Expression and biochemical characterization of the putative insulinase enzyme PF11_0189 found in the Plasmodium falciparum genome.

PF11_0189 is a putative insulin degrading enzyme present in Plasmodium falciparum genome. The catalytic domain of PF11_0189 is about 27 kDa. Substrate specificity study shows PF11_0189 acts upon different types of proteins. The substrate specificity is found to be highest when insulin is used as a substrate. Metal dependency study shows highest dependency of PF11_0189 towards zinc metal for its proteolytic activity. Chelation of zinc metal with EDTA shows complete absence of PF11_0189 activity. Peptide inhibitors, P-70 and P-121 from combinatorial peptide library prepared against PF11_0189 show inhibition with an IC50 value of 4.8 µM and 7.5 µM respectively. A proven natural anti-malarial peptide cyclosporin A shows complete inhibition against PF11_0189 with an IC50 value of 0.75 µM suggesting PF11_0189 as a potential target for peptide inhibitors. The study implicates that PF11_0189 is a zinc metalloprotease involved in catalysis of insulin. The study gives a preliminary insight into the mechanism of complications arising from glucose abnormalities during severe malaria.

The structure of the TH/INS locus and the parental allele expressed are not conserved between mammals.

Parent-of-origin-specific expression of imprinted genes is critical for successful mammalian growth and development. Insulin, coded by the INS gene, is an important growth factor expressed from the paternal allele in the yolk sac placenta of therian mammals. The tyrosine hydroxylase gene TH encodes an enzyme involved in dopamine synthesis. TH and INS are closely associated in most vertebrates, but the mouse orthologues, Th and Ins2, are separated by repeated DNA. In mice, Th is expressed from the maternal allele, but the parental origin of expression is not known for any other mammal so it is unclear whether the maternal expression observed in the mouse represents an evolutionary divergence or an ancestral condition. We compared the length of the DNA segment between TH and INS across species and show that separation of these genes occurred in the rodent lineage with an accumulation of repeated DNA. We found that the region containing TH and INS in the tammar wallaby produces at least five distinct RNA transcripts: TH, TH-INS1, TH-INS2, lncINS and INS. Using allele-specific expression analysis, we show that the TH/INS locus is expressed from the paternal allele in pre- and postnatal tammar wallaby tissues. Determining the imprinting pattern of TH/INS in other mammals might clarify if paternal expression is the ancestral condition which has been flipped to maternal expression in rodents by the accumulation of repeat sequences.

Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle.

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.

Phylogenetic and transcriptomic characterization of insulin and growth factor receptor tyrosine kinases in crustaceans.

Receptor tyrosine kinases (RTKs) mediate the actions of growth factors in metazoans. In decapod crustaceans, RTKs are implicated in various physiological processes, such molting and growth, limb regeneration, reproduction and sexual differentiation, and innate immunity. RTKs are organized into two main types: insulin receptors (InsRs) and growth factor receptors, which include epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR), and platelet-derived growth factor receptor (PDGFR). The identities of crustacean RTK genes are incomplete. A phylogenetic analysis of the CrusTome transcriptome database, which included all major crustacean taxa, showed that RTK sequences segregated into receptor clades representing InsR (72 sequences), EGFR (228 sequences), FGFR (129 sequences), and PDGFR/VEGFR (PVR; 235 sequences). These four receptor families were distinguished by the domain organization of the extracellular N-terminal region and motif sequences in the protein kinase catalytic domain in the C-terminus or the ligand-binding domain in the N-terminus. EGFR1 formed a single monophyletic group, while the other RTK sequences were divided into subclades, designated InsR1-3, FGFR1-3, and PVR1-2. In decapods, isoforms within the RTK subclades were common. InsRs were characterized by leucine-rich repeat, furin-like cysteine-rich, and fibronectin type 3 domains in the N-terminus. EGFRs had leucine-rich repeat, furin-like cysteine-rich, and growth factor IV domains. N-terminal regions of FGFR1 had one to three immunoglobulin-like domains, whereas FGFR2 had a cadherin tandem repeat domain. PVRs had between two and five immunoglobulin-like domains. A classification nomenclature of the four RTK classes, based on phylogenetic analysis and multiple sequence alignments, is proposed.

Clinical, glycometric features and treatment in a family with monogenic diabetes due to a new mutation in the insulin gene.

