Reciprocal regulation of glycogen phosphorylase and glycogen synthase by insulin involving phosphatidylinositol-3 kinase and protein phosphatase-1 in HepG2 cells

The effect of insulin on glycogen synthesis and key enzymes of glycogen metabolism, glycogen phosphorylase and glycogen synthase, was studied in HepG2 cells. Insulin stimulated glycogen synthesis 1.83-3.30 fold depending on insulin concentration in the medium. Insulin caused a maximum of 65% decrease in glycogen phosphorylase 'a' and 110% increase in glycogen synthase activities in 5 min. Although significant changes in enzyme activities were observed with as low as 0.5 nM insulin level, the maximum effects were observed with 100 nM insulin. There was a significant inverse correlation between activities of glycogen phosphorylase 'a' and glycogen synthase 'a' (R² = 0.66, p < 0.001). Addition of 30 mM glucose caused a decrease in phosphorylase 'a' activity in the absence of insulin and this effect was additive with insulin up to 10 nM concentration. The inactivation of phosphorylase 'a' by insulin was prevented by wortmannin and rapamycin but not by PD98059. The activation of glycogen synthase by insulin was prevented by wortmannin but not by PD98059 or rapamycin. In fact, PD98059 slightly stimulated glycogen synthase activation by insulin. Under these experimental conditions, insulin decreased glycogen synthase kinase-3 activity by 30-50% and activated more than 4-fold particulate protein phosphatase-1 activity and 1.9-fold protein kinase B activity; changes in all of these enzyme activities were abolished by wortmannin. The inactivation of GSK-3 and activation of PKB by insulin were associated with their phosphorylation and this was also reversed by wortmannin. The addition of protein phosphatase-1 inhibitors, okadaic acid and calyculin A, completely abolished the effects of insulin on both enzymes. These data suggest that stimulation of glycogen synthase by insulin in HepG2 cells is mediated through the PI-3 kinase pathway by activating PKB and PP-1G and inactivating GSK-3. On the other hand, inactivation of phosphorylase by insulin is mediated through the PI-3 kinase pathway involving a rapamycin-sensitive p70^s6k and PP-1G. These experiments demonstrate that insulin regulates glycogen phosphorylase and glycogen synthase through (i) a common signaling pathway at least up to PI-3 kinase and bifurcates downstream and (ii) that PP-1 activity is essential for the effect of insulin.     

Non-hormonal and hormonal control of glycogen metabolism in isolated sheep liver cells

1. Control of glycogen metabolism by various substrates and hormones was studied in ruminant liver using isolated hepatocytes from fed sheep. 2. In these cells glucose appeared uneffective to stimulate glycogen synthesis whereas fructose and propionate activated glycogen synthase owing to (i) a decrease in phosphorylase a activity and (ii) changes in the intracellular concentrations of glucose 6-phosphate and adenine nucleotides. 3. The activation of hepatic glycogenolysis by glucagon and alpha 1-adrenergic agents was associated with increased phosphorylase a and decreased glycogen synthase activities. 4. The simultaneous changes in these two enzyme activities suggest that in sheep liver, activation of phosphorylase a is not a prerequisite step for synthase inactivation. 5. In sheep hepatocytes, in the presence of propionate and after a lag period, insulin activated glycogen synthase without affecting phosphorylase a. 6. This latter result suggests that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase. Insulin also antagonized glucagon effect on glycogen synthesis by counteracting the rise of cAMP.     

Purification and characterization of glycogen phosphorylase A and B from the freeze-avoiding gall moth larvae Epiblema scudderiana

