Liver Fructose Research Articles

Regulatory effect of calcium-binding protein isolated from rat liver cytosol on activation of fructose 1,6-diphosphatase by Ca2+-calmodulin.

The role of a calcium-binding protein (CaBP) isolated from rat liver cytosol was investigated in relation to the activation of hepatic fructose 1, 6-diphosphatase by Ca2+-calmodulin. Fructose 1, 6-diphosphatase activity in rat liver cytosol was markedly increased by addition of Ca2+ (1.0-5.0 μM) to the incubation mixture. This increase was completely inhibited in the presence of N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W-7 15 μM), an inhibitor of calmodulin. Added Ca2+ (5.0 μM)-increased cytosolic fructose 1, 6-diphosphatase activity was markedly enhanced by the coexistence of calmodulin (2.5 μg/ml). Further, fructose 1, 6-diphosphatase isolated from rabbit liver cytosol was activated by Ca2+-calmodulin. This activation was completely inhibited by CaBP (20 μg/ml) isolated from rat liver cytosol, though CaBP in the absence of calmodulin had no effect on liver fructose 1, 6-diphosphatase activity. The present data suggest that CaBP can modify the action of Ca2+-calmodulin in liver cells. It is proposed that CaBP, which may regulate Ca2+ effects on liver function, should be named calregulin.

Metabolic effects of oral fructose in the liver of fasted rats.

Twenty-four-hour-fasted rats were given fructose (4 g/kg) by gavage. Fructose absorption and the portal vein, aorta, and hepatic vein plasma fructose, glucose, lactate, and insulin concentrations as well as liver fructose and fructose 1-P, glucose, glucose 6-P, UDPglucose, lactate, pyruvate, ATP, ADP, AMP, inorganic phosphate (Pi), cAMP, and Mg2+, and glycogen synthase I and phosphorylase alpha were measured at 10, 20, 30, 40, 60 and 120 min after gavage. Liver and muscle glycogen and serum uric acid and triglycerides also were measured. Fifty-nine percent of the fructose was absorbed in 2 h. There were modest increases in plasma and hepatic fructose, glucose, and lactate and in plasma insulin. Concentrations in the portal vein, aorta, and hepatic vein plasma indicate rapid removal of fructose and lactate by the liver and a modest increase in production of glucose. The source of the increase in plasma lactate is uncertain. Hepatic glucose 6-P increased twofold; UDPglucose rose transiently and then decreased below the control level. Fructose 1-P increased linearly to a concentration of 3.3 mumol/g wet wt by 120 min. There was no change in ATP, ADP, AMP, cAMP, Pi, or Mg2+. Serum triglycerides and uric acid were unchanged. Glycogen synthase was activated by 20 min without a change in phosphorylase alpha. This occurred with a fructose dose that did not significantly increase either the liver glucose or fructose concentrations. Liver glycogen increased linearly after 20 min, and glycogen storage was equal in liver (38.4%) and muscle (36.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

Rapid regulation of L-type pyruvate kinase mRNA by fructose in diabetic rat liver.

The effect of fructose on the induction of L-type pyruvate kinase mRNA in diabetic rat liver was studied by using a cloned cDNA probe. Fructose feeding resulted in a 5- to 6-fold increase in the L-type enzyme mRNA level after 1 to 3 days. These changes were approximately proportional to the changes in the level of translatable mRNA of this enzyme. A significant increase in total cellular L-type enzyme mRNA level was observed within 2 h after fructose feeding and the level reached a maximum after 8 h. Dietary glycerol also markedly increased the L-type mRNA level. These alterations were essentially due to the changes in the cytosolic mRNA. Northern blot analysis of total cellular RNA revealed that two L-type enzyme mRNA species with molecular sizes of 2.1 and 3.6 kilobases were proportionally increased during the fructose induction. The two mRNA forms were found in immunopurified L-type enzyme mRNA and directed synthesis of the L-type subunit in vitro; they are therefore functional mature forms. In contrast, analysis of nuclear RNA showed five putative precursor RNA species for the enzyme, up to 9.4 kilobases in length, in the liver of fructose-fed rats, while no band of the RNA species was found in the nuclei of control liver. The changes in the number of bands of these RNA species and their intensities after fructose feeding preceded the changes in the level of total cellular L-type enzyme mRNA sequences. These results indicate that dietary fructose causes a rapid increase in the level of L-type pyruvate kinase mRNA sequences by acting at the nuclear level.

