Sympathetic but not sensory denervation stimulates white adipocyte proliferation

White adipocyte proliferation is a hallmark of obesity, but it largely remains a mechanistic mystery. We and others previously demonstrated that surgical denervation of white adipose tissue (WAT) triggers increases in fat cell number, but it is unknown whether this was due to preadipocyte proliferation or maturation of existing preadipocytes that allowed them to be counted. In addition, surgical denervation severs not only sympathetic but also sensory innervation of WAT. Therefore, we tested whether sympathetic WAT denervation triggers adipocyte proliferation using 5-bromo-2'-deoxyuridine (BrdU) as a marker of proliferation and quantified BrdU-immunoreactive (ir) cells that were co-labeled with AD-3-ir, an adipocyte-specific membrane protein marker. The unilateral denervation model was used for all experiments where Siberian hamster inguinal WAT (IWAT) was unilaterally denervated, the contralateral pad was sham denervated serving as a within-animal control, and then BrdU was injected systemically for 6 days. When IWAT was surgically denervated, severing both sympathetic and sensory nerves, tyrosine hydroxylase (TH)-ir, a sympathetic nerve marker, and calcitonin gene-related peptide (CGRP)-ir, a sensory nerve marker, were significantly decreased, and BrdU+AD-3-ir adipocytes were increased approximately 300%. When IWAT was selectively sensory denervated via local microinjections of capsaicin, a sensory nerve-specific toxin, CGRP-ir, but not TH-ir, was decreased, and BrdU+AD-3-ir adipocytes were unchanged. When IWAT was selectively sympathetically denervated via local microinjections of 6-hydroxy-dopamine, a catecholaminergic-specific toxin, TH-ir, but not CGRP-ir, was significantly decreased, and BrdU+AD-3-ir adipocytes were increased approximately 400%. Collectively, these data provide the first direct evidence that sympathetic nerves inhibit white adipocyte proliferation in vivo.     

Hypothermia inhibits osteoblast differentiation and bone formation but stimulates osteoclastogenesis

It has long been known that core body temperature declines with age, with temperatures of 35.5°C or below common in the elderly. However, the effects of temperature reduction on bone cell function and skeletal homeostasis have been little studied. We investigated the effects of mild hypothermia (35.5°C) and severe hypothermia (34°C) on bone-forming osteoblasts, and bone-resorbing osteoclasts. Formation of 'trabecular' bone structures by rat calvarial osteoblasts was reduced by 75% at 35.5°C and by 95% at 34°C after 14-16 days culture, compared to 37°C. In addition to reductions in osteoblast cell number, expression of mRNAs for Runx2, alkaline phosphatase, osteocalcin and type I collagen were also down-regulated in hypothermia. In contrast, formation of osteoclasts in mononuclear cell cultures derived from mouse marrow, showed a 1.5 to 2-fold stimulation in hypothermia; resorption pit formation was similarly increased. Taken together, these data show that hypothermia exerts reciprocal effects on bone cell function by retarding osteoblast differentiation and bone formation, whilst increasing osteoclastogenesis and thus resorption. These results suggest the possibility that hypothermia in the elderly could potentially have a direct, negative impact on bone metabolism.     

Proteomic differences between white and brown adipocytes

Although a series of protein levels from several protein pathways have been shown to differ between white (WA) and brown (BA) adipocytes, proteomic work on this subject with the exception of mitochondrial protein differences is limited. It was, therefore, the aim of the study to compare WA with BA soluble protein levels. Proteins were extracted from WA and BA and the soluble fraction was run on two-dimensional gel electrophoresis. Quantification of spot volume was carried out and protein spots, statistically different between groups (P < 0.01), were in-gel digested with trypsin and peptides were identified using nano-LC–ESI–MS/MS in the CID and ETD mode. Differences between selected proteins were evaluated by immunoblotting. A network was generated using the ingenuity pathway analysis. Five proteins, protein DJ-1, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, isocitrate dehydrogenase subunit alpha, electron transfer flavoprotein subunit alpha and immunoglobulin-binding protein 1, were increased in BA based on a gel-based proteomic method and differential expression was verified by immunoblotting. These individual proteins were represented by one spot each and sequence coverages were between 28 and 65 %. A network generated based on these results indicated a link to ubiquitination. Differential protein levels between WA and BA allow interpretation of previous work on adipocyte biochemistry and form the basis for future studies with genetic or pharmacological inhibition of these proteins accompanied by work on phenotype and adipocyte function.     

