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The Journal of Pharmacology and Experimental Therapeutics logoLink to The Journal of Pharmacology and Experimental Therapeutics
. 2024 Oct;391(1):30–38. doi: 10.1124/jpet.124.002188

The Effects of Eating a Traditional High Fat/High Carbohydrate or a Ketogenic Diet on Sensitivity of Female Rats to Morphine

Nina M Beltran 1, Alyssa N Parra 1, Ana Paulina Serrano 1, Jazmin Castillo 1, Isabella M Castro 1, Madeline K Elsey 1, Vanessa Minervini 1, Katherine M Serafine 1,✉
PMCID: PMC11415821  PMID: 39060162

Abstract

Patients diagnosed with obesity are prescribed opioid medications at a higher rate than the general population; however, it is not known if eating a high fat diet might impact individual sensitivity to these medications. To explore the hypothesis that eating a high fat diet increases sensitivity of rats to the effects of morphine, 24 female Sprague-Dawley rats (n = 8/diet) ate either a standard (low fat) laboratory chow (17% kcal from fat), a high fat/low carbohydrate (ketogenic) chow (90.5% kcal from fat), or a traditional high fat/high carbohydrate chow (60% kcal from fat). Morphine-induced antinociception was assessed using a warm water tail withdrawal procedure, during which latency (in seconds) for rats to remove their tail from warm water baths was recorded following saline or morphine (0.32–56 mg/kg, i.p.) injections. Morphine was administered acutely and chronically (involving 18 days of twice-daily injections, increasing in 1/4 log dose increments every 3 days: 3.2–56 mg/kg, i.p., to induce dependence and assess tolerance). The adverse effects of morphine (i.e., tolerance, withdrawal, and changes in body temperature) were assessed throughout the study. Acute morphine induced comparable antinociception in rats eating different diets, and all rats developed tolerance following chronic morphine exposure. Observable withdrawal signs and body temperature were also comparable among rats eating different diets; however, withdrawal-induced weight loss was less severe for rats eating ketogenic chow. These results suggest that dietary manipulation might modulate the severity of withdrawal-related weight loss in ways that could be relevant for patients.

Significant Statement

Eating a high fat/low carbohydrate (ketogenic) or a traditional high fat/high carbohydrate diet did not impact the pain-relieving or adverse effects of opioids (i.e., tolerance or withdrawal) in female rats. However, eating a ketogenic diet may have beneficial effects on opioid withdrawal-related weight loss. Individuals diagnosed with obesity taking opioids for pain-related conditions might therefore consider adopting a ketogenic diet when opioid administration is discontinued to potentially mitigate withdrawal-related weight loss.

Introduction

The misuse of opioid pain-relieving drugs (e.g., morphine, oxycodone, and fentanyl) is a prevalent health concern, with nearly 6 million individuals in the United States meeting the diagnostic criteria for opioid use disorder (https://www.samhsa.gov/data/sites/default/files/reports/rpt42731/2022-nsduh-annual-national-web-110923/2022-nsduh-nnr.htm#:∼:text=Among%20people%20aged%2012%20or%20older%20in%202022%2C%2059.8%20percent,in%20the%20past%20month%3B%208.3). Opioid-related deaths have increased nearly 23 times since 2013, contributing to over 70,000 drug overdose deaths in the United States in 2021 (https://www.cdc.gov/drugoverdose/deaths/opioid-overdose.html). Although opioid pain-relieving drugs can have actions at multiple receptors (Al-Hasani and Bruchas, 2011; Gupta et al., 2021), prescription pain relievers primarily exert their analgesic effects through agonist activity at the μ-opioid receptor, which also mediates their rewarding effects (Al-Hasani and Bruchas, 2011).

Continuous or chronic use of opioids can lead to the development of tolerance and dependence (Morgan and Christie, 2011). For example, with chronic use, tolerance to the pain-relieving effects of these drugs can develop, such that larger doses of drug are needed to achieve pain relief as compared with initial exposure (Morgan and Christie, 2011). Repeated use of opioids can also lead to physical dependence, which can contribute to relapse and overdose, and is evidenced by the presence of withdrawal symptoms following either the discontinuation of treatment or the administration of an opioid receptor antagonist (Kosten and George, 2002). Symptoms of opioid withdrawal in humans include diarrhea, nausea, vomiting, insomnia, and increased heart rate (Diagnostic and Statistical Manual of Mental Disorders [DSM-5], 2013). Opioid withdrawal can be modeled in rats and is characterized by observable behavioral and physiological effects, including (but not limited to) ptosis (upper eyelid droop), teeth chattering, wet dog shakes, paw tremors, diarrhea, and weight loss (Gerak et al., 2019). In addition to tolerance and withdrawal, other opioid-induced adverse effects include changes to thermoregulation (Benyamin et al., 2008; Rawls and Benamar, 2011). For example, small doses of morphine induce hyperthermia and large doses produce hypothermia in rats (Geller et al., 1983). Opioid-induced hyperthermia is induced by activation of the μ-opioid receptor, whereas hypothermia is associated with activation of the κ-opioid receptor (Rawls and Benamar, 2011).

