The mechanisms of carnosic acid attenuates tumor necrosis factor-α-mediated inflammation and insulin resistance in 3T3-L1 adipocytes
Chia-Wen Tsai1, Kai-Li Liu2, Yu-Ru Lin1, and Wen-Cheng Kuo1
1Department of Nutrition, China Medical University, Taichung, Taiwan.
2Department of Nutrition, Chung Shan Medical University, Taichung, Taiwan.
Correspondence: Dr. Chia-Wen Tsai, Department of Nutrition, China Medical University, Taichung, Taiwan; E-mail: [email protected]; Fax: +886-4-22062891.
Abbreviations: AP-1, activator protein-1; aP2, adipocyte protein2; CA, carnosic acid; ERK, extracellular signal-regulated kinase; GLUT 4, glucose transporter 4; HDAC 3, histone deacetylase 3; IκB, inhibitor-κB; IKK, IκB kinase, IL-6, interleukin-6; IRS-1, insulin receptor substrate-1; JNK, c-Jun NH2-terminal kinase; MAPK,
mitogen-activated protein kinase; MCP-1, monocyte chemoattractant protein-1; NF-κB , nuclear factor-kappa B; PARP, poly (ADP-ribose) polymerase; PPARγ, peroxisome proliferator activated receptor γ; TNF-α, tumor necrosis factor-α.
Scope: Insulin resistance has been linked to a low-grade chronic inflammatory response. Carnosic acid (CA), which is found in rosemary, has been reported to have antioxidant, anti-inflammation and anti-adipogenic properties. Here, we examined the effects of CA on inflammation and insulin resistance in 3T3-L1 adipocytes treated with tumor necrosis factor-α (TNF-α).
Methods and results: CA attenuated the TNF-α-induced mRNA expression of inflammatory genes, including interleukin-6 (IL-6) and monocyte chemoattractant protein-1. CA also attenuated the TNF-α-mediated activation of extracellular signal-related kinase, c-Jun NH2-terminal kinase, and c-Jun; the phosphorylation of inhibitor-κB (IκB) kinase (IKK)α/β; the phosphorylation and degradation of IκBα; the nuclear
translocation of p65; and the DNA binding activity of NF-κB and AP-1. CA or PP242 (an mTOR inhibitor) suppressed the TNF-α-induced protein expression of mTOR, P70S6K, eIF4E, and IL-6. Moreover, CA attenuated the TNF-α-mediated suppression of
peroxisome proliferator activated receptor γ, adiponectin, and adipocyte protein 2. CA reversed the TNF-α-mediated suppression of insulin-stimulated glucose uptake and the phosphorylation of Tyr632 insulin receptor substrate-1 (IRS-1), Akt, and FoxO1, but
decreased the TNF-α-induced phosphorylation of Ser307 IRS-1 and total FoxO1.
Conclusion: CA attenuates TNF-α-mediated inflammation via inhibition of NF-κB and AP-1 pathways and insulin resistance via Akt-dependent FoxO1 signaling in 3T3-L1 adipocytes.
Keywords: Carnosic acid / tumor necrosis factor-α / insulin resistance / inflammation / 3T3-L1 adipocytes 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1 Introduction
Obesity is associated with chronic low-grade inflammation that is believed to contribute to the development of insulin resistance [1]. Adipose tissue in obesity is a major site of increased macrophage infiltration and inflammatory response. Inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) are secreted by adipose tissue. MCP-1 recruits macrophages into the adipose tissue. Macrophages promote the proinflammatory state and lead to an impaired preadipocyte differentiation. The accumulated evidence indicated that macrophages correlate with insulin resistance in adipocytes by inhibiting the
expression of glucose transporter 4 (GLUT4) in 3T3-L1 adipocytes [2].
The expression of TNF-α is increased in the adipose tissue of obese insulin-resistant mice and humans [3, 4]. Furthermore, one study showed that neutralization of TNF-α improves the insulin sensitivity in genetically obese fa/fa rats [5]. TNF-α-deficient mice are protected against insulin resistance induced by a high-fat diet [6]. The inflammation caused by TNF-α triggers the activation of mitogen-activated protein kinases (MAPKs) such as extracellular signal-related kinase (ERK) and c-Jun-NH2 terminal kinase (JNK). The activation of ERK and JNK leads to serine phosphorylation of insulin receptor substrate-1 (IRS-1), which inhibits the normal tyrosine phosphorylation of IRS-1, thereby impairing insulin action [7-9]. Moreover, these kinases induce an inflammatory response through activation of the transcription factors nuclear factor-κB (NF-κB) and activator protein (AP)-1, which in turn promotes inflammatory gene expression [10, 11]. Inhibition of NF-κB caused by deletion of inhibitor-κB (IκB) kinase (IKK)α/β in hepatocytes from a transgenic mice model attenuates inflammatory gene expression and insulin resistance in 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
response to a high-fat diet [12].
The ligand-activated transcription factor peroxisome proliferator-activated receptor γ (PPARγ) plays an important role in the regulation of adipogenesis, lipid metabolism, and glucose homeostasis [13, 14]. PPARγ mediates the insulin-sensitizing effects of
thiazolidinediones, which are used in the treatment of type 2 diabetes [15]. PPARγ activation reduces T-lymphocyte infiltration into adipose tissue and improves glucose metabolism in obese mice [16]. PPARγ forms a heterodimer with the retinoid X receptor and binds to the PPAR response elements in the promoters of target genes such as adiponectin and adipocyte protein 2 (aP2).
The forkhead transcription factor FoxO1 is highly expressed in insulin-responsive tissues, including liver, adipose tissue, and muscle cells. In response to insulin, FoxO1 proteins are phosphorylated by Akt, which results in their translocation from the nucleus to the cytoplasm [17, 18]. Treatment of 3T3-L1 adipocytes with TNF-α reduces the Akt-dependent phosphorylation of FoxOl and increases the transcriptional activity of FoxOl [19]. In a recent study, transgenic mice overexpressing a dominant-negative form of FoxO1 in adipose tissue showed improved insulin sensitivity and glucose tolerance when fed a high-fat diet [20]. Other studies have shown that FoxO1 binds directly to PPARγ and represses its function [21]. The accumulated evidence supportsthat adipocytes from high-fat diet-induced insulin-resistant mice undergo reduced phosphorylation and
enhanced nuclear accumulation of FoxO1, which then decreases the expression of PPARγ target genes [15].
