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Biomedical subjects

M C Barber

Publications and source records attributed to M C Barber.

At least 37 records · Page 2Linked to original sources
PubMed1997

Hormonal control of insulin-like growth factor-binding protein-5 production in the involuting mammary gland of the rat.

We have demonstrated a 50-fold increase in the concentration of insulin-like growth factor-binding protein-5 (IGFBP-5) in milk after 2 days of mammary involution induced by removal of the suckling young. IGFBP-5 was identified by its immunoreactivity with an antiserum to IGFBP-5 and was shown by in situ hybridization to be synthesized by the secretory epithelial cells undergoing apoptosis. Smaller increases in IGFBP-2 and -4 messenger RNAs (mRNAs) were also evident, but neither protein could be detected on Western ligand blots of milk. Preliminary evidence failed to detect mRNAs for IGFBP-1, -3, or -6. The large increase in IGFBP-5 concentrations in milk from involuting mammary glands was inhibited by 90% if the dams received concurrent PRL injections for 2 days, but was unaffected by GH, progesterone, corticosterone, or an antiserum to insulin-like growth factor I (IGF-I). In lactating rats allowed to continue nursing their young, 17beta-estradiol failed to affect IGFBP-5 concentrations, whereas in animals that had half the teats sealed to prevent milk removal, IGFBP-5 concentrations increased 5- to 10-fold in the sealed gland compared with those in the contralateral gland where milk removal continued. The changes in IGFBP-5 concentrations in milk were accompanied by similar changes in steady state mRNA levels of IGFBP-5 in mammary tissue. We have previously shown that PRL inhibits apoptosis and involution of the mammary gland, whereas teat sealing has the opposite effect. We, therefore, propose that IGFBP-5 serves to inhibit IGF-I-mediated cell survival, but that it is normally suppressed by PRL and milk removal. Although IGFBP-5, when bound to extracellular matrix, augments the action of IGF, we believe that in the involuting mammary gland IGFBP-5 inhibits IGF action by interacting with casein micelles, which contain calcium phosphate nanoclusters, thereby preventing IGF interaction with IGF receptors. This is analogous to the interaction of IGFBP-5 with hydroxyapatite, which serves to sequester IGFs in bone. IGFBP-5 may, in fact, play a central role in inducing apoptosis, as it is also up-regulated in involuting prostate and thyroid glands as well as in atretic ovarian follicles.

Animals↗
PubMed1997

Repression of the acetyl-CoA carboxylase gene in ovine adipose tissue during lactation: the role of insulin responsiveness.

We have investigated the mechanisms whereby lipogenesis is markedly suppressed in adipose tissue depots of lactating sheep. Expression of the gene encoding acetyl-CoA carboxylase (ACC), the flux-determining enzyme of the lipogenic pathway, is reduced approximately threefold in both omental and subcutaneous adipose tissue depots during late pregnancy and remains so into lactation when compared with non-pregnant, non-lactating animals. By comparison, total ACC enzyme activity in these adipose depots is suppressed approximately 25- to 30-fold in lactation. Culture of explants from the subcutaneous depot of lactating sheep with insulin plus dexamethasone for 72 h resulted in an approximately sevenfold increase in ACC mRNA, a fivefold increase in total enzyme activity and a marked increase in the proportion of the enzyme in the active state when compared with explants cultured with no added hormones for the same period. However, there was a lag of between 32 and 48 h before marked induction of any of these parameters by insulin plus dexamethasone was observed. Induction of the alpha-tubulin gene paralleled that of the ACC gene, suggesting that cytoskeletal rearrangements are associated with the aquisition of sensitivity to insulin plus dexamethasone. These results demonstrate that the reduction in lipogenic capacity in ovine adipose tissue during lactation is related to repression of the ACC gene, both at the level of steady-state mRNA abundance and possibly at translation, as well as to suppression of the mechanisms that regulate the proportion of ACC in the active state, and these are further related to the marked insensitivity of these parameters to insulin plus dexamethasone in vitro.

