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Biomedical subjects

D J Flint

Publications and source records attributed to D J Flint.

At least 55 records · Page 3Linked to original sources
PubMed1995

Production of insulin-like growth factor binding protein (IGFBP), IGFBP-3 protease, and expression of IGF-I receptors by cells of the sheep immune system.

Single-cell suspensions of sheep thymus cells were cultured in serum-free medium with or without polyclonal activators (phytohaemagglutinin or concanavalin A) and the resultant conditioned medium was assayed for insulin-like growth factor binding protein (IGFBP) activity by binding of [125I]IGF-I, using charcoal to separate free from bound. All cultures produced IGFBP but mitogen stimulation significantly increased IGFBP concentrations, indicating production by lymphoid cells. Conditioned medium also degraded recombinant human [125I]IGFBP-3, suggesting IGFBP-3 protease production within the thymus. This degradation was inhibited by several protease inhibitors (phenylmethylsulphonyl fluoride, aprotinin, N-alpha-p-tosyl-L-lysine chloromethyl ketone), suggesting the presence of a serine protease. Cell surface [125I]IGF-I binding was demonstrated on cells from thymus, mesenteric lymph node, peripheral blood mononuclear cells and platelets. The [125I]IGF-I binding to platelets could be inhibited by unlabelled peptides, with relative potencies IGF-I > IGF-II >> insulin. Scatchard analysis of IGF-I competitive binding revealed a Kd of 266 pmol/l and approximately 40 receptor sites per cell. The high-affinity binding of IGF-I and competition by insulin suggested that the [125I]IGF-I binding was to an IGF-I receptor rather than to a membrane-associated IGFBP, to which insulin does not bind. These data provide further support for the role of the IGF-IGFBP axis in the immune system, particularly in relation to the thymus.

Animals↗
PubMed1995

Divergent regulation of rat adipocyte GLUT1 and GLUT4 glucose transporters by GH.

The effect of GH, in vivo, on the glucose transport systems of rat adipocytes has been investigated. Lowering of serum GH levels, by treatment of rats with an antiserum specific for rat GH (anti-rGH), significantly decreased serum levels of both IGF-I and insulin. Treatment with anti-rGH also increased glucose oxidation and the conversion of glucose to lipid by isolated adipocytes. Adipocyte glucose oxidation and lipid synthesis were measured in the presence of a limiting concentration of glucose and therefore reflect changes in glucose transport. Immunoblot analysis of adipocyte subcellular fractions revealed that anti-rGH induced an increase in the amount of the glucose transporters GLUT1 (1.6-fold) and GLUT4 (2.5-fold) present in plasma membranes and a decrease (39%) in the amount of GLUT4 present in low-density microsomal fractions. Lowering of serum GH also increased, by 36%, the amount of GLUT1 present in a total membrane fraction but had no such effect on GLUT4 levels. Replenishment of serum GH, by concurrent administration of ovine GH to rats, prevented all of these effects of anti-rGH. It was concluded that GH in vivo down-regulates the amount of both GLUT1 and GLUT4 present in rat adipocyte plasma membranes. This reflects a decrease in the total cellular levels of GLUT1 and modification of the subcellular distribution of GLUT4 and results in restriction of adipocyte glucose uptake.

Adipose Tissue↗
PubMed1995

Growth hormone and longitudinal bone growth in vivo: short-term effect of a growth hormone antiserum.

