PPARγ agonists induce a white-to-brown fat conversion through stabilization of PRDM16 protein

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Highlights? PPARγ full agonists induce a brown fat phenotype in subcutaneous WAT ? PRDM16 is required for the development of PPARγ agonist-inducible brow adipocytes ? PRDM16 and PPARγ agonists synergistically activate the brown fat gene program ? Browning effect is mediated through the enhanced stability of the PRDM16 protein     

Morphological stages of regeneration in the planarian Dugesia tigrina: A light and electron microscopic study

A precise sequence of four morphological stages of head regeneration in the planarian Dugesia tigrina has been determined by light and electron microscopy. Each stage is identified by a particular morphogenetic process: I, wound healing; II, blastema development; III, growth; IV, differentiation. A wound epidermis consisting of a thin, sheet-like layer of cells, rapidly forms from undamaged epidermal cells at the wound margin. The early blastema is comprised of neoblasts which mature into regeneration cells. The maturational changes include the appearance of a nucleolus, nuclear pores, and perinuclear dense aggregates of granulofibrillar material in these cells. These elements are not evident in the neoblasts of the younger blastema. No mitotic cells are encountered in the blastema or wound epidermis. Cytoplasmic expansion of the regeneration cells is correlated with the formation of numerous microtubules radiating from a juxtanuclear centrosphere. During differentiation of muscle cells, distended, granule-studded cisternae, having moderately fibrillar contents, are regularly disposed adjacent to small patches of myofilaments. Presumptive epidermal cells are recognized by prominent “islands” of finely fibrillar cytoplasm. These cytoplasmic zones persist for a time during definitive differentiation when Golgi bodies, vacuoles, mucous droplets, and rhabdites become evident. The newly formed epidermal cells become inserted among the cells of the wound epidermis. Thus, cells of both the blastema and of the wound epidermis contribute to the reconstituted epidermis.     

Biosynthetic regulation of monobutyrin, an adipocyte-secreted lipid with angiogenic activity

1-Butyrylglycerol (monobutyrin) is a novel angiogenic compound that is synthesized and secreted during the differentiation of 3T3-F442A preadipocytes into adipocytes. To study the regulation of monobutyrin biosynthesis we have developed an assay utilizing the lgycerol kinase enzyme from Cellulomonas to quantitate the levels of this compound in cell-conditioned medium. Analysis of several cultured cell types, including tumor cell lines, indicated that monobutyrin production is detectable only from adipocytes, reaching a steady-state concentration of approximately 1.0 microM in conditioned medium. Monobutyrin synthesis was demonstrated in vitro using [14C]butyryl-CoA with total homogenate or particulate fractions from adipocytes. Similar fractions from non-adipocyte cell lines failed to synthesize monobutyrin. This biosynthetic activity was shown to be distinct by substrate competition studies from the microsomal sn-glycerol-3-phosphate acyltransferase, whose activity is known to increase during adipocyte differentiation. The production of monobutyrin was hormonally regulated, as the addition of epinephrine to adipocytes caused a 10-fold increase in the amount of monobutyrin secreted. These results indicate that monobutyrin synthesis is adipocyte specific, occurs through an apparently novel particulate enzyme system, and is regulated in a hormone-dependent manner. The implications of these results for adipose physiology and angiogenesis are discussed.     

Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments.

Functional analysis of mammalian genes relies, in part, on targeted mutations generated by homologous recombination in mice. We have developed a strategy for adipose-specific inactivation of loxP-floxed gene segments. Transgenic mice have been established that express Cre recombinase under the control of the adipose-specific aP2 enhancer/promoter. Crossing of the aP2/ Cre mice with any loxP-floxed gene will facilitate its functional analysis in adipose tissue.     

Mutational Analysis of the Role of Rap1 in Regulating Cytoskeletal Function inDictyostelium

It was shown previously that increased expression of theras-relatedrap1gene inDictyostelium discoideumaltered cell morphology (Rebsteinet al., Dev. Genet.,1993, 14, 347–355). Vegetative Rap1 transformants were more flattened and spread than parental Ax2 cells and had increased F-actin near the cell periphery. In addition, starving Rap1 cells were inhibited in the rapid cell contraction that occurs upon refeeding with nutrient media. In this communication, we show that expression of Rap also markedly reduces the contraction response that occurs upon addition of azide to vegetative cells. The changes in cell morphology, the refeeding contraction response, and the azide contraction response have been used to analyze mutants of Rap1 generated by site-directed mutagenesis. The substitution G12V, predicted to increase the proportion of protein binding GTP, did not alter the effect of Rap on cell morphology or on its ability to inhibit the contraction response to azide, but modestly enhanced the ability of Rap1 to inhibit cell rounding in response to nutrient media. The substitution S17N, predicted to restrict the protein to the GDP-bound state, did not produce the flattened cell morphology and abolished the inhibitory effects of Rap in the two cell contraction assays. These results are consistent with a requirement of GTP binding for the Rap-induced effects. Transformants carrying the Rap-S17N protein had a more polar morphology than the parental Ax2 cells, suggesting the possibility that Rap-S17N interferes with the ability of endogenous Rap to regulate the cytoskeleton. Substitutions at amino acid 38, within the presumptive effector domain, reduced but did not abolish the effects of Rap1 on cell contraction, while the substitution T61Q had no effect on Rap1 activity. Taken together, the results suggest that Rap may have multiple regulatory effects on cytoskeletal function.     

A strategy for dual inhibition of the proteasome and fatty acid synthase with belactosin C-orlistat hybrids

The proteasome, a validated cellular target for cancer, is central for maintaining cellular homeostasis, while fatty acid synthase (FAS), a novel target for numerous cancers, is responsible for palmitic acid biosynthesis. Perturbation of either enzymatic machine results in decreased proliferation and ultimately cellular apoptosis. Based on structural similarities, we hypothesized that hybrid molecules of belactosin C, a known proteasome inhibitor, and orlistat, a known inhibitor of the thioesterase domain of FAS, could inhibit both enzymes. Herein, we describe proof-of-principle studies leading to the design, synthesis and enzymatic activity of several novel, β-lactone-based, dual inhibitors of these two enzymes. Validation of dual enzyme targeting through activity-based proteome profiling with an alkyne probe modeled after the most potent inhibitor, and preliminary serum stability studies of selected derivatives are also described. These results provide proof of concept for dual targeting of the proteasome and fatty acid synthase-thioesterase (FAS-TE) enabling a new approach for the development of drug-candidates with potential to overcome resistance.