Biomedical subjects

D E Moller

Publications and source records attributed to D E Moller.

At least 37 records · Page 2Linked to original sources

PubMed2000

Both coactivator LXXLL motif-dependent and -independent interactions are required for peroxisome proliferator-activated receptor gamma (PPARgamma) function.

Nuclear receptor activation is dependent on recruitment of coactivators, including CREB-binding protein (CBP/p300) and steroid receptor coactivator-1 (SRC-1). A three-dimensional NMR approach was used to probe the coactivator binding interface in the peroxisome proliferator-activated receptor gamma (PPARgamma) ligand binding domain (LBD). In the presence of a CBP peptide, peaks corresponding to 20 residues in helices 3, 4, 5, and 12 of the LBD were attenuated. Alanine mutants revealed that K301A, V315A, Y320A, L468A, and E471A were required for binding of both CBP and SRC-1 and for cell-based transcription. Several additional amino acids in helix 4 of the PPARgammaLBD were defective with respect to CBP recruitment, but retained relatively normal SRC-1 recruitment. Thus these amino acid residues may be important determinants of specificity for nuclear receptor LBD interactions with discrete coactivator molecules.

Animals↗

PubMed2000

Activation of peroxisome proliferator-activated receptor gamma does not inhibit IL-6 or TNF-alpha responses of macrophages to lipopolysaccharide in vitro or in vivo.

We have investigated the potential use of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists as anti-inflammatory agents in cell-based assays and in a mouse model of endotoxemia. Human peripheral blood monocytes were treated with LPS or PMA and a variety of PPARgamma agonists. Although 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) at micromolar concentrations significantly inhibited the production of TNF-alpha and IL-6, four other high affinity PPARgamma ligands failed to affect cytokine production. Similar results were obtained when the monocytes were allowed to differentiate in culture into macrophages that expressed significantly higher levels of PPARgamma or when the murine macrophage cell line RAW 264.7 was used. Furthermore, saturating concentrations of a potent PPARgamma ligand not only failed to block cytokine production, but also were unable to block the inhibitory activity of 15d-PGJ2. Thus, activation of PPARgamma does not appear to inhibit the production of cytokines by either monocytes or macrophages, and the inhibitory effect observed with 15d-PGJ2 is most likely mediated by a PPARgamma-independent mechanism. To examine the anti-inflammatory activity of PPARgamma agonists in vivo, db/db mice were treated with a potent thiazolidinedione that lowered their elevated blood glucose and triglyceride levels as expected. When thiazolidinedione-treated mice were challenged with LPS, they displayed no suppression of cytokine production. Rather, their blood levels of TNF-alpha and IL-6 were elevated beyond the levels observed in control db/db mice challenged with LPS. Comparable results were obtained with the corresponding lean mice. Our data suggest that compounds capable of activating PPARgamma in leukocytes will not be useful for the treatment of acute inflammation.

Administration, Oral↗

PubMed2000

Potential role of TNF-alpha in the pathogenesis of insulin resistance and type 2 diabetes.

Tumor necrosis factor alpha (TNF-alpha) has well-described effects on lipid metabolism in the context of acute inflammation, as in sepsis. Recently, increased TNF-alpha production has been observed in adipose tissue derived from obese rodents or human subjects and TNF-alpha has been implicated as a causative factor in obesity-associated insulin resistance and the pathogenesis of type 2 diabetes. Thus, current evidence suggests that administration of exogenous TNF-alpha to animals can induce insulin resistance, whereas neutralization of TNF-alpha can improve insulin sensitivity. Importantly, results from knockout mice deficient in TNF-alpha or its receptors have suggested that TNF-alpha has a role in regulating in vivo insulin sensitivity. However, the absence of TNF-alpha action might only partially protect against obesity-induced insulin resistance in mice. Multiple mechanisms have been suggested to account for these metabolic effects of TNF-alpha. These include the downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated free fatty acids via stimulation of lipolysis, and negative regulation of PPAR gamma, an important insulin-sensitizing nuclear receptor. Although current evidence suggests that neutralizing TNF-alpha in type 2 diabetic subjects is not sufficient to cause metabolic improvement, it is still probable that TNF-alpha is a contributing factor in common metabolic disturbances such as insulin resistance and dyslipidemia.

