Mitosis of Contact-Inhibited 3T3 Preadipocytes Precedes Chemically Induced Differentiation into Adipocytes

Russell, Thomas; Files, Nancye; Ingram, Marylou

doi:10.3181/00379727-173-41672

Cited by 7 publications

(7 citation statements)

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“…If the IGF-I binding data are expressed per dish of cells, the binding in tlie 3T3-LI adipocytes woiild be about twice that in the fibroblasts. This is consistent with previous observations that 31'3-Ll cell number is approximately doubled as part of the differentiation process [Russell et al, 1983;Hauner, 1990;Schmidt et al, 19901, with the cellular IGF-I rcccptor density remaining constant [Smith et al, 19881. Because chronic exposure of cells to pioglitazone is required to augment cell surface insulin receptor number, this suggests that pioglitazone inay increase insulin receptor synthesis rather than altering subcelhlar traiislocation of existing receptors. Tuiiicamycin, at 1 pgirnl or less, has only modest effects oil protein synthesis and receptor degradation, but inhibits proper glycosylatiori of riascent insulin receptors, thus preventing receptor translocation to the cell surface [Ronnett et al, 1984;Rosen et al, 1979;Reed et al, 19811.…”

Section: Discussionsupporting

confidence: 92%

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Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

Swanson

Bleasdale

1995

Drug Development Research

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Pioglitazone and other antidiabetic thiazolidinediones appear to overcome insulin resistance by affecting an unidentified early event in insulin action. Because attempts to determine changes in insulin receptors in tissues from thiazolidinedione-treated diabetic animals are complicated by drug-induced reduction of hyperinsulinemia and consequent receptor up-regulation, 3T3-LI cells were utilized here as an in vitro model system. After the excess insulin required for optimal differentiation of 3T3-LI adipocytes was removed from the culture medium, there was an increase in specific binding of insulin to the cell surface that was further augmented 25 and 47% in cells treated with pioglitazone for 12 and 24 h, respectively (EC,, about 0.6 pM, maximal at 5-25 FM). Pioglitazone increased the number of insulin receptors without changing binding affinity. Specific IGF-I binding was not affected by pioglitazone in the 3T3-LI cells. Glycerol 3-phosphate dehydrogenase activity, a marker of adipocyte differentiation, was also increased in a dose-dependent manner by pioglitazone (maximal at 10-25 pM). Tunicamycin, which inhibits the N-linked glycosylation of newly synthesized insulin receptors that is required for their translocation to the cell surface, decreased specific insulin binding by about 50% after 24 h, and this decrease remained unchanged when pioglitazone was simultaneously present. Pioglitazone increased not only cell surface insulin receptors, but also total cellular insulin receptors (quantitated either by specific insulin binding or by immunoblotting) and insulin receptor mRNA. In addition, among several structurally related thiazolidinediones there was a positive correlation between in vivo antihyperglycemic activity and their ability to increase insulin binding in vitro, suggesting a possible common molecular mechanism for these actions. o 1995 WiIey-Liss, Inc.

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Section: Discussionsupporting

confidence: 92%

“…P < 0.05 relative t o the corresponding control value. differentiation of 3T3-Ll cells, by inducing IGF-I-dependent mitogenesis [Smith et al, 1988;Russell et al, 1983;Hauner, 1990;Schmidt ct al., 19901. Pioglitazone had no effect on IGF-I binding in either 3T3-Ll fibroblasts or adipocytes.…”

Section: Discussionmentioning

confidence: 99%

Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

Swanson

Bleasdale

1995

Drug Development Research

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“…Treatment to confluent cultures with insulin/dexamethasone/isobutylmethyl xanthine promoted expression of differentiation markers within a few days [34,351. Under these conditions, pre-adipocytes undergo at least one round of cell division before the enzymes of the new phenotype appear [36,371. Among the inany differentiation markers [38 -401 glycerol-3-phosphate dehydrogenase as an essential catalyst for triacylglycerol synthesis usually increases > 50-fold in the course of pre-adipocyte conversion.…”

Section: Activitjj Qfpoi-v( Adp-ribose) Polymerase During Difliirentimentioning

confidence: 99%

Differentiation of 3T3‐L1 pre‐adipocytes induced by inhibitors of poly(ADP‐ribose) polymerase and by related noninhibitory acids

