CDK5 inhibition improves glucose uptake in insulin-resistant neuronal cells via ERK1/2 pathway.

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.

Nuclear co-repressor (NCoR) is required to maintain insulin sensitivity in C2 C12 myotubes.

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 C2 C12 . Myotubes as compared to myoblasts of C2 C12 cells were found to be more sensitive in response to insulin as increase in insulin-stimulated phosphorylation of AKT at serine 473 residue (pAKTS473 ) was significantly higher in myotubes. Incidentally, reduced protein level of NCoR coincided with differentiation of myoblasts into myotubes of C2 C12 cells. However, insulin stimulation per se failed to affect protein level of NCoR either in myoblasts or myotubes of C2 C12 cells. To assess the role of NCoR on insulin sensitivity, NCoR was transiently knocked down using siRNA in myotubes of C2 C12 . In fact, transient silencing of NCoR led to significant reduction in insulin-stimulated pAKTS473 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 C2 C12 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 C2 C12 myotubes. Collectively, data from present study show differential protein level of NCoR under different cell state (myoblast and myotubes) of C2 C12 cells and NCoR proves to be vital for maintaining insulin sensitivity in C2 C12 myotubes.

Potentiation of neuronal insulin signaling and glucose uptake by resveratrol: the involvement of AMPK.

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-3ß (GSK-3ß). 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-3ß phosphorylation and glucose uptake. Thus, we demonstrate that RSV potentiates insulin signaling and glucose uptake via AMPK activation in neuronal cells.

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani.

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.

Evidence for the presence of R250G mutation at the ATPase domain of topoisomerase II in an arsenite-resistant Leishmania donovani exhibiting a differential drug inhibition profile.

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.

Focal Adhesion Kinase contributes to insulin-induced actin reorganization into a mesh harboring Glucose transporter-4 in insulin resistant skeletal muscle cells.

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.

In vivo inhibition of focal adhesion kinase causes 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.

Arsenite resistance in Leishmania and possible drug targets.

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.

Induction of apoptosis-like cell death by pentamidine and doxorubicin through differential inhibition of topoisomerase II in arsenite-resistant L. donovani.

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.

Confocal microscopic investigation of tubulin distribution and effect of paclitaxel on posttranslationally modified tubulins in sodium arsenite resistant Leishmania donovani.

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.

Miltefosine induces apoptosis in arsenite-resistant Leishmania donovani promastigotes through mitochondrial dysfunction.

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.

Novobiocin induces apoptosis-like cell death in topoisomerase II over-expressing arsenite resistant Leishmania donovani.

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.

PPAR-gamma expression modulates insulin sensitivity in C2C12 skeletal muscle cells.

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression is very low in skeletal muscle cells, which is one of the most important target tissues for insulin and plays a predominant role in glucose homeostasis. It has recently been shown that muscle-specific PPAR-gamma deletion in mouse causes insulin resistance. However, it is likely that the observed effects might be due to secondary interaction in whole animal. The aim of the study was to explore the role of muscle PPAR-gamma in insulin sensitivity. We stably transfected C2C12 skeletal muscle cells with plasmids containing sense or antisense constructs of PPAR-gamma and examined the effect of modulation of PPAR-gamma expression in terms of glucose uptake. Effect was also examined in insulin-resistant C2C12 skeletal muscle cells. In transfected C2C12 cell line, the inhibition of PPAR-gamma expression (23.0 +/-0.005%) was observed to induce insulin resistance as determined by functional assessment of 2-deoxyglucose incorporation. Overexpression of PPAR-gamma (28.5 +/- 0.008%) produced an additional effect on insulin (100 nM) and Pioglitazone (50 microM), resulting in 42.7 +/- 3.5% increase in glucose uptake as against 29.2+/-2.8% in wild-type C2C12 skeletal muscle cells differentiated under normal (2% horse serum) condition. Under similar treatment, PPAR-gamma overexpressing cells resistant to insulin exhibited enhanced glucose uptake upto 60.7 +/- 4.08%, as compared to 23.8 +/- 5.1% observed in wild-type C2C12 skeletal muscle cells. These data demonstrate a direct involvement of PPAR-gamma in insulin sensitization of TZD action on skeletal muscle cells, and suggest that pharmacological overexpression of muscle PPAR-gamma gene in skeletal muscle might be a useful strategy for the treatment of insulin resistance.

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones.

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.

Possible mechanism of miltefosine-mediated death of Leishmania donovani.

Miltefosine causes leishmanial death, but the possible mechanism(s) of action is not known. The mode of action of miltefosine was investigated in vitro in Leishmania donovani promastigotes as well as in extra- and intracellular amastigotes. Here, we demonstrate that miltefosine induces apoptosis-like death in L. donovani based on observed phenomena such as nuclear DNA condensation, DNA fragmentation with accompanying ladder formation, and in situ labeling of DNA fragments by the terminal deoxyribonucleotidyltransferase-mediated dUTP-biotin nick end labeling method. Understanding of miltefosine-mediated death will facilitate the design of new therapeutic strategies against Leishmania parasites.

