Determining the Effects of Combined Liraglutide andPhentermine on Metabolic Parameters, Blood Pressure,and Heart Rate in Lean and Obese Male MiceStephanie E. Simonds,1 Jack T. Pryor,1,2 Frank H. Koegler,3 Alberte S. Buch-Rasmussen,1 Lauren E. Kelly,1

Kevin L. Grove,3 and Michael A. Cowley1

Diabetes 2019;68:683–695 | https://doi.org/10.2337/db18-1149

Liraglutide, a glucagon-like peptide 1 (GLP-1) receptoragonist, and phentermine, a psychostimulant structur-ally related to amphetamine, are drugs approved for thetreatment of obesity and hyperphagia. There is signif-icant interest in combination use of liraglutide andphentermine for weight loss; however, both drugs havebeen reported to induce systemic hemodynamicchanges, and as such the therapeutic window for thisdrug combination needs to be determined. To under-stand their impact on metabolic and cardiovascularphysiology, we tested the effects of these drugs aloneand in combination for 21 days in lean and obese malemice. The combination of liraglutide and phentermine,at 100 mg/kg/day and 10 mg/kg/day, respectively, pro-duced the largest reduction in body weight in both leanand diet-induced obese (DIO) mice, when comparedwith both vehicle and monotherapy-treated mice. Inlean mice, combination treatment at the aforementioneddoses significantly increased heart rate and reducedblood pressure, whereas in DIO mice, combination ther-apy induced a transient increase in heart rate and de-creased blood pressure. These studies demonstrate thatin obese mice, the combination of liraglutide and phen-termine may reduce body weight but only induce modestimprovements in cardiovascular functions. Conversely, inlean mice, the additional weight loss from combinationtherapy does not improve cardiovascular parameters.

Obesity is a serious disease, characterized by the accumu-lation of excess body fat. A recent study estimated thatworldwide, 108 million children and 604 million adults are

obese, and that these numbers are on the rise (1). Obesitysignificantly increases the risk of developing secondarydiseases, including type 2 diabetes and cardiovasculardiseases. Annually, four million deaths are attributedto obesity and of these, two-thirds are due to obesity-associated cardiovascular disease (1).

Obesity pharmacotherapy treatments act by reducingcaloric intake, increasing energy expenditure, and/or in-creasing energy excretion. Pharmacological manipulationof energy homeostasis may affect neurons, neuropeptides,autonomic nervous system branches, or brain regions thatare intrinsic to cardiovascular control (2). Therefore, drugsthat are marketed for obesity should be considered interms of their impact on the cardiovascular systems.

The glucagon-like peptide 1 (GLP-1) receptor agonist lir-aglutide and the psychostimulant phentermine are two phar-macotherapies currently approved for the treatment of obesityin many countries, and both act to reduce body weightthrough reduced food intake (3). Additionally, both thesecompounds influence the activity of the cardiovascular system.

GLP-1 is secreted from L cells of the ilium and colon. Inaddition to its potent ability to reduce blood glucose levels,GLP-1 increases postprandial satiety, inhibits gastric emp-tying, and influences the hedonic reward systems (4–10).The hypophagic effects of GLP-1 appear to be driven bycentral actions: intracerebroventricular administrationof GLP-1 reduces food intake; however, this effect islost in the presence of a central GLP-1 antagonist orwhen administered to GLP receptor2/2 knockout mice(4,11–21). In humans, the GLP-1 receptor agonist liraglu-tide is well tolerated and produces significant weight loss

1Metabolic Disease and Obesity Program, Monash Biomedicine Discovery Institute,and Department of Physiology, Monash University, Melbourne, Victoria, Australia2Woodrudge LTD, London, U.K.3Novo Nordisk Research Center, Seattle, WA

Corresponding author: Michael A. Cowley, michael.cowley@monash.edu

Received 23 October 2018 and accepted 17 January 2019

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db18-1149/-/DC1.

© 2019 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 68, April 2019 683

METABOLISM

through decreased food intake without increasing energyexpenditure over a sustained period (22,23). In rodents,GLP-1 receptor agonists can act acutely to increase bloodpressure and heart rate in a dose-dependent manner (24).Liraglutide treatment in patients with type 2 diabetes doesnot significantly alter noradrenaline concentration com-pared with placebo treatment (25,26).

However, in rodents (but not in humans), cardiacGLP-1 receptor activation can also have a hypotensiveaction through the stimulation of atrial natriureticpeptide (ANP) release in rodents (27,28). Clinical evi-dence suggests that despite causing acute increases inheart rate, chronic treatment with liraglutide lowerssystolic blood pressure, the risk of cardiovascular death,nonfatal myocardial infarction, and nonfatal stroke inpatients with type 2 diabetes and cardiovascular diseaserisk (28,29).

The U.S. Food and Drug Administration (FDA) ap-proved the use of the psychostimulant phentermine forthe treatment of obesity in 1959 (30). It is currently pre-scribed for short-term appetite suppression (,3 months).Compared with placebo, it significantly reduces bodyweight in obese individuals and is currently the mostwidely prescribed antiobesity therapy, in part due toits relatively low cost (31–33). Although reports suggestphentermine can increase blood pressure, numerous stud-ies demonstrate that when used for weight loss, phenter-mine does not adversely influence the cardiovascularsystem. In a recent human study, phentermine-treatedindividuals lost significantly more weight compared withplacebo, heart rate remained unchanged in both groups,and blood pressure decreased in the phentermine-treatedgroup (31,34–37). Obese patients treated with 7.5 or15 mg phentermine lost 6.65% and 7.38% body weight,respectively, after 28 weeks of treatment (38). Despitea similar change in body weight, phentermine 7.5 mgtreatment reduced mean systolic blood pressure by3.3 mmHg compared with 6.4 mmHg in 15 mg phenter-mine–treated obese patients (38). Phentermine has beenused in combination with other antiobesity therapies andwas part of the fenfluramine-phentermine combinationthat led to pulmonary hypertension and the withdrawal offenfluramine from the U.S. market (39). A combination ofphentermine and topiramate is marketed by Vivus and wasapproved in the U.S. as an antiobesity drug in late 2012. Asphentermine and liraglutide are both approved for weightmanagement, it is plausible that some patients will beprescribed both agents, although they are not yet approvedor licensed for combination therapy. This study aims todetermine the cardiovascular effects of the combination ofliraglutide plus phentermine while evaluating food intake,body weight, body fat, locomotor activity, and glucose ho-meostasis in a mouse model of diet-induced obesity.

