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Insulin signaling cascade in peripheral insulin-sensitive tissues regulates whole-body glucose metabolism. Any deregulation in this pathway leads to insulin resistance, ultimately leading to metabolic diseases like type 1 diabetes, type 2 diabetes, and obesity. Insulin signaling in the brain has also been studied for many decades and associated with many primary functions like maintenance of synaptic plasticity, regulation of cognition, and circadian rhythm. Importantly, neuronal insulin signaling has also been associated with the regulation of neuronal glucose uptake. Any impairment in neuronal insulin signaling affecting neuronal glucose uptake has been associated with neurodegenerative disorders like Alzheimer's disease, the process now being termed as type 3 diabetes. Since the criticality lies in proper signaling cascade, determining important points of deregulation is important. In this review, we have discussed some critical points of such deregulation, dividing them into two classes of enzymes: kinases and phosphatases. We have highlighted their individual roles in neuronal insulin signaling, along with their possible implications in neuronal insulin resistance. Future strategies targeting these nodes in neuronal insulin signaling might be helpful in exploring potential therapeutic opportunities to overcome neuronal insulin resistance and related neurodegenerative diseases.

Overexpression of PKCα has been linked to inhibit insulin signaling disrupting IRS-1 and Akt phosphorylations in skeletal muscle. PKCα inhibits IRS-1 and Akt phosphorylations, but not required for insulin-stimulated glucose transport in skeletal muscles. Inhibition of PKCα increased whereas in some studies decreased GLUT-4 levels at the plasma membrane in skeletal muscles and adipocytes. Controversial studies have reported opposite expression pattern of PKCα expression in insulin-resistant skeletal muscles. These findings indicate that the role of PKCα on insulin signaling is controversial and could be tissue specific. Evidently, studies are required to decipher the role of PKCα in regulating insulin signaling and preferably in other cellular systems. Utilizing neuronal cells, like Neuro-2a, SHSY-5Y and insulin-resistant diabetic mice brain tissues; we have demonstrated that PKCα inhibits insulin signaling, through IRS-Akt pathway in PP2A-dependent mechanism by an AS160-independent route involving 14-3-3ζ. Inhibition and silencing of PKCα improves insulin sensitivity by increasing GLUT-4 translocation to the plasma membrane and glucose uptake. PKCα regulates GSK3 isoforms in an opposite manner in insulin-sensitive and in insulin-resistant condition. Higher activity of PKCα aggravates insulin-resistant neuronal diabetic condition through GSK3β but not GSK3α. Our results mechanistically explored the contribution of PKCα in regulating neuronal insulin resistance and diabetes, which opens up new avenues in dealing with metabolic disorders and neurodegenerative disorders., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The beneficial effects of exercise on the proper functioning of the body have been firmly established. Multi-systemic metabolic regulation of exercise is the consequence of multitudinous changes that occur at the cellular level. The exercise responsome comprises all molecular entities including exerkines, miRNA species, growth factors, signaling proteins that are elevated and activated by physical exercise. Exerkines are secretory molecules released by organs such as skeletal muscle, adipose tissue, liver, and gut as a function of acute/chronic exercise. Exerkines such as FNDC5/irisin, Cathepsin B, Adiponectin, and IL-6 circulate through the bloodstream, cross the blood-brain barrier, and modulate the expression of important signaling molecules such as AMPK, SIRT1, PGC1α, BDNF, IGF-1, and VEGF which further contribute to improved energy metabolism, glucose homeostasis, insulin sensitivity, neurogenesis, synaptic plasticity, and overall well-being of the body and brain. These molecules are also responsible for neuroprotective adaptations that exercise confers on the brain and potentially ameliorate neurodegeneration. This review aims to detail important cellular and molecular species that directly or indirectly mediate exercise-induced benefits in the body, with an emphasis on the central nervous system., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: PP1γ is one of the isoforms of catalytic subunit of a Ser/Thr phosphatase PP1. The role of PP1γ in cellular regulation is largely unknown. The present study investigated the role of PP1γ in regulating neuronal insulin signaling and insulin resistance in neuronal cells. PP1 was inhibited in mouse neuroblastoma cells (N2a) and human neuroblastoma cells (SH-SY5Y). The expression of PP1α and PP1γ was determined in insulin resistant N2a, SH-SY5Y cells and in high-fat-diet-fed-diabetic mice whole-brain-lysates. PP1α and PP1γ were silenced by siRNA in N2a and SH-SY5Y cells and effect was tested on AKT isoforms, AS160 and GSK3 isoforms using western immunoblot, GLUT4 translocation by confocal microscopy and glucose uptake by fluorescence-based assay., Results: Results showed that, in one hand PP1γ, and not PP1α, regulates neuronal insulin signaling and insulin resistance by regulating phosphorylation of AKT2 via AKT2-AS160-GLUT4 axis. On the other hand, PP1γ regulates phosphorylation of GSK3β via AKT2 while phosphorylation of GSK3α via MLK3. Imbalance in this regulation results into AD-like phenotype., Conclusion: PP1γ acts as a linker, regulating two pathophysiological conditions, neuronal insulin resistance and AD. Video Abstract., (© 2023. The Author(s).)

Neuronal insulin resistance is a major risk for development of Alzheimer's Disease (AD). Studies already reported few kinases participating in neuronal insulin signaling connected with progression of AD pathogenesis, yet complete information is missing. α isoform of Protein Phosphatase-2C (PP2C) is a Ser/Thr phosphatase, only known in 3T3-L1 adipocytes as a positive regulator of insulin signaling. However, many aspects of its function in neuronal insulin signaling and insulin resistance are unidentified. Recently, we reported that PP2Cα positively regulates neuronal glucose uptake possibly by a mechanism of dephosphorylation of IRS-1 at Ser522 and by inactivating AMPK, exacerbating hyperinsulinemia mediated neuronal insulin resistance. Since PP2Cα affected neuronal insulin signaling and AD is connected to neuronal insulin resistance, in the present study, we studied the role of PP2Cα in regulating activities of both isoforms of GSK3α and GSK3β (one of the leading kinases for AD progression). The results led us to test the role of PP2Cα on AD hallmarks. Silencing of PP2Cα caused hyperphosphorylation of a potential kinase Tau, leading to NFT formation and increased Aβ deposition. Our study thereby demonstrates escalation of hyperinsulinemia mediated neuronal insulin resistance leading to AD-like pathogenesis by PP2Cα in vitro and hints a novel molecule, PP2Cα, linking AD pathogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)

PP2Cα is one of the newly identified isoforms of metal-dependent protein phosphatases (PPM). The role of this phosphatase in neuronal insulin signalling is completely unknown. In the present study, we show insulin-mediated rapid upregulation of a protein of the insulin signalling cascade, PP2Cα, in mouse N2a cells and human SH-SY5Y cells. By contrast, such PP2Cα upregulation is not observed in insulin-resistant conditions despite insulin stimulation. Here, we report that, under insulin-sensitive and insulin-resistant conditions, the translation of PP2Cα was regulated by insulin through c-Jun N-terminal kinase. PP2Cα in turn dephosphorylated a novel inhibitory site of insulin receptor substrate-1 at Ser522 and AMP-activated protein kinase, hence positively regulating neuronal insulin signalling and insulin resistance., (© 2022 Federation of European Biochemical Societies.)

