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. 2003 Mar 18;100(6):3155-60.
doi: 10.1073/pnas.0130116100. Epub 2003 Mar 6.

Liver-specific mRNA for Insig-2 down-regulated by insulin: implications for fatty acid synthesis

Daisuke Yabe  1 , Ryutaro Komuro, Guosheng Liang, Joseph L Goldstein, Michael S Brown
Affiliations

Liver-specific mRNA for Insig-2 down-regulated by insulin: implications for fatty acid synthesis

Daisuke Yabe et al. Proc Natl Acad Sci U S A. .

Abstract

Insig-1 and -2 are closely related proteins of the endoplasmic reticulum (ER) that block proteolytic activation of sterol regulatory element-binding proteins (SREBPs), transcription factors that activate the synthesis of cholesterol and fatty acids in liver and other organs. When cellular cholesterol levels are high, Insig proteins bind and trap SREBP cleavage-activating protein (SCAP), retaining it in the ER and preventing it from escorting SREBPs from ER to the site of proteolytic activation in the Golgi complex. Here, we report the discovery of a liver-specific transcript of Insig-2, designated Insig-2a. This transcript and the ubiquitous transcript, designated Insig-2b, differ through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Although the Insig-2a and -2b mRNAs encode identical proteins, they differ in patterns of regulation. Insig-2a is the predominant transcript in livers of fed animals, and it is selectively down-regulated by insulin. Insig-2a mRNA increases when mice are fasted, and it declines when they are refed. The transcript also increases in livers of rats whose insulin-secreting pancreatic beta cells have been destroyed by streptozotocin, and it is reduced when insulin is injected. The insulin-mediated fall in Insig-2a may allow SREBP-1c to be processed, thereby allowing insulin to stimulate fatty acid synthesis, even under conditions in which hepatic cholesterol levels are elevated.

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Figures

Figure 1
Figure 1
Alternative sequences at the 5′ end of Insig-2 mRNA from mouse liver. (A) Alternative promoter usage produces two transcripts, Insig-2a and -2b, in which two different first exons (1a and 1b) are spliced to a common exon-2 containing the initiation codon (ATG) for the Insig-2 protein. The positions of the probe used for the RNase protection assay and the primers used for real-time PCR are shown. (B) The nucleotide sequences of the first exons of mouse Insig-2a and -2b are shown together with the partial sequence of the common exon-2, which is shaded.
Figure 2
Figure 2
Expression of Insig-2a and -2b mRNAs in various mouse tissues. Total RNA from the indicated tissues of four C57BL/6J male mice (12 weeks of age) maintained on ad libitum diet was pooled. Aliquots of total RNA (10 μg) were hybridized for 10 min at 68°C to 32P-labeled complementary RNA probes for mouse Insig-2 as described in Materials and Methods. After digestion with RNase A/T1, protected fragments were separated by gel electrophoresis and exposed to film at −80°C for 24 h (Insig-2a and -2b). The Insig-2a in the liver was 11-fold more abundant than Insig-2b.
Figure 3
Figure 3
Reciprocal changes of Insig-2a and Insig-1 in livers of mice subjected to fasting and refeeding. Wild-type mice were subjected to a fasting and refeeding protocol, and total RNA from livers of four C57BL/6J male mice (12 weeks of age) was pooled and subjected to real-time PCR quantification as described in Materials and Methods. The nonfasted group (N) was maintained on ad libitum diet, the fasted group (F) was fasted 12 h, and the refed group (R) was fasted for 12 h and then refed a high-carbohydrate/low-fat diet for 12 h before study. Each value for the fasted and refed groups represents the amount of mRNA relative to that of the nonfasted group, which is arbitrarily defined as 1. FAS, fatty acid synthase. Values for plasma glucose and plasma insulin (mean ± SE) in the three groups of mice were as follows: nonfasted, 174 ± 14 mg/dl and 1.3 ± 0.7 ng/ml; fasted, 98 ± 13 mg/dl and 0.15 ± 0.01 ng/ml; and refed, 188 ± 10 mg/dl and 11 ± 1.2 ng/ml.
Figure 4
Figure 4
Changes in Insig-2a mRNA in livers of control rats and rats treated with STZ without or with insulin. Male Sprague–Dawley rats were treated with STZ for 36 h and then injected with insulin or vehicle for 6 h as described in Materials and Methods. Total RNAs isolated from livers of three male rats were pooled and subjected to real-time PCR quantification as described in Materials and Methods. Each value for the STZ-treated diabetic group (S) and STZ-treated diabetic group supplemented with insulin (S+I) represents the amount of mRNA relative to that of the control group (C). Values for plasma glucose (mean ± SE) in the three groups of rats were as follows: control group, 154 ± 2.5 mg/dl; STZ-treated group, 631 ± 26 mg/dl; STZ-treated group supplemented with insulin, 206 ± 47 mg/dl.
Figure 5
Figure 5
Insulin-mediated suppression of Insig-2a mRNA in primary rat hepatocytes. After pretreatment for 16 h with 1 nM insulin as described in Materials and Methods, cells were incubated for 12 h in the absence or presence of the indicated concentration of insulin (A) or for the indicated time in the presence of 100 nM insulin (B). Additions of insulin were made in a staggered fashion such that all samples were harvested at the same time. Total RNA was prepared from duplicate dishes, pooled, and subjected to real-time PCR quantification as described in Materials and Methods.
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