RIN-5F Cell Line Research Articles

Multifaceted therapeutic approach via thiazolidinedione-infused magnolol in chitosan nanoparticles targeting hyperlipidemia and oxidative stress in gestational diabetes mellitus in experimental mice.

Recent advancements in nanotechnology have sparked interest in the synthesis of chitosan nanoparticles and their potential applications in medicine. This study investigates the synthesis of chitosan nanoparticles infused with thiazolidinedione and magnolol (TZ/ML-ChNPs) and their therapeutic effects on gestational diabetes mellitus (GDM) in experimental mice. Using streptozotocin-induced diabetic pregnant mice as a model, the study examines the anti-diabetic effects of TZ/ML-ChNPs in vitro and explores possible mechanisms of action. Results show a notable decrease in α-amylase and α-glucosidase activities in TZ/ML-ChNPs-treated samples. Cytocompatibility and flow cytometry analysis in streptozotocin-induced diabetic pregnant mice conducted on RIN-5F cell line demonstrate the safety profile of TZ/ML-ChNPs. The primary objective of this research is to assess whether TZ/ML-ChNPs can mitigate hyperlipidemia and oxidative stress in diabetic pregnant mice. Chitosan nanoparticles with thiazolidinedione and magnolol have therapeutic effects that may be used in clinical and pharmaceutical applications.

Effect of bovine beta-casomorphins on rat pancreatic beta cells (RIN-5F) under glucotoxic stress

Beta-casomorphins (BCMs) are the bio-active peptides having opioid properties which are formed by the proteolytic digestion of β-caseins in milk. BCM-7 forms when A1 milk is digested in the small intestine due to a histidine at the 67th position in β-casein, unlike A2 milk, which has proline at this position and produces BCM-9. BCM-7 has further degraded into BCM-5 by the dipeptidyl peptidase-IV (DPP-IV) enzyme in the intestine. The opioid-like activity of BCM-7 is responsible for eliciting signaling pathways which enable a wide range of physiological effects. The aim of our study was to find out the differential role of BCMs (BCM-7, BCM-9 and BCM-5) on pancreatic β-cell proliferation, insulin secretion, and opioid peptide binding receptors from β-cells (RIN-5F cell line) in normal (5.5 mM) and high glucose (27.5 mM) concentrations. Our results showed that BCM-7/9/5 did not affect β-cell viability, proliferation, and insulin secretion at normal glucose level. However, at higher glucose concentration, BCMs significantly protected β-cells from glucotoxicity but did not affect the insulin secretion. Interestingly, in the presence of Mu-opioid peptide receptor antagonist CTOP, BCMs did not protect β-cells from glucotoxicity. The results suggest that BCMs protect β-cells from glucotoxicity via non-opioid mediated pathways because BCMs did not modulate the gene expression of the mu, kappa and delta opioid peptide receptors.

X Ray-Induced Insulinoma Cell Line Rin-5F Has a Novel Mutation Site, C.A1459G (P.T487A), in Death Domain Associated Protein (DAXX) Gene

A popular toxicological and pharmacological research cell line is the insulin-secreting pancreatic cell line Rin-5F. The cell line originates from insulinomas induced by X-ray exposure. The author of this report looked at the mutation status of the DAXX gene in the Rin-5F cell line clone.<em> </em>The complete DNA and RNA were extracted from the cultivated cells as well. Double-stranded cDNA was then synthesized using the RNA template. Sequencing was done using a 3730xl DNA Analyzer.<em> </em>In the present study, c.A1459G (p.T487A) in Exon 5 in the DAXX gene was detected in Rin-5F cell lines, one of the X-ray-induced insulinomas. An NCBI homology search reveals that the 487 amino acid site in rats is the 497 amino acid of humans, based on the genomic cDNA homology between rats and humans. In humans, the COSMIC database suggests that mutations involving 497 amino acids have not been detected in all human cancers. However, the mutation of 496 amino acids was detected in human stomach and colon cancers. This is the first account of the DAXX gene's state in a cell line created by exposure to X-rays. This may point to the need for additional data and research on unique gene alterations involved in the development of X-ray-induced insulinoma tumors.

