Human Pancreatic Islets React to Glucolipotoxicity by Secreting Pyruvate and Citrate.

Progressive decline in pancreatic beta-cell function is central to the pathogenesis of type 2 diabetes (T2D). Here, we explore the relationship between the beta cell and its nutritional environment, asking how an excess of energy substrate leads to altered energy production and subsequent insulin secretion. Alterations in intracellular metabolic homeostasis are key markers of islets with T2D, but changes in cellular metabolite exchanges with their environment remain unknown. We answered this question using nuclear magnetic resonance-based quantitative metabolomics and evaluated the consumption or secretion of 31 extracellular metabolites from healthy and T2D human islets. Islets were also cultured under high levels of glucose and/or palmitate to induce gluco-, lipo-, and glucolipotoxicity. Biochemical analyses revealed drastic alterations in the pyruvate and citrate pathways, which appear to be associated with mitochondrial oxoglutarate dehydrogenase (OGDH) downregulation. We repeated these manipulations on the rat insulinoma-derived beta-pancreatic cell line (INS-1E). Our results highlight an OGDH downregulation with a clear effect on the pyruvate and citrate pathways. However, citrate is directed to lipogenesis in the INS-1E cells instead of being secreted as in human islets. Our results demonstrate the ability of metabolomic approaches performed on culture media to easily discriminate T2D from healthy and functional islets.

Fluorescein-based sensors to purify human α-cells for functional and transcriptomic analyses.

Pancreatic α-cells secrete glucagon, an insulin counter-regulatory peptide hormone critical for the maintenance of glucose homeostasis. Investigation of the function of human α-cells remains a challenge due to the lack of cost-effective purification methods to isolate high-quality α-cells from islets. Here, we use the reaction-based probe diacetylated Zinpyr1 (DA-ZP1) to introduce a novel and simple method for enriching live α-cells from dissociated human islet cells with ~95% purity. The α-cells, confirmed by sorting and immunostaining for glucagon, were cultured up to 10 days to form α-pseudoislets. The α-pseudoislets could be maintained in culture without significant loss of viability, and responded to glucose challenge by secreting appropriate levels of glucagon. RNA-sequencing analyses (RNA-seq) revealed that expression levels of key α-cell identity genes were sustained in culture while some of the genes such as DLK1, GSN, SMIM24 were altered in α-pseudoislets in a time-dependent manner. In conclusion, we report a method to sort human primary α-cells with high purity that can be used for downstream analyses such as functional and transcriptional studies.

Long-term cultures of human pancreatic islets in self-assembling peptides hydrogels.

Human pancreatic islets transplantation is an experimental therapeutic treatment for Type I Diabetes. Limited islets lifespan in culture remains the main drawback, due to the absence of native extracellular matrix as mechanical support after their enzymatic and mechanical isolation procedure. Extending the limited islets lifespan by creating a long-term in vitro culture remains a challenge. In this study, three biomimetic self-assembling peptides were proposed as potential candidates to recreate in vitro a pancreatic extracellular matrix, with the aim to mechanically and biologically support human pancreatic islets, by creating a three-dimensional culture system. The embedded human islets were analyzed for morphology and functionality in long-term cultures (14-and 28-days), by evaluating ß-cells content, endocrine component, and extracellular matrix constituents. The three-dimensional support provided by HYDROSAP scaffold, and cultured into MIAMI medium, displayed a preserved islets functionality, a maintained rounded islets morphology and an invariable islets diameter up to 4 weeks, with results analogues to freshly-isolated islets. In vivo efficacy studies of the in vitro 3D cell culture system are ongoing; however, preliminary data suggest that human pancreatic islets pre-cultured for 2 weeks in HYDROSAP hydrogels and transplanted under subrenal capsule may restore normoglycemia in diabetic mice. Therefore, engineered self-assembling peptide scaffolds may provide a useful platform for long-term maintenance and preservation of functional human pancreatic islets in vitro.

Isolation and Purification of Human Pancreatic Islets.

