Effects of psilocybin on body weight, body composition, and metabolites in male and female mice

Abstract

There is growing interest in the therapeutic potential of psilocybin for the treatment of a wide variety of medical problems, and even for the promotion of wellbeing among healthy individuals. Interestingly, among the many proposed indications, both obesity and anorexia nervosa (AN) have been discussed. However, the effect of psilocybin on appetitive behavior and metabolism is not well known. Here, we report the effects of psilocybin on body weight, intake and output, body composition, and metabolic function among lean male and female wild-type mice. In the days immediately following treatment, both male and female mice receiving a single intraperitoneal dose of psilocybin were consistently heavier than saline controls, with no effect of psilocybin on intake or output. Co-administration of the 5-HT2A/2C receptor antagonist ketanserin had no effect on this outcome. Body composition analysis revealed that psilocybin significantly increased lean and water mass among males, with a similar trend among females. A metabolic panel revealed increased creatine kinase (CK), aspartate aminotransferase (AST), and chloride among male and female psilocybin treated mice. Together, these findings begin to investigate the potential mechanisms of psilocybin’s effects on body weight and metabolic measures. Such understanding will be critical for the safe, efficacious, and well-informed use of psilocybin in clinical and non-clinical settings.

Introduction

The therapeutic potential of psilocybin and similar “psychedelic” compounds has recently garnered significant attention. Though most work to date has focused on psilocybin’s utility for the treatment of major depression and substance use disorders, there is interest in exploring benefits for a wide range of medical conditions, including both obesity and anorexia nervosa (AN).¹ Regarding the use of psilocybin for treatment of AN, the first known study was a 1959 case report describing a female patient who experienced immediate and lasting improvement in anorexia symptoms following two doses of psilocybin 4 days apart.² More recently, a study of 10 patients with AN by Peck et al. found that a single dose of psilocybin 25 mg is safe and may reduce anxiety symptoms as well as eating disorder psychopathology.³ Similarly, a survey by Spriggs et al. of 50 individuals with an eating disorder diagnosis described significant improvements in depression and wellbeing scores after a psychedelic experience.⁴ Interestingly, in addition to this early evidence of potential benefit for the treatment of anorexia, others have proposed that psilocybin may be useful for the treatment of obesity.⁵ In support of this, Simonsson et al. used national survey data from the United States and found that individuals who report any lifetime use of a classic psychedelic are significantly less likely to be overweight or obese but no more or less likely to be underweight.⁶

Psilocybin is a pro-drug that is dephosphorylated by alkaline phosphatase and converted into the pharmacologically active metabolite psilocin.⁵ Mechanistically, it is best known as an agonist of 5-HT2A and 5-HT2C receptors, though more complicated and nuanced mechanisms have been proposed. An extensive prior literature has demonstrated that activation of central 5-HT2C receptors suppresses food intake while central 5-HT2A receptors also appear to modulate food intake and body weight. ^{7 8 9} Others, though, have proposed that benefits of treating anorexia with psychedelics may result from the drugs’ effects on brain circuits regulating cognitive flexibility and related psychological functions, rather than through direct action on metabolism or appetitive behavior.¹⁰

Preclinical studies of psilocybin are still limited. Only a few have assessed effects of psilocybin on body weight, appetitive behavior, or metabolism, and results are conflicting.¹¹ Huang et al. found that psilocybin at single doses of 0.1 and 5.0 mg/kg produced significant weight reduction in male Sprague-Dawley rats on a high-calorie diet, reduced consumption of the high calorie diet, and decreased central adiposity.¹² Conversely, Fadahunsi et al. showed a single dose of psilocybin 3 mg/kg altered the prefrontal cortex transcriptome, but had no long-lasting effects on food intake or body weight in diet-induced or genetic mouse models of obesity.⁵ The same study also found that subchronic “microdosing” had no effect on food intake or body weight and did not modulate outcomes when combined with other weight loss interventions. Higgins et al. found that “microdosing” did not alter body weight, but increased motivation to obtain a food reward in a progressive ratio task in food restricted male Sprague-Dawley rats.¹³ Using rats trained to obtain a food reward on a fixed ratio schedule, Rech et al. found that psilocybin resulted in a transient “pause” in conditioned responses.¹⁴

