Regulatory Peptides 122 (2004) 179 – 183 www.elsevier.com/locate/regpep
Acute insulin infusion decreases plasma ghrelin levels in uncomplicated obesity Frida Leonetti, Gianluca Iacobellis *, Maria Cristina Ribaudo, Alessandra Zappaterreno, Claudio Tiberti, Concetta Valeria Iannucci, Elio Vecci, Umberto Di Mario Endocrinology, Department of Clinical Sciences, University ‘‘La Sapienza’’, Rome, Italy Received 24 February 2004; accepted 9 June 2004 Available online 28 July 2004
Abstract Background: Plasma ghrelin levels have been shown to decrease after insulin infusion in lean subjects. Nevertheless, the mechanism of the suggested inhibitory effect of insulin on ghrelin is still unclear and no data about the effect of acute insulin infusion on plasma ghrelin concentration in obese subjects are available. Objective: We sight to evaluate plasma ghrelin concentration during an hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. Methods: 35 uncomplicated obese subjects, body mass index (BMI) 43.3 F 10.1 kg/ m2, 33 women and 2 men, mean age 34.9 F 10, with a history of excess fat of at least 10 years underwent euglycemic hyperinsulinemic clamp. Blood samples for ghrelin were performed at baseline and steady state of euglycemic insulin clamp. Results: Ghrelin concentrations decreased over time to 10.6 F 15% (range 2 – 39%) of baseline, from a mean of 205.53 F 93.79 pg/ml to 179.03 F 70.43 pg/ml during the clamp (95% CI, 10.69 to 36.44, P < 0.01). In a univariate linear regression analysis baseline plasma ghrelin levels were inversely correlated to BMI (r = 0.564, P = 0.04). A linear positive trend between whole body glucose utilization (MFFMkg index) and ghrelin reduction during the clamp was found (v2 3.05, p = 0.05). Conclusions: Our data seem to suggest that hyperinsulinemia during a euglycemic clamp is able to suppress plasma ghrelin concentrations in uncomplicated obesity. This effect appears to be positively related to insulin sensitivity. D 2004 Elsevier B.V. All rights reserved. Keywords: Ghrelin; Obesity; Insulin resistance
1. Introduction Ghrelin, the novel peptide hormone produced primarily by the stomach [1], is assumed to be involved in regulating food consumption and meal initiation [2– 5]. It has been demonstrated that ghrelin plasma levels significantly fall after a meal [6] while diet-induced weight loss produces an increase in ghrelin concentration [7]. In contrast, ghrelin concentrations are down-regulated in obesity [8] and food intake fails to decrease ghrelin levels in obese patients [9]. Ghrelin showed reciprocal changes to insulin in response to a meal, suggesting a relationship between the two * Corresponding author. Center for Human Nutrition, The University of Texas, Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390-9052, USA. Tel.: +1-214-648-2419; fax: +1-214-648-7150. E-mail address:
[email protected] (G. Iacobellis). 0167-0115/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.regpep.2004.06.014
hormones [4]. Plasma ghrelin levels have been shown to decrease after insulin infusion in lean subjects allowing to formulate the hypothesis that insulin could act as physiological and dynamic modulator of ghrelin [10]. Nevertheless, the mechanism of the suggested inhibitory effect of insulin on ghrelin levels is still unclear and no data about the effect of acute insulin infusion on plasma ghrelin concentration in obese subjects are available. Acute hyperinsulinemia, as well chronic high insulin levels, could suppress, at least in part, ghrelin concentrations in obese subjects. In this study we sight to evaluate plasma ghrelin concentration during a hyperinsulinemic euglycemic clamp in uncomplicated obese subjects. The hyperinsulinemic euglycemic clamp allows us to inject a similar amount of insulin, keeping an unchanged glucose levels. Young adult obese subjects, with a long-term disease and without confounding factors on insulin sensitivity, may be a good model to better understand the influence of insulin on ghrelin secretion.
