Jee Hyun An

Lower bone mineral density is associated with higher coronary calcification and coronary plaque burdens by multidetector row coronary computed tomography in pre- and postmenopausal women

Objectives  There is growing evidence for the association between bone mineral density (BMD) and vascular calcification, which is related to cardiovascular disease. Coronary multidetector row computed tomography (MDCT) is a noninvasive tool developed to evaluate coronary status precisely. We used MDCT to evaluate this association.

Transplantation of Mesenchymal Stem Cells Overexpressing RANK-Fc or CXCR4 Prevents Bone Loss in Ovariectomized Mice

Osteoporosis is a systemic skeletal disorder characterized by reduced bone mineral density (BMD) and increased risk of fracture. We studied the effects of transplantation of mesenchymal stem cells (MSCs) overexpressing receptor activator of nuclear factor-kappaB (RANK)-Fc and CXC chemokine receptor-4 (CXCR4) using retrovirus on ovariectomy (OVX)-induced bone loss in mice. Ten-week-old adult female C57BL/6 mice were divided into six groups as follows: Sham-operated mice treated with phosphate-buffered saline (PBS) (Sham-op + PBS); OVX mice intravenously transplanted with syngeneic MSCs overexpressing RANK-Fc-DsRED and CXCR4-GFP (RANK-Fc + CXCR4); RANK-Fc-DsRED and GFP (RANK-Fc + GFP); CXCR4-GFP and DsRED (CXCR4 + RED); DsRED and GFP (RED + GFP); or treated with PBS only (OVX + PBS). Measurement of BMD showed that introduction of RANK-Fc resulted in significant protection against OVX-induced bone loss compared to treatment with PBS (-0.1% versus -6.2%, P < 0.05) at 8 weeks after cell infusion. CXCR4 + RED group also significantly prevented bone loss compared to OVX + PBS group (2.7% versus -6.2%, P < 0.05). Notably, the effect of RANK-Fc + CXCR4 was greater than that of RANK-Fc + GFP (4.4% versus -0.1%, P < 0.05) while it was not significantly different from that in CXCR4 + RFP group (4.4% versus 2.7%, P = 0.055) at 8 weeks. Transplantation of MSCs with control virus (RED + GFP group) also resulted in amelioration of bone loss compared to OVX + PBS group (-1.7% versus -6.2%, P < 0.05). Fluorescence-activated cell sorting (FACS) and real-time quantitative PCR (qPCR) analysis for GFP from bone tissue revealed enhanced cell trafficking to bone by co-overexpression of CXCR4. In conclusion, we have demonstrated that intravenous transplantation of syngeneic MSCs overexpressing CXCR4 could promote increased in vivo cell trafficking to bone in OVX mice, which could in itself protect against bone loss but also enhance the therapeutic effects of RANK-Fc.

Vitamin K2 Supplementation Improves Insulin Sensitivity via Osteocalcin Metabolism: A Placebo-Controlled Trial

Undercarboxylated osteocalcin (ucOC) is reported to function as an endocrine hormone, affecting glucose metabolism in mice (1,2). Vitamin K, which converts ucOC to carboxylated osteocalcin (cOC), has been suggested to regulate glucose metabolism by modulating osteocalcin and/or proinflammatory pathway (3–5). We studied whether modulation of ucOC via vitamin K2 supplementation for 4 weeks affects β-cell function and/or insulin sensitivity in healthy young male subjects. Forty-two healthy young male volunteers received vitamin K2 (menatetrenone; 30 mg; Eisai Co., Japan) or placebo t.i.d. for 4 weeks. Frequently sampled intravenous glucose tolerance test was performed to determine insulin sensitivity index ( S i), acute insulin response to glucose (AIRg), and disposition index (DI) before and after treatment. Adiponectin, interleukin (IL)-6, C-reactive protein (CRP), ucOC, and cOC …

Current Status of Glycemic Control of Patients with Diabetes in Korea: The Fifth Korea National Health and Nutrition Examination Survey

