- Email: [email protected]
WITHDRAWN: Modulation of adipocyte function by the TGF-β family
Cytokine xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Modulation of adipocyte function by the TGF-β family Yuhang Qiao, Shozo Tomonaga, Masashi Suenaga, Tohru Matsui, Masayuki Funaba
⁎
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: TGF-β family Adipocyte Adipokine Metabolome analysis
Members of the transforming growth factor-β (TGF-β) family are known to regulate the commitment of mesenchymal stem cells to an adipocyte lineage and the differentiation of preadipocytes to adipocytes. However, the roles of TGF-β family members in mature adipocytes are not known. The present study explored the modulation of cell function in 3T3-L1 adipocytes treated with the TGF-β family members TGF-β1, activin A, activin B, bone morphogenetic protein (BMP) 4, and BMP7. These TGF-β family members modulated the expression of some adipokines; activin A upregulated leptin expression, and activin B downregulated the expression of plasminogen activator inhibitor-1. All examined TGF-β family members, except for activin B, increased the expression of resistin. The TGF-β family members also modulated the free amino acid content in cells. Insulin-induced reductions in the cellular content of arginine, glutamic acid, and phenylalanine were exaggerated by TGF-β1, activin A, and activin B. In contrast, the cellular glutamine content decreased with BMP4 or BMP7 treatment, inhibiting the response to insulin. In addition, although the cellular citric acid cycle metabolite contents were not generally affected by TGF-β family members alone, activin A, activin B, BMP4, and BMP7 potentiated the insulin-induced accumulation of the metabolites. The present results suggest that TGF-β family members regulate adipocyte function by modulating adipokine expression and cell metabolism in differentiated adipocytes.
1. Introduction
the uptake of glucose [35,5] and amino acids [35] by adipocytes. Our previous study evaluated changes in adipocyte metabolite content in response to insulin by a metabolomic analysis; insulin increased the citric acid cycle metabolites in a 2-h cycle and decreased the free amino acid contents of the cells [29]. Because the metabolite content in cells reflects the net results of insulin-mediated cell responses, these results are useful in understanding the global modulation of adipocyte metabolism induced by insulin. Because there is cross-talk between components of the TGF-β and other signaling pathways in many biological systems [14,24], Guo and Wang (2009), the action of insulin on adipocyte metabolism may be modulated by TGF-β family members. The objective of this study was to clarify the role of TGF-β family members in the function of differentiated adipocytes. We evaluated the effects of TGF-β1, activin A, activin B, BMP4, and BMP7 on the expression levels of genes related to adipocyte function. In addition, cellular metabolite contents were explored to determine whether the insulin-mediated cell responses were modulated by TGF-β family members.
Adipocytes accumulate excess energy as triacylglycerol through lipogenesis and provide fatty acids to peripheral tissues, depending on the energy status of the tissues, through lipolysis [31]. In addition to regulating energy metabolism, adipocytes maintain homeostasis throughout the body by producing adipokines such as adiponectin, leptin, resistin, and plasminogen activator inhibitor-1 (Pai-1) [45,19]. Members of the transforming growth factor-β (TGF-β) family regulate a wide variety of biological processes, including adipogenesis (Derynck et al., 2008). Bone morphogenetic protein (BMP) 2 and BMP4 induce commitment of mesenchymal stem cells to an adipocyte lineage [43,17]. BMP4 expression in preadipocytes is required for differentiation to adipocytes [39,38], whereas TGF-β and activin A inhibit the differentiation of preadipocytes [9,16]. Despite well-established evidence of the role of members of the TGF-β family in adipogenesis, little information is available on the modulation of adipocyte function by members of the TGF-β family. Epidemiological studies have shown the relationship between expression levels of TGF-β family members in adipose tissue and adiposity [1,8,50,49,28], which may indicate (dys) regulation of adipocyte function by members of the TGF-β family. Insulin is known to modulate adipocyte function. Insulin activates
⁎
Corresponding author at: Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan. E-mail address: [email protected] (M. Funaba).
http://dx.doi.org/10.1016/j.cyto.2017.05.011 Received 26 January 2017; Received in revised form 1 May 2017; Accepted 12 May 2017 1043-4666/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Qiao, Y., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.05.011
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
2. Materials and methods
area of 2-isopropylmalic acid, an GC–MS internal control, and normalized by the protein concentration [29]. The content of each metabolite in cells untreated with insulin or a TGF-β family member, i.e., control cells, was set at 100.
2.1. Cell culture The 3T3-L1 preadipocytes used in this study were cultured and differentiated as described previously [29]. The cells were grown and maintained in growth medium, i.e., Dulbecco’s modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. Two days after the cells reached confluence (day 0), cells were cultured in a growth medium supplemented with differentiation inducers (IBMX [0.5 mM], dexamethasone [0.25 μM], and insulin [10 μg/ml]) for 2 days, followed by cultivation in a growth medium supplemented with insulin (5 μg/ml). By day 8, more than 95% of 3T3L1 cells had differentiated into adipocytes, based on appearance. To evaluate the changes in gene expression of TGF-β family receptors over time, cells were harvested on days −4, −2, −1, 0, 1, 2, 4, 6, and 8. Gene expression was examined by real-time reverse transcriptionquantitative PCR (RT-qPCR). To evaluate the lipid accumulation and expression of genes related to adipocyte function, TGF-β family members were added to the growth medium supplemented with insulin for 48 h, i.e., days 8–10, at the following concentrations: 200 pM for TGF-β1 (Becton Dickinson, Franklin Lakes, NJ, USA) and 3 nM for activin A, activin B, BMP4, and BMP7 (R & D Systems, Minneapolis, MN, USA). Because members of the TGF-β family function as local factors, plasma level of the TGF-β family does not necessarily reflect physiological concentration in affected cells. Concentrations of TGF-β1 (200 pM), activin A (3 nM), activin B (3 nM), BMP4 (3 nM) and BMP7 (3 nM) are within the range that is frequently used in cell culture studies [9,46,26,48,12,47]. On day 10, lipid accumulation was evaluated by Oil Red O staining, and images were obtained by scanning stained wells (GT-9400UF; Epson, Tokyo, Japan). In addition, the expression of genes related to adipocyte differentiation and function was evaluated by RT-qPCR. For the metabolomic analyses, 3T3-L1 adipocytes on day 8 were cultured in DMEM without insulin for 24 h, i.e., days 8–9, followed by treatment with or without insulin (10 μg/ml) and with TGF-β family members for 2 h or 6 h. TGF-β family members were applied in the concentrations given above. We previously showed that the cellular citric acid cycle metabolite contents exhibited periodic changes with a 2-h cycle [29]. It is possible that the TGF-β family members modulate insulin-induced cellular contents in a time-dependent manner. Therefore, we evaluated the metabolite contents in adipocytes treated with insulin and TGF-β family members for either 2 h or 6 h. In addition, at 6 h, the expression levels of genes related to amino acid catabolism were evaluated by RT-qPCR.
2.4. Data analysis To visualize changes in the cellular metabolite content in response to insulin and TGF-β family members, a heat map was prepared and a hierarchical cluster analysis was performed as described previously [29]. The threshold for grouping was set at a correlation coefficient of 0.9 in the dendrogram, and it was grouped to be as few as possible. 2.5. Statistical analyses Data are expressed as the mean ± standard error (SE). The expression levels of genes were log-transformed to provide an approximation of a normal distribution before analysis. Data used in the timecourse analysis of the expression of receptors in the TGF-β family were evaluated by one-way ANOVA, followed by a Tukey-Kramer test. For the expression of adipokines and genes related to adipogenesis and adipocyte function, data were analyzed by one-way ANOVA, considered factor was TGF-β family members. When the effect was significant (P < 0.05), the differences between control cells and TGF-β familytreated cells were examined by Dunnett’s test. For metabolite contents, data were also analyzed by two-way ANOVA. Factors considered in the analysis were insulin, TGF-β family members, and the interaction between insulin and TGF-β family members. When the effect of insulin or the interaction between insulin and TGF-β family members was significant, comparisons between insulin-treated or -untreated cells treated with the respective TGF-β family member were performed by the Student’s t-test. When the effect of TGF-β family members or the interaction of insulin and TGF-β family members was significant, comparisons between cells untreated with a TGF-β family member, as the reference, and TGF-β family member-treated cells that were either treated or untreated with insulin were also performed using the Dunnett’s test. 3. Results 3.1. Expression of receptors for TGF-β family members during adipogenesis At first, we examined the changes in expression levels of receptors for TGF-β family members during adipogenesis in 3T3-L1 cells (Fig. 1). Generally, expression levels of the receptors increased steadily from day −4 to day 0; specifically, the expression levels of Tβr2, Actr2a, and Actr2b on day 0 were significantly higher than those on day −4. Thereafter, the expression levels of these receptors numerically, but not significantly, decreased, except for the expression level of Actr2b on day 1. Expression of Alk1 and Alk6 were not detected during adipogenesis of 3T3-L1 cells (data not shown). Because the receptors that transmit signals from TGF-βs, activins, and BMPs [14,22] were expressed in differentiated adipocytes on day 8, we determined that 3T3-L1 adipocytes were suitable for an examination of the role of these TGF-β family members in adipocytes.
2.2. RNA isolation and RT-qPCR Total RNA isolation, cDNA synthesis, and RT-qPCR were conducted as described by Asano et al. [3]. The oligonucleotide primers for activin receptor type II (Actr2) a, Actr2b, activin receptor-like kinases (Alk) 1 to Alk6, BMP receptor type II (Bmpr2), fatty acid-binding protein (Fabp) 4, Pai-1, peroxisome proliferator-activated receptor (Ppar) γ2, resistin, and TGF-β receptor type II (Tβr2) were described previously [25,26,48]. The other oligonucleotide primers are presented in Supplementary Table 1. The cycle threshold (Ct) value was determined, and the abundance of gene transcripts was analyzed using the ▵▵Ct method, with normalization to the level of TATA-binding protein (Tbp) expression [11]. The expression level on day −4 in cells untreated with insulin or any member of the TGF-β family was set at 100.
3.2. Modulation of the expression of genes related to adipocyte differentiation and function by TGF-β family members Thirty-three members of the TGF-β family are recognized in mammals [24]. Among them, we evaluated the exogenous effects of TGF-β1, activin A, activin B, BMP4, and BMP7. TGF-β family consists of three subgroups, i.e., TGF-β group, activin group and BMP group [14,22]. TGF-β1 is frequently used as a member of TGF-β subgroup, whereas activin A is used as a representative member of activin subgroup. Furthermore, BMP4 and BMP7 are examined as BMP that
2.3. Metabolomic analysis A metabolomic analysis was performed by gas chromatographymass spectrometry (GC–MS) analysis as described previously [29]. The relative contents of metabolites were calculated as a ratio to the peak 2
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
ab ab
ab b
ab ab
ab
100 0
2
day
4
6
8
F
Tβr2 800
a
600
ab
400
c
ab ab
abc
bc
bc
-4 -2 0
c
2
day
4
2
day
4
6
8
ab ab
400 200
b ab
ab ab
ab
-4 -2 0
G
800
ab
600 400
b
ab
ab ab
b
ab
ab
0 6
a
600
8
Actr2a a
1000
200
800
ab
0 -4 -2 0
relative mRNA level
relative mRNA level
E
0
200 0
-4 -2 0
200
400
relative mRNA level
200
600
D
Alk4
2
day
4
6
ab
100
bc
c bc
abc abc
c
2
day
4
6
8
200 100
-4 -2 0
0 -4 -2 0
300
8
300 200
400
H
ab
400
Alk5 500
0
Actr2b a
500
relative mRNA level
a ab
300
800
relative mRNA level
400
C
Alk3
relative mRNA level
B
Alk2
relative mRNA level
relative mRNA level
A
2
day
4
6
8
Bmpr2 500
a
400
ab ab ab
300 200 100
b
ab
ab ab
ab
0 -4 -2 0
2
day
4
6
8
-4 -2 0
2
day
4
6
8
Fig. 1. Expression of receptors for TGF-β family members during adipogenesis in 3T3-L1 cells. The 3T3-L1 preadipocytes were differentiated into adipocytes. Expression levels of Alk2 (A), Alk3 (B), Alk4 (C), Alk5 (D), Tβr2 (E), Actr2a (F), Actr2b (G), and Bmpr2 (H) were examined by RT-qPCR. The expression levels were normalized to that of Tbp, and the expression level in cells on day −4 was set to 100. Data are shown as the mean ± SE (n = 4). a, b, c: means that do not have a common letter differ significantly (P < 0.05).