The active a and inactive b forms of glycogen phosphorylase from cold-hardy larvae of the gall moth, Epiblema scudderiana, were purified using DEAE⁺ ion exchange and 3-5-AMP-agarose affinity chromatography. Maximum activities for glycogen phosphorylases a and b were 6.3±0.74 and 2.7±0.87 mol glucose-1-P·min^-1·g wet weight^-1, respectively, in -4°C-acclimated larvae. Final specific activities of the purified enzymes were 396 and 82 units·mg protein^-1, respectively. Both enzymes were dimers with native molecular weights of 215000±18000 for glycogen phosphorylase a and 209000±15000 for glycogen phosphorylase b; the subunit molecular weight of both forms was 87000±2000. Both enzymes showed pH optima of 7.5 at 22°C and a break in the Arrhenius relationship with a two- to four-fold increase in activation energy below 10°C. Michaelis constant values for glycogen at 22°C were 0.12±0.004 mg·ml^-1 for glycogen phosphorylase a and 0.87±0.034 mg·ml^-1 for glycogen phosphorylase b; the Michaelis constant for inorganic phosphate was 6.5±0.07 mmol·l^-1 for glycogen phosphorylase a and 23.6 mmol·l^-1 for glycogen phosphorylase b. Glycogen phosphorylase b was activated by adenosine monophosphate with a K _a of 0.176±0.004 mmol·l^-1. Michaelis constant and K _a values decreased by two- to fivefold at 5°C compared with 22°C. Glycerol had a positive effect on the Michaelis constant for glycogen for glycogen phosphorylase a at intermediate concentrations (0.5 mol·l^-1) but was inhibitory to both enzyme forms at high concentrations (2 mol·l^-1). Glycerol production as a cryoprotectant in E. scudderiana larvae is facilitated by the low temperature-simulated glycogen phosphorylase b to glycogen phosphorylase a conversion and by positive effects of low temperature on the kinetic properties of glycogen phosphorylase a. Enzyme shut-down when polyol synthesis is complete appears to be aided by strong inhibitory effects of glycerol and KCl on glycogen phosphorylase b.Abbreviations E _a activation energy - GPa glycogen phosphorylase a - GPb glycogen phosphorylase b - h Hill coefficient - I ₅₀ concentration of inhibitor that reduces enzymes velocity by 50% - K _a concentration of activator that produces half-maximal activation of enzyme activity - K _m Michaelis-Menten substrate affinity constant - MW molecular weight - PEG polyethylene glycol - P_i morganic phosphate - SDS PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - V _max enzyme maximal velocity     

Insulin regulation of hepatic glycogen synthase and phosphorylase.

The relative roles of insulin and glucose in the regulation of hepatic glycogen synthase and phosphorylase were studied in hepatocytes from fed rats. Elevation of extra-cellular glucose led to a rapid decrease in phosphorylase a activity followed by a slower increase in glycogen synthase I activity. A reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose was observed; following initial glucose-induced inactivation of phosphorylase, there was a highly significant linear inverse relationship between residual phosphorylase activity and glycogen synthase activation. Insulin led to a further decrease in phosphorylase activity and a 30-50% additional increase in glycogen synthase activity over that caused by glucose. The effects of insulin required the presence of glucose and served to augment acute glucose stimulation of glycogen synthase and inhibition of phosphorylase. Insulin did not perturb the reciprocal and coordinate relationship between phosphorylase inactivation and synthase activation in response to glucose. The results suggest that the ability of insulin to activate hepatic glycogen synthase can be entirely accounted for by its ability to inactivate phosphorylase.     

Long term regulation of glycogen metabolizing enzymes by insulin in H4 hepatoma cells

Insulin alone at concentrations of less than 1 to 5 uU/ml increased the enzyme activities of glycogen synthase, synthase phosphatase, phosphorylase, and phosphorylase phosphatase in hepatoma H4 cells in culture in the presence and absence of serum. Increase in total and active forms of glycogen synthase and phosphorylase were observed. Cycloheximide blocked the action of insulin on glycogen synthase, glycogen synthase phosphatase and phosphorylase phosphatase. The enzymes with the exception of glycogen synthase phosphatase were expressed with greater hormonal sensitivity in the absence as compared to the presence of serum in terms of hormone concentration required and or time of onset.These results demonstrate that these glycogen metabolizing enzymes are under long term control by insulin, with glycogen synthase being the most sensitive of the enzymes studied to the action of the hormone.Supported by grants from NIH AM 14334 and AM 22125 (University of Virginia Diabetes Research and Training Center) and by a grant from Lilly Research Lab, and the March of Dimes     

Control of glycogen synthase and phosphorylase by insulin in rat adipocytes which is unrelated to the concentration of cyclic AMP

