NPY and Y receptors: lessons from transgenic and knockout models

Neuropeptides Neuropeptides 38 (2004) 189–200 www.elsevier.com/locate/npep

Review

NPY and Y receptors: lessons from transgenic and knockout models Shu Lin, Dana Boey, Herbert Herzog

*

Neurobiology Program, Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst Sydney, NSW 2010, Australia Received 2 April 2004; accepted 21 May 2004

Abstract 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. Ó 2004 Elsevier Ltd. All rights reserved.

Contents

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1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190

2.

Generation of transgenic models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. NPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. PP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

190 190 192

3.

Generation of knockout models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. NPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192 192

4.

Y receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

192

5.

Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. NPY transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. NPY transgenic rats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.3. PP transgenic mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. NPY knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.5. Y1 receptor knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.6. Y2 knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.7. Y4 receptor knockout mice . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.8. Y5 receptor knockout . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

193 193 193 194 194 195 196 197 197

Corresponding author. Tel.: +61-2-9295-8296; fax: +61-2-9295-8281. E-mail address: [email protected] (H. Herzog).

0143-4179/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.npep.2004.05.005

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6.

Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

197

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction NPY is widely expressed in the central and peripheral nervous system and has been shown to play an important role in numerous physiological processes (Hokfelt et al., 1998). The related family members peptide YY (PYY) and pancreatic polypeptide (PP) are produced by L type cells in the small intestine and colon or in F type cells in the pancreas, respectively (Hazelwood, 1993). They are known to have effects on gut motility, pancreatic and gall bladder secretion. Additionally, PYY and PP can also access specific Y receptors in the hypothalamus and the brain stem, again further influencing pancreatic and gastric secretion and intestinal motility via modulation of vagal output. Two more copies of a PYY related gene and one copy of a PP related gene have been found in the human genome (Couzens et al., 2000). However, their functionality remains to be tested. NPY and PYY have identical affinity for all known Y receptors (Y1, Y2, Y4, Y5, and in mouse also Y6), with PP preferring the Y4 receptor (Blomqvist and Herzog, 1997; Naveilhan et al., 1998; Parker and Herzog, 1999). All Y-receptors belong to the G-protein coupled receptor family and their activation causes inhibitory responses such as the inhibition of cAMP accumulation. The large number of Y-receptors and the availability of only a few specific synthetic agonists and antagonists has made it difficult in the past to delineate the function of the NPY system. Furthermore, almost all of the known antagonists have prob-

lems in regard to solubility and oral availability, reducing their value for in vivo experiments. Many questions regarding the functional contributions of the different Y receptors to a particular physiological process therefore remain unanswered. Transgenic over-expressing and knockout animal models are valid alternatives to these pharmacological approaches. Over the last eight years, studies describing the over-expression of NPY and PP in transgenic mice, and NPY overexpression in rats and the generation of NPY, Y1, Y2, Y4 and Y5 receptor deficient mice have been reported. The analysis of the phenotypes of all these animal models has revealed significant and distinct roles of each gene in a variety of different physiological processes. A summary of the major phenotypes found in NPY and Y-receptor transgenic and knockout models are shown in Tables 1 and 2 with a more detail description of the models following below.

2. Generation of transgenic models 2.1. NPY Two NPY overexpressing mice models (Inui et al., 1998; Thiele et al., 1998) and one NPY overexpressing rat model (Michalkiewicz and Michalkiewicz, 2000) have been generated and analysed. Only a modest 115% increase in NPY immunoreactivity could be detected in the transgenic mice generated by Inui et al., which are on a BDF1 background.

Table 1 Major phenotypes observed in transgenic models Targeted gene

Species

Examined behaviour/parameter

Behavioral effect

References

NPY

Rat

Anxiety-related behaviors Spatial memory (Morris Water maze) Locomotion Food intake/body weight Heart rate/mean arterial pressure Vascular resistance Bradykardia/hypotension

# # $ $ $ " #

Thorsell et al. (2000)

Michalkiewicz et al. (2001)

NPY

Mouse

Voluntary ethanol intake Sensitivity to ethanol induced sedation

# "

Thiele et al. (1998)

PP

Mouse

Food intake Body weight Adiposity

# # #

Ueno et al. (1999)

$: unaltered, #: decrease dor absent, ": increased.

