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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Protein tyrosine phosphatase-1B (PTP-1B) has been implicated in the negative regulation of insulin signaling. Disruption of the mouse homolog of the gene encoding PTP-1B yielded healthy mice that, in the fed state, had blood glucose concentrations that were slightly lower and concentrations of circulating insulin that were one-half those of their PTP-1B+/+ littermates. The enhanced insulin sensitivity of the PTP-1B-/- mice was also evident in glucose and insulin tolerance tests. The PTP-1B-/- mice showed increased phosphorylation of the insulin receptor in liver and muscle tissue after insulin injection in comparison to PTP-1B+/+ mice. On a high-fat diet, the PTP-1B-/- and PTP-1B+/- mice were resistant to weight gain and remained insulin sensitive, whereas the PTP-1B+/+ mice rapidly gained weight and became insulin resistant. These results demonstrate that PTP-1B has a major role in modulating both insulin sensitivity and fuel metabolism, thereby establishing it as a potential therapeutic target in the treatment of type 2 diabetes and obesity.

Common obesity is primarily characterized by resistance to the actions of the hormone leptin. Mice deficient in protein tyrosine phosphatase 1B (PTP1B) are resistant to diabetes and diet-induced obesity, prompting us to further define the relationship between PTP1B and leptin in modulating obesity. Leptin-deficient (Lep(ob/ob)) mice lacking PTP1B exhibit an attenuated weight gain, a decrease in adipose tissue, and an increase in resting metabolic rate. Furthermore, PTP1B-deficient mice show an enhanced response toward leptin-mediated weight loss and suppression of feeding. Hypothalami from these mice also display markedly increased leptin-induced Stat3 phosphorylation. Finally, substrate-trapping experiments demonstrate that leptin-activated Jak2, but not Stat3 or the leptin receptor, is a substrate of PTP1B. These results suggest that PTP1B negatively regulates leptin signaling, and provide one mechanism by which it may regulate obesity.

The reversible tyrosine phosphorylation of proteins, modulated by the coordinated actions of protein-tyrosine kinases and protein-tyrosine phosphatases (PTPs), regulates the cellular response to a wide variety of stimuli. It is established that protein kinases possess discrete sets of substrates and that substrate recognition is often dictated by the presence of consensus phosphorylation sites. Here, we have extended this concept to the PTPs and demonstrated that (E/D)-pY-pY-(R/K) is a consensus substrate recognition motif for PTP1B. We have shown that JAK2 and TYK2 are substrates of PTP1B and that the substrate recognition site within theses kinases is similar to the site of dephosphorylation previously identified within the insulin receptor. A substrate-trapping mutant of PTP1B formed a stable interaction with JAK2 and TYK2 in response to interferon stimulation. Expression of wild type or substrate-trapping mutant PTP1B inhibited interferon-dependent transcriptional activation. Finally, mouse embryo fibroblasts deficient in PTP1B displayed subtle changes in tyrosine phosphorylation, including hyperphosphorylation of JAK2. The closely related JAK family member, JAK1, which does not match the consensus dephosphorylation site, was not recognized as a substrate. These data illustrate that PTP1B may be an important physiological regulator of cytokine signaling and that it may be possible to derive consensus substrate recognition motifs for other members of the PTP family, which may then be used to predict novel physiological substrates.

All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.

3T3-L1 preadipocytes require cAMP for maximal differentiation. Microarray analysis reveals that the integrins alpha5 and alpha6 are coordinately regulated by cAMP. alpha5 expression is gradually diminished during adipogenesis, whereas alpha6 is increased. Overexpression of alpha5 in preadipocytes results in enhanced proliferation and attenuated differentiation. Conversely, alpha6 overexpression is without effect. The GTPase Rac is normally inhibited during differentiation. However, overexpression of integrin alpha5 increases Rac activity. Constitutively active but not dominant-negative Rac inhibits differentiation when overexpressed in preadipocytes, implying its role downstream of alpha5 integrin in maintaining preadipocytes in an undifferentiated state. Moreover, alpha6 integrin is critically involved in clustering growth-arrested preadipocytes on basement membrane Matrigel. Perturbation of such clustering enhances Rho activity and promotes growth-arrested preadipocytes to reenter the cell cycle. These findings demonstrate a role for integrin alpha6 in connecting morphogenesis with signaling processes leading to terminal differentiation.

It is shown that RalA is regulated by a Ral GAP complex (RGC 1/2) in insulin action and links PI 3-kinase signaling to RalA activation. Akt phosphorylates the complex and inhibits its function, resulting in increased RalA activity and glucose uptake.