Genetic variability affects the development of brown adipocytes in white fat but not in interscapular brown fat.

Cold exposure induces brown adipocytes in retroperitoneal fat (RP) of adult A/J mice but not in C57BL/6J (B6) mice. In contrast, induction of the mitochondrial uncoupling protein 1 gene (Ucp1) in interscapular brown adipose tissue (iBAT) shows no strain dependence. We now show that unlike iBAT, in which Ucp1 was expressed in the fetus and continued throughout life, in RP, Ucp1 was transiently expressed between 10 and 30 days of age and then disappeared. Similar to the lack of genetic variation in the expression of Ucp1 in iBAT during cold induction of adult mice, no genetic variation in Ucp1 expression in iBAT was detected during development. In contrast, UCP1-positive multilocular adipocytes, together with corresponding increases in Ucp1 expression, appeared in RP at 10 days of age in A/J and B6 mice, but with much higher expression in A/J mice. At 20 days of age, brown adipocytes represent the major adipocyte present in RP of A/J mice. The disappearance of brown adipocytes by 30 days of age suggested that tissue remodeling occurred in RP. Genetic variability in Ucp1 expression could not be explained by variation in the expression of selective transcription factors and signaling molecules of adipogenesis. In summary, the existence of genetic variability between A/J and B6 mice during the development of brown adipocyte expression in RP, but not in iBAT, suggests that developmental mechanisms for the brown adipocyte differentiation program are different in these adipose tissues.

The regulation of fatty acid synthase by STAT5A.

Growth hormone (GH) diminishes adipose tissue mass in vivo and decreases expression and activity of fatty acid synthase (FAS) in adipocytes. GH and prolactin (PRL) are potent activators of STAT5 and exert adipogenic and antiadipogenic effects in adipocytes. In this study, we demonstrate that GH and PRL decrease the mRNA and protein levels of FAS in 3T3-L1 adipocytes. We present evidence that indicates that FAS is repressed at the level of transcription. In addition, PRL responsiveness was shown to exist between -1,594 and -700 of the rat FAS promoter. Moreover, responsiveness to PRL was abolished with mutation of a site at position -908 to -893, which we have shown to bind STAT5A in a PRL-dependent manner. Taken together, these data strongly suggest that PRL directly represses expression of FAS in adipocytes through STAT5A binding to the -908 to -893 site. Furthermore, our results indicate that STAT5A has an antilipogenic function in adipocytes and may contribute to the regulation of energy balance.

Effects of leukemia inhibitory factor on 3T3-L1 adipocytes.

Leukemia inhibitory factor (LIF) is a member of the gp130 cytokine family and signals through the receptor complex of gp130 and the LIF receptor (LIFR) to activate the JAK/STAT signaling cascade. Since LIF activates STATs 1 and 3 in adipocytes, we examined the effects of LIF on 3T3-L1 adipocytes. Our studies clearly demonstrate that LIF treatment had minimal effects on adipocyte differentiation as judged by marker gene expression, but did inhibit triacylglyceride (TAG) accumulation during adipogenesis. Acute treatment with LIF resulted in increased expression of suppressors of cytokine signaling-3 (SOCS3) and CCAAT/enhancer-binding protein-delta (C/EBPdelta) mRNA in 3T3-L1 adipocytes. Moreover, the upregulation of C/EBPdelta correlated with binding to three sites in the C/EBPdelta promoter by LIF-activated protein complexes that contained STAT1 and not STAT3. Chronic treatment with LIF resulted in decreased protein levels of sterol regulatory element binding protein-1 (SREBP1) and fatty acid synthase (FAS), but had no effect on the expression of other adipocyte marker proteins or on TAG levels in mature 3T3-L1 adipocytes. LIF had a small effect on insulin-stimulated glucose uptake in 3T3-L1 adipocytes, but did not cause insulin resistance following chronic treatment. These findings indicate that LIF has similar and distinct effects in comparison with the effects of other gp130 cytokines on cultured fat cells. In summary, our results support a role for LIF in the regulation of proteins involved in lipid synthesis and in the modulation of signal transduction pathways in 3T3-L1 adipocytes.

