Hormonal effects on glycogen metabolism in isolated hepatocytes of a freeze-tolerant frog.

To determine whether specific hormonal responses were involved in the production of cryoprotectant (glucose) by liver of the freeze tolerant wood frog, Rana sylvatica, metabolically active hepatocytes were isolated in reasonable yields (mean 20.1 +/- 1.30% SEM, n = 29) by in situ liver perfusion with collagenase. Freshly isolated cells from autumn-collected frogs contained large amounts of glycogen (650 mumol glucosyl units/g packed cells) and produced glucose from this endogenous reserve at a rate of 10 mumol g-1 hr-1 at 0 degrees. Glucose output from cells was highly responsive to the addition of hormones; rates of glucose release increased 2.1-, 1.7-, and 1.7-fold with the addition of 10(-7) M bovine glucagon, 10(-7) M epinephrine, and 5 x 10(-6) M dibutyryl-cyclic AMP, respectively. Norepinephrine, 5-hydroxytryptamine, and bovine insulin were without effect at 0.1 microM/l. Hormone stimulation of glucose release was correlated with an increase in both the total activity and the percentage a of glycogen phosphorylase in hepatocytes. However, none of the hormones tested affected the kinetic properties of hepatocyte pyruvate kinase, suggesting the absence of covalent modification control of the enzyme. The data indicate that the freezing-stimulated production of large quantities of glucose as a cryoprotectant by R. sylvatica liver does not involve qualitative differences in the hormonal control of liver glycogenolysis, compared with other lower vertebrates. However, quantitative differences were seen, such as the much greater phosphorylase activity, 4.38 +/- 0.33 mumol min-1 g-1 packed cells, in freshly isolated R. sylvatica hepatocytes compared with 0.36 +/- 0.06 mumol min-1 g-1 in Rana pipiens hepatocytes.     

alpha-adrenergic stimulation mediates glucose uptake through phosphatidylinositol 3-kinase in rat heart

We examined whether insulin and catecholamines share common pathways for their stimulating effects on glucose uptake. We perfused isolated working rat hearts with Krebs-Henseleit buffer containing [2-3H]glucose (5 mmol/L, 0.05 microCi/mL) and sodium oleate (0.4 mmol/L). In the absence or presence of the phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin (3 micromol/L), we added insulin (1 mU/mL), epinephrine (1 micromol/L), phenylephrine (100 micromol/L) plus propranolol (10 micromol/L, selective alpha-adrenergic stimulation), or isoproterenol (1 micromol/L) plus phentolamine (10 micromol/L, selective beta-adrenergic stimulation) to the perfusate. Cardiac power was found to be stable in all groups (between 8.07+/-0.68 and 10.7+/-0. 88 mW) and increased (25% to 47%) with addition of epinephrine, but not with selective alpha- and beta-adrenergic stimulation. Insulin and epinephrine, as well as selective alpha- and beta-receptor stimulation, increased glucose uptake (the following values are in micromol/[min. g dry weight]: basal, 1.19+/-0.13; insulin, 3.89+/-0.36; epinephrine, 3.46+/-0.27; alpha-stimulation, 4.08+/-0.40; and beta-stimulation, 3.72+/-0.34). Wortmannin completely inhibited insulin-stimulated and selective alpha-stimulated glucose uptake, but it did not affect the epinephrine-stimulated or selective beta-stimulated glucose uptake. Sequential addition of insulin and epinephrine or insulin and alpha-selective stimulation showed additive effects on glucose uptake in both cases. Wortmannin further blocked the effects of insulin on glycogen synthesis. We conclude that alpha-adrenergic stimulation mediates glucose uptake in rat heart through a PI3-K-dependent pathway. However, the additive effects of alpha-adrenergic stimulation and insulin suggest 2 different isoforms of PI3-K, compartmentation of PI3-K, potentiation, or inhibition by wortmannin of another intermediate of the alpha-adrenergic signaling cascade. The stimulating effects of both the alpha- and the beta-adrenergic pathways on glucose uptake are independent of changes in cardiac performance.     

