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Reversal of dexamethasone induced insulin resistance in 3T3L1 adipocytes by 3[beta]-taraxerol of Mangifera indica.

ARTICLE INFO

Keywords:

3[beta]-Taraxerol

Insulin resistant model

Dexamethasone induced insulin resistance

GLUT4

PI3K

ABSTRACT

Aim: The present study investigates the efficacy of Mangifera indica ethyl acetate extract (MIEE) and its bioactive compound, 313-taraxerol in the reversal of dexamethasone (DEX) induced insulin resistance in 3[beta]-adipocytes.

Main methods: MIEE and 3[beta]-taraxerol were evaluated for their ability to restore impaired glucose uptake and, expression of molecular markers in the insulin signaling pathway induced by DEX in 3T3L1 adipocytes using 2-deoxy-D-[1 -[.sup.3]H] glucose uptake assay and ELISA.

Key findings: An insulin resistant model has been developed using a glucocorticoid. DEX on 3T3L1 adipocytes. Insulin resistant condition was observed at 24 h of DEX induction wherein a maximum degree of resistance of about 50% was measured based on inhibition of glucose uptake, which was confirmed using cytotoxicity analysis. The developed model of insulin resistance was studied in comparison to positive control rosiglitazone. DEX induced inhibition of glucose uptake and the expression of insulin signaling markers GLUT4 and PI3K were found to be restored by 3[beta]-taraxerol and MIEE, thus delineating its mechanism of action in the reversal of insulin resistance.

Significance: 3[beta]-Taraxerol effectively restored DEX induced desensitization via restoration of P13K and GLUT4 expression. To conclude, since 3[beta]-taraxerol exhibits significant effect in reversing insulin resistance it can be further investigated as an insulin resistance reversal agent.

[c] 2012 Elsevier GmbH. All rights reserved.

Introduction

Insulin resistance can be described as defective utilization of metabolites in insulin targeted tissues, which is manifested in two pathophysiological states, Non-Insulin-Dependent Diabetes Mellitus (NIDDM) and Obesity. In both these conditions, adipocytes become desensitized to the biological effects of insulin, leading to a decrease in the ability of insulin to stimulate glucose transport. Therefore, adipocytes serve as an amenable experimental system for studying insulin resistance providing insights toward understanding the molecular mechanisms underlying the metabolic disorder. In the current study, 3T3L1 adipocytes that demonstrate the indices of insulin action were rendered resistant using dexamethasone (DEX), a glucocorticoid. Induction of insulin resistance by DEX is through increasing the hepatic glucose production and protein degradation, protein synthesis and decreasing peripheral glucose transport and utilization. Excessive occurrence of DEX is a frequently observed clinical problem in insulin resistant state (Niels and Jorgensen 2009). DEX induced insulin resistance in 3T31.1 adipocytes is a validated model with its physiological relevance investigated using in vivo experiments (Sakoda et al. 2000).

The aim of developing an in vitro insulin resistance model was to understand the effect of insulin resistance enabling identification of suitable agents that can reverse the condition of insulin resistance. In this study, 313-taraxerol a triterpenoid (Fig. 1) isolated from ethyl acetate extract of Mangifera indica (MIEE) was assessed for its effect on the reversal of insulin resistance as it showed promising anti-diabetic effect in an insulin sensitive model of 3T3L1 adipocytes (Sangeetha et al. 2010). To gain additional insight into the mechanism by which DEX induces insulin resistance and the pathway undertaken by MIEE and 3[beta]-taraxerol to reverse the insulin resistance; a detailed protein profiling was performed for the major markers involved in insulin signaling which was compared with the classical insulin sensitive model (uninduced 3T3L1 adipocytes).

Chemicals and reagents

Cell culture medium DMEM supplemented with glutamine (2 mM), and 10% heat inactivated fetal calf serum was purchased from GIBCO BRL (USA). 2-Deoxy-o-[1-[.sup.3]H] glucose was obtained from Amersham Pharmacia Biotech (UK). Insulin and rosiglitazone were purchased from Sigma. MTT assay kit was purchased from Promega (USA). Antibodies like IR-13, PI3K, PKB, GLUT4, PTP 1B, p-GSK3[beta] and p-PKB were procured from Santa Cruz Biotechnologies.