Considering the rapidly growing interest in psychedelic medicine – including specifically for the treatment of obesity and eating disorders – these mixed findings from a limited preclinical literature highlight a need for additional studies to clarify the effects of psychedelics on body weight and appetitive behaviors. Here, we report the effects of psilocybin on body weight, intake and output, body composition, and serum metabolites in lean male and female wild type C57BL/6J mice. We also directly investigated the role of 5-HT2A and 5-HT2C receptor by combining psilocybin with a 5-HT2A and 5-HT2C receptor antagonist ketanserin. We found that psilocybin treatment increased body weight among both male and female mice in a manner that may be independent of 5-HT2A and 5-HT2C receptors. Body composition analysis revealed an increase in water mass that correlated closely with changes in body weight. A metabolic panel to assess organ function and electrolytes known to influence water balance revealed increased levels of creatine kinase, aspartate aminotransferase (AST), and chloride following psilocybin treatment.

Methods

Animals

Male and female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were randomly assigned to experimental groups. All mice were 7–9 weeks old at the beginning of each experiment with no differences in body weights across groups at the beginning of each experiment (Fig. S1). Mice were group housed by sex with 4–5 mice per cage in a temperature and humidity controlled room. Mice were maintained on a 12 h:12 h light-dark cycle with lights on at 6 am and lights off at 6 pm and provided with ad libitum access to water and standard chow diet (Chow; Envigo 2018, 18% kcal from fat, 24% kcal from protein, 58% kcal from carbohydrates). A total of 85 male and 40 female mice were used in this study. Group sizes within individual experiments ranged from 5 to 21 mice and are noted in the Figure Legends. All mice were weighed daily at the beginning of the light cycle. At the completion of each experiment, mice were killed by rapid decapitation. All protocols were approved by the Animal Care and Use Committee of the Johns Hopkins University School of Medicine.

Drugs and injections

Psilocybin (#7437–001) was obtained from the National Institute on Drug Abuse Drug Supply Program. Ketanserin was obtained from MilliporeSigma.

In Experiments 1 and 3, mice received a single intraperitoneal injection of either sterile saline (100 uL) or psilocybin (1 mg/kg in a total volume of 100 uL sterile saline). To confirm drug delivery, 15 minutes after injection, the head twitch response was measured during a 10-minute monitoring period (Fig. 1a & c). During this monitoring period, mice were briefly moved to clean cages and individually video recorded before being returned to the home cage. Their activity was then scored by a trained experimenter for the total number of head twitches.¹⁵

Fig. 1.

(a & c) Psilocybin-treated male and female mice had significantly more head twitch responses during the 10-minute monitoring period. ****P < 0.0001. (b & d) Psilocybin resulted in an acute increase in body weight among both male and female mice. Male (Saline n=10, Psilocybin n=10); Female (Saline n=10, Psilocybin n=10). *P < 0.05, **P < 0.01. (e & g) Male and female mice treated with saline and psilocybin had significantly more head twitch responses during the 10-minute monitoring period. Ketanserin blocked the effects of psilocybin on the head twitch response. ****P < 0.0001. (f & h) Psilocybin resulted in an acute increase in body weight among both male and female mice and the 5-HT_2A/2C antagonist ketanserin had no significant effect. Male (Saline/Saline n=21, Ketanserin/Saline n=13, Saline/Psilocybin n=13, Ketanserin/Psilocybin n=12); Female (Saline/Saline n=5, Ketanserin/Saline n=5, Saline/Psilocybin n=5, Ketanserin/Psilocybin n=5). *P < 0.05, ****P < 0.0001 main effect of psilocybin. Data represent mean ± SEM.

