Retinoblastoma protein phosphorylation via PI 3-kinase and mTOR pathway regulates adipocyte differentiation

In the early phase of adipocyte differentiation, transient increase of DNA synthesis, called clonal expansion, and transient hyperphosphorylation of retinoblastoma protein (Rb) are observed. We investigated the role of these phenomena in insulin-induced adipocyte differentiation of 3T3-L1 cells. Insulin-induced clonal expansion, Rb phosphorylation and adipocyte differentiation were all inhibited by the PI 3-kinase inhibitors and rapamycin, but not the MEK inhibitor, whereas the MEK inhibitor, but not PI 3-kinase inhibitors or rapamycin, decreased c-fos induction. We conclude that insulin induces hyperphosphorylation of Rb via PI 3-kinase and mTOR dependent pathway, which promotes clonal expansion and adipocyte differentiation of 3T3-L1 cells.     

Coordinate regulation of the biosynthesis of ATP-citrate lyase and malic enzyme during adipocyte differentiation. Studies on 3T3-L1 cells

The biosynthesis and degradation of two lipogenic enzymes were studied during the differentiation of 3T3-L1 preadipocytes into adipocytes. The activity and mass of malic enzyme, rose by an order of magnitude during adipocyte development and the enzyme accounted for 0.3% of the cytosol protein in mature fat cells. Similarly, the activity and amount of ATP-citrate lyase increased approximately 7-fold during the adipose conversion. The relative rates of synthesis of the two enzymes were less than or equal to 0.02% in preadipocytes, but increased sharply as the cells began to differentiate. Maximal steady state rates of malic enzyme and ATP-citrate lyase synthesis in 3T3-L1 adipocytes were 13- and 8-fold higher, respectively, than the basal rates in preadipocytes. In contrast, the half-lives of malic enzyme (67 h) and ATP-citrate lyase (47 h) were not altered during adipocyte development. Thus, accelerated rates of enzyme synthesis account for the differentiation-dependent accumulation of the two lipogenic enzymes. Increased rates of malic enzyme, ATP-citrate lyase, and fatty acid synthetase biosynthesis are expressed in a highly coordinated manner during adipocyte differentiation and are associated with parallel elevations in the levels of translatable mRNAs for these enzymes.     

Regulation of 3T3-L1 fibroblast differentiation by prostacyclin (prostaglandin I2)

Prostaglandin biosynthesis and prostaglandin-stimulated cyclic AMP accumulation were studied in 3T3-L1 fibroblasts as they differentiated into adipocytes. Incubation of 3T3-L1 membranes with [1-14C]prostaglandin H2, and subsequent radio-TLC analysis, showed that prostacyclin (prostaglandin I2) is the principal enzymatically synthesized prostaglandin in this cell line. Confirmation of the radiochemical data was obtained by demonstrating the presence of 6-keto-prostaglandin F1 alpha, the stable hydrolysis product of prostaglandin I2, by gas chromatography-mass spectrometry. In support of previous work, indomethacin, the prostaglandin endoperoxide synthetase (EC 1.14.99.1) inhibitor, accelerated 3T3-L1 differentiation. More importantly, the incubation of 3T3-L1 cells with insulin and the prostaglandin I2 synthetase inhibitor 9,11-azoprosta-5,13-dienoic acid (azo analog I) also enhanced the rate of cellular differentiation, even though this compound does not inhibit the synthesis of other prostaglandins. The repeated addition of exogenous prostaglandin I2 to 3T3-L1 cells inhibited insulin- and indomethacin-mediated differentiation. When 3T3-L1 cells were exposed to various prostaglandins and the cyclic AMP levels were measured, prostaglandin I2 proved to be the most potent stimulator of cyclic AMP accumulation, followed by prostaglandin E1 greater than prostaglandin H2 much greater than prostaglandin E2, while prostaglandin D2 was inactive. As 3T3-L1 cells differentiate, the ability of prostaglandin I2 or prostaglandin H2 to stimulate cyclic AMP accumulation progressively diminishes. It is suggested that 3T3-L1 differentiation may be controlled by the rate of prostaglandin I2 synthesis and/or sensitivity of the adenylate cyclase to prostaglandin I2.     

