Taurine regulates insulin release from pancreatic beta cell lines

Background

Pancreatic β-cells release insulin via an electrogenic response triggered by an increase in plasma glucose concentrations. The critical plasma glucose concentration has been determined to be ~3 mM, at which time both insulin and GABA are released from pancreatic β-cells. Taurine, a β-sulfonic acid, may be transported into cells to balance osmotic pressure. The taurine transporter (TauT) has been described in pancreatic tissue, but the function of taurine in insulin release has not been established. Uptake of taurine by pancreatic β-cells may alter membrane potential and have an effect on ion currents. If taurine uptake does alter β-cell current, it might have an effect on exocytosis of cytoplasmic vesicle. We wished to test the effect of taurine on regulating release of insulin from the pancreatic β-cell.

Methods

Pancreatic β-cell lines Hit-TI5 (Syrian hamster) and Rin-m (rat insulinoma) were used in these studies. Cells were grown to an 80% confluence on uncoated cover glass in RPMI media containing 10% fetal horse serum. The cells were then adapted to a serum-free, glucose free environment for 24 hours. At that time, the cells were treated with either 1 mM glucose, 1 mM taurine, 1 mM glucose + 1 mM taurine, 3 mM glucose, or 3 mM glucose + 1 mM taurine. The cells were examined by confocal microscopy for cytoplasmic levels of insulin.

Results

In both cell lines, 1 mM glucose had no effect on insulin levels and served as a control. Cells starved of glucose had a significant reduction (p<0.001) in the level of insulin, but this level was significantly higher than all other treatments. As expected, the 3 mM glucose treatment resulted in a statistically lower (p<0.001) insulin level than control cells. Interestingly, 1 mM taurine also resulted in a statistically lower level of insulin (p<0.001) compared to controls when either no glucose or 1 mM glucose was present. Cells treated with 1 mM taurine plus 3 mM glucose showed a level of insulin similar to that of 3 mM glucose alone.

Conclusions

Taurine administration can alter the electrogenic response in β-cell lines, leading to a change in calcium homeostasis and a subsequent decrease in intracellular insulin levels. The consequence of these actions could represent a method of increasing plasma insulin levels leading to a decrease in plasma glucose levels.

     

The role of TRPM2 in pancreatic β-cells and the development of diabetes

TRPM2 is a Ca²⁺-permeable non-selective cation channel that can be activated by adenosine dinucleotides, hydrogen peroxide, or intracellular Ca²⁺. The protein is expressed in a wide variety of cells, including neurons in the brain, immune cells, endocrine cells, and endothelial cells. This channel is also well expressed in β-cells in the pancreas. Insulin secretion from pancreatic β-cells is the primary mechanism by which the concentration of blood glucose is reduced. Thus, impairment of insulin secretion leads to hyperglycemia and eventually causes diabetes. Glucose is the principal stimulator of insulin secretion. The primary pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive K⁺ (K_ATP) channel to voltage-gated Ca²⁺ channel (VGCC)-mediated pathway. Increases in the intracellular Ca²⁺ concentration are necessary for insulin secretion, but VGCC is not sufficient to explain [Ca²⁺]_i increases in pancreatic β-cells and the resultant secretion of insulin. In this review, we focus on TRPM2 as a candidate for a [Ca²⁺]_i modulator in pancreatic β-cells and its involvement in insulin secretion and development of diabetes. Although further analyses are needed to clarify the mechanism underlying TRPM2-mediated insulin secretion, TRPM2 could be a key player in the regulation of insulin secretion and could represent a new target for diabetes therapy.     

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.     

The role of autophagy in pancreatic β-cell and diabetes

Pancreatic β-cells play a key role in glucose homeostasis in mammals. Although large-scale protein synthesis and degradation occur in pancreatic β-cells, the mechanism underlying dynamic protein turnover in β-cells remains largely unknown. We found low-level constitutive autophagy in β-cells of C57BL/6 mice fed a standard diet; however, autophagy was markedly upregulated in mice fed a high-fat diet. β-cells of diabetic db/db mice contained large numbers of autophagosomes, compared with non-diabetic db/misty controls. The functional importance of autophagy was analyzed using β-cell-specific Atg7 knockout mice. Autophagy-deficient mice showed degeneration of β-cells and impaired glucose tolerance with reduced insulin secretion. While a high-fat diet stimulated β-cell autophagy in control mice, it induced a profound deterioration of glucose intolerance in β-cell autophagy-deficient mutants, partly because of the lack of a compensatory increase in β-cell mass. These results suggest that the degradation of unnecessary cellular components by autophagy is essential for maintenance of the architecture and function of β-cells. Autophagy also serves as a crucial element of stress responses to protect β-cells under insulin resistant states. Impairment of autophagic machinery could thus predispose individuals to type 2 diabetes.     

