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1.
Appetite ; 128: 197-204, 2018 09 01.
Article in English | MEDLINE | ID: mdl-29920323

ABSTRACT

There is evidence that oat ß-glucan lowers appetite and ad libitum eating; however, not all studies are consistent, and the underpinning mechanisms are not entirely understood. We investigated the effects of 4 g high molecular weight (MW) oat ß-glucan on ad libitum eating, subjective appetite, glycemia, insulinemia and plasma GLP-1 responses in 33 normal-weight subjects (22 female/11 male, mean age (y): 26.9 ±â€¯1.0, BMI (kg/m2): 23.5 ±â€¯0.4). The study followed a randomised double-blind, cross-over design with subjects fed two test breakfasts with and without oat ß-glucan followed by an ad libitum test meal on two different days. Blood samples and ratings for subjective appetite were collected postprandially at regular time intervals. Oat ß-glucan increased feelings of fullness (p = 0.048) and satiety (p = 0.034), but did not affect energy and amount eaten at the ad libitum test meal. There was a treatment by time interaction for plasma GLP-1, plasma insulin and blood glucose. GLP-1 was significantly reduced at 90 min (p = 0.021), blood glucose at 30 min (p = 0.008) and plasma insulin at 30 and 60 min (p = 0.002 and 0.017, respectively) following the oat ß-glucan breakfast when compared with the control breakfast. Four grams of high MW oat ß-glucan lowers appetite but not ad libitum eating and beneficially modulates postprandial glycaemia, it does however, not increase plasma GLP-1 secretion.


Subject(s)
Appetite/drug effects , Avena , Breakfast/physiology , Eating/drug effects , beta-Glucans/administration & dosage , Adult , Blood Glucose/metabolism , Cross-Over Studies , Double-Blind Method , Female , Glucagon-Like Peptide 1/blood , Healthy Volunteers , Humans , Insulin/blood , Male , Postprandial Period , Satiation/drug effects
2.
Biochim Biophys Acta ; 1840(1): 136-45, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24005237

ABSTRACT

BACKGROUND: Molecular mechanisms of toxicity and cell damage were investigated in the novel human beta cell line, 1.1B4, after exposure to proinflammatory cytokines - IL-1ß, IFN-γ, TNF-α. METHODS: MTT assay, insulin radioimmunoassay, glucokinase assay, real time reverse transcription PCR, western blotting, nitrite assay, caspase assay and comet assay were used to investigate mechanisms of cytokine toxicity. RESULTS: Viability of 1.1B4 cells decreased after 18h cytokine exposure. Cytokines significantly reduced cellular insulin content and impaired insulin secretion induced by glucose, alanine, KCl, elevated Ca(2+), GLP-1 or forskolin. Glucokinase enzyme activity, regulation of intracellular Ca(2+) and PDX1 protein expression were significantly reduced by cytokines. mRNA expression of genes involved in secretory function - INS, GCK, PCSK2 and GJA1 was downregulated in cytokine treated 1.1B4 cells. Upregulation of transcription of genes involved in antioxidant defence - SOD2 and GPX1 was observed, suggesting involvement of oxidative stress. Cytokines also upregulated transcriptions of NFKB1 and STAT1, which was accompanied by a significant increase in NOS2 transcription and accumulation of nitrite in culture medium, implicating nitrosative stress. Oxidative and nitrosative stresses induced apoptosis was evident from increased % tail DNA, DNA fragmentation, caspase 3/7 activity, apoptotic cells and lower BCL2 protein expression. CONCLUSIONS: This study delineates molecular mechanisms of cytokine toxicity in 1.1B4 cells, which agree with earlier observations using human islets and rodent beta cells. GENERAL SIGNIFICANCE: This study emphasizes the potential usefulness of this cell line as a human beta cell model for research investigating autoimmune destruction of pancreatic beta cells.


Subject(s)
Apoptosis , Cytokines/pharmacology , Inflammation Mediators/pharmacology , Insulin-Secreting Cells/drug effects , Islets of Langerhans/drug effects , Antioxidants/metabolism , Blotting, Western , Calcium/metabolism , Caspases/genetics , Caspases/metabolism , Cell Proliferation , Cells, Cultured , Comet Assay , Glucokinase/metabolism , Humans , Insulin/metabolism , Insulin-Secreting Cells/metabolism , Insulin-Secreting Cells/pathology , Interferon-gamma/pharmacology , Interleukin-1beta/pharmacology , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Oxidative Stress/drug effects , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Necrosis Factor-alpha/pharmacology
3.
Islets ; 5(4): 170-7, 2013.
Article in English | MEDLINE | ID: mdl-23985558

