Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

AIMS/HYPOTHESIS: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility, which we explore here by focusing on the imprinted gene Nnat (encoding neuronatin [NNAT]), which is required for normal insulin synthesis and secretion. METHODS: Single-cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing enhanced GFP under the control of the Nnat enhancer/promoter regions were generated for FACS of beta cells and downstream analysis of CpG methylation by bisulphite sequencing and RNA-seq, respectively. Animals deleted for the de novo methyltransferase DNA methyltransferase 3 alpha (DNMT3A) from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 AM and Cal-590 AM. Insulin secretion was measured using homogeneous time-resolved fluorescence imaging. RESULTS: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic datasets demonstrated the early establishment of Nnat-positive and -negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a subpopulation specialised for insulin production, and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialisation. CONCLUSIONS/INTERPRETATION: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes. DATA AVAILABILITY: The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD048465.

Angiotensin I-converting enzyme type 2 expression is increased in pancreatic islets of type 2 diabetic donors.

AIMS: Angiotensin I-converting enzyme type 2 (ACE2), a pivotal SARS-CoV-2 receptor, has been shown to be expressed in multiple cells, including human pancreatic beta-cells. A putative bidirectional relationship between SARS-CoV-2 infection and diabetes has been suggested, confirming the hypothesis that viral infection in beta-cells may lead to new-onset diabetes or worse glycometabolic control in diabetic patients. However, whether ACE2 expression levels are altered in beta-cells of diabetic patients has not yet been investigated. Here, we aimed to elucidate the in situ expression pattern of ACE2 in Type 2 diabetes (T2D) with respect to non-diabetic donors which may account for a higher susceptibility to SARS-CoV-2 infection in beta-cells. MATERIAL AND METHODS: Angiotensin I-converting enzyme type 2 immunofluorescence analysis using two antibodies alongside insulin staining was performed on formalin-fixed paraffin embedded pancreatic sections obtained from n = 20 T2D and n = 20 non-diabetic (ND) multiorgan donors. Intensity and colocalisation analyses were performed on a total of 1082 pancreatic islets. Macrophage detection was performed using anti-CD68 immunohistochemistry on serial sections from the same donors. RESULTS: Using two different antibodies, ACE2 expression was confirmed in beta-cells and in pancreas microvasculature. Angiotensin I-converting enzyme type 2 expression was increased in pancreatic islets of T2D donors in comparison to ND controls alongside with a higher colocalisation rate between ACE2 and insulin using both anti-ACE2 antibodies. CD68+ cells tended to be increased in T2D pancreata, in line with higher ACE2 expression observed in serial sections. CONCLUSIONS: Higher ACE2 expression in T2D islets might increase their susceptibility to SARS-CoV-2 infection during COVID-19 in T2D patients, thus worsening glycometabolic outcomes and disease severity.

A direct look at the dysfunction and pathology of the ß cells in human type 2 diabetes.

ß cells uniquely produce and secrete insulin under the control of several, integrated signals, to maintain blood glucose concentrations within a narrow physiological interval. ß cell failure is key to the onset and progression of type 2 diabetes, due to impaired function and reduced mass. In this review we focus on several features of human ß cell dysfunction and pathology in type 2 diabetes, as revealed by direct assessment of isolated islet traits and examination of pancreatic tissue from organ donors, surgical samples or autoptic specimens. Insulin secretion defects and pathology findings are discussed in relation to some of the major underlying mechanisms, to also provide clues for conceiving better prevention and treatment of type 2 diabetes by targeting the pancreatic ß cells.

The Role of Beta Cell Recovery in Type 2 Diabetes Remission.

Type 2 diabetes (T2D) has been considered a relentlessly worsening disease, due to the progressive deterioration of the pancreatic beta cell functional mass. Recent evidence indicates, however, that remission of T2D may occur in variable proportions of patients after specific treatments that are associated with recovery of beta cell function. Here we review the available information on the recovery of beta cells in (a) non-diabetic individuals previously exposed to metabolic stress; (b) T2D patients following low-calorie diets, pharmacological therapies or bariatric surgery; (c) human islets isolated from non-diabetic organ donors that recover from "lipo-glucotoxic" conditions; and (d) human islets isolated from T2D organ donors and exposed to specific treatments. The improvement of insulin secretion reported by these studies and the associated molecular traits unveil the possibility to promote T2D remission by directly targeting pancreatic beta cells.

