Vanadium and insulin increase adiponectin production in 3T3-L1 adipocytes

Pharmacological Research 54 (2006) 30–38

Vanadium and insulin increase adiponectin production in 3T3-L1 adipocytes Andre P. Seale ∗ , Lucia A. de Jesus, Min-Chul Park, Yang-Sun Kim Proteonik Inc., Gyeonggi Technopark, Room 911, 1271-11 Sa-1-Dong, Sangnok-Gu, Ansan 425-170, South Korea Accepted 30 January 2006

Abstract Both adiponectin, an adipokine secreted by adipocytes, and vanadium compounds, have been extensively shown to enhance insulin sensitivity in vivo and in vitro. In this study we examined whether insulin and vanadyl sulfate (VS) affected adiponectin release and cell content from 3T3-L1 adipocytes, and whether they acted through a similar signaling pathway. Adiponectin cell content, but not release, consistently increased in cells treated with insulin (100 nM) and VS (10 and 50 ␮M) after 24 h. On the other hand, VS-induced adiponectin release only occurred after 4 days of incubation. The protein kinase B (PKB) inhibitor, NL-71-101, decreased both insulin and VS-induced adiponectin cell content, while neither wortmannin nor LY 294002, inhibitors of phosphatidylinositol 3-kinase (PI3-K), attenuated insulin or VS-induced adiponectin cell content. Furthermore, VS-induced adiponectin accumulation occurred in the presence of AGL2263, an insulin receptor (IR) inhibitor. These studies provide the first evidence that vanadium could exert its insulin sensitizing effects through the stimulation of adiponectin through a PKB-dependent transduction pathway. © 2006 Elsevier Ltd. All rights reserved. Keywords: Vanadium; Adiponectin; Adipocyte; Insulin; Signal transduction

1. Introduction Type 2 diabetes, a metabolic disorder that is reaching epidemic proportions in most countries, affects over 150 million adults worldwide and its incidence is expected to double over the next 25 years [1]. The first reported use of the trace metal vanadium to treat diabetes mellitus dates back to 1899 [2], and since then numerous studies have described the in vivo and in vitro anti-diabetic effects of vanadium salts and compounds (for recent reviews, see [3–5]). Vanadium salts have been shown to mimic the biological actions of insulin such as decreasing plasma glucose levels in diabetic mice models [6-8], and promoting the glucose transporter protein 4 (GLUT-4) translocation and glucose uptake in adipose and muscle cells [9–12]. Currently, the main mode of action of vanadium is believed to occur through the inhibition of the phosphatase PTP-1B [13,14]. Adipocytes play a crucial role in energy storage and homeostasis by converting free fatty acids into triglycerides and by producing a variety of adipokines, and therefore provide a very useful model for addressing questions related to insulin signal-

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Corresponding author. Tel.: +82 31 500 4070; fax: +82 31 500 4074. E-mail address: [email protected] (A.P. Seale).

1043-6618/$ – see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2006.01.013

ing and the interface between sugar and fat metabolism [15]. While much is known about the effects of vanadium on the sugar metabolism and insulin signaling pathway in adipocytes, less attention has been devoted to the possible effects it could have on adipokines, such as the recently discovered ACRP-30 or adiponectin [16]. Several factors have been shown to regulate adiponectin expression and release, including stimulation by thiazolidinedione, a peroxisome proliferator-activated receptor ␥ (PPAR␥) agonist, and ionomycin, a calcium ionophore, and inhibition by tumor necrosis factor (TNF)-␣, interleukin-6 (IL-6), and glucocorticoids [17–21]. The effects of vanadium on adiponectin synthesis and release have not been examined to date, but based on the similarity to insulin with respect to actions on cell signaling and metabolism, we hypothesized that insulin and vanadium would share a similar effect and transduction pathway. However, the role of insulin in regulating adiponectin secretion is not very clear. The effect of insulin on short-term (1 h) adiponectin release in cultured 3T3-L1 adipocytes has been shown to be stimulatory [22,23], whereas the effect on adiponectin gene expression on the same cell type after 16 h was inhibitory [24]. In the present study we examined the effects of insulin and vanadium on adiponectin release and cell content from 3T3-L1 adipocytes. Furthermore, with the use of pharmacological inhibitors, we addressed the involvement of insulin

