Expression and regulation of glycogen phosphorylase in preneoplastic and neoplastic hepatic lesions in rats

Glycogen phosphorylase (PHO) was demonstrated immunocytochemically and enzyme histochemically in cryostat sections of liver from rats treated for 7 weeks with N-nitrosomorpholine (120 mg/l and 200 mg/l drinking water) and from untreated controls. The activity and distribution of PHO protein were studied in normal liver and correlated with morphologically defined stages of hepatic tumour development. In normal liver the amount of enzyme protein, as visualized by the immunoperoxidase method using antibodies against phosphorylase, showed some heterogeneity within the liver lobule. The intralobular and intracellular distribution of PHO protein was the same as that of glycogen, namely coarse and granular in periportal hepatocytes and very fine in perivenular cells. In glycogen storage foci the amount of PHO protein was increased. In contrast, PHO activity was generally decreased. In other preneoplastic and neoplastic lesions such as mixed cell foci, neoplastic nodules and hepatocellular carcinomas, PHO protein was increased in all glycogen-loaded cells while PHO activity was reduced. In all glycogen-poor and basophilic cells, both PHO protein and PHO activity were decreased or absent. It was concluded that the decrease in PHO activity in glycogen storage foci was not the direct consequence of genetic changes leading to a loss in enzyme protein but was due to a defect in the cascade of phosphorylation processes resulting in active PHO. Alteration in gene expression leading to a loss of PHO protein was a late event in the process of hepatocarcinogenesis.     

Effect of partial hepatectomy on the glycogen level in hepatocytes in the portal and central lobule zones in cirrhotically-changed rat liver

Using cytophotometric method, the content of glycogen was studied in hepatocytes of the portal and central zones of a liver lobule in norm, in cirrhosis, and 1, 3, and 6 months after a partial hepatectomy of the normal and cirrhotic rat liver. As we showed earlier, glycogen content in cirrhotic liver hepatocytes rose 2-3-fold, along with obvious impairment of glycogen metabolic heterogeneity in these. In cirrhotic liver glycogen dominates in the central zone, whereas in norm more glycogen is observed in the portal one. The objective of this study was to find out to what degree a partial hepatectomy of cirrhotic liver may promote recovery of the metabolic glycogen heterogeneity in hepatocytes. Glycogen was determined in hepatocytes, using a quantitative variant of PAS-reaction on sections of the material obtained from serial supravital punctate liver biopsies. Glycogen amount in hepatocytes of different liver lobule zones was determined by an image analyzer technique that allows to bring together the cytophotometric analysis of the substance with its localization in a particular liver lobule. Results of these studies have shown that a partial hepatectomy of cirrhotic liver promotes restoration of the hepatocyte metabolic heterogeneity in the liver lobule.     

Long term phenobarbital administration does not promote the multiplication of hepatocytes replicating after single cyproterone acetate administration

Cyproterone acetate (CPA) is a synthetic antiandrogenic compound which is widely used in clinic but suspected to be hepatocarcinogenic. CPA is also mitogenic in rat liver. Using genetic labeling of dividing cells, we examined whether hepatocytes dividing in response to acute CPA administration could give rise to preneoplastic foci after administration of a tumor promoter: phenobarbital. CPA was administered orally to rats and dividing hepatocytes were genetically labeled using retroviral vectors carrying the beta-galactosidase gene. After labeling rats were given phenobarbital for 10 months and sacrificed. The presence of beta-galactosidase labeled hepatocytes as well as preneoplastic hepatocytes was assessed by immunohistochemistry. Genetic labeling of hepatocytes was obtained in all animals. At the end of phenobarbital administration, no hepatic tumors were observed. Preneoplastic foci were not increased in treated animals as compared to control rats. Moreover beta-galactosidase positive hepatocytes were never detected in preneoplastic foci. Finally, the size of the beta-galactosidase positive clusters was smaller in treated animals as compared to control rats. We conclude that acute CPA administration is not carcinogenic in rat liver and does not initiate preneoplastic hepatocytes capable to give rise to foci after phenobarbital promotion. Therefore the mitogenic property of CPA is distinct from its putative carcinogenic activity. Finally, analysis of the size of beta-galactosidase positive cells clusters demonstrate that phenobarbital does not induce hepatocyte proliferation in rats.     

