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.     

Carnosic acid and carnosol inhibit adipocyte differentiation in mouse 3T3-L1 cells through induction of phase2 enzymes and activation of glutathione metabolism

In the previous studies, we reported that carnosic acid (CA) and carnosol (CS) originating from rosemary protected cortical neurons by activating the Keap1/Nrf2 pathway, which activation was initiated by S-alkylation of the critical cysteine thiol of the Keap1 protein by the “electrophilic” quinone-type of CA or CS. Here, we found that CA and CS inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes. In contrast, other physiologically-active and rosemary-originated compounds were completely negative. These actions seemed to be mediated by activation of the antioxidant-response element (ARE) and induction of phase2 enzymes. This estimation is justified by our present findings that only CA and CS among rosemary-originated compounds significantly activated the ARE and induced the phase2 enzymes. Next, we performed cDNA microarray analysis in order to identify the gene(s) responsible for these biological actions and found that phase2 enzymes (Gsta2, Gclc, Abcc4, and Abcc1), all of which are involved in the metabolism of glutathione (GSH), constituted 4 of the top 5 CA-induced genes. Furthermore, CA and CS, but not the other compounds tested, significantly increased the intracellular level of total GSH. Thus, we propose that the stimulation of GSH metabolism may be a critical step for the inhibition of adipocyte differentiation in 3T3-L1 cells and suggest that pro-electrophilic compounds such as CA and CS may be potential drugs against obesity-related diseases.     

Alpha-lipoic acid attenuates adipocyte differentiation and lipid accumulation in 3T3-L1 cells via AMPK-dependent autophagy

Aims

During the adipocyte differentiation, some intracellular organelles are degraded and instead lipid droplets are gradually accumulated in the cytoplasm for energy storage. Autophagy, a self-eating process, has been implicated in the removal of intracellular components in adipogenesis, but its mechanism is poorly understood. In this work we examined how α-lipoic acid modulates the autophagic process during the adipocyte differentiation.

Main methods

3T3-L1 pre-adipocytes were differentiated in the medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Lipid contents in adipocytes were determined by Oil-Red O staining. Autophagy was evaluated by Western blotting, accumulation of acidic vacuoles in cells.

Key findings

We observed that formation of LC3-II, an indicative marker for autophagy, was greatly down-regulated at the beginning stage of differentiation, but it was gradually increased with respect to earlier differentiation time. In addition, ATG5-12 conjugates were similarly produced, and acidic autophagic vacuoles were greatly elevated at the earlier stages of differentiation. Furthermore, α-lipoic acid deteriorated the intracellular accumulation of lipid droplets by blocking the production of acidic autophagic vacuoles, LC3-II, and other autophagy-related proteins during the adipocyte differentiation and influenced expression of adipocyte-stimulating factors. It also specifically suppressed activation of AMPK, an essential modulator for autophagy, at the earlier step of adipocyte differentiation.

Significance

These data suggest that α-lipoic acid significantly attenuates adipocyte differentiation via the direct modulation of intracellular degradation process and consequently decrease intracellular fat deposit of adipocytes.     

Trigonelline attenuates the adipocyte differentiation and lipid accumulation in 3T3-L1 cells

Trigonelline is a natural alkaloid mainly found in Trigonella Foenum Graecum (fenugreek) Fabaceae and other edible plants with a variety of medicinal applications. Therefore, we investigated the molecular mechanism of trigonelline (TG) on the inhibition of adipocyte differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline suppressed lipid droplet accumulation in a concentration (75 and 100 μM) dependent manner. Treatment of adipocyte with of TG down regulates the peroxisome proliferator-activated receptor (PPARγ) and CCAAT element binding protein (C/EBP-α) mRNA expression, which leads to further down regulation of other gene such as adiponectin, adipogenin, leptin, resistin and adipocyte fatty acid binding protein (aP2) as compared with respective control cells on 5th and 10th day of differentiation. Further, addition of triognelline along with troglitazone to the adipocyte attenuated the troglitazone effects on PPARγ mediated differentiation and lipid accumulation in 3T3-L1 cells. Trigonelline might compete against troglitazone for its binding to the PPARγ. In addition, adipocyte treated with trigonelline and isoproterenol separately. Isoproterenol, a lipolytic agent which inhibits the fatty acid synthase and GLUT-4 transporter expression via cAMP mediated pathway, we found that similar magnitude response of fatty acid synthase and GLUT-4 transporter expression in trigonelline treated adipocyte. These results suggest that the trigonelline inhibits the adipogenesis by its influences on the expression PPARγ, which leads to subsequent down regulation of PPAR-γ mediated pathway during adipogenesis. Our findings provide key approach to the mechanism underlying the anti-adipogenic activity of trigonelline.     

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.     

Progestins stimulate the differentiation of 3T3-L1 preadipocytes

The effect of progesterone on the differentiation of the 3T3-L1 preadipocytes was investigated and compared with other sex steroids (estradiol and testosterone), with cortisol, with the synthetic progestin R5020 and with the progestin/glucocorticoid antagonist RU38486. At 10⁻⁸ M, progesterone stimulated the activity of glycerol-3-phosphate dehydrogenase and triglyceride deposition. Progesterone, R5020, cortisol, and RU38486 increased triglycerides about 2-fold at 10⁻⁷ M. Only minimal effects were observed with testosterone and estradiol even at 10⁻⁶ M. When the cells were cultured in presence of 10⁻⁵ M metyrapone the effect of progesterone was unchanged, suggesting that the progesterone was not metabolized to a glucocorticoid. Progesterone, R5020 and RU38486 competed efficiently with [³H]dexamethasone for the glucocorticoid receptor in 3T3-L1 cytosol. These results indicate a significant, reproducible dose-dependent effect of progestins on differentiation of the preadipocytes, which appears to be mediated via the glucocorticoid receptor.     

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.     

Phenylenediamine derivatives induce GDF-15/MIC-1 and inhibit adipocyte differentiation of mouse 3T3-L1 cells

Phenylenediamine derivatives can function as a hydrogen donor and reportedly exert various biological actions including cytoprotective effects against oxidative stress, possibly by acting as an antioxidant. Previous studies showed that feeding of such compounds to mice reduced their body weight, but the precise mechanism remains unknown at present. Here, we found that these compounds inhibited the in vitro differentiation of mouse preadipocytes, 3T3-L1 cells, into adipocytes, suggesting that, at least in part, reduced generation of adipocytes might contribute to the observed weight loss in mice. Next, we performed array analysis and found that the expression of GDF-15/MIC-1, which is a TGFβ superfamily cytokine, and Trib 3, an intracellular downstream effector of the cytokines, was up-regulated by these derivatives. Thus, we identified the compounds as inducers of GDF-15/MIC-1 and suggest that such induction may have led to inhibition of adipocyte differentiation, which could account for the weight-loss effect of these compounds.     

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.     

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.