Arginase 2 and Polyamines in Human Pancreatic Beta Cells: Possible Role in the Pathogenesis of Type 2 Diabetes.

Arginase 2 (ARG2) is a manganese metalloenzyme involved in several tissue specific processes, from physiology to pathophysiology. It is variably expressed in extra-hepatic tissues and is located in the mitochondria. In human pancreatic beta cells, ARG2 is downregulated in type 2 diabetes. The enzyme regulates the synthesis of polyamines, that are involved in pancreas development and regulation of beta cell function. Here, we discuss several features of ARG2 and polyamines, which can be relevant to the pathophysiology of type 2 diabetes.

Differential CpG methylation at Nnat in the early establishment of beta cell heterogeneity.

Aims/hypothesis: Beta cells within the pancreatic islet represent a heterogenous population wherein individual sub-groups of cells make distinct contributions to the overall control of insulin secretion. These include a subpopulation of highly-connected 'hub' cells, important for the propagation of intercellular Ca2+ waves. Functional subpopulations have also been demonstrated in human beta cells, with an altered subtype distribution apparent in type 2 diabetes. At present, the molecular mechanisms through which beta cell hierarchy is established are poorly understood. Changes at the level of the epigenome provide one such possibility which we explore here by focussing on the imprinted gene neuronatin (Nnat), which is required for normal insulin synthesis and secretion. Methods: Single cell RNA-seq datasets were examined using Seurat 4.0 and ClusterProfiler running under R. Transgenic mice expressing eGFP under the control of the Nnat enhancer/promoter regions were generated for fluorescence-activated cell (FAC) sorting of beta cells and downstream analysis of CpG methylation by bisulphite and RNA sequencing, respectively. Animals deleted for the de novo methyltransferase, DNMT3A from the pancreatic progenitor stage were used to explore control of promoter methylation. Proteomics was performed using affinity purification mass spectrometry and Ca2+ dynamics explored by rapid confocal imaging of Cal-520 and Cal-590. Insulin secretion was measured using Homogeneous Time Resolved Fluorescence Imaging. Results: Nnat mRNA was differentially expressed in a discrete beta cell population in a developmental stage- and DNA methylation (DNMT3A)-dependent manner. Thus, pseudo-time analysis of embryonic data sets demonstrated the early establishment of Nnat-positive and negative subpopulations during embryogenesis. NNAT expression is also restricted to a subset of beta cells across the human islet that is maintained throughout adult life. NNAT+ beta cells also displayed a discrete transcriptome at adult stages, representing a sub-population specialised for insulin production, reminiscent of recently-described "ßHI" cells and were diminished in db/db mice. 'Hub' cells were less abundant in the NNAT+ population, consistent with epigenetic control of this functional specialization. Conclusions/interpretation: These findings demonstrate that differential DNA methylation at Nnat represents a novel means through which beta cell heterogeneity is established during development. We therefore hypothesise that changes in methylation at this locus may thus contribute to a loss of beta cell hierarchy and connectivity, potentially contributing to defective insulin secretion in some forms of diabetes.

A single-cell human islet interactome atlas identifies disrupted autocrine and paracrine communications in type 2 diabetes.

A sensible control of hormone secretion from pancreatic islets requires concerted inter-cellular communications, but a comprehensive picture of the whole islet interactome is presently missing. Single-cell transcriptomics allows to overcome this and we used here a single-cell dataset from type 2 diabetic (T2D) and non-diabetic (ND) donors to leverage islet interaction networks. The single-cell dataset contains 3046 cells classified in 7 cell types. The interactions across cell types in T2D and ND were obtained and resulting networks analysed to identify high-centrality genes and altered interactions in T2D. The T2D interactome displayed a higher number of interactions (10 787) than ND (9707); 1289 interactions involved beta cells (1147 in ND). High-centrality genes included EGFR, FGFR1 and FGFR2, important for cell survival and proliferation. In conclusion, this analysis represents the first in silico model of the human islet interactome, enabling the identification of signatures potentially relevant for T2D pathophysiology.