Janßen

Hilz

1989

European Journal of Biochemistry

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To analyze a possible involvement of ADP-ribosylation reactions in 3T3-LI pre-adipocyte differentiation, ADP-ribosyltransferase activities is permeabilized cells as well as endogenous amounts of protein-bound monoand poly(ADP-ribose) residues were determined. Also, in vivo labeling with [3H]adenosine of ADP-ribose residues linked to high-mobility-group (HMG) proteins was performed. As an additional probe, the effects of ADPribosylation inhibitors and non-inhibitory analogs were studied. Basal and total poly(ADP-ribose) polymerase activities markedly increased prior to the appearance of the differentiation marker glycerol-3-phosphate dehydrogenase. Despite these apparent changes in activity, however, neither protein-bound poly(ADP-ribose) residue nor mono(ADP-ribosyl) groups in histones, nor the NAD content, changed significantly under these conditions. Furthermore, although HMG protein-associated [3H]ADP-ribose was reduced in differentiating [3H]adenosinelabeled cells, the data suggest altered precursor pool labeling rather than a specific decrease in ADP-ribosylated HMG proteins. Non-participation of ADP-ribosylation reactions in 3T3-LI differentiation is further supported by experiments with inhibitors and non-inhibitory analogs. Benzamide at 0.3 -3 mM per SP without effect on differentiation, was able to induce specific gene expression when combined with insulin (10-l 2 -M). Similar effects were seen with benzoate as well as with nicotinamide, 3-aminobenzamide and their corresponding acids. The data indicate that benzaiiiide and analogs have profound effects on chromatin functions that are not mediated by ADP-ribosylation reactions Eukaryotic cells contain an enzyme, poly(ADP-ribose) polymerase, which uses NAD to modify nuclear proteins by homopolymeric ADP-ribose. In addition, modification of acceptor polypeptides by single ADP-ribosyl groups [mono-(ADP-ribosyl)ation] has been found. Nuclear poly(ADPribosy1)ation appears to be linked to processes like DNA excision repair and carcinogenesis (for reviews see [l -31). The reaction has also been implicated in various differentiation processes [3,4], although opposite changes in different systems have been reported. Thus, in some differentiating cell lines, a rise of poly(ADP-ribose) as a prerequisite for differentiation was deduced mainly from experiments with inhibitors [5 -81, while in others a transient decrease of poly(ADP-ribose) polymerase activity as a regulatory signal was postulated [9 -121. Conflicting data were presented even for one and the same system: reports on an apparent decrease in poly(ADPribose) polymerase activity during 3T3-LI pre-adipocyte differentiation [13] and an inducing action of polymerase inhibitors [14] contrast with observations of a prevention of differentiation by nicotinamide or benzamide [15]. This led us to analyze the ADP-ribosylation status in vivo during 3T3-Ll

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“…Russell et al (14) have recently demonstrated that 3T3 preadipocytes undergo at least one complete round of replication in response to MIX, DEX, and fetal calf serum. This response does not occur when a nonconverting line, C2, is treated similarly.…”

Section: Discussionmentioning

confidence: 99%

“…It was suggested that some event occurred during the final division(s) prior to confluence of the monolayer which "primed" the cells to convert to adipocytes when they were exposed to the initiating factors for conversion. It was found, however, that when 3T3-Ll cells are grown to confluency and then treated with culture medium containing insulin or fetal calf serum (FCS) as well as additives such as methyl isobutyl xanthine (MIX) and dexamethasone (DEX), the cells undergo at least one round of DNA replication prior to adipocyte conversion (14).…”

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confidence: 99%

Flow cytometric analysis of DNA replication during the differentiation of 3T3‐L1 preadipocytes

et al. 1985

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We have employed a flow cytometric method of monitoring DNA synthesis in order to study the stimulation of DNA replication during the induction of adipocyte differentiation in the preadipocyte cell line When confluent monolayers of this cell line are treated with a mixture of methyl isobutyl xanthine, dexamethasone, and insulin (MDI), they undergo at least one round of cell division, which is followed by the de novo synthesis of many enzymes that are involved in the formation of lipid. If the MDI-treated cells are incubated in iododeoxyuridine (1dUrd)-or bromodeoxyuridine (BrdUrd)-containing medium and subsequently stained with antibodies specific for these base analogues and with propidium iodide (PI), the kinetics of the induction of replication can be monitered. No differences in the patterns of IdUrd incorporation versus PI staining were observed between exponentially growing 3T3-Ll cells and those that had been stimulated to replicate DNA with MDI. In addition, we developed a flow cytometric (FCM) method for staining fatty acid synthetase and localizing the antigen in the G1 phase. We have thus demonstrated the feasibility of this methodology for correlating by FCM the production of enzymes such as fatty acid synthetase with IdUrd and BrUrd incorporation. The technique should permit studies of the inhibition of differentiation of adipocytes by halogenated pyrimidines.Key terms: Adipocyte differentiation, antiBrUrd monoclonal antibody, fatty acid synthetase, flow cytometry Cellular differentiation is best studied in culture, where specific molecular processes can be more or less isolated from other phenomena that are not directly related to the specific differentiation program. When certain cell lines that have been cloned from Swiss 3T3 mouse fibroblast cells are treated in culture with various compounds, they are induced to become adipocytelike cells and to produce copious amounts of lipid (9). During this process of adipocyte conversion, many enzymes involved in lipid biosynthesis, such as pyruvate carboxylase, acetyl CoA carboxylase, ATP citrate lyase, and fatty acid synthetase (1,4,7,11), as well as some of their mRNAs are synthesized de novo (2,5).In previous studies of this phenomenon (61, three of these enzymes-fatty acid synthetase, acetyl CoA carboxylase, and pyruvate carboxylase-were visualized by immunofluorescence in cells that had been stimulated to differentiate. The enzymes could be located by this method several days before the appearance of fat globules. Tnterestingly, the fluorescence was observed in cells that appeared to be in "clones", i.e., cells which were adjacent, and in clusters of 2,4,8, or 16 cells per cluster. It was suggested that some event occurred during the final division(s) prior to confluence of the monolayer which "primed" the cells to convert to adipocytes when they were exposed to the initiating factors for conversion. It was found, however, that when 3T3-Ll cells are grown to confluency and then treated with culture medium containing insulin or fetal calf serum (FCS)...

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Mitosis of Contact-Inhibited 3T3 Preadipocytes Precedes Chemically Induced Differentiation into Adipocytes

Cited by 7 publications

References 0 publications

Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

Antidiabetic agent pioglitazone increases insulin receptors on 3T3‐L1 adipocytes

Differentiation of 3T3‐L1 pre‐adipocytes induced by inhibitors of poly(ADP‐ribose) polymerase and by related noninhibitory acids

Flow cytometric analysis of DNA replication during the differentiation of 3T3‐L1 preadipocytes

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