肝细胞的异质性：葡萄糖和酮体在大鼠肝脏的代谢

用大鼠肝脏门静脉或肝静脉周围的肝细胞来研究葡萄糖和酮体生成的区域分布。肝细胞通过毛地黄皂苷－胶原酶灌流技术分离。门静脉周围肝细胞的ｒ谷氨酰转肽酶的活性比肝静脉周围肝细胞高２．４倍;而谷氨酰胺合成酶的活性则相反,肝静脉周围肝细胞高出５６倍。门静脉周围肝细胞的内源性葡萄糖合成比肝静脉周围肝细胞高１．５７倍。给予刺激葡萄糖异性的底物,门静脉周围肝细胞的葡萄糖合成则增加１．７－２．１倍。肝静脉周围肝细胞的     

胰岛素对葡萄糖-6-磷酸酶基因转录的调控

葡萄糖－６－磷酸酶（Ｇｌｕｃｏｓｅ－６－ｐｈｏｓ－ｐｈａｔａｓｅ,Ｇ６Ｐａｓｅ,Ｅ．Ｃ．３．１．３．９）是一种膜结合酶,主要存在于肝和肾细胞中的内质网膜及核膜上,其生物功能是催化葡萄糖异生和糖原分解两个代谢途径中由葡萄糖－６－磷酸到葡萄糖的水解反应,是调节生物体内血糖水平的关键酶之一。胰岛素（Ｉｎｓ）通过调控Ｇ６Ｐａｓｅ而调节血糖水平,近年的研究表明,Ｉｎｓ可以通过调控相关酶的基因转录来实现其相应的生理功能。１．Ｇ６Ｐａｓｅ的分子生物学研究肝微粒体Ｇ６Ｐａｓｅ酶系包括活性部分位于内质网腔表面的Ｇ…     

肝静脉与门静脉血液中哪个血糖浓度高

门静脉内流的血液和肝静脉内流的血液中,哪个葡萄糖浓度高?一种意见是肝静脉中的要高于门静脉,一种意见则认为是肝静脉中的要比门静脉中的低。一个问题,引起两种截然不同的答案,本文就这个问题,谈一点看法。肝脏是维持血糖浓度相对恒定,以保证全身组织细胞能量供应的重要代谢器官。与此相适应的肝脏的血液供应就不同于其它器官。与肝脏相连的血管有三条:入肝的肝动脉和门静脉;出肝的肝静脉.由腹主动脉分支的肝动脉是肝脏的营养血管,内流富含氧和养料的血液,供肝细胞更新利用。门静脉——两端都连着毛细血管的静脉,它接受来自脾、胰、胆囊和胃、小肠、大肠的毛     

葡萄糖苷转移酶生产菌种的筛选

用甲基葡萄糖及麦芽糖为底物,对５０多株黑曲霉α－葡萄糖苷酶活性作了测定,结果表明各菌株对这二类底物之水解能力并不一致,甲基葡萄糖水解活性高的,其麦芽糖水解能力未必也高。通过ＴＬＣ测定这些菌株生成异麦芽低聚糖之能力,结果表明甲基葡萄糖和麦芽糖为底物的水解能力并不能正常反映转苷反应的能力。酶活力高的,生成异麦芽低聚糖的能力未必比活性低的为强,造成上述结果的原因,或许是胞外酶中存在着仅有水解作用的α－葡     

败血症大鼠肝细胞核Ca^2＋转运功能的改变

本实验观察败血症时肝细胞核钙转运的变化。早期败血症（结扎盲肠及穿刺后,９ｈ）大鼠肝细胞和肝细胞核钙含量分别增加２０％和３６％（Ｐ〈０．０５）。败血症大鼠肝细胞核Ｃａ＾２＋－ＡＴＰａｓｅ活性增加９４％（Ｐ〈０．０１）,核＾４５Ｃａ＾２＋转运显著增强（增加３２％,Ｐ〈０．０１）。核＾４５Ｃａ＾２＋转运与Ｃａ＾２＋－ＡＴＰａｓｅ活性呈明显正相关（ｒ＝０．９１４,Ｐ〈０．０１）。加入钙调素显著刺激而加入钙     