ABSTRACT

The novel human-derived pancreatic ß-cell line, 1.1B4 exhibits insulin secretion and ß-cell enriched gene expression. Recent investigations of the cellular responses of this novel cell line to lipotoxicity and cytokine toxicity revealed similarities to primary human ß cells. The current study has investigated the responses of 1.1B4 cells to chronic 48 and 72 h exposure to hyperglycemia to probe mechanisms of human ß-cell dysfunction and cell death. Exposure to 25 mM glucose significantly reduced insulin content (p<0.05) and glucokinase activity (p<0.01) after 72 h. Basal insulin release was unaffected but acute secretory response to 16.7 mM glucose was impaired (p<0.05). Insulin release stimulated by alanine, GLP-1, KCl, elevated Ca (2+) and forskolin was also markedly reduced after exposure to hyperglycemia (p<0.001). In addition, PDX1 protein expression was reduced by 58% by high glucose (p<0.05). Effects of hyperglycemia on secretory function were accompanied by decreased mRNA expression of INS, GCK, PCSK1, PCSK2, PPP3CB, GJA1, ABCC8, and KCNJ11. In contrast, exposure to hyperglycemia upregulated the transcription of GPX1, an antioxidant enzyme involved in detoxification of hydrogen peroxide and HSPA4, a molecular chaperone involved in ER stress response. Hyperglycemia-induced DNA damage was demonstrated by increased % tail DNA and olive tail moment, assessed by comet assay. Hyperglycemia-induced apoptosis was evident from increased activity of caspase 3/7 and decreased BCL2 protein. These observations reveal significant changes in cellular responses and gene expression in novel human pancreatic 1.1B4 ß cells exposed to hyperglycemia, illustrating the usefulness of this novel human-derived cell line for studying human ß-cell biology and diabetes.


Subject(s)
Glucose/pharmacology , Hyperglycemia/metabolism , Insulin-Secreting Cells/drug effects , Apoptosis/drug effects , Cell Line , DNA Damage , Endoplasmic Reticulum Stress/drug effects , Glucokinase/metabolism , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/metabolism
4.
Biol Chem ; 394(7): 909-18, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23492555

ABSTRACT

The novel insulin-secreting human pancreatic ß-cell line, 1.1B4, demonstrates stability in culture and many of the secretory functional attributes of human pancreatic ß-cells. This study investigated the cellular responses of 1.1B4 cells to lipotoxicity. Chronic 18-h exposure of 1.1B4 cells to 0.5 mm palmitate resulted in decreased cell viability and insulin content. Secretory responses to classical insulinotropic agents and cellular Ca2+ handling were also impaired. Palmitate decreased glucokinase activity and mRNA expression of genes involved in secretory function but up-regulated mRNA expression of HSPA5, EIF2A, and EIF2AK3, implicating activation of the endoplasmic reticulum stress response. Palmitate also induced DNA damage and apoptosis of 1.1B4 cells. These responses were accompanied by increased gene expression of the antioxidant enzymes SOD1, SOD2, CAT and GPX1. This study details molecular mechanisms underlying lipotoxicity in 1.1B4 cells and indicates the potential value of the novel ß-cell line for future research.


Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum Stress/physiology , Gene Expression Regulation/physiology , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Palmitates/pharmacology , Blotting, Western , Cell Line , Cell Survival/physiology , Comet Assay , Endoplasmic Reticulum Chaperone BiP , Endoplasmic Reticulum Stress/genetics , Humans , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/enzymology , RNA, Messenger/chemistry , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Statistics, Nonparametric
5.
J Endocrinol ; 214(3): 257-65, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22685334

ABSTRACT

Formation of pseudoislets from rodent cell lines has provided a particularly useful model to study homotypic islet cell interactions and insulin secretion. This study aimed to extend this research to generate and characterize, for the first time, functional human pseudoislets comprising the recently described electrofusion-derived insulin-secreting 1.1B4 human ß-cell line. Structural pseudoislets formed readily over 3-7 days in culture using ultra-low-attachment plastic, attaining a static size of 100-200 µm in diameter, corresponding to ~6000 ß cells. This was achieved by decreases in cell proliferation and integrity as assessed by BrdU ELISA, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, and lactate dehydrogenase assays. Insulin content was comparable between monolayers and pseudoislets. However, pseudoislet formation enhanced insulin secretion by 1·7- to 12·5-fold in response to acute stimulation with glucose, amino acids, incretin hormones, or drugs compared with equivalent cell monolayers. Western blot and RT-PCR showed expression of key genes involved in cell communication and the stimulus-secretion pathway. Expression of E-Cadherin and connexin 36 and 43 was greatly enhanced in pseudoislets with no appreciable connexin 43 protein expression in monolayers. Comparable levels of insulin, glucokinase, and GLUT1 were found in both cell populations. The improved secretory function of human 1.1B4 cell pseudoislets over monolayers results from improved cellular interactions mediated through gap junction communication. Pseudoislets comprising engineered electrofusion-derived human ß cells provide an attractive model for islet research and drug testing as well as offering novel therapeutic application through transplantation.


