Metabolic actions of glucagon-family hormones in liver

This review addresses direct and indirect metabolic actions of hormones co-encoded in the preproglucagon gene of fishes. Emphasis is placed on a critical analysis of the effects of glucagon and glucagon-like peptide (GLP) and the current knowledge of the respective modes of action is reviewed. In mammals GLPs exert no direct metabolic actions. In fish liver, GLP and glucagon act on similar targets of intermediary metabolism by enhancing flux through glycogenolysis, lipolysis and gluconeogenesis. Increases in substrate oxidation are not uniform. Hormonal activation of glycogen phosphorylase and triglyceride lipase and inhibition of pyruvate kinase are implicated in these actions. Hormone-dependent hyperglycemia, depletion of hepatic glycogen and increases in free fatty acids are noticeablein vivo. Glucagon also activates hepatic amino acid uptake and ammonia excretion. Glucagon actions are accompanied by large increases in hepatic cAMP and increased phosphorylation of pyruvate kinase. Metabolic effects measured after GLP administration are associated with minor, if any, increases in cAMP and effects on pyruvate kinase are variable. We hypothesize that different hepatic receptors with differing modes of intracellular message transduction are involved in glucagon and GLP actions while targetting identical metabolic routes. Responses of different species of fish cover a wide spectrum, and variation of response with the circannual cycle of experimental animals makes comparisons of results, even within one species, difficult.     

The role of glycogen phosphorylase in the regulation of glycogenolysis by insulin and glucagon in isolated eel (Anguilla rostrata) hepatocytes

The effects of porcine, scombroid, and salmon insulins, and bovine and anglerfish glucagons on glycogen depletion and glycogen phosphorylase (GPase) activities were examined in freshly isolated American eel (Anguilla rostrata) hepatocytes. Eel liver GPase in crude homogenates was activated (increase in % GPase a) by phosphorylating conditions and was rapidly inactivated (less than 1 h) when a phosphatase inhibitor (fluoride) was absent. Caffeine inhibits, and AMP activates, the b form of GPase consistent with their effects on rat liver GPase. Both mammalian and fish glucagons increased glucose production in eel hepatocytes, but had more ambiguous effects on glycogen levels and GPase activities. The magnitude of bovine glucagon effects were dependent on the initial glycogen content of the cells; only at glycogen concentrations less than approximately 70 μmoles.g⁻¹ did glucagon significantly increase % GPase a. Anglerfish glucagon significantly increased cyclic AMP (cAMP) concentrations by 90% at 10⁻⁷ M, but had no effects at 10⁻⁹ M and 10⁻⁸ M. Scombroid and salmon insulins maintained hepatocyte glycogen concentrations and decreased glucose production, with these effects more pronounced at low (10⁻⁹ to 10⁻⁸ M) rather than high (10⁻⁷ M) hormone concentrations. Porcine and salmon insulins decreased total GPase and % GPase a activities, and salmon insulin decreased CAMP levels, but only at 10⁻⁸ M (by 44%). Glycogen is, therefore, depleted by glucagon and maintained by insulin in freshly isolated American eel hepatocytes, and these changes are accomplished, at least in part, by changes in the activities of GPase. Changes in cAMP do not explain all of the observed hormone effects.     

Effects of cortisol on hepatic carbohydrate metabolism and responsiveness to hormones in the sea raven,Hemitripterus americanus

The sea raven, Hemitripterus americanus, is a sit-and-wait, low metabolic rate, marine teleost. The objective of this study was to determine i) whether cortisol implantation (50 mg. kg^-1) for 7 days altered hepatocyte metabolism, and hepatocyte responsiveness to epinephrine, glucagon and insulin, and ii) whether 8 weeks of food-deprivation modified the above response. Cortisol implantation significantly increased hepatocyte total glucose production and oxidation from alanine compared to the sham group. There was no cortisol effect on glycogen breakdown, suggesting that the activation of other pathways, including gluconeogenesis, are required to account for the increased glucose production. Epinephrine-mediated (10^-5M) glycogen breakdown and insulin-mediated (10^-8M) total glucose production were enhanced in hepatocytes of cortisol implanted sea ravens, but there were no change in any glucagon (10^-7M) effects. The enhanced glycogen breakdown in the absence of similar increases in total glucose production with epinephrine indicates mobilization of carbohydrate reserves for endogenous use by the liver. Food-deprivation for 8 weeks significantly decreased condition factor, plasma cortisol concentration and liver glycogen content in the sea raven, but had no effect on plasma glucose concentration. Hepatocyte total glucose production and flux rates from alanine increased significantly with food-deprivation. Moreover, food-deprivation increased responsiveness of total hepatocyte glucose production to the actions of glucagon and insulin, but not to epinephrine; none of these effects were modified by cortisol implantation. Our results indicate that cortisol in the sea raven exerts both a direct and an indirect or permissive effect on hepatocyte metabolism by modifying hepatocyte responsiveness to epinephrine and insulin stimulation. Cortisol implantation did not modify the effects of glucagon or food-deprivation in this species.     

