4.2.1 Anthropometric measurements and laboratory assays (Studies I-III) Height was measured without shoes to the nearest 0.5 cm, and weight in light clothing. BMI was calculated as weight (kg) divided by height (m) squared. Fasting laboratory measurements included plasma glucose and insulin, lipids, lipoproteins, and mass spectrometry metabolomics (Metabolon, Durham, NC). An OGTT was
performed to evaluate glucose tolerance. Plasma glucose was measured by an enzymatic hexokinase photometric assay (Konelab Systems Reagents, Thermo Fischer Scientific, Vantaa, Finland), insulin by immunoassay (ADVIA Centaur Insulin IRI, no 02230141, Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA), and serum ALT by an enzymatic photometric test (Konelab Reagent System, Thermo Fisher Scientific, Vantaa, Finland). Plasma adiponectin was measured using ELISA (human adiponectin ELISA kit; Linco Research, St. Charles, MI), and high sensitivity C-reactive protein (hs-CRP) was assayed using kinetic immunoturbidimetry (near infrared particle immunoassay; IMMAGE Immunochemistry System; Beckman Coulter, Fullerton, CA). Total TAG, plasma FFAs, high-density lipoprotein cholesterol (HDLC), and low-density lipoprotein cholesterol (LDLC) were measured using enzymatic colorimetric tests (Konelab Systems Reagents; Thermo Fisher Scientific, Vantaa, Finland).
4.2.2 OGTT (Studies I-III)
In Study I the OGTT (75 g glucose) included seven time-points (0, 15, 30, 45, 60, 90 and 120 min). Glucose, insulin and lactate levels were measured at all time-points.
We calculated the relative increases in lactate levels compared with the fasting lactate. In Studies II-III OGTT included three time-points (0, 30 and 120 min). The Matsuda insulin sensitivity index (ISI) was calculated based on glucose and insulin measurements in an OGTT as previously described (Stancáková et al., 2012).
4.2.3 Metabolomics analysis (Study II-III)
Metabolites were measured by using Metabolon Inc.’s untargeted Discovery HD4 platform based on Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectroscopy (UPLC-MS/MS) (Metabolon, Morrisville, NC, USA). In brief, several recovery standards were added prior to the first step in the extraction process for quality control purposes. Extracted water samples served as process blanks, and standards that were carefully chosen not to interfere with the measurement of endogenous compounds were spiked into every analyzed sample, which allowed instrument performance monitoring and aided chromatographic alignment.
Experimental samples were randomized across the platform run with quality control samples spaced evenly. Raw data were extracted, peak-identified and quality control processed using Metabolon’s hardware and software, and peaks quantified using area-under-the-curve. Compounds were identified by comparison to library entries of purified standards or recurrent unknown entities. The average percent of imputed metabolites among all metabolites was 5%. The determination of the metabolites was performed in three batches. A total of 857 unique metabolites were included in current statistical analysis. The sub-classification of the lipids was based on the Human Metabolome Database (http://www.hmdb.ca).
4.2.4 Genotyping (Studies I-III)
We genotyped the genetic variants of rs738409, rs58542926, rs641738, rs780094, rs2126259, rs3761472, rs72613567, rs4880, rs7946 and rs1137101 using specific TaqMan assays (ThermoFisher) in a 7500 Real-Time Polymerase Chain Reaction (PCR) System (Applied Biosystems) or the Sequenom iPlex Gold SBE assay at the National Human Genome Research Institute at the National Institutes of Health as previously described (Stancáková et al., 2012).
4.2.5 Cell culture methods (Study I)
HepG2 cells (ATCC, HB-8065) were cultured in Dulbecco’s modified Eagle medium (DMEM; 4.5 g/L glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin; LONZA) supplemented with 10% fetal bovine serum (FBS; GIBCO).
Cells were seeded at 0.2–0.3 × 106 cells/ml. Cryopreserved human primary hepatocytes (HPH) cells from a single non-diabetic donor were purchased from Biopredic International and cultured on collagen I coated 24-well plates at 0.8 × 106 cells/ml following the recommended protocol36,37. Both HepG2 and HPH cells were incubated on 24 well plates at 37 °C in 5% CO2 in a humidified incubator overnight before further manipulations.
HepG2 cells were co-transfected with plasmids expressing human GCK (SC127236, Origene) and GKRP (SC119244, Origene) or GCK and the control vector pCMV6-XL4 (Origene) in 1:0, 1:1 and 1:3 plasmid molar ratios (GCK to GCKR; GCK to pCMV6-XL4). Forty-eight hours after transfection the cells were serum starved overnight in low glucose DMEM (1 g/L glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin) followed by stimulation with glucose at the indicated concentrations in DMEM (supplemented only with 2 mM glutamine). L-lactate assay was performed according to the manufacturer´s instructions (ab65331, Abcam).
HPH cells were transfected with the control vector, or the vectors expressing GCK or GKRP using the Tergefect-Hepatocyte transfection kit (Tergeting Systems).
The cells expressed the constructs for 72 hours before conducting the stimulation with glucose and lactate assay as described for HepG2 cells. The concentration of proteins was measured with BCA protein assay. Samples were prepared with NuPage LDS sample buffer (Life Technologies) and loaded into 4–12% NuPAGE Bis-Tris gels (Life Technologies) for gel electrophoresis. Next, gel-immobilized proteins were transferred to polyvinylidene fluoride (PVDF) membranes (GE Healthcare) for immunodetection. A mouse monoclonal antibody (RRID:AB_2107650; sc74552, Santa Cruz) and a rabbit polyclonal antibody (RRID:AB_2232078; ab 37796, Abcam) were used for GKRP and GCK detection, respectively. ß-actin was used as loading control and it was detected with a goat polyclonal antibody (RRID:AB_630836;
sc1616-R, Santa Cruz). Anti-mouse (NA931V, GE Health care), anti-rabbit (NA934V,
GE Health Care) and anti-goat (sc-2020, Santa Cruz) HRP-conjugated IgGs were used accordingly for the detection of the bands by chemiluminiscence (ECL Plus, Pierce). For image acquisition, an Image Quant RT-ECL equipment was used. Band quantitation was carried out with the Gels tool from ImageJ.
HepG2 cells were transfected with a plasmid expressing green fluorescence protein (GFP) or a plasmid expressing human FOXA2 with Lipofectamine 3000. GFP was used as over-expression control. All constructs were expressed for 48 h before overnight serum starvation. Since glucagon induces FOXA2 activation through acetylation, we used a HAT inhibitor II (abcam) as a control treatment for 2 h. For stimulation of HPH, we adapted a protocol previously reported to evaluate the effect of glucagon on FOXA2 activation. After glucagon stimulation, both HepG2 and HPH cells were subject to total RNA extraction with an RNeasy Mini Kit. GCKR mRNA and RPLP0 mRNA expression were measured with specific TaqMan gene expression assays (Thermo Fisher Scientific) in a 7500 Real-Time PCR System (Applied Biosystems).