Successful islet isolation is the key to islet transplantation in diabetic patients. However, islet isolation is a technically complex and time-consuming manual process. Optimizing the islet isolation process can improve islet yield and quality, reduce operators, and thus reduce costs.The isolation and purification of human islets include pancreas acquisition and preservation, pancreas digestion, islet purification, islet culture, and islet quality identification. Briefly, after the duodenum was removed, the pancreas was trimmed, the main pancreatic duct was intubated at the distal end of the pancreatic head, collagenase was injected into the pancreatic duct, and the perfused pancreatic tissue was cut and then digested in a Ricordi chamber. A digestion temperature of 37 °C was continuously used to assess the number of samples and the integrity of the lysed and released islets. At the end of the digestion process, collect the digested tissue in a 500 mL centrifuge tube prefilled with 25 mL of cold (4 °C) human serum albumin and centrifuge twice at 150 g for 3 min. After mixing with UW solution as islet storage solution, put it on ice (shake occasionally to prevent clumping) after 30 min. Digested pancreatic tissue was centrifuged at 2200 rpm for 5 min in a COBE 2991 cell processor to isolate islets from exocrine tissue using a continuous density gradient. The purified islet fractions were washed twice in HBSS supplemented with 10% human serum albumin and finally collected in CMRL1066 medium supplemented with the corresponding liquid. The purity of purified islets was calculated by DTZ staining, the survival rate of islets was calculated by FDA/PI staining, and islet function was determined by in vitro glucose-stimulated insulin secretion test.

Microwell culture platform maintains viability and mass of human pancreatic islets.

Background: Transplantation of the human pancreatic islets is a promising approach for specific types of diabetes to improve glycemic control. Although effective, there are several issues that limit the clinical expansion of this treatment, including difficulty in maintaining the quality and quantity of isolated human islets prior to transplantation. During the culture, we frequently observe the multiple islets fusing together into large constructs, in which hypoxia-induced cell damage significantly reduces their viability and mass. In this study, we introduce the microwell platform optimized for the human islets to prevent unsolicited fusion, thus maintaining their viability and mass in long-term cultures. Method: Human islets are heterogeneous in size; therefore, two different-sized microwells were prepared in a 35 mm-dish format: 140 µm × 300 µm-microwells for <160 µm-islets and 200 µm × 370 µm-microwells for >160 µm-islets. Human islets (2,000 islet equivalent) were filtered through a 160 µm-mesh to prepare two size categories for subsequent two week-cultures in each microwell dish. Conventional flat-bottomed 35 mm-dishes were used for non-filtered islets (2,000 islet equivalent/2 dishes). Post-cultured islets are collected to combine in each condition (microwells and flat) for the comparisons in viability, islet mass, morphology, function and metabolism. Islets from three donors were independently tested. Results: The microwell platform prevented islet fusion during culture compared to conventional flat bottom dishes, which improved human islet viability and mass. Islet viability and mass on the microwells were well-maintained and comparable to those in pre-culture, while flat bottom dishes significantly reduced islet viability and mass in two weeks. Morphology assessed by histology, insulin-secreting function and metabolism by oxygen consumption did not exhibit the statistical significance among the three different conditions. Conclusion: Microwell-bottomed dishes maintained viability and mass of human islets for two weeks, which is significantly improved when compared to the conventional flat-bottomed dishes.

Sex Differences in the Molecular Programs of Pancreatic Cells Contribute to the Differential Risks of Type 2 Diabetes.

Epidemiology studies demonstrate that women are at a significantly lower risk of developing type 2 diabetes (T2D) compared to men. However, the molecular basis of this risk difference is not well understood. In this study, we examined the sex differences in the genetic programs of pancreatic endocrine cells. We combined pancreas perifusion data and single-cell genomic data from our laboratory and from publicly available data sets to investigate multiple axes of the sex differences in the human pancreas at the single-cell type and single-cell level. We systematically compared female and male islet secretion function, gene expression program, and regulatory principles of pancreatic endocrine cells. The perifusion data indicate that female endocrine cells have a higher secretion capacity than male endocrine cells. Single-cell RNA-sequencing analysis suggests that endocrine cells in male controls have molecular signatures that resemble T2D. In addition, we identified genomic elements associated with genome-wide association study T2D loci to have differential accessibility between female and male delta cells. These genomic elements may play a sex-specific causal role in the pathogenesis of T2D. We provide molecular mechanisms that explain the differential risk of T2D between women and men. Knowledge gained from our study will accelerate the development of diagnostics and therapeutics in sex-aware precision medicine for diabetes.

Nanovesicles From Lactobacillus johnsonii N6.2 Reduce Apoptosis in Human Beta Cells by Promoting AHR Translocation and IL10 Secretion.

