BRIN-BD11 Cells Research Articles

In vitro insulinotropic actions of extracts of Gnetum africanum welw and effects on glucose homeostasis in mice with diet-induced obesity-diabetes

IntroductionGnetum africanum leaf is consumed as a vegetable in many parts of Africa. Many ethnobotanical surveys indicate its usage in the management of inflammation-related diseases (such as diabetes). However, mechanisms underlying its antidiabetic actions are poorly understood. MethodsThis study conducted phytochemical analysis and investigated mechanisms underlying anti-diabetic actions of aqueous extracts of G. africanum in cellular and high fat-fed mice models of diabetes. Reversed phase high performance liquid chromatography (HPLC) analysis, qualitative phytochemical screening and the estimation of the total flavonoid and total phenolic content were conducted. Mechanisms underlying insulinotropic actions of the extracts as well as its effects on cytotoxicity, cell viability, intracellular calcium and membrane potential were assessed. In vivo actions were evaluated in mice with diet-induced obesity-diabetes. ResultsG. africanum has total phenolic content of 1.24±0.23GE/g and total flavonoid content of 1.54±0.06QE/g. HPLC analysis revealed the presence of the presence of atropine, vicenin 3, vicenin 2, alpha-chaconine, isoswertisin and solanidine. The extract stimulated non-toxic, concentration-dependent insulin-release from BRIN-BD11 cells (1.3-fold to 3.3-fold, P < 0.05 to P < 0.001). As glucose concentration increased from 1.1 mM to 5.6 mM and 5.6 mM to 16.7 mM, these effects increased by 2.0-fold (P < 0.001) and 1.2-fold (P < 0.05) respectively. Insulinotropic effects increased in cells depolarised with KCl (30 mM, 1.3-fold, P < 0.05). These effects reduced in the presence of diazoxide (300 µM, 67 %, P < 0.001), verapamil (50 nM, 50 %, P < 0.001), and absence of extracellular calcium (55 %, P < 0.001). Membrane potential (48 %, P < 0.05) and intracellular calcium (34 %, P < 0.05) increased in cells incubated with the extract. The extract improved glucose tolerance (22.2 %, P < 0.01 at 150 mg kg-1 bw; 35.1 %, P < 0.001 at 300 mg kg-1 bw) and plasma insulin levels (1.4-fold, P < 0.05 at 150 mg kg-1 bw; 2.9-fold, P < 0.001 at 300 mg kg-1 bw) in high fat fed mice in a dose-dependent manner. ConclusionThese results suggest a role for the KATP-dependent pathway in G. africanum insulinotropic actions and encourage further development of the therapeutic potential of the extract.

Potential of fermented kuini and ceri Terengganu beverages in enhancing insulin secretion in pancreatic cells

Kuini (Mangifera odorata) and ceri Terengganu (Lepisanthes fruticosa) are well-known indigenous fruit in Malaysia. Recently, the use of these fruits is gaining attention due to their high nutrient value. In this study, an antidiabetic activity was selected to be tested on the functional beverages from both fruits. A total of two products have been formulated based on the fermentation of kuini puree and ceri Terengganu juice. The study was conducted through an in vitro assay by using pancreatic cells (BRIN BD11). The pancreatic cells were incubated with KRB (negative control) or KRB-containing products (10-1000 μg/mL) or KRB containing glibenclamide (10-1000 μg/mL). Insulin concentration was determined using Rat INS (Insulin) ELISA Kit. Results showed that both fermented kuini and ceri Terengganu beverages have the ability to increase insulin secretion from BRIN BD11 cells as compared to the control. As for fermented ceri Terengganu beverage, the highest insulin secretion was observed at a concentration of 1000 µg/mL (3.51-fold of increment). Whereas for fermented kuini beverage, the highest insulin secretion was observed at a concentration of 500 µg/mL (3.38 -fold of increment). In conclusion, both fermented beverages have the potential to enhance insulin secretion from pancreatic cells. Further study is needed to be conducted through an in vivo experiment to verify the antidiabetic action of these fermented beverages.

Diterpenoids and Their Glycosides from the Stems of Tinospora crispa with Beta-Cell Protective Activity.

Seven previously undescribed diterpenoids, tinocrisposides A-D (1-4) and borapetic acids A (5), B (6), and C (7), together with 16 known compounds, were isolated from the stem of Tinospora crispa (Menispermaceae). The structures of the new isolates were elucidated by spectroscopic and chemical methods. The β-cell protective effect of the tested compounds was examined on insulin-secreting BRIN-BD11 cells under dexamethasone treatment. Diterpene glycosides 12, 14-16, and 18 presented a substantial protective effect on BRIN-BD11 cells treated with dexamethasone in a dose-dependent manner. Compounds 4 and 17 with two sugar moieties exhibited clear protective effects on β-cells.

