Nesfatin-1 is a recently discovered multifunctional metabolic hormone abundantly expressed in the pancreatic islets. The main objective of this study is to characterize the direct effects of nesfatin-1 on insulin secretion in vitro using MIN6 cells and islets isolated from C57BL/6 mice. We also examined the expression of the nesfatin-1 precursor protein, nucleobindin 2 (NUCB2) mRNA, and nesfatin-1 immunoreactivity (ir) in the islets of normal mice and in the islets from mice with streptozotocin-induced type 1 diabetes and diet-induced obese (DIO) mice with type 2 diabetes. Nesfatin-1 stimulated glucose-induced insulin release in vitro from mouse islets and MIN6 cells in a dose-dependent manner. No such stimulation in insulin secretion was found when MIN6 cells/islets were incubated with nesfatin-1 in low glucose. In addition, a fourfold increase in nesfatin-1 release from MIN6 cells was observed following incubation in high glucose (16.7 mM) compared to low glucose (2 mM). Furthermore, we observed a significant reduction in both NUCB2 mRNA expression and nesfatin-1-ir in the pancreatic islets of mice with type 1 diabetes, while a significant increase was observed in the islets of DIO mice. Together, our findings indicate that nesfatin-1 is a novel insulinotropic peptide and that the endogenous pancreatic islet NUCB2/nesfatin is altered in diabetes and diet-induced obesity.
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- Abstract: Islets x
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Ronald Gonzalez, Benjamin K Reingold, Xiaodong Gao, Mandeep P Gaidhu, Robert G Tsushima and Suraj Unniappan
M Nasu, T Sugimoto, H Kaji and K Chihara
Although there is clinical evidence showing that combined therapy with parathyroid hormone (PTH) and estrogen is additively effective in increasing the bone mass of patients with osteoporosis, the mechanism of the interaction between these hormones remains unclear. The present study was performed to determine whether estrogen would affect osteoblast proliferation and function modulated by PTH in human osteoblastic SaOS-2 cells. Human PTH-(1-34) significantly inhibited [(3)H]thymidine (TdR) incorporation, which was attenuated by 24 h pretreatment with 10(-10) to 10(-7) M 17 beta-estradiol (17 beta-E(2)) in a concentration-dependent manner. PTH significantly stimulated alkaline phosphatase (ALP) activity, collagen synthesis and type-1 procollagen mRNA expression after pretreatment with 17 beta-E(2 )in these cells. Tamoxifen, an anti-estrogen, antagonized these 17 beta-E(2)-induced effects. Pretreatment with insulin-like growth factor-I (IGF-I) mimicked estrogen action, and coincubation of 3 microg/ml anti-IGF-I antibody antagonized the effects of 17 beta-E(2 )as well as those of IGF-I. In the presence of 17 beta-E(2 )pretreatment, PTH strongly stimulated IGF-binding protein (IGFBP)-5 mRNA expression in these cells, and recombinant IGFBP-5 increased type-1 procollagen mRNA expression and ALP activity. In conclusion, estrogen attenuates PTH-induced inhibition of osteoblast proliferation and PTH stimulates osteoblast function in the presence of estrogen pretreatment. IGF-I and/or IGFBP-5 seemed to be involved in the estrogen-induced modulation of PTH action on osteoblast proliferation and function.
Bethany P Cummings, Ahmed Bettaieb, James L Graham, Kimber Stanhope, Fawaz G Haj and Peter J Havel
There is a need to identify strategies for type 2 diabetes prevention. Therefore, we investigated the efficacy of pioglitazone and alogliptin alone and in combination to prevent type 2 diabetes onset in UCD-T2DM rats, a model of polygenic obese type 2 diabetes. At 2 months of age, rats were divided into four groups: control, alogliptin (20 mg/kg per day), pioglitazone (2.5 mg/kg per day), and alogliptin+pioglitazone. Non-fasting blood glucose was measured weekly to determine diabetes onset. Pioglitazone alone and in combination with alogliptin lead to a 5-month delay in diabetes onset despite promoting increased food intake and body weight (BW). Alogliptin alone did not delay diabetes onset or affect food intake or BW relative to controls. Fasting plasma glucose, insulin, and lipid concentrations were lower and adiponectin concentrations were threefold higher in groups treated with pioglitazone. All treatment groups demonstrated improvements in glucose tolerance and insulin secretion during an oral glucose tolerance test with an additive improvement observed with alogliptin+pioglitazone. Islet histology revealed an improvement of islet morphology in all treatment groups compared with control. Pioglitazone treatment also resulted in increased expression of markers of mitochondrial biogenesis in brown adipose tissue and white adipose tissue, with mild elevations observed in animals treated with alogliptin alone. Pioglitazone markedly delays the onset of type 2 diabetes in UCD-T2DM rats through improvements of glucose tolerance, insulin sensitivity, islet function, and markers of adipose mitochondrial biogenesis; however, addition of alogliptin at a dose of 20 mg/kg per day to pioglitazone treatment does not enhance the prevention/delay of diabetes onset.
