Les concentrations plasmatiques d’insuline et de glucose

Un rapport sur les concentrations plasmatiques d’insuline et de glucose, tutoriel & guide de travaux pratiques en pdf.

Methods

All authors had access to the study data and reviewed and approved the final manuscript. The present study is a cross-sectional analysis of data from 127 male subjects who participated in intestinal gene expression studies in our laboratory.14, 18-21 These studies were approved by the Laval University Ethical Review Committee, and written consent was obtained from all subjects.

Study subjects

The participants who composed the present sample were healthy and exhibited various degrees of IR. None of the subjects had diabetes, acute gastrointestinal, hepatic or renal disease, symptomatic CVD, monogenic hyperlipidemia, acute inflammatory state (evidenced by fasting C-reactive protein levels >10 mg/dL),22 history of cancer, uncontrolled arterial hypertension, or recent history of drug or alcohol abuse. Prior to data collection, all subjects were free from lipid-lowering medication for at least 6 weeks and had a stable weight for more than 3 months.

Intestinal biopsies and extraction and quantification of total RNA

In all subjects, duodenal biopsies were collected from the second portion of the duodenum during gastroduodenoscopy after a 12 h fast. The procedure was conducted in the fasting state and limited to the second portion of the duodenum to avoid complications (e.g. pulmonary aspiration, perforation). Samples (3 x 3 mm) were collected using single-use biopsy forceps, immediately flash-frozen in liquid nitrogen and stored at -80°C before RNA extraction. Intestinal tissue samples were homogenized in 1 mL of Qiazol (Qiagen, Hilden, Germany). RNA was extracted using an RNeasy kit (Qiagen, Hilden, Germany). To eliminate any contaminating DNA, biopsies were treated with an RNase-free DNase set. Total RNA extraction and quantitative real-time polymerase chain reaction (PCR) were performed using standard procedures as previously described.20 Primer sequences and gene descriptions are available in Table 1. The expression of the housekeeping gene glucose-6-phosphate-dehydrogenase (G6PD) was used as a reference. The intestinal expression of ACS1 and SAR1B was quantified in n=95 and n=84 subjects, respectively, whereas the expression of SREBP1c, FATP4, FABP2, ACS1, DGAT1, DGAT2, MTP, ApoB and G6PD was quantified in all subjects (n=127). Gene expression measurements were performed by the CHU de Québec-Université Laval Research Center Gene Expression Platform (Quebec, Canada).
The validity of the intestinal gene expression model used in the present study was demonstrated in previous studies by the inter-relationship of the expression of coregulated genes.14, 18-21, 23 The validity of the intestinal gene expression model is also supported by positive associations between the expression of the gene and the mass of the protein in biopsy samples as previously reported.14, 23 Finally, the fact that the changes observed in intestinal gene expression following pharmaceutical,18, 23 or nutritional19, 21 interventions reflect the expected homeostatic mechanisms also validates this model.

Biochemical Measurements

A blood sample was obtained from all subjects after a delay ranging from 0 to 48 hours from the gastroduodenoscopy. Blood samples were collected after a 12-hour fast from an antecubital vein in tubes containing disodium EDTA and benzamidine (0.03 %).24 Blood lipids were measured using enzymatic methods and ultracentrifugation as previously described.25 Glucose levels were measured using colorimetry, and insulin levels were dosed using electrochemiluminescence (Roche Diagnostics, Indianapolis, IN, USA).

Dietary assessment

In 89 subjects, assessment of dietary intake was conducted using a validated food frequency questionnaire, which assessed food intake from the four weeks preceding the gastroduodenoscopy.26, 27 In the other 38 subjects, the diet on the days preceding clinical measurements was fully controlled, and the diet composition was measured using Nutrition Data System for Research software (University of Minnesota, MN, USA).

In Vivo Stable Isotope Kinetics

An in vivo stable isotope study to assess postprandial TRL apoB-48 intravascular kinetics was conducted within 24 to 48 h of the gastroduodenoscopy in a subgroup of 75 subjects. Subjects underwent a primed-constant infusion of L-[5,5,5-D3]leucine. Starting at 0700, subjects were maintained in a constant fed state by eating 30 small, identical snacks every half hour for 15 hours, each containing 1/30th of their estimated daily food intake based on the Harris-Benedict equation. Two snack types were used during the experimental protocol. One provided 35.1 % of total caloric intake from fat, and the other provided 41.1 % of total caloric intake from fat. At 1000, L-[5,5,5-D3]leucine (10 μmol/kg body weight) was injected as a bolus intravenously and then by continuous infusion (10 μmol/kg body weight/h) over a 12-h period. Blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 11, and 12 h.

