Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin
Luke J. Engelking, … , Michael S. Brown, Guosheng Liang
Luke J. Engelking, … , Michael S. Brown, Guosheng Liang
Published September 1, 2006
Citation Information: J Clin Invest. 2006;116(9):2356-2365. https://doi.org/10.1172/JCI28988.
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Severe facial clefting in Insig-deficient mouse embryos caused by sterol accumulation and reversed by lovastatin

Abstract

Insig-1 and Insig-2 are regulatory proteins that restrict the cholesterol biosynthetic pathway by preventing proteolytic activation of SREBPs and by enhancing degradation of HMG-CoA reductase. Here, we created Insig–double-knockout (Insig-DKO) mice that are homozygous for null mutations in Insig-1 and Insig-2. After 18.5 days of development, 96% of Insig-DKO embryos had defects in midline facial development, ranging from cleft palate (52%) to complete cleft face (44%). Middle and inner ear structures were abnormal, but teeth and skeletons were normal. The animals were lethargic and runted; they died within 1 day of birth. The livers and heads of Insig-DKO embryos overproduced sterols, causing a marked buildup of sterol intermediates. Treatment of pregnant mice with the HMG-CoA reductase inhibitor lovastatin reduced sterol synthesis in Insig-DKO embryos and reduced the pre-cholesterol intermediates. This treatment ameliorated the clefting syndrome so that 54% of Insig-DKO mice had normal faces, and only 7% had cleft faces. We conclude that buildup of pre-cholesterol sterol intermediates interferes with midline fusion of facial structures in mice. These findings have implications for the pathogenesis of the cleft palate component of Smith-Lemli-Opitz syndrome and other human malformation syndromes in which mutations in enzymes catalyzing steps in cholesterol biosynthesis produce a buildup of sterol intermediates.

Authors

Luke J. Engelking, Bret M. Evers, James A. Richardson, Joseph L. Goldstein, Michael S. Brown, Guosheng Liang

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Figure 7

Prevention of craniofacial defects inInsig -DKO embryos after feeding of lovastatin to pregnant females.

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Prevention of craniofacial defects inInsig ...
Insig-1+/–Insig-2–/– mice were mated with each other, and mated females were identified by the presence of a postcopulatory vaginal plug. Mated females were fed ad libitum a regular chow diet (red bars) or a chow diet containing 0.2% (wt/wt) lovastatin (blue bars) from 5.5 to 18.5 dpc. (A) Embryos from 50 litters in both dietary groups were collected by caesarean section at 18.5 dpc and analyzed for craniofacial morphology by 2 independent observers prior to genotyping by PCR. The 50 litters in the control chow diet group contained a total of 341 embryos, of which 78 had the Insig-DKO genotype (same data as in Table 1). The 50 litters in the lovastatin-fed group contained a total of 361 embryos, of which 76 had the DKO genotype. The embryos from both groups were harvested in 5 different experiments over a 6-month period in which 5–19 pregnant females in both dietary groups were studied concurrently. The mean ± SEM for body weights of lovastatin-treated DKO embryos was 0.69 ± 0.01 g as compared with 1.08 ± 0.01 g for lovastatin-treated control embryos. (B–E) Three Insig-DKO embryos from lovastatin-fed females represented in A were processed for histological analysis. One embryo (B and D) showed a cleft palate phenotype (not corrected) with middle and inner ear abnormalities, and 2 embryos showed normal craniofacial phenotype (corrected; 1 of which is shown in C and E). Arrows indicate the lateral margins of the palatal shelves. pa, palate; pca, pars canalicularis; pco, pars cochlearis; s, stapes; sa, stapedial artery. Scale bars: 0.5 mm.