Correlation of Plasma and Salivary Osteocalcin Levels with Nascent Metabolic Syndrome Components with and Without Pre/Diabetes Biochemical Parameters / Correlación de los niveles de osteocalcina en plasma y saliva con los componentes del síndrome metabólico naciente con y sin parámetros bioquímicos de prediabetes

Objectives: This study aimed to compare and correlate plasma and salivary levels of cardiometabolic risk biomarkers’ of pharmacotherapy (appraised using colorimetric assays), adiposity, and atherogenicity indices. Methods: 61 Nascent MetS subjects vs. 30 lean normoglycemic and healthy controls were recruited in Family Medicine outpatient clinics/Jordan University Hospital (a referral medical center). Fasting blood and saliva specimens were collected. Clinical and anthropometric variables were determined along with atherogenecity and adiposity indices. Results: Among nascent MetS (metabolic syndrome) recruits, almost half were normoglycemic, 43% were prediabetic and 8% were diabetic. Pronouncedly Glycemic (FPG and Alc) and lipid parameters (TG, HDL-C and non-HDL-C), adiposity indices (BMI, WHR, WtHR, Conicity-index, BAI, LAP, VAI) and atherogenicity indices (AIP, TC/HDL-C, LDL-C/HDL-C, non-HDL-C/HDL-C and TG/HDL-C) were higher in the nascent MetS group (P<0.05 vs. controls). Markedly among the plasma cardiometabolic risk biomarkers (P<0.05 vs. controls) in the nascent MetS group, adipolin, cathepsin S, ghrelin, irisin, LBP, leptin, and osteocalcin were higher but plasma FGF1 levels were oddly lower. Significantly (P<0.05 vs. controls) nascent MetS –linked salivary levels of adipolin and LBP were higher as opposed to the lower cathepsin S. Only osteocalcin, amongst 9 metabolic risk biomarkers studied, had remarkably significant correlation between plasma and saliva levels, in both total sample and MetS patients (P<0.05). Markedly in the nascent MetS only group, both plasma and salivary osteocalcin correlated with FPG and A1c (P<0.05); salivary osteocalcin correlated with BMI and LAP (P<0.05). Likewise, in the total sample plasma osteocalcin correlated significantly with BMI, BAI, WHt R, SBP, DBP, TG, LAP, VAI, TG/HDL-C and AIP (P<0.05), while salivary osteocalcin had substantial correlations only with FPG and A1c (P<0.05). Conclusion: Association of nascent MetS-related plasma and salivary osteocalcin levels and clinical characteristics and indices propagate salivary osteocalcin as a non-invasive marker for clinical control of MetS-/preDM.(AU)

Hepatic glycogenolysis is determined by maternal high-calorie diet via methylation of Pygl and it is modified by oteocalcin administration in mice.

OBJECTIVE: Accumulating evidence indicates that an adverse perinatal environment contributes to a higher risk of metabolic disorders in the later life of the offspring. However, the underlying molecular mechanisms remain largely unknown. Thus, we investigated the contribution of maternal high-calorie diet and osteocalcin to metabolic homeostasis in the offspring. METHODS: Eight-week-old C57Bl/6N female mice were mated with age-matched males and allocated randomly to three groups: a normal-diet (ND) or a high-fat, high-sucrose diet group, which was administered either saline (control) or GluOC (10 ng/g body mass) from the day of mating to that of delivery, and the dams were fed a ND after the delivery. Pups weaned at 24 days after birth were analyzed. RESULTS: A maternal high-fat, high-sucrose diet during pregnancy causes metabolic disorders in the liver of the offspring via hypermethylation of the Pygl gene, encoding glycogen phosphorylase L, which mediates hepatic glycogenolysis. The reduced expression of Pygl induced by the maternal diet causes the hepatic accumulation of glycogen and triglyceride in the offspring, which remains in adulthood. In addition, the administration of uncarboxylated osteocalcin during pregnancy upregulates Pygl expression via both direct CREBH and ATF4 and indirect epigenomic pathways, mitigating the maternal diet-induced obesity and abnormal glucose and lipid metabolism in adulthood. CONCLUSIONS: We propose that maternal energy status is reflected in the hepatic glycogenolysis capacity of the offspring via epigenetic modification of Pygl and uncarboxylated osteocalcin regulates glycogenolysis.

