Interleukin 17A: a Janus-faced regulator of osteoporosis.

Interleukin (IL)-17A is a well-described mediator of bone resorption in inflammatory diseases, and postmenopausal osteoporosis is associated with increased serum levels of IL-17A. Ovariectomy (OVX) can be used as a model to study bone loss induced by estrogen deficiency and the role of IL-17A in osteoporosis development has previously been investigated using various methods to inhibit IL-17A signaling in this model. However, the studies show opposing results. While some publications reported IL-17A as a mediator of OVX-induced osteoporosis, others found a bone-protective role for IL-17 receptor signaling. In this study, we provide an explanation for the discrepancies in previous literature and show for the first time that loss of IL-17A has differential effects on OVX-induced osteoporosis; with IL-17A being important for cortical but not trabecular bone loss. Interestingly, the decrease in trabecular bone after OVX in IL-17A knock-out mice, was accompanied by increased adipogenesis depicted by elevated leptin levels. Additionally, the bone marrow adipose tissue expanded, and the bone-turnover decreased in ovariectomized mice lacking IL-17A compared to ovariectomized WT mice. Our results increase the understanding of how IL-17A signaling influences bone remodeling in the different bone compartments, which is of importance for the development of new treatments of post-menopausal osteoporosis.

Increased bone mass in a mouse model with low fat mass.

Mice with impaired acute inflammatory responses within adipose tissue display reduced diet-induced fat mass gain associated with glucose intolerance and systemic inflammation. Therefore, acute adipose tissue inflammation is needed for a healthy expansion of adipose tissue. Because inflammatory disorders are associated with bone loss, we hypothesized that impaired acute adipose tissue inflammation leading to increased systemic inflammation results in a lower bone mass. To test this hypothesis, we used mice overexpressing an adenoviral protein complex, the receptor internalization and degradation (RID) complex that inhibits proinflammatory signaling, under the control of the aP2 promotor (RID tg mice), resulting in suppressed inflammatory signaling in adipocytes. As expected, RID tg mice had lower high-fat diet-induced weight and fat mass gain and higher systemic inflammation than littermate wild-type control mice. Contrary to our hypothesis, RID tg mice had increased bone mass in long bones and vertebrae, affecting trabecular and cortical parameters, as well as improved humeral biomechanical properties. We did not find any differences in bone formation or resorption parameters as determined by histology or enzyme immunoassay. However, bone marrow adiposity, often negatively associated with bone mass, was decreased in male RID tg mice as determined by histological analysis of tibia. In conclusion, mice with reduced fat mass due to impaired adipose tissue inflammation have increased bone mass.

ERα expression in T lymphocytes is dispensable for estrogenic effects in bone.

Estrogen treatment has positive effects on the skeleton, and we have shown that estrogen receptor alpha (ERα) expression in cells of hematopoietic origin contributes to a normal estrogen treatment response in bone tissue. T lymphocytes are implicated in the estrogenic regulation of bone mass, but it is not known whether T lymphocytes are direct estrogen target cells. Therefore, the aim of this study was to determine the importance of ERα expression in T lymphocytes for the estrogenic regulation of the skeleton using female mice lacking ERα expression specifically in T lymphocytes (Lck-ERα-/-) and ERαflox/flox littermate (control) mice. Deletion of ERα expression in T lymphocytes did not affect bone mineral density (BMD) in sham-operated Lck-ERα-/- compared to control mice, and ovariectomy (ovx) resulted in a similar decrease in BMD in control and Lck-ERα-/- mice compared to sham-operated mice. Furthermore, estrogen treatment of ovx Lck-ERα-/- led to an increased BMD that was indistinguishable from the increase seen after estrogen treatment of ovx control mice. Detailed analysis of both the appendicular (femur) and axial (vertebrae) skeleton showed that both trabecular and cortical bone parameters responded to a similar extent regardless of the presence of ERα in T lymphocytes. In conclusion, ERα expression in T lymphocytes is dispensable for normal estrogenic regulation of bone mass in female mice.

Roles of transactivating functions 1 and 2 of estrogen receptor-alpha in bone.

