Patterns of Gene Expression in Drosophila InsP3 Receptor Mutant Larvae Reveal a Role for InsP3 Signaling in Carbohydrate and Energy Metabolism

Background: The Inositol 1,4,5-trisphosphate receptor (InsP3R) is an InsP3 gated intracellular Ca 2+-release channel. Characterization of Drosophila mutants for the InsP3R has demonstrated that InsP3-mediated Ca 2+ release is required in Drosophila larvae for growth and viability. Methodology/Principal Findings: To understand the molecular basis of these growth defects a genome wide microarray analysis has been carried out with larval RNA obtained from a strong InsP3R mutant combination in which 1504 independent genes were differentially regulated with a log 2 of fold change of 1 or more and P,0.05. This was followed by similar transcript analyses from InsP3R mutants where growth defects were either suppressed by introduction of a dominant suppressor or rescued by ectopic expression of an InsP3R transgene in the Drosophila insulin like peptide-2 (Dilp2) producing cells. Conclusions/Significance: These studies show that expression of transcripts related to carbohydrate and amine metabolism is altered in InsP 3 receptor mutant larvae. Moreover, from a comparative analysis of genes that are regulated in the suppressed and rescued conditions with the mutant condition, it appears that the organism could use differen

Striking reduction of amyloid plaque burden in an Alzheimer's mouse model after chronic administration of carmustine

BACKGROUND: Currently available therapies for Alzheimer's disease (AD) do not treat the underlying cause of AD. Anecdotal observations in nursing homes from multiple studies strongly suggest an inverse relationship between cancer and AD. Therefore, we reasoned that oncology drugs may be effective against AD. METHODS: We screened a library of all the FDA-approved oncology drugs and identified bis-chloroethylnitrosourea (BCNU or carmustine) as an effective amyloid beta (Aβ) reducing compound. To quantify Aβ levels, Chinese hamster ovary (CHO) cells stably expressing amyloid precursor protein 751WT (APP751WT) called 7WD10 cells were exposed to different concentrations of BCNU for 48 hours and the conditioned media were collected. To detect Aβ the conditioned media were immunoprecipitated with Ab9 antibody and subjected to immunoblot detection. Amyloid plaques were quantified in the brains of a mouse model of AD after chronic exposure to BCNU by thoflavin S staining. RESULTS: BCNU decreased normalized levels of Aβ starting from 5 μM by 39% (P < 0.05), 10 μM by 51% (P < 0.01) and 20 μM by 63% (P < 0.01) in CHO cells compared to a control group treated with butyl amine, a structural derivative of BCNU. Interestingly, soluble amyloid precursor protein α (sAPPα) levels were increased to 167% (P < 0.01) at 0.5 μM, 186% (P < 0.05) at 1 μM, 204% (P < 0.01) at 5 μM and 152% (P < 0.05) at 10 μM compared to untreated cells. We also tested the effects of 12 structural derivatives of BCNU on Aβ levels, but none of them were as potent as BCNU. BCNU treatment at 5 μM led to an accumulation of immature APP at the cell surface resulting in an increased ratio of surface to total APP by 184% for immature APP, but no change in mature APP. It is also remarkable that BCNU reduced Aβ generation independent of secretases which were not altered up to 40 μM. Interestingly, levels of transforming growth factor beta (TGFβ) were increased at 5 μM (43%, P < 0.05), 10 μM (73%, P < 0.01) and 20 μM (92%, P < 0.001). Most significantly, cell culture results were confirmed in vivo after chronic administration of BCNU at 0.5 mg/kg which led to the reduction of Aβ40 by 75% and amyloid plaque burden by 81%. Conversely, the levels of sAPPα were increased by 45%. CONCLUSIONS: BCNU reduces Aβ generation and plaque burden at non-toxic concentrations possibly through altered intracellular trafficking and processing of APP. Taken together these data provided unequivocal evidence that BCNU is a potent secretase-sparing anti-Aβ drug. See related commentary article here http://www.biomedcentral.com/1741-7015/11/8