Regulation and intracellular localization of the biotin holocarboxylase synthetase of 3T3-L1 cells

A quantitative assay has been developed to measure holocarboxylase synthetase activity in cellular extracts. This assay was based on measuring the incorporation of [3H]biotin of high specific activity (4.3 Ci/mmol) into purified rat liver apopyruvate carboxylase. With this assay, holocarboxylase synthetase in 3T3-L1 mouse fibroblasts has been monitored. During the differentiation of this cell from a fibroblast to an adipocyte, holocarboxylase synthetase activity was found to increase threefold, while pyruvate carboxylase activity rose 20-fold. The results suggest a possible relationship between the activity of the holocarboxylase synthetase and the level of the biotin-dependent carboxylases within the mammalian cell. Utilizing digitonin fractionation. the intracellular distribution of this enzyme has also been examined. In the 3T3-L1 cell, the large majority (approximately 70%) of the total holocarboxylase synthetase activity was found in the cytosolic compartment.     

Induction of succinyl-coenzyme A:3-oxoacid coenzyme A-transferase during differentiation of 3T3-L1 cells

Differentiation of confluent 3T3-L1 preadipocytes to adipocytes in the presence of dexamethasone and 1-methyl-3-isobutylxanthine for 7 days resulted in a 4-fold increase in the incorporation of acetoacetate-carbon into fatty acids and in the activity of 3-oxoacid CoA-transferase, which catalyzes the first committed step in the conversion of acetoacetate to acetoacetyl-CoA. The increase in enzyme activity was due to an increase in the cellular content of the enzyme, as determined by immunoprecipitation of 3-oxoacid CoA-transferase from 3T3-L1 preadipocytes and adipocytes with rabbit antiserum specific for the rat brain enzyme. The 4-fold increase in enzyme activity was accompanied by a 2.7-fold increase in the average relative rate of synthesis of 3-oxoacid CoA-transferase (between Days 4 and 7). Additionally, the half-life of the enzyme increased 1.9-fold relative to the half-life of total protein, indicating that changes in both synthesis and degradation of 3-oxoacid CoA-transferase are responsible for alterations in its activity. Previous studies on the turnover of other enzymes that are induced during differentiation of 3T3-L1 cells have assigned changes in enzyme synthesis as the primary or sole mechanism for changes in enzyme activity. This report provides the first documentation that both enzyme synthesis and degradation play a role in regulating the enzyme activity of an enzyme during differentiation of 3T3-L1 cells.     

Co-treatment with dexamethasone and octanoate induces adipogenesis in 3T3-L1 cells

We report here that octanoate, a medium chain fatty acid, induces adipocyte differentiation in 3T3-L1 cells by co-treatment with dexamethasone, although octanoate has been known not to stimulate 3T3-L1 adipogenesis. A low concentration of exogenous glucose prevented 3T3-L1 adipogenesis induced by 1-methyl 3-isobutylxanthine, dexamethasone, and insulin (MDI) treatment (a common protocol for adipocyte differentiation). In contrast, co-treatment with dexamethasone and octanoate (D-OCT) induced adipogenesis under the same conditions. These findings imply that octanoate, rather than glucose, is the source of accumulated lipids in D-OCT-induced adipogenesis. D-OCT increased expression of the differentiation markers peroxisome proliferator-activated receptor (PPAR)gamma2 and caveolin-1. A specific inhibitor of p38 mitogen-activated protein (MAP) kinase inhibited D-OCT-induced adipogenesis. These results suggest that the p38 MAP kinase pathway followed by up-regulation of PPARgamma2 may be involved in 3T3-L1 adipocyte differentiation induced by D-OCT, as well as by MDI.     

