Sudipta Maitra

Fetuin-A acts as an endogenous ligand of TLR4 to promote lipid-induced insulin resistance.

Toll-like receptor 4 (TLR4) has a key role in innate immunity by activating an inflammatory signaling pathway. Free fatty acids (FFAs) stimulate adipose tissue inflammation through the TLR4 pathway, resulting in insulin resistance. However, current evidence suggests that FFAs do not directly bind to TLR4, but an endogenous ligand for TLR4 remains to be identified. Here we show that fetuin-A (FetA) could be this endogenous ligand and that it has a crucial role in regulating insulin sensitivity via Tlr4 signaling in mice. FetA (officially known as Ahsg) knockdown in mice with insulin resistance caused by a high-fat diet (HFD) resulted in downregulation of Tlr4-mediated inflammatory signaling in adipose tissue, whereas selective administration of FetA induced inflammatory signaling and insulin resistance. FFA-induced proinflammatory cytokine expression in adipocytes occurred only in the presence of both FetA and Tlr4; removing either of them prevented FFA-induced insulin resistance. We further found that FetA, through its terminal galactoside moiety, directly binds the residues of Leu100–Gly123 and Thr493–Thr516 in Tlr4. FFAs did not produce insulin resistance in adipocytes with mutated Tlr4 or galactoside-cleaved FetA. Taken together, our results suggest that FetA fulfills the requirement of an endogenous ligand for TLR4 through which lipids induce insulin resistance. This may position FetA as a new therapeutic target for managing insulin resistance and type 2 diabetes.

Albendazole induces apoptosis in adults and microfilariae of Setaria cervi.

Setaria cervi, a filarial nematode of cattle, inhabits in the peritoneal cavity and has been used as a suitable model for screening antifilarial agents. Albendazole (ABZ), a tubulin-disrupting benzimidazole (BZ) and a potent microfilaricide binds to β-tubulin, is causing structural impairment of cytoskeleton and worm death. Our present study has revealed that exposure of microfilaria (Mf) and adult to gradually increasing concentration of ABZ leads to a dose-dependent gradual impairment of their motility followed by early death in vitro. We found extreme cellular disturbances in ABZ-treated worms characterized by nucleosomal DNA laddering and chromatin condensation. However, in the treated Mf no nucleosomal DNA laddering was found although presence of TUNEL reactive DNA was evident, thus indicating an apoptotic pathway independent of DNA fragmentation. We present data from molecular studies to provide evidence for ABZ-induced apoptosis in Mf and adult worms of S. cervi.

Mechanism of lipid induced insulin resistance: activated PKCε is a key regulator.

Fatty acids (FAs) are known to impair insulin signaling in target cells. Accumulating evidences suggest that one of the major sites of FAs adverse effect is insulin receptor (IR). However, the underlying mechanism is yet unclear. An important clue was indicated in leptin receptor deficient (db/db) diabetic mice where increased circulatory FAs was coincided with phosphorylated PKCε and reduced IR expression. We report here that central to this mechanism is the phosphorylation of PKCε by FAs. Kinase dead mutant of PKCε did not augment FA induced IRβ downregulation indicating phosphorylation of PKCε is crucial for FA induced IRβ reduction. Investigation with insulin target cells showed that kinase independent phosphorylation of PKCε by FA occurred through palmitoylation. Mutation at cysteine 276 and 474 residues in PKCε suppressed this process indicating participation of these two residues in palmitoylation. Phosphorylation of PKCε endowed it the ability to migrate to the nuclear region of insulin target cells. It was intriguing to search about how translocation of phosphorylated PKCε occurred without having canonical nuclear localization signal (NLS). We found that F-actin recognized phospho-form of PKCε and chaperoned it to the nuclear region where it interact with HMGA1 and Sp1, the transcription regulator of IR and HMGA1 gene respectively and impaired HMGA1 function. This resulted in the attenuation of HMGA1 driven IR transcription that compromised insulin signaling and sensitivity.

Participation of PI3-kinase/Akt signalling in insulin stimulation of p34cdc2 activation in zebrafish oocyte: phosphodiesterase 3 as a potential downstream target.

Exposure of fully grown oocytes to growth factors (insulin/IGFs) initiates various signalling cascades that culminate to final stages of oocyte maturation. Regulation of signalling pathways during growth factor-induced meiosis resumption in fish is not well characterized. Here we studied the participation of PI3K/Akt signalling pathway during recombinant human insulin (rh-insulin)-induced meiotic maturation in zebrafish (Danio rerio) oocytes. Priming of defolliculated oocytes in vitro with rh-insulin promotes germinal vesicle breakdown (GVBD) in a dose- and time-dependent manner, an effect sensitive to translation but not transcription inhibition. More than 80% of the oocytes underwent GVBD due to 0.8IU/ml rh-insulin within 10h of incubation and the kinetics of p34cdc2 kinase activation corresponded well with GVBD data. PI3K inhibitors, wortmannin and LY294002 blocked insulin, but not 17α, 20β-DHP-induced GVBD. Immunoblot analyses of oocyte extract revealed that phospho-PI3K (p85α) was up regulated within 30-60 min of insulin stimulation followed by phospho-Akt (Ser473) at 60-120 min. Though PI3K/Akt phosphorylation was largely unaffected, pre-incubation with phosphodiesterase (PDE) inhibitors, IBMX and cilostamide, but not rolipram completely blocked rh-insulin-induced p34cdc2 activation and GVBD. These results suggest that PDE3 may be one potential downstream target to PI3K/Akt signalling necessary for rh-insulin-induced GVBD in zebrafish.

