Masahiro Nagasawa

Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was monitored by measuring translocation of MARCKS-GFP. Insulin was measured by radioimmunoassay. mRNA for T1R2, T1R3, and gustducin was expressed in MIN6 cells. In these cells, artificial sweeteners such as sucralose, succharin, and acesulfame-K increased insulin secretion and augmented secretion induced by glucose. Sucralose increased biphasic increase in [Ca2+]c. The second sustained phase was blocked by removal of extracellular calcium and addition of nifedipine. An inhibitor of inositol(1, 4, 5)-trisphophate receptor, 2-aminoethoxydiphenyl borate, blocked both phases of [Ca2+]c response. The effect of sucralose on [Ca2+]c was inhibited by gurmarin, an inhibitor of the sweet taste receptor, but not affected by a Gq inhibitor. Sucralose also induced sustained elevation of [cAMP]c, which was only partially inhibited by removal of extracellular calcium and nifedipine. Finally, mouse islets expressed T1R2 and T1R3, and artificial sweeteners stimulated insulin secretion. Conclusions Sweet taste receptor is expressed in β-cells, and activation of this receptor induces insulin secretion by Ca2+ and cAMP-dependent mechanisms.

Regulation of Calcium-Permeable TRPV2 Channel by Insulin in Pancreatic β-Cells

OBJECTIVE—Calcium-permeable cation channel TRPV2 is expressed in pancreatic β-cells. We investigated regulation and function of TRPV2 in β-cells. RESEARCH DESIGN AND METHODS—Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse β-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence. RESULTS—In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a β-cell line derived from islets obtained from a β-cell–specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse β-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose. CONCLUSIONS—TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on β-cells.

Chemotactic peptide fMetLeuPhe induces translocation of the TRPV2 channel in macrophages

The present study was conducted to characterize the regulation and function of TRPV2 in macrophages. Among six members of the TRPV family channels, only the expression of TRPV2 was detected in macrophages. We then determined localization of TRPV2 using TtT/M87 macrophages transfected with TRPV2‐EGFP. In serum‐free condition, most of the TRPV2 signal was located in the cytoplasm and colocalized with the endoplasmic reticulum marker. Treatment with serum induced translocation of some of the TRPV2‐EGFP to the plasma membrane. Serum‐induced translocation was blocked by transfection of short‐form TRPV2 (s‐TRPV2) lacking a pore‐forming region and the sixth transmembrane domain. Addition of a chemotactic peptide formyl Met‐Leu‐Phe (fMLP) also induced translocation of TRPV2‐EGFP to the plasma membrane. The fMLP‐induced translocation was blocked by an inhibitor of PI 3‐kinase, LY294002, and pertussis toxin. Whole‐cell patch clamp analysis showed a Cs+ current in the TtT/M87 cell, which was blocked by an addition of ruthenium red and transfection of either s‐TRPV2 or siRNA for TRPV2. fMLP increased the Cs+ current. fMLP induced a rapid and sustained elevation of cytoplasmic Ca2+ ([Ca2+]C), the sustained phase of which was abolished by removal of extracellular calcium. The sustained elevation of [Ca2+]C was also blocked by ruthenium red, and transfection of either s‐TRPV2 or siRNA. Finally, fMLP‐induced migration of macrophage was blocked by ruthenium red or transfection of s‐TRPV2. These results suggest that fMLP induces translocation of TRPV2 from intracellular compartment to the plasma membrane, and this translocation is critical for fMLP‐induced calcium entry. J. Cell. Physiol. 210: 692–702, 2007.

Bimodal role of conventional protein kinase C in insulin secretion from rat pancreatic β cells

The present study was conducted to evaluate the role of conventional protein kinase C (PKC) in calcium‐evoked insulin secretion. In rat β cells transfected with green fluorescent protein‐tagged PKC‐α (PKC‐α–EGFP), a depolarizing concentration of potassium induced transient elevation of cytoplasmic free calcium ([Ca2+]c), which was accompanied by transient translocation of PKC‐α–EGFP from the cytosol to the plasma membrane. Potassium also induced transient translocation of PKC‐θ–EGFP, the C1 domain of PKC‐γ and PKC‐ɛ–GFP. A high concentration of glucose induced repetitive elevation of [Ca2+]c and repetitive translocation of PKC‐α–EGFP. Diazoxide completely blocked both elevation of [Ca2+]c and translocation of PKC‐α–EGFP. We then studied the role of conventional PKC in calcium‐evoked insulin secretion using rat islets. When islets were incubated for 10 min with high potassium, Gö‐6976, an inhibitor of conventional PKC, and PKC‐α pseudosubstrate fused to antennapedia peptide (Antp‐PKC19–31) increased potassium induced secretion. Similarly, insulin release induced by high glucose for 10 min was enhanced by Gö‐6976 and Antp‐PKC19–31. However, when islets were stimulated for 60 min with high glucose, both Gö‐6976 and Antp‐PKC19–31 reduced glucose‐induced insulin secretion. Similar results were obtained by transfection of dominant‐negative PKC‐α using adenovirus vector. Taken together, PKC‐α is activated when cells are depolarized by a high concentration of potassium or glucose. Conventional PKC is inhibitory on depolarization‐induced insulin secretion per se, but it also augments glucose‐induced secretion.

