Exogenous Ketosis and Muscle Protein Synthesis During Exercise Recovery

A parallel group design will be used for this randomized double-blind, placebo-controlled study in healthy adults to investigate the effects of ketone monoester on myofibrillar protein synthesis rates during post-exercise recovery. There are 4 groups in this trial, including one ketone group (0.36g/kg body weight) (KET), one protein group (25 g whey protein) (PRO), a combination of ketone and protein (KET+PRO), and a placebo group (flavoured water) (CON). A total of 48 participants will be enrolled in this trial (n=12 per group).

The study will include a screening visit (visit 1), 10-repetition maximum (10-RM) testing (visit 2), where participants' 10-RM will be determined for the exercise protocol, and the experimental trial (visit 3).

During the experimental trials, participants will arrive to the laboratory in a fasted state, and a prime dose of the L-\[ring-2H5\]-phenylalanine will be administered followed by a continuous infusion at a rate 0.05 μmol/kg of body weight/min. Then, participants will perform a unilateral lower-body resistance exercise, consisting of 8 sets of 10 reps of unilateral leg extension at 90% of their 10-RM with 90 seconds rest in between sets. Following exercise, the nutritional treatment will be administered.

Arterialized blood will be collected at baseline and 13 postprandial timepoints across 9 hours for plasma amino acid, glucose, and insulin quantitation.

Additionally, changes in capillary blood β-HB concentration will be assessed throughout the trial by collecting capillary blood samples at baseline and 10 postprandial timepoints. Finally, muscle biopsy samples from both the exercised leg and the rested leg will be collected prior to beverage intake and at the 5-hour mark of the postprandial post-recovery period to assess myofibrillar protein synthesis rates.

• Ketone Monoester (KET) (Dietary supplement): \- Ketone monoester supplement (R)-3-hydroxybutyl (R)-3-hydroxybutyrate based on participants' body weight (0.36g/kg body weight). The ketone brand name: delta G Oxford Ketone Ester
• Ketone Monoester + Whey Protein (KET+PRO) (Dietary supplement): * Ketone monoester supplement (R)-3-hydroxybutyl (R)-3-hydroxybutyrate based on participants' body weight (0.36g/kg body weight) * 25g Whey Protein * L-\[ring-2H5\]-phenylalanine tracer (enriched to 4%)
• Whey Protein (PRO) (Dietary supplement): * 25g Whey Protein * L-\[ring-2H5\]-phenylalanine tracer (enriched to 4%)
• Flavour matched placebo (CON) (Dietary supplement): \- Flavoured water (non-caloric bitter + citrus flavours)
• Resistance exercise (Other): \- 8 sets of 10 reps at 90% of 10- repetition maximum (10-RM) of unilateral leg extension with 90 seconds rest in between sets.

Primary outcomes

• Myofibrillar fractional synthesis rate [0 - 5 hours in the postprandial period.]: Quantification of changes in basal myofibrillar fractional synthetic rate (%/hour) in the rested and exercised limbs.

