Anthony J. Acton

Serum Leptin, Parathyroid Hormone, 1,25-Dihydroxyvitamin D, Fibroblast Growth Factor 23, Bone Alkaline Phosphatase, and Sclerostin Relationships in Obesity

BACKGROUND Obesity is associated with hyperparathyroidism and increased bone mass and turnover, but their pathogeneses are unclear. AIMS Our aim was to determine in obesity interrelationships among serum levels of leptin, the mineral-regulating hormones, bone turnover markers, and sclerostin. METHODS This case-control study was performed in 20 women having bariatric surgery and 20 control women matched for race and age. Anthropometrics and fasting serum biochemistries were measured in controls and in bariatric patients the morning of surgery. RESULTS Body mass index (48.9 vs. 25.4 kg/m(2)), weight (128.6 vs. 71.9 kg), serum leptin (74.6 vs. 25.2 ng/ml), PTH (44.5 vs. 28.8 pg/ml), fibroblast growth factor 23 (FGF23) (42.4 vs. 25.9 pg/ml), and bone alkaline phosphatase (BAP) (25.8 vs. 17.5 U/liter) were higher, but height (162.3 vs. 167.7 cm) and 1,25-dihydroxyvitamin D (1,25D) (39.2 vs. 48.7 pg/ml) were lower in bariatric surgery patients than controls. There was no difference in serum sclerostin, amino-terminal collagen cross-links, 25-hydroxyvitamin D (25D), calcium, phosphate, and creatinine between groups. In the combined sample, leptin was positively related to PTH, FGF23, and BAP but not to 1,25D or sclerostin. Multiple regression analysis demonstrated that PTH was predicted by leptin and Ca (R(2) = 0.39); 1,25D by 25D, FGF23, and phosphate (R(2) = 0.43); FGF23 by leptin and 1,25D (R(2) = 0.27); BAP by leptin, PTH, and Ca (R(2) = 0.39); and sclerostin by leptin and PTH (R(2) = 0.20). CONCLUSIONS Women having bariatric surgery had higher leptin, PTH, FGF23, and BAP and lower 1,25D than controls. Leptin predicted the serum levels of PTH, 1,25D, and FGF23, the mineral-regulating hormones, and BAP, a bone formation marker, in women with body mass index ranging from 13.9-65.8 kg/m(2). The results suggest that leptin has an endocrine or paracrine effect on PTH and FGF23 production and that PTH may be one of the signals in obesity that leads to increased bone mass.

Role of Sox9 in growth factor regulation of articular chondrocytes

Chondrogenic polypeptide growth factors influence articular chondrocyte functions that are required for articular cartilage repair. Sox9 is a transcription factor that regulates chondrogenesis, but its role in the growth factor regulation of chondrocyte proliferation and matrix synthesis is poorly understood. We tested the hypotheses that selected chondrogenic growth factors regulate sox9 gene expression and protein production by adult articular chondrocytes and that sox9 modulates the actions of these growth factors. To test these hypotheses, we delivered insulin‐like growth factor‐I (IGF‐I), fibroblast growth factor‐2 (FGF‐2), bone morphogenetic protein‐2 (BMP‐2) and/or bone morphogenetic protein‐7 (BMP‐7), or their respective transgenes to adult bovine articular chondrocytes, and measured changes in sox9 gene expression and protein production. We then knocked down sox9 gene expression with sox9 siRNA, and measured changes in the expression of the genes encoding aggrecan and types I and II collagen, and in the production of glycosaminoglycan, collagen and DNA. We found that FGF‐2 or the combination of IGF‐I, BMP‐2, and BMP‐7 increased sox9 gene expression and protein production and that sox9 knockdown modulated growth factor actions in a complex fashion that differed both with growth factors and with chondrocyte function. The data suggest that sox9 mediates the stimulation of matrix production by the combined growth factors and the stimulation of chondrocyte proliferation by FGF‐2. The mitogenic effect of the combined growth factors and the catabolic effect of FGF‐2 appear to involve sox9‐independent mechanisms. Control of these molecular mechanisms may contribute to the treatment of cartilage damage. J. Cell. Biochem. 116: 1391–1400, 2015.

