LEM-PCR: a method for determining relative transcript isoform proportions using real-time PCR without a standard curve.

Many genes express multiple transcript isoforms generated by alternative splicing of mRNA. Using real-time PCR, it is straightforward to determine the relative expression level of each isoform independently. However, it is less trivial to determine the relative proportions of different isoforms in a cDNA sample. The relative proportions of different isoforms can be important, as a small change in a highly abundant transcript may be more relevant than a large change in a minimally expressed transcript. Currently, determining the relative proportions of isoforms requires the construction of a standard curve using recombinant plasmid DNA or genomic DNA. As recombinant or genomic DNA standards often amplify with different efficiencies to cDNA samples, they may give under- or overestimations of isoform abundances. The method described in this article uses a titration curve generated from the same cDNA samples measured in the experiment. By using samples with different levels of separate isoforms, it is possible to derive linear equations which, when solved, allow the determination of the proportion of each isoform within the samples under study.

Secreted frizzled-related protein 1 regulates adipose tissue expansion and is dysregulated in severe obesity.

AIM: The Wnt/ß-catenin signaling network offers potential targets to diagnose and uncouple obesity from its metabolic complications. In this study, we investigate the role of the Wnt antagonist, secreted frizzled-related protein 1 (SFRP1), in promoting adipogenesis in vitro and adipose tissue expansion in vivo. METHODS: We use a combination of human and murine, in vivo and in vitro models of adipogenesis, adipose tissue expansion and obesity-related metabolic syndrome to profile the involvement of SFRP1. RESULTS: SFRP1 is expressed in both murine and human mature adipocytes. The expression of SFRP1 is induced during in vitro adipogenesis, and SFRP1 is preferentially expressed in mature adipocytes in human adipose tissue. Constitutive ectopic expression of SFRP1 is proadipogenic and inhibits the Wnt/ß-catenin signaling pathway. In vivo endogenous levels of adipose SFRP1 are regulated in line with proadipogenic states. However, in longitudinal studies of high-fat-diet-fed mice, we observed a dynamic temporal but biphasic regulation of endogenous SFRP1. In agreement with this profile, we observed that SFRP1 expression in human tissues peaks in patients with mild obesity and gradually falls in morbidly obese subjects. CONCLUSIONS: Our results suggest that SFRP1 is an endogenous modulator of Wnt/ß-catenin signaling and participates in the paracrine regulation of human adipogenesis. The reduced adipose expression of SFRP1 in morbid obesity and its knock-on effect to prevent further adipose tissue expansion may contribute to the development of metabolic complications in these individuals.

Tumour necrosis factor-alpha inhibits adipogenesis via a beta-catenin/TCF4(TCF7L2)-dependent pathway.

Tumour necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is a potent negative regulator of adipocyte differentiation. However, the mechanism of TNF-alpha-mediated antiadipogenesis remains incompletely understood. In this study, we first confirm that TNF-alpha inhibits adipogenesis of 3T3-L1 preadipocytes by preventing the early induction of the adipogenic transcription factors peroxisome proliferator-activated receptor-gamma (PPARgamma) and CCAAT/enhancer binding protein-alpha (C/EBPalpha). This suppression coincides with enhanced expression of several reported mediators of antiadipogenesis that are also targets of the Wnt/beta-catenin/T-cell factor 4 (TCF4) pathway. Indeed, we found that TNF-alpha enhanced TCF4-dependent transcriptional activity during early antiadipogenesis, and promoted the stabilisation of beta-catenin throughout antiadipogenesis. We analysed the effect of TNF-alpha on adipogenesis in 3T3-L1 cells in which beta-catenin/TCF signalling was impaired, either via stable knockdown of beta-catenin, or by overexpression of dominant-negative TCF4 (dnTCF4). The knockdown of beta-catenin enhanced the adipogenic potential of 3T3-L1 preadipocytes and attenuated TNF-alpha-induced antiadipogenesis. However, beta-catenin knockdown also promoted TNF-alpha-induced apoptosis in these cells. In contrast, overexpression of dnTCF4 prevented TNF-alpha-induced antiadipogenesis but showed no apparent effect on cell survival. Finally, we show that TNF-alpha-induced antiadipogenesis and stabilisation of beta-catenin requires a functional death domain of TNF-alpha receptor 1 (TNFR1). Taken together these data suggest that TNFR1-mediated death domain signals can inhibit adipogenesis via a beta-catenin/TCF4-dependent pathway.

