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Prenatal caffeine exposure induces liver developmental dysfunction in offspring rats
in Journal of Endocrinology*(B He and Y Wen contributed equally to this work)
We previously showed that prenatal caffeine exposure (PCE) induces intrauterine growth retardation (IUGR) and high susceptibility to nonalcoholic fatty liver disease in offspring rats, and the underlying mechanisms are associated with fetal overexposure to maternal glucocorticoids. Herein, we aimed to verify whether PCE disrupts liver development before and after birth and explore its possible programming mechanism. In vivo, reduced fetal weights and increased IUGR rates were accompanied by fetal liver developmental dysfunction in PCE rats. Increased fetal serum corticosterone and decreased insulin-like growth factor 1 (IGF1) levels were observed. Both male and female fetal livers exhibited increased glucocorticoid function-related gene (Gr/C/ebpα) expression and inhibited IGF1 signaling pathway (Igf1/Igf1r/Akt2) expression. At PW6, the levels of serum corticosterone and glucocorticoid function-related genes in PCE offspring livers were decreased, while serum IGF1 and liver IGF1 signaling pathway expression were increased, accompanied by obvious catch-up growth and enhanced liver function. Furthermore, in PCE adult offspring under chronic stress, serum corticosterone and liver Gr/C/ebpα expression levels were elevated, while the serum IGF1 and liver IGF1 signaling pathway levels were decreased. In vitro, cortisol (not caffeine) upregulated GR and C/EBPα expression and downregulated IGF1R expression. The IGF1R expression downregulated by cortisol was partially reversed by GR or C/EBPα knockdown. In conclusion, PCE-induced liver developmental dysfunction in fetal rats and catch-up growth in IUGR offspring. The mechanisms may be closely associated with GR/C/EBPα upregulation and IGF1/IGF1R signaling pathway downregulation in the fetal liver, caused by intrauterine programming of the liver glucocorticoid–IGF1 axis induced by glucocorticoid overexposure.
Downloadable materials
- Supplementary Table 1. The oligonucleotide primers and PCR annealing temperature used in quantitative real-time
- Supplementary Table 2. Rat oligonucleotide primers and PCR annealing temperature used in quantitative real-time
- Supplementary Table 3. Oligonucleotide primers and PCR conditions in ChIP-PCR.
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Schematic illustration of animal treatment. From gestational day (GD) 9–20, pregnant Wistar rats were administrated intragastrically with caffeine (30, 60 and 120 mg/kg × day). At postnatal week (PW) 10, one rat was randomly selected from each litter for exposure to chronic stress for 2 weeks. The animals were killed at GD20, PW6 or PW12.
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Effects of prenatal caffeine exposure (PCE) on the body weight, intrauterine growth retardation (IUGR) rate and the liver structure of the fetal rats on gestational day 20. (A) Fetal body weight. (B) IUGR rate (%). (C) H&E staining of fetal liver. (D) Immunostaining of Ki67. (E) Quantification of Ki67 expression. (F) Ultrastructure of the fetal liver (transmission electron microscopy, ×8,000). n = 12 pregnant rats. n = 5 sections of each group were selected for H&E; n = 5 sections of each group were selected, and five random fields of each section were scored with respect to Ki67 expression. Scale bar = 100 μm. Mean ± s.e.m., **P < 0.01 vs the control. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
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Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of hepatic development- and glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis-related genes in fetal rats on gestational day 20. (A and B) ELISA of serum CORT and IGF1 concentration. (C, D and E) mRNA expression of liver development-promoting genes, including proliferating cell nuclear antigen (Pcna), hepatocyte nuclear factor 4α (Hnf4α) and albumin (Alb). (F and G) mRNA expression of liver development-suppressing genes, including cysteine-containing aspartate-specific protease-3 (Casp-3) and alpha-fetoprotein (Afp). (H, I, J, K and L) mRNA expression of liver GC-IGF1 axis-related genes, including glucocorticoid receptor (Gr), CCAAT enhancer-binding protein α (C/ebpα), Igf1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). n = 12 pooled samples from 12 different litters, Mean ± s.e.m., *P < 0.05, **P < 0.01 vs. the control.
