10%(+) Reduction in Testosterone After Glucose and Whey Protein Shakes - Is the Classic #BB Shake Anti-Anabolic?

We're talking about a cross-over study in adolescent subjects and acute changes but that's neither the only nor the most relevant reason you don't have to be afraid of the bodybuilding staple, now. In fact, a closer look at the data seems to suggest that we're talking about a 'protein-anabolic decrease in testosterone'... sounds odd? Well, here's how it may relate to your #androgenReceptors (AR) and eventually your gainz, irrespective of your age, by the way.

I have previously addressed the possible ill effects of very high protein intakes on your testosterone levels - in particular, when those intakes are combined with a caloric deficit and, accordingly, reduced intakes of glucose and fat (re-read "True or False - High or Low Protein Intakes Have Profound Influence on Testosterone, SHBG, Estrogen, Cortisol & Co?" | here)... Now, a new study in Clinical Endocrinology (Schwartz 2019) shows that testosterone ⇆ protein/carbohydrate interactions trigger significant acute decreases in serum testosterone levels in those whose testosterone levels should be soaring: adolescent males.

Read more about studies involving TRT/HRT & co on suppversity.com:

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Needless to say that this raises the question: Does that mean that a classic #bodybuilding staple, the high carb, high protein post-workout shake, will ruin your 'muscle building hormone' (learn more)?

The study Schwartz et al. conducted in twenty‐three adolescent males (12‐18 years old; only those with testosterone levels indicating mid-late puberty were included in the analysis) measured the levels testosterone, as well as luteinizing hormone (LH), GLP‐1 (active), ghrelin (acylated), glucose, insulin and subjective appetite prior (0) and at 20, 35 and 65 minutes after the consumption of a test-beverage; a test beverage that contained...

"...either 1 g of glucose monohydrate (BioShop Canada Inc, Burlington, Ontario, Canada) or 1 g of protein (plain whey protein isolate; BiPro USA) per kg of body weight [...] A noncalorie drink was used as control" (Schwartz 2019).

With a protein content of 90.4% (5.7% moisture, 2.2% ash, 1.18% fat and 0.6% carbohydrates), these test beverages were eventually only 'almost' isocaloric, though: 3.74 kcal/kg body weight for the protein and 4 kcal/kg body weight for the sugar shake is yet not far enough apart to invalidate the study results and the flavor was standardized:

"All beverages were flavoured with 1.5 mL of chocolate extract (Vanilla Food Company) to account for the flavour differences and mixed with 500 mL of water, similar to previous protocols.The whey protein and control beverages were sweetened with 0.2 g sucralose (Tate & Lyle) in order to match sweetness with the glucose beverage. Sucralose was chosen as it has been shown to have no effect on postprandial plasma glucose or insulin. Test beverages were prepared the evening before the study and refrigerated in order to be served chilled the following morning. Participants were served the drink in a large covered opaque cup through a straw" (Schwartz 2019).

In the 3xAM sessions that took place, each after a 12h fast, all participants of this cross over trial had to consume the randomly selected beverages (protein, glucose, control) within 5 minutes. In order to wash away any potential aftertaste, they topped that off with 50 mL of plain water.

Testosterone level changes from baseline to 60 min after ingesting the glucose/protein beverage in pre-early puberty (n = 8) and mid-late puberty (n = 13) | results of a previous study by Schwartz (2015).

Where does the idea of reduced T in adolescents even come from, anyway? Schwartz et al. actually did the study under review as a follow-up to their 2015 study in which they observed an acute decrease in serum testosterone after the consumption of a mixed glucose and protein beverage in order to identify whether glucose and protein, each on its own would have similar or the same ill effects on male adolescents postprandial testosterone levels as the researchers observed them in 2015. Reductions as high as -20% in male adolescents in the mid-late phase of puberty (see Figure on the left).

Since the scientists also speculated that these liquid snacks would have different effects on the subjects appetite and, more importantly, ad-libitum food intake, the boys/young men were fed an ad libitum pizza meal after the final blood draw. In that, the "[p]articipants were instructed to eat [pizza] during the next 20 minutes until comfortably full. Based on prescreening participant preferences" (Schwartz 2019 | Pepperoni pizza (87 g) contained 9 g protein, 6 g fat and 23 g carbohydrates for a total energy content of 180 kcal); three‐cheese pizza (81 g) contained 10 g protein, 6 g fat and 23 g carbohydrate for a total energy content of 180 kcal).

Significant differences in terms of the number of slices of pepperoni and/or three-cheese pizza were not observed. Neither in form of treatment nor baseline body weight. 

In other words, with ~1,300 kcal the pizza love (or rather food intake) of the boys was not influenced by either the beverage or the boys' weight status (F = 2.23, P = 0.14). That's in contrast to the testosterone levels which differed significantly when the scientists compared the testosterone response of overweight and normal-weight adolescents (Figure 1.B).

Figure 1: Differential effects of treatments by weight status (A), overall effect of weight status on plasma testosterone (B).

In that, Figure 1.B seems to suggest that lean individuals (mean BMI = 21.1 ± 0.9 kg/m²) are more susceptible to the detrimental effects of protein/carbohydrate shakes than overweight/obese ones (mean BMI = 29.8 ± 1.2 kg/m²). In fact, though, the differential effects of treatments by weight status that are plotted in Figure 1.A, as well as the lack of an asterisk below the open "normal weight" bar in Figure 1.B tell you that the ostensibly large changes in testosterone the scientists observed in the 12 normal-weight subjects were overall non-significant.

So, being overweight or obese seems in fact to modulate the effects of glucose and protein beverages on adolescents' ... as it is common for every extra pound you carry, negatively.

Table 1:  Baseline levels of appetite‐ and sex‐related hormones (Schwartz 2019); interestingly enough, the level of these metabolically relevant measures didn't change differently for normal- vs. overweight subjects in response to either PRO or GLU.

Does that mean that being fat sucks? Well, let's check if any of the metabolic parameters can explain the difference: At least based on the changes that were reported in the FT, that was not the case - while insulin, GLP1, and ghrelin increased, increased and decreased in response to the beverages, they did so to a similar extent in all adolescents. More importantly, though, ...

...there were no differences in the insulin, GLP1 and/or ghrelin response when comparing the protein vs. glucose beverages.

In the absence of treatment effects on the satiety and hunger hormones, it is no longer that surprising that the scientists didn't observe measurable (and significant) effects on the subjects' pizza intake (reported further towards the beginning of this article) - and that despite the fact that all participants' subjective appetite was decreased after the glucose (no, not the protein) beverage (p = 0.0198 for control and p = 0.0247 for protein).

So what's the main takeaway message, then? Is it "Boys love pizza, no matter what?" 

Well, that could be one takeaway, but I think that Schwartz et al. are right to point towards three other results when it comes to the takeaway messages:

• both, glucose and protein shakes acutely lower the testosterone levels of adolescent males, 
• but the effect is not mediated by the macronutrient composition of the liquid meals, 

Moreover, the levels of testosterone the scientists measured in their young subjects' blood or, rather, the changes that were induced by the protein and glucose beverages did not correlate with the regulation of appetite or food intake. The latter was, however, what the scientists had expected when they planned this follow-up to their previously mentioned 2015 study.

This is not an outlier study and similar effects can be expected in older men: The study at hand has by no means produced revolutionary new evidence. In fact, it rather adds to the results of previous research and the testosterone decline(s) that were observed in Caronia et al. 2013 in men who consumed 75g of glucose and Schwartz' previously referenced study in overweight/obese adolescents from 2015, for example (see previous infobox).

The underlying mechanisms of these effects, however, are still not clear. As the scientists from the University of Toronto point out, the changes may be initiated ....

Table 2: Relationships between testosterone and luteinizing hormone with dependent measures (Δ from baseline means).

"by the intake of glucose or amino acids, particularly leucine, which stimulates rapamycin (mTOR) signalling and subsequent protein synthesis" and/or their "inhibitory effect on adenosine monophosphate‐activated protein kinase (AMPK)," (Schwartz 2019)... 

which should - theoretically - have increased the androgen receptor (AR) mRNA expression (Shen et al. 2014 observed the opposite effect, i.e. AMPK up = AR down in prostate cancer cells and it's likely similar effects will occur in skeletal muscle).

Your androgen receptor status may not just determine how much muscle you gain - the data from Morton et al. seems to suggest that it even determines if you make visible muscle gains, at all (learn more in my August 2018 article with the title "If the Androgen Receptor Response to Training Determines Your Gainz, the Question is: How Can You Optimize 'ur AR Density? Training-, Diet-, and Supplement-Effects Reviewed".