Cell culture and differentiation

3T3L1 cells were cultured in DMEM supplemented with 2 mM glutamine, antibiotics (penicillin 120 U/ml, streptomycin 75 [micro]g/ml, gentamycin 160 [micro]g/ml, amphotericin B 3 [micro]g/m1) and 10% FCS. The cell cultures were maintained at 37 [degrees]C in a humidified incubator with 5% [CO.sub.2]. Preadipocytes were seeded in 24-well plates and after confluence the cells were differentiated using DMEM containing 0.5 mM 3-isobuty1-1-methylxanthine, 0.25 [micro]M DEX and 5 [micro]g/mlof insulin for 48 h, followed by their maintenance in DMEM containing 10 [micro]g/m1 insulin alone for 72 h as described (Student et al. 1980). The cells were finally maintained in DMEM containing 10% FCS for 24 h. Finally the differentiated 3T3L1 adipocytes were used for the following studies.

Development of insulin-resistant model using 3T3L1 adipocytes

Insulin-resistant model was developed through DEX induction as explained (Sakoda et al. 2000). The 3T3L1 adipocytes were completely differentiated as mentioned above and then induced with 100 nM DEX for the specified time periods of 12, 24, and 48h respectively. The period of insulin resistance was analyzed based on the maximum inhibition in glucose uptake. Glucose uptake experiments were performed as described (Kamei et al. 2002) with slight modifications. Cells were stimulated with insulin (10 nM) for 15 min followed by the addition of 0.5 [micro]Ci/well of 2-deoxy-D[1-[.sup.3]H]-glucose in KRPH buffer (118 mM NaCl, 5 mM KCI, 1.3 mM Ca[Cl.sub.2], 1.2 mM MgS[O.sub.4], 1.2 mM K[H.sub.2] P[ O.sub.4] and 30 mM HEPES-pH 7.4) for 45 min at 37 [degrees]C. Cells were then washed with KRPH and later lysed using 0.1% SDS. The lysates were quantitated using scintillation counter and the results expressed as % glucose uptake with respect to their controls (i.e., uninduced 3T3L1 adipocytes as insulin sensitive control and dexamethasone induced 3T3L1 adipocytes as insulin resistant control). The time point at which maximum inhibition in glucose uptake observed was considered as the insulin-resistant model.

Validation of insulin-resistant model

The insulin-resistant model was validated using rosiglitazone for which the fully differentiated 3T3L1 adipocytes were induced with DEX for 2411 followed by treatment with 50 [micro]M rosiglita-zone for specified time periods (12, 24 and 48h respectively). This was followed by the measurement of insulin-stimulated glucose uptake as described (Kamei et al. 2002) and the observed effects were compared with rosiglitazone treated normal 3T3L1 adipocytes (insulin-sensitive cells).

Effect of MIEE and 3[beta]-taraxerol on glucose uptake on

insulin-sensitive and insulin-resistant 3T3L1 adipocytes

The effect of MIEE and 3[beta]-taraxerol on glucose uptake in insulin-sensitive (uninduced 3T3L1 adipocytes) (Sangeetha et al. 2010) and insulin-resistant model (DEX induced 3T3L1 adipocytes) was studied as described. Both the uninducal and DEX induced cells were treated with MIEE and 313-taraxerol for 24 h. Glucose uptake experiments were performed as described earlier (Kamei et al. 2002) with slight modifications. The uptake effects of MIEE and 313-taraxerol on both the models were compared with the effects of positive control rosiglitazone (50[micro]M) on both the models.

Cytotoxic assessment

The cytotoxic effect of DEX induction at different time points such as 12,24 and 48 h was assessed by quantifying LDH release and comparing it with untreated controls, respectively. Likewise, MIEE and 3[beta]-taraxerol were assessed for their cytotoxic effect on the insulin-sensitive (uninduced) and insulin-resistant (DEX induced) model using MIT reagent as described by Gayathri et al. (2007). The assay was performed after 24 h of MIEE and 3[beta]-taraxerol treatment at different concentrations (10 [micro]g, 100 ng and 1 ng/ml). LDH released was measured at 492 nm which was directly proportional to the cell death or cell lysis.

