3T3-L1 preadipocyte differentiation and poly(ADP-ribose) synthetase

Differentiation of 3T3-L1 preadipocytes, induced by methyl-isobutylxanthine (MIX), dexamethasone (DEX), and insulin, results in cells with the morphological and biochemical characteristics of adipocytes. Following incubation of 3T3-L1 cells with MIX, DEX, and insulin, poly(ADP-ribose) synthetase activity decreased abruptly, remained low for several hours and then increased; this rise was delayed by readdition of MIX, DEX, and insulin. The transient reduction in poly(ADP-ribose) synthetase activity in 3T3-L1 cells occurred prior to the appearance of the adipocyte phenotype induced by the above agents. It was not observed when preparations were assayed in the presence of DNase I, indicating that poly(ADP-ribose) synthetase activity was masked following treatment with MIX, DEX, and insulin. The change in synthetase activity represents the earliest alteration of a specific enzyme yet detected during the differentiation of 3T3-L1 cells. It appears to be differentiation specific since nondifferentiating 3T3-C2 control cells did not exhibit changes in poly(ADP-ribose) synthetase activity when treated with MIX, DEX, and insulin. The transient reduction in activity may be an early event in differentiation which reflects changes in chromatin structure.     

Expression profiling during adipocyte differentiation of 3T3-L1 fibroblasts

The 3T3-L1 cell line is a well-established and commonly used in vitro model to assess adipocyte differentiation. Over the course of several days confluent 3T3-L1 cells can be converted to adipocytes in the presence of an adipogenic cocktail. Changes in gene expression were measured by DNA microarrays at three time points (24 h, 4 days, and 1 week) during the course of differentiation from preadipocytes to mature adipocytes. Several functional categories of genes were affected by adipocyte conversion. In addition, seven genes were found to be commonly altered by 5-fold or more by adipocyte conversion at all three time points. Lipocalin 2, haptoglobin, serum amyloid A3, stearoyl-CoA desaturase, and 11beta-hydroxysteroid dehydrogenase 1 were induced while actin alpha2 and procollagen VIII alpha1 were suppressed by adipocyte differentiation. Further study of the regulation of these genes and pathways will lead to an increased understanding of the biochemical pathways involved in adipocyte differentiation and possibly to the identification of new therapeutic targets for treatment of obesity and other metabolic diseases.     

Polyamines are needed for the differentiation of 3T3-L1 fibroblasts into adipose cells

When non-proliferating 3T3-L1 fibroblasts were stimulated to differentiate into adipose cells, there was a dramatic increase in the intracellular level of the polyamine, spermidine. Addition of α-difluoromethylornithine, an inhibitor of polyamine biosynthesis, depleted the cellular polyamines and prevented triglyceride accumulation and differentiation. The inhibitory effect of α-difluoromethylorinithine was completely abolished by provision of spermidine or putrescine. This suggests that polyamines are needed in the processes of differentiation as well as their established requirement for cell growth.     

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.     

Changes of pyridine nucleotide levels during adipocyte differentiation of mouse 3T3-L1 cells.

The levels of NAD and NADP were measured in 3T3-L1 cells during a differentiation from preadipocytes to adipocytes. The cells were grown in the ordinary medium and differentiated in the medium by adding dexamethasone, 1-methyl-3-isobutylxanthine, and insulin for 2 days, and then they were grown in the medium by adding only insulin for another 8 days to accumulate fat. The levels of cellular NAD and NADP increased abruptly with days after differentiation, and the levels of NAD and NADP reached maximum at day 7, and at day 10 the values were decreased compared with the maximum values. These results suggest that expression of the pyridine nucleotide biosynthesis genes is induced in the differentiation process.     