Carnosic acid (CA), a rosemary (Rosmarinus officinalis) phenolic diterpene, has been reported to possess multiple biological properties, such as antioxidative, anti-44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66
inflammatory, and anti-adipogenic activities [22-24]. Additional studies have revealed that CA inhibits lipid peroxidation [25] and protects red cells against oxidative hemolysis [26]. The anti-inflammatory property of CA has been shown in vivo and in vitro. In vivo, CA inhibits the inflammation induced by phorbol 12-myristate 13-acetate through down-regulation of the mRNA expression of IL-1β and TNF-α in mice ear. In vitro, CA reduces the overproduction of nitric oxide by lipopolysaccharides in RAW 264.7 macrophages [27]. A recent study has also shown that CA inhibited skin inflammatory responses by decreased the production of IL-6, MCP-1, and TNF-α [28]. Although CA has anti-inflammatory effects, it is not known whether CA modulates the anti-inflammatory response in adipocytes. Moreover, interest has been growing in the physiologic properties of CA, not only because of its inflammatory action but also because of its possible anti-adipogenic property. In ob/ob mice, CA significantly prevents weight gain and reduces visceral adiposity and the levels of serum triglyceride and cholesterol. In addition,
intraperitoneal glucose tolerance tests have shown that CA significantly improves glucose tolerance [29]. Proinflammatory cytokines induced by macrophages are related to the pathogenesis of insulin resistance by mediating glucose uptake [2]. How this compound can have the protective effects against inflammation and insulin resistance and the mechanism of action of these effects in 3T3-L1 adipocytes is not yet well understood. Therefore, we investigated the extent to which CA attenuated TNF-α mediated
inflammation and insulin resistance in 3T3-L1 adipocytes. 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86
2 Materials and methods 2.1 Materials
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless specified otherwise. Dulbecco’s Modified Eagle Medium and penicillin/streptomycin were
obtained from Gibco Laboratory (Gaithersburg, MD). Fetal calf serum was from Hyclone (Logan, UT). Trizol reagent was ordered from Invitrogen (Carlsbad, CA). RNase
inhibitor, oligo-dT, deoxynucleotide triphosphate, and Moloney murine leukemia virus reverse transcriptase were from Promega (Madison, WI). All antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) unless specified otherwise.
Horseradish peroxidase–conjugated goat anti-rabbit IgG and goat anti-mouse IgG were from PerkinElmer Life Sciences (Boston, MA). Rabbit anti-goat IgG was from R&D Systems Inc. (Minneapolis, MN).
2.2 Cell culture
3T3-L1 preadipocytes were obtained from Bioresources Collection and Research Center (BCRC, Hsin-Chu, Taiwan) and cultured in DMEM supplemented with 18 mM sodium bicarbonate, 1×105 units/L penicillin, 100 mg/L streptomycin and 10% FCS in a
humidified atmosphere of 5% CO2 at 37℃. For all studies, cells between passages 8 and
15 were used. After they reached confluence, the 3T3-L1 cells were allowed to grow for an additional 24 h. And then, cell differentiation was initiated by using differentiation medium I (included 10 μg/L insulin, 0.5 mM IBMX, and 0.25 μM dexamethasone) for 48 h. The medium was then replaced with DMEM supplemented with 10 μg/L insulin (differentiation medium II) and changed every other day for the following 6 d. 3T3-L1 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109
adipocytes were pretreated with 0.1% DMSO or with 1, 5, 10 or 20 μM CA for 24 h, and were then cotreated with 5 ng/mL TNF-α for 15 min or 24 h. For insulin-treated cells, 3T3-L1 adipocytes were treated with 10 nM insulin for the lasted 30 min. Moreover, cells were switched to a serum-free culture medium for 4 h before CA treatment.
2.3 Primary hepatocytes isolation and culture.
Male Sprague-Dawley rats were purchased from LASCO (BioLASCO Taiwan Co., Ltd.) and were used for hepatocyte isolation when aged 7 to 8 weeks old. Rats were treated in compliance with the Guide for the Care and Use of Laboratory Animals [30].
Hepatocyteswere isolated by a two-step collagenase perfusion method. Cell viability was >90% as determined by trypan blue exclusion.The isolated hepatocytes were suspended in RPMI-1640 medium containing 10 mM HEPES, 1 105 unit/L penicillin, 100 mg/L
streptomycin, 0.1 mM dexamethasone, and 1% ITS+. The cells were plated in 35-mm
plastic tissueculture dishes (Falcon, Franklin Lakes, NJ) precoated with rat tail collagen I at a density of 1 × 106 cells per dish and were incubated at 37oC in a humidified
atmosphere of 5% CO2 and 95% air. After culturing for 24 h, hepatocytes were pretreated
with 0.1% DMSO or with 10 μM CA for 24 h, and were then cotreated with 20 ng/mL TNF-α for 24 h. For insulin-treated cells, hepatocytes were treated with 10 nM insulin for the lasted 30 min.
2.4 RNA isolation and RT-PCR
Total RNA of 3T3-L1 adipocytes was extracted by using Trizol reagent. We used 1 μg of total RNA for the synthesis of first-stand cDNA. The sequences for the RT-PCR primers 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
were as follows: for adiponectin (forward: 5’-GTGCAGGTTGGATGGCAGGCA-3’; reverse: AGTGACGCGGGT CTCCAGCC-3’); aP2 (forward:
AAGACAGCTCCTCCTCGAAGGTT-3’; reverse:
5’-GCGTAAATGGGGATTTGGTCACCA-3’); MCP-1 (forward: 5’-AGGTCCCTG TCATGCTTCTG-3’; reverse: 5’-TCTGGACCCATTCCTTCTTG-3’ ); IL-6 (forward: 5’-GGTGACAACCACGGCCTTCCC-3’; reverse: 5’-GCCACTCCTTCTGTGACTC CAGC-3’); PPARγ (forward: 5’-TTTTCAAGGGTGCCAGTTTC-3’; reverse: 5’-AA TCCTTGGCCCTCTGAGAT-3’); GAPDH (forward: 5’-GACGTGCCGCCTGGAGA AA-3’; reverse 5’-GGGGGCCGAGTTGGGATAG-3’). The reaction mixture was incubated for 1 cycle at 42 °C for 15 min, 99 °C for 5 min, and 4 °C for 10 min. The PCR reactions were performed as follows: 5 min at 94 °C; 30 cycles of 40 s at 94 °C, 40 s at 60 °C, and 120 s at 72 °C; and a final extension of 5 min at 68 °C. The PCR products were resolved in a 1%-agarose gels containing 40 mM Tris/20 mM glacial acetic acid/2 mM EDTA buffer.