Amy R Quinn, Cynthia L Blanco, Carla Perego, Giovanna Finzi, Stefano La Rosa, Carlo Capella, Rodolfo Guardado-Mendoza, Francesca Casiraghi, Amalia Gastaldelli, Marney Johnson, Edward J Dick Jr and Franco Folli
Erratic regulation of glucose metabolism including hyperglycemia is a common condition in premature infants and is associated with increased morbidity and mortality. The objective of this study was to examine histological and ultrastructural differences in the endocrine pancreas in fetal (throughout gestation) and neonatal baboons. Twelve fetal baboons were delivered at 125 days (d) gestational age (GA), 140d GA, or 175d GA. Eight animals were delivered at term (185d GA); half were fed for 5 days. Seventy-three nondiabetic adult baboons were used for comparison. Pancreatic tissue was studied using light microscopy, confocal imaging, and electron microscopy. The fetal and neonatal endocrine pancreas islet architecture became more organized as GA advanced. The percent areas of α-β-δ-cell type were similar within each fetal and newborn GA (NS) but were higher than the adults (P<0.05) regardless of GA. The ratio of β cells within the islet (whole and core) increased with gestation (P<0.01). Neonatal baboons, which survived for 5 days (feeding), had a 2.5-fold increase in pancreas weight compared with their counterparts killed at birth (P=0.01). Endocrine cells were also found in exocrine ductal and acinar cells in 125, 140 and 175d GA fetuses. Subpopulation of tissue that coexpressed trypsin and glucagon/insulin shows the presence of cells with mixed endo–exocrine lineage in fetuses. In summary, the fetal endocrine pancreas has no prevalence of a α-β-δ-cell type with larger endocrine cell percent areas than adults. Cells with mixed endocrine/exocrine phenotype occur during fetal development. Developmental differences may play a role in glucose homeostasis during the neonatal period and may have long-term implications.
RH McCusker and J Novakofski
Zinc (Zn(2+)), a multifunctional micronutrient, was recently shown to lower the affinity of cell-associated insulin-like growth factor (IGF) binding protein (IGFBP)-3 and IGFBP-5 for both IGF-I and IGF-II, but to increase the affinity of the cell surface type 1 IGF receptor (IGF-1R) for the same two ligands. However, there is a need for data concerning the effects of Zn(2+) on soluble IGFBPs and the type 2 IGF receptor (IGF-2R). In the current work, we demonstrate that Zn(2+) affects the affinity of IGFBP-5 secreted by myoblasts but not IGFBP-4. Zn(2+), at physiological levels, depressed binding of both IGF-I and IGF-II to IGFBP-5, affecting (125)I-IGF-I more than (125)I-IGF-II. Both (125)I-IGF-I and (125)I-IGF-II bound to high and low affinity sites on IGFBP-5. Zn(2+) converted the high affinity binding sites of IGFBP-5 into low affinity binding sites. An IGF-I analog, (125)I-R(3)-IGF-I, did not bind to the soluble murine IGFBP-5. Zn(2+) also decreased the affinity of the IGF-2R on L6 myoblasts. In contrast, Zn(2+) increased IGF-I, IGF-II and R(3)-IGF-I binding to the IGF-1R by increasing ligand binding affinity on both P(2)A(2a)-LISN and L6 myoblasts. Soluble IGFBP-5 and IGFBP-4 depressed the binding of (125)I-IGF-I and (125)I-IGF-II to the IGF-1R, but did not affect binding of (125)I-R(3)-IGF-I. By depressing the association of the IGFs with soluble IGFBP-5, Zn(2+) partitioned (125)I-IGF-I and (125)I-IGF-II from soluble IGFBP-5 onto cell surface IGF-1Rs. This effect is not seen when soluble L6-derived IGFBP-4 is present in extracellular fluids. We introduce a novel mechanism by which the trace micronutrient Zn(2+) may alter IGF distribution, i.e. Zn(2+) acts to increase IGF-1R binding at the expense of IGF binding to soluble IGFBP-5 and the IGF-2R.
Jun-ichi Eiki, Kaori Saeki, Norihiro Nagano, Tomoharu Iino, Mari Yonemoto, Yoko Takayenoki-Iino, Satoru Ito, Teruyuki Nishimura, Yoshiyuki Sato, Makoto Bamba, Hitomi Watanabe, Kaori Sasaki, Sumika Ohyama, Akio Kanatani, Toshio Nagase and Toshihiko Yada
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca2+ release from intracellular stores, but to extracellular Ca2+ influx. The response was inhibited by the presence of either L-type Ca2+ channel blockers or K+ ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Gαs- nor Gαq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca2+ influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.
