Insulin resistance due to lipid-induced signaling defects could be ...

Mol Cell Biochem (2010) 336:97–107 DOI 10.1007/s11010-009-0257-4

Insulin resistance due to lipid-induced signaling defects could be prevented by mahanine Anindita Biswas • Sushmita Bhattacharya • Suman Dasgupta • Rakesh Kundu • Sib Sankar Roy Bikas C. Pal • Samir Bhattacharya

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Received: 8 March 2009 / Accepted: 15 September 2009 / Published online: 14 October 2009 Ó Springer Science+Business Media, LLC. 2009

Abstract It is well known that free fatty acids (FFAs) play a key role in implementing insulin resistance and type 2 diabetes. Resources of chemical compounds that intervene the derogatory effect of FFAs are indeed very limited. We have isolated mahanine, a carbazole alkaloid, from the leaves of Murraya koenegii that prevented palmitate-induced inhibition of insulin-stimulated phosphorylation of IRb, PI3K, PDK1, and Akt in L6 myotubes. This was also reflected in the palmitate-induced inhibition of insulinstimulated [3H] 2-DOG uptake by L6 myotubes, where palmitate adverse effect was significantly blocked by mahanine. Previous reports indicated that one of the major targets of lipid-induced damage in insulin signaling pathway resulting impairment of insulin sensitivity is insulin receptor (IR). Here, we have observed that palmitate significantly increased pPKCe in both cytosol and nuclear region of L6 myotubes in comparison to control. Translocation of pPKCe to the nucleus was associated with the impairment of HMGA1, the architectural transcription factor of IR gene and all these were reversed by mahanine. Palmitate-induced activation of IKK/IjB/NF-jB pathway was also attenuated

A. Biswas S. Bhattacharya S. Dasgupta R. Kundu S. Bhattacharya (&) Cellular and Molecular Endocrinology Laboratory, Department of Zoology, School of Life Science, Visva-Bharati (A Central University), Santiniketan 731235, West Bengal, India e-mail: [email protected] S. S. Roy Molecular Endocrinology Laboratory, Indian Institute of Chemical Biology, 4-Raja S.C. Mullick Road, Kolkata 700032, India B. C. Pal Medicinal Chemistry Laboratory, Indian Institute of Chemical Biology, 4-Raja S.C. Mullick Road, Kolkata 700032, India

by mahanine. Taken together, mahanine showed encouraging possibility to deal with lipid induced insulin resistance. In order to examine it further, mahanine was administered on nutritionally induced type 2 diabetic golden hamsters; it significantly improved hyperglycemia in all the treated animals. Our results, therefore, suggest that mahanine acts on two important sites of lipid induced insulin resistance (i) impairment of IR gene expression and (ii) activation of NF-jB pathway, thus, showing promise for its therapeutic choice for type 2 diabetes. Keywords Insulin resistance Insulin signaling Free fatty acids NF-jB PKCe Mahanine

Introduction It is well known that free fatty acids (FFAs) are major player in promoting the loss of insulin sensitivity causing insulin resistance and type 2 diabetes [1–5]. Several reports demonstrated that increased plasma FFA level contributes to the development of insulin resistance. Patients with type 2 diabetes often display signs of abnormal lipid metabolism where excess of plasma FFA levels reduces insulin-stimulated glucose uptake thus impairs insulin sensitivity [6–8]. Lowering of increased plasma FFA level in diabetic subjects causes improvement of insulin sensitivity [9]. Incubation of isolated muscle strips or cultured muscle cells with FFA-decreased insulin-stimulated glucose uptake [2, 3, 6]. These reports clearly suggest that greater deposition of lipids in insulin target tissues promote loss of insulin sensitivity thus causing insulin resistance. Several reports indicate that FFA disrupts insulin signaling pathway. Lowering of glucose transport by FFA is associated with the inhibition of insulin-stimulated IRS-1

