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B-cell-activating factor deficiency attenuates high-fat diet-induced glucose intolerance by potentiating adipose tissue function
Accepted Manuscript B-cell-activating factor deficiency attenuates high-fat diet-induced glucose intolerance by potentiating adipose tissue function Bobae Kim, Myoung-Sool Do, Chang-Kee Hyun PII:
S0006-291X(15)30329-6
DOI:
10.1016/j.bbrc.2015.07.099
Reference:
YBBRC 34311
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 17 July 2015 Accepted Date: 20 July 2015
Please cite this article as: B. Kim, M.-S. Do, C.-K. Hyun, B-cell-activating factor deficiency attenuates high-fat diet-induced glucose intolerance by potentiating adipose tissue function, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.07.099. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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B-cell-activating factor deficiency attenuates high-fat diet-
function Bobae Kim, Myoung-Sool Do and Chang-Kee Hyun*
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induced glucose intolerance by potentiating adipose tissue
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School of Life Science, Handong Global University, Pohang, Gyungbuk 791-708, Korea
* Corresponding author at: School of Life Science, Handong Global University, Pohang, Gyungbuk 791-708, Korea. Tel: +82-54-260-1361; Fax: +82-54-260-1319.
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E-mail address: [email protected] (C-.K. Hyun).
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Abstract
B-cell-activating factor (BAFF) has recently been demonstrated to be expressed in
metabolic regulation.
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adipocytes and up-regulated by high-fat diet feeding, indicating a possible role in Here we show that glucose tolerance was significantly improved
in high-fat diet-fed BAFF knockout (BAFF-/-) mice. BAFF-/- mice revealed higher levels
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of glucose transporter expression and insulin-stimulated Akt phosphorylation in brown adipose tissue compared to wild type controls. Expression levels of mitochondrial ND5
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and genes involved in lipid metabolism were significantly elevated in brown adipose tissue of BAFF-/- mice, and this enhancement was found to be mediated by FGF21 and leptin. It was also observed that expression of IL-10 and foxp3 was increased in adipose tissues, as well as PPARγ activity in white adipose tissue. Our findings suggest that
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diabetes.
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suppression of BAFF could have a therapeutic potential for prevention of type 2
Key words: B-cell-activating factor; glucose tolerance; brown adipose tissue; FGF21;
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leptin; IL-10
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1. Introduction
B-cell-activating factor (BAFF) is a cytokine that belongs to tumor necrosis factor
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(TNF) ligand family protein, which has been shown to play an important role in the proliferation and differentiation of B cells, and interacts with three different receptors: BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA), and transmembrane
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activator and CAML interactor (TACI) [1]. Although the expression of BAFF is primarily detected in immunocytes, a variety of cell types also have been known to
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produce BAFF [2]. Several studies have reported that mature adipocytes also produce BAFF and its receptors [3], and through this finding, impact of BAFF on energy metabolism is being questioned.
There have been a number of studies describing the role of BAFF in energy
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metabolism. Hamada et al. reported that levels of BAFF were significantly increased in serum and visceral adipose tissue of high-fat diet-fed mice relative to normal control mice [4]. They also found that treatment with recombinant BAFF protein impaired
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insulin-mediated glucose uptake in 3T3-L1 adipocytes. BAFF plasma levels were significantly correlated with body fat and dietary composition in obese Central-
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European Caucasian individuals [5], and the levels were also observed to be significantly higher in Japanese patients with nonalcoholic fatty liver disease [6]. Studies on the effect of BAFF on energy metabolism have shown us that BAFF and its receptors are closely related to the physiological activity of adipose tissues: BAFF is not only produced by adipocytes [3,4] but also associated with the differentiation process of adipose-derived stem cells towards adipocyte [7]. Moreover, Kawasaki et al. reported that BAFF-R knockout (BAFF-R-/-) mice were protected from high-fat diet-induced
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adiposity and showed improved insulin sensitivity [8]. In their study, no difference of visceral fat weight between high-fat diet-fed BAFF-R-/- and wild type mice was observed but the inguinal fat weight was significantly lower in BAFF-R-/- mice.
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Although these recent studies clearly illustrated that BAFF is linked to metabolic status and involved in the impairment of insulin sensitivity, questions remain in the elucidation of the role of BAFF in the development of adiposity and insulin resistance.
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In this study, we investigated the impact of BAFF on the onset of glucose intolerance using BAFF knockout (BAFF-/-) mice fed with high-fat diet. Insulin resistance in BAFFmice was significantly improved despite of diet-induced weight gain, which was
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attributed by up-regulated gene expression of lipid metabolism in brown adipose tissue mediated by FGF21 and leptin. It was also found that IL-10 expression and PPARγ activity in white adipose tissue was enhanced, resulting in enhanced glucose tolerance.
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Our study indicates that BAFF is a critical adipokine for the development of glucose intolerance, suggesting that blocking BAFF could be a therapeutic approach for the
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prevention and treatment of type 2 diabetes.
2. Materials and methods
2.1. Animals
Male C57BL/6J wild type (WT) mice and BAFF-/- mice were purchased from Central Lab. Animal Inc. (Seoul, Korea), and The Jackson Laboratory (Bar Harbor, ME; stock number 010572), respectively. Systemic BAFF knockout mice were generated by
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insertion of the tailless human CDs reporter gene into the BAFF locus. BAFF gene deletion was confirmed by baff mRNA expression levels in spleen, epididymal and brown adipose tissue, and BAFF protein level in spleen (Fig. 1A). Six-week-old males
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were maintained under a 12 h light:dark cycle at a constant temperature of 22 ± 1°C and humidity of 45 ± 10%. To stabilize all metabolic conditions, eleven-week-old male mice were fed normal chow diet (2018S, Harlan Laboratories, Indianapolis, IN) and
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individually housed in cages for a week. After the stabilization, mice were switched to the high-fat diet (HFD) containing 60% kcal from fat (D12493, Research Diets Inc., NJ)
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for 6 weeks.
Mice were fasted for 4 h, and sacrificed. Tissues of liver, spleen, inguinal fat, epididymal fat, brown fat and quadriceps muscle were harvested, snap-frozen in liquid
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nitrogen, and stored at -75°C until processed for RNA and protein analysis.
2.2. Glucose tolerance test
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After 4 weeks of high-fat feeding, mice were fasted for 16 h and followed by intraperitoneal injection of glucose (2 g/kg). Blood samples were obtained by tail-
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bleeding, and blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 min after glucose injection by Accu-Check Go (Roche Diagnostics GmbH, Basel, Switzerland).
2.3. Western blotting
Frozen tissues were homogenized and protein-extracted as described previously [9]. Antibodies against phospho-Akt (Ser473), total Akt, β-actin (Cell signaling technology,
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Beverly, MA), BAFF (R&D systems, Minneapolis, MN), phospho-PPARγ (Ser112) (Abcam, Cambridge, UK), phoshpo-STAT3 (Tyr705) and total STAT3 (Cambridge Bioscience, Cambridge, UK) were used as primary antibodies, followed by the
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appropriate IgG-horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by ECL.
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2.4. Real-time RT PCR
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Total RNA extraction, reverse transcription, and quantitative PCR were performed as described previously [9]. Relative expression level of mRNAs was calculated by the ∆∆Ct method using 36B4 (Arbp) as the reference control. Primer sequences are available
upon
request:
Acyl-CoA
oxidase
1
(Acox1),
BAFF,
carnitine
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palmitoyltransferase 1 (CPT1), fatty acid synthase (FAS), fibroblast growth factor 21 (FGF21), forkhead box P3 (Foxp3), glucose transporter I (GLUT1), GLUT4, interleukin-10 (IL-10), IL-12, leptin, mitochondrially encoded NADH dehydrogenase 5
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(ND5), peroxisome proliferator-activated receptor α (PPARα), PPARγ coactivator 1α
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(PGC1α), stearoyl-CoA desaturase-1 (SCD1), uncoupling protein 1 (UCP1).
2.5. ELISA
Measurement of serum leptin was performed with commercial ELISA kits (R&D systems) according to the manufacturer’s instruction.
2.6. Statistical analysis
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All data were presented as mean ± SD. Comparisons of two groups were performed
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by two-tailed Student’s t-test. p values < 0.05 were considered as statistically significant.
