Syntaxin 4 Eenrichment in β-cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetic (T1D) and non-obese diabetic (NOD) mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell-specific STX4-expressing NOD mice (NOD-iβSTX4).<b> </b>Of NOD-iβSTX4 mice, 73% had sustained normoglycemia versus <20% of control NOD (NOD-Ctrl) mice, by 25 weeks of age. At 12 weeks of age, prior to diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with those of NOD-Ctrl mice. Single-cell RNA‐sequencing revealed that islets from NOD-iβSTX4 had markedly reduced IFNƔ signaling and TNFα signaling via NF-ĸB in islet β-cells, including reduced expression of the chemokine CCL5; CD4⁺ Treg cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

Syntaxin 4 Eenrichment in β-cells Prevents Conversion to Autoimmune Diabetes in Non-Obese Diabetic (NOD) Mice

Syntaxin 4 (STX4), a plasma membrane-localized SNARE protein, regulates human islet β-cell insulin secretion and preservation of β-cell mass. We found that human type 1 diabetic (T1D) and non-obese diabetic (NOD) mouse islets show reduced β-cell STX4 expression, consistent with decreased STX4 expression as a potential driver of T1D phenotypes. To test this hypothesis, we generated inducible β-cell-specific STX4-expressing NOD mice (NOD-iβSTX4).<b> </b>Of NOD-iβSTX4 mice, 73% had sustained normoglycemia versus <20% of control NOD (NOD-Ctrl) mice, by 25 weeks of age. At 12 weeks of age, prior to diabetes conversion, NOD-iβSTX4 mice demonstrated superior whole-body glucose tolerance and β-cell glucose responsiveness than NOD-Ctrl mice. Higher β-cell mass and reduced β-cell apoptosis were also detected in NOD-iβSTX4 pancreata compared with those of NOD-Ctrl mice. Single-cell RNA‐sequencing revealed that islets from NOD-iβSTX4 had markedly reduced IFNƔ signaling and TNFα signaling via NF-ĸB in islet β-cells, including reduced expression of the chemokine CCL5; CD4⁺ Treg cells were also enriched in NOD-iβSTX4 islets. These results provide a deeper mechanistic understanding of STX4 function in β-cell protection and warrant further investigation of STX4 enrichment as a strategy to reverse or prevent T1D in humans or protect β-cell grafts.

Angiopoietins stimulate pancreatic islet development from stem cells

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into functional islets holds immense potential to create an unlimited source of islets for diabetes research and treatment. A continuous challenge in this field is to generate glucose-responsive mature islets. We herein report a previously undiscovered angiopoietin signal for in vitro islet development. We revealed, for the first time, that angiopoietins, including angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) permit the generation of islets from iPSCs with elevated glucose responsiveness, a hallmark of mature islets. Angiopoietin-stimulated islets exhibited glucose synchronized calcium ion influx in repetitive glucose challenges. Moreover, Ang2 augmented the expression of all islet hormones, including insulin, glucagon, somatostatin, and pancreatic polypeptide; and β cell transcription factors, including NKX6.1, MAFA, UCN3, and PDX1. Furthermore, we showed that the Ang2 stimulated islets were able to regulate insulin exocytosis through actin-filament polymerization and depolymerization upon glucose challenge, presumably through the CDC42-RAC1-gelsolin mediated insulin secretion signaling pathway. We also discovered the formation of endothelium within the islets under Ang2 stimulation. These results strongly suggest that angiopoietin acts as a signaling molecule to endorse in vitro islet development from iPSCs.

Respiratory Parameters for the Classification of Dysfunctional Insulin Secretion by Pancreatic Islets

The development of obesity and type 2 diabetes (T2D) has been associated with impaired mitochondrial function. In pancreatic beta (β) cells, mitochondrial energy metabolism plays a central role in triggering and controlling glucose-stimulated insulin secretion (GSIS). Here, we have explored whether mitochondrial bioenergetic parameters assessed with Seahorse extracellular flux technology can quantitatively predict insulin secretion. We metabolically stressed male C57BL/6 mice by high-fat feeding (HFD) and measured the glucose sensitivity of islet respiration and insulin secretion. The diet-induced obese (DIO) mice developed hyperinsulinemia, but no pathological secretory differences were apparent between isolated DIO and chow islets. Real-time extracellular flux analysis, however, revealed a lower respiratory sensitivity to glucose in DIO islets. Correlation of insulin secretion with respiratory parameters uncovers compromised insulin secretion in DIO islets by oxidative power. Normalization to increased insulin contents during DIO improves the quantitative relation between GSIS and respiration, allowing to classify dysfunctional properties of pancreatic insulin secretion, and thereby serving as valuable biomarker for pancreatic islet glucose responsiveness and health.

