Gabriele V. Ronnett

Neuropeptide Y functions as a neuroproliferative factor

Neuropeptide Y (NPY) has a number of functions in mammalian physiology. Here we identify a role for NPY in promoting proliferation of postnatal neuronal precursor cells. NPY is synthesized in the postnatal olfactory epithelium by sustentacular cells, previously proposed to function only in structural support. Mice with a targeted deletion of NPY contain half as many dividing olfactory neuronal precursor cells as do controls. Furthermore, NPY-deficient mice develop significantly fewer olfactory neurons by adulthood. NPY acts on multipotent neuronal precursor or basal cells to activate rapidly and transiently the extracellular signal-regulated kinase (ERK)1/2 subgroup of mitogen-activated protein kinases. The NPY Y1 receptor subtype appears to mediate this effect. The ability of NPY to induce neuronal precursor proliferation is mediated by protein kinase C (PKC), indicating an upstream PKC-dependent activation of ERK1/2. These results indicate that NPY may regulate neuronal precursor proliferation in the adult mammal.

Nitric oxide mediates the formation of synaptic connections in developing and regenerating olfactory receptor neurons

Nitric oxide (NO) is a diffusible free radical that functions as a second messenger and neurotransmitter. NO synthase (NOS) is highly and transiently expressed in neurons of the developing olfactory epithelium during migration and establishment of primary synapses in the olfactory bulb. NOS is first expressed at E11 in cells of the presumptive nervous layer of the olfactory placode. NOS immunoreactivity persists in the descendants of these cells that differentiate into embryonic olfactory receptor neurons (ORNs). Olfactory NOS expression in the ORN and in its afferents rapidly declines after birth and is undetectable by P7. Following bulbectomy, NOS expression is rapidly induced in the regenerating ORN and is particularly enriched in their outgrowing axons. Immunoblot and Northern blot analyses similarly demonstrate an induction of NOS protein and mRNA expression, respectively, the highest levels of which coincide with peaks of ORN regeneration. These data argue against a role for NO in odorant-sensitive signal transduction, but suggest a prominent function for NO in activity-dependent establishment of connections in both developing and regenerating olfactory neurons.

C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity

C75, a known inhibitor of fatty acid synthase is postulated to cause significant weight loss through decreased hypothalamic neuropeptide Y (NPY) production. Peripherally, C75, an α-methylene-γ-butyrolactone, reduces adipose tissue and fatty liver, despite high levels of malonyl-CoA. To investigate this paradox, we studied the effect of C75 on fatty acid oxidation and energy production in diet-induced obese (DIO) mice and cellular models. Whole-animal calorimetry showed that C75-treated DIO mice had a 50% greater weight loss, and a 32.9% increased production of energy because of fatty acid oxidation, compared with paired-fed controls. Etomoxir, an inhibitor of carnitine O-palmitoyltransferase-1 (CPT-1), reversed the increased energy expenditure in DIO mice by inhibiting fatty acid oxidation. C75 treatment of rodent adipocytes and hepatocytes and human breast cancer cells increased fatty acid oxidation and ATP levels by increasing CPT-1 activity, even in the presence of elevated concentrations of malonyl-CoA. Studies in human cancer cells showed that C75 competed with malonyl-CoA, as measured by CPT-1 activity assays. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, loss of adipose mass, and resolution of fatty liver. The pharmacological stimulation of CPT-1 activity is a novel finding. The dual action of the C75 class of compounds as fatty acid synthase inhibitors and CPT-1 agonists has therapeutic implications in the treatment of obesity and type II diabetes.

AMPK in the brain: its roles in energy balance and neuroprotection.

Adenosine monophosphate‐activated protein kinase (AMPK) senses metabolic stress and integrates diverse physiological signals to restore energy balance. Multiple functions are indicated for AMPK in the CNS. While all neurons sense their own energy status, some integrate neuro‐humoral signals to assess organismal energy balance. A variety of disease states may involve AMPK, so determining the underlying mechanisms is important. We review the impact of altered AMPK activity under physiological (hunger, satiety) and pathophysiological (stroke) conditions, as well as therapeutic manipulations of AMPK that may improve energy balance.

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptins Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptins anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptins signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptins anorectic signaling cascade.

Neuroprotective Effects of Adenosine Monophosphate-Activated Protein Kinase Inhibition and Gene Deletion in Stroke

Background and Purpose— 5′ adenosine monophosphate-dependent protein kinase (AMPK) acts as a metabolic sensor. AMPK is elevated under ischemic conditions, but the role of AMPK in ischemic brain remains controversial. In this study, we examined the effects of AMPK inhibition using both pharmacological and genetic approaches in an in vivo stroke model. Methods— Focal stroke was induced by reversible middle cerebral artery occlusion in male wild-type mice as well as mice deficient in one of the isoforms of the catalytic subunit of AMPK, AMPK α-1 or α-2. Results— AMPK inhibition was neuroprotective after focal stroke. Mice deficient in AMPK α-2 demonstrated significantly smaller infarct volumes compared with wild-type littermates, whereas deletion of AMPK α-1 had no effect. Phosphorylation of a major upstream regulator of AMPK, LKB1, was also induced in stroke brain. Conclusions— AMPK activation is detrimental in a model of focal stroke. The AMPK catalytic isoform α-2 contributes to the deleterious effects of AMPK activation. AMPK inhibition leads to neuroprotection even when these agents are administered poststroke.