L. johnsonii N6.2 releases nano-sized vesicles (NVs) with distinct protein and lipid contents. We hypothesized that these NVs play a central role in the delivery of bioactive molecules that may act as mechanistic effectors in immune modulation. In this report, we observed that addition of NVs to the human pancreatic cell line ßlox5 reduced cytokine-induced apoptosis. Through RNAseq analyses, increased expression of CYP1A1, CYP1B1, AHRR, and TIPARP genes in the aryl hydrocarbon receptor (AHR) pathways were found to be significantly induced in presence of NVs. AHR nuclear translocation was confirmed by confocal microscopy. The role of NVs on beta cell function was further evaluated using primary human pancreatic islets. It was found that NVs significantly increased insulin secretion in presence of high glucose concentrations. These increases positively correlated with increased GLUT6 and SREBF1 mRNA and coincided with reduced oxidative stress markers. Furthermore, incubation of NVs with THP-1 macrophages promoted the M2 tolerogenic phenotype through STAT3 activation, expression of AHR-dependent genes and secretion of IL10. Altogether, our findings indicate that bacterial NVs have the potential to modulate glucose homeostasis in the host by directly affecting insulin secretion by islets and through the induction of a tolerogenic immune phenotype.

Single-Cell Transcriptomics Links Loss of Human Pancreatic ß-Cell Identity to ER Stress.

The maintenance of pancreatic islet architecture is crucial for proper ß-cell function. We previously reported that disruption of human islet integrity could result in altered ß-cell identity. Here we combine ß-cell lineage tracing and single-cell transcriptomics to investigate the mechanisms underlying this process in primary human islet cells. Using drug-induced ER stress and cytoskeleton modification models, we demonstrate that altering the islet structure triggers an unfolding protein response that causes the downregulation of ß-cell maturity genes. Collectively, our findings illustrate the close relationship between endoplasmic reticulum homeostasis and ß-cell phenotype, and strengthen the concept of altered ß-cell identity as a mechanism underlying the loss of functional ß-cell mass.

Cathepsin C Regulates Cytokine-Induced Apoptosis in ß-Cell Model Systems.

Emerging evidence suggests that several of the lysosomal cathepsin proteases are genetically associated with type 1 diabetes (T1D) and participate in immune-mediated destruction of the pancreatic ß cells. We previously reported that the T1D candidate gene cathepsin H is downregulated by pro-inflammatory cytokines in human pancreatic islets and regulates ß-cell function, apoptosis, and disease progression in children with new-onset T1D. In the present study, the objective was to investigate the expression patterns of all 15 known cathepsins in ß-cell model systems and examine their role in the regulation of cytokine-induced apoptosis. Real-time qPCR screening of the cathepsins in human islets, 1.1B4 and INS-1E ß-cell models identified several cathepsins that were expressed and regulated by pro-inflammatory cytokines. Using small interfering RNAs to knock down (KD) the cytokine-regulated cathepsins, we identified an anti-apoptotic function of cathepsin C as KD increased cytokine-induced apoptosis. KD of cathepsin C correlated with increased phosphorylation of JNK and p38 mitogen-activated protein kinases, and elevated chemokine CXCL10/IP-10 expression. This study suggests that cathepsin C is a modulator of ß-cell survival, and that immune modulation of cathepsin expression in islets may contribute to immune-mediated ß-cell destruction in T1D.

The potential of the incorporated collagen microspheres in alginate hydrogel as an engineered three-dimensional microenvironment to attenuate apoptosis in human pancreatic islets.

BACKGROUND: Tissue engineering is considered as a promising tool for remodeling the native cells microenvironment. In the present study, the effect of alginate hydrogel and collagen microspheres integrated with extracellular matrix components were evaluated in the decrement of apoptosis in human pancreatic islets. MATERIALS/METHODS: For three-dimensional culture, the islets were encapsulated in collagen microspheres, containing laminin and collagen IV and embedded in alginate scaffold for one week. After that the islets were examined in terms of viability, apoptosis, genes and proteins expression including BAX, BCL2, active caspase-3, and insulin. Moreover, the islets function was evaluated through glucose-induced insulin and C-peptide secretion assay. In order to evaluate the structure of the scaffolds and the morphology of the pancreatic islets in three-dimensional microenvironments, we performed scanning electron microscopy. RESULTS: Our findings showed that the designed hydrogel scaffolds significantly improved the islets viability using the reduction of activated caspase-3 and TUNEL positive cells. CONCLUSIONS: The reconstruction of the destructed matrix with alginate hydrogels and collagen microspheres might be an effective step to promote the culture of the islets.

