Metallothioneins regulate the adipogenic differentiation of 3T3-L1 cells via the insulin signaling pathway

<div><p>Knockout of metallothionein (MT) genes contributes to a heavier body weight in early life and the potential to become obese through the intake of a high fat diet (HFD) in mice. It has thus been suggested that MT genes regulate the formation of adipose tissue, which would become the base for later HFD-induced obesity. We evaluated the fat pads of mice during the lactation stage. The fat mass and adipocyte size of MT1 and MT2 knockout mice were greater than those of wild type mice. Next, we assayed the ability of small interfering RNA (siRNA) to silence MT genes in the 3T3-L1 cell line. The expressions of MT1 and MT2 genes were transiently upregulated during adipocyte differentiation, and the siRNA pretreatment led to the suppression of the expression of both MT mRNAs and proteins. The MT siRNA promoted lipid accumulation in adipocytes and caused proliferation of post-confluent preadipocytes; these effects were suppressed by an inhibitor of phosphatidylinositol 3-kinase (LY294002). In addition, MT siRNA promoted insulin-stimulated phosphorylation of Akt, a downstream kinase of the insulin signaling pathway. Enhanced lipid accumulation in 3T3-L1 cells resulting from MT-gene silencing was inhibited by pretreatment with an antioxidant, <i>N</i>-acetylcysteine, used as a substitute for antioxidant protein MTs. These results suggest that interference in MT expression enhanced the activation of the insulin signaling pathway, resulting in higher lipid accumulation in 3T3-L1 adipocytes.</p></div

White adipose tissues (WATs) of five-day-old wild type (WT) and MTKO mice.

<p>Dorsal-ventral subcutaneous fat pads were excised from five-day-old mice. (A) Representative histology images of WAT in five-day-old WT and MTKO mice. WATs were larger in MTKO mice than in WT mice. Bar = 100 μm. (B) WAT weight and (C) body weight of five-day-old WT and MTKO mice. (D) Percent WAT weight was calculated by dividing fat mass by body weight. Values are presented as the mean ± SD (<i>n</i> = 5). ** represents a <i>P</i> value of < 0.01.</p

Scheme of a potential mechanism for the regulation of the insulin/Akt signaling pathway by MTs.

<p>The adipogenic differentiation of 3T3-L1 preadipocytes into adipocytes is induced with differentiation-inducing medium (DIM) containing insulin, dexamethasone (DEX), and IBMX. ROS activate insulin/Akt signaling and promote mitotic clonal expansion. Metallothioneins (MTs), which are induced by DEX treatment, may regulate insulin-Akt signaling by scavenging the ROS. IR, insulin receptor; GR, glucocorticoid receptor.</p

Effect of MT gene silencing and MT knockout on Akt phosphorylation.

<p>Cell lysates and tissue extracts were separated on 10% gels using SDS-PAGE. Immunoblotting was performed with the indicated antibodies. The phosphorylation ratios of Akt (pAkt) proteins or the expression ratios of PPARγ to β-actin were calculated by densitometric analyses of immunoreactive bands. (A) After siRNA pretreatment, the cells were pre-incubated with 1 μM DEX and 0.5 mM IBMX for 2 h and incubated in serum-free medium for 1 h. Then, the serum-free medium in the presence or absence of 10 nM insulin was added to the cells for 30 min. Open columns are cells untreated with insulin and closed columns are cells treated with 10 nM insulin. Data are expressed as the mean ± SD (<i>n</i> = 3). * and ** represent <i>P</i> values of < 0.05 and < 0.01, respectively. (B) Extracts from the adipose tissue of five-day-old mice were subjected to electrophoresis on SDS-PAGE gel. Open columns are WT mice and closed columns are MTKO mice. Data are expressed as the mean ± SD (<i>n</i> = 5). * represents <i>P</i> values of < 0.05. (C) ADCs derived from the adipose tissues of five-day-old WT and MTKO mice (two individuals of each genotype, Case #1 and Case #2) were incubated with 1 nM insulin in serum-free medium for the indicated time.</p

Effect of MTs siRNA on lipid accumulation in 3T3-L1 adipocytes.

<p>3T3-L1 preadipocytes were treated with 50 nM control RNA or MTs siRNA 24 h before the induction of differentiation. On day 2, the culture medium was changed to medium supplemented with 1 μg/ml insulin. On or after day 4, the medium was replaced every 2 days. Natural lipids in the fixed cells were stained with Oil Red O. (A) Representative images of Oil Red O staining of 3T3-L1 adipocytes pretreated with control RNA (control) or MTs siRNA. (B) Absorbance of Oil Red O was measured, and Oil Red O retention is expressed as a percentage relative to the control RNA-treated cells. Data are expressed as the mean ± SD (<i>n</i> = 5). ** represents a <i>P</i> value of < 0.01.</p

Effects of MTs siRNA on the expression of MTs and adipocyte-related genes and proteins.

