Glucokinase (GCK) in diabetes: from molecular mechanisms to disease pathogenesis.

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.

Advanced human embryo research beyond the 14-day limit: A bioethical perspective from the Muslim world.

Advancements in in vitro human embryo research prompt a reconsideration of the 14-day rule, highlighting the integration of global religious perspectives, particularly Islam. Through analyzing classical Muslim scholars' perspectives and modern interdisciplinary Islamic bioethical discussions, we advocate extending the 14-day limit to at least 40 days, with specified conditions.

Deletion of RFX6 impairs iPSC-derived islet organoid development and survival, with no impact on PDX1+/NKX6.1+ progenitors.

AIMS/HYPOTHESIS: Homozygous mutations in RFX6 lead to neonatal diabetes accompanied by a hypoplastic pancreas, whereas heterozygous mutations cause MODY. Recent studies have also shown RFX6 variants to be linked with type 2 diabetes. Despite RFX6's known function in islet development, its specific role in diabetes pathogenesis remains unclear. Here, we aimed to understand the mechanisms underlying the impairment of pancreatic islet development and subsequent hypoplasia due to loss-of-function mutations in RFX6. METHODS: We examined regulatory factor X6 (RFX6) expression during human embryonic stem cell (hESC) differentiation into pancreatic islets and re-analysed a single-cell RNA-seq dataset to identify RFX6-specific cell populations during islet development. Furthermore, induced pluripotent stem cell (iPSC) lines lacking RFX6 were generated using CRISPR/Cas9. Various approaches were then employed to explore the consequences of RFX6 loss across different developmental stages. Subsequently, we evaluated transcriptional changes resulting from RFX6 loss through RNA-seq of pancreatic progenitors (PPs) and endocrine progenitors (EPs). RESULTS: RFX6 expression was detected in PDX1+ cells in the hESC-derived posterior foregut (PF). However, in the PPs, RFX6 did not co-localise with pancreatic and duodenal homeobox 1 (PDX1) or NK homeobox 1 (NKX6.1) but instead co-localised with neurogenin 3, NK2 homeobox 2 and islet hormones in the EPs and islets. Single-cell analysis revealed high RFX6 expression levels in endocrine clusters across various hESC-derived pancreatic differentiation stages. Upon differentiating iPSCs lacking RFX6 into pancreatic islets, a significant decrease in PDX1 expression at the PF stage was observed, although this did not affect PPs co-expressing PDX1 and NKX6.1. RNA-seq analysis showed the downregulation of essential genes involved in pancreatic endocrine differentiation, insulin secretion and ion transport due to RFX6 deficiency. Furthermore, RFX6 deficiency resulted in the formation of smaller islet organoids due to increased cellular apoptosis, linked to reduced catalase expression, implying a protective role for RFX6. Overexpression of RFX6 reversed defective phenotypes in RFX6-knockout PPs, EPs and islets. CONCLUSIONS/INTERPRETATION: These findings suggest that pancreatic hypoplasia and reduced islet cell formation associated with RFX6 mutations are not due to alterations in PDX1+/NKX6.1+ PPs but instead result from cellular apoptosis and downregulation of pancreatic endocrine genes. DATA AVAILABILITY: RNA-seq datasets have been deposited in the Zenodo repository with accession link (DOI: https://doi.org/10.5281/zenodo.10656891 ).

Identifying miRNA Signatures Associated with Pancreatic Islet Dysfunction in a FOXA2-Deficient iPSC Model.

The pathogenesis of diabetes involves complex changes in the expression profiles of mRNA and non-coding RNAs within pancreatic islet cells. Recent progress in induced pluripotent stem cell (iPSC) technology have allowed the modeling of diabetes-associated genes. Our recent study using FOXA2-deficient human iPSC models has highlighted an essential role for FOXA2 in the development of human pancreas. Here, we aimed to provide further insights on the role of microRNAs (miRNAs) by studying the miRNA-mRNA regulatory networks in iPSC-derived islets lacking the FOXA2 gene. Consistent with our previous findings, the absence of FOXA2 significantly downregulated the expression of islet hormones, INS, and GCG, alongside other key developmental genes in pancreatic islets. Concordantly, RNA-Seq analysis showed significant downregulation of genes related to pancreatic development and upregulation of genes associated with nervous system development and lipid metabolic pathways. Furthermore, the absence of FOXA2 in iPSC-derived pancreatic islets resulted in significant alterations in miRNA expression, with 61 miRNAs upregulated and 99 downregulated. The upregulated miRNAs targeted crucial genes involved in diabetes and pancreatic islet cell development. In contrary, the absence of FOXA2 in islets showed a network of downregulated miRNAs targeting genes related to nervous system development and lipid metabolism. These findings highlight the impact of FOXA2 absence on pancreatic islet development and suggesting intricate miRNA-mRNA regulatory networks affecting pancreatic islet cell development.

