Isolation and characterization of the retinoblastoma protein from fish.

The retinoblastoma (Rb) gene represents the first tumor suppressor gene characterized. The encoded protein, pRb, plays a crucial role in cell cycle control, preventing malignant cell proliferation. Recently, homologues of the Rb gene have been isolated in fish and the pocket domain, which is central to Rb function, was conserved. In our studies, using coelocanth (Latimeria chalumnae), rainbow trout (Oncorhynchus mykiss), medaka (Oryzias latipes) and English sole (Parophrys vetulus), we have developed a simple protocol for the isolation of the Rb tumor suppressor protein and determined its' tissue and cellular localization. Fish Rb proteins display apparent molecular weights in the range of 100-110 kDa, similar to the human pRb. The protein was detected in all tissues examined, consistent with the proteins' universal role in cellular signalling. An interesting pattern of immunoreactive bands was detected in each of the cells' two main compartments, suggesting differential proteolysis. Immuno-analysis of the pRb in trout liver tumor material revealed an additional Rb reactive product that was absent in normal liver cell extracts.

Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas.

We have cloned a medaka homolog of the human retinoblastoma (Rb) susceptibility gene. The medaka Rb cDNA encodes a predicted protein of 909 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the medaka Rb cDNA is highly conserved in regions of functional importance. An antibody raised against an epitope of the human pRb recognizes the protein product of the medaka Rb gene, detecting a 105 kDa protein in all tissues examined and at differential levels for the stages of embryonic development studied. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated a preliminary investigation of the molecular etiology of chemically-induced retinoblastoma. The mutational alterations characterized suggest that medaka may provide a novel model and, thus, provide additional insight into the human retinoblastoma condition.

Effect of ring fluorination on the pharmacology of hallucinogenic tryptamines.

A series of fluorinated analogues of the hallucinogenic tryptamines N,N-diethyltryptamine (DET), 4-hydroxy-N,N-dimethyltryptamine (4-OH-DMT, psilocin), and 5-methoxy-DMT was synthesized to investigate possible explanations for the inactivity of 6-fluoro-DET as a hallucinogen and to determine the effects of fluorination on the molecular recognition and activation of these compounds at serotonin receptor subtypes. The target compounds were evaluated using in vivo behavioral assays for hallucinogen-like and 5-HT(1A) agonist activity and in vitro radioligand competition assays for their affinity at 5-HT(2A), 5-HT(2C), and 5-HT(1A) receptor sites. Functional activity at the 5-HT(2A) receptor was determined for all compounds. In addition, for some compounds functional activity was determined at the 5-HT(1A) receptor. Hallucinogen-like activity, evaluated in the two-lever drug discrimination paradigm using LSD-trained rats, was attenuated or abolished for all of the fluorinated analogues. One of the tryptamines, 4-fluoro-5-methoxy-DMT (6), displayed high 5-HT(1A) agonist activity, with potency greater than that of the 5-HT(1A) agonist 8-OH-DPAT. The ED(50) of 6 in the two-lever drug discrimination paradigm using rats trained to discriminate the 5-HT(1A) agonist LY293284 was 0.17 micromol/kg, and the K(i) at [(3)H]8-OH-DPAT-labeled 5-HT(1A) receptors was 0.23 nM. The results indicate that fluorination of hallucinogenic tryptamines generally has little effect on 5-HT(2A/2C) receptor affinity or intrinsic activity. Affinity at the 5-HT(1A) receptor was reduced, however, in all but one example, and all of the compounds tested were full agonists but with reduced functional potency at this serotonin receptor subtype. The one notable exception was 4-fluoro-5-methoxy-DMT (6), which had markedly enhanced 5-HT(1A) receptor affinity and functional potency. Although it is generally considered that hallucinogenic activity results from 5-HT(2A) receptor activation, the present results suggest a possible role for involvement of the 5-HT(1A) receptor with tryptamines.

Thieno[3,2-b]- and thieno[2,3-b]pyrrole bioisosteric analogues of the hallucinogen and serotonin agonist N,N-dimethyltryptamine.

