ZnT8 Loss of Function Mutation Increases Resistance of Human Embryonic Stem Cell-Derived Beta Cells to Apoptosis in Low Zinc Condition.

The rare SLC30A8 mutation encoding a truncating p.Arg138* variant (R138X) in zinc transporter 8 (ZnT8) is associated with a 65% reduced risk for type 2 diabetes. To determine whether ZnT8 is required for beta cell development and function, we derived human pluripotent stem cells carrying the R138X mutation and differentiated them into insulin-producing cells. We found that human pluripotent stem cells with homozygous or heterozygous R138X mutation and the null (KO) mutation have normal efficiency of differentiation towards insulin-producing cells, but these cells show diffuse granules that lack crystalline zinc-containing insulin granules. Insulin secretion is not compromised in vitro by KO or R138X mutations in human embryonic stem cell-derived beta cells (sc-beta cells). Likewise, the ability of sc-beta cells to secrete insulin and maintain glucose homeostasis after transplantation into mice was comparable across different genotypes. Interestingly, sc-beta cells with the SLC30A8 KO mutation showed increased cytoplasmic zinc, and cells with either KO or R138X mutation were resistant to apoptosis when extracellular zinc was limiting. These findings are consistent with a protective role of zinc in cell death and with the protective role of zinc in T2D.

Single-cell RNA transcriptome landscape of hepatocytes and non-parenchymal cells in healthy and NAFLD mouse liver.

Nonalcoholic fatty liver disease (NAFLD) is a global health-care problem with limited therapeutic options. To obtain a cellular resolution of pathogenesis, 82,168 single-cell transcriptomes (scRNA-seq) across different NAFLD stages were profiled, identifying hepatocytes and 12 other non-parenchymal cell (NPC) types. scRNA-seq revealed insights into the cellular and molecular mechanisms of the disease. We discovered a dual role for hepatic stellate cells in gene expression regulation and in the potential to trans-differentiate into myofibroblasts. We uncovered distinct expression profiles of Kupffer cells versus monocyte-derived macrophages during NAFLD progression. Kupffer cells showed stronger immune responses, while monocyte-derived macrophages demonstrated a capability for differentiation. Three chimeric NPCs were identified including endothelial-chimeric stellate cells, hepatocyte-chimeric endothelial cells, and endothelial-chimeric Kupffer cells. Our work identified unanticipated aspects of mouse with NAFLD at the single-cell level and advanced the understanding of cellular heterogeneity in NAFLD livers.

Tissue-specific activation of gene expression by the Synergistic Activation Mediator (SAM) CRISPRa system in mice.

CRISPR-based transcriptional activation is a powerful tool for functional gene interrogation; however, delivery difficulties have limited its applications in vivo. Here, we created a mouse model expressing all components of the CRISPR-Cas9 guide RNA-directed Synergistic Activation Mediator (SAM) from a single transcript that is capable of activating target genes in a tissue-specific manner. We optimized Lipid Nanoparticles and Adeno-Associated Virus guide RNA delivery approaches to achieve expression modulation of one or more genes in vivo. We utilized the SAM mouse model to generate a hypercholesteremia disease state that we could bidirectionally modulate with various guide RNAs. Additionally, we applied SAM to optimize gene expression in a humanized Transthyretin mouse model to recapitulate human expression levels. These results demonstrate that the SAM gene activation platform can facilitate in vivo research and drug discovery.

Reduced replication fork speed promotes pancreatic endocrine differentiation and controls graft size.

Limitations in cell proliferation are important for normal function of differentiated tissues and essential for the safety of cell replacement products made from pluripotent stem cells, which have unlimited proliferative potential. To evaluate whether these limitations can be established pharmacologically, we exposed pancreatic progenitors differentiating from human pluripotent stem cells to small molecules that interfere with cell cycle progression either by inducing G1 arrest or by impairing S phase entry or S phase completion and determined growth potential, differentiation, and function of insulin-producing endocrine cells. We found that the combination of G1 arrest with a compromised ability to complete DNA replication promoted the differentiation of pancreatic progenitor cells toward insulin-producing cells and could substitute for endocrine differentiation factors. Reduced replication fork speed during differentiation improved the stability of insulin expression, and the resulting cells protected mice from diabetes without the formation of cystic growths. The proliferative potential of grafts was proportional to the reduction of replication fork speed during pancreatic differentiation. Therefore, a compromised ability to enter and complete S phase is a functionally important property of pancreatic endocrine differentiation, can be achieved by reducing replication fork speed, and is an important determinant of cell-intrinsic limitations of growth.

Discordance between GLP-1R gene and protein expression in mouse pancreatic islet cells.

