Gene expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in a poorly ketogenic mammal: effect of starvation during the neonatal period of the piglet

Abstract

The low ketogenic capacity of pigs correlates with a low activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase. To identify the molecular mechanism controlling such activity, we isolated the pig cDNA encoding this enzyme and analysed changes in mRNA levels and mitochondrial specific activity induced during development and starvation. Pig mitochondrial synthase showed a tissue-specific expression pattern. As with rat and human, the gene is expressed in liver and large intestine; however, the pig differs in that mRNA was not detected in testis, kidney or small intestine. During development, pig mitochondrial HMG-CoA synthase gene expression showed interesting differences from that in the rat: (1) there was a 2–3 week lag in the postnatal induction; (2) the mRNA levels remained relatively abundant through the suckling–weaning transition and at maturity, in contrast with the fall observed in rats at similar stages of development; and (3) the gene expression was highly induced by fasting during the suckling, whereas no such change in mitochondrial HMG-CoA synthase mRNA levels has been observed in rat. The enzyme activity of mitochondrial HMG-CoA synthase increased 27-fold during starvation in piglets, but remained one order of magnitude lower than rats. These results indicate that post-transcriptional mechanism(s) and/or intrinsic differences in the encoded enzyme are responsible for the low activity of pig HMG-CoA synthase observed throughout development or after fasting.

Isolation of pig mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene promoter: characterization of a peroxisome proliferator-responsive element

“…The PCR product was cloned taking advantage of an EcoRI restriction site introduced in the anchor sequence and AMP5 primers [19]. The insert of the resulting plasmids (pSIM 2, pSIM 3, pSIM6) were sequenced to determine the transcription start site.…”

“…In the 5h end amplification [32], a RACE kit from Clontech was used as previously described [19]. The PCR product was cloned taking advantage of an EcoRI restriction site introduced in the anchor sequence and AMP5 primers [19].…”

“…600 000 recombinants of the pig genomic DNA library were plated (30 000 plaque-forming units\150 mm-diameter dish) and transferred to nylon membranes. Recombinants were screened with $#P-labelled double-strand pSIM3 cDNA, which encodes for the 5h end (417 bp) of pig liver mitochondrial HMGCoA synthase cDNA [19], and three bacteriophages were isolated. The λGPMS clone (22 kb insert) was the only one that also hybridized with an EcoRI\HindIII 5h fragment (111 bp probe) of pSIM3 clone, and it was taken through three rounds of plaque purification.…”

“…Thus the suckling pig has a limited capacity for liver ketogenesis [14][15][16][17][18], and liver mRNA levels and the activity [16,19] of mitochondrial HMG-CoA synthase are very low in this period. In addition, while fasting of suckled rat pups does not lead to an increase in mitochondrial HMG-CoA synthase mRNA levels [20], starvation of 2-week-old piglets elicits a large stimulation of liver mRNA and activity [19], raises ketogenesis from long-chain fatty acids in itro [14] and increases circulating ketone bodies [21]. These starvation-associated changes in pig mitochondrial HMG-CoA synthase gene expression correlate with increased plasma non-esterified fatty acid (NEFA) levels, without significant changes of insulin or glucagon levels [19], suggesting that PPAR could also trans-activate the pig mitochondrial HMG-CoA synthase gene.…”

Comparative Proteomic Analysis of Liver Mitochondrial Proteins Derived from Cloned Adult Pigs Reconstructed with Meishan Pig Fibroblast Cells and European Pig Enucleated Oocytes

“…The HMG-CoA synthase family is a mitochondrial enzyme that catalyzes the first reaction of ketogenesis, a metabolic pathway that provides lipid-derived energy for various organs during times of carbohydrate deprivation, such as fasting. The enzyme activity of HMG-CoA synthase increased 27-fold during starvation in piglets [36]. In our study, none of the SCNT pigs showed morphological abnormalities, and they all had with normal reproductive ability.…”