When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G1͞S checkpoint synchronously as evidenced by the expression͞ activation of cdk2-cyclin-E͞A, turnover of p27͞kip1, hyperphosphorylation of Rb, translocation of cyclin D1 from nuclei to cytoplasm and GSK-3␤ from cytoplasm to nuclei, and incorporation of [ 3 H]thymidine into DNA. As the cells cross the G1͞S checkpoint, C͞EBP␤ acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinase͞extra-cellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.cell cycle ͉ adipogenesis ͉ 3T3-L1 preadipocyte ͉ C͞EBP␣ ͉ PPAR␥

The increase of adipose tissue mass associated with obesity is due in part to an increase in the number of adipocytes. This hyperplasia results from recruitment of pluripotent stem cells present in the vascular stroma of adipose tissue. A model cell culture system has been developed that recapitulates this process both ex vivo and in vivo. After treatment of pluripotent C3H10T1͞2 stem cells with bone morphogenic protein 4 (BMP4) during proliferation followed by differentiation inducers at growth arrest, the cells synchronously enter S phase and undergo mitotic clonal expansion, a hallmark of preadipocyte differentiation. Upon exiting the cell cycle, these cells express adipocyte markers and acquire adipocyte characteristics at high frequency. C3H10T1͞2 cells treated with BMP4 in cell culture and implanted s.c. into athymic mice develop into tissue indistinguishable from adipose tissue in normal fat depots. We interpret the findings as evidence that BMP4 is capable of triggering commitment of pluripotent C3H10T1͞2 stem cells to the adipocyte lineage.adipose tissue ͉ differentiation ͉ bone morphogenic protein 4 ͉ mitotic clonal expansion ͉ development

Hormonal induction of growth-arrested 3T3-L1 preadipocytes triggers a signaling cascade that culminates in adipogenesis. CCAAT͞ enhancer-binding protein (C͞EBP)␤ is expressed immediately but gains DNA-binding activity only after a long lag as the cells synchronously begin mitotic clonal expansion (MCE). After MCE, a process required for adipogenesis, C͞EBP␤ activates expression of C͞EBP␣ and peroxisome proliferator-activated receptor ␥, which then transcriptionally activate genes that produce the adipocyte phenotype. When mouse embryo fibroblasts (MEFs) are subjected to the same differentiation protocol, a subset of the MEFs undergoes a similar program of events. Similar to 3T3-L1 preadipocytes, the MEFs reenter the cell cycle (as indicated by the synchronous expression of cyclin A) and undergo MCE as evidenced by the incorporation of BrdUrd into DNA and the formation of mitotic foci of cells that undergo adipogenesis. C͞EBP␤ is expressed immediately after induction but exhibits delayed acquisition of DNAbinding activity followed by expression of adipocyte markers and the accumulation of cytoplasmic triglyceride. MEFs from C͞EBP␤(؊͞؊) mice, however, neither undergo MCE nor differentiate into adipocytes. Forced expression of C͞EBP␤ (LAP) but not dominant-negative C͞EBP␤ (LIP) in C͞EBP␤(؊͞؊) MEFs restores MCE, expression of adipocyte markers, and the capacity to form mitotic foci of cells that undergo adipogenesis. These findings demonstrate that expression of C͞EBP␤ is a prerequisite for MCE in the adipocyte-differentiation program.3T3-L1 preadipocyte ͉ mouse embryo fibroblasts ͉ C͞EBP␣ ͉ PPAR␥ ͉ cell cycle A fter induction of differentiation, postconfluent, growtharrested 3T3-L1 preadipocytes synchronously reenter the cell cycle, undergo several rounds of mitotic clonal expansion (MCE), and then express genes that produce the adipocyte phenotype (1-4). Recent evidence (5, 6) has established that MCE is required for the progression of the differentiation program. Immediately (within 2-4 h) after induction CCAAT͞ enhancer-binding protein (C͞EBP)␤ is expressed but unable to bind DNA and thus cannot activate the regulatory genes responsible for terminal differentiation. Only after a long lag period (10-12 h) does C͞EBP␤ acquire DNA-binding activity (7). Acquisition of binding activity occurs as the cells synchronously reenter the cell cycle, traverse the G 1 -S checkpoint, and begin MCE (7). Coincident with the acquisition of DNA-binding activity, C͞EBP␤ binds to centromeres through consensus C͞EBP-binding sites in centromeric satellite DNA (7). After acquiring DNA-binding activity C͞EBP␤ activates transcription of the C͞EBP␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥) genes mediated by C͞EBP regulatory elements in their promoters (8-10). Together, C͞EBP␣ and PPAR␥ coordinately activate the transcription of genes that give rise to the adipocyte phenotype (1, 3, 4). Since both C͞EBP␣ and PPAR␥ are antimitotic (11)(12)(13)(14), the timing of this gain of function by C͞EBP␤ is critical, because premature e...

