Dietary-Induced Obesity, Hepatic Cytochrome P450, and Lidocaine Metabolism: Comparative Effects of High-Fat Diets in Mice and Rats and Reversibility of Effects With Normalization of Diet

Journal Pre-proof Dietary-induced obesity, hepatic cytochrome P450 and lidocaine metabolism: Comparative effects of high-fat diets in mice and rats and reversibility of effects with normalization of diet Hamdah M. Al Nebaihi, Rami Al Batran, John R. Ussher, Zaid H. Maayah, Ayman O.S. El-Kadi, Dion R. Brocks PII:

S0022-3549(19)30749-X

DOI:

https://doi.org/10.1016/j.xphs.2019.11.007

Reference:

XPHS 1802

To appear in:

Journal of Pharmaceutical Sciences

Received Date: 23 September 2019 Revised Date:

2 November 2019

Accepted Date: 7 November 2019

Please cite this article as: Al Nebaihi HM, Al Batran R, Ussher JR, Maayah ZH, El-Kadi AOS, Brocks DR, Dietary-induced obesity, hepatic cytochrome P450 and lidocaine metabolism: Comparative effects of high-fat diets in mice and rats and reversibility of effects with normalization of diet, Journal of Pharmaceutical Sciences (2019), doi: https://doi.org/10.1016/j.xphs.2019.11.007. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.

Dietary-induced obesity, hepatic cytochrome P450 and lidocaine metabolism: Comparative effects of high-fat diets in mice and rats and reversibility of effects with normalization of diet

Hamdah M. Al Nebaihi, Rami Al Batran, John R. Ussher, Zaid H. Maayah, Ayman O.S. El-Kadi and Dion R. Brocks** Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada

** Correspondence to: Dion R. Brocks, Ph.D. Professor and Associate Dean Faculty of Pharmacy and Pharmaceutical Sciences 2-142 H Katz Group Centre for Pharmacy and Health Research, University of Alberta Edmonton, AB Canada T6G 2E1 E-mail: [email protected] Phone: (780) 492-2953

Keywords: High-fat diet, dealkylation, microsomal metabolism, liver Acknowledgments: Ms. Al Nebaihi was supported by a Scholarship from the Kingdom of Saudi Arabia. None of the authors have any conflicts of interest to report in conjunction with this work. Access to the data used to generate the summary data within this manuscript is attached as Supplementary material (uncropped blots). Further data may be obtained by contacting the corresponding author.

Abstract

The effects of a high-fat diet on mRNA and protein of cytochrome P450 (CYP) enzymes in rats and mice, and its impact on lidocaine deethylation to its main active metabolite, monoethylglycinexylidide (MEGX) in rats were investigated. The effect of a change in diet from high-fat to standard diet was also evaluated. Plasma biochemistry, mRNA, protein expression for selected CYP and the activity of lidocaine deethylation was determined. The high-fat diet curtailed the activity and the expression the majority of CYPs (CYP1A2, CYP3A1, CYP2C11, CYP2C12 and CYP2D1), mRNA levels (Cyp1a2, Cyp3a2) and MEGX maximal formation rate (Vmax). Mice showed complimentary results in their protein expressions of cyp3a and 1a2. Switching the diet back to standard chow in rats for 4 wk reverted the expression levels of mRNA and protein back to normal levels as well as the maximum formation rates of MEGX. Female and male rodents showed similar patterns in CYP expression and lidocaine metabolism in response to the diets, although MEGX formation was faster in male rats. In conclusion, diet induced obesity caused general decreases in CYP isoforms in rat, but also in mice. The effects were shown to be reversible in rats by normalizing the diet.

Introduction Lidocaine is commonly used for the treatment of cardiac arrhythmias in emergency situations and as a local anesthetic due to its sodium blocking properties.1 More recently lidocaine has been found to have analgesic properties when plasma concentrations are sufficiently high.2 Lidocaine is primarily metabolized in the liver through cytochrome P450 (CYP) to several metabolites including its pharmacological active dealkylated metabolite, monoethylglycinexylidide (MEGX).3,4 This metabolite possesses up to 90% the antiarrhythmic potency of lidocaine.5 CYP3A4 and CYP1A2 are the enzymes mainly responsible for the formation of MEGX in humans.4,6 A lengthening of lidocaine t½ has been reported in healthy subjects and surgical patients with increases in body mass, which has possible implications in lidocaine dosing to the overweight and obese.7,8 The combination of over-consumption of energy-dense foods and a sedentary lifestyle are the primary causes of the global epidemic of obesity.9 Atherosclerosis, diabetes, hypertension, hyperlipidemia, depression and osteoarthritis are often ancillary conditions coupled with overweight and obesity.10 Most of these comorbidities are accompanied with a higher than normal usage rate of medications. In the administration of lidocaine during surgery, or as an antiarrhythmic agent, clinicians must take into account individual characteristics of the patient. Because of the possibility of altered pharmacokinetics (clearance, volume of distribution and half-life) in the presence of obesity, this may provide a challenge in the tailoring the dosage, something that remains incompletely understood.11 Male rats given diets rich in fructose and/or fats exhibit a downregulation in the expression of liver enzymes involved in the metabolism of drugs.10 Here we evaluated the

impact of a high-fat (HF) diet on selected cytochrome P450 including those known to be involved in lidocaine metabolism. Lidocaine was used as a model drug to examine functional changes in metabolizing efficiency by quantifying the rates of MEGX formation by liver microsomes. Because a major intervention used to reduce excess weight is a change in diet to one of reduced calorie content, we examined if changes caused by HF diet could be reversible by switching to a standard diet. Male and female rats may differ in their responses to diets, so we also examined the outcomes of HF diet and reversibility of diet in both sexes of rats. To provide for an interspecies comparison, we also examined the possibility of changes in liver CYP expression in mice after HF diets.

