Postprandial leucine and insulin responses and toxicological effects of a novel whey protein hydrolysate-based supplement in rats

More than 150 million US residents consume dietary supplements and many of those are products including whey protein, creatine, and branched-chain amino acids (BCAAs) [1]. Of the numerous marketed dietary supplements, it is well known that whey protein supplementation augments resistance training adaptations [2]. Moreover, recent evidence suggests that the consumption of whey protein elicits the greatest appearance of essential amino acids and insulin and is thus the seemingly most influential known protein source capable of augmenting muscle anabolism [2‐4]. Whey protein is commercially categorized by concentration or by degree of hydrolysate [5]. Whey protein concentrate (WPC) may contain 29% to 89% total protein by volume, with the remaining kcal coming from carbohydrates and lipids, whereas whey protein isolate (WPI) composition typically exceeds 90% total protein by volume [5]. WPH is enzymatically hydrolyzed in order to obtain smaller peptide fractions from its parent WPC or WPI source and is thought to undergo more rapid gastrointestinal absorption kinetics thus potentially improving amino acid bioavailability. In support of this hypothesis, data from Tang et al. [3] indicate that circulating leucine levels were greater with ingestion of WPH versus soy or casein at 30 minutes post ingestion in humans. Power et al. [6] studied the serum insulin, phenylalanine and total branched chain amino acid responses of ingesting 45 g of WPI or WPH after an overnight fast in humans. Of the measured variables, these authors reported that WPH elicited a statistically greater phenylalanine response compared to WPI [6]. Thus, there is still conflicting evidence as to whether or not WPH elicits a more favorable serum anabolic response (i.e., greater insulin and leucine values) relative to other whey protein forms. Furthermore, limited evidence to our knowledge has compared the postprandial effects that exist between a whey protein isolate relative to a hydrolyzed whey protein derived from WPI [7]. Data comparing the effects of different protein sources on serum amino acid and hormone concentrations typically examine these phenomena after overnight fasting period, which is not applicable to those who consume supplemental protein between meals. Lockwood et al. [8] studied the effects of ingesting 60 g/day of WPH versus two different whey protein concentrate supplements on body composition after 8 weeks of progressive resistance training. The authors discovered that all three protein forms similarly affected total body muscle mass, strength, anaerobic endurance and blood lipids. However, the authors did not analyze the acute feeding serum responses [8]. Therefore, while WPH may elicit transient increases in circulating leucine and insulin relative to other protein sources, data is lacking with regard to how a WPH-based supplement affects these variables in the post-absorptive state.

Clarity is also warranted with regard to whether or not weeks-to-months of whey protein supplementation yield adverse health effects (i.e., kidney and/or liver damage). Large-scale human studies have demonstrated that higher protein intakes seemingly exert no adverse effects on markers of renal or liver function [9, 10]. There are, however, equivocal safety concerns brought about through the internet and media regarding the prolonged effects of consuming copious amounts of dietary protein whether it is through high protein foods or protein supplements [11]. Likewise, there is the imminent possibility that whey protein supplement users disregard and supersede the recommended dosages and combine whey with other dietary supplement ingredients. Therefore, multiple dosages of protein supplements should be thoroughly investigated for safety of consumption.

Animal models offer a variety of advantages compared to humans to study how mammals physiologically cope with nutritional interventions. Specifically, animals’ diets can be tightly regulated, multiple tissues can be dissected and analyzed, and supplement adherence can be assured. Therefore, the purpose of the current study was two-fold: aim 1) to use a rat model to compare the post-prandial insulin and leucine responses between a novel WPH-based supplement versus a WPI powder in rats that were in the post-absorptive state, and aim 2) to perform a thorough toxicological analysis on rats that were fed low, medium, and high doses of the novel WPH-based supplement over a 30-day period in order to examine the safety of chronically consuming this protein source. We hypothesized that the tested WPH-based supplement would exhibit a superior insulin response when compared to the insulin response of WPI. Likewise, we hypothesized that leucine and insulin responses to the WPH-based protein would be superior to WPI based upon previous literature suggesting that the hydrolysis process potentially increases the digestibility of WPH [7]. Finally, we hypothesized that the supplement would not elicit adverse health effects on the measured health parameters on rats following a 30-day supplementation period.

Our data suggest that the tested WPH-based supplement: a) exerted a transient rise in post-prandial leucine and a subsequent insulin rise relative to WPI during a post-absorptive (not fasted) state at the low dose; and b) did not adversely affect markers of kidney, liver, and or other health markers after a 30-day feeding period, while decreasing food intakes in a dose-dependent fashion. Together, these data regarding serum responses to the tested WPH-based supplement can be considered to be a promising lead for future experiments, which would aim to continue examining the physiological effects that WPH-based protein sources exhibit on other tissues such as skeletal muscle and adipose tissue.