Background The Korea National Health and Nutrition Examination Survey (KNHANES) III (2005) reported that 22.9% of individuals with diabetes have a glycated hemoglobin (HbA1c) <6.5% and that 43.5% have an HbA1c <7%. We investigated the levels of glycemic control and the factors associated with glycemic control using data from the KNHANES V (2010 to 2012). Methods Subjects with diabetes diagnosed by a physician or those taking antidiabetic medications were classified as individuals with known diabetes. Of 1,498 subjects aged ≥30 years with diabetes, we excluded 157 individuals who were missing HbA1c data. A total of 1,341 subjects were included in the final analysis. Results The prevalence of known diabetes was 7.7% (n=1,498, estimated to be 2.32 million people). The proportions of well-controlled diabetes meeting a HbA1c goal of <6.5% and <7% were 27% and 45.6%, respectively. HbA1c increased as the duration of diabetes increased. HbA1c in subjects with a duration of diabetes ≤5 years was lower than in subjects with a duration >5 years. HbA1c in the group taking only oral hypoglycemic agents (OHAs) was significantly lower than that in the group administered only insulin or OHA and insulin in combination. In logistic regression analysis, a longer duration of diabetes, insulin use and the absence of chronic renal failure were associated with HbA1c levels >6.5%. Conclusion The level of adequate glycemic control was similar to but slightly improved compared with previous levels. The glycemic control of long-standing diabetes patients is more difficult even though they receive insulin treatment.

Enhanced mitochondrial biogenesis contributes to Wnt induced osteoblastic differentiation of C3H10T1/2 cells

Mitochondria play a key role in cell physiology including cell differentiation and proliferation. We investigated the changes of mitochondrial biogenesis during Wnt-induced osteoblastic differentiation of murine mesenchymal C3H10T1/2 cells. Scanning electron microscopy demonstrated that activation of Wnt signaling by Wnt-3A conditioned medicum (CM) resulted in significant increase in the number of mitochondria in C3H10T1/2 cells. In addition, the induction of alkaline phosphatase (ALP) activities by Wnt-3A CM was accompanied by significant increase in mitochondrial mass (p<0.05), mitochondrial membrane potential (p<0.05), intracellular reactive oxygen species production (p<0.05), resting oxygen consumption rate (p<0.05), cellular ATP content (p< or =0.05) and mtDNA copy number (p<0.05) compared to the cells with control CM (L292-CM) treatment. Moreover, co-treatment with Dkk-1 or WIF-1, both of which are Wnt inhibitors, abrogated the Wnt-3A-induced ALP activities as well as mitochondrial biogenesis markers. Upregulation of mitochondrial biogenesis by overexpression of mitochondrial transcription factor A (Tfam) significantly enhanced Wnt-induced osteogenesis as measured by ALP activities. In contrast, inhibition of mitochondrial biogenesis by treatment with Zidovudine (AZT) resulted in significant inhibition of ALP activities. Finally, ALP activities in human osteosarcoma cell line devoid of mitochondrial DNA (rho(0) cells) was significantly suppressed both in basal and Wnt-3A stimulated state compared to those from mitochondria-intact cells (rho+ cells). As a mechanism for Wnt-mediated mitochondrial biogenesis, we found that Wnt increased the expression of PGC-1alpha, a critical molecules in mitochondrial biogenesis, through Erk and p38 MAPK pathway independent of beta-catenin signaling. We also found that increased mitochondrial biogenesis is in turn positively regulating TOPflash reporter activity as well as beta-catenin levels. To summarize, mitochodrial biogenesis is upregulated by Wnt signaling and this upregulation contributes to the osteoblastic differentiation of mouse mesenchymal C3H10T1/2 cells.

Changes of microRNA profile and microRNA-mRNA regulatory network in bones of ovariectomized mice.