Hormonal regulation of renal inorganic phosphate transport in the winter flounder, Pleuronectes americanus

Intestinal uptake and renal excretion are the primary determinants of inorganic phosphate (P_i) balance in teleosts. In general, teleost kidneys may either reabsorb filtered P_i or secrete excess P_i into the urine. Primary monolayer cultures of flounder (Pleuronectes americanus) renal proximal tubule epithelium (PTCs) have helped identify several hormones that may participate in conservation or excretion of P_i. Mounted in Ussing chambers, the monolayer cultures can be used to assay transepithelial P_i transport. Several factors, including metabolic acidosis, elevation of plasma [P_i], salmon stanniocalcin, salmon somatolactin and mammalian prolactin, have now been shown to alter transepithelial P_i transport in winter flounder PTCs. Salmon stanniocalcin (STC) stimulated P_i luminal-to-peritubular transport (reabsorption) at a dosage of 12.5–50 ng/ml (0.25–1.0 nM). Net P_i transport changed within 30 min and progressively increased from slight net secretion in untreated controls to net reabsorption after 3 h. The target and function of somatolactin have been uncertain. In our hands salmon somatolactin (sSL) also stimulated P_i reabsorption by flounder PTCs in a dose-dependent manner at physiological levels of the hormone (12.5 ng/ml). Net P_i transport was significantly altered by sSL within 2 h after the initial exposure. Neither sSL nor STC had any effect on transepithelial Ca²⁺ transport. The effects of both sSL and STC were mimicked by forskolin, whereas H-89, a highly specific protein kinase A inhibitor, significantly decreased the effects of the hormones as well as forskolin-induced P_i reabsorption. Furthermore, the production and release of cAMP were increased more than two-fold following exposure to STC or sSL. The data indicate that STC and sSL directly stimulate net renal P_i reabsorption by a cAMP-dependent pathway. In addition, mammalian prolactin greatly, and salmon growth hormone slightly, increased net P_i reabsorptive flux, whereas salmon prolactin had no effect. These results appear to be related to the location of the cysteine disulfide bonds within the molecular structure. Although somatolactin and stanniocalcin may both stimulate renal P_i conservation, their actions may be related to different physiological conditions.     

中华绒螯蟹雄性生殖系统生化组成及精子代谢

通过对中华绒螯蟹肝胰腺及生殖系统各组织生化成分和乳酸脱氢酶(LDH)活性的测定发现:总碳水化合物的含量以副性腺及输精管内容物(含精荚和精浆)较高,为33.456mg·g-1湿重和21.537mg·g-1湿重,肝胰腺、精荚及精巢的含量较低;碳水化合物中的糖原和葡萄糖含量分别以输精管内容物和肝胰腺最高,分别为0.385mg·g-1湿重和5.866mg·g-1湿重,精巢和肝胰腺中的糖原含量太低而无法检测到,精荚葡萄糖含量最低;蛋白质含量以肝胰腺最高,其余各部分含量显著低于肝胰腺;脂类含量以精巢和肝胰腺最高,其中的甘油三酯仅存于肝胰腺和精巢,且肝胰腺含量显著高于精巢。乳酸脱氢酶活性以贮精囊最高,精荚仅为中等,这可能与精荚壁的通透性有关,而精浆和肝胰腺显著低于其它组织。结果表明,中华绒螯蟹能量代谢主要物质是脂类和蛋白质;精子代谢的能量物质为碳水化合物,并由精浆提供,而其代谢的方式以厌氧代谢为主。     

鱼类的葡萄糖感知与糖代谢调节研究进展

为加深对鱼类糖的感知与代谢调控的认识,本文综述了鱼类的葡萄糖感知与摄食调控、糖代谢调控等领域的研究进展。鱼类的下丘脑不仅是中枢葡萄糖感受器所处的主要部位,同时也是食欲调节中枢。leptin、ghrelin、CCK、NPY等内分泌因子均能调控鱼类对糖的感知与摄食。另一方面,鱼类糖代谢受到胰岛素、GLP-1、ghrelin、CCK、NPY、SS等内分泌因子和糖、脂、蛋白质等营养素的双重调节。尽管鱼类对糖的利用能力低于陆生动物,但鱼体内亦存在较完善的糖的感知、摄食与代谢调节机制。因此,将来的重点工作应在于研究鱼类中枢神经系统整合营养和内分泌等信号的机制,研究草食性鱼类、杂食性鱼类在糖耐受力及糖异生调控机制上与肉食性鱼类存在的差异。     

The effects of temperature on benzo[a]pyrene metabolism and adduct formation in the gulf toadfish,Opsanus beta