Effect of pregnancy on isolation efficiency and in vitro proliferation of equine peripheral-blood derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes.

Altered Regulation of the Glucose Transporter GLUT3 in PRDX1 Null Cells Caused Hypersensitivity to Arsenite.

Targeting tumour metabolism through glucose transporters is an attractive approach. However, the role these transporters play through interaction with other signalling proteins is not yet defined. The glucose transporter SLC2A3 (GLUT3) is a member of the solute carrier transporter proteins. GLUT3 has a high affinity for D-glucose and regulates glucose uptake in the neurons, as well as other tissues. Herein, we show that GLUT3 is involved in the uptake of arsenite, and its level is regulated by peroxiredoxin 1 (PRDX1). In the absence of PRDX1, GLUT3 mRNA and protein expression levels are low, but they are increased upon arsenite treatment, correlating with an increased uptake of glucose. The downregulation of GLUT3 by siRNA or deletion of the gene by CRISPR cas-9 confers resistance to arsenite. Additionally, the overexpression of GLUT3 sensitises the cells to arsenite. We further show that GLUT3 interacts with PRDX1, and it forms nuclear foci, which are redistributed upon arsenite exposure, as revealed by immunofluorescence analysis. We propose that GLUT3 plays a role in mediating the uptake of arsenite into cells, and its homeostatic and redox states are tightly regulated by PRDX1. As such, GLUT3 and PRDX1 are likely to be novel targets for arsenite-based cancer therapy.

Genome-wide differential expression profiling of long non-coding RNAs in FOXA2 knockout iPSC-derived pancreatic cells.

BACKGROUND: Our recent studies have demonstrated the crucial involvement of FOXA2 in the development of human pancreas. Reduction of FOXA2 expression during the differentiation of induced pluripotent stem cells (iPSCs) into pancreatic islets has been found to reduce α-and ß-cell masses. However, the extent to which such changes are linked to alterations in the expression profile of long non-coding RNAs (lncRNAs) remains unraveled. METHODS: Here, we employed our recently established FOXA2-deficient iPSCs (FOXA2-/- iPSCs) to investigate changes in lncRNA profiles and their correlation with dysregulated mRNAs during the pancreatic progenitor (PP) and pancreatic islet stages. Furthermore, we constructed co-expression networks linking significantly downregulated lncRNAs with differentially expressed pancreatic mRNAs. RESULTS: Our results showed that 442 lncRNAs were downregulated, and 114 lncRNAs were upregulated in PPs lacking FOXA2 compared to controls. Similarly, 177 lncRNAs were downregulated, and 59 lncRNAs were upregulated in islet cells lacking FOXA2 compared to controls. At both stages, we observed a strong correlation between lncRNAs and several crucial pancreatic genes and TFs during pancreatic differentiation. Correlation analysis revealed 12 DE-lncRNAs that strongly correlated with key downregulated pancreatic genes in both PPs and islet cell stages. Selected DE-lncRNAs were validated using RT-qPCR. CONCLUSIONS: Our data indicate that the observed defects in pancreatic islet development due to the FOXA2 loss is associated with significant alterations in the expression profile of lncRNAs. Therefore, our findings provide novel insights into the role of lncRNA and mRNA networks in regulating pancreatic islet development, which warrants further investigations. Video Abstract.

Pancreatic ß-cell heterogeneity in adult human islets and stem cell-derived islets.

Recent studies reported that pancreatic ß-cells are heterogeneous in terms of their transcriptional profiles and their abilities for insulin secretion. Sub-populations of pancreatic ß-cells have been identified based on the functionality and expression of specific surface markers. Under diabetes condition, ß-cell identity is altered leading to different ß-cell sub-populations. Furthermore, cell-cell contact between ß-cells and other endocrine cells within the islet play an important role in regulating insulin secretion. This highlights the significance of generating a cell product derived from stem cells containing ß-cells along with other major islet cells for treating patients with diabetes, instead of transplanting a purified population of ß-cells. Another key question is how close in terms of heterogeneity are the islet cells derived from stem cells? In this review, we summarize the heterogeneity in islet cells of the adult pancreas and those generated from stem cells. In addition, we highlight the significance of this heterogeneity in health and disease conditions and how this can be used to design a stem cell-derived product for diabetes cell therapy.

