Resistance training exercises for obese and non-obese individuals living in high-altitude regions utilizing biochemical markers-A controlled trial.

BACKGROUND: High-altitude disease prevalence varies according to types of exposure and the effects of hypoxic and hypobaric environments, with the result that people at high altitudes present many different physiological responses. AIMS: The research aims to analyze the effects of resistance training (RT) exercises at high altitudes on obese subjects and to explain the determinants that make obese people more susceptible to various chronic illnesses such as diabetes mellitus, hypertension, asthma, etc. METHODS: This study recruited 225 subjects living in the high-altitude region of Aseer, Saudi Arabia, and divided them into three groups. The first two groups consisted of obese people, out of which one group received RT and one did not. The third group consisted of average-weight individuals, according to their BMI, who received RT. Biochemical parameters were checked for all three groups before commencing with the RT and at the 4th and 8th week to measure the effects of the exercise. RESULTS: Mean and standard deviations of the demographic variables: age was 34.2 ± 8.9 years, weight was 69.3 ± 8.5 kg, and height was 1.6 ± 0.06 meters. RT had a significant effect on the total levels of cholesterol, triglycerides, HDL-cholesterol, LDL-cholesterol, adiponectin, interleukin-6, and testosterone. Post-hoc comparisons using the Fisher's Least Significant Difference test indicated that the mean scores between the groups differed significantly. CONCLUSION: Our findings show that RT would be a useful and practical substitute to improve the health status of obese patients. It helps to decrease body fat and to improve lipid profiles and hormonal control.

Expression of Sulf1 and Sulf2 in cartilage, bone and endochondral fracture healing.

SULF1/SULF2 enzymes regulate cell signalling that impacts the growth and differentiation of many tissues. To determine their possible role in cartilage and bone growth or repair, their expression was examined during development and bone fracture healing using RT-PCR and immunochemical analyses. Examination of epiphyseal growth plates revealed differential, inverse patterns of SULF1 and SULF2 expressions, with the former enriched in quiescent and the latter in hypertrophic chondrocyte zones. Markedly higher levels of both SULFs, however, were expressed in osteoblasts actively forming bone when compared with proliferating pre-osteoblasts in the periosteum or the entombed osteocytes which express the lowest levels. The increased expression of Sulf1 and Sulf2 in differentiating osteoblasts was further confirmed by RT-PCR analysis of mRNA levels in rat calvarial osteoblast cultures. SULF1 and SULF2 were expressed in most foetal articular chondrocytes but down-regulated in a larger subset of cells in the post-natal articular cartilage. Unlike adult articular chondrocytes, SULF1/SULF2 expression varied markedly in post-natal hypertrophic chondrocytes in the growth plate, with very high SULF2 expression compared with SULF1 apparent during neonatal growth in both primary and secondary centres of ossification. Similarly, hypertrophic chondrocytes expressed greatly higher levels of SULF2 but not SULF1 during bone fracture healing. SULF2 expression unlike SULF1 also spread to the calcifying matrix around the hypertrophic chondrocytes indicating its possible ligand inhibiting role through HSPG desulphation. Higher levels of SULF2 in both developing and healing bone closely correlated with parallel increases in hedgehog signalling analysed by ptc1 receptor expression.

Inhibition of the spindle assembly checkpoint kinase TTK enhances the efficacy of docetaxel in a triple-negative breast cancer model.

