The role of nitric oxide signaling in food intake; insights from the inner mitochondrial membrane peptidase 2 mutant mice

Abstract

Reactive oxygen species have been implicated in feeding control through involvement in brain lipid sensing, and regulating NPY/AgRP and pro-opiomelanocortin (POMC) neurons, although the underlying mechanisms are unclear. Nitric oxide is a signaling molecule in neurons and it stimulates feeding in many species. Whether reactive oxygen species affect feeding through interaction with nitric oxide is unclear. We previously reported that Immp2l mutation in mice causes excessive mitochondrial superoxide generation, which causes infertility and early signs of aging. In our present study, reduced food intake in mutant mice resulted in significantly reduced body weight and fat composition while energy expenditure remained unchanged. Lysate from mutant brain showed a significant decrease in cGMP levels, suggesting insufficient nitric oxide signaling. Thus, our data suggests that reactive oxygen species may regulate food intake through modulating the bioavailability of nitric oxide.

Keywords: ADSC, adipose-derived stromal cells; AMPK, AMP-activated protein kinase.; AgRP, agouti related protein; CART, cocaine- and amphetamine-regulated transcript; CYC1, cytochrome c1; Energy expenditure; Food intake; GPD2, mitochondrial glycerol phosphate dehydrogenase; Immp2l; Immp2l, IMP2 inner mitochondrial membrane peptidase-like; Mutant mice; NO, nitric oxide; NOS, nitric oxide synthase; NPY, neuropeptide Y; Nitric oxide; POMC, pro-opiomelanocortin; ROS, reactive oxygen species; Superoxide; UCP2, uncoupling protein 2; cGMP, cyclic guanosine monophosphate.

Graphical abstract

Fig. 1

Reduced body weight and adiposity of Immp2l mutant mice. (A) Body weight of female mice at different ages. Ad-lib indicates fed ad libitum. (B) Body weight of male mice. (C) Body weight and fat composition of 16-month-old female mice. Total fat composition was determined by MRI. Means±SEM. for n≥5 animals per group are represented. *, ** and ***, p<0.05, p<0.01 and p<0.0001 respectively by Bonferroni post-tests ((A), (C) and (D)) and Tukey post-tests (B) following ANOVA.

Fig. 2

Reduced food intake in mutant mice. (A) Accumulative food intake during 24 h. (B) Comparing food intake in 4-h blocks. (C) Number of meals in 4-h blocks. (D) Time spent on feeding in 4-h blocks. (E) Food intake of fasted mice during 9:00−13:00. (F) Food intake of fasted mice during 16:00−20:00. (G) Quantitative RT-PCR revealed similar hypothalamic expression of genes involved in feeding control. 4-month-old female mice were assayed. Means±SEM for n≥5 animals per group are represented. *, ** and ***, p<0.05, p<0.01 and p<0.0001, respectively, by Bonferroni post-tests following ANOVA.

Fig. 3

Pair-feeding equalized the body weight and fat composition of control and mutant mice. (A) Body weight and adiposity of mutant mice and normal mice fed the food consumed by mutant mice. MRI was conducted on female mice at the age of 6 months (n=5 for each group). (B) Fewer and smaller ADSC colonies were obtained from fat tissue of mutant mice. (C) Fewer cells could be obtained from mutant ADSC after in vitro culture. For (B) and (C), normal mice were pair-fed. Data represented three pairs of mice assayed at the age of 22−25 months.

Fig. 4

Normal energy expenditure of mutant mice. (A) Similar body weight of mice in metabolism analysis. (B) Mutant mice have reduced respiratory exchange ratio (RER). (C) Profile of oxygen consumption. (D) Profile of carbon dioxide production. (E) Profile of total energy expenditure. (F) Total energy expenditure during light and dark cycle. (G) Profile of ambulatory activity. (H) Total ambulatory activity during light and dark cycle. (I) Rectal temperature. Means±SEM for n≥5 animals per group are represented. *, ** and ***, p<0.05, p<0.01 and p<0.0001, respectively, by Bonferroni post-tests following ANOVA.

Fig. 5

Analysis of NO signaling and AMPK phosphorylation. (A) Mutant mice have decreased hypothalamic cGMP concentration. (B) Quantitative RT-PCR analysis of hypothalamic expression of genes encoding nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC). (C) Mutant mice had decreased free hypothalamic nitrate and nitrite content. Means±SEM for n≥5 animals per group are represented. (D) Western blotting analysis of AMPK phosphorylation. (E) Relative expression of phosphorylated AMPK by integrated density of ImageJ software. For (A)−(C) and (E), * and **, p<0.05 and p<0.01, respectively, by t-tests.

Fig. 6

Immp2l mutation in ob/ob mice did not significantly affect their obesity. Picture was taken on male mice at the age of 9 weeks.