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and IRS1-associated PI3K phosphorylation [5, 10] that affects the downstream signal molecules such as Akt and Glut4 [11, 12]. It has been shown that some PKC isoforms may have regulatory effect on insulin signaling pathway. Lipid infusion in rats and humans impairs insulin-stimulated glucose disposal into the muscle and activated certain PKC isoforms concomitantly [13–15]. It has also been demonstrated that lipid-induced NF-jB activation has been associated with insulin resistance and type 2 diabetes [15, 16]. FFA has been shown to activate NF-jB and its translocation to the nucleus that compromised insulin sensitivity in skeletal muscle cells; inhibitors of IKK/IjB/NF-jB pathway prevent FFA-induced impairment of insulin activity [17]. A link between NF-jB and insulin resistance is probably maintained by inflammatory cytokines. Incubation of skeletal muscle cells with FFA increased IL-6 gene expression and secretion through activation of NF-jB and PKC which causes insulin signaling defects [18]. We have recently demonstrated that PKCe plays a major role in FFA-induced insulin resistance by downregulating insulin receptor (IR) gene expression [19–21]. A current report from our laboratory showed that FFA-induced IR gene downregulation is associated with increased NF-jB protein level which in turn triggers its own gene expression [22]. Therapeutic choice for insulin resistance and type 2 diabetes is indeed very limited. Thaiazolidinedione (TZD) classes of compounds are potent agonists for peroxisome proliferator-activated receptor c (PPARc) which regulates adipocyte gene expression and differentiation [23, 24]. TZDs reduce lipid-induced insulin resistance by inducing the expression of adipocyte PPARc target genes involved in lipid metabolism i.e., adiponectin and CD36 [25]. Although TZDs are effective in clinical use, they have certain adverse effects like fluid retention; liver toxicity, congestive heart failure, and weight gain [25–27]. During the regular screening procedure to search for chemical compounds from plant sources that repairs the loss of insulin sensitivity, we obtained an interesting carbazole alkaloid compound, mahanine, isolated and purified from Murraya koenegii leaves which effectively improved FFAinduced insulin resistance in skeletal muscle cells, one of the major insulin target cells. Mahanine intervenes to FFAs impairment of insulin signaling pathway by reversing the downregulation of IR and NF-jB activation due to FFA.

Materials and methods Reagents All tissue culture materials were obtained from Gibco-BRL, Life Technologies Inc., Gaithersburg, MD 20884-9980, USA. Methyl palmitate and porcine insulin were purchased

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from Sigma chemical company, St Louis, MO, USA. [3H] 2-deoxyglucose (Specific activity: 12.0 Ci/mmol) was from GE Healthcare Biosciences Ltd., Kowloon, HK. Antibodies utilized included anti-IRb (anti-rabbit), anti-IRS1 (antirabbit), anti-HMGA1 (anti-rabbit), anti-pTyr (anti-mouse, PY99), anti-pPI3K (anti-goat, Tyr 508), anti pPDK1 (antirabbit, Ser 241), anti-pAkt 1/2/3 (anti-rabbit, Ser 473), antipGlut4 (anti-goat, Ser 488), anti-pPKCe (anti-rabbit, Ser 729), anti-pNF-jB p65 (anti-rabbit, Ser 536), anti-pIKKa/b (anti-goat, Thr 23), anti-pIjBa (anti-rabbit, Ser 32) and all the corresponding non-phosphorylated antibodies were purchased from Santa Cruz Biotechnology Inc., USA. Alkaline phosphatase conjugated anti-rabbit, anti-goat, and anti-mouse secondary antibodies were also purchased from Santa Cruz Biotechnology Inc., USA. All other chemicals were from Sigma Chemical Co. USA. Extraction and purification of mahanine from Murraya koenigii Murraya koenigii (Rutaceae) leaves were collected from different areas of West Bengal, India and around 3 kg of fresh leaves were extracted with methanol in a mixture blender. The methanol extract was concentrated to dryness (188 g), suspended in water and successively extracted with ethyl acetate and n-butanol. The ethyl acetate part was concentrated and dried to give residue (70 g), chromatographed on silica gel (700 g) using petroleum ether–chloroform (100:0 to 0:100) as an eluent to give four fractions in order of elution. The fourth fraction (2.9 g) was repeatedly chromatographed on silica gel [solvent: petrol–chloroform (9:1)] and crystallization in petrol gave mahanine (0.25 g). It is identified by comparing its physical data and its infrared (IR), nuclear magnetic resonance 1H NMR, 13C NMR, and mass spectral data with those of an authentic sample. In vivo treatments All in vivo experimental procedures were approved by the Animal Ethics Committee of IICB, Kolkata. Male golden hamsters weighing approximately similar range were conditioned at 25 ± 2°C with a 12-h day–night cycle and fed a standard diet ad libitum. An insulin resistant obese hamster model was produced by a high-fat diet for 100 days by following a previously described method [28]. In percent of total energy, the high-fat diet consisted of 32.5% lard, 32.5% corn oil, 20% sucrose, and 15% protein, whereas the standard diet contained 57.3% carbohydrate, 18.1% protein, and 4.5% fat. The energy content of the standard diet was 15 kJ/g and the high-fat diet was 26 kJ/g. The hamsters were divided initially into two groups, the control containing six animals (fed standard diet) and hamsters fed high-fat diet (HFD) having 12 animals. Six hamsters from