Incubation of adipocytes in glucose-free medium with adrenocorticotrophic hormone, epinephrine, isoproterenol, or norepinephrine increased the concentration of cyclic AMP and the percentage of phosphorylase a activity, and decreased the percentage of glycogen synthase I activity. Glucose was essentially without effect on glycogen synthase or phosphorylase in either the presence or absence of epinephrine. Although glucose potentiated the action of insulin to activate glycogen synthase, the hexose did not enhance the effectiveness of insulin in the presence of epinephrine. Likewise, glucose did not increase the ability of insulin to oppose the activation of phosphorylase by epinephrine.The activation of glycogen synthase by insulin was not associated with a decrease in the concentration of cyclic AMP. Insulin partially blocked the rise in cyclic AMP due to isoproterenol, adrenocorticotrophic hormone, and norepinephrine. The maximum effects of isoproterenol on glycogen synthase and phosphorylase were observed when the concentration of cyclic AMP was increased twofold. However, insulin clearly opposed the changes in enzyme activity produced by isoproterenol (and also adrenocorticotrophic hormone, epinephrine and norepinephrine) even though concentrations of cyclic AMP were still increased three- to fourfold. Nicotinic acid opposed the increases in cyclic AMP due to adrenocorticotrophic hormone, isoproterenol and norepinephrine to the same extent as insulin; however, nicotinic acid was ineffective in opposing the activation of phosphorylase and inactivation of glycogen synthase produced by these agents. Thus, it is unlikely that the effects of insulin on glycogen synthase and phosphorylase result from an action of the hormone to decrease the concentration of cyclic AMP.     

香蒲对不同浓度Pb~(2+)胁迫的生理应答及其细胞超微结构变化

通过室内水培试验,研究了不同浓度Pb2+(0、0.25、0.50、1.00和2.00mmol·L-1)胁迫对东方香蒲根和叶中Pb含量、叶绿素含量、丙二醛(MDA)含量、抗氧化酶(SOD、CAT和POD)活性以及亚细胞结构的影响。结果显示:(1)随着外源Pb2+浓度的增加,Pb在香蒲根和叶中的积累量均显著高于对照,且Pb在根中的含量明显高于叶中,并与外源Pb2+浓度呈显著正相关关系。(2)香蒲叶片中的叶绿素a和叶绿素b含量随着外源Pb2+浓度的增加呈先升后降趋势,均在处理浓度为0.50mmol·L-1时达到峰值。(3)胁迫处理叶片的MDA含量与对照相比变化不显著,但根中MDA含量呈显著下降趋势。(4)叶片中SOD活性在1.00mmol·L-1 Pb2+处理时达到峰值,然后下降,但始终高于对照,CAT和POD活性则均低于对照组;根中SOD活性除1.00mmol·L-1 Pb2+处理组外均显著低于对照组,CAT和POD活性分别在0.25和0.50mmol·L-1 Pb2+处理时达到峰值,然后随处理Pb2+浓度升高而下降。(5)电镜观察发现,Pb2+胁迫使香蒲叶细胞中叶绿体被膜破裂,类囊体膨胀、破损;根和叶细胞中的线粒体被膜均破裂、内腔空泡化,细胞核核膜破损、核仁消失、染色质凝集。研究表明,Pb2+胁迫致使东方香蒲根、叶生理代谢失衡,亚细胞结构出现不可逆的损伤,这为从分子水平研究Pb2+作用的具体机理以及香蒲在重金属污染修复中的应用提供了依据。     

Interactions between insulin and alpha 1-adrenergic agents in the regulation of glycogen metabolism in isolated hepatocytes

