K. E. Govoni

The multi-functional role of insulin-like growth factor binding proteins in bone

The insulin-like growth factor (IGF) system is an important regulator of bone formation. The IGFs (IGF-I and IGF-II) are the most abundant growth factors produced by bone, and are regulated by their six high affinity binding proteins (IGFBPs). The IGFBPs are produced by osteoblasts and are responsible for transporting the IGFs and extending their half-lives. In general, IGFBP-1, -2, -4, and -6 inhibit and IGFBP-3 and –5 stimulate osteoblast function. IGFBP-4 and -5 are the most abundant IGFBPs produced by osteoblasts, and therefore they are the primary focus of this review. IGFBP-5 is an important stimulator of bone formation and may also function independently of IGFs. IGFBP-4 inhibits osteoblast function by sequestering IGF and preventing it from binding to its receptor. This review focuses on the specific IGF-dependent and IGF-independent roles of the IGFBPs in bone formation, as well as their potential mechanisms of action. In addition, discussion of the regulation of the IGFBPs by post-translational modification (i.e., proteolysis) has been included. Studies on the regulation of production and actions of IGFBPs suggest that the IGFBP system in bone is pleiotropic and capable of serving multiple effector inputs from systemic and local sources.

Genetic evidence that thyroid hormone is indispensable for prepubertal insulin-like growth factor–I expression and bone acquisition in mice

Understanding how bone growth is regulated by hormonal and mechanical factors during early growth periods is important for optimizing the attainment of peak bone mass to prevent or postpone the occurrence of fragility fractures later in life. Using genetic mouse models that are deficient in thyroid hormone (TH) (Tshr−/− and Duox2−/−), growth hormone (GH) (Ghrhrlit/lit), or both (Tshr−/−; Ghrhrlit/lit), we demonstrate that there is an important period prior to puberty when the effects of GH are surprisingly small and TH plays a critical role in the regulation of skeletal growth. Daily administration of T3/T4 during days 5 to 14, the time when serum levels of T3 increase rapidly in mice, rescued the skeletal deficit in TH‐deficient mice but not in mice lacking both TH and GH. However, treatment of double‐mutant mice with both GH and T3/T4 rescued the bone density deficit. Increased body fat in the TH‐deficient as well as TH/GH double‐mutant mice was rescued by T3/T4 treatment during days 5 to 14. In vitro studies in osteoblasts revealed that T3 in the presence of TH receptor (TR) α1 bound to a TH response element in intron 1 of the IGF‐I gene to stimulate transcription. In vivo studies using TRα and TRβ knockout mice revealed evidence for differential regulation of insulin‐like growth factor (IGF)‐I expression by the two receptors. Furthermore, blockade of IGF‐I action partially inhibited the biological effects of TH, thus suggesting that both IGF‐I–dependent and IGF‐I–independent mechanisms contribute to TH effects on prepubertal bone acquisition.

Age-Related Changes of the Somatotropic Axis in Cloned Holstein Calves

Abstract To determine if the development of the somatotropic axis in somatic clones (clones) is similar to that in heifers produced by artificial insemination (controls), serum samples were collected every 30 min for 6 h, once per month, for 7 mo from 4 clones generated from a 13-yr-old cow and from 4 age-matched controls. Average concentrations of growth hormone (GH) were not different between clones and controls, and GH concentrations declined over time in controls. Average concentrations of insulin-like growth factor I (IGF-I) were less in clones than controls, and IGF-I concentrations increased over time in both groups. Concentrations of IGF-binding protein 3 (IGFBP-3) were greater in controls than in clones and did not change over time. Average IGFBP-2 concentrations did not change over time and were not different between clones and controls. Clones and controls were challenged with GH-releasing hormone (GHRH) (3 μg/100 kg body weight) and somatostatin (somatotropin release-inhibiting factor [SRIF]) (1.87 and 5 μg/100 kg body weight) at 14 mo of age. GHRH-induced GH secretion was greater and SRIF inhibition of GHRH-induced GH was less in clones than in controls. We speculate that some of the differences between clones and controls in concentrations of GH, IGF-I, and IGFBP-3 may be related to the genetic merit of the animals. Although there were differences in concentrations of components of the somatotropic axis between these clones and their age-matched controls, the values recorded were all within the range reported for calves of similar ages.

