Abstract
HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. In addition to cholesterol, Hmgcr activity is also required for synthesizing nonsterol isoprenoids, such as dolichol, ubiquinone, and farnesylated and geranylgeranylated proteins. Here, we investigated the effects of Hmgcr inhibition on nonsterol isoprenoids in the liver. We have generated new genetic models to acutely delete genes in the mevalonate pathway in the liver using AAV-mediated delivery of Cre-recombinase (AAV-Cre) or CRISPR/Cas9 (AAV-CRISPR). The genetic deletion of Hmgcr by AAV-Cre resulted in extensive hepatocyte apoptosis and compensatory liver regeneration. At the biochemical level, we observed decreased levels of sterols and depletion of the nonsterol isoprenoids, dolichol and ubiquinone. At the cellular level, Hmgcr-null hepatocytes showed ER stress and impaired N-glycosylation. We further hypothesized that the depletion of dolichol, essential for N-glycosylation, could be responsible for ER stress. Using AAV-CRISPR, we somatically disrupted dehydrodolichyl diphosphate synthase subunit (Dhdds), encoding a branch point enzyme required for dolichol biosynthesis. Dhdds-null livers showed ER stress and impaired N-glycosylation, along with apoptosis and regeneration. Finally, the combined deletion of Hmgcr and Dhdds synergistically exacerbated hepatocyte ER stress. Our data show a critical role for mevalonate-derived dolichol in the liver and suggest that dolichol depletion is at least partially responsible for ER stress and apoptosis upon potent Hmgcr inhibition.
Highlights
HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins
This line was crossed with FLP-transgenic mice and backcrossed to the C57BL/6J background, resulting in the generation of a mouse line harboring the exon 5 of Hmgcr flanked by loxP sites (Hmgcrfl/fl) (Fig. 1A)
The Hmgcr mRNA level was found to be significantly decreased in livers from Hmgcr liver-specific KO (LSKO) mice compared with control mice (Fig. 1D)
Summary
HMG-CoA reductase (Hmgcr) is the rate-limiting enzyme in the mevalonate pathway and is inhibited by statins. HMGCR activity is regulated by AMP-activated protein kinase (AMPK)-mediated phosphorylation, which can decrease cholesterol biosynthesis in low energy conditions [11]. Heterozygous mice appeared normal and showed unaffected cholesterol biosynthesis, due to compensatory upregulation of wild-type allele [12] Since this initial study, Hmgcr has been conditionally deleted in a number of tissues including the adipose [13], T-cells [14], skeletal muscle [15], myeloid cells [16], and the liver [17]. Hmgcr LSKO mice were generated by crossing floxed animals with the Albumin-Cre transgenic line These animals developed hepatic steatosis with apoptosis, hypoglycemia, and eventually liver failure and death. The gradual nature of Cre excision in this model allows ample time for physiological compensation, making it difficult to study direct effects
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