A novel peptide isolated from Aphonopelma chalcodes tarantula venom with benefits on pancreatic islet function and appetite control

Proof-of-concept for therapeutic application of venom-derived compounds in diabetes is exemplified by the incretin mimetic, exenatide, originally extracted from the saliva of the venomous Heloderma suspectum lizard. In this regard, we have isolated and sequenced a novel 28 amino acid peptide named Δ-theraphotoxin-Ac1 (Δ-TRTX-AC1) from venom of the Mexican Blond tarantula spider Aphonopelma chalcodes, with potential therapeutic benefits for diabetes. Following confirmation of the structure and safety profile of the synthetic peptide, assessment of enzymatic stability and effects of Δ-TRTX-AC1 on in vitro beta-cell function were studied, alongside potential mechanisms. Glucose homeostatic and satiety actions of Δ-TRTX-AC1 alone, and in combination with exenatide, were then assessed in C57BL/6 mice. Synthetic Δ-TRTX-AC1 was shown to adopt a characteristic inhibitor cysteine knot (ICK)-like structure and was non-toxic to beta-cells. Δ-TRTX-AC1 evoked glucose-dependent insulin secretion from BRIN BD11 cells with bioactivity confirmed in murine islets. Insulin secretory potency was established to be dependent on KATP and Ca2+ channel beta-cell signalling. In addition, Δ-TRTX-AC1 enhanced beta-cell proliferation and provided significant protection against cytokine-induced apoptosis. When injected co-jointly with glucose in mice at a dose of 250 nmol/kg, Δ-TRTX-AC1 decreased blood-glucose levels and evoked a significant satiating effect. Moreover, whilst Δ-TRTX-AC1 did not enhance exenatide induced benefits on glucose homeostasis, the peptide significantly augmented exenatide mediated suppression of appetite. Together these data highlight the therapeutic potential of tarantula spider venom-derived peptides, such as Δ-TRTX-Ac1, for diabetes and related obesity.

Antioxidant, Cytotoxic, and Insulinotropic Activities of Several Leaves Extracts of Medicinal Plants

The prevalence of diabetes mellitus and cancer is increasing; thus, research into efficient treatments utilizing active compounds derived from medicinal plants has focused on these diseases. Through the agro maritime 4.0 approach, medicinal plants are explored in the archipelago of Indonesia, particularly on Tinjil Island, Banten Province. The medicinal plants identified on the island include Morinda citrifolia, Terminalia catappa, and Gnetum gnemon. Therefore, this study aimed to evaluate the in vitro of aqueous extracts of leaves of those three plant species. All aqueous extracts were analyzed for total phenolic content and further tested for antioxidant activity using the DPPH method (2,2-diphenyl-1-picrylhydrazyl), MTT cytotoxic activity (3-[4,5-dimethylthiazole-2-yl]-2- 5-diphenyl-tetrazolium-bromide) in MCF-7- (ATCC HTB 22) and Burkitt’s Lymphoma Raji (ATCC CCL 86) cells, and insulinotropic activity in pancreatic BRIN BD11 cells. The results showed that the total phenolic content of T. catappa was significantly higher (9.21 ± 2.49 mg GAE/g extract sample) compared to M. citrifolia (3.00 ± 0.35 mg GAE/g) and G. gnemon (7.47 ± 0.33 mg GAE/g). Compared to the other two extracts, T. catappa extract has the best DPPH antioxidant activity of IC50 7.44 ± 0.77 µg/mL (p&lt;0.05). MTT cytotoxic activity in all samples did not inhibit the proliferation of Raji cells but did the proliferation of MCF-7 cells. The IC50 for the best cytotoxic activity was shown in M. citrifolia (8.06 µg/mL). T. catappa triggered insulin secretion at 62.5 µg/mL with the highest insulin concentration (54.55 mg/mL). The aqueous extract of T. catappa leaves shows potential as an antioxidant and insulinotropic agent, while M. citrifolia leaves have a cytotoxic effect with anticancer potential.

Antimicrobial, cytotoxic, and insulin-releasing activities of the amphibian host-defense peptide ocellatin-3N and its L-lysine-substituted analogs.

