Activation of inositol phosphate formation by circulating noradrenaline but not by sympathetic nerve stimulation with a similar increase of glucose release in perfused rat liver

In the isolated rat liver perfused in situ, stimulation of the nerve bundles around the hepatic artery and portal vein caused an increase of glucose and lactate output and a reduction of perfusion flow. These changes could be inhibited completely by alpha-receptor blockers. The possible involvement of inositol phosphates in the intracellular signal transmission was studied. 1. In cell-suspension experiments, which were performed as a positive control, noradrenaline caused an increase in glucose output and, in the presence of 10 mM LiCl, a dose-dependent and time-dependent increase of inositol mono, bis and trisphosphate. 2. In the perfused rat liver 1 microM noradrenaline caused an increase of glucose and lactate output and in the presence of 10 mM LiCl a time-dependent increase of inositol mono, bis and trisphosphate that was comparable to that observed in cell suspensions. 3. In the perfused rat liver stimulation of the nerve bundles around the portal vein and hepatic artery caused a similar increase in glucose and lactate output to that produced by noradrenaline, but in the presence of 10 mM LiCl there was a smaller increase of inositol monophosphate and no increase of inositol bis and trisphosphate. These findings are in line with the proposal that circulating noradrenaline reaches every hepatocyte, causing a clear overall increase of inositol phosphate formation and thus calcium release from the endoplasmic reticulum, while the hepatic nerves reach only a few cells causing there a small local change of inositol phosphate metabolism and thence a propagation of the signal via gap junctions.     

Mechanism of action of sympathetic hepatic nerves on carbohydrate metabolism in perfused rat liver

In the perfused rat liver stimulation of the hepatic nerves around the portal vein and the hepatic artery was previously shown to increase glucose output, to shift lactate uptake to output, to decrease and re-distribute intrahepatic perfusion flow and to cause an overflow of noradrenaline into the hepatic vein. The metabolic effects could be caused directly via nerve hepatocyte contacts or indirectly by the hemodynamic changes and/or by noradrenaline overflow from the afferent vasculature into the sinusoids. Evidence against the indirect modes of nerve action is presented. Reduction of perfusion flow by lowering the perfusion pressure from 2 to 1 ml X min-1 X g-1--as after nerve stimulation--or to 0.35 ml X min-1 X g-1--far beyond the nerve stimulation-dependent effect--did not change glucose output and lowered lactate uptake only slightly. Only re-increase of flow to 2 ml X min-1 X g-1 enhanced glucose and lactate release transiently due to washout of glucose and lactate accumulated in parenchymal areas not perfused during low perfusion flow. In chemically sympathectomized livers nerve stimulation decreased perfusion flow almost normally but without changing the intrahepatic microcirculation; yet it enhanced glucose and lactate output only insignificantly and caused noradrenaline overflow of less than 10% of normal. Conversely, in the presence of nitroprussiate (III) nerve stimulation reduced overall flow only slightly without intrahepatic redistribution but still increased glucose and lactate output strongly and caused normal noradrenaline overflow.(ABSTRACT TRUNCATED AT 250 WORDS)     

Signal propagation via gap junctions, a key step in the regulation of liver metabolism by the sympathetic hepatic nerves.