Gut microbiota-derived propionate mediates the neuroprotective effect of osteocalcin in a mouse model of Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder with no absolute cure. The evidence of the involvement of gut microbiota in PD pathogenesis suggests the need to identify certain molecule(s) derived from the gut microbiota, which has the potential to manage PD. Osteocalcin (OCN), an osteoblast-secreted protein, has been shown to modulate brain function. Thus, it is of interest to investigate whether OCN could exert protective effect on PD and, if yes, whether the underlying mechanism lies in the subsequent changes in gut microbiota. RESULTS: The intraperitoneal injection of OCN can effectively ameliorate the motor deficits and dopaminergic neuronal loss in a 6-hydroxydopamine-induced PD mouse model. The further antibiotics treatment and fecal microbiota transplantation experiments confirmed that the gut microbiota was required for OCN-induced protection in PD mice. OCN elevated Bacteroidetes and depleted Firmicutes phyla in the gut microbiota of PD mice with elevated potential of microbial propionate production and was confirmed by fecal propionate levels. Two months of orally administered propionate successfully rescued motor deficits and dopaminergic neuronal loss in PD mice. Furthermore, AR420626, the agonist of FFAR3, which is the receptor of propionate, mimicked the neuroprotective effects of propionate and the ablation of enteric neurons blocked the prevention of dopaminergic neuronal loss by propionate in PD mice. CONCLUSIONS: Together, our results demonstrate that OCN ameliorates motor deficits and dopaminergic neuronal loss in PD mice, modulating gut microbiome and increasing propionate level might be an underlying mechanism responsible for the neuroprotective effects of OCN on PD, and the FFAR3, expressed in enteric nervous system, might be the main action site of propionate. Video abstract.

Uncarboxylated osteocalcin ameliorates hepatic glucose and lipid metabolism in KKAy mice via activating insulin signaling pathway.

Osteocalcin, expressed in osteoblasts of the bone marrow, undergoes post-translational carboxylation and deposits in mineralized bone matrix. A portion of osteocalcin remains uncarboxylated (uncarboxylated osteocalcin, GluOC) that is released into blood where it functions as a hormone to regulate insulin secretion and insulin sensitivity. As insulin resistance is closely associated with metabolic syndrome, this study is aimed to elucidate how GluOC regulates glucose and lipid metabolism in KKAy mice, an animal model displaying obese, hyperglycemia, hyperinsulinemia, insulin resistance, and hepatic steatosis. GluOC (3, 30 ng/g per day, ig) was orally administered to female KKAy mice for 4 weeks. Whole-body insulin sensitivity, glucose metabolism, hepatic steatosis, dyslipidemia were examined using routine laboratory assays. We found that GluOC administration significantly enhanced insulin sensitivity in KKAy mice by activating hepatic IRß/PI3K/Akt pathway and elevated the whole-body insulin sensitivity with decreased FPI and HOMA-IR index. Furthermore, GluOC administration alleviated hyperglycemia through suppressing gluconeogenesis and promoting glycogen synthesis in KKAy mice and in cultured hepatocytes in vitro. Moreover, GluOC administration dose-dependently ameliorated dyslipidemia and attenuated hepatic steatosis in KKAy mice by inhibiting hepatic de novo lipogenesis and promoting fatty-acid ß-oxidation. These results demonstrate that GluOC effectively enhances hepatic insulin sensitivity, improves hyperglycemia and ameliorates hepatic steatosis in KKAy mice, suggesting that GluOC could be a promising drug candidate for treating metabolic syndrome.

Synergistic effect of extracellularly supplemented osteopontin and osteocalcin on stem cell proliferation, osteogenic differentiation, and angiogenic properties.