Evaluation of the expression of insulin signaling markers on insulin sensitive and insulin resistant model using ELISA

MIEE, 3[beta]-taraxerol and rosiglitazone treated insulin-sensitive and insulin-resistant cell lysates were prepared (Sangeetha et al. 2010) and ELISA experiments were performed (Jung et al. 2007). Equal amounts of cell lysates (10 [micro]g of protein) were applied to Nunc MAXI SORB 96-well plates in carbonate/bicarbonate buffer (15 mM [Na.sub.2][CO.sub.3] and 35 mM NaHC[O.sub.3], pH 9.6) at 4 [degrees]C and incubated for overnight. Next, the plates were washed with PBS containing 0.05% Tween 20, and then specific antibodies for IR-[beta], PI3K, p-PKB, p-GSK313 and GLUT4 respectively were added to the wells and incubated for 1 h at room temperature (25 [degrees]C). The wells were washed again and later, alkaline phosphatase conjugated secondary antibody specific for each of the primary antibody was added and incubated for 1 h. After the incubation period, secondary antibody was removed and then wells were finally washed and incubated with pNP. The expression and phosphorylation of the proteins mentioned above were quantified by measuring absorbance at 410 nm.

Statistical analysis

All data are expressed as mean [+ or -]S.E. The statistical significance between means of the independent groups was analyzed using one way ANNOVA and p value of less than 0.05 was considered to be statistically significant.

Results

Time course analysis of DEX induction on 3T3L1 adipocytes

Time course analysis was performed for 12, 24 and 4811 of DEX (100 nM) treatment and assessed for insulin resistance based on inhibition of glucose uptake. In DEX induced cells (insulin-resistant model), desensitization of insulin-stimulated glucose uptake was maximally achieved to about 50% at 24 h of treatment in comparison to the uninduced cells (insulin-sensitive model). At 12 and 48 h of induction a minimal inhibition of glucose uptake was observed, indicating negligible resistance (Fig. 2). Hence further experiments were carried out with 24h of DEX induction.

Effect of DEX induction on cytotoxicity

To confirm that the inhibition of glucose uptake was clue to insulin resistance and not due to the cytotoxic effect of DEX, cell viability assay was performed at different time periods (12, 24 and 48 h) of DEX induction. At 12 and 24 h of the study, DEX did not exhibit any cytotoxicity to the cells confirming that the inhibitory effect on glucose uptake was due to the resistance it imposes to the cells (Fig. 3). However at 48 h, 40% cytotoxicity was observed.

Validation of DEX induced insulin-resistant model

To validate the DEX induced insulin-resistant model and to confirm that the resistance created by it can be reverted on treatment with a suitable insulin sensitizing agent; rosiglitazone was used as positive control in the study. To optimize the time period at which rosiglitazone potentially reversed insulin resistance, unin-duced as well as DEX induced (24h) 3T3L1 adipocytes were treated with 50 [micro]M of rosiglitazone for 12, 24 and 48h. Glucose uptake analysis was performed and compared with their respective controls. Rosiglitazone exhibited maximum reversal (2.6-fold) of dexamethasone induced insulin resistance after 24h of its treatment in insulin-resistant cells as compared to 12 and 48 h where the reversal effect was lower. In insulin-sensitive cells, rosiglita-zone showed almost equivalent effect of about 0.9-fold at 24 and 48 h but at 12 h an increase of about 1.2-fold in glucose uptake was observed (Fig. 4). Rosiglitazone showed an optimum insulin resistance reversal effect at 24 h of treatment, and hence was used as a standard for further studies.

