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. 2019 Dec 5;14(12):e0225604.
doi: 10.1371/journal.pone.0225604. eCollection 2019.

Hyperglycemia induces key genetic and phenotypic changes in human liver epithelial HepG2 cells which parallel the Leprdb/J mouse model of non-alcoholic fatty liver disease (NAFLD)

Robin C Su  1 , Apurva Lad  1 , Joshua D Breidenbach  1 , Thomas M Blomquist  2 , William T Gunning  2 , Prabhatchandra Dube  1 , Andrew L Kleinhenz  1 , Deepak Malhotra  1 , Steven T Haller  1   3 , David J Kennedy  1   3
Affiliations

Hyperglycemia induces key genetic and phenotypic changes in human liver epithelial HepG2 cells which parallel the Leprdb/J mouse model of non-alcoholic fatty liver disease (NAFLD)

Robin C Su et al. PLoS One. .

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a growing global health concern. With a propensity to progress towards non-alcoholic steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma, NAFLD is an important link amongst a multitude of comorbidities including obesity, diabetes, and cardiovascular and kidney disease. As several in vivo models of hyperglycemia and NAFLD are employed to investigate the pathophysiology of this disease process, we aimed to characterize an in vitro model of hyperglycemia that was amenable to address molecular mechanisms and therapeutic targets at the cellular level. Utilizing hyperglycemic cell culturing conditions, we induced steatosis within a human hepatocyte cell line (HepG2 cells), as confirmed by electron microscopy. The deposition and accumulation of lipids within hyperglycemic HepG2 cells is significantly greater than in normoglycemic cells, as visualized and quantified by Nile red staining. Alanine aminotransferase (ALT) and alkaline phosphatase (ALP), diagnostic biomarkers for liver damage and disease, were found to be upregulated in hyperglycemic HepG2 cells as compared with normoglycemic cells. Suppression of CEACAM1, GLUT2, and PON1, and elevation of CD36, PCK1, and G6PK were also found to be characteristic in hyperglycemic HepG2 cells compared with normoglycemic cells, suggesting insulin resistance and NAFLD. These in vitro findings mirror the characteristic genetic and phenotypic profile seen in Leprdb/J mice, a well-established in vivo model of NAFLD. In conclusion, we characterize an in vitro model displaying several key genetic and phenotypic characteristics in common with NAFLD that may assist future studies in addressing the molecular mechanisms and therapeutic targets to combat this disease.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Hyperglycemic HepG2 cells exhibit lipid accumulation.
A. H&E histology reveals successive passaging of HepG2 cells in hyperglycemic conditions show progressive phenotypic changes as compared with normoglycemic HepG2 cells. B. Brightfield microscopy also reveal these phenotypic changes within hyperglycemic HepG2 cells as compared with normoglycemic cells. C. Representative cultured cells examined at the ultrastructural level by electron microscopy demonstrate accumulations of numerous lipid vacuoles (red arrows) and water influx (clear cytoplasmic inclusion) in hyperglycemic HepG2 cells in contrast to cells grown in normoglycemic culture media.
Fig 2
Fig 2. Nile red staining for triglycerides is elevated in hyperglycemic HepG2 cells.
A. Fluorescent imaging reveals increased Nile red staining in hyperglycemic HepG2 cells as compared with normoglycemic cells. B. Brightfield microscopy with fluorescent overlay reveals the accumulation of lipid within hyperglycemic HepG2 cells as compared with normoglycemic cells. C. Quantification of Nile red fluorescence reveals elevated levels in hyperglycemic HepG2 cells as compared with normoglycemic cells. Data presented indicate the mean ± SEM (n = 3 wells per group). *p<0.05 vs. normoglycemic group.
Fig 3
Fig 3. qPCR analysis shows trends in hyperglycemic HepG2 cells consistent with NAFLD.
A. CEACAM1 gene expression is depressed in hyperglycemic HepG2 cells as compared with normoglycemic cells. B. CD36 gene expression is elevated in hyperglycemic HepG2 cells as compared with normoglycemic cells. C. ALT and ALP gene expression is elevated in hyperglycemic HepG2 cells as compared with normoglycemic cells. Data presented indicate the mean ± SEM (n = 3 per group). **p<0.01 vs. normoglycemic group.
Fig 4
Fig 4. qPCR analysis shows trends in hyperglycemic HepG2 cells consistent with insulin resistance.
A. PCK1 and G6PC gene expression is elevated in hyperglycemic HepG2 cells compared with normoglycemic HepG2 cells. B. GLUT2 and C. PON1 gene expression is depressed in hyperglycemic HepG2 cells compared with normoglycemic HepG2 cells. Data presented indicate the mean ± SEM (n = 3 per group). *p<0.05, ***p<0.001, ****p<0.0001 vs. normoglycemic group.
Fig 5
Fig 5. MTT and LDH assay for HepG2 cells reveal no significant differences between cell conditions.
A. MTT assay demonstrated no differences in mitochondrial function between hyperglycemic and normoglycemic HepG2 cells. B. LDH assay demonstrated no differences in cytotoxicity between hyperglycemic and normoglycemic HepG2 cells.
Fig 6
Fig 6. Oil Red O staining is elevated in livers of Leprdb/J mice.
Livers from Leprdb/J mice revealed increased Oil Red O staining as compared with livers from WT C57 mice.
Fig 7
Fig 7. qPCR analysis shows trends in Leprdb/J mice consistent with NAFLD.
A. CEACAM1 gene expression is depressed in Leprdb/J mouse livers as compared with WT C57 mouse livers B. CD36 gene expression is elevated in Leprdb/J mouse livers as compared with WT C57 mouse livers C. ALT and ALP gene expression is elevated in Leprdb/J mouse livers as compared with WT C57 mouse livers. Data presented indicate the mean ± SEM (n = 5–6 per group). **p<0.01, ***p<0.001, and ****p<0.0001 vs. WT C57 group.
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