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PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

ABSTRACT: Proliferation and differentiation of preadipocytes, and other cell types, is accompanied by an increase in glucose uptake. Previous work showed that a pulse of high glucose was required during the first 3 days of differentiation in vitro, but was not required after that. The specific glucose metabolism pathways required for adipocyte differentiation are unknown. Herein, we used 3T3-L1 adipocytes as a model system to study glucose metabolism and expansion of the adipocyte metabolome during the first 3 days of differentiation. Our primary outcome measures were GLUT4 and adiponectin, key proteins associated with healthy adipocytes. Using complete media with 0 or 5 mM glucose, we distinguished between developmental features that were dependent on the differentiation cocktail of dexamethasone, insulin, and isobutylmethylxanthine alone or the cocktail plus glucose. Cocktail alone was sufficient to activate the capacity for 2-deoxglucose uptake and glycolysis, but was unable to support the expression of GLUT4 and adiponectin in mature adipocytes. In contrast, 5 mM glucose in the media promoted a transient increase in glucose uptake and glycolysis as well as a significant expansion of the adipocyte metabolome and proteome. Using genetic and pharmacologic approaches, we found that the positive effects of 5 mM glucose on adipocyte differentiation were specifically due to increased expression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3), a key regulator of glycolysis and the ancillary glucose metabolic pathways. Our data reveal a critical role for PFKFB3 activity in regulating the cellular metabolic remodeling required for adipocyte differentiation and maturation.

SUBMITTER: Griesel BA

PROVIDER: S-EPMC8205188 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundAdipose tissue is a critical regulator of lipid storage and endocrine function. Impairment of the recruitment of new adipocytes in the adipose tissue is associated with ectopic fat accumulation, diabetes and insulin resistance. Torreya nucifera, an evergreen conifer that grows in warm temperate climates, has been found to exert beneficial effects against inflammation, infection and diabetes. However, the molecular mechanisms responsible for these effects at the cellular level remain unknown. This study aimed to investigate effects of Torreya nucifera seed oil (TNSO) on 3T3-L1 adipocyte differentiation and its underlying regulatory mechanism.MethodsTo investigate the effects of TNSO on adipocyte differentiation, 3T3-L1 cells were induced to differentiate for 5 days in the presence of 0.75 μL/mL TNSO. Oil Red O staining and an assay for intracellular triglyceride were performed to determine the extent of lipid accumulation in 3T3-L1 cells. To elucidate the underlying mechanism of TNSO, adipogenic gene expression was analyzed using quantitative real-time PCR. Moreover, we monitored TNSO-derived activation of PPARγ and STAT3 with 3T3-L1 reporter cell lines engineered to secrete Gaussia luciferase upon the interaction of a transcription factor to its DNA binding element.ResultsOil Red O staining revealed that TNSO improved the differentiation of 3T3-L1 preadipocytes into mature adipocytes. The mRNA levels of adipogenic genes, including adiponectin, fatty acid synthase (FAS) and adipocyte fatty acid-binding protein (FABP4), were upregulated and intracellular triglyceride levels increased upon TNSO treatment. We also established that adipocyte differentiation was improved by TNSO-derived activation of PPARγ and STAT3.ConclusionsOur results suggest that TNSO improves adipocyte differentiation by regulating the activation of adipogenic transcription factors, indicating that it may serve as a potential treatment strategy for adipocyte dysfunction.

Project description:Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic stimulation.

Project description:BackgroundBoth glucocorticoid receptor and peroxisome proliferator-activated receptor-γ (PPARγ) play a critical role in adipocyte differentiation. Mifepristone is not only an antagonist of the glucocorticoid receptor but also an agonist of PPARγ. Therefore, the present study investigated the effect of mifepristone on adipocyte differentiation.MethodsMouse 3T3-L1 cells were used as a model for adipocyte differentiation. The lipid droplet formation was evaluated with Bodipy493/503 staining and the expression of adipocyte markers [adiponectin and adipocyte fatty acid binding protein-4 (Fabp4)] was evaluated with quantitative PCR and immunoblot analyses for indication of adipocyte differentiation. siRNA and neutralizing antibodies were used to elucidate the molecular mechanism of mifepristone-induced adipocyte differentiation. Luciferase reporter assay was used to examine the effect of mifepristone on the promoter activity of PPAR-response element (PPRE). The DNA microarray analysis was used to characterize the transcriptome of the mifepristone-induced adipocytes. In vivo adipogenic effect of mifepristone was examined in mice.ResultsMifepristone not only enhanced adipocyte differentiation induced by the conventional protocol consisting of insulin, dexamethasone and 3-isobutyl-1-methylxanthine but also induced adipocyte differentiation alone, as evidenced by lipid droplets formation and induction of the expression of adiponectin and Fabp4. These effects were inhibited by an adiponectin-neutralizing antibody and a PPARγ antagonist. Mifepristone activated the promoter activity of PPRE in a manner sensitive to PPARγ antagonist. A principal component analysis (PCA) of DNA microarray data revealed that the mifepristone-induced adipocytes represent some characteristics of the in situ adipocytes in normal adipose tissues to a greater extent than those induced by the conventional protocol. Mifepristone administration induced an increase in the weight of epididymal, perirenal and gluteofemoral adipose tissues.ConclusionsMifepristone alone is capable of inducing adipocyte differentiation in 3T3-L1 cells and adipogenesis in vivo. PPARγ plays a critical role in the mifepristone-induced adipocyte differentiation. Mifepristone-induced adipocytes are closer to the in situ adipocytes than those induced by the conventional protocol. The present study proposes a single treatment with mifepristone as a novel protocol to induce more physiologically relevant adipocytes in 3T3-L1 cells than the conventional protocol.

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PFKFB3-dependent glucose metabolism regulates 3T3-L1 adipocyte development.

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