Cell-to-cell communication via gap junctions has been proposed to be involved in the metabolic actions of sympathetic liver nerves in the rat. The effects of hepatic nerve stimulation and noradrenaline-, PGF2 alpha- and glucagon infusion on glucose metabolism and perfusion flow were studied in perfused rat liver in the absence and presence of the gap junctional inhibitors, heptanol, carbenoxolone and (4 beta)phorbol 12-myristate 13-acetate (4 beta PMA). (i) Stimulation of the hepatic nerve plexus increased glucose output, decreased flow and caused an overflow of noradrenaline into the hepatic vein. (ii) Heptanol completely inhibited not only the nerve stimulation-dependent metabolic and hemodynamic alterations but also the noradrenaline overflow. Thus the heptanol-dependent inhibitions were caused primarily by a strong impairment of transmitter release. (iii) Carbenoxolone inhibited the effects of neurostimulation on glucose metabolism partially by about 50%, whereas it left perfusion flow and noradrenaline overflow essentially unaltered. (iv) 4 beta PMA reduced the nerve stimulation-dependent enhancement of glucose release by about 80% but the noradrenaline-dependent increase in glucose output only by about 30%; the increase in glucose release by PGF2 alpha and by glucagon remained essentially unaltered. 4 beta PMA reduced the nerve stimulation-dependent decrease in portal flow by about 35% but did not affect the noradrenaline-and PGF2 alpha-elicited alterations, nor did it alter noradrenaline overflow. The results allow the conclusion that gap junctional communication plays a major role in the regulation of hepatic carbohydrate metabolism by sympathetic liver nerves, but not by circulating noradrenaline, PGF2 alpha or glucagon.     

Intracellular mechanism of action of sympathetic hepatic nerves on glucose and lactate balance in perfused rat liver

In rat liver perfused in situ stimulation of the nerve plexus around the hepatic artery and the portal vein caused an increase in glucose output and a shift from lactate uptake to output. The effects of nerve stimulation on some key enzymes, metabolites and effectors of carbohydrate metabolism were determined and compared to the actions of glucagon, which led to an increase not only of glucose output but also of lactate uptake. 1. Nerve stimulation caused an enhancement of the activity of glycogen phosphorylase a to 300% and a decrease of the activity of glycogen synthase I to 40%, while it left the activity of pyruvate kinase unaltered. Glucagon, similarly to nerve action, led to a strong increase of glycogen phosphorylase and to a decrease of glycogen synthase; yet in contrast to the nerve effect it lowered pyruvate kinase activity clearly. 2. Nerve stimulation increased the levels of glucose 6-phosphate and of fructose 6-phosphate to 200% and 170%, respectively; glucagon enhanced the levels to about 400% and 230%, respectively. The levels of ATP and ADP were not altered, those of AMP were increased slightly by nerve stimulation. 3. Nerve stimulation enhanced the levels of the effectors fructose 2,6-bisphosphate and cyclic AMP only slightly to 140% and 125%, respectively; glucagon lowered the level of fructose 2,6-bisphosphate to 15% and increased the level of cyclic AMP to 300%. 4. In calcium-free perfusions the metabolic responses to nerve stimulation showed normal kinetics, if calcium was re-added 3 min before, but delayed kinetics, if it was re-added 2 min after the onset of the stimulus. The delay may be due to the time required to refill intracellular calcium stores. The hemodynamic alterations dependent on extracellular calcium were normal in both cases. The activation of glycogen phosphorylase, the inhibition of glycogen synthase and the increase of glucose 6-phosphate can well explain the enhancement of glucose output following nerve stimulation. The unaltered activity of pyruvate kinase and the marginal increase of fructose 2,6-bisphosphate cannot be the cause of the nerve-stimulation-dependent shift from lactate uptake to output. The very slight increase of the level of cyclic AMP after nerve stimulation cannot elicit the observed activation of glycogen phosphorylase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Possible involvement of eicosanoids in the actions of sympathetic hepatic nerves on carbohydrate metabolism and hemodynamics in perfused rat liver

In isolated rat liver perfused at constant pressure with Krebs-Henseleit buffer containing 5 mM glucose, 2 mM lactate, 0.2 mM pyruvate and 0.1% bovine serum albumin, perivascular nerve stimulation (20 V, 20 Hz, 2 ms) and infusion of ATP (100 microM), noradrenaline (1 microM) or arachidonic acid (100 microM) caused an increase in glucose and lactate output and a reduction of perfusion flow. The metabolic effects of nerve stimulation but not those of ATP and noradrenaline were inhibited strongly by the phospholipase A2 inhibitor bromophenacyl bromide (BPB, 20 microM) and the cyclooxygenase inhibitor indomethacin (Indo, 20 microM) and only slightly by the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 20 microM). In contrast, the hemodynamic effects not only of nerve stimulation but also of ATP and noradrenaline were inhibited strongly by BPB and Indo and slightly by NDGA. The metabolic and hemodynamic actions of arachidonate were inhibited specifically by Indo. These results suggest that the effects of nerve stimulation were at least partially mediated or modulated by eicosanoids, especially by prostanoids.     

