scholarly journals Genetically Engineered Insulin Mediated by Glucose Transporter-2 (GLUT2) Promoter for the Biosynthesis of Insulin in Rat Hepatocytes: Insulin Gene Therapy

2021 ◽  
Author(s):  
Gehad Abdallah
Keyword(s):  
Gene Therapy ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Glucose Sensing ◽  
Genetically Engineered ◽  
Insulin Gene ◽  
Site Directed Mutagenesis ◽  
Target Cells ◽  
Insulin Production ◽  
Glucose Transporter 2

Abstract Background: Expression of insulin in hepatocytes by hepatic gene therapy is a promising treatment of diabetes. The conversion of immature proinsulin to mature insulin occurs only in cells that contain the enzymes responsible for the cleavage of proinsulin to insulin.Results: I engineered rat proinsulin with the sites of cleavage (Furin Cleavable Sites) using site directed mutagenesis for removal of C-peptide to form the two chains A and B for mature insulin production. This engineered proinsulin was constructed into a non-viral expressing vector and regulated by glucose transporter-2 promoter to control the amount of mature insulin expressed, and to modulate the amount of glucose found in hepatocytes. The mature, active and regulated expressed insulin was secreted according to the amount of glucose regulated by the glucose transporter-2 promoter. Concolusion: For successful hepatic insulin gene therapy, insulin production must be tightly coupled to glucose concentration. Hepatocytes are excellent target cells for insulin gene therapy since, they are similar to pancreatic beta cells, they have the ability to rapidly adapt to blood glucose concentrations as they possess glucose-sensing components, such as Glucose Transporter-2.

scholarly journals Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2

2000 ◽  
Vol 164 (3) ◽  
pp. 277-286 ◽  
Author(s):  
A Petryk ◽  
D Fleenor ◽  
P Driscoll ◽  
M Freemark
Keyword(s):  
Gene Expression ◽  
Beta Cell ◽  
Gene Transcription ◽  
Glucose Transporter ◽  
Insulin Gene ◽  
Luciferase Reporter ◽  
Insulin Production ◽  
Glucose Transporter 2 ◽  
Insulin Gene Expression ◽  
Insulin Gene Transcription

Previous studies have shown that lactogenic hormones stimulate beta-cell proliferation and insulin production in pancreatic islets. However, all such studies have been conducted in cells incubated in medium containing glucose. Since glucose independently stimulates beta-cell replication and insulin production, it is unclear whether the effects of prolactin (PRL) on insulin gene expression are exerted directly or through the uptake and/or metabolism of glucose. We examined the interactions between glucose and PRL in the regulation of insulin gene transcription and the expression of glucose transporter-2 (glut-2) and glucokinase mRNAs in rat insulinoma (INS-1) cells. In the presence of 5.5 mM glucose, the levels of preproinsulin and glut-2 mRNAs in PRL-treated cells exceeded the levels in control cells (1.7-fold, P<0.05 and 2-fold, P<0.05 respectively). The maximal effects of PRL were noted at 24-48 h of incubation. PRL had no effect on the levels of glucokinase mRNA. The higher levels of glut-2 mRNA were accompanied by an increase in the number of cellular glucose transporters, as demonstrated by a 1. 4- to 2.4-fold increase in the uptake of 2-deoxy-d-[(3)H]glucose in PRL-treated INS-1 cells (P<0.001). These findings suggested that the insulinotropic effect of PRL is mediated, in part, by induction of glucose transport and/or glucose metabolism. Nevertheless, even in the absence of glucose, PRL stimulated increases in the levels of preproinsulin mRNA (3.4-fold higher than controls, P<0.0001) and glut-2 mRNA (2-fold higher than controls, P<0.01). These observations suggested that PRL exerts glucose-independent as well as glucose-dependent effects on insulin gene expression. Support for this hypothesis was provided by studies of insulin gene transcription using INS-1 cells transfected with a plasmid containing the rat insulin 1 promoter linked to a luciferase reporter gene. Glucose and PRL, alone and in combination, stimulated increases in cellular luciferase activity. The relative potencies of glucose (5.5 mM) alone, PRL alone, and glucose plus PRL in combination were 2.2 (P<0.001), 3.4 (P<0.01), and 7.9 (P<0.0001) respectively. Our findings suggest that glucose and PRL act synergistically to induce insulin gene transcription.

