Isolation and subfractionation on ficoll gradients of adult rat hepatocytes. Size, morphology, and biochemical characteristics of cell fractions

The recirculating perfusion of adult rat liver with a Ca-++-free Hanks' solution produces a release of the adhesiveness of cells and a cleaving of the desmosomes. The addition of collagenase and hyaluronidase to the perfusion medium leads to complete dissociation of the liver tissue into a mixture of isolated cells and cell cords in which the hepatocytes remain connected with specific junctional differentiations, namely the gap and tight junctions. Individual cells are released by submitting the suspension of cell trabeculae to a gentle rolling. The gap junctions are ruptured at least in one of the two adjacent cells and remain generally attached to the other cell taking with them a small portion of cytoplasm. This technique of isolation of hepatocytes yields about 60-65% of the parenchymal cells contained in a liver; endothelial cells and other cells of the connective tissue are not recovered. The ultrastructural preservation of the isolated hepatocytes is excellent and the glucose-6-phosphatase activity, confined to the endoplasmic reticulum, appears unaltered in most cells. Protein, DNA and RNA recovery in the preparations of isolated hepatocytes is satisfactory, amounting to 70% of that found in liver homogenate; glycogen, the most labile component examined, is partly lost or degraded during the manipulations. Cell diameters measured by different methods confirm the preservation of the original volume of the in situ hepatocytes and the presence of more than one type of parenchymal cell. By submitting this heterogeneous cell population to an isopycnic density gradient centrifugation, two types of hepatocytes can be distinguished: the light hepatocytes, with a mean diameter of 20.5 mum and a mean density of 1.10, are characterized by an extended smooth- walled endoplasmic reticulum entrapping dispersed alpha-glycogen particles; the heavy hepatocytes, with a mean diameter of 19.0 mum and a mean density of 1.14, present a relatively reduced compartment of smooth endoplasmic reticulum, but large accumulations of glycogen. It is suggested that the cell fraction of low density is enriched in centrolobular cells and the high density fraction in perilobular hepatocytes.     

Effect of ischemia-reperfusion on the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity in human liver allograft.

In order to examine glucose metabolism in liver grafts after cold ischemia and reperfusion, the heterogeneous lobular distribution pattern of glycogen content and glucose-6-phosphatase activity was studied using histochemical methods. The characteristic heterogeneous lobular distribution pattern of glycogen and glucose-6-phosphatase was maintained after preservation and reperfusion. However, it appeared that glycogen content decreased in both periportal and centrilobular hepatocytes after reperfusion. The glycogen decrease was higher in periportal hepatocytes. Glucose-6-phosphatase activity was maintained after reperfusion in most of the cases in periportal hepatocytes. In centrilobular hepatocytes, more cases showed a decrease in enzyme activity. It is suggested that ischemia-reperfusion mainly affects the glycogen content in both periportal and centrilobular hepatocytes and that centrilobular glucose-6-phosphatase activity is more sensitive to ischemia-reperfusion injury than periportal hepatocytes.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : I. Glucose-6-Phosphatase Distribution In Situ

The distribution of glucose-6-phosphatase activity in rat hepatocytes during a period of rapid endoplasmic reticulum differentiation (4 days before birth-1 day after birth) was studied by electron microscope cytochemistry. Techniques were devised to insure adequate morphological preservation, retain glucose-6-phosphatase activity, and control some other possible artifacts. At all stages examined the lead phosphate deposited by the cytochemical reaction is localized to the endoplasmic reticulum and the nuclear envelope. At 4 days before birth, when the enzyme specific activity is only a few per cent of the adult level, the lead deposit is present in only a few hepatocytes. In these cells a light deposit is seen throughout the entire rough-surfaced endoplasmic reticulum. At birth, when the specific activity of glucose-6-phosphatase is approximately equal to that of the adult, nearly all cells show a positive reaction for the enzyme and, again, the deposit is evenly distributed throughout the entire endoplasmic reticulum. By 24 hr postparturition all of the rough endoplasmic reticulum, and in addition the newly formed smooth endoplasmic reticulum, contains heavy lead deposits; enzyme activity at this stage is 250% of the adult level. These findings indicate that glucose-6-phosphatase develops simultaneously within all of the rough endoplasmic reticulum membranes of a given cell, although asynchronously in the hepatocyte population as a whole. In addition, the enzyme appears throughout the entire smooth endoplasmic reticulum as the membranes form during the first 24 hr after birth. The results suggest a lack of differentiation within the endoplasmic reticulum with respect to the distribution of glucose-6-phosphatase at the present level of resolution.     

Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics

The enzyme glucose-6-phosphatase (G-6-Pase) catalyzes the hydrolysis of glucose-6-phosphate (G-6-P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G-6-Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three-part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G-6-Pase. The assessment of G-6-Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (P_i) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G-6-Pase with potential clinical importance. The format of the three-part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease.     

The molecular basis of the type 1 glycogen storage diseases.

Microsomal glucose-6-phosphatase catalyses the last step in liver glucose production. Glucose-6-phosphatase deficiency, now termed type 1 glycogen storage disease, was first described almost 40 years ago but until recently very little was known about the molecular basis of the various type 1 glycogen storage diseases. Recently we have shown that at least six different proteins are needed for normal glucose-6-phosphatase activity in liver. Four of the proteins have been purified and three cloned. Study of the type 1 glycogen storage diseases has stimulated investigations of the mechanisms of small molecule transport across the endoplasmic reticulum membrane and demonstrated the existence of novel endoplasmic reticulum transport proteins for glucose and phosphate.     

A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells

A diploid epithelial cell line (termed WB-F344) was isolated from the liver of an adult male Fischer-344 rat and the phenotypic characteristics of the cells were studied. These cells measure approximately two-fifths the volume of freshly isolated hepatocytes. They are histochemically negative for glucose-6-phosphatase and weakly positive for gamma-glutamyl transpeptidase. They produce extensive intercellular reticulin fibers which stain immunocytochemically for fibronectin, and they synthesize both alpha-fetoprotein and albumin, but they do not accumulate glycogen particles. Ultrastructurally, they are polygonal cells with numerous intercellular desmosomes and nexus junctions, and they are partially surrounded by basement membrane-like material. Cytoplasmic organelles include few, but sometimes dilated profiles of rough endoplasmic reticulum, lysosomes, abundant free ribosomes, sparse smooth endoplasmic reticulum and Golgi membranes, microbodies, and small, pleomorphic mitochondria. They express A and C isozymes of aldolase, K isozyme of pyruvate kinase, LDH2 to LDH5 isozymes of lactate dehydrogenase, and 'fetal liver'-type alkaline phosphatase isozyme. When compared with the phenotypes of isolated and purified normal hepatocytes, biliary epithelial (ductular) cells and 'oval' cells isolated from livers treated with chemical carcinogens, the phenotypic properties of the liver epithelial cell line in culture most resemble those of the 'oval' cells.     

Phenobarbital-induced alterations in the activities of the transport and hydrolytic components of the glucose-6-phosphatase system in smooth and rough subfractions of the rat hepatic endoplasmic reticulum

We have examined the influence of the phenobarbital-induced proliferation of the hepatic endoplasmic reticulum (ER) on the activities of the components of the glucose-6-phosphatase system, i.e., the enzyme, the glucose-6-P translocase (T1), and the phosphate translocase (T2). Young male rats were injected ip twice daily for 4 days with 4 mg/100 g body wt of phenobarbital (PB) or an equivalent volume of saline solution. On the fifth day, the rats were killed and smooth (SER) and rough (RER) fractions of the ER were isolated from liver homogenates. Kinetic constants for glucose-6-P hydrolysis by the system and enzyme were determined and used to calculate the kinetic constants for glucose-6-P transport. T2 activity was approximated by assaying the pyrophosphatase activity at pH 6.0 in intact microsomes. Three times more SER protein was recovered from livers of PB-treated rats. PB-treatment did not alter total liver enzyme activity, but total liver T1 activity was decreased to 59% of the control value. Maximal specific activities of the system, enzyme and T1 were all reduced by PB treatment to 44% of control values in the RER and to 68% of control values in the SER. PB treatment reduced the apparent activity of T2 in RER and SER to 35 and 49% of the respective control values. In the SER from both groups of rats, T1 activity or apparent T2 activity divided by enzyme activity was about 55% of the corresponding ratio in the RER. Our analysis of these data suggests that the lower activities of T1 and T2 in the smooth ER are the results of suppression by some intrinsic component localized in the smooth membrane. Accordingly, the reduction in total liver T1 activity and, therefore, system activity in PB-treated rats reflects the redistribution of the glucose-6-P translocase from the RER to the more abundant SER membrane where it is less active. The possibility is discussed that a higher cholesterol content within the SER membrane is responsible for the lower transport activities.     