The synthesis and biological activity of 6-[2-(N, N-dimethylamino)ethyl]-4H-thieno[3,2-b]pyrrole (3a) and 4-[2-(N, N-dimethylamino)ethyl]-6H-thieno[2,3-b]pyrrole (3b), thienopyrroles as potential bioisosteres of N,N-dimethyltryptamine (1a), are reported. Hallucinogen-like activity was evaluated in the two-lever drug discrimination paradigm using LSD- and DOI-trained rats. Neither 3a nor 3b substituted for LSD or DOI up to doses of 50 micromol/kg. By comparison, 1a fully substituted in LSD-trained rats. However, 3a and 3b fully substituted for the 5-HT1A agonist LY293284 ((-)-(4R)-6-acetyl-4-(di-n-propylamino)-1,3,4, 5-tetrahydrobenz[c,d]indole). Both 3a and 3b induced a brief "serotonin syndrome" and salivation, an indication of 5-HT1A receptor activation. At the cloned human 5-HT2A receptor 3b had about twice the affinity of 3a. At the cloned human 5-HT2B and 5-HT2C receptors, however, 3a had about twice the affinity of 3b. Therefore, thiophene lacks equivalence as a replacement for the phenyl ring in the indole nucleus of tryptamines that bind to 5-HT2 receptor subtypes and possess LSD-like behavioral effects. Whereas both of the thienopyrroles had lower affinity than the corresponding 1a at 5-HT2 receptors, 3a and 3b had significantly greater affinity than 1a at the 5-HT1A receptor. Thus, thienopyrrole does appear to serve as a potent bioisostere for the indole nucleus in compounds that bind to the serotonin 5-HT1A receptor. These differences in biological activity suggest that serotonin receptor isoforms are very sensitive to subtle changes in the electronic character of the aromatic systems of indole compounds.

Triiodothyronine treatment increases substrate cycling between pyruvate carboxylase and malic enzyme in perfused rat liver.

The relative roles of pyruvate kinase and malic enzyme in substrate cycling between pyruvate and oxaloacetate were examined in perfused livers of 24-hour-fasted normal and triiodothyronine (T3)-treated rats using an inhibitor of malic enzyme (hydroxymalonate). Livers were perfused for 60 minutes in a recirculating system with [3-13C]alanine (10 mmol/L, 99% 13C-enriched). The combined flux through pyruvate kinase plus malic enzyme relative to pyruvate carboxylase flux was assessed by the 13C-enrichment ratio of alanine C2 to glucose C5 in the perfusate, determined with 13C and 1H nuclear magnetic resonance (NMR) spectroscopy. In normal rat livers, the relative carbon flux through pyruvate kinase plus malic enzyme to pyruvate carboxylase was 0.18 +/- 0.04, and increased to 0.44 +/- 0.08 (P < .05) in the T3-treated group. After addition of hydroxymalonate, this relative carbon flux was unchanged in normal rat livers, but decreased to 0.15 +/- 0.04 (P < .01) in the T3-treated group, suggesting that the increased carbon flux in T3-treated livers was caused by increased flux through malic enzyme. Malic enzyme activity increased from 0.36 +/- 0.05 U/g liver in normal livers to 2.51 +/- 0.50 U/g liver (P < .05) in the T3-treated group, whereas there was no effect of T3 treatment on pyruvate kinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of biliary epithelial cells from rainbow trout liver.

Lectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment of coverslips coated with Phaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts with hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributed to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their responses to toxic and carcinogenic agents.

Long-term primary culture of epithelial cells from rainbow trout Oncorhynchus mykiss) liver.

Long-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (> 97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a "spindle cell," consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged. The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

Substrate cycling between pyruvate and oxaloacetate in awake normal and 3,3'-5-triiodo-L-thyronine-treated rats.