A double-blind controlled field trial of doxycycline and albendazole in combination for the treatment of bancroftian filariasis in India

In a placebo controlled field trial, the effects of doxycycline (200mg/day) for 23 days followed by doxycycline (200mg/day) in combination with albendazole (ABZ) (400mg/day) for 7 days on depletion of Wolbachia endobacteria from Wuchereria bancrofti and microfilaricidal activity were studied in 68 patients (34 males and 34 females) from West Bengal, India. The drugs in combination (i.e., doxycycline+ABZ) provided the best efficacy by totally eliminating the circulating microfilaria (mf) (in 42% cases) on day 365 with (99.8%, P<0.05) suppression even on day 365 post-treatment compared to both exclusive doxycycline (69%, P<0.05) and ABZ (89%, P<0.05) groups. Thus, our results have established that a 30-day course of doxycycline in combination with a 7-day course of ABZ is sufficient to ensure long-term reduction in mf level by depleting Wolbachia from worm tissues. Doxycycline combined with ABZ led to a greater reduction in mf density in blood at 4 months (post-treatment) in comparison to doxycycline or ABZ alone. There were significant differences between the three treatments after 12 months (post-treatment). Further, the impact of a 7-day regimen of ABZ was surprisingly good in reducing mf compared to doxycycline-alone group. Adverse reactions were mild. A 30-day course of doxycycline and ABZ in combination is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against microfilaremia.

Ambient and Elevated Temperature Geopolymerization Behaviour of Class F Fly Ash

Geopolymerization is the process, where synthetic alkali activated alumino-silicate material is produced by reaction of a solid alumino-silicate with a highly concentrated aqueous alkali hydroxide or silicate solution. Depending on the raw material selection and processing conditions, geopolymers can exhibit a wide variety of properties and characteristics, including high compressive strength, fast or slow setting, acid resistance, and fire resistance. Fly ash, a by-product of coal fired power plant, is produced during combustion of coal. Fly ash is a powdery, aluminosilicate material along with minor impurities which is suitable for geopolymer reaction. The geopolymerization behaviour of class F fly ash has been studied in a temperature range of 27°-55°C using isothermal conduction calorimeter with different alkali concentrations. It was observed that the rate of reaction got intensified and increased with temperature. Compressive strength of the resulting geopolymer was also found to increase with temperature more significantly during early days. Attempt has been made to correlate the reaction with structure and properties.

Participation of cAMP-dependent protein kinase and MAP kinase pathways during Anabas testudineus oocyte maturation.

Possible involvement of cyclic nucleotide dependent protein kinase (PKA) and MAP kinase (MAPK) pathways during oocyte maturation in Anabas testudineus was investigated. Pre-incubation with phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX), inhibited 17α, 20β-DHP-induced GVBD dose dependently. PKA inhibitor, H89 could induce resumption of meiosis independent of 17α, 20β-DHP, in dose and duration dependent manner. The maximum response was obtained with the dose of 10 μM of H89 and 95% of cells underwent GVBD within 18 h. Moreover, stimulation with 17α, 20β-DHP inhibited endogenous PKA activity significantly within first hour and this effect was attenuated by PDE inhibitor IBMX at all time points. The pattern of PKA inhibition corresponded well with kinetics of histone H1 kinase activation and p34cdc2 phosphorylation. These results suggest physiological relevance of cAMP/PKA signaling in perch oocytes undergoing G2/M transition. MAPK was demonstrated as two distinct isoforms (ERK1 and ERK2) which resolved in the range of 42-44 kDa in immunoblot. Though total protein content did not show significant variation, H89 stimulation was able to stimulate phosphorylation of ERK1/2 from 5h onwards and the strongest response was observed between 10 and 18 h. MEK inhibitor, U0126 completely blocked PKA inhibition induced MAPK activation and GVBD. In addition, inhibition of endogenous PKA by a more selective peptide inhibitor [PKI-(6-22)-amide] was sufficient to resume GVBD and MAPK activation in intact perch oocytes. Also, significant ERK1/2 phosphorylation could be stimulated in cell-free extracts of perch oocytes supplemented with PKI-(6-22)-amide. The results suggest an interaction between cAMP/PKA and MAPK pathways in mediating meiosis resumption in perch oocyte.

High cAMP attenuation of insulin-stimulated meiotic G2-M1 transition in zebrafish oocytes: interaction between the cAMP-dependent protein kinase (PKA) and the MAPK3/1 pathways.

