1Laboratory of Physiology, Medical School, 2Laboratory of Biochemistry, Department of Chemistry, 3Department of Internal Medicine, Medical School, University of Ioannina, Ioannina, Greece
OBJECTIVE: The investigation of the
association between retinol-binding protein 4 (RBP4) and
lipoproteins in subjects with hypertriglyceridemia. DESIGN: Forty-six obese or
overweight hypertriglyceridemic patients were studied at baseline and 20 of
them underwent a hypocaloric low-fat diet for 3 months.
RESULTS:
Plasma RBP4 levels were positively correlated with serum triglycerides (TG) in the
subgroup of patients with TG <200 mg/dL (r=0.453, p=0.039) and negatively
correlated with TG in patients with TG ≥200 mg/dL (r=-0.487, p=0.019). In the subgroup with TG
<200 mg/ dL, subjects with circulating RBP4 above the median 46
mg/L had higher levels of intermediate density lipoprotein-cholesterol (IDL-C),
low-density lipoprotein-cholesterol (LDL-C) and apolipoprotein B (ApoB), while
these differences were absent in patients with TG ≥200 mg/dL. The associations of percentage changes of circulating
RBP4 with the percentage changes of LDL-C, very low-density
lipoprotein-cholesterol (VLDL-C) and ApoB were positive after the first month
and 3 months of diet for patients with baseline TG <200 mg/dL, while no
correlations existed for patients with TG ≥200 mg/dL.
CONCLUSIONS: The positive association between
circulating RBP4 and ApoB-containing lipoproteins in a steady
metabolic state, as well as during a hypocaloric diet, appears to be attenuated
in patients with very high TG.
Apolipoprotein B, Diet, Obesity, Retinol-binding protein 4, Triglycerides
INTRODUCTION
Retinol-binding protein 4 (RBP4), a transport protein for vitamin A, is synthesized mainly by the hepatocytes and secondly by the adipose tissue.1 Plasma RBP4 levels are upregulated in insulin resistant states associated with obesity, while RBP4 also induces insulin resistance.1,2 Furthermore, elevated circulating RBP4 has been associated with the development of cardiovascular disease.3-6 Circulating RBP4 has been shown to be positively correlated with serum triglycerides (TG) and low-density lipoprotein-cholesterol (LDL-C) and negatively with high-density lipoprotein-cholesterol (HDL-C).1,7 Among these associations the strongest and the most consistently reported has been the association with TG.1,8 Moreover, serum RBP4 levels have been shown to increase TG in mice.9 These data indicate that RBP4 are possibly associated with TG metabolism. In the present study we investigated the association of RBP4 with various lipid parameters in subjects with obesity-related hypertriglyceridemia at baseline and during dietary intervention.
MATERIALS AND METHODS
Subjects
In the present study 46 subjects were recruited. They attended the obesity outpatient clinic of the University of Ioannina, Greece. Inclusion criteria were: body mass index (BMI) ≥27 Kg/m2 and hypertriglyceridemia (TG ≥150 mg/dL). Exclusion criteria were: age less than 18 years old, pregnancy, breastfeeding, kidney disease, liver disease, gastrointestinal disease, malignancy, any endocrine disorder or metabolic disease other than obesity or type 2 diabetes mellitus (T2DM), alteration of body weight (BW) by up to 5% of the initial BW during the last 3 months, any state of stress or systemic inflammation, taking any one of the following drugs within 3 weeks before the start of the study: hypolipidemic agents, antidiabetics, drugs for weight loss, β-blockers or thiazides. Diagnosis of T2DM was reasonably excluded by asking medical history and assessing the values of fasting serum glucose and HbA1c.
