Times Of Starvation Research Articles

RNA-seq analysis of small intestine transcriptional changes induced by starvation stress in piglets

Piglets may experience a variety of stress injuries, but the molecular regulatory mechanisms underlying these injuries are not well understood. In this study, we analysed the ileum of Large White (LW) and Mashen (MS) piglets at different times of starvation using chemical staining and transcriptome analysis. The intestinal barrier of piglets was damaged after starvation stress, but the intestinal antistress ability of MS piglets was stronger than LW piglets. A total of 8021 differentially expressed genes (DEGs) were identified in two breeds. Interestingly, the immune capacity (CHUK, TLR3) of MS piglets increased significantly after short-term starvation stress, while energy metabolism (NAGS, PLA2G12B, AGCG8) was predominant in LW piglets. After long-term starvation stress, the level of energy metabolism (PLIN5, PLA2G12B) was significantly increased in MS piglets. The expression of immune (HLA-DQB1, IGHG4, COL3A1, CD28, LAT) and disease (HSPA1B, MINPPI, ADH1C, GAL3ST1) related genes were significantly increased in two breeds of piglets. These results suggest that short-term stress mainly enhances immunity and energy metabolism in piglets, while long-term starvation produces greater stress on piglets, making it difficult for them to compensate for the damage to their bodies through self-regulation. This information can help improve the stress resistance of piglets through molecular breeding.

Glycolytically impaired Drosophila glial cells fuel neural metabolism via β-oxidation

Neuronal function is highly energy demanding and thus requires efficient and constant metabolite delivery by glia. Drosophila glia are highly glycolytic and provide lactate to fuel neuronal metabolism. Flies are able to survive for several weeks in the absence of glial glycolysis. Here, we study how Drosophila glial cells maintain sufficient nutrient supply to neurons under conditions of impaired glycolysis. We show that glycolytically impaired glia rely on mitochondrial fatty acid breakdown and ketone body production to nourish neurons, suggesting that ketone bodies serve as an alternate neuronal fuel to prevent neurodegeneration. We show that in times of long-term starvation, glial degradation of absorbed fatty acids is essential to ensure survival of the fly. Further, we show that Drosophila glial cells act as a metabolic sensor and can induce mobilization of peripheral lipid stores to preserve brain metabolic homeostasis. Our study gives evidence of the importance of glial fatty acid degradation for brain function, and survival, under adverse conditions in Drosophila.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Medieval surgery (eleventh-thirteenth century): barber surgeons and warfare surgeons in France.

From the eleventh to the thirteenth century, three major events marked surgery in France: the appearance of an epidemic linked to ergotism which led to numerous amputations, the emergence of barber surgeons for civilian practice, and the organization of war surgery for the First Crusade. If a certain separation between medicine and surgery had appeared at the beginning of the Middle Ages, it would have been from 1215 (at the time of the Lateran Council) that the real separation between medicine and surgery was made. Before this date, the surgery was done by the clergy (monks) who had some experience of surgical practice. After the Council de Lateran, the barbers will take charge of the surgery. In the Middle Ages, the first cause of amputation was linked to poisoning by ergot of rye, intoxication due to the ingestion of contaminated bread. Due to ergotamine-related vasoconstriction, many patients will die with a frequency ranging from 10 to 20% in times of starvation. For survivors, gangrenes will lead to amputations made at the beginning by monks, then by the barber surgeons after 1215. With the name of barber surgeon, barbers are initially responsible for small surgical procedures, then for amputations given the epidemics linked to ergotism which led to numerous amputations. By this practice, they will acquire the knowledge of anatomy and the knowledge of the surgery. Apart from this civilian practice of surgery carried out by barber surgeons, the military practice of surgery will appear with the conquests of England and the start of the crusades which will require a more professional organization of surgery. After 1371, as only barbers surgeons had some surgical practice, their knowledge surpassed knowledge of the university. This supremacy of the barber surgeon over the surgeons of the university will be demonstrated by Ambroise Paré.

