Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Tshz1 regulates pancreatic beta cell maturation.

International audienceThe homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic β-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional β-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of β-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1(+/-) mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1(+/-) islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing β-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes

Mitofusins Mfn1 and Mfn2 are required to preserve glucose- but not incretin-stimulated beta cell connectivity and insulin secretion

Mitochondrial glucose metabolism is essential for stimulated insulin release from pancreatic beta cells. Whether mitofusin gene expression, and hence mitochondrial network integrity, is important for glucose or incretin signalling has not previously been explored. Here, we generated mice with beta cell-selective, adult-restricted deletion of the mitofusin genes Mfn1 and Mfn2 (βMfn1/2 dKO). βMfn1/2 dKO mice displayed elevated fed and fasted glycaemia and a >five-fold decrease in plasma insulin. Mitochondrial length, glucose-induced polarisation, ATP synthesis, cytosolic and mitochondrial Ca2+ increases were all reduced in dKO islets. In contrast, oral glucose tolerance was more modestly affected in βMfn1/2 dKO mice and GLP-1 or GIP receptor agonists largely corrected defective GSIS through enhanced EPAC-dependent signalling. Correspondingly, cAMP increases in the cytosol, as measured with an Epac-camps based sensor, were exaggerated in dKO mice. Mitochondrial fusion and fission cycles are thus essential in the beta cell to maintain normal glucose, but not incretin, sensing. These findings broaden our understanding of the roles of mitofusins in beta cells, the potential contributions of altered mitochondrial dynamics to diabetes development and the impact of incretins on this process