A hybrid construct of decellularized matrix and fibrin for differentiating adipose stem cells into insulin-producing cells, an optimized in vitro assessment.

The generation of insulin-producing cells (IPCs) is an attractive approach for replacing damaged ß cells in diabetic patients. In the present work, we introduced a hybrid platform of decellularized amniotic membrane (dAM) and fibrin encapsulation for differentiating adipose tissue-derived stem cells (ASCs) into IPCs. ASCs were isolated from healthy donors and characterized. Human AM was decellularized, and its morphology, DNA, collagen, glycosaminoglycan (GAG) contents, and biocompatibility were evaluated. ASCs were subjected to four IPC differentiation methods, and the most efficient method was selected for the experiment. ASCs were seeded onto dAM, alone or encapsulated in fibrin gel with various thrombin concentrations, and differentiated into IPCs according to a method applying serum-free media containing 2-mercaptoethanol, nicotinamide, and exendin-4. PDX-1, GLUT-2 and insulin expression were evaluated in differentiated cells using real-time PCR. Structural integrity and collagen and GAG contents of AM were preserved after decellularization, while DNA content was minimized. Cultivating ASCs on dAM augmented their attachment, proliferation, and viability and enhanced the expression of PDX-1, GLUT-2, and insulin in differentiated cells. Encapsulating ASCs in fibrin gel containing 2 mg/ml fibrinogen and 10 units/ml thrombin increased their differentiation into IPCs. dAM and fibrin gel synergistically enhanced the differentiation of ASCs into IPCs, which could be considered an appropriate strategy for replacing damaged ß cells.

Mechanically reinforced hydrogel vehicle delivering angiogenic factor for beta cell therapy.

Type 1 diabetes mellitus (T1DM) is a chronic disease affecting millions worldwide. Insulin therapy is currently the golden standard for treating T1DM; however, it does not restore the normal glycaemic balance entirely, which increases the risk of secondary complications. Beta-cell therapy may be a possible way of curing T1DM and has already shown promising results in the clinic. However, low retention rates, poor cell survival, and limited therapeutic potential are ongoing challenges, thus increasing the need for better cell encapsulation devices. This study aimed to develop a mechanically reinforced vascular endothelial growth factor (VEGF)-delivering encapsulation device suitable for beta cell encapsulation and transplantation. Poly(l-lactide-co-ε-caprolactone) (PLCL)/gelatin methacryloyl (GelMA)/alginate coaxial nanofibres were produced using electrospinning and embedded in an alginate hydrogel. The encapsulation device was physically and biologically characterised and was found to be suitable for INS-1E beta cell encapsulation, vascularization, and transplantation in terms of its biocompatibility, porosity, swelling ratio and mechanical properties. Lastly, VEGF was incorporated into the hydrogel and the release kinetics and functional studies revealed a sustained release of bioactive VEGF for at least 14 days, making the modified alginate system a promising candidate for improving the beta cell survival after transplantation.

Update on the transdifferentiation of pancreatic cells into functional beta cells for treating diabetes.

The increasing global prevalence and associated comorbidities need innovative approaches for type 2 diabetes mellitus (T2DM) prevention and treatment. Genetics contributes significantly to T2DM susceptibility, and genetic counseling is significant in detecting and informing people about the diabetic risk. T2DM is also intricately linked to overnutrition and obesity, and nutritional advising is beneficial to mitigate diabetic evolution. However, manipulating pancreatic cell plasticity and transdifferentiation could help beta cell regeneration and glucose homeostasis, effectively contributing to the antidiabetic fight. Targeted modulation of transcription factors is highlighted for their roles in various aspects of pancreatic cell differentiation and function, inducing non-beta cells' conversion into functional beta cells (responsive to glucose). In addition, pharmacological interventions targeting specific receptors and pathways might facilitate cell transdifferentiation aiming to maintain or increase beta cell mass and function. However, the mechanisms underlying cellular reprogramming are not yet well understood. The present review highlights the primary transcriptional factors in the endocrine pancreas, focusing on transdifferentiation as a primary mechanism. Therefore, islet cell reprogramming, converting one cell type to another and transforming non-beta cells into insulin-producing cells, depends, among others, on transcription factors. It is a promising fact that new transcription factors are discovered every day, and their actions on pancreatic islet cells are revealed. Exploring these pathways associated with pancreatic development and islet endocrine cell differentiation could unravel the molecular intricacies underlying transdifferentiation processes, exploring novel therapeutic strategies to treat diabetes. The medical use of this biotechnology is expected to be achievable within a short time.

