The fat cell adenylate cyclase system. Characterization and manipulation of its bimodal regulation by GTP.

GTP evoked both an activatory and an inhibitory response from adipocyte adenylate cyclase. This paper describes the persistence of the bimodal response under a variety of assay conditions. Additionally, manipulations are described which eliminate one or other of these actions. Treatment of adipocyte plasma membranes with cholera toxin A1 peptide and NAD+ abolishes the inhibitory phase of GTP action while preserving the activating phase. Treatment of the membranes with p-hydroxymercuriphenylsulfonic acid eliminates the activatory phase while maintaining the inhibitory processes mediated by GTP in adipocytes normally coexist and operate through different pathways since either phase can be abolished leaving the other intact. Adenosine and its purine-modified analogs inhibit fat cell adenylate cyclase in the GTP inhibitory phase (Londos, C., Cooper, D. M. F., Schlegel, W., and Rodbell, M. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5362-5366). When this effect of GTP is abolished by either cholera toxin or Gpp(NH)p pretreatment, the inhibitory action of adenosine analogs is also lost. These data suggest a central role for GTP in mediating both activation and inhibition of adenylate cyclase by agents which act through cell surface receptors.     

Effects of clofibrate on the human fat cell adenylate cyclase system.

The effects of chlofibrate on the adenylate cyclase system of human adipocytes were studied. Clofibrate reduced basal as well as hormone-NaF)stimulated adenylate cyclase activities to about the same extent (45% inhibition at 1 mg/ml clofibrate). The relative extent of hormonal stimulation was not altered by this compound. The inhibitory action of clofibrate was non-competitive with respect to the substrate ATP and cofactors (Mg2+-ions). Inhibition of enzyme activity was detectable after 2.5 min. Our results suggest that the antilipolytic activity of clofibrate is mediated via inhibition of the catalytic subunit of the fat cell adenylate cyclase.     

A modified perifused incubation system for isolated fat-cells. Investigation of intermediary metabolism by perifusion.

Perifused fat-cells showed similar values for acylglycerol glycerol synthesis from glucose with insulin and for the effects of added palmitate to those in normal incubations and those reported in the literature. Fatty acid synthesis was lower in perifused cells compared with normal incubations, and there was a net release of fatty acids only with the perifused fat-cells. Hence fluxes of metabolites were different in the two incubation systems, and the perifusion system enables the investigation of the flux of metabolites under conditions which may more closely resemble those in vivo.     

Differential effects of sulfhydryl reagents on activation and deactivation of the fat cell hexose transport system.

A rapid filtration method was used to measure initial rates of 3-O-[3H]methylglucose uptake and thus estimate hexose transport system activity in isolated white fat cells. Insulin markedly stimulated the transport system activity and its effect was rapidly and completely reversible. In addition, such oxidants as vitamin K5 (50 muM), hydrogen peroxide (4mM), methylene blue (50 muM), and diamide (20 mM) also maximally activated 3-O-methylglucose transport and their effects were not additive to those of maximal concentrations of insulin. These oxidants had no effect on total cellular ATP levels under these conditions. Hexose transport system activity in either the presence or absence of these stimulatory agents was uniformly sensitive to inhibition by cytochalasin B. Treatment of fat cells with either 0.5 mM N-ethylmaleimide or 3 mM dithio(bis)nitrobenzoic acid abolished the ability of insulin or oxidants to activate hexose transport system activity. Control transport activity was not significantly influenced by these agents. Fat cells treated with dithio(bis)nitrobenzoic acid completely regained the ability to respond to insulin or vitamin K5 after removal of the agent by washing in low concentrations of reductant. Elevated rates of transport due to prior incubation of cells with insulin or vitamin K5 were completely resistant to inhibition by subsequent addition of N-ethylmaleimide or dithio(bis)nitrobenzoic acid. Deactivation of the hormone-stimulated transport system could be achieved by washing cells free of insulin or by destruction of insulin-receptor interaction by trypsin. N-Ethylmaleimide effectively blocked deactivation of insulin-stimulated transport system activity, while dithio(bis)nitrobenzoic acid was without effect. These results suggest that distinct cellular components mediate activation versus deactivation of the fat cell hexose transport system. N-Ethylmaleimide, which effectively penetrates fat cells, inhibits both processes while the layer, more polar dithio(bis)nitrobenzoic acid blocks activation but not deactivation of this transport system.     