Stimulatory effect of vasoactive intestinal peptide on glycogenolysis and gluconeogenesis in isolated rat hepatocytes: antagonism by insulin

We have studied the effect of Vasoactive Intestinal Peptide (VIP) on glycogenolysis and gluconeogenesis (as measured by the conversion of [U-14C]pyruvate into glucose) in hepatocytes isolated from fed rats. The influence of VIP on glycogen phosphorylase alpha and pyruvate kinase activities, as well as on cAMP levels, was also evaluated. In addition, the possible antagonism of insulin on these VIP-mediated effects was investigated. VIP enhanced both glycogenolysis and gluconeogenesis in a dose-dependent manner. At 10(-6) M VIP, both processes were increased 2-fold as compared to the basal values; the calculated half-maximal stimulatory concentrations were 2.5 x 10(-8) M and 4 x 10(-8) M, respectively. VIP also caused a dose-dependent activation of glycogen phosphorylase and inactivation of pyruvate kinase. At 10(-6) M VIP, glycogen phosphorylase a was increased 3-fold and pyruvate kinase activity was reduced by 46%. The addition of 10(-7) M VIP to the incubation medium caused a 2-fold increase of basal cAMP levels. All these VIP-mediated effects were markedly blocked by the presence of 10(-8) M insulin. As compared to glucagon (10(-7) M) the potency of an equimolar concentration of VIP, in terms of stimulation of gluconeogenesis, inactivation of pyruvate kinase, and activation of glycogen phosphorylase ranged from 35-45%. Our results indicate that VIP increases hepatic glucose output through the stimulation of both glycogenolysis and gluconeogenesis. These effects seem to be mediated by a cAMP-dependent mechanism.     

Mechanism of adrenergic stimulation of hepatic ketogenesis

The effects of alpha- and beta-adrenergic stimulation on ketogenesis were examined in freshly isolated rat hepatocytes in order to determine which alpha- or beta-adrenergic stimulation is involved in the enhancement of ketogenesis. In the presence of 0.3 mmol/L (U-14C)-palmitate, epinephrine, norepinephrine, and phenylephrine at 500 ng/mL increased ketogenesis by 25% (16.0 +/- 0.17 v 12.8 +/- 0.13 nmol/mg protein per hour), 20% (15.3 +/- 0.28) and 20% (15.4 +/- 0.36), respectively. However, isoproterenol even at 1 microgram/mL did not stimulate ketogenesis. Phentolamine (5 micrograms/mL) almost completely abolished the effect of epinephrine on ketogenesis (13.7 +/- 0.30 v 16.0 +/- 0.17) but propranolol did not inhibit the stimulation by epinephrine (15.6 +/- 0.38 v 16.0 +/- 0.17). Trifluoperazine (10 mumol/L), presumably an inhibitor of calcium-dependent protein kinase, abolished the effect of epinephrine (13.6 +/- 0.22 v 16.0 +/- 0.17). These results indicate that catecholamines increase ketogenesis predominantly through the alpha-adrenergic system independent of cyclic AMP, and calcium-dependent protein kinase is thought to be involved in the activation of ketogenesis. On the other hand, glucagon stimulated ketogenesis with an increase of cyclic AMP, which was not inhibited by alpha- and beta-adrenergic antagonists. Alpha-adrenergic stimulation increased hepatic glycogenolysis much more at much lower concentrations when compared with ketogenesis. Stimulation of ketogenesis by catecholamines seemed to be less sensitive and responsive compared with hepatic glycogenolysis.     