Scaling Insulin-Producing Cells by Multiple Strategies.

In the quest to combat insulin-dependent diabetes mellitus (IDDM), allogenic pancreatic islet cell therapy sourced from deceased donors represents a significant therapeutic advance. However, the applicability of this approach is hampered by donor scarcity and the demand for sustained immunosuppression. Human induced pluripotent stem cells are a game-changing resource for generating synthetic functional insulin-producing ß cells. In addition, novel methodologies allow the direct expansion of pancreatic progenitors and mature ß cells, thereby circumventing prolonged differentiation. Nevertheless, achieving practical reproducibility and scalability presents a substantial challenge for this technology. As these innovative approaches become more prominent, it is crucial to thoroughly evaluate existing expansion techniques with an emphasis on their optimization and scalability. This manuscript delineates these cutting-edge advancements, offers a critical analysis of the prevailing strategies, and underscores pivotal challenges, including cost-efficiency and logistical issues. Our insights provide a roadmap, elucidating both the promises and the imperatives in harnessing the potential of these cellular therapies for IDDM.

Mouse islet-derived stellate cells are similar to, but distinct from, mesenchymal stromal cells and influence the beta cell function.

AIMS: Evidence is accumulating of the therapeutic benefits of mesenchymal stromal cells (MSCs) in diabetes-related conditions. We have identified a novel population of stromal cells within islets of Langerhans - islet stellate cells (ISCs) - which have a similar morphology to MSCs. In this study we characterize mouse ISCs and compare their morphology and function to MSCs to determine whether ISCs may also have therapeutic potential in diabetes. METHODS: ISCs isolated from mouse islets were compared to mouse bone marrow MSCs by analysis of cell morphology; expression of cell-surface markers and extracellular matrix (ECM) components; proliferation; apoptosis; paracrine activity; and differentiation into adipocytes, chondrocytes and osteocytes. We also assessed the effects of co-culture with ISCs or MSCs on the insulin secretory capacity of islet beta cells. RESULTS: Although morphological similar, ISCs were functionally distinct from MSCs. Thus, ISCs were less proliferative and more apoptotic; they had different expression levels of important paracrine factors; and they were less efficient at differentiation down multiple lineages. Co-culture of mouse islets with ISCs enhanced glucose induced insulin secretion more effectively than co-culture with MSCs. CONCLUSIONS: ISCs are a specific sub-type of islet-derived stromal cells that possess biological behaviors distinct from MSCs. The enhanced beneficial effects of ISCs on islet beta cell function suggests that they may offer a therapeutic target for enhancing beta cell functional survival in diabetes.

Stress-induced pseudokinase TRB3 augments IL1ß signaling by interacting with Flightless homolog 1.

