|Year : 2012 | Volume
| Issue : 3 | Page : 326-332
Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?
Joseph Alex Davis1, Suchitra Sharma2, Shivani Mittra2, S Sujatha3, Anil Kanaujia4, Gyanesh Shukla4, Chandrakant Katiyar5, BS Lakshmi6, Vinay Sheel Bansal2, Pradip Kumar Bhatnagar7
1 Department of Pharmacology, New Drug Discovery Research, Ranbaxy Laboratories Ltd.; Daiichi Sankyo Life Science Research Centre in India (RCI), Daiichi Sankyo India Pharma Private Ltd., Village Sarhaul, Sector-18, Gurgaon, Haryana, India
2 Department of Pharmacology, New Drug Discovery Research, Ranbaxy Laboratories Ltd., Gurgaon, Haryana, India
3 Centre for Biotechnology, Anna University, Chennai; Biotechnology, School of Bioengineering, SRM University, Kattankulathur, Tamil Nadu, India
4 Herbal Research, New Drug Discovery Research, Ranbaxy Laboratories Ltd.; Daiichi Sankyo Life Science Research Centre in India (RCI), Daiichi Sankyo India Pharma Private Ltd., Village Sarhaul, Sector-18, Gurgaon, Haryana, India
5 Herbal Research, New Drug Discovery Research, Ranbaxy Laboratories Ltd., Gurgaon, Haryana, India
6 Centre for Biotechnology, Anna University, Chennai, Tamil Nadu, India
7 Department of Pharmacology, Herbal Research and Analytical Chemistry, New Drug Discovery Research, Ranbaxy Laboratories Ltd.; Daiichi Sankyo Life Science Research Centre in India (RCI), Daiichi Sankyo India Pharma Private Ltd., Village Sarhaul, Sector-18, Gurgaon, Haryana, India
|Date of Submission||19-Aug-2011|
|Date of Decision||02-Jan-2012|
|Date of Acceptance||31-Mar-2012|
|Date of Web Publication||17-May-2012|
Joseph Alex Davis
Department of Pharmacology, New Drug Discovery Research, Ranbaxy Laboratories Ltd.; Daiichi Sankyo Life Science Research Centre in India (RCI), Daiichi Sankyo India Pharma Private Ltd., Village Sarhaul, Sector-18, Gurgaon, Haryana
Source of Support: None, Conflict of Interest: None
Aim: The mechanism of action of Annona squamosa hexane extract in mediating antihyperglycemic and antitriglyceridimic effect were investigated in this study.
Materials and Methods: The effects of extract on glucose uptake, insulin receptor-β (IR-β), insulin receptor substrate-1 (IRS-1) phosphorylation and glucose transporter type 4 (GLUT4) and phosphoinositide 3-kinase (PI3 kinase) mRNA expression were studied in L6 myotubes. The in vitro mechanism of action was tested in protein-tyrosine phosphatase 1B (PTP1B), G-protein-coupled receptor 40 (GPR40), silent mating type information regulation 2 homolog 1 (SIRT1) and dipeptidyl peptidase-IV (DPP-IV) assays. The in vivo efficacy was characterized in ob/ob mice after an oral administration of the extract for 21 days.
Results: The effect of extract promoted glucose uptake, IR-β and IRS-1 phosphorylation and GLUT4 and PI3 kinase mRNA upregulation in L6 myotubes. The extract inhibited PTP1B with an IC 50 17.4 ΅g/ml and did not modulate GPR40, SIRT1 or DPP-IV activities. An oral administration of extract in ob/ob mice for 21 days improved random blood glucose, triglyceride and oral glucose tolerance. Further, the extract did not result in body weight gain before and after treatment (29.3 vs. 33.6 g) compared to rosiglitazone where significant body weight gain was observed (28.4 vs. 44.5 g; *P<0.05 after treatment compared to before treatment).
Conclusion: The results suggest that Annona squamosa hexane extract exerts its action by modulating insulin signaling through inhibition of PTP1B.