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receptor (IR), phosphatidylinositol 3-kinase (PI3-K) and protein kinase B (PKB) in both insulin and vanadium-induced alterations of adiponectin cell content. 2. Materials and methods 2.1. Reagents Vanadyl sulfate (VS), sodium orthovanadate and insulin were purchased from Sigma (St. Louis, USA). Bismaltolato oxovanadium (BMOV) was kindly provided by Prof. Cheong-Soo Hwang (Dankook University). Wortmannin, NL71-101, potassium bisperoxo(1,10-phenanthroline)oxovanadate V (bpV(Phen)), AGL-2263, LY294002, TNF-␣ and murine ACRP-30 antibody were obtained from Calbiochem-Merck (Darmstadt, Germany). The polyclonal ACRP-30 antibody, which was generated using two peptides (EDDVTTTEELAPALV; FTYDQYQEKNVDQA) corresponding to amino acids 18–32 and 187–200 of mouse ACRP-30, recognizes the 30 kDa adiponectin in mouse serum [16] and secreted from 3T3-L1 adipocytes [22]. The mouse monoclonal anti-␤-actin was purchased from Sigma (St. Louis, USA). Secondary antibodies and ECL chemiluminescence reagents were purchased from Amersham Biosciences (Piscataway, USA). Western blot semidry apparatus, Laemmli sample buffer, BioRad protein assay dye reagent and Criterion gels were from Bio-Rad (Hercules, USA). 2.2. 3T3-L1 cell culture and differentiation 3T3-L1 mouse fibroblasts were purchased from American Type Culture Collection and cultured according to the provider’s established protocol (ATTC® , Manassas, USA). Fibroblasts were cultured in Dulbecco’s-modified Eagle’s medium (DMEM) containing 4.5 g/l d-glucose with 10% heat-innactivated fetal bovine serum (FBS, Sigma, St. Louis, USA) at 37 ◦ C and 5% CO2 atmosphere. Cells were sub-cultured after reaching 70% confluence, every 2 days. For differentiation into adipocytes, 500 ␮l of fibroblast-containing media were plated per well of 24-well plates at approximately 4 × 104 cells/ml. Cells were incubated until reaching confluency, and incubated for another 2 days with D-MEM enriched with 0.5 mM 3isobuthyl-1-methyl-xantine (IBMX, Sigma, St. Louis, USA), 0.25 ␮M dexamethasone and 1 ␮g/ml insulin. Medium was then supplemented with insulin only, and adipocytes grew for two additional days. After this period, adipocytes were kept in DMEM medium supplemented only with 10% FBS, for another 4–7 days until experimentation. To minimize sample variability, only wells in which over 90% of the cells showed fat accumulation, and where cell coverage on the plate exceeded 85%, were used in experiments. At the end of 24 h experimental periods, adipocyte viability in control, insulin and VStreated samples averaged above 90% as estimated by Trypan blue (0.2%) exclusion test. Serum-free D-MEM medium was employed during all experimental incubations unless otherwise noted.