ATF3 inhibits adipocyte differentiation of 3T3-L1 cells

The global spread of highly pathogenic avian influenza A H5N1 viruses raises concerns about more widespread infection in the human population. Pre-pandemic vaccine for H5N1 clade 1 influenza viruses has been produced from the A/Viet Nam/1194/2004 strain (VN1194), but recent prevalent avian H5N1 viruses have been categorized into the clade 2 strains, which are antigenically distinct from the pre-pandemic vaccine. To understand the antigenicity of H5N1 hemagglutinin (HA), we produced a neutralizing monoclonal antibody (mAb12-1G6) using the pre-pandemic vaccine. Analysis with chimeric and point mutant HAs revealed that mAb12-1G6 bound to the loop (amino acid positions 140-145) corresponding to an antigenic site A in the H3 HA. mAb12-1G6 failed to bind to the mutant VN1194 HA when only 3 residues were substituted with the corresponding residues of the clade 2.1.3.2 A/Indonesia/5/05 strain (amino acid substitutions at positions Q142L, K144S, and S145P), suggesting that these amino acids are critical for binding of mAb12-1G6. Escape mutants of VN1194 selected with mAb12-1G6 carried a S145P mutation. Interestingly, mAb12-1G6 cross-neutralized clade 1 and clade 2.2.1 but not clade 2.1.3.2 or clade 2.3.4 of the H5N1 virus. We discuss the cross-reactivity, based on the amino acid sequence of the epitope.     

Expression of c-raf-1 and A-raf-1 during differentiation of 3T3-L1 preadipocyte fibroblasts into adipocytes

The objective of this study was to examine the expression of the c-raf-1 and A-rat-1 protooncogenes during differentiation of 3T3-L1 preadipocytes into adipocytes. At confluence, prior to initiation of differentiation c-raf and A-raf steady state mRNA levels were low. Expression of c-raf and A-raf began to increase 72 hours following initiation of differentiation by treatment with differentiation medium, reaching a maximum increase of 3 to 6-fold and 3 to 4-fold respectively by 190 hours. The increase of c-raf and A-raf steady state message levels occurred concomitant with the onset of differentiation as indicated by increased levels of glycerol-3-phosphate dehydrogenase mRNA. These changes were compared with those for several other protooncogene mRNAs including c-myc, c-fos, H-ras and histone H3. These results are the first to show increase expression of the raf protooncogenes during terminal differentiation rather than in association with proliferation.     

Regulation of tristetraprolin during differentiation of 3T3-L1 preadipocytes

Tristetraprolin is a zinc-finger-containing RNA-binding protein. Tristetraprolin binds to AU-rich elements of target mRNAs such as proto-oncogenes, cytokines and growth factors, and then induces mRNA rapid degradation. It was observed as an immediate-early gene that was induced in response to several kinds of stimulus, such as insulin and other growth factors and stimulators of innate immunity such as lipopolysaccharides. We observed that tristetraprolin was briefly expressed during a 1-8 h period after induction of differentiation in 3T3-L1 preadipocytes. Detailed analysis showed that tristetraprolin mRNA expression was stimulated by fetal bovine serum and differentiation inducers, and was followed by rapid degradation. The 3'UTR of tristetraprolin mRNAs contain adenine- and uridine-rich elements. Biochemical analyses using RNA pull-down, RNA immunoprecipitation and gel shift experiments demonstrated that adenine- and uridine-rich element-binding proteins, HuR and tristetraprolin itself, were associated with tristetraprolin adenine- and uridine-rich elements. Functional characterization confirmed that tristetraprolin negatively regulated its own expression. Thus, our results indicated that the tight autoregulation of tristetraprolin expression correlated with its critical functional role in 3T3-L1 differentiation.     

Redistribution of clathrin-coated vesicle adaptor complexes during adipocytic differentiation of 3T3-L1 cells

Mechanisms for intracellular retention of proteins are induced during adipocytic differentiation of 3T3-L1 cells. To investigate the potential role of clathrin lattices in these retention processes, we performed a morphological and biochemical analysis of coated vesicle components in 3T3-L1 cells. Optical sectioning and image restoration revealed a marked increase in the staining of clathrin and beta adaptins in the perinuclear region of cells with differentiation. In addition, predominance of beta (subunit of the AP-2, plasma membrane adaptor) over beta' (subunit of the AP-1, Golgi adaptor) adaptin was observed in immunoblots of clathrin-coated vesicles purified from nondifferentiated fibroblasts, and this ratio was reversed in coated vesicles purified from differentiated adipocytes. These results indicate that the relative abundance of TGN-derived clathrin lattices increases markedly during adipocytic differentiation. Subcellular fractionation indicated that cytosolic AP-1 and AP-2 adaptors comprised approximately 70% of the total cellular adaptor pool. Interestingly, neither the concentration nor the relative ratio of cytosolic AP-1 to AP-2 adaptors increased significantly during differentiation. These data suggest that the increase in TGN-derived lattices results from differentiation-induced mechanisms for enhanced assembly or stabilization of adaptors on Golgi membranes. Interestingly, double- immunofluorescence microscopy also revealed that whereas extensive colocalization between clathrin and beta adaptins occurred both in fibroblasts and adipocytes, structures stained only with anti-adaptin antibody could be detected. Taken together these results suggest that membranes coated with adaptors, but not clathrin, can exist in these cells.     