Addition of insulin to liver cells from fed rats incubated in the absence of other hormones resulted in a 2-fold increase in glycogen synthase activity. This direct effect of insulin has been characterized and compared with the antagonism by insulin of alpha 1-adrenergic effects on glycogen metabolism. The activation of glycogen synthase by insulin developed slowly (20-25 min) and was most effective when the enzyme was partially preactivated by glucose. With glucose concentrations above 15 mM the effects of insulin and glucose were additive. In contrast to glucose, which caused inverse changes in phosphorylase and glycogen synthase activity, insulin activated glycogen synthase without affecting phosphorylase a. Treatment of hepatocytes with phenylephrine led to an activation of phosphorylase and inactivation of glycogen synthase, which could be partially blocked by insulin. This antagonistic effect of insulin was rapid (complete within 5 min of insulin addition) and showed an identical time course for both enzymes. The activation of glycogen synthase by insulin and inactivation by phenylephrine both resulted principally from alterations in the Vmax. Insulin added alone did not alter the basal cytosolic free Ca2+ concentration, which was 160 nM as measured with Quin 2 as an intracellular Ca2+ indicator. Both the magnitude and the initial rate of cytosolic free Ca2+ increase induced by phenylephrine were reduced by about 50% in cells pretreated with insulin. It is concluded that the direct activation of glycogen synthase by insulin is mediated by a glycogen synthase-specific kinase or phosphatase, whereas insulin antagonizes the effects of alpha 1-agonists by interfering with their ability to elevate cytosolic free Ca2+.     

The effect of streptozotocin-induced diabetes on glycogen metabolism in rat kidney and its relationship to the liver system.

The effects in kidney of streptozotocin-induced diabetes and of insulin supplementation to diabetic animals on glycogen-metabolizing enzymes were determined. Kidney glycogen levels were approximately 30-fold higher in diabetic animals than in control or insulintreated diabetic animals. The activities of glycogenolytic enzymes i.e., phosphorylase (both a and b), phosphorylase kinase, and protein kinase were not significantly altered in the diabetic animals. Glycogen synthase (I form) activity decreased in the diabetic animals whereas total glycogen synthase (I + D) activity significantly increased in these animals. The activities were restored to control values after insulin therapy. Diabetic animals also showed a 3-fold increase in glucose 6-phosphate levels. These data suggest that higher accumulation of glycogen in kidneys of diabetic animals is due to increased amounts of total glycogen synthase and its activator glucose 6-phosphate.     

Triiodo-L-thyronine stimulates glycogen synthesis in rat hepatocyte cultures

The hyperthyroid state is associated with low hepatic glycogen levels, but paradoxically with a high activity of glycogen synthase and low activity of glycogen phosphorylase. We determined the effects of triiodo-L-thyronine (T₃) on glycogen synthesis and glycogen synthase activity in rat hepatocytesin vitro. Culture of rat hepatocytes with T₃ (100 nM–1 M) for 16 h–40 h increases glycogen synthesis from glucose and gluconeogenic precursors. The stimulation of glycogen synthesis by T₃ was associated with an increase in the activity of glycogen synthase and was additive with the long-term effects of insulin but not with the short-term stimulation of glycogen synthesis by insulin. Culture of hepatocytes with T₃ (at concentrations up to 1 M) did not affect the responsiveness of glycogen synthesis to short-term stimulation by insulin but culture with 10 M-T₃ decreased the responsiveness to insulin without affecting the basal rate. It is suggested that the high activity of glycogen synthase in the hyperthyroid state is due to a direct effect of T₃ on the hepatocyte, but the low hepatic glycogen content is probably due to either secondary metabolite and/or endocrine changes or to impaired responsiveness to insulin. T₃ may have an anabolic role in the control of hepatic glycogen storage in the euthyroid postprandial state. (Mol Cell Biol120: 151–158, 1993)Abbreviations T₃ triiodo-L-thyronine     

Glucagon and glucose as major regulators of glycogen metabolism in primary cultured rat hepatocytes

The short-term controls of glycogen synthase [EC 2.4.1.11] and glycogen phosphorylase [EC 2.4.1.1] by major regulators, such as insulin, glucose, catecholamine, and glucagon, were compared in a simple, yet organized experimental system, i.e., adult rat hepatocytes in primary culture. Glycogen synthase was activated by glucose markedly and dose-dependently (5-40 mM), but insulin alone (1 X 10(-8) M) activated this enzyme only two-fold. Therefore, activation of the enzyme by the two regulators together was mostly due to activation by glucose. Glucagon at a concentration of 5 X 10(-10) M suppressed this activation almost completely. Glucagon at this concentration activated phosphorylase considerably and this activation was slightly inhibited by insulin. Phenylephrine also activated phosphorylase, and this activation was inhibited by phenoxybenzamine or prazosin, suggesting that activation by catecholamine is through the alpha 1-adrenergic receptor. Similarly a high concentration of glucose diminished the effects of glucagon and phenylephrine. These results suggest that in rat liver, glycogen metabolism is controlled mainly by glucagon, catecholamine, and glucose; the former two activate phosphorylase and inactivate synthase, while glucose activates synthase strongly and inactivates phosphorylase partially. Insulin plays a minor role in both reactions. Thus, the liver is primarily an organ for glucose production, which is regulated by hormones, not for glycogen storage, which is increased only by a high glucose concentration in the portal blood.     