In Experiment 2, mice first received an intraperitoneal injection of either sterile saline (100 uL) or ketanserin (2 mg/kg in a total volume of 100 uL sterile saline). After 30 minutes, mice received a second intraperitoneal injection of either sterile saline (100 uL) or psilocybin (1 mg/kg in a total volume of 100 uL sterile saline). To confirm drug delivery, 15 minutes after injection, the head twitch response was measured during a 10-minute monitoring period (Fig. 1e & g).

Food and water intake

Five days after treatment, a subset of mice was moved to clean, divided cages with no bedding. In the divided cages, mice were co-housed but separated by clear, porous barriers for a 24-hour period. Water bottles and food in hoppers were weighed prior to and following the 24-hour period to measure food and water intake. Food spillage was collected, dried and weighed, and accounted for in the food intake measurement. As a measure of output, fecal boli were counted after the 24-hour period.

Tissue collection

Retro-orbital blood was collected under isoflurane anesthesia into EDTA-coated tubes (except as noted below for serum chemistry panel) and mice were euthanized by decapitation. Blood was centrifuged at 4 °C and plasma collected and stored at −80 °C for later analysis as described below. In experiment 3, interscapular brown adipose tissue and posterior subcutaneous white adipose tissue was dissected as previously described then immediately weighed.¹⁶ In the same experiment, whole carcasses were then stored at −20 °C for body composition analysis as described below.

Body composition

Body composition analysis was conducted 6 days after treatment to correspond with the final body weight measurement and metabolic analyses. Prior to body composition analysis, mouse carcasses were weighed. Lean, adipose, total water, and free water amounts were determined using EchoMRI-100H analysis (EchoMRI, Houston, TX) according to manufacturer protocols.

Plasma Leptin

Plasma leptin was measured in plasma samples collected when mice were euthanized 6 days following drug treatment. An enzyme-linked immunosorbent assay (ELISA) (Millipore, Burlington, MA) was used according to manufacturer protocols. Samples were measured in duplicate and the assay was read on a SpectraMax Microplate reader (Molecular Devices) at 450 nm. The intra-assay and inter-assay CVs were 1.1% and 3.0%, respectively.

Serum chemistry panel

At the end of Experiment 3, retro-orbital blood was collected, and serum was isolated using standard serum separator tubes (Zhejiang Gongdong Medical Technology Co., Zhejiang. China). Chloride, aspartate aminotransferase (AST), bicarbonate, creatine kinase, alanine transaminase (ALT), bilirubin, sodium, cholesterol, potassium, alkaline phosphatase (ALP), glucose, creatinine, phosphorus, blood urea nitrogen (BUN), albumin, protein, calcium, and globulin were all measured as part of a serum chemistry panel performed within 24 hours of collection at IDEXX BioAnalytics, (North Grafton, MA). Chemistry assay was performed on Beckman Coulter analyzers and were validated for serum matrix according to manufacturer protocols.

Statistical analysis

All statistical analyses were completed using Prism 9 (GraphPad, Boston, MA). Data are expressed as mean ± standard error of the mean (SEM). For body weight analyses, differences between groups were assessed by mixed model ANOVA with ‘time,’ ‘sex,’ and ‘treatment’ as factors. Otherwise, differences between groups were assessed by t-test or factorial ANOVA with ‘sex,’ and ‘treatment’ as factors. Correlations were assessed by Pearson correlation coefficient. For all statistical tests, P < 0.05 was considered significant.

Results

In lean, young adult C57BL/6J male mice, a single intraperitoneal dose of psilocybin (1 mg/kg) resulted in an acute increase in body weight over the course of six days post injection (main effect of time P < 0.01; main effect of psilocybin P < 0.05) (Fig. 1b). Psilocybin resulted in a similar increase in body weight among lean, young adult C57BL/6J female mice (main effect of time P < 0.01; main effect of psilocybin P < 0.05) (Fig. 1d). While all mice gained weight post-treatment, female mice gained weight more quickly (time*sex P < 0.01). There was no significant sex by drug interaction.

