The Adrenal Gland and Pancreatic Islets - A Beneficial Endocrine Alliance.

Intraportal islet transplantation in patients with type 1 diabetes enables restoration of glucose-regulated insulin secretion. However, several factors hamper a widespread application and long-term success: chronic hypoxia, an inappropriate microenvironment and suppression of regenerative and proliferative potential by high local levels of immunosuppressive agents. Therefore, the identification of alternative and superior transplant sites is of major scientific and clinical interest. Here, we aim to evaluate the adrenal as an alternative transplantation site. The adrenal features a particular microenvironment with extensive vascularization, anti-apoptotic and pro-proliferative, anti-inflammatory and immunosuppressive effects. To validate this novel transplantation site, an in vitro co-culture system of adrenal cells and pancreatic islets was established and viability, islet survival, functional potency and antioxidative defense capacity were evaluated. For in vivo validation, an immune-deficient diabetic mouse model for intra-adrenal islet transplantation was applied. The functional capacity of intra-adrenally grafted islets to reverse diabetes was compared to a standard islet transplant model and measures of engraftment such as vascular integration were evaluated. The presence of adrenal cells positively impacted on cell metabolism and oxidative stress. Following transplantation, we could demonstrate enhanced islet function in comparison to standard models with improved engraftment and superior re-vascularization. This experimental approach allows for novel insights into the interaction of endocrine systems and may open up novel strategies for islet transplantation augmented through the bystander effect of other endocrine cells or the active factors secreted by adrenal cells modulating the microenvironment.

Mechanistic Insights into Ferroptotic Cell Death in Pancreatic Islets.

Ferroptosis was recently identified as a non-apoptotic, iron-dependent cell death mechanism that is involved in various pathologic conditions. There is first evidence for its significance also in the context of islet isolation and transplantation. Transplantation of pancreatic human islets is a viable treatment strategy for patients with complicated diabetes mellitus type 1 (T1D) that suffer from severe hypoglycemia. A major determinant for functional outcome is the initial islet mass transplanted. Efficient islet isolation procedures and measures to minimize islet loss are therefore of high relevance. To this end, better understanding and subsequent targeted inhibition of cell death during islet isolation and transplantation is an effective approach. In this study, we aimed to elucidate the mechanism of ferroptosis in pancreatic islets. Using a rodent model, isolated islets were characterized relating to the effects of experimental induction (RSL3) and inhibition (Fer1) of ferroptotic pathways. Besides viability, survival, and function, the study focused on characteristic ferroptosis-associated intracellular changes such as MDA level, iron concentration and the expression of ACSL4. The study demonstrates that pharmaceutical induction of ferroptosis by RSL3 causes enhancement of oxidative stress and leads to an increase of intracellular iron, zinc and MDA concentration, as well as the expression of ACSL4 protein. Consequently, a massive reduction of islet function, viability, and survival was found. Fer1 has the potential to inhibit and attenuate these cellular changes and thereby protect the islets from cell death.

A transcriptional cross species map of pancreatic islet cells.

OBJECTIVE: Pancreatic islets of Langerhans secrete hormones to regulate systemic glucose levels. Emerging evidence suggests that islet cells are functionally heterogeneous to allow a fine-tuned and efficient endocrine response to physiological changes. A precise description of the molecular basis of this heterogeneity, in particular linking animal models to human islets, is an important step towards identifying the factors critical for endocrine cell function in physiological and pathophysiological conditions. METHODS: In this study, we used single-cell RNA sequencing to profile more than 50'000 endocrine cells isolated from healthy human, pig and mouse pancreatic islets and characterize transcriptional heterogeneity and evolutionary conservation of those cells across the three species. We systematically delineated endocrine cell types and α- and ß-cell heterogeneity through prior knowledge- and data-driven gene sets shared across species, which altogether capture common and differential cellular properties, transcriptional dynamics and putative driving factors of state transitions. RESULTS: We showed that global endocrine expression profiles correlate, and that critical identity and functional markers are shared between species, while only approximately 20% of cell type enriched expression is conserved. We resolved distinct human α- and ß-cell states that form continuous transcriptional landscapes. These states differentially activate maturation and hormone secretion programs, which are related to regulatory hormone receptor expression, signaling pathways and different types of cellular stress responses. Finally, we mapped mouse and pig cells to the human reference and observed that the spectrum of human α- and ß-cell heterogeneity and aspects of such functional gene expression are better recapitulated in the pig than mouse data. CONCLUSIONS: Here, we provide a high-resolution transcriptional map of healthy human islet cells and their murine and porcine counterparts, which is easily queryable via an online interface. This comprehensive resource informs future efforts that focus on pancreatic endocrine function, failure and regeneration, and enables to assess molecular conservation in islet biology across species for translational purposes.

