Epigenetic change in IGF2R is associated with fetal overgrowth after sheep embryo culture.

Manipulation or non-physiological embryo culture environments can lead to defective fetal programming in livestock. Our demonstration of reduced fetal methylation and expression of ovine IGF2R suggests pre-implantation embryo procedures may be vulnerable to epigenetic alterations in imprinted genes. This highlights the potential benefits of epigenetic diagnostic screening in developing embryo procedures.

Regulators of adipocyte precursor cells.

Lean and adipose tissue growth are two of the most important targets for manipulation in commercial livestock. Adipose tissue growth occurs by both hyperplasia and hypertrophy. The processes involved in adipocyte hypertrophy are relatively well understood but much less is known about adipocyte hyperplasia. The mature adipocyte has little capacity for cell division and the hyperplastic capacity of adipose tissue resides in a population of fibroblast-like adipocyte precursor cells. The origin of these cells and the processes involved in their commitment to the adipocyte lineage is not known. Growth factors, in particular the bone morphogenetic proteins (BMP), are likely to be involved in regulating commitment to the adipocyte lineage. In vitro studies have shown that once committed to the adipocyte lineage, the proliferation and differentiation of, adipocyte precursor cells is regulated by a number of different growth factors. A number of these growth factors are expressed in adipocyte precursor cells in vitro and may have an autocrine-paracrine role. Others, such as epidermal growth factor (EGF), are more likely to have an endocrine role. The precise role that each growth factor plays in regulating adipocyte development in vivo is poorly understood. The chick is a useful experimental system with which to study the precise function of growth factors in adipocyte development.

Decreased muscle cell proliferation in chicks with a deletion in the GH receptor gene.

The increase in muscle weight in neonatal animals is a consequence of increased protein accretion and DNA content. GH increases protein accretion but direct effects of GH on myogenic cell proliferation have not been demonstrated. Sex-linked dwarfism in the chick is caused by mutation or deletion in the GH receptor gene and has provided a useful model to study the physiological consequences of GH insensitivity. This study determined the consequences of GH receptor gene mutation on muscle cell proliferation in vivo. Northern and Southern blotting and PCR analysis revealed restriction fragment length polymorphism patterns and a 1.7 kb deletion of the intracellular domain of the GH receptor gene in commercial dwarf broiler chicks, similar to the Connecticut strain in which there is a dysfunctional GH receptor. Cell proliferation was measured in muscle sections from normal and dwarf chicks after incorporation of 5-bromo-2'-deoxyuridine (BrdU; 25 mg/kg) in vivo at 2, 5 and 13 days of age. Incorporation of BrdU into nuclei was measured in frozen sections, counterstained with propidium iodide to estimate the total number of nuclei by quantitative image analysis, and the labelling index was calculated. Paraffin-embedded sections of breast muscle were stained using an anti-human IGF-I polyclonal antibody. Expression of IGF-I mRNA in muscle from each genotype at 5 days of age was measured by RNAse protection assay. The labelling index was similar in 2-day-old chicks from both genotypes (normal, 20.14 +/- 2.39%; dwarf, 19.79 +/- 5.83%). By day 5 the labelling index had decreased but was significantly higher (P < 0.02) in normal (12.53 +/- 3.36%) compared with the dwarf (6.25 +/- 1.39%). By 13 days of age, there was a further decrease in labelling index but no difference between the groups (normal, 4.92 +/- 1.28%; dwarf, 4.96 +/- 1.51%). IGF-I mRNA was expressed and IGF-I peptide was identified in muscle sections but there was no difference between genotypes. The results show that cell division in breast muscle in vivo is high in neonatal chicks but it declines with increasing age. The absence of a functional GH receptor in the dwarf is associated with a greater decline in DNA synthesis and suggests that GH may directly affect a proportion of cells, since there was no difference in IGF-I mRNA or peptide.

Regulation of chick muscle satellite cells by fibroblast growth factors: interaction with insulin-like growth factor-I and heparin.

