NEUROPEPTIDE Y6 RECEPTORS ARE CRITICAL REGULATORS OF ENERGY METABOLISM
Ernie Yulyaningsih
A thesis in fulfilment of the requirements for the degree of Doctor of Philosophy
St Vincent’s Clinical School Faculty of Medicine
February 2014 Table of Contents
GENERAL INTRODUCTION ...... 2
Obesity ...... 3
Regulators of energy homeostasis...... 3
Neuropeptide Y ...... 4
The Y1 receptor ...... 6
The Y2 receptor ...... 9
The Y4 receptor ...... 16
The Y5 receptor ...... 21
The Y6 receptor ...... 25
Hypothesis and overall aims of the study ...... 28
EXPERIMENTAL PROCEDURES ...... 29
Generation and genotyping of the Y6-/--LacZ mouse ...... 30
Animal experiments ...... 31
RNA extraction ...... 31
Reverse transcription PCR ...... 32
Analysis of body weight and composition ...... 33
Spontaneous feeding studies ...... 34
Fasting and re-feeding studies ...... 35
Indirect calorimetry ...... 35
Glucose tolerance tests ...... 36
Tissue collection ...... 36
Oil Red O staining ...... 37
Serum assays ...... 38 i ß-galactosidase staining ...... 38
Colocalization studies with VIP and AVP ...... 40
Analysis of diurnal variations in serum hormone levels ...... 42
PP, PYY and PYY3-36 injection study ...... 43
Statistical analyses ...... 44
RESULTS ...... 46
Generation and validation of the Y6 knockout mouse model ...... 47
Y6-/- mice are viable and have normal fertility ...... 49
Y6-/- mice exhibit reduced body weight and lean mass and a late onset
increase in adiposity ...... 51
Y6-/- mice displayed increased liver weight and increased hepatic lipid
deposition ...... 55
Reduced body weight and preferential fat accumulation in Y6-/- mice is
independent of feeding behavior ...... 57
Reduced body weight in Y6-/- mice could be explained by metabolic
alterations ...... 60
Y6 receptors regulate glucose homeostasis in mice ...... 68
Y6-/- mice show exacerbated diet-induced obesity ...... 72
Y6-/- mice showed normal serum testosterone levels ...... 80
Y6 receptors are co-expressed in VIP neurons in the hypothalamic
suprachiasmatic nucleus ...... 81
Y6-/- mice have reduced serum IGF-1 and blunted corticosterone rhythm
...... 84
DISCUSSION ...... 89
FUTURE DIRECTIONS ...... 102 ii REFERENCES ...... 105
iii LIST OF FIGURES
FIGURE 1. Generation and validation of the Y6 knockout mouse model..…48
FIGURE 2. Y6-/- mice on a normal chow diet exhibit reduced body weight and
lean mass and a late onset increase in adiposity………………...53
FIGURE 3. Y6-/- mice displayed increased liver weight and increased lipid
deposition in the liver…………………………………………...56
FIGURE 4. Reduced body weight and preferential fat accumulation in Y6-/-
mice on a normal chow diet is independent of feeding
behavior…………………………………………………………58
FIGURE 5. Reduced body weight and reduced adiposity in 14-week-old Y6-/-
mice on a normal chow diet could be explained by metabolic
alterations……………………………………………………….62
FIGURE 6. Preferential fat accumulation in 23-week-old Y6-/- mice on a
normal chow diet is independent of feeding behavior but could be
explained by metabolic alterations……………………………...66
FIGURE 7. Impact of Y6 receptor deletion on glucose homeostasis in mice on
a normal chow diet……………………………………………...70
FIGURE 8. Mice lacking Y6 receptors showed exacerbated diet-induced
obesity………………………………………………………...... 75
FIGURE 9. Effects of Y6 receptor deletion on food intake in mice maintained
on a high-fat diet………………………………………………...77
FIGURE 10. Effects of Y6 receptor deletion on energy metabolism and
physical activity in mice maintained on a high-fat diet…………78
FIGURE 11. Glucose metabolism was significantly impaired in Y6-/- mice fed a
high-fat diet……………………………………………………..79
iv FIGURE 12. Deletion of Y6 receptors did not alter serum testosterone
levels…………………………………………………………….80
FIGURE 13. Y6 receptors are expressed in VIP-expressing neurons in the
hypothalamic suprachiasmatic nucleus…………………………82
FIGURE 14. Loss of Y6 receptor signalling is associated with reduced serum
IGF-1 levels and blunted rhythms in serum corticosterone and
light-phase feeding……………………………………………...85
FIGURE 15. Y6 receptors are activated by pancreatic polypeptide in vivo…..87
FIGURE 16. Schematic representation Y6 receptor signalling in the regulation
of energy homeostasis…………………………………………100
v LIST OF TABLES
TABLE 1. Genotype frequency, litter size, mortality and percentage of male
offspring born from heterozygous (Y6+/-), homozygous (Y6-/-) and
WT (Y6+/+) breeding pairs………………………………………50
TABLE 2. Resting metabolic rate in male Y6-/- and wildtype (WT) mice on a
normal chow diet………………………………………………..64
vi LIST OF ABBREVIATIONS
Abbreviation Description
A α-MSH alpha melanocyte stimulating hormone AgRP Agouti related peptide ANOVA analysis of variance AP area postrema ARC arcuate (ARC) AVP vasopression-associated neurophysin
B BAT brown adipose tissue BDNF brain-derived neurotrophic factor BSA bovine serum albumin
C cAMP cyclic AMP CART cocaine- and amphetamine-regulated transcript CRH corticotropin-releasing hormone CV calorific value
D DEPC diethylpyrocarbonate DMH dorsomedial hypothalamic nucleus DXA dual energy X-ray absorptiometry DVC dorsal vagal complex
E e epididymal (white adipose tissues) ELISA enzyme-linked immunosorbent assay ES embryonic stem
F FLS-ob/ob Shionogy ob/ob mice with fatty liver
G G418 geneticin GABA γ-amino butyric acid GAPDH glyceraldehyde 3-phosphate dehydrogenase GHRH growth hormone releasing hormone Gi/Go inhibitory G-proteins GRH gonadotropin-releasing hormone
H hPP human PP HPA hypothalamic-pituitary-adrenal axis
vii I i inguinal (white adipose tissues) i.c.v. intracerebroventricular IGF-1 insulin-like growth factor 1 i.p. intraperitoneal i.v. intravenous
K kcal kilocalories
L LHA lateral hypothalamic area
M m mesenteric (white adipose tissues)
N neoR neomycin resistance NPY Neuropeptide Y NTS nucleus tractus solitarus
P PBS phosphate-buffered saline PFA paraformaldehyde pNPY2-36 porcine NPY2-36 POMC pro-opiomelanocortin PP pancreatic polypeptide PVN paraventricular nuclei PYY peptide YY
R r retroperitoneal (white adipose tissues) RER respiratory exchange ratio RIA radioimmunoassay rPP rat PP
S SCN suprachiasmatic
V VCO2 volume of carbon dioxide production VIP vasoactive intestinal peptide VMH ventromedial hypothalamus VO2 volume of oxygen consumption VPAC2 vasoactive intestinal peptide receptor 2
W WT wildtype
viii Y Y6-/- Homozygous deletion of Y6 receptor Y6+/- Heterozygous deletion of Y6 receptors
ix
The contents of this thesis are a modified version of 2 manuscripts:
British Journal of Pharmacology (163: 1170–1202, 2011)
NPY RECEPTORS AS POTENTIAL TARGETS FOR ANTI-OBESITY DRUG
DEVELOPMENT
Ernie Yulyaningsih, Lei Zhang, Herbert Herzog and Amanda Sainsbury
and
Cell Metabolism (19: 58-72, 2014)
PANCREATIC POLYPEPTIDE CONTROLS ENERGY HOMEOSTASIS VIA
NPY6R SIGNALLING IN THE SUPRACHIASMATIC NUCLEUS IN MICE
Ernie Yulyaningsih, Kim Loh, Shu Lin, Jackie Lau, Lei Zhang, Yanchuan Shi,
Britt Berning, Ronaldo Enriquez, Frank Driessler, Laurence Macia, Ee Khor,
Yue Qi, Paul Baldock, Amanda Sainsbury and Herbert Herzog
1
GENERAL INTRODUCTION
2 Obesity
The prevalence of obesity has increased dramatically in the Australia and around the world (ABS, 2007/2008; Flegal et al., 2012; Xi et al., 2012). At present, 61% of the Australian population is overweight or obese, putting them at increased risk of type 2 diabetes, cardiovascular diseases, hypertension, stroke and other chronic yet preventable diseases (ABS, 2007/2008). Shedding 5-10% of their excess weight could improve the health of many overweight and obese people.
However, lifestyle intervention often proves challenging despite the effort and perseverance of such individuals, and this is partly due to compensatory physiological factors that are activated during weight loss and which collectively inhibit further weight and fat loss and promote regain at the expense of lean tissues such as muscle and bone (Sainsbury and Zhang, 2010, 2011). Thus, understanding the mechanisms controlling body weight regulation is paramount to fighting the obesity epidemic, as it will ultimately lead to the development of new pharmacological agents to inhibit the compensatory responses to weight loss.
Regulators of energy homeostasis
Several neurotransmitters and hormones, acting in or on specific neuronal pathways, have been identified as being pivotal to the regulation of energy homeostasis. These signals are integrated in the hypothalamus of the brain and act in concert to regulate appetite and energy balance (Gautron and Elmquist,
2011; Herzog, 2003; Sainsbury et al., 2002a; Suzuki et al., 2010). The hypothalamus expresses a number of orexigenic and anorexigenic neuropeptides that stimulate and inhibit food intake, respectively, including neuropeptide Y
3 (NPY), agouti related peptide (AgRP), orexin, alpha melanocyte stimulating hormone (α-MSH), derived from the pro-opiomelanocortin (POMC) gene, melanin-concentrating hormones and cocaine- and amphetamine-regulated transcript (CART) (Schwartz et al., 2000). Among them, NPY, a 36 amino acid neurotransmitter, is considered to be the most potent orexigenic stimulant (Clark et al., 1984; Stanley and Leibowitz, 1985).
Neuropeptide Y
NPY is part of a family of structurally related peptides that also includes the hormones peptide YY (PYY) and pancreatic polypeptide (PP) (Larhammar,
1996; Tatemoto et al., 1982). Whereas PYY and PP are mainly expressed in gastrointenstinal tissues (Ekblad and Sundler, 2002), NPY is most abundantly expressed in the brain (Allen et al., 1983; Chronwall et al., 1985). In the human brain, NPY expression is highly concentrated in the basal ganglia and in the limbic system, particularly in the nucleus accumbens, as well as in the hypothalamus, a key brain area regulating energy homeostasis and appetite
(Adrian et al., 1983). The expression pattern of NPY in the human brain closely resembles its central distribution in rats and mice (Adrian et al., 1983; Gehlert et al., 1987; Morris, 1989), in which most of the investigations into NPY functions have been carried out.
Chronic central administration of NPY to rats and mice induces hyperphagia, decreases energy expenditure, activates lipogenic enzymes in the liver and adipose tissues, collectively contributing to the development of obesity (Clark et al., 1984; Lin et al., 2006; Raposinho et al., 2001; Stanley et al., 1986; Stanley
4 and Leibowitz, 1985; Tiesjema et al., 2007; Zarjevski et al., 1993). On the other hand, neutralization of endogenous NPY with NPY antibody induces a dose- dependent inhibition of feeding in rats (Dube et al., 1994), demonstrating an important role for NPY in the mediation of normal feeding.
NPY, PYY and PP signal through a family of G-protein coupled receptors known as Y receptors, of which there are 5 (Blomqvist and Herzog, 1997). These Y receptors couple to pertusis toxin-sensitive inhibitory G-proteins (Gi/Go), activation of which brings about inhibitory responses such as inhibition of adenylate cyclase and subsequent inhibition of intracellular cyclic AMP (cAMP) accumulation (Herzog et al., 1992; Motulsky and Michel, 1988; Zhu et al.,
1992). Additionally, activation of Y receptors has been shown to inhibit K+ and to stimulate Ca2+ channels (Aakerlund et al., 1990; Bleakman et al., 1991; Sun et al., 2001; Xiong and Cheung, 1994), thereby imparting significant effects on cellular polarization and electrical signalling.
Members of the Y receptor family– Y1, Y2, Y4, Y5 and Y6 – show distinct tissue distribution profiles and binding affinities to NPY, PP and PYY. In central and peripheral sites where they are expressed, Y receptors mediate the diverse and overlapping physiological effects of these peptides, including body weight regulation (Asakawa et al., 2003; Heshka et al., 2006; Kuo et al., 2007; Lin et al.,
2009; Moriya et al., 2010; Puopolo et al., 2009; Sainsbury et al., 2010;
Shimazaki et al., 2007; Smith et al., 2009; Ueno et al., 1999; Yang et al., 2008;
Yukioka et al., 2006; Zhang et al., 2010b; Zhang et al., 2010c), circadian rhythmicity (Dyzma et al., 2010; Edelsbrunner et al., 2009), hormone secretion
(Kalra et al., 1992; Kalra et al., 1990), water homeostasis (Larsen et al., 1993)
5 and blood pressure regulation (Herzog, 2003; Pedrazzini et al., 1993; Pedrazzini et al., 1998). Given that dysregulation in some of these processes are known to be associated with a variety of disorders such as obesity and anorexia (Turek et al., 2005; Winterer et al., 1985), manipulation of the Y receptors is attractive not only for research applications but also as therapeutic agents in clinical settings.
The Y1 receptor
Initial studies using various peptide fragments (Wahlestedt et al., 1986) pointed to the existence of two major forms of Y receptors, designated Y1 and Y2, the former of which was the first to be successfully cloned (Herzog et al., 1992). Y1 receptors consist of 384 amino acids (Herzog et al., 1992) and are pharmacologically distinguished from Y2 receptors by their ability to bind
[Leu31,Pro34]NPY (Fuhlendorff et al., 1990; Wahlestedt et al., 1990). Indeed, ligand affinity analysis of a successfully cloned orphan G-protein-coupled receptor (Eva et al., 1990) led to the identification of Y1, which binds the NPY family of ligands with the following rank order of potency: NPY = PYY >
[Leu31,Pro34]NPY >> PP > PYY13-36 (Herzog et al., 1992; Larhammar et al.,
1992).
In line with the previously reported distribution of NPY and predicted NPY binding sites (Adrian, 1978), Y1 receptor expression, detected using in situ hybridization and immunohistochemistry, was found in several thalamic nuclei, in the hippocampus, various amygdaloid nuclei and in the hypothalamus of the rat and mouse (Eva et al., 1990; Kishi et al., 2005; Kopp et al., 2002). Evaluation of peripheral tissues revealed Y1 receptor mRNA in the colon (Goumain et al.,
6 1998), pancreatic cells (Morgan et al., 1998) and in the visceral adipose tissues of rats (Yang et al., 2008). In humans, the Y1 receptor is expressed in the epithelium and mucosal nerves of the colon, in the kidney, adrenal gland, heart, and placenta (Wharton et al., 1993). Centrally, moderate levels of Y1 receptor mRNA were detected in the caudate nucleus, putamen, nucleus accumbens, amygdaloid nuclei and arcuate (ARC) and paraventricular nuclei (PVN) of the hypothalamus of human brain (Jacques et al., 1996).
Suggestive of a role of Y1 receptors in the pathophysiology of human obesity, a polymorphism in the untranslated region of the Y1 gene was associated with lower fasting triglyceride and significantly higher plasma high-density lipoprotein concentrations in 306 obese subjects (Blumenthal et al., 2002).
Moreover, pharmacological blockade of the Y1 receptor in rodents by central administration of the Y1 antagonists BIBP3226 (Kask et al., 1998), LY357897
(Hipskind et al., 1997) or 1229U91 (Kanatani et al., 1996) resulted in significant attenuation of feeding, as did the administration of antisense oligodeoxynucleotides against Y1 receptors to the ventromedial hypothalamus
(VMH) of rats (Lopez-Valpuesta et al., 1996). The Y1 receptor was also demonstrated to mediate the stimulatory effect of NPY on the proliferation of primary cultures of rat pre-adipocytes and 3T3-L1 pre-adipocytes in vitro (Yang et al., 2008). Furthermore, chronic activation of Y1 receptors by central administration of NPY or a Y1-selective agonist to mice for 6 days led to significant increases in body weight and fat accumulation with a concomitant reduction in fat oxidation in the absence of hyperphagia (Henry et al., 2005).
Taken together, these data support a role of central and peripheral Y1 receptors in the development of obesity.
