Clinical study design
Participants were enrolled between February 2019 and September 2019 at the Maastricht University Medical Center (MUMC+), the Netherlands, and the last subject completed in February 2020. Two dropouts were reported during the study (Fig. 2). The protocol was reviewed and approved by the Medical Ethical Review Committee of the MUMC + (Netherlands Trial Register ID: NTR7426) and conducted in accordance with the declaration of Helsinki. All participants were informed about the nature and risk of the experimental procedures before their written informed consent was obtained.
Sixteen male and postmenopausal females diagnosed with T2D for at least 1.5 years, participated in the study. Participants underwent medical screening to check eligibility. Inclusion criteria were 40–75 y of age, BMI of 25–38 kg/m2, relatively well-controlled T2D (HbA1C5 kg in the last three months), participation in physical activity ≥ 3 times a week, insulin treatment, and MRI contra-indications.
The study had a randomized, double-blind, placebo-controlled, crossover design (Fig. 2). Each participant underwent 2 intervention arms, which involved daily administration of 4.8 g/m2/d NaPB or placebo. The participants were randomly assigned to receive either the NaPB or the placebo treatment, separated by a washout period of 6 to 8 weeks via controlled randomization. After 2 weeks, all participants underwent several measurements to evaluate patients’ metabolic health. Three days before the start of these measurements, participants were instructed to refrain from strenuous physical activities and to continue their antidiabetic medication with the last dose taken on the evening before the hyperinsulinemic-euglycemic clamp test. Throughout the study, patients were asked to maintain their habitual diet and regular physical activity pattern.
The study medication Pheburane (Lucane Pharma, Paris, France provided by Eurocept International, Ankeveen, The Netherlands) contained 483 mg/g NaPB and inactive ingredients (sucrose, maize starch, sodium, hypromellose, ethylcellulose N7, macrogel 1500 and povidone K25). The placebo (produced by Tiofarma, Oud-Beijerland, the Netherlands) only contained the inactive ingredients of Pheburane. The daily dose of 4.8 g/m2/day (NaPB and placebo) was below the minimal, clinically prescribed (9.9–13.0 g/m2/day NaPB), to prevent reaching the maximally allowed dose of 20g/day, due to high body surface area of the overweight/obese participants and the development of unwanted side effects. The study medication was administered in the form of granules, taken orally 3 times a day, divided into 3 equal amounts and given with breakfast, lunch and dinner. The granules could be directly swallowed with a drink (e.g. water, fruit juices) or sprinkled on solid foods (e.g. mashed potatoes, yoghurt). Administration of 4.8 g/m²/day NaPB was well tolerated and no adverse events or side effects were reported throughout the study.
Overview of specified outcomes
The primary outcome was peripheral insulin sensitivity, measured by the hyperinsulinemic-euglycemic clamps, expressed as the change in insulin-stimulated glucose disposal rate minus baseline (ΔRd).
Secondary outcomes were ex vivo mitochondrial oxidative capacity in skeletal muscle, measured with high-resolution respirometry expressed as O2 flux, substrate oxidation, assessed with indirect calorimetry and fat accumulation in muscle and liver measured with proton magnetic resonance spectroscopy (1H-MRS). Fasting blood samples were collected to determine levels of BCAA and their intermediates, insulin, triglycerides, FFA and glucose. In addition, phenylbutyrate levels were determined by LCMS to check compliance to the intervention.
2-step hyperinsulinemic-euglycemic clamp
A two-step hyperinsulinemic-euglycemic clamp with co-infusion of D-[6.6-2H2] Glucose tracer (0.04 mg kg−1 min−1) started in the morning of day 15 at 06:30 to assess hepatic and whole-body insulin sensitivity. After a pre-infusion of D-[6.6-2H2] glucose tracer (0.04 mg/kg/min) for 3 h (basal phase), low dose insulin was infused at 10 mU m−2 min−1 for 3 h to assess hepatic insulin sensitivity (low insulin phase), with a subsequently raise in insulin concentration to 40 mU m−2 min−1 for 2.5 h (high insulin phase) to determine peripheral insulin sensitivity. Blood was frequently sampled to measure glucose concentration in arterialized blood. In addition, 20% glucose (enriched with D-[6.6-2H2] glucose tracer) was co-infused at a variable rate to maintain euglycemia (~6.0 mmol/L). During the last 30 min of each phase, blood samples were collected at 10 minutes interval to determine glucose tracer kinetics and indirect calorimetry was performed to measure substrate oxidation. Steele’s single pool non-steady state equations were used to calculate the rate of glucose appearance (Ra) and disappearance (Rd)46. Volume of distribution was assumed to be 0.160 l/kg for glucose. The change in insulin-stimulated glucose disposal (ΔRd) was calculated by the difference between Rd measured under insulin-stimulated condition and basal conditions. Endogenous glucose production (EGP) was calculated as Ra minus exogenous glucose infusion rate. Hepatic insulin sensitivity was calculated as the percentage of EGP suppression during the low and high insulin phase. Nonoxidative glucose disposal (NOGD) was calculated as Rd minus carbohydrate oxidation, determined with indirect calorimetry. Isotopic enrichment of plasma glucose was determined by electron ionization gas chromatography-mass spectrometry as described previously47.
Before and during the clamp test, indirect calorimetry was performed to measure energy expenditure and substrate utilization. Gas exchange was measured by open-circuit respirometry with an automated ventilated hood system (Omnical, Maastricht, the Netherlands) for 30 min. The Weir equation48 was used to calculate whole-body resting energy expenditure from measurements of oxygen consumption and carbon dioxide production. Carbohydrate, fat and protein oxidation rates were calculated according to Frayn49 and nitrogen was measured in 24 h collected urine samples.
