Anna Coppola

UCP2 mediates ghrelin’s action on NPY/AgRP neurons by lowering free radicals

The gut-derived hormone ghrelin exerts its effect on the brain by regulating neuronal activity. Ghrelin-induced feeding behaviour is controlled by arcuate nucleus neurons that co-express neuropeptide Y and agouti-related protein (NPY/AgRP neurons). However, the intracellular mechanisms triggered by ghrelin to alter NPY/AgRP neuronal activity are poorly understood. Here we show that ghrelin initiates robust changes in hypothalamic mitochondrial respiration in mice that are dependent on uncoupling protein 2 (UCP2). Activation of this mitochondrial mechanism is critical for ghrelin-induced mitochondrial proliferation and electric activation of NPY/AgRP neurons, for ghrelin-triggered synaptic plasticity of pro-opiomelanocortin-expressing neurons, and for ghrelin-induced food intake. The UCP2-dependent action of ghrelin on NPY/AgRP neurons is driven by a hypothalamic fatty acid oxidation pathway involving AMPK, CPT1 and free radicals that are scavenged by UCP2. These results reveal a signalling modality connecting mitochondria-mediated effects of G-protein-coupled receptors on neuronal function and associated behaviour.

Prolylcarboxypeptidase regulates food intake by inactivating α-MSH in rodents

The anorexigenic neuromodulator alpha-melanocyte-stimulating hormone (alpha-MSH; referred to here as alpha-MSH1-13) undergoes extensive posttranslational processing, and its in vivo activity is short lived due to rapid inactivation. The enzymatic control of alpha-MSH1-13 maturation and inactivation is incompletely understood. Here we have provided insight into alpha-MSH1-13 inactivation through the generation and analysis of a subcongenic mouse strain with reduced body fat compared with controls. Using positional cloning, we identified a maximum of 6 coding genes, including that encoding prolylcarboxypeptidase (PRCP), in the donor region. Real-time PCR revealed a marked genotype effect on Prcp mRNA expression in brain tissue. Biochemical studies using recombinant PRCP demonstrated that PRCP removes the C-terminal amino acid of alpha-MSH1-13, producing alpha-MSH1-12, which is not neuroactive. We found that Prcp was expressed in the hypothalamus in neuronal populations that send efferents to areas where alpha-MSH1-13 is released from axon terminals. The inhibition of PRCP activity by small molecule protease inhibitors administered peripherally or centrally decreased food intake in both wild-type and obese mice. Furthermore, Prcp-null mice had elevated levels of alpha-MSH1-13 in the hypothalamus and were leaner and shorter than the wild-type controls on a regular chow diet; they were also resistant to high-fat diet-induced obesity. Our results suggest that PRCP is an important component of melanocortin signaling and weight maintenance via control of active alpha-MSH1-13 levels.

Leptin potentiates IFN-γ-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A.1

Leptin, a pleiotropic hormone believed to regulate body weight, has recently been associated with inflammatory states and immune activity. Here we have studied the effect of leptin on expression of IFN‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2), both prominent markers of macrophage activation, using the murine macrophage J774A.1 cell line. After 24 h of incubation, leptin (1–10 μg ml−1) potently synergized with IFN‐γ (100 U ml−1) in nitric oxide (NO) release, evaluated as nitrite and nitrate (NOx), and prostaglandin E2 (PGE2) production in culture medium. The observed increase of NO and PGE2 was related to enhanced expression of the respective inducible enzyme isoforms, measured in mRNA and protein by RT–PCR and Western blot analysis, respectively. When cells were stimulated only with leptin, a weak induction of NO and PGE2 release and of the expression of related inducible enzymes was observed. Moreover IFN‐γ increased the expression of the functional form of leptin receptor (Ob‐Rb) and this effect was potentiated by leptin in a concentration‐dependent manner. These data suggest that macrophages, among the peripheral immune cells, represent a target for leptin and confirm the relevance of this hormone in the pathophysiology of inflammation.