7
In keeping with a potential role in the aetiology or treatment of obesity, germline ablation of Y1 receptors in genetically obese leptin-deficient ob/ob mice led to a significant improvement in the obesity syndrome, as characterized by reductions in hyperphagia and body weight gain, demonstrating a role of Y1 in mediating the action of leptin deficiency and the associated elevation in hypothalamic
NPY-ergic transmission (Pralong et al., 2002). Paradoxically, while deletion of the Y1 receptor in male mice led to a reduction in body weight that persists for up to 20 weeks of age (Pralong et al., 2002), we and others have demonstrated that the absence of Y1 receptors in mice led to the development of mild late- onset increases in adiposity without hyperphagia, and that this effect of Y1 receptor loss is more pronounced in female mice (Baldock et al., 2007; Kushi et al., 1998; Zhang et al., 2010b), suggesting sexual dimorphism in Y1 receptor function (Zammaretti et al., 2007). Interestingly, loss of Y1 receptors in mice led to hyperinsulinemia and an altered ability to secrete insulin in response to glucose, and these effects may contribute to the obesity of Y1 deficient animals
(Kushi et al., 1998). Whereas specific adult-onset deletion of Y1 receptors in the hypothalamus did not alter any aspects of energy homeostasis (Baldock et al.,
2007), knockdown of Y1 receptors specifically in peripheral tissues led to a significant increase in lipid oxidation that was associated with protection against diet-induced obesity in the absence of changes in feeding behavior or insulin levels (Zhang et al., 2010b), demonstrating an important role of peripheral Y1 receptors in the regulation of energy balance. Collectively, these results reveal likely functional redundancy of Y receptors that may be compensated throughout development after single gene deletion, and emphasize the need for Y1-specific
8 agonists or antagonists to further delineate Y1 receptor functions in central and peripheral sites.
The Y2 receptor
The Y2 receptor is a 381 amino acid protein that is highly conserved between species, with more than 90% identity between orders of mammals and about 80% identity when comparing mammals and chicken (Berglund et al., 2003; Gerald et al., 1996). Interestingly, whereas the Y2 gene is localized in close proximity to the Y1 and Y5 gene cluster on chromosome 4q31-32 of humans, it has only approximately 30% overall amino acid identity to the Y1 receptor (Ammar et al.,
1996; Gerald et al., 1995). Pharmacologically, the Y2 receptor binds to NPY and
PYY with equally high affinity, but with low affinity for PP (Blomqvist and
Herzog, 1997; Michel et al., 1998), and is distinguished from the Y1 receptor by its high affinity to C-terminus fragment of NPY or PYY. Thus, NPY or PYY lacking 2, 13, 18 or even 22 amino acids from the N-terminus, resulting from pharmacological or endogenous protease processing (Mentlein, 1999; Unniappan et al., 2006), can bind to human, rat and mouse Y2 receptor with high to moderate affinity (Michel et al., 1998).
The Y2 receptor is mainly located presynaptically and is involved in the suppression of transmitter release (Colmers et al., 1991). In the central nervous system, Y2 receptor mRNA can be found within the hippocampus, hypothalamus and amygdala, as well as in specific nuclei of the brain stem (Parker and Herzog,
1999). Consistent with the high level of Y2 mRNA, autoradiography reveals that the Y2 receptor is the most prominent Y-receptor expressed in the central
9 nervous system, representing approximately two thirds of the total binding capacity for NPY (Lin et al., 2005). The Y2 receptors are also widely expressed in the peripheral tissues such as adipose tissues, liver and skeletal muscle
(Goumain et al., 1998; Kuo et al., 2007; Shi et al., 2011). Not surprisingly, Y2 receptors are involved in a large number of physiological functions induced by
NPY family peptides such as angiogenesis (Zukowska-Grojec et al., 1998), vasoconstriction (Malmstrom, 2001; Pheng et al., 1999), effects on gastric emptying (Chen et al., 1997), circadian rhythm (Golombek et al., 1996; Gribkoff et al., 1998; Huhman et al., 1996) as well as in modulating emotional and stress- coping behaviours (Heilig, 2004). Importantly, activation of Y2 receptors has been linked to the induction of satiety, thus generating great interest in the anti- obesity potential of Y2 receptor agonism with compounds such as PYY3-36
(Scott et al., 2005). This is in accordance with the particularly high levels of Y2 receptor expression found in the ARC of the hypothalamus and the area postrema
(AP) of the brain stem (Parker and Herzog, 1999), both areas known to have a semi-permeable blood brain barrier (Broadwell and Brightman, 1976), thus making Y2 receptors in these regions accessible to circulating PYY and PYY3-
36.
The notion of Y2 receptor agonism as a potential anti-obesity treatment is supported by results from studies demonstrating that peripheral administration of
PYY3-36 reduces food intake and/or body weight in animals and humans
(Batterham et al., 2003a; Batterham et al., 2002). Peripheral administration of
PYY3-36 in the concentration range normally seen postprandially dose- dependently inhibited feeding in 24 hour fasted and freely feeding rats prior to the onset of the dark phase (Batterham et al., 2002). These results are supported
10 by other reports showing similar effects in fasted non-obese rats and mice
(Adams et al., 2004; Challis et al., 2004; Martin et al., 2004; Pittner et al., 2004).
Moreover, the anorectic effect of intraperitonally-administered PYY3-36 was abolished in mice deficient of Y2 receptors (Batterham et al., 2002), or in wild type rodents in which PYY3-36 was co-administered with the Y2 receptor antagonist BIIE0246 (Abbott et al., 2005; Scott et al., 2005; Talsania et al.,
2005), demonstrating a Y2 receptor-dependent mechanism for PYY3-36- mediated anorexic effects.
In chronic settings, administration of PYY3-36 or Y2 receptor agonists to obese rodent models – such as diet-induced obese mice or rats (Adams et al., 2006;
Ortiz et al., 2007; Pittner et al., 2004; Vrang et al., 2006), ob/ob mice and fa/fa rats (Pittner et al., 2004) – has also been demonstrated to dose-dependently reduce body weight and/or adiposity (Adams et al., 2006; Ortiz et al., 2007;
Pittner et al., 2004; Vrang et al., 2006). These effects of chronic PYY3-36 administration were associated with a transient reduction in food intake and a significantly reduced respiratory exchange ratio (Adams et al., 2006; van den
Hoek et al., 2007), suggesting that enhanced lipid oxidation induced by PYY3-36 may contribute to its effects to reduce body weight and adiposity. Indeed, intravenous infusion of PYY3-36 in lean and obese subjects increases thermogenesis and lipolysis in association with a reduced respiratory exchange ratio, suggestive of enhanced lipid oxidation (Sloth et al., 2007a). These effects are consistent with a report that high post-prandial levels of PYY are associated with increased lipid oxidation as indicated by a reduction in respiratory quotient value (Guo et al., 2006). Thus, long-term alterations in circulating PYY levels can have long-term consequences on energy balance via effects on food intake
11 and / or metabolic processes (Adams et al., 2006; Guo et al., 2006; Sloth et al.,
2007b; van den Hoek et al., 2007).
In line with the impact of exogenous PYY3-36 administration, small increases in endogenous PYY expression in transgenic mice induces marked resistance to diet-induced obesity and significant attenuation of the metabolic syndrome of genetically obese ob/ob mice in the absence of changes in body weight or basal and fasting-induced food intake (Boey et al., 2008). Additionally, overexpression of PYY in ob/ob mice led to increased body temperature, enhanced hypothalamic expression of thyrotropin-releasing hormone mRNA and decreased brown adipose tissue depot weight, suggesting PYY-induced activation of the hypothalamo-pituitary-thyroid axis and increased thermogenic activity (Boey et al., 2008). In keeping with this, PYY3-36 injection was shown to increase serum levels of thyrotropin in fasted rats (Oliveira et al., 2006). On the other hand, absence of PYY in mice on a C57Bl/6 background led to progression towards obesity with (Batterham et al., 2006) or without hyperphagia (Boey et al., 2006;
Wortley et al., 2007). Interestingly, increases in basal and glucose-induced serum insulin levels were observed in PYY knockout mice (Boey et al., 2006), raising the possibility of a role for hyperinsulinemia in the development of increased adiposity associated with PYY deletion. Together, these findings imply that PYY may have long-term benefits to reduce excess adiposity and ameliorate metabolic abnormalities associated with obesity through mechanisms independent of effects on food intake, notably stimulation of thyroid function.
The physiological effects of Y2 receptor antagonism on energy homeostasis were further evaluated using Y2-deficient mice. The first study of a germline Y2
12 receptor knockout mouse reported increased food intake, fat mass and body weight accompanied with leptin resistance as indicated by an attenuated response to leptin in female mice (Naveilhan et al., 1999). Another study using an independently-produced Y2 deficient mouse model showed that female germline
Y2 receptor knockout mice also had increased food intake, however, with reduced body weight, whereas male Y2 knockout mice had transiently reduced food intake and a sustained decrease in body weight associated with decreased adiposity at 16 weeks of age (Sainsbury et al., 2002b; Zhang et al., 2010c). The discrepancies between these germline knockout models may arise from the different background of the two mouse strains as well as the different strategies used to target the Y2 gene, hence affecting the completeness of Y2 deletion
(Herzog, 2003). Newer evidence from conditional Y2 receptor knockout models suggests that interpretation of changes in body weight and body composition observed in different Y2 receptor knockout models needs to consider the possibility of differential effects of central versus peripheral, and/or hypothalamic versus non-hypothalamic, effects of this Y receptor. Thus, adult- onset hypothalamus-specific Y2 receptor deletion – induced by injection of a recombinant adeno-associated viral vector expressing Cre-recombinase into the hypothalamus of adult Y2lox/lox mice – led to significant increases in daily food intake, weight gain and fat gain (Shi et al., 2010). Furthermore, specific deletion of Y2 receptors expressed only in NPY-expressing neurons in adult mice resulted in a significant increase in NPY mRNA expression with a concomitant decrease in POMC mRNA expression in the ARC (Shi et al., 2010), providing direct evidence that Y2 receptors on NPY-ergic neurons act as an auto-receptor regulating NPY expression and directly or indirectly influencing neighbouring
POMC neurons in the ARC. Importantly, female mice with conditional Y2
13 receptor deletion in hypothalamic NPY-ergic neurons showed increased adipose tissue mass, hepatic steatosis and a greater capacity for fatty acid synthesis in muscle (Shi et al., 2010), demonstrating the obesogenic effect of selective blockade of Y2 receptor signalling in NPY neurons. Collectively, findings from conditional Y2 receptor knockout models are consistent with the notion that
PYY3-36 and other Y2 receptor agonists can act as a satiety and anti-obesogenic factor by interacting with hypothalamic Y2 receptors, and that lack of hypothalamic Y2 signalling results in increased food intake, fat gain and weight gain. These findings also suggest that lack of Y2 receptor signalling in non- hypothalamic tissues (for instance, in adipose tissue) could contribute to weight loss, as was observed in one (Sainsbury et al., 2003; Sainsbury et al., 2002c;
Zhang et al., 2010c) but not all (Naveilhan et al., 1999) germline Y2 receptor knockout models.
Absence of Y2 receptors in ob/ob mice attenuated the increased adiposity, hyperinsulinemia and hyperglycemia typical of ob/ob mice without affecting food intake or body weight gain (Naveilhan et al., 2002; Sainsbury et al., 2002c).
Y2 receptor deletion has also been shown to confer protection against obesity and associated metabolic conditions induced by high fat feeding (Sainsbury et al., 2006) and chronic corticosterone administration (Sainsbury et al., 2002b).
Furthermore, increased body weight and adiposity in female mice due to ovariectomy, a model mimicking menopause in women, was normalized by global Y2 receptor ablation but not by hypothalamic-specific Y2 receptor deletion (Allison et al., 2006). These studies suggest that the anti-obesity effects of peripheral Y2 receptor deletion may outweigh any possible obesogenic effects produced by blocking the anorectic and weight-reducing effects of Y2 agonists in
14 the hypothalamus, and thus that Y2 receptor antagonism may overall be more beneficial than Y2 agonism for treating obesity.
It is important to note that studies with Y2 receptor knockout models have also revealed a significant role for Y2 receptors in the regulation of bone metabolism.
Germline Y2 receptor knockout mice have a two-fold increase in trabecular bone volume as well as greater trabecular number and thickness compared with control mice, an effect due to increased osteoblast activity and an increased rate of bone mineralization and formation (Baldock et al., 2002). The increased bone mass in germline Y2 receptor knockout mice coincides with the reduced fat mass in these animals, suggesting potential energy partitioning changes between fat and lean tissues caused by Y2 receptor deletion. In support of this hypothesis, Y2 receptor deletion abolishes the fasting-induced reduction in activity of the hypothalamo-pituitary-somatotropic axis (Lin et al., 2007) and restores the low serum levels of insulin-like growth factor-1 in ob/ob mice (Sainsbury et al.,
2006), suggesting a role of Y2 signalling in regulating activity of the somatotropic axis, activation of which is known to promote the accretion of lean mass at the expense of fat mass (Ho et al., 1996). This regulation of the somatotropic axis by Y2 receptors is likely to occur in the hypothalamus, since
Y2 receptors have been shown to colocalize with growth hormone releasing hormone (GHRH) neurons in the ARC and VMH, and hypothalamus-specific Y2 receptor deletion prevented fasting-induced inhibition of hypothalamic GHRH expression (Lin et al., 2007). Interestingly, hypothalamus-specific Y2 receptor deletion recapitulated the high bone mass phenotype observed in germline Y2 knockout mice (Baldock et al., 2002), demonstrating the key role of hypothalamic Y2 receptors in regulating bone metabolism. Importantly, the
15 potential of hypothalamic Y2 receptors as a target for novel anabolic treatments for osteoporosis was demonstrated in a study showing that hypothalamic Y2 receptor deletion in gonadectomised sex-hormone deficient adult male and female mice prevented ongoing bone loss, an effect attributable to activation of an anabolic osteoblastic bone formation that counterbalances the persistent elevation of bone resorption seen in gonadectomised wild type animals (Allison et al., 2006). These studies suggest that whereas Y2 receptor agonists such as
PYY3-36 that can access the hypothalamus may be beneficial in treating obesity, they are likely to have detrimental effects on bone mass in the long-term, and analyses of bone density should be incorporated into trials of Y2 agonists.
The Y4 receptor
The Y4 receptor is substantially different from other Y receptors, sharing only
30% primary sequence identity with them (Darby et al., 1997; Yan et al., 1996).
The conservation of Y4 between human and mouse (76%) is also relatively low when compared against Y1 (94%) and Y2 (94%) receptors, suggesting rapid evolutionary divergence of this receptor. Despite this, rat and mouse Y4 receptors show similar pharmacology and tissue expression profiles to their human homolog. While the Y4 receptor exhibits affinity for all three members of the NPY family, it preferentially binds to PP and shows improved binding affinity to NPY and PYY analogues that incorporate PP residues, namely
[Pro34]PYY and [Leu31,Pro34]NPY (Bard et al., 1995; Darby et al., 1997). In situ hybridization on rat brain has revealed the presence of Y4 receptor mRNA in the brain stem, specifically in the AP, in the dorsal motor nucleus of the vagus nerve, and in the nucleus tractus solitarus (NTS) (Larsen and Kristensen, 1997; Parker
16 and Herzog, 1999). Consistent with this mRNA expression, Whitcomb and colleagues described a dense population of high-affinity PP receptors in the dorsal vagal complex (DVC) of the caudal brain stem of rats (Whitcomb et al.,
1990), the primary site receiving afferent neuronal signals from peripheral organs and also containing an incomplete blood-brain barrier, permitting the entry of small peptide hormones (Pardridge, 1983). The presence of Y4 receptors has also been reported by in situ hybridization and immunocytochemistry techniques in the lateral hypothalamic area (LHA), specifically in orexin-containing neurons
(Campbell et al., 2003). Northern blot analyses showed that in humans, Y4 receptors are predominantly expressed in peripheral tissues such as the heart, gastrointestinal tract, skeletal muscle, pancreas, testis and uterus (Bard et al.,
1995; Yan et al., 1996). Significant amounts of Y4 receptor mRNA, however, are also present in the human brain, with the highest level of expression being observed in the hypothalamus (Bard et al., 1995).
Elucidation of the function of Y4 receptors has been facilitated by pharmacological agents and transgenic and knockout mice. Overexpression of
PP, the endogenous ligand for Y4 receptors, reduces body weight and adiposity in association with reduced food intake and a decreased rate of gastric emptying in mice on a lean background (Ueno et al., 1999), while peripheral administration of exogenous PP to wildtype mice produced a dose-dependent reduction in food intake accompanied by a decreased rate of gastric emptying, increased energy expenditure, increased colonic muscle contraction and increased faecal output
(Asakawa et al., 1999; Asakawa et al., 2003; Balasubramaniam et al., 2006;
Moriya et al., 2010). Similarly, in genetically obese ob/ob mice, PP administration reduced food intake and body weight gain while increasing
17 energy expenditure and reducing the rate of gastric emptying (Asakawa et al.,
2003; Katsuura et al., 2002). Moreover, repeated administration of PP improved the insulin resistance and hyperlipidemia of ob/ob mice as well as of Shionogy ob/ob mice with fatty liver (FLS-ob/ob mice), and attenuated the liver enzyme abnormalities of the latter model (Asakawa et al., 2003). These findings suggest that PP promotes negative energy balance, likely via effects on appetite and gut function.