The angiogenic inhibitor TNP-470 decreases caloric intake and weight gain in high fat fed mice

The angiogenic inhibitor TNP‐470 attenuates high‐fat diet‐induced obesity; however, it is not clear how the compound alters energy balance to prevent weight gain. Five‐week‐old C57BL/6J mice were fed high‐fat diet (45% energy from fat) for 6.5 weeks and treated with TNP‐470 (20 mg/kg body weight; n = 7) or vehicle (saline; n = 7). Control mice (n = 8) received standard chow and sham injection. TNP‐470 mice initially gained weight, but by day 5 body weight was significantly less than high‐fat fed (HFF) mice and not different from that of chow‐fed mice, an effect maintained to the end of the study (28.6 ± 0.6 vs. 22.4 ± 0.6 and 22.2 ± 0.5 g). Percent body fat was reduced in TNP‐470 compared to HFF mice, but was greater than that of chow mice (34.0 ± 1.5, 23.9 ± 1.5, and 17.0 ± 1.4%, P < 0.05). Food intake in TNP‐470‐treated mice was less (P < 0.05) than that in HFF mice by day 5 of treatment (2.5 ± 0.1 vs. 2.8 ± 0.1 g/mouse/day) and remained so to the end of the study. Twenty‐four hours energy expenditure was greater (P < 0.05) in TNP‐470 than HFF or chow mice (7.05 ± 0.07 vs. 6.69 ± 0.08 vs. 6.79 ± 0.09 kcal/kg/h), an effect not explained by a difference in energy expended in locomotion. Despite normalization of body weight, TNP‐470 mice exhibited impaired glucose tolerance (area under the curve 30,556 ± 1,918 and 29,290 ± 1,584 vs. 24,421 ± 903 for TNP, HFF, and chow fed, P < 0.05). In summary, the angiogenic inhibitor TNP‐470 attenuates weight gain in HFF mice via a reduction in caloric intake and an increase in energy expenditure.

Systemic and cerebral iron homeostasis in ferritin knock-out Mice

Ferritin, a 24-mer heteropolymer of heavy (H) and light (L) subunits, is the main cellular iron storage protein and plays a pivotal role in iron homeostasis by modulating free iron levels thus reducing radical-mediated damage. The H subunit has ferroxidase activity (converting Fe(II) to Fe(III)), while the L subunit promotes iron nucleation and increases ferritin stability. Previous studies on the H gene (Fth) in mice have shown that complete inactivation of Fth is lethal during embryonic development, without ability to compensate by the L subunit. In humans, homozygous loss of the L gene (FTL) is associated with generalized seizure and atypical restless leg syndrome, while mutations in FTL cause a form of neurodegeneration with brain iron accumulation. Here we generated mice with genetic ablation of the Fth and Ftl genes. As previously reported, homozygous loss of the Fth allele on a wild-type Ftl background was embryonic lethal, whereas knock-out of the Ftl allele (Ftl-/-) led to a significant decrease in the percentage of Ftl-/- newborn mice. Analysis of Ftl-/- mice revealed systemic and brain iron dyshomeostasis, without any noticeable signs of neurodegeneration. Our findings indicate that expression of the H subunit can rescue the loss of the L subunit and that H ferritin homopolymers have the capacity to sequester iron in vivo. We also observed that a single allele expressing the H subunit is not sufficient for survival when both alleles encoding the L subunit are absent, suggesting the need of some degree of complementation between the subunits as well as a dosage effect.