Comparative use of fructose and glucose in human liver and fibroblastic cell cultures.

The effect of fructose as a substitute for glucose in cell culture media was investigated in human skin fibroblast and liver cell cultures. Cells were grown for between 2 and 10 days in identical flasks in four different media, containing 5.5 mmol X 1-1 and 27.5 mmol X 1-1 glucose and fructose, respectively. In the presence of fructose, cell growth was stimulated, but less in liver cells than fibroblasts. At Day 6, increases were observed in [3H]thymidine incorporation, protein levels, and amino acid consumption, and a reduction was noted in ATP levels. In media containing 5.5 mmol X 1-1 glucose or fructose, consumption of fructose was four times lower than that of glucose at Day 3 and did not rise until Day 6. In fructose media, the lactate production was very low (four to five times less than that of glucose) and the pH values were always higher. Some findings were different for the fibroblasts and liver cells, owing to the specific characteristics of these two cell types in culture; this applied especially to the effects of glucose and fructose concentrations of 27.5 mmol X 1-1. Several possible explanations for the stimulation of cell growth in fructose medium were discussed.

Mechanism of action of 2,5-anhydro-D-mannitol in hepatocytes. Effects of phosphorylated metabolites on enzymes of carbohydrate metabolism.

Isolated rat hepatocytes convert 2,5-anhydromannitol to 2,5-anhydromannitol-1-P and 2,5-anhydromannitol-1,6-P2. Cellular concentrations of the monophosphate and bisphosphate are proportional to the concentration of 2,5-anhydromannitol and are decreased by gluconeogenic substrates but not by glucose. Rat liver phosphofructokinase-1 phosphorylates 2,5-anhydromannitol-1-P; the rate is less than that for fructose-6-P but is stimulated by fructose-2,6-P2. At 1 mM fructose-6-P, bisphosphate compounds activate rat liver phosphofructokinase-1 in the following order of effectiveness: fructose-2,6-P2 much greater than 2,5-anhydromannitol-1,6-P2 greater than fructose-1,6-P2 greater than 2,5-anhydroglucitol-1,6-P2. High concentrations of fructose-1,6-P2 or 2,5-anhydromannitol-1,6-P2 inhibit phosphofructokinase-1. Rat liver fructose 1,6-bisphosphatase is inhibited competitively by 2,5-anhydromannitol-1,6-P2 and noncompetitively by 2,5-anhydroglucitol-1,6-P2. The AMP inhibition of fructose 1,6-bisphosphatase is potentiated by 2,5-anhydroglucitol-1,6-P2 but not by 2,5-anhydromannitol-1,6-P2. Rat liver pyruvate kinase is stimulated by micromolar concentrations of 2,5-anhydromannitol-1,6-P2; the maximal activation is the same as for fructose-1,6-P2. 2,5-Anhydroglucitol-1,6-P2 is a weak activator. 2,5-Anhydromannitol-1-P stimulates pyruvate kinase more effectively than fructose-1-P. Effects of glucagon on pyruvate kinase are not altered by prior treatment of hepatocytes with 2,5-anhydromannitol. Pyruvate kinase from glucagon-treated hepatocytes has the same activity as the control pyruvate kinase at saturating concentrations of 2,5-anhydromannitol-1,6-P2 but has a decreased affinity for 2,5-anhydromannitol-1,6-P2 and is not stimulated by 2,5-anhydromannitol-1-P. The inhibition of gluconeogenesis and enhancement of glycolysis from gluconeogenic precursors in hepatocytes treated with 2,5-anhydromannitol can be explained by an inhibition of fructose 1,6-bisphosphatase, an activation of pyruvate kinase, and an abolition of the influence of phosphorylation on pyruvate kinase.

Identification of the highly reactive sulfhydryl group of pig kidney fructose 1,6-bisphosphatase at cysteine 128.