Subject(s)
Cell Fusion/methods , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/physiology , Insulin/metabolism , Islets of Langerhans Transplantation , Tissue Engineering/methods , Amino Acids/pharmacology , Cell Communication/drug effects , Cell Communication/physiology , Cell Culture Techniques/methods , Cell Line, Transformed , Cell Line, Tumor , Cell Proliferation , Gap Junctions/physiology , Glucose/pharmacology , Hormones/pharmacology , Humans , Insulin Secretion , Insulin-Secreting Cells/drug effects , Keto Acids/pharmacology , Transcriptome
6.
J Biol Chem ; 286(25): 21982-92, 2011 Jun 24.
Article in English | MEDLINE | ID: mdl-21515691

ABSTRACT

Three novel human insulin-releasing cell lines designated 1.1B4, 1.4E7, and 1.1E7 were generated by electrofusion of freshly isolated of human pancreatic beta cells and the immortal human PANC-1 epithelial cell line. Functional studies demonstrated glucose sensitivity and responsiveness to known modulators of insulin secretion. Western blot, RT-PCR, and immunohistochemistry showed expression of the major genes involved in proinsulin processing and the pancreatic beta cell stimulus-secretion pathway including PC1/3, PC2, GLUT-1, glucokinase, and K-ATP channel complex (Sur1 and Kir6.2) and the voltage-dependent L-type Ca(2+) channel. The cells stained positively for insulin, and 1.1B4 cells were used to demonstrate specific staining for insulin, C-peptide, and proinsulin together with insulin secretory granules by electron microscopy. Analysis of metabolic function indicated intact mechanisms for glucose uptake, oxidation/utilization, and phosphorylation by glucokinase. Glucose, alanine, and depolarizing concentrations of K(+) were all able to increase [Ca(2+)](i) in at least two of the cell lines tested. Insulin secretion was also modulated by other nutrients, hormones, and drugs acting as stimulators or inhibitors in normal beta cells. Subscapular implantation of the 1.1B4 cell line improved hyperglycemia and resulted in glucose lowering in streptozotocin-diabetic SCID mice. These novel human electrofusion-derived beta cell lines therefore exhibit stable characteristics reminiscent of normal pancreatic beta cells, thereby providing an unlimited source of human insulin-producing cells for basic biochemical studies and pharmacological drug testing plus proof of concept for cellular insulin replacement therapy.


Subject(s)
Cell Fusion/methods , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Adult , Animals , Calcium/metabolism , Cell Line , Cell Proliferation , Clone Cells/cytology , Clone Cells/drug effects , Clone Cells/metabolism , Diabetes Mellitus, Experimental/complications , Epithelial Cells/cytology , Female , Gene Expression Regulation/drug effects , Glucose/metabolism , Glucose/pharmacology , Glucose Transport Proteins, Facilitative/metabolism , Humans , Hyperglycemia/complications , Hyperglycemia/pathology , Hyperglycemia/therapy , Insulin Secretion , Insulin-Secreting Cells/drug effects , Intracellular Space/drug effects , Intracellular Space/metabolism , KATP Channels/metabolism , Mice , Oxidation-Reduction , Protein Transport/drug effects
7.
Diabetes Metab Res Rev ; 26(7): 525-33, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20718077