Although opioid misuse continues to be problematic, another prevalent public health concern is obesity. Overconsumption of high fat or high sugar foods can contribute to the development of obesity (https://www.cdc.gov/nchs/products/databriefs/db360.html; Thaker, 2017). Approximately 27% of patients diagnosed with obesity are prescribed opioids for long-term use (https://www.cdc.gov/nchs/products/databriefs/db360.html; Stokes et al., 2020), for conditions such as osteoarthritis, fibromyalgia, and lower back pain (Stokes et al., 2019). There are a variety of current treatments for obesity, including pharmacotherapeutic approaches as well as dietary manipulations (https://www.cdc.gov/nchs/products/databriefs/db360.html; Ruban et al., 2019). One dietary approach that has been increasing in popularity for weight loss is a diet high in fat but very low in carbohydrates, known as a ketogenic diet (Moreno et al., 2014; Batch et al., 2020). To date, it is not known if the effects of opioid drugs like morphine might be impacted by dietary manipulation in the context of eating a traditional high fat/high carbohydrate or ketogenic diet. Understanding this potential impact is particularly critical for patients who identify as women; for whom obesity diagnoses and opioid prescriptions are even more prevalent, as compared with those who identify as men (https://www.cdc.gov/nchs/products/databriefs/db360.html; Goetz et al., 2021).

It has been well established that sensitivity to the effects of other drugs (i.e., psychomotor stimulants) can be modulated by the type and amount of food consumed. For example, rats eating a high fat/high carbohydrate chow are more sensitive than rats eating a low fat chow to the locomotor-stimulating effects of cocaine and methamphetamine (Baladi et al., 2012, 2015; Ramos et al., 2020). However, there are limited reports exploring the impact of dietary manipulation on general nociception (pain sensation) (Ziegler et al., 2005; Ruskin et al., 2013) and the antinociceptive effects of opioids (Nealon et al., 2018; Trinko et al., 2023). To address this gap, the present study tested the hypothesis that female rats eating high fat/high carbohydrate chow would be more sensitive to the antinociceptive and adverse effects of morphine as compared with rats eating a ketogenic or low fat (i.e., standard) laboratory chow.

Materials and Methods

In the present study, 24 female Sprague-Dawley rats (Envigo, Indianapolis, IN) arrived on postnatal day (PND) 20 and were housed individually in an environmentally controlled room under a 12:12 hour light/dark cycle with free access to water and standard (low fat) laboratory chow (Envigo Teklad 7912; 17% kcal from fat). Starting on PND 23–25, rats were randomly assigned (n = 8/group) to either continue to eat the standard chow or were provided a high fat/high carbohydrate chow (Envigo Teklad 06414; 60% kcal from fat) or a ketogenic chow (Envigo Teklad 96355; 90.5% kcal from fat; see Table 1 for nutritional content). All rats had free access to their respective chows for the duration of the study except during the experimental testing procedures outlined below (that did not last longer than 2 hours on a given day). Body weight and food consumption were recorded daily for individual rats during 0800 and 1000 hours throughout the duration of the study, beginning on PND 96 and continuing until 6 days after morphine discontinuation. Experiments were conducted in accordance with the Institutional Animal Care and Use Committee at The University of Texas at El Paso, and in accordance with the 2011 Guide for Care and Use of Laboratory Animals (Institute of Laboratory, Animal Resources on Life Sciences, the National Research Council, and the National Academy of Sciences).

TABLE 1.

Nutritional contents for the dietary conditions broken down by % kcal for carbohydrates (% fiber in g/kg), protein, and fat

Feeding Conditions Carbohydrates Protein Fat
Standard 58% (13.7% g/kg fiber) 25% 17%
High fat/high carbohydrate 21.4% (6.6% g/kg fiber) 18.3% 60.3%
High fat/low carbohydrate (ketogenic) 0.3% (8.8% g/kg fiber) 9.2% 90.5%
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Procedures

Warm Water Tail Withdrawal.

To examine morphine’s effects on thermal pain, rats were tested using a warm water tail withdrawal procedure, during which three water baths (EW-12105-84, Cole-Parmer, Vernon Hills, IL) were maintained at constant temperatures (40°C, 50°C, or 55°C) throughout the experiment. The latency in seconds for rats to remove their tails from water maintained at each temperature was recorded using a stopwatch. Sessions comprised of 15-minute cycles, with an injection of saline given at the start of the first cycle. After each injection, each rat was returned to its home cage for 13 minutes. Next, each rat was positioned within the experimenter’s hands such that 5 cm of the tail was lowered into a water bath. Three water temperatures were tested in a randomized order and separated by 15 seconds. The maximum possible latency was 15 seconds for each water temperature. Saline injections were administered in the first cycle, followed by cumulative doses of morphine in the subsequent cycles (i.e., 0.32, 1, 3.2, 10, 17.8 mg/kg; intraperitoneal), with all injections occurring at the start of each 15-minute cycle. However, the experiment only progressed to the 6th cycle (i.e., rats only received the final cumulative dose of 17.8 mg/kg) if 10 mg/kg morphine was not sufficient at inducing at least an 80% of the maximum possible effect (MPE), determined for individual rats.

