Expression of specific white adipose tissue genes in denervation-induced skeletal muscle fatty degeneration.

Denervation of skeletal muscle results in rapid atrophy with loss of contractile mass and/or progressive degeneration of muscle fibers which are replaced to a greater or lesser degree by connective and fatty tissues. In this study, we show that denervated rabbit muscles are transformed into a white adipose tissue, depending on their fiber types. This tissue does express LPL, G3PDH and particularly the ob gene, a white adipose tissue-specific marker, and does not express the brown adipose tissue molecular marker UCP1 mRNA.

Cloning of rabbit prolactin cDNA and prolactin gene expression in the rabbit mammary gland.

cDNA clones coding for rabbit prolactin were isolated from a pituitary library using a rat prolactin RNA probe. One cDNA contained 873 bases including the entire coding sequence of rabbit prolactin, its signal peptide and the 5' and 3' untranslated regions of 44 and 145 nucleotides respectively. The deduced amino acid sequence of the cloned prolactin cDNA presented a 93-78% identity with mink, porcine and human prolactins. The prolactin gene transcription was investigated by RT-PCR analysis in several organs of midlactating New Zealand White rabbits. The ectopic transcription of the prolactin gene was examined in more detail in the mammary gland. A strong PCR signal was detected in the mammary gland of virgin does and was also observed during pregnancy and at the beginning of lactation. This PCR signal was very weak in mid-lactating and absent in post-weaning mammary gland.

In vivo effects of a treatment with antibodies to adipocyte plasma membranes in the rabbit.

Antibodies against rabbit adipocyte plasma membranes were injected in 6-week-old rabbits. Controls received normal IgG. Animals were killed 1, 2, 5 or 9 weeks after treatment. Body weight and food intake were reduced significantly until the 7th week for the live weight and the 5th week for the intake. Whatever the anatomical location considered, adipose tissue was markedly reduced: -75% for week 1 and -20% for week 9 respectively for the total adipose mass. Cell volume and enzymatic activities of G3PDH, LPL and LDH were highly decreased during the first 2 weeks after treatment. Simultaneously the plasma levels of triglycerides and plasma free fatty acids were increased. As shown by others in the rat, it is possible to induce a long-term fatness reduction in the rabbit by treatment with antibodies to adipocyte plasma membranes. The cytotoxic effects of antibodies have also been discussed.

Effects of a chronic GRF treatment on lambs having low or normal birth weight.

The effects of a long term treatment with human GRF(1-29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 micrograms/kg body weight) of hGRF(1-29) NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvent only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P less than .001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P less than .05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P less than .05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P less than .05) under GRF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Differentiation of rabbit adipocyte precursor cells in a serum-free medium.

A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (beta-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells.

Differentiation of rabbit adipocyte precursors in primary culture.

A primary culture system was used to study the adipose conversion of adipocyte precursors derived from the stromal-vascular fraction of perirenal adipose tissue of rabbit fetuses Differentiation was assessed by the development of glycerol-3-phosphate dehydrogenase, Acid:CoA ligase and lipoprotein lipase activities. Stromal-vascular cells were not able to differentiate when maintained in a medium supplemented with fetal calf serum or with rabbit serum. In contrast, differentiation was induced when the medium was supplemented with rabbit plasma. It also occurred when the growth phase was performed in serum provided that the serum was replaced by plasma when the cultures reached confluence. Supplementation of the culture medium with mesenteric lymph or chylomicrons as lipid sources greatly enhanced both lipid accumulation and the level of enzymatic markers of adipocyte differentiation. Following confluence in serum, cell proliferation ceased almost completely. In contrast, cells in the presence of plasma continued to proliferate, leading to a higher cell density at the time of adipocyte differentiation. These results suggest a positive effect of plasma on the post-confluent mitoses of susceptible cells. To our knowledge, it is the first time that such a difference between plasma and serum has been shown for the differentiation of adipocytes, using an homologous system. These studies also demonstrate that rabbit adipocyte precursors differentiating in primary culture show both similarities to and differences from the adipocytes of cell lines or cell precursors obtained from other animal species.

[Effects of GRF 1-29 in normal and hypotrophic lambs]. / Effects du GRF 1-29 chez l'agneau normal et hypotrophique.

Injections of hGRF rapidly stimulated GH secretion in lamb and this response was significantly higher in hypotrophic than in normal lambs. The treatment had an effect on body composition reducing the retention of lipids and the energy level and increasing calcium and phosphorus content. The results showed that GRF affected both metabolism and skeletal growth of ruminants, stimulating specially the production of Sm-C and proliferation of chondrocytes.

Secretory profiles and production rate of growth hormone in ruminant lambs.

Secretory profiles and production rates of growth hormone (GH) were determined in 6 ruminant lambs during winter. The mean GH concentrations (3.78 +/- 2.17 ng/ml) calculated were based upon blood sampling obtained every 3 min using a withdrawal pump. Body clearance (0.162 +/- 0.031 1/h/kg) was calculated from bolus intravenous oGH administration. The data were analysed by non-linear regression analysis; a bicompartmental model was selected to describe the data. production rate was 14.6 +/- 7.98 micrograms/kg/24 h. It has been emphasized that the experimental design used gave an accurate estimate of GH production rate.

Effects of hypothalamic hormones (GRF, TRH, somatostatin) and insulin-like growth factor I on growth hormone secretion from prepubertal male lamb pituitary cultures.