Effect of bemythyl on carbohydrate metabolism in cirrhotic rat liver

Effect of actoprotector bemitil (2-ethylthiobenzimidazole hydrobromide) on glycogen content and activities of glycogen synthase, glycogen phosphorylase, and glucose-6-phosphatase was studied in cirrhotically altered rat liver. The contents of glycogen and its fraction were determined a cytofluorimetrically (Kudryavtseva et al., 1974). In cirrhosis, the total glycogen content in hepatocytes increases by nearly 3 times, while the amount of a stable fraction of glycogen rises by 7.5 times. Glucose-6-phosphatase activity fell to the level of 25% compare to the norm. Activities of glycogen synthase and glycogen phosphorylase in the cirrhotic liver did not differ from the norm. In cirrhotically altered liver, bemitil produced a decrease in the total glycogen content due to a decrease in glycogen synthase activity in an increase in glucose-6-phosphatase and glycogen phosphorylase activities. The above results suggest a favorable effect of bemitil on cirrhotic liver.     

Studies on the effect of paraquat on glycogen mobilization in liver of common carp (Cyprinus carpio L.)

1. A herbicide, paraquat (1,1'dimethyl-4,4'-bipyridilium-dichloride) was administered to carp in 0.5-10.0 ppm concentrations, respectively, and blood sugar level, glucose-6-phosphatase and glycogen phosphorylase activities of liver were determined. 2. Paraquat treatment caused an increase of blood sugar level and enhanced phosphorylase and glucose-6-phosphatase activities. 3. Paraquat can induce alterations in endoplasmic reticulum that might contribute to the changes in glucose-6-phosphatase activity, resulting in an increase of blood glucose level and/or all the effects can be attributed to a high level of circulating epinephrine produced by paraquat toxicosis.     

Molecular pathology of glucose-6-phosphatase

It was known in the 1950s that hepatic microsomal glucose-6-phosphatase plays an important role in the regulation of blood glucose levels. All attempts since then to purify a single polypeptide with glucose-6-phosphatase activity have failed. Until recently, virtually nothing was known about the molecular basis of glucose-6-phosphatase or its regulation. Recent studies of the type 1 glycogen storage diseases, which are human genetic deficiencies that result in impaired glucose-6-phosphatase activity, have greatly increased our understanding of glucose-6-phosphatase. Glucose-6-phosphatase has been shown to comprise at least five different polypeptides, the catalytic subunit of glucose-6-phosphatase with its active site situated in the lumen of the endoplasmic reticulum; a regulatory Ca2+ binding protein; and three transport proteins, T1, T2, and T3, which respectively allow glucose-6-phosphate, phosphate, and glucose to cross the endoplasmic reticulum membrane. Purified glucose-6-phosphatase proteins, immunospecific antibodies, and improved assay techniques have led to the diagnosis of a variety of new type 1 glycogen storage diseases. Recent studies of the type 1 glycogen storage diseases have led to a much greater understanding of the role and regulation of each of the glucose-6-phosphatase proteins.     

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.     

Loss of endoplasmic reticulum membrane integrity: an image analysis of the glucose-6-phosphatase system in human hepatocyte.

Histochemical and cytochemical methods induce a loss of endoplasmic reticulum (ER) membrane integrity in hepatocytes. In order to evaluate the degree of ER membrane integrity, glucose-6-phosphatase (G6P-A) was localized in light and electron microscopy using glucose-6-phosphate (G6P) and mannose-6-phosphate (M6P) as substrates. In case of ER membrane alteration, M6P diffuses inside the ER and is hydrolysed by a non-specific phosphohydrolase. G6P and M6P hydrolysis was quantified with image analysis methods. In light microscopy, the ratio of reaction of M6P hydrolysis/G6P hydrolysis gave 75% of non specific reaction. In electron microscopic study this ratio was about 30%. These results showed that enzyme localization methods in electron microscopy produced less ER membrane alteration than light microscopic methods.     

High glucose-6-phosphatase activity in non-pigmented epithelial cells of rabbit ciliary body.

