A possible substitute for ephedrine is albuterol, a common and accessible bronchodilator used to treat asthma and a selective β2‐adrenergic agonist. It was previously shown to increase metabolic rate (oxygen consumption) and lipolysis when given by inhalation at four times the therapeutic dose of 200 µg ( 8 , 9 ). Unfortunately, albuterol given at this dose in the aerosolized form invariably causes tachycardia. However, the swallowed fraction does not cause this side effect, and thus an orally given pill form of albuterol seems to be a safer form of administration ( 10 ). A comprehensive investigation into the metabolic effects of a combination therapy of caffeine and albuterol has not been completed. Therefore, the aim of these studies was to determine whether the combination treatment of albuterol and caffeine would act synergistically to effectively stimulate lipolysis and increase resting metabolic rate.

Caffeine is a well‐known stimulator of lipolysis and metabolic rate. It increases intracellular cyclic adenosine monophosphate (cAMP) levels, which are key to regulating lipolysis in white adipose tissue (WAT). Elevated cAMP results in increased triacylglyceride breakdown and increased circulating free fatty acids (FFA) ( 5 ). Caffeine acts as a competitive inhibitor of phosphodiesterase, the enzyme that breaks down cAMP within the adipocyte ( 6 ). Caffeine also stimulates the sympathetic nervous system ( 5 ) and activates the β2‐adrenergic receptors on WAT through the neural release of norepinephrine. The activation of the β2‐adrenergic receptors results in activation of adenylate cyclase, promotion of cAMP production, and lipolysis. While caffeine has proven to raise resting energy expenditure and increase fatty acid turnover, most of the mobilized FFAs are re‐esterified, and significant weight loss cannot be achieved ( 5 ). To solve this problem, researchers focused their attention on finding a drug that could increase metabolic rate to complement the effects of caffeine.

Chronic diseases such as hypertension and diabetes are commonly treated with combination drug therapies, but the development of such treatments for obesity is still in its infancy ( 1 ). Recent obesity research has focused on finding drugs that will increase lipolysis and metabolic rate. One such combination, caffeine and ephedrine, initially showed much promise. Studies showed that subjects treated with a caffeine/ephedrine combination exhibited greater loss of body fat compared to those treated with placebo ( 1 , 2 ). This combination was subsequently sold as an unregulated dietary supplement using ephedra, a naturally occurring food consisting of four isomers, the most potent being ephedrine. However, in 2004, the US FDA banned the sale of ephedra‐containing supplements due to concerns over the cardiovascular risks associated with their use ( 3 , 4 ). To find an optimal replacement, the mechanism of action for both caffeine and ephedra/ephedrine needs to be considered.

All data were analyzed using the PROC MIXED procedure of the statistical software package SAS® version 9.3 (SAS Institute, Cary, NC). Data from experiment 1 were analyzed by analysis of variance. Experiment 2 investigated 8 treatments (dose combinations of albuterol and caffeine) in an 8 × 8 Latin square study design with 8 subjects observed across 8 weeks in a balanced arrangement of the 8 treatment‐dose combinations whereby each subject received each of the treatment‐dose combinations exactly once and each treatment‐dose combination was given exactly once in each week. Since this study was a pilot trial, no calculations were done to determine the number of participants required to provide nominal 80% power for detecting specific treatment differences. Changes from assessment time 0 were viewed as repeated measurements across assessment times (60, 120, and 180 min) and modeled as effects of albuterol doses, caffeine doses, weeks and assessment times. Demographics, adverse events and any other non‐normally distributed data were analyzed by chi squared test. Data from experiments 3 and 4 were analyzed similarly using analogous mixed effects statistical models. Energy expenditure data were analyzed using analysis of covariance with lean body mass as a covariate. Statistical significance was defined as P ≤ 0.05.