Blood-Based Proteomic Profiling Identifies Potential Biomarker Candidates and Pathogenic Pathways in Dementia.

Dementia is a progressive and debilitating neurological disease that affects millions of people worldwide. Identifying the minimally invasive biomarkers associated with dementia that could provide insights into the disease pathogenesis, improve early diagnosis, and facilitate the development of effective treatments is pressing. Proteomic studies have emerged as a promising approach for identifying the protein biomarkers associated with dementia. This pilot study aimed to investigate the plasma proteome profile and identify a panel of various protein biomarkers for dementia. We used a high-throughput proximity extension immunoassay to quantify 1090 proteins in 122 participants (22 with dementia, 64 with mild cognitive impairment (MCI), and 36 controls with normal cognitive function). Limma-based differential expression analysis reported the dysregulation of 61 proteins in the plasma of those with dementia compared with controls, and machine learning algorithms identified 17 stable diagnostic biomarkers that differentiated individuals with AUC = 0.98 ± 0.02. There was also the dysregulation of 153 plasma proteins in individuals with dementia compared with those with MCI, and machine learning algorithms identified 8 biomarkers that classified dementia from MCI with an AUC of 0.87 ± 0.07. Moreover, multiple proteins selected in both diagnostic panels such as NEFL, IL17D, WNT9A, and PGF were negatively correlated with cognitive performance, with a correlation coefficient (r2) ≤ -0.47. Gene Ontology (GO) and pathway analysis of dementia-associated proteins implicated immune response, vascular injury, and extracellular matrix organization pathways in dementia pathogenesis. In conclusion, the combination of high-throughput proteomics and machine learning enabled us to identify a blood-based protein signature capable of potentially differentiating dementia from MCI and cognitively normal controls. Further research is required to validate these biomarkers and investigate the potential underlying mechanisms for the development of dementia.

iPSC-Derived Pancreatic Progenitors Lacking FOXA2 Reveal Alterations in miRNA Expression Targeting Key Pancreatic Genes.

Recently, we reported that forkhead box A2 (FOXA2) is required for the development of human pancreatic α- and ß-cells. However, whether miRNAs play a role in regulating pancreatic genes during pancreatic development in the absence of FOXA2 expression is largely unknown. Here, we aimed to capture the dysregulated miRNAs and to identify their pancreatic-specific gene targets in pancreatic progenitors (PPs) derived from wild-type induced pluripotent stem cells (WT-iPSCs) and from iPSCs lacking FOXA2 (FOXA2-/-iPSCs). To identify differentially expressed miRNAs (DEmiRs), and genes (DEGs), two different FOXA2-/-iPSC lines were differentiated into PPs. FOXA2-/- PPs showed a significant reduction in the expression of the main PP transcription factors (TFs) in comparison to WT-PPs. RNA sequencing analysis demonstrated significant reduction in the mRNA expression of genes involved in the development and function of exocrine and endocrine pancreas. Furthermore, miRNA profiling identified 107 downregulated and 111 upregulated DEmiRs in FOXA2-/- PPs compared to WT-PPs. Target prediction analysis between DEmiRs and DEGs identified 92 upregulated miRNAs, predicted to target 1498 downregulated genes in FOXA2-/- PPs. Several important pancreatic TFs essential for pancreatic development were targeted by multiple DEmiRs. Selected DEmiRs and DEGs were further validated using RT-qPCR. Our findings revealed that FOXA2 expression is crucial for pancreatic development through regulating the expression of pancreatic endocrine and exocrine genes targeted by a set of miRNAs at the pancreatic progenitor stage. These data provide novel insights of the effect of FOXA2 deficiency on miRNA-mRNA regulatory networks controlling pancreatic development and differentiation.

p53 Inhibition in Pancreatic Progenitors Enhances the Differentiation of Human Pluripotent Stem Cells into Pancreatic ß-Cells.