Skeletal muscle biopsies
In the morning on day 15, before the start of the clamp test, a muscle biopsy was obtained from the m.vastus lateralis under local anesthesia (1% lidocaine without epinephrine), according to the technique of Bergström et al.50. A portion of muscle tissue was directly frozen in isopentane and stored at −80 °C until further analysis. Another portion was immediately placed in ice-cold preservation medium and processed for high resolution respirometry.
High-resolution respirometry in permeabilized muscle fibers
A small portion of the muscle biopsy sample was immediately placed in ice-cold biopsy preservation medium (BIOPS; OROBOROS Instruments, Innsbruck, Austria). Muscle fibers were permeabilized with saponin according to the technique of Veksler et al.51. After permeabilization, muscle fibers were transferred into ice-cold mitochondrial respiration buffer (MiRO5; OROBOROS Instruments). Then, permeabilized muscle fibers (~2.5 g wet weight) were used for ex vivo high-resolution respirometry (Oxygraph, OROBOROS Instruments) by measuring oxygen consumption rate upon addition of several substrates. In every protocol applied, first, 4.0 mM malate was added to obtain state 2 respiration followed by addition of 1.0 mM octanoyl-carnitine or in the presence of 5 mM pyruvate. In addition, 2 mM ADP with 10 mM glutamate was added to obtain ADP-driven state 3 respiration of complex I. Then 10 mM succinate was added to obtain state 3 respiration by activating both complex I and II. Finally, 1.0 mM carbonylcyanide p- trifluoromethoxyphenylhydrozone (FCCP) was added (in stepwise titration) to evaluate maximum respiratory capacity.
Magnetic resonance spectroscopy: IHL and IMCL content
On day 14, directly after the BodPod measurement, participants also underwent proton magnetic 1H-MRS to quantify intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) content on a 3 T whole body scanner (Achieva 3T-X, Philips Healthcare, Best, the Netherlands). IHL and hepatic fatty acid composition was quantified as previously described52. Values were corrected for T2 relaxation (T2 water: 26.3 ms and T2 CH2: 57.8 ms) and given as ratios of CH2 peak relative to the sum of CH2 resonance and the unsuppressed water peak (in %). IMCL was measured in the m. tibialis anterior of the left leg, as previously described53. Values are given as T1- and T2-corrected ratios of the CH2 peak54 relative to the unsuppressed water peak (in %). Due to analytical problems only 13 participants could be included in the analyzes of IMCL.
After the MRS measurements, in the late afternoon of day 14 of each intervention arm, participants consumed a standardized dinner before they went into the respiration chamber: a small room with a bed, toilet, TV and computer. During the overnight stay (for 12 hours) in this chamber, oxygen consumption and carbohydrate production were measured continuously in sampled room air. Sleep metabolic rate (SMR), substrate oxidation and sleep respiration quotient (RQ) were measured using direct calorimetry equipment (Omnical, Maastricht, the Netherlands). SMR was calculated as the lowest average 3-h energy expenditure during the sleep. At 6 AM the next morning, participants were woken up and left the respiration chamber.
Venous blood samples were taken throughout the study in which routine medical laboratory analysis were performed (Tables 1 and 2). The metabolites phenylbutyrate, BCAA, BCKA and 3-HIB were analyzed in plasma by LC-MS, as previously described23.
To extract metabolites from serum samples, 100 μl − 20° 40:40:20 methanol:acetonitrile:water (extraction solvent) was added to 5 μl of serum sample and incubated in −20 °C for 1 hour, followed by vortexing and centrifugation at 16,000 × g for 10 min at 4 °C. The supernatant (first extract) was transferred to a new tube. Then, 50 μl extraction solution was added to resuspend the pellet, followed by vortexing and centrifugation at 16,000 × g for 10 min at 4 °C. The supernatant (second extract) was combined with the first extract. Then, 3 μl among the 150 μl extract was loaded to LC-MS. A quadrupole-orbitrap mass spectrometer (Q Exactive, Thermo Fisher Scientific, San Jose, CA) operating in negative or positive ion mode was coupled to hydrophilic interaction chromatography via electrospray ionization and used to scan from m/z 70 to 1000 at 1 Hz and 75,000 resolutions. LC separation was on a XBridge BEH Amide column (2.1 mm × 150 mm, 2.5 μm particle size, 130 Å pore size; Waters, Milford, MA) using a gradient of solvent A (20 mM ammonium acetate, 20 mM ammonium hydroxide in 95 :5 water: acetonitrile, pH 9.45) and solvent B (acetonitrile). Data were analyzed using the MAVEN software55. Isotope labeling was corrected for natural 13C abundance56. Flow rate was 150 μL/min. The LC gradient was: 0 min, 85% B; 2 min, 85% B; 3 min, 80% B; 5 min, 80% B; 6 min, 75% B; 7 min, 75% B; 8 min, 70% B; 9 min, 70% B; 10 min, 50% B; 12 min, 50% B; 13 min, 25% B; 16 min, 25% B; 18 min, 0% B; 23 min, 0% B; 24 min, 85% B; 30 min, 85% B. Autosampler temperature is 5 °C, and injection volume is 3 μL.
On day 14 of each intervention period, participants were advised to have a lunch at 12:00 and to remain fasted until they arrived at the research unit. In the afternoon, participants underwent a body composition measurement with the BodPod ® (Cosmed, California, USA). Body mass and body volume were assessed as previously described57.
All results were normally distributed and presented as mean ± SE. The intervention effect was analyzed using the paired student t-test and correlations by using Pearson’s correlation coefficient. Statistics were performed using SPSS 26.0 for Mac and a two-sided p < 0.05 was considered statistically significant.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.