Release of Soluble Insulin Receptor From Neurons by Cerebrospinal Fluid From Patients With Neurocognitive Dysfunction and HIV Infection

Previously, we found that high levels of soluble insulin receptor (sIR) in the cerebrospinal fluid (CSF) of an HIV-infected women cohort were associated with the presence and severity of HIV-associated neurocognitive disorders (HAND). In this study we investigated if CSF from this population, HIV-1 Tat, and selected cytokines induces sIR secretion from human neuronal cells. Twenty-three (23) HIV-seropositive women stratified by cognitive status and five HIV- seronegative women were evaluated. Soluble IR levels were measured in the extracellular medium of neuronal cells (SH-SY5Y) that were exposed (for 24 h) to the CSF of patients. The levels of sIR, HIV-1 Tat, and cytokine levels (IL-2, IL4, IL-6, IFNγ, TNFα, and IL-10) were quantified in the CSF of participants by ELISA and flow cytometry. Neuronal secretion of sIR was measured after exposure (24 h) to HIV-1 Tat (0.5–250 nM), or specific cytokines. The effects of TNFα and HIV-1 Tat on sIR secretion were also evaluated in the presence of R7050 (TNFα antagonist; 10 nM). Neurons exposed to the CSF of HIV-infected women had higher sIR levels according to the severity of neurocognitive impairment of the participant. Increased CSF sIR levels were associated with the presence and severity of HAND and were positively correlated with CSF HIV-1 Tat levels in HIV-infected women with cognitive impairment. CSF levels of IL-2, IFNγ, and TNFα were significantly increased with HAND. However, only TNFα (5 pg/mL) and HIV-1 Tat (100 nM) induced a significant increase in neuronal sIR secretion after 24 h exposure, an effect that was antagonized when each were combined with R7050. Our data suggests that TNFα and HIV-1 Tat from the CSF of HIV-infected women may regulate the secretion of sIR from neuronal cells and that the effect of HIV-1 Tat on sIR secretion may depend on TNFα receptor activation

Soluble and Cell-Associated Insulin Receptor Dysfunction Correlates with Severity of HAND in HIV-Infected Women

Blood sugar metabolism abnormalities have been identified in HIV-infected individuals and associated with HIV-associated neurocognitive disorders (HAND). These abnormalities may occur as a result of chronic HIV infection, long-term use of combined antiretroviral treatment (CART), aging, genetic predisposition, or a combination of these factors, and may increase morbidity and mortality in this population.To determine if changes in soluble and cell-associated insulin receptor (IR) levels, IR substrate-1 (IRS-1) levels, and IRS-1 tyrosine phosphorylation are associated with the presence and severity of HAND in a cohort of HIV-seropositive women.This is a retrospective cross-sectional study using patient database information and stored samples from 34 HIV-seropositive women and 10 controls without history of diabetes from the Hispanic-Latino Longitudinal Cohort of Women. Soluble IR subunits [sIR, ectodomain (α) and full-length or intact (αβ)] were assayed in plasma and CSF samples by ELISA. Membrane IR levels, IRS-1 levels, and IRS-1 tyrosine phosphorylation were analyzed in CSF white cell pellets (WCP) using flow cytometry. HIV-seropositive women had significantly increased levels of intact or full-length sIR in plasma (p<0.001) and CSF (p<0.005) relative to controls. Stratified by HAND, increased levels of full-length sIR in plasma were associated with the presence (p<0.001) and severity (p<0.005) of HAND. A significant decrease in IRS-1 tyrosine-phosphorylation in the WCP was also associated with the presence (p<0.02) and severity (p<0.02) of HAND.This study provides evidence that IR secretion is increased in HIV-seropositive women, and increased IR secretion is associated with cognitive impairment in these women. Thus, IR dysfunction may have a role in the progression of HAND and could represent a biomarker for the presence and severity of HAND

Angiotensins and Huntington’s Disease: A Study on Immortalized Progenitor Striatal Cell Lines