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Effects of prenatal caffeine exposure (PCE) on the body weights, body weight gain rate and liver histology in offspring rats. (A and B) Body weights and body weight gain rates from postnatal week 1 (PW1) to 12 in male offspring. (C and D) Body weights and body weight gain rates from postnatal week 1 (PW1) to 12 in female offspring. (E) H&E staining of liver in male and female offspring at PW6. (F) Immunostaining of Ki67 in male and female offspring at PW6. (G) Quantification of Ki67 expression in male and female offspring at PW6. n = 8 offspring from 8 pregnant rats. n = 5 sections of each group were selected for H&E, and five random fields of each section scored with respect to Ki67 expression. Scale bar = 100 μm, mean ± s.e.m., *P < 0.05, **P < 0.01 vs the control. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
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Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of hepatic development-and glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis-related genes in offspring rats at postnatal week 6 (PW6). (A, B and C) mRNA expression of liver development-promoting genes, including proliferating cell nuclear antigen (Pcna), hepatocyte nuclear factor 4α (Hnf4α) and albumin (Alb). (D and E) mRNA expression of liver development-suppressing genes, including cysteine-containing aspartate-specific protease-3 (Casp-3) and alpha-fetoprotein (Afp). (F and G) ELISA analysis of serum CORT and IGF1 concentration. (H, I, J, K and L) mRNA expression of liver GC-IGF1 axis-related genes, including glucocorticoid receptor (Gr), CCAAT enhancer binding protein α (C/ebpα), Igf1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). n = 8 offspring from 8 pregnant rats. Mean ± s.e.m., * P < 0.05, ** P < 0.01 vs the control.
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Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of glucocorticoid-insulin like growth factor 1 (GC-IGF1) axis-related genes in offspring rats at postnatal week 12 (PW12) without or with chronic stress. (A and B) ELISA analysis of serum CORT and IGF1 concentrations in offspring rats at PW12 without chronic stress. (C, D, E, F and G) mRNA expression of liver GC-IGF1 axis-related genes in offspring rats at PW12 without chronic stress, including glucocorticoid receptor (Gr), CCAAT enhancer binding protein α (C/ebpα), IGF1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). (H and I) ELISA analysis of serum CORT and IGF1 concentrations in offspring rats at PW12 with chronic stress. (J, K, L, M and N) mRNA expression of liver GC-IGF1 axis-related genes, including Gr, C/ebpα, Igf1, Igf1r and Akt2, in rat offspring at PW12 with chronic stress. n = 8 offspring from 8 pregnant rats. Mean ± s.e.m., *P < 0.05, **P < 0.01 vs the control.
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Effects of caffeine or cortisol treatment for 24 h on cell viability and the expression levels of insulin-like growth factor 1 receptor (IGF1R), glucocorticoid receptor (GR) and CCAAT enhancer binding protein α (C/EBPα) in L02 cells. (A) Cell viability after caffeine treatment. (B) Cell viability after cortisol treatment. (C) mRNA expression of IGF1R after caffeine treatment. (D, E and F) mRNA expression of IGF1R, glucocorticoid receptor (GR) and CCAAT enhancer binding protein α (C/EBPα) after cortisol treatment. (G) Protein expression of GR and C/EBPA detected by Western blotting after cortisol treatment. (H) Protein expression of nuclear GR and C/EBPA detected by Western blotting after cortisol treatment. (I) mRNA expression of IGF1R after cortisol and GR siRNA treatment. (J) mRNA expression of IGF1R after cortisol and C/EBPα siRNA treatment. (K) Chromatin immunoprecipitation (ChIP)-PCR assay for the enrichment of C/EBPα at the IGF1R promoter after cortisol and C/EBPα siRNA treatment. IgG served as a negative control. All experiments were performed at least three times. Mean ± s.e.m., *P < 0.05, **P < 0.01 vs compared with the untreated cells.
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Prenatal caffeine exposure (PCE)-induced liver developmental toxicity and the related glucocorticoid-insulin-like growth factor 1 (GC-IGF1) mechanism. C/EBPα, CCAAT enhancer-binding protein α; GR, glucocorticoid receptor; IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; NAFLD, non-alcoholic fatty liver disease. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
Schematic illustration of animal treatment. From gestational day (GD) 9–20, pregnant Wistar rats were administrated intragastrically with caffeine (30, 60 and 120 mg/kg × day). At postnatal week (PW) 10, one rat was randomly selected from each litter for exposure to chronic stress for 2 weeks. The animals were killed at GD20, PW6 or PW12.
Effects of prenatal caffeine exposure (PCE) on the body weight, intrauterine growth retardation (IUGR) rate and the liver structure of the fetal rats on gestational day 20. (A) Fetal body weight. (B) IUGR rate (%). (C) H&E staining of fetal liver. (D) Immunostaining of Ki67. (E) Quantification of Ki67 expression. (F) Ultrastructure of the fetal liver (transmission electron microscopy, ×8,000). n = 12 pregnant rats. n = 5 sections of each group were selected for H&E; n = 5 sections of each group were selected, and five random fields of each section were scored with respect to Ki67 expression. Scale bar = 100 μm. Mean ± s.e.m., **P < 0.01 vs the control. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of hepatic development- and glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis-related genes in fetal rats on gestational day 20. (A and B) ELISA of serum CORT and IGF1 concentration. (C, D and E) mRNA expression of liver development-promoting genes, including proliferating cell nuclear antigen (Pcna), hepatocyte nuclear factor 4α (Hnf4α) and albumin (Alb). (F and G) mRNA expression of liver development-suppressing genes, including cysteine-containing aspartate-specific protease-3 (Casp-3) and alpha-fetoprotein (Afp). (H, I, J, K and L) mRNA expression of liver GC-IGF1 axis-related genes, including glucocorticoid receptor (Gr), CCAAT enhancer-binding protein α (C/ebpα), Igf1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). n = 12 pooled samples from 12 different litters, Mean ± s.e.m., *P < 0.05, **P < 0.01 vs. the control.
