Methylation of <Emphasis Type="Italic">Cdkn1c</Emphasis> may be involved in the regulation of tooth development through cell cycle inhibition

Cdkn1c, a member of the Cip/Kip cyclin-dependent kinase inhibitor family, is critically involved in regulating cell cycle and cellular differentiation during development in mammals. However, the functional role of Cdkn1c and the underlying mechanisms by which Cdkn1c affects odontogenesis remain largely unknown. In our study, we found that Cdkn1c expression dynamically changes from embryonic day 11.5 (E11.5) to postnatal day 3 (P3), and exhibits tissue-specific expression profiles. Evaluation of CDKN1C protein by immunohistochemistry and western blot, revealed that CDKN1C protein expression peaks at P3 and then is reduced at P5 and P7. Interestingly, we observed that CDKN1C expression is higher in immature odontoblasts than preodontoblasts, is lower in mature odontoblasts, and is practically absent from ameloblasts. We evaluated cell cycle progression to further investigate the mechanisms underlying CDKN1C-mediated regulation of odontogenesis, and found that pRB, cyclin D1 and CDK2 expression decreased from P1 to P3, and reduced at P5 and P7. In addition, we observed increased methylation of KvDMR1 at P1 and P3, and reduced KvDMR1 methylation at P5 and P7. However, the methylation levels of Cdkn1c-sDMR were relatively low from P1 to P7. In summary, we demonstrated that Cdkn1c expression and methylation status may be involved in early postnatal tooth development through regulating the cell cycle inhibition activity of Cdkn1c. Notably, Cdkn1c expression and methylation may associate with cell cycle exit and differentiation of odontoblasts.     

Inducible beige adipocytes improve impaired glucose metabolism in interscapular BAT-removal mice

Inducible beige adipocytes are emerging as an interesting issue in obesity and metabolism research. There is a neglected possibility that brown adipocytes are equally activated when external stimuli induce the formation of beige adipocytes. Thus, the question is whether beige adipocytes have the same functions as brown adipocytes when brown adipose tissue (BAT) is lacking. This question has not been well studied. Therefore we determine the beneficial effects of beige adipocytes upon cold challenge or CL316243 treatments in animal models of interscapular BAT (iBAT) ablation by surgical denervation. We found that denervated iBAT were activated by cold exposure and CL316243 treatments. The data show that beige adipocytes partly contribute to the improvement of impaired glucose metabolism resulting from denervated iBAT. Thus, we further used iBAT-removal animal models to abolish iBAT functions completely. We found that beige adipocytes upon cold exposure or CL316243 treatments improved impaired glucose metabolism and enhanced glucose uptake in iBAT-removal mice. The insulin signaling was activated in iBAT-removal mice upon cold exposure. Both the activation of insulin signaling and up-regulation of glucose transporter expression were observed in iBAT-removal mice with CL316243 treatments. The data show that inducible beige adipocytes may have different mechanisms to improve impaired glucose metabolism. Inducible beige adipocytes can also enhance energy expenditure and lipolytic activity of white adipose tissues when iBAT is lacking. We provide direct evidences for the beneficial effect of inducible beige adipocytes in glucose metabolism and energy expenditure in the absence of iBAT in vivo.     

MiR-499/PRDM16 axis modulates the adipogenic differentiation of mouse skeletal muscle satellite cells

Obesity is associated with increased risks of diverse diseases; brown adipose tissue (BAT) can increase energy expenditure and protect against obesity by increasing the decomposition of white adipose tissue (WAT) to enhance the non-coupled oxidative phosphorylation of fatty acid in adipocytes and contributes to weight loss. However, BAT is abundant in only small rodents and newborn humans, but not in adults. PRDM16 is a key factor that induces the differentiation of skeletal muscle precursors to brown adipocytes and simultaneously inhibits myogenic differentiation. In the present study, we set insulin-induced skeletal muscle satellite cells (SMSCs) adipogenic differentiation model, as confirmed by the contents of adipogenic markers PRDM16, UCP1 and PGC1α and myogenic markers MyoD1 and MyoG. We selected miR-499 as candidate miRNA, which might regulate PRDM16 to affect SMSCs adipogenic differentiation. Possibly through directly binding to PRDM16 3′-UTR, miR-499 negatively regulated PRDM16 expression and hindered SMSCs adipogenic differentiation by reducing adipogenic markers PRDM16, UCP1 and PGC1α and increasing myogenic markers MyoD1 and MyoG. PRDM16 overexpression could partially reverse the effect of miR-499 on the above markers and SMSCs adipogenic differentiation. Taken together, miR-499/PRDM16 axis can affect the balance between SMSC myogenic and adipogenic differentiation, targeting miR-499 to rescue PRDM16 expression, thus promoting SMSCs adipogenic differentiation may be a promising strategy for obesity treatment.     

