Insulin-like growth factor-1 coordinately induces the expression of fatty acid and cholesterol biosynthetic genes in murine C2C12 myoblasts

Abstract

Background: We present evidence that a major aspect of the mechanism of acute signal transduction regulation by insulin-like growth factor-1 (IGF-1) in cultured murine myoblasts is associated with a broad perturbation of many components of cholesterol and fatty acid biosynthetic pathways.

Results: We have used microarray transcriptional analysis to examine the acute effects of IGF-1 on global patterns of gene expression in C2C12 myoblasts and have identified approximately 157 genes that are up-regulated and 75 genes down-regulated from 2- to 6-fold after treatment with IGF-1. Of the up-regulated genes, 19 genes are associated with cholesterol biosynthesis and 5 genes specify aspects of fatty acid biosynthesis. In addition 10 recognized transcription factors are significantly induced by IGF-1 at 1 hour.

Conclusion: The SREBPs, important regulators of fatty acid and cholesterol biosynthesis, operate via a post-transcriptional route and no significant transcriptional induction was observed in the 4 hr of IGF-1 treatment. Since there are no prior reports of significant and coordinated perturbations of fatty acid and cholesterol biosynthetic pathways with IGF-1 in muscle cells, these findings provide a substantive expansion of our understanding of IGF-1 action and the signal transduction pathways mediated by it, its variants and insulin.

Figure 1

Global changes in gene expression following exposure of murine myoblasts to IGF-1. The number of genes that are either up- or down-regulated following acute exposure of C2C12 myoblasts to IGF-1 is shown (Fig. 1A). More genes are up- than down-regulated by IGF-1 at all the time points studied. Several, early induced genes are transcription factors and few of these are consistently expressed at all times. More genes are induced or repressed at the later times indicating secondary affects on the expression of a larger number of target genes, following the early surge in transcription factor expression. Figures 1 B-C show the temporal pattern of up- and down-regulated genes following exposure of murine myoblasts to IGF-1. Data from Venn diagrams show that several early expressed genes are transcription factors. Only 12 and 2 genes are up- (Fig. 1B) or down-(Fig. 1C) regulated, respectively, at all time points of acute IGF-1 treatment. At least 37 genes are induced at 2 hrs and remain so at 4 hrs. In contrast the expression of only 16 genes is repressed at 2 hrs and this remains so at 4 hrs. A tabulated list of genes for each of these groups is given elsewhere (see Tables 1 and 2 and also Additional files 1 and 2). Genes considered here and in other sections represent a 2-fold or greater change with respect to the zero time point.

Figure 2

Expression profiles of fatty acid and cholesterol biosynthetic pathway genes following exposure of murine myoblasts to IGF-1. The expression values (derived from microarray studies) are plotted as relative change over untreated control (100) values for Fatty acid synthase (Fas), ATP citrate lyase (Acly), Acyl CoA synthetase (Acs) (Fig. 2 A; Fatty acid desaturase (Fads), sterol C5 desaturase (SC5d), and Stearoyl CoA desaturase 1 (Scd1) (Fig. 2B). Data shows a two-fold or greater induction with IGF-1 treatment for all these genes (Fig. 2 A-B). Relative gene expression profiles are also shown for the genes involved in cholesterol biosynthesis (Fig 2C–F), namely HMG CoA synthase 1 (Hmgcs1) and HMG CoA reductase (Hmgcr) (Fig. 2C); Mevalonate (diphospho) decarboxylase (Mvd), Mevalonate kinase (Mk) and Phosphomevalonate kinase (Pmk) (Fig. 2D). Profiles are also shown for Cyp51, Hydroxysteroid dehydrogenase 17 beta (Hsd17b7), Lanosterol synthase (Lss) (Fig. 2E) and for the Low density Lipoprotein receptor (Ldlr), START domain 4 (Startd4) and cholesterol 25-hydroxylase (M25oh) genes (Fig. 2F). The genes involved in fatty acid and cholesterol biosynthesis are coordinately induced by IGF-1, though subtle variations exist in the time and extent of induction. In general, the induction is about 3–4 fold.

Figure 3

Genes involved in the regulation of fatty acid and cholesterol biosynthetic pathways. Microarray data show no significant change in SREBP-1 and -2 gene expression with IGF-1 treatment (Fig. 3A). However, when cycloheximide was added in conjunction with IGF-1 (IGF-1+CHX) to block nascent protein synthesis, both genes were activated to low and comparable extents, suggesting a similar pattern of regulation via 'derepression' (Fig. 3B). This level of 'derepression' was not seen with CHX treatment alone (data not shown). Insig2, Scap, S1p and S2p, are genes associated with SREBP-mediated lipogenesis, of these only Insig2 expression is moderately (1.6-fold) up-regulated (Fig 3C).

Figure 4

Qantitative-PCR based assay for selective fatty acid, cholesterol biosynthetic pathway and regulatory genes following IGF-1 treatment. The relative abundance of mRNA as compared to untreated control samples was assayed by q-PCR and plotted on a histogram for 6 genes involved in the cholesterol biosynthetic pathway, namely for HMG CoA synthase 1 (Hmgcs1), HMG CoA reductase (Hmgcr), (Mevalonate kinase (Mk/Mvk1), Cytochrome P450 51 (Cyp51), Lanosterol synthetase (Lss), and Dehydrocholestrol reductase (7Dhcr) (Fig. 4 A–F); 2 genes involved in fatty acid biosynthesis, namely Fatty Acid synthase (Fas) and stearoyl-Coenzyme A desaturase 1 (Scd1)(Fig. 4G and 4H); and 3 regulatory genes involved in fatty acid and cholesterol biosynthesis, namely SREBP-1 and -2, and Sterol Cleavage Activator Protein (SCAP) (Fig. 4J, 4K and 4L). The beta-Actin mRNA was assayed here as a control (Fig. 4I). In addition, 6 other genes unrelated to the fatty acid or cholesterol pathways were assayed; namely Chac1, Klf10, Ier2, Socs3, Shh and Dusp1 (Fig. 4 M-R, respectively) and the data show that the expression profile for all of these genes is similar to patterns obtained from our microarray experiments. The pattern obtained is in general agreement with the Affymetrix GeneChip data for these genes, with the exception of SREBP-1 where no change was detected with the microarray study. The extent of induction obtained with q-PCR, however, is greater than the microarray hybridization signals, probably due to the greater dynamic range observed with PCR amplification.