The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic ß-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1+/NKX6.1+ MPCs and pancreatic ß-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and ß-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-µ (PFT-µ) at the MPC stage resulted in a significant increase in the number of PDX1+/NKX6.1+ cells and a reduction in the number of CHGA+/NKX6.1- cells. Further differentiation of MPCs treated with PFT-µ into pancreatic ß-cells showed that PFT-µ treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-µ treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal ß-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal ß-cells.

Reciprocal interplays between MicroRNAs and pluripotency transcription factors in dictating stemness features in human cancers.

The interplay between microRNAs (miRNAs) and pluripotency transcription factors (TFs) orchestrates the acquisition of cancer stem cell (CSC) features during the course of malignant transformation, rendering them essential cancer cell dependencies and therapeutic vulnerabilities. In this review, we discuss emerging themes in tumor heterogeneity, including the clonal evolution and the CSC models and their implications in resistance to cancer therapies, and then provide thorough coverage on the roles played by key TFs in maintaining normal and malignant stem cell pluripotency and plasticity. In addition, we discuss the reciprocal interactions between miRNAs and MYC, OCT4, NANOG, SOX2, and KLF4 pluripotency TFs and their contributions to tumorigenesis. We provide our view on the potential to interfere with key miRNA-TF networks through the use of RNA-based therapeutics as single agents or in combination with other therapeutic strategies, to abrogate the CSC state and render tumor cells more responsive to standard and targeted therapies.

Flavin Adenine Dinucleotide (FAD) and Pyridoxal 5'-Phosphate (PLP) Bind to Sox9 and Alter the Expression of Key Pancreatic Progenitor Transcription Factors.

Cofactor flavin adenine dinucleotide (FAD), a compound with flavin moiety and a derivative of riboflavin (vitamin B2), is shown to bind to Sox9 (a key transcription factor in early pancreatic development) and, subsequently, induce a large increase in markers of pancreatic development, including Ngn3 and PTF1a. Pyridoxal 5'-phosphate (PLP), the active form of vitamin B6, also binds to Sox9 and results in a similar increase in pancreatic development markers. Sox9 is known to be specifically important for pancreatic progenitors. Previously, there was no known link between FAD, PLP, or other co-factors and Sox9 for function. Thus, our findings show the mechanism by which FAD and PLP interact with Sox9 and result in the altered expression of pancreatic progenitor transcription factors involved in the pancreas development.

Identification of distinct circulating microRNAs in acute ischemic stroke patients with type 2 diabetes mellitus.

Stroke is the second leading cause of global mortality and continued efforts aim to identify predictive, diagnostic, or prognostic biomarkers to reduce the disease burden. Circulating microRNAs (miRNAs) have emerged as potential biomarkers in stroke. We performed comprehensive circulating miRNA profiling of ischemic stroke patients with or without type 2 diabetes mellitus (T2DM), an important risk factor associated with worse clinical outcomes in stroke. Serum samples were collected within 24 h of acute stroke diagnosis and circulating miRNAs profiled using RNA-Seq were compared between stroke patients with T2DM (SWDM; n = 92) and those without T2DM (SWoDM; n = 98). Our analysis workflow involved random allocation of study cohorts into discovery (n = 96) and validation (n = 94) datasets. Five miRNAs were found to be differentially regulated in SWDM compared to SWoDM patients. Hsa-miR-361-3p and -664a-5p were downregulated, whereas miR-423-3p, -140-5p, and -17-3p were upregulated. We also explored the gene targets of these miRNAs and investigated the downstream pathways associated with them to decipher the potential pathways impacted in stroke with diabetes as comorbidity. Overall, our novel findings provide important insights into the differentially regulated miRNAs, their associated pathways and potential utilization for clinical benefits in ischemic stroke patients with diabetes.

Loss of FOXA2 induces ER stress and hepatic steatosis and alters developmental gene expression in human iPSC-derived hepatocytes.