S. Lin et al. / Neuropeptides 38 (2004) 189–200

191

Table 2 Major phenotypes observed in NPY and Y-receptor knockout models Targeted gene

Examined behaviour/parameter

Behavioral effect

References

NPY

Food intake/body weight Fasting induced re-feeding Leptin sensitivity Elevated plus-maze Locomotor activity Acoustic startle Seizure susceptibility Hot plate (latency) Inhibitory avoidance (memory) Endocrine function Ethanol consumption Sensitivity to ethanol induced sedation

$ # " $ $ " " " $ $ " #

Erickson et al. (1996a,b) Bannon et al. (2000)

NPY induced Blood pressure Basal blood pressure and heart rate NA/angiotensin II induced Blood pressure Body weight Fat mass Food intake NPY induced feeding Fasting induced re-feeding Voluntary ethanol intake Sensitivity to ethanol Algesia Neurogenesis

# $ $ " (>30 weeks) " # or $ # or $ # " # " #

Pedrazzini et al. (1998)

Food intake/body weight NPY induced feeding Fasting induced re-feeding Heart rate Blood pressure Activity Body weight gain Food intake Adiposity Fertility Heart rate Blood Pressure Anxiety

" $ # " $ # # " # $ $ $ #

Bone Mass

"

Y4

Food intake Body weight Adiposity Bone mass Fertility Heart rate Blood pressure

# # # $ $ # #

Sainsbury et al. (2002a,b,c); Smith-White et al. (2002a,b)

Y5 receptor

Food intake and body weight Fasting induced re-feeding Leptin response Adiposity NPY induced feeding

" (old), $ (young) $ $ " # or $

Marsh et al. (1998)

Sensitivity to kainic induced seizures Antiepileleptic effect of NPY

" $

Y1 receptor

Y2 receptor

Bannon et al. (2000) Erickson et al. (1996a); Baraban et al. (1997) Bannon et al. (2000) Erickson et al. (1997) Thiele et al. (1998)

Pedrazzini et al. (1998) Kushi et al. (1998) Kanatani et al. (2000)

Thiele et al. (2002) Naveilhan et al. (2001c) Howell et al. (2003) Naveilhan et al. (1999)

Sainsbury et al. (2002a) Sainsbury et al. (2002b)

Smith-White et al. (2002a) Redrobe et al. (in press) Tschenett et al. (2003) Baldock et al. (2002)

Marsh et al. (1998) Kanatani et al. (2000)/(2001) Marsh et al. (1999) Guo et al. (2002)

$: unaltered, #: decrease dor absent, ": increased.

The model produced by Thiele et al. showed approximately 5 times higher NPY mRNA levels with abundant NPY protein expression in the cortex, amyg-

dala and hippocampus but interestingly not in the arcuate nucleus of the hypothalamus. This NPY transgenic mouse line is on FVB background.

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A 14.5 kb fragment that contained the entire rat NPY gene including 5 kb of 50 and 1.5 kb of 30 flanking sequences was used to generate the transgenic NPY rat line on a Sprague–Dawley background. Significant increases of NPY mRNA levels centrally were only found in the CA1-2 region of the hippocampus, which were confirmed by increased NPY peptide levels in these regions. However, significant increases in NPY levels were also found in peripheral tissues including kidney, lung, spleen and heart. 2.2. PP In addition, a transgenic mouse model on a mixed BDF1xC57BL/6 background overexpressing the PP gene product has been produced (Ueno et al., 1999). Expression of the PP transgene is specific to the pancreas with strong increases in immunoreactivity in islets. Serum levels of PP reached up to 20 times higher levels in the transgenic animals compared to wildtypes and a 50% mortality rate was observed in these mice within 2 weeks after birth.

3. Generation of knockout models 3.1. NPY The era of knockout mice in the NPY field was started by Erickson and colleagues in 1996 with the generation of a NPY knockout mouse line (Erickson et al., 1996a). The targeting vector was designed to replace the coding sequence of the pre-pro-NPY peptide with the lacZ gene to verify the expression and functional activity of the LacZ gene in place of it. The mice are reported in a subsequent publication as being on a 129/SvCp-J background (Erickson et al., 1996b). However, these NPY knockout mice made available to other researches have also been back-crossed onto a C57BL/6 background (Bannon et al., 2000).