RESEARCH DESIGN AND METHODS

All animal procedures were approved by the MonashUniversity Animal Ethics Committee. All mice were housed

in a controlled environment. The light period was from7:00 A.M. to 7:00 P.M., and the dark period was 7:00 P.M. to7:00 A.M. Temperature and humidity remained constant.Mouse food was available ad libitum, and mice weremonitored daily.

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

RadiotelemetryRadiotelemetry probes were implanted as previously de-scribed (40). In brief, TA11PA-C10 Data Science Interna-tional radiotelemetry probes were implanted into the leftcarotid artery under isoflurane anesthesia. After surgery,body weight and food intake were measured daily whilemice recovered for 2 weeks. After this period, baselinerecordings were measured (this included baseline bloodpressure and heart rate), and an intraperitoneal glucosetolerance test (GTT) was performed as previously de-scribed (41) as was a DEXA scan (40).

After this, all mice received single, combination, orvehicle drug treatments, administered via intraperitonealor subcutaneous injection daily for 21 days as outlined inthe treatment table (Table 1). Liraglutide was provided byNovo Nordisk. This study used a submaximal dose ofliraglutide. Phentermine-HCl was purchased from Sigma-Aldrich (P8653). Body weight and food intake weremeasured daily, and blood pressure, heart rate, andactivity were measured continuously. At the conclusionof this 21-day period, a final GTT was performed, anda DEXA scan was performed on diet-induced obese (DIO)mice.

Analysis and Recording of DataCardiovascular recordings were collected on the Ponemahversion 6.31 software for 1 min, every 10 min for 21 days.Daily averaged data were all the data collected over that24-h period. Light and dark period averages were all datacollected throughout that 12-h period. Change data werethe difference in treatment data compared with baselinefor the same time period. Percentage change was calculatedas the change divided by the baseline value, for the

Table 1—Treatment composition

Treatments Abbreviation

Saline s.c. + saline i.p. S+S

Liraglutide (100 mg/kg/day s.c.) + saline i.p. L+S

Saline s.c. + phentermine (10 mg/kg/day i.p.) S+P

Liraglutide (100 mg/kg/day s.c.) + phentermine(10 mg/kg/day i.p.) L+P

First treatment was administered subcutaneously, and thesecond treatment was administered intraperitoneally.

684 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

corresponding time period, multiplied by 100. This wasonly used for body weight analysis due to the largedifference existing between lean and DIO mice at baseline.

The glucose tolerance was compared as area under thecurve (AUC) throughout the GTT. A comparison was madebetween the same mice at baseline and after the 21-daytreatment period.

Data are presented as mean 6 SEM. The significancebetween groups is outlined in Table 2. The significancecompared with baseline is outlined on graphs by color.Comparisons between treatment groups are outlined inblack by the symbols expressed in Table 2.

RESULTS

Body Weight in Lean MiceAt baseline, no difference in body weight in lean mice wasobserved. Herein, comparisons for each group will be drawnrespective to its own baseline and to S+S-treated (salines.c. + saline i.p.) mice at day 21. S+S-treated animals wereweight stable throughout the treatment period. The L+S-treated (liraglutide [100 mg/kg/day s.c.] + saline i.p.) ani-mals lost an average of 4.56 1.2% bodyweight, whereas theS+P-treated (saline s.c. + phentermine [10 mg/kg/day i.p.])mice lost an average of 6.2 6 2.0% (Fig. 1A and Supple-mentary Fig. 1A). The combination of L+P (liraglutide[100 mg/kg/day s.c.] + phentermine [10 mg/kg/day i.p.])resulted in significantly more weight loss (9.2 6 0.8%),compared with the liraglutide alone or phentermine alone–treated group (Fig. 1A).

Body Weight in DIO MiceNo difference in body weight between DIO treatmentgroups was found at baseline (Supplementary Fig. 1B).At day 21 of treatment, the S+S treatment group hadincreased their body weight by 13 6 3.5% (Fig. 1B). The L+S-treated mice lost an average of 7.56 1.7% body weightfrom baseline by day 21 of treatment. The S+P-treated DIOanimals lost an average of 8.7 6 2.7% body weight, whichwas not significantly different from the L+S-treated animals.The L+P-treated group lost 20 6 2.7% of body weight from

baseline, significantly more weight than the L+S, S+P, andS+S-treated groups (Fig. 1B and Supplementary Fig. 1B).

Body Weight: Lean Versus DIO MiceThe majority of drug treatments produced significantlygreater weight loss in DIO mice compared with lean mice(Fig. 1A and B). The L+S treatment as of day 21 oftreatment caused a weight loss of 4.5% in lean micecompared with 7.5% in DIO mice. The S+P treatmentcaused a weight loss of 6.2% in lean mice comparedwith 8.7% in DIO mice (Fig. 1A and B). The combinationwith L+P caused the greatest weight loss in both lean (9%)and DIO (20%) mice. In combination, liraglutide andphentermine resulted in significantly greater weight lossthan either the L+S or S+P-treated lean and DIO animals(Fig. 1A and B).

Body Fat: DIO MiceDEXA scans (Supplementary Fig. 1C) were performed onthe DIOmice at days 0 and 21 and showed that weight lossin all treatment groups was primarily due to the loss ofbody fat. In the DIO L+P treatment group, 9.2 g of body fatwas lost compared with 4 g in the S+P treatment group and6.2 g in the L+S treatment group (Supplementary Fig. 1C).Lean body mass did not significantly decrease in anytreatment group (data not shown).

Food Intake in Lean MiceCompared with baseline, all treatment groups showeda transient decrease in food intake. However, therewere no significant differences between the drug-treatedgroups (Supplementary Fig. 1D).

Food Intake in DIO MiceAll drug treatment groups significantly decreased foodintake in the DIO mice with a similar effect comparedwith S+S-treated DIO mice. The one exception to this wasL+P, which had a significant decrease in food intake whencompared with the L+S group (Fig. 1C and SupplementaryFig. 1E).