Activity of glucose-6-phosphatase during liver regeneration]

During hepatic regeneration a drop in the liver glycogen content along with a lower blood glucose level have been observed. These data are difficult to correlate with the rise of blood glucagon and the drop of insulin shown at the same times after partial hepatectomy. Therefore, liver glucose-6-phosphatase activity has been studied at 1.5, 4, 15 and 24 h, since that enzyme is involved in the release of glucose from the cell. 4 and 15 h after partial hepatectomy a remarkable decrease in glucose-6-phosphatase activity is observed. These results are discussed in view of the higher metabolic demand of regenerating liver.     

Intercellular communication in normal and regenerating rat liver: a quantitative analysis

We have compared intercellular communication in the regenerating and normal livers of weanling rats. The electrophysiological studies were conducted at the edge of the liver, and we have found that here as elsewhere in the liver there is a dramatic decrease in the number and size of gap junctions during regeneration. The area of hepatocyte membrane occupied by gap junctions is reduced 100-fold 29-35 h after hepatectomy. By combining observations made with the scanning electron microscope with our freeze fracture data we have estimated the number of "communicating interfaces" (areas of contact between hepatocytes that include at least one gap junction) formed by hepatocytes in normal and regenerating liver. In normal liver a hepatocyte forms gap junctions with every hepatocyte it contacts (approximately 6). In regenerating liver a hepatocyte forms detectable gap junctions with, on average, only one other hepatocyte. Intercellular spread of fluorescent dye and electric current is reduced in regenerating as compared with normal liver. The incidence of electric coupling is reduced from 100% of hepatocyte pairs tested in control liver to 92% in regenerating liver. Analysis of the spatial dependence of electronic potentials indicates a substantial increase in intercellular resistance in regenerating liver. A quantitative comparison of our morphological and physiological data is complicated by tortuous pattern of current flow and by inhomogeneities in the liver during regeneration. Nevertheless we believe that our results are consistent with the hypothesis that gap junctions are aggregates of channels between cell interiors.     

Activation of glycogenolysis by stimulation of the hepatic nerves in perfused livers of guinea pig and tree shrew as compared to rat: differences in the mode of action

A study on the metabolic and hemodynamic actions of hepatic nerve stimulation in the perfused liver of guinea pig and tree shrew as compared to rat was performed, since the density of liver innervation was reported to be different. 1) Nerve stimulation resulted in an increase in glucose release and decrease in lactate uptake or in a shift to output as well as a decrease in portal flow in all three species. The change in glucose output was very similar, that in lactate balance and flow was smaller in tree shrew than in guinea pig and rat. Apparently, the metabolic and hemodynamic changes did not reflect the different densities of liver innervation. 2) The overflow of the neurotransmitter noradrenaline into the hepatic vein differed very clearly in the three animals. In the guinea pig and tree shrew the maximal increase in noradrenaline concentration measured in the effluent was about 6-7-fold higher than in the rat. 3) The content of noradrenaline in the liver in vivo was about five-fold higher in the guinea pig and again another four-fold higher in the tree shrew than in the rat. The contents of adrenaline and dopamine were very low in comparison to those of noradrenaline. The different hepatic noradrenaline contents of the three species investigated are in line with the anatomical findings on the different innervation density. 4) Inhibitors of eicosanoid synthesis reduced the nerve stimulation-dependent metabolic and hemodynamic alterations in guinea pig liver as in rat liver indicating a similar mechanism in these species. Apparently, prostaglandins might be involved as mediators or modulators of nerve actions also in the more densely innervated guinea pig liver and not only in the less densely innervated rat liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acute effects of interleukin 1 alpha and 6 on intermediary metabolism in freshly isolated rat hepatocytes