Animals↗

PubMed2000

New approaches in the treatment of type 2 diabetes.

Type 2 diabetes is a chronic metabolic derangement that results from defects in both insulin action and secretion. New thiazolidinedione insulin sensitizers have been recently launched. New approaches with mechanisms different from current therapies are being explored, including novel ligands of peroxisome proliferator-activated receptor, glucagon receptor antagonists, dipeptidyl peptidase IV inhibitors, and insulin receptor activators.

Diabetes Mellitus, Type 2↗

PubMed2000

Evidence for peroxisome proliferator-activated receptor (PPAR)alpha-independent peroxisome proliferation: effects of PPARgamma/delta-specific agonists in PPARalpha-null mice.

Peroxisome proliferators are a diverse group of compounds that cause hepatic hypertrophy and hyperplasia, increase peroxisome number, and on chronic high-dose administration, lead to rodent liver tumorigenesis. Various lines of evidence have led to the conclusion that these agents induce their pleiotropic effects exclusively via agonism of peroxisome proliferator-activated receptor (PPAR)alpha, a member of the steroid receptor superfamily involved in the regulation of fatty acid metabolism. Recently, agonists of two other members of this receptor family have been identified. PPARgamma is predominantly expressed in adipocytes where it mediates differentiation; PPARdelta is a widely expressed orphan receptor with yet unresolved physiologic functions. In the course of characterizing newer PPAR ligands, we noted that highly selective PPARgamma agonists or dual PPARgamma/PPARdelta agonists, lacking apparent murine PPARalpha agonist activity, cause peroxisome proliferation in CD-1 mice. We therefore made use of PPARalpha knockout mice to investigate whether these effects resulted from agonism of PPARalpha by these agents at very high dose levels or whether PPARgamma (or PPARdelta) agonism alone can result in peroxisome proliferation. We report here that several parameters linked to the hepatic peroxisome proliferation response in mice that were seen with these agents resulted from PPARalpha-independent effects.

Animals↗

PubMed1999

Cyclo-oxygenase-2-derived prostacyclin mediates embryo implantation in the mouse via PPARdelta.

We have demonstrated previously that cyclo-oxygenase-2 (COX2), the rate-limiting enzyme in the biosynthesis of prostaglandins (PGs), is essential for blastocyst implantation and decidualization. However, the candidate PG(s) that participates in these processes and the mechanism of its action remain undefined. Using COX2-deficient mice and multiple approaches, we demonstrate herein that COX2-derived prostacyclin (PGI2) is the primary PG that is essential for implantation and decidualization. Several lines of evidence suggest that the effects of PGI2 are mediated by its activation of the nuclear hormone receptor PPARdelta, demonstrating the first reported biologic function of this receptor signaling pathway.

Animals↗

PubMed1999

Discovery of a small molecule insulin mimetic with antidiabetic activity in mice.

Insulin elicits a spectrum of biological responses by binding to its cell surface receptor. In a screen for small molecules that activate the human insulin receptor tyrosine kinase, a nonpeptidyl fungal metabolite (L-783,281) was identified that acted as an insulin mimetic in several biochemical and cellular assays. The compound was selective for insulin receptor versus insulin-like growth factor I (IGFI) receptor and other receptor tyrosine kinases. Oral administration of L-783,281 to two mouse models of diabetes resulted in significant lowering in blood glucose levels. These results demonstrate the feasibility of discovering novel insulin receptor activators that may lead to new therapies for diabetes.

Adenosine Triphosphate↗

PubMed1999

L-764406 is a partial agonist of human peroxisome proliferator-activated receptor gamma. The role of Cys313 in ligand binding.