A high demand for functional bone grafts is being observed worldwide, especially due to the increased life expectancy. Osteoinductive components should be incorporated into functional bone grafts, accelerating cell recruitment, cell proliferation, angiogenesis, and new bone formation at a defect site. Noncollagenous bone matrix proteins, especially osteopontin (OPN) and osteocalcin (OC), have been reported to regulate some physiological process, such as cell migration and bone mineralization. However, the effects of OPN and OC on cell proliferation, osteogenic differentiation, mineralization, and angiogenesis are still undefined. Therefore, we assessed the exogenous effect of OPN and OC supplementation on human bone marrow mesenchymal stem/stromal cells (hBM MSC) proliferation and osteogenic differentiation. OPN dose-dependently increased the proliferation of hBM MSC, as well as improved the angiogenic properties of human umbilical vein endothelial cells (HUVEC) by increasing the capillary-like tube formation in vitro. On the other hand, OC enhanced the differentiation of hBM MSC into osteoblasts and demonstrated an increase in extracellular calcium levels and alkaline phosphatase activity, as well as higher messenger RNA levels of mature osteogenic markers osteopontin and osteocalcin. In vivo assessment of OC/OPN-enhanced scaffolds in a critical-sized defect rabbit long-bone model revealed no infection, while new bone was being formed. Taken together, these results suggest that OC and OPN stimulate bone regeneration by inducing stem cell proliferation, osteogenesis and by enhancing angiogenic properties. The synergistic effect of OC and OPN observed in this study can be applied as an attractive strategy for bone regeneration therapeutics by targeting different vital cellular processes.

Androgen receptor gene polymorphisms and maximal fat oxidation in healthy men. A longitudinal study / Polimorfismo del gen del receptor de andrógenos y oxidación máxima de grasa en hombres sanos. Estudio longitudinal

Introduction: Androgens play a major role in fat oxidation; however, the effects of androgens depend, among other factors, on the intrinsic characteristics of the androgen receptor (AR). Lower repetitions of CAG and GGN polymorphism appear to have a protective effect on fat accumulation in the transition from adolescent to mid-twenties. Whether a similar protective effect is present later in life remains unknown. The aims of this study were: a) to evaluate if extreme CAG and GGN repeat polymorphisms of the androgen receptors influence body fat mass, its regional distribution, resting metabolic rate (RMR), maximal fat oxidation capacity (MFO) and serum leptin, free testosterone and osteocalcin in healthy adult men; and b) to determine the longitudinal effects on fat tissue accumulation after 6.4 years of follow-up. Methods: CAG and GGN repeats length were measured in 319 healthy men (mean ± standard deviation [SD]: 28.3 ± 7.6 years). From these, we selected the subjects with extreme short (CAGS ≤ 19; n = 7) and long (CAGL ≥ 24; n = 10) CAG repeats, and the subjects with short (GGNS ≤ 22; n = 9) and long (GGNL ≥ 25; n = 10) GGN repeats. Body composition was assessed by DXA and serum levels of leptin, free testosterone and osteocalcin by ELISA. After 6.4 years of follow-up, DXA was repeated, and resting metabolic rate (RMR), MFO and VO2max determined by indirect calorimetry. Results: CAGS and CAGL subjects had similar RMR and accumulated comparable amounts of fat tissue over 6.4 ± 1.0 years of follow-up. However, CAGL had higher MFO and total lean mass than CAGS (p < 0.05). Men with GGNS accumulated greater amount of total fat mass than men with GGNL, particularly in the trunk region seven years later. This concurred with a greater MFO in the GGNL group (p < 0.05), who accumulated less fat mass. Free testosterone was associated with MFO in absolute values (r = 0.45; p < 0.05) and MFO per kg of lower extremity lean mass per height squared (r = 0.35; p < 0.05). Conclusions: CAG and GGN repeat polymorphisms may influence muscle fat oxidation capacity and may have a role in the accumulation of fat over the years (AU)

