Short-term protein restriction with weight gain doesn't mimic CR in mice

Dean Pomerleau · August 29, 2015

Since Michael Rae posted a link on the official CR Society blog to the this series of three papers [1][2][3] from the same study on Protein Restriction (PR) vs. Calorie Restriction (CR) which James Cain posted in last week's weekly research updates (thanks James for this service!), I figured I'd take a look to see what the hubbub is about.

While it was relatively short term (3 months), it appears to be a very well-conducted and informative study. They first had a 14-day baseline period where they fed the mice ad lib a standard mice chow diet of 20% protein, 70% carbs and 10% fat (by energy). Then, when the mice reached 20 weeks of age (early adulthood in humans), they were put on the various diets. One had 24-hour ad lib access to the same baseline diet (24AL) and one group had 12-hour ad lib access to the same baseline diet (12AL), during the normal time mice eat (at night). So right off the bat this is interesting because it investigates some degree of time-restricted feeding (although over the course of the study the 24AL mice gained more weight and fat than the 12AL mice, and not much is made in the papers about differences between these two groups).

In addition, there were 5 different groups of of CR mice with 0, 10, 20, 30 and 40% lower food intake than their own individual intake measured over the 14-day baseline period. There were also three levels of protein restriction, down from the baseline 20% to 16, 14 and 12% protein, which matched the quantity of protein consumed by the 20, 30 and 40% CR mice. The diet of the protein-restricted mice was supplemented with extra carbs to bring their total calorie intake up to their individual calorie intake during the ad lib baseline period. In other words, the protein-restricted mice were eating their AL level of calories, but getting a reduced quantity of protein to matched the amount of protein in the three most restricted CR groups. So it was a pretty cool design.

Like the 12AL controls, the CR and PR groups were fed during the 12-hour dark period, although the mice in the CR groups (being hungrier) probably scarfed down their food faster than the 12AL and the PR groups, which were getting fed an ad lib quantity of food, meaning the CR mice ate during a narrower time window than the other groups...

They followed this protocol for three months and then sacrificed the mice and measured a boatload of biomarkers reported in these three papers.

Long story short, they found that compared with the 12AL control mice, the CR mice exhibited all the usual hallmarks of effective CR, including reduced body weight, fat mass and organ weight (except for digestive system weight, which was increased!), lower leptin, tumor necrosis factor-alpha, and IGF-1, improved glucose tolerance and insulin sensitivity, lower "investment in reproduction" (measured via urine protein markers) and lower body temperature. Interestingly, if anything the authors observed an improvement in bone health of the CR mice relative to the 12AL controls, which was nice to see. They said:

[O]ur results offer no evidence that three months of CR, even at the 40% level, had a negative effect on bone composition or mechanics. In fact one could conclude that there was a beneficial effect of CR on bone mass in the current study.

They report that in general the changes in biomarkers of the CR mice correlated most with changes in individual body weight (morphology changes), so the more weight the mice lost, the greater the biomarker changes they observed, (somewhat) independent of the level of CR they were subjected to. I found this interesting since it indirectly speaks to a question of interest to me, which is whether its the calories or the resulting weight loss (equivalent to net energy deficit?) that matters when it comes to CR benefits.

In contrast to the beneficial biomarker changes seen in the CR mice, the mice fed the diets with ad lib calories but restricted protein didn't lose weight, and didn't exhibit any of the biomarker changes observed in the CR groups, at least to a statistically significant degree. In fact, by the end of the 3 months, the protein restricted mice weighed significantly more, and had significantly more fat mass than even the 12AL control mice! Further, the amount of weight gain and fat mass gain was proportional to the degree of protein restriction. On the surface, this extra weight & fat gain in the PR mice relative to controls seems surprising, since the PR groups of mice were given the same number of calories as they individually consumed during the AL baseline, and the 12AL mice had free access to food during the same 12-hour period as the PR mice. Here is how the authors explained this seeming paradox:

As PR increased the animals became fatter over the three month manipulation period. Since the animals were provided with the same total calorie intake as during the baseline period and the same intake across the different PR groups, the most likely reason for this effect on fatness was that reducing the levels of protein in the diet reduced the specific dynamic action (SDA) of the diet, which is known to be greatest for the protein component [71]. Thus while gross energy intake remained constant the net metabolizable energy increased as the protein level declined. This would lead to surplus energy above requirements that the animals could deposit as fat. Since the extra fat in the 40PR group amounted to just over 1 g (39.5 kJ), this was equivalent to less than 0.3 kJ/day over the 90 day experiment and hence entirely consistent in magnitude with an alteration in the level of SDA.

The unfamiliar (to me) term Specific Dynamic Action (SDA) is another name for a more familiar concept, the Thermic Effect of Food (TEF). In short, when food is metabolized, some of the energy is wasted as heat, rather than converted to useable energy. More energy is wasted as heat when metabolizing protein as compared with carbs (or fat). Since the PR mice were eating less protein and more carbs, they were getting more useable energy from their food, which resulted in weight and fat gain over the course of the study, even relative to ad lib fed controls (which also gained a lot of weight and fat, BTW).

So the failure of this study to replicated the previous results (see this thread for some discussion) that PR can mimic many of the biomarker changes associated with CR is not as surprising as one might initially have thought. The discrepancy can be explained by the fact that the PR mice in this study gained a lot of weight and fat, and the negative effects of these gains were enough to trump and obviate any improvements in the measured biomarkers that might have otherwise been observed from protein restriction.

The fact that the PR mice weren't worse off than the AL controls with respect to these biomarkers, despite being significantly fatter, might be interpreted as weak evidence in support of the health benefits of a low protein, high carb diet, but that would be a stretch.

Instead, the major takeaway message from this one on the subject of protein restriction seems to be that becoming obese on a low protein, high (refined) carb diet is not a particularly good recipe for health or longevity. Not quite as informative a study as I'd originally hoped...

--Dean

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1. Oncotarget. 2015 Jun 30;6(18):15902-30.

The effects of graded levels of calorie restriction: I. impact of short term calorie and protein restriction on body composition in the C57BL/6 mouse.

Mitchell SE1, Tang Z1, Kerbois C1, Delville C1, Konstantopedos P1, Bruel A1, Derous D1, Green C1, Aspden RM2,Goodyear SR2, Chen L3, Han JJ4, Wang Y5, Promislow DE6, Lusseau D1, Douglas A1, Speakman JR1,5.

Author information

Abstract

Faced with reduced levels of food, animals must adjust to the consequences of the shortfall in energy. We explored how C57BL/6 mice withdrew energy from different body tissues during three months of food restriction at graded levels up to 40% (calorie restriction: CR). We compared this to the response to equivalent levels of protein restriction (PR) without a shortfall in calories. Under CR there was a dynamic change in body mass over 30 days and thereafter it stabilized. The time to reach stability was independent of the level of restriction. At the end of three months whole body dissections revealed differential utilization of the different tissues. Adipose tissue depots were the most significantly utilized tissue, and provided 55.8 to 60.9% of the total released energy. In comparison, reductions in the sizes of structural tissues contributed between 29.8 and 38.7% of the energy. The balance was made up by relatively small changes in the vital organs. The components of the alimentary tract grew slightly under restriction, particularly the stomach, and this was associated with a parallel increase in assimilation efficiency of the food (averaging 1.73%). None of the changes under CR were recapitulated by equivalent levels of PR.

KEYWORDS:

Gerotarget; body composition; calorie restriction; dietary restriction; food intake; protein restriction

PMID: 26079539 [PubMed - in process] Free full text

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Short-term protein restriction with weight gain doesn't mimic CR in mice

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