Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Dietary fat type alters glucose metabolism in isolated rat hepatocytes

Dietary fat type can influence the regulation of carbohydrate metabolism in multiple tissue types. The influence of feeding high-fat (40% of kilocalories) diets containing either menhaden oil (MO) or coconut oil (CO) on hepatic glycogenolytic and gluconeogenic capacities was studied in isolated rat hepatocytes. Estimates of both glycogenolytic and gluconeogenic capacities were performed on hepatocytes isolated from fed and fasted animals, respectively. In MO-fed animals, both basal and hormone-stimulated rates of glucose production were significantly greater than those in CO-fed animals. However, both groups displayed a similar maximal increase in glucose production above basal for glucagon and epinephrine (2.3- and 1.9-fold, respectively). Basal rates of adenosine 3′,5′-cyclic phosphate (cAMP) production were not different between groups whereas glucagon-stimulated cAMP production was increased twofold in the MO-fed group. In both MO and CO groups, the addition of 10 nM insulin reduced glucose production in fed animals to similar absolute rates. In animals fasted for 24 hours, gluconeogenic capacity was estimated using 10 mM pyruvate, lactate, or glycerol. Glucose production from all substrates was significantly greater in CO-fed animals. In addition to increased gluconeogenic rates, maximal phosphoenolpyruvate carboxykinase (PEPCK) activity was increased in the CO-fed group. Insulin reduced glucose production in both dietary groups, but the absolute rate of glucose production was 28% greater in the CO-fed group relative to the MO-fed group. In summary, dietary fat type can markedly influence the regulation of hepatic glucose metabolism in multiple metabolic pathways. MO feeding promoted glycogenolysis and sensitivity to insulin whereas CO feeding favored gluconeogenesis and reduced insulin sensitivity.     

Difference in glucose sensitivity of liver glycolysis and glycogen synthesis. Relationship between lactate production and fructose 2,6-bisphosphate concentration.

Incubation of isolated rat hepatocytes from fasted rats with 0-6 mM-glucose caused an increase in [fructose 2,6-bisphosphate] (0.2 to about 5 nmol/g) without net lactate production. A release of 3H2O from [3-3H]glucose was, however, detectable, indicating that phosphofructokinase was active and that cycling occurred between fructose 6-phosphate and fructose 1,6-bisphosphate. A relationship between [fructose 2,6-bisphosphate] and lactate production was observed when hepatocytes were incubated with [glucose] greater than 6 mM. Incubation with glucose caused a dose-dependent increase in [hexose 6-phosphates]. The maximal capacity of liver cytosolic proteins to bind fructose 2,6-bisphosphate was 15 nmol/g, with affinity constants of 5 X 10(6) and 0.5 X 10(6) M-1. One can calculate that, at 5 microM, more than 90% of fructose 2,6-bisphosphate is bound to cytosolic proteins. In livers of non-anaesthetized fasted mice, the activation of glycogen synthase was more sensitive to glucose injection than was the increase in [fructose 2,6-bisphosphate], whereas the opposite situation was observed in livers of fed mice. Glucose injection caused no change in the activity of liver phosphofructokinase-2 and decreased the [hexose 6-phosphates] in livers of fed mice.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Glutamine metabolism in rat hepatocytes. Stimulation by a nonmetabolizable analog of leucine