Insulin-sensitizing thiazolidinedione (TZD) compounds are high affinity ligands for a member of the nuclear receptor family, peroxisome proliferator-activated receptor (PPAR) gamma. A scintillation proximity assay for measurement of 3H-radiolabeled TZD binding to human PPARgamma under homogeneous conditions was developed. Using this approach, a novel non-TZD compound (L-764406) was shown to be a potent (apparent binding IC50 of 70 nM) PPARgamma ligand. Preincubation of PPARgamma with L-764406 prevented binding of the [3H]TZD, suggesting a covalent interaction with the receptor; in addition, structurally related analogues of L-764406, which would be predicted not to interact with PPARgamma in a covalent fashion, did not displace [3H]TZD binding to PPARgamma. Covalent binding of L-764406 was proven by an observed molecular weight shift of a tryptic PPARgamma ligand binding domain (LBD) peptide by mass spectrometric analysis. A specific cysteine residue (Cys313 in helix 3 of hPPARgamma2) was identified as the attachment site for this compound. In protease protection experiments, the liganded receptor adopted a typical agonist conformation. L-764406 exhibited partial agonist activity in cells expressing a chimeric receptor containing the PPARgamma LBD and a cognate reporter gene and also induced the expression of the adipocyte-specific gene aP2 in 3T3-L1 cells. In contrast, L-764406 did not exhibit activity in cells transfected with chimeric receptors containing PPARalpha or PPARdelta LBDs. The partial agonist properties of L-764406 were also evident in a co-activator association assay, indicating that the increased transcription in cells was co-activator mediated. Thus, L-764406 is a novel non-TZD ligand for PPARgamma and is also the first known partial agonist for this receptor. The results suggest a critical functional role for Cys313, and helix 3, in contributing to ligand binding and subsequent agonist-induced conformational changes.

Amino Acid Sequence↗

PubMed1999

Novel peroxisome proliferator-activated receptor (PPAR) gamma and PPARdelta ligands produce distinct biological effects.

The peroxisome proliferator-activated receptors (PPARs) include three receptor subtypes encoded by separate genes: PPARalpha, PPARdelta, and PPARgamma. PPARgamma has been implicated as a mediator of adipocyte differentiation and the mechanism by which thiazolidinedione drugs exert in vivo insulin sensitization. Here we characterized novel, non-thiazolidinedione agonists for PPARgamma and PPARdelta that were identified by radioligand binding assays. In transient transactivation assays these ligands were agonists of the receptors to which they bind. Protease protection studies showed that ligand binding produced specific alterations in receptor conformation. Both PPARgamma and PPARdelta directly interacted with a nuclear receptor co-activator (CREB-binding protein) in an agonist-dependent manner. Only the PPARgamma agonists were able to promote differentiation of 3T3-L1 preadipocytes. In diabetic db/db mice all PPARgamma agonists were orally active insulin-sensitizing agents producing reductions of elevated plasma glucose and triglyceride concentrations. In contrast, selective in vivo activation of PPARdelta did not significantly affect these parameters. In vivo PPARalpha activation with WY-14653 resulted in reductions in elevated triglyceride levels with minimal effect on hyperglycemia. We conclude that: 1) synthetic non-thiazolidinediones can serve as ligands of PPARgamma and PPARdelta; 2) ligand-dependent activation of PPARdelta involves an apparent conformational change and association of the receptor ligand binding domain with CREB-binding protein; 3) PPARgamma activation (but not PPARdelta or PPARalpha activation) is sufficient to potentiate preadipocyte differentiation; 4) non-thiazolidinedione PPARgamma agonists improve hyperglycemia and hypertriglyceridemia in vivo; 5) although PPARalpha activation is sufficient to affect triglyceride metabolism, PPARdelta activation does not appear to modulate glucose or triglyceride levels.

Adipocytes↗

PubMed1999

Increased amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals.

We have recently demonstrated that insulin activates farnesyltransferase (FTase) and thereby increases the amounts of cellular farnesylated p21Ras in 3T3-L1 fibroblasts, adipocytes and vascular smooth muscle cells. We postulated that hyperinsulinaemia might considerably increase the the cellular pool of farnesylated p21Ras available for activation by other growth factors. To examine the role of in vivo hyperinsulinaemia in regulating farnesylated p21Ras, we measured the amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals. Liver, aorta, and skeletal muscle of ob/ob mice, and mice made obese and hyperinsulinaemic by injection of gold-thioglucose contained greater amounts of farnesylated p21Ras than tissues of their lean normoinsulinaemic counterparts. Similarly, farnesylated p21Ras was increased (67 vs. 35 % in control animals, p<0.01) in the livers of hyperinsulinaemic Zucker rats (fa/fa). Reduction of hyperinsulinaemia by exercise training (2 h/day for 7-8 weeks) resulted in decreases in the amounts of farnesylated p21Ras in these animals. Increased farnesylated p21Ras in hyperinsulinaemic animals reflected increasing increments in the activity of FTase in ob/ob mice (2-fold increase) and fa/fa Zucker rats (3.5-fold increase), while the total amounts of Ras proteins remained unchanged. In contrast to insulin-resistant hyperinsulinaemic animals, denervated insulin-resistant rat soleus muscle (in the presence of normoinsulinaemia) showed normal amounts of farnesylated p21Ras. In summary, these data confirm increased amounts of farnesylated p21Ras in tissues of hyperinsulinaemic animals.

