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Publications and source records attributed to P Grimaldi.
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The Ob17 cell line was established from the epididymal fat pad of ob/ob mice. Chronic exposure to physiological concentrations of insulin led to a net increase in lipogenic enzyme activities (EC50 = 1 to 3 nM). Proinsulin exerted the same effects at higher concentrations (EC50 = 10 to 30 nM). The mitogenic effects of both hormones appeared only at supraphysiological concentrations (EC50 = 300 nM). We conclude this was a net dissociation between the lipogenic and mitogenic effects of insulin during differentiation of the Ob17 cells.
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An active fraction present in fetal calf serum (adipose conversion factor(s) or ACF) controls the formation of fat cell clusters observed during the adipose conversion of Ob17 cells. This conclusion is based upon microscopic examinations, autoradiographic experiments, and activity levels of characteristic enzyme markers. ACF has no effect on the doubling time of exponentially-growing cells but is required in the first few days of the resting phase. ACF has been partially purified; it is a low mol. wt. component (less than 6000-8000), proteas -insensitive, thermostable at neutral pH but not at strongly acidic pH. An active fraction recovered from bovine pituitary extract shows properties similar to those of ACF. Therefore, ACF plays the role of a mitogenic factor specific for cells susceptible to conversion to adipose cells.
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The adipose conversion of ob 17 preadipose cells can be irreversibly blocked when prostaglandin F2alpha is included post-confluence for a minimum of 24 h in insulin-containing media. Prostaglandins E1 and E2 are inactive. The lack of adipose conversion is accompanied by the maintenance of a fusiform cell shape, by a slow increase in cell number and by a potent rise in de novo prostaglandin synthesis; it is paralleled by the absence of the characteristic phenotypes of adipose conversion. The multiple effects of prostaglandin F2alpha are dose-dependent, with half-maximal concentrations ranging from 10 to 40 nM. The absence of differentiation and the high rate of prostaglandin synthesis in the presence of prostaglandin F2alpha are likely a consequence of a sustained growth, as also observed with other growth-promoting agents (bovine retinal extract and cat serum). Indomethacin, while suppressing endogenous prostaglandin synthesis, is unable to reverse the long-term and multiple effects of prostaglandin F2alpha. Although adipose conversion normally follows a decrease in prostaglandin production (R. Négrel and G. Ailhaud, Biochem. Biophys. Res. Commun. (1981) 98, 768-777), these results indicate that both events can be dissociated.
Conversion of ob 17 preadipocytes to mature adipose cells is accelerated by addition of clofenapate or of indomethacin, in either the absence or presence of insulin. General stimulation of triacylglycerol-pathway enzymes is observed, as well as dramatic increase in endogenous fatty-acid synthesis. This increase is a function of drug concentration and exposure time. In contrast to indomethacin, the continuous presence of clofenapate after the cells reached confluence was required to observe the effects on adipose conversion. Growth of ob 17 fibroblasts in the presence of 5-bromo-2'-deoxyuridine normally prevents their differentiation to adipose cells. Addition of either clofenapate or indomethacin to these cells at confluence overrides this block. The effects of hypolipidemic drugs such as clofenapate observed on a long-term basis in vitro are consistent with the results of studies on adipose tissue in vivo.
Serum concentrations of immunoreactive somatomedin C, GH, and PRL were determined on samples obtained from 16 adult subjects throughout the day. Samples were collected by a portable continuous flow, blood withdrawal pump. Somatomedin C concentrations were stable during waking hours [1.13 +/- 0.09 U/ml (mean +/- SE)] but fell after the onset of sleep to a minimum value of 0.85 +/- 0.08 U/ml. After awakening, the concentration rose progressively to a mean of 1.26 +/- 0.10 U/ml between 1100-1200 h. These changes were statistically significant. There were no sex differences in the sleep-related decrement. The secretion of GH showed the expected surge in the first hours of sleep, and PRL concentrations were higher in the late phases of sleep or immediately after awakening. In sleep reversal experiments, three subjects who were deprived of sleep had a modest, slightly delayed fall in somatomedin C but no secretory peaks of GH or PRL. After the delayed onset of sleep, somatomedin C declined sharply as human GH concentrations rose.
Cells of a preadipocyte clonal line (ob 17) isolated from epididymal fat pad of ob/ob mouse possess high-affinity binding sites for triiodothyronine. A single class of sites was found on growing and early confluent cells (KD 0.14 +/- 0.025 nM ; 5,000 +/- 600 sites per cell). A two-fold increase in the number if T3 binding sites occurs during adipose conversion, with no significant change in KD values. The order of potency of structural analogs to compete with 125I-T3 is in favor of nuclear binding sites. A correlation was obtained 3 between this order of potency and the ability of the analogs, included on a long-term basis to confluent cells, to increase 14 C-acetate incorporation into lipids, suggesting an enhancement of de novo fatty acid synthesis, This hypothesis was supported by increased activity levels of fatty acid synthetase after chronic exposure to 1.5 nM triiodothyronine. Under these conditions activity levels of acid:CoA ligase and glycerol-3-phosphate dehydrogenase were also increased significantly. Inclusion of bromodeoxyuridine as a differentiation-blocking agent in the culture medium of growing cells decreases drastically the T3 effects, favoring the role of the latter hormone as amplifier of specific phenotypes expressed during adipose conversion. These results show that ob17 cell line should be an useful tool to study the role of thyroid hormones in the regulation of fatty acid synthesis and esterification in adipose cells.