Introducción: los andrógenos juegan un papel importante en la oxidación de grasas; sin embargo, el efecto de los andrógenos depende, entre otros factores, de las características intrínsecas del receptor de andrógenos (RA). Un menor número de repeticiones CAG y GGN del RA parecen tener un efecto protector sobre la acumulación de grasa en la transición de la adolescencia hasta la veintena. Se desconoce si adelante en la vida persiste un efecto protector similar. Los objetivos de este estudio fueron: a) evaluar si repeticiones extremas de los polimorfismos CAG y GGN del RA influyen sobre la masa grasa corporal, su distribución regional, la tasa metabólica en reposo (RMR), la máxima oxidación de grasas (MFO) y la concentración sérica de leptina, testosterona libre y osteocalcina en hombres sanos; y b) determinar los efectos longitudinales sobre la acumulación de grasa después de 6.4 años de seguimiento. Métodos: la longitud de las repeticiones de CAG y GGN fueron medidas en 319 hombres sanos (media ± desviación estándar [SD]: 28,3 ± 7,6 años). De estos, seleccionamos los sujetos con repeticiones del CAG extremas cortas (CAGS ≤ 19; n = 7) y largas (CAGL ≥ 24; n = 10), y los sujetos con repeticiones del GGN extremas cortas (GGNS ≤ 22; n = 9) y largas (GGNL ≥ 25; n = 10). Se evaluaron la composición corporal mediante DXA y los niveles séricos de leptina, testosterona libre y osteocalcina por ELISA. Tras 6.4 años de seguimiento el DXA fue repetido, y la tasa metabólica en reposo (RMR), máxima oxidación de grasas (MFO) y VO2max fueron determinados mediante calorimetría indirecta. Resultados: los grupos CAGS y CAGL fueron comparables en RMR y cantidad de tejido graso tras 6,4 ± 1,0 años de seguimiento. Sin embargo, el grupo CAGL tuvo mayor MFO y masa libre de grasa que el grupo CAGS (p < 0,05). Los hombres con GGNS acumularon mayor cantidad de masa grasa total que los hombres con GGNL, particularmente en la región del tronco siete años después. Esto concordó con un mayor MFO en el grupo GGNL (p < 0,05), que acumuló menos masa grasa. La testosterona libre se asoció con el MFO en valores absolutos (r = 0,45; p < 0,05) y con MFO expresado por kg de masa libre de grasa de las piernas al cuadrado (r = 0,35; p < 0,05). Conclusiones: las repeticiones del polimorfismo del CAG y GGN pueden influenciar la capacidad muscular de oxidación de grasas y pueden tener un rol en la acumulación de grasa con los años (AU)

Assessment of bone repair in critical-size defect in the calvarium of rats after the implantation of tricalcium phosphate beta (ß-TCP).

OBJECTIVES: Evaluating the osteoconductive property of tricalcium phosphate beta (ß-TCP) in comparison to that of inorganic bovine bone for repair in a critical-size defect in the rat calvarium. MATERIALS AND METHODS: Critical-size defects of 7mm were made with a trephine in the calvaria of 48 Wistar rats. The animals were divided into four groups, and the defects in each group were filled with tricalcium phosphate beta (ß-TCP), inorganic bovine bone (Bio-Oss), autogenous bone, or left empty. The animals were euthanized at two different time points (30 and 60days post-operation). All defects were recovered with a absorbable membrane of bovine cortical bone. Histological, histometric, and immunohistochemical (osteocalcin) assessments were carried out at 30 and 60days post-operation. RESULTS: At 30days post-operation, all groups showed areas of bone formation, predominantly when autogenous grafts were used. However, there were no statistically significant differences between the treatment groups (p>0.05). After 60days, there were similarities in the bone formation patterns between the ß-TCP (26.32±) and Bio-Oss (17.35±) groups (p=0.549). In terms of the immunohistochemical assessment of osteocalcin, the clot group showed light to moderate staining at 30 and 60days. The autogenous group showed moderate staining at 30days and moderate to intense staining after 60days. The Bio-Oss group showed light to moderate staining after 30days and intense staining at 60days. The ß-TCP group showed moderate staining at 30 and 60days post-operation. CONCLUSION: ß-TCP is a good osteoconductive material with similar effects to those of inorganic bovine bone graft and is suitable for utilization in the repair of bone defects.

Maternal oral administration of osteocalcin protects offspring from metabolic impairment in adulthood.

OBJECTIVE: Maternal diet during pregnancy has been found to influence the health of offspring. However, strategies for modulation of maternal energy metabolism without an adverse effect on the fetus have remained limited. It was recently shown that oral administration of uncarboxylated osteocalcin (GluOC) improves metabolic status in adult female mice. Whether maternal GluOC administration during gestation might improve the metabolic status of offspring was investigated. METHODS: Female C57BL/6 mice were fed a normal diet (ND) or high-fat, high-sucrose diet (HFS) and were given saline or GluOC by oral administration during pregnancy. The resulting offspring were in turn assigned to ND- or HFS-fed groups immediately after weaning, and their body weight, glucose metabolism, serum lipid parameters, and level of adipose tissue inflammation were subsequently assessed. RESULTS: Maternal HFS feeding during gestation had adverse effects on glucose and lipid parameters, body weight, and adipose tissue inflammation in female offspring fed the same diet, and these effects were attenuated by maternal oral GluOC administration. CONCLUSIONS: Maternal oral administration of GluOC protects HFS-fed female offspring from metabolic disorders induced by maternal obesity.

Autophagic dysfunction is improved by intermittent administration of osteocalcin in obese mice.