Growing evidence shows the possibility of a role of microRNAs (miRNA) in regulating bone mass. We investigated the change of miRNAs and mRNA expression profiles in bone tissue in an ovariectomized mice model and evaluated the regulatory mechanism of bone mass mediated by miRNAs in an estrogen‐deficiency state. Eight‐week‐old female C3H/HeJ mice underwent ovariectomy (OVX) or sham operation (Sham‐op), and their femur and tibia were harvested to extract total bone RNAs after 4 weeks for microarray analysis. Eight miRNAs (miR‐127, ‐133a, ‐133a*, ‐133b, ‐136, ‐206, ‐378, ‐378*) were identified to be upregulated after OVX, whereas one miRNA (miR‐204) was downregulated. Concomitant analysis of mRNA microarray revealed that 658 genes were differentially expressed between OVX and Sham‐op mice. Target prediction of differentially expressed miRNAs identified potential targets, and integrative analysis using the mRNA microarray results showed that PPARγ and CREB pathways are activated in skeletal tissues after ovariectomy. Among the potential candidates of miRNA, we further studied the role of miR‐127 in vitro, which exhibited the greatest changes after OVX. We also studied the effects of miR‐136, which has not been studied in the context of bone mass regulation. Transfection of miR‐127 inhibitor has enhanced osteoblastic differentiation in UAMS‐32 cells as measured by alkaline phosphatase activities and mRNA expression of osteoblast‐specific genes, whereas miR‐136 precursor has inhibited osteoblastic differentiation. Furthermore, transfection of both miR‐127 and miR‐136 inhibitors enhanced the osteocyte‐like morphological changes and survival in MLO‐Y4 cells, whereas precursors of miR‐127 and ‐136 have aggravated dexamethasone‐induced cell death. Both of the precursors enhanced osteoclastic differentiation in bone marrow macrophages, indicating that both miR‐127 and ‐136 are negatively regulating bone mass. Taken together, these results suggest a novel insight into the association between distinct miRNAs expression and their possible role through regulatory network with mRNAs in the pathogenesis of estrogen deficiency–induced osteoporosis.

Osteoblast‐targeted overexpression of PPARγ inhibited bone mass gain in male mice and accelerated ovariectomy‐induced bone loss in female mice

PPARγ has critical role in the differentiation of mesenchymal stem cells into adipocytes while suppressing osteoblastic differentiation. We generated transgenic mice that overexpress PPARγ specifically in osteoblasts under the control of a 2.3‐kb procollagen type 1 promoter (Col.1‐PPARγ). Bone mineral density (BMD) of 6‐ to 14‐week‐old Col.1 − PPARγ male mice was 8% to 10% lower than that of their wild‐type littermates, whereas no difference was noticed in Col.1‐PPARγ female mice. Col.1‐PPARγ male mice exhibited decreased bone volume (45%), trabecular thickness (23%), and trabecular number (27%), with a reciprocal increase in trabecular spacing (51%). Dynamic histomorphometric analysis also revealed that bone‐formation rate (42%) and mineral apposition rate (32%) were suppressed significantly in Col.1‐PPARγ male mice compared with their wild‐type littermates. Interestingly, osteoclast number and surface also were decreased by 40% and 58%, respectively, in Col.1‐PPARγ male mice. In vitro whole‐marrow culture for osteoclastogenesis also showed a significant decrease in osteoclast formation (approximately 35%) with the cells from Col.1‐PPARγ male mice, and OPG/RANKL ratio was reduced in stromal cells from Col.1‐PPARγ male mice. Although there was no significant difference in BMD in Col.1‐PPARγ female mice up to 30 weeks, bone loss was accelerated after ovariectomy compared with wild‐type female mice (−3.9% versus −6.8% at 12 weeks after ovariectomy, p < .01), indicating that the effects of PPARγ overexpression becomes more evident in an estrogen‐deprived state in female mice. In conclusion, in vivo osteoblast‐specific overexpression of PPARγ negatively regulates bone mass in male mice and accelerates estrogen‐deficiency‐related bone loss in female mice.

Factors predicting therapeutic efficacy of combination treatment with sitagliptin and metformin in type 2 diabetic patients: the COSMETIC study

Objective  We assessed the predictive parameters for therapeutic efficacy of initial combination therapy with sitagliptin and metformin in drug‐naïve type 2 diabetic patients.