Impact of DEX induction on insulin signaling markers involved in glucose transport

The study attempts to delineate the effect of DEX on various molecular switches involved in glucose transport and establish their link with the insulin-resistance condition. The cells were treated with DEX for different time periods (12, 24, 48 h) and the total cell lysates quantified (semi-quantitatively) and assessed for their expression and phosphorylation status of the major insulin signaling proteins such as IR[beta], PTP1B, PI3K and GLUT4 using ELISA. Induction with DEX at various time points showed no reduction in the IR[beta] expression compared to the uninducecl cells, although, PTP1B expression levels were significantly reduced at all the time points. Analysis of DEX administered cell lysates of 3T3L1 adipocytes for PI3K expression showed a slightly decreased expression at 12h compare to 24h (0.52-fold) and 48 h (0.62-fold). On analyzing the role of DEX in regulating GLUT 4 expression, it was observed that 24 h treatment with DEX was able to cause maximum impairment of GLUT 4 exhibiting 0.42-fold decrease compared to untreated cells, whereas, only 0.19 and 0.22-fold decrease was observed at 12 hand 48 h. No significant decrease in PKB and GSK3[beta] phosphorylation on induction with DEX was observed at all the three time points (12, 24 and 48h) compared to the uninduced control (Fig. 5a).

Effect of rosiglitazone on the insulin signaling proteins in the insulin sensitive and insulin resistant model

The effects of rosiglitazone (insulin sensitizing positive control) on the insulin signaling proteins such as IR[beta], PTPIB, PI3K, PKB, GSK3[beta] and GLUT4 in the insulin sensitive and dexamethsone induced insulin resistant model of 3T3L1 adipocytes were assessed using ELISA experiments. From the results it was inferred that in the insulin sensitive model, rosiglitazone showed a significant increase in expression of the insulin signaling markers such as IR, PI3K, PKB, GSK313 and GLUT 4 but there was no significant change in the expression of PTP1B levels. However in the insulin resistant model, rosiglitazone showed a promising effect on the restoration of the expression of PI3K and GLUT4 which was impaired by dexamethasone treatment. Whereas the effect of rosiglitazone on the expression of IR, PTP1B, PKB, and GSK3[beta] in the insulin resistant model were considered to be insignificant as dexamethsone did not have any deleterious effect on these molecular targets (Fig. 5b).

Comparative assessment of the glucose uptake potential of MIEE and 3f3-taraxerol on the insulin-sensitive and insulin-resistant model of 3T3L1 adipocytes

To elucidate the role of MIEE and 3[beta]-taraxerol on reversal of insulin resistance, a dose response analysis was performed in an insulin-resistant model. The reversal effects were compared with MIEE and3[beta]-taraxerol treated insulin-sensitive model (Sangeetha et al. 2010). Inhibition of glucose uptake by DEX induction was effectively restored by MIEE in a dose dependant manner (Fig. 6a). The restoration effect on glucose uptake was optimum to about 166 [+ or -] 15% at 100 ng/m1 of MIEE in the insulin-resistant model (DEX induced) compared to the insulin-resistant control. Likewise 313-taraxerol also showed a better response on insulin resistance reversal that was optimum at 100 ng/ml to about 153 [+ or -] 5% (Fig. 6b) comparable to rosiglitazone (50 [micro],M) which exhibited a restoration in glucose uptake to about 250 [+ or -] 7% in the insulin resistant model. Hence it is inferred that like rosiglitazone both MIEE and 3[beta]-taraxerol exhibited potential reversal of DEX induced insulin resistance evident through the restoration of glucose uptake exhibited in insulin-resistant models.

Effect of MIEE and 3[beta]-taraxerol on cytotoxicity in insulin sensitive and insulin resistant 3T3L1 adipocytes

The cytotoxic effect of MIEE and 3[beta]-taraxerol on both the models of study was assessed using cytotoxicity assay. MIEE showed negligible effect on toxicity whereas cytotoxic effect of less than 25% was reported at the highest concentration tested for 3[beta]-taraxerol thus confirming its non-toxic nature even in insulin-resistant model (DEX induced) (Fig. 7).

