Autophagic Protein Degradation Research Articles

Induction of Lysosomal Dilatation, Arrested Autophagy, and Cell Death by Chloroquine in Cultured ARPE-19 Cells

To characterize and investigate the mechanism of chloroquine (CQ) retinotoxicity in human retinal pigment epithelium-derived ARPE-19 cells. Cultured ARPE-19 cells were exposed to 10 to 250 μM CQ, and cell death was quantified using a lactate dehydrogenase release assay. Autophagy was studied in ARPE-19 cells transfected with GFP-LC3. Lysosomes in living cells were stained and observed by live-cell confocal microscopy. After exposure to CQ, ARPE-19 cells developed cytosolic vacuoles within 1 hour and underwent cell lysis within 24 hours. The levels of LC3-II, beclin-1 and, p62, as well as the number GFP-LC3- and RPF-LC3-positive autophagic vacuoles (AVs), increased after CQ treatment, indicating that autophagy was activated. However, lysosomal staining revealed that almost all AVs were separate from lysosomes; thus, fusion between AVs and lysosomes was completely blocked. In addition, the levels of ubiquitinated proteins and GFP-mHttp aggregates in ARPE-19 cells were increased by CQ, providing further evidence that autophagic degradation was inhibited. CQ induces vacuole formation and cell death in ARPE-19 cells. Initially, vacuoles developed from enlarged lysosomes, followed by the activation of upstream steps in the autophagy pathway and the formation of LC3-positive AVs. Because CQ blocked the fusion of AVs with lysosomes, autophagic protein degradation was inhibited, indicating that CQ-induced retinotoxicity may be caused by the accumulation of potentially toxic ubiquitinated proteins.

Abstract 4844: Dual effect of Akt on autophagy in pancreatic cancer cells

Abstract Background & Aims: Key steps in autophagic protein degradation are formation of autophagosomes (induction) and their fusion with lysosomes (progression). Akt is a major anti-apoptotic factor in cancer cells; however, its role in autophagy in general, and in pancreatic cancer (PaCa) in particular, is poorly understood. We recently showed that phosphatases PHLPP-I and -II are key negative regulators of Akt in PaCa cells. The present study aimed to elucidate the effects of Akt and PHLPPs on individual steps of autophagy and the role of autophagy in the prosurvival function of Akt in PaCa cells. Methods: We measured apoptosis by DNA fragmentation and caspase-3 activity; autophagy, by conversion of cytosolic LC3-I to autophagic LC3-II (with immunoblot) and LC3 immunofluorescence; autophagy induction, in cells pretreated with NH4Cl to block autophagy progression; autophagosomal fusion with lysosomes, by co-localization of the lysosomal marker LAMP-1 with LC3-II; and efficiency of protein degradation, by level of p62 protein, a specific autophagy target. Results: Akt inactivation by AktVIII inhibitor or siRNA, or by overexpression of PHLPP-I and -II all stimulated autophagic vacuole formation in MiaPaCa-2 and PANC-1 cells. Akt inactivation greatly facilitated LC3-I/LC3-II conversion both in the presence and absence of NH4Cl, indicating increased autophagy induction. At the same time, Akt inactivation inhibited autophagosomal fusion with lysosomes and efficiency of protein degradation, indicating decreased autophagy progression. Thus, Akt has dual effect on autophagy in PaCa cells: it inhibits autophagy induction but promotes autophagosomal fusion with lysosomes and hence autophagy progression. The overall effect of Akt is decreased accumulation of autophagic vacuoles, i.e., inhibition of autophagic death. To study the role of autophagy in apoptosis, we used 3-methyladenine or beclin siRNA to inhibit autophagy induction, and bafilomycin or chloroquine to block the fusion of autophagosomes with lysosomes. All these treatments stimulated apoptosis in PaCa cells, indicating the anti-apoptotic role of autophagy. In cells with inactivated Akt, blocking autophagy induction (e.g., with 3-methyladenine) further greatly stimulated apoptosis. This indicates that the ability of Akt to inhibit autophagy induction greatly diminishes its anti-apoptotic function. Differently, blocking autophagosomal fusion with lysosomes had little effect on apoptosis in cells in which Akt was inactivated, likely because in these cells autophagy progression was already inhibited. Conclusions: Akt inhibition hampers autophagy progression, thus promoting vacuolization and autophagic cell death. At the same time, Akt inhibition greatly stimulates autophagy induction, thus counteracting apoptosis. Therefore, to maximally stimulate apoptosis in PaCa cells we propose to apply Akt inhibitors in combination with inhibitors of autophagy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4844.