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2.3. Measurement of stored and released adiponectin Prior to experimentation, fully differentiated adipocytes were washed four times with serum-free D-MEM, including a 2 h pre-incubation period in control conditions. Adipocytes were then incubated in serum-free D-MEM medium containing the appropriate treatments for the designated times, as outlined in the results. At termination, medium was collected and kept at −20 ◦ C until analysis. Cells were pelleted, sonicated in 300 ␮l lysis buffer (1% NP-40, 15 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) containing 1:1000 type III protease inhibitor cocktail (Calbiochem-Merck, Darmstadt, Germany) and 200 ␮M sodium orthovanadate, and kept at −20 ◦ C. Sub-samples (7.5 ␮l) of media and cell lysates were solubilized in an equivalent volume of Laemmli sample buffer containing 100 mM DTT and heated at 99 ◦ C for 5 min. Proteins were separated by 10–20% Tris–HCl sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose membranes in buffer containing 20% methanol, 0.3% Tris base and 0.2 M glycine. Membranes were blocked with PBS-T containing 1% proprietary blocking reagent (Amersham Biosciences, Piscataway, USA), incubated for at least 1 h at room temperature with murine ACRP-30 polyclonal antibody (1:5000), washed with PBS-T and incubated with anti-rabbit-IgG-horseradish peroxidase-conjugated secondary antibody (1:10,000). Prior to detection, blots were washed with PBS-T and developed using ECL chemiluminescence reagents according to manufacturer’s instructions (Amersham Biosciences, Piscataway, USA). Protein content of untreated adipocytes within a plate, detected using BioRad protein assay dye reagent and quantified using a BSA standard curve, were compared to determine the extent of well-to-well variation, and whether this variation could compromise the ability to detect adiponectin release and cell content among groups. There was no significant variation in protein or adiponectin content among untreated wells (data not shown), therefore statistical differences observed between treatments throughout the study unlikely result from variations in cell number among culture wells alone. Sample loading and transfer were controlled by reprobing membranes with mouse monoclonal anti␤-actin. Intensity was quantified by measuring backgroundsubtracted pixel densities using ChemiDoc XRS imaging system with Quantity One software (Bio-Rad, Hercules, USA). All membranes were loaded with two protein standard markers, SeeBlue® Plus2 and Magic MarkTM XP western standard (Invitrogen, Carlsbad, USA). From both medium and cell lysate samples, all bands detected with ACRP-30 antibody were localized close to 30 kDa, indicating that full adiponectin monomers were consistently analyzed. Bands representing ␤-actin (42 kDa) and IR-␤ (90 kDa) were located closest to 40 and 98 kDa standards, respectively. 2.4. Measurement of insulin receptor activation Membranes from selected experiments were stripped with a solution containing 2% SDS, 62.5 mM Tris–HCl pH 6.7 and 100 mM 2-mercaptoethanol for 1 h at 70 ◦ C under constant shak-

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ing. After washing twice with PBS-T, membranes were incubated with 1% proprietary blocking reagent for 1 h, followed by 1:2000 rabbit polyclonal insulin receptor beta (IR-␤) antibody (Santa Cruz Biotechnology, Santa Cruz, USA) prepared in 1% blocking reagent. Addition of secondary antibody and band quantification proceeded as described for adiponectin detection. Membranes were stripped once more as described above, and incubated with 1:500 mouse monoclonal phosphotyrosine antibody (4G10, Upstate, New York, USA). Bands were quantified using Quantity One software and aligned with those detected with anti IR-␤. Results were displayed as the backgroundsubtracted pixel density ratio of phosphotyrosine over IR-␤ as described previously [13].

2.5. Statistical analysis For all adiponectin release and cell content, individual sample values were expressed as percent change from the mean of control samples within a given experiment, where, excluding the addition or not of experimental treatments, all samples were processed identically from cell culture to data acquisition. In the case of cell content data, sample values were normalized by ␤-actin prior to percent change calculations. Data was then combined from identical experiments and statistical comparisons were carried out. Comparisons between treatments were performed using a one-way ANOVA, followed by Fisher’s least significant difference test to determine individual differences

Fig. 1. Dose response of VS and insulin on adiponectin release and cell content. Effects of 1, 10 and 100 nM insulin on adiponectin release were measured at 1 h (A), 24 h (B) and adiponectin cell content was measured after 24 h (C). Effects of 1–100 ␮M VS on adiponectin release at 1 h (D), 24 h (E) and cell content (F). Representative adiponectin band images are shown above each respective treatment bar, followed by ␤-actin band images, which were used to normalize cell content data. Molecular weight standard band sizes are indicated in between band images. Bars represent mean percent change from control ± standard error (S.E.), n = 4–6 for 1 h release and cell content determinations and n = 6–10 for 24 h release. *** P < 0.001, ** P < 0.01, * P < 0.05.