Interaction of salmon glucagon, glucagon-like peptide, and epinephrine in the stimulation of phosphorylase a activity in fish isolated hepatocytes

The simultaneous addition of epinephrine and salmon glucagon to catfish (Ictalurus melas) and trout (Salmo gairdneri) hepatocytes did not induce greater increases in glycogen phosphorylase a activity and in glucose release than those caused by epinephrine alone. The effects of epinephrine are greater than those of glucagon. Propranolol added to the hormonal pool blocked the epinephrine effects. In trout cells, epinephrine and glucagon-like peptide (GLP) had similar effects and when they were added simultaneously the stimulation of metabolic indices was higher compared to that obtained with either epinephrine or GLP. However, the effects were not additive. In the presence of epinephrine plus GLP the inhibitory effect of propranolol was not evident, due to the effect induced by GLP, on which propranolol was not effective. This may indicate that epinephrine masks the GLP effect. Results could mean that epinephrine and glucagon-family peptides act in catfish and trout hepatocytes through different receptors on the same pathway leading to glycogen phosphorylase a activation.     

The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes

Aims/hypothesis An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. Materials and methods We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. Results Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. Conclusions/interpretation In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.     

THE EFFECT OF VASOPRESSIN INFUSION ON GLUCOSE METABOLISM IN MAN

Studies on intact animals and isolated rat hepatocytes have shown that arginine vasopression (AVP) stimulates glycogen phosphorylase to break down glycogen and raise plasma glucose concentrations. Since no similar work has been performed on healthy human adults, the effect of moderate (25 pmol/min) and high (75 pmol/min) dose AVP infusion on plasma glucose, intermediary metabolites, glucose kinetics, and circulating glucagon and insulin concentrations was investigated. After AVP infusion, plasma glucose rose from 4.9 +/- 0.1 to a peak of 5.7 +/- 0.2 mmol/l (P less than 0.001), but no changes in blood lactate, pyruvate, alanine, glycerol or 3-hydroxybutyrate concentrations were observed. The glucose rise was accounted for entirely by an increase in the rate of appearance of glucose from 11.19 +/- 0.43 to 13.38 +/- 0.63 mu mol/kg/min (P less than 0.001). Infusion of AVP also increased plasma glucagon concentrations from 38 +/- 8 to 79 +/- 20 pg/l (P less than 0.01). The hyperglycaemic effect of AVP may be mediated solely by stimulation of glucagon release, but we cannot exclude direct stimulation of glycogen phosphorylase activity.     

Lack of effect of islet amyloid polypeptide on hepatic glucose output in the in situ-perfused rat liver.

Islet amyloid polypeptide (IAPP), a novel peptide isolated from islet amyloid deposits in patients with insulinoma and non-insulin-dependent diabetes mellitus (NIDDM), has been reported to be cosecreted with insulin from pancreatic beta cells and to inhibit glucose uptake and glycogen synthesis in muscle tissue in vitro. We investigated the effects of the synthesized, rat-amidated form of IAPP on hepatic glucose output, and IAPP extraction, using an in situ flow-through perfusion system in rats to elucidate the actions of IAPP on the liver. The IAPP (10(-8) mol/L) alone had no effects on the hepatic glucose release. Infusion of 6 x 10(-11) mol/L glucagon alone resulted in an expected elevation in glucose production (30.0 +/- 1.7 mumol/35 min/g liver). Insulin (3 x 10(-10) mol/L) submaximally decreased the glucagon-stimulated glucose production to 73% (from 30.0 +/- 1.7 to 22.0 +/- 1.4 mumol/35 min/g liver; n = 7, P less than .01). A simultaneous infusion of 10(-8) mol/L IAPP did not influence the glucagon-stimulated glucose production (27.6 +/- 1.2 mumol/35 min/g liver) or the insulin-dependent inhibition of glucagon-stimulated glucose production (22.6 +/- 1.3 mumol/35 min/g liver). IAPP extraction by the liver in a single passage was minimal, in contrast to approximately 50% hepatic insulin extraction. These results indicate that IAPP does not play any important role in modulating glycogen metabolism in the liver.     