4. Y receptors Shortly following the generation of NPY knockout mice, the genes encoding the Y1, Y2, Y4 and Y5 receptor have all been inactivated using various gene-targeting techniques. Five laboratories have reported the generation of Y1 receptor deficient mice (Howell et al., 2003; Kanatani et al., 2000; Kushi et al., 1998; Naveilhan et al., 2001c; Pedrazzini et al., 1998). Two of the constructs are designed to interrupt the coding sequence with either a ‘IRES Tau–LacZ–Neo’ (Naveilhan et al., 2001c) or a neo cassette (Pedrazzini et al., 1998), whereas the two other constructs also delete parts of the gene including the start codon (Kushi et al., 1998;

Kanatani et al., 2000). The fifth report describes the removal of the entire coding sequence of the Y1 receptor gene employing the cre/loxP technology (Howell et al., 2003). The Y1 knockout strains generated by Naveilhan are on a mixed 129SV/Balb/c background and all other Y1 knockout mice are on a mixed 129Sv/C57BL/6 background. Two different strategies were used to generate Y2 knockout mice. In one, the coding sequence of the Y2 receptor was disrupted with a cassette containing the ‘IRES Tau–LacZ–Neo’ genes but leaving the translation initiation codon intact (Naveilhan et al., 1999). The second approach used the cre/loxP technology subsequently deleting the entire coding sequence of the Y2 receptor (Sainsbury et al., 2002a). The first model is on a mixed 129Sv x Balb/c background and the second is on a mixed 129SvJ/C57BL/6 background. So far only one publication describes the generation of Y4 knockout mice (Sainsbury et al., 2002c). These mice are generated by the cre/loxP technology allowing the removal of the entire coding sequence of the gene. Two different models of Y5 knockout mice are known (Kanatani et al., 2000; Marsh et al., 1998). The first targeting construct for the Y5 receptor gene was designed to replace the coding sequence with a Tau–LacZ cassette, also eliminating the initiation start codon. The second construct replaced the entire Y5 receptor gene with a neo selection marker. All three strains of mice are on a mixed 129SvJ/C57BL/6 background. Conditional Y receptor knockout mice lines have also been generated which includes mouse lines carrying a Y1lox=lox , Y2lox=lox or Y4lox=lox floxed allele (Sainsbury et al., 2002a). Gene deletion has been induced by two different methods. The first one by crossing chimeric mice with transgenic mice that express the Cre-recombinase under an oozyte specific promoter leading to the deletion of the gene in the first cell stage (Schenk et al., 1995). And the second one in the adult mice injecting Cre-recombinase expressing adenovirus in the hypothalamus (Sainsbury et al., 2002a). Furthermore, interbreeding single homozygous mutant mice deficient of NPY, the Y-receptor(s), agoutirelated peptide (AgRP) or leptin genes has generated double knockout models (see Table 3). So far double knockouts for NPY
=
, ob/ob (Erickson et al., 1996b); NPY
=
, AgRP
=
(Qian et al., 2002); NPY
=
, Ay (Hollopeter et al., 1998); Y1
=
, ob/ob (Pralong et al., 2002); Y2
=
, ob/ob (Naveilhan et al., 2002; Sainsbury et al., 2002b); Y4
=
, ob/ob (Sainsbury et al., 2002c); Y5
=
, ob/ob (Marsh et al., 1998) and Y1
=
, Y2
=
(Naveilhan et al., 2001b), Y2
=
, Y4
=
(Sainsbury et al., 2003) have been reported. All these double knockout mutants are viable and show varying degrees of improvement to the obese, diabetic and infertile phenotype of the leptin deficient mice, indicating the important role of NPY as a downstream mediator of leptin function.

S. Lin et al. / Neuropeptides 38 (2004) 189–200

193

Table 3 Double knockout models of neuropeptide Y family members Targeted gene

Examined behaviour/parameter

Behavioral effect

References

NPY/AgRP

Food intake Body weight Adiposity

$ $ $

Qian et al. (2002)

NPY/ob

Food intake Body weight Adiposity Fertility

# # # "

Erickson et al. (1996a,b)

NPY/Ay

Food intake Body weight Adiposity

$ $ $

Hollopeter et al. (1998)

NPY/Galanin

Food intake Body weight Adiposity High Fat diet induced obesity

" " " "

Hohmann et al. (2004)

Y1/Y2

Nociception

"

Naveilhan et al. (2001a,b,c)

Y2/Y4

Food intake Body weight Adiposity Bone mass

# # # "

Sainsbury et al. (2003)

Y1/ob

Food intake Body weight Adiposity Fertility

$ # # "

Pralong et al. (2002)

Y2/ob

Food intake Body weight Adiposity Fertility

$ $ # $

Sainsbury et al. (2002a,b,c) Naveilhan et al. (2002)

Y4/ob

Body weight Adiposity Nociception

" " #

Sainsbury et al. (2002a,b,c)

Y5/ob

Food intake Body weight Adiposity Fertility

$ $ $ $

Marsh et al. (1998)

$: unaltered, #: decrease dor absent, ": increased.

