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Mitochondria are essential to all higher forms of life. Every animal and plant depends on these small intracellular structures. Mitochondria have multiple tasks: Since they generate most of the cell’s biochemical energy, they are referred to as the powerhouses of the cell. In addition, they are responsible for producing and breaking down amino acids and fats. They also regulate cellular death, called apoptosis.–As a result, the spectrum of diseases that are linked to mitochondrial defects is wide, ranging from severe muscular and nervous disorders to neurodegenerative diseases as well as all symptoms of aging.–“It was by pure chance that we discovered this completely new control mechanism of mitochondrial function,” says first author Deniz Senyilmaz, who works in Aurelio Teleman’s group at the German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). In collaboration with colleagues from Cambridge, Teleman and his team had planned to investigate the metabolism of long-chain fatty acids. For this purpose, the researchers bred flies whose cells were unable to produce stearic acid, a fatty acid that is composed of 18 carbon atoms. Animals with this defect did not develop beyond the pupal stage and were not viable afterwards.–Teleman and his team were curious to find out why this happened. They then discovered a highly complex biological control mechanism that regulates the fusion — as well as fragmentation — of mitochondria and, hence, the performance of these organelles.–The key element in this control mechanism is the transferrin receptor, which binds stearic acid. “For the first time in biological research, we have found out that stearic acid, which up until now has been believed to be simply a metabolic product, also has signaling function,” says Teleman. The researchers demonstrated that mitochondrial control via stearic acid works not only in flies but also in the HeLa human cancer cell line.–When the researchers added stearic acid to fly food, the animals’ mitochondria fused; when they kept fatty acid levels low, the organelles fragmented. “If using stearic acid as a food additive improves the performance of normal mitochondria, then it might do the same in pathogenically dysfunctional mitochondria,” [F6]Teleman explained, describing their experimental approach.–The researchers studied flies that exhibit Parkinson’s-like symptoms resulting from a mitochondrial defect in the PINK and Parkin proteins and are recognized as a model system for studying this neurodegenerative disease. When the affected animals were fed stearic acid with their food, their motor skills and energy balance improved and they survived for much longer.–“This opens up the fascinating possibility of using a food additive to alleviate symptoms in patients with mitochondrial disease,” says Teleman. “However, this still is a dream of the future, because we do not yet know whether human cells respond in the same way as fly cells do to increased quantities of stearic acid in the diet. Our diet naturally contains much more stearic acid than fly food does. Therefore, a further increase might not make any more difference.”–Story Source-The above post is reprinted from materials provided by German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). Journal Reference-Deniz Senyilmaz, Sam Virtue, Xiaojun Xu, Chong Yew Tan, Julian L. Griffin, Aubry K. Miller, Antonio Vidal-Puig, Aurelio A. Teleman. Regulation of mitochondrial morphology and function by stearoylation of TFR1. Nature, 2015; DOI: 10.1038/nature14601 —–German Cancer Research Center (Deutsches Krebsforschungszentrum, DKFZ). “Fatty acid increases performance of cellular powerhouse: Fundamentally new biological signaling pathway discovered.” ScienceDaily. ScienceDaily, 28 July 2015. <www.sciencedaily.com/releases/2015/07/150728101220.htm>.
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Leucine Modulates Mitochondrial Biogenesis and SIRT1-AMPK Signaling in C2C12 Myotubes
Chunzi Liang,1 Benjamin J. Curry,2 Patricia L. Brown,1 and Michael B. Zemel3
 
1Department of Nutrition, University of Tennessee, Knoxville, 1215 W. Cumberland Avenue, 229 Jessie Harris Building, Knoxville, TN 37996-1920, USA
2Ension, Inc., 11020 Solway School Road, Suite 108, Knoxville, TN 37931, USA
3NuSirt Biopharma, 11020 Solway School Road, Suite 109, Knoxville, TN 37931, USA
 
Received 19 June 2014; Accepted 17 September 2014; Published 7 October 2014
 
Academic Editor: Robert B. Rucker
 
Previous studies from this laboratory demonstrate that dietary leucine protects against high fat diet-induced mitochondrial impairments and stimulates mitochondrial biogenesis and energy partitioning from adipocytes to muscle cells through SIRT1-mediated mechanisms. Moreover, β-hydroxy-β-methyl butyrate (HMB), a metabolite of leucine, has been reported to activate AMPK synergistically with resveratrol in C2C12 myotubes. Therefore, we hypothesize that leucine-induced activation of SIRT1 and AMPK is the central event that links the upregulated mitochondrial biogenesis and fatty acid oxidation in skeletal muscle. Thus, C2C12 myotubes were treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), EX527 (SIRT1 inhibitor, 25 μM), and Compound C (AMPK inhibitor, 25 μM) alone or in combination to determine the roles of AMPK and SIRT1 in leucine-modulation of energy metabolism. Leucine significantly increased mitochondrial content, mitochondrial biogenesis-related genes expression, fatty acid oxidation, SIRT1 activity and gene expression, and AMPK phosphorylation in C2C12 myotubes compared to the controls, while EX527 and Compound C markedly attenuated these effects. Furthermore, leucine treatment for 24 hours resulted in time-dependent increases in cellular NAD+, SIRT1 activity, and p-AMPK level, with SIRT1 activation preceding that of AMPK, indicating that leucine activation of SIRT1, rather than AMPK, is the primary event.
 
1. Introduction—
Impaired mitochondrial function in skeletal muscle is one of the major predisposing factors to metabolic diseases, such as insulin resistance, type 2 diabetes, and cardiovascular diseases [1]. Indeed, lower mitochondrial content and decreased expression of oxidative enzymes are observed in patients with type 2 diabetes [2]. SIRT1 and AMP-activated protein kinase (AMPK) are known to promote mitochondrial biogenesis and oxidative capacity and prevent the mitochondrial dysfunction in skeletal muscle [3, 4].
 
