Honokiol: A non-adipogenic PPARγ agonist from nature

Background

Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are clinically used to counteract hyperglycemia. However, so far experienced unwanted side effects, such as weight gain, promote the search for new PPARγ activators.

Methods

We used a combination of in silico, in vitro, cell-based and in vivo models to identify and validate natural products as promising leads for partial novel PPARγ agonists.

Results

The natural product honokiol from the traditional Chinese herbal drug Magnolia bark was in silico predicted to bind into the PPARγ ligand binding pocket as dimer. Honokiol indeed directly bound to purified PPARγ ligand-binding domain (LBD) and acted as partial agonist in a PPARγ-mediated luciferase reporter assay. Honokiol was then directly compared to the clinically used full agonist pioglitazone with regard to stimulation of glucose uptake in adipocytes as well as adipogenic differentiation in 3T3-L1 pre-adipocytes and mouse embryonic fibroblasts. While honokiol stimulated basal glucose uptake to a similar extent as pioglitazone, it did not induce adipogenesis in contrast to pioglitazone. In diabetic KKAy mice oral application of honokiol prevented hyperglycemia and suppressed weight gain.

Conclusion

We identified honokiol as a partial non-adipogenic PPARγ agonist in vitro which prevented hyperglycemia and weight gain in vivo.

General significance

This observed activity profile suggests honokiol as promising new pharmaceutical lead or dietary supplement to combat metabolic disease, and provides a molecular explanation for the use of Magnolia in traditional medicine.     

A proline-type fullerene derivative inhibits adipogenesis by preventing PPARγ activation

Obesity and its associated metabolic diseases represent some of the most rapidly expanding health issues worldwide, and, thus, the development of a novel chemical compound to suppress adipogenesis is strongly expected. We herein investigated the effects of water-soluble fullerene derivatives: a bis-malonic acid derivative and three types of proline-type fullerene derivatives, on adipogenesis using NIH-3T3 cells overexpressing PPARγ. One of the proline-type fullerene derivatives (P3) harboring three carboxy groups significantly inhibited lipid accumulation and the expression of adipocyte-specific genes, such as aP2, induced by the PPARγ agonist rosiglitazone. On the other hand, the bis-malonic acid derivative (M) and the 2 other proline-type fullerene derivatives (P1, P2), which have two carboxy groups, had no effect on PPARγ-mediated lipid accumulation or the expression of aP2. P3 fullerene also inhibited lipid accumulation induced by the combined stimulation with 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, and insulin in 3T3-L1 preadipocytes. During the differentiation of 3T3-L1 cells into adipocytes, P3 fullerene did not affect the expression of C/EBPδ, C/EBPβ, or PPARγ, but markedly inhibited that of aP2 mRNA. These results suggest that P3 fullerene exhibits anti-obesity activity by preventing the activation of PPARγ.     

Extent and high frequency of a short conversion between the human Aγ and Gγ fetal globin genes

Summary Southern blotting and DNA sequencing after polymerase chain reaction (PCR) amplification provide evidence for the frequent occurrence (in 7 out of 24 chromosomes) of a short conversion GA in the 3 end of the human fetal A globin gene. This short conversion is characterized by the presence, 3 nucleotides downstream from the termination codon of the A gene, of the TCAC sequence that is normally present at the equivalent position at the 3 end of the G gene; it is therefore identical to a conversion already described. Interestingly, we have found that this conversion is associated with the presence of theHindIII polymorphic restriction site in the A IVS2, occuppying an equivalent position in both the G and A genes. Our observations strengthen the hypothesis that the presence of the HindIII polymorphic restriction site in A IVS2 and the presence of the sequence TCAC at the 3 end of the A gene might be the result of a single conversion event.     

Functional signaling of membrane-bound TL1A induces IFN-γ expression

TL1A, a TNF member implicated in autoimmune diseases, is a transmembrane protein that is processed to release soluble TL1A (TL1A-S). TL1A-S induces a Th1 response, although the functional significance of membrane-bound TL1A (TL1A-M) remains unknown. We generated TL1A-M expression in HEK-293 cells capable of binding DR3-Fc. Co-incubating IL-12/IL-18-primed CD4⁺ T cells with HEK-293 cells expressing TL1A-M induced 3-fold increase in IFN-γ that was blocked by anti-TL1A Ab. These results demonstrate that TL1A-M can bind death domain receptor 3 (DR3) through cell-cell contact to induce downstream IFN-γ secretion enhancement. Anti-TL1A antibodies designed to treat immune diseases should be verified to block both endogenous TL1A forms.     

