Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation

Abstract

Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Hydrodynamic gene delivery of an Igf2 plasmid used for transient Igf2 overexpression employing codon-optimized plasmid DNA resulted in a strong induction of hepatic Igf2 expression. The exogenously delivered Igf2 had no influence on endogenous Igf2 expression. The downstream kinase AKT was activated in Igf2 animals. Decreased ALT levels mirrored the cytoprotective effect of IGF2. Serum cholesterol was increased and sulfo-phospho-vanillin colorimetric assay confirmed lipid accumulation in Igf2-livers while no signs of inflammation were observed. Interestingly, hepatic cholesterol and phospholipids, determined by thin layer chromatography, and free cholesterol by filipin staining, were specifically increased. Lipid droplet (LD) size was not changed, but their number was significantly elevated. Furthermore, free cholesterol, which can be stored in LDs and has been reported to be critical for steatosis progression, was elevated in Igf2 overexpressing mice. Accordingly, Hmgcr/HmgCoAR was upregulated. To have a closer look at de novo lipid synthesis we investigated expression of the lipogenic transcription factor SREBF1 and its target genes. SREBF1 was induced and also SREBF1 target genes were slightly upregulated. Interestingly, the expression of Cpt1a, which is responsible for mitochondrial fatty acid oxidation, was induced. Hepatic IGF2 expression induces a fatty liver, characterized by increased cholesterol and phospholipids leading to accumulation of LDs. We therefore suggest a causal role for IGF2 in hepatic lipid accumulation.

Keywords: NASH; fatty liver; hydrodynamic gene delivery; insulin-like growth factor 2 (IGF2); lipid droplets.

Figure 1

Plasmid sequence and vector maps. The sequence of the Igf2 insert is shown. Restriction sites are highlighted in gray.

Figure 2

Characterization of the IGF2 overexpression model. (A) Exogenous Igf2 mRNA after hydrodynamic gene delivery of the Igf2 plasmid (Igf2) compared to the control plasmid (co), real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA. (B) Endogenous Igf2 mRNA levels, real-time RT-PCR (co, n = 10; Igf2, n = 11, independent two-sample t-test), were normalized to 18S mRNA and co. (C) Western blot analysis of phosphorylated and total AKT protein levels (co, n = 8; Igf2, n = 9, independent two-sample t-test), densitometric data were normalized to α-tubulin and co. (D) Liver weight on day 7 after plasmid injection (co, n = 10; Igf2, n = 11, independent two-sample t-test). (E) Serum parameters 2 days (glucose) or 7 days (other parameters) after plasmid injection. Glucose and triglycerides (co, n = 10; Igf2, n = 11, independent two-sample t-test). Cholesterol, HDL, AST, and ALT (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). All results are presented as mean ± SEM.

Figure 3

IGF2 induces lipid accumulation without inflammation. (A) Representative Scharlach Red staining for lipids (red staining) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were counterstained with hematoxylin. Original magnification was 200 × or 400 × (each group, n = 4). (B) Quantification of total lipids via sulfo-phospho-vanillin colorimetric assay. Igf2 animals normalized to co animals (co, n = 10; Igf2, n = 11, independent two-sample t-test). (C) mRNA levels of the macrophage marker Emr1 (F4/80), real-time RT-PCR (co, n = 10; Igf2, n = 11, independent two-sample t-test). Data were normalized to 18S mRNA and co. (D) Quantification of lipid classes by TLC: triglycerides (TG), cholesterol (CH), ceramides (CER), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC) in Igf2 animals normalized to co animals (co, n = 10; Igf2, n = 11, independent two-sample t-test). All results are presented as mean ± SEM.

Figure 4

IGF2 induces lipid droplet formation but has no influence on lipid droplet size. (A) Representative LD540 fluorescence staining for lipid droplets (LD, yellow droplets) in animals injected with Igf2 plasmid (Igf2) compared to the control plasmid (co), nuclei were stained with DAPI (blue) (scale bar: 20 μm) (upper panel). Five randomly selected pictures per sample were collected and analyzed with ImageJ. Counted LDs were normalized to the nuclei count (lower panel, left). Total LD area was normalized to the nuclei count (lower panel, middle). Total LD size was estimated as the mean area of all counted LDs (lower panel, right) (co, n = 10; Igf2, n = 11, independent two-sample t-test). (B) Representative filipin fluorescence staining for free cholesterol (black droplets) in Igf2 and co animals (co, n = 10; Igf2, n = 11, arrows designate examples for free cholesterol). (C) Levels of Hmgcr mRNA, real-time RT-PCR (co, n = 10; Igf2, n = 11, Wilcoxon rank sum test). Data were normalized to 18S mRNA and co. All results are presented as mean ± SEM.

Figure 5

Igf2 induces lipogenesis. (A) Lipogenic transcription factor Srebf1c mRNA (left) and SREBF1 protein (right) compared to the control plasmid (co). Left: real-time RT-PCR data (co, n = 10; Igf2, n = 11, independent two-sample t-test) were normalized to 18S mRNA and co. Right: Western blot analysis of SREBF1 (co, n = 8; Igf2, n = 9, independent two-sample t-test), densitometric data were normalized to α-tubulin and co. (B) Expression of SREBF1 target genes Fasn, Elovl6, Acaca, Scd, Gck determined by real-time RT-PCR (co, n = 10; Igf2, n = 11), normalized to 18S mRNA. Data are shown as fold of co. (c-f) Mlxipl (C), Nr1h3 (D), Cpt1 (E), Ppara (F) mRNA expression, determined by real-time RT-PCR data (co, n = 10; Igf2, n = 11, independent two-sample t-test), normalized to 18S mRNA and co. All results are presented as mean ± SEM.