High intra-cellular cyclic nucleotide (cAMP) ensures prophase-I arrest and prevent steroid-induced meiotic G2-M1 transition in full-grown oocytes; however, relatively less information is available for cAMP regulation of growth factor-stimulated signalling events in the oocyte model. Here using zebrafish oocytes, we show that priming with dibutyryl cAMP (dbcAMP) or cAMP modulators, e.g. adenylate cyclase activator, forskolin or phosphodiesterase inhibitors (IBMX/cilostamide) block insulin action on germinal vesicle breakdown (GVBD) and histone H1 kinase activation. Though high cAMP priming attenuates insulin-induced MAPK3/1 (ERK1/2) phosphorylation (activation), following 2h of insulin stimulation it fails to block MAPK activation and GVBD. Further, insulin stimulation promotes down regulation of phospho-PKAc (inactivation) and PKA inhibition by H89/PKI-(6-22)-amide overcomes negative regulation by cAMP and induces GVBD and MAPK activation. Moreover, MEK1/2 inhibitor U0126 has no influence on H89-induced GVBD; however, it delays GVBD response in insulin-stimulated oocytes. MAPK activation by okadaic acid (OA) promotes GVBD; however, high dbcAMP abrogates OA action suggesting cross-talk between cAMP/PKA and MAPK-mediated signalling pathways may contribute significantly in maturing zebrafish oocyte.

Releasing prophase arrest in zebrafish oocyte: synergism between maturational steroid and Igf1

Binding of 17β-estradiol (E2) to novel G-protein coupled receptor, Gper1, promotes intra-oocyte adenylyl cyclase activity and transactivates epidermal growth factor receptor to ensure prophase-I arrest. Although involvement of either membrane progestin receptor (mPR) or Igf system has been implicated in regulation of meiosis resumption, possibility of concurrent activation and potential synergism between 17α,20β-dihydroxy-4-pregnen-3-one (DHP)- and Igf-mediated signalling cascades in alleviating E2 inhibition of oocyte maturation (OM) has not been investigated. Here using zebrafish (Danio rerio) defolliculated oocytes, we examined the effect of DHP and Igf1, either alone or in combination, in presence or absence of E2, on OM in vitro. While priming of denuded oocytes with E2 blocked spontaneous maturation, co-treatment with DHP (3 nM) and Igf1 (10 nM), but not alone, reversed E2 inhibition and promoted a robust increase in germinal vesicle breakdown (GVBD). Although stimulation with either Igf1 or DHP promoted Akt phosphorylation, pharmacological inhibition of PI3K/Akt signalling prevented Igf1-induced GVBD but delayed DHP action till 4-5 h of incubation. Moreover, high intra-oocyte cAMP attenuates both DHP and Igf1-mediated OM and co-stimulation with DHP and Igf1 could effectively reverse E2 action on PKA phosphorylation. Interestingly, data from in vivo studies reveal that heightened expression of igf1, igf3 transcripts in intact follicles corresponded well with elevated phosphorylation of Igf1r and Akt, mPRa immunoreactivity, PKA inhibition and accelerated GVBD response just prior to ovulation. This indicates potential synergism between maturational steroid and Igf1 which might have physiological relevance in overcoming E2 inhibition of meiosis resumption in zebrafish oocytes.

Endocrine and paracrine regulation of meiotic cell cycle progression in teleost oocytes: cAMP at the centre of complex intra-oocyte signalling events.

Participation of major endocrine and/or local autocrine/paracrine factors and potential interplay between apparently disparate intra-oocyte signalling events during maintenance and withdrawal of meiotic prophase arrest has been an area of active research in recent years. Studies on oocyte maturation have contributed substantially in the discovery of some of the most important biochemical and cellular events like functional significance of novel membrane-associated steroid receptors, elucidation of maturation promoting factor (MPF), cytostatic factor (CSF) and other signalling cascades that entrain the cell cycle clock to hormonal stimuli. While follicular estrogen has largely been implicated in maintenance of prophase arrest, involvement of maturational steroid and membrane progestin receptor in resumption of meiotic G2-M1 transition in piscine oocytes has been shown earlier. Moreover, detection of ovarian IGF system, maturational gonadotropin stimulation of IGF ligands and potential synergism between maturational steroid and IGF1 in zebrafish oocytes are most recent advancements. Though endocrine/paracrine regulation of cyclic nucleotide-mediated signalling events in meiotic cell cycle progression is well established, involvement of PI3K/Akt signalling cascade has also been reported in fish, amphibian and mammalian oocytes. The major objective of this overview is to describe how fish oocytes maintain high cAMP/PKA activity and how steroid- and/or growth factor-mediated signalling cascade regulate this pathway for the withdrawal of meiotic arrest. Moreover, special emphasis is placed on some recent findings on interaction of PKA with some of the MPF-regulating components (e.g., synthesis of cyclin B or MEK/MAPK signalling cascade) for the maintenance of prophase arrest.