Haruka Okamoto

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.

Genetic Deletion of Trb3, the Mammalian Drosophila tribbles Homolog, Displays Normal Hepatic Insulin Signaling and Glucose Homeostasis

Trb3, a mammalian homolog of Drosophila tribbles, was proposed as a suppressor of Akt activity, predominantly in conditions of fasting and diabetes. Given these prior studies, we sought to determine whether Trb3 plays a major role in modulating hepatic insulin sensitivity. To answer this question, we produced mice in which a lacZ reporter was knocked into the locus containing the gene Trib3, resulting in a Trib3 null animal. Trib3 expression analyses demonstrated that the Trib3 is expressed in liver, adipose tissues, heart, kidney, lung, skin, small intestine, stomach, and denervated, but not normal, skeletal muscle. Trib3−/− mice are essentially identical to their wild-type littermates in overall appearance and body composition. Phenotypic analysis of Trib3−/− mice did not detect any alteration in serum glucose, insulin, or lipid levels; glucose or insulin tolerance; or energy metabolism. Studies in Trib3−/− hepatocytes revealed normal Akt and glycogen synthase kinase- 3β phosphorylation patterns, glycogen levels, and expressions of key regulatory gluconeogenic and glycolytic genes. These data demonstrate that deletion of Trib3 has minimal effect on insulin-induced Akt activation in hepatic tissue, and, as such, they question any nonredundant role for Trb3 in the maintenance of glucose and energy homeostasis in mice.

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.

Analysis of knockout mice suggests a role for VGF in the control of fat storage and energy expenditure

BackgroundPrevious studies of mixed background mice have demonstrated that targeted deletion of Vgf produces a lean, hypermetabolic mouse that is resistant to diet-, lesion-, and genetically-induced obesity. To investigate potential mechanism(s) and site(s) of action of VGF, a neuronal and endocrine secreted protein and neuropeptide precursor, we further analyzed the metabolic phenotypes of two independent VGF knockout lines on C57Bl6 backgrounds.ResultsUnlike hyperactive VGF knockout mice on a mixed C57Bl6-129/SvJ background, homozygous mutant mice on a C57Bl6 background were hypermetabolic with similar locomotor activity levels to Vgf+/Vgf+ mice, during day and night cycles, indicating that mechanism(s) other than hyperactivity were responsible for their increased energy expenditure. In Vgf-/Vgf- knockout mice, morphological analysis of brown and white adipose tissues (BAT and WAT) indicated decreased fat storage in both tissues, and decreased adipocyte perimeter and area in WAT. Changes in gene expression measured by real-time RT-PCR were consistent with increased fatty acid oxidation and uptake in BAT, and increased lipolysis, decreased lipogenesis, and brown adipocyte differentiation in WAT, suggesting that increased sympathetic nervous system activity in Vgf-/Vgf- mice may be associated with or responsible for alterations in energy expenditure and fat storage. In addition, uncoupling protein 1 (UCP1) and UCP2 protein levels, mitochondrial number, and mitochondrial cristae density were upregulated in Vgf-/Vgf- BAT. Using immunohistochemical and histochemical techniques, we detected VGF in nerve fibers innervating BAT and Vgf promoter-driven reporter expression in cervical and thoracic spinal ganglia that project to and innervate the chest wall and tissues including BAT. Moreover, VGF peptide levels were quantified by radioimmunoassay in BAT, and were found to be down-regulated by a high fat diet. Lastly, despite being hypermetabolic, VGF knockout mice were cold intolerant.ConclusionWe propose that VGF and/or VGF-derived peptides modulate sympathetic outflow pathways to regulate fat storage and energy expenditure.

Glucagon Receptor Blockade With a Human Antibody Normalizes Blood Glucose in Diabetic Mice and Monkeys

Antagonizing glucagon action represents an attractive therapeutic option for reducing hepatic glucose production in settings of hyperglycemia where glucagon excess plays a key pathophysiological role. We therefore generated REGN1193, a fully human monoclonal antibody that binds and inhibits glucagon receptor (GCGR) signaling in vitro. REGN1193 administration to diabetic ob/ob and diet-induced obese mice lowered blood glucose to levels observed in GCGR-deficient mice. In diet-induced obese mice, REGN1193 reduced food intake, adipose tissue mass, and body weight. REGN1193 increased circulating levels of glucagon and glucagon-like peptide 1 and was associated with reversible expansion of pancreatic α-cell area. Hyperglucagonemia and α-cell hyperplasia was observed in fibroblast growth factor 21-deficient mice treated with REGN1193. Single administration of REGN1193 to diabetic cynomolgus monkeys normalized fasting blood glucose and glucose tolerance and increased circulating levels of glucagon and amino acids. Finally, administration of REGN1193 for 8 weeks to normoglycemic cynomolgus monkeys did not cause hypoglycemia or increase pancreatic α-cell area. In summary, the GCGR-blocking antibody REGN1193 normalizes blood glucose in diabetic mice and monkeys but does not produce hypoglycemia in normoglycemic monkeys. Thus, REGN1193 provides a potential therapeutic modality for diabetes mellitus and acute hyperglycemic conditions.

