Mittwoch, 27. April 2016

Iron: focus on epidemiology and current studies relevant to CVD and all-cause mortality

Although, I believe that iron is more of a player in carcinogenesis than cardiovascular disease (CVD) I would like to do a brief review of the latter anyway. So far, the best evidence suggests that modest iron excess may promote atherosclerosis but not other types of CVD. But does it really?

1. Ferritin associates with all-cause mortality - does it matter?

2. Evidence against the iron-atherosclerosis hypothesis: ferritin & CVD incidence
3. Hemochromatosis: a special casse

4. Special epidemiology - beyond ferritin and CVD incidence

5. What are the problems with epidemiology?
6. The solution: best biomarker, best study design

7. Other studies of interest - biogerontology
 
8. References


1. Ferritin associates with all-cause mortality - does it matter?
A recent study by Ellervik et al. (2014) found a positive association of ferritin with total mortality (n~9000, 23y follow-up). They also performed a "meta-analysis", but in the end only three studies were included (4).
"...we conducted a meta analysis of our own study and 2 additional previous population-based studies on risk of increased total mortality according to ferritin concentrations in quartiles or tertiles, indirectly testing the hypothesis that using quartiles or tertiles for risk prediction hides an increased risk because the highest quartiles or tertiles often include part of the reference interval for ferritin concentrations, thereby diluting risk estimates."

Indeed, given these tertiles any significant result is unlikely using this approach:
"lowest tertile T1 (range 1–55 ug/L), second tertile T2 (56–126 ug/L), and highest tertile T3 (127–1524 ug/L)."
The normal range is 12-150 ng/mL or up to 300 for males. For comparison, male participants in the largest iron reduction study ever performed reached 50-70ng/ml and lower seemed better (10).

The critical discussion of the Ellervik study by several letters and editorials reminded me just how complicated iron-CVD epidemiology is (4a-4e). Below follow a few key Points.

Montag, 25. April 2016

Life extension - Calories or protein?

There are some major controverises left in the calorie restriction (CR) field: modest or null results in wild-dervied mice and ILSXISS strains, human epidemiology, rhesus monkey studies and recently CR vs. protein restriction (PR) (cf. IGF1 vs other pathways).

Recently, people started to wonder if protein restriction may drive the effect of CR in rodents. Speakman et al. provide a beautifully succint review and meta-analysis of the CR vs PR debate. A must read for everyone in the field, since Speakman is generally known for his diligent work.

Some background: Calorie restriction (CR) was discovered in the 1920s but Clive McCay's rigoros studies from the 30s are credited with starting the field of biogerontologic CR. Nevertheless the question whether CR or PR is more important was unresolved at that time, but after a few decades the consensus emerged that CR is not due to protein restriction. Many relevant papers were published including those by Ross et al. in the 1960s and Masoro et al. in the 1980s. However, when life extending essential amino acid restriction was discovered in the late 80s/early 90s, and secondly Linda Partridge (and many others) showed that insect lifespans are more responsive to PR than CR, this reignited the debate about mammalian CR vs PR.
Recent work on mice has involved varying the protein to carbohydrate ratio in ad libitum fed mice and then plotting their responses including lifespan in the context of a two-dimensional framework defined by the balance of intake of protein to non-protein components ( Solon-Biet et al., 2014). This massive experiment involved feeding mice 25 different diets that varied in their protein to non-protein ratios, and following cohorts of them until they died. The data clearly [and controversially] indicated that the key factor influencing longevity, as was observed in insects, was the protein to non-protein (carbohydrate) ratio in the diet. As the protein to carbohydrate ratio declined, lifespan increased, recapitulating the same finding detailed above (Ross, 1961).
To rephrase, Solon-Biet claim that protein restriction was the exclusive determinant of lifespan and not CR. This claim will be challenged by Speakman et al.

Methods. A shout-out to the free science crowd:
Occasionally (n = 4 papers) we could not extract data on the diet compositions, either because the full text of the papers were hidden behind pay-walls, or the specialised diet manufacturer appeared to be no longer in business, and the paper only cited the source of the diet rather than its composition. 
Speakman excluded studies with a null result in their analysis because they seek to explain changes in lifespan across macronutrient levels. There is nothing to explain if the experiment didn't work, even if these experiments have some relevance to CR per se. So they excluded the important studies on wild-derived mice and in the ILSXISS strains.