Glutaredoxin 5 regulates osteoblast apoptosis by protecting against oxidative stress

There is now increasing evidence which suggests an important role for reactive oxygen species (ROS) in the pathogenesis of osteoporosis. However, little is known on the molecular components of the oxidative stress pathway or their functions in bone. In this study, we evaluated the role and mechanism of action of glutaredoxin (Grx) 5, a protein that is highly expressed in bone. Osteoblasts were transfected with Grx5 siRNA and treated with hydrogen peroxide (H(2)O(2)). Grx5 siRNA treatment increased apoptosis while Grx5 overexpression protected MC3T3-E1 cells against H(2)O(2) induced apoptosis and ROS formation. Grx5 deficiency results in impaired biogenesis of Fe-S cluster in yeast. Accordingly, activity of mitochondrial aconitase, whose activity is dependent on Fe-S cluster, decreased in Grx5 siRNA treated cells. Since reduced formation of Fe-S cluster would lead to increased level of free iron, a competitive inhibitor of manganese superoxide dismutase (MnSOD), we measured MnSOD activity in Grx5 deficient osteoblasts and found MnSOD activity was significantly reduced. The consequence of long term inhibition of Grx5 on osteoblast apoptosis was evaluated using lentiviral shRNA technology. Grx5 shRNA cells exhibited higher caspase activity and cardiolipin oxidation in the presence of H(2)O(2). MnSOD activity was rescued by the addition of MnCl(2) to Grx5 shRNA osteoblasts in the presence of H(2)O(2). Our findings are consistent with the hypothesis that Grx5 is an important determinant of osteoblast apoptosis and acts via a molecular pathway that involves regulation of ROS production, cardiolipin oxidation, caspase activity, Fe-S cluster formation, and MnSOD activity.

T-Box 3 Negatively Regulates Osteoblast Differentiation by Inhibiting Expression of Osterix and Runx2

T‐box (Tbx)3, a known transcriptional repressor, is a member of a family of transcription factors, which contain a highly homologous DNA binding domain known as the Tbx domain. Based on the knowledge that mutation of the Tbx3 gene results in limb malformation, Tbx3 regulates osteoblast proliferation and its expression increases during osteoblast differentiation, we predicted that Tbx3 is an important regulator of osteoblast cell functions. In this study, we evaluated the consequence of transgenic overexpression of Tbx3 on osteoblast differentiation. Retroviral overexpression increased Tbx3 expression >100‐fold at the mRNA and protein level. Overexpression of Tbx3 blocked mineralized nodule formation (28 ± 8 vs. 7 ± 1%) in MC3T3‐E1 cells. In support of these data, alkaline phosphatase (ALP) activity was reduced 33–70% (P < 0.05) in both MC3T3‐E1 cells and primary calvaria osteoblasts overexpressing Tbx3. In contrast, Tbx3 overexpression did not alter ALP activity in bone marrow stromal cells. Tbx3 overexpression blocked the increase in expression of key osteoblast marker genes, ALP, bone sialoprotein, and osteocalcin that occurs during normal osteoblast differentiation, but had little or no effect on expression of proliferation genes p53 and Myc. In addition, Tbx3 overexpression abolished increased osterix and runx2 expression observed during normal osteoblast differentiation, but the change in Msx1 and Msx2 expression over time was similar between control and Tbx3 overexpressing cells. Interestingly, osterix and runx2, but not Msx1 and Msx2, contain Tbx binding site in the regulatory region. Based on these data and our previous findings, we conclude that Tbx3 promotes proliferation and suppresses differentiation of osteoblasts and may be involved in regulating expression of key transcription factors involved in osteoblast differentiation. J. Cell. Biochem. 106: 482–490, 2009.

Prepubertal OVX increases IGF-I expression and bone accretion in C57BL/6J mice

It is generally well accepted that the pubertal surge in estrogen is responsible for the rapid bone accretion that occurs during puberty and that this effect is mediated by an estrogen-induced increase in growth hormone (GH)/insulin-like growth factor (IGF) action. To test the cause and effect relationship between estrogen and GH/IGF, we evaluated the consequence of ovariectomy (OVX) in prepubertal mice (C57BL/6J mice at 3 wk of age) on skeletal changes and the GH/IGF axis during puberty. Contrary to our expectations, OVX increased body weight (12-18%), bone mineral content (11%), bone length (4%), bone size (3%), and serum, liver, and bone IGF-I (30-50%) and decreased total body fat (18%) at 3 wk postsurgery. To determine whether estrogen is the key ovarian factor responsible for these changes, we performed a second experiment in which OVX mice were treated with placebo or estrogen implants. In addition to observing similar results compared with our first experiment, estrogen treatment partially rescued the increased body weight and bone size and completely rescued body fat and IGF-I levels. The increased bone accretion in OVX mice was due to increased bone formation rate (as determined by bone histomorphometry) and increased serum procollagen peptide. In conclusion, contrary to the known estrogen effect as an initiator of GH/IGF surge and thereby pubertal growth spurt, our findings demonstrate that loss of estrogen and/or other hormones during the prepubertal growth period effect leads to an increase in IGF-I production and bone accretion in mice.