ABSTRACT

BACKGROUND: Pseudoislet studies have concentrated on single beta-cell lines or a combination of insulin and glucagon-secreting cells, overlooking the potential role of somatostatin in insulin release. This study sought to evaluate a heterotypic pseudoislet model containing insulin- (MIN6), glucagon- (αTC1.9) and somatostatin (TGP52)-secreting cells of mouse origin and to compare these pseudoislets with traditional monolayer preparations. METHODS: Cellular viability (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays), proliferation (5-bromo-2-deoxyuridine ELISA), hormone content and functional insulin release in response to a variety of stimuli were measured. Differential expression of E-cadherin, connexin 36 and connexin 43 was assessed by reverse transcriptase-polymerase chain reaction and Western blot to determine a possible role for adherens in insulin release from these pseudoislets. RESULTS: All pseudoislet cells displayed reduced proliferation coupled with an increase in cell death which may contribute to their static size in culture. While MIN6 and TGP52 cells expressed E-cadherin and showed sustained or improved hormone content when configured as pseudoislets, αTC1.9 lacked E-cadherin and contained less glucagon following pseudoislet formation. MIN6 and αTC1.9 cells expressed connexin 36, but not connexin 43 and TGP52 cells expressed connexin 43 only. In the presence of Alanine, Arginine and glucagon-like peptide-1, heterotypic pseudoislet cultures secreted levels of insulin that were comparable to that of MIN6 pseudoislets. In addition, pseudoislets comprising all three cell lines released more insulin into the surrounding culture medium than MIN6 pseudoislets when studied over a 1-week period. CONCLUSIONS: The current model may prove useful in studying the role of islet cell interactions in the release of insulin from pancreatic islets.


Subject(s)
Glucagon-Secreting Cells/metabolism , Glucagon/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Somatostatin-Secreting Cells/metabolism , Somatostatin/metabolism , Alanine/metabolism , Animals , Arginine/metabolism , Cadherins/analysis , Cell Communication , Cell Proliferation , Cell Survival , Cells, Cultured , Connexin 43/analysis , Connexins/analysis , Glucagon-Like Peptide 1/metabolism , Glucagon-Secreting Cells/cytology , Glucose/metabolism , Insulin Secretion , Insulin-Secreting Cells/cytology , Mice , Somatostatin-Secreting Cells/cytology , Gap Junction delta-2 Protein
8.
Pancreas ; 39(7): 1016-23, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20467348

ABSTRACT

OBJECTIVES: Cellular communication is required for normal patterns of insulin secretion from ß cells. Experiments using isolated islets of Langerhans are hampered by lack of supply and the consuming isolation process. Pseudoislets comprising clonal cells have emerged as an alternative to study islet-cell interactions and insulin secretion. The current study compared MIN6 pseudoislets and freshly isolated mouse islets. METHODS: Insulin content and release were measured by insulin radioimmunoassay. Reverse transcription polymerase chain reaction and Western blot analysis of adhesion molecule expression were performed on MIN6 monolayers and pseudoislets. MIN6 cellular proliferation and viability were measured by 5-bromo-2-deoxyuridine (BrdU) enzyme-linked immunosorbent assay, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and lactate dehydrogenase assays. RESULTS: Mouse islets were found to have greater insulin content than pseudoislets. However, insulin release was comparable between the 2 groups. With the use of MIN6 monolayers as a control, the expression of the adhesion molecule E-cadherin and connexin 36 were found to be enhanced in cells cultured as pseudoislets. Moreover, connexin 43 was shown to be absent from MIN6 cells irrespective of configuration. Finally, MIN6 pseudoislets seem able to manage their rate of proliferation with apoptosis resulting in a static size in the culture for extended periods. CONCLUSIONS: The current study found that MIN6 pseudoislets share many important functional and molecular features with islets of Langerhans.


Subject(s)
Cell Communication , Insulin/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Animals , Cadherins/analysis , Cell Proliferation , Cell Survival , Cells, Cultured , Insulin Secretion , Mice
9.
Pancreas ; 36(3): 294-301, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18362844

ABSTRACT

OBJECTIVES: Prevention of pancreatic beta-cell destruction combined with preservation of insulin secretory function is an important goal for cell-based diabetes therapy. This study describes the generation and characteristics of toxin-resistant beta-cells. METHODS: By using iterative exposures to ninhydrin, a new class of robust ninhydrin-tolerant insulin-secreting BRIN-BD11 ninhydrin-tolerant (BRINnt) cells was generated. Low- and high-passage BRINnt cells were used to evaluate beta-cell function and tolerance against toxins in comparison with native BRIN-BD11 cells. Differences in viability, gene expression, insulin secretory function, antioxidant enzyme activity, DNA damage, and DNA repair efficiency were compared. RESULTS: BRIN-BD11 ninhydrin-tolerant cells exhibited resistance toward ninhydrin and hydrogen peroxide but not streptozotocin (STZ). Both total superoxide dismutase (SOD) and catalase enzyme activities of BRINnt cells were significantly enhanced, and ninhydrin-induced DNA damage was decreased. BRIN-BD11 ninhydrin-tolerant cells also exhibited enhanced DNA repair efficiency. However, this was accompanied by loss of secretagogue-induced insulin release, decreased cellular insulin content, and deficits in insulin and glucose transporter 2 gene expression. Prolonged culture of BRINnt cells in the absence of ninhydrin reversed the degenerated function of BRINnt cells but restored ninhydrin susceptibility. CONCLUSIONS: These data illustrate dissociation between beta-cell toxin resistance and secretory function, indicating difficulties in generation of robust and well-functioning cells using this approach.