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3. Results
3.1. BAFF deficiency improves glucose tolerance under HFD condition despite
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increased adiposity
After 6 weeks of normal chow diet (ND) or HFD feeding, BAFF-/- mice showed significantly increased weight gain compared to their wild-type (WT) control mice (Fig.
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1B). The quantitative changes in tissue weight revealed differences between various types of tissues. BAFF-/- mice on HF diet had significantly increased weights of adipose tissues and liver and decreased weights of quadriceps and spleen compared to WT
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counterparts (Fig. 1C). Interestingly, however, no substantial difference in the weight of epididymal adipose tissue was observed between wild-type and BAFF-/- mice, which
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was different from not only brown adipose tissue but also other types of white adipose tissues.
To test whether BAFF deficiency affects insulin sensitivity, glucose tolerance test was carried out in ND- or HFD-fed WT and BAFF-/- mice. Despite the increased adiposity, the HFD-fed BAFF-/- mice exhibited significantly improved glucose tolerance compared to HFD-fed WT mice (Fig. 1D). Notably, this improvement effect was observed only under HFD feeding condition, but not under ND feeding condition.
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To confirm that activation of insulin signaling led to improved glucose tolerance, we analyzed insulin-stimulated phosphorylation of Akt in epididymal white adipose tissue, brown adipose tissue and quadriceps muscle. Akt phosphorylation was significantly
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higher in brown adipose tissue of BAFF-/- mice compared to the wild-type mice (Fig. 1E), but unexpectedly not in epididymal adipose tissue and quadriceps (data not shown).
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3.2. BAFF deficiency changes mRNA expression of genes involved in glucose and lipid
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metabolism in brown adipose tissue
Brown adipose tissue is a thermogenic organ which stores nutrients as lipids and dissipates their energy as heat. To examine how BAFF deficiency modulates metabolism in brown adipose tissue, we analyzed the altered gene expression for
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glucose and lipid metabolism. Brown adipose tissue of HFD-fed BAFF-/- mice displayed a significant increase in mRNA expression of glucose transporters, GLUT4 and GLUT1, relative to WT controls (Fig. 2A). Since brown adipose tissue consumes lipids to
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generate heat through uncoupling respiration, mRNA expression levels of a mitochondrial gene ND5 and genes involved in thermogenesis such as PGC1α, PPARα
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and UCP1 were also measured. HFD-fed BAFF-/- mice had significantly higher gene expression of ND5 and PGC1α, the master regulator of UCP1-mediated thermogenesis in brown adipose tissue, relative to WT control mice (Fig. 2B). Expression of the nuclear-encoded mitochondrial thermogenic genes, PPARα and UCP1, was also modestly elevated in HFD-fed BAFF-/- mice relative to WT controls even though the statistical significances were not observed. To examine the effect of BAFF deficiency on lipid metabolism in adipose tissues,
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expression of genes involved in lipogenesis, FAS and SCD1, and fatty acid oxidation, Acox1 and CPT1, in epididymal and brown adipose tissue were measured and compared. On HFD diet, BAFF-/- mice had higher levels of expression of FAS, SCD1,
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Acox1 and CPT1 in brown adipose tissue, compared to WT controls, with or without significance (Fig. 2C). However, epididymal adipose tissue was different from brown adipose tissue, showing that expression of lipogenic genes was suppressed whereas
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expression of fatty acid oxidation genes was up-regulated in BAFF-/- mice relative to WT controls. To better understand the mechanism underlying the effect of BAFF
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deficiency on metabolism in brown adipose tissue, we analyzed gene expression of FGF21 which plays significant roles in lipid oxidation and browning of white adipose tissue and thermogenesis in brown adipose tissue. Notably, HFD-fed BAFF-/- mice had significantly higher expression of FGF21 in epididymal and brown adipose tissues,
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while there was no difference in the liver between BAFF-/- and WT mice (Fig. 2D).
adipose tissues
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3.3. BAFF deficiency enhances both leptin expression and STAT3 phosphorylation in
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Leptin is an adipokine which regulates food intake and energy expenditure in an endocrine manner. The mRNA expression levels of leptin were significantly higher in epididymal, inguinal and brown adipose tissues of HFD-fed BAFF-/- mice compared to WT controls (Fig. 3A). Commensurate with their increased mRNA expression, BAFF-/mice had significantly higher level of serum leptin than WT controls (Fig. 3B). Based on the fact that leptin action is mediated by Jak/STAT pathway, higher leptin expression was corroborated by the finding that STAT3 phosphorylation in brown adipose tissue of
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BAFF-/- mice was significantly higher than WT controls (Fig. 3C).
3.4. BAFF deficiency changes IL-10/IL-12 mRNA expression and enhances PPARγ
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activity in adipose tissues
Accumulation of pro-inflammatory immune cells and their cytokines in adipose
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tissue is considered a focal point in the progression of insulin resistance. We observed that the mRNA expression of anti-inflammatory cytokine IL-10 was significantly
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increased, whereas the expression of IL-12, a pro-inflammatory cytokine, was suppressed in both epididymal and brown adipose tissues of HFD-fed BAFF-/- mice (Fig. 4A). Unexpectedly, however, there were no significant differences in the expression of other anti-inflammatory cytokines except IL-10, including IL-4, IL-1ra, and IL-13, as
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well as of other pro-inflammatory cytokines except IL-12, including TNFα, IL-6, IFNγ, MCP-1 (data not shown). On the other hand, it was observed that the expression level of foxp3, a marker of regulatory T cells induced by anti-inflammatory cytokines, was
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higher in BAFF-/- mice than WT controls (Fig. 4B). We next analyzed the status of PPARγ phosphorylation at serine 112 to examine BAFF
deficiency
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whether
enhances
PPARγ
activity
by
reducing
PPARγ
phosphorylation in adipose tissue under HFD feeding condition. The level of PPARγ phosphorylation was significantly reduced in epididymal adipose tissue of BAFF-/- mice relative to WT controls (Fig. 4C) without any change in the mRNA expression level (data not shown). This result is consistent with the observation of improved glucose tolerance despite higher body weight gain in HFD-fed BAFF-/- mice compared to WT controls.
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4. Discussion
Recent studies have shown that mature adipocytes produce BAFF, which has been identified as an adipokine with metabolic effects [3-5]. The aim of this study was to
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elucidate the role of BAFF deficiency in metabolic regulation through exploring the
tolerance in HFD-fed BAFF-/- mice.
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molecular mechanism underlying the effect of BAFF depletion on improved glucose
In this study, HFD-fed BAFF-/- mice showed a significant improvement in insulin sensitivity relative to their WT control mice (Fig. 1D), which was partly consistent with a previous report that BAFF-R-/- mice were protected against HFD-induced obesity and
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insulin resistance [8]. BAFF-/- mice had improved glucose tolerance under HFD-fed condition, but, unlike BAFF-R-/- mice, were not protected against HFD-induced weight gain (Fig. 1B). These different responses of BAFF-/- and BAFF-R knockout mice to
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HFD feeding could be accounted for the fact that the action of BAFF is mediated by not only BAFF-R but also other receptors on adipocytes and adipose-tissue derived
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mesenchymal cells, such as BCMA, TACI and Fn14 [1-3]. The body weight gain of HFD-fed BAFF-/- mice was observed to be attributed mainly by the increase in tissue weights of white adipose tissues except epididymal adipose tissue (Fig. 1C), which is explicable by the decreased level of lipogenesis gene expression specifically in epididymal adipose tissue but not in other adipose tissues of BAFF-/- mice relative to the WT controls (Fig. 2C). The improved glucose tolerance in HFD-fed BAFF-/- mice was corroborated by
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observation of an increased level of insulin-stimulated Akt phosphorylation in brown adipose tissue (Fig. 1E). We unexpectedly found that other insulin-responsive tissues including white adipose tissue, skeletal muscle and liver did not show PI3K-dependent
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activation of insulin signaling and were not responsible for the insulin-sensitizing effect of BAFF depletion. Along with enhanced translocation of GLUT4 indicated by increased Akt activation, GLUT4 and GLUT1 mRNA expression levels were both
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found to be significantly increased in brown adipose tissue of BAFF-/- mice compared with WT controls (Fig. 2A), which might account for the enhanced glucose uptake [10].