肾上腺素和葡萄糖对3T3-L1脂肪细胞水孔蛋白mRNA表达的影响

目的：通过细胞培养研究肾上腺素、葡萄糖对成熟脂肪细胞水孔蛋白（aquaporin adipose,AQPap）基因表达的影响。了解AQPap基因表达的影响因素，探讨其在肥胖及糖尿病发病中所起作用。方法：用不同浓度（10^-6mol/L、10^－7mol/L、10^-8mol/L、10^-9mol/L）的肾上腺素刺激诱导分化第9天的3T3－L1细胞6h，另以不同浓度的葡萄糖（5．6mmol/L、11．2mmol/L、16．8mmol/L、33．6mmol/L）刺激细胞48h。提取细胞RNA。运用半定量RT－PCR技术检测AQPap mRNA表达量的变化。结果：与对照组相比，给予不同浓度的肾上腺素刺激分化成熟的3T3－L1细胞，其AQPap mRNA的表达量变化，差异无显著性意义（P＞0．05）。 葡萄糖浓度的升高使培养细胞AQPap mRNA的表达量显著增强（P＜0．05，16．8mmol/L葡萄糖刺激培养细胞48h,AQPap mRNA表达量约是对照组（葡萄糖浓度为5．6mmol/L）的3倍。结论：肾上腺素是一种脂解激素，它对脂肪细胞AQPap mRNA的表达无影响；高糖状态下AQPap mRNA表达明显增强。     

Lack of effect of beta-endorphin on basal or glucagon-stimulated hepatic glucose production in vitro

beta-Endorphin appears to have effects on hepatic glucose production in vivo. In order to determine whether beta-endorphin modulates glucose production directly, the effects of beta-endorphin on isolated rat hepatocytes were investigated. This permitted isolation of the effects of beta-endorphin from hormonal and/or neuronal influences. A significant dose-related stimulatory effect of glucagon (10(-10) to 10(-6) mol/L) on both hepatic glucose production and glycogen phosphorylase a activity was demonstrated. No effect of either physiologic (10(-11) to 10(-9) mol/L) or supraphysiologic (10(-6) mol/L) concentrations of beta-endorphin on these parameters, under basal or glucagon-stimulated conditions, could be detected. These results suggest that reported in vivo effects of beta-endorphin to inhibit hepatic glucose production were either indirect or centrally mediated.     

Alpha-receptor restriction of coronary blood flow during atrial fibrillation

The effects of atrial fibrillation (AF) on coronary circulation before and after alpha-receptor blockade were studied in 14 anesthetized, open-chest dogs. AF was induced by electrical stimulation of the left atrial appendage; identical rhythmic heart rates were adjusted by left atrial pacing. During atrial pacing, coronary vascular resistance (CVR) was 0.97 +/- 0.10 mm Hg X min X 100 g/ml (resistance units [RU]), coronary blood flow (CBF) 125 +/- 14 ml/min X 100 g, and oxygen saturation 30 +/- 2%; plasma epinephrine was 193 +/- 42 pg/ml and norepinephrine 584 +/- 111 pg/ml. During AF, CVR was higher (1.16 +/- 0.11 RU, p less than 0.0005), whereas CBF (92 +/- 9 ml/min X 100 g, p less than 0.001) and coronary sinus oxygen saturation (24 +/- 2%, p less than 0.0025) were lower than during atrial pacing. When AF was induced, epinephrine increased to 333 +/- 98 pg/ml (p less than 0.05) and norepinephrine to 1,005 +/- 214 pg/ml (p less than 0.005). The large increase in plasma catecholamines suggested an activation of the sympathoadrenal system during AF. In addition, the alpha-receptor blocker phenoxybenzamine (10 mg/kg, intravenously) abolished the differences in CVR, CBF and oxygen saturation between AF and atrial pacing. The data suggest that the decrease in CBF and increase in CVR during experimentally induced AF are caused by coronary vasoconstriction, mediated by sympathetic activation of alpha receptors in the coronary vascular bed.     

