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Diabetes Type 1 and Type 2 are widely occurring diseases. In spite of a vast amount of biomedical literature about diabetic processes in general, links to certain biological processes are only becoming evident these days. One such area of biology is the sulfation of small molecules, such as steroid hormones or metabolites from the gastrointestinal tract, as well as larger biomolecules, such as proteins and proteoglycans. Thus, modulating the physicochemical propensities of the different sulfate acceptors, resulting in enhanced solubility, expedited circulatory transit, or enhanced macromolecular interaction. This review lists evidence for the involvement of sulfation pathways in the maintenance of functional pancreatic beta-cell mass and the implications for diabetes, grouped into various classes of sulfated biomolecule. Complex heparan sulfates might play a role in the development and maintenance of beta-cells. The sulfolipids sulfatide and sulfo-cholesterol might contribute to beta-cell health. In beta-cells, there are only very few proteins with confirmed sulfation on some tyrosine residues, with the IRS4 molecule being one of them. Sulfated steroid hormones, such as estradiol-sulfate and vitamin-D-sulfate, may facilitate downstream steroid signaling in beta-cells, following de-sulfation. Indoxyl sulfate is a metabolite from the intestine, that causes kidney damage, contributing to diabetic kidney disease. Finally, from a technological perspective, there is heparan sulfate, heparin, and chondroitin sulfate, that all might be involved in next-generation beta-cell transplantation. Sulfation pathways may play a role in pancreatic beta-cells through multiple mechanisms. A more coherent understanding of sulfation pathways in diabetes will facilitate discussion and guide future research.

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Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.

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Dysfunction or loss of pancreatic ß cells can cause insulin deficiency and impaired glucose regulation, resulting in conditions like type 2 diabetes. The ATP-binding cassette transporter A1 (ABCA1) plays a key role in the reverse cholesterol transport system, and its decreased expression is associated with pancreatic ß cell lipotoxicity, resulting in abnormal insulin synthesis and secretion. Increased glutamate release can cause glucotoxicity in ß cells, though the detailed mechanisms remain unclear. This study investigated the effect of N-methyl-D-aspartic acid (NMDA) on ABCA1 expression in INS-1 cells and primary pancreatic islets to elucidate the signaling mechanisms that suppress insulin secretion. Using Western blotting, microscopy, and biochemical analyses, we found that NMDA activated the mitogen-activated protein kinase (MEK)-dependent pathway, suppressing ABCA1 protein and mRNA expression. The MEK-specific inhibitor PD98059 restored ABCA1 promoter activity, indicating the involvement of the extracellular signal-regulated kinase (MEK/ERK) pathway. Furthermore, we identified the liver X receptor (LXR) as an effector transcription factor in NMDA regulation of ABCA1 transcription. NMDA treatment increased cholesterol and triglyceride levels while decreasing insulin secretion, even under high-glucose conditions. These effects were abrogated by treatment with PD98059. This study reveals that NMDA suppresses ABCA1 expression via the MEK/ERK/LXR pathway, providing new insights into the pathological suppression of insulin secretion in pancreatic ß cells and emphasizing the importance of investigating the role of NMDA in ß cell dysfunction.

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This review seeks to address major gaps and delays between our rapidly evolving body of knowledge on type 2 diabetes and its translation into real-world practice. Through updated and improved best practices informed by recent evidence and described herein, we stand to better attain A1c targets, help preserve beta cell integrity and moderate glycemic variability, minimize treatment-emergent hypoglycemia, circumvent prescribing to "treatment failure," and prevent long-term complications. The first topic addressed in this review concerns updates in the 2023 and 2024 diabetes treatment guidelines for which further elaboration can help facilitate integration into routine care. The second concerns advances in diabetes research that have not yet found their way into guidelines, though they are endorsed by strong evidence and are ready for real-world use in appropriate patients. The final theme addresses lingering misconceptions about the underpinnings of type 2 diabetes-fundamental fallacies that continue to be asserted in the textbooks and continuing medical education upon which physicians build their approaches. A corrected and up-to-date understanding of the disease state is essential for practitioners to both conceptually and translationally manage initial onset through late-stage type 2 diabetes.