2.5 Western blotting
Cell lysates was appliedto 10% SDS-PAGE gels and was electrophoretically transferred to PVDF (Millipore, Bedford, MA). The nonspecific binding sites on the membranes were blocked at 4°C overnight with 50 g/L nonfat dry milkand were then incubated with primary antibodies (diluted 1:500-1000) against JNK1/2, ERK1/2, p38, Akt, FoxO1, IRS1, phospho-JNK1/2, phospho-ERK1/2, phospho-p38, phospho-Akt, phospho-FoxO1, phospho-IRS1, GLUT 4, PPARγ, and actin at 4°C overnight. Then, the membrane was incubated with the secondary peroxidase-conjugated anti-goat, anti-rabbit or anti-mouse 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155
IgG at room temperature for 1 h. The bands were detected by using an enhanced chemiluminescence kit (PerkinElmer Life Science, Boston, MA) and were quantitated with an ImageGauge (FujiFilm LAS-4000, Japan).
2.6 Preparation of nuclear extracts
3T3-L1 adipocytes were washed twice with cold PBS followed by scraping from the dishes with 500 μL PBS. Cell homogenates were centrifuged at 2,000 xg for 5 min. The cell pellet was allowed to swell on ice for 15 min after the addition of 400 μL of
hypotonic buffer (10 mM HEPES, 10 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM
DTT, 0.5% Nonidet P-40, 4 mg/L leupeptin, 20 mg/L aprotinin, and 0.2 mM PMSF). After centrifugation at 7,000 xg for 15 min, pellets containing crude nuclei were resuspended in 50 μL of hypertonic buffer containing 10 mM HEPES, 400 mM KCl, 1 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 10% glycerol, 4 mg/L leupeptin, 20 mg/L aprotinin, and 0.2 mM PMSF and incubatedfor an additional 30 min on ice. The samples were then obtained by centrifugation at 20,000 xg for 15 min and were frozen at −80 °C until the western blotting or EMSA was performed.
2.7 Preparation of membrane extracts
3T3-L1 adipocytes were washed twice with cold PBS followed by scraping from the dishes with 500 μL PBS. Cell homogenates were centrifuged at 2,000 xg for 5 min. The cell pellet was added to 100 μL of buffer A (100 mM Tris-HCl, 30 mM Na3VO4, 100 mM
MgCl2, 100 mM EDTA, 100 mM EGTA, 0.5 mM DTT, 250 mM sucrose, 1 mg/L
leupeptin, and 50 mM PMSF) and homogenized with a Dounce homogenizer. The 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178
homogenate was centrifuged at 800 xg for 10 min at 4℃. The supernatant was
centrifuged at 32,000 xg for 70 min at 4℃ to pellet the crude membrane. The cell pellet was allowed to swell on ice for 60 min after the addition of 100 μL of buffer B containing 100 mM Tris-HCl, 30 mM Na3VO4, 100 mM MgCl2, 100 mM EDTA, 100 mM EGTA,
0.5 mM DTT, 1 mg/L leupeptin, 100 mM NaF, and 0.1% Triton X-100. The samples were obtained by centrifugation at 32,000 xg for 70 min. This pellet was resuspended in PPB and frozen at −80 °C until the Western blotting was performed.
2.8 Electrophoretic mobility shift assay
The Light-ShiftTM Chemiluminescent electrophoretic mobility shift assay Kit (Pierce
Chemical Co., Rockford, IL) and synthetic biotin-labeled double-stranded AP-1 consensus oligonucleotides (forward: 5’- CGCTTGATGACTC
AGCCGGAA -3’; reverse: 5’- TTCCGGC TGAGTCATCAAGCG -3’); NF-κB (forward: 5’- AGTTGAGGGGACTTTCCC AGGC -3’; reverse: 5’- GCCTGGG AAAGTCCC CTCAACT -3’) were used to measure the AP-1, and NF-κB nuclear protein-DNA binding activity. Nuclear extract (6-10 μg), poly(dI-dC), and biotin-labeled double -stranded AP-1, and NF-κB oligonucleotides were mixed with the binding buffer to a final volume of 20 μL and were incubated at room temperature for 30 min. In addition, the unlabeled double-stranded oligonucleotides and mutant double-stranded oligonucleotides were used to confirm the protein binding specificity. The nuclear protein-DNA complex was separated by electrophoresis on a 6% tris-borate-EDTA polyacrylamide gel and was then electrotransferred to a Hybond-N+ nylon membrane (GE
Healthcare, Buckinghamshamshire, UK). Next, the membranes were cross-linked by UV 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201
light for 10 min and were then treated with streptavidin- horseradish peroxidase for 20 min, and the nuclear protein-DNA bands were developed with a SuperSignal West Pico kit (Pierce, Rockford, USA).
2.9 Glucose uptake assay
Uptake of 2-deoxy-D-[3H]glucose was assessed by use of a modified version of a
protocol previously described [31]. Briefly, cells were changed in Krebs Ringer HEPES buffer containing 121 mM NaCl, 4.9 mM KCl, 1.2 mM MgSO4, 0.33 mM CaCl2, and 12
mM HEPES. Four hours later, the cells were pretreated with 10 μM CA for 24 h and then co-treated with 5 ng/mL TNF-α for 24 h. The culture medium was removed and replaced with 900 μL Krebs Ringer HEPES buffer with or without 10 nM insulin for 60 min at 37℃. Cells were then incubated in the Krebs Ringer HEPES buffer with 100 mM 2-deoxyglucose and 10 Ci/mM 2-deoxy-D-[3H]glucose for 5 min at 37℃. After incubation,
the cells were quickly washed three times with ice-cold Krebs Ringer HEPES buffer containing 25 mM glucose. The cells were lysed in 0.1% SDS Krebs Ringer HEPES buffer for 10 min and were then solubilized in liquid scintillation fluid. Incorporated radioactivity was determined by using a scintillation counter (MicroBeta, Perkin-Elmer, MA).
2.10 Statistical analysis
Data are expressed as means ± SD. Data were analyzed by using analysis of variance (SAS Institute, Cary, NC). The significance of the difference among mean values was determined by one-way analysis of variance followed by Tukey’s test. Comparisons 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224
between two groups were made by using Student’s t-test. A value of P<0.05 was considered to be significant.
225 226
3 Results
3.1 Effect of CA on the cell viability of 3T3-L1 adipocytes in the presence or absence of TNF-α
The MTT assay was used to evaluate whether the concentrations of CA used in the presence or absence of TNF-α caused cell damage in 3T3-L1 adipocytes. There were no adverse effects on the growth of 3T3-L1 adipocytes up to a concentration of 20 μM CA in the presence or absence of TNF-α (data not shown).