Neurons from mouse models of Huntington’s disease (HD) exhibit altered electrophysiological properties, potentially contributing to neuronal dysfunction and neurodegeneration. The renin–angiotensin system (RAS) is a potential contributor to the pathophysiology of neurodegenerative diseases. However, the role of angiotensin II (Ang II) and angiotensin (1-7) has not been characterized in HD. We investigated the influence of Ang II and angiotensin (1-7) on total potassium current using immortalized progenitor mutant huntingtin-expressing (Q111) and wild-type (Q7) cell lines. Measurements of potassium current were performed using the whole cell configuration of pCLAMP. The results showed that (1) the effect of Ang II administered to the bath caused a negligible effect on potassium current in mutant Q111 cells compared with wild-type Q7 cells and that intracellular administration of Ang II reduced the potassium current in wild type but not in mutant cells; (2) the small effect of Ang II was abolished by losartan; (3) intracellular administration of Ang II performed in mutant huntingtin-expressing Q111 cells revealed a negligible effect of the peptide on potassium current; (4) flow cytometer analysis indicated a low expression of Ang II AT1 receptors in mutant Q111 cells; (5) mutant huntingtin-expressing striatal cells are highly sensitive to Ang (1-7) and that the effect of Ang (1-7) is related to the activation of Mas receptors. In conclusion, mutant huntingtin-expressing cells showed a negligible effect of Ang II on potassium current, a result probably due to the reduced expression of AT1 receptors at the surface cell membrane. In contrast, administration of Ang (1-7) to the bath showed a significant decline of the potassium current in mutant cells, an effect dependent on the activation of Mas receptors. Ang II had an intracrine effect in wild-type cells and Ang (1-7) exerted a significant effect in mutant huntingtin-expressing striatal cells

Anti-Breast Cancer Potential of Quercetin via the Akt/AMPK/Mammalian Target of Rapamycin (mTOR) Signaling Cascade.

The Akt/adenosine monophosphate protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway has emerged as a critical signaling nexus for regulating cellular metabolism, energy homeostasis, and cell growth. Thus, dysregulation of this pathway contributes to the development of metabolic disorders such as obesity, type 2diabetes, and cancer. We previously reported that a combination of grape polyphenols (resveratrol, quercetin and catechin: RQC), at equimolar concentrations, reduces breast cancer (BC) growth and metastasis in nude mice, and inhibits Akt and mTOR activities and activates AMPK, an endogenous inhibitor of mTOR, in metastatic BC cells. The objective of the present study was to determine the contribution of individual polyphenols to the effect of combined RQC on mTOR signaling. Metastatic BC cells were treated with RQC individually or in combination, at various concentrations, and the activities (phosphorylation) of AMPK, Akt, and the mTOR downstream effectors, p70S6 kinase (p70S6K) and 4E binding protein (4EBP1), were determined by Western blot. Results show that quercetin was the most effective compound for Akt/mTOR inhibition. Treatment with quercetin at 15μM had a similar effect as the RQC combination in the inhibition of BC cell proliferation, apoptosis, and migration. However, cell cycle analysis showed that the RQC treatment arrested BC cells in the G1 phase, while quercetin arrested the cell cycle in G2/M. In vivo experiments, using SCID mice with implanted tumors from metastatic BC cells, demonstrated that administration of quercetin at 15mg/kg body weight resulted in a ~70% reduction in tumor growth. In conclusion, quercetin appears to be a viable grape polyphenol for future development as an anti BC therapeutic

Effect of individual or combined RQC on Akt activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined Res, Quer, and Cat (RQC) at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle or 15μM quercetin. Cells were lysed immediately following treatment for 15min, and western blotted for total or active (phospho-Akt^Ser473) Akt. <b>(A)</b> Relative Akt activity (phospho-Akt/Akt) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. <b>(B)</b> Relative Akt activity of MDA-MB-231 cells following vehicle or 15μM quercetin. <b>(C)</b> Relative Akt activity of MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle, as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of quercetin on the growth of MDA-MB-231 mammary fat pad tumors.

<p>GFP-MDA-MB-231 cells (0.5x10⁶) were inoculated at the mammary fat pad of SCID mice. One week after inoculation, mice were treated with vehicle, or quercetin at 15 or 45mg/kg BW three times a week by intraperitoneal injection. Fluorescence images of the mammary fatpad tumors were acquired once a week. <b>(A)</b> Average relative tumor area (N = 12) as a function of days following injection. Relative tumor area was calculated as the area of fluorescence, measured by fluorescence intensity on each day of imaging as a function of the fluorescence intensity of the same tumor on day 1. <b>(B)</b> Representative digital images of MDA-MB-231 tumors following vehicle, or quercetin at 15 or 45mg/kg BW at 13 weeks, followed by the quantification of tumor growth. N = 12±SEM, asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle. <b>(C)</b> Relative SCID mice weight following vehicle, or quercetin at 15 or 45mg/kg BW treatments.</p

Effect of combined RQC or individual quercetin on breast cancer cell apoptosis.