5. Phenotypes 5.1. NPY transgenic mice NPY transgenic mice generated by Inui et al. with only 18% net increase in arcuate NPY levels do not show any change in food intake or bodyweight. However, an obese phenotype with transiently increased food intake can be induced in these mice once they are put on a sucrose loaded diet (Kaga et al., 2001). At approximately 1 year of age these mice also develop hyperglycemia and hyperinsulinemia although without altered glucose excursion (Kaga et al., 2001) confirming that elevated hypothalamic NPY expression levels are important for the development of obesity.

No reports in regard to the effects of feeding and bodyweight are available for the second NPY overexpressing model generated by Thiele et al. (1998). However, these NPY-overexpressing mice show a significant reduction in voluntary ethanol consumption and a higher sensitivity to the effects of ethanol compared to control mice (Thiele et al., 1998). 5.2. NPY transgenic rats No effects on bodyweight or food intake have been reported from rats overexpressing NPY. However, the elevated NPY expression in the transgenic NPY rat model influences a variety of behavioral characteristics such as anxiety (Thorsell et al., 2000). For example, restraint stress causes a significant reduction in

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exploratory behavior in the elevated plus maze in wildtype rats but has no effect on this behavior in the NPY overexpressing rats. In the punished drinking test, NPY transgenic rats show an increase in the number of drinking episodes compared to control animals (Thiele et al., 1998). Under basal conditions, seizure susceptibility in NPY transgenic rats is normal (Vezzani et al., 2002). However, icv injection of kainic acid or stimulation by electrical kindling shows a significant reduction in the number and duration of electroencephalographic seizures in the NPY transgenic rats when compared to wild-type rats. Furthermore, transgenic rats are less susceptible to epileptogenesis than wild-type littermates. NPY levels in the transgenic rats are also elevated in peripheral tissues including heart and blood vessels (Michalkiewicz et al., 2001). There is no direct effect on basal arterial blood pressure or heart rate due to elevated NPY levels, however, total vascular resistance was significantly increased. Noradrenaline induced blood pressure increases were also significantly elevated in the transgenic animals. 5.3. PP transgenic mice Interestingly, overexpression of PP in mice leads to the development of a lean phenotype with a reduction of fat mass which is more pronounced in male than in female transgenic PP mice (Ueno et al., 1999). More importantly, these mice exhibit a significant decrease in food intake, during both the dark and the light phase. Effects on the reduced rate of gastric emptying and the reduction in food intake are reversible by ip injection of PP-antiserum. There are no significant effects on water intake or oxygen consumption in these mice. These results confirm the role of PP as a postprandial satiety signal, which most likely acts on brain stem Y4 receptors to modulate sympathetic output. A summary of all effects of the transgenic rat and mouse models is given in Table 1. 5.4. NPY knockout mice Initial studies on the NPY knockout mice generated by Erickson et al. (1996a) did not show a reduction in food intake or bodyweight under normal conditions, although they showed hyperphagic behavior after fasting. However, leptin treatment reduced food intake and bodyweight to a greater extent in the NPY
=
mice compared to the controls. Interestingly, study on these mice back-crossed onto a C57BL/6 background by Bannon et al. (2000) showed a significant reduction of food intake after 24 or 48 h fasting again suggesting an important role of NPY in feeding behavior. Interventions such as high fat diet did not appear to influence bodyweight or food intake in the NPY

knockout mice (Hollopeter et al., 1998). Interestingly however, although the high fat diet increases the weight of their fat pad depots in NPY knockout mice, the actual bodyweight of these mice on the high fat diet is not significantly different from the chow fed controls. Surprisingly, no difference in food intake, bodyweight or fat pad weight between the NPY knockout mice and the wild-type control mice can be detected after chemical lesions in the arcuate nucleus of the hypothalamus via mono-sodium-glutamate injection into neonates. Similarly, ip injections of gold-thio-glucose in adult mice which destroys neurons selectively in the ventral medial hypothalamus (VMH), also known as the satiety center, did not reveal significant differences between NPY knockout mice and controls (Hollopeter et al., 1998). More obvious are the effects of NPY deficiency on the regulation of food intake and energy homeostasis once the NPY knockout mice are crossed onto the leptin deficient ob/ob background. These double knockout mice show a significant reduction of the severe obese phenotype of ob/ob mice accompanied by reduced food intake, increased energy expenditure and improved serum parameters influencing the development of diabetes (Erickson et al., 1996b). This also shows that central NPY is acting downstream of leptin. Other studies crossing the NPY null allele onto obese mouse models did not lead to a significant reduction in the obese phenotype. For example, double knockout of NPY and the yellow obese mouse (Ay ), which has a defect in the melanocortin 4 receptor pathway, did not improve the obese phenotype (Hollopeter et al., 1998). Another model with decreased energy expenditure due to impaired BAT function when crossed onto the NPY deficient background also did not improve the obesity phenotype (Hollopeter et al., 1998). The effects of NPY deficiency have also been investigated in a mouse model which lacks the gene encoding agouti-related peptide (AgRP), an orexigenic peptide acting as an endogenous MC-4 antagonist, which is found to be co-expressed in arcuate NPY neurons (Qian et al., 2002). Again this NPY/AgRP double mutation, like the other two models, does not appear to influence feeding behavior or weight gain under normal conditions suggesting that compensatory mechanism may have been activated. Interestingly, double mutant mice lacking NPY and galanin, eat significantly more and are heavier (30%) than wild-type mice (Hohmann et al., 2004). They also responded to a high-fat diet by gaining more weight than wild-type mice and are unable to regulate their weight normally after a change in diet. Leptin, insulin, and glucose levels are elevated and chronic leptin treatment causes greater weight loss in the mutant mice than in wild-type mice. One of the more obvious phenotypes seen in young adult NPY knockout mice is the increase (30%) in mild seizures when exposed to an unfamiliar environment

