The regulation by growth hormone of lipoprotein lipase gene expression is mediated by c-fos protooncogene.

GH has been previously shown in Ob1771 adipose cells to activate transiently the expression of c-fos gene by a protein kinase-C-dependent pathway and to modulate, at last in part by a protein kinase-C-dependent pathway, the expression of the lipoprotein lipase (LPL) gene. In Ob1771 cells exposed to GH, under conditions where protein synthesis is inhibited by cycloheximide, the modulation of LPL gene expression is prevented, suggesting that synthesis of trans-acting factor(s) is required to modulate LPL gene expression. The present results indicate the involvement of c-Fos protein in this modulation; this involvement is supported by various lines of evidence: 1) upon GH stimulation, the increase in c-fos mRNA content is followed by the emergence of c-Fos protein within the nucleus, and this emergence precedes the increase in LPL mRNA content; 2) in GH-treated Ob1771 cells, exposure to antisense sof oligonucleotides abolishes the synthesis of c-Fos protein; and 3) at the same time, the increase in LPL mRNA content and LPL activity does not occur, whereas sense fos oligonucleotides show no effect. It is concluded that c-Fos protein plays an intermediary role in the modulation of LPL gene expression by GH.

Processing and secretion of tumor necrosis factor alpha in endotoxin-treated Mono Mac 6 cells are dependent on phorbol myristate acetate.

Lipopolysaccharide (LPS, endotoxin) is a potent stimulator of tumor necrosis factor alpha (TNF alpha) synthesis and secretion in mouse macrophage tumor cells (Golenbock, D. T., Hampton, R. Y., Qureshi, N., Takayama, K., and Raetz, C. H. R. (1991) J. Biol. Chem. 266, 19490-19498). In contrast, addition of LPS (10 ng/ml) to human monomyelocytic (Mono Mac 6) cells induces very little production of TNF alpha, as judged by immunoassay of the growth medium. When 30 ng/ml 4-beta-phorbol-12-myristate 13-acetate (PMA) is added together with LPS, large amounts of TNF alpha are secreted. PMA alone is inactive. Maximal TNF alpha levels in the medium are achieved at 1 ng/ml of LPS. Protein kinase C inhibitors, such as H7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine), staurosporine, and sphingosine, reduce TNF alpha secretion stimulated by PMA. The effect of PMA has been investigated at each stage of TNF alpha biogenesis. Treatment of Mono Mac 6 cells with LPS alone results in rapid, transient, and full expression of TNF alpha mRNA. Concomitant addition of PMA does not increase TNF alpha mRNA synthesis any further, but it prolongs the half-life of TNF alpha mRNA about 3-fold. However, mRNA stabilization does not account for the striking effect of PMA on TNF alpha secretion. Analysis of TNF alpha synthesis and secretion by immunoprecipitation indicates that LPS alone is fully effective in stimulating the formation of the intracellular 26-kDa TNF alpha precursor. LPS alone is not sufficient to allow processing of the precursor and secretion of mature 17-kDa TNF alpha. The rate of TNF alpha secretion observed immediately after the addition of PMA to LPS-pretreated cells is similar to the maximum rate from LPS/PMA-treated cells, but without the lag observed in cells after being exposed to LPS and PMA simultaneously. In summary, PMA is required for the completion of TNF alpha precursor processing and secretion in LPS-treated human Mono Mac 6 cells, whereas murine RAW cells are able to complete the terminal steps of TNF alpha processing in the absence of PMA.

Lipoprotein lipase stored in adipocytes and muscle cells is a cryptic enzyme.

The status of lipoprotein lipase (LPL) has been examined in different cell types (adipose, skeletal muscle, and heart muscle cells) and different tissues (adipose, muscle, and cardiac tissues) from mouse, rat, and human. Cell and secreted activities were compared in cycloheximide-, heparin-treated cells present in culture. A gross underestimation of cell LPL activity was found; excess of LPL over substrate and/or apolipoprotein C-II was excluded as well as inhibition by cell component(s) or detergent molecules used to disrupt membrane structures in the cell lysates. Unmasking of LPL activity occurred upon dilution: the higher the concentration of LPL, the higher were the dilution factor and the concentration of heparin required to reach a plateau of activity. This maximal value was found to be identical to that determined in the secretion medium, indicating that the cell LPL activity can be determined in toto. The unmasking effect of dilution upon LPL activity was extended to adipose, muscle, and cardiac tissues from rat and to adipose tissues from mouse and human. In agreement with previous results (Vannier et al., 1989, J. Biol. 264: 13199-13205), our results are in favor of LPL as being cryptic within the cell. A model is proposed, in which potentially active LPL molecules are present as aggregates in various membrane compartments. It is concluded that the determination of the pool size of catalytically active cell LPL has to be estimated in vitro under the appropriate conditions described herein.