Abstract 85: Chloroquine potentiates the 5-fluorouracil-induced cell cycle arrest on HT-29 colon cancer cells

Abstract Introduction Autophagy is a self-degradation process that is essential for cell survival, development, and homeostasis. During autophagy, cytoplasmic organelles and proteins damaged by the cellular stress, such as starvation, hypoxia and chemotherapy, are recycled and converted into more essential proteins. Thus, induction of autophagy in cancer cells may result in cell adaptation against anticancer agents, consequently leading to resistance to therapy. Chloroquine (CQ), a worldwide used anti-malarial drug, is a lysosomotropic drug, also known to inhibit autophagy by impairing the autophagic protein degradation. On the other hand, 5-Fluorouracil (5-FU) is the most common chemotherapeutic agent for colorectal cancer, which inhibits DNA synthesis and induces apoptosis and/or cell cycle arrest in colon cancer cells. In the present study, we aimed to investigate the induction of autophagy as a mechanism of resistance of colon cancer cells to 5-FU-based chemotherapy, and the role of autophagy inhibition, by CQ, in potentiating the effect of 5-FU, especially focusing on cell cycle arrest. Procedure The human colon cancer cell line HT-29 was used. Cells were treated with CQ and/or 5-FU at various concentrations, and the changes in the proliferative activity were investigated, especially focusing on the induction of autophagy, the changes of cell cycle, and the development of apoptosis. The proliferative ability of HT-29 was analyzed by the MTS assay. The cell cycle was evaluated by flow-cytometry after staining of cells with PI, and autophagy was quantified by the analysis of the formation of acidic vesicular organelles (AVOs) by flow-cytometry after staining of cells with acridine orange (AO), and additionally, by the analysis of the expression of LC3-II by Western blot. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. Finally, to evaluate the long-term viability of the cells, the colony formation assay was performed. Results Treatment of HT-29 with 5-FU resulted in significant inhibition of the proliferative activity, compared with untreated cells. Treatment of the cells with CQ prior to exposure to 5-FU, potentiated the inhibitory effect. And it was dependent on the increase of the percentage of cells in G0/G1-phase, and the simultaneous decrease of cells in G2/M-phase. Also a partial increase of apoptotic cells was observed. These effects were dependent on the decreased expression of cyclin-dependent kinase-2 and the increased expression of p21Cip1, p27Kip1, important regulators of the cell cycle progression from G1 to S phase. Therefore, pre-treatment of HT-29 with the autophagy inhibitor, CQ, resulted in potentiation of the G0/G1 phase cell cycle arrest induced by 5-FU. Conclusion: The combination therapy using inhibitor of autophagy, such as CQ, should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 85.

Review: Autophagy in neurodegeneration: firefighter and/or incendiarist?