Using hepatocytes in suspension, freshly isolated from adult male fed rats, we studied the acute influence of recombinant human interleukins 1 alpha, 2 and 6 on glycogen and fatty acid metabolism. By far the largest effects were observed with interleukin-1 alpha: short incubations (up to 60 min) sufficed to depress glycogen synthesis in a dose-dependent manner, while the rates of glycogenolysis and glycolysis were increased as indicated by the release of glucose and lactate. Interleukin-6 acted similarly, though being much less effective on a molar basis, whereas interleukin-2 only caused a small increase in lactate production. In hepatocytes from 24h-starved rats interleukin-1 alpha caused a minor stimulation of gluconeogenesis. Although neither fatty acid synthesis nor oxidation of fatty acids in quiescent hepatocytes from fed rats was significantly affected by interleukins, interleukin-1 alpha was able to cause appreciable inhibition of fatty acid synthesis in hepatocytes from regenerating liver (isolated 22h after partial hepatectomy). It is concluded (i) that interleukins, in particular interleukin-1 alpha, acutely promote hepatic glucose release, and (ii) that transition of adult hepatocytes from a quiescent into a proliferatory state allows the occurrence of rapid effects of interleukin-1 alpha on fatty acid metabolism.     

Direct control of glycogen metabolism in the perfused rat liver by the sympathetic innervation

The mode of action of hepatic nerves on the metabolism of carbohydrates was studied in the rat liver perfused in situ. 1. Electrical stimulation of the nerve bundles around the hepatic artery and the portal vein resulted in an increase of glucose and lactate output, an enhancement of phosphorylase a activity and a decrease of portal flow. 2. Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation without affecting the metabolic alterations. 3. Phentolamine or an extracellular calcium level below 300 mumol x 1(-1) abolished both hemodynamic and metabolic changes after nerve stimulation, while propranolol or atropine were without effect. 4. Norepinephrine infusion mimicked nerve stimulation only at the highly unphysiological concentration of 0.1 microM; it was not effective at a concentration of 0.01 microM, which might be reached in the sinusoidal blood due to an overflow from intrahepatic synapses. The present results suggest that, in rat liver, glycogen breakdown is regulated by alpha-sympathetic nerves directly rather than indirectly via hemodynamic changes or via norepinephrine overflow.     

Role of extracellular calcium in the metabolic and hemodynamic actions of sympathetic nerve stimulation, noradrenaline and prostaglandin F2 alpha in perfused rat liver. Differential inhibition by nifedipine and verapamil

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) caused an increase in glucose and lactate output, a decrease in flow and an overflow of noradrenaline into the hepatic vein. Noradrenaline (1 microM) (NA) and prostaglandin F2 alpha (5 microM) (PGF2 alpha), which are implicated as mediators of nerve action, elicited similar effects. 1) All actions of nerve stimulation and the hemodynamic but not the metabolic effects of noradrenaline and PGF2 alpha were largely dependent on extracellular calcium. 2) The dihydropyridine type calcium antagonist nifedipine (5 microM) inhibited the hemodynamic but not the metabolic actions of nerve stimulation, NA and PGF2 alpha, while the phenylalkylamine type calcium antagonist verapamil (5 microM) had no effect. These findings allow the following conclusions: Calcium influx into I nerve endings, necessary for the release of neurotransmitter, II parenchymal cells, for the display of metabolic effects induced by nerve stimulation, and III the actions of NA and PGF2 alpha, do not appear to be mediated by the normal affinity nifedipine- or the verapamil-sensitive channels. Calcium influx into vascular smooth muscle and/or endothelial cells for the display of hemodynamic action induced by nerve stimulation and the NA and PGF2 alpha effects, appear to occur through nifedipine-sensitive but verapamil-insensitive channels.     