On the other hand, mice lacking Y4 receptors do not exhibit the obese phenotypes that might have been expected given the effects of PP administration to reduce energy balance as described above. Indeed, germline Y4 receptor knockout mice on a lean background exhibit reduced food intake, reduced adiposity and / or reduced body weight (Sainsbury et al., 2002d). It is postulated that compensatory mechanisms for the global loss of Y4 receptors throughout development may contribute to the conflicting phenotype of PP transgenic and
Y4 deficient mice. Indeed, circulating PP concentrations were significantly elevated in the germline Y4 receptor knockout mice, to levels comparable with
PP-over-expressing mice (Sainsbury et al., 2002d; Ueno et al., 1999). Thus in the absence of Y4 receptors, excess PP may activate other receptors besides Y4, such as Y5, albeit with lower affinity, thereby inducing PP-like effects. Similar to the lack of obesity in Y4 receptor knockout mice on a lean background, germline Y4 receptor knockout does not reduce the hyperphagia, increased body weight nor excess adiposity of genetically obese leptin-deficient ob/ob mice, although it does rescue the infertility and depressed activity of the hypothalamo- pituitary-gonadotropic axis of these mice (Sainsbury et al., 2002d). These findings suggest that whereas PP can reduce food intake and energy balance in
18 lean and obese mice, signalling through Y4 receptors does not contribute to the hyperphagia and obesity of leptin-deficient mice, in which other mechanisms such as increased Y1 and Y5 signalling may be at play. On the other hand, although PP is not expressed in the brain, the enhanced NPY signalling known to occur in the brain of ob/ob mice – as well as in normal animals during energy deficit – (Sainsbury and Zhang, 2010) may lead to Y4-mediated down-regulation of the gonadotropic axis and prevention of pregnancy under conditions of low energy supply (Sainsbury et al., 2010).
The parasympathetic nervous system and the Y4 receptor appear to be key mediators of PP-induced physiological effects. The effects of peripheral PP administration to mice were abolished by vagotomy or deficiency of Y4 receptors (Asakawa et al., 2003; Lin et al., 2009; Moriya et al., 2010), demonstrating the critical role of the vagus nerve and Y4 receptors in mediating effects of PP. Similarly in humans, insulin-induced PP release was attenuated in vagotomised patients with duodenal ulcers or by the administration of atropine, a competitive antagonist for the muscarinic acetylcholine receptor (Schwartz,
1978). Circulating PP was demonstrated to cross the blood brain barrier
(Whitcomb et al., 1990) through a semi-permeable region in the AP (Broadwell and Brightman, 1976) and to bind to Y4 receptors in the DVC (Katsuura et al.,
2002; Whitcomb et al., 1990). It is thought that Y4 agonism in this area of the brain stem may mediate the transmission of afferent neuronal activity, leading to autonomic regulation of gastrointestinal functions. Indeed, peripheral administration of PP was shown to activate several different types of orexigenic and anorexigenic hypothalamic neurons in wildtype but not in Y4 receptor deficient mice, emphasizing the involvement of Y4 receptor signalling.
19 Importantly, these PP-activated neurons include the orexigenic brain-derived neurotrophic factor (BDNF) and orexin as well as the anorexigenic protein product of the POMC gene, -MSH, suggesting that PP may up-regulate anorexigenic and down-regulate orexigenic pathways in the central nervous system (Lin et al., 2009; Sainsbury et al., 2010). Consistent with this, hypothalamic mRNA levels of the orexigenic NPY, ghrelin and orexin in the hypothalamus were reduced while that of the anorexigenic urocortin and POMC were elevated in response to Y4 agonism with PP in mice (Asakawa et al., 2003;
Lin et al., 2009; Sainsbury et al., 2010). Notably, these changes in mRNA expression in response to PP injection were observed in wildtype, but not in Y4 receptor deficient mice, demonstrating the critical role of the Y4 receptor in these processes (Lin et al., 2009; Sainsbury et al., 2010). Thus Y4 agonism by PP may regulate food intake by suppressing orexigenic pathways and stimulating anorexigenic pathways.
In keeping with an effect of PP to induce negative energy balance, low circulating PP levels are associated with conditions of obesity in both children
(Reinehr et al., 2006; Zipf et al., 1981) and adults (Glaser et al., 1988; Lassmann et al., 1980; Marco et al., 1980), whereas fasting and post-prandial PP levels are significantly elevated in the circulation of patients with anorexia nervosa
(Alderdice et al., 1985; Kinzig et al., 2007; Takeno et al., 1990). These results indicate that PP is involved in the pathophysiology of obesity and may be a suitable anti-obesogenic agent. Indeed, short-term intravenous (i.v.) administration of PP to lean human subjects led to sustained suppression of appetite and food intake for up to 24 hours post-infusion (Batterham et al.,
2003b; Jesudason et al., 2007). Additionally, an i.v. infusion of PP that restored
20 basal and meal-stimulated PP levels in people with obesity or obesity due to
Prader-Willi syndrome attenuated the hyperphagia seen in these people, possibly through enhanced satiation (Berntson et al., 1993). More importantly from a clinical perspective, administration of PP at doses that reduces food intake does not induce nausea in humans, unlike PYY (Batterham et al., 2003b; Jesudason et al., 2007; le Roux et al., 2008). These reports, along with results from rodent studies, have highlighted the potential benefit of PP as a satiety-promoting agent and have suggested that agonism of Y4 receptors may be a potential anti-obesity strategy.
The Y5 receptor
The Y5 receptor was first isolated and cloned from rat hypothalamic cDNA on the speculation that an additional Y receptor subtype existed that was pharmacologically similar to but distinct from the Y1 receptor and was able to mediate feeding responses to NPY that are not mediated by Y1 (Gerald et al.,
1996; Hu et al., 1996; Wahlestedt and Reis, 1993).
Consistent with its hypothesized role in feeding, in situ hybridization histochemistry showed high levels of Y5 mRNA in areas of the rat brain that are implicated in the regulation of feeding, including the LHA and overlapping with
Y1 mRNA expression in the ARC, PVN and suprachiasmatic (SCN) nuclei of the hypothalamus (Gerald et al., 1996; Parker and Herzog, 1999). Y5 mRNA was also reported in the amygdala, an area of the brain that is primarily involved in memory processing and emotional functions, suggesting a possible role for this
Y receptor in mediating emotional aspects of feeding behaviour (Gerald et al.,
21 1996). In the mouse brain, like in the rat brain, Y5 receptor mRNA was also detected in the ARC, adding further weight to the functional candidature of Y5 in the mediation of feeding behaviour (Naveilhan et al., 1998). In contrast to its high transcript levels however, competitive binding assays on rat brain slices using the Y1- and Y5-preferring ligand 125I-[Leu32,Pro34]PYY in the presence of
Y1 and Y4-blockade by BIBP3226 or GW1229 demonstrated a low density of
Y5 receptor translation in hypothalamic neurons relative to other areas that exhibited higher levels of Y5-specific binding, including the ventral hippocampus, area AP and NTS (Dumont et al., 1998). Immunohistochemistry for Y5 receptor promoter-controlled Cre expression revealed strong expression in the mouse SCN, PVN and dorsomedial hypothalamic nucleus (DMH) (Oberto et al., 2007). Additionally, Y5-immunoreactive neurons were detected in abundance in the hippocampus and hypothalamus, where they overlap with neurons expressing gonadotropin-releasing hormone (GRH), neurophysins, corticotropin-releasing hormone (CRH), and γ-amino butyric acid (GABA)
(Campbell et al., 2001; Grove et al., 2000). Taken together, these findings show that Y5 mRNA is expressed in numerous regions of the mouse and rat brain, albeit protein expression and Y5 binding in hypothalamic nuclei appears to be relatively low.
Interestingly, the Y1 and Y5 receptor genes are transcribed in opposite directions from a common promoter region on human chromosome 4q31-q32 (Herzog et al., 1997; Nakamura et al., 1997). This close proximity of the two Y receptor genes suggests that they have evolved from a gene duplication event (Herzog et al., 1997). Furthermore, the transcription of both genes from opposite strands of the same DNA sequence suggests that transcriptional activation of one will have
22 an effect on expression of the other (Herzog et al., 1997). Since both Y1 and Y5 receptors are known to play an important role in the regulation of food intake and energy homeostasis (Brothers and Wahlestedt, 2010; Kalra and Kalra, 2004), the coordinate expression of their specific genes may be important in the modulation of NPY-ergic activity (Herzog et al., 1997).
Pharmacologically, human NPY, PYY and PP (hNPY, hPYY, hPP) are equally potent at activating Y5 receptors and inhibiting foskolin-stimulated cAMP synthesis in transfected cells (Gerald et al., 1996; Hu et al., 1996). Y5 receptors are also activated by various analogues of NPY and PYY, including hPYY3-36, porcine NPY2-36 (pNPY2-36) and p[Leu31, Pro34]NPY, but not by peptide fragments or analogues that are poor orexigenic agents, such as pNPY13-36
(Gerald et al., 1996; Hu et al., 1996). Furthermore, [D-Trp32]NPY was demonstrated to be a weak but selective Y5 agonist, effectively producing a 4.5- fold increase in food intake after central administration of a dose of 2 nmol per rat when compared to empty-treated control rats (Gerald et al., 1996).
Additionally, activation of Y5 receptors by intracerebroventricular (i.c.v.) administration of 3 nmol [D-Trp32]NPY in satiated Long-Evans rats led to dose- dependant depression of body temperature and a significant reduction in energy expenditure (Hwa et al., 1999), further confirming a critical role of this receptor in the regulation of energy homeostasis. In support of this, intracerebroventricular administration of phosphothioate end-protected antisense oligodeoxynucleotides against Y5 receptors led to the inhibition of spontaneous, fasting-induced, and NPY-induced feeding in rats (Schaffhauser et al., 1997;
Tang-Christensen et al., 1998). Interestingly, although showing a blunted response to centrally administered hNPY- and hPYY3-36-induced feeding
23 (Marsh et al., 1998), Y5-/- mice develop late-onset obesity and significant hyperphagia in association with down regulation of the anorexigenic POMC gene and up-regulation of NPY expression in the hypothalamus under fasting conditions (Higuchi et al., 2008; Kanatani et al., 2000; Marsh et al., 1998), suggesting that potential compensatory effects of germline Y5 deletion may mask the true role of Y5 receptors in the regulation of energy homeostasis. For instance, it is possible that enhanced activation of Y1 receptors in Y5-/- mice can compensate for the lack of Y5 receptor signalling. Indeed, co-administration of
NPY and 1229U91, a potent Y1 and Y4 receptor antagonist and agonist, respectively, to Y5-/- mice completely abolished NPY-induced hyperphagia, leading to the conclusion that the central effects of NPY are mediated by both Y1 and Y5 receptors (Marsh et al., 1998). Furthermore, the modest or lack of improvement in the obesity syndrome of genetically obese ob/ob mice by separate inactivation of Y1 or Y5 receptors, respectively (Marsh et al., 1998;
Pralong et al., 2002) supports the notion that interaction between Y1 and Y5 receptors exists in the regulation of feeding behavior and energy homeostasis
(Kanatani et al., 2000).
Collectively, these results demonstrate the role of Y5 receptors in the central mediation of feeding behavior, and imply that development of highly specific and highly potent antagonists to one or both of the Y1 or Y5 receptors could be an attractive avenue to pursue for the medical management of obesity. However, the predominantly central distribution of Y1 and Y5 receptors presents a challenge for the design of a compound able to permeate the blood brain barrier.
24 The Y6 receptor
Despite the large body of literature relating to the functions of Y1, Y2, Y4 and
Y5 receptors, a limited number of reports deal with the Y6 receptor. This paucity of information is perhaps owing to the fact that the Y6 receptor gene is truncated in primate species, including humans (Gregor et al., 1996a; Matsumoto et al.,
1996; Rose et al., 1997) and is absent from the rat (Burkhoff et al., 1998). The human Y6 receptor gene is located on chromosome 5q31(Gregor et al., 1996a;
Lutz et al., 1997) and contains a single-base deletion between the putative 6th and
7th transmembrane domains, resulting in a frame shift mutation and an early stop codon (Gregor et al., 1996a; Matsumoto et al., 1996; Rose et al., 1997). This finding suggests that the human Y6 receptor gene is a pseudogene, an inactivated product of gene duplication (Starback et al., 2000). The predicted protein encoded by the human Y6 receptor gene contains 290 amino acids and lacks the
7th transmembrane domain and the 3rd extracellular loop (Gregor et al., 1996a;
Matsumoto et al., 1996; Rose et al., 1997). Cloning of the human Y6 receptor in its mutated or corrected form, the latter containing a single base insertion to restore the open reading frame, failed to produce specific binding to 125I-bound
NPY, PP or PYY (Gregor et al., 1996a; Rose et al., 1997). This negative result suggests that the human Y6 receptor is not translated or expressed or that it does not participate in ligand binding.
Although human Y6 may not bind NPY, PP or PYY, it is noteworthy that Y6 mRNA was found in several human tissues including the heart and skeletal muscle, where the transcript was particularly abundant, as well as in the brain, gastrointestinal tissues and adrenal glands (Burkhoff et al., 1998; Gregor et al.,
1996b; Matsumoto et al., 1996). These findings raise questions as to the function
25 of Y6 in humans. Emerging studies now demonstrate active roles of pseudogenes, some of which have been shown to be expressed (Kandouz et al.,
2004; Lai et al., 2008; Muro et al., 2011; Pink et al., 2011; Poliseno et al., 2010a;
Poliseno et al., 2010b; Thiele et al., 2000; Zhang et al., 2006). Importantly, the human Y6 gene is a candidate locus that has been linked to hypertension
(Chitbangonsyn et al., 2003) due to its location near a chromosomal region previously found to be associated with differences in systolic blood pressure
(Krushkal et al., 1999; Krushkal et al., 1998). It is thus possible that Y6 mRNA or protein may mediate important functions.
In contrast to its apparent lack of expression in primates, humans and rats, the Y6 receptor gene is present in non-truncated form in mice and rabbits (Burkhoff et al., 1998; Matsumoto et al., 1996). The function of Y6 in these species is unknown. The mouse Y6 receptor is a 371-amino-acid protein that contains all 7 transmembrane domains characteristic of a G-protein coupled receptor (Burkhoff et al., 1998; Gregor et al., 1996a; Matsumoto et al., 1996; Weinberg et al., 1996).
In the mouse, Y6 receptors are expressed in developing embryo, skeletal muscle, kidney and testis (Burkhoff et al., 1998; Gregor et al., 1996b). Additionally, in situ hybridization located Y6 receptor expression in discrete regions of the mouse hypothalamus, including the VMH (Weinberg et al., 1996), a brain region critical to the regulation of energy homeostasis (Xu et al., 2010). The mouse Y6 receptor has been suggested to be an endogenous receptor to PP, based on the observation that human and rat PP (hPP and rPP, respectively) displaced specific binding of 125I-rPP from Y6 receptors that had been transiently expressed on
COS-7 cells (Gregor et al., 1996a). In these experiments moreover, PP had a higher affinity for Y6 receptors than other members of the NPY peptide family
26 or their analogues (Gregor et al., 1996a). These findings suggest that Y6 may have Y4-like qualities based on its affinity for PP, which preferentially activates
Y4 receptors (Gregor et al., 1996a). In contrast to this, a Y1-receptor-like pharmacology has been proposed for Y6 receptors based on the rank order of affinity of various NPY analogues to Y6 receptors stably expressed in Chinese hamster ovary cells in competitive binding assays against 125I-PYY (Weinberg et al., 1996). Further adding to the lack of consensus over the endogenous Y6 ligand, a separate study described a unique pharmacological profile of Y6 receptors distinct from that of other Y receptors (Mullins et al., 2000). This was achieved by assessing both the affinity and ability of various NPY analogues to inhibit forskolin-stimulated cAMP production in human embryonic kidney cells stably expressing mouse Y6 receptors (Mullins et al., 2000). The lack of consistent results arising from these in vitro studies make it difficult to ascertain the true pharmacology and potential physiological role of the Y6 receptor.
27 Hypothesis and overall aims of the study
Functional evaluation of individual Y receptors and manipulation of NPY and related peptide signalling pathways in developing therapeutic leads for obesity, diabetes and related disorders commonly rely on animal studies, a large number of which are conducted in mice. However, these studies frequently overlook the existence of a functional Y6 receptor in mice and its potential interaction with various Y-receptor agonists and antagonists. These factors are likely to present the potential for false interpretation of drug evaluation results that are not only costly, but potentially also detrimental for human health. Additionally, while the function of the human Y6 receptor, if any, is currently unknown, recent findings suggest that the human Y6 gene may have important functions as a pseudo gene.
Together, these findings assert the need for a comprehensive understanding of
NPY signalling pathways, inclusive of the Y6 receptor.
Since Y6 receptors have previously been demonstrated to be expressed in tissues that are important for energy homeostasis, namely the hypothalamic VMH and the skeletal muscle in mice, and based on its pharmacological profile in vitro, I hypothesize that the Y6 receptor plays an important role in the regulation of energy balance in the mouse. In this study I thus investigated the function of Y6 receptors in the regulation of energy homeostasis by studying the metabolic characteristics of germline knockout mice lacking Y6 receptors (Y6-/-). To gain further insights into the possible physiological functions of Y6 receptors in mice,
I used our Y6-/- mouse model in order to determine physiological ligands for mouse Y6 receptors in vivo.