Keywords: Annona squamosa , type 2 diabetes mellitus, insulin mimetics, PTP1B inhibitor, OGTT, ob/ob mice
|How to cite this article:|
Davis JA, Sharma S, Mittra S, Sujatha S, Kanaujia A, Shukla G, Katiyar C, Lakshmi B S, Bansal VS, Bhatnagar PK. Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?. Indian J Pharmacol 2012;44:326-32
|How to cite this URL:|
Davis JA, Sharma S, Mittra S, Sujatha S, Kanaujia A, Shukla G, Katiyar C, Lakshmi B S, Bansal VS, Bhatnagar PK. Antihyperglycemic effect of Annona squamosa hexane extract in type 2 diabetes animal model: PTP1B inhibition, a possible mechanism of action?. Indian J Pharmacol [serial online] 2012 [cited 2017 Mar 26];44:326-32. Available from: http://www.ijp-online.com/text.asp?2012/44/3/326/96304
| » Introduction|| |
Type 2 diabetes is a metabolic disorder, primarily characterized by insulin resistance, insulin deficiency and hyperglycemia. Given its prevalence and complexity, there is a growing need for novel strategies and effective therapeutic approaches to treat type 2 diabetes mellitus. Although many antidiabetic agents are available, much attention has been paid recently to discover natural products mainly due to their less toxicity and side effects compared to non-herbal synthetic counterparts. , Ayurvedic texts also describing various antidiabetic medications of plant products as single or in combinations, and many indigenous Indian medicinal plants have been reported to be useful in managing diabetes. 
Annona squamosa (Family: Annonaceae), also known as custard apple or sugar-apple, has been shown to possess wide range of biological activities such as anti-lipidemic,  anti-tumor,  antimicrobial,  antithyroidal  and antidiabetic activity. , Though antidiabetic activity of Annona squamosa has been demonstrated in various animal models, ,, the molecular mechanisms by which the extract improves glucose lowering and the mechanism of action are not known. Further, pharmacological studies using herbal extracts invariably used either alloxan or streptozotocin to induce diabetic conditions. , However, the chemically-induced diabetic animal models may suffer from toxicity in different organs besides their cytotoxicity on β cells, development of hyperglycemia due to insulin insufficiency rather than insulin resistance and difficulties in performing long-term experiments due to instability of chemicals may lead to reversibility of diabetic conditions.  In fact, streptozotocin and alloxan have been widely used as diabetogenic agents for their ability to produce free radicals in the body, cut DNA chains in β cells of pancreas causing necrosis, causing decreased production of insulin leading to severe hypoinsulinemia and hyperglycemia, similar to type 1 diabetes in humans.  Thus, animal models, mimicking type 2 diabetic conditions, are suitable to evaluate herbal extracts or its constituents. The present study utilizes ob/ob mouse exhibiting type 2 diabetic phenotypes such as insulin resistance, hyperglycemia, impaired glucose tolerance and severe hyperinsulinemia. 
In view of the above, the current study was designed to demonstrate the ability of Annona squamosa leaf hexane extract to stimulate insulin signaling events in L6 myotubes, an appropriate in vitro model and to assess its glucose and triglyceride lowering effect in type 2 diabetes genetic model (ob/ob mice). Also, attempts were made to elucidate the possible mechanism of action of the extract using battery of antidiabetic targets, including PTP1B.
| » Materials and Methods|| |
Chemicals and Reagents
All cell culture media and supplements were procured from Life Technologies Inc. Insulin and cytochalasin B were obtained from Sigma-Aldrich. 2-deoxy-D-[1- 3 H] glucose (2-DG) were purchased from Board of Radiation and Isotope Technology (BRIT), Mumbai. Trizol reagent, AMLV reverse transcriptase, dNTP and Taq polymerase were obtained from GIBCO, BRL, USA. Human recombinant PTP1B and RK-682 were purchased from Biomol.
Culture and Differentiation of L6 Myotubes
Culture and differentiation of L6 myotubes were carried out as described earlier.  The differentiated myotubes were used for the study.