Effects of MIEE and 3[beta]-taraxerol on expression of PI3K and GLUT 4 in insulin-sensitive and insulin-resistant model of 3T3L1 adipocytes

MIEE and 3[beta]-taraxerol treated cell lysates of insulin sensitive and resistant models were quantified for their MIK and GLUT 4 protein expressions using ELISA. In the insulin-sensitive model, 313-taraxerol showed an enhanced effect of 1.9, 1.8 and 1.9-fold respectively at 12, 24 and 48 h. Whereas. MIEE caused a significant 1.7-fold increase in P131K expression at 12 and 24 h compared to untreated controls. The expression was even sustained at 48 h time point to about 1.4-fold but was less compared to 12 and 24 h (Fig. 7). However in the insulin-resistant model, DEX treated cells showed almost 50% inhibition in P13K expression. This inhibition in expression of PI3K was restored on treatment with MIEE and 313-taraxerol. MIEE induction caused an increased expression of P13K at 12, 24 and 48h with an optimum effect of 2.3-fold being observed at 24 h indicating effective restoration (Fig. 8). Likewise, 313-taraxerol induction showed a much greater effect with 2.6-fold increase in expression compared to insulin-resistant control (DEX control). Thus, it can be inferred that MlEE and 3[beta]-taraxerol exhibit significant effects in insulin-sensitive model in addition to restoration of P13K in insulin-resistant model.

Assessment of the effect of MIEE and 3[beta]-taraxerol for their role on GLUT4 protein expression in the insulin-sensitive and resistant 3T3L1 cells showed that both MIEE and 3[beta]-taraxerol exhibit a significant time dependant increase of about 1.5 and 1.6-fold at 12 and 24 h respectively compared to uninduced control which showed a basal level expression. But at 48 h a slight decline in the GLUT4 expression was observed in insulin-sensitive 3T3L1 adipocytes (uninduce(1) (Fig. 9).

In the insulin resistant model, DEX treatment caused a drastic inhibition in GLUT4 expression to about 50% compared to unin-duced control. The decrease in the GLUT4 levels was effectively restored on treatment with MIEE and 3[beta]-taraxerol. From the time course analysis performed with MIEE and 3[beta]-taraxerol at 12, 24 and 48 h, it is clear that MIEE shows a time dependant increase in expression of GLUT4 of 1.5, 1.6 and 1.8 respectively (Fig. 9). Upon 3[beta]-taraxerol induction, restoration of GLUT 4 protein was found to be optimum at 24h time point with 1.8-fold increase in its expression.

Discussion

Insulin resistance is a major cause for macro-metabolic complications like diabetes and obesity, and agents with potential insulin resistance reverting effects could serve as drugs for the treatment of various metabolic disorders. To identify compounds with potential insulin resistance reverting property, an in vitro model that could actually mimic the state of insulin resistance and reflect the pathophysiological progression of the disease condition would be beneficial. Thus, the present study was designed to develop an in vitro model mimicking insulin resistance using 3T3L1 adipocytes and to exploit it for the screening and identification of molecules with potential insulin resistance reverting properties. The insulin resistant model of 3T3L1 adipocytes was developed using DEX, a glucocorticoid, and was utilized for assessing the insulin resistance reverting potential of MIEE and 3[beta]-taraxerol (sensitizers of glucose transport and storage).

DEX induced model of insulin resistance has been well established and has lot of relevance to clinical insulin resistant condition in vivo, since it mimics exactly the condition of insulin resistance which can be reversed on treatment with a suitable agent. Numerous studies have reported the possible mechanisms of action for dexamethasone induced insulin resistance. One possibility is the down regulation of insulin receptor substrate 1 (IRS1 ) expression (Turnbow et al. 1994, 1995), that plays a major role in the activation of phosphatidylinositol 3-kinase (PI3K), which is essential for GLUT4 translocation. The other possibility is dexamethasone induced impairment of GLUT4 translocation step independent of insulin signaling supporting the fact that glucocorticoids inhibit not only insulin-induced but also hypoxia induced GLUT4 translocation to the cell surface in skeletal muscle (Weinstein et al. 1995). GLUT1 decrease rather than the inhibition of PI3K activation resulting from a decreased IRS-1 content may be involved in the dexamethasone-induced decrease in basal glucose transport activity which is likely to underlie impaired glucose transporter regulation (Sakoda et al. 2000). In addition, studies have demonstrated that most factors causing insulin resistance impair the early steps of insulin signaling leading to P131< activation (Saad et al. 1992: Folli et al. 1993). However each of these findings seems to report different molecular mechanism of action of dexamethasone induced insulin resistance, the present study was performed to decipher the actual role of the insulin signaling targets on dexamethasone treatment in 3T3L1 adipocytes.

