Integration of whole-body [18F]FDG PET/MRI with non-targeted metabolomics can provide new insights on tissue-specific insulin resistance in type 2 diabetes

Alteration of various metabolites has been linked to type 2 diabetes (T2D) and insulin resistance. However, identifying significant associations between metabolites and tissue-specific phenotypes requires a multi-omics approach. In a cohort of 42 subjects with different levels of glucose tolerance (normal, prediabetes and T2D) matched for age and body mass index, we calculated associations between parameters of whole-body positron emission tomography (PET)/magnetic resonance imaging (MRI) during hyperinsulinemic euglycemic clamp and non-targeted metabolomics profiling for subcutaneous adipose tissue (SAT) and plasma. Plasma metabolomics profiling revealed that hepatic fat content was positively associated with tyrosine, and negatively associated with lysoPC(P-16:0). Visceral adipose tissue (VAT) and SAT insulin sensitivity (Ki), were positively associated with several lysophospholipids, while the opposite applied to branched-chain amino acids. The adipose tissue metabolomics revealed a positive association between non-esterified fatty acids and, VAT and liver Ki. Bile acids and carnitines in adipose tissue were inversely associated with VAT Ki. Furthermore, we detected several metabolites that were significantly higher in T2D than normal/prediabetes. In this study we present novel associations between several metabolites from SAT and plasma with the fat fraction, volume and insulin sensitivity of various tissues throughout the body, demonstrating the benefit of an integrative multi-omics approach.


S1. MoDentify
MoDentify identifies modules of metabolites associated with a selected outcome, that was Mvalue in this case 1 . It gains statistical power from metabolomics cohorts originating from multiple tissues. In this case, we used MoDentify to compute partial correlations of pairs of metabolites for SAT and plasma, while regressing out the other metabolites. Partially correlated metabolites were then represented into a network which was used as the base to identify functional modules of metabolites for the unified set of tissues. A score maximization algorithm was used to "walk" the network and to identify modules that are significantly associated with M-value at FDR<0.1, while controlling for BMI, WHR, age and sex. MoDentify identified two modules that we visualized in Cytoscape (Supplementary Figure S5).

S2. Metabolic Profiling
Information about reagents, solvents, standards, reference and tuning standards, and stable isotopes internal standards are displayed in the Supplementary Note -1.4 Metabolic profiling solvents.

Sample Preparation
Sample preparation of plasma was performed according to A et al. 2 . In detail, 900µL of extraction buffer (90/10 v/v methanol: water) including internal standards for both GC-MS and LC-MS (Supplementary Note -1.4 Metabolic profiling solvents) were added to 100µL of serum. The samples were shaken at 30Hz for two minutes in a mixer mill and proteins were precipitated at +4°C on ice. Afterwards, the samples were centrifuged at +4°C, 14000 rpm, for 10 minutes. The supernatants, 200µL for both batches of the LC-MS analysis, and 200µL and 50µL, respectively for the two batches of the GCMS analysis, were transferred to micro vials and evaporated to dryness in a SpeedVac concentrator.
SAT samples were extracted as follows: 500µL of 2/1 (v/v) CHCl3: methanol (including D4-Cholic Acid) and, 100µL water (including 13C9-phenylalanine) and two tungsten beads were added to each sample (18-23mg). The samples were shaken at 30Hz for 3 minutes. The tungsten beads were removed, and the samples were left standing at room temperature for 30 minutes. Samples were centrifuged at 14000 rpm, +4°C for 3 minutes and 80µL of the aqueous phase was transferred to Eppendorf tubes. 320µL methanol (including D6-salicylic acid) was added to the Eppendorf tubes, whereupon remaining proteins were precipitated at -20°C for 1 hour. The samples were centrifuged for 10 minutes at 14000 rpm, +4°C and 50µL supernatant was taken out to GC vials and 200µL for LC-MS. Solvents were evaporated and the samples were stored at -80°C until analysis.
The remaining supernatants of each tissue were pooled and used to create tissue-specific quality control (QC) samples. Tandem mass spectrometry (MS/MS) analysis for LC-MS was performed on the QC samples for identification purposes. Samples were analysed in tissuedependent batches according to a randomized run order on both GC-MS and LC-MS.

GC-MS
Derivatization and GC-MS analysis were performed as described previously 2 . SAT samples were derivatized in a final volume of 30µL rather than 90µL which was used for plasma 3 .