Findings:
The idea was to derive a dose-response curve for CR w/o PR and contrast it with a curve for CR + PR.
The effect of ‘CR with PR’  [n~67]on rodent lifespan does not differ from the effect of ‘CR without PR’ [n~8]... the overlap of the fitted regressions was striking, and it is clear that this is not a situation where there is a trend for an effect that fails to reach significance because of the high variation in the responses within treatments.

Speakman and colleagues describe the problem with Solon-Biet et al. (2014) which is that in their hands 30% CR shortended lifespan in the B6 strain, in contrast with the vast majority of all studies ever performed (dozens!) Intriguingly, Solon-Biet et al. achieved CR via cellulose addition which could have reduced hunger signalling which is essential to the life extension effects. Thus it seems that Speakman et al. speculate along the same lines as I did when I first discussed Solon-Biet after a first glance review. Furthermore, "the mice ingested fewer calories, they did so while ingesting almost twice as much mass of food". Hence I would speculate that excess cellulose could be harmful, but I'm not sure about the natural cellulose content of mouse diets, much less if natural is optimal (4).

A second quick glance at the literature makes me wonder again if there is something wrong with the Solon-Biet paper. Kokkonen et al (1988) for instance claim that dietary dilution via cellulose extends lifespan (I have no full access to ref. 2 using two different university accounts, who doesn't love paywalls?). A review of all studies using cellulose to induce CR might be useful esp. when combined with macronutrient data.

"Effect of percentage protein composition of the diet on lifespan" in ad libitum animals
If 18-26E% protein is taken as the baseline an extension of LS is seen especially with lower intakes of protein. The lowest studied intake was 4E% of protein.

The authors believe that:
...the impact of CR plus PR on lifespan is a completely different phenomenon from PR alone. Hence, the effect of reducing food intake on lifespan in rodents acts only via the restriction of calorie intake. This conclusion is consistent with the fact that the morphological, physiological and behavioural responses of mice to CR with PR (between 0 and 40% restriction) are completely different to the responses of mice to PR alone over the equivalent range (20% down to 12% protein) ...
it is also clear that there is an independent impact of dietary protein reduction on lifespan, but it operates over a different range of restriction (50 to 85%: relative to a reference intake of 18–26% protein in the diet) than that over which CR is effective (10–65% relative to ad libitum intake), and has a much smaller impact. Hence, reducing protein levels by 80% (from 20% to 4%) increases median lifespan by about 15%, while reducing calories by half this amount (40%) increases median lifespan by on average twice as much (30%). 
The finding is a little surprising if I am reading it correctly, because PR seems to be exclusively beneficial under ad libitum conditions. This non-additivity is confusing: if CR and PR both led to a graded response via e.g. mTOR or IGF-1 reduction we would expect them to be additive. However, it is interesting to note that acute CR leads to a decrease in IGF-1 proportional to the reduction in calories (5), but protein restriction does not.

And a further caution against taking invertebrate studies too seriously:
...[there may be] a major methodological difference between studies on insects and rodents. In particular, rodents are generally provided with less food when on restriction, but insects are primarily manipulated by giving them food diluted with indigestible components, and hence, paradoxically, despite being called on ‘restriction’, they eat a greater mass of food.
A cautious conclusion: low/reduced protein is probably still beneficial as are other macros
First of all, there is evidence that reduced n3 fatty acid intake is relevant to CR in rodents, which was acknowledged by Speakman. Unfortunately, there was only study so far. Furthermore, the Situation in rodents is different from humans. In rodents CR leads to reduced IGF1 but in humans moderate protein restriction is absolutely essential for this reduction (cf Fontana work).

References
1. Speakman, J. R., Mitchell, S. E., & Mazidi, M. (2016). Calories or protein? The effect of dietary restriction on lifespan in rodents is explained by calories alone. Experimental Gerontology.

2.
Kokkonen, G. C., & Barrows, C. H. (1988). The effect of dietary cellulose on life span and biochemical variables of male mice. Age, 11(1), 7-9.

3.
Solon-Biet, S. M., McMahon, A. C., Ballard, J. W. O., Ruohonen, K., Wu, L. E., Cogger, V. C., ... & Gokarn, R. (2014). The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice. Cell metabolism, 19(3), 418-430.
4.
Anderson, R. L., Owens, J. W., & Timms, C. W. (1992). The toxicity of purified cellulose in studies with laboratory animals. Cancer letters, 63(2), 83-92.