28
EXPERIMENTAL PROCEDURES
29 Generation and genotyping of the Y6-/--LacZ mouse
3 breeding pairs of homozygote germline Y6-/--LacZ mice were purchased from
Deltagene, USA. Briefly, disruption and ablation of the Y6 receptor was achieved by inserting a selection cassette containing lacZ and a neomycin resistance (neoR) genes into the Y6 coding exon. This vector was inserted into an embryonic stem (ES) cell line from the 129/SvEvBrd mouse strain. Positively targeted ES cell clones were identified by resistance against geneticin (G418) and were subsequently injected into blastocysts from mice on a mixed C57BL/6–
129SvJ background.
Genotyping of Y6-/- mice was performed by PCR analysis using primers from
Sigma-Aldrich, St Louis, MO, USA. The location of primer binding sites on the
Y6 receptor gene (GenBank: U58367.1) are shown in Figure 1A. Presence of the
Y6 receptor gene was determined using the Y6 receptor gene-specific primers,
GS(E) (5’-GGCCAAACATCCACTGAGGATACAC-3’) and GS(TE) (5’-
AGATGTCAGAGAGGGACAGGTTGGC-3’). Absence of the Y6 receptor gene was confirmed using the combination of GS(TE) and a primer targeting the selection cassette, NEO(T) (5’- GGGGATCGATCCGTCCTGTAAGTCT-3’).
PCR analysis was performed using 35 cycles with 30 seconds of denaturing at
96°C and 30 seconds of elongation at 78°C. Homozygous deletion (Y6-/-) was confirmed when a PCR product was obtained with the GS(TE)/NEO(T) but not with the GS(TE)/GS(E) primer pair. A PCR product resulting from
GS(TE)/GS(E) but not from the GS(TE)/NEO(T) primer pair indicates a homozygous WT. A mouse was determined to be heterozygous for the Y6
30 deletion (Y6+/-) when a PCR product was obtained with both GS(TE)/GS(E) and
GS(TE)/NEO(T) primer pairs.
Animal experiments
All animal care and experiments were approved by the Garvan/St. Vincent's
Animal Ethics Committee and were conducted in accordance with relevant guidelines and regulations. Male mice were used for all experiments, except for data presented in Figure 13. Mice were housed under a controlled temperature of 22C and a 12-hour light cycle (lights on from 07:00 to 19:00 hours) with standardized environmental enrichment. Unless otherwise stated, all animals had ad libitum access to water and a normal chow diet (6% kilo calories (kcal)) from fat, 21% kcal from protein, 71% kcal from carbohydrate, 2.6 kcal/g, Gordon’s
Specialty Stock Feeds, Yanderra, NSW, Australia). A subset of animals was fed a high fat diet (43% kcal from fat, 17% kcal from protein, 40% kcal from carbohydrate, 4.8 kcal/g, Specialty Feeds, Glen Forrest, WA, Australia) for 16 weeks starting from 7-8 weeks of age.
RNA extraction
To analyze the tissue distribution of Y6 mRNA expression and to confirm deletion of Y6 receptors in Y6-/- mice, animals were killed by cervical dislocation followed by decapitation and tissue collection. All tissues were snap frozen in liquid nitrogen and stored at -80ºC for subsequent mRNA analysis as described below. Brains were collected and hypothalami were dissected and stored separately. A 5-cm segment of the intestine was collected from the gastro- duodenal junction, and the intestinal contents were removed by passing saline through the intestinal tube. White adipose tissue depots (right inguinal, right
31 retroperitoneal and mesenteric), intrascapular brown adipose tissue, liver (the left hepatic lobe), testis, pancreas, femur and calvaria were collected. Whole tissues were homogenized in 2 ml TRI® reagent (Invitrogen, CA, USA). Chloroform
(500 µl) was added and samples were vortexed for 15 seconds. Supernatant was collected after centrifugation at 12000 g for 10 minutes at 4ºC. An aliquot of the aqueous phase was mixed with an equal volume of isopropanol and incubated at room temperature for 15 minutes followed by centrifugation at 12000 g for 10 minutes at 4ºC. Supernatant was removed from the resulting RNA pellet, which was then rinsed with 75% ethanol in diethylpyrocarbonate (DEPC)-treated water.
Samples were centrifuged at 10000 g for 5 minutes at 4ºC. Supernatant was removed and the RNA pellet was air dried for 10 minutes and resuspended in 30
µl DEPC-treated water that had been preheated to 55ºC. After overnight incubation at -80ºC, RNA quality and concentration was determined using the
NanoDrop® ND-1000 Spectrophotometer (ThermoScientific, Wilmington, DE,
USA).
Reverse transcription PCR
Synthesis of cDNA was performed using the QuantiTect Reverse Transcription kit (QIAGEN GmbH, Hilden, Germany). cDNA synthesis was preceded by elimination of genomic DNA by incubation of 1 µg of RNA in 12 µL total volume containing 2 µL of the gDNA Wipeout Buffer (QIAGEN GmbH, Hilden,
Germany) at 42ºC for 2 minutes. A 6-µL master mix solution containing reverse transcriptase, oligo-dT, Mg2+ and dNTPs was then added to each RNA sample and reverse transcription was carried out at 42ºC for 15 minutes. The reaction was terminated by a 3-minute incubation at 95ºC.
32
PCR reactions were preformed for 35 cycles with denaturing at 94°C and extension at 72°C. Mouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to control for variations between samples. PCR was performed with GoTag® Green master mix (Promega, Madison, WI, USA) using 2 µL of cDNA per 25 µL total volume. To determine the expression of Y6 receptor and GAPDH, the following primers and annealing temperatures were used: Y6, 5’-GGAGGGATGGTTATTGTGAC-3’ and 5’-
GTTGTTGCTCTTGCCACTGG-3’ at an annealing temperature of 56˚C;
GAPDH, 5’- ACTTTGTCAAGCTCATTTCC-3’ and 5’-
TGCAGCGAACTTTATTGATG-3’ at an annealing temperature of 57˚C.
Products of 367 and 269 bp were expected from amplification using the specified primers for Y6 and GAPDH, respectively.
Analysis of body weight and composition
Body weight was determined once a week at the same time each week from 6 weeks of age onwards, unless otherwise stated. To determine whole-body lean and fat masses, mice were anesthesized by inhalation of 3% isoflurane and then scanned using dual energy X-ray absorptiometry (DXA) (Lunar Piximus II mouse densitometer; GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK) as previously described (Baldock et al., 2009; Zhang et al., 2010c). DXA scans were performed on normal chow-fed animals at 9, 15, 21 and 24 weeks of age.
High fat diet-fed mice underwent DXA scanning at 7, 14 and 21 weeks of age, corresponding to 2, 7 and 14 weeks following the commencement of the high fat diet.
33 Spontaneous feeding studies
Measurements of spontaneous food intake in chow-fed mice were made at 15 and 24 weeks of age to examine feeding behaviour before and after the development of age-induced increase in adiposity, respectively. Similarly, for mice on the high fat diet, spontaneous food intake was measured at 10 and 20 weeks of age, or after 3 and 13 weeks on a high fat diet, respectively, to examine feeding behaviour before and after the development of diet-induced obesity.
Mice were transferred from group housing on soft bedding to individual cages with a single paper towel in the bottom of the cage and allowed to acclimatize for at least 2 days. Mice were given ad libitum access to their assigned diet. Body weight, the weight of food in the hopper, the weight of food left on the cage floor and fecal output were recorded over four consecutive days. Food consumed was calculated as the weight of food taken from the hopper minus the weight of food spillage on the cage floor.
A separate cohort of animals was used to determine spontaneous feeding behavior during the light and dark phases. Four chow-fed wildtype (WT) and 4 chow-fed Y6-/- mice were housed individually as described above and allowed to acclimatize for at least 2 days. At 16 and 23 weeks of age, body weight and hopper weight were recorded at 2, 4, 8 and 12 hours after onset of the light phase over 6 consecutive days, and the changes in hopper weight were taken as food intake during the time interval. Differences in hopper weight between 2 to 12 hours and 12 to 2 hours after the onset of the light phase were referred to as food eaten during the light and dark phases, respectively. Additionally, body weight
34 and the weight of food in the food hoppers were recorded weekly from 16 to 24 weeks of age for this cohort of mice in order to calculate cumulative food intake.
Fasting and re-feeding studies
The effect of fasting on body weight and food intake was examined in WT and
Y6-/- mice maintained on their normal chow or high fat diet. As described previously (Shi et al., 2011; Zhang et al., 2010a), food was removed from the cages for 24 hours, after which time mice were given free access to their usual diet. Hopper weight, spillage, actual food intake and fecal output were recorded at 1, 2 and 24 hours following re-feeding as described above. Body weight was recorded at all time points, including at 24 hours prior to re-feeding.
Indirect calorimetry
In order to determine energy expenditure, respiratory exchange ratio (RER) and physical activity, 14- and 23-week old mice fed on a normal chow diet, or 22- week old mice that had been maintained on the high fat diet for 15 weeks, were transferred to individual cages (20.1 x 10.1 x 12.7 cm) in an eight chamber open- circuit calorimeter (Oxymax Series; Columbus Instruments, Columbus, OH,
USA). Temperature was maintained at 22C, with an airflow of 0.6 L/min. Mice were acclimatized to the cages for 24 hours before beginning 24-hour recording of oxygen consumption (VO2) and carbon dioxide production (VCO2). RER was calculated as VCO2 ÷ VO2. Energy expenditure (kilocalories of heat produced) was calculated as calorific value (CV) x VO2, where CV = 3.815 + 1.232 x RER as previously published (Melgar et al., 2007). Lean mass-normalized energy expenditure was calculated as energy expenditure divided by lean mass, which
35 was determined immediately following the completion of indirect calorimetry using DXA as described above. Physical activity was also measured, using an
OPTO-M3 sensor system (Columbus Instruments, Columbus, OH, USA), whereby ambulatory counts were a record of consecutive adjacent photo beam breaks in the horizontal space. Data for the 24-h monitoring period was presented as hourly averages for VO2, VCO2, RER, and energy expenditure as well as hourly summation for ambulatory activities. The calorimeter was calibrated before each use using highly pure primary gas standards (O2 and CO2).
Glucose tolerance tests
Food was removed from the hopper and mice were transferred to new cages with fresh bedding before overnight fasting (16-18 hours). The injected glucose solution was prepared by diluting a sterile solution of 50% w/v glucose
(Pharmalab, Lane Cove, NSW, Australia) to 10% using sterile physiological saline. Mice received intraperitoneal (i.p.) administration of glucose at a dose of
1 mg/kg body weight in a volume of 10 µl/g body weight. Blood glucose levels were assessed using blood taken from the tip of the tail at 0, 15, 30, 60 and 90 minutes after glucose administration using the Accu-chek® Go glucometer
(Roche, Dee Why, NSW, Australia). Blood samples were subsequently kept at room temperature before centrifugation at 13000 rpm for 2 minutes. Serum was collected and stored at -20°C for subsequent insulin assay as described below.
Tissue collection
At 6-10 hours after onset of the light phase, chow-fed mice at 15 or 24 weeks of age as well as high fat-fed mice at 24 weeks of age were culled by cervical
36 dislocation followed by decapitation for collection of trunk blood. Glucose levels were assessed in trunk blood using the Accu-chek® Go glucometer (Roche, Dee
Why, NSW, Australia). Trunk blood was allowed to clot at room temperature, centrifuged and the resulting serum was collected and stored at –20 C for subsequent hormone analyses as described below. White adipose tissue depots
(right inguinal (i), right epididymal (e), right retroperitoneal (r) and mesenteric
(m)), intrascapular brown adipose tissue (BAT) and liver were excised and weighed. The left hepatic lobe was collected and frozen in liquid nitrogen and stored at -80C for subsequent lipid quantification as described below.
Oil Red O staining
To measure hepatic lipid infiltration, frozen livers were mounted in embedding medium (Tissue-Tek OCT compound, Sakura Finetek USA, Torrance, CA,
USA) and cryostat sectioned at 6 µm and thaw-mounted on charged slides
(SuperFrost® Plus, Menzel-Glaser, Braunschweig, Germany). Sections were fixed for 30 minutes in 4% PFA at 4C, washed in 3 changes of 50% isopropanol, and then stained for 10 minutes with a solution of 1.2% Oil Red O in 60% isopropanol. The solution had been previously filtered on a 0.45 µm filter
(MILLEX®-HA, Millipore, Carrigtwohill, Ireland). To quantify lipid infiltration in the liver, Oil Red O stained sections were assessed on a grey scale for stained pixels within a defined frame using a light microscope (Leica, Heerbrugg,
Switzerland) at 40x magnification.
37 Serum assays
Serum insulin, testosterone, insulin-like growth factor 1 (IGF-1) and corticosterone levels were measured using the following kits: an Ultra Sensitive
Mouse Insulin enzyme-linked immunosorbent assay (ELISA) kit (Crystal Chem,
Downers Grove, IL, USA) or a Rat/Mouse Insulin ELISA kit (Linco Research,
St. Charles, MO, USA), a testosterone radioimmunoassay (RIA) kit (MP
Biomedicals Germany GmbH, Eschwege, Germany), an IGF-1 RIA kit
(Bioclone Australia Pty Limited, Marrickville, NSW, Australia), and a corticosterone RIA kit (MP Biomedicals Germany GmbH, Eschwege, Germany) in accordance with the manufacturers’ specifications and as previously described
(Sainsbury et al., 2003).
ß-galactosidase staining
As mentioned above, ablation of the Y6 receptor gene was achieved by insertion of a selection cassette containing the lacZ gene into the coding region of the Y6 gene. Therefore, the presence of one or two alleles in Y6+/- or Y6-/- mice, respectively, drives the expression of ß-galactosidase, a product of the lacZ gene, in tissues where Y6 receptors are normally expressed. Thus to determine the distribution of Y6 receptor expression, the presence of ß-galactosidase was detected by incubating tissues with a chromogenic substrate, X-gal (Promega,
Alexandria, NSW, Australia), which yields an insoluble blue dye upon conversion by ß-galactosidase to 5-bromo-4-chloro-3-hydroxyindole.
For detection of Y6 receptor deletion/expression in the brain, mice were killed by cervical dislocation followed by decapitation. Brains were collected and immediately frozen on dry ice and stored in -80°C. Brains were embedded in
38 optimal cutting temperature (OCT) medium (Tissue-Tek OCT compound, Sakura
Finetek USA, Torrance, CA, USA) and cryosectioned at 20 µm. Coronal brain sections from the olfactory bulb to the brain stem were collected and thaw mounted on charged slides (SuperFrost® Plus, Menzel-Glaser, Braunschweig,
Germany). Slides were processed for β -galactosidase histochemistry by 2- minute incubation in a solution containing 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA in 0.1 M phosphate buffer, followed by 2 washes in a solution containing 2 mM MgCl2, 0.1% sodium deoxycholate, 0.02% NP40 (2 minutes each). Finally the slides were incubated overnight at 37C in a ß-galactosidase staining solution containing 1 mg/ml X-Gal (Promega, Alexandria, NSW,
Australia), 0.03 mM spermidine, 0.2 mM NaCl, 5 mM K3Fe(CN)6 and 5 mM
K4Fe(CN)6). All solutions were filter-sterilized (0.2 μm filter) and pH-adjusted to 7.3 with filtered NaOH. The color reaction was stopped by washing slides in a
PBS solution. Slides were then eosin counterstained by a 1-minute incubation in
1% eosin (Eosin Y, Sigma-Aldrich, St Louis, MO, USA) in an 85% ethanol solution and rinsed in distilled water. Slides were dehydrated through graded solutions of ethanol (50%, 70%, 90%, 95% and 100%) followed by incubation in
100% xylene and cover-slipping with Eukitt mounting media (O. Kindler GmbH,
Freiburg, Germany). To visualize Y6 receptors deletion/expression in the brain, images of ß-galactosidase-stained sections were captured using a light microscope (Leica, Heerbrugg, Switzerland). Blue-stained neurons indicate neurons expressing Y6 receptors.
39 Colocalization studies with VIP and AVP
To determine the chemical identity of neurons expressing Y6 receptors, mice were anaesthetized by i.p. administration of ketamine (Ketamav 100, Mavlab,
Slacks Creek, QLD, Australia) and xylazine (Ilium xylazil-20, Troy Laboratories
Pty Limited, Smithfield, NSW, Australia) at a dose of 200 mg/kg and 40 mg/kg body weight, respectively, in a volume of 20 µl/g body weight. The chest cavity was opened and an incision was made at the tip of the left ventricle. A blunted
21-gauged needle was inserted into the incision and clamped in place. The right atrium was cut and 50 ml of sterile isotonic sodium chloride solution (Baxter,
Healthcare, Pty. Ltd., Toongabbie, NSW, Australia) at 4°C was passed into the coronary circulation, followed by 50 mL of 4% paraformaldehyde (PFA)
(ProSciTech, Thuringowa, QLD, Australia) in PBS at 4°C. All solutions were delivered manually using a 50 mL syringe (BD Plastipak, Becton Dickinson,
North Ryde, Australia).