HFD group were separated on 100 days to orally administer mahanine (0.6 mg/kg body weight) for 15 days. Blood was collected from the hamsters for the estimation of serum glucose level by enzymatic GOD-POD method (Autospan, Span diagnostics, Surat, India). Cell culture and treatments L6 myotubes were procured from the National Centre for Cell Science, Pune, India and were cultured in a similar manner by a previously described method from our laboratory [21, 22]. Confluent cells were treated without (control) or, with 0.75 mM palmitate (lipid-containing media was prepared by conjugation of free fatty acid with bovine serum albumin as described by us previously [19] or with palmitate plus mahanine (35 lg/ml) and was incubated for 6 h. Mahanine-treated cells were pretreated with mahanine for 2 h, then a 4-h incubation with palmitate. After termination of incubations cells were pelleted, resuspended in lysis buffer (1% NP-40, 20 mM HEPES (pH 7.4), 2 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 1 lg/ml leupeptin, 1 lg/ml aprotinin, 1 lg/ml pepstatin, and 1 mM PMSF) and sonicated on ice for 10 min. Cell lysates were centrifuged for 10 min at 10,000g and protein concentrations were determined by the method of Lowry et al. [29]. Cytosolic and nuclear fractions were prepared by following a previously described method [30]. [3H] 2-Deoxyglucose uptake [3H] 2-Deoxyglucose (Amersham Biosciences, USA) uptake in L6 myotubes was conducted as described by us previously [22]. L6 myotubes were serum starved overnight in Kreb’s Ringer Phosphate (KRP) buffer (12.5 mM HEPES, pH 7.4, 120 mM NaCl, 6 mM KCl, 1.2 mM MgSO4, 1 mM CaCl2, 0.4 mM NaH2PO4, and 0.6 mM Na2HPO4) supplemented with 0.2% bovine serum albumin. Cells were incubated separately for 6 h without or with 0.75 mM palmitate in presence or absence of mahanine (35 lg/ml) followed by 30 min incubation with porcine insulin (100 nM). Incubations in the absence of any of these chemicals were taken as the control. [3H] 2- deoxyglucose (0.4 nmol/ml) was added to each incubation 5 min before termination of incubation. Uptake was stopped by washing cells thrice with ice-cold KRP buffer in the presence of 0.3 mM phloretin to correct the glucose uptake data from simple diffusion and non specific trapping of radioactivity. Cells were harvested with trypsin (0.25%)-EDTA (0.5mM), solubilized with 1% NP-40, and [3H] 2-deoxyglucose (2-DOG) was measured in a Liquid Scintillation counter (Perkin Elmer, Tricarb 2800 TR).

