Evidence of growth hormone effect on plasma leptin in diet-induced obesity and diet-resistant rats
Article Category: Brief communication (original)
Publicado en línea: 01 oct 2019
Páginas: 219 - 228
DOI: https://doi.org/10.1515/abm-2019-0023
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© 2018 Likitnukul et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.
White adipose tissue is considered the major leptin-secreting organ. The mechanisms regulating leptin production and secretion are complex and depend on multiple factors. Plasma leptin is well correlated with fat mass in both lean and obese animals [1, 2]. Plasma leptin and its pattern have been investigated in many animals, including laboratory rodents and humans [3, 4, 5]. In adult rats, plasma leptin shows basal level without peak in light period [4, 6, 7]. This plasma leptin level could be influenced mainly by nutritional status (i.e., feeding and fasting), glucose and free fatty acids (FFAs), and hormonal control (i.e., insulin and catecholamines). Feeding and fasting models suggested important roles of plasma insulin, glucose, and FFA in the diurnal patterns of plasma leptin [3, 4, 8, 9, 10]. In addition to these well-known effects of insulin on plasma leptin, growth hormone (GH) has also been demonstrated to influence plasma leptin [11, 12, 13, 14, 15]. With longterm GH treatment, lower plasma leptin has been demonstrated in accordance with decreased fat mass [16, 17]. This article aimed to study the short-term GH effect on plasma leptin, which was independently of the altered body adiposity. This point was interesting for 3 main reasons. First, obesity is associated with attenuated plasma GH, in which GH has long been considered as hormonal treatment in obesity and metabolic syndrome [18, 19, 20]. Second, short-term GH treatment decreased food intake (FI) in laboratory rodents and goats [14, 15], which may associate with elevated plasma leptin in this period. Finally, the metabolic effects of GH are closely related to and influenced by insulin and leptin [21]. The latter argument appeared to be the major cause of the discrepancy in the short-term GH treatment on plasma leptin [11, 12, 13, 14, 15]. To study whether fat mass influenced the effect of GH on plasma leptin, the current experiments investigated the short-term effects of GH treatment on 2 different amounts of adipose tissue mass from control and obese rats. In addition, this study emphasized basal plasma leptin , which refers to the light phase plasma leptin in
White adipose tissue is considered the major leptin-secreting organ. The mechanisms regulating leptin production and secretion are complex and depend on multiple factors. Plasma leptin is well correlated with fat mass in both lean and obese animals [1, 2]. Plasma leptin and its pattern have been investigated in many animals, including laboratory rodents and humans [3, 4, 5]. In adult rats, plasma leptin shows basal level without peak in light period [4, 6, 7]. This plasma leptin level could be influenced mainly by nutritional status (i.e., feeding and fasting), glucose and free fatty acids (FFAs), and hormonal control (i.e., insulin and catecholamines). Feeding and fasting models suggested important roles of plasma insulin, glucose, and FFA in the diurnal patterns of plasma leptin [3, 4, 8, 9, 10]. In addition to these well-known effects of insulin on plasma leptin, growth hormone (GH) has also been demonstrated to influence plasma leptin [11, 12, 13, 14, 15]. With longterm GH treatment, lower plasma leptin has been demonstrated in accordance with decreased fat mass [16, 17]. This article aimed to study the short-term GH effect on plasma leptin, which was independently of the altered body adiposity. This point was interesting for 3 main reasons. First, obesity is associated with attenuated plasma GH, in which GH has long been considered as hormonal treatment in obesity and metabolic syndrome [18, 19, 20]. Second, short-term GH treatment decreased food intake (FI) in laboratory rodents and goats [14, 15], which may associate with elevated plasma leptin in this period. Finally, the metabolic effects of GH are closely related to and influenced by insulin and leptin [21]. The latter argument appeared to be the major cause of the discrepancy in the short-term GH treatment on plasma leptin [11, 12, 13, 14, 15]. To study whether fat mass influenced the effect of GH on plasma leptin, the current experiments investigated the short-term effects of GH treatment on 2 different amounts of adipose tissue mass from control and obese rats. In addition, this study emphasized basal plasma leptin , which refers to the light phase plasma leptin in