Interfering with Autophagy: The Opposing Strategies Deployed by Legionella pneumophila and Coxiella burnetii Effector Proteins.

Autophagy is a fundamental and highly conserved eukaryotic process, responsible for maintaining cellular homeostasis and releasing nutrients during times of starvation. An increasingly important function of autophagy is its role in the cell autonomous immune response; a process known as xenophagy. Intracellular pathogens are engulfed by autophagosomes and targeted to lysosomes to eliminate the threat to the host cell. To counteract this, many intracellular bacterial pathogens have developed unique approaches to overcome, evade, or co-opt host autophagy to facilitate a successful infection. The intracellular bacteria Legionella pneumophila and Coxiella burnetii are able to avoid destruction by the cell, causing Legionnaires’ disease and Q fever, respectively. Despite being related and employing homologous Dot/Icm type 4 secretion systems (T4SS) to translocate effector proteins into the host cell, these pathogens have developed their own unique intracellular niches. L. pneumophila evades the host endocytic pathway and instead forms an ER-derived vacuole, while C. burnetii requires delivery to mature, acidified endosomes which it remodels into a large, replicative vacuole. Throughout infection, L. pneumophila effectors act at multiple points to inhibit recognition by xenophagy receptors and disrupt host autophagy, ensuring it avoids fusion with destructive lysosomes. In contrast, C. burnetii employs its effector cohort to control autophagy, hypothesized to facilitate the delivery of nutrients and membrane to support the growing vacuole and replicating bacteria. In this review we explore the effector proteins that these two organisms utilize to modulate the host autophagy pathway in order to survive and replicate. By better understanding how these pathogens manipulate this highly conserved pathway, we can not only develop better treatments for these important human diseases, but also better understand and control autophagy in the context of human health and disease.

Abstract MP39: Autophagy Regulates β-hydroxybutyrate Synthesis To Prevent High-salt Induced Vascular Damage

Overconsumption of dietary salt is unequivocally linked with end organ damage. We have recently observed that a high salt (HS) diet induces liver dysfunction in the form of impaired synthesis of ketone body, β-hydroxybutyrate (βOHB), in Dahl salt-sensitive rats. However, the molecular mechanisms as to why βOHB is decreased after consuming a HS diet remains unknown, as well as if it contributes to vascular dysfunction. We hypothesized that a HS diet (2%) for 8 weeks would decrease systemic levels of βOHB in Dahl salt-resistant (R) rats and this would be associated with endothelial dysfunction. Furthermore, reconstitution of βOHB bioavailability by giving 1,3-Butanediol (1,3-BD; 20% v/v ) in the drinking water would improve endothelial function in HS fed Dahl R. As hypothesized, a HS diet decreased serum concentration (mM) of βOHB [Low salt (LS), non-fasting (NF): 0.31±0.02 vs. fasting (F): 0.96±0.04*; HS, NF: 0.33±0.02 vs. F: 0.77±0.04*#, *p<0.05 vs. NF, #p<0.05 vs. LS-F]. A HS diet decreased the sensitivity of isolated mesenteric resistance arteries (MRA) to acetylcholine (ACh), and 1,3-BD treatment prevented this endothelial dysfunction (LogEC50, Dahl R-LS: -7.35±0.05 vs. Dahl R-HS: -7.19±0.07* vs. Dahl R-HS+1,3-BD: -7.42±0.10, p<0.05). Furthermore, when MRA were incubated with indomethacin, only Dahl R-HS presented with a decreased sensitivity to ACh (LogEC50, HS: -7.19±0.07 vs. HS+indomethacin: -7.66±0.08*, p<0.05), indicating that 1,3-BD treatment decreased cyclooxygenase-induced vascular inflammation (LogEC50, 1,3-BD -7.42±0.10 vs. 1,3-BD +indomethacin: -7.61±0.09, p>0.05). Evolutionarily, autophagy functions to mobilize nutrients in times of starvation, including ketone bodies such as βOHB. Therefore, we hypothesized that inhibition of autophagy would decrease βOHB. Treatment with lysosomal alkalizer chloroquine (CQ), impeded the starvation-induced increase in βOHB (NF: 0.38±0.02 vs. F: 1.11±0.09* vs. F+CQ: 0.78±0.19*#, *p<0.05 vs NF, # p<0.05 vs F). In summary, these data reveal that a HS diet decreases liver-derived βOHB, independent of high blood pressure, and is protective against HS-induced vascular damage. We suggest that autophagy could be a novel mechanism underlying the vascular protective effects of βOHB.

