Ippei Kanazawa

Serum Osteocalcin Level Is Associated with Glucose Metabolism and Atherosclerosis Parameters in Type 2 Diabetes Mellitus

CONTEXT Recent animal studies showed that osteocalcin action is related to not only bone metabolism but also glucose metabolism and fat mass. We investigated the relationship between two bone formation markers, serum osteocalcin and bone-specific alkaline phosphatase, and glucose metabolism, serum adiponectin, and the amount of fat mass as well as atherosclerosis parameters in men and postmenopausal women with type 2 diabetes. METHODS A total of 179 men and 149 postmenopausal women were recruited consecutively, and radiographic and biochemical characteristics were collected. Brachial-ankle pulse wave velocity (baPWV) and intima-media thickness (IMT) were evaluated as the parameters of atherosclerosis. RESULTS Multiple regression analysis adjusted for age, duration of diabetes, body mass index, and serum creatinine showed that osteocalcin negatively correlated with fasting plasma glucose and hemoglobin A(1c) in both men and postmenopausal women (P < 0.05) and with percent fat, baPWV, and IMT in men (P < 0.05). Osteocalcin positively correlated with total adiponectin in postmenopausal women (P < 0.001). After additional adjustments for systolic blood pressure, low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, hemoglobin A(1c), and Brinkmann index, osteocalcin still significantly and negatively correlated with baPWV and IMT in men. In contrast, osteocalcin did not correlate with fasting C-peptide, and bone-specific alkaline phosphatase did not correlate with any variable in either men or postmenopausal women. CONCLUSIONS Serum osteocalcin is associated with glucose and total adiponectin levels, fat mass, and atherosclerosis parameters in patients with type 2 diabetes, suggesting that osteocalcin is important for not only bone metabolism but also glucose and fat metabolism.

Serum undercarboxylated osteocalcin was inversely associated with plasma glucose level and fat mass in type 2 diabetes mellitus

SummaryAlthough recent animal studies have shown that undercarboxylated osteocalcin acts as a hormone regulating glucose metabolism and fat mass, little is known about the relationships in humans. We reported here for the first time that undercarboxylated osteocalcin were associated with glucose/fat metabolism in patients with type 2 diabetes.IntroductionRecent studies have shown that undercarboxylated osteocalcin (ucOC) acts as a hormone regulating glucose metabolism and fat mass. We investigated the relationship between ucOC as well as other bone turnover markers [serum OC, bone-specific alkaline phosphatase (BAP), and urinary N-terminal cross-linked telopeptide of type-I collagen] versus serum levels of glucose, fasting serum C-peptide, and adiponectin as well as the amount of fat mass in type 2 diabetes.MethodsA total of 180 men and 109 postmenopausal women were consecutively recruited, and radiographic and biochemical characteristics were collected. Fat mass was measured by dual X-ray absorptiometry (DXA) and computed tomography (CT).ResultsIn men, ucOC negatively correlated with percent trunk fat (%trunk fat; by DXA) and visceral/subcutaneous fat ratio (by CT) as well as fasting plasma glucose and HbA1c (at least p < 0.05). Multiple regression analysis showed that these associations were still significant independent of age, duration of diabetes, body stature, and renal function as well as glucose or fat metabolism, whereas BAP, another bone formation marker, did not correlate with any variable. On the other hand, although ucOC also negatively correlated with %fat and %trunk fat as well as HbA1c (at least p < 0.05) in postmenopausal women, we found no significant association in multiple regression analysis.ConclusionsThese findings suggest that ucOC is associated with plasma glucose level and fat mass in men with type 2 diabetes.

Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

BackgroundAdiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells.ResultsAdiponectin receptor type 1 (AdipoR1) mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase) was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR), in the cells. AdipoR1 small interfering RNA (siRNA) transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM) in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml) also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively.ConclusionTaken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

Metformin enhances the differentiation and mineralization of osteoblastic MC3T3-E1 cells via AMP kinase activation as well as eNOS and BMP-2 expression

It is unclear whether metformin, one of the anti-hyperglycemic agents commonly used for type 2 diabetes, could affect bone formation through activation of AMP-activated protein kinase (AMPK). In order to clarify this issue, we investigated the effects of metformin on the differentiation and mineralization of osteoblastic MC3T3-E1 cells as well as intracellular signal transduction. Metformin (50 microM) significantly increased collagen-I and osteocalcin mRNA expression, stimulated alkaline phosphatase activity, and enhanced cell mineralization. Moreover, metformin significantly activated AMPK in dose- and time-dependent manners, and induced endothelial nitric oxide synthase (eNOS) and bone morphogenetic protein-2 (BMP-2) expressions. Supplementation of Ara-A (0.1mM), a specific AMPK inhibitor, significantly reversed the metformin-induced eNOS and BMP-2 expressions. Our findings suggest that metformin can induce the differentiation and mineralization of osteoblasts via activation of the AMPK signaling pathway, and that this drug might be beneficial for not only diabetes but also osteoporosis by promoting bone formation.