Effects of prenatal caffeine exposure (PCE) on the body weights, body weight gain rate and liver histology in offspring rats. (A and B) Body weights and body weight gain rates from postnatal week 1 (PW1) to 12 in male offspring. (C and D) Body weights and body weight gain rates from postnatal week 1 (PW1) to 12 in female offspring. (E) H&E staining of liver in male and female offspring at PW6. (F) Immunostaining of Ki67 in male and female offspring at PW6. (G) Quantification of Ki67 expression in male and female offspring at PW6. n = 8 offspring from 8 pregnant rats. n = 5 sections of each group were selected for H&E, and five random fields of each section scored with respect to Ki67 expression. Scale bar = 100 μm, mean ± s.e.m., *P < 0.05, **P < 0.01 vs the control. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of hepatic development-and glucocorticoid-insulin-like growth factor 1 (GC-IGF1) axis-related genes in offspring rats at postnatal week 6 (PW6). (A, B and C) mRNA expression of liver development-promoting genes, including proliferating cell nuclear antigen (Pcna), hepatocyte nuclear factor 4α (Hnf4α) and albumin (Alb). (D and E) mRNA expression of liver development-suppressing genes, including cysteine-containing aspartate-specific protease-3 (Casp-3) and alpha-fetoprotein (Afp). (F and G) ELISA analysis of serum CORT and IGF1 concentration. (H, I, J, K and L) mRNA expression of liver GC-IGF1 axis-related genes, including glucocorticoid receptor (Gr), CCAAT enhancer binding protein α (C/ebpα), Igf1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). n = 8 offspring from 8 pregnant rats. Mean ± s.e.m., * P < 0.05, ** P < 0.01 vs the control.
Effects of prenatal caffeine exposure (PCE) on serum corticosterone (CORT) content and mRNA expression levels of glucocorticoid-insulin like growth factor 1 (GC-IGF1) axis-related genes in offspring rats at postnatal week 12 (PW12) without or with chronic stress. (A and B) ELISA analysis of serum CORT and IGF1 concentrations in offspring rats at PW12 without chronic stress. (C, D, E, F and G) mRNA expression of liver GC-IGF1 axis-related genes in offspring rats at PW12 without chronic stress, including glucocorticoid receptor (Gr), CCAAT enhancer binding protein α (C/ebpα), IGF1, insulin-like growth factor 1 receptor (Igf1r) and protein kinase B beta (Akt2). (H and I) ELISA analysis of serum CORT and IGF1 concentrations in offspring rats at PW12 with chronic stress. (J, K, L, M and N) mRNA expression of liver GC-IGF1 axis-related genes, including Gr, C/ebpα, Igf1, Igf1r and Akt2, in rat offspring at PW12 with chronic stress. n = 8 offspring from 8 pregnant rats. Mean ± s.e.m., *P < 0.05, **P < 0.01 vs the control.
Effects of caffeine or cortisol treatment for 24 h on cell viability and the expression levels of insulin-like growth factor 1 receptor (IGF1R), glucocorticoid receptor (GR) and CCAAT enhancer binding protein α (C/EBPα) in L02 cells. (A) Cell viability after caffeine treatment. (B) Cell viability after cortisol treatment. (C) mRNA expression of IGF1R after caffeine treatment. (D, E and F) mRNA expression of IGF1R, glucocorticoid receptor (GR) and CCAAT enhancer binding protein α (C/EBPα) after cortisol treatment. (G) Protein expression of GR and C/EBPA detected by Western blotting after cortisol treatment. (H) Protein expression of nuclear GR and C/EBPA detected by Western blotting after cortisol treatment. (I) mRNA expression of IGF1R after cortisol and GR siRNA treatment. (J) mRNA expression of IGF1R after cortisol and C/EBPα siRNA treatment. (K) Chromatin immunoprecipitation (ChIP)-PCR assay for the enrichment of C/EBPα at the IGF1R promoter after cortisol and C/EBPα siRNA treatment. IgG served as a negative control. All experiments were performed at least three times. Mean ± s.e.m., *P < 0.05, **P < 0.01 vs compared with the untreated cells.
Prenatal caffeine exposure (PCE)-induced liver developmental toxicity and the related glucocorticoid-insulin-like growth factor 1 (GC-IGF1) mechanism. C/EBPα, CCAAT enhancer-binding protein α; GR, glucocorticoid receptor; IGF1, insulin-like growth factor 1; IGF1R, IGF1 receptor; NAFLD, non-alcoholic fatty liver disease. A full colour version of this figure is available at https://doi.org/10.1530/JOE-19-0066.
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