The insulinotropic actions of glucagon-like peptide 1 receptor (GLP-1R) in ß-cells have made it a useful target to manage type 2 diabetes. Metabolic stress reduces ß-cell sensitivity to GLP-1, yet the underlying mechanisms are unknown. We hypothesized that Glp1r expression is heterogeneous among ß-cells and that metabolic stress decreases the number of GLP-1R-positive ß-cells. Here, analyses of publicly available single-cell RNA-Seq sequencing (scRNASeq) data from mouse and human ß-cells indicated that significant populations of ß-cells do not express the Glp1r gene, supporting heterogeneous GLP-1R expression. To check these results, we used complementary approaches employing FACS coupled with quantitative RT-PCR, a validated GLP-1R antibody, and flow cytometry to quantify GLP-1R promoter activity, gene expression, and protein expression in mouse α-, ß-, and δ-cells. Experiments with Glp1r reporter mice and a validated GLP-1R antibody indicated that >90% of the ß-cells are GLP-1R positive, contradicting the findings with the scRNASeq data. α-cells did not express Glp1r mRNA and δ-cells expressed Glp1r mRNA but not protein. We also examined the expression patterns of GLP-1R in mouse models of metabolic stress. Multiparous female mice had significantly decreased ß-cell Glp1r expression, but no reduction in GLP-1R protein levels or GLP-1R-mediated insulin secretion. These findings suggest caution in interpreting the results of scRNASeq for low-abundance transcripts such as the incretin receptors and indicate that GLP-1R is widely expressed in ß-cells, absent in α-cells, and expressed at the mRNA, but not protein, level in δ-cells.

Heterogeneity of human pancreatic ß-cells.

BACKGROUND: Human pancreatic ß-cells are heterogeneous. This has been known for a long time and is based on various functional and morphological readouts. ß-Cell heterogeneity could reflect fixed subpopulations with distinct functions. However, recent pseudotime analysis of large-scale RNA sequencing data suggest that human ß-cell subpopulations may rather reflect dynamic interchangeable states characterized by low expression of genes involved in the unfolded protein response (UPR) and low insulin gene expression, low UPR and high insulin expression or high UPR and low insulin expression. SCOPE OF REVIEW: This review discusses findings obtained by single-cell RNA sequencing combined with pseudotime analysis that human ß-cell heterogeneity represents dynamic interchangeable functional states. The physiological significance and potential implications of ß-cell heterogeneity in the development and progression of diabetes is highlighted. MAJOR CONCLUSIONS: The existence of dynamic functional states allow ß-cells to transition between periods of high insulin production and UPR-mediated stress recovery. The recovery state is important since proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum a stressful event. The transition of ß-cells between dynamic states is likely controlled at multiple levels and influenced by the microenvironment within the pancreatic islets. Disturbances in the ability of the ß-cells to transition between periods of high insulin biosynthesis and UPR-mediated stress recovery may contribute to diabetes development. Diabetes medications that restore the ability of the ß-cells to transition between the functional states should be considered.

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets.

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Effects of Noncanonical Base Pairing on RNA Folding: Structural Context and Spatial Arrangements of G·A Pairs.

Noncanonical base pairs play important roles in assembling the three-dimensional structures critical to the diverse functions of RNA. These associations contribute to the looped segments that intersperse the canonical double-helical elements within folded, globular RNA molecules. They stitch together various structural elements, serve as recognition elements for other molecules, and act as sites of intrinsic stiffness or deformability. This work takes advantage of new software (DSSR) designed to streamline the analysis and annotation of RNA three-dimensional structures. The multiscale structural information gathered for individual molecules, combined with the growing number of unique, well-resolved RNA structures, makes it possible to examine the collective features deeply and to uncover previously unrecognized patterns of chain organization. Here we focus on a subset of noncanonical base pairs involving guanine and adenine and the links between their modes of association, secondary structural context, and contributions to tertiary folding. The rigorous descriptions of base-pair geometry that we employ facilitate characterization of recurrent geometric motifs and the structural settings in which these arrangements occur. Moreover, the numerical parameters hint at the natural motions of the interacting bases and the pathways likely to connect different spatial forms. We draw attention to higher-order multiplexes involving two or more G·A pairs and the roles these associations appear to play in bridging different secondary structural units. The collective data reveal pairing propensities in base organization, secondary structural context, and deformability and serve as a starting point for further multiscale investigations and/or simulations of RNA folding.

Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon Receptor Inhibition.

Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver-human α-cell endocrine feedback loop.

Gene Signature of the Human Pancreatic ε Cell.