Autophagy is an intracellular bulk degradation system that is found ubiquitously in eukaryotes. Autophagy is responsible for the degradation of most long-lived proteins and some organelles. Cytoplasmic constituents, including organelles, are sequestered into double-membrane autophagosomes, which subsequently fuse with lysosomes where their contents are degraded. This system has been implicated in various physiological processes including protein and organelle turnover, stress response, cellular differentiation, programmed cell death and pathological conditions. Defects in the autophagy machinery might have several consequences, as they have been associated with neurodegenerative disease and different forms of cancer. Thus, autophagy occupies a crucial position within the cell's metabolism, and its modulation may represent an alternative therapeutic strategy in several pathological settings including stroke, Alzheimer's, Huntington's, Parkinson's diseases and cancer. Recently, research has begun to identify some characteristics of neuronal autophagy. The results suggest that autophagy may provide a neuroprotective mechanism. However, there is evidence showing that dysfunction of autophagy in certain pathological situations can trigger and mediate programmed cell death. Autophagy has also been defined as prime suspect cause of non-apoptotic cellular demise. However, there is now mounting evidence that autophagy and apoptosis share several common regulatory elements that are crucial in any attempt to understand the dual role of autophagy in cell death and cell survival. It will be of fundamental importance to dissect whether autophagy is primarily a strategy for survival or whether autophagy can also be a part of a cell death programme and thus contribute to cell death. Many questions are open. Is autophagy a direct death execution pathway? Is autophagy an innocent bystander? Is autophagy a defence mechanism or just a scavenger or self-clearance tool in the cell? A profound understanding of the biological effects and the mechanisms underlying autophagy in neurones might be helpful in seeking effective new treatments for neurodegenerative diseases. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurone.

Quantitative Metabolome Profiling of Colon and Stomach Cancer Microenvironment by Capillary Electrophoresis Time-of-Flight Mass Spectrometry

Most cancer cells predominantly produce energy by glycolysis rather than oxidative phosphorylation via the tricarboxylic acid (TCA) cycle, even in the presence of an adequate oxygen supply (Warburg effect). However, little has been reported regarding the direct measurements of global metabolites in clinical tumor tissues. Here, we applied capillary electrophoresis time-of-flight mass spectrometry, which enables comprehensive and quantitative analysis of charged metabolites, to simultaneously measure their levels in tumor and grossly normal tissues obtained from 16 colon and 12 stomach cancer patients. Quantification of 94 metabolites in colon and 95 metabolites in stomach involved in glycolysis, the pentose phosphate pathway, the TCA and urea cycles, and amino acid and nucleotide metabolisms resulted in the identification of several cancer-specific metabolic traits. Extremely low glucose and high lactate and glycolytic intermediate concentrations were found in both colon and stomach tumor tissues, which indicated enhanced glycolysis and thus confirmed the Warburg effect. Significant accumulation of all amino acids except glutamine in the tumors implied autophagic degradation of proteins and active glutamine breakdown for energy production, i.e., glutaminolysis. In addition, significant organ-specific differences were found in the levels of TCA cycle intermediates, which reflected the dependency of each tissue on aerobic respiration according to oxygen availability. The results uncovered unexpectedly poor nutritional conditions in the actual tumor microenvironment and showed that capillary electrophoresis coupled to mass spectrometry-based metabolomics, which is capable of quantifying the levels of energy metabolites in tissues, could be a powerful tool for the development of novel anticancer agents that target cancer-specific metabolism.

The α‐glucosidase inhibitor acarbose prevents obesity and simple steatosis in sequestosome 1/A170/p62 deficient mice

Sequestosome 1 (SQSTM1)/A170/p62 plays an important role in membrane-receptor mediated signal transduction and autophagic protein degradation. Although the mechanism involved is not clear, sqstm1 gene knockout (KO) mice develop mature-onset obesity and insulin resistance, leading to type II diabetes. KO mice show accumulation of fat in white adipose tissue and the liver when fed a standard diet. Acarbose is an alpha-glucosidase inhibitor that improves insulin sensitivity and decreases postprandial hyperglycemia, and it is used to treat type 2 diabetes. We examined whether or not dietary acarbose prevented obesity and simple steatosis in KO mice. Wild-type (WT) and KO mice were fed a standard diet with or without acarbose (0.8% w/w) from 15-25 weeks of age. The body weight and the fat content of adipose tissue and the liver were measured, and changes of lipid metabolism in these tissues were assessed from gene expression. Acarbose treatment suppressed weight gain and the development of hepatic steatosis in KO mice. Acarbose treatment up-regulated hepatic expression of the pparalpha, ucp-2, and abca1 genes, as well as srebp1c, pparalpha, and ppargamma in adipose tissue. In WT mice, however, acarbose treatment had little influence on weight gain and gene expression. The results of this study suggest that long-term administration of acarbose is effective for prevention of obesity and simple steatosis in SQSTM1-KO mice.