Gene Therapy for Diabetes Mellitus

1997 ◽  
Vol 92 (4) ◽  
pp. 321-330 ◽  
Author(s):  
Kevin Docherty
Keyword(s):  
Gene Therapy ◽  
Cell Lines ◽  
Ex Vivo ◽  
Genetically Engineered ◽  
Insulin Gene ◽  
Diabetic Patients ◽  
Β Cell ◽  
Type 2 Diabetic Patients ◽  
Glucose Stimulated Insulin Secretion

1. This review describes experimental approaches to test the feasibility of using gene therapy to administer insulin to type 1 and type 2 diabetic patients. Two approaches, i.e. ex vivo and in vivo transfer of the insulin gene, are described. 2. Substantial progress has been made in recent years in engineering glucose-responsive β-cell lines that have been genetically engineered to proliferate or differentiate in response to appropriate extracellular signals. 3. Non-β-cell lines have been engineered to constitutively secrete insulin at a constant rate. These cells may improve glycaemic control in patients over longer periods when used in combination with insulin injections. Engineering glucose-stimulated insulin secretion in such cells has proved extremely difficult and several genes may be required. 4. In vivo transfer of the insulin gene to animals results in improved control of diabetes. However, for safety reasons this approach may have limited use in the treatment of diabetes in humans.

Glucose transporter-2 (GLUT2) promoter mediated transgenic insulin production reduces hyperglycemia in diabetic mice

FEBS Letters ◽  
2005 ◽  
Vol 579 (25) ◽  
pp. 5759-5764 ◽  
Author(s):  
Brant R. Burkhardt ◽  
Mathew J. Parker ◽  
Y. Clare Zhang ◽  
Sihong Song ◽  
Clive H. Wasserfall ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Diabetic Mice ◽  
Insulin Production ◽  
Glucose Transporter 2

Proper mTORC1 Activity Is Required for Glucose Sensing and Early Adaptation in Human Pancreatic β Cells

2020 ◽  
Author(s):  
Qicheng Ni ◽  
Jiaxi Song ◽  
Yichen Wang ◽  
Jiajun Sun ◽  
Jing Xie ◽  
...  
Keyword(s):  
Glucose Transporter ◽  
Fasting Glucose ◽  
Dynamic Change ◽  
Human Islets ◽  
Glucose Sensing ◽  
Β Cells ◽  
Β Cell ◽  
Strong Negative Correlation ◽  
Glucose Levels ◽  
Glucose Transporter 2

Abstract Context The mechanistic target of rapamycin complex I (mTORC1) is crucial for β-cell identity and function in rodents. However, its possible relevance to the physiopathology of diabetes in humans remains unclear. Objective This work aimed to understand the participation of mTORC1 in human β cells in prediabetes and diabetes. Design We evaluated the PS6 immunofluorescence intensity in islets of pancreatic sections from 12 nondiabetic (ND), 11 impaired fasting glucose (IFG), and 11 glycemic-controlled type 2 diabetic (T2D) individuals. We also assessed the dynamic change of mTORC1 activity in β cells of db/db mice with new-onset diabetes. Results There exists intercellular heterogeneity of mTORC1 activities in human islets. Islet mTORC1 activity was independently and positively correlated with FBG in ND, but not in IFG and T2D. Moreover, we did not detect significant change in mTORC1 activities between T2D and ND. Of note, the islet mTORC1 activities were significantly higher in IFG than in ND. We further stratified IFG individuals according to their islet PS6 levels and found that IFG-PS6high exhibited remarkably higher urocortin3 and glucose transporter 2 expression in their β cells compared to IFG-PS6low. Consistently, we also detected a significant increase in mTORC1 activities in prediabetic db/db mice compared to nondiabetic littermates. Interestingly, mTORC1 activities determined β-cell adaptation or failure in db/db mice: A strong negative correlation was found between islet mTORC1 activities and fasting glucose levels in db/db mice during their diabetes progression. Conclusions Our finding highlights a dynamic islet mTORC1 response in β-cell adaption/failure in human T2D.

N-glycosylation modulates the membrane sub-domain distribution and activity of glucose transporter 2 in pancreatic beta cells

2013 ◽  
Vol 434 (2) ◽  
pp. 346-351 ◽  
Author(s):  
Kazuaki Ohtsubo ◽  
Shinji Takamatsu ◽  
Congxiao Gao ◽  
Hiroaki Korekane ◽  
Tsutomu M. Kurosawa ◽  
...  
Keyword(s):  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Glucose Transporter 2

scholarly journals The Glucose Transporter 2 Undergoes Plasma Membrane Endocytosis and Lysosomal Degradation in a Secretagogue-Dependent Manner