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Glucokinase (GCK), a key enzyme in glucose metabolism, plays a central role in glucose sensing and insulin secretion in pancreatic ß-cells, as well as glycogen synthesis in the liver. Mutations in the GCK gene have been associated with various monogenic diabetes (MD) disorders, including permanent neonatal diabetes mellitus (PNDM) and maturity-onset diabetes of the young (MODY), highlighting its importance in maintaining glucose homeostasis. Additionally, GCK gain-of-function mutations lead to a rare congenital form of hyperinsulinism known as hyperinsulinemic hypoglycemia (HH), characterized by increased enzymatic activity and increased glucose sensitivity in pancreatic ß-cells. This review offers a comprehensive exploration of the critical role played by the GCK gene in diabetes development, shedding light on its expression patterns, regulatory mechanisms, and diverse forms of associated monogenic disorders. Structural and mechanistic insights into GCK's involvement in glucose metabolism are discussed, emphasizing its significance in insulin secretion and glycogen synthesis. Animal models have provided valuable insights into the physiological consequences of GCK mutations, although challenges remain in accurately recapitulating human disease phenotypes. In addition, the potential of human pluripotent stem cell (hPSC) technology in overcoming current model limitations is discussed, offering a promising avenue for studying GCK-related diseases at the molecular level. Ultimately, a deeper understanding of GCK's multifaceted role in glucose metabolism and its dysregulation in disease states holds implications for developing targeted therapeutic interventions for diabetes and related disorders.

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Despite the fact that environmental pollution has been implicated in the global rise of diabetes, the research on the impact of emerging pollutants such as novel flame retardants remains limited. In line with the shift towards the use of non-animal approaches in toxicological testing, this study aimed to investigate the effects of two novel flame retardants tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) and triphenyl phosphate (TPhP) in rat (INS1E) and human (NES2Y) pancreatic beta-cell lines. One-week exposure to 1 µM and 10 µM TDCIPP and TPhP altered intracellular insulin and proinsulin levels, but not the levels of secreted insulin (despite the presence of a statistically insignificant trend). The exposures also altered the protein expression of several factors involved in beta-cell metabolic pathways and signaling, including ATP citrate lyase, isocitrate dehydrogenase 1, perilipins, glucose transporters, ER stress-related factors, and antioxidant enzymes. This study has brought new and valuable insights into the toxicity of TDCIPP and TPhP on beta-cell function and revealed alterations that might impact insulin secretion after more extended exposure. It also adds to the scarce studies using in vitro pancreatic beta-cells models in toxicological testing, thereby promoting the development of non-animal testing strategy for identifying pro-diabetic effects of chemical pollutants.

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Type 1 Diabetes (T1D) is a chronic metabolic disease resulting from autoimmune destruction of pancreatic beta cells. Beta cells activate a variety of stress responses during the development of T1D, including senescence, which involves cell cycle arrest, prosurvival signaling and a proinflammatory secretome termed the senescence-associated secretory phenotype (SASP). We previously identified growth and differentiation factor 15 (GDF15) as a major SASP factor in human islets and human EndoC-ßH5 beta cells in a model of DNA damage-mediated senescence that recapitulates features of senescent beta cells in T1D. Soluble GDF15 has been shown to exert protective effects on human and mouse beta cells during various forms of stress relevant to T1D, therefore we hypothesized that secreted GDF15 may play a prosurvival role during DNA damage-mediated senescence in human beta cells. We found that elevated GDF15 secretion was associated with endogenous senescent beta cells in an islet preparation from a T1D donor, supporting the validity of our DNA damage model. Using antibody-based neutralization, we found that secreted endogenous GDF15 was not required for senescent human islet or EndoC cell viability. Rather, neutralization of GDF15 led to reduced expression of specific senescence-associated genes, including GDF15 itself and the prosurvival gene BCL2L1. Taken together, these data suggest that SASP factor GDF15 is not required to sustain senescent human islet viability, but it is required to maintain senescence-associated transcriptional responses.