Suppression of NADPH oxidase 2 substantially restores glucose-induced dysfunction of pancreatic NIT-1 cells

Defects in insulin secretion by pancreatic cells and/or decreased sensitivity of target tissues to insulin action are the key features of type 2 diabetes. It has been shown that excessive generation of reactive oxygen species (ROS) is linked to glucose-induced β-cell dysfunction. However, cellular mechanisms involved in ROS generation in β-cells and the link between ROS and glucose-induced β-cell dysfunction are poorly understood. Here, we demonstrate a key role of NADPH oxidase 2 (NOX2)-derived ROS in the deterioration of β-cell function induced by a high concentration of glucose. Sprague-Dawley rats were fed a high-fat diet for 24 weeks to induce diabetes. Diabetic rats showed increased glucose levels and elevated ROS generation in blood, but decreased insulin content in pancreatic β-cells. In vitro, increased ROS levels in pancreatic NIT-1 cells exposed to high concentrations of glucose (33.3 mmol·L(-1)) were associated with elevated expression of NOX2. Importantly, decreased glucose-induced insulin expression and secretion in NIT-1 cells could be rescued via siRNA-mediated NOX2 reduction. Furthermore, high glucose concentrations led to apoptosis of β-cells by activation of p38MAPK and p53, and dysfunction of β-cells through phosphatase and tensih homolog (PTEN)-dependent Jun N-terminal kinase (JNK) activation and protein kinase B (AKT/PKB) inhibition, which induced the translocation of forkhead box O1 and pancreatic duodenal homeobox-1, followed by reduced insulin expression and secretion. In conclusion, NOX2-derived ROS could play a critical role in high glucose-induced β-cell dysfunction through PTEN-dependent JNK activation and AKT inhibition.     

The role of protein kinase CK2 in the regulation of the insulin production of pancreatic islets

An appropriate regulation of the insulin production and secretion in pancreatic β-cells is necessary for the control of blood glucose homeostasis. The pancreatic duodenal homeobox factor-1 (Pdx-1) is among the various factors and signals which are implicated in the regulation of the insulin synthesis and secretion in the pancreatic β-cells. Recently, we identified Pdx-1 as a substrate for protein kinase CK2. Since CK2 is implicated in the regulation of many different cellular signaling pathways we now asked whether it might also be involved in the regulation of the insulin regulation in β-cells. Here, we show that insulin treatment of β-cells resulted in an elevated CK2 kinase activity. On the other hand down-regulation of CK2 activity by quinalizarin led to an elevated level of insulin. These results demonstrate that CK2 is implicated in the insulin regulation on pancreatic β-cells.     

Islet-derived multipotential cells/progenitor cells

The pancreatic β-cell has a pivotal role in the regulation of glucose homeostasis; its death leads to type I diabetes. Neogenesis of β-cells, the differentiation of β-cells from non-β-cells, could be an important mechanism of islet cell repopulation. To examine the ability of the adult pancreas to generate new β-cells, we characterized the phenotype of β precursor cells in embryos and then determined that cells expressing embryonic traits appeared in islets of adult mouse pancreas following deletion of preexisting insulin cells by streptozotocin, a specific β-cell toxin. These precursor cells generated new β-cells (NBCs) that repopulated the islets. The number of NBCs increased dramatically after restoration of normoglycemia by insulin therapy. Future studies will seek to identify the source of the NBCs and to examine the mechanisms that lead to their differentiation.     

GABA coordinates with insulin in regulating secretory function in pancreatic INS-1 β-cells

Pancreatic islet β-cells produce large amounts of γ-aminobutyric acid (GABA), which is co-released with insulin. GABA inhibits glucagon secretion by hyperpolarizing α-cells via type-A GABA receptors (GABA(A)Rs). We and others recently reported that islet β-cells also express GABA(A)Rs and that activation of GABA(A)Rs increases insulin release. Here we investigate the effects of insulin on the GABA-GABA(A)R system in the pancreatic INS-1 cells using perforated-patch recording. The results showed that GABA produces a rapid inward current and depolarizes INS-1 cells. However, pre-treatment of the cell with regular insulin (1 μM) suppressed the GABA-induced current (I(GABA)) by 43%. Zinc-free insulin also suppressed I(GABA) to the same extent of inhibition by regular insulin. The inhibition of I(GABA) occurs within 30 seconds after application of insulin. The insulin-induced inhibition of I(GABA) persisted in the presence of PI3-kinase inhibitor, but was abolished upon inhibition of ERK, indicating that insulin suppresses GABA(A)Rs through a mechanism that involves ERK activation. Radioimmunoassay revealed that the secretion of C-peptide was enhanced by GABA, which was blocked by pre-incubating the cells with picrotoxin (50 μM, p<0.01) and insulin (1 μM, p<0.01), respectively. Together, these data suggest that autocrine GABA, via activation of GABA(A)Rs, depolarizes the pancreatic β-cells and enhances insulin secretion. On the other hand, insulin down-regulates GABA-GABA(A)R signaling presenting a feedback mechanism for fine-tuning β-cell secretion.     

