Liraglutide, a glucagon-like peptide 1 (GLP-1) receptor agonist, and phentermine, a psychostimulant structurally related to amphetamine, are drugs approved for the treatment of obesity and hyperphagia. There is significant interest in combination use of liraglutide and phentermine for weight loss; however, both drugs have been reported to induce systemic hemodynamic changes, and as such the therapeutic window for this drug combination needs to be determined. To understand their impact on metabolic and cardiovascular physiology, we tested the effects of these drugs alone and in combination for 21 days in lean and obese male mice. The combination of liraglutide and phentermine, at 100 μg/kg/day and 10 mg/kg/day, respectively, produced the largest reduction in body weight in both lean and diet-induced obese (DIO) mice, when compared with both vehicle and monotherapy-treated mice. In lean mice, combination treatment at the aforementioned doses significantly increased heart rate and reduced blood pressure, whereas in DIO mice, combination therapy induced a transient increase in heart rate and decreased blood pressure. These studies demonstrate that in obese mice, the combination of liraglutide and phentermine may reduce body weight but only induce modest improvements in cardiovascular functions. Conversely, in lean mice, the additional weight loss from combination therapy does not improve cardiovascular parameters.

Obesity is a serious disease, characterized by the accumulation of excess body fat. A recent study estimated that worldwide, 108 million children and 604 million adults are obese, and that these numbers are on the rise (1). Obesity significantly increases the risk of developing secondary diseases, including type 2 diabetes and cardiovascular diseases. Annually, four million deaths are attributed to obesity and of these, two-thirds are due to obesity-associated cardiovascular disease (1).

Obesity pharmacotherapy treatments act by reducing caloric intake, increasing energy expenditure, and/or increasing energy excretion. Pharmacological manipulation of energy homeostasis may affect neurons, neuropeptides, autonomic nervous system branches, or brain regions that are intrinsic to cardiovascular control (2). Therefore, drugs that are marketed for obesity should be considered in terms of their impact on the cardiovascular systems.

The glucagon-like peptide 1 (GLP-1) receptor agonist liraglutide and the psychostimulant phentermine are two pharmacotherapies currently approved for the treatment of obesity in many countries, and both act to reduce body weight through reduced food intake (3). Additionally, both these compounds influence the activity of the cardiovascular system.

GLP-1 is secreted from L cells of the ilium and colon. In addition to its potent ability to reduce blood glucose levels, GLP-1 increases postprandial satiety, inhibits gastric emptying, and influences the hedonic reward systems (410). The hypophagic effects of GLP-1 appear to be driven by central actions: intracerebroventricular administration of GLP-1 reduces food intake; however, this effect is lost in the presence of a central GLP-1 antagonist or when administered to GLP receptor−/− knockout mice (4,1121). In humans, the GLP-1 receptor agonist liraglutide is well tolerated and produces significant weight loss through decreased food intake without increasing energy expenditure over a sustained period (22,23). In rodents, GLP-1 receptor agonists can act acutely to increase blood pressure and heart rate in a dose-dependent manner (24). Liraglutide treatment in patients with type 2 diabetes does not significantly alter noradrenaline concentration compared with placebo treatment (25,26).

However, in rodents (but not in humans), cardiac GLP-1 receptor activation can also have a hypotensive action through the stimulation of atrial natriuretic peptide (ANP) release in rodents (27,28). Clinical evidence suggests that despite causing acute increases in heart rate, chronic treatment with liraglutide lowers systolic blood pressure, the risk of cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke in patients with type 2 diabetes and cardiovascular disease risk (28,29).

The U.S. Food and Drug Administration (FDA) approved the use of the psychostimulant phentermine for the treatment of obesity in 1959 (30). It is currently prescribed for short-term appetite suppression (<3 months). Compared with placebo, it significantly reduces body weight in obese individuals and is currently the most widely prescribed antiobesity therapy, in part due to its relatively low cost (3133). Although reports suggest phentermine can increase blood pressure, numerous studies demonstrate that when used for weight loss, phentermine does not adversely influence the cardiovascular system. In a recent human study, phentermine-treated individuals lost significantly more weight compared with placebo, heart rate remained unchanged in both groups, and blood pressure decreased in the phentermine-treated group (31,3437). Obese patients treated with 7.5 or 15 mg phentermine lost 6.65% and 7.38% body weight, respectively, after 28 weeks of treatment (38). Despite a similar change in body weight, phentermine 7.5 mg treatment reduced mean systolic blood pressure by 3.3 mmHg compared with 6.4 mmHg in 15 mg phentermine–treated obese patients (38). Phentermine has been used in combination with other antiobesity therapies and was part of the fenfluramine-phentermine combination that led to pulmonary hypertension and the withdrawal of fenfluramine from the U.S. market (39). A combination of phentermine and topiramate is marketed by Vivus and was approved in the U.S. as an antiobesity drug in late 2012. As phentermine and liraglutide are both approved for weight management, it is plausible that some patients will be prescribed both agents, although they are not yet approved or licensed for combination therapy. This study aims to determine the cardiovascular effects of the combination of liraglutide plus phentermine while evaluating food intake, body weight, body fat, locomotor activity, and glucose homeostasis in a mouse model of diet-induced obesity.