The Pennington Biomedical Research Center Animal Care and Use Committee approved the animal protocols. Rats were individually housed in shoe‐box cages under controlled conditions (12 h light‐dark cycle, 22°C, 55% humidity). Male Sprague‐Dawley (Harlan, Indianapolis, IN) rats (8‐9 weeks old) were fed high fat (60% kcal from fat, D12492, Research Diets, New Brunswick, NJ) diet for 4 weeks. For experiment 3, forty rats were randomized to 4 treatment groups and continued on high‐fat diet for 4 weeks: 1) saline/saline, 2) saline/albuterol, 3) saline/caffeine, and 4) caffeine/albuterol. For experiment 4, sixty rats were randomized to 2 treatment groups and continued on high‐fat diet for 8 weeks: 1) saline/albuterol and 2) caffeine/albuterol. All treatments were administered by intraperitoneal injection twice a day. Albuterol was administered at 0.125 mg/kg and caffeine was administered at 3.12 mg/kg. The dose of albuterol and caffeine was the rodent equivalent of the human dose of albuterol 4 mg and caffeine 100 mg three times a day based on the metabolic mass equation (3). Body composition was measured by Nuclear Magnetic Resonance (Minispec LF90 NMR analyzer, Bruker Optics, Billerica, MA). Weight and food intake were recorded every 2 days. In experiment 3, the animals were placed in a metabolic chamber (Phenomaster, TSE Systems, Chesterfield, MO) at the end of the 4‐week treatment period for 3 days to measure oxygen consumption, respiratory exchange ratio, and activity levels while the treatment to which they were assigned was continued. Total activity was measured using the Phenomaster system (TSE Systems, Chesterfield MO). Chambers had evenly spaced infrared beam grids emitted along the X, Y, and Z axis, and the system sensed and quantified total beam breaks caused by movements of the animal.

The study was a randomized, double‐blind, crossover study comparing the effects of: 1) albuterol 2 mg‐placebo, 2) albuterol 4 mg‐placebo, 3) placebo‐caffeine 100 mg, 4) placebo‐caffeine 200 mg, 5) albuterol 2 mg‐caffeine 100 mg, 6) albuterol 2 mg‐caffeine 200 mg, 7) albuterol 4 mg‐caffeine 100 mg, and 8) albuterol 4 mg‐caffeine 200 mg. Subjects enrolled in the trial completed eight test days, each separated by 1 week. Before each visit the subjects fasted from 9 pm the prior night, refrained from strenuous physical activity for 24 h, ate their normal diet, and refrained from alcohol or caffeine‐containing beverages for 48 h. On each visit, the subjects had blood pressure, pulses and temperature recorded and rested for 30 min prior to measurement of the resting metabolic rate (RMR) and respiratory quotient (RQ) at 0, 60, 120, and 180 min. Resting metabolic rate and respiratory quotient were measured by indirect calorimetry using a ventilated hood system (DeltaTrac II metabolic monitor, Datex, Helsinki, Finland). During this procedure a transparent hood was placed over the subject's head and the amount of oxygen consumed and carbon dioxide exhaled was analyzed by the system. After the baseline measurements were taken, subjects were given their treatment consisting of two pills to swallow, and the baseline measurements were repeated the last 30 min out of each hour for 3 h.

The study was approved by the Pennington Biomedical Research Center Institutional Review Board. Eight healthy males and females between the ages of 18 and 50 years with a body mass index between 19 and 40 kg/m 2 were recruited from the greater Baton Rouge, LA area. Subjects who were pregnant, breast feeding, smoking, using nicotine products, taking regular medication or taking any medication known to alter metabolic rate like asthma medications or β‐adrenergic blocking drugs were specifically excluded. Women of child‐bearing potential who did not agree to use an effective method of contraception (abstinence, barrier methods, intrauterine devices, or hormonal methods of contraception) were also excluded. All subjects signed a written informed consent prior to initiation of study procedures.