WNT10B mutations in human obesity.

AIMS/HYPOTHESIS: Recent studies suggest that wingless-type MMTV integration site family, member 10B (WNT10B) may play a role in the negative regulation of adipocyte differentiation in vitro and in vivo. In order to determine whether mutations in WNT10B contribute to human obesity, we screened two independent populations of obese subjects for mutations in this gene. SUBJECTS AND METHODS: We studied 96 subjects with severe obesity of early onset (less than 10 years of age) from the UK Genetics of Obesity Study and 115 obese Italian subjects of European origin. RESULTS: One proband with early-onset obesity was found to be heterozygous for a C256Y mutation, which abrogated the ability of WNT10B to activate canonical WNT signalling and block adipogenesis and was not found in 600 control alleles. All relatives of the proband who carried this allele were either overweight or obese. Three other rare missense variants were found in obese probands, but these did not clearly cosegregate with obesity in family studies and one (P301S), which was found in three unrelated subjects with early-onset obesity, had normal functional properties. CONCLUSIONS/INTERPRETATION: These mutations represent the first naturally occurring missense variants of WNT10B. While the pedigree analysis in the case of C256Y WNT10B does not provide definitive proof of a causal link of this variant with obesity, the finding of a non-functioning WNT10B allele in a human family affected by obesity should encourage further study of this gene in other obese populations.

Characterisation of receptor-specific TNFalpha functions in adipocyte cell lines lacking type 1 and 2 TNF receptors.

Tumour necrosis factor-alpha (TNFalpha) is a multifunctional cytokine that exerts a myriad of biological actions in numerous different tissues including adipocytes through its two distinct cell surface receptors. To address the role of each TNF receptor in the biological actions of TNFalpha in adipocytes, we have developed four new preadipocyte cell lines. These were established from wild type controls (TNFR1(+/+)R2(+/+)) and from mice lacking TNFR1 (TNFR1(-/-)), TNFR2 (TNFR2(-/-)) or both (TNFR1(-/-)R2(-/-)). All four new cell lines can fully differentiate to form mature adipocytes, under appropriate culture conditions, as judged by cell morphology, expression of multiple adipogenic markers and the ability to mediate agonist-stimulated lipolysis and insulin-stimulated glucose transport. In wild type (TNFR1(+/+)R2(+/+)) and TNFR2(-/-) adipocytes, TNFalpha stimulated lipolysis and inhibited insulin-stimulated glucose transport as well as insulin receptor autophosphorylation. In contrast, these activities were completely lost in the TNFR1(-/-)R2(-/-) and TNFR1(-/-) cells. Taken together, these studies demonstrate that TNFalpha-induced lipolysis, as well as inhibition of insulin-stimulated glucose transport are predominantly mediated by TNFR1 and that the presence of TNFR2 is not necessary for these functions. This new experimental system promises to be useful in dissecting the molecular pathways activated by each TNF receptor in mediating the biological functions of TNFalpha in differentiated adipocytes.

Obesity: Adipogenesis and Insulin Resistance.

This meeting focused on the recent developments in the fields of adipogensis and insulin resistance. In particular, the molecular mechanisms presented were those that are likely to be relevant as drug discovery targets for the treatment of obesity and diabetes. New compounds included the LIFR antagonist, hLIF05, and LG- 100641, a selective antagonist of PPARgamma but with insulin sensitizing actions in adipocytes. The small insulin sensitizers discussed were CLX-0901, isolated from plant extracts and currently in phase I clinical trials, TLK-17411, a small synthetic compound and S-15261, which increases insulin sensitivity.

Transmembrane tumor necrosis factor (TNF)-alpha inhibits adipocyte differentiation by selectively activating TNF receptor 1.