decreased by activin B (Fig. 2C).
is frequently used in cell culture studies to evaluate the role of BMP subgroup. In addition, activin B is suggested to relate to obesity [8]. Thus, the present study examined effects of TGF-β1, activin A, activin B, BMP4 and BMP7 as the TGF-β family members. These ligands use the receptors shown in Fig. 1; TGF-β1 uses Alk5 and Tβr2, and activin A and activin B signal via Alk4 and Actr2a. In addition, BMP4 and BMP7 transmit their signals via Alk2/3, Actr2a, and Bmpr2 [14,22]. To explore roles of the TGF-β family members in adipocytes, 3T3-L1 adipocytes were treated with the individual ligands for 2 days each. Oil Red O staining, which indicates lipid accumulation, was not affected by the TGF-β family members (Supplementary Fig. 1A). Fabp4, a carrier protein for fatty acids, is highly expressed in mature adipocytes [42]; here, Fabp4 expression was unaffected by the TGF-β family members (Supplementary Fig. 1B). Pparγ2 regulates expression of functional genes in mature adipocytes, and the activity of Pparγ2 is related to its expression level [44]. Consistent with the expression of Fabp4, a gene regulated by Pparγ2, the TGF-β family members did not modify the expression level of Pparγ2 either (Supplementary Fig. 1B). Furthermore, the TGF-β family members did not affect the expression levels of genes related not only to lipolysis but also to fatty acid synthesis (Supplementary Fig. 2); adipose triglyceride lipase (Atgl) and hormonesensitive lipase (Hsl) catalyze lipolysis, whereas acetyl-CoA carboxykinase (Acc) and fatty acid synthase (Fas) promote lipogenesis. We also examined adipokine expression in adipocytes treated with the TGF-β family members (Fig. 2). Some adipokines have beneficial effects on health by improving insulin sensitivity; others have harmful effects by promoting insulin resistance and atherosclerosis. For example, adiponectin and leptin increase insulin sensitivity, whereas resistin can induce insulin resistance [37]. Furthermore, Pai-1 has been implicated in the onset of atherosclerosis through the inhibition of fibrinolysis, which is mediated by plasmin inhibition [10,2]. Activin A significantly increased leptin expression (Fig. 2A). In contrast, adiponectin expression was not affected by treatment with any of the TGF-β family members (Supplementary Fig. 3). All of the TGF-β family members, except for activin B, significantly increased the expression of resistin (Fig. 2B). In addition, Pai-1 expression was significantly
3.3. Modulation of metabolite contents by the TGF-β family members treated for 2 h Previously, we revealed that citric acid cycle metabolite contents showed time-dependent changes (oscillations) in response to insulin [29]. In addition, cellular free amino acids generally decreased in response to insulin [29]. Cellular metabolite content in response to insulin and TGF-β family members was next evaluated in 3T3-L1 adipocytes. The metabolomic analysis identified a total of 46 compounds in 3T3-L1 adipocytes treated with insulin and TGF-β family members for either 2 h or 6 h. The hierarchical clustering analyses of metabolite contents indicated that 46 metabolites detected in 2-h samples could be classified into 6 groups, when a correlation coefficient in the dendrogram was set to 0.9; group 1–6 consisted of 1, 3, 2, 2, 21 and 17 metabolites, respectively (Fig. 3). Relative content of a metabolite classified into group 1, i.e., glutamine, exhibited decrease in response to insulin, and the effects of TGF-β1, activin A, and activin B were minimal. In contrast, treatment with BMP4 or BMP7 in the absence of insulin decreased the cellular content, so that the repressive effect of insulin on the cellular content was undetectable. The content of metabolites classified in group 2, i.e., arginine, glutamic acid and phenylalanine, indicated that insulin slightly decreased these contents in the absence of TGF-β family members, but treatment with TGF-β1, activin A, or activin B induced a significant reduction in these contents in response to insulin; in contrast, BMP4 and BMP7 blunted the response to insulin (Fig. 4A–C). Metabolites in group 3, i.e., aspartic acid and cysteine, exhibited that insulin decreased cellular contents, and TGF-β1, activin A, or activin B did not affect the modulation of these cellular contents by insulin. BMP7, but not BMP4, significantly decreased these contents in the absence of insulin, so that insulin did not decrease the cellular content (Fig. 4D and E). The metabolites of group 4 showed that insulin decreased cellular contents, and TGF-β1, activin A, or activin B did not affect the modulation of these cellular contents by insulin. In 3
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Leptin
150 100 50
Ligand: -
Resistin
BMP7
* BMP4
0
Activin A
Ligand: -
200
Activin B
0
250
TGF-β1
100
relative mRNA level
*
BMP7
BMP7
BMP4
Ligand: -
Activin B
0
Activin A
100
BMP4
200
*
*
200
Activin A
300
*
Activin B
*
300
TGF-β1
relative mRNA level
400
TGF-β1
relative mRNA level
C
B
A
Pai-1
Fig. 2. Expression of adipokines in 3T3-L1 adipocytes treated with TGF-β family members. The 3T3-L1 adipocytes were treated with the indicated members of the TGF-β family for 2 d. Expression levels of leptin (A), resistin (B) and Pai-1 (C) were examined by RT-qPCR. The expression levels were normalized to that of Tbp, and expression in the control group was set to 100. Data are shown as the mean ± SE (n = 4). *: P < 0.05, significantly different from the expression level in cells not treated with a TGF-β family member.
and malic acid were categorized as group 6, and the cellular contents were significantly increased by insulin in the presence of at least one member of TGF-β family, except for TGF-β1 (Fig. 5A–C). Relative contents of the other amino acids and citric acid cycle metabolites in response to insulin and the TGF-β family are shown in Supplementary Figs. 4 and 5, respectively. Furthermore, the contents of the other 24 metabolites in cells treated with insulin and TGF-β family members for 2 h are presented in Supplementary Fig. 6.
contrast, BMP4 and BMP7 did not decrease cellular content of metabolites in group 4 in response to insulin. The metabolites classified into group 5 and group 6 indicate that effect of insulin alone was minimal. As for metabolites in group 5, TGF-β1, activin A, or activin B elicited insulin-induced reduction of the cellular content significantly, whereas BMP4 and BMP7 did not modulate responsiveness to insulin. In group 6, insulin tended to increase metabolites in the presence of TGF-β family. Metabolites of citric acid cycle such as succinic acid, fumaric acid
TGF-β family: - TGF-β1 ActA Insulin: - + - + - +
ActB
BMP4
BMP7
- + - + - +
category Q F R E D C A V T I S L K P Y W
group
① ② ③ ④
⑤
C G A
F M
S
⑥
-3 -2 -1 0 1 2 3 Fig. 3. Hierarchical clustering of metabolites detected in 3T3-L1 adipocytes treated with insulin and TGF-β family for 2 h. The 3T3-L1 adipocytes treated with or without insulin and a TGF-β family member for 2 h. Cellular metabolites were comprehensively analyzed by metabolomic analyses, and the relative abundance of metabolites is shown as a heat map. Red: compounds involved in glycolysis and the citric acid cycle; white: sugar or sugar-related compounds; blue: amino acid, amino acid-related compounds, or amines; purple: bases or baserelated compounds; green: free fatty acids; black: low molecular organic acids or acid compounds. A, C, F, M, and S in red indicate aconitic acid, citric acid, fumaric acid, malic acid, and succinic acid, respectively. In addition, blue characters denote amino acids in single-letter code. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Arginine 200
*
150
*
*
P <0.001 0.69 0.03
Ins Fam Ins×Fam
P 0.64 0.98 0.59
Ins Fam Ins×Fam
100
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
0
TGF-β family:
TGF-β1
50
None
arbitrary unit
A
6h
B
Glutamic acid P <0.001 0.25 0.004
Ins Fam Ins×Fam
150
*
*
ActA
100
Ins Fam Ins×Fam
*
*
TGF-β1
arbitrary unit
200
P 0.03 0.41 0.06
50
2h
BMP7
BMP4
ActB
6h Phenylalanine
200
*
150
*
*
*
Ins Fam Ins×Fam
P <0.001 0.60 0.009
Ins Fam Ins×Fam
P 0.37 0.58 0.20
100
BMP7
BMP4
ActB
ActA
GF-β1
None
BMP7
BMP4
ActB
ActA
0
TGF-β family:
GF-β1
50
None
arbitrary unit
C
ActA
TGF-β1
None
BMP7
BMP4
ActB
None
0
TGF-β family:
Fig. 4. Effect of treatment with insulin and TGF-β family member on cellular content of free amino acids in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 2 h or 6 h. The cellular arginine (A), glutamic acid (B), phenylalanine (C), aspartic acid (D), and cysteine (E) contents were quantified, with the content in cells untreated with insulin and a TGF-β family member for each time point set at 100. Black bars: cells untreated with insulin; hatched bars: cells treated with insulin. Data are shown as the mean ± SE (n = 3). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin. †: P < 0.05, significantly different from the content in cells untreated with a TGF-β family member, and either treated or untreated with insulin.
acid categorized in group 2 (Fig. 5A). These results suggest different effect of insulin and TGF-β family on cellular content of metabolites depending on the duration of treatment. The differential pattern of metabolite contents in response to insulin and TGF-β family was also detected on free amino acids; for example, cellular contents of arginine, glutamic acid and phenylalanine categorized as group 5 were generally unchanged by insulin and TGF-β family (Fig. 4A–C); amino acids except for aspartic acid and cysteine (group 4) and glutamine (group 6) were categorized in group 6 (Fig. 6). Cellular aspartic acid and cysteine contents were numerically and/or significantly decreased by insulin in
3.4. Modulation of metabolite contents by the TGF-β family members treated for 6 h The hierarchical clustering analyses of metabolite contents also indicated 6 groups in 6-h samples; group 1–6 consisted of 2, 5, 1, 2, 34 and 2 metabolites, respectively (Fig. 6). The metabolites in group 1, i.e., fumaric acid and malic acid, indicate that insulin clearly increased cellular contents, and effects of TGF-β family and the interaction of insulin and TGF-β family were not significant (Fig. 5B and C). Similar results on ANOVA were also detected on cellular content of succinic 5
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Aspartic acid
D arbitrary unit
250 Ins Fam Ins×Fam
200 150
*
*
100
P <0.001 0.03 0.01
Ins Fam Ins×Fam
P 0.19 0.32 0.57
*
*
50
†
2h
BMP7
BMP4
6h
Cysteine 500 Ins Fam Ins×Fam
400
P <0.001 <0.001 <0.001
*
Ins Fam Ins×Fam
P 0.03 0.33 0.43
300
*
200
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
† ActB
TGF-β family:
*
*
ActA
0
TGF-β1
100
*
None
arbitrary unit
E
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
None
0
TGF-β family:
6h Fig. 4. (continued)
4. Discussion
the presence of activin A or activin B (Fig. 4D and E). Relative contents of the other amino acids and citric acid cycle metabolites in response to insulin and the TGF-β family treated for 6 h are shown in Supplementary Figs. 7 and 8, respectively. Furthermore, the contents of the other 24 metabolites in cells treated with insulin and TGF-β family members for 6 h are presented in Supplementary Fig. 9.
