Plasma levels of leptin and ghrelin and their correlation with BMI, and circulating lipids and glucose in obese Tunisian women

After approval of the National Ethics Committee for the implementation of this study, we recruited 40 obese women and 20 female controls. They were matched by age. Obese women were recruited from nursing unit “C” of the National Institute of Nutrition and Food Technology (INNTA). A woman was considered obese when her body mass index (BMI) was greater than 30. Women selected for the survey were informed in advance about study objectives and showed no pathology or metabolic complication other than obesity. The healthy control group was recruited from women accompanying patients and who also agreed to participate in the survey. As motivation, they received results of their conventional laboratory tests, all gave their written consent to participate. It should be noted that, at the beginning of our investigation, we had the agreement of 45 women who were considered healthy after physical examination by the team physician, but 25 women withdrew during the blood taking phase.

We used a form to collect anthropometric data. Each woman was weighed 2 consecutive times using a calibrated scale and their height was measured. The average of these measurements was calculated. Blood samples were taken from an antecubital vein after a 12 hour fast. The measured biochemical variables were: total cholesterol (TC), triglycerides (TG), high-density lipoprotein-cholesterol (HDL-C), fasting blood glucose, and low-density lipoprotein-cholesterol (LDL-C). Blood glucose determination was by an enzymatic glucose oxidase method using a Beckman kit on Beckman Synchron CX9 analyzer. TGs were determined using a Beckman Synchron Cx7 kit. (Beckman Coulter, Brea, CA, USA). TC was measured using a cholesterol oxidase kit adapted to a Beckman Synchron CX9 analyzer. The HDL-cholesterol was determined using a Randox kit with a Beckman Synchron CX9. LDL-cholesterol was calculated using the Friedwald formula [LDL = CT – (TG/5 + HDL-C) with the condition TG < 4 g/L]. Ghrelin was measured using a radioimmunoassay (GHRA-88HK, Linco Research, Inc, MO, USA) for active ghrelin with a sensitivity of 7.8 pg/mL. Leptin was measured using a radioimmunoassay (HL-81HK, Millipore) with a sensitivity ranging from 0.5 ng/mL to 100 ng/mL.

Data were analyzed with SPSS software, version 11.5 (SPSS Inc, Chicago, IL, USA). Comparisons of distributions of two numeric variables were conducted using a nonparametric Mann–Whitney U test. Correlations were tested using a Pearson correlation coefficient and a Spearman rank–correlation coefficient.

To determine the threshold-value of ghrelin that increases the risk of obesity, we have established a receiver operating characteristics (ROC) curve. After verifying that the area under the curve was significant (>0.50), we have identified the value of ghrelin, which maximizes the pair “sensitivity–specificity”. We calculated the odds ratio, associated with this value.

Multivariate backward linear regression analysis was conducted with ghrelin as a dependent variable and BMI, HDL-cholesterol, LDL-cholesterol, glucose, and leptin as independent variables. For all tests, P < 0.05 was considered significant.

We found a significant positive correlation between plasma levels of leptin and BMI in the two groups (obese: r = 0.61, P < 0.001; controls: r = 0.95, P < 0.001). These results are consistent with those of Sanlier [11] and other authors [13-20]. The correlation was consistent with that found by Ibrahim et al. [21] and Ezam et al. [22].

Our results confirm the existence of a significant negative correlation between plasma levels of ghrelin and BMI for both groups studied (obese: r = –0.41, P = 0.01; controls: r = –0.61, P = 0.005). These results are consistent with those of Tschop et al. [23], Monti et al. [17], Koc et al. [19], Hamed et al. [24], Suematsu et al. [25], Purnell et al. [26], Al-Hakeim and Ali [27], Saad et al. [28], and Stepien et al. [29] (in a group of hypertensive subjects with class II and III obesity). Indeed, ghrelin is synthesized in inverse proportion to energy balance: the highest values are found in malnourished patients, lower values in lean subjects, and the lowest values were observed in obese patients. This apparently challenging adaptive response to inhibiting appetite depends on the deregulation of the energy balance [30]. However, Eizadi et al. [31] showed that ghrelin levels are independent of BMI. This is consistent with our multivariate analysis in obese women, which showed that there is no direct correlation between these two variables, suggesting that the correlation established in the univariate analysis is dependent on other variables. Indeed, changes in BMI are accompanied by proportional changes in leptin and insulin. In the obese, there is a downregulation of ghrelin, which correlates inversely with leptin and insulin [22, 27]. The interference of these variables explains the lack of direct correlation between ghrelin and BMI in the multivariate analysis.

