Phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme A reductase by microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase

Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent M_r of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-³²P]ATP produces a unique band containing ³²P bound to protein which migrates at the same R_f as the reductase subunit. Incubation of ³²P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound ³²P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, ³²P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one ³²P-labeled phosphopeptide with the same R_f as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed.     

Isolation and immunological characterization of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) has been isolated from human liver utilizing HMG-CoA affinity chromatography. The apparent monomer molecular weight of purified human HMG-CoA reductase by SDS-gel electrophoresis was 53,000, and the oligomeric molecular weight determined by sucrose density centrifugation was 104,000. A monospecific antibody prepared against rat liver HMG-CoA reductase inhibited the enzymic activity of microsomal and purified human liver enzyme and formed a single immunoprecipitin line by radial immunodiffusion. These results represent the initial isolation and characterization of human liver HMG-CoA reductase.     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.     

Influence of mevalonate kinase on studies of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase

The nature of the MgATP-dependent inactivator of 3-hydroxy-3-methylglutaryl coenzyme A reductase has been studied. Several observations suggest that reductase inactivator preparations from both microsomes and cytosol possess mevalonate kinase activity. (1) Reductase inactivator (reductase kinase) activity copurified with mevalonate kinase activity. (2) Inactivator activity was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, known to be potent inhibitors of mevalonate kinase. (3) Addition of an excess of mevalonate completely prevented inhibition of reductase activity. (4) Formation of phosphomevalonate fully accounted for the decreased amount of mevalonate formed in the presence of inactivator and MgATP. (5) When reductase activity was measured by NADPH oxidation, no inhibition was observed. Clearly, the presence of mevalonate kinase in reductase inactivator preparations can lead to misinterpretations concerning whether reductase activity is regulated by phosphorylation-dephosphorylation. In this paper, we present several methods and approaches which can be used to critically evaluate this possibility.     

Reversible inactivation of 3-hydroxy-3-methylglutaryl coenzyme A reductase: reductase kinase and mevalonate kinase are separate enzymes

Reductase kinase and mevalonate kinase are separated by: a) ammonium sulfate fractionation; b) chromatography on agarose-Procion Red HE3B; and c) chromatography on DEAE-Sephacel. Fractions containing only reductase kinase reversibly inactivated microsomal or homogeneous HMG-CoA reductase. Fractions containing only mevalonate kinase revealed artifactual reductase kinase activity in the absence of EDTA or mevalonic acid; however, addition of EDTA or mevalonate before reductase assay completely blocked any apparent decline in HMG-CoA reductase activity. Under these conditions no dephosphorylation (reactivation) was observed by phosphatase. The combined results demonstrate unequivocally that reductase kinase and mevalonate kinase are two different enzymes and inactivation of HMG-CoA reductase is catalyzed by ATP-Mg-dependent reductase kinase.     

Analysis of the coordinate expression of 3-hydroxy-3-methylglutaryl coenzyme A synthase and reductase activities in Chinese hamster ovary fibroblasts

Decreased activities of both 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase are observed in the presence of sterol in the Chinese hamster ovary (CHO) fibroblast. In three different genotypes of CHO cell mutants resistant to 25-hydroxycholesterol both enzyme activities exhibit a decreased response to 25-hydroxycholesterol compared to wild-type cells. Permanently repressed levels of both HMG CoA synthase and HMG CoA reductase activities are observed in another CHO mutant, phenotypically a mevalonate auxotroph. Mevinolin, a competitive inhibitor of HMG CoA reductase, has no effect on HMG CoA synthase activity measured in vitro. Incubation of CHO cells with sublethal concentrations of mevinolin produces an inhibition of the conversion of [14C]acetate to cholesterol and results in elevated levels of both HMG CoA synthase and HMG CoA reductase activities. Studies of CHO cells in sterol-free medium supplemented with cycloheximide indicate that continuous protein synthesis is not required for the maximal expression of HMG CoA synthase activity and provide an explanation for the lack of temporal similarity between HMG CoA synthase and reductase activities after derepression. These results support the hypothesis of a common mode of regulation for HMG CoA synthase and HMG CoA reductase activities in CHO fibroblasts.     