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amongst treatments. Significance level was set at 95% (P < 0.05). Calculations and analysis were performed using Minitab Statistical Software Package (State College, USA). 3. Results 3.1. Adiponectin cell content but not release is increased by insulin and vanadium in a dose-related manner after 24 h In the present studies insulin was employed to clarify its effects on adiponectin release and synthesis over 24 h and to serve as a positive control for all vanadium experiments. In the following experiment, adipocytes were treated with 1, 10 or 100 nM insulin and adiponectin release was measured after 1 and 24 h of incubation. Likewise, to test the direct effects of vanadium on adiponectin release and cell content, cells were treated with 1, 10, 50 or 100 ␮M of VS over the same period. Consistent with earlier reports, insulin significantly stimulated adiponectin release in a dose-dependent matter in 1 h (Fig. 1A). However, insulin elicited a reverse trend on adiponectin release after 24 h of incubation, causing a significant stimulation (P < 0.001) at the lowest dose, and no effect at 10 nM and at 100 nM (Fig. 1B). Adiponectin cell content after 24 h was significantly increased by 10 and 100 nM insulin to over 150% of non-treated controls (Fig. 1C). Unlike insulin, vanadium did not change adiponectin release after 1 h (Fig. 1D) or 24 h (Fig. 1E). However, similar to that observed with insulin treatments, adiponectin cell content increased after treatment with VS (Fig. 1F). Furthermore, the effects of sodium orthovanadate (NaOV), bismaltolato oxovanadium (BMOV) and potassium bisperoxo(1,10-phenanthroline)oxovanadate (bpV(Phen)), other widely employed vanadium compounds, on adiponectin cell content were investigated. To varying extents, all vanadium compounds tested increased adiponectin cell content, with VS stimulating 200% of control (Fig. 2). Together, these results indicate that while insulin and VS do not share the same effects on adiponectin release, they were both able to significantly and consistently increase the amount of stored adiponectin after 24 h. Based on these data, adiponectin cell content will be shown and discussed in subsequent experiments. The insulin and vanadium doses for these experiments are based on a commonly employed stimulatory dose in the case of insulin (100 nM) or two effective doses in the case of VS (10 and 50 ␮M). 3.2. Vanadium-induced increase in adiponectin cell content does not depend on insulin receptor activation The involvement of insulin receptor (IR) phosphorylation in insulin and vanadium-mediated adiponectin accumulation was examined by measuring tyrosine phosphorylation and by employing AGL-2263 (AGL), a potent substrate-competitive inhibitor of IR [25]. Although AGL (5 ␮M) alone showed a tendency to increase adiponectin cell content, it significantly inhibited (P < 0.001) insulin-induced adiponectin accumulation (Fig. 3A). However, AGL was not as effective in preventing VSinduced adiponectin accumulation (Fig. 3B), suggesting that

Fig. 2. Effects of insulin (100 nM), and 50 ␮M of the vanadium compounds, VS, NaOV, BMOV and Bpv(phen), on adiponectin cell content after 24 h. Representative adiponectin and respective ␤-actin band images are shown above each treatment. Molecular weight standard band sizes are indicated to the right of band images Bars represent mean percent change from control ± S.E., n = 4–8. *** P < 0.001, ** P < 0.01, * P < 0.05.

