Insulin resistance is associated with altered amino acid metabolism and adipose tissue dysfunction in normoglycemic women

Insulin resistance is associated adiposity, but the mechanisms are not fully understood. In this study, we aimed to identify early metabolic alterations associated with insulin resistance in normoglycemic women with varying degree of adiposity. One-hundred and ten young and middle-aged women were divided into low and high IR groups based on their median HOMA-IR (0.9 ± 0.4 vs. 2.8 ± 1.2). Body composition was assessed using DXA, skeletal muscle and liver fat by proton magnetic resonance spectroscopy, serum metabolites by nuclear magnetic resonance spectroscopy and adipose tissue and skeletal muscle gene expression by microarrays. High HOMA-IR subjects had higher serum branched-chain amino acid concentrations (BCAA) (p < 0.05 for both). Gene expression analysis of subcutaneous adipose tissue revealed significant down-regulation of genes related to BCAA catabolism and mitochondrial energy metabolism and up-regulation of several inflammation-related pathways in high HOMA-IR subjects (p < 0.05 for all), but no differentially expressed genes in skeletal muscle were found. In conclusion, in normoglycemic women insulin resistance was associated with increased serum BCAA concentrations, down-regulation of mitochondrial energy metabolism and increased expression of inflammation-related genes in the adipose tissue.

Prodigy, GE Lunar Corp., Madison, WI USA). Two repeated measurements of FM and FFM showed a coefficient of variation (CV) of 2.2% and 1.0% respectively in this study 2 .
Abdominal region and liver were scanned using a 1.5 Tesla MR-scanner (GE Sigma CV/i, General Electric Healthcare, Waukesha, WI, USA). Abdominal visceral adipose tissue (VAT) was quantified from a single slice image at the level of the L2-L3 intervertebral disc using the OsiriX software (OsiriX Foundation, Geneva, Switzerland). The results were converted into tissue fat mass in kilograms taking into account slice thickness and an adipose tissue density of 0.9196 g/ml 3 . Liver fat content was assessed by 1HMRS with a PRESS sequence and was analyzed using the Linear Combination of Model spectra software which is generally considered to be the gold standard for in-vivo spectroscopy analysis 4 .
Muscle intra-myocellular lipid (IMCL) and extra-myocellular lipid (EMCL) from the tibialis anterior muscle were measured using a similar 1H MRS method with a surface coil placed over the middle part of the muscle 5 . In order to obtain maximal IMCL and EMCL separation the tibialis anterior muscle was aligned as good as possible with the direction of the magnetic field and the voxel was placed parallel to the muscle fibers 5 .

Fitness test
Maximum oxygen uptake (VO2max, ml/kg/min) was assessed by bicycle ergometer. During tests, heart rates were assessed using ECG and respiratory gases and ventilation was measured using respiratory gas analyzer VIASYS (Healthcare Inc. USA). A specialist physician was responsible for monitoring ECG and blood pressure responses during the test and recording subject's signs and symptoms throughout the test.

Biochemical assessments
Blood samples were collected in the morning between 7:00 and 9:00 am after an overnight fasting. Plasma glucose and non-esterified fatty acids (NEFA) were assessed by KONELAB 20XTi analyzer (Thermo Fischer Scientific inc.Waltham, MA, USA). Plasma insulin was assessed by chemiluminescent immunoassay using the IMMULITE analyzer (Diagnostic Products Corporation, Los Angeles). The intra-and inter-assay CVs were 3.4% and 2.0% for glucose, 11% and 3.4% for insulin, and 7.4% and 8.4% for NEFA, respectively. The HOMA-IR index (homeostatic model assessment of insulin resistance) was calculated as (fasting glucose x fasting insulin/22.5). Serum leptin was assessed using human leptin (ELISA; Diagnostic Systems Laboratories, Inc., Webster, TX). Total adiponectin was measured by an enzyme immunoassay method using the Quantikine human total adiponectin/Acrp30 immunoassay (R&D Systems, Minneapolis, MN). Inter-and intra-assay coefficients of variation (CVs) were 2.2% and 2.7% for leptin, 3.3% and 4.3% for adiponectin, respectively

Insulin sensitivity
Whole-body insulin sensitivity was determined by the 75-g oral glucose tolerance test (OGTT). The test was performed for a subset of subjects (n=24) and insulin sensitivity index (Matsuda index) was calculated using the 0, 60 min and 120 min time points according to Matsuda and DeFronzo 6 .

Serum NMR spectroscopy
All serum samples were analyzed using a high-throughput serum NMR metabonomics platform; the experimental protocols including sample preparation and NMR spectroscopy have been described in detail elsewhere 7 . This methodology has recently been applied in various large-scale epidemiological and genetic studies 8,9 . The NMR metabolomics methodology provides comprehensive quantitative information on various amino acids, glycolysis intermediates, fatty acid composition and degree of saturation and lipoprotein subclass distributions. Altogether 130 metabolites were assessed.

Subcutaneous adipose tissue biopsies
Twenty-four participants agreed to donate subcutaneous adipose tissue biopsies, which were obtained under local anesthesia after overnight fasting. A region 5 cm lateral from the umbilicus either to the left side or right side was sterilized. A small intracutaneous injection was made, and 2 ml of a local anesthetic agent (lidocaine) was injected. After 5 min, anesthesia was confirmed, skin was sterilized again and 16 G, 40 mm needle, was then adapted to a 50-mL syringe and 10ml of 0.9% sodium-chloride was aspirated. Approximately two-third of the length of the needle was inserted into the subcutaneous fat, and 5 ml of 0.9% sodium chloride was injected. The needle piston was then pulled back maximally and released until it was locked by a stopper, thereby creating a vacuum. Tissue resistance was created by gripping the abdominal skin with one hand while the other hand rotated the needle throughout the tissue in back and forth motion. Once the tissue was aspirated by the syringe, the needle was withdrawn, and the piston was removed. The adipose tissue samples were washed with saline solution, and were immediately frozen in liquid nitrogen and stored at -80°C.

