PPARγ activation mitigates glucocorticoid receptor-induced excessive lipolysis in adipocytes via homeostatic crosstalk.

Proper balance between lipolysis and lipogenesis in adipocytes determines the release of free fatty acids (FFA) and glycerol, which is crucial for whole body lipid homeostasis. Although, dysregulation of lipid homeostasis contributes to various metabolic complications such as insulin resistance, the regulatory mechanism remains elusive. This study clarified the individual and combined roles for glucocorticoid receptor (GCR) and peroxisome proliferator-activated receptor (PPAR)γ pathways in lipid metabolism of adipocytes. In mature 3T3-L1 adipocytes, GCR activation using dexamethasone upregulated adipose triglyceride lipase (ATGL) and downregulated phosphoenolpyruvate carboxykinase (PEPCK), resulting in enhanced glycerol release into the medium. In contrast, PPARγ ligand pioglitazone modestly upregulated ATGL and hormone sensitive lipase (HSL), but markedly enhanced PEPCK and glycerol kinase (GK), thereby suppressed glycerol release. Dexamethasone showed permissive like effect on PPARγ target genes including perilipin A and aP2, therefore co-administration of dexamethasone and pioglitazone demonstrated synergistic upregulation of these enzymes excepting PEPCK, of which downregulation by dexamethasone was abolished by pioglitazone to the level above control. Thus, the excessive glycerol release was prevented as the net outcome of the co-administration. Consistently, the bodipy stain demonstrated that dexamethasone reduced the amount of cytosolic lipid, which was preserved in co-treated adipocytes. Moreover, silencing of PPARγ suppressed the synergistic effects of co-treatment on the lipolytic and lipogenic genes, and therefore the GCR pathway indeed involves PPARγ. In conclusion, crosstalk between GCR and PPARγ is largely synergistic but counter-regulatory in lipogenic genes, of which enhancement prevents excessive glycerol and possibly FFA release by glucocorticoids into the circulation.

Increase in tumor suppressor Arf compensates gene dysregulation in in vitro aged adipocytes.

Deterioration of adipocyte function due to increased oxidative stress predisposes patients to metabolic disorders in advanced age. However, the roles of tumor suppressors in such conditions remain largely unknown. Therefore, we aimed to address their dynamics in aged adipocytes using a long-term culture model. We compared 3T3-L1 adipocytes at 17-19 days (long-term) with those at 8-10 days (short-term) after initiation of adipogenic induction for mimicking 'aged' and 'young' adipocytes, respectively. H2O2 release and dihydroethidium (DHE) staining was increased, while superoxide dismutase (SOD) activity was reduced in long-term cultured adipocytes, which is suggestive of enhanced oxidative stress in this group. Moreover, qRT-PCR revealed increased mRNAs of Nox4 (a subunit of NADPH oxidase complex), Ccl2 (a proinflammatory chemokine) and Il6 [a marker of senescence-associated secretory phenotype (SASP)] along with decreased levels of Pparγ, Adipoq and Slc2a4 (genes related to glucose metabolism). These alterations were associated with increased expression of the tumor suppressors alternate-reading-frame protein p19Arf (Arf) and p16Ink4a. However, silencing of Arf reduced mRNAs of Adipoq and Slc2a4 and enhanced release of Il6. The effect was opposite in Arf overexpressing adipocytes, which showed reduced superoxide production as assessed with DHE staining and SOD activity. Western blots showed that Arf negatively regulates the phosphorylation of Akt. Luciferase assay in Hela cells additionally suggested that Arf negatively regulates Il6 transcriptional activity through a PI3 K/Akt mediated pathway. These findings strongly suggest that the enhanced Arf expression in oxidative stress plays compensatory protective roles against aging-related dysregulation of gene expression in adipocytes.

Eicosapentaenoic acid upregulates VEGF-A through both GPR120 and PPARγ mediated pathways in 3T3-L1 adipocytes.

Vascular endothelial growth factor-A (VEGF-A) released from adipocytes promotes angiogenesis; and thereby ameliorates the local hypoxia-induced adipose inflammation and insulin resistance. Here, we newly found that eicosapentaenoic acid (EPA) upregulated both mRNA expression and release of VEGF-A in mature 3T3-L1 adipocytes. Silencing mRNA of G-protein coupled receptor 120 (GPR120) and specific inhibition of peroxisome proliferator-activated receptor γ (PPARγ) by GW9662 respectively attenuated the EPA-induced augmentation of VEGF-A release by adipocytes. Furthermore, transfection of GPR120 gene alone and PPARγ gene alone to HEK293 cells respectively increased the promoter activity of VEGF-A as assessed by luciferase reporter assay, which was further augmented when both genes were co-transfected. Promoter deletion analysis and chromatin immunoprecipitation assay revealed that co-transfection of GPR120 enhanced EPA-induced PPARγ binding to PPAR-response element in VEGF-A promoter region. Thus, by the synchronized activation of a membrane receptor GRP120 and a nuclear receptor PPARγ, EPA enhances VEGF-A production in adipocytes.