Ketones can become the major fuel source for the heart but do not increase cardiac efficiency.

Ketones have been proposed to be a 'thrifty' fuel for the heart and increasing cardiac ketone oxidation can be cardioprotective. However, it is unclear how much ketone oxidation can contribute to energy production in the heart, nor whether increasing ketone oxidation increases cardiac efficiency. Therefore, our goal was to determine to what extent high levels of the ketone body, β-hydroxybutyrate (βOHB), contributes to cardiac energy production, and whether this influences cardiac efficiency. Isolated working mice hearts were aerobically perfused with palmitate (0.8 mM or 1.2 mM), glucose (5 mM) and increasing concentrations of βOHB (0, 0.6, 2.0 mM). Subsequently, oxidation of these substrates, cardiac function, and cardiac efficiency were assessed. Increasing βOHB concentrations increased myocardial ketone oxidation rates without affecting glucose or fatty acid oxidation rates where normal physiological levels of glucose (5 mM) and fatty acid (0.8 mM) are present. Notably, ketones became the major fuel source for the heart at 2.0 mM βOHB (at both low or high fatty acid concentrations), with the elevated ketone oxidation rates markedly increasing tricarboxylic acid (TCA) cycle activity, producing a large amount of reducing equivalents and finally, increasing myocardial oxygen consumption. However, the marked increase in ketone oxidation at high concentrations of βOHB was not accompanied by an increase in cardiac work, suggesting that a mismatch between excess reduced equivalents production from ketone oxidation and cardiac adenosine triphosphate production. Consequently, cardiac efficiency decreased when the heart was exposed to higher ketone levels. We demonstrate that while ketones can become the major fuel source for the heart, they do not increase cardiac efficiency, which also underscores the importance of recognizing ketones as a major fuel source for the heart in times of starvation, consumption of a ketogenic diet or poorly controlled diabetes.

β‐Hydroxybutyrate (βOHB) Activates Gpr109a to Contribute to the Anti‐vascular Aging Effect of Autophagy

