Serum retinol binding protein 4 in patients with familial partial lipodystrophy

Results RBP4-log levels were lower in patients with FPLD in comparison to control group (1.52 ± 0.32 vs 1.84 ± 0.25, p = 0.029). A statistical trend was observed between Waist-to-Hip Ratio and RBP4-log (r = - 0.44, p = 0.054). Conclusion RBP4 levels are decreased in FPLD. Keywords Retinol Binding Protein 4 Lipodystrophy Insulin Resistance Introduction Retinol Binding Protein 4 (RBP4) was initially known as the specific transport protein for retinol (vitamin A) in the circulation [1] . In the last few years, several manuscripts have also linked this protein to obesity and insulin resistance (IR). More specifically, supporting evidence can be found linking RBP4 levels and anthropometric indicators of overweight and/or fat distribution, and metabolic abnormalities such as triglyceride levels, systolic blood pressure and low levels of high-density lipoprotein cholesterol [1,2] . However, studies have not conclusively demonstrated whether RBP4 plays a causal role in IR or acts only as a biomarker for Diabetes Mellitus (DM) and IR. Lipodystrophies are an interesting group of clinical disorders that are most often characterized by lipoatrophy or selective loss of adipose tissue. Familial Partial Lipodystrophy, Dunnigan variety (FPLD) is characterized by a progressive, gradual loss of subcutaneous adipose tissue and severe IR [3] . The combination of diminished adipose tissue and IR, makes FPLD an unique disease to investigate whether RBP4 is linked to the amount of adipose tissue, IR, or both. The aim of this study was to determine RBP4 levels in patients with FPLD and compare these with controls as well as to investigate its relationship with anthropometric and metabolic parameters. Patients and Methods Participants Ten (10) patients with FPLD were invited to participate in the study. All participants were carefully examined by an experienced endocrinologist. The protocol was approved by the Ethics Committee of the Institution. A written informed consent was obtained from each patient or a legal tutor, after the procedures involved in the study were fully explained. The diagnosis of FPLD was confirmed by genetic evaluation (ABI Prism 3100) provided by the Molecular Endocrinology Laboratory of Paulista Medical School. All patients had a missense mutation in the LMNA gen. In 90% of the patients, the mutation identified was at codon 482. A control group (9 healthy individuals) was also carefully and sequentially selected in order to match the lipodystrophic group by gender, Body Mass Index (BMI) and age. Anthropometrical examination All participants had the following anthropometrical data registered: body weight (Kg), height (m), Body Mass Index (BMI), waist circumference (WC), waist-to-hip ratio (WHR) and blood pressure. BMI was calculated as weight in kilograms divided by the square of height in meters (kg/m 2 ). Waist circumference was determined at the midpoint between the lowest rib and the iliac crest. WHR was defined as the ratio of waist girth to the largest circumference of the hips, measured at the trochanter major. Laboratory evaluation Fasting blood sample were collected in appropriate tubes and assays for plasma glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-c), low-density lipoprotein cholesterol (LDL-c), very low-density lipoprotein cholesterol (VLDL-c) and triglycerides were performed using an automated analyzer (Cobas Integra 400, Roche Diagnosis). Serum RBP4 concentrations were measured by an enzyme-linked immunosorbent assay (ELISA) kit (BioSource International, Inc., Camarillo, CA). The ELISA system had an intra-assay coefficient of variation of 2.6 – 9.2% and an interassay coefficient of variation of 3.4 – 10.2%. Serum leptin were measured by Radioimmunoassay (Linco Research, Missouri, USA), both kindly provided by Sergio Franco Medical Diagnosis. Statistical analysis Statistical analysis was performed with GraphPad InStat 3.00 for Windows 95 (GraphPad Software, San Diego, California, USA). The unpaired t test was used for parametric variables and Mann-Whitney for non-parametric variables. The strength of the linear relationship between two continuous variables was evaluated by means of the Pearson's correlation coefficient or Spearman's correlation coefficient. RBP4 values were converted to logarithm to correct for the dispersion of the variable. The level of statistical significance was 5%. Results Ten patients with FPLD were evaluated in our study. Seven patients had the diagnosis of type 2 DM and 5 were on insulin therapy. Three patients