Following PFA perfusion, brains were collected and left in 4% PFA in PBS overnight at 4°C. Brains were transferred to a 30% D+ glucose (Sigma-Aldrich,
St Louis, MO, USA) solution in PBS and incubated for 16-18 hours at 4°C for cryoprotection. Brains were removed, rid of excess glucose solution by briefly blotting on absorbent paper, and frozen on a cold plate in a -80°C freezer for 7 minutes. Brains were then embedded in OCT medium (Tissue-Tek OCT compound, Sakura Finetek USA, Torrance, CA, USA). Coronal brain sections at
35 µm were cut with a cryostat, and sections containing the suprachiasmatic nucleus (SCN) (Bregma -0.34 mm through to -0.70 mm (Franklin and Paxinos,
1997)) were collected in mesh rings in a 6-well plate containing phosphate- buffered saline (PBS) at room temperature.
40
Brain sections were first processed for ß-galactosidase staining as described above in order to localize cells in which Y6 receptors are normally expressed, as indicated by an insoluble blue dye resulting from X-gal cleavage by ß- galactosidase, a product of lacZ gene expression. Brain sections were then washed in 0.2% Triton® X-100 (Sigma-Aldrich, St Louis, MO, USA) in 1x PBS solution (3 x 5 minutes) and incubated in 3% H2O2 in 53% ethanol for 30 minutes on an orbital shaker to deactivate endogenous peroxidase. Sections were washed in 0.2% Triton® X-100 in 1x PBS solution (3 x 5 minutes) and then incubated in a PBS solution containing 2% horse serum, 0.1% bovine serum albumin (BSA) and 0.2% Triton® X-100. Sections were subsequently washed in
0.02% Triton® X-100 in PBS (3 x 5 minutes) and transferred into a tube containing one of the following primary antibodies: mouse anti vasoactive intestinal peptide (VIP) (diluted 1:4000) (Abcam, Cambridge, UK) or mouse anti vasopression-associated neurophysin (AVP) (diluted 1:1000) (A gift from H
Gainer, Bathesda, MD, USA). Primary antibodies were diluted as specified above in phosphate-buffered horse serum (PBH) (0.1% 100 µg/ml BSA, 2% horse serum, 0.2% Triton® X-100), and sections were incubated for 16-18 hours at room temperature on an orbital shaker. Sections were washed in 0.2% Triton®
X-100 in PBS solution (3 x 5 minutes) and incubated in a PBH solution containing goat anti-mouse IgG-Biotin (diluted 1:250) (Sigma-Aldrich, St Louis,
MO, USA) or goat anti-rabbit IgG-Biotin (diluted 1:250) (Sigma-Aldrich, St
Louis, MO, USA) for 2 hours at room temperature on an orbital shaker. Sections were washed in 0.2% Triton® X-100 in PBS solution (3 x 5 minutes), and secondary antibody bound on tissue sections was conjugated with ExtrAvidin®-
Peroxidase (diluted 1:250) (Sigma-Aldrich, St Louis, MO, USA) in PBH by
41 incubation for 2 hours at room temperature on an orbital shaker, followed by washing with 0.02% Triton® X-100 in PBS (3 x 5 minutes). Sections were then incubated in either liquid diaminobenzine (DAB+, Dako, Glostrup, Denmark) or
Vector® NovaREDTM substrate kit for peroxidase (Vector Laboratories,
Burlingame, CA, USA) for two minutes in order to visualize secondary antibody binding. Sections were washed in two changes of PBS solution and mounted on charged slides (SuperFrost® Plus, Menzel-Glaser, Braunschweig, Germany).
Slides were allowed to dry overnight prior to cover slipping with an aqueous mounting agent (Aquatex®, Merck, Darmstadt, Germany).
To visualize cells in the SCN which co-express Y6 receptors and VIP or AVP, images of double-stained sections were captured using a light microscope (Leica,
Heerbrugg, Switzerland) at 40x magnification. Blue-stained neurons indicate neurons expressing Y6 receptors; brown- or red-stained neurons indicate neurons expressing VIP or AVP, respectively. Neurons expressing both Y6 receptors and
VIP or AVP are identified by dark brown or dark red staining, respectively.
Analysis of diurnal variations in serum hormone levels
To determine circadian variations in serum corticosterone concentrations, Y6-/- and WT animals were culled at 0, 6, 12 and 18 hours after onset of the light phase. Mice were culled by cervical dislocation followed by decapitation for collection of trunk blood. Serum corticosterone levels were assessed from serum as described above.
42 PP, PYY and PYY3-36 injection study
To identify potential endogenous activators of Y6 receptors, I examined neuronal activation in response to i.p. administration of Y receptor ligands in Y6-/- and
WT mice. Since Y6 receptors are expressed in the SCN, I examined the expression of an early neuronal activation marker, c-Fos, in this region of the brain. Mice were injected with 250 µl of sterile physiological saline daily for 5 consecutive days prior to study in order to acclimatize them to the injection procedure and thereby reduce background c-Fos expression due to stress, as previously described (Halatchev and Cone, 2005). On the day of the experiment, food was removed from the hopper and mice were transferred to clean cages without food at 5 hours after onset of the light phase. The purpose of this step was to reduce neuronal activation due to feeding (Fan et al., 2004). Mice received i.p. administration of 200 µg/kg of either pancreatic polypeptide (PP), peptide YY (PYY) or PYY3-36 in a volume of 10 µl/g body weight of saline.
Control mice were injected with saline vehicle only. Mice were anaesthetized with i.p. administration of ketamine (Ketamav 100, Mavlab, Slacks Creek, QLD,
Australia) and xylazine (Ilium xylazil-20, Troy Laboratories Pty Limited,
Smithfield, NSW, Australia) at a dose of 200 mg/kg and 40 mg/kg body weight, respectively, in a volume of 20 µl/g body weight. At 30 minutes following peptide or vehicle injection, mice were perfused with sterile isotonic sodium chloride solution and 4% PFA (ProSciTech, Thuringowa, QLD, Australia) in
PBS at 4°C as described above.
Brains were collected and processed for immunohistochemistry as described above. Brain sections were incubated with rabbit polyclonal primary antibody against c-Fos (diluted 1:2500) (Santa Cruz Biotechnology, Santa Cruz, CA,
43 USA) and then with biotinylated goat anti-rabbit IgG secondary antibody
(diluted 1:250) (Sigma-Aldrich, St Louis, MO, USA). Sections were incubated in
ExtrAvidin®-Peroxidase for 2 hours and liquid diaminobenzine (DAB+, Dako,
Glostrup, Denmark) for two minutes to visualize the expression of c-Fos. After cover slipping, brains were visualized and images were captured using light microscopy (Leica, Heerbrugg, Switzerland) at 10x magnification. C-Fos expression was quantified in brain sections containing the SCN (Bregma -0.34 mm through to -0.70 mm (Franklin and Paxinos, 1997)). The number of c-Fos positive neurons within a constant and defined frame was counted using
ImageJ64 software (National Institute of Health, USA).
Statistical analyses
All data are expressed as mean ± standard error of the mean. Genotype distribution in litters resulting from heterozygous Y6 breeding pairs were compared to the expected Mendelian ratio using a Chi-squared test. Litter sizes, percentage of male offspring and mortality rates in pups born to WT, homozygous knockout and heterozygous breeding pairs were analysed using one-way analysis of variance (ANOVA).
A two-tailed student’s t test was used to test difference between 2 groups of mice for parameters such as liver weight and lipid content and serum IGF-1 or testosterone. Differences among groups of mice were assessed by one or two way ANOVA or repeated measures ANOVA followed by Bonferroni posthoc- tests when overall significance was detected by ANOVA. Statistical analyses
44 were performed with Prism software (GraphPad Software, Inc, LaJolla, CA,
USA). Differences were regarded as statistically significant if P < 0.05.
45
RESULTS
46 Generation and validation of the Y6 knockout mouse model
A schematic of the targeting strategy from the commercial available germline
Y6-deficient (Y6-/-) mouse model is shown in (Figure 1A). The insertion of a lacZ-IRES-neoR selection cassette into the coding region of the y6 gene results in disruption of a major part of the y6 gene-coding region. PCR was used to confirm integration of the selection cassette and ablation of the y6 gene. Forward primers located either at the start site of the y6 gene (GS(E)) or in the selection cassette (NEO(T)) were combined with the gene-specific primer (GS(TE)), which binds downstream of the integration site as shown on Figure 1A. A homozygous Y6-/- mouse was identified by the absence of the 312 bp WT fragment and the presence of a larger fragment (~520-540 bp) (Figure 1B).
In order to determine the distribution of Y6 receptors in WT mice and to confirm successful deletion of Y6 receptors in our knockout mice, I performed RT-PCR in various tissues using a primer pair described in the Experimental Procedures which amplifies a 347-bp region of mouse y6 mRNA (Figure 1C). I detected Y6 receptor expression in the hypothalamus, testis and in the inguinal white adipose tissues of WT mice as previously reported (Burkhoff et al., 1998; Weinberg et al., 1996), but not in Y6-/- mice, indicating successful deletion of the Y6 receptor.
47
Figure 1. Generation and validation of the Y6 knockout mouse model.
(A) Targeted germline deletion of the Y6 receptor was achieved by insertion of a lacZ-IRES-neoR cassette into the coding sequence of the y6 gene. Positions of primers used for genotyping are indicated on the wildtype (WT, Y6+) and knockout (Y6-) alleles. (B) To confirm deletion of Y6 receptors, genotyping was performed using GS(E)/GS(TE) or Neo(T)/GS(TE) primer pairs to amplify WT or knockout (Y6-/-) alleles, respectively. (C) Y6 receptor mRNA is present in WT hypothalamus, testis and inguinal white adipose tissues (i), but not in the retroperitoneal (r), mesenteric (m) white adipose tissue depots or in the brown adipose tissues (b). Y6 receptor mRNA is absent from corresponding tissues in
Y6-/- mice as determined by RT-PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as a housekeeping gene.
48 Y6-/- mice are viable and have normal fertility
Breeding of heterozygous Y6 (Y6+/-) mice resulted in live offspring with a frequency of genotypes that was not significantly different from the expected
Mendelian ratio (Table 1). Since Y6 receptors are expressed in the testis, I investigated whether deletion of this receptor has an impact on fertility.
Homozygous Y6-/- breeding pairs produced litters of a similar size to those of
WT and heterozygous breeding pairs (Table 1), indicating that deletion of the Y6 receptors was not likely to affect fertility. Additionally, mortality rate and gender ratio of pups born from heterozygous and homozygous breeding pairs were not significantly different from those of homozygous WT breeding pairs (Table 1), further suggesting that disruption of Y6 receptor expression in mice does not impact upon reproductive fitness.
49 Genotype frequency % male Breeding pair N Litter size Mortality +/+ +/- -/- Y6 Y6 Y6 offspring
8 breeding pairs Heterozygous +/- 20.75% 50.73% 28.51% 7.25 ± 0.507 9.17% 48.04% 20 litters ±0.032 ±0.043 ±0.042 ± 0.069 ± 0.052
11 breeding pairs Homozygous -/- N/A N/A 100% 7.11 ± 0.46 6.53% 49.73% 36 litters ± 3.268 ± 3.642
18 breeding pairs Homozygous +/+ 100% N/A N/A 7.76 ± 0.27 3.41% 51.49% 72 litters ± 1.026 ± 0.023
p=0.9119 p=0.3799 p=0.3387 p=0.7847
Table 1. Genotype frequency, litter size, mortality and percentage of male offspring born from heterozygous (Y6+/-), homozygous (Y6-/-) and WT (Y6+/+) breeding pairs. Data are means ± SEM.
50 Y6-/- mice exhibit reduced body weight and lean mass and a late onset increase in adiposity
Mice heterozygous and homozygous for the Y6 receptor deletion are viable.
Apart from being smaller, Y6-/- mice are morphologically indistinguishable from
WT control mice (Figure 2A). On a normal chow diet, mice lacking Y6 receptors showed reduced body weights relative to WT mice from 6 to 23 weeks of age but showed no significant difference from WT body weights at 24 weeks of age (Figure 2B). The catching-up of body weight in Y6-/- mice was accompanied by a large increase in body weight gain between 23 and 24 weeks of age, likely owing to reduced handling-stress in the absence of experimentation at this time. To assess whether the reduction in body weight was associated with changes in body composition, I performed whole body DXA analyses in Y6-/- and WT mice at 9, 15, 20, and 24 weeks of age. At all ages investigated, Y6-/- mice showed markedly reduced lean body mass when compared to WT mice and when expressed as absolute weight (Figure 2C). Lean body mass as a percent of body weight was also significantly lower in 20- and 24-week-old Y6-/- than in
WT mice (Figure 2D), suggesting a disproportionate reduction in lean mass in the knockouts. Deletion of Y6 receptors also led to a transient reduction in absolute fat mass at 15 weeks of age (Figure 2C). On the other hand, at 20 or 24 weeks of age, Y6-/- mice showed greater adiposity relative to age-matched WT controls, either when expressed as absolute mass (Figure 2C) or as a percent of body weight (Figure 2D). Consistent with the results obtained from whole body
DXA scans, the absolute or relative weights of dissected white adipose tissues depots from the inguinal (i), epididymal (e), retroperitoneal (r) and mesenteric
(m) sites were significantly reduced in the absence of Y6 receptors in 15-week-
51 old mice, as were the absolute and relative weights of the brown adipose tissue
(b) (Figure 2E-F). In contrast to this early reduction in adiposity, the weights of dissected white and brown adipose tissue depots were markedly and significantly greater in 24-week-old Y6-/- compared to WT mice (Figure 2G-H).
52
Figure 2. Y6-/- mice on a normal chow diet exhibit reduced body weight and lean mass and a late onset increase in adiposity. (A) A representative photograph of 15-week old WT and Y6-/- mice. (B) Y6-/- mice displayed significantly reduced body weight compared to their WT counterparts (n=8 or more mice per group). (C) Whole-body dual energy X-ray absorptiometry
(DXA) scans revealed that reduced lean body mass in Y6-/- compared to WT mice was accompanied by a transient reduction in fat mass at 15 weeks of age
53 followed by significant elevations in fat mass at 20 and 24 weeks of age (n=8 or more mice per group). (D) Relative to body weights, the reduction in lean mass and increases in adiposity in 20- and 24-week old Y6-/- relative to WT mice were significant (n=8 or more mice per group). (E-F) Absolute and relative (as a percent of body weight) weights of white adipose tissues from the inguinal (i), epidydimal (e), mesenteric (m) and retroperitoneal (r) sites, and intrascapular brown adipose tissue (b) depots, were significantly reduced in Y6-/- compared to
WT mice at 15 weeks of age (n=13 or more mice per group). (G-H) Relative to
WT controls, 24-week-old Y6-/- mice showed significantly greater weights of white and brown adipose tissue depots, whether fat mass was expressed in absolute values or as a percent of body weight (n=5 or more mice per group).
Data are means ± SEM of the number of male mice shown in parentheses.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus WT.
54 Y6-/- mice displayed increased liver weight and increased hepatic lipid deposition
Obesity is associated with ectopic lipid deposition in various tissues including the heart (Zhou et al., 2000), pancreas (Pinnick et al., 2008) and liver (Fraulob et al., 2010). I found a significant increase in liver weight relative to body weight in chow-fed Y6-/- when compared to WT mice at 15 weeks of age (Figure 3A).
Associated with this, I found a significantly greater amount of lipid in livers from
Y6-/- mice relative to WT control livers at 15 weeks of age (Figure 3B), as determined by Oil Red O staining (Figure 3C-F). Since Y6-/- mice showed reduced adiposity at 15 weeks of age (Figure 2C-F), this result suggests that ectopic fat accumulation in the liver precedes the development of age-induced fat accumulation in Y6-/- mice.
Collectively, these results demonstrate that deficiency of Y6 receptors in mice leads to attenuation of body weight through initial suppression of both lean and fat mass. While lean body mass remains markedly reduced up to 24 weeks of age, mice lacking Y6 receptors showed normalization of body weight in association with significant increases in adiposity, reminiscent of sarcopenic obesity.
55
Figure 3. Y6-/- mice displayed increased liver weight and increased lipid deposition in the liver.
(A) Liver weight was significantly greater in 15-week-old chow-fed Y6-/- relative to age-matched WT mice on the same diet (n=11 or more mice per group). (B)
Liver sections from 15-week-old chow-fed Y6-/- mice showed greater lipid infiltration than those of age-matched WT animals, as determined by the quantity of Oil Red O staining (n=4 mice per group). (C-D) Representative Oil Red O- stained liver sections from WT and Y6-/- mice. (E-F) Representative Oil Red O- and hematoxylin-stained liver sections from WT and Y6-/- mice. Data are means
± SEM of the number of male mice shown in parentheses. *P<0.05, ***P<0.001 versus WT.
