Enhancement of differentiation of cultured adipogenic cells (TA1) by pertussis toxin.

Differentiation of adipocytes is controlled by a variety of hormones and growth factors. To investigate the possible role of GTP-binding proteins (G proteins) in the process of adipose conversion, we studied the effect of pertussis toxin on differentiation of the fibroblast/adipocyte cell line (TA1). Pertussis toxin potentiated dexamethasone- and indomethacin-induced adipocyte differentiation in a time- and dose-dependent fashion. Addition of dibutyryl cAMP or forskolin inhibited adipose conversion, indicating that an abolishment of inhibitory control of adenylate cyclase is not responsible for the action of pertussis toxin. The B oligomer of the toxin did not mimic the effect of the holotoxin. Pertussis toxin catalyzed ADP-ribosylation of 40,000 molecular mass protein of the membrane fraction was dose-dependently inhibited by the pretreatment of the cells with the toxin. These results indicate the possible involvement of pertussis toxin-sensitive G proteins in adipogenesis.     

Hormonal regulation of the differentiation of rat adipocyte precursor cells in primary culture

A reproducible cell culture system is described that allows the study of adipose conversion in fibroblast-like cells isolated by collagenase digestion of epididymal and perirenal adipose tissue from male rats weighing 70-200 g. Adipose conversion as measured by lipid accumulation and increase in glycerophosphate dehydrogenase (GPDH) activity during differentiation strongly depends on the density at which cells are inoculated and starts only when cells are confluent and when physiological amounts of corticosterone and insulin are added. beta-Estradiol, testosterone, thyroxine, triiodothyronine, and growth hormone do not affect the differentiation process. Methylisobutylxanthine added during the first 2 days after confluence, added with insulin and corticosterone, potentiates the effect of insulin on GPDH activity and accelerates triglyceride accumulation. The effect of methyl-isobutylxanthine seems to be mediated by increased cyclic AMP concentrations, inasmuch as it may be replaced by forskolin.     

Differentiation and function of rat adipocyte precursor cells in primary culture

Precursor cells to adipocytes were purified from the epididymal fat pads of small rats and studied in primary culture. A culture system in which substrate and cofactors were not rate-limiting for complete adipocyte conversion was used by utilizing an agarose feeding-layer. Detachment of cells from the culture dish was prevented by addition of a viscous layer of culture medium, containing methyl cellulose. This system allowed quantitation and definite characterization of formed adipocytes, defined as cells accumulating a lipid droplet >20 micro m in diameter. The cells could be subcultured but then gradually lost their adipocyte conversion ability. Age of the donor depressed the adipocyte conversion which, however, never seemed to stop completely. Prostaglandin E(1) and F(2alpha) had no definite effect in the physiological concentration range while indomethachin possibly had a weak inhibitory effect. Insulin, heparin, and isobutylmethylxanthine increased adipocyte formation. Development of characteristic adipocyte functions with time was examined. Lipoprotein lipase activity was very low in the isolated precursor cells before culture, but developed in culture at confluence and was a thousand-fold higher within a few days. At this peak lipoprotein lipase activity was 50-fold higher than in mature adipocytes from the same donor animal. Triglyceride synthesis from glucose peaked in parallel but never reached the value of mature adipocytes and very little fatty acid was synthesized. Hormone-sensitive glycerol release developed at confluence and reached the level of activity of mature adipocytes. This study and previous work have indicated a role for the cyclase system in the development of adipocytes from precursor cells. Dibutyrylcyclic AMP caused an enhancement of lipoprotein lipase activity and adipocyte conversion. In suspension media, the nucleotide caused inhibition. These results are compatible with an effect of the nucleotide, not directly on lipoprotein lipase and cell determination, but via events taking place at confluence associated with cell to cell interactions. In comparison with previously described cells from an established cell line which undergo adipose conversion (3T3 cells), the cells described in the present work, like adipocytes, showed more metabolic activity in pathways for fatty acid incorporation from exogenous lipid sources (lipoprotein lipase activity) than from de novo synthesis. Furthermore, host-factors could be followed such as in the age- and site-dependence of adipocyte formation. Physiological stimuli such as insulin, lipid substrate, and heparin had effects on adipocyte formation. It was therefore concluded that this cell preparation has a potential of yielding information of physiological significance in studies of the regulation of adipocyte multiplication.-Bj?rntorp, P., M. Karlsson, P. Pettersson, and G. Sypniewska. Differentiation and function of rat adipocyte precursor cells in primary culture.     