Interleukin-1ß is one of the most potent inducers of beta cell inflammation in the lead-up to type 1 diabetes. We have previously reported that IL1ß-stimulated pancreatic islets from mice with genetic ablation of stress-induced pseudokinase TRB3(TRB3KO) show attenuated activation kinetics for the MAP3K MLK3 and JNK stress kinases. However, JNK signaling constitutes only a portion of the cytokine-induced inflammatory response. Here we report that TRB3KO islets also show a decrease in amplitude and duration of IL1ß-induced phosphorylation of TAK1 and IKK, kinases that drive the potent NF-κB proinflammatory signaling pathway. We observed that TRB3KO islets display decreased cytokine-induced beta cell death, preceded by a decrease in select downstream NF-κB targets, including iNOS/NOS2 (inducible nitric oxide synthase), a mediator of beta cell dysfunction and death. Thus, loss of TRB3 attenuates both pathways required for a cytokine-inducible, proapoptotic response in beta cells. In order to better understand the molecular basis of TRB3-enhanced, post-receptor IL1ß signaling, we interrogated the TRB3 interactome using coimmunoprecipitation followed by mass spectrometry to identify immunomodulatory protein Flightless homolog 1 (Fli1) as a novel, TRB3-interacting protein. We show that TRB3 binds and disrupts Fli1-dependent sequestration of MyD88, thereby increasing availability of this most proximal adaptor required for IL1ß receptor-dependent signaling. Fli1 sequesters MyD88 in a multiprotein complex resulting in a brake on the assembly of downstream signaling complexes. By interacting with Fli1, we propose that TRB3 lifts the brake on IL1ß signaling to augment the proinflammatory response in beta cells.

Metabolic stress-induced human beta-cell death is mediated by increased intracellular levels of adenosine.

Introduction: High intracellular concentrations of adenosine and 2'-deoxyadenosine have been suggested to be an important mediator of cell death. The aim of the present study was to characterize adenosine-induced death in insulin-producing beta-cells, at control and high glucose + palmitate-induced stress conditions. Methods: Human insulin-producing EndoC-betaH1 cells were treated with adenosine, 2'-deoxyadenosine, inosine and high glucose + sodium palmitate, and death rates using flow cytometry were studied. Results: We observed that adenosine and the non-receptor-activating analogue 2-deoxyadenosine, but not the adenosine deamination product inosine, promoted beta-cell apoptosis at concentrations exceeding maximal adenosine-receptor stimulating concentrations. Both adenosine and inosine were efficiently taken up by EndoC-betaH1 cells, and inosine counteracted the cell death promoting effect of adenosine by competing with adenosine for uptake. Both adenosine and 2'-deoxyadenosine promptly reduced insulin-stimulated production of plasma membrane PI(3,4,5)P3, an effect that was reversed upon wash out of adenosine. In line with this, adenosine, but not inosine, rapidly diminished Akt phosphorylation. Both pharmacological Bax inhibition and Akt activation blocked adenosine-induced beta-cell apoptosis, indicating that adenosine/2'-deoxyadenosine inhibits the PI3K/Akt/BAD anti-apoptotic pathway. High glucose + palmitate-induced cell death was paralleled by increased intracellular adenosine and inosine levels. Overexpression of adenosine deaminase-1 (ADA1) in EndoC-betaH1 cells, which increased Akt phosphorylation, prevented both adenosine-induced apoptosis and high glucose + palmitate-induced necrosis. ADA2 overexpression not only failed to protect against adenosine and high glucose + palmitate-activated cell death, but instead potentiated the apoptosis-stimulating effect of adenosine. In line with this, ADA1 overexpression increased inosine production from adenosine-exposed cells, whereas ADA2 did not. Knockdown of ADA1 resulted in increased cell death rates in response to both adenosine and high glucose + palmitate. Inhibition of miR-30e-3p binding to the ADA1 mRNA 3'-UTR promoted the opposite effects on cell death rates and reduced intracellular adenosine contents. Discussion: It is concluded that intracellular adenosine/2'-deoxyadenosine regulates negatively the PI3K pathway and is therefore an important mediator of beta-cell apoptosis. Adenosine levels are controlled, at least in part, by ADA1, and strategies to upregulate ADA1 activity, during conditions of metabolic stress, could be useful in attempts to preserve beta-cell mass in diabetes.

Differentially expressed microRNAs during the differentiation of muscle-derived stem cells into insulin-producing cells, a promoting role of microRNA-708-5p/STK4 axis.