The metabolic effects of beta-(+/-)-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH), a nonmetabolizable analog of leucine and known activator of glutamate dehydrogenase, were studied in hepatocytes isolated from fed and fasted rats. With glutamine as substrate, BCH stimulated in a concentration-dependent manner urea synthesis in both physiological states and glucose formation in hepatocytes from fasted rats. Despite the much higher rates of ureagenesis in the fasted animals, the degree of stimulation by BCH, over 2-fold, was similar. The effect of the drug was specific for glutamine since the rates of urea synthesis from NH4Cl, alanine, and asparagine were essentially unaltered. The stimulation of glutamine catabolism by BCH led to a decrease in the content of intracellular glutamine. The redox states of the mitochondrial and cytosolic nicotinamide adenine dinucleotides remained unaltered. In hepatocytes isolated from fasted rats and incubated with 5 mM glutamine the BCH-induced increases in urea, ammonia, and the amino acids, glutamate, aspartate, and alanine, accounted fully for the 2.4-fold rise in glutamine utilization. The stimulatory effects of BCH and glucagon on the formation of glucose, urea, and 14CO2 from [U-14C]glutamine were additive. Aminooxyacetate, and inhibitor of transaminases, neither blocked glutamine catabolism (as measured by the sum of urea, ammonia, and glutamate) nor prevented its activation by BCH. It is suggested that, in isolated hepatocytes, BCH-induced stimulation of glucose and urea formation from glutamine results from activation of glutaminase by a mechanism which is distinct from that of glucagon.     

Ammonia overloading in hepatocytes isolated from liver of fetal and adult rats

Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.     

The effect of A23187 on glucose production from methylglyoxal and pyruvate in isolated murine hepatocytes.

1. A23187 increased the glucose production from methylglyoxal in isolated hepatocytes, and maximal stimulation was obtained at 10(-6) M. The effect of A23187 was dependent on the presence of Ca2+. 2. Glucose production from pyruvate (less than 1 mM) in isolated hepatocytes was stimulated by A23187 in the presence of 2.5 mM Ca2+ and was depressed at pyruvate concentrations above 1 mM. Both the virtual Km and the virtual Vmax of glucose production from pyruvate were decreased by A23187.     

In vivo and in vitro effects of beta-endorphin on glucose metabolism in the rat

The effects of beta-endorphin (beta-Ep) on plasma glucose levels in rats and on glucose metabolism in isolated rat liver cells were examined. Intravenous injection of beta-Ep (5 micrograms/100 g BW) into ether-anaesthetized rats resulted in prompt and sustained hyperglycaemia with increases in the plasma glucagon and somatostatin levels and decrease in the plasma insulin level. When liver cells isolated from fed rats were incubated in the presence of beta-Ep at concentrations of 6 X 10(-8) M to 6 X 10(-7) M, glucose release into the medium increased within 15 min in a dose-related manner. Time course experiments showed that beta-Ep increased the level of cyclic AMP within 3 min. Significant increase in gluconeogenesis in liver cells isolated from fasted rats was also observed on addition of 10(-7) M beta-Ep in the presence of 10 mM L-lactate. These results suggest that the hyperglycaemia induced by beta-Ep may be caused, at least in part, by the effects of beta-Ep on releases of pancreatic hormones and glucose production in liver cells.     

Effect of metal ion catalyzed oxidation of rifamycin SV on cell viability and metabolic performance of isolated rat hepatocytes

The effect of rifamycin SV on metabolic performance and cell viability was studied using isolated hepatocytes from fed, starved and glutathione (GSH) depleted rats. The relationships between GSH depletion, nutritional status of the cells, glucose metabolism, lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) production in the presence of rifamycin SV and transition metal ions was investigated. Glucose metabolism was impaired in isolated hepatocytes from both fed and starved animals, the effect is dependent on the rifamycin SV concentration and is enhanced by copper (II). Oxygen consumption by isolated hepatocytes from starved rats was also increased by copper (II) and a partial inhibition due to catalase was observed. Cellular GSH levels which decrease with increasing the rifamycin SV concentration were almost depleted in the presence of copper (II). A correlation between GSH depletion and LDH leakage was observed in fed and starved cells. Catalase induced a slight inhibition of the impairment of gluconeogenesis, GSH depletion and LDH leakage in starved hepatocytes incubated with rifamycin SV, iron (II) and copper (II) salts. Lipid peroxidation measured as MDA production by isolated hepatocytes was also augmented by rifamycin SV and copper (II), especially in hepatic cells isolated from starved and GSH depleted rats. Higher cytotoxicity was observed in isolated hepatocytes from fasted animals when compared with fed or GSH depleted animals. It seems likely that in addition to GSH level, there are other factors which may have an influence on the susceptibility of hepatic cells towards xenobiotic induced cytotoxicity.     