3T3 Cells↗

PubMed1999

Differential regulation of MAP kinase, p70(S6K), and Akt by contraction and insulin in rat skeletal muscle.

To study the effects of contractile activity on mitogen-activated protein kinase (MAP kinase), p70 S6 kinase (p70(S6K)), and Akt kinase signaling in rat skeletal muscle, hindlimb muscles were contracted by electrical stimulation of the sciatic nerve for periods of 15 s to 60 min. Contraction resulted in a rapid and transient activation of Raf-1 and MAP kinase kinase 1, a rapid and more sustained activation of MAP kinase and the 90-kDa ribosomal S6 kinase 2, and a dramatic increase in c-fos mRNA expression. Contraction also resulted in an apparent increase in the association of Raf-1 with p21Ras, although stimulation of MAP kinase signaling occurred independent of Shc, IRS1, and IRS2 tyrosine phosphorylation or the formation of Shc/Grb2 or IRS1/Grb2 complexes. Insulin was considerably less effective than contraction in stimulating the MAP kinase pathway. However, insulin, but not contraction, increased p70(S6K) and Akt activities in the muscle. These results demonstrate that contraction-induced activation of the MAP kinase pathway is independent of proximal steps in insulin and/or growth factor-mediated signaling, and that contraction and insulin have discordant effects with respect to the activation of the MAP kinase pathway vs. p70(S6K) and Akt. Of the numerous stimulators of MAP kinase in skeletal muscle, contractile activity emerges as a potent and physiologically relevant activator of MAP kinase signaling, and thus activation of this pathway is likely to be an important molecular mechanism by which skeletal muscle cells transduce mechanical and/or biochemical signals into downstream biological responses.

Animals↗

PubMed1999

Inhibition of adipocyte differentiation by HIV protease inhibitors.

Patients with AIDS who are receiving therapy with HIV protease inhibitors have been widely reported to be afflicted with a syndrome characterized by lipodystrophy (fat redistribution favoring the accumulation of abdominal and cervical adipose tissue), hyperlipidemia, and insulin resistance. HIV protease inhibitors have been suggested to have a direct role in modulating adipocyte differentiation. To address this hypothesis, several HIV protease inhibitors were studied for their ability to either augment or inhibit the differentiation of murine 3T3-L1 preadipocytes. Dose-responsive inhibition of adipogenesis by several protease inhibitors was noted as measured by reduced triglyceride accumulation and attenuated induction of three differentiation marker genes -- aP2, lipoprotein lipase, and Adipo Q. Potential mechanisms for altered adipocyte function, including direct binding to PPARgamma or inhibition of PPARgamma-mediated gene transcription were effectively excluded.

3T3 Cells↗

PubMed1998

Sex hormone-binding globulin mediates prostate androgen receptor action via a novel signaling pathway.

Estradiol (E2) and 5alpha-androstan-3alpha,17beta-diol (3alpha-diol) have been implicated in prostate hyperplasia in man and dogs, but neither of these steroids bind to androgen receptors (ARs). Recently, we reported that E2 and 3alpha-diol stimulated generation of intracellular cAMP via binding to a complex of sex hormone-binding globulin (SHBG) and its receptor (R(SHBG)) on prostate cells. We speculated that this pathway, involving steroids normally found in the prostate, was involved in the indirect activation of ARs. Using the dog as a model to test this hypothesis in normal prostate, we investigated whether E2, 3alpha-diol, and SHBG stimulated the production of the androgen-responsive protein, arginine esterase (AE), the canine equivalent of human prostate-specific antigen. In cultured dog prostate tissue preincubated with SHBG, E2 and 3alpha-diol stimulated AE activity. These effects were blocked by hydroxyflutamide, an AR antagonist, and by 2-methoxyestradiol, a competitive inhibitor of E2 and 3alpha-diol binding to SHBG. In the absence of exogenous steroids and SHBG, AE also was significantly increased by treatment with forskolin or 8-Bromoadenosine-cAMP. These observations support the hypothesis that in normal prostate, E2 and 3alpha-diol can amplify or substitute for androgens, with regard to activation of the AR via the R(SHBG) by a signal transduction pathway involving cAMP. Because both E2 and 3alpha-diol are involved in the pathogenesis of benign prostatic hyperplasia in dogs and implicated in benign prostatic hyperplasia in man, antagonism of the prostatic SHBG pathway may offer a novel and attractive therapeutic target.