A significant positive correlation was found between leptin and glucose levels in the two groups (obese: r = 0.41, P = 0.008; controls: r = 0.53, P = 0.017). These findings are consistent with the findings of Hamed et al. [24] in obese patients with diabetes. However, our results are in discordance with several studies. Indeed, Martins et al. [32] did not identify any significant correlation between leptin and glucose. They concluded that hyperinsulinemia and insulin resistance strongly influence leptin. They even found that leptin is the best predictive parameter of insulin resistance in both sexes. Similarly, Taghdir et al. [33] found no significant correlation between leptin and fasting plasma glucose in the groups studied, and found a significant positive correlation between leptin and insulin, and leptin and insulin resistance in the control group. In the same context, Baban et al. [34] concluded that there was a significant positive correlation between circulating levels of fasting leptin and insulin. Silha et al. [13] have demonstrated a significant positive correlation between leptin and fasting insulin and a clear link between leptin and fasting glucose without having a significant correlation.

Our data also showed a significant negative correlation between circulating levels of ghrelin and glucose in the obese group. These results are consistent with those found by Purnell et al. [26] and Kellokoski et al. [35]. By contrast, Eizadi et al. [36] and Zwirska-Korczala et al. [37] failed to find a correlation between these two variables. This discrepancy may be related to differences between the various populations studied. Despite the lack of concordance between the results of our univariate analysis and data from the literature, this direct correlation persisted after the completion of the multivariate analysis.

Circulating levels of leptin showed a significant positive correlation with LDL-cholesterol in both groups (obese: r = 0.48, P = 0.011; controls: r = 0.61, P = 0.005). Haluzik et al. [38] has established no correlation between leptin and LDL-cholesterol both in a hyperlipidemic group and control group. We found that the level of leptin correlates negatively with HDL-cholesterol. This correlation was significant for both groups studied (obese: r = –0.48, P = 0.001; control group: r = –0.50, P = 0.024). These results are consistent with those found by Ibrahim et al. [21], Ezam et al. [22], Saad et al. [28], and Haluzik et al. [38].

Plasma levels of LDL-cholesterol are negatively correlated with those of ghrelin, but significant only for the obese group (r = –0.42, P = 0.011). These results were consistent with findings by Ezam et al. [22] and Saad et al. [28] in their control group (nonobese/nondiabetic, but not in nondiabetic/obese and diabetic/obese groups). The correlation between ghrelin levels and LDL-cholesterol could be explained by presence of hyperglycemia in the obese group. This suggests the existence of insulin resistance. However, the size of the control group (n = 20) may also explain the lack of significant correlation. In addition, multivariate analysis has verified the existence of a direct correlation between ghrelin and LDL-cholesterol. Indeed, a direct link between these two variables is not well established, but Kellokoski et al. [35] explained it by the potential ability of ghrelin to increase the binding of oxidized LDL in macrophages and monocytes, thereby inducing a decrease in its free level. Moreover, we found a significant positive correlation between plasma levels of ghrelin and HDL-cholesterol and for the two groups (obese: r = 0.64, P = 0.001; controls: r = 0.48, P = 0.032). These results are consistent with those found by Ezam et al. [22], Purnell et al. [26], Al-Hakeim and Ali [27], Saad et al. [28], and Nogueira et al. [39]. Multivariate analysis found that there is a direct correlation between ghrelin and HDL-cholesterol levels. The mechanism of these two influencing variables is not known, but Beaumont et al. [40] have demonstrated that ghrelin may bind to HDL-cholesterol particles, which serve as its circulatory carrier, and which would explain this correlation.

We found that leptin was significantly and negatively correlated with ghrelin in both groups (obese: r = –0.5, P = 0.001; controls: r = –0.58, P = 0.007). These findings are consistent with those of Tschöp et al. [23] and Monti et al. [17] in a population of 233 people (107 men and 126 women) ranging in age from 23 to 75 years, and Ko et al. [19]. Krzyzanowska-Swiniarska et al. [41] found a negative correlation between ghrelin and leptin in an obese noninsulin-resistant group. The insulin-resistant obese group shows no correlation as such. The presence of this correlation in the noninsulin-resistant obese group and its absence in the obese insulin-resistant group is consistent with results found in multivariate analysis, which did not result in a correlation between leptin and ghrelin. Leptin is synthesized in proportion to the state of adiposity and it acts by inhibiting neuropeptide Y/agouti related neuropeptide and proopiomelanocortin neurons by stimulating the cocaine- and amphetamine-regulated transcript. Ghrelin levels decreased in obesity suggest that there is a downregulation of ghrelin as an adaptive response to obesity [23]. With the presence of such a correlation, leptin appears to be involved in the negative regulation of ghrelin. A limitation of our study is that we did not measure insulin. However, insulin seems to be involved in the pathophysiology of obesity and correlates with the parameters we studied.