All animal procedures were approved by the Monash University Animal Ethics Committee. All mice were housed in a controlled environment. The light period was from 7:00 a.m. to 7:00 p.m., and the dark period was 7:00 p.m. to 7:00 a.m. Temperature and humidity remained constant. Mouse food was available ad libitum, and mice were monitored daily.

Male C57Bl/6J mice were placed on diets at 6 weeks of age and remained on either chow (4.8% fat, mouse and rat rodent chow diet; Specialty Feeds, Glen Forrest, Australia) or high-fat (43% fat, SF04-001; Specialty Feeds, Western Australia, Australia) diets for 20 weeks. At 26 weeks of age, mice were individually housed and surgery to implant a radiotelemetry probe was conducted.

Radiotelemetry

Radiotelemetry probes were implanted as previously described (40). In brief, TA11PA-C10 Data Science International radiotelemetry probes were implanted into the left carotid artery under isoflurane anesthesia. After surgery, body weight and food intake were measured daily while mice recovered for 2 weeks. After this period, baseline recordings were measured (this included baseline blood pressure and heart rate), and an intraperitoneal glucose tolerance test (GTT) was performed as previously described (41) as was a DEXA scan (40).

After this, all mice received single, combination, or vehicle drug treatments, administered via intraperitoneal or subcutaneous injection daily for 21 days as outlined in the treatment table (Table 1). Liraglutide was provided by Novo Nordisk. This study used a submaximal dose of liraglutide. Phentermine-HCl was purchased from Sigma-Aldrich (P8653). Body weight and food intake were measured daily, and blood pressure, heart rate, and activity were measured continuously. At the conclusion of this 21-day period, a final GTT was performed, and a DEXA scan was performed on diet-induced obese (DIO) mice.

Table 1

Treatment composition

TreatmentsAbbreviation
Saline s.c. + saline i.p. S+S 
Liraglutide (100 μg/kg/day s.c.) + saline i.p. L+S 
Saline s.c. + phentermine (10 mg/kg/day i.p.) S+P 
Liraglutide (100 μg/kg/day s.c.) + phentermine (10 mg/kg/day i.p.) L+P 
TreatmentsAbbreviation
Saline s.c. + saline i.p. S+S 
Liraglutide (100 μg/kg/day s.c.) + saline i.p. L+S 
Saline s.c. + phentermine (10 mg/kg/day i.p.) S+P 
Liraglutide (100 μg/kg/day s.c.) + phentermine (10 mg/kg/day i.p.) L+P 

First treatment was administered subcutaneously, and the second treatment was administered intraperitoneally.

Analysis and Recording of Data

Cardiovascular recordings were collected on the Ponemah version 6.31 software for 1 min, every 10 min for 21 days. Daily averaged data were all the data collected over that 24-h period. Light and dark period averages were all data collected throughout that 12-h period. Change data were the difference in treatment data compared with baseline for the same time period. Percentage change was calculated as the change divided by the baseline value, for the corresponding time period, multiplied by 100. This was only used for body weight analysis due to the large difference existing between lean and DIO mice at baseline.

The glucose tolerance was compared as area under the curve (AUC) throughout the GTT. A comparison was made between the same mice at baseline and after the 21-day treatment period.

Data are presented as mean ± SEM. The significance between groups is outlined in Table 2. The significance compared with baseline is outlined on graphs by color. Comparisons between treatment groups are outlined in black by the symbols expressed in Table 2.

