Ob1771 Cells Research Articles

Transcriptional control of the expression of lipoprotein lipase gene by growth hormone in preadipocyte Ob1771 cells.

A direct and modulating effect of growth hormone (GH) on the regulation of the lipoprotein lipase (LPL) gene has been shown in preadipocyte Ob1771 cells. Growth hormone acts as a modulator within the physiological range of concentrations and regulates the abundance of the two species of LPL mRNAs (3.3 and 3.7 kb) in a differentiation-dependent manner, the stimulation factor being between 4- and 7-fold. The regulation of LPL gene expression by GH is rapid (2 to 8 h) and similar for both mRNA species. It is reversible and takes place primarily at a transcriptional level. Parallel increases of LPL mRNAs, LPL protein, and LPL activity are observed. The expression of both cellular and secreted activities is stimulated by GH. The role of GH is mediated, at least in part, by means of activation of protein kinase C. In the presence of 4-beta-phorbol-12-myristate 13-acetate (PMA), a parallel increase of LPL mRNA content and LPL activity is observed at half the values obtained upon stimulation by GH. The kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) abolishes completely the PMA-induced accumulation but decreases only by half that induced by GH. Like H7, staurosporine, polymixin B, and sphingosine inhibit only by half the stimulatory effect of GH on the expression of the LPL gene. These results show for the first time a rapid regulation of the LPL gene expression at a transcriptional level. Ob1771 cells should be helpful in gaining some insights in the promoter function of the LPL gene and the trans-acting factors involved in its regulation.

Inhibition by serum components of the expression of lipoprotein lipase gene upon stimulation by growth hormone

Growth hormone regulates in a positive way the expression of the lipoprotein lipase gene at a transcriptional level in preadipocyte Ob1771 cells. Inhibition by serum components of this expression was investigated upon stimulation by growth hormone. Low-molecular weight, lipid-soluble components (a serum lipid extract, corticosteroïds and oleic acid) and high-molecular weight, hydrophilic components (TGF-β and those present in delipidated serum) were inhibitory. Inhibition of the expression of LPL mRNAs and that of LPL activity were parallel. It is concluded that the regulation of the expression of LPL gene occurs likely at a transcriptional level and that a balance between multiple effectors present in serum are active in an opposite manner.

Cloning and regulation of a mRNA specifically expressed in the preadipose state

A cDNA library of Ob1771 preadipocytes was constructed, and a cDNA clone designated pOb24 was isolated by differential screening. The pOb24 mRNA, 6 kilobases in length, rose sharply in early differentiating Ob1771 and 3T3-F442A cells and decreased thereafter. In mouse adipose tissue, it was present at a high level in stromal-vascular cells (containing adipose precursor cells) and at a low level in mature adipocytes. Thus, pOb24 mRNA appears to be both in vitro and in vivo an unique marker of the preadipose state, i.e. of cell commitment during adipose cell differentiation. In contrast to glycerol-3-phosphate dehydrogenase mRNA, the emergence of pOb24 mRNA in Ob1771 cells required neither growth hormone or triiodothyronine as obligatory hormones nor insulin as a modulating hormone. Comparative studies of the expression of pOb24 and dihydrofolate reductase genes during the cell cycle suggest that arrest at the G1/S boundary was critical for the entry into the preadipose state. Tumor necrosis factor and transforming growth factor-beta were able to induce a large decrease of pOb24 mRNA level in growth-arrested Ob1771 cells. This decrease was shown to be only confined to early differentiating, glycerol-3-phosphate dehydrogenase negative cells as no decrease of pOb24 mRNA level was observed in glycerol-3-phosphate dehydrogenase positive cells. This result suggests that signals generated by tumor necrosis factor and transforming growth factor-beta have no effect on a commitment-related gene in late differentiated cells.

Growth hormone stimulates c-fos gene expression by means of protein kinase C without increasing inositol lipid turnover.

Growth hormone (GH) is required for the terminal differentiation of preadipose Ob1771 cells that have entered the differentiation program as evidenced by the expression of early marker genes (pOb24 and lipoprotein lipase). Induction of c-fos mRNA within 15 min and induction of insulin-like growth factor I mRNA within a few hours take place in response to GH. The role of GH is mediated, at least in part, by means of the activation of protein kinase C, as shown by the inhibition of epidermal growth factor binding and by the expression of the c-fos gene, and is thus analogous to the action of prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate in this respect. However, in contrast to that of the c-fos gene, the regulation of insulin-like growth factor I gene expression by GH is not mediated by means of the activation of protein kinase C, and, in line with this, prostaglandin F2 alpha and 4 beta-phorbol-12,13-didecanoate were ineffective. GH and prostaglandin F2 alpha were able to stimulate the formation of diacyglycerol within a few seconds, but GH did not elicit an accumulation of inositol phosphates, in contrast to that generated by prostaglandin F2 alpha. We conclude that the transduction signal of GH action in c-fos mRNA induction is the formation of diacylglycerol and that the mechanism whereby GH can activate protein kinase C is associated with a phospholipase C-mediated hydrolysis of glycerophospholipids other than inositol phospholipids.