Therapeutic Agents from Herbal Source to Address Diabetes Type 2 Problems

To understand the molecular mechanism involved in insulin resistance and type 2 diabetes is basically necessary to identify the precise target molecules from plants having antidiabetic property. To investigate the precise target in respect of FFA induced impairment leading to insulin resistance and type 2 diabetes, further investigation was carried out taking IR as the major target. Elevated plasma free fatty acids (FFAs) are known to be associated with insulin resistance and type 2 diabetes mellitus. How FFAs oppose insulin action is still not very clear but a few reports suggest their interference in the insulin signaling pathways. FFA also impairs a number of PKC isoforms, which ultimately reduce insulin-signaling leading to reduced glucose uptake. 2-Deoxyglucose uptake by 3T3L1 adipocytes and L6 skeletal muscle cells incubated with different free fatty acids showed that palmitic acid among other free fatty acids is the key player in inhibiting insulin signaling pathway. Insulin augmented signals starting from the activation of insulin receptor (IR) to Glut 4 translocation was inhibited by palmitate. Insulin stimulated IR phosphorylation was significantly reduced by palmitate treatment. Insulin signaling proteins downstream to IR, which get consequently phosphorylated following IR tyrosine kinase phosphorylation on insulin stimulation, also showed significant inhibition due to palmitate treatment. Inhibition of IR, IRS1, PI3 kinase and Akt/PKB phosphorylation, which are markers of insulin resistance, was observed in palmitate fed rats. Therefore, we could develop a rat model for nutritionally induced insulin resistance. Rats fed with palmitate showed inhibition in phosphorylation of IR, IRS1, PI3 kinase and Akt/PKB, all of which were considered as important markers for insulin resistance, indicating that palmitate-fed rats can be used as a model for nutritionally induced insulin resistance and type 2 diabetes. In insulin signal transduction pathway, PDK1 is the upstream kinase that directly phosphorylates downstream substrates such as Akt/PKB and PKC isoforms. Insulin stimulated phosphorylation of Akt/PKB was inhibited by palmitate along with PDK1 phosphorylation. Since phosphorylation of PKC is also dependent on PDK1 it was expected to be inhibited. On the contrary PKC show PDK1 independent phosphorylation due to FFA. This was really surprising, as in vitro incubation of skeletal muscle cells with palmitate increased PKC phosphorylation while PDK1 phosphorylation was decreased. Inhibition of PDK1 by palmitate was reflected on Akt phosphorylation whose phosphorylation was inhibited in palmitate treated cells, Data demonstrated a significant decrease in IR mRNA in palmitate treated muscle cells. Western blot analysis also showed a decrease in IR protein level as a consequence of decreased IR gene expression in palmitate incubated cells as compared to the control. These results suggested that fatty acid-induced insulin resistance is related to reduced IR gene expression. HMGA1 plays a critical regulatory role in IR gene transcription. Phosphorylation of HMGA1 restricts its movement towards IR promoter leading to inhibition of IR gene transcription, and palmitate activates the PKC phosphorylation, which in turn phosphorylates HMGA1 leading to downregulation of IR gene expression. Our observations show the involvement of one of the possible pathways involved in fatty acid induced insulin resistance. Free fatty acid (i.e. Palmitate) induced PDK1 independent PKC phosphorylation; pPKC was then translocated to the nucleus and phosphorylated HMGA1 that restricts its movement to IR gene promoter. Experimental evidences with V1 supported this contention. Phosphorylated HMGA1 interfered with the reactivation of IR promoter causing downregulation of IR gene expression and reduced IR copies on the cell surface thus reducing insulin sensitivity. After availing all these experimental results in relation to FFA induced insulin resistance, the process of searching for antidiabetic activity of medicinal plants of Indian origin begun. A specific fraction or molecules were tested to find whether there was any intervention in the molecular pathway of insulin insensitivity or not. Extract from two plants, i) Pueraria tuberosa and ii) Aloe vera were found to improve FFA induced impairment when insulin activity was determined in terms of 3H-2 deoxyglucose uptake by 3T3L1 adipocytes. However, Aloe vera activity was far less as compared to P. tuberosa. Therefore, only P tuberosa was studied further. HPLC purified closely related structures from Pueraria tuberosa were identified by 2D-NMR and Mass spectrometry and these are LPA4, LPA5, Soyasaponin1 and Kudzusaponin1.LPA4 is different from the others in having -L-rhamnopyranosyloxy moiety and therefore its improvement in terms of insulin activity is due to the presence of this chemical character.The crude extract from Aloe vera was also examined for antidiabetic property. 2-deoxyglucose uptake showed that both crude extract and sub fractions increases the insulin sensitivity in comparison to palmitate treated cells. Our results demonstrated that palmitate-induced reduction of insulin augmented 3H-2-DOG uptake by adipocyte could be prevented by LPA4. The inhibitory effect of palmitate on insulin-stimulated IRβ tyrosine kinase phosphorylation and Akt phosphorylation was waived by LPA4. The inhibition of IR gene expression by palmitate was also reduced by LPA4

Fatty acid Represses Insulin Receptor Gene Expression by Impairing HMGA1 through Protein Kinase Ce

It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKCe was also inhibited. Surprisingly, phosphorylation of cytosolic PKCe was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKCe translocation inhibitor peptide, eV1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKCe migration to implement palmitate effect. Experimental evidences indicate that phospho-PKCe adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity

Comparative analysis of use of porous orbital implant with mucus membrane graft and dermis fat graft as a primary procedure in reconstruction of severely contracted socket