So, does it all come back to androgen receptors? Unlike acute changes in testosterone, the androgen receptor density on your muscles has recently been shown to significantly affect the gains of resistance trained men. I discussed the corresponding study at length in a SuppVersity article from August 2018. What I may or may not have highlighted enough in that context is that...

...any increase in androgen receptor (AR) expression leads to greater testosterone uptake by the muscle tissue which lowers plasma testosterone levels... and that probably to an extent similar to what was observed in the study at hand!

This may also explain why neither glucose nor protein has ever been shown to have anti-anabolic effects. In fact, if their isolated and combined consumption in form of a beverage does indeed increase the AR receptor expression and lower the levels of circulating testosterone only by increasing the amount of T that's bound to receptors, that's even more evidence that, on the endocrine level, both nutrients exert pro- not anti-anabolic effects. Accordingly, the notion that the study at hand would support low-carb keto- vs. classic high protein + low-fat-diets for bodybuilders and anyone else striving for a muscular physique would be fundamentally flawed... but let's not jump to conclusions, here! It will be up to other, long(er)-term studies that include resistance training regimen and more relevant outcomes (=changes in body composition) to say which dietary pattern is best for your gainz - as of now, it seems to make less of a difference than people on either side of the nutritional divide want to make you believe - that is, assuming that the dietary protein and energy intake are equal in a low vs. high carb diet | Comment!

References:

• Caronia, Lisa M., et al. "Abrupt decrease in serum testosterone levels after an oral glucose load in men: implications for screening for hypogonadism." Clinical Endocrinology 78.2 (2013): 291-296.
• Schwartz, Alexander, et al. "Acute decrease in serum testosterone after a mixed glucose and protein beverage in obese peripubertal boys." Clinical Endocrinology 83.3 (2015): 332-338.
• Schwartz, Alexander, et al. "Acute decrease in plasma testosterone and appetite after either glucose or protein beverages in adolescent males." Clinical Endocrinology (2019).
• Shen, Min, et al. "The interplay of AMP‐activated protein kinase and androgen receptor in prostate cancer cells." Journal of cellular physiology 229.6 (2014): 688-695.

Will Adding 100mg of GABA to Your Whey Turn You into GH-ulk? RCT Finds 10x Higher Total Lean Mass Gainz, BUT...

Sometimes study results just seem too good to be true... I mean, who cares about total lean (including organ) mass when everything you can show off: strength, arms, and legs didn't grow appropriately? You don't? Well, the study is still interesting. Or did you anticipate that, in young-to-middle-aged training noobs GABA probably has to be cycled to keep its effects on growth hormone (GH)?

I've previously told you about #GABA, I also highlighted that it's questionable whether its use as a growth hormone (#GH) booster is actually going to yield visible improvements in your physique... on the other hand, the GH response to workouts was - next to cortisol - the only hormonal correlate West et al. (see Figure 1, in previous article) observed in the best and IMHO only decently well powered long(er)-term study investigating what Stuart Philips called "a hormonal ghost" (Philips 2012), i.e. the contribution of the transient increase in growth hormone, testosterone, IGF1, etc. in the hour/s after a workout.

Now, GABA has been found to trigger similarly transient increases in GH as resistance training. Unlike the effects of straight exogenous human growth hormone (#HGH), the effects of GABA on muscle protein synthesis have not yet been studied in humans... not yet? Yes, this means it has been done.

Learn more about building muscle and strength while losing fat with www.suppversity.com

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In their latest paper, scientists from the R&D department of Pharma Foods International report on the results of an experiment in which they examined "the effects of oral GABA plus whey protein supplementation on muscular hypertrophy in men after progressive resistance training" (Sakashita 2019). In that, you guessed it, ...

 the not exactly 100% unbiased scientists hypothesized that "GABA administration with post-exercise protein supplementation may enhance training-induced muscle hypertrophy concomitant with elevated resting plasma GH concentrations" (ibid.)

To test the hypothesis, the scientists conducted a randomized double-blind parallel-group design study involving N=21 healthy, non-exercising men (26 - 48 years) who were randomized to receive

• whey protein isolate (Lactocrystal®) at a daily dosage of 10 g) or the same
• whey protein isolate  + 100 mg of GABA (80% pure Pharma GABA® produced by fermentation) 

daily for 12 weeks. With regard to the way the supplement was consumed, the scientists write:

"Subjects were instructed to ingest the supplements within 15 min of training, or before sleep on a non-exercise day. Each supplement was dissolved in 150 mL water immediately before ingestion." 

The whey protein powder was a whey protein isolate (Lactocrystal®; Nippon Shinyaku Co., Ltd, Kyoto, Japan); the GABA powder was produced through natural fermentation using a specific lactic acid bacteria strain (Pharma GABA®; 80% purity; Pharma Foods International Co., Ltd, Kyoto, Japan).

If you want to stack something w/ your whey protein, it should be creatine - especially if you're a vegan (elderly) trainee as you've learned in my 2017 article "Creatine Non-Responder? Age+Meat Intake - Determinants of Creatine's Effect on PCr (±200%) + Probably Performance".

How did they train? Here's the authors' summary: "The resistance training program included five upper-body and lower-body exercises: leg press, leg extension, leg curl, chest press, and pull down (LifeFitness, Schiller Park, IL, USA). Both groups performed the same training program. Training sessions were completed within 60 min and included the following: 5 min, warm-up (ergometer cycling); 45 min, resistance exercise; and 10 min, stretching exercise.

This training program was performed twice per week in a fitness club with training equipment and included one unsupervised and one professional trainer-supervised session per week. All resistance exercises were performed in three sets (12 repetitions; 2 - 3 min rest periods).

[...] Exercise intensity was set at 50% maximal strength for the first week and then raised to 60% after week 1. Weights were progressively increased in 2.5 - 5 kg intervals when the prescribed repetitions could be completed. The trainer ensured that subjects provided maximum effort and intensity for all supervised training sessions. Subjects were instructed to only perform exercises as part of the training program; those who missed three exercise sessions were excluded from the analysis" (ibid.) - whether the non-preregistered study was adequately powered was not reported, by the way ... 🤔

The main outcomes were the 12-week increases in maximal strength which were not tested directly, but calculated by dividing weight by percentage of RM, as well as body composition (fat-free mass, fat mass, and total body mass), which was assessed for all subjects using dual-energy X-ray (#DXA) absorptiometry, and plasma GH concentrations, which were measured using a commercially available enzyme-linked immunosorbent assay for human GH.

As it's common for any training + supplementation study, not all subjects made it to the finish line: Five subjects excluded from the analysis because of personal reasons (WP, n = 1; WP + GABA, n = 1), not completing 90% of supplement intake requirements (WP, n = 1), and not completing 90% of exercise sessions (WP, n = 1; WP + GABA, n = 1). Thus, 21 subjects completed the 12-week program (WP, n = 10, 40.1 ± 7.9 years; WP + GABA, n = 11, 38.8 ± 5.7 years). For all of them, it can be said that:

Neither the training nor the supplements significantly changed either the energy content or the macronutrient composition of the subjects' diets!

What the combination of resistance training + 10g of whey that was consumed on top of rather marginal protein intakes of 1g/kg body weight per day did, though, was to trigger significant changes in body composition - total lean mass to be precise.

Figure 1: Change in body composition after 12 weeks as measured using dual-energy X-ray absorptiometry. (a) Whole body fat-free mass and (b) arm and leg lean mass. Values represent the mean ± standard error for 10 subjects (WP) or 11 subjects (WP + GABA). *P < 0.05 vs. the WP group (Sakashita 2019).

Now, that's per se not surprising. After all, we're talking about training noobs, but what is surprising (at least to me) is both the statistical significance as well as the absolute size of the differences in lean mass gains in the "whey only" vs. "whey plus GABA" group, you can see in Figure 1.

Figure 2: Max. strength increased significantly in both groups, but there was absolutely no effect of GABA - not even on leg presses (p = 0.040), bros - and it happens to be in line with the observations in West 2012: no interaction between GH and strength (Sakashita 2019).

A significant time effect was also observed for the strength parameters which increased

Two additional observations are interesting and potentially worth considering: the difference between total extra lean mass gains and arm and leg lean mass gains (Figure 1), and the decline of the GH boosting effects of GABA over time (Figure 3).