Lithium inhibits hepatic gluconeogenesis and phosphoenolpyruvate carboxykinase gene expression.

Incubation of isolated hepatocytes from fasted rats with 20 mM LiCl for 1 h decreased glucose production from lactate, pyruvate, and alanine. In addition, phosphoenolpyruvate carboxykinase (PEPCK) gene expression in FTO-2B rat hepatoma cells was inhibited by treatment with LiCl. Lithium was also able to counteract the increased PEPCK mRNA levels caused by both Bt2cAMP and dexamethasone, in a concentration-dependent manner. A chimeric gene containing the PEPCK promoter (-550 to +73) linked to the amino-3-glycosyl phosphotransferase (neo) structural gene was transduced into FTO-2B cells using a Moloney murine leukemia virus-based retrovirus. In these infected cells, 20 mM LiCl decreased both the concentration of neo mRNA transcribed from the PEPCK-neo chimeric gene and mRNA from the endogenous PEPCK gene. Lithium also inhibited the stimulatory effect of Bt2cAMP and dexamethasone on both genes. The stability of neo mRNA was not altered by lithium, since in cells infected with retrovirus containing only the neo gene transcribed via the retroviral 5'-LTR and treated with 20 mM LiCl, no change in neo mRNA levels was observed. The intraperitoneal administration of LiCl to rats caused a decrease in hepatic PEPCK mRNA, indicating that lithium could also modify gene expression in vivo. The effects of lithium were not due to an increase in the concentration of insulin in the blood but were correlated with an increase in hepatic glycogen and fructose 2,6-bisphosphate levels. These results indicate that lithium ions, at concentrations normally used therapeutically for depression in humans, can inhibit glucose synthesis in the liver by a mechanism which can selectively modify the expression of hepatic phosphoenolpyruvate carboxykinase.     

Synergistic inhibition of glucagon-induced effects on hepatic glucose metabolism in the presence of insulin and a cAMP antagonist

Inhibition of hepatic glycogenolysis by an intracellular inhibitor of cAMP-dependent protein kinase in glucagon-stimulated hepatocytes was potentiated by insulin. When hepatocytes isolated from fed rats were treated with 0.3 nM glucagon, which activates glycogen breakdown half-maximally, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate [Rp-cAMPS), a cAMP antagonist, inhibited glucose production half-maximally at 3 microM. A 10-fold lower concentration of antagonist was required to half-maximally inhibit glucose production in the presence of 10 nM insulin, which alone produced only 15% inhibition. Under the same experimental conditions, the maximal effect of (Rp)-cAMPS was also potentiated. In addition, the increase in the concentration of glucagon required for half-maximal activation of phosphorylase activity and inactivation of glycogen synthase activity in the presence of minimally effective concentrations of insulin and (Rp)-cAMPS were clearly synergistic. It is postulated that the synergism observed is a consequence of action at several enzymatic sites leading to, and including, alteration of the phosphorylation state of the two rate-limiting enzymes in glycogen metabolism.     

Stimulation of glycogen synthesis by proglycosyn (LY177507) by isolated hepatocytes of normal and streptozotocin diabetic rats

