Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.     

Effects of deflazacort and cortisone on cellular proliferation in the rat thymus

Deflazacort is a relatively new glucocorticoid with significant immunosuppressant activity and presumably fewer side effects. The present study was designed to compare the effects of deflazacort on the proliferative activity of thymus cells and thymolysis with the growth inhibition. We treated Long-Evans rats for nine days with cortisone (CORT, 5.0 mg/day), deflazacort (DFZ, 0.15 mg/day), and control vehicle (CTRL). Animals were sacrificed 1 hour after injection of bromodeoxyuridine (BrdUrd) on day 10. BrdUrd-labeled thymic cells were quantified without knowledge of treatment. A Labeling Index (LI), expressed as the number of BrdUrd BrdUrd-labeled cells per 100 total cells and the Numerical Density (ND), expressed as the total number of cells per 100 microm(2) were calculated. Treatment with either glucocorticoid resulted in a significant and equal decrease of thymus weight, indicating a marked reduction in total immunogenic tissue. A general alteration of thymic histological structure occurred in the CORT group. The LI was not different between CTRL and DFZ groups, 6.9 and 7.9% of cells were labeled respectively. In the CORT group, the LI was 2.5%. With respect to Numerical Density, the CTRL group had the greatest value (14.6 +/- 0.4 cells/100 microm(2)), with the DFZ (12.3 +/- 0.06 cells/100 microm(2)) and CORT groups being significantly lower (10.4 +/- 0.5 cells/100 microm(2)). Although regression analysis of thymus weight pointed to bioequivalence of the glucocorticoid dosages used, BrdUrd-labeling raised the possibility that the cells still present in the thymus of DFZ-treated animals retained, at least partially, their normal capacities for proliferation.     

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70(S6k) kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.     

胃缺血-再灌注对大鼠胃黏膜细胞凋亡和增殖的影响

本研究采用大鼠胃缺血-再灌注（gastricischemia-reperfusion,GI-R）模型（夹闭腹腔动脉30 min后再灌注）,通过组织学、免疫组化等方法,研究GI-R不同时间（0、0.5、1、3、6、24、48、72 h）对胃黏膜细胞凋亡和增殖的影响.结果发现,单纯缺血30 min胃黏膜损伤较轻,再灌注后损伤逐渐加重,胃黏膜的凋亡细胞迅速增加,而增殖细胞迅速减少;至再灌注后1 h达高峰;之后胃黏膜开始修复,凋亡细胞逐渐减少,增殖细胞逐渐增加;至再灌注后24 h胃黏膜细胞增殖达高峰;再灌注后72 h胃黏膜基本恢复正常.上述结果提示,在GI-R中,胃黏膜损伤主要由再灌注引起,凋亡细胞增加;然后胃黏膜启动自我修复机制,增殖细胞逐渐取代损伤细胞,3 d左右就可基本修复,表明胃黏膜细胞具有很强的自我修复能力.     

Relationship between cytoplasmic pH and proliferation during exponential growth and cellular quiescence

Flow cytometry was used to measure intracellular pH (pHi) on an individual cell basis during exponential and plateau phases of growth. In all three cell lines examined a range of pHi values was associated with exponential growth. When cells from the extremes of the pHi distribution were sorted using a fluorescence-activated cell sorter and then restained for cellular DNA content, it was found that the higher pHi values were associated with enrichment of the S, G2, and M phases of the cell cycle, with a corresponding increase in the percentage of G1 cells at the lower pH1 range, suggesting cell-cycle dependence of pHi. It has been shown previously (I. W. Taylor and P. Hodson, 1984, J. Cell Physiol. 121, 517) that PMC-22 human melanoma cells are capable of entering a distinct pH-dependent quiescent state in response to the acidification of the growth medium which occurs naturally during growth to plateau phase. Simultaneous measurement of pHi and external pH showed that under these conditions pHi was maintained at control values down to an external pH of approximately 6.5, below which cytoplasmic acidification took place. This fall in pHi coincided with the onset of the transition to quiescence. Individual quiescent cells (defined by failure to incorporate bromodeoxyuridine during a 24-h exposure) could not be identified as such on the basis of a low pHi, suggesting that the probability of cell cycling is reduced by lowering pHi. Those cells which remained in cycle showed a markedly reduced rate of DNA synthesis, but a cell-cycle phase distribution similar to that in exponential growth, indicating that prolongation of all cell-cycle phases is an additional factor influencing overall population growth. The external pH at which both of these effects on cell proliferation kinetics took place in vitro is similar to that which occurs regionally within solid tumors, suggesting that pH effects could play a significant role in determining tumor cell growth in vivo.     

Effects of growth regulators on inflorescence proliferation of Bambusa edulis

The effects of various growth regulators in Bambusa edulis inflorescence proliferation were studied. Cytokinin is essential for inflorescence proliferation. Thidiazuron (TDZ) had been the most efficient cytokinin to induce inflorescence proliferation. The optimal TDZ concentration was 0.01–0.1 mg l^–1. Inflorescences did not proliferate in media containing auxin, gibberellic acid (GA₃), abscisic acid (ABA), or 1-amino- cyclopropane-1-carboxylic acid (ACC) alone. In TDZ-containing medium, the proliferation ratio decreased when the naphthaleneacetic acid (NAA) concentration higher than 5 mg l^–1.     

