In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

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Until recently, intracellular triacylglycerols (TAG) stored in the form of cytoplasmic lipid droplets have been considered to be only passive “energy conserves”. Nevertheless, degradation of TAG gives rise to a pleiotropic spectrum of bioactive intermediates, which may function as potent co-factors of transcription factors or enzymes and contribute to the regulation of numerous cellular processes. From this point of view, the process of lipolysis not only provides energy-rich equivalents but also acquires a new regulatory function. In this review, we will concentrate on the role that fatty acids liberated from intracellular TAG stores play as signaling molecules. The first part provides an overview of the transcription factors, which are regulated by fatty acids derived from intracellular stores. The second part is devoted to the role of fatty acid signaling in different organs/tissues. The specific contribution of free fatty acids released by particular lipases, hormone-sensitive lipase, adipose triacylglycerol lipase and lysosomal lipase will also be discussed.

Acetaminophen or paracetamol (APAP) overdose is a common cause of liver injury. Silymarin (SLM) is a hepatoprotective agent widely used for treating liver injury of different origin. In order to evaluate the possible beneficial effects of SLM, Balb/c mice were pretreated with SLM (100 mg/kg b.wt. per os) once daily for three days. Two hours after the last SLM dose, the mice were administered APAP (300 mg/kg b.wt. i.p.) and killed 6 (T6), 12 (T12) and 24 (T24) hours later. SLM-treated mice exhibited a significant reduction in APAP-induced liver injury, assessed according to AST and ALT release and histological examination. SLM treatment significantly reduced superoxide production, as indicated by lower GSSG content, lower HO-1 induction, alleviated nitrosative stress, decreased p-JNK activation and direct measurement of mitochondrial superoxide production in vitro. SLM did not affect the APAP-induced decrease in CYP2E1 activity and expression during the first 12 hrs. Neutrophil infiltration and enhanced expression of inflammatory markers were first detected at T12 in both groups. Inflammation progressed in the APAP group at T24 but became attenuated in SLM-treated animals. Histological examination suggests that necrosis the dominant cell death pathway in APAP intoxication, which is partially preventable by SLM pretreatment. We demonstrate that SLM significantly protects against APAP-induced liver damage through the scavenger activity of SLM and the reduction of superoxide and peroxynitrite content. Neutrophil-induced damage is probably secondary to necrosis development.

Z, Oliyarnyk O, Kazdova L. Metformin prevents ischemia reperfusion-induced oxidative stress in the fatty liver by attenuation of reactive oxygen species formation. Am J Physiol Gastrointest Liver Physiol 309: G100 -G111, 2015. First published June 4, 2015; doi:10.1152/ajpgi.00329.2014.-Nonalcoholic fatty liver disease is associated with chronic oxidative stress. In our study, we explored the antioxidant effect of antidiabetic metformin on chronic [high-fat diet (HFD)-induced] and acute oxidative stress induced by short-term warm partial ischemia-reperfusion (I/R) or on a combination of both in the liver. Wistar rats were fed a standard diet (SD) or HFD for 10 wk, half of them being administered metformin (150 mg·kg body wt Ϫ1 ·day Ϫ1 ). Metformin treatment prevented acute stress-induced necroinflammatory reaction, reduced alanine aminotransferase and aspartate aminotransferase serum activity, and diminished lipoperoxidation. The effect was more pronounced in the HFD than in the SD group. The metformin-treated groups exhibited less severe mitochondrial damage (markers: cytochrome c release, citrate synthase activity, mtDNA copy number, mitochondrial respiration) and apoptosis (caspase 9 and caspase 3 activation). Metformin-treated HFD-fed rats subjected to I/R exhibited increased antioxidant enzyme activity as well as attenuated mitochondrial respiratory capacity and ATP resynthesis. The exposure to I/R significantly increased NADH-and succinate-related reactive oxygen species (ROS) mitochondrial production in vitro. The effect of I/R was significantly alleviated by previous metformin treatment. Metformin downregulated the I/R-induced expression of proinflammatory (TNF-␣, TLR4, IL-1␤, Ccr2) and infiltrating monocyte (Ly6c) and macrophage (CD11b) markers. Our data indicate that metformin reduces mitochondrial performance but concomitantly protects the liver from I/R-induced injury. We propose that the beneficial effect of metformin action is based on a combination of three contributory mechanisms: increased antioxidant enzyme activity, lower mitochondrial ROS production, and reduction of postischemic inflammation. metformin; oxidative stress; mitochondrial respiration; liver injury; 31 P MR spectroscopy METFORMIN IS AN ORAL ANTIHYPERGLYCEMIC drug widely used in the treatment of Type 2 diabetes (T2D) (7). There is evidence that metformin also protects against oxidative stress in various tissues, although the antioxidant activity of metformin is not well understood. It has been shown that hyperglycemia is associated with increased reactive oxygen species (ROS) formation due to superoxide overproduction from the mitochondrial electron transport chain as a consequence of increased glycolysis (8). It has been hypothesized that the antioxidative effect of metformin is simply the consequence of its glucoselowering effect and subsequent decrease in superoxide anion production (3). Nevertheless, metformin has also displayed antioxidative characteristics in several models of oxidative stress without hyperglycemia (2,19,20,2...