Fructose 1,6-bisphosphatases contain a highly reactive cysteine residue, the reactivity of which is influenced by ligands that bind at the catalytic and at the allosteric AMP sites of the enzyme. Nevertheless, the sulfhydryl group appears to be proximal to these sites and not a functional component of either. Modification of pig kidney fructose 1,6-bisphosphatase with three reagents, 5,5'-dithiobis-(2-nitrobenzoic acid), iodoacetamide, and phenacyl bromide, yields derivatives with similar properties, thus suggesting that the same residue was modified in each case. The modified enzymes exhibited: (a) higher Vmax when Mn2+ was used as the activating cation; (b) decreased activity in the presence of nonsaturating Mg2+ concentrations; (c) no change in sensitivity toward AMP inhibition. Automated Edman degradation of a tryptic peptide containing radioactive carboxamidomethylcysteine showed the sequence of residues Gly-111-Arg-140 of pig kidney fructose 1,6-bisphosphatase. The modified residue was shown to be cysteine-128, and the same cysteine residue was alkylated when the enzyme was reacted with phenacyl bromide. Cysteine-128 is also present in rat and sheep liver fructose 1,6-bisphosphatase and a long stretch of the sequence around this reactive cysteine residue is highly conserved.

Identification of the in vivo and in vitro phosphorylation sites of rat liver fructose 1,6-bisphosphatase.

Rat liver fructose 1,6-bisphosphatase appears to be unique in that it extends 24-26 residues beyond the COOH-terminal amino acid of other mammalian fructose 1,6-bisphosphatases and this extension contains phosphorylation sites. Using as a frame of reference the 335-residue sequence of pig kidney fructose 1,6-bisphosphatase (Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, R. L. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 7161-7165), the rat liver enzyme would extend to residue 361. Limited proteolysis in the COOH-terminal region of the molecule with chymotrypsin, trypsin, or both sequentially, led us to establish that the phosphorylation sites are located at Ser residues 341 and 356. The in vitro phosphorylation of purified rat liver fructose 1,6-bisphosphatase by the catalytic subunit of cyclic AMP-dependent protein kinase results in modification at both residues, although the major site of phosphorylation (61%) is at Ser-341. In contrast, rat liver fructose 1,6-bisphosphatase purified from animals that had been injected with [32P] phosphate contains most of the label (81%) at Ser-356.

Interaction of rabbit liver cathepsin M and fructose 1,6-bisphosphatase converting enzyme with their endogenous inhibitors

The stoichiometry of complex formation between two lysosomal proteinases from rabbit liver, cathepsin M and fructose 1,6-bisphosphatase converting enzyme (CE), and their respective endogenous inhibitors was studied by the equilibrium gel penetration method. In each case the molecular weight of the complex was found to be the sum of the molecular weights of the proteinase and its inhibitor, indicating the formation of 1:1 complexes. From the reappearance of proteinase activity on dilution, it is concluded that complex formation is reversible. Localization of the proteinase activities on the outer surface of the lysosomes was confirmed in these experiments by the inhibition of this proteinase activity on addition of inhibitors to intact lysosomes. The digestion by subtilisin of rabbit liver aldolase and rabbit liver fructose 1,6-bisphosphatase, the endogenous substrates for the lysosomal proteinases, was unaffected by the inhibitors.

Inhibition of gluconeogenesis and glycogenolysis by 2,5-anhydro-D-mannitol.

2,5-Anhydro-D-mannitol (100 to 200 mg/kg) decreased blood glucose by 17 to 58% in fasting mice, rats, streptozotocin-diabetic mice, and genetically diabetic db/db mice. Serum lactate in rats was elevated 56% by 2,5-anhydro-D-mannitol, but this could be prevented by dichloroacetate (200 mg/kg) or thiamin (200 mg/kg). In hepatocytes from fasted rats, 1 mM 2,5-anhydro-D-mannitol inhibited gluconeogenesis from a mixture of alanine, lactate, and pyruvate. It also inhibited glucose production and stimulated lactate formation from glycerol or dihydroxyacetone. Glycogenolysis in hepatocytes from fed rats was markedly inhibited by 1 mM 2,5-anhydro-D-mannitol both in the presence or absence of 1 microM glucagon. 2,5-Anhydro-D-mannitol can be phosphorylated by fructokinase or hexokinase to the 1-phosphate and then by phosphofructokinase to the 1,6-bisphosphate. Rat liver glycogen phosphorylase was inhibited by 2,5-anhydro-D-mannitol 1-phosphate (apparent Ki = 0.66 +/- 0.09 mM) but was little affected by 2,5-anhydro-D-mannitol 1,6-bisphosphate. Rat liver phosphoglucomutase was inhibited by 2,5-anhydro-D-mannitol 1-phosphate (apparent Ki = 2.8 +/- 0.2 mM), whereas 2,5-anhydro-D-mannitol 1,6-bisphosphate served as an alternative activator (apparent K alpha = 7.0 +/- 0.5 microM). Rabbit liver pyruvate kinase was activated by 2,5-anhydro-D-mannitol 1,6-bisphosphate (apparent K alpha = 9.5 +/- 0.9 microM), whereas rabbit liver fructose 1,6-bisphosphatase was inhibited by 2,5-anhydro-D-mannitol 1,6-bisphosphate (apparent Ki = 3.6 +/- 0.3 microM). The phosphate esters of 2,5-anhydro-D-mannitol would, therefore, be expected to inhibit glycogenolysis and gluconeogenesis and stimulate glycolysis in liver.