Substrate cycling between pyruvate and oxaloacetate was assessed in awake 24-h fasted normal and triiodothyronine (T3)-treated rats. After a 20- or 60-min infusion of [3-13C]alanine (99% enriched, 12 mg/min) the 13C enrichments of liver glucose and alanine carbons were analyzed by 13C and 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. Substrate cycling from phosphoenolpyruvate to pyruvate [via pyruvate kinase (PK)] and from oxaloacetate to pyruvate [via malic enzyme (ME)] relative to the pyruvate carboxylase (PC) flux [i.e., (PK+ME)/PC] was assessed by the ratio of the 13C enrichment of C-2 alanine relative to that in C-5 glucose. In the normal rats (PK+ME)/PC was 0.26 +/- 0.07 (n = 7, t = 20 min) and 0.37 +/- 0.08 (n = 4, t = 60 min). In the T3-treated rats the (PK+ME)/PC increased four- to fivefold to 1.03 +/- 0.19 (n = 8, t = 20 min) and to 1.83 +/- 0.19 (n = 3, t = 60 min) (P < 0.05 vs. normal rats). The liver enzyme activity of PK did not change with T3 treatment (normal 14.22 +/- 5.25 U/g liver vs. T3 treated 13.40 +/- 1.10 U/g liver), whereas both the enzyme activity ratio of PK (normal 0.47 +/- 0.15 vs. T3 treated 0.77 +/- 0.03, P < 0.05) and the activity of ME (normal 0.89 +/- 0.30 U/g liver vs. T3 treated 4.25 +/- 0.60 U/g liver, P < 0.05) increased with T3 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured trout liver cells: utilization of substrates and response to hormones.

The characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [14C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetaminophen toxicity in cultured trout liver cells. I. Morphological alterations and effects on cytochrome P450 1A1.

To better characterize the hepatotoxicity of acetaminophen, the effects of this drug were investigated on isolated liver cells from a species relatively resistant to acetaminophen toxicity, rainbow trout. At high concentrations of acetaminophen (2-10 mM), pathologic effects were detected, including loss of lactate dehydrogenase from cells, disruption of cell-cell aggregation, cell death, and distinctive alterations in cell morphology, as demonstrated by light and electron microscopic examination. Most striking was the acetaminophen-induced rearrangement of mitochondria, which were clustered adjacent to the nucleus and rarely seen at cell periphery. The endoplasmic reticulum was also altered by acetaminophen treatment. In the middle portion of the cytoplasm, parallel arrays of endoplasmic reticulum cisternae were abundant; however, the peripheral cytoplasm was restricted to vesicular profiles of endoplasmic reticulum. Although nuclei in acetaminophen-treated cells displayed peripheral heterochromatin aggregation, acetaminophen did not produce detectable DNA fragmentation, in contrast to effects reported for mouse liver cells. Thus DNA fragmentation does not appear to be required for acetaminophen to manifest cytotoxic effects. In addition, immunohistochemical studies indicated that toxic concentrations of acetaminophen which altered the endoplasmic reticulum helped maintain cytochrome P450 1A1 in liver cells from beta-naphthoflavone-induced trout.

Acetaminophen toxicity in cultured trout liver cells. II. Maintenance of cytochrome P450 1A1.

Acetaminophen was demonstrated to maintain cytochrome P450 1A1 (P450 1A1) in isolated rainbow trout liver cells cultured in serum-free medium. This novel finding was characterized in detail. Cultured trout liver cells retained their ability to respond to typical 1A1 inducers in vitro; induction of ethoxyresorufin O-deethylase (EROD) activity was readily demonstrated by exposing liver cells from control trout to beta-naphthoflavone (BNF), Aroclor 1254, or 7,12-dimethylbenz[a]anthracene. BNF was the most potent inducer studied and was used in further experiments. High levels of EROD activity, immunoreactive 1A1, and 1A1 mRNA were expressed in liver cells prepared from trout pretreated with BNF. However, all of these 1A1-specific indicators rapidly declined when cells from BNF-treated trout were placed in culture, and BNF in culture medium was not effective in maintaining EROD activity. Immunohistochemical studies suggested that addition of acetaminophen to liver cells prepared from BNF-induced trout helped maintain elevated levels of P450 1A1. Total cytochrome P450, EROD activity and immunoreactive P450 1A1 were retained in liver cells from BNF-induced trout by the addition of acetaminophen, in a dose-dependent manner. The concentrations of acetaminophen most effective in maintaining P450 1A1 produced cytotoxic effects, including vesiculation of endoplasmic reticulum. Furthermore, the acetaminophen maintenance of P450 1A1 was primarily attributed to elevated levels of P450 1A1 mRNA. In contrast to BNF, acetaminophen was not capable of inducing 1A1 in liver cells prepared from control trout. This is the first report to demonstrate that acetaminophen can help maintain P450 1A1 and that this effect is exerted at the level of P450 1A1 mRNA.