Table 2

Statistical analysis comparison legend for explanation of two-way ANOVA results on graphs

Gray Purple Liraglutide (100 μg/kg/day) + saline vs. Saline + saline 
Purple Red Saline + saline vs. Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) 
Purple Black Saline + saline vs. Saline + phentermine (10 mg/kg/day) 
Gray Red Liraglutide (100 μg/kg/day) + saline vs. Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) 
Gray Black Liraglutide (100 μg/kg/day) + saline vs. Saline + phentermine (10 mg/kg/day) 
Red Black Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) vs. Saline + phentermine (10 mg/kg/day) 
Gray Purple Liraglutide (100 μg/kg/day) + saline vs. Saline + saline 
Purple Red Saline + saline vs. Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) 
Purple Black Saline + saline vs. Saline + phentermine (10 mg/kg/day) 
Gray Red Liraglutide (100 μg/kg/day) + saline vs. Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) 
Gray Black Liraglutide (100 μg/kg/day) + saline vs. Saline + phentermine (10 mg/kg/day) 
Red Black Liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) vs. Saline + phentermine (10 mg/kg/day) 

@P < 0.05, saline + saline vs. liraglutide (100 μg/kg/day) + saline.

!P < 0.05 saline + saline vs. liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day).

<P < 0.05, saline + saline vs. saline + phentermine (10 mg/kg/day).

$P < 0.05, liraglutide (100 μg/kg/day) + saline vs. liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day).

%P < 0.05, liraglutide (100 μg/kg/day) + saline vs. saline + phentermine (10 mg/kg/day).

>P < 0.05, liraglutide (100 μg/kg/day) + phentermine (10 mg/kg/day) vs. saline + phentermine (10 mg/kg/day).

Body Weight in Lean Mice

At baseline, no difference in body weight in lean mice was observed. Herein, comparisons for each group will be drawn respective to its own baseline and to S+S-treated (saline s.c. + saline i.p.) mice at day 21. S+S-treated animals were weight stable throughout the treatment period. The L+S-treated (liraglutide [100 μg/kg/day s.c.] + saline i.p.) animals lost an average of 4.5 ± 1.2% body weight, whereas the S+P-treated (saline s.c. + phentermine [10 mg/kg/day i.p.]) mice lost an average of 6.2 ± 2.0% (Fig. 1A and Supplementary Fig. 1A). The combination of L+P (liraglutide [100 μg/kg/day s.c.] + phentermine [10 mg/kg/day i.p.]) resulted in significantly more weight loss (9.2 ± 0.8%), compared with the liraglutide alone or phentermine alone–treated group (Fig. 1A).

Figure 1

A: Percentage change in body weight in lean mice from baseline throughout 21-day treatment period; n = 8–11. B: Percentage change in body weight in DIO mice from baseline throughout 21-day treatment period; n = 7–9. C: Cumulative food intake, from start of treatment in DIO mice from baseline throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Figure 1

A: Percentage change in body weight in lean mice from baseline throughout 21-day treatment period; n = 8–11. B: Percentage change in body weight in DIO mice from baseline throughout 21-day treatment period; n = 7–9. C: Cumulative food intake, from start of treatment in DIO mice from baseline throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Body Weight in DIO Mice

No difference in body weight between DIO treatment groups was found at baseline (Supplementary Fig. 1B). At day 21 of treatment, the S+S treatment group had increased their body weight by 13 ± 3.5% (Fig. 1B). The L+S-treated mice lost an average of 7.5 ± 1.7% body weight from baseline by day 21 of treatment. The S+P-treated DIO animals lost an average of 8.7 ± 2.7% body weight, which was not significantly different from the L+S-treated animals. The L+P-treated group lost 20 ± 2.7% of body weight from baseline, significantly more weight than the L+S, S+P, and S+S-treated groups (Fig. 1B and Supplementary Fig. 1B).

Body Weight: Lean Versus DIO Mice

The majority of drug treatments produced significantly greater weight loss in DIO mice compared with lean mice (Fig. 1A and B). The L+S treatment as of day 21 of treatment caused a weight loss of 4.5% in lean mice compared with 7.5% in DIO mice. The S+P treatment caused a weight loss of 6.2% in lean mice compared with 8.7% in DIO mice (Fig. 1A and B). The combination with L+P caused the greatest weight loss in both lean (9%) and DIO (20%) mice. In combination, liraglutide and phentermine resulted in significantly greater weight loss than either the L+S or S+P-treated lean and DIO animals (Fig. 1A and B).

Body Fat: DIO Mice

DEXA scans (Supplementary Fig. 1C) were performed on the DIO mice at days 0 and 21 and showed that weight loss in all treatment groups was primarily due to the loss of body fat. In the DIO L+P treatment group, 9.2 g of body fat was lost compared with 4 g in the S+P treatment group and 6.2 g in the L+S treatment group (Supplementary Fig. 1C). Lean body mass did not significantly decrease in any treatment group (data not shown).