It has been shown that extracellular leucine availability, with or without exercise, increases muscle protein synthesis rates [3, 14‐17]. Likewise, the insulinogenic effects of whey have been posited to potentially aid in augmenting muscle protein synthesis in an mTORC1-dependent fashion independent of intramuscular mRNA expression patterns [18], although this effect has been suggested to be more permissive rather than stimulatory [14]. In agreement with previous evidence, our data demonstrates that WPH has been shown to be insulinogenic at one hour following feeding in humans [3], albeit their data was collected after an overnight fast. The mechanism whereby whey elicits its superior insulinogenic effects relative to other protein sources may be related to unidentified bioactive peptides and/or its amino acid profile; specifically arginine [19]. However, both protein sources in our study possessed nearly similar amounts of arginine (WPH-based supplement: 470 mg per human serving, WPI = 510 mg). Nonetheless, our data suggests that WPH may be superiorly insulinogenic relative to an undigested whey protein source; an effect which we speculate could be due either: a) its superior effect in stimulating the transient increase in postprandial serum leucine given that leucine has been shown to stimulate insulin secretion [20], or b) the presence of unidentified bioactive peptides that occur due to the hydrolysis process which stimulate pancreatic insulin secretion. In regards to the later, Morifuji et al. [21] have determined that dipeptides from WPH stimulate muscle glucose uptake via PI3-kinase and protein kinase C (PKC) pathways. Therefore, existing evidence in the literature, demonstrates that WPH-based peptides exhibit significant physiological effects on the pancreas warrants future research into elucidating mechanisms that drive these phenomena.

As mentioned previously, WPH has been shown to elicit a transient leucine spike in the serum, although this effect has only been shown under fasting conditions and when comparing WPH to casein and soy [3]; of note WPI and WPH have been examined for branched chain amino acid responses, but not leucine responses explicitly [7]. Fasting rats for 12 hours prior to feeding them a high-protein test meal yielded serum leucine concentrations that were 60% lower than the rats in our study after 3 hours of food removal [22] which implies that our animals were in a post-absorptive (not fasted) state. However, we chose to examine the leucine responses between the WPH-based versus WPI after a 3-h food withdrawal with the notion that humans would likely consume the whey protein-based supplement prior to or following an exercise bout within 3–6 hours of consuming a meal, as most humans eat throughout the wake cycle. Therefore, this is the first report to our knowledge demonstrating that subjects in the post-absorptive state exhibit greater leucine and subsequent insulin responses when ingesting a hydrolyzed whey protein source versus a native whey protein isolate.

We also report that 30 days of chronic supplementation with a WPH-based supplement in rodents aged 62 days old when study began: a) causes no apparent adverse health effects on the kidneys and/or liver, b) does not affect brain and/or heart weights, c) does not affect circulating clinical chemistry and whole blood markers, and d) does not alter body composition. As mentioned previously, studies in healthy humans have demonstrated that higher protein intakes seemingly exert no adverse effects on markers of renal or liver function [9, 10]. Resistance training studies have also determined that increasing protein intakes for two months did not negatively impact serum clinical chemistry markers related to kidney and liver damage [23, 24]. However, concern still exists in the medical literature regarding the potential negative effects that protein supplementation exerts on liver [11, 25] and kidney physiology [25, 26]. While limited data exists on the safety of chronic whey protein supplementation, little data to our knowledge has utilized a rodent model whereby liver and kidney tissues were morphologically examined for lesions following chronic feeding. One recent study [27] did determine that 18 days of WPI consumption offset liver toxicity caused by the concomitant administration of a pro-oxidant agent (dimethylnitrosamine). Interestingly, we determined that only the water condition presented a greater incidence of liver damage (> 21 hepatocellular mitoses) relative to the WPH-supplemented conditions. We speculate that WPH or whey protein supplementation in general supplementation could indeed be hepatoprotective. Of note, the WPH supplement contained Rhodiola rosea extract which is a well-known adaptogen that confers hepatoprotective (i.e., antioxidant and antilipidemic) effects in db/db mice [28]. Whether it is the WPH fraction and/or the Rhodiola rosea extract in the WPH-based supplement, we conclude that the WPH-based supplement used in our study does not exacerbate liver damage when administered in very high doses and could, instead, confer hepatoprotective effects.