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2. Materials and methods 2.1. Subjects We selected 35 obese subjects (body mass index (BMI) >30 kg/m2), 33 women and 2 men, mean age 34.9 F 10, BMI 43.3 F 10.1 kg/m2, with a history of excess fat of at least 10 years, who were all screened from our Day Hospital. All obese subjects had normal fasting plasma glucose ( < 110 mg/dl), normal glucose tolerance ( 2-h glucose of oral glucose tolerance test (OGTT) < 140 mg/dl), normal resting arterial blood pressure (systolic, < 140 mm Hg; diastolic, < 90 mm Hg for at least three measurements), normal serum level of total cholesterol ( < 220 mg/dl) and triglycerides ( < 170 mg/dl), HDL higher than 50 mg/dl for women and 40 mg/dl for men, and normal thyroid hormones. None had any evidence of cardiovascular, hepatic, renal, respiratory and other metabolic diseases by routine history, physical examination and laboratory screening tests (plasmatic creatinine, aspartate aminotransferase, alanine aminotransferase and glutamyl transferase, red and white cells count) and no subject was taking any medication. All the subjects had stable body weight, as defined by a change of no more than 5% in body weight during the preceding 3 months. This study was conducted in accordance with the guidelines proposed in The Declaration of Helsinki and has been approved by the review committee of ‘‘ La Sapienza’’ University. All subjects gave their informed consent before the study began.
correction for changes in glucose levels in a distribution volume of 250 ml/kg. The M value was adjusted by kilograms of fat-free mass (M/FFMkg index). 3.1. Plasma ghrelin evaluation Blood samples for ghrelin were performed at baseline and at 120 min of euglycemic insulin clamp. Samples were stored at 4 jC during the collection period, after which plasma was stored at 80 jC until the time of the assay. 3.2. Analytical procedures Plasma ghrelin was determined in duplicate by radioimmunoassay (RIA) kits (Ghrelin Human RIA Kit Catalog No: RK-031-30, Phoenix Pharmaceuticals). Phoenix’s Ghrelin Human RIA Kit detects full-length, des-octanoyl human ghrelin, including Ser3-octanoyl and Ser3-des-octanoyl ghrelins. Plasma glucose was determined by the glucose oxidase method [Autoanalyzer, Beckman Coulter, Fullerton, CA; coefficient of variation (CV), 1.9 F 0.2%]. Plasma total cholesterol (CV, 3.4 F 0.2%), high-density lipoprotein cholesterol (CV, 3.7 F 0.4%), and triglycerides (CV, 3.1 F 0.5%) concentrations were measured using enzymatic kits (OrthoClinical Diagnostic, Milan, Italy). Blood samples for plasma insulin measurements were collected in heparinized tubes. After centrifugation, plasma insulin concentrations were determined with a commercial RIA kit (CV, 3.0 F 0.3%) (Linco Research, St. Louis).
3. Experimental
3.3. Anthropometric measurements
In order to inject a similar amount of insulin with unchanged glucose levels, we performed hyperinsulinemic euglycemic clamp [11] in all obese subjects, after 10 – 12 h overnight fast, at 9:00 a.m. A polyethylene cannula was inserted into an antecubital vein for the infusion. A second catheter was inserted into the antecubital vein controlaterally to determine plasma glucose and insulin concentrations. Insulin was continuously infused at the rate of 4.0 mU/kg min for 5 min, 2.0 mU/kg min for 5 min and 1.0 mU/kg min for 110 min. The plasma glucose concentration was measured every 5 min after the start of the insulin infusion and a variable infusion of 20% glucose was adjusted based on the negative feedback principle to maintain the plasma glucose level at fasting plasma glucose with a coefficient of variation of < 5%. Plasma samples were collected every 20 min for determination of insulin concentrations. The steady state of the test was considered the interval between 60 and 120 min. In these standard conditions, glucose infusion requirement in order to maintain euglycemia equals whole body insulin mediated glucose disposal, indicating the degree of insulin sensitivity. Whole body glucose utilization (M) was calculated from the infusion rate of exogenous glucose during the second hour of the insulin clamp period after
Weight (to the nearest 0.1 kg) and height (to the nearest 0.5 cm) were measured while the subjects were fasting and wearing only their undergarments. BMI was calculated as body weight divided by height squared and was used as a marker of obesity. 3.4. Measurement of body fat distribution Body composition was evaluated by electrical bioimpedance. Fat-free mass (FFM) was calculated as the difference between body weight and fat mass (FM).
