The Metabolic Effects of β-hydroxybutyrate on Working Skeletal Muscle

Study Overview
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Brief Summary
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The goal of this clinical trial is to test ketone bodies in healthy elderly and young individuals. The main question it aims to answer are: • Do ketone bodies improve skeletal muscle function? Participants will ingest a ketone monoester and skeletal muscle function will then be evaluated by: Special magnetic imaging techniques Intravenous infusion of tracer-marked nutrients Performance tests on a ergometer bike and in a dynamometer Researchers will compare the outcomes between within the young and elderly groups and between the young and the elderly group to investigate if age has an effect on the outcomes.

Detailed Description
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BACKGROUND With ageing, skeletal muscles metabolism changes and muscle function declines. This may lead to muscle weakness and increased risk of developing metabolic diseases. Ketone bodies, namely 3-hydroxybutyrate (3-OHB), is an energy substrate that may change the metabolism and improve efficiency of skeletal muscles in a setting of ageing. OBJECTIVE The study aims to investigate the effects of beta-hydroxybutyrate ingested as a monoester on skeletal muscle function and metabolism during muscle work in young and elderly individuals. DESIGN Healthy young (20-25 years) and elderly (65-85 years) untrained males will be paired based on age corrected VO2-max. Participants will be evaluated in a double blinded cross-over design on two study days: One day with ketone ester ingestion (D-beta-hydroxybutyrate/D-1,3-butanediol; KetoneAid Pro KE4), one day with ingestion of a volume and calorie and taste matched placebo (lipid emulsion). Blood ketone levels will be kept elevated through a sipping protocol. During both conditions a low glucose dose will be continuously infused to block physiological ketogenesis. The order of the study days will be randomized and interspaced by at least 4 weeks. On experimental days, participants meet fasted to perform voluntary contractions with tibialis anterior muscles in a MR compatible dynamometer while oxidative capacity, ATP generation, intramuscular pH, fatiguability and work efficiency is evaluated through 31P-MR spectroscopy. After, participants will bike at a fixed intensity ( 45% of Wmax) while lipid fluxes and glucose oxidation rates are measured by palmitate- and glucose tracer infusions and carbamide-corrected indirect calorimetry over 60 min. This was initially intended for both young and elderly individuals, but only involves the young group, as the elderly group had difficulties completing 60 min cycling. Lastly on the study days, participants will perform a cycling test as a measure of performance starting at 70 % of Wmax for 5 min thereafter increasing by 10 % of Wmax every 1 min until exhaustion. Muscle biopsies are obtained before (both groups) and just following fixed intensity cycle work (only young group). Adipose tissue biopsies are collected at the beginning of the experimental day (both groups) before ketone/placebo ingestion and after the constant load cycle work (only young group). Blood samples are performed throughout the day to assess substrate levels, hormones and for proteomics analysis. Before each study day, participant's activity level is measured for 7 days by accelerometry (ActiGraph). Participants will log their diet 3 days prior to each experimental day. Participants are asked to ingest a similar diet 2 days before both experimental days and to withstand from strenuous exercise 2 days prior to both experimental days. Participants are asked not to make any significant changes to their lifestyle while taking part in the study.

Official Title
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The Metabolic Effects of β-hydroxybutyrate on Working Skeletal Muscle

Conditions
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Aging, Metabolism Disorder, Ketonemia, Muscle Disorder

Intervention / Treatment
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* Dietary Supplement: D-β-hydroxybutyrate/D 1,3 butanediol monoester


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Male 20-25 years old (n = 12) OR male 65-85 years old (n = 12) BMI range: 19 to 27 Stable weight (< 5% change over last 6 months) Less than 3 x 60 min of structured exercise per week. Exclusion Criteria: Medication that affect energy metabolism. Non-MR-compatible metals or electric devices in the body. Anaemia or bleeding disorders. Heart, lung or other disease that affects the subjects ability to exercise. Smoking. Drug abuse. Lack of compliance. Known allergy towards local anaesthetics. Any condition that the principal investigator considers unsuitable for the subject's ability to complete the study.