the IR is not critically required by VS to exert an effect on adiponectin accumulation. Direct examination of tyrosine phosphorylation revealed that insulin, but not VS (10 ␮M), elicited an increase in phosphotyrosine (p-tyr) activity relative to IR␤ (Fig. 3C). Taken together, these results indicate that, unlike insulin, the signaling cascade involved in vanadium-induced adiponectin accumulation does not depend on the activation of IR. 3.3. Vanadium and insulin-induced increases in adiponectin cell content are not dependent on PI3-K The involvement of PI3-K in mediating both VS and insulin mediated adiponectin accumulation was examined with the PI3K inhibitors wortmannin and LY294002 (LY). Treatment with wortmannin (500 nM) alone significantly increased (P < 0.01) adiponectin cell content, to a level similar to that induced by insulin (100 nM) and VS (50 ␮M, Fig. 4A). There was no significant difference in adiponectin cell content between insulin or VS-treated groups with or without wortmannin. Treatment with LY (10 ␮M) alone, however, did not change adiponectin accumulation (Fig. 4B). Surprisingly, treatment with insulin combined with LY induced adiponectin accumulation to a level significantly higher (P < 0.05) than that induced by insulin alone. This synergistic effect with LY was not seen in VS (10 ␮M) treated cells. Adiponectin release from the same cells after 24 h did not change between groups treated with insulin or VS, containing or not, wortmannin or LY (data not shown). While the lack of an inhibitory effect of wortmannin and LY suggest that PI3-K may not be critically involved in mediating insulin and VS-induced adiponectin accumulation, stimulation by wortmannin alone also suggest that PI3-K may

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Fig. 4. Effects of the PI3-K inhibitors (A) wortmannin (W, 500 nM) and (B) LY294002 (LY, 10 ␮M) on insulin (100 nM) and VS-induced (50 and 10 ␮M, for A and B, respectively) adiponectin cell content after 24 h. Representative adiponectin with respective ␤-actin band images are shown above each treatment bar. Molecular weight standard band sizes are indicated to the right of band images. Bars represent mean percent change from control ± S.E., n = 6–8. *** P < 0.001, ** P < 0.01, compared with control and †† P < 0.05 compared with either insulin-treated groups; (ns) non-significant.

be suppressive towards adiponectin accumulation under basal conditions. Fig. 3. Effects of the insulin receptor inhibitor AGL2263 (AGL, 5 ␮M) after 24 h on (A) insulin-induced (100 nM) adiponectin cell content and (B) VS-induced (10 ␮M) adiponectin cell content. Representative adiponectin with respective ␤-actin band images are shown above each treatment bar. Molecular weight standard band sizes are indicated to the right of band images. Bars represent mean percent change from control ± S.E., n = 8. *** P < 0.001, ** P < 0.01, * P < 0.05 compared with control and ††† P < 0.001 compared between insulintreated groups; (ns) non-significant. (C) Insulin receptor phosphorylation in response to either insulin (100 nM) or VS (10 ␮M) after 24 h. Bars represent the ratio of tyrosine phosphorylation over IR-␤ ± S.E., n = 3–5. ** P < 0.01. Membranes were probed with anti-p-tyr, followed by IR-␤. Representative IR-␤ and p-tyr bands are shown above each respective treatment.

3.4. Vanadium and insulin-induced increases in adiponectin cell content are dependent on PKB We examined whether PKB, also known as Akt, is involved in mediating insulin or VS-induced adiponectin accumulation with NL-71-101 (NL), a specific inhibitor of PKB. While both 10 and 50 ␮M VS were tested in this experiment, we report only the higher dose, as results were similar. Although basal adiponectin cell content was increased by NL, both insulin and VS-induced increases in adiponectin cell content were significantly inhibited by the PKB inhibitor (P < 0.05 and <0.01, respectively; Fig. 5). On the other hand, adiponectin release after 24 h in the presence

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used as a negative control. Insulin and VS-induced adiponectin release gradually increased over time, reaching the first significant elevation with respect to controls after 3 and 4 days, respectively (Fig. 6A). Adiponectin release was strongly inhibited by TNF-␣, reaching near undetectable levels after 3 days. Adiponectin cell content after 4 days was stimulated by both insulin or VS, reaching over 150% of control (Fig. 6B). Adiponectin content was significantly lower (P < 0.001) in cells treated with TNF-␣.