The aged phenotype is classically viewed as the accumulation of cellular debris and dysfunctional organelles. Although waste products are an inevitable consequence of normal cellular metabolism, multiple systems are devoted to its repair, clearance, or recycling. Autophagy is the constitutively active catabolic process responsible for cellular degradation. Autophagic activity has also been implicated as a modulator of longevity, as well as inversely related with chronological vascular aging and premature vascular aging associated with hypertension. However, the mechanisms by which autophagy exerts anti‐vascular aging effects are still unknown. Evolutionarily, autophagy functions to mobilize nutrients in times of starvation. Specifically, hepatic autophagy releases free fatty acids from triglycerides, which are oxidized to generate ketone bodies. Therefore, we hypothesized that fasting would increase the systemic expression of ketone body β‐hydroxybutyrate (βOHB) in Dahl salt‐sensitive rats fed a low salt‐diet, and this would be prevented by inhibition of autophagy with chloroquine (CQ). As hypothesized, fasting increased serum βOHB, and treatment with CQ blocked this increase (mmol/L, non‐fasting: 0.38±0.01 vs. fasting: 1.01±0.06* vs. fasting+CQ: 0.45±0.04, *p<0.05) (Figure 1). Next we questioned whether βOHB could potentially contribute to the anti‐vascular aging effect of autophagy. Previously, we have observed that βOHB is a potent vasodilator via endothelial potassium channels. However, it is not known if βOHB acts as an agonist or antagonist to any specific receptor to mediate endothelium‐dependent relaxation. To help answer this question we needed to switch to a genetic knockout model, as pharmacological antagonists for the βOHB receptors, Gpr109a and Gpr41, do not exist. βOHB activates Gpr109a, whereas it is generally recognized as an antagonist of Gpr41. Therefore, we hypothesized that vascular relaxation to βOHB would be significantly attenuated in isolated mesenteric resistance arteries from Gpr109a−/− mice, but not Gpr41−/− mice. As hypothesized, vasodilatory effect of βOHB was significantly blunted in Gpr109a−/− mice, but not Gpr41−/− mice [Emax (%relaxation), WT: 75±2 vs. Gpr109a−/−: 53±3* vs. Gpr41−/−: 73±1, *p<0.05] (Figure 2). Overall, these data reveal that ketone body βOHB is stimulated by autophagy and that activation of Gpr109a by βOHB can cause vasodilation of isolated resistance arteries. Significantly, our data define a novel mechanism that underlies the anti‐vascular aging effect of autophagy.Support or Funding InformationAmerican Heart Association (18POST34060003) and National Institutes of Health (R00GM118885 and R01HL143082).Fasting (24 h) stimulates serum βOHB expression and this is prevented by autophagy inhibition (chloroquine 50 mg/kg i.p.; CQ). n=6–10. One way ANOVA: *p<0.05.Figure 1βOHB mediates vasodilation partially via Gpr109a, and not Gpr41. Concentration response curves to βOHB. n=2–4. Two‐way ANOVA: *p<0.05. vs. wild type (WT).Figure 2

Phosphoinositides in autophagy: current roles and future insights.

Today, the importance of autophagy in physiological processes and pathological conditions is undeniable. Initially, autophagy merely was described as an evolutionarily conserved mechanism to maintain metabolic homeostasis in times of starvation; however, in recent years it is now apparent that autophagy is a powerful regulator of many facets of cellular metabolism, that its deregulation contributes to various human pathologies, including cancer and neurodegeneration, and that its modulation has considerable potential as a therapeutic approach. Different lipid species, including sphingolipids, sterols, and phospholipids, play important roles in the various steps of autophagy. In particular, there is accumulating evidence indicating the minor group of phospholipids called the phosphoinositides as key modulators of autophagy, including the signaling processes underlying autophagy initiation, autophagosome biogenesis and maturation. In this review, we discuss the known functions to date of the phosphoinositides in autophagy and attempt to summarize the kinases and phosphatases that regulate them as well as the proteins that bind to them throughout the autophagy program. We will also provide examples of how the control of phosphoinositides and their metabolizing enzymes is relevant to understanding many human diseases.

Abstract 066: Beta-Hydroxybutyrate Synthesis Contributes to the Anti-Vascular Aging Effect of Autophagy