Enhancement of tenogenic differentiation of rat tendon-derived stem cells by biglycan

Biglycan (BGN) has been identified as one of the critical components of the tendon-derived stem cells (TDSCs) niche and may be related to tendon formation. However, so far, no study has demonstrated whether the soluble BGN could induce the tenogenic differentiation of TDSCs in vitro. The aim of this study was to investigate the effect of BGN on the tenogenic differentiation of TDSCs. The proliferation and tenogenic differentiation of TDSCs exposed to different concentrations of BGN (0, 50, 100, and 500 ng/ml) were determined by the live/dead cell staining assay, CCK-8 assay, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot analysis. The BGN signaling pathway of TDSCs (with and without 50 ng/ml of BGN) was determined by western blot analysis and qRT-PCR analysis. At a concentration of 50 ng/ml, BGN increased the expression of the tenogenic markers THBS-4 and TNMD at both the messenger RNA (mRNA) and protein levels. Meanwhile, 50 ng/ml of BGN inhibited the expression of the chondrogenic and osteogenic markers SOX9, ACN, and RUNX2 at both the mRNA and protein levels. Moreover, BGN (50 ng/ml) affected the expression of the components of the extracellular matrix of TDSCs. Additionally, BGN activated the Smad1/5/8 pathway as indicated by an increase in phosphorylation and demonstrated by inhibition experiments. Upregulation in the gene expression of BMP-associated receptors (BMPRII, ActR-IIa, and BMPR-Ib) and Smad pathway components (Smad4 and 8) was observed. Taken together, BGN regulates tenogenic differentiation of TDSCs via BMP7/Smad1/5/8 pathway and this regulation may provide a basic insight into treating tendon injury.     

Action of insulin modulated by pertussis toxin in rat adipocytes

We studied the effect of pertussis toxin (PT) treatment on the ability of insulin to inhibit lipolysis and to stimulate glucose oxidation in isolated rat adipocytes. In cells maximally modified by PT (100% ADP ribosylation of a 41-kdalton protein in membranes), the ability of insulin to inhibit lipolysis stimulated either by PT alone or in combination with a catecholamine was abolished. In cells wherein ADP ribosylation was submaximal (about 67% modification), a small but variable antilipolytic action of insulin could still be detected. In cells maximally modified by PT, both basal and insulin-stimulated glucose oxidation were markedly reduced (to 10-15% of control levels). However, relative to the basal oxidation level, the fold stimulation by insulin in PT-treated cells was equivalent to the fold stimulation in control cells. Nonetheless, PT treatment caused a rightward shift in the dose-response curve for insulin-stimulated glucose oxidation as well as a small reduction in insulin binding. Our results point strongly not only to a link between the inhibitory guanine nucleotide regulatory complex (Gi) and the antilipolytic action of insulin but also to a link between the Gi complex and the overall regulation of glucose metabolism in adipocytes.     

Interaction of Bordetella pertussis with mast cells, modulation of cytokine secretion by pertussis toxin

Together with macrophages and dendritic cells, mast cells have recently been shown to interact with certain pathogenic bacteria and present microbial antigens to the immune system. We show here that Bordetella pertussis can adhere to and be phagocytosed by mast cells. In addition, mast cells are able to process and present B. pertussis antigens to T lymphocytes. Furthermore, exposure of mast cells to B. pertussis induced the release of the proinflammatory cytokines tumour necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). The release of IL-6 was strongly reduced by pertussis toxin expressed by B. pertussis . The production of IL-10, but not that of IL-4, by mast cells was also inhibited by pertussis toxin. Depletion of mast cells in vivo resulted in significant reduction of early TNF-α production in bronchoalveolar lavage (BAL) fluids of B. pertussis -infected mice. These data suggest that mast cells may play a role in the induction of immune responses against B. pertussis through the release of cytokines, especially TNF-α.     