S. Lin et al. / Neuropeptides 38 (2004) 189–200

(Erickson et al., 1996a). More so, when challenged with a convulsing agent such as penetetraxole, a GABA antagonist, hyperexcitability is strongly increased in NPY
=
mice with 80% developing motor convulsions compared to only 30% of control mice. Furthermore, the severity of the seizures is increased in the NPY
=
mice and the onset of the seizures is significantly reduced. Investigations into behavioral phenotypes on NPY
=
mice revealed a significant increase in an anxiogenic-like phenotype of these mice (Bannon et al., 2000). In the open field test, NPY
=
mice spend significantly less time in the center area of the apparatus compared to the control animals. However, the total distance traveled by NPY
=
and wild-type mice was not different suggesting no influence on locomoter activity. Experiments investigating the acoustic startle response showed that the NPY knockout mice have a significant increase in amplitude indicating greater anxiety (Bannon et al., 2000). Voluntary ethanol consumption is inversely related to NPY levels, as NPY
=
mice exhibit significant higher ethanol consumption and lower sensitivity to ethanol compared to control mice with NPY-overexpressing mice showing opposite effects (Thiele et al., 1998). The lack of NPY in NPY
=
mice causes an exaggerated autotomy, a self-mutilation behavior possibly related to pain sensation, in agreement with the analgesic effects of NPY (Shi et al., 1998). Alterations in the levels of Y1 and especially Y2 receptor mRNAs were observed in the spinal cord of NPY-deficient mice, suggesting that these two Y receptors are the major receptors involved in the analgesic effect of NPY. 5.5. Y1 receptor knockout mice Y1 receptors are widely expressed in the central nervous system including the major nuclei in the hypothalamus thought to be responsible for the regulation of food intake and energy homeostasis. Intracerebroventricular (icv) injection of the Y1/Y5 preferring ligand Leu31 /Pro34 NPY into rodents strongly stimulates feeding behavior. Interestingly, Y1 receptor knockout models do not display any major abnormalities in regard to food intake or bodyweight, although there are subtle changes seen in all Y1 receptor knockout mice lines analysed. For example, the Y1 receptor knockout mice described by Pedrazzini et al. (1998) show a slightly reduced food intake, both in freely fed and NPY-induced feeding mice. More importantly, fasting induced re-feeding is strongly decreased in these mice. Interestingly, in older Y1 knockout mice an obese phenotype appears to develop, which is more pronounced in females than in males. These mice also show hyperinsulinemia associated with increased adipose tissue glucose utilization and glycogen synthesis (Burcelin et al., 2001).

