Ubiquitination is a post-translational modification (PTM) that is essential for balancing numerous physiological processes. To enable delineation of protein ubiquitination at a site-specific level, we generated an antibody, denoted UbiSite, recognizing the C-terminal 13 amino acids of ubiquitin, which remain attached to modified peptides after proteolytic digestion with the endoproteinase LysC. Notably, UbiSite is specific to ubiquitin. Furthermore, besides ubiquitination on lysine residues, protein N-terminal ubiquitination is readily detected as well. By combining UbiSite enrichment with sequential LysC and trypsin digestion and high-accuracy MS, we identified over 63,000 unique ubiquitination sites on 9,200 proteins in two human cell lines. In addition to uncovering widespread involvement of this PTM in all cellular aspects, the analyses reveal an inverse association between protein N-terminal ubiquitination and acetylation, as well as a complete lack of correlation between changes in protein abundance and alterations in ubiquitination sites upon proteasome inhibition.

Adipocyte precursor cells give raise to two major cell populations with different physiological roles: white and brown adipocytes. Here we demonstrate that the retinoblastoma protein (pRB) regulates white vs. brown adipocyte differentiation. Functional inactivation of pRB in wild-type mouse embryo fibroblasts (MEFs) and white preadipocytes by expression of simian virus 40 large T antigen results in the expression of the brown fat-specific uncoupling protein 1 (UCP-1) in the adipose state. Retinoblastoma gene-deficient ( Rb – / – ) MEFs and stem cells, but not the corresponding wild-type cells, differentiate into adipocytes with a gene expression pattern and mitochondria content resembling brown adipose tissue. pRB-deficient MEFs exhibit an increased expression of the Forkhead transcription factor Foxc2 and its target gene cAMP-dependent protein kinase regulatory subunit RIα, resulting in increased cAMP sensitivity. Suppression of cAMP-dependent protein kinase activity in Rb –/– MEFs blocked the brown adipocyte-like gene expression pattern without affecting differentiation per se . Immunohistochemical studies revealed that pRB is present in the nuclei of white but not brown adipocyte precursor cells at a developmental stage where both cell types begin to accumulate lipid and brown adipocytes express UCP-1. Furthermore, pRB rapidly undergoes phosphorylation upon cold-induced neodifferentiation and up-regulation of UCP-1 expression in brown adipose tissue. Finally, down-regulation of pRB expression accompanies transdifferentiation of white into brown adipocytes in response to β3-adrenergic receptor agonist treatment. We propose that pRB acts as a molecular switch determining white vs. brown adipogenesis, suggesting a previously uncharacterized function of this key cell cycle regulator in adipocyte lineage commitment and differentiation.

BackgroundThe uncoupling protein 1 (UCP1) is a hallmark of brown adipocytes and pivotal for cold- and diet-induced thermogenesis.Methodology/Principal FindingsHere we report that cyclooxygenase (COX) activity and prostaglandin E2 (PGE2) are crucially involved in induction of UCP1 expression in inguinal white adipocytes, but not in classic interscapular brown adipocytes. Cold-induced expression of UCP1 in inguinal white adipocytes was repressed in COX2 knockout (KO) mice and by administration of the COX inhibitor indomethacin in wild-type mice. Indomethacin repressed β-adrenergic induction of UCP1 expression in primary inguinal adipocytes. The use of PGE2 receptor antagonists implicated EP4 as a main PGE2 receptor, and injection of the stable PGE2 analog (EP3/4 agonist) 16,16 dm PGE2 induced UCP1 expression in inguinal white adipose tissue. Inhibition of COX activity attenuated diet-induced UCP1 expression and increased energy efficiency and adipose tissue mass in obesity-resistant mice kept at thermoneutrality.Conclusions/SignificanceOur findings provide evidence that induction of UCP1 expression in white adipose tissue, but not in classic interscapular brown adipose tissue is dependent on cyclooxygenase activity. Our results indicate that cyclooxygenase-dependent induction of UCP1 expression in white adipose tissues is important for diet-induced thermogenesis providing support for a surprising role of COX activity in the control of energy balance and obesity development.

Adipocytes play a central role in whole-body energy homoeostasis. Complex regulatory transcriptional networks control adipogensis, with ligand-dependent activation of PPARγ (peroxisome proliferator-activated receptor γ) being a decisive factor. Yet the identity of endogenous ligands promoting adipocyte differentiation has not been established. Here we present a critical evaluation of the role of LOXs (lipoxygenases) during adipocyte differentiation of 3T3-L1 cells. We show that adipocyte differentiation of 3T3-L1 preadipocytes is inhibited by the general LOX inhibitor NDGA (nordihydroguaiaretic acid) and the 12/15-LOX selective inhibitor baicalein. Baicalein-mediated inhibition of adipocyte differentiation was rescued by administration of rosiglitazone. Treatment with baicalein during the first 4 days of the differentiation process prevented adipocyte differentiation; supplementation with rosiglitazone during the same period was sufficient to rescue adipogenesis. Accordingly, we demonstrate that adipogenic conversion of 3T3-L1 cells requires PPARγ ligands only during the first 4 days of the differentiation process. We show that the baicalein-sensitive synthesis of endogenous PPARγ ligand(s) increases rapidly upon induction of differentiation and reaches a maximum on days 3–4 of the adipocyte differentiation programme. The conventional platelet- and leucocyte-type 12(S)-LOXs and the novel eLOX-3 (epidermis-type LOX-3) are expressed in white and brown adipose tissue, whereas only eLOX-3 is clearly expressed in 3T3-L1 cells. We suggest that endogenous PPARγ ligand(s) promoting adipocyte differentiation are generated via a baicalein-sensitive pathway involving the novel eLOX-3.