Rats were habituated to the procedure with saline injections on PND 97–99 and PND 104–106 (1 week apart). One week later, acute morphine dose–response curves were generated twice (1 week apart; on PND 111–113 and PND 118–120). Before chronic morphine administration, rats were tested with saline injections on PND 187–189, and a final acute morphine dose–response curve was generated 1 week later on PND 194–196. Beginning the day after this final acute morphine dose–response curve was generated (on PND 195–197), rats began the chronic morphine administration protocol. Specifically, rats received twice-daily intraperitoneal injections of morphine at 0800 hours and 1800 hours starting with 3.2 mg/kg and increasing in 1/4 log increments every 3 days up to 56 mg/kg, for a total of 18 days. On the third day of treatment with 56 mg/kg (day 18 of chronic administration on PND 212–214), morphine-induced antinociception dose–response curves were generated to assess the development of tolerance in the warm water tail withdrawal procedure. Starting at 0700 hours, saline was injected during the first cycle followed by cumulative doses of morphine (i.e., 3.2, 10, 32, and 56 mg/kg). During this procedure, rats received a cumulative dose of 56 mg/kg at approximately 0800 hours, and at 1800 hours that same day, rats received their final dose of 56 mg/kg morphine.

Withdrawal.

To examine the effects of morphine discontinuation (i.e., nonprecipitated withdrawal) rats were kept in their home cage with bedding in the same room where warm water tail withdrawal observations previously occurred. Starting on PND 213–215 (the day after the chronic administration protocol and final evening injection of morphine), at 0800 and 1800 hours and continuing for the next 5 days (through PND 217–219), rats received saline instead of morphine (i.e., nonprecipitated withdrawal). Observable withdrawal signs were recorded for each rat beginning 30, 60, and 90 minutes after the injection of saline at 0800 hours. Body weight and vocalization were recorded during handling, and 13 observational signs of withdrawal (ptosis, teeth chattering, tongue protrusion, salivation, lacrimation, chromodacryorrhea, jumping, abdominal writhing, wet dog shake, rearing, paw biting, paw tremor, and diarrhea) were scored as present or absent during four 15-second intervals, separated by 15 seconds. If a particular sign was observed during at least one interval, it was recorded as present for the observation period. Thus, the maximum score for each observation period was 14 (mirroring the methodology used in Gerak et al. [2019]). Signs of withdrawal from the three observation periods were added together and averaged within dietary group. Although observable withdrawal signs were assessed for 5 days following morphine discontinuation, a final body weight was also collected on day 6 following morphine discontinuation (PND 218–220).

Body Temperature.

Changes in body temperature were also measured at the end of each warm water tail withdrawal observation cycle, following saline or morphine injections. Body temperature in degrees Celsius (°C) was recorded using a rectal thermometer (PhysiTemp Instruments, Clifton, NJ) (Minervini et al., 2018; Gerak et al., 2019).

Drugs

Morphine sulfate was purchased from Sigma-Aldrich (St. Louis, MO) and was dissolved in 0.9% saline and injected intraperitoneally at a volume of 1 mg/kg body weight.

Data Analyses

Body weight (in g) and food consumption (in g and kcal) collected throughout the duration of the study (beginning the day before morphine was tested acutely [PND 194–196] to the day after the last of five consecutive withdrawal observations [PND 218–220]) were analyzed using a two-way mixed model ANOVA with diet and day as factors. For each individual rat, warm water tail withdrawal latencies were first converted to a % of the maximum possible effect (MPE; 15 seconds) according to the following formula: [(test latency – control latency)/(15 seconds – control latency)] × 100% and then were averaged for each group (i.e., diet). Across groups, data were analyzed using a two-way mixed model ANOVA with diet and dose as factors. ED50 values were determined for individual subjects using only the linear portion of the antinociception dose–response curves, which included the largest dose for which tail-withdrawal latency remained below 25%, the smallest dose for which tail-withdrawal exceeded 75%, and all doses in between. ED50 values were then analyzed using a two-way mixed model ANOVA with diet and week as factors. Observable withdrawal signs were analyzed using a two-way mixed model ANOVA with day and diet as factors. To examine withdrawal-related weight loss, body weight during the withdrawal procedure was further analyzed as a % change in body weight for rats eating different diets normalized based on body weight examined on the day of the warm water tail withdrawal procedure during the chronic morphine administration protocol using a two-way mixed model ANOVA with diet and day as factors. Change in body temperature, calculated as the difference from each cycle as compared with the first cycle (saline) was analyzed using a two-way mixed model ANOVA with diet and cycle as factors. Individual group comparisons were made using Tukey’s multiple comparisons when appropriate, with statistical significance set at P < 0.05.