Cyclic AMP-mediated control of lipogenic enzyme synthesis during adipose differentiation of 3T3 cells

During the adipose differentiation of 3T3-F442A cells, there is an increase in the synthesis of numerous proteins, including the lipogenic enzymes glycerophosphate dehydrogenase, fatty acid synthetase and malic enzyme. Agents that increase cAMP content (Dibutyryl cAMP, theophylline, and isoproterenol) are known to induce lipolysis in fat cells; but the same agents are shown here to reduce the synthesis of the lipogenic enzymes during adipose differentiation. The extent of reduction depends on the agent used and differs for the three enzymes; fatty acid synthetase is most sensitive and its synthesis can be suppressed completely. In contrast to their effects on lipogenic enzyme synthesis, these agents do not affect morphological changes or the synthesis of several other proteins, of which some increase and others (such as actin) decrease during the differentiation. The effects of the agents on the synthesis of lipogenic enzymes are not dependent on lipolysis, since they take place to the same degree in cells not permitted to accumulate triglyceride. Translation in vitro of mRNA isolated from cells treated with the agents promoting cAMP accumulation indicates that the levels of functional mRNA for lipogenic enzymes are reduced. We conclude that, in addition to its activation of lipolysis, cAMP reduces specifically mRNA accumulation for lipogenic enzymes. These results also demonstrate the independent control of morphological change and enzyme synthesis during adipose differentiation.     

Stearoyl-CoA desaturase 2 is required for peroxisome proliferator-activated receptor gamma expression and adipogenesis in cultured 3T3-L1 cells

Based on recent evidence that fatty acid synthase and endogenously produced fatty acid derivatives are required for adipogenesis in 3T3-L1 adipocytes, we conducted a small interfering RNA-based screen to identify other fatty acid-metabolizing enzymes that may mediate this effect. Of 24 enzymes screened, stearoyl-CoA desaturase 2 (SCD2) was found to be uniquely and absolutely required for adipogenesis. Remarkably, SCD2 also controls the maintenance of adipocyte-specific gene expression in fully differentiated 3T3-L1 adipocytes, including the expression of SCD1. Despite the high sequence similarity between SCD2 and SCD1, silencing of SCD1 did not down-regulate 3T3-L1 cell differentiation or gene expression. SCD2 mRNA expression was also uniquely elevated 44-fold in adipose tissue upon feeding mice a high fat diet, whereas SCD1 showed little response. The inhibition of adipogenesis caused by SCD2 depletion was associated with a decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA and protein, whereas in mature adipocytes loss of SCD2 diminished PPARgamma protein levels, with little change in mRNA levels. In the latter case, SCD2 depletion did not change the degradation rate of PPARgamma protein but decreased the metabolic labeling of PPARgamma protein using [(35)S]methionine/cysteine, indicating protein translation was decreased. This requirement of SCD2 for optimal protein synthesis in fully differentiated adipocytes was verified by polysome profile analysis, where a shift in the mRNA to monosomes was apparent in response to SCD2 silencing. These results reveal that SCD2 is required for the induction and maintenance of PPARgamma protein levels and adipogenesis in 3T3-L1 cells.     

Effects of sterculic acid on stearoyl-CoA desaturase in differentiating 3T3-L1 adipocytes

The effects of sterculic acid on cell size, adiposity, and fatty acid composition of differentiating 3T3-L1 adipocytes are correlated with stearoyl-CoA desaturase (SCD) expression (mRNA and protein levels) and enzyme activity. Fluorescence-activated cell scanning (FACS) analysis showed that adipocytes differentiated with methylisobutylxanthine, dexamethasone, and insulin (MDI) plus 100 microM sterculic acid comprised a population of predominantly large cells with reduced adiposity compared to MDI-treated cells. Although both groups had similar amounts of total fat, their fatty acid profiles were strikingly different: MDI-treated cells had high levels of the unsaturated palmitoleic (Delta(9)-16:1) and oleic (Delta(9)-18:1) acids, whereas the cells cultured with MDI plus sterculic acid accumulated palmitic (16:0) and stearic (18:0) acids together with a marked reduction in Delta(9)-16:1. Although the cells treated with MDI plus sterculic acid had similar levels of scd1 and scd2 mRNAs and antibody-detectable SCD protein as the MDI-treated cells, the SCD enzyme activity was inhibited more than 90%. The accumulation of 16:0 and 18:0, together with normal levels of fatty acid synthase (FAS) and aP2 mRNAs, shows that de novo synthesis and elongation of fatty acids, as well as cell differentiation, were not affected by sterculic acid. Because of the increase in cell size in the sterculic acid-treated cells, the insulin-stimulated 2-deoxyglucose (2-DOG) uptake was determined. Compared to MDI-treated cells, the 2-DOG uptake in the cells treated with sterculic acid was not affected. These results indicate that sterculic acid directly inhibits SCD activity, possibly by a turnover-dependent reaction, without affecting the processes required for adipocyte differentiation, scd gene expression or SCD protein translation.     

黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响

目的 研究黄芩素对3T3-L1小鼠前脂肪细胞分化及对脂肪酸合成酶活性的影响。方法 油红O染色法测定细胞分化速度;分光光度法测定脂肪酸合成酶活性。结果 黄芩素对3T3-L1小鼠前脂肪细胞向脂肪细胞分化以及对脂肪酸合成酶活性有抑制作用。结论 黄芩素通过阻断脂肪合成而抑制小鼠前脂肪细胞分化;黄芩素具有开发成减肥药物的潜力。     

Application of a DNA double labelling method for the flow cytometric analysis of recruitment of non-cycling cells in a mixed population of P and Q cells

Abstract. In this paper we describe the application of a non-radioactive DNA double labelling and staining method to an analysis of cell proliferation kinetics by flow cytometry, aimed at the direct measurement of recruitment rates in cell cultures. The method is based on the application of two halogenated deoxyuridines: iododeoxyuri-dine (IdUrd) and chlorodeoxyuridine (CldUrd) which are incorporated into DNA synthesizing cells. By applying two commercially available monoclonal antibodies both deoxyuridines can be detected separately. To measure recruitment all proliferating cells in a plateau phase culture were labelled first with IdUrd applied during a time interval approximately equal to the cell cycle time. Subsequently, recruitment induced by a medium change was analysed by flow cytometric assessment of incorporation of CldUrd in cells which had not taken up IdUrd.
Experiments designed to determine the toxicity of continuous labelling with IdUrd in different concentrations and of pulse labelling with CldUrd showed that there was no effect on the progression of cells through the cell cycle. The aim of this study is to test the sensitivity of the procedure to detect changes in proliferation kinetics, in particular the entrance of resting cells into the S phase. Although the cell culture model used is very simple, the results demonstrate clearly that a low rate of recruitment can be detected. It is suggested that the procedure described here is specific and sensitive enough to quantify changes in cell proliferation in tumours induced by various treatments and has advantages over other methods, which measure recruitment indirectly, or directly by using two radioactive thymidines.     

Increase in fatty acid synthetase content of 3T3-L cells undergoing spontaneous and chemically induced differentiation to adipocytes.

3T3-L fibroblasts differentiate into adipose cells when maintained in a non-growing state. The specific activity of fatty acid synthetase of differentiated cells was 25--30-fold higher than that present in 3T3-L fibroblasts or in 3T3-C2 cells that possess an extremely low incidence of differentiation to adipocytes. The results of immunochemical analysis indicate that the increased specific activity of fatty acid synthetase in the differentiated cells is due to an increase in the cellular content of this enzyme. The rate of conversion of adipose cells was accelerated by brief exposure of confluent non-growing cultures of 3T3-L cells to 3-isobutyl-1-methylxanthine. This was accompanied by an increase in the specificity activity of fatty acid synthetase, which was also shown to be due to an increase in the cellular content of this enzyme. The continuous presence of 3-isobutyl-1-methylxanthine in the culture medium was not required to elicit the morphological and biochemical changes in 3T3-L cells that occurred many days after the removal of the inducer but earlier than the onset of spontanous differentiation.     

