Expression and function of Abcg4 in the mouse blood-brain barrier: role in restricting the brain entry of amyloid-ß peptide.

ABCG4 is an ATP-binding cassette transmembrane protein which has been shown, in vitro, to participate in the cellular efflux of desmosterol and amyloid-ß peptide (Aß). ABCG4 is highly expressed in the brain, but its localization and function at the blood-brain barrier (BBB) level remain unknown. We demonstrate by qRT-PCR and confocal imaging that mouse Abcg4 is expressed in the brain capillary endothelial cells. Modelling studies of the Abcg4 dimer suggested that desmosterol showed thermodynamically favorable binding at the putative sterol-binding site, and this was greater than for cholesterol. Additionally, unbiased docking also showed Aß binding at this site. Using a novel Abcg4-deficient mouse model, we show that Abcg4 was able to export Aß and desmosterol at the BBB level and these processes could be inhibited by probucol and L-thyroxine. Our assay also showed that desmosterol antagonized the export of Aß, presumably as both bind at the sterol-binding site on Abcg4. We show for the first time that Abcg4 may function in vivo to export Aß at the BBB, in a process that can be antagonized by its putative natural ligand, desmosterol (and possibly cholesterol).

The metabolic rate of cultured muscle cells from hybrid Coturnix quail is intermediate to that of muscle cells from fast-growing and slow-growing Coturnix quail.

Growth rate is a fundamental parameter of an organism's life history and varies 30-fold across bird species. To explore how whole-organism growth rate and the metabolic rate of cultured muscle cells are connected, two lines of Japanese quail (Coturnix coturnix japonica), one that had been artificially selected for fast growth for over 60 generations and a control line were used to culture myoblasts. In line with previous work, myoblasts from the fast growth line had significantly higher rates of oxygen consumption, glycolytic flux, and higher mitochondrial volume than myoblasts from the control line, indicating that an increase in growth rate is associated with a concomitant increase in cellular metabolic rates and that mitochondrial density contributes to the differences in rates of metabolism between the lines. We reared chicks from two hybrid lines with reciprocal parental configurations for growth rate to explore the effect of maternally inherited mitochondrial DNA on rates of growth and metabolism. Growth rates of chicks, cellular basal oxygen consumption, glycolytic flux, and mitochondrial volume in myoblasts from chicks from both reciprocal crosses were intermediate to the fast and control lines. This indicates that genes in the nucleus have a strong influence on metabolic rates at the cellular level, compared with maternally inherited mitochondrial DNA.

Cellular metabolic rate is influenced by life-history traits in tropical and temperate birds.

In general, tropical birds have a "slow pace of life," lower rates of whole-animal metabolism and higher survival rates, than temperate species. A fundamental challenge facing physiological ecologists is the understanding of how variation in life-history at the whole-organism level might be linked to cellular function. Because tropical birds have lower rates of whole-animal metabolism, we hypothesized that cells from tropical species would also have lower rates of cellular metabolism than cells from temperate species of similar body size and common phylogenetic history. We cultured primary dermal fibroblasts from 17 tropical and 17 temperate phylogenetically-paired species of birds in a common nutritive and thermal environment and then examined basal, uncoupled, and non-mitochondrial cellular O2 consumption (OCR), proton leak, and anaerobic glycolysis (extracellular acidification rates [ECAR]), using an XF24 Seahorse Analyzer. We found that multiple measures of metabolism in cells from tropical birds were significantly lower than their temperate counterparts. Basal and uncoupled cellular metabolism were 29% and 35% lower in cells from tropical birds, respectively, a decrease closely aligned with differences in whole-animal metabolism between tropical and temperate birds. Proton leak was significantly lower in cells from tropical birds compared with cells from temperate birds. Our results offer compelling evidence that whole-animal metabolism is linked to cellular respiration as a function of an animal's life-history evolution. These findings are consistent with the idea that natural selection has uniquely fashioned cells of long-lived tropical bird species to have lower rates of metabolism than cells from shorter-lived temperate species.

Discrete mechanisms of mTOR and cell cycle regulation by AMPK agonists independent of AMPK.

The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis--two crucial arms of cancer growth--AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.

Micromolar changes in lysophosphatidylcholine concentration cause minor effects on mitochondrial permeability but major alterations in function.