BACKGROUND: Triple-negative breast cancers (TNBC) are considered the most aggressive type of breast cancer, for which no targeted therapy exists at the moment. These tumors are characterized by having a high degree of chromosome instability and often overexpress the spindle assembly checkpoint kinase TTK. To explore the potential of TTK inhibition as a targeted therapy in TNBC, we developed a highly potent and selective small molecule inhibitor of TTK, NTRC 0066-0. RESULTS AND CONCLUSIONS: The compound is characterized by long residence time on the target and inhibits the proliferation of a wide variety of human cancer cell lines with potency in the same range as marketed cytotoxic agents. In cell lines and in mice, NTRC 0066-0 inhibits the phosphorylation of a TTK substrate and induces chromosome missegregation. NTRC 0066-0 inhibits tumor growth in MDA-MB-231 xenografts as a single agent after oral application. To address the effect of the inhibitor in breast cancer, we used a well-defined mouse model that spontaneously develops breast tumors that share key morphologic and molecular features with human TNBC. Our studies show that combination of NTRC 0066-0 with a therapeutic dose of docetaxel resulted in doubling of mouse survival and extended tumor remission, without toxicity. Furthermore, we observed that treatment efficacy is only achieved upon co-administration of the two compounds, which suggests a synergistic in vivo effect. Therefore, we propose TTK inhibition as a novel therapeutic target for neoadjuvant therapy in TNBC.

The anti-diabetic drug metformin does not affect bone mass in vivo or fracture healing.

SUMMARY: The present study shows no adverse effects of the anti-diabetic drug metformin on bone mass and fracture healing in rodents but demonstrates that metformin is not osteogenic in vivo, as previously proposed. INTRODUCTION: In view of the increased incidence of fractures in patients with type 2 diabetes mellitus (T2DM), we investigated the effects of metformin, a widely used T2DM therapy, on bone mass and fracture healing in vivo using two different rodent models and modes of metformin administration. METHODS: We first subjected 12-week-old female C57BL/6 mice to ovariectomy (OVX). Four weeks after OVX, mice received either saline or metformin administered by gavage (100 mg/kg/daily). After 4 weeks of treatment, bone micro-architecture and cellular activity were determined in tibia by micro-CT and bone histomorphometry. In another experiment, female Wistar rats aged 3 months were given only water or metformin for 8 weeks via the drinking water (2 mg/ml). After 4 weeks of treatment, a mid-diaphyseal osteotomy was performed in the left femur. Rats were sacrificed 4 weeks after osteotomy and bone architecture analysed by micro-CT in the right tibia while fracture healing and callus volume were determined in the left femur by X-ray analysis and micro-CT, respectively. RESULTS: In both models, our results show no significant differences in cortical and trabecular bone architecture in metformin-treated rodents compared to saline. Metformin had no effect on bone resorption but reduced bone formation rate in trabecular bone. Mean X-ray scores assessed on control and metformin fractures showed no significant differences of healing between the groups. Fracture callus volume and mineral content after 4 weeks were similar in both groups. CONCLUSIONS: Our results indicate that metformin has no effect on bone mass in vivo or fracture healing in rodents.

Mechanical loading-related changes in osteocyte sclerostin expression in mice are more closely associated with the subsequent osteogenic response than the peak strains engendered.

UNLABELLED: Osteocyte sclerostin is regulated by loading and disuse in mouse tibiae but is more closely related to subsequent local osteogenesis than the peak strains engendered. INTRODUCTION: The purpose of this study was to assess the relationship between loading-related change in osteocyte sclerostin expression, local strain magnitude, and local bone modeling/remodeling. METHODS: The right tibiae of 19-week-old female C57BL/6 mice were subjected to non-invasive, dynamic axial loading and/or to sciatic neurectomy-induced disuse. The sclerostin status of osteocytes was evaluated immunohistochemically, changes in bone mass by micro-computed tomography, new bone formation by histomorphometry, and loading-induced strain by strain gauges and finite element analysis. RESULTS: In cortical bone of the tibial shaft, loading engendered strains of similar peak magnitude proximally and distally. Proximally, sclerostin-positive osteocytes decreased and new bone formation increased. Distally, there was neither decrease in sclerostin-positive osteocytes nor increased osteogenesis. In trabecular bone of the proximal secondary spongiosa, loading decreased sclerostin-positive osteocytes and increased bone volume. Neither occurred in the primary spongiosa. Disuse increased sclerostin-positive osteocytes and decreased bone volume at all four sites. Loading reversed this sclerostin upregulation to a level below baseline in the proximal cortex and secondary spongiosa. CONCLUSION: Loading-related sclerostin downregulation in osteocytes of the mouse tibia is associated preferentially with regions where new bone formation is stimulated rather than where high peak strains are engendered. The mechanisms involved remain unclear, but could relate to peak surface strains not accurately reflecting the strain-related osteogenic stimulus or that sclerostin regulation occurs after sufficient signal processing to distinguish between local osteogenic and non-osteogenic responses.