195

A reduced locomotor activity and decreased metabolic rate has been suggested to be responsible for this phenotype. The Y1 knockout mice generated by Kushi et al. (1998) and Kanatani et al. (2000) show a similar phenotype with no change in basal food intake, mild hyperinsulinemia, late onset obesity and a significant increase in fat mass, particularly in the female knockout mice. Kushi et al. also found increased levels of uncoupling protein 1 (UCP-1) in brown adipose tissue and reduced levels of UCP-2 in white adipose tissue suggesting a decrease in energy expenditure. No information in regards to food intake and body weight changes is yet available from the other Y1 knockout models (Howell et al., 2003; Naveilhan et al., 2001c). The discrepancies found between the icv injection studies in wild-type mice and rats and the phenotype of the Y1 receptor knockout mice is most likely due to redundancies in the system leading to compensation during development. However, the significantly reduced food intake seen in Y1 knockout mice after icv injection of NPY and analogues clearly show that NPY stimulated food intake is mediated through this receptor (Kanatani et al., 2000). In addition, Y1-receptor deficient mice also carrying a nonfunctional leptin gene (Y1
=
, ob/ob) show a significantly reduced body weight in both males and female double knockout mice compared to ob/ob mice (Pralong et al., 2002). The main reason for the reduced bodyweight seen in these mice is a reduction in hyperphagia. Y1
=
, ob/ob double knockout mice generated by our laboratory also display a significant reduced obese phenotype including significantly reduced fat mass and improved metabolic parameters such as insulin and glucose. Y1 receptor deficient animals have been reported to have increased pituitary levels of Inteinizing hormone and increased seminal vesicle size after 48 h of starvation (Pedrazzini et al., 1998). Daily injections of leptin into juvenile Y1
=
female mice causes an advancement in puberty compared to wild-type mice, which is accompanied by an increase in uterus weight. An improved function of the gonadotropic axis is also seen in Y1
=
, ob/ob double knockout mutant mice (Pralong et al., 2002). Both, pituitary luteinizing hormone levels and seminal vesicle weight was increased two-fold in the double knockout animals compared to ob/ob mice. The vasoconstrictor action of NPY is completely abolished in Y1 knockout mice (Pedrazzini et al., 1998), as is the strong potentiation of noradrenaline action on blood pressure. However, basal blood pressure and heart rate are unaffected in these mice. Reduced anti-nociception and plasma extravasation has been shown in mice deficient in the Y1 receptor (Naveilhan et al., 2001c). Exposure to acute thermal, cutaneous and visceral chemical pain causes hyperalgesia and mechanical hypersensitivity in Y1 knockout

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mice. The release of substance P, a known mediator of pain related functions, is also compromised in these Y1 receptor knockout mice. Another study has found that the major vasoactive activity of NPY in the cutaneous microvasculature acts to decrease blood flow via Y1 receptors (Chu et al., 2003). In addition, this study does not confirm a potent proinflammatory activity of NPY in the cutaneous microvasculature. However, the Y1 receptor has been implicated in mediating NPY’s effects on sedation (Naveilhan et al., 2001a). Interestingly, the Y1 receptor is also responsible for mediating voluntary alcohol consumption, as mice that lack the Y1 receptor show increased consumption of ethanol when compared with wild-type mice (Thiele et al., 2002). Male Y1
=
mice were found to be less sensitive to the sedative effects of alcohol with a more rapid recovery from ethanol-induced sleep compared to wild-type mice. Very recently, a new role for NPY in neuro-regeneration has been discovered (Hansel et al., 2001). That this is a Y1 receptor mediated effect has been shown in vitro and in vivo as a significant reduction in cell proliferation and number of newly generated neurons are found in Y1 knockout brain preparations compared to wild-type controls (Howell et al., 2003). 5.6. Y2 knockout mice Y2 receptors are widely expressed in the CNS with particular high level found in an area of the arcuate nucleus with a permeable blood brain barrier. This makes the Y2 receptor accessible to circulating factors and an ideal candidate for mediating peripheral signals on the regulation of energy homeostasis (King et al., 2000; Baskin et al., 1999; Broberger et al., 1997). This has recently been confirmed with the discovery of the endogenous Y2 agonist PYY(3-36) that is released from the gastrointestinal tract postprandially (Batterham et al., 2002). When injected peripherally at a physiological dose, PYY(3-36) inhibits food intake and reduces weight gain in wildtype mice but fails to do so in Y2 knockout mice (Batterham et al., 2002). The germline Y2 knockout model described by Sainsbury et al. (2002a) shows reduced bodyweight gain and adiposity in male mice with food intake unaltered in male and increased in female knockout mice. However, the refeeding response after starvation is strongly elevated in both genders. Naveilhan et al. (1999) describes a Y2 receptor knockout phenotype of increased body weight, food intake and fat deposition accompanied with an attenuated response to leptin in female mice. However, NPY induced food intake and the re-feeding responses after 24-h starvation were normal. The different backgrounds of the two mouse strains as well as the different targeting strategy of the Y2 gene may explain some of the differences.