Poor maternal nutrition inhibits muscle development in ovine offspring

BackgroundMaternal over and restricted nutrition has negative consequences on the muscle of offspring by reducing muscle fiber number and altering regulators of muscle growth. To determine if over and restricted maternal nutrition affected muscle growth and gene and protein expression in offspring, 36 pregnant ewes were fed 60%, 100% or 140% of National Research Council requirements from d 31 ± 1.3 of gestation until parturition. Lambs from control-fed (CON), restricted-fed (RES) or over-fed (OVER) ewes were necropsied within 1 d of birth (n = 18) or maintained on a control diet for 3 mo (n = 15). Semitendinosus muscle was collected for immunohistochemistry, and protein and gene expression analysis.ResultsCompared with CON, muscle fiber cross-sectional area (CSA) increased in RES (58%) and OVER (47%) lambs at 1 d of age (P < 0.01); however at 3 mo, CSA decreased 15% and 17% compared with CON, respectively (P < 0.01). Compared with CON, muscle lipid content was increased in OVER (212.4%) and RES (92.5%) at d 1 (P < 0.0001). Muscle lipid content was increased 36.1% in OVER and decreased 23.6% in RES compared with CON at 3 mo (P < 0.0001). At d 1, myostatin mRNA abundance in whole muscle tended to be greater in OVER (P = 0.07) than CON. Follistatin mRNA abundance increased in OVER (P = 0.04) and tended to increase in RES (P = 0.06) compared with CON at d 1. However, there was no difference in myostatin or follistatin protein expression (P > 0.3). Phosphorylated Akt (ser473) was increased in RES at 3 mo compared with CON (P = 0.006).ConclusionsIn conclusion, maternal over and restricted nutrient intake alters muscle lipid content and growth of offspring, possibly through altered gene and protein expression.

Insulin-Like Growth Factor-I Molecular Pathways in Osteoblasts: Potential Targets for Pharmacological Manipulation

The insulin-like growth factors (IGFs) are the most abundant growth factors stored in bone and produced by osteoblasts. IGF-I is an important regulator of osteoblast function and required for optimal bone development and maintenance. IGF-I can act in an endocrine, paracrine or autocrine manner and is regulated by a family of six IGF binding proteins (IGFBPs). The IGFBPs are often found bound to IGF-I in the circulation or complexed with IGF-I in osteoblasts. IGFBP-3 and -5 are known stimulators of IGF-I actions, whereas IGFBP-1, -2, -4 and -6 are known inhibitors of IGF-I action in bone. Once IGF-I binds to its receptor (type 1 IGF receptor) it initiates a complex signaling pathway including the phosphoinositol 3-kinase (PI3-K)/3-PI-dependent kinase (PDK)-1/Akt pathway and the Ras/Raf/mitogen-activated protein (MAP) kinase pathway which stimulate cell function and/or survival. Based on the critical role for IGF-I in osteoblasts, it is a logical candidate for anabolic therapy. However, systemic administration of IGF-I is not cell specific and a limited number of long term experiments have been completed to date. Several recent findings indicate that many of the IGFBPs and specific proteins in the IGF-I signaling pathways are also potent anabolic factors in regulating osteoblast function. This review will focus on the role of these factors in mediating IGF-I action in osteoblasts and how they may serve as potential targets to stimulate osteoblast function and bone formation.

Transabdominal ultrasound for detection of pregnancy, fetal and placental landmarks, and fetal age before Day 45 of gestation in the sheep