OBJECTIVE: Stem cell therapy is a promising approach for diabetes via promoting the differentiation of insulin-producing cells (IPCs). This study aimed to screen the differentially expressed miRNAs (DEmiRNAs) during the differentiation of muscle-derived stem cells (MDSCs) into IPCs, and uncover the underlying function and mechanism of a specific DEmiRNA, miR-708-5p. METHODS: MDSCs were successfully isolated from the leg muscle of rats, and were induced for IPCs differentiation through a five-stage protocol. miRNA microarray assay was performed for screening DEmiRNAs during differentiation. The features of MDSCs-derived IPCs were identified by qRT-PCR, flow cytometry, and immunofluorescence staining. The targeting of STK4 by miR-708-5p was examined by luciferase assay. The protein expression of STK4, YAP1, and p-YAP1 was determined by Western blot and immunofluorescence staining. RESULTS: MDSCs were successfully isolated and differentiated into IPCs. A total of 12 common DEmiRNAs were obtained during five-stage differentiation. Among them, miR-708-5p that highly expressed in MDSCs-derived IPCs was selected. Overexpression of miR-708-5p upregulated some key transcription factors (Pdx1, Ngn3, Nkx2.2, Nkx6.1, Gata4, Gata6, Pax4, and Pax6) involving in IPCs differentiation, and increased insulin positive cells. In addition, STK4 was identified as the target gene of miR-708-5p. miR-708-5p overexpression downregulated the expression of STK4 and the downstream phosphorylated YAP1. CONCLUSIONS: There were 12 DEmiRNAs involved in the differentiation of MDSCs into IPCs. miR-708-5p promoted MDSCs differentiation into IPCs probably by targeting STK4-mediated Hippo-YAP1 signaling pathway.

TAZ promotes PDX1-mediated insulinogenesis.

Transcriptional co-activator with PDZ-binding motif (TAZ) is a key mediator of the Hippo signaling pathway and regulates structural and functional homeostasis in various tissues. TAZ activation is associated with the development of pancreatic cancer in humans, but it is unclear whether TAZ directly affects the structure and function of the pancreas. So we sought to identify the TAZ function in the normal pancreas. TAZ defect caused structural changes in the pancreas, particularly islet cell shrinkage and decreased insulin production and ß-cell markers expression, leading to hyperglycemia. Interestingly, TAZ physically interacted with the pancreatic and duodenal homeobox 1 (PDX1), a key insulin transcription factor, through the N-terminal domain of TAZ and the homeodomain of PDX1. TAZ deficiency decreased the DNA-binding and transcriptional activity of PDX1, whereas TAZ overexpression promoted PDX1 activity and increased insulin production even in a low glucose environment. Indeed, high glucose increased insulin production by turning off the Hippo pathway and inducing TAZ activation in pancreatic ß-cells. Ectopic TAZ overexpression along with PDX1 activation was sufficient to produce insulin in non-ß-cells. TAZ deficiency impaired the mesenchymal stem cell differentiation into insulin-producing cells (IPCs), whereas TAZ recovery restored normal IPCs differentiation. Compared to WT control, body weight increased in TAZ-deficient mice with age and even more with a high-fat diet (HFD). TAZ deficiency significantly exacerbated HFD-induced glucose intolerance and insulin resistance. Therefore, TAZ deficiency impaired pancreatic insulin production, causing hyperglycemia and exacerbating HFD-induced insulin resistance, indicating that TAZ may have a beneficial effect in treating insulin deficiency in diabetes.

G protein-coupled receptors as regulators of pancreatic islet functionality.

Glucose homeostasis is maintained by hormones secreted from different types of pancreatic islets and its dysregulation can result in diseases including diabetes mellitus. The secretion of hormones from pancreatic islets is highly complex and tightly controlled by G protein-coupled receptors (GPCRs). Moreover, GPCR signaling may play a role in enhancing islet cell replication and proliferation. Thus, targeting GPCRs offers a promising strategy for regulating the functionality of pancreatic islets. Here, available RNAseq datasets from human and mouse islets were used to identify the GPCR expression profile and the impact of GPCR signaling for normal islet functionality is discussed.