56 Reduced body weight and preferential fat accumulation in Y6-/- mice is independent of feeding behavior
I next sought to determine if the reduction in body weight and altered body composition in Y6-/- mice could be explained by changes in feeding behaviour.
Since NPY exerts anxiolytic effects (Wahlestedt et al., 1993), and since ablation of Y1, Y2 and Y4 receptors in mice leads to increased food grinding and spillage behavior (Baldock et al., 2007; Sainsbury et al., 2006), I first analyzed feeding behavior of Y6-/- and WT mice by taking into account food spilled on the cage floor. When measured over 4 consecutive days, I found that spontaneous food intake and spillage did not differ significantly between genotypes at 15 (Figure
4A) nor at 24 weeks of age (Figure 4B). Despite this, Y6-/- mice displayed significantly greater fecal output at 15 but not at 24 weeks of age relative to WT animals (Figure 4C-D). This result suggests that reduced food efficiency, possibly owing to delayed maturity in the gastrointestinal tract, may contribute to the decreased body weight, lean mass and adiposity seen in these mice at this earlier age. When feeding was evaluated after a 24-hour fast at 13 weeks of age, food intake, spillage (Figure 4E), and fecal output (Figure 4F) were not significantly different between Y6-/- and WT mice. Since food spillage was not significantly different between Y6-/- and WT mice, I monitored food intake as the amount of food removed from the hopper from ages 16 to 24 weeks of age. As such, I report no significant difference between Y6-/- and WT mice with respect to cumulative food consumption, either when expressed as absolute weight of food (Figure 4G) or as a percent of body weight (Figure 4H).
57
Figure 4. Reduced body weight and preferential fat accumulation in Y6-/- mice on a normal chow diet is independent of feeding behavior. Food consumption and spillage in Y6-/- mice were not significantly different from that of wildtype (WT) mice at 15 (n=5 mice per group) and 24 weeks of age (n=3 mice per group). (C) Fecal output at 15 weeks of age was significantly elevated in Y6-/- relative to WT mice (n=5 mice per group). (D) Fecal output did not differ
58 significantly different between 24-week-old Y6-/- and WT mice (n=3 mice per group). (E-F) 24-hour fasting-induced food intake, spillage and fecal output were similar between 13-week-old Y6-/- and WT mice (n=12 mice per group). (G-H)
Cumulated food intake from 16 to 24 weeks of age did not differ significantly between Y6-/- and WT mice when analysed as absolute weight or as a percentage of body weight (n=4 mice per group). Data are means ± SEM of the number of male mice shown in parentheses. *P<0.05 versus WT.
59 Reduced body weight in Y6-/- mice could be explained by metabolic alterations
I further investigated energy expenditure, physical activity and respiratory exchange ratio (RER), all of which influence energy balance and body composition. Using indirect calorimetry, I found that 14-week-old chow-fed Y6-/- mice exhibit a significant increase in energy expenditure, either when expressed as absolute values (Figure 5A) or when normalized for the reduced lean body mass of Y6-/- mice (Figure 5B). Associated with this, 14-week-old Y6-/- mice exhibited significant hyperactivity during both the light and the dark phase when compared to WT controls (Figure 5C).
Since muscle atrophy or lean muscle degeneration is often associated with reduced physical activity (Kamei et al., 2004; MacArthur et al., 2008), and since
Y6-/- mice exhibited hyperactivity despite reduced lean body mass, I investigated muscles of differing fibre composition. I did not find any change in weight of the soleus muscle (composed of predominantly slow twitching fibres) or the gastrocnemius muscle (composed of predominantly fast twitching fibres) of the hindlimb in Y6-/- mice relative to WT controls (0.008 ± 0.0007 g versus 0.008 ±
0.0007 g for soleus muscle, P>0.999, and 0.141 ± 0.001 g versus 0.149 ± 0.005 g, P=0.1354, mean ± SEM of 4 male mice per group), suggesting that there were other factors besides altered muscle weight that contributed to the reduction in lean body mass without impairing physical activity.
Besides this increase in physical activity, I investigated whether changes in resting metabolic rate could contribute to the observed increase in energy
60 expenditure in Y6-/- mice, by evaluating energy expenditure corresponding to the hour of least activity. I found no significant difference in recorded energy expenditure during “rest” between Y6-/- and WT mice at 14 weeks of age (Table
2). However, Y6-/- mice are constantly hyperactive throughout the 24 hours of measurement (Figure 5C), and recorded ambulatory counts used to determine metabolic rate during “rest” were markedly greater in Y6-/- than in WT mice
(Table 2). Thus I further assessed resting metabolic rate by extrapolating regression lines of energy expenditure to physical activity as shown in Figure 5
D-E and as previously published (Bjursell et al., 2008; Zhang et al., 2010c). I found that the correlation coefficients were similar between WT and Y6-/- mice
(Table 2), indicating that an increase in physical activity is accompanied by an equal increase in energy expenditure in both genotypes. Additionally, resting energy expenditure, estimated by extrapolating the regression lines to the point when activity level is zero, was not significantly different between 14-week-old
Y6-/- and WT mice (Table 2). These results indicate that the absence of Y6 receptors in 14-week-old mice does not promote the elevation of basal metabolic rate, and that the increase in energy expenditure observed in these knockout mice was due to the associated increase in physical activity.
At 14 weeks of age, Y6-/- mice demonstrated a significant elevation in RER relative to WT values, indicative of a reduced preference for lipid utilization for whole-body energy production (Figure 5F). Whilst the elevated RER in Y6-/- mice may reflect the reduced adiposity in 15-week-old Y6-/- mice (Figure 2C-F), an RER value of greater than 1.0, as seen in Y6-/- mice between 22:00 to 02:00 hours (Figure 5F), is associated with de novo lipogenesis (Elia and Livesey,
1988; Schutz, 2004). Since an increase in RER has been demonstrated to predict
61 weight gain (Marra et al., 2004; Marra et al., 1998; Seidell et al., 1992; Weinberg et al., 1996; Weinsier et al., 1995; Zurlo et al., 1990), these data are in line with the development of late-onset increases in adiposity in Y6-/- mice.
Figure 5. Reduced body weight and reduced adiposity in 14-week-old Y6-/- mice on a normal chow diet could be explained by metabolic alterations. (A)
14-week-old Y6-/- mice demonstrated significantly greater energy expenditure relative to WT mice as determined by indirect calorimetry. (B) Adjusted for lean body mass, the significant elevation in energy expenditure in 14-week-old Y6-/- relative to WT mice was more pronounced. (C) Increased energy expenditure in
62 Y6-/- versus WT mice was accompanied by significantly greater physical activity levels. (D-E) Correlation analysis between physical activity (x-axis) and energy expenditure (y-axis) performed using hourly data from indirect calorimetry for individual WT and Y6-/- mice at 14 weeks of age. Functions generated from the trend lines were used to calculate the y-axis intercept as an indication of resting metabolic rate as shown on Table 2. (F) 14-week-old Y6-/- mice showed significantly increased respiratory exchange ratio relative to WT controls, particularly during the dark phase, indicative of a reduced preference for fat as an oxidative fuel source. Data are means ± SEM of 9 male mice per genotype.
Significant effects of genotype are indicated by *P<0.05. Significant time and genotype interaction effects are indicated by ΨP<0.05, ΨΨP<0.01,
ΨΨΨP<0.001.
63 Recorded values Extrapolated values Age group Genotypes Activity level Energy expenditure Correlation coefficient Energy expenditure (weeks) (ambulatory counts) (x10- 2kcal/g lean mass) (x10-1) (x10- 2kcal/g lean mass)
WT, n=9 13.22 ± 8.095 1.87 ± 0.077 6.979 ± 0.281 2.019 ± 0.074 14 Y6-/-, n=9 71.11 ± 38.44 * 2.03 ± 0.125 6.524 ± 0.516 2.141 ± 0.122
p=0.0453 0.2866 p=0.4504 p=0.4036
WT, n=8 24.00 ± 10.17 1.681 ± 0.069 6.400 ± 0.840 1.784 ± 0.0626 23 Y6-/-, n=9 50.33 ± 13.36 2.015 ± 0.106 * 7.159 ± 0.517 2.098 ± 0.076**
p=0.1305 p=0.0218 p=0.4423 p=0.0066
Table 2. Resting metabolic rate in male Y6-/- and wildtype (WT) mice on a normal chow diet.
Data are means ± SEM for the indicated numbers of 14- and 23-week-old mice. Recorded activity levels and the associated energy expenditure are based on the lowest hourly ambulatory counts recorded for individual mice during 24-hour indirect calorimetry measurements and the corresponding energy expenditure, respectively. This value of recorded energy expenditure is used as an estimate of resting metabolic rate. Correlation analysis between physical activity (x-axis) and energy expenditure (y-axis) was performed using hourly data from indirect calorimetry for individual mice. Functions generated from the trend line were used to calculate the y-axis intercept as an indication of resting metabolic rate. * p<0.05, ** p<0.01 versus WT. 64 In contrast to the decreased RER seen in 14-week-old knockout mice, secondary to the age-induced increase in adiposity, 23-week old Y6-/- mice displayed a significant reduction in RER relative to WT mice (Figure 6A), suggesting an increased preference for lipid as an oxidative fuel source. Consistent with the increased energy expenditure and physical activity seen in 14-week-old Y6-/- versus WT mice, 23-week-old Y6-/- also demonstrated significant increases in energy expenditure, at least when expressed per gram of lean mass if not in absolute values (Figure 6B-C), as well as an increase in physical activity, significantly so during the light phase (particularly in the first hour) (Figure 6D).
Furthermore, resting energy expenditure, either estimated as the value observed at the point of least activity or by extrapolation from regression analysis as shown on Figure 6E-F, was significantly elevated in Y6-/- relative to WT mice at
23 weeks of age (Table 2). These data suggest that, in addition to increased physical activity, an increase in basal metabolic rate is an important contributor to the increased total energy expenditure observed in 23-week-old Y6-/- mice.
Taken together, the reduction in body weight and the transient reduction in adiposity in mice lacking Y6 receptors is likely to be a consequence of negative energy balance resulting from increased energy expenditure, driven by increased physical activity or increased resting metabolic rate, without compensatory hyperphagia and the possible presence of nutrient malabsorption. Furthermore, an adaptation to increase fuel efficiency in response to the chronic negative energy balance in Y6-/- mice may account for the development of late-onset obesity.
65 Figure 6. Preferential fat accumulation in 23-week-old Y6-/- mice on a normal chow diet is independent of feeding behavior but could be explained by metabolic alterations. (A) At 23 weeks of age, Y6-/- mice showed significantly reduced respiratory exchange ratio relative to WT mice, indicating increased fat oxidation possibly resulting from increased availability of lipid as a metabolic fuel source. (B) Energy expenditure was significantly greater during the light phase in 23-week-old Y6-/- relative to WT mice. (C) Energy expenditure per gram of lean body mass was significantly greater in Y6-/- mice than in WT controls at 23 weeks of age. (D) This was associated with significantly greater physical activity during the light phase in Y6-/- versus WT control mice. (E-F)
Correlation analysis between physical activity (x-axis) and energy expenditure
66 (y-axis) performed using hourly data from indirect calorimetry for individual WT and Y6-/- mice at 23 weeks of age. Functions generated from the trend lines were used to calculate the y-axis intercept as an indication of resting metabolic rate as shown on Table 2. Data are means ± SEM of 8 or more male mice per group.
Significant effects of genotype are indicated by **P<0.01, ***P<0.001.
Significant time and genotype interaction effects are indicated by ΨP<0.05,
ΨΨP<0.01, ΨΨΨP<0.001.
67 Y6 receptors regulate glucose homeostasis in mice
Skeletal muscle is a major site of insulin-mediated glucose disposal, and a reduction in lean mass is associated with impairment in glucose homeostasis and insulin sensitivity (Dulloo, 2009; Tennese and Wevrick, 2011). Since Y6-/- mice demonstrated a significant reduction in lean body mass, I proposed that glucose metabolism might be impaired in these mice. Measurements of fasted and non- fasted blood glucose levels showed that 15-week old Y6-/- mice exhibited normoglycemia (Figure 7A). Despite this, Y6-/- mice at 15 weeks of age exhibit reduced serum insulin concentrations relative to WT values, significantly so in the non-fasted condition (Figure 7B), suggesting improved insulin action on glucose clearance. Indeed, in response to i.p. glucose administration, 15-week old Y6-/- mice exhibited significantly reduced blood glucose levels (Figure 7C) despite significantly lower serum insulin concentrations (Figure 7D) relative to corresponding values in age-matched WT mice. These results indicate that deletion of Y6 receptors in mice led to improved glucose tolerance and insulin action.
While fasted and non-fasted blood glucose levels were similar between 24-week- old Y6-/- and WT mice (Figure 7E), the heightened fat accumulation in Y6-/- mice of this age was accompanied by a significant elevation in non-fasting serum insulin concentrations relative to WT values (Figure 7F), suggesting decreased insulin action in aged knockouts. Despite this, I did not observe any significant deterioration in glucose tolerance in knockouts at 24 weeks of age. Indeed, blood glucose and serum insulin levels in response to i.p. glucose injection were similar between 24-week-old Y6-/- and WT mice (Figure 7G, H). These data suggest
68 that the effect of Y6 receptor deletion to improve glucose tolerance that was observed at 15 weeks of age was abolished by the age-induced increase in adiposity.
69
Figure 7. Impact of Y6 receptor deletion on glucose homeostasis in mice on a normal chow diet. (A) Blood glucose levels were not significantly different between male Y6-/- and WT (WT) mice at 15 weeks of age (n=13 or more mice per group). (B) Serum insulin levels were reduced in 15-week-old Y6-/- relative to WT mice, significantly so under fasted conditions (n=11 or more mice per group). (C-D) In response to i.p. glucose injection (1 mg/kg), 15-week-old Y6-/-
70 mice showed significantly lower blood glucose levels despite significantly reduced serum insulin levels when compared to WT animals (n=5 mice per group). (E) Blood glucose levels in 24-week-old Y6-/- mice were not significantly different from WT control values (n=5 or more mice per group). (F) Y6-/- mice displayed significantly higher serum insulin level under non-fasted conditions at
24-weeks of age compared to WT mice (n=5 or more mice per group). (G-H) At
24 weeks of age, blood glucose and serum insulin levels in response to i.p. glucose injection (1 mg/kg) were not significantly different between WT and Y6-
/- mice (n=10 mice per group). Data are means ± SEM of the number of male mice shown in parentheses. *P<0.05, ****P<0.0001 versus WT.
71 Y6-/- mice show exacerbated diet-induced obesity
I next assessed the effect of Y6 receptor deletion in mice on a high fat diet
(HFD), which was introduced at 7 to 8 weeks of age and was continued for 16 weeks. After 16 weeks of HFD feeding, WT and Y6-/- mice weighed significantly more than their age-matched counterparts maintained on a normal chow diet
(41.1 ± 1.5 g versus 33.4 ± 0.7 g in WT, P<0.0001 and 41.5 ± 0.8 g versus 29.7 ±
0.9 g in Y6-/-, P<0.001). Further, whole-body adiposity in HFD-fed WT and Y6-/- mice, as determined by DXA, were over 2.5 fold greater in HFD-fed mice than corresponding values in normal chow-fed mice of the same genotype (11.8 ± 0.9 g versus 4.7 ± 0.4 g in WT, P<0.001 and 18.2 ± 0.8 g versus 6.0 ± 0.4 g in Y6-/- mice, P<0.001). Together, these results confirm the obesogenic effect of the HFD used in this study.
Relative to WT mice, Y6-/- mice exhibited significantly lower starting body weights (Figure 8A), but displayed significantly greater body weight gain on the
HFD (Figure 8B). As a result, after 8 weeks on the HFD – when animals were
15 weeks of age – Y6-/- mice showed similar body weights to age-matched WT controls (Figure 8A), showing that the normalization of body weight in 24 week- old chow-fed Y6-/- mice seen in Figure 2B was accelerated by the obesogenic diet.
Consistent with the effect of Y6 receptor deletion in mice maintained on a normal chow diet (Figure 2C-D), Y6-/- mice fed a HFD displayed reduced lean body mass at all ages investigated, as determined by whole body DXA scans and when expressed as either absolute weight or as a percent of body weight (Figure
72 8C-D). When compared to WT controls, Y6-/- mice showed significantly higher adiposity at 7 and 14 weeks after the commencement of HFD feeding, whether fat weight was expressed as absolute values (Figure 8C) or as a percentage of body weight (Figure 8D). This greater adiposity as determined by DXA was further confirmed by significantly greater absolute and relative masses of dissected white and brown adipose tissue depots in Y6-/- mice compared to those of WT mice after 16 weeks on the HFD (Figure 8E, F). These data show that together with the suppression of lean body mass, the impact of Y6 receptor deficiency in enhancing adiposity in mice is exacerbated by a HFD.