SIRT1, a nicotinamide adenine dinucleotide- (NAD+-) dependent deacetylase, is the key enzyme that mediates caloric restriction- (CR-) induced longevity in mammals [5]. By sensing intracellular NAD+/NADH ratio, SIRT1 regulates target gene expression via changing acetylation status of histones of transcriptional factors, such as peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), tumor suppressor p53 (p53), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and forkhead box O3 (FOXO3) [6], resulting in modulation of wide range of cellular fundamental processes, including DNA repairing, energy metabolism, and cell apoptosis [7, 8]. Overexpression and activation of SIRT1 protect against high fat diet- (HFD-) induced metabolic abnormalities in mice, such as insulin resistance, glucose intolerance, and liver steatosis, without extending their lifespan [9, 10]. Therefore, small molecules that could activate SIRT1 and mimic the CR impacts have drawn considerable attention.—
Leucine, a branched-chain amino acid (BCAA), plays a distinct role in energy metabolism in addition to its pivotal function in protein synthesis [11, 12]. For example, leucine promotes energy partitioning from adipocytes to muscle cells, leading to decreased lipid storage in adipocytes and increased fat utilization in muscle cells [13]. Leucine administration increases insulin sensitivity and glucose tolerance by promoting glucose uptake and fatty acid oxidation in skeletal muscle in HFD-fed mice [14–17]. In fact these effects are mediated partially through SIRT1-dependent pathway, as Sirt1 knockout significantly attenuates these effects [18, 19]. Further, recent data indicate that leucine can directly activate SIRT1 by promoting the enzyme affinity for its substrates and NAD+ [18], resulting in elevated mitochondrial biogenesis and fatty acid oxidation in both adipocytes and myotubes [20, 21].–HMB, a minor metabolite of leucine, has been reported to stimulate AMPK phosphorylation synergistically with metformin, resulting in significant increases in insulin sensitivity and glucose tolerance in mice [22]. Similar to SIRT1, AMPK is an evolutionary conserved enzyme and acts as an energy status sensor via intracellular AMP or AMP/ATP ratio in eukaryotes [3]. In response to nutrient restriction, activated AMPK promotes a cell catabolic shift with increased ATP production to rescue the cellular fuel crisis [23]. Furthermore, phosphorylated AMPK is highly associated with SIRT1 activation in both in vivo and in vitro studies [5, 24], and part of these two enzymes signaling pathways are overlapped [25].
 
These findings provide a mechanistic framework for leucine-modulation of mitochondrial biogenesis [21, 26]. We hypothesize that leucine activation of SIRT1 and AMPK is the major event that regulates fatty acid oxidation and mitochondrial biogenesis in skeletal muscle. Accordingly, we examined the effects of leucine, valine (branched-chain amino acid control), and alanine (nonbranched chain amino acid control) on mitochondrial content, mitochondrial biogenesis-related gene expression, SIRT1 activity, and AMPK phosphorylation in C2C12 myotubes. In addition, we used EX-527 (SIRT1 selective inhibitor) and Compound C (specific AMPK inhibitor) to probe the roles of each enzyme in leucine-modulation of energy metabolism in C2C12 myotubes.
2. Materials and Methods
2.1. Cell Culture
 
C2C12 myoblast cells were seeded at a density of 1.2 106 cells per well in 6-well plates and incubated in Dulbecco’s modified eagle medium (DMEM) containing 4.5 g/L D-glucose, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin at 37°C and 5% CO2. After the cells reach 90% confluence, the medium was switched to a standard differentiation medium (DMEM supplemented with 2% horse serum and 1% penicillin-streptomycin) for 2 to 4 days. The differentiation medium was changed every other day to allow 90% of the cells to fully form myotubes (3–5 days later) before additional treatments began.–The dosages of reagents were 0.5 mM for leucine, 0.5 mM for alanine, 0.5 mM for valine, 100 nM for resveratrol, 25 μM for EX527, 25 μM for Compound C, and 50 μM for AICAR. The incubation lengths were from 1 to 48 hours as indicated in the figure legend. 2.2. RNA Extraction and Quantitative Real-Time PCR (RT-PCR) Analyses—-Total RNA was extracted from C2C12 myotubes using Ambion Totally RNA Isolation Kit (Ambion, Inc., Austin, TX, USA) according to the manufacturers’ instructions. The RNA content was determined using NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). RNA quality was assessed by the 260 nm/280 nm ratio (1.8–2.0) and 260 nm/230 nm ratio (2.0). The mRNA expression of selected genes related to mitochondrial biogenesis, including Sirt1, Sirt3, PGC-1α, cytochrome c oxidase subunit 5b (Cox5b), heat shock cognate protein 1 (Hspd 1), and Cox2, was analyzed using a TaqMan Universal PCR Master Mix kit (Applied Biosystem) according to the manufacturers’ instructions. The primers and probes sets were obtained from Applied Biosystems TaqMan Gene Expression Assays primers and probe set collection. The quantitative RT-PCR reactions were carried out in 96-well format using ABI 7300HT instrument (Applied Biosystem) according to the instructions. Mouse 18S ribosomal RNA was used as the housekeeping gene. Data for each gene was normalized to 18S and presented as a ratio to the transcript of interest to 18S.-2.3. SIRT1 Activity Measurement–SIRT1 Fluorometric Drug Discovery Kit (BML-AK555, ENZO Life Science International, Inc., PA, USA) was used to measure SIRT1 activity in C2C12 myotubes, following the manufacturer instruction. In this assay, SIRT1 activity is determined by the degree of deacetylation of a standardized substrate that contains an acetylated lysine residue. This Fluor de Lys substrate is a peptide containing amino acids 379–382 of human p53 (Arg-His-Lys-Lys [Ac]) and serves as a direct target for SIRT1. SIRT1 activity is proportionally related to the degree of deacetylation of Lys-382 and the corresponding fluorescence signal changes.
 
Cell lysates were harvested by homogenizing cells in RIPA buffer (Sigma-Aldrich, MO, USA), which contains protease inhibitor cocktail (MP Biomedicals LLC, Solon, OH, USA) (100 : 1 v/v). After 5 seconds of ultrasonication on ice, the cell lysates were centrifuged at 12,000 ×g for 5 minutes. The supernatant was used for SIRT1 activity assessment and other experiments. According to the protocol, 5 μL of cell lysate was used for the endogenous SIRT1 activity detection. Samples were incubated in a phosphate-buffered saline solution with peptide substrate (25 μM) and NAD+ (500 μM) at 37°C on a horizontal shaker for 45 minutes. The deacetylation reaction was stopped with the addition of the stop solution (2 mM nicotinamide) and developer that binds to the deacetylated lysine to form a fluorophore. Following 10 minutes of incubation at 37°C, fluorescence intensity was measured using Glomax Multi Detection System (Promega, WI, USA), with excitation and emission wavelengths of 360 nm and 450 nm, respectively. Resveratrol (100 mM) and suramin sodium (25 mM) were used as positive and negative controls, respectively. To normalize the data of SIRT1 activity, concentrations of the sample cellular protein were measured via BCA-assay (Thermo Scientific Inc, Waltham, MA, USA). Data for each sample SIRT1 activity is presented as a ratio to the protein content.
2.4. Cellular NAD+
 
NAD+ was measured in C2C12 myotubes using a colorimetric assay (Cayman Chemical Company, Ann Arbor, MI, USA) that uses an alcohol dehydrogenase reaction to reduce NAD+ in cell lysates to NADH and the NADH is used to reduce a tretrazolium salt substrate (WST-1) to formazan. Formazan absorbance, measured at 450 nm, is proportional to the NAD+ in the cell lysate.
2.5. Fatty Acid Oxidation
 