A putative role for platelet-derived PPARγ in vascular homeostasis demonstrated by anti-PPARγ induction of bleeding, thrombocytopenia and compensatory megakaryocytopoiesis

Widely known for its role in adipogenesis and energy metabolism, PPARγ also plays a role in platelet function. To further understand functions of platelet-derived PPARγ, we produced rabbit polyclonal (PoAbs) and mouse monoclonal (MoAbs) antibodies against PPARγ 14mer/19mer peptide-immunogens. Unexpectedly, our work produced two key findings. First, MoAbs but not PoAbs produced against PPARγ peptide-immunogens displayed antigenic crossreactivity with highly conserved PPARα and PPARβ/δ. Similarly, Santa Cruz PoAb sc-7196 was monospecific for PPARγ while MoAb sc-7273 crossreacted with PPARα and PPARβ/δ. Second, immunized rabbits and mice exhibited unusual pathology including cachexia, excessive bleeding, and low platelet counts leading to thrombocytopenia. Spleens from immunized mice were fatty, hemorrhagic and friable. Although passive administration of anti-PPARγ PoAbs failed to induce experimental thrombocytopenia, megakaryocytopoiesis was induced 4-8-fold in mouse spleens. Similarly, marrow megakaryocytopoiesis was enhanced 1.8-4-fold in immunized rabbits. These peptide-immunogens are 100% conserved in human, rabbit and mouse; thus, immune-mediated platelet destruction via crossreactivity with platelet-derived PPARγ likely caused bleeding, thrombocytopenia, and compensatory megakaryocytopoiesis. Such overt pathology would cause significant problems for large-scale production of anti-PPARγ PoAbs. Furthermore, a major pitfall associated with MoAb production against closely related molecules is that monoclonicity does not guarantee monospecificity, an issue worth further scientific scrutiny.     

Conserved antagonism between JMJD2A/KDM4A and HP1γ during cell cycle progression

The KDM4/JMJD2 family of histone demethylases is amplified in human cancers. However, little is known about their physiologic or tumorigenic roles. We have identified a conserved and unappreciated role for the JMJD2A/KDM4A H3K9/36 tridemethylase in cell cycle progression. We demonstrate that JMJD2A protein levels are regulated in a cell cycle-dependent manner and that JMJD2A overexpression increased chromatin accessibility, S phase progression, and altered replication timing of specific genomic loci. These phenotypes depended on JMJD2A enzymatic activity. Strikingly, depletion of the only C. elegans homolog, JMJD-2, slowed DNA replication and increased ATR/p53-dependent apoptosis. Importantly, overexpression of HP1γ antagonized JMJD2A-dependent progression through S phase, and depletion of HPL-2 rescued the DNA replication-related phenotypes in jmjd-2(-/-) animals. Our findings describe a highly conserved model whereby JMJD2A regulates DNA replication by antagonizing HP1γ and controlling chromatin accessibility.     

A haplotype-linked four base pair deletion upstream of the Aγ globin gene coincides with decreased gene expression

Summary During normal human development, a switch is classically observed in the relative expression of the two globin genes, the G/A ratio varying from 70/30 at birth to 40/60 by the end of the first year. An exception to this developmental pattern is linked to the presence of an XmnI restriction site at a position — 158 to the Cap site of the G gene. Another exception is observed in individuals homozygous for two easily detectable variations of the A gene: the presence of a threonine residue at codon 75 and a HindIII site within the second intron. A 4-bp deletion has been described around position — 225 in some thalassemic patients presenting with these variations. In this study, we find this deletion to be haplotypelinked in a series of 156 individuals of various ethnic origins and presenting with various normal and pathological phenotypes. In sickle cell patients heterozygous for this 4-bp deletion, the relative expression of the A genes on the two chromosomes can be measured by estimating the AT and AI chains, the former always being synthesized at a lower rate. These results suggest a functional role for the deleted sequence.     

Discovery of INT131: A selective PPARγ modulator that enhances insulin sensitivity

PPARγ is a member of the nuclear hormone receptor family and plays a key role in the regulation of glucose homeostasis. This Letter describes the discovery of a novel chemical class of diarylsulfonamide partial agonists that act as selective PPARγ modulators (SPPARγMs) and display a unique pharmacological profile compared to the thiazolidinedione (TZD) class of PPARγ full agonists. Herein we report the initial discovery of partial agonist 4 and the structure–activity relationship studies that led to the selection of clinical compound INT131 (3), a potent PPARγ partial agonist that displays robust glucose-lowering activity in rodent models of diabetes while exhibiting a reduced side-effects profile compared to marketed TZDs.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.     

Cyclin G-associated kinase regulates protein phosphatase 2A by phosphorylation of its B'γ subunit

Protein phosphatase 2A (PP2A) bearing the B’γ (= B’α/B56γ1/PR61γ) subunit is recruited to dephosphorylation targets by cyclin G. We demonstrate here that cyclin G-associated kinase (GAK), a component of the GAK/B’γ/cyclin G complex, directly phosphorylates the B’γ-Thr104 residue and regulates PP2A activity. Indeed, an anti-B’γ-pT104 antibody detected immunofluorescence signals at the chromosome and centrosome during mitosis; these signals were reduced by siRNA-mediated GAK knockdown. After DNA damage by γ-irradiation, the chromosome signals formed foci that colocalized with a DNA double-strand break (DSB) marker H2AX-pS139 (γH2AX) and CHK2-pT68. Moreover, B’γ-pT104 enhanced PP2A holoenzyme assembly and PP2A activity, as shown by the results of an in vitro phosphatase assay. These results suggest a novel role for GAK as a regulator of dephosphorylation events under the control of the PP2A B’γ subunit.     

