Dual Roles Of Glucocorticoids In Glycogen Metabolism Of Primary Cultured Rat Hepatocytes

Background: Glucocorticoids describe a series of steroid-like compounds capable of influencing metabolism and exerting a clinically useful antiinflammatory effect. It is essential for life and regulates or supports a variety of important cardiovascular, metabolic, immunologic, and homeostatic functions. Especially in stress reaction, they help to enhance the resistance to the stimulus. They play important roles in the metabolism of carbohydrates, which is the reason why they are so called. They increase plasma glucose by stimulating the gluconeogenesis particularly in the liver and inhibit the uptake and utilization of glucose by peripheral tissues. It was also considered that glucocorticoids, which provide long-term protection against stress, promote glycogen synthesis and thus prime the liver for acute glycogenolytic stress signals. However, questions are raised that whether they still stimulate glycogen deposition when glucose are imperatively needed in stress.Objective: This study was to investigate the effects of glucocorticoids on glycogen metabolism in primary cultured hepatocytes and their possible mechanism.Methods: Hepatocytes were isolated by the method of Selgen with some modification. Then hepatocytes were cultured in 12-well plates in William's Medium with 10% FBS or, in case of hormonal treatments, in serum-free William's Medium. After incubation with or without dexamethasone as indicated time, hepatocytes were collected and treated in different ways for the purposes. The contents of glycogen in hepatocytes were determined when liberated glucosyl units from glycogen followed by measuring glucose using a standard fluorimetric enzymatic glucose assay. The activity of phosphorylase a and glycogen synthase were analysised in enzyme kinetics assay. Protein concentration was measured by BCA (bicinchoninic acid) Protein Assay. Cytoplasmic cyclic AMP (cAMP) level was determined by a radioimmunoassay, and intracellular free calcium concentration was measured in fluorescence analyses.Results: Glucocorticoids could modulate glycogen metabolism of hepatocytes in a bidirectional way. Dexamethasone decrease the glycogen contents of hepatocytes in higher concentration (10^-8 - 10^-5 M) and increase them in lower concentration (10^-10 - 10^-9 M). When the hepatocytes were treated with dexamethasone in a high concentration (10^-6 M), the glycogen contents were lower than the control 2 h later, but higher than the control 72 h or later. Treating the hepatocytes with dexamethasone (10^-6 M) with or without CHX (10^-5 M) for 6 h, the down-regulating effect can't be blocked by CHX. When the hepatocytes were treated with dexamethasone in a high concentration (10^-6 M), the phosphorylase a activity was higher than the control 15 min later, but lower than the control 6 h or later. And the glycogen synthase activity was decreased when treated with dexamethasone at the concentration of 10^-6 M 6 hs later. The hepatocytes intracellular cAMP concentration was increased when treated with dexamethasone at the concentration of 10^-6 M 6 hs later, and the cytoplasmic free calcium concentration in hepatocytes was raised rapidly when treated with dexamethasone at the concentration of 10^-6 M.Conclusion: Our results demonstrate that glucocorticoids have dual roles in the glycogen metabolism of hepatocytes. GCs could decrease the glycogen contents of hepatocytes in the higher concentration and in the relatively shorter time. We infer that these effects may have important physiological significance in the roles in the stress reaction of glucocorticoids.