Bone turnover is not influenced by serum 25-hydroxyvitamin D in pubertal healthy black and white children ☆

Low serum 25-hydroxyvitamin D [25 (OH) D] is common in healthy children particularly in blacks. However, serum 25 (OH) D concentrations for optimal bone turnover in children is unknown and few data exist that describe effects of increasing serum 25 (OH) D on bone turnover markers during puberty. The purpose of this study was to determine the relationships between serum 25 (OH) D and changes in serum 25 (OH) D and bone turnover in white and black pubertal adolescents. Bone turnover markers were measured in 318 healthy boys and girls from Georgia (34°N) and Indiana (40°N) who participated in a study of oral vitamin D(3) supplementation (0 to 4000 IU/d). Serum 25 (OH) D, osteocalcin, bone alkaline phosphatase, and urine N-telopeptide cross-links were measured at baseline and 12 weeks. Relationships among baseline 25 (OH) D and bone biomarkers, and between changes over 12 weeks were determined and tested for effects of race, sex, latitude, and baseline 25 (OH) D. Median 25 (OH) D was 27.6 ng/mL (n=318, range 10.1-46.0 ng/mL) at baseline and 34.5 ng/mL (n=302, range 9.7-95.1 ng/mL) at 12 weeks. Neither baseline nor change in 25 (OH) D over 12 weeks was associated with bone turnover. The lack of association was not affected by race, sex, latitude, or baseline serum 25 (OH) D. Serum 25 (OH) D in the range of 10-46 ng/mL appears to be sufficient for normal bone turnover in healthy black and white pubertal adolescents.

An Examination of Non-Formalin-Based Fixation Methods for Xenopus Embryos

Despite the growing availability of non‐formalin‐based fixatives, the vast majority of researchers in developmental biology continue to fix embryos and tissue in 4% paraformaldehyde. This fixation method has proven useful for both immunohistochemistry and in situ hybridization, yet working with paraformaldehyde has distinct disadvantages in its toxicity and the short shelf life of prepared solutions. In a search for viable alternative fixatives, we have evaluated two non‐formalin‐based commercial products, FineFIX (Milestone Microwave Laboratory System) and NOTOXhisto® (Scientific Device Laboratory). These products were tested side‐by‐side with a commonly used 4% paraformaldehyde solution (MEMPFA) on Xenopus laevis embryos and assayed using whole mount immunohistochemistry and whole mount in situ hybridization. The results indicate that NOTOXhisto® can be used as a substitute for MEMPFA in both tested Xenopus protocols with no loss of sensitivity or tissue morphology. Developmental Dynamics 233:1464–1469, 2005.

Serum fibroblast growth factor 23, serum iron and bone mineral density in premenopausal women

Fibroblast growth factor 23 (FGF23) circulates as active protein and inactive fragments. Low iron status increases FGF23 gene expression, and iron deficiency is common. We hypothesized that in healthy premenopausal women, serum iron influences C-terminal and intact FGF23 concentrations, and that iron and FGF23 associate with bone mineral density (BMD). Serum iron, iron binding capacity, percent iron saturation, phosphorus, and other biochemistries were measured in stored fasting samples from healthy premenopausal white (n=1898) and black women (n=994), age 20-55years. Serum C-terminal and intact FGF23 were measured in a subset (1631 white and 296 black women). BMD was measured at the lumbar spine and femur neck. Serum phosphorus, calcium, alkaline phosphatase and creatinine were lower in white women than black women (p<0.001). Serum iron (p<0.0001) and intact FGF23 (p<0.01) were higher in white women. C-terminal FGF23 did not differ between races. Phosphorus correlated with intact FGF23 (white women, r=0.120, p<0.0001; black women r=0.163, p<0.01). However, phosphorus correlated with C-terminal FGF23 only in black women (r=0.157, p<0.01). Intact FGF23 did not correlate with iron. C-terminal FGF23 correlated inversely with iron (white women r=-0.134, p<0.0001; black women r=-0.188, p<0.01), having a steeper slope at iron <50mcg/dl than ≥50mcg/dl. Longitudinal changes in iron predicted changes in C-terminal FGF23. Spine BMD correlated with iron negatively (r=-0.076, p<0.01) in white women; femur neck BMD correlated with iron negatively (r=-0.119, p<0.0001) in black women. Both relationships were eliminated in weight-adjusted models. BMD did not correlate with FGF23. Serum iron did not relate to intact FGF23, but was inversely related to C-terminal FGF23. Intact FGF23 correlated with serum phosphorus. In weight-adjusted models, BMD was not related to intact FGF23, C-terminal FGF23 or iron. The influence of iron on FGF23 gene expression is not important in determining bone density in healthy premenopausal women.

Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy.

Mutations in the ferritin light chain (FTL) gene cause the neurodegenerative disease neuroferritinopathy or hereditary ferritinopathy (HF). HF is characterized by a severe movement disorder and by the presence of nuclear and cytoplasmic iron-containing ferritin inclusion bodies (IBs) in glia and neurons throughout the central nervous system (CNS) and in tissues of multiple organ systems. Herein, using primary mouse embryonic fibroblasts from a mouse model of HF, we show significant intracellular accumulation of ferritin and an increase in susceptibility to oxidative damage when cells are exposed to iron. Treatment of the cells with the iron chelator deferiprone (DFP) led to a significant improvement in cell viability and a decrease in iron content. In vivo, iron overload and DFP treatment of the mouse model had remarkable effects on systemic iron homeostasis and ferritin deposition, without significantly affecting CNS pathology. Our study highlights the role of iron in modulating ferritin aggregation in vivo in the disease HF. It also puts emphasis on the potential usefulness of a therapy based on chelators that can target the CNS to remove and redistribute iron and to resolubilize or prevent ferritin aggregation while maintaining normal systemic iron stores.

Genetic and Environmental Influences on the Prospective Correlation Between Systemic Inflammation and Coronary Heart Disease Death in Male Twins

Objective— Because of lack of evidence, we aimed to examine to what degree low-grade systemic inflammation and coronary heart disease (CHD) death shared common genetic and environmental substrates. Approach and Results— From the 41-year prospective National Heart, Lung, and Blood Institute Twin Study, we included 950 middle-aged male twins at baseline (1969–1973). Low-grade systemic inflammation was measured with plasma levels of interleukin-6 (IL-6) and C-reactive protein. Univariate and bivariate structural equation models were used, adjusted for a risk score for CHD death. The score-adjusted heritability was 19% for IL-6, 27% for C-reactive protein, and 22% for CHD death. The positive phenotypic correlation of IL-6 with CHD death (r adjusted=0.27; 95% confidence interval [CI], 0.08–0.43) was driven by additive genetic factors (contribution [relative contribution], 0.30 [111%]) but attenuated by unique environment (–0.03 [–11%]). The genetic correlation between IL-6 and CHD death was 0.74 (95% CI, 0.21–1.00), whereas the unique environmental correlation was –0.05 (95% CI, –0.35 to 0.25). The proportion of genetic variance for CHD death shared with that for IL-6 was 74%. The phenotypic correlation of C-reactive protein with CHD death (r adjusted=0.10; 95% CI, –0.02 to 0.22) was explained by additive genetic factors (0.20 [149%]) but was attenuated by the unique environment (–0.09 [–49%]). The genetic correlation of C-reactive protein with CHD death was 0.63 (95% CI, –0.07 to 1.00), whereas the unique environmental correlation was –0.07 (95% CI, –0.29 to 0.17). Conclusions— Low-grade systemic inflammation, measured by IL-6, and long-term CHD death share moderate genetic substrates that augment both traits.

The apéritif effect: Alcohol's effects on the brain's response to food aromas in women.

Consuming alcohol prior to a meal (an apéritif) increases food consumption. This greater food consumption may result from increased activity in brain regions that mediate reward and regulate feeding behavior. Using functional magnetic resonance imaging, we evaluated the blood oxygenation level dependent (BOLD) response to the food aromas of either roast beef or Italian meat sauce following pharmacokinetically controlled intravenous infusion of alcohol.