TGF-β family members are known to modulate adipogenesis; TGFβ1 and activin A act as potent inhibitors of adipogenesis, whereas BMP4 and BMP7 stimulate adipogenesis [9,30,16,17]. In contrast, the function of TGF-β family members in differentiated adipocytes is largely unknown. The present study revealed the following: (1) Differentiated adipocytes expressed receptors for TGF-β family members. (2) The TGFβ family members modulated the expression of adipokines, with activin A increasing leptin expression and activin B decreasing expression of Pai-1. In addition, resistin expression increased in response to all TGF-β family members except activin B. (3) Time-dependent effects of the TGF-β family members on cellular metabolite contents were detected; treatment with TGF-β1, activin A, or activin B for 2 h, but not 6 h, exaggerated the reductions in arginine, glutamic acid, and phenylalanine contents by insulin, whereas BMP4 and BMP7 blunted the insulininduced reduction in amino acid contents. (4) Treatment with activin A, activin B, BMP4, or BMP7 for 2 h potentiated the cellular accumulation of succinic acid, fumaric acid, and malic acid in response to insulin. Our results indicate that the TGF-β family members also modulated adipocyte function in a ligand- and time-dependent manner. The multifunctions of the TGF-β family depending on cell stage may enable to regulate metabolism in adipose tissues discretely. Previous studies have indicated that TGF-β1 released from adipose tissue is positively correlated with body mass index [13], and a positive correlation between adipose TGF-β1 expression and body mass index was also observed [1]. Furthermore, the expression level of inhibin βA, a constituent of activin A, was significantly higher in the fat deposits of obese humans than in those of lean humans [50]. The present study
3.5. Modulation of expression level of genes related to amino acid catabolism by the TGF-β family members Provided that free amino acid contents were modulated by insulin and TGF-β family, we examined the expression levels of genes related to amino acid catabolism (Fig. 7). Aspartate aminotransferase (Aat) catalyzes the reversible transfer of α-amino groups between aspartic acid and glutamic acid [36]. Branched chain aminotransferase 2 (Bcat2) catalyzes catabolism of branched chain amino acids to branched chain α-keto acids, which are further metabolized to acyl-CoA by branched chain ketoacid dehydrogenase (Bckdh) [6]. Insulin increased the expression of Aat-1 (cytosolic Aat) in the absence of a TGF-β family member, whereas the expression levels of Bcat2 and Bckdha (α polypeptide of Bckdh) decreased with insulin. Treatment with TGFβ1, activin A, activin B, or BMP4 decreased the insulin-induced Aat-1 expression, and activin A also downregulated the expression of Aat-1, even without insulin. In addition, TGF-β1 and BMP7 decreased and increased the expression of Aat-2 and Bcat2, respectively, in the presence of insulin.
6
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Succinic acid
A
Ins Fam Ins×Fam
200
P 0.03 0.03 0.76
* *
P <0.001 0.87 0.20
*
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
0
TGF-β family:
6h
B
Fumaric acid 600
arbitrary unit
*
Ins Fam Ins×Fam
100
None
arbitrary unit
300
Ins Fam Ins×Fam
400
P <0.001 0.02 0.83
*
*
*
*
Ins Fam Ins×Fam
P <0.001 0.52 0.29
* 200
*
*
*
2h
BMP7
BMP4
ActB
6h Malic acid
C 800
Ins Fam Ins×Fam
600
P <0.001 0.04 0.91
*
*
P <0.001 0.53 0.30
2h
BMP7
BMP4
ActB
ActA
TGF-β1
* None
ActB
*
BMP7
*
BMP4
* ActA
TGF-β1
0
Ins Fam Ins×Fam
*
200
TGF-β family:
*
*
400
None
arbitrary unit
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
None
0
TGF-β family:
6h
Fig. 5. Effect of treatment with insulin and TGF-β family members on citric acid cycle metabolite contents in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 2 h. The cellular succinic acid (A), fumaric acid (B), and malic acid (C) contents were quantified, with the content in cells untreated with insulin and a TGF-β family member for each time point set at 100. Black bars: cells untreated with insulin; hatched bars: cells treated with insulin. Data are shown as the mean ± SE (n = 3). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin.
growth factors potentially act as undesirable adipokines in terms of their health effects. In the present study, Pai-1 expression was not affected by TGF-β in 3T3-L1 adipocytes; TGF-β has been shown to induce Pai-1 expression in several cell types [21,25,20,26]. In addition, previous studies also showed that expression level of Pai-1 was clearly increased in 3T3-L1 adipocytes treated with TGF-β for 3 h [27], which was dose-dependent of TGF-β [32]. However, the gene induction of Pai-1 was transient, and TGF-β treatment for 24 h hardly affected Pai-1 expression [32]. The present study examined gene expression in 3T3-L1 adipocytes treated with TGF-β family for 2 d. Chronic treatment with TGF-β1 is likely to lead to the failure of Pai-1 gene induction in 3T3-L1 adipocytes.
indicated that TGF-β1 and activin A increased expression of resistin in adipocytes. Because the expression level of resistin in adipose tissue was found to be higher in obese humans than in lean humans [33], the increased expression of resistin may have been mediated by TGF-β1 and activin A. However, unlike the effects in mice [18], non-adipocyte resident inflammatory cells mainly contribute to resistin expression in adipose tissues in humans [33]. Therefore, the contribution of resistin expression in adipocytes, induced by TGF-β1 or activin A, may be minor in human adipose tissues. Nevertheless, because resistin has been implicated in various diseases, including diabetes and cardiovascular disease [37,34], TGF-β1 and activin A produced in the adipose tissues of obese subjects may increase resistin expression in adipocytes; these 7
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
TGF-β family: - TGF-β1 ActA Insulin: - + - + - +
ActB
BMP4
BMP7
- + - + - +
category F M C S
D C A F K R G I V T P S L Y
group
① ② ③ ④
⑤
A
E W Q
⑥
-3 -2 -1 0 1 2 3 Fig. 6. Hierarchical clustering of metabolites detected in 3T3-L1 adipocytes treated with insulin and TGF-β family for 6 h. The 3T3-L1 adipocytes treated with or without insulin and a TGF-β family member for 6 h. Cellular metabolites were comprehensively analyzed by metabolomic analyses, and the relative abundance of metabolites is shown as a heat map. Red: compounds involved in glycolysis and the citric acid cycle; white: sugar or sugar-related compounds; blue: amino acid, amino acid-related compounds, or amines; purple: bases or baserelated compounds; green: free fatty acids; black: low molecular organic acids or acid compounds. A, C, F, M, and S in red indicate aconitic acid, citric acid, fumaric acid, malic acid, and succinic acid, respectively. In addition, blue characters denote amino acids in single-letter code. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
also higher in obese subjects than in lean subjects [8], which may reflect a defensive reaction to the harmful effects of obesity. Leptin is another adipokine that regulates adipose tissue mass as well as metabolic, reproductive, neuroendocrine, and immune functions [15,23]. Recessive mutations in the leptin gene are associated
100
P Ins <0.001 Fam <0.001 Ins×Fam 0.10
* †
†
P Ins <0.001 Fam 0.07 Ins×Fam 0.91
* †
* *
Ins Fam Ins×Fam
* *
* *
Aat-1
Bcat2
Bckdha
BMP7
ActB
BMP4
ActA
None
TGF-β1
BMP7
ActB
BMP4
ActA
None
Aat-2
TGF-β1
BMP7
ActB
BMP4
ActA
None
TGF-β1
BMP7
ActB
BMP4
ActA
None
TGF-β1
0
TGF-β family:
P 0.64 0.06 0.40
BMP7
* * * † † † †
P 0.800 0.002 0.32
ActB
*
Ins Fam Ins×Fam
BMP4
200
*
ActA
300
P Ins <0.001 Fam <0.001 Ins×Fam 0.02
None
relative mRNA level
400
TGF-β1
Unlike activin A, activin B did not increase resistin expression; rather, it decreased Pai-1 expression. Pai-1 is linked to the onset of atherosclerosis, as well as insulin resistance [10,2]. Thus, the activin Bmediated response in adipocytes could be beneficial to health. The expression level of inhibin βB, a constituent molecule of activin B, was
Bckdhb
Fig. 7. Effect of treatment with insulin and TGF-β family members on expression level of genes related to amino acid catabolism in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 6 h. Expression levels of Aat-1, Aat-2, Bcat2, Bckdha, and Bckdhb examined by RT-qPCR. Expression levels normalized to that of Tbp, and the expression level in the control group was set at 100. Data are shown as the mean ± SE (n = 4). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin. †: P < 0.05, significantly different from the content in cells untreated with a TGF-β family member, and either treated or untreated with insulin.
8
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
with extreme obesity in mice and in some humans, and leptin has been used successfully to treat obesity, indicating an anti-obesity role for leptin [15,23]. The present study revealed that activin A increased the expression of leptin in adipocytes. Therefore, activin A may enhance the expression of molecules with anti-obesity activities, which suggests that activin A is a desirable adipokine for health. The physiological roles of activin A in adipose tissues should be evaluated in future studies, because it simultaneously increases the expression of resistin and has adverse effects on health, as described above. Insulin stimulates fatty acid synthesis partly by increasing the expression of fatty acid synthase in adipocytes [40,4]. Thus, the results showing that treatment with insulin transiently (∼2 h) led to general decrease in cellular amino acid contents might be a response, in part, to the stimulation of amino acid catabolism for fatty acid synthesis. In fact, the expression of Aat-1 increased in response to insulin. However, altered expression of Aat-1 could not explain the modulation of insulininduced amino acid contents by TGF-β family members. Insulin induced the upregulation of Aat-1 expression in adipocytes, except for that in activin A-treated cells, but insulin did not affect the cellular amino acid contents in BMP4- or BMP7-treated cells. TGF-β family members may post-transcriptionally regulate genes related to amino acid catabolism. Alternatively, considering that insulin also stimulates protein synthesis in adipocytes [41], the insulin-induced reduction of free amino acids may result, in part, from the stimulation of protein synthesis; TGF-β family may modulate protein synthesis. Insulin significantly increased the citric acid cycle metabolite contents in adipocytes in response to treatment with activin A, activin B, BMP4, or BMP7 for 2 h. In contrast, treatment with TGF-β family members for 6 h did not affect the insulin-induced accumulation of the same metabolites. These results suggest that tested members of the TGFβ family, except for TGF-β1, transiently enhanced the response to insulin. Our previous study revealed that the citric acid cycle metabolite contents of adipocytes exhibited periodic changes with a 2-h cycle [29]. Thus, TGF-β family members appear to modulate insulin-induced oscillations in adipocytes. The present study unbiasedly and comprehensively explored regulatory changes in metabolite content in response to TGF-β family in the presence or absence of relatively high concentration of insulin (10 μg/mL) in 3T3-L1 adipocytes; changes in metabolite content in 3T3L1 adipocytes treated with 20 nM (0.12 μg/mL) of insulin were comparable with those in 3T3-L1 treated with 10 μg/mL of insulin (data not shown). Future studies are needed to know the mechanism underlying the regulation. Cross-talk between insulin and TGF-β family may lead to discrete regulation of cell metabolism; insulin modulated TGF-β signaling in NMuMG epithelial cells and mouse embryo fibroblasts [7], In addition, effects of the TGF-β family on cell response should be clarified in the presence of low dose of insulin.
[2] [3]
[4] [5]
[6] [7]
[8]
[9]
[10] [11]
[12]
[13] [14] [15] [16] [17]
[18]
[19] [20]
[21]
[22] [23]
[24]
Conflict of interest [25]
All authors have no conflict of interest. Acknowledgement
[26]
This work was supported by a Grant-in-Aid for Scientific Research (26450442) from The Japan Society for the Promotion of Science.
[27]
Appendix A. Supplementary material
[28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2017.05.01.