P-450(11beta)-dependent conversion of androgen to estrogen, the aromatase reaction

Purified bovine adrenal P-450(11)beta has been shown to act as an aromatase which catalyzes conversion of 19-oxoandrostenedione to estrone. No conversions took place when any one of the required components such as NADPH, NADPH:adrenodoxin reductase, adrenodoxin and P-450(11)beta was omitted from the complete reconstituted system. P-450scc, another mitochondrial P-450 obtained from adrenal cortex, did not substitute for the P-450(11)beta in the aromatase reaction. These results show that P-450(11)beta is able to catalyze a series of reaction which can generate adrenal estrogen through androstenedione and its 19-hydroxy- and 19-oxo-derivatives. The P-450(11)beta-dependent reaction appears to be quite different from the placental aromatase reaction in that the latter is catalyzed by a microsomal P-450.     

N-acetylcysteine inhibits kinase phosphorylation during 3T3-L1 adipocyte differentiation

Objectives: Reports investigating the effects of antioxidants on obesity have provided contradictory results. We have previously demonstrated that treatment with the antioxidant N-acetylcysteine (NAC) inhibits cellular triglyceride (Tg) accumulation as well as total cellular monoamine oxidase A (MAOA) expression in 3T3-L1 mature adipocytes (Calzadilla et al., Redox Rep. 2013;210–218). Here we analyzed the role of NAC on adipogenic differentiation pathway.

Methods: Assays were conducted using 3T3-L1 preadipocytes (undifferentiated cells: CC), which are capable of differentiating into mature adipocytes (differentiated cells: DC). We studied the effects of different doses of NAC (0.01 or 1?mM) on DC, to evaluate cellular expression of phospho-JNK½ (pJNK½), phospho-ERK½ (pERK½) and, mitochondrial expression of citrate synthase, fumarate hydratase and MAOA.

Results: Following the differentiation of preadipocytes, an increase in the expression levels of pJNK½ and pERK½ was observed, together with mitotic clonal expansion (MCE). We found that both doses of NAC decreased the expression of pJNK½ and pERK½. Consistent with these results, NAC significantly inhibited MCE and modified the expression of different mitochondrial proteins.

Discussion: Our results suggested that NAC could inhibit Tg and mitochondrial protein expression by preventing both MCE and kinase phosphorylation.     

SNARE expression and distribution during 3T3-L1 adipocyte differentiation

Differentiation of 3T3-L1 cells into adipocytes presupposes the expression of the glucose transporter isoform GLUT4 and the acquisition of insulin-dependent GLUT4 translocation from intracellular storage vesicles to plasma membrane. This ability to translocate GLUT4 depends on the presence of a set of proteins of the SNARE category that are essential in the fusion step. The expression and levels of some of these SNARE proteins are altered during 3T3-L1 differentiation. Levels of the v-SNARE protein cellubrevin and of the t-SNARE protein syntaxin 4 were increased in this process in parallel to GLUT4. However, the levels of SNAP-23, another t-SNARE, were maintained during differentiation. Immunofluorescence images of SNAP-23 showed the initial distribution of this protein in a perinuclear region before differentiation and its redistribution towards plasma membrane in the adipocyte form. These results suggest a capital role in the expression levels and cellular distribution, during 3T3-L1 differentiation, of SNARE proteins involved in the late steps of GLUT4 translocation.     

Lithium ion reversibly inhibits inducer-stimulated adipose conversion of 3T3-L1 cells

Adipose conversion of 3T3-L1 cells by inducers (dexamethasone, 1-methyl-3-isobutylxanthine and insulin) was inhibited by LiCl at concentrations from 2 to 20 mM. The effect of LiCl was reversible and the inhibited cells were converted to adipocytes when stimulated after the removal of LiCl. Inhibition by LiCl of adipose conversion was accompanied with a blockage of the enhanced [3H]thymidine incorporation and cellular proliferation that occurred before the adipocyte phenotype was expressed. Of the cations tested, only Li+ had these effects.