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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.


Effect of BmPer gene knockdown on BmN cell growth.(a) Cell growth velocity measured by MTT method. (b) Cells were transferred to a coverslip when they reached 70% confluence. After the cells were completely adherent (at least 12 h) the diameters of the nuclei were measured (average of the longest and shortest diameters). (c) Cell nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI) and cell membranes stained with Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (showing cell morphology). WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 and **p ≤ 0.01 (n = 3 plates, observations and statistical analyses were repeated at least five times). Bar = 50 μm.
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f2: Effect of BmPer gene knockdown on BmN cell growth.(a) Cell growth velocity measured by MTT method. (b) Cells were transferred to a coverslip when they reached 70% confluence. After the cells were completely adherent (at least 12 h) the diameters of the nuclei were measured (average of the longest and shortest diameters). (c) Cell nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI) and cell membranes stained with Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (showing cell morphology). WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 and **p ≤ 0.01 (n = 3 plates, observations and statistical analyses were repeated at least five times). Bar = 50 μm.

Mentions: Per-KD cells showed distinctly different phenotypic traits from WT cells. Cell growth rate measured by MTT showed that Per-KD cells grew slower than WT cells; the growth velocity of Per-KD cells was decreased by about 30% within 4 h of measurement (Fig. 2a). Staining of cell membranes and nuclei also revealed that Per-KD cells were smaller than WT cells (Fig. 2b), and the average diameter of WT nuclei stained with DAPI was 10.5 μm, compared with 7 μm in Per-KD nuclei (p ≤ 0.05) (Fig. 2b,c).


Inhibition of expression of the circadian clock gene Period causes metabolic abnormalities including repression of glycometabolism in Bombyx mori cells
Effect of BmPer gene knockdown on BmN cell growth.(a) Cell growth velocity measured by MTT method. (b) Cells were transferred to a coverslip when they reached 70% confluence. After the cells were completely adherent (at least 12 h) the diameters of the nuclei were measured (average of the longest and shortest diameters). (c) Cell nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI) and cell membranes stained with Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (showing cell morphology). WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 and **p ≤ 0.01 (n = 3 plates, observations and statistical analyses were repeated at least five times). Bar = 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Effect of BmPer gene knockdown on BmN cell growth.(a) Cell growth velocity measured by MTT method. (b) Cells were transferred to a coverslip when they reached 70% confluence. After the cells were completely adherent (at least 12 h) the diameters of the nuclei were measured (average of the longest and shortest diameters). (c) Cell nuclei stained with 4′-6-diamidino-2-phenylindole (DAPI) and cell membranes stained with Dil (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) (showing cell morphology). WT, wild-type BmN cells; Per-KD, BmPer knockdown BmN cells. *p ≤ 0.05 and **p ≤ 0.01 (n = 3 plates, observations and statistical analyses were repeated at least five times). Bar = 50 μm.
Mentions: Per-KD cells showed distinctly different phenotypic traits from WT cells. Cell growth rate measured by MTT showed that Per-KD cells grew slower than WT cells; the growth velocity of Per-KD cells was decreased by about 30% within 4 h of measurement (Fig. 2a). Staining of cell membranes and nuclei also revealed that Per-KD cells were smaller than WT cells (Fig. 2b), and the average diameter of WT nuclei stained with DAPI was 10.5 μm, compared with 7 μm in Per-KD nuclei (p ≤ 0.05) (Fig. 2b,c).

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.