Results

Body Weight and Food Consumption

Average (±S.E.M.) body weight in g was assessed daily for rats eating different diets throughout the entire experiment (i.e., beginning the day before acute morphine dose–response curves were generated, continuing throughout the chronic morphine administration protocol, and for 6 days following morphine discontinuation) (Fig. 1). A two-way mixed model ANOVA revealed a significant main effect of day (F [25,525] = 15.22; P < 0.0001), and a significant day by diet interaction effect (F [50,525] = 5.548; P < 0.0001); however, no main effect of diet was revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that 2 and 3 days after morphine discontinuation (days 20 and 21 following the 18-day chronic administration protocol), rats lost a significant amount of body weight regardless of dietary group (P < 0.0001). That is, there were no differences in body weight during morphine administration protocol for rats eating different diets, and rats in all groups lost a significant amount of weight when morphine was discontinued (Fig. 1).

Fig. 1.

Fig. 1.

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Mean (±S.E.M.) body weight (g) collected during the morphine administration protocol and discontinuation from morphine (withdrawal) in female rats eating standard chow (closed symbols), high fat/high carbohydrate chow (open symbols), and ketogenic chow (shaded symbols). Vertical axis: body weight in g. Horizontal axis: day in study. Rats were tested acutely with morphine the day before receiving twice-daily injections of morphine. Rats began twice daily injections on day 1, continuing until day 18 of the study (increasing in quarter log doses every 3 days). On day 18, tolerance was assessed using a warm water tail withdrawal procedure. Morphine administration was discontinued on day 19, and observable withdrawal signs were collected for 5 days (until day 23 of the study). A final body weight was recorded on day 24 of the study. Two and three days after morphine discontinuation (day 20 and 21 following the 18-day chronic administration protocol), rats in all groups lost a significant amount of body weight (P < 0.0001).

Average (±S.E.M.) food consumption in g and kcal was assessed daily throughout the study. For food consumption in g, a two-way mixed model ANOVA revealed a significant main effect of day (F [25,525] = 4.515; P < 0.0001), a significant main effect of diet (F [2,21] = 29.23; P < 0.0001), and a significant day by diet interaction effect (F [50,525] = 1.731; P = 0.0020). Tukey’s multiple comparisons tests revealed that rats eating standard chow consumed more g of food daily as compared with rats eating high fat chow throughout the study (P < 0.0001). Next, g consumed were converted to kcal consumed. For food consumption in kcal, a two-way mixed model ANOVA revealed a significant main effect of day (F [25,525] = 2.57; P < 0.0001), a significant main effect of diet (F [2,21] = 42.62; P < 0.0001); however, no significant day by diet interaction effects were revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that rats eating ketogenic chow consumed more kcal on average than rats eating standard and high fat chow (P < 0.0001). That is, rats eating standard chow consumed more g daily throughout the study as compared with rats eating high fat and ketogenic chow (Fig. 2). In addition, rats eating ketogenic chow consumed more kcal daily throughout the study as compared with rats eating standard and high fat chow (Fig. 2).

Fig. 2.

Fig. 2.

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Mean (±S.E.M.) food consumed (g and kcal) collected during the morphine administration protocol and discontinuation from morphine (withdrawal) in female rats eating standard chow (closed symbols), high fat/high carbohydrate chow (open symbols), and ketogenic chow (shaded symbols). Vertical axis: food consumption in g and kcal. Horizontal axis: day in study. Throughout the study, rats eating standard chow consumed significantly more food in g than rats eating high fat/high carbohydrate chow (P < 0.0001), and rats eating ketogenic chow consumed significantly more food in kcal than rats eating standard and high fat chow (P < 0.0001).

Warm Water Tail Withdrawal.

Average (±S.E.M.) withdrawal latency for rats to remove their tails baths set to 40°C, 50°C, and 55°C was assessed. Rats were initially tested with saline only during PND 97–99 and PND 104–106, and initial acute morphine tests occurred on PND 97–99 and PND 104–106. The % MPE for these first acute morphine dose–response curves were not statistically different from the % MPE for acute morphine tests obtained later (during PND 194–196) (P > 0.05). Therefore, statistical comparisons between acute and chronic morphine tests included only the acute morphine test that occurred when rats were older (collected on PND 194–196) rather than the test data from when rats were younger (see Table 2 for timeline). One week before acute morphine testing (PND 181–183), rats were habituated to the procedure in a session comprised of six 15-minute cycles, with an injection of saline given at the start of each cycle. A two-way mixed model ANOVA revealed no significant main effect of cycle, diet, nor any cycle by diet interaction effect for the latency in seconds for rats to remove their tails from all water bath temperatures after saline injections (data not shown; P > 0.05). That is, the withdrawal latency for rats to remove their tails was comparable for rats in all dietary groups, for all three water temperatures following saline injections. The average (±S.E.M.) latencies when rats were tested in a session of consecutive saline injections was 14.94 ± 0.26 seconds, 4.96 ± 0.96 seconds, and 2.6 ± 0.52 seconds at temperatures of warm water baths set to 40°C, 50°C, and 55°C, respectively.

TABLE 2.

Timeline of tests and PND range throughout the study

Day 0 and onward represent tests for statistical comparisons.