We have examined the regulation of GH secretion from monolayer cultures of prepubertal male lamb anterior pituitary cells. Growth hormone-releasing factor (GRF 1-44) stimulated GH release in a dose-related manner: the maximal effective dose was 10(-10) M, which caused a 500% increase in basal GH secretion, while the half-maximal effect was reached with a dose of 2.5 x 10(-11) M (ED50). Thyrotropin-releasing hormone (TRH) also elicited a dose-dependent stimulation of GH secretion, although it was approximately 1000 times less potent than GRF. GRF and TRH did not have additive or synergistic effects on GH secretion. Somatostatin (SRIF) at a concentration of 10(-7) M maximally inhibited basal GH release to 40% of that of the control; the ED50 was 2.0 x 10(-9) M. Moreover, 10(-7) M SRIF blocked the stimulation of GH secretion induced by 10(-8) M GRF. However, when the cells were incubated with these two peptides at an identical concentration (10(-8) M), GH secretion was stimulated significantly above control values. When added at the same concentration (10(-7) M, TRH ans SRIF nullified their respective effects. A dose of 100 ng/ml of synthetic IGF-I was without effect on basal GH release, but significantly decreased 10(-9) M GRF-induced stimulation of GH secretion. these data indicate that in prepubertal male lambs: the stimulatory effect of GRF is predominant over the inhibitory effect of SRIF, somatostatin inhibits TRH stimulation of GH secretion in vitro, and IGF-I may control GH secretion by modulating GRF effects at the pituitary level.

Characteristics of growth hormone response to the administration of growth hormone-releasing hormone (GRF) in the lamb.

Human growth hormone releasing hormone (GRF 1-44 or GRF 1-29) was administered to lambs at two different physiological stages (suckling: 5-6 week-old and weaned: 14-15 week-old) when growth hormone (GH) secretory patterns were different: suckling lambs exhibited flat basal GH profiles (5-10 ng/ml) while the weaned lambs had frequent spontaneous episodes of GH release (15-65 ng/ml). The iv injection of GRF evoked an immediate release of GH. In each case, plasma GH levels reached a maximum 1-4 min after the injection. The secretory spike was multiphasic and lasted 30-60 min. Administration of GRF (0.1 microgram/kg) in weaned lambs induced GH pulses with an amplitude comparable to that of endogenous peaks. The induction of a GH peak occurred even when a spontaneous peak immediately preceded the GRF injection. Also, spontaneous peaks were observed during the hour following a GRF-induced GH peak. In suckling lambs, GRF injected intravenously as a single bolus in a range of 0.01 to 0.5 microgram/kg (2 to 100 pmoles/kg) stimulated GH release in a dose-dependent manner. Chronic administration of GRF (0.75 nmole GRF 1-44 or GRF 1-29 per kg twice daily for 21 days) in newborn lambs increased significantly (p less than 0.001) the acute response to GRF during the course of the treatment. GH response to GRF 1-44 and GRF 1-29 was the same. These data show that lambs are highly responsive to GRF action during both suckling and weaning and suggest that there is no in vivo desensitization of the pituitary gland after acute or chronic GRF administration.

Effects of TRH and GRF administration on GH, TSH, T4 and T3 secretion in the lamb.

The effects of various amounts of thyrotropin-releasing hormone (TRH) injected subcutaneously or intravenously (alone or in combination with growth hormone-releasing factor: GRF 1-44) on growth hormone (GH), thyroid-stimulating hormone (TSH), thyroxine (T4) and triiodothyronine (T3) were studied in the plasma of 2-week, 2-month and 3-month old lambs. After subcutaneous TRH administration, increases in plasma TSH, T4 and T3 levels were equivalent, whatever the amount of TRH used (1,2,5 or 10 micrograms/kg). These responses lasted longer after 5 and 10 micrograms/kg. After intravenous TRH administration in 2-week old lambs, the maximal increase in plasma TSH levels occurred after the injection of 0.25 microgram/kg. However, plasma T4 and T3 responses were not different, whatever the amount used. As previously, the amount of TRH affected the duration of these responses more than the magnitude of the pituitary-thyroid axis response. Whatever the injection route, amount used or animal age, TRH alone did not increase GH secretion in lambs. However, it slightly delayed the GH response to GRF. GRF did not affect the response of TSH and T4 to TRH; however it could inhibit T3 increase. In conclusion, in contrast to results obtained in calves by Hodate et al. (1985), TRH did not enhance GH secretion in lambs but, as expected, induced sharp increases in plasma thyroid hormone levels. Its classification as a "growth factor" is therefore questionable, at least in lambs.

Reevaluation of lipolytic activity of growth hormone in rabbit adipocytes.

The lipolytic activities of porcine pituitary fractions and purified growth hormone (GH) from human (h), porcine (p), ovine (o) and rabbit (Rb) origin as well as ovine placental lactogen (oPL), were compared to that of ACTH on rabbit adipocytes. All the GH preparations and oPL were equivalent in inhibiting the binding of labelled oGH to liver plasma membranes from pregnant rabbits. ACTH, and to a lesser extent porcine pituitary fractions and hGH, stimulated free fatty acid production by isolated adipocytes. The sensitivity of the adipocytes to these factors was increased when adenosine deaminase was added to the incubation medium. But, RbGH, pGH, oGH and oPL had no effect. We conclude that GH is not directly involved in the control of lipolysis in rabbit adipocytes and that the effect of hGH is rather due to a contamination of this preparation by other pituitary factors.