Table 2—Statistical analysis comparison legend for explanation of two-way ANOVA results on graphs

@ Gray Purple Liraglutide (100 mg/kg/day) + saline vs. Saline + saline

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

, Purple Black Saline + saline vs. Saline + phentermine (10 mg/kg/day)

$ Gray Red Liraglutide (100 mg/kg/day) + saline vs. Liraglutide (100 mg/kg/day) +phentermine (10 mg/kg/day)

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

. Red Black Liraglutide (100 mg/kg/day) +phentermine (10 mg/kg/day)

vs. Saline + phentermine (10 mg/kg/day)

@P , 0.05, saline + saline vs. liraglutide (100 mg/kg/day) + saline. !P , 0.05 saline + saline vs. liraglutide (100 mg/kg/day) + phentermine(10 mg/kg/day). ,P , 0.05, saline + saline vs. saline + phentermine (10 mg/kg/day). $P , 0.05, liraglutide (100 mg/kg/day) + saline vs.liraglutide (100 mg/kg/day) + phentermine (10 mg/kg/day). %P , 0.05, liraglutide (100 mg/kg/day) + saline vs. saline + phentermine(10 mg/kg/day). .P , 0.05, liraglutide (100 mg/kg/day) + phentermine (10 mg/kg/day) vs. saline + phentermine (10 mg/kg/day).

diabetes.diabetesjournals.org Simonds and Associates 685

Figure 1—A: Percentage change in body weight in lean mice from baseline throughout 21-day treatment period; n = 8–11. B: Percentagechange in body weight in DIO mice from baseline throughout 21-day treatment period; n = 7–9. C: Cumulative food intake, from start oftreatment in DIO mice from baseline throughout 21-day treatment period; n = 7–9. Mean 6 SEM, two-way AVOVA, Sidak multiplecomparisons 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, L1Ptreatment compared to own baseline; black asterisks, S1P treatment compared to own baseline.

686 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

Food Intake: Lean Versus DIO MiceThe effect of treatments on food intake was notably differentbetween lean andDIOmice. Both liraglutide and phenterminealone and in combination significantly reduced food intake inDIO mice; however, this substantial effect was not observedin lean mice (Fig. 1C and Supplementary Fig. 1D and E).

Activity in Lean MiceCompared with the S+S group, the L+S treatment groupshowed no increase in activity in lean mice (Fig. 2A). TheS+P group had significantly increased activity comparedwith S+S-treated lean mice. Mice that received thecombination L+P had significantly elevated activity levels

Figure 2—A: Activity (24 h) in leanmice from baseline throughout 21-day treatment period; n = 8–11.B: Light period (12 h) activity in leanmicefrom baseline throughout 21-day treatment period; n = 8–11. C: Dark period (12 h) activity in lean mice from baseline throughout 21-daytreatment 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 baselinethroughout 21-day treatment period; n = 7–9. Mean6SEM, two-way AVOVA, Sidakmultiple 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, L1P treatment compared toown baseline; black asterisks, S1P treatment compared to own baseline.

diabetes.diabetesjournals.org Simonds and Associates 687

compared with those in the S+S and L+S groups. Interest-ingly, the majority of the increase in activity in these groupswas during the normally inactive light period, with nosignificant change during the dark period (Fig. 2B and C).

Activity in DIO MiceIn DIO mice, both the S+S and L+S treatments had noeffect on activity (Fig. 2D). The S+P group had a signif-icant increase in activity compared with baseline and S+S and L+S-treated DIO mice (Fig. 2A). The combinationDIO treatment group L+P had increased levels of ac-tivity compared with the L+S group (Fig. 2D). Theactivity change was substantially greater in the 12-hlight period as compared with the 12-h dark period (Fig.2E and F).

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

Glucose Tolerance in Lean MiceIn lean mice, the AUC after GTTs at both baseline and after21 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).

Glucose Tolerance in DIO MiceIn DIO mice, the improvement in AUC during a GTT wasseen in L+S and L+P treatment groups (Fig. 3B).

Heart Rate in Lean MiceCompared with the change in heart rate of the S+S-treatedmice, all other treatment groups had a significantly largerchange in heart rate (Fig. 4A). All treatment groups exceptthe S+P had increased heart rate immediately after theonset of treatment, whereas the increase in the S+P groupbecame evident several days after the start of treatment(Fig. 4A). Compared with L+S or S+P, the combination ofphentermine with liraglutide significantly increased heartrate further in lean mice (Fig. 4A). The change in heart ratewas greatest in the 12-h light period (Supplementary Fig.2A) compared with the 12-h dark period (SupplementaryFig. 2B).

Heart Rate in DIO MiceIn DIO mice, L+S-treated mice had a tendency for in-creased heart rate compared with S+S treatment, but thisdid not reach statistical significance (Fig. 4B). In the first4 days of treatment, DIO mice treated with S+P hada significantly reduced heart rate compared with S+S,L+S, and L+P-treated mice (Fig. 4B). In DIO L+P-treatedmice, heart rate was significantly elevated at timescompared with the S+S-treated DIO mice (Fig. 4B).

Heart Rate: Lean Versus DIO MiceDIO mice had a significantly higher basal heart rate thanlean mice (Fig. 4A and B). No difference in the averagepercentage change of heart rate was evident in the L+S(4.1% vs. 4.1%) or S+P (3.1% vs. 20.45%) treated leanand DIO mice. The percentage change in peak (greatestrate) heart rate in L+P-treated lean mice was 19% vs.6.8% in DIO mice. Much of the difference between thechanges in heart rate of lean and DIO mice occurredduring the 12-h light period (Fig. 4A and B and Supple-mentary Fig. 2A).

Systolic Blood Pressure in Lean MiceAt day 21, systolic blood pressure in the L+S-treated lean mice was significantly reduced comparedwith baseline and S+S-treated mice (Fig. 5A). The S+P-treated lean mice had a significantly greater (increased)

Figure 3—A: Average AUC during GTT in lean mice at baseline andafter 21 days of treatment. n = 8–11, mean6 SEM, repeated Studentt 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 AUCduring GTT in DIO mice at baseline and after 21 days of treatment.n = 7–9, mean6 SEM, repeated Student t test between baseline andafter 21 days of treatment. *P , 0.05 and **P , 0.01, baseline vs.after 21 days of treatment.