[29] [30]
References
[31]
[1] M.C. Alessi, D. Bastelica, P. Morange, B. Berthet, I. Leduc, M. Verdier, O. Geel, I. Juhan-Vague, Plasminogen activator inhibitor 1, transforming growth factor-β1,
9
and BMI are closely associated in human adipose tissue during morbid obesity, Diabetes 49 (2000) 1374–1380. M.C. Alessi, M. Poggi, I. Juhan-Vague, Plasminogen activator inhibitor-1, adipose tissue and insulin resistance, Curr. Opin. Lipidol. 18 (2007) 240–245. H. Asano, T. Yamada, O. Hashimoto, T. Umemoto, R. Sato, S. Ohwatari, Y. Kanamori, T. Terachi, M. Funaba, T. Matsui, Diet-induced changes in Ucp1 expression in bovine adipose tissues, Gen. Comp. Endocrinol. 184 (2013) 87–92. L.H. Baumgard, G.J. Hausman, M.V. Sanz Fernandez, Insulin: pancreatic secretion and adipocyte regulation, Domest. Anim. Endocrinol. 54 (2016) 76–84. J. Bolinder, A. Lindblad, P. Engfeldt, P. Arner, Studies of acute effects of insulin-like growth factors I and II in human fat cells, J. Clin. Endocrinol. Metab. 65 (1987) 732–737. J.T. Brosnan, M.E. Brosnan, Branched-chain amino acids: enzyme and substrate regulation, J. Nutr. 136 (1 Suppl) (2006) 207S–211S. E.H. Budi, B.P. Muthusamy, R. Derynck, The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors, Sci. Signal. 8 (2015) ra96. L.M. Carlsson, P. Jacobson, A. Walley, P. Froguel, L. Sjöström, P.A. Svensson, K. Sjöholm, ALK7 expression is specific for adipose tissue, reduced in obesity and correlates to factors implicated in metabolic disease, Biochem. Biophys. Res. Commun. 382 (2009) 309–314. L. Choy, R. Derynck, Transforming growth factor-β inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function, J. Biol. Chem. 278 (2003) 9609–9619. C. Dellas, D.J. Loskutoff, Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease, Thromb. Haemost. 93 (2005) 631–640. E.M. Duran, P. Shapshak, J. Worley, A. Minagar, F. Ziegler, S. Haliko, I. MoleonBorodowsky, P.A. Haslett, Presenilin-1 detection in brain neurons and FOXP3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase PCR using the deltadeltaCt method, Front Biosci. 10 (2005) 2955–2965. M. Elsen, S. Raschke, N. Tennagels, U. Schwahn, T. Jelenik, M. Roden, T. Romacho, J. Eckel, BMP4 and BMP7 induce the white-to-brown transition of primary human adipose stem cells, Am. J. Physiol. Cell Physiol. 306 (2014) C431–C440. J.N. Fain, D.S. Tichansky, A.K. Madan, Transforming growth factor β 1 release by human adipose tissue is enhanced in obesity, Metabolism 54 (2005) 1546–1551. X.H. Feng, R. Derynck, Specificity and versatility in TGF- β signaling through Smads, Annu. Rev. Cell Dev. Biol. 21 (2005) 659–693. J.M. Friedman, Leptin at 14 y of age: an ongoing story, Am. J. Clin. Nutr. 89 (2009) 973S–979S. S. Hirai, M. Yamanaka, H. Kawachi, T. Matsui, H. Yano, Activin A inhibits differentiation of 3T3-L1 preadipocyte, Mol. Cell. Endocrinol. 232 (2005) 21–26. H. Huang, T.J. Song, X. Li, L. Hu, Q. He, M. Liu, M.D. Lane, Q.Q. Tang, BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage, Proc. Natl. Acad Sci. USA 106 (2009) 12670–12675. K.H. Kim, K. Lee, Y.S. Moon, H.S. Sul, A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation, J. Biol. Chem. 276 (2001) 11252–11256. N. Klöting, M. Blüher, Adipocyte dysfunction, inflammation and metabolic syndrome, Rev. Endocr. Metab. Disord. 15 (2014) 277–287. R.M. Kortlever, J.H. Nijwening, R. Bernards, Transforming growth factor-β requires its target plasminogen activator inhibitor-1 for cytostatic activity, J. Biol. Chem. 283 (2008) 24308–24313. M. Laiho, F.M. Weis, F.T. Boyd, R.A. Ignotz, J. Massagué, Responsiveness to transforming growth factor-β (TGF-β) restored by genetic complementation between cells defective in TGF-β receptors I and II, J. Biol. Chem. 266 (1991) 9108–9112. K. Miyazono, Y. Kamiya, M. Morikawa, Bone morphogenetic protein receptors and signal transduction, J. Biochem. 147 (2010) 35–51. H.S. Moon, M. Dalamaga, S.Y. Kim, S.A. Polyzos, O.P. Hamnvik, F. Magkos, J. Paruthi, C.S. Mantzoros, Leptin's role in lipodystrophic and nonlipodystrophic insulin-resistant and diabetic individuals, Endocr. Rev. 34 (2013) 377–412. M. Morikawa, R. Derynck, K. Miyazono, TGF-β and the TGF-β family: contextdependent roles in cell and tissue physiology, Cold Spring Harb Perspect Biol. 8 (5) (2016), http://dx.doi.org/10.1101/cshperspect.a021873 pii: a021873. M. Murakami, T. Ikeda, T. Saito, K. Ogawa, Y. Nishino, K. Nakaya, M. Funaba, Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor- β, activin A and microphthalmia-associated transcription factor, Cell. Signal. 18 (2006) 256–265. M. Murakami, H. Kawachi, K. Ogawa, Y. Nishino, M. Funaba, Receptor expression modulates the specificity of transforming growth factor- β signaling pathways, Genes Cells 14 (2009) 469–482. M. Pandey, D.J. Loskutoff, F. Samad, Molecular mechanisms of tumor necrosis factor-α-mediated plasminogen activator inhibitor-1 expression in adipocytes, FASEB J. 19 (2005) 1317–1319. S.W. Qian, Y. Tang, X. Li, Y. Liu, Y.Y. Zhang, H.Y. Huang, R.D. Xue, H.Y. Yu, L. Guo, H.D. Gao, Y. Liu, X. Sun, Y.M. Li, W.P. Jia, Q.Q. Tang, BMP4-mediated brown fatlike changes in white adipose tissue alter glucose and energy homeostasis, Proc. Natl. Acad. Sci. USA 110 (2013) E798–E807. Y. Qiao, S. Tomonaga, T. Matsui, M. Funaba, Modulation of the cellular content of metabolites in adipocytes by insulin, Mol. Cell. Endocrinol. 424 (2016) 71–80. A. Rebbapragada, H. Benchabane, J.L. Wrana, A.J. Celeste, L. Attisano, Myostatin signals through a transforming growth factor β-like signaling pathway to block adipogenesis, Mol. Cell. Biol. 23 (2003) 7230–7242. E.D. Rosen, B.M. Spiegelman, Adipocytes as regulators of energy balance and glucose homeostasis, Nature 444 (2006) 847–853.
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Biochem. 81 (2012) 715–736. [43] Q.Q. Tang, T.C. Otto, M.D. Lane, Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage, Proc. Natl. Acad. Sci. USA 101 (2004) 9607–9611. [44] P. Tontonoz, B.M. Spiegelman, Fat and beyond: the diverse biology of PPAR γ, Annu. Rev. Biochem. 77 (2008) 289–312. [45] M.E. Trujillo, P.E. Scherer, Adipose tissue-derived factors: impact on health and disease, Endocr. Rev. 27 (2006) 762–778. [46] Y.H. Tseng, E. Kokkotou, T.J. Schulz, T.L. Huang, J.N. Winnay, C.M. Taniguchi, T.T. Tran, R. Suzuki, D.O. Espinoza, Y. Yamamoto, M.J. Ahrens, A.T. Dudley, A.W. Norris, R.N. Kulkarni, C.R. Kahn, New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure, Nature 454 (2008) 1000–1004. [47] R. Xue, Y. Wan, S. Zhang, Q. Zhang, H. Ye, Y. Li, Role of bone morphogenetic protein 4 in the differentiation of brown fat-like adipocytes, Am. J. Physiol. Endocrinol. Metab. 306 (2014) E363–E372. [48] H. Yoshida, Y. Kanamori, H. Asano, O. Hashimoto, M. Murakami, T. Kawada, T. Matsui, M. Funaba, Regulation of brown adipogenesis by the Tgf-β family: involvement of Srebp1c in Tgf-β- and Activin-induced inhibition of adipogenesis, Biochem. Biophys. Acta. 1830 (2013) 5027–5035. [49] N. Zamani, C.W. Brown, Emerging roles for the transforming growth factor- β superfamily in regulating adiposity and energy expenditure, Endocr. Rev. 32 (2011) 387–403. [50] L.E. Zaragosi, B. Wdziekonski, P. Villageois, M. Keophiphath, M. Maumus, T. Tchkonia, V. Bourlier, T. Mohsen-Kanson, A. Ladoux, C. Elabd, M. Scheideler, Z. Trajanoski, Y. Takashima, E.Z. Amri, D. Lacasa, C. Sengenes, G. Ailhaud, K. Clément, A. Bouloumie, J.L. Kirkland, C. Dani, Activin a plays a critical role in proliferation and differentiation of human adipose progenitors, Diabetes 59 (2010) 2513–2521.
[32] F. Samad, K. Yamamoto, M. Pandey, D.J. Loskutoff, Elevated expression of transforming growth factor-β in adipose tissue from obese mice, Mol. Med. 3 (1997) 37–48. [33] D.B. Savage, C.P. Sewter, E.S. Klenk, D.G. Segal, A. Vidal-Puig, R.V. Considine, S. O'Rahilly, Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-γ action in humans, Diabetes 50 (2001) 2199–2202. [34] D.R. Schwartz, M.A. Lazar, Human resistin: found in translation from mouse to man, Trends Endocrinol. Metab. 22 (2011) 259–265. [35] M. Shimizu, F. Torti, R.A. Roth, Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation, Biochem. Biophys. Res. Commun. 137 (1986) 552–558. [36] S. Sookoian, C.J. Pirola, Alanine and aspartate aminotransferase and glutaminecycling pathway: their roles in pathogenesis of metabolic syndrome, World J. Gastroenterol. 18 (2012) 3775–3781. [37] C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes, Nature 409 (2001) 307–312. [38] M. Suenaga, N. Kurosawa, H. Asano, Y. Kanamori, T. Umemoto, H. Yoshida, M. Murakami, H. Kawachi, T. Matsui, M. Funaba, Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation, Cytokine 64 (2013) 138–145. [39] M. Suenaga, T. Matsui, M. Funaba, BMP Inhibition with dorsomorphin limits adipogenic potential of preadipocytes, J. Vet. Med. Sci. 72 (2010) 373–377. [40] H.S. Sul, M.J. Latasa, Y. Moon, K.H. Kim, Regulation of the fatty acid synthase promoter by insulin, J. Nutr. 130 (2000) 315S–320S. [41] S.X. Tan, K.H. Fisher-Wellman, D.J. Fazakerley, Y. Ng, H. Pant, J. Li, C.C. Meoli, A.C. Coster, J. Stöckli, D.E. James, Selective insulin resistance in adipocytes, J. Biol. Chem. 290 (2015) 11337–11348. [42] Q.Q. Tang, M.D. Lane, Adipogenesis: from stem cell to adipocyte, Annu. Rev.
