Dual-specificity phosphatases regulate mitogen-activated protein kinase signaling in adipocytes in response to inflammatory stress.

Obesity is a strong predictor of heart disease, insulin resistance, and type II diabetes. Chronic, low-grade inflammation links obesity and insulin resistance through mitogen-activated protein kinase (MAPK) signaling pathways. Upstream kinases activate MAPK signaling, while MAPK-specific dual-specificity phosphatases (DUSPs) act as key modulators and controllers of MAPK deactivation (i.e. dephosphorylation). Using tumor necrosis factor α (TNFα) in 3 T3-L1 adipocytes as a model of inflammation, we report that TNFα-mediated induction of Dusp1, Dusp8 and Dusp16 modulated the transient regulation of MAPK (i.e., ERK, JNK, and p38) phosphorylation and subsequent inflammatory gene expression. All three MAPKs examined were phosphorylated in preadipocytes and adipocytes in response to TNFα, where signaling magnitude and duration were phenotype-specific. Moreover, TNFα increased mRNA abundance of DUSPs in preadipocytes and adipocytes in a phenotype-specific manner, concomitant with dephosphorylation of MAPKs. RNA interference (RNAi)-mediated knockdown of Dusp1, Dusp8 and Dusp16 increased signaling magnitude and duration of ERK, JNK, and p38 that subsequently resulted in significant increases in MAPK-dependent inflammatory gene expression of MCP-1, IL-6, and Cox-2 in response to TNFα. This study highlights important roles for DUSPs as integral components of MAPK signaling and adipocyte inflammatory gene expression.

Mitogen-Dependent Regulation of DUSP1 Governs ERK and p38 Signaling During Early 3T3-L1 Adipocyte Differentiation.

Knowledge concerning mechanisms that control proliferation and differentiation of preadipocytes is essential to our understanding of adipocyte hyperplasia and the development of obesity. Evidence has shown that temporal regulation of mitogen-activated protein kinase (MAPK) phosphorylation and dephosphorylation is critical for coupling extracellular stimuli to cellular growth and differentiation. Using differentiating 3T3-L1 preadipocytes as a model of adipocyte hyperplasia, we examined a role for dual-specificity phosphatase 1 (DUSP1) on the timely modulation of MAPK signaling during states of growth arrest, proliferation, and differentiation. Using real-time reverse transcription PCR (qRT-PCR), we report that DUSP1 is induced during early preadipocyte proliferation concomitant with ERK and p38 dephosphorylation. As deactivation of ERK and p38 is essential for the progression of adipocyte differentiation, we further showed that de novo mRNA synthesis was required for ERK and p38 dephosphorylation, suggesting a role for "inducible" phosphatases in regulating MAPK signaling. Pharmacological and genetic inhibition of DUSP1 markedly increased ERK and p38 phosphorylation during early adipocyte differentiation. Based on these findings, we postulated that loss of DUSP1 would block adipocyte hyperplasia. However, genetic loss of DUSP1 was not sufficient to prevent preadipocyte proliferation or differentiation, suggesting a role for other phosphatases in the regulation of adipogenesis. In support of this, qRT-PCR identified several MAPK-specific DUSPs induced during early (DUSP2, -4, -5, & -6), mid (DUSP4 & -16) and late (DUSP9) stages of adipocyte differentiation. Collectively, these data suggest an important role for DUSPs in regulating MAPK dephosphorylation, with an emphasis on DUSP1, during early adipogenesis.

Modulation of IL-27 in adipocytes during inflammatory stress.

OBJECTIVE: While it is well established that adipose tissue-derived inflammation plays an important role in the pathogenic mechanisms linking obesity with metabolic dysfunction, the inflammatory mediators involved have not been fully elucidated. Here, we explored IL-12 family cytokines with a focus on IL-27 during obesity-induced inflammation in mice and cultured adipocytes (ADs) following exposure to inflammatory stimuli. METHODS: Relative mRNA abundance of IL-12 cytokines was assessed by reverse transcription polymerase chain reaction (RT-PCR) in genetically obese B6-ob/ob mice as well as C57BL/6J mice fed a high-fat diet and in ADs following exposure to inflammatory stimuli. Protein secretion of cytokines into culture media was assessed by ELISA, and the biological outcome of IL-27 stimulation was assessed by RT-PCR and immunoblotting. RESULTS: Heterodimeric subunits constituting IL-27 were significantly induced in obese mice. While all IL-12 genes were markedly induced by inflammatory stress in cultured ADs, IL-27 protein was the only cytokine secreted into culture media in response to inflammatory stress. Cultured ADs also responded to IL-27 stimulation with divergent outcomes that were dependent on the inflammatory milieu of target cells. CONCLUSIONS: These findings support the premise of autocrine/paracrine mechanisms involving IL-27 in ADs under conditions of inflammatory stress that may link obesity with inflammatory diseases.