Regulation of basal and insulin-stimulated glycogen synthesis in cultured hepatocytes. Inverse relationship to glycogen content

Cultured rat hepatocytes were used to characterize the relationship between cellular glycogen content and the basal rate, as well as response to insulin of glycogen synthesis. Depending on the concentration of medium glucose, glycogen-depleted monolayers accumulated glycogen between 24 and 48 h of culture up to the fed in vivo level. Insulin at 100 nM stimulated glycogen deposition 20-fold at 1 mM and 1.5-fold at 50 mM glucose. The rate of further glycogen storage decreased with time and increasing glycogen content. In hepatocytes preincubated with 1-50 mM glucose during 24-48 h, short-term basal and insulin-dependent incorporation of 10 mM [14C]glucose into glycogen was inversely related to the actual cellular glycogen content. This was not due to different intracellular dilution of the label, since the specific radioactivity of UDP-glucose was similar in all groups. 125I-Insulin binding indicated that insulin receptors were also not involved in this phenomenon. An inverse relationship was also found between glycogen content and the stimulation of glycogen synthase I activity by insulin, whereas the basal activity of the enzyme was dissociated from the rate of incorporation of [14C]glucose. Basal net glycogen deposition at 10 mM glucose was also inversely related to cellular glycogen; however, no such relation was evident in the presence of insulin due to the overlapping inhibition of glycogenolysis. These studies suggest that the glycogen-mediated inhibition of the activation of glycogen synthase I is operative in the cultured hepatocyte and leads to an apparent inverse relationship between the actual glycogen content and basal as well as insulin-dependent glycogenesis.     

叶施NO对NaCl胁迫下红砂幼苗叶片和根系中氮代谢酶及营养物质的影响

以当年生红砂(Reaumuria soongorica)幼苗为材料,采用盆栽实验,考察叶面喷施不同浓度(0、0.01、0.10、0.25、0.50、1.00 mmol·L^-1)NO供体硝普钠 (SNP) 对NaCl(300 mmol·L^-1)胁迫下红砂根、叶中可溶性蛋白、游离氨基酸和硝态氮含量,以及谷氨酰胺合成酶(GS)、谷氨酸合酶(GOGAT)、硝酸还原酶(NR)活性的影响,并采用主成分分析和隶属函数法筛选NO对NaCl胁迫缓解效应的氮代谢指标和最佳NO浓度,以探讨外源NO对NaCl 胁迫下红砂缓解效应的氮代谢响应机制。结果表明：(1)在300 mmol·L^-1 NaCl胁迫处理下,红砂幼苗根、叶中可溶性蛋白、硝态氮含量以及GS、GOGAT、NR活性均比对照显著下降。(2)外源NO能显著提高盐胁迫下红砂叶、根中GS、GOGAT、NR活性和硝态氮含量,增加根中可溶性蛋白和游离氨基酸含量。(3)NR和GOGAT活性可用于评价NO对NaCl胁迫下红砂幼苗的缓解作用,外源NO(SNP)对红砂幼苗在NaCl胁迫下的缓解效果强弱表现为0.25 mmol·L^-1> 0.50 mmol·L^-1> 0.10 mmol·L^-1> 1.00 mmol·L^-1> 0.01 mmol·L^-1。研究发现,300 mmol·L^-1 NaCl胁迫显著抑制了红砂幼苗氮代谢,外源NO(SNP)有助于提高盐胁迫下红砂NR活性,加快硝态氮转化为铵态氮,促进红砂叶片和根中GS/GOGAT对转化物的同化,从而增强红砂幼苗的耐盐性,并以0.25 mmol·L^-1SNP处理时缓解作用最佳;NR和GOGAT活性可作为NO缓解盐胁迫的评价指标。     

