Rapid analysis of Saposhnikovia divaricate decoction metabolism in rats by UHPLC-Q-TOFMS and multivariate statistical analysis.

Saposhnikovia divaricata is a commonly used traditional Chinese medicine in treating various diseases such as pyrexia, rheumatism and headache. So far, there have been few reports on the metabolism of orally administered Saposhnikovia divaricate decoction (SDD), hindering further study on its bioactive components and their pharmacological characteristics. In the present study, ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOFMS) was used coupled with principal component analysis (PCA) and partial least squared discriminant analysis (PLS-DA) to rapidly discover and identify the metabolites of SDD. According to the result of PLS-DA, a total of 139 ions of interest including 87 positive ions and 52 negative ions were extracted as SDD-related xenobiotics in urine. Finally, 12 and 65 compounds were identified as absorbed parent components and metabolites of SDD, respectively. Among them, 40 new metabolites were reported for the first time. Our results suggested that hydrolysis, hydroxylation, glucuronidation and sulfation are the major metabolic pathways of chromones, while hydroxylation, hydrogenation and sulfation are the main metabolic pathways of coumarins. This study is the first to explore the absorption and metabolism of SDD using UHPLC-Q-TOFMS, with results providing a basis for further study of its pharmacokinetics and discovery of its bioactive components.

Isolation and identification of three new chromones from the leaves of Pimenta dioica with cytotoxic, oestrogenic and anti-oestrogenic effects.

CONTEXT: Pimenta dioica (L.) Merr. (Myrtaceae) is used in Costa Rican traditional medicine for women's health. Our previous work showed that P. dioica extracts were oestrogenic. OBJECTIVES: This work identifies phytochemicals from P. dioica that are responsible for the plant's oestrogen-like activities. MATERIALS AND METHODS: P. dioica leaves were collected in Costa Rica in 2005. Fractions resulting from chromatographic separation of a methanol extract were tested at 50 µg/mL in a competitive oestrogen receptor-binding assay. Active compounds were isolated by HPLC and identified by NMR and MS. Pure compounds were tested at 1 µM in the oestrogen-responsive SEAP reporter gene assay. The effects on cell viability, cytotoxicity and apoptosis were investigated in breast cancer (MCF-7 and SK-BR3) and gastric cancer (AGS and NCI-N87) cell lines using the ApoTox-Glo and Caspase-Glo assays and qPCR. RESULTS: Quercitrin and three new chromones, including a 2-phenoxychromone, 6,8-di-C-methylcapillarisin (1) were isolated and identified. Compound 1 caused a 6.2-fold increase in SEAP expression at 1 µM (p < 0.05). This activity was blocked by the ER antagonist ICI 182,780. Compound 2 caused a 6.0-fold increase in SEAP, inhibited the growth of MCF-7, AGS and NCI-N87 cells (IC50 54.27, 38.13 and 51.22 µg/mL, respectively), and induced apoptosis via caspase 8 and increased the Bax/Bcl-2 mRNA ratio in MCF-7 cells. Compound 3 was anti-oestrogenic in MCF-7 cells. DISCUSSION AND CONCLUSIONS: Compounds from P. dioica have oestrogenic, anti-oestrogenic and cytotoxic effects that may explain the ethnomedical use of this plant.

Preparative separation of TL1-1 from Daldinia eschscholzii extract by macroporous resin and evaluation of its antimicrobial activities.

2,3-Dihydro-5-hydroxy-2-methylchromen-4-one (TL1-1) has already been reported to exhibit significant activities such as cytotoxicity, antifungal activity and growth inhibitory activity. In order to simply and efficiently separate TL1-1 from crude extracts of Daldinia eschscholzii on a large-preparative scale, XAD-16 resin was selected from ten types of resin based on its superior adsorption and desorption performance. Adsorption equilibrium data for this resin fitted well with pseudo-first order kinetics and the Freundlich model, which were elucidated from kinetic experiments and adsorption isotherms. Under optimized conditions, the purity of TL1-1 increased from 19.21% (w/w) in the crude extract, to 84.64% (w/w) in the final product, with a recovery yield of 75.06% (w/w) by a one-step treatment. Moreover, in a large-scale separation, the purity and recovery of TL1-1 was 80.33% and 72.02% (w/w), respectively. These results demonstrated that a simple adsorption-desorption strategy, using XAD-16 resin, was efficient, which also highlighted its potential for the future large-scale purification and preparation of TL1-1. In addition, studies showed that the purified TL1-1 exhibited moderate antibacterial activity against Ralstonia solanacearum.