For study of the origin of glucose in the aqueous humor, glucose-6-phosphatase (G6Pase) and hexokinase activities, and glycogen, were cytochemically examined in the ciliary body (CB) of rabbit. G6Pase activity was also assayed biochemically. The staining reaction for G6Pase activity was strong in the non-pigmented epithelium (NPE) in the pars plana and tips of ciliary processes in the region containing large ciliary pockets within the pars plicata. NPE cells contained abundant reaction product for G6Pase activity in the endoplasmic reticulum (ER) and nuclear envelope. However, NPE in other regions of the CB and pigmented epithelium (PE) of CB, and other areas surrounding the anterior and (PE) of CB, and other areas surrounding the anterior and posterior chambers, showed weak or no G6Pase staining reaction. Biochemical G6Pase activity in the whole ciliary body was relatively high. Both NPE and PE in the pars plana and the tips showed strong staining reaction for hexokinase activity but no staining for glycogen. Furthermore, NPE cells in the tips bore large aggregates of smooth ER and many Golgi apparati. These suggest that the high G6Pase activity in NPE cells in the pars plana and the tips is related to glucose release into the aqueous humor.     

Features of the effect of bemethyl on glycogen metabolism in hepatocytes of pathological changed human liver

Effect of actoprotector bemithyl (2-ethylthiobenzimidazole hydrobromide) on glycogen metabolism in hepatocytes of patients with chronic hepatitis and liver cirrhosis was investigated. Using cytofluorimetric method, the content of glycogen and its fractions in isolated hepatocytes was measured. The treatment with bemithyl resulted in a decrease in glycogen levels in hepatocytes, and in a marked restoration of fractional glycogen composition as compared to the basic therapy. Besides, it was established that the degree of glycogen decrease in cells of patients with chronic hepatitis depended on the increase of glucose-6-phosphatase activity (r = 0.75, P < 0.05), and on the levels of glycogen in hepatocytes prior to bemitil treatment (r = = 0.87, P < 0.01). Positive changes in glycogen metabolism after bemithyl treatment are pronounced in patients with chronic hepatitis. These positive alterations take place simultaneously with the conservation of basic structural disturbances in the liver parenchyma. However, even in this case, the indices of glycogen metabolism do not reach the normal levels.     

The flavonoid silibinin decreases glucose-6-phosphate hydrolysis in perfused rat hepatocytes by an inhibitory effect on glucose-6-phosphatase.

BACKGROUND/AIMS: The flavonoid silibinin has been reported to be beneficial in several hepatic disorders. Recent evidence also suggests that silibinin could be beneficial in the treatment of type 2 diabetes, owing to its anti-hyperglycemic properties. However, the mechanism(s) underlying these metabolic effects remains unknown. METHODS: The effects of silibinin on liver gluconeogenesis were studied by titrating hepatocytes from starved rats with sub-saturating concentrations of various exogenous substrates in a perifusion system. Hepatocytes from fed rats were also used to investigate glycogenolysis from endogenous glycogen. The effect of silibinin on glucose-6-phosphatase kinetics was determined in intact and permeabilized rat liver microsomes. RESULTS: Silibinin induced a dose-dependent inhibition of gluconeogenesis associated with a potent decrease in glucose-6-phosphate hydrolysis. This effect was demonstrated whatever the gluconeogenic substrates used, i.e. dihydroxyacetone, lactate/pyruvate, glycerol and fructose. In addition, silibinin decreased the glucagon-induced stimulation of both gluconeogenesis and glycogenolysis, this being associated with a reduction of glucose-6-phosphate hydrolysis. Silibinin inhibits glucose-6-phosphatase in rat liver microsomes in a concentration-dependent manner that could explain the decrease in glucose-6-phosphate hydrolysis seen in intact cells. CONCLUSION: The inhibitory effect of silibinin on both hepatic glucose-6-phosphatase and gluconeogenesis suggests that its use may be interesting in treatment of type 2 diabetes.     

DIFFERENTIATION OF ENDOPLASMIC RETICULUM IN HEPATOCYTES : II. Glucose-6-Phosphatase in Rough Microsomes

Electron microscope cytochemical localization of glucose-6-phosphatase in the developing hepatocytes of fetal and newborn rats indicates that the enzyme appears simultaneously in all the rough endoplasmic reticulum of a cell, although asynchronously within the hepatocyte population as a whole. To confirm that the pattern of cytochemical deposits reflects the actual distribution of enzyme sites, a method to subfractionate rough endoplasmic reticulum was developed. The procedure is based on the retention of the cytochemical reaction product (precipitated lead phosphate) within freshly prepared rough microsomes reacted in vitro with glucose-6-phosphate and lead ions. Lead phosphate increases the density of the microsomes which have glucose-6-phosphatase activity and thereby makes possible their separation from microsomes lacking the enzyme; separation is obtained by isopycnic centrifugation on a two-step density gradient. The procedure was applied to rough microsomes isolated from rats at several stages during hepatocyte differentiation and the results obtained agree with those given by cytochemical studies in situ. Before birth, when only some of the cells react positively for glucose-6-phosphatase, only a commensurate proportion of the rough microsome fraction can be rendered dense by the enzyme reaction. At the time of birth and in the adult, when all cells react positively, practically all microsomes acquire deposit and become dense after reaction. Thus, the results of the microsome subfractionation confirm the cytochemical findings; the enzyme is evenly distributed throughout all the endoplasmic reticulum of a cell and there is no regional differentiation within the rough endoplasmic reticulum with respect to glucose-6-phosphatase. These findings suggest that new components are inserted molecule-by-molecule into a pre-existing structural framework. The membranes are thus mosaics of old and new molecules and do not contain large regions of entirely "new" membrane in which all of the components are newly synthesized or newly assembled.     