Tumor necrosis factor alpha (TNFalpha) is a potent cytokine with multiple biological activities and exists in two forms as follows: a 17-kDa soluble form that is a cleaved product of the 26-kDa transmembrane form (mTNFalpha). It has been suggested that the transmembrane form of TNFalpha is mainly responsible for localized responses via cell-cell contact. Here, we have examined the activities of transmembrane TNFalpha in cultured adipocytes. A non-cleavable transmembrane form of TNFalpha (mTNFDelta1-9K11E) was expressed in several preadipocyte cell lines using retroviral gene transfer. In wild type preadipocytes carrying both TNF receptors, expression of mTNFDelta1-9K11E resulted in inhibition of the differentiation program. The extent of this varied depending on the nature and strength of the adipogenic stimuli. The TNF receptor responsible for this function was determined by expressing mTNFDelta1-9K11E in preadipocyte cell lines lacking either TNF receptor 1 (TNFR1), 2 (TNFR2), or both. In order to confirm the results in the same cellular background, TNF receptors were also reconstituted in the cell lines lacking corresponding receptors. These experiments demonstrated that TNFR1 was necessary and sufficient for mediating mTNFDelta1-9K11E-induced inhibition of adipogenesis and that this action was similar to that of soluble TNFalpha. In conclusion, our results indicate that mTNFDelta1-9K11E is biologically active in cultured adipocytes and can alter the adipogenic program of these cells by selectively activating TNFR1. This may have physiological implications where local TNFalpha actions are thought to be generated at sites such as adipose tissue.

The role of TNF alpha in adipocyte metabolism.

Tumor necrosis factor-alpha (TNF alpha) is a multifunctional cytokine which exerts a myriad of biological actions in different tissues and species. Many of these actions can perturb the normal regulation of energy metabolism. In adipose tissue, in particular, TNF alpha has been demonstrated to regulate or interfere with adipocyte metabolism at numerous sites including transcriptional regulation, glucose and fatty acid metabolism and hormone receptor signaling. The implications of these perturbations in disease states and the current understanding of the molecular mechanisms utilised by TNF alpha are discussed herein.

Roles for adenosine ribose hydroxyl groups in cyclic adenosine 5'-diphosphate ribose-mediated Ca2+ release.

Cyclic adenosine diphosphate ribose (cADPR) is a naturally occurring and potent Ca2+-mobilizing agent. Structural analogues are currently required as pharmacological tools for the investigation of this topical molecule, but modifications to date have concentrated primarily upon the purine ring. Two novel dehydroxylated analogues of cADPR have now been prepared from chemically synthesized nicotinamide adenine dinucleotide (NAD+) precursors modified in the ribose moiety linked to adenine. ADP-ribosyl cyclase of Aplysia californica catalyzed the conversion of 2'A-deoxy-NAD+ and 3'A-deoxy-NAD+ into the corresponding 2'A-deoxy-cADPR and 3'A-deoxy-cADPR analogues, respectively. These analogues were used to assess the effect of 2'- and 3'-hydroxyl group deletion in the adenosine ribose moiety of cADPR on the Ca2+-releasing potential of cADPR. These compounds were found to have comparatively markedly different activities as agonists for Ca2+ mobilization in sea urchin egg homogenate. 2'A-Deoxy-cADPR is similar to cADPR, whereas 3'A-deoxy-cADPR is at least 100-fold less potent, indicating that the 3'A-hydroxyl group, but not the 2'A-hydroxyl group, is essential for calcium releasing activity. EC50 values recorded were 32 nM, 58 nM, and 5 microM for cADPR, 2'A-deoxy-cADPR, and 3'A-deoxy-cADPR, respectively. Moreover, 200 nM 2'A-deoxy-cADPR was required to desensitize the cADPR-sensitive Ca2+ channel to a subsequent addition of 100 nM cADPR, but 20 microM 3'A-deoxy-cADPR was required to produce the same desensitizing effect. This is in accordance with the 100-fold lower potency exhibited by the latter analogue. To further investigate the importance of the 3'-hydroxyl group, we have also synthesized 3'A-O-methyl-cADPR, in which the 3'-hydroxyl group of adenosine has been methylated and its ability potentially to donate a hydrogen atom in a hydrogen bond has been removed. Although inactive in releasing Ca2+, 3'A-O-methyl-cADPR inhibited cADPR-induced Ca2+ release in a dose-dependent manner with an approximate IC50 value of 5 microM, whereas 3'-O-methyladenosine had no effect. This further supports the requirement of a 3'-OH group for Ca2+ releasing activity. In addition, however, it suggests that this group may not be crucial for ligand-receptor recognition. Thus, replacement of the hydrogen atom of the hydroxyl with a methyl group effects a change of activity from an agonist to an antagonist of cADPR-induced Ca2+ release. Two other analogues with modifications in the 2' and/or 3' positions, 3'-cADPR phosphate and 2',3'-cyclic-cADPR phosphate, were synthesized and tested for their Ca2+-mobilizing activity in sea urchin egg homogenates. Both analogues were inactive with respect to both agonistic and antagonistic activities on the cADPR-sensitive Ca2+ release mechanism. These are the first steps toward a wider structure-activity relationship for cADPR, and this is the first study to implicate a crucial role for the adenosine ribose hydroxyl groups of cADPR in the biological activity of this cyclic nucleotide. Additionally, this is the first report of a cADPR receptor antagonist that is not modified at the 8-position of the purine ring.