Animals↗

PubMed1998

Role of skeletal muscle in thiazolidinedione insulin sensitizer (PPARgamma agonist) action.

Thiazolidinedione (TZD) insulin sensitizers are specific agonists of peroxisome proliferator activated receptor (PPAR)gamma. However, their mechanism of action and the in vivo target tissue(s) that mediate insulin sensitization remain poorly defined. Although PPARgamma messenger RNA expression has been reported in skeletal muscle, the expression of PPARgamma within myocytes in intact muscle tissue has not been examined. An antipeptide PPARgamma antibody was generated; immunohistochemistry was then used to demonstrate that PPARgamma is present within nuclei of myocytes [in both skeletal (white and red fibers) and cardiac tissue (rodent and human)]. The effect of insulin sensitizer treatment on muscle insulin action was studied using ob/ob mice after 4 days dosing with a potent (6 nM PPARgamma Kd) TZD (10 mg/kg x day). 2-deoxyglucose (2-DOG) uptake was then assessed in freshly isolated soleus muscles from lean vs. ob/ob vs. TZD-treated ob/ob mice. In lean mouse muscles, 2-DOG uptake was stimulated by 82%, 95%, 165% (with 25, 100, 2000 microU/ml insulin); muscles from ob/ob were severely insulin resistant (<80% stimulation with 2000 microU/ml insulin). Muscles from TZD-treated ob/ob displayed a normal insulin response with 100 (71%) or 2000 (158%) microU/ml insulin. Additional studies were performed using ZDF rats treated with/without TZD for 7 days. In vivo 2-DOG glucose uptake into soleus, gastrocnemius, and diaphragm muscles was measured during euglycemic-hyperinsulinemic clamp. Compared with lean rats, muscle 2-DOG uptake in ZDF was reduced by 52% (soleus) or 71% (diaphragm). Partial (40-60%) normalization of the reduced 2-DOG uptake was evident in TZD-treated ZDF rats. In contrast to the effect of in vivo treatment on muscle insulin action, preincubation of isolated soleus muscles from naive lean or ob/ob mice for 5 h with 100 nM TZD did not affect insulin-stimulated 2-DOG uptake. We conclude: 1) PPARgamma is expressed in myocytes within skeletal and cardiac muscle. 2) In vivo activation of PPARgamma by treatment of insulin-resistant mice/rats with a potent TZD corrects impaired muscle insulin action. 3) The lack of a direct effect on muscle after 5 h in vitro TZD incubation suggests that changes in insulin action may require a longer duration of PPARgamma activation or that improved muscle insulin sensitivity may result from an indirect in vivo effect of PPARgamma activation (e.g. changes in systemic lipid metabolism).

Animals↗

PubMed1998

Peroxisome proliferator-activated receptors gamma and alpha mediate in vivo regulation of uncoupling protein (UCP-1, UCP-2, UCP-3) gene expression.