Cytoplasmic retention of polyglutamine-expanded androgen receptor ameliorates disease via autophagy in a mouse model of spinal and bulbar muscular atrophy.

The nucleus is the primary site of protein aggregation in many polyglutamine diseases, suggesting a central role in pathogenesis. In SBMA, the nucleus is further implicated by the critical role for disease of androgens, which promote the nuclear translocation of the mutant androgen receptor (AR). To clarify the importance of the nucleus in SBMA, we genetically manipulated the nuclear localization signal of the polyglutamine-expanded AR. Transgenic mice expressing this mutant AR displayed inefficient nuclear translocation and substantially improved motor function compared with SBMA mice. While we found that nuclear localization of polyglutamine-expanded AR is required for SBMA, we also discovered, using cell models of SBMA, that it is insufficient for both aggregation and toxicity and requires androgens for these disease features. Through our studies of cultured motor neurons, we further found that the autophagic pathway was able to degrade cytoplasmically retained expanded AR and represents an endogenous neuroprotective mechanism. Moreover, pharmacologic induction of autophagy rescued motor neurons from the toxic effects of even nuclear-residing mutant AR, suggesting a therapeutic role for autophagy in this nucleus-centric disease. Thus, our studies firmly establish that polyglutamine-expanded AR must reside within nuclei in the presence of its ligand to cause SBMA. They also highlight a mechanistic basis for the requirement for nuclear localization in SBMA neurotoxicity, namely the lack of mutant AR removal by the autophagic protein degradation pathway.

GFP‐LC3 labels organised smooth endoplasmic reticulum membranes independently of autophagy

Disruption of autophagy leads to accumulation of intracellular multilamellar inclusions morphologically similar to organised smooth endoplasmic reticulum (OSER) membranes. However, the relation of these membranous compartments to autophagy is unknown. The purpose of this study was to test whether OSER plays a role in the autophagic protein degradation pathway. Here, GFP-LC3 is shown to localise to the OSER membranes induced by calnexin expression both in transiently transfected HEK293 cells and in mouse embryo fibroblasts. In contrast to GFP-LC3, endogenous LC3 is excluded from these membranes under normal conditions as well as after cell starvation. Furthermore, YFP-Atg5, a protein essential for autophagy and known to reside on autophagic membranes, is excluded from the calnexin-positive inclusion structures. In cells devoid of Atg5, a protein essential for autophagy and known to reside on autophagic membranes, colocalisation of calnexin with GFP-LC3 within the multilamellar bodies is preserved. I show that calnexin, a protein enriched in the OSER, is not subject to autophagic or lysosomal degradation. Finally, GFP-LC3 targeting to these membranes is independent of its processing and insensitive to drugs modulating autophagic and lysosomal protein degradation. These observations are inconsistent with a role of autophagic/lysosomal degradation in clearance of multilamellar bodies comprising OSER. Furthermore, GFP-LC3, a fusion protein widely used as a marker for autophagic vesicles and pre-autophagic compartments, may be trapped in this compartment and this artefact must be taken into account if the construct is used to visualise autophagic membranes. J. Cell. Biochem. 107: 86–95, 2009. © 2009 Wiley-Liss, Inc.

Characterization of CAA0225, a Novel Inhibitor Specific for Cathepsin L, as a Probe for Autophagic Proteolysis