Characterization of oxytocin receptors on isolated rat fat cells.

The binding of 3H-labelled neurohypophyseal nonapeptide hormone, oxytocin, to isolated rat fat cells has been measured under conditions where this compound elicits the known activation of glucose oxidation by these cells, called "insulin-like" action. Uptake by the cells of the [3H]peptide as a function of various concentrations of the hormone in the medium indicated the presence of two classes of binding sites with different apparent affinities and capacities. The sites of the first type exhibit a rather high affinity, but low capacity, for oxytocin (5 nM; 3 X 10(4) sited per cell) and appear to be saturable under a reversible process. Evaluation of dose-response relationships suggest that they may be directly related to the measured biological response (i.e. activation of the glucose to 14CO2 conversion). Competition experiments show that [3H]oxytocin binding to the cells remains constant within a large range of insulin concentrations. The apparent capacity of different hormone analogs to compete with oxytocin for binding to this class of receptors has been evaluated and compared with the measured insulin-like activity of these different compounds. The sites of the second category have significantly lower affinity, but higher capacity for oxytocin, and were found to be not saturable under the experimental conditions. [3H]Oxytocin uptake by ghosts prepared from the isolated fat cells showed striking similarities to the binding process described for whole cells, although the affinity and total capacity of the former were found to be slightly lower. The basal and adrenalin-stimulated adenylate cyclase of these fractions appeared to be unaffected by various concentrations of oxytocin. It is concluded that there may exist on the rat fat cell membranes a discrete number of oxytocin receptors possessing high specificity for oxytocin and exhibiting affinities and kinetic behaviour similar to those of other characterized oxytocin receptors. They are believed to be independent of the other hormonal receptors of the rat fact cells.     

Application of cell co-culture system to study fat and muscle cells

Animal cell culture is a highly complex process, in which cells are grown under specific conditions. The growth and development of these cells is a highly unnatural process in vitro condition. Cells are removed from animal tissues and artificially cultured in various culture vessels. Vitamins, minerals, and serum growth factors are supplied to maintain cell viability. Obtaining result homogeneity of in vitro and in vivo experiments is rare, because their structure and function are different. Living tissues have highly ordered complex architecture and are three-dimensional (3D) in structure. The interaction between adjacent cell types is quite distinct from the in vitro cell culture, which is usually two-dimensional (2D). Co-culture systems are studied to analyze the interactions between the two different cell types. The muscle and fat co-culture system is useful in addressing several questions related to muscle modeling, muscle degeneration, apoptosis, and muscle regeneration. Co-culture of C2C12 and 3T3-L1 cells could be a useful diagnostic tool to understand the muscle and fat formation in animals. Even though, co-culture systems have certain limitations, they provide a more realistic 3D view and information than the individual cell culture system. It is suggested that co-culture systems are useful in evaluating the intercellular communication and composition of two different cell types.     

Alpha-adrenoceptor-mediated glucose release from perifused catfish hepatocytes.

In fish liver catecholamines bind to beta-adrenoceptors (AR) and increase glucose release via cAMP augmentation. Alpha1-AR have recently been shown to mediate IP3 and Ca2+ elevation in catfish and eel hepatocytes, although their coupling to a physiological response has remained doubtful. We have perifused isolated catfish hepatocytes in Bio-Gel P4 columns with epinephrine in the presence of prazosin and/or propranolol, alpha- and beta-AR antagonists, respectively. Ten nM epinephrine stimulated glucose release approximately 3-fold, and this effect was completely antagonized by the simultaneous presence of both alpha- and beta-AR blockers. The two AR antagonists separately inhibited about one-third and two-third of the total stimulation, respectively. Through alpha-AR occupancy, epinephrine provoked a significant increase of glucose release whereas no stimulation was detected in Ca2+-depleted hepatocytes. Glucose release was strongly elevated by both ionomycin and dibutyryl cAMP. These results represent the first direct evidence that alpha-AR transduction pathway is involved in epinephrine-induced glucose release from fish hepatocytes.     