Endocrinology ◽  
2009 ◽  
Vol 150 (9) ◽  
pp. 4056-4064 ◽  
Author(s):  
June Chunqiu Hou ◽  
Dumaine Williams ◽  
Jérôme Vicogne ◽  
Jeffrey E. Pessin
Keyword(s):  
Plasma Membrane ◽  
Insulin Secretion ◽  
Cell Surface ◽  
Glucose Transporter ◽  
Cell Function ◽  
Glucose Sensing ◽  
Dependent Manner ◽  
Β Cells ◽  
Rapid Degradation ◽  
Glucose Transporter 2

Abstract In β-cells of the pancreas, the glucose transporter (GLUT)-2 facilitative glucose transporter protein is localized to the plasma membrane and functions as part of the glucose sensing mechanism for the stimulation of insulin secretion. We observed that expressed GLUT2 protein in the cultured Min6B1 cell line undergoes enhanced endocytosis at high extracellular glucose concentrations that stimulate insulin secretion. Moreover, the internalized GLUT2 protein undergoes rapid degradation induced by chronic high-glucose or arginine stimulation but does not undergo plasma membrane recycling or accumulation in any microscopically apparent intracellular membrane compartment. The rapid degradation of GLUT2 was prevented by lysosomal inhibition (chloroquine) concomitant with the accumulation of GLUT2 in endomembrane structures. In contrast, neither endocytosis nor the lack of internal membrane localized GLUT2 remained completely unaffected by proteosomal inhibition (lactacystin) or an heat shock protein-90 inhibitor (geldanamycin). Moreover, the endocytosis and degradation of GLUT2 was specific for β-cells because expression of GLUT2 in 3T3L1 adipocytes remained cell surface localized and did not display a rapid rate of degradation. Together, these data demonstrate that hyperglycemia directly affects β-cell function and activates a trafficking pathway that results in the rapid endocytosis and degradation of the cell surface GLUT2 glucose transporter.

scholarly journals Gangliosides modulate insulin secretion by pancreatic beta cells under glucose stress

Glycobiology ◽  
2020 ◽  
Vol 30 (9) ◽  
pp. 722-734 ◽  
Author(s):  
Richard Jennemann ◽  
Sylvia Kaden ◽  
Martina Volz ◽  
Viola Nordström ◽  
Silke Herzer ◽  
...  
Keyword(s):  
Insulin Secretion ◽  
Glucose Uptake ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Specific Model ◽  
Downstream Signaling ◽  
Glucose Levels ◽  
Glucose Transporter 2 ◽  
Lower Glucose

Abstract In pancreatic beta cells, the entry of glucose and downstream signaling for insulin release is regulated by the glucose transporter 2 (Glut2) in rodents. Dysfunction of the insulin-signaling cascade may lead to diabetes mellitus. Gangliosides, sialic acid-containing glycosphingolipids (GSLs), have been reported to modulate the function of several membrane proteins.Murine islets express predominantly sialylated GSLs, particularly the simple gangliosides GM3 and GD3 having a potential modulatory role in Glut2 activity. Conditional, tamoxifen-inducible gene targeting in pancreatic islets has now shown that mice lacking the glucosylceramide synthase (Ugcg), which represents the rate-limiting enzyme in GSL biosynthesis, displayed impaired glucose uptake and showed reduced insulin secretion. Consequently, mice with pancreatic GSL deficiency had higher blood glucose levels than respective controls after intraperitoneal glucose application. High-fat diet feeding enhanced this effect. GSL-deficient islets did not show apoptosis or ER stress and displayed a normal ultrastructure. Their insulin content, size and number were similar as in control islets. Isolated beta cells from GM3 synthase null mice unable to synthesize GM3 and GD3 also showed lower glucose uptake than respective control cells, corroborating the results obtained from the cell-specific model. We conclude that in particular the negatively charged gangliosides GM3 and GD3 of beta cells positively influence Glut2 function to adequately respond to high glucose loads.

Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic beta cells

Development ◽  
1998 ◽  
Vol 125 (12) ◽  
pp. 2213-2221 ◽  
Author(s):  
L. Sussel ◽  
J. Kalamaras ◽  
D.J. Hartigan-O'Connor ◽  
J.J. Meneses ◽  
R.A. Pedersen ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Large Population ◽  
Islet Amyloid ◽  
Cell Markers ◽  
Glucose Transporter 2 ◽  
Alpha Beta