羊草细胞变异系HR20－8的氨基酸组成水平与脯氨酸含成代谢变化的研究

离体培养,筛选获得羊草（Ａｎｅｕｒｏｌｅｐｉｄｉｕｍｃｈｉｎｅｎｓｅ（Ｔｒｉｎ．）Ｋｉｔａｇ．）抗羟脯氨酸（ＨＹＰ）细胞变异系ＨＲ２０－８,其愈伤组织中游离氨基酸和蛋白质氨基酸含量均发生了较大的变化,与供体对照比较,分别提高２．３５倍和１．４０倍,其中的脯氨酸组分在变异系体内表现异常增加,游离和蛋白质中的脯氨酸组分分别提高６．６倍和３．０倍,对脯氨酸合成途径中的γ－谷氨酸激酶的特性分析表明,变异系ＨＲ２０－８细胞内γ－谷氨酸激酶的活性比供体提高２．５倍。     

甘蓝型油菜子油分的积累与某些生理变化关系的研究

油菜种子发育过程中,其内部的生理代谢过程发生了规律性的变化。伴随着种子的发育进程,６－磷酸葡萄糖脱氢酶、异柠檬酸裂解酶、异柠檬酸脱氢酶和琥珀酸脱氢酶的活性均有不同程度的增强。在油分旺盛合成期,６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均达到了最大值,而此时,异柠檬酸脱氢酶和琥珀酸脱氢酶的活属于匀增加较慢;在种子的不同发育时期,高含油量品系的６－磷酸葡萄糖脱氢酶和异柠檬酸裂解酶的活性均高于低含油量的     

植酸钠对黑曲霉柠檬酸发酵产酸的促进效应

研究了植酸钠对黑曲霉柠檬酸发酵产酸的促进效应。在葡萄糖全合成培养基中添加１％的植酸钠,可使产酸比对照提高２．４倍;在薯粉、玉米粉等天然培养基中添加１％植酸钠,柠檬酸产量分别提高１．７倍和１．３倍。酶活性测定分析表明,植酸钠对柠檬酸代谢途径中的几种关键酶的活性有影响。     

新生牛肝脏再生刺激物质的部分纯化及生物学特性

新生牛肝胞浆液中存在刺激肝细胞DNA合成的物质,经过一定的分离提纯,可使其杂蛋白含量降低770倍,特异活性提高26.7倍。在无血清培养条件下,可使成年原代大鼠肝细胞和肝源性肿瘤细胞DNA合成分别增加5倍及7.2倍左右。HSS为耐热的蛋白质,一定的酸碱及变性剂可使活性丧失,主要活性成分的分子量在43—67KD之间,但小于10KD的物质亦具有一定的活性。该实验结果提示:HSS很可能是正常肝细胞分裂与再生时共同的调节因子。     

柞树林下菌根真菌对碳、氮营养的利用

研究了柞树林下菌根真菌对Ｃ、Ｎ营养的利用状况．结果表明,供试菌种对Ｃ源的利用较为广泛,葡萄糖、果糖为其最适碳源,平均生长量比对照高出４．４倍;供试菌种对有机氮的利用优于无机氮,平均生长量比无机氮源高出１．６倍,硝态无机氮源中平均生长量为对照的２．５倍,对铵态氮的利用较差,平均生长量仅为对照的２．２倍     

Glucagon regulation of gluconeogenesis and ketogenesis in periportal and perivenous rat hepatocytes. Heterogeneity of hormone action and of the mitochondrial redox state.

Hepatocytes isolated from the periportal or perivenous zones of livers of fed rats were used to study the long-term (14 h) and short-term (2 h) effects of glucagon on gluconeogenesis and ketogenesis. Long-term culture with glucagon (100 nM) resulted in a greater increase (P less than 0.01) in gluconeogenesis in periportal than in perivenous cells (93 +/- 16 versus 30 +/- 14 nmol/h per mg of protein; 72% versus 30% increase), but short-term incubation (2 h) with glucagon resulted in similar stimulation in the two cell populations. Rates of ketogenesis (acetoacetate and D-3-hydroxybutyrate production) were not significantly higher in periportal cells cultured without glucagon, compared with perivenous cells. However, after long-term culture with glucagon, the periportal cells had a significantly higher rate of ketogenesis (from either palmitate or octanoate as substrate), but a lower 3-hydroxybutyrate/acetoacetate production ratio, suggesting a more oxidized mitochondrial NADH/NAD+ redox state despite the higher rate of beta-oxidation. Periportal hepatocytes had a higher activity of carnitine palmitoyltransferase but a lower activity of citrate synthase than did perivenous cells. These findings suggest that: (i) glucagon elicits greater long-term stimulation of gluconeogenesis in periportal than in perivenous hepatocytes maintained in culture; (ii) after culture with glucagon, the rates of ketogenesis and the mitochondrial redox state differ in periportal and perivenous hepatocytes.     