3.2 CA inhibits TNF-α-mediated inflammatory gene expression
To examine whether TNF-α-mediated inflammatory gene expression was affected by CA in 3T3-L1 adipocytes, cells were pretreated with 1, 5, 10, and 20 μM CA and mRNA expression was assessed by RT-PCR. The mRNA expression of IL-6 and MCP-1 was significantly induced by α for 24 h, and CA dose-dependently attenuated this TNF-α-mediated mRNA expression (Fig. 1A). The MAPK signaling pathway plays a crucial role in TNF-α-induced inflammatory gene expression [32]. Therefore, we attempted to determine the impact of CA on MAPK activation. 3T3-L1 adipocytes were pretreated with 1, 5, 10, and 20 μM CA for 24 h and were then co-treated with TNF-α for 15 min. Immunoblot analysis showed that TNF-α increased the phosphorylation of JNK and ERK but not of p38 (Fig. 1B). Pretreatment with CA attenuated the TNF-α-mediated activation of ERK and JNK in a dose-dependent manner.
3.3 CA attenuates TNF-α-induced activation of the NF-κB and AP-1 signaling pathways 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249
As shown in Fig. 2A, TNF-α induced IKKα/β phosphorylation, and the activation of IKKα/β was significantly attenuated by CA pretreatment. In addition, CA suppressed the phosphorylation and degradation of IκBα caused by TNF-α. Nuclear translocation of p65 was induced by TNF-α, and this effect was attenuated by CA (Fig. 2B). Electrophoretic mobility shift assay indicated that TNF-α increased NF-κB binding activity (Fig. 2C). Pretreatment of cells with CA decreased the activation of NF-κB binding. The specificity of the DNA-protein interaction for NF-κB was demonstrated by a competitive assay with 200-fold excess of unlabeled double-stranded oligonucleotide (cold) and also with a mutant double-stranded oligonucleotide (mut). Similar to these data, CA suppressed the TNF-α-induced activation of c-Jun and AP-1 binding (Fig. 2C and 2D).
3.4 CA suppresses TNF-α-induced inflammation via mTOR
Studies have shown that the mTOR pathway is associated with obesity and insulin resistance [33, 34]. Activation of mTOR plays an important role in TNF-α-induced inflammatory cascades [35]. Upon TNF-α treatment, the phosphorylation of mTOR, p70S6K (Thr421/Ser424), p70S6K (Thr389), eIF4E, and Ser307 IRS-1 was increased
when compared with controls (Fig. 3A). However, these effects were attenuated by treatment with CA and PP242 (an inhibitor of mTOR). The results also indicated that CA and PP242 inhibited the mRNA expression of IL-6 and MCP-1 (Fig. 3B). Moreover, the protein expression of IL-6 was suppressed by CA and PP242 (Fig. 3C).
3.5 CA attenuates the TNF-α-mediated suppression of PPARγ expression
TNF-α treatment decreased the mRNA expression of PPARγ and the PPARγ target genes 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272
aP2 and adiponectin (Fig. 4). However, CA reversed the mRNA inhibition of PPARγ, aP2,
and adiponectin by TNF-α.
3.6 CA restores TNF-α-induced impairment of insulin sensitivity
Treatment of 3T3-L1 adipocytes with insulin or CA alone increased the glucose uptake by 311% and 48%, respectively (Fig. 5A). However, TNF-α treatment suppressed glucose uptake by 26%. TNF-α treatment suppressed insulin-stimulated glucose uptake by 59%. When cells were pretreated with CA, the glucose uptake impaired by TNF-α was restored. We next studied whether CA improved glucose uptake by promoting the
translocation of glucose transporter 4 (GLUT4) from the cytoplasm to the membrane. Immunoblot analysis revealed that insulin or CA increased GLUT4 membrane
translocation, but treatment with TNF-α decreased membrane GLUT4. Moreover, CA restored the insulin-stimulated GLUT4 membrane translocation that was inhibited by TNF-α (Fig. 5B).
3.7 CA enhances the insulin signaling impaired by TNF-α.
As shown in Fig. 6, the phosphorylation of Tyr632 IRS-1, Akt, and FoxO1 was increased,
whereas the phosphorylation of Ser307 IRS-1 as well as total FoxO1 were decreased by
treatment with insulin or CA. The expression of these proteins induced by insulin was reversed by TNF-α. Pretreatment of cells with CA attenuated the TNF-α-induced impairment of insulin signaling.
3.8 CA improves the insulin signaling impaired by TNF-α through the PI3K/Akt 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295
pathway
To further explore whether the PI3K/Akt pathway is involved in the improvement by CA of the insulin signaling impaired by TNF-α in 3T3-L1 adipocytes, we used a PI3K inhibitor (LY 294002). The immunoblot results showed that LY 294002 increased total FoxO1 and decreased phosphorylation of Akt and FoxO1, PPARγ, glucose uptake, and GLUT4 (Fig. 7).
3.9 CA enhances the insulin signaling impaired by TNF-α in primary hepatocytes To investigate whether the protective effect of CA also on hepatocytes, we treated primary culture of hepatocytes with CA for 24 h followed by 24 h of TNF-α cotreatment to induce insulin resistance. As same as the results in 3T3-L1 adipocytes, insulin
treatment increased the phosphorylation of Tyr632 IRS-1, Akt, and FoxO1 in primary
hepatocytes (Fig. 8A). Cells cultured with insulin in the presence of TNF-α decreased the phosphorylation of Tyr632 IRS-1, Akt, and FoxO1, whereas increased the phosphorylation
of Ser307 IRS-1 and total FoxO1. Pretreatment of cells with CA attenuated the
α-induced impairment of insulin signaling. Moreover, the glucose uptake impaired by TNF-α was restored in cells were pretreated with CA (Fig. 8B). Pretreatment of cells with CA increased TNF-α-inhibited glucose uptake by 122%.