GM13133 is a negative regulator in mouse white adipocytes differentiation and drives the characteristics of brown adipocytes

Obesity is tightly associated with the disturbance of white adipose tissue storing excess energy. Thermogenic adipocytes (brown and beige) exert a critical role of oxidizing nutrients at the high rates through non‐shivering thermogenesis. The recruitment of brown characteristics in white adipocytes, termed browning, has been considered as a promising strategy for treating obesity and associated metabolic complications. Recently, long noncoding RNAs play a crucial role in regulating tissue development and participating in disease pathogenesis, yet their effects on the conversion of white into brown‐like adipocytes and thermogenic function were not totally understood. Here, we identified a mouse brown adipose specific expressed lncRNA, termed GM13133. Moreover, a considerable amount of GM13133 is expressed in adipocytes and actively modulated by cold, β₃‐adrenergic agonist and cAMP stimuli, implying a potential role in the conversion from white to brown adipocytes. Overexpression of GM13133 did not affect the proliferation of mouse white pre‐adipocytes, but inhibited white adipocyte differentiation by decreasing lipid accumulation. The forced expression of GM13133 also significantly drove the conversion of white into brown‐like adipocytes with the enhanced mitochondrial biogenesis and the induced expression of brown adipocytes specific markers. A global mRNA analysis further indicated the possible regulatory role of cAMP signaling pathway in GM13133 mediated white‐to‐brown adipocytes conversion. Our results identified a lncRNA‐mediated modulation in primary mouse white adipocyte differentiation and indicate the functional significance of GM13133 in promoting browning of white adipocytes and maintenance of thermogenesis, further providing a potential strategy to treating obesity.     

Interleukin-1alpha stimulates the differentiation of melanocytes but inhibits the proliferation of melanoblasts from neonatal mouse epidermis

Interleukin (IL)-1alpha is one of the important cytokines involved in regulating immunological reactions in the mouse skin. However, it is not known whether IL-1alpha regulates the proliferation and differentiation of mouse epidermal melanocytes. In this study, to investigate the role of IL-1alpha in the regulation of the proliferation and differentiation of mouse epidermal melanocytes, IL-1alpha was supplemented to serum-free primary cultures of epidermal cell suspensions from the initiation of the primary culture (keratinocytes and melanoblasts-melanocytes) as well as to pure cultures of melanoblasts-melanocytes (keratinocyte-depleted cultures, after 14 days), and its effect was tested. IL-1alpha inhibited the proliferation of undifferentiated melanoblasts irrespective of the presence or absence of keratinocytes, whereas the cytokine inhibited the proliferation of differentiated melanocytes only in the presence of keratinocytes. Moreover, IL-1alpha induced the differentiation of melanocytes and, in addition, stimulated tyrosinase activity, melanin synthesis, and dendritogenesis of melanocytes irrespective of the presence or absence of keratinocytes. These results suggest that IL-1alpha is involved in inhibiting the proliferation of neonatal murine epidermal melanoblasts and in stimulating the differentiation, melanogenesis, and dendritogenesis of melanocytes. The results also suggest that IL-1alpha inhibits the proliferation of differentiated melanocytes in cooperation with keratinocyte-derived factors.     

Lithium inhibits brown adipocyte differentiation

Lithium impairs the appearance of the characteristic morphology of brown adipocytes and downregulates the expression of marker genes of brown adipocyte differentiation. These effects are dose-dependent and are more pronounced when exposure of preadipocytes to lithium is initiated at early stages of differentiation. Although lithium reduces the expression of genes common to both white and brown adipocytes [fatty acid binding protein aP2 (aP2/FABP) or peroxisome proliferating activated receptor gamma], genes expressed differentially in brown adipocytes, i.e., uncoupling protein 1, PPAR gamma coactivator-1alpha, and peroxisome proliferating activated receptor alpha, are particularly sensitive to lithium treatment-dependent downregulation. Brown adipocytes appear as preferential targets of the inhibitory action of lithium on adipocyte differentiation.     

Programming human pluripotent stem cells into white and brown adipocytes

The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.     