Evidence for different forms of rat liver fructose 1,6-bisphosphatase

Purified liver fructose 1,6-bisphosphatase exhibits different forms upon isoelectric focusing. The enzyme focused at pH 5.75, 5.60, and 5.44. Treatment of the enzyme preparation with the catalytic subunit of cAMP-dependent protein kinase and ATP altered the isoelectric focusing profile such that the bands at 5.75 and 5.60 were diminished, the band at 5.44 increased, and two new bands appeared at 5.30, and 5.18. Fructose 1,6-bisphosphatase may be present in rat liver in different forms, one of which is phosphorylated as the enzyme is isolated.

Synergistic effect of AMP and fructose 2,6-bisphosphate on the protection of fructose 1,6-bisphosphatase against inactivation by trypsin.

The rate of inactivation of chicken liver fructose 1,6-bisphosphatase by trypsin is reduced if the digestive reaction is conducted in the presence of AMP or fructose 2,6-bisphosphate. The effects of these 2 compounds are synergistic. Although fructose 1,6-bisphosphate does not protect the enzyme against tryptic inactivation, it can enhance the effect of AMP. Selective modification of the AMP allosteric site of fructose 1,6-bisphosphatase with pyridoxal-P and NaBH4 renders the enzyme more resistant to tryptic inactivation, but the modified enzyme is no longer responsive to the protective effect of AMP.

The interaction of fructose 2,6-bisphosphate and AMP with rat hepatic fructose 1,6-bisphosphatase.

The binding of the inhibitory ligands fructose 2,6-bisphosphate and AMP to rat liver fructose 1,6-bisphosphatase has been investigated. 4 mol of fructose-2,6-P2 and 4 mol of AMP bind per mol of tetrameric enzyme at pH 7.4. Fructose 2,6-bisphosphate exhibits negative cooperatively as indicated by K'1 greater than K'2 greater than K'3 greater than or equal to K'4 and a Hill plot, the curvature of which indicates K'2/K'1 less than 1, K'3/K'2 less than 1, and K'4/K'3 = 1. AMP binding, on the other hand, exhibits positive cooperativity as indicated by K'1 less than K'2 less than K'3 less than K'4 and an nH of 2.05. Fructose 2,6- and fructose 1,6-bisphosphates enhance the binding of AMP as indicated by an increase in the intrinsic association constants. At pH 9.2, where fructose 2,6-bisphosphate and AMP inhibition of the enzyme are diminished, fructose 2,6-bisphosphate binds with a lower affinity but in a positively cooperative manner, whereas AMP exhibits half-sites reactivity with only 2 mol of AMP bound per mol of tetramer. Ultraviolet difference spectroscopy confirmed the results of these binding studies. The site at which fructose 2,6-bisphosphate binds to fructose 1,6-bisphosphatase has been identified as the catalytic site on the basis of the following. 1) Fructose 2,6-bisphosphate binds with a stoichiometry of 1 mol/mol of monomer; 2) covalent modification of the active site with acetylimidazole inhibits fructose 2,6-bisphosphate binding; and 3) alpha-methyl D-fructofuranoside-1,6-P2 and beta-methyl D-fructofuranoside-1,6-P2, substrate analogs, block fructose 2,6-bisphosphate binding. We propose that fructose 2,6-bisphosphate enhances AMP affinity by binding to the active site of the enzyme and bringing about a conformational change which may be similar to that induced by AMP interaction at the allosteric site.