10
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Modulation of adipocyte function by the TGF-β family Yuhang Qiao, Shozo Tomonaga, Masashi Suenaga, Tohru Matsui, Masayuki Funaba
⁎
Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
A R T I C L E I N F O
A B S T R A C T
Keywords: TGF-β family Adipocyte Adipokine Metabolome analysis
Members of the transforming growth factor-β (TGF-β) family are known to regulate the commitment of mesenchymal stem cells to an adipocyte lineage and the differentiation of preadipocytes to adipocytes. However, the roles of TGF-β family members in mature adipocytes are not known. The present study explored the modulation of cell function in 3T3-L1 adipocytes treated with the TGF-β family members TGF-β1, activin A, activin B, bone morphogenetic protein (BMP) 4, and BMP7. These TGF-β family members modulated the expression of some adipokines; activin A upregulated leptin expression, and activin B downregulated the expression of plasminogen activator inhibitor-1. All examined TGF-β family members, except for activin B, increased the expression of resistin. The TGF-β family members also modulated the free amino acid content in cells. Insulin-induced reductions in the cellular content of arginine, glutamic acid, and phenylalanine were exaggerated by TGF-β1, activin A, and activin B. In contrast, the cellular glutamine content decreased with BMP4 or BMP7 treatment, inhibiting the response to insulin. In addition, although the cellular citric acid cycle metabolite contents were not generally affected by TGF-β family members alone, activin A, activin B, BMP4, and BMP7 potentiated the insulin-induced accumulation of the metabolites. The present results suggest that TGF-β family members regulate adipocyte function by modulating adipokine expression and cell metabolism in differentiated adipocytes.
1. Introduction
the uptake of glucose [35,5] and amino acids [35] by adipocytes. Our previous study evaluated changes in adipocyte metabolite content in response to insulin by a metabolomic analysis; insulin increased the citric acid cycle metabolites in a 2-h cycle and decreased the free amino acid contents of the cells [29]. Because the metabolite content in cells reflects the net results of insulin-mediated cell responses, these results are useful in understanding the global modulation of adipocyte metabolism induced by insulin. Because there is cross-talk between components of the TGF-β and other signaling pathways in many biological systems [14,24], Guo and Wang (2009), the action of insulin on adipocyte metabolism may be modulated by TGF-β family members. The objective of this study was to clarify the role of TGF-β family members in the function of differentiated adipocytes. We evaluated the effects of TGF-β1, activin A, activin B, BMP4, and BMP7 on the expression levels of genes related to adipocyte function. In addition, cellular metabolite contents were explored to determine whether the insulin-mediated cell responses were modulated by TGF-β family members.
Adipocytes accumulate excess energy as triacylglycerol through lipogenesis and provide fatty acids to peripheral tissues, depending on the energy status of the tissues, through lipolysis [31]. In addition to regulating energy metabolism, adipocytes maintain homeostasis throughout the body by producing adipokines such as adiponectin, leptin, resistin, and plasminogen activator inhibitor-1 (Pai-1) [45,19]. Members of the transforming growth factor-β (TGF-β) family regulate a wide variety of biological processes, including adipogenesis (Derynck et al., 2008). Bone morphogenetic protein (BMP) 2 and BMP4 induce commitment of mesenchymal stem cells to an adipocyte lineage [43,17]. BMP4 expression in preadipocytes is required for differentiation to adipocytes [39,38], whereas TGF-β and activin A inhibit the differentiation of preadipocytes [9,16]. Despite well-established evidence of the role of members of the TGF-β family in adipogenesis, little information is available on the modulation of adipocyte function by members of the TGF-β family. Epidemiological studies have shown the relationship between expression levels of TGF-β family members in adipose tissue and adiposity [1,8,50,49,28], which may indicate (dys) regulation of adipocyte function by members of the TGF-β family. Insulin is known to modulate adipocyte function. Insulin activates
⁎
Corresponding author at: Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa Oiwakecho, Kyoto 606-8502, Japan. E-mail address: [email protected] (M. Funaba).
http://dx.doi.org/10.1016/j.cyto.2017.05.011 Received 26 January 2017; Received in revised form 1 May 2017; Accepted 12 May 2017 1043-4666/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Qiao, Y., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.05.011
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
2. Materials and methods
area of 2-isopropylmalic acid, an GC–MS internal control, and normalized by the protein concentration [29]. The content of each metabolite in cells untreated with insulin or a TGF-β family member, i.e., control cells, was set at 100.
2.1. Cell culture The 3T3-L1 preadipocytes used in this study were cultured and differentiated as described previously [29]. The cells were grown and maintained in growth medium, i.e., Dulbecco’s modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics. Two days after the cells reached confluence (day 0), cells were cultured in a growth medium supplemented with differentiation inducers (IBMX [0.5 mM], dexamethasone [0.25 μM], and insulin [10 μg/ml]) for 2 days, followed by cultivation in a growth medium supplemented with insulin (5 μg/ml). By day 8, more than 95% of 3T3L1 cells had differentiated into adipocytes, based on appearance. To evaluate the changes in gene expression of TGF-β family receptors over time, cells were harvested on days −4, −2, −1, 0, 1, 2, 4, 6, and 8. Gene expression was examined by real-time reverse transcriptionquantitative PCR (RT-qPCR). To evaluate the lipid accumulation and expression of genes related to adipocyte function, TGF-β family members were added to the growth medium supplemented with insulin for 48 h, i.e., days 8–10, at the following concentrations: 200 pM for TGF-β1 (Becton Dickinson, Franklin Lakes, NJ, USA) and 3 nM for activin A, activin B, BMP4, and BMP7 (R & D Systems, Minneapolis, MN, USA). Because members of the TGF-β family function as local factors, plasma level of the TGF-β family does not necessarily reflect physiological concentration in affected cells. Concentrations of TGF-β1 (200 pM), activin A (3 nM), activin B (3 nM), BMP4 (3 nM) and BMP7 (3 nM) are within the range that is frequently used in cell culture studies [9,46,26,48,12,47]. On day 10, lipid accumulation was evaluated by Oil Red O staining, and images were obtained by scanning stained wells (GT-9400UF; Epson, Tokyo, Japan). In addition, the expression of genes related to adipocyte differentiation and function was evaluated by RT-qPCR. For the metabolomic analyses, 3T3-L1 adipocytes on day 8 were cultured in DMEM without insulin for 24 h, i.e., days 8–9, followed by treatment with or without insulin (10 μg/ml) and with TGF-β family members for 2 h or 6 h. TGF-β family members were applied in the concentrations given above. We previously showed that the cellular citric acid cycle metabolite contents exhibited periodic changes with a 2-h cycle [29]. It is possible that the TGF-β family members modulate insulin-induced cellular contents in a time-dependent manner. Therefore, we evaluated the metabolite contents in adipocytes treated with insulin and TGF-β family members for either 2 h or 6 h. In addition, at 6 h, the expression levels of genes related to amino acid catabolism were evaluated by RT-qPCR.
2.4. Data analysis To visualize changes in the cellular metabolite content in response to insulin and TGF-β family members, a heat map was prepared and a hierarchical cluster analysis was performed as described previously [29]. The threshold for grouping was set at a correlation coefficient of 0.9 in the dendrogram, and it was grouped to be as few as possible. 2.5. Statistical analyses Data are expressed as the mean ± standard error (SE). The expression levels of genes were log-transformed to provide an approximation of a normal distribution before analysis. Data used in the timecourse analysis of the expression of receptors in the TGF-β family were evaluated by one-way ANOVA, followed by a Tukey-Kramer test. For the expression of adipokines and genes related to adipogenesis and adipocyte function, data were analyzed by one-way ANOVA, considered factor was TGF-β family members. When the effect was significant (P < 0.05), the differences between control cells and TGF-β familytreated cells were examined by Dunnett’s test. For metabolite contents, data were also analyzed by two-way ANOVA. Factors considered in the analysis were insulin, TGF-β family members, and the interaction between insulin and TGF-β family members. When the effect of insulin or the interaction between insulin and TGF-β family members was significant, comparisons between insulin-treated or -untreated cells treated with the respective TGF-β family member were performed by the Student’s t-test. When the effect of TGF-β family members or the interaction of insulin and TGF-β family members was significant, comparisons between cells untreated with a TGF-β family member, as the reference, and TGF-β family member-treated cells that were either treated or untreated with insulin were also performed using the Dunnett’s test. 3. Results 3.1. Expression of receptors for TGF-β family members during adipogenesis At first, we examined the changes in expression levels of receptors for TGF-β family members during adipogenesis in 3T3-L1 cells (Fig. 1). Generally, expression levels of the receptors increased steadily from day −4 to day 0; specifically, the expression levels of Tβr2, Actr2a, and Actr2b on day 0 were significantly higher than those on day −4. Thereafter, the expression levels of these receptors numerically, but not significantly, decreased, except for the expression level of Actr2b on day 1. Expression of Alk1 and Alk6 were not detected during adipogenesis of 3T3-L1 cells (data not shown). Because the receptors that transmit signals from TGF-βs, activins, and BMPs [14,22] were expressed in differentiated adipocytes on day 8, we determined that 3T3-L1 adipocytes were suitable for an examination of the role of these TGF-β family members in adipocytes.
2.2. RNA isolation and RT-qPCR Total RNA isolation, cDNA synthesis, and RT-qPCR were conducted as described by Asano et al. [3]. The oligonucleotide primers for activin receptor type II (Actr2) a, Actr2b, activin receptor-like kinases (Alk) 1 to Alk6, BMP receptor type II (Bmpr2), fatty acid-binding protein (Fabp) 4, Pai-1, peroxisome proliferator-activated receptor (Ppar) γ2, resistin, and TGF-β receptor type II (Tβr2) were described previously [25,26,48]. The other oligonucleotide primers are presented in Supplementary Table 1. The cycle threshold (Ct) value was determined, and the abundance of gene transcripts was analyzed using the ▵▵Ct method, with normalization to the level of TATA-binding protein (Tbp) expression [11]. The expression level on day −4 in cells untreated with insulin or any member of the TGF-β family was set at 100.
3.2. Modulation of the expression of genes related to adipocyte differentiation and function by TGF-β family members Thirty-three members of the TGF-β family are recognized in mammals [24]. Among them, we evaluated the exogenous effects of TGF-β1, activin A, activin B, BMP4, and BMP7. TGF-β family consists of three subgroups, i.e., TGF-β group, activin group and BMP group [14,22]. TGF-β1 is frequently used as a member of TGF-β subgroup, whereas activin A is used as a representative member of activin subgroup. Furthermore, BMP4 and BMP7 are examined as BMP that
2.3. Metabolomic analysis A metabolomic analysis was performed by gas chromatographymass spectrometry (GC–MS) analysis as described previously [29]. The relative contents of metabolites were calculated as a ratio to the peak 2
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
ab ab
ab b
ab ab
ab
100 0
2
day
4
6
8
F
Tβr2 800
a
600
ab
400
c
ab ab
abc
bc
bc
-4 -2 0
c
2
day
4
2
day
4
6
8
ab ab
400 200
b ab
ab ab
ab
-4 -2 0
G
800
ab
600 400
b
ab
ab ab
b
ab
ab
0 6
a
600
8
Actr2a a
1000
200
800
ab
0 -4 -2 0
relative mRNA level
relative mRNA level
E
0
200 0
-4 -2 0
200
400
relative mRNA level
200
600
D
Alk4
2
day
4
6
ab
100
bc
c bc
abc abc
c
2
day
4
6
8
200 100
-4 -2 0
0 -4 -2 0
300
8
300 200
400
H
ab
400
Alk5 500
0
Actr2b a
500
relative mRNA level
a ab
300
800
relative mRNA level
400
C
Alk3
relative mRNA level
B
Alk2
relative mRNA level
relative mRNA level
A
2
day
4
6
8
Bmpr2 500
a
400
ab ab ab
300 200 100
b
ab
ab ab
ab
0 -4 -2 0
2
day
4
6
8
-4 -2 0
2
day
4
6
8
Fig. 1. Expression of receptors for TGF-β family members during adipogenesis in 3T3-L1 cells. The 3T3-L1 preadipocytes were differentiated into adipocytes. Expression levels of Alk2 (A), Alk3 (B), Alk4 (C), Alk5 (D), Tβr2 (E), Actr2a (F), Actr2b (G), and Bmpr2 (H) were examined by RT-qPCR. The expression levels were normalized to that of Tbp, and the expression level in cells on day −4 was set to 100. Data are shown as the mean ± SE (n = 4). a, b, c: means that do not have a common letter differ significantly (P < 0.05).
decreased by activin B (Fig. 2C).