Requirement and role of arachidonic acid in the differentiation of pre-adipose cells

The terminal adipose differentiation of Ob1771 cells, characterized by glycerol-3-phosphate dehydrogenase activity and triacylglycerol accumulation, was studied in serum-free hormone-supplemented medium containing growth hormone, tri-iodothyronine, insulin, transferrin and fetuin. Arachidonic acid was able to substitute for a crude adipogenic fraction isolated from fetal bovine serum but not for growth hormone or tri-iodothyronine. Arachidonic acid was also able to increase in a rapid and dramatic manner cyclic AMP production; moreover it was able to amplify the adipose conversion promoted by other agents elevating cyclic AMP concentrations and to induce inositol phospholipid breakdown. Both phorbol 12-myristate 13-acetate, a protein kinase C activator and ionomycin, a Ca2+-mobilizing agent, showed potent synergy with agents elevating cyclic AMP concentrations for the promotion of adipose conversion, whereas 8-bromo cyclic GMP and 4 alpha-phorbol 12,13-dibutyrate were ineffective. The triggering of both the cyclic AMP and inositol phospholipid pathways was accompanied by a single round of cell division, and within a few days all the cells became differentiated. Similar results were obtained, after exposure to arachidonic acid, with preadipose 3T3-F442A cells and with rat adipose precursor cells in primary culture. The availability of arachidonic acid from intracellular stores and/or of exogenous origin should play a major role for the onset of critical mitoses leading to terminal differentiation in pre-adipose cells.

Relationship in adipose cells between the presence of receptor sites for high density lipoproteins and the promotion of reverse cholesterol transport

The role of a high-affinity receptor site for high-density lipoproteins (HDL) has been investigated in parental Ob1771 adipose cells and their transformed counterparts after transfer of the complete early region of polyoma virus (Ob17PY cells). Binding of ApoA I, ApoA II and HDL 3 occurs in Ob1771 cells and derived membranes, whereas no binding is observed in Ob17PY cells and derived membranes. After thymidine block, growth-arrested Ob17PY cells become able to bind ApoA I, ApoA II and HDL 3; this recovery is prevented in actinomycin D- or cycloheximide-treated cells. In contrast to ApoA I, ApoA II or HDL 3 binding, both growing and growth-arrested Ob17PY cells do show receptor activities for low density lipoproteins and transferrin, respectively, which are similar in affinity and maximal capacity. Following cholesterol accumulation which takes place in the presence of LDL cholesterol, subsequent exposure to HDL 3 or ApoA I promotes cholesterol efflux from Ob1771 cells and growth-arrested Ob17PY cells but not from growing Ob17PY cells. These results show that the presence of a high-affinity receptor site for HDL in intact adipose cells is required for the promotion of reverse cholesterol transport.

Acute regulation of insulin-like growth factor-I gene expression by growth hormone during adipose cell differentiation.

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide that is thought to play, under the control of growth hormone, a role in fetal development as well as post-natally. The direct effect of growth hormone on the regulation of the expression of IGF-I gene was examined in adipose Ob1771 cells. Growth hormone regulates the abundance of multiple species of IGF-I mRNAs of 15, 7.5, 1.5 and 0.8 kb in a differentiation-dependent manner. The regulation of IGF-I gene expression is strikingly rapid (less than 2 h), reversible and takes place primarily at transcriptional level. Thus growth hormone can increase the cellular content of IGF-I mRNA encoding for a protein which could be involved in a paracrine/autocrine action during adipose tissue development.

Cholesterol efflux from cultured adipose cells is mediated by LpA I particles but not by LpA I:A II particles

Cholesterol efflux was studied in cultured adipose cells after preloading with LDL cholesterol. Long-term exposure to LpA I and LpA I:A II particles isolated from the HDL fraction showed that LpA I particles only were able to promote cholesterol efflux. Liposomes containing different ApoA I ApoA II molar ratios were tested: the larger the proportion of ApoA I, the faster the ability to remove cholesterol from Ob1771 cells. Dose-response curves showed that LpA I particles were active within a physiological range of concentrations, whereas LpA I:A II particles had no effect at all concentrations. The results are in favour of LpA I particles being the active components of the HDL fraction for the promotion of cholesterol efflux and suggest that LpA I particles and LpA I:A II particles represent distinct metabolic entities.

Role of spermidine in the expression of late markers of adipose conversion. Effects of growth hormone.