The Protective Action of Metformin against Pro-Inflammatory Cytokine-Induced Human Islet Cell Damage and the Mechanisms Involved.

Metformin, a drug widely used in type 2 diabetes (T2D), has been shown to protect human ß-cells exposed to gluco- and/or lipotoxic conditions and those in islets from T2D donors. We assessed whether metformin could relieve the human ß-cell stress induced by pro-inflammatory cytokines (which mediate ß-cells damage in type 1 diabetes, T1D) and investigated the underlying mechanisms using shotgun proteomics. Human islets were exposed to 50 U/mL interleukin-1ß plus 1000 U/mL interferon-γ for 48 h, with or without 2.4 µg/mL metformin. Glucose-stimulated insulin secretion (GSIS) and caspase 3/7 activity were studied, and a shotgun label free proteomics analysis was performed. Metformin prevented the reduction of GSIS and the activation of caspase 3/7 induced by cytokines. Proteomics analysis identified more than 3000 proteins in human islets. Cytokines alone altered the expression of 244 proteins (145 up- and 99 down-regulated), while, in the presence of metformin, cytokine-exposure modified the expression of 231 proteins (128 up- and 103 downregulated). Among the proteins inversely regulated in the two conditions, we found proteins involved in vesicle motility, defense against oxidative stress (including peroxiredoxins), metabolism, protein synthesis, glycolysis and its regulation, and cytoskeletal proteins. Metformin inhibited pathways linked to inflammation, immune reactions, mammalian target of rapamycin (mTOR) signaling, and cell senescence. Some of the changes were confirmed by Western blot. Therefore, metformin prevented part of the deleterious actions of pro-inflammatory cytokines in human ß-cells, which was accompanied by islet proteome modifications. This suggests that metformin, besides use in T2D, might be considered for ß-cell protection in other types of diabetes, possibly including early T1D.

Correction to 'Integration of single-cell datasets reveals novel transcriptomic signatures of ß-cells in human type 2 diabetes'.

[This corrects the article DOI: 10.1093/nargab/lqaa097.].

Spatiotemporal Correlation Spectroscopy Reveals a Protective Effect of Peptide-Based GLP-1 Receptor Agonism against Lipotoxicity on Insulin Granule Dynamics in Primary Human ß-Cells.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are being used for the treatment of type 2 diabetes (T2D) and may have beneficial effects on the pancreatic ß-cells. Here, we evaluated the effects of GLP-1R agonism on insulin secretory granule (ISG) dynamics in primary ß-cells isolated from human islets exposed to palmitate-induced lipotoxic stress. Islets cells were exposed for 48 h to 0.5 mM palmitate (hereafter, 'Palm') with or without the addition of a GLP-1 agonist, namely 10 nM exendin-4 (hereafter, 'Ex-4'). Dissociated cells were first transfected with syncollin-EGFP in order to fluorescently mark the ISGs. Then, by applying a recently established spatiotemporal correlation spectroscopy technique, the average structural (i.e., size) and dynamic (i.e., the local diffusivity and mode of motion) properties of ISGs are extracted from a calculated imaging-derived Mean Square Displacement (iMSD) trace. Besides defining the structural/dynamic fingerprint of ISGs in human cells for the first time, iMSD analysis allowed to probe fingerprint variations under selected conditions: namely, it was shown that Palm affects ISGs dynamics in response to acute glucose stimulation by abolishing the ISGs mobilization typically imparted by glucose and, concomitantly, by reducing the extent of ISGs active/directed intracellular movement. By contrast, co-treatment with Ex-4 normalizes ISG dynamics, i.e., re-establish ISG mobilization and ability to perform active transport in response to glucose stimulation. These observations were correlated with standard glucose-stimulated insulin secretion (GSIS), which resulted in being reduced in cells exposed to Palm but preserved in cells concomitantly exposed to 10 nM Ex-4. Our data support the idea that GLP-1R agonism may exert its beneficial effect on human ß-cells under metabolic stress by maintaining ISGs' proper intracellular dynamics.

Pro-Inflammatory Cytokines Induce Insulin and Glucagon Double Positive Human Islet Cells That Are Resistant to Apoptosis.