The endocrine pancreas is organized into clusters of cells called islets of Langerhans comprising four well-defined cell types: alpha beta, delta and PP cells. While recent genetic studies indicate that islet development depends on the function of an integrated network of transcription factors, the specific roles of these factors in early cell-type specification and differentiation remain elusive. Nkx2.2 is a member of the mammalian NK2 homeobox transcription factor family that is expressed in the ventral CNS and the pancreas. Within the pancreas, we demonstrate that Nkx2.2 is expressed in alpha, beta and PP cells, but not in delta cells. In addition, we show that mice homozygous for a null mutation of Nkx2.2 develop severe hyperglycemia and die shortly after birth. Immunohistochemical analysis reveals that the mutant embryos lack insulin-producing beta cells and have fewer glucagon-producing alpha cells and PP cells. Remarkably, in the mutants there remains a large population of islet cells that do not produce any of the four endocrine hormones. These cells express some beta cell markers, such as islet amyloid polypeptide and Pdx1, but lack other definitive beta cell markers including glucose transporter 2 and Nkx6.1. We propose that Nkx2.2 is required for the final differentiation of pancreatic beta cells, and in its absence, beta cells are trapped in an incompletely differentiated state.

scholarly journals Effects of Sclerocarya birrea Stem-Bark Extracts on Glucose Uptake, Insulin Synthesis and Expression of Selected Genes Involved in the Synthesis and Secretion of Insulin in Rat Insulinoma Pancreatic Beta Cells

2020 ◽  
Vol 32 (9) ◽  
pp. 2195-2202
Author(s):  
A.H. KGOPA ◽  
L.J. SHAI ◽  
M.A. MOGALE
Keyword(s):  
Glucose Uptake ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Stem Bark ◽  
Hexane Extract ◽  
Insulin Synthesis ◽  
Sclerocarya Birrea ◽  
Glucose Transporter 2 ◽  
Rat Insulinoma

The present study reported the effects of Sclerocarya birrea stem-bark (SBSB) extracts on glucose uptake, insulin synthesis and the expression of glucose transporter 2 (GLUT2), glucokinase, pancreatic duodenal homeobox-1 (PDX-1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) and pre-proinsulin genes in rat insulinoma (RIN)-m5F pancreatic beta cells. The amount of glucose takenup by RIN-m5F cells was measured using a glucose oxidase-based assay kit. Intracellular and secreted insulin were measured using an enzyme linked immunoassay kit. Pre-proinsulin gene expression was determined using the conventional polymerase chain reaction (PCR) technique, while the expressions of GLUT2, glucokinase, PDX-1 and MafA genes were evaluated using quantitative real-time PCR technique. Of the four SBSB extracts investigated in the study, only the SBSB hexane extract positively affected all the study variables in RIN-m5F cells compared with the DMSO control. Thus, the SBSB hexane extract contains phytochemicals capable of enhancing insulin synthesis partly through up-regulation of the expression of GLUT2, glucokinase, PDX-1, MafA and pre-proinsulin genes.

scholarly journals 1-Palmitoyl-2-Linoleoyl-3-Acetyl-rac-Glycerol Attenuates Streptozotocin-Induced Pancreatic Beta Cell Damage by Promoting Glucose Transporter 2 Endocytosis

2019 ◽  
Vol 39 (21) ◽  
Author(s):  
Jimin Kim ◽  
Joo Heon Kim ◽  
Ki-Young Sohn ◽  
Sun Young Yoon ◽  
Jae Wha Kim
Keyword(s):  
Beta Cell ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Glucose Transporter ◽  
Cell Damage ◽  
Serum Insulin ◽  
Pancreatic Beta Cell ◽  
Ros Generation ◽  
Glucose Transporter 2 ◽  
Beta Cell Line

ABSTRACT Streptozotocin (STZ) is widely used to induce diabetic rodent models. It is specifically toxic to pancreatic beta cells and causes severe destruction and dysfunction. We investigated the effect of 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) on an STZ-induced diabetic mouse model. PLAG attenuated the glucose increase and maintained serum insulin at levels similar to those seen with control mice. In pancreatic beta cell line INS-1, STZ-induced cell apoptosis and intracellular reactive oxygen species (ROS) generation were significantly reduced to nearly normal levels after PLAG treatment. Glucose transporter 2 (GLUT2) localization analyses and glucose uptake assays showed that PLAG accelerated GLUT2 internalization, which ameliorated excessive entry of glucose, as well as STZ. STZ-induced cytotoxic effects were significantly reduced in PLAG-treated groups. The biological activity of PLAG was further confirmed in GLUT2-silenced cells, and the specificity of PLAG was verified using its derivative 1-palmitoyl-2-linoleoyl-3-hydroxyl-rac-glycerol (PLH). Our results suggest that PLAG may be a useful agent for protecting beta cells in the setting of excessive glucose influx.

Sign in / Sign up

Export Citation Format

Share Document