Reorganization of a novel vimentin-associated protein in 3T3-L1 cells during adipose conversion

We have found that the antibody A2, a marker for the capsule of steroidogenic lipid droplets, reacts with an intermediate filament-associated protein, P₂₀₀, in 3T3-L1 preadipocytes. Supporting evidence came from the colocalization pattern of P₂₀₀ with vimentin in double label experiments. The association of P₂₀₀ with vimentin was further confirmed by its copurification with vimentin after high salt extraction and colocalization of these two proteins in high salt-extracted and vinblastine-treated cells. In preadipocytes this protein was distributed on the vimentin filament network. At the early stage of adipose conversion, this protein was found to encircle nascent lipid droplets ranging from 0.1 to 0.2 μm, accompanied with a decreased distribution on the vimentin filament system. This infers a possible translocation of P₂₀₀ from the vimentin filaments to the droplet surface. Meanwhile, the vimentin filaments remained in a normal distribution in the cytoplasm and were apparently not associated with the nascent droplet. The association of vimentin filaments to droplet surfaces became prominent in lipid droplets larger than 0.2 μm, forming a typical vimentin cage. Immunogold staining also confirmed the translocation of P₂₀₀ immunoreactivity from the droplet surface to the vimentin cage. The relocation of P₂₀₀ from the cytoplasmic vimentin filaments to the droplet surface prior to the formation of the vimentin cage, as well as the reorganization of this protein in the vimentin cage, suggests a stabilizing role in the lipid droplet formation and an inducing function of this protein in the formation of the vimentin cage. J. Cell. Biochem. 67:84–91, 1997. © 1997 Wiley-Liss, Inc.     

Identification of novel putative membrane proteins selectively expressed during adipose conversion of 3T3-L1 cells

Fat tissue plays a critical role in the regulation of energy metabolism. Here we report the proteomic identification of a novel _fa_t tissue-specific _l_ow molecular weight _p_rotein (Falp) which responds to insulin. Falp is preferentially expressed in adipocytes but not in preadipocytes, as shown by two-dimensional gel electrophoresis. Northern blot analysis shows that the Falp gene is predominantly expressed in brown and white fat tissues, but not in any other tissues examined. Human homologs of mouse Falp are found to exist as two alternatively spliced isoforms, which share the same N-terminus but have different C-termini. Both human and mouse Falp contain a conserved putative transmembrane domain. Immunofluorescent analyses of 3T3-L1 adipocytes show that Falp protein strictly localizes at a compact perinuclear membrane compartment. Treatment of cells with insulin induces the redistribution of Falp into numerous discrete spotty structures spreading throughout the cytoplasm. Whereas the function of Falp is currently unclear, its tissue specific expression and the responsiveness to insulin suggest that Falp might be involved in a process specifically restricted to adipose tissue function, such as vesicular transport and protein secretion.     

The induction of adipose conversion by bezafibrate in 3T3-L1 cells. Synergism with dibutyryl-cAMP

The induction of adipose conversion in 3T3-L1 cells by bezafibrate (Brandes, R., Hertz, R. Arad R., Naishtat S., Weil, S. and Bar-Tana, J. (1987) Life Sci., 40, 935-941) was enhanced by dibutyryl-cAMP as well as forskolin, theophylline or isobutylmethylxanthine added to the incubation medium together with the bezafibrate inducer. The synergistic effect of bezafibrate and dibutyryl-cAMP resulted in enhancing the expression of late markers of adipose conversion, e.g., lipid accumulation or glycerol-3-phosphate dehydrogenase activity and its mRNA. This enhanced expression of late markers was reflected in shortening the time period required for their first appearance as well as increasing their yield during the course of adipose conversion. By following the accumulation of glutamine synthetase mRNA serving as an early marker for adipose conversion, the synergistic effect of bezafibrate and dibutyryl-cAMP was already evident as early as 5 h following their addition to confluent 3T3-L1 cells. Hence, the induction of adipose conversion by bezafibrate in 3T3-L1 cells appears to involve an early event which is rate-limited by the availability of intracellular cAMP.     

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.     

Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells

We showed that the synthesis and secretion of type IV collagen, entactin, and laminin were enhanced when adipose conversion of 3T3-L1 cells at confluence was stimulated by hormones (Y. Aratani and Y. Kitagawa (1988) J. Biol. Chem. 263, 16163-16169). Ascorbic acid phosphate (Asc-P) stimulated the synthesis and secretion of type IV collagen and other collagens from both 3T3-L1 preadipocytes and adipocytes. The synthesis and secretion of laminin and entactin were not affected by Asc-P. The continuous addition of Asc-P stimulated cell growth and increased cell density at confluence 1.3-fold. Concomitantly, Asc-P remarkably accelerated the emergence of lipoprotein lipase, glycerophosphate dehydrogenase, and Oil Red O-stainable lipid droplets. These findings suggest an important role for type IV collagen in adipocyte differentiation.     