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Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

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Genetic polymorphisms in nuclear respiratory factor-1 (Nrf1), a key transcriptional regulator of nuclear-encoded mitochondrial proteins, have been linked to diabetes. Homozygous deletion of Nrf1 is embryonic lethal in mice. Our goal was to generate mice with ß-cell-specific reduction in NRF1 function to investigate the relationship between NRF1 and diabetes. We report the generation of mice expressing a dominant-negative allele of Nrf1 (DNNRF1) in pancreatic ß-cells. Heterozygous transgenic mice had high fed blood glucose levels detected at 3 wks of age, which persisted through adulthood. Plasma insulin levels in DNNRF1 transgenic mice were reduced, while insulin sensitivity remained intact in young animals. Islet size was reduced with increased numbers of apoptotic cells, and insulin content in islets by immunohistochemistry was low. Glucose-stimulated insulin secretion in isolated islets was reduced in DNNRF1-mice, but partially rescued by KCl, suggesting that decreased mitochondrial function contributed to the insulin secretory defect. Electron micrographs demonstrated abnormal mitochondrial morphology in ß-cells. Expression of NRF1 target genes Tfam, Tfb1m and Tfb2m, and islet cytochrome c oxidase and succinate dehydrogenase activities were reduced in DNNRF1-mice. Rescue of mitochondrial function with low level activation of transgenic c-Myc in ß-cells was sufficient to restore ß-cell mass and prevent diabetes. This study demonstrates that reduced NRF1 function can lead to loss of ß-cell function and establishes a model to study the interplay between regulators of bi-genomic gene transcription in diabetes.

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Closed-loop control systems for precise control of therapeutic gene expression are promising candidates for personalized treatment of chronic ailments such as diabetes. Pancreatic iß-cells are engineered with blue-light-inducible melanopsin to drive rapid insulin release by vesicular secretion from intracellular stores. In this work, a glucose-operated widget (GLOW) is designed as a component of a closed-loop control system for diabetes treatment by employing a probe that emits blue fluorescence in a glucose-concentration-dependent manner as a real-time glucose sensor to precisely control insulin release from these iß-cells. As proof-of-concept of the complete control system, the probe is encapsulated together with iß-cells in alginate-poly-(L-lysine) hydrogel-microbeads(400 µm in diameter and containing about 500 cells) called GLOWiß (GLOW with iß-cells), are subcutaneously implanted into type-1-diabetic (T1D) mice. Illumination by UV-A light at 390 nm results in glucose-concentration-dependent blue-light emission from the probe at 445 nm that in turn induces glucose-concentration-dependent insulin release from the iß-cells in a fully reversible manner. Activation of the injected GLOWiß at 390 nm for 15 min effectively restores normoglycemia within 60-120 min in a closed-loop manner in these diabetic mice. The system is robust, as normoglycemia is well maintained by daily activation for at least 7 days.

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Glucose plays a key role in shaping pancreatic ß cell function. Thus, deciphering the mechanisms by which this nutrient stimulates ß cells holds therapeutic promise for combating ß cell failure in type 2 diabetes (T2D). ß Cells respond to hyperglycemia in part by rewiring their mRNA metabolism, yet the mechanisms governing these changes remain poorly understood. Here, we identify a requirement for the RNA-binding protein PCBP2 in maintaining ß cell function basally and during sustained hyperglycemic challenge. PCBP2 was induced in primary mouse islets incubated with elevated glucose and was required to adapt insulin secretion. Transcriptomic analysis of primary Pcbp2-deficient ß cells revealed impacts on basal and glucose-regulated mRNAs encoding core components of the insulin secretory pathway. Accordingly, Pcbp2-deficient ß cells exhibited defects in calcium flux, insulin granule ultrastructure and exocytosis, and the amplification pathway of insulin secretion. Further, PCBP2 was induced by glucose in primary human islets, was downregulated in islets from T2D donors, and impacted genes commonly altered in islets from donors with T2D and linked to single-nucleotide polymorphisms associated with T2D. Thus, these findings establish a paradigm for PCBP2 in governing basal and glucose-adaptive gene programs critical for shaping the functional state of ß cells.