Among the total population, 20 participants underwent a hypocaloric low-fat diet for 3 months. A dietician, taking into account each patient’s basal energy requirements and on an estimation of the subject’s typical activity level, prescribed an individualized low-fat diet promoting a 500 to 1000 kcal reduction in daily energy intake. The administered diets consisted of a mean of 1471±382 kcal/day (ranging from 1085 to 2000 kcal/day depending on the initial BW). The daily distribution of nutrients during the study was as follows: carbohydrates 52.4±3.5%, fat 27.8±2.6% (monounsaturated 15.4±1.7%, polyunsaturated 7.3±1.2% and saturated fatty acids 5.1±1.0%) and protein 19.8±1.2%. There were no differences in diet composition between the study groups. At the end of the 3-month period, the patients were consuming significantly less carbohydrates and saturated fatty acids as well as more monounsaturated fatty acids and n-3 polyunsaturated fatty acids compared with their baseline diet. All patients were asked to attend the clinic monthly during the treatment in order to assess diet compliance.
Anthropometric measurements and collection of venous blood samples, after an overnight fast of at least 12 h, were performed at baseline, after 1 month and after 3 months of treatment. Plasma samples were stored at -80 °C until analysis.
Measurement of RBP4
Plasma RBP4 was analyzed using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (ALPCO DIAGNOSTICS), following the manufacturer’s instructions, as previously described.10
LDL subclass analysis
LDL subclass analysis was performed electrophoretically by use of high-resolution 3% polyacrylamide gel tubes and the Lipoprint LDL System (Quantimetrix, Redondo Beach, CA), as we have previously described.10 After electrophoresis, very low-density lipoprotein (VLDL) remained at the origin [retention factor (Rf) = 0.0], HDL migrated to the front (Rf=1.0). In between, several bands can be detected: MID bands C, B, and A, which correspond mainly to intermediate-density lipoprotein (IDL), as well as up to 7 LDL bands. The LDL-1 and LDL-2 bands correspond to large buoyant LDL particles, whereas bands LDL-3 to LDL-7 correspond to sdLDL particles. A detailed description of the methods used for the measurement of LDL subclasses can be found in an expert consensus document of the “European panel on LDL subclasses”.11
Routine biochemical measurements
Total serum cholesterol (TC), HDL-C and TG were measured by enzymatic methods, as previously described.10,12 Non-HDL-cholesterol was calculated as TC-HDL-C. Serum apolipoprotein (Apo) A-I and ApoB levels were measured with a Behring Holding GmbH analyzer (Liederbach, Germany).
Serum Creatinine (Cr) levels were determined by standard laboratory methods. The Modification of Diet in Renal Disease (MDRD) formula was used for the estimation of glomerular filtration rate (eGFR).13 Body surface area (BSA) was calculated by body weight (BW) and height (H) using the Du Bois formula: BSA = 0.007184 × BW0.425 × H0.725 (BSA is in m2, BW is in kg, and H is in cm).14
Statistical analysis
All statistical analyses were performed using the SPSS 16.0 statistical package for Windows (SPSS Inc., 1989-2007). The Kolmogorov-Smirnov test was used to verify the normality of the distributions of the parameters of interest. Normally distributed data were expressed as means±SD. Parameters with skewed distribution were reported as median (range). The paired t-test, independent t-test and Pearson’s correlation analysis were performed for normally distributed parameters, whereas the Mann-Whitney U test and Spearman’s correlation analysis were performed for non-normally distributed parameters. Linear regression analysis was used for the assessment of the relationship between circulating RBP4 and lipid parameters after adjustment for gender and cGFR. A two-tailed p value <0.05 was considered significant.
RESULTS
Baseline
Participants were 50±14 years old (24 males and 22 females) and their BMI was 36.5±7.3 Kg/m2. Plasma RBP4 levels were higher in males compared with females (54.7±14.2 mg/L vs 42.2±13.6 mg/L, p=0.005). Circulating RBP4 was positively correlated with Cr (r = 0.367, p = 0.014). Although circulating RBP4 was not correlated with eGFR, it was negatively correlated with corrected GFR for BSA (cGFR) (r=-0.311, p=0.040).
Plasma RBP4 levels were not correlated with TGin all patients. Circulating RBP4 was positively correlated with TG in the subgroup of patients with TG <200 mg/dL (r=0.453, p=0.039) and negatively correlated with TG in the subgroup of patients with TG ≥200 mg/dL (r=-0.487, p=0.019).
