Blocking ß1/ß2-Adrenergic Signaling Reduces Dietary Fat Absorption by Suppressing Expression of Pancreatic Lipase in High Fat-Fed Mice.

We investigated whether ß-adrenergic antagonists attenuates dietary fat absorption through the regulation of pancreatic lipase (PNLIP) expression in pancreatic acinar cells in the context of high fat diet feeding. Male six-week-old C57BL/6 mice were assigned into an ad libitum fed control diet (CON) and a high fat diet (HIGH). Within each diet group, subgroups of mice were treated with vehicle (VEH) or propranolol, a ß-adrenergic antagonist (BB). Over 12 weeks, body weight gain observed in HIGHVEH was mitigated in HIGHBB (+103% vs. +72%). Increase in fecal fat amount observed in HIGHVEH was further increased in HIGHBB. Increase in PNLIP expressions observed in HIGHVEH pancreatic tissues was abolished in HIGHBB. PNLIP expression in mouse primary pancreatic acinar cells and 266-6 cell lines increased with isoproterenol treatment, which was blocked by propranolol. Isoproterenol increased PNLIP expression in a cAMP/protein kinase A/ cyclic AMP response element binding protein (CREB)-dependent manner. CREB directly bound to the CRE on the mouse PNLIP promoter and transactivated PNLIP expression. These results suggest that sympathetic activation increases dietary fat absorption through the upregulation of PNLIP expression and that a ß-adrenergic antagonist attenuates obesity development partly through the downregulation of PNLIP expression and inhibition of dietary fat absorption in the context of high fat diet feeding.

Propranolol attenuates calorie restriction- and high calorie diet-induced bone marrow adiposity.

We investigated the effects of ß-adrenergic activation on bone marrow adiposity and on adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). C57BL/6 mice were subjected to a control (CON), high calorie (HIGH) or low calorie (LOW) diet for 12 weeks. In each group, mice were treated with vehicle (VEH) or propranolol. The number of adipocytes per area bone marrow was increased in LOWVEH and HIGHVEH mice compared with CONVEH mice, which was attenuated by propranolol. Isoproterenol increased lipid droplet accumulation and adipogenic marker gene expression in 3T3-L1 preadipocytes and mouse BMSCs, which were blocked by propranolol. Conditioned medium obtained from MC3T3-E1 osteoblasts suppressed adipogenic differentiation of 3T3-L1 cells, which was significantly attenuated by treatment of MC3T3-E1 cells with isoproterenol. These data suggest that ß-adrenergic activation enhances bone marrow adipogenesis via direct stimulation of BMSCs adipogenesis and indirect inhibition of osteoblast anti-adipogenic potential.

Propranolol, a ß-adrenergic antagonist, attenuates the decrease in trabecular bone mass in high calorie diet fed growing mice.

We investigated the effects of high calorie and low calorie diets on skeletal integrity, and whether ß-adrenergic blockade (BB) attenuates bone loss induced by dietary calorie alteration. Male 6-week-old C57BL/6 mice were assigned to either an ad-lib fed control diet (CON), a high calorie diet (HIGH), or a low calorie diet (LOW) group. In each diet group, mice were treated with either vehicle (VEH) or propranolol, a ß-adrenergic antagonist. Over 12-weeks, ß-blockade mitigated body weight and fat mass increases induced by the high calorie diet. Femoral trabecular bone mineral density and the expression levels of osteogenic marker genes in bone marrow cells were reduced in HIGHVEH and LOWVEH mice, and BB significantly attenuated this decline only in HIGH mice. In summary, the magnitude of bone loss induced by low calorie diet was greater than that caused by high calorie diet in growing mice, and ß-blockade mitigated high calorie diet-induced bone loss.

TNF-α upregulates sclerostin expression in obese mice fed a high-fat diet.