Increased sensitivity to anticancer drugs and decreased inflammatory response in mice lacking the multidrug resistance-associated protein.

The multidrug resistance-associated protein (MRP) mediates the cellular excretion of many drugs, glutathione S-conjugates (GS-X) of lipophilic xenobiotics and endogenous cysteinyl leukotrienes. Increased MRP levels in tumor cells can cause multidrug resistance (MDR) by decreasing the intracellular drug concentration. The physiological role or roles of MRP remain ill-defined, however. We have generated MRP-deficient mice by using embryonic stem cell technology. Mice homozygous for the mrp mutant allele, mrp-/-, are viable and fertile, but their response to an inflammatory stimulus is impaired. We attribute this defect to a decreased secretion of leukotriene C4 (LTC4) from leukotriene-synthesizing cells. Moreover, the mrp-/- mice are hypersensitive to the anticancer drug etoposide. The phenotype of mrp-/- mice is consistent with a role for MRP as the main LTC4-exporter in leukotriene-synthesizing cells, and as an important drug exporter in drug-sensitive cells. Our results suggest that this ubiquitous GS-X pump is dispensable in mice, making treatment of MDR with MRP-specific reversal agents potentially feasible.

Congenital jaundice in rats with a mutation in a multidrug resistance-associated protein gene.

The human Dubin-Johnson syndrome and its animal model, the TR(-) rat, are characterized by a chronic conjugated hyperbilirubinemia. TR(-) rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homolog of the human multidrug resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR(-) rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR(-) phenotype.

Non-genetic factors affecting pre-weaning growth and morphometric traits in Assam Hill goat.

AIM: This study aimed to determine the genetic and non-genetic factors affecting pre-weaning body weight (BW) and morphometry in Assam Hill goat along with the genetic parameters. MATERIALS AND METHODS: The detailed information in respect of BW and body measurements of 960 animals at birth and 3 months of age belonging to three different populations of Assam Hill goat maintained at field units, namely, Batabari, Nahira, and Tetelia under "All India Coordinated Research Project on Goat Improvement" were utilized in the present study. The data were analyzed using least squares technique. RESULTS: The least squares means for BW, height at withers (HW), heart girth (HG), and body length (BL) were 1.166±0.008 kg, 26.198±0.070 cm, 26.695±0.096 cm, and 29.482±0.119 cm at birth and 4.590±0.083 kg, 36.850±0.105 cm, 40.741±0.115 cm, and 39.703±0.108 cm at 3 months of age, respectively. Location had a significant effect on BW, HW, and BL at both birth and 3 months and on HG at 3 months of age. Season of birth exerted significant effect only on BL at birth, whereas the significant effect of sex was observed on HG and BL at 3 months of age. The heritability estimates for BW and body measurements were moderate indicating the scope of selection. The phenotypic and genetic correlations among BWs and body measurements at birth and 3 months of age were positive in direction and high in magnitude. CONCLUSION: On the basis of the present findings, it could be concluded that the weaning weight of kids can be considered for the selection of parent stock to increase productivity and eventually the economic efficiency. Further, animals with higher body measurements at initial phases of growth will perform better with respect to even BW at later stages of growth.

Basolateral localization and export activity of the human multidrug resistance-associated protein in polarized pig kidney cells.