In vitro and functional investigation reveals the curative effect of thymoquinone from black cumin-loaded chitosan nanoparticles on streptozotocin induced paediatric diabetes

Diabetic ketoacidosis (DKA) is regarded to be a communal complication of both type 1 and type 2 diabetes mellitus in children and adolescents. Successful therapy of DKA in children requires prompt diagnosis, strict monitoring of medical indicators, and prompt action. Thymoquinone (Tq) from black cumin loaded chitosan nanoparticles (ChNPs) intend to assess an effective agent to overcome this problem. XRD, FTIR, SEM, and TEM were used in the physicochemical analysis. Enzymatic activity of α-amylase and α-glucosidase was used in in vitro tests of anti-diabetic efficacy. Protecting insulin against enzyme breakdown is a crucial part of the insulin delivery mechanism. In the STZ-induced diabetes RIN-5F cell line, the anti-apoptotic capability of Tq-ChNPs was demonstrated through the NF-κB mediated apoptotic pathway. The combination of thymoquinone and chitosan NPs demonstrated that a wide variety of incredibly effective substances to elevate their curative effects, thus contributing to the growth of clinical and pharmaceutical fields.

In Vitro Antidiabetic and Antioxidant Effects of Different Extracts of Catharanthus roseus and Its Indole Alkaloid, Vindoline.

The Catharanthus roseus plant has been used traditionally to treat diabetes mellitus. Scientific evidence supporting the antidiabetic effects of this plant’s active ingredient-vindoline has not been fully evaluated. In this study, extracts of C. roseus and vindoline were tested for antioxidant activities, alpha amylase and alpha glucosidase inhibitory activities and insulin secretory effects in pancreatic RIN-5F cell line cultured in the absence of glucose, at low and high glucose concentrations. The methanolic extract of the plant showed the highest antioxidant activities in addition to the high total polyphenolic content (p < 0.05). The HPLC results exhibited increased concentration of vindoline in the dichloromethane and the ethylacetate extracts. Vindoline showed noticeable antioxidant activity when compared to ascorbic acid at p < 0.05 and significantly improved the in vitro insulin secretion. The intracellular reactive oxygen species formation in glucotoxicity-induced cells was significantly reduced following treatment with vindoline, methanolic and the dichloromethane extracts when compared to the high glucose untreated control (p < 0.05). Plant extracts and vindoline showed weaker inhibitory effects on the activities of carbohydrate metabolizing enzymes when compared to acarbose, which inhibited the activities of the enzymes by 80%. The plant extracts also exhibited weak alpha amylase and alpha glucosidase inhibitory effects.

Green synthesis of gold nanoparticle using Eclipta alba and its antidiabetic activities through regulation of Bcl-2 expression in pancreatic cell line

Causes of diabetes could be associated with many dysfunctions regarding the regulation of insulin and progression of pancreatic beta cells. Phytonanotherapy provides a symbiotic properties of metal nanoparticles and plants as they exhibit medicinal benefits which may be biologically equivalent for synthetic drugs. The present study focused on rapid and ecofriendly biosynthesis of gold nanoparticles using Eclipta alba (EA-AuNPs) and evaluated its pharmacological efficacy against pathogenic bacteria and streptozotocin (STZ) induced apoptosis in RIN-5F cell line. The bioreduction of auric chloride was attained through capping or stabilizing molecules offered by the phytoconstituents present in Eclipta alba. The green synthesized EA-AuNPs was characterized by spectrometric studies, such as UV–vis spectroscopy, X-ray diffraction analysis (XRD), Fourier transform infrared spectroscopic (FTIR) study, dynamic light scattering (DLS), and by microscopic studies such as high-resolution transmission electron microscopy (TEM), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The synthesized EA-AuNPs showed surface plasmon resonance band at 536 nm and the particle was spherical about 26 nm with a good zeta potential value of −12mv. The anti-apoptotic potential of EA-AuNPs was proved by evaluating the NF-κB mediated apoptotic pathway in STZ-induced diabetes in RIN-5F cell line. We further showed the free radical scavenging potential of EA-AuNPs and stabilized EA-AuNPs demonstrated strong antibacterial activity against human pathogenic bacteria. Therefore, we conclude that Eclipta alba has a good potential to synthesize AuNPs and the synthesized EA-AuNPs are promising for biomedical applications in the field of nanomedicine.

Insulinotropic and antidiabetic effects of β-caryophyllene with l-arginine in type 2 diabetic rats.