In fact, if you scrutinize the data in Figure 3, you will realize that timing is the only relevant inter-group difference - the direct comparison of the increases didn't yield significant results (even if it may initially look like that).

You may have to cycle GABA to see benefits: Before we draw some final conclusions, though, let me briefly address what I consider to be an important but easily overlooked implication of the study at hand: if it even works, you probably need to cycle GABA, as its effects wear off within 1-2 months and it's not clear if they return after another 2 months of abstinence... since you'll by then be no newby, anymore, it's questionable whether this 2nd 'cycle' will yield significant results, at all - even in the less relevant "total lean mass" category. Ah, ... and with the age of the study's participants ranging from the early-20s to the mid-40s, the corresponding "GABA-cycling"-trial should also investigate whether the need for and/or optimal timing of these cycles depends on biological, not just on training age.

What? Ok, let's rephrase that: (a) both WP + GABA + training and WP only + training lead to statistically significant increases in growth hormone, but (b) it took 8 weeks vs. 4 weeks in the WP only group for these changes to reach statistical significance over the baseline levels in this group - and again (c) an inter-group difference was not observed at any point during the study due to the high variability of the GH levels.

Figure 3: Resting plasma growth hormone concentrations. Values represent the mean ± standard error for 10 subjects (WP) or 11 subjects (WP + GABA). *P < 0.05 vs. baseline of the WP + GABA group; **P < 0.05 vs. baseline of the WP group.

So what do we make of these results, then?  Well, let's first address the usual confounders: (a) the subjects were untrained, results may differ for a trained population (the amount of lean mass gained, for example, is likely to be smaller), (b) protein nutrition is beyond current recommendations for strength trainees (up to 3g/kg | ISSN), hence different overall gains and inter-group differences may be expected from studies in populations with higher protein intakes.

Weight lifting shoes don't make a difference and "elevation masks" (those things that simply impair your ability to breath) work... they increase your cerebral oxygenation - don't rely on the potentially misleading abstracts, alone... read SuppVersity - and this 2019 research update, in particular!

Now for the exciting question: Does this study show that daily GABA supplementation helps to enhance exercise-induced muscle hypertrophy? The answer is more straight forward than you'd expect: No, it doesn't!

The study demonstrates that there are beneficial effects in terms of total lean mass. Since the latter includes organ mass and the subjects' "organ-free" arms and legs didn't grow faster, the scientists are right to limit their conclusion to "daily GABA supplementation may help enhance exercise-induced muscle hypertrophy" (my emphasis in Sakashita 2019).

Disappointed? Well, the mere fact that the Japanese scientists observed a difference in hypertrophy-related outcomes in response to the co-supplementation with GABA, at all, is newsworthy, IMHO. We do yet have to avoid to be carried away by impressive relative increases of only semi-relevant study outcomes. After all, anything that goes beyond the conclusions that there's some interaction with total lean body mass (but not the typical hypertrophy targets of resistance training: arms and legs) going on would be a misinterpretation (see red Box on cycling) for another caveat). To go beyond these two fundamental statements is speculation...

... so let's speculate! 

As the authors highlight GABA could "enhance muscle hypertrophy [due] to its ability to increase basal GH concentrations"; and, at first, the data seems to support that... "at first", i.e. until you realize that significant inter-group differences in the GH levels between the WP and the WP + GABA group were not detected at any time-point of the study.

Accordingly, it makes sense look further to potential "gain mediators" that were not measured in the study at hand... which ones? Well, what about improvements in sleep quality and fatigue reduction as they have been previously reported by Yamatsu et al. (2016) three years ago? As the authors of the study at hand readily themselves admit, we'd have to do a second study to investigate potential effects on sleep, HRV, subjective stress, and training motivation, which could all be affected by GABA supplementation. Moreover, studies in trained individuals and with more appropriate protein intakes (>2g/d vs. ~1g/day) are warranted and the scientists should make sure that the 1kg increase in lean mass is not a consequence of increased glycogen storage (learn why), which could be a side effect of the interactions between GABA and glucose metabolism (Gomez 1999; Wan 2015) and or endocrine functions other than growth hormone (Erdö 1990; Gladkevich 2006; )... so, in essence: little do we know, but we are intrigued, right? Don't worry, I will keep you posted here and in the SuppVersity Facebook News at www.facebook.com/SuppVersity | Comment!

References:

• Erdö, Sándor L., and Joachim R. Wolff. "γ‐Aminobutyric acid outside the mammalian brain." Journal of neurochemistry 54.2 (1990): 363-372.
• Gladkevich, A., et al. "The peripheral GABAergic system as a target in endocrine disorders." Autonomic Neuroscience 124.1-2 (2006): 1-8.
• Gomez, Rosane, et al. "GABA agonists differentially modify blood glucose levels of diabetic rats." The Japanese Journal of Pharmacology 80.4 (1999): 327-331.
• Phillips, Stuart M. "Strength and hypertrophy with resistance training: chasing a hormonal ghost." European journal of applied physiology 112.5 (2012): 1981-1983.
• Sakashita, Maya, et al. "Oral Supplementation Using Gamma-Aminobutyric Acid and Whey Protein Improves Whole Body Fat-Free Mass in Men After Resistance Training." Journal of Clinical Medicine Research 11.6 (2019): 428-434.
• Wan, Yun, Qinghua Wang, and Gerald J. Prud’homme. "GABAergic system in the endocrine pancreas: a new target for diabetes treatment." Diabetes, metabolic syndrome and obesity: targets and therapy 8 (2015): 79.
• West, Daniel WD, and Stuart M. Phillips. "Associations of exercise-induced hormone profiles and gains in strength and hypertrophy in a large cohort after weight training." European journal of applied physiology 112.7 (2012): 2693-2702.
• Yamatsu, Atsushi, et al. "Effect of oral γ-aminobutyric acid (GABA) administration on sleep and its absorption in humans." Food science and biotechnology 25.2 (2016): 547-551.

Beta-Alanine, Widely Used, but Rarely Tested for Safety!? Individual Studies Find Serum / Muscle Taurine is Reduced by >20%, However the Totality of Evidence Suggests...

Reductions in muscle and especially serum taurine have indeed also been observed in humans, but the fact that they do not occur in lower dose studies and cannot be observed consistently in studies using higher dosages (6g) suggests that they shouldn't be a problem for the average BA user.

Regular SuppVersity readers will be familiar with the way(s) in which taurine (#TAU) and beta-alanine seem to both complement and antagonize each other. Beta-alanine, in particular, has been found to deplete muscular (and other tissue) taurine stores - a problem, generations of scientists have used to study the ill health effects of taurine-deficiency.

While studies have never reported clinical taurine depletion in response to beta-alanine supplements, we have to consider the possibility that ...

If it works (no runs + high intensity+volume exercise) bicarbonate is the king of H+buffers:

Caffeine + Bicarb Make Champions

Bicarb + Asp = Muscle Magic!?

NaCHO3 & Leg Days're a Breeze

+100% Anaerobic Endurance

Bicarb Buffers Creatine

Instant 14% HIIT Boost

... the corresponding studies (a) were not long enough in duration, didn't use (b) the same crazy amounts of beta-alanine (#BA) of which I am sure that some bros out there are taking it and (c) - most importantly - won't manifest before literally the last taurine molecules have been bumped by BA.

Note: A possible lack of histidine to recombine w/ BA is probably not a problem given the high protein intakes of the average beta-alanine supplementing athlete/gymrat.

Now, a recent study from the University of Sao Paulo (Dolan 2019) cannot fully address all of issues a-c, but the systematic risk assessment and meta-analysis can provide us with an overview of what human and animal studies that investigated an isolated, oral, β-alanine supplementation strategy can tell us so far about the following 5 safety primary outcomes

• side effects reported during longitudinal trials, 
• side effects reported during acute trials, 
• effect of supplementation on circulating health-related biomarkers, 
• effect of supplementation on skeletal muscle taurine and histidine concentration, and 
• safety-related outcomes from animal trials. 

For the analysis, the quality of evidence for outcomes was ascertained using the Grading of Recommendations Assessment Development and Evaluation (GRADE) framework, and all quantitative data were meta-analyzed using multilevel models grounded in Bayesian principles.
The first at least somewhat surprising result in Dolan et al.'s recently published paper  (2019) is the mere number of studies they came up with:

101 human and 50 animal studies were included in the study. Tingling was the only persistently reported "side effect".