Diminished autophagy leads to the accumulation of debris, dysfunctional organelles, and consequently, cellular aging. Premature vascular aging occurs in hypertension. In fact, many factors that contribute to the deterioration of vascular function as we age are accelerated in hypertension. Previously, we have reported decreased autophagic activity in arteries from hypertensive rats and reconstituting autophagy ameliorated several vascular aging phenotypes. However, the mechanisms by which autophagy exerts anti-vascular aging effects are still unknown. Evolutionarily, autophagy functions to mobilize micronutrients in times of starvation. As a result, autophagy has also been recognized as a mediator of fatty acid oxidation and ketogenesis, predominantly from the liver. Therefore, we hypothesized that autophagy induction by fasting would increase the expression of ketone body beta-hydroxybutyrate (βOHB) in the livers of Wistar-kyoto rats and this would be prevented by systemic inhibition autophagy (3-methyadenine). As hypothesized, fasting increased βOHB, and this was prevented by 3-methyadenine (mmol/L, non-fasting: 0.43±0.04 vs. fasting: 0.58±0.04 vs. fasting+3-methyadenine: 0.35±0.01, p<0.05). Next we questioned whether βOHB could contribute to the anti-vascular aging effect of autophagy and hypothesized that βOHB administered directly to isolated arteries would cause endothelium-dependent relaxation. To test this hypothesis, we performed concentration-response curves to βOHB in mesenteric resistance arteries from Dahl salt-sensitive rats. We observed that βOHB was a potent endothelium-dependent vasodilator (%Relaxation, βOHB+endothelium-intact: 53±6 vs. βOHB+endothelium-denuded: 24±5, p<0.05). Inhibition of endothelium-derived vasodilators revealed that this relaxation was mediated by potassium channels (%Relaxation, βOHB: 53±6 vs. βOHB+tetraethylammonium: 19±9, p<0.05), but not nitric oxide (%Relaxation, βOHB: 53±6 vs. βOHB+L-NAME: 44±15, p>0.05). Overall, these data reveal βOHB as a novel endothelium-dependent vasodilator synthesized by the liver due to induction of autophagy. We conclude that βOHB synthesis is a mechanism by which autophagy exerts anti-vascular aging effects in hypertension.

SAT-167 Intra-Islet Ghrelin Signaling Does Not Regulate Insulin Secretion from Adult Mice

Ghrelin is a 28-peptide hormone that was originally discovered as a ligand for the growth hormone secretagogue receptor (GHSR) and noted for its roles in food intake and feeding behavior. Beyond this, ghrelin may also regulate glucose tolerance and insulin secretion. In times of starvation, ghrelin defends against hypoglycemia. Contrastingly, ghrelin removal increases insulin secretion and improves glucose tolerance in metabolically-stressed mice. Though ghrelin is predominantly produced by stomach X/A cells, it is also made in the pancreas. Within the pancreatic islet, ghrelin is produced by ε-cells and GHSR is predominantly expressed in somatostatin-secreting δ-cells. This positioning suggests that, locally, islet ghrelin could reach interstitial levels that are higher than detected in circulation and act in a paracrine manner to inhibit insulin secretion. Additionally, GHSR has high constitutive activity and may act in the absence of ghrelin. We hypothesized that a ghrelin-GHSR paracrine axis within the islet modulates insulin secretion and tested this using parallel in vivo and ex vivo approaches in genetic rodent models lacking ghrelin (Ghrl-/-) or GHSR (GHSR-/-). Glycemic responses in GHSR-/- and GHSR+/+male mice did not differ after i.p. glucose tolerance tests (IPGTT) after a 6- or 16-hr fast, oral mixed meal tolerance test (MMTT), or insulin sensitivity during insulin tolerance test (ITT). Female GHSR-/- mice had impaired glucose tolerance after 6-hr fast IPGTT compared to GHSR+/+ (glucose AUC, p=0.01) but did not differ from controls during 16-hr fasted IPGTT, MMTT, or ITT. These data suggest constitutive GHSR activity does not mediate in vivo glucose tolerance. Ghrl-/- and Ghrl+/+ male mice did not differ in glycemia in response to IPGTTs, MMTT, or ITT. Female Ghrl-/- mice showed a slight improvement compared to Ghrl+/+ during MMTT (glucose AUC, p=0.04) but did not differ in other tests. Thus, endogenous ghrelin does not substantially alter glucose tolerance. Given that many factors can affect in vivo glucose tolerance, we perifused isolated islets. Here, exogenous ghrelin robustly decreased glucose-stimulated insulin secretion (GSIS, p<0.001 at 16.7 mM glucose) in a GHSR-dependent manner. Insulin secretion from Ghrl-/- or GHSR-/- islets did not differ from their wild type controls during a glucose ramp or in response to alanine or GLP-1 stimulation. Ex vivo, islet ghrelin and constitutive GHSR activity do not appear to mediate insulin secretion in the paradigms we tested. Collectively, our data provide strong evidence against a paracrine ghrelin-GHSR axis in mediating insulin secretion in lean, chow-fed adult mice. Islet ghrelin and GHSR may impact insulin secretion at earlier stages of development, during times of extreme nutritional stress (e.g., starvation or overnutrition), or in the transition from fasting to fed state.

