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. 2013 Oct 10;8(10):e75917.
doi: 10.1371/journal.pone.0075917. eCollection 2013.

GCN2 in the Brain Programs PPARγ2 and Triglyceride Storage in the Liver During Perinatal Development in Response to Maternal Dietary Fat

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Free PMC article

GCN2 in the Brain Programs PPARγ2 and Triglyceride Storage in the Liver During Perinatal Development in Response to Maternal Dietary Fat

Xu Xu et al. PLoS One. .
Free PMC article

Abstract

The liver plays a central role in regulating lipid metabolism and facilitates efficient lipid utilization and storage. We discovered that a modest increase in maternal dietary fat in mice programs triglyceride storage in the liver of their developing offspring. The activation of this programming is not apparent, however, until several months later at the adult stage. We found that the perinatal programming of adult hepatic triglyceride storage was controlled by the eIF2α kinase GCN2 (EIF2AK4) in the brain of the offspring, which stimulates epigenetic modification of the Pparγ2 gene in the neonatal liver. Genetic ablation of Gcn2 in the offspring exhibited reduced hepatic triglyceride storage and repressed expression of the peroxisome proliferator-activated receptor gamma 2 (Pparγ2) and two lipid droplet protein genes, Fsp27 and Cidea. Brain-specific, but not liver-specific, Gcn2 KO mice exhibit these same defects demonstrating that GCN2 in the developing brain programs hepatic triglyceride storage. GCN2 and nutrition-dependent programming of Pparγ2 is correlated with trimethylation of lysine 4 of histone 3 (H3K4me3) in the Pparγ2 promoter region during neonatal development. In addition to regulating hepatic triglyceride in response to modest changes in dietary fat, Gcn2 deficiency profoundly impacts the severity of the obese-diabetic phenotype of the leptin receptor mutant (db/db) mouse, by reducing hepatic steatosis and obesity but exacerbating the diabetic phenotype. We suggest that GCN2-dependent perinatal programming of hepatic triglyceride storage is an adaptation to couple early nutrition to anticipated needs for hepatic triglyceride storage in adults. However, increasing the hepatic triglyceride set point during perinatal development may predispose individuals to hepatosteatosis, while reducing circulating fatty acid levels that promote insulin resistance.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gcn2 deficient mice display reduced hepatic triglyceride but increased serum triglyceride levels.
(A). A comparison of phenotypic and metabolic parameters between wild type (WT) and Gcn2 KO (KO) mice, 8 months of age, in random fed state and in response to starvation (Data expressed as a percentage of KO mice to WT mice (see Table S1 for parameter values). (B). Triglyceride content in the VLDL/LDL and HDL fractions of the serum of wild type (WT) and Gcn2 KO (KO) mice (mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (C). Cholesterol content in the VLDL/LDL and HDL fractions of the serum in mice of the indicated genotypes (mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (D). Serum leptin concentration of mice of indicated genotypes (mean ± SEM, n≥8, *p<0.05 Gcn2 KO vs. WT). (E). Daily food intake of mice of the indicated genotypes fed ad libitum for one week (mean ± SEM, n≥8).
Figure 2
Figure 2. GCN2 regulates basal expression of PPARγ2 and lipid droplet proteins FSP27 and CIDEA in the liver but not the fasting response.
(A). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of wild type (WT) and Gcn2 KO (KO) mice, 8 months of age, (normalized to WT mice, mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (B). Expression of Pparγ1, Pparγ2, Fsp27 and Cidea mRNAs in livers of random fed, 24 hr-fasted and refed wild type (WT) and Gcn2 KO (KO) mice (normalized to WT mice, mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT).