7-Deaza-8-bromo-cyclic ADP-ribose, the first membrane-permeant, hydrolysis-resistant cyclic ADP-ribose antagonist.

Cyclic ADP-ribose (cADPR) is a putative second messenger that has been demonstrated to mobilize Ca2+ in many cell types. Its postulated role as the endogenous regulator of ryanodine-sensitive Ca2+ release channels has been greatly supported by the advent and use of specific cADPR receptor antagonists such as 8-NH2-cADPR (Walseth, T. F., and Lee, H. C. (1993) Biochim. Biophys. Acta 1178, 235-242). However, investigations of the role of cADPR in physiological responses, such as fertilization, stimulus-secretion coupling, and excitation-contraction coupling, have been hindered by the susceptibility of cADPR receptor antagonists to hydrolysis and the need to introduce these molecules into cells by microinjection or patch clamp techniques. We have recently reported on the discovery of a poorly hydrolyzable analogue of cADPR, 7-deaza-cADPR (Bailey, V. C., Sethi, J. K., Fortt, S. M., Galione, A., and Potter, B. V. L. (1997) Chem. Biol. 4, 41-51) but this, like cADPR, is an agonist of ryanodine-sensitive Ca2+ release channels. We therefore explored the possibility of combining antagonistic activity with that of hydrolytic resistance and now report on the biological properties of the first hydrolysis-resistant cADPR receptor antagonist, 7-deaza-8-bromo-cADPR. In addition this compound has the advantage of being membrane-permeable. Together these properties make this hybrid molecule the most powerful tool to date for studying cADPR-mediated Ca2+ signaling in intact cells.

7-Deaza cyclic adenosine 5'-diphosphate ribose: first example of a Ca(2+)-mobilizing partial agonist related to cyclic adenosine 5'-diphosphate ribose.

BACKGROUND: Cyclic adenosine 5'-diphosphate ribose (cADPR), a naturally occurring metabolite of nicotinamide adenine dinucleotide (NAD+), mobilizes Ca2+ from non-mitochondrial stores in a variety of mammalian and invertebrate tissues. It has been shown that cADPR activates ryanodine-sensitive Ca(2+)-release channels, working independently of inositol 1,4,5-trisphosphate (IP3) to mobilize intracellular Ca2+ stores. In some systems, cADPR has been shown to be more potent than IP3. The chemo-enzymatic synthesis of structurally modified analogues of cADPR can provide pharmacological tools for probing this new Ca(2+)-signaling pathway. In this work, we describe the synthesis and evaluation of a structural mimic of cADPR with different Ca(2+)-releasing properties. RESULTS: 7-Deaza cyclic adenosine 5'-diphosphate ribose (7-deaza cADPR), a novel cADPR analogue modified in the purine ring, was synthesized and its ability to release Ca2+ from non-mitochondrial pools in homogenates made from sea urchin eggs was investigated. 7-Deaza cADPR was more effective in releasing Ca2+ than cADPR, but it only released approximately 66% of the Ca2+ released by a maximal concentration of cADPR. It was also more resistant to hydrolysis than cADPR. If we administered increasing concentrations of 7-deaza cADPR at the same time as a maximal concentration of cADPR, the induction of Ca2+ release by cADPR was antagonized. CONCLUSIONS: 7-Deaza cADPR has a Ca(2+)-release profile consistent with that of a partial agonist, and it is the first reported example of such a compound to act at the cADPR receptor. The imidazole ring of cADPR is clearly important in stimulating the Ca(2+)-release machinery, and the present results demonstrate that structural modification of a site other than position 8 of the purine ring can affect the efficacy of Ca2+ release. 7-Deaza cADPR represents a significant step forwards in designing modulators of the cADPR signaling pathway.

