Insulin-like growth factor-1 receptor signaling in 3T3-L1 adipocyte differentiation requires lipid rafts but not caveolae

Previously, we have found that lipid rafts/caveolae were essential for insulin-like growth factor-1 (IGF-1) receptor signaling during 3T3-L1 preadipocytes differentiation induction. However, it was not identified as to which of the membrane lipid-ordered microdomains mediates the receptor signal. Using small double-stranded RNA-mediated interference (RNAi), we successfully suppressed the caveolin-1 protein expression. In cells stably transfected with vector expressing small interfering RNA (siRNA) fragment, no caveolin-1 protein or caveola was detected. On the other hand, removal of caveolin-1 did not affect the caveolinless lipid rafts or the localization of IGF-1 receptor in lipid rafts on plasma membrane. IGF-1 receptor signal transduction and induced cellular differentiation were normal in RNAi cells with only lipid rafts. Furthermore, these IGF-1 receptor signaling events were still sensitive to the cholesterol-binding reagents. Thus, our results suggest that lipid rafts are sufficient for IGF-1 receptor signaling and the recruitment of signal molecules by caveolin-1 is not essential for IGF-1 receptor signaling.     

Lipid rafts/caveolae are essential for insulin-like growth factor-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction

Lipid rafts/caveolae are found to be essential for insulin-like growth factor (IGF)-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction. In 3T3-L1 cells, IGF-1 receptor is located in lipid rafts/caveolae of the plasma membrane and can directly interact with caveolin-1, the major protein component in caveolae. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with cholesterol-binding reagent, beta-methylcyclodextrin or filipin, blocks the IGF-1 receptor signaling in 3T3-L1 preadipocyte. Both hormonal induced adipocyte differentiation and mitotic clonal expansion are inhibited by lipid rafts/caveolae disruption. However, a nonspecific lipid binding reagent, xylazine, does not affect adipocyte differentiation or mitotic clonal expansion. Further studies indicate that lipid rafts/caveolae are required only for IGF-1 receptor downstream signaling and not the activation of receptor itself by ligand. Thus, our results suggest that localization in lipid rafts/caveolae and association with caveolin enable IGF-1 receptor to have a close contact with downstream signal molecules recruited into lipid rafts/caveolae and transmit the signal through these signal molecule complexes.     

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.     

Prolactin receptor signaling is essential for perinatal brown adipocyte function: a role for insulin-like growth factor-2

Background

The lactogenic hormones prolactin (PRL) and placental lactogens (PL) play central roles in reproduction and mammary development. Their actions are mediated via binding to PRL receptor (PRLR), highly expressed in brown adipose tissue (BAT), yet their impact on adipocyte function and metabolism remains unclear.

Methodology/Principal Findings

PRLR knockout (KO) newborn mice were phenotypically characterized in terms of thermoregulation and their BAT differentiation assayed for gene expression studies. Derived brown preadipocyte cell lines were established to evaluate the molecular mechanisms involved in PRL signaling on BAT function. Here, we report that newborn mice lacking PRLR have hypotrophic BAT depots that express low levels of adipocyte nuclear receptor PPARγ2, its coactivator PGC-1α, uncoupling protein 1 (UCP1) and the β3 adrenoceptor, reducing mouse viability during cold challenge. Immortalized PRLR KO preadipocytes fail to undergo differentiation into mature adipocytes, a defect reversed by reintroduction of PRLR. That the effects of the lactogens in BAT are at least partly mediated by Insulin-like Growth Factor-2 (IGF-2) is supported by: i) a striking reduction in BAT IGF-2 expression in PRLR KO mice and in PRLR-deficient preadipocytes; ii) induction of cellular IGF-2 expression by PRL through JAK2/STAT5 pathway activation; and iii) reversal of defective differentiation in PRLR KO cells by exogenous IGF-2.

Conclusions

Our findings demonstrate that the lactogens act in concert with IGF-2 to control brown adipocyte differentiation and growth. Given the prominent role of brown adipose tissue during the perinatal period, our results identified prolactin receptor signaling as a major player and a potential therapeutic target in protecting newborn mammals against hypothermia.     

Protein kinase B beta/Akt2 plays a specific role in muscle differentiation

Insulin-like growth factors positively regulate muscle differentiation through activation of the phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt) signaling pathway. Here, we compare the role of the two closely related alpha (Akt1) and beta (Akt2) isoforms of PKB in muscle differentiation. During differentiation of C2.7 or L6D2 myoblasts, PKBbeta was up-regulated whereas expression of PKBalpha was unaltered. Although the two isoforms were found active in both myoblasts and myotubes, cell fractionation experiments indicated that they displayed distinct subcellular localizations in differentiated cells with only PKBbeta localized in the nuclei. In a transactivation assay, PKBbeta (either wild-type or constitutively active) was more efficient than PKBalpha in activating muscle-specific gene expression. Moreover, microinjection of specific antibodies to PKBbeta inhibited differentiation of muscle cells, whereas control or anti-PKBalpha antibodies did not. On the other hand, microinjection of the anti-PKBalpha antibodies caused a block in cell cycle progression in both non muscle and muscle cells, whereas anti-PKBbeta antibodies had no effect. Taken together, these results show that PKBbeta plays a crucial role in the commitment of myoblasts to differentiation that cannot be substituted by PKBalpha.     