is frequently used in cell culture studies to evaluate the role of BMP subgroup. In addition, activin B is suggested to relate to obesity [8]. Thus, the present study examined effects of TGF-β1, activin A, activin B, BMP4 and BMP7 as the TGF-β family members. These ligands use the receptors shown in Fig. 1; TGF-β1 uses Alk5 and Tβr2, and activin A and activin B signal via Alk4 and Actr2a. In addition, BMP4 and BMP7 transmit their signals via Alk2/3, Actr2a, and Bmpr2 [14,22]. To explore roles of the TGF-β family members in adipocytes, 3T3-L1 adipocytes were treated with the individual ligands for 2 days each. Oil Red O staining, which indicates lipid accumulation, was not affected by the TGF-β family members (Supplementary Fig. 1A). Fabp4, a carrier protein for fatty acids, is highly expressed in mature adipocytes [42]; here, Fabp4 expression was unaffected by the TGF-β family members (Supplementary Fig. 1B). Pparγ2 regulates expression of functional genes in mature adipocytes, and the activity of Pparγ2 is related to its expression level [44]. Consistent with the expression of Fabp4, a gene regulated by Pparγ2, the TGF-β family members did not modify the expression level of Pparγ2 either (Supplementary Fig. 1B). Furthermore, the TGF-β family members did not affect the expression levels of genes related not only to lipolysis but also to fatty acid synthesis (Supplementary Fig. 2); adipose triglyceride lipase (Atgl) and hormonesensitive lipase (Hsl) catalyze lipolysis, whereas acetyl-CoA carboxykinase (Acc) and fatty acid synthase (Fas) promote lipogenesis. We also examined adipokine expression in adipocytes treated with the TGF-β family members (Fig. 2). Some adipokines have beneficial effects on health by improving insulin sensitivity; others have harmful effects by promoting insulin resistance and atherosclerosis. For example, adiponectin and leptin increase insulin sensitivity, whereas resistin can induce insulin resistance [37]. Furthermore, Pai-1 has been implicated in the onset of atherosclerosis through the inhibition of fibrinolysis, which is mediated by plasmin inhibition [10,2]. Activin A significantly increased leptin expression (Fig. 2A). In contrast, adiponectin expression was not affected by treatment with any of the TGF-β family members (Supplementary Fig. 3). All of the TGF-β family members, except for activin B, significantly increased the expression of resistin (Fig. 2B). In addition, Pai-1 expression was significantly
3.3. Modulation of metabolite contents by the TGF-β family members treated for 2 h Previously, we revealed that citric acid cycle metabolite contents showed time-dependent changes (oscillations) in response to insulin [29]. In addition, cellular free amino acids generally decreased in response to insulin [29]. Cellular metabolite content in response to insulin and TGF-β family members was next evaluated in 3T3-L1 adipocytes. The metabolomic analysis identified a total of 46 compounds in 3T3-L1 adipocytes treated with insulin and TGF-β family members for either 2 h or 6 h. The hierarchical clustering analyses of metabolite contents indicated that 46 metabolites detected in 2-h samples could be classified into 6 groups, when a correlation coefficient in the dendrogram was set to 0.9; group 1–6 consisted of 1, 3, 2, 2, 21 and 17 metabolites, respectively (Fig. 3). Relative content of a metabolite classified into group 1, i.e., glutamine, exhibited decrease in response to insulin, and the effects of TGF-β1, activin A, and activin B were minimal. In contrast, treatment with BMP4 or BMP7 in the absence of insulin decreased the cellular content, so that the repressive effect of insulin on the cellular content was undetectable. The content of metabolites classified in group 2, i.e., arginine, glutamic acid and phenylalanine, indicated that insulin slightly decreased these contents in the absence of TGF-β family members, but treatment with TGF-β1, activin A, or activin B induced a significant reduction in these contents in response to insulin; in contrast, BMP4 and BMP7 blunted the response to insulin (Fig. 4A–C). Metabolites in group 3, i.e., aspartic acid and cysteine, exhibited that insulin decreased cellular contents, and TGF-β1, activin A, or activin B did not affect the modulation of these cellular contents by insulin. BMP7, but not BMP4, significantly decreased these contents in the absence of insulin, so that insulin did not decrease the cellular content (Fig. 4D and E). The metabolites of group 4 showed that insulin decreased cellular contents, and TGF-β1, activin A, or activin B did not affect the modulation of these cellular contents by insulin. In 3
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Leptin
150 100 50
Ligand: -
Resistin
BMP7
* BMP4
0
Activin A
Ligand: -
200
Activin B
0
250
TGF-β1
100
relative mRNA level
*
BMP7
BMP7
BMP4
Ligand: -
Activin B
0
Activin A
100
BMP4
200
*
*
200
Activin A
300
*
Activin B
*
300
TGF-β1
relative mRNA level
400
TGF-β1
relative mRNA level
C
B
A
Pai-1
Fig. 2. Expression of adipokines in 3T3-L1 adipocytes treated with TGF-β family members. The 3T3-L1 adipocytes were treated with the indicated members of the TGF-β family for 2 d. Expression levels of leptin (A), resistin (B) and Pai-1 (C) were examined by RT-qPCR. The expression levels were normalized to that of Tbp, and expression in the control group was set to 100. Data are shown as the mean ± SE (n = 4). *: P < 0.05, significantly different from the expression level in cells not treated with a TGF-β family member.
and malic acid were categorized as group 6, and the cellular contents were significantly increased by insulin in the presence of at least one member of TGF-β family, except for TGF-β1 (Fig. 5A–C). Relative contents of the other amino acids and citric acid cycle metabolites in response to insulin and the TGF-β family are shown in Supplementary Figs. 4 and 5, respectively. Furthermore, the contents of the other 24 metabolites in cells treated with insulin and TGF-β family members for 2 h are presented in Supplementary Fig. 6.
contrast, BMP4 and BMP7 did not decrease cellular content of metabolites in group 4 in response to insulin. The metabolites classified into group 5 and group 6 indicate that effect of insulin alone was minimal. As for metabolites in group 5, TGF-β1, activin A, or activin B elicited insulin-induced reduction of the cellular content significantly, whereas BMP4 and BMP7 did not modulate responsiveness to insulin. In group 6, insulin tended to increase metabolites in the presence of TGF-β family. Metabolites of citric acid cycle such as succinic acid, fumaric acid
TGF-β family: - TGF-β1 ActA Insulin: - + - + - +
ActB
BMP4
BMP7
- + - + - +
category Q F R E D C A V T I S L K P Y W
group
① ② ③ ④
⑤
C G A
F M
S
⑥
-3 -2 -1 0 1 2 3 Fig. 3. Hierarchical clustering of metabolites detected in 3T3-L1 adipocytes treated with insulin and TGF-β family for 2 h. The 3T3-L1 adipocytes treated with or without insulin and a TGF-β family member for 2 h. Cellular metabolites were comprehensively analyzed by metabolomic analyses, and the relative abundance of metabolites is shown as a heat map. Red: compounds involved in glycolysis and the citric acid cycle; white: sugar or sugar-related compounds; blue: amino acid, amino acid-related compounds, or amines; purple: bases or baserelated compounds; green: free fatty acids; black: low molecular organic acids or acid compounds. A, C, F, M, and S in red indicate aconitic acid, citric acid, fumaric acid, malic acid, and succinic acid, respectively. In addition, blue characters denote amino acids in single-letter code. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Arginine 200
*
150
*
*
P <0.001 0.69 0.03
Ins Fam Ins×Fam
P 0.64 0.98 0.59
Ins Fam Ins×Fam
100
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
0
TGF-β family:
TGF-β1
50
None
arbitrary unit
A
6h
B
Glutamic acid P <0.001 0.25 0.004
Ins Fam Ins×Fam
150
*
*
ActA
100
Ins Fam Ins×Fam
*
*
TGF-β1
arbitrary unit
200
P 0.03 0.41 0.06
50
2h
BMP7
BMP4
ActB
6h Phenylalanine
200
*
150
*
*
*
Ins Fam Ins×Fam
P <0.001 0.60 0.009
Ins Fam Ins×Fam
P 0.37 0.58 0.20
100
BMP7
BMP4
ActB
ActA
GF-β1
None
BMP7
BMP4
ActB
ActA
0
TGF-β family:
GF-β1
50
None
arbitrary unit
C
ActA
TGF-β1
None
BMP7
BMP4
ActB
None
0
TGF-β family:
Fig. 4. Effect of treatment with insulin and TGF-β family member on cellular content of free amino acids in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 2 h or 6 h. The cellular arginine (A), glutamic acid (B), phenylalanine (C), aspartic acid (D), and cysteine (E) contents were quantified, with the content in cells untreated with insulin and a TGF-β family member for each time point set at 100. Black bars: cells untreated with insulin; hatched bars: cells treated with insulin. Data are shown as the mean ± SE (n = 3). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin. †: P < 0.05, significantly different from the content in cells untreated with a TGF-β family member, and either treated or untreated with insulin.
acid categorized in group 2 (Fig. 5A). These results suggest different effect of insulin and TGF-β family on cellular content of metabolites depending on the duration of treatment. The differential pattern of metabolite contents in response to insulin and TGF-β family was also detected on free amino acids; for example, cellular contents of arginine, glutamic acid and phenylalanine categorized as group 5 were generally unchanged by insulin and TGF-β family (Fig. 4A–C); amino acids except for aspartic acid and cysteine (group 4) and glutamine (group 6) were categorized in group 6 (Fig. 6). Cellular aspartic acid and cysteine contents were numerically and/or significantly decreased by insulin in
3.4. Modulation of metabolite contents by the TGF-β family members treated for 6 h The hierarchical clustering analyses of metabolite contents also indicated 6 groups in 6-h samples; group 1–6 consisted of 2, 5, 1, 2, 34 and 2 metabolites, respectively (Fig. 6). The metabolites in group 1, i.e., fumaric acid and malic acid, indicate that insulin clearly increased cellular contents, and effects of TGF-β family and the interaction of insulin and TGF-β family were not significant (Fig. 5B and C). Similar results on ANOVA were also detected on cellular content of succinic 5
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Aspartic acid
D arbitrary unit
250 Ins Fam Ins×Fam
200 150
*
*
100
P <0.001 0.03 0.01
Ins Fam Ins×Fam
P 0.19 0.32 0.57
*
*
50
†
2h
BMP7
BMP4
6h
Cysteine 500 Ins Fam Ins×Fam
400
P <0.001 <0.001 <0.001
*
Ins Fam Ins×Fam
P 0.03 0.33 0.43
300
*
200
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
† ActB
TGF-β family:
*
*
ActA
0
TGF-β1
100
*
None
arbitrary unit
E
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
None
0
TGF-β family:
6h Fig. 4. (continued)
4. Discussion
the presence of activin A or activin B (Fig. 4D and E). Relative contents of the other amino acids and citric acid cycle metabolites in response to insulin and the TGF-β family treated for 6 h are shown in Supplementary Figs. 7 and 8, respectively. Furthermore, the contents of the other 24 metabolites in cells treated with insulin and TGF-β family members for 6 h are presented in Supplementary Fig. 9.