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Brown adipose tissue exerts beneficial effects on insulin sensitivity and glucose and lipid metabolism by dissipating energy in the form of heat [11]. Consistent with higher weight of brown adipose tissue in HFD-fed BAFF-/- mice compared to their WT controls, increased expression of genes related to lipogenesis such as SCD1 and FAS,
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was observed in BAFF-/- mice (Fig. 2C). Increased expressions of mitochondrial ND5 gene and thermogenic genes PGC1α, PPARα and UCP1 in brown adipose tissue of BAFF-/- mice (Fig. 2B) indicates that mitochondrial biogenesis and thermogenesis were
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enhanced by BAFF depletion. We also found that the BAFF-/- mice had significantly higher expression levels of the fatty acid oxidation genes such as Acox1 and CPT1,
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compared to WT mice. Taken together, our findings suggest that BAFF depletion, under HFD-fed condition, leads to increases in glucose uptake, lipogenesis, and fatty acid oxidation, triggering brown adipose tissue to be metabolically active. FGF21 is a peptide hormone produced mainly in the liver and adipose tissues, which is known to mediate the thermogenic response of brown adipose tissue to cold and adrenergic stimulation [12]. It has also been reported that FGF21 reduces blood glucose, insulin, and lipid levels and increases total energy expenditure and physical activity in diet-
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induced obese mice [13]. In our study, FGF21 mRNA expression was significantly increased in adipose tissues of BAFF-/- mice whereas there was no difference in the liver between BAFF-/- and WT mice (Fig. 2D). This result suggests that BAFF deficiency
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enhances glucose tolerance by potentiating adipose tissue function, causing an increase in FGF21 production that leads to enhanced metabolic activity of brown fat in BAFF-/mice.
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We also found that BAFF depletion enhanced leptin expression in both white and brown adipose tissue under HFD-fed condition, which was corroborated by the
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observation of higher levels of leptin mRNA and serum leptin in BAFF-/- mice compared to WT controls (Fig. 3A and 3B). It has been reported that elevated levels of circulating leptin result in the enhancement of glucose utilization by inducing UCP1 expression and STAT phosphorylation in brown adipose tissue [14,15]. We observed
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that STAT3 phosphorylation in brown adipose tissue of BAFF-/- mice was significantly increased compared to WT controls (Fig. 3C). This observation lends further support to the hypothesis that BAFF depletion might also augment the metabolic activity of brown
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adipose tissue through leptin-mediated STAT3 activation. Obesity is a chronic state of low-grade inflammation with progressive immune cell
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infiltration into adipose tissue [16]. Cytokines produced from infiltrating immune cells modulate adipose tissue inflammation and consequential insulin resistance [17]. IL-10, is an anti-inflammatory cytokine that is also known to enhance B cell activation, proliferation and differentiation. Recent studies have reported that IL-10 protects mice against obesity and insulin resistance and reduces immune cell infiltration [18]. In this study, we found that mRNA expression of IL-10 was significantly higher while IL-12 expression was decreased in both epididymal and brown adipose tissue of BAFF-/- mice
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compared to WT controls (Fig. 4A). Since transcription of IL-12, a pro-inflammatory cytokine mainly produced by phagocytic cells and involved in regulating T cell response, is elevated by HFD feeding and repressed by IL-10 [19,20], the suppression
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of IL-12 gene expression strongly supports IL-10 action in BAFF-/- mice. Regulatory T (Treg) cells have been reported to attenuate inflammation and obesity-induced insulin resistance by IL-10 production [21]. We found that the expression level of foxp3, a
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specific marker of Treg cell population, was significantly increased in BAFF-/- mice relative to WT controls (Fig. 4B). Additionally, we observed a significantly reduced
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phosphorylation of PPARγ at serine 112 in white adipose tissue of HFD-fed BAFF-/mice compared to WT controls (Fig. 4C), indicating a significant enhancement of PPARγ activity. Phosphorylation at Ser112 of PPARγ is known to repress its transcriptional activity by hindering ligand binding and altering cofactor recruitment,
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and genetically modified mice carrying mutation of S112A showed improved insulin sensitivity [22,23]. Taken together with the beneficial changes in cytokine production, this might possibly be one cause for the activation of brown adipose tissue, thereby
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contributing to the enhancement of glucose tolerance of BAFF-/- mice. In summary, the results of this study suggest that BAFF deficiency prevents mice
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from HFD-induced glucose intolerance by potentiating adipose tissue function, particularly through enhanced activities of brown adipose tissue. Our data demonstrate that the protective effect of BAFF depletion is mediated by FGF21 and leptin, which might be also attributed to up-regulation of IL-10 and foxp3 and enhancement of PPARγ activity. Furthermore, these findings illustrate that BAFF is an adipokine which mediates the onset of glucose intolerance, and blocking BAFF could stand as a valid therapeutic approach that attenuates the progression of insulin resistance.
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References
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[1] F. Mackay, P. Schneider, Cracking the BAFF code. Nat. Rev. Immunol. 9 (2009) 491-502.
[2] M. Zonca, P. Mancheño-Corvo, O. Delarosa, S. Mañes. D. Büscher, E. Lombardo, L.
SC
Planelles, APRIL and BAFF proteins increase proliferation of human adiposederived stem cells through activation of Erk1/2 MAP kinase. Tissue Eng. Part A. 18
M AN U
(2012) 852-859
[3] V. Alexaki, G. Notas, V. Pelekanou, M. Kampa, M. Valkanou, P. Theodoropoulos, E.N. Stathopoulos, A. Tsapis, E. Castanas, Adipocytes as immune cells: differential expression of TWEAK, BAFF, and APRIL and their receptors (Fn14, BAFF-R,
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TACI, and BCMA) at different stages of normal and pathological adipose tissue development. J. Immunol. 183 (2009) 5948-5956 [4] M. Hamada, M. Abe, T. Miyake, K. Kawasaki, J. Tada, S. Furukawa, B. Matsuura,
EP
Y. Hiasa, M. Onji, B cell-activating factor controls the production of adipokines and induces insulin resistance. Obesity 19 (2011) 1915-1922
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[5] J. Bienertova-Vasku, P. Bienert, F. Zlamal, F. Tomandl, M. Forejt, M. Tomandlova, M. Vavrina, J. Kudelková, Z. Splichal, A. Vasku, B-cell activating factor (BAFF)-a new factor linking immunity to diet? Cent. Eur. J. Med. 7 (2012) 275-283
[6] T. Mihyke, M. Abe, Y. Tokumoto, M. Kirooka, S. Furukawa, T. Kumagi, M. Hamada, K. Kawasadi, F. Tada, T. Ueda, Y. Hiasa, B. Matsuura, M. Onji, B cellactivating factor is associated with the histological severity of nonalcoholic fatty liver disease. Hepatol. Int. 7 (2013) 539-547
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[7] H. Wa, D. Han, Z. Jiang, D. Zhao, M. Liu, X. Xu, X. Liu, L. Yang, X. Ji, M. Wang, S. Zhang, Equine adipose-derived stem cell (ASC) expresses BAFF and its
adipocyte. Int. Immunopharmacol. 18 (2014) 365-372
RI PT
receptors, which may be associated with the differentiation process of ASC towards
[8] K. Kawasaki, M. Abe, F. Tada, Y. Tokumoto, S. Chen, T. Miyake, S. Furukawa, B. Matsuura, Y. Hiasa, M. Onji, Blockade of B-cell-activating factor signaling
sensitivity. Lab. Invest. 93 (2013) 311-321
SC
enhances hepatic steatosis induced by a high-fat diet and improves insulin
M AN U
[9] E. Kim, J.H. Lee, J.M. Ntambi, C.K. Hyun, Inhibition of stearoyl-CoA desaturase1 activates AMPK and exhibits beneficial lipid metabolic effects in vitro, Eur. J. Pharmacol. 672 (2011) 38-44.
[10] M.A. Herman, B.B. Kahn, Glucose transport and sensing in the maintenance of
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glucose homeostasis and metabolic harmony. J. Clin. Invest. 116 (2006) 1767-1775. [11] A. Bartelt, J. Heeren, Adipose tissue browning and metabolic health. Nat. Rev. Endocrinol. 10 (2014) 24-36.