Proliferation and differentiation of human adipocyte precursor cells: Differences between the preperitoneal and subcutaneous compartments

Human adipocyte precursor cells (APC) have been characterized in their proliferation and differentiation potential from subcutaneous, omental, and mesenteric depots, mostly from morbidly obese patients. Cells from the preperitoneal adipose compartment have not been characterized yet, least of all when obtained from normal weight subjects. The aim was to compare proliferation and differentiation of subcutaneous (SC) and preperitoneal (PP) APC derived from adipose tissue in healthy subjects with different body mass. SC and PP adipose tissue was obtained during surgery of inguinal hernias in five healthy non‐obese subjects and three obese otherwise healthy men. APC, obtained by collagenase digestion, were cultured. Proliferation was assayed by cell counting and differentiation by oil red O staining and flow cytometry using Nile Red staining. Proliferation of SC was higher than PP APC. Such differences between both compartments were even higher in APC obtained from obese patients. Conversely PP APC differentiated earlier in vitro compared with SC cells. These results agree with published data on fat cell proliferation. However regarding differentiation, our data show that APC from deeper depots (in this case PP) differentiate earlier than subcutaneous APC. This is different to previous studies performed in mesenteric or omental adipose tissue. J. Cell. Biochem. 111: 659–664, 2010. © 2010 Wiley‐Liss, Inc.     

ADP-ribosylation of transducin by pertussis toxin

Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is [32P]ADP-ribosylated by pertussis toxin and [32P]NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and 32 kDa. The amino terminus of both 38- and 32-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The 32-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of [32P]ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased [32P]ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed [32P]ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma. The amino terminus of T alpha, while it does not contain the pertussis toxin ADP-ribosylation site, appeared critical to its reactivity.     

Inhibition by islet-activating protein, pertussis toxin, of retinoic acid-induced differentiation of human leukemic (HL-60) cells

Human promyelocytic leukemic (HL-60) cells were induced to differentiate into neutrophil- or macrophage-like cells by incubation of the cells with retinoic acid, dibutyryl cyclic AMP (Bt2cAMP) or phorbol 12-myristate 13-acetate (PMA). Differentiation was determined by an increase in the percentage of morphologically mature cells. The retinoic acid-induced differentiation of HL-60 cells was, but the Bt2cAMP- or PMA-induced one was not, inhibited by prior exposure of the cells to islet-activating protein (IAP), pertussis toxin. The IAP-induced inhibition was correlated with the toxin-catalyzed ADP-ribosylation of a membrane GTP-binding protein with a molecular mass of 40 kDa. Thus, the IAP-substrate GTP-binding protein appears to be involved in the retinoic acid-induced differentiation of HL-60 cells.     

Studies on the expression of adipocyte-specific cell surface antigens during the differentiation of adipocyte precursor cells in vitro

Using species and cell specific antiadipocyte sera an immunoprecipitation procedure was developed which allowed the nature of adipocyte cell surface antigens to be investigated. Analysis of immunoprecipitates from mature adipocyte plasma membranes of rat, ox and chicken and similar 125I-labelled membranes revealed the presence of specific externally disposed adipocyte specific antigens which were also species specific. For mature cells the specific antigens had molecular weights of 124,000, 92,000 and 59,000 in the case of the rat, 87,000 in the case of the ox and 56,000, 47,000 and 37,000 in the case of the chicken. None of these antigens were cross immunoprecipated by antisera to non-homologous adipocytes. The presence of the antigens at the surface of differentiating rat while adipocyte precursor cells was demonstrated using a labelled-second antibody cellular immunoassay and the expression of this reactivity revealed to be an early event in the differentiation programme of the cells. The increase in cell surface immunoreactivity during the differentiation of the cells was shown to be dependent upon the expression of two of the antigens previously shown to be markers of the mature adipocyte phenotype. The functional identity and possible role of these antigens in the control of adipocyte differentiation in vitro and in vivo now becomes accessible to investigation experimentally.     

Bone morphogenetic proteins inhibit adipocyte differentiation by bone marrow stromal cells

