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Inhibition of expression of the circadian clock gene Period causes metabolic abnormalities including repression of glycometabolism in Bombyx mori cells

View Article: PubMed Central - PubMed

ABSTRACT

Abnormalities in the circadian clock system are known to affect the body’s metabolic functions, though the molecular mechanisms responsible remain uncertain. In this study, we achieved continuous knockdown of B. mori Period (BmPer) gene expression in the B. mori ovary cell line (BmN), and generated a Per-KD B. mori model with developmental disorders including small individual cells and slow growth. We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism. Amino acids that used glucose metabolites as a source were also down-regulated, while lipid metabolism and nucleotide metabolism were significantly up-regulated. Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism, as well as to metabolites such as aspartate, alanine, and xanthine in other pathways. Further validation experiments showed that the activities of the key enzymes of glucose metabolism, hexokinase, phosphofructokinase, and citrate synthase, were significantly decreased and transcription of their encoding genes, as well as that of pyruvate kinase, were also significantly down-regulated. We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism, and may be associated with changes in cellular amino acid metabolism, and in cell growth and development.

No MeSH data available.


Knockdown efficiency of BmPer gene.(a) BmPer transcripts were analyzed by qRT-PCR with BmRp49 as an internal control. (b) BmPER protein levels determined by western blot with BmTublin protein as an internal control. WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 (repeated three times).
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f1: Knockdown efficiency of BmPer gene.(a) BmPer transcripts were analyzed by qRT-PCR with BmRp49 as an internal control. (b) BmPER protein levels determined by western blot with BmTublin protein as an internal control. WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 (repeated three times).

Mentions: Screening revealed that BmPer gene expression was continuously knocked down in B. mori ovary cells transfected with a recombinant vector expressing the interference sequence of the BmPer gene (Per-KD). BmPer gene transcription in Per-KD cells, determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), was downregulated by about 40% compared with wild type (WT) cells (Fig. 1a), while BmPER protein levels were significantly reduced by about 80%, according to western blotting (Fig. 1b). BmPer gene knockdown thus significantly reduced both gene and protein expression of Per. We investigated transcription of the genes encoding proteins involved in circadian clock oscillation, BmCRY1 (Cryptochrome1), BmCRY2 (Cryptochrome2), BmTIM (Timeless), BmCLK (Clock), and BmCYC (Cycle), and showed that rhythms in expression levels of all five genes were decreased or even absent after synchronization with dexamethasone (Dex), with expression levels of BmTim and BmClk genes being significantly down-regulated (Figure S2). These results indicated that continuous down-regulation of BmPer gene transcription and protein translation in Per-KD cells further disrupted the transcription of genes encoding the circadian oscillators, suggesting that circadian signal outputs may be affected in Per-KD cells.


Inhibition of expression of the circadian clock gene Period causes metabolic abnormalities including repression of glycometabolism in Bombyx mori cells
Knockdown efficiency of BmPer gene.(a) BmPer transcripts were analyzed by qRT-PCR with BmRp49 as an internal control. (b) BmPER protein levels determined by western blot with BmTublin protein as an internal control. WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 (repeated three times).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385517&req=5

f1: Knockdown efficiency of BmPer gene.(a) BmPer transcripts were analyzed by qRT-PCR with BmRp49 as an internal control. (b) BmPER protein levels determined by western blot with BmTublin protein as an internal control. WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 (repeated three times).
Mentions: Screening revealed that BmPer gene expression was continuously knocked down in B. mori ovary cells transfected with a recombinant vector expressing the interference sequence of the BmPer gene (Per-KD). BmPer gene transcription in Per-KD cells, determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), was downregulated by about 40% compared with wild type (WT) cells (Fig. 1a), while BmPER protein levels were significantly reduced by about 80%, according to western blotting (Fig. 1b). BmPer gene knockdown thus significantly reduced both gene and protein expression of Per. We investigated transcription of the genes encoding proteins involved in circadian clock oscillation, BmCRY1 (Cryptochrome1), BmCRY2 (Cryptochrome2), BmTIM (Timeless), BmCLK (Clock), and BmCYC (Cycle), and showed that rhythms in expression levels of all five genes were decreased or even absent after synchronization with dexamethasone (Dex), with expression levels of BmTim and BmClk genes being significantly down-regulated (Figure S2). These results indicated that continuous down-regulation of BmPer gene transcription and protein translation in Per-KD cells further disrupted the transcription of genes encoding the circadian oscillators, suggesting that circadian signal outputs may be affected in Per-KD cells.

View Article: PubMed Central - PubMed

ABSTRACT

Abnormalities in the circadian clock system are known to affect the body’s metabolic functions, though the molecular mechanisms responsible remain uncertain. In this study, we achieved continuous knockdown of B. mori Period (BmPer) gene expression in the B. mori ovary cell line (BmN), and generated a Per-KD B. mori model with developmental disorders including small individual cells and slow growth. We conducted cell metabolomics assays by gas chromatography/liquid chromatography-mass spectrometry and showed that knockdown of BmPer gene expression resulted in significant inhibition of glycometabolism. Amino acids that used glucose metabolites as a source were also down-regulated, while lipid metabolism and nucleotide metabolism were significantly up-regulated. Metabolite correlation analysis showed that pyruvate and lactate were closely related to glycometabolism, as well as to metabolites such as aspartate, alanine, and xanthine in other pathways. Further validation experiments showed that the activities of the key enzymes of glucose metabolism, hexokinase, phosphofructokinase, and citrate synthase, were significantly decreased and transcription of their encoding genes, as well as that of pyruvate kinase, were also significantly down-regulated. We concluded that inhibition of the circadian clock gene BmPer repressed glycometabolism, and may be associated with changes in cellular amino acid metabolism, and in cell growth and development.

No MeSH data available.