Interferon αβ-induced abnormalities in adipocytes of suckling mice

The modification induced by interferon (IFN) in brown adipose tissue (BAT) was studied by high spatial resolution magnetic resonance imaging (MRI), histology, and transmission electron microscopy (TEM). In IFN-treated mice, at MRI, the interscapular BAT was slightly enlarged and showed non-homogeneous areas of lipid accumulation. The thickness of the subcutaneous white adipose tissue was reduced with respect to control mice. In the liver, MRI showed a lipid accumulation. In IFN-treated mice, by light microscopy, brown adipocytes showed a larger lipid deposit with respect to control mice. At TEM, in BAT, the mitochondria were reduced in number, smaller and the number of cristae was also significantly reduced with respect to the controls (9.1 ± 1.5 vs 20.1 ± 1.9, P < 0.01). The inclusions in the mitochondrial matrix were significantly less numerous in IFN-treated than in control animals (1.9 ± 0.7 vs 0.9 ± 0.7 for mitochondrial section, P < 0.01). Abnormalities of endoplasmic reticulum described in hepatocytes were not found in brown adipocytes of IFN-treated mice. The present work demonstrates that, in the BAT of sucking mice, IFN-treatment induces morphologic alterations and that brown adipocytes have MRI and TEM features resembling those found in the lipid laden BAT of aged animals.     

Eicosapentaenoic acid regulates brown adipose tissue metabolism in high-fat-fed mice and in clonal brown adipocytes

Brown adipose tissue (BAT) plays a key role in energy expenditure through its specialized thermogenic function. Therefore, BAT activation may help prevent and/or treat obesity. Interestingly, subcutaneous white adipose tissue (WAT) also has the ability to differentiate into brown-like adipocytes and may potentially contribute to increased thermogenesis. We have previously reported that eicosapentaenoic acid (EPA) reduces high-fat (HF)-diet-induced obesity and insulin resistance in mice. Whether BAT mediates some of these beneficial effects of EPA has not been determined. We hypothesized that EPA activates BAT thermogenic program, contributing to its antiobesity effects. BAT and WAT were harvested from B6 male mice fed HF diets supplemented with or without EPA. HIB 1B clonal brown adipocytes treated with or without EPA were also used. Gene and protein expressions were measured in adipose tissues and H1B 1B cells by quantitative polymerase chain reaction and immunoblotting, respectively. Our results show that BAT from EPA-supplemented mice expressed significantly higher levels of thermogenic genes such as PRDM16 and PGC1α and higher levels of uncoupling protein 1 compared to HF-fed mice. By contrast, both WATs (subcutaneous and visceral) had undetectable levels of these markers with no up regulation by EPA. HIB 1B cells treated with EPA showed significantly higher mRNA expression of PGC1α and SIRT2. EPA treatment significantly increased maximum oxidative and peak glycolytic metabolism in H1B 1B cells. Our results demonstrate a novel and promising role for EPA in preventing obesity via activation of BAT, adding to its known beneficial anti-inflammatory effects.     