Substrate mobilization and hormonal changes in rainbow trout (Oncorhynchus mykiss,L.) and common carp (Cyprinus carpio,L.) during deep hypoxia and subsequent recovery

Common carp (at 20°C) and rainbow trout (at 15°C) were fitted with an indwelling cannula in the dorsal aorta. The fish were exposed to a controlled decline of waterpO₂ followed by 90 min deep hypoxia at 0.3 kPa (carp) or 4.8 kPa (trout). Thereafter, normoxic recovery was monitored in both species for 48 h. At regular intervals blood samples were analysed for glucose, lactate, free fatty acids, adrenaline, noradrenaline and cortisol. The oxygen restriction was maximal in both species and resulted in a significant increase of plasma lactate levels. In carp, adrenaline, noradrenaline and cortisol levels increased to 2, 50, and 753 ng·ml^-1 respectively during anoxia, whereas in trout these hormones increased to 12, 8 and 735 ng·ml^-1 respectively during hypoxia. In hypoxic trout, the plasma levels of glucose (3 mol·l^-1) were increased modestly whereas levels of free fatty acids (0.25 mmol·l^-1) were decreased to 0.15 mmol·l^-1. In carp, however, a marked and prolonged hyperglycaemia (from 5 to 10 mmol·l^-1) and a significant continuous depression of plasma levels of free fatty acids (from 0.4 to 0.2 mmol·l^-1) were observed indicating a difference in metabolic organization. It is suggested that hyperglycaemia is likely to be the result of hepatic glycogenolysis, stimulated by circulating catecholamines and a stimulation of gluconeogenesis by cortisol during recovery. The mechanism for the decline of plasma levels of free fatty acids is most probably a reduction of lipolytic activity, which appears to be an adaptation to hypoxia.     

Control mechanisms in the acceleration of hepatic glycogen degradation during hypoxia

Hepatic glycogen metabolism in aerobic and hypoxic conditions has been assessed with respect to glycogenolysis, phosphorylase a activity and nucleotide content. Insulin did not inhibit glycogen breakdown nor stimulate lipogenesis in the aerobic perfused liver.Partial ischaemia induced glycogen breakdown, release of glucose and changes in nucleotide content in the perfused liver. Phosphorylase a content increased within 2 min in response to total ischaemia, in vivo and in the perfused liver. This change was paralleled by an increase in hepatic AMP. Glycogen synthase a activity decreased, as did the hepatic content of both cyclic AMP and cyclic GMP.     

外源亚精胺对盐胁迫下白刺幼苗叶片抗氧化酶系统的影响

为进一步阐明盐生植物白刺耐盐性与多胺的关系,通过水培试验研究了叶面喷施亚精胺(Spd)对不同浓度NaCl胁迫下西伯利亚白刺幼苗叶片丙二醛(MDA)和超氧阴离子(O2)产生速率,以及抗氧化物酶系统和根系活力的影响.结果表明:叶面喷施0.1 mmol·L1 Spd 5 d后,可显著提高100和200 mmol·L1 NaCl胁迫下白刺幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性以及根系活力,降低了叶片MDA含量和O2的产生速率;而在0、50、300 mmol·L-1 NaC1处理下,外施Spd对白刺幼苗叶片上述指标无显著影响.研究结果证实,在100～200 mmol·L-1 NaCl胁迫范围内,外施亚精胺可能通过增强体内保护酶活性来显著降低活性氧水平,有效减轻盐胁迫对盐生植物白刺幼苗造成的过氧化伤害,从而增强白刺对盐环境的适应性.     