The human multidrug resistance-associated protein MRP confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Recent evidence indicates that MRP can also transport glutathione S-conjugates across membranes. To study the transport properties of MRP in intact cells, we have expressed human MRP cDNA in the polarized pig kidney epithelial cell line LLC-PK1. MRP mainly localized to the basolateral plasma membrane of these cells, and not to the apical membrane, as determined by immunocytochemistry using confocal laser scanning and electron microscopy. In accordance with this localization, MRP caused increased transport of the glutathione S-conjugate S-(2, 4-dinitrophenyl)-glutathione and of the anticancer drug daunorubicin to the basal side of the epithelial cell layer. Sulfinpyrazone and probenecid, known inhibitors of multispecific organic anion transport, inhibited this basolateral transport, but not the apical transport of daunorubicin mediated by the apically localized human MDR1 P-glycoprotein in MDR1-transfected LLC-PK1 cells. Probenecid and sulfinpyrazone may therefore be useful lead compounds for the development of clinical reversal agents specific for MRP-mediated drug resistance.

Immunochemical detection of the multidrug resistance-associated protein MRP in human multidrug-resistant tumor cells by monoclonal antibodies.

We have generated rat and murine monoclonal antibodies against multidrug resistance-associated protein (MRP), a M(r) 180,000-195,000 membrane glycoprotein involved in a non-P-glycoprotein multidrug resistance of human tumor cells. The antibodies were raised against two different segments of MRP and found to be suitable for protein blot analyses, immunohistochemical and cytochemical studies, as well as flow cytometry of permeabilized cells. The antibodies do not cross-react with the human P-glycoproteins. Immunocytochemistry using MRP-overexpressing tumor cells of different histogenetic origins showed that MRP is predominantly located in the plasma membrane. Immunoelectron microscopy confirmed the plasma membrane location of MRP. The MRP antibodies provide a sensitive and specific tool for studies on MRP-mediated multidrug resistance.

Analysis of the expression of MRP, the gene for a new putative transmembrane drug transporter, in human multidrug resistant lung cancer cell lines.

Human cells can become multidrug resistant (MDR) by an increase in the activity of the MDR1 P-glycoprotein or by other, as yet unknown mechanisms, referred to as non-P-glycoprotein mediated MDR (non-Pgp MDR). S. P. C. Cole et al. [Science (Washington DC), 258: 1650-1654, 1992] recently reported that in two cell lines non-Pgp MDR was associated with the overexpression of a new putative membrane transporter gene, MRP. Using an RNase protection assay we have analyzed the expression of MRP in non-Pgp MDR sublines of the human lung cancer cell lines SW-1573 (non-small cell lung cancer) and GLC4 (small cell lung cancer). In all of ten SW-1573 derived lines examined the MRP mRNA level was equal to that in the parental line, whereas MRP was 25-fold overexpressed in a resistant subline of GLC4. We conclude that overexpression of MRP cannot account for all forms of non-Pgp MDR.

MEK and PI3K-AKT inhibitors synergistically block activated IL7 receptor signaling in T-cell acute lymphoblastic leukemia.

We identified mutations in the IL7Ra gene or in genes encoding the downstream signaling molecules JAK1, JAK3, STAT5B, N-RAS, K-RAS, NF1, AKT and PTEN in 49% of patients with pediatric T-cell acute lymphoblastic leukemia (T-ALL). Strikingly, these mutations (except RAS/NF1) were mutually exclusive, suggesting that they each cause the aberrant activation of a common downstream target. Expressing these mutant signaling molecules-but not their wild-type counterparts-rendered Ba/F3 cells independent of IL3 by activating the RAS-MEK-ERK and PI3K-AKT pathways. Interestingly, cells expressing either IL7Ra or JAK mutants are sensitive to JAK inhibitors, but respond less robustly to inhibitors of the downstream RAS-MEK-ERK and PI3K-AKT-mTOR pathways, indicating that inhibiting only one downstream pathway is not sufficient. Here, we show that inhibiting both the MEK and PI3K-AKT pathways synergistically prevents the proliferation of BaF3 cells expressing mutant IL7Ra, JAK and RAS. Furthermore, combined inhibition of MEK and PI3K/AKT was cytotoxic to samples obtained from 6 out of 11 primary T-ALL patients, including 1 patient who had no mutations in the IL7R signaling pathway. Taken together, these results suggest that the potent cytotoxic effects of inhibiting both MEK and PI3K/AKT should be investigated further as a therapeutic option using leukemia xenograft models.