Confluent Ob1771 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxyal bis(guanylhydrazone), were dependent on putrescine addition for the expression of glycerol-3-phosphate dehydrogenase and acyl-CoA synthetase, which behaved as late markers of adipose conversion. A similar dependence was observed with drug-treated Ob17MT18 and 3T3-F442A preadipocyte cells, but not with non-differentiating 3T3-C2 cells. Studies in drug-treated Ob1771 cells at the mRNA level showed that the parallel expression of mRNAs encoding for glycerol-3-phosphate dehydrogenase and an homologue of serine proteinases of Mr 28,000 [Cook, Groves, Min & Spiegelman (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 6480-6484] was also dependent on putrescine addition. Double-isotope experiments with [14C]putrescine and [3H]spermidine, as well as analysis of the polyamine content in drug-treated Ob1771 cells under various conditions, demonstrate after putrescine addition that the expression of late markers of adipose conversion was highly correlated with a 2-fold increase in the intracellular concentration of spermidine. No correlation was observed with changes in the intracellular concentrations of putrescine and spermine. Long-term exposure of untreated Ob1771 cells to growth hormone, which led to the expression of late markers of adipose conversion [Doglio, Dani, Grimaldi & Ailhaud (1986) Biochem. J. 238, 123-129] was also accompanied by the same increase in spermidine concentration, which attained values identical with those determined in drug-treated cells supplemented with putrescine. This observation suggests that the permissive effect of growth hormone on the terminal differentiation of adipose cells might e related to changes in the intracellular concentration of spermidine.

Growth hormone regulation of the expression of differentiation-dependent genes in preadipocyte Ob1771 cells.

The adipose conversion of Ob1771 preadipocytes, during exposure to medium containing bovine serum and supplemented with growth hormone, is accompanied by the acquisition of phenotypic markers and the increased accumulation of specific mRNAs. The expression of lipoprotein lipase, and that of unidentified pOb24 and pGH3 mRNAs, are early events which are independent of growth hormone supplementation. By contrast, the late expression of mRNAs encoding for glycerol-3-phosphate dehydrogenase and p422 protein (a myelin-P2 homologue) and that of glycerol-3-phosphate dehydrogenase activity require the presence of growth hormone. The abundance of beta-actin mRNA does not change during differentiation. Runoff transcription by nuclei isolated from untreated or growth hormone-treated cells reveal little or no change in the rates of transcription of pOb24, pGH3 and beta-actin mRNAs. By contrast, the transcription rate of the p422 gene increases markedly (greater than 6-fold) in nuclei of growth hormone-treated cells. However, the p422 mRNA is more abundant than would be predicted by its nuclear transcription alone, suggesting, in Ob1771 cells exposed to growth hormone, that there is a post-transcriptional level of control. These results indicate that the permissive role of growth hormone during adipose cell differentiation is related to terminal events only and that its effects can be seen both at the protein and mRNA level. These results strongly suggest that an increased rate of specific transcription is primarily responsible for the accumulation of mRNAs during exposure to growth hormone.

Expression of the phosphoenolpyruvate carboxykinase gene and its insulin regulation during differentiation of preadipose cell lines

The adipose conversion of Ob1771 and 3T3-F442A preadipose cells is accompanied by the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. The PEPCK mRNA is absent from growing, undifferentiated Ob1771 and 3T3-F442A cells as well as from non-differentiating 3T3-C2 cells. It is present in differentiated Ob1771 and 3T3-F442A cells as well as in liver, kidney and white adipose tissue from mouse. Transcriptional run-off measurements in nuclei isolated from undifferentiated and differentiated Ob1771 and 3T3-F442A cells reveal that the PEPCK gene transcription is activated during differentiation. Studies of the time course of changes indicate that the emergence of PEPCK mRNA takes place in parallel to mRNA encoding for a 28 kDa protein (28 K mRNA) but later than that encoding for glycerol-3-phosphate dehydrogenase (GPDH mRNA). Insulin leads to an increase in the content of PEPCK and GPDH mRNAs with half-maximally effective concentrations of 0.5 and 5 nM for GPDH mRNA and PEPCK mRNA, respectively. Thus, in contrast to rat hepatoma cells, insulin exerts in adipose cells a positive regulation on the expression of the PEPCK gene.

Coupling of growth arrest and expression of early markers during adipose conversion of preadipocyte cell lines

After growth arrest at the entry of the S phase of the cell cycle, Ob1771 and 3T3-F442A cells, but not 3T3-C2 cells, accumulate lipoprotein lipase and pOb24 mRNA that are early markers of adipose conversion. Removal of the single- or double-thymidine block when cultured cells are present at low density leads first to DNA synthesis and growth resumption, then to a continuous proliferation and a rapid disappearance of these markers. By contrast, growth-arrested Ob1771 cells reinoculated at high density undergo a single round of cell division, maintain high levels of early marker(s) and acquire with time both glycerol-3-phosphate dehydrogenase and lipids. Thus, depending upon the conditions in culture, growth-arrested cells can undergo either a dedifferentiation leading to a loss of early markers or a terminal differentiation leading to the acquisition of late markers.