Adult male Wistar rats, aged 12 weeks, were purchased from National Laboratory Animal Center, Mahidol University, and individually housed in conventional hanging cages with stainless steel wire mesh floors (33 cm
Characteristics of the control, HC-R, and HC-O rats in the current experiments
| Control (n = 12) | HC-R (n = 9) | HC-O (n = 12) | |
|---|---|---|---|
| Initial BW (g) | 441.02 ± 2.93a | 420.19 ± 7.58b | 447.33 ± 5.40a |
| Final BW (g) | 503.68 ± 3.41b | 508.27 ± 7.73b | 574.52 ± 7.98a |
| BW gain (g) | 62.67 ± 1.93c | 88.08 ± 4.68b | 127.19 ± 5.80a |
| BW gain/day | 1.57 ± 0.05c | 2.20 ± 0.12b | 3.18 ± 0.14a |
| (g/day) | |||
| Body fat mass (%) | 11.25 ± 0.30c | 12.92 ± 0.41b | 14.97 ± 0.31a |
| AUC-IPGTT | 24667 ± 1303b | 24304 ± 1198b | 28411 ± 1242a |
a–cSignificant difference between groups,
AUC-IPGTT, area under the curve from intraperitoneal glucose tolerance test; BW, body weight; HC-O, hypercaloric diet obese; HC-R, hypercaloric diet resistance
This experiment aimed at determining the effect of short-term exogenous GH treatment on basal, meal-induced and fasting plasma leptin in control (n = 12) and HC-R (n = 9) and HC-O rats (n = 12). All rats from each group were randomly divided into 2 subgroups and treated with either normal saline or GH injection (n = 6; for control and HC-O rats, n = 4 and 5; for saline-treated and GH-treated HC-R rats, respectively). The GH-treated rats were injected subcutaneously with recombinant human GH (GenHeal®; Shanghai United Cell Biotechnology Co., Ltd., Shanghai, China) 1 mg/kg twice per day (at 0800 and 1600 h) for 3 days. The 3-day GH treatment was selected based on the effect of GH on basal plasma leptin from our pilot study. Blood sample (0.3 mL) was collected from the ventral tail artery at 24 and 32 h after the first GH injection for basal plasma leptin measurement, because the results from our pilot study indicated that GH decreased basal plasma leptin at 32–36 h under light period. Meal-induced plasma leptin was investigated at the beginning of dark phase on day 3 after the first GH injection. Rats were fasted for 2 h (1600 h) before dark onset. At the onset of dark phase (1800 h), food was provided; then, the rats were allowed to access the food for 2 h [20]. Blood samples (0.3 mL) for meal-induced plasma leptin were collected before light off (premeal, 1800 h), which was considered as basal leptin after 32 h from the first injection and after eating for 2 h (post-meal, 2000 h). Meal-induced plasma leptin was calculated from the post- and premeal difference of plasma leptin (leptin difference). After meal-induced plasma leptin experiment, food was removed, and then the rats were fasted for another 16 h. At the mid-light phase of day 3, blood samples were collected for the measurement of fasting plasma glucose (Gfasting), insulin (Ifasting), FFAfasting, and leptin, as shown in
Figure 1
The experimental procedure is demonstrated. After the obesity induction period, IPGTT was performed before GH or saline treatment (at day -7). Then, the rats were injected with either GH or saline for 3 days and blood samples were collected for plasma hormones and FFA measurement. The white and black bars indicate light and dark phases, respectively (12h/12h light/dark cycle).
FFA, free fatty acid; GH, growth hormone; IPGTT, intraperitoneal glucose tolerance test
Glucose tolerance test was used to determine the representative whole-body insulin sensitivity after obesity induction period. The rats were fasted for 16 h before intraperitoneal glucose tolerance test (IPGTT) evaluation. Blood samples were collected by tail-clipping technique for blood glucose measurement (Accu-Chek® Performa; Roche Diagnostic GmbH, Mannheim, Germany) before glucose administration (time 0). The 50% glucose solution (A.N.B. Laboratories Co., Ltd., Bangkok, Thailand) was injected intraperitoneally (2 g/kg BW). Blood glucose was measured at 15, 30, 60, 90, and 120 min after glucose injection.
Plasma leptin levels were measured by ELISA kits (EZRL-83K; Merck Millipore, Massachusetts, USA). The intra-assay coefficient of variability (CV) for this measurement was 3.46%, and the inter-assay CV was 6.97%. Plasma insulin was measured with a commercial ELISA kit (EZRMI-13K; Merck Millipore, Massachusetts, USA). The intra-assay CV for this measurement was 2.43%. Plasma FFA was measured with colorimetric assay kit (ab65341; Abcam, Cambridge, UK).
