Skeletal muscle metabolism was studied by measuring arterio-venous concentration differences

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1 Supplemental Data Dual stable-isotope experiment Skeletal muscle metabolism was studied by measuring arterio-venous concentration differences across the forearm, adjusted for forearm blood flow (FBF) (1). After collection of an arterialized and deep venous (contralateral arm) blood sample for background enrichment at t-90 (90min before meal ingestion), a continuous intravenous infusion (0.035 µmol min -1 per kg body weight) of the stable isotope tracer [ 2 H 2 ] -palmitate complexed to albumin (97% enrichment: Cambridge Isotope Laboratories, Andover, MA, USA) was started using a calibrated infusion pump (IVAC560 pump; IVAC corporation, San Diego, CA USA). Fasting blood sampling was started after 1h of tracer infusion to allow for isotopic equilibration to occur. Arterialized and deep venous blood samples were collected simultaneously at three baseline time points (t-30, t-15 and t0) and at several time points (t30, t60, t90, t120, t180 and t240) after consumption (<5 min) of a standardized liquid high-fat mixed-meal (t0) (2.6 MJ, consisting of 61E% fat (35.5E% SFA, 18.8E% MUFA and 1.7E% PUFA), 33E% carbohydrate and 6E% protein), containing 200 mg [ U- 13 C] -palmitate (98% enrichment; Cambridge Isotope Laboratories). Skeletal muscle biopsies ( mg) were taken from m. vastus lateralis under local anesthesia using the Bergström technique with suction during fasting conditions and at the end of the postprandial period (t240) to determine intramuscular TAG, DAG, FFA, and phospholipid (PL) content, their degree of saturation, as well as the fractional synthetic rates (FSR) of TAG, DAG, and PL (1), as described below. Skeletal muscle lipids Skeletal muscle biopsies were lyophilized and dissected free of extramyocellular lipid, blood, and connective tissue. Total lipids were extracted from 10-20mg muscle sample using chloroform-

2 methanol (2:1 vol/vol) and internal standards, and thereafter evaporated under nitrogen at 37 C. The extracted lipids were separated into FFA, DAG, TAG, and PL by thin-layer chromatography and transferred into tubes for methylation. The TAG and DAG fractions were methylated by adding 1 ml of toluene-methanol BF3-methanol (14%) (20%-55%-25% vol/vol), and were incubated in capped tubes for 30 min at 100 C. The PL fraction was methylated by adding 1 ml of BF3-methanol (14%) and incubation in capped tubes for 90min at 100 C. The FFA fraction was methylated by adding 1 ml of methanol BF3 methanol (14%) (50%-50% vol/vol) and incubation in capped tubes at room temperature for 15min. After incubation, 2ml pentane was added to the samples, vortexed, and centrifuged (1,000 g, 5min, 20 C), followed by isolation of pentane extracts (upper phase) and evaporation under nitrogen at 30 C. Finally, the residues were dissolved in iso-octane and concentrations of FAs in the fractions were determined using an analytical GC. Stable isotope enrichment of the lipid fractions was determined by measuring the 13 C-to- 12 C ratios on a gas chromatography continuous-flow isotope ratio-mass spectrometer (Finnigan MAT-252). Tracer enrichment To determine tracer enrichment in plasma FFA and TAG, we extracted total lipids from the plasma using chloroform/methanol 2:1. The FFA and TAG fractions were separated by thin-layer chromatography and derivatized to their methyl esters for the analysis of plasma palmitate. Plasma fractions were analyzed for the 13 C/ 12 C ratio in a gas chromatography continuous-flow isotope ratio-mass spectrometer (Finnigan MAT-252 GC-IRMS, Bremen, Germany) and for enrichment of [ 2 H 2 ] (Finnigan Incos-XL GC-MS, Bremen, Germany). The methyl ester of palmitate contains 17 carbon atoms and therefore, the tracer/tracee ratio of palmitate was corrected for the extra methyl group. Plasma palmitate concentrations (µmol min -1 ) were