The bone morphogenetic proteins were originally identified based on their ability to induce ectopic bone formation in vivo and have since been identified as members of the transforming growth factor-β gene superfamily. It has been well established that the bone morphogenetic cytokines enhance osteogenic activity in bone marrow stromal cells in vitro. Recent reports have described how bone morphogenetic proteins inhibited myogenic differentiation of bone marrow stromal cells in vitro. In vivo, bone marrow stromal cells differentiate along the related adipogenic pathway with advancing age. The current work reports the inhibitory effects of the bone morphorphogenetic proteins on adipogenesis in a multipotent murine bone marrow stromal cell line, BMS2. When exposed to bone morphogenetic protein-2, the pre-adipocyte BMS2 cells exhibited the expected induction of the osteogenic-related enzyme, alkaline phosphatase. Following induction of the BMS2 cells with adipogenic agonists, adipocyte differentiation was assessed by morphologic, enzymatic, and mRNA markers. Flow cytometric analysis combined with staining by the lipophilic fluorescent dye, Nile red, was used to quantitate the extent of lipid accumulation within the BMS2 cells. By this morphologic criteria, the bone morphogenetic proteins inhibited adipogenesis at concentrations of 50 to 500 ng/ml. This correlated with decreased levels of adipocyte specific enzymes and mRNAs. The BMS2 pre-adipocytes constitutively expressed mRNA encoding bone morphogenetic protein-4 and this was inhibited by adipogenic agonists. Together, these findings demonstrate that bone morphogenetic proteins act as adipogenic antagonists. This supports the hypothesis that adipogenesis and osteogenesis in the bone marrow microenvironment are reciprocally regulated.     

Incomplete functional differentiation of HL-60 leukemic cells by synthetic lipopeptides. Partial inhibition by pertussis toxin of enhanced superoxide formation.

In human neutrophils, the synthetic lipopeptide, N-palmitoyl-S-[2,3- bis(palmitoyloxy-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-( S)-lysyl-(S) -lysyl-(S)-lysine [Pam3CysSer(Lys)4], activates NADPH-oxidase catalyzed superoxide (O2-) formation through pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms (Seifert, R., Schultz, G., Richter-Freund, M., Metzger, J., Wiesmüller, K.-H., Jung, G., Bessler, W. G. & Hauschildt, S. (1990) Biochem. J. 267, 795-802). We studied the effects of lipopeptides on differentiation of HL-60 leukemic cells. Pam3CysSer(Lys)4 enhanced phorbol-12-myristate-13-acetate-induced O2- formation (presumably through the expression of components of NADPH oxidase) in a concentration-dependent manner with a half-maximal effect at 100 ng/ml and a maximum at 1 microgram/ml. The effect of the lipopeptide was evident after 24 h and reached a plateau after 48 h. (2S,6S)-2-Palmitoylamino-6,7- bis(palmitoyloxy)heptanoyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S) -lysyl-(S)-lysine enhanced O2- formation as well. The effects of Pam3CysSer(Lys)4 were potentiated by dibutyryl cAMP, dimethyl sulfoxide, retinoic acid, 1,25-dihydroxyvitamin D3, interferon-gamma and tumor-necrosis-factor-alpha. Pertussis toxin, but not its B-oligomer, partially inhibited enhanced O2- formation induced by Pam3CysSer(Lys)4. O2- formation induced by arachidonic acid and gamma-hexachlorocyclohexane were more sensitive to inhibition by pertussis toxin than O2- formation induced by phorbol 12-myristate 13-acetate. Enhanced O2- formation induced by dibutyryl cAMP was not affected by pertussis toxin. Unlike ATP, histamine, prostaglandin E1 and the beta-adrenergic agonist, isoproterenol, Pam3CysSer(Lys)4 did not increase cytosolic Ca2+ [( Ca2+]i) in undifferentiated HL-60 cells. Histamine but not lipopeptides stimulated high-affinity GTPase of guanine-nucleotide-binding proteins in membranes of undifferentiated HL-60 cells. In Pam3CysSer(Lys)4-differentiated HL-60 cells, the responsiveness to the [Ca2+]i-increasing agonists, N-formyl-L-methionyl-L-leucyl-L-phenylalanine, C5a and leukotriene B4, was increased, whilst the responsiveness to prostaglandin E1 and isoproterenol was decreased. Pam3CysSer(Lys)4 did not inhibit proliferation of HL-60 cells but decreased transferrin receptor expression and increased C3bi receptor expression. Pertussis toxin did not affect proliferation and expression of transferrin and C3bi receptors. Dibutyryl cAMP was considerably more effective than Pam3CysSer(Lys)4 at inducing alterations in the above parameters. Our results suggest that (a) Pam3CysSer(Lys)4 induces incomplete functional differentiation of HL-60 cells through a mechanism which does not depend on a rise in [Ca2+]i and is different from that of other differentiation-inducing substances and (b) the mechanism by which Pam3CysSer(Lys)4 induces differentiation involves pertussis-toxin-sensitive and pertussis-toxin-insensitive mechanisms.     

