Synergy analysis reveals association between insulin signaling and desmoplakin expression in palmitate treated HepG2 cells

Abstract

The regulation of complex cellular activities in palmitate treated HepG2 cells, and the ensuing cytotoxic phenotype, involves cooperative interactions between genes. While previous approaches have largely focused on identifying individual target genes, elucidating interacting genes has thus far remained elusive. We applied the concept of information synergy to reconstruct a "gene-cooperativity" network for palmititate-induced cytotoxicity in liver cells. Our approach integrated gene expression data with metabolic profiles to select a subset of genes for network reconstruction. Subsequent analysis of the network revealed insulin signaling as the most significantly enriched pathway, and desmoplakin (DSP) as its top neighbor. We determined that palmitate significantly reduces DSP expression, and treatment with insulin restores the lost expression of DSP. Insulin resistance is a common pathological feature of fatty liver and related ailments, whereas loss of DSP has been noted in liver carcinoma. Reduced DSP expression can lead to loss of cell-cell adhesion via desmosomes, and disrupt the keratin intermediate filament network. Our findings suggest that DSP expression may be perturbed by palmitate and, along with insulin resistance, may play a role in palmitate induced cytotoxicity, and serve as potential targets for further studies on non-alcoholic fatty liver disease (NAFLD).

Figure 1. Joint expression of gene pairs with different information synergy.

Blue and red points represent nontoxic and toxic samples, respectively. From top to bottom: Gene pairs with positive synergy jointly discriminate the phenotypes (indicated by the black dashed lines – joint information), whereas either of the genes alone provide little information in discriminating the phenotypes (indicated by the data points projected to the axes – marginal information). In contrast, genes with zero information synergy appear to be independent of the phenotypes, both jointly and marginally. Gene pairs with negative information synergy are marginally informative on the phenotype, such that either of the genes can provide information on the phenotype, but the additional information provided by the other gene does not enhance the discrimination of the phenotype, i.e. information provided by both genes about the phenotype is redundant.

Figure 2. Sub-network for insulin signaling genes and their neighbor genes in the synergy network.

Yellow nodes represent insulin signaling genes, and pink, green, blue and black nodes represent the neighbor genes connecting to one, two, three and more insulin signaling genes, respectively. The fifteen insulin genes, the top three neighbor genes (in Table 2) and the four genes connecting to more than three genes were labeled with gene names for reference.

Figure 3. Immuno-fluorescence images of HepG2 cells stained for DSP (green) and cell nuclei (blue) obtained using confocal microscopy (see methods).

Scale bars represent 50 µm. A) Untreated, control cells grown in regular growth media. B) Cells treated with palmitate for 48 hours show decrease in DSP levels C) Cells treated with palmitate for 48 hours and recovered in normal growth media for 72 hours show partial recovery of DSP expression. Cells treated with palmitate for 48 hours and recovered in growth media with insulin for D) 24 hours and E) 48 hours show partial recovery, whereas for F) 72 hours show complete recovery of DSP expression.

Figure 4. Quantitative effects of palmitate and insulin treatment on DSP expression (see methods).

Bars indicate relative expression of DSP under various treatment conditions. Palmitate treatment significantly decreases DSP expression where as subsequent treatment with insulin restores DSP expression. Lines represent pairs of condition where changes in DSP level are significant. * indicates significant difference (p<0.05) from control cells. ** indicates significant difference (p<0.05) from palmitate (48 hours) treated cells.

Figure 5. The expressions of gene pairs, namely of insulin signaling genes and DSP.

Shown are three pairs of insulin signaling genes connected to DSP in the synergy analysis (top) and three pairs obtained from the differential correlation analysis (bottom). Red and blue points represent toxic (treated with palmitate) and nontoxic samples, respectively.