This study describes the effect of acidic and basic fibroblast growth factor (FGF) on DNA synthesis in chick satellite cells in vitro and interactions with insulin-like growth factor-I (IGF-I) and exogenous heparin. Basic bFGF stimulated incorporation of [3H]thymidine into DNA with a half-maximum concentration (ED50) of 3.23 +/- 0.33 pmol/l, more than 500-fold more potent than acidic FGF (ED50 = 2.13 +/- 0.5 nmol/l). Both bFGF and IGF-I allowed the cells to traverse the cell cycle with an approximate length of the G1 phase of 12 h. When cells were incubated with bFGF and IGF-I together their effects on DNA synthesis were additive rather than synergistic throughout the full concentration range. Incubation of satellite cells with low concentrations of heparin (ng/ml) to mimic the effect of endogenous heparan sulphate proteoglycan caused a small increase in DNA synthesis, whereas higher concentrations (microgram/ml) inhibited DNA synthesis in a dose-related manner. A low concentration of heparin increased DNA synthesis at the highest concentration of bFGF, but high doses of heparin inhibited the response to bFGF throughout the dose-response curve but without altering the ED50. RNAse protection assay showed the expression of bFGF mRNA in proliferating cells which appeared to decrease on differentiation. The results suggest that aspects of neonatal muscle development are regulated by interactions between autocrine/paracrine growth factors such as IGF-I and bFGF, perhaps IGF-I derived from the circulation, and components of the extracellular matrix. Concentrations of the matrix components may change throughout the neonatal period and into adulthood and have an important effect on the regulatory role played by the growth factors.

Molecular events in adipocyte development.

Adipocyte hyperplasia occurs by the proliferation and differentiation of adipocyte precursor cells or preadipocytes. Although the process of commitment to the adipocyte lineage is poorly understood, a great deal of information has accumulated about the processes and regulatory mechanisms involved in preadipocyte differentiation. The differentiation of preadipocytes is known to be characterized by increased transcription of a number of specific genes. AP-1 and C/EBP binding sites within these genes have been identified as important regulatory sequences. In addition, a specific enhancer sequence has been shown to confer adipose tissue specificity. This article will review the changes in gene transcription that occur during preadipocyte differentiation and how these are regulated. The potential role of autocrine/paracrine acting factors in the proliferation and differentiation of the preadipocyte is also discussed.

Regulation of adipocyte precursor DNA synthesis by acidic and basic fibroblast growth factors: interaction with heparin and other growth factors.

The development of adipose tissue is dependent on the growth and differentiation of fibroblast-like adipocyte precursor cells. Culture of adipocyte precursor cells in vitro has provided an ideal system for identifying potential regulators of proliferation and differentiation. We have demonstrated that both acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF) stimulate chicken adipocyte precursor DNA synthesis in a dose-dependent manner up to a concentration of 100 micrograms aFGF/l and 1 microgram bFGF/l. The effect of bFGF was biphasic, so that in incubations with 25 micrograms bFGF/l, DNA synthesis was not significantly different from controls. In the presence of heparin, stimulation of DNA synthesis at 25 micrograms bFGF/l was 1.6-fold greater than at a concentration of 1 microgram bFGF/l. Addition of heparin to incubations containing aFGF reduced the concentration required for maximum stimulation of DNA synthesis to 1 microgram/l. Cells incubated with aFGF (1-100 micrograms/l) in combination with insulin-like growth factor-I (IGF-I), platelet-derived growth factor, transforming growth factor-alpha or transforming growth factor-beta 1 (TGF-beta 1) exhibited a marked synergistic increase in DNA synthesis. This was also the case when 1 microgram bFGF/l was used, but at a concentration of 25 micrograms bFGF/l synergy was only seen with IGF-I and TGF-beta 1. These results suggest that both basic and acidic FGF are potentially important regulators of adipocyte hyperplasia and that their effect is modulated by constituents of the extracellular matrix and the presence of other growth factors.

Comparison of the rates of proliferation of adipocyte precursor cells derived from two lines of chicken which differ in their rates of adipose tissue development.

1. Putative adipocyte precursor cells were isolated from the white adipose tissue of young broiler and layer chickens and cultured in vitro. 2. The cells from both sources were shown to have the characteristics of adipocyte precursor cells. On reaching confluence, lipoprotein lipase activity was induced and the cells from both strains accumulated large amounts of lipid in the presence of chicken serum. 3. Measurement of cell number over time in culture and calculation of cell doubling times showed that cells from broilers proliferated at a faster rate than those derived from layer-strain chickens. This was the case whether primary or secondary cell cultures were used. Primary cultures of broiler cells had a doubling time of 22 h versus 39 h for layer cells. 4. The contribution of such a difference in proliferative rate to the differential rate of adipose tissue growth between broiler and layer strains observed in vivo is discussed.

Multiple growth factor mRNAs are expressed in chicken adipocyte precursor cells.

We have examined the expression of growth factor genes in primary cultures of chicken adipocyte precursors. RNA was extracted from proliferating and differentiated cells, reversed transcribed and amplified by PCR using gene specific primers. The identity of the PCR products was confirmed by restriction mapping. We show, for the first time, constitutive expression of TGF-beta 2, TGF-beta 3, TGF-beta 4 and bFGF genes in chicken adipocyte precursors. We also detect GH-independent, but differentiation-dependent IGF-I gene expression. The synthesis and action of these growth factors supports the hypothesis that they act as autocrine and/or paracrine regulators of adipocyte precursor cell proliferation and differentiation.