The phenacylimidazolium compound LY177507 was shown by Harris et al. (Harris, R. A., Yamanuchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J., and Stephens, T. W. (1989) J. Biol. Chem. 264, 14674-14680) to stimulate glycogen synthesis greatly in isolated rat hepatocytes. We extended studies with this compound, designated proglycosyn (Yamaguchi, K., Stephens, T. W., Chikadar, K., Depaoli-Roach, A., And Harris, R. A. (1991) Diabetes 40, (Suppl. 1) 102 (abstr.] employing hepatocytes from normal and streptozotocin diabetic rats. Proglycosyn is more effective than amino acids in stimulating glycogen synthesis. In cells incubated with glucose, lactate, or dihydroxyacetone the effect of glutamine and proglycosyn was synergistic. In cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, the stimulation by the two agonists was additive. Proglycosyn diverted the gluconeogenic flux from glucose to glycogen. The maximal rates of glycogen deposition attained in the presence of glutamine and proglycosyn from cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, where about 80 and 110 mumols/h/g of liver, respectively. Proglycosyn depressed glycogenolysis in hepatocytes of fed rats and stimulated glycogen synthesis from lactate and dihydroxyacetone. The incorporation of [U-14C]glucose and [U-14C]lactate in these cells occurred in the presence of glycogen breakdown or exceeded net production, indicating the occurrence of recycling of glycogen in hepatocytes of fed rats. Hepatocytes from fasted streptozotocin diabetic rats contained high levels of glycogen. Glycogenolysis was markedly depressed by proglycosyn. Glycogen synthesis from lactate and dihydroxyacetone in these cells was stimulated by glutamine and proglycosyn in a fashion similar to that in cells from fasted control rats, and the rates of glycogen synthesis were similar in cells of control and diabetic rats. With glucose as sole substrate, glutamine did not stimulate glycogen synthesis. When both agonists were present, there was a marked synergism and substantial glycogen formation. Streptozotocin diabetic rats prior to the onset of cachexia have a normal capacity for glycogen synthesis.     

Role of a guanine nucleotide-binding regulatory protein in the hydrolysis of hepatocyte phosphatidylinositol 4,5-bisphosphate by calcium-mobilizing hormones and the control of cell calcium. Studies utilizing aluminum fluoride

Treatment of isolated hepatocytes with NaF produced a concentration-dependent activation of phosphorylase, inactivation of glycogen synthase, efflux of Ca2+, rise in cytosolic free Ca2+ ([Ca2+]i), increase in myo-inositol-1,4,5,-P3 levels, decrease in phosphatidylinositol-4,5-P2 levels, and increase in 1,2-diacylglycerol levels. These changes were evident within 1 min and maximum at 2-5 min. Maximum effects on Ca2+ efflux, [Ca2+]i, glycogen synthase, and phosphorylase were observed with 15 mM NaF, whereas myo-inositol-1,4,5-P3 and 1,2-diacylglycerol levels were maximally stimulated by 50 mM NaF. The levels of intracellular cAMP were decreased by NaF (up to 10 mM) in the absence or presence of glucagon (0.1-1 nM) or forskolin (2 microM). The effects of low doses of NaF (2-15 mM) to inhibit basal or glucagon-stimulated cAMP accumulation, mobilize Ca2+, activate phosphorylase, and inactivate glycogen synthase were all potentiated by AlCl3. This potentiation was abolished by the Al3+ chelator deferoxamine. These results illustrate that AlF4- can mimic the effects of Ca2+-mobilizing hormones in hepatocytes and suggest that the coupling of the receptors for these hormones to the hydrolysis of phosphatidylinositol-4,5-P2 to myo-inositol 1,4,5-P3 is through a guanine nucleotide-binding regulatory protein. This is because AlF4- is known to modulate the activity of other guanine nucleotide regulatory proteins (Ni, Ns, and transducin).     

Short-term stimulation of net glycogen production by insulin in rat hepatocytes

Isolated liver cells from 24 h starved rats were incubated in Krebs-Ringer buffer containing 4% albumin. In the presence of 10, 20 and 30 mM glucose, addition of insulin stimulated net glycogen production by 52, 39 and 20%, respectively. 2 . 10(-9) M insulin was required for half-maximal stimulation. Increases of glycogen production and of glycogen synthase a activity were observed after 15-30 min of incubation with insulin. The stimulatory effect of insulin was additive to that of lithium. In agreement with the literature, insulin antagonized the inhibitory action of suboptimal doses of glucagon on glycogen deposition whereby a decrease of glucagon-elevated cyclic AMP levels was observed. In addition, we found that insulin also decreased the basal cyclic AMP levels in the absence of added glucagon by 22%. It is concluded that physiological concentrations of insulin stimulate net glycogen deposition in hepatocytes from fasted rats; the decrease of basal cyclic AMP levels upon insulin addition may play a role in the mechanism of the hormone action.     

