Human stem cell models: lessons for pancreatic development and disease.

A comprehensive understanding of mechanisms that underlie the development and function of human cells requires human cell models. For the pancreatic lineage, protocols have been developed to differentiate human pluripotent stem cells (hPSCs) into pancreatic endocrine and exocrine cells through intermediates resembling in vivo development. In recent years, this differentiation system has been employed to decipher mechanisms of pancreatic development, congenital defects of the pancreas, as well as genetic forms of diabetes and exocrine diseases. In this review, we summarize recent insights gained from studies of pancreatic hPSC models. We discuss how genome-scale analyses of the differentiation system have helped elucidate roles of chromatin state, transcription factors, and noncoding RNAs in pancreatic development and how the analysis of cells with disease-relevant mutations has provided insight into the molecular underpinnings of genetically determined diseases of the pancreas.

A Network of microRNAs Acts to Promote Cell Cycle Exit and Differentiation of Human Pancreatic Endocrine Cells.

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

Interrogating islets in health and disease with single-cell technologies.

BACKGROUND: Blood glucose levels are tightly controlled by the coordinated actions of hormone-producing endocrine cells that reside in pancreatic islets. Islet cell malfunction underlies diabetes development and progression. Due to the cellular heterogeneity within islets, it has been challenging to uncover how specific islet cells contribute to glucose homeostasis and diabetes pathogenesis. Recent advances in single-cell technologies and computational methods have opened up new avenues to resolve islet heterogeneity and study islet cell states in health and disease. SCOPE OF REVIEW: In the past year, a multitude of studies have been published that used single-cell approaches to interrogate the transcriptome and proteome of the different islet cell types. Here, we summarize the conclusions of these studies, as well as discuss the technologies used and the challenges faced with computational analysis of single-cell data from islet studies. MAJOR CONCLUSIONS: By analyzing single islet cells from rodents and humans at different ages and disease states, the studies reviewed here have provided new insight into endocrine cell function and facilitated a high resolution molecular characterization of poorly understood processes, including regeneration, maturation, and diabetes pathogenesis. Gene expression programs and pathways identified in these studies pave the way for the discovery of new targets and approaches to prevent, monitor, and treat diabetes.

Pseudotemporal Ordering of Single Cells Reveals Metabolic Control of Postnatal ß Cell Proliferation.

Pancreatic ß cell mass for appropriate blood glucose control is established during early postnatal life. ß cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of ß cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of ß cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative ß cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature ß cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal ß cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal ß cells and paves the way for the identification of novel therapeutic targets to stimulate ß cell regeneration.

Advances in ß cell replacement and regeneration strategies for treating diabetes.

In the past decade, new approaches have been explored that are aimed at restoring functional ß cell mass as a treatment strategy for diabetes. The two most intensely pursued strategies are ß cell replacement through conversion of other cell types and ß cell regeneration by enhancement of ß cell replication. The approach closest to clinical implementation is the replacement of ß cells with human pluripotent stem cell-derived (hPSC-derived) cells, which are currently under investigation in a clinical trial to assess their safety in humans. In addition, there has been success in reprogramming developmentally related cell types into ß cells. Reprogramming approaches could find therapeutic applications by inducing ß cell conversion in vivo or by reprogramming cells ex vivo followed by implantation. Finally, recent studies have revealed novel pharmacologic targets for stimulating ß cell replication. Manipulating these targets or the pathways they regulate could be a strategy for promoting the expansion of residual ß cells in diabetic patients. Here, we provide an overview of progress made toward ß cell replacement and regeneration and discuss promises and challenges for clinical implementation of these strategies.

The Deubiquitylase MATH-33 Controls DAF-16 Stability and Function in Metabolism and Longevity.

FOXO family transcription factors are downstream effectors of Insulin/IGF-1 signaling (IIS) and major determinants of aging in organisms ranging from worms to man. The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response, and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

A systems view of epigenetic networks regulating pancreas development and ß-cell function.

The development of the pancreas and determination of endocrine cell fate are controlled by a highly complex interplay of signaling events and transcriptional networks. It is now known that an interconnected epigenetic program is also required to drive these processes. Recent studies using genome-wide approaches have implicated epigenetic regulators, such as DNA and histone-modifying enzymes and noncoding RNAs, to play critical roles in pancreas development and the maintenance of cell identity and function. Furthermore, genome-wide analyses have implicated epigenetic changes as a casual factor in the pathogenesis of diabetes. In the future, genomic approaches to further our understanding of the role of epigenetics in endocrine cell development and function will be useful for devising strategies to produce or manipulate ß-cells for therapies of diabetes.

A Krüppel-like factor downstream of the E3 ligase WWP-1 mediates dietary-restriction-induced longevity in Caenorhabditis elegans.

The HECT ubiquitin E3 ligase WWP-1 is a positive regulator of lifespan in response to dietary restriction (DR) in Caenorhabditis elegans. However, substrates of WWP-1 for ubiquitylation in the DR pathway have not yet been identified. Here we identify the C. elegans Krüppel-like factor, KLF-1, as an essential and specific regulator of DR-induced longevity and a substrate for ubiquitylation by WWP-1. Knockdown of klf-1 suppresses the extended lifespan of both DR animals and wwp-1-overexpressing animals, indicating that KLF-1 functions within the same pathway as WWP-1. In addition, overexpression of klf-1 in the intestine is sufficient to extend the lifespan of WT animals on an ad libitum diet, and requires wwp-1 or pha-4/FoxA. We demonstrate that WWP-1 directly interacts with KLF-1 and mediates multiple monoubiquitylation of KLF-1 in vitro and in cellulo. Our data support a model in which modulation of KLF-1 by WWP-1 regulates diet-restriction-induced longevity.

Using the ubiquitin-modified proteome to monitor protein homeostasis function.

The ubiquitin system is essential for the maintenance of proper protein homeostasis function across eukaryotic species. Although the general enzymatic architecture for adding and removing ubiquitin from substrates is well defined, methods for the comprehensive investigation of cellular ubiquitylation targets have just started to emerge. Recent advances in ubiquitin-modified peptide enrichment have greatly increased the number of identified endogenous ubiquitylation targets, as well as the number of sites of ubiquitin attachment within these substrates. Herein we evaluate current strategies using mass-spectrometry-based proteomics to characterize ubiquitin and ubiquitin-like modifications. Using existing data, we describe the characteristics of the ubiquitin-modified proteome and discuss strategies for the biological interpretation of existing and future ubiquitin-based proteomic studies.

A conserved ubiquitination pathway determines longevity in response to diet restriction.

Dietary restriction extends longevity in diverse species, suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses. Here we show a role for the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in Caenorhabditis elegans in response to dietary restriction. We find that overexpression of wwp-1 in worms extends lifespan by up to 20% under conditions of ad libitum feeding. This extension is dependent on the FOXA transcription factor pha-4, and independent of the FOXO transcription factor daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, the loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signalling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Furthermore, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for diet-restriction-induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate diet-restriction-induced longevity.