Subject(s)
Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/physiology , Insulin/metabolism , Ninhydrin/pharmacology , Animals , Antioxidants/metabolism , Catalase/metabolism , Clone Cells , DNA Damage , DNA Repair/drug effects , Drug Resistance , Gene Expression/drug effects , Glucose Transporter Type 2/genetics , Hydrogen Peroxide/toxicity , Insulin Secretion , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Rats , Streptozocin/toxicity , Superoxide Dismutase/metabolism
10.
Endocrine ; 32(1): 20-9, 2007 Aug.
Article in English | MEDLINE | ID: mdl-17992598

ABSTRACT

Since streptozotocin (STZ) exhibits beta-cell toxicity, mediated through diverse mechanisms, multiple toxin resistance can be expected in insulin-secretory cells rendered STZ-resistant. RINm5F, but not all cell lines surviving STZ treatment, possess higher insulin content than native parental cells and additional tolerance against alloxan. To understand the impact of STZ tolerant cell selection on toxin resistance and insulin-secretory function, STZ-resistant BRIN-BD11 cells were generated by iterative acute exposure to 20 mM STZ. These cells, denoted BRINst cells, exhibited resistance to toxic challenges from STZ, H(2)O(2), and ninhydrin. Insulin content and both glucose and arginine-stimulated insulin secretion were significantly enhanced in BRINst cells. The toxin-resistance of BRINst cells was gradually lost during continuous cultivation without STZ challenge. However, enhanced insulin secretory capacity at high passage in BRINst cells persisted. Although total SOD activity was decreased, catalase activity was increased and appeared to be important for the ninhydrin and STZ resistance of BRINst cells. This was associated with reductions of both STZ- and ninhydrin-induced DNA damage, although DNA repair was abolished. Further characterization of cells exhibiting multiple toxin tolerance and an enhanced insulin secretory function could provide useful lessons for understanding of beta-cell survival.


Subject(s)
Antioxidants/metabolism , DNA Repair/physiology , Drug Resistance, Multiple/physiology , Insulin-Secreting Cells/metabolism , Signal Transduction/physiology , Animals , Antibiotics, Antineoplastic/pharmacology , Cell Culture Techniques , DNA Damage , Glucose/metabolism , Hydrogen Peroxide/pharmacology , Insulin/biosynthesis , Insulin/metabolism , Insulin Secretion , Ninhydrin/pharmacology , Oxidants/pharmacology , Radiation-Sensitizing Agents/pharmacology , Rats , Streptozocin/pharmacology
11.
Diabetes Metab Res Rev ; 23(4): 324-34, 2007 May.
Article in English | MEDLINE | ID: mdl-17089371

ABSTRACT

BACKGROUND: Plasma homocysteine levels may be elevated in poorly controlled diabetes with pre-existing vascular complications and/or nephropathy. Since homocysteine has detrimental effects on a wide diversity of cell types, the present study examined the effects of long-term homocysteine exposure on the secretory function of clonal BRIN-BD11 beta-cells. METHODS: Acute insulin secretory function, cellular insulin content and viability of BRIN-BD11 cells were assessed following long-term (18 h) exposure to homocysteine in culture. RT-PCR and Western blot analysis were used to determine the expression of key beta-cell genes and proteins. Cells were cultured for a further 18 h without homocysteine to determine any long-lasting effects. RESULTS: Homocysteine (250-1000 micromol/L) exposure reduced insulin secretion at both moderate (5.6 mmol/L) and stimulatory (16.7 mmol/L) glucose by 48-63%. Similarly, insulin secretory responsiveness to stimulatory concentrations of alanine, arginine, 2-ketoisocaproate, tolbutamide, KCl, elevated Ca2+, forskolin and PMA, GLP-1, GIP and CCK-8 were reduced by 11-62% following culture with 100-250 micromol/L homocysteine. These inhibitory effects could not simply be attributed to changes in cellular insulin content, cell viability, H2O2 generation or any obvious alterations of gene/protein expression for insulin, glucokinase, GLUT2, VDCC, or Kir6.2 and SUR1. Additional culture for 18 h in standard culture media after homocysteine exposure restored secretory responsiveness to all agents tested. CONCLUSION: These findings suggest that long-term exposure to high homocysteine levels causes a reversible impairment of pancreatic beta-cell insulinotropic pathways. The in vivo actions of hyperhomocysteinaemia on islet cell function merit investigation.