Effects of transforming growth factor-alpha on chicken adipocyte precursor cells in vitro.

The hyperplastic capacity of adipose tissue resides in a group of fibroblast-like adipocyte precursor cells. There is evidence to suggest that their proliferation and differentiation is regulated by insulin-like growth factor-I (IGF-I) and transforming growth factor-beta (TGF-beta) but there is less information about other growth factors which may also participate in adipocyte precursor cell hyperplasia. Transforming growth factor-alpha (TGF-alpha) is a 50 amino acid polypeptide which has been shown to stimulate proliferation in both neoplastic and normal cell types acting through the epidermal growth factor (EGF) receptor. We have studied the regulation of DNA synthesis and the activity of lipoprotein lipase by TGF-alpha in chicken adipocyte precursor cells in vitro. Both TGF-alpha and EGF stimulated incorporation of [3H]thymidine into DNA in a dose-dependent manner. TGF-alpha was approximately 180-fold more potent than EGF. Addition of TGF-alpha in combination with IGF-I, TGF-beta 1 or platelet-derived growth factor produced a synergistic increase in DNA synthesis. Short-term incubation with TGF-alpha reduced lipoprotein lipase activity by 23%. These results show that TGF-alpha is a potent mitogen in these adipocyte precursor cells and can inhibit their differentiation in vitro and may participate in the regulation of adipose tissue development in vivo.

Adipose tissue lipogenesis and fat deposition in leaner broiler chickens.

Rates of hepatic lipogenesis and secretion of plasma triglyceride-rich lipoproteins in 6- to 7-wk-old broiler chickens were similar to the overall rate of fat deposition in these birds, although approximately 20% of [14C]-labeled VLDL was oxidized to CO2 within 8 h. Only 6-7% of VLDL and portomicron triglyceride was taken up by the abdominal fat pad, but this proportion of total triglyceride flux could account for about 80-85% of the total fatty acids accumulating in that depot. The rate of lipogenesis in adipose tissue was much lower than that in the liver, but it could account for much of the remaining fatty acids. Lipogenesis from [14C]acetate in cultured chicken adipocytes was markedly inhibited by adding VLDL as an exogenous source of fatty acids. However, adipose tissue lipogenesis was not increased in vivo by reduction of plasma lipoprotein flux by genetic selection, by the feeding of a high protein diet or by immunological intervention. The results confirm that adipose tissue lipogenesis makes only a small contribution to adipose tissue growth in normal broilers. Its importance does not increase in response to the reductions in hepatic lipogenesis that accompany genetic or nutritional manipulation of body composition.

Identification of chicken (Gallus domesticus) adipocyte plasma membrane and differentiation specific proteins using SDS-PAGE and western blotting.

1. Affinity-purified adipocyte membrane proteins were used to raise antisera in two sheep. 2. Using one of the antisera 15 proteins were identified as being adipocyte specific by comparison on Western blots of plasma membrane proteins from various tissues. 3. Of these 15 proteins eight appeared to be present only in mature adipocytes and not in the adipocyte precursor. 4. In the presence of guinea pig complement the two antisera raised were cytotoxic to adipocytes and their precursors. 5. Characterization and further study of these adipocyte differentiation specific proteins will provide valuable information about the process of adipocyte differentiation.

Regulation of DNA synthesis in chicken adipocyte precursor cells by insulin-like growth factors, platelet-derived growth factor and transforming growth factor-beta.

Adipose tissue growth can occur by both hypertrophy and hyperplasia. The capacity for adipocyte hyperplasia in vivo resides in a population of fibroblast-like adipocyte precursor cells but the regulation of the proliferation of these cells by growth factors has not been well characterized. This study was designed to determine the effects of the insulin-like growth factors (IGF-I and IGF-II), platelet-derived growth factor (PDGF) and transforming growth factor-beta 1 (TGF-beta 1) added alone or together on the proliferation of primary adipocyte precursor cells in vitro. Adipocyte precursor cell proliferation measured by [3H]thymidine incorporation into DNA was stimulated by all of these growth factors and was particularly marked with PDGF. IGF-I or IGF-II added together with TGF-beta 1 produced a greater than additive response and the effect of PDGF was synergistic with that of IGF-I at certain concentrations. Stimulation of proliferation of some cell types by TGF-beta has been linked to the secondary production of PDGF but the evidence we have suggests that this is unlikely in chicken adipocyte precursors. DNA synthesis in response to TGF-beta 1 required only a short exposure to the peptide, and conditioned medium from chicken adipocyte precursor cells previously exposed to TGF-beta had no effect on DNA synthesis when added to fresh batches of cells. Addition of TGF-beta 1 together with PDGF produced a synergistic effect whereas an additive effect would be expected if PDGF mediated the effect of TGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of transforming growth factor beta 1 and basic fibroblast growth factor on lipoprotein lipase activity in primary cultures of chicken (Gallus domesticus) adipocyte precursors.

