A fluorescent reporter assay of differential gene expression response to insulin in hepatocytes

• Published in: American Journal of Physiology: Cell Physiology Vol. 317; no. 1; pp. C143 - C151
• Main Authors: McKimpson, Wendy M, Accili, Domenico
• Format: Journal Article
• Language: English
• Published: United States American Physiological Society 01.07.2019
• Subjects: AKT protein; Animals; Cell Line, Tumor; Cell Separation - methods; Enzyme Activation; Fatty Acid Synthases - genetics; Fatty-acid synthase; Flow Cytometry; Forkhead protein; Forkhead Transcription Factors - genetics; Forkhead Transcription Factors - metabolism; Gene expression; Gene Expression Regulation - drug effects; Genes, Reporter; Hepatocytes; Hepatocytes - drug effects; Hepatocytes - metabolism; Insulin; Insulin - pharmacology; Insulin resistance; Liver; Luminescent Proteins - biosynthesis; Luminescent Proteins - genetics; Metabolic pathways; Methods in Cell Physiology; Mice; Microscopy, Fluorescence; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt - metabolism; Ribonucleic acid; RNA; Time Factors; Transcription factors; Transfection; insulin sensitivity; cell heterogeneity; hepatocyte; fatty acid synthase; Foxo1
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00504.2018

Insulin regulates multiple hepatic metabolic pathways in a seemingly heterogeneous manner. To understand this heterogeneity, we hypothesized that different subpopulations of hepatocytes have different sensitivity to insulin. To test this hypothesis, we developed a fluorescent reporter in which the insulin-responsive fatty acid synthase (FAS) promoter drove expression of a time-dependent fluorescent protein ("timer") and characterized timer expression in flow-sorted cell populations. In Hepa1c1c7 and AML12 hepatocytes, we found that different cell populations express distinct timer fluorescence following insulin treatment, consistent with cellular heterogeneity in the response to insulin. RNA measurements indicated an enrichment of forkhead box O transcription factors in cells with a greater response to insulin. Moreover, we found evidence of increased Akt activation. These data are consistent with a heterogeneous cellular response to insulin and raise the possibility that these different subpopulations underlie the peculiar pathophysiology of hepatic insulin resistance.