Stimulation of hepatic glycogen synthesis by amino acids.

Abstract

Hepatocytes isolated from livers of fasted rats form little glycogen from glucose or lactate at concentrations below 20 mM. Glycogen is formed in substantial quantities at a glucose concentration of 60 mM. In the presence of 10 mM glucose, 20-30% as much glycogen as glucose-is formed from fructose, sorbitol, or dihydroxyacetone. The addition of either glutamine, alanine, or asparagine stimulates the formation of glycogen from lactate 10-to 40-fold. The formation of glucose and glycogen is then about equal, and glycogen deposition in hepatocytes is similar to rates attained in vivo after fasted rats are refed. The amino acids stimulate 1.5-to 2-fold glycogen synthesis from fructose, and 2-to 4-fold synthesis from dihydroxyacetone. Ammonium chloride is about one-half as effective as amino acids in stimulating glycogen synthesis when glucose with lactate are substrates. It increased glycogen synthesis 25-50% from fructose but inhibited synthesis from dihydroxyacetone plus glucose. Isolated rat hepatocytes form glucose from numerous substrates at a rate similar to that in vivo, and such cells have served in recent years as a major preparation for the study of gluconeogenesis. However, attempts to obtain hepatocytes capable of efficient glycogen synthesis have not been successful. We set out to develop a hepatocyte preparation capable of synthesis, from common gluconeogenic substrates at moderate concentrations, similar to that obtained in vivo (1-2 ,tmol of glucose equivalents per min/g) within 1 hr after refeeding fasted rats. We report here that certain amino acids stimulate glycogen formation from lactate to this extent. Our findings suggest that certain amino acids or their derivatives may have a major role in the regulation of glycogen synthesis. METHODSMale rats of the Sprague-Dawley strain which weighed 180-220 g were fasted for 24 hr. The rats were used for hepatocyte preparation without further treatment, or they were injected intraperitoneally with a solution, 1 ml/100 g of body weight, of either 5% glucose plus 5% fructose, or 1 mg/100 g of body weight of prednisolone succinate. They were anesthesized with nembutal and operated on 30 min after the injection of sugar and 1 hr after that of prednisolone.Hepatocytes were prepared as previously described (1) except that the bile duct and adjacent vein were not ligated prior to cannulation of the portal vein. The yield from a 200 g rat was 6-8 ml of washed and lightly packed cells. Between 0.04 and 0.06 ml of cells were incubated in 3 ml of Krebs bicarbonate buffer, usually for 1 hr, in an atmosphere of 95% 02-5% CO2, at 380. Incubations were terminated by injecting perchloric acid to a 3% final concentration. Each substrate combination was incubated in duplicate, and results between them nearly always agreed to within 10%. Enzymatic procedures were used to determine glucose, fructose and lactate (1), urea and ammonia (2), glutamate (3), glutamine (4), asparagine and aspartate (5), and alanine (6) spectrophotometrically. Glycogen was determined acco...

Effect of insulin on glycogen and protein synthesis in monolayer cultures of hepatocytes from normal and alloxan diabetic rats

“…Hems et al [1] demonstrated that the presence of gluconeogenic precursors stimulate glycogen synthesis in the peffused liver of the starved rat. More recently, Katz et al [19] reported an effect of amino acids on glycogen synthesis in isolated hepatocytes. However, addition of insulin resulted in an increased glycogen synthesis in cultured parenchymal cells from both normal and diabetic rats.…”

Stimulation of rat liver glycogen synthesis by the adenosine kinase inhibitor 5-iodotubercidin

“…As shown in Figures 3(a) and 3(c), glycogen formation was totally suppressed by 2-chloroadenosine, and this inhibition was accompanied by an increase in cyclic AMP. However, the inhibition of glycogen synthesis caused if the effects of ltu and amino acids It is well documented that amino acids stimulate glycogen synthesis from glucose and gluconeogenic precursors [7][8][9][10][11], and we determined whether Itu acts in a similar manner. Promotion of glycogen synthesis by added amino acids appears to be due to hepatocyte swelling after their uptake and the accumulation of intracellular catabolites such as glutamate and aspartate [34][35][36][37].…”

“…We had evidence that the known stimulating effect of amino acids on glycogen synthesis [7][8][9][10][11] may be due to a purine compound, since the increase in glycogen production by amino acids is strongly blocked by inhibitors of purine synthesis [8] and because this inhibition is correlated with a decrease of the formation of adenine nucleotides [12]. Adenosine has been reported to stimulate glycogen synthesis in vivo [13] and glycogen synthase activity in isolated liver preparations [1 1,14].…”

Evidence against Glycogen Cycling of Gluconeogenic Substrates in Various Liver Preparations

“…The low insulin/glucagon ratio used has previously been reported to stimulate net glucose output in perfused rat liver (33). Livers were perfused in a nonrecirculating set-up, and samples of 2 ml were drawn simultaneously from the inflow and outflow of the liver at t ϭ 0, 5,10,15,20,25,30,35, and 40 min. At t ϭ 40 min, 0.1 Ci/ml [U- 14 C]lactate (Amersham Biosciences) was added to the Krebs-Ringer buffer, and the perfusion was changed to a recirculating set-up (200 ml).…”