Inhibition of hormonal activation of hepatic phosphorylase by chlorpropamide: evidence for an intracellular site of drug action

Phosphorylase a activity was measured in hepatocytes from fed rats, some of which received ip chlorpropamide injections for 5 days preceding death (20 mg/100 g BW X day for 5 days). Chlorpropamide treatment significantly depressed basal phosphorylase a activity and lessened the increments in the activity of this enzyme induced by 10(-10) -10(-8) M glucagon and arginine vasopressin. The reductions in phosphorylase a activity after treatment with chlorpropamide were more than sufficient to explain the accompanying decreases in hepatic glucose production. Since glucagon and arginine vasopressin stimulate alternate pathways of phosphorylase activation and since chlorpropamide antagonizes both hormones, it is likely that the drug acts at or distal to the intracellular site (phosphorylase kinase) at which the two activation pathways converge.     

Adrenergic modulation of human polymorphonuclear leukocyte activation. Potentiating effect of adenosine.

The activation of polymorphonuclear leukocytes (PMN) is an important step in the development of tissue damage associated with inflammatory and ischemic conditions. Catecholamines have been reported to inhibit PMN functions, but the high concentrations required cast doubt on their actual relevance as a defense mechanism. We report here that adenosine, which is actively released in ischemic conditions, potentiates the effect of epinephrine and reduces the minimal active concentration required to inhibit PMN activation by at least two orders of magnitude. Epinephrine caused a dose-related reduction of chemiluminescence, superoxide anion generation, enzyme release (lysozyme and beta-glucuronidase), and adhesion to endothelial cell (EC) monolayers in human PMN activated by N-formyl-methionyl-leucyl-phenyl-alanine (fMLP). This effect was only apparent at 10(-7) to 10(-6) mol/L. As expected, adenosine caused dose-dependent reductions of superoxide anion production and PMN adhesion to EC. Adenosine and epinephrine combined had an additive effect on PMN superoxide production and adhesion to EC. The minimal effective concentration of epinephrine in combination with 10(-8) mol/L adenosine was in the range of 10(-10) to 10(-9) mol/L. In contrast, adenosine inhibited only slightly enzyme release and did not significantly enhance the inhibition by epinephrine on this parameter. Studies with adenosine analogs suggested that the potentiating effect of adenosine was mediated by A2 receptors. The mechanism of potentiation was not related to additive effect on intracellular cyclic adenosine monophosphate levels. Epinephrine's ability to modulate PMN activation and the potentiating effect of adenosine may constitute a form of physiologic protection against tissue injury in inflammatory and ischemic processes.     

Endothelin-1-stimulated glucose production in vitro in the isolated perfused rat liver.

The effect of endothelin-1 (ET-1) on production rates of glucose, lactate and cyclic adenosine monophosphate (cAMP) was studied in isolated rat livers perfused in a non-recirculating system. Continuous infusion of ET-1 (0.5 to 10 nmol/L) resulted in a dose-dependent increase in hepatic glucose production, reaching a maximum of 7.56 +/- 1.04 mumols.min-1.100 g bodyweight (BW)-1 at 10(-9) mol/L ET-1 versus 1.32 +/- 0.13 mumols.min-1.100 g BW-1 (P less than .01) after 60 minutes in control experiments. The integral ET-1-induced glucose release (37 +/- 20 mumols.g liver-1) was accompanied by a 15% decrease in hepatic glycogen content (basal, 116 +/- 12, after ET-1, 99 +/- 8 mumols glucose.g liver-1, P less than .05), while 10(-9) mol/L ET-1 affected neither hepatic lactate nor cAMP release versus control experiments. ET-1-induced glucose output was abolished during nominally Ca(2+)-free perfusions (-41.3 +/- 65.2 mumols.100 g BW-1, P less than .01), but was unaffected by 10(-6) mol/L verapamil and only slightly attenuated by 10(-6) mol/L nicardipine (376.3 +/- 101.4, NS, and 244.4 +/- 70.0 mumols.100 g BW-1, P less than .05, respectively). Dantrolene (10(-5) mol/L), an inhibitor of Ca2+ release from the endoplasmic reticulum, reduced glucose release elicited by 10(-9) mol/L ET-1 to 241.1 +/- 57.3 mumols.100 g BW-1 (P less than .05). Pharmacological concentrations of insulin (1 U/L) were required to inhibit ET-1-dependent glucose release by 59% (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovine placental lactogen inhibits glucagon-induced glycogenolysis in fetal rat hepatocytes