We screened a series of new epoxysuccinyl peptides for the development of a lysosomal cathepsin L-specific inhibitor. Among the compounds tested, (2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide} (compound CAA0225) was the most potent inhibitor of cathepsin L. CAA0225 inhibited rat liver cathepsin L with IC50 values of 1.9 nM, but not rat liver cathepsin B (IC50, >1000-5000 nM). To assess the contribution of cathepsin L to lysosomal proteolysis, we evaluated autophagy, which is the process of lysosomal self-degradation of cell constituents. In HeLa and Huh-7 cells cultured under nutrient-deprived conditions CAA0225 significantly inhibited degradation of long-lived proteins; however, the magnitude of inhibition was comparable to that in the presence of CA-074-OMe, which is a cathepsin B-specific inhibitor. Thus the contributions of cathepsin L and cathepsin B to autophagic protein degradation of cytoplasmic proteins are nearly equal. During autophagy, microtubule-associated protein IA/IB light chain 3-II (LC3-II) and gamma-aminobutyric acid (A) receptor-associated protein (GABARAP)-II, which are specific markers of autophagosomal membranes that engulf cytoplasmic components, also undergo degradation upon fusion of autophagosomes with lysosomes. CAA0225 effectively inhibited degradation of LC3-II and GABARAP, whereas CA-074-OMe had only a marginal effect on their levels. Therefore we conclude that cathepsin L does not play a general role in the degradation of proteins in the lumen of autophagosomes, but rather is involved specifically in the degradation of autophagosomal membrane markers.

Ubiquitin signals autophagic degradation of cytosolic proteins and peroxisomes

Autophagy is responsible for nonspecific, bulk degradation of cytoplasmic components. Recent work has revealed also that there is specific, autophagic degradation of polyubiquitinated protein aggregates, whose buildup occurs during neurodegenerative disease. Here, we report that simple mono-ubiquitination of normally long-lived cytoplasmic substrates is sufficient to target these substrates for autophagic degradation in mammalian cells. That is, upon their ubiquitination, both small [i.e., red fluorescent protein (RFP)] and large (i.e., peroxisomes) substrates are efficiently targeted to autophagosomes and then degraded within lysosomes upon autophagosome-lysosome fusion. This targeting requires the ubiquitin-binding protein, p62, and is blocked by the Class III phosphatidylinositol 3-kinase (PI3K) inhibitor, 3-methyladenine (3-MA), or by depletion of the autophagy-related-12 (Atg12) protein homolog. Mammalian cells thus use a common pathway involving ubiquitin and p62 for targeting diverse types of substrates for autophagy.

Induction of incomplete autophagic response by hepatitis C virus via the unfolded protein response†

Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis.

Can Lipofuscin Accumulation Be Prevented?

During normal autophagic degradation of mitochondria and iron-containing proteins in lysosomes, iron is released intralysosomally where it may react with hydrogen peroxide forming hydroxyl radicals (Fenton reaction). Depending on their rate of formation, these highly reactive radicals can cross-link intralysosomal material, leading to lipofuscin formation, or destabilize the lysosomal membrane, which induces apoptosis/necrosis. Since the sensitivity of lysosomes to oxidative stress can be manipulated by altering the intralysosomal level of redox-active iron, it follows that lipofuscin formation might also be influenced. It is suggested that pulse doses of iron chelators that easily penetrate membranes could be used to diminish lipofuscinogenesis.

Role of Hrs in maturation of autophagosomes in mammalian cells

Autophagy is an evolutionarily conserved system responsible for the degradation of cellular components and contributes to the increasing of amino acid pool, organelle turnover, and elimination of intracellular bacteria. The molecular process of autophagy is still unclear. Here we demonstrate that Hrs, a master regulator in endosomal protein sorting, plays critical roles for the autophagic degradation of non-specific proteins and Streptococcus pyogenes. We found that Hrs containing FYVE domain is localized to autophagosomes. Hrs depletion resulted in a significant decrease in the number of mature autophagosomes (autophagolysosomes) detected by the co-localization of autophagosome marker LC3 and lysosome marker LAMP-1. In contrast, formation of the primary autophagosome, detected by LC3 immunoblotting and lysosomal degradation of non-specific proteins, were not significantly altered by Hrs depletion. Based on these results, we propose a novel function of Hrs, as a crucial player in the maturation of autophagosomes.

Low postabsorptive net protein degradation in male cancer patients: Lack of sensitivity to regulatory amino acids?