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Immunoprecipitation In order to observe insulin receptor b (IRb) tyrosine phosphorylation, palmitate and palmitate plus mahaninetreated L6 myotubes were incubated with insulin (100 nM) for 30 min following a previously described method by us [19]. Incubation in the absence of any of these chemicals was considered as the control. Cells were lysed by sonication in lysis buffer (as described above) followed by centrifugation at 10,000g for 10 min. Supernatant was collected and 200 lg of protein was incubated overnight at 4°C with 2 lg IR b antibody. To each tube around 50 ll of protein A-sepharose was then added and incubated at 4°C for 2 h followed by centrifugation at 10,000g. Immunecomplexed IRb was resuspended in 500 ll of 0.1% CHAPS in PBS, washed thoroughly and subjected to SDSPAGE followed by immunoblotting with anti-phosphotyrosine antibody (anti-mouse; 1:1,000). Reverse transcription–polymerase chain reaction Total RNA was extracted from control and treated L6 myotubes using Tri-reagent (Sigma-Aldrich) and RT–PCR was conducted using RT kit from First strand cDNA synthesis kit, Fermentas Life Sciences, Revert AidTM, Hanover, MD, USA, following a previous description from this laboratory [19, 21]. The following primers [31] were used to amplify the insulin receptor cDNA sequence and b actin for internal control: IR forward: 50 -ACTGACCTCATGCGCATGTGCT GG-30 Reverse: 50 -GCCCGTTTTTCTTGCCTCCGTTCAT-30 ; b actin forward: 50 -TGACGGGGTCACCCACACTGT GCCCATCTA-30 Reverse: 50 -CTAGAAGCATTTGCGGTGGACGATGG AGGG-30 . Electrophoresis and immunoblotting Control and treated cell lysates (60 lg) were resolved on 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA 01730) through transfer buffer (25 mM Tris, 193 mM glycine, 20% methanol, pH 8.5) for 1.5 h by following our previous description [19, 21, 22]. Electrophoresis was carried out at 90 V constant voltage. Membranes were incubated with 10% Blocking buffer (20mM Tris base, 137mM NaCl, 1mM HCl, 0.1% Tween 20, and 10% non-fat milk) for 1 h followed by incubation with primary antibodies (1:1,000) for 4 h. Bound primary antibodies were visualized using corresponding secondary antibodies (alkaline phosphatase conjugated) at 1:1,000

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dilutions for 2 h and were developed with corresponding substrates BCIP/NBT. Chromatin immunoprecipitation assay L6 myotubes were incubated for 6 h without (control) or with 0.75 mM palmitate or palmitate plus mahanine. On termination of incubation, cells were fixed with 1% formaldehyde and incubated for 10 min at 37°C. Chromatin Immunoprecipitation (ChIP) assay was performed using a ChIP assay kit (Upstate, Temecula, CA, USA) according to the manufacturer’s protocol using anti-HMGA1 antibody. For amplification of the immunoprecipitated DNA, the following primers for the human IR-promoter sequence [32] has been used Forward: 50 -AACCACCTCGAGTCACCAAAA-30 and Reverse: 50 -AGAGAGAGGGAAAGCTTGCAG- 30 The PCR products were resolved on ethidium bromide stained 1.5% agarose gel and image was captured by BioRad gel documentation system using Quantity One software. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) where the F value indicated significance, means were compared by Duncun’s multiple range post hoc test. All values were means ± SEM of at least three independent observations.

Results Attenuation of insulin sensitivity by palmitate due to its signaling defects could be reversed by mahanine Insulin signaling cascade that ultimately leads to the uptake of glucose into the target cells is well known. Binding of insulin to insulin receptor (IR) a-subunit in the extracellular domain causes phosphorylation of tyrosine kinase in IR-b and thus initiates the signaling pathway. In order to observe whether impairment of insulin signaling mechanism by palmitate could be prevented by mahanine, we incubated L6 myotubes with insulin or insulin plus palmitate in the absence or presence of mahanine. Insulin signaling was augmented by insulin from upstream to downstream through the phosphorylation of IRb, PI3K, PDK1, and Akt while addition of palmitate significantly (P \ 0.001) attenuated this stimulation. Mahanine reversed palmitate inhibitory effect on insulin signaling (Fig. 1a, b, c, d). Figure 2a demonstrates that insulin-stimulated [3H] 2-DOG uptake by L6 myotubes was inhibited by palmitate,