The effect of ovine placental lactogen (oPL) on glucagon-stimulated glycogenolysis was studied in cultured hepatocytes from 20-day-old fetal rats. Pretreatment of hepatocytes with oPL (0.5-5 micrograms/ml) significantly attenuated the inhibitory effect of glucagon on glycogen synthesis. Hepatocytes exposed to glucagon alone at 1, 5, and 20 nM incorporated 32.0, 43.2, and 62.1% less [14C]glucose into glycogen than control hepatocytes. However, hepatocytes pretreated for 1 h with oPL (1 microgram/ml) and then exposed to the same concentration of glucagon incorporated only 5.8, 9.2, and 22.1% less [14C]glucose than control cells (P less than 0.01 vs. glucagon alone). In cells preincubated for 24 h in medium containing [14C]glucose, glucagon reduced cellular [14C]glycogen and total glycogen content by 47.1 and 51.0% while oPL increased total cellular glycogen content by 105.8% and attenuated the glycogen-degradative effect of glucagon. While oPL alone had no effect on basal phosphorylase a (Pa) activity, oPL (1-10 micrograms/ml) caused a 15.3-91.6% inhibition of glucagon-stimulated Pa activity (P less than 0.01). The maximal inhibition by oPL of glucagon-stimulated Pa activity occurred within 2 min of exposure to oPL, and the effect was not blocked by cycloheximide. oPL also caused a 49.4-95.0% inhibition of (Bu)2cAMP-stimulated Pa activity (P less than 0.01), suggesting that the inhibitory effect of oPL on glucagon action is exerted, at least in part, at a site distal to the intracellular accumulation of cAMP. Insulin (1 microM) reduced basal Pa activity, abolished the stimulation of Pa activity by glucagon, and markedly attenuated the stimulation of Pa by (Bu)2-cAMP. These studies demonstrate that oPL acutely inhibits glycogen degradation in fetal rat hepatocytes and suggest that oPL promotes glycogen storage in fetal liver both by antagonizing the glycogenolytic effects of glucagon and by stimulating fetal hepatic glycogenesis.     

Alpha 2-adrenergic stimulation counteracts the metabolic effects of vasoactive intestinal peptide in isolated rat enterocytes

The purpose of this investigation was to determine if alpha 2-adrenergic receptor activation suppresses the effects of vasoactive intestinal peptide (VIP) on glucose and fatty acid metabolism in isolated rat enterocytes. VIP (10(-7) M) produced an acute 40% inhibition of glucose oxidation and a 115% stimulation of palmitate oxidation. Addition of epinephrine (10(-6) M) together with propranolol (10(-6) M) to avoid interference with beta-adrenoreceptors did not affect basal glucose and palmitate oxidation, but suppressed the effects of VIP by more than 90%. The inhibition by VIP of pyruvate dehydrogenase and acetylcoenzyme-A carboxylase, the enzymes considered as the target sites for the neuropeptide, was also abolished by epinephrine. We assumed that epinephrine acted through the stimulation of alpha 2-adrenergic receptors, since 1) epinephrine suppressed VIP-induced accumulation of cAMP in the cells; and 2) the alpha 2-agonist clonidine (10(-6) M) reproduced epinephrine effects, whereas they were abolished by the alpha 2-antagonist yohimbine (10(-6) M). These findings strengthen the assumption that VIP controls glucose and fatty acid metabolism by a cAMP-dependent mechanism and demonstrate, for the first time, that the contribution of substrates as energy fuel is under dual neurohormonal regulation by VIP and catecholamines in isolated rat enterocytes.     