Figure 3
Figure 3. GCN2 regulation of liver and serum triglycerides and expression of PPARγ2 and lipid droplet proteins FSP27 and CIDEA in the liver is dependent upon dietary fat.
(A). Liver triglyceride (TG) content of mice of indicated genotypes after Low Fat Chow (LFC) and Medium Fat Chow (MFC) feeding (mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (B). Serum triglyceride (TG) level of mice of indicated genotypes after LFC and MFC feeding (mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (C). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of mice of indicated genotypes fed with LFC and MFC (normalized to wildtype mice on LFC, mean ± SEM, n = 4, *p<0.05 Gcn2 KO vs. WT).
Figure 4
Figure 4. A block in leptin signaling amplifies the HiS/LoH triglyceride phenotype of Gcn2 mice and hyperglycemia of the db/db mice.
(A). Body weight (B) Blood glucose (C) Serum triglyceride (D) Liver triglyceride of Gcn2;db DKO (Gcn2 −/−; db/db) and db/db (Gcn2+/+;db/db) mice (mean ± SEM, n = 4, *p<0.05, Gcn2;db DKO versus db/db mice). (E). Oil red O staining of representative liver sections of Gcn2;db DKO mice and (F) db/db mice. (G). H&E staining of representative liver sections of Gcn2;db DKO mice and (H) db/db mice. Unstained empty areas indicated formation of lipid droplets. (I). mmunofluorescent (IF) staining of CIEDA proteins (green) in representative liver sections of Gcn2;db DKO mice and (J) db/db mice. Nuclei are stained with DAPI (blue). (K). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of mice of indicated genotypes (normalized to db/db mice, mean ± SEM, n = 8, *p<0.05, Gcn2;db DKO vs. db/db). (L). PPARγ and FSP27 protein western blotting from whole liver lysates of mice of indicated genotypes.
Figure 5
Figure 5. The nutritional programming of PPARγ2, FSP27, and CIDEA by GCN2 is fixed during perinatal development and resistant to nutritional changes at the adult stage.
(A). Pparγ1, Pparγ2, Fsp27 and Cidea mRNA levels relative to Gapdh mRNA in livers of 3 weeks, 6 months and 8 months old wild type (WT) and Gcn2 KO (KO) mice (mean ± SEM, n = 4, *p<0.05 Gcn2 KO vs. WT). (B). Illustration of time points for diet shift experiments. Upper panel illustrates the diet shift from MFC diet to LFC occurred two months of age. Lower panel illustrates the diet shift from MFC to LFC diet occurred at weaning (3 weeks of age). Mice in both experiments were euthanized at 8 months of age for analysis of serum and hepatic triglycerides and gene expression. (C). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of mice of indicated genotypes, which were reared on MFC chow diet from conception to 2 months of age and then switched to the lower fat chow diet (LFC) for additional 6 months (normalized to WT mice, mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (D). Expression of Pparγ2, Fsp27, Cidea mRNAs in livers of mice of indicated genotypes, which were reared on MFC chow diet before weaning and then switched to the lower fat chow diet (LFC) until 8 months old (normalized to WT mice, mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT). (E). Liver triglyceride content in mice of indicated genotypes, which were reared on MFC chow diet before weaning and then switched to the lower fat chow diet (LFC) until 8 months old (normalized to WT mice, mean ± SEM, n = 8, *p<0.05 Gcn2 KO vs. WT).
Figure 6
Figure 6. The PPARγ agonist rosiglitazone partially reverses the effect of GCN2 deficiency on liver and serum triglycerides and lipid droplet protein expression.