The bone-sparing effect of estrogen is primarily mediated via estrogen receptor-α (ERα), which stimulates target gene transcription through two activation functions (AFs), AF-1 in the N-terminal and AF-2 in the ligand binding domain. To evaluate the role of ERα AF-1 and ERα AF-2 for the effects of estrogen in bone in vivo, we analyzed mouse models lacking the entire ERα protein (ERα(-/-)), ERα AF-1 (ERαAF-1(0)), or ERα AF-2 (ERαAF-2(0)). Estradiol (E2) treatment increased the amount of both trabecular and cortical bone in ovariectomized (OVX) WT mice. Neither the trabecular nor the cortical bone responded to E2 treatment in OVX ERα(-/-) or OVX ERαAF-2(0) mice. OVX ERαAF-1(0) mice displayed a normal E2 response in cortical bone but no E2 response in trabecular bone. Although E2 treatment increased the uterine and liver weights and reduced the thymus weight in OVX WT mice, no effect was seen on these parameters in OVX ERα(-/-) or OVX ERαAF-2(0) mice. The effect of E2 in OVX ERαAF-1(0) mice was tissue-dependent, with no or weak E2 response on thymus and uterine weights but a normal response on liver weight. In conclusion, ERα AF-2 is required for the estrogenic effects on all parameters evaluated, whereas the role of ERα AF-1 is tissue-specific, with a crucial role in trabecular bone and uterus but not cortical bone. Selective ER modulators stimulating ERα with minimal activation of ERα AF-1 could retain beneficial actions in cortical bone, constituting 80% of the skeleton, while minimizing effects on reproductive organs.

Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2.

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-alpha expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the beta-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17beta-estradiol (E(2)) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E(2) was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E(2) suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E(2) inhibition. E(2) increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E(2) was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GHJAKSTAT pathway, in which SOCS-2 plays a central mechanistic role.

Fluoride and urea chewing gums in an intra-oral experimental caries model.

The aim of the present investigation was to evaluate the effect of sugar-free chewing gums containing fluoride (F) and urea in an intra-oral experimental caries model. Placebo chewing gums (without any active ingredient) and no gum served as controls. Fifteen subjects participated in a cross-over, single-blind study. Demineralised enamel and dentine blocks were embedded in circular plastic discs and bonded to the buccal surfaces of the lower canines and first premolars. The discs were removed and analysed using transversal microradiography after each of the six 4-week periods during which the subjects used either test or placebo products or no product. The results revealed that frequent use of sugar-free chewing gum is sufficient to inhibit further demineralisation of previously demineralised enamel and dentine specimens in the oral cavity. Comparing F, urea and placebo gums, the data showed that there was little or no difference between the products, except for an inhibitory effect on the chewing side of the dentition after using F chewing gums.

A transgenic model to determine the physiological role of liver-derived insulin-like growth factor I.

Insulin-like growth factor-I (IGF-I) has important growth promoting and metabolic effects and is expressed in virtually every tissue of the body. The highest expression is found in the liver but the physiological role of liver-derived IGF-I is unknown. It has been difficult to separate the endocrine effects of liver-derived IGF-I from the autocrine/paracrine effects of locally produced IGF-I in peripheral tissues. Therefore, we have developed a mouse model with a liver-specific inducible deletion of the IGF-I gene. The liver-IGF-I deficient mouse have dramatically reduced (>80%) serum IGF-I levels, demonstrating that the major part of serum IGF-I is liver-derived. Surprisingly, liver-IGF-I deficient mice demonstrate a normal appendicular skeletal growth up to at least 12 months of age despite the dramatic decrease in circulating IGF-I levels, indicating that liver-derived IGF-I is not required for appendicular skeletal growth. However, the adult axial skeletal growth is clearly reduced in the liver-IGF-I deficient mice. Furthermore, the amount of cortical bone is reduced due to decreased radial growth of the cortical bone while the amount of trabecular bone is unchanged in the liver-IGF-I deficient mice. The decreased levels of circulating IGF-I are associated with increased serum levels of growth hormone (GH), indicating a role for liver-derived IGF-I in the negative feedback regulation of GH secretion. Measurements of factors regulating GH-secretion in the pituitary and in the hypothalamus revealed an increased expression of growth hormone releasing hormone (GHRH) and growth hormone secretagogue (GHS) receptors in the pituitary of liver-IGF-I deficient mice. This in turn results in an increased sensitivity to systemically administered GHRH and GHS, demonstrating that the regulatory action of liver-derived IGF-I on GH secretion is at the pituitary rather than at the hypothalamic level. The liver is an important metabolic organ and liver-IGF-I deficient mice are markedly hyperinsulinemic and yet normoglycemic, consistent with an adequately compensated insulin resistance. Interestingly, liver-IGF-I deficient mice display a reduced age-dependent fat mass accumulation compared with control mice. In conclusion, liver-derived IGF-I is important for carbohydrate- and lipid-metabolism and for the regulation of GH-secretion at the pituitary level. Furthermore, it regulates adult axial skeletal growth and cortical radial growth while it is not required for appendicular skeletal growth.