Relationship between Expression of Chalcone Synthase Genes and Chromones in Artificial Agarwood induced by Formic Acid Stimulation Combined with Fusarium sp. A2 Inoculation.

Agarwood (gaharu) is a fragrant resin produced in the heartwood of resinous Gyrinops and Aquilaria species. Artificial agarwood samples were obtained from Aquilaria sinensis (Lour.) Gilg using formic acid (FA) stimulation combined with Fusarium sp. A2 inoculation. The relationship between the expression of chalcone synthase genes (CHS) and dynamic changes in chromone content was explored in resin-deposited parts of the trunks of A. sinensis. CHS gene expression levels were detected by qRT-PCR analysis. The chemical composition of agarwood obtained from the heartwood of A. sinensis before and within 1 year after induction was determined by GC-MS. After induction with FA stimulation combined with F. sp. A2 inoculation, the CHS1 gene showed relatively high expression, whereas the CHS2 gene showed low expression. The relative gene expression level of CHS1 peaked at 12 months, with a 153.1-fold increase, and the dominant period of the CHS2 gene expression was 10 months with a 14.13-fold increase. Moreover, chromones were not detected until after 2 months, and a large proportion of chromone compounds were detected after 4 months. Chromone content increased with time and peaked at 12 months. CHS1 gene expression was significantly correlated with 6-hydroxy-2-(2-phenylethyl)chromone accumulation, and CHS2 gene expression was significantly correlated with 5-hydroxy-6-methoxy-2-(2-phenylethyl)chromone accumulation. CHS gene expression was extremely sensitive to FA stimulation combined with F. sp. A2 inoculation and responded to late-onset injury. CHS genes expression also preceded the chromone accumulation. This work laid the foundation for studies on the mechanism by which genes regulate chromone biosynthesis pathways during the formation of agarwood resin in A. sinensis.

Salinity stress induces the production of 2-(2-phenylethyl)chromones and regulates novel classes of responsive genes involved in signal transduction in Aquilaria sinensis calli.

BACKGROUND: Agarwood, is a resinous portion derived from Aquilaria sinensis, has been widely used in traditional medicine and incense. 2-(2-phenylethyl)chromones are principal components responsible for the quality of agarwood. However, the molecular basis of 2-(2-phenylethyl)chromones biosynthesis and regulation remains almost unknown. Our research indicated that salt stress induced production of several of 2-(2-phenylethyl)chromones in A. sinensis calli. Transcriptome analysis of A. sinensis calli treated with NaCl is required to further facilitate the multiple signal pathways in response to salt stress and to understand the mechanism of 2-(2-phenylethyl)chromones biosynthesis. RESULTS: Forty one 2-(2-phenylethyl)chromones were identified from NaCl-treated A. sinensis calli. 93 041 unigenes with an average length of 1562 nt were generated from the control and salt-treated calli by Illmunina sequencing after assembly, and the unigenes were annotated by comparing with the public databases including NR, Swiss-Prot, KEGG, COG, and GO database. In total, 18 069 differentially expressed transcripts were identified by the transcriptome comparisons on the control calli and calli induced by 24 h or 120 h salinity stress. Numerous genes involved in signal transduction pathways including the genes responsible for hormone signal transduction, receptor-like kinases, MAPK cascades, Ca(2+) signal transduction, and transcription factors showed clear differences between the control calli and NaCl-treated calli. Furthermore, our data suggested that the genes annotated as chalcone synthases and O-methyltransferases may contribute to the biosynthesis of 2-(2-phenylethyl)chromones. CONCLUSIONS: Salinity stress could induce the production of 41 2-(2-phenylethyl)chromones in A. sinensis calli. We conducted the first deep-sequencing transcriptome profiling of A. sinensis under salt stress and observed a large number of differentially expressed genes in response to salinity stress. Moreover, salt stress induced dynamic changes in transcript abundance for novel classes of responsive genes involved in signal transduction, including the genes responsible for hormone signal transduction, receptor-like kinases, MAPK cascades, Ca(2+) signal transduction, and transcription factors. This study will aid in selecting the target genes to genetically regulate A. sinensis salt-stress signal transduction and elucidating the biosynthesis of 2-(2-phenylethyl)chromones under salinity stress.

