Effect of l-arginine on energy metabolism, skeletal muscle and brown adipose tissue in South Asian and Europid prediabetic men: a randomised double-blinded crossover study

Aims/hypothesis Individuals of South Asian origin are at increased risk of developing type 2 diabetes mellitus and associated comorbidities compared with Europids. Disturbances in energy metabolism may contribute to this increased risk. Skeletal muscle and possibly also brown adipose tissue (BAT) are involved in human energy metabolism and nitric oxide (NO) is suggested to play a pivotal role in regulating mitochondrial biogenesis in both tissues. We aimed to investigate the effects of 6 weeks of supplementation with l-arginine, a precursor of NO, on energy metabolism by BAT and skeletal muscle, as well as glucose metabolism in South Asian men compared with men of European descent. Methods We included ten Dutch South Asian men (age 46.5 ± 2.8 years, BMI 30.1 ± 1.1 kg/m2) and ten Dutch men of European descent, that were similar with respect to age and BMI, with prediabetes (fasting plasma glucose level 5.6–6.9 mmol/l or plasma glucose levels 2 h after an OGTT 7.8–11.1 mmol/l). Participants took either l-arginine (9 g/day) or placebo orally for 6 weeks in a randomised double-blind crossover study. Participants were eligible to participate in the study when they were aged between 40 and 55 years, had a BMI between 25 and 35 kg/m2 and did not have type 2 diabetes. Furthermore, ethnicity was defined as having four grandparents of South Asian or white European origin, respectively. Blinding of treatment was done by the pharmacy (Hankintatukku) and an independent researcher from Leiden University Medical Center randomly assigned treatments by providing a coded list. All people involved in the study as well as participants were blinded to group assignment. After each intervention, glucose tolerance was determined by OGTT and basal metabolic rate (BMR) was determined by indirect calorimetry; BAT activity was assessed by cold-induced [18F]fluorodeoxyglucose ([18F]FDG) positron emission tomography–computed tomography scanning. In addition, a fasting skeletal muscle biopsy was taken and analysed ex vivo for respiratory capacity using a multisubstrate protocol. The primary study endpoint was the effect of l-arginine on BAT volume and activity. Results l-Arginine did not affect BMR, [18F]FDG uptake by BAT or skeletal muscle respiration in either ethnicity. During OGTT, l-arginine lowered plasma glucose concentrations (AUC0–2 h − 9%, p < 0.01), insulin excursion (AUC0–2 h − 26%, p < 0.05) and peak insulin concentrations (−26%, p < 0.05) in Europid but not South Asian men. This coincided with enhanced cold-induced glucose oxidation (+44%, p < 0.05) in Europids only. Of note, in skeletal muscle biopsies several respiration states were consistently lower in South Asian men compared with Europid men. Conclusions/interpretation l-Arginine supplementation does not affect BMR, [18F]FDG uptake by BAT, or skeletal muscle mitochondrial respiration in Europid and South Asian overweight and prediabetic men. However, l-arginine improves glucose tolerance in Europids but not in South Asians. Furthermore, South Asian men have lower skeletal muscle oxidative capacity than men of European descent. Funding This study was funded by the EU FP7 project DIABAT, the Netherlands Organization for Scientific Research, the Dutch Diabetes Research Foundation and the Dutch Heart Foundation. Trial registration: ClinicalTrials.gov NCT02291458. Electronic supplementary material The online version of this article (10.1007/s00125-018-4752-6) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

followed by two experimental days and the last gift of L-arginine was taken the evening before the study days. During the first day an individualized cooling protocol followed by for quantification of BAT volume and activity was performed (2). On the second day, a fasting skeletal muscle biopsy was taken from the vastus lateralis muscle. Furthermore, an oral glucose tolerance test (OGTT) was performed and body composition was determined by means of dual x-ray absorptiometry (DEXA, Discovery A, Hologic, Bedford, MA, USA).
Subjects were instructed to refrain from heavy physical exercise, caffeine and alcohol intake 48h before the first experimental day, and standardized evening meals were prescribed the day before each experimental day.
Two Europid subjects did not complete the study. One subject due to abdominal complains during the first supplementation period (after deblinding upon consultation with an independent physician this appeared to be L-arginine) and one subject because he moved abroad. Both subjects were replaced and baseline characteristics are based on calculations excluding these subjects.

