Organ donor pancreases for the study of human islet cell histology and pathophysiology: a precious and valuable resource

Direct in vivo assessment of pancreatic islet-cells for the study of the pathophysiology of diabetes in humans is hampered by anatomical and technological hurdles. To date, most of the information that has been generated is derived from histological studies performed on pancreatic tissue from autopsy, surgery, in vivo biopsy or organ donation. Each approach has its advantages and disadvantages (as summarised in this commentary); however, in this edition of Diabetologia, Kusmartseva et al (https://doi.org/10.1007/s00125-017-4494-x) provide further evidence to support the use of organ donor pancreases for the study of human diabetes. They show that length of terminal hospitalisation of organ donors prior to death does not seem to influence the frequency of inflammatory cells infiltrating the pancreas and the replication of beta cells. These findings are reassuring, demonstrating the reliability of this precious and valuable resource for human islet cells research

Pancreatic Beta Cell Identity in Humans and the Role of Type 2 Diabetes

Pancreatic beta cells uniquely synthetize and release insulin. Specific molecular, functional as well as ultrastructural traits characterize their insulin secretion properties and survival phenotype. In this review we focus on human islet/beta cells, and describe the changes that occur in type 2 diabetes and could play roles in the disease as well as represent possible targets for therapeutical interventions. These include transcription factors, molecules involved in glucose metabolism and insulin granule handling. Quantitative and qualitative insulin release patterns and their changes in type 2 diabetes are also associated with ultrastructural features involving the insulin granules, the mitochondria, and the endoplasmic reticulum

Response of young and adult birds to the same environmental variables and different spatial scales during post breeding period

Context: How do young birds achieve spatial knowledge about the environment during the initial stages of their life? They may follow adults, so gaining social information and learning; alternatively, young birds may acquire knowledge of the environment themselves by experiencing habitat and landscape features. If learning is at least partially independent of adults then young birds should respond to landscape composition at finer spatial scale than adults, who possess knowledge over a larger area. Objectives: We studied the responses of juvenile, immature and adult Caspian Gull Larus cachinnans to the same habitat and landscape variables, but at several spatial scales (ranging from 2.5 to 15\ua0km), during post-breeding period. Methods: We surveyed 61 fish ponds (foraging patches) in southern Poland and counted Caspian gulls. Results: Juvenile birds responded at finer spatial scales to the factors than did adults. Immature birds showed complicated, intermediate responses to spatial scale. The abundance of juvenile birds was mostly correlated with the landscape composition (positively with the cover of corridors and negatively with barriers). Adult abundance was positively related to foraging patch quality (fish stock), which clearly required previous spatial experience of the environment. The abundance of all age classes were moderately correlated with each other indicating that social behaviour may also contribute to the learning of the environment. Conclusions: This study shows that as birds mature, they respond differently to components of their environment at different spatial scales. This has considerable ecological consequences for their distribution across environments

Histopathology and ex vivo insulin secretion of pancreatic islets in gestational diabetes: A case report

Gestational diabetes (GD) results from insufficient endogenous insulin supply. No information is available on features of islet cells in human GD. Herein, we describe several properties of islets from a woman with GD. Immunohistochemical stainings and EM analyses were performed on pancreatic samples. Islet isolation was achieved by enzymatic dissociation and density gradient centrifugation. Ex vivo insulin secretion was studied in response to fuel secretagogues. Control islets were obtained from matched non-pregnant, non-diabetic women. Total insulin positive area was lower in GD, mainly due to the presence of smaller islets. β-cell apoptosis and the presence of Ki67 positive islet cells were similar in GD and controls, whereas the amount of insulin positive cells in or close to the ducts was decreased in GD. Ex vivo insulin secretion did not differ between GD and non-pregnant, non-diabetic islets. These findings suggest that in this case of human GD there might mainly be a defect of β-cell amount, not due to increased apoptosis, but possibly to insufficient regeneration

Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition

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. Graphical Abstract

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P < 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Human beta cell dysfunction induced by different lipoglucotoxic conditions may be transient or persistent and associates with specific transcriptomic changes which are shared in type 2 diabetes