Autophagic (lysosomal) and proteasomic protein degradation are important regulatory mechanisms in the homeostasis of muscle mass, that may be profoundly disturbed in cancer and other wasting syndromes. Due to the inhibiting effect of amino acids and insulin, net proteolysis is restricted to the fasted state, and in autophagy certain amino acids have been identified as 'regulatory' in the rat, including leucine, tyrosine, phenylalanine, methionine, and histidine (i.e. LYFMH). The present cross-sectional study assessed postabsorptive net protein catabolism in male cancer patients as well as in healthy male volunteers, to analyse its relation to such 'regulatory amino acids'. Postabsorptive amino acid exchange rates across the leg were determined in patients with gastrointestinal cancer (GIC, n=47) or renal cell carcinoma (RCC, n=15), age-matched (n=33), and young male control subjects (n=42). Both groups of cancer patients revealed a significantly lower postabsorptive net protein catabolism than control subjects. Furthermore, in the control subjects, the postabsorptive net protein catabolism was found to be inversely and significantly correlated with the arterial concentrations of the 8 amino acids YSHMFGI and L which include 5 of the 'regulatory amino acids'. Cancer patients, in contrast, revealed no such significant correlations. These results may indicate i) that postabsorptive net protein catabolism in skeletal muscle of healthy subjects may be sensitive to amino acids which reportedly regulate autophagy and ii) that such amino acid-sensitive mechanism of protein catabolism may be disturbed in cancer patients.

A Sorting Nexin PpAtg24 Regulates Vacuolar Membrane Dynamics during Pexophagy via Binding to Phosphatidylinositol-3-Phosphate

Diverse cellular processes such as autophagic protein degradation require phosphoinositide signaling in eukaryotic cells. In the methylotrophic yeast Pichia pastoris, peroxisomes can be selectively degraded via two types of pexophagic pathways, macropexophagy and micropexophagy. Both involve membrane fusion events at the vacuolar surface that are characterized by internalization of the boundary domain of the fusion complex, indicating that fusion occurs at the vertex. Here, we show that PpAtg24, a molecule with a phosphatidylinositol 3-phosphate-binding module (PX domain) that is indispensable for pexophagy, functions in membrane fusion at the vacuolar surface. CFP-tagged PpAtg24 localized to the vertex and boundary region of the pexophagosome-vacuole fusion complex during macropexophagy. Depletion of PpAtg24 resulted in the blockage of macropexophagy after pexophagosome formation and before the fusion stage. These and other results suggest that PpAtg24 is involved in the spatiotemporal regulation of membrane fusion at the vacuolar surface during pexophagy via binding to phosphatidylinositol 3-phosphate, rather than the previously suggested function in formation of the pexophagosome.

Mechanisms responsible for regulation of branched-chain amino acid catabolism

The branched-chain amino acids (BCAAs) are essential amino acids and therefore must be continuously available for protein synthesis. However, BCAAs are toxic at high concentrations as evidenced by maple syrup urine disease (MSUD), which explains why animals have such an efficient oxidative mechanism for their disposal. Nevertheless, it is clear that leucine is special among the BCAAs. Leucine promotes global protein synthesis by signaling an increase in translation, promotes insulin release, and inhibits autophagic protein degradation. However, leucine’s effects are self-limiting because leucine promotes its own disposal by an oxidative pathway, thereby terminating its positive effects on body protein accretion. A strong case can therefore be made that the proper leucine concentration in the various compartments of the body is critically important for maintaining body protein levels beyond simply the need of this essential amino acid for protein synthesis. The goal of the work of this laboratory is to establish the importance of regulation of the branched chain α-ketoacid dehydrogenase complex (BCKDC) to growth and maintenance of body protein. We hypothesize that proper regulation of the activity state of BCKDC by way of its kinase (BDK) and its phosphatase (BDP) is critically important for body growth, tissue repair, and maintenance of body protein. We believe that growth and protection of body protein during illness and stress will be improved by therapeutic control of BCKDC activity. We also believe that it is possible that the negative effects of some drugs (PPARα ligands) and dietary supplements (medium chain fatty acids) on growth and body protein maintenance can be countered by therapeutic control of BCDKC activity.