Experimental Materials Lidocaine HCl was obtained from AstraZeneca (Mississauga, ON, Canada) as Xylocaine® 2% for injection USP. Nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt hydrate (NADPH) and monoethylglycinexylidide (MEGX) were supplied by Sigma Aldrich (Oakville, ON, Canada). General laboratory chemicals were purchased from Fisher Scientific (Ottawa, ON. Canada). Isoflurane USP 99.9% was purchased from Pharmaceutical Partners of Canada (Richmond Hill, ON, Canada). The Supermix qScript™ cDNA Synthesis Kits was supplied by QuantaBio (Mississauga, ON, Canada). The 96-well optical reaction plates, SYBR Green and optical adhesive films were purchased from Applied Biosystems by Thermo Fisher Scientific (Carlsbad, CA). Real-time PCR primers for metabolic enzymes were synthesized and purchased

by Integrated DNA Technologies, Inc. (Coralville, IA). TRIzol reagent and UltraPure distilled water were purchased from Invitrogen (Carlsbad, CA). Page Ruler Plus Protein Ladder was from Thermo Scientific (Grand Island, NY), and bovine serum albumin from Fisher Scientific (Ottawa, ON, Canada). Bromophenol blue, ammonium persulfate, β-mercaptoethanol, acrylamide, N,N,N’,N’ tetramethylethylenediamine, and nitrocellulose membrane were purchased from Bio-Rad Laboratories (Hercules, CA). Primary antibodies for rat cytochrome P450 (CYP)1A2 and CYP3A1 were purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA) while CYP2C11, CYP2C12, CYP2D1, and CYP2E1 were purchased from abcam (Toronto, ON, Canada). The rat anti-CYP1A2, 3A1 and 2E1 were also cross reactive with mouse cyp3a41, cyp1a2, and cyp2e1, respectively. Secondary antibodies (anti-mouse and anti-rabbit IgG peroxidase) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Chemiluminescence Western blotting detection reagents were from GE Healthcare Life Sciences (Piscataway, NJ). Anti-rat polyclonal CYP3A2, CYP3A1, and CYP1A2 antibody raised in rabbit (suitable for Immunohistochemistry) were purchased from Abcam (Toronto, ON, Canada). Non-immune rabbit serum was provided by Science Animal Support Services (SASS) at the University of Alberta (Edmonton, AB, Canada).

Animals and Treatments All experimental procedures involving animals were approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee. Male and female Sprague-Dawley rats of 3-4 wk of age were purchased from The University of Alberta Science Animal Support

Services (Edmonton, AB, Canada). The rats weighed 100 to 140 g when received. The rats were housed 2 per cage in a temperature-controlled room with 12-h dark/light cycle.

An overview of the experimental design is shown in Figure 1. Rats (n=8 total for each sex) were started on a 14 wk course of 45% HF rat chow (Harlan Teklad diet TD.06415, Frederick, MD) with water (dietary compositions shown in Table 1). At the end of the 14 wk period, half of the animals were euthanized under isoflurane. In the remaining rats, the diet was switched to normal (standard) rat chow (fat content 13.4% Kcal, PicoLab® LabDiet, St. Louis, MO). Four wk after, those rats were euthanized. For each of the 14 and 18 wk groups, control rats (n=4 each) were included where the diet was 13.4% fat content. Free access to food and fluids was permitted throughout. Rat weights from the start of the study, and food consumption from 10 to 18 wk, were measured once or twice weekly. Table 1 lists the dietary compositions of each diet.

After euthanasia (following a 6 h period of food restriction but with water ad libitum), blood was collected in heparinized tubes and plasma being separated by centrifugation at 3000 g for 10 min. Livers were also collected. All samples were kept at -80˚C until being used for biochemical assay, enzyme kinetics and protein determinations.

To provide for an interspecies assessment, we had available to us livers from mice which were given a HF diet, and from diabetic mice. The animals were 5-6 wk-old C57Bl/6J male and female mice (Jackson Laboratory, Bar Harbor, ME; stock no. 000664; Tg (ACTA1-cre/Esr1*)2Kesr). Mice

were lean-diet fed or high-fat-fed (obese). The compositions are shown in Table 1 and were obtained from Research Diets (New Brunswick, NJ; lean diet, D12450J and high-fat with 60% lard, D12492). During the dietary phase of T2D mice, they were administered a single injection of streptozotocin 90 mg/kg during week 5.12,13 The dietary phase in all mice lasted 10 wk followed by euthanasia (Intraperitoneal injection of sodium pentobarbital, Cambridge, ON) and collection of liver.

Plasma Biochemistry Fasting plasma samples were assayed for cholesterol, triglyceride, glucose, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) using an EasyRA assay kit purchased from Cypress Diagnostics Inc. (Campbellville, ON, Canada) and the EasyRA Analyzer (Bedford, MA). All samples were measured in duplicate, and the average of the values was used for statistical data analysis. The plasma level of leptin was assayed by Eve Technology Corporation (Calgary, AB, Canada). Insulin was measured using “Rat Insulin ELISA” that was procured from ALPCO (Salem, NH).