Trimethylguanosine synthase 1 is a novel regulator of pancreatic beta-cell mass and function.

Type 2 diabetes is a metabolic disorder associated with abnormal glucose homeostasis and is characterized by intrinsic defects in ß-cell function and mass. Trimethylguanosine synthase 1 (TGS1) is an evolutionarily conserved enzyme that methylates small nuclear and nucleolar RNAs and that is involved in pre-mRNA splicing, transcription, and ribosome production. However, the role of TGS1 in ß-cells and glucose homeostasis had not been explored. Here, we show that TGS1 is upregulated by insulin and upregulated in islets of Langerhans from mice exposed to a high-fat diet and in human ß-cells from type 2 diabetes donors. Using mice with conditional (ßTGS1KO) and inducible (MIP-CreERT-TGS1KO) TGS1 deletion, we determined that TGS1 regulates ß-cell mass and function. Using unbiased approaches, we identified a link between TGS1 and endoplasmic reticulum stress and cell cycle arrest, as well as and how TGS1 regulates ß-cell apoptosis. We also found that deletion of TGS1 results in an increase in the unfolded protein response by increasing XBP-1, ATF-4, and the phosphorylation of eIF2α, in addition to promoting several changes in cell cycle inhibitors and activators such as p27 and Cyclin D2. This study establishes TGS1 as a key player regulating ß-cell mass and function. We propose that these observations can be used as a stepping-stone for the design of novel strategies focused on TGS1 as a therapeutic target for the treatment of diabetes.

A single-cell atlas of de novo ß-cell regeneration reveals the contribution of hybrid ß/δ-cells to diabetes recovery in zebrafish.

Regeneration-competent species possess the ability to reverse the progression of severe diseases by restoring the function of the damaged tissue. However, the cellular dynamics underlying this capability remain unexplored. Here, we have used single-cell transcriptomics to map de novo ß-cell regeneration during induction and recovery from diabetes in zebrafish. We show that the zebrafish has evolved two distinct types of somatostatin-producing δ-cells, which we term δ1- and δ2-cells. Moreover, we characterize a small population of glucose-responsive islet cells, which share the hormones and fate-determinants of both ß- and δ1-cells. The transcriptomic analysis of ß-cell regeneration reveals that ß/δ hybrid cells provide a prominent source of insulin expression during diabetes recovery. Using in vivo calcium imaging and cell tracking, we further show that the hybrid cells form de novo and acquire glucose-responsiveness in the course of regeneration. The overexpression of dkk3, a gene enriched in hybrid cells, increases their formation in the absence of ß-cell injury. Finally, interspecies comparison shows that plastic δ1-cells are partially related to PP cells in the human pancreas. Our work provides an atlas of ß-cell regeneration and indicates that the rapid formation of glucose-responsive hybrid cells contributes to the resolution of diabetes in zebrafish.

Hyperbaric oxygen treatment improves pancreatic ß­cell function and hepatic gluconeogenesis in STZ­induced type­2 diabetes mellitus model mice.