FOXA2 has been known to play important roles in liver functions in rodents. However, its role in human hepatocytes is not fully understood. Recently, we generated FOXA2 mutant induced pluripotent stem cell (FOXA2-/-iPSC) lines and illustrated that loss of FOXA2 results in developmental defects in pancreatic islet cells. Here, we used FOXA2-/-iPSC lines to understand the role of FOXA2 on the development and function of human hepatocytes. Lack of FOXA2 resulted in significant alterations in the expression of key developmental and functional genes in hepatic progenitors (HP) and mature hepatocytes (MH) as well as an increase in the expression of ER stress markers. Functional assays demonstrated an increase in lipid accumulation, bile acid synthesis and glycerol production, while a decrease in glucose uptake, glycogen storage, and Albumin secretion. RNA-sequencing analysis further validated the findings by showing a significant increase in genes associated with lipid metabolism, bile acid secretion, and suggested the activation of hepatic stellate cells and hepatic fibrosis in MH lacking FOXA2. Overexpression of FOXA2 reversed the defective phenotypes and improved hepatocyte functionality in iPSC-derived hepatic cells lacking FOXA2. These results highlight a potential role of FOXA2 in regulating human hepatic development and function and provide a human hepatocyte model, which can be used to identify novel therapeutic targets for FOXA2-associated liver disorders.

Directed differentiation of human pluripotent stem cells into epidermal keratinocyte-like cells.

The human pluripotent stem cell (hPSC) differentiation has allowed for the generation of in vitro models to study human diseases in a dish. This protocol describes the generation of keratinocyte-like cells from hPSCs in chemically defined media. Treating hPSCs with retinoic acid and BMP4 induced the generation of keratinocyte progenitors, which further differentiated into mature keratinocytes in the presence of calcium. The keratinocytes generated with this protocol could be used to study keratinocyte biology, drug screening, and skin-related diseases. For complete details on the use and execution of this protocol, please refer to Ali et al. (2020).

iPSCs derived from insulin resistant offspring of type 2 diabetic patients show increased oxidative stress and lactate secretion.

BACKGROUND: The genetic factors associated with insulin resistance (IR) are not well understood. Clinical studies on first-degree relatives of type 2 diabetic (T2D) patients, which have the highest genetic predisposition to T2D, have given insights into the role of IR in T2D pathogenesis. Induced pluripotent stem cells (iPSCs) are excellent tools for disease modeling as they can retain the genetic imprint of the disease. Therefore, in this study, we aimed to investigate the genetic perturbations associated with insulin resistance (IR) in the offspring of T2D parents using patient-specific iPSCs. METHODS: We generated iPSCs from IR individuals (IR-iPSCs) that were offspring of T2D parents as well as from insulin-sensitive (IS-iPSCs) individuals. We then performed transcriptomics to identify key dysregulated gene networks in the IR-iPSCs in comparison to IS-iPSCs and functionally validated them. RESULTS: Transcriptomics on IR-iPSCs revealed dysregulated gene networks and biological processes indicating that they carry the genetic defects associated with IR that may lead to T2D. The IR-iPSCs had increased lactate secretion and a higher phosphorylation of AKT upon stimulation with insulin. IR-iPSCs have increased cellular oxidative stress indicated by a high production of reactive oxygen species and higher susceptibility to H2O2 -induced apoptosis. CONCLUSIONS: IR-iPSCs generated from offspring of diabetic patients confirm that oxidative stress and increased lactate secretion, associated with IR, are inherited in this population, and may place them at a high risk of T2D. Overall, our IR-iPSC model can be employed for T2D modeling and drug screening studies that target genetic perturbations associated with IR in individuals with a high risk for T2D.

Toward Precision Medicine with Human Pluripotent Stem Cells for Diabetes.

Although genome profiling provides important genetic and phenotypic details for applying precision medicine to diabetes, it is imperative to integrate in vitro human cell models, accurately recapitulating the genetic alterations associated with diabetes. The absence of the appropriate preclinical human models and the unavailability of genetically relevant cells substantially limit the progress in developing personalized treatment for diabetes. Human pluripotent stem cells (hPSCs) provide a scalable source for generating diabetes-relevant cells carrying the genetic signatures of the patients. Remarkably, allogenic hPSC-derived pancreatic progenitors and ß cells are being used in clinical trials with promising preliminary results. Autologous hiPSC therapy options exist for those with monogenic and type 2 diabetes; however, encapsulation or immunosuppression must be accompanied with in the case of type 1 diabetes. Furthermore, genome-wide association studies-identified candidate variants can be introduced in hPSCs for deciphering the associated molecular defects. The hPSC-based disease models serve as excellent resources for drug development facilitating personalized treatment. Indeed, hPSC-based diabetes models have successfully provided valuable knowledge by modeling different types of diabetes, which are discussed in this review. Herein, we also evaluate their strengths and shortcomings in dissecting the underlying pathogenic molecular mechanisms and discuss strategies for improving hPSC-based disease modeling investigations.