Insulin resistance model was developed based on time course analysis that was performed using 100 nM DEX induction. The extent of insulin resistance exhibited was expressed based on the inhibition in glucose uptake. Desensitization in insulin-stimulated glucose uptake was maximally achieved to about 50% at 24 h of DEX treatment compared to the uninduced i.e., insulin-sensitive cells coinciding with Hyun et al. (2004), who have reported a 50% reduction in insulin-sensitive glucose uptake during insulin-resistant state. DEX did not impose any cytotoxicity to the cells confirming that the inhibitory effect on glucose uptake was due to the resistance it imposes to the cells. But at 48 h, 40% cytotoxicity was observed coinciding with earlier work (Chrysis et al. 2005) wherein they had hypothesized that DEX induces apoptosis in chondrocytes after 48 h.

DEX induced insulin-resistant model was validated using rosiglitazone, a thiazolidinedione as a positive control. Thiazo-lidinedione derivatives (TZD) are a family of insulin-sensitizing drugs that have been widely reported for reversing DEX induced insulin resistance both in vitro and in vivo (Anil Kumar and Marita 2000; Morita et al. 2001). TZD and DEX act as ligands for nuclear receptor and TZDs antagonize the action of DEX by completely preventing the deleterious effects of DEX on glucose tolerance and insulin sensitivity (Willi et al. 2002a). However there are several evidences suggesting that TZDs can reverse insulin resistance induced by glucocorticoids in rats (Willi et al. 2002b). Rosiglitazone belongs to the TZD group and has been widely reported to improve insulin sensitivity in insulin-resistant rat models (Zhao et al. 2009). Hence in the present study, rosiglitazone was used for validation of the developed model of resistance. In the time course analysis of the effect of rosiglitazone (50 [micro]M) in the insulin resistant cells, TZD was found to reverse dexamethasone induced insulin resistance at 24h.

The defects in insulin signaling in T2D patients have been characterized to involve the impairment of IRS1 phosphorylation, PI3K activity, and glucose transport activity as a consequence of functional defects, whereas insulin receptor tyrosine phosphorylation, mitogen-activated protein kinase (MAPK) phosphorylation, and glycogen synthase activity appears to be normal (Krook et al. 2004). Likewise, in animal models of insulin resistance the expression and activation of PI31< and GLUT 4 is decreased correlating with defective insulin signaling confirming the consistent role of these alterations in insulin resistance (Nathalie Rosenblatt-Velin et al. 2004). This study attempts to delineate the effect of DEX on these molecular switches and trying to establish their link with insulin-resistance condition.

The influence of DEX induction on insulin sensitivity was assessed initially at the receptor level by evaluating 1R13 and PTP1B as they are the primary determinants of insulin-dependent glucose uptake. It was observed that there was no reduction in the IR13 expression on DEX induction at all the three time points compared to the insulin-sensitive control (uninduced cells). But PTP1B expression levels were significantly reduced at all the time points which were consistent to reports stating that there is a decreased expression of PTP1B in DEX induced insulin resistance leading to the conclusion that DEX induced insulin resistance could be due to a defect at the post binding or post receptor site (Zabolotny et al. 2008). Hence, post receptor targets like P13K and GLUT 4, which are the major molecular switches involved in glucose transport process were evaluated. A time dependant decrease in P13K expression was observed which was low at 12 h and significant at 24 and 48 h compared to uninduced controls. Singleton et al. (2000) have stated that DEX induced overexpression of the P13K subunit p85alpha, competes with the complete P13K heterodimer for binding at insulin receptor substrate-1, inhibiting P13K activation. Hence it has been hypothesized that decrease in expression of P13K on DEX induction could be due to the competitive binding of p85alpha subunit thereby, hindering the expression of P13K leading to perturbed glucose uptake.