688 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

change in systolic blood pressure compared with L+Sand the combination L+P treatments. L+P-treated leanmice had a significantly reduced systolic blood pressurecompared with baseline and the S+S treatment group(Fig. 5A).

Systolic Blood Pressure in DIO MiceCompared with S+S-treated DIO mice, L+S treatment inDIO mice significantly reduced systolic blood pressure aswell as compared with baseline. S+P-treated DIO micehad a significantly increased change in systolic bloodpressure at times compared with L+S and the combina-tion of L+P (Fig. 5C). L+P treatment reduced systolic

blood pressure compared with S+S, and no differencebetween L+P and L+S-treated DIO mice was evident (Fig.5C).

Systolic Blood Pressure: Lean Versus DIO MiceThe largest decrease in systolic blood pressure in lean miceoccurred in themice treated with L+P (210.4%), comparedwith only a 3.8% decrease in systolic blood pressure inrespective treated DIO mice. In DIO mice, the largestpercentage change in systolic blood pressure was observedin the L+S-treated mice (28.5%). This compares to a 6.9%decrease in systolic blood pressure in the L+S-treated leangroup (Fig. 5A and C).

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 frombaseline of DIO mice throughout 21-day treatment period; n = 7–9. Mean6 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, L1P treatment compared to own baseline. BPM, beats per minute.

diabetes.diabetesjournals.org Simonds and Associates 689

Diastolic Blood Pressure in Lean MiceL+S and L+P-treated lean mice had reduced diastolic bloodpressure from baseline and compared with the S+P-treatedmice (Fig. 5B).

Diastolic Blood Pressure in DIO MiceThe change in diastolic blood pressure in L+S and L+P-treated DIO mice was significantly lower compared withthe S+P-treated mice (Fig. 5D).

DISCUSSION

The FraminghamHeart Study estimated that 78% of primaryhypertension in men and 65% in women can be attributed toexcess body weight (42). Cardiovascular disease is responsiblefor the death of two-thirds of individuals with obesity (1). Ittherefore follows that drugs designed to elicit weight lossshould avoid adverse cardiovascular effects.

Preclinical rodent studies have shown that GLP-1 re-ceptor agonists can increase blood pressure and heart rate.

Clinical data show that chronic treatment with liraglutidecan reduce blood pressure and increase heart rate (24,43),yet when used at recommended doses, liraglutide caninduce weight loss in individuals with obesity, withoutadversely increasing cardiovascular risk (29).

Phentermine’s hypertensive effects are the result ofincreased synaptic noradrenaline release and sympatheticnervous system activation (44). However, in recent stud-ies, phentermine treatment in patients with obesityresulted in weight loss and decreases in systolic bloodpressure (38,44). The Endocrine Society advises that sym-pathomimetics including phentermine are not to be usedfor weight loss in patients with uncontrolled hypertensionor a history of heart disease (45).

The combination of phentermine and fenfluraminenot only can lead to valvuopathy (reason for FDA with-drawal) but can also increase blood pressure and heart rate(44,46,47). Combination therapy with phentermine andtopiramate is currently available for weight loss. This

Figure 5—A: Change from baseline in systolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. B: Change frombaseline in diastolic blood pressure of lean mice throughout 21-day treatment period; n = 8–11. C: Change from baseline in systolic bloodpressure of DIO mice throughout 21-day treatment period; n = 7–9. D: Change from baseline in diastolic blood pressure of DIO micethroughout 21-day treatment period; n = 7–9. Mean 6 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, L1P treatment comparedto own baseline; black asterisks, S1P treatment compared to own baseline.

690 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

combination when given at the highest acceptable dosescan increase blood pressure and heart rate, despite de-creasing body weight (38,48).

As individuals aim to induce the greatest weight losspossible, it is expected that people may combine the use ofliraglutide and phentermine even though this combinationis not FDA approved. The data presented here demon-strate that liraglutide and phentermine both alone and incombination can significantly reduce body weight in bothlean and DIO mice. These studies also demonstrate theactions of these drugs on the activity of the cardiovascularsystem.

The administration of liraglutide alone in both lean andDIO mice induced modest weight loss compared withvehicle-treated mice. The dose of liraglutide used in thesestudies was a submaximal weight loss dose; this dose waschosen to enable further weight loss when combined. Inliraglutide alone–treated groups in both lean and DIOmice, weight loss occurred most rapidly in the first5 days and then occurred at a reduced rate for the restof the treatment period. This stabilizing of body weightloss after liraglutide alone treatment is believed to be dueto adaptation by the body and is observed in numerousliraglutide treatment studies in numerous animal modelsas well as with other anorexigenic agents (49–52). Therewas no significant difference in the final weight lossinduced by liraglutide alone or phentermine alone in eitherthe lean or DIO treated mice. The only difference betweenthe two treatment groups was the rate of weight loss;weight loss was slower in the phentermine alone–treatedmice. In lean mice, after 21 days of treatment with thecombination dose of liraglutide and phentermine, thegreatest proportion of body weight loss had occurredcompared with all other lean treatment groups: 9% ofbody weight from baseline, this being a significant re-duction relative to baseline. The percentage change in bodyweight was subadditive, that is, less than the numericaddition of the effects of liraglutide alone added to theeffects of phentermine alone. That the combination treat-ment in lean mice did not result in additive effects on bodyweight may be because some of the weight loss actions ofboth liraglutide and phentermine are through reducedfood intake. Similar to lean mice, the DIO mice treatedwith the combination of liraglutide and phentermine hadthe largest reduction in body weight among the DIOgroups (19.8% body weight compared with baseline byday 21 of treatment). This weight loss could be consideredsynergistic, as the sum of the total weight loss of animalstreated with liraglutide alone or phentermine alone wasless than the total weight loss of the combination liraglu-tide and phentermine treatment group. The liraglutide andphentermine–treated DIO mice lost double the weightof 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 dueto a significant reduction in body fat. Although lean masswas also decreased, this reduction did not reach statistical

significance. Reasons for the increased effectiveness ofcombination liraglutide and phentermine treatment inDIO compared with lean mice may be due to complexcentral and peripheral metabolic factors resisting the drug-induced weight loss in lean animals, factors that may bediminished in the obese state.