(A). Fsp27 and Cidea mRNA levels in livers relative to Gapdh mRNA of Gcn2 KO (KO) and wild type (WT) mice after rosiglitazone feeding rosiglitazone or vehicle treated Medium Fat chow diet (MFC) for 3 weeks (mean ± SEM, n = 3–7, *p<0.05 Fsp27 and Cidea Gcn2 KO rosiglitazone vs. MFC, *p<0.05 Cidea Gcn2 WT rosiglitazone vs. MFC, *p<0.05 Fsp27 and Cidea rosiglitazone treated Gcn2 KO vs. WT). (B). Liver triglyceride of Gcn2 KO (KO) and wild type (WT) mice after rosiglitazone feeding 3 weeks (mean ± SEM, n = 3–7, *p = 0.08 Gcn2 KO rosiglitazone vs. MFC, *p<0.05 rosiglitazone treated Gcn2 KO vs. WT).(C). Serum triglyceride of Gcn2 KO (KO) and wild type (WT) mice after rosiglitazone feeding 3 weeks (mean ± SEM, n = 3–7, *p<0.05 Gcn2 KO rosiglitazone vs. MFC, *p<0.05 Gcn2 WT rosiglitazone vs. MFC, *p<0.05 rosiglitazone treated Gcn2 KO vs. WT).
Figure 7
Figure 7. GCN2 programming of PPARγ2 during perinatal development is associated with histone 3 trimethylation.
(A). ChIP assay of H3K4me3, H3K9me3 and H3K27me3 on Pparγ2 promoters in livers of 3 weeks old Gcn2 KO (KO) and wild type (WT) mice. −2.0 kb, −0.3 kb, exon1, +0.5 kb indicated locations of primers used for PCR quantification of ChIP assays in the Pparγ2 promoter region. (Mean ± SEM, n = 4, *p<0.05 Gcn2 KO vs. WT). (B). ChIP assay of H3K4me3, H3K9me3 and H3K27me3 on Pparγ1 promoters in livers of 3 weeks old Gcn2 KO (KO) and wild type (WT) mice. −0.75 kb, −0.1 kb, +0.5 kb indicated locations of primers used for PCR quantification of ChIP assays in the Pparγ1 promoter region. (Mean ± SEM, n = 4). (C). ChIP assay of H3K4me3, H3K9me3 and H3K27me3 on Pparγ2 promoters in livers of 8 months old Gcn2 KO (KO) and wild type (WT) mice. −2.0 kb, −0.3 kb, exon1, +0.5 kb indicated locations of primers used for PCR quantitation of ChIP assays in Pparγ2 promoter regions. (mean ± SEM, n = 4, *p<0.05 Gcn2 KO vs. WT).
Figure 8
Figure 8. GCN2 expressed in the brain is responsible for PPARγ2, FSP27, and CIDEA and triglyceride storage programming in the liver.
(A). Liver triglyceride of liver-specific Gcn2 knockout mice (LiGcn2 KO), brain-specific Gcn2 knockout (BrGcn2 KO) and wildtype (WT) mice (mean ± SEM, n = 8, *p<0.05, BrGcn2 KO vs. WT). Total expression of wildtype Gcn2 mRNA was reduced by approximately 65% in the liver of the LiGcn2 KO mice and 74.7% in the brain of the BrGcn2 KO mice. Residual expression of Gcn2 mRNA in tissues that Gcn2 has been specifically deleted is likely to be due to expression of Gcn2 mRNA in minor cell type for which the Cre-driver transgene is not expressed. No reduction of wild-type Gcn2 mRNA was seen in the liver of the BrGcn2KO mice. (B). Serum triglyceride of mice of indicated genotypes (mean ± SEM, n = 8, *p<0.05, BrGcn2 KO vs. WT). (C). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of liver-specific knockout (LiGcn2 KO) and wildtype (WT) mice (normalized to WT mice, mean ± SEM, n = 8, *p<0.05, LiGcn2 KO vs. WT).(D). Expression of Pparγ1, Pparγ2, Fsp27, Cidea, Plin1, Plin2 and Sgms2 mRNAs in livers of brain-specific knockout mice (BrGcn2 KO) and wildtype (WT) mice (normalized to WT mice, mean ± SEM, n = 8, *p<0.05, BrGcn2 KO vs. WT).
Figure 9
Figure 9. Model for regulation and programming of hepatic triglyceride storage.
Dietary triglycerides increase free fatty acids (FA) in circulation that are taken up by the liver and acutely activate the Pparγ2 gene that in turn induces the expression of the lipid droplet genes Fsp27 and Cidea and increases triglyceride storage. During perinatal development GCN2 in the developing brain senses lipid concentrations provided maternally and signals the liver to program Pparγ2 for future triglyceride storage levels. GCN2 programming of Pparγ2 will specifically dictate the future expression of FSP27 and CIDEA and hepatic triglyceride storage but not that of adipose tissue.

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