EP
[12] F.M. Fisher, S. Kleiner, N. Douris, E.C. Fox, R.J. Mepani, F. Verdeguer, J. Wu, A. Kharitonenkov, J.S. Flier, E. Maratos-Flier, B.M. Spiegelman, FGF21 regulates
AC C
PGC-1α and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 26 (2012) 271-281.
[13] J. Xu, D.J. Lloyd, C. Hale, H, Stanislaus, M. Chen, G. Sivits, S. Vonderfecht, R. Hecht, Y.S. Li, R.A. Lindberg, J.L. Chen, D.Y. Jung, Z. Zhang, H.J. Ko, J.K. Kim, M.M. Véniant, Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58 (2009), 250-259.
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[14] J. Rouru, I. Cusin, K.E. Zakrzewska, B. Jeanrenaud and F. Rohner-Jeanrenaud, Effects of intravenously infused leptin on insulin sensitivity and on the expression of uncoupling proteins in brown adipose tissue. Endocrinology 140 (1999) 3688-
RI PT
3692.
[15] P. Bendinelli, P. Maroni, F. Pecori Giraldi, R. Piccoletti, Leptin activates Stat3, Stat1 and AP-1 in mouse adipose tissue. Mol. Cell. Endocrionol. 168 (2000) 11-20.
SC
[16] M.F. Gregor, G.S. Hotamisligil, Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29 (2011) 415-445.
M AN U
[17] K.A. Harford, C.M. Reynolds, F.C. McCillicuddy, H.M. Roche, Fats, inflammation and insulin resistance: insights to the role of macrophage and T-cell accumulation in adipose tissue. Proc. Nutr. Soc. 70 (2011) 408-417.
[18] M.A. Febbraio, Role of interleukins in obesity: implications for metabolic disease.
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Trends Endocrinol. Metab. 25 (2014) 312-319.
[19] M. Aste-Amezaga, X. Ma, A. Sartori, G. Trinchieri, Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10. J. Immunol. 160 (1998) 5936-5944.
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[20] T. Kobayashi, K. Matsuoka, S.Z. Sheikh, S.M. Russo, Y. Mishima, C. Collins, E.F. deZoeten, C.L. Karp, J.P.Y. Ting, R.B. Sartor, S.E. Plevy, IL-10 regulates IL-12b
AC C
expression via histone deacetylation: implications for intestinal macrophage
homeostasis. J. Immunol. 189 (2012) 1792-1799.
[21] B.C. Lee, J. Lee, Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim. Biophys. Acta 1842 (2014) 446-462.
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[22] M. Ahmadian, J.M. Suh, N. Hag, C. Liddle, A.R. Atkins, M. Downes, R.M. Evans, PPARγ signaling and metabolism: the good, the bad and the future. Nat Med. 99 (2013) 557-566
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[23] S.M. Rangwala, B. Rhoades, J.S. Shapiro, A.S. Rich, J.K. Kim, G.I. Shulman, K.H. Kaestner, M.A. Lazar, Genetic modulation of PPARγ phosphorylation regulates
AC C
EP
TE D
M AN U
SC
insulin sensitivity. Dev. Cell 5 (2003) 657-663.
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Figure Legends
Fig. 1. BAFF-/- mice shows enhanced glucose tolerance in HFD-fed condition despite of
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increased obesity. BAFF-/- and C57BL/6J mice were fed a normal or high-fat diet for 6 weeks. (A) BAFF knockout proved by real-time PCR in spleen and adipose tissues (n=6), and then verified by immunoblotting in spleen. CBB stands for Coomassie
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Brilliant Blue staining. (B) Changes of body weight for 6 weeks of HFD or ND feeding (n=8). (C) Changes of tissue weight after 6-week HFD feeding (n=8). (D) Changes of
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glucose level at 5-week of HFD feeding (n=6). The blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 after intraperitoneal injection of glucose (2 g/kg). (E) Effect of BAFF-/- on insulin-stimulated Akt phosphorylation. After 4-h fasting and acute insulin stimulation by intraperitoneal insulin administration (0.75 U/kg) for 10 min,
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mice were sacrificed, and brown adipose tissue was rapidly excised. Proteins were extracted from the tissue for SDS-PAGE-immunoblot analysis. Data represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 between HFD-fed wild-type and HFD-fed
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BAFF-/- mice. #p < 0.05, ##p < 0.01, and ###p < 0.001 between ND-fed wild-type and ND-fed BAFF-/- mice. chow+/+: ND-fed WT mice, chow-/-: ND-fed BAFF-/- mice,
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HF+/+: HFD-fed WT mice, HF-/-: HFD-fed BAFF-/- mice, EAT: epididymal adipose tissue, SAT: subcutaneous adipose tissue, MAT: mesenteric adipose tissue, BAT: brown adipose tissue.
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Fig. 2. BAFF deficiency alters expression of genes involved in glucose and lipid metabolism. (A) Effect of BAFF deficiency on expression of glucose transporter genes. Total RNA were extracted from brown adipose tissue of mice and gene expression
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levels were analyzed. (B) Effect of BAFF deficiency on mRNA expression associated with mitochondrial thermogenesis and fatty acid oxidation in brown adipose tissue. (C) Effect of BAFF deficiency on mRNA expression associated with lipogenesis and lipid
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oxidation in epididymal and brown adipose tissue. (D) Effect of BAFF deficiency on FGF21 mRNA expression in adipose tissues and the liver. All genes are normalized with
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mRNA expression level of Arbp. Data represent means ± SD for 6 mice per group. *p < 0.05 and **p < 0.01 between HFD-fed WT and HFD-fed BAFF-/- mice. HF+/+: HFD-
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fed WT mice, HF-/-: HFD-fed BAFF-/- mice.
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Fig. 3. BAFF deficiency enhances both leptin expression and STAT3 phosphorylation in adipose tissues. (A) Effect of BAFF deficiency on leptin mRNA expression in adipose tissues. Total RNA were extracted from epididymal, subcutaneous, and brown adipose
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tissue of mice and leptin mRNA expression levels were analyzed (n=6). The mRNA expression level of leptin is normalized with mRNA expression level of Arbp. (B) Serum concentration of leptin quantified by ELISA (n=4). Serum sample was diluted
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20-fold with dilution buffer, and analyzed according to the manufacturer’s protocol. (C) Effect of BAFF deficiency on STAT3 phosphorylation in brown adipose tissue. Proteins
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were extracted from the tissue for SDS-PAGE-immunoblot analysis. Data represent means ± SD. *p < 0.05 and **p < 0.01 between HFD-fed WT and HFD-fed BAFF-/mice. HF+/+: HFD-fed WT mice, HF-/-: HFD-fed BAFF-/- mice, EAT: epididymal
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adipose tissue, SAT: subcutaneous adipose tissue, BAT: brown adipose tissue.
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Fig. 4. BAFF deficiency alters expression of IL-10 and IL-12 and increases PPARγ activity in adipose tissues. (A) Effect of BAFF deficiency on mRNA expression level of IL-10 and IL-12 in adipose tissues. Total RNA were extracted from epididymal and
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brown adipose tissues and gene expression levels were analyzed (n=6). All genes are normalized with mRNA expression level of Arbp. (B) Effect of BAFF deficiency on foxp3 mRNA expression. (C) Effect of BAFF deficiency on PPARγ phosphorylation in
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white adipose tissue. Proteins were extracted from the tissue and analyzed by immunoblotting. Data represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001
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between HFD-fed WT and HFD-fed BAFF-/- mice. HF+/+: HFD-fed WT mice, HF-/-:
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HFD-fed BAFF-/- mice, EAT: epididymal adipose tissue, BAT: brown adipose tissue.
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Fig. 1.
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Research Highlights :
1. BAFF deficient mice show improved glucose tolerance under HFD feeding condition.
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2. Akt phosphorylation was increased in brown adipose tissue (BAT).
3. Expression of genes involved in thermogenesis and lipid metabolism was enhanced in BAT. 4. Expression of IL-10, foxp3, FGF21 and leptin was enhanced in adipose tissues.
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5. Inhibitory phosphorylation of PPARγ was reduced in adipose tissue.
