Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Insulin regulation of glycogen synthase phosphatase in primary cultures of hepatocytes

Activation of glycogen synthase in the perfused rat liver is defective in severely diabetic rats. In the present study, activation of glycogen synthase by glucose and increased incorporation of [14C]glucose into glycogen by insulin are defective in hepatocytes isolated from alloxan diabetic rats. Acute activation of glycogen synthase in hepatocytes isolated from diabetic rats was restored by treatment of the rats with insulin in vivo. Restoration of synthase activation was not achieved by incubation of hepatocytes in the presence of insulin in vitro for up to 12 h. When isolated hepatocytes from diabetic rats were placed in primary culture in a serum-free defined medium over a 3-day period, glycogen synthesis was partially restored by cortisol and triiodothyronine and dramatically increased by insulin. Concomitant with restoration of [14C]glycogen synthesis was an insulin-mediated increase in glycogen synthase I and synthase phosphatase activity. Restoration of regulation of glycogen synthesis in primary cultures of hepatocytes from diabetic rats by insulin required the presence of cortisol and triiodothyronine. Primary cultures of hepatocytes from normal rats did not require triiodothyronine for insulin to effect glycogenesis over a 3-day period. These data demonstrate that insulin acts in a chronic manner in concert with other hormones to control synthase phosphatase activity, an effect which may be influencing acute control of hepatic glycogen synthesis.     

Biochemical and ultrastructural examination of cryopreserved hepatocytes in rat

We investigated the ultrastructure and metabolic capabilities of isolated rat hepatocytes after cryopreservation using 1.5 M Me₂SO as protectant and a slow cool/fast thaw regime. Ultrastructural assessment of the cryopreserved population revealed only approximately 10% of cells with normal morphology. Conjugation of bilirubin by the cryopreserved cells was reduced to 20% of that seen in unfrozen hepatocytes and there was a net loss of glycogen measured in cryopreserved cells incubated in conditions which stimulated glycogen synthesis by unfrozen cells. These results are in contrast to other reports in which cryopreserved hepatocytes have been successfully used for transplantation to reverse hepatic insufficiency.     

Effect of "vilon" on cirrhotically changed rat liver. Liver regeneration, and status of glycogen-forming function of hepatocytes

Effects of a dipeptide preparation "Vilon" on rehabilitation of functional activity of hepatocytes and regeneration of the cirrhotically altered rat liver were studied. The liver cirrhosis was produced by poisoning of rats for 4 months with carbon tetrachloride (CCl4). On the end of the poisoning with CCl4, one group of animals was not submitted to any further actions, whereas animals of the other group were injected "Vilon" (1.7 micrograms/kg) daily for 5 days. On smears of isolated hepatocytes, contents of total glycogen (TG), and its labile and stable fractions (LF and SF) were determined in addition to cell ploidy levels and the total protein content. In liver homogenates, activities of glucose-6-phosphatase (G6P), glycogen synthase (GS), and glycogen phosphorylase (GP) were measured. In 2 weeks after the drug application, G6P activity being reduced in cirrhosis 1.2 times, elevated under effect of "Vilon". In non-treated rats the contents of TG and its fractions and of G6P activity remained at the level characteristic of the cirrhotic liver prior to "Vilon" administration. In both groups of rats, GP and GS activities in the cirrhotically altered liver did not differ from their control values throughout the entire experiment. "Vilon" has been shown to exert a weak stimulating effect on regeneration of the cirrotically altered rat liver: in hepatocytes of the second group of rats the total protein content and ploidy levels were higher than those in the first group by 4.7 and 11.5%, respectively.     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of glucose in cells from diabetic rats involves protein synthesis