Regulation of expression of the genome of bacteriophage M13. Gene V protein regulated translation of the mRNAs encoded by genes I, III, V and X.

With the aid of a binary plasmid in vivo testsystem it was demonstrated that the single-stranded DNA binding protein encoded by gene V of bacteriophage M13 not only regulates the synthesis of its cognate DNA replication proteins at the level of translation, but also of the assembly proteins and the coat proteins encoded by genes I and II, respectively. Furthermore, gene V protein functions as a translational autoregulator of its own synthesis. Comparison of the mRNA levels of genes I and X in the presence and absence of wild-type gene V protein indicated that gene V protein augments the physical stability of these mRNAs. The expression of the Escherichia coli beta-galactosidase gene and of a gene X mutant containing a deletion in the nontranslated mRNA leader sequence was not influenced by gene V protein, lending support to the conclusion that gene V protein exerts its regulatory effect via a specific nucleotide sequence in the leader sequences of the respective M13 mRNAs. We conclude that gene V protein functions as a master regulatory protein of the expression and replication of the M13 genome.

Classical and novel forms of multidrug resistance and the physiological functions of P-glycoproteins in mammals.

In this paper, we review recent work on multidrug resistance (MDR) in Amsterdam. We have generated mice homozygous for a disruption of one of their P-glycoprotein (Pgp) genes. The mutations do not interfere with viability or fertility, showing that these Pgps have no indispensable role in early development or metabolism. Mice homozygous for a disruption of their mdr2 gene, however, develop liver disease and this appears to be due to their complete inability to secrete phospholipids into bile. This suggests that the mdr2 Pgp (and, by inference, its human MDR3 homologue) is essential for translocating phospholipids through the hepatocyte canalicular membrane in which this Pgp is located. These and other results show the importance of the genetic approach for studying drug metabolism. MDR is not only caused by increased activity of Pgps. When the human non-small cell lung carcinoma cell line SW-1573 is selected in vitro for low level doxorubicin resistance, the resistant variants are nearly always multidrug resistant, but this is not due to increased Pgp activity. Only when resistance is pushed to higher levels does activation of the MDR1 Pgp gene occur. This suggests that clinically relevant levels of drug resistance in some cells may be caused predominantly by non-Pgp-mediated drug resistance mechanisms. The protein responsible for MDR in the SW-1573 cells has not yet been identified and experiments are in progress to find the gene encoding it.

Expression of the multidrug resistance-associated protein (MRP) in acute and chronic leukemias.

We determined the expression of the multidrug resistance-associated protein (MRP), a new putative transmembrane drug transporter, in peripheral blood cells from healthy volunteers as well as from 60 patients with acute or chronic leukemia, using an RNase protection assay. MRP appeared to be ubiquitously expressed at low levels in all nonmalignant hemopoietic cell types, reflecting its basal constitutive expression. In acute myelocytic leukemia (AML) (n = 16), one of nine untreated patients and two of seven patients with prior chemotherapy showed significant hyperexpression of MRP. In chronic lymphocytic leukemia (CLL) (n = 21), either treated (n = 8) or untreated (n = 13), a high percentage (15 of 21: 71% had relatively high expression levels of the MRP gene. In contrast, low MRP expression levels were detected in acute lymphocytic leukemia (n = 14), and in chronic myelocytic leukemia (n = 9). DNA analysis by Southern blotting did not reveal amplification of the MRP gene in the leukemia samples, including those with elevated MRP mRNA levels. We conclude that relatively high expression of MRP is occasionally observed in AML and at high frequency in CLL, irrespective of treatment, probably due to transcriptional activation and/or increased mRNA stability.

Expression of the multidrug resistance-associated protein (MRP) gene in human cancers.