Purpose: The purpose of our study is to present a surgical technique of primary porous orbital ball implantation with overlying mucus membrane graft (MMG) for reconstruction of severely contracted socket and to evaluate prosthesis retention and motility in comparison to dermis fat graft (DFG). Study Design: Prospective comparative study. Materials and Methods: A total of 24 patients of severe socket contracture (Grade 2-4 Krishna′s classification) were subdivided into two groups, 12 patients in each group. In Group I, DFG have been used for reconstruction. In Group II, porous polyethylene implant with MMG has been used as a primary procedure for socket reconstruction. In Group I DFG was carried out in usual procedure. In case of Group II, vascularized scar tissues were separated 360° and were fashioned into four strips. A scleral capped porous polyethylene implant was placed in the intraconal space and four strips of scar tissue were secured to the scleral cap and extended part overlapped the implant to make a twofold barrier between the implant and MMG. Patients were followed-up as per prefixed proforma. Prosthesis motility and retention between the two groups were measured. Results: In Group I, four patients had recurrence of contracture with fall out of prosthesis. In Group II stable reconstruction was achieved in all the patients. In terms of prosthesis motility, maximum in Group I was 39.2% and Group II, was 59.3%. The difference in prosthesis retention (P = 0.001) and motility (P = 0.004) between the two groups was significant. Conclusion: Primary socket reconstruction with porous orbital implant and MMG for severe socket contracture is an effective method in terms of prosthesis motility and prosthesis retention

Involvement of novel PKC isoforms in FFA induced defects in insulin signaling

Involvement of novel PKCs (nPKCs) in the negative regulation of insulin-signaling pathway is a current interest of many workers investigating the cause for insulin resistance and type 2 diabetes. Free fatty acids (FFAs) are recently shown to be the major players in inducing insulin resistance in insulin target cells. They are also found to be involved in activating nPKCs associated with the impairment of insulin sensitivity. In this overview, we describe PKC δ, θ and ε linked to the FFA induced damage of insulin-signaling molecules

A Lupinoside prevented fatty acid induced inhibition of insulin sensitivity in 3T3 L1 adipocytes

The decrease in insulin sensitivity to target tissues or insulin resistance leads to type 2 diabetes mellitus, an insidious disease threatening global health. Numerous evidences made free fatty acids (FFAs) responsible for insulin resistance and type 2 diabetes. We demonstrate here that the damage of insulin acitivity by a free fatty acid, palmitate could be prevented by a lupinoside. An incubation of 3T3 L1 adipocytes with a FFA i.e. palmitate inhibited insulin stimulated uptake of 3H-2 deoxyglucose (2 DOG) significantly. Addition of a lupinoside purified from Pueraria tuberosa, lupinoside PA4 (LPA4) strongly prevented this inhibition. We then examined insulin signaling pathway where palmitate significantly inhibited insulin stimulated phosphorylation of Insulin receptor tyrosine kinase, IRS 1and PI3 kinase, PDK1 and Akt/PKB. LPA4 rescued this inhibition of signaling molecule by palmitate. Insulin mediated translocation of Glut4, the glucose transporter in insulin target cells, was effectively blocked by palmitate while, LPA4 waived this block. Administration of LPA4 to nutritionally induced diabetic rats significantly reduced the increase in plasma glucose. All these indicate LPA4 to be a potentially therapeutic agent for insulin resistance and type 2 diabetes

2-CDA increases Aβ and CTF levels in CHO cells stably expressing WT APP751 (7WD10 cells).

<p>(A) 7WD10 cells were exposed to different concentrations of 2-CDA for 48 h and the conditioned medium was immunoprecipitated for Aβ and detected by immunoblots using NuPAGE 4–12% bis-tris gel. The lysates were used to detect c-terminal fragments (CTFs) using NuPAGE gels. APP as well as actin as a loading control were separated using 10% bis-acrylamide gels. (B) Quantitation by image J revealed increased levels of Aβ at 1.0 µM (69% (p<0.01), 5.0 µM (84%, p<0.01) and 10.0 µM (107%, p<0.01) compared to controls. Values are expressed as percentage change from controls. CTF levels were also significantly increased at 0.5 µM (278%, p<0.05), 1.0 µM (302%, p<0.01), 5.0 µM (302%, p<0.01) and 10.0 µM (301%, p<0.01) compared to controls. For all samples, n = 4. *, p<0.05, **, p<0.01, versus control by analysis of variance (ANOVA) followed by post-hoc test by Dunnett multiple comparisons.</p

2-CDA is cytotoxic at high concentrations.

<p>(A) LDH release was measured as an indicator of cytotoxicity in HEK 293 cells and N2A cells after incubation with different concentrations of 2-CDA as indicated. Significant cytotoxicity was observed at 1.0 µM (p<0.05), 10.0 µM (p<0.001), 80.0 µM (p<0.001) and 240.0 µM (p<0.001) in HEK 293 cells. Similarly in N2A cells significant toxicity was observed at 10.0 µM (p<0.001), 80.0 µM (p<0.001) and 240.0 µM (p<0.001). (B) MTT reduction assay, however indicated significant toxicity only in HEK 293 cells at 10.0 µM (p<0.05), 80.0 µM (p<0.001) and 240.0 µM (p<0.001), but 2-CDA was nontoxic in N2A cells even at high concentrations. (C) the calculated DCK activity was almost one-fold higher in the brain compared to kidneys. But kidneys showed almost three-fold increased 5′-NTases activity compared to whole brain tissue. For all samples in A and B, n = 4. *, p<0.05, **, p<0.01, ***, p<0.001 versus controls by ANOVA followed by Tukey-Kramer multiple comparison test.</p