Yuko Tagaya

Nucleobindin-2 Is a Positive Modulator of EGF-Dependent Signals Leading to Enhancement of Cell Growth and Suppression of Adipocyte Differentiation

Nucleobindin-2 is a 420-amino-acid EF-hand calcium-binding protein that undergoes proteolytic processing to generate an 82-amino-acid amino-terminal peptide termed nesfatin-1. To determine whether nucleobindin-2 has any biological function, nucleobindin-2 was either overexpressed or knocked down by short hairpin RNA in cultured CHO cells expressing the human insulin and epidermal growth factor (EGF) receptors (CHO/IE) and in 3T3-L1 cells. Reduction in nucleobindin-2 expression inhibited EGF-stimulated MAPK kinase (S217/S221) and Erk phosphorylation (T202/Y204). In contrast, there was no significant effect on EGF-stimulated EGF receptor phosphorylation, EGF receptor internalization, or 52-kDa Shc and c-Raf phosphorylation. Although kinase suppressor of Ras-1 and protein phosphatase 2A expression was not changed, intracellular calcium concentrations and PP2A activity was significantly increased in nucleobindin-2 knocked-down cells. Concomitant with these alterations in EGF-stimulated signaling, cell proliferation was significantly reduced in nucleobindin-2 knocked-down cells. Moreover, reduced nucleobindin-2 expression in 3T3-L1 preadipocytes resulted in a greater extent of 3T3-L1 cell adipocyte differentiation. Taken together, these data indicate that nucleobindin-2 regulates EGF-stimulated MAPK kinase/Erk signaling, cell proliferation, and adipocyte differentiation.

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin-2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.

Effect of dapagliflozin on colon cancer cell [Rapid Communication]

Dapagliflozin is a SGLT2 (Sodium/Glucose cotransporter 2) inhibitor that reduces circulating glucose levels in type 2 diabetic patients by blocking the SGLT2-dependent reabsorption of glucose in the kidney. Dapagliflozin is metabolized by UGT1A9 (UDP Glucuronosyltransferase 1 family, Polypeptidase A9), suppressing its SGLT2 inhibitor activity. However little information is available on whether dapagliflozin acts in the absence of dapagliflozin metabolism. Treatment with 0.5μM dapagliflozin significantly reduced the number of HCT116 cells, which express SGLT2 but not UGT1A9. This was independent of SGLT2 inhibition, as the SGLT2 inhibitor phlorizin had no effect. Dapagliflozin also enhanced Erk phosphorylation but without changing levels of uncleaved and cleaved PPAR and uncleaved caspase-3, suggesting that the cause of the decrease in HCT116 cell number was apoptosis independent cell death. Taken together, these data indicate a new potential role for dapagliflozin as an anticancer reagent in tumor cell populations that do not express UGT1A9.

Effect of carbohydrate counting using bolus calculators on glycemic control in type 1 diabetes patients during continuous subcutaneous insulin infusion

The present study examined the long‐term efficacy of insulin pump therapy for type 1 diabetes patients when carried out using carbohydrate counting with bolus calculators for 1 year. A total of 22 type 1 diabetes patients who had just started continuous subcutaneous insulin infusion were examined and divided into two groups: one that was educated about carbohydrate counting using bolus calculators (n = 14); and another that did not use bolus calculators (n = 8). After 1 year, the hemoglobin A1c levels of the patient group that used bolus calculators decreased persistently and significantly (P = 0.0297), whereas those of the other group did not. The bodyweight, total daily dose of insulin and bolus percentage of both groups did not change. Carbohydrate counting using bolus calculators is necessary to achieve optimal and persistent glycemic control in patients undergoing continuous subcutaneous insulin infusion.

Syntaxin4 Interacting Protein (Synip) Binds Phosphatidylinositol (3,4,5) Triphosphate

The insulin responsive Glut4 transport vesicles contain the v-SNARE protein Vamp2 that associate with the plasma membrane t-SNARE protein Syntaxin 4 to drive insulin-stimulated Glut4 translocation in skeletal muscle and adipocytes. The syntaxin 4 interacting protein (Synip) binds to syntaxin 4 in the basal state and dissociates in the insulin-stimulated state allowing for the subsequent binding of Vamp2 containing Glut4 vesicles and fusion with the plasma membrane. In this study, we have found that Synip binds phosphatidylinositol 3,4,5-triphosphate (PIP3), but not phosphatidylinositol 3 phosphate (PIP) or phosphatidylinositol 3,4-biphosphate (PIP2) through the Synip WW domain as deletion of this domain (Synip ΔWW) failed to bind PIP3. Over-expressed Synip ΔWW in 3T3L1 adipocytes reduced the basal levels of Glut4 at the plasma membrane with no effect on the binding to syntaxin 4 in vitro. Subcellular fractionation demonstrated that the amount of Synip ΔWW at the PM was decreased in response to insulin in 3T3L1 adipocytes whereas the amount of Synip WT increased. These data suggest that in the presence of insulin, the dissociated Synip remains anchored to the plasma membrane by binding to PIP3.