In a more specific approach to investigate the functional role of the Y2 receptor in energy homeostasis, conditional Y2 receptor knockout mice were generated. The deletion of the Y2 receptor in the hypothalamus of these adult mice showed a significant decrease in bodyweight and a significant increase in food intake (Sainsbury et al., 2002a). However, over a period of 4 weeks after Y2 deletion, the effect on body weight and food intake subsides. The transience in the observed effects on food intake and bodyweight in the hypothalamus-specific Y2 knockout mice compared to germline Y2 knockout mice, underlines the importance of conditional models of gene deletion, as developmental, secondary, or extra-hypothalamic mechanisms may mask such effects in germline knockouts. Crossing the Y2
=
mice onto the ob/ob background attenuates the increased adiposity, hyperinsulinemia, hyperglycemia, and increased hypothalamo–pituitary– adrenal (HPA) axis activity of ob/ob mice without affecting food intake and bodyweight gain (Sainsbury et al., 2002b). Interestingly, the reduction in white adipose tissue mass in Y2
=
, ob/ob double knockout mice is compensated for by increased lean mass. Two studies describe anxiety-related behavior changes in Y2
=
mice (Redrobe et al., 2003; Tschenett et al., 2003). Behavioral tests such as the elevated plus maze and the open field test demonstrate that Y2
=
mice have reduced anxiety suggesting that the Y2 receptor has an inhibitory role on the anxiolytic-like effects of NPY. Removal of the Y2 receptor does not affect basal blood pressure, however, heart rate is increased in these mice (Naveilhan et al., 1999; Smith-White et al., 2002a). Furthermore, vagotomy in control mice causes an increase in heart rate, however, this parameter remains unchanged in Y2
=
mice, confirming that the pre-synaptic located Y2 receptors are responsible for the NPY mediated attenuation of parasympathetic activity to the heart. NPY acts as a potent angiogenic factor in vivo, but fails to induce angiogenesis in Y2 receptor knockout mice (Ekstrand et al., 2003). Furthermore, NPY-induced aortic sprouting and in vivo Matrigel capillary formation were also decreased by 50% in Y2 knockout mice. Similarly, spontaneous and NPY-induced re-vascularization of ischemic gastrocnemius muscles were reduced in Y2R-null mice (Lee et al., 2003). Y2-receptors, together with Y1 and Y4 receptors are located on intrinsic neurons and colon epithelia. Deletion of the Y2 receptor showed that a significant amount of electrogenic ion transport in isolated mouse colonic mucosa was abolished (Chu et al., 2003). Another very interesting and unexpected phenotype discovered in the Y2 receptor deficient mice is a two-fold increase in trabecular bone volume compared with control mice (Baldock et al., 2002). Moreover, this study demonstrates that central Y2 receptors are crucial for

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this process, since selective deletion of hypothalamic Y2 receptors in mature conditional Y2 knockout mice results in an identical increase in bone mass within 5 weeks of Y2 receptor deletion.

5.7. Y4 receptor knockout mice Germline deletion of the Y4 receptor generates mice which show reduced food intake and significantly reduced body weight (Sainsbury et al., 2002c). Plasma levels for PP, the proposed endogenous high affinity ligand for this Y receptor, was strongly elevated and white adipose tissue mass was reduced in male knockout mice. The increases in plasma PP levels in these animals, which reached similar values as seen in the lean PP transgenic animals (Ueno et al., 1999), is the most likely explanation for the reduced food intake and body weight seen in this mice. Interestingly, ablation of the Y4 receptor in ob/ob mice does not improve the obese and diabetic phenotype of these mice. However, it restores fertility to 100% in male mice and improves fertility in female double knockout mice by 50% (Sainsbury et al., 2002c). The significant improvement in fertility in Y4
=
; ob/ob double knockout mice confirms a major role of the Y4 receptor in reproduction. Y4
=
mice have elevated GnRH expression in forebrain neurons accompanied with increased serum testosterone levels in male Y4
=
mice (Sainsbury et al., 2002c). In addition, female Y4
=
mice show a strong advancement in mammary gland development with significant increases in ductile branching in both virgin and in pregnant animals (Sainsbury et al., 2002c). Double knockout mutant mice missing the Y2 and the Y4 receptor show a strong elevation in feeding behavior (Sainsbury et al., 2003). However, Y2
=
, Y4
=
double knockout mice retained a lean phenotype, with reduced body weight, white adipose tissue mass, leptinemia and insulinemia suggesting that increases in thermogenesis and locomotor activity might compensate for the elevated energy intake. Furthermore, bone volume was also increased three-fold in these Y2
=
, Y4
=
double knockout mice, associated with enhanced osteoblastic activity. This suggests a synergistic interaction between Y2 and Y4 pathways to regulate bone volume and adiposity. Interestingly, mice missing the Y4 receptor display significantly lower basal blood pressure compared to control mice, have a smaller heart and a significantly reduced heart weight (Smith-White et al., 2002b). Injection of NPY evokes an increase in blood pressure in control mice but this response is attenuated in Y4 receptor knockout mice. Vagotomy increased heart rate in both Y4
=
and control mice, however, this increase is significantly lower in the Y4
=
mice (Smith-White et al., 2002b).