Detection of pregnancy during early gestation is advantageous for flock breeding management. Transabdominal ultrasound is a practical and efficient approach for monitoring pregnancy and fetal growth in small ruminants. However, there is limited information using the transabdominal technique before Day 45 of gestation in sheep. Therefore, our objective was to determine how accurately transabdominal ultrasound could be used to detect pregnancy, to identify pregnancy landmarks, and to quantify fetal length before Day 45 in ewes. Multiparous Western White-faced ewes (n = 99) were estrus synchronized and exposed to one of four Dorset rams. The day a ewe was marked by a ram was considered Day 0 of gestation. Ewes not remarked by Day 20 were separated for ultrasonography. To detect pregnancy and landmarks, ewes were scanned three times per week between Day 26.0 ± 0.3 (mean ± standard error) and Day 40.0 ± 0.2. A single technician performed all scans in the right nonhaired abdominal pit using a real-time portable Eazi-Scan machine and a 5-MHz linear rectal transducer. All data were analyzed using the MIXED procedure in SAS (with repeated measures where appropriate). Because of rebreeding activity, 113 ultrasound periods were initiated. The specificity and positive predictive value were 100% during the entire study. The accuracy, sensitivity, and negative predictive value of ultrasound scanning were greater than 90% beginning at Day 33 ± 1. On average, pregnancy (n = 85) was detected at Day 28.7 ± 0.4 and nonpregnancy (n = 28) at Day 25.5 ± 0.6. Three early fetal losses were identified at Day 39.7 ± 0.7. In pregnant ewes (n = 82), the overall accuracy of fetal counting was 78%. The first observance of an enlarged uterus (P = 0.05) and pregnancy (P = 0.03) was detected earlier when multiple fetuses were developing compared with singletons. Placentome evagination was first observed earlier in triplets compared with twins and singletons (P = 0.02). Fetal length increased with day of gestation (P < 0.0001) but not fetal number (P = 0.72). A fetal number by day of gestation interaction (P = 0.01) indicated differences in fetal length at Day 29 ± 1 and Day 32 ± 1. These data demonstrate that a portable ultrasound using the transabdominal technique can be used to accurately determine pregnancy, identify landmarks indicative of gestation, and estimate fetal age, before Day 45 of gestation in sheep.

The effects of poor maternal nutrition during gestation on postnatal growth and development of lambs12

Poor maternal nutrition can affect the growth and development of offspring, which may lead to negative consequences in adult life. We hypothesized that lambs born to poorly nourished ewes would have reduced growth rate and increased fat deposition, with corresponding changes in the somatotropic axis, and leptin, insulin and glucose concentrations. Ewes ( = 36; 12/treatment) were assigned 1 of 3 diets; 100% (CON), 60% (RES), or 140% (OVER) of NRC requirements for TDN at d 31 of gestation until parturition. One lamb per ewe ( = 35; 11 to 12 per treatment) was used; 18 lambs were euthanized at d 1, and 17 were fed the same diet for 3 mo and then euthanized. Lamb crown rump length (CRL), heart girth, BW, and BCS were measured, and blood samples were collected at d 1 and then at weekly intervals until euthanasia. Averaged from d 1 until 3 mo, lambs from OVER ewes were larger compared with lambs born to CON ewes (BW [16.97 vs. 15.44 kg ± 0.60; = 0.09], ADG [0.23 vs. 0.21 ± 0.01 kg/d; = 0.01], and CRL [68.9 vs. 66.1 ± 0.80 cm; = 0.02]). On a BW basis, heart weight from lambs from RES (0.18 kg ± 0.03; = 0.03) ewes was greater than that of CON lambs (0.15 kg ± 0.03). Backfat thickness was reduced in RES lambs (0.11 ± 0.06; ≤ 0.04) compared with CON (0.20 ± 0.06) and OVER (0.26 ± 0.06) lambs. Concentrations of IGF-I at 3 mo and IGFBP-3 from weaning (d 56 of age) to 3 mo of age tended to be greater ( ≤ 0.06) in OVER lambs (334 ± 66 ng/mL and 175 ± 11 arbitrary units [AU], respectively) than CON lambs (149 ± 66 ng/mL and 140 ± 11 AU, respectively). At 3 mo, leptin was greater in OVER lambs compared with RES lambs (1.24 vs. 0.78 ± 0.13 ng/mL; < 0.05). Over time, average insulin concentrations were greater in OVER and RES lambs than CON lambs (0.49 and 0.49 vs. 0.33 ± 0.05 ng/mL; ≤ 0.02). However, concentrations of GH, IGFBP-2, glucose, triglycerides, and total cholesterol were not different ( > 0.10) between treatment groups. During in vivo glucose tolerance test, baseline insulin concentrations were 68% and 85% greater ( 0.01), respectively, in RES and OVER lambs compared with CON lambs. Similarly, the glucose:insulin ratio was greater in RES and OVER lambs compared with CON lambs ( 0.01). Thus, in this experiment, poor maternal nutrition during gestation influenced body size, organ growth, fat accumulation, and concentrations of IGF-I, IGFBP-3, leptin, and insulin of offspring during the first 3 mo of age.