The interaction of fructose 2,6-bisphosphate with an allosteric site of rat liver fructose 1,6-bisphosphatase

Rat liver fructose 1,6-bisphosphatase can be protected against partial inactivation by N-ethylmaleimide by low concentrations of fructose 2,6-bisphosphate or high concentrations of fructose 1,6-bisphosphate. The partially inactivated enzyme has a much reduced sensitivity to high substrate inhibition and has lost the sigmoid component of the inhibition by fructose 2,6-bisphosphate; this compound is a simple linear competitive inhibitor of the modified enzyme. The results suggest that fructose 2,6-bisphosphate can bind to the enzyme at two distinct sites, the catalytic site and an allosteric site. High levels of fructose 1,6-bisphosphate probably inhibit by binding to the allosteric site.

On the mechanism of inhibition of neutral liver fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate.

The inhibitory effect of fructose 2,6-biphosphate on fructose 1,6-bisphosphatase was reinvestigated in order to solve the apparent contradiction between competition with the substrate and the synergism with AMP, a strictly noncompetitive inhibitor. The effect of fructose 2,6-bisphosphate was compared to that of other ligands of the enzyme, which, like the substrate and methyl (alpha + beta)fructofuranoside 1,6-bisphosphate bind to the active site or which, like AMP, bind to an allosteric site. An increase in temperature or pH, or the presence of sulfosalicylate, lithium or higher concentrations of magnesium as well as partial proteolysis by subtilisin increased [I]0.5 for fructose 2,6-bisphosphate and AMP without affecting Km. With the exception of the pH change, all these conditions were also without effect on the affinity of the enzyme for the competitive inhibitor, methyl (alpha + beta)fructofuranoside 1,6-bisphosphate. These observations can be explained by assuming that fructose 2,6-bisphosphate has no affinity for the active site of fructose 1,6-bisphosphatase but binds to an allosteric site which is different from the AMP site. Fructose 2,6-bisphosphate is therefore classified as an allosteric competitive inhibitor and a model is proposed which explains its synergism with AMP as well as the various cooperative effects.

Hysteretic behavior of rat liver fructose 1,6-bisphosphatase induced by zinc ions

The measurement of the time dependency of the activity of rat liver fructose 1,6-bisphosphatase shows that the enzyme under certain conditions exhibits kinetic hysteretics. After addition of the substrate, the enzyme is initially in a state characterized by a “high” K m of about 2 μ m. During the reaction the enzyme is converted in a slow process to a low K m form ( K m is about 0.5 μ m). The transition is accompanied by a decrease in V. It is concluded that the hysteretic behavior is caused by binding of the Zn 2+ substrate complex to the enzyme. The earlier reported effect of glucagon treatment on the activity of fructose 1,6-bisphosphate ( O. D. Taunton, F. B. Stifel, H. L. Greene, and R. H. Herman (1974) J. Biol. Chem. 249, 7228–7239 ) was reinvestigated, taking into account the hysteretic behavior. Under conditions where the pyruvate kinase activity is decreased by glucagon injection, no activity change of fructose 1,6-bisphosphatase is observed. It can be suggested that for studies concerning the effects of incubation or hormone treatment on fructose 1,6-bisphosphatase, the complex kinetics of the rat liver enzyme has to be taken into account.

On the mechanism of inhibition of fructose 1,6-bisphosphatase by fructose 2,6-bisphosphate

The inhibition of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) by fructose 2,6-bisphosphate (Fru-2,6-P 2) is shown to be competitive with the substrate, fructose 1,6-bisphosphate (Fru-1,6-P 2), with K i for Fru-2,6-P 2 of approximately 0.5 μ m. Binding of Fru-2,6-P 2 to the catalytic site is confirmed by the fact that it protects this site against modification by pyridoxal phosphate. Inhibition by Fru-2,6-P 2 is enhanced in the presence of a noninhibitory concentration (5 μ m) of the allosteric inhibitor AMP and decreased by modification of the enzyme by limited proteolysis with subtilisin. Fru-2,6-P 2, unlike the substrate Fru-1,6-P 2, protects the enzyme against proteolysis by subtilisin or lysosomal proteinases.

Rat and rabbit liver mRNAs code for fructose 1,6-bisphosphatases that differ in molecular weight

Summary Fructose 1,6-bisphosphatase was synthesized in vitro using Poly(A)+ RNA isolated from rat and rabbit liver and the specific translation products were identified by immunoprecipitation using antibody raised against the individual purified proteins, followed by electrophoresis on polyacrylamide gels in the presence of sodium dodecyl sulfate. The in vitro synthesis products were found to have subunit molecular weights which were identical to the corresponding purified proteins; 36,000 and 40,000 for the rabbit and rat enzymes, respectively. The rat liver enzyme synthesized in vitro could be converted to a form with a molecular weight nearly identical to that of the rabbit liver enzyme by treatment with trypsin. The results demonstrate that the difference in molecular weight between rat and rabbit liver fructose 1,6-bisphosphatase subunits is not due to limited proteolytic modification of the rabbit liver enzyme during purification. It is proposed that the rabbit liver enzyme in vivo lacks the COOH-terminal phosphorylation site which is present on the rat liver enzyme.