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S0006-291X(15)30329-6
DOI:
10.1016/j.bbrc.2015.07.099
Reference:
YBBRC 34311
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 17 July 2015 Accepted Date: 20 July 2015
Please cite this article as: B. Kim, M.-S. Do, C.-K. Hyun, B-cell-activating factor deficiency attenuates high-fat diet-induced glucose intolerance by potentiating adipose tissue function, Biochemical and Biophysical Research Communications (2015), doi: 10.1016/j.bbrc.2015.07.099. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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B-cell-activating factor deficiency attenuates high-fat diet-
function Bobae Kim, Myoung-Sool Do and Chang-Kee Hyun*
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induced glucose intolerance by potentiating adipose tissue
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School of Life Science, Handong Global University, Pohang, Gyungbuk 791-708, Korea
* Corresponding author at: School of Life Science, Handong Global University, Pohang, Gyungbuk 791-708, Korea. Tel: +82-54-260-1361; Fax: +82-54-260-1319.
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E-mail address: [email protected] (C-.K. Hyun).
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Abstract
B-cell-activating factor (BAFF) has recently been demonstrated to be expressed in
metabolic regulation.
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adipocytes and up-regulated by high-fat diet feeding, indicating a possible role in Here we show that glucose tolerance was significantly improved
in high-fat diet-fed BAFF knockout (BAFF-/-) mice. BAFF-/- mice revealed higher levels
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of glucose transporter expression and insulin-stimulated Akt phosphorylation in brown adipose tissue compared to wild type controls. Expression levels of mitochondrial ND5
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and genes involved in lipid metabolism were significantly elevated in brown adipose tissue of BAFF-/- mice, and this enhancement was found to be mediated by FGF21 and leptin. It was also observed that expression of IL-10 and foxp3 was increased in adipose tissues, as well as PPARγ activity in white adipose tissue. Our findings suggest that
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diabetes.
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suppression of BAFF could have a therapeutic potential for prevention of type 2
Key words: B-cell-activating factor; glucose tolerance; brown adipose tissue; FGF21;
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leptin; IL-10
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1. Introduction
B-cell-activating factor (BAFF) is a cytokine that belongs to tumor necrosis factor
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(TNF) ligand family protein, which has been shown to play an important role in the proliferation and differentiation of B cells, and interacts with three different receptors: BAFF receptor (BAFF-R), B-cell maturation antigen (BCMA), and transmembrane
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activator and CAML interactor (TACI) [1]. Although the expression of BAFF is primarily detected in immunocytes, a variety of cell types also have been known to
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produce BAFF [2]. Several studies have reported that mature adipocytes also produce BAFF and its receptors [3], and through this finding, impact of BAFF on energy metabolism is being questioned.
There have been a number of studies describing the role of BAFF in energy
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metabolism. Hamada et al. reported that levels of BAFF were significantly increased in serum and visceral adipose tissue of high-fat diet-fed mice relative to normal control mice [4]. They also found that treatment with recombinant BAFF protein impaired
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insulin-mediated glucose uptake in 3T3-L1 adipocytes. BAFF plasma levels were significantly correlated with body fat and dietary composition in obese Central-
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European Caucasian individuals [5], and the levels were also observed to be significantly higher in Japanese patients with nonalcoholic fatty liver disease [6]. Studies on the effect of BAFF on energy metabolism have shown us that BAFF and its receptors are closely related to the physiological activity of adipose tissues: BAFF is not only produced by adipocytes [3,4] but also associated with the differentiation process of adipose-derived stem cells towards adipocyte [7]. Moreover, Kawasaki et al. reported that BAFF-R knockout (BAFF-R-/-) mice were protected from high-fat diet-induced
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adiposity and showed improved insulin sensitivity [8]. In their study, no difference of visceral fat weight between high-fat diet-fed BAFF-R-/- and wild type mice was observed but the inguinal fat weight was significantly lower in BAFF-R-/- mice.
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Although these recent studies clearly illustrated that BAFF is linked to metabolic status and involved in the impairment of insulin sensitivity, questions remain in the elucidation of the role of BAFF in the development of adiposity and insulin resistance.
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In this study, we investigated the impact of BAFF on the onset of glucose intolerance using BAFF knockout (BAFF-/-) mice fed with high-fat diet. Insulin resistance in BAFFmice was significantly improved despite of diet-induced weight gain, which was
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/-
attributed by up-regulated gene expression of lipid metabolism in brown adipose tissue mediated by FGF21 and leptin. It was also found that IL-10 expression and PPARγ activity in white adipose tissue was enhanced, resulting in enhanced glucose tolerance.
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Our study indicates that BAFF is a critical adipokine for the development of glucose intolerance, suggesting that blocking BAFF could be a therapeutic approach for the
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prevention and treatment of type 2 diabetes.
2. Materials and methods
2.1. Animals
Male C57BL/6J wild type (WT) mice and BAFF-/- mice were purchased from Central Lab. Animal Inc. (Seoul, Korea), and The Jackson Laboratory (Bar Harbor, ME; stock number 010572), respectively. Systemic BAFF knockout mice were generated by
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insertion of the tailless human CDs reporter gene into the BAFF locus. BAFF gene deletion was confirmed by baff mRNA expression levels in spleen, epididymal and brown adipose tissue, and BAFF protein level in spleen (Fig. 1A). Six-week-old males
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were maintained under a 12 h light:dark cycle at a constant temperature of 22 ± 1°C and humidity of 45 ± 10%. To stabilize all metabolic conditions, eleven-week-old male mice were fed normal chow diet (2018S, Harlan Laboratories, Indianapolis, IN) and
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individually housed in cages for a week. After the stabilization, mice were switched to the high-fat diet (HFD) containing 60% kcal from fat (D12493, Research Diets Inc., NJ)
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for 6 weeks.
Mice were fasted for 4 h, and sacrificed. Tissues of liver, spleen, inguinal fat, epididymal fat, brown fat and quadriceps muscle were harvested, snap-frozen in liquid
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nitrogen, and stored at -75°C until processed for RNA and protein analysis.
2.2. Glucose tolerance test
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After 4 weeks of high-fat feeding, mice were fasted for 16 h and followed by intraperitoneal injection of glucose (2 g/kg). Blood samples were obtained by tail-
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bleeding, and blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 min after glucose injection by Accu-Check Go (Roche Diagnostics GmbH, Basel, Switzerland).
2.3. Western blotting
Frozen tissues were homogenized and protein-extracted as described previously [9]. Antibodies against phospho-Akt (Ser473), total Akt, β-actin (Cell signaling technology,
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Beverly, MA), BAFF (R&D systems, Minneapolis, MN), phospho-PPARγ (Ser112) (Abcam, Cambridge, UK), phoshpo-STAT3 (Tyr705) and total STAT3 (Cambridge Bioscience, Cambridge, UK) were used as primary antibodies, followed by the
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appropriate IgG-horseradish peroxidase-conjugated secondary antibody. Proteins were visualized by ECL.
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2.4. Real-time RT PCR
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Total RNA extraction, reverse transcription, and quantitative PCR were performed as described previously [9]. Relative expression level of mRNAs was calculated by the ∆∆Ct method using 36B4 (Arbp) as the reference control. Primer sequences are available
upon
request:
Acyl-CoA
oxidase
1
(Acox1),
BAFF,
carnitine
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palmitoyltransferase 1 (CPT1), fatty acid synthase (FAS), fibroblast growth factor 21 (FGF21), forkhead box P3 (Foxp3), glucose transporter I (GLUT1), GLUT4, interleukin-10 (IL-10), IL-12, leptin, mitochondrially encoded NADH dehydrogenase 5
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(ND5), peroxisome proliferator-activated receptor α (PPARα), PPARγ coactivator 1α
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(PGC1α), stearoyl-CoA desaturase-1 (SCD1), uncoupling protein 1 (UCP1).
2.5. ELISA
Measurement of serum leptin was performed with commercial ELISA kits (R&D systems) according to the manufacturer’s instruction.
2.6. Statistical analysis
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All data were presented as mean ± SD. Comparisons of two groups were performed
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by two-tailed Student’s t-test. p values < 0.05 were considered as statistically significant.
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3. Results
3.1. BAFF deficiency improves glucose tolerance under HFD condition despite
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increased adiposity
After 6 weeks of normal chow diet (ND) or HFD feeding, BAFF-/- mice showed significantly increased weight gain compared to their wild-type (WT) control mice (Fig.