1. The effect of TGF-beta and bFGF on lipoprotein lipase activity in chicken adipocyte precursors was investigated. 2. Lipoprotein lipase activity was reduced by up to 80% by incubation with TGF-beta whereas bFGF had no effect. 3. Contrary to that found with the 3T3-L1 preadipocyte cell line it was not necessary for TGF-beta to be present prior to the start of differentiation in order to be effective. 4. Incubation of adipocyte precursors with actinomycin D abolished the effect of TGF-beta suggesting that synthesis of a protein effector is required. 5. These results indicate differences in responsiveness to TGF-beta and bFGF between primary chicken adipocyte precursors and some preadipocyte cell lines.

Cytotoxic antibodies to chicken adipocytes and their precursors: lack of tissue specificity.

1. Antisera raised in sheep against chicken adipocyte plasma membranes recognised adipocyte, liver and red blood cell membranes in enzyme immunoassays (EIA). 2. These antisera were cytotoxic when incubated with both adipocytes and their precursors grown in culture, and with red blood cells. 3. Adsorption of antisera with liver membranes completely abolished the response to liver and red blood cell membranes in the EIA. The response in adipocytes was reduced to only about 6% of the titre present in the unadsorbed sample. 4. Adsorption of antisera with liver membranes abolished the cytotoxic response to red blood cells and to adipocyte precursors, but some response to adipocytes still remained. 5. These results are in contrast to the rat for which adipocyte specific antisera were obtained which represented about 75% of the unadsorbed titre.

The effects of macrophage-derived cytokines on lipid metabolism in chicken (Gallus domesticus) hepatocytes and adipocytes.

1. The effects of avian and mammalian cytokines on avian lipid metabolism were compared using cultured chicken hepatocytes and adipocytes. 2. Conditioned medium from an endotoxin-stimulated chicken macrophage cell line was used as a source of chicken cytokines. Incubation of chicken adipocytes with conditioned medium greatly decreased their lipoprotein lipase activity. 3. Inhibition of lipoprotein lipase synthesis in similar experiments in mammals has been attributed to the effects of TNF-alpha and/or IL-1, but recombinant human TNF-alpha and IL-1 had no effect on lipoprotein lipase activity in chicken adipocytes. 4. Conditioned medium from chicken macrophages produced a 2-fold increase in lipogenesis in chicken adipocytes but had no effect on lipogenesis in chicken hepatocytes. 5. The results point to major differences between mammals and birds in the way that lipid metabolism responds to cytokines and provide further evidence that mammalian cytokines are ineffective in birds.

Biochemical indicators of fatness in meat-type chickens: lack of correlation between lipoprotein lipase activity in post-heparin plasma and body fat.

1. Possible relationships between fatness and lipoprotein lipase activity in adipose tissue and plasma from heparinised birds were examined in 7-week-old male and female broilers. 2. Total lipoprotein lipase activity in abdominal fat was significantly correlated (r = 0.5) with fat pad weight, but there was no correlation between specific activity of the enzyme and fat pad weight. 3. Lipoprotein lipase activity in post-heparin plasma showed no correlation with either abdominal fat or total body fat content. 4. The results indicate that measurements of lipoprotein lipase activity in biopsy samples or in post-heparin plasma are of no value in predicting the fat content of live birds.

Effect of Escherichia coli endotoxin on tissue lipoprotein lipase activities in chickens.

1. Four-week-old broiler chickens were injected intravenously with from 0.01 to 1 mg of E. coli endotoxin/kg body weight or with saline. 2. At all doses used endotoxin markedly depressed food intake and lipoprotein lipase activities in muscle and adipose tissue within 8 h. Heart lipoprotein lipase activity was significantly depressed only at doses of 0.1 mg endotoxin/kg body weight or greater. 3. Treatment of birds with 0.3 mg endotoxin/kg body weight reduced post-heparin lipoprotein lipase activity to 0.13 of that in control birds in 8 h. 4. Endotoxin generally depressed plasma very-low-density lipoprotein concentration. Plasma non-esterified fatty acid concentration was significantly elevated only in birds given 1 mg endotoxin/kg body weight. 5. Fatty acid synthetase activity in the liver of endotoxin-treated birds was significantly lower than in control birds 16 h after administration of endoxin, but not after 8 h. 6. These results show that tissue lipoprotein lipase activity in birds is very responsive to E. coli endotoxin, as in mammals. Hypertriglyceridaemia occurs only occasionally in endotoxin-treated chickens, most probably because of the particularly close relationship between food intake and hepatic lipoprotein synthesis in birds.