Subject(s)
Homocysteine/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Animals , Cell Line , Dose-Response Relationship, Drug , Gene Expression/drug effects , Glucose/administration & dosage , Glucose/pharmacology , Homocysteine/administration & dosage , Insulin Secretion , Rats , Stimulation, Chemical , Time Factors
12.
Biol Chem ; 387(7): 941-7, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16913844

ABSTRACT

Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic beta-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic beta-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35-100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific beta-cell genes and releasing insulin.


Subject(s)
Cell Differentiation/drug effects , Embryo, Mammalian/drug effects , Gastric Inhibitory Polypeptide/pharmacology , Insulin/metabolism , Islets of Langerhans/metabolism , Stem Cells/drug effects , Animals , Base Sequence , Cell Line , DNA Primers , Embryo, Mammalian/cytology , Immunohistochemistry , Insulin Secretion , Islets of Langerhans/cytology , Mice , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/cytology
13.
Diabetes ; 54(8): 2436-46, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16046312

ABSTRACT

Glucose-dependent insulinotropic polypeptide (gastric inhibitory polypeptide [GIP]) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. In this study, we investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-related diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult ob/ob mice received once-daily intraperitoneal injections of saline vehicle or (Pro3)GIP over an 11-day period. Nonfasting plasma glucose levels and the overall glycemic excursion (area under the curve) to a glucose load were significantly reduced (1.6-fold; P < 0.05) in (Pro3)GIP-treated mice compared with controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P < 0.05) and insulin (1.5-fold; P < 0.05) responses to feeding. These changes were associated with significantly enhanced (1.6-fold; P < 0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP reduced pancreatic insulin content (1.3-fold; P < 0.05) and partially corrected the obesity-related islet hypertrophy and beta-cell hyperplasia of ob/ob mice. These comprehensive beneficial effects of (Pro3)GIP were reversed 9 days after cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Gastric Inhibitory Polypeptide/administration & dosage , Insulin Resistance , Islets of Langerhans/pathology , Obesity/complications , Receptors, Gastrointestinal Hormone/antagonists & inhibitors , Animals , Blood Glucose/analysis , Body Weight/drug effects , Diabetes Mellitus, Type 2/etiology , Eating/drug effects , Food , Glucose Intolerance/drug therapy , Glycated Hemoglobin/analysis , Hyperplasia , Insulin/analysis , Insulin/blood , Islets of Langerhans/chemistry , Kinetics , Mice , Mice, Obese , Receptors, Gastrointestinal Hormone/drug effects
14.
Biol Chem ; 385(9): 845-52, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15493880

ABSTRACT

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone secreted by endocrine K-cells in response to nutrient absorption. In this study we have utilized a specific and enzymatically stable GIP receptor antagonist, (Pro3)GIP, to evaluate the contribution of endogenous GIP to insulin secretion and glucose homeostasis in mice. Daily injection of (Pro3)GIP (25 nmol/kg body weight) for 11 days had no effect on food intake or body weight. Non-fasting plasma glucose concentrations were significantly raised (p<0.05) by day 11, while plasma insulin concentrations were not significantly different from saline treated controls. After 11 days, intraperitoneal glucose tolerance was significantly impaired in the (Pro3)GIP treated mice compared to control (p<0.01). Glucose-mediated insulin secretion was not significantly different between the two groups. Insulin sensitivity of 11-day (Pro3)GIP treated mice was slightly impaired 60 min post injection compared with controls. Following a 15 min refeeding period in 18 h fasted mice, food intake was not significantly different in (Pro3)GIP treated mice and controls. However, (Pro3)GIP treated mice displayed significantly elevated plasma glucose levels 30 and 60 min post feeding (p<0.05, in both cases). Postprandial insulin secretion was not significantly different and no changes in pancreatic insulin content or islet morphology were observed in (Pro3)GIP treated mice. The observed biological effects of (Pro3)GIP were reversed following cessation of treatment for 9 days. These data indicate that ablation of GIP signaling causes a readily reversible glucose intolerance without appreciable change of insulin secretion.