BACKGROUND: Osteoblast-specific secreted osteocalcin has been considered as an important regulator of energy and glucose metabolism, however, the causative role and clinical potential of osteocalcin implicated in insulin resistance remains not fully understood. METHODS: Osteocalcin was administered intermittently in vivo and in vitro, and metabolic parameters, autophagy and insulin signaling were assessed. RESULTS: The intermittent injections of osteocalcin in mice fed high-fat diet resulted in decreased body weight gain, fat-pad weight gain, serum triglycerides, serum-free fatty acid, blood glucose, insulin level and partial normalization of glucose tolerance relative to the mice fed high-fat diet and received vehicle injections. Meanwhile, the intermittent administration of osteocalcin not only led to the alleviation of autophagic dysfunction and endoplasmic reticulum (ER) stress, but also contributed to the restoration of the impaired insulin signaling in adipose tissue and skeleton muscle of mice consumed the high-fat diet. In accordance with these findings in vivo, osteocalcin treatment also displayed a protective impact on adipocytes and myocytes against tunicamycin- or palmitate-induced ER stress and autophagy dysfunction in an XBP-1-independent manner, with these effects of osteocalcin being reversed by inhibition of mammalian target of rapamycin (mTOR) or nuclear factor-κB (NF-κB). CONCLUSIONS: Intermittent administration of osteocalcin efficiently reversed the attenuated autophagy and ER stress, and restored the impaired insulin sensitivity in cellular and mice models of insulin resistance. Our findings provide new insights into the clinical potential of osteocalcin in metabolic homeostasis, and suggest an innovative strategy for the treatment against diabetes, obesity and metabolic syndrome.

The effects of muscle contraction and recombinant osteocalcin on insulin sensitivity ex vivo.

UNLABELLED: We tested whether GPRC6A, the putative receptor of undercarboxylated osteocalcin (ucOC), is present in mouse muscle and whether ucOC increases insulin sensitivity following ex vivo muscle contraction. GPPRC6A is expressed in mouse muscle and in the mouse myotubes from a cell line. ucOC potentiated the effect of ex vivo contraction on insulin sensitivity. INTRODUCTION: Acute exercise increases skeletal muscle insulin sensitivity. In humans, exercise increases circulating ucOC, a hormone that increases insulin sensitivity in rodents. We tested whether GPRC6A, the putative receptor of ucOC, is present in mouse muscle and whether recombinant ucOC increases insulin sensitivity in both C2C12 myotubes and whole mouse muscle following ex vivo muscle contraction. METHODS: Glucose uptake was examined in C2C12 myotubes that express GPRC6A following treatment with insulin alone or with insulin and increasing ucOC concentrations (0.3, 3, 10 and 30 ng/ml). In addition, glucose uptake, phosphorylated (p-)AKT and p-AS160 were examined ex vivo in extensor digitorum longus (EDL) dissected from C57BL/6J wild-type mice, at rest, following insulin alone, after muscle contraction followed by insulin and after muscle contraction followed by recombinant ucOC then insulin exposure. RESULTS: We observed protein expression of the likely receptor for ucOC, GPRC6A, in whole muscle sections and differentiated mouse myotubes. We observed reduced GPRC6A expression following siRNA transfection. ucOC significantly increased insulin-stimulated glucose uptake dose-dependently up to 10 ng/ml, in differentiated mouse C2C12 myotubes. Insulin increased EDL glucose uptake (∼30 %, p < 0.05) and p-AKT and p-AKT/AKT compared with rest (all p < 0.05). Contraction prior to insulin increased muscle glucose uptake (∼25 %, p < 0.05), p-AKT, p-AKT/AKT, p-AS160 and p-AS160/AS160 compared with contraction alone (all p < 0.05). ucOC after contraction increased insulin-stimulated muscle glucose uptake (∼12 % p < 0.05) and p-AS160 (<0.05) more than contraction plus insulin alone but without effect on p-AKT. In the absence of insulin and/or of contraction, ucOC had no significant effect on muscle glucose uptake. CONCLUSIONS: GPRC6A, the likely receptor of osteocalcin (OC), is expressed in mouse muscle. ucOC treatment augments insulin-stimulated skeletal muscle glucose uptake in C2C12 myotubes and following ex vivo muscle contraction. ucOC may partly account for the insulin sensitizing effect of exercise.

Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation. We previously showed that the effect of GluOC on insulin secretion is mediated largely by glucagon-like peptide-1 (GLP-1) secreted from the intestine in response to GluOC exposure. We have now examined the effect of oral administration of GluOC on glucose utilization as well as the fate of such administered GluOC in mice. Long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level and improved glucose tolerance in mice without affecting insulin sensitivity. It also increased the fasting serum insulin concentration as well as the ß-cell area in the pancreas. A small proportion of orally administered GluOC reached the small intestine and remained there for at least 24h. GluOC also entered the general circulation, and the serum GLP-1 concentration was increased in association with the presence of GluOC in the intestine and systemic circulation. The putative GluOC receptor, GPRC6A was detected in intestinal cells, and was colocalized with GLP-1 in some of these cells. Our results suggest that orally administered GluOC improved glucose handling likely by acting from both the intestinal lumen and the general circulation, with this effect being mediated in part by stimulation of GLP-1 secretion. Oral administration of GluOC warrants further study as a safe and convenient option for the treatment or prevention of metabolic disorders.

Osteocalcin induces growth hormone/insulin-like growth factor-1 system by promoting testosterone synthesis in male mice.

Osteocalcin has been shown to enhance testosterone production in men. In the present study, we investigated the effects of osteocalcin on testosterone and on induction of the growth hormone/insulin-like growth factor-1 axis. Osteocalcin injection stimulated growth, which could be inhibited by castration. In addition, osteocalcin induced testosterone secretion in testes both in vivo and in vitro. Using real-time polymerase chain reaction and Western blotting, we showed that growth hormone expression was significantly increased in the pituitary after osteocalcin injection (p<0.05). Growth hormone expression in CLU401 mouse pituitary cells was also significantly stimulated (p<0.05) by osteocalcin-induced MA-10 cells. Osteocalcin injection also promoted hepatic expression of growth hormone receptor and insulin-like growth factor-1 (p<0.05), as demonstrated by real-time polymerase chain reaction and Western blotting. Similarly, osteocalcin-induced MA-10 cells promoted growth hormone receptor and insulin-like growth factor-1 expression in NCTC1469 cells. These results suggest that the growth-stimulating activities of osteocalcin are mediated by testicular testosterone secretion, and thus provide valuable information regarding the regulatory effects of osteocalcin expression on the growth hormone/insulin-like growth factor-1 axis via reproductive activities.

Intermittent injections of osteocalcin improve glucose metabolism and prevent type 2 diabetes in mice.

The uncarboxylated form of the osteoblast-specific secreted molecule osteocalcin is a hormone favoring glucose handling and increasing energy expenditure. As a result, the absence of osteocalcin leads to glucose intolerance in mice, while genetically modified mice with an increase in uncarboxylated osteocalcin are protected from type 2 diabetes and obesity. Here, we tested in the mouse the therapeutic potential of intermittent administration of osteocalcin. We found that daily injections of osteocalcin at either 3 or 30 ng/g/day significantly improved glucose tolerance and insulin sensitivity in mice fed a normal diet. This was attributable, in part, to an increase in both ß-cell mass and insulin secretion. When mice were fed a high-fat diet (HFD), daily injections of osteocalcin partially restored insulin sensitivity and glucose tolerance. Moreover, mice treated with intermittent osteocalcin injections displayed additional mitochondria in their skeletal muscle, had increased energy expenditure and were protected from diet-induced obesity. Finally, the hepatic steatosis induced by the HFD was completely rescued in mice receiving osteocalcin daily. Overall, these results provide evidence that daily injections of osteocalcin can improve glucose handling and prevent the development of type 2 diabetes.

Effect of an isoflavones-containing red clover preparation and alkaline supplementation on bone metabolism in ovariectomized rats.