Regulation of rat liver fructose 2,6-bisphosphatase.

An enzyme activity that catalyzes the hydrolysis of phosphate from the C-2 position of fructose 2,6-bisphosphate has been detected in rat liver cytoplasm. The S0.5 for fructose 2,6-bisphosphate was about 15 microM and the enzyme was inhibited by fructose 6-phosphate (Ki 40 microM) and activated by Pi (KA 1 mM). Fructose 2,6-bisphosphatase activity was purified to homogeneity by specific elution from phosphocellulose with fructose by specific elution from phosphocellulose with fructose 6-phosphate and had an apparent molecular weight of about 100,000, 6-phosphofructo 2-kinase activity copurified with fructose 2,6-bisphosphatase activity at each step of the purification scheme. Incubation of the purified protein with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in the incorporation of 1 mol of 32P/mol of enzyme subunit (Mr = 50,000). Concomitant with this phosphorylation was an activation of the fructose 2,6-bisphosphatase and an inhibition of the 6-phosphofructo 2-kinase activity. Glucagon addition to isolated hepatocytes also resulted in an inhibition of 6-phosphofructo 2-kinase and activation of fructose 2,6-bisphosphatase measured in cell extracts, suggesting that the hormone regulates the level of fructose 2,6-bisphosphate by affecting both synthesis and degradation of the compound. These findings suggest that this enzyme has both phosphohydrolase and phosphotransferase activities i.e. that it is bifunctional, and that both activities can be regulated by cAMP-dependent phosphorylation.

Localization of two lysosomal proteinases on the external surface of the lysosomal membrane

Two enzymes present in rabbit liver extracts that catalyze the limited proteolysis of rabbit liver fructose 1,6-bisphosphatase (EC 3.1.3.11) and rabbit liver fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) have now been identified as lysosomal proteinases. This identification was based on their cosedimentation with cathepsin C (EC 3.4.14.1) in the particulate fractions isolated from livers of normal and Triton WR1339-injected rabbits. These proteinases, designated Fru-P 2ase-converting enzyme 2 (CE2) and cathepsin M, respectively, are partially associated with lysosomal membranes. The membrane-bound activity is selectively destroyed by incubation of the intact lysosomes with soluble or immobilized trypsin. The results suggest that the CE2 and cathepsin M activities are located in part on the outer lysosomal surface and that modification of cytosolic proteins by these proteinases may occur without entry of the proteins into the lysosomes.

Response of rainbow trout (Salmo gairdneri) to increased levels of available carbohydrate in practical trout diets.

1. The physiological response of rainbow trout (Salmo gairdneri) reared on different levels of available carbohydrate in practical trout diets having the same levels of energy and nitrogen for 16-24 weeks was determined. 2. Weight gain was significantly reduced in trout reared on the highest level of available carbohydrate, 210 g cerelose (alpha-glucose) kg, and there was a significant linear regression (R2 0.88 of dietary carbohydrate on weight gain. 3. Liver: body-weight values and liver glycogen levels increased in relation to increased dietary carbohydrate. 4. Liver glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity increased and liver phosphoenolpyruvate carboxykinase (EC 4.1.1.32) activity decreased per kg body-weight of fish with increasing dietary carbohydrate. However, no significant effect was noted on the activity of these liver enzymes above a dietary cerelose level of 140 g/kg. 5. Liver fructose diphosphatase (EC 3.1.3.11) activity increased with increasing dietary carbohydrate has been interpreted as meaning a recycling of triosephosphate to glucose-6-phosphate. 6. Dietary carbohydrate level had no significant effect on the liver pyruvate kinase (EC 2.7.1.40) activity, the rate of glucose utilization or the percentage conversion of [14C]alanine to glucose in the plasma of trout. 7. The results indicate that rainbow trout have a limited ability to adapt to increased dietary carbohydrate and a level in excess of 140 g/kg of the diet is not efficiently utilized.