<p>3T3-L1 preadipocytes were treated with 50 nM control RNA (open circle) or MTs siRNA (closed circle) 24 h before the addition of DIM (day -1). Total RNA was isolated on day 0, 1, 4, and 8, and the expressions of (A) <i>mt1</i>, (B) <i>mt2</i>, (C) <i>pparγ</i>, (D) <i>fabp4</i>, (E) <i>cebpβ</i>, and (F) <i>cebpδ</i>, were determined using real-time PCR. All mRNA levels were normalized to the expression level of the <i>36B4</i> gene and are shown as fold induction from the control RNA-treated cells on day 0. Data are expressed as the mean ± SD (<i>n</i> = 3). ^**<i>P</i> < 0.01, compared with control RNA-treated cells at the same time point. (G) Cell lysates from the control RNA-treated cells (control) and the MTs siRNA-treated cells (MTs siRNA) were obtained at the indicated points. The protein expression of PPARγ, FABP4, C/EBPβ, C/EBPδ, and FAS (with 36B4 as a loading control) during the adipogenic differentiation of 3T3-L1 cells was determined using immunoblotting.</p

Effect of MTs siRNA on BrdU incorporation during post-confluent mitosis, known as mitosis clonal expansion.

<p>After siRNA pretreatment for 24 h, 3T3-L1 preadipocytes were stimulated with DIM for 0 or 24 h followed by exposure to 10 μM BrdU for 2 h. (A) The BrdU incorporated into replicating DNA was detected using an anti-BrdU primary antibody and fluorescein-conjugated secondary antibody using a fluorescence microscope (green). All nuclei were counterstained with Hoechest 33258 (blue). Bar = 200 μm. (B) The number of BrdU-positive cells is expressed as a percentage of Hoechst-stained nuclei. (C) and (D) After siRNA pretreatment for 24 h, 3T3-L1 preadipocytes were not treated (VC, vehicle control (DMSO)), or were treated with the indicated inhibitors (50 μM PD98059 (PD), 50 μM LY294002 (LY), or H89) for 30 min. (C) After treatment, the cells were stimulated with DIM for 24 h. The number of BrdU-positive cells is expressed as a percentage of the number of MTs siRNA-treated cells without inhibitor treatment. (D) After treatment, 3T3-L1 preadipocytes were induced to differentiate for eight days, as described in the Materials and Methods section. Natural lipids in the fixed cells were stained with Oil Red O. The absorbance of Oil Red O was measured, and Oil Red O retention is expressed as a percentage of the number of MTs siRNA-treated cells without inhibitor treatment. (B), (C), and (D) Open columns are control RNA-pretreated cells, and closed columns are MTs siRNA-pretreated cells. Data are expressed as the mean ± SD (B and C, <i>n</i> = 3; D, <i>n</i> = 5). (B) ** <i>P</i> < 0.01. (C) and (D) * <i>P</i> < 0.05 and ** <i>P</i> < 0.01, compared to MTs siRNA-treated cells without inhibitor treatment.</p

Effect of standard DIM and three separate components on MT expression.

<p>3T3-L1 preadipocytes were treated with DIM or three components separately (1 μg/ml insulin (INS), 1 μM DEX, and 0.5 mM IBMX). A thiazolidinedione, i.e., pioglitazone (PTZ), was also used at a concentration of 1 μM. (A) The expression levels of <i>mt1</i> mRNA (open columns) and <i>mt2</i> mRNA (filled columns) are shown as percentages relative to the cells cultured in DMEM + 10% FBS (cont.). Data are expressed as the mean ± SD (<i>n</i> = 3). ^**<i>P</i> < 0.01, compared with the control cells. (B) The expressions of the MT proteins were detected using anti-MT1 and MT2 total proteins followed by Alexa Fluor 488-conjugated secondary antibody (Green). Negative control (NC); staining without the primary antibody. Nuclei were stained with Hoechst 33258 (blue). Bar = 40 μm.</p

Effects of MTs siRNA on the expression of MT1 and MT2 total proteins.

<p>3T3-L1 preadipocytes were treated with 50 nM control RNA or MTs siRNA 24 h before the addition of DIM (day -1). The cells were fixed with 4% paraformaldehyde on day 0, 1, and 2, and the expression of MT proteins was detected using a primary antibody against mouse MT1 and MT2 total proteins followed by Alexa Fluor 488-conjugated secondary antibody (Green). Nuclei were stained with Hoechst 33258 (blue). Bar = 40 μm.</p

Relative myosin heavy chain isoform (MyHC1, 2A, 2X, and 2B) mRNA expression in EDL and soleus muscles from soybean oil-fed (open bars), fish oil-fed (filled bars), and lard-fed (gray bars) rats.

<p>Values are calculated based on the ΔCt method (where Ct is the threshold cycle) [25], using the following formula: 2^{-(Ct(target gene) – Ct(β-actin))}, which gives the relative target gene expression compared to β-actin expression. The values are means ± SE for six rats. Relative MyHC2X mRNA expression levels in the EDL differed significantly (p < 0.05) between soybean oil-fed and fish oil-fed rats. Different superscripts indicate a significant difference between two groups (p < 0.05, one-way ANOVA; post hoc: Tukey–Kramer multiple-comparison test).</p