Autocrine insulin-like growth factor II inhibits beta-casein mRNA expression in a mammary cell line.

A hallmark of mammary cell differentiation is the induction of beta-casein mRNA expression. A mouse mammary epithelial cell line (COMMA-1D) was treated with insulin, hydrocortisone (HC), and prolactin (Prl) at concentrations (50, 500, and 20 ng/ml, respectively) that resulted in less than half-maximal beta-casein mRNA expression. The cells secreted insulin-like growth factor (IGF)-II (106 pg/ml per 24 h) in the condition media under these conditions. Replacement of insulin with rhIGF-II (150 ng/ml) resulted in significantly less beta-casein mRNA expression. Long-Arg IGF-I (50 ng/ml) was similar to insulin in terms of its ability to induce differentiation, but its activity differed from that of insulin in that it also induced cell proliferation. When the two receptor-specific IGF-II analogs, Arg54,55IGF-II and Leu27IGF-II, were used in studies, only at high concentrations (150 ng/ml) was either analog capable of stimulating any beta-casein mRNA expression. When autocrine IGF-II was immuno-neutralized or bound by the addition of rhIGF binding protein-3 (IGFBP-3)beta-casein mRNA expression was enhanced seven-fold and three-fold, respectively. Exogenous application of IGF-II to counteract the IGF-II mAb stimulation resulted in increased cellular growth and reduced differentiation. We conclude that autocrine IGF-II inhibits mammary cell differentiation and that the blockage of autocrine IGF-II benefits mammary cell differentiation.

Expression of inducible nitric oxide synthase in spontaneous bovine bronchopneumonia.

The expression of inducible nitric oxide synthase (iNOS), major histocompatibility class II molecules (MHC-II), CD68, and the calcium-binding proteins S100A8 and S100A9 (also called MRP8 and MRP14, respectively) was assessed in lung tissues from cattle that succumbed to pneumonia. Expression patterns of these markers were related to the types of lung lesion. iNOS expression was only observed in lungs infected with Arcanobacterium pyogenes or Pasteurella haemolytica but not in lungs from cattle with subacute chronic interstitial pneumonia and acute interstitial pneumonia due to Escherichia coli infection. High levels of iNOS were expressed by cells (probably leukocytes) surrounding necrotic foci. Occasionally, iNOS was expressed by intraalveolar macrophages in viable parenchyma, by leukocytes within the airways, and by some chondrocytes in the supporting cartilage of bronchi. Cells expressing MHC-II were distributed relatively evenly throughout areas of inflammation and did not display any clear association with necrotic foci. Cell types expressing MHC-II included type II alveolar epithelial cells, spindle-shaped cells of the interstitium, cells in bronchus-associated lymphoid tissue, and leukocytes in lymph and blood vessels but largely excluded iNOS-positive cells. Likewise, CD68-positive cells were rarely positive for iNOS and were not confined to the areas surrounding necrotic tissue. As with MHC-II and CD68, there was little if any coexpression of iNOS and either of the S100 proteins tested. Thus, in cattle with necrotizing bronchopneumonia, iNOS-expressing cells were largely restricted to the cellular zone surrounding necrotic areas.

Increases in insulin-like growth factor binding protein-2 accompany decreases in proliferation and differentiation when porcine muscle satellite cells undergo multiple passages.