Much less to anyone's surprise who has ever felt "the tingles", paraesthesia was the most commonly reported side effect of oral BA supplementation. With an 8.9-fold increase in odds of "tingling" and a crazy variability [95% credible interval (CrI): 2.2, 32.6] this odd feeling was - and that's good news - also the only reported side effect.

Taurine deficiency: If you're asking yourself why you should care about taurine deficiency, the following list of possible consequence may come handy: impaired vision, central nervous system and cardiac function; reduced bile flow, hence impaired fat digestion, and high blood lipids, impaired metabolism and elimination of toxins; reduced antioxidant defenses; impaired passage of sodium, potassium and possibly calcium and magnesium ions into and out of cells, immune imbalances; reduced muscular performance, etc.

This observation is in line with the lack of differences in terms of the participants' dropout rates when the scientists compared the active (#BA) to the placebo (#PLA) treatment; the tingles are, after all, not that bad and have been avoided in many trials by supplementation timing BA with foods and/or splitting larger into multiple smaller dosages to avoid that they would be messing with blinding the study participants to the treatment (BA or PLA).

As far as common "safety markers" are concerned, β-Alanine supplementation caused a small increase in circulating alanine aminotransferase concentration (#ALT | effect size, ES: 0.274, CrI: 0.04, 0.527), although mean data remained well within clinical reference ranges. 

The small increase in ALT is not a problem - exercise alone will increase it much more as you've learned here. 

Now, while this sounds problematic, ALT is eventually just an enzyme that metabolizes alanine, and - as I explained in "Three Reasons Why Your Doctor May Falsely Believe Your Kidney, Liver or Heart Were Damaged" (Moussa 2015), in detail - not a valuable tool for diagnosing liver damage, in particular in athletes.

Hence, the more important, unquestionably health-relevant and, at least on my part, long-awaited result of the meta-analysis of human data is this:

The scientists found no evidence of a main effect of β-alanine supplementation on skeletal muscle taurine (ES: 0.156; 95% CrI: −0.38, 0.72) or histidine (ES: −0.15; 95% CrI: −0.64, 0.33) concentration. 

You can see a forest plot displaying the effect of β-alanine supplementation on skeletal muscle taurine concentration in humans in Figure 1. As usual, the study-specific intervals represent individual effect size estimates and sampling error, while the diamond represents the pooled estimate generated with Bayesian inference along with the 95% credible interval (95% CrI). This analysis included 83 observations (63 β-alanine/18 placebo) - in short: There's no measurable effect according to the meta-analysis at hand.

Figure 1: Even if you didn't read the rest of this article an effect size of 0.156 with confidence levels ranging from -0.38 to +0.72 should qualm your worries over the taurine depleting effects of appropriately dosed (max. 3-6g/d) beta-alanine supplements - even if individual studies such as Blancquaert et al. report >25% reductions in plasma taurine.

What about the outliers, i.e. Blancquaert et al. (2017) and Harris et al. (2010)? Well, even though both studies found potentially relevant effect sizes, the confidence intervals tell you that the effects were at least so heterogeneous that it would be stupid to argue that they provide limited but relevant evidence that there could be an issue, after all - and that's despite the fact that the statistically at best borderline significant relative reduction in plasma and muscle taurine (p = 0.063 and p = 0.156, respectively) Blanquaert et al. observed was quite large, i.e. -25% in plasma and -13% in skeletal muscle after 23 days on a rather high dose of beta-alanine of 6g/day (see data in Figure 2).

Figure 2: If the Blancquaert human study, the data of which I've used to calculate the relative change in plasma (blue) and muscle (orange) levels of beta-alanine, histidine, and taurine in response to 6g/d of beta-alanine, was the only study we had, it may provide reason for concern... not just for taurine, but even more so for histidine of which the subjects in the BA group consumed a relatively normal amount of 2.1g/d.

So there is reason to worry - at least for people who can't live without worries? Well, as previously hinted at, rodent studies clearly demonstrate that overdoing it on beta-alanine can deplete your taurine levels. However, to achieve a significant reduction of taurine the minimal "daily dose was ≥3% β-alanine in drinking water" (Dolan 2019). The human equivalent of this dosage may mirror the practice of some bros who still believe in the "more helps more"-principle, but the totality of the evidence seems to suggest that within the recommended dosing scheme of 3-6g/d issues w/ local or systemic taurine deficiency shouldn't be an issue... in the short run: long-term studies (years vs. days and months) are imho still warranted.

Looking for what in theory should be the optimal H+-buffer stack? Look no further! Simply (re)view my 2018 article on #BA + #bicarbonate | more

So, what's the verdict then? Within commonly applied (performance enhancing) dosing schemes of 1.5-6-5g/d beta-alanine (#BA) is likely to cause the tingles (if administered as a bolus). Moreover, BA could trigger small and physiologically irrelevant increases in the alanine-metabolizing (liver-) enzyme ALT (learn more about those and how the enzyme is by no means liver-specific, here). The depletion of your cellular taurine stores, on the other hand, doesn't seem to be an issue if you don't dose your supplements based on bro-logic, i.e. "if some's good more is going to get me even more jacked!".

In other words, potentially serious side effects due to the depletion of taurine (and/or histidine, which is even more unlikely given the overall high protein intakes of the average BA consumer) must be feared only by those morons among you whose daily beta-alanine intake is more than 10 times the regular human dose of ~3-6g/d | Comment on Facebook!

References:

• Blancquaert, Laura, et al. "Effects of histidine and β-alanine supplementation on human muscle carnosine storage." Med Sci Sports Exerc 49.3 (2017): 602-609.
• Dolan, Eimear, et al. "A Systematic Risk Assessment and Meta-Analysis on the Use of Oral β-Alanine Supplementation." Advances in Nutrition (2019).
• Harris, Roger C., et al. "Simultaneous Changes In Muscle Carnosine and Taurine During and Following Supplementation with b-alanine." Medicine & Science in Sports & Exercise 42.5 (2010): 107.
• Saunders, Bryan, et al. "24-Week β-alanine ingestion does not affect muscle taurine or clinical blood parameters in healthy males." European journal of nutrition (2018): 1-9.

Calories Count, but Steps Count Too! From 10 to 5k Steps/d W/Out Effect on Body Composition if Energy Intake is Mildly Reduced | Unlike Fatness, the Fitness Worsens, Though

Trust me, once you try to match your intake to your alleged expenditure via apps and trackers you WILL fail if you're also working out regularly - so don't extrapolate the results to: "Oh, I'll just have that Pizza + IceCream, I burned 1,000kcal extra in the gym, I can afford it" - you can't out-exercise overeating!

Those of you who're friends with me on Facebook may have seen the link I posted to an article about an epic total bed rest study (yes, including bedpans, etc.) that's currently recruiting volunteers. If it was not for the good of humanity - Mission to Mars etc. you know - I guess, studies in which you have to stay lying at a decline angle in bed for a whopping two months wouldn't even pass the scrutiny of the ethics committee of (in this case) NASA... but I am digressing and things were significantly less bad and much more realistic in a recent study from the University of Missouri.

With this new study, Winn et al. (2019) wanted to quantify the ill effects of physical inactivity on glucose metabolism and energy balance; and ascertain that energy restriction (yes, calories count) does, as the researchers speculated based on previous studies, indeed blunt these adverse manifestations.

What will affect your energy expenditure and how much energy do you expend?

Intermittent Fasting Boosts Energy Exp. (EE)

3 Revelations About EE While Lifting

How Dieting Reduces Your EE via the CNS

Calculate Your RMR Accurately (+Spreadsheet)

Synergistic or Antagonistic for Max EE

Up the Volume to Up Your Energy Expenditure

To give the study an extra-twist and shut the "calories don't count"-crybabies up, the US researchers decided to go beyond a mere reduction of the energy intake and fed their subjects [inclusion criteria]...

"1) males and females between 18 and 45 yr of age; 2) body mass index (BMI) <28 kg/m² = normal or underweight; 3) no known cardiovascular, kidney, or liver disease; 4) no history of surgery for weight loss and weight stable for prior 3 months (weight change <3 kg); and 5) physically active individuals (90 min of primarily whole body aerobic physical activity >3 d/wk and taking greater than 10,000 steps/day) assessed via accelerometers," (Winn 2019)

... two different diets. More specifically, the subjects were assigned to either the 'regular Western diet' (64% carbohydrate, 20% fat, 16% protein) or "high" protein diet containing "only" 50% of the energy in form of carbohydrate, 20% fat, and a whopping 30% as protein [yeah, I know the crybabies are not going to happy with that, but I cannot change the study design and carb-restrict the "higher protein" group retrospectively, sorry ;-].