When monitored for 24 hours, there was no significant effect of psilocybin on food intake, water intake, or fecal boli output among male (Fig. S2a, c, and e) or female (Fig. S2b, d, and f) mice.

To investigate the role of 5-HT_2A and 5-HT_2C receptors in mediating psilocybin’s effects, a separate cohort of male and female mice were pre-treated with the 5-HT_2A and 5-HT_2C receptor antagonist ketanserin (2 mg/kg, i.p.) prior to treatment with psilocybin. Consistent with the prior cohort, a single intraperitoneal dose of psilocybin (1 mg/kg) resulted in an acute increase in body weight over the course of six days post injection among male and female mice (main effect of time P < 0.01; main effect of psilocybin P < 0.01) (Fig. 1f and e). While all mice gained weight post-treatment, female mice gained weight more quickly (time*sex P < 0.01). There was no significant sex by drug interaction. There was also no effect of ketanserin on body weight among male or female mice. When monitored for 24 hours after injection, there were no significant effects of ketanserin, psilocybin, or sex on food intake, water intake, or fecal boli output among male (Fig. S3a, c, and e) or female (Fig. S3b, d, and f) mice.

Among male mice, body composition analysis conducted 6 days after treatment demonstrated that psilocybin was associated with significantly increased lean mass excluding water (P < 0.05) as well as an increase in total water (P < 0.05) but not free water (Fig. 2a–c). Among female mice, psilocybin had no effect on lean or water mass, but there was a non-significant trend towards an increase in total water (P = 0.05) (Fig. 2e–g). Psilocybin had no effect on fat mass in either male (Fig. 2d) or female (Fig. 2h) mice. Pearson correlation coefficient revealed a significant positive correlation between body weight and lean mass among saline treated mice (R² = 0.6879, P = 0.005); this correlation was strengthened among psilocybin treated mice (R² = 0.8898, P < 0.0001) (Fig. 3a). Pearson correlation coefficient also revealed a significant positive correlation between body weight and total water among saline treated mice (R² = 0.6695, P = 0.0006); this correlation was strengthened among psilocybin treated mice (R² = 0.8878, P < 0.0001) (Fig. 3b). When assessing the relationship between body weight and fat mass, there was a significant positive correlation among saline treated mice (R² = 0.7461, P = 0.0001), that weakened among psilocybin treated mice (R² = 0.3714, P = 0.027) (Fig. 3c).

Fig. 2.

(a-d) In male mice, psilocybin increased lean mass, which was associated with an increase in water mass. Psilocybin had no effect on fat mass or free water. Male (Saline n=13, Psilocybin n=13). *P < 0.05. (e-h) In female mice, psilocybin resulted in a trend towards increased water mass. Psilocybin had no effect on fat mass or free water. Female (Saline n=10, Psilocybin n=10). Data represent mean ± SEM.

Fig. 3.

(a & b) Body weight was significantly correlated with lean mass and water mass in both saline and psilocybin treated groups, though the strength of the correlations were stronger among psilocybin treated mice. (c) Body weight was significantly correlated with fat mass among saline and psilocybin treated mice, though the strength of the correlation was weaker among psilocybin. Male (Saline n=13, Psilocybin n=13).

The observation that psilocybin acutely increases body weight without affecting adiposity was further supported by direct measurement of brown adipose tissue and subcutaneous white adipose tissue in male (Fig. 4a–b) and female mice (Fig. 4d–e). Measurement of circulating leptin was used as an indirect indicator of total body adiposity.¹⁷ Psilocybin had no effect on plasma leptin in male (Fig. 4c) or female (Fig. 4f) mice.

Fig. 4.

(a-f) Among male and female mice, psilocybin had no effect on brown adipose tissue weight or subcutaneous white adipose tissue weight. Male (Saline n=15, Psilocybin n=15); Female (Saline n=5, Psilocybin n=5). Psilocybin had no effect on plasma leptin in male or female mice. Male (Saline n=10, Psilocybin n=11); Female (Saline n=7, Psilocybin n=10). Data represent mean ± SEM.