Food Intake in Lean Mice

Compared with baseline, all treatment groups showed a transient decrease in food intake. However, there were no significant differences between the drug-treated groups (Supplementary Fig. 1D).

Food Intake in DIO Mice

All drug treatment groups significantly decreased food intake in the DIO mice with a similar effect compared with S+S-treated DIO mice. The one exception to this was L+P, which had a significant decrease in food intake when compared with the L+S group (Fig. 1C and Supplementary Fig. 1E).

Food Intake: Lean Versus DIO Mice

The effect of treatments on food intake was notably different between lean and DIO mice. Both liraglutide and phentermine alone and in combination significantly reduced food intake in DIO mice; however, this substantial effect was not observed in lean mice (Fig. 1C and Supplementary Fig. 1D and E).

Activity in Lean Mice

Compared with the S+S group, the L+S treatment group showed no increase in activity in lean mice (Fig. 2A). The S+P group had significantly increased activity compared with S+S-treated lean mice. Mice that received the combination L+P had significantly elevated activity levels compared with those in the S+S and L+S groups. Interestingly, the majority of the increase in activity in these groups was during the normally inactive light period, with no significant change during the dark period (Fig. 2B and C).

Figure 2

A: Activity (24 h) in lean mice from baseline throughout 21-day treatment period; n = 8–11. B: Light period (12 h) activity in lean mice from baseline throughout 21-day treatment period; n = 8–11. C: Dark period (12 h) activity in lean mice from baseline throughout 21-day treatment period; n = 8–11. D: Activity (24 h) in DIO mice from baseline throughout 21-day treatment period; n = 7–9. E: Light period (12 h) activity in DIO mice from baseline throughout 21-day treatment period; n = 7–9. F: Dark period (12 h) activity in DIO mice from baseline throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Figure 2

A: Activity (24 h) in lean mice from baseline throughout 21-day treatment period; n = 8–11. B: Light period (12 h) activity in lean mice from baseline throughout 21-day treatment period; n = 8–11. C: Dark period (12 h) activity in lean mice from baseline throughout 21-day treatment period; n = 8–11. D: Activity (24 h) in DIO mice from baseline throughout 21-day treatment period; n = 7–9. E: Light period (12 h) activity in DIO mice from baseline throughout 21-day treatment period; n = 7–9. F: Dark period (12 h) activity in DIO mice from baseline throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Activity in DIO Mice

In DIO mice, both the S+S and L+S treatments had no effect on activity (Fig. 2D). The S+P group had a significant increase in activity compared with baseline and S+S and L+S-treated DIO mice (Fig. 2A). The combination DIO treatment group L+P had increased levels of activity compared with the L+S group (Fig. 2D). The activity change was substantially greater in the 12-h light period as compared with the 12-h dark period (Fig. 2E and F).

Activity: Lean Versus DIO Mice

The average 24-h activity between the S+S and L+S-treated lean and DIO mice was not significantly different (Fig. 2A–F). The majority of increased activity occurred in the 12-h light period in both lean and DIO mice of S+P and L+P-treated groups (Fig. 2A–F).

Glucose Tolerance in Lean Mice

In lean mice, the AUC after GTTs at both baseline and after 21 days of treatment is reported in Fig. 3A. Both L+S and L+P treatment groups had improved glucose tolerance, compared with baseline (Fig. 3A).

Figure 3

A: Average AUC during GTT in lean mice at baseline and after 21 days of treatment. n = 8–11, mean ± SEM, repeated Student t test between baseline and after 21 days of treatment. *P < 0.05 and **P < 0.01, baseline vs. after 21 days of treatment. B: Average AUC during GTT in DIO mice at baseline and after 21 days of treatment. n = 7–9, mean ± SEM, repeated Student t test between baseline and after 21 days of treatment. *P < 0.05 and **P < 0.01, baseline vs. after 21 days of treatment.

Figure 3

A: Average AUC during GTT in lean mice at baseline and after 21 days of treatment. n = 8–11, mean ± SEM, repeated Student t test between baseline and after 21 days of treatment. *P < 0.05 and **P < 0.01, baseline vs. after 21 days of treatment. B: Average AUC during GTT in DIO mice at baseline and after 21 days of treatment. n = 7–9, mean ± SEM, repeated Student t test between baseline and after 21 days of treatment. *P < 0.05 and **P < 0.01, baseline vs. after 21 days of treatment.

Glucose Tolerance in DIO Mice

In DIO mice, the improvement in AUC during a GTT was seen in L+S and L+P treatment groups (Fig. 3B).

