Masahiro Emoto

Myosin motor Myo1c and its receptor NEMO/IKK-γ promote TNF-α–induced serine307 phosphorylation of IRS-1

Tumor necrosis factor-α (TNF-α) signaling through the IκB kinase (IKK) complex attenuates insulin action via the phosphorylation of insulin receptor substrate 1 (IRS-1) at Ser307. However, the precise molecular mechanism by which the IKK complex phosphorylates IRS-1 is unknown. In this study, we report nuclear factor κB essential modulator (NEMO)/IKK-γ subunit accumulation in membrane ruffles followed by an interaction with IRS-1. This intracellular trafficking of NEMO requires insulin, an intact actin cytoskeletal network, and the motor protein Myo1c. Increased Myo1c expression enhanced the NEMO–IRS-1 interaction, which is essential for TNF-α– induced phosphorylation of Ser307–IRS-1. In contrast, dominant inhibitory Myo1c cargo domain expression diminished this interaction and inhibited IRS-1 phosphorylation. NEMO expression also enhanced TNF-α–induced Ser307–IRS-1 phosphorylation and inhibited glucose uptake. In contrast, a deletion mutant of NEMO lacking the IKK-β–binding domain or silencing NEMO blocked the TNF-α signal. Thus, motor protein Myo1c and its receptor protein NEMO act cooperatively to form the IKK–IRS-1 complex and function in TNF-α–induced insulin resistance.

DOC2B: A Novel Syntaxin-4 Binding Protein Mediating Insulin-Regulated GLUT4 Vesicle Fusion in Adipocytes

OBJECTIVE— Insulin stimulates glucose uptake in skeletal muscle and adipose tissues primarily by stimulating the translocation of vesicles containing a facilitative glucose transporter, GLUT4, from intracellular compartments to the plasma membrane. The formation of stable soluble N-ethyl-maleimide–sensitive fusion protein [NSF] attachment protein receptor (SNARE) complexes between vesicle-associated membrane protein-2 (VAMP-2) and syntaxin-4 initiates GLUT4 vesicle docking and fusion processes. Additional factors such as munc18c and tomosyn were reported to be negative regulators of the SNARE complex assembly involved in GLUT4 vesicle fusion. However, despite numerous investigations, the positive regulators have not been adequately clarified. RESEARCH DESIGN AND METHODS— We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin-4 was assessed by the yeast two-hybrid screening system, immunoprecipitation, and in vitro glutathione S-transferase (GST) pull-down experiments. Cell surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b. RESULTS— Herein, we show that DOC2b, a SNARE-related protein containing double C2 domains but lacking a transmembrane region, is translocated to the plasma membrane upon insulin stimulation and directly associates with syntaxin-4 in an intracellular Ca2+-dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca2+-interacting domain mutant DCO2b or knockdown of DOC2b inhibited, insulin-stimulated glucose uptake. CONCLUSIONS— These findings indicate that DOC2b is a positive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes.

CS-045, a new oral antidiabetic agent, stimulates fructose-2,6-bisphosphate production in rat hepatocytes.

Fructose-2,6-bisphosphate is a potent activator of 6-phosphofructo-1-kinase, a key enzyme in glycolysis. We previously revealed that sulfonylureas stimulate fructose-2,6-bisphosphate production in the rat liver by activating 6-phosphofructo-2-kinase. In the present study, we show that CS-045, a new antidiabetic agent, activated 6-phosphofructo-2-kinase and raised fructose-2,6-bisphosphate levels in dispersed rat hepatocytes. This action was time- and dose-dependent. Ten micromolar CS-045 raised the fructose-2,6-bisphosphate content linearly to the submaximal level in 20 min. Dose dependency was observed in the range of 1-30 microM. Thirty micromolar CS-045 completely reversed the inhibitory effect of 0.1 nM glucagon on fructose-2,6-bisphosphate production. CS-045 activated 6-phosphofructo-2-kinase by decreasing the Km value for the substrate (fructose-6-phosphate) without affecting the Vmax. The combination of suboptimal doses of CS-045 and tolbutamide increased fructose-2,6-bisphosphate content more than that induced by each agent alone. These results indicate that CS-045 may reduce plasma glucose by facilitating glycolysis in the liver.

DOC2b is a SNARE regulator of glucose-stimulated delayed insulin secretion

Insulin secretion is precisely regulated by blood glucose with unique biphasic pattern. The regulatory mechanism of the second-phase insulin release is unclear. In this study, we report that DOC2b (double C2 domain protein isoform b), a SNARE related protein, was associated with insulin vesicles and translocated to plasma membrane within several minutes upon high-glucose stimulation followed by an interaction with syntaxin4, but not syntaxin1. This binding specificity and the time course of DOC2b translocation were suitable for the regulation of second-phase insulin release. Increased DOC2b expression enhanced glucose-stimulated insulin secretion. In contrast, silencing DOC2b inhibited delayed release of insulin, without affecting rapid (approximately 7min) phase secretion. Interestingly, DOC2b had no effects on KCl-triggered insulin release. These data suggest that DOC2b may be a regulator for delayed (second-phase) insulin secretion in MIN6 cells.