Nicotinamide inhibits cyclic ADP-ribose-mediated calcium signalling in sea urchin eggs.

Cyclic ADP ribose (cADPR) is a potent Ca(2+)-releasing agent, and putative second messenger, the endogenous levels of which are tightly regulated by synthetic (ADP-ribosyl cyclases) and degradative (cADPR hydrolase) enzymes. These enzymes have been characterized in a number of mammalian and invertebrate tissues and their activities are often found on a single polypeptide. beta-NAD+, cGMP and nitric oxide (NO) have been reported to mobilize Ca2+ in the sea urchin egg via the cADPR-mediated pathway. We now report that in sea urchin egg homogenates, nicotinamide inhibits the Ca(2+)-mobilizing action of beta-NAD+, cGMP and NO, but has no effect on cADPR-induced Ca2+ release. Moreover, nicotinamide inhibits cGMP-induced regenerative Ca2+ waves in the intact sea urchin egg. By successfully separating the cADPR-metabolizing machinery from that which releases Ca2+, we have shown that nicotinamide inhibits cADPR-mediated Ca2+ signalling at the level of cADPR generation. Importantly, nicotinamide had no effect upon the hydrolysis of cADPR, and its selective action on cyclase activity was supported by its inhibition of purified Aplysia ADP-ribosyl cyclase, which does not exhibit detectable hydrolytic activity. The action of nicotinamide in blocking Ca2+ release by beta-NAD+, cGMP and NO strongly suggests that these agents act as modulators of cADPR synthesis rather than to sensitize calcium release channels to cADPR.

Nitric oxide-induced mobilization of intracellular calcium via the cyclic ADP-ribose signaling pathway.

Cyclic adenosine diphosphate ribose (cADPR) is a potent endogenous calcium-mobilizing agent synthesized from beta-NAD+ by ADP-ribosyl cyclases in sea urchin eggs and in several mammalian cells (Galione, A., and White, A. (1994) Trends Cell Biol. 4, 431 436). Pharmacological studies suggest that cADPR is an endogenous modulator of Ca2+-induced Ca2+ release mediated by ryanodine-sensitive Ca2+ release channels. An unresolved question is whether cADPR can act as a Ca2+-mobilizing intracellular messenger. We show that exogenous application of nitric oxide (NO) mobilizes Ca2+ from intracellular stores in intact sea urchin eggs and that it releases Ca2+ and elevates cADPR levels in egg homogenates. 8-Amino-cADPR, a selective competitive antagonist of cADPR-mediated Ca2+ release, and nicotinamide, an inhibitor of ADP-ribosyl cyclase, inhibit the Ca2+-mobilizing actions of NO, while, heparin, a competitive antagonist of the inositol 1,4,5-trisphosphate receptor, did not affect NO-induced Ca2+ release. Since the Ca2+-mobilizing effects of NO can be mimicked by cGMP, are inhibited by the cGMP-dependent-protein kinase inhibitor, Rp-8-pCPT-cGMPS, and in egg homogenates show a requirement for the guanylyl cyclase substrate, GTP, we suggest a novel action of NO in mobilizing intracellular calcium from microsomal stores via a signaling pathway involving cGMP and cADPR. These results suggest that cADPR has the capacity to act as a Ca2+-mobilizing intracellular messenger.

Inhibition of psychrotrophic organisms by propionicin PLG-1, a bacteriocin produced by Propionibacterium thoenii.

