Aya Osaki

Fasting concentrations of nesfatin-1 are negatively correlated with body mass index in non-obese males

Background  We recently identified a novel anorexigenic protein, nesfatin‐1, which is processed from nesfatin/nucleobindin‐2 (NUCB2). However, the clinical importance of this protein has not been determined.

Enhanced expression of nesfatin/nucleobindin-2 in white adipose tissue of ventromedial hypothalamus-lesioned rats.

Nesfatin-1, an anorexigenic protein, is ubiquitously expressed in the body. However, the exact mechanism underlying the in vivo regulation of production of nesfatin/nucleobindin-2 (NUCB2), a precursor protein of nesfatin-1, is unknown. We investigated the influence of modulation of autonomic nerve activity by a ventromedial hypothalamus (VMH) lesion and the subsequent effect on nesfatin/NUCB2 production in rat tissues innervated by the peripheral nervous system. Nesfatin/NUCB2 is strongly expressed in the pancreas and liver, moderately expressed in subcutaneous and visceral fat tissues and interscapular brown adipose tissue (iBAT), but is weakly expressed in the skeletal muscles. Our study results showed that the VMH lesion in VMH-lesioned rats did not affect nesfatin/NUCB2 expression in the pancreas, liver, skeletal muscle, and iBAT; however, the protein expression was significantly high in both subcutaneous and visceral fat tissues. In addition, continuous peripheral administration of carbachol for 5 days did not affect nesfatin/NUCB2 expression, but chemical sympathectomy using 6-hydroxydopamine mimicked the effect of VMH lesion by showing significantly high nesfatin/NUCB2 expression in the subcutaneous fat tissues. These results show that VMH lesion can modulate the autonomic nervous system activity and balance and increase nesfatin/NUCB2 expression in white adipose tissues of rats. Further, this action may be mediated via inhibition of the sympathetic nerve activity.

Peripheral administration of nesfatin-1 increases blood pressure in mice

Anorexigenic protein, nesfatin-1, processed from its precursor protein, nesfatin/nucleobindin-2 (NUCB2), by prohormone convertases, is found in the paraventricular nucleus of the hypothalamus.1 Peripherally administered nesfatin-1 or its mid-segment section also inhibits feeding behavior.2 On the other hand, nesfatin/NUCB2 is ubiquitously expressed in peripheral tissues, including the white adipose tissue.3,4 Production of nesfatin/ NUCB2 in the white adipose tissue is regulated by the sympathetic nervous system.4 Bioactive substances released from adipose tissue are involved in the central regulation of metabolism.5 Nesfatin/NUCB2 and nesfatin-1, released from the adipose tissue, appear to be involved in the transmission of the anorexigenic signal from the periphery to the brain. Recently, intracerebroventricular infusion of nesfatin-1 was demonstrated to activate sympathetic nerve activities;6 intracerebroventricular administration of nesfatin-1also increases mean blood pressure (MBP).6 However, the issue of whether nesfatin/NUCB2, produced in the peripheral tissues, is physiologically involved in regulating blood pressure remains to be clarified. We have demonstrated a physiological role of nesfatin/NUCB2 in regulating blood pressure. Male ICR (Institute of Cancer Research) mice were obtained from Charles Rivers Japan, Tokyo, Japan. Recombinant murine nesfatin-1 (25 nmol per dose; Bachem AG, Bubendorf, Switzerland) was subcutaneously injected into male ICR mice. Mice were pretreated with intraperitoneal administration of 10 mg kg 1 phentolamine (SigmaAldrich, St Louis, MO, USA)7 or 1mg kg 1 propranolol (Sigma-Aldrich)8 30 min prior to the administration of 25 nmol nesfatin-1. A non-invasive blood pressure monitor (Model MK-2000ST, Muromachi Kikai, -40 -30 -20 -10 0 10 20 30 40 50

Nesfatin/Nucleobindin-2 (NUCB2) and Glucose Homeostasis.

Anorexigenic protein, nesfatin/nucleobindin-2 (NUCB2) is ubiquitously expressed in peripheral tissues including white adipose tissue. Nesfatin/NUCB2 is selectively expressed in pancreatic β-cell, but not β-cells, β-cells, PP-cells.Starvation significantly increased circulating nesfatin-1 concentrations, and refeeding restores the increase by starvation. There is an obvious dissociation in changes between nesfatin-1 releases from pancreatic β-cells and circulating nesfatin-1, and an increase of nesfatin-1 from pancreatic islets may not be reflected to circulating concentrations of nesfatin-1.Nesfatin-1 may be a novel insulinotropic peptide, since endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity. Nesfatin-1 may also contribute to the improvement of insulin sensitivity in hyperglycemic state. An increase of circulating nesfatin-1may shift glucose uptake to peripheral organs from skeletal muscles to adipocytes. Nesfatin-1 may involve the feedback system from adipocytes to the hypothalamus via general circulation, and from the hypothalamus to adipocytes via sympathetic nervous system. The details of those molecular mechanisms should be clarified by future studies including the analysis of gene targeted animals.

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.

Possible Involvement of Anorexigenic Protein, Nesfatin/Nucleobindin-2 (NUCB2) in Blood Pressure Regulation by Regulating Water Reabsorption in Renal Collecting Duct

Nesfatin/nucleobindin-2 (NUCB2), a precursor protein of anorexigenic protein, nesfatin-1, is ubiquitously expressed in the body. The finding that peripheral administration of nesfatin-1 increases blood pressure indicates a possible involvement of nesfatin-1 in the regulation of blood pressure. The present studies were undertaken to investigate a possible involvement of nesfatin/NUCB2 in the regulation of blood pressure. The immunoreactivity against nesfatin/NUCB2 was selectively found in vascular endothelial cells of aorta, pulmonary artery and renal artery, cardiac muscle and skeletal striated muscle cells, but not in vascular smooth muscle cells at all. Furthermore, the immunoreactivity against nesfatin/NUCB2 was selectively found in renal collecting duct cells, which contain aquaporin (AQP)-2 and/or epithelial sodium channel (ENaC). In medullary collecting ducts, cells expressing nesfatin/ NUCB2 co-expressed AQP-2, but did not co-expressed AQP-2 in renal cortical collecting ducts. On the other hand, collecting ductal cells expressing ENaC were totally compatible with those expressing nesfatin/NUCB2 in both renal medullary and cortical collecting ducts. Thus, there is a possibility that nesfatin/NUCB2 is involved in the regulation of blood pressure through increased water reabsorption in the kidney.

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.

Renal threshold for glucose reabsorption predicts diabetes improvement by sodium-glucose cotransporter 2 inhibitor therapy

In the present study we examined the efficacy of sodium‐glucose cotransporter 2 inhibitors on improvement of glycated hemoglobin (HbA1c) in comparison with the renal threshold for glucose reabsorption in patients with type 2 diabetes mellitus. Patients visited the hospital once a month for a regular follow‐up examination with the determination of blood glucose and HbA1c levels, and urinary glucose concentration from spot urine samples. Patient samples were compared before and after ipragliflozin administration. We defined the renal threshold for glucose reabsorption as the lowest blood glucose level that correlated with the first detectable appearance of urine glucose. These data showed a significant negative correlation between improvement of HbA1c level and renal threshold for glucose reabsorption in patients treated with the sodium‐glucose cotransporter 2 inhibitor. These findings show that patients who have a higher renal threshold for glucose reabsorption can be expected to more effectively respond to sodium‐glucose cotransporter 2 inhibitor therapy in terms of lowering HbA1c levels.