The control of longitudinal growth is poorly understood but GH is considered to be one of the major hormones regulating postnatal growth. However, there is dispute as to whether it has a direct or indirect action. To study the role of GH we used a polyclonal antiserum to rat GH and investigated changes in cell proliferation and enzyme activities associated with bone formation and resorption during longitudinal growth. IGF-I levels were measured by two independent RIAs, DNA synthesis by bromodeoxyuridine incorporation followed by immunocytochemistry and enzyme activities were quantified in situ by microdensitometry. After 1 day the percentage of chondrocytes undergoing DNA synthesis within the proliferative zone was reduced but no other parameters were affected. By day 4 the labelling index was the same as in pair-fed animals but the number of chondrocytes synthesising DNA was reduced as was the total width of the growth plate and that of the proliferative zone. Alkaline phosphatase (associated with mineralisation) was unchanged but glucose 6-phosphate dehydrogenase activity (associated with cell proliferation) was decreased. Osteoclastic tartrate-resistant acid phosphatase activity (associated with bone resorption) was also significantly reduced. Similar changes were apparent after 10 days. At no time was the circulating level of IGF-I decreased. These data suggest that, during longitudinal growth, GH affects the number of proliferating chondrocytes but not the percentage of cells undergoing DNA synthesis, indicating that its primary role may be on the commitment of prechondrocytes to a proliferative state. Furthermore, while GH does not seem to have any effect on skeletal mineralisation it may stimulate osteoclastic resorption of the primary spongiosa.

Acid Phosphatase↗
PubMed1995

Effects of growth hormone and ovariectomy on performance, serum hormones, insulin-like growth factor-binding proteins, and muscle fiber properties of prepubertal Friesian heifers.

Sixteen prepubertal Friesian heifers were used to examine the effect of bovine growth hormone (GH) and ovariectomy (OVX) at 2.5 mo of age (2 x 2 factorial design) on growth, carcass quality, and fiber types, capillarization, and metabolic potentials of the longissimus muscle, and serum concentrations of estradiol-17beta (E2beta), insulin, GH, IGF-I, and IGF-binding proteins (IGFBP). Treatment with GH (15 mg/d) started at 147 +/- 3 kg BW and lasted for 15 wk. Heifers were fed a mixed roughage-based diet. Growth hormone increased ADG (P < .001), improved gain:feed (P < .007), and had a small but positive influence on lean accretion. Growth hormone reduced fat thickness (P < .009), carcass fat trim (P < .009) and i.m. fat (P < .09). Ovariectomy did not affect performance but increased dressing percentage (P < .03), full rib weight (P < .003), and fat thickness (P < .04). Ovariectomy reduced E2beta (P < .001) and insulin (P < .02), and increased the 32-kDa IGFBP (IGFBP-2) (P < .09). Growth hormone treatment increased GH, IGF-I, the 28-kDa IGFBP, and the 40- to 43-kDa IGFBP (IGFBP-3) (P < .004 or P < .001). Neither GH nor ovariectomy affected the proportion and relative area of the individual muscle fiber types, but GH tended to increase type I fiber area (P < .10). Number of capillaries per fiber increased in OVX GH-treated heifers (GH x OVX interaction, P < .02). Activities of citrate synthetase were higher in GH-treated (P < .05) and OVX (P < .02) heifers, indicating increased oxidative capacity of the longissimus muscle. The effects of GH on performance and carcass fattening were in accordance with the observed hormonal changes. When slaughter occurs before puberty, ovariectomy has no effect on performance, only few effects on carcass quality, and small effects on hormone concentrations.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1994

Evidence that growth hormone stimulates milk synthesis by direct action on the mammary gland and that prolactin exerts effects on milk secretion by maintenance of mammary deoxyribonucleic acid content and tight junction status.