Transgenic mice overexpressing secreted frizzled-related proteins (sFRP)4 under the control of serum amyloid P promoter exhibit low bone mass but did not result in disturbed phosphate homeostasis

Secreted frizzled-related protein-4 (sFRP4) is a member of secreted modulators of Wnt signaling pathways and has been recognized to play important role in the pathogenesis of oncogenic osteomalacia as a potential phosphatonin. To investigate the role of sFRP4 in bone biology and phosphorus homeostasis in postnatal life, we generated transgenic mice that overexpress sFRP4 under the control of the serum amyloid P promoter (SAP-sFRP4), which drives transgene expression postnatally. Serum phosphorus level and urinary phosphorus excretion were slightly lower and higher, respectively, in SAP-sFRP4 compared to wild-type (WT) littermate, but the difference did not reach statistical significance. However, renal Na(+/-)/Pi co-transporter (Npt) 2a and 1alpha-hydroxylase gene expression were up-regulated in SAP-sFRP4 mice. In addition, the level of serum 1,25-dihydroxyvitamin D(3) was higher in SAP-sFRP4 mice. At 5 weeks of age, bone mineral density (BMD) in SAP-sFRP4 was similar to that in WT. However, with advancing age, SAP-sFRP4 mice gained less BMD so that areal BMD of SAP-sFRP4 mice was significantly lower compared to WT at 15 weeks of age. Histomorphometric analysis of proximal tibia showed that trabecular bone volume (BV/TV) and thickness (Tb.Th) were significantly lower in SAP-sFRP4 mice. There was no evidence of osteomalacia in histological analysis. Our data do not support the role of sFRP4 per se as a phosphatonin but suggest that sFRP4 negatively regulates bone formation without disrupting phosphorus homeostasis.

Osteoblast-Targeted Overexpression of TAZ Increases Bone Mass In Vivo

Osteoblasts are derived from mesenchymal progenitors. Differentiation to osteoblasts and adipocytes is reciprocally regulated. Transcriptional coactivator with a PDZ-binding motif (TAZ) is a transcriptional coactivator that induces differentiation of mesenchymal cells into osteoblasts while blocking differentiation into adipocytes. To investigate the role of TAZ on bone metabolism in vivo, we generated transgenic mice that overexpress TAZ under the control of the procollagen type 1 promoter (Col1-TAZ). Whole body bone mineral density (BMD) of 6- to 19-week-old Col-TAZ mice was 4% to 7% higher than that of their wild-type (WT) littermates, whereas no difference was noticed in Col.1-TAZ female mice. Microcomputed tomography analyses of proximal tibiae at 16 weeks of age demonstrated a significant increase in trabecular bone volume (26.7%) and trabecular number (26.6%) with a reciprocal decrease in trabecular spacing (14.2%) in Col1-TAZ mice compared with their WT littermates. In addition, dynamic histomorphometric analysis of the lumbar spine revealed increased mineral apposition rate (42.8%) and the serum P1NP level was also significantly increased (53%) in Col.1-TAZ mice. When primary calvaria cells were cultured in osteogenic medium, alkaline phosphatase (ALP) activity was significantly increased and adipogenesis was significantly suppressed in Col1-TAZ mice compared with their WT littermates. Quantitative real-time polymerase chain reaction analyses showed that expression of collagen type 1, bone sialoprotein, osteocalcin, ALP, osterix, and Runx2 was significantly increased in calvaria cells from Col1-TAZ mice compared to their WT littermates. In vitro, TAZ enhanced Runx2-mediated transcriptional activity while suppressing the peroxisome proliferator-activated receptor gamma signaling pathway. TAZ also enhanced transcriptional activity from 3TP-Lux, which reflects transforming growth factor-beta (TGF-β)-mediated signaling. In addition, TAZ enhanced TGF-β-dependent nuclear translocation of Smad2/3 and Smad4. Taken together, these results suggest that TAZ positively regulates bone formation in vivo, which seems to be mediated by enhancing both Runx2 and TGF-β signaling.