A comprehensive serum chemistry panel to assess organ function and electrolytes known to influence water balance found that psilocybin treatment was associated with increased creatine kinase (P < 0.05), aspartate aminotransferase (AST) (P < 0.05), and chloride (P < 0.05) among male and female mice. Psilocybin treatment also significantly decreased bicarbonate among male mice (P < 0.05) and significantly decreased total bilirubin among female mice (P < 0.05) (Table 1–2 and Fig. 5a–h). There were also non-significant trends towards increased alanine transaminase (ALT) among male mice (P = 0.09) (Table 1) and decreased potassium among female mice (P = 0.05) (Table 2).

Table 1.

Male Mice Serum Chemistry (Saline n=15, Psilocybin n=15).

Metabolite	Saline Average	PSBN Average	P-value
Chloride (mmol/L)	106.8 (±0.86)	110.7 (±0.85)	P = 0.0032, **
AST (U/L)	110.6 (±9.93)	155.40 (±10.86)	P = 0.0050, **
Bicarbonate TCO2 (mmol/L)	17.07 (±0.45)	15.27 (±0.53)	P = 0.015, *
Creatine Kinase (U/L)	1121 (±135.1)	1587 (±150.6)	P = 0.029, *
ALT (U/L)	42.13 (±5.91)	57.53 (±6.61)	P = 0.093, ns
Total Bilirubin (mg/dL)	0.31 (±0.02)	0.27 (±0.02)	P = 0.14, ns
Conjugated Bilirubin (mg/dL)	0.01 (±0.009)	0	P = 0.15, ns
Sodium (mmol/L)	142.3 (±0.99)	144 (±0.64)	P = 0.16, ns
Cholesterol (mg/dL)	103.8 (±3.83)	97.47 (±3.7)	P = 0.24, ns
Potassium (mmol/L)	7.35 (±0.2)	7.76 (±0.29)	P = 0.27, ns
Unconjugated Bilirubin (mg/dL)	0.30 (±0.02)	0.27 (±0.02)	P = 0.33, ns
ALP (U/L)	84.33 (±2.15)	81.2 (±2.46)	P = 0.35, ns
Glucose (mg/dL)	182 (±5.03)	188.6 (±5.23)	P = 0.37, ns
Creatinine (mg/dL)	0.13 (±0.02)	0.15 (±0.02)	P = 0.42, ns
NA/K Ratio	19.38 (±0.74)	18.71 (±0.7)	P = 0.52, ns
Phosphorus (mg/dL)	7.99 (±0.17)	7.88 (±0.14)	P = 0.61, ns
BUN/Creatinine Ratio	201.5 (±19.81)	192.9 (±21.79)	P = 0.77, ns
ALB/GLOB ratio	1.59 (±0.02)	1.60 (±0.01)	P = 0.79, ns
Albumin (g/dL)	2.95 (±0.04)	2.96 (±0.04)	P = 0.80, ns
BUN (mg/dL)	26.27 (±0.83)	26.53 (±1.12)	P = 0.85, ns
Total Protein (g/dL)	4.8 (±0.05)	4.81 (±0.06)	P = 0.87, ns
Calcium (mg/dL)	8.59 (±0.04)	8.6 (±0.08)	P = 0.88, ns
Globulin (g/dL)	1.85 (±0.02)	1.85 (±0.03)	P > 0.99, ns

Table 2.

Female Mice Serum Chemistry (Saline n=5, Psilocybin n=5).