Both PRL and GH play a role in maintaining lactation in the rat, although GH can only maintain pup weight gain at around 50% of the control value, whereas PRL can maintain weight gain close to 90% in the absence of GH. In this study we examined the effects of PRL and GH deficiency (using bromocriptine and an antiserum to rat GH) on milk yield and composition in lactating rats. Treatment with bromocriptine to suppress PRL secretion for 48 h led to a 57% decrease in milk yield with a concomitant decrease in milk protein and lactose yields, but no decrease in fat output. This led to the production of milk with a lower lactose concentration but increased concentrations of protein and particularly fat (increased 100%), which suggests that GH serves an auxiliary role by maintaining an energy-rich milk for the neonate when PRL secretion is reduced. This decrease in milk synthesis was accompanied by decreases in total mammary DNA content and increased milk sodium concentrations. The latter indicates the opening of tight junctions between mammary epithelial cells, which normally occurs during dedifferentiation and involution of the mammary gland. This suggests that PRL maintains milk synthesis at least in part by inhibiting epithelial cell loss and maintaining cellular differentiation. A deficiency in GH, by contrast, caused only a small decrease (24%) in milk yield and had no effect on the major constituents of milk or on milk sodium concentrations or total mammary DNA content. When animals were made deficient in both PRL and GH, however, there was a further marked decrease (88%) in milk volume along with the yields of all major milk constituents, confirming our previous findings that PRL and GH are the major regulators of milk synthesis. Recent studies have indicated that GH exerts direct effects on mammary gland growth, but its actions on milk secretion have been proposed to be mediated indirectly via insulin-like growth factor-I (IGF-I). We, therefore, inhibited lactation by inducing PRL and GH deficiency for 48 h and then attempted to reinitiate it by administering GH either systemically or by local oil-based implants into the mammary gland. Oil-based GH implants were as effective in stimulating milk secretion in the treated (but not contralateral, control) gland as was systemic GH treatment. Thus, GH does act directly on the mammary gland to stimulate milk synthesis, although this does not rule out the possibility that GH acts by stimulating local production of IGF-I.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Several insulin-like growth factor-I analogues and complexes of insulin-like growth factors-I and -II with insulin-like growth factor-binding protein-3 fail to mimic the effect of growth hormone upon lactation in the rat.

Lactation was suppressed in rats using a combined treatment of bromocriptine (to reduce prolactin concentrations) and a specific antiserum to rat GH administered twice daily for 2 days. When milk production had ceased, as determined by litter weight loss and the absence of milk in the stomachs of pups, attempts were made to reinitiate lactation using prolactin, GH, insulin-like growth factor-I (IGF-I) precomplexed to recombinant human IGF-binding protein-3 (hIGFBP-3) or IGF-I plus IGF-II precomplexed to hIGFBP-3. Despite the fact that all treatments except prolactin led to increases in serum IGFs and IGFBP-3, only prolactin and GH provoked the reinitiation of milk production as determined by increased litter weight gain, milk in the stomach of pups and a significant increase in the weight of the mammary glands. Since the mammary gland has been shown to produce IGFBPs which may inhibit IGF action we also tested three IGF-I analogues, R3-IGF-I, Long-IGF-I and Long-R3-IGF-I. R3-IGF-I has a single amino acid substitution (Glu to Arg) at position 3 whereas Long-IGF-I has a 13 amino acid N-terminal extension. These modifications dramatically reduce the ability of these analogues to bind to IGFBPs although they remain active at the IGF-I receptor. Such IGF analogues would therefore be expected to be active irrespective of the production of inhibitory IGFBPs. However, none was effective in reinitiating lactation, even at doses which have been shown to be biologically effective in terms of nitrogen retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Effects of a polyclonal antiserum to rat growth hormone on circulating insulin-like growth factor (IGF)-I and IGF-binding protein concentrations and the growth of muscle and bone.

A polyclonal antiserum to rat GH (anti-rGH) injected into rats for 3 or 8 weeks markedly reduced the weight, total protein and RNA content of muscles of the hind limb. These effects were prevented when bovine GH (bGH) was administered simultaneously. In a second experiment, the effects of 8 weeks of treatment with anti-rGH on the growth of the whole body, muscle and bone were investigated. Body weights of rats were decreased by 58% by treatment with anti-rGH; muscle weights were reduced by slightly more than the decrease in body weight (by 64%, 65% and 61% respectively for plantaris, soleus and gastrocnemius). The weight of the tibia was decreased by 54%, its length was decreased by 23%, cortical width and overall width were reduced by 26% and 18% respectively, suggesting a possible role for GH in osteoclastic activity. Serum total insulin-like growth factor-I (IGF-I) concentrations were decreased by 80-90% in both experiments by anti-rGH; these changes were prevented in the first experiment by concurrent treatment with anti-rGH and bGH. The serum IGF-binding protein-3 (IGFBP-3) concentration was also decreased by anti-rGH in experiment 1 (by 86%); the response of the 28-32 kDa IGFBPs was smaller (-35%), and was restored to control values by simultaneous injection of bGH. Western immunoblotting using an antiserum to IGFBP-2 showed that there was a marked decrease from neonatal to adult stages which was independent of anti-rGH treatment. This clearly demonstrated a dissociation of the reciprocal relationship supposed to exist between IGFBPs-2 and -3. The 24 kDa IGFBP-4 was unaffected by anti-rGH but replacement therapy with bGH doubled its concentration. Although the effects on body and muscle weight were prevented when rats were given anti-rGH and bGH simultaneously, the possibility of mediation by other hormones cannot be precluded.

