Deciphering hepatocellular responses to metabolic and oncogenic stress

Abstract

Each cell type responds uniquely to stress and fractionally contributes to global and tissue-specific stress responses. Hepatocytes, liver macrophages (MΦ), and sinusoidal endothelial cells (SEC) play functionally important and interdependent roles in adaptive processes such as obesity and tumor growth. Although these cell types demonstrate significant phenotypic and functional heterogeneity, their distinctions enabling disease-specific responses remain understudied. We developed a strategy for the simultaneous isolation and quantification of these liver cell types based on antigenic cell surface marker expression. To demonstrate the utility and applicability of this technique, we quantified liver cell-specific responses to high-fat diet (HFD) or diethylnitrosamine (DEN), a liver-specific carcinogen, and found that while there was only a marginal increase in hepatocyte number, MΦ and SEC populations were quantitatively increased. Global gene expression profiling of hepatocytes, MΦ and SEC identified characteristic gene signatures that define each cell type in their distinct physiological or pathological states. Integration of hepatic gene signatures with available human obesity and liver cancer microarray data provides further insight into the cell-specific responses to metabolic or oncogenic stress. Our data reveal unique gene expression patterns that serve as molecular “fingerprints” for the cell-centric responses to pathologic stimuli in the distinct microenvironment of the liver. The technical advance highlighted in this study provides an essential resource for assessing hepatic cell-specific contributions to metabolic and oncogenic stress, information that could unveil previously unappreciated molecular mechanisms for the cellular crosstalk that underlies the continuum from metabolic disruption to obesity and ultimately hepatic cancer.

Methodology for measuring oxidative capacity of isolated peroxisomes in the Seahorse assay

“…To selectively eliminate mitochondria from our isolated fractions, we included an additional purification step that leveraged the MACS column purification system. The MACS purification system has been successfully used by our laboratory to isolate individual populations of cells, and presented a viable option that could be similarly applied to organelles [ 28 ]. We carefully selected carnitine palmitoyl transferase 1B (Cpt1B) as an integral outer mitochondrial membrane protein that could be exploited to specifically capture mitochondria while allowing peroxisomes to remain unaltered in the purification scheme.…”

Calcium/calmodulin-dependent protein kinase kinase 2 regulates hepatic fuel metabolism

“…For the CaMKK2 LKO transcriptome response, all genes were ranked by the fold-change between the CaMKK2 LKO and the CaMKK2 f/f sample groups. Normalized Enrichment Score (NES) and adjusted q-values were computed utilizing the GSEA method, based on 1000 random permutations of the ranked genes as previously described [ 32 , 59 , 60 ]. Enriched pathways were determined using the gene set collections at the Molecular Signature Database (MSigDB, http://www.broadinstitute.org/gsea/msigdb/ ) using a hypergeometric distribution (p < 0.05).…”