Cellular fatty acid oxidation was measured using [3H]-palmitate, as described in our previous studies [13]. C2C12 cells in 12-well plates were washed with 2 mL of cold PBS solution twice and incubated in 1 mL of Hank’s basic salt solution containing 0.5 mg/mL BSA, 22 μM-unlabeled palmitate plus 5 μM [3H]-palmitate (32.4 mCi/μm) for 2 hours. All of the reaction solutions were collected from each well, and then 200 μL of 10% trichloroacetic acid and 70 μL 6 N NaOH were added in the solution. Mixtures were then removed from each well and placed in corresponding poly-prep chromatography columns with 1.5 mL Dowex-1 overnight. The 3H2O that passed through the column was collected into a scintillation vial, and radioactivity was measured with a liquid scintillation counter. The protein level of each well was measured using BCA-assay (Thermo Scientific Inc., Waltham, MA, USA) and was used to normalize the palmitate oxidation data.
2.6. Western Blotting
 
Primary antibodies for total AMPK, phospho-AMPK (Thr172), total ACC (Acetyl-CoA Carboxylase), and phospho-ACC (Ser79) were obtained from Cell Signaling Technology Inc. (Danvers, MA, USA). Horseradish peroxidase- (HRP-) conjugated goat anti-rabbit secondary antibody was obtained from Thermo Scientific Inc. (Waltham, MA, USA).
 
Following the indicated treatments, C2C12 myotubes were washed twice with ice cold PBS, and the total cell lysates were prepared using RIPA buffer plus protease/phosphatase inhibitor cocktails with 100 : 1 : 1 (v/v/v, ratio) (Sigma-Aldrich). Following a 10-minute centrifugation at 14,000 ×g, the supernatants were collected for the determination of protein content using BCA assay kit (Thermo Scientific Inc., Waltham, MA, USA) and western blotting. Equal amounts of total cell lysates (20 μg) were loaded to 10% SDS-PAGE (10 cm × 10 cm, Criterion precast gel, Bio-Rad Laboratories, Hercules, CA, USA) and transferred to PVDF membrane (polyvinylidene difluoride membrane) (Bio-Rad, Hercules, CA). The membrane was incubated in 25 mL blocking buffer (1 TBS, 0.1% Tween-20 with 5% w/v nonfat dry milk) for 1 hour at room temperature. Then the membrane was incubated in TBST containing 5% dry milk with primary antibody (1 : 1000) with gentle agitation at 4°C overnight, washed three times with TBST, and incubated with TBST containing rabbit HRP-conjugated secondary antibody (1 : 5000) for 1 hour at RT. Bound antibodies were visualized by chemiluminescence (ECL Western Blotting Substrate, Thermo Scientific) and membranes were exposed to X-ray films (Phenix Research Product, Candler, NC) for protein band detection. The films were scanned using an HP Scanjet 39070 (Palo Alto, CA 94304) and stored as tagged image file format (TIFF) at 300 dpi. The protein bands were quantified by densitometry using BioRad ChemiDoc instrumentation and software of Image Lab 4.0 (Bio-Rad Laboratories).
2.7. Measurement of Mitochondrial Contents
 
Mitochondrial abundance in C2C12 myotubes was assessed by 10-N-nonyl acridine orange (NAO) dye (Life Sciences, PA, USA) according to manufacturer’s instruction. After desired treatment, cells in 96-well plates were treated with 10 M NAO dye, following 2-hour incubation at 37°C in the dark. NAO is not fluorescent, but it can be oxidized into the fluorescent-NAO by oxidative species and accumulated in mitochondrial membrane. The absorbance in each well was measured at 570 nm wavelengths (Promega, WI, USA) and normalized with cellular protein level. The image of mitochondria was taken using a Nikon Eclipse Ti-E Ti-E Fluorescence microscope (Nikon Metrology, Inc., US) equipped with an automated stage and a 20x objective. A 3 × 3 large image scan was taken in each of 5 random fields by multichannel capture (channel 1: excitation/emission = 488/517, channel 2: excitation/emission = 550/567 nm).
2.8. Statistical Analysis
 
Data is presented as means ± standard deviation (SD). Levene’s test was used to determine homogeneity of variance among groups using SPSS 21.0 statistical software (IBM, Armonk, NY) and where necessary natural log transformation was performed before analysis. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) using least significant difference when equal variance was assumed, and Games-Howell test was used when equal variance was not assumed. The independent sample t-test was used to compare two conditions. Differences were considered statistically significant at .
3. Results -3.1. Leucine Treatment Induced the Mitochondrial Biogenesis in C2C12 Myotubes
 
Leucine significantly increased mitochondria content in C2C12s compared to alanine and valine () (Figure 1(a)). These effects were accompanied by increases in mRNA levels of PGC-1α (198%, ) and SIRT3 (167%, ) (Figure 1(b)). SIRT1 activity () and fatty acid oxidation () in the C2C12 myotubes were significantly elevated by leucine compared to the control groups (Figures 1(c) and 1(d), resp.).
fig1
Figure 1: Leucine treatment induces mitochondrial biogenesis and SIRT1 enzymatic activity in C2C12 myotubes. (a) Mitochondrial content was quantitated with NAO dye (10 μM) 48 hours after treatment with leucine (0.5 mM), alanine (0.5 mM), and valine (0.5 mM); (b) mRNA expression levels of PGC-1α and Sirt3 with the same treatments were evaluated by quantitative RT-PCR. The relative mRNA expression was normalized to 18S and expressed as dark bars for PGC-1α and grey bars for Sirt3. (c) Cellular SIRT1 activity and (d) palmitate oxidation were measured after treatment for 48 hours. The results were normalized to cellular protein level for each sample. Data are mean ± SE (). Significantly different from controls with .3.2. SIRT1 Is Required for Leucine-Induced Mitochondria Biogenesis in C2C12 Myotubes–We used a selective SIRT1 inhibitor (EX527) to determine the role of SIRT1 in leucine-induced mitochondrial biogenesis. Leucine increased mitochondrial biogenesis as demonstrated by significant increases in mitochondrial content (), palmitate oxidation () and expression of mitochondrial biogenesis-related gene markers PGC-1α(), SIRT3 (), and COX5b () (Figures 2(a), 2(c) and 2(d), dark panel), and these effects were markedly attenuated by EX527 administration (Figures 2(a), 2(c) and 2(d), grey panel). Comparing the relative Sirt1 expression, leucine and resveratrol (positive control) markedly increased Sirt1 mRNA level (); the SIRT1 inhibitor (EX527) plus leucine treatment () revealed the same pattern (Figure 2(b)).–fig2-Figure 2: Leucine improves mitochondrial biogenesis in C2C12 myotubes in a SIRT1-dependent manner. (a) Mitochondrial content was measured using NAO (10 μM) dye after 48-hour leucine (dark bars), leucine plus SIRT1 inhibitor (EX527 25 μM; grey bars) for 48 hours in C2C12 myotubes. (b, c) SIRT1 activity and mitochondrial biogenesis- related genes (PGC-1α, Sirt3, and COX5b) mRNA levels were measured after the same treatments. The relative SIRT1 activity was normalized to cellular protein level, and mRNA level was normalized to housekeeper gene 18S. (b) Dark bars are DMSO control, grey bars are EX527. (c) Dark bars are PGC-1α, grey bars are Sirt3; striped bars are COX5b. (d) Palmitate oxidation level was detected after the same treatment, and the results were normalized to cellular protein for each sample. Data are mean ± SE (). Different letters indicate significant differences within a given variable. Dark bars are DMSO control and grey bars are EX527. Significantly different from controls, and significantly different from control and EX527 groups with .
3.3. Leucine Stimulates Phosphorylation of AMPK in a SIRT1-Dependent Manner–Six hours of leucine treatment resulted in a 3-fold increase in AMPK phosphorylation in the C2C12 myotubes, which was significantly different from baseline, valine, and alanine. Consistent with this observation, phosphorylation of ACC, a downstream target enzyme of AMPK, was also elevated by leucine compared to the controls () (Figure 3(a)), while EX527 treatment resulted in corresponding suppression of AMPK phosphorylation () (Figure 3(b)), indicating the necessity of SIRT1 for leucine-induced AMPK activation.
fig3–Figure 3: Leucine-induced phosphorylation of AMPK and ACC requires SIRT1 in C2C12 myotubes. (a) C2C12 myotubes were serum starved overnight and treated with leucine (0.5 mM), alanine (0.5 mM), valine (0.5 mM), and DMSO for 6 hours. The cell lysates were assessed by western blotting analysis with specific antibodies against phosphor-AMPKα (Thr 172), phosphor-ACC (Ser 79), total AMPKα (Thr 172), and beta-actin. Integrated density values for the p-AMPK and p-ACC were normalized to total-AMPK band density and represented as dark or gray bars. (b) C2C12 myotubes were treated with 0.2% FBS medium overnight and then treated with leucine (0.5 mM), resveratrol (100 nM), and leucine plus EX527 (25 μM) for 6 hours. Whole cell lysates were prepared and detected by western blotting with specific antibodies against phosphor-AMPKα, AMPKα, and beta-actin. Integrated density value for phosphor-AMPK was normalized to total-AMPK. Significantly different from controls with .–3.4. Leucine Stimulates SIRT1 Activity, Phosphorylation of AMPK, and Cellular NAD+ in a Time-Dependent Manner
 