Genistein inhibits differentiation of primary human adipocytes

Genistein, a major soy isoflavone, has been reported to exhibit antiadipogenic and proapoptotic potential in vivo and in vitro. It is also a phytoestrogen which has high affinity to estrogen receptor beta. In this study, we determined the effect of genistein on adipogenesis and estrogen receptor (ER) alpha and beta expression during differentiation in primary human preadipocytes. Genistein inhibited lipid accumulation in a dose-dependent manner at concentrations of 6.25 microM and higher, with 50 microM genistein inhibiting lipid accumulation almost completely. Low concentrations of genistein (3.25 microM) increased cell viability and higher concentrations (25 and 50 microM) decreased it by 16.48+/-1.35% (P<.0001) and 50.68+/-1.34% (P<.0001). Oil Red O staining was used to confirm the effects on lipid accumulation. The inhibition of lipid accumulation was associated with inhibition of glycerol-3-phosphate dehydrogenase activity and down-regulation of expression of adipocyte-specific genes, including peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, glycerol-3-phosphate dehydrogenase, adipocyte fatty acid binding protein, fatty acid synthase, sterol regulatory element-binding protein 1, perilipin, leptin, lipoprotein lipase and hormone-sensitive lipase. These effects of genistein during the differentiation period were associated with down-regulation of ERalpha and ERbeta expression. This study adds to the elucidation of the molecular pathways involved in the inhibition of adipogenesis by phytoestrogens.     

Albumin inhibits adipogenesis and stimulates cytokine release from human adipocytes

Bovine serum albumin (BSA) is commonly used in adipocyte experiments as a binding protein for fat-soluble substances. Therefore, it is of interest to investigate whether BSA per se is influencing the functioning of human adipocytes in vitro. In the present study, we investigated the potential of BSA to affect the proliferation and differentiation capacity of human preadipocytes. BSA was found to inhibit adipose differentiation in a dose-dependent manner (being significant at concentrations of 2.5 µM), whereas proliferation was not affected. We further investigated the effect of BSA on the secretory function of adipocytes focusing on the release of selected cytokines. Preadipocytes and freshly isolated adipocytes incubated with BSA secreted significantly higher amounts of IL-6, -8, and -10, and TNF- compared with cells incubated without BSA. The effects on cytokine secretion seemed to reside at the level of gene expression because BSA increased TNF- and IL-6 mRNA in a dose-dependent manner. The results of the present study indicate that the presence of BSA in the culture medium has considerable effects on adipocyte function in vitro. These effects should be carefully considered for in vitro studies of adipose tissue. adipose differentiation     

Huang-Qi San ameliorates hyperlipidemia with obesity rats via activating brown adipocytes and converting white adipocytes into brown-like adipocytes

BackgroundBrown adipose tissue (BAT) activation is a promising therapeutic target to treat hyperlipidemia with obesity. Huang-Qi San (HQS), an traditional Chinese medicine, can ameliorate hyperlipidemia with obesity, but its mechanism of action (MOA) is not understood.PurposeTo articulate the MOA for HQS with animal models.MethodsThe main chemical constituents of HQS were identified by high-performance liquid chromatography (HPLC) based assay. Hyperlipidemia with obesity rat models induced by high-fat diet were employed in the study. The levels of the fasting plasma glucose (FPG), triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol (HDL-C) were measured to evaluate the ability of HQS to ameliorate hyperlipidemia with obesity. Pathological analyses of organs were conducted with Oil Red O staining, hematoxylin-eosin (H&E) staining and transmission electron microscopy. The expression of mRNAs related to thermogenic genes, fatty acid oxidation-related genes and mitochondria biogenic genes were examined by quantitative real-time PCR. The protein expressions of uncoupling protein 1 (UCP1) were investigated by immunohistochemistry and western blot. Simultaneously, the protein expression of PR domain containing 16 (PRDM16), ATP synthase F1 subunit alpha (ATP5A) was detected by western blot.ResultsHQS ameliorates metabolic disorder, lipid ectopic deposition, obesity and maintained glucose homeostasis in hyperlipidemia with obesity rats. HQS can significantly increase the number of mitochondria and reduced the size of the intracellular lipid droplets in BAT, and increase the expression of BAT activation-related genes (UCP1, PGC1α, PGC1β, Prdm16, CD137, TBX1, CPT1a, PPARα, Tfam, NRF1 and NRF2) in vivo. Furthermore, UCP1, PRDM16 and ATP5A proteins of BAT were increased.ConclusionHQS can activate BAT and browning of S-WAT (subcutaneous white adipose tissue) through activating the PRDM16/PGC1α/UCP1 pathway, augmenting mitochondrial biogenesis and fatty acid oxidation to increase thermogenesis and energy expenditure, resulting in a significant amelioration of hyperlipidemia with obesity. Therefore, HQS is an effective therapeutic medicine for the treatment of hyperlipidemia with obesity.     

