Fat/Vessel-derived Secretory Protein (Favine)/CCDC3 Is Involved in Lipid Accumulation

We previously identified a novel gene encoding Favine/CCDC3 (NCBI protein entry {"type":"entrez-protein","attrs":{"text":"NP_083080","term_id":"58037351","term_text":"NP_083080"}}NP_083080), a possible secretory factor, the mRNA of which is highly expressed in adipose tissue and the aorta. The Favine mRNA levels are increased in the course of differentiation of rat primary adipocytes and are more elevated in the adipose tissue of genetically obese and diet-induced obese mice than in lean mice. However, its biological function has not yet been elucidated until now. Here, we tested the hypothesis that Favine is involved in lipid metabolism in adipocytes. We found that overexpression of Favine promoted 3T3-L1 adipocyte differentiation. To further investigate the function of Favine in vivo, we generated Favine knock-out (KO) mice. Favine KO mice exhibited a lean phenotype as they aged. The weights of white adipose tissue and liver were less, and adipocyte size was smaller in Favine KO mice compared with wild-type littermates (WT). Expression levels of lipogenic genes, such as fatty-acid synthase (FAS), acetyl-CoA carboxylase α (ACC1), and diacylglycerol O-acyltransferase-2 (Dgat2), were decreased in adipose tissue of Favine KO mice. In 1-year-old mice, Favine deficiency decreased the number of inflammatory cells in white adipose tissue and diminished hepatic steatosis. In vitro, deficiency of Favine attenuated differentiation of primary adipocytes. Taken together, these data demonstrate that Favine has adipogenic and lipogenic effects on adipocytes.     

Adipose Hypothermia in Obesity and Its Association with Period Homolog 1, Insulin Sensitivity,and Inflammation in Fat

Visceral fat adiposity plays an important role in the development of metabolic syndrome. We reported previously the impact of human visceral fat adiposity on gene expression profile of peripheral blood cells. Genes related to circadian rhythm were highly associated with visceral fat area and period homolog 1 (PER1) showed the most significant negative correlation with visceral fat area. However, regulation of adipose Per1 remains poorly understood. The present study was designed to understand the regulation of Per1 in adipose tissues. Adipose Per1 mRNA levels of ob/ob mice were markedly low at 25 and 35 weeks of age. The levels of other core clock genes of white adipose tissues were also low in ob/ob mice at 25 and 35 weeks of age. Per1 mRNA was mainly expressed in the mature adipocyte fraction (MAF) and it was significantly low in MAF of ob/ob mice. To examine the possible mechanisms, 3T3-L1 adipocytes were treated with H₂O₂, tumor necrosis factor-α (TNF-α), S100A8, and lipopolysaccharide (LPS). However, no significant changes in Per1 mRNA level were observed by these agents. Exposure of cultured 3T3-L1 adipocytes to low temperature (33°C) decreased Per1 and catalase, and increased monocyte chemoattractant protein-1 (Mcp-1) mRNA levels. Hypothermia also worsened insulin-mediated Akt phosphorylation in 3T3-L1 adipocytes. Finally, telemetric analysis showed low temperature of adipose tissues in ob/ob mice. In obesity, adipose hypothermia seems to accelerate adipocyte dysfunction.     

Up-regulation of adipogenin, an adipocyte plasma transmembrane protein, during adipogenesis

Until now, the various proteins highly expressed in adipose tissues have been identified and characterized by traditional gene cloning techniques. However, methods of computer analysis have been developed to compare the levels of expression among various tissues, and genes whose expression levels differ significantly between tissues have been found. Among these genes, we report on the possible function of a new adipose-specific gene, showed higher expression in adipose tissue through ‘Search Expression’ on Genome Institute of Norvartis Research Foundation (GNF) SymAtlas v0.8.0. This database has generated and analyzed gene expression of each gene in diverse samples of normal tissues, organs, and cell lines. This newly discovered gene product was named adipogenin because of its role in stimulating adipocyte differentiation and development. Adipogenin mRNA was highly expressed in four different fat depots, and exclusively expressed in adipocytes isolated from adipose tissues. The level of adipogenin mRNA was up-regulated in the subcutaneous and visceral adipose tissues of mice fed a high-fat diet compared to those on the control diet. The expression of adipogenin mRNA is dramatically elevated during adipocyte differentiation of 3T3-L1 cells. Troglitazone, which up-regulated peroxisome proliferators-activated receptor γ2 (PPAR-γ2) expression, increased adipogenin mRNA expression, although this gene was down-regulated by retinoic acid. Confocal image analyses of green-fluorescent protein-adipogenin (pEGFP-adipogenin) transiently expressed in 3T3-L1 adipocytes showed that adipogenin was strictly localized to membranes and was absent from the cytosol. Moreover, small interfering RNA (siRNA) mediated a reduction of adipogenin mRNA in 3T3-L1 cells and blocked the process of adipocyte differentiation. These results indicate that adipogenin, an adipocyte-specific membrane protein, may be involved with adipogenesis, as one of the regulators of adipose tissue development.Yeon-Hee Hong and Daisuke Hishikawa contributed equally to this work     