Pancreatic beta-cell failure is determined by the interplay of genetic and acquired factors and represent the key event that leads to the development and progression of type 2 diabetes (T2D). The cause of this derangement is not completely clarified. Work in vivo shows evidence that alleviation of metabolic stress, through low calorie diet, administration of glucose-lowering drugs or bariatric surgery, can improve beta-cell function. Studies in vitro demonstrated that prolonged exposure to saturated fatty acids (lipotoxicity), high glucose (glucotoxicity) or combinations thereof (lipoglucotoxicity) may contribute to beta-cell failure, possibly via endoplasmic reticulum (ER) stress, oxidative stress, loss of identity and/or other mechanisms. In addition, the molecular mechanisms underlying the persistence or transience of human beta-cell defects are still to be investigated to unveil to which extent the functional and molecular modifications (and possible recovery) in non-diabetic (ND) islets reflect those of islets from T2D individuals. The present thesis aimed first to assess the direct impact of different lipoglucotoxic treatments on human beta-cell function, and then to evaluate if the deleterious effects were persistent or reversible after washout. Finally, the associated transcriptomic changes were analyzed and compared with T2D islet gene expression signature. Islets obtained from 26 ND organ donors were cultured in M199 medium, containing 5.5 mmol/l glucose, for 2 days (D2); then batches of islets were cultured for 2 additional days (D4) either in absence (ctrl) or in the presence of: 0.5 palmitate (P), 11.1 mmol/l glucose (g), 22.2 mmol/l glucose (G), 0.5 mmol/l palmitate + 11.1 mmol/l glucose (P+g), 0.5 mmol/l palmitate + 22.2 mmol/l glucose (P+G), 1.0 mmol/l palmitate + oleate, (1:2 molar ratio, P+O), 1.0 mmol/l palmitate + oleate + 11.1 mmol/l glucose (P+O+g), and 1.0 mmol/l palmitate + oleate + 22.2 mmol/l glucose (P+O+G). At D4, islets were washed and incubated with plain M199 medium for 4 additional days (D8 control and D8 washout, according to the incubation condition). Furthermore, 28 ND and 58 T2D islet preparations were studied. Glucose-stimulated insulin secretion (GSIS) from all the preparations was assessed, and islets were also prepared for RNA extraction and, in selected cases, for histology. No significant change occurred in GSIS with ctrl, g, P+O and P+O+g islets throughout the study period. However, GSIS at D4 declined (p<0.05 or less) with P, G, P+g, P+G and P+O+G exposure. Normalization of GSIS was observed at D8 washout vs D4 with P, G and P+g, but not with P+G and P+O+G. For P, G and P+G conditions, islet transcriptome and genome features were analyzed by RNA-sequencing and eQTL, respectively, to unveil the molecular mechanisms underlying beta-cell damage and its reversal. For the conditions where islets were exposed to P (functionally impaired at D4 and rescued at D8 washout), treated islets compared to control islets at D4 had 646 differentially (FDR<0.05) expressed genes (272 up- and 374 downregulated). Of these, 595 were protein-coding genes (248 up- and 347 downregulated by palmitate), including genes involved in lipid metabolism, inflammation and other cell functions. Enrichment analysis identified several P-modified functional categories including upregulation of unfolded protein response, acyl-CoA biosynthesis and fatty acid metabolism and others. Comparison of D8 washout vs D4 palmitate-treated islets identified 714 genes differentially expressed (167 up- and 547 down-). Of these, 656 were protein-coding (142 up- and 514 down-) comprising genes with a role in lipid/glucose metabolism, transcription, inflammation, beta-cell function and others. The Enrichment Map regarding D8 washed out islets showed downregulation of fatty acid metabolism and upregulation of carbohydrate catabolic processes. As for human islets exposed to G (impaired GSIS at D4 with recovery after washout), the comparison of G-exposed islets vs control islets at D4 showed 50 differentially expressed genes (38 up- and 12 down-); among these, 42 were protein-coding (32 up- and 10 downregulated by high glucose) including genes involved in metabolic pathways, gated channel activity and other cell functions. Enrichment analysis showed inhibition of cell junction and metabolic processes. D8 washout vs D4 G-treated islets RNA-seq data comparison resulted in 341 differentially expressed genes (81 up- and 260 down-regulated). Of these, 320 were protein-coding (70 up- and 250 down-), also involved in extracellular organization, establishment of protein localization to ER and glycerophospholipid metabolism. The relative Enrichment Map showed downregulation of response to wounding, myeloid cell differentiation and chemotaxis. More profound changes in islet transcriptome were observed with combined P+G (beta-cell dysfunction at D4 and persistence after washout). D4 P+G-exposed islets resulted in differential expression of 1,498 genes (756 up- and 742 down-). Of these, 1,386 were protein-coding (699 up- and 687 down-) and they were mainly related to transcription, inflammation, cell turnover, ion channels/transporters, mitochondrial function, and redox balance. The Enrichment Map showed clustering of interrelated gene-sets upregulated for most and comprising the unfolded protein response, protein degradation, mRNA splicing regulation and ER stress-induced apoptosis. The RNA-seq data of human islets at P+G D8 washout vs D4 treated identified only 322 genes differentially expressed (120 up- and 202 down-). Of them, 292 were protein-coding (102 up- and 190 down-). Finally, the molecular changes associated with persistent or transient beta-cell insulin secretion defects were correlated with those of human islets from T2D donors compared with ND donors. The Rank-Rank Hypergeometric Overlap (RRHO) approach was used, which allows to compare differentially expressed transcriptomes between independent experiments. Overall, the RRHO analyses showed that persistent or transient human beta-cell dysfunction induced by metabolic stress was accompanied by specific gene expression signatures that were shared with T2D, with the greatest concordant overlap between conditions that induce beta-cell dysfunction and fail to recover after washout, namely P+G and P+O+G. These results, obtained during the PhD course, show that certain lipoglucotoxic conditions may induce persistent or reversible beta-cell dysfunction, depending on the type, concentration and combination of the stressors. This associates with specific molecular changes that overlap with T2D islet traits. Identification of novel mechanisms responsible for human beta-cell functional deterioration and rescue, which are shared in T2D, could provide novel insights into T2D pathogenesis and should foster the development of improved beta-cell specific therapeutic approaches