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Objective: Osteoporosis and obesity are complex diseases with a strong genetic component. Bone mineral density (BMD) and body mass index (BMI) linkage studies identified a locus at 1q21-23, where the interleukin-6 receptor (IL6R) gene is located. The IL6R and the gp130 receptors are the mediators of IL6 action. Serum levels of IL6 and sIL6R (the soluble form of IL6R) are higher in several diseases such as osteoporosis or obesity. Variants at IL6R have been associated with BMI and obesity. However, IL6R is an as-yet-unexplored osteoporosis candidate gene. Design: In the present study we analysed two polymorphisms in the IL6R promoter, K1435 C/T (rs3887104) and K208 G/A (rs4845617), and the Asp358Ala polymorphism (rs8192284), in relation to both BMD and BMI in a cohort of 559 postmenopausal Spanish women. Results: The promoter polymorphisms, K1435 C/T and K208 G/A were associated with femoral neck (FN) BMD (PZ0.011 and PZ0.025 respectively). The C-A and T-G promoter haplotypes were also associated with FN BMD. Additionally, the Asp358Ala variant was associated with lumbar spine BMD (PZ0.038). Finally, the K208 G/A polymorphism and the C-G and C-A haplotypes were associated with BMI and obesity, where GG was the risk genotype (PZ0.033 for BMI; PZ0.010 for obesity). Conclusion: These data suggest that variants in the IL6R gene are not only involved in the determination of BMI but also relevant for the determination of BMD. The IL6R gene may belong to the growing list of genes known to be involved in both phenotypes.

Stimulation of bone formation is a key therapeutic target in osteoporosis. Runx2 is a runt domain transcription factor essential to osteoblast differentiation, bone remodeling, and fracture healing. Runx2 knockout mice exhibit a complete lack of ossification, while overexpression of this gene in transgenic mice results in an osteoporotic phenotype. Thus, RUNX2 is a good candidate for the genetic determination of osteoporosis. In this association study, the effects of the -330 G/T polymorphism in promoter 1 and the -1025 T/C polymorphism (rs7771980) in promoter 2 of RUNX2 were tested in relation to lumbar spine (LS) and femoral neck (FN) bone mineral density (BMD) in a cohort of 821 Spanish postmenopausal women. The minor allele frequencies for the two polymorphisms were 0.15 and 0.07, respectively. The two polymorphisms, located more than 90 kb apart, were not in linkage disequilibrium (D' = 0.27, r (2) = 0.028). In an ANCOVA test adjusting by weight, height, age, and years since menopause, the -330 G/T polymorphism was not associated with any of the phenotypes analyzed, while we found the -1025 T/C polymorphism to be associated with FN BMD (p = 0.001). In particular, individuals carrying the TC genotype had higher mean adjusted FN BMD values than those bearing the TT genotype. Our results highlight the importance of this RUNX2 promoter 2 polymorphism in FN BMD determination.

The RANKL/RANK/OPG pathway is essential for bone remodeling regulation. Many hormones and cytokines are involved in regulating gene expression in most of the pathway components. Moreover, any deregulation of this pathway can alter bone metabolism, resulting in loss or gain of bone mass. Whether osteoblasts from osteoporotic and nonosteoporotic patients respond differently to cytokines is unknown. The aim of this study was to compare the effect of interleukin (IL)-1beta, proftaglandin E(2) (PGE(2)), and transforming growth factor-beta1 (TGF-beta1) treatments on OPG and RANKL gene expression in normal (n = 11) and osteoporotic (n = 8) primary osteoblasts. OPG and RANKL mRNA levels of primary human osteoblastic (hOB) cell cultures were assessed by real-time PCR. In all cultures, OPG mRNA increased significantly in response to IL-1beta treatment and decreased in response to TGF-beta1 whereas PGE(2) treatment had no effect. RANKL mRNA levels were significantly increased by all treatments. Differences in OPG and RANKL responses were observed between osteoporotic and nonosteoporotic hOB: in osteoporotic hOB, the OPG response to IL-1beta treatment was up to three times lower (P = 0.009), whereas that of RANKL response to TGF-beta1 was five times higher (P = 0.002) after 8 h of treatment, as compared with those in nonosteoporotic hOBs. In conclusion, osteoporotic hOB cells showed an anomalous response under cytokine stimulation, consistent with an enhanced osteoclastogenesis resulting in high levels of bone resorption.

Undernutrition elicited by a low-protein diet determines a marked reduction of hypophyseal activity and affects the function of the respective target organs. The objective of the present investigation was to study the ultrastructural and quantitative immunohistochemical changes of the different pituitary cell populations in undernourished monkeys that had been previously shown to have significant changes in craniofacial growth. Twenty Saimiri sciureus boliviensis monkeys of both sexes were used. The animals were born in captivity and were separated into two groups at one year of age, i.e., control and undernourished animals. The monkeys were fed ad libitum a 20% (control group) and a 10% (experimental group) protein diet for two years. Pituitaries were processed for light and electron microscopy. The former was immunolabeled with anti-GH, -PRL, -LH, -FSH, -ACTH, and -TSH sera. Volume density and cell density were measured using an image analyzer. Quantitative immunohistochemistry revealed a decrease in these parameters with regard to somatotrophs, lactotrophs, gonadotrophs and thyrotrophs from undernourished animals compared to control ones. In these populations, the ultrastructural study showed changes suggesting compensatory hyperfunction. On the contrary, no significant changes were found in the morphometric parameters or the ultrastructure of the corticotroph population. We conclude that in undernourished monkeys the somatotroph, lactotroph, gonadotroph, and thyrotroph cell populations showed quantitative immunohistochemical changes that can be correlated with ultrastructural findings.

LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. LRP5 is an important regulator of osteoblast growth and differentiation, affecting bone mass in vertebrates. Whether common variations in LRP5 are associated with normal BMD variation or osteoporotic phenotypes is of great relevance. We used a haplotype-based approach to search for common disease-associated variants in LRP5 in a cohort of 964 Spanish postmenopausal women. Twenty-four SNPs were selected, covering the LRP5 region, including the missense changes p.V667M and p.A1330V. The SNPs were genotyped and evaluated for association with BMD at the lumbar spine (LS) or femoral neck (FN) and with osteoporotic fracture, at single SNP and haplotype levels, by regression methods. Association with LS BMD was found for SNP 1, rs312009, located in the 5Ј-flanking region (p ‫ס‬ 0.011, recessive model). SNP 6, rs2508836, in intron 1, was also associated with BMD, both at LS (p ‫ס‬ 0.025, additive model) and FN (p ‫ס‬ 0.031, recessive model). Two polymorphisms were associated with fracture: SNP 11, rs729635, in intron 1, and SNP 15, rs643892, in intron 5 (p ‫ס‬ 0.007 additive model and p ‫ס‬ 0.019 recessive model, respectively). Haplotype analyses did not provide additional information, except for haplotype "GC" of the block located at the 3Јend of the gene. This haplotype spans intron 22 and the 3Ј untranslated region and was associated with FN BMD (p ‫ס‬ 0.029, one copy of the haplotype versus none). In silico analyses showed that SNP 1 (rs312009) lies in a putative RUNX2 binding site. Electro-mobility shift assays confirmed RUNX2 binding to this site.

Osteoporosis is a complex disease involving many putative genetic factors. Association analysis of functional SNPs in candidate genes is an important tool for their identification. However, this approach is affected by limited power, population stratification, and other drawbacks that lead to discordant results. Replication in independent cohorts is essential. We performed association analyses of three functional polymorphisms previously associated with bone phenotypes--namely, Ala222Val in MTHFR, Ile1062Val in LRP6, and -13910C>T in LCT--in a cohort of 944 postmenopausal Spanish women, all of them with lumbar spine (LS) bone mineral density (BMD) data and most with femoral neck (FN) BMD and fracture data. We found significant differences between genotypes only for the MTHFR polymorphism and vertebral factures, with an OR of 2.27 (95% CI 1.17-4.38) for the TT vs. CC/CT genotypes, P = 0.018. We present genotype and allele frequency data for LCT -13910C>T for a Spanish population, where the T allele (conferring lactase persistence) has a frequency of 38.6%. Genotype frequencies were consistent with observed clines in Europe and with the prevalence of lactase nonpersistence. The LCT -13910C>T polymorphism was significantly associated with height and weight, such that T allele carriers were 0.88 cm taller (95% CI 0.08-1.59 cm, P = 0.032, adjusted by age) than CC individuals and TT homozygotes were 1.91 kg heavier than CC/CT individuals (95% CI 0.11-3.71 kg, P = 0.038, adjusted by age). In conclusion, no significant association was observed between the studied polymorphisms and LS BMD or FN BMD in postmenopausal Spanish women, and only MTHFR Ala222Val was associated with vertebral fractures.

Inhibition of prolactin (PRL) secretion has been previously shown in pituitaries from male and female hamsters exposed to short photoperiods. The purpose of the present study was to analyze the possible quantitative immunohistochemical and ultrastructural changes of PRL cells in male golden hamsters undergoing regression and spontaneous recrudescence, correlating the morphological findings with circulating PRL levels. Thus, adult male golden hamsters were exposed for 8, 16, 22 and 28 weeks to either short photoperiods (SP: 6 h light, 18 h darkness) or long photoperiods (LP: 14 h light, 10 h darkness). Pituitaries were processed for both light and electron microscopy, and serum levels of PRL were assessed by radioimmunoassay (RIA). Volume density (VD = cell area/reference area) and cell density (CD = number of cells/reference area) of lactotropes were measured with an image analysis system (Imaging Technology, Software Optimas 5.2). One hundred lactotropes were recorded for measuring several ultrastructural parameters. When analyzing the lactrotrope cell population from animals submitted to SP, the VD and CD were found to be significantly (p < 0.05) diminished with respect to those of the groups submitted to LP at weeks 8, 16 and 22. However, at week 28 a spontaneous recrudescence appeared. The lactotropes from animals submitted to LP for 8, 16, 22 and 28 weeks exhibited numerous large electrondense secretory granules. The rough endoplasmic reticulum (RER) presented some flat cisternae and numerous free ribosomes. Animals submitted to SP for 8 weeks showed a number of medium and large secretory granules, and the RER exhibited mainly numerous free ribosomes. In those animals submitted to SP for 16 and 22 weeks, lactotropes were found smaller and showed small and medium-sized secretory granules decreased in number. The Golgi complex exhibited some immature granules and dilated cisternae, while the RER did not present differences with respect to the 8-week SP group. The hamsters submitted to SP for 28 weeks presented medium and large secretory granules, and the RER exhibited dilated irregular cisternae. The ultrastructural morphometric parameters showed a decrease (p < 0.05) in the number of secretory granules and exocytotic profiles, as well as a diminution (p < 0.05) in the areas of Golgi complex, RER, secretory granules, and individual mean area of secretory granule during the 16 and 22 weeks of SP. Serum PRL levels were severely reduced under SP. This decrease was greater at 16 weeks than at 8 weeks of exposure. When hamsters were kept for 22 weeks under SP, PRL levels started their recovery. Thus, at the end of the experiment, the PRL values returned to near those of LP group. In summary, we found quantitative immunohistochemical and ultrastructural, as well as biochemical changes that suggest an inhibition of synthesis, storage and release of PRL in male golden hamsters submitted to SP, with a spontaneous recrudescence appearing at the end of the study.