Study of lactoferrin gene expression in human and mouse adipose tissue,human preadipocytes and mouse 3T3-L1 fibroblasts. Association with adipogenic and inflammatory markers

Lactoferrin is considered an epithelial protein present in different gland secretions. Administration of exogenous lactoferrin is also known to modulate adipogenesis and insulin action in human adipocytes. Here, we aimed to investigate lactoferrin gene expression (real-time polymerase chain reaction) and protein (enzyme-linked immunosorbent assay) levels in human (n=143) and mice adipose tissue samples, in adipose tissue fractions and during human preadipocyte and 3T3-L1 cell line differentiation, evaluating the effects of inducers (rosiglitazone) and disruptors (inflammatory factors) of adipocyte differentiation. Lactoferrin (LTF) gene and protein were detectable at relatively high levels in whole adipose tissue and isolated adipocytes in direct association with low-density lipoprotein-related protein 1 (LRP1, its putative receptor). Obese subjects with type 2 diabetes and increased triglycerides had the lowest levels of LTF gene expression in subcutaneous adipose tissue. Specifically, LTF gene expression was significantly increased in adipocytes, mainly from lean subjects, increasing during differentiation in parallel to adipogenic genes and gene markers of lipid droplets. The induction or disruption of adipogenesis led to concomitant changes (increase and decrease, respectively) of lactoferrin levels during adipocyte differentiation also in parallel to gene markers of adipogenesis and lipid droplet development. The administration of lactoferrin led to autopotentiated increased expression of the LTF gene. The decreased lactoferrin mRNA levels in association with obesity and diabetes were replicated in mice adipose tissue. In conclusion, this is the first observation, to our knowledge, of lactoferrin gene expression in whole adipose tissue and isolated adipocytes, increasing during adipogenesis and suggesting a possible contribution in adipose tissue physiology through LRP1.     

Metallothionein gene expression and secretion in white adipose tissue

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.     

RASSF6 Expression in Adipocytes Is Down-Regulated by Interaction with Macrophages

Macrophage infiltration into adipose tissue is associated with obesity and the crosstalk between adipocytes and infiltrated macrophages has been investigated as an important pathological phenomenon during adipose tissue inflammation. Here, we sought to identify adipocyte mRNAs that are regulated by interaction with infiltrated macrophages in vivo. An anti-inflammatory vitamin, vitamin B6, suppressed macrophage infiltration into white adipose tissue and altered mRNA expression. We identified >3500 genes whose expression is significantly altered during the development of obesity in db/db mice, and compared them to the adipose tissue mRNA expression profile of mice supplemented with vitamin B6. We identified PTX3 and MMP3 as candidate genes regulated by macrophage infiltration. PTX3 and MMP3 mRNA expression in 3T3-L1 adipocytes was up-regulated by activated RAW264.7 cells and these mRNA levels were positively correlated with macrophage number in adipose tissue in vivo. Next, we screened adipose genes down-regulated by the interaction with macrophages, and isolated RASSF6 (Ras association domain family 6). RASSF6 mRNA in adipocytes was decreased by culture medium conditioned by activated RAW264.7 cells, and RASSF6 mRNA level was negatively correlated with macrophage number in adipose tissue, suggesting that adipocyte RASSF6 mRNA expression is down-regulated by infiltrated macrophages in vivo. Finally, this study also showed that decreased RASSF6 expression up-regulates mRNA expression of several genes, such as CD44 and high mobility group protein HMGA2. These data provide novel insights into the biological significance of interactions between adipocytes and macrophages in adipose tissue during the development of obesity.     

Circadian clock rhythms in different adipose tissue model systems

ABSTRACT

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.     

