Y1 and Y5 Receptors Are Both Required for the Regulation of Food Intake and Energy Homeostasis in Mice

Neuropeptide Y (NPY) acting in the hypothalamus is one of the most powerful orexigenic agents known. Of the five known Y receptors, hypothalamic Y1 and Y5 have been most strongly implicated in mediating hyperphagic effects. However, knockout of individual Y1 or Y5 receptors induces late-onset obesity – and Y5 receptor knockout also induces hyperphagia, possibly due to redundancy in functions of these genes. Here we show that food intake in mice requires the combined actions of both Y1 and Y5 receptors. Germline Y1Y5 ablation in Y1Y5−/− mice results in hypophagia, an effect that is at least partially mediated by the hypothalamus, since mice with adult-onset Y1Y5 receptor dual ablation targeted to the paraventricular nucleus (PVN) of the hypothalamus (Y1Y5Hyp/Hyp) also exhibit reduced spontaneous or fasting-induced food intake when fed a high fat diet. Interestingly, despite hypophagia, mice with germline or hypothalamus-specific Y1Y5 deficiency exhibited increased body weight and/or increased adiposity, possibly due to compensatory responses to gene deletion, such as the decreased energy expenditure observed in male Y1Y5−/− animals relative to wildtype values. While Y1 and Y5 receptors expressed in other hypothalamic areas besides the PVN – such as the dorsomedial nucleus and the ventromedial hypothalamus – cannot be excluded from having a role in the regulation of food intake, these studies demonstrate the pivotal, combined role of both Y1 and Y5 receptors in the mediation of food intake

Unaltered energy expenditure and decreased physical activity in chow-fed mice with adult-onset hypothalamus-specific Y1Y5 receptor deletion.

<p>(A–L) 24-hour time course of energy expenditure (A–D), physical activity (E–H) and respiratory exchange ratio (I–L) in male and female adult-onset hypothalamus-specific Y1Y5 receptor knockout (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice at 19 and 36 weeks of age. Energy expenditure was adjusted for lean mass by analysis of covariance (ANCOVA). Adjusted energy expenditure was presented at a common lean mass of 21.629 g (males) and 16.252 g (females) at 19 weeks of age and 22.762 g (males) and 18.865 g (females) at 36 weeks of age. Open and filled horizontal bars indicate light and dark phases, respectively. Plotted values are means ± SEM of 5–12 standard chow-fed mice per group. *P<0.05 or **P<0.01 for Y1Y5^Hyp/Hyp versus Y1Y5^lox/lox mice of the same age and sex.</p

Reduced ARC NPY expression and increased body weight in young Y1Y5^−/− mice on chow.

<p>(A–B) Bright field photomicrographs of coronal brain sections from 15 week old male wildtype control (WT, A) and germline Y1Y5 receptor knockout (Y1Y5^−/−, B) mice after <i>in situ</i> hybridisation for NPY mRNA. Scale bar  = 25 µm. Abbreviations: 3V; third ventricle, ARC, arcuate nucleus of the hypothalamus. (C–D) Body weight from 5 to 15 weeks of age in male (C) and 5 to 13 weeks of age in female (D) WT or Y1Y5^−/− mice. Plotted values are means ± SEM of 4–21 standard chow-fed mice per group. ***P<0.001 for Y1Y5^−/− versus WT mice of the same age and sex.</p

No effect of adult-onset hypothalamus-specific Y1Y5 receptor deletion on energy homeostasis on a chow diet.

<p>(A–H) Spontaneous (A, C, E, G) and accumulated (accum) 24-hour fasting-induced food intake (B, D, F, H), normalised to body weight, in adult-onset hypothalamus-specific Y1Y5 receptor deficient (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice at 18 and 34 weeks of age. (I–J) Body weight from 12 to 36 weeks of age in male (I) and female (J) Y1Y5^Hyp/Hyp or Y1Y5^lox/lox control mice. ‘PVN injection’ refers to the time of induction of gene deletion by injection of a Cre-recombinase-expressing viral vector into the paraventricular nucleus of the hypothalamus. (K–P) Fat and lean masses as a percentage of body weight (%BW), measured using dual energy x-ray absorptiometry, in male (K, O) and female (M, P) Y1Y5^Hyp/Hyp and Y1Y5^lox/lox mice at 19, 27 and 36 weeks of age. Weight (as %BW) of dissected white (WAT) and brown adipose tissue (BAT) depots at the end of the study in male (L) and female (N) Y1Y5^Hyp/Hyp and Y1Y5^lox/lox mice at 36 weeks of age. Abbreviations: i, inguinal; g, gonadal; m, mesenteric; r, retroperitoneal; total, summed weight of i, g, m and r WAT depots. Plotted values are means ± SEM of 5–12 standard chow-fed mice per group.</p