4. Statistical analysis Data are presented as mean F standard deviation (SD). We used paired t test with the calculation of 95% CI to estimate the differences between plasma ghrelin during the clamp. Unpaired t test was used to assess the difference on baseline ghrelin between obese subjects with different BMI. Simple linear regression analysis was carried out using standard techniques. v2 test for trend was used to assess a linear trend between M index and ghrelin reduction during
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the clamp. Two-tailed p < 0.05 indicated statistical significance. We used GraphPad InStat software (San Diego, CA, USA) for statistical analysis.
5. Results The anthropometric and clinical characteristics of all subjects studied are summarized in Table 1. 5.1. Baseline Baseline ghrelin concentration was 205.53 F 93.79 pg/ ml in all obese subjects. Obese subjects with BMI < 40 kg/ m2 (n = 12) showed baseline ghrelin levels higher than subjects with BMI >40 kg/m2 (n = 23) (250.46 F 94.84 pg/ ml vs. 174.78 F 71.41) (95% CI, 15.95 to 142.04, P < 0.05). Baseline insulin and glucose levels were 26.5 F 15.5 AU/ ml and 86.2 F 10.9 mg/dl, respectively (Table 1).
Fig. 1. Glucose, insulin and ghrelin variation during the clamp. Plasma ghrelin concentrations decreased over time to 10.6 F 15% (range 2 – 39%) of baseline, from a mean of 205.53 F 93.79 to 179.03 F 70.43 pg/ml during the clamp ( P < 0.01).
P = 0.04). A linear trend between MFFMkg index and D ghrelin reduction during the clamp was found (v2 3.05, p = 0.05). No correlation between baseline ghrelin and age, fasting insulin, fasting glucose and fat mass was observed.
5.2. Hyperinsulinemic euglycemic clamp
6. Discussion
The whole body glucose utilization (MFFMkg index) of uncomplicated obese subjects was 7.94 F 3.36 mg/FFMkg/ min. Coefficient of variation of blood glucose was less than 4% in each clamp study throughout the test. Exogenous insulin increased circulating insulin concentrations, from a baseline of 26.5 F 15.5 to 149.8 F 33.7 AU/ml. Ghrelin concentrations decreased over time to 10.6 F 15% (range 2– 39%) of baseline, from a mean of 205.53 F 93.79 to 179.03 F 70.43 pg/ml during the clamp (95% CI, 10.69 to 36.44, P < 0.01) (Fig. 1). No difference on glucose levels during the clamp was observed.
Our data show a significant decrease of plasma ghrelin levels during a euglycemic hyperinsulinemic clamp in uncomplicated obesity. To our knowledge, no previous findings on the effect of acute hyperinsulinemia on plasma ghrelin concentrations in uncomplicated obese subjects were described before. The relationship between insulin and ghrelin remains debatable and controversial. Some studies have shown the inhibitor action of insulin [10] on plasma ghrelin levels in normal subjects, whereas others have not [12]. The mechanism of the inhibitory effect of insulin on ghrelin secretion is still unclear. It was suggested that insulin could act directly on ghrelin-secreting cells [10] or that the physiological post-prandial increase of insulin suppresses plasma ghrelin levels. Changes in plasma insulin concentration have been observed to be associated with swift and reciprocal changes in plasma ghrelin concentrations. In fact, euglycemic clamp study reported that plasma ghrelin decreased by 90 min and continued to be suppressed for 15 min after the insulin infusion was discontinued in lean subjects [10]. On the other hand, it has been recently shown that only prolonged and supraphysiological hyperinsulinemia resulted in suppressed plasma ghrelin levels in lean subjects [13]. In that study, the authors observed a reduction in plasma ghrelin values only during pharmacological hyperinsulinemia, suggesting that sustained insulin concentrations would be required to suppress ghrelin in lean subjects. They concluded that insulin at physiological concentrations is not responsible for the reduction in ghrelin in the healthy lean subjects. In contrast, no effect on plasma ghrelin by administration of insulin lispro has been reported either. Probably, this finding may be better explained with the short-acting insulin preparation.