Defective acute regulation of hepatic glycogen synthase by glucose and insulin, caused by severe insulin deficiency, can be corrected in adult rat hepatocytes in primary culture by inclusion of insulin, triiodothyronine, and cortisol in a chemically defined serum-free culture medium over a 3-day period (Miller, T. B., Jr., Garnache, A. K., Cruz, J., McPherson, R. K., and Wolleben, C. (1986) J. Biol. Chem. 261, 785-790). Using primary cultures of hepatocytes isolated from normal and diabetic rats in the same serum-free chemically defined medium, the present study addresses the effects of cycloheximide and actinomycin D on the chronic actions of insulin, triiodothyronine, and cortisol to facilitate the direct effects of glucose on the short-term activation of glycogen synthase. The short-term presence (1 h) of the protein synthesis blockers had no effect on acute activation of glycogen synthase by glucose in primary hepatocyte cultures from normal rats. Normal cells maintained in the presence of cycloheximide or actinomycin D for 2 and 3 days exhibited unimpaired responsiveness to glucose activation of synthase. The protein synthesis inhibitors were effective at blocking the restoration of glucose activation of synthase in diabetic cells in media which restored the activation in their absence. Restoration of glycogen synthase phosphatase activity by insulin, triiodothyronine, and cortisol in primary cultures of diabetic hepatocytes was also blocked by cycloheximide or actinomycin D. These data clearly demonstrate that restoration of acute glycogen synthase activation by glucose and restoration of glycogen synthase phosphatase activity in primary cultures of hepatocytes from adult diabetic rats are dependent upon the synthesis of new protein.     

Glycogen distribution in rat hepatocyte populations after a single exposure to 4-dimethylaminoazobenzine and subsequent injections of phenobarbital

7 day after a single interperitoneal injection of carcinogen 4-dimethylaminoazobenzen (DAB), a little number of cells with high glycogen contents was found in parallel with a decreased glycogen content in most isolated hepatocytes. 1.5 months after DAB injection, the normal distribution of glycogen content was seen restored in hepatocytes. The treatment of rats with phenobarbital (6 PhB injections 7 days after DAB application) blocked the restoration of the normal glycogen distribution. 2 months after the last PhB injection (3 months after DAB injection) an increased glycogen content was found in the smallest hepatocytes.     

Critical evaluation of methods used for the isolation of rat liver hepatocytes for metabolic studies.

Hepatocytes were isolated from normal fed, fasted and alloxan diabetic animals. The best cell preparations were obtained by using low concentrations of collagenase (10–20 mg) and exposing the liver for a very short period of time (10–15 min). Addition of hyaluronidase significantly decreased the glycogen content of the isolated hepatocytes. Glucagon (10⁻¹²M) stimulated glycogenesis in hepatocytes containing high glycogen whereas, in cells containing low glycogen much higher concentration of glucagon was needed (10⁻⁹M). Addition of insulin (100 μunits) stimulated both glycogen and protein synthesis in isolated hepatocytes containing high glycogen. Under these conditions glycogen synthase activity was stimulated by 40%. Incorporation of ¹⁴C phenylalanine into protein was linear for only 3–4 hr in cells containing low glycogen whereas, in cells containing high glycogen incorporating was linear for 8–10 hr. These studies suggest that intracellular glycogen plays an important role in the hormonal regulation of metabolism in hepatocytes.     

Cytofluorimetric study of the glycogen content in hepatocytes of varying ploidy in adult rats]

A combined method, that allows measuring glycogen and DNA contents in one of the same cell, was applied for quantitative determination of these in mono- and binucleate hepatocytes with different ploidy obtained from adult rats. The mean glycogen content was shown to increase proportionally to the genome number within the changes of the hepatocyte ploidy from 2 to 8c.     

不同粘附剂对大鼠肝实质细胞体外培养的影响

摘要 目的：比较鼠尾胶与多聚赖氨酸对大鼠肝实质细胞体外培养的影响。方法：分别采用鼠尾胶与多聚赖氨酸包被同一块培养板,然后将从大鼠肝脏中分离出来的肝实质细胞,接种到包被好的培养板中。于接种前（0 h）,接种后体外培养24 h、72 h显微镜下观察细胞贴壁与形态变化情况。结果：接种前（0 h）可见新鲜分离的肝实质细胞呈圆形,明亮,有立体感,轮廓完整,层次清楚;体外培养24 h后两种粘附剂包被的同一块培养板中,均可观察到肝实质细胞正常生长,且细胞形态由圆形转变为多角形,并且融合聚集,胞体变平整,贴壁情况区别不大;培养72 h后细胞间开始出现连接,大部分肝细胞呈现出双核或多核,并且多聚赖氨酸包被的培养板中可见大量肝细胞呈岛屿状,已完全贴壁于培养板上。结论：多聚赖氨酸作为包被材料更有利于肝实质细胞贴壁生长以及保持细胞固有形态。     