The host-defense peptide ocellatin-3N (GIFDVLKNLAKGVITSLAS.NH2 ), first isolated from the Caribbean frog Leptodactylus nesiotus, inhibited growth of clinically relevant Gram-positive and Gram-negative bacteria as well as a strain of the major emerging yeast pathogen Candida parapsilosis. Increasing cationicity while maintaining amphipathicity by the substitution Asp4 →Lys increased potency against the microorganisms by between 4- and 16-fold (MIC ≤3 μM) compared with the naturally occurring peptide. The substitution Ala18 →Lys and the double substitution Asp4 →Lys and Ala18 →Lys had less effects on potency. The [D4K] analog also showed 2.5- to 4-fold greater cytotoxic potency against non-small-cell lung adenocarcinoma A549 cells, breast adenocarcinoma MDA-MB-231 cells, and colorectal adenocarcinoma HT-29 cells (LC50 values in the range of 12-20 μM) compared with ocellatin-3N but was less hemolytic to mouse erythrocytes. However, the peptide showed no selectivity for tumor-derived cells [LC50 = 20 μM for human umbilical vein endothelial cells (HUVECs)]. Ocellatin-3N and [D4K]ocellatin-3N stimulated the release of insulin from BRIN-BD11 clonal β-cells at concentrations ≥1nM, and [A18K]ocellatin-3N, at concentrations ≥0.1nM. No peptide stimulated the release of lactate dehydrogenase at concentrations up to 3 μM, indicating that plasma membrane integrity had been preserved. The three peptides produced an increase in intracellular [Ca2+ ] in BRIN-BD11 cells when incubated at a concentration of 1μM. In view of its high insulinotropic potency and relatively low hemolytic activity, the [A18K] ocellatin analog may represent a template for the design of agents with therapeutic potential for the treatment of patients with type 2 diabetes.

Vanillic acid potentiates insulin secretion and prevents pancreatic β-cells cytotoxicity under H2O2-induced oxidative stress.

Oxidative stress is known to impair cellular functions and, therefore, plays a significant role in the pathophysiology of various diseases, including diabetes. The persistently elevated glucose levels may cause enhanced mitochondrial reactive oxygen species generation, which in turn can damage the pancreatic β-cells. In this study, we have investigated the effect of vanillic acid on preventing H2O2-induced β-cells death and retaining its insulin secretion potentiating effect in the presence of H2O2. The insulin secretion from the BRIN-BD11 cells was quantified using ELISA-based assays. The viability of the cells was assessed by estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay and DAPI staining. The expression levels of apoptotic and antioxidant proteins were estimated by western blot experiments. Vanillic acid protected pancreatic β-cells viability and function under the H2O2 oxidative stress condition. The Erk1/2 activation appears to play an important role in vanillic acid potentiated insulin secretion and protection of the β-cells in the presence of H2O2. Vanillic acid pretreated cells exhibited enhanced expression of antioxidant enzymes such as catalase and SOD-2 and reduced the expression of proapoptotic markers such as BAX and BAD. In addition, it also enhanced the expression of oxidative stress-sensitive transcription factor Nrf-2 and cell survival protein Akt. The present study shows that vanillic acid potentiates insulin secretion and protects pancreatic β-cells from H2O2-induced oxidative stress.

Polyphenol-Rich Leaf of Annona squamosa Stimulates Insulin Release from BRIN-BD11 Cells and Isolated Mouse Islets, Reduces (CH2O)n Digestion and Absorption, and Improves Glucose Tolerance and GLP-1 (7-36) Levels in High-Fat-Fed Rats.

Annona squamosa, commonly known as custard apple, is traditionally used for the treatment of various diseases including diabetes, cardiovascular disease (CVD), and gastritis. This study was undertaken to investigate the effects of an ethanolic (80% v/v) extract of A. squamosa (EEAS) leaves in vitro on insulin secretion from clonal pancreatic BRIN BD11 β-cells and mouse islets, including mechanistic studies on the effect of EEAS on membrane potential and intracellular calcium ion concentration. Additional in vitro glucose-lowering actions were assessed. For in vivo studies, high-fat-fed (HFF) obese/normal rats were selected. EEAS increased insulin secretion in vitro in a dose-dependent manner. This effect was linked to β-cell membrane depolarisation and cytoplasmic Ca2+ influx. In the presence of isobutyl methylxanthine (IBMX), tolbutamide, or KCl, the insulin-releasing effect of EEAS was increased, suggesting its effect was also mediated via a KATP-independent pathways. EEAS inhibited insulin glycation, glucose absorption, and DPP-IV enzyme activity in vitro and enhanced glucose uptake and insulin action in 3T3L1 cells. In vivo, gut motility, food intake, glucose tolerance, plasma insulin, and active GLP-1 (7-36) levels were improved, whereas plasma DPP-IV levels were reduced in HFF rats. EEAS attenuated the absorption of sucrose and glucose as well as decreased serum glucose levels after sucrose loading and in situ intestinal perfusion in non-diabetic rats. Rutin, proanthocyanidin, and squafosacin G were putatively identified as the anti-hyperglycaemic phytomolecules in EEAS using HPLC followed by LC-MS analysis. This study illustrates the potential of A. squamosa and its phytoconstituents as a source of potential antidiabetic agents.