Modulation of glucagon effects by changes in extracellular pH and calcium

We have examined the influence of extracellular pH and calcium concentration on the action of glucagon on isolated rat hepatocytes, perfused liver or plasma membrane preparations. Incubation of rat hepatocytes with 10 nM glucagon at pH 7.4 caused an immediate increase in cAMP concentrations (8-fold), and this rise was almost 50% lower at acidic extracellular pH (6.9). This effect of pH could not be explained by an alteration of the hormone binding to its receptor for glucagon concentrations higher than 1 nM. The effect of acidosis on cAMP production was still present with non-hormonal effectors, such as 10 microM Gpp[NH]p, 30 microM forskolin or 10 mM NaF. This suggests a direct action of acidosis on the regulatory component Ns and/or on the catalytic subunit of adenylate cyclase. Acidic pH also depressed mitochondrial processes responsive to glucagon (NAD(P)H fluorescence, glutamine breakdown). Whatever the experimental model, calcium appeared to be required for maximal stimulation of cAMP production by glucagon. On perfused rat liver, glycogenolysis was depressed in the absence of extracellular calcium in the perfusate. In isolated hepatocytes, the stimulation of phosphorylase alpha activity by glucagon was modulated by extracellular calcium concentrations lower than 0.2 mM. This suggests that, although glucagon action is chiefly cAMP-mediated, its effect on calcium mobilization (affecting various cellular process, including cAMP production itself) should also be taken into account. This work also confirmed the importance of calcium in the stimulation of mitochondrial metabolism of glutamine by glucagon.     

Role of microtubules in insulin and glucagon stimulation of amino acid transport in isolated rat hepatocytes

The effects of the microtubule inhibitor, colchicine, on insulin or glucagon stimulation of alpha-amino[1-14C]-isobutyric acid (AIB) transport were investigated in isolated hepatocytes from normal fed rats. Under all conditions tested, AIB uptake appeared to occur through two components of transport: a low affinity (Km approximately 50 mM) component and a high affinity (Km approximately 1 mM) component. Within 2 h of incubation, insulin and glucagon, at maximal concentrations, increase AIB (0.1 mM) uptake by 2- to 3-fold and 4- to 6-fold, respectively. Colchicine, at the low concentration of 5 X 10(-7) M, slightly reduces basal AIB transport, decreases by 80% the simulatory effect of insulin, and diminishes by 40% the stimulatory effect of either glucagon or dibutyryl cAMP. Kinetic analysis of AIB influx indicates that the drug inhibits the increase in Vmax of a high affinity (Km approximately 1 mM) component of transport stimulated by insulin or glucagon, without affecting the kinetic parameters of a low affinity component of transport (Km approximately 50 mM). Various short term hormonal effects of insulin and glucagon (changes in glucose, urea, and lactate production) were found not to be modified by the drug. Vinblastine elicits similar changes as colchicine on AIB uptake. Lumicolchicine, a colchicine analogue that does not bind to tubulin, has no effect. The concentration of colchicine (10(-7) M) required for half-maximal inhibition of hormone-stimulated AIB transport is in the appropriate range for specific microtubule disruption. These data suggest that microtubules are involved in the regulation of the insulin or glucagon stimulation of AIB transport in isolated rat hepatocytes.     