Subject(s)
Blood Glucose/drug effects , Gastric Inhibitory Polypeptide/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Receptors, Gastrointestinal Hormone/antagonists & inhibitors , Animals , Blood Glucose/metabolism , Homeostasis/drug effects , Homeostasis/physiology , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans/pathology , Mice , Receptors, Gastrointestinal Hormone/physiology
15.
Pharmacol Res ; 50(1): 41-6, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15082027

ABSTRACT

Clonal insulin-secreting BRIN-BD11 cells were used to examine effects of chronic 72-144 h exposure to the sulphonylureas tolbutamide and glibenclamide on insulin release, cellular insulin content, and mRNA levels of the Kir6.2 and SUR1 subunits of the beta-cell K(ATP) channel. Chronic exposure for 72-144 h to 5-100 microM tolbutamide and glibenclamide resulted in a time- and concentration-dependent irreversible decline in sulphonylurea-induced insulin secretion. In contrast, the decline in cellular insulin content induced by chronic exposure to high concentrations of sulphonylureas was readily reversible. Chronic exposure to tolbutamide or glibenclamide had no effect upon transcription of the Kir6.2 or SUR1 subunits of the pancreatic beta-cell K(ATP) channel. Whilst further studies are required to understand the precise nature of the chronic interactions of sulphonylurea with the insulin exocytotic mechanism, these observations may partially explain the well-known progressive failure of sulphonylurea therapy in type 2 diabetes.


Subject(s)
Glyburide/pharmacology , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Pancreas/metabolism , Potassium Channels, Inwardly Rectifying/biosynthesis , Potassium Channels, Inwardly Rectifying/genetics , Tolbutamide/pharmacology , ATP-Binding Cassette Transporters , Animals , Cells, Cultured , Clone Cells , Gene Expression Regulation/drug effects , Insulin/analysis , Insulin Secretion , Multidrug Resistance-Associated Proteins , Pancreas/drug effects , Potassium Channels, Inwardly Rectifying/drug effects , RNA, Messenger/biosynthesis , Rats , Receptors, Drug , Reverse Transcriptase Polymerase Chain Reaction , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors
16.
Endocr Res ; 30(1): 61-8, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15098920

ABSTRACT

The B vitamin nicotinamide (NIC), commonly known as niacin, is currently in trial as a potential means of preventing Type 1 diabetes in first-degree relatives of affected individuals. Sodium butyrate (BUT) a common dietary micronutrient has also been reported to have beneficial effects on the differentiation and function of pancreatic beta cells. Cultured rat insulin-secreting BRIN-BD11 cells were used to investigate the effects of 3 days exposure to NIC (10 mM) and BUT (1 mM) both alone and in combination on beta cell function. Culture with NIC and/or BUT resulted in reduction of growth, insulin content and basal insulin secretion. BUT additionally decreased cell viability whilst NIC had no significant effect. Treatment with either agent abolished beta cell glucose sensitivity but insulin secretory responsiveness to a wide range of beta cell stimulators, including a depolarizing concentration of K+, elevation of Ca2+ and activation of adenylate cyclase and protein kinase C, were enhanced. These data illustrate that long term exposure to NIC and BUT has both positive and negative effects on the function of insulin-secreting cells.


Subject(s)
Butyrates/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Niacinamide/pharmacology , Animals , Cell Division/drug effects , Cell Survival/drug effects , Clone Cells , Diabetes Mellitus, Type 1/prevention & control , Glucose/metabolism , Insulin Secretion , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Rats , Time Factors
17.
Pancreas ; 28(4): 364-8, 2004 May.
Article in English | MEDLINE | ID: mdl-15097851

ABSTRACT

The ultratrace elements vanadate, tungstate, and molybdate exhibit significant antihyperglycemic effects in both type 1 and 2 diabetic animals, but possible effects on the function of pancreatic beta cells are understudied. In the present study, clonal BRIN BD11 cells were cultured for 3 days with each ultratrace element to establish doses lacking detrimental effects on viable beta cell mass. Vanadate treatment (4 micromol/L) had no effect on cellular insulin content but improved glucose-induced insulin secretory responsiveness. However, insulin secretion mediated by PKA and PKC activation was desensitized in vanadate-treated cells. Culture with tungstate (300 micromol/L) and molybdate (1 mmol/L) increased cellular insulin content and enhanced basal insulin release and the responsiveness to glucose and a wide range of other secretagogues. These observations suggest significant effects of ultratrace elements on pancreatic beta cells that may contribute to their antihyperglycemic action.