Association between nesfatin-1 levels and metabolic improvements in severely obese patients who underwent biliopancreatic derivation with duodenal switch

CONTEXT Nesfatin-1 is a neuroendocrine peptide with potent anorexigenic activity in rodents. The potential role of nesfatin-1 on the regulation of energy balance, metabolic functions and inflammation is currently debated in obese humans. In the present study, nesfatin-1 fluctuations and their associations with metabolic factors were investigated in severely obese patients who underwent biliopancreatic diversion with duodenal switch (BPD/DS) and severely obese controls (SOC). BASIC PROCEDURES Sixty severely obese patients who underwent BPD/DS and 15 SOC (matched for BMI and age) were included in the study. Associations between nesfatin-1 levels and body composition, glucose metabolism, lipid profile as well as inflammatory markers were evaluated at baseline and over a post-surgery12-month (12M) period. MAIN FINDINGS Body weight was reduced at 6M and at 12M in BPD/DS patients (P<0.001). Nesfatin-1 levels were reduced at 6M (women: P<0.05) and at 12M (men and women; P<0.001) in BPD/DS patients. At baseline, nesfatin-1 levels negatively correlated with weight, fat (FM) and fat-free mass (FFM) in the whole population (combined BPD/DS and SOC patients). At 12M, nesfatin-1 concentrations positively correlated with weight, FM, fasting insulin, insulin resistance, total cholesterol, LDL-cholesterol, triglyceride and apoB values. At 12M, % changes in nesfatin-1 were positively associated with% changes in weight, FM, FFM, fasting insulin, insulin resistance, total cholesterol, LDL-cholesterol, apoB and C-reactive protein. CONCLUSION Nesfatin-1 levels decrease following BPD/DS-induced weight loss and are significantly associated with parameters of metabolic health.

Assessment of factors determining an HbA1c concentration ≤ 7.5% in patients with type 1 diabetes

Establishing an optimal insulin regimen is crucial for maintaining glycemic control in patients with type 1 diabetes (T1D). The aim of the present study was to determine the insulin dose required to achieve an HbA1c concentration ≤7.5% in Japanese patients with T1D.

Interstitial pneumonia during administration of dipeptidyl peptidase-4 inhibitors.

Recognizing that case reports do not document a causal protein-A and surfactant protein-D were above the relationship, we report three clinical cases in which individuals receiving dipeptidyl peptidase (DPP)-4 inhibitor treatment developed interstitial pneumonia, with increases in the high molecular weight glycoprotein Krebs von den Lungen-6 (KL-6; normal range <500 U/mL). Case 1 was an 80-year-old Japanese female treated with alogliptin for type 2 diabetes mellitus and suffering dry cough. Serum levels of KL-6 (Fig. 1a) and surfactant

APPL1 promotes glucose uptake in response to mechanical stretch via the PKCζ-non-muscle myosin IIa pathway in C2C12 myotubes.

Expression of adaptor protein, phosphotyrosine interaction, pleckstrin homology domain, and leucine zipper containing 1 (APPL1) promoted glucose transporter 4 (GLUT4) translocation and glucose uptake in adipose and muscle tissues in response to stimulation with insulin, adiponectin, or exercise. In response to mechanical stretch, knockdown of APPL1 in C2C12 myotubes suppressed glucose uptake. APPL1-induced increased glucose uptake was mediated by protein kinase C (PKC) ζ but not AKT, AMPK, or calmodulin-dependent protein kinase. In myotubes overexpressing APPL1, PKCζ was phosphorylated and translocated to the plasma membrane (PM) in response to mechanical stretch. Phosphorylated PKCζ co-immunoprecipitated with protein phosphatase 2A (PP2A) under basal conditions, but dissociated upon myotube stretching. Moreover, stretch-induced phosphorylated PKCζ co-immunoprecipitated with non-muscle myosin IIa. Blebbistatin, an inhibitor of myosin II ATPase activity, suppressed APPL1-mediated stretch-induced glucose uptake and PKCζ translocation. Taken together these data demonstrate that in response to mechanical stretch, APPL1 enhances glucose uptake by modulating the activation and localization of PKCζ, as well as its functional interaction with both PP2A and myosin IIa. These findings support a new function for non-muscle myosin IIa in differentiated myotubes.

Interstitial pneumonia during administration of DPP4 inhibitors

Recognizing that case reports do not document a causal protein-A and surfactant protein-D were above the relationship, we report three clinical cases in which individuals receiving dipeptidyl peptidase (DPP)-4 inhibitor treatment developed interstitial pneumonia, with increases in the high molecular weight glycoprotein Krebs von den Lungen-6 (KL-6; normal range <500 U/mL). Case 1 was an 80-year-old Japanese female treated with alogliptin for type 2 diabetes mellitus and suffering dry cough. Serum levels of KL-6 (Fig. 1a) and surfactant