Mice deficient in group 1b phospholipase A2 have decreased plasma lysophosphatidylcholine and increased hepatic oxidation that is inhibited by intraperitoneal lysophosphatidylcholine injection. This study sought to identify a mechanism for lysophosphatidylcholine-mediated inhibition of hepatic oxidative function. Results showed that in vitro incubation of isolated mitochondria with 40-200µM lysophosphatidylcholine caused cyclosporine A-resistant swelling in a concentration-dependent manner. However, when mitochondria were challenged with 220µM CaCl2, cyclosporine A protected against permeability transition induced by 40µM, but not 80µM lysophosphatidylcholine. Incubation with 40-120µM lysophosphatidylcholine also increased mitochondrial permeability to 75µM CaCl2 in a concentration-dependent manner. Interestingly, despite incubation with 80µM lysophosphatidylcholine, the mitochondrial membrane potential was steady in the presence of succinate, and oxidation rates and respiratory control indices were similar to controls in the presence of succinate, glutamate/malate, and palmitoyl-carnitine. However, mitochondrial oxidation rates were inhibited by 30-50% at 100µM lysophosphatidylcholine. Finally, while 40µM lysophosphatidylcholine has no effect on fatty acid oxidation and mitochondria remained impermeable in intact hepatocytes, 100µM lysophosphatidylcholine inhibited fatty acid stimulated oxidation and caused intracellular mitochondrial permeability. Taken together, these present data demonstrated that LPC concentration dependently modulates mitochondrial microenvironment, with low micromolar concentrations of lysophosphatidylcholine sufficient to change hepatic oxidation rate whereas higher concentrations are required to disrupt mitochondrial integrity.

Development of a small-molecule screening method for inhibitors of cellular response to myostatin and activin A.

Myostatin, a member of the transforming growth factor (TGF)-ß family of secreted ligands, is a strong negative regulator of muscle growth. As such, therapeutic inhibitors of myostatin are actively being investigated for their potential in the treatment of muscle-wasting diseases such as muscular dystrophy and sarcopenia. Here, we sought to develop a high-throughput screening (HTS) method for small-molecule inhibitors that target myostatin. We created a HEK293 stable cell line that expresses the (CAGA)12-luciferase reporter construct and robustly responds to signaling of certain classes of TGF-ß family ligands. After optimization and miniaturization of the assay to a 384-well format, we successfully screened a library of compounds for inhibition of myostatin and the closely related activin A. Selection of some of the tested compounds was directed by in silico screening against myostatin, which led to an enrichment of target hits as compared with random selection. Altogether, we present an HTS method that will be useful for screening potential inhibitors of not only myostatin but also many other ligands of the TGF-ß family.

Small-molecule inhibitors targeting G-protein-coupled Rho guanine nucleotide exchange factors.

The G-protein-mediated Rho guanine nucleotide exchange factor (GEF)-Rho GTPase signaling axis has been implicated in human pathophysiology and is a potential therapeutic target. By virtual screening of chemicals that fit into a surface groove of the DH-PH domain of LARG, a G-protein-regulated Rho GEF involved in RhoA activation, and subsequent validations in biochemical assays, we have identified a class of chemical inhibitors represented by Y16 that are active in specifically inhibiting LARG binding to RhoA. Y16 binds to the junction site of the DH-PH domains of LARG with a ∼80 nM K(d) and suppresses LARG catalyzed RhoA activation dose dependently. It is active in blocking the interaction of LARG and related G-protein-coupled Rho GEFs with RhoA without a detectable effect on other DBL family Rho GEFs, Rho effectors, or a RhoGAP. In cells, Y16 selectively inhibits serum-induced RhoA activity and RhoA-mediated signaling, effects that can be rescued by a constitutively active RhoA or ROCK mutant. By suppressing RhoA activity, Y16 inhibits mammary sphere formation of MCF7 breast cancer cells but does not affect the nontransforming MCF10A cells. Significantly, Y16 works synergistically with Rhosin/G04, a Rho GTPase activation site inhibitor, in inhibiting LARG-RhoA interaction, RhoA activation, and RhoA-mediated signaling functions. Thus, our studies show that Rho GEFs can serve as selective targets of small chemicals and present a strategy of dual inhibition of the enzyme-substrate pair of GEF-RhoA at their binding interface that leads to enhanced efficacy and specificity.

Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases.

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho-specific inhibitor, Rhosin. Rhosin contains two aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells, Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity, Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily-specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase.

Small-molecule targeting of proliferating cell nuclear antigen chromatin association inhibits tumor cell growth.