Monogenic diabetes caused by changes in the gene that encodes insulin (INS) is a very rare form of monogenic diabetes (<1%). The aim of this work is to describe the clinical and glycaemic control characteristics over time from four members of a family diagnosed with monogenic diabetes with the novel mutation: c.206del,p.(Gly69Aalfs*62) located in exon 3 of the gene INS. 75% are females, with debut in adolescence and negative autoimmunity. In all cases, C-peptide is detectable decades after diagnosis (>0.6ng/ml). Currently, patients are being treated either with insulin in a bolus-basal regimen, oral antidiabetics or hybrid closed loop system. Monogenic diabetes due to mutation in the INS is an entity with heterogeneous presentation, whose diagnosis requires high suspicion and presents an important clinical impact. Given the lack of standards in this regard, therapy must be individualized, although insulin therapy could help preserve beta cell functionality in these subjects.

Loss of Preproinsulin Interaction with Signal Recognition Particle Activates Protein Quality Control, Decreasing mRNA Stability.

Many insulin gene variants alter the protein sequence and result in monogenic diabetes due to insulin insufficiency. However, the molecular mechanisms of various disease-causing mutations are unknown. Insulin is synthesized as preproinsulin containing a signal peptide (SP). SPs of secreted proteins are recognized by the signal recognition particle (SRP) or by another factor in a SRP-independent pathway. If preproinsulin uses SRP-dependent or independent pathways is still debatable. We demonstrate by the use of site-specific photocrosslinking that the SRP subunit, SRP54, interacts with the preproinsulin SP. Moreover, SRP54 depletion leads to the decrease of insulin mRNA and protein expression, supporting the involvement of the RAPP protein quality control in insulin biogenesis. RAPP regulates the quality of secretory proteins through degradation of their mRNA. We tested five disease-causing mutations in the preproinsulin SP on recognition by SRP and on their effects on mRNA and protein levels. We demonstrate that the effects of mutations are associated with their position in the SP and their severity. The data support diverse molecular mechanisms involved in the pathogenesis of these mutations. We show for the first time the involvement of the RAPP protein quality control pathway in insulin biogenesis that is implicated in the development of neonatal diabetes caused by the Leu13Arg mutation.

The Concentrations of Interleukin-6, Insulin, and Glucagon in the Context of Obesity and Type 2 Diabetes and Single Nucleotide Polymorphisms in IL6 and INS Genes.

Obesity and diabetes are a problem of modern medicine. Although the environmental factors contributing to the development of these diseases are widely known, research into genetic factors is still ongoing. At the same time, the role of inflammation in the pathophysiology of obesity and diabetes is increasingly emphasized. Therefore, the purpose of this study was to investigate the influence of two selected polymorphisms (rs1800795 and rs3842729) on the development of obesity and type 2 diabetes. In this study, 118 participants were examined, including a control group (nonobese and nondiabetic group), an obese group, and a diabetic group. Genotype analysis was performed using the PCR-RFLP method. It has been shown that in patients with the G/G genotype within the rs1800795 polymorphism (IL6), the chance of developing type 2 diabetes is several times lower compared to patients with the G/C and C/C genotypes. However, the rs3842729 polymorphism (INS) does not directly affect the risk of obesity or type 2 diabetes (T2D), although elevated insulin concentrations have been observed in obese and diabetic patients. These results confirm the impact of the rs1800795 polymorphism on the development of diabetes; however, this relationship is more complex and requires further research on other factors.

Novel gene-based therapeutic approaches for the management of hepatic complications in diabetes: Reviewing recent advances.

Diabetes mellitus is a chronic metabolic disorder marked by hyperglycemia and systemic complications, including hepatic dysfunction, significantly contributing to disease progression and morbidity. This article reviews recent advances in gene-based therapeutic strategies targeting hepatic complications in diabetes, offering a promising approach for precision medicine by addressing underlying molecular mechanisms. Traditional treatments for hepatic complications in diabetes often manage symptoms rather than molecular causes, showing limited efficacy. Gene-based therapies are poised to correct dysfunctional pathways and restore hepatic function. Fundamental gene therapy approaches include gene silencing via small interfering RNAs (siRNAs) to target hepatic glucose production, lipid metabolism, and inflammation. Viral vectors can restore insulin sensitivity and reduce oxidative stress in diabetic livers. Genome editing, especially CRISPR-Cas9, allows the precise modification of disease-associated genes, offering immense potential for hepatic complication treatment. Strategies using CRISPR-Cas9 to enhance insulin receptor expression and modulate aberrant lipid regulatory genes are explored. Safety challenges in gene-based therapies, such as off-target effects and immune responses, are discussed. Advances in nanoparticle-based delivery systems and targeted gene editing techniques offer solutions to enhance specificity and minimize adverse effects. In conclusion, gene-based therapeutic approaches are a transformative direction in managing hepatic complications in diabetes. Further research is needed to optimize efficacy, safety, and long-term outcomes. Nevertheless, these innovative strategies promise to improve the lives of individuals with diabetes by addressing hepatic dysfunction's genetic root causes.