Secondary outcomes

• Time-course data for plasma enrichments (in moles percent excess) of L-[ring-2H5]-phenylalanine [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma-free L-\[ring-2H5\]-phenylalanine enrichment measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for total amino acid concentration [3 hours pre-prandial to 5 hours postprandial]: Incremental area under the curve (iAUC) for plasma concentration of total amino acids (μmol/L) including: free leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, arginine, glutamine, glycine, alanine, serine, glutamic acid, aspartic acid, asparagine, tyrosine, cysteine, proline (combined), measured at baseline and across the 3-hour pre-prandial period and 5-hour postprandial period.
• Time-course data for total amino acid concentration [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma concentration of total amino acid concentration (μmol/L) including: free leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, arginine, glutamine, glycine, alanine, serine, glutamic acid, aspartic acid, asparagine, tyrosine, cysteine, proline (combined), measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for essential amino acid concentration [3 hours pre-prandial to 5 hours postprandial]: Incremental area under the curve (iAUC) for plasma essential amino acid concentration (μmol/L) including free leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan (combined), measured at baseline and across the 3-hour pre-prandial period and 5-hour postprandial period.
• Time-course data for essential amino acid concentration [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma concentration of essential amino acid (μmol/L) including: free leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan (combined), measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for leucine concentration [3 hours pre-prandial to 5 hours postprandial]: Incremental area under the curve (iAUC) for plasma leucine concentration (μmol/L) measured at baseline and across the 3-hour pre-prandial period and 5-hour postprandial period.
• Time-course data for leucine concentration [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma concentration of leucine (μmol/L) measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for glucose concentration [3 hours pre-prandial to 5 hours post-prandial]: Plasma glucose concentration (mmol/L) and its corresponding incremental area under the curve (iAUC), measured at baseline and across the 3-hour pre-prandial period and 5-hour postprandial period.
• Time-course data for glucose concentration [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma concentration of glucose (mmol/L) measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for insulin concentration [3 hours pre-prandial to 5 hours post-prandial]: Incremental area under the curve (iAUC) for plasma concentration of insulin (pmol/L), measured at baseline and across the 3-hour pre-prandial period and 5-hour postprandial period.
• Time-course data for insulin concentration [Baseline, 3 hours pre-prandial, and 5 hours into the postprandial period.]: Changes in plasma concentration of insulin (pmol/L) measured at 14 time points (t = -180, -120, -60, 0, 15, 30, 45, 60, 90, 120, 150, 180, 240, 300 minute).
• Incremental area under the curve for β-HB concentrations [1 hour pre-prandial to 5 hours postprandial]: Capillary blood β-OHB concentration (mmol/L) and its corresponding incremental area under the curve (iAUC) measured during the pre-prandial period and postprandial period.
• Time-course data for β-HB concentrations [1 hour pre-prandial to 5 hours in the post-prandial period]: Changes in capillary blood β-OHB concentration (mmol/L) measured at 10 time points (t = -60, 0, 30, 60, 90, 120, 150, 180, 240, 300 minute).
• Changes in the phosphorylation status of anabolic signaling molecules [0 and 5 hours of the postprandial period.]: Western blot analysis will be used to measure the changes in the basal phosphorylation status of anabolic signaling molecules, including p-mTORC1 (Ser2448), p-p70S6K (Thr389), p-Akt (Ser473), p-4E-BP1 (Thr37/46), and p-rpS6 (Ser240/244), p-ERK1(Thr202/Tyr204), and p-ERK2 (Thr185/Tyr187), in the exercised and the rested limbs.

Sex: All, ages: 18 Years to 40 Years, accepts healthy volunteers

Inclusion Criteria:

* Healthy adult female or male participants who are 18-40 years of age (inclusive) BMI >18.5 and <30.0 kg/m2
* Moderately active (i.e., ≥ 1 session of lower-body weightlifting /week for the previous 2 months).
* Has maintained stable use of medication and supplements (which are not limited by the exclusion criteria), stable dietary and lifestyle habits, and stable body weight, for the last 3 months prior to screening and agree to maintain them throughout the study.
* Be willing to entirely avoid alcohol consumption 48hr prior to the experimental test day.
* Willing and able to agree to the requirements and restrictions of this study, be willing to give voluntary consent, be able to understand and read the questionnaires, and carry out all study-related procedures.

Exclusion Criteria:

* Females who are lactating or pregnant
* Females using third-generation oral contraceptives (including: Desogen®, Ortho-Cept, Ortho-Cyclen, Ortho Tri-Cyclen) as these are known to affect protein metabolism in females.
* Individuals with metabolic disorders including: Type I or Type II diabetes
* Individuals with a history of thrombosis / cardiovascular disease
* Individuals who use of anticoagulants
* Individuals with musculoskeletal / orthopedic disorders
* Individuals with knee injuries (i.e., ACL injuries).
* Individuals who have used tobacco products within the last 6 months
* Individuals with a history of neuromuscular problems
* Chronic usage of medications known to modulate skeletal muscle metabolism (i.e. corticosteroids, hormone replacement therapy (HRT), and over-the-counter supplements including creatine monohydrate) in the last 6 months.
* Individuals with allergies to milk proteins (whey or casein).
* Individuals with lactose intolerance
* Individuals with Phenylketonuria (PKU)
* Previous participation in amino acid tracer studies.
* Adherence to a vegetarian or vegan diet
* Current use of ketone supplements or adherence to a ketogenic diet
* Individuals who train more than ≥ 5 sessions of lower-body weightlifting /week for the previous 4 months

• Tyler Churchward-Venne, PhD, Principal investigator, Department of Kinesiology and Physical Education, McGill University