5.,
Mitchell, S. E., Delville, C., Konstantopedos, P., Hurst, J., Derous, D., Green, C., ... & Lusseau, D. (2015). The effects of graded levels of calorie restriction: II. Impact of short term calorie and protein restriction on circulating hormone levels, glucose homeostasis and oxidative stress in male C57BL/6 mice. Oncotarget, 6(27), 23213-23237.

Montag, 11. Januar 2016

Cliffnotes: Molecular targets downstream of CR

A very brief list and not a comprehensive one follows. I focus on direct evidence from KO models and lifespan studies, because this is the most robust study design. I don't care for surrogate endpoints, with the exception of pathology. The most common study design can be summarized thusly: When you combine "Factor X" with CR, does this affect the magnitude of LS extension from CR? Where X is either a gain of function (e.g. other LS extension) or loss of function (usually KO) intervention.

A. Direct evidence
B. Indirect evidence
C. Speculative or parallel pathways

A. Direct evidence

GH/IGF1

Montag, 14. Dezember 2015

Optimizing resource use - Chronic toxicity and preclinical studies

This post is related to the idea of "Optimizing resource use by outsourcing of lifespan research" to pet owners and zoos.

What should we study? We're clueless!
We don't understand aging very well. That's the reason why we need to perform both naive screens with novel chemical matter (more on that below) as well as large, high-throughput screens with drugs that target plausible pathways. But what are the plausible pathways if we are clueless?

Well, it's not quite as bad as you may think, since we do have some basic paradigms in aging research. Bluntly put, there's for instance "anti-growth" as well as "mimic CR/dwarfism" (= which usually amounts to an "anti-growth" paradigm). The idea that diminished signalling through a multitude of growth pathways extends longevity has been shown to be true in scores of studies (just recently, ref. 1, 2).

Preclinical studies: how about worms and flies?
Preclinical safety studies presently include cell culture work in human cells as well as rodent models (usually rat) and non-rodent models. The latter often includes dogs and primates (5). From a basic science perspective, we should consider mandating chronic "safety" testing in invertebrates. This would be very useful to biogerontologists, as we can expect to find a reasonable number of life extending drugs by pure chance. On the other hand, it is not clear if such "safety" testing has any meaning for human toxicity. An alternative would be to compel companies to give out the drugs (in a blinded fashion to prevent IP problems) and for the government to perform lifespan testing. This method would work particularly well to test the already available drug libraries. (As of today no free market based incentive exists to develop anti-aging therapies. We won't consider the details here.)

Indeed thousands of failed drug candidates must exist and represent a large untapped source for biogerontology. Modern cancer drugs are particularly promising since they target various growth-related pathways. In 2015 "According to the Pharmaceutical Research and Manufacturers of America (PhRMA), 771 new drugs and vaccines are in development by US companies" just to treat cancer (6). It is difficult to find high quality lists with advanced drugs, but there's one up to date list for lung cancer (7). These lists may come in handy for "smaller" projects like the NIA's ITP. It might also be helpful to think in terms of drug targets and then prioritize the most plausible targets and drugs (8).

Samstag, 21. November 2015

Resources for biogerontologists


Suggestions for Animal husbandry

List of lifespan extension...
Genetic (1, 2). No good online resource or database available. "Lifespan Observations Database" is outdated
Other interventions

AnAGE database: lists maxLS for different species

(to be continued)


References
1. Ladiges W, Van Remmen H, Strong R, et al. Lifespan extension in genetically modified mice. Aging Cell. 2009;8(4):346–352
http://onlinelibrary.wiley.com/doi/10.1111/j.1474-9726.2009.00491.x/full

2. Liao, C. Y., & Kennedy, B. K. (2014). Mouse models and aging: longevity and progeria. Mouse Models of the Nuclear Envelopathies and Related Diseases, 109, 249-285.

Donnerstag, 19. November 2015

Cliffnotes: anti-aging effects GHRKO, what is the target tissue?

GHRKO - Growth hormone receptor knpck-out robustly extends lifespan, but how? Is it through lower IGF1, lowere GH, both and which tissue are the targets?