Increase in glucose and lactate output and perfusion resistance by stimulation of hepatic nerves in isolated perfused rat liver: role of alpha 1-, alpha 2-, beta 1- and beta 2-receptors.

Rat liver was perfused in situ via the portal vein without recirculation: 1) Electrical stimulation of the nerve bundles around hepatic artery and portal vein increased glucose and lactate output, reduced flow and caused an overflow of noradrenaline into the hepatic vein. The alpha-agonist phenylephrine also augmented glucose and lactate output and lowered flow with an ED50 of about 1 microM, while the beta-agonist isoproterenol increased glucose output but reduced lactate output with an ED50 of about 0.2 microM and left flow unaltered. 2) The alpha 1-receptor antagonist prazosin (KI at alpha 1-sites approximately 1 nM, at alpha 2-sites approximately 100 nM) inhibited the nerve stimulation-dependent increase in glucose and lactate output and reduction of flow with an ID50 of about 1 nM, while the alpha 2-receptor antagonist yohimbine (KI at alpha 2-sites approximately 10 nM, at alpha 1-sites approximately 1500 nM) was inhibitory only with an ID50 of about 400 nM. 10 nM prazosin clearly reduced the nerve actions, completely blocked the effects of 1 microM phenylephrine and left the effects of 0.2 microM isoproterenol unaltered. 10 nM yohimbine did not affect the nerve actions nor the effects of phenylephrine or isoproterenol. 3) The beta 1-receptor antagonist metoprolol (KI at beta 1-sites approximately 100 nM, at beta 2-sites approximately 1.2 microM) at 10 microM concentrations did not interfere with the nerve stimulation-dependent increase in glucose and lactate output or the decrease in flow. It did not have any specific alpha-antagonistic influence either on the changes brought about by 1 microM phenylephrine; however, it blocked the beta 2-mediated increase in glucose output by isoproterenol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unspecific inhibition by the calmodulin antagonist calmidazolium and the intracellular calcium antagonist TMB-8 of the actions of sympathetic hepatic nerves and noradrenaline on glucose balance and flow in perfused rat liver

In perfused rat liver hepatic nerve stimulation (10 Hz, 2 ms) (NS) increased glucose and lactate output, decreased flow and was accompanied by an overflow of noradrenaline into the hepatic vein. These effects were dependent on extracellular and partly on intracellular calcium. Infusion of noradrenaline (1 microM) (NA) elicited similar effects. 1) Calmidazolium at 1, 2 and 5 microM caused an increase in basal glucose output and a decrease and intrahepatic redistribution of flow after a lag of 30, 20 and 5 min, respectively. 2) After 5 min of 1 microM calmidazolium, i.e. before it altered basal metabolism and flow, the actions of NS and NA remained unaltered. 3) After 40 min of 1 microM calmidazolium, i.e. after it had just begun to alter basal metabolism and flow, NS caused a decrease in glucose and lactate output rather than an increase and the metabolic effects of NA were strongly reduced whereas the hemodynamic changes of both stimuli were not altered. 4) TMB-8 at 25, 50 and 100 microM caused a transient increase in lactate output and a decrease and intrahepatic redistribution of flow after a lag of 5 min only at 100 microM concentrations. 5) The effects of NS were inhibited already by 25 microM TMB-8 which reduced NA release whereas the effects of NA were not influenced. Thus, calmidazolium and TMB-8 did not act as a calmodulin and intracellular calcium antagonist, respectively, but had unspecific "side effects" in the complex system of the perfused liver. The antagonists cannot be used to study the role of intracellular calcium in intact organs.     