Valsartan ameliorates the constitutive adipokine expression pattern in mature adipocytes: a role for inverse agonism of the angiotensin II type 1 receptor in obesity.

Angiotensin (Ang) II receptor blockers (ARBs) alleviate obesity-related insulin resistance, which suggests an important role for the Ang II type 1 receptor (AT1R) in the regulation of adipocytokines. Therefore, we treated mature 3T3-L1 adipocytes with 50 µmol l(-1) of valsartan, a selective AT1R blocker without direct agonism to peroxisome proliferator-activated receptor (PPAR)-γ. In the absence of effective concentrations of Ang II, unstimulated mature adipocytes expressed and secreted high levels of interleukin (IL)-6. This constitutive proinflammatory activity was attenuated by the suppression of extracellular signal-regulated kinase phosphorylation by valsartan but was unaffected by the Ang II type 2 receptor blocker PD123319. COS7 cells co-transfected with AT1R and IL-6, which expressed NF-κB but lacked PPAR-γ, showed no constitutive but substantial ligand-dependent IL-6 reporter activity, which was counteracted by valsartan. Valsartan preserved cytosolic IκB-α and subsequently reduced nuclear NF-κB1 protein expression in mature adipocytes. Interestingly, valsartan did not increase PPAR-γ messenger RNA expression per se but enhanced the transcriptional activity of PPAR-γ in mature adipocytes; this enhancement was accompanied by upregulation of the PPAR coactivator (PGC)-1α. Moreover, T0090907, a PPAR-γ inhibitor, increased IL-6 expression, and this increase was attenuated by valsartan. Indeed, addition of valsartan without direct PPAR-γ agonism increased adiponectin production in mature adipocytes. Together, the findings indicate that valsartan blocks the constitutive AT1R activity involving the NF-κB pathway that limits PPAR-γ activity in mature adipocytes. Thus, inverse agonism of AT1R attenuates the spontaneous proinflammatory response and enhances the constitutive insulin-sensitizing activities of mature adipocytes, which may underlie the beneficial metabolic impacts of ARBs.

Mifepristone promotes adiponectin production and improves insulin sensitivity in a mouse model of diet-induced-obesity.

The steroid receptor antagonist mifepristone is used as an anti-cancer agent, eliciting both cytostatic and cytotoxic effects on malignant cells. However, the metabolic effects of long-term treatment with mifepristone have remained unclear. The effects of mifepristone on insulin sensitivity and adiponectin secretion were evaluated both in in vivo and in vitro. First, we explored the effects of mifepristone, on metabolic functions in obese mice receiving a high-fat diet. When these mice were fed mifepristone, they exhibited a marked improvement in insulin sensitivity, attenuated hepatic injury, and decreased adipocyte size, compared with mice that received only the high-fat diet. Intriguingly, mifepristone-treated mice showed significantly elevated plasma adiponectin levels. Second, we tested the effects of mifepristone on differentiated 3T3-L1 adipocytes in vitro. When differentiated adipocytes were treated with mifepristone for 48 h, adiponectin was upregulated at both mRNA and protein levels. Collectively, these results reveal novel actions of mifepristone on metabolic functions, in vivo and in vitro, in which the drug exerts antidiabetic effects associated with an upregulation in adiponectin-secretion.

Pioglitazone promotes preadipocyte proliferation by downregulating p16(Ink4a).

Pioglitazone, a synthetic ligand of peroxisome proliferator-activated receptor (PPAR)γ, causes preadipocyte proliferation through a mechanism which still remains elusive. Here, to address the mechanism, we investigated the effects of PPARγ and pioglitazone on the kinetics of cyclin-dependent kinase inhibitors, especially with p16(Ink4a) (p16) centered, by employing 3T3-L1 preadipocytes. Pioglitazone promoted preadipocyte proliferation by increasing S and G(2)/M cell-cycle entry, which was accompanied by decreased p16 mRNA expression. PPARγ overexpression along with the luciferase reporter assay confirmed that PPARγ was crucial for the downregulation of p16 mRNA transcription, and that the action was augmented by pioglitazone. Thus, pioglitazone exerted cell-cycle dependent promoting effect on preadipocyte proliferation, of which mechanisms include p16-downregulation through PPARγ.

Implementation of a novel PCR based method for detecting malaria parasites from naturally infected mosquitoes in Papua New Guinea.