Subject(s)
Hypoglycemic Agents/pharmacology , Insulin/metabolism , Islets of Langerhans/metabolism , Trace Elements/pharmacology , Animals , Cell Line , Cell Survival/drug effects , Dose-Response Relationship, Drug , Exocytosis , Insulin Secretion , Islets of Langerhans/drug effects , Kinetics , Molybdenum/pharmacology , Rats , Tungsten Compounds/pharmacology , Vanadates/pharmacology
18.
Cell Biol Int ; 28(1): 69-73, 2004.
Article in English | MEDLINE | ID: mdl-14759771

ABSTRACT

Glucagon-like peptide-1(7-36)amide (GLP-1) is a key insulinotropic hormone with the reported potential to differentiate non-insulin secreting cells into insulin-secreting cells. The short biological half-life of GLP-1 after cleavage by dipeptidylpeptidase IV (DPP IV) to GLP-1(9-36)amide is a major therapeutic drawback. Several GLP-1 analogues have been developed with improved stability and insulinotropic action. In this study, the N-terminally modified GLP-1 analogue, N-acetyl-GLP-1, was shown to be completely resistant to DPP IV, unlike native GLP-1, which was rapidly degraded. Furthermore, culture of pancreatic ductal ARIP cells for 72 h with N-acetyl-GLP-1 indicated a greater ability to induce pancreatic beta-cell-associated gene expression, including insulin and glucokinase. Further investigation of the effects of stable GLP-1 analogues on beta-cell differentiation is required to assess their potential in diabetic therapy.


Subject(s)
Glucagon/analogs & derivatives , Glucagon/pharmacology , Islets of Langerhans/metabolism , Peptide Fragments/pharmacology , Animals , Base Sequence , Cell Differentiation , Cell Line, Tumor , Gene Expression/drug effects , Glucagon/chemistry , Glucagon/genetics , Glucagon-Like Peptide 1 , Glucagon-Like Peptides , Islets of Langerhans/cytology , Molecular Sequence Data , Pancreatic Ducts/cytology , Peptide Fragments/genetics , Peptide Fragments/metabolism , Rats
19.
Diabetes ; 52(2): 492-8, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12540626

ABSTRACT

The presence and biological significance of circulating glycated insulin has been evaluated by high-pressure liquid chromatography (HPLC), electrospray ionization mass spectrometry (ESI-MS), radioimmunoassay (RIA), receptor binding, and hyperinsulinemic-euglycemic clamp techniques. ESI-MS analysis of an HPLC-purified plasma pool from four male type 2 diabetic subjects (HbA(1c) 8.1 +/- 0.2%, plasma glucose 8.7 +/- 1.3 mmol/l [means +/- SE]) revealed two major insulin-like peaks with retention times of 14-16 min. After spectral averaging, the peak with retention time of 14.32 min exhibited a prominent triply charged (M+3H)(3+) species at 1,991.1 m/z, representing monoglycated insulin with an intact M(r) of 5,970.3 Da. The second peak (retention time 15.70 min) corresponded to native insulin (M(r) 5,807.6 Da), with the difference between the two peptides (162.7 Da) representing a single glucitol adduct (theoretical 164 Da). Measurement of glycated insulin in plasma of type 2 diabetic subjects by specific RIA gave circulating levels of 10.1 +/- 2.3 pmol/l, corresponding to approximately 9% total insulin. Biological activity of pure synthetic monoglycated insulin (insulin B-chain Phe(1)-glucitol adduct) was evaluated in seven overnight-fasted healthy nonobese male volunteers using two-step euglycemic-hyperinsulinemic clamps (2 h at 16.6 micro g x kg(-1) x min(-1), followed by 2 h at 83.0 micro g x kg(-1) x min(-1); corresponding to 0.4 and 2.0 mU x kg(-1) x min(-1)). At the lower dose, the exogenous glucose infusion rates required to maintain euglycemia during steady state were significantly lower with glycated insulin (P < 0.01) and approximately 70% more glycated insulin was required to induce a similar rate of insulin-mediated glucose uptake. Maximal responses at the higher rates of infusion were similar for glycated and control insulin. Inhibitory effects on endogenous glucose production, insulin secretion, and lipolysis, as indicated by measurements of C-peptide, nonesterified free fatty acids, and glycerol, were also similar. Receptor binding to CHO-T cells transfected with human insulin receptor and in vivo metabolic clearance revealed no differences between glycated and native insulin, suggesting that impaired biological activity is due to a postreceptor effect. The present demonstration of glycated insulin in human plasma and related impairment of physiological insulin-mediated glucose uptake suggests a role for glycated insulin in glucose toxicity and impaired insulin action in type 2 diabetes.


Subject(s)
Blood Glucose/metabolism , Diabetes Mellitus, Type 2/blood , Insulin/analogs & derivatives , Insulin/blood , Animals , Binding, Competitive , CHO Cells , Chromatography, High Pressure Liquid , Cricetinae , Glucose Clamp Technique , Glycated Hemoglobin/analysis , Glycosylation , Humans , Hyperinsulinism/blood , Insulin/administration & dosage , Insulin/isolation & purification , Insulin/metabolism , Insulin/pharmacology , Male , Spectrometry, Mass, Electrospray Ionization
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