Isolation of Liver Microsomes Isolation of microsomal protein from liver was performed as previously described.14,15 The tissues were homogenized in cold sucrose solution (0.25 M) then centrifuged at 12,000 rpm. The supernatants were further centrifuged at 100,000 g for 1 h. The resulting pellets were resuspended in cold (4°C) sucrose and protein quantified using the Lowry method.16

Real-Time PCR RNA extraction approach was adopted as previously described.10 In brief, total RNA from liver tissues was extracted by 1 mL of TRIzol reagent per 100 mg of tissue to homogenize frozen samples. Then, the homogenate solution was collected into separate microcentrifuge tube and 200 µL of chloroform was added to each sample and shaken vigorously for 15 s. Followed by centrifugation at 14,000 ˣ g for 20 min at 4 °C. The resultant supernatants were transferred into microcentrifuge tubes, and 500 µL of isopropyl alcohol was added for each tube followed by mixing for 10 s and then incubated at -20°C. After 2 h of incubation, samples were centrifuged at 14,000 × g for 10 min at 4°C, followed by removal of the supernatant. Then 500 µL of 75% ethanol in diethyl bicarbonate treated water was used to wash the formed pellet. Thereafter, samples were centrifuged at 12000 ˣ g and 4 °C for 10 min to precipitate the RNA . The samples were subjected to vortex mixing for a few seconds and incubated at 60°C in a water bath for 15 min. All samples were stored at -80°C until further analysis. The total RNA was quantified by measuring the UV absorbance at 260 nm. The quality and the purity of the isolated RNA was determined by measuring the absorption ratios at 260/280 nm.

For cDNA Synthesis the first-strand cDNA was made using Supermix qScript™ cDNA Synthesis Kits according to manufacturer’s instructions. In brief, 1.5 µg of total RNA from each tissue sample was added to a mixture of 1 µL of qScript Reverse Transcriptase and 4 µL of qScript Reaction mix (5X), then Nuclease Free Water was added to reach 20 µL in total mixture.

Real-time PCR was applied for quantitative analysis of specific mRNA expression. It was performed on the ABI Prism 7500 System (Applied Biosystems, Foster City, CA) using 96-well optical reaction plates. The 25 µL reaction mixture contained 0.075 µL of 10 µM forward primer (Table 2) and 0.075 µL of 10 µM reverse primers (40 nM final concentration of each primer), 12.5 µL of SYBR Green PCR Master mix, 10.35 µL of nuclease-free water, and 2 µL of cDNA sample. The final reaction mixture was kept at 22°C for 5 min and then heated to 42°C for 30 min. Thereafter, the samples were heated for 85°C for 5 min followed by cooling process to 4°C.

The 2-Δ(ΔCT) method as described in Applied Biosystems User Bulletin No. 2 and explained further by Livak and Schmittgen was employed to analyze the real-time PCR data.19 All data are presented as fold change in gene expression normalized to β-actin (the endogenous reference gene) and relative to the untreated control.

Western Blot Analysis After separating proteins from liver tissue following the previously published method,10 Western blot samples were prepared by adding 2X loading buffer which contains 0.5 M Tris HCL (pH 6.8), 10% sodium dodecyl sulfate (SDS), 1.5% bromophenol blue, glycerol and B meracptoethanol to protein samples and then heated for 5 min at 100°C to denature the proteins. Proteins in each denatured sample were separated by 10% SDS-polyacrylamide gel (SDS-PAGE) and electrophoretically transferred to a nitrocellulose membrane. Then, membranes were blocked for an hour at room temperature by 5% BSA diluted in Tris-Buffered Saline Tween-20 containing 250 mM Tris-base, 1.5 M sodium chloride, 30 mM potassium

chloride, and 0.1% Tween-20. Thereafter, the blocking solution was discarded, and the blots were rinsed once in a washing buffer (0.1% Tween-20 in Tris-buffered saline). Subsequently, the membranes were incubated with primary antibody overnight at 4°C. The primary antibody solution was removed, and blots were washed (3 washes for 5 min each), followed by incubation with secondary antibody for 1 h at room temperature. Rinsing with washing buffer (3 washes for 5 min each) was then applied to remove the extra secondary antibody. Lastly, the protein bands were detected using enhanced chemiluminescence. The intensity of protein bands was quantified using Image-J software (National Institutes of Health, Bethesda, MD, http://rsb.info.nih.gov/ij).

Metabolism of Lidocaine to MEGX The formation kinetics of MEGX were characterized after exposing lidocaine to liver microsomes from control and HF groups of male and female rats. The stock lidocaine HCl solution was diluted in HPLC grade water to provide a nominal concentration of 100, 250, 1000, 2000, 5000 and 10000 μM. The water was then evaporated to dryness in vacuo and reconstituted in incubation mixture which was composed of 5 mM magnesium chloride hexahydrate dissolved in 0.1 mM potassium phosphate buffer (pH=7.4). Each incubation, in final volume of 0.25 mL, contained 1 mg/mL of protein from liver microsomes from each rat. The reaction was started with the addition of 1 mM NADPH after a 5 min pre-equilibration period. All incubations were performed at 37ºC in a shaking (50 rpm) water bath for 10 min. The reaction was ended by addition of 0.2 mL of 1 M NaOH. Internal standard, procainamide, was then added followed by extraction and assayed for MEGX.