It has been more than a decade since the discovery of a novel class of phosphatase, the Pleckstrin Homology (PH) domain Leucine-rich repeat Protein Phosphatases (PHLPP). Over time, they have been recognized as crucial regulators of various cellular processes, such as memory formation, cellular survival and proliferation, maintenance of circadian rhythm, and others, with any deregulation in their expression or cellular localization causing havoc in any cellular system. With the ever-growing number of downstream substrates across multiple tissue systems, a web is emerging wherein the central point is PHLPP. A slight nick in the normal signaling cascade of the two isoforms of PHLPP, namely PHLPP1 and PHLPP2, has been recently found to invoke a variety of neurological disorders including Alzheimer's disease, epileptic seizures, Parkinson's disease, and others, in the neuronal system. Improper regulation of the two isoforms has also been associated with various disease pathologies such as diabetes, cardiovascular disorders, cancer, musculoskeletal disorders, etc. In this review, we have summarized all the current knowledge about PHLPP1 (PHLPP1α and PHLPP1β) and PHLPP2 and their emerging roles in regulating various neuronal signaling pathways to pave the way for a better understanding of the complexities. This would in turn aid in providing context for the development of possible future therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that there are no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: The aim of the present study was to determine the role of individual PHLPP isoforms in insulin signaling and insulin resistance in neuronal cells., Methods: PHLPP isoforms were either silenced or overexpressed individually, and the effects were observed on individual Akt isoforms, AS160 and on neuronal glucose uptake, under insulin sensitive and resistant conditions. To determine PHLPP regulation itself, we tested effect of scaffold protein, Scribble, on PHLPP isoforms and neuronal glucose uptake., Results: We observed elevated expression of both PHLPP1 and PHLPP2 in insulin resistant neuronal cells (Neuro-2A, mouse neuroblastoma; SHSY-5Y, human neuroblastoma) as well as in the whole brain lysates of high-fat-diet mediated diabetic mice. In insulin sensitive condition, PHLPP isoforms differentially affected activation of all Akt isoforms, wherein PHLPP1 regulated serine phosphorylation of Akt2 and Akt3, while PHLPP2 regulated Akt1 and Akt3. This PHLPP mediated Akt isoform specific regulation activated AS160 affecting glucose uptake. Under insulin resistant condition, a similar trend of results were observed in Akt isoforms, AS160 and glucose uptake. Over-expressed PHLPP isoforms combined with elevated endogenous expression under insulin resistant condition drastically affected downstream signaling, reducing neuronal glucose uptake. No compensation was observed amongst PHLPP isoforms under all conditions tested, indicating independent roles and pointing towards possible scaffolding interactions behind isoform specificity. Silencing of Scribble, a scaffolding protein known to interact with PHLPP, affected cellular localization of both PHLPP1 and PHLPP2, and caused increase in glucose uptake., Conclusions: PHLPP isoforms play independent roles via Scribble in regulating Akt isoforms differentially, affecting AS160 and neuronal glucose uptake. Video abstract., (© 2022. The Author(s).)

Protein phosphorylation is an important post-translational modification that regulates several cellular processes including insulin signaling. The evidences so far have already portrayed the importance of balanced actions of kinases and phosphatases in regulating the insulin signaling cascade. Therefore, elucidating the role of both kinases and phosphatases are equally important. Unfortunately, the role of phosphatases is less studied as compared to kinases. Since brain responds to insulin and insulin signaling is reported to be crucial for many neuronal processes, it is important to understand the role of neuronal insulin signaling regulators. Ser/Thr phosphatases seem to play significant roles in regulating neuronal insulin signaling. Therefore, in this review, we discussed the involvement of Ser/Thr phosphatases in regulating insulin signaling and insulin resistance in neuronal system at the backdrop of the same phosphatases in peripheral insulin sensitive tissues., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Role of CDK5 and its inhibition in various neuronal processes and functions are well established. However, role of CDK5 and its inhibition in neuronal insulin-signaling and-resistance is not yet explored. In the present study, we investigated the effect of CDK5 inhibition in neuronal insulin signaling, specifically insulin-stimulated glucose uptake. CDK5 expression in neuro-2a cells was increased under insulin-resistant state, developed by chronic treatment of insulin, confirming the crucial role of CDK5 in insulin resistance in neuronal cells. However, whether increased expression of CDK5 in hyperinsulinemia-mediated insulin-resistant conditions is a cause or a consequence, is still an unanswered question. We showed that CDK5 inhibition did not affect basal insulin signaling; however, insulin-stimulated glucose uptake enhanced in insulin-resistant cells. Moreover, CDK5 inhibition could improve glucose uptake, the ultimate outcome of insulin signaling, in insulin-resistant neuro-2a cells. We first time showed that CDK5 inhibition by roscovitine could ameliorate insulin resistance and increase glucose uptake in neuronal cells via ERK1/2 pathway. Our study provides intriguing insights about the effect of CDK5 inhibition on neuronal insulin resistance and opens up a new paradigm to develop new therapeutic strategies for neuronal insulin resistance and associated pathophysiological conditions., (© 2021 International Federation for Cell Biology.)