Circadian Lipidomics: Analysis of Lipid Metabolites Around the Clock.

Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.

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.

SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic ß-Cells and in the Human Pancreas Microvasculature.

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing ß-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human ß-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the ß-cell line EndoC-ßH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct ß-cell virus tropism.

Functional loss of pancreatic islets in type 2 diabetes: How can we halt it?

The loss of beta-cell functional mass is a necessary and early condition in the development of type 2 diabetes (T2D). In T2D patients, beta-cell function is already reduced by about 50% at diagnosis and further declines thereafter. Beta-cell mass is also reduced in subjects with T2D, and islets from diabetic donors are smaller compared to non-diabetic donors. Thus, beta-cell regeneration and/or preservation of the functional islet integrity should be highly considered for T2D treatment and possibly cure. To date, the available anti-diabetes drugs have been developed as "symptomatic" medications since they act to primarily reduce elevated blood glucose levels. However, a truly efficient anti-diabetes medication, capable to prevent the onset and progression of T2D, should stop beta-cell loss and/or promote the restoration of fully functional beta-cell mass, independently of reducing hyperglycemia and ameliorating glucotoxicity on the pancreatic islets. This review provides a view of the experimental and clinical evidence on the ability of available anti-diabetes drugs to exert protective effects on beta-cells, with a specific focus on human pancreatic islets and clinical trials. Potential explanations for the lack of concordance between evidence of beta-cell protection in vitro and of persistent amelioration of beta-cell function in vivo are also discussed.

A Versatile Model of Microfluidic Perifusion System for the Evaluation of C-Peptide Secretion Profiles: Comparison Between Human Pancreatic Islets and HLSC-Derived Islet-Like Structures.

A robust and easy-to-use tool for the ex vivo dynamic evaluation of pancreatic islet (PI) function is essential for further development of novel cell-based therapeutic approaches to treating diabetes. Here, we developed four different glucose perifusion protocols (GPPs) in a microfluidic perifusion system (MPS), based entirely on commercially available components. After validation, the GPPs were used to evaluate C-peptide secretion profiles of PIs derived from different donors (healthy, obese, and type 2 diabetic) and from human liver stem-cell-derived islet-like structures (HLSC-ILS). Using this device, we demonstrated that PIs derived from healthy donors displayed a physiological C-peptide secretion profile as characterized by the response to (a) different glucose concentrations, (b) consecutive pulses of high-glucose concentrations, (c) a glucose threshold ranging from 5-8 mM, and (d) a constant high-glucose perifusion in a biphasic manner. Moreover, we were able to detect a dysregulated secretion profile in PIs derived from both obese and type 2 diabetes mellitus (T2DM) donors. Finally, we also evaluated the kinetic secretion profiles of HLSC-ILS, demonstrating that, nonetheless, with a lower amplitude of secretion compared to PI derived from healthy donors, they were already glucose-responsive on day seven post-differentiation. In conclusion, we have provided evidence that our MPS is a versatile device and may represent a valuable tool to study insulin-producing cells in vitro.

Cytoprotective effects of ginsenoside Rd on apoptosis-associated cell death in the isolated human pancreatic islets.

Ginsenoside Rd (GS-Rd), one of the main pharmacologically active components of ginseng, has shown the potential to stabilize mitochondrial membrane integrity and decrease apoptotic death in neuronal and non-neuronal cells. The present study aimed to evaluate the effect of this bioactive molecule on the apoptosis-associated cell death in human pancreatic islets. In this regard human pancreatic islets were isolated and grouped for the treatment with GS-Rd. The isolated islets were treated with different concentrations of GS-Rd. After 24 and 72 h of incubation, the islets were evaluated in terms of viability, BAX, BCL2, and insulin gene expression, BAX, BCL2, and caspase-3 protein expression, apoptosis, and glucose-induced insulin/C-peptide secretion. Our results revealed the islet survival was significantly decreased in the control group after 72 h of incubation. However, GS-Rd inhibited the progress of the islet death in the treated groups. TUNEL staining revealed that the preventive effect of this molecule was caused by the inhibition of apoptosis-associated death. In this regard, the activation of caspase-3 was down-regulated in the presence of GS-Rd. GS-Rd did not exhibit undesirable effects on glucose-induced insulin and C-peptide stimulation secretion. In conclusion, GS-Rd inhibited the progress of death of cultured human pancreatic islets by diminishing the apoptosis of the islet cells.