All active treatments reduced food intake, which wouldbe expected to cause weight loss. Additionally, some treat-ments increased locomotor activity, which would furtherdrive weight loss. Although we did not assess thermo-genesis in these studies, others have demonstrated thatliraglutide and GLP-1 receptor activation increases ther-mogenesis (53,54). One major difference between lean andDIO mice was the way body weight reduced. In DIO mice,food intake was significantly reduced in the liraglutidealone, phentermine alone, and additively in the combinedliraglutide and phentermine–treated DIO mice. Addition-ally, activity was increased in the DIO mice treated withboth phentermine alone and liraglutide and phenterminein combination, compared with vehicle treatment. It isestablished that phentermine can increase physical activityin rats (55–57), whereas liraglutide has previously shownno clear effect on activity. However, in lean mice, whilesimilar effects on activity were observed with phenterminealone and liraglutide–phentermine combination treat-ment, food intake in all three experimental groups wasnot significantly inhibited. Most notably, reduction was inthe combined liraglutide and phentermine–treated mice,but cumulative food intake was not different betweentreatment groups. This absence of sustained reductionsin food intake in lean mice shows that the induced weightloss must be due to other mechanisms, including increasedactivity, which was observed in the studies described here,or through increased metabolic rate or increased thermo-genesis. Indeed, previous studies have shown that GLP-1receptor agonists can increase brown adipose tissue tem-perature (54). The greater reduction in food intake intreated DIO mice may be one mechanism behind theincreased effectiveness of combination therapy in DIOmice compared with lean mice.

One notable finding of the combination therapy in bothlean and DIOmice was that liraglutide was not attenuatingthe 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 thatthe combination therapy allows the additional weightloss, as compared with liraglutide alone treatment, viaa different mechanism than reduced food intake alone. Fora long-term sustainable weight loss option, this would bean additive benefit. In our studies, animals were not givenextra activity equipment, e.g., a running wheel to induceweight loss and stimulate the increase in activity. If thiswas to be incorporated, the weight loss induced may haveequated to a much greater value in mice. In this study, theincreased change in activity occurred most notably in thelight period, when mice should be sleeping. As such, it

diabetes.diabetesjournals.org Simonds and Associates 691

would be expected that weight loss in response to activityin humans is likely to be varied, because humans areunlikely to exercise when they usually sleep.

Impact on Heart RateThe LEADER (Liraglutide Effect and Action in Diabetes:Evaluation of Cardiovascular Outcome Results) study hasshown that liraglutide (1.8 mg s.c. once daily) significantlyprovides a cardiovascular benefit on the three-point majoradverse cardiovascular events scale. This includes thenumber of deaths being reduced in patients with type2 diabetes and high cardiovascular risk, even thoughliraglutide can increase heart rate (29,59,60). At the sub-maximal dose used in the studies described here, wedemonstrate that liraglutide-only treatment in lean miceincreased heart rate compared with baseline and comparedwith vehicle-treated mice despite weight loss, which is inaccordance with previous findings (24,43). The increasein heart rate caused by liraglutide alone was significantlyless than the increase caused by combining liraglutide andphentermine. Hence the combination of liraglutide andphentermine produced an additive increase in heart ratein lean mice. Liraglutide and phentermine treatment to-gether significantly increased heart rate in lean mice fromday 1 throughout the total treatment period. The increasewas 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 leanmice, combination caused a synergistic increase in heartrate. During the initial phase of treatment until day 5 oftreatment, the increase in heart rate was evenly elevated inboth the 12-h dark and 12-h light periods. As the treat-ment period progressed, the increased heart rate could beobserved more in the 12-h light period. This was associatedwith increased activity during the light phase, a time whenmice are relatively inactive (61). If we consider the effect ofthe combination therapy on body weight and on heart rate,we can conclude that the combination therapy appears tobe effective in lean mice in causing weight loss; however,treatment does cause a 20% increase in heart rate frombaseline in this model.

Heart rate was also elevated in the DIO mice treatedwith the combination of liraglutide and phenterminecompared with both vehicle- and phentermine alone–treated DIO mice (heart rate was not significantly changedin liraglutide alone–treated DIO mice). Compared with thelean mice treated with the combination of liraglutide andphentermine, the increase in heart rate in the DIO micewas only half as great. Lean mice have a significantly lowerbasal heart rate compared with DIOmice; the 20% increaseoccurs as the heart rate reaches the same basal beats perminute as DIO mice and the increase in percentage did notappear as great in DIO mice. From this data, it could beinferred that lean mice have lower sympathetic nerveactivity as has been seen in obese humans (62).

This is likely because the baseline heart rate of DIOmice started at an elevated rate due to the mice already

displaying hypertension and tachycardia. Heart rate wasincreased in both the dark and light periods. We canconclude that the combination is effective for weightloss in obese mice but does increase heart rate. If thesestudies were continued and weight continued to be lost,the heart rate may be improved but further research wouldneed to be undertaken to discover the long-term impact ofthe combination therapies.

Effects on Blood PressureClinical studies have conclusively demonstrated thatchronic treatment with liraglutide decreases systolic bloodpressure (23,43,59,63). However preclinical studies haveshown that GLP-1 agonists can increase blood pressure inrats (24). In the studies described here, liraglutide alonedecreased systolic blood pressure in lean and obese mice.This decrease in systolic blood pressure occurred alongwith a decrease in body weight. Phentermine alone did notdecrease systolic blood pressure, despite similar bodyweight reductions in lean and DIO mice, and at day 21,phentermine alone–treated lean and DIO mice had highersystolic blood pressure than the combination-treated lir-aglutide and phentermine groups. By day 21, liraglutideand phentermine–treated DIO mice had double the weightloss compared with all other treatment groups, yet nochange from baseline systolic blood pressure was observed.A similar pattern of effects was seen in diastolic bloodpressure and mean arterial blood pressure. These datashow that the combination of liraglutide plus phentermineproduces more than additive weight loss but does notreduce blood pressure more than liraglutide alone.

