Yurong Xin

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, β, 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 β 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.

ANGPTL3 blockade with a human monoclonal antibody reduces plasma lipids in dyslipidemic mice and monkeys

Angiopoietin-like protein 3 (ANGPTL3) is a circulating protein synthesized exclusively in the liver that inhibits LPL and endothelial lipase (EL), enzymes that hydrolyze TGs and phospholipids in plasma lipoproteins. Here we describe the development and testing of a fully human monoclonal antibody (REGN1500) that binds ANGPTL3 with high affinity. REGN1500 reversed ANGPTL3-induced inhibition of LPL activity in vitro. Intravenous administration of REGN1500 to normolipidemic C57Bl/6 mice increased LPL activity and decreased plasma TG levels by ≥50%. Chronic administration of REGN1500 to dyslipidemic C57Bl/6 mice for 8 weeks reduced circulating plasma levels of TG, LDL-cholesterol (LDL-C), and HDL-cholesterol (HDL-C) without any changes in liver, adipose, or heart TG contents. Studies in EL knockout mice revealed that REGN1500 reduced serum HDL-C through an EL-dependent mechanism. Finally, administration of a single dose of REGN1500 to dyslipidemic cynomolgus monkeys caused a rapid and pronounced decrease in plasma TG, nonHDL-C, and HDL-C. REGN1500 normalized plasma TG levels even in monkeys with a baseline plasma TG greater than 400 mg/dl. Collectively, these data demonstrate that neutralization of ANGPTL3 using REGN1500 reduces plasma lipids in dyslipidemic mice and monkeys, and thus provides a potential therapeutic agent for treatment of patients with hyperlipidemia.

Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

Significance Pancreatic islets are complex structures composed of four cell types whose primary function is to maintain glucose homeostasis. Owing to the scarcity and heterogeneity of the islet cell types, little is known about their individual gene expression profiles. Here we used the Fluidigm C1 platform to obtain high-quality gene expression profiles of each islet cell type from mice. We identified cell-type–specific transcription factors and pathways providing previously unrecognized insights into genes characterizing islet cells. Unexpectedly, our data uncover technical limitations with the C1 Fluidigm cell capture process, which should be considered when analyzing single-cell transcriptomics data. This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type–specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system.

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.)

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.

GPR17 gene disruption does not alter food intake or glucose homeostasis in mice

Significance Hypothalamic agouti-related peptide (AGRP) neurons control food intake and body weight. G protein-coupled receptor 17 (GPR17) was recently shown to be expressed in these neurons and controls their activity, thereby reducing body weight and food intake in mice. In the current study, we demonstrate that Gpr17-deficient mice have normal hypothalamic and circulating AGRP levels. Body weight, food intake, and glucose homeostasis appear normal in the GPR17-deficient mice. The current data do not validate GPR17 as a therapeutic target for obesity or type 2 diabetes. G protein-coupled receptor 17 (GPR17) was recently reported to be a Foxo1 target in agouti-related peptide (AGRP) neurons. Intracerebroventricular injection of GPR17 agonists induced food intake, whereas administration of an antagonist to the receptor reduced feeding. These data lead to the conclusion that pharmacological modulation of GPR17 has therapeutic potential to treat obesity. Here we report that mice deficient in Gpr17 (Gpr17−/−) have similar food intake and body weight compared with their wild-type littermates. Gpr17−/− mice have normal hypothalamic Agrp mRNA expression, AGRP plasma levels, and metabolic rate. GPR17 deficiency in mice did not affect glucose homeostasis or prevent fat-induced insulin resistance. These data do not support a role for GPR17 in the control of food intake, body weight, or glycemic control.

Single-Cell RNAseq Reveals That Pancreatic β-Cells From Very Old Male Mice Have a Young Gene Signature

Aging improves pancreatic β-cell function in mice. This is a surprising finding because aging is typically associated with functional decline. We performed single-cell RNA sequencing of β-cells from 3- and 26-month-old mice to explore how changes in gene expression contribute to improved function with age. The old mice were healthy and had reduced blood glucose levels and increased β-cell mass, which correlated to their body weight. β-Cells from young and old mice had similar transcriptome profiles. In fact, only 193 genes (0.89% of all detected genes) were significantly regulated (≥2-fold; false discovery rate < 0.01; normalized counts > 5). Of these, 183 were down-regulated and mainly associated with pathways regulating gene expression, cell cycle, cell death, and survival as well as cellular movement, function, and maintenance. Collectively our data show that β-cells from very old mice have transcriptome profiles similar to those of young mice. These data support previous findings that aging is not associated with reduced β-cell mass or functional β-cell decline in mice.

Loss of SFRP4 Alters Body Size, Food Intake, and Energy Expenditure in Diet-Induced Obese Male Mice

Secreted frizzled-related protein 4 (SFRP4) is an extracellular regulator of the wingless-type mouse mammary tumor virus integration site family (WNT) pathway. SFRP4 has been implicated in adipocyte dysfunction, obesity, insulin resistance, and impaired insulin secretion in patients with type 2 diabetes. However, the exact role of SFRP4 in regulating whole-body metabolism and glucose homeostasis is unknown. We show here that male Sfrp4(-/-) mice have increased spine length and gain more weight when fed a high-fat diet. The body composition and body mass per spine length of diet-induced obese Sfrp4(-/-) mice is similar to wild-type littermates, suggesting that the increase in body weight can be accounted for by their longer body size. The diet-induced obese Sfrp4(-/-) mice have reduced energy expenditure, food intake, and bone mineral density. Sfrp4(-/-) mice have normal glucose and insulin tolerance and β-cell mass. Diet-induced obese Sfrp4(-/-) and control mice show similar impairments of glucose tolerance and a 5-fold compensatory expansion of their β-cell mass. In summary, our data suggest that loss of SFRP4 alters body length and bone mineral density as well as energy expenditure and food intake. However, SFRP4 does not control glucose homeostasis and β-cell mass in mice.

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

Summary 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.

Glucagon contributes to liver zonation

Significance The lobules are the functional units of the liver. They consist of 15–25 layers of hepatocytes with specialized metabolic functions and gene expression patterns relative to their position along the lobule, a phenomenon referred to as metabolic zonation. The Wnt/β-catenin pathway regulates hepatocyte function but how the zonation is controlled to meet the metabolic demands of the liver is unclear. Glucagon regulates hepatic function. We now demonstrate that glucagon contributes to liver zonation by interacting and opposing the actions of the Wnt/β-catenin pathway. Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal function. Wnt/β-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/β-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/β-catenin. The glucagon and Wnt/β-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/β-catenin signaling pathway.