The results were analyzed by 2-way analysis of variance (ANOVA). In addition, for the analysis of basal plasma leptin at 2 different time points either saline or GH, the test was performed by 2-way repeated-measures ANOVA. The significant main effects were followed up using a Bonferroni posttest. A significant mean difference was set at
After 24 and 32 h of the first GH injection, Basal plasma leptin levels were not significantly different in control rats (
The effect of short-term GH treatment on basal plasma leptin at 24 and 32 h after GH injection
| Time after the first injection | Basal plasma leptin (ng/mL) | ||
|---|---|---|---|
| Control | HC-R | HC-O | |
| Saline | |||
| 24 h | 16.98 ± 0.7 | 18.93 ± 2.7 | 21.16 ± 1.0 |
| 32 h | 17.94 ± 1.2 | 18.47 ± 1.9 | 29.20 ± 1.8* |
| GH | |||
| 24 h | 16.89 ± 0.8 | 15.74 ± 0.9 | 17.00 ± 1.8 |
| 32 h | 17.44 ± 2.5 | 18.38 ± 0.8 | 21.55 ± 2.0*,# |
*Effect of time on basal plasma leptin in HC-O rats,
#Effect of GH on basal plasma leptin in saline vs GH treatment at 32 h,
GH, growth hormone; HC-O, hypercaloric diet obese; HC-R, hypercaloric diet resistance
Figure 2
The effect of short-term GH treatment on body adiposity and leptin per fat mass. (
*The leptin per fat mass of GH-treated HC-O rats was significantly lower than that from saline treatment,
GH, growth hormone; HC-O, hypercaloric diet obese; HC-R, hypercaloric diet resistance
Figure 3
The effect of short-term GH treatment on meal-induced plasma leptin. (
*The energy intake of HC-O rats was significantly different from others,
GH, growth hormone; HC-O, hypercaloric diet obese; HC-R, hypercaloric diet resistance
In 16-h fasting condition, fasting effect on plasma leptin level was shown as leptin difference. The effect of GH on the fasting effect on plasma leptin was significantly difference (
Figure 4
The effect of short-term GH treatment on the fasting effect on plasma leptin (leptin difference). There was a significant difference of the effect of GH treatment in the control and HC-R groups.
*GH effect is significantly different from saline treatment,
GH, growth hormone; HC-R, hypercaloric diet resistance
Figure 5
The effect of short-term GH treatment on the surrogate indexes for insulin receptor sensitivity. (
*GH effect is significantly different from saline treatment,
Adipo-IR, adipose tissue insulin resistance; GH, growth hormone; HC-R, hypercaloric diet resistance; HOMA-IR, homeostasis model of insulin resistance.
The effect of short-term GH treatment on fasting plasma insulin, glucose, and FFA
| Control | HC-R | HC-O | |
|---|---|---|---|
| Insulin (ng/mL) | |||
| Saline | 2.96 ± 0.5 | 2.39 ± 0.7 | 5.12 ± 0.8 |
| GH | 6.09 ± 1.0† | 6.26 ± 1.4† | 6.92 ± 1.2† |
| Glucose (mg/dL) | |||
| Saline | 131.50 ± 6.8 | 126.00 ± 6.0 | 131.50 ± 4.0 |
| GH | 135.20 ± 3.0 | 126.80 ± 12.8 | 123.30 ± 7.7 |
| FFA (mmol/L) | |||
| Saline | 0.55 ± 0.13 | 0.29 ± 0.06 | 0.60 ± 0.14 |
| GH | 0.45 ± 0.10 | 0.81 ± 0.12 | 0.67 ± 0.17 |
†GH effect on fasting plasma insulin in all groups,
FFA, free fatty acid; GH, growth hormone; HC-O, hypercaloric diet obese;
HC-R, hypercaloric diet resistance
Our data showed that the short-term GH treatment decreased basal plasma leptin only in HC-O rats, which suggested that the short-term GH effect on basal plasma leptin partly depends on body adiposity. The results revealed that the short-term GH administration could not alter adipose tissue mass in all rats. Interestingly, GH decreased normalized plasma leptin to body fat mass only in HC-O rats. However, the short-term GH treatment could not influence meal-induced plasma leptin in all rats. In addition, GH administration could attenuate fasting effect on plasma leptin in the control and HC-R groups.
The main objective of the current experiment was to investigate the short-term effect of GH treatment on plasma leptin in different paradigms. By using GH as exogenous hormonal stimulus, the current findings revealed the different mechanisms controlling basal plasma leptin. In the current experiment, we measured basal plasma leptin during light period. After 24 h of the first injection, GH had no effect on basal plasma leptin. However, after 32 h of the first injection, plasma leptin from GH treatment was lower than that from saline injection in the HC-O group. It should be noted that all rats cannot access to food for 1 h before light off during the maintenance period every day. Since all rats were fasted for only 2 h before the light off (at 32 h after the first injection), the premeal plasma leptin level is considered to be within the range of basal plasma leptin [3, 4]. This interpretation agrees with our pilot study for the GH effect on basal plasma leptin. Our results are consistent with the chronological study of acute GH treatment on basal plasma leptin in healthy humans [12]. Unfortunately, many experiments in rodents were investigated for long-term GH effect on plasma leptin or cross-sectional information [11, 25, 26]. This study suggests that the amount of adipose tissue apparently influences the effect of GH on basal plasma leptin. The previous studies demonstrated that leptin content and secretion rate have been correlated well with the fat mass and the size of adipocyte [27, 28]. However, the reason that GH decreased basal plasma leptin levels and normalized plasma leptin to body fat mass in which specifically occurred in HC-O rats remains unknown (see the following section). In rodents, the diurnal pattern of plasma leptin has been well demonstrated. Plasma leptin is maintained at its basal level during the light phase and gradually increases after the first meal of dark onset [3, 4]. The reason that explained the higher plasma leptin at 32 h than that of 24 h in HC-O rats was unknown. Although many experiments have been performed to study nighttime plasma leptin, the mechanisms that control basal plasma leptin are less studied and remain unclear. Previous studies demonstrated that basal leptin content in adipose tissue depends on new leptin synthesis. The pathway of leptin biosynthesis was apparently regulated by a mechanism independent of insulin action [29]. Taken together, the
Next, we demonstrated that the short-term GH treatment had no effect on meal-induced plasma leptin in all rats. Meal-induced plasma leptin has been well studied and was related to the nighttime plasma leptin in the rodent. The single peak of nighttime plasma leptin in rodents could be influenced strongly by eating behavior [3, 4]. When food was provided only in the light phase, diurnal rhythm of plasma leptin was reversed according to eating activity [4]. Insulin and glucose were important determinants of meal-induced plasma leptin [28, 30]. For instance, plasma leptin after glucose administration was dependent on high plasma insulin [30]. We propose from our previous result that the mechanism of GH effect on basal plasma leptin may be independent on insulin-mediated plasma leptin. The current results support, but not prove, our hypothesis and further inform that GH treatment had no effect on meal-induced plasma leptin (or insulin-stimulating leptin secretion) in HC-O rats during the stage of ample energy. The previous report provided an association between the amount of energy intake and meal-induced plasma leptin [22]. In addition, our results revealed that HC-O rats had the greatest energy intake among all groups, which was related to meal-induced plasma leptin.
Fasting plasma leptin appears to share a similar mechanism with basal and meal-induced plasma leptin. First, the leptin secretion during fasting derived mainly from the basal pool of leptin vesicle [29]. Second, decreased plasma leptin during fasting apparently occurred in part from the absence of the signal from insulin-stimulating leptin secretion [30, 31]. During fasting, decreased plasma insulin concentration as well as decreased glucose uptake and adipose tissue oxidation have been well demonstrated [32, 33]. In addition, lipolysis is the main biochemical pathway in adipocytes to provide FFA as an energy source [34]. Although intracellular FFA has been demonstrated to attenuate insulin-stimulated leptin secretion [8], the mechanism apparently fits the stage of ample energy with the presence of insulin and glucose rather than the stage of fasting [8, 29, 31]. With 16-h fasting condition, plasma leptin was decreased in saline-treated control and HC-R rats, which was significantly different from GH-treated control and HC-R rats. This result was apparently due to the GH effect, which could attenuate fasting effect. This attenuation effect may be explained indirectly by the effect of GH on plasma insulin. However, for HC-O rats, the fasting effect was similar as control rats but not statistical significance (
In conclusion, the current results revealed the short-term effect of GH on plasma leptin levels. GH could decrease basal plasma leptin, which appeared to associate with body adiposity. GH did not produce this effect on meal-induced plasma leptin. In addition, GH could attenuate the fasting effect on plasma leptin.