Impact on Glycemic ControlAs expected, liraglutide improved glycemic control(29). In lean mice, phentermine did not improve glu-cose tolerance despite modest weight loss. The combi-nation of liraglutide and phentermine improvedglucose tolerance more than liraglutide alone, whichis consistent with this being secondary to the addi-tional weight loss. In obese mice, liraglutide aloneimproved glucose tolerance more than other treatmentgroups, whereas phentermine alone produced no ben-efit on glucose tolerance relative to vehicle-treated DIOmice. This was despite a weight loss similar to theliraglutide alone–treated DIO mice. The combinationof liraglutide and phentermine had an effectivenesssimilar to the liraglutide alone–treated DIO mice, im-proving glucose tolerance compared with vehicle-trea-ted animals. Hence the combination of liraglutide andphentermine improved glycemic control and increasedweight loss.

LimitationsOne limitation to this study is that a submaximal dose ofliraglutide was used. Liraglutide is prescribed at 1.8 mg forthe treatment of diabetes or 3.0 mg for the treatment ofobesity daily; however, many physicians acknowledge that

692 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

patients do not reach full dose for a variety of reasons. Inour preclinical study, maximal doses of the individualdrugs would have minimalized the weight loss possiblewith the combination and lead to a premature studytermination. In clinical usage, the liraglutide dose is ti-trated up over a period of weeks, slowly increasing to themaximum dose to increase compliance with the drugtreatment regimen. In contrast, in this study, the doseof liraglutide was not titrated. The extent to which resultspresented here can be generalized to humans remainsunclear. We hope that our results will guide the designof further clinical studies to address similar questions.Specifically, studies addressing systemic hemodynamics inpatients receiving either or both drugs are needed.

ConclusionsThe combination of liraglutide and phentermine producedsubstantial body weight loss in both lean and DIO micecompared with animals treated with either phentermine orliraglutide alone. In DIO mice, the body weight reductionassociated with combination treatment was more thanadditive, i.e., synergistic. In DIO mice, weight loss wasdriven by a reduction in food intake and an increase inlocomotor activity, whereas decreased food intake was lessresponsible for body weight reductions in lean mice. Thecombination of liraglutide and phentermine had an addi-tive tachycardic effect, and this was especially pronouncedin lean mice. Compared with phentermine-only treatment,combination therapy was associated with reduced bloodpressure, yet these decreases in blood pressure were not aslarge as would have been expected based on the extent ofweight loss. Treatment with liraglutide alone produceda nonsignificant increase in heart rate, whereas phenter-mine increased locomotor activity even in the presence ofliraglutide. Thus, it appears that phentermine augmentsthe weight loss caused by liraglutide in DIO mice and doesnot modulate the effects of liraglutide on heart rate andblood pressure.

Funding. This work was supported by the National Heart Foundation ofAustralia (S.E.S.) and the National Health and Medical Research Council ofAustralia (S.E.S. and M.A.C.).Duality of Interest. This study was supported by Novo Nordisk ResearchCenter Seattle (S.E.S. and M.A.C.). S.E.S., J.T.P., L.E.K., and M.A.C. are share-holders in Integrated Physiology Services, which provides services to NovoNordisk. F.H.K. and K.L.G. are employees of Novo Nordisk Research CenterSeattle. M.A.C. is a consultant to Novo Nordisk and Valeant/iNova, which marketliraglutide and phentermine, respectively, in Australia. No other potential conflictsof 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 experimentsand contributed reagents/animals. A.S.B.-R. conducted experiments. L.E.K. pre-pared the manuscript. K.L.G. and M.A.C. designed experiments, contributedreagents/animals, and prepared the manuscript. M.A.C. is the guarantor of thiswork and, as such, had full access to all the data in the study and takesresponsibility for the integrity of the data and the accuracy of the data analysis.

References1. Afshin A, Forouzanfar MH, Reitsma MB, et al.; GBD 2015 Obesity Collab-orators. Health effects of overweight and obesity in 195 countries over 25 years. NEngl J Med 2017;377:13–272. Simonds SE, Cowley MA, Enriori PJ. Leptin increasing sympathetic nerveoutflow in obesity: a cure for obesity or a potential contributor to metabolicsyndrome? Adipocyte 2012;1:177–1813. Hocking S, Dear A, Cowley MA. Current and emerging pharmacotherapies forobesity in Australia. Obes Res Clin Pract 2017;11:501–5214. Jelsing J, Vrang N, Hansen G, Raun K, Tang-Christensen M, Knudsen LB.Liraglutide: short-lived effect on gastric emptying – long lasting effects on bodyweight. Diabetes Obes Metab 2012;14:531–5385. Larsen PJ, Fledelius C, Knudsen LB, Tang-Christensen M. Systemic ad-ministration of the long-acting GLP-1 derivative NN2211 induces lasting andreversible weight loss in both normal and obese rats. Diabetes 2001;50:2530–25396. Raun K, von Voss P, Knudsen LB. Liraglutide, a once-daily human glucagon-like peptide-1 analog, minimizes food intake in severely obese minipigs. Obesity(Silver Spring) 2007;15:1710–17167. Raun K, von Voss P, Gotfredsen CF, Golozoubova V, Rolin B, Knudsen LB.Liraglutide, a long-acting glucagon-like peptide-1 analog, reduces body weightand food intake in obese candy-fed rats, whereas a dipeptidyl peptidase-IV in-hibitor, vildagliptin, does not. Diabetes 2007;56:8–158. Pannacciulli N, Le DS, Salbe AD, et al. Postprandial glucagon-like peptide-1(GLP-1) response is positively associated with changes in neuronal activity of brainareas implicated in satiety and food intake regulation in humans. Neuroimage2007;35:511–5179. Farr OM, Sofopoulos M, Tsoukas MA, et al. GLP-1 receptors exist in theparietal cortex, hypothalamus and medulla of human brains and the GLP-1 an-alogue liraglutide alters brain activity related to highly desirable food cues inindividuals with diabetes: a crossover, randomised, placebo-controlled trial. Di-abetologia 2016;59:954–96510. Ten Kulve JS, Veltman DJ, van Bloemendaal L, et al. Liraglutide reduces CNSactivation in response to visual food cues only after short-term treatment inpatients with type 2 diabetes. Diabetes Care 2016;39:214–22111. Tang-Christensen M, Larsen PJ, Göke R, et al. Central administration of GLP-1-(7-36) amide inhibits food and water intake in rats. Am J Physiol 1996;271:R848–R85612. Turton MD, O’Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in thecentral regulation of feeding. Nature 1996;379:69–7213. Flint A, Raben A, Astrup A, Holst JJ. Glucagon-like peptide 1 promotes satietyand suppresses energy intake in humans. J Clin Invest 1998;101:515–52014. Rüttimann EB, Arnold M, Hillebrand JJ, Geary N, Langhans W. Intramealhepatic portal and intraperitoneal infusions of glucagon-like peptide-1 reducespontaneous meal size in the rat via different mechanisms. Endocrinology 2009;150:1174–118115. Barrera JG, Jones KR, Herman JP, D’Alessio DA, Woods SC, Seeley RJ.Hyperphagia and increased fat accumulation in two models of chronic CNSglucagon-like peptide-1 loss of function. J Neurosci 2011;31:3904–391316. Scrocchi LA, Brown TJ, MaClusky N, et al. Glucose intolerance but normalsatiety in mice with a null mutation in the glucagon-like peptide 1 receptor gene.Nat Med 1996;2:1254–125817. Hare KJ, Vilsbøll T, Asmar M, Deacon CF, Knop FK, Holst JJ. The gluca-gonostatic and insulinotropic effects of glucagon-like peptide 1 contribute equallyto its glucose-lowering action. Diabetes 2010;59:1765–177018. Nauck MA, Niedereichholz U, Ettler R, et al. Glucagon-like peptide 1 inhibitionof gastric emptying outweighs its insulinotropic effects in healthy humans. Am JPhysiol 1997;273:E981–E988