Protein kinase A suppresses the differentiation of 3T3-L1 preadipocytes

cAMP and protein kinase A （PKA） are widely known as signaling molecules that are important for the induction of adipogenesis. Here we show that a strong increase in the amount of cAMP inhibits the adipogenesis of 3T3-L1 fibroblast cells. Stimulation of PKA activity suppresses adipogenesis and, in contrast, inhibition of PKA activity markedly accelerates the adipogenic process. As adipogenesis progresses, there is a significant increase in the expression level of PKA regulatory and a corresponding decrease in PKA activity. Moreover, treatment of 3T3-L1 cells with epidermal growth factor （EGF） stimulates PKA activity and blocks adipogenesis. Inhibition of PKA activity abolishes this suppressive effect of EGF on adipogenesis. Moreover, asubunits ctivation of PKA induces serine/threonine phosphorylation, reduces tyrosine phosphorylation of insulin receptor substrate 1 （IRS-1） and the association between PKA and IRS-1. Taken together, our study demonstrates that PKA has a pivotal role in the suppression of adipogenesis, cAMP at high concentrations can suppress adipogenesis through PKA activation. These findings could be important and useful for understanding the mechanisms of adipogenesis and the relevant physiological events.     

Prostanoid EP4 receptor is involved in suppression of 3T3-L1 adipocyte differentiation

Prostaglandins (PGs) have been shown to play various roles in adipogenesis. In this study, we investigated on which PGE receptor subtypes are involved in the inhibition of 3T3-L1 preadipocyte differentiation. The triglyceride content of cells, used as an index of differentiation, was decreased when PGE(2), the FP-agonist fluprostenol or dibutyryl cAMP, was exogenously added to differentiation cocktails. 3T3-L1 preadipocyte cells express mRNAs for the prostanoid EP4, FP, and IP receptors. PGE(2) and the EP4 agonist AE1-329 increased cAMP levels in preadipocytes in a dose-dependent manner. AE1-329 suppressed the expression induction of differentiation marker genes such as resistin and peroxisome proliferator-activated receptor-gamma. The inhibitory effect of PGE(2) but not that of fluprostenol was reversed by the addition of the EP4 antagonist AE3-208. AE3-208 mimicked the differentiation-promoting effects of indomethacin. These results suggest that the EP4 receptor mediates the suppressive action of PGE(2) in 3T3-L1 adipocyte differentiation.     

The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.     

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.     

Vanillin induces adipocyte differentiation in 3T3-L1 cells by activating extracellular signal regulated kinase 42/44

AimsTo investigate the effect of vanillin, a dietary component, on adipocyte differentiation and the mechanism involved in the process using 3T3-L1 murine preadipocytes.Main methodsThe effect of vanillin on adipocyte differentiation was detected by Oil Red O analysis. The activation of extracellular signal regulated kinase 42/44 (ERK 42/44), Akt, expression of the key regulator of adipocyte differentiation peroxisome proliferators-activated receptor (PPARγ) and its target gene glucose transporter 4 (GLUT4) were detected by western blotting. Glucose uptake assay was used to determine the insulin sensitivity of adipocytes differentiated by vanillin treatment. To confirm the role of ERK 42/44 and Akt, Oil Red O analysis was performed with cells differentiated in the presence or absence of ERK inhibitor U0126 or Akt kinase 1/2 inhibitor.Key findingsVanillin induced adipocyte differentiation in 3T3-L1 cells in a dose dependent manner and also increased the expression levels of PPARγ and its target gene GLUT4. The adipocytes differentiated by vanillin exhibited insulin sensitivity as demonstrated by a significant increase in glucose uptake. Vanillin treatment activated the phosphorylation of ERK 42/44 during the initial phase of adipocyte differentiation but there was no significant change in the Akt phosphorylation status.SignificanceThe data show that vanillin induces adipocyte differentiation in 3T3-L1 cells by activating ERK42/44 and these adipocytes are insulin sensitive in nature.     

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.     

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.     

c-Crk, a substrate of the insulin-like growth factor-1 receptor tyrosine kinase, functions as an early signal mediator in the adipocyte differentiation process

Differentiation of 3T3-L1 preadipocytes into adipocytes is induced by a combination of inducers, including a glucocorticoid, an agent that elevates cellular cAMP, and a ligand of the insulin-like growth factor-1 receptor. Previous studies have implicated protein-tyrosine phosphatase (PTPase) HA2, a homologue of PTPase 1B, in the signaling cascade initiated by the differentiation inducers. Vanadate, a potent PTPase inhibitor, blocks adipocyte differentiation at an early stage in the program, but has no effect on the mitotic clonal expansion required for differentiation. Exposure of preadipocytes to vanadate along with the inducing agents led to the accumulation of pp35, a phosphotyrosyl protein that is a substrate for PTPase HA2. pp35 was purified to homogeneity and shown by amino acid sequence and mass analyses of tryptic peptides to be c-Crk, a known cytoplasmic target of the insulin-like growth factor-1 receptor tyrosine kinase. Transfection of 3T3-L1 preadipocytes with a c-Crk antisense RNA expression vector markedly reduced c-Crk levels and prevented differentiation into adipocytes. Studies with C3G, a protein that binds to the SH3 domain in c-Crk, showed that phosphorylation of c-Crk rendered the SH3 domain inaccessible to C3G. Taken together, these findings indicate that locking c-Crk in the phosphorylated state with vanadate prevents its participation in the signaling system that initiates adipocyte differentiation.