Sclerostin decreases bone mass by antagonizing the Wnt signaling pathway. We examined whether obesity-induced bone loss is associated with the expression of sclerostin. Five-week-old male mice were assigned to one of two groups (n = 10 each) and fed either a control diet (10% kcal from fat; CON) or a high-fat diet (60% kcal from fat; HF) for 12 weeks. Thex final body weight and whole body fat mass of the HF mice were higher than those of the CON mice. The distal femur cancellous bone mineral density and bone formation rate was lower in HF mice than in CON mice. The percent erosion surface was higher in the HF mice than the CON mice. The serum levels and femoral osteocytic protein expression levels of tumor necrosis factor-α (TNF-α) were significantly higher in HF mice than in CON mice. Sclerostin mRNA levels and osteocytic sclerostin protein levels in femoral cortex were also higher in HF mice than in CON mice. Sclerostin expression in MLO-Y4 osteocytes increased with TNF-α treatment, and TNF-α-induced sclerostin expression was blocked by the inhibition of NF-κB activation. Chromatin immunoprecipitation and a luciferase reporter assay demonstrated that NF-κB directly binds to the NF-κB binding elements on the mouse sost promoter and stimulates sclerostin expression. These results support a model in which, in the context of obesity or other inflammatory diseases that increase the production of TNF-α, TNF-α upregulates the expression of sclerostin through NF-κB signaling pathway, thus contributing to bone loss.

Tumor necrosis factor-α enhances the transcription of Smad ubiquitination regulatory factor 1 in an activating protein-1- and Runx2-dependent manner.

Smad ubiquitination regulatory factor 1 (Smurf1) is an E3 ubiquitin ligase that negatively regulates osteoblast differentiation. Although tumor necrosis factor-α (TNF-α) has been shown to increase Smurf1 expression, the details of the regulatory mechanisms remain unclear. Here, we investigated the molecular mechanism by which TNF-α stimulates Smurf1 expression in C2C12 and primary cultured mouse calvarial cells. TNF-α treatment rapidly induced the activation of NF-κB and MAPKs. Smurf1 induction by TNF-α was blocked by the inhibition of JNK or ERK, while the inhibition of NF-κB and p38 MAPK had no effect on Smurf1 induction. TNF-α treatment or c-Jun overexpression enhanced the activity of a luciferase reporter that contained a 2.7 kb mouse Smurf1 promoter sequence. Site-directed mutagenesis of the Smurf1 reporter and chromatin immunoprecipitation analysis demonstrated that the activating protein-1 (AP-1) binding motif at -922 bp on the mouse Smurf1 promoter mediated TNF-α/JNK/AP-1-stimulated Smurf1 transcription. Interestingly, Smurf1 expression was not observed in Runx2-null mouse calvarial cells. When Runx2 was ectopically expressed in these cells, the basal and TNF-α-induced expression of Smurf1 was restored. Overexpression of Runx2 transactivated the Smurf1 promoter in a dose-dependent manner. Reporter and chromatin immunoprecipitation assays demonstrated that the Runx2-binding motif at -202 bp functioned in Runx2-mediated Smurf1 expression. ERK activation by TNF-α treatment or constitutively active MEK1 overexpression increased Smurf1 expression in a Runx2-dependent manner. These results suggest that the JNK/AP-1 and ERK/Runx2 signaling pathways mediate TNF-α-dependent Smurf1 transcription.

Myeloid Elf-1-like factor stimulates adipogenic differentiation through the induction of peroxisome proliferator-activated receptor γ expression in bone marrow.

Myeloid Elf-1 like factor (MEF) is one of the Ets transcription factors known to regulate cell proliferation and differentiation. A previous report has shown that osteoblast-specific MEF transgenic mice (Col1a1-MEF TG mice) have low bone mass but high bone marrow adiposity. In the present study, we explored a previously unappreciated mechanism whereby MEF promotes adipogenesis in bone marrow. An adipogenic colony-forming unit assay showed that bone marrow cells derived from Col1a1-MEF TG mice had a higher adipogenic differentiation potential compared to those from wild-type. The levels of adipogenic marker genes expression in 3T3L1 cells were higher when co-cultured with Col1a1-MEF TG bone marrow cells than with wild-type cells. MC3T3-E1 preosteoblasts transfected with MEF secreted higher levels of 15-deoxy-delta (12, 14)-prostaglandin J(2), a potent endogenous ligand of peroxisome proliferator-activated receptor γ (PPARγ), under adipogenic conditions. MEF overexpression increased the adipogenic marker genes expression including PPARγ and lipid droplet accumulation in MC3T3-E1 preosteoblasts and 3T3L1 preadipocytes. Endogenous MEF expression levels increased as adipocyte differentiation proceeded. Knockdown of MEF by siRNA suppressed expression levels of adipogenic marker genes including PPARγ. MEF directly bound to the MEF binding element on the mouse PPARγ promoter, transactivating promoter activity. Immunohistochemical staining of tibia sections demonstrated that bone lining cells and bone marrow cells express higher levels of PPARγ protein in Col1a1-MEF TG mice than in wild-type mice. These results suggest that MEF transactivates PPARγ expression, which, in turn, enhances adipogenic differentiation. Furthermore, MEF overexpressing osteoblasts secrete higher levels of adipogenic factors, creating a marrow microenvironment that favors adipogenesis.