The number of subjects seems ridiculously low, no? You're right it's not exactly a large-scale study, but the sample size requirements for this study were calculated based on a type I error rate of 0.05; and according to this calculation an N = 10 subject (paired) design should yield a satisfactory 83% power to detect a 30% change in postprandial insulin AUC with an effect size of 1.0 - that's everything but ideal, but if we're looking for effects that are large enough to be physiologically relevant, this 'accuracy' should be about good enough to make some general statements.

Figure 1: Experimental design and physical activity/energy expenditure. Healthy physically active adults (n = 10) defined as exceeding 10,000 steps per day, completed (A) two periods of physical inactivity while consuming either a control diet or higher-protein diet in a randomized crossover design. Average B) daily steps and (C) energy expenditure (total and physical activity). Data are means ± SEM.  *P < 0.05 vs active. Horizontal arrows represent the number of days for a given data assessment, whereas vertical arrows indicate testing on a single day in the laboratory. Days 1 to 10 were “free-living.” White bars reflect the “active” phase and gray bars represent the ‘inactive’ period. CGMS, continuous glucose monitoring system; BP, blood pressure; DEXA, dual x-ray absorptiometry; EE, energy expenditure. n = 10/condition.

All in all, ten healthy adults met criteria 1-5 (see quote in the previous paragraph). They had a mean age of 24 ± 1 yr and regular activity level of 10,000+ steps per day. Now, these specimen may not necessarily be representative of 'the average Westerner' who's (meanwhile) overweight and mostly sedentary, but the beauty is...

... the cohort of N=10 young adults is small, but it's more representative of the average SuppVersity reader than participants in the average activity <> obesity study.

As you can see in Figure 1, the study used a cross-over design so that, after a 4-week washout those in the "high protein" group were switched to control diet and vice versa, which was then consumed for another 10 days of reduced activity (5,000 vs. 10,000+ steps per day).

As the smaller figures from the bottom tell you the ActiGraph GTX3 data Walk4Life™ told the researchers that the subjects 'managed' to move significantly less, with the average subject taking slightly less than the targeted 5,000 steps per day.

In a similar vein, the scientists' were able to confirm that the subjects actually hit their energy deficit goal of -400kcal/day (measured by the doubly labeled water technique | Chomistek 2017).

Why -400kcal? Well, the specific reduction in energy intake, i.e. 400kcal below maintenance was selected to "account for reduced energy expenditure associated with inactivity" (Winn 2019). 

With another 5 subjects completing the 10-day inactivity cycles consuming 35% excess of their basal energy requirements, the scientists hoped to be able to understand the interaction of sedentary behavior, energy intake and expenditure, and the subject's glucose management by comparing their primary outcome, the subjects gluco-metabolic response to their combined diet + physical activity intervention.

Table 1: Metabolic Implications of Diet and Energy Intake during Physical Inactivity (Winn 2019).

To this ends, Winn et al. used continuous glucose monitoring and OGTT blood collections at 10, 20, 30, 60, 90, 120, 150, and 180 min after ingestion of the oral glucose solution and compared the restricted control and "high protein" diets to the positive control condition (overfeeding + inactivity).

Why only N=5 subjects in the positive (overfeeding + inactivity) control condition? Well after intervention #1 (energy reduction), only N=5 subjects volunteered to participate in another 10-day study... and that despite the fact that the extra calories were primarily comprised of dessert-like foods that were provided by research staff (those left the subjects with extra ~880 kcal/day (macros of the diet 50% carbohydrate, 43% fat, and 7% protein).

Figure 2: Yearly Holiday Weight Gain (Helander 2016)

"What on earth? Why only 7% protein in the overfeeding study?" What I like about the study at hand is that the researchers have addressed all these questions and explain that "[...t]his behavior [meaning to eat an even 'junkier' diet] is typical of the Western holiday seasons, which are characterized by periods of physical inactivity and overconsumption of calories creating a net positive energy balance" (Winn 2019).

Figure 3: In an energy-balanced scenario, medium-intensity exercise does not improve your insulin sensitivity on the day after your workout. High-intensity interval training, on the other hand, works even if it doesn't induce an energy deficit (Fisher 2019).

Calories count - for the benefits of exercise, too! Alongside the study under review, a new paper from Fisher et al. was published that shows that the improvement you see if you move more (including moderate intensity exercise) is also mostly a function of an energy deficit. In the corresponding experiment, the scientists assessed the effect of 8 to 16 wk of aerobic exercise training on the insulin sensitivity (SI) of untrained women under rigorously controlled energy-balanced conditions and found that only high-intensity interval (HII | 84% VO2peak) training had energy-independent beneficial effects on the N= 28 untrained female study participants.

The ladies' insulin sensitivity was assessed 22 h after either a medium-intensity cycling (MIC | 50% VO2peak) or the previously referenced HII workout using a hyperinsulinemic–euglycemic clamp. During the whole procedure, the participants were in a whole-room indirect calorimeter during each condition, and food intake was adjusted to ensure energy balance across 23 h before each clamp, the scientists can be sure that "[t]here were no significant differences in acute energy balance between each condition" (Fisher 2019). By excluding the influence of an energy deficit, the scientist were thus able to show that medium-intensity cycling, alone - in the absence of a caloric deficit or at least changes in the energy balance will not improve the insulin sensitivity of untrained, still normal-weight (BMI < 28, but >37% body fat) women; or, as the scientists interpret their own results...

"the reported improvements in SI [insulin sensitivity] in response to chronic exercise training may be mediated in part by shifts in energy balance" (Fisher 2019).

That sounds like bad news, but we should not forget that Fisher et al. were also able to show that "an acute bout of HII exercise may increase SI even in the context of energy balance" (Fisher 2019). Plus, all exercise groups saw increases in physical fitness (VO2max, p > 0.05 for inter-group differences); a benefit of which the study by Winn et al. suggests that it can be undone by only 10 days of limited physical activity - even if the reduced energy expenditure is accounted for.

Another strength of the study is its decently tight control and the fact that we know that the subjects actually went from their initial 12,154 ± 308 steps per day to a rather meager 4275 ± 269 steps per day (P < 0.05) during the 10-day intervention study... Figure 4 shows the primary outcome, the measures of glucose metabolism.

Figure 4: Effect of physical inactivity on glucose tolerance and indices of insulin sensitivity/resistance in response to a control diet and higher-protein diet. Physically active and physically inactive (A) glucose, (B) insulin, and (C) NEFA curves with corresponding 3-h AUC (inset) after a 75-g oral glucose challenge during the control diet and higher-protein diet conditions. (D) Two-hour glucose and (E) 2-h insulin during the OGTT. (F) HOMA-IR. Data are means ± SEM. *P < 0.05 vs Active. Two-way ANOVA with activity and diet as factors was used for statistical comparisons. Post hoc comparisons with Tukey correction were run when a significant main effect was observed. n = 10/condition. (Winn 2019)

And what do the graphs tell us? Well, as long as you reduce your energy intake appropriately, taking less than half your usual steps per day does not impair your glucose metabolism - that is, allegedly, over a very short time-frame.

You want to do some damage? Don't adapt your energy intake to your (new) sedentary lifestyle!

Table 2: Age, body composition, and blood chemistry in the "holiday" (=reduced activity + extra 800kcal energy intake) experiment (Winn 2019).

As Table 2 on the right shows, the inertness of reduced activity in an isocaloric/deficit scenario is in contrast to the consequences of the "holiday lifestyle" (sit around and eat trash) that was simulated in experiment #2. When the energy intake was increased and the physical activity was reduced, there were ill effects on body weight (+1kg) and BMI, body fat and (+0.7%), fat mass (0.8 kg), as well as a significant worsening of glucose, insulin, C-peptide, and the clearance of insulin (learn more) by the liver...

Similar, albeit less pronounced, effects can be expected for lower mismatches of energy intake and expenditure as they can easily occur when people become progressively more sedentary without adapting their diet (the "I've always had three slices of toast for breakfast"-phenomenon) - over months+years most of us accumulate a mismatch and hence risk fat gain and diabetes.

Unfortunately, the experiment #2 in the study at hand was not designed to investigate the effects of macronutrients, which could be significant, even if it didn't do shit in energy balance/deficit, where  the scientists didn't observe "higher protein" magic in form of differential effects on glucose management or additional effects on body composition, aerobic capacity, and energy expenditure. What the high protein diet in experiment #1 did do, though, was to lower the level of triglycerides, which increased by 15mg/dL (+21%) on the control diet and declined by 27mg/dL (-26% | albeit from higher baseline levels) in the "high protein" group.

