This cross-sectional study included 30 obese premenopausal black and white women, matched for age (30–45 years) and BMI (≥30 kg/m2), with no known diseases, not pregnant or lactating, and who consumed <20 g alcohol/day. The study was undertaken in accordance with the guidelines of The Declaration of Helsinki and approved by the University of Cape Town Faculty of Health Sciences Human Research Ethics Committee. Participants gave written informed consent prior to participation.
A questionnaire was administered to measure family history of type 2 diabetes, smoking, alcohol and dietary intake (food frequency) . Physical activity was measured using actigraphy (ActiGraph LLC, Pensacola, FL, USA). Fat mass, fat-free mass (FFM), abdominal visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) areas were measured by dual energy x-ray absorptiometry (DXA, Discovery-W, software 188.8.131.52; Hologic, Bedford, MA, USA).
Fasting blood samples were drawn for metabolites, insulin and adipocytokines before a standard 75 g OGTT. On another day, a two-step euglycaemic (±5 mmol/l), hyperinsulinaemic clamp, with 6,6-[2H2]glucose isotope label was performed, with a 3 h low-dose insulin infusion (10 mU m−2 min−1), followed by a 2 h higher dose insulin infusion (40 mU m−2 min−1), with samples drawn and respiratory exchange ratio (RER) measured (Quark RMR, Cosmed, Rome, Italy) in the last 30 min of each period. Serum metabolites, insulin and adipocytokines were measured using standard techniques (Electronic Supplementary Material [ESM] Methods) and 6,6-[2H2]glucose was measured using Agilent 6890 gas chromatograph and analysed using ChemStation software (Agilent Technologies, Palo Alto, CA, USA).
Hepatic, and intra- (IMCL) and extra-myocellular lipid (EMCL), and total lipid content of the soleus and tibialis anterior (TA) muscles of the calf were measured by 1H-magnetic resonance spectroscopy (MRS) and MRI, respectively, using a 3 Tesla scanner (GE Healthcare, Global Diagnostic Imaging, Pewaukee, WI, USA).
A biopsy was taken from the vastus lateralis muscle from which RNA was extracted and the expression of genes (ESM Table 1) was measured using the Applied Biosystems 7900HT Fast Real-time PCR system using standard cycling conditions (Applied Biosystems, Foster City, CA, USA) and expressed relative to β2 microglobulin.
Differences in participant characteristics were compared using χ
2 analysis, one-way analysis of variance and/or covariance, adjusting for fat mass index (FMI), which takes into account differences in height and fat mass between groups. Bivariate associations were explored using Pearson’s correlation coefficients, which informed multiple regression analyses that included an interaction term (ethnicity × independent variable). Data were analysed using STATA version 11.1 (StataCorp, College Station, TX, USA).