Suppression of hypothalamic deiodinase type II activity blunts TRH mRNA decline during fasting

Fasting is characterized by disrupted thyroid feedback, with suppressed levels of thyroid hormones and paraventricular thyrotropin releasing hormone (TRH). We found that third ventricle administration of the deiodinase inhibitor, iopanoic acid, dose‐dependently reduced deiodinase type II (DII) activity selectively in the hypothalamus. This suppression of DII by iopanoic acid during fasting prevented elevated DII activity and blunted the decline in hypothalamic TRH mRNA levels. Because fasting‐induced elevation in hypothalamic DII activity is paralleled by increased hypothalamic T3 concentration, our study suggests that T3 formation by DII in the hypothalamus is the cause of disrupted thyroid feedback during fasting.

Leptin induces nitric oxide synthase type II in C6 glioma cells: Role for nuclear factor-κB in hormone effect

Astrocytes in the CNS produce inflammatory mediators in response to several stimuli and cytokines. Here we investigated the in vitro effect of leptin on inducible nitric oxide synthase (iNOS) expression in a glioma cell line (C6). After hormone stimulation, culture media were analysed for accumulated stable oxidation products of NO (NO2(-) and NO3(-), designated as NO(x)), cellular RNA was extracted to determine iNOS mRNA level by RT-PCR and cellular lysates were prepared for protein expression. Leptin induced a concentration-dependent increase of NO release, related to iNOS induction. This effect was potentiated by IFN-gamma, or TNF-alpha, or IFN-gamma plus IL-1beta. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, as well as the specific proteasome inhibitor MG132, blocked leptin-induced iNOS. The role of NF-kappaB was also confirmed by time course studies on degradation of IkappaB-alpha, which began to degrade 5 min after treatment with leptin and returned to basal level after 30-60 min. Pre-incubation of cells with MG132 inhibited leptin-induced IkappaB-alpha degradation. These results confirm the pro-inflammatory role of leptin and identify it as a potential up-regulator of cytokine-induced inflammatory response in the CNS.

Hormonal regulation of the arcuate nucleus melanocortin system.

Over the past century, the hypothalamus has emerged as one of the critical sites involved in energy homeostasis. Degeneration studies in rats performed some six decades ago, first led to identifying hypothalamic subregions controlling food intake and body weight. The idea that the central nervous system (CNS), and the hypothalamus in particular, are key in metabolism regulation was reinforced by the discovery of leptin in 1994. Since the identification of leptin, enormous progress has been made in the understanding of the regulation of hypothalamic and extrahypothalamic brain regions that control food intake and energy expenditure by peripheral signals such as hormones. An important challenge is to decipher these complicated interactions between peripheral signals and neuronal circuits to better understand the etiology of metabolic disorders and to identify opportunities to intervene with pharmacological treatment. In this review, we focus on the hormonal regulation of the neuronal circuits of the arcuate nucleus of the hypothalamus: the melanocortin system.

Branched-chain amino acids alter neurobehavioral function in rats

Recently, we have described a strong association of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) with obesity and insulin resistance. In the current study, we have investigated the potential impact of BCAA on behavioral functions. We demonstrate that supplementation of either a high-sucrose or a high-fat diet with BCAA induces anxiety-like behavior in rats compared with control groups fed on unsupplemented diets. These behavioral changes are associated with a significant decrease in the concentration of tryptophan (Trp) in brain tissues and a consequent decrease in serotonin but no difference in indices of serotonin synaptic function. The anxiety-like behaviors and decreased levels of Trp in the brain of BCAA-fed rats were reversed by supplementation of Trp in the drinking water but not by administration of fluoxetine, a selective serotonin reuptake inhibitor, suggesting that the behavioral changes are independent of the serotonergic pathway of Trp metabolism. Instead, BCAA supplementation lowers the brain levels of another Trp-derived metabolite, kynurenic acid, and these levels are normalized by Trp supplementation. We conclude that supplementation of high-energy diets with BCAA causes neurobehavioral impairment. Since BCAA are elevated spontaneously in human obesity, our studies suggest a potential mechanism for explaining the strong association of obesity and mood disorders.