Interaction of sympathomimetics and insulin with hepatic glucose production by isolated perfused rat livers: effects of continuous versus pulsatile infusion

To elucidate the efficacy of continuous vs. intermittent exposure to epinephrine, phenylephrine, and insulin, hepatic glucose production was monitored in isolated perfused rat livers (means +/- SE, n = 6 each). To this end livers of fed rats were perfused with 5 mM glucose Krebs-Ringer buffer in a nonrecirculating system. Using this model it was shown that intermittent exposure (3 min on/off period, dose reduction -50%) to epinephrine (0.4 microM, alpha + beta-agonist) and phenylephrine (5 microM, alpha-agonist) elicited an almost identical rise in hepatic glucose production [epinephrine: 0.72 +/- 0.08 mmol/(86 min X 100 g BW); phenylephrine: 0.68 +/- 0.07 mmol/(86 min X 100 g BW) as their continuous administration (epinephrine: 0.78 +/- 0.06 mmol/(86 min X 100 g BW); phenylephrine: 0.74 +/- 0.09 mmol/(86 min X 100 g BW)]. Inhibition by insulin (100 mU/liter) given either continuously or intermittently (3 min on/off intervals; dose reduction -50%) was equipotent for epinephrine- and phenylephrine-stimulated hepatic glucose production. When the off period was doubled to 6 min, thereby reducing the total insulin dose to 33%, no significant suppression of epinephrine- and phenylephrine-stimulated hepatic glucose production was observed. From this we conclude that 1) the effect on hepatic glucose production of pulsatile (3 min on/off, dose reduction 50%) and continuous administration is equipotent for the respective action of epinephrine, phenylephrine as well as of insulin; and 2) insulin is more effective (P less than 0.02) in inhibiting hepatic glucose production stimulated by an alpha-agonist (phenylephrine; 5.0 microM) than in counteracting alpha + beta-agonist action (epinephrine; 0.4 microM). The characteristics of hepatic glucose release as stimulated by alpha- and/or beta-adrenergic agonists and its inhibition by continuously or intermittently infused insulin were simulated and described by a computer model. Thereby, no qualitative difference could be demonstrated in alpha- vs. beta-adrenergic agonists action on stimulated hepatic glucose production.     

Endothelium-derived nitric oxide modulates vascular action of aldosterone in renal arteriole

We have recently demonstrated that aldosterone causes nongenomic vasoconstriction by activating phospholipase C (PLC) in the preglomerular afferent arteriole (Af-Art). In the present study, we tested the hypothesis that endothelium modulates this vasoconstrictor action by releasing nitric oxide (NO). In addition, to study the post-PLC mechanism, we examined possible contributions of phosphoinositol hydrolysis products. Rabbit Af-Arts were microperfused at 60 mm Hg in vitro, and increasing doses of aldosterone (10(-10) to 10(-8) mol/L) were added to the bath and lumen. Aldosterone caused dose-dependent vasoconstriction (within 10 minutes); significant (P<0.01) constriction was observed from 5x10(-9) mol/L, and at 10(-8) mol/L, intraluminal diameter decreased by 29%+/-3% (n=9). Disrupting the endothelium augmented vasoconstriction; significant constriction was observed from 10(-10) mol/L, and at 10(-8) mol/L, the diameter decreased by 38%+/-2% (n=6). NO synthesis inhibition reproduced this augmentation (n=7). Pretreatment with chelerythrine (10(-6) mol/L), a protein kinase C (PKC) inhibitor, slightly attenuated the constriction; aldosterone at 10(-8) mol/L now decreased the diameter by 18%+/-3% (n=7). However, in Af-Arts treated with thapsigargin (10(-6) mol/L) or dantrolene (3x10(-5) mol/L), which blocks inositol 1,4,5-triphosphate (IP3)-induced intracellular calcium release, aldosterone at 10(-8) mol/L decreased the diameter by only 9%+/-1% (n=6) or 9%+/-2% (n=5), respectively. These results demonstrate that in the Af-Art endothelium-derived NO modulates vasoconstrictor actions of aldosterone that are mediated by the activation of both IP3 and PKC pathways. Such vasoconstrictor actions of aldosterone may contribute to the development or aggravation of hypertension by elevating renal vascular resistance in cardiovascular diseases associated with endothelium dysfunction.     