Altered blood glucose responses to glucose and insulin after adult-onset hypothalamus-specific Y1Y5 receptor deletion.

<p>(A–H) Intraperitoneal glucose tolerance tests (1 g/kg) were conducted in 24-hour fasted male (A, B, E, F) and female (C, D, G, H) adult-onset hypothalamus-specific Y1Y5 receptor deficient (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice at 18 or 35 weeks of age. (I–P). Intraperitoneal insulin tolerance tests (0.5 U/kg) were conducted in 6-hour fasted male (I, J, M, N) and female (K, L, O, P) Y1Y5^Hyp/Hyp or control Y1Y5^lox/lox mice at 18 or 35 weeks of age. Plotted values are means ± SEM of 5–12 chow or high fat diet (HFD)-fed mice per group. *P<0.05 or **P<0.01 for Y1Y5^Hyp/Hyp versus Y1Y5^lox/lox mice of the same age, sex and diet.</p

Effect of adult-onset hypothalamus-specific Y1Y5 receptor deletion on weight regain after a 24-hour fast.

<p>Body weight measurements were taken at the time points of fasting-induced food intake measurements in male (A, C, E, G) and female (B, D, F, H) adult-onset hypothalamus-specific Y1Y5 receptor knockout (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice, at both 17 and 34 weeks of age and on a standard chow (Chow) or a high fat diet (HFD). Data are represented as a percent of pre-fasting body weight (%basal). Absolute pre-fasting body weights at 17 weeks of age were Y1Y5^lox/lox male  = 25.4±0.6 g (standard chow), 28.0±0.6 g (HFD); Y1Y5^−/− male  = 26.1±0.9 g (standard chow), 28.9±0.9 g (HFD); Y1Y5^lox/lox female  = 20.1±0.5 g (standard chow), 21.6±1.0 g (HFD); Y1Y5^−/− female  = 20.1±0.6 g (standard chow), 21.6±0.8 g (HFD), and at 34 weeks of age were Y1Y5^lox/lox male  = 30.0±1.3 g (standard chow), 34.0±1.5 g (HFD); Y1Y5^−/− male  = 31.2±1.6 g (standard chow), 40.2±1.6 g (HFD); Y1Y5^lox/lox female  = 24.7±0.7 g (standard chow), 27.9±1.5 g (HFD); Y1Y5^−/− female  = 27.1±2.0 g (standard chow), 29.9±1.3 g (HFD). Plotted values are means ± SEM of 4–21 mice per group. *P<0.05 for Y1Y5^Hyp/Hyp versus Y1Y5^lox/lox mice of the same age, sex and diet.</p

Increased body weight/adiposity despite reduced food intake in Y1Y5^−/− mice on chow.

<p>(A–H) Spontaneous (A, C, E, G) and accumulated (accum) 24-hour fasting-induced food intake (B, D, F, H), normalised to body weight, in male and female germline Y1Y5 receptor knockout (Y1Y5^−/−) and wildtype control (WT) mice at 18 and 34 weeks of age. (I–J) Body weight from 16 to 36 weeks of age in male (I) and female (J) Y1Y5^−/− or WT mice. (K–P) Fat and lean masses as a percentage of body weight (%BW), measured using dual energy x-ray absorptiometry, in male (K, O) and female (M, P) Y1Y5^−/− and WT mice at 20 and 36 weeks of age. Weight (as %BW) of dissected white (WAT) and brown adipose tissue (BAT) depots at the end of the study in male (L) and female (N) Y1Y5^−/− and WT mice at 36 weeks of age. Abbreviations: i, inguinal; g, gonadal; m, mesenteric; r, retroperitoneal; total, summed weight of i, g, m and r WAT depots. Plotted values are means ± SEM of 5–12 standard chow-fed mice per group. *P<0.05, **P<0.01 or ***P<0.001 for Y1Y5^−/− versus WT mice of the same age and sex.</p

Obesity despite hypophagia after adult-onset hypothalamus-specific Y1Y5 deletion in mice on a high fat diet.