Ages Eligible for Study
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Minimum Age: 20 Years
Maximum Age: 85 Years

Sexes Eligible for Study
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Male

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: Two-day cross-over design.
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: First Ketone, then placebo<br>Ingestion of ketone monoester D-β-hydroxybutyrate / D 1,3 butanediol monoester on first experimental day and ingestion of a fat placebo drink on the second experimental day. | Dietary Supplement: D-β-hydroxybutyrate/D 1,3 butanediol monoester<br>* Ketone monoester<br>|
| Experimental: First placebo, then ketone<br>Ingestion of a fat placebo drink on the first experimental day followed by ingestion of ketone monoester D-β-hydroxybutyrate / D 1,3 butanediol monoester on the second experimental day. | Dietary Supplement: D-β-hydroxybutyrate/D 1,3 butanediol monoester<br>* Ketone monoester<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Work efficiency | External work performed by the ankle during dorsiflexion per ATP consumed. ATP consumption is assessed by 31P-MRS while external force is measured by the dynamometer. | Over 60 minutes on each of the two experimental days. | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Oxidative capacity | Assessed by 31P-MRS as phosphocreatine resynthesis. | Over 20 minutes at each of the two experimental days. | 
| Glucose oxidation rates (only young group) | Assessed by measuring tracer dilution from blood samples obtained during 3H-glucose infusion while cycling at a fixed intensity. | At 10 minute intervals over the last 30 min of the 90 min fixed intensity cycling on each experimental day. | 
| Palmitate flux (only young group) | Assessed by measuring tracer dilution from blood samples obtained during 3H-palmitate infusion while cycling at a fixed intensity. | At 10 minute intervals over the last 30 min of the 90 min fixed intensity cycling on each experimental day. | 
| ATP generation | ATP generated from different pathways (glycolytic, oxidative, phosphocreatine) assessed by 31P-MRS. | Over 30 minutes at each of the two experimental days. | 
| Mitochondrial function | Assessed by high-resolution respirometry on muscle biopsies | Just before and immediately after the constant load cycling at each of the two experimental days. | 
| Intramuscular pH | Measurements at rest rest and during work assessed by 31P-MRS | Over 60 minutes at each of the two experimental days. | 
| Blood 3-OHB | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Blood glucose | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Blood free fatty acids | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Cycle performance | Assessed by a performance test on a bike ergometer with incremental load. The test continues until failure. The test outcome is the power generated at time of failure. | 5-8 minutes during each of the two experimental days | 
| Rating of perceived exertion (only young group) | Subjective measure of exertion during cycling evaluated by reporting on a number assessment scale (Borg scale (6-20)). | After 30, 60 and 90 minutes of fixed intensity cycling and just after the incremental performance test. | 
| Tibialis anterior fatigue | Tibialis anterior fatigue assessed by the dynamometer during 3 minutes of dorsiflexion at a fixed resistance. | Over 3 minutes on each of the two experimental days. | 
| Blood growth hormone | Blood concentration measured by blood sampling | During experimental days | 
| Blood insulin | Blood concentration measured by blood sampling | During each of the two experimental days | 
| AMPK phosphorylation (only young group) | From muscle biopsies | Just before and immediately after the constant load cycling at each of the two experimental days. | 
| Blood glucagon | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Blood pH | Assessed from arterial blood samples | During each of the two experimental days | 
| Blood catecholamines | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Blood cortisol | Blood concentration measured by blood sampling | During each of the two experimental days | 
| Intramuscular lipid content | Concentration measured in muscle biopsies | At the beginning and at the end of each of the two experimental days | 
| Intramuscular glycogen content | Concentration measured in muscle biopsies | Just before and immediately after the constant load cycling at each of the two experimental days. |