Phytochemical constituents and in vitro radical scavenging activity of different Aloe species.

The phytochemical profile of Aloe barbadensis Mill. and Aloe arborescens Mill. was investigated using colorimetric assays, triple quadrupole and time-of-flight mass spectrometry, focusing on phenolic secondary metabolites in the different leaf portions. Hydroxycinnamic acids, several characteristic anthrones and chromones, the phenolic dimer feralolide and flavonoids such as flavones and isoflavones were identified. The stable radical DPPH test and the ORAC assay were then used to determine the in vitro radical scavenging. The outer green rind was the most active, while the inner parenchyma was much less effective. The 5-methylchromones aloesin, aloeresin A and aloesone were the most active among the pure secondary metabolites tested. The results suggest that several compounds are likely to contribute to the overall radical scavenging activity, and indicate that leaf portion must be taken into account when the plant is used for its antioxidant properties.

Production of rohitukine in leaves and seeds of Dysoxylum binectariferum: an alternate renewable resource.

CONTEXT: Rohitukine is an important precursor for the synthesis of potential anticancer drugs flavopiridol (Sanofi-Aventis) and P-276-00 (Piramal Healthcare Limited, Mumbai, India). Trunk bark of Dysoxylum binectariferum (Roxb.) Hook. f. ex Bedd. (Meliaceae) is the widely used source for isolation of rohitukine. However, removal of trunk bark threatens the survival of the tree. OBJECTIVE: To investigate the amount of rohitukine accumulated in other tissues of D. binectariferum. MATERIALS AND METHODS: Rohitukine standard was isolated from leaves of D. binectariferum. Its purity was ascertained using HR-MS and NMR. Crude extracts were prepared from different tissues of D. binectariferum. Rohitukine content in all the tissues was quantified by HPLC. RESULTS: Rohitukine accumulates in a significant amount in seeds, trunk bark, leaves, twigs, and fruits of D. binectariferum. Seeds have the highest rohitukine content (2.42%, dry weight) followed by trunk bark (1.34%, dry weight), leaves (1.064%, dry weight), twigs (0.844% dry weight), and fruits (0.4559% dry weight). DISCUSSION AND CONCLUSION: Seeds and leaves of D. binectariferum could be used as alternate renewable sources for isolation of rohitukine.

Pharmacokinetic and metabolism studies of rohitukine in rats by high performance liquid-chromatography with tandem mass spectrometry.

A sensitive, selective, and rapid high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C18 (5 µ, 4.6 × 150 mm) column, and isocratic elution with ammonium acetate buffer (pH4; 10 mM):methanol (08:92, v/v) at a flow rate of 0.6 mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100 µL plasma. The parent→product ion transitions (MRM) for analyte and IS were 306.1→245.1 m/z and 180.1→138.1 m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5-500 ng/mL for rohitukine, with a fast run time of 4.5 min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <±10% and precision (% RSD) of <±12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30 days of storage in a freezer at -70±10°C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague-Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.

Rohitukine, a chromone alkaloid and a precursor of flavopiridol, is produced by endophytic fungi isolated from Dysoxylum binectariferum Hook.f and Amoora rohituka (Roxb).Wight & Arn.

Rohitukine, a chromone alkaloid, has gained considerable international attention in recent years because of its novel semi-synthetic derivative, flavopiridol and P-276-00. Both these molecules are in advanced stages of clinical development and trial for cancer treatment. Recently, flavopiridol was approved as an orphan drug for treatment of chronic lymphocytic leukemia cancer. The natural occurrence of rohitukine is restricted to only four plant species, Amoora rohituka and Dysoxylum binectariferum (both from the Meliaceae family) and from Schumanniophyton magnificum and Schumanniophyton problematicum (both from the Rubiaceae family). Recently, an endophytic fungi isolated from D. binectariferum was reported to produce rohitukine in culture. In this study, we report the production of rohitukine and its subsequent attenuation by endophytic fungi, Fusarium oxysporum (MTCC-11383), Fusarium oxysporum (MTCC-11384) and Fusarium solani (MTCC-11385), all isolated from D. binectariferum and Gibberella fujikuroi (MTCC-11382) isolated from Amoora rohituka. The fungal rohitukine which was analyzed by HPLC, LC-MS and LC-MS/MS was identical to reference rohitukine and that produced by the plant. The rohitukine content in the mycelial samples ranged from 192.78µg to 359.55µg100g(-1) of dry weight of and in broth it ranged from 14.10 to 71.90µg100ml(-1). In all the fungal cultures, the production declined from first to fourth sub-culture. Studies are underway to unravel the mechanism by which the fungi produce the host metabolite in culture.