The ability of deep eutectic solvent systems to extract bioactive compounds from apple pomace

The objective of this study was to examine the bioactivity of extracts from apple pomace obtained by non-conventional green extraction methods (DES systems). Bioactivity was antioxidant capacity and ability to stimulate insulin secretion from pancreatic beta-cells. The antioxidant capacity of extracts was examined using the DPPH and the FRAP assay. Impact of the extracts on cell viability and insulin secretion were examined using the BRIN-BD11 cell line. ChCl:EG(1:4) extracts resulted in high antioxidant capacity in the DPPH assay (80.1% inhibition versus 11.3%). Extracts obtained from the classical systems demonstrated an ability to promote insulin secretion significantly higher than the positive control, p < 0.05. ChCl:EG(1:4) extracts stimulated insulin secretion to a lesser extent. Overall, the data provides evidence for the potential of DES systems to extract bioactive compounds from apple pomace that have relevance for metabolic health. Further optimisation of the extraction procedures should be tailored to the desired bioactive properties.

Insulin secretory and antidiabetic actions of Heritiera fomes bark together with isolation of active phytomolecules.

In folklore, Heritiera fomes (H. fomes) has been extensively used in treatment of various ailments such as diabetes, cardiac and hepatic disorders. The present study aimed to elucidate the antidiabetic actions of hot water extract of H. fomes (HWHF), including effects on insulin release from BRIN BD11 cells and isolated mouse islets as well as glucose homeostasis in high-fat-fed rats. Molecular mechanisms underlying anti-diabetic activity along with isolation of active compounds were also evaluated. Non-toxic concentrations of HWHF stimulated concentration-dependent insulin release from isolated mouse islets and clonal pancreatic β-cells. The stimulatory effect was potentiated by glucose and isobutyl methylxanthine (IBMX), persisted in presence of tolbutamide or a depolarizing concentration of KCl but was attenuated by established inhibitors of insulin release such as diazoxide, verapamil, and Ca2+ chelation. HWHF caused depolarization of the β-cell membrane and increased intracellular Ca2+. The extract also enhanced glucose uptake and insulin action in 3T3-L1 differentiated adipocytes cells and significantly inhibited in a dose-dependent manner starch digestion, protein glycation, DPP-IV enzyme activity, and glucose diffusion in vitro. Oral administration of HWHF (250 mg/5ml/kg b.w.) to high-fat fed rats significantly improved glucose tolerance and plasma insulin responses and it inhibited plasma DPP-IV activity. HWHF also decreased in vivo glucose absorption and intestinal disaccharidase activity while increasing gastrointestinal motility and unabsorbed sucrose transit. Compounds were isolated from HWHF with similar molecular weights to quercitrin (C21 H20 O11) ranging from 447.9 to 449.9 Da which stimulated the insulin release in vitro and improved both glucose tolerance and plasma insulin responses in mice. In conclusion, H. fomes and its water-soluble phytochemicals such as quercitrin may exert antidiabetic actions mediated through a variety of mechanisms which might be useful as dietary adjunct in the management of type 2 diabetes.

