Quick Notes: Neuropeptidy Y, calorie restriction and aging (Draft)

Here's a short and unpolished intro to this topic. We're going to discuss the following article:

Sci Rep. 2014 Mar 31;4:4517. doi: 10.1038/srep04517.
A key role for neuropeptide Y in lifespan extension and cancer suppression via dietary restriction.
Chiba et al.

Sample size is this study's major weaknes
sample size WT-AL (n=48), NPY-AL (n=25), WT-CR (n=42), NPY-CR (n=24).
"As suspected from the survival curves, WT and Npy−/− mice seemed to respond differently to the DR diet in terms of lifespan extension (p = 0.0578 [Genotype × Diet])....The small numbers of mice in the longevity study also limit our conclusions."
The rule for a successful study should be 40-50 mice per group. Unfortunately, the most important groups in this study were the smallest.

A second huge problem is the lifespan (LS) of controls:
The strain used was 129S1/SvImJ or closely related. Median lifespan of their mice was ~840d, single outlier maximum LS below 1000d. A good rule is "900/1200" for healthy mice - 900 days mean LS and 1200d for 10%-survivorship. All other studies can be considered as using unhealthy or short-lived (strains of) mice.

In this particular instance, mean lifespan matches or surpasses values reported for the 129S1/SvImJ strain (approx. 820-880) (ref. 1). Maximum lifespan, on the other hand, is unacceptably low both compared to the vigorous black 6 strain as well as 129S1 mice kept by others (2). Mean lifespan of the last 20% surviving mice in a well kept colony of 129S1/SvImJ mice has been reported to be above 1000d (2).

Strain background
"A limitation of this study is the fact that the genetic backgrounds of the Npy/− (Npytm1Rpa/J, approximate to 129S1/SvImJ) and WT (129S6/SvEvTac) mice differed..."

Now, given those limitations, we can discuss the study, but the data is seriously called into question. The NPY hypothesis is sound, as we know from the introduction, but this paper does not make the most convincing case in favour of it.

A. Results in more Detail
NPY KO almost completely abrogated the effects of CR on cancer and lifespan.
Pathology report: lymphoma and hepatic cancer, but not lung cancer, were reduced by CR. This effect was completely negated by NPY KO.

No effect on Insulin, IGF1, mTOR signalling, leptin, adiponectin, glucocorticoids or body temperature. Amazing! Could it be that none of these matter for the lifespan extension by CR? Let's not get too excited yet.

Effects on stress resistance
Liver microarray data suggests that NPY KO reduces expression of genes involve in stress resistance and particularly xenobiotic metabolism.

This was confirmed in vivo:
"We assessed survival rates of 6-month-old male mice from different experimental groups subjected to oxidative stress induced by administration of 3-nitropropionic acid, an inhibitor of mitochondrial respiratory complex II...WT-DR mice exhibited notable stress resistance in comparison to WT-AL mice, as indicated by the significantly increased survival rate (p = 0.0009 by log-rank test[...]). The DR effect was diminished in Npy/− mice (p = 0.0612 by log-rank test[...]); therefore, Npy may also be involved in the stress resistance induced by DR." [again, no significant difference due to sample size]

B. Conclusions
This study is consistent with the idea that the effect of CR is primarily dependent on NPY. It is also consistent with the idea that the effect of CR on lifespan is largely due to cancer inhibition. If this were true it would mean that pathways currently considered important are completely irrelevant (mTOR, GH/IGF, ...). Since NPY-KO abrogated the effect of CR on cancer and lifespan, but did not affect most of these biomarkers. Unfortunately, the study did not test if age-related non-cancer pathologies were decreased. It is still entirely possible that the anti-cancer effect of CR is necessary but not sufficient to extend mouse lifespan; and that this effect hinges almost entirely on NPY signalling.

Put another way: chemoprevention may be dependent upon NPY, but the anti-aging effect of CR could be independent.

Finally, this paper considerably strengthens the hypothesis that "multi-stress resistance" is central to lifespan extension, at least in rodents. The study does not completely rule out other mechanisms due to its small sample size and the peculiarities of mice as an aging model.

C. How does NPY mediate this effect?
The authors write that "Npy could act in the liver via the sympathetic nerves and the circulation.", but give no explanation and provide no citations.
Is it via circulating Neuropeptide Y? Another circulating endocrine mediator whose release is triggered by NPY? NPY as a neurotransmitter in the autonomic nervous system?

Given the effects on "stress resistance" related genes, one has to wonder if NPY and NRF2 signalling are interlinked (c.f. ref. 3 which I have not read in full).

Aging Cell. 2009 Jun;8(3):277-87. doi: 10.1111/j.1474-9726.2009.00478.x. Epub 2009 Apr 9.
Aging in inbred strains of mice: study design and interim report on median lifespans and circulating IGF1 levels.
Yuan R, Tsaih SW, Petkova SB, Marin de Evsikova C, Xing S, Marion MA, Bogue MA, Mills KD, Peters LL, Bult CJ, Rosen CJ, Sundberg JP, Harrison DE, Churchill GA, Paigen B.

2. ILAR J. 2011;52(1):4-15.
Mice as a mammalian model for research on the genetics of aging.
Yuan R1, Peters LL, Paigen B.

3. Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2325-30. doi: 10.1073/pnas.0712162105. Epub 2008 Feb 19. Nrf2 mediates cancer protection but not prolongevity induced by caloric restriction. Pearson et al.