Hypoxic enhancement of type IV collagen secretion accelerates adipose conversion of 3T3-L1 fibroblasts

Hypoxic modulation of collagen metabolism appears to be related to pathogenesis of many diseases such as fibrosis of connective tissue after injury and scleroderma. Since most of our understanding of how procollagen assembles within the cell has come from studies on cells cultured under normoxia, it may not be helpful for the etiology of the diseases observed in peripheral tissues under hypoxic conditions. As an experimental model for the hypoxic modulation of collagen metabolism, we cultured 3T3-L1 fibroblasts under low partial oxygen pressure and found that hypoxia enhances secretion of type IV collagen 10-fold and accelerates adipose conversion of the cells. The enhanced secretion of type IV collagen was not accompanied by an appreciable increase of alpha1(IV) and alpha2(IV) mRNAs. Prolyl 4-hydroxylase alpha increased only 3-fold under hypoxia. We suggest that hypoxia creates an environment of prolyl 4-hydroxylase alpha(2)beta(2) tetramers favorable for the folding of type IV procollagen which has many interruptions of the Gly-Xaa-Yaa repeat.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Dihydrocytochalasin B promotes adipose conversion of 3T3 cells

Differentiation of preadipose 3T3-F442A cells into adipose cells is accelerated by the addition of dihydrocytochalasin B. The effect of the drug on 3T3-C2 cells is more marked: these cells are practically unable to differentiate in the absence of H2CB but a long-term exposure to the drug enables the cells to accumulate lipid droplets in medium supplemented with fetal calf serum and insulin. During their differentiation under these conditions the 3T3-C2 cells develop markers typical of adipose cells: glycerophosphate dehydrogenase, ATP-citrate lyase, fatty acid synthetase and glycerophosphate acyltransferase.     

Endothelin-1 inhibits adipogenic differentiation of 3T3-L1 preadipocytes

The effect of endothelin (ET)-1 on the adipogenic differentiation of 3T3-L1 preadipocytes was examined. Cellular morphology and lipoprotein lipase activity were used as differentiation markers. ET-1 inhibited the hormone-induced adipogenic differentiation of 3T3-L1 preadipocytes morphologically and biochemically in a dose-dependent manner. These findings promote ET-1 as a potent inhibitor of adipogenic differentiation, playing an important role in cellular differentiation of preadipocytes and making it a significant regulator of lipid metabolism.     

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.     

Endothelin-1 inhibits resistin secretion in 3T3-L1 adipocytes

Resistin is an adipocyte-derived hormone whose role in the development of insulin resistance is controversial. Endothelin-1 (ET-1) is a 21 amino acid peptide demonstrated to possess vasoconstrictor, positive inotropic, mitogenic, and metabolic properties. In numerous disease states, including congestive heart failure, obesity, and diabetes, elevated levels of ET-1 have been reported and are thought to contribute to the pathology of the disease. A recent study demonstrated that ET-1 induces the expression and stimulates the secretion of the adipose tissue-derived hormone leptin. However, the effect of ET-1 on resistin secretion has not been determined. To characterize the effect of ET-1 on resistin secretion, 3T3-L1 fibroblasts were differentiated into adipocytes and allowed to mature for 14 days. Cells were incubated for 24h with ET-1 (1-100 nM), insulin (1-100 nM), insulin+ET-1 (100 nM I+E) or the appropriate vehicle or antagonist. At the end of the incubation period, resistin secretion was determined in the media by immunoblotting and densitometric analysis. ET-1 (1-100 nM) significantly decreased basal resistin secretion by 49% (1 nM), 43% (10nM), and 59% (100 nM). Insulin (1-100 nM) produced a concentration-dependent increase in resistin secretion from 3T3-L1 adipocytes (1 nM-42%, 10nM-55%, and 100 nM-86% vs. control). Insulin-stimulated resistin secretion (100 nM) was almost completely inhibited (94%) by ET-1 (100 nM). The effects of ET-1 on resistin protein secretion were inhibited by co-incubation with the ET(A) receptor antagonist BQ-610. In conclusion, our studies demonstrate that basal and hormonal stimulation of resistin secretion by insulin are inhibited by ET-1. Such findings demonstrate that resistin secretion is regulated in a similar manner to other adipose tissue factors, including leptin, in 3T3-L1 adipocytes. In addition, our findings suggest that vascular factors such as ET-1 may regulate whole body energy metabolism through adipocyte-derived hormones, including leptin and resistin.     

Growth hormone and the adipose conversion of 3T3 cells

Cultured preadipose 3T3 cells are induced or enabled to undergo adipose conversion in the presence of an extract of pituitary gland. Adipogenic activity is found in standard growth hormone preparations derived from different species. Further purification of rat growth hormone by several methods does not remove its adipogenic activity. Human growth hormone synthesized in Escherichia coli is also effective. Adipogenic activity is not associated with other pituitary polypeptides. Since growth hormone acts on preadipose cells in the absence of any other cell type, a mechanism exists for the direct participation of the pituitary gland in the regulation of this form of mesenchymal differentiation.