Assessment of Lipotoxic Endoplasmic Reticulum (ER) Stress in Nonalcoholic Steatohepatitis (NASH)

Abstract

Hepatocyte lipotoxicity is a hallmark of nonalcoholic steatohepatitis (NASH), and lipid induced liver injury occurs, in part, via activation of endoplasmic reticulum (ER) stress. Consequently, the unfolded protein response (UPR) is initiated, driven by three key ER transmembrane proteins, resulting in downstream responses that are dynamic and interconnected. Thus, careful interrogation of these pathways is required to investigate the complex role of ER stress in NASH. Herein, we describe different mechanisms of, and in vitro assays for assessment of lipotoxic ER stress in mouse hepatocytes.

Keywords: Apoptosis; C/EBP homologous binding protein; Free fatty acid; Palmitic acid; Unfolded protein response; X-box binding protein 1.

Figure 1.. Suggested timeline for assessing lipotoxic ER stress in mouse hepatocytes in vitro.

After induction of lipotoxicity in vitro by application of palmitic acid, different readouts of ER stress can be examined at different time points as indicated. Readouts depicted in this figure can be assayed with techniques listed in Table 1.

Figure 2.. Representative examples of ER stress responses to palmitic acid in primary mouse hepatocytes.

(A) Western blotting for expression of BiP, ATF4, phospho-eIF2α and CHOP in an immortalized mouse hepatocyte (IMH) cell line treated with vehicle (isopropyl alcohol), palmitic acid (400 μM), and tunicamycin (1.2 μM) as a positive control, for the indicated time periods. (B) QIAxcel capillary electrophoresis images of total and spliced Xbp1 in primary mouse hepatocytes treated with vehicle (isopropyl alcohol), thapsigargin, tunicamycin and palmitic acid; 18s is included as the housekeeping control.