Beta-caryophyllene (BCP) is a flavoring agent, whereas l-arginine (LA) is used as a food supplement. They possess insulinotropic and β cell regeneration activities, respectively. We assessed the antidiabetic potential of BCP, LA, and its combination in RIN-5F cell lines and diabetic rats. Ex vivo studies were carried out for glucose uptake and absorption of the combination of BCP with LA. The results indicated that the combination of BCP with LA showed a significant decrease in glucose absorption and an increase in its uptake in tissues and also an increase in insulin secretion in RIN-5F cells. The combination treatment of BCP with LA showed a significant reduction in glucose, lipid levels, and oxidative stress in pancreatic tissue when compared with the diabetic group. Furthermore, the combination of BCP with LA normalized glucose tolerance and pancreatic cell damage in diabetic rats. In conclusion, the combinational treatment showed significant potentials in the treatment of type 2 diabetes mellitus. PRACTICAL APPLICATIONS: Type 2 diabetes mellitus is the most prevalent chronic metabolic disorder affecting a large population. Beta-caryophyllene is a CB2 receptor agonist shown to have insulinotropic activity. l-Arginine is a food supplement that possesses beta-cell regeneration property. The combination of BCP with LA could work as a potential therapeutic intervention, considering the individual pharmacological activities of each. We evaluated the antidiabetic activity of the combination of BCP with LA in diabetic rats using ex vivo and in vitro experimentations. Results from the study revealed that the combination of BCP with LA showed a significant (p<.001) reduction in glucose and lipid levels as compared to individual treatment. In vitro study also supports the diabetic potential of the combination of BCP with LA in the glucose-induced insulin secretion in RIN-5F cell lines. The study indicates a therapeutic approach to treat T2DM by BCP and LA combination as food and dietary supplement.

Antidiabetic Activity of Gold Nanoparticles Synthesized Using Wedelolactone in RIN-5F Cell Line.

We synthesized the gold nanoparticles (AuNPs) using wedelolactone (WDL) and characterized them using UV-visible spectroscopy, fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopic (SEM), transmission electron microscopic (TEM), energy dispersive X-ray diffraction, and atomic force microscopic (AFM) studies. The electronic spectrum exhibited an absorption peak at 535 nm. The FT-IR results proved that WDL was stabilized on the surface of AuNPs by acting as a capping or reducing agent. The crystalline structure was affirmed by XRD pattern and the spherical shape of WDL-AuNPs was evidenced by SEM, TEM, and AFM. The synthesized WDL-AuNPS were evaluated for anti-diabetic activity in pancreatic RIN-5F cell lines. In vitro results showed that WDL-AuNPs did not only improve the insulin secretion affected by di-(2-ethylhexyl) phthalate (DEHP), but also the cell viability in RIN5F cells. WDL-AuNPs treatment modulates the pro-apoptotic proteins and anti-apoptotic proteins expression to prevent the cells undergoing apoptosis in DEHP-exposed RIN-5F cells. The exposure of DEHP causes an increase in ROS production and lipid peroxidation levels. The free radical scavenging and antioxidant properties of WDL-AuNPs increase the deleterious effect caused by DEHP. On the other side, WDL-AuNPs increase mRNA expressions of insulin-signaling proteins in RIN-5F cells. This study concludes that WDL-AuNPs can be successfully used to regulate the expression of Bcl-2 family proteins, reduce lipid peroxidation, and to improve the secretion of antioxidants and insulin through the GLUT2 pathway in RIN-5F cell lines.

Antidiabetic Activity of Phytosaponin in STZ-Induced Type I Diabetes in Rats

Objective: The study was focused on antidiabetic activity of saponin isolated from the fruits of Momordica dioica against STZ induced Type I diabetic mellitus in rats. Methods: Saponin was isolated from methanolic extract of fruits of M. dioica and purification was achieved through fractionation method of TLC. Overnight fasting, for Type I diabetes was induced in rats by I.P. of STZ dose of 65 mg/kg b.w. During the study, body weight and fasting blood glucose level were taken. At the end of study, animals in all groups were sacrificed and biochemical parameters; Lipid profile, HbA1c, Serum insulin, pancreatic insulin, histology of pancreas, and in-vitro insulin secretion activity and proliferation of beta cells in RIN-5F cell lines were performed. Results and conclusion: Significant antidiabetic activity of saponin Momordica dioica (SMD) observed in the present investigation could be the result of decreased blood glucose levels, improved body mass, showed improvisation in lipid profile. The diabetic rats were validated by their higher HbA1c levels as compared the normal control but the treated diabetic group showed significantly decreased in the HbA1c levels. Similarly the increase in serum insulin and pancreatic insulin may facilitate to prevent diabetic complication. Diabetic pancreas of rats showed degeneration of beta cell density and disruption of normal architecture, whereas treatment group showed reversible beta cell degeneration. SMD was found to possess insulinotropic activity when rat insulinoma cell line was treated with it. Proliferation of RIN-5F was found to increase at low doses but tend to decrease with accelerating doses.

