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.

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

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.

Endothelial glycocalyx coats healthy vascular endothelium and plays an important role in vascular homeostasis. Although cerebral capillaries are categorized as continuous, as are those in the heart and lung, they likely have specific features related to their function in the blood brain barrier. To test that idea, brains, hearts and lungs from C57BL6 mice were processed with lanthanum-containing alkaline fixative, which preserves the structure of glycocalyx, and examined using scanning and transmission electron microscopy. We found that endothelial glycocalyx is present over the entire luminal surface of cerebral capillaries. The percent area physically covered by glycocalyx within the lumen of cerebral capillaries was 40.1 ± 4.5%, which is significantly more than in cardiac and pulmonary capillaries (15.1 ± 3.7% and 3.7 ± 0.3%, respectively). Upon lipopolysaccharide-induced vascular injury, the endothelial glycocalyx was reduced within cerebral capillaries, but substantial amounts remained. By contrast, cardiac and pulmonary capillaries became nearly devoid of glycocalyx. These findings suggest the denser structure of glycocalyx in the brain is associated with endothelial protection and may be an important component of the blood brain barrier.

Growth and differentiation factor 8 (GDF8) is a TGF-β superfamily member, and negative regulator of skeletal muscle mass. GDF8 inhibition results in prominent muscle growth in mice, but less impressive hypertrophy in primates, including man. Broad TGF-β inhibition suggests another family member negatively regulates muscle mass, and its blockade enhances muscle growth seen with GDF8-specific inhibition. Here we show that activin A is the long-sought second negative muscle regulator. Activin A specific inhibition, on top of GDF8 inhibition, leads to pronounced muscle hypertrophy and force production in mice and monkeys. Inhibition of these two ligands mimics the hypertrophy seen with broad TGF-β blockers, while avoiding the adverse effects due to inhibition of multiple family members. Altogether, we identify activin A as a second negative regulator of muscle mass, and suggest that inhibition of both ligands provides a preferred therapeutic approach, which maximizes the benefit:risk ratio for muscle diseases in man.

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.

Nonstandard abbreviations used: acetylated LDL (acLDL); insulin receptor (IR); IR substrate 2 (IRS-2); LDL receptor (LDLR); oxidized LDL (oxLDL); scavenger receptor A (SR-A); scavenger receptor BI (SR-BI); Tricine-buffered saline (TBS). Conflict of interest:The authors have declared that no conflict of interest exists. IntroductionThe complications of atherosclerosis in coronary, cerebral, and peripheral arteries are the major reason for hospital admission in diabetics and account for about 80% of the mortality of this condition (1). Although diabetes is often associated with dyslipidemia (2, 3), the excess risk is disproportionate to lipid abnormalities and the root causes of accelerated atherosclerosis in diabetes are not well understood. Some evidence suggests that insulin resistance, commonly seen in overweight individuals with the metabolic syndrome, is an important cause of both diabetes and increased atherosclerosis risk (4). Insulin resistance has been related to dyslipidemia, hypertension, and hypercoagulability, all factors that may promote atherosclerosis (2, 3). Potentially insulin resistance could also be important at the arterial cellular level (5). Defective insulin signaling in endothelial cells leads to impaired NO activity (6), and this is probably proatherogenic. Macrophages also express insulin receptors (IRs) (7), but the relationship of insulin signaling to macrophage foam cell formation has received little attention. Thiazolidinediones, activators of PPAR-γ, have emerged as an important class of drugs in the treatment of type II diabetes. PPAR-γ activators markedly improve insulin resistance and decrease atherosclerosis in mouse models, and may also decrease atherosclerosis in humans (8-11). Although the in vivo mode of action of PPAR-γ activators is not well understood, PPAR-γ is highly expressed in adipocytes and macrophages (11), suggesting these cells could be important targets. Paradoxically, however, PPAR-γ activators increase the expression of the proatherogenic molecule CD36 in cultured macrophages (12). These proatherogenic effects of PPAR-γ activators may be counteracted by an increase in ABCA1, a macrophage transporter that promotes efflux of cholesterol to lipid-poor apolipoproteins, perhaps offsetting increased uptake of modified low-density lipoprotein (LDL) via CD36 (11,13,14). However, the importance of this mechanism is uncertain, and some studies have failed to find any effect of PPAR-γ activators on ABCA1 levels or lipid efflux in macrophages (15, 16).Obese (ob/ob) mice are genetically deficient in leptin and have been widely used as a model of insulin resistance and diabetes (17). In this study, we made the initial observation that macrophages from ob/ob mice have increased uptake of modified LDL, due to post-transcriptional upregulation of CD36 protein. Unexpectedly, the increase in CD36 is caused by defective insulin signaling in macrophages, suggesting that macrophage insulin resistance promotes foam cell formation. Consistent with this hypothesis, in vivo treatment...

The role of different tissues in insulin action and their contribution to the pathogenesis of diabetes remain unclear. To examine this question, we have used genetic reconstitution experiments in mice. Genetic ablation of insulin receptors causes early postnatal death from diabetic ketoacidosis. We show that combined restoration of insulin receptor function in brain, liver, and pancreatic β cells rescues insulin receptor knockout mice from neonatal death, prevents diabetes in a majority of animals, and normalizes adipose tissue content, lifespan, and reproductive function. In contrast, mice with insulin receptor expression limited to brain or liver and pancreatic β cells are rescued from neonatal death, but develop lipoatrophic diabetes and die prematurely. These data indicate, surprisingly, that insulin receptor signaling in noncanonical insulin target tissues is sufficient to maintain fuel homeostasis and prevent diabetes.