Type­2 diabetes mellitus (T2DM) causes several complications that affect the quality of life and life span of patients. Hyperbaric oxygen therapy (HBOT) has been used to successfully treat several diseases, including carbon monoxide poisoning, ischemia, infections and diabetic foot ulcer, and increases insulin sensitivity in T2DM. The present study aimed to determine the effect of HBOT on ß­cell function and hepatic gluconeogenesis in streptozotocin (STZ)­induced type­2 diabetic mice. To establish a T2DM model, 7­week­old male C57BL/6J mice were fed a high­fat diet (HFD) and injected once daily with low­dose STZ for 3 days after 1­week HFD feeding. At the 14th week, HFD+HBOT and T2DM+HBOT groups received 1­h HBOT (2 ATA; 100% pure O2) daily from 5:00 to 6:00 p.m. for 7 days. The HFD and T2DM groups were maintained under normobaric oxygen conditions and used as controls. During HBOT, the 12­h nocturnal food intake and body weight were measured daily. Moreover, blood glucose was measured by using a tail vein prick and a glucometer. After the final HBO treatment, all mice were sacrificed to conduct molecular biology experiments. Fasting insulin levels of blood samples of sacrificed mice were measured by an ultrasensitive ELISA kit. Pancreas and liver tissues were stained with hematoxylin and eosin, while immunohistochemistry was performed to determine the effects of HBOT on insulin resistance. TUNEL was used to determine the effects of HBOT on ß­cell apoptosis, and immunoblotting was conducted to determine the ß­cell apoptosis pathway. HBOT notably reduced fasting blood glucose and improved insulin sensitivity in T2DM mice. After HBOT, ß­cell area and ß­cell mass in T2DM mice were significantly increased. HBOT significantly decreased the ß­cell apoptotic rate in T2DM mice via the pancreatic Bcl­2/caspase­3/poly(ADP­ribose) polymerase (PARP) apoptosis pathway. Moreover, HBOT improved the morphology of the liver tissue and increased hepatic glycogen storage in T2DM mice. These findings suggested that HBOT ameliorated the insulin sensitivity of T2DM mice by decreasing the ß­cell apoptotic rate via the pancreatic Bcl­2/caspase­3/PARP apoptosis pathway.

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced ß-cell damage via inhibition of neutral ceramidase.

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to ß-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on ß-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic ß-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced ß-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced ß-cell injury by promoting ceramide synthesis.

Let7b-5p inhibits insulin secretion and decreases pancreatic ß-cell mass in mice.

MicroRNAs are crucial regulators for the development, mass and function of pancreatic ß-cells. MiRNA dysregulation is associated with ß-cell dysfunction and development of diabetes. The members of let7 family are important players in regulating cellular growth and metabolism. In this study we investigated the functional role of let7b-5p in the mouse pancreatic ß-cells. We generated pancreatic ß-cell-specific let7b-5p transgenic mouse model and analyzed the glucose metabolic phenotype, ß-cells mass and insulin secretion in vivo. Luciferase reporter assay, immunofluorescence staining and western blot were carried out to study the target genes of let7b-5p in ß-cells. Let7b-5p overexpression impaired the insulin production and secretion of ß-cells and resulted impaired glucose tolerance in mice. The overexpressed let7b-5p inhibited pancreatic ß-cell proliferation and decreased the expression of cyclin D1 and cyclin D2. Our findings demonstrated that let7b-5p was critical in regulating the proliferation and insulin secretion of pancreatic ß-cells.

Gut Metabolite Trimethylamine N-Oxide Protects INS-1 ß-Cell and Rat Islet Function under Diabetic Glucolipotoxic Conditions.

Serum accumulation of the gut microbial metabolite trimethylamine N-oxide (TMAO) is associated with high caloric intake and type 2 diabetes (T2D). Impaired pancreatic ß-cell function is a hallmark of diet-induced T2D, which is linked to hyperglycemia and hyperlipidemia. While TMAO production via the gut microbiome-liver axis is well defined, its molecular effects on metabolic tissues are unclear, since studies in various tissues show deleterious and beneficial TMAO effects. We investigated the molecular effects of TMAO on functional ß-cell mass. We hypothesized that TMAO may damage functional ß-cell mass by inhibiting ß-cell viability, survival, proliferation, or function to promote T2D pathogenesis. We treated INS-1 832/13 ß-cells and primary rat islets with physiological TMAO concentrations and compared functional ß-cell mass under healthy standard cell culture (SCC) and T2D-like glucolipotoxic (GLT) conditions. GLT significantly impeded ß-cell mass and function by inducing oxidative and endoplasmic reticulum (ER) stress. TMAO normalized GLT-mediated damage in ß-cells and primary islet function. Acute 40µM TMAO recovered insulin production, insulin granule formation, and insulin secretion by upregulating the IRE1α unfolded protein response to GLT-induced ER and oxidative stress. These novel results demonstrate that TMAO protects ß-cell function and suggest that TMAO may play a beneficial molecular role in diet-induced T2D conditions.