Subjecting cloned porcine myogenic satellite cells to multiple passages leads to decreased rates of cell division and myotube formation. Because IGF have been implicated in the regulation of muscle cell proliferation and differentiation, the present study was conducted to characterize secretion of IGF-I and IGF-binding proteins (IGFBP) in cultures of cloned porcine satellite cells at two stages of multiple passaging. To this end, we obtained a single porcine satellite cell clone that demonstrated relatively high capacities for cellular proliferation and differentiation into myotubes at the fifth passage but that had greatly diminished capacities for proliferation and myotube formation by the seventh passage. The predominant IGFBP secreted by this satellite cell clone was immunologically identified as IGFBP-2, and quantities of it were increased in medium from seventh-passage cultures. Quantities of IGF-I in medium were determined with a newly developed "titration" radioimmunoassay in which interference from IGFBP was minimized by adding a range of saturating quantities of IGF-II. Medium IGF-I concentrations in seventh-passage cultures were also increased relative to the fifth-passage cultures when expressed per unit of DNA. It is hypothesized that the observed increase of IGF-I in medium likely resulted from protective sequestration of IGF-I by IGFBP-2 rather than from enhanced IGF-I secretion. In summary, these data suggest that multiple passaging of cloned porcine satellite cells results in increased secretion of IGFBP-2, which is associated with depressed cell proliferation and myotube formation, perhaps because the increased IGFBP-2 sequestered IGF-I and reduced its bioactivity.

Arginine supplementation increases weight gain, depresses antibody production, and alters circulating leukocyte profiles in preruminant calves without affecting plasma growth hormone concentrations.

The hypothesis that dietary L-arginine (L-Arg) supplementation would increase growth hormone (GH) secretion and antibody production in preruminant calves was tested. Sixteen newborn calves were randomly assigned to either Arg+ or Arg- treatment groups. Both groups were fed a single dose of Colostrx within 6 h after birth followed by milk replacer twice daily until weaning. Beginning with the Colostrx feeding, calves in the Arg+ group were supplemented with L-arginine at 500 mg kg x BW(-1) x d(-1), and the Arg- group received equivalent, but unsupplemented, diets. All calves were immunized against keyhole limpet hemocyanin (KLH) on d 4 and received a booster vaccination on d 14. The Arg+ treatment increased (P < .05) plasma L-Arg and urea concentrations an average of 2.8-fold and 26%, respectively, during the 4-wk supplementation period. Average daily gain (ADG) of Arg+ calves was increased (P < .10) during wk 1, 3, and 5 of life. The Arg+ treatment depressed (P < .05) total and KLH-specific IgG concentrations in plasma and caused a decrease (P < .01) in circulating leukocyte numbers. Differential counts revealed that the decrease in circulating leukocyte numbers was due to decreases in absolute numbers of lymphocytes, monocytes, and neutrophils. The Arg+ diet did not affect mean plasma GH concentrations during the first 3 wk of life, but GH mean concentrations were decreased (P < .01) during wk 4 due to depressed (P < .10) pulse amplitudes. The decrease in GH mean concentrations during wk 4 was paralleled by lower (P < .10) plasma IGF binding protein-3 concentrations. These data show that supplementary L-Arg does not increase plasma GH concentrations, but it increases ADG, depresses KLH antibody production, and alters circulating leukocyte populations in preruminant calves.

Targeted expression of des(1-3) human insulin-like growth factor I in transgenic mice influences mammary gland development and IGF-binding protein expression.

To test the hypothesis that insulin-like growth factor I (IGF-I) regulates mammary gland development and lactation, the expression of both human (h) IGF-I and des(1-3)hIGF-I was targeted to the mammary gland in transgenic mice using a novel exon replacement strategy and the rat whey acidic protein (rWAP) gene regulatory sequences. Both transgenes expressed a 0.7-kilobase messenger RNA (mRNA). The abundance of WAP-IGE-I and WAP-DES mRNA on day 10 of lactation ranged from 0.2-1.0% and 0.2-13% of the endogenous mouse WAP mRNA, respectively. For WAP-DES mice, transgene expression was greatest from midpregnancy throughout lactation. Western blot analysis showed the presence of correctly processed hIGF-I in milk from these transgenic mice. This hIGF-I was capable of stimulating protein synthesis in cultured rat L6 myoblasts. Ligand blotting indicated changes in mammary gland secretion of IGFBP in response to WAP-DES expression. Histological analysis of mammary tissue from mice overexpressing des(1-3)hIGF-I showed incomplete mammary involution, ductile hypertrophy, and loss of secretory lobules associated with increased deposition of collagen. These changes are believed to occur through autocrine and paracrine effects of des(1-3)-hIGF-I on both epithelial and stromal cells.