Evidence that physiologic levels of circulating estrogens and neonatal sex-imprinting modify postpubertal hepatic microsomal 3-hydroxy-3-methylglutaryl coenzyme a reductase activity

Ontact, but sham-operated female rats had 2- to 3-fold higher levels of hepatic 3-hydroxy-3-methylglutary CoA reductase activity than their male couterparts (15–21.5 vs. 6.7–8.7 nmol mevalonate/mg protein per h). The activity of the hepatic enzyme declined to about the same relative degree (40–60%) in male and female rats that were gonadectomized after puberty (53 days of age) and killed 5 weeks later. Implantion of silastic capsules containing 17β-estradiol increased the level of hepatic 3-hydroxy-3-methylglutaryl CoA reductase to levels found in sham-operated controls. In rats that were gonadectomized in infacny (12 h old) and killed 7–8 weeks later, the level of enzyme activity was not altered in females, but it was increased from 60–240% in males. Consequently, following neonatal gonadectomy, male-female differences in enzyme activity were no longer apparent. Implantation of islastic capsules containing estradiol in neonatally gonadectomized rats resulted in a doubling of enzyme activity in both males and females. Ovariectomy reduced plasma estrogen levels, but implantation of estradiol in gonadectomized males and behavioral characteristics. We found in confirmation of an earlier study [20], that in comparison to females, the higher body weight of males and presumably their increased food intake, was also dependent on sex imprinting that occured prior to birth. This observation takes on particular significance in view of the recent report that the amount and quality of food eaten during infancy exerted a long lasting effect on the post-pubertal regulation of 3-hydroxy-3-methyl-glutaryl CoA reductase activity [21,22] and bile acid synthesis [23]. Thus, while a direct effect of neonatal sex imprinting on the regulation of 3-hydroxy-3-methyglutaryl CoA reductase activity is still possible, more indirect mechanisms [24] should also be considered.     

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme a reductase: Analysis of 32P-labeled,inactivated enzyme

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg²⁺ and [γ-³²P]ATP, then solubilized and purified to homogeneity. The ³²P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, ³²P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg²⁺ and ATP.     

Plastid 3-hydroxy-3-methylglutaryl coenzyme A reductase has distinctive kinetic and regulatory features: Properties of the enzyme and positive phytochrome control of activity in pea seedlings

Plastid 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase (mevalonate:NADP oxidoreductase [acylating CoA] EC 1.1.1.34) differs from the cytosolic (microsomal) reductase in pH optimum and apparent K_m for RS-HMG-CoA. Values for the plastid and cytosolic enzyme (brackets) are: pH optimum 7.9 (6.9); apparent K_mRS-HMG-CoA, 0.77 μm (160 μm). Hence the plastid and cytosolic enzymes appear to be different species and not simply compartmented forms of the same protein. The plastid reductase is membrane bound, optimally active only in the presence of dithiothreitol, and specifically requires NADPH; in these respects it is similar to the cytosolic enzyme. In dark-grown seedlings irradiated with red light plastid reductase activity increases to 139% of controls after 20 min, approximately double after 1.75 h, and subsequently declines to a new steady state higher than controls. Far-red reversal studies indicate phytochrome (Pfr) mediation. Reversal can only be demonstrated with very brief (1.5 min) red irradiation followed immediately by far red. It is concluded that Pfr does not act by binding to the enzyme, but that the regulatory mechanism is closely linked to the primary action of Pfr. The rapid Pfr stimulation indicates that this is an early event in the phytochrome control of chloroplast development. The response time and light effects on plastid isoprenoids (photosynthetic and hormonal) also suggest that the regulation of this enzyme is associated with the coordinate control of chloroplast and leaf development by phytochrome. The present positive Pfr control of the plastid reductase contrasts with the previously reported negative Pfr control of the cytosolic reductase.     

Regulation of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from pea seedlings: rapid posttranslational phytochrome-mediated decrease in activity and in vivo regulation by isoprenoid products.

Brief red light irradiation (5 min) of etiolated pea seedlings causes a 40 to 50% decline in microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity, and far red reversal experiments indicate phytochrome mediation. The response is apparent at the earliest assay time, 5 min after irradiation, hence there is little or no lag period; a substantial change occurs within 10 min, and a 24% decrease at 1 h. Activity remains low for about 24 h. The response half-time is about 25 min. Cordycepin affects activity only after 3 h; cycloheximide inhibits only 6% at 1 h and has no effect on activity for at least 20 to 30 min after it blocks protein synthesis. It is concluded that phytochrome regulates reductase activity indirectly through a posttranslational mechanism which causes a stable change in enzyme activity; there is no indication that phytochrome acts by binding directly to the reductase. The decline in reductase activity following irradiation, or cycloheximide treatment, does not follow first-order kinetics. Mixing experiments suggest increased levels of a reductase inactivator in irradiated tissues. The low reductase activity in green seedlings is increased by treatment with dibutyryl-cyclicAMP. Abscisic acid and cholesterol applied to etiolated seedlings reduce activity of the enzyme but gibberellic acid has no effect. However, abscisic acid and cholesterol added to reaction mixtures do not inhibit activity. The metabolic consequences of the rapid light-induced enzyme response may trigger, or contribute to, later biochemical responses previously assumed to be under more direct phytochrome control.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.     

Rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: A comparison and immunological study of purified solubilized preparations,and alteration of enzyme levels by cholestyramine feeding

Some properties of various preparations of solubilized 3-hydroxy-3-methylglutaryl CoA reductase from rat liver are described. One, prepared by solubilization with deoxycholate, has been brought to a level of purity such that only a sińgle component is detected by polyacrylamide gel electrophoresis. A second preparation, solubilized by high salt concentration and heat treatment, has also been purified to a high level of purity so that only minor contaminants are detected. The deoxycholate-solubilized 3-hydroxy-3-methylglutaryl CoA reductase has a molecular weight of 197,000–202,000. Electrophoresis of both preparations treated with mercaptoethanol on polyacrylamide gels in the presence of sodium dodecyl sulfate revealed one band with a molecular weight of 65,000. The data are consistent with the trimeric structure consisting of three polypeptide chains of apparently identical molecular weight. An antiserum to the deoxycholate-solubilized preparation has been prepared. Despite major differences among these preparations in specific activity, in stability to cold, and in the requirement of high salt concentration for preservation, both samples react in the same manner to the antibody and are immunologically indistinguishable. A preparation solubilized by freeze-thawing also reacts with the antiserum. Possible reasons for the variations in specific activity are considered, and it is concluded that specific activity changes cannot be reliably related to protein concentration unless the protein is isolated.Application of the immunological assay to an analysis of the effect of feeding cholestyramine to rats shows that compared to normals the diurnal cycle is unchanged but the rate of enzyme protein synthesis in the cholestyramine-fed rats is greatly accelerated. However, the first-order rate constant for degradatation of enzyme protein remains essentially unchanged throughout the falling phases of the cycle. The specific activity relationships of the enzyme protein of cholestyramine-fed rats appear to be altered when compared to that of normally fed controls.     

Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity

The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.     

Rapid,high-yield purification of rat liver 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase

A rapid method for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase from the livers of cholestyramine-fed rats is reported. The procedure involves a sequence of separations on affinity chromatography columns consisting of Blue Dextran-Sepharose, agarose-CoA, and agarose-HMG-CoA. The advantage of this method is its flexibility in scavenging enzyme that might be lost during purification, resulting in a yield of homogeneous reductase (specific activity approximately 10,000 nmol/min/mg protein) as high as 50%, which is at least twice that previously reported.     

Biochemical properties of short- and long-chain rat liver microsomal trans-2-enoyl coenzyme A reductase

This study describes the biochemical properties of the rat hepatic microsomal NADPH-specific short-chain enoyl CoA reductase and NAD(P)H-dependent long-chain enoyl CoA reductase. Of the substrates tested, crotonyl CoA and trans-2-hexenoyl CoA are reduced by the short-chain reductase only in the presence of NADPH. The trans-2-octenoyl CoA and trans-2-decenoyl CoA appear to undergo reduction to octanoate and decanoate, respectively, catalyzed by both enzymes; 64% conversion of the C8:1 is catalyzed by the short-chain reductase, while 36% conversion is catalyzed by the long-chain enzyme. For the C10:1 substrate, 45% is converted by the short-chain reductase, while 55% is reduced by the long-chain reductase. trans-2-Hexadecenoyl CoA is a substrate for the long-chain enoyl CoA reductase only. Reduction of C4 and C6 enoyl CoA's was unaffected by bovine serum albumin (BSA), whereas BSA markedly stimulated the conversion of C10 and C16 enoyl CoA's to their respective saturated product. Reduction rates as a function of microsomal protein concentration, incubation time, pH, and cofactors are reported including the apparent Km and Vmax for substrates and cofactors. In general, the apparent Km's for the substrates ranged from 19 to 125 microM. The apparent Vmax for the short-chain enoyl CoA reductase was greatest with trans-2-hexenoyl CoA, having a turnover of 65 nmol/min/mg microsomal protein, while the apparent Vmax for the long-chain enzyme was greatest with trans-2-hexadecenoyl CoA, having a turnover of 55 nmol/min/mg microsomal protein. With respect to electron input, NADPH-cytochrome P-450 reductase, either alone, mixed with phospholipid, or incorporated into phospholipid vesicles, possessed no enoyl CoA reductase activity. Cytochrome c did not affect the NADPH-dependent conversion of the trans-2-enoyl CoA. In addition, anti-NADPH-cytochrome P-450 reductase IgG did not inhibit the reduction of trans-2-hexadecenoyl CoA in hepatic microsomes. Finally, the NADPH-specific short-chain and NAD(P)H-dependent long-chain enoyl CoA reductases were solubilized and completely separated from NADPH-cytochrome P-450 reductase by employing DE-52 column chromatography. These studies demonstrate the noninvolvement of NADPH-cytochrome P-450 reductase in either the short-chain (13) or long-chain enoyl CoA reductase system. Thus, the role of NADPH-cytochrome P-450 reductase in the microsomal elongation of fatty acids appears to be at the level of the first reduction step.     