Stearoyl-CoA desaturase enzyme 1 inhibition reduces glucose utilization for de novo fatty acid synthesis and cell proliferation in 3T3-L1 adipocytes

Stearoyl-CoA desaturase enzyme 1 (SCD1) is a lipogenic enzyme that is upregulated in obesity, insulin resistance, and cancer. Since glucose is a substrate for both de novo fatty acid synthesis and deoxyribose synthesis, we hypothesized that SCD1 affects these multiple synthetic pathways through changes in glucose utilization. This study determined glucose utilization for fatty acid synthesis and cell proliferation in 3T3-L1 preadipocytes during SCD1 inhibition. The effects of SCD1 on cellular metabolism as mediated by its monounstaurated fatty acid products (palmitoleate and oleate) were also observed. 3T3-L1 preadipocytes underwent differentiation induction in conjunction with one of the following treatments for 4 days: (A) no treatment, (B) SCD1 inhibitor CGX0290, (C) CGX0290 + palmitoleate, or (D) CGX0290 + oleate. All cells received medium with 50 % [U¹³C]-glucose. Cells were harvested on day 7 for studies of fatty acid metabolism, tricarboxylic acid (TCA) cycle activities, and deoxyribose synthesis. CGX0290 decreased fatty acid desaturation, glucose utilization for fatty acid synthesis (acetyl-CoA enrichment), and de novo synthesis. CGX0290 treatment also led to decreased cell density through increased cell death. Further analysis showed that deoxyribose new synthesis and oxidative pentose phosphate pathway activity were unchanged, while non-oxidative transketolase pathway activity was stimulated. Palmitoleate and oleate supplementation each partially ameliorated the effects of CGX0290. In 3T3-L1 cells, SCD1 promotes glucose utilization for fatty acid synthesis. In cell proliferation, SCD1 may promote cell survival, but does not impact the oxidative pathway of deoxyribose production. These effects may be mediated through the production of palmitoleate and oleate.     

Estrogen regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and acetyl-CoA carboxylase in xenopus laevis

Administration of estradiol-17 beta to male Xenopus laevis evokes the proliferation of the endoplasmic reticulum and the Golgi apparatus and the synthesis and secretion by the liver of massive amounts of the egg yolk precursor phospholipoglycoprotein, vitellogenin. We have investigated the effects of estrogen on three key regulatory enzymes in lipid biosynthesis, 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase, the major regulatory enzyme in cholesterol and isoprenoid synthesis, and acetyl-CoA carboxylase and fatty acid synthetase, which regulate fatty acid biosynthesis. HMG-CoA reductase activity and cholesterol synthesis increase in parallel following estrogen administration. Reductase activity in estrogen stimulated Xenopus liver cells peaks at 40-100 times the activity observed in control liver cells. The increased rate of reduction of HMG-CoA to mevalonic acid is not due to activation of pre-existing HMG-CoA reductase by dephosphorylation, as the fold induction is unchanged when reductase from control and estrogen-stimulated animals is fully activated prior to assay. The estrogen-induced increase of fatty acid synthesis is paralleled by a 16- to 20-fold increase of acetyl-CoA carboxylase activity, indicating that estrogen regulates fatty acid synthesis at the level of acetyl-CoA carboxylase. Fatty acid synthetase activity was unchanged during the induction of fatty acid biosynthesis by estrogen. The induction of HMG-CoA reductase and of acetyl-CoA carboxylase by estradiol-17 beta provides a useful model for regulation of these enzymes by steroid hormones.     