Permanent neonatal diabetes-causing insulin mutations have dominant negative effects on beta cell identity.

OBJECTIVE: Heterozygous coding sequence mutations of the INS gene are a cause of permanent neonatal diabetes (PNDM), requiring insulin therapy similar to T1D. While the negative effects on insulin processing and secretion are known, how dominant insulin mutations result in a continued decline of beta cell function after birth is not well understood. METHODS: We explored the causes of beta cell failure in two PNDM patients with two distinct INS mutations using patient-derived iPSCs and mutated hESCs. RESULTS: we detected accumulation of misfolded proinsulin and impaired proinsulin processing in vitro, and a dominant-negative effect of these mutations on beta-cell mass and function after transplantation into mice. In addition to anticipated ER stress, we found evidence of beta-cell dedifferentiation, characterized by an increase of cells expressing both Nkx6.1 and ALDH1A3, but negative for insulin and glucagon. CONCLUSIONS: These results highlight a novel mechanism, the loss of beta cell identity, contributing to the loss and functional failure of human beta cells with specific insulin gene mutations.

Sex- and time-specific associations of obesity with glycaemic traits: A two-step multivariate Mendelian randomization study.

AIM: To assess the sex- and time-specific causal effects of obesity-related anthropometric traits on glycaemic traits. MATERIALS AND METHODS: We used univariate and multivariate Mendelian randomization to assess the causal associations of anthropometric traits (gestational variables, birth weight, childhood body mass index [BMI], BMI, waist-to-hip ratio [WHR], BMI-adjusted WHR [WHRadj BMI]) with fasting glucose and insulin in Europeans from the Early Growth Genetics Consortium (n ≤ 298 142), the UK Biobank, the Genetic Investigation of Anthropometric Traits Consortium (n ≤ 697 734; females: n ≤ 434 794; males: n ≤ 374 754) and the Meta-Analyses of Glucose and Insulin-related traits Consortium (n ≤ 151 188; females: n ≤ 73 089; males: n ≤ 67 506), adjusting for maternal genetic effects, smoking, alcohol consumption, and age at menarche. RESULTS: We observed a null association for gestational variables, a negative association for birth weight, and positive associations for childhood BMI and adult traits (BMI, WHR, and WHRadj BMI). In female participants, increased birth weight causally decreased fasting insulin (betaIVW , -0.07, 95% confidence interval [CI] -0.11 to -0.03; p = 1.92 × 10-3 ), but not glucose levels, which was annulled by adjusting for age at menarche. In male participants, increased birth weight causally decreased fasting glucose (betainverse-variance-weighted (IVW) , -0.07, 95% CI -0.11 to -0.03; p = 3.22 × 10-4 ), but not insulin levels. In time-specific analyses, independent effects of birth weight were absent in female participants, and were more pronounced in male participants. Independent effects of childhood BMI were attenuated in both sexes; independent effects of adult traits differed by sex. CONCLUSIONS: Our findings provide evidence for causal and independent effects of sex- and time-specific anthropometric traits on glycaemic variables, and highlight the importance of considering multiple obesity exposures at different time points in the life course.

Diversity and Evolutionary Analysis of Venom Insulin Derived from Cone Snails.

Cone snails possess a diverse array of novel peptide toxins, which selectively target ion channels and receptors in the nervous and cardiovascular systems. These numerous novel peptide toxins are a valuable resource for future marine drug development. In this review, we compared and analyzed the sequence diversity, three-dimensional structural variations, and evolutionary aspects of venom insulin derived from different cone snail species. The comparative analysis reveals that there are significant variations in the sequences and three-dimensional structures of venom insulins from cone snails with different feeding habits. Notably, the venom insulin of some piscivorous cone snails exhibits a greater similarity to humans and zebrafish insulins. It is important to emphasize that these venom insulins play a crucial role in the predatory strategies of these cone snails. Furthermore, a phylogenetic tree was constructed to trace the lineage of venom insulin sequences, shedding light on the evolutionary interconnections among cone snails with diverse diets.