Cytofluorimetric research on the content of glycogen and its fractions in the hepatocytes of patients with liver cirrhosis of different etiologies]

By cytofluorometric method, a study was made of the total glycogen and its two fractions in liver parenchymal cells both in the donors (20 men) and in patients with cirrhosis of different etiology (39 men). The examination was performed on preparations--smears of isolated hepatocytes, obtained from the live functional liver biopsies. The quantitative analysis has shown an increase in the total glycogen content in hepatocytes of patients with cirrhosis by 3 times compared to the norm, and this increase is independent on the etiology of liver cirrhosis. To study the mechanism of the discovered glycogenosis, the activity of key enzymes of glycogenolyses was determined. It was shown that glucose-6-phosphatase and glycogen-phosphorylase activity in the liver with cirrhosis was lower than in the norm. The most considerable changes were shown in hepatocytes of patients with liver cirrhosis in fractional glycogen composition and, even more significant, in the content of a hard soluble fraction. The hard soluble fraction portion was higher in hepatocytes of the patients with liver cirrhosis of alcohol etiology. The quantitative analysis of glycogen fraction contents in liver cells may be the best marker in the differential diagnosis of symptomless elapsing liver cirrhosis.     

Direct differentiation of human embryonic stem cells to hepatocyte-like cells exhibiting functional activities

The utilization of human hepatocytes for biomedical research, drug discovery, and treatment of liver diseases is hindered by the limited availability of donated livers and the variability of their derived hepatocytes. Human embryonic stem cells (hESCs) are pluripotent and provide a unique, unlimited resource for human hepatocytes. However, differentiation of hESCs to hepatocytes remains a challenge. We have developed a multistage procedure by which hESCs can be directly differentiated to hepatocyte-like cells without embryoid body formation and the requirement of sodium butyrate. The hESC-derived hepatocyte-like cells (HLCs) exhibited characteristic hepatocyte morphology, expressed hepatocyte markers, including alpha-fetoprotein, albumin, and hepatocyte nuclear factor 4alpha, and possessed hepatocyte-specific activities, such as p450 metabolism, albumin production, glycogen storage, and uptake and excretion of indocyanine green. Hepatocyte growth factor was found to play a positive role in promoting hepatocyte differentiation. Our differentiation system has shown that hESCs can be differentiated to hepatocyte-like cells capable of executing a range of hepatocyte functions. Therefore, it presents a proof-of-principle of potential applications of using the hESC-derived hepatocytes. Additionally, the hESC-derived HLCs provide a unique model to study the mechanisms involved in human hepatocyte differentiation and liver function.     

Histochemical identification of preneoplastic hepatocellular lesions in the human liver

The activity of gamma-glutamyltranspeptidase (GGT) as well as glycogen storage within hepatocytes from cirrhotic human liver were investigated by histochemical means. In 4 cases out of 9, many enzyme-altered, PAS positive dysplastic 'foci' were seen. Such lesions may be considered as possible precursors of hepatoma, as already reported in rat liver during chemical carcinogenesis. Among the preneoplastic liver markers, GGT positivity seems the most useful one to be added to the routine histological examination of liver biopsies.     

Morphometric and cytophotometric nuclear analysis of altered hepatocyte foci induced by N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1) in liver of Wistar rats

The progressive morphological changes in the liver during neoplastic transformation have been studied by histological, cytophotometric and morphometric methods in male Wistar rats treated with two carcinogens: N-nitrosomorpholine (NNM) and aflatoxin B1 (AFB1). Cytophotometric and morphometric analysis of hepatocyte nuclei using Feulgen-stained tissue sections were performed in morphologically normal hepatic parenchyma and in early preneoplastic foci composed of altered hepatocytes. Foci of clear cells, mixed cells and large basophilic cells possessed a ploidy distribution similar to the surrounding non-transformed parenchyma, while the small hyperbasophilic cell foci were predominantly diploid. These findings confirm that the foci composed of PAS-negative, small hyperbasophilic cells with an unique diploid content may represent one of the earliest stages in the neoplastic transformation.     

