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1.
Cytotechnology ; 75(1): 27-38, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36713065

ABSTRACT

The unlimited proliferation capacity of embryonic stem cells (ESCs) coupled with their capability to differentiate into several cell types makes them an attractive candidate for studying the molecular mechanisms regulating self-renewal and transition from pluripotent state. Although the roles of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase family (PFKFB1-4) in cell survival, proliferation, and differentiation in tumor cells have been studied, their role in mouse ESC (mESC) biology is currently unkown. In the current study, Pfkfb isoenzyme expressions were analyzed in R1 and J1 mESCs that were cultured in the presence and absence of leukemia inhibitory factor (LIF). We report that expression of the Pfkfb3 isoenzyme was markedly increased when mESCs were promoted to differentiate upon LIF removal. We then demonstrated that Pfkfb3 silencing induced the differentiation marker Brachyury suggesting that Pfkfb3 may be required for the regulation of mesodermal differentiation of mESCs. Furthermore, we show that the increase in Pfkfb3 expression is required for the growth of early differentiated mESCs. Although these results provide important insights into the early differentiation of mESCs with regard to Pfkfb expressions, further mechanistic studies will be needed for understanding the pathways and mechanisms involved in regulation of proliferation and early differentiation of mESCs through Pfkfb3.

2.
Turk J Med Sci ; 53(6): 1565-1573, 2023.
Article in English | MEDLINE | ID: mdl-38813509

ABSTRACT

Background/aim: Type 1 diabetes mellitus (T1DM) is caused by the autoimmune-mediated destruction of insulin-producing cells (IPCs) and still has no effective cure. Better understanding of the molecular mechanisms involved in the differentiation of embryonic stem cells (ESCs) into IPCs may help us improve the therapeutic strategies for treating T1DM. 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (Pfkfb1-4) are key regulators of glucose metabolism. Although Pfkfb3 has been shown to be required for the growth of early differentiated mouse ESCs (mESCs), more studies are needed to further assess the roles of Pfkfb isoenzymes in embryonic development and differentiation, particularly into specific cell types. In this study, we aimed to elucidate the changes in the expression of Pfkfb isoenzymes on the differentiation of mESCs into IPCs. Materials and methods: A 3-step protocol was used to differentiate R1 and J1 mESCs into IPCs. The changes in the gene expression of MafA, MafB, Ins2, and Nkx6.1 (IPC specific markers) and Pfkfb1-4 were analyzed using real-time quantitative polymerase chain reaction (qPCR). Insulin expression and secretion were determined by immunofluorescence (IF) staining and the enzyme linked immunosorbent assay (ELISA), respectively. Results: Upon differentiation, the IPC specific markers in differentiated cells were upregulated. Continued differentiation was confirmed by the development of insulin-positive islet-like clusters that secreted insulin in response to glucose uptake. Expressions of the Pfkfb2 and Pfkfb3 isoenzymes were markedly increased in various stages of differentiation. Conclusion: These findings suggest that Pfkfb2 and Pfkfb3 may impact the differentiation of mESCs into IPCs and the regulation of the insulin response to glucose levels. This study also lays a foundation for researchers to further probe the roles of Pfkfb isoenzymes on the differentiation of mESCs into IPCs and may open new avenues for regenerative medicine.


Subject(s)
Cell Differentiation , Isoenzymes , Mouse Embryonic Stem Cells , Phosphofructokinase-2 , Animals , Mice , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/enzymology , Isoenzymes/metabolism , Isoenzymes/genetics , Mouse Embryonic Stem Cells/metabolism , Mouse Embryonic Stem Cells/cytology , Phosphofructokinase-2/metabolism , Phosphofructokinase-2/genetics
3.
Mol Cell Biochem ; 470(1-2): 115-129, 2020 Jul.
Article in English | MEDLINE | ID: mdl-32415418

ABSTRACT

Tumor cells increase glucose metabolism through glycolysis and pentose phosphate pathways to meet the bioenergetic and biosynthetic demands of rapid cell proliferation. The family of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (PFKFB1-4) are key regulators of glucose metabolism via their synthesis of fructose-2,6-bisphosphate (F2,6BP), a potent activator of glycolysis. Previous studies have reported the co-expression of PFKFB isozymes, as well as the mRNA splice variants of particular PFKFB isozymes, suggesting non-redundant functions. Majority of the evidence demonstrating a requirement for PFKFB activity in increased glycolysis and oncogenic properties in tumor cells comes from studies on PFKFB3 and PFKFB4 isozymes. In this study, we show that the PFKFB2 isozyme is expressed in tumor cell lines of various origin, overexpressed and localizes to the nucleus in pancreatic adenocarcinoma, relative to normal pancreatic tissue. We then demonstrate the differential intracellular localization of two PFKFB2 mRNA splice variants and that, when ectopically expressed, cytoplasmically localized mRNA splice variant causes a greater increase in F2,6BP which coincides with an increased glucose uptake, as compared with the mRNA splice variant localizing to the nucleus. We then show that PFKFB2 expression is required for steady-state F2,6BP levels, glycolytic activity, and proliferation of pancreatic adenocarcinoma cells. In conclusion, this study may provide a rationale for detailed investigation of PFKFB2's requirement for the glycolytic and oncogenic phenotype of pancreatic adenocarcinoma cells.