Propionibacterium thoenii strain P127, which produces the bacteriocin propionicin PLG-1, was grown in a skim milk medium and produced bacteriocin in that medium. No bacteriocin activity was detected in skim milk medium in which strain P127-1, a bacteriocin-negative variant of strain P127, had been grown. Five psychrotrophic spoilage or pathogenic organisms (one strain each of Listeria monocytogenes, Pseudomonas fluorescens, Vibrio parahaemolyticus, Yersinia enterocolitica, and one strain of Corynebacterium sp.) were incubated for 24 h in laboratory medium, nonfermented skim milk, and skim milk that had been fermented by strain P127 or P127-1. Strains were inhibited only in the skim milk fermented by strain P127, as evidenced by loss in numbers of viable cells after 24 h at 10 degrees C and less growth than in other media after 24 h at optimal growth temperatures. Growth of selected strains was delayed or slowed during prolonged incubation (21 d) at 10 degrees C. Propionicin PLG-1 shows promise as a preservative for food products.

Effect of dietary fat on the in vitro hepatotoxicity of paracetamol.

In the present study, we have examined the effect of dietary fat on paracetamol-induced liver injury in an in vitro rat liver slice model. Rats were fed, for 7-10 days, diets containing either butter or polyunsaturated vegetable margarine, two fat sources commonly consumed in the human diet. Liver slices were then exposed to paracetamol for 2 hr and further incubated for 4 hr without paracetamol. Cell damage in the slices was quantified at 6 hr by measuring leakage of lactate dehydrogenase, increase in water content and potassium loss. Covalent binding of radioactive paracetamol to liver and the membrane fatty acid composition of the liver were also measured. Liver slices from rats fed butter diets were significantly more sensitive to the toxic effects of paracetamol than those from margarine fed rats. The membrane lipid composition of the livers also reflected the differing fatty acid content of the two diets.

Effect of paracetamol on mitochondrial membrane function in rat liver slices.

The effect of paracetamol on mitochondrial function was studied using rat liver slices. Changes in the potential of the mitochondrial and plasma membrane were monitored using [3H]-triphenylmethylphosphonium (TPMP+) and [14C]thiocyanate (SCN-) probes, respectively. Liver slices were exposed to 10 mM paracetamol for various time periods (0-360 min) after loading with TPMP+. The release of TPMP+ which correlates with a decrease in the mitochondrial membrane potential became significant after 30 min incubation with 10 mM paracetamol. The change in the mitochondrial membrane potential was shown to be independent of cytochrome P450 activity. No significant change in plasma membrane potential was observed, until the release of lactate dehydrogenase (LDH) had begun, 4 hr after exposure, reflecting the ultimate stages of cell injury by paracetamol. These results suggest that paracetamol elicits a direct effect on the mitochondrial function before cell injury develops and adds further evidence to the role of mitochondria in paracetamol toxicity.

Reversible acquisition of a host cell surface membrane antigen by Trypanosoma cruzi.

The ability of Trypanosoma cruzi, the etiological agent of Chagas' disease to acquire host cell surface antigen was tested. Parasites emerging after intracellular replication in WOS sarcoma monolayers expressed a sarcoma-associated surface antigen. This antigen was deleted from these parasites after replication in the MNS control monolayer, which does not express WOS sarcoma-associated surface antigen, or by replication in cell-free medium. This type of reversible acquisition of host surface antigen has not been previously described in T. cruzi or other intracellular protozoan parasites.

Characterization of human sarcoma antigen S3.

An antigen common to human sarcomas, S3, has been further characterized. It is antigenically distinct from human blood-group substances A and B and from heterophile antigens such as Forssman, infectious mononucleosis and serum sickness antigens. Whilst S3 antigen preparations may contain small amounts of CEA and AFP there is no correlation between S3 antigen and the presence or amount of these known tumour-associated substances. S3 antibody can be fully absorbed with guinea-pig kidney but not boiled beef or SRBC. S3, therefore, is a heterophile substance which has not previously been identified. A seroepidemiological survey confirms that S3-antibody prevalence is significantly increased in persons with a wide variety of malignant disease, as well as in family members of patients with sarcoma.