Body fat content can be predicted in vivo in mice using a modified dual-energy X-ray absorptiometry technique.

The introduction of transgenic mice as animal models in medical research has increased the need for methods to study the phenotype of mice. The aim of the present study was to develop and evaluate a method for in vivo prediction of fat content in living mice. We combined a modified dual-energy X-ray technique with an image analysis procedure. This combined procedure calculates the percentage of fat area, defined as the percentage of the total area of the mice consisting of >50% fat. A high correlation between the percentage of fat area and dissected adipose tissue was seen in both male and female mice (males, r = 0.92, P < 0.001; females, r = 0.88, P < 0.001). A high correlation was also seen between the percentage of fat area and serum levels of leptin (males, r = 0.95, P < 0.001; females, r = 0.86, P < 0.001). An additional experiment demonstrated a very strong correlation between the percentage of fat area and total body fat as determined by chemical extraction (r = 0.97, P < 0.001). In summary, the percentage of fat area, as measured with the dual-energy X-ray/image combined procedure, provides a good in vivo estimation of total body fat content in mice.

Metabolic functions of liver-derived (endocrine) insulin-like growth factor I.

Until now it has been difficult to determine the relative importance of locally produced (autocrine/paracrine) versus systemically derived (endocrine) insulin-like growth factor I (IGF-I) in the intact organism. We recently eliminated IGF-I production in the livers of mice using the Cre/loxP recombination system. These mice displayed a reduction in serum IGF-I levels of more than 80%, but demonstrated normal body growth, suggesting that autocrine/paracrine-acting IGF-I, but not endocrine-acting IGF-I, regulates body growth. Long-term metabolic studies of mice in which IGF-I production had been inactivated in the liver, have shown that the mice have decreased fat mass, but increased serum levels of insulin and cholesterol. Despite the marked increase in plasma insulin following glucose administration, the glucose elimination was not altered in these animals. Thus, the mice showed an adequately compensated insulin resistance. In conclusion, liver-derived or endocrine IGF-I is not required for post-natal statural growth, but seems to be of vital importance for normal carbohydrate and lipid metabolism.

Liver-derived IGF-I regulates GH secretion at the pituitary level in mice.

We have reported that liver-specific deletion of IGF-I in mice (LI-IGF-I-/-) results in decreased circulating IGF-I and increased GH levels. In the present study, we determined how elimination of hepatic IGF-I modifies the hypothalamic-pituitary GH axis to enhance GH secretion. The pituitary mRNA levels of GH releasing factor (GHRF) receptor and GH secretagogue (GHS) receptor were increased in LI-IGF-I-/- mice, and in line with this, their GH response to ip injections of GHRF and GHS was increased. Expression of mRNA for pituitary somatostatin receptors, hypothalamic GHRF, somatostatin, and neuropeptide Y was not altered in LI-IGF-I-/- mice, whereas hypothalamic IGF-I expression was increased. Changes in hepatic expression of major urinary protein and the PRL receptor in male LI-IGF-I-/- mice indicated an altered GH release pattern most consistent with enhanced GH trough levels. Liver weight was enhanced in LI-IGF-I-/- mice of both genders. In conclusion, loss of liver-derived IGF-I enhances GH release by increasing expression of pituitary GHRF and GHS receptors. The enhanced GH release in turn affects several liver parameters, in line with the existence of a pituitary-liver axis.

The somatomedin hypothesis revisited in a transgenic model.

Studies of insulin-like growth factor I (IGF-I) gene knockout mice models have clearly shown that IGF-I is necessary for prenatal as well as postnatal body growth in mice. Clinical studies of a patient with an IGF-I gene defect which caused complete absence of IGF-I, verified that it is important for intrauterine and postnatal growth. Recent studies of mice with liver-specific and inducible IGF-I gene knockout indicated that liver-derived IGF-I is not necessary for postnatal body growth, although serum IGF-I levels are decreased by more than 80% in these mice. Therefore, extrahepatic IGF-I is sufficient for maintenance of postnatal body growth in mice. Further investigations are needed to assess whether liver-derived circulating IGF-I is essential for other biological functions.

Liver-derived IGF-I is of importance for normal carbohydrate and lipid metabolism.