Quality of life and metabolic outcomes after total pancreatectomy and simultaneous islet autotransplantation.

Background: Pancreas surgery remains technically challenging and is associated with considerable morbidity and mortality. Identification of predictive risk factors for complications have led to a stratified surgical approach and postoperative management. The option of simultaneous islet autotransplantation (sIAT) allows for significant attenuation of long-term metabolic and overall complications and improvement of quality of life (QoL). The potential of sIAT to stratify a priori the indication for total pancreatectomy is yet not adequately evaluated. Methods: The aim of this analysis was to evaluate the potential of sIAT in patients undergoing total pancreatectomy to improve QoL, functional and overall outcome and therefore modify the surgical strategy towards earlier and extended indications. A center cohort of 24 patients undergoing pancreatectomy were simultaneously treated with IAT. Patients were retrospectively analyzed regarding in-hospital and overall mortality, postoperative complications, ICU stay, hospital stay, metabolic outcome, and QoL. Results: Here we present that all patients undergoing primary total pancreatectomy or surviving complicated two-stage pancreas resection and receiving sIAT show excellent metabolic outcome (33% insulin independence, 66% partial graft function; HbA1c 6,1 ± 1,0%) and significant benefit regarding QoL. Primary total pancreatectomy leads to significantly improved overall outcome and a significant reduction in ICU- and hospital stay compared to a two-stage completion pancreatectomy approach. Conclusions: The findings emphasize the importance of risk-stratified pancreas surgery. Feasibility of sIAT should govern the indication for primary total pancreatectomy particularly in high-risk patients. In rescue completion pancreatectomy sIAT should be performed whenever possible due to tremendous metabolic benefit and associated QoL.

Viral infiltration of pancreatic islets in patients with COVID-19.

Metabolic diseases are associated with an increased risk of severe COVID-19 and conversely, new-onset hyperglycemia and complications of preexisting diabetes have been observed in COVID-19 patients. Here, we performed a comprehensive analysis of pancreatic autopsy tissue from COVID-19 patients using immunofluorescence, immunohistochemistry, RNA scope and electron microscopy and detected SARS-CoV-2 viral infiltration of beta-cells in all patients. Using SARS-CoV-2 pseudoviruses, we confirmed that isolated human islet cells are permissive to infection. In eleven COVID-19 patients, we examined the expression of ACE2, TMPRSS and other receptors and factors, such as DPP4, HMBG1 and NRP1, that might facilitate virus entry. Whereas 70% of the COVID-19 patients expressed ACE2 in the vasculature, only 30% displayed ACE2-expression in beta-cells. Even in the absence of manifest new-onset diabetes, necroptotic cell death, immune cell infiltration and SARS-CoV-2 viral infection of pancreatic beta-cells may contribute to varying degrees of metabolic dysregulation in patients with COVID-19.

Enhanced differentiation of functional human T cells in NSGW41 mice with tissue-specific expression of human interleukin-7.

Humanized mouse models have become increasingly valuable tools to study human hematopoiesis and infectious diseases. However, human T-cell differentiation remains inefficient. We generated mice expressing human interleukin-7 (IL-7), a critical growth and survival factor for T cells, under the control of murine IL-7 regulatory elements. After transfer of human cord blood-derived hematopoietic stem and progenitor cells, transgenic mice on the NSGW41 background, termed NSGW41hIL7, showed elevated and prolonged human cellularity in the thymus while maintaining physiological ratios of thymocyte subsets. As a consequence, numbers of functional human T cells in the periphery were increased without evidence for pathological lymphoproliferation or aberrant expansion of effector or memory-like T cells. We conclude that the novel NSGW41hIL7 strain represents an optimized mouse model for humanization to better understand human T-cell differentiation in vivo and to generate a human immune system with a better approximation of human lymphocyte ratios.