Studies on gluconeogenesis and ketogenesis in isolated hepatocytes from alloxan diabetic rats.

Gluconeogenesis and ketogenesis were studied in isolated hepatocytes obtained from normal and alloxan diabetic rats. Insulin treatment maintained near-normal blood glucose levels and caused an increase in glycogen deposition. The third day after insulin withdrawal the rats displayed a diabetic syndrome marked by progressive hyperglycemia and glycogen depletion. Net glucose production in liver cells isolated from alloxan diabetic rats progressively increased with time up to 72 hr after the last in vivo insulin injection. Maximal glucose production was observed at 72 hr with 10 mM alanine, lactate, pyruvate, or fructose. Glucose production decreased at 96 hr. The same pattern was observed with the incorporation of labeled bicarbonate into glucose. Ketogenesis in liver cells and hepatic lipid content also peaked at 72 hr.     

Control of ketogenesis in the perfused rat liver by the sympathetic innervation

The regulation of ketogenesis by the hepatic nerves was investigated in the rat liver perfused in situ. Electrical stimulation of the hepatic nerves around the portal vein and the hepatic artery caused a reduction of basal ketogenesis owing to a decrease in acetoacetate release to 30% with essentially no change in 3-hydroxybutyrate release. At the same time, as observed before [Hartmann et al. (1982) Eur. J. Biochem. 123, 521-526], nerve stimulation increased glucose output, shifted lactate uptake to output and decreased perfusion flow. Ketogenesis from oleate, which enters the mitochondria via the carnitine system, was also lowered after nerve stimulation owing to a decrease of acetoacetate release to 30% with no alteration in 3-hydroxybutyrate release. Ketogenesis from octanoate, which enters the mitochondria independently of the carnitine system, was decreased after nerve stimulation as a result of a drastic decrease of acetoacetate output to 15% and a less pronounced decrease of 3-hydroxybutyrate release to 65%. Noradrenaline mimicked the metabolic nerve effects on ketogenesis only at the highly unphysiological concentration of 0.1 microM under basal conditions and in the presence of oleate as well as partly in the presence of octanoate. It was essentially not effective at a concentration of 0.01 microM, which might be reached in the sinusoids owing to overflow from the hepatic vasculature. Sodium nitroprusside prevented the hemodynamic changes after nerve stimulation; it did not affect the nerve-dependent reduction of ketogenesis under basal conditions and in the presence of oleate, yet it diminished the nerve effect on octanoate-dependent ketogenesis. Phentolamine clearly reduced the metabolic and hemodynamic nerve effects, while propranolol was without effect. The present data suggest that hepatic ketogenesis was inhibited by stimulation of alpha-sympathetic liver nerves directly rather than indirectly via hemodynamic changes or noradrenaline overflow from the vessels and that the site of regulation should be mainly intramitochondrial.     

Mechanism of action of cysteinyl leukotrienes on glucose and lactate balance and on flow in perfused rat liver. Comparison with the effects of sympathetic nerve stimulation and noradrenaline