The ghrelin-producing ε cell represents the fifth endocrine cell type in human pancreatic islets. The abundance of ε cells in adult pancreas is extremely low, which has hampered the investigation on the molecular pathways regulating the development and the function of this cell type. In this study, we explored the molecular features defining the function of pancreatic ε cells isolated from adult nondiabetic donors using single-cell RNA sequencing technology. We focus on transcription factors, cell surface receptors, and genes involved in metabolic pathways that contribute to regulation of cellular function. Furthermore, the genes that separate ε cells from the other islet endocrine cell types are presented. This study expands prior knowledge about the genes important for ε cell functioning during development and provides a resource to interrogate the transcriptome of this rare human islet cell type.

Gene Signature of Proliferating Human Pancreatic α Cells.

Pancreatic α cells proliferate at a low rate, and little is known about the control of this process. Here we report the characterization of human α cells by large-scale, single-cell RNA sequencing coupled with pseudotime ordering. We identified two large subpopulations and a smaller cluster of proliferating α cells with increased expression of genes involved in cell-cycle regulation. The proliferating α cells were differentiated, had normal levels of GCG expression, and showed no signs of cellular stress. Proliferating α cells were detected in both the G1S and G2M phases of the cell cycle. Human α cells proliferate at a fivefold higher rate than human ß cells and express lower levels of the cell-cycle inhibitors CDKN1A and CDKN1C. Collectively, this study provides the gene signatures of human α cells and the genes involved in their cell division. The lower expression of two cell-cycle inhibitors in human α cells could account for their higher rate of proliferation compared with their insulin-producing counterparts.

Mice harboring the human SLC30A8 R138X loss-of-function mutation have increased insulin secretory capacity.

SLC30A8 encodes a zinc transporter that is primarily expressed in the pancreatic islets of Langerhans. In ß-cells it transports zinc into insulin-containing secretory granules. Loss-of-function (LOF) mutations in SLC30A8 protect against type 2 diabetes in humans. In this study, we generated a knockin mouse model carrying one of the most common human LOF mutations for SLC30A8, R138X. The R138X mice had normal body weight, glucose tolerance, and pancreatic ß-cell mass. Interestingly, in hyperglycemic conditions induced by the insulin receptor antagonist S961, the R138X mice showed a 50% increase in insulin secretion. This effect was not associated with enhanced ß-cell proliferation or mass. Our data suggest that the SLC30A8 R138X LOF mutation may exert beneficial effects on glucose metabolism by increasing the capacity of ß-cells to secrete insulin under hyperglycemic conditions.

Pseudotime Ordering of Single Human ß-Cells Reveals States of Insulin Production and Unfolded Protein Response.

Proinsulin is a misfolding-prone protein, making its biosynthesis in the endoplasmic reticulum (ER) a stressful event. Pancreatic ß-cells overcome ER stress by activating the unfolded protein response (UPR) and reducing insulin production. This suggests that ß-cells transition between periods of high insulin biosynthesis and UPR-mediated recovery from cellular stress. We now report the pseudotime ordering of single ß-cells from humans without diabetes detected by large-scale RNA sequencing. We identified major states with 1) low UPR and low insulin gene expression, 2) low UPR and high insulin gene expression, or 3) high UPR and low insulin gene expression. The latter state was enriched for proliferating cells. Stressed human ß-cells do not dedifferentiate and show little propensity for apoptosis. These data suggest that human ß-cells transition between states with high rates of biosynthesis to fulfill the body's insulin requirements to maintain normal blood glucose levels and UPR-mediated recovery from ER stress due to high insulin production.

A Protein-Truncating HSD17B13 Variant and Protection from Chronic Liver Disease.

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).

Glucagon contributes to liver zonation.

Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal function. Wnt/ß-catenin signaling controls metabolic zonation by activating genes in the perivenous hepatocytes, while suppressing genes in the periportal counterparts. We now demonstrate that glucagon opposes the actions of Wnt/ß-catenin signaling on gene expression and metabolic zonation pattern. The effects were more pronounced in the periportal hepatocytes where 28% of all genes were activated by glucagon and inhibited by Wnt/ß-catenin. The glucagon and Wnt/ß-catenin receptors and their signaling pathways are uniformly distributed in periportal and perivenous hepatocytes and the expression is not regulated by the opposing signal. Collectively, our results show that glucagon controls gene expression and metabolic zonation in the liver through a counterplay with the Wnt/ß-catenin signaling pathway.

Delta-like Ligand-4-Notch Signaling Inhibition Regulates Pancreatic Islet Function and Insulin Secretion.

Although Notch signaling has been proposed as a therapeutic target for type-2 diabetes, liver steatosis, and atherosclerosis, its direct effect on pancreatic islets remains unknown. Here, we demonstrated a function of Dll4-Notch signaling inhibition on the biology of insulin-producing cells. We confirmed enhanced expression of key Notch signaling genes in purified pancreatic islets from diabetic NOD mice and showed that treatment with anti-Dll4 antibody specifically abolished Notch signaling pathway activation. Furthermore, we showed that Notch inhibition could drive proliferation of ß-islet cells and confer protection from the development of STZ-induced diabetes. Importantly, inhibition of the Dll4 pathway in WT mice increased insulin secretion by inducing the differentiation of pancreatic ß-islet cell progenitors, as well as the proliferation of insulin-secreting cells. These findings reveal a direct effect of Dll4-blockade on pancreatic islets that, in conjunction with its immunomodulatory effects, could be used for unmet medical needs hallmarked by inefficient insulin action.