Anti-inflammatory effects of miRNA-146a induced in adipose and periodontal tissues

MicroRNA (miRNA) plays an important role in diverse cellular biological processes such as inflammatory response, differentiation and proliferation, and carcinogenesis. miR-146a has been suggested as a negative regulator of the inflammatory reaction. Although, it has been reported as expressed in inflamed adipose and periodontal tissues, however, miR-146a's inhibitory effects against inflammatory response in both the tissues, are not well understood. Therefore, in this study, the inhibitory effects of miR-146a on both adipose and periodontal inflammation, was investigated. In vitro study has revealed that miR-146a transfection into either adipocytes or gingival fibroblasts, has resulted in a reduced cytokine gene expression, observed on co-culturing the cells with macrophages in the presence of lipopolysaccharides (LPS), in comparison to the control miRNA transfected. Similarly, miR-146a transfection into macrophages resulted in a reduced expression of TNF-α gene and protein in response to LPS stimulation. In vivo study revealed that a continuous intravenous miR-146a administration into mice via tail vein, protected the mice from developing high-fat diet-induced obesity and the inflammatory cytokine gene expression was down-regulated in both adipose and periodontal tissues. miR-146a appeared to be induced by macrophage-derived inflammatory signals such as TNF-α by negative feed-back mechanism, and it suppressed inflammatory reaction in both adipose and periodontal tissues. Therefore, miR-146a could be suggested as a potential therapeutic molecule and as a common inflammatory regulator for both obesity-induced diabetes and related periodontal diseases.     

URB is abundantly expressed in adipose tissue and dysregulated in obesity

Dysregulated production of adipocytokines in obesity is involved in the development of metabolic syndrome. URB/DRO1 contains N-terminal signal sequence and is thought to play a role in apoptosis of tumor cells. In the present study, we investigated the expression pattern of URB mRNA in adipose tissue and secretion from cultured adipocytes. In human and mouse, URB mRNA was predominantly expressed in adipose tissue and was downregulated in obese mouse models, such as ob/ob, KKAy, and diet-induced obese mice. In 3T3L1 adipocytes, insulin, TNF-α, H₂O₂ and hypoxia decreased URB mRNA level. This regulation was similar to that for adiponectin and opposite to MCP-1. URB protein was secreted in media of URB cDNA-stably transfected cells and endogenous URB was detected in media of cultured human adipocytes. In conclusion, the expression pattern of URB suggests its role in obesity and the results suggest that URB is secreted, at least in part, from adipocytes.     

Thermogenic Ability of Uncoupling Protein 1 in Beige Adipocytes in Mice

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.     

Distinct hypoxic regulation of preadipocyte factor-1 (Pref-1) in preadipocytes and mature adipocytes

Preadipocyte factor-1 (Pref-1) is a secretory soluble protein, which exerts pleiotropic effects on maintenance of cancer stem cell characteristics and commitment of mesenchymal stem cell lineages by inhibiting adipogenesis. Observations that obesity renders the microenvironment of adipose tissues hypoxic and that hypoxia inhibits adipogenesis lead us to investigate whether hypoxia increases the expression of anti-adipogenic Pref-1 in preadipocytes, mature adipocytes, and adipose tissues from obese mouse. In 3T3-L1 preadipocytes, hypoxia induces Pref-1 by a hypoxia-inducible factor 1 (HIF-1)-dependent mechanism accompanied by increase in the levels of the active histone mark, acetylated H3K9/14 (H3K9/14Ac). Adipogenesis increased the levels of the heterochromatin histone mark, trimethylated H3K27 (H3K27me3), whereas it decreased the levels of H3K4me3 and H3K9/14Ac euchromatin marks of the mouse Pref-1 promoter. However, differently from preadipocytes, in mature adipocytes hypoxia failed to reverse the repressive epigenetic changes of Pref-1 promoter and to increase its expression. Short term (8 weeks) high fat diet (HFD) increased HIF-1α protein in subcutaneous and epididymal adipose tissues, but did not increase Pref-1 expression. Unlike in 3T3-L1 preadipocytes, HIF-1α did not increase Pref-1 expression in adipose tissues in which mature adipocytes constitute the main population. Interestingly, long term (35 weeks) HFD increased Pref-1 in serum but not in obese adipose tissues. This study suggests that Pref-1 is an endocrine factor which is synergistically increased by obesity and age.     