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pre-treatment with mahanine rescued the cells from the adverse effect of palmitate indicating a preventive effect of mahanine. In order to observe whether mahanine also produce a curative effect, we incubated L6 myotubes with palmitate for 8 h and at 4 h it was co-incubated with mahanine. Post-treatment of mahanine significantly reduced palmitate adverse effect on insulin-stimulated [3H] 2-DOG uptake (Fig. 2b) suggesting its curative effect. Phosphorylation of Glut4, the glucose transporter protein, in L6 myotubes was markedly increased due to insulin which was blocked by palmitate and mahanine prevented this inhibition (Fig. 2c). These findings indicate that impairment of insulin signaling cascade by palmitate adversely affects insulin-stimulated 2-DOG uptake; mahanine significantly improves this situation by intervening the palmitate-induced damage of insulin signaling molecules. Palmitate-induced downregulation of IR expression was reversed by mahanine Our previous reports showed that phospho-PKCe is associated with the FFA-induced downregulation of IR gene and protein, and that may be one of the reasons for which decrease of insulin sensitivity occurs [12, 21]. Fig. 3a demonstrates that in control cells, non-phospho form of PKCe was prevalent in the cytosol and it was negligible in the nuclear region, palmitate treatment marginally reduced cytosolic PKCe but this was statistically insignificant. Mahanine did not produce any noticeable change of PKCe in palmitate-incubated cells. In contrast, phospho-PKCe (pPKCe) was significantly higher in palmitate-treated cells (P \ 0.001 C vs. P) than its non-phospho form and a significant amount was detected in the nuclear region, addition of mahanine to palmitate-incubated cells markedly reduced pPKCe both in the cytosol and nuclear region (P \ 0.001, P vs. P ? Ma, Fig. 3b). Our previous report demonstrated that only pPKCe is translocated into the nuclear region while its non-phospho form is restricted in the cytosol [21]. An important contention of our earlier report is the involvement of pPKCe in the alteration of HMGA1, an architectural transcription factor of IR gene, its impairment may adversely affect IR gene expression. Figure 4a demonstrates that eV1 restricted the migration of pPKCe to the nucleus and addition of mahanine to this incubation indicates a considerable reduction of palmitate-induced PKCe phosphorylation. That translocation of pPKCe to the nuclear region is associated with HMGA1 expression was shown in Fig. 4b where reduction of HMGA1 expression due to palmitate was blocked by eV1. Mahanine treatment with palmitate also showed improvement of HMGA1 expression presumably because of its


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Fig. 1 Mahanine prevents palmitate-induced damage of insulin signaling molecules. L6 myotubes were incubated for 6 h without (C-Control) or with insulin (I) or insulin plus palmitate (I ? P) or insulin plus palmitate plus mahanine (I ? P ? Ma). Insulin was added 30 min prior to the termination of the incubation. On termination of incubations cells were lysed by sonication and subjected to immunoprecipitation and immunoblot analysis. In the case of A, cell lysates were centrifuged for 10 min at 10,000g and 200 lg of supernatant protein was incubated overnight at 4°C with 2 lg IRb antibody. The immunocomplex was pelleted with protein

A-agarose by centrifuging at 10,000g for 10 min, this was washed thoroughly in PBS and resuspended in SDS-PAGE sample buffer. It was then boiled and resolved in 10% SDS-PAGE followed by immunoblot with anti-pTyr antibody. For B, C and D, 60 lg protein from each cell lysate was resolved in SDS-PAGE and immunoblotted with non-phospho or phospho-specific antibodies against PI3K or pPI3K (b), PDK1 or pPDK1 (c) and Akt or pAkt (d). Densitometric analysis carried out by using ImageJ software. Each value is the Mean ± SEM of three individual experiments. *P \ 0.001 (I vs. I ? P), # P \ 0.001 (I ? P vs. I ? P ? Ma)

inhibitory effect on PKCe phosphorylation. Palmitate affected decrease of HMGA1 binding to IR promoter indicating attenuation of promoter activity (Fig. 4c). This decrease of HMGA1 binding is possibly due to the dearth of HMGA1 level in response to palmitate as indicated from Fig. 4b. Inhibition of HMGA1 binding due to palmitate could be prevented by mahanine as mahanine reduced the adverse effect of palmitate. Since HMGA1 regulates IR gene transcription, we examined IRb gene expression by RT–PCR analysis. The results were of similar nature, palmitate attenuation effect was opposed by mahanine (Fig. 4d).