The exacerbated body weight gain and adipose tissue accumulation in Y6-/- mice on a HFD occurred in the presence of transient hyperphagia that was observed at
3 but not at 13 weeks after introduction of the HFD (i.e. at 10 but not at 20 weeks of age), significantly so when food intake was expressed as a percent of body weight (Figure 9A-B). In contrast, food intake of Y6-/- mice was significantly reduced relative to that of WT controls after 13 weeks on the HFD (i.e. at 20 weeks of age), whether food intake was assessed as absolute weight or when adjusted for body weight (Figure 9A-B). Reflective of the changes in absolute food consumption (Figure 9A-B), fecal output was significantly reduced after 10 but not 3 weeks of high fat feeding in Y6-/- relative to WT mice (Figure 9C). In line with the differences in spontaneous feeding behaviour, while 11-week-old
Y6-/- and WT mice did not show any differences in fasting-induced re-feeding or the associated fecal output after 4 weeks on the HFD (Figure 9D-F), after 14 weeks on the HFD, 21-week-old Y6-/- mice exhibited significant hypophagia in response to fasting (Figure 9G-H), and significantly reduced fecal output
(Figure 9I) relative to WT animals. The transient increase in food intake seen at
73 3 weeks after commencement of the HFD (i.e. at 10 weeks of age) may contribute to the accelerated body weight gain in HFD-fed Y6-/- mice.
Additionally, since food intake at the later time point was significantly reduced, deletion of Y6 receptors in mice may lead to increased metabolic efficiency that contributes to the greater body weight gain seen in HFD-fed Y6-/- mice.
Using indirect calorimetry, I report that after 14 weeks on the HFD (and at 21 weeks of age), Y6-/- mice did not exhibit any difference in absolute energy expenditure when compared to WT controls (Figure 10A). When adjusted for the difference in lean body mass, however, energy expenditure was markedly and significantly greater in HFD-fed Y6-/- than in WT mice (Figure 10B). This increase in energy expenditure was more pronounced than that seen in chow-fed
Y6-/- mice at either the younger or the older age (Figure 5A-B, 6B-C). Unlike in chow-fed animals (Figure 5C,F, 6A,D), I did not detect any significant differences in physical activity (Figure 10C) nor RER (Figure 10D) between
HFD-fed Y6-/- and WT mice. These results show that the increases in body weight gain and fat accumulation in Y6-/- mice on the HFD is not due to reduced metabolism.
After 12 weeks on the HFD (i.e. at 19 weeks of age), Y6-/- mice displayed normoglycemia (Figure 11A) and elevated serum insulin levels relative to WT controls, significantly so in the non-fasted state (Figure 11B). When assessed at
13 weeks after commencement of the HFD (i.e. at 20 weeks of age), Y6-/- mice displayed markedly higher blood glucose (Figure 11C) and serum insulin levels
(Figure 11D) in response to i.p. glucose injection, signifying impaired glucose metabolism. Taken together, our data demonstrate that deficiency in Y6
74 receptors in mice exacerbates diet-induced obesity and promotes the associated abnormalities in glucose homeostasis.
Figure 8. Mice lacking Y6 receptors showed exacerbated diet-induced obesity. (Erondu et al.) Relative to wildtype (WT) animals, Y6-/- mice showed increased body weight gain during 16 weeks on a high fat diet (HFD) from age 7 to 23 weeks of age, whether expressed as absolute weight or as a percent of starting body weight. (C-D) As determined by dual energy X-ray absorptiometry
(DXA) scans and when expressed as absolute weight or a percent of body weight, Y6-/- mice showed significantly reduced lean body mass relative to WT mice, while fat mass was markedly greater in knockout than in WT mice at 7 and
14 weeks after commencement of the high fat diet (at 14 and 21 weeks of age).
75 (E-F) Weight of white adipose tissues from the inguinal (i), epidydimal (e), mesenteric (m) and retroperitoneal (r) sites, and intrascapular brown adipose tissue (b) depots in male Y6-/- mice maintained on a HFD for 16 weeks were significantly greater than those of WT control mice, whether expressed as absolute weight or as a percentage of body weight. Data are means ± SEM of 9 male mice per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus
WT.
76
Figure 9. Effects of Y6 receptor deletion on food intake in mice maintained on a high-fat diet
(A) Whereas absolute food intake was similar in mice of both genotypes after 3 weeks on the high fat diet (HFD, 10 weeks of age), Y6-/- mice showed significantly reduced food intake after 13 weeks of high fat feeding (20 weeks of age) when compared to wildtype (WT) controls. (B) Food intake data from (A) expressed as a percent of body weight. (C) Reflective of the changes in food intake, fecal output was significantly reduced after 13 weeks of high fat feeding
(20 weeks of age) in Y6-/- relative to WT mice. (D-F) 24-hour fasting-induced food intake, expressed in absolute terms and as a percent of body weight, and fecal output were similar between Y6-/- and WT mice after 4 weeks of on the
HFD (i.e. at 11 weeks of age). (G-I) Food intake in absolute terms and relative to body weight as well as fecal output following 24-hour fasting were significantly decreased in 20-week-old Y6-/- mice relative to WT controls after 14 weeks on
77 the HFD. Data are means ± SEM of 9 male mice per group. *P<0.05, **P<0.01,
****P<0.0001 versus WT.
Figure 10. Effects of Y6 receptor deletion on energy metabolism and physical activity in mice maintained on a high-fat diet.
(A) When measured after 12 weeks on the high fat diet (HFD), indirect calorimetry revealed similar energy expenditure in Y6-/- and WT mice. (B) When data from (A) was adjusted for the difference in lean body mass, energy expenditure in Y6-/- mice was significantly greater than that of WT mice. (C-D)
Physical activity and respiratory exchange ratio were not significantly different between Y6-/- and WT mice. Data are means ± SEM of 9 male mice per group.
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 versus WT.
78
Figure 11. Glucose metabolism was significantly impaired in Y6-/- mice fed a high-fat diet.
(A) After 12 weeks on the high fat diet, Y6-/- mice showed similar blood glucose levels to WT controls, with (B) serum insulin levels under non-fasted conditions being significantly elevated in Y6-/- relative to WT control values. (C-D)
Compared to WT controls, Y6-/- mice that had been on the high fat diet for 13 weeks showed impaired glucose tolerance in response to 1 mg/kg i.p. glucose injection in association with significantly increased serum insulin levels. Data are means ± SEM of 9 male mice per group. *P<0.05, **P<0.01, ***P<0.001,
****P<0.0001 versus WT.
79 Y6-/- mice showed normal serum testosterone levels
In Figure 1C I showed strong expression of Y6 receptors in WT hypothalamus and testis. I thus asked whether loss of Y6 receptors in these tissues might contribute to the metabolic phenotype of Y6-/- mice. Circulating testosterone is released from Leydig cells of the testes, and reduction in circulating testosterone levels is strongly linked to reduced lean body mass (Brown et al., 2009; Sattler et al., 2009; Sinha-Hikim et al., 2002; Sinha-Hikim et al., 2003) and obesity
(Nettleship et al., 2007; Pi et al., 2008; Tsai et al., 2000). However, the absence of Y6 receptors in mice did not lead to a reduction in serum testosterone levels
(Figure 12).
Figure 12. Deletion of Y6 receptors did not alter serum testosterone levels.
Non-fasted serum testosterone levels in 15-week-old male Y6-/- mice on a normal chow diet was not significantly different from that of wildtype (WT) controls.
Data are means ± SEM of 8 or more male mice per group.
80 Y6 receptors are co-expressed in VIP neurons in the hypothalamic suprachiasmatic nucleus
In order to elucidate possible pathways via which lack of Y6 receptors may regulate energy balance and body composition in mice, I next determined the chemical identity of Y6-expressing neurons in the hypothalamus. As targeted deletion of Y6 receptors was achieved by inserting a selection cassette containing the lacZ gene into the y6 gene coding region, I was able to visualize the in situ expression of Y6 receptors by performing lacZ staining as previously described
(Choi et al., 2009). However, one potential caveat to studying the distribution of
Y6 receptors using a reporter such as lacZ is the possibility of reporter infidelity, which may lead to the over- or under-reporting of Y6 receptor expression. A reporter-independent method such as in situ hybridization would offer a greater insight to the distribution of Y6 receptors in peripheral tissues, but for the current study, lacZ staining was a suitable tool for the identification of Y6 expression sites within the brain.
LacZ staining in rostral to caudal coronal brain sections spanning the olfactory bulb to the brain stem reveals that Y6 receptor expression is restricted to the hypothalamic suprachiasmatic nucleus (Figure 13A-F), with no apparent staining in other brain regions (data not shown).
Immunohistochemical staining with antibodies against neuropeptides known to be expressed in the SCN (Reghunandanan et al., 1993) revealed doubly labeled neurons expressing lacZ (thereby revealing cells that normally express Y6 receptors) as well as being immunoreactive for vasoactive intestinal peptide
81 (VIP) (Figure 5G-H). This suggests that Y6 receptors are expressed in VIP- expressing neurons. In contrast, I did not detect lacZ co-expression with another predominant SCN neuropeptide; arginine vasopressin (AVP) (Figure 5I-J).
Figure 13. Y6 receptors are expressed in VIP-expressing neurons in the hypothalamic suprachiasmatic nucleus
(A-F) Representative photographs of coronal brains sections in anterior to posterior order showing the distribution of Y6 receptors as indicated by lacZ 82 expression in the mouse suprachiasmatic nucleus (SCN) (Bregma -0.34 mm through to -0.70 mm (Franklin and Paxinos, 1997))(G-H) Representative photographs showing co-expression of lacZ and vasoactive intestinal peptide
(VIP)-immunoreactive neurons in the SCN of Y6-/- mice. Representative photographs showing the absence of lacZ and arginine vasopressin (AVP) colocalization in the SCN of Y6-/- mice. Female mice were used for these experiments to reduce the number of animals used in this research, after it was established that the distribution of Y6 receptors in the brain did not differ between the sexes.
83 Y6-/- mice have reduced serum IGF-1 and blunted corticosterone rhythm
Having established that Y6 receptors are co-expressed with VIP in neurons of the
SCN, I hypothesized that the metabolic phenotypes of Y6-/- mice may be explained by impairment in VIP signalling. VIP is a key regulator of the growth hormone axis (Asnicar et al., 2002; Niewiadomski et al., 2008), circadian rhythm
(Aton et al., 2005; Brown et al., 2007; Pantazopoulos et al., 2010; Piggins and
Cutler, 2003) and glucocorticoid secretion (Loh et al., 2008). Strikingly, Y6-/- mice showed significantly reduced serum IGF-1 levels relative to WT controls
(Figure 14A), which is in line with the reductions in body weight and lean body mass seen in these animals. Additionally, loss of Y6 receptors in mice led to blunting of the diurnal variations in serum corticosterone levels seen in WT mice
(Figure 14B). The dampened corticosterone rhythm seen in Y6-/- mice is apparently not due to impaired secretion, since fasting elicited comparable increases in serum corticosterone levels in mice of both genotypes, although Y6-/- mice showed significantly reduced serum corticosterone levels compared to WT controls under both fed and fasted conditions (Figure 14C). Since the timing of food intake is linked to corticosterone secretion (Le Minh et al., 2001; Sheward et al., 2007; Stokkan et al., 2001) and has been shown to be abrogated in mice with disrupted VIP signalling (Bechtold et al., 2008), I investigated food consumption during light and dark phases and found significantly reduced daytime feeding in mice lacking Y6 receptors, with no significant difference between genotypes with respect to dark-phase food intake (Figure 14D-E). As low serum IGF-1 levels are etiologically linked to low lean body mass and increased fat mass (Sainsbury and Zhang, 2011), our data provide evidence that
84 the reduced lean mass and late-onset or diet-induced increases in adiposity in mice lacking Y6 receptors may result from reduced IGF-1 levels.
Figure 14. Loss of Y6 receptor signalling is associated with reduced serum
IGF-1 levels and blunted rhythms in serum corticosterone and light-phase feeding.
(A) Fasted Y6-/- mice on a normal chow diet exhibited reduced serum insulin-like growth factor-1 (IGF-1) levels relative to wild type (WT) values (n=6 or more male mice per group). (B) Serum corticosterone levels assessed in trunk blood at
6-hourly intervals reveals a blunted corticosterone rhythm in Y6-/- mice when compared to WT controls (n=3 or more male mice per group). (C) Y6-/- and WT mice showed increased serum corticosterone levels in response to fasting, with significantly higher levels in knockout than in WT animals (n=6 or more male mice per group). (D-E) Light and dark-phase food intake shown in white and black bars, respectively, were measured for 6 consecutive days. Relative to WT mice, light-phase feeding was significantly reduced in Y6-/- mice at 16 and 23 weeks of age (n=5 per group). Data are means ± SEM of the number of male mice shown in parentheses. *P<0.05, **P<0.01 versus WT. 85 Y6 receptors are activated by pancreatic polypeptide in mice
To address our aim of identifying potential physiological ligands for Y6 receptors in mice, I examined the expression of c-Fos, a marker of early neuronal activation, in the SCN of Y6-/- and WT mice in response to i.p. administration of either PP, peptide YY (PYY), PYY3-36 or saline.
Whereas injection of PYY or PYY3-36 did not elicit significant increases in c-
Fos expression in the SCN relative to that in saline-injected WT controls (Figure
15A), I observed a significant increase in c-Fos immunoreactivity in the SCN of
WT mice at 30 minutes following i.p. PP injection (Figure 15A-C). In contrast to this WT response, injection of PP into Y6-/- mice did not produce any increase in c-Fos immunoreactivity relative to saline-injected mice of the same genotype
(Figure 15A, D-E). This finding indicates that PP-induced neuronal activation in the mouse SCN is mediated by Y6 receptors.
86
Figure 15. Y6 receptors are activated by pancreatic polypeptide in vivo. (A)
Increased expression of c-Fos, a marker of early neuronal activation, was detected by immunohistochemistry in the suprachiasmatic nucleus (SCN) at 30 minutes after i.p. injection of pancreatic polypeptide (PP) (200 µg/kg) but not peptide YY (PYY) (200 µg/kg) or PYY3-36 (200 µg/kg) in (WT) mice relative to saline-injected controls. This effect of i.p. PP injection on SCN c-Fos
87 immunoreactivity was not seen in Y6-/- mice (n=3 or more mice per group). (B-
E) Representative photographs of coronal brain sections of WT and Y6-/- SCN at
30 minutes after i.p. saline or PP injections. Data are means ± SEM of the number of female mice shown in parentheses. *P<0.05, ****P<0.0001 versus
WT.
88
DISCUSSION
89 This work showed that the Y6 receptor is a key regulator of energy homeostasis and body composition, because germline deletion of Y6 receptors in male mice resulted in reduced body weight and reduced lean body mass in association with a late onset increase in adiposity that was exacerbated by a high-fat diet. These changes may be due to increased energy expenditure (as determined by indirect calorimetry) which is at least in part due to hyperactivity in the absence of hyperphagia, in response to which body weight was recovered through disproportionate promotion of fat accumulation. These metabolic consequences of Y6 receptor deletion may arise from disrupted VIP signalling in the SCN, since I showed that Y6 receptors are co-expressed in VIP-expressing neurons in this hypothalamic nucleus. Lending further support to this notion, the effects of
Y6 receptor deletion mimics previously known effects of ablating VIP or its receptor, vasoactive intestinal peptide receptor 2 (VPAC2), namely reduced function of the growth hormone axis as indicated by reduced lean body mass and serum IGF-1 levels in Y6 receptor knockout mice, as well as blunted diurnal variations in serum corticosterone levels and reduced light-phase feeding.
Additionally, our findings implicate the Y6 receptor as an endogenous mediator of PP action, since exogenous administration of PP, but not PYY or PYY3-36, activated Y6-expressing neurons in the SCN, and this effect was absent in Y6 knockout mice. Taken together, this work demonstrates the critical importance of
Y6 receptors in the regulation of energy homeostasis. Future studies into hypothalamic control of physiological functions such as energy balance in mice, notably when involving Y receptor ligands, must take into account the possible contribution of Y6 receptors to observed effects.
90 Associated with the reduction in lean body mass, normal chow-fed Y6-/- mice demonstrated increased ambulatory movement or physical activity. This result was unexpected since muscle atrophy or lean muscle degeneration is often associated with reduced physical activity (Kamei et al., 2004; MacArthur et al.,
2008) and conversely, increased lean body mass is associated with increased exercise tolerance (Hamrick et al., 2006). Despite this, several studies show associations that are opposite to this pattern of observations. For instance, increased lean body mass in MC4R-/- mice was associated with reduced exercise performance (Braun et al., 2012), while reduction in lean body mass in MCK-
CnA transgenic mice was linked to elevated exercise endurance (Jiang et al.,
2010). These disconnects between lean body mass and physical activity could be explained by a number of factors such as differences in muscle fibre composition or function, cardiac output, and muscle mitochondrial content. (Braun et al.,
2012; Jiang et al., 2010). Interestingly, the deletion of Y6 receptors did not change the weight of the predominantly slow-twitching soleus or the predominantly fast-twitching gastrocnemius muscle of the hindlimb, suggesting that there were other factors besides muscle weight that contributed to the reduction in lean body mass without impairing physical activity.