A validated assay for lidocaine20 was adapted to measure MEGX in the microsomal samples. Validation of the assay for MEGX was determined using a range of MEGX concentrations from 0.24 to 77.6 µM of MEGX in microsomal incubation buffer. Each concentration had five replicates. The recovery of MEGX and IS after liquid-liquid extraction was determined by comparing the mean area ratio (MEGX/IS) response of samples spiked before extraction to that extracts of blanks spiked with the analyte post extraction at three levels (0.73, 4.9, and 19.4 µM) (five replicates for each).

Inhibition of MEGX Formation To gain insight into the CYP isoforms contributing to MEGX formation, 20 µL/mL concentration of the CYP3A1, CYP1A2, and CYP3A2 antibodies were added separately to the liver microsomal mixture incubate containing 0.5 mg/mL non-pooled microsomal protein (n=4) for control male and female rats. The degree of inhibition was determined by comparing the metabolite formed in the presence of antibody to that of matching controls containing non-immune rabbit serum in place of the antibody.

Statistical and Data Analysis Compiled data is shown as mean±SD unless indicated. To determine the kinetic constants for MEGX formation by liver microsomal preparations, Michaelis-Menten models were fitted to MEGX formation rates using the GRG nonlinear algorithm in the Solver routine of Microsoft Excel (Redmond WA). The total sum of squares, regression coefficient and the Akaike information criteria were used to judge the quality of fit and to guide model selection.

A one-way ANOVA followed by post-hoc test was used to analyze multiple comparisons. (ANOVA with Bonferroni correction, p<0.05). Student’s paired or unpaired Student’s t- tests were used as appropriate to assess the significance of differences between two groups. Linear regression was used to explore relationships between enzyme kinetics and protein expression. SigmaPlot 11.0 (Systat software, Inc. Chicago, IL) and Microsoft Excel (Microsoft, Redmond, WA) were used in statistical analysis of data. The level of significance was set at p < 0.05.

Results Body Metrics Rats After the end of the 14 wk dietary phase, there were significant increases in the body weights of rats fed HF diet compared to control rats (Figure 2). The weights of the HF groups began to differ from control rats by week 4 and week 3 for male and female rats, respectively, and remained higher up to 14 wk (P< 0.05) (Figure 2). These differences increased consistently with time. By the end of 14 wk, the final average body weights were 656 and 841 g for the control and HF male groups respectively, and 355 and 427 g for the control and HF female groups, respectively (Figure 2). Similar results were observed for up to 14 wk in the 18 wk study. The body weights of HF groups increased significantly compared to control group in both male and female rats. The average weights were 695.25 g in control and 974.25 g in obese males (P< 0.05) while in control females was 345 g and 424.25 in obese female group (P< 0.05) . The significant difference between the control and HF groups were observed in week 3 and week 4 for male and female rats, respectively.

When the diet was switched to standard chow at 14 wk, the weights of HF male rats plateaued showing a significant different in the weight compared to control group. For the body masses of HF female rats, the weights actually diminished after the HF diet was switched to standard chow. Their final weights closely matched those of the control female rats by the end of 18 wk study (P> 0.05) (371 mg control vs 375 mg HF group) (Figure 2).

Mice The obese and diabetic male mice (Figure 3) had significantly higher weights compared to control mice (47.2 g and 40.8 g vs 26. 8 g, respectively). The obese male mice also had a significantly higher weight than diabetic mice. Obese female mice were also significantly heavier than lean-fed mice. The obese female mice gained significant weight compared to control group.

Calorie Intake in Rats Males tended to have higher daily food intake than females in all groups where overall males consumed about 35% more food than females. Control rats consumed larger amounts of food compared to HF rats. However in terms of Kcal per day, the intake was significantly lower in the control vs. the HF animals in the initial 14 wk of the experiments (P< 0.05). Using the linear trapezoidal rule for estimating overall calorie intake versus time, the HF diet delivered 22% and 10% more calories to the HF fed male and female rats, respectively. After switching to standard chow in the last 4 wk, there was a noticeable reduction in calorie intake for both male (28% reduction) and female rats (36% reduction) (Figure 2). Between 14 and 18 wk, the HF female

group showed about 34% reduction in overall calorie intake compared to control group which was also reflected by a reduction in food intake of the standard diet. In HF males, however, although calorie intake was lower after the switch compared to before the switch, there was no apparent difference after 14 wk in calorie intake of the HF group rats compared to their control counterparts (Figure 2).

Plasma Biochemistry in Rats HF diet led to increases in leptin (Table 3) and was also associated with a significant increase in plasma cholesterol in male rats. In female rats, however, the increase in lipids was primary in the triglyceride fraction with only a slight elevation in cholesterol. These changes generally reverted back to normal levels when the standard diet was implemented after 14 wk of feeding. The AST:ALT ratio was significantly higher in obese female rats (Table 3).

Only livers were available from the mice, so plasma biochemical measures were not possible for them.

CYP Expression Rats After 14 wk on HF diet, there were a number of reductions in protein and mRNA expression observed. In male rats, several enzymes including CYP3A1, CYP1A2, CYP2C11, and CYP2D1 were markedly reduced compared to control rats (reduction by 84%, 59%, 69% and 63%,

respectively) (Figure 4A). Similarly, mRNA levels of CYP1A2 and CYP3A2 were reduced by 33% and 53%, respectively, compared to control rats (Figure 4A). In female rats, both the protein expression of CYP3A1, 1A2 and CYP2C12 as well as the mRNA level of CYP1A2 and CYP3A2 were diminished significantly compared to control groups (Figure 5A). The protein expression of CYP3A1 was reduced by 60% while CYP1A2 and CYP2C12 were reduced just over 40%. The reduction level of mRNA for CYP3A2 and 1A2 was 52% and 37%, respectively. In contrast, the mRNA level of CYP2C12 was more than three times higher than control female. The protein expression of CYP 2E1 did not change in obese male and female rats compared to control group (Figures 4A and 5A).