Saline Test 1 Saline Test 2 Acute Test 1 Acute Test 2 Saline Test 3 Acute Test 3 (0.32–17.8 mg/kg morphine) Twice-Daily Injections (3.2–56 mg/kg morphine) Tolerance Test (3.2–56 mg/kg morphine) Non-Precipitated Withdrawal
PND 97–99 PND 104–106 PND 111–113 PND 118–120 PND 187–189 PND 194–196 PND 195–214 PND 212–214 PND 213–219
Day −97 Day −90 Day −83 Day −76 Day −7 Day 0 Days 1–18 Day 18 Days 19–24
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The warm water baths set to 40°C and 55°C were used as control temperatures, and the antinociceptive effects of morphine at 50°C are shown (Fig. 3). At 50°C, latencies increased as the dose of acute morphine increased across cycles. A two-way mixed model ANOVA of % MPE revealed a significant main effect of dose (F [5,92] = 145.0; P < 0.0001); however, no main effect of diet nor a dose by diet interaction effect was revealed (P > 0.05) for this final acute morphine test (PND 194–196). Tukey’s multiple comparisons tests revealed that the latency in seconds for rats to remove their tails from 50°C water was significantly increased following cumulative doses of 10 mg/kg and 17.8 mg/kg morphine as compared with saline for rats eating standard chow, high fat chow, and ketogenic chow (P < 0.0001). That is, at larger doses of morphine, rats left their tails in the 50°C water longer, but this did not vary based on dietary group (Fig. 3).

Fig. 3.

Fig. 3.

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Mean (±S.E.M.) withdrawal latency (seconds) for female rats eating standard chow (closed symbols [A, B]), high fat/high carbohydrate chow (open symbols [A]), and ketogenic chow (shaded symbols [B]) to remove their tails from 50°C water after acute morphine (left) or following chronic administration morphine (right). Gray lines represent the acute dose–response curves, and black lines represent the dose–response curves generated on the last day of the chronic morphine administration protocol (day 18). Vertical axis: % maximum possible effect. Horizontal axis: morphine mg/kg. Rats in all groups had comparable latencies when tested with acute morphine, or following chronic morphine administration. Further, all rats developed tolerance, as demonstrated by greater ED50 values after chronic morphine administration as compared with ED50 values calculated from acute morphine exposure (P < 0.0001).

Following the chronic morphine administration protocol, morphine dose–response curves were again generated on PND 212–214. A two-way mixed model ANOVA of % MPE revealed a significant main effect of dose (F [4,80] = 30.16; P < 0.0001) and a significant dose by diet interaction effect (F [8,80] = 2.365; P = 0.0244); however, no main effect of diet was revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that following the cumulative dose of 56 mg/kg of morphine, rats eating ketogenic chow kept their tail in the 50°C water significantly longer than rats eating standard chow (P = 0.0246). That is, whereas all rats left their tails in the 50°C water longer following increasing doses of morphine, rats eating ketogenic chow left their tail in the water longer as compared with rats eating standard chow (Fig. 3).

To examine differences between the morphine-induced antinociception tests that took place when morphine was administered acutely (on PND 194–196) versus chronically (i.e., to examine the development of tolerance on PND 212–214), a two-way mixed model ANOVA analyzing log ED50 values revealed a significant main effect of week (F [1,20] = 25.14; P < 0.0001); however, no diet or diet by week interaction effects were revealed (P > 0.05). Tukey’s multiple comparisons tests revealed significantly larger ED50 values for rats following chronic morphine administration protocol as compared with when morphine was administered acutely (P < 0.0001). That is, after the chronic morphine administration protocol, morphine-induced antinociception dose–response curves shifted rightward 7.1-fold for rats eating high fat chow and ketogenic chow, and 7.4-fold for rats eating standard chow. That is, regardless of diet, the magnitude of tolerance that developed to the antinociceptive effects of morphine was comparable.

Withdrawal.

Average (±S.E.M.) withdrawal signs were collapsed across the three observation periods observed for 5 days beginning the day of the final examination of morphine-induced antinociception (that occurred after 18-day chronic morphine administration protocol; on PND 213–219), after which morphine injections were replaced with saline (i.e., when the discontinuation of morphine began). A two-way mixed model ANOVA revealed a significant main effect of day (F [5,105] = 7.074; P < 0.0001); however, no main effects of diet nor any day by diet interaction effects were revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that, as compared with 1 day following morphine discontinuation, observable withdrawal signs for all groups of rats were significantly increased 3–5 days after morphine discontinuation (P < 0.05). That is, there were no group differences in withdrawal signs in rats eating different diets; however, rats in all groups displayed more signs of withdrawal after 3 days of morphine discontinuation (Fig. 4).

Fig. 4.

Fig. 4.

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Mean (±S.E.M.) withdrawal signs were observed in female rats eating standard chow (closed symbols), high fat/high carbohydrate chow (open symbols), and ketogenic chow (shaded symbols). Vertical axis: withdrawal signs. Horizontal axis: days post morphine discontinuation. Withdrawal signs were not different for rats eating different diets (P > 0.05). There were significantly more signs of withdrawal observed on day 3 as compared with day 1 and day 2 of morphine discontinuation (P < 0.05).