The effect of hexane extract on cell viability was assessed by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). Briefly, L6 myotubes were seeded in 24 well plates with a cell density of 2500 in each well for differentiation, followed by treatment with different concentrations of the hexane extract at 1, 5 and 10 μg/ml and MTT for 4 h followed by the addition of 100% dimethyl sulfoxide (DMSO) to lyse the cells and solubilize the formazan crystals. The samples were transferred to 96 well plates and read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced was directly proportional to the number of viable cells. The viable cells were determined by trypan blue exclusion test using the following formula:
% viable cells = cell number × dilution factor × 104 cells/ml
2-DG Uptake Assay
L6 myotubes cells, grown in 24-well plate (Corning, NY), were subjected to glucose uptake as reported.  Briefly, differentiated myotubes were serum starved for 5 h and incubated with different concentrations of hexane extract (1 pg - 1 μg/ml) for 18 h. After incubation, cells were rinsed once with HEPES-buffered Krebs Ringer phosphate solution (118 mM NaCl, 5 mM KCl, 1.3 mM CaCl 2 , 1.2 mM MgSO 4 , 1.2 mM KH 2 PO 4 and 30 mM HEPES pH 7.4) and subsequently incubated for 15 min in HEPES-buffered solution containing 0.5 mCi/ml 2-DG. The assay was terminated by rapidly aspirating the media, and the cells were washed thrice with ice cold HEPES buffer solution. The cell-associated radioactivity was determined by cell lysis in 0.1 N NaOH, neutralized with 0.1 N HCl and counted in liquid scintillation counter. Aliquot from each well was quantified for protein using the BCA protein assay kit (Pierce). To study the effect in presence of insulin, 100 nM insulin was added to the hexane extract-treated cells for 15 min prior to the assay. The differentiated cells were pre-incubated with 100 ng hexane extract in presence of 10 μM cytochalasin B before the assay. 2-DG uptake values were corrected for non-specific uptake in the presence of 10 μM cytochalasin B (5% - 10% of total uptake). All the assays were performed twice in duplicates for consistency.
Annona squamosa leaves procured from reliable source were authenticated by a botanist in New Drug Discovery Research, Ranbaxy Laboratories Ltd., India. Since A. squamosa aqueous extract from different laboratories have shown anti-diabetic activity, ,, we also attempted aqueous extract preparation using bioassay-guided fractionation. The fractions were screened in 2-DG uptake assay. As the fractions did not show significant glucose uptake, we tried different solvents such as hexane, methanol, petroleum ether. Hexane extract was found to give good response in the bioassay, which was used in the study. The detailed report of the fractionation of standardized bioactive extracts of A. squamosa have been reported elsewhere. 
Preparation of Hexane Extract
Powdered Annona squamosa leaves (1 kg) were macerated with 10 liters of hexane for 16 hours in the extractor, filtered and the extract stored in a container. The process was repeated twice. The hexane extracts were combined and concentrated to 1/5 th under reduced pressure at low temperature. The extract was poured into stainless steel trays and dried in high vacuum oven at room temperature for about 16 - 18 hours. The yield was around 3% - 4%. For in vitro studies, % stock solution (w/v) was made in 100% DMSO. The working solutions were made in the appropriate buffers with 1% DMSO final concentrations. For the in vivo studies, freshly prepared hexane extract was suspended in 0.25% carboxy methyl cellulose as per the dose.
IR-ß and IRS-1 Tyrosine Phosphorylation Assays
The differentiated L6 cells were treated with the hexane extract for 18 h and insulin (100 nM) for 15 min. Thereafter, cells were washed once with ice-cold PBS and lysed in 1 ml of lysis buffer (50 mM Hepes, 150 mM NaCl, 10 mM EDTA, 10 mM Na 4 P 2 O 7 , 1 mM sodium orthovanadate, 50 mM NaF, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1% triton X-100 pH 7.4). The lysates were centrifuged, and the supernatant was incubated separately with 2 μg of rabbit anti-insulin receptor antibody (BD Biosciences, San Jose, CA) and 2 μg of mouse anti-IRS-1 antibody (BD Biosciences, San Jose, CA) for 12 h at 4°C. The immunocomplex was precipitated by 50 μl of protein A-Sepharose beads, washed 3 times with 500 μl lysis buffer and resolved in 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a PVDF membrane. The membrane was blocked in TBST (25 mM Tris-HCl, pH 8.0, 125 mm NaCl, 0.1% Tween 20) containing 5% skimmed milk for 1 h and then incubated with rabbit anti-phosphotyrosine (primary) antibody (1:1000 dilution) for 1 h at 25°C. The blot was washed extensively with TBST and further incubated with secondary anti-rabbit antibody, conjugated to horseradish peroxidise (HRP) (1:4000 dilution). After further washing with TBST, the blots were developed using enhanced chemiluminescence (ECL kit, Amersham).