5.3. Correlates of ghrelin In a univariate linear regression analysis baseline plasma ghrelin levels were inversely correlated to BMI (r = 0.564, Table 1 Clinical parameters in uncomplicated obese subjects Age (years) Weight (kg) Height (m) BMI (kg m2) Fasting glucose (mg/dl) Fasting insulin (AU) Total cholesterol (mg/dl) HDL cholesterol (mg/dl) LDL cholesterol (mg/dl) Triglycerides (mg/dl) Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg)
34.9 F 10.0 114.7 F 20.4 1.63 F 0.09 43.3 F 10.1 86.2 F 10.9 26.5 F 15.5 188.1 F 25.3 54.2 F 11.6 91.5 F 55.2 93.0 F 38.9 123.5 F 9.7 79.7 F 6.1
Fasting glucose (Glucose); fasting insulin (Insulin); Total cholesterol (Tot cholesterol); HDL Cholesterol (HDL); LDL cholesterol (LDL); Systolic Blood Pressure (Syst BP); Diastolic Blood Pressure (Diast BP).
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In our study, plasma insulin levels during the clamp seem to be within physiological values for subjects with a severe obesity degree. In fact, it is well known that high post-prandial plasma insulin levels occurred in severe obese subjects. Moreover, obesity is known to be associated with low baseline plasma ghrelin concentrations and our results confirmed this finding [8]. Even in this case, it could be speculated that high insulin levels frequently observed in severe obese subjects could suppress, at least in part, baseline ghrelin concentrations in these subjects. This action could be addressed to reduce food intake in obese subjects. In this study we evaluated a number of uncomplicated obese subjects with a severe degree of obesity. BMI resulted, in agreement with other reports, as the major determinant of ghrelin [8]. Hence, the occurrence of a high BMI could also explain the finding of low plasma ghrelin levels in our obese subjects. The low baseline ghrelin levels could justify the weak, but significant, decrease of ghrelin levels during the clamp. At the same time, the reduction of ghrelin concentration, despite its low baseline level, during insulin infusion could provide an interesting information about the effect of insulin on ghrelin secretion in severe obesity. We observed a wide range of percentage of reduction (2– 39%) suggesting and confirming a great variability of circulating ghrelin concentrations [13]. Insulin sensitivity degree appears to be a weak determinant of ghrelin response to insulin infusion during the clamp. Nevertheless, we have observed that obese subjects with preserved insulin sensitivity tend to show a higher percentage of reduction in ghrelin levels during the clamp in comparison with subjects with important insulin resistance state. It could be speculated that insulin resistance condition could play an additional role in the weak inhibitory response of ghrelin after insulin infusion and, besides the high BMI, in the low baseline ghrelin levels observed in obese subjects. Furthermore, the presence of severe but uncomplicated obese subjects allows us to correctly evaluate the effect of insulin sensitivity, without confounding factors, on ghrelin secretion. Ghrelin was not significantly related to fasting insulin in our study. We have recently shown a lack of correlation between insulin and ghrelin in severe obese subjects who underwent laparoscopic gastric bypass and gastric banding [14]. On the other hand, our finding seems to be consistent with some studies [15,16] and partially in disagreement with others performed on children or adolescents [17,18]. In conclusion, our data seem to suggest that hyperinsulinemia during a euglycemic clamp is able to suppress plasma ghrelin concentrations in uncomplicated obesity. This effect appears to be related, although weakly, to the ability of insulin to stimulate glucose uptake and then to the insulin sensitivity. However, the mechanism of the inhibitory action of insulin on plasma ghrelin in obesity is far from being completely clear.
6.1. Limitations of the study The absence of a control group of normal-weighted subjects does not allow to completely assess the pathophysiological mechanism of the inhibitory action of insulin on plasma ghrelin in obesity. However, the study describes, for the first time, the effect of acute hyperinsulinemia on ghrelin in obese subjects without any comorbidities. A small number of men were in this study. This is probably due to the difficulty of enrolling men with uncomplicated obesity. Hence, the evaluation of a possible influence of gender on ghrelin response to acute hyperinsulinemia in obese subjects will require further investigations.
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