BAd-CRISPR: Inducible gene knockout in interscapular brown adipose tissue of adult mice

CRISPR/Cas9 has enabled inducible gene knockout in numerous tissues; however, its use has not been reported in brown adipose tissue (BAT). Here, we developed the brown adipocyte CRISPR (BAd-CRISPR) methodology to rapidly interrogate the function of one or multiple genes. With BAd-CRISPR, an adeno-associated virus (AAV8) expressing a single guide RNA (sgRNA) is administered directly to BAT of mice expressing Cas9 in brown adipocytes. We show that the local administration of AAV8-sgRNA to interscapular BAT of adult mice robustly transduced brown adipocytes and ablated expression of adiponectin, adipose triglyceride lipase, fatty acid synthase, perilipin 1, or stearoyl-CoA desaturase 1 by >90%. Administration of multiple AAV8 sgRNAs led to simultaneous knockout of up to three genes. BAd-CRISPR induced frameshift mutations and suppressed target gene mRNA expression but did not lead to substantial accumulation of off-target mutations in BAT. We used BAd-CRISPR to create an inducible uncoupling protein 1 (Ucp1) knockout mouse to assess the effects of UCP1 loss on adaptive thermogenesis in adult mice. Inducible Ucp1 knockout did not alter core body temperature; however, BAd-CRISPR Ucp1 mice had elevated circulating concentrations of fibroblast growth factor 21 and changes in BAT gene expression consistent with heat production through increased peroxisomal lipid oxidation. Other molecular adaptations predict additional cellular inefficiencies with an increase in both protein synthesis and turnover, and mitochondria with reduced reliance on mitochondrial-encoded gene expression and increased expression of nuclear-encoded mitochondrial genes. These data suggest that BAd-CRISPR is an efficient tool to speed discoveries in adipose tissue biology.     

A diurnal rhythm in glucose uptake in brown adipose tissue revealed by in vivo PET-FDG imaging

Using a micro-positron emission tomography (PET)/computerized tomography scanner, we have measured (18)F-fluorodeoxyglucose (FDG) uptake in interscapular brown adipose tissue (iBAT) in C57Bl/6 mice at intervals across a 24-h light-dark cycle. Our data reveal a strong 24-h profile of glucose uptake of iBAT, peaking at ~9 h into the light phase of the 12-h light, 12-h dark day. BAT is increasingly gaining attention as being involved in metabolic phenotypes and obesity, where BAT, as observed by PET analysis, negatively correlates with obesity and age. Conversely, animals that show perturbations in circadian clocks, behavior, and physiology show metabolic phenotypes. The observation of a 24-h rhythm in glucose uptake in iBAT makes this tissue a candidate site of interaction between metabolic and circadian systems.     

Role of sympathetic activity in controlling the expression of vascular endothelial growth factor in brown fat cells of lean and genetically obese rats

The thermogenic activity of brown adipose tissue (BAT) is heavily dependent on high perfusion, through its dense vascular system. Angiogenesis must go hand-in-hand with BAT functions, but little is known about the factors controlling it. In the present study we demonstrate that: (a) vascular endothelial growth factor (VEGF) is synthesised and released in brown adipocytes in culture; (b) VEGF mRNA isoforms and protein appear in dispersed mature brown adipocytes and whole tissue; (c) VEGF expression is increased in BAT from cold-exposed rats, and in cultured brown adipocytes exposed to noradrenaline and the β₃-adrenoceptor agonists; (e) BAT from genetically obese (fa/fa) rats exhibits reduced expression of VEGF as well as a change in the ratio of mRNA isoforms. It is concluded that sympathetic control of VEGF expression via noradrenaline acting on β₃-adrenoceptors plays a major role in developmental and adaptive angiogenesis, and defects in this contribute to the reduced thermogenic capacity of BAT in genetic obesity.     

Pmch-Deficiency in Rats Is Associated with Normal Adipocyte Differentiation and Lower Sympathetic Adipose Drive