Interestingly, all the observed changes in the protein and mRNA expression in male and female were reversible when the standard diet was implemented after 14 wk of HF diet (Figure 4B) (Figure 5B).

Mice In male and female mice, the liver protein expression of cyp3a41 was significantly reduced in obese and diabetic males and obese females whilst the cyp2e1 was markedly increased. The expression of cyp1a2 was reduced significantly in obese male and female mice (Figure 8).

Assay of MEGX The chromatograms showed that MEGX and lidocaine peaks were well separated and free of interference (Figure 6). The accuracy and precision of the MEGX validation for the HPLC assay

were within acceptable ranges of accuracy and precision. Bias was <20% at the lowest concentration measured (0.73 µM) and <15% at all concentrations above that. The coefficient of variation was < 10% for all concentrations. The recovery of MEGX was >98%.

Hepatic microsomal metabolism of lidocaine to MEGX As lidocaine concentrations increased, there was an increase in the formation rate of MEGX (Figure 7). It was found that the kinetic profile of MEGX formation versus concentration conformed best to the simple Michaelis-Menten model.

=



× +

Where V is the velocity rate of MEGX formation, Vmax is the maximal formation rate of MEGX, km is the affinity constant, [S] is lidocaine concentration. The estimated km, Vmax, and intrinsic clearance (CLint) were determined in liver (Table 4). The obesity induced in our male and female rats at 14 wk led to significant reductions in the Vmax of MEGX formation compared to control rats (Table 4) (Figure 7). There was also a significant reduction in the CLint in male rats and a trend to the same in female rats, but no difference in km. Four wk after normalization of diet in the HF rats, however, there were no differences noticed between control and HF rats in any of the kinetic parameters.

Metabolizing enzymes determination Antibodies to CYP3A2, 3A1, and 1A2 were used to gain insight into the primary CYP involved in the metabolism of lidocaine by rat liver microsomes. No significant reductions were observed in

the formation of MEGX with co-exposure to CYP3A antibody under non-saturated concentrations (0.5 mM lidocaine) (Figure 8). However, treating the liver microsomes with antiCYP1A2 diminished the formation of MEGX significantly by 25-29% (Figure 8).

The reduction in lidocaine concentrations was also measured in this experiment. It was noticed that there was no difference between control male and female rats in lidocaine consumption, where 30% of the total amount of lidocaine added was gone after 15 min of incubation. This meant that of the total amount of lidocaine metabolized presumably by CYP-mediated metabolism, that 4.9% and 0.97% of the recovered metabolite was MEGX.

The relationships between CYP protein-normalized band densities in the microsomes from all control and HF rats were explored. CYP3A1 was observed to show significant positive relationships with the observed Vmax of MEGX formation for both sexes. The relationship for CYP1A2 was not significant in male rats, but when an outlier differing more than 40% in Vmax from all of the other rats was removed, the differences became significant for female rats. Neither CYP2C11 nor 2C12 correlated with MEGX formation rates.

Discussion The male rat HF model was previously described10 and was designed to simulate the Western diet, here with a diet rich in fat. The rats compared to control rats experienced a significant increase in body mass (Figure 2). The diet was not overtly diabetic in nature (no changes in plasma glucose) although as seen here there were increases in mean plasma cholesterol and leptin (Table 3). There was also a trend in these rats towards an increase in insulin (Table 3). The diet was also associated with reductions in a number of drug metabolizing enzymes. In this respect, the current results were as previously seen although here we also looked at the effect of the HF diet on CYP2E1 and 1A2. Although CYP2E1 was not significantly affected by the HF diet, CYP1A2 was significantly reduced (Figure 4A). Authors of a more recent paper21 fed male rats a HF diet for 12 wk and duplicated many of our previous findings of CYP protein expression. They likewise noted no change in CYP2E1 and a decrease in CYP1A2 (Figures 4 and 5). They also reported a decrease in CYP2B1 and in a number of uridyl glucuronyltransferases.

A previous investigation

22

of the effects of HF diet on female rats mostly focussed on

transporter expression, mostly at the level of mRNA. Although 13 wk of HF feeding to female rats led to an increase in body mass, it was much more modest that seen in male rats (Figure 2).10 We likewise observed that after 14 wk of HF diet there was a much less pronounced increase in the body mass of females than males (Figure 2). Schemmel et al. (1970)

23

had

studied the effects of HF diets on the body weights of seven rat strains including SpragueDawley and likewise found that males gained much more weight than females. In terms of plasma biochemistry females had higher triglyceride with HF, something that did not occur in

males. Although the body mass did not increase as much as males, we nevertheless observed that the protein expressions of CYP were generally in line with what was seen in the male rats, with the exception of no change in CYP2D1 in female rats (whereas in males there was a decrease) (Figure 5A). In general there was a concordance between protein and mRNA relative expressions of CYP isoforms in females with the exception of CYP2C12 where in HF mRNA increased but protein decreased (Figure 5A). The decrease in CYP2C12 protein may mean that there is increase the degradation of CYP2C12 protein at posttranslational level (decrease protein t½). The increase in mRNA might be due to changes in the levels of estrogen and growth hormone by obesity.24 It is of note that Ghoneim et al. (2015)22 found a slight increase in the protein expression of female CYP3A2 in HF fed rats, a protein we did not examine here.

