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Role of protein synthesis in the carbohydrate-induced changes in the activities of acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase in cultured rat hepatocytes.

ABSTRACT: Changes in the activities of acetyl-CoA carboxylase and HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase were studied in primary cultures of adult-rat hepatocytes after exposure of the cells to insulin and/or carbohydrates. To determine the contribution of protein synthesis to changes in enzyme activity, the relative rate of synthesis of each enzyme was measured and the amount of translatable mRNA coding for the enzymes was determined by translation in vitro and immunoprecipitation. Addition of insulin to the culture medium increased the activities of acetyl-CoA carboxylase and HMG-CoA reductase by approx. 4- and 3-fold respectively. Although similar increases in the relative rate of synthesis of each protein and template activity were noted, initial increases in the activity of each enzyme occurred before any changes in protein synthesis were observed, suggesting the involvement of post-translational modification of enzyme activity in addition to changes in protein synthesis. The addition of fructose to the culture medium, in the absence of insulin, increased the activity of the carboxylase and the reductase approx. 3-fold, similar to the effects of insulin. However, the effect of fructose was to increase the rate of synthesis and the amount of translatable mRNA coding for acetyl-CoA carboxylase, whereas the increase in the activity of HMG-CoA reductase was not accompanied by any changes in the rate of synthesis or template activity. The effects of fructose could not be mimicked by glucose unless insulin was also present in the culture medium. Similar to observations in vitro, the injection of insulin or the feeding of a high-fructose diet to rats made diabetic by the injection of streptozotocin produced an increase in the activities of acetyl-CoA carboxylase and HMG-CoA reductase, and only the increase in the activity of the carboxylase was accompanied by an increase in the amount of translatable mRNA coding for the enzyme. The results are discussed in terms of the effects of fructose on the synthesis of enzymes involved in lipogenesis.

SUBMITTER: Spence JT

PROVIDER: S-EPMC1144925 | biostudies-other | 1985 May

REPOSITORIES: biostudies-other

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Project description:BackgroundPolyadenylation is a crucial process that terminates mRNA molecules at their 3'-ends. It has been observed that alternative polyadenylation (APA) can generate multiple transcripts from a single gene locus, each with different polyadenylation sites (PASs). This leads to the formation of several 3' untranslated regions (UTRs) that vary in length and composition. APA has a significant impact on approximately 60-70% of eukaryotic genes and has far-reaching implications for cell proliferation, differentiation, and tumorigenesis.ResultsIn this study, we conducted long-read, single-molecule sequencing of mRNA from peanut seeds. Our findings revealed that over half of all peanut genes possess over two PASs, with older developing seeds containing more PASs. This suggesting that the PAS exhibits high tissue specificity and plays a crucial role in peanut seed maturation. For the peanut acetyl-CoA carboxylase A1 (AhACCA1) gene, we discovered four 3' UTRs referred to UTR1-4. RT-PCR analysis showed that UTR1-containing transcripts are predominantly expressed in roots, leaves, and early developing seeds. Transcripts containing UTR2/3 accumulated mainly in roots, flowers, and seeds, while those carrying UTR4 were constitutively expressed. In Nicotiana benthamiana leaves, we transiently expressed all four UTRs, revealing that each UTR impacted protein abundance but not subcellular location. For functional validation, we introduced each UTR into yeast cells and found UTR2 enhanced AhACCA1 expression compared to a yeast transcription terminator, whereas UTR3 did not. Furthermore, we determined ACC gene structures in seven plant species and identified 51 PASs for 15 ACC genes across four plant species, confirming that APA of the ACC gene family is universal phenomenon in plants.ConclusionOur data demonstrate that APA is widespread in peanut seeds and plays vital roles in peanut seed maturation. We have identified four 3' UTRs for AhACCA1 gene, each showing distinct tissue-specific expression patterns. Through subcellular location experiment and yeast transformation test, we have determined that UTR2 has a stronger impact on gene expression regulation compared to the other three UTRs.

Project description:Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1M-NM-^T acs2M-NM-^T S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h-1 were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.21 h-1) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While, for the first time, demonstrating that yeast ACS can be fully replaced by alternative reactions, this study demonstrates that further modifications are needed to achieve optimal in vivo efficiencies of the supply of acetyl-CoA as product precursor. To investigate the impact of introduced changes in native pathway of cytosolic acetyl-CoA formation in S. cerevisiae, a DNA microarray-based transcriptome analysis was performed on aerobic or anaerobic, glucose-limited chemostat cultures.

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Role of protein synthesis in the carbohydrate-induced changes in the activities of acetyl-CoA carboxylase and hydroxymethylglutaryl-CoA reductase in cultured rat hepatocytes.

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