A20 controls expression of beta-cell regulatory genes and transcription factors

TNFAIP3 encodes a zinc finger protein called A20, which has potent anti-inflammatory and anti-apoptotic properties. A20 promotes beta-cell survival and protects against islet graft rejection in experimental models. The current study sought to investigate the mechanisms underlying the protective role of A20 in the pancreatic beta-cell. Two islet cell types were used for experiments: the insulin-secreting BRIN-BD11 cell line and human islet cells. A20 was silenced using siRNA against TNFAIP3, and knockdown was confirmed by qPCR and immunostaining of cells. Cell viability, cytotoxicity and apoptosis were assessed using the ApotoxGlo assay. Glucose-stimulated insulin secretion and production of inflammatory cytokines (TNFa, IL1b and IFNg) were measured by ELISA. Expression of beta-cell regulatory genes (Abcc8, Kcnj11, Kcnq1, Gck, Scl2a2) and transcription factors (Hnf1a, Pdx1, Nkx6.1, Ngn3) was determined by qPCR. A20 deficiency increased apoptosis, impaired glucose-induced insulin secretion, and reduced expression of beta-cell regulatory genes and transcription factors. Addition of recombinant A20 normalized gene expression profiles. TNFa, IL1b and IFNg were elevated in A20 deficient cells and found to independently elicit changes in gene expression. Analysis of PCR array data suggests that A20 action in the beta cell is largely, although not exclusively, driven by the P65 subunit of NF-kB. The current report demonstrates a role for A20 in controlling beta-cell integrity and survival, which likely results from the regulation of inflammatory signalling. Of particular note is the impact that A20 deficiency has on the expression of transcription factors regulating the maturation and normal function of beta cells.

Chitosan/Gelatin/PVA Scaffolds for Beta Pancreatic Cell Culture.

Chitosan scaffolds based on blending polymers are a common strategy used in tissue engineering. The objective of this study was evaluation the properties of scaffolds based on a ternary blend of chitosan (Chi), gelatin (Ge), and polyvinyl alcohol (PVA) (Chi/Ge/PVA), which were prepared by cycles of freeze-thawing and freeze-drying. It then was used for three-dimensional BRIN-BD11 beta-cells culturing. Weight ratios of Chi/Ge/PVA (1:1:1, 2:2:1, 2:3:1, and 3:2:1) were proposed and porosity, pore size, degradation, swelling rate, compressive strength, and cell viability analyzed. All ternary blend scaffolds structures are highly porous (with a porosity higher than 80%) and interconnected. The pore size distribution varied from 0.6 to 265 μm. Ternary blends scaffolds had controllable degradation rates compared to binary blend scaffolds, and an improved swelling capacity of the samples with increasing chitosan concentration was found. An increase in Young’s modulus and compressive strength was observed with increasing gelatin concentration. The highest compressive strength reached 101.6 Pa. The MTT assay showed that the ternary blends scaffolds P3 and P4 supported cell viability better than the binary blend scaffold. Therefore, these results illustrated that ternary blends scaffolds P3 and P4 could provide a better environment for BRIN-BD11 cell proliferation.

Physicochemical, Nutritional and In Vitro Antidiabetic Characterisation of Blue Whiting (Micromesistiuspoutassou) Protein Hydrolysates.

Protein hydrolysates from low-value underutilised fish species are potential sources of high-quality dietary protein and health enhancing peptides. Six blue whiting soluble protein hydrolysates (BW-SPH-A_F), generated at industrial scale using different hydrolysis conditions, were assessed in terms of their protein equivalent content, amino acid profile and score and physicochemical properties in addition to their ability to inhibit dipeptidyl peptidase IV (DPP-IV) and stimulate the secretion of insulin from BRIN-BD11 cells. Furthermore, the effect of simulated gastrointestinal digestion (SGID) on the stability of the BW-SPHs and their associated in vitro antidiabetic activity was investigated. The BW-SPHs contained between 70–74% (w/w) protein and all essential and non-essential amino acids. All BW-SPHs mediated DPP-IV inhibitory (IC50: 2.12–2.90 mg protein/mL) and insulin secretory activity (2.5 mg/mL; 4.7 to 6.4-fold increase compared to the basal control (5.6 mM glucose alone)). All BW-SPHs were further hydrolysed during SGID. While the in vitro DPP-IV inhibitory and insulin secretory activity mediated by some BW-SPHs was reduced following SGID, the activity remained high. In general, the insulin secretory activity of the BW-SPHs were 4.5–5.4-fold higher than the basal control following SGID. The BW-SPHs generated herein provide potential for anti-diabetic related functional ingredients, whilst also enhancing environmental and commercial sustainability.