Effects of atractyloside and palmitoyl coenzyme A on calcium transport in cardiac mitochondria.

Palmitoyl CoA (PCoA) and the adenine translocase inhibitor atractyloside (ATR) appear to produce a similar effect in discharging accumulated calcium from cardiac mitochondria. Although mitochondrial respiration is stimulated upon addition of either PCoA or ATR to preparations preloaded with calcium, the effect is not the same as that produced by classical uncouplers. PCoA and ATR also do not interfere with respiration-supported calcium uptake by mitochondria. The presence of exogenous ATP can prevent the calcium discharging effects of PCoA or ATR. Carnitine will prevent the PCoA calcium discharging effect, but has no effect on ATR-induced discharge. It is suggested the PCoA may act at a site on or near the adenine translocase, perhaps through allosteric interaction, to produce an efflux of calcium from mitochondria. The results also suggest that the internal adenine nucleotide pool plays a significant role in mitochondrial calcium retention.     

Transport of coenzyme A in plant mitochondria

Oxoglutarate oxidation by purified potato mitochondria which had been stored at low temperature for 48 h or longer was stimulated by added coenzyme A. Exogenous coenzyme A was accumulated by potato mitochondria, both freshly prepared and aged, in a manner sensitive to uncouplers and low temperature. Coenzyme A was concentrated approximately 10-fold in the matrix under steady-state conditions. This coenzyme A uptake followed saturation kinetics with an apparent Km of 0.2 mM and a V of 4-6.5 nmol min-1 mg-1 protein, suggesting carrier-mediated transport. This transport was insensitive to an inhibitor of NAD+ transport. It is suggested that plant mitochondria possess a specific carrier for the net accumulation of coenzyme A.     

3-Hydroxy-3-methylglutaryl CoA reductase of Hevea latex: The occurrence of a heat-stable activator in the C-serum

The effect of the C-serum (the cytosol) on the activity of 3-hydroxy-3-methylglutaryl CoA reductase in the latex of $\text{Hevea}\text{brasiliensis}$ was investigated. Depending on the clone from which the latex was obtained, the C-serum was found to depress or activate or have little effect on the enzyme activity. Boiling the C-serum however, resulted in a consistent activation effect in all the clones examined. Optimal activation was obtained with 20 μl boiled C-serum. Dialysis or EDTA (40 mM) treatment of the boiled C-serum did not diminish the activation effect. Although not essential, dithiothreitol complemented the activation effect of the boiled C-serum and the optimal concentration was 10 mM. Trypsin digestion of the boiled C-serum resulted in the complete loss of the activation effect. The activator in the boiled C-serum was salted out by ammonium sulphate at 25 – 100% saturation. Hevein had no effect on reductase activity.     

Regulation of ribonucleotide reductase activity in intact mammalian cells

An intact cell assay system based upon Tween-80 permeabilization was used to investigate the regulation of ribonucleotide reductase activity in Chinese hamster ovary cells. Models used to explain the regulation of the enzyme have been based upon work carried out with cell-free extracts, although there is concern that the properties of such a complex enzyme would be modified by extraction procedures. We have used the intact cell assay system to evaluate, within whole cells, the current model of ribonucleotide reductase regulation. While some of the results agree with the proposals of the model, others do not. Most significantly, it was found that ribonucleotide reductase within the intact cell could simultaneously bind the nucleoside triphosphate activators for both CDP and ADP reductions. According to the model based upon studies with cell-free preparations, the binding of one of these nucleotides should exclude the binding of others. Also, studies on intracellular enzyme activity in the presence of combinations of nucleotide effectors indicate that GTP and perhaps dCTP should be included in a model for ribonucleotide reductase regulation. For example, GTP has the unique ability to modify through activation both ADP and CDP reductions, and synergistic effects were obtained for the reduction of CDP by various combinations of ATP and dCTP. In general, studies with intact cells suggest that the in vivo regulation of ribonucleotide reductase is more complex than predicted from enzyme work with cell-free preparations. A possible mechanism for the in vivo regulation of ribonucleotide reductase, which combines observations of enzyme activity in intact cells and recent reports of independent substrate-binding subunits in mammalian cells is discussed.     

Effects of glucose oligomers on the glycogen levels of isolated hepatocytes

Isolated rat hepatocytes appear able to utilize glucose oligomers of the maltose series for the synthesis of glycogen. This utilization is expressed as a stabilization of the glycogen levels during incubation, and also as an incorporation of 14C into glycogen from 14C-labeled glucose polymers. The extent of utilization is only a fraction of that observed with glucose, and it does not appear to be affected by insulin. No extracellular hydrolysis of the polymers seems to be required prior to their uptake by the isolated hepatocytes.