Rat livers were perfused at constant pressure via the portal vein with media containing 5 mM glucose, 2 mM lactate and 0.2 mM pyruvate. 1. Leukotrienes C4 and D4 enhanced glucose and lactate output and reduced perfusion flow to the same extent and with essentially identical kinetics. They both caused half-maximal alterations (area under the curve) of carbohydrate metabolism at a concentration of about 1 nM and of flow at about 5 nM. The leukotriene-C4/D4 antagonist CGP 35949 B inhibited the metabolic and hemodynamic effects of 5 nM leukotrienes C4 and D4 with the same efficiency, causing 50% inhibition at about 0.1 microM. 2. Leukotriene C4 elicited the same metabolic and hemodynamic alterations with the same kinetics as leukotriene D4 in livers from rats pretreated with the gamma-glutamyltransferase inhibitor, acivicin. 3. The calcium antagonist, nifedipine, at a concentration of 50 microM did not affect the metabolic and hemodynamic changes caused by 5 nM leukotriene D4. The smooth-muscle relaxant, nitroprussiate, at a concentration of 10 microM reduced flow changes, without significantly affecting the metabolic alterations. 4. Leukotriene D4 not only reduced flow; it also caused an intrahepatic redistribution of flow, restricting some areas from perfusion. Thus, leukotrienes increased glucose and lactate output directly in the accessible parenchyma and, in addition, indirectly by washout from restricted areas during their reopening upon termination of application. 5. The phospholipase A2 inhibitor, bromophenacyl bromide, but not the cyclooxygenase inhibitor, indomethacin, at a concentration of 20 microM reduced the metabolic and hemodynamic effects of 5 mM leukotriene D4. 6. Stimulation of the sympathetic hepatic nerves with 2-ms rectangular pulses at 20 Hz and infusion of 1 microM noradrenaline increased glucose and lactate output and decreased flow, similar to 10 nM leukotrienes C4 and D4. The kinetics of the metabolic and hemodynamic changes caused by the leukotrienes differed, however, from those due to nerve stimulation and noradrenaline. 7. The leukotriene-C4/D4 antagonist, CGP 35949 B, even at very high concentrations (20 microM) inhibited the metabolic and hemodynamic alterations caused by nerve stimulation or noradrenaline infusion only slightly and unspecifically.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormone and substrate regulation of glycogen accumulation in primary cultures of rat hepatocytes.

Hormonal and substrate regulation of hepatic glycogen accumulation was evaluated in primary cultures of hepatocytes prepared from 1-day-fasted rats. Hepatocytes were cultured in media containing 5 mM-glucose and 10 mM-lactate and then exposed to 100 nM-dexamethasone for 4 h before an increase in glucose concentration and the addition of insulin. When this protocol was used to mimic the post-prandial state in vivo, net glycogen accumulation (over 2 h) and insulin (10 nM) effects were linear at physiological (5-10 mM) and supraphysiological (20-30 mM) glucose concentrations. To define the role of substrates in glycogen accumulation, hepatocytes were incubated in a buffered salt solution containing 10 mM-glucose and either 10 mM-lactate or 5 mM-glutamine, or both. In the absence of hormones, net glycogen accumulation was increased by 59%, 83%, and 127% by the addition of lactate, glutamine, and lactate plus glutamine respectively, compared with incubations with glucose alone, and 6-fold in the presence of substrates, insulin and dexamethasone. Labelling with [3-3H]glucose and [U-14C]glucose showed that in the absence of hormones approx. 50% of glycogen formation came from glucose via the direct pathway and the remainder from glucose via the indirect pathway or from non-glucose precursors, or both. Insulin-dependent enhancement of glycogen formation is through stimulation of both the direct and indirect pathways, and dexamethasone-dependent stimulation occurs through stimulation of both these pathways of glycogen formation from glucose as well as from non-glucose precursors. Lactate serves as a gluconeogenic C3 precursor for the observed enhanced glycogen formation, whereas glutamine-dependent enhancement of glycogen accumulation occurs primarily through a stimulation of the direct and indirect pathways of glycogen formation from glucose.     

Control of oxygen uptake, microcirculation and glucose release by circulating noradrenaline in perfused rat liver