Cell density dependent morphological changes in adult rat hepatocytes during primary culture

In order to gain morphological insights about the cell density dependency, hepatocytes cultured at a low cell density (less than about 0.1 X 10(5) nuclei (cm2)-1) and at a high cell density (greater than about 1 X 10(5) nuclei (cm2)-1) were examined ultrastructurally 24 h after plating (just prior to the beginning of DNA synthesis). The results were as follows: (i) glycogen rosettes disappeared completely in low density culture as compared with sections from an intact liver. In contrast, glycogen rosettes were still present in high density culture. (ii) Polysomes seemed increased in low density culture in comparison with those seen in sections from an intact liver and from the high density culture. (iii) In low density culture, the shape of mitochondria deviated from that of hepatocytes in an intact liver and the mitochondria often lost a characteristic close contact with rough endoplasmic reticulum (rough ER). (iv) In low density culture, bundles of filamentous structure were detected, which were not found in an intact liver or high density culture. The following features were found only in high density culture; (v) numerous villous cytoplasmic protrusions developed along the area facing adjacent cells, and seemed to intertwine with each other, and (vi) between the hepatocytes, only abortive junctions were found. These results indicate that the hepatocytes cultured at a low density express most of the characteristics of the hepatocytes in a regenerating liver and the features of the cells cultured at a high density are very similar to those of the hepatocytes in sections from an intact liver.     

Zonal expression of StARD1 and oxidative stress in alcoholic-related liver disease

Alcoholic-related liver disease (ALD) is one of the leading causes of chronic liver disease and morbidity. Unfortunately, the pathogenesis of ALD is still incompletely understood. StARD1 has emerged as a key player in other etiologies of chronic liver disease, and alcohol-induced liver injury exhibits zonal distribution. Here, we report that StARD1 is predominantly expressed in perivenous (PV) zone of liver sections from mice-fed chronic and acute-on-chronic ALD models compared to periportal (PP) area and is observed as early as 10 days of alcohol feeding. Ethanol and chemical hypoxia induced the expression of StARD1 in isolated primary mouse hepatocytes. The zonal-dependent expression of StARD1 resulted in the accumulation of cholesterol in mitochondria and increased lipid peroxidation in PV hepatocytes compared to PP hepatocytes, effects that were abrogated in PV hepatocytes upon hepatocyte-specific Stard1 KO mice. Transmission electron microscopy indicated differential glycogen and lipid droplets content between PP and PV areas, and alcohol feeding decreased glycogen content in both areas while increased lipid droplets content preferentially in PV zone. Moreover, transmission electron microscopy revealed that mitochondria from PV zone exhibited reduced length with respect to PP area, and alcohol feeding increased mitochondrial number, particularly, in PV zone. Extracellular flux analysis indicated lower maximal respiration and spared respiratory capacity in control PV hepatocytes that were reversed upon alcohol feeding. These findings reveal a differential morphology and functional activity of mitochondria between PP and PV hepatocytes following alcohol feeding and that StARD1 may play a key role in the zonal-dependent liver injury characteristic of ALD.     

中国大鲵肝脏的超微结构

应用透射电镜对中国大鲵的肝脏进行了超微结构研究.观察表明,大鲵肝不具肝小叶,与其他脊椎动物有所不同.肝细胞含有单个卵圆形的核;细胞质内含有粗面内质网、高尔基囊泡、线粒体、糖原颗粒和脂滴等细胞器和内含物.胆小管由两个相邻肝细胞质膜凹陷围成,而肝血窦则由内皮细胞的胞质构成.胆小管腔和窦周隙内浸润许多由肝细胞发出的微绒毛结构.还发现了枯否氏细胞和贮脂细胞.还讨论了中国大鲵肝脏的一般形态结构特点.     

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.     

Morphology and function of cultured hepatocytes isolated from rats with experimental toxic hepatitis

The goal of the study was to examine the morphology and function of primary hepatocytes isolated from rats with toxic hepatitis induced by a combination of CCl₄ and ethanol. Fluorescent immunocytochemical analysis demonstrated that normal and pathologic hepatocytes in culture formed actin cytoskeleton, cell-cell, and cell-matrix contacts. In this investigation, the morphology of mitochondria and their localization in hepatocytes was assayed with Rhodamine 123 staining. Glycogen and DNA contents in cultured hepatocytes were determined by fluorescent cytometry. It was found that the ploidy of hepatocytes isolated from normal and injured livers were different. Cells were maintained in culture for 5 days and no changes in ploidy distribution were observed. The glycogen content was 50% higher in the experimental group than the control one; it was decreased in control and cirrhotic hepatocytes treated with collagenase. Intact hepatocytes accumulated glycogen within 3 days; the glycogen level remained low in pathologic hepatocytes.     