Macroalgal protein hydrolysates from Palmaria palmata influence the \u2018incretin effect\u2019 in vitro via DPP-4 inhibition and upregulation of insulin, GLP-1 and GIP secretion

PurposeThis study investigated metabolic benefits of protein hydrolysates from the macroalgae Palmaria palmata, previously shown to inhibit dipeptidylpeptidase-4 (DPP-4) activity in vitro.MethodsPreviously, Alcalase/Flavourzyme-produced P. palmata protein hydrolysate (PPPH) improved glycaemia and insulin production in streptozotocin-induced diabetic mice. Here the PPPH, was compared to alternative Alcalase, bromelain and Promod-derived hydrolysates and an unhydrolysed control. All PPPH’s underwent simulated gastrointestinal digestion (SGID) to establish oral bioavailability. PPPH’s and their SGID counterparts were tested in pancreatic, clonal BRIN-BD11 cells to assess their insulinotropic effect and associated intracellular mechanisms. PPPH actions on the incretin effect were assessed via measurement of DPP-4 activity, coupled with GLP-1 and GIP release from GLUTag and STC-1 cells, respectively. Acute in vivo effects of Alcalase/Flavourzyme PPPH administration on glucose tolerance and satiety were assessed in overnight-fasted mice.ResultsPPPH’s (0.02–2.5 mg/ml) elicited varying insulinotropic effects (p < 0.05–0.001). SGID of the unhydrolysed protein control, bromelain and Promod PPPH’s retained, or improved, bioactivity regarding insulin secretion, DPP-4 inhibition and GIP release. Insulinotropic effects were retained for all SGID-hydrolysates at higher PPPH concentrations. DPP-4 inhibitory effects were confirmed for all PPPH’s and SGID counterparts (p < 0.05–0.001). PPPH’s were shown to directly influence the incretin effect via upregulated GLP-1 and GIP (p < 0.01–0.001) secretion in vitro, largely retained after SGID. Alcalase/Flavourzyme PPPH produced the greatest elevation in cAMP (p < 0.001, 1.7-fold), which was fully retained post-SGID. This hydrolysate elicited elevations in intracellular calcium (p < 0.01) and membrane potential (p < 0.001). In acute in vivo settings, Alcalase/Flavourzyme PPPH improved glucose tolerance (p < 0.01–0.001) and satiety (p < 0.05–0.001).ConclusionBioavailable PPPH peptides may be useful for the management of T2DM and obesity.

248-OR: Type 1 Diabetes Risk Variants Alter the Transcriptional Profile of Beta Cells

GWAS studies have identified a number of SNPs connected to the development of type 1 diabetes (T1D). It is unclear how these variants confer risk for T1D development and whether this is based on direct effects on the beta cell, or whether the systemic inflammatory environment is detrimental to the beta cell. Using CRISPR mediated homology directed repair (Life Technologies), T1D risk variants rs10517086 and rs1534422 were introduced into the BRIN-BD11 beta cell line and the Jurkat T-cell line. Experiments were conducted in BRIN-BD11 cells expressing each variant and in native BRIN-BD11 cells treated with conditioned media from Jurkat cells expressing each variant. ApoTox GloTM (Promega) was used to assess cell viability, cytotoxicity and apoptosis. In all instances, cell viability and cytotoxicity were unchanged following the introduction of the two variants. However, rs1534422 significantly (P&amp;lt;0.01) increased apoptosis in BRIN-BD11 cells. This was not observed when BRIN-BD11 cells were treated with Jurkat condition media. In all instances, modest reductions (P&amp;lt;0.05) in glucose-stimulated insulin secretion were observed (Insulin ELISA, Mercodia). Expression of beta cell markers (ABCC8, KCNJ11, KCNQ1, SCL2A2, GCK, HNF1A, PDX1, NKX6.1 NGN3) was examined by qPCR (Roche Lightcycler). Irrespective of whether BRIN-BD11 cells were exposed to Jurkat conditioned media or not, induction of rs10517086 or rs1534422 caused significant (P&amp;lt;0.05-0.001) reductions in the expression of all genes examined with two exceptions: both HNF1A and ABCC8 were significantly upregulated (P&amp;lt;0.001). The transcriptional profiles of BRIN-BD11 cells were remarkably similar regardless of whether rs10517086 and rs1534422 were directly induced in BRIN-BD11 cells or whether they were exposed to conditioned media from Jurkat cells expressing the variants. This suggests that soluble secreted factors arising from the expression of these variants may drive the changes observed in the function of BRIN-BD11 cells. Disclosure W. Ratajczak: None. S. Atkinson: None. C. Kelly: None. Funding European Union Regional Development Fund; European Union Sustainable Competitiveness Programme for Northern Ireland; Northern Ireland Public Health Agency; Ulster University; Northern Ireland Department of Employment and Learning

In Vitro and In Vivo Effects of Palmaria palmata Derived Peptides on Glucose Metabolism

