Dana Boey

NPY and Y receptors: lessons from transgenic and knockout models

Neuropeptide Y (NPY) in the central nervous system is a major regulator of food consumption and energy homeostasis. It also regulates blood pressure, induces anxiolysis, enhances memory retention, affects circadian rhythms and modulates hormone release. Five Y receptors (Y1, Y2, Y4, Y5 and Y6) are known to mediate the action of NPY and its two other family members, peptide YY (PYY) and pancreatic polypeptide (PP). Increased NPY signaling due to elevated NPY expression in the hypothalamus leads to the development of obesity and its related phenotypes, Type II diabetes and cardiovascular disease. Dysregulation in NPY signaling also causes alterations in bone formation, alcohol consumption and seizure susceptibility. The large number of Y receptors has made it difficult to delineate their individual contributions to these physiological processes. However, recent studies analysing NPY and Y receptor overexpressing and knockout models have started to unravel some of the different functions of these Y receptors. Particularly, the use of conditional knockout models has made it possible to pinpoint a specific function to an individual Y receptor in a particular location.

Peptide YY ablation in mice leads to the development of hyperinsulinaemia and obesity

Aims/hypothesisObese people exhibit reduced circulating peptide YY (PYY) levels, but it is unclear whether this is a consequence or cause of obesity. We therefore investigated the effect of Pyy ablation on energy homeostasis.MethodsBody composition, i.p. glucose tolerance, food intake and hypothalamic neuropeptide expression were determined in Pyy knock-out and wild-type mice on a normal or high-fat diet.ResultsPyy knock-out significantly increased bodyweight and increased fat mass by 50% in aged females on a normal diet. Male chow-fed Pyy−/− mice were resistant to obesity but became significantly fatter and glucose-intolerant compared with wild-types when fed a high-fat diet. Pyy knock-out animals exhibited significantly elevated fasting or glucose-stimulated serum insulin concentrations vs wild-types, with no increase in basal or fasting-induced food intake. Pyy knock-out decreased or had no effect on neuropeptide Y expression in the arcuate nucleus of the hypothalamus, and significantly increased proopiomelanocortin expression in this region. Male but not female knock-outs exhibited significantly increased growth hormone-releasing hormone expression in the ventromedial hypothalamus and significantly elevated serum IGF-I and testosterone levels. This sex difference in activation of the hypothalamo–pituitary somatotrophic axis by Pyy ablation may contribute to the resistance of chow-fed male knock-outs to late-onset obesity.Conclusions/interpretationPYY signalling is important in the regulation of energy balance and glucose homeostasis, possibly via regulation of insulin release. Therefore reduced PYY levels may predispose to the development of obesity, particularly with ageing or under conditions of high-fat feeding.

A fundamental bimodal role for neuropeptide Y1 receptor in the immune system

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY) in the central nervous system is a major regulator of numerous physiological functions, including stress. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five Y receptors (Y1, Y2, Y4, Y5, and y6). Using Y1-deficient (Y1−/−) mice, we showed that Y1−/− T cells are hyperresponsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signaling through Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, Y1−/− mice were resistant to T helper type 1 (Th1) cell–mediated inflammatory responses and showed reduced levels of the Th1 cell–promoting cytokine interleukin 12 and reduced interferon γ production. This defect was due to functionally impaired antigen-presenting cells (APCs), and consequently, Y1−/− mice had reduced numbers of effector T cells. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. Our findings uncover a sophisticated molecular mechanism regulating immune cell functions that can lead to stress-induced immunosuppression.

Hypothalamic Control of Bone Formation: Distinct Actions of Leptin and Y2 Receptor Pathways

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti‐osteogenic and Y2−/− anabolic pathways in modulating bone formation.