4. Discussion

Fig. 5. Effects of the PKB inhibitor NL-71-101 (NL, 10 ␮M) on insulin and VS-induced adiponectin cell content. Both insulin (100 nM) and VS-induced (50 ␮M) adiponectin cell content are inhibited by NL after 24 h. Representative adiponectin with respective ␤-actin band images are shown above each treatment. Molecular weight standard band sizes are indicated to the right of band images. Bars represent mean percent change from control ± S.E., n = 11–16. *** P < 0.001, ** P < 0.01, compared with control, and †† P < 0.001, † P < 0.05 compared with either VS or insulin-treated groups.

of NL was strongly inhibited, with or without insulin and VS (data not shown). A strong inhibition of insulin and VS-induced adiponectin cell content by this inhibitor, suggests that PKB plays a critical role in the signaling cascade involved in triggering the insulin and VS-induced adiponectin synthesis and accumulation. 3.5. Adiponectin release is increased by insulin and vanadium after 3 days Despite the lack of a strong prevailing effect of insulin or VS on adiponectin release after 24 h, the consistent increase in adiponectin cell content suggested a long time-course of action. In this experiment, adipocytes were treated with insulin (100 nM) and VS (10 ␮M) for 4 days, and TNF-␣ (50 nM) was

Adiponectin has been shown to play a pivotal role in energy metabolism and type 2 diabetes. Recently described actions for adiponectin include reduction in tissue triglyceride content, enhancement of insulin signaling and consequent reversal of insulin resistance state in lipoatrophic mice [21,26]. Also possessing anti-diabetic activity, vanadium salts and conjugated compounds, have been previously described as insulin-mimics or insulin-enhancers in a variety of models, based on several biological actions similar to those elicited by insulin, which mainly result in the improvement of glucose homeostasis at the cellular and systemic levels [3]. In the present study we describe the stimulatory effect of insulin and vanadium on adiponectin accumulation in 3T3-L1 adipocytes, while examining the timecourse and downstream pathway involved in their action. Based on the similarity of cellular effects elicited by vanadium and insulin, it is not surprising to find that, like insulin, vanadium can stimulate adiponectin release and accumulation. Insulin (100 nM) has previously been reported to stimulate adiponectin release within 1 h [22,23]. Adiponectin gene expression, however, was inhibited by the same dose of insulin after 16 h [24]. While the present insulin-induced adiponectin release data is consistent with previous short-term (1 h) observations, longer-term experiments designed for establishing concordance between gene expression and protein content and release patterns may be required. In the present study insulin did not maintain increased adiponectin release after 24 h, when compared with controls, although cellular accumulation after the

Fig. 6. Long-term effects of insulin (100 nM), VS (10 ␮M) and TNF-␣ (50 nM) on adiponectin release and cell content. (A) Adiponectin release is increased after 3 and 4 days of treatment with insulin and VS, respectively, while TNF-␣ consistently inhibits release from day 1. (B) Adiponectin cell content is increased by insulin and VS, and inhibited by TNF-␣ after 4 days of incubation. Representative adiponectin with respective ␤-actin band images are shown above each treatment bar. Molecular weight standard band sizes are indicated to the right of band images. Symbols and bars represent mean percent change from control ± S.E., n = 8 (A) and n = 4 (B). *** P < 0.001, ** P < 0.01, * P < 0.05 compared with control.