To determine the interplay between SIRT1 and AMPK, we measured the cellular NAD+ level, SIRT1 activity, and phosphorylation of AMPK at time points: 0, 1, 4, 6, 12, and 24 hours by leucine treatment in C2C12 myotubes. Leucine increased SIRT1 activity at 1 (), 12 () and 24 hours () compared to the baseline (Figure 4(a)). However, no change was observed for the NAD+ content and p-AMPK level during the first 4 hours. NAD+ level was elevated almost twofold higher at time points 4 () and 24 hours () compared to baseline level and otherwise remained low (Figure 4(b)); the levels of p-AMPK were markedly increased and stayed high from 4 to 24 hours () (Figure 4(c)).
fig4
Figure 4: Leucine stimulates SIRT1 activity, AMPK phosphorylation, and cellular NAD+ in a time-dependent manner. C2C12 myotubes were serum starved overnight and treated with leucine (0.5 mM). Cell lysate was collected and analyzed for cellular SIRT1 activity, western blotting of p-AMPK and cellular NAD+ levels at indicated certain time points. (a) SIRT1 activity. (b) Cellular NAD+. Both SIRT1 activity and NAD+ level were normalized to cellular protein for each sample. (c) Phosphorylation level of AMPK was detected using western blotting following the same time course in C2C12 cells, with resveratrol serving as positive control. Data are mean ± SE (). Different letters indicate significant differences between dark or gray bars. Significantly different from point 0, and significantly different from time point 1.
3.5. Leucine-Induced Mitochondrial Biogenesis in C2C12 Myotubes Requires AMPK
 
We next examined whether AMPK also mediates leucine’s impacts on mitochondrial biogenesis in C2C12 myotubes. As shown in Figure 5, leucine treatment markedly increased the mitochondrial component genes expression (Figure 5(a), dark columns), Hspd1 () and COX2 (). Similar effects were found for genes encoding mitochondrial biogenesis regulatory proteins, [PGC-1α (), Sirt1 ()] and component proteins [Cox5b ()], (Figure 5(b), dark columns), while Compound C treatment markedly impaired all these inductions (Figure 5, grey panels).
fig5
Figure 5: Leucine-induced mitochondrial biogenesis in C2C12 myotubes requires AMPK. (a) C2C12 myotubes were treated with leucine (0.5 mM), AICAR (20 μM), and Compound C (25 μM) for 24 hours. mtDNA levels of the cells were analyzed by the mitochondrial markers gene expression, Hspd1 and COX2, using real-time PCR. (b) Sirt1 and mitochondrial biogenesis related mRNA level of PGC-1α and COX5b were evaluated also by RT-PCR after treating with leucine and Compound C for 24 hours was measured; all the mRNA levels were normalized to 18S housekeeping gene. Data are mean ± SE (). Dark bars are vehicle control; grey bars are Compound C. Significantly different from controls. Significant Compound C effects.
4. Discussion–These data indicate that leucine stimulates significant muscular metabolic changes, including SIRT1 activation, AMPK phosphorylation, and mitochondrial biogenesis in C2C12 myotubes. These changes may contribute to leucine’s beneficial effects on energy metabolism and insulin sensitivity in both animal and human models [17, 19, 27, 28].–A previous clinical trial has shown that high dairy intake (rich in BCAAs) induces significant suppression of reactive oxygen species (ROS) and inflammatory stress, indicated by decreased plasma tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1) levels [19]. Doubling leucine intake in mice has been found to reverse multiple HFD-induced metabolic abnormalities, including glucose intolerance, hepatic steatosis, and inflammation [11]. These effects are accompanied by corresponding increases in mitochondrial oxidative capacity and mitochondrial content. Since mitochondrial dysfunction and mitochondrial content loss are directly linked to the development of metabolic disorders [1, 29], increased mitochondrial biogenesis appears to rescue part of these obesity-related abnormalities [30].–Consistent with our previous studies [13], here we show that 0.5 mM leucine treatment, which is comparable to the plasma leucine concentration achieved by a leucine-rich diet [31], can markedly increase mitochondrial content, mitochondrial biogenesis-related gene expression, and fatty acid oxidation in C2C12 myotubes, compared to valine and alanine.
 