In order to examine the effects of temperature on benzo[a]pyrene (BaP) metabolism and adduct formation in the absence of the effects of temperature on uptake, gulf toadfish,Opsanus beta, were given a dose of 0.05 mg/kg³H-BaPvia caudal vein cannulae at their acclimation temperatures. (18 or 28°C) or following an acute temperature change (18 to 28°C or 28 to 18°C). After 72h, BaP-derived radioactivity was detected in all tissues examined and, as in otherin vivo studies of fish, the highest levels were found in the bile, the liver and the kidney. Temperature did not affect the total amount of BaP metabolized and excreted to the bile, but there were significant quantitative differences between temperature treatments in the classes of Phase I metabolites accumulated. Fish acclimated to high temperature accumulated more BaP triols and tetrols (breakdown products of highly carcinogenic BaP diol epoxides) than fish acclimated at low temperature regardless of exposure temperature: the proportion of biliary metabolites as tetrols and triols in each of the four temperature treatments (acclimation: exposure temperature), 28:18, 28:28, 18:18 and 18:28°C were 21.3±3.6, 58.1±6.1, 14.2±1.8 and 20.9±3.2% (mean±SEM, n=4), respectively. Significant quantities of BaP-DNA and BaP-hemoglobin adducts were detected; however, only the amounts of BaP-DNA adducts showed sensitivity to temperature. As predicted from our metabolite data, high acclimation or exposure temperature led to a significant increase in the amount of BaP-DNA adducts formed: adduct formation in the temperature treatments, 28:18, 28:28, 18:18 and 18:28°C were 342±52, 526±51, 155±42 and 252±55 fg BaP/ µg DNA (mean±SEM, n=4), respectively. These results are discussed in the context of mechanisms of high temperature-enhancement of carcinogenesis in fish.     

Nutritional and environmental regulation of the synthesis of highly unsaturated fatty acids and of fatty-acid oxidation in Atlantic salmon (Salmo salar L.) enterocytes and hepatocytes

The objective of this work was to determine whether highly unsaturated fatty acid (HUFA) synthesis and fatty-acid oxidation in Atlantic salmon (Salmo salar L.) intestine was under environmental and/or seasonal regulation. Triplicate groups of salmon were grown through a full two-year cycle on two diets containing either fish oil (FO) or a diet with 75% of the FO replaced by a vegetable oil (VO) blend containing rapeseed, palm, and linseed oils. At key points in the life cycle fatty acyl desaturation/elongation (HUFA synthesis) and oxidation activity were determined in enterocytes and hepatocytes using [1−¹⁴C]18:3n−3 as substrate. As observed previously, HUFA synthesis in hepatocytes reached a peak at seawater transfer and declined thereafter, with activity consistently greater in fish fed the VO diet. In fish fed FO, HUFA synthesis in enterocytes in the freshwater stage was at a level similar to that in hepatocytes. HUFA synthesis in enterocytes increased rapidly after seawater transfer, however, and remained high for some months after transfer before decreasing to levels that were again similar to those observed in hepatocytes. Enterocyte synthesis of HUFA was usually higher in fish fed the VO diet than in those fed the FO diet. Oxidation of [1−¹⁴C]18:3n−3 in hepatocytes from fish fed FO tended to decrease during the freshwater phase but then increased steeply, peaking just after transfer before decreasing during the remaining seawater phase. At the peak in oxidation activity around seawater transfer, activity was significantly lower in fish fed VO than in fish fed FO. In enterocytes, oxidation of [1−¹⁴C]18:3 in fish fed FO reached a peak in activity just before seawater transfer. In fish fed VO, except for high activity at nine months the pattern was similar to that obtained in enterocytes from fish fed FO, with high activity around seawater transfer and declining activity in seawater. In conclusion, fatty acid metabolism in intestinal cells seemed to be under dual nutritional and environmental or seasonal regulation. Temporal patterns of oxidation of fatty acids were usually similar in the two cell types, but HUFA synthesis in enterocytes peaked over the summer seawater phase rather than at transfer, as with hepatocytes, suggesting the possibility of different regulatory cues.     

The effects of temperature on the uptake and metabolism of benzo[a]pyrene in isolated gill cells of the gulf toadfish,Opsanus beta