CCAAT enhancer-binding protein (C͞EBP)␤, C͞EBP␣, and peroxisome proliferator activated receptor (PPAR)␥ act in a cascade where C͞EBP␤ activates expression of C͞EBP␣ and PPAR␥, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C͞EBP␤ is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3␤ enters the nucleus, which correlates with hyperphosphorylation of C͞EBP␤ and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C͞EBP␤ show that phosphorylation of Thr-188 by mitogen-activating protein kinase ''primes'' C͞EBP␤ for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3␤, acquisition of DNA-binding function, and transactivation of the C͞EBP␣ and PPAR␥ genes. The delayed transactivation of the C͞EBP␣ and PPAR␥ genes by C͞EBP␤ appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C͞EBP␣ and PPAR␥ are antimitotic.3T3-L1 preadipocyte ͉ cell cycle ͉ differentiation ͉ mitotic clonal expansion C CAAT enhancer-binding protein ␤ (C͞EBP␤) is expressed early in the adipocyte differentiation program, first initiating mitotic clonal expansion (MCE) (1, 2) and later activating expression of C͞EBP␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥) (3-6), pleiotropic activators of adipocyte genes (3,4,7,8). Both MCE and expression of C͞EBP␣ and PPAR␥ are required for differentiation (1, 2, 9). When treated with differentiation inducers, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle, undergo approximately two rounds of mitosis, then exit the cell cycle and enter the terminal stages of differentiation. Transcriptional activation of the C͞EBP␣ and PPAR␥ genes is induced by the interaction of C͞EBP␤ with C͞EBP regulatory elements in these gene promoters (3-6).Although expression of C͞EBP␤ occurs within 2 h of induction of differentiation, acquisition of DNA-binding activity and thus transcription of the C͞EBP␣ and PPAR␥ genes are delayed (10). Acquisition of DNA-binding activity begins after a long lag (Ϸ14 h), concurrent with the entry of S phase at the onset of MCE and transcription of the C͞EBP␣ and PPAR␥ genes (ref. 10 and Fig. 1). This lag appears necessary, because C͞EBP␣ and PPAR␥ are antimitotic (11-15), and their premature expression would otherwise prevent the MCE required for differentiation. To elucidate the mechanism by which C͞EBP␤ acquires DNAbinding activity, we considered the possibility that covalent modification of C͞EBP␤ occurs during this time window.Several lines of evidence indicated that C͞EBP␤ can be phosphorylated in vitro by a variety of kinases, including PKA (16), PKC (16), mitogen-activated protein kinase (MAPK) (17), and Ca 2ϩ -calmodulin-dependent kinase II (18). ...

Previous studies showed that exposure of C3H10T1͞2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1͞2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. 5-azacytidine ͉ adipogenesis ͉ determination ͉ mesenchymal stem cell ͉ preadipocyte

The forkhead factor Foxo1 (or FKHR) was identified in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor (PPAR) ␥-interacting protein.

The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g. adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.

The concept of using cholinesterase bioscavengers for prophylaxis against organophosphorous nerve agents and pesticides has progressed from the bench to clinical trial. However, the supply of the native human proteins is either limited (e.g., plasma-derived butyrylcholinesterase and erythrocytic acetylcholinesterase) or nonexisting (synaptic acetylcholinesterase). Here we identify a unique form of recombinant human butyrylcholinesterase that mimics the native enzyme assembly into tetramers; this form provides extended effective pharmacokinetics that is significantly enhanced by polyethylene glycol conjugation. We further demonstrate that this enzyme (but not a G117H/E197Q organophosphorus acid anhydride hydrolase catalytic variant) can prevent morbidity and mortality associated with organophosphorous nerve agent and pesticide exposure of animal subjects of two model species.countermeasures | nonconventional warfare agents | organophosphorous pesticides | protein engineering | transgenic plants B utyrylcholinesterase (BChE) is the major cholinesterase (ChE) in the serum of humans (1, 2). Although the closely related enzyme acetylcholinesterase (AChE) is well described as the primary synaptic regulator of cholinergic transmission, a definitive physiological role for BChE has not yet been demonstrated (3). BChE is catalytically promiscuous and hydrolyzes not only acetylcholine (ACh), but also longer-chain choline esters (e.g., butyrylcholine, its preferred substrate, and succinylcholine) and a variety of non-choline esters, such as acetylsalicylic acid (aspirin) and cocaine (4, 5). Moreover, BChE binds most environmentally occurring ChE inhibitors as well as man-made organophosphorous (OP) pesticides and nerve agents (NAs) (6, 7-10).The systemic biodistribution and affinity for ChE inhibitors allow endogenous BChE to provide broad-spectrum protection against various toxicants by their sequestration before they reach cholinergic synapses. However, under realistic high-dose exposure scenarios, BChE serum levels are too low to afford adequate protection, resulting in persistent cholinergic excitation due to irreversible inhibition of AChE and subsequent accumulation of ACh. Sublethal manifestations of this state include unregulated exocrine secretion and gastrointestinal hypermotility. Death usually results from unregulated stimulation at neuromuscular junction leading to hemodynamic instability and tetanic contraction of the respiratory muscles (11,12).Current OP poisoning therapy consists of atropine for muscarinic ACh receptor blockade and diazepam for symptomatic management of convulsions (12). Additionally, oxime therapy with 2-pralidoxime (2-PAM) can effectively reactivate some but not all OP-AChE adducts (13)(14)(15). This standard therapeutic approach can reduce mortality, but insufficiently prevents the incapacitation associated with OP toxicity (12, 16).Prophylaxis by administration of exogenous ChEs has proven successful in reducing OP-associated morbidity and mortality, but requires the availability of rel...