PYY transgenic mice are protected against diet-induced and genetic obesity

The gut-derived hormone, peptide YY (PYY) reduces food intake and enhances satiety in both humans and animals. Obese individuals also have a deficiency in circulating peptide YY, although whether this is a cause or a consequence of obesity is unclear. Our aims were to determine whether peptide YY (PYY) over-expression may have therapeutic effects for the treatment of obesity by altering energy balance and glucose homeostasis. We generated PYY transgenic mice and measured body weight, food intake, temperature, adiposity, glucose tolerance, circulating hormone and lipid concentrations and hypothalamic neuropeptide levels (neuropeptide Y; proopiomelanocortin, and thyrotropin-releasing hormone) under chow and high-fat feeding and after crossing these mice onto the genetically obese leptin-deficient ob/ob mouse background. PYY transgenic mice were protected against diet-induced obesity in association with increased body temperature (indicative of increased thermogenesis) and sustained expression of thyrotropin-releasing hormone in the paraventricular nucleus of the hypothalamus. Moreover, PYY transgenic mice crossed onto the genetically obese ob/ob background had significantly decreased weight gain and adiposity, reduced serum triglyceride levels and improved glucose tolerance compared to ob/ob controls. There was no effect of PYY transgenic over expression on basal or fasting-induced food intake measured at 11-12 weeks of age. Together, these findings suggest that long-term administration of PYY, PYY-like compounds or agents that stimulate PYY synthesis in vivo can reduce excess adiposity and improve glucose tolerance, possibly via effects on the hypothalamo-pituitary-thyroid axis and thermogenesis.

Low serum PYY is linked to insulin resistance in first-degree relatives of subjects with type 2 diabetes

Low circulating peptide YY (PYY) levels are reported in obese and type II diabetic subjects and results from PYY knockout animals suggests that PYY deficiency may have a causative role in the etiology of obesity and type 2 diabetes. Here, our aims were to determine whether people with a genetic predisposition to developing type 2 diabetes and obesity differ from otherwise similar subjects without such family history, in fasting or meal-related PYY levels, fasting insulin, insulin secretion (HOMA-B) and insulin sensitivity. We also investigated whether PYY ablation affects the intrinsic ability of islets to secrete insulin, which may be a contributing factor to the hyperinsulinemia observed in PYY knockout mice. Healthy female first-degree relatives of people with type 2 diabetes were matched for age, gender and BMI to control subjects but had significantly lower insulin sensitivity (p<0.05). Relatives also had significantly lower fasting serum PYY levels than controls (p<0.05), but their PYY response to a high fat meal (4250 kJ, 73% fat) was not significantly different. Fasting PYY level correlated positively with glucose infusion rate (r=0.713, p=0.002) and fasting adiponectin (r=0.5, p=0.02). Islets of Langerhans from PYY knockout mice were found to hypersecrete insulin in response to 25 mM glucose (p<0.05). These data demonstrate that lack of PYY enhances insulin secretion from the Islets of Langerhans and that low fasting PYY levels are associated with insulin resistance in humans. Together, these findings suggest that low circulating levels of PYY could contribute to hyperinsulinemia and insulin resistance, and possibly contribute to subsequent development of obesity and type 2 diabetes.

Compensatory changes in [125I]-PYY binding in Y receptor knockout mice suggest the potential existence of further Y receptor(s).

Gene knockout approaches have helped to better understand the functions of the different Y receptors. However, some results obtained from these knockout mice are unexpected and differ from the results of pharmacological intervention experiments. One possible explanation for this is that germ-line gene deletion of a particular Y receptor can influence expression and function of the remaining Y receptors. Here we show that such compensation in mRNA and protein expression does occur in Y receptor single, double and triple knockout models. Radio-ligand binding experiments using [(125)I]-PYY revealed significant up- and down-regulation of remaining Y receptor binding sites in various Y receptor knockout models compared to results from control mice employing Y receptor preferring agonist or antagonists for displacement of the radio-ligand. The most obvious change can be seen in the hippocampus of Y(1) knockout mice, where the level of the remaining Y receptors is strongly down-regulated. In Y(2) knockout mice no such trend can be seen, however, the expression pattern is significantly changed with a strong up-regulation of [(125)I]-PYY specific binding in the dentate gyrus. Interestingly, this pattern was also seen in Y(1)Y(2)Y(4) triple knockout mice. Y(5) receptor mRNA was approximately 20% higher in the hippocampus and dentate gyrus in the triple knockout mice compared to wild-type controls, while Y(6) mRNA expression could not be detected. However, competition binding experiments in Y(1)Y(2)Y(4) triple knockout mice with the Y(5) receptor preferring ligands [Leu(31), Pro(34)] NPY and [A(31), Aib(32)] NPY were able to replace only approximately 50% of [(125)I]-PYY binding in the dentate gyrus suggesting the existence of further yet unidentified Y receptor(s).