PI3 Kinase and GLUT4 mRNA Expression Analysis
Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out as described previously.  Briefly, the differentiated L6 myotubes were incubated for 18 h at 37°C with the hexane extract (10 and 100 ng/ml) and lysed in total RNA isolation reagent Trizol. Proteins were extracted with chloroform, and total RNA was precipitated with isopropanol. The RNA precipitate was washed with 70% ethanol and resuspended in 50 μl of diethylpyrocarbonate-treated water. Reverse transcription was carried out to obtain cDNA using 200 units of avian reverse transcriptase and 200 ng/μl oligo d[T]. For PCR, following primers were used: PI3 kinase forward, 5′-TGA CGC TTT CAA ACG CTA TC-3′; reverse, 5′-CAG AGA GTA CTC TTG CAT TC-3′ (246-bp); GLUT4 forward, 5′-CGG GAC GTG GAG CTG GCC GAG GAG-3′; Reverse 5′-CCC CCT CAG CAG CGA GTG A-3′ (318-bp); GAPDH forward, 5′-CCA CCC ATG GCA AAT TCC ATG GCA-3′; reverse, 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′ (588-bp). For PCR reaction, 1 μl of the cDNA mixture was added to a PCR reaction mix consisting of 10 x PCR buffer, 2 mM dNTP, 10 pM of paired primers, 2 units of Taq polymerase and distilled water in a total volume of 50 μl. The reaction mixture was placed in a PCR thermal cycler for 35 cyclic reactions. PCR products were run on 1.5% agarose gels, stained with ethidium bromide and photographed. The intensity of the bands was quantified using Biorad Gel Doc system.
Cell-free and Cell-based Assays for Mechanism of Action
Hexane extract was tested for its ability to modulate various antidiabetic targets. GPR40 assay was performed in GPR40 over-expressing CHO cells.  SIRT1 assay was performed using human recombinant SIRT1.  DPP-IV assay was performed using citrated human plasma as enzyme source.  PTP1B assay was carried out using recombinant human PTP1B (25 ng/well) in sodium acetate buffer, pH 5.5 containing 1 mM DTT, 1 mM EDTA 0.5% Igepal and 5 mM p-nitrophenyl phosphate in 96-well format in absence and presence of varying concentrations of the hexane extract. The product p-nitrophenol was measured at 405 nm and IC 50 calculated. The enzyme reaction was carried out at 30°C for 20 min in dark and arrested by 1N NaOH. RK682 was used as a positive control for PTP1B enzyme inhibition. (IC 50 = 100 μM).
Animals and Extract Treatment
Ob/ob mice (8-10 weeks old, either sex), procured from in-house animal breeding facility, Ranbaxy Research Laboratories Ltd., were provided standard laboratory chow (Harlan Teklad, Oxon, UK) and water ad lib. The animals were maintained at a controlled temperature (24 ± 2°C) and illumination to provide a light dark cycle of 12 h, till the end of experimentation. All experiments were conducted according to the Guidelines of Experimental Animal Care issued by the Committee for Purpose of Control and Supervision of Experiments on Animals (CPCSEA). The animals grouped, based on their random blood glucose, were divided into 3 groups (4 male + 4 female in each group). First group was given vehicle (0.25% carboxy methyl cellulose, p.o), once daily. Second group was given rosiglitazone (10 mg/kg, once daily p.o), a PPAR-γ agonist as a standard compound. Hexane extract was freshly prepared with vehicle and administered twice daily (08.00 AM and 8.00 PM) for 21 days (500 mg/kg, b.i.d. p.o). The dose selection was based on the toxicity study in C57BL/6j mice (4 groups of 6 each; 3 male and 3 female), where the animals were treated with varying amounts (250, 500, 1000 and 2000 mg/kg, p.o) of the extract for 7 days. The mice were monitored for behavioral changes, food consumption and body weight changes. The behavior of the treated mice appeared normal, no death was noted and body weight was normal at all doses tested. For the present study, 500 mg/kg, b.i.d. p.o was selected since 500 mg/kg, p.o did not produce significant glucose lowering effect as reported earlier.  Before extract treatment, body weights of animals were recorded and monitored throughout the treatment period (21 days). During the treatment period, the animals were monitored for behavioral changes, food consumption and body weight changes (for 21 days) and found no undesirable side effects. Blood samples were collected before starting the treatment and on day 21 to measure blood glucose, triglyceride and total cholesterol levels. Blood samples were collected by orbital sinus bleeding under light ether anesthesia. On day 22, overnight fasted ob/ob mice in all the 3 groups were challenged with oral glucose (2 g/ kg), followed by blood collection at 0, 15, 30, 60 and 120 min. Plasma glucose concentrations were measured and % AUC of ∆blood glucose 0-120 min calculated from the oral glucose tolerance curves using GraphPad Prism software, Version 4.02 (GraphPad Software, San Diego, CA).
