Metabolic Consequences of TGFb Stimulation in CulturedPrimary Mouse Hepatocytes Screened from Transcript Data with ModeScore

Abstract

TGFb signaling plays a major role in the reorganization of liver tissue upon injury and is an important driver of chronic liver disease. This is achieved by a deep impact on a cohort of cellular functions. To comprehensively assess the full range of affected metabolic functions, transcript changes of cultured mouse hepatocytes were analyzed with a novel method (ModeScore), which predicts the activity of metabolic functions by scoring transcript expression changes with 987 reference flux distributions, which yielded the following hypotheses. TGFb multiplies down-regulation of most metabolic functions occurring in culture stressed controls. This is especially pronounced for tyrosine degradation, urea synthesis, glucuronization capacity, and cholesterol synthesis. Ethanol degradation and creatine synthesis are down-regulated only in TGFb treated hepatocytes, but not in the control. Among the few TGFb dependently up-regulated functions, synthesis of various collagens is most pronounced. Further interesting findings include: down-regulation of glucose export is postponed by TGFb, TGFb up-regulates the synthesis capacity of ketone bodies only as an early response, TGFb suppresses the strong up-regulation of Vanin, and TGFb induces re-formation of ceramides and sphingomyelin.

Figure 1

A Average expression values of metabolic genes (upon mapping to HepatoNet1) vs. all other genes, by expression profile (C = control, T = TGFβ treated). In B, the set of metabolic genes is split into genes encoding enzymes, transporters, and selected excretion proteins. Error bars indicate average standard deviation of the 3 repeat experiments. C and D show differences of average expressions. Red bars indicate down-regulation and green bars indicate up-regulation. Either 2 time points in the control experiment are compared (e.g., C1 h/24 h) or the same time point of control vs. treated sample (e.g., C/T 24 h). P-values refer to the probability that the averages of the two series are equal, determined with the Welch’s t test [18] and the average RNA abundance values. The error bars refer to the average of standard deviation of the repeats, not the standard deviation of the gene abundances (which would be huge in this diagram).

Figure 2

(A) Regulation of the degradation cascade of phenylalanine and tyrosine; (B) Regulation of selected collagens and a promoter. Red bars indicate down-regulation and green bars indicate up-regulation. Either 2 time points in the control experiment (e.g., C1h/24 h) or the same time point of control vs. TGFβ treated sample (e.g., C/T 24 h) are compared. Error bars indicate average standard deviation of 3 independent experiments. P-values refer to the probability that there is equal expression of two respective probe values, as determined with the Welch’s t test [18] in 3 independent experiments.

Figure 3

Regulation of genes involved in ethanol degradation (A); and bilirubin conjugation (B). Among the genes encoding alcohol and aldehyde dehydrogenase only those are selected that show a sufficient expression in hepatocytes and activity on ethanol/ethanal.

Figure 4

Regulation of genes involved in urea synthesis. Panel (A) shows genes essential for urea synthesis, whereas panel (B) shows genes used for the supply of precursors.

Figure 5

Regulation of genes involved in cholesterol synthesis.

Figure 6

Regulation of genes involved in glucose release from glycogen.

Figure 7

Regulation of genes involved in synthesis of β-hydroxybutyrate. Panel A shows comparisons of the 1 h and 24 h time points only, panel B shows comparisons involving the 6 h time point.

Figure 8

(A) Regulation of creatine synthesis and of some solitude genes; (B) Regulation of selected genes encoding TGFβ, its receptors and related proteins.