Table 1 shows the values of circulating lipoproteins in the subgroups of patients with plasma RBP4 levels below or above the median 46 mg/L in all patients as well as in subjects with TG < or ≥200 mg/ dL. In all patients, subjects with circulating RBP4 above the median 46 mg/L had higher levels of IDL-C, LDL-C, nonHDL-C and ApoB and lower levels of ApoE compared with subjects with circulating RBP4 below 46 mg/L. There was a tendency for higher levels of TC and VLDL-C in subjects with circulating RBP4 above 46 mg/L.
In the subgroup of patients with TG <200 mg/ dL, subjects with circulating RBP4 above 46 mg/L had higher levels of IDL-C, LDL-C and nonHDL-C and lower levels of Lp(a) compared with subjects with circulating RBP4 below 46 mg/L. There was a tendency for higher levels of ApoB in subjects with circulating RBP4 above 46 mg/L. Linear regression analysis showed that plasma RBP4 levels were positively correlated with VLDL-C after adjustment for gender and cGFR (β=1.779, p=0.013).
In the subgroup of patients with TG ≥200 mg/ dL, subjects with serum RBP4 levels above 46 mg/L had lower levels of TG, sdLDL-C and ApoE compared with subjects with circulating RBP4 below 46 mg/L.
Dietary treatment
BMI was significantly decreased from baseline (36.2±5.7 Kg/m2) after first month (35.7±4.6 Kg/m2, p <0.001), as well as after 3 months of diet (34.9±5.1 Kg/m2, p <0.001). Circulating RBP4 decreased after 3 months of diet (from 51.9±13.8 to 45.7±14.7, p=0.03). Table 2 shows the associations between the percentage change of plasma RBP4 levels after 1 month of treatment and the percentage changes of IDL-C, LDL-C, VLDL-C, sdLDL-C and ApoB in the total of subjects who underwent dietary treatment and in the subgroups of patients with baseline TG above or below 200 mg/dL. Table 3 shows similar data to Table 2 regarding the 3 months of diet. The associations of percentage changes of circulating RBP4 with the percentage changes of IDL-C, LDL-C, VLDL-C, sdLDL-C and ApoB were positive over the first month and 3 months of diet for patients with baseline TG <200 mg/dL, while no correlations existed for patients with baseline TG ≥200 mg/dL, except for IDL-C during first month and sdLDL-C for 3 months.
DISCUSSION
The present study showed that the association between circulating RBP4 and TG was characterized by a biphasic mode, being positive for TG <200 mg/dL and negative for TG ≥200 mg/dL. Moreover, the positive association between RBP4 and ApoB-containing lipoproteins in a steady metabolic state, as well as during the hypocaloric low-fat diet, was found to be attenuated in subjects with TG ≥200 mg/dL.
The relationship between RBP4 and ApoB-containing lipoproteins
The current study demonstrated that circulating RBP4 was positively correlated with serum levels of the ApoB-containing lipoproteins LDL-C, IDL-C and VLDL-C. Importantly, this relationship was shown not only at baseline but also during a hypocaloric low-fat diet. Indeed, our study group has demonstrated that circulating RBP4 is possibly associated more consistently and strongly with the metabolism of the ApoB-containing lipoproteins than the metabolism of the ApoA-I-containing lipoprotein HDL.1,10 RBP4 has been found to induce not only the enhancement of hepatic production of ApoB-containing lipoproteins but also the decrease in catabolism of ApoB-containing lipoproteins through the downregulation of the LDL receptor.15 To our knowledge, the association between circulating RBP4 and IDL-C has not previously been investigated.