Identification of transcription factors involved in the transcriptional regulation of the CXCL12 gene in rat pancreatic insulinoma Rin-5F cell line.

Diabetes is characterized by a deficit in the number of functional pancreatic β-cells. Understanding the mechanisms that stimulate neogenesis of β-cells should contribute to improved maintenance of β-cell mass. Chemokine CXCL12 has recently become established as a novel β-cell growth factor, however the mechanisms controlling its expression require clarification. We investigated the proteins involved in the transcriptional regulation of the rat β-cell CXCL12 gene (Cxcl12). Using the electrophoretic mobility shift assay and chromatin immunoprecipitation, we established the in vitro and in vivo binding of C/EBPβ, C/EBPα, STAT3, p53, FOXO3a, and HMG I/Y to the Cxcl12 promoter. Co-immunoprecipitation experiments revealed protein-protein interactions between YY1 and PARP-1, FOXO3a and PARP-1, Sp1 and PARP-1, p53 and PARP-1, C/EBPβ and PARP-1, YY1 and p53, YY1 and FOXO3a, p53 and FOXO3a, Sp1 and FOXO3a, C/EBPβ and FOXO3a, C/EBPα and FOXO3a, Sp1 and STAT3. Our data lay the foundation for research into the interplay of signaling pathways that determine the β-cell Cxcl12 expression profile.

Manufacturing of Insulin-Secreting Spheroids with the RIN-5F Cell Line Using a Shaking Culture Method

Background There have been many efforts to find methods to increase insulin production by islets or modified cells. Commercially available established cell lines can be a good source of artificial islets. We manufactured sphere-shaped cell clusters composed of insulin-secreting cells from the commercially available RIN-5F cell line. Methods To generate artificial islets with insulin-secretion functions, we used the RIN-5F cell line. When cells cultured in RPMI-1640 medium containing 10% fetal bovine serum reached near confluency, they were trypsinized for suspension culture at high density, using a horizontal shaker. The cells were maintained for 5 days under 5% CO 2 with humidification. Next, the media from the RIN cell spheroid culture was collected over 5 consecutive days to test for insulin secretion. Results Spheroids of artificial islets exhibited an oval shape with an approximate size of 94.13 ± 20.41 μm on day 5 during the shaking culture. Abnormal outgrowth of spheroids was not observed. Terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling–positive cells were not detected among the overall spheroids, including the core position. Insulin secretion, measured by enzyme-linked immunosorbent assay, was well maintained in the culture media over 5 days after spheroid formation. Conclusion This result suggested that a culture method with shaking can be applied to commercially available established cell lines to generate artificial islets, which might be used for a bioartificial pancreas.

CrmA and Bcl-2 protect a cell line derived from islet of Langerhans, RIN-5F from hypoxia-induced apoptosis

Encapsulated islet cells have potential for use in cellular therapy for type I diabetes, but cell survival under hypoxia during revascularization is a serious problem lessening the effect of cellular therapy. To overcome the difficulty in cell survival under hypoxia, we have introduced the anti-apoptotic genes, crmA and bcl-2, into a RIN-5F cell line derived from rat islet of Langerhans cells, named RIN-5F-CrmA and RIN-5F-Bcl-2, respectively. RT-PCR and immunoblotting showed that RIN-5F-CrmA cells stably expressed crmA transcripts and that RIN-5F-Bcl-2 cells stably expressed Bcl-2, respectively. Over-expression of these anti-apoptotic genes prolonged the culture period under hypoxic conditions ( pO 2 2%). In hypoxia, Rin-5F cell lines produced less insulin, but over-expression of crmA significantly reduced the decrease of insulin synthesis in hypoxic culture. These results indicate that both CrmA and Bcl-2 are effective for inhibiting cell death under hypoxia, and suggest that CrmA would be superior to Bcl-2 because of the improved insulin production in hypoxia. This anti-apoptotic approach would have important therapeutic implications for successful transplantation of encapsulated islet cells.

