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.

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.

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.

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.

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.

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.

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.

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.