The presence of islet cells double positive for insulin and glucagon (Ins+/Glu+) has been described in the pancreas from both type 2 (T2D) and type 1 (T1D) diabetic subjects. We studied the role of pro-inflammatory cytokines on the occurrence, trajectory, and characteristics of Ins+/Glu+ cells in human pancreatic islets. Pancreas samples, isolated islets, and dispersed islet cells from 3 T1D and 11 non-diabetic (ND) multi-organ donors were studied by immunofluorescence, confocal microscopy, and/or electron microscopy. ND islet cells were exposed to interleukin-1ß and interferon-γ for up to 120 h. In T1D islets, we confirmed an increased prevalence of Ins+/Glu+ cells. Cytokine-exposed islets showed a progressive increase of Ins+/Glu+ cells that represented around 50% of endocrine cells after 120h. Concomitantly, cells expressing insulin granules only decreased significantly over time, whereas those containing only glucagon granules remained stable. Interestingly, Ins+/Glu+ cells were less prone to cytokine-induced apoptosis than cells containing only insulin. Cytokine-exposed islets showed down-regulation of ß-cell identity genes. In conclusion, pro-inflammatory cytokines induce Ins+/Glu+ cells in human islets, possibly due to a switch from a ß- to a ß-/α-cell phenotype. These Ins+/Glu+ cells appear to be resistant to cytokine-induced apoptosis.

Integration of single-cell datasets reveals novel transcriptomic signatures of ß-cells in human type 2 diabetes.

Pancreatic islet ß-cell failure is key to the onset and progression of type 2 diabetes (T2D). The advent of single-cell RNA sequencing (scRNA-seq) has opened the possibility to determine transcriptional signatures specifically relevant for T2D at the ß-cell level. Yet, applications of this technique have been underwhelming, as three independent studies failed to show shared differentially expressed genes in T2D ß-cells. We performed an integrative analysis of the available datasets from these studies to overcome confounding sources of variability and better highlight common T2D ß-cell transcriptomic signatures. After removing low-quality transcriptomes, we retained 3046 single cells expressing 27 931 genes. Cells were integrated to attenuate dataset-specific biases, and clustered into cell type groups. In T2D ß-cells (n = 801), we found 210 upregulated and 16 downregulated genes, identifying key pathways for T2D pathogenesis, including defective insulin secretion, SREBP signaling and oxidative stress. We also compared these results with previous data of human T2D ß-cells from laser capture microdissection and diabetic rat islets, revealing shared ß-cell genes. Overall, the present study encourages the pursuit of single ß-cell RNA-seq analysis, preventing presently identified sources of variability, to identify transcriptomic changes associated with human T2D and underscores specific traits of dysfunctional ß-cells across different models and techniques.

Persistent or Transient Human ß Cell Dysfunction Induced by Metabolic Stress: Specific Signatures and Shared Gene Expression with Type 2 Diabetes.

Pancreatic ß cell failure is key to type 2 diabetes (T2D) onset and progression. Here, we assess whether human ß cell dysfunction induced by metabolic stress is reversible, evaluate the molecular pathways underlying persistent or transient damage, and explore the relationships with T2D islet traits. Twenty-six islet preparations are exposed to several lipotoxic/glucotoxic conditions, some of which impair insulin release, depending on stressor type, concentration, and combination. The reversal of dysfunction occurs after washout for some, although not all, of the lipoglucotoxic insults. Islet transcriptomes assessed by RNA sequencing and expression quantitative trait loci (eQTL) analysis identify specific pathways underlying ß cell failure and recovery. Comparison of a large number of human T2D islet transcriptomes with those of persistent or reversible ß cell lipoglucotoxicity show shared gene expression signatures. The identification of mechanisms associated with human ß cell dysfunction and recovery and their overlap with T2D islet traits provide insights into T2D pathogenesis, fostering the development of improved ß cell-targeted therapeutic strategies.

Author Correction to: Gene Patents in Canada: Is There a New Legal Landscape?

An error was subsequently identified in the article, and the following correction should be noted.

Gene Patents in Canada: Is There a New Legal Landscape?