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5.8. Y5 receptor knockout ICV injections of NPY analogs that prefer the Y5 receptor in mice and rats suggested a major role for this Y-receptor in the regulation of food intake. However, germline deletion of this receptor does not confirm this observation (Marsh et al., 1998). Mice lacking the Y5 receptor feed and grow normally, however, develop late onset obesity (>30 weeks). This is accompanied with increases in food intake and body weight. Y5 knockout mice do not show any changes in fasting-induced refeeding, although responses to icv NPY or NPY analogs are either reduced or absent. Similar results have been obtained in Y5 knockout mice generated by Kanatani et al. (2000). No beneficial consequences to food intake or body weight are observed in the Y5
=
, ob/ob double knockout animals (Marsh et al., 1998). In both genders food intake, bodyweight and adiposity was not different from ob/ob mice. Consistent with the lack of any improvement in body weight gain and obesity is the lack of effects on the reproductive axis in the Y5
=
and the Y5
=
/ob/ob double knockout animals, suggesting that this Y receptor does not play a major role in fertility. Y5
=
mice have been reported to be more sensitive to kainic acid-induced seizures but do not exhibit spontaneous seizure-like activity (Marsh et al., 1999). In a different study it was shown that NPY-ergic inhibition of excitatory CA3 synaptic transmission is absent (Guo et al., 2002). However, the effect on seizure susceptibility in Y5
=
is depend on the genetic background as mice on a 129/SvJ background show much greater responses to kainic acid-induced seizures compared to mice on a mixed 129/SvJ x C57BL/6 background. The background of the mice also appears to be important on the effects of alcohol consumption. Y5 receptor knockout mice on an inbred 129/SvEv background show normal ethanol-induced locomotor activity and normal voluntary ethanol consumption, however, injection of ethanol increases sleep duration (Thiele et al., 1998).

6. Conclusions Transgenic and knockout models are powerful tools for studying the functions of genes. This is especially the case in systems like the NPY family, which consists of 5 different receptor and 3 ligand genes being involved in the regulation of numerous physiological processes. Knockout models are also an invaluable source for testing the specificity of agonists and antagonist or to determine the specificity of antibodies against the different receptors. Important new insights have been revealed with the help of these NPY and Y-receptor knockout models in

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many areas including food intake, energy homeostasis, reproduction, anxiety, epilepsy and cardiovascular regulation. In addition, new functions of the NPY system have been identified such as the central regulation of bone formation and in neuronal regeneration. It has also became clear that Y4 receptors are not only responsive to PP but also other NPY family members. However, there are also unexpected differences between the findings in the transgenic models and previous pharmacological studies. One example being the relatively ‘normal’ feeding phenotype of the NPY
=
mouse, considering the massive increase in food intake and bodyweight gain observed after acute and chronic NPY administration. There are obviously limitations for both approaches, with the lack of highly selective agonists and antagonists being one example. The redundancy in the system could lead to compensatory changes in knockout animals during development. One other factor consistently overlooked is that icv injection of NPY activates all Y receptors at various different sites in the brain at the same time. Not all of these receptors are involved in the regulation of energy homeostasis. Stimulation of all Y receptors in many different locations at once might not represent a ‘normal’ physiological response and might not show the true functional consequence of the activation of a particular Y-receptor pathway. Germline deletion causes the loss of function of a particular gene in all tissues and the observed phenotype is compiled by the sum of all lost function. Loss of function in one tissue can also produce secondary effects not directly linked to the actual function of the deleted gene. Therefore, it will be necessary to employ more sophisticated strategies in the future. Particularly, the use of knock-in strategies, which allows the modification rather then the complete inactivation of receptor or ligand functions should be considered. Furthermore, the generation of conditional knockout models for all Y receptors and ligands will avoid the problems of developmental influences on the phenotypes. This model will also allow the selectively deletion of genes in a defined tissue or nucleus of an adult animal, thereby avoiding complication due to whole body ablation. There are still transgenic and knockout models for PYY and PP as well as the Y6 receptor missing. Although the Y6 receptor is considered to be a pseudogene in primates, it is functional in the mouse system and therefore remains an important target for analysis. As expected, phenotypes described for specific Y receptor knockout mice generated by different investigators are not always consistent. Many reasons could explain these discrepancies with the variations in targeting strategies and mouse strain background being the more plausible ones. However, holding and test conditions (e.g. light cycles, single or group housing, number of mice per cage, food composition, time of day when tests are performed, way of sacrificing animals, etc.) can

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