Secondary metabolites from Eupenicillium parvum and their in vitro binding affinity for human opioid and cannabinoid receptors.

Phytochemical investigation of the soil microfungus Eupenicillum parvum led to the isolation of two new compounds: a chromone derivative euparvione (1) and a new mycophenolic derivative euparvilactone (2), as well as thirteen known compounds. The structures of the new compounds were elucidated by means of extensive IR, NMR, and MS data and by comparison of data reported in the literature. The structure of the known compound 6 was confirmed by X-ray crystallography. Several isolated compounds were evaluated for in vitro binding assays using opioid receptors (subtypes δ, κ, and µ) and cannabinoid receptors (CB1 and CB2). Compound 10 displayed the best selective µ-opioid receptor and CB1 receptor binding affinities showing values of 47% and 52% at a 10 µM concentration, respectively. These findings provide insight into the potential therapeutic utility of this class of compounds.

Influence of growing conditions on metabolite profile of Ammi visnaga umbels with special reference to bioactive furanochromones and pyranocoumarins.

The medicinal plant Ammi visnaga is a valuable source of furanochromones and pyranocoumarins used as vasodilator agents. Its ability to germinate under unfavourable growth conditions, such as saline soil and hypoxia characterizing clay soils and marshes ecosystems, prompted us to qualitatively characterize secondary metabolites in umbels of A. visnaga plants grown under different conditions (in field, hydroponically controlled, and contrasted by salinity and/or hypoxia) by HPLC-ESI/IT/MS(n) analysis. Subsequently, the quantitative analysis of the bioactive compounds, above all furanochromones and pyranocoumarins, was carried out by HPLC-ESI/QqQ/MS/MS. The results show the influence of growing conditions on the quali-quantitative profile of A. visnaga secondary metabolites and evidence that hydroponic culture leads to increased level of A. visnaga active principles. Furthermore, two furanochromones never reported before were identified and characterized by 1D- and 2D-NMR analysis.

Biocatalytic conversion of aloeresin A to aloesin.

Leaf exudates from Aloe species, such as the Southern African Aloe ferox, are used in traditional medicines for both humans and livestock. This includes aloesin, a skin bleaching product that inhibits the synthesis of melanin. Aloesin, (a C-glycoside-5-methylchromone) can be released from aloeresin A, an ester of aloesin, through hydrolysis. The objective of the current study was to identify an enzymatic hydrolysis method for converting aloeresin A to aloesin, resulting in increased concentrations of aloesin in the aloe bitters extract. More than 70 commercially available hydrolytic enzymes were screened for the conversion of aloeresin A. An esterase (ESL001-02) from Diversa, a lipase (Novozym 388) and a protease (Aspergillus oryzae) preparation were identified during screening as being capable of providing conversion of pure aloeresin A, with the protease giving the best conversion (~100%). It was found that a contaminating enzyme in Novo 388 was responsible for the conversion of aloeresin A to aloesin. This contaminating enzyme, possibly a protease, was able to give almost complete conversion using crude aloe bitters extract, doubling the concentration of aloesin in aloe bitters extract via the hydrolysis of aloeresin A.

Anti-AIDS agents 87. New bio-isosteric dicamphanoyl-dihydropyranochromone (DCP) and dicamphanoyl-khellactone (DCK) analogues with potent anti-HIV activity.

