Justin K. Kane

Nicotine administration enhances NPY expression in the rat hypothalamus

Epidemiological studies have shown an inverse relationship between cigarette smoking and body weight. In rodents, a negative correlation between nicotine and body weight has been reported, but this observation was largely derived from studies where relatively high doses of nicotine ( approximately 12 mg/kg/day) were used. In the current study, we showed that a negative relationship also holds for low doses of nicotine that are comparable to that consumed by average human smokers (<6 mg/kg/day). We also demonstrated that 14 days of nicotine administration (4 mg/kg/day) reduced average daily food intake by 19.5% (P<0.01) in the free-feeding nicotine-treated group compared to saline controls. No significant differences in body weight were detected between the nicotine-treated and pair-fed groups. To determine whether the effects of nicotine on food intake and body weight were related to neuropeptide Y (NPY) expression, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and radioimmunoassay were utilized to measure NPY mRNA and peptide levels in various regions of the hypothalamus. Significantly higher levels of NPY mRNA (ca. 20-50%) and peptide (ca. 24-69%) were only detected in the nicotine-treated groups. In addition, significantly higher NPY contents were also obtained in two hypothalamic areas of pair-fed control animals. In summary, our data suggest that the pharmacological effects of nicotine on food intake and body weight may be mediated by changes in hypothalamic NPY levels, a neuropeptide that is pivotal to the hypothalamic regulation of food intake.

Region-specific transcriptional response to chronic nicotine in rat brain

Even though nicotine has been shown to modulate mRNA expression of a variety of genes, a comprehensive high-throughput study of the effects of nicotine on the tissue-specific gene expression profiles has been lacking in the literature. In this study, cDNA microarrays containing 1117 genes and ESTs were used to assess the transcriptional response to chronic nicotine treatment in rat, based on four brain regions, i.e. prefrontal cortex (PFC), nucleus accumbens (NAs), ventral tegmental area (VTA), and amygdala (AMYG). On the basis of a non-parametric resampling method, an index (called jackknifed reliability index, JRI) was proposed, and employed to determine the inherent measurement error across multiple arrays used in this study. Upon removal of the outliers, the mean correlation coefficient between duplicate measurements increased to 0.978+/-0.0035 from 0.941+/-0.045. Results from principal component analysis and pairwise correlations suggested that brain regions studied were highly similar in terms of their absolute expression levels, but exhibited divergent transcriptional responses to chronic nicotine administration. For example, PFC and NAs were significantly more similar to each other (r=0.7; P<10(-14)) than to either VTA or AMYG. Furthermore, we confirmed our microarray results for two representative genes, i.e. the weak inward rectifier K(+) channel (TWIK-1), and phosphate and tensin homolog (PTEN) by using real-time quantitative RT-PCR technique. Finally, a number of genes, involved in MAPK, phosphatidylinositol, and EGFR signaling pathways, were identified and proposed as possible targets in response to nicotine administration.

Hypothalamic orexin-A binding sites are downregulated by chronic nicotine treatment in the rat.

Chronic nicotine treatment (4 mg/kg per day; 14 days) significantly reduced the affinity and density of orexin-A binding sites in the anterior hypothalamus of rat brain. There was a significantly lower sensitivity of orexin-A binding to orexin peptides, to the related secretin and pituitary adenylate cyclase activating peptide, and to unrelated neuropeptide Y (NPY). This change correlated with selective downregulation of a fraction of hypothalamic NPY Y(1) receptors. In previous studies, we have demonstrated an increase in the levels of orexin-A peptide and NPY in discrete hypothalamic areas upon nicotine treatment. This finding contradicts an expected increase in the production of these orexigenic peptides in a model where an inverse relationship is observed between food consumption and nicotine treatment. This study provides a possible explanation to this inconsistency in that a decrease in affinity of orexin-A binding could reduce neural orexin signaling, which may contribute to decreased food intake observed in smokers and animals chronically treated with nicotine.

Regulation of feeding-associated peptides and receptors by nicotine.

Although numerous epidemiological studies have provided convincing evidence for the inverse association between tobacco smoking and body weight, the molecular mechanisms underlying this relationship are not well-understood. Nicotine, as a potent secretagogue, could be expected to influence the levels and expression of many classes of neurotransmitters, as well as of cell-membrane constituents linked to neurotransmission, including signal transducers and related effectors. A potentially major group of candidate molecules that could be involved in feeding-related actions of nicotine are the numerous neuropeptides and peptide hormones shown in the past two decades to regulate food intake and energy expenditure. These could include neuropeptide Y (NPY), orexins, leptins, and uncoupling proteins (UCPs). Some of these peptides were already shown to respond to nicotine treatment in terms of regulation of levels and of activity at the level of cell-membrane receptors. The primary objective of this review is to summarize our current understanding of the regulatory effects of nicotine on the food intake and energy expenditure as related to the expression levels of leptin, NPY, orexin, uncoupling proteins, and of NPY and orexin receptors.