After switching the HF diet to standard diet, the average body mass of male rats plateaued but remained higher in the HF rats (Figure 2). In contrast the body weight of female rats dropped significantly to match the weight of the control group (p>0.05) (Figure 2). Both sexes were observed after restoring the standard diet for 4 wk to show restoration of normal (standarddiet fed) protein expressions for all CYP tested (Figure 4B) (Figure 5B). To our knowledge this is the first report to show such a reversal with respect to drug metabolizing enzymes. What is notable about the results is that even though the male rats were still relatively “obese” with the same body mass after the additional 4 wk of standard diet (Figure 2), they were more metabolically “normal” based on their CYP expression patterns and plasma biochemistry. These observations are matched by humans with excess BMI, where some are metabolically abnormal, yet others are much more like healthy-lean BMI subjects.25,26

An interspecies comparison of the effects of a HF diet was gleaned from the mice. Unlike rats, cyp2e1 in obese male and female mice was significantly higher than the lean mice (Figure 9). These findings were in the line with those reported earlier in the protein expressions of ob/ob female mice27 and in male NMRI mice rendered obese by injection of monosodium glutamate for several days in the postnatal period.28 One study was conducted on rats where there was a significant induction in the hepatic expression of CYP2E1. However, those rats were given a very aggressive diet of fat (70% lard, 20% protein without carbohydrate) that markedly elevated the level of ketone bodies, which are known to induce of CYP2E1.29,30 The mice studied here were also given a relatively higher fat content in their diet compared to the rats which might explain the difference between the two species in 2E1 expression in DIO rodents (Table 1). Induction of CYP2E1 has been observed in diabetic rats.31 Diabetes in mice has been associated with changes in the expression of cyp2e1, but the data have been somewhat conflicting, possibly due to the manner in which the diabetes was experimentally induced and when after diabetes induction that measures were made.32,33

The observation of decreased 3A isoforms were seen in HF rats and mice, and diabetic mice. Similar to the rats in response to HF diet, male and female mice showed reductions in the expression of cyp1a2.

Similarly, Elam et al, revealed that CYP1A2 gene expression was

significantly reduced in the livers of morbidly obese women.34 In people with non-alcoholic fatty liver disease (NAFLD), a condition associated with obesity, significant reductions were observed in the protein expression of CYP3A4 in the liver.35,36 The metabolism of certain drugs

primarily metabolized by CYP3A4 were significantly reduced in obese patients compared to healthy subjects.37-39 Similar findings were observed in hepatocytes from patients with NAFLD, who had reduced expression levels of a number of drug metabolizing enzymes including CYP3A4, 1A2 and 2C isoforms.40

Interestingly, in NAFLD patients before gastroplasty, elevated CYP2E1 activities were noted. This is similar to the mice which experienced huge weight gain (Figure 3).29 Our rats, which gained proportionately much less weight (Figure 2), did not experience an increase in CYP2E1, but also did not have NAFLD given the lack of increase in liver function enzymes (Table 3). It is likely given the magnitude of weight gain that our mice had NAFLD, but we cannot confirm since plasma was lacking from those animals.

Some of the mRNA responses to the treatments were not matched by protein expression (Figures 4A and 5A). For example, compared to control female rats the mRNA expression of CYP2C12 in obese females was more than 3-fold higher, but protein expression was lower (Figure 5A). Such a discordance between mRNA and protein expression is attributable to alterations in translational efficiency of specific protein or transcriptional rate of target protein.41

With respect to enzyme kinetics of MEGX formation, in line with the decreases in CYP expressions (Figures 4A and 5A), there were decreases in Vmax associated with the HF diet at 14 wk in both sexes (Figure 7), Also, in line with the CYP protein measures (Figures 4B and 5B),

switching the diet to standard diet for 4 wk caused normalization of lidocaine metabolism (Figure 7). Thus, the protein expression matched the function of the protein. To gain insight into other aspects of lidocaine metabolism in the rat, we also looked at the effects of immunoinhibition and relationships between protein band densities and Vmax (Figure 8). The involvement of CYP1A2 was implicated by the reduction in MEGX formation with coincubation of the antibody in both sexes. Although the CYP3A1 and 3A2 antibodies had no effect, but there was a significant relationship for 3A1 protein expression and MEGX Vmax, The lack of an effect of the 3A antibodies may be related to the knowledge that MEGX is sequentially metabolized to other metabolites by CYP isoforms such as glycinexylidide. Hence inhibiting CYP3A might perhaps not only decrease its formation from lidocaine, but perhaps also inhibit its sequential metabolism, leading to no difference in this metabolite in a closed microsomal system.

In microsomes isolated from male rat liver, it has been reported that MEGX maximum formation rate was 4.84 nmol/min/mg which is consistent to what we found in our male rats (4.6 nmol/min/mg)42. The sex difference seen in MEGX formation in the immunoinhibition experiment (Figure 8) was observed here as well as in the microsomal experiments used to identify kinetic constants (Table 4). Differences between adult male and female rats in hepatic CYP3A expression could explain the higher metabolism rates seen in males.43 What was surprising upon first glance was that of the total amount of lidocaine consumed (30%), only 4.9 and 0.97% were recovered as MEGX in the control incubations used in the immunoinhibition experiments. 4-hydroxyxylidine is the major metabolite of lidocaine recovered in urine of humans after lidocaine administration. This would require MEGX to be sequentially

metabolized presumably by CYP isoforms. Hence this low percentage is possibly indicative of sequential metabolism occurring in our incubations, which also suggests that the Vmax measured in our metabolite recovery vs. lidocaine concentration experiment is an underestimate of the true value. Metabolism of lidocaine to MEGX occurs not only by CYP1A2 and 3A, but also to 2B1.44 Although it was suggested that purified CYP2C was important in rat for MEGX formation,43 we saw no significant correlation in complete microsomes between Vmax of MEGX formation and CYP2C11 or CYP2C12 protein levels. Nevertheless, each of these were decreased by the HF diet which likely contributes to the lower Vmax of MEGX formation after 14 wk of HF treatment.