Blood Chemistry Analysis
Plasma samples were analyzed for triglyceride, glucose and total cholesterol using commercial diagnostic kits (RANBAXY DIAGNOSTICS, DELHI) and Automated Biochemical AutoAnalyzer (DADE BEHRING, USA). All the animal studies have been approved by the Institutional Animal Ethics Committee (IAEC Approval No. 84/05 dt. 19/09/2005)
Glucose uptake data represented the average of 2 individual experiments done in duplicates. The EC 50 values and 95% confidence intervals (95% C.I.) were determined from sigmoidal curves of dose response of glucose uptake by stimulated by hexane extract. Significance of differences in random plasma glucose and triglycerides was analyzed by performing one-way analysis of variance (ANOVA), followed by Dunnett's multiple comparison test between control and treated groups. The statistical difference before and after treatment was analyzed by paired t-test. Differences in AUC values of blood glucose during glucose tolerance test between vehicle control and different treatment groups (rosiglitazone and hexane extract) were determined by ANOVA, followed by Dunnett type multiple comparison tests. P value of <0.05 was considered statistically significant. All the statistical analysis was carried out using GraphPad Prism software, Version 4.02 (GraphPad Software, San Diego, CA).
| » Results|| |
Effect of Annona Squamosa Extract on 2-DG Uptake
Differentiated L6 myotubes when treated with different doses of hexane extract did not show cytotoxic effect with more that 95% viable cells at 10 μg/ml treatment (data not shown). Differentiated myotubes after incubation with hexane extract were analyzed for glucose uptake. Hexane extract stimulated glucose uptake in a dose-dependent manner with an EC 50 39 ng/ml. When the hexane extract was tested in presence of insulin at 100 nM, there was no augmentation of glucose uptake (EC 50 = 40 ng/ml) [Figure 1]a. Further, glucose uptake was not potentiated by rosiglitazone (50 μM) compared to hexane extract treatment alone (data not shown).
|Figure 1: Effect of Annona squamosa hexane extract on insulin signaling pathway in differentiated L6 myotubes (a) 2-deoxy-D-[1-3H] glucose uptake; Results are expressed as mean of two experiments done in duplicates. (b) IR-b phosphorylation-Lane 1: control (untreated); lane 2: rosiglitazone (50 μM), lane 3: insulin (100 nM), lane 4: hexane extract (100 ng), lane 5: hexane extract (10 ng) and (c) IRS-1 phosphorylation-Lane 1: control (untreated); lane 2: insulin (100 nM), lane 3: rosiglitazone (50 μM), lane 4: hexane extract (100 ng), lane 5: hexane extract (10 ng)|
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Effect of Hexane Extract on IR-ß and IRS-1 Phosphorylation
Next, we examined the effect of hexane extract on the phosphorylation events in proximal steps of insulin signaling pathway. As shown in [Figure 1]b, hexane extract stimulated IR-β phosphorylation 2 to 3 fold at 10 and 100 ng. Both insulin and rosiglitazone at 100 nM and 50 μM also increased the phosphorylation of IR-b 1.5 to 2.5 fold. Since IR-β autophosphorylation is a pre-requisite for insulin signaling, the effect of hexane extract on IRS-1 phosphorylation was also checked. As shown in [Figure 1]c, hexane extract stimulated IRS-1 phosphorylation ~ 3 fold at both 10 and 100 ng, whereas insulin and rosiglitazone (100 nM and 50 μM, respectively) stimulated ~ 2 fold. The intensity of the phosphorylated bands was quantified using Biorad Gel Doc system.
Effect of Hexane Extract on PI3 Kinase and GLUT4 mRNA Expression
To further investigate the effect of hexane extract on insulin signaling pathway, hexane extract treated myotubes were analyzed for PI3 kinase and GLUT4 expression at transcript level by semi-quantitative RT-PCR. Hexane extract treatment showed an increased expression of PI3 kinase (at 100 ng/ml) comparable to insulin and untreated control. However, 10 ng/ml as well as rosiglitazone treatment had no detectable effect on upregulation of PI3 kinase mRNA expression as compared to untreated control cells [Figure 2]a. Likewise, GLUT4 mRNA expression was also upregulated at both 10 and 100 ng/ml [Figure 2]b. Densitometric scanning revealed that GLUT4 mRNA is upregulated approximately 4 fold at both concentrations compared over untreated control cells and more prominent compared to rosiglitazone or insulin treatment (2 fold and 3 fold, respectively). GAPDH was used as an internal control.