Primary human adipose tissue was obtained from patients undergoing liposuction, paniculectomy, or bariatric surgery. Tissue was processed by LaCell LLC (New Orleans, LA) to isolate and cryopreserve preadipocytes. At the time of assay, preadipocytes were differentiated into adipocytes. Differentiated human adipocytes adherent to the bottom of a 96‐well plate were washed and treated with media containing 1) albuterol 7 ng/ml, 2) albuterol 17 ng/ml, 3) caffeine 3 μg/ml 4) caffeine 10 μg/ml 5) albuterol 7 ng/ml and caffeine 3 μg/ml 6) albuterol 17 ng/ml, and caffeine 3 μg/ml 7) albuterol 7 ng/ml and caffeine 10 μg/ml 8) albuterol 17 ng/ml and caffeine 10 μg/ml. Cells were incubated with their respective treatments at 37°C, 5% CO 2 for 3 hours. Isoproterenol 1 μM was used as a positive control. After incubation, a 50 μl aliquot was removed and combined with 50 μl glycerol reagent to create a colorimetric assay dependent on the amount of glycerol released during incubation with test compounds. Samples were read at 540 nm and results were calculated using a standard curve. Eight replicates were conducted for each condition.

Because rats treated with caffeine/albuterol appeared to gain more lean mass than rats treated with albuterol alone in Experiment ( 3 ), an 8‐week study was undertaken to assess whether these effects would be more pronounced with longer‐term treatment. Rats treated with caffeine albuterol gained more lean mass by week 8 compared to albuterol alone (63.9 vs. 60.3g, P = 0.03, Figure 4 A). When analyzed by the change in percent lean mass, significant differences were detected at weeks 2, 6, 7, and 8 (Figure 4 B). Rats treated with caffeine albuterol also gained less fat mass compared to albuterol alone (Figure 4 C). This difference was statistically significant by week 5 and persisted through week 8 (17.4 vs. 20.1 g, P = 0.004). When analyzed by change in percent fat mass, significant differences could be detected beginning at week 3 (Figure 4 D).

Rats treated with saline/albuterol or caffeine/albuterol gained significantly more weight compared to the saline control group ( P = 0.04, Figure 3 A). This was not explained by changes in food intake or activity as only the caffeine/saline group experienced a significant increase in activity ( P = 0.02, Figure 3 B and 3 C). The differences in weight were largely explained by changes in body composition. Rats treated with saline/albuterol or caffeine/albuterol gained an additional 16.6 and 20.2 g of lean mass, respectively, compared with the saline control group ( P < 0.001, Figure 3 D). Treatment with caffeine or caffeine/albuterol also reduced fat accretion ( P = 0.006, Figure 3 D). Rats treated with albuterol alone exhibited a trend for increased energy expenditure during the dark phase ( P = 0.07, Figure 3 E). Respiratory exchange ratio was similar among all groups (Figure 3 F).

Two doses of caffeine and albuterol increased energy expenditure more than two times the respective caffeine monotherapy. Increased energy expenditure was 2.87 fold higher with combination therapy of 2 mg albuterol and 100 mg caffeine compared to 100 mg caffeine alone (104.7 vs. 36.5 kcal). Increased energy expenditure was 4.80 fold higher with combination therapy of 4 mg albuterol and 100 mg caffeine compared to 100 mg caffeine alone (175.2 vs. 36.5 kcal, P < 0.0001). Although there was evidence that combination therapy was significantly better than monotherapy, there was no clear evidence of a dose‐response of the combinations or evidence of an albuterol/caffeine synergy on metabolic rate (see Figure 2 ).

Discussion

Previous studies on adipocytes have shown that the combined treatment of catecholamines and caffeine display synergy and yield substantial increases in cAMP production (6). Furthermore, it has been established that α2‐adrenergic receptors inhibit WAT lipolysis, which would give a selective β‐agonist an advantage in stimulating lipolysis (11). Thus, we wanted to determine if the combination of a more selective β‐agonist, albuterol, and caffeine would display synergy through a larger increase in cAMP resulting in a greater increase in glycerol production, an end product of lipolysis, than albuterol alone. Our first experiment measured glycerol production of in vitro adipocytes treated with different doses of caffeine, albuterol, and a caffeine/albuterol combination (Figure 1). Other than the 10 μg/ml dose of caffeine, all of the treatments yielded an increase in glycerol of ∼30%. Surprisingly, treatment with caffeine/albuterol combinations did not result in any additive or synergistic effects on glycerol production compared to caffeine alone. This result is inconsistent with an experiment conducted by Butcher et al. (6), which found that cultured adipocytes treated with a combination of caffeine and the α and β‐agonist epinephrine yielded a synergistic increase in cAMP. Because the mechanism of lipolysis relies on cAMP, we expected to see greater results in glycerol production with the use of a more selective β‐agonist combined with caffeine than with the β‐agonist alone. It is important to note that this in vitro model does not account for caffeine's potential effects on metabolic rate in a whole organism. Thus, conclusions on the potential of albuterol/caffeine as a weight loss therapy cannot be drawn from this experiment alone.