In post-surgical patients, lidocaine was observed to have a positive serum half-life vs. BMI relationship.8 Likewise in obese and lean body weight healthy subjects, it was reported that there was no change in lidocaine CL but an increase in volume of distribution (Vd), leading to an increase in the half-life of the drug.7 The doses administered the subjects were not body weight-normalized. Interestingly when the authors weight-normalized there was no difference in Vd between obese and non-obese. However they did not report the weight-normalized total body CL. In doing so using the reported mean body weights, a notable reduction in the mean CL of lidocaine becomes apparent in males (obese 11.5 vs. control 19.5 mL/min/kg) and females (obese 11.3 vs. control 19.7 mL/min/kg). This is consistent with our observations in the HF rats Vmax in both sexes and CLint in male after 14 wk (Table 4). A decrease in CYP3A4 or 1A2 would be expected to alter the pharmacokinetics of lidocaine in patients with moderate levels of hepatic extraction, such as might occur in post-surgical patients8. The addition of another

inhibitory-interacting drug could possibly enhance the strength of such an interaction in obese subjects with decreased CYP expression.

In conclusion, diet-induced obesity was associated with reduction in expressions of a number of proteins involved in drug elimination by liver in male and female rodents which also gave rise to a reduction in the efficiency of lidocaine deethylation in rat. Adopting a leaner diet was seen to cause normalization of these enzymes and in lidocaine metabolism, even when the body mass was elevated, as was the case for the male rats. Mice fed a HF diet, similar to rats, experiences decrease in many of the same CYP isoforms studied in the rat. The findings also seemed to parallel CL values reported for lidocaine in obese vs. non-obese humans. The findings of reduced drug metabolism in obesity requires further examination because it has ramifications in the safe and effective dose regimens that should be used in the pharmacologic treatment of patients in the clinic. Studies examining the pharmacokinetics of lidocaine and its metabolites in obese and lean rats and humans are warranted to establish the relevance of these findings to the pharmacokinetics of lidocaine.

Legends to Figures Figure 1. Experimental design. Figure 2. Mean±SD of weight gain throughout the study, and daily calorie and food intake over the last few weeks of the study. The span of time over which the body weights of HF vs. Control rats differed are indicated. Upper panels show data for the 14 wk dietary study, and Lower panels depict the results from the 18 wk study. HF denotes high-fat. Data are from four rats per group. The black vertical arrow (lower panels) depicts diet switching from HF to standard diet for HF groups. Figure 3. Mean+SE of weight gain in the beginning and the end the study for both male and female mice. Figure 4. Effect of high-fat diet (A) and switching to standard caloric diet (B) on metabolizing enzyme CYP expression in male rat liver. The mRNA was measured using real-time PCR (top panels), with expression normalized to the housekeeping gene mRNA (β-actin) and relative to the normalized ratio from the control rat mean message of 4 rats. Target protein from each rat sample (bottom panels) was determined using Western blot, with the intensity of bands being normalized to the band density of β-actin, the housekeeping protein. On each day of the protein determination, a control protein determination was performed from rats given a lean diet. Columns represent the mean β-actin-normalized band intensities of 4 rats. The bands under each panel show the results from 3 representative rats (target protein and corresponding β-actin bands). Data are presented as fold of control (mean + SE). *p < 0.05 compared to the corresponding control using unpaired Student t-test.

Figure 5. Effect of high-fat diet (A) and switching to standard caloric diet (B) on metabolizing enzyme CYP expression in female rat liver. The mRNA was measured using real-time PCR (top panels), with expression normalized to the housekeeping gene mRNA (β-actin) and relative to the normalized ration from the control rat mean message of 4 rats. Target protein from each rat sample (bottom panels) was determined using Western blot, with the intensity of bands being normalized to the band density of β-actin, the housekeeping protein. On each day of the protein determination, a control protein determination was performed from rats given a lean diet. Columns represent the mean β-actin-normalized band intensities of 4 rats. The bands under each panel show the results from 3 representative rats (target protein and corresponding β-actin bands). Data are presented as fold of control (mean + SE). *p < 0.05 compared to the corresponding control using unpaired Student t-test. Figure 6. HPLC-UV chromatograms (from bottom up) of blank incubation buffer, incubation buffer spiked with MEGX, incubation buffer spiked with lidocaine, and after microsomal incubation (100 µM lidocaine) with NADPH. Procainamide is the internal standard.

Figure 7. Formation rates of MEGX from lidocaine in liver microsomes from female and male rats (mean ±SD, n=4). Top panels depict the MEGX formation rates in lean and obese animals after 14 wk on high-fat (HF) diet and lower panels show the formation rates in lean and obese rats four wk after the HF diet was switched to standard diet. The best fits to the one enzyme simple Michaelis-Menten are shown as dotted and solid lines between the data points.