Genetic lineage tracing identifies adaptive mechanisms of pancreatic islet ß cells in various mouse models of diabetes with distinct age of initiation.

During the pathogenesis of type 1 diabetes (T1D) and type 2 diabetes (T2D), pancreatic islets, especially the ß cells, face significant challenges. These insulin-producing cells adopt a regeneration strategy to compensate for the shortage of insulin, but the exact mechanism needs to be defined. High-fat diet (HFD) and streptozotocin (STZ) treatment are well-established models to study islet damage in T2D and T1D respectively. Therefore, we applied these two diabetic mouse models, triggered at different ages, to pursue the cell fate transition of islet ß cells. Cre-LoxP systems were used to generate islet cell type-specific (α, ß, or δ) green fluorescent protein (GFP)-labeled mice for genetic lineage tracing, thereinto ß-cell GFP-labeled mice were tamoxifen induced. Single-cell RNA sequencing (scRNA-seq) was used to investigate the evolutionary trajectories and molecular mechanisms of the GFP-labeled ß cells in STZ-treated mice. STZ-induced diabetes caused extensive dedifferentiation of ß cells and some of which transdifferentiated into a or δ cells in both youth- and adulthood-initiated mice while this phenomenon was barely observed in HFD models. ß cells in HFD mice were expanded via self-replication rather than via transdifferentiation from α or δ cells, in contrast, α or δ cells were induced to transdifferentiate into ß cells in STZ-treated mice (both youth- and adulthood-initiated). In addition to the re-dedifferentiation of ß cells, it is also highly likely that these "α or δ" cells transdifferentiated from pre-existing ß cells could also re-trans-differentiate into insulin-producing ß cells and be beneficial to islet recovery. The analysis of ScRNA-seq revealed that several pathways including mitochondrial function, chromatin modification, and remodeling are crucial in the dynamic transition of ß cells. Our findings shed light on how islet ß cells overcome the deficit of insulin and the molecular mechanism of islet recovery in T1D and T2D pathogenesis.

Molecular engineering of insulin for recombinant expression in yeast.

Since the first administration of insulin to a person with diabetes in 1922, scientific contributions from academia and industry have improved insulin therapy and access. The pharmaceutical need for insulin is now more than 40 tons annually, half of which is produced by recombinant secretory expression in Saccharomyces cerevisiae. We discuss how, in this yeast species, adaptation of insulin precursors by removable structural elements is pivotal for efficient secretory expression. The technologies reviewed have been implemented at industrial scale and are seminal for the supply of human insulin and insulin analogues to people with diabetes now and in the future. Engineering of a target protein with removable structural elements may provide a general approach to yield optimisation.

Sex-specific causality of MRI-derived body compositions on glycaemic traits: Mendelian randomization and observational study.

AIM: To investigate the sex-specific causality of body compositions in type 2 diabetes and related glycaemic traits using Mendelian randomization (MR). MATERIALS AND METHODS: We leveraged sex-specific summary-level statistics from genome-wide association studies for three adipose deposits adjusted for body mass index and height, including abdominal subcutaneous adipose tissue, visceral adipose tissue (VATadj) and gluteofemoral adipose tissue (GFATadj), measured by MRI (20 038 women; 19 038 men), and fat mass-adjusted appendicular lean mass (ALMadj) (244 730 women; 205 513 men) in the UK Biobank. Sex-specific statistics of type 2 diabetes were from the Diabetes Genetics Replication and Meta-analysis Consortium and those for fasting glucose and insulin were from the Meta-analyses of Glucose and Insulin-related Traits Consortium. Univariable and multivariable MR (MVMR) were performed. We also performed MR analyses of anthropometric traits and genetic association analyses using individual-level data of body composition as validation. RESULTS: Univariable MR analysis showed that, in women, higher GFATadj and ALMadj exerted a causally protective effect on type 2 diabetes (GFATadj: odds ratio [OR] 0.59, 95% confidence interval [CI; 0.50, 0.69]; ALMadj: OR 0.84, 95% CI [0.77, 0.91]) and VATadj to be riskier in glycaemic traits. MVMR showed that GFATadj retained a robust effect on type 2 diabetes (OR 0.57, 95% CI [0.42, 0.77]; P = 2.6 × 10-4 ) in women, while it was nominally significant in men (OR 0.58, 95% CI [0.35, 0.96]; P = 3.3 × 10-2 ), after adjustment for ASATadj and VATadj. MR analyses of anthropometric measures and genetic association analyses of glycaemic traits confirmed the results. CONCLUSIONS: Body composition has a sex-specific effect on type 2 diabetes, and higher GFATadj has an independent protective effect on type 2 diabetes in both sexes.