19. Verdich C, Flint A, Gutzwiller JP, et al. A meta-analysis of the effect ofglucagon-like peptide-1 (7-36) amide on ad libitum energy intake in humans. JClin Endocrinol Metab 2001;86:4382–4389

diabetes.diabetesjournals.org Simonds and Associates 693

20. Nauck MA, Kemmeries G, Holst JJ, Meier JJ. Rapid tachyphylaxis of theglucagon-like peptide 1-induced deceleration of gastric emptying in humans.Diabetes 2011;60:1561–156521. Sisley S, Gutierrez-Aguilar R, Scott M, D’Alessio DA, Sandoval DA, Seeley RJ.Neuronal GLP1R mediates liraglutide’s anorectic but not glucose-lowering effect. JClin Invest 2014;124:2456–246322. van Can J, Sloth B, Jensen CB, Flint A, Blaak EE, Saris WH. Effects of theonce-daily GLP-1 analog liraglutide on gastric emptying, glycemic parameters,appetite and energy metabolism in obese, non-diabetic adults. Int J Obes 2014;38:784–79323. Astrup A, Rössner S, Van Gaal L, et al.; NN8022-1807 Study Group. Effects ofliraglutide in the treatment of obesity: a randomised, double-blind, placebo-controlled study. Lancet 2009;374:1606–161624. Yamamoto H, Lee CE, Marcus JN, et al. Glucagon-like peptide-1 receptorstimulation increases blood pressure and heart rate and activates autonomicregulatory neurons. J Clin Invest 2002;110:43–5225. Nauck MA, El-Ouaghlidi A, Hompesch M, Jacobsen J, Elbroend B. No im-pairment of hypoglycaemia counterregulation via glucagon with the long-actingGLP-1 derivative, NN2211, in subjects with type 2-diabetes (Abstract). Diabetologia2003;46(Suppl. 2):A28526. Nauck MA, El-Ouaghlidi A, Hompesch M, Jacobsen J, Elbroend B. No im-pairment of hypoglycemia counterregulation via glucagon with NN2211, a GLP-1derivative, in subjects with type 2-diabetes (Abstract). Diabetes 2003;52(Suppl. 1):A12827. Kim M, Platt MJ, Shibasaki T, et al. GLP-1 receptor activation and Epac2 linkatrial natriuretic peptide secretion to control of blood pressure. Nat Med 2013;19:567–57528. Lovshin JA, Barnie A, DeAlmeida A, Logan A, Zinman B, Drucker DJ.Liraglutide promotes natriuresis but does not increase circulating levels of atrialnatriuretic peptide in hypertensive subjects with type 2 diabetes. Diabetes Care2015;38:132–13929. Marso SP, Daniels GH, Brown-Frandsen K, et al.; LEADER Steering Com-mittee; LEADER Trial Investigators. Liraglutide and cardiovascular outcomes intype 2 diabetes. N Engl J Med 2016;375:311–32230. Go RE, Hwang KA, Kim SH, et al. Effects of anti-obesity drugs, phentermineand mahuang, on the behavioral patterns in Sprague-Dawley rat model. Lab AnimRes 2014;30:73–7831. Weintraub M, Hasday JD, Mushlin AI, Lockwood DH. A double-blind clinicaltrial in weight control. Use of fenfluramine and phentermine alone and incombination. Arch Intern Med 1984;144:1143–114832. Stafford RS, Radley DC. National trends in antiobesity medication use. ArchIntern Med 2003;163:1046–105033. Hendricks EJ, Rothman RB, Greenway FL. How physician obesityspecialists use drugs to treat obesity. Obesity (Silver Spring) 2009;17:1730–173534. Hendricks EJ, Greenway FL, Westman EC, Gupta AK. Blood pressure andheart rate effects, weight loss and maintenance during long-term phenter-mine pharmacotherapy for obesity. Obesity (Silver Spring) 2011;19:2351–236035. Lorber J. Obesity in childhood. A controlled trial of anorectic drugs. Arch DisChild 1966;41:309–31236. Douglas A, Douglas JG, Robertson CE, Munro JF. Plasma phentermine levels,weight loss and side-effects. Int J Obes 1983;7:591–59537. Vallé-Jones JC, Brodie NH, O’Hara H, O’Hara J, McGhie RL. A comparativestudy of phentermine and diethylpropion in the treatment of obese patients ingeneral practice. Pharmatherapeutica 1983;3:300–30438. Aronne LJ, Wadden TA, Peterson C, Winslow D, Odeh S, Gadde KM.Evaluation of phentermine and topiramate versus phentermine/topiramate ex-tended-release in obese adults. Obesity (Silver Spring) 2013;21:2163–217139. Connolly HM, Crary JL, McGoon MD, et al. Valvular heart diseaseassociated with fenfluramine-phentermine. N Engl J Med 1997;337:581–588