Purification of an apolipoprotein A binding protein from mouse adipose cells.

A protein recognizing apolipoproteins AI, AII and AIV was purified from cultured mouse adipose cells of the Ob17MT18 clonal line. Apolipoprotein A binding sites were solubilized in the presence of proteinase inhibitors using the non-denaturating detergent CHAPS. Chromatography of the soluble extract on DEAE-Trisacryl was followed by immunoaffinity chromatography of the complex apolipoprotein AI-binding proteins on anti-(apolipoprotein AI) coupled to Sepharose 4B and then by h.p.l.c. on an RP-Select B column. A 1400-fold purification over the starting crude homogenate was achieved. The purified material contained two proteins that were both able to bind apolipoproteins AI, AII and AIV, but not low-density lipoprotein. Glycopeptidase F treatment showed the existence of a single protein bearing either N-linked high-mannose or complex oligosaccharide chains. The purified material showed an apparent molecular mass of 80 +/- 9 kDa by h.p.l.c. on a TSKG 3000 SW column. Rabbit polyclonal antibodies directed against the purified material revealed two protein bands of 80 and 92 kDa after SDS/PAGE under reducing conditions and immunoblotting. These bands were undetectable in growing Ob17PY cells previously shown not to bind the various apolipoproteins A and not to undergo cholesterol efflux, whereas they were conspicuous in growth-arrested Ob17PY cells which have recovered these properties.

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, corticosteroids and oleic acid) and high-molecular weight, hydrophilic components (TGF-beta 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.

Biosynthesis of lipoprotein lipase in cultured mouse adipocytes. I. Characterization of a specific antibody and relationships between the intracellular and secreted pools of the enzyme.

Polyclonal antibodies have been raised in rabbits against homogeneous lipoprotein lipase (LPL) purified from the media of adipose 3T3-F442A cells. The antibody is able to inhibit the apolipoprotein C-II-dependent activity of LPL, to immunoprecipitate LPL under nondenaturating conditions from media and cellular extracts. A dot-blot immunoassay of secreted LPL is also described (range 0.1-0.7 milliunits). The secretion potential mu, taken as the ratio of total releasable activity or antigen to initial cellular activity or antigen, was determined. This was shown in cells treated with heparin and cycloheximide to be equal to 1 for LPL antigen but significantly greater than 1 for LPL activity assayed under standard conditions. No LPL was actually degraded within the cells. A dramatic enhancement of the intracellular activity was induced by a mere dilution of detergent-treated cell lysates with no change in LPL antigen. The total intracellular activity reached a plateau at a value which now became identical to that obtained in the medium of cells exposed to heparin and cycloheximide. The existence of an inhibitor of LPL activity has been excluded as well as that of an increase in the catalytic activity of LPL during its secretion, before or after exposure to heparin. Our results indicate a systematic underestimation of LPL intracellular activity and suggest that LPL is present within intracellular cisternae in a cryptic state. This potential activity can be fully unmasked in vitro. In agreement with other data (Vannier, C., and Ailhaud, G., (1989) J. Biol. Chem. 264, 13206-13216), our results appear to exclude the existence of a reservoir of catalytically inactive LPL molecules within adipose cells.

Short-term stimulation by insulin of lipoprotein lipase secretion in adipose cells.

The spontaneous secretion of lipoprotein lipase has been examined in adipose cells of mouse Ob17, Ob17SA and 3T3-F442A clonal lines as well as in rat adipose cells in primary culture. Striking differences are observed both in serum-free and serum-supplemented media, rat adipose cells and 3T3-F442A cells being the most active. Insulin from 10(-11) M to 10(-9) M was able to modulate the rate of LPL secretion from 2- to 4-fold. The stimulatory effect of insulin on this process occurred within 30 min in cells treated or not with cycloheximide. It is concluded that insulin is able to modulate the rate of LPL secretion independently of the synthesis of new enzyme molecules on a short-term basis and within a physiological range of concentrations.