Three synthetic peptides, ILAP, LLAP and MAGVDHI, derived from a Palmaria palmata protein hydrolysate were assessed for their antidiabetic potential in vitro and in vivo. In addition to inhibiting dipeptidyl peptidase-IV in a cell-based in situ assay all three peptides significantly increased the half-life of the incretin hormone glucagon-like peptide-1 (GLP-1). ILAP and LLAP mediated a significant increase (p < 0.001) in insulin secretion from BRIN-BD11 cells compared to the glucose control, while MAGVDHI had no insulinotropic activity at an eqimolar concentration (10–6 M). A significant increase in the concentration of cyclic adenosine monophosphate production in BRIN-BD11 cells mediated by ILAP (p < 0.001) and LLAP (p < 0.01) compared to the basal control, would indicate that insulin secretion may be mediated by membrane based activation. Furthermore, ILAP and LLAP acted as glucose-dependent insulinotropic polypeptide (GIP) secretagogues, stimulating a significant increase (p < 0.01) in the concentration of GIP released from enteroendocrine STC-1 cells compared to the glucose control. When tested in vivo in healthy male NIH Swiss mice, ILAP and LLAP, mediated a significant increase (p < 0.01) in plasma insulin and decrease (p < 0.05) in blood glucose, respectively, compared to the control. MAGVDHI mediated a significant (p < 0.001) sustained reduction in food intake in food deprived trained mice. These results demonstrate that the Palmaria palmata peptides studied herein have prospective antidiabetic activity and have the potential to act as agents that can be used alone or in combination with drugs, to aid in the prevention and management of Type 2 diabetes mellitus.

In vitro dipeptidyl peptidase IV inhibitory activity and in situ insulinotropic activity of milk and egg white protein digests.

Dietary proteins are involved in the regulation of glucose homeostasis by different mechanisms. Food protein digestion products are reported to inhibit dipeptidyl peptidase IV (DPP-IV), induce incretin secretion or directly exert an insulinotropic effect in pancreatic β-cells. This study illustrates the DPP-IV inhibitory activity of gastric and intestinal digests of casein, whey and egg white proteins determined in vitro, using Gly-Pro-AMC, and in situ using non-differentiated Caco-2 cells. Comparable trends in the DPP-IV inhibitory profiles were obtained by these two methods although the extent of inhibition in situ was consistently lower than the inhibition observed in vitro. Casein intestinal digests and whey protein gastric and intestinal digests showed potent DPP-IV inhibitory activities in Caco-2 cells with IC50 values ranging from 0.8 to 1.2 mg mL-1. The absorbed fraction of the intestinal digests from whey and egg white protein induced insulin secretion in BRIN-BD11 cells when determined using a two-tiered cellular model (Caco-2 and BRIN-BD11). However, the gastric digests from the same substrates showed no insulin secretion. This may be related to limited trans-epithelial transport through the Caco-2 monolayer of the gastric digestion products. However, both, gastric and intestinal digests were able to induce insulin secretion in BRIN-BD11 cells when the monolayer was composed of a co-culture of STC-1 and Caco-2 cells. This result may be attributed to the activation of STC-1 cells and subsequent incretin secretion, induced by the gastric digest, as shown by an enhanced intracellular calcium uptake.

Amylase-Dependent Regulation of Glucose Metabolism and Insulin/Glucagon Secretion in the Streptozotocin-Induced Diabetic Pig Model and in a Rat Pancreatic Beta-Cell Line, BRIN-BD11.

The current study was aimed at highlighting the role of blood pancreatic amylase in the regulation of glucose homeostasis and insulin secretion in a porcine model of streptozotocin- (STZ-) induced diabetes and in a rat pancreatic beta-cell line, BRIN-BD11. Blood glucose, plasma insulin, and glucagon levels were measured following a duodenal glucose tolerance test (IDGTT), in four pigs with STZ-induced type 2 diabetes (T2D pigs) and in four pigs with STZ-induced type 1 diabetes (T1D pigs). Four intact pigs were used as the control group. The effect of amylase supplementation on both acute and chronic insulin secretion was determined in a BRIN-BD11 cell line. The amylase infusion had no effect on the glucose utilization curve or glucagon levels in the healthy pigs. However, a significant lowering of insulin release was observed in healthy pigs treated with amylase. In the T2D pigs, the glucose utilization curve was significantly lowered in the presence of amylase, while the insulin response curve remained unchanged. Amylase also significantly increased glucagon release during the IDGTT in the T2D and T1D pigs, by between 2- and 4-fold. Amylase did not affect the glucose utilization curve in the T1D pigs. Amylase supplementation significantly decreased both acute and chronic insulin secretion in the BRIN-BD11 cells. These data confirm our previous observations and demonstrate the participation of pancreatic amylase in glucose absorption/utilization. Moreover, the present study clearly highlights the direct impact of pancreatic blood amylase on insulin secretion from pancreatic beta-cells and its interactions with insulin and glucagon secretion in a porcine model.