Effects of insulin and insulin‐like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin‐like growth factor 1 (IGF‐1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF‐1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF‐1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF‐1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF‐1 promote osteoblast differentiation and mineralization in vitro, IGF‐1, but not insulin, can induce osteoblast proliferation. The IGF‐1‐induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF‐1‐mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3‐kinase inhibitor. Osteocalcin, an osteoblast‐specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose‐ and time‐dependent manner after insulin treatment, whereas it was decreased with IGF‐1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF‐1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of fibroblast growth factor and nerve growth factor on the cellular proliferation and functional activity of isolated islands of Langerhans

The effects of fibroblast growth factor (FGF) and nerve growth factor (NGF) on DNA synthesis and insulin secretion were studied in 4-5-day cultures of the isolated neonatal rat islets. FGF (0.1 ng/ml) stimulated significantly the incorporation of 3H-thymidine into DNA of the isolated islets, but failed to change either insulin content in the islets or the rate of insulin secretion. NGF (0.1-1000 ng/ml) did not affect the above parameters. The responses of the islets of Langerhans to increasing concentrations of glucose and isobutylmethylxanthine were not modified after prolonged exposure to NGF. The role of FGF and NGF in the regulation of proliferation and secretory process in pancreatic islet cells is discussed.     

The influence of epidermal growth factor and insulin on proliferation and DNA synthesis in ciliates Tetrahymena pyriformis

The influence of the epidermal growth factor (EGE) (10(-8) M), insulin (10(-6) M) and EGF (10(-8) M) in combination with insulin (10(-6) M) on proliferation and DNA synthesis in the nuclei of ciliates Tetrahymena pyriformis GL was studied. Insulin and EGF, known to stimulate growth of many types of mammalian cells revealed a mitogen influence on the unicellular eukaryotes. This effect involves stimulation of DNA synthesis, rising synchronization of cell division (upon the influence of EGF), and increase in cell number during the exponential growth. The mitogen effect may be evoked by cell progression in G1-phase under the action of growth factors and, consequently by earlier entry of cells into S-phase of the first cell cycle. Insulin repressed division of cells that entered into the generative cycle. These cells were delayed in late S-phase and G2-phase of the cycle. Part of these cells perished, while other cells could successively overcome the cell block to start their division by the 4th hours of cultivation. A collateral cytotoxic effect of insulin was found, being most prominent in early periods of Tetrahymena cultivation.     

Effects of human growth hormone on erythrocyte insulin binding in growth hormone deficient children

In this paper, the effect of acute human growth hormone (GH) administration on erythrocyte insulin binding in GH deficient children (N = 6) was studied. Following GH (0.25 U/kg) administration, the blood levels of GH peaked within 4 to 8 h and returned to basal levels 24 h later. However, the changes in somatomedin activity, free fatty acid (FFA), urea, blood glucose and 125I-insulin binding to erythrocyte were observed around 24 h following the injection, and there was a converse relationship between maximum percent 125I-insulin binding (IBmax) and FFA (P less than 0.02). By Scatchard analysis it was found that the decrease in IBmax is mainly due to the change in the number of insulin receptors. These results suggest that GH may possibly affect the insulin binding to erythrocyte indirectly through metabolic changes as a result of hormonal changes in GH deficient children.     

In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation

For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl. The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine). In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline. During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur. The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6%. Addition of foetal calf serum to the basal medium favours cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%). Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium. These results are discussed with respect to the problem of cell proliferation control during limb regeneration.     

Relative influence of testosterone and insulin in the regulation of prostatic cell proliferation and growth

Prostatic hyperplasia is a common problem of the aged men population. Recent experimental and clinical studies provide sufficient evidence that apart from androgens, insulin also plays an important role in the pathogenesis of prostatic hyperplasia. The present study was aimed to investigate the relative influence of testosterone and insulin on the cellular proliferation and prostatic growth. Effect of testosterone on the prostate of hypoinsulinemic, and glandular injection of insulin-receptor antagonist S961 on the prostate of castrated Sprague-Dawley rat (220 ± 10 g) was examined. Significant decrease in the weight of the ventral prostate was observed in the streptozotocin-induced hypoinsulinemic rats (∼6 fold), which is restored by the intervention of testosterone. Although, glandular injection of S961 did not led to any change in the frequency of proliferating cell nuclear antigen (PCNA) positive cells in normal rats, significant decrease was observed in the castrated rats. Castration led to increase in the frequency of the caspase-3 and the TUNEL positive cells in the ventral prostate. Further, long-term (6 weeks) administration of S961 induced significant decrease in the weight of the ventral prostate. Results of the present study provide that both testosterone and insulin promote prostatic cell proliferation and change in the level of either of the hormone results in the destabilization of cellular equilibrium, and modulation of the insulin-receptor signaling in the prostate may provide an alternative strategy for the treatment of prostatic enlargement. Further, studies are required to better understand the interplay between these hormones in the regulation of prostatic growth.     

Effects of EGF and PMA on the growth and proliferation of IEC-6 cells

Proliferation of an epithelial line (IEC-6) derived from the crypts of rat jejunum was induced with epidermal growth factor (EGF). EGF enhanced synthesis of protein, RNA, and DNA in a dose-dependent manner. Protein synthesis increased within 6-12 hours of exposure to EGF and remained elevated for 72 hours. Maximal 3H-thymidine incorporation occurred 48 hours after addition of EGF. The stimulatory effect of EGF on 3H-thymidine incorporation was two-fold greater in serum-free media than in media containing fetal calf serum (FCS). In contrast to EGF, phorbol-12-myristate-13-acetate (PMA) decreased 3H-thymidine uptake by IEC-6 cells and had no effect on either protein synthesis or RNA synthesis. EGF did not alter protein kinase-C activity in IEC-6 cells whereas PMA induced enzyme activity: activity was translocated from cytosol to membrane. Moreover, the EGF-associated increase in 3H-thymidine uptake was not altered by amiloride. These data suggest protein kinase-C activation may not be involved in the proliferation of IEC-6 cells.