Associations between components of the metabolic syndrome versus bone mineral density and vertebral fractures in patients with type 2 diabetes

The association of bone with the metabolic syndrome and its features, visceral fat accumulation or insulin resistance, remains unclear. We determined visceral and subcutaneous fat areas (V and S) by computed tomography on 187 men (28-83 years) and 125 postmenopausal women (46-82 years) with type 2 diabetes. Men whose V was 100 cm(2) or more had significantly lower urinary N-terminal cross-linked telopeptide of type-I collagen (p=0.005), higher femoral neck bone mineral density (FN-BMD) (p=0.004), and lower prevalence of vertebral fractures (VFs) (p=0.04) than controls. Fat mass, V, S, and lean body mass positively correlated with FN-BMD in men and with lumbar (L) and FN-BMD in women. When adjusted for weight, these correlations became negative. Urinary C-peptide positively correlated with FN-BMD in both genders. Multivariate logistic regression analysis adjusted for age, height, weight, L-BMD, duration of diabetes, and diabetes therapies identified V in men and urinary C-peptide in women as factors inversely associated with the presence of VFs [odds ratio (OR)=0.61 per SD increase, p=0.04, and OR=0.32, p=0.01, respectively]. These findings suggest that, of the components of the metabolic syndrome, body fat in gravity and hyperinsulinemia could increase FN-BMD in diabetic subjects. Visceral fat in men and hyperinsulinemia in women may protect against VFs independent of weight, L-BMD, diabetes duration, or therapies.

Serum osteocalcin level is positively associated with insulin sensitivity and secretion in patients with type 2 diabetes

OBJECTIVE Bone is being recognized as an endocrine organ. Although previous animal studies showed that osteocalcin stimulated the expression of insulin in islets and of adiponectin in adipocytes with increased insulin secretion and sensitivity, the associations of serum osteocalcin with those parameters remain unclear in humans. METHODS In this cross-sectional study, we employed 101 postmenopausal women and 152 men with type 2 diabetes, who have not taken drugs for diabetes or osteoporosis. We also examined 75 g oral glucose tolerance test (OGTT) in 18 postmenopausal women and 20 men who visited our clinic for medical check-up for diabetes. RESULTS In both postmenopausal women and men, multiple regression analysis adjusted for age, body mass index, and serum creatinine showed that serum osteocalcin level was significantly and negatively associated with fasting plasma glucose, HbA(1c), %Trunk fat, and homeostasis model assessment (HOMA) for insulin resistance (p<0.05), and positively with HOMA for beta-cell function (p<0.05). In addition, significant positive association of serum osteocalcin level with serum adiponectin level was found in postmenopausal women (p<0.05), but not in men. In the OGTT examinations, subjects were divided into tertiles by their serum osteocalcin levels in each gender. Postmenopausal women in the lowest tertile showed hyperglycemia and hyperinsulinemia compared to those in the highest tertile after oral glucose loading (p<0.05). Men in the lowest tertile also exhibited hyperinsulinemia (p<0.05), while hyperglycemia was not found. CONCLUSION These findings indicate that serum osteocalcin level is positively associated with insulin sensitivity and secretion in Japanese patients with type 2 diabetes.

Relationships between serum adiponectin levels versus bone mineral density, bone metabolic markers, and vertebral fractures in type 2 diabetes mellitus

BACKGROUND Although, adiponectin might be associated with bone metabolism, the relationships between serum adiponectin and bone mineral density (BMD) as well as vertebral fracture in type 2 diabetes are still unclear. OBJECTIVE AND METHODS We investigated the relationships between each of serum total and high molecular weight (HMW) adiponectin versus BMD, bone markers, and the presence of vertebral fractures in a total of 231 men and 170 post-menopausal women with type 2 diabetes. RESULTS Multiple regression analysis adjusted for age, duration of diabetes, BMI, serum creatinine, and HbA(1c) showed that serum total adiponectin was negatively correlated with BMD at the total, lumbar spine, and femoral neck (r=-0.165, P<0.05; r=-0.187, P<0.05; and r=-0.136, P<0.05 respectively) and positively with urinary N-terminal cross-linked telopeptide of type-I collagen in men (r=0.148, P<0.05), and that serum HMW adiponectin was negatively correlated with BMD at the lumbar spine (r=-0.146, P<0.05). Multivariate logistic regression analysis adjusted for the parameters described above showed that total adiponectin was associated with the presence of vertebral fractures in men (odds ratio (OR)=1.396, 95% confidential interval (CI) 1.020-1.911 per s.d. increase, P<0.05), and both total and HMW adiponectin were associated with moderate or severe vertebral fractures (OR=1.709, 95% CI 1.048-2.787 per s.d. increase, P<0.05 and OR=1.810, 95% CI 1.112-2.946 per s.d. increase, P<0.05 respectively), but not in post-menopausal women. CONCLUSIONS Serum adiponectin could be associated with BMD and turnover and clinically useful for assessing the risk of vertebral fractures in type 2 diabetic men.