TGF-β family members are known to modulate adipogenesis; TGFβ1 and activin A act as potent inhibitors of adipogenesis, whereas BMP4 and BMP7 stimulate adipogenesis [9,30,16,17]. In contrast, the function of TGF-β family members in differentiated adipocytes is largely unknown. The present study revealed the following: (1) Differentiated adipocytes expressed receptors for TGF-β family members. (2) The TGFβ family members modulated the expression of adipokines, with activin A increasing leptin expression and activin B decreasing expression of Pai-1. In addition, resistin expression increased in response to all TGF-β family members except activin B. (3) Time-dependent effects of the TGF-β family members on cellular metabolite contents were detected; treatment with TGF-β1, activin A, or activin B for 2 h, but not 6 h, exaggerated the reductions in arginine, glutamic acid, and phenylalanine contents by insulin, whereas BMP4 and BMP7 blunted the insulininduced reduction in amino acid contents. (4) Treatment with activin A, activin B, BMP4, or BMP7 for 2 h potentiated the cellular accumulation of succinic acid, fumaric acid, and malic acid in response to insulin. Our results indicate that the TGF-β family members also modulated adipocyte function in a ligand- and time-dependent manner. The multifunctions of the TGF-β family depending on cell stage may enable to regulate metabolism in adipose tissues discretely. Previous studies have indicated that TGF-β1 released from adipose tissue is positively correlated with body mass index [13], and a positive correlation between adipose TGF-β1 expression and body mass index was also observed [1]. Furthermore, the expression level of inhibin βA, a constituent of activin A, was significantly higher in the fat deposits of obese humans than in those of lean humans [50]. The present study
3.5. Modulation of expression level of genes related to amino acid catabolism by the TGF-β family members Provided that free amino acid contents were modulated by insulin and TGF-β family, we examined the expression levels of genes related to amino acid catabolism (Fig. 7). Aspartate aminotransferase (Aat) catalyzes the reversible transfer of α-amino groups between aspartic acid and glutamic acid [36]. Branched chain aminotransferase 2 (Bcat2) catalyzes catabolism of branched chain amino acids to branched chain α-keto acids, which are further metabolized to acyl-CoA by branched chain ketoacid dehydrogenase (Bckdh) [6]. Insulin increased the expression of Aat-1 (cytosolic Aat) in the absence of a TGF-β family member, whereas the expression levels of Bcat2 and Bckdha (α polypeptide of Bckdh) decreased with insulin. Treatment with TGFβ1, activin A, activin B, or BMP4 decreased the insulin-induced Aat-1 expression, and activin A also downregulated the expression of Aat-1, even without insulin. In addition, TGF-β1 and BMP7 decreased and increased the expression of Aat-2 and Bcat2, respectively, in the presence of insulin.
6
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Succinic acid
A
Ins Fam Ins×Fam
200
P 0.03 0.03 0.76
* *
P <0.001 0.87 0.20
*
2h
BMP7
BMP4
ActB
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
0
TGF-β family:
6h
B
Fumaric acid 600
arbitrary unit
*
Ins Fam Ins×Fam
100
None
arbitrary unit
300
Ins Fam Ins×Fam
400
P <0.001 0.02 0.83
*
*
*
*
Ins Fam Ins×Fam
P <0.001 0.52 0.29
* 200
*
*
*
2h
BMP7
BMP4
ActB
6h Malic acid
C 800
Ins Fam Ins×Fam
600
P <0.001 0.04 0.91
*
*
P <0.001 0.53 0.30
2h
BMP7
BMP4
ActB
ActA
TGF-β1
* None
ActB
*
BMP7
*
BMP4
* ActA
TGF-β1
0
Ins Fam Ins×Fam
*
200
TGF-β family:
*
*
400
None
arbitrary unit
ActA
TGF-β1
None
BMP7
BMP4
ActB
ActA
TGF-β1
None
0
TGF-β family:
6h
Fig. 5. Effect of treatment with insulin and TGF-β family members on citric acid cycle metabolite contents in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 2 h. The cellular succinic acid (A), fumaric acid (B), and malic acid (C) contents were quantified, with the content in cells untreated with insulin and a TGF-β family member for each time point set at 100. Black bars: cells untreated with insulin; hatched bars: cells treated with insulin. Data are shown as the mean ± SE (n = 3). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin.
growth factors potentially act as undesirable adipokines in terms of their health effects. In the present study, Pai-1 expression was not affected by TGF-β in 3T3-L1 adipocytes; TGF-β has been shown to induce Pai-1 expression in several cell types [21,25,20,26]. In addition, previous studies also showed that expression level of Pai-1 was clearly increased in 3T3-L1 adipocytes treated with TGF-β for 3 h [27], which was dose-dependent of TGF-β [32]. However, the gene induction of Pai-1 was transient, and TGF-β treatment for 24 h hardly affected Pai-1 expression [32]. The present study examined gene expression in 3T3-L1 adipocytes treated with TGF-β family for 2 d. Chronic treatment with TGF-β1 is likely to lead to the failure of Pai-1 gene induction in 3T3-L1 adipocytes.
indicated that TGF-β1 and activin A increased expression of resistin in adipocytes. Because the expression level of resistin in adipose tissue was found to be higher in obese humans than in lean humans [33], the increased expression of resistin may have been mediated by TGF-β1 and activin A. However, unlike the effects in mice [18], non-adipocyte resident inflammatory cells mainly contribute to resistin expression in adipose tissues in humans [33]. Therefore, the contribution of resistin expression in adipocytes, induced by TGF-β1 or activin A, may be minor in human adipose tissues. Nevertheless, because resistin has been implicated in various diseases, including diabetes and cardiovascular disease [37,34], TGF-β1 and activin A produced in the adipose tissues of obese subjects may increase resistin expression in adipocytes; these 7
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
TGF-β family: - TGF-β1 ActA Insulin: - + - + - +
ActB
BMP4
BMP7
- + - + - +
category F M C S
D C A F K R G I V T P S L Y
group
① ② ③ ④
⑤
A
E W Q
⑥
-3 -2 -1 0 1 2 3 Fig. 6. Hierarchical clustering of metabolites detected in 3T3-L1 adipocytes treated with insulin and TGF-β family for 6 h. The 3T3-L1 adipocytes treated with or without insulin and a TGF-β family member for 6 h. Cellular metabolites were comprehensively analyzed by metabolomic analyses, and the relative abundance of metabolites is shown as a heat map. Red: compounds involved in glycolysis and the citric acid cycle; white: sugar or sugar-related compounds; blue: amino acid, amino acid-related compounds, or amines; purple: bases or baserelated compounds; green: free fatty acids; black: low molecular organic acids or acid compounds. A, C, F, M, and S in red indicate aconitic acid, citric acid, fumaric acid, malic acid, and succinic acid, respectively. In addition, blue characters denote amino acids in single-letter code. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
also higher in obese subjects than in lean subjects [8], which may reflect a defensive reaction to the harmful effects of obesity. Leptin is another adipokine that regulates adipose tissue mass as well as metabolic, reproductive, neuroendocrine, and immune functions [15,23]. Recessive mutations in the leptin gene are associated
100
P Ins <0.001 Fam <0.001 Ins×Fam 0.10
* †
†
P Ins <0.001 Fam 0.07 Ins×Fam 0.91
* †
* *
Ins Fam Ins×Fam
* *
* *
Aat-1
Bcat2
Bckdha
BMP7
ActB
BMP4
ActA
None
TGF-β1
BMP7
ActB
BMP4
ActA
None
Aat-2
TGF-β1
BMP7
ActB
BMP4
ActA
None
TGF-β1
BMP7
ActB
BMP4
ActA
None
TGF-β1
0
TGF-β family:
P 0.64 0.06 0.40
BMP7
* * * † † † †
P 0.800 0.002 0.32
ActB
*
Ins Fam Ins×Fam
BMP4
200
*
ActA
300
P Ins <0.001 Fam <0.001 Ins×Fam 0.02
None
relative mRNA level
400
TGF-β1
Unlike activin A, activin B did not increase resistin expression; rather, it decreased Pai-1 expression. Pai-1 is linked to the onset of atherosclerosis, as well as insulin resistance [10,2]. Thus, the activin Bmediated response in adipocytes could be beneficial to health. The expression level of inhibin βB, a constituent molecule of activin B, was
Bckdhb
Fig. 7. Effect of treatment with insulin and TGF-β family members on expression level of genes related to amino acid catabolism in 3T3-L1 adipocytes. The 3T3-L1 adipocytes were treated or untreated with insulin and a TGF-β family member for 6 h. Expression levels of Aat-1, Aat-2, Bcat2, Bckdha, and Bckdhb examined by RT-qPCR. Expression levels normalized to that of Tbp, and the expression level in the control group was set at 100. Data are shown as the mean ± SE (n = 4). *: P < 0.05, significantly different from the content in cells treated with the respective TGF-β family member, but not with insulin. †: P < 0.05, significantly different from the content in cells untreated with a TGF-β family member, and either treated or untreated with insulin.
8
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
with extreme obesity in mice and in some humans, and leptin has been used successfully to treat obesity, indicating an anti-obesity role for leptin [15,23]. The present study revealed that activin A increased the expression of leptin in adipocytes. Therefore, activin A may enhance the expression of molecules with anti-obesity activities, which suggests that activin A is a desirable adipokine for health. The physiological roles of activin A in adipose tissues should be evaluated in future studies, because it simultaneously increases the expression of resistin and has adverse effects on health, as described above. Insulin stimulates fatty acid synthesis partly by increasing the expression of fatty acid synthase in adipocytes [40,4]. Thus, the results showing that treatment with insulin transiently (∼2 h) led to general decrease in cellular amino acid contents might be a response, in part, to the stimulation of amino acid catabolism for fatty acid synthesis. In fact, the expression of Aat-1 increased in response to insulin. However, altered expression of Aat-1 could not explain the modulation of insulininduced amino acid contents by TGF-β family members. Insulin induced the upregulation of Aat-1 expression in adipocytes, except for that in activin A-treated cells, but insulin did not affect the cellular amino acid contents in BMP4- or BMP7-treated cells. TGF-β family members may post-transcriptionally regulate genes related to amino acid catabolism. Alternatively, considering that insulin also stimulates protein synthesis in adipocytes [41], the insulin-induced reduction of free amino acids may result, in part, from the stimulation of protein synthesis; TGF-β family may modulate protein synthesis. Insulin significantly increased the citric acid cycle metabolite contents in adipocytes in response to treatment with activin A, activin B, BMP4, or BMP7 for 2 h. In contrast, treatment with TGF-β family members for 6 h did not affect the insulin-induced accumulation of the same metabolites. These results suggest that tested members of the TGFβ family, except for TGF-β1, transiently enhanced the response to insulin. Our previous study revealed that the citric acid cycle metabolite contents of adipocytes exhibited periodic changes with a 2-h cycle [29]. Thus, TGF-β family members appear to modulate insulin-induced oscillations in adipocytes. The present study unbiasedly and comprehensively explored regulatory changes in metabolite content in response to TGF-β family in the presence or absence of relatively high concentration of insulin (10 μg/mL) in 3T3-L1 adipocytes; changes in metabolite content in 3T3L1 adipocytes treated with 20 nM (0.12 μg/mL) of insulin were comparable with those in 3T3-L1 treated with 10 μg/mL of insulin (data not shown). Future studies are needed to know the mechanism underlying the regulation. Cross-talk between insulin and TGF-β family may lead to discrete regulation of cell metabolism; insulin modulated TGF-β signaling in NMuMG epithelial cells and mouse embryo fibroblasts [7], In addition, effects of the TGF-β family on cell response should be clarified in the presence of low dose of insulin.
[2] [3]
[4] [5]
[6] [7]
[8]
[9]
[10] [11]
[12]
[13] [14] [15] [16] [17]
[18]
[19] [20]
[21]
[22] [23]
[24]
Conflict of interest [25]
All authors have no conflict of interest. Acknowledgement
[26]
This work was supported by a Grant-in-Aid for Scientific Research (26450442) from The Japan Society for the Promotion of Science.
[27]
Appendix A. Supplementary material
[28]
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cyto.2017.05.01.
