Acute effects of a ketone monoester, whey protein, or their coingestion on mTOR trafficking and protein-protein colocalization in human skeletal muscle

Abstract

We recently demonstrated that acute oral ketone monoester intake induces a stimulation of postprandial myofibrillar protein synthesis rates comparable to that elicited following the ingestion of 10 g whey protein or their coingestion. The present investigation aimed to determine the acute effects of ingesting a ketone monoester, whey protein, or their coingestion on mechanistic target of rapamycin (mTOR)-related protein-protein colocalization and intracellular trafficking in human skeletal muscle. In a randomized, double-blind, parallel group design, 36 healthy recreationally active young males (age: 24.2 ± 4.1 yr) ingested either: 1) 0.36 g·kg^-1 bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 2) 10 g whey protein (PRO), or 3) the combination of both (KET + PRO). Muscle biopsies were obtained in the overnight postabsorptive state (basal conditions), and at 120 and 300 min in the postprandial period for immunofluorescence assessment of protein translocation and colocalization of mTOR-related signaling molecules. All treatments resulted in a significant (Interaction: P < 0.0001) decrease in tuberous sclerosis complex 2 (TSC2)-Ras homolog enriched in brain (Rheb) colocalization at 120 min versus basal; however, the decrease was sustained at 300 min versus basal (P < 0.0001) only in KET + PRO. PRO and KET + PRO increased (Interaction: P < 0.0001) mTOR-Rheb colocalization at 120 min versus basal; however, KET + PRO resulted in a sustained increase in mTOR-Rheb colocalization at 300 min that was greater than KET and PRO. Treatment intake increased mTOR-wheat germ agglutinin (WGA) colocalization at 120 and 300 min (Time: P = 0.0031), suggesting translocation toward the fiber periphery. These findings demonstrate that ketone monoester intake can influence the spatial mechanisms involved in the regulation of mTORC1 in human skeletal muscle.NEW & NOTEWORTHY We explored the effects of a ketone monoester (KET), whey protein (PRO), or their coingestion (KET + PRO) on mTOR-related protein-protein colocalization and intracellular trafficking in human muscle. All treatments decreased TSC2-Rheb colocalization at 120 minutes; however, KET + PRO sustained the decrease at 300 min. Only PRO and KET + PRO increased mTOR-Rheb colocalization; however, the increase at 300 min was greater in KET + PRO. Treatment intake increased mTOR-WGA colocalization, suggesting translocation to the fiber periphery. Ketone bodies influence the spatial regulation of mTOR.

Keywords: Rheb; TSC2; immunofluorescence; ketone bodies; β-hydroxybutyrate.

Graphical abstract

Figure 1.

Tuberous sclerosis complex 2 (TSC2) and mechanistic target of rapamycin (mTOR) colocalization with Ras homolog enriched in brain (Rheb) in the basal state and in response to 0.36 g·kg⁻¹ bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 10 g whey protein concentrate (PRO), or the combination of both (KET + PRO) at 120 and 300 min in the postprandial period. Immunofluorescence quantification of mTOR/TSC2 (red) and Rheb (green) colocalization, displayed as a composite image (merge) (n = 12/condition). Yellow/orange regions represent TSC2 and Rheb (A) mTOR and Rheb colocalization (C). Each panel represents one subject from KET, PRO, and KET + PRO across the experimental time course. Group data are quantified and reported as TSC2-Rheb colocalization (B) and mTOR-Rheb colocalization (D). Scale bar represents 100-µm area. All data are presented as individual participant data along with means ± SD and were analyzed using two-way repeated measures ANOVA. Means with different letters are different within each treatment (P < 0.05). *Different from KET at same time point (P < 0.05). †Different from PRO at same time point (P < 0.05).

Figure 2.

Lysosome-associated membrane 2 (LAMP2) and wheat germ agglutinin (WGA) colocalization with mechanistic target of rapamycin (mTOR) in the basal state and in response to 0.36 g·kg⁻¹ bodyweight of the ketone monoester (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KET), 10 g whey protein concentrate (PRO), or the combination of both (KET + PRO) at 120 and 300 minutes in the postprandial period. Immunofluorescence quantification of mTOR (red) and LAMP2/Rheb (green), and mTOR and WGA (blue) colocalization, displayed as a composite image (merge) (n = 12/condition). Yellow/orange regions represent mTOR and LAMP2 colocalization (A) and pink/magenta regions represent mTOR and WGA colocalization (C). Each panel represents one subject from KET, PRO, KET + PRO across the experimental time course. Group data are quantified and reported as mTOR-LAMP2 colocalization (B) and mTOR-WGA colocalization (D). Scale bar represents 100-µm area. All data are presented as individual participant data along with means ± SD and were analyzed using two-way repeated measures ANOVA. In B, means with different letters are different within each treatment (P < 0.05). In D, means with different letters are different (P < 0.05).