Protective effect of estradiol on hepatocytic oxidative damage

AIM: To examine the protective effect of estradiol on the cultured hepatocytes under oxidative stress.METHODS: Hepatocytes of rat were isolated by using perfusion method, and oxidative stress wes induced by a serum-free medium and FeNTA. MDA level was determined with TBA method. Cell damage was assessed by LDH assay. Apoptosis of hepatocytes was assessed with cytoflowmetric analysis. Expression of Bcl-xl in cultured hepatocytes was detected by Western blot.The radicalscavenging activity of estradiol was valued by its ability to scavenge the stable free radical of DDPH.RESULTS: Oxidative stress increased LDH (from 168 ± 25 x10-6 IU. cell 1 to 780 ± 62 x 10-6 IU. cell-1 ) and MDA(from 0.28 ±0.07 x 10-6 nmol. cell-1 to 1. 35 ± 0.12 × 10-6 nmol. cell-1 ) levelsin cultured hepatocyte, and estradiol inhibited both LDH andMDA production in a dose dependent manner. In thepresence of estradiol t0-6 mol. L-1, 107 mol. L-1 and 10-8 mol.L-1 ,the LDH levels are 410 ± 53 × 10-6 IU. cell-1 ( P ＜ 0.01 vsoxidative group), 530 ± 37 × 10-6 IU. cell-1 ( P ＜ 0. 01 vsoxidative group), 687 ± 42 x 10-6 IU. cell-1 ( P ＜ 0.05 vsoxidative group) respectively, and the MDA level are 0.71 ±0.12 x 10-6 nmol. cell-1 ( P ＜ 0.01 vs oxidative group), 0.97 ± 0.11 × 10-6 nmol. cell-1 ( P ＜ 0.01 vs oxidative group) and 1.27 ±0. 19 x 10-6 nmol. cell-1 respectively. Estradiol suppressedapoptosis of hepatocytes induced by oxidative stress,administration of estradiol (10-6 mol/ L)decreased theapoptotic rate of hepatocytes under oxidative stress from 18.6 ± 1.2% to 6.5 ± 2.5%, P ＜ 0.01. Bcl-xl expression wasrelated to the degree of liver cell damage due to oxidativestress, and estradiol showed a protective action.CONCLUSION: Estmdiol protects hepatocytes from oxidativedamage by means of its antioxidant activity.     

Divergent effects of epinephrine and prostaglandin E2 on glucose-induced insulin secretion from perifused rat islets

The impact of the catecholamine epinephrine and the postulated inhibitory second messenger prostaglandin E(2) (PGE(2)) on the kinetics and magnitude of glucose-induced insulin secretion were compared and contrasted. In agreement with a number of studies, epinephrine was a most effective antagonist of glucose-induced insulin secretion. Dose-response studies using 8 to 10 mmol/L glucose as stimulant established that levels as low as 1 to 10 nmol/L of the catecholamine were effective at inhibiting release. Glucose (20 mmol/L) caused an approximately 25-fold increase in insulin secretion, an effect that was completely abolished by 1 micromol/L epinephrine. Under conditions where it completely abolished 20 mmol/L glucose-induced insulin release, epinephrine (1 micromol/L) reduced, but did not abolish, the stimulatory effect of glucose on phospholipase C activation. Chronic 3-hour exposure to 10 mmol/L glucose alone desensitized the islet to subsequent stimulation by glucose. Despite its ability to completely suppress secretion to 10 mmol/L glucose, epinephrine failed to protect the islet from hyperglycemia-induced desensitization. In sharp contrast to epinephrine, PGE(2) at levels ranging from 1 to 10 micromol/L had no discernible adverse effect on 10 mmol/L glucose-induced secretion. These findings suggest that multiple mechanisms contribute to the inhibitory impact of epinephrine on release and, in conjunction with other studies, cast serious doubt on the concept that PGE(2) plays any significant inhibitory role in the regulation of glucose-induced secretion.