长爪沙鼠肝细胞体外分离培养体系的建立

目的建立长爪沙鼠原代肝细胞分离培养体系。方法以雄性长爪沙鼠为供体,采用组织消化法和Seglen两步灌流法分离肝细胞,以台盼蓝染色检测细胞得率和活率,过碘酸-希夫氏反应(PAS)鉴定肝细胞,倒置显微镜观察肝细胞形态变化,并使用含有多种细胞因子的培养基维持培养。结果组织消化法和Seglen两步灌流法平均每只长爪沙鼠可分别获得肝细胞(1.33±0.34)×107个、(3.97±1.15)×107个,细胞活率分别为(29.4±6.05)%、(80.3±4.56)%,这两种方法在细胞得率及活率方面存在显著差异。肝细胞内因有大量的糖原颗粒,经PAS染色后被染成红色。结果表明肝细胞在贴壁后72 h内,肝细胞形态发生显著变化。结论采用胶原酶经肝门静脉灌流分离肝细胞是一种高效获得肝细胞的方法。各种细胞因子有利于维持肝细胞在体外的生长分化,长爪沙鼠原代肝细胞分离培养体系的建立将为肝脏相关疾病研究和防治药物的开发提供技术支持。     

Morphometric and cytochemical characteristics of the hepatocytes isolated from the liver of rats after a single exposure to 4,dimethylaminoazobenzene

Hepatocytes have been characterized that were isolated with sodium perchlorate from the livers of rats both intact and given a single injection of a carcinogen--4-dimethylaminoazobenzene. A decreased number of big hepatocytes (600-900 mkm2) and the appearance of small hepatocytes (75-120 mkm2) were observed 7 days after the carcinogen injection. An excessive accumulation of glycogen was shown in certain hepatocytes. By the 30th day the picture was nearly normal.     

Studies on the differential response to insulin on the stimulation of amino acid incorporation into protein in isolated hepatocytes containing different levels of glycogen.

Effect of insulin on amino acid incorporation into protein by isolated rat liver hepatocytes was studied. A two to three-fold increase in the incorporation of U-¹⁴C-Leucine and U-¹⁴C-Phenylalanine into protein by insulin (100 μUnits) was observed in isolated hepatocytes containing high glycogen. This effect was abolished by the addition of glucagon (3 × 10^?6M). No stimulation in amino acid incorporation by insulin was observed when isolated hepatocytes contained low or no glycogen. Electron micrographs of incubated cells show that in the presence of insulin more normal parallel strands of polyribosomes are maintained as compared to control cell preparation.     

Microarray analysis of gene expression of mouse hepatocytes of different ploidy

Polyploidisation in hepatocytes has been associated with many physiologic and pathologic processes such as proliferation, metabolism, regeneration, aging, and cancer. We studied gene expression patterns in hepatocytes of different ploidy. Primary hepatocytes were obtained from mice of different ages: young (4-6 weeks old), adult (8-10 weeks old), and older (22-24 weeks old). Diploid (2N), tetraploid (4N), and octoploid (8N) hepatocytes were isolated for studies using a high-density mouse genome microarray. No major changes of gene expression patterns between hepatocytes of different ploidy were found. Fifty genes were identified as differentially expressed in the diploid and tetraploid populations, but the changes were less than twofold either way. Four genes (Gas2, Igfbp2, Nr1i3, and Ccne2) were differentially expressed in tetraploid and octoploid cells. This was confirmed in two age groups, "adult" and "older," but once again the factors were less than twofold and the expressions of Gas2 and Igfbp2 were more different between age groups than between ploidy classes. Our results show that polyploid hepatocytes are stable and "normal" without aberrant gene expression, unlike what is thought for cancer cells. By contrast to megakaryocytes, hepatocyte polyploidisation is not a differentiation step associated with major changes in gene expression. Our data support the hypothesis that hepatocyte polyploidisation is a protective mechanism against oxidative stress that occurs via a controlled process throughout growth and aging where binucleation is important.     

Glycogen metabolism in adult rat liver parenchymal cell primary cultures.