Subject(s)
Adenocarcinoma/enzymology , Glycolysis , Pancreas/enzymology , Pancreatic Neoplasms/enzymology , Phosphofructokinase-2/physiology , Adenocarcinoma/pathology , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Proliferation , Cytoplasm/metabolism , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Gene Silencing , HeLa Cells , Humans , Isoenzymes/genetics , Isoenzymes/physiology , Pancreatic Neoplasms/pathology , Phenotype , Phosphofructokinase-2/genetics , RNA Splicing , RNA, Messenger/metabolism
4.
Biochem Biophys Res Commun ; 484(3): 687-693, 2017 03 11.
Article in English | MEDLINE | ID: mdl-28161638

ABSTRACT

Transforming growth factor ß1 (TGFß1) is a well-established inducer of the epithelial-mesenchymal transition (EMT) that is essential for the acquisition of malignant properties, such as invasion, in tumor cells. Although recent studies suggest that the EMT in tumor cells is associated with reprogramming of energy metabolism and TGFß1 has been shown to stimulate glycolysis in multiple primary cell lines, little is known about TGFß1's effect on glycolysis and glycolytic regulators in transformed cells. Given the known regulatory role of 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase-3 (PFKFB3) in glycolysis and association of glycolytic activity with malignant features such as invasion, we sought to investigate whether TGFß1 regulates PFKFB3 expression and if PFKFB3 is involved in the TGFß1-mediated increase in the invasive ability of the Panc1 cell cline-a well-established model of TGFß1-initiated EMT. Herein we demonstrate that TGFß1 induces PFKFB3 expression and stimulates glycolysis in Panc1 cells. We also show that siRNA silencing of PFKFB3 prevents the stimulation of glycolysis and in vitro invasive ability of Panc1 cells by TGFß1. Furthermore, PFKFB3 silencing suppresses the TGFß1-mediated induction of the Snail protein, suggesting that PFKFB3 is required for the regulation of Snail expression by TGFß1. Taken together, our study identifies PFKFB3 as a key TGFß1 effector protein that mediates TGFß1's effect on Snail expression, invasion, and glycolysis.


Subject(s)
Glucose/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Peptide Fragments/metabolism , Phosphofructokinase-2/metabolism , Transforming Growth Factor beta1/metabolism , Cell Line, Tumor , Cell Movement , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Neoplasm Invasiveness , Signal Transduction , Snail Family Transcription Factors/metabolism
5.
J Vet Med Sci ; 79(3): 602-607, 2017 Mar 23.
Article in English | MEDLINE | ID: mdl-28111374

ABSTRACT

The main objectives of our study were to measure the major adipokines adiponectin, leptin and resistin in goat milk, to assess their interrelationships and to assess their relationships with the plasma and serum concentrations of total protein, cholesterol, total lipids, plasma C-reactive protein (CRP), milk somatic cell count (SCC), milk total aerobic colony and lactobacillus count, and milk components in lactating Saanen goats. The study was performed on eighteen lactating Saanen goats. Milk and blood samples were collected on days 20, 35, 50, 65 and 80 of lactation postpartum. The milk and plasma adiponectin levels on days 50, 65 and 80 postpartum were significantly higher than those on day 20. The milk and plasma leptin levels were lower on day 20 than on days 35, 50, 65 and 80. The milk concentrations of these major adipokines were positively intercorrelated. The milk and plasma concentrations of these three adipokines were also positively correlated. The plasma CRP concentrations correlated positively with milk leptin and resistin concentrations and inversely with milk adiponectin concentration. Milk adiponectin concentration was inversely related with its SCC. These data confirm that adiponectin, leptin and resistin are present in goat milk. The milk concentrations of these three adipokines were interrelated and interacted with the general inflammatory marker, CRP. The inverse relationship between milk adiponectin concentrations and its SCC suggests that variations in milk adiponectin might be involved in the udder health of lactating goats, but clinical trials are needed to support this hypothesis.


Subject(s)
Adipokines/analysis , Milk/chemistry , Animals , Bacteria, Aerobic/isolation & purification , Bacterial Load , C-Reactive Protein/analysis , Cholesterol/blood , Female , Goats , Lactation/metabolism , Lactobacillus/isolation & purification , Milk/cytology , Milk/microbiology , Turkey
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