In vivo evaluation of GG2–GG1/A2 element activity in the insulin promoter region using the CRISPR–Cas9 system

The insulin promoter is regulated by ubiquitous as well as pancreatic β-cell-specific transcription factors. In the insulin promoter, GG2–GG1/A2–C1 (bases − 149 to − 116 in the human insulin promoter) play important roles in regulating β-cell-specific expression of the insulin gene. However, these events were identified through in vitro studies, and we are unaware of comparable in vivo studies. In this study, we evaluated the activity of GG2–GG1/A2 elements in the insulin promoter region in vivo. We generated homozygous mice with mutations in the GG2–GG1/A2 elements in each of the Ins1 and Ins2 promoters by CRISPR–Cas9 technology. The mice with homozygous mutations in the GG2–GG1/A2 elements in both Ins1 and Ins2 were diabetic. These data suggest that the GG2–GG1/A2 element in mice is important for Ins transcription in vivo.

In-silico discovery of antidiabetic drug potential of Balanites aegyptiaca leaf’s phenolic compounds

<i>Balanites aegyptiaca</i> leaf is very effective in managing diabetes and rich in phenolic compounds. However, the modes of action of the phytochemicals are mainly unknown. Thus, the present in silico drug discovery study on some phenolic compounds was designed to evaluate potential mechanisms of action of the antihyperglycemic phytochemicals of <i>B. aegyptiaca</i> leaf extract. The study deployed in silico drug-like studying techniques such as; predicted activity spectra of substances (PASS), molecular docking, prediction of adsorption, distribution, metabolism, excretion, and toxicity (ADMET), Lipinski’s rule of 5 (PLOP). The study reveals six compounds with good drug-like properties: cLogp, hydrogen bond donor/acceptor (<5/ < 10), and molar refractivity. In addition, ADMET and drug properties like kinase inhibitors, ion channel modulators, and nuclear receptors were positive for the compounds. Each phenolic compound showed one or more antidiabetic activities like insulin promoter, insulin sensitizer and inhibitors of α-amylase and α-glucosidase. Docking result predicted that the phenolic compounds inhibited either α-amylase or α-glucosidase while one of the compounds; 2-methoxy-4-(1-propenyl)-phenol inhibited both α-amylase and α-glucosidase with binding energies of -4.4 and -4.2 kcal/mol against -3.8 and -4.8 kcal/mol by Acarbose. The study revealed that phenolic compounds from <i>B. aegyptiaca</i> leaf possessed drug-like properties, including the ability to interact with α-amylase and α-glucosidase, a vital target protein in the management of diabetes mellitus. The data from the in silico study is a step toward the pharmaceutical discovery of the antidiabetic drug potential of <i>B. aegyptiaca</i> leaf.

Prostaglandin EP3 Receptor signaling is required to prevent insulin hypersecretion and metabolic dysfunction in a non-obese mouse model of insulin resistance

When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gαz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion.

Clinical and Functional Characteristics of a Novel KLF11 Cys354Phe Variant Involved in Maturity-Onset Diabetes of the Young

Background. Mutations in human KLF11 may lead to the development of maturity-onset diabetes of the young 7 (MODY7). This occurs due to impaired insulin synthesis in the pancreas. To date, the clinical and functional characteristics of the novel KLF11 mutation c.1061G > T have not yet been reported. Methods. Whole-exon sequencing was used to screen the proband and family members with clinical suspicion of the KLF11 variant. Luciferase reporter assays were used to investigate whether the KLF11 variant binds to the insulin promoter. Real-time PCR, western blotting, and glucose-stimulated insulin secretion (GSIS) analysis were used to analyze the KLF11 variant that regulates insulin expression and insulin secretion activity in beta cell lines. The Freestyle Libre H (Abbott Diabetes Care Ltd) was used to dynamically monitor the proband daily blood glucose levels. Results. Mutation screening for the whole exon genes identified a heterozygous KLF11 (c.1061G > T) variant in the proband, her mother, and her maternal grandfather. Cell-based luciferase reporter assays using wild-type and mutant transgenes revealed that the KLF11 (c.1061G > T) variant had impaired insulin promoter regulation activity. Moreover, this variant was found to impair insulin expression and insulin secretion in pancreatic beta cells. The proband had better blood glucose control without staple food intake ( p < 0.05 ). Conclusions. Herein, for the first time, we report a novel KLF11 (c.1061G > T) monogenic mutation associated with MODY7. This variant has impaired insulin promoter regulation activity and impairs insulin expression and secretion in pancreatic beta cells. Therefore, administering oral antidiabetic drugs along with dietary intervention may benefit the proband.

Co-opting regulation bypass repair (CRBR) as a gene correction strategy for monogenic diseases

With the development of CRISPR/Cas9-mediated gene editing technologies, correction of disease- causing mutations has become possible. However, current gene correction strategies preclude mutation repair in post-mitotic cells of human tissues, and a unique repair strategy must be designed and tested for each and every mutation that may occur in a gene. We have developed a novel gene correction strategy, Co-opting Regulation Bypass Repair (CRBR), which can repair a spectrum of mutations in mitotic or post-mitotic cells and tissues. CRBR utilizes the non-homologous end-joining (NHEJ) pathway to insert a coding sequence (CDS) and transcription/translation terminators targeted upstream of any CDS mutation and downstream of the transcriptional promoter. CRBR gene repair results in simultaneous co-option of the endogenous regulatory region and bypass of the genetic defect. We demonstrated the potential of CRBR strategy for human gene therapy by rescuing a mouse model of the Wolcott-Rallison syndrome (WRS) with permanent neonatal diabetes caused by either large deletion or nonsense mutation in the PERK (EIF2AK3) gene. Additionally, we expressed a GFP CDS-terminator cassette that was integrated downstream of the human insulin promoter in cadaver pancreatic islets of Langerhans which paves the way for autologous cell-tissue replacement therapy for gene repair in beta cells.

