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.

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.

NKX6.1 transcription factor: a crucial regulator of pancreatic ß cell development, identity, and proliferation.

Understanding the biology underlying the mechanisms and pathways regulating pancreatic ß cell development is necessary to understand the pathology of diabetes mellitus (DM), which is characterized by the progressive reduction in insulin-producing ß cell mass. Pluripotent stem cells (PSCs) can potentially offer an unlimited supply of functional ß cells for cellular therapy and disease modeling of DM. Homeobox protein NKX6.1 is a transcription factor (TF) that plays a critical role in pancreatic ß cell function and proliferation. In human pancreatic islet, NKX6.1 expression is exclusive to ß cells and is undetectable in other islet cells. Several reports showed that activation of NKX6.1 in PSC-derived pancreatic progenitors (MPCs), expressing PDX1 (PDX1+/NKX6.1+), warrants their future commitment to monohormonal ß cells. However, further differentiation of MPCs lacking NKX6.1 expression (PDX1+/NKX6.1-) results in an undesirable generation of non-functional polyhormonal ß cells. The importance of NKX6.1 as a crucial regulator in MPC specification into functional ß cells directs attentions to further investigating its mechanism and enhancing NKX6.1 expression as a means to increase ß cell function and mass. Here, we shed light on the role of NKX6.1 during pancreatic ß cell development and in directing the MPCs to functional monohormonal lineage. Furthermore, we address the transcriptional mechanisms and targets of NKX6.1 as well as its association with diabetes.

Identification of a LIF-Responsive, Replication-Competent Subpopulation of Human ß Cells.

The beta (ß)-cell mass formed during embryogenesis is amplified by cell replication during fetal and early postnatal development. Thereafter, ß cells become functionally mature, and their mass is maintained by a low rate of replication. For those few ß cells that replicate in adult life, it is not known how replication is initiated nor whether this occurs in a specialized subset of ß cells. We capitalized on a YAP overexpression system to induce replication of stem-cell-derived ß cells and, by single-cell RNA sequencing, identified an upregulation of the leukemia inhibitory factor (LIF) pathway. Activation of the LIF pathway induces replication of human ß cells in vitro and in vivo. The expression of the LIF receptor is restricted to a subset of transcriptionally distinct human ß cells with increased proliferative capacity. This study delineates novel genetic networks that control the replication of LIF-responsive, replication-competent human ß cells.

Differentiation of human pluripotent stem cells into two distinct NKX6.1 populations of pancreatic progenitors.

BACKGROUND: The expression of a specific combination of transcription factors (TFs) in the multipotent progenitor cells (MPCs) is critical for determining pancreatic cell fate. NKX6.1 expression in PDX1+ MPCs is required for functional ß cell generation. We have recently demonstrated the generation of a novel population of human pluripotent stem cell (hPSC)-derived MPCs that exclusively express NKX6.1, independently of PDX1 (PDX1-/NKX6.1+). Therefore, the aim of this study was to characterize this novel population to elucidate its role in pancreatic development. METHODS: The hPSCs were exposed to two differentiation protocols to generate MPCs that were analyzed using different techniques. RESULTS: Based on the expression of PDX1 and NKX6.1, we generated three different populations of MPCs, two of them were NKX6.1+. One of these NKX6.1 populations coexpressed PDX1 (PDX1+/NKX6.1+) which is known to mature into functional ß cells, and an additional novel population did not express PDX1 (PDX1-/NKX6.1+) with an undefined role in pancreatic cell fate. This novel population was enriched using our recently established protocol, allowing their reorganization in three-dimensional (3D) structures. Since NKX6.1 induction in MPCs can direct them to endocrine and/or ductal cells in humans, we examined the coexpression of endocrine and ductal markers. We found that the expression of the pancreatic endocrine progenitor markers chromogranin A (CHGA) and neurogenin 3 (NGN3) was not detected in the NKX6.1+ 3D structures, while few structures were positive for NKX2.2, another endocrine progenitor marker, thereby shedding light on the origin of this novel population and its role in pancreatic endocrine development. Furthermore, SOX9 was highly expressed in the 3D structures, but cytokeratin 19, a main ductal marker, was not detected in these structures. CONCLUSIONS: These data support the existence of two independent NKX6.1+ MPC populations during human pancreatic development and the novel PDX1-/NKX6.1+ population may be involved in a unique trajectory to generate ß cells in humans.

Obesity susceptibility loci in Qataris, a highly consanguineous Arabian population.

OBJECTIVES: In Qataris, a population characterized by a small size and a high rate of consanguinity, between two-thirds to three-quarters of adults are overweight or obese. We investigated the relevance of 23 obesity-related loci in the Qatari population. METHODS: Eight-hundred-four individuals assessed to be third generation Qataris were included in the study and assigned to 3 groups according to their body mass index (BMI): 190 lean (BMI < 25 kg/m(2)); 131 overweight (25 kg/m(2) ≤ BMI < 30 kg/m(2)) and 483 obese (BMI ≥ 30 kg/m(2)). Genomic DNA was isolated from peripheral blood and genotyped by TaqMan. RESULTS: Two loci significantly associated with obesity in Qataris: the TFAP2B variation (rs987237) (A allele versus G allele: chi-square = 10.3; P = 0.0013) and GNPDA2 variation (rs10938397) (A allele versus G allele: chi-square = 6.15; P = 0.013). The TFAP2B GG genotype negatively associated with obesity (OR = 0.21; P = 0.0031). Conversely, the GNDPA2 GG homozygous genotype associated with higher risk of obesity in subjects of age < 32 years (P = 0.0358). CONCLUSION: We showed a different genetic profile associated with obesity in the Qatari population compared to Western populations. Studying the genetic background of Qataris is of primary importance as the etiology of a given disease might be population-specific.