296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313
4 Discussion
Obesity-associated inflammation is characterized by the production of inflammatory cytokines in adipose tissues, which plays an important role in the development of insulin resistance. Several phenolic compounds commonly found in vegetables and fruits are reported to prevent inflammatory signaling and insulin resistance. Among those, grape powder extract was shown to attenuate lipopolysaccharide-mediated inflammation by decreasing the activation of MAPK, NF-κB, and AP-1 in human macrophages [36]. In another study, luteolin improved insulin resistance through activation of PPARγ
transcriptional activity in 3T3-L1 adipocytes [37]. CA has been shown to suppress NF-κB luciferase-induced by macrophages and to up-regulate adiponectin secretion in 3T3-L1 adipocytes [38]. In ob/ob mice, CA reduced hepatocyte lipid accumulation and inhibited expression of inflammatory cytokines such as MCP-1 and IL-1β. Moreover, CA reduced lipid accumulation in HepG2 liver cells was associated with reduced expression of epithelium growth factor receptor and mitogen-activated protein kinase [39]. Oh et al. (2012) indicated that CA decreased the production of IL-6, MCP-1, and TNF-α in
keratinocyte HaCaT cells and RAW264.7 cells is associated the Syk/Src pathway [28]. In the present study, we showed that the attenuation of TNF-α-induced inflammation by CA may be partly attributed to its inhibition of the activation of ERK, JNK, NF-κB, and mTOR (Fig. 1-3), which led to down-regulation of IL-6 and MCP-1 expression.
Moreover, CA stimulated glucose uptake and improved insulin resistance by decreasing FoxO1 and increasing the PPARγ pathway (Figs. 4-7).
Several studies have been conducted on the insulin signaling pathway. Generally, insulin binds to the insulin receptor and increases tyrosine phosphorylation of insulin 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336
receptor substrates, which in turn induces activation of PI3K and Akt, GLUT4 translocation to the plasma membrane, and glucose uptake [10, 40]. However,
inflammatory cytokines such as TNF-α activate MAPK, AP-1, and NF-κB pathways and induce serine phosphorylation of IRS, thereby causing impaired insulin signaling
cascades. Studies have indicated that TNF-α activates NF-κB, which in turn enhances the gene expression of protein tyrosine phosphatase-1B, a negative regulator of insulin signaling, and induces dephosphorylation of tyrosine residues on IRS-1 [41]. In the present study, treatment of adipocytes with TNF-α increased the activation of ERK, JNK, AP-1, and NF-κB and subsequently led to increased inflammatory gene expression and insulin resistance (Fig. 1-2). CA inhibited the activity of IKK and the degradation of IκB and the NF-κB binding activity induced by TNF-α (Fig. 2). CA also prevented TNF-α-mediated insulin resistance in 3T3-L1 adipocytes, possibly by inhibiting activation of ERK, JNK, and NF-κB and phosphorylation of Ser307 IRS-1 (Fig. 6).
It has been reported that TNF-α-activated IKKβ directly inhibits tuberous sclerosis complex 1, which then increases mTOR activation [42]. Recently, the TNF-α-activated mTOR pathway was shown to be associated with increased inflammation and insulin resistance via serine phosphorylation of IRS-1 [33, 43]. The mechanism is involved with the activation of p70S6K and the activation of JNK through the induction of the
unfolded-protein response [44, 45]. The activation of mTOR leads to the phosphorylation of p70S6K and eIF4E binding proteins, which then induces protein synthesis [46] . Studies have shown that resveratrol suppresses phosphorylated mTOR and S6RP induced by TNF-α and further reduces the protein expression of IL-1β in 3T3/NIH fibroblasts [47]. In this study, we showed that CA reduced TNF-α-stimulated phosphorylation of 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359
mTOR, p70S6K, eIF4E, and Ser307 IRS-1 and the protein expression of IL-6 (Fig. 3). This
finding suggests that the anti-inflammatory effect of CA may be partly attributed to its inhibition of mTOR.
Our findings suggest that the suppression of PPARγ by TNF-α was reversed by CA. PPARγ is a nuclear receptor that is important for adipogenesis and insulin sensitivity. A clinical study has indicated that PPARγ is a therapeutic target for the prevention and treatment of insulin resistance and obesity-associated inflammation [48]. Activation of NF-κB, ERK, and JNK by TNF-α has been reported to inhibit the transcriptional activity of PPARγ [49, 50]. Furthermore, ERK and JNK were reported to inhibit PPARγ function by direct phosphorylation of Ser112 in PPARγ, and this phosphorylation was shown to
result in inhibition of PPARγ transactivation [51]. In the presence of TNF-α, histone deacetylase 3 (HDAC3) is the co-repressor for PPARγ and is translocated to the nucleus [52]. Studies have reported that HDAC3 combines to the heterodimer of PPARγ and retinoid X receptor and then decreases PPARγ transcriptional activity, leading to insulin resistance [53, 54].
Four isoforms of the FoxO transcription factor family, FoxO1, FoxO3, FoxO4, and FoxO6, have been identified in mammals. FoxO1 is the most abundant FoxO isoform in insulin-responsive tissues such as liver, adipose tissue, and muscle cells, and is negatively regulated by insulin stimulation [55]. Insulin treatment leads to the activation of Akt and subsequent phosphorylation of FoxO1 at Thr24, Ser256, and Ser316 [56]. Phosphorylated
FoxO1 is then excluded from the nucleus and sequestered in the cytosol, where it associates with 14-3-3 proteins, which explains the effect of insulin on inhibition of FoxO1 transactivation [57]. The increased nuclear accumulation of FoxO1 in insulin-360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382
resistant mice lowers the expression of PPARγ target genes [15]. The results of one study support that FoxO1 (TTGTTTAC) binds to the DNA response element located at 310 to 270 bp in the PPARγ promoter and decreases PPARγ expression [21]. Moreover, Ito et al. (2009) indicated that FoxO1 increases the inflammatory gene expression of MCP-1 and IL-6 by inducing C/EBPβ in TNF-α-treated adipocytes [19]. In agreement with these reports, our data showed that exposure of 3T3-L1 adipocytes to TNF-α increased the amount of FoxO1. Treatment with insulin or CA alone increased phosphorylation of FoxO1 but decreased total FoxO1. Moreover, the insulin signaling impairment induced by TNF-α was attenuated by CA (Fig. 6).
The results of the present study indicate that CA attenuates TNF-α-mediated
inflammation and insulin resistance, which is likely through the inhibition of ERK, JNK, NF-κB, and mTOR; the reduction of multiple signaling pathways including
Akt-dependent FoxO1 signaling; and the activation of the PPARγ signaling pathway.
The authors have declared no conflict of interest. 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397
5 References
[1] Fernandez-Veledo, S., Nieto-Vazquez, I., Vila-Bedmar, R., Garcia-Guerra, L., et al., Molecular mechanisms involved in obesity-associated insulin resistance: therapeutical approach. Arch Physiol Biochem 2009, 115, 227-239.