A role for peroxisome proliferator-activated receptors, PPAR gamma and PPAR alpha, as regulators of energy homeostasis and lipid metabolism, has been suggested. Recently, three distinct uncoupling protein isoforms, UCP-1, UCP-2, and UCP-3, have also been identified and implicated as mediators of thermogenesis. Here, we examined whether in vivo PPAR gamma or PPAR alpha activation regulates the expression of all three UCP isoforms. Rats or lean and db/db mice were treated with PPAR gamma [thiazolidinedione (TZD)] or PPAR alpha (WY-14643) agonists, followed by measurement of messenger RNAs (mRNAs) for UCP-1, UCP-2, and UCP-3 in selected tissues where they are expressed. TZD treatment (AD 5075 at 5 mg/kg x day) of rats (14 days) increased brown adipose tissue (BAT) depot size and induced the expression of each UCP mRNA (3x control levels for UCP-1 and UCP-2, 2.5x control for UCP-3). In contrast, UCP-2 and UCP-3 mRNA levels were not affected in white adipose tissue or skeletal muscle. Chronic (30 days) low-dose (0.3 mg/kg x day) TZD treatment induced UCP-1 mRNA and protein in BAT (2.5x control). In contrast, chronic TZD treatment (30 mg/kg x day) suppressed UCP-1 mRNA (>80%) and protein (50%) expression in BAT. This was associated with further induction of UCP-2 expression (>10-fold) and an increase in the size of lipid vacuoles, a decrease in the number of lipid vacuoles in each adipocyte, and an increase in the size of the adipocytes. TZD treatment of db/db mice (BRL 49653 at 10 mg/kg x day for 10 days) also induced UCP-1 and UCP-3 (but not UCP-2) expression in BAT. PPAR alpha is present in BAT, as well as liver. Treatment of rats or db/db mice with WY-14643 did not affect expression of UCP-1, -2, or -3 in BAT. Hepatic UCP-2 mRNA was increased (4x control level) in db/db and lean mice, although this effect was not observed in rats. Thus, in vivo PPAR gamma activation can induce expression of UCP-1, -2, and -3 in BAT; whereas chronic-intense PPAR gamma activation may cause BAT to assume white adipose tissue-like phenotype with increased UCP-2 levels. PPAR alpha activation in mice is sufficient to induce liver UCP-2 expression.

Adipose Tissue, Brown↗

PubMed1998

Nuclear receptors have distinct affinities for coactivators: characterization by fluorescence resonance energy transfer.

UNLABELLED: Ligand-dependent interactions between nuclear receptors and members of a family of nuclear receptor coactivators are associated with transcriptional activation. Here we used fluorescence resonance energy transfer (FRET) as an approach for detecting and quantitating such interactions. Using the ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARgamma) as a model, known agonists (thiazolidinediones and delta12, 14-PGJ2) induced a specific interaction resulting in FRET between the fluorescently labeled LBD and fluorescently labeled coactivators [CREB-binding protein (CBP) or steroid receptor coactivator-1 (SRC-1)]. Specific energy transfer was dose dependent; individual ligands displayed distinct potency and maximal FRET profiles that were identical when results obtained using CBP vs. SRC-1 were compared. In addition, half-maximally effective agonist concentrations (EC59s) correlated well with reported results using cell-based assays. A site-directed AF2 mutant of PPARgamma (E471A) that abrogated ligand-stimulated transcription in transfected cells also failed to induce ligand-mediated FRET between PPARgamma LBD and CBP or SRC-1. Using estrogen receptor (ERalpha) as an alternative system, known agonists induced an interaction between ERalpha LBD and SRC-1, whereas ER antagonists disrupted agonist-induced interaction of ERalpha with SRC-1. In the presence of saturating agonist concentrations, unlabeled CBP or SRC-1 was used to compete with fluorescently labeled coactivators with saturation kinetics. Relative affinities for the individual receptor-coactivator pairs were determined as follows: PPARgamma-CBP = ERalpha-SRC-1 > PPARgamma-SRC-1 >> ERalpha-CBP. CONCLUSIONS: 1) FRET-based coactivator association is a novel approach for characterizing nuclear receptor agonists or antagonists; individual ligands display potencies that are predictive of in vivo effects and distinct profiles of maximal activity that are suggestive of alternative receptor conformations. 2) PPARgamma interacts with both CBP and SRC-1; transcriptional activation and coactivator association are AF2 dependent. 3) Nuclear receptor LBDs have distinct affinities for individual coactivators; thus, PPARgamma has a greater apparent affinity for CBP than for SRC-1, whereas ERalpha interacts preferentially with SRC-1 but very weakly with CBP.

Animals↗

PubMed1997

Functional antagonism between CCAAT/Enhancer binding protein-alpha and peroxisome proliferator-activated receptor-gamma on the leptin promoter.