Molecular messengers of olfaction

Our knowledge of olfactory signal transduction has been greatly clarified by several recent advances. Molecular cloning has revealed a large family of putative odorant receptors localized to olfactory epithelium that display a seven-transmembrane-domain motif suggesting an association with G proteins. Very potent and rapid enhancement of both adenylyl cyclase and phosphoinositide turnover has been demonstrated in response to odorants both in isolated olfactory cilia and primary olfactory receptor neuronal cultures. A Ca(2+)-calmodulin-dependent phosphodiesterase has been localized to olfactory cilia. A key role for Ca2+ is evident from many investigations. More recently, odorants have also been shown to affect the levels of cGMP in olfactory receptor neurons. The involvement of multiple second messengers may provide mechanisms for both fine-tuning and desensitization of olfaction.

Developing a head for energy sensing: AMP-activated protein kinase as a multifunctional metabolic sensor in the brain

The 5′‐adenosine monophosphate‐activated protein kinase (AMPK) is a metabolic and stress sensor that has been functionally conserved throughout eukaryotic evolution. Activation of the AMPK system by various physiological or pathological stimuli that deplete cellular energy levels promotes activation of energy restorative processes and inhibits energy consumptive processes. AMPK has a prominent role not only as a peripheral sensor of energy balance, but also in the CNS as a multifunctional metabolic sensor. Recent work suggests that AMPK plays an important role in maintaining whole body energy balance by coordinating feeding behaviour through the hypothalamus in conjunction with peripheral energy expenditure. In addition, brain AMPK is activated by energy‐poor conditions induced by hypoxia, starvation, and ischaemic stroke. Under these conditions, AMPK is activated as a protective response in an attempt to restore cellular homeostasis. However in vivo, it appears that the overall consequence of activation of AMPK is more complex than previously imagined, in that over‐activation may be deleterious rather than neuroprotective. This review discusses recent findings that support the role of AMPK in brain as a multidimensional energy sensor and the consequences of its activation or inhibition under physiological and pathological states.

Expression of neuron-specific beta-III tubulin during olfactory neurogenesis in the embryonic and adult rat

The olfactory neuroepithelium retains the unique capacity to produce a new set of mature neurons every three to four weeks from a precursor population situated at the base of the epithelium. It is not known however, whether developing olfactory neurons in the adult rat follow the same program that is initiated embryonically. By tracking the expression of beta-III tubulin (by immunoreactivity to TuJ-1, an isoform-specific antibody) throughout embryogenesis, we have demonstrated a commitment to the olfactory neuron lineage in a subset of cells in the embryonic olfactory placode and followed their development into adulthood. We have also shown that this developmental pattern of beta-III tubulin expression is recapitulated in neurons undergoing a synchronized neurogenic response to either physical or chemical lesion in the adult neuroepithelium. The embryonic expression pattern reported here is similar to, but earlier than that reported for other markers of developing neurons, such as growth-associated protein-43 and neural cell adhesion molecule. The results of these studies suggest the retention of a conserved neurogenic program from embryonic to adult life in the olfactory neuron and, in addition, support the use of a readily accessible system such as the regenerating olfactory neuroepithelium as an alternative means of studying genes which may be crucial to normal neuronal development.

Fatty Acid Synthase Inhibition Activates AMP-Activated Protein Kinase in SKOV3 Human Ovarian Cancer Cells

Fatty acid synthase (FAS), the enzyme responsible for the de novo synthesis of fatty acids, is highly expressed in ovarian cancers and most common human carcinomas. Inhibition of FAS and activation of AMP-activated protein kinase (AMPK) have been shown to be cytotoxic to human cancer cells in vitro and in vivo. In this report, we explore the cytotoxic mechanism of action of FAS inhibition and show that C93, a synthetic FAS inhibitor, increases the AMP/ATP ratio, activating AMPK in SKOV3 human ovarian cancer cells, which leads to cytotoxicity. As a physiologic consequence of AMPK activation, acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid synthesis, was phosphorylated and inhibited whereas glucose oxidation was increased. Despite these attempts to conserve energy, the AMP/ATP ratio increased with worsening cellular redox status. Pretreatment of SKOV3 cells with compound C, an AMPK inhibitor, substantially rescued the cells from C93 cytotoxicity, indicating its dependence on AMPK activation. 5-(Tetradecyloxy)-2-furoic acid, an ACC inhibitor, did not activate AMPK despite inhibiting fatty acid synthesis pathway activity and was not significantly cytotoxic to SKOV3 cells. This indicates that substrate accumulation from FAS inhibition triggering AMPK activation, not end-product depletion of fatty acids, is likely responsible for AMPK activation. C93 also exhibited significant antitumor activity and apoptosis against SKOV3 xenografts in athymic mice without significant weight loss or cytotoxicity to proliferating cellular compartments such as bone marrow, gastrointestinal tract, or skin. Thus, pharmacologic FAS inhibition selectively activates AMPK in ovarian cancer cells, inducing cytotoxicity while sparing most normal human tissues from the pleiotropic effects of AMPK activation.