IGF-I is important for postnatal body growth and exhibits insulin-like effects on carbohydrate metabolism. The function of liver-derived IGF-I is still not established, although we previously demonstrated that liver-derived IGF-I is not required for postnatal body growth. Mice whose IGF-I gene in the liver was inactivated at 24 days of age were used to investigate the long-term role of liver-derived IGF-I for carbohydrate and lipid metabolism. Serum levels of leptin in these mice were increased by >100% at 3 months of age, whereas the fat mass of the mice was decreased by 25% at 13 months of age. The mice became markedly hyperinsulinemic and yet normoglycemic, indicating an adequately compensated insulin resistance. Furthermore, they had increased serum levels of cholesterol. We conclude that liver-derived IGF-I is of importance for carbohydrate and lipid metabolism.

Possible roles of insulin-like growth factor in regulation of physiological and pathophysiological liver growth.

BACKGROUND/AIMS: Almost all circulating insulin-like growth factor-1 (IGF-1) is produced and secreted from the liver. However, the possible role of IGF-1 in local regulation of liver functions including liver growth is unclear. In the present study, we investigated the role of IGF-1 on liver growth in vivo and in hepatic stellate cell function in vitro. RESULTS: Liver-specific knock-out of the IGF-1 gene by use of the cre-loxP system caused enhanced liver growth, possibly reflecting increased growth hormone (GH) secretion due to decreased negative feedback by IGF-1. Studies on cultured rat hepatic stellate cells (HSC) showed that IGF-1 and hepatocyte-conditioned medium (PCcM) time- and dose-dependently increased hepatocyte growth factor (HGF) mRNA and HGF immunoreactivity. IGF-1 and PCcM also enhanced DNA synthesis in the HSC cultures. The PCcM did not contain bioactive IGF-1 and was also able to stimulate proliferation when prepared under serum- and hormone-free conditions. CONCLUSION: In vivo results show that IGF-1 is not essential for normal growth of the intact liver. The in vitro results indicate that both IGF-1 and IGF- 1-independent factor(s) from hepatocytes can stimulate HGF production by HSC. It remains to be investigated whether these effects are of importance for liver regeneration or pathological conditions.

How to improve oral fluoride retention?

This paper reviews current ideas relating to oral fluoride retention after topical applications of various fluoride preparations. The oral fluoride reservoirs, which serve as a store for fluoride, gradually releasing its content into saliva, will be discussed. The present paper also deals with fluoride kinetics in the oral cavity. The answer to the title of the paper has, however, not yet been found but remains a goal for researchers studying caries prophylaxis worldwide in the search for possible ways of increasing and improving oral fluoride retention.

The influence of rinsing routines on fluoride retention after toothbrushing.

OBJECTIVE: An important determinant of the anticaries effect of brushing with a fluoride dentifrice is the retention of fluoride in the oral fluids. This study measured the influence of various post-brushing rinsing routines on fluoride retention. SETTING: The Faculty of Odontology, Göteborg University, Sweden. SUBJECTS: Twenty-one healthy dental students, aged 23 +/- 2 yr. DESIGN: A randomised, cross-over protocol, comparing various post-brushing rinsing procedures, with the following variables: volume of rinsing water (5 mL vs. 10 x 3 mL) and duration of rinsing (10 vs. 60 s). Samples of whole saliva, interdental fluid and interdental plaque were analysed for fluoride content. RESULTS: The duration of rinsing did not influence fluoride retention in whole saliva, interdental fluid or interdental plaque. Rinsing with a higher volume of water substantially decreased fluoride retention in all samples. CONCLUSION: Brushing with a fluoride dentifrice should be followed by rinsing procedures which enhance the retention of fluoride. Rinsing with large volumes of water decreases fluoride retention in the oral fluids, and should be discouraged.

Autoantibodies against the angiotensin receptor (AT1) in patients with hypertension.

Sera from patients with malignant essential hypertension (n = 14), malignant secondary hypertension mainly attributable to renovascular diseases (n = 12) and renovascular diseases without malignant hypertension (n = 11) and from normotensive healthy blood donors (n = 35) were studied for the presence of autoantibodies against G-protein-coupled cardiovascular receptors. Autoantibodies against the angiotensin II receptor (AT1) were detected in 14, 33, 18 and 14% of patients with malignant essential hypertension, malignant secondary hypertension, renovascular diseases and control patients, respectively. Sensitivity of the enzyme immunoassay was assessed as 5 microg/ml IgG. Patients did not show antibodies against bradykinin (B2) or angiotensin II subtype 2 (AT2) receptors. Autoantibodies affinity-purified from positive patients localized AT receptors in Chinese hamster ovary transfected cells, and displayed a positive chronotropic effect on cultured neonatal rat cardiomyocytes. These results demonstrate the existence of autoantibodies against a functional extracellular domain of human AT1 receptors in patients with malignant hypertension, and suggest that these autoantibodies might be involved in the pathogenesis of malignant hypertension.