The relationship between RBP4 and TG in patients with TG <200 mg/dL
The positive association between circulating RBP4 and TG has consistently been reported in studies investigating subjects with variable TG levels, including normal as well as high TG levels.7,8 This relationship is possibly causal since treatment with RNA oligonucleotide against RBP4 was shown to reduce TG levels in mice.9 Vergès et al found that circulating RBP4 in patients with T2DM was negatively correlated with indirect VLDL-apoB fractional catabolic rate (FCR), which represents the VLDL delipidation toward IDL, while there was no significant association with direct VLDL-apoB FCR, which reflects the direct VLDL removal from plasma through receptor-mediated particle uptake.16
The relationship of RBP4 with lipoproteins in patients with TG ≥200 mg/dL
The negative association between circulating RBP4 and TG in patients with TG ≥200 mg/dL of the current study was accompanied by the dissociation between circulating RBP4 and ApoB-containing lipoproteins. A possible explanation for these data is the downregulation of plasma RBP4 levels in patients with TG ≥200 mg/dL. Further studies are needed to confirm these findings and elucidate the underlying mechanisms. The dissociation between circulating RBP4 and ApoB-containing lipoproteins in patients with considerable hypertriglyceridemia that was found in the present study implies that the adverse impact of RBP4 on lipoprotein metabolism may be important only in patients without considerable hypertriglyceridemia. In this context, it is prudent to evaluate the effects of RBP4 on lipoprotein metabolism only in patients without considerable hypertriglyceridemia, at least in initial studies.
The negative relationship between circulating RBP4 and serum ApoE levels in patients with TG ≥200 mg/dL of the current study may be explained by the high serum levels of ApoE, which is carried by remnants of triglyceride-rich lipoproteins in subjects with hypertriglyceridemia.17 The relationship between RBP4 and ApoE has not been investigated in any previous study.
Study strengths and limitations
Strengths of this study include the investigation for the first time of the relationship of RBP4 with lipoproteins in patients who had exclusively hypertriglyceridemia and not in mixed populations with variable TG levels. Secondly, taking into account the many factors that influence circulating RBP4, including renal or liver impairment and drugs affecting metabolism, the present study excluded patients with all these conditions.1 However, an important limitation of the majority of studies investigating RBP4 was that they did not take into account all these factors. Thirdly, the current study applied a direct measurement of all ApoB-containing lipoproteins through lipoprotein electrophoresis. However, most of the studies investigating the relationship between RBP4 and LDL-C assessed LDL-C by its indirect calculation using the Friedewald equation [LDL-C = TC - (HDL-C + TG/5)], which is less accurate than the direct measurement of LDL-C, especially in subjects with considerable hypertriglyceridemia, as is the case in the present study.18 Moreover, LDL-C calculated through the Friedewald equation represents a crude estimation of the sum of directly measured LDL-C and IDL-C and thus it is not a highly accurate estimation of true LDL-C.
The results of the present study should be interpreted in light of some limitations. Firstly, the number of patients was not large enough, thus decreasing the statistical power of the study to detect significant associations of circulating RBP4 with serum lipoprotein levels. Therefore, taking into account the small number of study participants, firm conclusions cannot be drawn regarding the relationship between circulating RBP4 and ApoB-containing lipoproteins in subjects with hypertriglyceridemia. Secondly, the relationships between RBP4 and lipoproteins that were found in the current study cannot confirm the existence of causal mechanisms underlying these associations.
In conclusion, the present study showed that circulating RBP4 was positively correlated with serum levels of ApoB-containing lipoproteins in a steady metabolic state, as well as during a hypocaloric low-fat diet in overweight or obese patients with hypertriglyceridemia. This relationship appears to be attenuated in patients with TG ≥200 mg/dL. Further well designed studies with a greater number of patients are needed to confirm these results and elucidate the underlying mechanisms.
CONFLICT OF INTEREST
The authors declare that there is no conflict of interest.
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Address for correspondence:
Dimitrios N Kiortsis, MD, PhD, Professor of Physiology, Laboratory of Physiology, Medical School, University of Ioannina, 45110 Ioannina, Greece; Tel.: +30 2651007551, Fax: +30 2651007850, E-mail: dkiorts@cc.uoi.gr
Received: 05-12-2015, Accepted: 22-02-2016