BACKGROUND: Detection of Plasmodium species in mosquitoes is important for designing vector control studies. However, most of the PCR-based detection methods show some potential limitations. The objective of this study was to introduce an effective PCR-based method for detecting Plasmodium vivax and Plasmodium falciparum from the field-caught mosquitoes of Papua New Guinea. METHODS: A method has been developed to concurrently detect mitochondrial cytochrome b (Cyt b) of four human Plasmodium species using PCR (Cytb-PCR). To particularly discriminate P. falciparum from P. vivax, Plasmodium ovale and Plasmodium malariae, a polymerase chain reaction-repeated fragment length polymorphism (PCR-RFLP) has further been developed to use with this method. However, due to limited samples number of P. ovale and P. malariae; this study was mainly confined to P. vivax and P. falciparum. The efficiency of Cytb-PCR was evaluated by comparing it with two 'gold standards' enzyme linked immunosorbent assay specific for circumsporozoite protein (CS-ELISA) using artificially infected mosquitoes; and nested PCR specific for small subunit ribosomal RNA (SSUrRNA) using field caught mosquitoes collected from three areas (Kaboibus, Wingei, and Jawia) of the East Sepic Province of Papua New Guinea. RESULTS: A total of 90 mosquitoes were artificially infected with three strains of Plasmodium: P. vivax-210 (n = 30), P. vivax-247 (n = 30) and P. falciparum (n = 30). These infected mosquitoes along with another 32 unfed mosquitoes were first checked for the presence of Plasmodium infection by CS-ELISA, and later the same samples were compared with the Cytb-PCR. CS-ELISA for P. vivax-210, P. vivax-247 and P. falciparum detected positive infection in 30, 19 and 18 mosquitoes respectively; whereas Cytb-PCR detected 27, 16 and 16 infections, respectively. The comparison revealed a close agreement between the two assays (kappa = 0.862, 0.842 and 0.894, respectively for Pv-210, Pv-247 and P. falciparum groups). It was found that the eight CS-ELISA-positive mosquitoes detected negative by Cytb-PCR were false-positive results. The lowest detection limit of this Cytb-PCR was 10 sporozoites. A highly concordance result was also found between nested PCR and Cytb-PCR using 107 field caught mosquitoes, and both tests concordantly detected P. falciparum in an Anopheles punctulatus mosquito collected from Kaboibus. Both tests thus suggested an overall sporozoite rate of 0.9% (1/107) in the study areas. Subsequently, PCR-RFLP efficiently discriminated P. falciparum from P. vivax for all of the Cytb-PCR positive samples. CONCLUSION: A single step PCR based method has been introduced here that is highly sensitive, efficient and reliable for identifying P. vivax and P. falciparum from mosquitoes. The reliability of the technique was confirmed by its ability to detect Plasmodium as efficiently as those of CS-ELISA and nested PCR. Application of the assay offers the opportunity to detect vector species of Papua New Guinea and may contribute for designing further vector control programmes.

Genetic diversity in two sibling species of the Anopheles punctulatus group of mosquitoes on Guadalcanal in the Solomon Islands.

BACKGROUND: The mosquito Anopheles irenicus, a member of the Anopheles punctulatus group, is geographically restricted to Guadalcanal in the Solomon Islands. It shows remarkable morphological similarities to one of its sibling species, An. farauti sensu stricto (An. farauti s.s.), but is dissimilar in host and habitat preferences. To infer the genetic variations between these two species, we have analyzed mitochondrial cytochrome oxidase subunit II (COII) and nuclear ribosomal internal transcribed spacer 2 (ITS2) sequences from Guadalcanal and from one of its nearest neighbours, Malaita, in the Solomon Islands. RESULTS: An. farauti s.s. was collected mostly from brackish water and by the human bait method on both islands, whereas An. irenicus was only collected from fresh water bodies on Guadalcanal Island. An. irenicus is distributed evenly with An. farauti s.s. (Phi SC = 0.033, 0.38%) and its range overlaps in three of the seven sampling sites. However, there is a significant population genetic structure between the species (Phi CT = 0.863, P < 0.01; Phi ST = 0.865, P < 0.01 and FST = 0.878, P < 0.01). Phylogenetic analyses suggest that An. irenicus is a monophyletic species, not a hybrid, and is closely related to the An. farauti s.s. on Guadalcanal. The time estimator suggests that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal within 29,000 years before present (BP). An. farauti s.s. expanded much earlier on Malaita (texp = 24,600 BP) than the populations on Guadalcanal (texp = 16,800 BP for An. farauti s.s. and 14,000 BP for An. irenicus). CONCLUSION: These findings suggest that An. irenicus and An. farauti s.s. are monophyletic sister species living in sympatry, and their populations on Guadalcanal have recently expanded. Consequently, the findings further suggest that An. irenicus diverged from the ancestral An. farauti s.s. on Guadalcanal.