Characterization of the calcium-dependent proteolytic system in a mouse muscle cell line

Many studies have demonstrated that the calcium-dependent proteolytic system (calpains and calpastatin) is involved in myoblast differentiation. It is also known that myogenic differentiation can be studied in vitro. In the present experiments, using a mouse muscle cell line (C₂C₁₂) we have analyzed both the sequences of appearance and the expression profiles of calpains 1, 2, 3 and calpastatin during the course of myoblast differentiation. Our results mainly show that the expression of ubiquitous calpains (calpain 1 and 2) and muscle-specific calpain (calpain 3) at the mRNAs level as well as at the protein level do not change significantly all along this biological process. In the same time, the specific inhibitor of ubiquitous calpains, calpastatin, presents a stable expression at mRNAs level as well as protein level, all along myoblast to myotube transition. A comparison with other myogenic cells is presented.     

Characterization of the calcium signaling system in the submandibular cell line SMG-C6

Establishment of salivary cell lines retaining normal morphological and physiological characteristics is important in the investigation of salivary cell function. A submandibular gland cell line, SMG-C6, has recently been established. In the present study, we characterized the phosphoinositide (PI)-Ca2+ signaling system in this cell line. Inositol 1,4,5-trisphosphate(1,4,5-IP3) formation, as well as Ca2+ storage, release, and influx in response to muscarinic, alpha1-adrenergic, P2Y-nucleotide, and cytokine receptor agonists were determined. Ca2+ release from intracellular stores was strongly stimulated by acetylcholine (ACh) and ATP, but not by norepinephrine (NA), epidermal growth factor (EGF), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFalpha). Consistently, 1, 4,5-IP3 formation was dramatically stimulated by ACh and ATP. ACh-stimulated cytosolic free Ca2+ concentration [Ca2+]i increase was inhibited by ryanodine, suggesting that the Ca2+-induced Ca2+ release mechanism is involved in the ACh-elicited Ca2+ release process. Furthermore, ACh and ATP partially discharged the IP3-sensitive Ca2+ store, and a subsequent exposure to thapsigargin (TG) induced further [Ca2+]i increase. However, exposure to TG depleted the store and a subsequent stimulation with ACh or ATP did not induce further [Ca2+]i increase, suggesting that ACh and ATP discharge the same storage site sensitive to TG. As in freshly isolated submandibular acinar cells, exposure to ionomycin and monensin following ACh or TG induced further [Ca2+]i increase, suggesting that IP3-insensitive stores exist in SMG-C6 cells. Ca2+ influx was activated by ACh, ATP, or TG, and was significantly inhibited by La3+, suggesting the involvement of store-operated Ca2+ entry (SOCE) pathway. These results indicate that in SMG-C6 cells: (i) Ca2+ release is triggered by muscarinic and P2Y-nucleotide receptor agonists through formation of IP3; (ii) both the IP3-sensitive and -insensitive Ca2+ stores are present; and (iii) Ca2+ influx is mediated by the store-operated Ca2+ entry pathway. We conclude that Ca2+ regulation in SMG-C6 cells is similar to that in freshly isolated SMG acinar cells; therefore, this cell line represents an excellent SMG cell model in terms of intracellular Ca2+ signaling.     

