Hepatic steatosis does not cause insulin resistance in people with familial hypobetalipoproteinaemia

We included seven male patients with documented FHBL and hepatic steatosis and seven healthy controls. We were not able to recruit more participants because of the low prevalence of FHBL and the strict inclusion criteria. Four of the FHBL patients had the same mutation in the APOB gene (11712delC). Two of these patients were brothers. The mutations identified in the other FHBL patients were 2534delA, Q1309X and 2783delC, respectively [17]. Patients and controls were matched for age, sex, BMI and WHR. Persons who performed regular exercise above sedentary level and those who were regularly using >3 units of alcohol per day or any recreational drug during the last 30 days were excluded from participation. Participants did not have any somatic illnesses, nor did they use supplements or medication influencing glucose or lipid metabolism. Oral glucose tolerance tests were within the normal range (<7.8 mmol/l) according to the criteria of the ADA [18]. All control participants had a normal routine blood examination.

After an overnight fast, participants were admitted to the metabolic ward of the Academic Medical Center at 07:30 hours. Prior to the study day, all participants consumed at least 250 g of carbohydrates for 3 days and refrained from vigorous exercise for 1 week. A catheter was inserted into an antecubital vein for infusion of stable isotope tracers, insulin and glucose. Another catheter was inserted into a contralateral hand vein and kept in a thermoregulated (60°C) Plexiglas box for sampling of arterialised venous blood. Saline was infused as NaCl 0.9% at a rate of 50 ml/h to sustain catheter patency. [6,6-²H₂]Glucose and [1,1,2,3,3-²H₅]glycerol were infused as tracers (>99% enriched; Cambridge Isotopes, Andover, MA, USA) to study glucose kinetics and lipolysis (total triacylglycerol hydrolysis), respectively.

At time 0 (08:00 hours) blood samples were drawn for determination of background enrichments. A primed continuous infusion of isotopes was then started ([6,6-²H₂]glucose and [1,1,2,3,3-²H₅]glycerol, both at a rate of 0.11 μmol kg⁻¹ min⁻¹, with a priming dose equivalent to 80 min of infusion) and continued until the end of the study. After an equilibration time of 2.5 h, three blood samples were taken for the measurement of isotope enrichments and one for the measurement of glucoregulatory hormones and NEFA. Thereafter, a two-step hyperinsulinaemic–euglycaemic clamp was started. A continuous infusion of insulin (Actrapid 100 U/ml; Novo Nordisk Farma, Alphen aan de Rijn, the Netherlands) was started for 2 h and 10 min at the rate of 20 mU [m² body surface area]⁻¹ min^–1, followed by an infusion of insulin at a rate of 60 mU [m² body surface area]⁻¹ min^–1 for another 2 h and 10 min. Plasma glucose levels were measured every 5 min at the bedside. Glucose was infused as 20% glucose at a variable rate, to maintain a plasma glucose concentration of 5.0 mmol/l. [6,6-²H₂]Glucose was added to the 20% glucose solution to achieve glucose enrichments of 1% to approximate the values for enrichment reached in plasma and thereby minimise changes in isotopic enrichment due to changes in the infusion rate of exogenous glucose [19]. During the last 40 min of both hyperinsulinaemic periods, blood samples were drawn at 10 min intervals for determination of isotope enrichments, glucoregulatory hormones and NEFA. During the study day, participants remained fasted but were allowed to drink water.

Body composition was measured by bioelectrical impedance analysis (Maltron BF906; Maltron, Rayleigh, UK). Peripheral and trunk fat mass were quantified by dual-energy X-ray absorptiometry (QDR-4500W, software version whole body v8.26A: 5, Hologic, Bedford, MA, USA).

For determination of visceral and abdominal fat mass, a standardised single slice abdominal CAT-scan was performed through the level of the fourth lumbar vertebra (MX8000, Brilliance, Philips, Best, the Netherlands).

Oxygen consumption (\( \dot{V}{{\text{O}}_{{2}}} \)) and CO₂ production (\( \dot{V}{\text{C}}{{\text{O}}_{{2}}} \)) were measured with the ventilated hood technique (model 2900; Sensormedics, Anaheim, CA, USA). \( \dot{V}{{\text{O}}_{{2}}} \) and \( \dot{V}{\text{C}}{{\text{O}}_{{2}}} \) were measured continuously during the final 30 min of the basal state and during the final 30 min of step 2 of the hyperinsulinaemic–euglycaemic clamp. The mean values of \( \dot{V}{{\text{O}}_{{2}}} \) and \( \dot{V}{\text{C}}{{\text{O}}_{{2}}} \) during the final 20 min were used for the calculation of glucose and fat oxidation.

IHTG content was measured by ¹H-MRS. We hypothesised that the reduced delivery of VLDL triacylglycerols to peripheral tissues in FHBL might be associated with reduced storage of triacylglycerols in skeletal muscle. Therefore, we also measured intramyocellular lipid content in the soleus muscle. Measurements were performed after an overnight fast, within a 2 week time frame before or after the clamp test. For logistical reasons, scanning was performed at two separate sites with two different scanners. ¹H-MRS spectra were acquired using a 3.0 T Magnetom Trio (Siemens, Erlangen, Germany) and a 3.0 T Intera (Philips, Best, the Netherlands). Identical scanning parameters were used in both situations. During the measurements, participants remained in the supine position within the MRI scanner.