We determined the expression of a newly recognized drug resistance gene, the multidrug resistance-associated protein (MRP) gene, [Cole et al., Science (Washington DC), 258: 1650-1654, 1992], in normal human tissues and in >370 human tumor biopsies using a quantitative RNase protection assay and immunohistochemistry. MRP mRNA appeared to be ubiquitously expressed at low levels in all normal tissues, including peripheral blood, the endocrine glands (adrenal and thyroid), striated muscle, the lymphoreticular system (spleen and tonsil), the digestive tract (salivary gland, esophagus, liver, gall bladder, pancreas, and colon), the respiratory tract (lung), and the urogenital tract (kidney, bladder, testis, and ovary). The human cancers analyzed could be divided into three groups with regard to MRP expression. Group 1 consists of tumors that often exhibit high to very high MRP mRNA levels (e.g., chronic lymphocytic leukemia). Group 2 comprises the tumors that often exhibit low, but occasionally exhibit high MRP mRNA expression (e.g., esophagus squamous cell carcinoma, non-small cell lung cancer, and acute myelocytic leukemia). Group 3 comprises the tumors with predominantly low levels of MRP mRNA, comparable to the levels found in normal tissues (e.g., other hematological malignancies, soft tissue sarcomas, melanoma, and cancers of the prostate, breast, kidney, bladder, testis, ovary, and colon). Using the MRP-specific mAbs MRPr1 and MRPm6, we confirmed the elevated MRP mRNA levels in tumor tissues by immunohistochemistry. We conclude that hyperexpression of MRP is observed in several human cancers, and that additional studies are needed to assess the clinical relevance of MRP.

Estrogen enhances the stimulation of bone collagen synthesis by loading and exogenous prostacyclin, but not prostaglandin E2, in organ cultures of rat ulnae.

The shafts of ulnae from 110 g male rats were cultured, and after a period of 5 h preincubation one of each pair of bones was either loaded cyclically (500 g, 1 Hz, 8 minutes) to produce physiologic strains (-1300 mu epsilon) or treated with exogenous prostacyclin (PGI2) or prostaglandin E2 (10(-6) M, 8 minutes) in the presence or absence of 17 beta-estradiol (10(-8) M). PGI2, PGE2, and loading stimulated almost immediate increases in glucose 6-phosphate dehydrogenase (G6PD) activity in osteocytes and osteoblasts. This increase was uniform throughout the section with exogenous PGs in the medium but was related to local strain magnitude in loading. Elevated G6PD levels in response to loading and PGI2 persisted for 18 h, by which time, ALP activity in surface osteoblasts was elevated and [3H]proline incorporation into collagen increased. PGE2 produced similar immediate and sustained increases in G6PD activity and [3H]proline incorporation after 18 h but no change in ALP activity. Bones cultured for 18 h with 17 beta-estradiol increased their [3H]proline incorporation, as did those loaded, and treated with PGI2 and PGE2. Loading and PGI2 but not PGE2 produced similar proportional increases in [3H]proline incorporation above the increased baseline of estradiol alone. These results suggest that estrogen and loading together produce a greater osteogenic response than either separately. If so, estrogen withdrawal would result in a rapid fall in bone mass to establish a new equilibrium appropriate to the reduced effectiveness of the loading-related stimulus. Such a fall in bone mass is a characteristic feature of estrogen withdrawal at the menopause.

Early strain-related changes in cultured embryonic chick tibiotarsi parallel those associated with adaptive modeling in vivo.