Metabolite	Saline Average	PSBN Average	P-value
AST (U/L)	69.8 (±6.45)	89.4 (±2.16)	P = 0.0204, *
Chloride (mmol/L)	108.6 (±0.75)	110.8 (±0.49)	P = 0.0393, *
Creatine Kinase (U/L)	323.4 (±52.32)	533.4 (±53.19)	P = 0.0227, *
Total Bilirubin (mg/dL)	0.24 (±0.025)	0.16 (±0.025)	P = 0.0497, *
Potassium (mmol/L)	7.2 (±0.205)	6.32 (±0.33)	P = 0.054, ns
Unconjugated Bilirubin (mg/dL)	0.22 (±0.02)	0.16 (±0.025)	P = 0.094, ns
Phosphorus (mg/dL)	8.28 (±0.42)	7.72 (±0.22)	P = 0.15, ns
Creatinine (mg/dL)	0.08 (±0.02)	0.04 (±0.025)	P = 0.24, ns
BUN (mg/dL)	23.6 (±0.87)	25.0 (±0.84)	P = 0.28, ns
Conjugated Bilirubin (mg/dL)	0.02 (±0.02)	0	P = 0.35, ns
Bicarbonate TCO2 (mmol/L)	15.4 (±1.12)	14 (±0.89)	P = 0.36, ns
Sodium (mmol/L)	145.0 (±1.45)	146.2 (±0.58)	P = 0.46, ns
Cholesterol (mg/dL)	92 (±3.77)	90 (±2.74)	P = 0.68, ns
Calcium (mg/dL)	8.34 (±0.093)	8.38 (± 0.037)	P = 0.70, ns
Albumin (g/dL)	2.96 (±0.051)	2.98 (±0.058)	P = 0.80, ns
ALP (U/L)	129.6 (±7.88)	127.2 (±6.54)	P = 0.82, ns
BUN/Creatinine Ratio	242.5 (±7.5)	245.0 (±15)	P = 0.87, ns
ALT (U/L)	22.8 (±1.16)	22.6 (±1.17)	P = 0.91, ns
Glucose (mg/dL)	175.0 (±9.88)	173.8 (±9.58)	P = 0.93, ns
Globulin (g/dL)	1.56 (±0.025)	1.56 (±0.025)	P > 0.99, ns
Total Protein (g/dL)	4.52 (±0.074)	4.3 (±0.074)	P > 0.99, ns
ALB/GLOB ratio	1.9	1.9	P = 1

Fig. 5.

(a-h) Among male and female mice, psilocybin resulted in increased creatine kinase, AST, and chloride. Psilocybin also resulted in decreased bicarbonate in male mice and decreased total bilirubin in female mice. Male (Saline n=15, Psilocybin n=15); Female (Saline n=5, Psilocybin n=5). *P < 0.05, **P < 0.01. Data represent mean ± SEM.

Discussion

In this study, we examined the effects of a single dose of psilocybin on body weight, body composition and food intake in lean male and female wild type mice. In addition, we assessed the effect of ketanserin, a 5HT-2A and −2C antagonist on psilocybin-associated outcomes. We found that administration of a single intraperitoneal dose of psilocybin to wild type, young adult mice produced a subtle increase in body weight that persisted for at least 6 days post-injection. Based on body composition analysis, this change in body weight was associated with increased lean mass, which was contributed to by an increase in water weight. In a subset of 60 mice, we found no effect of psilocybin or ketanserin on food intake, water intake, or fecal boli output when measured over a 24-hour period. However, the relatively small sample size, the limited time of measurement, and potential stress effects of temporarily moving mice from social housing to divided cages may limit generalizability of this data. While others have found that a single dose of psilocybin had no effect on intake across several mouse strains and dietary conditions⁵, additional studies are needed. When mice were pretreated with a single dose of the 5-HT_2A and 5-HT_2C receptor antagonist ketanserin immediately prior to psilocybin treatment, the effects of psilocybin on body weight were still evident suggesting that the increased weight gain was not dependent on full availability of the 5-HT_2A or 5-HT_2C receptors.

In this study, both male and female mice were tested. We observed subtle sex differences in some outcomes, including a transient, non-statistically significant decrease in body weight among male mice that did not receive psilocybin (Fig. 1f). This may have been caused by a stress response to injections, however, this was not observed in females and the overall impact of psilocybin on body weight remained similar in males and females. To further explore the impact of psilocybin on systems that can affect weight and metabolism, we also assessed the effects of psilocybin on serum indicators of major organ function and electrolytes known to influence water balance; this resulted in several novel observations.

To date, most work related to psilocybin has focused on its psychedelic effects and potential use as a therapeutic for a variety of neuropsychiatric disorders. Relatively few studies have assessed effects on body weight, metabolism, or other major organ systems. What limited work has been done appears to have mixed results. A study by Huang et al. demonstrated potential weight-loss properties of psilocybin in obese male rats fed a high-calorie diet when rats treated with daily intraperitoneal injections of psilocybin at doses of 0.1 mg/kg or 5 mg/kg over 27 consecutive weekdays.¹² The contrast between this study and ours may suggest that a single dose of psilocybin has different effects compared to chronic, repeated administration. The contrast, alternatively, could represent species-specific effects, or perhaps weight-dependent or metabolic state-dependent effects. It is also possible that the low (0.1 mg/kg) and high (5 mg/kg) individual doses used by Huang et al. may result in different effects than the intermediate dose used in our study, consistent with other data suggesting that, at least for some outcomes, psilocybin has a non-linear dose response curve.¹¹ Another study done by Fadahunsi et al. showed a single intraperitoneal dose of psilocybin 3 mg/kg broadly altered expression of genes associated with neuronal plasticity in the prefrontal cortex but had no acute or long-lasting effects on food intake or body weight in obese mice. This dose of psilocybin did reduce sucrose-preference in lean mice but did not produce any effects to counter binge-like eating behaviors. Fadahunsi et al. also found that sub-chronic microdosing of psilocybin at a dose of 0.3 mg/kg daily for 7 days had no significant effects on body weight and did not enhance GLP-1 or diet-induced weight loss.⁵ Again, these results present a conflicted picture of psilocybin’s effects on body weight and metabolism. The doses and preclinical models used by Fadahunsi et al. differ from those used by Huang and those used in the current study, which may again suggest that psilocybin has dose-dependent, strain-dependent, or metabolic state-dependent effects.

Regarding our specific finding that psilocybin appears to increase body weight which may be related to an increase in water weight, we found no significant difference in water intake. However, our period of measurement was limited to 24 hours. When measuring over a 4-day period, Fadahunsi et al observed a significant increase in both food and water intake following psilocybin administration at a dose of 3 mg/kg.

We found that psilocybin treated mice had elevated levels of creatine kinase (CK) and aspartate aminotransferase (AST) among both males and females, which could indicate muscle injury.¹⁸ Expectedly, the levels of these metabolites differed between males and females.^19,20,21 Among male mice, alanine aminotransferase (ALT) was also elevated. ALT and AST elevation could also suggest liver injury, providing an alternative explanation. ²² However, previous mouse studies have used plasma CK, AST, and ALT activity as indicators of muscle tissue injury.²³ Muscle injury and related consequences following use of psychedelic mushrooms appear rare but have been previously reported. Markus et al. described a case of a 25-year-old male with elevated CK, AST, and ALT after the intake of the mushroom psilocybe cubensis.²⁴ The patient experienced acute renal failure due to rhabdomyolysis. In another case, Mariella et al. describe elevated CK in an individual following ingestion of a massive dose of psilocybin, though this particular finding was confounded by the intramuscular injection of an antipsychotic medication, which can also cause increased CK.²⁵ There are other case studies that show a link between consumption of other, non-psychedelic mushrooms and the development of muscle injury. For example, Bedry et al. reported that between 1992 and 2000, 12 patients, including seven women and five men, were hospitalized due to severe rhabdomyolysis after eating the wild mushroom Tricholoma Equestre.²⁶ Elevated CK levels were also observed when mice were exposed to T. equestre.²⁶

In the current study, psilocybin also elevated levels of chloride in both male and female mice. Elevated chloride has been linked to loss of bicarbonate through the renal tract ²⁷, which was observed in psilocybin treated male mice. This, along with the reduction in bilirubin among psilocybin treated female mice and the other observed trends towards changes in ALT and potassium, further emphasizes that psilocybin may cause homeostatic disruptions that are currently poorly understood. Collectively, these results strongly suggest the need for additional investigation of toxicology and mechanisms of action of psilocybin.