The data herein demonstrate that the improvement of fatty acid oxidation and mitochondrial content by leucine is accompanied by increased SIRT1 activity in C2C12 cells. SIRT1 has been demonstrated to play significant roles in leucine’s effects on energy metabolism. In Macotela’s study [11], leucine restores HFD-reduced hepatic NAD+ and SIRT1 expression back to normal levels. Similarly, Li et al. demonstrate that leucine increases SIRT1 expression and decreases acetylation level of PGC-1α, resulting in attenuation of HFD-induced mitochondrial dysfunction, insulin resistance, and obesity in mice [32]. Furthermore, Sun and Zemel found that leucine induces mitochondrial biogenesis in muscle cells by stimulating the expression of PGC-1α and NRF-1 via a SIRT1-dependent pathway [26]. These findings, along with the observations reported here, are in agreement with our recent work that leucine could activate SIRT1 enzyme through allosteric interaction in adipocytes and myotubes [25].
 
To establish whether or not SIRT1 is required for leucine-induced mitochondrial biogenesis, EX527, a selective SIRT1 enzyme inhibitor, was used to treat the cells in combination with leucine. EX527 significantly attenuated leucine-induced mitochondrial content, mitochondrial biogenesis-related genes expression, and fatty acid oxidation in C2C12 myotubes. The observations reported here are consistent with Price’s work,in which SIRT1 knockout completely blocked resveratrol-induced mitochondrial biogenesis and β-oxidation in skeletal muscle [33], further supporting the essential roles of SIRT1. However, the leucine-induced Sirt1 gene expression was not affected by EX527, possibly due to the unique inhibition mechanism of EX527 on SIRT1 catalytic activity [34].
 
 
We also found that AMPK phosphorylation, which is elevated in response to metabolic stress [35], was also increased by leucine in C2C12 myotubes. This change might help to explain the increased fatty acid oxidation in the cells. Similarly, in mice, leucine supplementation has been reported to activate AMPK synergistically with resveratrol and metformin, resulting in increased insulin sensitivity and glucose tolerance [22]. On the other hand, Compound C, an inhibitor of AMPK, markedly blocked leucine’s effects on mitochondrial biogenesis, indicating that like SIRT1 elevated mitochondrial biogenesis and fatty acid oxidation by leucine requires AMPK in C2C12 myotubes.
 
Notably, we found that leucine-induced AMPK phosphorylation was markedly blocked by EX527, suggesting that AMPK might serve as a downstream target of SIRT1. In support of this concept, Price et al. reported that SIRT1 activation is required for AMPK phosphorylation and improvement of mitochondrial function via deacetylation and activation of LKB1, a primary upstream kinase of AMPK [33, 36], while Park et al. found that resveratrol activates SIRT1 via an indirect pathway involving calmodulin-dependent protein kinase kinase β (Camkkβ) and AMPK activation [36]. Currently available evidences suggest that AMPK and SIRT1 display mutual interactions with each other; AMPK could activate SIRT1 by increasing cellular NAD+ level via promoting expression of nicotinamide phosphoribosyltransferase (Nampt), a rate-limiting enzyme in NAD+ biosynthesis; however, SIRT1 can also directly deacetylate and activate LKB1, resulting in the activation and phosphorylation of AMPK [37].
 
Our time-course data suggest that SIRT1 may be the initial target of leucine. SIRT1 activity was increased within the first hour of leucine treatment, while cellular NAD+ and p-AMPK levels remained unchanged. Considering that the increased Sirt1 mRNA and SIRT1 activity level occurred at some time after the leucine treatment for 24 hours, it is possible that SIRT1 activity is elevated by leucine first, and then activation of AMPK is a subsequent event, which may be responsible for the further SIRT1 activation at the later time points.
 
Our data may also reflect dose-dependent effects of leucine treatment. For example, high-dose leucine infusion and supplementation have been shown to induce insulin resistance and glucose intolerance in both human and animal models [38, 39], possibly via activation of mammalian target of rapamycin- (mTOR-) insulin receptor substrate 1 (IRS-1) signaling pathways [40]. In contrast, modest increases in leucine intake, sufficient to induce plasma leucine elevations to ~0.5 mM, significantly reduced obesity-related oxidative and inflammatory stress, resulting in improvement of insulin sensitivity in humans [19]. Similarly, Vaughan et al. found that leucine in the 0.1–0.5 mM range induces a dose-dependent increases of PGC-1α expression, leading to significant elevated mitochondrial density and oxidative capacity in skeletal muscle cells [17]. Consistent with these evidences, we found comparable levels of leucine promoted mitochondrial biogenesis and fatty acid oxidation in C2C12 myotubes.
 
There are several limitations to this study. One of them is the use of the Fleur de Lys assay to measure SIRT1 activity. Studies have challenged the validity of the assay, as some of them have found that sirtuin-activating compounds (STACs) only increased SIRT1 activity by using fluorophore-tagged substrates but not the matching nontagged peptides, which also might explain why the activation can be found exclusively in vitro but not in vivo [41, 42]. According to Gertz et al., the fluorophore can act synergistically with STACs to promote binding between substrates and SIRT1 enzyme [43]. Furthermore, evidence suggests that resveratrol-induced SIRT1 activation is actually mediated through an indirect signaling pathway involved in cAMP phosphodiesterases (PDE) and AMPK in vivo [36]. However, Hubbard et al. recently provided more evidences to support the allosteric binding and activation theory between STACs and SIRT1. They found that specific hydrophobic motifs in SIRT1 substrates and a single amino acid (Glu230) in SIRT1 enzyme mediate the structure change during the deacetylation [44]. As a highly hydrophobic amino acid, leucine might directly activate SIRT1 through conformation change. Indeed, recent evidence demonstrates that leucine exerts direct effects on SIRT1 kinetics by decreasing 50% km for NAD+ and substrates. With the presence of leucine and HMB, lower concentration of resveratrol is required for the activation of SIRT1 [45]. Therefore, further experiments using fluorophore-free substrates to measure the SIRT1 activity are needed to elucidate the exact pathways of leucine-activated SIRT1. A second limitation is lack of data assessing the cellular acetylation status of LKB1 and PGC-1α, as well as Nampt phosphorylation and expression.
 
In summary, with the present work, we demonstrate that leucine improves mitochondrial biogenesis and fatty acid oxidation in C2C12 myotubes through SIRT1 and AMPK-dependent pathway, with secondary activation of AMPK mediated by SIRT1 (Figure 6).
239750.fig.006
Figure 6: Proposed mechanism of leucine-induced mitochondrial biogenesis. In C2C12 myotubes, leucine treatment leads to activation of SIRT1. SIRT1 then deacetylates and activates LKB1, which subsequently induces AMPK phosphorylation and activation. In turn, activated AMPK could promote SIRT1 activation via intracellular NAD+ level by changing expression and activity of Nampt. Activated AMPK and SIRT1 further activate PGC-1α via phosphorylation and deacetylation, resulting in elevated mitochondrial biogenesis and oxidative function.
Conflict of Interests
 
The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgments
 
The authors thank Drs. Ling Zhao and Antje Bruckbauer for technical support in cell culture and SIRT1 activity measurement.
References
 