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same period was consistently stimulated. The same pattern was seen in adipocytes treated with VS for 24 h. The stimulation of adiponectin cell content was also observed after treatment with other vanadium salts and conjugates, which is consistent with recent observations made in vivo [27], but did not show further increase after combining VS (10 ␮M) with insulin (100 nM, data not shown). When taken together with release data, an increase in insulin and VS-mediated adiponectin cell content relative to control after 24 h indicates an increase in accumulation, possibly as a result of increased synthesis, given that release was relatively unchanged between treatments and control during the same period. This increase in accumulation was consistent with an increase in adiponectin release over the course of 4 days. The long-term increase in adiponectin release induced by insulin suggests the presence of a positive feedback mechanism where the chronic presence of high insulin levels, which are known to induce insulin resistant states, can trigger the release of adiponectin, which is known to increase the sensitivity of cells to insulin. This scenario is consistent with the observation that full length adiponectin is needed to promote reduced plasma glucose levels without stimulating insulin secretion [28]. Based on the relative cell content of adiponectin after 24 h of incubation, pharmacological interventions were applied to identify the main mediators in the signaling process of longterm insulin and VS-mediated adiponectin accumulation, and whether signaling mediators activated by these two treatments differed from one another. The precise mechanism by which vanadium elicits all insulin-like and anti-diabetic effects in 3T3L1 adipocytes remains poorly understood. Because vanadium salts and compounds are strong inhibitors of protein phosphatases, such as PTP-1B [13,14,29], it has been suggested that its downstream effects are a consequence of the decrease in PTP-1B-mediated dephosphorylation of the IR-␤ subunit. As a result, protein tyrosine kinase (PTK) would become activated and phosphorylate IR substrates (IRS), in a process that is similar to that of direct IR stimulation by insulin. This has been demonstrated to occur in a dose-dependant manner with vanadate treatment in cardiomyocytes [30]. At 10 ␮M NaOV, however, IR-␤ was not stimulated, consistent with our observations using VS at the same dose in 3T3-L1 cells. Furthermore, while the insulin receptor inhibitor, AGL-2263, significantly attenuated insulin-induced adiponectin accumulation, it did not decrease VS-induced adiponectin cell content. This would suggest that, at this dose and incubation period, VS would mainly exert its downstream effects leading to adiponectin accumulation through mediators distal to IR. Evidence that vanadium could exert downstream effects independently from IR activation, has been previously shown with regards to glycogen synthesis in Chinese hamster ovary cells overexpressing human insulin receptor [31] and rat adipocytes [32]. Despite the complexity of pathways and physiological actions triggered by insulin, evidence indicates that vanadium exerts its effects by enhancing the activation of IRS-1 and the associated PI3-K [11,31]. Insulin and vanadium-induced adiponectin cellular accumulation, however, are longer-term processes, requiring gene upregulation and protein synthesis,

and may involve several pathways. For example, Fasshauer et al. [24] have reported that PI3-K and MAPK were partially involved in mediating the insulin-induced inhibition of adiponectin gene expression. In the present study, both inhibitors of PI3-K, wortmannin and LY94002, failed to attenuate the insulin or vanadium-induced increase in adiponectin cell content, indicating that PI3-K activation may not be critical in this process. However, inhibition of PI3-K alone was enough to stimulate adiponectin accumulation at 24 h, suggesting that PI3-K may, under basal conditions, have inhibitory control over adiponectin production. On the other hand, by employing pulse-chase determinations in 3T3-L1 adipocytes, Bogan and Lodish [22] reported that both wortmannin and LY94002 blocked insulin-induced adiponectin secretion, although this determination was done within 2 h. Taken together, these results would suggest that while short-term insulin-induced release of stored adiponectin is mediated by a PI3-K-dependent pathway, long-term accumulation is not. Most studies support an important role of wortmannin and LY94002-sensitive PI3-K activity in a variety of insulin and vanadium induced cellular effects (for review see [3]), although some reports have shown that certain insulin-like effects mediated by vanadium, such as glucose uptake and lipid synthesis can occur independently from wortmannin-sensitive PI3-K activation [33,34]. Furthermore, using skeletal muscle, Mohammad et al. [35] concluded that BMOV does not act via the stimulation of the PI3-K pathway in vivo. However, because PKB has also been implicated in mediating vanadium [30] and insulin-induced glucose uptake [36], PI3-K and PKB have been generally regarded as vital mediators of insulin-like effects within the same signaling pathway. In the present study, both insulin and VS-induced adiponectin cell content were significantly inhibited in the presence of the PKB inhibitor, NL-71-101. The requirement of PKB for adiponectin accumulation is consistent with its ability to regulate the FOXO family of forkhead transcription factors [37], which are indirectly responsible for the expression of a wide range of proteins, including adiponectin [38]. On the other hand, the apparent lack of dependency on PI3-K suggests the presence of a mechanism that can bypass this kinase to activate PKB. It has been reported that leucine may allow insulin activation of the PKB/mTOR pathway in normal rat adipocytes treated with wortmannin [39], and that two independent mechanisms for ceramide-induced inhibition of insulin-stimulated PKB activation occur downstream of PI3-K in 3T3-L1 pre-adipocytes [40]. Furthermore, angiotensin II has been shown to activate PKB in mesangial cells, by an arachidonic acid-dependent pathway which is independent of PI3-K [41]. The extent of how similar mechanisms could be operating in insulin and VS-stimulated adiponectin cell content, however, would require further investigation. Additionally, direct examination of the activation of insulin signaling kinases would provide further information on the extent of PI3-K and PKB dependence during insulin and VSmediated adiponectin stimulation, as well as the involvement of different pathways. In summary, both vanadium and insulin can increase adiponectin cell content and long-term release in fully differ-