Next, withdrawal-induced weight loss was examined. The % change in body weight for rats eating different diets was normalized based on body weight examined on the last day (e.g., day 18; on PND 212–214) of the chronic morphine administration protocol. A two-way mixed model ANOVA revealed a significant main effect of day (F [5,105] = 2.203; P < 0.0001), a significant main effect of diet (F [2,21] = 6.208; P = 0.00746), and a significant day by diet interaction effect (F [10,105] = 2.203; P = 0.0230). Tukey’s multiple comparisons tests revealed that, as compared with 1 day following morphine discontinuation, body weight for all groups of rats significantly decreased 2 days after morphine discontinuation (P < 0.0001). Further, as compared with day 4 following morphine discontinuation, body weight for all groups significantly increased 5 days after morphine discontinuation (P < 0.0001). Tukey’s multiple comparisons tests also revealed that body weight (examined as a % change from the day before morphine discontinuation took place) was significantly greater for rats eating ketogenic chow as compared with rats eating standard chow on day 4 (P = 0.012), day 5 (P = 0.0015), and day 6 (P = 0.0003) after morphine discontinuation. That is, rats eating ketogenic chow lost less weight after 4–6 days following discontinuation from morphine, as compared with rats eating standard chow (Fig. 5).

Fig. 5.

Fig. 5.

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Mean (±S.E.M.) % change in body weight during withdrawal, calculated for female rats eating standard chow (closed symbols), high fat/high carbohydrate chow (open symbols), and ketogenic chow (shaded symbols). Data are normalized based on body weight calculated on the day prior to morphine discontinuation. Vertical axis: % change in body weight. Horizontal axis: days post morphine discontinuation. Although body weights slightly decreased for rats in all groups immediately after morphine discontinuation, rats eating ketogenic chow gained significantly more weight back by days 4–6 postmorphine discontinuation as compared with rats eating standard chow (P < 0.05).

Body Temperature.

Average °C (±S.E.M.) changes in body temperature following saline or morphine injections collected during the warm water tail withdrawal procedure were assessed for rats eating different diets (Fig. 6). A two-way mixed model ANOVA revealed that there were no main effects of cycle, diet, nor any cycle by diet interaction effects regarding body temperature change following six consecutive saline injections (i.e., during habituation, 1 week before morphine was tested [P > 0.05]). That is, without drug administration, body temperature was not different for rats eating different diets. In contrast, a two-way mixed model ANOVA revealed a significant main effect of cycle (F [4,76] = 11.00; P < 0.0001) following acute morphine administration; however, no main effect of diet nor any cycle by diet interaction effects were revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that body temperature decreased or became significantly cooler after cumulative doses of 1–17.8 mg/kg morphine for all groups (P < 0.05). That is, increasing cumulative doses of morphine during the warm water tail withdrawal procedure decreased body temperature when assessed acutely. Further, a two-way mixed model ANOVA revealed a significant main effect of cycle (F [3,63] = 24.26; P < 0.0001) following the chronic morphine administration protocol; however, no main effect of diet nor any cycle by diet interaction effects were revealed (P > 0.05). Tukey’s multiple comparisons tests revealed that body temperature increased or became significantly warmer after cumulative doses of 3.2–56 mg/kg morphine for all groups (P < 0.05). That is, when rats were given morphine chronically, cumulative doses of morphine assessed in the warm water tail withdrawal procedure increased body temperature.

Fig. 6.

Fig. 6.

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Mean (±S.E.M.) change in body temperature for female rats eating standard chow (closed symbols), high fat/high carbohydrate chow (open symbols), and ketogenic chow (shaded symbols), calculated as the difference from temperature assessed during the first cycle (saline) when rats received either five consecutive saline injections (A), saline followed by cumulative doses of morphine administered acutely (B), and saline followed by cumulative doses of morphine administered following an 18 day chronic morphine administration protocol (C). Vertical axis: change in body temperature °C. Horizontal axis: cycle. There were no group differences in body temperature throughout the study (P > 0.05). However, for all rats, morphine induced significant hypothermia when administered acutely (B) and induced significant hyperthermia following chronic morphine administration (C) (P < 0.05).