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[F1]Quercetin is a sulphur based antioxidant found in onions and garlic—- Quercetin may protect the body’s endogenous Deoxyribonucleic Acid (DNA) from breakage from Ferric Iron-initiated Hydrogen Peroxide lipid peroxidation (by chelating (removing) Ferric Iron from the body).
[F2]Breast Cancer. references
– Quercetin may facilitate the apoptosis (cellular death) of Cervical Cancer cells. references
– Quercetin may help to prevent Colon Cancer by inhibiting the ability of Epidermal Growth Factor (EGF) to stimulate the growth of Colon Cancer cells and by inhibiting DNA damage in Colon cells (colonocytes). references
 
– Quercetin may inhibit the synthesis of Deoxyribonucleic Acid (DNA) in Leukemia (including Promyelocytic Leukemia) cells (thereby preventing the replication of Leukemia cells). references
– Quercetin may help to prevent Lung Cancer. references
– Quercetin may inhibit the growth and metastasis of Melanoma. references
– Mouth Cancer references
 
– Ovarian Cancer references
– Quercetin may help to prevent Pancreatic Cancer and may stimulate the apoptosis of Pancreatic Cancer cells. references
– Quercetin may help to prevent Prostate Cancer (by inhibiting the expression of Androgen Receptors, Ornithine Decarboxylase and Prostate-Specific Antigen). references
– Squamous Cell Carcinoma references
 
– Quercetin may inhibit the growth of Stomach Cancer. references
– Quercetin may reduce the carcinogenicity of Heterocyclic Aromatic Amines (HAAs) (by interfering with the ability of p450 Enzymes to activate HAAs). references
 
Quercetin may suppress many forms of Detrimental Fungi. references
Quercetin may reduce Inflammation: references
 
– Quercetin may inhibit the excessive release of Acid Hydrolases from Lysosomes. references
– Quercetin may inhibit the production and release of Histamine by Basophils. references
– Quercetin may stabilize the Cell Membranes of Mast Cells, causing Mast Cells to become less reactive to the Antigens that are implicated in Allergies and decreases the ease with which they release their stored Histamine and Serotonin. references
 
– Quercetin may inhibit the production of inflammatory Series 4 Leukotrienes and Prostaglandin E2 within the body.
 
Quercetin may inhibit Helicobacter pylori. references
Quercetin may prevent many infections caused by Viruses: references
 
– Quercetin may help to suppress the HIV virus (the Virus that causes Acquired Immune Deficiency Syndrome (AIDS) – part of Quercetin’s ability to suppress the HIV virus stems from the inhibition of the Viral Reverse Transcriptase enzyme. references
– Quercetin may suppress the Epstein-Barr Virus. references
– Quercetin may help to prevent and alleviate the Common Cold (due to its ability to suppress Rhinoviruses).
 
– Quercetin may suppress the Herpes Simplex Virus Type 1. references
– Quercetin may suppress many strains of Influenza Viruses. references
– Quercetin may suppress Parainfluenza Viruses. references
– Quercetin may suppress Picornaviruses. [more info]
– Quercetin may suppress Polio Viruses. references
 
– Quercetin may suppress the Respiratory Syncytial Virus. references
– Quercetin may suppress Rhinoviruses. [more info]
 
[F3]Meaning the ratio of the fat may need to increase in order to reduce the leakage and or reducing the solute you mixing into the fat-
[F4]One type of Liposome
[F5]Single layer type of liposome
[F6]Primary Functions of Stearic Acid in Cell Culture Systems:
Long-term energy storage: energy derived from NADPH and ATP is stored in fatty acids. Fatty acids are esterified to a glycerol backbone to form a group of compounds known as mono-, di- and tri- glycerides (neutral fats). Energy is released when fatty acids are degraded.
Fatty acids are precursors of other molecules: prostaglandins, prostacyclins, thromboxanes, phospho-lipids, glycolipids, and vitamins.
Structural elements: fatty acids are important constituents of cell structures such as the membranes.
Stearic Acid is a direct precursor of the n-9 unsaturated fatty acid, Oleic acid.
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From: H S Truman
 
Political Correctness is a doctrine, recently fostered by a delusional, illogical minority and
promoted by a sick mainstream media, which holds forth the proposition that it is entirely
possible to pick up a piece of shit by the clean end!
 
 
 
 
Show of the Month August 8 2015
 
Xenotransplantation History and Development
 
Xenozoonosis
 
Nanoparticles-What are they
 
Rapid aging of the thymus linked to decline in free radical defenses
 
New vitamin B3 pathway identified
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Xenotransplantation History and Development
A timeline featuring some of the key events in xenotransplantation from the early 1900s until 2011.
1902
Reconnecting blood vessels for organ transplants
Alexis Carrel at the Rockefeller Institute in New York describes how blood vessels could be reconnected in transplanted organs. Carrel receives the Nobel prize for this work in 1912.
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1912/carrel.htmlExternal Link
1902–1923
First attempts at organ xenotransplants
Transplants with pig, goat, sheep and monkey organs are attempted, but all fail, with patients surviving only hours or days after transplantation. No further animal to human transplants are tried again until 1963, after immunosuppressing drugs are developed.
1944
Immune system causes transplant rejection
Peter Medawar from the University of London shows that transplants are failing because of an immune system reaction.
1954
First successful human to human transplant
First successful human to human transplant of a kidney between identical twin brothers.
1960
Acquired immune tolerance
Peter Medawar receives the Nobel prize for discovering that it is possible to induce tolerance to transplanted tissue.
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1960/medawar.htmlExternal Link
1960
First immunosuppressive drugs identified
A number of researchers independently demonstrate that a drug called 6-MP can delay rejection of tissue and organs transplanted between the same species.
1963
Baboon kidney transplant
Baboon kidneys are transplanted into six patients in Denver by Dr Thomas Starzl. The patients survive between 19–98 days.
1963
Chimpanzee kidney transplant
Chimpanzee kidneys are transplanted into 13 patients by Keith Reemtsma at Tulane University in Louisiana. One patient survives for 9 months.
1964
Chimpanzee heart transplant
The first animal to human heart transplant is carried out by James Hardy at the University of Mississippi, but it fails rapidly.
1969–1974
Chimpanzee liver transplant
The world’s first chimpanzee liver transplants are done on three children between 1969 and 1974 but none of them survives for more than 2 weeks.
1977
Baboon and chimpanzee hearts used as back-up pumps
Christiaan Barnard uses baboon and chimpanzee hearts as temporary back-up pumps in two patients with heart failure after surgery, but the treatment does not help the patients survive.
1978
Pig skin used to treat burns patients
Burns patients treated with pig skin grafts have faster healing times and less pain than patients treated with standard paraffin gauze dressings.
1984
Baboon heart transplant in baby
Baby Fae, an infant born with a severe heart defect, receives a baboon heart, but only lives for 20 days after the transplant.
1992–1993
Baboon to human liver transplant
Dr Thomas Starzl transplants baboon livers into two patients. One of the patients survives for 70 days with little evidence of rejection.
1995
Transgenic pigs prevent transplant rejection
Dr David White in Cambridge, UK, creates transgenic pigs that have a human protein to prevent their tissues and organs being rejected by the immune system. Several other labs investigate similar strategies.
1995
Baboon to human bone marrow transplant for HIV
Jeff Getty, a patient infected with human immunodeficiency virus (HIV), receives baboon bone marrow to treat his illness. Baboon bone marrow has a natural resistance to HIV. His symptoms improve for a while, but the baboon cells die after about 2 weeks.
1996
Pig cell transplant for type 1 diabetes
Living Cell Technologies (formerly Diacrin) transplants encapsulated pig islet cells into type 1 diabetic patient Michael Helyer. The treatment is successful and allows Michael to reduce insulin injections.
Get information sheet: Trialling pig cell transplants
1997
Pig nerve cell transplants for Parkinson’s disease
Foetal pig nerve cells are used to treat patients with Parkinson’s disease with some success.
Get video clip: Xenotransplantation saves Jim
1997
Pig liver used to keep patient alive
Robert Pennington, a 20-year-old with liver failure, is kept alive by passing his blood through transgenic pig livers, which had been genetically modified so they would not be recognised by the recipient’s immune system. This procedure is carried out for 7 hours over 3 days until a suitable liver becomes available. This procedure is done a few weeks before a worldwide ban on xenotransplants.
Get video clip: Xenotransplantation saves Robert
1997
Worldwide ban on all xenotransplantation
Concerns about the risk of infecting human recipients with animal endogenous retroviruses lead to a worldwide ban or moratorium on animal to human transplants. Pig endogenous retrovirus (PERV) is of particular concern.
Get video clip: Pig cell transplants and PERV
1997–1999
Risk of infectious disease assessed
Several groups publish findings showing no evidence of PERV infection in human recipients of pig tissues.
2000–2011
Ban on xenotransplantation is lifted in some countries
The ban on xenotransplantation is lifted in some countries and applications for trials with xenotransplants are assessed on a case-by-case basis.
2007–2011
Clinical trials of pig cell transplants continue
Russia, New Zealand and Argentina all approve clinical trials of pig cells for the treatment of type 1 diabetes.
Get focus story: Pig cell transplants
 