The Akt isoforms-AS160-GLUT4 axis is the primary axis that governs glucose homeostasis in the body. The first step on the path to insulin resistance is deregulated Akt isoforms. This could be Akt isoform expression, its phosphorylation, or improper isoform-specific redistribution to the plasma membrane in a specific tissue system. The second step is deregulated AS160 expression, its phosphorylation, improper dissociation from glucose transporter storage vesicles (GSVs), or its inability to bind to 14-3-3 proteins, thus not allowing it to execute its function. The final step is improper GLUT4 translocation and aberrant glucose uptake. These processes lead to insulin resistance in a tissue-specific way affecting the whole-body glucose homeostasis, eventually progressing to an overt diabetic phenotype. Thus, the relationship between these three key proteins and their proper regulation comes out as the defining axis of insulin signaling and -resistance. This review summarizes the role of this central axis in insulin resistance and disease in a new light., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The aim of the present study was to determine the role of Akt isoforms in insulin signaling and resistance in neuronal cells. By silencing Akt isoforms individually and in pairs, in Neuro-2a and HT22 cells we observed that, in insulin-sensitive condition, Akt isoforms differentially reduced activation of AS160 and glucose uptake with Akt2 playing the major role. Under insulin-resistant condition, phosphorylation of all isoforms and glucose uptake were severely affected. Over-expression of individual isoforms in insulin-sensitive and resistant cells differentially reversed AS160 phosphorylation with concomitant reversal in glucose uptake indicating a compensatory role of Akt isoforms in controlling neuronal insulin signaling. Post-insulin stimulation Akt2 translocated to the membrane the most followed by Akt3 and Akt1, decreasing glucose uptake in the similar order in insulin-sensitive cells. None of the Akt isoforms translocated in insulin-resistant cells or high-fat-diet mediated diabetic mice brain cells. Based on our data, insulin-dependent differential translocation of Akt isoforms to the plasma membrane turns out to be the key factor in determining Akt isoform specificity. Thus, isoforms play parallel with predominant role by Akt2, and compensatory yet novel role by Akt1 and Akt3 to regulate neuronal insulin signaling, glucose uptake, and insulin-resistance., (© 2021. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Protein kinase C alpha (PKCα) is known to participate in various signaling pathways due to its ubiquitous and dynamic characteristics. Previous studies report that PKCα abrogates peripheral insulin resistance, and recent publications show that it takes part in regulating Alzheimer's disease (AD). Based on evidence in the literature, we have highlighted how many of the substrates of PKCα in its signal transduction cascades are common in AD and diabetes and may have the capability to regulate both diseases simultaneously. Signaling pathways crosslinking these two diseases by PKCα have not been explored. Understanding the complexities of PKCα interactions with common molecules will deepen our understanding of its regulation of relevant pathophysiologies and, in the future, may broaden the possibility of using PKCα as a therapeutic target., Competing Interests: Declaration of Interests The authors declare no conflict of interest., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Type-2 Diabetes is associated with one of the co-morbidities due to SARS-Coronavirus 2 (SARS-Cov2) infection. Clinical studies show out of control glucose levels in SARS-Cov2 infected patients with type-2 diabetes. There is no experimental evidence suggesting aberrant molecular pathway(s) that explains why SARS-Cov2 infected patients with type-2 diabetes have uncontrolled glucose homeostasis and are co-morbid. In this article, we have highlighted major proteins involved in SARS-Cov2 infection, like, ACE 2, proteases like, TMPRSS2, Furin and their connectivity to insulin signaling molecules like, PI3K, Akt, AMPK, MAPK, mTOR, those regulate glucose homeostasis and the possible outcome of that cross-talk. We also raised concerns about the effect of anti-SARS-Cov2 drugs on patients with type-2 diabetes with reference to insulin signaling and the outcome of their possible cross-talk. There are no studies to decipher the possibilities of these obvious cross-talks. The major objective of this article is to urge the scientific community to explore the possibility of determining whether derangement of insulin signaling could be one of the possible causes of the patients with type-2 diabetes being co-morbid due to SARS-Cov2 infection., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Tankyrase, a member of poly (ADP-ribose) polymerase (PARP) family, regulates various cellular pathways including wnt signaling, telomere maintenance and mitosis, has become a prime target for the development of cancer therapeutics. Inhibition of tankyrase, which leads to its increased cellular accumulation, reveal the role of tankyrase in the regulation of Glucose transporter type 4 (GLUT4) translocation and glucose homeostasis in peripheral insulin responsive tissues. While in adipocytes inhibition of tankyrase improves insulin sensitivity and glucose uptake, its inhibition in skeletal muscle leads to development of insulin resistance. Evidently further studies are required to determine the broader perspective of tankyrase in other cellular systems in regulating insulin signaling and insulin resistance. Role of tankyrase in neuronal tissues/cells has not been tested. In the present study, we investigated the effect of tankyrase inhibition in insulin-sensitive and insulin-resistant Neuro-2a cells. Here, we report that XAV939 treatment, a tankyrase inhibitor, improves insulin-stimulated glucose uptake in insulin-sensitive as well as in insulin-resistant neuronal cells via AMP-activated protein kinase (AMPK) - AKT Substrate of 160 kDa (AS160) mediated pathway without affecting the phosphorylation/activation of AKT. AMPK inhibition by Compound C repressed XAV939 treatment mediated increase in glucose uptake, confirming the role of tankyrase in glucose uptake via AMPK. We show for the first time that inhibition of tankyrase significantly improves glucose uptake and insulin sensitivity of insulin-resistant neuronal cells via AMPK-AS160 mediated pathway. Our study demonstrates new mechanistic insights of tankyrase mediated regulation of insulin sensitivity as well as glucose uptake in neuronal cells., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Protein kinase C (PKC) family of enzymes is known to be a feedback regulator of insulin signalling pathway in peripheral insulin-responsive tissues. Insulin signalling is reported to be required for maintaining cognitive abilities in brain. PKCs are involved in innumerable neuronal processes including differentiation, apoptosis, survival, maintaining synaptic plasticity, long-term potentiation and memory formation. In the present study, we made an attempt to elucidate the role of PKC, if any, in regulating insulin signalling and insulin resistance in Neuro-2a (N2a) cells in vitro. We show that phorbol 12-myristate 13-acetate (PMA) -activated PKC inhibited Akt activation in neuronal cell, N2a. In the process of inhibiting Akt, PMA-activated PKC decreased downstream insulin signalling proteins like Akt substrate 160 kDa (AS160) and glycogen synthase kinase (GSK3β), followed by a decrease of glucose uptake in N2a cells. PKC activation caused insulin resistance in N2a cells and worsened the resistant state of already insulin-resistant cells. Hence, our study demonstrated that the activation of PKC attenuates insulin signalling cascade and make N2a cells insulin-resistant.