Heart Rate in Lean Mice

Compared with the change in heart rate of the S+S-treated mice, all other treatment groups had a significantly larger change in heart rate (Fig. 4A). All treatment groups except the S+P had increased heart rate immediately after the onset of treatment, whereas the increase in the S+P group became evident several days after the start of treatment (Fig. 4A). Compared with L+S or S+P, the combination of phentermine with liraglutide significantly increased heart rate further in lean mice (Fig. 4A). The change in heart rate was greatest in the 12-h light period (Supplementary Fig. 2A) compared with the 12-h dark period (Supplementary Fig. 2B).

Figure 4

A: Change in heart rate from baseline of lean mice throughout 21-day treatment period; n = 8–11. B: Change in heart rate from baseline of DIO mice throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline. BPM, beats per minute.

Figure 4

A: Change in heart rate from baseline of lean mice throughout 21-day treatment period; n = 8–11. B: Change in heart rate from baseline of DIO mice throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; <P < 0.05, S+S vs. S+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline. BPM, beats per minute.

Heart Rate in DIO Mice

In DIO mice, L+S-treated mice had a tendency for increased heart rate compared with S+S treatment, but this did not reach statistical significance (Fig. 4B). In the first 4 days of treatment, DIO mice treated with S+P had a significantly reduced heart rate compared with S+S, L+S, and L+P-treated mice (Fig. 4B). In DIO L+P-treated mice, heart rate was significantly elevated at times compared with the S+S-treated DIO mice (Fig. 4B).

Heart Rate: Lean Versus DIO Mice

DIO mice had a significantly higher basal heart rate than lean mice (Fig. 4A and B). No difference in the average percentage change of heart rate was evident in the L+S (4.1% vs. 4.1%) or S+P (3.1% vs. −0.45%) treated lean and DIO mice. The percentage change in peak (greatest rate) heart rate in L+P-treated lean mice was 19% vs. 6.8% in DIO mice. Much of the difference between the changes in heart rate of lean and DIO mice occurred during the 12-h light period (Fig. 4A and B and Supplementary Fig. 2A).

Systolic Blood Pressure in Lean Mice

At day 21, systolic blood pressure in the L+S-treated lean mice was significantly reduced compared with baseline and S+S-treated mice (Fig. 5A). The S+P-treated lean mice had a significantly greater (increased) change in systolic blood pressure compared with L+S and the combination L+P treatments. L+P-treated lean mice had a significantly reduced systolic blood pressure compared with baseline and the S+S treatment group (Fig. 5A).

Figure 5

A: Change from baseline in systolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. B: Change from baseline in diastolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. C: Change from baseline in systolic blood pressure of DIO mice throughout 21-day treatment period; n = 7–9. D: Change from baseline in diastolic blood pressure of DIO mice throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Figure 5

A: Change from baseline in systolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. B: Change from baseline in diastolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. C: Change from baseline in systolic blood pressure of DIO mice throughout 21-day treatment period; n = 7–9. D: Change from baseline in diastolic blood pressure of DIO mice throughout 21-day treatment period; n = 7–9. Mean ± SEM, two-way AVOVA, Sidak multiple comparisons test. @P < 0.05, S+S vs. L+S; !P < 0.05, S+S vs. L+P; $P < 0.05, L+S vs. L+P; %P < 0.05, L+S vs. S+P; >P < 0.05, L+P vs. S+P. Red asterisks, L+P treatment compared to own baseline; black asterisks, S+P treatment compared to own baseline.

Systolic Blood Pressure in DIO Mice

Compared with S+S-treated DIO mice, L+S treatment in DIO mice significantly reduced systolic blood pressure as well as compared with baseline. S+P-treated DIO mice had a significantly increased change in systolic blood pressure at times compared with L+S and the combination of L+P (Fig. 5C). L+P treatment reduced systolic blood pressure compared with S+S, and no difference between L+P and L+S-treated DIO mice was evident (Fig. 5C).

Systolic Blood Pressure: Lean Versus DIO Mice

The largest decrease in systolic blood pressure in lean mice occurred in the mice treated with L+P (−10.4%), compared with only a 3.8% decrease in systolic blood pressure in respective treated DIO mice. In DIO mice, the largest percentage change in systolic blood pressure was observed in the L+S-treated mice (−8.5%). This compares to a 6.9% decrease in systolic blood pressure in the L+S-treated lean group (Fig. 5A and C).

Diastolic Blood Pressure in Lean Mice

L+S and L+P-treated lean mice had reduced diastolic blood pressure from baseline and compared with the S+P-treated mice (Fig. 5B).

