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Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development.

ABSTRACT: Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.

SUBMITTER: Wicheanvonagoon S

PROVIDER: S-EPMC1144008 | biostudies-other | 1984 Jul

REPOSITORIES: biostudies-other

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Project description:ObjectivesThyroid hormone (T3) and high glucose concentrations are critical components of β-cell maturation and function. In the present study, we asked whether T3 and glucose signaling pathways coordinately regulate transcription of genes important for β-cell function and proliferation.MethodsRNA-seq analysis was performed on cadaveric human islets from five different donors in response to low and high glucose concentrations and in the presence or absence of T3. Gene expression was also studies in sorted human β-cells, mouse islets and Ins-1 cells by RT-qPCR. Silencing of the thyroid hormone receptors (THR) was conducted using lentiviruses. Proliferation was assessed by ki67 immunostaining in primary human/mouse islets. Chromatin immunoprecipitation and proximity ligation assay were preformed to validate interactions of ChREBP and THR.ResultsWe found glucose-mediated expression of carbohydrate response element binding protein alpha and beta (ChREBPα and ChREBPβ) mRNAs and their target genes are highly dependent on T3 concentrations in rodent and human β-cells. In β-cells, T3 and glucose coordinately regulate the expression of ChREBPβ and PCK1 (phosphoenolpyruvate carboxykinase-1) among other important genes for β-cell maturation. Additionally, we show the thyroid hormone receptor (THR) and ChREBP interact, and their relative response elements are located near to each other on mutually responsive genes. In FACS-sorted adult human β-cells, we found that high concentrations of glucose and T3 induced the expression of PCK1. Next, we show that overexpression of Pck1 together with dimethyl malate (DMM), a substrate precursor, significantly increased β-cell proliferation in human islets. Finally, using a Cre-Lox approach, we demonstrated that ChREBPβ contributes to Pck1-dependent β-cell proliferation in mouse β-cells.ConclusionsWe conclude that T3 and glucose act together to regulate ChREBPβ, leading to increased expression and activity of Pck1, and ultimately increased β-cell proliferation.

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Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development.

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