Skeletal muscle biopsies
Twenty-four participants agreed to donate skeletal muscle biopsies, which were obtained under local anesthesia after overnight fasting. Biopsies were obtained from the vastus lateralis dx muscle with a 5-mm Bergström biopsy needle, midway between the patella and greater trochanter. A region and the optimum depth for muscle biopsy were confirmed by ultrasound imaging. The skin of the identified location was sterilized and 4 ml of local anesthetic agent (lidocaine) was injected in to the procedure area. A cooling pack was then applied on the location. After 10 minutes, anesthesia was confirmed, skin was sterilized again and small stab incision was made with surgical scalpel. Subsequently, the biopsy needle attached to a syringe was introduced perpendicularly into the incision. The piston was then pulled back maximally creating a vacuum and sample was obtained. After the muscle biopsy was obtained, pressure was applied to the incision site for hemostasis. The muscle sample was cleaned of any visible connective and adipose tissue as well as blood and was frozen immediately in liquid nitrogen (−180°C) and stored at −80°C.

RNA extraction
Total RNA was extracted from biopsies using the FastPrep system (MP Biomedicals, France) and the RNeasy Lipid Tissue Mini Kit (QIAGEN, Gaithersburg, MD, USA) according to manufacturer's instructions. Total RNA was digested on column with the RNase-free DNase set (QIAGEN) during RNA isolation. The quality of the total RNA was studied using a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and Experion Automated Electrophoresis Station (BioRad, Hercules, CA, USA). The total RNA was amplified and processed using the Gene Chip 3´ IVT Express Kit (Affymetrix, Santa Clara, CA, USA) and hybridized on Affymetrix Human Genome U219 Array Plates. The samples of this study have been submitted to Array Express. Array Express data are fully accessible in E-MTAB-2649.

Microarray analysis
The total RNA was amplified and processed using the GeneChip 3´IVT Express Kit (Affymetrix, Santa Clara, CA, USA) and hybridized on Affymetrix Human Genome U219 Array Plates as described previously 10 . Microarray data was pre-processed by the Robust Multiarray Averaging (RMA) algorithm in the R package affy 11-13 . Differentially expressed genes (DEG) were identified with the limma R package utilizing linear modeling and empirical Bayes methods. Raw p-values were adjusted using the Benjamini and Hochberg multiple adjustment method 14 .

Gene enrichment analysis
The enriched Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways for a given gene set were calculated using the R packages GOStats and KEGG.db. In the enrichment analysis, all human ENSEMBL genes were used as a background gene group and categories with a p-value lower than 0.05 were considered significantly enriched. Genes related to HOMA-IR were identified using the following two criteria: Genes were differentially expressed in our DEGanalysis between the low HOMA-IR and high HOMA-IR groups with adjusted p-value <0.05 or genes fold change >= 2 between the low and high HOMA-IR groups.
For the gene pathways derived from KEGG enrichment analysis, the mean-centroid value representing the "activity" of the regulated part of the pathway was computed by normalizing the expression levels of all subset genes to a mean of zero and a variance of 1 across all individuals. The mean centroids have previously been shown to correlate with various metabolic and physiologic parameters 15,16 , and may therefore be used to assess gene expression patterns that are associated with metabolic diseases.

Protein extraction from skeletal muscle biopsies and Western blot analysis
Muscle biopsies were homogenized in ice-cold lysis buffer [20 mM Tris-HCl (pH 7.4), 1 mM EDTA, 150mM NaCl, 100 mM β-glycerophosphate, 1 mM Na3VO4, 1 mM DTT, 1% Triton-X-100], supplemented with protease and phosphatase inhibitors inhibitors (Sigma Aldrich, St Louis, MO, USA). Thirty to sixty micrograms of muscle lysate samples were separated by SDS-Page using 4-20% gradient gels on Criterion electrophoresis cell (Bio-Rad Laboratories, Richmond, CA). Proteins were transferred to nitrocellulose membranes at 300-mA constant current on ice at 4ºC. Membranes were blocked in TBS containing 5% nonfat dry milk for 1 hour at room temperature (RT), and then probed overnight at 4ºC with primary antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) (p-Akt, p-IRβ and p-AS160), Sigma-Aldrich (anti-GAPDH) and Abcam (MitoProfile® Total OXPHOS Rodent WB Antibody Cocktail, Abcam, Cambridge, MA, USA). All antibodies were diluted 1:1.000 (except anti-GAPDH (housekeeping, which was diluted 1:40.000) in TBS containing 5% nonfat dry milk. Membranes were then washed with TBS containing 0.1% Tween-20 (TBS-T) followed by 1 hour incubation with the secondary antibody. Odyssey anti-rabbit IRDye 800 and Odyssey anti-mouse IRDye 600 (LI-COR Biosciences, Lincoln, NE, USA) were used as a secondary antibody. Blots were visualized and quantified using Odyssey CLX Infrared Imager of Li-COR and manufacturer's software. When reprobing was needed, the membranes were incubated in 0.2 M NaOH for 10 min at RT, washed with TBS and reprobed with appropriate antibodies. All samples were run in the same gel to minimize the variability and the quantitative results for each protein were normalized to GAPDH.  Table S2. General characteristics and serum metabolites in normal weight individuals stratified by low and high HOMA-IR groups (MIXED model estimated marginal means with 95% confidence intervals are given taking into account genetic similarity and shared environment (daughter and mother) and contrast estimates' p-values were used to localize the significant differences between the two groups and group by generation interaction).