Modeling Congenital Adrenal Hyperplasia and Testing Interventions for Adrenal Insufficiency Using Donor-Specific Reprogrammed Cells.

Adrenal insufficiency is managed by hormone replacement therapy, which is far from optimal; the ability to generate functional steroidogenic cells would offer a unique opportunity for a curative approach to restoring the complex feedback regulation of the hypothalamic-pituitary-adrenal axis. Here, we generated human induced steroidogenic cells (hiSCs) from fibroblasts, blood-, and urine-derived cells through forced expression of steroidogenic factor-1 and activation of the PKA and LHRH pathways. hiSCs had ultrastructural features resembling steroid-secreting cells, expressed steroidogenic enzymes, and secreted steroid hormones in response to stimuli. hiSCs were viable when transplanted into the mouse kidney capsule and intra-adrenal. Importantly, the hypocortisolism of hiSCs derived from patients with adrenal insufficiency due to congenital adrenal hyperplasia was rescued by expressing the wild-type version of the defective disease-causing enzymes. Our study provides an effective tool with many potential applications for studying adrenal pathobiology in a personalized manner and opens venues for the development of precision therapies.

Favorable outcome of experimental islet xenotransplantation without immunosuppression in a nonhuman primate model of diabetes.

Transplantation of pancreatic islets for treating type 1 diabetes is restricted to patients with critical metabolic lability resulting from the need for immunosuppression and the shortage of donor organs. To overcome these barriers, we developed a strategy to macroencapsulate islets from different sources that allow their survival and function without immunosuppression. Here we report successful and safe transplantation of porcine islets with a bioartificial pancreas device in diabetic primates without any immune suppression. This strategy should lead to pioneering clinical trials with xenotransplantation for treatment of diabetes and, thereby, represents a previously unidentified approach to efficient cell replacement for a broad spectrum of endocrine disorders and other organ dysfunctions.

Production of high-quality islets from goettingen minipigs: Choice of organ preservation solution, donor pool, and optimal cold ischemia time.

BACKGROUND: The transplantation of porcine islets into man might soon become reality for patients with type 1 diabetes mellitus. Therefore, porcine islets of high quality and quantity, and a scalable isolation process with strict quality control will be an unconditional prerequisite to enable the best possible transplantation graft. In this study, we provide a comparative study evaluating islet isolation outcome and in vitro survival based upon donor age, organ preservation solution (OPS), and cold ischemia time (CIT). METHODS: Goettingen minipigs of younger age (1 year) and retired breeder animals (3.5 years) were studied. Pancreata were harvested according to the standards of human organ retrieval including in situ cold perfusion with either Custodiol® -HTK or Belzer® UW solution. Pancreatic tissue was characterized by quantification of apoptotic cells. Islet isolations were performed according to a modified Ricordi method, and isolation outcome was assessed by determining islet particle numbers (IP), islet equivalents (IEQ), and isolation factor (IF). Isolated islets were cultured for 24 and 48 h for the assessment of in vitro survival. RESULTS: Islet viability was significantly higher in Custodiol® -HTK preserved pancreas organs compared to Belzer® UW. Furthermore, organs harvested from retired breeder preserved in Custodiol® -HTK resulted in stable islet isolation yields even after prolonged CIT and showed superior survival rates of islets in vitro compared to the Belzer® UW group. Younger porcine donor organs resulted generally in lower islet yield and survival rates. CONCLUSIONS: In summary, Custodiol® -HTK solution should be preferred over Belzer® UW solution for the preservation of pancreata from porcine origin. Custodiol® -HTK allows for maintaining islet viability and promotes reproducible isolation outcome and survival even after longer CIT. The usage of retired breeder animals over young animals for islet isolation is highly advisable to yield high quality and quantity.

Diabetes induction by total pancreatectomy in minipigs with simultaneous splenectomy: a feasible approach for advanced diabetes research.