For the measurement of IMCL content, the soleus muscle of the right leg was positioned within the homogeneous volume of the magnet. Scout images were acquired in order to position the volume of interest 15 cm beneath the tibia plateau. Two-dimensional chemical shift imaging MRS data were collected using a point-resolved spectroscopy sequence (PRESS) with the following parameters: repetition time (TR) 1,100 ms, echo time (TE) 30 ms, 10 mm slice thickness, 32 × 32 matrix size, field of view 16 × 16 cm, acquisition time 13 min, one acquisition. Spectra with and without water suppression were obtained. A number of voxels ranging from 5 to 27 inside the soleus were selected for further analysis and were processed using the freely available 3DiCSI package (version 1.9.11; Columbia University, New York, NY, USA). The number of voxels selected for analysis was similar between the two sites. Chemical shifts were reported using water as the internal standard at 4.65 ppm. Average spectra were then processed using specialised computer software (jMRUI 2.2) [20]. Three peaks were line-fitted: IMCL (CH₂) and extramyocellular lipid content (CH₂ and CH₃) peaks. Lipid content was calculated from the peak areas of IMCL CH₂ (methylene) at 1.3 ppm. IMCL contents were then expressed as the percentage of water content. In one FHBL patient quantification of IMCL content was impossible because of a large extramyocellular lipid content.

IHTG content was obtained using single-voxel ¹H-MRS, using a body array coil as the transmitter and phased surface coils as receivers. MRS measurements were acquired during breathhold, using single-voxel stimulated acquisition mode (TE/TR 20/3,000 ms, six acquisitions). Volumes of interest in the liver were located away from major vascular structures and bile ducts. Voxel size was 27 mm³. The water and fat resonance peaks, located at 4.65 and 1.3 ppm, were integrated using jMRUI software [20] and relative fat content was expressed as the ratio of the fat peak area over the cumulative water and fat peak areas. Calculated peak areas of water and fat were corrected for T2 relaxation (T2_water, 34 ms; T2_fat, 68 ms) [21] and the percentage hepatic fat content was calculated according to Szczepaniak et al. [22].

Plasma glucose concentrations were measured with the glucose oxidase method using a Biosen C-line plus glucose analyser (EKF Diagnostics, Barbleben/Magdeburg, Germany). Plasma NEFA concentrations were determined with an enzymatic colorimetric method (NEFA-C test kit; Wako Chemicals, Neuss, Germany) with intra-assay variation of 1%, inter-assay variation of 4–15% and a detection limit of 0.02 mmol/l. [6,6-²H₂]Glucose enrichment (tracer-to-tracee ratio) was measured as described by Ackermans et al. [23]. [6,6-²H₂]Glucose enrichment intra-assay variation was 0.5–1% with an inter-assay variation of 1% and a detection limit of 0.04%. [1,1,2,3,3-²H₅]Glycerol enrichment was determined as described earlier [24]. Intra-assay variation was 1–3% for glycerol and 4% for [1,1,2,3,3-²H₅]glycerol; inter-assay variation was 2–3% for glycerol and 7% for [1,1,2,3,3-²H₅]glycerol.

Insulin and cortisol were determined on an Immulite 2000 system (Diagnostic Products, Los Angeles, CA, USA). Insulin was measured with a chemiluminescent immunometric assay with intra-assay variation of 3–6%, inter-assay variation of 4–6% and detection limit of 15 pmol/l. Cortisol was measured with a chemiluminescent immunoassay with intra-assay variation of 7–8%, inter-assay variation of 7–8% and a detection limit of 50 nmol/l. Glucagon was determined with the Linco 125I RIA (Linco Research, St Charles, MO, USA) with an intra-assay variation of 9–10%, inter-assay variation of 5–7% and detection limit of 15 ng/l.

HOMA of insulin resistance (HOMA-IR) was calculated using the formula described previously by Matthews et al. [25]. Endogenous glucose production (EGP) and peripheral glucose uptake (rate of disappearance [R _d]) were calculated using the modified forms of the Steele equations as described previously [19, 26]. EGP and R _d were expressed as μmol (kg FFM)⁻¹ min⁻¹ (FFM, fat-free mass). Insulin clearance was calculated as the rate of insulin infusion (mU [m² body surface area]⁻¹ min⁻¹) divided by the mean plasma insulin concentration during the clamp [27]. Total triacylglycerol hydrolysis/lipolysis (glycerol turnover) was calculated using formulas for steady-state kinetics adapted for stable isotopes [24, 26] and was expressed as μmol kg⁻¹ min⁻¹.

Resting energy expenditure (REE), glucose oxidation and fat oxidation rates were calculated from \( \dot{V}{{\text{O}}_{{2}}} \) and \( \dot{V}{\text{C}}{{\text{O}}_{{2}}} \) as reported previously [28]. Non-oxidative glucose disposal was calculated as the difference between total glucose disposal and glucose oxidation.

All data were analysed with non-parametric tests. Comparisons between groups were performed using the Mann–Whitney U test. SPSS version 14.0.2 (SPSS, Chicago, IL, USA) was used for statistical analysis. Data are presented as median (minimum–maximum). In the Electronic supplementary material (ESM) data are also presented as mean ± SEM.