Amino Acids as Regulators and Components of Nonproteinogenic Pathways

Amino acids are not only important precursors for the synthesis of proteins and other N-containing compounds, but also participate in the regulation of major metabolic pathways. Glutamate and aspartate, for example, are components of the malate/aspartate shuttle and their concentrations control the rate of mitochondrial oxidation of glycolytic NADH. Glutamate also controls the rate of urea synthesis, not only as the precursor of ammonia and aspartate, but as substrate for synthesis of N-acetylglutamate, the essential activator of carbamoyl-phosphate synthase. This mechanism allows large variations in urea synthesis at relatively constant ammonia concentrations. Increases in intracellular amino acid concentration increase cell volume. Cell swelling per se has anabolic effects on protein, carbohydrate and lipid metabolism: enhanced synthesis of macromolecules compensates for increases in intracellular osmolarity. Mechanisms responsible for cell swelling-induced changes in pathway fluxes include changes in intracellular ion concentrations and in signal transduction. Specific amino acids (e.g., leucine) stimulate protein synthesis and inhibit (autophagic) protein degradation independent of changes in cell volume because they stimulate mTOR (mammalian target of rapamycin), a protein kinase, which is one of the components of a signal transduction pathway used by insulin. When the cellular energy state is low, stimulation of mTOR by amino acids is prevented by activation of AMP-dependent protein kinase. Amino acid-dependent signaling also promotes insulin production by beta-cells. This further adds to the anabolic properties of amino acids. It is concluded that amino acids are important regulators of major metabolic pathways.

Autophagy in neurons: a review.

Macroautophagy is a process of regulated turnover of cellular constituents that occurs during development and under conditions of stress such as starvation. Defects in autophagy have serious consequences, as they have been linked to neurodegenerative disease, cancer, and cardiomyopathy. This process, which exists in all eukaryotic cells, is tightly controlled, but in extreme cases results in the death of the cell. While major insights into the molecular and biochemical pathways involved have come from genetic studies in yeast, little is known about autophagic pathways in mammalian cells, particularly in neurons. Recently, research in neuronal culture models has begun to identify some characteristics of neuronal macroautophagy. The results suggest that macroautophagy in neurons may provide a neuroprotective mechanism. Here, we review the defining characteristics of autophagy with special attention to its role in neurodegenerative disorders, and recent efforts to delineate the pathway of autophagic protein degradation in neurons.

Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficient mice.

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danon's disease is associated with the accumulation of autophagic material in striated myocytes.

Inhibition of hepatocytic autophagy by adenosine, adenosine analogs and AMP.

Autophagy, measured in isolated rat hepatocytes as the sequestration of electroinjected [3H]raffinose, was moderately (17%) inhibited by adenosine (0.4 mM) alone, but more strongly (85%) in the presence of the adenosine deaminase inhibitor, 2'-deoxycoformycin (50 microM), suggesting that metabolic deamination of adenosine limited its inhibitory effectiveness. The adenosine analogs, 6-methylmercaptopurine riboside and N6,N6-dimethyladenosine, inhibited autophagy by 89% and 99%, respectively, at 0.5 mM, probably reflecting the adenosine deaminase-resistance of their 6-substitutions. 5-Iodotubercidin (10 microM), an adenosine kinase inhibitor, blocked the conversion of adenosine to AMP and largely abolished the inhibitory effects of both adenosine and its analogs, indicating that AMP/nucleotide formation was required for inhibition of autophagy. Inhibition by adenosine of autophagic protein degradation, measured as the release of [14C]valine from prelabelled protein, was similarly potentiated by deoxycoformycin and prevented by iodotubercidin. Inhibition of autophagy by added AMP, ADP or ATP (0.3-1 mM) was, likewise, potentiated by deoxycoformycin and prevented by iodotubercidin, suggesting dephosphorylation to adenosine and intracellular re-phosphorylation to AMP. Suppression of autophagy by AMP may be regarded as a feedback inhibition of autophagic RNA degradation, or as an aspect of the general down-regulation of energy-requiring processes that occurs under conditions of ATP depletion, when AMP levels are high.