Studies on vinblastine-induced autophagocytosis in mouse liver. IV. Origin of membranes

The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Studies on vinblastine-induced autophagocytosis in mouse liver

Summary The origin of the limiting membranes of autophagic vacuoles (AV) in mouse hepatocytes was studied by cytochemical techniques. Autophagocytosis was induced by an intraperitoneal injection of vinblastine (50 mg/kg). The marker enzymes used were adenosine triphosphatase for the plasma membrane, glucose-6-phosphatase for the endoplasmic reticulum and thiamine pyrophosphatase for the Golgi apparatus and the endoplasmic reticulum. All the three enzymes showed a characteristic localization in both control and vinblastine-treated hepatocytes. The space between the limiting membranes of a few apparently newly formed AV's showed weak glucose-6-phosphatase activity. Neither adenosine triphosphatase nor thiamine pyrophosphatase activities were observed on or between the AV membranes. It was suggested that endoplasmic reticulum membranes may be used as a source of AV membranes in hepatocytes. The lack of glucose-6-phosphatase activity in the limiting membranes even of most of the newly formed AV's suggests a transformation process of the membranes destined to form AV, during which the enzyme activity characteristic for endoplasmic reticulum may disappear from them.     

Glycogen synthesizing function of hepatocytes in rats with liver cirrhosis after treatment with chorionic gonadotropin]

Using rat liver hepatocytes, methods of cytofluorimetry (Kudryavtseva et al., 1974) and biochemistry were applied to comparative studies of the total glycogen content, including its labile (LF) and stable (SF) fractions, and activities of glucose-6-phosphatase, glycogen phosphorylase and glycogensynthetase in these. The liver hepatocytes were examined in norm, and under conditions of CCl4 poisoning of rats, both 6 months after a chronic poisoning, and 1, 3 and 6 months following poisoning cessation. All the experimentally poisoned rats were divided into two conventional groups: rats of one group received, apart from poisoning, a complex treatment with chorionic gonadotropin (CG); the other group rats received, no treatment. The material used for examination was obtained from serial functional biopsies of each experimental animal. It has been shown that under cirrhosis the content of the total glycogen in hepatocytes increased by 3 times, and that of its SF even by 9.7 times. The treatment with CG for 1 month resulted in its reducing to the norm, and 3 to 6 months treatments normalized contents of both the glycogen fractions. In the group of non-treated rats no similar changes were registered. Besides, in the cirrotic rats the activity of glucose-6-phosphatase was shown to increase by 4 times. After CG treatment it was seen to decrease by 3 times. Thus, CG may be regarded as an optimum and more effective agent for restoring abnormalities in cirrotic liver, compared to some other stimulating factors, such as hepatectomy (Kudryavtseva et al., 1996) or rich-carbohydrate diet (Kudryavtseva et al., 1998).     

Effect of thyroxine on hepatic glucose-6-phosphatase activity and glycogen content of toad (Bufo melanostictus) and Lata fish (Ophicephalus punctatus) at different stages of life

The effects of thyroxine (T4) on hepatic glucose-6-phosphatase activity and glycogen content in toad (Bufo melanostictus) and Lata fish (Ophicephalus punctatus) were studied in order to show the difference, if any in the enzyme activity and glycogen metabolism in their liver. Thyroxine injections (1 microgram/g) for five consecutive days caused a reduction in hepatic glucose-6-phosphatase activity and glycogen content in toads of immature, juvenile and adult stages. In contrast, Lata fish of different stages showed an enhancement of hepatic glucose-6-phosphatase activity after T4 treatment (1 microgram/g, 5 injections). The liver glycogen content in Lata fish of different age groups was found to be reduced after T4 injections, but not so much as in the toad.