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

Genome wide association studies revealed that variation in the Melatonin Receptor 1B gene (MTNR1B) is associated with insulin and glucose concentrations. Here we show that the risk genotype of this SNP predicts future type 2 diabetes (T2D) in two large prospective studies. Specifically, the risk genotype was associated with impairment of early insulin response to both oral and intravenous glucose and with faster deterioration of insulin secretion over time. We also show that the Melatonin Receptor 1B mRNA is expressed in human islets, and immunocytochemistry confirms that it is primarily localized in β-cells in islets. Non-diabetic individuals carrying the risk allele and patients with T2D showed increased expression of the receptor in islets. Insulin release from clonal β-cells in response to glucose was inhibited in the presence of melatonin. These data suggest that the circulating hormone melatonin, which is predominantly released from the pineal gland in the brain, is involved in the pathogenesis of T2D. Given the increased expression of Melatonin Receptor 1B in individuals at risk of T2D, the pathogenic effects are likely exerted via a direct inhibitory effect on β-cells. In view of these results, blocking the melatonin ligand-receptor system could be a therapeutic avenue in T2D.

Genetic variants in the gene encoding for transcription factor-7-like 2 (TCF7L2) have been associated with type 2 diabetes (T2D) and impaired β cell function, but the mechanisms have remained unknown. We therefore studied prospectively the ability of common variants in TCF7L2 to predict future T2D and explored the mechanisms by which they would do this. Scandinavian subjects followed for up to 22 years were genotyped for 3 SNPs (rs7903146, rs12255372, and rs10885406) in TCF7L2, and a subset of them underwent extensive metabolic studies. Expression of TCF7L2 was related to genotype and metabolic parameters in human islets. The CT/TT genotypes of SNP rs7903146 strongly predicted future T2D in 2 independent cohorts (Swedish and Finnish). The risk T allele was associated with impaired insulin secretion, incretin effects, and enhanced rate of hepatic glucose production. TCF7L2 expression in human islets was increased 5-fold in T2D, particularly in carriers of the TT genotype. Overexpression of TCF7L2 in human islets reduced glucose-stimulated insulin secretion. In conclusion, the increased risk of T2D conferred by variants in TCF7L2 involves the enteroinsular axis, enhanced expression of the gene in islets, and impaired insulin secretion.

No abstract

SummaryThe arrangement of β cells within islets of Langerhans is critical for insulin release through the generation of rhythmic activity. A privileged role for individual β cells in orchestrating these responses has long been suspected, but not directly demonstrated. We show here that the β cell population in situ is operationally heterogeneous. Mapping of islet functional architecture revealed the presence of hub cells with pacemaker properties, which remain stable over recording periods of 2 to 3 hr. Using a dual optogenetic/photopharmacological strategy, silencing of hubs abolished coordinated islet responses to glucose, whereas specific stimulation restored communication patterns. Hubs were metabolically adapted and targeted by both pro-inflammatory and glucolipotoxic insults to induce widespread β cell dysfunction. Thus, the islet is wired by hubs, whose failure may contribute to type 2 diabetes mellitus.