Garvey et at. (1989) have stated that early time periods of DEX induction (2 h) impairs the ability of insulin to translocate intracellular glucose transporters to the cell surface and with more chronic exposure (24h), depletes the total number of cellular transporters. In the current study, the role of DEX in regulating GLUT4 expression was investigated wherein 24 h of its treatment exhibited maximum impairment of GLUT 4. The decrease in GLUT 4 expression appeared to be less at 12 and 48 h correlating with the glucose uptake. This coincides with earlier reports wherein chronic DEX exposure (24 h) decreased plasma membrane and low density microsomal transporters by 30-50% in both basal and insulin-stimulated cells and depleted transporters by 43% in a total cellular membrane fraction (Garvey et al. 1989) in addition to showing a diminished expression of GLUT 4 in fat cells of rat models (Coderre et al. 1996).

No significant decrease in PKB and GSK3[beta] phosphorylation was observed on DEX induction at all the time points of study. Therefore it could be inferred that the effect of DEX on glucose uptake inhibition is independent of PKB and GSK3[beta] phosphorylation at earlier time points (Ruzzin et at. 2005). Buren et al. (2008) have demonstrated that chronic DEX treatment for 12 days impairs insulin-stimulated PKB and GSK3P phosphorylation in skeletal muscles of male Wistar rats contributing to insulin resistance. Therefore the study clearly shows that short term (acute) administration of DEX does not alter the IR-[beta], PKB and GSK3[beta] expression levels but significant time dependent reduction of P13K and GLUT 4 expressions were observed correlating with a decreased glucose uptake effect.

The current study was attempted to decipher whether 3[beta]-taraxerol administration could reverse the dexamethasone induced impairment in glucose uptake. 3[beta]-Taraxerol is a triterpenoicl isolated from the ethyl acetate extract of the leaves of Mangifera indica (MIEE) and has been reported to possess significant effect on insulin stimulated glucose uptake through P13K dependent activation reported in our earlier work (Sangeetha et al. 2010). MIEE and 3[beta]-taraxerol administration improved DEX impaired insulin stimulated glucose uptake. However, promising but only partial restoration of glucose uptake was observed for both MIEE and 3[beta]-taraxerol in the DEX induced cells compared to its effect observed in the uninduced cells (insulin sensitive cells) reported in our earlier work (Sangeetha et al. 2010). The level of restoration of DEX induced inhibition in glucose uptake was equivalent for MIEE and 3[beta]-taraxerol (i.e., 150 [+ or -] 5% at their optimum concentration 10[micro]g/ml and 100 ng/m1 respectively). This indicates that 3[beta]-taraxerol is the actual bioactive compound that is responsible for the insulin resistance reversal effect exhibited by the extract confirming the enrichment of activity on isolation.

To investigate the mechanism of action in the reversal of glucose transport in DEX induced insulin-resistant model, the effect of MIEE and 3[beta]-taraxerol on insulin signaling in the insulin-resistant model (DEX induced) was assessed and compared with insulin-sensitive (uninduced) control. It has been well established that insulin-stimulated redistribution of the insulin responsive glucose transporter GLUT4, from intracellular storage sites to the plasma membrane depends on the production of P1(3,4,5)P3 by the P13K (Khan and Pessin 2002). P13K exists in the cytosol as a dimer of the regulatory p85 subunit and catalytic p110[alpha] subunit. Recruitment of the regulatory subunit brings the catalytic p110ot subunit to the plasma membrane, wherein it catalyzes the phosphorylation of the 3' position in the inositol ring of phosphoinositide (P1) lipids. Biological blockade of the PI3K signaling pathway, including expression of dominant negative mutants, inhibit insulin-stimulated GLUT4 translocation and glucose uptake (Okada et al. 1994; Kotani et al. 1995). This evidence suggests that the activation of P13K and the formation of P1(3,4,5)P3 is necessary to mediate insulin-stimulated GLUT4 translocation. Hence the effect of MIEE and 3[beta]-taraxerol in restoring the DEX induced P13K expression was evaluated. In the insulin-sensitive model, compared to untreated control 313-taraxerol showed an enhanced P13K expression that was much greater than the expression levels observed for MIEE. In the insulin-resistant model. DEX treated cells (insulin-resistant control) showed a 50% inhibition in P13K expression. This inhibition in P13K expression was restored on treatment with MIEE and 3[beta]-taraxerol. MIEE induction caused an increased expression of P13K at all the three time points of study (12, 24 and 48 h) with an optimum effect observed at 24h. Likewise, 3[beta]-taraxerol induction showed a much greater effect with 2.6-fold increase in expression compared to insulin-resistant control. Thus, it can be inferred that MIEE and 3[beta]-taraxerol exhibit significant effects in insulin-sensitive model in addition to restoration of P13K in insulin-resistant model.