Autopoietic Influence Hierarchies in Pancreatic ß Cells.

ß cells are biologically essential for humans and other vertebrates. Because their functionality arises from cell-cell interactions, they are also a model system for collective organization among cells. There are currently two contradictory pictures of this organization: the hub-cell idea pointing at leaders who coordinate the others, and the electrophysiological theory describing all cells as equal. We use new data and computational modeling to reconcile these pictures. We find via a network representation of interacting ß cells that leaders emerge naturally (confirming the hub-cell idea), yet all cells can take the hub role following a perturbation (in line with electrophysiology).

Butyrate and Class I Histone Deacetylase Inhibitors Promote Differentiation of Neonatal Porcine Islet Cells into Beta Cells.

Neonatal porcine islets-like clusters (NPICCs) are a promising source for cell therapy of type 1 diabetes. Freshly isolated NPICCs are composed of progenitor cells and endocrine cells, which undergo a maturation process lasting several weeks until the normal beta cell function has developed. Here, we investigated the effects of short-chain fatty acids on the maturation of islet cells isolated from two to three day-old piglets. NPICCs were cultivated with acetate, butyrate and propionate (0-2000 µM) for one to eight days. Incubation with butyrate resulted in a significant upregulation of insulin gene expression and an increased beta cell number, whereas acetate or propionate had only marginal effects. Treatment with specific inhibitors of G-protein-coupled receptor GPR41 (ß-hydroxybutyrate) and/or GPR43 (GPLG0974) did not abolish butyrate induced insulin expression. However, incubation of NPICCs with class I histone deacetylase inhibitors (HDACi) mocetinostat and MS275, but not selective class II HDACi (TMP269, MC1568) mimicked the butyrate effect on beta cell differentiation. Our study revealed that butyrate treatment has the capacity to increase the number of beta cells, which may be predominantly mediated through its HDAC inhibitory activity. Butyrate and specific class I HDAC inhibitors may represent beneficial supplements to promote differentiation of neonatal porcine islet cells towards beta cells for cell replacement therapies.

Development of an orally delivered GLP-1 receptor agonist through peptide engineering and drug delivery to treat chronic disease.

Peptide therapeutics are increasingly used in the treatment of disease, but their administration by injection reduces patient compliance and convenience, especially for chronic diseases. Thus, oral administration of a peptide therapeutic represents a significant advance in medicine, but is challenged by gastrointestinal instability and ineffective uptake into the circulation. Here, we have used glucagon-like peptide-1 (GLP-1) as a model peptide therapeutic for treating obesity-linked type 2 diabetes, a common chronic disease. We describe a comprehensive multidisciplinary approach leading to the development of MEDI7219, a GLP-1 receptor agonist (GLP-1RA) specifically engineered for oral delivery. Sites of protease/peptidase vulnerabilities in GLP-1 were removed by amino acid substitution and the peptide backbone was bis-lipidated to promote MEDI7219 reversible plasma protein binding without affecting potency. A combination of sodium chenodeoxycholate and propyl gallate was used to enhance bioavailability of MEDI7219 at the site of maximal gastrointestinal absorption, targeted by enteric-coated tablets. This synergistic approach resulted in MEDI7219 bioavailability of ~ 6% in dogs receiving oral tablets. In a dog model of obesity and insulin resistance, MEDI7219 oral tablets significantly decreased food intake, body weight and glucose excursions, validating the approach. This novel approach to the development of MEDI7219 provides a template for the development of other oral peptide therapeutics.