Nicotine, Body Weight and Potential Implications in the Treatment of Obesity

Obesity is an epidemic problem in the U. S. and many other industrialized nations. Historically, the drugs used for the treatment of obesity generally targeted small molecule neurotransmitters. As research grows to decipher the underlying molecular mechanisms behind energy homeostasis, it is becoming evident that the modulating effects of neuropeptides also are critical in the regulation of appetite and metabolism. The search for drugs to modify these monoaminergic and peptidergic pathways may eventually prove successful in the treatment of obesity. While tobacco smoking has long been used as one strategy to maintain a lower body weight, especially in female smokers, its adverse associations with addiction and disease overshadow its potential use as an antiobesity agent. Potential pharmacological effects of nicotine could be better understood as the intricacies of the nicotinic acetylcholine receptor are revealed. The objective of this review is threefold: first is to provide an overview of the physiological effects of nicotine on body weight while focusing on the drugs that are available as antiobesity and smoking cessation agents. Second is to provide the present status of the nicotinic acetylcholine receptor as it relates to energy homeostasis and its potential as an effective treatment modality for obesity. Third is to present the current knowledge with respect to nicotines effects on energy homeostatic and reward related pathways at the molecular level. A better understanding of the regulatory mechanisms underlying the pharmacological effects of nicotine on body weight will provide insights in identification of potential targets for the development of appropriate medicines in the treatment of obesity.

Microarray Technology and its Application on Nicotine Research

Since its development, microarray technique has revolutionized almost all fields of biomedical research by enabling high-throughput gene expression profiling. Using cDNA microarrays, thousands of genes from various organisms have been examined with respect to differentiation/development, disease diagnosis, and drug discovery. Nevertheless, research on nicotine using cDNA microarrays has been rather limited. Therefore, it is our intention in this article to report the findings of our cDNA microarray study on nicotine. We first present an overview of the microarray technology, particularly focusing on the factors related to microarray design and analysis. Second, we provide a detailed description of several newly identified biological pathways in our laboratory, such as phosphatidylinositol signaling and calcium homeostasis, which are involved in response to chronic nicotine administration. Additionally, we illustrate how comparisons between microarray studies help identify candidate genes that potentially may explain the observed inverse association between smoking and schizophrenia. Lastly, given the early stage of microarray research on nicotine, we elaborate on the need for an efficient analysis of genetic networks to further enhance our understanding of the mechanisms involved in nicotine abuse and addiction.

Agonist internalization by cloned Y1 neuropeptide Y (NPY) receptor in Chinese hamster ovary cells shows strong preference for NPY, endosome-linked entry and fast receptor recycling.

In Chinese hamster ovary (CHO) cells expressing the cloned guinea-pig Y1 receptor, the saturable, receptor-linked internalization of NPY (NPY)-related peptides showed the rank order of human/rat neuropeptide Y (hNPY)>pig/rat peptide YY (pPYY)>=(Pro(34))human PYY>(Leu(31),Pro(34))hNPY>(Leu(31),Pro(34))hPYY>>BVD-11 (a selective Y1 antagonist). All agonists accessed similar numbers of Y1 sites in particulates from disrupted cells, with relatively small affinity variation. The rate of internalization could significantly depend on the overall interactivity of the agonist peptide (reflected in sensitivity to chaotropic agents, as well as in the level of non-saturable binding and internalization). Concentration-dependent inhibition of the agonist-driven CHO-Y1 internalization was found with filipin III (a cholesterol-complexing macrolide), and confirmed with inhibitors of clathrin lattice formation, phenylarsine oxide (PAO) and sucrose. In the concentration range affecting Y1 internalization, none of the above treatments or agents significantly alter agonist affinity for Y1 cell surface or particulate receptors. Largely similar responses to the above inhibitors were observed in CHO-Y1 cells for internalization of human transferrin. Internalization of CHO-Y1 receptor apparently is driven by NPY in strong preference to other naturally encountered agonists. At 37 degrees C, most of the internalized receptor is rapidly recycled through endosome-like membrane elements, detectable in Percoll gradients.

Internalization of neuropeptide Y Y1 and Y5 and of pancreatic polypeptide Y4 receptors is inhibited by lithium in preference to sodium and potassium ions

The receptor-linked internalization of [125I] human neuropeptide Y (NPY) in Chinese hamster ovary (CHO) cells expressing the guinea-pig Y1 receptors or in human endometrial carcinoma-1B (Hec-1B) cells expressing the human Y5 receptor, as well as the receptor-linked internalization of human pancreatic polypeptide (hPP) receptor expressed in CHO cells, is selectively inhibited by low molarities of the Li+ cation. The Na+ and K+ cations decreased the receptor-linked internalization of agonist peptides only at high molar inputs, and largely in proportion to the reduction of cell surface binding of Y ligand peptides, dependent on ion concentration and the type of Y receptor examined. With particulates isolated from disrupted cells, there was no preferential inhibition by Li+ relative to Na+ in the binding of type-specific ligand peptides to Y receptors of any type. The observed difference could be connected to the known ability of Li+ to modify active conformations of signal transducers, which may also directly or indirectly affect the internalization motors. The decrease in the rate of Y receptor internalization by Li+ also points to a possible alteration of Y receptor signaling in vivo by lithium at acute therapeutically employed dose levels.