[29] [30]
References
[31]
[1] M.C. Alessi, D. Bastelica, P. Morange, B. Berthet, I. Leduc, M. Verdier, O. Geel, I. Juhan-Vague, Plasminogen activator inhibitor 1, transforming growth factor-β1,
9
and BMI are closely associated in human adipose tissue during morbid obesity, Diabetes 49 (2000) 1374–1380. M.C. Alessi, M. Poggi, I. Juhan-Vague, Plasminogen activator inhibitor-1, adipose tissue and insulin resistance, Curr. Opin. Lipidol. 18 (2007) 240–245. H. Asano, T. Yamada, O. Hashimoto, T. Umemoto, R. Sato, S. Ohwatari, Y. Kanamori, T. Terachi, M. Funaba, T. Matsui, Diet-induced changes in Ucp1 expression in bovine adipose tissues, Gen. Comp. Endocrinol. 184 (2013) 87–92. L.H. Baumgard, G.J. Hausman, M.V. Sanz Fernandez, Insulin: pancreatic secretion and adipocyte regulation, Domest. Anim. Endocrinol. 54 (2016) 76–84. J. Bolinder, A. Lindblad, P. Engfeldt, P. Arner, Studies of acute effects of insulin-like growth factors I and II in human fat cells, J. Clin. Endocrinol. Metab. 65 (1987) 732–737. J.T. Brosnan, M.E. Brosnan, Branched-chain amino acids: enzyme and substrate regulation, J. Nutr. 136 (1 Suppl) (2006) 207S–211S. E.H. Budi, B.P. Muthusamy, R. Derynck, The insulin response integrates increased TGF-β signaling through Akt-induced enhancement of cell surface delivery of TGF-β receptors, Sci. Signal. 8 (2015) ra96. L.M. Carlsson, P. Jacobson, A. Walley, P. Froguel, L. Sjöström, P.A. Svensson, K. Sjöholm, ALK7 expression is specific for adipose tissue, reduced in obesity and correlates to factors implicated in metabolic disease, Biochem. Biophys. Res. Commun. 382 (2009) 309–314. L. Choy, R. Derynck, Transforming growth factor-β inhibits adipocyte differentiation by Smad3 interacting with CCAAT/enhancer-binding protein (C/EBP) and repressing C/EBP transactivation function, J. Biol. Chem. 278 (2003) 9609–9619. C. Dellas, D.J. Loskutoff, Historical analysis of PAI-1 from its discovery to its potential role in cell motility and disease, Thromb. Haemost. 93 (2005) 631–640. E.M. Duran, P. Shapshak, J. Worley, A. Minagar, F. Ziegler, S. Haliko, I. MoleonBorodowsky, P.A. Haslett, Presenilin-1 detection in brain neurons and FOXP3 in peripheral blood mononuclear cells: normalizer gene selection for real time reverse transcriptase PCR using the deltadeltaCt method, Front Biosci. 10 (2005) 2955–2965. M. Elsen, S. Raschke, N. Tennagels, U. Schwahn, T. Jelenik, M. Roden, T. Romacho, J. Eckel, BMP4 and BMP7 induce the white-to-brown transition of primary human adipose stem cells, Am. J. Physiol. Cell Physiol. 306 (2014) C431–C440. J.N. Fain, D.S. Tichansky, A.K. Madan, Transforming growth factor β 1 release by human adipose tissue is enhanced in obesity, Metabolism 54 (2005) 1546–1551. X.H. Feng, R. Derynck, Specificity and versatility in TGF- β signaling through Smads, Annu. Rev. Cell Dev. Biol. 21 (2005) 659–693. J.M. Friedman, Leptin at 14 y of age: an ongoing story, Am. J. Clin. Nutr. 89 (2009) 973S–979S. S. Hirai, M. Yamanaka, H. Kawachi, T. Matsui, H. Yano, Activin A inhibits differentiation of 3T3-L1 preadipocyte, Mol. Cell. Endocrinol. 232 (2005) 21–26. H. Huang, T.J. Song, X. Li, L. Hu, Q. He, M. Liu, M.D. Lane, Q.Q. Tang, BMP signaling pathway is required for commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage, Proc. Natl. Acad Sci. USA 106 (2009) 12670–12675. K.H. Kim, K. Lee, Y.S. Moon, H.S. Sul, A cysteine-rich adipose tissue-specific secretory factor inhibits adipocyte differentiation, J. Biol. Chem. 276 (2001) 11252–11256. N. Klöting, M. Blüher, Adipocyte dysfunction, inflammation and metabolic syndrome, Rev. Endocr. Metab. Disord. 15 (2014) 277–287. R.M. Kortlever, J.H. Nijwening, R. Bernards, Transforming growth factor-β requires its target plasminogen activator inhibitor-1 for cytostatic activity, J. Biol. Chem. 283 (2008) 24308–24313. M. Laiho, F.M. Weis, F.T. Boyd, R.A. Ignotz, J. Massagué, Responsiveness to transforming growth factor-β (TGF-β) restored by genetic complementation between cells defective in TGF-β receptors I and II, J. Biol. Chem. 266 (1991) 9108–9112. K. Miyazono, Y. Kamiya, M. Morikawa, Bone morphogenetic protein receptors and signal transduction, J. Biochem. 147 (2010) 35–51. H.S. Moon, M. Dalamaga, S.Y. Kim, S.A. Polyzos, O.P. Hamnvik, F. Magkos, J. Paruthi, C.S. Mantzoros, Leptin's role in lipodystrophic and nonlipodystrophic insulin-resistant and diabetic individuals, Endocr. Rev. 34 (2013) 377–412. M. Morikawa, R. Derynck, K. Miyazono, TGF-β and the TGF-β family: contextdependent roles in cell and tissue physiology, Cold Spring Harb Perspect Biol. 8 (5) (2016), http://dx.doi.org/10.1101/cshperspect.a021873 pii: a021873. M. Murakami, T. Ikeda, T. Saito, K. Ogawa, Y. Nishino, K. Nakaya, M. Funaba, Transcriptional regulation of plasminogen activator inhibitor-1 by transforming growth factor- β, activin A and microphthalmia-associated transcription factor, Cell. Signal. 18 (2006) 256–265. M. Murakami, H. Kawachi, K. Ogawa, Y. Nishino, M. Funaba, Receptor expression modulates the specificity of transforming growth factor- β signaling pathways, Genes Cells 14 (2009) 469–482. M. Pandey, D.J. Loskutoff, F. Samad, Molecular mechanisms of tumor necrosis factor-α-mediated plasminogen activator inhibitor-1 expression in adipocytes, FASEB J. 19 (2005) 1317–1319. S.W. Qian, Y. Tang, X. Li, Y. Liu, Y.Y. Zhang, H.Y. Huang, R.D. Xue, H.Y. Yu, L. Guo, H.D. Gao, Y. Liu, X. Sun, Y.M. Li, W.P. Jia, Q.Q. Tang, BMP4-mediated brown fatlike changes in white adipose tissue alter glucose and energy homeostasis, Proc. Natl. Acad. Sci. USA 110 (2013) E798–E807. Y. Qiao, S. Tomonaga, T. Matsui, M. Funaba, Modulation of the cellular content of metabolites in adipocytes by insulin, Mol. Cell. Endocrinol. 424 (2016) 71–80. A. Rebbapragada, H. Benchabane, J.L. Wrana, A.J. Celeste, L. Attisano, Myostatin signals through a transforming growth factor β-like signaling pathway to block adipogenesis, Mol. Cell. Biol. 23 (2003) 7230–7242. E.D. Rosen, B.M. Spiegelman, Adipocytes as regulators of energy balance and glucose homeostasis, Nature 444 (2006) 847–853.
Cytokine xxx (xxxx) xxx–xxx
Y. Qiao et al.
Biochem. 81 (2012) 715–736. [43] Q.Q. Tang, T.C. Otto, M.D. Lane, Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage, Proc. Natl. Acad. Sci. USA 101 (2004) 9607–9611. [44] P. Tontonoz, B.M. Spiegelman, Fat and beyond: the diverse biology of PPAR γ, Annu. Rev. Biochem. 77 (2008) 289–312. [45] M.E. Trujillo, P.E. Scherer, Adipose tissue-derived factors: impact on health and disease, Endocr. Rev. 27 (2006) 762–778. [46] Y.H. Tseng, E. Kokkotou, T.J. Schulz, T.L. Huang, J.N. Winnay, C.M. Taniguchi, T.T. Tran, R. Suzuki, D.O. Espinoza, Y. Yamamoto, M.J. Ahrens, A.T. Dudley, A.W. Norris, R.N. Kulkarni, C.R. Kahn, New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure, Nature 454 (2008) 1000–1004. [47] R. Xue, Y. Wan, S. Zhang, Q. Zhang, H. Ye, Y. Li, Role of bone morphogenetic protein 4 in the differentiation of brown fat-like adipocytes, Am. J. Physiol. Endocrinol. Metab. 306 (2014) E363–E372. [48] H. Yoshida, Y. Kanamori, H. Asano, O. Hashimoto, M. Murakami, T. Kawada, T. Matsui, M. Funaba, Regulation of brown adipogenesis by the Tgf-β family: involvement of Srebp1c in Tgf-β- and Activin-induced inhibition of adipogenesis, Biochem. Biophys. Acta. 1830 (2013) 5027–5035. [49] N. Zamani, C.W. Brown, Emerging roles for the transforming growth factor- β superfamily in regulating adiposity and energy expenditure, Endocr. Rev. 32 (2011) 387–403. [50] L.E. Zaragosi, B. Wdziekonski, P. Villageois, M. Keophiphath, M. Maumus, T. Tchkonia, V. Bourlier, T. Mohsen-Kanson, A. Ladoux, C. Elabd, M. Scheideler, Z. Trajanoski, Y. Takashima, E.Z. Amri, D. Lacasa, C. Sengenes, G. Ailhaud, K. Clément, A. Bouloumie, J.L. Kirkland, C. Dani, Activin a plays a critical role in proliferation and differentiation of human adipose progenitors, Diabetes 59 (2010) 2513–2521.
[32] F. Samad, K. Yamamoto, M. Pandey, D.J. Loskutoff, Elevated expression of transforming growth factor-β in adipose tissue from obese mice, Mol. Med. 3 (1997) 37–48. [33] D.B. Savage, C.P. Sewter, E.S. Klenk, D.G. Segal, A. Vidal-Puig, R.V. Considine, S. O'Rahilly, Resistin/Fizz3 expression in relation to obesity and peroxisome proliferator-activated receptor-γ action in humans, Diabetes 50 (2001) 2199–2202. [34] D.R. Schwartz, M.A. Lazar, Human resistin: found in translation from mouse to man, Trends Endocrinol. Metab. 22 (2011) 259–265. [35] M. Shimizu, F. Torti, R.A. Roth, Characterization of the insulin and insulin-like growth factor receptors and responsitivity of a fibroblast/adipocyte cell line before and after differentiation, Biochem. Biophys. Res. Commun. 137 (1986) 552–558. [36] S. Sookoian, C.J. Pirola, Alanine and aspartate aminotransferase and glutaminecycling pathway: their roles in pathogenesis of metabolic syndrome, World J. Gastroenterol. 18 (2012) 3775–3781. [37] C.M. Steppan, S.T. Bailey, S. Bhat, E.J. Brown, R.R. Banerjee, C.M. Wright, H.R. Patel, R.S. Ahima, M.A. Lazar, The hormone resistin links obesity to diabetes, Nature 409 (2001) 307–312. [38] M. Suenaga, N. Kurosawa, H. Asano, Y. Kanamori, T. Umemoto, H. Yoshida, M. Murakami, H. Kawachi, T. Matsui, M. Funaba, Bmp4 expressed in preadipocytes is required for the onset of adipocyte differentiation, Cytokine 64 (2013) 138–145. [39] M. Suenaga, T. Matsui, M. Funaba, BMP Inhibition with dorsomorphin limits adipogenic potential of preadipocytes, J. Vet. Med. Sci. 72 (2010) 373–377. [40] H.S. Sul, M.J. Latasa, Y. Moon, K.H. Kim, Regulation of the fatty acid synthase promoter by insulin, J. Nutr. 130 (2000) 315S–320S. [41] S.X. Tan, K.H. Fisher-Wellman, D.J. Fazakerley, Y. Ng, H. Pant, J. Li, C.C. Meoli, A.C. Coster, J. Stöckli, D.E. James, Selective insulin resistance in adipocytes, J. Biol. Chem. 290 (2015) 11337–11348. [42] Q.Q. Tang, M.D. Lane, Adipogenesis: from stem cell to adipocyte, Annu. Rev.
10