While young Y6-/- mice exhibited negative energy balance, indicated by reductions in body weight, adiposity and lean body mass – possibly mediated by increased energy expenditure without any increase in absolute food intake, at an older age or on a high fat diet knockouts exhibited increased adiposity with reduced lean body mass, reminiscent of sarcopenic obesity (Evans, 1995; Kamei et al., 2004). The age- or diet-induced obesity of Y6-/- mice may be a consequence of the negative energy balance in early life, because long-term
91 energy deficit is thought to lead to a disproportionate preference for fat accumulation at the expense of lean body mass (Dulloo and Girardier, 1990;
Escriva et al., 2007; Faulks et al., 2006; Li et al., 2010; Redman et al., 2007).
This phenomenon has been termed ‘catch up fat’ or ‘catch up growth’ (Dulloo,
2009). Indeed, re-feeding adult rats that had previously undergone chronic energy restriction resulted in the restoration of body weight and enhanced efficiency of energy utilization, with energy being directed towards fat accretion but not protein synthesis (Dulloo and Girardier, 1990; Ozelci et al., 1978;
Summermatter et al., 2008). Similarly, experimentally imposed chronic energy restriction in adult mice led to enhanced adiposity despite reduced body weight
(Li et al., 2010), as well as induction of a 3-fold increase in adipose tissue fatty acid synthesis (Bruss et al., 2010). In humans, a similar preference for fat accretion at the expense of lean body mass is evident after experimental starvation (Keys, 1946), in patients recovering from anorexia nervosa (Mayer et al., 2005; Orphanidou et al., 1997), and in hypermetabolic states such as cancer
(Cao et al., 2010; Loprinzi et al., 1993; Talvensaari et al., 1996a; Talvensaari et al., 1996b), acquired immune deficiency syndrome (Kotler et al., 1990) and
Parkinson’s disease (Dulloo and Montani, 2005). In the absence of any observed increase in absolute energy intake in Y6-/- mice of any age or dietary condition, the age-induced elevation in fat accumulation in Y6-/- mice must have resulted from other factors, such as alterations in metabolic fuel preference. Indeed, a shift in fuel utilization that favours the oxidation of carbohydrate and preferentially diverts ingested lipid to adipose depots has been shown to reduce the energetic cost of body weight regain in response to caloric restriction
(MacLean, 2005; MacLean et al., 2006). In addition, increased carbohydrate oxidation and decreased lipid oxidation, as indicated by an elevated RER,
92 predicts body weight gain in obese or non-obese rodents and human alike (Marra et al., 2004; Marra et al., 1998; Seidell et al., 1992). In line with increased carbohydrate oxidation, decreased lipid oxidation and increased de novo lipogenesis contributing to the late-onset or exacerbated diet-induced obesity of
Y6 deficient mice, I observed a significant elevation in RER in Y6-/- relative to
WT mice, with values actually exceeding 1.0, signifying endogenous fatty acid synthesis (MacLean et al., 2006; Schutz, 2004). Thus, while chronic negative energy balance due to hyperactivity and increased energy expenditure without hyperphagia probably contributed to the reduced body weight of Y6-/- mice, it likely also contributed to the age-induced and diet-induced increases in adiposity observed in these animals.
Deletion of Y6 receptors improves glucose homeostasis in young animals but did not protect against deterioration of glucose tolerance and hyperinsulinemia associated with increased adiposity in older age or on a high fat diet. Y6-/- mice showed reduced lean mass, a major site for insulin-induced glucose disposal. In the absence of any apparent change in muscle fibre composition, namely in the weight of the insulin-responsive soleus or gastrocnemius muscles, it might be expected that Y6-/- mice have impaired insulin sensitivity in skeletal muscle that is compensated for by increased glucose disposal in other tissues such as liver and adipose tissue. In the liver, insulin is known to promote the storage of glucose in the form of glycogen and the synthesis of fatty acid, which is circulated and serves as a substrate for triglyceride synthesis in adipose tissue.
High concentrations of free fatty acid in the blood has previously been reported to contribute to excessive accumulation of lipids in the liver (hepatic steatosis)
(Kahn and Flier, 2000; Zivkovic et al., 2007), a condition linked to the
93 pathogenesis of hepatic insulin resistance, increased hepatic gluconeogenesis, and fasting hyperglycemia (Mayerson et al., 2002; Petersen et al., 2005; Samuel et al., 2004). In line with this, 15-week-old Y6-/- mice demonstrated improved tolerance to exogenous glucose in association with increased liver weight and hepatic lipid deposition. This was followed by the deterioration in glucose metabolism in concert with age- or diet-induced increase in adiposity, which likely resulted from the exhaustion of liver and saturation of adipose tissue as compensatory organs for glucose disposal in Y6-/- mice. Additionally, while increased adiposity itself is a risk factor for insulin resistance and type 2 diabetes, previous reports have also outlined the link between catch up growth and the metabolic syndrome. Indeed, asymmetrical growth favoring fat accumulation over fat-free mass observed in children born small for gestational age (Dulloo, 2008, 2009; Dulloo et al., 2006a; Dulloo et al., 2006b; Hales and
Barker, 2001; Meas et al., 2008; Ravelli et al., 1976) and in long term survivors of childhood cancer (Garmey et al., 2008; Talvensaari et al., 1996a; Talvensaari et al., 1996b; van Waas et al., 2010) is intimately associated with an increased prevalence of glucose intolerance, insulin resistance or type 2 diabetes later in life. One mechanism linking catch up fat accretion to development of the metabolic syndrome may be a shift of glucose utilization from skeletal muscle to adipose tissue, since reduced insulin-stimulated glucose utilization in skeletal muscle with concomitant insulin hyperresponsiveness in white adipose tissue was reported in studies of catch up fat (Cettour-Rose et al., 2005; Summermatter et al., 2009). Hence, while improvement in glucose metabolism appears to be a primary feature of Y6 receptor deletion, the predisposition to eventual development of increased adiposity with age or on a high fat diet could override this metabolic effect of Y6 receptor deficiency in mice.
94
I propose that Y6 receptor deletion may mediate at least part of its effects on body composition via alterations in VIP signalling in the hypothalamic SCN, since the central expression of Y6 is exclusively confined to this region of the brain, in cells that co-express VIP. The SCN contains the master circadian clock in mammals; it generates distinct temporal changes in biological processes such as core body temperature, hormone secretion and activity (Huang et al., 2011).
Disruptions in circadian clock function, whether by genetic or environmental insults, can result in metabolic dysfunction. For example, mutation of either of two key regulators of the circadian clock – circadian locomotor output cycles kaput (CLOCK) or brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) – has been shown to not only disrupt circadian rhythmicity, manifesting in loss of normal daily hormonal and feeding patterns, but to also predispose mice to metabolic diseases (Marcheva et al.,
2010; Rudic et al., 2004; Turek et al., 2005). As such, it is possible that the disrupted circadian rhythm observed in our Y6-/- mice, as evidenced by blunted daily fluctuations in serum corticosterone levels and reduced light-phase feeding, may have contributed to the metabolic abnormalities of these mice. VIP is a critical regulator of circadian rhythm in the SCN, with deletion of VIP or its receptor, VPAC2, leading to loss of circadian rhythmicity and synchrony in SCN neurons (Aton et al., 2005; Colwell et al., 2003; Hannibal et al., 2011; Harmar et al., 2004; Kalamatianos et al., 2004). Disrupted VIP signalling in Y6-/- mice may have contributed to the low serum IGF-1 levels and blunted corticosterone rhythm observed in these animals. Indeed, VIP is an important regulator of the growth hormone axis, since VIP stimulated growth hormone release in rats in vivo (Bluet-Pajot et al., 1987), in cultured bovine adenohypophysial cells
95 (Soliman et al., 1995) and in perifused bovine adenohypophysis (Hashizume and
Kanematsu, 1990), and loss of VIP or VPAC2 in mice led to reduced growth in association with reduced circulating IGF-1 levels (Asnicar et al., 2002;
Niewiadomski et al., 2008). Additionally, VIP is an essential regulator of circadian rhythmicity in the hypothalamic-pituitary-adrenal axis
(Balasubramaniam et al.) (Loh et al., 2008), since VIP is contained in the neuronal projections emanating from the SCN and projecting to the PVN, a hypothalamic nucleus important for transmitting circadian signal to the HPA axis
(Dai et al., 1997; Teclemariam-Mesbah et al., 1997). While the PVN contain neurons that regulate the release of several key hormones from the pituitary, it is also known to be an important central site for integration of sympathetic nerve activity (Swanson and Kuypers, 1980; Swanson and Sawchenko, 1980). Indeed, a subset of parvocellular neurons of the PVN project directly to the dorsal vagal complex and to the spinal cord (Swanson and Kuypers, 1980). Hence, while the activation of Y6 receptors on VIP neurons could influence energy metabolism through hormonal changes, it could also influence peripheral energy balance through both the autonomic nervous system. Additionally, VIP has been shown to promote corticosteroid production and secretion (Alexander and Sander, 1994;
Bodnar et al., 1997; Cunningham and Holzwarth, 1988) and diurnal variation in
ACTH and corticosterone levels were lost in VIP-deficient animals (Loh et al.,
2008). Thus, impairment in function of the growth hormone axis as well as impaired circadian rhythmicity resulting from defective neuronal – possibly VIP
– signalling pathways in the SCN could have contributed to the reduction in lean body mass and the increased predisposition for elevated adiposity in Y6-/- mice.
96 This study provides in vivo evidence that Y6 receptors are not involved in the regulation of food intake, because absolute food intake was unaltered by Y6 deletion under a variety of circumstances (fasting-induced and spontaneous food intake on a chow or high fat diet, as well as cumulative food intake measured from the age of 16 to 23 weeks). These findings extend previous pharmacological studies which suggested that Y6 was not involved in the regulation of appetite (Iyengar et al., 1999; Lin et al., 2005; Mullins et al., 2000).
Although I showed that the anorexigenic hormone PP activated neurons in the hypothalamic SCN via a Y6 receptor-dependent process, this PP-Y6-SCN pathway does not appear to influence food intake; rather it may mediate other physiological effects of PP. PP is produced in pancreatic islets and its postprandial release into the circulation is associated with the promotion of satiety (Asakawa et al., 2003; Ueno et al., 1999). Furthermore, administration of exogenous PP, or transgenic over-expression of PP in mice, is known to reduce body weight and adiposity in association with reductions in feeding and gastric emptying as well as an increase in energy expenditure (Asakawa et al., 1999;
Asakawa et al., 2003; Katsuura et al., 2002; Moriya et al., 2010; Ueno et al.,
1999; Zhang et al., 2010c). I propose that the PP-Y6-SCN pathway may mediate the anti adipogenic effect of PP. In the absence of Y6 receptors, these functions of PP are blunted, thereby leading to the impairment in long-term homeostatic regulation of energy balance, explaining the late-onset or exacerbated diet- induced increase in adiposity of Y6-/- mice. Although it cannot be ruled out that
PP may activate Y6 receptors on white adipose tissues, other physiological effects of central or peripheral PP are just as likely to be mediated by other Y receptors besides Y6. For instance, we recently showed that PP reduces food intake predominantly via stimulation of the anorexigenic -MSH signalling 97 pathway, and that this effect is mediated by direct action on local Y4 receptors within the hypothalamic arcuate nucleus (ARC) (Lin et al., 2009). Indeed, it is not uncommon for different effects of a Y receptor ligand to be mediated by different Y receptors, as illustrated by the distinct involvement of Y2 but not Y4 receptors on the growth hormone axis, while Y4, but not Y2, regulate the reproductive axis (Lin et al., 2007). An alternate explanation for the lack of effect of Y6 ablation on energy intake despite Y6 being activated by the anorexigenic hormone PP is the possibility that germline Y6 deletion was compensated for by the up-regulation of other Y receptors or their endogenous ligands, as has been described for Y1, Y2 or Y1Y2Y4 deletion (Lin et al., 2005;
Wittmann et al., 2005). For instance, up-regulation of endogenous PP production in response to Y6 deletion, as has been reported in Y4-/- mice (Sainsbury et al.,
2002d), might contribute to the lack of immediate obese phenotype. This might have been expected of Y6-/- mice in which PP action on Y6 was blocked, because
PP over-expressing mice exhibited a metabolic phenotype similar to that of Y6-/- mice, including lowered body weight and reduced fat mass, as well as lowered glucose-induced insulin secretion (Ueno et al., 1999). Hence, the role of Y6 receptors on the regulation of energy balance may occur through mediation of the effects of PP on adiposity but not on feeding behavior.
In summary, this work showed that the Y6 receptor is a critical regulator of energy homeostasis and body composition, with germline deletion of Y6 receptors leading to reduced body weight, inhibition of lean body mass and an age-dependent increase in adiposity that is exacerbated by high fat feeding.
Although Y6 receptors are not likely to affect appetite control, these metabolic phenotypes of Y6-/- mice may occur through the influence of Y6 receptors on
98 metabolic rate, circadian rhythm and the growth hormone axis, possibly mediated at least partially by VIP signalling in the hypothalamic SCN. Under physiological conditions, Y6-mediated control of energy metabolism may occur through a PP-dependent pathway, possibly during food intake-triggered events in which PP secretion is stimulated (Adrian et al., 1978; Schwartz, 1978).
Collectively, these data suggest a model for Y6 receptor function that is illustrated on Figure 16. Release of PP from the pancreas activates signalling pathways in VIP neurons in the SCN via Y6 receptors, resulting in stimulation of the growth hormone axis, reduced adiposity and reduced metabolic rate, hence explaining the opposite changes in these parameters in Y6 receptor deficient mice. Additionally, this proposed PP-Y6-VIP signalling event also regulate circadian rhythm and glucose metabolism. Concurrently, PP stimulates α-MSH signalling pathway via Y4 receptors in the ARC to evoke an anorexigenic response in vivo (Lin et al., 2009). Future studies in mice on the function and possible clinical utility of Y receptors and their ligands as drug targets must take into account this new knowledge that Y6 receptors play an important role in the regulation of energy homeostasis.
99
Figure 16. Schematic representation Y6 receptor signalling in the regulation of energy homeostasis. Circulating pancreatic polypeptide (PP) activates vasoactive intestinal peptide (VIP) signalling pathways in the suprachiasmatic nucleus (SCN) via Y6 receptors (orange arrow), resulting in stimulation of the growth hormone axis, reduced adiposity and reduced metabolic rate (red arrows), hence explaining the opposite changes in these parameters in Y6 receptor deficient mice. Additionally, this proposed PP-Y6-VIP signalling event also regulates circadian rhythm and glucose metabolism (blue arrows). Concurrently,
PP stimulates α-MSH signalling pathway via Y4 receptors in the ARC (purple 100 arrow) to evoke an anorexigenic response in vivo. The blue and orange clouds represent factors that were activated and inhibited, respectively, by either Y4 or Y6 receptor signalling
101
FUTURE DIRECTIONS
102 The overall goal of this thesis was to determine the function of Y6 receptors in the regulation of energy homeostasis in mice. Through a series of experiments involving metabolic characterization of Y6 knockout mice, in vivo pharmacological assays and immunohistochemical studies, I established several key foundations that describe the role of Y6 receptors in mice and lead to new avenues to be pursued in future work.
Y6 receptors and circadian regulations
The current study reveals that Y6 receptors are distributed in the hypothalamic
SCN, an area of the brain that is implicated in the regulation of circadian rhythms and is associated with effects on metabolic control. In fact, deletion of Y6 receptors in the mouse led to reduced circadian oscillations in serum corticosterone and reduced light-phase feeding, pointing towards circadian dysregulation that may contribute towards the metabolic defects observed in Y6-/- mice. These results not only warrant further investigation into the role of Y6 receptors in the regulation of circadian rhythm, but they also call for detailed analysis of the daily fluctuations in orexigenic and anorexigenic peptides such as
NPY, AgRP, orexin, α-MSH, CCK that could contribute to the reduction in daytime feeding and overall phenotype seen in the Y6-/- mouse model.
The role of Y6 receptors in glucose homeostasis
In the mouse, absence of Y6 receptors leads to a significant improvement in
glucose homeostasis that deteriorated secondary to the development of obesity.
In light of the known effects of changes in lean body mass or fat mass to
positively or negatively influence glucose tolerance (Chen et al., 2000; Holt et
al., 2009), it is likely that the changes in glucose tolerance seen in the Y6-/-
103 model are related to the changes in their body composition. However, this does
not exclude the possibility that Y6 deficiency has direct effects on glucose
homeostasis, and this remains to be further elucidated.
The role of Y6 receptors in human
The overall goal of biomedical research ultimately lies in the improvement of human health and living condition. Hence, it is important to determine whether the Y6 receptor, which in human is expressed as a truncated GPCR, plays a functional role in this species. A metabolic characterization of a mouse model expressing humanized Y6 receptors would help shed light on the function of the human Y6 receptor, if any, and could potentially demonstrate that the RNA transcribed from this gene either by itself or translated as a shortened receptor molecule has a biological role.
104
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