Reduction of LDL cholesterol by a monoclonal antibody to PCSK9 in rodents and nonhuman primates

Abstract Aim: PCSK9 regulates serum LDL cholesterol (LDL-C) by binding hepatic LDL receptor (LDLR) and targeting it to the lysosome for degradation. Fully human monoclonal antibodies were generated against PCSK9 to block its interaction with LDLR and decrease serum LDL-C. Materials & methods: A high affinity human monoclonal antibody, REGN727/SAR236553 (REGN727), was isolated using VelocImmune® technology and evaluated for effects in relevant animal models. Results: After 8 weeks on a high carbohydrate diet, humanized Pcsk9hum/hum mice had elevated serum PCSK9 and LDL-C levels. REGN727 effectively reduced LDL-C back to prediet levels. Administration of REGN727 to hyperlipidemic Pcsk9hum/humLdlr -/+ mice led to significantly reduced LDL‑C and increased hepatic LDLR levels. Furthermore, administration of a single intravenous dose of REGN727 to normal cynomolgus monkeys reduced serum LDL-C levels by up to 75% for >;20 days. Conclusion: Based on these preclinical findings, REGN727 may provide an effective treatment for hypercholesterolemia. Further clinical investigations are ongoing.

Glucagon receptor inhibition normalizes blood glucose in severe insulin-resistant mice.

Significance Insulin and glucagon are key hormones controlling blood glucose levels. Insulin binding to its receptor promotes glucose disposal in peripheral tissues and suppresses hepatic glucose output. Patients with inactivating mutations in their insulin receptors experience severe insulin resistance and uncontrolled diabetes. No effective therapy is available. Here we demonstrate that glucagon receptor (GCGR) blockade with monoclonal antibody normalized blood glucose in a mouse model of extreme insulin resistance and hyperglycemia. A surprising finding was that compensatory expansions of α- and β-cell masses in settings of inhibited glucagon and insulin signaling occurred at normal glucose levels. The data show that GCGR antibody inhibition represents a potential therapeutic option for patients with extreme insulin-resistance syndromes. Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and β-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic β-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled β-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.

Angptl4 does not control hyperglucagonemia or α-cell hyperplasia following glucagon receptor inhibition

Significance Glucagon supports glucose homeostasis by stimulating hepatic glucose output. Inhibition of glucagon signaling has drawn much attention because of potential implications for diabetes treatment. It is well established that inhibition of glucagon signaling effectively lowers blood glucose but results in compensatory glucagon hypersecretion and expansion of pancreatic α-cell mass. It was recently proposed that Angptl4, an inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance, links glucagon receptor inhibition to α-cell proliferation. Here we confirm that Angptl4 is a powerful regulator of plasma triglycerides, but not of hyperglucagonemia or α-cell hyperplasia. We observed an increase in plasma amino acids in humans following administration of a glucagon receptor-blocking antibody, confirming preclinical findings indicate that amino acids mediate the compensatory α-cell response. Genetic disruption or pharmacologic inhibition of glucagon signaling effectively lowers blood glucose but results in compensatory glucagon hypersecretion involving expansion of pancreatic α-cell mass. Ben-Zvi et al. recently reported that angiopoietin-like protein 4 (Angptl4) links glucagon receptor inhibition to hyperglucagonemia and α-cell proliferation [Ben-Zvi et al. (2015) Proc Natl Acad Sci USA 112:15498–15503]. Angptl4 is a secreted protein and inhibitor of lipoprotein lipase-mediated plasma triglyceride clearance. We report that Angptl4−/− mice treated with an anti-glucagon receptor monoclonal antibody undergo elevation of plasma glucagon levels and α-cell expansion similar to wild-type mice. Overexpression of Angptl4 in liver of mice caused a 8.6-fold elevation in plasma triglyceride levels, but did not alter plasma glucagon levels or α-cell mass. Furthermore, administration of glucagon receptor-blocking antibody to healthy individuals increased plasma glucagon and amino acid levels, but did not change circulating Angptl4 concentration. These data show that Angptl4 does not link glucagon receptor inhibition to compensatory hyperglucagonemia or expansion of α-cell mass, and that it cannot be given to induce such secretion and growth. The reduction of plasma triglyceride levels in Angptl4−/− mice and increase following Angptl4 overexpression suggest that changes in plasma triglyceride metabolism do not regulate α-cells in the pancreas. Our findings corroborate recent data showing that increased plasma amino acids and their transport into α-cells link glucagon receptor blockage to α-cell hyperplasia.

Activin A more prominently regulates muscle mass in primates than does GDF8

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.

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.