Mahanine-inhibited palmitate augmentation of NF-jB activation NF-jB is known to play a critical role in implementing the pathogenesis in insulin resistance and type 2 diabetes. FFA was shown to activate NF-jB and its nuclear translocation which impaired insulin sensitivity in skeletal muscle cells; inhibitors of IKK/IjB/NF-jB pathway prevented FFAinduced impairment of insulin sensitivity [17]. It could be seen from Fig. 5a, b and c that palmitate greatly stimulated (P \ 0.001) IKK, IjB, and NF-jB phosphorylation suggesting an activation of NF-jB pathway; addition

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Fig. 2 Palmitate inhibition of insulin activity was prevented by mahanine. (a) L6 myotubes were incubated for 6 h without (C) or with insulin (I) or insulin plus palmitate (I ? P) or pre-treated with mahanine and then incubated with palmitate and insulin (I ? P ? Ma); insulin and [3H] 2-DOG was added 30 min and 5 min prior to the termination of incubation respectively. Cells were washed 3 times with ice-cold KRP buffer, lysed and solubilized with 1% NP-40 followed by the determination of [3H] 2-DOG uptake in a liquid scintillation counter. b L6 myotubes were incubated in a similar manner as described above except the duration of incubation

was 8 h and in one group of palmitate treated cells mahanine was added at 4 h and then terminated at 8 h. Cells were washed, lysed and [3H] 2-DOG uptake was measured in a liquid scintillation counter. c L6 myotubes incubated similarly as mentioned above were lysed and immunoblotted with anti-pGlut4 antibody, the procedure was described under materials and methods. b-actin was used as loading control. Densitometric analysis was done using ImageJ software. Each value is the Mean ± SEM of three individual experiments. * P \ 0.001 (I vs. I ? P), # P \ 0.001 (I ? P vs. I ? P ? Ma)

of mahanine to these incubations significantly inhibited (P \ 0.001) palmitate-induced activation of NF-jB cascade.

the mahanine administered group. Since the time of mahanine treatment for 15 days, HFD group hamsters did not show significant differences in body weight or blood sugar levels in comparison to the HFD animals of 100 days, those data were not represented in Fig. 6. It would be interesting to note that there was no significant reduction of body weight in mahanine treated hamsters over the HFD (from 254 ± 22 g to 246 ± 19 g) indicating mahanine effect may not be related to the reduction of FFA level.

Nutritionally induced diabetic golden hamsters showed improvement due to mahanine treatment Golden hamsters were fed high-fat diet (HFD) for 100 days along with a control group having normal diet. Figure 6 demonstrates that there was significant increase in body weight due to fat-enriched diet (125 ± 5 g at 0 day to 254 ± 22 g after 100 days) as compared to the control (123 ± 8 g at 0 day to 130 ± 8 g after 100 days). There was also a significant increase in the blood glucose level in the HFD than the control animals (from 82 ± 8 mg/dl in the control to 232 ± 34 mg/dl in HFD on 100 days treatment). 15 days treatment of mahanine (0.6 mg/kg body weight/day fed orally) strikingly reduced blood glucose level from 232 ± 34 mg/dl in HFD to 101 ± 23 mg/dl in

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Discussion Several chemical compounds have been examined for the amelioration of insulin resistance and type 2 diabetes. Except thiazolidinedione (TZD) classes of compounds, none other showed significant effect on the loss of insulin sensitivity. TZDs improve insulin sensitivity by lowering


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Fig. 3 Mahanine inhibits palmitate induced translocation of phosphorylated PKCe from cytosol to nuclear region. L6 myotubes were incubated for 6 h without (C) or with palmitate (P) or palmitate plus mahanine (P ? Ma). Cytosolic and nuclear fractions were isolated and subjected to SDS-PAGE followed by immunoblot with anti-PKCe

antibody (a) or with anti-pPKCe antibody (b). b-actin was used as loading control. Densitometric analysis was done using ImageJ software. Each value is the Mean ± SEM of three individual experiments. * P \ 0.001 (C vs. P), # P \ 0.001 (P vs. P ? Ma)

Fig. 4 Palmitate induced pPKCe inhibited HMGA1 binding to IR promoter that affects downregulation of IR gene expression which was reversed by mahanine. a L6 myotubes were incubated without or with palmitate (P) or with palmitate plus eV1 or palmitate plus eV1 plus mahanine (Ma) or palmitate plus mahanine and probed with antipPKCe antibody or b anti-HMGA1 antibody. c L6 myotubes were incubated without or with palmitate or palmitate plus mahanine; incubations were terminated by adding 1% formaldehyde. Chromatin immunoprecipitation assay was performed by immunoprecipitating HMGA1 bound IR-promoter region using anti-HMGA1 antibody.