were from the same family (sisters). A statistical trend was observed in the comparison of the absolute values of RBP4. However, the dispersion of these results might compromise interpretation of these data. On the other hand, RBP4-log was found to be decreased in FLPD in comparison to control group. Furthermore, leptin was also decreased in patients with FLPD and several metabolic parameters showed in Table 1 were also significantly different in patients in FPLD. Correlation analysis was used to investigate the relationship among RBP4 levels and metabolic parameters. An inverse and significant relationship was found only between RBP4 log and DBP (r = -0.46, p = 0.043) and trends observed between RBP4 log and SBP (r = -0.40, p = 0.082) and HDL cholesterol (r = 0.40, p = 0.082). These correlations were in patients who were receiving treatment for hypertension and dyslipidemia. No correlation was found between RBP4 log and leptin (r = 0.34, p = 0.14), BMI (r = -0.13, p = 0.57), age (r = 0.05, p = 0.83), waist (r = -0.25, p = 0.29), and triglycerides (r = -0.24, p = 0.30). Correlation analysis was also used to identify whether RBP4 correlated independently with WHR and metabolic parameters. A significant correlation was found between WHR and SBP (r = 0.68, p = 0.0012), DBP (r = 0.65, p = 0.0022) and triglycerides (r = 0.74, p = 0.0003). Also, an inverse and significant relationship was found between WHR and HDL cholesterol (r = -0.63, p = 0.0038). Only a statistical trend was observed between WHR and RBP4 log (r = -0.44, p = 0.054) ( Fig. 1 ). Linear regression analysis was used to identify which variable (RBP4 or WHR) independently correlates with metabolic parameters. After analysis, only WHR continue to independently correlate with DBP (p = 0.02), SBP (p = 0.0064), triglycerides (p = 0.02) and HDL cholesterol (p = 0.01). Discussion Retinol Binding Protein 4 (RBP4) is one of the many metabolic markers identified in the last few years. Although several studies demonstrated that RBP4 levels correlated directly with adipose tissue distribution and IR, these results are still a matter of debate. Familial Partial Lipodystrophy (FPLD) is a well-known disease and characterized by an almost complete absence of peripheral adipose tissue and severe IR. The most important findings of this study were: (i) – patients with FPLD presented significantly lower levels of RBP4 and leptin, despite the same BMI of a control group and (ii) – an unexpected correlation was observed among RBP4 levels and some important metabolic variables, including SBP, DBP and HDL cholesterol, although the last 2 did not reached statistical significance. The most important finding of this study was to show that patients with FPLD showed significantly lower levels of both RPB4 and leptin compared to the control group with similar BMI. An unexpected negative correlation was also observed between RBP4 levels and DBP and trends observed between RBP4 and SBP and HDL cholesterol. Others have not found a relationship between RBP4 levels and the quantity and distribution of adipose tissue [2,4] . We observed an inverse trend between RBP4 and WHR, but not BMI, which could suggest that fat distribution may reflect RBP4 levels rather than total body fat as shown by other reports [4,5] whereas an inverse relationship has been found between RBP4 and waist circumference, body fat and BMI in overweight subjects [6] . Accordingly, the identification of low RBP4 levels in a condition characterized mainly by disturbed fat distribution is consistent with the concept that adipose tissue distribution, not just total fat, may play a role in circulating RBP4 levels. The identification of an inverse significant relationship between RBP4 levels and DBP and negative trends between RBP4 and SBP and WHR, as well as positive trends between RBP4 and HDL cholesterol was a new finding and contrary to most of the studies published so far [1,2] . Two hypotheses can be advanced by these findings: (i) – There is no relationship between RBP4 levels and IR. Retinol Binding Protein 4 levels only reflect the amount of adipose tissue and are an indirect measurement of the effect of fat in IR. This hypothesis was partially confirmed since after multivariate analysis, the effect of RBP4 levels in metabolic parameters was not significant. The only variable that independently correlated with metabolic alterations was WHR; and (ii) – in lipodystrophyc patients, RBP4 levels are not the main determinant of IR. The abnormal fat distribution in these patients might led to several other metabolic modifications (an increase in efflux