|Figure 2: Effect of Annona squamosa hexane extract on (a) GLUT4 mRNA expression (b) PI3 kinase mRNA expression in differentiated L6 myotubes. Lane 1: control (untreated); lane 2: rosiglitazone - 50 μM, lane 3: insulin -100 nM, lane 4: hexane extract-100 ng, lane 5: hexane extract-10 ng, lane 6: PCR negative|
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Mechanism of Action
The hexane extract was tested for their ability to modulate (activation or inhibition) battery of antidiabetic targets. The hexane extract inhibited human PTP1B in a dose-dependent manner with an IC 50 17.4 μg/ml [Figure 3]. However, hexane extract did not activate GPR40 or SIRT1 and inhibit DPP-IV even at 30 μg/ml (data not shown).
|Figure 3: Dose-response inhibition of PTP1B by hexane extract. Inhibition of PTP1B activity was measured using human recombinant PTP1B enzyme. Each point represents average of two experiments performed in triplicate|
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Effect of Hexane Extract on Random Blood Glucose, Triglyceride, Cholesterol and Glucose Excursion
As in vitro experiments have demonstrated hexane extract's ability to stimulate insulin signaling events (IR-β and IRS-1 phosphorylation, PI3 kinase and GLUT4 mRNA expression) in parallel with glucose uptake, the effect of hexane extract on blood glucose, triglyceride and cholesterol has been tested in ob/ob mice. An oral administration of hexane extract for 21 days caused significant reduction in random glucose (27.7%; P<0.01 vs. control) comparable to rosiglitazone treatment (32.3%; P<0.01 vs. control) [Figure 4]a. Further, hexane extract showed a significant reduction in plasma triglyceride comparable to rosiglitazone (30.5% and 33.5%, respectively; P<0.01 vs. control) [Figure 4]b. However, neither rosiglitazone nor hexane extract had an effect on total cholesterol level [Table 1]. Stimulation of insulin signaling and glucose uptake by hexane extract should result in plasma glucose lowering in type 2 diabetes. To test this hypothesis, we examined the effect of hexane extract on plasma glucose lowering in ob/ob mice. As shown in [Figure 4]c, oral administration of the hexane extract (500 mg, b.i.d.) for 21 days, exhibited significant glucose lowering in OGTT (P<0.05 vs. control at 30, 60 and 120 min). The effect was comparable to rosiglitazone, dosed at 10 mg/ kg for 21 days [Figure 4]c; P<0.05 vs. control at 30, 60 and 120 min). Further, the extract did not result in body weight gain compared to rosiglitazone where significant body weight gain was observed ([Table 1]; P<0.05 before and after treatment).
|Figure 4: Effect of hexane extract and rosiglitazone on (a) random blood glucose, (b) triglycerides (b), and (c) glucose lowering in OGTT after 21 days of treatment in ob/ob mice. Data are shown as mean ± S.E.M (n = 8). Hexane extract and rosiglitazone **P<0.01 vs. control for random blood glucose and triglyceride and *P<0.05 for OGTT|
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|Table 1: Effect of 21 day treatment of hexane extract and rosiglitazone on fasting blood glucose, post-prandial glucose, total cholesterol, triglycerides and body weight|
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| » Discussion|| |
Agents, stimulating insulin signaling events through activation of PI3 kinase for the regulation of glucose turnover,  could be useful in the treatment of type 2 diabetes. The present study was carried out to evaluate the effects of Annona squamosa hexane extract on insulin signaling events using appropriate cell model and animal model. L6 cell line (rat skeletal muscle) was chosen as cell model since skeletal muscle accounts for more than 75% whole body insulin-stimulated glucose uptake in mammals.  Hexane extract stimulated glucose uptake in a dose-dependent manner, and the effect was comparable to insulin and rosiglitazone, a PPARγ agonist,  which may be attributed to translocation of GLUT4 from insulin-responsive vesicles to the plasma membrane.  Since insulin and PPARγ agonists are known to increase glucose uptake in L6 myotubes,  their potentiating effect on the hexane extract was also studied. However, neither insulin nor rosiglitazone potentiate the effect of hexane extract on glucose uptake, suggesting their non-synergic and non-sensitizing roles respectively. Further, 2-DG uptake was specific to glucose transporter and not by simple diffusion as hexane extract-stimulated 2-DG uptake was completely blocked by cytochalasin B at 10 μM (data not shown). Mechanistically, herbal anti-diabetic agents exert their action as insulin mimetics, insulin secretogogues, and stimulants of glucose utilization or through miscellaneous mechanisms.  Based on the glucose uptake data, we hypothesized that Annona squamosa hexane extract may exert their action through modulation of insulin signaling, thereby potentiating glucose disposal and utilization in different tissues.