The ob gene product, leptin, is a major hormonal regulator of appetite and fat cell mass. Recent work has suggested that the antidiabetic agents, the thiazolidinediones (TZ), which are also high affinity ligands of peroxisome proliferator-activated receptor-gamma (PPARgamma), inhibit leptin expression in rodents. To examine the effects of this class of drug on the leptin gene in adipocytes we performed Northern analysis on primary rat adipocytes cultured in the presence or absence of TZ. TZ reduced leptin mRNA levels by 75%. To determine whether this effect was mediated at the transcriptional level, we isolated 6510 base pairs of 5'-flanking sequence of the leptin promoter and studied reporter constructs in primary rat adipocytes and CV-1 cells. Sequence analysis demonstrated the presence of a consensus direct repeat with a 1-base-pair gap site between -3951 and -3939 as well as a consensus CCAAT/enhancer binding protein (C/EBP) site between -55 and -47. Our functional analysis in transfected primary rat adipocytes demonstrates that, despite the presence of a canonical direct repeat with a 1-base-pair gap site, TZ alone decreases reporter gene expression of leptin promoter constructs ranging from -6510 to +9 to -65 to +9. In CV-1 cells, which contain endogenous PPARgamma, TZ treatment alone had little effect on these constructs. However, TZ treatment did inhibit C/EBPalpha-mediated transactivation of the leptin promoter. This down-regulation of leptin reporter constructs mapped to a -65 to +9 promoter fragment which binds C/EBPalpha in gel-mobility shift assays but does not bind PPARgamma2 alone or as a heterodimer with 9-cis-retinoic acid receptor. Conversely, the promoter (-5400 to +24 base pairs) of the aP2 gene, another adipocyte-specific gene, was induced 7.3-fold by TZ. Co-transfection with C/EBPalpha minimally stimulated the aP2 promoter from basal levels but notably blocked activation by TZ. These data indicate that PPARgamma and C/EBPalpha can functionally antagonize each other on at least two separate promoters and that this mechanism may explain the down-regulation of leptin expression by thiazolidinediones.

Adipocytes↗

PubMed1997

Targeted disruption of the tumor necrosis factor-alpha gene: metabolic consequences in obese and nonobese mice.

To address the hypothesis that tumor necrosis factor (TNF)-alpha has a role in obesity-associated insulin resistance or the regulation of in vivo lipid metabolism, mice with targeted disruption of the TNF-alpha gene were generated and studied. The absence of TNF-alpha protein in TNF-null (-/-) mice was confirmed. Lean or obese (gold-thioglucose [GTG]-injected) homozygous (-/-) mice were compared with lean or obese age- and sex-matched wild-type (+/+) mice derived from the same line at 13, 19, and 28 weeks of age. The following parameters were significantly affected in lean -/- versus +/+ mice: Body weight was not affected until week 28 (decreased by 14%); epididymal fat pad weight also decreased (25%) at this time, as did percentage body fat (16%), while percentage body protein was increased 13%. Fed plasma insulin levels decreased 47% (28 weeks), triglyceride levels decreased (all three ages; maximum 35% at 19 weeks), and fed plasma leptin decreased 33% (28 weeks). Fasting glucose was slightly (10%) reduced, but the glucose response to an oral glucose tolerance test (OGTT) was not affected. There was a trend (NS) toward increased total adipose tissue lipoprotein lipase in -/- versus +/+ mice. GTG-treatment resulted in obese -/- and +/+ mice with equal mean body weights (42 and 58% increased weight versus lean mice). The following parameters were significantly different in obese -/- mice: fasting plasma glucose decreased 13% (28 weeks), fed plasma insulin decreased 67% (28 weeks), and insulin response to OGTT was decreased by 50%. For both groups of obese mice, glucose levels during the OGTT were substantially increased compared with those in lean mice; however, mean stimulated glucose levels were 20% lower in obese -/- versus +/+ mice. We conclude 1) that TNF-alpha functions to regulate plasma triglycerides and body adiposity and 2) that although TNF-alpha contributes to reduced insulin sensitivity in older or obese mice, the absence of TNF-alpha is not sufficient to substantially protect against insulin resistance in the GTG hyperphagic model of rodent obesity.

Animals↗