The aim of this study was to test the combined effect of a quality-controlled red clover extract (RCE) standardized to contain 40% isoflavones by weight (genistein, daidzein, biochanin A, and formononetin present as hydrolyzed aglycones) together with a modified alkaline supplementation on bone metabolic and biomechanical parameters in an experimental model of surgically-induced menopause. Sprague-Dawley female rats were maintained under controlled standard conditions of light and fed with conventional food of standard calcium content and no alfalfa or soybean components. Rats were randomized into four groups: Group A represented normal rats (sham operated) while three other groups were ovariectomized (OVX) and fed for three months as follows: standard food (group B), 6 mg/kg/day food mixed with RCE (Group C), or given 6 mg/kg/day of RCE plus a modified alkaline supplementation (BP) through a nasogastric tube at a dose of 16 mg (group D). The animals were killed 90 days after surgery. As compared to group B, RCE or RCE + BP treatments brought about significantly higher level of estradiol and mitigated the weight loss of the uterus and improved maximum load of the femoral neck. Osteocalcin level showed an over 65% increase in group B but both RCE and RCE + BP treatments prevented such abnormality with a significantly better result in RCE + BP group which virtually normalized such parameter as well as urinary excretion of DPD. Group C and D reduced the over 20% loss of bone mineral density and bone mineral content/body weight ratio observed in untreated post-ovariectomy group. Untreated ovariectomy caused about 48% decrease of cancellous bone mass in the femoral neck while this abnormality was prevented at similar extent by both RCE and RCE + BP treatments. Ovariectomy determined an over 80% increase of bone alkaline phosphatase (BALP) level but both RCE and RCE + BP treatments significantly mitigated such variable. The BALP decrease yielded by the combined RCE + BP treatment was statistically lower than RCE alone. Taken together these data show that red clover preparation in dosages amenable to clinical practice do improve OVX-induced osteoporosis while a mild metabolic alkalosis might further synergize some therapeutic aspects.

Cotargeting tumor and tumor endothelium effectively inhibits the growth of human prostate cancer in adenovirus-mediated antiangiogenesis and oncolysis combination therapy.

Tumor-endothelial interaction contributes to local prostate tumor growth and distant metastasis. In this communication, we designed a novel approach to target both cancer cells and their "crosstalk" with surrounding microvascular endothelium in an experimental hormone refractory human prostate cancer model. We evaluated the in vitro and in vivo synergistic and/or additive effects of a combination of conditional oncolytic adenovirus plus an adenoviral-mediated antiangiogenic therapy. In the in vitro study, we demonstrated that human umbilical vein endothelial cells (HUVEC) and human C4-2 androgen-independent (AI) prostate cancer cells, when infected with an antiangiogenic adenoviral (Ad)-Flk1-Fc vector secreting a soluble form of Flk1, showed dramatically inhibited proliferation, migration and tubular formation of HUVEC endothelial cells. C4-2 cells showed maximal growth inhibition when coinfected with Ad-Flk1-Fc and Ad-hOC-E1, a conditional replication-competent Ad vector with viral replication driven by a human osteocalcin (hOC) promoter targeting both prostate cancer epithelial and stromal cells. Using a three-dimensional (3D) coculture model, we found that targeting C4-2 cells with Ad-hOC-E1 markedly decreased tubular formation in HUVEC, as visualized by confocal microscopy. In a subcutaneous C4-2 tumor xenograft model, tumor volume was decreased by 40-60% in animals treated with Ad-Flk1-Fc or Ad-hOC-E1 plus vitamin D3 alone and by 90% in a combined treatment group, compared to untreated animals in an 8-week treatment period. Moreover, three of 10 (30%) pre-established tumors completely regressed when animals received combination therapy. Cotargeting tumor and tumor endothelium could be a promising gene therapy strategy for the treatment of both localized and metastatic human prostate cancer.

[Regulation of bone mineralization by parathyroid hormone].

In randomized clinical trials, parathyroid hormone (PTH) showed potent anabolic effects on the lumbar spine and decreased the risk of incident vertebral fractures dramatically. Although the anabolic effect of PTH on cortical bone in the femoral neck is still unclear, it should be demonstrated in further clinical studies. Concurrent or sequential therapies of PTH and anti-resorptive agents will be one of the major issues of treatment for osteoporosis in the future.

Marcadores bioquímicos de remodelado óseo: aspectos descriptivos / Biochemical markers of bone turnover: descriptives aspects

La osteoporosis posmenopáusica es un problema de salud pública mundial, y su principal causa es la deficiencia estrogénica. El metabolismo óseo presenta dos actividades opuestas: la formación de hueso nuevo por el osteoblasto y la degradación o resorción de hueso viejo por el osteoclasto. Dado que estas actividades cursan con el vertido a la circulación de una serie de productos, los denominados marcadores bioquímicos de remodelado óseo, nosotros podemos estimar la tasa de ambos procesos determinando los valores séricos o urinarios de estos productos en el laboratorio. Estos marcadores son una poderosa herramienta para el ginecólogo, ya que su determinación es fácil de realizar, es barata, puede ser repetida a menudo y refleja el metabolismo óseo en un momento dado. En los últimos años se han desarrollado marcadores más específicos como los telopéptidos del colágeno tipo I (CTx y NTx), y se están introduciendo métodos automatizados para su determinación que disminuyen la variabilidad intra e interensayo, aunque existen diversas fuentes de variabilidad que deben tenerse en cuenta a la hora de evaluar su uso (AU)