Trypanosomes of the genus Leishmania swim by undulating motions of a single flagellum driven by axonemal dynein ATPases, essential for parasite survival and infectivity. The flagellum possesses two waveforms; flagellar (tip-to-base) responsible for forward movements and ciliary (base-to-tip) possibly responsible for reorientation in response to changes in surroundings. However, the role of dyneins in regulating the two waveforms remains unknown. Moreover, the unpredictable nature of the parasite ciliary waveform makes it difficult to study. We have previously reported a detergent-extracted, ATP-reactivated model ideal for investigating flagellar motility regulation in Leishmania that allows one to generate reactivated Leishmania flagella with constitutively beating ciliary waves in presence of cyclic-AMP. Here, using three dynein inhibitors [erythro-9-(2-hydroxy-3-nonyl) adenine, ciliobrevin A and vanadate] we investigated the role of dyneins in regulating the two waveforms of Leishmania. Using high speed videomicroscopy we observed differential inhibition of beat frequencies and waveforms of flagellar and ciliary beats in live (in vivo) and ATP-reactivated (in vitro) parasites. Beat frequency of flagellar waveform was more strongly reduced than ciliary waveform. Surprisingly, inhibition of the ciliary waveform led to an altered phenotype with the distal half of the flagellum paralysed. ATPase assays confirmed that dynein activity of flagellar cells was more strongly inhibited compared to ciliary cells irrespective of the mechanism of inhibition. Possibly the two different waveforms are an outcome of changes in the mechanical properties of axonemal dyneins present at the tip of the flagellum that contains a sliding resistive structure. Our study suggests that dyneins responsible for the two waveforms in Leishmania bear different structural and functional conformations. Moreover, during ciliary beating, there is heterogeneity along the flagellum., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Mitogen-activated protein kinases (MAPKs) have been demonstrated to regulate flagellar/ciliary motility of spermatozoa and miracidia of Schistosoma mansoni. However, the role of MAPKs in mediating flagella-driven motility of Leishmania donovani is unexplored. We investigated the function of MAPKs in motility regulation of L. donovani using pharmacological inhibitors and activators of various MAPKs and fast-capture videomicroscopy. Our studies have revealed that the inhibitor of p38 MAPK, PD 169316, significantly affected various motility parameters such as flagellar beat frequency, parasite swimming speed, waveform of the flagellum and resulted in reduced parasite motility. Together, our results suggest that a MAPK, similar to human p38 MAPK, is implicated in flagellar motility regulation of L. donovani., (Copyright © 2017 Elsevier Inc. All rights reserved.)

We have previously reported the involvement of cyclic AMP in regulating flagellar waveforms in Leishmania. Here, we investigated the roles of calcium, calmodulin, and calcineurin in flagellar motility regulation in L. donovani. Using high-speed videomicroscopy, we show that calcium-independent calmodulin and calcineurin activity is necessary for motility in Leishmania. Inhibition of calmodulin and calcineurin induced ciliary beats interrupting flagellar beating in both live (in vivo) and ATP-reactivated (in vitro) parasites. Our results indicate that signaling mediated by calmodulin and calcineurin operates antagonistically to cAMP signaling in regulating the waveforms of Leishmania flagellum. These two pathways are possibly involved in maintaining the balance between the two waveforms, essential for responding to environmental cues, survival, and infectivity.

Axonemal dyneins are members of AAA+ proteins involved in force generation and are responsible for flagellar motility in eukaryotes. In this study, we characterized the effects of ciliobrevin A (CbA), a dynein ATPase inhibitor, on flagella driven motility of the protozoan parasite Leishmania donovani. Using fast-capture video microscopy, we observed that CbA decreased flagellar beat frequency of swimming parasites in a concentration-dependent manner. Beat frequency of live and reactivated L. donovani decreased by approximately 89% and 41% respectively in the presence of 250μM CbA. This inhibition was lost when CbA was removed, suggesting its effects were reversible. CbA also altered wavelength and amplitude of the flagellum of live parasites. Waveform analysis of live and reactivated L. donovani revealed that CbA significantly affected flagellar waveform by inducing non-uniform bends with the flagellum beating away from the cell axis. These results suggest that CbA sensitive dynein ATPases possibly are responsible for power generation and waveform maintenance of the flagellum of L. donovani. This ability to inhibit axonemal dyneins also emphasizes the use of dynein inhibitors as valuable tools in studying functional roles of axonemal dyneins of flagellated eukaryotes., (Copyright © 2017. Published by Elsevier B.V.)

We have recently reported P21-activated kinase 2 (PAK2), a serine/threonine kinase as a negative regulator of neuronal glucose uptake and insulin sensitivity. Resveratrol (RSV), a natural polyphenol with anti-oxidative, anti-inflammatory and anti-diabetic properties, regulates PAK2 activity in HepG2 and ESC-B5 cell apoptosis. However, regulation of PAK2 by RSV in neuronal insulin signaling pathway, if any, is still unknown. In the present study, RSV treatment significantly increased PAK2 activity under insulin-sensitive and insulin-resistant condition, along with a marked decrease in glucose uptake in differentiated N2A cells. Pretreatment with AMPK inhibitor, followed by RSV treatment resulted in reduction in PAK2 activity whereas glucose uptake showed an increase. However, pretreatment with Akt inhibitor and then RSV exposure significantly increased PAK2 activity, with a corresponding decrease in glucose uptake. RSV treatment increased AMPK activity and decreased Akt activity. In conclusion, RSV negatively regulates neuronal glucose uptake and insulin sensitivity via PAK2., (Copyright © 2017. Published by Elsevier Inc.)

Nuclear co-repressor (NCoR) regulates peripheral insulin sensitivity; however, its role in modulating insulin sensitivity in skeletal muscle remains elusive. Present study investigated protein expression and effect of NCoR on insulin sensitivity in murine skeletal muscle cell line C 2 C 12 . Myotubes as compared to myoblasts of C 2 C 12 cells were found to be more sensitive in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKT S473 ) was significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into myotubes of C 2 C 12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or myotubes of C 2 C 12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA in myotubes of C 2 C 12 . In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKT S473 and impaired glucose uptake. This observation is in contrast to published studies where NCoR has been reported to negatively regulate insulin signaling cascade. Furthermore, transient silencing of NCoR failed to improve insulin sensitivity in chronic hyperinsulinemia-induced insulin-resistant model of C 2 C 12 cells. Importantly, inhibition of lysosomal protein degradation pathway using ammonium chloride restored protein level of NCoR but failed to increase glucose uptake in serum-starved C 2 C 12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state (myoblast and myotubes) of C 2 C 12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C 2 C 12 myotubes., (© 2016 International Federation for Cell Biology.)