The influence of hepatoprotector 2-ethylthiobenzimidazole hydrobromide (bemithyl) on the content of glycogen in cirrhotic rat liver hepatocytes located in various microenvironments

Using absorption and fluorescent cytophotometry methods, glycogen contents were studied in hepatocytes located in liver lobules and in hepatocytes, which make the general population of these cells in normal and cirrhotic rat liver. In cirrhosis, the content of glycogen in hepatocytes located in lobules obviously rises in comparison with the norm, but to a lesser degree, than in hepatocytes making the general population of these cells in cirrhotic liver. The content of glycogen in hepatocytes, located in lobules of pathologically changed liver in bemithyl treated rats, did not differ from the norm. At the same time, the glycogen content in hepatocytes, representing the general population of these cells in cirrhotically altered bemithyl injected rat liver, remained higher than in the norm. The data obtained indicate that distinctions in particular cell microinvironment, obviously present in cirrhotic liver, render essential influence on hepatocyte functional activity.     

Study of carbohydrate metabolism in glycogen storing cell lines derived from cultured rat hepatocytes

Some aspects of carbohydrate metabolism were investigated in three non-malignant, glycogen storing, cell lines derived from a primary culture of rat hepatocytes, and in the Morris hepatoma 3924 cells. The three cell lines show biochemical alterations which are, to a large extent, similar to those found in the hepatoma cells: increased activity of glycolytic enzymes and decreased activity of gluconeogenetic enzymes. An increase of glucose-6-phosphate dehydrogenase activity is also found. The three cell lines, as the Morris hepatoma cells, actively convert glucose into lactate under the in vitro conditions of culture. Fructose is not taken up as quickly as glucose and galactose is not metabolized. As compared with normal hepatocytes, the three cell lines have altered metabolism and growth behaviour. They largely resemble the preneoplastic cells appearing in rat liver at the early stages of experimental carcinogenesis.     

Cytofluorimetric research on the glycogen content of the hepatocytes in patients with various forms of alcoholic liver lesions

By cytofluorometry employing the cytofluorometric PAS reaction, a study was made of the total glycogen and of its two fractions in liver parenchymal cells, both in the norm and in patients with chronic alcoholism (alcoholic steatosis, chronic alcoholic hepatitis, and mixed forms of alcoholic-viral hepatitis, viral hepatitis with steatosis and also viral hepatitis). The examination was performed on preparations-smears of isolated hepatocytes, obtained from the live puncture liver biopsies. The quantitative analysis has shown the increase in the total glycogen content in hepatocytes of patients with alcoholic hepatitis in comparison with the norm and with chronic viral hepatitis. The transition from a reverse stage--alcoholic steatosis--to alcoholic hepatitis was accompanied by a sharp increase in the total glycogen content and by an obvious change in the ratio of glycogen fractions, towards the hard soluble fraction in liver cells. The quantitative analysis of glycogen fractions in liver cells of patients with chronic alcoholic disease may be an appreciated marker of differential diagnostics of different stages and forms of alcoholic liver disease.     

Effects of adrenalectomy on hormone action on hepatic glucose metabolism. Impaired glucagon activation of glycogen phosphorylase in hepatocytes from adrenalectomized rats.

The effects of adrenalectomy on glucagon activation of liver glycogen phosphorylase and glycogenolysis were studied in isolated hepatocytes. Adrenalectomy resulted in reduced responsiveness of glycogenolysis and phosphorylase to glucagon activation. Stimulation of cAMP accumulation and cAMP-dependent protein kinase activity by glucagon was unaltered in cells from adrenalectomized rats. Adrenalectomy did not alter the proportion of type I and type II protein kinase isozymes in liver, whereas this was changed by fasting. Activation of phosphorylase kinase by glucagon was reduced in hepatocytes from adrenalectomized rats, although the half-maximal effective concentration of glucagon was unchanged. No difference in phosphorylase phosphatase activity between liver cells from control and adrenalectomized rats was detected. Glucagon-activated phosphorylase declined rapidly in hepatocytes from adrenalectomized rats, whereas the time course of cAMP increase in response to glucagon was normal. Addition of glucose (15 mM) rapidly inactivated glucagon-stimulated phosphorylase in both adrenalectomized and control rat hepatocytes. The inactivation by glucose was reversed by increasing glucagon concentration in cells from control rats, but was accelerated in cells from adrenalectomized rats. It is concluded that impaired activation of phosphorylase kinase contributes to the reduced glucagon stimulation of hepatic glycogenolysis in adrenalectomized rats. The possible role of changes in phosphorylase phosphatase is discussed.     