Discussion

The present study examined the antinociceptive and adverse effects (i.e., tolerance, withdrawal, and changes in body temperature) of morphine in female rats eating different diets. Rats eating the high fat/high carbohydrate chow were hypothesized to be more sensitive to all the effects of morphine, as compared with rats eating standard or ketogenic chow. However, in the present study, the antinociceptive effects of morphine were comparable for rats in all dietary groups (Fig. 3). These results are consistent with literature describing the effectiveness of morphine for patients with and without an obesity diagnosis, some of whom might consume higher fat diets (Patanwala et al., 2014). These results are also consistent with previous work assessing morphine-induced antinociception using the hot plate procedure in female mice fed either a high fat/high carbohydrate or standard chow (Nealon et al., 2018). However, in the same report, female mice fed a high fat chow were more sensitive to the antinociceptive effects of morphine using the tail-flick procedure as compared with mice eating standard chow (Nealon et al., 2018). Further, male and female mice fed a high fat/low carbohydrate (ketogenic) chow were more sensitive to the antinociceptive effects of oxycodone as compared with controls using the hot-plate procedure (Trinko et al., 2023). An additional report demonstrated that rats eating hydrogenated vegetable fat (Crisco) were more sensitive to the antinociceptive effects of opioids as compared with rats eating standard chow using the tail-flick procedure (Kanarek et al., 1997). It is possible that the use of these different antinociception assays across experiments (e.g., hot plate, tail flick, or warm water tail withdrawal) might have contributed to the discordant results between studies. For example, while hot plate, tail flick and warm water tail withdrawal assays all examine thermal pain, both the specific physical responses (i.e., paw removal, tail flick, and tail withdrawal) and the pain-induction stimuli (i.e., hot plate, light, and warm water) are different between assays. Further, the physiological mechanisms underlying these unique physiological responses also differ. For example, tail-flick and warm water tail withdrawal responses are mediated spinally, whereas paw removal in the hot plate assay is mediated supraspinally (Gårdmark et al., 1998; Nealon et al., 2018). These differences might contribute to the differences observed across reports using different assays.

Individuals with chronic pain frequently require repeated administration of pain-relieving medications, which can lead to opioid dependence and tolerance (Chou et al., 2020). The development of tolerance to opioid drugs can lead to patients requiring larger doses to achieve pain relief, and in turn, increase the likelihood of adverse effects emerging. In the present study, rats underwent a chronic morphine administration protocol for 18 days to assess tolerance and induce dependence. After this chronic morphine administration protocol, morphine-induced antinociception dose–response curves shifted over 7-fold to the right for rats in all dietary groups. That is, all rats developed comparable tolerance to morphine following chronic administration (Fig. 3), despite consuming different diets. In one prior report, female mice eating high fat/high carbohydrate chow developed greater tolerance to morphine in the tail flick procedure, but were less tolerant to morphine in the hot plate procedure, as compared with rats eating a low fat chow (Nealon et al., 2018). The present study assessed tolerance at only one time point (e.g., at the end of the chronic morphine administration protocol). Therefore, we were unable to assess whether tolerance developed at the same rate for groups of rats eating different diets. To assess this, future studies should include probing for tolerance at several time points throughout the 18-day chronic morphine administration protocol.

In the present study, withdrawal signs were recorded beginning on the last day of the chronic morphine administration protocol to control for being observed, which may elicit stress or exploratory responses on the first day of observations (McDonald and Siegel, 1998). Withdrawal signs were assessed for 5 consecutive days to compare signs among the dietary groups. Withdrawal signs following morphine discontinuation were most prominent on day 3 and 4 (i.e., 3 and 4 days after morphine was discontinued) (Fig. 4), which is somewhat inconsistent with previous studies examining nonprecipitated withdrawal in male rats (Gerak et al., 2019). However, there are known sex differences regarding the severity of nonprecipitated withdrawal following chronic morphine administration (Bobzean et al., 2019). For example, in a prior study, fewer spontaneous withdrawal signs were observed for female rats as compared with male rats, but the presence of withdrawal signs persisted longer for females (Bobzean et al., 2019). In this study, there were no significant differences regarding observable withdrawal signs for female rats eating different diets (Fig. 4). However, in a previous study, lean, food restricted rats eating a high fat/high sugar diet, experienced fewer observable signs of withdrawal as compared with rats with free access to the high fat/high sugar diet, following naloxone administration (e.g., precipitated withdrawal) (Bocarsly et al., 2011). In the present study, nonprecipitated withdrawal was examined, rather than precipitated withdrawal, which might have yielded different results (see also Trinko et al., 2023). That is, it is well established that the withdrawal syndrome produced following opioid receptor antagonist administration is much more robust than observable signs of withdrawal measured following morphine discontinuation (Gerak et al., 2019; Ayoub et al., 2021). The lack of dietary group differences in the present report could be an artifact of the relatively little overall withdrawal signs observed across all subjects (Fig. 4). Therefore, the utilization of a procedure that allows for the observation of a more robust withdrawal syndrome (e.g., precipitated withdrawal) might facilitate the ability to detect small differences between groups of rats eating different diets, should such differences exist. That said, a recent report revealed no significant differences in observable withdrawal to oxycodone precipitated by naloxone in female rats (Trinko et al., 2023). Another methodological consideration is the duration of observation during which withdraw signs were scored. For example, in some reports, the observation duration was much longer than in the present study (e.g., longer observation intervals between doses of opioids; see Seaman and Collins [2021] for an example). A longer observation duration yields a larger snapshot of observable signs of withdrawal, which could also facilitate an improved ability to detect small group differences, if they exist. As such, both type of withdrawal (precipitated vs. nonprecipitated) as well as observation interval duration are important methodological considerations for future studies.