The recovered DNA was used as template for PCR analysis with primers of IRb promoter as described in the materials and method section. d From the similar sets of incubation RNA was extracted and IRb mRNA level was determined by RT-PCR. PCR amplification was performed using IRb primers (mentioned in the materials and method) taking gapdh as an internal control. Densitometric analysis was carried out by using ImageJ software. Each value is the Mean ± SEM of three individual experiments. * P \ 0.001 (C vs. P), # P\0.001 (P vs. P ? Ma)

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Fig. 5 Mahanine abrogates palmitate induced activation of NF-jB through IKK-IjB pathway. Levels of palmitate induced phosphorylation of IKK (a), IrBa (b) and NF-jB (c) were determined in the L6 myotubes incubated in the presence (P ? Ma) or absence (P) of mahanine using antipIKK, anti-pIrBa and anti-pNFjB antibodies. b-actin was used as loading control. Densitometric analysis was done using ImageJ software. Each value is the Mean ± SEM of three individual experiments. * P \ 0.001 (C vs. P), ** P \ 0.01 (P vs. P ? Ma), # P\0.001 (P vs. P ? Ma)

Fig. 6 Effect of mahanine on the improvement of high blood glucose level in nutritionally induced diabetic hamsters. Hamsters fed with high fat diet (HFD) for 100 days and six out of twelve hamsters from that group were administered mahanine (0.6 mg/kg body wt) through oral gavage for 15 days. After completion of treatment, hamsters

were weighed and blood was collected for estimation of serum glucose by enzymatic GOD-POD method. Each value is the Mean ± SEM of the six hamsters. * P \ 0.001 (C vs. HFD), # P \ 0.001 (HFD vs. HFD ? Ma)

plasma FFA levels [33–36], they exert this effect through the activation of PPARc [23, 37] which is highly expressed in adipose tissue [38]. PPARc activation in turn regulates number of adipocyte gene expressions in adipocytes which promote FFA entry [39, 40] and reduced FFA release from the adipocytes [41]. These together substantially reduce circulatory FFA level leading to the improvement of insulin sensitivity. Although TZDs are clinically very effective, they

are found to be associated with increased development of edema, congestive heart failure [42, 43], weight gain and decrease in hemoglobin and hematocrit values [44, 45]. Hence, alternative therapeutic choice becomes imminent. In the present investigation, we have observed that mahanine, a carbazole alkaloid, effectively reversed fatty acid-induced insulin resistance by blocking palmitate-induced downregulation of insulin receptor gene expression and NF-jB

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activation that improves IR tyrosine kinase phosphorylation in response to insulin. Therefore, mahanine showed a promise in ameliorating the problem of insulin resistance. Prior to initiate the study presented here, we investigated whether mahanine can reduce circulatory FFA levels in nutritionally induced diabetic hamsters as mahanine remarkably decreased the increased blood glucose levels in these hamsters. We did not find a significant difference in circulatory levels of FFA between the control and mahanine-treated hamsters (data not shown). This experiment gave us a clue that decrease of blood glucose level by mahanine may not be through the pathway of TZDs; it might be improving insulin signaling defects due to FFA and that in turn reversed palmitate-induced insulin resistance. Several reports indicate that FFA adversely affects insulin signaling molecules thus cause insulin resistance [2, 3, 5, 14]. We have found that palmitate abrogated phosphorylation of major insulin signaling molecules starting from IR tyrosine kinase to Akt and these are reversed by mahanine indicating mahanine effect is associated with the FFA-induced insulin signaling defects. Question is—whether mahanine action is on upstream signal that causes an inhibition of the downstream molecules or it acts at several points of signaling cascade. Probability is more on the upstream molecule(s). Going through the earlier reports it seems possible that IR could be a primary target of FFA. Psammomys obesus, commonly known as sand rat, is a well-accepted model for nutritionally induced diabetes, the insulin resistance in this rat is considered as an innate characteristic. A PKC isoform, PKCe, has been found to be overexpressed in the skeletal muscle of this type 2 diabetic rat which is associated with the degradation of IRs [46]. A previous report also indicated involvement of PKC in decreasing tyrosine kinase receptors [47]. We have recently demonstrated that FFA-induced defects in insulin signaling in skeletal muscle cell is linked to IR gene downregulation where PKCe plays a significant role [19]. PKCe has been found to play a supportive role in fat induced hepatic insulin resistance by associating with IR, knocking down of PKCe expression could prevent rats from this pathogenesis [48]. Homozygous IR null mice die shortly after birth due to extreme insulin resistance [49, 50]. All these information imply that in FFA-induced insulin resistance, downregulation of IR is an important upstream event and its link with PKCe may be an important target for a therapeutic compound. Interestingly, mahanine showed encouraging results, it reversed palmitate inhibition of IR expression and revives insulin activity as monitored by 2-DOG uptake. A previous report by us demonstrated that translocation of FFA-induced pPKCe from cytosol to nuclear region in skeletal muscle cells is a key factor in relation to IR degradation as blocking of this translocation inhibits downregulation of IR gene. Translocation of pPKCe is