Contrary to the one referenced study examining the effects of whey protein on liver histopathology markers in rodents [27], our study is seemingly the first to suggest that 30 days of feeding a range of WPH-based protein dosages to rats does not negatively impact kidney damage/toxicology markers and/or circulating markers of kidney function (i.e., creatinine and blood urea nitrogen). Rats in the high dose condition consuming 6 human equivalent doses per day (would be equivalent to an additional 120 g of protein in humans) increased daily protein intakes up to 21.7 g/kg/day. Additionally, 30-days of creatine feeding present within the WPH-based supplement did not adversely affect the examined health markers; for the high dose condition this would be equivalent to a human consuming 15 g/d of creatine. Therefore, our 30-day study is in agreement with other literature which continues to refute speculation that whey protein [9, 10] and/or creatine supplementation [29] negatively impacts kidney function and/or elicits kidney damage in animals that do not possess pre-existing kidney issues.

Interestingly, animals that were gavage-fed three and six human equivalent doses per day of the WPH-based supplement for 30 days consumed less total kilocalories per day relative to animals that consumed one human-equivalent dose and water over this time frame. Multiple studies have established that whey protein may exert satiating effects and reduce adiposity in rats [30, 31]. In explaining this effect, authors from the later study propose that whey-derived proteins do elicit a satiating effect through the enhanced secretion of gut neuropeptides including cholecystokinin (CCK) or glucagon-like peptide-1 (GLP-1). Thus, this effect might have been observed in our study although examining circulating CCK and GLP-1 was beyond the scope of our investigation. With regard to body composition alterations, however, the feeding intervention in our study did not confer changes in body fat in the protein supplemented conditions. Likewise, the feeding intervention did not increase DXA lean body mass which has been demonstrated in the aforementioned rodent study that chronically fed rats whey protein over a 25-day period [31]. However, that Pichon et al. [31] used dissection methods to assess body composition whereas our DEXA method may introduce a larger degree of error which could have obscured our findings. Furthermore, we cannot rule out the hypothesis that consuming higher protein diets over longer periods (i.e., years to decades in humans) reduces adiposity and enhances and/or maintains muscle mass during maturation and subsequent aging in humans, respectively.

It is also noteworthy mentioning that there are limitations to the current study. First, rodents were examined instead of humans with regards to studying leucine, insulin, and toxicological responses to these whey protein sources. It should be noted, however, that rats and humans seem to respond similarly to whey protein as it has been shown to increase circulating leucine and markers of muscle protein synthesis following exercise in both species [3, 32]. Thus, we hypothesize that human responses will likely be similar when examining the physiological effects of WPH versus WPI supplements. With regard to the current toxicology study, it should be noted that only 5 animals were examined per condition over a 30-day feeding period. In parallel to our study, however, there are other recent studies examining the toxicological effects of other compounds which have similarly studied 6 animals per condition [33, 34]. Creatine monohydrate (equivalent to 2.5 g/dose for humans) is also a major ingredient in the WPH-based supplement. However, creatine monohydrate does not alter glucose tolerance or insulin sensitivity and is not insulinogenic nor does it affect circulating leucine concentrations [35]. With regard to other major ingredients present in the WPH-based supplement, L-citrulline has not been shown to impact circulating insulin and/or leucine levels [36], although vitamin C has been shown to reduce insulin in type II diabetes patients over chronic supplementation periods [37], and L-lysine may stimulate insulin secretion from pancreatic beta cells [38]. Therefore, beyond the active biopeptides that exist in the WPH formulation, other ingredients may have influenced the insulin response. Finally, while we examined the postprandial circulating leucine response to a WPH-based supplement versus WPI, it remains unknown as to whether or not potential unknown biologically active peptide fragments that occur during the whey hydrolysis process spike in the bloodstream after feeding relative to WPI [this aspect of food science is reviewed in [39]. In this regard, future animal and/or human studies should pursue this exciting and unexplored nutraceutical research area in order to determine if WPH supplementation with exercise confer positive skeletal muscle anabolic responses due to potential increases in circulating bioactive peptide fragments relative to other protein sources.

In summary, our rodent feeding model uniquely found that the WPH-based supplement elicited greater transient leucine with a subsequent increased insulin response relative to the WPI. Given these data in conjunction with the recent data demonstrating that WPH may possess biologically active peptide fragments [5], it will be of future interest to compare the anabolic effects of WPI- versus WPH-based supplements surrounding resistance training and/or the effect of WPH-based supplements in persons with diminished insulin secretion. Our 30-day feeding rodent model suggests that WPH-based supplements are safe to consume for one month in rats and may confer satiating effects which reduced total food intake, albeit the relatively short-term feeding study did not unveil significant alterations in total fat mass between the administered dosages. In this regard, longer-term human studies might be performed in order to examine the potential weight regulatory effects that WPH-based products (i.e., meal-replacement shakes) may exhibit on overweight and obese populations.