GNAS gene is an important regulator of insulin secretory capacity in pancreatic ß-cells.

BACKGROUND: Type 2 diabetes (T2D) is a complex polygenic disease with unclear mechanism. In an attempt to identify novel genes involved in ß-cell function, we harness a bioinformatics method called Loss-of-function tool (LoFtool) gene score. METHODS: RNA-sequencing data from human islets were used to cross-reference genes within the 1st quartile of most intolerant LoFtool score with the 100th most expressed genes in human islets. Out of these genes, GNAS and EEF1A1 genes were selected for further investigation in diabetic islets, metabolic tissues along with their correlation with diabetic phenotypes. The influence of GNAS and EEF1A1 on insulin secretion and ß-cell function were validated in INS-1 cells. RESULTS: A comparatively higher expression level of GNAS and EEF1A1 was observed in human islets than fat, liver and muscle tissues. Furthermore, diabetic islets displayed a reduced expression of GNAS, but not of EEF1A, compared to non-diabetic islets. The expression of GNAS was positively correlated with insulin secretory index, GLP1R, GIPR and inversely correlated with HbA1c. Diabetic human islets displayed a reduced cAMP generation and insulin secretory capacity in response to glucose. Moreover, siRNA silencing of GNAS in INS-1 cells reduced insulin secretion, insulin content, and cAMP production. In addition, the expression of Insulin, PDX1, and MAFA was significantly down-regulated in GNAS-silenced cells. However, cell viability and apoptosis rate were unaffected. CONCLUSION: LoFtool is a powerful tool to identify genes associated with pancreatic islets dysfunction. GNAS is a crucial gene for the ß-cell insulin secretory capacity.

Polymerized laminin incorporation into alginate-based microcapsules reduces pericapsular overgrowth and inflammation.

Cell encapsulation coats cells with an artificial membrane to preserve their physical and functional integrity. Different approaches try to develop more functional and biocompatible materials to avoid cell loss after transplantation due to inflammatory reaction, one of the main causes for graft failure. In this study, the LN-Biodritin biomaterial, based on alginate, chondroitin sulfate, and laminin, previously developed by our group, was further improved by replacing laminin by polylaminin, an artificial laminin polymer with anti-inflammatory properties, generating the new biomaterial polyLN-Biodritin. Capsules containing polylaminin are stable, do not induce macrophage activation in vitro, and are also able to prevent macrophage activation by encapsulated human pancreatic islets in vitro, preserving their glucose-stimulated insulin secretion potential. In addition, when empty capsules containing polylaminin were implanted into immunocompetent mice, the inflammatory response towards the implant was attenuated, when compared with capsules without polylaminin. The results indicate that polylaminin incorporation leads to lower levels of pericapsular growth on the capsules surface, lower infiltration of cells into the peritoneal cavity, and lower production of proinflammatory cytokines, both at the implant site (interleukin-12p70 (IL-12p70), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interferon-γ (IFN-γ)) and systemically (IL-12p70 and TNF-α). Therefore, polylaminin incorporation into the microcapsules polymer attenuates the host posttransplantation immune response against implanted microcapsules, being likely to favor maintenance of engrafted encapsulated cells.

Amelioration of the apoptosis-mediated death in isolated human pancreatic islets by minocycline.

Minocycline functions as a therapeutic drug in different diseases because of its cytoprotective properties. In the present study, we examined the potential of minocycline to decrease the islet loss in pre-transplantation culture stage. Pancreatic islets were isolated from the deceased donors and treated by 0, 2, 10, and 20 µM minocycline for 24 and 72 h. After that, the incubated islets were evaluated for viability and function. Apoptosis markers including Bax, Bcl2, and caspase-3 were determined at gene and protein level. On the other hand, TUNEL assay was used to confirm apoptosis. The functionality of the islets was investigated using glucose-induced insulin and c-peptide secretion assay. After 72 h of incubation, the viability of human islet was drastically decreased, whereas supplementation with minocycline inhibited the cells death. In this regard, the expression of Bax and active Caspase-3 was downregulated, whereas the expression of Bcl2 was upregulated. These consequences suggest that pancreatic islets undergo apoptosis in vitro and minocycline can decelerate or inhibit this process. Our findings identified minocycline as a cytoprotective molecule for preventing human islets death in pre-transplantation culture.