BACKGROUND: Safe and reliable diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents and also widely used in large animal models of non-human primates and minipigs. However, uncertain efficacy, the potential of beta-cell regeneration, and relevant side effects are debatable aspects particularly in large animals. Therefore, we aimed to evaluate a surgical approach of total pancreatectomy combined with splenectomy for diabetes induction in an exploratory study in Goettingen minipigs. METHODS: Total pancreatectomy was performed in Goettingen minipigs (n = 4) under general anesthesia and endotracheal intubation. Prior to surgery, a central venous line was established for drug application and blood sampling. After median laparotomy, splenectomy was performed and the lobular pancreas was carefully dissected with particular attention to the duodenal vascular arcade. Close monitoring of blood glucose was initiated immediately after surgery by standard glucometer measurement or continuous glucose monitoring systems (CGMS). Exogenous insulin was given by multiple daily subcutaneous (s.c.) injections or via insulin pump systems (CSII). Complete endogenous insulin deficiency was confirmed by intravenous glucose tolerance test (ivGTT) and measurement of c-peptide. For establishing a suitable regimen for diabetes management, the animals were followed for 4-6 weeks. RESULTS: Following pancreatectomy and splenectomy, the animals showed a quick recovery from surgery and initial analgetic medication and volume substitution could be terminated within 24 h. A rapid increase in blood glucose was observed immediately following pancreatectomy necessitating insulin therapy. The induced exocrine insufficiency did not cause any clinical symptoms. Complete insulin deficiency could be confirmed in all animals by determination of negative c-peptide during glucose challenge. The two regimen of insulin treatment (multiple daily injections (MDI) and continuous subcutaneous insulin infusion (CSII)) were both feasible with respect to acceptable glycemic control whereas CSII was considerably advantageous in comfort and popularity for both animals and care takers. CONCLUSIONS: Surgical pancreatectomy in combination with splenectomy to facilitate access to the pancreas is a feasible model for efficient diabetes induction in minipigs. The procedure itself and postoperative animal care could be performed without complications in this exploratory study. Nevertheless, this approach requires well-equipped infrastructure, experienced and skilled surgeons and anesthesiologists and dedicated animal care takers. The impact of total pancreatectomy in combination with splenectomy on the digestive and immune system must be considered in the design and definition of end points of experimental diabetes and transplantation studies.

Image-based analysis of lipid nanoparticle-mediated siRNA delivery, intracellular trafficking and endosomal escape.

Delivery of short interfering RNAs (siRNAs) remains a key challenge in the development of RNA interference (RNAi) therapeutics. A better understanding of the mechanisms of siRNA cellular uptake, intracellular transport and endosomal release could critically contribute to the improvement of delivery methods. Here we monitored the uptake of lipid nanoparticles (LNPs) loaded with traceable siRNAs in different cell types in vitro and in mouse liver by quantitative fluorescence imaging and electron microscopy. We found that LNPs enter cells by both constitutive and inducible pathways in a cell type-specific manner using clathrin-mediated endocytosis as well as macropinocytosis. By directly detecting colloidal-gold particles conjugated to siRNAs, we estimated that escape of siRNAs from endosomes into the cytosol occurs at low efficiency (1-2%) and only during a limited window of time when the LNPs reside in a specific compartment sharing early and late endosomal characteristics. Our results provide insights into LNP-mediated siRNA delivery that can guide development of the next generation of delivery systems for RNAi therapeutics.

Transplantation of pancreatic islets to adrenal gland is promoted by agonists of growth-hormone-releasing hormone.

Here, we evaluate an alternative approach of preconditioning pancreatic islets before transplantation using a potent agonist of growth-hormone-releasing hormone (GHRH) to promote islet viability and function, and we explore the adrenal gland as an alternative transplantation site for islet engraftment. The endocrine microenvironment of the adrenal represents a promising niche with the unique advantages of exceptional high oxygen tension and local anti-inflammatory and immunosuppressive properties. GHRH agonists have been shown to promote islet graft survival and function, which may help to reduce the islet mass necessary to reverse diabetes. In the present study, the most potent GHRH agonist MR403 was tested on insulinoma cells, isolated rat islets, and adrenal ß-cell cocultures in vitro. GHRH receptor is expressed on both adrenal cells and islets. MR403 caused a significant increase in cell viability and proliferation and revealed an antiapoptotic effect on insulinoma cells. Viability of rat islets was increased after treatment with the agonist and in coculture with adrenal cells. Rat islets were transplanted into diabetic mice to the intraadrenal transplant site and compared with the classical transplants underneath the kidney capsule. Graft function and integration were tested by metabolic follow-up and immunohistochemical staining of intraadrenal grafts. A rapid decrease occurred in blood glucose levels in both models, and all animals reached normoglycemia within the first days after transplantation. Our studies demonstrated that the adrenal may be an attractive site for islet transplantation and that GHRH analogs might allow reduction of the islet mass needed to reverse a diabetic status.