Batch 1
1µL of the derivatized sample was injected in splitless mode by a CTC Combi Pal autosampler (CTC Analytics AG, Switzerland) into an Agilent 6890 gas chromatograph equipped with a 10m x 0.18mm fused silica capillary column with a chemically bonded 0.18 µm DB 5-MS UI stationary phase (J&W Scientific). The injector temperature was 270°C, the purge flow rate was 20mL/min and the purge was turned on after 60 seconds. The gas flow rate through the column was 1mL/min, the column temperature was held at 70°C for 2 minutes, then increased by 40°C/min to 320°C, and held there for 2 minutes. The column effluent was introduced into the ion source of a Pegasus III time-of-flight mass spectrometer, GC/TOFMS (Leco Corp., St Joseph, MI, USA). The transfer line and the ion source temperatures were 250°C and 200°C, respectively. Ions were generated by a 70eV electron beam at an ionization current of 2.0mA, and 30 spectra/s were recorded in the mass range m/z 50-800. The acceleration voltage was turned on after a solvent delay of 150 seconds. The detector voltage was 1500-2000V.

Batch 2
0.5µL of the derivatized sample was injected in splitless mode by a L-PAL3 autosampler (CTC Analytics AG, Switzerland) into an Agilent 7890B gas chromatograph equipped with a 10m x 0.18mm fused silica capillary column with a chemically bonded 0.18µm Rxi-5 Sil MS stationary phase (Restek Corporation, U.S.). The injector temperature was 270°C, the purge flow rate was 20mL/min and the purge was turned on after 60 seconds. The gas flow rate through the column was 1mL/min, the column temperature was held at 70°C for 2 minutes, then increased by 40°C/min to 320°C, and held there for 2 minutes. The column effluent was introduced into the ion source of a Pegasus BT time-of-flight mass spectrometer, GC/TOFMS (Leco Corp., St Joseph, MI, USA). The transfer line and the ion source temperatures were 250°C and 200°C, respectively. Ions were generated by a 70eV electron beam at an ionization current of 2.0mA, and 30 spectra/s were recorded in the mass range m/z 50-800. The acceleration voltage was turned on after a solvent delay of 150 seconds. The detector voltage was 1800-2300V.

LC-MS
The LC-MS was performed identically for both batches. Before LC-MS analysis the sample was re-suspended in 10+10µL methanol and water. Batches from the samples were first analysed in positive mode, next the instrument was switched to negative mode and a second injection of the samples was performed.
The chromatographic separation was performed on an Agilent 1290 Infinity UHPLC-system (Agilent Technologies, Waldbronn, Germany). 2µL of each sample was injected into an Acquity UPLC HSS T3, 2.1 x 50mm, 1.8µm C18 column in combination with a 2.1mm x 5mm, 1.8µm VanGuard precolumn (Waters Corporation, Milford, MA, USA) held at 40°C. The gradient elution buffers were A (H2O, 0.1% formic acid) and B (75/25 acetonitrile:2-propanol, 0.1% formic acid), and the flow-rate was 0.5mL/min. The compounds were eluted with a linear gradient consisting of 0.1-10% B over 2 minutes, B was increased to 99% over 5 minutes and held at 99% for 2 minutes; B was decreased to 0.1% for 0.3 minutes and the flow-rate was increased to 0.8mL/min for 0.5 minutes; these conditions were held for 0.9 minutes, after which the flow-rate was reduced to 0.5mL/min for 0.1 minutes before the next injection.
The compounds were detected with an Agilent 6550 Q-TOF mass spectrometer equipped with a jet stream electrospray ion source operating in positive or negative ion mode. The settings were kept identical between the modes, with the exception of the capillary voltage. A reference interface was connected for accurate mass measurements; the reference ions purine (4µM) and HP-0921 (Hexakis(1H, 1H, 3H-tetrafluoropropoxy)phosphazine) (1µM) were infused directly into the MS at a flow rate of 0.05mL/min for internal calibration, and the monitored ions were purine m/z 121.05 and m/z 119.03632; HP-0921 m/z 922.0098 and m/z 966.000725 for positive and negative mode respectively. The gas temperature was set to 150°C, the drying gas flow to 16L/min and the nebulizer pressure 35psig. The sheath gas temp was set to 350°C and the sheath gas flow 11L/min. The capillary voltage was set to 4000V in positive ion mode, and to