Animals↗
PubMed1993

Molybdenum-induced changes in the epiphyseal growth plate.

Molybdenum (Mo), at high concentrations, induces changes in the epiphyseal growth plate through its effects on copper (Cu) metabolism but it is unclear whether or not Mo can induce changes independent of its effects on copper status. To this end, the effect of Mo on longitudinal bone growth was examined in rats. Dietary Mo was given either as ammonium heptamolybdate or as ammonium tetrathiomolybdate, the latter producing a marked Cu deficiency. There was a significant reduction in longitudinal bone growth in both groups; however, growth plate width was increased only in the Cu-deficient animals due to an increase in the width of the zone of transitional/hypertrophic chondrocytes. Both glucose 6-phosphate dehydrogenase activity and cell proliferation (assessed by bromodeoxyuridine incorporation) were markedly decreased in the proliferating zone of the growth plate in both Mo-treated groups. These changes were not apparently related to changes in circulating vitamin D metabolites or insulin-like growth factor-1. The results indicate that excess Mo impairs cell proliferation within the growth plate, whereas the effects of copper deficiency are more related to chondrocyte differentiation. Thus, Mo can induce changes in longitudinal bone growth which are distinct from those resulting from Cu deficiency.

Alkaline Phosphatase↗
PubMed1993

Influence of growth hormone deficiency on growth and body composition in rats: site-specific effects upon adipose tissue development.

Neonatal female rats were treated for 3 weeks (short term) or 8 weeks (long term) with antiserum to rat GH (anti-rGH) with or without replacement therapy with recombinant bovine GH (bGH). Body weight gain and tail length were significantly suppressed within the first 3 weeks and were even more markedly suppressed when treatment was continued for 8 weeks. When treatment was stopped in short-term-treated animals the rate of body weight gain recovered, although without evidence of catch-up growth. These effects were all normalized by concurrent treatment with bGH. Long-term anti-rGH treatment caused a profound reduction (80%) in the number of differentiated adipocytes in two internal fat depots, whilst the subcutaneous depot was only moderately affected (20%). In contrast, after recovery from short-term treatment with anti-rGH, the internal depots were only marginally decreased in both weight and adipocyte numbers, whereas the subcutaneous depot was actually doubled in size compared with controls, due entirely to an increase in the number of differentiated adipocytes. These data clearly demonstrate for the first time that GH is required for the differentiation of adipocytes in vivo. In addition, the results demonstrate distinct effects at different anatomical sites and suggest that GH may be one factor responsible for the differences described in numerous metabolic parameters and hormonal sensitivities of adipose tissue derived from different locations within the body.

Adipose Tissue↗
PubMed1993

Regulation of serum insulin-like growth factor-I (IGF-I), hepatic growth hormone binding and IGF-I gene expression in the rat during pregnancy and lactation.