Six 3'R,4'R-di-O-(S)-camphanoyl-2',2'-dimethyldihydropyrano[2,3-f]chromone (DCP) and two 3'R,4'R-di-O-(S)-camphanoyl-(+)-cis-khellactone (DCK) derivatives were designed, synthesized, and evaluated for inhibition of HIV-1(NL4-3) replication in TZM-bl cells. 2-Ethyl-2'-monomethyl-1'-oxa- and -1'-thia-DCP (5a, 6a), as well as 2-ethyl-1'-thia-DCP (7a) exhibited potent anti-HIV activity with EC(50) values of 30, 38 and 54 nM and therapeutic indexes of 152.6, 48.0 and 100.0, respectively, which were better than or comparable to those of the lead compound 2-ethyl-DCP in the same assay. 4-Methyl-1'-thia-DCK (8a) also showed significant inhibitory activity with an EC(50) of 128 nM and TI of 237.9.

[Research progress of chromanone derivatives from Calophyllum].

Calophyllum plants have great pharmaceutical value. Some species of this genus are used in folk medicine to treat and cure diseases, such as toothache, rheumatism, diarrhea, chronic ulcers, skin infections, and wounds. The genus is known to be rich in chromanone derivatives, and some of these compounds have antiviral, antifungal, antibacterial, cytotoxic, and other activities. In order to come to a more complete understanding of chromanone derivatives and gain new bioactive constituents from Calophyllum, chemical structures, pharmacological activities, and biogenesis of chromanone derivatives from Calophyllum have been discussed in this review.

Characterization of the interaction between farrerol and bovine serum albumin by fluorescence and circular dichroism.

The binding of farrerol to bovine serum albumin (BSA) in aqueous solution was investigated by fluorescence quenching spectra, synchronous fluorescence spectra, circular dichroism (CD) and the three-dimensional (3D) fluorescence spectra at pH 7.40. The results of fluorescence titration indicated that farrerol could quench the intrinsic fluorescence of BSA in a static quenching way. The cause of showing upward curvy patterns in Stern-Volmer plots was analyzed. The binding sites number n and binding constant K using fluorescence quenching equation at 310 K were calculated. The binding distance and the energy transfer efficiency between farrerol and BSA were also obtained according to the theory of Förster's non-radiation energy transfer. The effect of some metal ions on the binding constant of farrerol with BSA was also studied. The effect of farrerol on the conformation of BSA was analyzed using CD, synchronous fluorescence spectra and three-dimensional (3D) fluorescence spectra under experimental conditions. Furthermore, the fluorescence displacement experiments indicated that farrerol could bind to the site I of BSA.

Regulation of CD1, Ki-67, PCNA mRNA expression, and Akt activation in estrogen-responsive human breast adenocarcinoma cell line, MCF-7 cells, by griffonianone C, an isoflavone derived from Millettia griffoniana.

CONTEXT: Millettia griffoniana Baill. (Fabaceae), which contains isoflavonoids like griffonianone C (Griff C), is commonly used in the folk medicine in Cameroon to treat various ailments. Possible health benefits of Griff C which include alleviation of menopausal symptoms, limitation of bone resorption, and lowering of the risks of cancer and cardiovascular diseases attracted our interest. OBJECTIVE: The effects of Griff C on the regulation of the expression of proliferation markers such as proliferating cell nuclear antigen (PCNA), cyclin D1 (CD1) and Ki-67 are investigated here. Its role in apoptosis or cell survival, through the phosphatidylinositol 3 kinase-Akt (PI3K-Akt) signaling pathway is further studied. MATERIALS AND METHODS: Semiquantitative real-time PCR was performed to analyze the effects of Griff C on gene expression in MCF-7 cells. Western blot analysis was used to assess the role of Griff C on the expression of phosphorylated Akt in MCF-7 cells. RESULTS: Griff C induced a 4.84-fold increase in the expression of Ki-67 mRNA at the concentration of 10(-8) M and a 3.90-fold increase of CD1 mRNA at 10(-7) M. Griff C slightly increased the phosphorylation of Akt at its serine 473 residue. Akt phosphorylation was inhibited by the PI3K inhibitor, LY294002, but not by the specific estrogen receptor antagonist, fulvestrant. DISCUSSION AND CONCLUSION: These findings suggest that Griff C can modulate proliferation of MCF-7 cells. Our results also suggest that Griff C can affect the PI3K-related signaling pathway. Thus, Griff C may exert part of its low proliferative and antiapoptotic effects by a nongenomic mode of action.