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1B). The quantitative changes in tissue weight revealed differences between various types of tissues. BAFF-/- mice on HF diet had significantly increased weights of adipose tissues and liver and decreased weights of quadriceps and spleen compared to WT
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counterparts (Fig. 1C). Interestingly, however, no substantial difference in the weight of epididymal adipose tissue was observed between wild-type and BAFF-/- mice, which
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was different from not only brown adipose tissue but also other types of white adipose tissues.
To test whether BAFF deficiency affects insulin sensitivity, glucose tolerance test was carried out in ND- or HFD-fed WT and BAFF-/- mice. Despite the increased adiposity, the HFD-fed BAFF-/- mice exhibited significantly improved glucose tolerance compared to HFD-fed WT mice (Fig. 1D). Notably, this improvement effect was observed only under HFD feeding condition, but not under ND feeding condition.
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To confirm that activation of insulin signaling led to improved glucose tolerance, we analyzed insulin-stimulated phosphorylation of Akt in epididymal white adipose tissue, brown adipose tissue and quadriceps muscle. Akt phosphorylation was significantly
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higher in brown adipose tissue of BAFF-/- mice compared to the wild-type mice (Fig. 1E), but unexpectedly not in epididymal adipose tissue and quadriceps (data not shown).
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3.2. BAFF deficiency changes mRNA expression of genes involved in glucose and lipid
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metabolism in brown adipose tissue
Brown adipose tissue is a thermogenic organ which stores nutrients as lipids and dissipates their energy as heat. To examine how BAFF deficiency modulates metabolism in brown adipose tissue, we analyzed the altered gene expression for
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glucose and lipid metabolism. Brown adipose tissue of HFD-fed BAFF-/- mice displayed a significant increase in mRNA expression of glucose transporters, GLUT4 and GLUT1, relative to WT controls (Fig. 2A). Since brown adipose tissue consumes lipids to
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generate heat through uncoupling respiration, mRNA expression levels of a mitochondrial gene ND5 and genes involved in thermogenesis such as PGC1α, PPARα
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and UCP1 were also measured. HFD-fed BAFF-/- mice had significantly higher gene expression of ND5 and PGC1α, the master regulator of UCP1-mediated thermogenesis in brown adipose tissue, relative to WT control mice (Fig. 2B). Expression of the nuclear-encoded mitochondrial thermogenic genes, PPARα and UCP1, was also modestly elevated in HFD-fed BAFF-/- mice relative to WT controls even though the statistical significances were not observed. To examine the effect of BAFF deficiency on lipid metabolism in adipose tissues,
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expression of genes involved in lipogenesis, FAS and SCD1, and fatty acid oxidation, Acox1 and CPT1, in epididymal and brown adipose tissue were measured and compared. On HFD diet, BAFF-/- mice had higher levels of expression of FAS, SCD1,
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Acox1 and CPT1 in brown adipose tissue, compared to WT controls, with or without significance (Fig. 2C). However, epididymal adipose tissue was different from brown adipose tissue, showing that expression of lipogenic genes was suppressed whereas
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expression of fatty acid oxidation genes was up-regulated in BAFF-/- mice relative to WT controls. To better understand the mechanism underlying the effect of BAFF
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deficiency on metabolism in brown adipose tissue, we analyzed gene expression of FGF21 which plays significant roles in lipid oxidation and browning of white adipose tissue and thermogenesis in brown adipose tissue. Notably, HFD-fed BAFF-/- mice had significantly higher expression of FGF21 in epididymal and brown adipose tissues,
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while there was no difference in the liver between BAFF-/- and WT mice (Fig. 2D).
adipose tissues
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3.3. BAFF deficiency enhances both leptin expression and STAT3 phosphorylation in
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Leptin is an adipokine which regulates food intake and energy expenditure in an endocrine manner. The mRNA expression levels of leptin were significantly higher in epididymal, inguinal and brown adipose tissues of HFD-fed BAFF-/- mice compared to WT controls (Fig. 3A). Commensurate with their increased mRNA expression, BAFF-/mice had significantly higher level of serum leptin than WT controls (Fig. 3B). Based on the fact that leptin action is mediated by Jak/STAT pathway, higher leptin expression was corroborated by the finding that STAT3 phosphorylation in brown adipose tissue of
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BAFF-/- mice was significantly higher than WT controls (Fig. 3C).
3.4. BAFF deficiency changes IL-10/IL-12 mRNA expression and enhances PPARγ
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activity in adipose tissues
Accumulation of pro-inflammatory immune cells and their cytokines in adipose
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tissue is considered a focal point in the progression of insulin resistance. We observed that the mRNA expression of anti-inflammatory cytokine IL-10 was significantly
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increased, whereas the expression of IL-12, a pro-inflammatory cytokine, was suppressed in both epididymal and brown adipose tissues of HFD-fed BAFF-/- mice (Fig. 4A). Unexpectedly, however, there were no significant differences in the expression of other anti-inflammatory cytokines except IL-10, including IL-4, IL-1ra, and IL-13, as
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well as of other pro-inflammatory cytokines except IL-12, including TNFα, IL-6, IFNγ, MCP-1 (data not shown). On the other hand, it was observed that the expression level of foxp3, a marker of regulatory T cells induced by anti-inflammatory cytokines, was
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higher in BAFF-/- mice than WT controls (Fig. 4B). We next analyzed the status of PPARγ phosphorylation at serine 112 to examine BAFF
deficiency
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whether
enhances
PPARγ
activity
by
reducing
PPARγ
phosphorylation in adipose tissue under HFD feeding condition. The level of PPARγ phosphorylation was significantly reduced in epididymal adipose tissue of BAFF-/- mice relative to WT controls (Fig. 4C) without any change in the mRNA expression level (data not shown). This result is consistent with the observation of improved glucose tolerance despite higher body weight gain in HFD-fed BAFF-/- mice compared to WT controls.
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4. Discussion
Recent studies have shown that mature adipocytes produce BAFF, which has been identified as an adipokine with metabolic effects [3-5]. The aim of this study was to
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elucidate the role of BAFF deficiency in metabolic regulation through exploring the
tolerance in HFD-fed BAFF-/- mice.
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molecular mechanism underlying the effect of BAFF depletion on improved glucose
In this study, HFD-fed BAFF-/- mice showed a significant improvement in insulin sensitivity relative to their WT control mice (Fig. 1D), which was partly consistent with a previous report that BAFF-R-/- mice were protected against HFD-induced obesity and
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insulin resistance [8]. BAFF-/- mice had improved glucose tolerance under HFD-fed condition, but, unlike BAFF-R-/- mice, were not protected against HFD-induced weight gain (Fig. 1B). These different responses of BAFF-/- and BAFF-R knockout mice to
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HFD feeding could be accounted for the fact that the action of BAFF is mediated by not only BAFF-R but also other receptors on adipocytes and adipose-tissue derived
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mesenchymal cells, such as BCMA, TACI and Fn14 [1-3]. The body weight gain of HFD-fed BAFF-/- mice was observed to be attributed mainly by the increase in tissue weights of white adipose tissues except epididymal adipose tissue (Fig. 1C), which is explicable by the decreased level of lipogenesis gene expression specifically in epididymal adipose tissue but not in other adipose tissues of BAFF-/- mice relative to the WT controls (Fig. 2C). The improved glucose tolerance in HFD-fed BAFF-/- mice was corroborated by
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observation of an increased level of insulin-stimulated Akt phosphorylation in brown adipose tissue (Fig. 1E). We unexpectedly found that other insulin-responsive tissues including white adipose tissue, skeletal muscle and liver did not show PI3K-dependent
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activation of insulin signaling and were not responsible for the insulin-sensitizing effect of BAFF depletion. Along with enhanced translocation of GLUT4 indicated by increased Akt activation, GLUT4 and GLUT1 mRNA expression levels were both
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found to be significantly increased in brown adipose tissue of BAFF-/- mice compared with WT controls (Fig. 2A), which might account for the enhanced glucose uptake [10].