JA Shaw, MI Delday, AW Hart, HM Docherty, CA Maltin and K Docherty
The objective of these studies was to evaluate human insulin gene expression following intramuscular plasmid injection in non-diabetic rats as a potential approach to gene therapy for diabetes mellitus avoiding the need for immunosuppression. A wild-type human preproinsulin construct and a mutant construct in which PC2/PC3 sites were engineered to form furin consensus sites were evaluated in in vitro transfections of hepatocyte (HepG2) and myoblast (C2C12/L6) cell lines, primary rat myoblasts, and dermal fibroblasts. In vivo gene transfer by percutaneous plasmid injection of soleus muscle +/- prior notexin-induced myolysis was assessed in rats. In vitro transfection of non-neuroendocrine cell lines and primary cultures with wild-type human preproinsulin resulted in secretion of predominantly unprocessed proinsulin. Employing the mutant construct, there was significant processing to mature insulin (HepG2, 95%; C2C12, 75%; L6, 65%; primary myoblasts, 48%; neonatal fibroblasts, 56%; adult fibroblasts, 87%). In rats aged 5 weeks, circulating human (pro)insulin was detected from 1 to 37 days following plasmid injection and the potential of augmenting transfection efficiency by prior notexin injection was demonstrated (wild-type processing, 87%; mutant, 90%). Relative hypoglycaemia was confirmed by HbA1C (saline, 5.5%; wild type, 5.1%; mutant, 5.1% (P<0.05)). Human (pro)insulin levels and processing (wild-type, 8%; mutant, 53%) were lower in rats aged 9 months but relative hypoglycaemia was confirmed by serum glucose at 10 days (saline, 6.4 mmol/l; wild-type, 6.0 mmol/l; mutant, 5.4 mmol/l). In conclusion, prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cells in vitro and in growing and mature rats following intramuscular plasmid injection.
Jean-Claude Henquin, Myriam Nenquin, Andras Szollosi, Atsutaka Kubosaki and Abner Louis Notkins
Islet antigen-2 (IA-2 or ICA 512) and IA-2β (or phogrin) are major autoantigens in type 1 diabetes. They are located in dense core secretory vesicles including insulin granules, but their role in β-cell function is unclear. Targeted disruption of either IA-2 or IA-2β, or both, impaired glucose tolerance, an effect attributed to diminution of insulin secretion. In this study, we therefore characterized the dynamic changes in cytosolic Ca2+([Ca2+]c) and insulin secretion in islets from IA-2/IA-2β double knockout (KO) mice. High glucose (15 mM) induced biphasic insulin secretion in IA-2/IA-2β KO islets, with a similar first phase and smaller second phase compared with controls. Since the insulin content of IA-2/IA-2β KO islets was ∼45% less than that of controls, fractional insulin secretion (relative to content) was thus increased during first phase and unaffected during second phase. This peculiar response occurred in spite of a slightly smaller rise in [Ca2+]c, could not be attributed to an alteration of glucose metabolism (NADPH fluorescence) and also was observed with tolbutamide. The dual control of insulin secretion via the KATP channel-dependent triggering pathway and KATP channel-independent amplifying pathway was unaltered in IA-2/IA-2β KO islets, and so were the potentiations by acetylcholine or cAMP (forskolin). Intriguingly, amino acids, in particular the cationic arginine and lysine, induced larger fractional insulin secretion in IA-2/IA-2β KO than control islets. In conclusion, IA-2 and IA-2β are dispensable for exocytosis of insulin granules, but are probably more important for cargo loading and/or stability of dense core vesicles.
R Kooijman, G T Rijkers and B J M Zegers
IGF-I stimulates the proliferation and differentiation of many cell types. In the case of T cells, IGF-I has been described to potentiate mitogen-induced DNA synthesis. We have addressed the working mechanism of IGF-I on T cell proliferation by measuring the effects of IGF-I on various stages of T cell activation. We found that IGF-I augmented the phytohaemagglutinin- and anti-CD3-induced interleukin-2 (IL2) production of human peripheral T cells before they enter the S phase of the cell cycle. Furthermore, the addition of IGF-I did not influence DNA synthesis of IL2-dependent growing T cells.
Journal of Endocrinology (1996) 149, 351–356
Kai-Chun Cheng, Ying-Xiao Li, Akihiro Asakawa, Miharu Ushikai, Ikuo Kato, Yuki Sato, Juei-Tang Cheng and Akio Inui
We aimed to characterize the effects of preptin on insulin secretion at the single-cell level, as well as the mechanisms underlying these changes, with respect to regulation by intracellular Ca2+ [Ca2+]i mobilization. This study assessed the effect of preptin on insulin secretion and investigated the link between preptin and the phospholipase C (PLC)/protein kinase C (PKC) pathway at the cellular level using fura-2 pentakis(acetoxymethyl) ester-loaded insulin-producing cells (Min 6 cells). Our results demonstrate that preptin promotes insulin secretion in a concentration-dependent manner. Using a PLC inhibitor (chelerythrine) or a PKC inhibitor (U73122) resulted in a concentration-dependent decrease in insulin secretion. Also, preptin mixed with IGF2 receptor (IGF2R) antibodies suppressed insulin secretion in a dose-dependent manner, which indicates that activation of IGF2R is mediated probably because preptin is a type of proIGF2. In addition, preptin stimulated insulin secretion to a similar level as did glibenclamide. The activation of PKC/PLC by preptin stimulation is highly relevant to the potential mechanisms for increase in insulin secretion. Our results provide new insight into the insulin secretion of preptin, a secreted proIGF2-derived peptide that can induce greater efficacy of signal transduction resulting from PLC and PKC activation through the IGF2R.