Proliferating cell nuclear antigen (PCNA), a potential anticancer target, forms a homotrimer and is required for DNA replication and numerous other cellular processes. The purpose of this study was to identify novel small molecules that modulate PCNA activity to affect tumor cell proliferation. An in silico screen of a compound library against a crystal structure of PCNA and a subsequent structural similarity search of the ZINC chemical database were carried out to derive relevant docking partners. Nine compounds, termed PCNA inhibitors (PCNA-Is), were selected for further characterization. PCNA-I1 selectively bound to PCNA trimers with a dissociation constant (K(d)) of ~0.2 to 0.4 µM. PCNA-Is promoted the formation of SDS-refractory PCNA trimers. PCNA-I1 dose- and time-dependently reduced the chromatin-associated PCNA in cells. Consistent with its effects on PCNA trimer stabilization, PCNA-I1 inhibited the growth of tumor cells of various tissue types with an IC(50) of ~0.2 µM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC(50), ~1.6 µM). Moreover, uptake of BrdU was dose-dependently reduced in cells treated with PCNA-I1. Mechanistically the PCNA-Is mimicked the effect of PCNA knockdown by siRNA, inducing cancer cell arrest at both the S and G(2)/M phases. Thus, we have identified a class of compounds that can directly bind to PCNA, stabilize PCNA trimers, reduce PCNA association with chromatin, and inhibit tumor cell growth by inducing a cell cycle arrest. They are valuable tools in studying PCNA function and may be useful for future PCNA-targeted cancer therapy.

Consumption of a high-fat diet induces central insulin resistance independent of adiposity.

Plasma insulin enters the CNS where it interacts with insulin receptors in areas that are related to energy homeostasis and elicits a decrease of food intake and body weight. Here, we demonstrate that consumption of a high-fat (HF) diet impairs the central actions of insulin. Male Long-Evans rats were given chronic (70-day) or acute (3-day) ad libitum access to HF, low-fat (LF), or chow diets. Insulin administered into the 3rd-cerebral ventricle (i3vt) decreased food intake and body weight of LF and chow rats but had no effect on HF rats in either the chronic or the acute experiment. Rats chronically pair-fed the HF diet to match the caloric intake of LF rats, and with body weights and adiposity levels comparable to those of LF rats, were also unresponsive to i3vt insulin when returned to ad libitum food whereas rats pair-fed the LF diet had reduced food intake and body weight when administered i3vt insulin. Insulin's inability to reduce food intake in the presence of the high-fat diet was associated with a reduced ability of insulin to activate its signaling cascade, as measured by pAKT. Finally, i3vt administration of insulin increased hypothalamic expression of POMC mRNA in the LF- but not the HF-fed rats. We conclude that consumption of a HF diet leads to central insulin resistance following short exposure to the diet, and as demonstrated by reductions in insulin signaling and insulin-induced hypothalamic expression of POMC mRNA.

Preferential induction of G1 arrest in androgen-responsive human prostate cancer cells by androgen receptor signaling antagonists DL3 and antiandrogen bicalutamide.

The purpose of this study was to further characterize cell growth-inhibitory effects of a recently identified androgen receptor (AR) signaling inhibitor 6-amino-2-[2-(4-tert-butyl-pnenoxy)-ethylsulfanyl]-1H-pyrimidin-4-one (DL3)(5) and antiandrogen bicalutamide (Bic). DL3 was more potent than Bic in induction of G1 arrest and reduction of G1-related cell cycle protein expression in AR-positive LNCaP cells. DL3, but not Bic, moderately inhibited growth of AR-negative PC-3 cells independent of G1 arrest. The data indicated that DL3 inhibit cell growth in both AR-dependent and -independent manners and is potentially a potent therapeutic agent for the management of advanced human prostate cancer.

Small molecule inhibition of HIV-1-induced MHC-I down-regulation identifies a temporally regulated switch in Nef action.

HIV-1 Nef triggers down-regulation of cell-surface MHC-I by assembling a Src family kinase (SFK)-ZAP-70/Syk-PI3K cascade. Here, we report that chemical disruption of the Nef-SFK interaction with the small molecule inhibitor 2c blocks assembly of the multi-kinase complex and represses HIV-1-mediated MHC-I down-regulation in primary CD4(+) T-cells. 2c did not interfere with the PACS-2-dependent trafficking of Nef required for the Nef-SFK interaction or the AP-1 and PACS-1-dependent sequestering of internalized MHC-I, suggesting the inhibitor specifically interfered with the Nef-SFK interaction required for triggering MHC-I down-regulation. Transport studies revealed Nef directs a highly regulated program to down-regulate MHC-I in primary CD4(+) T-cells. During the first two days after infection, Nef assembles the 2c-sensitive multi-kinase complex to trigger down-regulation of cell-surface MHC-I. By three days postinfection Nef switches to a stoichiometric mode that prevents surface delivery of newly synthesized MHC-I. Pharmacologic inhibition of the multi-kinase cascade prevents the Nef-dependent block in MHC-I transport, suggesting the signaling and stoichiometric modes are causally linked. Together, these studies resolve the seemingly controversial models that describe Nef-induced MHC-I down-regulation and provide new insights into the mechanism of Nef action.

Exploring the PI3K alpha and gamma binding sites with 2,6-disubstituted isonicotinic derivatives.