The effects of acclimation temperature and acute temperature change on the uptake and metabolism of the procarcinogen benzo[a]pyrene (BaP) by gill cells of the gulf toadfish, Opsanus beta, were examined. BaP was rapidly accumulated by isolated gill cells and uptake rates were directly proportional to BaP concentration in the medium (1 to 100 μg/ml). Uptake rates were higher in cells isolated from fish acclimated to 18°C when compared to cells from 28°C acclimated fish at all incubation temperatures. When cells were exposed to BaP at the respective acclimation temperatures of the fish, uptake rates were similar (0.14 ± 0.01 at 18°C and 0.12 ± 0.01 μg BaP/s/10 mg cells at 28°C). This finding is discussed in view of results which showed a partial compensation of membrane fluidity in plasma membranes isolated from fish from the two acclimation temperatures. At higher incubation temperatures, cells from fish acclimated to 18°C metabolized BaP at a greater rate than those at 28°C (49.6 ± 1.92 and 43.0 ± 2.24 μg/g/8h, respectively, at 23°C). Low but detectable activities of common biotransformation enzymes (aryl hydrocarbon hydroxylase, glutathione-S-transferase) and cytochrome P-450 content were found, however, no significant differences were evident between cells from fish acclimated to different temperatures. To whom to address correspondence     

发酵豆粕部分替代鱼粉后添加晶体赖氨酸对凡纳滨对虾肝胰腺代谢的转录调控

为研究发酵豆粕部分替代鱼粉后,饲料中赖氨酸(Lys)含量变化在凡纳滨对虾机体内的响应调控机制,在基础饲料中分别添加0%、0.25%、0.50%、0.75%和1.00%的晶体赖氨酸配制5种等氮等脂的实验饲料,记为Lys0、Lys25、Lys50、Lys75和Lys100。选择初始体重为(2.0±0.1) g的凡纳滨对虾在室内水泥池中进行56 d的养殖实验。饲养结束后对生长性能、肌肉粗蛋白和脂肪含量、肝胰腺健康程度差异显著的Lys0组和Lys75组对虾肝胰腺进行了代谢组学和转录组学分析。结果显示,以Lys0作对照,Lys75组共检测出28个差异代谢物,其中有2个代谢物下调,26个代谢物上调;对注释到KEGG数据库中7种差异代谢物进行信号通路分析,筛选出4条比较重要的代谢通路,分别为甘氨酸、丝氨酸和苏氨酸的代谢,花生四烯酸代谢,鞘脂类代谢和甘油磷脂代谢。Lys75组对虾肝胰腺中胆碱显著下调,γ-丁甜菜碱双加氧酶表达显著上调,己糖激酶基因和乙酰胆碱酯酶基因表达显著下调。研究表明,饲料赖氨酸水平的提高有助于对虾肝胰腺中脂肪酸的β氧化供能,起到节约蛋白质的作用,也能保护对虾肝胰腺健康,促进对虾生长...     

Influence of partial substitution of dietary fish oil by vegetable oils on the metabolism of [1-14C] 18:3n-3 in isolated hepatocytes of European sea bass (Dicentrarchus labrax L.)

The static or declining supply of fish oil from industrial fisheries demands the search of alternatives, such as plant (vegetable) oils, for diets in expanding marine aquaculture. Vegetable oils are rich in C₁₈ polyunsaturated fatty acids but devoid of the n-3 highly unsaturated fatty acids in fish oils. Previous studies, primarily with salmonids, have shown that including vegetable oils in their diets increased hepatocyte fatty acid desaturation. In the present study, we have investigated the effects of dietary partial substitution of fish oil (FO) with rapeseed oil (RO), linseed oil (LO) and olive oil (OO) on the desaturation /elongation and, -oxidation capacities of [1-¹⁴C]18:3n-3 in isolated hepatocytes from European sea bass (Dicentrarchus labrax L.), in a simultaneous combined assay. Fish were fed during 34 weeks with diets containing 100% FO, or RO, LO and OO, each included at 60% with the balance being met by FO, with no detrimental effect upon growth or survival. The highest total desaturation rates were found in hepatocytes of fish fed FO diet (0.52±0.08 pmol/h/mg protein) and OO diet (0.43±0.09 pmol/h/mg protein), which represented 3.2% and 2.7% of total [1-¹⁴C]18:3n-3 incorporated, respectively. In contrast, lowest desaturation rates were presented by hepatocytes of fish fed LO and RO diets (0.23±0.06 and 0.14±0.05 pmol/h/mg protein, respectively) represented 1.4% and 0.9% of total [1-¹⁴C]18:3n-3 incorporated, respectively. The rates of [1-¹⁴C]18:3n-3 β-oxidized were between 11-fold and 35-fold higher than desaturation. However, no significant differences were observed among β-oxidation activities in hepatocytes of fish fed any of the diets. The present study demonstrated that the European sea bass, as a carnivorous marine fish, presented a ‘marine’ fish pattern in the metabolism of 18:3n-3 to 20:5n-3 and 22:6n-3. This species appeared to have all the enzymic activities necessary to produce 22:6n-3 but presented only extremely low rates of fatty acid bioconversion. Furthermore, nutritional regulation of hepatocyte fatty acid desaturation was minimal, and dietary vegetable oils did not increase desaturase activities, and in RO and LO treatments the activity was significantly lower. This revised version was published online in July 2006 with corrections to the Cover Date.