Stimulation of IR-β/IRS-1 phosphorylation by insulin or insulin mimetic may activate two major intracellular signaling pathways, namely PI3 kinase dependent and mitogen-activated protein kinase pathways. , In the present study, Annona squamosa hexane extract stimulates IR-β and IRS-1 phosphorylation, a prerequisite for downstream signaling cascade linking glucose transport. Our finding is similar to insulin action in stimulating the phosphorylation events in the proximal steps of the signaling pathway, suggesting the presence of bioactive principles in the hexane extract, which may act like insulin to mediate glucose transport. Activation of PI3 kinase is one of the post-receptor event in the insulin signaling pathway mediating glucose transport  and the same was reported for insulin mimetics or insulin receptor activators.  Our study has shown that both PI3 kinase and GLUT4 mRNA expression are elevated on hexane extract treatment in L6 cells in parallel with dose-dependent increase in glucose uptake, which may be due to an increased translocation of GLUT4.  This finding further confirms that Annona squamosa hexane extract possesses bioactive principles, acting as insulin sensitizer or insulin receptor activator, to mediate the glucose transport in a PI3 kinase dependent pathway.
Though the anti-diabetic effect of Annona squamosa have been reported earlier, ,, no attempt was made to elucidate the possible molecular mechanisms, which bring the hypoglycemic effect. Our study has established that hexane extract inhibited PTP1B in a dose-dependent manner. PTP1B is implicated in negative regulation of insulin signaling by dephosphorylating IR-β and its substrates IRS-1 and IRS-2. Inhibition of its activity would be highly beneficial in the treatment of insulin resistance observed in type 2 diabetes.  Since earlier studies have revealed the ability of Annona squamosa extract to stimulate insulin in β cells in animal model,  we tested the effect of hexane extract on different targets modulating insulin secretion (insulin secretogogue). However, the extract failed to activate GPR40 or SIRT1 and inhibit DPP-IV, suggesting the absence of bioactive principles in the hexane extract, stimulating insulin secretion. The inability of the hexane extract to stimulate insulin secretion was also confirmed in RIN cells where upto 30 μg/ml had no effect on insulin secretion (data not shown). Thus, it is evident that Annona squamosa hexane extract has the ability to inhibit PTP1B, thereby activating insulin signaling events, promoting PI3 kinase-dependent glucose uptake.
As the earlier studies have employed either STZ or alloxan to induce diabetic conditions in rats, the phenotype may not reflect type 2 diabetes  and warranted appropriate animal models. In the present study, ob/ob mice model was chosen to evaluate in vivo efficacy of hexane extract. This model is considered to reflect type 2 diabetes conditions such as hyperglycemia, insulin resistance and impaired insulin signaling.  Hexane extract improved glucose lowering, and the effect was comparable to rosiglitazone. The hexane extract also exhibited triglyceride lowering effect, which is in agreement with earlier reports. , However, the extract had no effect on serum cholesterol in contrast to other reports where cholesterol lowering effect was reported. , The differential profiles of triglycerides and cholesterol may be attributed to different bioactive principles present in hexane extract in our study compared to aqueous or ethanol extract reported in the literature. Further, the extract had no effect on body weight compared to rosiglitazone where significant body weight gain was observed as reported earlier with this class of compounds. 
In conclusion, the present study demonstrated that Annona squamosa hexane extract improves insulin signaling in L6 cells. Treatment of ob/ob mice with the extract for 21 days significantly improves glucose and triglyceride lowering. The study also highlighted that PTP1B inhibition by the bioactive principle(s) in the extract may be one of the possible mechanisms bringing these effects. To our knowledge, this study is the first in demonstrating one of the possible mechanisms of Annona squamosa extract in in vitro system and mediating glucose lowering effect in genetic model for type 2 diabetes. Identification of potential molecules, present in hexane extract through bioassay-guided fractionation, might lead to the discovery of novel agents to treat type 2 diabetes.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]