Xenozoonosis
Xenozoonosis, also known as zoonosis or xenosis, is the transmission of infectious agents between species via xenograft. Animal to human infection is normally rare, but has occurred in the past. An example of such is the avian influenza, when an influenza A virus was passed from birds to humans.[27] Xenotransplantation may increase the chance of disease transmission for 3 reasons:
Implantation breaches the physical barrier that normally helps to prevent disease transmission,
The recipient of the transplant will be severely immunosuppressed; and
Human complement regulators (CD46, CD55, and CD59) expressed in transgenic pigs have been shown to serve as virus receptors, and may also help to protect viruses from attack by the complement system.[28]
Examples of viruses carried by pigs include porcine herpesvirus, rotavirus, parvovirus, and circovirus. Porcine herpesviruses and rotaviruses can be eliminated from the donor pool by screening, however others (such as parvovirus and circovirus) may contaminate food and footwear then re-infect the herd. Thus, pigs to be used as organ donors must be housed under strict regulations and screened regularly for microbes and pathogens. Unknown viruses, as well as those not harmful in the animal, may also pose risks (Takeuchi and George, 2000). Of particular concern are PERVS (porcine endogenous retroviruses), vertically transmitted microbes that embed in swine genomes. The risks with xenosis are twofold, as not only could the individual become infected, but a novel infection could initiate an epidemic in the human population. Because of this risk, the FDA has suggested any recipients of xenotransplants shall be closely monitored for the remainder of their life, and quarantined if they show signs of xenosis.[29]
Baboons and pigs carry myriad transmittable agents that are harmless in their natural host, but extremely toxic and deadly in humans. HIV is an example of a disease believed to have jumped from monkeys to humans. Researchers also do not know if an outbreak of infectious diseases could occur and if they could contain the outbreak even though they have measures for control. Another obstacle facing xenotransplants is that of the body’s rejection of foreign objects by its immune system. These antigens (foreign objects) are often treated with powerful immunosuppressive drugs that could, in turn, make the patient vulnerable to other infections and actually aid the disease. This is the reason the organs would have to be altered to fit the patients’ DNA (histocompatibility).–In 2005, the Australian National Health and Medical Research Council (NHMRC) declared an eighteen-year moratorium on all animal-to-human transplantation, concluding that the risks of transmission of animal viruses to patients and the wider community had not been resolved.[30] This was repealed in 2009 after an NHMRC review stated “… the risks, if appropriately regulated, are minimal and acceptable given the potential benefits.”, citing international developments on the management and regulation of xenotransplantation by the World Health Organisation and the European Medicines Agency.[31]
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Nanoparticles-What are they?
 
Nanoparticles can be divided into three groups: · Inorganic nanoparticles · Solid lipid nanoparticles · Polymer nanoparticles Inorganic nanoparticles is the generic term for several nanoparticles including for example metal oxide- and non-oxide ceramics, metals, calcium phosphate, gold, silicate and magnetic nanoparticles. So called “nanoshells” combine various inorganic elements or materials. They typically have a silicon core, which is sealed in an outer metallic cover. Polymer nanoparticles involve various natural or biocompatible synthetic polymers. They include rationally designed macromolecular drugs, polymerdrug and polymer-protein conjugates, polymeric micelles containing covalently bounded drugs, and polyplexes for DNA delivery. Polymer nanoparticles can be divided into nanospheres, which build a continuous polymer matrix–
and can be referred as “drug sponges” and nanocapsules, which consist of a polymer layer enclosing a fluid-filled cavity and are mimicking liposomes. Solid lipid nanoparticles combine the advantages but avoiding the disadvantages of other colloidal carriers have attracted increasing attention in recent years, and are regarded as an alternative carrier system to traditional colloidal systems, such as emulsions, liposomes and polymeric microparticles and nanoparticles.
How are they applied to drug delivery?–Nanoparticles are widely used in drug delivery where they can increase drug solubility and, additionally, can lead to controlled release and/or drug targeting. They are used in anti-cancer treatment, genedelivery, asthma inhalers, hormone delivery through the skin, drug delivery through the eye and in oral and vaccine delivery systems. A lot of companies employ nanoparticles in anti-cancer treatment.–Availability: The first United States approval of a product produced incorporating the NanoCrystal® technology occurred in August 2000. Key Players: Elan Pharmaceuticals (USA) SkyePharma (UK)
 
Nanocrystals-What are they?
Increasing the active surface area is the key to many applications of nanotechnologies, from improving automotive and industrial catalysts to improving the uptake of poorly soluble drugs in the human body. Nanocrystals are ground in special mills and the resulting drugs can be applied intravenously as nanosuspensions or bronchially through an inhaler. This small size enhances the surface/volume-ratio and bioavailability of almost insoluble pharmaceuticals. drug substance using a proprietary, wet-milling technique[F1]. The resulting particles of the drug are stabilized against agglomeration by surface adsorption of selected GRAS (Generally Regarded As Safe) stabilizers. The result is an aqueous dispersion of the drug substance that behaves like a solution, which can be processed into finished dosage forms for all routes of administration. The size of the particles allows for safe and effective passage through capillaries. [F2]NanoCrystal technology represents both an enabling technology for evaluating new chemical entities that exhibit poor water solubility and also a valuable tool for optimizing the performance of established drugs. NanoCrystal technology is of particular benefit for drugs with poor solubility in water. The process is also useful for moderately soluble drugs when a high concentration of drug in a low volume of fluid is desired.
 
