Inhibition of de novo ceramide synthesis by sirtuin-1 improves beta-cell function and glucose metabolism in type 2 diabetes.

AIMS: Obesity and type 2 diabetes (T2D) are major risk factors for cardiovascular (CV) diseases. Dysregulated pro-apoptotic ceramide synthesis reduces ß-cell insulin secretion, thereby promoting hyperglycaemic states that may manifest as T2D. Pro-apoptotic ceramides modulate insulin sensitivity and glucose tolerance while being linked to poor CV outcomes. Sirtuin-1 (SIRT1) is a NAD + -dependent deacetylase that protects against pancreatic ß-cell dysfunction; however, systemic levels are decreased in obese-T2D mice and may promote pro-apoptotic ceramide synthesis and hyperglycaemia. Herein, we aimed to assess the effects of restoring circulating SIRT1 levels to prevent metabolic imbalance in obese and diabetic mice. METHODS AND RESULTS: Circulating SIRT1 levels were reduced in obese-diabetic mice (db/db) as compared to age-matched non-diabetic db/+ controls. Restoration of SIRT1 plasma levels with recombinant murine SIRT1 for 4 weeks prevented body weight gain and improved glucose tolerance, insulin sensitivity, and vascular function in mice models of obesity and T2D. Untargeted lipidomics revealed that SIRT1 restored insulin secretory function of ß-cells by reducing synthesis and accumulation of pro-apoptotic ceramides. Molecular mechanisms involved direct binding to and deacetylation of Toll-like receptor 4 (TLR4) by SIRT1 in ß-cells, thereby decreasing the rate-limiting enzymes of sphingolipid synthesis SPTLC1/2 via AKT/NF-κB. Among patients with T2D, those with high baseline plasma levels of SIRT1 prior to metabolic surgery displayed restored ß-cell function (HOMA2-ß) and were more likely to have T2D remission during follow-up. CONCLUSION: Acetylation of TLR4 promotes ß-cell dysfunction via ceramide synthesis in T2D, which is blunted by systemic SIRT1 replenishment. Hence, restoration of systemic SIRT1 may provide a novel therapeutic strategy to counteract toxic ceramide synthesis and mitigate CV complications of T2D.

Innovative multidimensional models in a high-throughput-format for different cell types of endocrine origin.

The adrenal gland provides an important function by integrating neuronal, immune, vascular, metabolic and endocrine signals under a common organ capsule. It is the central organ of the stress response system and has been implicated in numerous stress-related disorders. While for other diseases, regeneration of healthy organ tissue has been aimed at such approaches are lacking for endocrine diseases - with the exception of type-I-diabetes. Moreover, adrenal tumor formation is very common, however, appropriate high-throughput applications reflecting the high heterogeneity and furthermore relevant 3D-structures in vitro are still widely lacking. Recently, we have initiated the development of standardized multidimensional models of a variety of endocrine cell/tissue sources in a new multiwell-format. Firstly, we confirmed common applicability for pancreatic pseudo-islets. Next, we translated applicability for spheroid establishment to adrenocortical cell lines as well as patient material to establish spheroids from malignant, but also benign adrenal tumors. We aimed furthermore at the development of bovine derived healthy adrenal organoids and were able to establish steroidogenic active organoids containing both, cells of cortical and medullary origin. Overall, we hope to open new avenues for basic research, endocrine cancer and adrenal tissue-replacement-therapies as we demonstrate potential for innovative mechanistic insights and personalized medicine in endocrine (tumor)-biology.

Growth of Epithelial Organoids in a Defined Hydrogel.

Epithelial organoids are simplified models of organs grown in vitro from embryonic and adult stem cells. They are widely used to study organ development and disease, and enable drug screening in patient-derived primary tissues. Current protocols, however, rely on animal- and tumor-derived basement membrane extract (BME) as a 3D scaffold, which limits possible applications in regenerative medicine. This prompted us to study how organoids interact with their matrix, and to develop a well-defined hydrogel that supports organoid generation and growth. It is found that soft fibrin matrices provide suitable physical support, and that naturally occurring Arg-Gly-Asp (RGD) adhesion domains on the scaffold, as well as supplementation with laminin-111, are key parameters required for robust organoid formation and expansion. The possibility to functionalize fibrin via factor XIII-mediated anchoring also allows to covalently link fluorescent nanoparticles to the matrix for 3D traction force microscopy. These measurements suggest that the morphogenesis of budding intestinal organoids results from internal pressure combined with higher cell contractility in the regions containing differentiated cells compared to the regions containing stem cells. Since the fibrin/laminin matrix supports long-term expansion of all tested murine and human epithelial organoids, this hydrogel can be widely used as a defined equivalent to BME.

Successful simultaneous islet-kidney transplantation using a steroid-free immunosuppression: two-year follow-up.

We report on the feasibility of a glucocorticoid-free immunosuppression (sirolimus, low-dose tacrolimus, and daclizumab) in simultaneous islet-kidney transplantation in nine patients with type 1 diabetes. There was one renal primary nonfunction. Renal function (n = 8) as assessed by creatinine and creatinine clearance over time was 103 +/- 6 micromol/L and 64 +/- 6 mL/min/1.73 m(2), respectively. Five out of six patients with >or= 2 islet transplantations became insulin independent. The mean HbA(1c) during the follow-up period for all patients after transplantation is 6.2 +/- 0.9% as compared with 8.7 +/- 1.9% prior to transplant. These results in patients with a median follow-up of 2.3 years suggest that kidney transplantation under a glucocorticoid-free immunosuppression is feasible, and that the rate of insulin independence of 80% can be achieved not only in patients with no or minimal diabetes complications, but also in patients with more advanced late complications and in conjunction with kidney transplantation.