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entiated 3T3-L1 adipocytes. This is the first study to provide evidence towards a PKB-dependent pathway for the stimulation of an adipokine elicited by both insulin and vanadium. Adiponectin stimulation could, at least in part, explain longterm insulin-sensitizing effects elicited by vanadium. Interestingly, while insulin is capable of eliciting similar long-term effects on adiponectin accumulation, it is also known to induce insulin resistance when used chronically. This apparent paradox could potentially favor the further development of vanadium compounds, especially in light of adiponectin replenishment becoming recognized as a novel treatment strategy for insulin resistance [42]. Nevertheless, confirmation of the present results with human adipocytes, as well as the characterization of in vivo effects of vanadium on insulin sensitivity, together with adiponectin levels and its isoform distribution will be required for advancing the field. Acknowledgements This research was supported by grants from the Center for Nanoscale Mechatronics & Manufacturing, one of 21st Century Frontier Research Programs, Ministry of Science and Technology, South Korea, and by Dankook University Research Fund for 2004, South Korea. References [1] Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care 2004;27:1047–53. [2] Lyonnet B, Martz X, Martin E. L’emploi therapeutique des derives du vanadium. Presse Med 1899;1:191. [3] Srivastava AK, Mehdi MZ. Insulino-mimetic and anti-diabetic effects of vanadium compounds. Diabet Med 2005;22:2–13. [4] Shechter Y, Goldwaser I, Mironchik M, Fridkin M, Gefel D. Historic perspective and recent developments on the insulin-actions of vanadium; toward developing vanadium-based drugs for diabetes. Coord Chem Rev 2003;237:3–11. [5] Mukherjee B, Patra B, Mahapatra S, Banerjee P, Tiwari A, Chatterjee M. Vanadium—an element of atypical biological significance. Toxicol Lett 2004;150:135–43. [6] Yuen VG, Orvig C, McNeill JH. Comparison of the glucose-lowering properties of vanadyl sulfate and bis(maltolato)oxovanadium(IV) following acute and chronic administration. Can J Physiol Pharmacol 1995;73:55–64. [7] Sakurai H, Tsuchiya K, Nukatsuka M, Sofue M, Kawada J. Insulinlike effect of vanadyl ion on streptozotocin-induced diabetic rats. J Endocrinol 1990;126:451–9. [8] Meyerovitch J, Farfel Z, Sack J, Shechter Y. Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats. Characterization and mode of action. J Biol Chem 1987;262:6658–62. [9] Green A. The insulin-like effect of sodium vanadate on adipocyte glucose transport is mediated at a post-insulin-receptor level. Biochem J 1986;238:663–9. [10] Mohammad A, Sharma V, McNeill JH. Vanadium increases GLUT4 in diabetic rat skeletal muscle. Mol Cell Biochem 2002;233:139–43. [11] O’Connor JC, Freund GG. Vanadate and rapamycin synergistically enhance insulin-stimulated glucose uptake. Metabolism 2003;52:666–74. [12] Rehder D, Costa Pessoa J, Geraldes CF, Castro MC, Kabanos T, Kiss T, et al. In vitro study of the insulin-mimetic behaviour of vanadium(IV, V) coordination compounds. J Biol Inorg Chem 2002;7:384– 96.

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