[2] Xie, L., Ortega, M. T., Mora, S., Chapes, S. K., Interactive changes between macrophages and adipocytes. Clin Vaccine Immunol 2010, 17, 651-659. [3] Vinolo, M. A., Rodrigues, H., Festuccia, W. T., Crisma, A., et al., Tributyrin
attenuates obesity-associated inflammation and insulin resistance in high-fat fed mice. Am J Physiol Endocrinol Metab 2012, 303, E272-82.
[4] Sakurai, T., Ogasawara, J., Kizaki, T., Ishibashi, Y., et al., Preventive and improvement effects of exercise training and supplement intake in white adipose tissues on obesity and lifestyle-related diseases. Environ Health Prev Med 2012, 17, 348-56.
[5] Hotamisligil, G. S., Shargill, N. S., Spiegelman, B. M., Adipose expression of tumor necrosis factor-alpha: direct role in obesity-linked insulin resistance. Science 1993, 259, 87-91.
[6] Uysal, K. T., Wiesbrock, S. M., Marino, M. W., Hotamisligil, G. S., Protection from obesity-induced insulin resistance in mice lacking TNF-alpha function. Nature 1997, 389, 610-614.
[7] Gual, P., Le Marchand-Brustel, Y., Tanti, J. F., Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. Biochimie 2005, 87, 99-109. [8] Boura-Halfon, S., Zick, Y., Phosphorylation of IRS proteins, insulin action, and
insulin resistance. Am J Physiol Endocrinol Metab 2009, 296, E581-591. 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420
[9] Tanti, J. F., Jager, J., Cellular mechanisms of insulin resistance: role of
stress-regulated serine kinases and insulin receptor substrates (IRS) serine phosphorylation. Current Opinion in Pharmacology 2009, 9, 753-762.
[10] Jin, L., Shi, G., Ning, G., Li, X., Zhang, Z., Andrographolide attenuates tumor necrosis factor-alpha-induced insulin resistance in 3T3-L1 adipocytes. Mol Cell Endocrinol 2011, 332, 134-139.
[11] Chuang, C. C., Bumrungpert, A., Kennedy, A., Overman, A., et al., Grape powder extract attenuates tumor necrosis factor alpha-mediated inflammation and insulin resistance in primary cultures of human adipocytes. J Nutr Biochem 2011, 22, 89-94. [12] Arkan, M. C., Hevener, A. L., Greten, F. R., Maeda, S., et al., IKK-beta links
inflammation to obesity-induced insulin resistance. Nat Med 2005, 11, 191-198. [13] Viana Abranches, M., Esteves de Oliveira, F. C., Bressan, J., Peroxisome
proliferator-activated receptor: effects on nutritional homeostasis, obesity and diabetes mellitus. Nutr Hosp 2011, 26, 271-279.
[14] Li, H. X., Xiao, L., Wang, C., Gao, J. L., Zhai, Y. G., Review: Epigenetic regulation of adipocyte differentiation and adipogenesis. J Zhejiang Univ Sci B 2010, 11, 784-791.
[15] Fan, W., Imamura, T., Sonoda, N., Sears, D. D., et al., FOXO1 transrepresses peroxisome proliferator-activated receptor gamma transactivation, coordinating an insulin-induced feed-forward response in adipocytes. J Biol Chem 2009, 284, 12188-12197.
[16] Foryst-Ludwig, A., Hartge, M., Clemenz, M., Sprang, C., et al., PPARgamma activation attenuates T-lymphocyte-dependent inflammation of adipose tissue and 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443
development of insulin resistance in obese mice. Cardiovasc Diabetol 2010, 9, 64. [17] Salih, D. A., Brunet, A., FoxO transcription factors in the maintenance of cellular
homeostasis during aging. Curr Opin Cell Biol 2008, 20, 126-136.
[18] Ponugoti, B., Dong, G., Graves, D. T., Role of forkhead transcription factors in diabetes-induced oxidative stress. Exp Diabetes Res 2012, 2012, 939751. [19] Ito, Y., Daitoku, H., Fukamizu, A., Foxo1 increases pro-inflammatory gene
expression by inducing C/EBPbeta in TNF-alpha-treated adipocytes. Biochem Biophys Res Commun 2009, 378, 290-295.
[20] Nakae, J., Cao, Y., Oki, M., Orba, Y., et al., Forkhead transcription factor FoxO1 in adipose tissue regulates energy storage and expenditure. Diabetes 2008, 57, 563-576.
[21] Armoni, M., Harel, C., Karni, S., Chen, H., et al., FOXO1 represses peroxisome proliferator-activated receptor-gamma1 and -gamma2 gene promoters in primary adipocytes. A novel paradigm to increase insulin sensitivity. J Biol Chem 2006, 281, 19881-19891.
[22] Ibarra, A., Cases, J., Bily, A., He, K., et al., Importance of extract standardization and in vitro/ex vivo assay selection for the evaluation of antioxidant activity of botanicals: a case study on three Rosmarinus officinalis L. extracts. J Med Food 2010, 13, 1167-1175.
[23] Ibarra, A., Cases, J., Roller, M., Chiralt-Boix, A., et al., Carnosic acid-rich rosemary (Rosmarinus officinalis L.) leaf extract limits weight gain and improves cholesterol levels and glycaemia in mice on a high-fat diet. Br J Nutr 2011, 106, 1182-1189. [24] Harach, T., Aprikian, O., Monnard, I., Moulin, J., et al., Rosemary (Rosmarinus 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466
officinalis L.) leaf extract limits weight gain and liver steatosis in mice fed a high-fat diet. Planta Med 2010, 76, 566-571.
[25] Wijeratne, S. S., Cuppett, S. L., Potential of rosemary (Rosemarinus officinalis L.) diterpenes in preventing lipid hydroperoxide-mediated oxidative stress in Caco-2 cells. J Agric Food Chem 2007, 55, 1193-1199.
[26] Haraguchi, H., Saito, T., Okamura, N., Yagi, A., Inhibition of lipid peroxidation and superoxide generation by diterpenoids from Rosmarinus officinalis. Planta Med 1995, 61, 333-336.
[27] Mengoni, E. S., Vichera, G., Rigano, L. A., Rodriguez-Puebla, M. L., et al., Suppression of COX-2, IL-1beta and TNF-alpha expression and leukocyte
infiltration in inflamed skin by bioactive compounds from Rosmarinus officinalis L. Fitoterapia 2011, 82, 414-421.