Organism motility in an oxygenated shallow-marine environment 2.1 billion years ago

Evidence for macroscopic life in the Paleoproterozoic Era comes from 1.8 billion-year-old (Ga) compression fossils [Han TM, Runnegar B (1992) Science 257:232-235; Knoll et al. (2006) Philos Trans R Soc Lond B 361:1023-1038], Stirling biota [Bengtson S et al. (2007) Paleobiology 33:351-381], and large colonial organisms exhibiting signs of coordinated growth from the 2.1-Ga Francevillian series, Gabon. Here we report on pyritized string-shaped structures from the Francevillian Basin. Combined microscopic, microtomographic, geochemical, and sedimentologic analyses provide evidence for biogenicity, and syngenicity and suggest that the structures underwent fossilization during early diagenesis close to the sediment-water interface. The string-shaped structures are up to 6 mm across and extend up to 170 mm through the strata. Morphological and 3D tomographic reconstructions suggest that the producer may have been a multicellular or syncytial organism able to migrate laterally and vertically to reach food resources. A possible modern analog is the aggregation of amoeboid cells into a migratory slug phase in cellular slime molds at times of starvation. This unique ecologic window established in an oxygenated, shallow-marine environment represents an exceptional record of the biosphere following the crucial changes that occurred in the atmosphere and ocean in the aftermath of the great oxidation event (GOE).

Determining the Impact of Metabolic Nutrients on Autophagy.

Tumorigenesis relies on the ability of cancer cells to obtain necessary nutrients and fulfill increased energy demands associated with rapid proliferation. However, as a result of increased metabolite consumption and poor vascularization, most cancer cells must survive in a nutrient poor and high cellular stress microenvironment. Cancer cells undergo metabolic reprogramming to evade cell death and ensure proliferation; in particular, cancer cells utilize the catabolic process of autophagy. Autophagy creates an intracellular pool of metabolites by sequestering cytosolic macromolecules in double-membrane vesicles targeted for lysosomal degradation. During times of environmental stress and nutrient starvation, autophagy is upregulated through the dynamic interactions between two nutrient sensing proteins, AMP activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR), in cooperation with Unc-51 like autophagy activating kinase 1 (ULK1). In this way, a lack of metabolic nutrients plays a critical role in inducing autophagy, while the products of autophagy also serve as readily available fuel for the cell. In this chapter, we describe methods to visualize and quantify autophagy using a fluorescent sensor of autophagic membranes. Thus, the impact of specific nutrients on autophagy can be measured using live-cell fluorescent microscopy.

Estrogen and/or Estrogen Receptor α Inhibits BNIP3-Induced Apoptosis and Autophagy in H9c2 Cardiomyoblast Cells

The process of autophagy in heart cells maintains homeostasis during cellular stress such as hypoxia by removing aggregated proteins and damaged organelles and thereby protects the heart during the times of starvation and ischemia. However, autophagy can lead to substantial cell death under certain circumstances. BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a hypoxia-induced marker, has been shown to induce both autophagy and apoptosis. A BNIP3-docked organelle, e.g., mitochondria, also determines whether autophagy or apoptosis will take place. Estrogen (E2) and estrogen receptor (ER) alpha (ERα) have been shown to protect the heart against mitochondria-dependent apoptosis. The aim of the present study is to investigate the mechanisms by which ERα regulates BNIP3-induced apoptosis and autophagy, which is associated with hypoxic injury, in cardiomyoblast cells. An in vitro model to mimic hypoxic injury in the heart by engineering H9c2 cardiomyoblast cells to overexpress BNIP3 was established. Further, the effects of E2 and ERα in BNIP3-induced apoptosis and autophagy were determined in BNIP3 expressing H9c2 cells. Results from TUNEL assay and Immunoflourecense assay for LC3 puncta formation, respectively, revealed that ERα/E2 suppresses BNIP3-induced apoptosis and autophagy. The Western blot analysis showed ERα/E2 decreases the protein levels of caspase 3 (apoptotic marker), Atg5, and LC3-II (autophagic markers). Co-immunoprecipitation of BNIP3 and immunoblotting of Bcl-2 and Rheb showed that ERα reduced the interaction between BNIP3 and Bcl-2 or Rheb. The results confirm that ERα binds to BNIP3 causing a reduction in the levels of functional BNIP3 and thereby inhibits cellular apoptosis and autophagy. In addition, ERα attenuated the activity of the BNIP3 promoter by binding to SP-1 or NFκB sites.