The flagellum of parasitic trypanosomes is a multifunctional appendage essential for its viability and infectivity. However, the biological mechanisms that make the flagellum so dynamic remains unexplored. No method is available to access and induce axonemal motility at will to decipher motility regulation in trypanosomes. For the first time we report the development of a detergent-extracted/demembranated ATP-reactivated model for studying flagellar motility in Leishmania. Flagellar beat parameters of reactivated parasites were similar to live ones. Using this model we discovered that cAMP (both exogenous and endogenous) induced flagellar wave reversal to a ciliary waveform in reactivated parasites via cAMP-dependent protein kinase A. The effect was reversible and highly specific. Such an effect of cAMP on the flagellar waveform has never been observed before in any organism. Flagellar wave reversal allows parasites to change direction of swimming. Our findings suggest a possible cAMP-dependent mechanism by which Leishmania responds to its surrounding microenvironment, necessary for its survival. Our demembranated-reactivated model not only serves as an important tool for functional studies of flagellated eukaryotic parasites but has the potential to understand ciliary motility regulation with possible implication on human ciliopathies.

P21-activated kinases (PAKs) are recently reported as important players of insulin signaling and glucose homeostasis in tissues like muscle, pancreas and liver. However, their role in neuronal insulin signaling is still unknown. Present study reports the involvement of PAK2 in neuronal insulin signaling, glucose uptake and insulin resistance. Irrespective of insulin sensitivity, insulin stimulation decreased PAK2 activity. PAK2 downregulation displayed marked enhancement of GLUT4 translocation with increase in glucose uptake whereas PAK2 over-expression showed its reduction. Treatment with Akti-1/2 and wortmannin suggested that Akt and PI3K are mediators of insulin effect on PAK2 and glucose uptake. Rac1 inhibition demonstrated decreased PAK2 activity while inhibition of PP2A resulted in increased PAK2 activity, with corresponding changes in glucose uptake. Taken together, present study demonstrates an inhibitory role of insulin signaling (via PI3K-Akt) and PP2A on PAK2 activity and establishes PAK2 as a Rac1-dependent negative regulator of neuronal glucose uptake and insulin sensitivity., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Insulin resistance in brain is well-associated with pathophysiology of deficits in whole-body energy metabolism, neurodegenerative diseases etc. Among the seven sirtuins, SIRT2 is the major deacetylase expressed in brain. Inhibition of SIRT2 confers neuroprotection in case of Parkinson's disease (PD) and Huntington's disease (HD). However, the role of this sirtuin in neuronal insulin resistance is not known. In this study, we report the role of SIRT2 in regulating insulin-sensitivity in neuronal cells in vitro. Using approaches like pharmacological inhibition of SIRT2, siRNA mediated SIRT2 knockdown and over-expression of wild-type and catalytically-mutated SIRT2, we observed that downregulation of SIRT2 ameliorated the reduced activity of AKT and increased insulin-stimulated glucose uptake in insulin resistant neuro-2a cells. The data was supported by over expression of catalytically-inactive SIRT2 in insulin-resistant human SH-SY5Y neuronal cells. Data highlights a crucial role of SIRT2 in regulation of neuronal insulin sensitivity under insulin resistant condition., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania . However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena , the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp.

SIRT2 is primarily a cytoplasmic protein deacetylase and is abundantly expressed in metabolically active tissues like adipocytes and brain. However, its role, if any, in regulating insulin signaling in skeletal muscle cells, is not known. We have examined the role of SIRT2 in insulin-mediated glucose disposal in normal and insulin resistant C2C12 skeletal muscle cells in vitro. SIRT2 was over expressed in insulin resistant skeletal muscle cells. Pharmacological inhibition of SIRT2 increased insulin-stimulated glucose uptake and improved phosphorylation of Akt and GSK3β in insulin resistant cells. Knockdown of endogenous SIRT2 and over expression of catalytically-inactive SIRT2 mutant under insulin-resistant condition showed similar amelioration of insulin sensitivity. Our results suggest that down-regulation of SIRT2 improved insulin sensitivity in skeletal muscle cells under insulin-resistant condition. Previously it has been reported that down-regulation of SIRT1 and SIRT3 in C2C12 cells results in impairment of insulin signaling and induces insulin resistance. However, we have observed an altogether different role of SIRT2 in skeletal muscle. This implicates a differential regulation of insulin resistance by sirtuins which otherwise share a conserved catalytic domain. The study significantly directs towards future approaches in targeting inhibition of SIRT2 for therapeutic treatment of insulin resistance which is the major risk factor in Type 2 diabetes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Lipid and protein tyrosine phosphatase, phosphatase and tension homologue (PTEN), is a widely known negative regulator of insulin/phosphoinositide 3-kinase signaling. Down-regulation of PTEN is thus widely documented to ameliorate insulin resistance in peripheral tissues such as skeletal muscle and adipose. However, not much is known about its exact role in neuronal insulin signaling and insulin resistance. Moreover, alterations of PTEN in neuronal systems have led to discovery of several unexpected outcomes, including in the neurodegenerative disorder Alzheimer's disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. In addition, contrary to expectations, its neuron-specific deletion in mice resulted in development of diet-sensitive obesity. The present study shows that PTEN, paradoxically, positively regulates neuronal insulin signaling and glucose uptake. Its down-regulation exacerbates neuronal insulin resistance. The positive role of PTEN in neuronal insulin signaling is likely due to its protein phosphatase actions, which prevents the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), the kinases critically involved in neuronal energy impairment and neurodegeneration. Results suggest that PTEN acting through FAK, the direct protein substrate of PTEN, prevents ERK activation. Our findings provide an explanation for unexpected outcomes reported earlier with PTEN alterations in neuronal systems and also suggest a novel molecular pathway linking neuronal insulin resistance and AD, the two pathophysiological states demonstrated to be closely linked.