As for potential mechanisms, we found that pretreatment with ketanserin did not significantly alter psilocybin’s effects on body weight or body composition in our model, suggesting that the observed effects of psilocybin may be at least partially independent of 5-HT_2A and 5-HT_2C receptors. Clearly, there is strong evidence that serotonin receptor agonism, and 5-HT_2A receptor agonism, is essential in facilitating some, but not all the effects of psilocybin.¹¹ Findings such as these highlight the importance of exploring other potential mechanisms of action as most work to date has focused on psilocybin’s agonism of 5-HT_2A receptors in the brain. This observation also highlights the likelihood that different effects of the drugs may be due to different mechanisms, and some of those mechanisms may be dose or context dependent.

Though the current study further supports the feasibility of using translational models to study psychedelics and adds to an emerging body of literature suggesting that psilocybin impacts body weight and alters physiological systems outside the CNS, several limitations should be noted. First, findings like these, which have not been previously reported, should be interpreted cautiously until additional work can be done to both replicate the observations and determine the extent to which they are generalizable beyond the specific model used here. This study utilized lean male and female young adult, wild type C57BL/6J mice, but future work should evaluate similar outcomes in other mouse lines, other model organisms, and human populations. If these findings are species-specific, there will be a substantial need for more studies of psilocybin’s unique effects on humans compared to translational models. Regarding our assessment of potential sex differences, more work is needed to investigate consequences of the relatively subtle differences observed in this study. Additionally, it will be important to evaluate potential effects of age, repeated dosing, and long-term consequences of the drug. It is also possible that the observed effects of psilocybin are context dependent. That is, psilocybin may have different effects on lean versus obese versus underweight subjects. Providing some support for this, others have found evidence that psilocybin may have efficacy in treating both obesity and anorexia.^{1 2 3 4 6 28} Regarding the use of ketanserin, the 2 mg/kg dose has been used in other studies of C57BL/6J mice.²⁹ It is likely that ketanserin at this dose does not fully block psilocybin’s action at 5-HT_2A and 5-HT_2C receptors, and psilocybin is also clearly known to bind other serotonin receptor types. Regardless, the finding that ketanserin fails to fully prevent psilocybin’s effects underlines the importance of additional mechanistic work. Finally, regarding our observation that psilocybin selectively increases water weight in lean mice, data suggest that this is not driven by differences in intake, sodium homeostasis, or frank hepatic or renal dysfunction, but the underlying mechanism remains otherwise unclear. It is possible that the observed changes in CK, AST/ALT, chloride, and bicarbonate could contribute, though this will require further investigation.

In summary, our study indicates that a single dose of psilocybin may result in acute, relatively subtle weight-gain that is associated with an increase in total body water by a mechanism that may not be fully dependent of 5-HT_2A and 5-HT_2C receptor mediated pathways. These findings are particularly significant when considering psilocybin as a tool for treating eating disorders like anorexia nervosa, where early weight restoration is a key aspect of modern AN treatment and a predictor of long-term recovery.³⁰ At the same time, this effect of psilocybin on body weight may discourage its use as an effective treatment for obesity. Regardless, as interest in the clinical and recreational use of psilocybin rapidly expands, there is a clear need for in-depth understanding of the mechanisms of psilocybin, including drug effects outside the central nervous system, which is an area that has received little attention. Such work may have important consequences for the field of psychedelic medicine and the use of psychedelics among healthy individuals.

Highlights.

• A single dose of psilocybin was associated with increased body weight.

• Co-administration of ketanserin had no effect on this outcome.

• Psilocybin treatment was associated with increased lean and water mass.

• Psilocybin treatment increased CK, AST, and chloride.