by insulin. In contrast, glycogen deposition was stimulated by insulin and attenuated by glucagon; high insulin was also associated with a reduction in the ketone body ratio (acetoacetate/b-hydroxybutyrate). High levels of dexamethasone stimulated ketogenesis, but inhibited glycogen deposition at low insulin. Culture of cells with leptin for 24 hr, over the range of insulin,glucagon and dexamethasone concentrations had no effect on either glycogen deposition or ketogenesis. These data suggest that while adult porcine hepatocytes are indeed sensitive to hormonal manipulation, leptin has no direct influence on hepatic energy metabolism in swine. Key words: pig hepatocytes, ketogenesis, ketone body ratio (KBR), glycogen, leptin |