Immunohistochemical localization of facilitated-diffusion glucose transporters in rat pancreatic islets

The subcellular localization of five isoforms of facillitated-diffusion glucose transporters (GLUTs), from GLUT1 to GLUT5, in rat pancreatic islets was studied by immunohistochemistry using rabbit polyclonal antisera against mouse or rat GLUT peptides. Animals were perfusion-fixed with phosphate-buffered 4% paraformaldehyde and the pancreases were removed. Some specimens were embedded in paraffin, serially sectioned, and immunostained for glucagon, insulin, somatostatin, and the GLUTs for light microscopic observation. Others were prepared for immunoelectron microscopy by the post-embedding method. By these methods, GLUT2 immunostaining was observed on the lateral membranes of pancreatic β-cells, whereas GLUT3 immunoreaction was predominatly localized in the cytoplasm to β-cells and was not found in α-cells. In contrast, GLUT5 immunostaining was preferentially localized in the cytoplasm of α-cells compared to that of β-cells. However, GLUT1 and GLUT4 were either barely or not at all detectable in any cells. These results suggest that rat islets take up glucose by at least three different processes and that blood glucose levels could be modulated differentially by: a high Km glucose transporter, GLUT2, in β-cells; by a low Km glucose transporter, GLUT3, in β-cells; and by a low Km glucose transporter, GLUT5, in α-cells.     

Obestatin、Ghrelin与血糖调节

肥胖抑制素（obestatin）和生长激素释放肽（ghrelin）能互相拮抗,参与血糖的调节.其中obestatin与GPR-39（G-protein-coupled receptor 39）结合抑制摄食和胃肠排空、促进胰岛β细胞功能,影响胰岛素的分泌;而ghrelin与生长激素促分泌受体（GHSR1a）结合,促进食欲和胃肠排空,减少脂肪的利用,抑制胰岛细胞凋亡,调节胰岛素的分泌.但两者参与血糖调节的具体机制尚存在争议.     

Novel aspects on pancreatic beta-cell signal-transduction

Pancreatic β-cells release insulin in appropriate amounts in order to keep blood glucose levels within physiological limits. Failure to do so leads to the most common metabolic disorder in man, diabetes mellitus. The glucose-stimulus/insulin-secretion coupling represents a sophisticated interplay between glucose and a variety of modulatory factors. These factors are provided by the blood supply (such as nutrients, vitamins, incretins etc.), the nerval innervations, cell-cell contacts as well as by paracrine and autocrine feedback loops within the pancreatic islet of Langerhans. However, the underlying mechanisms of their action remain poorly understood.In the present mini-review we discuss novel aspects of selective insulin signaling in the β-cell and novel insights into the role of higher inositol phosphates in insulin secretion. Finally we present a newly developed experimental platform that allows non-invasive and longitudinal in vivo imaging of pancreatic islet/β-cell biology at single-cell resolution.     

Imaging glucose-regulated insulin secretion and gene expression in single islet β-cells

The mechanisms by which changes in glucose concentration regulate gene expression and insulin secretion in pancreatic islet β-cells are only partly understood. Here we describe the development of new technologies for examining these processes at the level of single living β-cells. We also present recent findings, made using these and other techniques, which implicate a role for adenosine 5′-monophosphate-activated protein kinase in glucose signaling in these cells.     

Distinct requirements for beta-catenin in pancreatic epithelial growth and patterning

Pancreatic exocrine and endocrine lineages arise from multipotent pancreatic progenitor cells (MPCs). Exploiting the mechanisms that govern expansion and differentiation of these cells could enhance efforts to generate β-cells from stem cells. Although our prior work indicates that the canonical Wnt signaling component β-catenin is required qualitatively for exocrine acinar but not endocrine development, precisely how this requirement plays out at the level of MPCs and their lineage-restricted progeny is unknown. In addition, the contribution of β-catenin function to β-cell development remains controversial. To resolve the potential roles of β-catenin in development of MPCs and β-cells, we generated pancreas- and pre-endocrine-specific β-catenin knockout mice. Pancreas-specific loss of β-catenin produced not only a dramatic reduction in acinar cell numbers, but also a significant reduction in β-cell mass. The loss of β-cells is due not to a defect in the differentiation of endocrine precursors, but instead correlates with an early and specific loss of MPCs. In turn, this reflects a novel role for β-catenin in maintaining proximal–distal patterning of the early epithelium, such that distal MPCs resort to a proximal, endocrine-competent “trunk” fate when β-catenin is deleted. Moreover, β-catenin maintains proximal–distal patterning, in part, by inhibiting Notch signaling. Subsequently, β-catenin is required for proliferation of both distal and proximal cells, driving overall organ growth. In distinguishing two distinct roles for β-catenin along the route of β-cell development, we suggest that temporally appropriate positive and negative manipulation of this molecule could enhance expansion and differentiation of stem cell-derived MPCs.     