Individualized cooling and PET-CT scanning
The individualized cooling protocol (2; 3) on the first experimental day was initiated at noon, after a 4-h fasting period. For this purpose, a cannula was inserted in the right antecubital vein for blood sampling during thermoneutral and mild cold conditions and injection of the CT scan was performed. The PET-CT imaging protocol started with a low-dose CT scan (120 kV, 30mAs), immediately followed by a static PET scan (6 to 7 bed positions, 4 min per bed position) covering the range from the skull to the abdomen.

PET-CT analysis
PET-CT scans were analyzed using PMOD software (version 3.0, PMOD Technologies, Zurich, Switzerland) by both the researcher (MJWH) and an experienced nuclear medicine physician liver and brain, as described previously (4). As such, we were able to compare [ 18 F]FDG uptake (calculated as SUVmean) between these tissues and between placebo and L-arginine interventions. Due to technical reasons, we could not perform a PET-CT scan in 1 of the South Asian subjects (after placebo treatment).

Ex vivo skeletal muscle respiration
The fasting skeletal muscle tissue, acquired at the second study day, was instantly placed in ice-cold preservation medium (BIOPS, OROBOROS Instruments, Innsbruck, Austria) and used for the preparation of permeabilized muscle fibers, as described previously (5).
Subsequently, oxygen consumption of these permeabilized muscle fibers (2.5-3.0 mg wet weight) was measured using an oxygraph (OROBOROS Instruments, Innsbruck, Austria), in essence according to Hoeks et al. (6). Respiration chambers were hyperoxygenated to ~360 μmol/l O2. State 2 respiration was initiated by addition of malate (4 mM) and octanoylcarnitine (4 mM). Subsequently, ADP was added to evaluate coupled (state 3) respiration. Coupled respiration was then maximized by subsequent addition of glutamate (10 mM) and succinate (10 mM). Finally, the chemical uncoupler FCCP was titrated to evaluate the maximal capacity of the electron transport chain (State U). The integrity of the outer mitochondrial membrane was assessed by addition of cytochrome C (20 μM) upon maximal coupled respiration. Respiration measurements that displayed a cytochrome C response >10% above maximal coupled respiration were excluded from analysis. All measurements were performed in quadruplicate.

Muscle biopsy analysis
Protein levels were determined by Western blotting according to standard procedures.

Oral glucose tolerance test
To assess glucose tolerance in participants, an OGTT was performed one hour following the muscle biopsy. A cannula was inserted in the antecubital vein for blood sampling and the participant drank 75 gram of dextrose dissolved in 300 mL of tap water. Blood was drawn at t = 0, 10, 20, 30, 40, 50, 60, 90 and 120 min. Blood samples were cooled on ice and centrifuged. Next, plasma was obtained, snap-frozen in liquid nitrogen and stored at -80° C until further analysis.

Plasma measurements
Plasma glucose, NEFA and triglycerides were determined with an automated spectrophotometer (ABX Pentra 400 autoanalyzer) by using enzymatic colorimetric kits.
Plasma glycerol concentrations were measured with an enzymatic assay (Enzytec Glycerol; Roche Biopharm) automated on a Cobas Fara spectrophotometric autoanalyzer (Roche Diagnostics). Insulin levels were analyzed by using commercially available radioimmunoassay kits (Human Insulin-specific Radioimmunoassay, Millipore Corporation).

Calculations and statistical analyses
During OGTT, AUC values were determined using the trapezoid rule (7). Incremental values were calculated by deducting the area below the baseline value from total AUCs. Insulin all subjects combined and the separate study groups (South Asians and Europids). For normally distributed data, two-sided independent sample t tests were used to compare finding between the study groups, and two-sided paired sample t tests were used to compare findings between placebo and L-arginine treatments. For not normally distributed data, Mann-Whitney U tests and Wicoxon signed rank tests were used, respectively. ANCOVA was used to correct parameters for fat free mass. Pearson correlations were used to identify correlations between variables. P-values<0.05 were considered statistically significant. Data are reported as mean±SEM.