The orexigenic neuropeptide melanin-concentrating hormone (MCH), a product of Pmch, is an important mediator of energy homeostasis. Pmch-deficient rodents are lean and smaller, characterized by lower food intake, body-, and fat mass. Pmch is expressed in hypothalamic neurons that ultimately are components in the sympathetic nervous system (SNS) drive to white and interscapular brown adipose tissue (WAT, iBAT, respectively). MCH binds to MCH receptor 1 (MCH1R), which is present on adipocytes. Currently it is unknown if Pmch-ablation changes adipocyte differentiation or sympathetic adipose drive. Using Pmch-deficient and wild-type rats on a standard low-fat diet, we analyzed dorsal subcutaneous and perirenal WAT mass and adipocyte morphology (size and number) throughout development, and indices of sympathetic activation in WAT and iBAT during adulthood. Moreover, using an in vitro approach we investigated the ability of MCH to modulate 3T3-L1 adipocyte differentiation. Pmch-deficiency decreased dorsal subcutaneous and perirenal WAT mass by reducing adipocyte size, but not number. In line with this, in vitro 3T3-L1 adipocyte differentiation was unaffected by MCH. Finally, adult Pmch-deficient rats had lower norepinephrine turnover (an index of sympathetic adipose drive) in WAT and iBAT than wild-type rats. Collectively, our data indicate that MCH/MCH1R-pathway does not modify adipocyte differentiation, whereas Pmch-deficiency in laboratory rats lowers adiposity throughout development and sympathetic adipose drive during adulthood.     

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.     

Uncoupling Protein 1 Decreases Superoxide Production in Brown Adipose Tissue Mitochondria

In thermogenic brown adipose tissue, uncoupling protein 1 (UCP1) catalyzes the dissipation of mitochondrial proton motive force as heat. In a cellular environment of high oxidative capacity such as brown adipose tissue (BAT), mitochondrial uncoupling could also reduce deleterious reactive oxygen species, but the specific involvement of UCP1 in this process is disputed. By comparing brown adipose tissue mitochondria of wild type mice and UCP1-ablated litter mates, we show that UCP1 potently reduces mitochondrial superoxide production after cold acclimation and during fatty acid oxidation. We address the sites of superoxide production and suggest diminished probability of “reverse electron transport” facilitated by uncoupled respiration as the underlying mechanism of reactive oxygen species suppression in BAT. Furthermore, ablation of UCP1 represses the cold-stimulated increase of substrate oxidation normally seen in active BAT, resulting in lower superoxide production, presumably avoiding deleterious oxidative damage. We conclude that UCP1 allows high oxidative capacity without promoting oxidative damage by simultaneously lowering superoxide production.     

GDF1 is a novel mediator of macrophage infiltration in brown adipose tissue of obese mice

We previously demonstrated a marked upregulation in the bone morphogenic protein (BMP)/growth differentiation factor (GDF) family member, GDF5, which is capable of promoting brown adipogenesis, in brown adipose tissue (BAT) of obese mice. In this study, we identified other GDF family members, besides GDF5 that are responsive to different obesogenic signals in BAT using inborn and acquired obesity animal models. In BAT from leptin-deficient ob/ob mice, GDF1 expression was preferentially downregulated, whereas the expression of several other genes in the BMP/GDF family, including GDF5, was upregulated. Moreover, in cultured brown adipocytes exposed to tunicamycin and hydrogen peroxide, at concentrations not affecting cellular viability, GDF1 expression was significantly downregulated. Recombinant GDF1 failed to significantly alter brown adipogenesis, despite the promoted phosphorylation of Smad1/5/8 in cultured brown adipocytes, but accelerated Smad1/5/8 phosphorylation with a concomitant increase in the number of migrating cells during exposure in a manner sensitive to activin-like kinase inhibitors in macrophagic RAW264.7 cells. Similarly, accelerated migration was observed in murine peritoneal macrophages exposed to GDF1. These results indicate that obesity could lead to predominant downregulation of GDF1 expression in BAT, which can modulate cellular migration through a mechanism relevant to activation of the downstream Smad signaling pathway in adjacent macrophages.     

Nuclear factor-κB is a common upstream signal for growth differentiation factor-5 expression in brown adipocytes exposed to pro-inflammatory cytokines and palmitate