A model was developed for the application of cyclic mechanical loads to 17 day embryonic chick tibiotarsi in culture. A single 20 minute period of intermittent loading at 0.4 Hz, producing physiologic peak strains and strain rates, resulted in two peak strain magnitude-related responses that were previously reported in vivo: (1) a rapid increase in glucose 6-phosphate dehydrogenase activity in osteoblasts and osteocytes and (2) increased RNA synthesis, as shown by increased incorporation of [3H]uridine into extracted RNA. The RNA response was detectable 8 h following loading but was more pronounced by 24 h. Both responses were blocked by indomethacin (10(-6) M). These results demonstrate that embryonic chick bones in organ culture exhibit cellular responses to loading similar to those previously identified in adult canine cancellous bone cultures in vitro and adult avian cortical bone in vivo. These findings are consistent with a sequence of events between loading and new bone formation that includes an immediate strain magnitude-related, prostanoid-dependent increase in activity of the pentose monophosphate shunt in osteoblasts and osteocytes, followed by a similarly strain magnitude-related increase in RNA synthesis over the subsequent 24 h.

Early responses to dynamic strain change and prostaglandins in bone-derived cells in culture.

Mechanical loading of bone explants stimulates prostaglandin E2 (PGE2) and prostacyclin (PGI2) release and increases glucose 6-phosphate dehydrogenase (G6PD) activity. This response is blocked by indomethacin and imitated by exogenous PGs. In the experiments reported here, primary cultures of rat long bone-derived osteoblast-like cells were exposed to a dynamic strain and exogenous PGs in the culture dish. Strain (3400 mu epsilon, 600 cycles, 1 Hz) caused an immediate release of PGI2 into the culture medium but had no effect on PGE2. Strain also caused an increase in G6PD activity per cell and an increase in the smallest transcript of insulin-like growth factor II (IGF-II) (IGF-II T3) but had no effect on the expression of transforming growth factor-beta1 (TGF-beta1). Indomethacin inhibited strain-induced release of PGI2 and suppressed strain-induced stimulation of IGF-II T3 transcript. PGI2 (1 microM) increased G6PD activity and mRNA levels of all three transcripts of IGF-II but had no effect on the mRNA levels of IGF-I or TGF-beta1. PGE2 (1 microM) stimulated G6PD activity and caused a marked increase in IGF-I and the largest transcript of IGF-II (IGF-II T1) but had no effect on the IGF-II transcripts T2 and T3 or on TGF-beta1 mRNA levels. These findings show similarities in response between osteoblast-like cells strained in monolayer culture and bone cells in loaded bone explants in situ. They provide support for a role for IGF-II and PGI2 in the early strain-related response of osteoblasts in loading-related bone modeling/remodeling.

Mechanical loading and sex hormone interactions in organ cultures of rat ulna.

The separate and combined effects of loading and 17 beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) on [3H]thymidine and [3H]proline incorporation were investigated in cultured ulna shafts from male and female rats. Ulnae were cultured and loaded to produce physiological strains in the presence or absence of 10(-8) M E2 or DHT. Loading engendered similar increases in incorporation of [3H]thymidine and [3H]proline in male and female bones. E2 engendered greater increases in incorporation in females than in males, and DHT greater increases in males than in females. In males E2 with loading produced increases in both [3H]thymidine and [3H]proline incorporation, which approximated to the arithmetic addition of the increases due to E2 and loading separately. In females E2 with loading produced increases greater than those in males, and substantially greater than the addition of the effects of E2 and loading separately. Loading with DHT in males also showed additional [3H]thymidine and [3H]proline incorporation. In females there was additional incorporation of [3H]proline, but not [3H]thymidine. The location of incorporation of [3H]thymidine and [3H] proline was consistent with their level of incorporation reflecting periosteal osteogenesis, in which case the early osteogenic effects of sex hormones are gender-specific when acting alone and in combination with loading. In males the effects of estrogen and testosterone add to, but do not enhance, the osteogenic responses to loading. In females testosterone with loading produces an additional effect on [3H]proline incorporation but no greater effect than loading alone on that of [3H]thymidine. In contrast, estrogen and loading together produce a greater effect than the sum of the two influences separately. Because premenopausal bone mass will have been achieved under the influence of loading and estrogen acting together, these findings suggest that the bone loss which follows estrogen withdrawal may result, at least in part, from reduction in the effectiveness of the loading-related stimulus on bone cell activity. This stimulus is normally responsible for maintaining bone mass and architecture.