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Cells of a clonal cell line (ob 17) isolated from the epididymal fat pad of ob/ob mouse possess insulin receptors. Their number was increased 1.5-fold after growth arrest, with no significant change in the Kd values of the "high affinity" sites determined by extrapolation of the high affinity portion of the curvilinear Scatchard plots. With chronic insulin exposure for 3 to 11 days after confluence, ob 17 cells showed a decrease in insulin receptor concentrations from 8,000 to 1,600 high affinity sites/cell (Kd from 0.45 to 1.10(-9) M) while similar levels of "low affinity" sites were found (80,000 to 100,000 sites/cell; Kd from 10(-8) to 3 x 10(-8) M). The loss of the high affinity binding sites is accompanied by the disappearance of the stimulatory effect by insulin of alpha-aminoisobutyrate uptake. Therefore, in contrast to 3T3-L1 fibroblasts, the ob 17 cells present, in culture, a self-modulation of insulin receptors and a loss of insulin sensitivity after chronic exposure to insulin.
The sleep-wake behaviour of LH, FSH, PRI, GH and TSH was studied in seven cryptorchid patients (four unilateral and three bilateral cryptorchids) average age 12 years and in nine normal pubertal boys of 13 years (mean age). Blood samples were collected by a continuous withdrawal pump, every hour, for 24 h. The hormonal concentration for every fraction of time was measured and related to the sleep (Sc-), wake (Wc-) and total 24 h period (Dc-). The integrated concentrations of the corresponding periods (IS, IW, ID) were calculated as well as their ratios (IS/IW; IS/ID%). For GH and TSH, the data obtained demonstrated no differences between cryptorchid and pubertal subjects. The PRL secretion in cryptorchid patients was moderately increased during the hours of nocturnal sleep. A normal pubertal sleep-wake rhythm was found for gonadotrophins in both groups of subjects. More marked levels of LH secretion were observed in cryptorchid boys compared to normal pubertals. The presence of a sleep-wake rhythm was also found in the cryptorchid patients and normal pubertal subjects in the P 1 stage. These data suggest that the CNS "programme" which controls the onset of puberty may be normal in cryptorchid patients.
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A clonal cell line that responds to insulin and to lipolytic hormones has been established from the epididymal fat pad of the C57BL/6J ob/ob mouse. This line, designated ob 17, has a doubling time of 12.5 or 19 hr in 10% or 1% fetal calf serum, respectively. It presents a heterogeneous chromosome number with 40% of the cells containing 35-44 chromosomes and expresses the characteristic H2-LA antigen. After cessation of growth, ob 17 cells accumulate droplets of triglycerides; this accumulation occurs to a significant extent even in the absence of insulin normally added after confluence. Lipoprotein lipase activity is negligible in exponentially growing cells but appears at its maximal level just after confluence with or without insulin. Acid:CoA ligase and acylCoA:diglyceride acyltransferase develop later than lipoprotein lipase. The appearance of lipolytic and lipogenic enzymes, but not of triglycerides, seems to be independent of the presence of lipoproteins or of unesterified fatty acids in the culture medium. Therefore, the differentiation program becomes operative when growth is arrested, and differentiation occurs, providing a source of exogenous lipids. Differentiated ob 17 cells in which endogenous triglycerides have been prelabeled on the fatty acid moiety do respond to epinephrine and corticotropin by release of radioactive fatty acid. This lipolytic response is counteracted by prior addition of insulin. The ob 17 cell line appears to be a useful model for study of growth and differentiation of adipose cells as compared to preadipocyte cell lines from the nongenetically obese mouse.
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Somatomedin was measured by rat bioassay in samples obtained from renal artery and vein, in order to study the role of this organ in the production of somatomedin activity. The results showed a significant difference, with an increase of the activity in the venous samples. According to the data reported in the literature with bioassay (low values) and radioreceptor assay (normal or high values) in nephropathic subjects, our results seem to suggest that the kidney mainly inactivates a specific somatomedin inhibitor.
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The ob17 cell line is a clonal line established from epididymal fat pads of C57 BL/6J ob/ob mice. After conversion into adipose-like cells, ob17 presents both the morphological and biochemical properties of mature rodent fat cells. The adipose conversion process is best represented by a stochastic model in which a pool of stem cells (adipoblasts) gives rise to clusters of adipose cells and to additional stem cells that remain in the population. The role of the different factors involved in the adipose conversion process of ob17 cells is discussed, i.e. 1) mitogenic factors, that enhance the number of committed cells (ACF or adipose conversion factor(s)) 2) lipogenic factors, that enhance the expression of adipocyte enzyme markers (insulin) and 3) adipogenic factors, that are obligatory requirements for adipose conversion (triiodothyronine, growth hormone and other pituitary factors).