Diastolic Blood Pressure in DIO Mice

The change in diastolic blood pressure in L+S and L+P-treated DIO mice was significantly lower compared with the S+P-treated mice (Fig. 5D).

The Framingham Heart Study estimated that 78% of primary hypertension in men and 65% in women can be attributed to excess body weight (42). Cardiovascular disease is responsible for the death of two-thirds of individuals with obesity (1). It therefore follows that drugs designed to elicit weight loss should avoid adverse cardiovascular effects.

Preclinical rodent studies have shown that GLP-1 receptor agonists can increase blood pressure and heart rate. Clinical data show that chronic treatment with liraglutide can reduce blood pressure and increase heart rate (24,43), yet when used at recommended doses, liraglutide can induce weight loss in individuals with obesity, without adversely increasing cardiovascular risk (29).

Phentermine’s hypertensive effects are the result of increased synaptic noradrenaline release and sympathetic nervous system activation (44). However, in recent studies, phentermine treatment in patients with obesity resulted in weight loss and decreases in systolic blood pressure (38,44). The Endocrine Society advises that sympathomimetics including phentermine are not to be used for weight loss in patients with uncontrolled hypertension or a history of heart disease (45).

The combination of phentermine and fenfluramine not only can lead to valvuopathy (reason for FDA withdrawal) but can also increase blood pressure and heart rate (44,46,47). Combination therapy with phentermine and topiramate is currently available for weight loss. This combination when given at the highest acceptable doses can increase blood pressure and heart rate, despite decreasing body weight (38,48).

As individuals aim to induce the greatest weight loss possible, it is expected that people may combine the use of liraglutide and phentermine even though this combination is not FDA approved. The data presented here demonstrate that liraglutide and phentermine both alone and in combination can significantly reduce body weight in both lean and DIO mice. These studies also demonstrate the actions of these drugs on the activity of the cardiovascular system.

The administration of liraglutide alone in both lean and DIO mice induced modest weight loss compared with vehicle-treated mice. The dose of liraglutide used in these studies was a submaximal weight loss dose; this dose was chosen to enable further weight loss when combined. In liraglutide alone–treated groups in both lean and DIO mice, weight loss occurred most rapidly in the first 5 days and then occurred at a reduced rate for the rest of the treatment period. This stabilizing of body weight loss after liraglutide alone treatment is believed to be due to adaptation by the body and is observed in numerous liraglutide treatment studies in numerous animal models as well as with other anorexigenic agents (4952). There was no significant difference in the final weight loss induced by liraglutide alone or phentermine alone in either the lean or DIO treated mice. The only difference between the two treatment groups was the rate of weight loss; weight loss was slower in the phentermine alone–treated mice. In lean mice, after 21 days of treatment with the combination dose of liraglutide and phentermine, the greatest proportion of body weight loss had occurred compared with all other lean treatment groups: 9% of body weight from baseline, this being a significant reduction relative to baseline. The percentage change in body weight was subadditive, that is, less than the numeric addition of the effects of liraglutide alone added to the effects of phentermine alone. That the combination treatment in lean mice did not result in additive effects on body weight may be because some of the weight loss actions of both liraglutide and phentermine are through reduced food intake. Similar to lean mice, the DIO mice treated with the combination of liraglutide and phentermine had the largest reduction in body weight among the DIO groups (19.8% body weight compared with baseline by day 21 of treatment). This weight loss could be considered synergistic, as the sum of the total weight loss of animals treated with liraglutide alone or phentermine alone was less than the total weight loss of the combination liraglutide and phentermine treatment group. The liraglutide and phentermine–treated DIO mice lost double the weight of the liraglutide and phentermine–treated lean mice, when measured in terms of percentage body weight. The weight loss that occurred in the DIO mice was due to a significant reduction in body fat. Although lean mass was also decreased, this reduction did not reach statistical significance. Reasons for the increased effectiveness of combination liraglutide and phentermine treatment in DIO compared with lean mice may be due to complex central and peripheral metabolic factors resisting the drug-induced weight loss in lean animals, factors that may be diminished in the obese state.