Evaluation of the Antidiabetic and Insulin Releasing Effects of A. squamosa, Including Isolation and Characterization of Active Phytochemicals.

Annona squamosa is generally referred to as a ‘custard apple’. Antidiabetic actions of hot water extract of Annona squamosa (HWAS) leaves together with isolation of active insulinotropic compounds were studied. Insulin release, membrane potential and intracellular Ca2+ were determined using BRIN-BD11 cells and isolated mouse islets. 3T3L1 adipocytes and in vitro models were used to determine cellular glucose uptake, insulin action, starch digestion, glucose diffusion, DPP-IV activity and glycation. Glucose intolerant high-fat fed rats were used for in vivo studies. Active compounds were isolated and characterized by HPLC, LCMS and NMR. HWAS stimulated insulin release from clonal β-cells and mouse islets. Using fluorescent indicator dyes and modulators of insulin secretion, effects could be attributed to depolarization of β-cells and influx of Ca2+. Secretion was stimulated by isobutylmethylxanthine (IBMX), tolbutamide or 30 mM KCl, indicating additional non-KATP dependent pathways. Extract stimulated cellular glucose uptake and insulin action and inhibited starch digestion, protein glycation, DPP-IV enzyme activity and glucose diffusion. Oral HWAS improved glucose tolerance and plasma insulin in high-fat fed obese rats. Treatment for 9 days with HWAS (250 mg/5 mL/kg), partially normalised energy intake, body weight, pancreatic insulin content, and both islet size and beta cell mass. This was associated with improved oral glucose tolerance, increased plasma insulin and inhibition of plasma DPP-IV activity. Isolated insulinotropic compounds, including rutin (C27H30O16), recapitulated the positive actions of HWAS on beta cells and in vivo glucose tolerance and plasma insulin responses. Annona squamosa is attractive as a dietary adjunct in treatment of T2DM and as a source of potential antidiabetic agents including rutin.

Costus pictus D. Don leaf extract stimulates GLP-1 secretion from GLUTag L-cells and has cytoprotective effects in BRIN-BD11 β-cells

Costus pictus D. Don, commonly known as insulin plant, is a traditional Indian antidiabetic herbal medicine with glucose-lowering and insulin secretory effects having been reported in animal models and humans with Type 2 diabetes. However, its effects on GLP-1 secretion from intestinal endocrine L-cells and potential metabolic and protective effects in insulin secreting pancreatic β-cells are not yet fully understood. This study is aimed to elucidate the effects of Costus pictus D. Don leaf extract (CPE) on L-cell function and GLP-1 secretion using the established murine GLUTag L-cell model and to investigate its potential cytoprotective effects against detrimental effects of palmitate and cytokines in pancreatic β-cells using BRIN-BD11cells. Costus pictus D. Don dried leaf powder was extracted by soxhlet method. Cell viability was determined by MTT assay. Changes in gene and protein expression were quantified by qPCR and western blotting, respectively. GLP-1 and insulin secretion were measured by ELISA. CPE significantly enhanced the percentage of viable BRIN-BD11 and GLUTag cells and protected BRIN-BD11cells against palmitate- and proinflammatory cytokine-induced toxicity. CPE enhanced acute GLP-1 secretion 6.4-16.3-fold from GLUTag cells at both low (1.1mM) and high (16.7mM) glucose (P<0.01) concentrations. Antioxidant (Nrf2, Cat & Gpx1) and pro-proliferative (Erk1 and Jnk1) gene expression were upregulated by 24h culture with CPE, while proinflammatory transcription factor NF-κB was downregulated. Diminished postprandial GLP-1 secretion and loss of insulin secreting β-cells are known contributors of T2DM. Our data suggests that CPE acutely stimulates GLP-1 secretion from L-cells. Long term exposure of the BRIN-BD11cells to CPE enhances cell number and may protect against palmitate and proinflammatory cytokines by activating multiple pathways. Thus, the current study suggests that the possible antidiabetic properties of CPE may be linked to enhanced GLP-1 secretion and β-cell protection which could be beneficial in the management of T2DM.