Plasma concentrations of vascular endothelial growth factor are associated with peripheral oedema in patients treated with thiazolidinedione

To the Editor Thiazolidinediones are widely used in patients with type 2 diabetes. In addition to lowering plasma glucose, this class of drugs may reduce the risk of cardiovascular diseases in patients with diabetes. Despite the beneficial effect of thiazolidinediones, their use is sometimes associated with peripheral oedema. This is the most frequent adverse effect leading to discontinued treatment with thiazolidinediones in Japan. However, the mechanism by which thiazolidinediones induce oedema remains to be established. Recently, Guan et al. reported that, in a mouse model, thiazolidinediones caused fluid retention through peroxisome proliferator-activated receptor-γ stimulation of renal salt absorption mediated by epithelial Na channel (ENaC) [1]. The authors speculated that an increase in plasma volume causes thiazolidinedione-induced peripheral oedema. We, however, doubt that this is the sole cause of oedema in humans. ENaC is a sodium channel located in collecting tubules. This channel is also the site of action of the hormone aldosterone, a mineralocorticoid that increases sodium reabsorption. Primary aldosteronism is a disease caused by hypersecretion of this hormone. Due to excess sodium reabsorption, the circulating blood volume increases. The resulting headache and hypertension are well documented. However, peripheral oedema does not usually develop [2]. This is a well-known outcome of this disease. Oedema, moreover, also does not develop as a result of the increased plasma volume in the syndrome of inappropriate secretion of antidiuretic hormone [3]. Antidiuretic horomone is a pituitary hormone that activates the water channel aquaporin 2 in collecting tubules, thereby increasing water reabsorption. These observations in relation to endocrine diseases suggest that blood volume expansion is not sufficient for peripheral oedema to develop. We would like to highlight another important factor that might also be involved in the process. Previously, we reported that thiazolidinediones increase the plasma levels of vascular endothelial growth factor (VEGF) [4]. Since VEGF is a potent vascular permeability factor, an increase in VEGF levels may contribute to thiazolidinedioneinduced oedema. However, because of the difficulty of quantifying the severity of oedema, no one has demonstrated a clear correlation between peripheral oedema and plasma VEGF levels. Assessing the presence of peripheral oedema by body weight can be misleading, because thiazolidinediones increase body fat mass [5] and circulating blood volume [1]. In our previous report, for example, a weak correlation between body weight and plasma VEGF concentration was observed only in female patients, but not in male patients or groups comprising both sexes [4]. Here, we demonstrate a strong correlation between clinically apparent peripheral oedema and plasma VEGF concentration. Oedema is defined as a clinically apparent increase in the interstitial fluid volume. Thus, we judged the existence of peripheral oedema by the presence of pitting after pressure was applied to the bilateral lower extremities. We re-evaluated the clinical data collected for our previous study [4]. Briefly, a total of 30 patients with type 2 diabetes Diabetologia (2006) 49:2217–2218 DOI 10.1007/s00125-006-0313-5

Identification of Glypican3 as a novel GLUT4-binding protein

Insulin stimulates glucose uptake in fat and muscle primarily by stimulating the translocation of vesicles containing facilitative glucose transporters, GLUT4, from intracellular compartments to the plasma membrane. Although cell surface externalization of GLUT4 is critical for glucose transport, the mechanism regulating cell surface GLUT4 remains unknown. Using a yeast two-hybrid screening system, we have screened GLUT4-binding proteins, and identified a novel glycosyl phosphatidyl inositol (GPI)-linked proteoglycan, Glypican3 (GPC3). We confirmed their interaction using immunoprecipitation and a GST pull-down assay. We also revealed that GPC3 and GLUT4 to co-localized at the plasma membrane, using immunofluorescent microscopy. Furthermore, we observed that glucose uptake in GPC3-overexpressing adipocytes was increased by 30% as compared to control cells. These findings suggest that GPC3 may play roles in glucose transport through GLUT4.

Characterization of the binding sites for [3H]glibenclamide in rat liver membranes.

The specific binding sites for sulfonylureas in the rat liver membrane fraction were demonstrated and characterized. [3H]Glibenclamide binding to the liver membrane was specific, time- and temperature-dependent, and reversible. Scatchard analysis showed a single class binding site. The dissociation constant (Kd) for glibenclamide was 1.1 microM and the binding capacity (Bmax) was 50 pmol/mg protein. [3H]Glibenclamide binding could be displaced by other sulfonylureas. Half-maximal inhibition of binding (IC50) for glimepiride, gliclazide, acetohexamide, tolbutamide and chlorpropamide was 4.2 microM, 74 microM, 0.33 mM, 0.60 mM, 1.2 mM, respectively. Each value is close to the reported blood concentration when a therapeutic dose of each drug is administered orally. The order of IC50 values is coincident with the order of potency of the clinical hypoglycemic effect of these drugs. We had shown that these concentrations of sulfonylureas stimulate 6-phosphofructo-2-kinase in the liver or hepatocytes and inhibit phosphoenolpyruvate carboxykinase in the hepatoma cells. The specific binding sites demonstrated here may play some roles when sulfonylureas affect carbohydrate metabolism in the liver.

PKC iota facilitates insulin-induced glucose transport by phosphorylation of SNARE regulator DOC2b

Double C2 domain protein b (DOC2b), one of the synaptotagmins, has been shown to translocate to the plasma membrane, and to initiate membrane‐fusion processes of vesicles containing glucose transporter 4 proteins on insulin stimulation. However, the mechanism by which DOC2b is regulated remains unclear. Herein, we identified the upstream regulatory factors of DOC2b in insulin signal transduction. We also examined the role of DOC2b on systemic homeostasis using DOC2b knockout (KO) mice.