Quantification and Isolation of ApoB-48

In 46 subjects, TRL apoB-100 and TRL apoB-48 were separated using SDS-PAGE according to standardized electrophoresis procedures, and densitometry was used to measure the relative
proportion of apoB-48. Three different time points were scanned to estimate the average
concentration of apoB-48 and to confirm steady states. In the other 29 subjects, the apoB-48 concentration in TRL was determined using a noncompetitive ELISA kit (Shibayagi Co. Ltd., Gunma, Japan). The assay was calibrated according to manufacturer’s instructions. The within assay variation was 3.5% and the between assay variation was 2.8-8.6%. Three different time points during the infusion protocol were also used to estimate the average concentration of apoB-48 and to confirm steady states. The two methods are highly correlated.20, 30, 31

Isotopic Enrichment Determinations

The isotopic enrichment of leucine in apoB-48 was determined using gas chromatography-mass spectrometry in 46 subjects and using liquid-chromatography with multiple reaction monitoring in 29 subjects. These two procedures have been previously described and are highly correlated.20, 31

Kinetic Analysis

The TRL apoB-48 fractional catabolic rate (FCR; pools/d) was derived using a multicompartmental model.32 We assumed a constant enrichment of the precursor pool and used the TRL apoB-48 plateau tracer/tracee ratio data as the forcing function to drive the appearance of tracer into apoB-48 32. Assuming that each subject remains in steady state with respect to apoB-48 metabolism during the study as previously shown 32, the FCR is equivalent to the fractional synthetic rate. The apoB-48 production rate (PR) was determined using the formula: PR (mg/kg/d) = [FCR ● apoB-48 concentration (mg/dL) ● plasma volume (L)]/body weight (kg)]. The plasma volume was estimated at 4.5 % of body weight. The SAAM II program (SAAM Institute, Seattle, WA, USA) was used to fit the model to the observed tracer data.

Minimal detectable association

Calculation of the minimal detectable association was conducted on the expected association between plasma insulin concentrations and duodenal expression of MTP as the primary outcome. Our calculation indicated that a sample size of 127 subjects would allow us to detect a correlation coefficient of 0.251 or greater with a power of 0.8 at a two-sided 0.05 significance level, with the conservative assumption that the standard deviation of insulin concentrations and MTP intestinal expression is 50 % of the mean. This calculation is consistent with a previous study by Gutierrez-Repiso et al.33, who found an inverse association between the fasting insulin concentrations and jejunal expression of MTP (standard β=-0.746) in 45 morbidly obese IR subjects.

Statistical Analyses

Non-normally distributed variables were transformed prior to Pearson’s correlation analyses. Pearson’s correlations were systematically adjusted for intestinal mRNA level of the G6PD housekeeping gene. The adaptive Holm-Bonferroni method was used to adjust Pearson’s correlation P values for multiple testing.34 This sequential stepdown approach is a rigorous and recognized technique to control for a family-wise error rate 34. The method ensures that the probability of ≥1 false discovery under the null hypothesis is fixed at the α level while maintaining high statistical power. P values are adjusted and evaluated against α<0.05.
Differences among subjects with plasma levels of insulin above and below the median were analyzed using mixed models. Only covariates with significant effects were maintained in the models as fixed effects. The same approach was used to compare subjects with plasma levels of glucose above and below the median.
Backward stepwise linear regression models were used to identify independent correlates of intestinal gene expression. The models were systematically adjusted for the duodenal expression of G6PD to limit the heterogeneity between samples. Statistical significance was considered P<0.05. Data were analyzed using SAS Studio University Edition v3.5 (SAS Institute Inc., Cary, NC, USA).

Results

Anthropometric and fasting biochemical characteristics of the 127 male subjects included in the study are presented in Table 2. Participants’ mean age (±SD) was 37.8 ± 10.9 years (range: 21.0 to 65.0 years). As a group, subjects exhibited features of IR with abdominal obesity (mean waist circumference: 107.0 ± 12.6 cm), hyperinsulinemia (mean insulin levels: 116 ± 45 ρmol/L) and normoglycemia (5.4 ± 0.5 mmol/L).
Figure 1 presents Pearson’s correlations between fasting plasma concentrations of insulin and glucose and the intestinal expression of key genes involved in chylomicron metabolism. Correlations were adjusted for age, BMI, waist circumference, glucose levels (for insulin), insulin levels (for glucose), and dietary intake of energy, fat and carbohydrates. Significant inverse associations were observed between plasma insulin levels and intestinal expression of ACS1 (r=-0.22; P=0.02), DGAT1 (r=-0.24; P=0.009), DGAT2 (r=-0.19; P=0.04) and MTP (r=-0.26; P=0.004). Insulin levels also tended to be inversely associated with expression of FATP4 (r=-0.17; P=0.06), FABP2 (r=-0.15; P=0.1) and ApoB (r=-0.17; P=0.06). No correlation was observed between plasma glucose concentrations and intestinal expression of key genes involved in chylomicron assembly and secretion. The HOMA-IR index was inversely associated with expression of DGAT1 (r=-0.20; P=0.03) and MTP (r=-0.18; P=0.05) and tended to be associated with expression of ACS1 (r=-0.18; P=0.07) and DGAT2 (r=-0.16; P=0.07) (data not shown).
To assess the magnitude of the associations between plasma concentrations of insulin or glucose and intestinal expression of key genes involved in chylomicron assembly and transport, subjects were first separated according to the median value of plasma insulin (113 ρmol/L). The mean insulin levels were 151 ± 33 ρmol/L and 80 ± 21 ρmol/L among subjects with high and low insulin levels, respectively. Both groups had identical mean plasma glucose concentrations (5.4 ± 0.5 mmol/L). As presented in Figure 2, downregulation of intestinal expression of ACS1 (Δ=-20.6 ± 8.9 %; P=0.03).