Hepatocellular Ballooning is Due to Highly Pronounced Glycogenosis Potentially Associated with Steatosis and Metabolic Reprogramming

Abstract

Background and aims: Hepatocellular ballooning is a common finding in chronic liver disease, mainly characterized by rarefied cytoplasm that often contains Mallory-Denk bodies (MDB). Ballooning has mostly been attributed to degeneration but its striking resemblance to glycogenotic/steatotic changes characterizing preneoplastic hepatocellular lesions in animal models and chronic human liver diseases prompts the question whether ballooned hepatocytes (BH) are damaged cells on the path to death or rather viable cells, possibly involved in neoplastic development.

Methods: Using specimens from 96 cirrhotic human livers, BH characteristics were assessed for their glycogen/lipid stores, enzyme activities, and proto-oncogenic signaling cascades by enzyme- and immunohistochemical approaches with serial paraffin and cryostat sections.

Results: BH were present in 43.8% of cirrhotic livers. Particularly pronounced excess glycogen storage of (glycogenosis) and/or lipids (steatosis) were characteristic, ground glass features and MDB were often observed. Decreased glucose-6-phosphatase, increased glucose-6-phosphate dehydrogenase activity and altered immunoreactivity of enzymes involved in glycolysis, lipid metabolism, and cholesterol biosynthesis were discovered. Furthermore, components of the insulin signaling cascade were upregulated along with insulin dependent glucose transporter glucose transporter 4 and the v-akt murine thymoma viral oncogene homolog/mammalian target of rapamycin signaling pathway associated with de novo lipogenesis.

Conclusions: BH are hallmarked by particularly pronounced glycogenosis with facultative steatosis, many of their features being reminiscent of metabolic aberrations documented in preneoplastic hepatocellular lesions in experimental animals and chronic human liver diseases. Hence, BH are not damaged entities facing death but rather viable cells featuring metabolic reprogramming, indicative of a preneoplastic nature.

Keywords: Carbohydrate metabolism; Chronic liver disease; Insulin signaling; Metabolic reprogramming; Preneoplasia.

Fig. 1. Serial paraffin sections of cirrhotic liver from a patient with alcoholic steatohepatitis.

(A) Parenchymal portion showing ballooned (right part) in contrast to normal hepatocytes (left part) (B) higher magnification showing many ballooned hepatocytes with clear or slightly eosinophilic (ground glass) cytoplasm and prominent nuclei (H&E), corresponding (D) to loss of expression of CK18; and (E) excessive storage of glycogen (PAS). Serial paraffin sections of cirrhotic liver from an HBV case are shown. Group of ballooned hepatocytes with typical clear cytoplasm, highly condensed nuclear chromatin (C, H&E) and massive glycogen content (F, PAS). Labeled scale bars are included in every picture.

Fig. 2. Serial paraffin sections of cirrhotic liver from an HBV case.

The portion of a cirrhotic nodule is shown containing several large ballooned hepatocytes with clear cytoplasm (A, B, H&E) excess glycogen storage (C, PAS) with fat vacuoles, sometimes also MDB in addition (A, H & E, arrows). Serial paraffin sections of cirrhotic liver from a patient with alcoholic steatohepatitis are shown. Several ballooned hepatocytes show clear cytoplasm and often also MDB (arrows) (D, H&E) positive for ubiquitin staining (E). In addition, eosinophilic GGH with variable glycogen content (F, PAS). Labeled scale bars are included in each picture.

Fig. 3. Findings in hepatocellular carcinomas with gylcogenos and steatosis.

A: Serial paraffin sections of glycogenotic clear-cell HCC from an HCV case. Tumor portions containing many ballooned clear cells (a, H&E) excessively storing glycogen (b, PAS), and showing gradual transitions from clear glycogenotic to glycogen poor, slightly basophilic tumor cells (c, H&E) occasionally containing MDB (arrows) (d, H&E). B: Serial paraffin sections of hepatocellular carcinoma with steatohepatitic features. Ballooning of clear neoplastic hepatocytes (a and square with higher magnification in b, H&E), occasionally containing MDB as demonstrated by ubiquitin staining (c), and showing excessive storage of glycogen, some fat vacuoles (d, PAS), and loss of cytokeratin 18 expression (e). Labeled scale bars are included in each picture.

Fig. 4. Serial semithin resin sections of cirrhotic liver from patients with HBV observed by microscopy. Ultrastructural findings.

(A) Ballooned hepatocytes with strong PAS-positive cytoplasm (a), corresponding to glycogen particles (G) in the cytoplasm (b), magnified segment square in (c). (d) Accumulation of glycogen as alpha particles in the cytoplasmic matrix and as beta particles in glycogenosome (*). (e) Combination of glycogen accumulation and lipid droplets. (f) MDB with tightly arranged fuzzy filaments. (B) Portions of GGH showing extended SER (a and square with higher magnification in b, arrows) alternating with layers of alpha or beta glycogen particles. Glycogen-rich hepatocytes with formation of unusual RER complexes (ergastoplasma pockets) which are poor in, or completely free of, glycogen particles but rich in ribosomes. c, and with higher magnification in d, arrows). Labeled scale bars are included in each picture. MDB, Mallory-Denk bodies.

Fig. 5. Serial cryostat sections of cryptogenic cirrhotic liver and normal liver specimens comparing histological and enzyme histochemical properties.

Cirrhotic liver nodule with ballooned hepatocytes (A, H&E) showing decreased activity of glucose-6-phosphatase (B) and increased activity of glucose-6-phosphate dehydrogenase (C) compared with normal liver parenchyma (D, H&E) (E and F). Labeled scale bars are included in each picture.

Fig. 6. Serial cryostat sections of part of a cirrhotic liver nodule from a HBV case.

Several ballooned hepatocytes (H&E) with excess stored glycogen (PAS) and/or fat (Oil red) and showing overexpression of glucose transporter GLUT4, insulin receptor, IRS1, the glycolytic enzymes IGLK, aldolase A, PKM2, lipogenic enzymes like SCD1, FASN, HMGCoAR and upregulation of the proto-oncogenic pathways AKT/mTOR, ras/raf-1 and PanERK. The labeled scale bar included in H&E picture is representative of all images. H&E, hematoxylin and eosin; PAS, periodic acid Schiff reaction; GLUT4, glucose transporter 4; IRS1, insulin receptor substrate 1; IR, insulin receptor; IGLK, iso-glukokinase; PKM2, pyruvate kinase 2; SCD1, stearoyl-CoA desaturase; FASN, fatty acid synthase; HMGCoAR, 3-hydroxy-3-methylglutaryl-CoA-reductase; AKT, v-akt murine thymoma viral oncogene homolog; ERK, extracellular related kinase.