Cyclic AMP (cAMP)-dependent processes are pivotal during the early stages of adipocyte differentiation. We show that exchange protein directly activated by cAMP (Epac), which functions as a guanine nucleotide exchange factor for the Ras-like GTPases Rap1 and Rap2, was required for cAMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of cAMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response.Adipocytes are derived from multipotent mesenchymal stem cells in a process involving commitment to the adipocyte lineage followed by terminal differentiation of the committed preadipocytes. The process is regulated via complex interaction of external and internal clues, where cell shape and cytoskeletal tension converging on regulation of Rho and Rhokinase activity have been demonstrated to play pivotal roles (48,63). Whereas our understanding of the early steps of lineage determination still is limited, regulatory cascades controlling terminal adipocyte differentiation have been elucidated in great detail, particularly the sequential action of different transcription factors culminating in the expression of adipocyte-specific genes (25,30,58). Much information on terminal adipocyte differentiation has been obtained using model cell lines such as 3T3-L1 and 3T3-F442A or mouse embryo fibroblasts (MEFs). In both MEFs and 3T3-L1 preadipocytes, terminal differentiation is initiated upon treatment with fetal calf serum, glucocorticoids, and high levels of insulin or physiological concentrations of insulin-like growth factor 1 (IGF-1). Factors that increase cellular cyclic AMP (cAMP), such as isobutylmethylxanthine (IBMX) or forskolin, strongly accelerate the initiation of the differentiation program (for review, see references 25 and 45).Elevation of cellular cAMP concentration has been associated with crucial events in the early program of differentiation, such as suppression of Wnt10b (5) and Sp1 (64) and induction of CCAAT/enhancer-binding protein ␤ (C/EBP␤) (10,29,70). Moreover, the transcriptional activity of peroxisome proliferator-activated receptor ␦ (PPAR␦) is regulated synergistically by ligands and cAMP (32). In addition, cAMP has been implicated in the production of endogenous PPAR␥ ligand(s) occurring during the initial stages of differentiation (46, 67). The cAMP-responsive element-binding protein (CREB) is a central transcriptional activator of the adipocyte differentiation program. Activated CREB induces expres...

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The nuclear receptor peroxisome proliferator-activated receptor ␥ (PPAR␥) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR␥, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differentiation has remained enigmatic. Previously, we showed that lipoxygenase (LOX) activity is involved in activation of PPAR␥ during the early stages of adipocyte differentiation. Of the seven known murine LOXs, only the unconventional LOX epidermis-type lipoxygenase 3 (eLOX3) is expressed in 3T3-L1 preadipocytes. Here, we show that forced expression of eLOX3 or addition of eLOX3 products stimulated adipogenesis under conditions that normally require an exogenous PPAR␥ ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPAR␥. Production of hepoxilins was increased transiently during the initial stages of adipogenesis. Furthermore, small interfering RNA-mediated or retroviral short hairpin RNA-mediated knockdown of eLOX3 expression abolished differentiation of 3T3-L1 preadipocytes. Finally, we demonstrate that xanthine oxidoreductase (XOR) and eLOX3 synergistically enhanced PPAR␥-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPAR␥ activators capable of promoting the early PPAR␥-dependent steps in the conversion of preadipocytes into adipocytes.

Bile acids (BAs) are powerful regulators of metabolism, and mice treated orally with cholic acid are protected from dietinduced obesity, hepatic lipid accumulation, and increased plasma triacylglycerol (TAG) and glucose levels. Here, we show that plasma BA concentration in rats was elevated by exchanging the dietary protein source from casein to salmon protein hydrolysate (SPH). Importantly, the SPH-treated rats were resistant to diet-induced obesity. SPH-treated rats had reduced fed state plasma glucose and TAG levels and lower TAG in liver. The elevated plasma BA concentration was associated with induction of genes involved in energy metabolism and uncoupling, Dio2, Pgc-1␣, and Ucp1, in interscapular brown adipose tissue. Interestingly, the same transcriptional pattern was found in white adipose tissue depots of both abdominal and subcutaneous origin. Accordingly, rats fed SPH-based diet exhibited increased whole body energy expenditure and heat dissipation. In skeletal muscle, expressions of the peroxisome proliferatoractivated receptor ␤/␦ target genes (Cpt-1b, Angptl4, Adrp, and Ucp3) were induced. Pharmacological removal of BAs by inclusion of 0.5 weight % cholestyramine to the high fat SPH diet attenuated the reduction in abdominal obesity, the reduction in liver TAG, and the decrease in nonfasted plasma TAG and glucose levels. Induction of Ucp3 gene expression in muscle by SPH treatment was completely abolished by cholestyramine inclusion. Taken together, our data provide evidence that bile acid metabolism can be modulated by diet and that such modulation may prevent/ameliorate the characteristic features of the metabolic syndrome.