Figure 8. Top panels show MEGX formation rates in the presence and absence of antibodies to CYP1A2 or CYP3A1 and 3A2 (* denotes difference from control incubations without antibody). Lower panels show regression analysis of CYP1A2 and 3A1 with each other and with the Vmax observed in microsomes from each rat. An apparent outlier is depicted for female rats as X. If this is removed the relationship for CYP1A2 becomes significant (r2 = 0.34) and relationship strengthened (r2 = 0.56) for CYP3A1. Figure 9. Effect of high-fat diet on the protein expression in the liver of obese male (A), female mice (B) and diabetic male mice (C). Data are presented as percent of control (mean±SE). *p < 0.05 compared to the corresponding control using unpaired Student’s t-test.

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Table 1: Composition of the dietary components u given to the rodents Component

Calories Provided % Rat diet

Protein Carbohydrate Fat

Standard Chow 3.35 Kcal/g 29.8 56.8 13.4

High-Fat Chow (HF) 4.6 Kcal/g 19 36 44.8

Mouse diet Protein Carbohydrate Fat

Lean Chow 3.85 Kcal/g 20 70 10

HF Chow 5.24 Kcal/g 20 20 60

Table 2: Rat primer sequences used for real-time PCR10,17,18 Gene Cyp1a2 Cyp3a2 Cyp2c11 Cyp2c12 β-actin

Forward Primer 5`-CGC CCA GAG CGG TTT CTT A- 3` 5`-GCT CTT GAT GCA TGG TTA AAG ATT TG- 3` 5` -CAC CAG CTA TCA GTG GAT TTG G- 3` 5` -TAT AAA CTC AAT ACG TTC TGA G- 3` 5` -CCA GAT CAT GTT TGA GAC CTT CAA - 3`

Reverse Primer 5`-TCC CAA GCC GAA GAG CAT C- 3` 5`-ATC ACA GAC CTT GCC AAC TCC TT- 3` 5` -GTC TGC CCT TTG CAC AGG AA- 3` 5` -TTT TAC ATT AAC TTC AGA AAC TG- 3` 5` -GTG GTA CGA CCA GAG GCA TAC A- 3`

Table 3: Plasma biochemistry results (geometric means, with the percent coefficient of variation of the geometric mean in parentheses).

Male rats

18-week dietary phase

14-week dietary phase

Analyte

a d

Insulin, ng/mL Glucose, mM AST:ALT ratio AST, U/L ALT, U/L Cholesterol, mg/dL Triglyceride, mg/dL Leptin, ng/mL Insulin, ng/mL Glucose, mM AST:ALT ratio AST, U/L ALT, U/L Cholesterol, mg/dL Triglyceride, mg/dL Leptin, ng/mL

Female rats

Control

HF

Control

HF

0.87 (26.7) 12.2 (10.2) 1.86 (31.1) 379.9 (40.1) 204.13 (83.5) 52.8 (22.9) 106.3 (16.8) 3.69 (34.9) 0.67 (27.1) 10.3 (11.9) 2.8 (21.9) 170.4 (6.31) 61.1 (18.9) 66.1 (7.5) 106.2 (22.4) 17.2 (28.7)

1.72 (81.8) 12.8 (5.3) d 4.01 (55.5) 131.8 (124.5) 32.8 (59.7) 93.4 (12.6) a,d 148.412 (52.5) 39.2 (131)a 0.91 (11.3) 10.6 (7.2) 3.3 (23.8) 148.1 (69.6) 44.7 (45.5) 69.4 (13.6) 125.4 (34.4) 10.6 (23.6)

0.98 (129) 10.1 (12.3) 1.8 (9.6) 87.9 (40.2) 47.8 (39.3) 58.7 (32.1) 111.6 (25.9) 4.25 (25.1) 0.78 (17.7) 9.2 (6.9) 2.2 (22.1) 113.27 (55.4) 49.27 (60) 50 (11.2) 147.8 (40.2) 9.21 (44.7)

1.91 (70.7) 10.5 (10.6) 3.1 (14.2) a 136.6 (46.3) 44.9 (40.9) 69.4 (16.2) 201.9 (7.5) a,d 29.5 (32.7)a,d 0.73 (38.8) 9.2 (11.4) 2.8 (26.4) 149.8 (66.6) 51.8 (68.0) 71.7 (9.8) a 95.7 (30.9) 4.02 (25.3)

P < 0.05 versus control of same gender (14 wk vs 14 wk and 18 wk vs 18 wk) P < 0.05 HF versus HF of same gender (14 wk vs 18 wk )

Table 4: Enzyme kinetic parameters (mean±SD) for MEGX formation by rat liver microsomes from four individual rats, based on fitting of the simple Michaelis Menten equation to the MEGX formation rates vs. lidocaine concentrations. Male rats 14 wk Control Vmax, nmol/min/mg protein Km, mM CLint, µL/min/mg protein a

Female rats 18 wk

Obese

Control

14 wk Obese

Control

18 wk Obese

Control

Obese

a

0.99±0.09

1.18±0.26

0.54±0.021

0.82±0.47

0.51±0.03

0.52±0.00

1.92±0.10

1.48±0.94

1.92±0.10

2.27±0.50

a

3.95±0.79

3.38±1.99

1.04±0.025 0.88±0.83

0.51±0.003 0.50±0.003

0.53±0.01

0.53±0.02

5.32±1.56a

7.55±1.55

6.25±3.38

3.91±1.00

7.62±0.30

2.68±0.85

Significantly differ from control rats.