All active treatments reduced food intake, which would be expected to cause weight loss. Additionally, some treatments increased locomotor activity, which would further drive weight loss. Although we did not assess thermogenesis in these studies, others have demonstrated that liraglutide and GLP-1 receptor activation increases thermogenesis (53,54). One major difference between lean and DIO mice was the way body weight reduced. In DIO mice, food intake was significantly reduced in the liraglutide alone, phentermine alone, and additively in the combined liraglutide and phentermine–treated DIO mice. Additionally, activity was increased in the DIO mice treated with both phentermine alone and liraglutide and phentermine in combination, compared with vehicle treatment. It is established that phentermine can increase physical activity in rats (5557), whereas liraglutide has previously shown no clear effect on activity. However, in lean mice, while similar effects on activity were observed with phentermine alone and liraglutide–phentermine combination treatment, food intake in all three experimental groups was not significantly inhibited. Most notably, reduction was in the combined liraglutide and phentermine–treated mice, but cumulative food intake was not different between treatment groups. This absence of sustained reductions in food intake in lean mice shows that the induced weight loss must be due to other mechanisms, including increased activity, which was observed in the studies described here, or through increased metabolic rate or increased thermogenesis. Indeed, previous studies have shown that GLP-1 receptor agonists can increase brown adipose tissue temperature (54). The greater reduction in food intake in treated DIO mice may be one mechanism behind the increased effectiveness of combination therapy in DIO mice compared with lean mice.

One notable finding of the combination therapy in both lean and DIO mice was that liraglutide was not attenuating the increased locomotor activity induced by phentermine, although this attenuation has been observed with exendin-4 (another GLP-1 receptor agonist) and amphetamine-induced hyperactivity (58). It also demonstrates that the combination therapy allows the additional weight loss, as compared with liraglutide alone treatment, via a different mechanism than reduced food intake alone. For a long-term sustainable weight loss option, this would be an additive benefit. In our studies, animals were not given extra activity equipment, e.g., a running wheel to induce weight loss and stimulate the increase in activity. If this was to be incorporated, the weight loss induced may have equated to a much greater value in mice. In this study, the increased change in activity occurred most notably in the light period, when mice should be sleeping. As such, it would be expected that weight loss in response to activity in humans is likely to be varied, because humans are unlikely to exercise when they usually sleep.

Impact on Heart Rate

The LEADER (Liraglutide Effect and Action in Diabetes: Evaluation of Cardiovascular Outcome Results) study has shown that liraglutide (1.8 mg s.c. once daily) significantly provides a cardiovascular benefit on the three-point major adverse cardiovascular events scale. This includes the number of deaths being reduced in patients with type 2 diabetes and high cardiovascular risk, even though liraglutide can increase heart rate (29,59,60). At the submaximal dose used in the studies described here, we demonstrate that liraglutide-only treatment in lean mice increased heart rate compared with baseline and compared with vehicle-treated mice despite weight loss, which is in accordance with previous findings (24,43). The increase in heart rate caused by liraglutide alone was significantly less than the increase caused by combining liraglutide and phentermine. Hence the combination of liraglutide and phentermine produced an additive increase in heart rate in lean mice. Liraglutide and phentermine treatment together significantly increased heart rate in lean mice from day 1 throughout the total treatment period. The increase was substantial, by as much as 100 beats per minute or ∼20% on some days compared with baseline of lean mice. Compared with liraglutide or phentermine alone in lean mice, combination caused a synergistic increase in heart rate. During the initial phase of treatment until day 5 of treatment, the increase in heart rate was evenly elevated in both the 12-h dark and 12-h light periods. As the treatment period progressed, the increased heart rate could be observed more in the 12-h light period. This was associated with increased activity during the light phase, a time when mice are relatively inactive (61). If we consider the effect of the combination therapy on body weight and on heart rate, we can conclude that the combination therapy appears to be effective in lean mice in causing weight loss; however, treatment does cause a 20% increase in heart rate from baseline in this model.

Heart rate was also elevated in the DIO mice treated with the combination of liraglutide and phentermine compared with both vehicle- and phentermine alone–treated DIO mice (heart rate was not significantly changed in liraglutide alone–treated DIO mice). Compared with the lean mice treated with the combination of liraglutide and phentermine, the increase in heart rate in the DIO mice was only half as great. Lean mice have a significantly lower basal heart rate compared with DIO mice; the 20% increase occurs as the heart rate reaches the same basal beats per minute as DIO mice and the increase in percentage did not appear as great in DIO mice. From this data, it could be inferred that lean mice have lower sympathetic nerve activity as has been seen in obese humans (62).

This is likely because the baseline heart rate of DIO mice started at an elevated rate due to the mice already displaying hypertension and tachycardia. Heart rate was increased in both the dark and light periods. We can conclude that the combination is effective for weight loss in obese mice but does increase heart rate. If these studies were continued and weight continued to be lost, the heart rate may be improved but further research would need to be undertaken to discover the long-term impact of the combination therapies.