[28] Oh, J., Yu, T., Choi, S. J., Yang, Y., et al., Syk/Src pathway-targeted inhibition of skin inflammatory responses by carnosic acid. Mediators Inflamm, 2012, 781375. [29] Wang, T., Takikawa, Y., Satoh, T., Yoshioka, Y., et al., Carnosic acid prevents
obesity and hepatic steatosis in ob/ob mice. Hepatol Res 2011, 41, 87-92.
[30] Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Guide for the Care and Use of Laboratory Animals. National Academy Press, Washington, DC; 1985.
[31] Shi, J., Kandror, K. V., Study of glucose uptake in adipose cells. Methods Mol Biol 2008, 456, 307-315.
[32] Kaminska, B., MAPK signalling pathways as molecular targets for
anti-inflammatory therapy--from molecular mechanisms to therapeutic benefits. Biochim 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489
Biophys Acta 2005, 1754, 253-262.
[33] Adochio, R., Leitner, J. W., Hedlund, R., Draznin, B., Rescuing 3T3-L1 adipocytes from insulin resistance induced by stimulation of Akt-mammalian target of
rapamycin/p70 S6 kinase (S6K1) pathway and serine phosphorylation of insulin receptor substrate-1: effect of reduced expression of p85alpha subunit of
phosphatidylinositol 3-kinase and S6K1 kinase. Endocrinology 2009, 150, 1165-1173.
[34] Weichhart, T., Costantino, G., Poglitsch, M., Rosner, M., et al., The TSC-mTOR signaling pathway regulates the innate inflammatory response. Immunity 2008, 29, 565-577.
[35] Lorne, E., Zhao, X., Zmijewski, J. W., Liu, G., et al., Participation of mammalian target of rapamycin complex 1 in Toll-like receptor 2- and 4-induced neutrophil activation and acute lung injury. Am J Respir Cell Mol Biol 2009, 41, 237-245. [36] Overman, A., Bumrungpert, A., Kennedy, A., Martinez, K., et al., Polyphenol-rich
grape powder extract (GPE) attenuates inflammation in human macrophages and in human adipocytes exposed to macrophage-conditioned media. Int J Obes (Lond) 2010, 34, 800-808.
[37] Ding, L., Jin, D., Chen, X., Luteolin enhances insulin sensitivity via activation of PPARgamma transcriptional activity in adipocytes. J Nutr Biochem 2010, 21, 941-947.
[38] Nagasaki, H., Yoshimura, T., Aoki, N., Real-time monitoring of inflammation status in 3T3-L1 adipocytes possessing a secretory Gaussia luciferase gene under the control of nuclear factor-kappa B response element. Biochem Biophys Res Commun 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512
2012, 420, 623-627.
[39] Wang, T., Takikawa, Y., Tabuchi, T., Satoh, T., et al., Carnosic acid (CA) prevents lipid accumulation in hepatocytes through the EGFR/MAPK pathway. J
Gastroenterol 2012, 47, 805-813.
[40] Khan, A. H., Pessin, J. E., Insulin regulation of glucose uptake: a complex interplay of intracellular signalling pathways. Diabetologia 2002, 45, 1475-1483.
[41] Zabolotny, J. M., Kim, Y. B., Welsh, L. A., Kershaw, E. E., et al., Protein-tyrosine phosphatase 1B expression is induced by inflammation in vivo. J Biol Chem 2008, 283, 14230-14241.
[42] Lee, D. F., Kuo, H. P., Chen, C. T., Hsu, J. M., et al., IKK beta suppression of TSC1 links inflammation and tumor angiogenesis via the mTOR pathway. Cell 2007, 130, 440-455.
[43] Ozes, O. N., Akca, H., Mayo, L. D., Gustin, J. A., et al., A phosphatidylinositol 3-kinase/Akt/mTOR pathway mediates and PTEN antagonizes tumor necrosis factor inhibition of insulin signaling through insulin receptor substrate-1. Proc Natl Acad Sci U S A 2001, 98, 4640-4645.
[44] Ozcan, U., Ozcan, L., Yilmaz, E., Duvel, K., et al., Loss of the tuberous sclerosis complex tumor suppressors triggers the unfolded protein response to regulate insulin signaling and apoptosis. Mol Cell 2008, 29, 541-551.
[45] Harrington, L. S., Findlay, G. M., Gray, A., Tolkacheva, T., et al., The TSC1-2 tumor suppressor controls insulin-PI3K signaling via regulation of IRS proteins. J Cell Biol 2004, 166, 213-223.
[46] Schaeffer, V., Abrass, C. K., Mechanisms and control of protein translation in the 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535
kidney. Am J Nephrol 2010, 31, 189-201.
[47] Zhu, X., Liu, Q., Wang, M., Liang, M., et al., Activation of Sirt1 by resveratrol inhibits TNF-alpha induced inflammation in fibroblasts. PLoS One 2011, 6, e27081. [48] Ricote, M., Glass, C. K., PPARs and molecular mechanisms of transrepression.
Biochim Biophys Acta 2007, 1771, 926-935.
[49] Camp, H. S., Tafuri, S. R., Regulation of peroxisome proliferator-activated receptor gamma activity by mitogen-activated protein kinase. J Biol Chem 1997, 272, 10811-10816.
[50] Ruan, H., Pownall, H. J., Lodish, H. F., Troglitazone antagonizes tumor necrosis factor-alpha-induced reprogramming of adipocyte gene expression by inhibiting the transcriptional regulatory functions of NF-kappaB. J Biol Chem 2003, 278, 28181-28192.
[51] Hosooka, T., Noguchi, T., Kotani, K., Nakamura, T., et al., Dok1 mediates high-fat diet-induced adipocyte hypertrophy and obesity through modulation of PPAR-gamma phosphorylation. Nat Med 2008, 14, 188-193.
[52] Ye, J., Regulation of PPARgamma function by TNF-alpha. Biochem Biophys Res Commun 2008, 374, 405-408.
[53] Sun, Z., Miller, R. A., Patel, R. T., Chen, J., et al., Hepatic Hdac3 promotes
gluconeogenesis by repressing lipid synthesis and sequestration. Nat Med 2012, 18, 934-42.
[54] Li, D., Wang, X., Ren, W., Ren, J., et al., High expression of liver histone deacetylase 3 contributes to high-fat-diet-induced metabolic syndrome by suppressing the PPAR-gamma and LXR-alpha-pathways in E3 rats. Mol Cell 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558
Endocrinol 2011, 344, 69-80.
[55] Dominy, J. E., Puigserver, P., Nuclear FoxO1 inflames insulin resistance. EMBO J 2010, 29, 4068-4069.