Mendelian randomization analyses suggest a causal role for circulating GIP and IL-1RA levels in homeostatic model assessment-derived measures of ß-cell function and insulin sensitivity in Africans without type 2 diabetes.

BACKGROUND: In vitro and in vivo studies have shown that certain cytokines and hormones may play a role in the development and progression of type 2 diabetes (T2D). However, studies on their role in T2D in humans are scarce. We evaluated associations between 11 circulating cytokines and hormones with T2D among a population of sub-Saharan Africans and tested for causal relationships using Mendelian randomization (MR) analyses. METHODS: We used logistic regression analysis adjusted for age, sex, body mass index, and recruitment country to regress levels of 11 cytokines and hormones (adipsin, leptin, visfatin, PAI-1, GIP, GLP-1, ghrelin, resistin, IL-6, IL-10, IL-1RA) on T2D among Ghanaians, Nigerians, and Kenyans from the Africa America Diabetes Mellitus study including 2276 individuals with T2D and 2790 non-T2D individuals. Similar linear regression models were fitted with homeostatic modelling assessments of insulin sensitivity (HOMA-S) and ß-cell function (HOMA-B) as dependent variables among non-T2D individuals (n = 2790). We used 35 genetic variants previously associated with at least one of these 11 cytokines and hormones among non-T2D individuals as instrumental variables in univariable and multivariable MR analyses. Statistical significance was set at 0.0045 (0.05/11 cytokines and hormones). RESULTS: Circulating GIP and IL-1RA levels were associated with T2D. Nine of the 11 cytokines and hormones (exceptions GLP-1 and IL-6) were associated with HOMA-S, HOMA-B, or both among non-T2D individuals. Two-stage least squares MR analysis provided evidence for a causal effect of GIP and IL-RA on HOMA-S and HOMA-B in multivariable analyses (GIP ~ HOMA-S ß = - 0.67, P-value = 1.88 × 10-6 and HOMA-B ß = 0.59, P-value = 1.88 × 10-5; IL-1RA ~ HOMA-S ß = - 0.51, P-value = 8.49 × 10-5 and HOMA-B ß = 0.48, P-value = 5.71 × 10-4). IL-RA was partly mediated via BMI (30-34%), but GIP was not. Inverse variance weighted MR analysis provided evidence for a causal effect of adipsin on T2D (multivariable OR = 1.83, P-value = 9.79 × 10-6), though these associations were not consistent in all sensitivity analyses. CONCLUSIONS: The findings of this comprehensive MR analysis indicate that circulating GIP and IL-1RA levels are causal for reduced insulin sensitivity and increased ß-cell function. GIP's effect being independent of BMI suggests that circulating levels of GIP could be a promising early biomarker for T2D risk. Our MR analyses do not provide conclusive evidence for a causal role of other circulating cytokines in T2D among sub-Saharan Africans.

An Artificial Intelligence Prediction Model of Insulin Sensitivity, Insulin Resistance, and Diabetes Using Genes Obtained through Differential Expression.

Insulin is a powerful pleiotropic hormone that affects processes such as cell growth, energy expenditure, and carbohydrate, lipid, and protein metabolism. The molecular mechanisms by which insulin regulates muscle metabolism and the underlying defects that cause insulin resistance have not been fully elucidated. This study aimed to perform a microarray data analysis to find differentially expressed genes. The analysis has been based on the data of a study deposited in Gene Expression Omnibus (GEO) with the identifier "GSE22309". The selected data contain samples from three types of patients after taking insulin treatment: patients with diabetes (DB), patients with insulin sensitivity (IS), and patients with insulin resistance (IR). Through an analysis of omics data, 20 genes were found to be differentially expressed (DEG) between the three possible comparisons obtained (DB vs. IS, DB vs. IR, and IS vs. IR); these data sets have been used to develop predictive models through machine learning (ML) techniques to classify patients with respect to the three categories mentioned previously. All the ML techniques present an accuracy superior to 80%, reaching almost 90% when unifying IR and DB categories.

