Matthew Wortman

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.

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

Significance Cancer cells reprogram their metabolism for optimal growth and survival. AMPK-activated protein kinase (AMPK) is a key energy sensor that controls many metabolic pathways including metabolic reprogramming. However, its role in cancer is poorly understood. Some studies claim that it has a tumor suppressor role while others show its protumor role. Two AMPK-activating compounds (including metformin, now in many clinical trials) are widely used to suppress cancer cell proliferation. We found that AMPK is abundantly expressed in high-grade gliomas and, in contrast to popular belief, these two AMPK activators suppressed glioma cell proliferation through unique AMPK-independent mechanisms. 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.

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. Insulins 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.

C75 inhibits food intake by increasing CNS glucose metabolism.

for NO surrounding the erythrocyte” has no bearing on the mechanism of hypoxic vasodilation and hyperoxic vasoconstriction. Further, such “barriers” are insignificant in smaller flow-regulating arterioles where RBCs contact the endothelium (and where eNOS is absent). We have found that native RBCs as well as RBCs containing 50 nM SNOHb (an amount Gladwin et al. measure in situ) mediate hypoxic vasodilation. These empirical observations refute all model-based arguments against the formation, processing and release of NO bioactivity from RBCs, including the contention by Gladwin et al. that SNOHb cannot exist in RBCs at physiologically relevant levels. Our results also refute the contention of Feelisch and coworkers made above that micromolar SNOHb concentrations are a “key element” of the mechanism of the activity of SNOHb. Overall, we feel that the arguments of Gladwin et al. reflect a misunderstanding of the dynamic interactions between hemoglobin and NO-related molecules, which comprise a non-linear network of reactions subject to allosteric modulation. It is not surprising, for example, that Gladwin et al. did not obtain the SNOHb levels that they had anticipated with high concentrations of inhaled NO gas administered at high flow rates. We have reported (and Herold and Rock have confirmed) that the relative yield of SNOHb formation is inversely related to the rate and the amount of NO delivered. Similarly, Gladwin et al. ascribe hypoxic vasodilation by SNOHb to a nonspecific effect of oxygen tension, rather than allosteric modulation, yet none of the studies they refer to actually tested for allostery in a physiologically relevant context. We have shown that only SNOHb and RBCs, and not other vasodilators, exhibit graded responses across the physiological range of oxygen tensions over which hemoglobin undergoes an allosteric transition; the oxygen-dependent behavior of SNOHb in this and other respects is distinct from that of other nitrosothiols.

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.

Cerebrospinal fluid and plasma β-endorphin in combat veterans with post-traumatic stress disorder

Abstract Opioid-mediated analgesia develops in experimental animals following traumatic stress and increased opioid-mediated analgesia has been observed in combat veterans with post-traumatic stress disorder (PTSD). These observations have led to the hypothesis that increased central nervous system (CNS) opioidergic activity exists in patients with PTSD. However, direct CNS data on opioid peptide concentrations and dynamics in patients with PTSD are lacking. We withdrew cerebrospinal fluid (CSF) via a flexible, indwelling subarachnoid catheter over a 6-h period and determined hourly CSF concentrations of immunoreactive β-endorphin (irβEND) in 10 well-characterized combat veterans with PTSD and nine matched normal volunteers. Blood was simultaneously withdrawn to obtain plasma for irβEND. PTSD symptom clusters, as measured by the CAPS, were correlated with neuroendocrine data. Mean CSF irβEND was significantly greater in patients with PTSD compared with normals and there was a negative correlation between the irβEND and PTSD intrusive and avoidant symptoms of PTSD. No intergroup difference between plasma ieβEND was found, nor was there a significant correlation between CSF and plasma irβEND. Immunoreactive β-lipotropin (irβLPH) and pro-opiomelanocortin (irPOMC), both precursors of βEND, were much more plentiful in human CSF than was β-endorphin itself, as has been previously reported. It remains to be determined whether the increased CNS opioid concentrations predate traumatic stress, thereby conferring a vulnerability to dissociative states and PTSD itself, or result from the trauma. The negative correlation between CSF irβEND and avoidant and intrusive symptoms suggests that CNS hypersecretion of opioids might constitute an adaptive response to traumatic experience. Poor correlation between CSF and plasma irβEND limits use of plasma measures to assess CNS opioid activity.

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 Kd 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.

Small Molecule Inhibition of HIV-1–Induced MHC-I Down-Regulation Identifies a Temporally Regulated Switch in Nef Action

Nef assembles a multi-kinase complex triggering MHC-I down-regulation. We identify an inhibitor that blocks MHC-I down-regulation, identifying a temporally regulated switch in Nef action from directing MHC-I endocytosis to blocking cell surface delivery. These findings challenge current dogma and reveal a regulated immune evasion program.

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 (Kd) 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 IC50 of ∼0.2 μM, whereas it affected the growth of nontransformed cells at significantly higher concentrations (IC50, ∼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 G2/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.

Continuous covariability of dopamine and serotonin metabolites in human cerebrospinal fluid

BACKGROUND Experiments in lower animals and humans have demonstrated the existence of functional interactions between serotonin and dopamine in neuronal tissue. However, the relationship between parameters of serotonin and dopamine neuronal activity over time within the central nervous system (CNS) of the individual human has not yet been established. METHODS We used continuous cerebrospinal fluid (CSF) sampling over 6 hours to test the hypothesis that the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) and the dopamine metabolite homovanillic acid (HVA) significantly covary in concentration over time. Two groups of normal volunteers (total n = 16) were studied at separate hospitals and CSF was assayed for 5-HIAA and HVA by high-performance liquid chromatography (HPLC). Three subjects underwent repeat CSF-withdrawal procedures after a 6-week interval. RESULTS Strong and sustained positive covariability in concentrations of HVA and 5-HIAA was observed in the CSF of individual humans. High intraindividual correlation coefficients were +0.897 and +0.871 in the two normal volunteer groups. The HVA to 5-HIAA concentration ratio in CSF was 2.2 +/- 0.7 with very little variability over intervals ranging from minutes to weeks. CONCLUSIONS The balance between CSF dopamine and serotonin metabolite concentrations remains relatively constant over time in healthy humans. Serial measures of CSF dopamine and serotonin metabolites within the same person could be an effective model in which to explore the interrelationships between these systems in various psychiatric syndromes, in response to drug treatment, and during provactive testing.