Characterization of the calpain/calpastatin system in human hemopoietic cell lines

As previously suggested by PCR analysis [R. DeTullio, R. Stifanese, F. Salamino, S. Pontremoli, E. Melloni, Characterization of a new p94-like calpain form in human lymphocytes, Biochem. J. 375 (2003) 689-696], a p94-like calpain was now established to be present in six different human cells resembling the various peripheral blood cell types. This protease resulted to be the predominant calpain isoforms whereas the conventional mu- and m-calpains are also expressed although at lower or almost undetectable amounts. The p94-like calpain has been identified by a specific mAb and displays unique features such as: Ca2+ requirement for half maximum activity around 30 microM; no autolytic conversion to a low Ca2+ requiring form and lower sensitivity to calpastatin inhibition. Following cell stimulation, the p94-like calpain undergoes inactivation, a process indicating that the protease is activated and participates in the cell responses to stimuli. The involvement of this protease isoform in immunocompetent cell activation is further supported by its partial recruitment on plasma membranes, the site of action of the conventional calpain forms. The amount of calpain translocated to the membranes correlates to the level of calpastatin which has been shown to control this process through the formation of a complex with calpain, which maintains the protease in the cytosol. These results provide new information on the calpain/calpastatin system expressed in immunocompetent cells and on the functional relationship between the p94-like calpain and the biological function of these cells.     

Conjugated linoleic acids promote human fat cell apoptosis.

Conjugated linoleic acids (CLAs) are conjugated dienoic isomers of linoleic acid. Some isomers have been shown to reduce fat mass in animal and cell culture models. However, controversial results were obtained in studies of supplementation of CLAs in human subjects. In order to get more insights into the direct effects of CLAs on human fat cells, we have studied the influence of cis-9, trans-11 CLA and trans-10, cis-12 CLA on the biology of human SGBS preadipocytes and adipocytes. Both CLA isomers equally inhibited the proliferation of preadipocytes in a dose-dependent manner. Continuous treatment with 1-10 microM trans-10, cis-12 CLA, and to a weaker extent cis-9, trans-11 CLA, inhibited accumulation of lipids during adipogenic differentiation. Treatment with higher doses of CLA induced apoptosis in preadipocytes, in differentiating cells, and adipocytes. The trans-10, cis-12 isomer had a higher apoptotic potency in adipocytes than cis-9, trans-11 CLA. Taken together, the treatment of human preadipocytes and adipocytes with physiological relevant concentrations of CLAs resulted in an impairment of proliferation and differentiation and induction of apoptosis. The trans-10, cis-12 isomer was more potent than the cis-9, trans-11 isomer. Further clinical studies are needed to evaluate the effects of CLAs on human fat mass and metabolism in vivo.     

Characterization of the cell wall and cell wall proteins of Chromatium vinosum.

Highly purified cell walls of Chromatium vinosum were isolated by differential centrifugation, with or without Triton X-100 extraction. The isolated material had a protein composition similar to that of cell walls obtained by sucrose density gradient centrifugation. Twenty-two proteins were reproducibly detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 42-kilodalton protein was shown to account for 65% of the total cell wall protein. The majority of cell wall proteins were solubilized in sodium dodecyl sulfate at room temperature; however, they existed as high-molecular-weight complexes unless heated to 45 degrees C or above. The cell wall contained one heat-modifiable protein which migrated with an apparent molecular weight of 37,400 when solubilized at 70 degrees C or below, but which migrated with an apparent molecular weight of 52,500 if solubilized at 100 degrees C. The electrophoretic mobility of three proteins was modified by 2-mercaptoethanol. The majority of C. vinosum cell wall proteins had isoelectric points between pH 4.5 and 5.5, and the 42-kilodalton protein focused at pH 4.9. No proteins were detected which were analogous to the lipoprotein or peptidoglycan-associated proteins of the Enterobacteriaceae. Nearest-neighbor analysis with a reducible, cross-linking reagent indicated that three proteins, including the 42-kilodalton protein, associated with themselves. Most of the cell wall proteins were partially accessible to proteases in both intact cells and isolated cell walls. Protease treatment of the whole cell or isolated cell wall digested approximately an 11,000-molecular-weight portion of the 42-kilodalton protein.