Stabilization, not polymerization, of microtubules inhibits the nuclear translocation of STATs in adipocytes.

Signal transducers and activators of transcriptions (STATs) are a family of latent transcription factors which are activated by a variety of growth factors and cytokines in many cell types. However, the mechanism by which these transcription factors translocate to the nucleus is poorly understood. The goal of this study was to determine the requirement of microfilaments and microtubules for cytokine induced STAT activation in cultured adipocytes. We used seven different actin-specific and microtubule-specific agents that are well-established effectors of these cytoskeletal networks. Our results clearly demonstrate that inhibition of microfilaments or the prevention of microtubule polymerization has no effect on the ability of STATs to be tyrosine phosphorylated or to translocate to the nucleus. However, we observed that paclitaxel, a microtubule stabilizer, resulted in a significant decrease in the nuclear translocation of STATs without affecting the cytosolic tyrosine phosphorylation of these transcription factors. In summary, our results demonstrate that the dynamic instability, but not the polymerization, of microtubules contributes to nuclear translocation of STAT proteins in adipocytes.

Effects of cardiotrophin on adipocytes.

Cardiotrophin (CT-1) is a naturally occurring protein member of the interleukin (IL)-6 cytokine family and signals through the gp130/leukemia inhibitory factor receptor (LIFR) heterodimer. The formation of gp130/LIFR complex triggers the auto/trans-phosphorylation of associated Janus kinases, leading to the activation of Janus kinase/STAT and MAPK (ERK1 and -2) signaling pathways. Since adipocytes express both gp130 and LIFR proteins and are responsive to other IL-6 family cytokines, we examined the effects of CT-1 on 3T3-L1 adipocytes. Our studies have shown that CT-1 administration results in a dose- and time-dependent activation and nuclear translocation of STAT1, -3, -5A, and -5B as well as ERK1 and -2. We also confirmed the ability of CT-1 to induce signaling in fat cells in vivo. Our studies revealed that neither CT-1 nor ciliary neurotrophic factor treatment affected adipocyte differentiation. However, acute CT-1 treatment caused an increase in SOCS-3 mRNA in adipocytes and a transient decrease in peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA that was regulated by the binding of STAT1 to the PPARgamma2 promoter. The effects of CT-1 on SOCS-3 and PPARgamma mRNA were independent of MAPK activation. Chronic administration of CT-1 to 3T3-L1 adipocytes resulted in a decrease of both fatty acid synthase and insulin receptor substrate-1 protein expression yet did not effect the expression of a variety of other adipocyte proteins. Moreover, chronic CT-1 treatment resulted in the development of insulin resistance as judged by a decrease in insulin-stimulated glucose uptake. In summary, CT-1 is a potent regulator of signaling in adipocytes in vitro and in vivo, and our current efforts are focused on determining the role of this cardioprotective cytokine on adipocyte physiology.

STAT 1 binds to the LPL promoter in vitro.

Interferon-gamma (IFNgamma) has been shown to decrease the expression and activity of lipoprotein lipase (LPL). Hence, we searched for IFNgamma sensitive binding sites within the murine LPL promoter. A region of the LPL promoter was identified that specifically binds nuclear, but not cytosolic, extracts isolated from IFNgamma-treated 3T3-L1 adipocytes. EMSA analysis revealed that two protein complexes bind to this site within the LPL promoter and supershift analysis demonstrated that both of these complexes contained STAT 1 proteins. In addition, we have shown that this effect is specific for IFNgamma, since LIF treatment, which also induces STAT 1, did not confer binding to this site. Interestingly, binding to this site within the LPL promoter could be effectively competed with a STAT 1 binding site that we previously identified in the PPARgamma2 promoter. Also, IFNgamma treatment resulted in decreased levels of LPL protein. In summary, we have identified a STAT 1 binding site within the murine LPL promoter which likely plays a role in the IFNgamma induced decrease of LPL expression.