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells

Aims/hypothesis Neonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells. Methods The effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations. Results NICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log2 fold change [Log2FC] > 1.6). An active TGFβ receptor (TGFBR)–SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFβ-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0 ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (Log2FC ≤ −2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area. Conclusions/interpretation Our results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells. Data availability The RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950 Graphical abstract

FFA2-, but not FFA3-agonists inhibit GSIS of human pseudoislets: a comparative study with mouse islets and rat INS-1E cells

The expression of short chain fatty acid receptors FFA2 and FFA3 in pancreatic islets raised interest in using them as drug targets for treating hyperglycemia in humans. This study aims to examine the efficacy of synthetic FFA2- and FFA3-ligands to modulate glucose-stimulated insulin secretion (GSIS) in human pseudoislets which display intact glucose responsiveness. The FFA2-agonists 4-CMTB and TUG-1375 inhibited GSIS, an effect reversed by the FFA2-antagonist CATPB. GSIS itself was not augmented by CATPB. The FFA3-agonists FHQC and 1-MCPC did not affect GSIS in human pseudoislets. For further drug evaluation we used mouse islets. The CATPB-sensitive inhibitory effect of 100 µM 4-CMTB on GSIS was recapitulated. The inhibition was partially sensitive to the Gi/o-protein inhibitor pertussis toxin. A previously described FFA2-dependent increase of GSIS was observed with lower concentrations of 4-CMTB (10 and 30 µM). The stimulatory effect of 4-CMTB on secretion was prevented by the Gq-protein inhibitor FR900359. As in human pseudoislets, in mouse islets relative mRNA levels were FFAR2 > FFAR3 and FFA3-agonists did not affect GSIS. The FFA3-agonists, however, inhibited GSIS in a pertussis toxin-sensitive manner in INS-1E cells and this correlated with relative mRNA levels of Ffar3 > > Ffar2. Thus, in humans, when FFA2-activation impedes GSIS, FFA2-antagonism may reduce glycemia.

1H MAS NMR of Donor Pancreatic Tissue is a Useful Predictor of Islet Viability Prior to Islet Isolation

A significant limitation and cost to any clinical islet program is the related to processing human pancreas and not recovering significant numbers of viable islets for clinical transplantation. The development of an assay system that could be utilized and provide an index of cell and tissue viability before islet isolation would provide a major impact on the scientific aspects of organ preservation and a huge cost saving to any clinical islet transplantation program.Metabolomic analysis by 1H MAS NMR was used to assess samples of donor pancreatic tissue taken prior to islet isolation. A significant correlation was observed between the ratio of the combined integrals of the sugar (3.5-4.5 ppm) and choline (3.0-3.5 ppm) regions to the integrals of the CH3 (0.9 ppm) and CH2 (1.3 ppm) peaks of the 1H MAS NMR spectra of pancreatic tissue samples taken prior to islet isolation and the glucose responsiveness, a measure of islet viability, of the isolated islets (P<0.05). The effect of the two-layer (University of Wisconsin solution/perfluorochemical [UW/PFC]) cold-storage method, previously shown to restore ischemically damaged pancreases by increasing oxygenation, was also studied using 1H MAS NMR spectroscopy. PFC recovery of the donor pancreas also correlated with an increase in the combined integrals of the sugar and choline regions to the CH3 and CH2 peaks of the 1H MAS NMR spectra (P<0.05). In addition, significant differences in the integrals of the sugar region and CH2 peaks were observed between the pre- and post-PFC samples (P<0.05). These results support the notion that specific metabolites observed in 1H MAS NMR can be used as a means to assess reversible/irreversible tissue damage and offers a means to assess donor pancreatic tissue prior to islet isolation for transplantation.

Stem Cell Therapy for Diabetes: Beta Cells versus Pancreatic Progenitors

Diabetes mellitus (DM) is one of the most prevalent metabolic disorders. In order to replace the function of the destroyed pancreatic beta cells in diabetes, islet transplantation is the most widely practiced treatment. However, it has several limitations. As an alternative approach, human pluripotent stem cells (hPSCs) can provide an unlimited source of pancreatic cells that have the ability to secrete insulin in response to a high blood glucose level. However, the determination of the appropriate pancreatic lineage candidate for the purpose of cell therapy for the treatment of diabetes is still debated. While hPSC-derived beta cells are perceived as the ultimate candidate, their efficiency needs further improvement in order to obtain a sufficient number of glucose responsive beta cells for transplantation therapy. On the other hand, hPSC-derived pancreatic progenitors can be efficiently generated in vitro and can further mature into glucose responsive beta cells in vivo after transplantation. Herein, we discuss the advantages and predicted challenges associated with the use of each of the two pancreatic lineage products for diabetes cell therapy. Furthermore, we address the co-generation of functionally relevant islet cell subpopulations and structural properties contributing to the glucose responsiveness of beta cells, as well as the available encapsulation technology for these cells.