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

Spexin mRNA and protein are widely expressed in rat tissues and associate with weight loss in rodents of diet-induced obesity. Its location in endocrine and epithelial cells has also been suggested. Spexin is a novel peptide that involves weight loss in rodents of diet-induced obesity. Therefore, we aimed to examine its expression in human tissues and test whether spexin could have a role in glucose and lipid metabolism in type 2 diabetes mellitus (T2DM). The expression of the spexin gene and immunoreactivity in the adrenal gland, skin, stomach, small intestine, liver, thyroid, pancreatic islets, visceral fat, lung, colon, and kidney was higher than that in the muscle and connective tissue. Immunoreactive serum spexin levels were reduced in T2DM patients and correlated with fasting blood glucose (FBG, r=-0.686, P<0.001), hemoglobin A1c (HbA1c, r=-0.632, P<0.001), triglyceride (TG, r=-0.236, P<0.001) and low density lipoprotein-cholesterol (LDL-C, r=-0.382, P<0.001). A negative correlation of blood glucose with spexin was observed during oral glucose tolerance test (OGTT). Spexin is intensely expressed in normal human endocrine and epithelial tissues, indicating that spexin may be involved in physiological functions of endocrine and in several other tissues. Circulating spexin levels are low in T2DM patients and negatively related to blood glucose and lipids suggesting that the peptide may play a role in glucose and lipid metabolism in T2DM.

Obesity is associated with a state of chronic low-grade inflammation, which contributes to insulin resistance and type 2 diabetes. However, the molecular mechanisms that link obesity to inflammation are not fully understood. Follistatin-like 1 (FSTL1) is a novel proinflammatory cytokine that is expressed in adipose tissue and secreted by preadipocytes/adipocytes. We aimed to test whether FSTL1 could have a role in obesity-induced inflammation and insulin resistance. It was found that FSTL1 expression was markedly decreased during differentiation of 3T3-L1 preadipocytes but reinduced by TNF-α. Furthermore, a significant increase in FSTL1 levels was observed in adipose tissue of obese ob/ob mice, as well as in serum of overweight/obese subjects. Mechanistic studies revealed that FSTL1 induced inflammatory responses in both 3T3-L1 adipocytes and RAW264.7 macrophages. The expression of proinflammatory mediators including IL-6, TNF-α, and MCP-1 was upregulated by recombinant FSTL1 in a dose-dependent manner, paralleled with activation of the IKKβ-NFκB and JNK signaling pathways in the two cell lines. Moreover, FSTL1 impaired insulin signaling in 3T3-L1 adipocytes, as revealed by attenuated phosphorylation of both Akt and IRS-1 in response to insulin stimulation. Together, our results suggest that FSTL1 is a potential mediator of inflammation and insulin resistance in obesity.