Multiple requirements for glycogen synthesis by hepatocytes isolated from fasted rats

Glycogen synthesis from various combinations of substrates by hepatocytes isolated from rats fasted 24 h was studied. As reported by Katz et al. (Katz, J., Golden, S., and Wals, P. A. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3433-3437), appreciable rates of glycogen synthesis occurred only in the presence of gluconeogenic precursors and one of several amino acids, which includes L-glutamine. L-Leucine had negligible effects on glycogen synthesis from 20 mM dihydroxyacetone and/or 15 mM glucose when L-glutamine was not added to the medium. In the presence of 10 mM L-glutamine, L-leucine greatly increased glycogen synthesis from these substrates. alpha-Ketoisocaproate was ineffective, as was oleate. NH4Cl depressed glycogen synthesis from 10 mM glucose plus 20 mM dihydroxyacetone in the absence of added L-glutamine and enhanced that in its presence, but these effects were weak compared to those of L-leucine. The amino acid analogues L-norvaline and L-norleucine exerted effects that were similar to those exerted by L-leucine. Under all conditions studied, cycloheximide and puromycin inhibited net glycogen synthesis. Cycloheximide did not stimulate gluconeogenesis from dihydroxyacetone, or phosphorylase in hepatocytes from starved rats, or glycogenolysis in hepatocytes from fed rats. Puromycin, however, stimulated glycogenolysis in hepatocytes from fed rats. Glycogen synthesis from 20 mM dihydroxyacetone proceeds with a pronounced initial lag phase that can be shortened by incubation of cells with glutamine plus leucine before addition of dihydroxyacetone. Concurrent measurements of glycogen synthesis, glycogen synthase, and gluconeogenesis under different conditions reveal that in addition to protein synthesis, activation of glycogen synthase, which must occur to allow glycogen synthesis in hepatocytes, requires a second component which can be satisfied by addition of dihydroxyacetone or fructose to the cells.     

Inhibition of glycogen synthesis in rat hepatocytes by medium Zn2+

In hepatocytes from fasted rats, Zn2+ in the range from 0 to 500 microM has relatively minor effects on gluconeogenesis from most substrates, or on ureagenesis from NH3. In hepatocytes from fed rats, Zn2+ does not affect glycogenolysis. In hepatocytes from fasted rats, in which glycogen is being actively synthesized using the substrate combination (Katz et al. (1976) Proc. Natl.Acad.Sci.USA 73,3433-3437) of glucose, lactate and glutamine (all 10mM), Zn2+ markedly inhibits glycogen synthesis, with total inhibition at 500 microM, and a half maximal effect in the range from 50 to 100 microM. Dipicolinate (pyridine 2,6-dicarboxylate), a zinc chelator, is about as effective as L-glutamine in activating glycogen synthesis with the substrate combination of dihydroxyacetone, lactate and glucose (all 10mM). This suggests the possible hypothesis that endogenous Zn2+ might control the rate of glycogen synthesis in vivo. However, alternate explanations such as metabolite accumulation are also possible, since dipicolinate causes inhibition of gluconeogenesis from L-lactate.     

Epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate stimulate lactate production and the pentose phosphate pathway in freshly isolated rat hepatocytes

Epidermal growth factor (EGF) and tetradecanoylphorbol acetate (TPA) rapidly stimulated the production of lactate by hepatocytes isolated from fed rats. Our results indicate that enzymes of both glycolysis and the pentose phosphate pathway are involved in these actions. EGF stimulated CO2 release from the 1-position of glucose, and caused a small but significant increase in pyruvate kinase activity. In addition, EGF caused a rise in fructose 1,6-bisphosphate and fructose 2,6-bisphosphate concentrations, indicating activation of phosphofructokinase. TPA did not alter the concentrations of these sugar phosphates, but did cause an increased lactate production and CO2 production from the 1-position of glucose similar to EGF. Furthermore, the EGF stimulation of lactate formation was independent of the presence of medium Ca2+. Phenylephrine stimulation of this process, in parallel incubations, was entirely dependent upon the presence of Ca2+ in the medium. We conclude that EGF stimulates glycolysis and the pentose phosphate pathway in isolated hepatocytes from fed rats. The duplication of these actions by TPA suggests that protein kinase C is a mediator of EGF action in hepatocytes.