We have previously demonstrated that genetic and acquired obesity similarly led to drastic upregulation in brown adipose tissue (BAT), rather than white adipose tissue, of expression of both mRNA and corresponding protein for the bone morphogenic protein/growth differentiation factor (GDF) member GDF5 capable of promoting brown adipogenesis. In this study, we evaluated expression profiles of GDF5 in cultured murine brown pre-adipocytes exposed to pro-inflammatory cytokines and free fatty acids (FFAs), which are all shown to play a role in the pathogenesis of obesity. Both interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were effective in up-regulating GDF5 expression in a concentration-dependent manner, while similar upregulation was seen in cells exposed to the saturated FFA palmitate, but not to the unsaturated FFA oleate. In silico analysis revealed existence of the putative nuclear factor-κB (NF-κB) binding site in the 5′-flanking region of mouse GDF5, whereas introduction of NF-κB subunits drastically facilitated both promoter activity and expression of GDF5 in brown pre-adipocytes. Chromatin immunoprecipitation analysis confirmed significant facilitation of the recruitment of NF-κB to the GDF5 promoter in lysed extracts of BAT from leptin-deficient ob/ob obese mice. Upregulation o GDF5 expression was invariably inhibited by an NF-κB inhibitor in cultured brown pre-adipocytes exposed to IL-1β, TNF-α and palmitate. These results suggest that obesity leads to upregulation of GDF5 expression responsible for the promotion of brown adipogenesis through a mechanism relevant to activation of the NF-κB pathway in response to particular pro-inflammatory cytokines and/or saturated FFAs in BAT.     

Conjugated linoleic acid reduces adiposity and increases markers of browning and inflammation in white adipose tissue of mice

The objective of this study was to examine the mechanism by which conjugated linoleic acid (CLA) reduces body fat. Young male mice were fed three combinations of fatty acids at three doses (0.06%, 0.2%, and 0.6%, w/w) incorporated into AIN76 diets for 7 weeks. The types of fatty acids were linoleic acid (control), an equal mixture of trans-10, cis-12 (10,12) CLA plus linoleic acid, and an equal isomer mixture of 10,12 plus cis-9, trans-11 (9,11) CLA. Mice receiving the 0.2% and 0.6% dose of 10,12 CLA plus linoleic acid or the CLA isomer mixture had decreased white adipose tissue (WAT) and brown adipose tissue (BAT) mass and increased incorporation of CLA isomers in epididymal WAT and liver. Notably, in mice receiving 0.2% of both CLA treatments, the mRNA levels of genes associated with browning, including uncoupling protein 1 (UCP1), UCP1 protein levels, and cytochrome c oxidase activity, were increased in epididymal WAT. CLA-induced browning in WAT was accompanied by increases in mRNA levels of markers of inflammation. Muscle cytochrome c oxidase activity and BAT UCP1 protein levels were not affected by CLA treatment. These data suggest a linkage between decreased adiposity, browning in WAT, and low-grade inflammation due to consumption of 10,12 CLA.     

Simultaneous telemetric monitoring of the circadian changes in core and BAT temperature in rats: Endogenous vasopressin may contribute to reduced BAT themogenesis and body temperature in the light phase of the circadian cycle

The purpose of the present study was to analyze simultaneously the temporal relationship between the changes of circadian rhythms of brown adipose tissue (BAT) thermogenesis and core temperature (T_c) by dual probe telemetric monitoring transmitters and to determine the role of endogenous arginine vasopressin (AVP) in the circadian rhythms of BAT temperature (T_BAT) and T_c in male rats. The key observations in this study are: (1) Increase in T_BAT commenced approximately 8 min before T_c increases at the start of transition from the light to dark phase. Whereas at the start of transition from the dark to light phase, decrease in T_BAT commenced approximately 3 min before T_c decreases. The data show that circadian changes of BAT thermogenesis do indeed play a significant role in the overall maintenance of the circadian rhythm of core temperature. (2) The plasma AVP level was significantly elevated when core temperature decreases during the light phase, suggesting that endogenous AVP is involved in thermoregulatory processes during the light phase. V1a receptor antagonist could elevate core and BAT temperature during the light period, suggesting that endogenous AVP, acting through V1a receptor, could be involved in tonic thermoregulatory processes.V1a receptor antagonist can increase the blood lipid metabolism, suggesting that the mechanism of endogenous AVP in tonic thermoregulatory processes during light period could involve the suppression of lipolysis in BAT and other peripheral tissues. In summary, this study demonstrated that endogenous vasopressin contributes to reduced BAT themogenesis and body temperature in the light phase of the circadian cycle.