Adiponectin Is Associated with Changes in Bone Markers during Glycemic Control in Type 2 Diabetes Mellitus

OBJECTIVE Although several experiments show that adiponectin is associated with bone metabolism, a relationship between adiponectin and bone markers is still unclear. We monitored chronological changes in hyperglycemia, serum adiponectin, and bone markers during glycemic control in type 2 diabetes and analyzed relationships among these parameters. SUBJECTS AND RESULTS A total of 50 Japanese patients with poorly controlled type 2 diabetes [initial hemoglobin A(1c) (HbA(1c)) = 10.0 +/- 2.5%] were recruited, and biochemical data were collected before and after glycemic control for a month. Of bone formation markers, bone-specific alkaline phosphatase was decreased with a mean change of -3.11 [95% confidence interval (CI), -5.03 to -1.20; P < 0.01], whereas osteocalcin (OC) was increased with a mean change of 1.94 (95% CI, 1.45-2.42; P < 0.001) and undercarboxylated OC (ucOC)/OC ratio was decreased with a mean change of -0.15 (95% CI, -0.27 to -0.03; P < 0.01). Although adiponectin level was not significantly different before and after glycemic control, baseline adiponectin level, but not HbA(1c), was positively correlated with changes in OC, ucOC, and urinary N-terminal cross-linked telopeptide of type I collagen (uNTX) (r = 0.30, P =0.04; r = 0.32, P = 0.03; and r = 0.36, P = 0.01, respectively). Changes in adiponectin were also negatively correlated with changes in OC and uNTX (r = -0.42, P < 0.01; and r = -0.38, P < 0.01, respectively). Changes in HbA(1c) were negatively correlated with changes in OC (r = -0.30, P = 0.03). CONCLUSION These findings show that treatments for hyperglycemia enhance OC level and suggest that serum adiponectin level before starting to compensate poorly controlled diabetics could predict the subsequent improvement of bone remodeling markers during glycemic control.

BMP/Wnt antagonists are upregulated by dexamethasone in osteoblasts and reversed by alendronate and PTH: potential therapeutic targets for glucocorticoid-induced osteoporosis.

We used osteoblastic MC3T3-E1 cells to clarify the mechanisms by which dexamethasone (Dex) suppresses osteoblast function, or alendronate or parathyroid hormone (PTH) alleviate it. Dex (10(-7)M) increased mRNA expression of bone morphogenetic protein (BMP) antagonists, follistatin and Dan, and of a Wnt antagonist, secreted frizzled-related protein-1 (sFRP-1) and a Wnt signal inhibitor, axin-2, while concomitantly decreased the expression of downstream molecules, Runx2 mRNA and beta-catenin protein. Pretreatments with alendronate (10(-8)M) or human PTH-(1-34) (10(-8)M) totally or partially antagonized not only the Dex-induced enhancement in mRNA expression of follistatin/Dan and sFRP-1/axin-2 but also the Dex-induced reduction in Runx2 mRNA expression and mineralization. These findings suggest that Dex suppresses the Wnt and BMP pathways as well as osteoblast function by enhancing the expression of BMP and Wnt antagonists, and bisphosphonate and PTH exert pharmacologic effects by canceling these processes.

Activation of AMP kinase and inhibition of Rho kinase induce the mineralization of osteoblastic MC3T3-E1 cells through endothelial NOS and BMP-2 expression

AMP-activated protein kinase (AMPK) and Rho kinase (ROK) are known to modulate the mevalonate pathway. Activation of AMPK suppresses 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase. ROK acts downstream of HMG-CoA reductase, and its inhibition exerts antiatherosclerosis effects. However, whether or not these enzymes are involved in bone metabolism is unclear. The present study was undertaken to investigate the effects of an AMPK activator, 5-aminoimidazole-4-carboxamide1-beta-d-ribonucleoside (AICAR), and a ROK inhibitor, fasudil hydrochrolide, on the mineralization of osteoblastic MC3T3-E1 cells. Real-time PCR and mineralization stainings revealed that both AICAR and fasudil significantly stimulated endothelial nitric oxide synthase (eNOS), bone morphogenetic protein-2 (BMP-2), and osteocalcin mRNA expression as well as mineralization in the cells. Supplementation of either mevalonate or geranyl-geranyl pyrophosphate, the downstream molecules of HMG-CoA reductase, or coincubation with either a nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester, or a BMP-2 antagonist, noggin, significantly reversed these AICAR-induced reactions. Western blot analysis showed that AICAR activated protein kinase B and extracellular signal-regulated kinase (ERK). ERK inhibitor significantly reversed the AICAR-induced increase in eNOS and BMP-2 mRNA expression. Measurement of ROK activities by enzyme-linked immunosorbent assay revealed that both AICAR and fasudil significantly suppressed the phosphorylation of the myosin-binding subunit of myosin phosphate, a ROK substrate. These findings suggest that the AMPK activator and the ROK inhibitor are able to stimulate the mineralization of osteoblasts through modulating the mevalonate pathway. These agents could be candidate drugs that promote bone formation for the treatment of osteoporosis.