Increase in membrane uptake of long-chain fatty acids early during preadipocyte differentiation.

An increase in early rates of oleate uptake, which reflected fatty acid (FA) entry into the cells, was apparent 2-3 days after confluence of differentiating BFC-1 preadipocytes. The increase was measured in cells kept without glucose and with arsenate, where greater than 95% of intracellular radioactivity was recovered as free unesterified oleate. Uptake of retinoic acid, a molecule structurally similar to long-chain FA, remained unaltered during cell differentiation.

Metabolism of oleic acid in differentiating BFC-1 preadipose cells.

Incorporation of [3H]oleate and [14C]glucose into cellular lipids was studied in the preadipose cell line BFC-1 to determine flux changes that accompany the adipose conversion process. Dilution of oleate by intracellular fatty acids (FA) was estimated from the 3H/14C incorporation ratios and from relating steady-state radioactivity in diglycerides to their measured cellular levels. The data indicated that exogenous FA mixed with less than 1% of endogenous FA on its pathway to esterification.

The adipocyte: relationships between proliferation and adipose cell differentiation.

The differentiation of adipose precursor cells can be divided into early and late events. Growth arrest at the G1/S boundary triggers the activation of early genes, i.e.

Regulation of phosphoenolpyruvate carboxykinase gene expression by insulin. Use of the stable transfection approach to locate an insulin responsive sequence.

H4IIE rat hepatoma cells were stably transfected with various phosphoenolpyruvate carboxykinase-chloramphenicol acetyltransferase (PEPCK-CAT) expression vectors. The regulation of the transfected genes was qualitatively similar to that of the endogenous PEPCK gene. CAT expression was increased in response to cAMP and dexamethasone and insulin overrode these effects at concentrations known to be effective in suppressing transcription of the endogenous gene.

Identification of a sequence in the PEPCK gene that mediates a negative effect of insulin on transcription.

Phosphoenolpyruvate carboxykinase (PEPCK) governs the rate-limiting step in gluconeogenesis. Glucocorticoids and adenosine 3',5'-monophosphate (cAMP) increase PEPCK gene transcription and gluconeogenesis, whereas insulin has the opposite effect. Insulin is dominant, since it prevents cAMP and glucocorticoid-stimulated transcription.

Coupling growth arrest and adipocyte differentiation.

The complete differentiation program of preadipose cells can be divided into early and late events. The expression of early markers takes place at growth arrest (G1/S boundary), whereas that of late markers, leading to terminal differentiation, takes place after a limited number of mitoses of early marker-containing cells. Only terminal differentiation requires the presence of growth hormone and triiodothyronine and results in the formation of triacylglycerol-filled, nondividing cells.

Genetic control of plasma membrane adhesion and fusion in Chlamydomonas gametes.

The final stages of gamete interaction in Chlamydomonas reinhardtii occur between the specialized plasma membrane organelles (mating structures) of opposite mating-type cells. A number of mutants affecting these interactions have been obtained in several laboratories; all have been defined as fusion-defective. This paper demonstrates that there are at least two genetically definable stages of mating-structure interaction: (1) the recognition/adhesion of mating structures, and (2) gamete fusion.

Characterization of mitochondrial-uncoupling protein in bovine fetus and newborn calf.

Development changes in the content of the mitochondrial-uncoupling protein (UCP) have been studied in adipose depots of bovine fetuses and a newborn calf as well as in adipose depots of newborn and aging lambs. The occurrence of UCP unique to brown adipose tissue (BAT) was investigated by GDP binding, photoaffinity labeling with 8-azidoadenosine 5'-triphosphate, and immunoblots using specific antibodies directed against rat UCP. A protein of 32,000 relative molecular weight was characterized in both species with properties similar to those of rodent UCP.

Expression of the mitochondrial uncoupling protein in brown adipocytes. Absence in brown preadipocytes and BFC-1 cells. Modulation by isoproterenol in adipocytes.

The expression of the uncoupling protein has been compared in cells of BFC-1 clonal line established from mouse brown adipose tissue (BAT) and in preadipocytes, as well as in adipocytes from mouse BAT, both in primary culture. The results of immunoblots show that, after one week in culture, adipocytes have a reduced level of the 32 kD protein. This level can be raised 2-3.

A preadipocyte clonal line from mouse brown adipose tissue. Short- and long-term responses to insulin and beta-adrenergics.