Isolated perfused lung extraction and HPLC-ESI-MS(n) analysis for predicting bioactive components of Saposhnikoviae Radix.

A novel strategy for predicting bioactive components in traditional Chinese material herb was proposed, using isolated perfused rat lung (IPL) extraction and high performance liquid chromatography\tandem mass spectrometry (HPLC-MS(n)) analysis. The hypothesis is that when the IPL is perfused with the extract of Saposhnikoviae Radix (ESR), the potential bioactive components in the ESR should selectively combine with the receptor or channel of lung, by changing the pH of perfused liquid, the combining components would be eluated and then detected by HPLC-ESI-MS(n). Five compounds were detected in the desorption eluate of IPL; among these compounds, two potential bioactive compounds, prim-O-glucosylcimifugin (2) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (4) were identified by comparing with the chromatography of the standard sample, and three other compounds, i.e. cimifugin (1), 5-O-methylvisamminol (3) and sec-O-glucosylhamaudol (5) were determined by analysis of the structure clearage characterization of mass spectrometry. The application of IPL extraction coupled with HPLC-ESI-MS(n) for predicting potential bioactive components of TCMs is rapid, convenient, operational, economic and reliable.

Central leptin action improves skeletal muscle AKT, AMPK, and PGC1 alpha activation by hypothalamic PI3K-dependent mechanism.

Central leptin action requires PI3K activity to modulate glucose homeostasis and peripheral metabolism. However, the mechanism behind this phenomenon is not clearly understood. We hypothesize that hypothalamic PI3K activity is important for the modulation of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, PGC1 alpha, and AKT in skeletal muscle (SM). To address this issue, we injected leptin into the lateral ventricle of rats. Hypothalamic JAK2 and AKT were activated by intracerebroventricular (ICV) injection of leptin in a time-dependent manner. Central leptin improved tolerance to glucose (GTT), increased PGC1 alpha expression, and AKT, AMPK, ACC and JAK2 phosphorylation in the soleus muscle. Previous ICV administration of either LY294002 or propranolol (IP) blocked these effects. We concluded that the activation of the hypothalamic PI3K pathway is important for leptin-induced AKT phosphorylation, as well as for active catabolic pathway through AMPK and PGC1 alpha in SM. Thus, a defective leptin signalling PI3K pathway in the hypothalamus may contribute to peripheral resistance to insulin associated to diet-induced obesity.

Salt stress induces production of melanin related metabolites in the phytopathogenic fungus Leptosphaeria maculans.

A search for stress metabolites produced by the plant pathogenic fungus Leptosphaeria maculans (asexual stage Phoma lingam) cultured in high salt medium led to the isolation and structure elucidation of two metabolites associated with melanin biosynthesis and cell melanization, a self-protection mechanism against salt stress. The chemical structures of the metabolites were deduced by detailed analysis of 1D and 2D NMR spectroscopic data and chemical transformations.

Enhanced PIP3 signaling in POMC neurons causes KATP channel activation and leads to diet-sensitive obesity.

Leptin and insulin have been identified as fuel sensors acting in part through their hypothalamic receptors to inhibit food intake and stimulate energy expenditure. As their intracellular signaling converges at the PI3K pathway, we directly addressed the role of phosphatidylinositol3,4,5-trisphosphate-mediated (PIP3-mediated) signals in hypothalamic proopiomelanocortin (POMC) neurons by inactivating the gene for the PIP3 phosphatase Pten specifically in this cell type. Here we show that POMC-specific disruption of Pten resulted in hyperphagia and sexually dimorphic diet-sensitive obesity. Although leptin potently stimulated Stat3 phosphorylation in POMC neurons of POMC cell-restricted Pten knockout (PPKO) mice, it failed to significantly inhibit food intake in vivo. POMC neurons of PPKO mice showed a marked hyperpolarization and a reduction in basal firing rate due to increased ATP-sensitive potassium (KATP) channel activity. Leptin was not able to elicit electrical activity in PPKO POMC neurons, but application of the PI3K inhibitor LY294002 and the KATP blocker tolbutamide restored electrical activity and leptin-evoked firing of POMC neurons in these mice. Moreover, icv administration of tolbutamide abolished hyperphagia in PPKO mice. These data indicate that PIP3-mediated signals are critical regulators of the melanocortin system via modulation of KATP channels.