Prevalência de alterações dos níveis plasmáticos de osteocalcina em indivíduos portadores do virus da imunodeficiência humana tipo 1 / Prevalence of changes in plasma levels of osteocalcin in individuals of the human immunodeficiency virus type 1

Pacientes infectados pelo HIV apresentam alterações no metabolismo ósseo e mineral aparentemente relacionadas à infecção. Osteopenia tem sido associada à terapia com inibidores de protease. Porém, interação do HIV com células do esqueleto e da matriz óssea, assim como, ativação crônica de células T e produção anormal de citocinas podem afetar a função de osteoblastos e osteoclastos mesmo antes do uso de terapia. O objetivo deste estudo foi o de investigar a influência da infecção pelo HIV nos níveis séricos do marcador de formação óssea osteocalcina. Realizamos um estudo secdonal no qual avaliamos 69 indivíduos portadores do HIV [ homens, 21 mulheres, idade média 1 (sd): 33 anos ± 4] antes do uso de terapia antiretroviral. Para fins de comparação, 50 indivíduos HIV negativos foram testados como controles. Com objetivo de analisar uma possível relação ntre os níveis séricos de osteocalcina e a severidade da doença o grupo HIV + foi agrupado - de acordo com seus níveis de linfócitos T CD4 - em três grupos de 23 indivíduos: Grupo 1 CD4 que 500, Grupo 2 CD4 entre 499 e 200 e Grupo 3 CD4 que 199. Níveis de osteocalcina foram mensurados por um ensaio imunométrico (DPC Corp., Los Angeles, CA) detectando a fração intacta da osteocalcina (1-49) com valores de referência variando de 3,1 a 13,7 ng/mI. Quando comparando mais de dois grupos, o teste de Kruskal-WaIIis foi utilizado. Quando uma diferença estatisticamente significante era encontrada o teste U, Mann-Whitney foi usado para determinar as diferenças entre cada par de grupos. Coeficientes de correlação foram calculados utilizando o teste de Spearman. Valores de P foram considerados significantes quando < 0,05. Redução de níveis sericos de osteocalcina foram encontrados em 43,5 % dos indivíduos HIV positivos e em 16% dos controles [ odds ratio (OR) 4,04; intervalo de confiança 95% 1,68-9,69]. N houve diferença estatisticamente significante nos níveis de osteocalcina, quando comparada...

Administration of osteocalcin accelerates orthodontic tooth movement induced by a closed coil spring in rats.

The effect of local administration of osteocalcin (OC) on experimental tooth movement was examined in the rat. The maxillary first molar was first moved mesially with an initial tipping force of 30 g with a closed-coil spring anchored to the incisor for 10 days (n = 48). Three experimental groups (n = 8) were injected with purified rat OC at doses of 0.1, 1, and 10 micrograms, respectively. The injection into the palatal bifurcation site of the first molar was repeated daily. The control groups (n = 8) were injected with rat serum albumin (10 micrograms), phosphate buffered saline (PBS), or were not injected. Tooth movement was evaluated daily by measuring the inter-cuspal distance between the first and the second molars on a precise plaster model. The cumulative tooth movement (mm) in the 1-microgram OC-injected groups was significantly more than that in all of the control groups on day 9. The rate of tooth movement (mm/day) showed periodical elevation, with high values on days 1, 4, 7, and 9. Acceleration of tooth movement by OC was significant in the early experimental period. Subsequently, acceleration of early tooth movement by OC was histologically evaluated (n = 40). Each of four animals from the control (PBS, n = 20) and the experimental (1 microgram OC, n = 20) groups was killed daily up to 5 days. A significantly larger number of osteoclasts accumulated on the mesial alveolar bone surface in the 1-microgram OC-injected group on day 3 than that observed in control group. These results suggest that administration of OC accelerates orthodontic tooth movement due to enhancement of osteoclastogenesis on the pressure side, primarily in the early experimental period.