40. Simonds SE, Pryor JT, Ravussin E, et al. Leptin mediates the increase inblood pressure associated with obesity. Cell 2014;159:1404–141641. Loh K, Fukushima A, Zhang X, et al. Elevated hypothalamic TCPTP inobesity contributes to cellular leptin resistance. Cell Metab 2011;14:684–69942. Garrison RJ, Kannel WB, Stokes J III, Castelli WP. Incidence and precursors ofhypertension in young adults: the Framingham Offspring study. Prev Med 1987;16:235–25143. Robinson LE, Holt TA, Rees K, Randeva HS, O’Hare JP. Effects of exenatide andliraglutide on heart rate, blood pressure and body weight: systematic review andmeta-analysis. BMJ Open 2013;3:e000198644. Inchiosa MA. Experience (mostly negative) with the use of sympathomimeticagents for weight loss. J Obes 2011;2011:pii:76458445. Apovian CM, Aronne LJ, Bessesen DH, et al.; Endocrine Society. Pharma-cological management of obesity: an Endocrine Society clinical practice guideline.J Clin Endocrinol Metab 2015;100:342–36246. Mark EJ, Patalas ED, Chang HT, Evans RJ, Kessler SC. Fatal pulmonaryhypertension associated with short-term use of fenfluramine and phentermine.N Engl J Med 1997;337:602–60647. Food and Drug Administration; Department of Health and Human Services.Determination that PONDIMIN (fenfluramine hydrochloride) tablets, 20 milligramsand 60 milligrams, and PONDEREX (fenfluramine hydrochloride) capsules, 20milligrams were withdrawn from sale for reasons of safety or effectiveness.Washington, DC, U.S. Govt. Printing Office, 2015, p. 58490–58491 (FDA-2015-N-3389)48. Jordan J, Astrup A, Engeli S, Narkiewicz K, Day WW, Finer N. Cardiovasculareffects of phentermine and topiramate: a new drug combination for the treatmentof obesity. J Hypertens 2014;32:1178–118849. Davies MJ, Bergenstal R, Bode B, et al.; NN8022-1922 Study Group. Efficacyof liraglutide for weight loss among patients with type 2 diabetes: the SCALEdiabetes randomized clinical trial. JAMA 2015;314:687–69950. Hansen G, Jelsing J, Vrang N. Effects of liraglutide and sibutramine on foodintake, palatability, body weight and glucose tolerance in the gubra DIO-rats. ActaPharmacol Sin 2012;33:194–20051. Hodge RJ, Paulik MA, Walker A, et al. Weight and glucose reduction observedwith a combination of nutritional agents in rodent models does not translate tohumans in a randomized clinical trial with healthy volunteers and subjects withtype 2 diabetes. PLoS One 2016;11:e015315152. Billes SK, Sinnayah P, Cowley MA. Naltrexone/bupropion for obesity: aninvestigational combination pharmacotherapy for weight loss. Pharmacol Res2014;84:1–1153. Heppner KM, Marks S, Holland J, et al. Contribution of brown adipose tissueactivity to the control of energy balance by GLP-1 receptor signalling in mice.Diabetologia 2015;58:2124–213254. Beiroa D, Imbernon M, Gallego R, et al. GLP-1 agonism stimulates brownadipose tissue thermogenesis and browning through hypothalamic AMPK. Di-abetes 2014;63:3346–335855. Baumann MH, Ayestas MA, Dersch CM, Brockington A, Rice KC, RothmanRB. Effects of phentermine and fenfluramine on extracellular dopamine andserotonin in rat nucleus accumbens: therapeutic implications. Synapse 2000;36:102–11356. Roth JD, Rowland NE. Efficacy of administration of dexfenflura-mine and phentermine, alone and in combination, on ingestive behaviorand body weight in rats. Psychopharmacology (Berl) 1998;137:99–10657. Roth JD, Trevaskis JL, Wilson J, et al. Antiobesity effects of the beta-cellhormone amylin in combination with phentermine or sibutramine in diet-inducedobese rats. Int J Obes 2008;32:1201–121058. Erreger K, Davis AR, Poe AM, Greig NH, Stanwood GD, Galli A. Exendin-4decreases amphetamine-induced locomotor activity. Physiol Behav 2012;106:574–57859. Sun F, Wu S, Guo S, et al. Impact of GLP-1 receptor agonists on blood pressure,heart rate and hypertension among patients with type 2 diabetes: a systematic reviewand network meta-analysis. Diabetes Res Clin Pract 2015;110:26–37

694 Effects of Liraglutide and Phentermine in Mice Diabetes Volume 68, April 2019

60. Kumarathurai P, Anholm C, Larsen BS, et al. Effects of liraglutide on heartrate and heart rate variability: a randomized, double-blind, placebo-controlledcrossover study. Diabetes Care 2017;40:117–12461. Gomez-Peralta F, Abreu C, Castro JC, et al. An association between lir-aglutide treatment and reduction in excessive daytime sleepiness in obesesubjects with type 2 diabetes. BMC Endocr Disord 2015;15:78

62. Vaz M, Jennings G, Turner A, Cox H, Lambert G, Esler M. Regionalsympathetic nervous activity and oxygen consumption in obese normotensivehuman subjects. Circulation 1997;96:3423–342963. Blackman A, Foster GD, Zammit G, et al. Effect of liraglutide 3.0 mg inindividuals with obesity and moderate or severe obstructive sleep apnea: theSCALE Sleep Apnea randomized clinical trial. Int J Obes 2016;40:1310–1319

diabetes.diabetesjournals.org Simonds and Associates 695