Don't make the unwarranted assumption that you just have to work out more to compensate for Pizzas & co. While this may work for additional intakes of 100-400kcal/d even the most active of us don't burn enough to "afford" eating the average sized extra meal | learn more.

So, I can get away with sitting around? For 10 days, you can, but the short nature of the study is not the only reason I would not throw away my step-counter, yet.

After all, the subjects' fitness deteriorated significantly within only 10 days with a VO2max going down by −1.8 ± 0.7 mL/kg/min (P < 0.05) - and that independent of the diet conditions, as a consequence of the prescribed decrease in steps.

How's that significant? Well, physical fitness - and VO2max is still one of the best all-around measures of that - has been convincingly linked to health & longevity (or your 'healthspan'). Hence, reducing your VO2max by ~2% in only 10 days is bad news (Ozemec 2018).

You don't care about health? Well, what about the latest data from Shad et al. (2019) whose recent one week study, in which eleven healthy men reduced the number of daily steps from ~13,000 to ~1,200 steps per day, reports a whopping -27% reduction in myofibrillar protein synthesis ... 😲 Ok, the study was not ideal as the subject's diets were not prescribed and the protein intake of the subjects dropped from 2.1g/kg to 1.8g/kg (p < 0.05) in the high physical activity (HPA >10,000) vs. step reduction (SR | <1,300) condition | Comment on Facebook! 

References:

• Chomistek, Andrea K., et al. "Physical Activity Assessment with the ActiGraph GT3X and Doubly Labeled Water." Medicine and science in sports and exercise 49.9 (2017): 1935-1944.
• Fisher, et al. "Acute Effects of Exercise Intensity on Insulin Sensitivity under Energy Balance." Medicine & Science in Sports & Exercise: May 2019 - Volume 51 - Issue 5 - p 988–994
• Ozemek, Cemal, et al. "An update on the role of cardiorespiratory fitness, structured exercise and lifestyle physical activity in preventing cardiovascular disease and health risk." Progress in cardiovascular diseases (2018).
• Shad et al. "One Week of Step Reduction Lowers Myofibrillar Protein Synthesis Rates in Young Men." Medicine & Science in Sports & Exercise: May 7, 2019 < ahead of print > 
• Winn, Nathan C., et al. "Metabolic Implications of Diet and Energy Intake during Physical Inactivity." Medicine and science in sports and exercise 51.5 (2019): 995-1005.

The Case Against Saccharin: Weight Gain in 12-Wk Human Study Only W/ 1st Gen. Sweetener or Sucrose | Plus: Have You Noticed That Coke 'Secretly' Pulled it Only Recently?

I had to change both, article and thumbnail repeatedly. Initially, I realized that the EU coke ZERO formula contained saccharin, while the US version didn't, then I checked on the official website and found (a) 'Zero' is no longer officially sold in Germany and the follow-up "Zero Sugar" doesn't contain saccharin in the US & EU 😲

If you follow the SuppVersity and/or myself on Facebook, you will have seen my link to an interview with the author of the "artificial sweeteners" <> "stroke" study that made the headlines lately.

In the interview, the lead author puts his results into perspective; a step that's absolutely missing from 99% of the press coverage ... worth reading, but unlike the study Tal Ben Moshe shared on the ISSN Facebook yesterday, not a new intervention study in living human beings - one that taught me several lessons in the history and spectrum of coke's sweetener compositions.

You can learn more about sweeteners at the SuppVersity

Aspartame & Your Microbiome - Not a Problem?

Sucralose 2018 Update #1 - Does it Make Us Fat?

Diet Soda Beats Water as Dieting Aid in RCT

Experiments Don't Support AS<>Obesity link

Sucralose Tricks 'Ur Energy Gauge - Implications?

Other Diet Soda Additives May be the Real Problem

The study by Kelly A Higgins and Richard D Mattes from the Purdue University a compares the effects of the consumption of 4 low-calorie sweeteners (LCSs) and sucrose (=regular sugar) on body weight, ingestive behaviors, and glucose tolerance over a 12-wk intervention in adults (18–60 y old) with overweight or obesity (body mass index 25–40 kg/m2)... and it yielded intriguing results, of which the researchers rightly point out that "[i]f substantiated through additional testing and confirmed through mechanistic studies, findings from this trial have implications for consumers, clinicians, policymakers, and the food industry" (Higgins & Mattes 2019).

Essentially the study demonstrates an obvious truth: One artificial sweetener is unlike the other.

While the 21st-century sweeteners sucralose and stevia display the anticipated benefits, the big granddaddy of artificial sweeteners, saccharin, seems to do just what epidemiological data suggests: saccharin will fatten you up. Really? Well, not to the extent the media would have it: If you take a look at the statistically significant absolute fat gain in Figure 1 (left), it turns out that their practical relevance is questionable, as long as we don't know that the fat gain progresses at the same pace over years.

Figure 1: Effects of 12 weeks on sucrose, saccharin, aspartame, RebA, and sucralose on total body fat (left, p = 0.032) and body fat percentage (right, p = 0.478 | Higgins 2019) - both are physiologically irrelevant for people at >37% body fat.

But there's more: Only the effects on total body weight in Figure 2 were statistically significant. The much more (health) relevant body fat % was not affected significantly by any of the four artificially sweetened beverages (p = 0.478).

'Tab' was the only diet coke to contain saccharin and coke had several reasons to pull it from the market - today you're most likely exposed via processed foods, sugar alternatives like Sweet'n'Low and protein powders & other supplements 🤮

LCS classic ➡ Saccharin is one of the least expensive LCS and, together with the similarly cheap cyclamate, it still constitutes the lion's share of LCS consumption worldwide (Sylvetsky 2016) - saccharin is the worldwide market leader based on sucrose equivalents and has a particularly strong presence in the tabletop packet market, primarily as Sweet ‘N’ Low™" (ibid).

Saccharin has an interesting history in the US with it being banned temporarily in the 1980s (that was the end of Coca Cola's T@b diet soda), being stigmatized with a "could cause cancer"-label in the late 1980s and early 1990s and finally being exonerated in 2000 (FDA).

'Exonerated' for a good reason? Well, the study at hand doesn't tell you anything about saccharin's carcinogenic effects as they were described by Cohen et al. (1986), but follow-up studies to the 80s research was unable to confirm the carcinogenic effects that were observed in early rodent studies... follow-up studies in monkeys that lasted up to 24 years.

This doesn't mean that the scientists' previously cited conclusion that the "findings from this trial have implications for consumers, clinicians, policymakers, and the food industry"... in 12 weeks, however, the physiological relevance of LCS consumption in overweight and obese individuals is somewhere between 'irrelevant' and 'of questionable relevance'.

More studies are warranted... longer studies in young(er), health(ier), and lean(er) subjects

Clearly, more actual research (no observational fortune-telling) is necessary to make firm conclusions with regard to the individual effects of specific types of artificial sweeteners on body weight and composition; but that's not 'news'. What is news or, as some may argue, the previously ignored 'elephant in the room', is the observation that the "beneficial effects of one LCS (e.g., sucralose) may be attenuated if combined with selected other LCSs," (Higgins 2019).

Figure 2: If you look at the 12-week body weight changes things don't look that impressive either - of particular importance: only the sugar group saw something akin to statistically significant linear weight gain (Higgins 2019).

IMHO this is the actually relevant 'news' the study at hand provides. Why's that? Well, the vast majority of products use combinations of various artificial sweeteners - products that are based on a single LCS are the exception, not the rule; and an ever-increasing number of products seeks to achieve a "more natural", sugar-like taste by combining three or more sweeteners (e.g. Coke Zero - 58 mg aspartame, 31 mg acesulfame-K, and 38 mg sucralose).

🤷‍♂️ Do you remember my 2016 article about the putative (interactive) effects of common food additives on diet sodas' effect on blood glucose management? No? Read it!

Let's briefly recap: The scientists started with the contemporary inconsistency of experimental research and observation, they recruited 154 pretty fat (>35% body fat) young-to-middle-aged men and women (18-16y), assessed the subjects food intake on 2x3 days (2 nonconsecutive weekdays and 1 weekend day) at the beginning and end of the 12 week intervention phase, respectively.