The effect of antioxidants on the production of pro-inflammatory cytokines and orthodontic tooth movement.

Orthodontic force causes gradual compression of the periodontal ligament tissues, which leads to local hypoxia in the compression side of the tissues. In this study, we investigated whether antioxidants exert a regulatory effect on two factors: the expression of pro-inflammatory cytokines in human periodontal ligament fibroblasts (PDLFs) that were exposed to mechanical compression and hypoxia and the rate of orthodontic tooth movement in rats. Exposure of PDLFs to mechanical compression (0.5-3.0 g/cm(2)) or hypoxic conditions increased the production of intracellular reactive oxygen species. Hypoxic treatment for 24 h increased the mRNA levels of IL-1ß, IL-6 and IL-8 as well as vascular endothelial growth factor (VEGF) in PDLFs. Resveratrol (10 nM) or N-acetylcysteine (NAC, 20 mM) diminished the transcriptional activity of hypoxiainducible factor-1 and hypoxia-induced expression of VEGF. Combined treatment with mechanical compression and hypoxia significantly increased the expression levels of IL-1ß, IL-6, IL-8, TNF-α and VEGF in PDLFs. These levels were suppressed by NAC and resveratrol. The maxillary first molars of rats were moved mesially for seven days using an orthodontic appliance. NAC decreased the amount of orthodontic tooth movement compared to the vehicle-treated group. The results from immunohistochemical staining demonstrated that NAC suppressed the expression of IL-1ß and TNF-α in the periodontal ligament tissues compared to the vehicle-treated group. These results suggest that antioxidants have the potential to negatively regulate the rate of orthodontic tooth movement through the down-regulation of pro-inflammatory cytokines in the compression sides of periodontal ligament tissues.

Hypoxia inducible factor-1α directly induces the expression of receptor activator of nuclear factor-κB ligand in periodontal ligament fibroblasts.

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1α (HIF-1α) protein in PDLFs. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL expression. To investigate further whether HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1α significantly increased RANKL promoter activity, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL promoter activity. Furthermore, mutations of putative HIF-1α binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1α increased the DNA binding of HIF-1α to RANKL promoter. These results suggest that HIF-1α mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs.

High extracellular calcium-induced NFATc3 regulates the expression of receptor activator of NF-κB ligand in osteoblasts.

Nuclear factor of activated T cell (NFAT) is a key transcription factor for receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. However, it is unclear whether NFAT plays a role in the expression of RANKL in osteoblasts. High extracellular calcium ([Ca(2+)](o)) increases intracellular calcium, enhances RANKL expression in osteoblasts/stromal cells, and induces osteoclastogenesis in a coculture of osteoblasts and hematopoietic bone marrow cells. Because intracellular calcium signaling activates the calcineurin/NFAT pathway, we examined the role of NFAT activation on high [Ca(2+)](o)-induced RANKL expression in MC3T3-E1 subclone 4 (MC4) cells. Among the family of NFAT transcription factors, expression of NFATc1 and NFATc3, but not NFATc2, NFATc4 or NFAT5, was observed in MC4 cells. High [Ca(2+)](o) increased the expression levels of NFATc1, NFATc3 and RANKL. Cyclosporin A and FK506, inhibitors of calcineurin phosphatase, blocked high [Ca(2+)](o)-induced expression of NFAT and RANKL. Knockdown of NFATc1 and NFATc3 by siRNA prevented high [Ca(2+)](o)-induced RANKL expression, whereas overexpression of NFATc1 and NFATc3 induced RANKL expression. Furthermore, overexpressed NFATc1 upregulated NFATc3 expression, but NFATc1 knockdown decreased NFATc3 expression. Chromatin immunoprecipitation and reporter assay results showed that NFATc3, but not NFATc1, directly binds to the RANKL promoter and stimulates RANKL expression. In summary, these results demonstrate that high [Ca(2+)](o) increases expression of RANKL via activation of the calcineurin/NFAT pathway in osteoblasts. In addition, high [Ca(2+)](o) induces the activation and expression of NFATc1; NFATc3 expression and activity are subsequently increased; and NFATc3 directly binds to the RANKL promoter to increase its expression.