of free fat acids to liver, for instance) that, independently of RBP4, leads to severe IR. It is also important to notice that other authors have recently suggested that RBP4 is not a useful marker of IR [7,8] . Further studies in lipodystrophy are necessary to clarify this issue. Our results also provide further support to the hypothesis that the pathogenesis of some lipodystrophies, including FPLD, may be linked to the retinoic acid pathway. It is currently known that the use of some drugs, especially protease inhibitors (i.e. Indinavir), may induce a FPLD-like condition [9] . This side effect can be partially explained by the inhibition of cytoplasmic retinoic acid binding protein (cRABP). CRABP is an intracellular protein involved in the transmission of the vitamin A-derived signal, which regulates genes responsible for lipid metabolism and adipocyte differentiation [10] . It seems reasonable to speculate that the decreased levels of a specific vitamin A transporter (namely RBP4) may also disrupt intracellular signaling and compromise adipocyte differentiation, leading to lipodystrophy. This study has a few limitations. First, methods to study insulin sensitivity (i.e. glucose clamping) were not done and Homeostasis Model Assessment (HOMA) was not studied because most of lipodystrophyc patients were patients already receiving insulin. On the other hand, the diagnosis of type 2 DM in 70% of the patients with FPLD provides an indirect evidence that IR is one of the main features of this condition. Second, a specific examination (i.e. computed tomography) was not used to investigate total body fat or fat distribution in our patients. However, leptin, a marker of adipose tissue amount, was also significantly decreased in FPLD, and this may strengthen the finding of our study. Thirdly, most of our patients were receiving treatment for dyslipidemia and hypertension which confounds interpretation and could tend to counter our results. Finally, only a small number of patients with FPLD was evaluated in this study and results should be carefully interpreted. In conclusion, patients with FPLD presented significantly lower levels of RBP4 in comparison to a control group. This finding supports the idea that RBP4 levels correlate with the amount of fat tissue. On the other hand, at least in this sample of FPLD, RBP4 levels correlated inversely with IR parameters. These correlations, however, were not upheld after adjustment for WHR. Taken together, these results could suggest that RBP4 levels may be regulated by fat distribution but are not an independent variable related to IR in lipodystrophic patients. Finally, it may also suggest a relationship between RAP and FPLD. References [1] Q. Yang T.E. Graham N. Mody F. Preitner OD. Peroni JM. Zabolotny Serum Retinol Binding Protein 4 Contributes to Insulin Resistance in Obesity and Type 2 Diabetes Nature 436 2005 356 362 [2] M. von Eynatten P.M. Humpert Retinol-binding protein-4 in experimental and clinical metabolic disease Expert Rev. Mol. Diagn. 8 2008 289 299 [3] R.A. Hegele T.R. Joy S.A. Al-Attar B.K. Rutt Lipodystrophies: windows on adipose biology and metabolism J. Lipid Res. 48 2007 1433 1444 [4] T.M. Barber M. Hazell C. Christodoulides SJ. Golding C. Alvey K. Burling Serum levels of retinol-binding protein 4 and adiponectin in women with polycystic ovary syndrome: associations with visceral fat but no evidence for fat mass-independent effects on pathogenesis in this condition J. Clin. Endocrinol. Metab. 93 2008 2859 2865 [5] S. Makino M. Fujiwara K. Suzukawa H. Handa T. Fujie Y. Ohtaka Visceral Obesity is Associated with the Metabolic Syndrome and Elevated Plasma Retinol Binding Protein-4 Level in Obstructive Sleep Apnea Syndrome Horm. Metab. Res. 41 2009 221 226 [6] A. Tschoner W. Sturm J. Engl S. Kaser M. Laimer E. Laimer Retinol-binding Protein 4, Visceral Fat, and the Metabolic Syndrome: Effects of Weight Loss Obesity 16 2008 2439 2444 [7] J.G. Lewis B.I. Shand C.M. Frampton P.A. Elder R.S. Scott Plasma retinol-binding protein is not a marker of insulin resistance in overweight subjects: a three year longitudinal study Clin. Biochem. 41 2008 1034 1038 [8] J.G. Lewis B.I. Shand P.A. Elder R.S. Scott Plasma retinol-binding protein is unlikely to be a useful marker of insulin resistance Diabetes Res. Clin. Pract. 80 2008 e13 e15 [9] J. Fuller A 39-Year-Old Man With HIV-Associated Lipodystrophy JAMA 300 9 2008 1056 1066 [10] Lenhard JM, Weiel JE, Paulik MA, Furfine ES. Stimulation of vitamin A (1) acid signalling by the HIV protease inhibitor indinavir. Biochem. Pharmacol. 2000;1; 59(9):1063-8.