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Brown adipose tissue exerts beneficial effects on insulin sensitivity and glucose and lipid metabolism by dissipating energy in the form of heat [11]. Consistent with higher weight of brown adipose tissue in HFD-fed BAFF-/- mice compared to their WT controls, increased expression of genes related to lipogenesis such as SCD1 and FAS,
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was observed in BAFF-/- mice (Fig. 2C). Increased expressions of mitochondrial ND5 gene and thermogenic genes PGC1α, PPARα and UCP1 in brown adipose tissue of BAFF-/- mice (Fig. 2B) indicates that mitochondrial biogenesis and thermogenesis were
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enhanced by BAFF depletion. We also found that the BAFF-/- mice had significantly higher expression levels of the fatty acid oxidation genes such as Acox1 and CPT1,
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compared to WT mice. Taken together, our findings suggest that BAFF depletion, under HFD-fed condition, leads to increases in glucose uptake, lipogenesis, and fatty acid oxidation, triggering brown adipose tissue to be metabolically active. FGF21 is a peptide hormone produced mainly in the liver and adipose tissues, which is known to mediate the thermogenic response of brown adipose tissue to cold and adrenergic stimulation [12]. It has also been reported that FGF21 reduces blood glucose, insulin, and lipid levels and increases total energy expenditure and physical activity in diet-
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induced obese mice [13]. In our study, FGF21 mRNA expression was significantly increased in adipose tissues of BAFF-/- mice whereas there was no difference in the liver between BAFF-/- and WT mice (Fig. 2D). This result suggests that BAFF deficiency
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enhances glucose tolerance by potentiating adipose tissue function, causing an increase in FGF21 production that leads to enhanced metabolic activity of brown fat in BAFF-/mice.
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We also found that BAFF depletion enhanced leptin expression in both white and brown adipose tissue under HFD-fed condition, which was corroborated by the
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observation of higher levels of leptin mRNA and serum leptin in BAFF-/- mice compared to WT controls (Fig. 3A and 3B). It has been reported that elevated levels of circulating leptin result in the enhancement of glucose utilization by inducing UCP1 expression and STAT phosphorylation in brown adipose tissue [14,15]. We observed
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that STAT3 phosphorylation in brown adipose tissue of BAFF-/- mice was significantly increased compared to WT controls (Fig. 3C). This observation lends further support to the hypothesis that BAFF depletion might also augment the metabolic activity of brown
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adipose tissue through leptin-mediated STAT3 activation. Obesity is a chronic state of low-grade inflammation with progressive immune cell
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infiltration into adipose tissue [16]. Cytokines produced from infiltrating immune cells modulate adipose tissue inflammation and consequential insulin resistance [17]. IL-10, is an anti-inflammatory cytokine that is also known to enhance B cell activation, proliferation and differentiation. Recent studies have reported that IL-10 protects mice against obesity and insulin resistance and reduces immune cell infiltration [18]. In this study, we found that mRNA expression of IL-10 was significantly higher while IL-12 expression was decreased in both epididymal and brown adipose tissue of BAFF-/- mice
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compared to WT controls (Fig. 4A). Since transcription of IL-12, a pro-inflammatory cytokine mainly produced by phagocytic cells and involved in regulating T cell response, is elevated by HFD feeding and repressed by IL-10 [19,20], the suppression
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of IL-12 gene expression strongly supports IL-10 action in BAFF-/- mice. Regulatory T (Treg) cells have been reported to attenuate inflammation and obesity-induced insulin resistance by IL-10 production [21]. We found that the expression level of foxp3, a
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specific marker of Treg cell population, was significantly increased in BAFF-/- mice relative to WT controls (Fig. 4B). Additionally, we observed a significantly reduced
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phosphorylation of PPARγ at serine 112 in white adipose tissue of HFD-fed BAFF-/mice compared to WT controls (Fig. 4C), indicating a significant enhancement of PPARγ activity. Phosphorylation at Ser112 of PPARγ is known to repress its transcriptional activity by hindering ligand binding and altering cofactor recruitment,
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and genetically modified mice carrying mutation of S112A showed improved insulin sensitivity [22,23]. Taken together with the beneficial changes in cytokine production, this might possibly be one cause for the activation of brown adipose tissue, thereby
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contributing to the enhancement of glucose tolerance of BAFF-/- mice. In summary, the results of this study suggest that BAFF deficiency prevents mice
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from HFD-induced glucose intolerance by potentiating adipose tissue function, particularly through enhanced activities of brown adipose tissue. Our data demonstrate that the protective effect of BAFF depletion is mediated by FGF21 and leptin, which might be also attributed to up-regulation of IL-10 and foxp3 and enhancement of PPARγ activity. Furthermore, these findings illustrate that BAFF is an adipokine which mediates the onset of glucose intolerance, and blocking BAFF could stand as a valid therapeutic approach that attenuates the progression of insulin resistance.
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References
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[1] F. Mackay, P. Schneider, Cracking the BAFF code. Nat. Rev. Immunol. 9 (2009) 491-502.
[2] M. Zonca, P. Mancheño-Corvo, O. Delarosa, S. Mañes. D. Büscher, E. Lombardo, L.
SC
Planelles, APRIL and BAFF proteins increase proliferation of human adiposederived stem cells through activation of Erk1/2 MAP kinase. Tissue Eng. Part A. 18
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(2012) 852-859
[3] V. Alexaki, G. Notas, V. Pelekanou, M. Kampa, M. Valkanou, P. Theodoropoulos, E.N. Stathopoulos, A. Tsapis, E. Castanas, Adipocytes as immune cells: differential expression of TWEAK, BAFF, and APRIL and their receptors (Fn14, BAFF-R,
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TACI, and BCMA) at different stages of normal and pathological adipose tissue development. J. Immunol. 183 (2009) 5948-5956 [4] M. Hamada, M. Abe, T. Miyake, K. Kawasaki, J. Tada, S. Furukawa, B. Matsuura,
EP
Y. Hiasa, M. Onji, B cell-activating factor controls the production of adipokines and induces insulin resistance. Obesity 19 (2011) 1915-1922
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[5] J. Bienertova-Vasku, P. Bienert, F. Zlamal, F. Tomandl, M. Forejt, M. Tomandlova, M. Vavrina, J. Kudelková, Z. Splichal, A. Vasku, B-cell activating factor (BAFF)-a new factor linking immunity to diet? Cent. Eur. J. Med. 7 (2012) 275-283
[6] T. Mihyke, M. Abe, Y. Tokumoto, M. Kirooka, S. Furukawa, T. Kumagi, M. Hamada, K. Kawasadi, F. Tada, T. Ueda, Y. Hiasa, B. Matsuura, M. Onji, B cellactivating factor is associated with the histological severity of nonalcoholic fatty liver disease. Hepatol. Int. 7 (2013) 539-547
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[7] H. Wa, D. Han, Z. Jiang, D. Zhao, M. Liu, X. Xu, X. Liu, L. Yang, X. Ji, M. Wang, S. Zhang, Equine adipose-derived stem cell (ASC) expresses BAFF and its
adipocyte. Int. Immunopharmacol. 18 (2014) 365-372
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receptors, which may be associated with the differentiation process of ASC towards
[8] K. Kawasaki, M. Abe, F. Tada, Y. Tokumoto, S. Chen, T. Miyake, S. Furukawa, B. Matsuura, Y. Hiasa, M. Onji, Blockade of B-cell-activating factor signaling
sensitivity. Lab. Invest. 93 (2013) 311-321
SC
enhances hepatic steatosis induced by a high-fat diet and improves insulin
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[9] E. Kim, J.H. Lee, J.M. Ntambi, C.K. Hyun, Inhibition of stearoyl-CoA desaturase1 activates AMPK and exhibits beneficial lipid metabolic effects in vitro, Eur. J. Pharmacol. 672 (2011) 38-44.
[10] M.A. Herman, B.B. Kahn, Glucose transport and sensing in the maintenance of
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glucose homeostasis and metabolic harmony. J. Clin. Invest. 116 (2006) 1767-1775. [11] A. Bartelt, J. Heeren, Adipose tissue browning and metabolic health. Nat. Rev. Endocrinol. 10 (2014) 24-36.
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[12] F.M. Fisher, S. Kleiner, N. Douris, E.C. Fox, R.J. Mepani, F. Verdeguer, J. Wu, A. Kharitonenkov, J.S. Flier, E. Maratos-Flier, B.M. Spiegelman, FGF21 regulates
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PGC-1α and browning of white adipose tissues in adaptive thermogenesis. Genes Dev. 26 (2012) 271-281.