The relative importance of endocrine versus autocrine/paracrine insulin-like growth factor-I in the regulation of body growth.

Body growth is regulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I). The classical somatomedin hypothesis of this regulation is that most IGF-I in the blood originates in the liver and that body growth is controlled by the concentration of IGF-I in the blood. We have recently abolished IGF-I production in the livers of mice by using the Cre/loxP recombination system. These mice displayed a more than 75% reduction in serum IGF-I associated with increased serum levels of GH. In contrast, they demonstrated a normal postnatal growth, indicating that extrahepatic, autocrine/paracrine-acting IGF-I is the main determinant of postnatal growth. Thus, the "classical" somatomedin hypothesis needs revision. We propose the "dual somatomedin hypothesis" according to which: (1) autocrine/paracrine IGF-I is the main determinant of postnatal body growth and (2) liver-derived, endocrine-acting, IGF-I exerts negative feedback on GH secretion and possibly also exerts other effects on carbohydrate and lipid metabolism.

Disproportional skeletal growth and markedly decreased bone mineral content in growth hormone receptor -/- mice.

Growth hormone (GH) is important for skeletal growth as well as for a normal bone metabolism in adults. The skeletal growth and adult bone metabolism was studied in mice with an inactivated growth hormone receptor (GHR) gene. The lengths of femur, tibia, and crown-rump were, as expected, decreased in GHR-/- mice. Unexpectedly, GHR-/- mice displayed disproportional skeletal growth reflected by decreased femur/crown-rump and femur/tibia ratios. GHR-/- mice demonstrated decreased width of the growth plates in the long bones and disturbed ossification of the proximal tibial epiphysis. Furthermore, the area bone mineral density (BMD) as well as the bone mineral content (BMC)/body weight were markedly decreased in GHR-/- mice. The decrease in BMC in GHR-/- mice was not due to decreased trabecular volumetric BMD but to a decreased cross-sectional cortical bone area In conclusion, GHR-/- mice demonstrate disproportional skeletal growth and markedly decreased bone mineral content.

Liver-derived insulin-like growth factor I (IGF-I) is the principal source of IGF-I in blood but is not required for postnatal body growth in mice.

The body growth of animals is regulated by growth hormone and IGF-I. The classical theory of this regulation is that most IGF-I in the blood originates in the liver and that body growth is controlled by the concentration of IGF-I in the blood. We have abolished IGF-I production in the livers of mice by using the Cre/loxP recombination system. These mice demonstrated complete inactivation of the IGF-I gene in the hepatocytes. Although the liver accounts for less than 5% of body mass, the concentration of IGF-I in the serum was reduced by 75%. This finding confirms that the liver is the principal source of IGF-I in the blood. However, the reduction in serum IGF-I concentration had no discernible effect on postnatal body growth. We conclude that postnatal body growth is preserved despite complete absence of IGF-I production by the hepatocytes.

Osteoprotegerin mRNA is increased by interleukin-1 alpha in the human osteosarcoma cell line MG-63 and in human osteoblast-like cells.

Osteoprotegerin (OPG) is a soluble receptor for the Osteoprotegerin-Ligand (OPGL) which is expressed on osteoblasts and mediates the signal for osteoclast differentiation. In the present study we demonstrate that OPG mRNA levels in MG-63 cells are increased in a dose-dependent manner after 8 h of treatment with IL-1 alpha (338 +/- 53% over control at 25 U/ml). Interleukin-6 (IL-6), under similar culture conditions, does not affect OPG mRNA levels. Time-course studies show that IL-1 alpha (25 U/ml) causes a transient increase of OPG mRNA levels in MG-63 cells, peaking after 4 h of treatment. An increase of the OPG transcript occurs in hOB cells at 0.5 h which is still present after 24 h of IL-1 alpha treatment. In MG-63 cells neither basal-nor IL-1 alpha-induced OPG mRNA levels are altered by the translational inhibitor cycloheximide. These results suggest that expression of OPG in osteoblasts may be regulated by IL-1 alpha.