NaCl胁迫对小黄花菜生长及相关生理指标的影响

为探讨小黄花菜的耐盐机理,选育良好的耐盐植物以缓解土壤盐渍化问题,该文选取小黄花菜(Hemerocallis minor)为试材,采用砂培法,研究不同浓度Na Cl(50、100、150、200、250 mmol·L~(-1))胁迫对小黄花菜的生长性状、细胞质膜透性和有机渗透调节物质含量等的影响。结果表明:(1)小黄花菜在100～150mmol·L~(-1)Na Cl胁迫时,损害初步显现,但不影响其存活;在Nacl浓度为200 mmol·L~(-1)以上时,小黄花菜生长被显著抑制,造成根系不发育、叶片受害、植株干物质积累显著不足,严重影响其生存状态。(2)在50～150mmol·L~(-1)盐渍环境下,叶片膜透性、MDA含量增幅较小,该浓度范围的Na Cl胁迫造成的膜脂损伤有限; 200mmol·L~(-1)以上浓度的Na Cl胁迫使得小黄花菜叶片的离子平衡无法继续维持,膜的选择透性丧失。(3)随着Na Cl浓度的增加,叶片中脯氨酸含量显著增加;在50～100 mmol·L~(-1)Nacl胁迫下,叶片可溶性糖含量在胁迫初期有所增加,在15 d时达到最大,胁迫后期开始下降;叶片中可溶性蛋白含量的变幅较为平缓,说明小黄花菜的主要渗透调节物质不是可溶性蛋白。该研究发现通过提高叶片膜透性,促进自身有机渗透调节物质的合成,能够在一定程度上缓解盐渍对植株的侵害,使得小黄花菜能够在50～100 mmol·L~(-1)的盐碱环境下正常生长。     

Altered liver glycogen metabolism in fed genetically obese mice.

The cyclic AMP and glycogen concentrations and the activities of phosphorylase kinase, phosphorylase a and glycogen synthase a were not different in livers from lean or ob/ob mice despite increased plasma glucose and insulin in the obese group. The liver water content was decreased by 10% in the obese mice. In hepatocytes isolated from lean mice and incubated with increasing glucose concentrations (14-112 mM), a sequential inactivation of phosphorylase and activation of glycogen synthase was observed. In hepatocytes from obese mice the inactivation of phosphorylase was not followed by an activation of synthase. The inactivation of phosphorylase occurred more rapidly and was followed by an activation of synthase in hepatocytes isolated from both groups of mice when in the incubation medium Na+ was replaced by K+ or when Ca2+ was omitted and 2.5 mM-EGTA included. The inactivation of phosphorylase and activation of synthase were not different in broken-liver-cell preparations from lean and obese animals. The re-activation of phosphorylase in liver filtrates in the presence of 0.1 microM-cyclic AMP and MgATP was inhibited by about 70% by EGTA and stimulated by Ca2+ and was always greater in preparations from ob/ob mice. The apparent paradox between the impairment of glycogen metabolism in isolated liver preparations and the situation in vivo in obese mice is discussed.     

Glucose, pyruvate and lactate efflux by the perfused liver of a teleost, Clarias batrachus during aniso-osmotic exposure

Glucose, lactate and pyruvate efflux by the perfused liver of the walking catfish, Clarias batrachus was studied during aniso-osmotic exposure. During hypo-osmotic exposure (−80 mOsmol l⁻¹, maintained with NaCl), glucose, lactate and pyruvate efflux by the perfused liver significantly decreased by 55, 19 and 16%, respectively. During hyper-osmotic exposure (+80 mOsmol l⁻¹, maintained with NaCl), efflux increased by 57, 12 and 18%, respectively. Similar effects of glucose, lactate and pyruvate efflux by the perfused liver was also seen when the anisotonicity of the medium was adjusted with mannitol instead of NaCl. The decrease of glucose, lactate and pyruvate efflux during hypo-osmotic exposure was correlated with the stimulation of glycogen synthesis and the reverse was true during hyper-osmotic exposure. These observations were supported by changes in glycogen phosphorylase a (GPase a) and glycogen synthase a (GSase a) activities. During hypo-osmotic exposure (−80 mOsmol l⁻¹), the GPase a activity decreased by 1.93 fold and GSase a activity increased by 1.63 fold, while during hyper-osmotic exposure (+80 mOsmol l⁻¹), the GPase a activity increased by 1.58 fold and GSase a activity decreased by 1.95 fold. The total activity of both the enzymes were not effected by a short term exposure to aniso-osmotic conditions, suggesting that the alterations in GPase a and GSase a activity were mainly due to changes of their phosphorylation status during aniso-osmotic exposure. A direct correlation exists between glucose efflux and the hydration status of the perfused liver. These alterations of glucose metabolism are probably necessary by this walking catfish to meet the different energy demand, and also for maintenance of glucose homeostasis under osmotic stress.