ECM microenvironment unlocks brown adipogenic potential of adult human bone marrow-derived MSCs

Key to realizing the diagnostic and therapeutic potential of human brown/brite adipocytes is the identification of a renewable, easily accessible and safe tissue source of progenitor cells, and an efficacious in vitro differentiation protocol. We show that macromolecular crowding (MMC) facilitates brown adipocyte differentiation in adult human bone marrow mesenchymal stem cells (bmMSCs), as evidenced by substantially upregulating uncoupling protein 1 (UCP1) and uncoupled respiration. Moreover, MMC also induced ‘browning’ in bmMSC-derived white adipocytes. Mechanistically, MMC creates a 3D extracellular matrix architecture enshrouding maturing adipocytes in a collagen IV cocoon that is engaged by paxillin-positive focal adhesions also at the apical side of cells, without contact to the stiff support structure. This leads to an enhanced matrix-cell signaling, reflected by increased phosphorylation of ATF2, a key transcription factor in UCP1 regulation. Thus, tuning the dimensionality of the microenvironment in vitro can unlock a strong brown potential dormant in bone marrow

TRP channels in brown and white adipogenesis from human progenitors:new therapeutic targets and the caveats associated with the common antibiotic, streptomycin

\u3cp\u3eTransient receptor potential (TRP) channels are polymodal cell sensors responding to diverse stimuli and widely implicated in the developmental programs of numerous tissues. The evidence for an involvement of TRP family members in adipogenesis, however, is scant. We present the first comprehensive expression profile of all known 27 human TRP genes in mesenchymal progenitors cells during white or brown adipogenesis. Using positive trilineage differentiation as an exclusion criterion, TRP polycystic (P)3, and TPR melastatin (M)8 were found to be uniquely adipospecific. Knockdown of TRPP3 repressed the expression of the brown fat signature genes uncoupling protein (UCP)-1 and peroxisome proliferator-activated receptor γ coactivator (PGC)-1α as well as attenuated forskolin-stimulated uncoupled respiration. However, indices of generalized adipogenesis, such as lipid droplet morphology and fatty acid binding protein (FAPB)-4 expression, were not affected, indicating a principal mitochondrial role of TRPP3. Conversely, activating TRPM8 with menthol up-regulated UCP-1 expression and augmented uncoupled respiration predominantly in white adipocytes (browning), whereas streptomycin antagonized TRPM8-mediated calcium entry, downregulated UCP-1 expression, and mitigated uncoupled respiration; menthol was less capable of augmenting uncoupled respiration (thermogenesis) in brown adipocytes. TRPP3 and TRPM8 hence appear to be involved in the priming of mitochondria to perform uncoupled respiration downstream of adenylate cyclase. Our results also underscore the developmental caveats of using antibiotics in adipogenic studies.-Goralczyk, A., van Vijven, M., Koch, M., Badowski, C., Yassin, M. S., Toh, S.-A., Shabbir, A., Franco-Obregón, A., Raghunath, M. TRP channels in brown and white adipogenesis from human progenitors: new therapeutic targets and the caveats associated with the common antibiotic, streptomycin.\u3c/p\u3

Optical visualisation of thermogenesis in stimulated single-cell brown adipocytes

The identification of brown adipose deposits in adults has led to significant interest in targeting this metabolically active tissue for treatment of obesity and diabetes. Improved methods for the direct measurement of heat production as the signature function of brown adipocytes (BAs), particularly at the single cell level, would be of substantial benefit to these ongoing efforts. Here, we report the first application of a small molecule-type thermosensitive fluorescent dye, ERthermAC, to monitor thermogenesis in BAs derived from murine brown fat precursors and in human brown fat cells differentiated from human neck brown preadipocytes. ERthermAC accumulated in the endoplasmic reticulum of BAs and displayed a marked change in fluorescence intensity in response to adrenergic stimulation of cells, which corresponded to temperature change. ERthermAC fluorescence intensity profiles were congruent with mitochondrial depolarisation events visualised by the JC-1 probe. Moreover, the averaged fluorescence intensity changes across a population of cells correlated well with dynamic changes such as thermal power, oxygen consumption, and extracellular acidification rates. These findings suggest ERthermAC as a promising new tool for studying thermogenic function in brown adipocytes of both murine and human origins

Angiogenesis in Balb/c mice under beta-carotene supplementation in diet

Angiogenesis is a process of new blood vessel formation from pre-existing ones. The most important steps in angiogenesis include detachment, proliferation, migration, homing and differentiation of vascular wall cells, which are mainly endothelial cells and their progenitors. The study focused on the effect of beta-carotene (BC) supplementation (12,000 mg/kg) in the diet on angiogenesis in Balb/c mice. Female Balb/c mice were fed for 5 weeks with two different diets: with BC or without BC supplementation. After 4 weeks of feeding, Balb/c mice were injected subcutaneously with two matrigel plugs with or without basic fibroblast growth factor (bFGF). Six days later, the animals were killed, and the matrigel plugs were used for immunohistochemical staining with CD31 antibody and for gene expression analysis. Microarray and Real-Time PCR data showed down-regulation of genes involved in proliferation and up-regulation of genes encoding inhibitors of apoptosis, proteins regulating cell adhesion, matrix-degrading enzymes and proteins involved in the VEGF pathway. The results of this study demonstrated that BC proangiogenic activity (with or without bFGF) in vivo seemed to be more significantly associated with cells’ protection from apoptosis and their stimulation of chemotaxis/homing than cell proliferation