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Lipotoxicity has been considered the main cause of pancreatic beta-cell failure during type 2 diabetes development. Lipid droplets (LD) are believed to regulate the beta-cell sensitivity to free fatty acids (FFA), but the underlying molecular mechanisms are largely unclear. Accumulating evidence points, however, to an important role of intracellular sphingosine-1-phosphate (S1P) metabolism in lipotoxicity-mediated disturbances of beta-cell function. In the present study, we compared the effects of an increased irreversible S1P degradation (S1P-lyase, SPL overexpression) with those associated with an enhanced S1P recycling (overexpression of S1P phosphatase 1, SGPP1) on LD formation and lipotoxicity in rat INS1E beta-cells. Interestingly, although both approaches led to a reduced S1P concentration, they had opposite effects on the susceptibility to FFA. Overexpression of SGPP1 prevented FFA-mediated caspase-3 activation by a mechanism involving an enhanced lipid storage capacity and prevention of oxidative stress. In contrast, SPL overexpression limited LD biogenesis, content, and size, while accelerating lipophagy. This was associated with FFA-induced hydrogen peroxide formation, mitochondrial fragmentation, and dysfunction, as well as ER stress. These changes coincided with the upregulation of proapoptotic ceramides but were independent of lipid peroxidation rate. Also in human EndoC-ßH1 beta-cells, suppression of SPL with simultaneous overexpression of SGPP1 led to a similar and even more pronounced LD phenotype as that in INS1E-SGPP1 cells. Thus, intracellular S1P turnover significantly regulates LD content and size and influences beta-cell sensitivity to FFA.

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One of the challenges in studying islet inflammation-insulitis-is that it is a transient phenomenon. Traditional reporting of the insulitis progression is based on cumulative, donor-averaged values of leucocyte density in the vicinity of pancreatic islets, that hinder intra- and inter-islet heterogeneity of disease progression. Here, we aimed to understand why insulitis is non-uniform, often with peri-insulitis lesions formed on one side of an islet. To achieve this, we demonstrated the applicability of network theory in detangling intra-islet multi-cellular interactions during insulitis. Specifically, we asked the question "What is unique about regions of the islet that interact with immune cells first". This study utilized the non-obese diabetic mouse model of type one diabetes and examined the interplay among α-, ß-, T-cells, myeloid cells, and macrophages in pancreatic islets during the progression of insulitis. Disease evolution was tracked based on the T/ß cell ratio in individual islets. In the early stage, we found that immune cells are preferentially interacting with α-cell-rich regions of an islet. At the islet periphery α-linked ß-cells were found to be targeted significantly more compared to those without α-cell neighbors. Additionally, network analysis revealed increased T-myeloid, and T-macrophage interactions with all ß-cells.

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BACKGROUND: Type 1 diabetes (T1D) is an autoimmune disease characterized by the specific destruction of insulin-producing beta cells in the pancreas by the immune system, including CD4 cells which orchestrate the attack and CD8 cells which directly destroy the beta cells, resulting in the loss of glucose homeostasis. SCOPE OF REVIEW: This comprehensive document delves into the complex interplay between the immune system and beta cells, aiming to shed light on the mechanisms driving their destruction in T1D. Insights into the genetic predisposition, environmental triggers, and autoimmune responses provide a foundation for understanding the autoimmune attack on beta cells. From the role of viral infections as potential triggers to the inflammatory response of beta cells, an intricate puzzle starts to unfold. This exploration highlights the importance of beta cells in breaking immune tolerance and the factors contributing to their targeted destruction. Furthermore, it examines the potential role of autophagy and the impact of cytokine signaling on beta cell function and survival. MAJOR CONCLUSIONS: This review collectively represents current research findings on T1D which offers valuable perspectives on novel therapeutic approaches for preserving beta cell mass, restoring immune tolerance, and ultimately preventing or halting the progression of T1D. By unraveling the complex dynamics between the immune system and beta cells, we inch closer to a comprehensive understanding of T1D pathogenesis, paving the way for more effective treatments and ultimately a cure.