Amino Acid Transporter Slc38a5 Controls Glucagon Receptor Inhibition-Induced Pancreatic α Cell Hyperplasia in Mice.

Glucagon supports glucose homeostasis by stimulating hepatic gluconeogenesis, in part by promoting the uptake and conversion of amino acids into gluconeogenic precursors. Genetic disruption or pharmacologic inhibition of glucagon signaling results in elevated plasma amino acids and compensatory glucagon hypersecretion involving expansion of pancreatic α cell mass. Recent findings indicate that hyperaminoacidemia triggers pancreatic α cell proliferation via an mTOR-dependent pathway. We confirm and extend these findings by demonstrating that glucagon pathway blockade selectively increases expression of the sodium-coupled neutral amino acid transporter Slc38a5 in a subset of highly proliferative α cells and that Slc38a5 controls the pancreatic response to glucagon pathway blockade; most notably, mice deficient in Slc38a5 exhibit markedly decreased α cell hyperplasia to glucagon pathway blockade-induced hyperaminoacidemia. These results show that Slc38a5 is a key component of the feedback circuit between glucagon receptor signaling in the liver and amino-acid-dependent regulation of pancreatic α cell mass in mice.

ANGPTL8 Blockade With a Monoclonal Antibody Promotes Triglyceride Clearance, Energy Expenditure, and Weight Loss in Mice.

Angiopoietin-like protein (ANGPTL)8 is a negative regulator of lipoprotein lipase-mediated plasma triglyceride (TG) clearance. In this study, we describe a fully human monoclonal antibody (REGN3776) that binds monkey and human ANGPTL8 with high affinity. Inhibition of ANGPTL8 with REGN3776 in humanized ANGPTL8 mice decreased plasma TGs and increased lipoprotein lipase activity. Additionally, REGN3776 reduced body weight and fat content. The reduction in body weight was secondary to increased energy expenditure. Finally, single administration of REGN3776 normalized plasma TGs in dyslipidemic cynomolgus monkeys. In conclusion, we show that blockade of ANGPTL8 with monoclonal antibody strongly reduced plasma TGs in mice and monkeys. These data suggest that inhibition of ANGPTL8 may provide a new therapeutic avenue for the treatment of dyslipidemia with beneficial effects on body weight.

PC1/3 Deficiency Impacts Pro-opiomelanocortin Processing in Human Embryonic Stem Cell-Derived Hypothalamic Neurons.

We recently developed a technique for generating hypothalamic neurons from human pluripotent stem cells. Here, as proof of principle, we examine the use of these cells in modeling of a monogenic form of severe obesity: PCSK1 deficiency. The cognate enzyme, PC1/3, processes many prohormones in neuroendocrine and other tissues. We generated PCSK1 (PC1/3)-deficient human embryonic stem cell (hESC) lines using both short hairpin RNA and CRISPR-Cas9, and investigated pro-opiomelanocortin (POMC) processing using hESC-differentiated hypothalamic neurons. The increased levels of unprocessed POMC and the decreased ratios (relative to POMC) of processed POMC-derived peptides in both PCSK1 knockdown and knockout hESC-derived neurons phenocopied POMC processing reported in PC1/3-null mice and PC1/3-deficient patients. PC1/3 deficiency was associated with increased expression of melanocortin receptors and PRCP (prolylcarboxypeptidase, a catabolic enzyme for α-melanocyte stimulating hormone (αMSH)), and reduced adrenocorticotropic hormone secretion. We conclude that the obesity accompanying PCSK1 deficiency may not be primarily due to αMSH deficiency.

RNA Sequencing of Single Human Islet Cells Reveals Type 2 Diabetes Genes.

Pancreatic islet cells are critical for maintaining normal blood glucose levels, and their malfunction underlies diabetes development and progression. We used single-cell RNA sequencing to determine the transcriptomes of 1,492 human pancreatic α, ß, δ, and PP cells from non-diabetic and type 2 diabetes organ donors. We identified cell-type-specific genes and pathways as well as 245 genes with disturbed expression in type 2 diabetes. Importantly, 92% of the genes have not previously been associated with islet cell function or growth. Comparison of gene profiles in mouse and human α and ß cells revealed species-specific expression. All data are available for online browsing and download and will hopefully serve as a resource for the islet research community.