Cytofluorometric study of the glycogen content in DNA-synthesizing and -nonsynthesizing hepatocytes in the regenerating liver

Using a combined cytochemical method that allows to determine glycogen, DNA and 3H-thymidine label in the same cell, glycogen amounts were measured both in 3H-TdR-marked and non-marked hepatocytes of the regenerating 3H-thymidine. During mitosis, the glycogen amount is reduced if compared with that in cells being in presynthetic phase. It is proposed that the decrease in glycogen content in the regenerating liver may partly depend on energetic expenses of cells that started DNA synthesis and mitotic division. The phenotypic expression of genes responsible for glycogen synthesis and splitting in di-, tetra- and octoploid hepatocytes of the regenerating liver liver was proportional to the corresponding values.     

Adenovirus-mediated transfer of the muscle glycogen phosphorylase gene into hepatocytes confers altered regulation of glycogen metabolism.

The muscle isozyme of glycogen phosphorylase is potently activated by the allosteric ligand AMP, whereas the liver isozyme is not. In this study we have investigated the metabolic impact of expression of muscle phosphorylase in liver cells. To this end, we constructed a replication-defective, recombinant adenovirus containing the muscle glycogen phosphorylase cDNA (termed AdCMV-MGP) and used this system to infect hepatocytes in culture. AMP-activatable glycogen phosphorylase activity was increased 46-fold 6 days after infection of primary liver cells with AdCMV-MGP. Despite large increases in phosphorylase activity, glycogen levels were only slightly reduced in AdCMV-MGP-infected liver cells compared to uninfected cells or cells infected with wild-type adenovirus. The lack of correlation of phosphorylase activity and glycogen content suggests that the liver cell environment can inhibit the muscle phosphorylase isozyme. This inhibition can be overcome, however, by addition of carbonyl cyanide m-chlorophenylhydrazone (CCCP), which increases AMP levels by 30-fold and causes a much larger decrease in glycogen levels in AdCMV-MGP-infected cells than in uninfected or wild-type adenovirus-infected controls. CCCP treatment also caused a preferential decrease in glycogen content relative to glucagon treatment in AdCMV-MGP-infected hepatocytes (74% versus 11%, respectively), even though the two drugs caused equal increases in phosphorylase a activity. Introduction of muscle phosphorylase into hepatocytes therefore confers a capacity for glycogenolytic response to effectors that is not provided by the endogenous liver phosphorylase isozyme. The remarkable efficiency of adenovirus-mediated gene transfer into primary hepatocytes and the demonstration of altered regulation of glycogen metabolism as a consequence of expression of a non-cognate phosphorylase isozyme may have implications for gene therapy of glycogen storage diseases.     

Fractionation of Rat Hepatocyte Subpopulations with Varying Metabolic Potential, Proliferative Capacity, and Retroviral Gene Transfer Efficiency

The liver contains hepatocytes with varying ploidy and gene expression. To isolate cells on the basis of ploidy for analyzing mechanisms concerning cell proliferation and differentiation, we used Percoll gradients to separate F344 rat hepatocyte subpopulations. Specific fractions were enriched in polyploid (H2 fraction) or diploid (H3 and H4 fractions) hepatocytes containing glycogen and glucose-6-phosphatase. H4 cells were relatively smaller with greater nuclear/cytoplasmic ratios, less complex cytoplasm, and higher serum albumin or ceruloplasmin biosynthetic rates. H2 fraction cells were larger with lesser nuclear/cytoplasmic ratio, more complex cytoplasm, and more cytochrome P450 activity. Phenotypic marking showed that H4 cells originated in zone one and H2 cells in zones two or three of the liver lobule. H4 cells showed much greater mitogenic responsiveness to human hepatocyte growth factor. Retroviral gene transfer, which requires both viral receptors and cellular DNA synthesis, was significantly more efficient in H4 cells. The findings indicated thatsmalldiploid andlargepolyploid hepatocytes show unique biological differences. The ability to isolate hepatocytes of varying maturity is relevant for mechanisms concerning liver growth control and hepatic gene expression.