Abstract. Incretins including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secreted from the small intestine after oral food ingestion

Incretins including glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secreted from the small intestine after oral food ingestion are currently recognized to stimulate insulin secretion from pancreatic β cells. We previously reported that p70 S6 kinase limits the tumor necrosis factor-α (TNF-α)-stimulated interleukin-6 (IL-6) synthesis in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of incretins on the TNF-α-induced IL-6 synthesis and the underlying mechanism in MC3T3‐E1 cells. GLP‐1 and GIP significantly upregulated both TNF-α-stimulated IL-6 release and mRNA levels. Wedelolactone, an inhibitor of IκB kinase, amplified the TNF-α‐induced IL‐6 release. GLP‐1 significantly attenuated the TNF-α-induced phosphorylation of IκB without affecting the phosphorylation of p70 S6 kinase. On the other hand, GLP-1 markedly induced the phosphorylation of cAMP response element-binding protein (CREB). H-89, an inhibitor of protein kinase A, significantly suppressed the enhancement by GLP-1 of TNF-α-stimulated IL-6 release. Dibutyryl cAMP, a permeable analogue of cAMP, which suppressed the TNF-α-induced IκB phosphorylation, amplified the IL‐6 release. These results strongly suggest that incretins upregulate the TNF-α-stimulated IL-6 synthesis in osteoblasts, and that the amplifying effect of incretin is exerted via reducing the IκB/NF-κB pathway through the adenylyl cyclase-cAMP system. Introduction Bone metabolism is strictly controlled by osteoblastic bone formation and osteoclastic bone resorption (1). Bone tissue is continuously regenerated through a process called bone remodeling (2). To maintain adequate quality and quantity of bone, formation and resorption are tightly coordinated. This bone remodeling process begins with osteoclastic bone resorption, followed by osteoblastic bone formation (1). Disruption of the bone remodeling process causes metabolic bone diseases such as osteoporosis or fracture healing distress. It is currently recognized that osteoblasts play a crucial role also in the regulation of bone resorption through the expression of the receptor activator of nuclear factor-κB (RANK) ligand in response to a variety of bone resorptive stimuli (3). It is firmly established that tumor necrosis factor‐α (TNF-α) is a multifunctional cytokine and plays a pivotal role in inflammation, infection and cancer (4). In bone metabolism, TNF-α reportedly induces the activation of RANK in osteoclasts through RANK ligand expression after binding to its specific receptors in osteoblasts, leading to osteoclast activation (5). In addition, it has been shown that TNF-α inhibits the differentiation of osteoblasts from precursor cells (6). Therefore, TNF-α is generally recognized as a potent bone resorptive agent. Regarding the intracellular signaling mechanism of TNF-α, it is well known that TNF-α induces activation of the inhibiting inhibitor of κB (IκB)/nuclear factor-κB (NF-κB) pathway (7). In our previous studies (8,9), we demonstrated that TNF-α stimulated the activation of p70 S6 kinase in osteoblast-like MC3T3-E1 cells, and that the activated p70 S6 kinase negatively regulated TNF-α-stimulated interleukin-6 (IL-6) synthesis. IL-6, a member of the glycoprotein 130 cytokine families (4,10), is well known to stimulate bone resorption and induce osteoclastgenesis (4). It has been shown that IL-6 acts on the osteoblast lineage to stimulate bone formation and maintain strength rather than promoting osteoclast formation (11). Additionally, IL-6 reportedly has a pivotal role in the process of bone fracture repair (12). Therefore, IL-6 secreted by osteoblasts is currently considered as an osteotropic modulator, leading to influencing bone formation under the Incretins amplify TNF-α-stimulated IL-6 synthesis in osteoblasts: Suppression of the IκB/NF-κB pathway KAZUHIKO FUJITA1,2, HARUHIKO TOKUDA2,3, NAOHIRO YAMAMOTO1,2, SHINGO KAINUMA1,2, TETSU KAWABATA1,2, GO SAKAI1,2, GEN KUROYANAGI1,2, RIE MATSUSHIMA-NISHIWAKI2, ATSUSHI HARADA4, OSAMU KOZAWA2 and TAKANOBU OTSUKA1 1Department of Orthopedic Surgery, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601; 2Department of Pharmacology, Gifu University Graduate School of Medicine, Gifu 501-1194; Departments of 3Clinical Laboratory/Biobank of Medical Genome Center and 4Orthopedic Surgery, National Center for Geriatrics and Gerontology, Obu, Aichi 474-8511, Japan Received November 2, 2016; Accepted February 8, 2017 DOI: 10.3892/ijmm.2017. 2892 Correspondence to: Dr Haruhiko Tokuda, Department of Clinical Laboratory/Biobank of Medical Genome Center, National Center for Geriatrics and Gerontology, 7-430 Morioka, Obu, Aichi 474-8511,