From the Pharmacogenomics Biobank of the University of Southern Denmark, healthy individuals were, in 2012, included based on their OCT1 genotype . All individuals were healthy with renal and hepatic functions within normal range as assessed by plasma creatinine and plasma aminotransferase. None had a history of alcohol abuse or took any medication, and all gave written informed consent to participate in the study. The genotypes of OCT1, OCT2 (also known as SLC22A2), MATE1 (also known as SLC47A1) and MATE2-K (also known as SLC47A2) including the haplotypes and diplotypes of the reduced-function alleles in OCT1 have previously been described .
The study was designed as a randomised crossover trial with a wash-out period of at least 4 weeks between the phases. Sealed envelopes were used for the randomisation procedure. The random allocation sequence was generated with assistance from the Good Clinical Practice (GCP) unit, Odense University Hospital, Denmark. The primary investigator enrolled the participants. To ensure that gluconeogenesis was the main contributor to glucose production, the healthy individuals were fasted for 42 h, after which hepatic glycogen stores are known to be almost completely exhausted . Moreover, the liver is the main gluconeogenic source when individuals are fasted for <60 h. However, as time goes by, the contribution from the kidneys will increase .
The individuals were admitted to the Department of Endocrinology, Odense University Hospital, Denmark, at 16:00 hours after 20 h of fast. The next day at 08:00 hours, after 36 h of fast, the experiment was initiated. Two catheters were inserted into contralateral antecubital veins. One of them was used for [3-3H]glucose tracer infusion and the other for collection of blood samples. The latter was placed and maintained in a heated plexiglass box for arterialisation of venous blood. A primed-constant intravenous infusion of [3-3H]glucose was initiated and continued throughout the next 6 h using a precision syringe pump (Harvard Apparatus, Natick, MA, USA). The ratio between priming dose and constant tracer infusion was 100:1. To achieve a common level of basal plasma-specific activity, the tracer infusion rate was adjusted for body surface area by adjustment of the infusate volume as previously described [18, 19]. Blood samples were collected at timed intervals (0, 1, 2, 3, 4, 5, 5.5 and 6 h) for determination of plasma cortisol, plasma NEFA, plasma glucagon, serum insulin and C-peptide. For plasma glucose, plasma lactate and plasma [3-3H]glucose activity, blood samples were collected every 15 min for the 6 h period. Both plasma glucose and lactate were measured bedside. Additional blood samples were immediately centrifuged; serum samples were stored at −80°C and plasma samples at −20°C until analysis. Indirect calorimetry was performed using a ParvoMedics TrueOne 2400 (Sandy, UT, USA) automated gas analysis system. After an equilibration period of 10 min, the average gas exchange rates recorded over the two 30-min steady-state periods (90–120 min and 330–360 min) were used to calculate rates of glucose oxidation, lipid oxidation, and respiratory exchange ratio (RER) and resting energy expenditure (REE) as previously described . The protein oxidation rate was estimated from urinary urea nitrogen excretion (1 g nitrogen = 6.25 g protein) and corrected for changes in pool size.
When the experiment and the sampling started in this phase, all individuals were in steady state with 1,000 mg metformin twice daily. For 7 days, they had ingested tablets of metformin (Metformin ‘Actavis’, Denmark, 500 mg) at 08:00 hours and 20:00 hours (Day 1: 500 mg a.m. and 500 mg p.m.; Day 2: 500 mg a.m., 1,000 mg p.m.; Days 3, 4, 5 and 6: 1,000 mg a.m. and 1,000 mg p.m.; Day 7: 1,000 mg a.m.). As in phase A, the individuals were admitted to the Department of Endocrinology, Odense University Hospital, Denmark, at 16:00 hours after 20 h of fast. The experiment was initiated the next day at 08:00 hours, after 36 h of fast. The experiment was performed as in phase A and the fast likewise ended after 42 h.
The study was conducted in accordance with the Helsinki Declaration and Good Clinical Practice and monitored by the GCP unit, Odense University Hospital, Odense, Denmark. It was approved by the Danish Health and Medicines Authority (J. no: 2011050747), the Danish Data Protection Agency (J. no. 2011-41-6231) and the Regional Committee on Biomedical Research Ethics of Southern Denmark (Project ID: S-20110082). It is registered in the European Clinical Trial Database (EudraCT no.: 2011-001696-39) and at ClinicalTrials.gov (registration no. NCT01400191).
Plasma glucose and lactate were measured using an ABL800 FLEX Analyzer (Radiometer, Copenhagen, Denmark). Serum insulin and C-peptide were measured using COBAS immunoassay platforms (Roche Diagnostics, IN, USA). Plasma NEFA concentration was measured using a colorimetric assay kit (Wako Chemicals, Richmond, VA, USA) using a COBAS FARA 2 Autoanalyzer (Roche Diagnostic, Rotkreuz, Switzerland). Serum cortisol was measured by a chemiluminescent method using an Immulite 2000 (DPC Cirrus, Los Angeles, CA, USA). The plasma glucagon concentration was determined by a validated antibody method previously described .
Statistical analysis and considerations
The demographic data are presented as medians with 25th to 75th percentiles; others are listed as means with 95% CIs unless otherwise specified. The difference in the EGP was, guided by qq-plots, found to follow a Gaussian distribution. Statistical inference of the primary endpoint for OCT1 diplotypes was analysed using one-way ANOVA. Paired t tests were used to determine significance between the two periods. A level of p < 0.05 was considered statistically significant. All statistical analyses were performed using STATA 11.0 (StataCorp, College Station, TX, USA).
The sample size calculation was based on the primary outcome represented by differences in HGP between individuals homozygous and heterozygous for reduced-function alleles in OCT1 (rs12208357, rs72552763, rs34130495 and rs34059508). Based on an interindividual coefficient of variance for HGP of 18% after 40 h of fast , it was estimated that a true difference of 25% could be detected, given a two-sided level of significance of 0.05 and a power of 80%, by using 12 individuals in each group and taking into account a dropout rate of 20%.
After a 2 h basal tracer equilibration period, calculation of glucose turnover rates for the following 4 h period was based on determinations of plasma glucose concentration and [3-3H]glucose activity as previously described . Rates of total glucose appearance (Ra) and glucose disposal (Rd) were calculated using Steele’s non-steady-state equations . In these calculations, the distribution volume of glucose was set to 200 ml/kg body weight and the pool fraction to 0.65 . In the final 4 h period, EGP was assumed to equal Ra. The in vivo glycolytic flux rates were calculated from the generation rates of plasma 3H2O from [3-3H]glucose, assuming that all tritium in the C-3 position was lost to water during the glycolytic process as previously described in detail . Non-oxidative glucose metabolism (NOGM) was calculated as the difference between Rd and glucose oxidation.
Genotyping, linkage disequilibrium, haplotype and diplotype inference
Genotyping of OCT1 including inferring the haplotypes and diplotypes of the reduced-function alleles in OCT1 has previously been described in detail in a separate publication .