BHLHE40, a third transcription factor required for insulin induction of SREBP-1c mRNA in rodent liver
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Co-immunoprecipitation of BHLHE40 phosphorylated at serine-383 (S383).
(A) Protocol for identifying the phosphoproteins that are induced by feeding and interact with both LXRα and C/EBPβ. Rats were either fasted for 48 hr, or fasted for 48 hr and then refed with a …
Figure 2
Relative amounts of mRNA for BHLHE40 (A), SREBP-1c (B), LXRα (C), and C/EBPβ (D), in livers of rats subjected to fasting and refeeding.
Male rats (age 2–3 months) were either fasted for 48 hr, or fasted for 48 hr and then refed with a high-carbohydrate diet for 6 hr. Total liver RNA from eight rats in each group was subjected to …
Figure 3
Co-immunoprecipitation of LXRα, C/EBPβ, and BHLHE40 in livers from fasted and refed rats.
Nuclear extracts were prepared from livers of rats subjected to 48 hr fasting (F) or 48 hr fasting followed by 6 hr refeeding (R). Immunoprecipitations were carried out with agarose beads conjugated …
Figure 4
ChIP assay to demonstrate BHLHE40 binding to the enhancer/promoter region of rat SREBP-1c gene.
(A) Schematic diagram of rat SREBP-1c enhancer/promoter region, showing the location of the two LXREs. Arrows denote the location of the primers used in the ChIP assays. (B) ChIP assay was performed …
Figure 5
Requirement for mTOR in induction in mRNAs encoding SREBP-1c (A) and BHLHE40 (B) in livers of rats subjected to fasting and refeeding.
Sixteen male rats (age 2–3 months) were fasted for 48 hr. Six hours prior to sacrifice, eight rats received an intraperitoneal injection of 20 mg/kg rapamycin, and the other eight received vehicle. …
Figure 6
Overexpression of BHLHE40 fails to overcome rapamycin-mediated inhibition of SREBP-1c induction by insulin in primary rat hepatocytes.
Rat hepatocytes were prepared and plated on day 0 as described in Materials and methods. At 2 hr after plating, cells were infected with or without lentivirus expressing rat BHLHE40 (Lenti-B40; 5 × …
Figure 7
Time course of induction of BHLHE40 and SREBP-1c mRNA in livers of refed rats (A–C) and in primary rat hepatocytes treated with insulin (D–F).
(A–C) Male rats (age, 2–3 months) were fasted for 48 hr and then refed with a high-carbohydrate diet for the indicated time, after which total RNA from the liver was subjected to quantitative …
Figure 8
Knockdown of BHLHE40 mRNA in rat hepatocytes reduces insulin-induced mRNA for SREBP-1c, but not mRNA for LXRα or C/EBPβ.
Hepatocytes from non-fasted male rats (age, 2–3 months) on a chow diet were prepared and plated on day 0. After attachment for 2 hr, cells were transfected with either 50 nM control siRNA …
Figure 9
Knockout of Bhlhe40 gene in mice decreases SREBP-1c mRNA in livers of refed animals.
(A) Immunoblot analysis of liver nuclear extracts from wild type (WT) and Bhlhe40−/− (KO) mice. Nuclear extracts (20 µg protein) were subjected to 4–12% SDS-PAGE, followed by immunoblot analysis …
Figure 10
AAV-mediated injection of rat BHLHE40 restores the decrease of SREBP-1c mRNA in livers of refed Bhlhe40 knockout mice.
WT and KO mice (age 6 wk; 4 mice/group) received tail vein injections of AAV encoding a control mRNA (AAV-EGFP), wild-type BHLHE40, or mutant BHLHE40(S383A). Three weeks after injection, mice were …
Tables
Table 1
Primer sequence for mRNA measurements
Primer sequences were custom synthesized by Integrated DNA Technologies, Coraville, IA.
| Gene (species) | Sequences for forward and reverse primers (5'–3') |
|---|---|
| 36B4 (rat) | Forward: TTCCCACTGGCTGAAAAGGT Reverse: CGCAGCCGCAAATGC |
| Srebp-1c (rat) | Forward: GACGACGGAGCCATGGATT Reverse: GGGAAGTCACTGTCTTGGTTGTT |
| C/Ebpβ (rat) | Forward: AAGCTGAGCGACGAGTACAAGA Reverse: GTCAGCTCCAGCACCTTGTG |
| Lxrα (rat) | Forward: TTCCCACGGATGCTAATGAA Reverse: GAATGGACGCTGCTCAAAGT |
| Bhlhe40 (rat) | Forward: GCTTCCAGGAAACCATTGGA Reverse: GGCTAGGAAGCTGGGCTTCT |
| Apob (mouse) | Forward: CGTGGGCTCCAGCATTCTA Reverse: TCACCAGTCATTTCTGCCTTTG |
| Srebp-1c (mouse) | Forward: GGAGCCATGGATTGCACATT Reverse: GGCCCGGGAAGTCACTGT |
| Bhlhe40 (mouse) | Forward: TGGTGATTTGTCGGGAAGAAA Reverse: ACGGGCACAAGTCTGGAAAC |
| Lxrα (mouse) | Forward: TCTGGAGACGTCACGGAGGTA Reverse: CCCGGTTGTAACTGAAGTCCTT |
| C/Ebpβ (mouse) | Forward: AAGCTGAGCGACGAGTACAAGA Reverse: GTCAGCTCCAGCACCTTGTG |
| Cyclophilin (mouse) | Forward: TGGAGAGCACCAAGACAGACA Reverse: TGCCGGAGTCGACAATGAT |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36826.013