Effects on Blood Pressure

Clinical studies have conclusively demonstrated that chronic treatment with liraglutide decreases systolic blood pressure (23,43,59,63). However preclinical studies have shown that GLP-1 agonists can increase blood pressure in rats (24). In the studies described here, liraglutide alone decreased systolic blood pressure in lean and obese mice. This decrease in systolic blood pressure occurred along with a decrease in body weight. Phentermine alone did not decrease systolic blood pressure, despite similar body weight reductions in lean and DIO mice, and at day 21, phentermine alone–treated lean and DIO mice had higher systolic blood pressure than the combination-treated liraglutide and phentermine groups. By day 21, liraglutide and phentermine–treated DIO mice had double the weight loss compared with all other treatment groups, yet no change from baseline systolic blood pressure was observed. A similar pattern of effects was seen in diastolic blood pressure and mean arterial blood pressure. These data show that the combination of liraglutide plus phentermine produces more than additive weight loss but does not reduce blood pressure more than liraglutide alone.

Impact on Glycemic Control

As expected, liraglutide improved glycemic control (29). In lean mice, phentermine did not improve glucose tolerance despite modest weight loss. The combination of liraglutide and phentermine improved glucose tolerance more than liraglutide alone, which is consistent with this being secondary to the additional weight loss. In obese mice, liraglutide alone improved glucose tolerance more than other treatment groups, whereas phentermine alone produced no benefit on glucose tolerance relative to vehicle-treated DIO mice. This was despite a weight loss similar to the liraglutide alone–treated DIO mice. The combination of liraglutide and phentermine had an effectiveness similar to the liraglutide alone–treated DIO mice, improving glucose tolerance compared with vehicle-treated animals. Hence the combination of liraglutide and phentermine improved glycemic control and increased weight loss.

Limitations

One limitation to this study is that a submaximal dose of liraglutide was used. Liraglutide is prescribed at 1.8 mg for the treatment of diabetes or 3.0 mg for the treatment of obesity daily; however, many physicians acknowledge that patients do not reach full dose for a variety of reasons. In our preclinical study, maximal doses of the individual drugs would have minimalized the weight loss possible with the combination and lead to a premature study termination. In clinical usage, the liraglutide dose is titrated up over a period of weeks, slowly increasing to the maximum dose to increase compliance with the drug treatment regimen. In contrast, in this study, the dose of liraglutide was not titrated. The extent to which results presented here can be generalized to humans remains unclear. We hope that our results will guide the design of further clinical studies to address similar questions. Specifically, studies addressing systemic hemodynamics in patients receiving either or both drugs are needed.

Conclusions

The combination of liraglutide and phentermine produced substantial body weight loss in both lean and DIO mice compared with animals treated with either phentermine or liraglutide alone. In DIO mice, the body weight reduction associated with combination treatment was more than additive, i.e., synergistic. In DIO mice, weight loss was driven by a reduction in food intake and an increase in locomotor activity, whereas decreased food intake was less responsible for body weight reductions in lean mice. The combination of liraglutide and phentermine had an additive tachycardic effect, and this was especially pronounced in lean mice. Compared with phentermine-only treatment, combination therapy was associated with reduced blood pressure, yet these decreases in blood pressure were not as large as would have been expected based on the extent of weight loss. Treatment with liraglutide alone produced a nonsignificant increase in heart rate, whereas phentermine increased locomotor activity even in the presence of liraglutide. Thus, it appears that phentermine augments the weight loss caused by liraglutide in DIO mice and does not modulate the effects of liraglutide on heart rate and blood pressure.

Funding. This work was supported by the National Heart Foundation of Australia (S.E.S.) and the National Health and Medical Research Council of Australia (S.E.S. and M.A.C.).

Duality of Interest. This study was supported by Novo Nordisk Research Center Seattle (S.E.S. and M.A.C.). S.E.S., J.T.P., L.E.K., and M.A.C. are shareholders in Integrated Physiology Services, which provides services to Novo Nordisk. F.H.K. and K.L.G. are employees of Novo Nordisk Research Center Seattle. M.A.C. is a consultant to Novo Nordisk and Valeant/iNova, which market liraglutide and phentermine, respectively, in Australia. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. S.E.S. designed and conducted experiments, contributed reagents/animals, analyzed data, and prepared the manuscript. J.T.P. analyzed data and prepared the manuscript. F.H.K. designed experiments and contributed reagents/animals. A.S.B.-R. conducted experiments. L.E.K. prepared the manuscript. K.L.G. and M.A.C. designed experiments, contributed reagents/animals, and prepared the manuscript. M.A.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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