Gluconeogenesis in periportal and perivenous hepatocytes of rat liver, isolated by a new high-yield digitonin/collagenase perfusion technique.

A technique is described which allows preparations of hepatocytes, enriched in either periportal or perivenous hepatocytes ('PP-cells' and 'PV-cells' respectively), in a yield of about 30-50% compared with control cell preparations. The liver is first perfused for 40-60s with digitonin (4 mg/ml) to destroy selectively either the periportal or the perivenous part of the microcirculatory unit, and then the remaining hepatocytes are isolated by the ordinary collagenase perfusion technique. In periportal cells the activities of alanine aminotransferase and pyruvate kinase were 29.4 and 18.7 mumol/min per mg of DNA respectively. The rate of gluconeogenesis was 0.402 mumol/min per mg of DNA. In perivenous cells the corresponding values were 9.55, 22.1 and 0.244 mumol/min per mg of DNA respectively. These data support the concept of a zonation of glucose metabolism within the microcirculatory unit of the liver, with the afferent part (periportal zone) having a 2-fold, more active gluconeogenesis than the efferent part (perivenous zone).     

Functional heterogeneity of periportal and perivenous hepatocytes

Periportal and perivenous hepatocytes differ in their content of many key enzymes and subcellular structures. The cells also receive different regulatory signals due to the gradients established during liver passage of oxygen, substrates and hormones. The signal heterogeneity is important not only for short-term regulation of metabolism but also for long-term control, i.e. the induction of liver cell heterogeneity. The zonal heterogeneity changes upon longer lasting physiological and pathological alterations of the metabolic situation such as starvation, diabetes or regeneration after partial hepatectomy; it develops only gradually during the first weeks of postnatal life. The model of 'metabolic zonation' proposes a functional specialization for the two zones: in the periportal zone oxidative energy metabolism with beta-oxidation and amino acid metabolism, ureagenesis, gluconeogenesis, cholesterol synthesis, bile formation and oxidation protection are the predominant activities, and in the perivenous zone glycolysis, liponeogenesis, ketogenesis, glutamine formation and biotransformation are the prevalent processes.     

Hypophysectomy does not alter the acinar zonation of gluconeogenesis or the mitochondrial redox state in rat liver.

The biochemical and functional heterogeneity of hepatocytes in different zones of the liver acinus may be related to the concentrations of hormones within the liver acinus. We examined the effects of hypophysectomy, which causes marked changes in plasma hormone levels and in activities of hepatic enzymes that are normally heterogeneously distributed, on the degree of metabolic zonation within the liver acinus. In hypophysectomized rats the activity of alanine aminotransferase was increased, but its normal zonation (predominance in the periportal zone) was preserved. The activity in cultured periportal and perivenous hepatocytes was increased by dexamethasone, but not by glucagon. Periportal hepatocytes from hypophysectomized rats expressed higher rates of gluconeogenesis in culture than did perivenous hepatocytes, irrespective of the absence or presence of dexamethasone, glucagon or insulin. Similar differences in rates of ketogenesis and in the mitochondrial redox state in response to glucagon were observed between periportal and perivenous hepatocytes from hypophysectomized rats as between cell populations from normal rats. Although hypophysectomy causes marked changes in hepatic enzyme activities, it does not alter the degree of zonation of alanine aminotransferase, gluconeogenesis or the mitochondrial redox state within the liver acinus.     

Zonation of glycogen and glucose syntheses, but not glycolysis, in rat liver.