20-Aminosteroids as a novel class of selective and complete androgen receptor antagonists and inhibitors of prostate cancer cell growth.

Here, the synthesis and the evaluation of novel 20-aminosteroids on androgen receptor (AR) activity is reported. Compounds 11 and 18 of the series inhibit both the wild type and the T877A mutant AR-mediated transactivation indicating AR antagonistic function. Interestingly, minor structural changes such as stereoisomers of the amino lactame moiety exhibit preferences for antagonism among wild type and mutant AR. Other tested nuclear receptors are only weakly or not affected. In line with this, the prostate cancer cell growth of androgen-dependent but not of cancer cells lacking expression of the AR is inhibited. Further, the expression of the prostate specific antigen used as a diagnostic marker is also repressed. Finally steroid 18 enhances cellular senescence that might explain in part the growth inhibition mediated by this derivative. Steroids 11 and 18 are the first steroids that act as complete AR antagonists and exhibit AR specificity.

NBBS isolated from Pygeum africanum bark exhibits androgen antagonistic activity, inhibits AR nuclear translocation and prostate cancer cell growth.

Extracts from Pygeum africanum are used in the treatment of prostatitis, benign prostatic hyperplasia (BPH) and prostate cancer (PCa). The ligand-activated human androgen receptor (AR) is known to control the growth of the prostate gland. Inhibition of human AR is therefore a major goal in treatment of patients. Here, we characterize the compound N-butylbenzene-sulfonamide (NBBS) isolated from P. africanum as a specific AR antagonist. This antihormonal activity inhibits AR- and progesterone receptor- (PR) mediated transactivation, but not the related human glucocorticoid receptor (GR) or the estrogen receptors (ERα or ERß). Importantly, NBBS inhibits both endogenous PSA expression and growth of human PCa cells. Mechanistically, NBBS binds to AR and inhibits its translocation to the cell nucleus. Furthermore, using a battery of chemically synthesized derivatives of NBBS we revealed important structural aspects for androgen antagonism and have identified more potent AR antagonistic compounds. Our data suggest that NBBS is one of the active compounds of P. africanum bark and may serve as a naturally occurring, novel therapeutic agent for treatment of prostatic diseases. Thus, NBBS and its derivatives may serve as novel chemical platform for treatment prostatitis, BPH and PCa.

The natural compound atraric acid is an antagonist of the human androgen receptor inhibiting cellular invasiveness and prostate cancer cell growth.

Extracts from Pygeum africanum are used in the treatment of prostatitis, benign prostatic hyperplasia and prostate cancer (Pca), major health problems of men in Western countries. The ligand-activated human androgen receptor (AR) supports the growth of the prostate gland. Inhibition of human AR by androgen ablation therapy and by applying synthetic anti-androgens is therefore the primary goal in treatment of patients. Here, we show that atraric acid (AA) isolated from bark material of Pygeum africanum has anti-androgenic activity, inhibiting the transactivation mediated by the ligand-activated human AR. This androgen antagonistic activity is receptor specific and does not inhibit the closely related glucocorticoid or progesterone receptors. Mechanistically, AA inhibits nuclear transport of AR. Importantly, AA is able to efficiently repress the growth of both the androgen-dependent LNCaP and also the androgen-independent C4-2 Pca cells but not that of PC3 or CV1 cells lacking AR. In line with this, AA inhibits the expression of the endogenous prostate specific antigen gene in both LNCaP und C4-2 cells. Analyses of cell invasion revealed that AA inhibits the invasiveness of LNCaP cells through extracellular matrix. Thus, this study provides a molecular insight for AA as a natural anti-androgenic compound and may serve as a basis for AA derivatives as a new chemical lead structure for novel therapeutic compounds as AR antagonists, that can be used for prophylaxis or treatment of prostatic diseases.