<p>(A–H) Spontaneous (A, C, E, G) and accumulated (accum) 24-hour fasting-induced food intake (B, D, F, H), normalised to body weight, in adult-onset hypothalamus-specific Y1Y5 receptor deficient (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice at 18 and 34 weeks of age. (I–J) Body weight from 12 to 36 weeks of age in male (I) and female Y1Y5^Hyp/Hyp or Y1Y5^lox/lox mice. Mice were on the high fat diet (HFD) from 16 weeks of age onwards. ‘PVN injection’ refers to the time of induction of gene deletion by injection of a Cre-recombinase-expressing viral vector into the paraventricular nucleus of the hypothalamus. (K–P) Fat and lean masses as a percentage of body weight (%BW), measured using dual energy x-ray absorptiometry, in male (K, O) and female (M, P) Y1Y5^Hyp/Hyp and Y1Y5^lox/lox mice at 19, 27 and 36 weeks of age. Weight (as %BW) of dissected white (WAT) and brown adipose tissue (BAT) depots at the end of the study in male (L) and female (N) Y1Y5^Hyp/Hyp and Y1Y5^lox/lox mice at 36 weeks of age. Abbreviations: i, inguinal; g, gonadal; m, mesenteric; r, retroperitoneal; total, summed weight of i, g, m and r WAT depots. Plotted values are means ± SEM of 5–12 HFD-fed mice per group. *P<0.05 or **P<0.01 for Y1Y5^Hyp/Hyp versus Y1Y5^lox/lox mice of the same age and sex.</p

Slower weight regain after a 24-hour fast in Y1Y5^−/− mice on chow.

<p>Body weight measurements were taken at the time points of fasting-induced food intake measurements in male (A, C) and female (B, D) germline Y1Y5 receptor knockout (Y1Y5^−/−) or wildtype control (WT) mice, at both 18 and 34 weeks of age. Data are presented as a percent of pre-fasting body weight (%basal). Absolute pre-fasting body weights at 18 weeks of age were WT male  = 25.4±0.6 g; Y1Y5^−/− male  = 29.3±1.8 g; WT female  = 19.7±0.5 g; Y1Y5^−/− female  = 20.1±0.4 g, and at 34 weeks of age, WT male  = 30.1±1.3 g; Y1Y5^−/− male  = 35.5±1.2 g; WT female  = 24.5±0.7 g; Y1Y5^−/− female  = 25.0±0.9 g. Plotted values are means ± SEM of 4–21 standard chow-fed mice per group. **P<0.01 or ***P<0.001 for Y1Y5^−/− versus WT mice of the same age and sex.</p

Increased energy expenditure/physical activity in high fat-fed mice with adult-onset hypothalamus-specific Y1Y5 receptor deletion.

<p>(A–L) 24-hour time course of energy expenditure (A–D), physical activity (E–H) and respiratory exchange ratio (I–L) in male and female adult-onset hypothalamus-specific Y1Y5 receptor knockout (Y1Y5^Hyp/Hyp) or control (Y1Y5^lox/lox) mice on a high fat diet (HFD) at 19 and 36 weeks of age. Energy expenditure was adjusted for lean mass by analysis of covariance (ANCOVA). Adjusted energy expenditure was presented at a common lean mass of 20.741 g (males) and 15.607 g (females) at 19 weeks of age and 21.636 g (males) and 17.320 g (females) at 36 weeks of age. Open and filled horizontal bars indicate light and dark phases, respectively. Plotted values are means ± SEM of 5–12 HFD-fed mice per group. *P<0.05 or ***P<0.001 for Y1Y5^Hyp/Hyp versus Y1Y5^lox/lox mice of the same age and sex.</p