In 2016, the Children's Hospital of Eastern Ontario (CHEO) announced the settlement of its patent lawsuit against US-based Transgenomic, Inc. At issue in the case was CHEO's ability to test for gene mutations associated with long QT syndrome (LQTS) that are described in Transgenomic's patents. CHEO challenged the patents as invalid, and Transgenomic ultimately agreed to license them on a royalty-free basis to CHEO and other healthcare institutions for LQTS testing and research. While widely celebrated in the media, the ethical rhetoric surrounding the settlement has at times obscured the practical and legal context in which it was made and will operate. Here, we provide a nuanced account of the events surrounding the settlement and its implications for research and clinical care. Although the settlement is remarkable for the transparency of its terms and its inclusion of a license intended to benefit unaffiliated test providers, we conclude that another significant implication of the settlement may be its elimination of the opportunity to clarify an increasingly confused area of Canadian law against a backdrop of continued international controversy surrounding the patenting of genes and gene-based diagnostic and therapeutic methods.

Patent disclosure requirements for therapeutic antibody patents.

INTRODUCTION: Therapeutic antibodies have grown to become an important product class within the biopharmaceutical market. A prerequisite to their commercialization is adequate patent protection. Disclosure requirements and the types of claims available in different jurisdictions can impact the scope of protection available for antibodies. Areas covered: A comparative review of statutory bases, patent office practices and selected decisions in Canada, the United States and the United Kingdom related to disclosure requirements is provided. Expert opinion: Differences in disclosure requirements exist in different jurisdictions which can impact the type of claims obtained and their survival when attacked in litigation. Including a wide variety of claim types is a key strategy to ensuring therapeutic antibodies are adequately protected. Method of use claims may provide advantages and broader protection in some circumstances and should also be considered.

Medicine patent pool--pharma philanthropy or PR?

Merck recently signed an agreement with The Medicines Patent Pool (MPP) to license intellectual property relating to pediatric formulations of its integrase HIV drug, raltegravir (Ral) (the 'Agreement'). The Agreement is alleged to clear the way for cheaper formulations for use in developing and some middle income countries and allows for the development of novel pediatric formulations of Ral as well as novel combinations. Merck's license is royalty free and under the terms of the Agreement, manufacturers anywhere in the world who meet the quality assurance criteria, can manufacture and sell pediatric versions of the drug in the licensed countries under the agreed conditions without paying a royalty to Merck. The Agreement covers at least 92 countries and MPP reports that 98.1% of children with HIV in the developing world live in the included countries. The Agreement has been criticized as a public relations exercise. The article asks if the criticism is justified and explores several aspects of the Agreement in addressing the question.

Solitary metachronous gastric metastasis from pulmonary adenocarcinoma: Report of a case.

INTRODUCTION: Gastric metastases from lung adenocarcinoma are rare and usually associated with disseminated disease. The great majority is asymptomatic and in few cases discovered during autopsy studies. Reports of single metachronous metastases during the lifetime are anecdotal. We describe a case of solitary gastric metastasis 5 years after lung surgery. PRESENTATION OF CASE: A 68-year-old male submitted in 2006 to right lobectomy for lung adenocarcinoma was referred at Emergency Room department in 01/2011 because of chronic epigastric pain. Radiologic and endoscopic evaluation showed a bulky lesion inside the stomach, originating from the muscular layer, suspected for GIST. He underwent a subtotal gastrectomy and the pathologic examination revealed an undifferentiated adenocarcinoma, positive for Thyroid Transcriptional Factor-1, Cytokeratin 7, AE 1/3 and CEA, confirming the pulmonary origin. DISCUSSION: At the time of diagnosis about 50% of lung cancer are metastatic, with survival rates of 1% at 5-year. Gastric metastasis is very rare; autopsy studies report an incidence of 0.2-0.5%. They develop in the submucosa, usually without any symptom and the diagnosis is incidental during the staging of primary cancer or the follow-up. There are no guidelines about surgical treatment; however few cases of long-term survival following the operation were reported. Pathologic diagnosis is difficult, but the immunohistochemical staining helps to recognize the primary origin. CONCLUSION: Solitary metachronous gastric metastasis from pulmonary adenocarcinoma is an exceptional event, but it could happen during the follow-up. It seems that a radical resection, in absence of systemic implants, might provide survival benefits in selected patients.