Parenchymal cells were isolated from adult rat liver with an enzyme perfusion technique. The single-cell suspension, representing 40-50% of the liver's hepatocytes was suspended in medium and maintained in primary culture for up to four days. The cells were found to carry out glycogen synthesis for the first eight hours in culture after which time the accumulated glycogen was gradually degraded. The ability of the liver cell cultures to accumulate glycogen was found to be dependent upon the metabolic state of the animal prior to cell isolation. Cells prepared during the feeding period from animals on the 8+16 feeding schedule had markedly different capacities for glycogen accumulation. Changes in glycogen metabolism were found to be due, in part, to changes in the fraction of cells involved in metabolism at any given time. High concentrations of glucose stimulated the cells to deposit glycogen but the response was reduced the longer the cells were in culture over a 3-day period. This loss of glycogen synthesizing capacity appears to be due to a decrease in glycogen synthetase activity. The activities of pyruvate kinase, hexokinase and aldolase also decrease during the culture period.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

Ploidy class-dependent metabolic changes in rat hepatocytes after partial hepatectomy

Glucose-6-phosphate dehydrogenase (G6PDH), succinate dehydrogenase (SDH) activity and the single-stranded RNA (ssRNA) content of isolated hepatocytes of different ploidy classes from adult male rats have been studied after partial hepatectomy using quantitative cytochemical means. The SDH activity and ssRNA content in all classes of hepatocytes are decreased during the first hours after operation followed by an increase above control values. The increase of both SDH activity and ssRNA content is significant only in the mononuclear diploid (MD) cells but not in the hepatocytes of higher ploidy classes and is related with the mitotic wave at 32 h after hepatectomy. After the mitotic wave, the values quickly return to normal levels. The G6PDH activity does not show any significant change in hepatocytes other than MD cells. In MD cells the G6PDH activity is elevated on a highly significant level up to a maximum value of 3.5 times the control value at 48 h after operation. The G6PDH activity in MD cells is returned to normal values within 14 days after operation. It is concluded that: 1. The MD cells show a distinct metabolic behaviour due to their function as stem cells of liver parenchyma and retain at least some of their fetal characteristics. 2. G6PDH activity is not a transformation-linked discriminant for neoplastic metabolism.     

Enhancement of glycogen concentrations in primary cultures of rat hepatocytes exposed to glucose and fructose.

Glycogen synthesis in isolated hepatocytes can occur from glucose both by a direct mechanism and by an indirect process in which glucose is first metabolized to C3 intermediates before use for glycogenesis via gluconeogenesis. We studied the incorporation into glycogen of glucose and the gluconeogenic substrate, fructose, in primary cultures of hepatocytes from fasted rats. In the presence of insulin, both glucose and fructose promoted net deposition of glycogen; however, fructose carbon was incorporated into glycogen to a greater extent than that from glucose. When glucose and fructose were administered simultaneously, the glycogenic utilization of glucose was stimulated 2-3-fold, and that of fructose was increased by about 50%. At constant hexose concentrations, the total incorporation of carbon, and the total accumulation of glycogen mass, from glucose and fructose when present together exceeded that from either substrate alone. Fructose did not change the relative proportion of glucose carbon incorporated into glycogen via the indirect (gluconeogenic) mechanism. The synergism of glucose and fructose in glycogen synthesis in isolated rat hepatocytes in primary culture appears to result from a decrease in the rate of degradation of newly deposited glycogen, owing to (i) decreased amount of phosphorylase a mediated by glucose and (ii) noncovalent inhibition of residual phosphorylase activity by some intermediate arising from the metabolism of fructose, presumably fructose 1-phosphate.     

无血清培养高密度乳猪肝细胞的形态和功能变化

本文研究了无血清培养高密度猪肝细胞的形态和功能变化。将分离的肝细胞以高密度(1×10~7/ml)培养在含激素、多种生长因子和营养成分的无血清培养基中,动态观察培养7天中肝细胞形态、活率、蛋白质合成功能、G-6-Pase活性、安定转化功能及LDH含量;同时以无血清培养低密度(5×10~5/ml)肝细胞作为对照组。研究结果表明:高密度培养的 肝细胞各项功能较低密度培养的肝细胞为低;高密度培养的肝细胞的形态、蛋白质合成功能在培养7天中保持稳定;活率随着培养时间的延长而下降,但均高于90％;安定转化功能在培养第2、3天最强;G-6-Pase活性在培养1天后明显下降,然后维持在较低水平;LDH含量在第1、2、3天较高。     

Primary rat hepatocyte culture on 3D nanofibrous polymer scaffolds for toxicology and pharmaceutical research

Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two‐dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell‐specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three‐dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen‐coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver‐cell‐specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver‐specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real‐time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well‐preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF‐4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re‐established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO‐1‐positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes. Biotechnol. Bioeng. 2011; 108:141–150. © 2010 Wiley Periodicals, Inc.