Supplemental Data for
FGFR4 Prevents Hyperlipidemia and Insulin Resistance but Underlies High Fat Diet-Induced Fatty Liver by X. Huang, et al.
Supplemental Table 1. Primer sequences used for real-time PCR
Gene / Sequence ID / Forward Primers / Reverse PrimersACC / NM_133360 / GGACAGACTGATCGCAGAGAAAG / TGGAGAGCCCCACACACA
CD36 / NM_007643 / TCATATTGTGCTTGCAAATCCAA / GCTTTACCAAAGATGTAGCCAGTGT
CYP7A / NM_007824 / AGCAACTAAACAACCTGCCAGTACTA / GTCCGGATATTCAAGGATGCA
FAS / NM_007988 / GCCTACTACATAGATTGCATC / TGTGTTCCACATCAAGAAACT
G6Pase / NM_008061 / GACTCCCAGGACTGGTTCAT / GGGCGTTGTCCAAACAGAAT
MCAD / NM_007382 / CCCGTTCCCTCTCATCAAAAG / ACACCCATACGCCAACTCTTCG
MTP / NM_008642 / CAAGCTCACGTACTCCACTGAAG / TCATCATCACCATCAGGATTCCT
PEPCK / NM_011044 / GTCTATGAAGCCCTCAGCT / AAGAAGGGTCGCATGGCAA
PPAR α / NM_011144 / GACAAGGCCTCAGGGTACCA / GCCGAATAGTTCGCCGAAA
PPAR γ / NM_011146 / AAGGCGAGGGCGATCTTG / ATCATTAAGGAATTCATGTCGTAGATGAC
SCD1 / NM_009127 / GGCCTGTACGGGATCATACTG / GGTCATGTAGTAGAAAATCCCGAAGA
SREBP1C / NM_011480 / TGCGTGGTTTCCAACATGAC / TGGCCTCATGTAGGAATACCCT
β actin / NM_007393 / AGGCCCAGAGCAAGAGAGGTA / GGGGTGTTGAAGGTCTCAAACA
Supplemental Methods
In vivo Insulin signaling analysis. Mice were fasted overnight and injected intraperitoneally with 5U/kg body weight of insulin. Mice were then sacrificed 5 minutes after insulin stimulation. Liver was removed and snap-frozen in liquid nitrogen and stored at -80 ºC until use. Liver samples were homogenized with RIPA buffer (1XPBS, 1% NP40, 0.5% sodium deoxycholate, 0.1% SDS). For immunoprecipitation, 2mg total protein was incubated with 2ug of anti-insulin receptor β subunit for 2 hrs at 4ºC on a rocker.Then protein A-Sepharose was added and incubated for overnight. The beads were then washed for 4 times. For western blot analysis, immunoprecipitates or 100 μg liver lysate were resolved on 8% SDS-PAGE and transferred to Hybond-P membrane which was incubated with the primary antibodies, washed 3 times and then incubated with the second antibodies conjugated to horseradish peroxidase (Bio-Rad). Bands were visualized by development with the ECL-Plus detection reagents (Amersham). Anti-insulin receptor β subunit antibodies were from Santa Cruz Biotechnology (SC-711). Anti-phosphotyrosine antibodies were from Upstate (05-321). Akt, p-Akt (ser473) antibodies were from Cell Signaling Technology (9271, 9272).
Supplemental Fig. 1. FGFR4 deficiency does not impair liver insulin signaling. Phosphorylation of insulin receptor (IR)and Akt after insulin stimulation. Liver tissue homogenate from 3-4 mice per group were pooled together and subjected to immunoprecipitation (IP) and immunoblotting (IB) using antibodies as indicated. The gels were scanned and quantified by densitometry. The ratio of intensities of the phosphorylated and total protein bands of WT mice on a normal diet was set as 1. Results are representative of 3 independent experiments.
Supplemental Fig. 2. Restoration of hepatic FGFR4 reversed expression of liver genes involved in lipid metabolism in FGFR4-/- mice. Liver gene expression levels were determined in the indicated mice on the indicated diet by quantitative real-time PCR analysis of steady-state mRNA levels and normalized to β-actin expression. Expression level of each gene in WT mice on a normal diet was set to 1. Data are mean ± SD of three independent experiments for each gene with 4 to 5 mice of each genotype on each dietary regimen. *p<0.05, **p<0.01 relative to the corresponding FGFR4-/- mice.
Supplemental Fig. 3. Hepatic FGFR4 does not affect glucose tolerance or insulin sensitivity.A,B: Plasma glucose levels were determined in mice overexpressing Alb-caFGFR4 and WT FVB strain at the indicated times after glucose and insulin administration respectively.C, D:Plasma glucose levels were determined in the FGFR4-/- x Alb-caFGFR4 hybrid and FGFR4-/- littermates at the indicated time after glucose and insulin administration respectively. 1 g glucose per kg body weight or 0.6 U insulin per kg body weight was administrated. Data are expressed as mean ± SD (n = 6-9 mice). N = normal diet; HF = high fat diet.