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.     

Glucagon-stimulated phosphorylation of rat liver glycogen synthase in isolated hepatocytes

Addition of glucagon (20 nM) to the isolated hepatocytes from 24-h starved male rats results in an inactivation of glycogen synthase. The A0.5 for glucose-6-P is increased 2-fold over the control but the S0.5 for UDP-glucose is not significantly affected. The glucagon-stimulated inactivation of glycogen synthase is also accompanied by a 60-120% increase in the phosphorylation of the synthase. Glycogen synthase labeled with 32P by incubation of the hepatocytes with [32P] PO4(3-) was recovered by immunoprecipitation and the resulting immunoprecipitate was subjected to tryptic digestion. Analysis of the 32P-labeled peptides reveals that the sites corresponding to those phosphorylated by cAMP-dependent protein kinase and glycogen synthase (casein) kinase-1 (Itarte, E., and Huang, K.-P. (1979) J. Biol. Chem. 254, 4052-4057) are rapidly phosphorylated in response to glucagon. These results demonstrate that glucagon not only triggers the activation of cAMP-dependent protein kinase through an increase in the intracellular level of cAMP but also, by an unknown mechanism, activates a Ca2+- and cAMP-independent protein kinase.     

Effect of deafferentation of the liver on hepatocytes located in various parts of the hepatic lobule

Taking into account the data on functional heterogeneity of hepatocytes, situating in various parts of the hepatic lobule, influence of deafferentation of the cat liver on changes in the size of hepatocytes and their nuclei, as well as contents of glycogen and nucleic acids in their cytoplasm have been investigated. The greatest decrease of the glycogen contents and the greatest increase of the nucleic acids and the nuclei volume take place in hepatocytes, situating around the central vein.     

The influence of prolonged di(2-ethylhexyl)phthalate treatment on the dolichol and dolichyl-P content of rat liver

Dolichols and glycosyl transferase activities were studied in rat liver fractions after treatment with the plasticizer di(2-ethylhexyl)phthalate, an inducer of peroxisomes and mitochondria. After a few weeks of treatment with 2% plasticizer in the diet, the amount of dolichol is more than doubled in the lysosomes but not in the microsomes while dolichyl-P decreased by 50% in the microsomes but not in the lysosomes. The isoprenoid pattern for dolichol and dolichyl-P, respectively, is modified to longer polyprenols in the two fractions as seen in the percent distribution of the individual isoprenes. Dolichyl-P and protein glycosylation by N-acetylglucosamine and mannose decreased considerably. Incubation with mixtures containing exogenous dolichyl-P did not increase protein glycosylation. Phthalate ester treatment for 2 years increased dolichol content above the control values even when the dose was decreased a hundred times, to 0.02%. The results demonstrate a compartmentalization of dolichol and dolichyl-P distribution, and the induction studies suggest that hepatocytes possess separate regulating mechanisms for these two compounds.     

The influence of sialoadenectomy, thymectomy and starvation on liver glycogen in the rat

Hepatic glycogen was assayed in young and adult rats subjected to sialoadenectomy and/or thymectomy and starvation. Sialoadenectomy in young, but not in adult rats caused the rats to stop feeding. In young, but not in adult sialoadenectomized and starved rats the glycogen level was notably higher than in unoperated and starved rats, indicating active participation of salivary glucagon in immature animals in hepatic glycogenolysis under conditions of starvation. Simultaneous sialoadenectomy and thymectomy caused glycogen depletion in the liver of young rats in spite of the absence of the salivary glands. Acceleration of glycogenolysis in these rats was not due to thymectomy, being probably a result of excessive secretion of adrenal catecholamines.     

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.     

Effect of epinephrine on glycogen phosphorylase-alpha in various preparations of rat liver

1. Glycogen phosphorylase-alpha, a commonly used index of cytosolic free calcium, was compared in various preparations of rat liver in the absence and presence of 0.1 microM epinephrine. 2. Total phosphorylase in isolated perfused livers and freshly-isolated hepatocytes were the same as that observed in liver in situ; however, phosphorylase-alpha was 50% higher in perfused liver and 80% higher in hepatocytes than activities measured in situ. Total phosphorylase was reduced approximately 50% in hepatocytes maintained in primary culture for 24 hr. 3. Epinephrine increased phosphorylase-alpha approximately 2-fold in livers perfused for 30 min but only about 20% in hepatocytes incubated for 30 min. After 90 min of perfusion or incubation, epinephrine increased phosphorylase-alpha nearly 4-fold in perfused livers and only 30% in isolated hepatocytes. The results suggest that amounts of free calcium and calcium-dependent coupling of adrenergic receptors to phosphorylase-alpha differ markedly between the intact liver and isolated hepatocytes.     

Dynamics of glycogen content in the normal and cirrhotic liver following glucose administration to starving rats

Using biochemical, cytofluorimetric and television cytophotometric methods, glycogen contents were studied in normal and cirrhotic rat liver at various intervals after glucose administration to fasting animals. The obtained data indicate that after a 48 h fasting glycogen contents in normal and cirrhotic liver are equally poor. A marked rise of glycogen content in cirrhotic liver was observed only 20-30 min after glucose administration to rats. It has been established that at all intervals after glucose administration to rats hepatocytes of the portal lobule zone, both in normal and in cirrhotic liver, accumulate more glycogen than those of the central zone. Again, the intensity of glycogen accumulation in cirrhotically altered liver is significantly lower than in normal liver, due, presumably, to a lower rate of glycogen synthesis in pathologically changed liver.     

A major locus (RN) affecting muscle glycogen content is located on pig Chromosome 15

The RN locus in pigs has a major effect on the amount of stored glycogen in white muscle and affects meat quality. The fully dominant RN ⁻ allele, associated with high glycogen content, occurs in the Hampshire breed. We have mapped the RN locus using a large half-sib family comprising one heterozygous RN ⁻/rn ⁺ Hampshire boar mated to homozygous rn ⁺/rn ⁺ Swedish Landrace × Swedish Yorkshire sows. The segregation at the RN locus was inferred from data on glycolytic potential and residual glycogen in white muscle which both showed clear bimodal distributions. Highly significant evidence for genetic linkage was obtained against microsatellite markers on Chromosome (Chr) 15. Multipoint analysis revealed the order Sw1111–8.0–S0088–10.6–RN–4.8–Sw936,Sw906 (recombination estimates are given as Kosambi cM). Comparative mapping data imply that the human homolog of RN is located on Chr 2q. Received: 18 April 1995 / Accepted: 16 June 1995     

Studies on the in vitro effects of insulin on glycogen synthesis and ultrastructure in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.