How are they applied to drug delivery?
For poorly water-soluble drug compounds, grinding them into nanoscale crystals increases the surface area of the compounds, which leads to an increase in dissolution rate. In one of the best known cases, Elan Pharmaceuticals’ NanoCrystal® technology, particles are small particles of drug substance, typically less than 1000 nanometers (nm) in diameter, which are produced by milling the
Availability: The first poly (ethylenglycol) (PEG)ylated proteins were approved by regulatory authorities for routine clinical use in the early 1990s. Key Players: StarPharma (USA) Enzon (USA) Teva Pharmaceuticals (Israel)
 
Polymer Therapeutics-What is it?
Polymer therapeutics differ from particle shaped drug delivery systems in their dimensions. They are molecular units with diameters of a few nanometres and can be subdivided into four groups: · Polymer drugs · Polymer drug conjugates · Polymer micelles · Dendrimers Early designs for a polymer therapeutic system involved attaching a water-soluble polymer to a drug through a selected linker molecule. Trapping low molecular weight drugs as polymer conjugates not only temporarily inactivates the drug, but also restricts their uptake by cells to endocytosis (the process whereby cells absorb material such as proteins from the outside by engulfing it with their cell membrane). As high molecular weight macromolecules of the drugs are unable to diffuse passively into cells, they are ‘engulfed’ as membrane-encircled sacs called vesicles, in which intracellular enzymes then set to [F3]work to release the drug. This means that the polymer-drug conjugate should be able to circulate longer in the body, potentially without the toxic side-effects associated with many drugs. With the appropriate biodegradable linker, and/or a cell-specific targeting group, it should be possible to deliver the drug direct to the target site.
 
How is it applied to drug delivery?
A drug can be covalently bound to the four groups above. They differ from other drug-delivery systems in which a drug is encapsulated or solubilised and are more akin to new chemical entities since chemical conjugation occurs (i.e the combined polymer and drug behave as a compound different from either component).
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Rapid aging of the thymus linked to decline in free radical defenses
A critical immune organ called the thymus shrinks rapidly with age, putting older individuals at greater risk for life-threatening infections. A study published August 6 in Cell Reports reveals that thymus atrophy may stem from a decline in its ability to protect against DNA damage from free radicals. The damage accelerates metabolic dysfunction in the organ, progressively reducing its production of pathogen-fighting T cells.–The findings suggest that common dietary antioxidants may slow thymus atrophy and could represent a promising treatment strategy for protecting older adults from infections.–“The thymus ages more rapidly than any other tissue in the body, diminishing the ability of older individuals to respond to new immunologic challenges, including evolving pathogens and the vaccines that may otherwise offer protection from them,[F1]” says senior study author Howard Petrie of the Scripps Research Institute. “We provide, for the first time, a mechanistic link between antioxidants and normal immune function, opening new avenues for potential treatment strategies that could improve immune defenses in the aging population.”–The thymus produces essential immune cells called T cells, which are continuously lost and must be replaced throughout life[F2]. But starting around the time of puberty, the thymus rapidly decreases in size and loses its capacity to produce enough new T cells. This loss is partially offset by the duplication of existing T cells, but the resulting population of cells becomes more and more biased toward memory T cells, which recognize pathogens from previous or ongoing infections. As a result, broad-spectrum immunity against new pathogens and protective immune responses elicited by new vaccines diminish with age.–[F3]The development of interventions to slow the progression of thymus atrophy has been limited by the lack of knowledge about the underlying mechanisms. The prevailing theory suggests that sex hormones play a key role, but this explanation does not account for the accelerated speed at which the thymus diminishes in size in comparison to other tissues. Moreover, the body of scientific evidence clearly indicates that other factors must be involved in age-related thymus atrophy.–To address this question, Petrie and first author Ann Griffith, currently at the University of Texas Health Science Center at San Antonio, developed a computational approach for analyzing the activity of genes in two major thymic cell types–stromal cells and lymphoid cells–in mouse tissues, which are very similar to human thymic tissues in terms of function and the properties of atrophy. They found that stromal cells were deficient in an antioxidant enzyme called catalase, resulting in the accumulation of free radical and metabolic damage.-To test whether catalase deficiency plays a causal role in thymus atrophy, the researchers performed genetic experiments to enhance catalase levels in mice. By 6 months of age, the size of the thymus of the genetically engineered mice was more than double that of normal mice. Moreover, mice that were treated with two common antioxidants from the time of weaning achieved nearly normal thymus size by 10 weeks of age.–Taken together, the findings provide support for the free-radical theory of aging, which proposes that reactive oxygen species such as hydrogen peroxide cause cellular damage that contributes to aging and a variety of age-related diseases. These toxic molecules, which form in cells as a natural byproduct of the metabolism of oxygen, have been linked to progressive atrophy in many organs and tissues as part of the normal aging process. However, these are generally slow, progressive processes that do not become apparent until late in life and often go mostly unnoticed.–“In the case of the thymus, atrophy is more rapid than other tissues, which we now show is a consequence of stromal catalase deficiency in the context of a highly metabolic environment designed to support the demands of T-cell proliferation,” Petrie says. “Our studies show that, rather than an idiosyncratic relationship to sex steroids, thymic atrophy represents the widely recognized process of accumulated cellular damage resulting from lifelong exposure to the oxidative byproducts of aerobic metabolism.[F4]”–In future studies, the researchers will investigate whether antioxidant supplementation improves the functioning of the thymus and the immune system during aging. If these studies provide support for this idea, then they could lead to the development of new clinical recommendations for the prevention or treatment of age-related thymus atrophy in humans.–Story Source-The above post is reprinted from materials provided by Cell Press. –Journal Reference-Griffith et al. Metabolic damage and premature thymus aging caused by stromal catalase deficiency. Cell Reports, August 2015 DOI: 10.1016/j.celrep.2015.07.008 –Cite This Page-Cell Press. “Rapid aging of the thymus linked to decline in free radical defenses.” ScienceDaily. ScienceDaily, 6 August 2015. <www.sciencedaily.com/releases/2015/08/150806133043.htm>.

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