Chronic palmitic acid-induced lipotoxicity correlates with defective trafficking of ATP sensitive potassium channels in pancreatic β cells

Lipotoxicity is associated with a high level of fatty acid accumulation in pancreatic β-cells. An overload of free fatty acids contributes to pancreatic β-cell apoptosis and dysfunction. Insulin secretion involves sequential ionic events upon glucose stimulation. ATP sensitive potassium (K_ATP) channels serve as glucose sensors and effectively initiate glucose-stimulated insulin secretion. This study investigated the effects of lipotoxicity on the trafficking of K_ATP channels in pancreatic β cells using chronic palmitic acid –injected mice and treated insulinoma cells. The chronic palmitic acid -injected mice displayed type II diabetic characteristics. The pancreatic sections of these mice exhibited a decrease in the expression of K_ATP channels. We then tested the time and dose effects of palmitic acid on the cell viability of INS-1 cells. We observed a significant decrease in the surface expression of K_ATP channels after 72 h of treatment with 0.4 mM palmitic acid. In addition, this treatment induced pancreatic β-cell apoptosis by increasing cleaved caspase 3 protein level. Our results demonstrated cotreatment with glibenclamide, the sulfonylurea compounds for type II diabetes mellitus, in palmitic acid -treated cells reduces cell death and recovers the glucose stimulated insulin secretion through increasing the surface expression of K_ATP channels. Importantly, glibenclamide also improved glucose tolerance, triglyceride concentration, and insulin sensitivity in the palmitic acid-injected mice. In conclusion, an increase in the surface expression of K_ATP channels restores insulin secretion, reduces pancreatic β-cell’s apoptosis, highlighting correct trafficking of K_ATP channels is important in survival of β-cells during lipotoxicity.     

Insulin contributes to fine-tuning of the pancreatic beta-cell response to glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion from pancreatic β-cells in a glucose-dependent manner. However, factors other than glucose that regulate the β-cell response to GLP-1 remain poorly understood. In this study, we examined the possible involvement of insulin and receptor tyrosine kinase signaling in regulation of the GLP-1 responsiveness of β-cells. Pretreatment of β-cells with HNMPA, an insulin receptor inhibitor, and AG1478, an epidermal growth factor receptor inhibitor, further increased the cAMP level and Erk phosphorylation in the presence of exendin-4 (exe-4), a GLP-1 agonist. When β-cells were exposed to a high concentration of glucose (25 mM), which stimulates insulin secretion, exe-4-induced cAMP formation declined gradually as exposure time was increased. This decreased cAMP formation was not observed in the presence of HNMPA. HNMPA was able to further increase the exe-4-induced insulin secretion when β-cells were exposed to high glucose for 18 h. Treatment of β-cells with insulin significantly decreased exe-4-induced cAMP formation in a dose-dependent manner. Lowering the phospho-Akt level by HNMPA or LY294002, a PI3K inhibitor, further augmented exe-4-induced cAMP formation and Erk phosphorylation. These results suggest that insulin contributes to fine-tuning of the β-cell response to GLP-1.     

Cellular hypoxia of pancreatic beta-cells due to high levels of oxygen consumption for insulin secretion in vitro

Cellular oxygen consumption is a determinant of intracellular oxygen levels. Because of the high demand of mitochondrial respiration during insulin secretion, pancreatic β-cells consume large amounts of oxygen in a short time period. We examined the effect of insulin secretion on cellular oxygen tension in vitro. We confirmed that Western blotting of pimonidazole adduct was more sensitive than immunostaining for detection of cellular hypoxia in vitro and in vivo. The islets of the diabetic mice but not those of normal mice were hypoxic, especially when a high dose of glucose was loaded. In MIN6 cells, a pancreatic β-cell line, pimonidazole adduct formation and stabilization of hypoxia-inducible factor-1α (HIF-1α) were detected under mildly hypoxic conditions. Inhibition of respiration rescued the cells from becoming hypoxic. Glucose stimulation decreased cellular oxygen levels in parallel with increased insulin secretion and mitochondrial respiration. The cellular hypoxia by glucose stimulation was also observed in the isolated islets from mice. The MIN6 cells overexpressing HIF-1α were resistant to becoming hypoxic after glucose stimulation. Thus, glucose-stimulated β-cells can become hypoxic by oxygen consumption, especially when the oxygen supply is impaired.