Till Death Do Us Part: The Marriage of Autophagy and Apoptosis.

Autophagy is a widely conserved catabolic process that is necessary for maintaining cellular homeostasis under normal physiological conditions and driving the cell to switch back to this status quo under times of starvation, hypoxia, and oxidative stress. The potential similarities and differences between basal autophagy and stimulus-induced autophagy are still largely unknown. Both act by clearing aberrant or unnecessary cytoplasmic material, such as misfolded proteins, supernumerary and defective organelles. The relationship between reactive oxygen species (ROS) and autophagy is complex. Cellular ROS is predominantly derived from mitochondria. Autophagy is triggered by this event, and by clearing the defective organelles effectively, it lowers cellular ROS thereby restoring cellular homeostasis. However, if cellular homeostasis cannot be reached, the cells can switch back and choose a regulated cell death response. Intriguingly, the autophagic and cell death machines both respond to the same stresses and share key regulatory proteins, suggesting that the pathways are intricately connected. Here, the intersection between autophagy and apoptosis is discussed with a particular focus on the role ROS plays.

Nutrient modulation in the management of disease-induced muscle wasting: evidence from human studies.

In addition to being essential for movement, skeletal muscles act as both a store and source of key macronutrients. As such, muscle is an important tissue for whole body homeostasis, undergoing muscle wasting in times of starvation, disease, and stress, for example, to provide energy substrates for other tissues. Yet, muscle wasting is also associated with disability, comorbidities, and mortality. As nutrition is so crucial to maintaining muscle homeostasis 'in health', it has been postulated that muscle wasting in cachexia syndromes may be alleviated by nutritional interventions. This review will highlight recent work in this area in relation to muscle kinetics, the acute metabolic (e.g. dietary protein), and longer-term effects of dietary interventions. Whole body and skeletal muscle protein synthesis invariably exhibit deranged kinetics (favouring catabolism) in wasting states; further, many of these conditions harbour blunted anabolic responses to protein nutrition compared with healthy controls. These derangements underlie muscle wasting. Recent trials of essential amino acid and protein-based nutrition have shown some potential for therapeutic benefit. Nutritional modulation, particularly of dietary amino acids, may have benefits to prevent or attenuate disease-induced muscle wasting. Nonetheless, there remains a lack of recent studies exploring these key concepts to make conclusive recommendations.

Chitosan nanoparticle-mediated co-delivery of shAtg-5 and gefitinib synergistically promoted the efficacy of chemotherapeutics through the modulation of autophagy