Very briefly, superficially:

Kopchick, Miller and others (1) have been running tests at two different sites to dig into an answer, but so far they haven't really uncovered any answers. So far they looked into liver, muscle and fat tissue.
An increase in survival and in maximal lifespan was detected in male MuGHRKO [muscle specific GHRKO] at UM mice, though not in a parallel experiment at OU and not in females at either test site. [OU, UM are the 2 different test sites/universities]
While removal of GH action in muscle of male mice results in features that are consistent with the hypothesis that blocking the anti-insulin activity of GH improved glucose homeostasis, the hypothesis that improved glucose homeostasis in MuGHRKO mice will improve lifespan remains questionable. However, we do know that removal of GHR in muscle did not shorten lifespan as discussed above. Since MuGHRKO mice were one of three lines simultaneously generated and studied by our laboratories, we can compare the effects of disrupting GHR in three insulin sensitive tissues (muscle, liver, and fat). Our previous work with liver- and fat-specific GHR gene disrupted mice indicates that lifespan does not always positively correlate with glucose homeostasis. For example, liver-specific disruption of the GHR (LiGHRKO) produces mice that have impaired glucose homeostasis [21, 22]. However, these mice have a normal lifespan as determined by two laboratories (OU and UM)[34]. Furthermore, fat-specific disruption of GHR (FaGHRKO) produces mice that have normal glucose homeostasis and these mice are short lived (List, Kopchick and Miller unpublished results at OU and UM). This suggests that other processes related to aging may have been altered (improved in LiGHRKO and impaired in FaGHRKO mice) to counteract the effect of glucose homeostasis on aging.
The authors state:
Collective data regarding muscle from MuGHRKO, global GHR−/−, Ames, and LiGHRKO mouse lines suggests that removing the indirect GH action, i.e. lowering IGF-1, may be more important in protection against musculoskeletal frailty.
Which to me doesn't quite add up (at a first glance). Another paper utilizing large, multi-site testing (2) found that IGF-1 itself has a small (but apprently real) effect on aging. For a few years now, I think, there has been mounting evidence that global GHRKO is somehow "better" than messing with IGF1 only. I recall quite clearly other controversial IGF1R papers (e.g. ref. 3).

I suppose lowered IGF1 could be more important in muscle, but on a whole body level there must be some beneficial effect of lowered GH.

References

1. Aging (Albany NY). 2015 Jul;7(7):500-12.
Removal of growth hormone receptor (GHR) in muscle of male mice replicates some of the health benefits seen in global GHR-/- mice.
List EO, Berryman DE, Ikeno Y, Hubbard GB, Funk K, Comisford R, Young JA, Stout MB, Tchkonia T, Masternak MM, Bartke A, Kirkland JL, Miller RA, Kopchick JJ.

2. Lorenzini A, Salmon AB, Lerner C, Torres C, Ikeno Y, Motch S, McCarter R, Sell C. Mice producing reduced levels of insulin-like growth factor type 1 display an increase in maximum, but not mean, life span. The journals of gerontology Series, A, Biological sciences and medical sciences. 2014;69:410–419.

3. Aging Cell. 2014 Feb;13(1):19-28. doi: 10.1111/acel.12145. Epub 2013 Sep 11.
Longevity effect of IGF-1R(+/-) mutation depends on genetic background-specific receptor activation.
Xu J1, Gontier G, Chaker Z, Lacube P, Dupont J, Holzenberger M.

Freitag, 30. Oktober 2015

Stress resistance, Proteostasis and Aging reviewed (2015)


Let's start with a brief literature review by Alper, Bronikowski and Harper 2015 (1). I have excerpted some particularly noteworthy passages.

CR does not lead to epigenetically stable programming, hence an ex vivo model of CR has been hard to come by:
Interestingly, cells grown from dietary models of life extension fail to show this correlation. More specifically, dermal fibroblasts from mice subjected to life-long caloric restriction (CR) or provided with a diet low in the essential amino acid methionine, were no more stress resistant to multiple cytotoxins relative to their normal-fed counterparts (Harper et al., 2006b). Caloric restriction is perhaps the most robust life-extending intervention known (Fontana and Partridge, 2015) while diets low in methionine have been repeatedly shown to increase longevity in both rats and mice (Perrone et al., 2013, Sun et al., 2009 and Miller et al., 2005). A clue to this apparent discrepancy comes from studies using conditioned media; or more specifically, cells exposed to media supplemented with serum collected from rodents undergoing CR are more stress resistant than are cells grown in the presence of normal media alone. This suggests the presence of specific circulating factors needed for the life extending effects of dietary restriction that are lost during the derivation and expansion of individual cell lines (de Cabo et al., 2003 and De Cabo et al., 2015).
Multi-stress resistance correlates with long lifespans but there are exceptions to the rule: