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Effect of adrenocorticotropic hormone on UCP1 gene expression in brown adipocytes

  • Hirendra M. Biswas EMAIL logo
Published/Copyright: April 4, 2017
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Journal of Basic and Clinical Physiology and Pharmacology
From the journal Journal of Basic and Clinical Physiology and Pharmacology Volume 28 Issue 3

Abstract

Background:

Like other tissues, adrenocorticotropic hormone (ACTH) can produce its effect on brown adipose tissue (BAT). This study was taken to understand the direct effect of ACTH action on thermogenin gene expression and possible relation with α receptors and caffeine with this hormone.

Methods:

Brown fat precursor cells were isolated from interscapular BAT of young mice and grown in culture. The cells were exposed to norepinephrine (NE) and other agents. Total RNA was isolated after harvesting the cells, and northern blot analysis was performed. Hybridization was performed with nick translated cDNA probes. Filters were exposed to film, and results were evaluated by scanning. Cyclic adenosine monophosphate (cAMP) was measured by using Amersham assay kit.

Results:

ACTH stimulates thermogenin gene expression in brown adipocytes. Initiation and maximum stimulations are observed with 0.01 μM and 10 μM (about 45%) of ACTH, respectively, in comparison to 0.1 μM of NE. Maximum response of cAMP is also observed with 10 μM of ACTH (about 64%). Studies with cirazoline and ACTH show that UCP1 mRNA expression is increased significantly with 10 μM of ACTH, whereas cAMP generation is decreased. In the presence of caffeine, ACTH increases cAMP generation and UCP1 gene expression more than twofold.

Conclusions:

ACTH stimulates thermogenin gene expression in cultured brown adipocytes. The complex interrelationship of ACTH with cirazoline indicates the possibility of relation between the activity of ACTH and α receptors in brown adipocytes. Further stimulation of cAMP generation and thermogenin gene expression is possible with ACTH in conjugation with caffeine and RO 20-1724.

Keywords: adrenocorticotropic hormone (ACTH); brown adipose tissue (BAT); caffeine; cirazoline; cyclic adenosine monophosphate (cAMP); UCP1 mRNA

Introduction

Brown adipose tissue (BAT) is an organ that is responsible for heat production in newborn infants and in adult animals. The heat production is governed by signals from the hypothalamus. These signals are relayed via the sympathetic nervous system and transmitted to the cells as norepinephrine (NE) stimulus leading to a series of events within the cells [1]. Besides being sensitive to NE, brown fat cells are also sensitive to some other hormones, such as insulin, glucagon, glucocorticoids and thyroid hormones [2].

An early report of Skala et al. [3] indicated that the stimulation of brown fat cells by adrenocorticotropic hormone (ACTH) was probably due to secondary effect of NE which is released from the sympathetic nerves. On the other hand, it was also reported that cold exposure increased the plasma level of ACTH and in adipocytes it also increased the lipolytic responsiveness to this hormone [4]. In 1984, York and Al-Baker [5] suggested that BAT function is enhanced by corticotrophin. It also showed that ACTH stimulates lipolysis [6] and increases BAT glucose transport [7] via fatty activation of mitochondrial thermogenesis. It is clear from the literature that the previous studies of BAT with ACTH were observed mostly on impure cell preparations. There is very little information in the literature about the effect of ACTH on cultured brown fat cells, and no report is available about the relation between the α adrenoreceptor and ACTH on these cells.

The present in vitro study was undertaken to investigate the possible effect of ACTH action on thermogenin gene expression in brown adipocytes and the nature and complexity of the receptors (specially the α receptor) which are involved in this hormone action. It also tries to investigate the mechanism involved in this effect at cyclic adenosine monophosphate (cAMP) level and the effects of some agents (such as caffeine and RO 20-1724) on brown adipocytes, related to the inhibition of phosphodiesterase enzyme and prolongation of hormonal activities.

Materials and methods

Animals

Four-week-old mice of NMRI strain were obtained from local supplier (A-lab, Stockholm) and kept at 21±2 °C with food and water ad libitum. The animals were killed with CO2. Experiments were carried out in Sweden. The procedures were approved by the Animal Ethics of the North Stockholm region.

Isolation of cells and culture

The precursor cells of brown fat were isolated from cervical, interscapular and axillary BAT depots of 4-week-old mice according to the method of Nechad et al. [8]. Tissues were combined from all these depots and incubated in Hepes-buffered solution [8] containing 200 units/mL crude collagenase type 2 (Sigma, St. Louis, MO, USA). The tissue was digested for 30 min at 37 °C in a plastic polypropylene test tube with 10 s vortexing every 5 min. It was then filtered through a 250 μm nylon screen. The filtrate was then placed on ice for 30 min to allow the mature brown fat cells and lipid droplets to float. Then the infranatant was filtered through a 25 μm nylon screen, and the precursor cells were pelleted by centrifugation for 10 min at 2000 rpm (Sigma 3E-1, St. Louis, Mo, USA). The supernatant was discarded, and the cells were washed with 2 mL of Dulbecco’s modified Eagle’s medium (DMEM, Flow, Sigma, St. Louis, MO, USA). The precursor cells were pelleted further by centrifugation for 10 min at 2000 rpm. The supernatant was discarded, and the pellets of cells were diluted with culture medium (0.5 mL/animal), consisting of DMEM supplemented with 10% newborn calf serum (Flow), 4 nM insulin (Actrapid Human, Novo, Copenhagen, Denmark), 10 mM Hepes (Flow), 4 mM glutamine (Flow), 50 IU of penicillin, 5 μg of streptomycin and 25 μg of sodium ascorbate (Kebo, Spanga, Sweden) per mL. A volume of 1.8 mL of culture medium was added to each culture well (35 mm diameter culture wells, Corning cell wells) before 0.2 mL of pooled final cell suspension was added. Routinely, about 36 wells (maximum) were inoculated with each cell suspension. The cells were grown at 37 °C in an atmosphere of 8% CO2 in air in Heraeus CO2-auto-zero B 5061 incubator. On the first and the third days after inoculation, the culture medium was removed and cells were washed with 2 mL DMEM before the addition of 2 mL of fresh medium. The medium was not changed on the day of the experiment.

Experiments

The experiments were performed with cells grown for 6 days (i.e. Confluent). The following agents were used for the stimulation of the cells: NE (Arterenol bitartrate salt, Sigma, 98%); ACTH (corticotrophin A, Sigma, 95 IU/mg); propranolol (Sigma); cirazoline (Synthelabo recherché, Paris, France); caffeine (Sigma) and RO 20-1724 (RBI, Natick, MA, USA). Caffeine and RO were dissolved in 40% alcohol. A 10 μL solution of the required concentration of the agents was used for stimulation of cells. All the experiments were performed at least three times, and each performed in duplicate.

Cell harvesting, RNA isolation and analysis

The cultured cells were harvested 4 h after addition of the agents to the wells. The cells were washed with 2 mL of ice-cold saline/well after discarding the medium, and then the cells were dissolved in 1 mL of hot guanidine-HCl extraction buffer, according to the method of Jacobsson et al. [9]. Total RNA was isolated as described previously [9, 10], and the RNA concentration was determined in a spectrophotometer (DU-50, Beckman Instruments, San diego, USA) at 260 nm. The 260/280 ratios were routinely around 1.8–2.0.

Northern blot analysis

Northern blot analysis was performed as previously described [9] in a 1.25% agarose gel containing 3% formaldehyde in 10 mM sodium phosphate buffer (pH 6.5), and then RNA was blotted to zeta-probe nylon membranes. The filters were prehybridized at 45 °C for at least 1 h and then hybridized with cDNA probes overnight at 45 °C. The probes had been labeled by nick translation (BRL nick translation kit, from Bethesda Research Laboratories, USA) and had specific activity of about 10–7 counts per minute/μg DNA. After hybridization, the filters were washed, dried and exposed to a phosphoimager screen at room temperature or Kodak X-omat film at −80 °C. The resulting autoradiogram was evaluated by scanning with laser densitometer (Molecular Dynamics, Madison, USA) or a phosphoimager (Molecular Dynamics, Madison, USA).

Measurement of cAMP

Measurement of cAMP was performed in the cultured cells after incubation for 30 min with the agents described in “Results” section. The culture medium was then discarded; 0.8 mL of 95% ethanol was added to each well, and it was collected into the Eppendorf tube. The wells were then washed with 0.4 mL of 70% ethanol. The combined suspensions were dried in a Speedvac centrifuge for 3–4 h at 55 °C. The dried samples were dissolved in 250 μL of Buffer 1 (Tris-EDTA) provided with cAMP 3H Assay System from Amersham and centrifuged in an Eppendorf centrifuge at 12,000 rpm for 5 min. Five-microliter aliquots of the supernatants were analyzed according to the description of the assay system.

Statistics

For statistical analysis, ANOVA and Tukey’s HSD post hoc test were performed in this study to evaluate the significance of changes of the experimental data. The results are shown in Figures and Tables as the mean±SE.

Results

For investigation of the role of ACTH on UCP gene expression, 0.1 μM of NE is used as a standard concentration of comparison for stimulation of transcription of UCP1 gene. This is because UCP1 gene expression is maximally stimulated with 0.1 μM NE [11].

The time-response curve of the level of cAMP after stimulation with NE and ACTH on brown fat cell cultures is shown in Figure 1. This curve shows that maximum response of cAMP generation is observed after 60 min stimulation with NE and 30 min stimulation with ACTH. The disappearance rate of cAMP from the cells was similar in both cases. Significant difference is observed in ACTH-stimulated cells (60, 120 and 240 min) in comparison to NE.

Figure 1: Time-response curve of cAMP level after stimulation of brown fat cell cultures with NE (0.1 μM) or ACTH (10 μM).Points are means±SE from three independent experiments in three different times, each performed in duplicate. The NE and ACTH responses were measured in parallel wells. Statistically significant difference is marked with asterisk when 10 μM of ACTH is compared with 0.1 μM of NE in each respective time period. *p<0.05, **p<0.001 (Student’s t-test) compared with NE.
Figure 1:

Time-response curve of cAMP level after stimulation of brown fat cell cultures with NE (0.1 μM) or ACTH (10 μM).

Points are means±SE from three independent experiments in three different times, each performed in duplicate. The NE and ACTH responses were measured in parallel wells. Statistically significant difference is marked with asterisk when 10 μM of ACTH is compared with 0.1 μM of NE in each respective time period. *p<0.05, **p<0.001 (Student’s t-test) compared with NE.

The induction of UCP1 mRNA in cultured brown fat cells in mice after 4 h stimulation of the cells with NE and different doses of ACTH is shown in Figure 2. In comparison to 0.1 μM of NE, about 45% response was observed after treatment with 10 μM concentration of ACTH. The dose-response curve of the effect of ACTH on brown fat cells demonstrate that 0.01 μM of ACTH was the minimum concentration to detect the UCP1 gene expression, and the maximum stimulation was observed with 10 μM concentration. All the results are calculated after deduction of control value which is 1.0±0.5 in Table 1, 3.0±1.0 for cAMP and 0.5±0.2 for UCP1 in Table 2, and 1.25±0.7 for cAMP and 1.0±0.25 for UCP1 in Table 3.

Figure 2: Northern blot of 5 μg of total RNA isolated from brown fat cell cultures after different treatments.Brown adipocyte cells are isolated from mice and grown for 6 days in culture. Four hours before harvesting, 0.1 μM of NE, 0.01, 0.1, 1.0, 2.0 and 10 μM of ACTH (A 0.01, A 0.1, A 1, A 2 and A 10) and distilled water (C) were added to the wells as indicated. Results are of three independent experiments in three different times, and each performed in duplicate.
Figure 2:

Northern blot of 5 μg of total RNA isolated from brown fat cell cultures after different treatments.

Brown adipocyte cells are isolated from mice and grown for 6 days in culture. Four hours before harvesting, 0.1 μM of NE, 0.01, 0.1, 1.0, 2.0 and 10 μM of ACTH (A 0.01, A 0.1, A 1, A 2 and A 10) and distilled water (C) were added to the wells as indicated. Results are of three independent experiments in three different times, and each performed in duplicate.

Table 1:

Effect of propranolol on NE and ACTH stimulated UCP1 mRNA expression in brown adipocytes.

NE

0.1 μM		Propranolol

10 μM		Propranolol+NEACTH

10 μM		Propranolol+ACTH
UCP1100.05.9534.243.654.4
mRNA±0.00±1.67±5.59±4.85±7.56

Results are ±SE of three independent experiments in three times, each performed in duplicate. All results are calculated in relation to the standard result of 0.1 μM NE (100%).

Table 2:

Effect of ACTH (1 μM and 10 μM) and cirazoline (10 μM) on cAMP and UCP1 mRNA expression in brown adipocytes.

ANOVA
ACTH

1 μM		ACTH

10 μM		Cirazoline

10 μM		Cirazoline+ACTH

1 μM		Cirazoline+ACTH

10 μM		F-valuep-Value
cAMP40.364.1a9 .822.337.5b13.3650.001
pmol±4.15±8.5±3.06±4.96±6.35
UCP130.643.05.0248.565.1b16.880.000
mRNA%±3.85±5.25±1.58±7.56±6.76

Results are ±SE of three independent experiments in three times, each performed in duplicate. All results are in relation to the standard result of 0.1 μM NE (100%). aSignificant compared with ACTH 1 μM, bSignificant compared with respective parameters of ACTH (post hoc test).

Table 3:

Effect of ACTH (10 μM), caffeine (1 μM) and RO 20-1724 (0.25 mM) on cAMP and UCP1 mRNA expression in brown adipocytes.

ANOVA
ACTH

10 μM		Caffeine

1 μM		RO 20-1724

0.25 mM		ACTH+CaffeineACTH+ROF-valuep-Value
cAMP60.615.26.1128.6a118.0a20.5960.000
pmol±11.4±4.66±0.33±18.3±17.0
UCP146.516.612.5113.9a101.3a13.3660.001
mRNA%±7.15±4.4±3.75±18.8±19.86

Results are mean±SE of three independent experiments in three times, each performed in duplicate. All results are calculated in relation to the standard result of 0.1 μM of NE (100%). aSignificant compared with respective parameters of ACTH (post hoc test).

The dose-response curve of cAMP production (Figure 3) indicates that the initiation of generation of cAMP is detected with 0.01 μM concentration of ACTH, and the highest response is observed with 10 μM concentration.

Figure 3: Dose-response curve of cAMP level from brown fat cell cultures after treatment with ACTH.Results are mean percent value in relation to 0.1 μM of NE. Points are mean±SE from three independent experiments, each performed in duplicate.
Figure 3:

Dose-response curve of cAMP level from brown fat cell cultures after treatment with ACTH.

Results are mean percent value in relation to 0.1 μM of NE. Points are mean±SE from three independent experiments, each performed in duplicate.

The dose-response for the effect of ACTH on brown fat cell cultures (Figure 4) demonstrate that 0.01 μM of ACTH was the minimum concentration to detect the UCP1 gene expression, and the maximum stimulation was observed with 10 μM concentration.

Figure 4: Dose-response curve of UCP1 mRNA expression from brown fat cell cultures after treatment with ACTH.Results are mean percent value in relation to 0.1 μM of NE. Points are mean±SE from three independent experiments, each performed in duplicate.
Figure 4:

Dose-response curve of UCP1 mRNA expression from brown fat cell cultures after treatment with ACTH.

Results are mean percent value in relation to 0.1 μM of NE. Points are mean±SE from three independent experiments, each performed in duplicate.

Figure 5 represents the relationship between the cAMP generation and UCP1 gene expression in ACTH-treated brown fat cultured cells. A good correlation is observed between the cAMP level and the UCP1 gene expression in BAT cells.

Figure 5: Relationship between cAMP levels and UCP1 mRNA levels in dose-response curve of ACTH.Points represent mean±SE of three independent experiments, each performed in duplicate. Based on data in Figures 3 and 4.
Figure 5:

Relationship between cAMP levels and UCP1 mRNA levels in dose-response curve of ACTH.

Points represent mean±SE of three independent experiments, each performed in duplicate. Based on data in Figures 3 and 4.

Table 1 represents the effect of propranolol (a nonselective antagonist of β receptors) on UCP1 mRNA of cultured brown fat cells. It is observed from this table that propranolol could not inhibit the effect of ACTH (10 μM) on brown fat cells, whereas it can block significantly the effect of NE on it. On the other hand, a slight increase of UCP1 mRNA expression is observed in brown adipocytes when treated with a combination of ACTH and propranolol.

Table 2 shows that the maximum response of cAMP generation (about 64%) by ACTH (10 μM) is observed when compared with NE (0.1 μM). The interactions of cirazoline (an α1 receptor agonist and α2 receptor antagonist) with ACTH are shown in Table 2. Cirazoline itself can stimulate cAMP slightly in brown fat cells. But the production of cAMP is significantly reduced in brown fat cells with cirazoline in combination with 1 μM and 10 μM concentration of ACTH .

Table 2 also shows that cirazoline can stimulate slightly the UCP1 mRNA, but in conjugation with 10 μM concentration of ACTH it increases significantly the thermogenin mRNA expression in brown fat cells. No such significant result is observed in 1 μM of ACTH-treated cells with cirazoline.

The effects of caffeine and RO 20-1724 on ACTH-stimulated cAMP and UCP1 mRNA are shown in Table 3. Addition of these two compounds in brown fat cells increases slightly the basal level of cAMP, but addition of ACTH with caffeine cAMP increases significantly more than twofold when compared with ACTH alone. This table also shows that addition of ACTH with RO 20-1724 increases significantly the cAMP level which is about nearly twofold in comparison to ACTH alone.

The UCP1 mRNA expression in brown fat cells is also increased slightly after treatment with these two compounds. This UCP1 mRNA expression is significantly increased when the cells are treated with ACTH in combination with caffeine and RO 20-1724 (Table 3). The UCP1 mRNA expression is increased about 2.5-fold when cells are treated with ACTH and caffeine in comparison to ACTH alone. This expression is also increased more than twofold in combined treatment with ACTH and RO 20-1724 when compared with ACTH (10 μM). These UCP1 mRNA expressions are comparable with respective cAMP production.

Discussion

Earlier it has been attempted by several groups of investigators to demonstrate the effects of ACTH on brown fat cells [2, 3, 5, 12]. In 1990, Marette and Bukowiecki [7] reported that ACTH with high concentration was able to increase respiration and glucose uptake in isolated brown adipocytes. Most of the previous experiments were carried out on impure cell preparations. Recently, van den Beukel et al. [13] showed that ACTH activates hamster BAT, while corticosterone largely inhibits ACTH-mediated BAT-activating effects at a peripheral level. The present study is performed on cultured brown fat cells. In this study, the expression of UCP1 mRNA shows that the effect of ACTH is dose dependent (Figure 2). This indicates the presence of independent cellular receptors for ACTH in BAT. The minimum expression of UCP1 mRNA is observed after treatment with 0.01 μM concentration of ACTH, whereas maximum response is observed with 10 μM concentration. Similar expression of UCP1 mRNA with 10 μM concentration was observed by Iwen et al. [14].

It is known that BAT contains both α (α1 and α2) and β adrenoreceptors (β1, β2 and β3). Stimulation of α1 and all the β receptors causes thermogenesis, whereas α2 receptor stimulation inhibits thermogenesis [15]. To evaluate the nature of the receptors involved in ACTH action on BAT, propranolol, a recognized β receptor antagonist, was used in this study (Table 1). Propranolol itself can slightly stimulate the UCP1 mRNA expression. Cultured BAT when incubated with propranolol (10 μM) together with NE and ACTH, propranolol could successfully inhibits only the NE stimulated UCP1 mRNA expression. On the other hand, no such inhibition on ACTH-generated UCP1 mRNA expression is observed. But the insignificant increase of UCP1 mRNA expression instead of inhibition with combination of ACTH and propranolol in comparison to ACTH alone is thought to be due to summation of the effects of ACTH and propranolol. Therefore, this finding of propranolol treatment would seem to favor the idea that the β receptors may not be involved in ACTH action. A similar type of experiment was also done by van Heerden and Oelofsen [16] with NE and ACTH. They had observed a complete abolishing effect of NE-generated lipolytic response in impure cell preparations of brown adipocytes after using atenolol, a β1 receptor antagonist of lipolysis, but not the lipolytic response of ACTH.

The combined effect of cirazoline (an α1 receptor agonist and α2 antagonist) and ACTH on both cAMP level and UCP1 mRNA expression (Table 2) is interesting. But it is very difficult to explain the exact cause of this significant reduction of cAMP level and increase of UCP1 mRNA in BAT. Table 2 shows that ACTH (10 μM) stimulates cAMP generation in BAT, and it is about 58% (approximately) after treatment with cirazoline in comparison to ACTH (10 μM) alone. Significant reduction of cAMP is also observed in the cells treated with 1 μM concentration of ACTH with cirazoline (55%) when compared with ACTH (1 μM) alone. Similar decreased level of cAMP with α1 receptor stimulation was also reported by Wu et al. [17] in rabbit myometrium.

α1 adrenoreceptors probably activate Gq proteins and phospholipase C leading to the formation of inositol 1,4,5-triphosphate (IP3) and diglycerides and then produces effect in the cells [12]. In brown adipocytes, this IP3 releases calcium from intracellular stores. This α1-induced increased calcium level can augment thermogenesis. But this thermogenic effect occurs through plasma membrane ionic events which appears to be minor [12]. On the other hand, α1-induced increase of calcium also activates a phosphodiesterase activity which in turn decreases cAMP levels [18]. Therefore, in our present investigation, combined treatment with ACTH and cirazoline (α1 agonist) leads to decrease of cAMP level in BAT and indicates the possibility of activation of phosphodiesterase activity.

On the other hand, the UCP1 mRNA expression with ACTH (10 μM) is about 43% (approximately) in comparison to NE (Table 2), whereas combined treatment of cirazoline (an α2 antagonist) and ACTH shows 65% (approximately) expression of UCP1 mRNA when compared with NE. The effect is more than 1.5 times when compared with ACTH (10 μM) alone. This result is thought to be due to inhibition of inhibitory effect of α2 receptor activity in BAT after treatment with cirazoline.

In BAT, α2 adrenoreceptors are coupled with Gi proteins [19]. Gi activation leads to inhibition of adenylyl cyclase [17]. Under these circumstances, it may be possible to get a stimulatory effect if this inhibition is able to withdraw with α2 receptor antagonist. In a previous study, it was observed in this laboratory that the combined effect of yohimbine (an α2 receptor antagonist) and NE can increase UCP1 mRNA expression significantly in comparison to NE alone (an unpublished observation). Increase of cAMP generation was also reported by Nakaki et al. [20] in isolated pancreatic tissue after treatment with yohimbine. In this experiment, the parallel stimulatory and inhibitory effects of cirazoline (an α1 agonist and α2 antagonist) on UCP1 mRNA expression and cAMP level in BAT after ACTH stimulation show higher and lower responses than the ACTH alone. In these responses, the physiological significance of the presence of two counteracting systems (agonistic and antagonistic) may depend on the balance between the stimulatory and inhibitory effects which allows the brown adipocytes to modulate its response to the external stimulation by ACTH. At present, it has not been identified that how and when the cell decides to use this modulatory power.

In the present experiment, two phosphodiesterase inhibitors, caffeine (also adenosine receptor antagonist) and RO 20-1724, have been used to differentiate the effects of two compounds on ACTH activity. It is well known that caffeine stimulates thermogenesis in vivo [21, 22] and in vitro [23].

The significant high level of cAMP in ACTH-treated cells (Table 3) indicates its accumulation within the brown fat cells, and this occurs due to the inhibition of phosphodiesterase by caffeine and RO 20-1724. Caffeine increases more than twofold (approximately) of cAMP generation with ACTH (10 μM) when compared with ACTH alone. Similar type of potentiating effect of the hormone and accumulation of cAMP in the target tissues were also reported by Diepvens et al. [24], who suggested that the possible mechanism of thermogenic effect of caffeine involves inhibiting phosphodiesterase-induced degradation of intracellular cAMP. It is assumed that this inhibition of phosphodiesterase activity and the antagonistic effect at the level of adenosine receptors by caffeine causes increase of cAMP generation in brown adipocytes. Anti-adenosine and inhibition of phosphodiesterase activity of methylxanthine were also reported by some other investigators [25]. The antagonism of A1 adenosine receptors with caffeine increases cAMP production, thereby increasing lipolysis which causes further increase of plasma concentration of triglyceride [26]. BAT thermogenesis is stimulated by caffeine [27]. So brown fat could be a target in the treatment of obesity and metabolic syndrome due to its significant capacity to dissipate energy and regulate triglyceride and glucose metabolism [28]. Not only thermogenesis but also inhibition of phosphodiesterase activity indicates the possibility of the role of caffeine against obesity and diabetes [29]. Ro 20-1724 also increases cAMP production significantly with ACTH in comparison to ACTH alone.

Recently, it was observed that NE-stimulated thermogenesis (both at cAMP level and UCP1 mRNA expression) can be increased significantly in the presence of caffeine and RO 20-1724 on cultured brown fat cells [30]. The present experiment shows that not only NE but also ACTH can augment caffeine-stimulated thermogenesis (Table 3). The stimulation of UCP1 mRNA expression is observed in brown adipocytes with caffeine and RO 20-1724 in combination with ACTH. The increase of thermogenin gene expression by caffeine (2.5-fold approximately) in conjugation with ACTH is also thought to be due to the inhibition of phosphodiesterase activity which accumulates cAMP within the brown fat cells. In obese mice, it was also reported that caffeine can upregulate the expression of uncoupling protein (UCP1 and UCP3) in BAT [31].

On the other hand, RO 20-1724 acts like caffeine, but it acts only at the level of phosphodiesterase [32]. Therefore, these results show that stimulation of thermogenesis by ACTH can be significantly augmented in presence of caffeine and RO 20-1724, and this may occur due to inhibition of phosphodiesterase enzyme and accumulation of intracellular cAMP.

Therefore, this present investigation identifies the direct effect of ACTH on UCP1 gene expression and on the generation of cAMP in brown adipocytes. Although the cells contain independent receptor mechanism for ACTH, the activity of this hormone may have some relation with the α receptors. This finding shows the complexity of α adrenergic receptor interactions in BAT containing adrenergic subtypes. Further stimulation of thermogenin gene expression is possible by the accumulation of cAMP within the cells with ACTH in the presence of caffeine and RO 20-1724. So, future study will show the possibility of the use of caffeine as thermogenic agent in conjugation with ACTH as well as in the treatment of obesity. Further elaborate study will identify the exact nature of the receptors in BAT and their mechanisms which are involved in ACTH action.

Corresponding author: Dr. Hirendra M. Biswas, Department of Physiology, Kathmandu Medical College, 184, Baburam Acharya sadak, Sinamangal, Kathmandu, Nepal, Phone: 00977 9721504578, 9860652080(Nepal), 0091 9874483054 (India)

Acknowledgments

The author is grateful to Prof. Barbara Cannon and Prof. Jan Nedregaard of the Department of Metabolic Research, The Wenner-Gren Institute, Stockholm University, 106 91 Stockholm, Sweden, for providing laboratory facilities by the grants from the Swedish Natural Science Research Council and for their help and valuable suggestions. The author would also like to thank Dr. Chanda Karki, Principal, Kathmandu Medical College, Kathmandu, Nepal, for her interest in research activities in this college.

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: This investigation was supported by grants from the Swedish Natural Science Research Council.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

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Received: 2016-2-17
Accepted: 2017-2-7
Published Online: 2017-4-4
Published in Print: 2017-5-1

©2017 Walter de Gruyter GmbH, Berlin/Boston

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Isolated heart models for studying cardiac electrophysiology: a historical perspective and recent advances
  4. Reproduction
  5. Sub-chronic indomethacin treatment and its effect on the male reproductive system of albino rats: possible protective role of black tea extract
  6. The effect of extracellular ATP on rat uterine contraction from different gestational stages and its possible mechanisms of action
  7. Cardiovascular Function
  8. Effect of cardiovascular and muscular endurance is not associated with stress fracture incidence in female military recruits: a 12-month follow up study
  9. Cardiovascular-Pulmonary Interactions
  10. Comparison of structured and unstructured physical activity training on predicted VO2max and heart rate variability in adolescents – a randomized control trial
  11. Oxidative Stress
  12. Aminoguanidine pretreatment prevents methotrexate-induced small intestinal injury in the rat by attenuating nitrosative stress and restoring the activities of vital mitochondrial enzymes
  13. Anti-aging effects of M2000 (β-D-mannuronic acid) as a novel immunosuppressive drug on the enzymatic and non-enzymatic oxidative stress parameters in an experimental model
  14. Metabolism
  15. Long-term exposure to a butter-rich diet induces mild-to-moderate steatosis in Chang liver cells and Swiss albino mice models
  16. Effect of adrenocorticotropic hormone on UCP1 gene expression in brown adipocytes
  17. Phytotherapy
  18. Antidiabetic effect of Ruta montana L. in streptozotocin-induced diabetic rats
  19. Ethnopharmacological investigation of the aerial part of Phragmites karka (Poaceae)
https://doi.org/10.1515/jbcpp-2016-0017
Keywords for this article
adrenocorticotropic hormone (ACTH); brown adipose tissue (BAT); caffeine; cirazoline; cyclic adenosine monophosphate (cAMP); UCP1 mRNA

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Isolated heart models for studying cardiac electrophysiology: a historical perspective and recent advances
  4. Reproduction
  5. Sub-chronic indomethacin treatment and its effect on the male reproductive system of albino rats: possible protective role of black tea extract
  6. The effect of extracellular ATP on rat uterine contraction from different gestational stages and its possible mechanisms of action
  7. Cardiovascular Function
  8. Effect of cardiovascular and muscular endurance is not associated with stress fracture incidence in female military recruits: a 12-month follow up study
  9. Cardiovascular-Pulmonary Interactions
  10. Comparison of structured and unstructured physical activity training on predicted VO2max and heart rate variability in adolescents – a randomized control trial
  11. Oxidative Stress
  12. Aminoguanidine pretreatment prevents methotrexate-induced small intestinal injury in the rat by attenuating nitrosative stress and restoring the activities of vital mitochondrial enzymes
  13. Anti-aging effects of M2000 (β-D-mannuronic acid) as a novel immunosuppressive drug on the enzymatic and non-enzymatic oxidative stress parameters in an experimental model
  14. Metabolism
  15. Long-term exposure to a butter-rich diet induces mild-to-moderate steatosis in Chang liver cells and Swiss albino mice models
  16. Effect of adrenocorticotropic hormone on UCP1 gene expression in brown adipocytes
  17. Phytotherapy
  18. Antidiabetic effect of Ruta montana L. in streptozotocin-induced diabetic rats
  19. Ethnopharmacological investigation of the aerial part of Phragmites karka (Poaceae)
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