Genome scan study of prostate cancer in Arabs: identification of three genomic regions with multiple prostate cancer susceptibility loci in Tunisians.

BACKGROUND: Large databases focused on genetic susceptibility to prostate cancer have been accumulated from population studies of different ancestries, including Europeans and African-Americans. Arab populations, however, have been only rarely studied. METHODS: Using Affymetrix Genome-Wide Human SNP Array 6, we conducted a genome-wide association study (GWAS) in which 534,781 single nucleotide polymorphisms (SNPs) were genotyped in 221 Tunisians (90 prostate cancer patients and 131 age-matched healthy controls). TaqMan SNP Genotyping Assays on 11 prostate cancer associated SNPs were performed in a distinct cohort of 337 individuals from Arab ancestry living in Qatar and Saudi Arabia (155 prostate cancer patients and 182 age-matched controls). In-silico expression quantitative trait locus (eQTL) analysis along with mRNA quantification of nearby genes was performed to identify loci potentially cis-regulated by the identified SNPs. RESULTS: Three chromosomal regions, encompassing 14 SNPs, are significantly associated with prostate cancer risk in the Tunisian population (P = 1 × 10-4 to P = 1 × 10-5). In addition to SNPs located on chromosome 17q21, previously found associated with prostate cancer in Western populations, two novel chromosomal regions are revealed on chromosome 9p24 and 22q13. eQTL analysis and mRNA quantification indicate that the prostate cancer associated SNPs of chromosome 17 could enhance the expression of STAT5B gene. CONCLUSION: Our findings, identifying novel GWAS prostate cancer susceptibility loci, indicate that prostate cancer genetic risk factors could be ethnic specific.

TNRC9 downregulates BRCA1 expression and promotes breast cancer aggressiveness.

Although the linkage between germline mutations of BRCA1 and hereditary breast/ovarian cancers is well established, recent evidence suggests that altered expression of wild-type BRCA1 might contribute to the sporadic forms of breast cancer. The breast cancer gene trinucleotide-repeat-containing 9 (TNRC9; TOX3) has been associated with disease susceptibility but its function is undetermined. Here, we report that TNRC9 is often amplified and overexpressed in breast cancer, particularly in advanced breast cancer. Gene amplification was associated with reduced disease-free and metastasis-free survival rates. Ectopic expression of TNRC9 increased breast cancer cell proliferation, migration, and survival after exposure to apoptotic stimuli. These phenotypes were associated with tumor progression in a mouse model of breast cancer. Gene expression profiling, protein analysis, and in silico assays of large datasets of breast and ovarian cancer samples suggested that TNRC9 and BRCA1 expression were inversely correlated. Notably, we found that TNRC9 bound to both the BRCA1 promoter and the cAMP-responsive element-binding protein (CREB) complex, a regulator of BRCA1 transcription. In support of this connection, expression of TNRC9 downregulated expression of BRCA1 by altering the methylation status of its promoter. Our studies unveil a function for TNRC9 in breast cancer that highlights a new paradigm in BRCA1 regulation.

Genome-Wide Association Studies (GWAS) breast cancer susceptibility loci in Arabs: susceptibility and prognostic implications in Tunisians.

Genome-wide Association Studies (GWAS) revealed novel genetic markers for breast cancer susceptibility. But little is known about the risk factors and molecular events associated with breast cancer in Arab Population. Therefore, we designed a broad study to investigate the susceptibility and prognostic implications of the GWAS breast cancer loci in the Tunisian population. In a cohort of 640 unrelated patients with breast cancer and 371 healthy control subjects, we characterized the variation of 9 single nucleotide polymorphisms (SNPs), namely rs1219648, rs2981582; rs8051542, rs12443621, and rs3803662; rs889312; rs3817198; rs13387042 and rs13281615. Only 5 out of 9 GWAS breast cancer loci were found to be significantly associated with breast cancer in Tunisians: The rs1219648 (G vs. A allele: OR = 1.36, P = 1 × 10(-3)) and rs2981582 (A vs. G allele: OR = 1.55, P = 3 × 10(-6)) of FGFR2 gene; the rs8051542 of the TNRC9 gene (T vs. C allele: OR = 1.40, P = 4 × 10(-4)); the rs889312 of the MAP3K1 gene (C vs. A allele: OR = 1.33, P = 3 × 10(-3)) and the rs13281615 located on 8q24 (G vs. A allele: OR = 1.21, P = 0.03). Homozygous variant genotypes of rs2981582 were strongly related to lymph node negative breast cancer (OR = 3.33, P = 6 × 10(-7)) and the minor allele of rs2981582 was associated with increased risk of ER+ tumors (OR = 1.57, P = 0.02; OR = 2.15, P = 0.001, for heterozygous and homozygous variant genotypes, respectively) and increased risk of distant metastasis development (OR = 2.30, P = 4 × 10(-3); OR = 3.57, P = 6 × 10(-5), for heterozygous and homozygous variant genotypes, respectively) in a dose dependent manner. The association for rs8051542 was stronger for high-grade SBR tumors (OR = 2.54, P = 2 × 10(-4)). GG genotype of rs13387042 on 2q35 showed a significant association with the risk of developing distant metastasis (OR = 1.94, P = 0.02). The G allele of rs1219648 in FGFR2 and the A allele of rs13387042 on 2q35 indicated a better prognosis by showing a significantly higher overall survival rates (P = 0.013 and P = 0.005, respectively). In conclusion, GWAS breast cancer FGFR2, TNRC9, MAP3K1, and 8q24 loci are associated with an increased risk of breast cancer and genetic variation in FGFR2 gene may predict the aggressiveness of breast cancer in Tunisians.