A homology model of the p110alpha catalytic subunit of PI3Kalpha was generated from the p110gamma crystal structure. Using this model, an isonicotinic scaffold was designed for chemically exploring the PI3Kalpha and gamma binding sites. A focused library of derivatives was synthesized and tested. The morpholine acids 5a and 5b proved to be the most potent analogs.

Neuropeptide Y prepares rats for scheduled feeding.

When neuropeptide Y (NPY) is administered centrally, meal-anticipatory responses are elicited. If an increase of endogenous NPY is a signal that heralds an imminent large caloric load, timed daily NPY injections may be expected to condition meal-anticipatory responses that facilitate ingestion. Rats received 4-h access to food beginning in the morning and then timed (1600 h), daily third-ventricular injections of NPY or saline for 7 days. On test day (day 8), animals received the conditioning drug (NPY or saline) or the opposite drug. Food was available immediately after injection on test day, and intake was measured. Rats conditioned with NPY and then given saline ate significantly more than rats conditioned with saline and then given saline; they ate the same amount as rats given NPY. Although they ate more, rats trained with NPY did not have changed plasma glucose, insulin, or ghrelin. These data suggest that NPY plays a role in mediating conditionable food-anticipatory responses that help to cope with the effects of large caloric loads.

Differential effects of adrenalectomy on melanin-concentrating hormone and orexin A.

Removal of glucocorticoids by adrenalectomy (ADX) reduces food intake and body weight in rodents and prevents excessive weight gain in many genetic and dietary models of obesity. Glucocorticoids play a key role to promote positive energy balance in normal and pathological conditions, at least in part, by altering the sensitivity to hypothalamic peptides. The hyperphagia after central neuropeptide Y administration, for example, is attenuated by ADX, and there is evidence that glucocorticoids influence both MCH and orexin A activity. In the present study, feeding responses to third ventricular MCH and orexin A were measured in rats after bilateral ADX or sham surgery. ADX rats were significantly less sensitive to the orexigenic action of third ventricular MCH, whereas orexin A-induced hyperphagia was unaffected. Replacement of corticosterone in the drinking water of ADX rats reversed the effects of ADX on MCH sensitivity. Although we found significant populations of glucocorticoid receptors in the lateral hypothalamus, none were colocalized with either MCH or orexin A-containing cell bodies. Furthermore, whereas ADX significantly reduced hypothalamic MCH and orexin gene expression, this could not be restored by glucocorticoids in the drinking water. Collectively, the present data suggest that glucocorticoids may promote food intake in part by potentiating the orexigenic actions of MCH without affecting the actions of orexin A and that glucocorticoids act indirectly to influence the effects of MCH on food intake.

Adrenalectomy alters the sensitivity of the central nervous system melanocortin system.

Removal of adrenal steroids by adrenalectomy (ADX) reduces food intake and body weight in rodents and prevents excessive weight gain in many genetic and dietary models of obesity. Thus, glucocorticoids appear to play a key role to promote positive energy balance in normal and pathological conditions. By comparison, central nervous system melanocortin signaling provides critical inhibitory tone to regulate energy balance. The present experiments sought to test whether glucocorticoids influence energy balance by altering the sensitivity to melanocortin receptor ligands. Because melanocortin-producing neurons are hypothesized to be downstream of leptin in a key weight-reducing circuit, we tested rats for their sensitivity to leptin and confirmed reports that the hypophagic response to third ventricular (i3vt) leptin is increased in ADX rats and is normalized by glucocorticoid replacement. Next we tested rats for their sensitivity to the melanocortin agonist melanotan II and found that, as for leptin, ADX enhanced the hypophagic response via a glucocorticoid-dependent mechanism. The central nervous system melanocortin system is unique in that it includes the endogenous melanocortin receptor antagonist, AgRP. The orexigenic effect of i3vt AgRP was absent in ADX rats and restored by glucocorticoid replacement. We conclude that the potent weight-reducing effects of ADX likely involve heightened responsiveness to melanocortin receptor stimulation.

Cloned mice have an obese phenotype not transmitted to their offspring.

Mammalian cloning using somatic cells has been accomplished successfully in several species, and its potential basic, clinical and therapeutic applications are being pursued on many fronts. Determining the long-term effects of cloning on offspring is crucial for consideration of future application of the technique. Although full-term development of animals cloned from adult somatic cells has been reported, problems in the resulting progeny indicate that the cloning procedure may not produce animals that are phenotypically identical to their cell donor. We used a mouse model to take advantage of its short generation time and lifespan. Here we report that the increased body weight of cloned B6C3F1 female mice reflects an increase of body fat in addition to a larger body size, and that these mice share many characteristics consistent with obesity. We also show that the obese phenotype is not transmitted to offspring generated by mating male and female cloned mice.