Focal adhesion kinase (FAK), a non-receptor protein kinase, is known to be a phosphatidyl inositol 3-kinase (PI3K) pathway activator and thus widely implicated in regulation of cell survival and cancer. In recent years FAK has also been strongly implicated as a crucial regulator of insulin resistance in peripheral tissues like skeletal muscle and liver, where decrease in its expression/activity has been shown to lead to insulin resistance. However, in the present study we report an altogether different role of FAK in regulation of insulin/PI3K signaling in neurons, the post-mitotic cells. An aberrant increase in FAK tyrosine phosphorylation was observed in insulin resistant Neuro-2a (N2A) cells. Downregulation of FAK expression utilizing RNAi mediated gene silencing in insulin resistant N2A cells completely ameliorated the impaired insulin/PI3K signaling and glucose uptake. FAK silencing in primary cortical neurons also showed marked enhancement in glucose uptake. The results thus suggest that in neurons FAK acts as a negative regulator of insulin/PI3K signaling. Interestingly, the available literature also demonstrates cell-type specific functions of FAK in neurons. FAK that is well known for its cell survival effects has been shown to be involved in neurodegeneration. Along with these previous reports, present findings highlight a novel and critical role of FAK in neurons. Moreover, as this implicates differential regulation of insulin/PI3K pathway by FAK in peripheral tissues and neuronal cells, it strongly suggests precaution while considering FAK modulators as possible therapeutics., (© 2012 Elsevier B.V. All rights reserved.)

Insulin signaling is extensively studied in peripheral tissues while comparatively understudied in neuronal cells. AMPK is considered to be a fuel gauge of our body and activation of the same has been reported to increase insulin sensitivity in skeletal muscles thereby increasing glucose transport. However its role in neuronal insulin signaling is not established yet. Here we report positive regulation of insulin signaling as well as glucose uptake by AICAR, a pharmacological activator of AMPK, in cultured Neuro-2a cells in vitro. Compound C, a specific AMPK inhibitor, completely blocked the potentiating effects of AICAR on insulin signaling and glucose uptake, thus suggesting that AMPK mediates effects of AICAR on insulin signaling. Our study provides valuable insight in understanding the role of AMPK in neuronal insulin signal transduction., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most common neurodegenerative disease worldwide. Pharmacological treatments presently available can slow down the progression of symptoms but can not cure the disease. Currently there is widening recognition that AD is closely associated with impaired insulin signaling and glucose metabolism in brain, suggesting it to be a brain-specific form of diabetes and so also termed as "type 3 diabetes". Hence investigating the role of pharmacological agents that could ameliorate neuronal insulin resistance merit attention in AD therapeutics, however the therapeutics for pathophysiological condition like neuronal insulin resistance itself is largely unknown. In the present study we have determined the effect of metformin on neuronal insulin resistance and AD-associated characteristics in an in vitro model of "type 3 diabetes" by differentiating neuronal cell line Neuro-2a under prolonged presence of insulin. We observed that prolonged hyperinsulinemic conditions in addition to generating insulin resistance also led to development of hallmark AD-associated neuropathological changes. Treatment with metformin sensitized the impaired insulin actions and also prevented appearance of molecular and pathological characteristics observed in AD. The results thus demonstrate possible therapeutic efficacy of peripheral insulin-sensitizer drug metformin in AD by its ability to sensitize neuronal insulin resistance. These findings also provide direct evidences linking hyperinsulinemia and AD and suggest a unique opportunity for prevention and treatment of "type 3 diabetes"., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Resveratrol (RSV), a polyphenolic phytoestrogen, has been shown to activate the serine/threonine kinase 5'-adenosine monophosphate-activated protein kinase (AMPK) and to stimulate insulin signaling and glucose uptake in skeletal muscle cells. A direct effect of RSV on neuronal insulin signaling, however, has not been demonstrated. Here, we report that RSV stimulates glucose uptake and potentiates insulin signaling in Neuro-2A (N2A) cells, which is characterized by the increased phosphorylation of protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β). Furthermore, RSV activates AMPK in N2A cells, which can be prevented using a specific pharmacological inhibitor, Compound C. Compound C abrogates RSV-induced Akt and GSK-3β phosphorylation and glucose uptake. Thus, we demonstrate that RSV potentiates insulin signaling and glucose uptake via AMPK activation in neuronal cells.

Hexane, chloroform and ethyl acetate extracts (100 microg/ml) of Alpinia galanga rhizomes exhibited significant activity in vitro against promastigotes of L. donovani. Twelve compounds namely, methyleugenol (1), p-coumaryl diacetate (2), 1'-acetoxychavicol acetate (3), 1'-acetoxyeugenol acetate (4), trans-p-acetoxycinnamyl alcohol (5), trans-3,4-dimethoxycinnamyl alcohol (6), p-hydroxybenzaldehyde (7), p-hydroxycinnamaldehyde (8), trans-p-coumaryl alcohol (9), galangin (10), trans-p-coumaric acid (11) and galanganol B (12) were isolated from these extracts. Of these, compounds 2, 3, 4 and 5 were found most active in vitro against promastigotes of L. donovani with IC50 values of 39.3, 32.9, 18.9 and 79.9 microM respectively. This is the first report of antileishmanial activity of the extracts and isolated constituents of A. galanga.

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.

Our laboratory has established a novel role of focal adhesion kinase (FAK) in vitro and in vivo, as a positive regulator of insulin signaling pathway. In vitro studies reported tyrosine dephosphorylation of FAK under insulin resistance in C2C12 skeletal muscle cells. A decrease in FAK tyrosine phosphorylation was also observed in skeletal muscle of insulin resistant Sprague-Dawley rats fed on high-fat-diet. Present study was undertaken to explore the cellular mechanism of FAK dephosphorylation under insulin resistance in C2C12 skeletal muscle cells. Here we report that PTEN and SHIP2, the phosphatases widely implicated as negative regulators of insulin signaling, to be responsible for dephosphorylation of FAK. Data propose that under insulin resistance upregulation of PTEN and SHIP2 act through changes in FAK phosphorylation to impair insulin signaling suggesting FAK to be a key mediator of PTEN and SHIP2 in the regulation of insulin signaling. Thus data elucidates a part of molecular mechanism of insulin resistance in skeletal muscle cells.

Resistance to operational drugs is a major barrier to successful antileishmanial chemotherapy that demands development of novel drug intervention strategies based on rational approaches. Model drug resistance phenotypes, such as arsenite resistance used in the current study, facilitate our understanding of the mechanism of drug resistance and assist in identifying new drug target(s). The current study was undertaken to investigate the sensitivity of topoisomerase II (topo II) of arsenite-sensitive (Ld-Wt) and -resistant (Ld-As20) Leishmania donovani to antileishmanial/anti-topo II agents. The effect of antileishmanial/anti-topo II drugs on partially purified topo II enzyme from Ld-Wt and Ld-As20 revealed differential inhibition of topo II decatenation activity for the two strains, with a lower amount of drug required to inhibit activity by 50% in Ld-Wt compared with Ld-As20. Comparison of topo II sequences from both strains indicated a point mutation, R250G, in the ATPase domain of the resistant strain. Furthermore, the Arg-250 of the ATPase domain of topo II was observed to be conserved throughout different species of Leishmania. Variation in the topo II gene sequence between Ld-Wt and Ld-As20 is envisaged to be responsible for the differential behaviour of the enzymes from the two sources.