We have investigated the cause of defective glycogen synthesis in hepatocyte preparations enriched with cells from the periportal or perivenous zones obtained by the methods of Lindros & Penttila [Biochem. J. (1985) 228, 757-760] and of Quistorff [Biochem. J. (1985) 229, 221-226]. A modified procedure which yields hepatocytes capable of consistent rates of glycogen synthesis is described, and the rates of glucose and glycogen syntheses and of glycolysis in hepatocytes from the two zones are compared. Glycogen synthesis in cells was greatly impaired by very low concentrations (0.01-0.05 mg/ml) of digitonin, which had little effect on glucose and protein syntheses and Trypan Blue exclusion. Cells exposed to such low concentrations of digitonin lose all their synthetic capacity and ability to exclude Trypan Blue when incubated with EGTA, which does not affect cells not exposed to digitonin. With a modified procedure based on this phenomenon, our study reveals that hepatocyte preparations enriched with cells from the periportal zone synthesized glucose from lactate and alanine at rates twice those by cells from the perivenous zone, whereas the rate of glycogen synthesis from C3 precursors in periportal cells was 4 times that in the perivenous preparations. With substrates entering the pathway at the triose phosphate level, gluconeogenesis in periportal-cell preparations was 20% higher, and glycogen synthesis was twice that in perivenous preparations. Glycolysis was studied by the formation of 3HOH from [2-3H]glucose, the yield of lactate, and the conversion of [14C]glucose into [14C]lactate. In cell preparations from both zones glycolysis by all criteria was negligible at 10 mM-glucose, but was substantial at higher concentrations. However, there was no difference between the zones. We confirm that the capacities for glucose and glycogen syntheses in periportal cells are higher than in perivenous cells, but that at physiological glucose concentrations there is negligible glycolysis in liver parenchyma in both zones. The metabolic pattern in the perivenous cells is not glycolytic.     

Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes.

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.     

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.     

Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture

Periportal and perivenous hepatocytes from rat liver were isolated by combined digitonin-collagenase perfusion, and gluconeogenesis, urea synthesis and fatty acid synthesis was measured both in freshly isolated cells and in primary culture. A periportal zonation of gluconeogenesis and urea synthesis of about 3 and 1.5 fold, respectively, was observed. This zonation persisted unchanged for 23 hours in culture under identical conditions of incubation for periportal and perivenous cells. Fatty acid synthesis was not zonated.     

Induction in primary culture of 'gluconeogenic' and 'glycolytic' hepatocytes resembling periportal and perivenous cells

Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1. Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity 'periportal'. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply 'perivenous'. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2. Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3. Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 muM dexamethasone) gluconeogenesis was 1.5-fold faster in the 'periportal' cells and glycolysis was 2.4-fold faster in the 'perivenous' cells. 4. Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in 'periportal' cells containing high phosphoenolpyruvate carboxykinase activity, not in the 'perivenous' cells with a low level of this enzyme. 5. A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in 'periportal' cells and glycolysis was eightfold faster in 'perivenous' cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.     

Zonation of the action of ethanol on gluconeogenesis and ketogenesis studied in the bivascularly perfused rat liver

Zonation of the actions of ethanol on gluconeogenesis and ketogenesis from lactate were investigated in the bivascularly perfused rat liver. Livers from fasted rats were perfused bivascularly in the antegrade and retrograde modes. Ethanol and lactate were infused into the hepatic artery (antegrade and retrograde) and portal vein. A previously described quantitative analysis that takes into account the microcirculatory characteristics of the rat liver was extended to the analysis of zone-specific effects of inhibitors. Confirming previous reports, gluconeogenesis and the corresponding oxygen uptake increment due to saturable lactate infusions were more pronounced in the periportal region. Arterially infused ethanol inhibited gluconeogenesis more strongly in the periportal region (inhibition constant = 3.99 ± 0.22 mM) when compared to downstream localized regions (inhibition constant = 8.64 ± 2.73 mM). The decrease in oxygen uptake caused by ethanol was also more pronounced in the periportal zone. Lactate decreased ketogenesis dependent on endogenous substrates in both regions, periportal and perivenous, but more strongly in the former. Ethanol further inhibited ketogenesis, but only in the periportal zone. Stimulation was found for the perivenous zone. The predominance of most ethanol effects in the periportal region of the liver is probably related to the fact that its transformation is also clearly predominant in this region, as demonstrated in a previous study. The differential effect on ketogenesis, on the other hand, suggest that the net effects of ethanol are the consequence of a summation of several partial effects with different intensities along the hepatic acini.