Contribution of glucocorticoid-mineralocorticoid receptor pathway on the obesity-related adipocyte dysfunction

AimsMineralocorticoid receptor (MR) blockade ameliorated insulin resistance with improvements in adipocytokine dysregulation, inflammation, and excess of reactive oxygen species (ROS) in obese adipose tissue and adipocytes, but its mechanism has not been clarified. The 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), producing active glucocorticoids, is highly expressed in adipocytes and glucocorticoids bind to MR with higher affinity than to glucocorticoid receptor (GR). We investigated whether glucocorticoids effect on adipocytokines and ROS through MR in adipocytes. In addition, fat distributions of MR and GR were investigated in human subjects.Methods and ResultsCorticoid receptors and their target genes were examined in adipose tissue of obese db/db mice. 3T3-L1 adipocytes were treated with glucocorticoids, H₂O₂, MR antagonist eplerenone (EP), GR antagonist RU486 (RU), MR-siRNA, and/or N-acetylcysteine. Human adipose tissues were obtained from seven patients who underwent abdominal surgery. The mRNA levels of MR and its target gene were higher in db/db mice than in control db/m + mice. In 3T3-L1 adipocytes, glucocorticoids, similar to H₂O₂, caused the dysregulation of mRNA levels of various genes related to adipocytokines and the increase of intracellular ROS. Such changes were rectified by MR blockade, not by GR antagonist. In human fat, MR mRNA level was increased in parallel with the increase of body mass index (BMI) and its increase was more significant in visceral fat, while there were no apparent correlations of GR mRNA level to BMI or fat distribution.ConclusionGlucocorticoid-MR pathway may contribute to the obesity-related adipocytokine dysregulation and adipose ROS.     

Deficiency of Interleukin-15 Confers Resistance to Obesity by Diminishing Inflammation and Enhancing the Thermogenic Function of Adipose Tissues

ObjectiveIL-15 is an inflammatory cytokine secreted by many cell types. IL-15 is also produced during physical exercise by skeletal muscle and has been reported to reduce weight gain in mice. Contrarily, our findings on IL-15 knockout (KO) mice indicate that IL-15 promotes obesity. The aim of this study is to investigate the mechanisms underlying the pro-obesity role of IL-15 in adipose tissues.MethodsControl and IL-15 KO mice were maintained on high fat diet (HFD) or normal control diet. After 16 weeks, body weight, adipose tissue and skeletal mass, serum lipid levels and gene/protein expression in the adipose tissues were evaluated. The effect of IL-15 on thermogenesis and oxygen consumption was also studied in primary cultures of adipocytes differentiated from mouse preadipocyte and human stem cells.ResultsOur results show that IL-15 deficiency prevents diet-induced weight gain and accumulation of lipids in visceral and subcutaneous white and brown adipose tissues. Gene expression analysis also revealed elevated expression of genes associated with adaptive thermogenesis in the brown and subcutaneous adipose tissues of IL-15 KO mice. Accordingly, oxygen consumption was increased in the brown adipocytes from IL-15 KO mice. In addition, IL-15 KO mice showed decreased expression of pro-inflammatory mediators in their adipose tissues.ConclusionsAbsence of IL-15 results in decreased accumulation of fat in the white adipose tissues and increased lipid utilization via adaptive thermogenesis. IL-15 also promotes inflammation in adipose tissues that could sustain chronic inflammation leading to obesity-associated metabolic syndrome.     

A Novel Role for Adipose Ephrin-B1 in Inflammatory Response

Aims

Ephrin-B1 (EfnB1) was selected among genes of unknown function in adipocytes or adipose tissue and subjected to thorough analysis to understand its role in the development of obesity.

Methods and Results

EfnB1 mRNA and protein levels were significantly decreased in adipose tissues of obese mice and such reduction was mainly observed in mature adipocytes. Exposure of 3T3-L1 adipocytes to tumor necrosis factor-α (TNF-α) and their culture with RAW264.7 cells reduced EFNB1 levels. Knockdown of adipose EFNB1 increased monocyte chemoattractant protein-1 (Mcp-1) mRNA level and augmented the TNF-α-mediated THP-1 monocyte adhesion to adipocytes. Adenovirus-mediated adipose EFNB1-overexpression significantly reduced the increase in Mcp-1 mRNA level induced by coculture of 3T3-L1 adipocytes with RAW264.7 cells. Monocyte adherent assay showed that adipose EfnB1-overexpression significantly decreased the increase of monocyte adhesion by coculture with RAW264.7 cells. TNF-α-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) was reduced by EFNB1-overexpression.

Conclusions

EFNB1 contributes to the suppression of adipose inflammatory response. In obesity, reduction of adipose EFNB1 may accelerate the vicious cycle involved in adipose tissue inflammation.     

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.