Neuropeptide Y resists excess loss of fat by lipolysis in calorie-restricted mice: a trait potential for the life-extending effect of calorie restriction

Abstract

Neuropeptide Y (NPY) is an orexigenic peptide that plays an essential role in caloric restriction (CR)-mediated lifespan extension. However, the mechanisms underlying the NPY-mediated effects in CR are poorly defined. Here, we report that NPY deficiency in male mice during CR increases mortality in association with lipodystrophy. NPY^-/- mice displayed a rapid decrease in body weight and fat mass, as well as increased lipolysis during CR. These alterations in fat regulation were inhibited by the lipolysis inhibitor, acipimox, a treatment associated with reduced mortality. The lipolytic/thermogenic signaling, β3-adrenergic receptor/hormone sensitive lipase, was markedly activated in white adipose tissue of NPY^-/- mice compared with that of NPY^+/+ mice, and thermogenesis was controlled by NPY under negative energy balance. These results demonstrate the critical role of NPY in the regulation of lipid metabolic homeostasis and survival via control of lipolysis and thermogenesis in a state of negative energy balance.

Keywords: adipose tissue; aging; calorie restriction; lipolysis; neuropeptide Y.

Figure 1

NPY deficiency increases mortality with reduction in body weight in CR mice, which is inhibited by ACM treatment. (A) mRNA expression level of Y1R was measured by qPCR in WAT. (B) Survival rate was measured by the log‐rank test (n = 18, upper panel). The dotted line indicates the percent survival at 400 days. (C) Food intake, (D) body weight, and (E–G) change of body weight were measured in NPY ^+/+ and NPY ^−/− mice. All data are presented as the mean ± SEM, n = 12–18 per group; *P < 0.05 and ***P < 0.001.

Figure 2

NPY ^−/− mice exhibit reduction in fat mass independent of adipogenic regulation during CR, which is inhibited by ACM treatment. (A, B) Percentage of total, visceral, and subcutaneous adipose tissue was determined by 3D micro‐CT. (C) mRNA expression levels of adiponetic and lipogenic genes were measured by qRT–PCR in eWAT. All data are presented as the mean ± SEM, n = 9–11 per group; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 3

NPY ^−/− mice exhibit reduction in adipocyte size during CR, which is inhibited by ACM treatment. Representative images from immunohistochemistry for H&E (hematoxylin and eosin) stain in eWAT and iWAT (upper panel), and the mean sizes of adipocytes (bottom panel). All data are presented as the mean ± SEM, n = 6 per group; *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 4

NPY deficiency increases p‐HSL (ser563), but it is inhibited by ACM in WAT of CR mice. Protein level of p‐HSL (ser563), HSL, ATGL, and CGI‐58 was measured by Western blotting in (A) eWAT and (B) iWAT of same mice (left panel) and quantified (right panel). All data are presented as the mean ± SEM; n = 6 per group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Figure 5

NPY deficiency increases Adrb3 expression, but it is moderately inhibited by ACM in WAT of CR mice. (A–C) mRNA expression level of Adrb1, 2, 3 in iWAT of same mice. (D–F) Correlation of Adrb1, 2, 3 mRNA levels in iWAT with WAT/BW ratio. mRNA expression of genes encoding (G) M1, M2 macrophage markers and (H) thermogenesis was measured by qRT–PCR in iWAT. All data are presented as the mean ± SEM; n = 5–6 per group. *P < 0.05 and ***P < 0.001.

Figure 6

Administration of NPY reduces thermogenesis in NPY ^−/− mice under fasted status. (A) Body temperature (B) mRNA expression level of Ucp1 in iWAT and iBAT. (C) NPY deficiency enhances lipolytic action through activation of Adrb3‐HSL signaling pathway, and ACM inhibits them under negative energy balance. NPY deficiency increases mortality accompanied by BW reduction and fat loss, and it may be due to the increase in lipolysis‐mediated thermogenic action. All data are presented as the mean ± SEM; n = 6 per group. *P < 0.05; ^### P < 0.001 vs. NPY ^−/− +Sa.