Background: Focal Adhesion Kinase (FAK) is recently reported to regulate insulin resistance by regulating glucose uptake in C2C12 skeletal muscle cells. However, the underlying mechanism for FAK-mediated glucose transporter-4 translocation (Glut-4), responsible for glucose uptake, remains unknown. Recently actin remodeling was reported to be essential for Glut-4 translocation. Therefore, we investigated whether FAK contributes to insulin-induced actin remodeling and harbor Glut-4 for glucose transport and whether downregulation of FAK affects the remodeling and causes insulin resistance., Results: To address the issue we employed two approaches: gain of function by overexpressing FAK and loss of function by siRNA-mediated silencing of FAK. We observed that overexpression of FAK induces actin remodeling in skeletal muscle cells in presence of insulin. Concomitant to this Glut-4 molecules were also observed to be present in the vicinity of remodeled actin, as indicated by the colocalization studies. FAK-mediated actin remodeling resulted into subsequent glucose uptake via PI3K-dependent pathway. On the other hand FAK silencing reduced actin remodeling affecting Glut-4 translocation resulting into insulin resistance., Conclusion: The data confirms that FAK regulates glucose uptake through actin reorganization in skeletal muscle. FAK overexpression supports actin remodeling and subsequent glucose uptake in a PI3K dependent manner. Inhibition of FAK prevents insulin-stimulated remodeling of actin filaments resulting into decreased Glut-4 translocation and glucose uptake generating insulin resistance. To our knowledge this is the first study relating FAK, actin remodeling, Glut-4 translocation and glucose uptake and their interrelationship in generating insulin resistance.

Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, has recently been implicated in the regulation of insulin resistance in vitro. However, its in vivo validation has not been attempted due to lethality of FAK knockout. Hence, to ascertain the role of FAK in the development of insulin resistance in vivo, we have down-regulated FAK expression by delivering FAK-specific small interfering RNA (siRNA) in mice using hydrodynamic tail vein injection. Here, we show for the first time that FAK silencing (57 +/- 0.05% in muscle and 80 +/- 0.08% in liver) exacerbates insulin signalling and causes hyperglycaemia (251.68 +/- 8.1 mg dl(-1)) and hyperinsulinaemia (3.48 +/- 0.06 ng ml(-1)) in vivo. FAK-silenced animals are less glucose tolerant and have physiological and biochemical parameters similar to that of high fat diet (HFD)-fed insulin-resistant animals. Phosphorylation and expression of insulin receptor substrate 1 (IRS-1) was attenuated by 40.2 +/- 0.03% and 35.2 +/- 0.6% in muscle and 52.3 +/- 0.04% and 40.2 +/- 0.03% in liver in FAK-silenced mice. Akt-Ser473-phosphorylation decreased in muscle and liver (50.3 +/- 0.03% and 70.2 +/- 0.02%, respectively) in FAK-silenced mice. This, in part, explains the mechanism of development of insulin resistance in FAK-silenced mice. The present study provides direct evidence that FAK is a crucial mediator of insulin resistance in vivo. Considering the lethality of FAK gene knockout the approach of this study will provide a new strategy for in vivo inhibition of FAK. Furthermore, the study should certainly motivate chemists to synthesize new chemical entities for FAK activation. This may shed light on new drug development against insulin resistance.

Pregarded as the second-most dreaded parasitic disease after malaria (WHO). Visceral leishmaniasis or kala-azar, caused by Leishmania donovani, is the most fatal form of leishmaniasis afflicting millions of people worldwide. No vaccination is available against leishmaniasis and fast spreading drug resistance in these parasitic organisms is posing a major medical threat. All these emphasize the need for new drugs and molecular targets along with reappraisal of existing therapeutics. Identification and characterization of cellular targets and answering the problem of drug resistance in Leishmania has always been the main thrust of protozoal research worldwide. Model drug resistance phenotypes against drugs, viz. arsenite (an antimony related metal ion, the first line of treatment against leishmaniasis), have been widely used to address and understand mechanism of drug resistance. The present discussion is an attempt to understand the different factors associated with arsenite resistance in Leishmania.

The current study has been undertaken to investigate the sensitivity of the topoisomerase II (topo II) of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani to an anti-leishmanial agent pentamidine and an anti-cancer drug doxorubicin. We demonstrate that the cross resistance to pentamidine and doxorubicin in Ld-As20, was in part implicated through differential inhibition of topo II in Ld-Wt and Ld-As20. Further, the treatment of promastigotes at drug concentrations inhibiting 50% of topo II activity inflicted a regulated cell death sharing several apoptotic features like externalization of phosphatidylserine, loss of mitochondrial membrane potential, cytochrome C release into the cytosol, activation of cellular proteases and DNA fragmentation. The cytotoxic potential of pentamidine and doxorubicin in L. donovani has been shown to be mediated through topoisomerase II inhibition and results in inciting programmed cell death process.

The affinity of arsenic towards the cytoskeleton leading to disturbance of tubulin polymerization is well known. Tubulin undergoes extensive posttranslational modifications which effect stability and dynamics of microtubules but little is known about the effect of antimicrotubule drugs on their distribution and function in kinetoplastid parasites such as Leishmania. The current study was undertaken to investigate the effect of continuous sodium arsenite exposure on the tubulin distribution profile in wild type and sodium arsenite resistant Leishmania donovani together with effect of paclitaxel, a tubulin-polymerizing agent, on that distribution using confocal microscopy. Immunofluorescence studies using specific monoclonal antibodies against alpha-tubulin and posttranslationally modified tubulins (acetylated and tyrosinated) have revealed distinct differences in the organization of microtubule arrays in wild type and sodium arsenite resistant L. donovani that is further affected by paclitaxel treatment. Microtubules are arranged in spiral arrays in wild type as compared to the longitudinal arrays in arsenite resistant L. donovani. The difference in microtubular structure organization may explain the parasite response to continuous drug pressure and illustrate the fundamental impact of arsenite on microtubules in arsenite resistant L. donovani.