Table 2: Qualitative overview of the treatments on four key parameters; even statistically significant effects were rather small (e.g. weight loss w/ sucralose − 0.78 kg; 

While we don't even know if that's important, yet, I think I should also point out that the trial which was pre-registered as NCT02928653 and had body weight, body composition, dietary
intake, EE, appetite, and glycemia as its main outcomes, excluded anyone who used artificial sweeteners regularly, i.e. more than once per week.

You don't remember the 'saccharin can give you cancer'-signs? You're either a European, a Millenial, or younger. The FDA pulled the cancer warning for saccharin in 2000.

Where do you find all the (natrium)saccharin that's used globally? Well, you will be surprised to hear that, but your risk of being exposed is much lower in the US than elsewhere 😲.

After Coca Cola and Co had to label both, the presence and amount of the sweetener that's disguised as E 954 in the EU, in the 1980s-90s, they the companies pulled the plug on the no-longer-marketable artificial sweetener (Note: Its cousin, E 952, aka cyclamate, has even been banned from the US market due to potentially carcinogenic effects and is still used widely in the EU).

Neither of these measures was taken in the EU, where Coca Cola ab-)used the lack of regulation and consumer-awareness until recently; 'recently' as in when they pulled the plug on Coke Zero, and the other "Zero" products.

Yeah, you read that right, I had to compare dozens of ingredient labels online, though, to realize that the Coca Cola Company silently pulled saccharin (and in the EU cyclamate) from their product line with the transition from "Coke Zero" to "Coke Zero Sugar" of which the latest (03/09) overview on their (German) websites informs you that the last remaining mainstream product that contains E954 aka saccharin is "Sprite Zero". So, there's no saccharin in the "new" Coke Zero Sugar - neither in the US nor in Europe... Shamed be he who thinks evil of it 🤔! *[note to conspiracy theorists: the Coca Cola company was not directly involved in the study at hand]

Coca Cola pulled it, US producers have avoided it ever since manufacturers had to label it as carcinogen in the late 20th century, only the European food and supplement industry has always loved and still loves its saccharin aka "E954"!

Things look different for supplements, though. Protein supplements, for example, often contain saccharin and, in Europe, cyclamate. So, you better check your supplement labels! At least those of you who buy European products will likely find both sodium-/natriumsaccharin (E954) and cyclamate (E952) on the label. Why? Well, mixing only "modern" sweeteners will not yield the "clean" sugary taste consumers are looking for - at least not until exceeds the consumers' maximal sweetness threshold (Heikel 2012). Other reasons to stick to the 1st generation sweeteners are their solubility, their effects on the texture of the final product, their heat stability etc. (Hutteau 1998).

In addition, the pre-registration lists sweetness perception and overall liking of the delivery beverage as secondary outcomes that were supposed to be evaluated.  Compliance was monitored via urinary para-aminobenzoic acid (PABA) was used to monitor participant compliance with the beverage intervention (more about PABA testing in Sharma 2014). So far so good, and here's what Higgins and Mattes (2019) found when it comes to ...

• compliance and taste preferences: within their compliance testing, all groups were similarly compliant; this seems to be logical in view of the fact that the scientists did not find a difference in perceived intensity of sweet, salty, sour, bitter, off-flavor, drying, bitter aftertaste, and off-flavor aftertaste between treatments (P ≥ 0.10) or between treatments over time (P ≥ 0.10)
• body weight: while there was a significant treatment-by-time effect (P = 0.01) for weight, only the sugar- and saccharin-guzzling group recorded significant weight gain. There was no statistically significant difference between the sugary and the saccharin sweetened beverages, but the "gains" were more consistent and marginally larger in the "sugar" (=sucrose) group (weight change = 1.85 ± 0.36 kg, P < 0.001; 1.18 ± 0.36 kg, P = 0.02, respectively | Figure 2).

🤷‍♂️ Note to the smart SuppVersity reader you are: The average Westerner will gain 'only' ~0.12–0.15kg in 12 weeks, anyway, i.e. without extra saccharin (Malhotra 2013)... there's a reason the subjects were overweight or obese, though, so it's not clear how representative the data from Mahotra et al (2013) is for the interpretation of the data in the study under review 🤣

• As you will have seen in Figures 1 & 2, already, the subjects in the aspartame and rebA groups also gained some weight over the 12-week study: a non-significant 0.73 ± 0.35 kg and 0.60 ± 0.36 kg, respectively. Sucralose, currently the most-vilified sweetener, on the other hand, delivered on the weight loss promises attributes like "diet" or "no sugar" on the packaging of artificially sweetened products imply... well, they 'delivered' a non-significant (P ≥ 0.07) reduction of body weight of −0.78 ± 0.36 kg - needless to say that the associated <1% reduction in body fat didn't turn the heavy-weight couch potatoes into fashion models.
• body composition: While BMI (and total weight) are pretty good proxies of body fatness in the general population, there's a reason why I focussed on the actual fat loss in the first graph I created to illustrate the results. As you can see in the corresponding figure (Figure 1), the results of the body fat analysis which was measured using DXA at baseline and after the 12-week intervention
  • increased significantly during the intervention among participants in the sucrose treatment ( = +1.35 ± 0.25 kg, P < 0.001) and was significantly higher for the sucrose group than for all LCSs (P ≤ 0.01). Moreover, ...
  • among the LCS groups, change in total fat mass was negative and significantly lower for the sucralose group than for the saccharin and aspartame groups (−0.79 ± 0.33 kg and −0.80 ± 0.33 kg, respectively; P ≤ 0.02). Moreover, ...
  • only the sucrose group saw significant increases in nasty android (belly) and unaesthetic gynoid fat mass (+0.16 ± 0.03 kg and +0.28 ± 0.05 kg, respectively; P < 0.001), while...
  • the already small increase in the fat depots around the waist didn't differ between sweetener groups; the gynoid fat which forms around the hips, breasts and thigh, on the other hand, increased more rapidly in the saccharin vs. rebA and sucralose groups [+0.18 ± 0.06 kg for both rebA (P = 0.006) and sucralose (P = 0.005)].
  Just like the body weight changes, these changes in body composition did not correlate with baseline body weight (Figure 1 uses the corrected data, already).
  
  The same (no effect of initial body weight/composition) held true for the subjects' fat-free mass (FMM), of which the scientists report increases all but the sucralose group (+0.84 ± 0.20 kg, P = 0.001 for sucrose; +0.70 ± 0.18 kg, P = 0.006 for saccharin; +0.63 ± 0.18 kg, P = 0.01 for aspartame). A clear pattern, however, cannot be distinguished. 
• energy intake: With no inter-group differences in energy intake at baseline (P ≥ 0.11), the scientists observed the expected increase energy intake in the sucrose group and a (non-expected) decrease of total energy intake in the sucralose group (P = 0.007 and 0.02, respectively). In that, ...
  • the mean reported energy intake for the sucrose treatment group was significantly higher than for the saccharin, aspartame, rebA, and sucralose treatment groups by 584 ± 162, 336 ± 160, 587 ± 164, and 553 ± 164 kcal, respectively (P ≤ 0.04). At week 12,
  • the energy intake was significantly higher for the sucrose group than for the saccharin, rebA, and sucralose treatment groups (P ≤ 0.004) and only tended to be greater than the aspartame treatment group (P = 0.07).
  As the authors rightly point out, "[t]he differences in energy intake between the sucrose and LCS groups can be attributed to the energy provided from the beverage". It is thus not surprising that, ...
  • when the energy provided by the beverage is removed from the reported energy intake, the treatment, and treatment-by-time main and interaction effects are no longer statistically significant (P = 0.37 and 0.96, respectively).
  In this context, it's certainly worth mentioning that a significant inter-group difference for the LCS was not observed for either the total or the adjusted intakes when saccharin, aspartame, rebA, and sucralose were compared. 

How reliable is the data? You already know that the scientists' adjustments for baseline weight didn't make a difference. Hence, it's unlikely that a rule of thumb like "the fatter, the better/worse" exists. But what about other typical confounders? Misreporting of dietary intakes, for example? Well, with "only 14.2% of the mean reported energy intakes [falling] within the Goldberg cutoffs", the scientists were quite happy with their subjects' ability (and willingness) to use their “Automated Self-Administered 24-hour Dietary Recall” (ASA24) correctly.