[13] J. Xu, D.J. Lloyd, C. Hale, H, Stanislaus, M. Chen, G. Sivits, S. Vonderfecht, R. Hecht, Y.S. Li, R.A. Lindberg, J.L. Chen, D.Y. Jung, Z. Zhang, H.J. Ko, J.K. Kim, M.M. Véniant, Fibroblast growth factor 21 reverses hepatic steatosis, increases energy expenditure, and improves insulin sensitivity in diet-induced obese mice. Diabetes 58 (2009), 250-259.
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[14] J. Rouru, I. Cusin, K.E. Zakrzewska, B. Jeanrenaud and F. Rohner-Jeanrenaud, Effects of intravenously infused leptin on insulin sensitivity and on the expression of uncoupling proteins in brown adipose tissue. Endocrinology 140 (1999) 3688-
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3692.
[15] P. Bendinelli, P. Maroni, F. Pecori Giraldi, R. Piccoletti, Leptin activates Stat3, Stat1 and AP-1 in mouse adipose tissue. Mol. Cell. Endocrionol. 168 (2000) 11-20.
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[16] M.F. Gregor, G.S. Hotamisligil, Inflammatory mechanisms in obesity. Annu. Rev. Immunol. 29 (2011) 415-445.
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[17] K.A. Harford, C.M. Reynolds, F.C. McCillicuddy, H.M. Roche, Fats, inflammation and insulin resistance: insights to the role of macrophage and T-cell accumulation in adipose tissue. Proc. Nutr. Soc. 70 (2011) 408-417.
[18] M.A. Febbraio, Role of interleukins in obesity: implications for metabolic disease.
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Trends Endocrinol. Metab. 25 (2014) 312-319.
[19] M. Aste-Amezaga, X. Ma, A. Sartori, G. Trinchieri, Molecular mechanisms of the induction of IL-12 and its inhibition by IL-10. J. Immunol. 160 (1998) 5936-5944.
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[20] T. Kobayashi, K. Matsuoka, S.Z. Sheikh, S.M. Russo, Y. Mishima, C. Collins, E.F. deZoeten, C.L. Karp, J.P.Y. Ting, R.B. Sartor, S.E. Plevy, IL-10 regulates IL-12b
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expression via histone deacetylation: implications for intestinal macrophage
homeostasis. J. Immunol. 189 (2012) 1792-1799.
[21] B.C. Lee, J. Lee, Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim. Biophys. Acta 1842 (2014) 446-462.
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[22] M. Ahmadian, J.M. Suh, N. Hag, C. Liddle, A.R. Atkins, M. Downes, R.M. Evans, PPARγ signaling and metabolism: the good, the bad and the future. Nat Med. 99 (2013) 557-566
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[23] S.M. Rangwala, B. Rhoades, J.S. Shapiro, A.S. Rich, J.K. Kim, G.I. Shulman, K.H. Kaestner, M.A. Lazar, Genetic modulation of PPARγ phosphorylation regulates
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insulin sensitivity. Dev. Cell 5 (2003) 657-663.
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Figure Legends
Fig. 1. BAFF-/- mice shows enhanced glucose tolerance in HFD-fed condition despite of
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increased obesity. BAFF-/- and C57BL/6J mice were fed a normal or high-fat diet for 6 weeks. (A) BAFF knockout proved by real-time PCR in spleen and adipose tissues (n=6), and then verified by immunoblotting in spleen. CBB stands for Coomassie
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Brilliant Blue staining. (B) Changes of body weight for 6 weeks of HFD or ND feeding (n=8). (C) Changes of tissue weight after 6-week HFD feeding (n=8). (D) Changes of
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glucose level at 5-week of HFD feeding (n=6). The blood glucose levels were measured at 0, 15, 30, 60, 90 and 120 after intraperitoneal injection of glucose (2 g/kg). (E) Effect of BAFF-/- on insulin-stimulated Akt phosphorylation. After 4-h fasting and acute insulin stimulation by intraperitoneal insulin administration (0.75 U/kg) for 10 min,
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mice were sacrificed, and brown adipose tissue was rapidly excised. Proteins were extracted from the tissue for SDS-PAGE-immunoblot analysis. Data represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001 between HFD-fed wild-type and HFD-fed
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BAFF-/- mice. #p < 0.05, ##p < 0.01, and ###p < 0.001 between ND-fed wild-type and ND-fed BAFF-/- mice. chow+/+: ND-fed WT mice, chow-/-: ND-fed BAFF-/- mice,
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HF+/+: HFD-fed WT mice, HF-/-: HFD-fed BAFF-/- mice, EAT: epididymal adipose tissue, SAT: subcutaneous adipose tissue, MAT: mesenteric adipose tissue, BAT: brown adipose tissue.
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Fig. 2. BAFF deficiency alters expression of genes involved in glucose and lipid metabolism. (A) Effect of BAFF deficiency on expression of glucose transporter genes. Total RNA were extracted from brown adipose tissue of mice and gene expression
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levels were analyzed. (B) Effect of BAFF deficiency on mRNA expression associated with mitochondrial thermogenesis and fatty acid oxidation in brown adipose tissue. (C) Effect of BAFF deficiency on mRNA expression associated with lipogenesis and lipid
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oxidation in epididymal and brown adipose tissue. (D) Effect of BAFF deficiency on FGF21 mRNA expression in adipose tissues and the liver. All genes are normalized with
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mRNA expression level of Arbp. Data represent means ± SD for 6 mice per group. *p < 0.05 and **p < 0.01 between HFD-fed WT and HFD-fed BAFF-/- mice. HF+/+: HFD-
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fed WT mice, HF-/-: HFD-fed BAFF-/- mice.
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Fig. 3. BAFF deficiency enhances both leptin expression and STAT3 phosphorylation in adipose tissues. (A) Effect of BAFF deficiency on leptin mRNA expression in adipose tissues. Total RNA were extracted from epididymal, subcutaneous, and brown adipose
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tissue of mice and leptin mRNA expression levels were analyzed (n=6). The mRNA expression level of leptin is normalized with mRNA expression level of Arbp. (B) Serum concentration of leptin quantified by ELISA (n=4). Serum sample was diluted
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20-fold with dilution buffer, and analyzed according to the manufacturer’s protocol. (C) Effect of BAFF deficiency on STAT3 phosphorylation in brown adipose tissue. Proteins
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were extracted from the tissue for SDS-PAGE-immunoblot analysis. Data represent means ± SD. *p < 0.05 and **p < 0.01 between HFD-fed WT and HFD-fed BAFF-/mice. HF+/+: HFD-fed WT mice, HF-/-: HFD-fed BAFF-/- mice, EAT: epididymal
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adipose tissue, SAT: subcutaneous adipose tissue, BAT: brown adipose tissue.
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Fig. 4. BAFF deficiency alters expression of IL-10 and IL-12 and increases PPARγ activity in adipose tissues. (A) Effect of BAFF deficiency on mRNA expression level of IL-10 and IL-12 in adipose tissues. Total RNA were extracted from epididymal and
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brown adipose tissues and gene expression levels were analyzed (n=6). All genes are normalized with mRNA expression level of Arbp. (B) Effect of BAFF deficiency on foxp3 mRNA expression. (C) Effect of BAFF deficiency on PPARγ phosphorylation in
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white adipose tissue. Proteins were extracted from the tissue and analyzed by immunoblotting. Data represent means ± SD. *p < 0.05, **p < 0.01, and ***p < 0.001
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between HFD-fed WT and HFD-fed BAFF-/- mice. HF+/+: HFD-fed WT mice, HF-/-:
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HFD-fed BAFF-/- mice, EAT: epididymal adipose tissue, BAT: brown adipose tissue.
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Fig. 1.
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Fig. 2.
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Fig. 3.
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Fig. 4.
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Research Highlights :
1. BAFF deficient mice show improved glucose tolerance under HFD feeding condition.
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2. Akt phosphorylation was increased in brown adipose tissue (BAT).
3. Expression of genes involved in thermogenesis and lipid metabolism was enhanced in BAT. 4. Expression of IL-10, foxp3, FGF21 and leptin was enhanced in adipose tissues.
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5. Inhibitory phosphorylation of PPARγ was reduced in adipose tissue.
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