3 analyzed on an analytical GC with ion flame detection using heptadecanoic acid as internal standard. Calculations The net flux of metabolites (labeled and unlabeled) across the forearm muscle was calculated by multiplying the arterio-venous concentration difference by forearm plasma flow (FPF). FPF was calculated by multiplying FBF with (1-hematocrite/100). A positive flux indicates net uptake across forearm muscle, whereas a negative flux indicates net release from the tissue. Fractional extraction of metabolites (in %) was calculated as the arteriovenous concentration difference divided by the arterial concentration (((A-V)/A)*100%). Fasting rate of appearance of FFA (Ra FFA ) (µmol kg -1 min -1 ) was calculated with Steele s equation for steady state, whereas Steele s single-pool non-steady state equations adapted for use with stable isotopes was used to calculate Ra FFA in the postprandial state (2). Labeled FFA and TAG concentrations were calculated as the product of tracer/tracee ratio (TTR) of [ 2 H 2 ] - and [ U- 13 C] -palmitate and the palmitate concentration in FFA and TAG, respectively. Total uptake of FA derived from lipoprotein lipase-mediated TAG hydrolysis was calculated as described previously, with the assumption of incomplete uptake of TAG-derived FA by forearm muscle (3). FSR of skeletal muscle TAG, DAG and PL were calculated by using skeletal muscle FFA as the precursor pool for lipid synthesis (4). The increase in TTR of [ U- 13 C] from fasting to 4h postprandial was divided by the average enrichment of skeletal muscle FFA from fasting and 4h postprandial, and is expressed as percentage per hour (%/h). The degree of saturation of skeletal muscle TAG, DAG, PL and FFA (%) was calculated by dividing the sum of saturated FA by the total amount of FA in a fraction. 9-desaturase activity was estimated as the proportion palmitoleic acid (C16:1n-7) to palmitic acid (C16:0), and as the proportion oleic acid (C18:1n-9) to stearic acid (C18:0). Total skeletal muscle TAG, DAG, PL and FFA contents were estimated as

4 the sum of the particular FA content of the assessed fraction. For comparison of postprandial responses, the area under the curve (AUC) or incremental AUC (iauc) was calculated for early (0-2h), mid (2-4h) and total (0-4h) postprandial phase. References 1. van Hees AM, Jans A, Hul GB, Roche HM, Saris WH, Blaak EE 2011 Skeletal muscle fatty acid handling in insulin resistant men. Obesity (Silver Spring) 19: Wolfe R 1992 Radioactive and Stable Isotope Tracers in Biomedicine: Principles and Practice of Kinetic Analysis. Wiley Liss: New York, NY 3. Bickerton AS, Roberts R, Fielding BA, Hodson L, Blaak EE, Wagenmakers AJ, Gilbert M, Karpe F, Frayn KN 2007 Preferential uptake of dietary Fatty acids in adipose tissue and muscle in the postprandial period. Diabetes 56: Guo Z, Jensen MD 1998 Intramuscular fatty acid metabolism evaluated with stable isotopic tracers. J Appl Physiol 84:

5 Supplemental Table 1 Fasting and postprandial metabolism before and after 26-wks treatment Forearm blood flow Fasting (ml 100ml -1 min -1 ) Postprandial (0-4h) Net flux across forearm muscle Glucose Fasting (µmol 100ml -1 min -1 ) Glycerol FFA [ 2 H 2 ]-palmitate FFA TAG [ 2 H 2 ]-palmitate TAG [U- 13 C]-palmitate TAG Placebo (n=12) Valsartan (n=14) P-value # Baseline 26 wks Baseline 26 wks 2.5± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.36 NA 1.22± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.50 NA 0.33± ± ± ± ± ± ± ± ± ± ±20.5* 3.7± ± ± ± ± ± ± ± ± ± ±1.47 NA 0.11± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.57 NA 0.28± ±0.25 *P<0.05 between placebo and valsartan at baseline. FFA, free fatty acids; TAG, triacylglycerol; NA, not applicable. # Valsartan (VAL) vs. placebo (PLB) using repeated-measures ANOVA with gender and glucose status as covariates (with Bonferroni post-hoc analysis). Values are mean ± SEM. Postprandial data are indicated as AUC/min. A positive flux indicates net uptake across forearm muscle, whereas a negative flux indicates net release

6 Supplemental Table 2 Skeletal muscle lipid content and composition under fasting conditions and fractional synthetic rate after a high-fat mixed-meal before and after 26-wks treatment TAG Total (μmol/g dry weight) Saturation (%) MUFA (%) PUFA (%) FSR (%/h) DAG Total (μmol/g dry weight) Saturation (%) MUFA (%) PUFA (%) FSR (%/h) PL Total (μmol/g dry weight) Saturation (%) MUFA (%) PUFA (%) FSR (%/h) FFA Total (μmol/g dry weight) Saturation (%) MUFA (%) PUFA (%) Placebo (n=12) Valsartan (n=14) P-value # Baseline 26 wks Baseline 26 wks 332.5± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.7 TAG, triacylglycerol; DAG, diacylglycerol; PL, phospholipids; FFA, free fatty acids; MUFA, monounsaturated fatty acids; PUFA, poly-unsaturated fatty acids; FSR, fractional synthetic rate. # Valsartan (VAL) vs. placebo (PLB) using repeated-measures ANOVA with gender and glucose status as covariates (with Bonferroni post-hoc analysis). Values are mean ± SEM

7 Figure legends Supplemental Figure 1: Arterialized plasma (A) glucose, (B) insulin, (C) FFA and (D) TAG concentrations under fasting conditions (t0) and after consumption of a high-fat mixed-meal before and after 26-wks treatment with valsartan (VAL) or placebo (PLB). PLB baseline: black circles, solid line; VAL baseline: black squares, solid line; PLB intervention: open circles, dashed line; VAL intervention: open squares, dashed line. Values are mean ± SEM.

8 Supplemental Figure 1