Curcumin Inhibits 3T3-L1 Preadipocyte Proliferation by Mechanisms Involving Post-transcriptional p27 Regulation.

Previous reports from our lab have shown that Skp2 is necessary for p27 degradation and cell cycle progression during adipocyte differentiation. Data presented here demonstrate that the anti-inflammatory, anti-obesity phytochemical curcumin blocked Skp2 protein accumulation during early adipocyte hyperplasia. In addition, curcumin dose-dependently induced p27 protein accumulation and G1 arrest of synchronously replicating 3T3-L1 preadipocytes. Of note, p27 protein accumulation occurred in the presence of decreased p27 mRNA suggesting a role for post-transcriptional regulation. In support of this hypothesis, curcumin markedly increased p27 protein half-life as well as attenuated ubiquitin proteasome activity suggesting that inhibition of targeted p27 proteolysis occurred through curcumin-mediated attenuation of Skp2 and 26S proteasome activity. While we observed no cytotoxic effects for curcumin at doses less than 20 µM, it is important to note an increase in apoptotic signaling at concentrations greater than 30 µM. Finally, data presented here demonstrate that the anti-proliferative effect of curcumin was critical for the suppression of adipocyte differentiation and the development of the mature adipocyte. Collectively, our data demonstrate that curcumin-mediated post-transcriptional accumulation of p27 accounts in part for the anti-proliferative effect observed in 3T3-L1 preadipocytes.

Impact of obesity on IL-12 family gene expression in insulin responsive tissues.

Mounting evidence has established a role for chronic inflammation in the development of obesity-induced insulin resistance, as genetic ablation of pro-inflammatory cytokines and chemokines elevated in obesity improves insulin signaling in vitro and in vivo. Recent evidence further highlights interleukin (IL)-12 family cytokines as prospective inflammatory mediators linking obesity to insulin resistance. In this study, we present empirical evidence demonstrating that IL-12 family related genes are expressed and regulated in insulin-responsive tissues under conditions of obesity. First, we report that respective mRNAs for each of the known members of this cytokine family are expressed within detectable ranges in WAT, skeletal muscle, liver and heart. Second, we show that these cytokines and their cognate receptors are divergently regulated with genetic obesity in a tissue-specific manner. Third, we demonstrate that select IL-12 family cytokines are regulated in WAT in a manner that is dependent on the developmental stage of obesity as well as the inflammatory progression associated with obesity. Fourth, we report that respective mRNAs for IL-12 cytokines and receptors are also expressed and divergently regulated in cultured adipocytes under conditions of inflammatory stress. To our knowledge, this report is the first study to systemically evaluated mRNA expression of all IL-12 family cytokines and receptors in any tissue under conditions of obesity highlighting select family members as potential mediators linking excess nutrient intake to metabolic diseases such as insulin resistance, diabetes and heart disease.

Impact of reference gene selection for target gene normalization on experimental outcome using real-time qRT-PCR in adipocytes.

BACKGROUND: With the current rise in obesity-related morbidities, real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become a widely used method for assessment of genes expressed and regulated by adipocytes. In order to measure accurate changes in relative gene expression and monitor intersample variability, normalization to endogenous control genes that do not change in relative expression is commonly used with qRT-PCR determinations. However, historical evidence has clearly demonstrated that the expression profiles of traditional control genes (e.g., ß-actin, GAPDH, α-tubulin) are differentially regulated across multiple tissue types and experimental conditions. METHODOLOGY/PRINCIPAL FINDINGS: Therefore, we validated six commonly used endogenous control genes under diverse experimental conditions of inflammatory stress, oxidative stress, synchronous cell cycle progression and cellular differentiation in 3T3-L1 adipocytes using TaqMan qRT-PCR. Under each study condition, we further evaluated the impact of reference gene selection on experimental outcome using examples of target genes relevant to adipocyte function and differentiation. We demonstrate that multiple reference genes are regulated in a condition-specific manner that is not suitable for use in target gene normalization. CONCLUSION/SIGNIFICANCE: Data are presented demonstrating that inappropriate reference gene selection can have profound influence on study conclusions ranging from divergent statistical outcome to inaccurate data interpretation of significant magnitude. This study validated the use of endogenous controls in 3T3-L1 adipocytes and highlights the impact of inappropriate reference gene selection on data interpretation and study conclusions.