The role of peptide YY in regulating glucose homeostasis

The gut-derived hormone peptide YY (PYY) is most commonly known for its effect on satiety, decreasing food intake and body weight in animals and humans. However, PYY is also involved in a wide range of digestive functions including regulating insulin secretion and glucose homeostasis. Over the last few years, there have been several interesting clinical and animal studies investigating the role of PYY in glucose homeostasis. This review aims to present an updated summary of findings over the last few decades highlighting the role of PYY in regulating insulin output and insulin sensitivity, and the potential mechanisms involved.

Distribution of prodynorphin mRNA and its interaction with the NPY system in the mouse brain.

Using radioactive in situ hybridisation, the distribution of prodynorphin mRNA in the brains of C57Bl/6 mice was systemically investigated, and double-labelling in situ hybridisation was used to determine the extent to which neuropeptide Y (NPY) and prodynorphin mRNAs were co-expressed. Our results demonstrate that prodynorphin mRNA expression in the mouse brain is localised at specific subregions of the olfactory bulb, cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, mesencephalon and myelencephalon. Among the regions displaying the most intense labelling were the olfactory tubercle, lateral septum (LS), caudate putamen (Cpu), central amygdaloid nucleus (Ce), paraventricular hypothalamic nucleus (PVN), supraoptic nucleus (SO), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), lateral reticular nucleus (LRt) and solitary tract nucleus (NTS). In the arcuate nucleus of the hypothalamus (Arc), double-labelling in situ hybridisation revealed that prodynorphin expressing neurons also contained NPY mRNA, with a co-localisation rate of approximately 88% in the lateral part of the Arc, and 79% in the dorsal part of the Arc, respectively, suggesting potential overlapping functions of these two neurotransmitters in feeding type behaviour.

Synergistic attenuation of obesity by Y2- and Y4-receptor double knockout in ob/ob mice

OBJECTIVE Neuropeptide Y regulates numerous processes including food intake, body composition, and reproduction by at least five different Y receptors. We previously demonstrated a synergistic interaction between Y2 and Y4 receptors in reducing adiposity in chow- or fat-fed Y2Y4-receptor double-knockout mice. In the present study, we investigated whether this synergy could reduce the massive obesity of leptin-deficient ob/ob mice. METHODS Mice with germline deletions of Y2 and Y4 receptors were crossed onto the ob/ob strain. Body weight was measured weekly until 15-18 wk of age before decapitation for collection of trunk blood and tissues. RESULTS Male and female Y24ob triple mutants showed highly significant reductions in body weight and white adipose tissue mass compared with ob/ob mice. This reduction in body weight was not evident in Y2ob or Y4ob double mutants, and the effect on adiposity was significantly greater than that seen in Y2ob or Y4ob mice. These changes were associated with significant attenuation of the increased brown adipose tissue mass and small intestinal hypertrophy seen in ob/ob mice and with normalization of the low circulating free thyroxine concentrations seen in female ob/ob mice and the high circulating corticosterone concentrations seen in male ob/ob mice. CONCLUSION These data reveal a synergistic interaction between Y2 and Y4 receptors in attenuating the massive obesity of ob/ob mice, possibly mediated by stimulation of thyroid function and inhibition of intestinal nutrient absorption. Dual pharmacologic antagonism of Y2 and Y4 receptors could help people to attain and maintain a healthy weight.