Altered expression, polymerisation and cellular distribution of alpha-/beta-tubulins and apoptosis-like cell death in arsenite resistant Leishmania donovani promastigotes.

Studies in mammalian systems have shown specific affinity of arsenite for tubulin proteins. The sodium m-arsenite (NaAsO2) resistant Leishmania donovani used in this study is resistant to 20 microM NaAsO2, which is a 13-fold increase in resistance compared to the wild type. Data presented in this study shows decreased expression of alpha- and beta-tubulin in wild type L. donovani promastigotes on exposure to NaAsO2 from 0.0016 to 5.0 microM (IC50 in the wild type strain) in a dose-dependent manner. alpha- and beta-tubulins in the resistant strain show decreased expression levels only at 65.0 microM NaAsO2 (IC50 in the resistant strain). Treatment with respective IC50 concentrations of NaAsO2 caused alterations in tubulin polymerisation dynamics and deregulated the cellular distribution of the microtubules in wild type and resistant strains. The NaAsO2-induced cell death exhibited characteristics of apoptosis-like DNA laddering and fragmentation in both the affected wild type and resistant cells. However, poly(ADP-ribose)polymerase cleavage was evident in the wild type strain but not in the resistant strain.

Combination of metformin and thiazolidindiones restore insulin signalling in insulin-resistant cultured myotubes.

We examined the effect of combination of thiazolidinediones (TZDs) and metformin on insulin-resistant skeletal muscle cells. The combined use of TZDs and metformin resulted in maximum tyrosine phosphorylation of insulin receptor (IR) and insulin receptor substrate-1 (IRS-1) at 12.5 microM of TZDs and 100 microM of metformin as compared to the maximum tyrosine phosphorylation of IR and IRS-1 achieved at 50 microM of TZDs or 400 microM of metformin. The glucose uptake was significantly high at the combination of lower concentration (12.5 microM of TZDs and 100 microM of metformin) as compared to the combination of higher concentration (50 microM of TZDs and 400 microM of metformin). Results demonstrated that (1) Additive effect on insulin sensitization can be achieved by a combination of TZDs and metformin at lower concentration; (2) combination of TZDs and metformin act on insulin signaling molecules in insulin resistance; (3) in vitro system has the potentiality to determine possible target molecule(s) and mechanism of action of drugs.

Involvement of c-Jun N-terminal kinase activities in skeletal muscle differentiation.

Previous studies on skeletal muscle differentiation showed that myogenesis is regulated by extracellular signal-regulated kinases (ERK-1/-2) and p38 mitogen activated kinase (MAPK) pathways. Present study shows that c-Jun NH2-terminal protein kinase (JNK) activities were up regulated during skeletal muscle differentiation in rat skeletal muscle L6E9 cells, as determined by Western immunoblot of differentiating cells probed with anti-phospho-JNK antibody. Inhibition of JNK activities by JNK inhibitor II drastically inhibited differentiation as determined by decreased myosin, myogenin expression and creatine kinase activity. The inhibition of the differentiation was regulated by apoptosis as determined by the detection of poly(ADP-ribose) polymerase (PARP) cleavage, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) positive cells when JNK activities were inhibited. Apoptosis was accompanied by marked expression and activation of c-Jun and p53 transcription factors. Taken together, our results indicate that basal JNK activities are essential for regulating skeletal muscle differentiation, and inhibition of JNK activation affects myogenesis by apoptosis dependent on c-Jun and p53 transcription factors.

P-glycoprotein inhibitors and their screening: a perspective from bioavailability enhancement.

Drug efflux pumps like P-glycoprotein (P-gp) and multidrug resistance (MDR) proteins were recognized to possess functional role in determining the pharmacokinetics of drugs administered by peroral as well as parenteral route. Advancements in molecular biology, to some extent, had revealed the structure, localization and functional role of P-glycoprotein and its mechanism of drug efflux. Broad substrate recognition by this protein and clinical implications of its inhibition has revolutionized cancer chemotherapy leading to design and development of novel P-glycoprotein inhibitors. In the recent times, the application of these inhibitors in improving peroral drug delivery has gained special interest. Inhibition of P-glycoprotein improves intestinal absorption and tissue distribution while reducing the substrate metabolism and its elimination. Eventually, various screening methodologies have been developed for determining the activity of P-glycoprotein, kinetics of drug transport and identification of substrates and inhibitors. In the present review, techniques used for screening P-glycoprotein inhibitors and the scope of these inhibitors in optimizing peroral drug absorption and pharmacokinetics are discussed along with a brief introduction to P-glycoprotein, its physiological function and active role in extrusion of drugs.