Cloning and Characterization of the Rat Complementary Deoxyribonucleic Acid and Gene Encoding the Neuroendocrine Peptide 7B2*

A 7B2 cDNA clone was isolated from a rat insulinoma cDNA library. The 1.1-kilobase (kb) cDNA insert contained 1 open reading frame of 630 nucleotides which encoded a 210-amino acid sequence. The deduced rat 7B2 precursor peptide of approximately 24 kDa would yield an approximately 21-kDa peptide upon cleavage of the signal sequence. The rat 7B2 nucleotide and amino acid sequences were highly homologous to those of mouse, human, pig, toad, and salmon. Northern analysis revealed 7B2 expression in rat pituitary and brain and in the PC12 and RIN5F cell lines. A DNA clone containing the 5' end of the rat 7B2 gene was isolated from a rat genomic DNA library. A 3.5-kb fragment isolated from this clone contained 1.5 kb of 5' nontranscribed DNA and 2 kb of the transcriptional unit. Nucleotide sequence and primer extension analyses revealed a TATA-like sequence approximately 25 basepairs up-stream of the transcription start site. Sequence analysis also revealed three putative cis-acting elements in the 5' flanking region. The second exon encoded the first 73 amino acids of the 7B2 prepeptide. Plasmids containing the 7B2 promoter fused with a reporter gene were transiently transfected into LAN-5 cells. Expression was evident only when the first intron was included with the 5' flanking sequences in the construct and only when cells were treated with forskolin or a phorbol ester. The rat 7B2 gene and cDNA will be valuable tools for the identification of factors that regulate 7B2 gene expression.

Origin of the Beta Cells of the Islets of Langerhans is Further Questioned by the Expression of Neuronal Intermediate Filament Proteins, Peripherin and NF-L, in the Rat Insulinoma RIN5F Cell Line

Intermediate filament proteins of the rat insulinoma RIN5F cell line were characterized. Two-dimensional gel analysis followed by immunostaining of proteins demonstrated that these cells express both peripherin and the low-molecular-mass neurofilament protein (NF-L); this was confirmed for peripherin by immunohistochemistry, peptide analysis and Northern blot. No expression of these proteins could be detected with these same methods either in the adult pancreas or in the tumor at the origin of the cell line, although such expression was apparent on sections of rat pancreas at embryonal day 16. These results were compared to those obtained on the rat pheochromocytoma PC12 cell line: expression in the adrenal medulla of the embryo, no expression either in the adult tissue or in the tumor, but solely in the derived cell line. The expression of neuronal intermediate filament proteins in the rat insulinoma RIN5F cell line is discussed in relation to its similarity in the rat pheochromocytoma PC12 cell line, and its meaning as to the developmental cell lineage; an ectodermal origin is suggested for the pancreatic islet cells.

Identification of the promoter sequences involved in the cell specific expression of the rat somatostatin gene.

DNA sequences containing the 5' flanking region of the rat somatostatin gene were linked to the coding sequence of the bacterial chloramphenicol acetyl transferase gene. This recombinant plasmid is active in expressing CAT activity in the neuronally derived, somatostatin producing CA-77 cell line. Deletion analyses of the somatostatin promoter show that the sequences proximal to position -60, relative to the cap site are required for expression of this promoter. A 4 base pair deletion of residues -46 through -43 within the somatostatin promoter results in a down mutation in vivo suggesting the existence of an element critical for the expression of the promoter in CA-77 cells. In addition, the somatostatin recombinant and its 5' deletion constructs preferentially express CAT activity in CA-77 cells, whereas only basal level of expression is observed in HeLa, BSC40, and RIN-5F cell lines, pointing to the cell specific nature of this promoter.

Control of insulin gene expression in pancreatic beta-cells and in an insulin-producing cell line, RIN-5F cells. I. Effects of glucose and cyclic AMP on the transcription of insulin mRNA.

To define the mechanism whereby glucose regulates islet insulin mRNA content, insulin gene transcription rates were determined in islets labeled with [3H]uridine at low (3.3) or high (17 mM) glucose. Glucose stimulated the transcription of total RNA almost 2-fold and insulin mRNA 5.6-fold. Addition of dibutyryl cAMP to islets in vitro could partially mimic the effect of glucose on insulin gene-specific transcription. In the insulin-producing RIN-5F cell line, glucose did not affect transcription, while cholera toxin acted as a secretagogue and increased total RNA and insulin gene-specific transcription as well. We conclude that glucose exerts a specific stimulatory effect on the transcription of the insulin gene(s) in normal islets and that this effect may be mediated in part by cAMP.