To examine withdrawal-related weight loss, body weight during the withdrawal procedure was further examined as a % change in body weight for rats eating different diets normalized based on body weight examined on the day of the warm water tail withdrawal procedure during chronic morphine administration. Compared with the last day of the chronic morphine administration protocol, rats in all groups lost a significant amount of body weight following morphine discontinuation (Fig. 5). Although there were no differences in weight loss between rats eating standard or high fat chow, rats eating ketogenic chow lost less weight during days 4–6 postmorphine discontinuation as compared with rats eating standard chow (Fig. 5). The mechanism(s) of action underlying observable withdrawal signs and withdrawal-related weight loss remain relatively understudied, and as such, it remains unclear why rats eating a ketogenic chow seemed to be more resilient to this weight loss as compared with other groups. However, the rats in this ketogenic group noticeably (although not statistically significantly) lost weight during the chronic morphine administration phase of the experiment (Fig. 1). This weight loss was not observed during chronic morphine administration for the other two dietary groups, and might contribute to the overall smaller delta change in weight between chronic morphine and withdrawal among the ketogenic diet fed animals (Fig. 5). Although this weight loss during chronic morphine was not statistically significant, it might be indicative of the ketogenetic diet fed-rats experiencing a greater stress response as a function of chronic morphine administration than the other two groups. For example, chronic morphine administration has been previously described as a chronic stressor (Houshyar et al., 2003) associated with reductions in body weight. Further, prior research has also identified that some dietary manipulations also serve as stressors, leading to elevations in stress hormones (i.e., adrenocorticotropin hormone) (Ryan et al., 2018). It is, therefore, possible that the nonsignificant but noticeable weight changes for the ketogenic rats during chronic morphine administration are a function of stress, induced either by the dietary manipulation, the chronic morphine, or a combination of these variables. This possibility should be further explored in future studies.

Although body weight can be a helpful assessment for examining the effects of eating different diets, there are other physiological assessments of metabolic function that could also be explored. For example, blood ketone concentrations can be assessed to determine if rats eating a ketogenic diet are in a state of ketosis, a metabolic state of metabolizing fat for energy rather than glucose (Gershuni et al., 2018). Although blood ketone concentrations were not evaluated here, if rats in the present study were in ketosis, this could have contributed to group differences observed in withdrawal-related weight loss. Previous work with rats eating the same ketogenic chow used in the present study reported increased concentrations of ketones after consuming the diet for several months without significant reductions in body weight as compared with rats eating standard chow (Granados-Rojas et al., 2020). However, regardless of whether ketosis was achieved, the present results do suggest that individuals diagnosed with obesity taking opioids chronically for pain-related conditions might be able to mitigate some of the potential for withdrawal-related weight loss by adopting a high fat/low carbohydrate (i.e., ketogenic) diet, when tapering or discontinuing opioid medications.

In the present study, rats experienced hypothermia following acute morphine administration, and hyperthermia following chronic morphine administration (Fig. 6). These results are like those from a previous study in mice (Nealon et al., 2018), demonstrating that acute morphine administration induced hypothermia in all rats, regardless of dietary condition (Fig. 6). After chronic morphine administration, body temperature for rats in all dietary groups increased following 32 mg/kg and 56 mg/kg morphine injections as compared with after saline injections (Fig. 6); consistent with previous studies (Mucha et al., 1987). These changes in body temperature were comparable among rats eating different diets when tested acutely and chronically with morphine (Fig. 6).

In conclusion, dietary manipulation did not impact sensitivity of rats to the antinociceptive effects of morphine. Further, there were no dietary group differences regarding the adverse effects of morphine (e.g., tolerance, withdrawal, and changes in body temperature), though rats eating ketogenic chow experienced less withdrawal-related weight loss than rats eating in other dietary groups. These results suggest that while dietary manipulation might not impact the analgesic effects of opioids, the severity of withdrawal-related weight loss might be mitigated by the consumption of a high fat/low carbohydrate (ketogenic) diet.

Data Availability

The authors declare that all the data supporting the findings of this study are contained within the paper.

Abbreviations

MPE

maximum possible effect

PND

postnatal day

Authorship Contributions

Participated in research design: Beltran, Minervini, Serafine.

Conducted experiments: Beltran, Parra, Serrano, Castillo, Castro, Elsey, Serafine.

Performed data analysis: Beltran, Minervini, Serafine.

Wrote or contributed to the writing of the manuscript: Beltran, Minervini, Serafine.

Footnotes

This work was supported in part by grants from National Institutes of Health National Institute of General Medical Sciences [Grants RL5GM118969, TL4GM118971, and UL1GM118970 (to M.K.E. and J.C.) and R16 GM149426-01]; National Institutes of Health National Institute on Drug Abuse [Grant R25DA033613] (to K.M.S., A.P.S., and A.N.P.); and National Institute on Drug Abuse Research Centers in Minority Institutions [Grant 3U54MD007592-29S4] (to K.M.S.).

No author has an actual or perceived conflict of interest with the contents of this article.

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