<p>Quiescent MDA-MB-231 (<b>A</b>) or MDA-MB-435 (<b>B</b>) cells in 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each, or Quercetin 15μM for 48h. Trypsinized cells were incubated with Annexin V conjugate and propidium iodide, and analyzed by flow cytometry, using a four-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA). Representative dot plots of 20,000 events/ treatment (N = 3), collected using Cell Quest software 3.3 (BD Biosciences, San Jose, CA) and analyzed using Flow Jo software vX.0.7 (BD Biosciences, San Jose, CA). Cell size and granularity were determined on forward versus side scatter (FSC vs. SSC). Annexin-V fluorescence was measured at 530/30 nm and Propidium Iodide at 585/42nm. The average percentage of early and late apoptotic cells obtained from Annexin V vs. Propidium Iodide dot plots are shown below. <b>(C)</b> Effect of combined RQC or individual quercetin on caspase 3/7 activity. Quiescent MDA-MB-231 cells in 96 well plate at 5% serum and phenol red-free media were treated with vehicle, combined RQC at 5μM each or quercetin 15μM for 48hr or 96hr. Then Caspase-Glo®3/7 reagent were added to each treatment, and after 1hr of incubation the luminescence was measured in a plate-reader luminometer. Relative luminescence to vehicle is shown, N = 3±SEM, asterisk indicates statistical significance (p≤0.05) when compared to vehicle.</p

Effect of combined RQC or individual quercetin on breast cancer cell migration.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were placed in the top of a transwell chamber with the bottom well containing: vehicle, combined resveratrol, quercetin, and catechin (RQC) at 5μM each, or 15μM quercetin in serum- and phenol red- free media. After 8h incubation, cells that migrated through the 8μM pore membrane were fixed, nuclei stained with Propidium Iodide and quantified. Percentage of migrated cells ± SEM for 15 microscopic fields/duplicate treatments for 3 independent experiments is presented. (<b>A)</b> Average cell migration of MDA-MB-231 cells treated with RQC or quercetin relative to vehicle. <b>(B)</b> Average cell migration of MDA-MB-435 cells treated with RQC or quercetin relative to vehicle.</p

Effect of quercetin on mTOR signaling in breast cancer cells.

<p>Quiescent MDA-MB-231 and MDA-MB-435 cells were treated with vehicle or quercetin at 15μM for 15min, lysed immediately, and western blotted for total or active (phospho-p70S6K^Thr389) p70S6K or (phospho-4EBP-1^Thr37/46) 4EBP-1. <b>(A)</b> Relative p70S6K activity (phospho-p70S6K/p70S6K) from MDA-MB-231 cells. <b>(B)</b> Relative p70S6K activity from MDA-MB-435 cells. <b>(C)</b> Relative 4EBP-1 activity (phospho-4EBP-1/4EBP-1) from MDA-MB-231 cells. <b>(D)</b> Relative 4EBP-1 activity from MDA-MB-435 cells. Representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p

Effect of individual or combined RQC on AMPK activity in breast cancer cells.

<p>Quiescent MDA-MB-231 cells were treated with vehicle, or 1, 3, 5, 9, or 15μM of resveratrol (Res), quercetin (Quer), catechin (Cat), or combined RQC at 3μM total (1μM each), 9μM total (3μM each), or 15μM total (5μM each). MDA-MB-435 cells were treated only with vehicle and 15μM quercetin. Cells were treated for 15 min, lysed immediately, and western blotted for total or active (phospho-AMPK^Thr172) AMPK. <b>(A)</b> Relative AMPK activity (phospho-AMPK/AMPK) of MDA-MB-231 cells following RQC or individual Res, Quer, or Cat at the indicated concentrations. Relative AMPK activity of <b>(B)</b> MDA-MB-231 cells following vehicle or 15μM quercetin, or <b>(C)</b> MDA-MB-435 cells following vehicle or 15μM quercetin. For <b>(B)</b> and <b>(C)</b>, representative western blots from 3 separate experiments are shown. Graphs show the analyses of the integrated densities of positive bands relative to vehicle as quantified from image J analysis. An asterisk indicates statistical significance (<i>p≤</i>0.05) when compared to vehicle.</p