GLUT4 is the primary effector for mediating glucose transport, with a genetic defect resulting in impaired insulin tolerance and defective glucose metabolism in skeletal muscle and adipose tissue (Zisman et al. 2000). In case of insulin resistance, reduced glucose transport in skeletal muscle is a major contributing factor responsible for reduced whole body glucose uptake and an overexpression of GLUT4 would exhibit glucose homeostasis (Wallberg-Henriksson and Zierath 2001). Thus, GLUT4 could be an ideal target for pharmacological intervention strategies to control glucose homeostasis. Hence, MIEE and 3[beta]-taraxerol were evaluated for their role in regulating GLUT4 expression in the insulin-sensitive and resistant 3T3L1 cells. Results indicate a basal level expression of GLUT4 which upon treatment with MIEE and 3[beta]-taraxerol was observed to increase significantly in a time dependant manner for insulin sensitive cells. Likewise, in the resistant model of 3T3L1 adipocytes, the drastic inhibition in GLUT4 expression induced by DEX was observed to be effectively restored on treatment with M1EE and 3[beta]-taraxerol respectively (Sangeetha etal. 2010). Hence it is evident that MIEE exhibits a time dependant increase in expression of GLUT4 and 3[beta]-taraxerol treatment leading to an optimum restoration of GLUT 4 protein at 24 h time point.

Conclusion

To summarize, upon induction with Dexamethasone, fully differentiated 3T3L1 adipocytes exhibit significant desensitization toward insulin stimulated glucose uptake. The possibility of this desensitization has also been shown to be due to an impairment in the insulin signaling targets such as GLUT 4 and P13K. However this desensitization induced by DEX was observed to be effectively restored by 3[beta]-taraxero1 isolated from Mangifera indica via the restoration of P13K and GLUT4 expression. Moreover the bioactive compound did not exhibit any cytotoxic effect on the DEX induced insulin resistant model.

Conflict of interest

No conflict of interest to disclose.

Abbreviations: DEX, dexamethasone: DMEM, Dulbecco's modified Eagle's medium; ELISA, enzyme linked immunosorbent assay; FCS, fetal calf serum; GLUT 4, glucose transporter 4; GS, glycogen synthase; GSK313, glycogen synthase kinase beta; IR 13, insulin receptor beta; IRS1, insulin receptor substrate1; KRBH. Krebs ringers phosphate buffer; LDH, lactate dehydrogenase; MIEE. Mangifera indica ethyl acetate extract; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliurn bromide; PI3K, phosphoinositide 3-kinase; PKB, protein kinase B; pNPP, p-nitrophenyl phosphate; PTP1B, protein tyrosine phosphatase 18; T2D, type 2 diabetes.

* Corresponding author. Tel.: +91 4422350772: fax: +91 4422350299.

E-mail address: lakshmibs@annauniv.edu (B.S. Lakshmi).

0944-7113/$--see front matter [c] Elsevier GmbH. All rights reserved hattp://dx.doi.org/10.1016/j.phymed.2012.11.0101

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K.N. Sangeetha, K. Shilpa, P. Jyothi Kumari, B.S. Lakshmi *

Centre for Biotechnology, Anna University. Chennai, India
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Author:Sangeetha, K.N.; Shilpa, K.; Kumari, Jyothi P.; Lakshmi, B.S.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Feb 15, 2013
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