Isolated trout liver cells: establishing short-term primary cultures exhibiting cell-to-cell interactions.

Composition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120 X g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

DNA repair synthesis in isolated rainbow trout liver cells.

Isolated trout liver cells were treated with lysolecithin to produce an in situ system for characterizing DNA repair in teleosts. In this preparation, the integrity of the plasma membrane is altered, nuclei remain intact, and the concentrations of dNTPs and nucleotide analogs, which normally do not penetrate intact plasma membranes, can be controlled. Following lysolecithin treatment, 50% of the total cellular protein and nearly 75% of total lactate dehydrogenase activity was released from the liver cells. Microscopic examination indicated that the integrity of the plasma membrane of trout hepatocytes was disrupted by lysolecithin; however, smaller nonhepatocytic liver cells were resistant to the disrupting effects of this detergent. Bleomycin induced DNA repair synthesis in lysolecithin-treated cells, as demonstrated by CsCl gradient analysis of 5-bromo, 2'-deoxyuridine, 5'-triphosphate-labeled DNA. Optimal conditions for bleomycin-induced DNA repair synthesis in lysolecithin-treated trout liver cells were considerably different from that in lysolecithin-treated mammalian cells. Bleomycin-induced DNA repair synthesis was lower in lysolecithin-treated trout liver cells than in lysolecithin-treated mammalian cells at identical concentrations of 2'-deoxyribonucleoside, 5'-triphosphates (dNTPs), suggesting the decreased sensitivity of trout cells in unscheduled DNA synthesis assays can be attributed to factors other than differences in dNTP pools. Bleomycin-induced DNA repair synthesis in trout hepatocytes was shown to be very sensitive to inhibition by 2', 3'-dideoxythymidine, 5'-triphosphate and was resistant to inhibition by cytosine arabinoside, 5'-triphosphate, butylphenyldeoxyguanosine, 5'-triphosphate and aphidicolin. These observations indicate repair of bleomycin-induced DNA damage in trout cells occurs through a mechanism similar to that in mammalian cells, utilizing DNA polymerase beta.

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: I. Process concept and enzyme binding studies for pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae.

A process using ligand-coupled particles in aqueous polyethylene glycol-dextran two-phase polymer systems was developed to achieve a highly selective, scaleable biochemical separation process. Product protein is bound to the ligand-coupled particles that quantitatively distribute to the polyethylene glycol-rich upper phase. Other proteins and contaminants partition preferentially to the dextran-rich lower phase.The process offers significant advantages over affinity partitioning here the ligand is coupled to the backbone of a polyethylene glycol polymer. These advantages include a much wider diversity of ligands that can be coupled to particles and more effective confinement of the ligand in the process. Affinity partition with ligands coupled to particles is more amenable to scale-up than is affinity chromatography. A variety of commercially available Sepharose-based particles are suitable for this process. Homogenates from Saccharomyces cerevisiae, which is genetically altered to overproduce pyruvate kinase, and Cibacron blue F3G-A-coupled Sepharose particles are used as a model system for the process. Binding studies with/without aqueous two-phase systems show that the formation of a two-phase system after the adsorption equilibrium is reached does not affect the apparent dissociation constant. Binding of protein to ligand-coupled particles is more rapid in single-phase systems than in the polymer two-phase system. Single-phase binding eliminates the mass transfer resistance associated with redistribution of product protein from the dextran-rich bottom phase to the polyethylene glycol-rich top phase.

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae.

The novel approach of using aqueous two-phase systems for the elution of protein from ligand-coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G-A-coupled Sepharose CL6B (Blue-Sepharose) particles as a model system. The ligand-coupled particles distribute quantitatively to the polyethylene glycol-(PEG-) rich top phase and the recovered enzymes partition selectively to the dextran-(DEX-) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue-Sepharose particles using PEG8000-DEXT500 aqueous two-phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two-phase systems. Recovery of pyruvate kinase, which is bound to ligand-coupled particles, in the DEX-rich bottom phase of aqueous two-phase systems can be up to 95% in one-step operations. The mixing time of ligand-coupled particles with aqueous two-phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two-phase systems also influence the recovery of pyruvate kinase from ligand-coupled particles. The recovered enzymes in the DEX-rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand-coupled particles are added advantages of the process using aqueous two-phase systems. Preliminary studies with goat polyclonal anti-pyruvate kinase-coupled Sepharose particles indicate that the process also may be applicable when a high-affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.