Identification of a Novel Chloride Channel Expressed in the Endoplasmic Reticulum, Golgi Apparatus, and Nucleus

MID-1 is a Saccharomyces cerevisiae gene encoding a stretch-activated channel. UsingMID-1 as a molecular probe, we isolated rat cDNA encoding a protein with four putative transmembrane domains. This gene encoded a protein of 541 amino acids. We also cloned the human homologue, which encoded 551 amino acids. Messenger RNA for this gene was expressed abundantly in the testis and moderately in the spleen, liver, kidney, heart, brain, and lung. In the testis, immunoreactivity of the gene product was detected both in the cytoplasm and the nucleus. When expressed in Chinese hamster ovary cells, the gene product was located in intracellular compartments including endoplasmic reticulum and the Golgi apparatus. When microsome fraction obtained from the transfected cells, but not from mock-transfected cells, was incorporated into the lipid bilayer, an anion channel activity was detected. Unitary conductance was 70 picosiemens in symmetric 150 mm KCl solution. We designated this gene Mid-1-related chloride channel (MCLC). MCLC encodes a new class of chloride channel expressed in intracellular compartments.

Return of the glucoreceptor: Glucose activates the glucose-sensing receptor T1R3 and facilitates metabolism in pancreatic β-cells

Subunits of the sweet taste receptor, namely T1R2 and T1R3, are expressed in mouse pancreatic islets. Quantitatively, the expression of messenger ribonucleic acid for T1R2 is much lower than that of T1R3, and immunoreactive T1R2 is in fact undetectable. Presumably, a homodimer of T1R3 could function as a signaling receptor. Activation of this receptor by adding an artificial sweetener, sucralose, leads to an increase in intracellular adenosine triphosphate ([ATP]c). This increase in [ATP]c is observed in the absence of ambient glucose. Sucralose also augments elevation of [ATP]c induced by methylsuccinate, a substrate for mitochondria. Consequently, activation of T1R3 promotes metabolism in mitochondria and increases [ATP]c. 3‐O‐Methylglucose, a non‐metabolizable analog of glucose, also increases [ATP]c. Conversely, knockdown of T1R3 attenuates elevation of [ATP]c induced by glucose. Hence, glucose promotes its own metabolism by activating T1R3 and augmenting ATP production. Collectively, a homodimer of T1R3 functions as a cell surface glucose‐sensing receptor and participates in the action of glucose on insulin secretion. The glucose‐sensing receptor T1R3 might be the putative glucoreceptor proposed decades ago by Niki et al. The glucose‐sensing receptor is involved in the action of glucose and modulates glucose metabolism in pancreatic β‐cells.

Diverse signaling systems activated by the sweet taste receptor in human GLP-1-secreting cells

Sweet taste receptor regulates GLP-1 secretion in enteroendocrine L-cells. We investigated the signaling system activated by this receptor using Hutu-80 cells. We stimulated them with sucralose, saccharin, acesulfame K and glycyrrhizin. These sweeteners stimulated GLP-1 secretion, which was attenuated by lactisole. All these sweeteners elevated cytoplasmic cyclic AMP ([cAMP]c) whereas only sucralose and saccharin induced a monophasic increase in cytoplasmic Ca(2+) ([Ca(2+)]c). Removal of extracellular calcium or sodium and addition of a Gq/11 inhibitor greatly reduced the [Ca(2+)]c responses to two sweeteners. In contrast, acesulfame K induced rapid and sustained reduction of [Ca(2+)]c. In addition, glycyrrhizin first reduced [Ca(2+)]c which was followed by an elevation of [Ca(2+)]c. Reductions of [Ca(2+)]c induced by acesulfame K and glycyrrhizin were attenuated by a calmodulin inhibitor or by knockdown of the plasma membrane calcium pump. These results indicate that various sweet molecules act as biased agonists and evoke strikingly different patterns of intracellular signals.