Potential confounding due to ineffective blinding is possible and most likely for aspartame (69% v. 50-57% of the subjects in the aspartame group correctly assumed that they had consumed LCS). None of these numbers is yet far enough off the 50% that would signify that the subjects were absolutely clueless as to which drink they had been given (the sucrose drink was correctly identified by only 42%). The observed effects can thus neither be blamed on the distinctive(ly chemical) taste of any of the sweeteners or being able to say if one's beverage was sweetened with sugar or LCS, as the likelihood of correctly identifying the beverage sweetener was not different between treatment groups (P = 0.44).

For the physical activity and energy expenditure (EE), the scientists also found "no significant treatment effect or treatment-by-time interaction" (P = 0.56 and 0.17, respectively)" (ibid.) - this goes for all aspects of the Baecke index for work, sport, or leisure physical activity (P ≥ 0.05) and was to be expected in the absence of an exercise intervention.

• In other words: Irrespective of whether the energy from the beverages was included or not, there was no effect of the type of LCS on the subjects' energy intake "at any time point" (ibid.). That is intriguing as the subjects in the saccharin group reported significantly greater hunger ratings than all other treatment groups (P ≤ 0.03); and, in a similar vein,  the subjects in the saccharin group were the only ones to report an increased desire to eat (DTE) and prospective consumption ratings.

The authors summarize their own results as follows: "Sucrose and saccharin consumption significantly increased body weight compared with aspartame, rebA, and sucralose and weight change was directionally negative and lower for sucralose compared with saccharin, aspartame, and rebA" (ibid.) For the Purdue University researchers, the most important message is yet not that the subjects gained weight, but rather that it's unwarranted to study LCSs as a group, when each of them seems to have individual effects.

What all sweeteners have in common, on the other hand, is ...

... that saccharin, aspartame, sucralose, and rebA lack the often claimed effect on energy balance: the scientists were unable to detect the consistent increase in energy intake sweetener - in that, it's particularly important to highlight once more that a significant increase in energy intake was not observed in the saccharin group, either... or, as the scientists have it: "no association between energy intake and saccharin use was observed despite increases in hunger, DTE, and prospective consumption" (Higgins & Mattes 2019).  Accordingly, we must assume that there were "metabolic changes unrelated to energy intake" (Higgins & Mattes 2019). So what could be the explanation?

• insulin & glucose management: The results of studies assessing the effects of saccharin on our cephalic phase insulin response (CPIR) is inconsistent, compared to studies in other sweeteners it does yet seem more likely for saccharin to trigger/modulate the insulin response than for any other sweetener. So, is it insulin? Not impossible, but not proven either... and the lack of effect on the subjects' response to the oral glucose test (glucose AUC and insulin release didn't differ) makes it seem unlikely that insulin mediates either the increased desire to eat and weight gain in the saccharin group of the 12-week study at hand.
• dissociation between taste and energy content: The aforementioned changes in insulin could, if they are accompanied by corresponding changes in GIP, GLP, PYY and other 'satiety hormones' mess with our bodies' ability to guesstimate the true energy content of foods. If that was the case, though, we should see significantly increased intakes in the study at hand - that those were not measured in spite of the presence of increased hunger, could yet be a result of underreporting - no matter what the scientists' analysis using the Goldberg criteria.

No, it's not clear that 'this is the nasty effect on the microbiome'. I've addressed the fearmongering news about sweeteners interactions with the microbiome before (Moussa 2015;  Moussa 2017) and am not going to do that again. If it even exists, it would not be saccharin-specific, though, since even your "holy stevia" will mess with the composition of your gut microbiota. Indeed, a saccharin-specific effect as it occurred in the study at hand rather provides further evidence that general LCS-triggered changes in the microbiome are not the missing metabolic link to explain the observational link between sweetener intake and diabesity.

• leaky gut: As previously pointed out, it is unlikely that the weight gain was mediated by saccharin-specific changes in the gut microbiome. Only recently, though, Santos et al. (2018) have been able to show that, in contrast to acesulfame K, aspartame, and sucralose, saccharin seems to possess the nasty ability to reduce the integrity of the human gut integrity by "[increasing] paracellular permeability and decreas[ing the] transepithelial electrical resistance (TEER) via a non-cytotoxic mechanism" (Santos 2018).
  

  

  Figure 3: Saccharin has uniquely detrimental effects on the transepithelial electrical resistance (TEER, let) in Caco-2 monolayers and increases the amount of FITC-dextran that leaked into the scientists' petri-dish guts through the Caco-2 cell layer that simulates your gut lining into the lower compartment of the culture (Santos 2018).

  The underlying mechanism here is, as you would expect it, inflammation, with saccharin elevating the NF-κB activation in the Caco-2 cells of the gut lining which will, in turn, initiate the ubiquitination of the tight junction protein claudin-1 and the subsequent breakdown of the (simulated) gut lining. 

Eventually, we are thus far from having a conclusive answer to the important question/s: If saccharin is unique and if so, why is that!? What is clear, is that ...

"[t]he present findings are consistent with the positive association in epidemiological and rodent trials [which c]ollectively suggests [that] saccharin stands apart from the other popular LCSs in that it may actually contribute to weight gain" (Higgins & Mattes 2019). 

If the mechanism is indeed an inflammatory one, it is yet at least astonishing that Higgins & Mattes et al. didn't observe significant effects on total, android, and gynoid fat mass. More importantly,...

"[...] other health outcomes often related to adiposity, such as glucose tolerance or serum lipid concentrations, were also nonsignificant" (Higgins & Mattes 2019).

If that would stay this way in a long(er)-term studies, is one out of many questions future studies will have to address; studies that should also follow up on the potential weight loss effect of sucralose, by the way - we shouldn't forget, after all, that the consumption of this dreaded 3rd generation sweetener did not significantly alter body weight from baseline, but it was the only sweetener to trigger a measurable downward trend in body weight in the study at hand.

What I have learned writing this article is that it's not really easy to find out if you were, have been, or are exposed to saccharin, because the answer depends not just on the products you consume, but also on (a) whether you bought them in the US or Europe, (b) if they have been formulated before the year 2000 or after manu-facturers were no longer required to put the carcinogen warning on saccharin containing products in the US, and (c) if you consume Coke Zero from discounters like ALDI and other copy cats or the new original Coke Zero Sugar of which I realized only after doing a lot of research that (03/19) it does no longer contain saccharin... neither in the US, nor in Europe...🤔 So, does that mean that I believe that saccharine makes you fat and gives you cancer? Not really, even with the publication of the study under review there's not enough evidence to substantiate the claim that saccharin (let alone other LCSs) fuel the global diabesity epidemic.

So what's the verdict then? Firstly, Higgins' & Mattes' paper exonerates sucralose and rebaudioside A. For the former, the study results even suggest beneficial effects on weight and body fat in the absence of dietary or other lifestyle change. This does, however, require confirmation from longer-term studies in a metabolically (+body-composition-wise) more diverse population.

Secondly, the results Higgins & Mattes present implicate saccharin, one of the "first generation" sweeteners. That's interesting because it seems to support my non-systematic cumulative review of the evidence regarding the link between artificial sweeteners and body weight gain, where - even among the rodent studies - those using saccharin seemed more likely to detect an effect than (e.g.) those using aspartame or sucralose.

This leads us to the third take-home message, which is that the study adds to the evidence that not all sweeteners are created the same. As obvious as that may be, self-proclaimed "experts" still tend to lump them all together into one category of 'sweet poison' you have to avoid at all costs - whenever you read something like that or, even worse, statements like "Stevia is ok, because it is 'natural' but all the rest is 'chemical' poison", you better treat what you've just read like you should treat the stuff on naturalnews.com and the other pseudo-science websites: 🚮.

With that being said: Your final question probably is - "Will saccharine make me fat?" I am sorry to say that but if I answered this question with a simple yes or no, you'd have to trash this article, as well. The paper by Higgins & Mattes has added to the evidence that it could do, however, the only mechanistic explanation the study offers (increased appetite) is, at the same time, refuted by the scientists' observation that the energy intake didn't change significantly in response to saccharine. This leaves us with the new "leaky gut" by Santos et al. and a potentially individual increase in inflammation that could promote weight gain even in the absence of significant changes in energy intake | Leave a comment on Facebook!

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• Higgins, Kelly A & Mattes, Richard D. "A randomized controlled trial contrasting the effects of 4 low-calorie sweeteners and sucrose on body weight in adults with overweight or obesity." The American Journal of Clinical Nutrition (2019): nqy381.
• Hutteau, F., et al. "Physicochemical and psychophysical characteristics of binary mixtures of bulk and intense sweeteners." Food Chemistry 63.1 (1998): 9-16.
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