The effect of noradrenaline on oxygen uptake, on periportal and perivenous oxygen tension at surface acini, on microcirculation and on glucose output were studied in isolated rat livers perfused at constant flow with Krebs-Henseleit-hydrogen carbonate buffer containing 5mM glucose and 2mM lactate. Noradrenaline at 1 microM concentration caused a decrease in oxygen uptake, while at 0.1 microM it led to an increase. Both high and low doses of noradrenaline decreased the tissue surface oxygen tension in periportal and - after a transient rise - in perivenous areas. Noradrenaline at an overall constant flow caused an increase of portal pressure and an alteration of the intrahepatic distribution of the perfusate: at the surface of the liver and in cross sections infused trypan blue led to only a slightly heterogeneous staining after a low dose of noradrenaline but to a clearly heterogeneous staining after a high dose. Both high and low doses of noradrenaline stimulated glucose release. All effects could be inhibited by the alpha-blocking agent phentolamine. In conclusion, control of hepatic oxygen consumption by circulating noradrenaline is a complex result of opposing hemodynamic and metabolic components: the microcirculatory changes inhibit oxygen uptake; they dominate after high catecholamine doses. The metabolic effects include a stimulation of oxygen utilization; they prevail at low catecholamine levels. The noradrenergic control of glucose release is also very complex, involving direct, metabolic and indirect, hemodynamic components.     

Hepatic lactate uptake versus leg lactate output during exercise in humans.

The exponential rise in blood lactate with exercise intensity may be influenced by hepatic lactate uptake. We compared muscle-derived lactate to the hepatic elimination during 2 h prolonged cycling (62 +/- 4% of maximal O(2) uptake, (.)Vo(2max)) followed by incremental exercise in seven healthy men. Hepatic blood flow was assessed by indocyanine green dye elimination and leg blood flow by thermodilution. During prolonged exercise, the hepatic glucose output was lower than the leg glucose uptake (3.8 +/- 0.5 vs. 6.5 +/- 0.6 mmol/min; mean +/- SE) and at an arterial lactate of 2.0 +/- 0.2 mM, the leg lactate output of 3.0 +/- 1.8 mmol/min was about fourfold higher than the hepatic lactate uptake (0.7 +/- 0.3 mmol/min). During incremental exercise, the hepatic glucose output was about one-third of the leg glucose uptake (2.0 +/- 0.4 vs. 6.2 +/- 1.3 mmol/min) and the arterial lactate reached 6.0 +/- 1.1 mM because the leg lactate output of 8.9 +/- 2.7 mmol/min was markedly higher than the lactate taken up by the liver (1.1 +/- 0.6 mmol/min). Compared with prolonged exercise, the hepatic lactate uptake increased during incremental exercise, but the relative hepatic lactate uptake decreased to about one-tenth of the lactate released by the legs. This drop in relative hepatic lactate extraction may contribute to the increase in arterial lactate during intense exercise.     

Concurrent changes in sinusoidal expression of laminin and affinity of hepatocytes to laminin during rat liver regeneration.

Distribution of fibronectin, laminin, and collagens type I, III, IV, and V in the lobular regions of regenerating rat liver was studied by indirect immunofluorescence. Little or no laminin was detected in sham-operated controls throughout the experimental period, while it was detected in sinusoids of regenerating liver as early as 6 h after partial hepatectomy (PH). After reaching a maximum at 24 h, it decreased and was barely detectable 6 days after PH. Changes in the other extracellular matrix (ECM) proteins were evident 3 days after PH, but not earlier than 24 h. Hepatocytes isolated from regenerating rat livers were tested in a short term assay for attachment to the substrates coated with the ECM proteins. The attachment of hepatocytes to laminin substrates increased 12 h after PH, reached a maximum at 24 h, and decreased to the control level 6 days after PH, while that of the control remained constant. The attachment to fibronectin substrates was not different between regenerating livers and controls at any time point. The attachment to collagen did not change earlier than 24 h after PH, but increased slightly 3 days after PH. Primary rat hepatocytes cultured on the substrates coated with the ECM proteins were determined for replicative DNA synthesis in response to epidermal growth factor. Both in normal liver and in regenerating liver 24 h after PH, laminin was one of the most effective substrates in supporting the responsiveness of hepatocytes to the growth stimulus. Taken together, these results suggest the importance of hepatocyte-laminin interaction during the early stage of liver regeneration possibly in growth stimulation of hepatocytes and/or maintenance of hepatocyte-specific functions.