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associated with the impairment of HMGA1, the architectural transcription factor of IR gene and this causes a reduction in IR gene expression [21]. In this communication, it has been shown that in control cells there is a prevalence of non-phospho PKCe in the cytosol while in palmitate-treated cells it is the phospho form which not only increased significantly but also translocated to the nuclear region and this coincided with the attenuation of the HMGA1 expression and its phosphorylation. Mahanine inhibits phosphorylation of PKCe and thus abrogates its entry into the nuclear region. Impairment of HMGA1 by FFA appears to be associated with its effect on PKCe. A recent report showed that tissues from type 2 diabetic patients have reduced HMGA1 expression along with the attenuation of IR expression [51]. Although underlying mechanism of mahanine action is yet unclear, its effect on PKCe and HMGA1 provides an encouraging possibility in relation to its use as a therapeutic choice. Another interesting aspect of mahanine is its inhibition of NF-jB activity. NF-jB plays a significant role in imposing the pathogenesis in insulin resistance and type 2 diabetes [16, 52]. FFA promotes activation and nuclear localization of NF-jB in skeletal muscle cells and inhibitors of IKK/IjB/ NF-jB pathway prevent FFA-induced impairment of insulin activity. Indications are there that FFA induces insulin resistance through the involvement of activated NF-jB and novel PKCs [18, 53, 54]. A current report by us demonstrated that palmitate induces NF-jB expression and activation through the mediation of pPKCe [22]. Based on this background, we examined mahanine effect on NF-jB in IKK/IjB/NF-jB pathway in insulin resistant skeletal muscle cells where insulin signaling molecules were impaired by palmitate. Mahanine significantly inhibited the activation of NF-jB by palmitate. It is, therefore, intriguing to observe that mahanine acts on two targets of FFA-induced insulin resistance (i) it impedes downregulation of IR expression and (ii) attenuates the activation of NF-jB due to palmitate. From our present experimental results, it is not possible to clearly determine whether mahanine effect is direct or not. It appears mahanine acts through the inhibition of PKCe phosphorylation which restricts its mobility toward nucleus and that blocks HMGA1 impairment. Underlying mechanism of mahanine inhibitory effect on PKCe phosphorylation is yet unclear. Since, in vitro experiments with mahanine showed encouraging possibility of therapeutic use, we performed in vivo experiments with diabetic golden hamsters which is one of the ideal animal models because of its closeness to human being in comparison to rat. Almost a two fold increase in blood sugar levels occurs due to fatenriched diet for a prolong period which was consistently decreased in diabetic hamsters to near control level on mahanine treatment. Taken together, our observations with mahanine demonstrated that mahanine intervenes two

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important sites of palmitate-induced damage i.e., downregulation of IR expression and NF-jB activation in skeletal muscle cells that results in the improvement of hyperglycemia in diabetic golden hamsters indicating its promise as a therapeutic choice for dealing with insulin resistance and type 2 diabetes. Acknowledgments Authors are grateful to the Head, Department of Zoology, Visva-Bharati University and the Director, Indian Institute of Chemical Biology for extending all facilities for this present investigation. The authors are also grateful to the Department of Science & Technology (Grant No. VI-D&P/137/06-07/TDT), Ministry of Science & Technology, New Delhi for financial assistance. S. Dasgupta and R. Kundu are NET qualified candidates and they are thankful to the University Grants Commission, New Delhi for providing research fellowship.


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