Generation of megakaryocytes and platelets from preadipocyte cell line 3T3-L1, but not the parent cell line 3T3, in vitro

Platelets are produced from megakaryocytes (MKs), although the pathway leading from stem cells to MK lineages are not yet fully understood. Recently, we reported to obtain abundant MKs and platelets from human subcutaneous adipose tissues. Adipose tissues contain various cell types, most of which are lineage cells from mesenchymal or adipocyte-derived stem cells, distinct from hematopoietic cells. To identify the cells responsible for the differentiation MK lineages in adipose tissues, this study examined whether the preadipocyte cell line 3T3-L1 and fibroblast cell line 3T3 differentiated into MK lineages in vitro. Cells were cultured in megakaryocyte lineage induction medium. By day 4, most of 3T3 cell-derived cells leaded to cell death. In contrast, 3T3-L1-derived cells on days 8 showed to have typical characterizations of MK lineages in analyses for specific marker, DNA ploidy, transmission electro micrograph. 3T3-L1-derived platelet-sized cells on day 12 could be stimulated by ADP and PAR4-activating peptide. This study clearly shows in vitro differentiation from 3T3-L1 cells, not from 3T3 cells, into MK lineages.     

An association between glutamine synthetase activity and adipocyte differentiation in cultured 3T3-L1 cells

Confluent 3T3-L1 Swiss mouse fibroblasts acquired morphological and biochemical characteristics of adipocytes when maintained in medium containing 10% calf serum and added insulin. Identical cultures maintained in the absence of added insulin did not differentiate into adipocytes. Incubation of confluent cultures for 48 h with 0.25 μm dexamethasone and 0.5 mm 1-methyl-3-isobutylxanthine yielded subsequent adipocyte differentiation when the culture medium contained 10% fetal calf serum. In contrast, differentiation did not occur when similarly treated cultures were maintained in medium containing 10% calf serum. The increase in glutamine synthetase which occurred during adipocyte differentiation was closely associated with an increased rate of triglyceride synthesis from acetate, with increased protein, and with increases in the activities of glycerol-3-P dehydrogenase and glucose-6-P dehydrogenase. Glutamine synthetase activity remained undetectable in insulin-treated confluent 3T3-C2 cells maintained under conditions which yielded high glutamine synthetase activity in 3T3-L1 cells. (3T3-C2 cells did not differentiate into adipocytes.) Glutamine accumulated in the culture medium of 3T3-L1 adipocytes, but it did not accumulate in the medium from identically treated 3T3-C2 cells. A half-maximal increase in glutamine synthetase specific activity occurred at a culture medium insulin concentration of 10 ng/ml. Neither adipocyte differentiation nor the rise in glutamine synthetase activity were substantially altered by maintaining confluent cultures in medium lacking added glutamine. Incubation of confluent 3T3-L1 cultures with 3 mml-methionine sulfone, a reversible inhibitor of glutamine synthetase, increased by two-fold both the activity and the cellular content of glutamine synthetase. Incubation of confluent 3T3-L1 cultures with 4 mml-glutamine and l-methionine-dl-sulfoximine, an irreversible inhibitor of glutamine synthetase activity, decreased glutamine synthetase activity to less than 5% of the activity in control cultures; however, neither cellular content of the enzyme nor synthesis rate of the enzyme were substantially altered. In the presence of added glutamine, neither methionine sulfone nor methionine sulfoximine had a significant effect on phenotypic adipocyte conversion. By contrast, when confluent cultures were incubated with methionine sulfoximine and no added glutamine, glutamine synthetase remained absent and there was no evidence of adipocyte conversion. Our data indicate (1) that added insulin is required for adipocyte differentiation of 3T3-L1 cells maintained in medium containing calf serum, (2) that glutamine synthetase activity increases during adipocyte conversion regardless of the culture conditions employed to achieve differentiation, and (3) that glutamine synthetase activity may be required for adipocyte differentiation when cultures are maintained in medium lacking added glutamine.     

Lack of coordinated regulation of lipogenic enzymes in a human breast cell line, SKBr3

A human breast cell line has been identified which contains prodigious levels of fatty acid synthetase but has a very low capacity for lipogenesis from glucose, lactate or acetate. The fatty acid synthetase from this cell line appears to be structurally and functionally normal, and the low lipogenic capacity of the cells appears to be due to the low activities of other lipogenic enzymes, notably acetyl-CoA carboxylase. Thus, the SKBr3 cell line appears to lack the long-term coordinated control of acetyl-CoA carboxylase and fatty acid synthetase commonly observed in normal lipogenic tissues.