Acetyl-CoA Carboxylase↗
PubMed1996

The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion.

We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH-deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland.

Animals↗
PubMed1995

Cloning and characterisation of multiple acetyl-CoA carboxylase transcripts in ovine adipose tissue.

A full-length ovine acetyl-CoA carboxylase-encoding cDNA (ACC) has been cloned from adipose tissue and completely sequenced. The open reading frame of 7041 nucleotides (nt) is highly homologous to the previously cloned human, rat, chicken, yeast and algal ACC (85, 89, 82, 54 and 54% identity, respectively). Transcript heterogeneity was found in the 5' and 3' untranslated regions (UTR) resulting in ACC transcripts in the range of 9.0 kb to 9.4 kb. Heterogeneity at the 5' end was generated by the insertion of a 47-nt sequence, resulting in transcripts with either 272 or 319 nt in the 5'-UTR. Heterogeneity at the 3' end was the result of the use of different polyadenylation signals. RNase protection analysis demonstrated that shorter transcripts containing 1635 nt predominated over longer transcripts of 2065 nt in the 3'-UTR.

Acetyl-CoA Carboxylase↗
PubMed1994

Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation.

'Expressed' and 'total' activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in 'total' PK-A activity. The 'expressed' activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel 'total' PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.

Affinity Labels↗
PubMed1994

Growth hormone inhibition of lipogenesis in sheep adipose tissue: requirement for gene transcription and polyamines.

The chronic inhibitory effect of growth hormone (GH) on lipogenesis in sheep adipose tissue explants was investigated in an in vitro tissue culture system. In the absence of other hormones, GH caused a decrease in the rate of lipogenesis after 6 h of culture. In contrast, when lipogenesis was stimulated by the presence of insulin plus dexamethasone, GH again decreased lipogenesis but after a lag of at least 12 h. Actinomycin D, an inhibitor of gene transcription, prevented the effect of GH on lipogenesis in both the absence and presence of insulin plus dexamethasone. Actinomycin D added to tissue previously incubated for 6 h in the presence of GH alone prevented further decline in lipogenesis over the next 5 h, suggesting that transcription of a short-lived mediator protein is required for the GH effect to occur. An increase in ornithine decarboxylase activity was detected in explants exposed to GH, reaching a peak after 12 h incubation; this was prevented by actinomycin D. Methylglyoxal bis-(guanylhydrazone), an inhibitor of polyamine biosynthesis, partially alleviated the effect of GH on lipogenesis; this was reversed by addition of spermidine. However, spermidine did not reverse the effects of actinomycin D, implicating a short-lived protein in addition to ornithine decarboxylase in the action of GH. In the absence of other hormones GH had no effect on either the expressed (initial) or total activity of acetyl-CoA carboxylase, but GH prevented the increase in both expressed and total activities of the enzyme induced by insulin plus dexamethasone.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyl-CoA Carboxylase↗
PubMed1993

Isolation of a goat acetyl-CoA carboxylase complementary DNA and effect of milking frequency on the expression of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland.

1. Using the polymerase chain reaction we have isolated a partial complementary DNA for goat acetyl-CoA carboxylase which is 90 and 82% homologous to the published rat and chicken complementary DNA sequences, respectively. 2. Frequent milking causes an upregulation of the acetyl-CoA carboxylase and fatty acid synthase genes in goat mammary gland that parallels the increase in the respective enzyme activities. 3. The sequence for goat acetyl-CoA carboxylase is in the EMBL data base, Accession Number Z17803.

Acetyl-CoA Carboxylase↗
PubMed1992

Growth-hormone-prolactin interactions in the regulation of mammary and adipose-tissue acetyl-CoA carboxylase activity and gene expression in lactating rats.