Translocation of calcium-permeable TRPV2 channel to the podosome: Its role in the regulation of podosome assembly

The present study was conducted to investigate localization and function of TRPV2 channel in a mouse macrophage cell line, TtT/M87. We infected an adenovirus vector encoding TRPV2 tagged with c-Myc in the extracellular domain. Immunoreactivity of c-Myc epitope exposed to the cell surface formed a ring structure, which was colocalized with markers of the podosome, namely β-integrin, paxillin and Pyk2. The ring structure was also observed in TRPV2-GFP-expressing cells using total internal reflection fluorescent microscopy. Addition of formyl-Met-Leu-Phe (fMLP) increased the number of podosome and increased the intensity of the TRPV2 signal associated with the podosome. Measurement of subplasmalenmal free calcium concentration ([Ca(2+)](pm)) revealed that [Ca(2+)](pm) was elevated around the podosome. fMLP further increased [Ca(2+)](pm) in this region, which was abolished by a TRPV2 inhibitor ruthenium red. Phosphorylated Pyk2 was detected in fMLP-treated cells, and knockdown of TRPV2 reduced the expression of phospho-Pyk2. Introduction of dominant-negative Pyk2 or knockdown of TRPV2 increased the number of podosome. Conversely, elevation of [Ca(2+)](pm) by the addition of ionomycin reduced the number of podosome. These results indicate that TRPV2 is localized abundantly in the podosome and increases [Ca(2+)](pm) by the podosome. The elevation of [Ca(2+)](pm) is critical to regulate assembly of the podosome.

Dictyostelium differentiation‐inducing factor‐1 induces glucose transporter 1 translocation and promotes glucose uptake in mammalian cells

The differentiation‐inducing factor‐1 (DIF‐1) is a signal molecule that induces stalk cell formation in the cellular slime mold Dictyostelium discoideum, while DIF‐1 and its analogs have been shown to possess antiproliferative activity in vitro in mammalian tumor cells. In the present study, we investigated the effects of DIF‐1 and its analogs on normal (nontransformed) mammalian cells. Without affecting the cell morphology and cell number, DIF‐1 at micromolar levels dose‐dependently promoted the glucose uptake in confluent 3T3‐L1 fibroblasts, which was not inhibited with wortmannin or LY294002 (inhibitors for phosphatidylinositol 3‐kinase). DIF‐1 affected neither the expression level of glucose transporter 1 nor the activities of four key enzymes involved in glucose metabolism, such as hexokinase, fluctose 6‐phosphate kinase, pyruvate kinase, and glucose 6‐phosphate dehydrogenase. Most importantly, stimulation with DIF‐1 was found to induce the translocation of glucose transporter 1 from intracellular vesicles to the plasma membranes in the cells. In differentiated 3T3‐L1 adipocytes, DIF‐1 induced the translocation of glucose trasporter 1 (but not of glucose transporter 4) and promoted glucose uptake, which was not inhibited with wortmannin. These results indicate that DIF‐1 induces glucose transporter 1 translocation and thereby promotes glucose uptake, at least in part, via a inhibitors for phosphatidylinositol 3‐kinase/Akt‐independent pathway in mammalian cells. Furthermore, analogs of DIF‐1 that possess stronger antitumor activity than DIF‐1 were less effective in promoting glucose consumption, suggesting that the mechanism of the action of DIF‐1 for stimulating glucose uptake should be different from that for suppressing tumor cell growth.

Glucose Evokes Rapid Ca2+ and Cyclic AMP Signals by Activating the Cell-Surface Glucose-Sensing Receptor in Pancreatic β-Cells.

Glucose is a primary stimulator of insulin secretion in pancreatic β-cells. High concentration of glucose has been thought to exert its action solely through its metabolism. In this regard, we have recently reported that glucose also activates a cell-surface glucose-sensing receptor and facilitates its own metabolism. In the present study, we investigated whether glucose activates the glucose-sensing receptor and elicits receptor-mediated rapid actions. In MIN6 cells and isolated mouse β-cells, glucose induced triphasic changes in cytoplasmic Ca2+ concentration ([Ca2+]c); glucose evoked an immediate elevation of [Ca2+]c, which was followed by a decrease in [Ca2+]c, and after a certain lag period it induced large oscillatory elevations of [Ca2+]c. Initial rapid peak and subsequent reduction of [Ca2+]c were independent of glucose metabolism and reproduced by a nonmetabolizable glucose analogue. These signals were also blocked by an inhibitor of T1R3, a subunit of the glucose-sensing receptor, and by deletion of the T1R3 gene. Besides Ca2+, glucose also induced an immediate and sustained elevation of intracellular cAMP ([cAMP]c). The elevation of [cAMP]c was blocked by transduction of the dominant-negative Gs, and deletion of the T1R3 gene. These results indicate that glucose induces rapid changes in [Ca2+]c and [cAMP]c by activating the cell-surface glucose-sensing receptor. Hence, glucose generates rapid intracellular signals by activating the cell-surface receptor.