Sorcin stimulates Activation Transcription Factor 6α (ATF6) transcriptional activity

ABSTRACTLevels of the transcription factor ATF6α, a key mediator of the unfolded protein response, that provides cellular protection during the progression endoplasmic reticulum (ER) stress, are markedly reduced in the pancreatic islet of patients with type 2 diabetes and in rodent models of the disease, including ob/ob and high fat-fed mice. Sorcin (gene name SRI) is a calcium (Ca2+) binding protein involved in maintaining ER Ca2+ homeostasis.We have previously shown that overexpressing sorcin under the rat insulin promoter in transgenic mice was protective against high fat diet-induced pancreatic beta cell dysfunction, namely preserving intracellular Ca2+ homeostasis and glucose-stimulated insulin secretion during lipotoxic stress. Additionally, sorcin overexpression was apparently activating ATF6 signalling in MIN6 cells despite lowering ER stress.Here, in order to investigate further the relationship between sorcin and ATF6, we describe changes in sorcin expression during ER and lipotoxic stress and changes in ATF6 signalling after sorcin overexpression or inactivation, both in excitable and non-excitable cells.Sorcin mRNA levels were significantly increased in response to the ER stress-inducing agents thapsigargin and tunicamycin, but not by palmitate. On the contrary, palmitate caused a significant decrease in sorcin expression as assessed by both qRT-PCR and Western blotting despite inducing ER stress. Moreover, palmitate prevented the increase in sorcin expression induced by thapsigargin. In addition, sorcin overexpression significantly increased ATF6 transcriptional activity, whereas sorcin inactivation decreased ATF6 signalling. Finally, sorcin overexpression increased levels of ATF6 immunoreactivity and FRET imaging experiments following ER stress induction by thapsigargin showed a direct sorcin-ATF6 interaction.Altogether, our data suggest that sorcin down-regulation during lipotoxicity may prevent full ATF6 activation and a normal UPR during the progression of obesity and insulin resistance, contributing to beta cell failure and type 2 diabetes.

Placental growth factor in beta cells plays an essential role in gestational beta-cell growth

ObjectivePancreatic beta cells proliferate in response to metabolic requirements during pregnancy, while failure of this response may cause gestational diabetes. A member of the vascular endothelial growth factor family, placental growth factor (PlGF), typically plays a role in metabolic disorder and pathological circumstance. The expression and function of PlGF in the endocrine pancreas have not been reported and are addressed in the current study.Research design and methodsPlGF levels in beta cells were determined by immunostaining or ELISA in purified beta cells in non-pregnant and pregnant adult mice. An adeno-associated virus (AAV) serotype 8 carrying a shRNA for PlGF under the control of a rat insulin promoter (AAV–rat insulin promoter (RIP)–short hairpin small interfering RNA for PlGF (shPlGF)) was prepared and infused into mouse pancreas through the pancreatic duct to specifically knock down PlGF in beta cells, and its effects on beta-cell growth were determined by beta-cell proliferation, beta-cell mass and insulin release. A macrophage-depleting reagent, clodronate, was coapplied into AAV-treated mice to study crosstalk between beta cells and macrophages.ResultsPlGF is exclusively produced by beta cells in the adult mouse pancreas. Moreover, PlGF expression in beta cells was significantly increased during pregnancy. Intraductal infusion of AAV–RIP–shPlGF specifically knocked down PlGF in beta cells, resulting in compromised beta-cell proliferation, reduced growth in beta-cell mass and impaired glucose tolerance during pregnancy. Mechanistically, PlGF depletion in beta cells reduced islet infiltration of trophic macrophages, which appeared to be essential for gestational beta-cell growth.ConclusionsOur study suggests that increased expression of PlGF in beta cells may trigger gestational beta-cell growth through recruited macrophages.

Multifaceted secretion of htNSC-derived hypothalamic islets induces survival and antidiabetic effect via peripheral implantation in mice

We report that mouse hypothalamic stem/progenitor cells produce multiple pancreatic, gastrointestinal and hypothalamic peptides in addition to exosomes. Through cell sorting and selection according to insulin promoter activity, we generated a subpopulation(s) of these cells which formed 3D spherical structure with combined features of hypothalamic neurospheres and pancreatic islets. Through testing streptozotocin-induced pancreatic islet disruption and fatal diabetes, we found that peripheral implantation of these spheres in mice led to remarkable improvements in general health and survival in addition to a moderate antidiabetic effect, and notably these pro-survival versus metabolic effects were dissociable to a significant extent. Mechanistically, secretion of exosomes by these spheres was essential for enhancing survival while production of insulin was important for the antidiabetic effect. In summary, hypothalamic neural stem/progenitor cells comprise subpopulations with multifaceted secretion, and their derived hypothalamic islets can be implanted peripherally to enhance general health and survival together with an antidiabetic benefit.