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We describe a scalable method for the robust generation of 3D pancreatic islet-like organoids from human pluripotent stem cells using suspension bioreactors. Our protocol involves a 6-stage, 20-day directed differentiation process, resulting in the production of 104-105 organoids. These organoids comprise α- and ß-like cells that exhibit glucose-responsive insulin and glucagon secretion. We detail methods for culturing, passaging, and cryopreserving stem cells as suspended clusters and for differentiating them through specific growth media and exogenous factors added in a stepwise manner. Additionally, we address quality control measures, troubleshooting strategies, and functional assays for research applications.

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Pancreatic ß-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1ß) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent ß-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1ß-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.

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Stem cell-derived islets (SC-islets) are not only an unlimited source for cell-based therapy of type 1 diabetes but have also emerged as an attractive material for modeling diabetes and conducting screening for treatment options. Prior to SC-islets becoming the established standard for disease modeling and drug development, it is essential to understand their response to various nutrient sources in vitro. This study demonstrates an enhanced efficiency of pancreatic endocrine cell differentiation through the incorporation of WNT signaling inhibition following the definitive endoderm stage. We have identified a tri-hormonal cell population within SC-islets, which undergoes reduction concurrent with the emergence of elevated numbers of glucagon-positive cells during extended in vitro culture. Over a 6-week period of in vitro culture, the SC-islets consistently demonstrated robust insulin secretion in response to glucose stimulation. Moreover, they manifested diverse reactivity patterns when exposed to distinct nutrient sources and exhibited deviant glycolytic metabolic characteristics in comparison to human primary islets. Although the SC-islets demonstrated an aberrant glucose metabolism trafficking, the evaluation of a potential antidiabetic drug, pyruvate kinase agonist known as TEPP46, significantly improved in vitro insulin secretion of SC-islets. Overall, this study provided cell identity dynamics investigation of SC-islets during prolonged culturing in vitro, and insights into insulin secretagogues. Associated advantages and limitations were discussed when employing SC-islets for disease modeling.

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Our understanding of type 2 diabetes (T2D) has evolved dramatically. Advances have upended entrenched dogmas pertaining to the onset and progression of T2D, beliefs that have prevailed from the early era of diabetes research-and continue to populate our medical textbooks and continuing medical education materials. This review article highlights key insights that lend new governing principles for gold standard management of T2D. From the historical context upon which old beliefs arose to new findings, this article outlines evidence and perspectives on beta cell function, the underlying defects in glucoregulation, the remediable nature of T2D, and, the rationale supporting the shift to complication-centric prescribing. Practical approaches translate this rectified understanding of T2D into strategies that fill gaps in current management practices of prediabetes through late type 2 diabetes.

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Currently, there are more than 10 million patients with diabetes mellitus in Japan. Therefore, the need to explore the pathogenesis of diabetes and the complications leading to its cure is becoming increasingly urgent. Pathological examination of pancreatic tissues from patients with type 2 diabetes reveals a decrease in the volume of beta cells because of a combination of various stresses. In human type 2 diabetes, islet amyloid deposition is a unique pathological change characterized by proinflammatory macrophage (M1) infiltration into the islets. The pathological changes in the pancreas with islet amyloid were different according to clinical factors, which suggests that type 2 diabetes can be further subclassified based on islet pathology. On the other hand, diabetic peripheral neuropathy is the most frequent diabetic complication. In early diabetic peripheral neuropathy, M1 infiltration in the sciatic nerve evokes oxidative stress or attenuates retrograde axonal transport, as clearly demonstrated by in vitro live imaging. Furthermore, islet parasympathetic nerve density and beta cell volume were inversely correlated in type 2 diabetic Goto-Kakizaki rats, suggesting that diabetic peripheral neuropathy itself may contribute to the decrease in beta cell volume. These findings suggest that the pathogenesis of diabetes mellitus and diabetic peripheral neuropathy may be interrelated.

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