Aims/hypothesis: On the basis of our previous studies, we investigated the possible role of focal adhesion kinase (FAK) in the development of insulin resistance in skeletal muscle, a major organ responsible for insulin-stimulated glucose uptake., Materials and Methods: Insulin-resistant C2C12 skeletal muscle cells were transfected with FAK wild-type or FAK mutant plasmids, knocked down using small interfering RNA (siRNA), and their effects on the levels and activities of insulin-signalling molecules and on glucose uptake were determined., Results: A significant decrease in tyrosine phosphorylation of FAK in insulin-resistant C2C12 cells was observed. A similar decrease was observed in skeletal muscle obtained from insulin-resistant Sprague-Dawley rats fed a high-fat diet. Increased levels of FAK in insulin-resistant C2C12 skeletal muscle cells increased insulin sensitivity and glucose uptake. These effects were reversed by an increase in the level of kinase activity mutant FAK or suppression of endogenous FAK by siRNA. FAK was also found to interact downstream with insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase C and glycogen synthase kinase 3beta, leading to translocation of glucose transporter 4 and resulting in the regulation of glucose uptake., Conclusions/interpretation: The present study provides strong evidence that the modulation of FAK level regulates the insulin sensitivity of skeletal muscle cells. The results demonstrate a direct role of FAK in insulin-resistant skeletal muscle cells for the first time.

The control of leishmaniasis in absence of vaccine solely depends on the choice of chemotherapy. The major hurdle in successful leishmanial chemotherapy is emergence of drug resistance. Miltefosine, the first orally administrable anti-leishmanial drug, has shown the potential against drug-resistant strains of Leishmania. However, there are discrepancies regarding the involvement of P-glycoprotein (Pgp) and sensitivity of miltefosine in multiple drug-resistant (MDR) cell lines that overexpress Pgp in Leishmania. To address this, the effect of miltefosine in arsenite-resistant Leishmania donovani (Ld-As20) promastigotes displaying an MDR phenotype and overexpressing Pgp-like protein was investigated in the current study. Results indicate that Ld-As20 is sensitive to miltefosine. Miltefosine induces process of programmed cell death in Ld-As20 in a time-dependent manner as determined by cell shrinkage, externalization of phosphatidylserine and DNA fragmentation. Miltefosine treatment leads to loss of mitochondrial membrane potential and the release of cytochrome C with consequent activation of cellular proteases. Activation of cellular proteases resulted in activation of DNase that damaged kinetoplast DNA and induced dyskinetoplasty. These data indicate that miltefosine causes apoptosis-like death in arsenite-resistant L. donovani.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor superfamily known to regulate adipocyte differentiation. However, its role in skeletal muscle differentiation is not known. To investigate possible involvement of PPARgamma in skeletal muscle differentiation, we modulated its expression in C2C12 mouse skeletal muscle cells by stable transfection with sense or antisense plasmid constructs of PPARgamma cDNA. Phenotypic observations and biochemical analysis of different myogenic markers showed that altered expression of PPARgamma inhibited the formation of myotubes, as well as expression of muscle-specific myogenic proteins including myogenin, MyoD and creatine kinase activity. Together, we show that critical expression of PPARgamma is required for skeletal muscle cells differentiation.

The present study focuses on the physical and chemical stability of insulin under iontophoretic conditions using HPLC, SDS-PAGE, RIA and biological assay. Influence of pH, concentration of insulin, current strength and duration of current application on the stability of insulin was studied. Anodal iontophoresis at pH 7.4 caused more than 80% degradation of insulin, while the degradation was minimal at pH 3.6. The degradation was not influenced by insulin concentration, but increase in current strength above 0.75 mA/cm2 or application of current for 12 h (at 0.5 mA/cm2) led to 80 and 20% degradation respectively. All the samples showed biological activity comparable to intact insulin.

Aims/hypothesis: Miltefosine, the first oral anti-leishmanial drug, is reported to inhibit phosphatidylinositol 3-kinase (PI3K)/Akt activity in carcinoma cell lines. Inhibition of the PI3K/Akt pathway is known to result in insulin resistance. Therefore, we investigated whether miltefosine has any deleterious effect(s) on insulin sensitivity in L6E9 skeletal muscle cells., Materials and Methods: L6E9 myotubes were treated with miltefosine and its effect was observed on insulin-signalling proteins such as Akt, PI3K, insulin receptor-beta, IRS-1, c-Jun N-terminal kinase, p38 and glycogen synthase kinase beta, as well as on glucose uptake., Results: Miltefosine caused skeletal muscle insulin resistance in vitro by interfering with the insulin-signalling pathway and inhibiting insulin-stimulated glucose uptake., Conclusions/interpretation: Miltefosine may contribute to the risk of type 2 diabetes and needs further clinical exploration.

In this study the anti-leishmanial activity and anti-microtubule effects of paclitaxel, trifluralin and a combination of paclitaxel and trifluralin have been tested in a wild type and sodium arsenite-resistant strain of Leishmania donovani. Both paclitaxel and trifluralin have been shown to be effective in limiting parasite growth. Specific alterations in morphology, tubulin polymerization dynamics, post-translational modifications and cellular distribution of the tubulins have been confirmed to be a part of the intracellular anti-microtubule-events that occur in arsenite-resistant L. donovani in response to these agents, ultimately leading to death of the parasite. DNA analyses of the drug-treated wild type and arsenite-resistant strains revealed an apoptosis-like death in response to paclitaxel and the combination but not to trifluralin. Data provide valuable information for further development of chemotherapeutic strategies based on anti-microtubule agents against drug resistant Leishmania parasites.

Leishmaniasis affects millions of people worldwide every year. Lack of effective vaccination, co-infection with other dreaded diseases like AIDS and generation of drug resistant strains demand immediate attention into this neglected area of research. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which shows a 13-fold increase in resistance compared with wild type. Here we report that the arsenite resistant strain of L. donovani promastigotes shows cross-resistance to novobiocin, a catalytic inhibitor of topoisomerase II, with IC50 value of 320 microg ml-1 as compared with 242 microg ml-1 for wild type L. donovani. Leishmanicidal action of novobiocin induces dose- and time-dependent increase in cell death. Treatment with IC50 of novobiocin caused morphological and biochemical changes which lead to induction of cell death exhibiting characteristic features of metazoan apoptosis. Phosphatidylserine externalization, cytochrome C release to cytoplasm, activation of caspases, oligonucleosomal DNA fragmentation and in situ labelling of condensed and fragmented nuclei in both wild type and arsenite resistant L. donovani promastigotes strongly suggest the apoptosis-like mode of cell death. Cross-resistance to novobiocin in arsenite resistant strain has been correlated to over-expression of topoisomerase II and substantiated by differential inhibition of enzyme activity in wild type and arsenite resistant L. donovani.