[56] Brent, M. M., Anand, R., Marmorstein, R., Structural basis for DNA recognition by FoxO1 and its regulation by posttranslational modification. Structure 2008, 16, 1407-1416.
[57] Tzivion, G., Dobson, M., Ramakrishnan, G., FoxO transcription factors; Regulation by AKT and 14-3-3 proteins. Biochim Biophys Acta 2011, 1813, 1938-1945.
559 560 561 562 563 564 565 566 567
Figure 1. CA attenuates TNF-α-mediated inflammatory gene expression in 3T3-L1
adipocytes. (A) 3T3-L1 adipocytes were pretreated with 0.1% DMSO or with 1, 5, 10, or 20 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 24 h. mRNA
levels of IL-6 and MCP-1 were determined by RT-PCR. (B) The protein levels of phospho (p) or total ERK, JNK, and p38 were determined by immunoblotting. The bands were quantified by densitometry, and the level in control cells was regarded as 1. Values are the mean (SD) of three independent experiments. Groups without a common letter differ significantly (p <0.05).
Figure 2. CA attenuates TNF-α-induced activation of NF-κB and AP-1 in 3T3-L1 adipocytes. 3T3-L1 adipocytes were pretreated with 0.1% DMSO or with 10 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 1 or 3 h. Cultures were then harvested to determine the protein concentration of (A) IKKα/β, IκBα, and (B) nuclear p65 (1 h) by immunoblot assay. (C) Nuclear extracts were prepared to measure NF-κB (1 h) and AP-1 (3 h) binding activity by electrophoretic mobility shift assay. Unlabeled double-stranded oligonucleotides (cold) and mutant double-stranded oligonucleotides (mut) were added for the specificity assay. (D) Protein levels of phospho (p) or total c-Jun (3 h) were determined by immunoblot assay. Changes in protein expression were measured by densitometry. Data were normalized to β-actin expression. The level in control cells was set at 1. Values are the mean (SD) of three independent
experiments. Groups without a common letter differ significantly (p < 0.05).
Figure 3. CA suppresses TNF-α-induced inflammation via mTOR in 3T3-L1 adipocytes. Cells were treated with 0.1% DMSO or with 10 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 3 h (A) or 24 h (B). PP242 (20 nM) was added 1 h before TNF-α treatment. 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590
(A) Cultures were then harvested to determine the protein concentrations of phospho (p) or total mTOR, P70S6K, eIF4E, and Ser307 p-IRS-1 by immunoblotting. (B) mRNA levels of IL-6 and
MCP-1 were determined by RT-PCR. (C) Protein levels of IL-6 were determined by
immunoblotting. The bands were quantified by densitometry, and the level in control cells was regarded as 1. Values are the mean (SD) of three independent experiments. Groups without a common letter differ significantly (p <0.05).
Figure 4. CA attenuates TNF-α-mediated suppression of PPARγ expression in 3T3-L1
adipocytes. Cells were pretreated with 0.1％ DMSO or with 1, 5, 10, or 20 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 24 h. Cultures were harvested to determine mRNA concentrations of PPARγ, adiponectin, and aP2 by RT-PCR. The mRNA levels of the
bands were quantified by densitometry, and the level in control cells was regarded as 1. Values are the mean (SD) of three independent experiments. Groups without a common letter differ significantly (p < 0.05).
Figure 5. CA restores TNF-α-attenuated insulin sensitivity in 3T3-L1 adipocytes. Cultures were pretreated with 0.1% DMSO or with 10 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 24 h. Insulin (10 nM) was added at the last 60 min. (A) Cultures were then harvested to determine the glucose uptake. Values are the mean±SD of three independent
experiments. Data were analyzed by using Student’s t-test. (B) The protein expression of GLUT4 was determined by immunoblotting. The protein levels of the bands were quantified by
densitometry. Data were normalized to clathrin expression. The level in control cells was set at 1. Values are the mean (SD) of three independent experiments. Data were analyzed by using 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613
Student’s t-test. * p < 0.05 compared with the control. ＃p < 0.05 compared with insulin
treatment. ＆p < 0.05 compared with insulin plus TNF-α.
Figure 6. CA attenuates TNF-α-mediated suppression of the insulin signaling pathway in 3T3-L1 adipocytes. Cultures were pretreated with 0.1% DMSO or with 10 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 24 h. Insulin (10 nM) was added at the last 30 min. Cultures were then harvested to determine the protein concentrations of phospho (p) or total IRS-1, Akt, and FoxO1 by immunoblotting. The protein levels of the bands were quantified by densitometry. The level in control cells was set at 1. Data were analyzed by using Student’s t-test. * p < 0.05 compared with the control. ＃p < 0.05 compared with insulin treatment. ＆p < 0.05
compared with insulin plus TNF-α.
Figure 7. CA improves insulin signaling impaired by TNF-α through the PI3K/Akt pathway. Cultures were pretreated with 10 μM CA for 24 h and were then treated with or without 5 ng/mL TNF-α for 24 h. Insulin (10 nM) was added at the last 30 or 60 min. LY294002 was added to cells for 1 h before TNF-α treatment. (A) Cultures were then harvested to determine the protein concentrations of PPARγ and phospho (p) or total Akt and FoxO1 by immunoblotting. (B) Cultures were then harvested to determine glucose uptake. (C) The protein expression of GLUT4 was determined by immunoblotting. Data were analyzed by using Student’s t-test. * p < 0.05 compared with insulin plus TNF-α and CA. One representative experiment out of three independent experiments is shown.
Figure 8. CA attenuates TNF-α-mediated suppression of the insulin signaling pathway in 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 629 630 631 632 633 634 635 636
primary hepatocytes. Cultures were pretreated with 0.1% DMSO or with 10 μM CA for 24 h and were then treated with or without 20 ng/mL TNF-α for 24 h. Insulin (10 nM) was added at the last 30 or 60 min. (A) Cultures were then harvested to determine the protein concentrations of phospho (p) or total IRS-1, Akt, and FoxO1 by immunoblotting. The protein levels of the bands were quantified by densitometry. The level in control cells was set at 1. Values are the mean (SD) of three independent experiments. (B) Cultures were then harvested to determine the glucose uptake. Values are the mean±SD of three independent experiments. Data were analyzed by using Student’s t-test. * p < 0.05 compared with the control. ＃p < 0.05 compared with insulin
treatment. ＆p < 0.05 compared with insulin plus TNF-α.
637 638 639 640 641 642 643 644 645 646 647 648
