Improved oxygenation dramatically alters metabolism and gene expression in cultured primary mouse hepatocytes

Abstract

Primary hepatocyte culture is an important in vitro system for the study of liver functions. In vivo, hepatocytes have high oxidative metabolism. However, oxygen supply by means of diffusion in in vitro static cultures is much less than that by blood circulation in vivo. Therefore, we investigated whether hypoxia contributes to dedifferentiation and deregulated metabolism in cultured hepatocytes. To this end, murine hepatocytes were cultured under static or shaken (60 revolutions per minute) conditions in a collagen sandwich. The effect of hypoxia on hepatocyte cultures was examined by metabolites in media and cells, hypoxia-inducible factors (HIF)-1/2α western blotting, and real-time quantitative polymerase chain reaction for HIF target genes and key genes of glucose and lipid metabolism. Hepatocytes in shaken cultures showed lower glycolytic activity and triglyceride accumulation than static cultures, compatible with improved oxygen delivery and mitochondrial energy metabolism. Consistently, static cultures displayed significant HIF-2α expression, which was undetectable in freshly isolated hepatocytes and shaken cultures. Transcript levels of HIF target genes (glyceraldehyde 3-phosphate dehydrogenase [Gapdh], glucose transporter 1 [Glut1], pyruvate dehydrogenase kinase 1 [Pdk1], and lactate dehydrogenase A [Ldha]) and key genes of lipid metabolism, such as carnitine palmitoyltransferase 1 (Cpt1), apolipoprotein B (Apob), and acetyl-coenzyme A carboxylase 1 (Acc1), were significantly lower in shaken compared to static cultures. Moreover, expression of hepatocyte nuclear factor 4α (Hnf4α) and farnesoid X receptor (Fxr) were better preserved in shaken cultures as a result of improved oxygen delivery. We further revealed that HIF-2 signaling was involved in hypoxia-induced down-regulation of Fxr. Conclusion: Primary murine hepatocytes in static culture suffer from hypoxia. Improving oxygenation by simple shaking prevents major changes in expression of metabolic enzymes and aberrant triglyceride accumulation; in addition, it better maintains the differentiation state of the cells. The shaken culture is, therefore, an advisable strategy for the use of primary hepatocytes as an in vitro model. (Hepatology Communications 2018;2:299-312).

Figure 1

Static culture promotes lipid accumulation and glycolysis in primary mouse hepatocytes. (A) Representative light microscopy images of hepatocytes in static or shaken culture 20 hours after attachment. (B) Triglyceride or (C) glycogen of freshly isolated hepatocytes and hepatocytes after overnight in static and shaken culture were assayed. Overnight medium samples of primary mouse hepatocytes in static and shaken culture were collected for enzymatic determination of (D) glucose, (E) L‐lactate, and (F) pyruvate. (G) The lactate and pyruvate results were used to calculate the lactate‐to‐pyruvate ratio. Results are representative of three independent experiments of triplicate determinations. The data (B‐G) represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviation: n.s., not significant.

Figure 2

Activation of HIF signaling causes lipid accumulation in statically cultured primary mouse hepatocytes. Protein lysates of primary mouse hepatocytes after overnight static and shaken culture were subjected to immunoblot for (A) HIF‐1α, (B) HIF‐2α, and (C) the HIF‐target GLUT1. (D) Lipid from overnight static and shaken hepatocyte cultures in triplicate was extracted and enzymatically determined. Results are representative of three independent experiments. The data in D represent mean ± SD. **P < 0.01, ***P < 0.001. Abbreviation: IB, immunoblot.

Figure 3

Shaken cultures reverted increased expression of glycolysis‐related genes in static cultures. Transcript levels of HIF‐responsive genes and glycolysis‐related genes were examined in freshly isolated primary mouse hepatocytes and overnight static and shaken cultures by quantitative reverse‐transcription polymerase chain reaction. (A) Glut1, (B) Gapdh, (C) Pdk1, (D), Ldha, (E) Gck. Results are representative of three independent experiments of triplicate determinations. The data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviation: n.s., not significant.

Figure 4

Effect of the shaken culture on expression of genes related to lipid metabolism. Transcript levels of genes related to lipid metabolism were examined in freshly isolated primary mouse hepatocytes and overnight static and shaken cultures by quantitative reverse‐transcription polymerase chain reaction. (A) Cpt1a, (B) Apob, (C) Fsp27, (D), Acc1, (E) Ppara, (F) Pparg. Results are representative of three independent experiments of triplicate determinations. The data represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviation: n.s., not significant.

Figure 5

Expression of Hnf4α and Fxr in static and shaken cultures of primary mouse hepatocytes. Transcript levels of (A) Hnf4α and (B) Fxr were examined in freshly isolated primary mouse hepatocytes and overnight static and shaken cultures by quantitative reverse‐transcription polymerase chain reaction. (C) Immunoblot for FXR in freshly isolated primary mouse hepatocyte and overnight static and shaken cultures. (D) Immunoblot was quantified by ImageJ. Results are representative of at least two independent experiments of triplicate determinations. The data (A, B, and D) represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviations: DMSO, dimethyl sulfoxide; IB, immunoblot; n.s., not significant.