The factors and mechanisms responsible for the reciprocal changes in lipogenesis in rat mammary gland and adipose tissue during the lactation cycle have been investigated. Lactation decreased the activation status and mRNA concentration of acetyl-CoA carboxylase in adipose tissue. Litter removal decreased the mRNA concentration of acetyl-CoA carboxylase in the mammary gland and increased the enzyme's mRNA concentration and activation status in adipose tissue. Lowering serum prolactin concentration in lactating rats decreased the amount of mammary acetyl-CoA carboxylase mRNA and increased that of adipose tissue, and increased the activation status of the enzyme in adipose tissue. Decreasing serum growth hormone (GH) alone had little effect on acetyl-CoA carboxylase in lactating rats, although it did lower pup growth rate and serum concentration of insulin-like growth factor-I. Lowering serum GH concentration exacerbated the effects of decreasing serum prolactin on mammary-gland (but not adipose-tissue) acetyl-CoA carboxylase mRNA and further increased the rise in activation status of the adipose-tissue enzyme induced by decreasing serum prolactin. Changes in acetyl-CoA carboxylase mRNA in both mammary and adipose tissue were paralleled by changes in total enzyme activity except after litter removal, when there was a disproportionately large decrease in total enzyme activity of the mammary gland. Thus prolactin has a major and GH a minor role in the regulation of acetyl-CoA carboxylase activity during lactation. Changes in mammary activity in response to prolactin and GH are primarily due to alterations in gene transcription, whereas adaptation in adipose tissue involves both changes in gene transcription and activation status.

Acetyl-CoA Carboxylase↗
PubMed1992

Effects of insulin, hydrocortisone and prolactin on cell morphology, growth and survival of mammary organoids from mid-pregnant mice cultured on collagen gels.

Mammary epithelial organoids consisting of groups of lobular-alveolar acini were prepared from mid-pregnant mice and cultured for 24, 48, 96 and 192 hr on attached collagen gels in the presence of combinations of insulin, hydrocortisone and prolactin. The organoids rapidly attached to the gels and with all the combinations of hormones used colonies of cells spread out as a monolayer from the organoids within 48 hr. Although colony formation continued for up to 192 hr in culture, the maintenance of parental organoid structure after 96 and 192 hr was strongly favoured when hydrocortisone was present in the culture medium. The presence of hydrocortisone produced a dose-dependent increase in the amount of organoid DNA associated with the collagen substratum but decreased the rate of DNA synthesis by the organoids, as measured by the incorporation of labelled thymidine into DNA, in a dose-dependent manner under these conditions. The results suggest that the presence of hydrocortisone minimised the loss of cells from the collagen matrix in these cultures.

Animals↗
PubMed1992

The role of growth hormone, prolactin and insulin-like growth factors in the regulation of rat mammary gland and adipose tissue metabolism during lactation.

Inhibition of prolactin secretion with bromocriptine and neutralization of GH action with a specific antiserum to rat GH (rGH) were used to explore the modes of action of GH and prolactin in maintaining lactation in the rat. Treatment of dams with anti-rGH caused a small reduction in litter weight gain whilst bromocriptine reduced litter weight gain by 50%. When both treatments were combined, however, milk yield ceased completely and this was accompanied by a wide variety of effects on mammary lipid metabolism including decreases in the mRNA concentrations of acetyl CoA carboxylase, fatty acid synthase, malic enzyme and lipoprotein lipase. Activities of acetyl CoA carboxylase and lipoprotein lipase were also significantly reduced. Reciprocal changes were evident in adipose tissue with increases in acetyl CoA carboxylase and lipoprotein lipase activities. In conjunction with a decreased lipolytic response to noradrenaline in adipose tissue of animals given the combined treatment of bromocriptine and anti-rGH, this represented a co-ordinated series of changes to reduce lipid synthesis in the mammary gland and enhance lipogenesis and triglyceride storage in adipose tissue as milk production ceased. All of these effects could be prevented in part by concurrent treatment with GH, but insulin-like growth factor-I (IGF-I) and IGF-II failed to affect any of the parameters measured.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