Clinical characteristics and genetic analysis of a child with specific type of diabetes mellitus caused by missense mutation of GATA6 gene. / 一例GATA6åŸºå› å˜å¼‚å¼•èµ·å„¿ç«¥ç‰¹æ®Šç±»åž‹ç³–å°¿ç— çš„ä¸´åºŠç‰¹ç‚¹åŠåŸºå› å˜å¼‚åˆ†æž.

A 2-year-old boy was admitted to Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine in Nov 30th, 2018, due to polydipsia, polyphagia, polyuria accompanied with increased glucose levels for more than 2 weeks. He presented with symmetrical short stature [height 81 cm (－2.2 SD), weight 9.8 kg (－2.1 SD), body mass index 14.94 kg/m2 (P10-P15)], and with no special facial or physical features. Laboratory results showed that the glycated hemoglobin A1c was 14%, the fasting C-peptide was 0.3 ng/mL, and the islet autoantibodies were all negative. Oral glucose tolerance test showed significant increases in both fasting and postprandial glucose, but partial islet functions remained (post-load C-peptide increased 1.43 times compared to baseline). A heterozygous variant c.1366C>T (p.R456C) was detected in GATA6 gene, thereby the boy was diagnosed with a specific type of diabetes mellitus. The boy had congenital heart disease and suffered from transient hyperosmolar hyperglycemia after a patent ductus arteriosus surgery at 11 months of age. Insulin replacement therapy was prescribed, but without regular follow-up thereafter. The latest follow-up was about 3.5 years after the diagnosis of diabetes when the child was 5 years and 11 months old, with the fasting blood glucose of 6.0-10.0 mmol/L, and the 2 h postprandial glucose of 17.0-20.0 mmol/L.

Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".

Type 1 diabetes mellitus (T1D) is an autoimmune disease caused by the destruction of insulin-producing ß-cells in the pancreas by cytotoxic T-cells. To date, there are no drugs that can prevent the development of T1D. Insulin replacement therapy is the standard care for patients with T1D. This treatment is life-saving, but is expensive, can lead to acute and long-term complications, and results in reduced overall life expectancy. This has stimulated the research and development of alternative treatments for T1D. In this review, we consider potential therapies for T1D using cellular regenerative medicine approaches with a focus on CRISPR/Cas-engineered cellular products. However, CRISPR/Cas as a genome editing tool has several drawbacks that should be considered for safe and efficient cell engineering. In addition, cellular engineering approaches themselves pose a hidden threat. The purpose of this review is to critically discuss novel strategies for the treatment of T1D using genome editing technology. A well-designed approach to ß-cell derivation using CRISPR/Cas-based genome editing technology will significantly reduce the risk of incorrectly engineered cell products that could behave as a "Trojan horse".

Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes.

Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D.

Exploring Large MAF Transcription Factors: Functions, Pathology, and Mouse Models with Point Mutations.

Large musculoaponeurotic fibrosarcoma (MAF) transcription factors contain acidic, basic, and leucine zipper regions. Four types of MAF have been elucidated in mice and humans, namely c-MAF, MAFA, MAFB, and NRL. This review aimed to elaborate on the functions of MAF transcription factors that have been studied in vivo so far, as well as describe the pathology of human patients and corresponding mouse models with c-MAF, MAFA, and MAFB point mutations. To identify the functions of MAF transcription factors in vivo, we generated genetically modified mice lacking c-MAF, MAFA, and MAFB and analyzed their phenotypes. Further, in recent years, c-MAF, MAFA, and MAFB have been identified as causative genes underpinning many rare diseases. Careful observation of human patients and animal models is important to examine the pathophysiological mechanisms underlying these conditions for targeted therapies. Murine models exhibit phenotypes similar to those of human patients with c-MAF, MAFA, and MAFB mutations. Therefore, generating these animal models emphasizes their usefulness for research uncovering the pathophysiology of point mutations in MAF transcription factors and the development of etiology-based therapies.