EDTA stimulates cleavage stage bovine embryo development in culture but inhibits blastocyst development and differentiation

Culture of bovine zygotes in medium SOFaa supplemented with 100 microM EDTA significantly increased cleavage rates during the first 72 hr of development compared to development in SOFaa. However, continued culture in the presence of EDTA for a further 72 hr (total of 6 days of culture) resulted in significantly reduced development to the morulae/blastocyst and blastocyst stages compared to culture without EDTA. Highest rates of development to the morulae/blastocyst stage (56.5%) and to the blastocyst stage (43.2%) were achieved when zygotes were cultured for 72 hr with EDTA before transfer to medium SOFaa without EDTA. Resultant blastocysts also had significantly increased blastocyst cell number and ICM cell number compared to those cultured without EDTA in the first 72 hr. EDTA was shown to inhibit glycolytic activity of the cleavage stage embryo, thereby preventing the premature stimulation of glycolysis and enhancing development. However, EDTA should not be used for the later stage embryo as the inhibition of glycolysis reduces energy production at the blastocyst stage and significantly inhibits inner cell mass development.     

Raf-1 activation stimulates proliferation and inhibits IGF-stimulated differentiation in L6A1 myoblasts.

Our previous work has demonstrated that the insulin-like growth factors (IGFs), acting through a single receptor, stimulate both proliferation and differentiation of L6A1 myoblasts. This unique model system has enabled us to closely examine the switch that regulates these two opposing responses. We have previously shown, using specific inhibitors of the IGF-I signal transduction pathway, that the mitogenic response is mediated by the Ras/Raf/MAP kinase pathway and the myogenic response by the PI 3-kinase/p70s6k pathway (Coolican SA, Samuel DS, Ewton DZ, McWade FJ, Florini JR, J Biol Chem 1997; 272: 6653-62). In that study we found that PD098059, an inhibitor of MEK activation, inhibited the proliferative response, but dramatically enhanced IGF-stimulated differentiation which was associated with elevation of p70s6k activity. Since there have been reports of elevation of Raf-1 activity in PD098059-treated L6 myoblasts, and stimulation of p70s6k activity in cells expressing an activated Raf-1, it was important to determine whether or not Raf-1 elevation plays a role in the myogenic response. To test this, we have transfected L6A1 myoblasts with delta Raf-1:ER, an estradiol-regulated form of oncogenic Raf-1. We found that activation of Raf-1 by estradiol resulted in increased phosphorylation of p42 and p44 MAP kinases and stimulation of proliferation. In contrast, Raf-1 activation inhibited all measured aspects of the myogenic response: myogenin expression, creatine kinase elevation, and fusion of myoblasts to form myotubes. In addition, we found no elevation of p70s6k activity upon Raf-1 activation. These results indicate the following: (1) stimulation of myogenic differentiation by PD098059 treatment is not simply due to the elevation of Raf-1, (2) Raf-1 has a positive role in the MAP kinase pathway and myoblast proliferation, and (3) Raf-1 activation inhibits myogenesis, possibly by forcing cells to remain in the proliferative state.     

A novel brown adipocyte-enriched long non-coding RNA that is required for brown adipocyte differentiation and sufficient to drive thermogenic gene program in white adipocytes

The thermogenic activities of brown and beige adipocytes can be exploited to reduce energy surplus and counteract obesity. Recent RNA sequencing studies have uncovered a number of long noncoding RNAs (lncRNAs) uniquely expressed in white and brown adipose tissues (WAT and BAT), but whether and how these lncRNAs function in adipogenesis remain largely unknown. Here, we report the identification of a novel brown adipocyte-enriched LncRNA (AK079912), and its nuclear localization, function and regulation. The expression of AK079912 increases during brown preadipocyte differentiation and in response to cold-stimulated browning of white adipocytes. Knockdown of AK079912 inhibits brown preadipocyte differentiation, manifested by reductions in lipid accumulation and down-regulation of adipogenic and BAT-specific genes. Conversely, ectopic expression of AK079912 in white preadipocytes up-regulates the expression of genes involved in thermogenesis. Mechanistically, inhibition of AK079912 reduces mitochondrial copy number and protein levels of mitochondria electron transport chain (ETC) complexes, whereas AK079912 overexpression increases the levels of ETC proteins. Lastly, reporter and pharmacological assays identify Pparγ as an upstream regulator of AK079912. These results provide new insights into the function of non-coding RNAs in brown adipogenesis and regulating browning of white adipocytes.