Yeast cell debris and protein partitioning in the poly(ethylene glycol)-poly(vinyl alcohol) biphasic system.

Yeast cells, cell debris and protein partitioning have been investigated in the poly(ethylene glycol) (PEG) 8000/poly(vinyl alcohol) (PVA) 10,000 system. Cells and cell debris partition into the lower (PVA) phase over the pH range 4.8-7.5, and with up to 0.37 M KCl at pH 5.9. Protein partitioning is more pH-dependent in the PEG/PVA system than in the PEG/dextran system, and a significant fraction of the total protein is found at the interface at lower pH values. Significant, rapid purification of overproduced pyruvate kinase in a PEG/PVA system containing Blue Sepharose CL-6B particles is demonstrated.

Quantitative analysis of glycogen repletion by nuclear magnetic resonance spectroscopy in the conscious rat.

In order to directly determine the amount of label exchange that occurs in the tricarboxylic cycle from labeled alanine and lactate after the ingestion of a glucose load [1-13C]glucose was administered by continuous intraduodenal infusion to awake catheterized rats to achieve steady state jugular venous glycemia (160 mg/dl) for 180 min. Liver was freeze-clamped at 90 and 180 min, and perchloric acid extracts of the liver were subjected to 13C and 1H nuclear magnetic resonance analysis. Dilution in the oxaloacetate pool was determined by comparing the intrahepatic 13C enrichments of C2, C3 positions of glutamate with the C2, C3 positions of alanine and lactate. In addition steady state flux equations were derived for calculation of relative fluxes through pyruvate dehydrogenase/TCA cycle flux and pyruvate kinase flux/total pyruvate utilization. After glucose ingestion in a 24-h fasted rat direct conversion of glucose was responsible for 34% of glycogen. The intrahepatic dilution factor for labeled pyruvate in the oxaloacetate pool was 2.4. Using this factor, alanine and lactate contributed approximately 55% to glycogen formation. Pyruvate dehydrogenase flux ranged between 24 and 35% of total acetyl-coenzyme A (CoA) production and pyruvate kinase flux relative to total pyruvate utilization was approximately 40%.

Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity.

Homogeneous liver pyruvate kinase was reacted with different sulfhydryl reagents, which included o-iodosobenzoate, 5',5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Activity determinations of the treated enzyme made with and without Fru(1,6)P2 indicate that the protein contains two sulfhydryl groups per subunit important to its properties, one more accessible than the other. Fru(1,6)P2 added to mixtures prevented loss of activity obtained with o-iodosobenzoate and 5',5'-dithiobis(2-nitrobenzoic acid). It appears that Fru(1,6)P2 does not interfere with the reaction of the reagent with the sulfhydryl group, but prevents an ensuing conformational change, which leads to changes in the enzyme's properties.

Inability of glucagon to regulate glycogen metabolism in rat hepatocytes isolated after fasting and refeeding high-carbohydrate diets.

Studies are described which demonstrate that the ability of glucagon, epinephrine, and dibutyryl-cAMP to stimulate glycogenolysis is impaired in rat hepatocytes isolated from animals starved for 24 h and then refed a sucrose-rich diet or refed standard rat chow. The impaired regulation of glycogenolysis by glucagon was observed within 24 h after refeeding and persisted for at least 3 days. The inability of glucagon to stimulate glycogen breakdown in the refed condition appeared to be due to a suppressed activation of glycogen phosphorylase and phosphorylase b kinase by the hormone. The capacity of glucagon to regulate pyruvate kinase and glycolysis was not altered by refeeding, suggesting that the defect lies beyond interaction of the hormone at its receptor. Prolonged incubation of hepatocytes from refed rats was accompanied by depletion of glycogen reserves and was accompanied by restoration of hormonal stimulation of glycogenolysis. Addition of glycogen to cell-free extracts was found to inhibit phosphorylase b kinase but not phosphorylase. The findings of this investigation are consistent with the interpretation that high levels of glycogen present of liver after refeeding may lead to a diminished activity of phosphorylase b kinase and its hormonal regulation.