A phosphoinositide 3-kinase-γ inhibitor, AS605240 prevents bleomycin-induced pulmonary fibrosis in rats

Phosphoinositide 3-kinase-γ (PI3Kγ) has been identified to play the critical roles in inflammatory cells activation and recruitment in multiply inflammatory diseases and it promised to be a prospective target for relevant inflammatory diseases therapy. AS605240, a selective PI3Kγ inhibitor, has been proved effective on several inflammatory diseases. In this study, we investigated the protective effect of AS605240 on bleomycin-induced pulmonary fibrosis in rats. Our results showed that orally administration of AS605240 significantly prevented lung inflammation and reduced collagen deposition. AS605240 also inhibited augmented expression of TNF-α and IL-1β induced by bleomycin instillation. Moreover, the mRNA levels of TNF-α and IL-1β in lung were remarkably suppressed. Histological assessment found that AS605240 reduced the expression of TGF-β₁ and prevented T lymphocytes infiltration to lung. Phospho-Akt level in inflammatory cells by blocking PI3Kγ was down-regulated and the inhibition of Akt phosphorylation was further confirmed by Western blot. Our findings illustrated that AS605240 was effective for preventing pulmonary fibrosis by suppressing inflammatory cells recruitment and production of inflammatory cytokines. These findings also suggest that PI3Kγ may be a useful target in treating inflammation diseases and AS605240 may represent a promising novel agent for the future therapy of pulmonary fibrosis.     

γ Irradiation of Staphylococcal Enterotoxin B

The inactivation of enterotoxin B by γ irradiation was studied by use of single-and double-gel-diffusion assay techniques. Enterotoxin B (99+% purity) was suspended either in 0.04 m Veronal buffer (pH 7.2) or in milk, dispensed and heat-sealed in borosilicate glass vials, and irradiated essentially at 21 to 26 C with a cobalt-60 source. Parallel titrations of irradiated enterotoxin B in Veronal buffer were made by use of gel-diffusion and cat assay procedures to establish the relative sensitivity of these two assay procedures to irradiated enterotoxin. Results were identical. A dose of 5 Mrad was required to reduce an enterotoxin B concentration of 31 μg/ml in Veronal buffer to less than 0.7 μg/ml. When milk was used as a vehicle, a dose of 20 Mrad was needed to inactivate a 30 μg/ml concentration of enterotoxin B to less than 0.5 μg/ml. With Veronal buffer and milk as vehicles, the D values (dose required to inactivate 90%) for enterotoxin B inactivation were 2.7 and 9.7 Mrad, respectively.     

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

Insulin resistance, tissue inflammation, and adipose tissue dysfunction are features of obesity and Type 2 diabetes. We generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knockout (AKO) mice to investigate the function of NCoR in adipocyte biology, glucose and insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and clamp studies demonstrated enhanced insulin sensitivity in liver, muscle, and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies NCoR as an adaptor protein that enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD-treated state.     

IL-17A synergizes with IFN-γ to upregulate iNOS and NO production and inhibit chlamydial growth

IFN-γ-mediated inducible nitric oxide synthase (iNOS) expression is critical for controlling chlamydial infection through microbicidal nitric oxide (NO) production. Interleukin-17A (IL-17A), as a new proinflammatory cytokine, has been shown to play a protective role in host defense against Chlamydia muridarum (Cm) infection. To define the related mechanism, we investigated, in the present study, the effect of IL-17A on IFN-γ induced iNOS expression and NO production during Cm infection in vitro and in vivo. Our data showed that IL-17A significantly enhanced IFN-γ-induced iNOS expression and NO production and inhibited Cm growth in Cm-infected murine lung epithelial (TC-1) cells. The synergistic effect of IL-17A and IFN-γ on Chlamydia clearance from TC-1 cells correlated with iNOS induction. Since one of the main antimicrobial mechanisms of activated macrophages is the release of NO, we also examined the inhibitory effect of IL-17A and IFN-γ on Cm growth in peritoneal macrophages. IL-17A (10 ng/ml) synergizes with IFN-γ (200 U/ml) in macrophages to inhibit Cm growth. This effect was largely reversed by aminoguanidine (AG), an iNOS inhibitor. Finally, neutralization of IL-17A in Cm infected mice resulted in reduced iNOS expression in the lung and higher Cm growth. Taken together, the results indicate that IL-17A and IFN-γ play a synergistic role in inhibiting chlamydial lung infection, at least partially through enhancing iNOS expression and NO production in epithelial cells and macrophages.