Regulation of sterol regulatory element binding proteins in livers of fasted and refed mice

Abstract

Hepatic lipid synthesis is known to be regulated by food consumption. In rodents fasting decreases the synthesis of cholesterol as well as fatty acids. Refeeding a high carbohydrate/low fat diet enhances fatty acid synthesis by 5- to 20-fold above the fed state, whereas cholesterol synthesis returns only to the prefasted level. Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate genes involved in cholesterol and fatty acid synthesis. Here, we show that fasting markedly reduces the amounts of SREBP-1 and -2 in mouse liver nuclei, with corresponding decreases in the mRNAs for SREBP-activated target genes. Refeeding a high carbohydrate/low fat diet resulted in a 4- to 5-fold increase of nuclear SREBP-1 above nonfasted levels, whereas nuclear SREBP-2 protein returned only to the nonfasted level. The hepatic mRNAs for fatty acid biosynthetic enzymes increased 5- to 10-fold above nonfasted levels, a pattern that paralleled the changes in nuclear SREBP-1. The hepatic mRNAs for enzymes involved in cholesterol synthesis returned to the nonfasted level, closely following the pattern of nuclear SREBP-2 regulation. Transgenic mice that overproduce nuclear SREBP-1c failed to show the normal decrease in hepatic mRNA levels for cholesterol and fatty acid synthetic enzymes upon fasting. We conclude that SREBPs are regulated by food consumption in the mouse liver and that the decline in nuclear SREBP-1c upon fasting may explain in part the decrease in mRNAs encoding enzymes of the fatty acid biosynthetic pathway.

Figure 1

Immunoblot analysis of SREBP-1 (lanes 1–3) and SREBP-2 (lanes 4–6) in membranes (A) and nuclear extracts (B) from livers of mice in a nonfasted state (lanes 1 and 4), fasted for 24 h (lanes 2 and 5), or fasted for 24 h and refed a high carbohydrate/low fat diet for 12 h (lanes 3 and 6). For each group, livers from five mice shown in Table 1 were pooled, and aliquots (30 μg) of membranes and nuclear extracts were subjected to SDS/PAGE and electrophoretically transferred to a nitrocellulose filter. Immunoblot analysis was carried out with 5 μg/ml rabbit anti-mouse SREBP-1 IgG (lanes 1–3) or SREBP-2 IgG (lanes 4–6). Bound antibodies were visualized as described in the text. Filters were exposed to film for 60 s for SREBP-1 (lanes 1–3) and 90 s for SREBP-2 (lanes 4–6). P and N denote the precursor and cleaved nuclear forms of SREBP-1 or SREBP-2, respectively.

Figure 2

Changes in SREBP-1 protein in the livers of mice at various times after fasting (A) and refeeding a high carbohydrate/low fat diet after 24-h fast (B). Livers from three 8-wk-old SJL male mice were pooled for each treatment group, and aliquots (30 μg) of membranes (upper gels) and nuclear extracts (lower gels) were subjected to SDS/PAGE and electrophoretically transferred to a nitrocellulose filter. Immunoblot analysis was carried out with 5 μg/ml rabbit anti-mouse SREBP-1 IgG. Bound antibodies were visualized as described in the text. Filters were exposed to film for 60 s. P and N denote the precursor and nuclear cleaved forms of SREBP-1, respectively.

Figure 3

Amounts of various mRNAs in livers from nonfasted mice (N), mice fasted for 24 h (F), or mice fasted for 24 h and refed a high carbohydrate/low fat diet for 12 h (R) as measured by Northern blot hybridization. The mice are described in Table 1. Total RNA was isolated from five mice in each treatment group, pooled, and subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe. The amount of radioactivity in each band was quantified by exposure of the filters to a Bio-Imaging Analyzer with BAS1000 MacBus software (Fuji Medical Systems). The fold change in each mRNA relative to that of nonfasted mice was calculated after correction for loading differences by using the 28S ribosomal RNA as a loading control. These values are shown below each blot.

Figure 4

RNase protection assay for SREBP-1a and SREBP-1c mRNAs from livers of nonfasted mice (lanes 1 and 4), mice fasted for 24 h (lanes 2 and 5), or mice fasted for 24 h and refed a high carbohydrate/low fat diet for 12 h (lanes 3 and 6). The animals are described in Table 1. Pooled total RNA was isolated from five mice in each treatment group, and a 15-μg aliquot was hybridized to ³²P-labeled cRNA probes for mouse SREBP-1a, SREBP-1c, and β-actin for 10 min at 68°C. After RNase digestion, the protected fragments were separated by gel electrophoresis and exposed to film with an intensifying screen for 8 h at −80°C. The gels were also quantified in a PhosphorImager as described in the text. The fold change in each mRNA relative to that of the nonfasted mice was calculated after correction for loading differences, using β-actin mRNA as a loading control.

Figure 5

Immunoblot analysis of transgenic human SREBP-1c436 (lanes 1–4), endogenous mouse SREBP-1 (lanes 5–8), and endogenous mouse SREBP-2 (lanes 9–12) in membranes (A) and nuclear extracts (B) from livers of wild-type (WT) and TgSREBP-1c436 (Tg) mice in the nonfasted state or after fasting for 6 h as indicated. For each group, livers from mice shown in Table 2 were pooled, and aliquots (30 μg) of membranes and nuclear extracts were subjected to SDS/PAGE and electrophoretically transferred to a nitrocellulose filter. Immunoblot analysis was carried out using 5 μg/ml rabbit anti-human SREBP-1 IgG (lanes 1–4), anti-mouse SREBP-1 IgG (lanes 5–8), or anti-mouse SREBP-2 IgG (lanes 9–12) as the primary antibody. The anti-mouse SREBP-1 IgG antibody is specific for mouse SREBP-1 and does not cross-react with the human SREBP-1 transgene product. Bound antibodies were visualized as described in the text. Filters were exposed to film for 15 s for the truncated human SREBP-1, 25 s for mouse SREBP-1, and 90 s for mouse SREBP-2.

Figure 6

Amounts of various mRNAs in livers from wild-type (WT) and TgSREBP-1c436 (Tg) mice in the nonfasted state (N) or fasted (F) for 6 h as measured by blot hybridization. The mice used in this experiment are described in Table 2. Total RNA was isolated, pooled, and subjected to electrophoresis and blot hybridization with the indicated ³²P-labeled probe. The amount of radioactivity in each band was quantified as described in the text. The fold change in each mRNA relative to that of wild-type nonfasted mice was calculated after correction for loading differences by using the 28S ribosomal RNA as a loading control. These values are shown below each blot.