BackgroundAutophagy reportedly plays vital and complex roles in many diseases. During times of starvation or energy deficiency, autophagy will occur at higher levels to provide cells with the nutrients or energy necessary to survive in stressful conditions. Some anti-cancer drugs induce protective autophagy and reduce cell apoptosis. Autophagy can adversely affect apoptosis, and blocking autophagy will increase the sensitivity of cells to apoptosis signals.MethodsWe designed chitosan nanoparticles (NPs) to promote the co-delivery of gefitinib (an anti-cancer drug) and shRNA-expressing plasmid DNA that targets the Atg-5 gene (shAtg-5) as an autophagy inhibitor to improve anti-cancer effects and autophagy mediation.ResultsThe results showed that when compared to treatment with a single drug, chitosan NPs were able to facilitate the intracellular distribution of NPs, and they improved the transfection efficiency of gene in vitro. The co-delivery of gefitinib and shAtg-5 increased cytotoxicity, induced significant apoptosis through the prohibition of autophagy, and markedly inhibited tumor growth in vivo.ConclusionsThe co-delivery of gefitinib/shAtg-5 in chitosan NPs produced superior anti-cancer efficacy via the internalization effect of NPs, while blocking autophagy with shAtg-5 enhanced the synergistic antitumor efficacy of gefitinib.

From Wasting to Obesity: The Contribution of Nutritional Status to Immune Activation in HIV Infection.

The impact of human immunodeficiency virus (HIV) infection on innate and adaptive immune activation occurs in the context of host factors, which serve to augment or dampen the physiologic response to the virus. Independent of HIV infection, nutritional status, particularly body composition, affects innate immune activation through a variety of conditions, including reduced mucosal barrier defenses and microbiome dysbiosis in malnutrition and the proinflammatory contribution of adipocytes and stromal vascular cells in obesity. Similarly, T-cell activation, proliferation, and cytokine expression are reduced in the setting of malnutrition and increased in obesity, potentially due to adipokine regulatory mechanisms restraining energy-avid adaptive immunity in times of starvation and exerting a paradoxical effect in overnutrition. The response to HIV infection is situated within these complex interactions between host nutritional health and immunologic function, which contribute to the varied phenotypes of immune activation among HIV-infected patients across a spectrum from malnutrition to obesity.

Autophagy exacerbates caspase-dependent apoptotic cell death after short times of starvation

Autophagy is generally regarded as a mechanism to promote cell survival. However, autophagy can occasionally be the mechanism responsible of cell demise. We have found that a concomitant depletion of glucose, nutrients and growth factors provoked cell death in a variety of cell lines. This death process was contingent upon caspase activation and was mediated by BAX/BAK proteins, thus indicating its apoptotic nature and the engagement of an intrinsic pathway. In order to abrogate autophagy, 3-methyladenine (3-MA), BECLIN-1 siRNA and Atg5 knock-out (Tet-Off type) approaches were alternatively employed. Irrespective of the procedure, at short times of starvation, we found that the ongoing autophagy was sensitizing cells to the permeabilization of the mitochondrial outer membrane (MOMP), caspase activation and, therefore, apoptosis. On the contrary, at longer times of starvation, autophagy displayed its characteristic pro-survival effect on cells. As far as we know, we provide the first experimental paradigm where time is the only variable determining the final outcome of autophagy. In other words, we have circumscribed in time the shift transforming autophagy from a cell death to a protection mechanism. Moreover, at short times, starvation-driven autophagy exacerbated the apoptotic cell death caused by several antitumor agents. In agreement with this fact, their apoptotic effects were greatly diminished by autophagy inhibition. The implications of these facts in tumor biology will be discussed.

Techniques for the Detection of Autophagy in Primary Mammalian Cells.

Autophagy is a lysosomal catabolic pathway responsible for the degradation of cytoplasmic constituents. Autophagy is primarily a survival pathway for recycling cellular material in times of nutrient starvation, and in response to hypoxia, endoplasmic reticulum stress, and other stresses, regulated through the mammalian target of rapamycin pathway. The proteasomal pathway is responsible for degradation of proteins, whereas autophagy can degrade cytoplasmic material in bulk, including whole organelles such as mitochondria (mitophagy), bacteria (xenophagy), or lipids (lipophagy). Although signs of autophagy can be present during cell death, it remains controversial whether autophagy can execute cell death in vivo. Here, we will introduce protocols for detecting autophagy in mammalian primary cells by using western blots, immunofluorescence, immunohistochemistry, flow cytometry, and imaging flow cytometry.