Effects of dexamethasone on multiplication and differentiation of rat adipose precursor cells

The effects of dexamethasone (DEX) on adipose precursor cells from rat adipose tissue were studied in primary culture. When added from the beginning of culture in media containing untreated fetal calf serum (SM), serum treated with charcoal to remove steroid hormones (CSM), or serum-free medium (SFM), DEX inhibited cellular growth. Lipoprotein lipase (LPL) as well as glycerophosphate dehydrogenase (GPDH) activities, markers of cellular differentiation, were also inhibited, except in CSM where LPL was stimulated. When added after cellular confluence, however, DEX had opposite effects and now stimulated cellular differentiation. This effect was highly dependent on insulin. These studies demonstrate that DEX affects adipose precursor cells in several ways, depending on the type of culture medium, the time period of exposure, and the presence of insulin.     

Production of cell mass and pertussis toxin by Bordetella pertussis.

The cultivation of Bordetella pertussis affects production of pertussis toxin and biomass. Comparison of batch mode, chemostat operation and pHstat-turbidostatic control showed that productivities for the continuous process were greater than that for the batch operation. Continuous operation in balanced growth at the maximum specific growth rate, provided by the pHstat, resulted in the maximum specific production rate. Because of the strong association of pertussis toxin synthesis and cell growth, the concentration of toxin in the effluent of the continuous processes was greater than the maximum obtained in the batch bioprocess. An expanded Luedeking-Piret model of product formation kinetics fits the observed chemostat data and demonstrates that the production of pertussis toxin from the culture of B. pertussis is predominantly growth associated.     

Differentiation of rabbit adipocyte precursor cells in a serum-free medium

Summary A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (β-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells. This work was supported by grant 4388 from the Institut National de la Recherche Agronomique, France.     

Effect of pertussis toxin on hormonal responsiveness of rat hepatocytes

The ureogenic action of epinephrine in hepatocytes from normal adult rats is mediated through activation of alpha 1-adrenoceptors. beta-Adrenoceptors in addition to alpha 1-adrenoceptors, became involved in mediating this effect in cells from animals treated with pertussis toxin. The accumulation of cyclic-AMP in response to epinephrine or isoproterenol was markedly increased in hepatocytes from pertussis-treated rats as compared to that observed in control cells. The accumulation of cyclic-AMP due to glucagon was also increased. It is suggested that pertussis toxin may release a constraint on adenylate cyclase activity by blocking the inhibitory coupling mechanism (Ni) or some other entity involved in the regulation of the activity of this enzyme.     

Thyroliberin action in pituitary cells is not inhibited by pertussis toxin.

The effects of pertussis toxin on the responses of rat pituitary-tumour (GH) cells to thyrotropin-releasing hormone (thyroliberin, TRH) were examined. Treatment of cells with pertussis toxin did not alter the affinity or concentration of TRH receptors, or the sensitivity of the TRH receptor to inhibition by guanine nucleotides. TRH caused an increase in low-Km GTPase activity in membrane-containing fractions from both control and pertussis-toxin-treated cells. TRH stimulation of inositol phosphate formation was insensitive to pertussis toxin. TRH caused a biphasic increase in the concentrations of cytosolic free Ca2+ as monitored by intracellularly trapped Quin 2, and this increase was the same in control and toxin-treated cultures. The toxin did not alter the increase in prolactin and growth-hormone (somatotropin) release stimulated by TRH or shift the TRH dose-response curve, and it did not affect the TRH-induced rise in prolactin synthesis measured over 24 h. However, pertussis toxin did block the ability of somatostatin and muscarinic agonists to inhibit prolactin and growth-hormone secretion stimulated by vasoactive intestinal peptide when analysed under the same conditions as those in which the TRH system was unaffected. These data indicate that the guanine nucleotide effects on TRH binding and activity are not mediated by Ni, but possibly by another member of the family of guanine-nucleotide-dependent regulatory proteins.     

Effect of pertussis toxin on water metabolism in the rat

Pertussis toxin administered to rats resulted in a polyuric state that was due to enhanced renal water excretion. Pertussis toxin also induced a defect in renal water conservation. These abnormalities in renal water excretion could not be attributed to polydipsia, impaired synthesis and/or release of arginine vasopressin or renal tubular dysfunction with solute diuresis. No evidence of pertussis toxin-induced renal tubular damage was present. These results indicate that pertussis toxin induces nephrogenic diabetes insipidus and this defect occurs at the level of the renal collecting tubule.