It was previously reported that the combination of 20 mg ephedrine and 200 mg caffeine yielded synergy during measurement of energy expenditure in six healthy and lean human subjects (2). For this reason, it was expected that the combination of albuterol and caffeine would also have synergistic properties in increasing resting metabolic rate in humans. According to our data, the resting metabolic rate for all treatments increased from baseline. However, our results did not indicate synergy between albuterol and caffeine in increasing metabolic rate. Response trends were inconsistent across combined doses of albuterol and caffeine. Our study was limited in that it only examined a small number of subjects due to the exploratory nature of the study. Differences in the mechanism of action between ephedrine and albuterol may have also contributed to the lack of synergy. Ephedrine and albuterol differ in that ephedrine stimulates both α‐ and β‐adrenergic receptors, while albuterol is a selective β2‐adrenergic receptor agonist. Since α‐adrenergic stimulators cause vasoconstriction inducing an acute increase in pulse rate and blood pressure, we anticipated and saw an absence of this effect with albuterol. Due to this difference in receptor stimulation, it is possible that synergy between ephedrine and caffeine on energy expenditure may be due to additional stimulation of α1‐ or β1‐adrenergic receptors.

Experiment 3 yielded some interesting and unexpected changes in body composition. The saline/albuterol and caffeine/albuterol treatment groups actually gained more weight than the saline control group. The caffeine/saline and caffeine/albuterol groups had reduced body fat gain. These data indicate that the differences in body weight were largely due to an increase in lean body tissue with albuterol treatment. The combination of caffeine and albuterol did not increase the lean body tissue significantly more than the albuterol treatment individually in experiment 3, but did in experiment 4 where group size was larger and treatment duration was longer (8 weeks vs. 4 weeks). These data provide further support for the muscle building effect of albuterol that has been found in previous studies (12, 13). The increase in skeletal muscle mass is believed to be a result of both an increase in muscle protein synthesis and a decrease in protein degradation (14, 15). A previous investigation of clenbuterol, a similar β2 agonist, proposed that the increase in muscle protein synthesis is mediated by an increase in muscle sensitivity to insulin, which has a primary role in protein synthesis (15). Further study is still needed to better evaluate the potential of albuterol as a treatment to change body composition in humans.

The combination of caffeine and albuterol might be most relevant to treatment of pediatric obesity. Currently, orlistat is the only medication approved for obesity treatment in adolescents, and it has embarrassing side effects such as fecal urgency and oily stools. Albuterol is an inexpensive generic medication approved for the treatment of asthma in children age ≥6 years and caffeine is not only a food, but is an inexpensive approved nonprescription medication for the treatment of drowsiness in children ≥12 years. Our data indicate that the combination of caffeine and albuterol can increase lean mass and decrease fat mass during growth and weight gain. Thus, a medication combination that increases lean mass, decreases fat mass, and does so without changing food intake might be a welcome option for physicians confronted with the challenge of treating childhood and adolescent obesity. Parents may be reluctant to act as food police and many pediatricians are concerned about restricting food intake during growth. Further research would be needed to determine safety and efficacy of caffeine and albuterol.

In summary, caffeine and albuterol increased lipolysis in vitro and metabolic rate in humans, but not synergistically. Albuterol treatment resulted in a trend for increased metabolic rate in rats, and caffeine did not increase the effect of albuterol alone. However, the addition of caffeine to albuterol enhanced the treatment's ability to increase lean body mass while decreasing fat mass without changing food intake in rats, and it deserves further exploration as a potential treatment for pediatric obesity.