An apparent GH resistance occurs in pregnancy, since GH concentrations in serum are reported to be normal or elevated, whereas serum IGF-I falls to very low levels. To determine whether this GH resistance is manifest at the level of the hepatic GH receptor or in the ability of GH to initiate IGF-I gene expression, we have determined hepatic IGF-I mRNA expression, circulating IGF-I and hepatic GH binding during various stages of pregnancy and lactation in the rat. The concentration of IGF-I in serum fell from 37 +/- 5 nmol/l (means +/- S.E.M.) in virgin rats to 17 +/- 1 nmol/l in rats in late pregnancy, recovered in early lactation (31 +/- 3 nmol/l) but was again significantly lower than in virgin animals by mid-lactation (22 +/- 3 nmol/l). Hepatic GH binding did not vary significantly during pregnancy but showed a small significant decrease in early lactation when expressed per mg membrane protein. When expressed as GH binding per liver, however, there were no significant changes in GH binding at any stage. Liver weight increased significantly between virgin and early pregnant animals (7.1 +/- 0.2 g compared with 9.2 +/- 0.5 g respectively, P < 0.01) and continued to increase up to late lactation (14.3 +/- 0.4 g).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Effects of growth hormone and an antiserum to rat growth hormone on serum IGF-I and muscle protein synthesis and accretion in the rat.

Rats were injected twice daily for up to 10 days with GH or with a polyclonal antiserum to rat GH, commencing at 21-22 days of age. Administration of bovine or human GH (1mg/day) improved whole body growth rates by 22% and 29% respectively. Plantaris muscle mass was also increased, by 7 and 14% respectively. Anti-GH injected twice daily resulted in a 7% decrease in body weight at 4 days and a 10% reduction by 10 days. Similar decreases were observed in the total protein content of plantaris and soleus muscles. The decrease in the fractional rate of protein synthesis was proportionately greater than the decline in protein content in plantaris muscle whereas in the soleus no change in the rate of protein synthesis was observed, suggesting that the effect on this muscle was due to an increase in the rate of protein degradation. Serum total IGF-I was unchanged by treatment with either GH or anti-GH while the amount of hepatic IGF-I mRNA was also unaffected by anti-GH injection. These data are consistent with a direct effect of GH or an effect mediated by an autocrine/paracrine mechanism of action on muscle but do not support a role for serum total IGF-I as an endocrine mediator of GH action.

Animals↗
PubMed1992

The use of multiple-pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti-(recombinant bovine growth hormone) sera. Comparison with predicted regions of immunogenicity and location within the three-dimensional structure of the molecule.

A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth-hormone (rbGH) molecule recognised by three polyclonal guinea-pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea-pig antisera, 3 or 4, very-well-defined major continuous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea-pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring alpha-helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea-pig antiserum. Comparison of the four guinea-pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson-Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods. In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous epitopes defined by different anti-rbGH sera, there are large regions of overlap defining immunogenic core regions within the molecule. We believe that studies of this nature, together with further understanding of antigen processing and peptide presentation to immune cells, may have a role to play in the development of candidate peptide vaccines.

Amino Acid Sequence↗
PubMed1992

Regional and interspecific differences in the ligand binding properties of beta-adrenergic receptors of individual white adipose tissue depots in the sheep and rat.

The ligand binding of beta-adrenergic receptors was characterized in the omental, subcutaneous and popliteal adipose tissue depots in the sheep, and the lumbar and parametrial depots in the rat. Displacement of [125I]iodocyanopindolol ([125I]ICYP) binding to sheep adipose tissue membranes by the beta-adrenergic antagonists CGP 20712A and ICI 118,551 (beta 1- and beta 2-antagonists, respectively) suggested only a single ligand binding site predominantly beta 2 in character in all three depots, but revealed differences in affinity for these ligands between depots. Lactation increased beta-receptor ligand binding in subcutaneous and omental but not popliteal sheep adipose tissue depots, but had no effect on beta-adrenergic receptor affinities for CGP 20712A and ICI 118,551 in the three sheep adipose tissue depots studied. In rat adipocyte membranes, displacement of [125I]ICYP by CGP 20712A and ICI 118,551 was biphasic, indicating high and low affinity sites; displacement profiles differed markedly between lumbar and parametrial depots, the former having a preponderance of beta 1-like receptors and the latter a preponderance of beta 2-like receptors. The properties of the beta-adrenergic receptor binding site thus show species and depot specific differences.

Adipose Tissue↗