A clonal cell line has been established from the interscapular brown adipose tissue (BAT) of the C57 BL/6J +/+ mouse. The line, designated BFC-1, is aneuploid and exhibits both morphological and biochemical properties characteristics of mature adipocytes. Adipose conversion begins after confluence and is accompanied by an early emergence of lipoprotein lipase; a later emergence of glycerol-3-phosphate dehydrogenase and acid: CoA ligase; an increase in the average triglyceride content.

Adipose conversion of ob17 cells and hormone-related events.

The ob17 preadipocyte clonal line has been established from the adipocyte fraction of the epididymal fat pads of adult C57 BL/6J ob/ob mice. In vivo, injection of ouabain-resistant mutant cells (ob 17OR11 cell line) into athymic mice is followed by the formation of fat pads containing ouabain-resistant mature fat cells. In vitro, ob17 cells develop after confluence biochemical and morphological characteristics of adipocytes.

[Lipoprotein lipase and adipocyte differentiation].

Some hormonal factors, possibly involved in the proliferation and differentiation of adipose precursor cells in vivo, have been characterized in vitro using different preadipocyte cell lines established from rodent adipose tissue. The process of adipose conversion has also been studied using these cell lines; in this process, stem cells (adipoblasts) were committed at any cell division during the growth phase. At confluence, committed cells (preadipocytes) underwent a limited number of mitoses and differentiated into adipose cells, whereas the uncommitted cells remained as stem cells in the cell population.

Specific contact between mating structure membranes observed in conditional fusion-defective Chlamydomonas mutants.

Mating interactions between opposite mating type gametes in Chlamydomonas reinhardi can be conditionally manipulated by the use of mutants which control different stages in this process. Two independently isolated, sex-limited mutants (expressed only in mating type (-) (mt-] gam-10 and gam-11, have been shown to display a temperature-sensitive defect in mating. Gametes of these mutants can agglutinate, signal, and participate in a specific contact between their activated mating structures and the mating structures of wild-type mt+ gametes but are unable to fuse at the restrictive temperature.

Modulation of lipid-synthesizing enzymes by insulin in differentiated ob17 adipose-like cells.

ob17 cells convert into adipose-like cells when maintained in the presence of physiological concentrations of insulin and tri-iodothyronine. After this conversion, insulin removal from differentiated ob17 cells gives within 24-48 h a large decrease in fatty acid synthetase, glycerol 3-phosphate dehydrogenase and acid:CoA ligase activities, as well as in the rate of fatty acid synthesis determined by [14C]acetate incorporation into lipids. All parameters are restored by insulin addition to initial values within 24-48 h.

Mutational disruption of the 9 + 2 structure of the axoneme of Chlamydomonas flagella.

A mutant of Chlamydomonas, gam-5, which has short but variable-length, paralysed flagella has been characterized using electron microscopy and genetics. The flagellar axoneme shows varying degrees of disorganization of its 9 + 2 structure, and the structures that attach to the microtubule pairs may be wholly or partly missing or, if present, abnormal. Flagellar length is inversely related to temperature, and gametes of this mutant are generally incapable of mating at higher temperatures.

Hormonal requirements for growth and differentiation of ob17 preadipocyte cells in vitro.

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.

Establishment of a preadipocyte cell line from the epididymal fat pad of the lean C57 BL/6J mouse--long term effects of insulin and triiodothyronine on adipose conversion.

A clonal cell line has been established from the epididymal fat pad of the C57 BL/6J +/? mouse. This line, designated HGFu, is aneuploid and exhibits both morphological and biochemical properties characteristic of mature adipocytes. Adipose conversion begins after confluence and is accompanied by (a) an early emergence of lipoprotein lipase, (b) an increase in the incorporation of [14C]acetate into lipids and in the activities of acid:CoA ligase and glycerol-3-phosphate dehydrogenase, (c) a 27- to 35-fold increase in the average triglyceride content per cell.

Lipogenic and mitogenic effects of insulin during conversion of Ob17 cells to adipose-like cells.

Chronic exposure to insulin of confluent cells of a preadipocyte clonal line (Ob17) leads to an acceleration of their development into adipose cells. The short-term effects of insulin have been examined by the stimulation of [14C] alpha-aminoisobutyrate uptake and the long-term effects by the increase in the activity levels of several lipogenic enzymes and in the intracellular triacylglycerol content. These metabolic effects of insulin occur within a physiological range of concentrations (EC50 congruent to 1 nM).

Establishment of a human cell line after transformation by a plasmid containing the early region of the SV40 genome.

A human clonal cell line, designated TAH9, has been established from cells of adipose tissue by fusion with E. coli protoplasts containing a recombinant plasmid carrying the early genes of Simian virus 40. The establishment of the cell line is preceeded by a delayed growth crisis.