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. 2021 Mar 9;4(2):757–764. doi: 10.1021/acsptsci.0c00213

Peripherally Selective CB1 Receptor Antagonist Improves Symptoms of Metabolic Syndrome in Mice

Nayaab Khan 1, Lucas Laudermilk 1, Jalen Ware 1, Taylor Rosa 1, Kelly Mathews 1, Elaine Gay 1, George Amato 1, Rangan Maitra 1,*
PMCID: PMC8033769  PMID: 33860199

Abstract

graphic file with name pt0c00213_0006.jpg

Metabolic syndrome (MetS) is a complex disorder that stems from the additive effects of multiple underlying causes such as obesity, insulin resistance, and chronic low-grade inflammation. The endocannabinoid system plays a central role in appetite regulation, energy balance, lipid metabolism, insulin sensitivity, and β-cell function. The type 1 cannabinoid receptor (CB1R) antagonist SR141716A (rimonabant) showed promising antiobesity effects, but its use was discontinued due to adverse psychiatric events in some users. These adverse effects are due to antagonism of CB1R in the central nervous system (CNS). As such, CNS-sparing CB1R antagonists are presently being developed for various indications. In this study, we report that a recently described compound, 3-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[6-(difluoromethoxy)pyridin-3-yl]urea (RTI1092769), a pyrazole based weak inverse agonist/antagonist of CB1 with very limited brain exposure, improves MetS related complications. Treatment with RTI1092769 inhibited weight gain and improved glucose utilization in obese mice maintained on a high fat diet. Hepatic triglyceride content and steatosis significantly improved with treatment. These phenotypes were supported by improvement in several biomarkers associated with nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). These results reinforce the idea that CB1 antagonists with limited brain exposure should be pursued for MetS and other important indications.

Keywords: CB1, peripheral, antagonist, diabetes, steatosis, NAFLD

Introduction

Over 40% of Americans are obese (https://www.cdc.gov/obesity/data/adult.html) and thus are at high risk of developing metabolic syndrome (MetS). MetS is a complex illness that is often associated with insulin resistance and type 2 diabetes mellitus, liver diseases including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), cardiovascular diseases, and higher incidence of premature death.1,2 Lifestyle modification comprising diet and exercise is often the first approach to manage MetS. However, long-term adherence and compliance are often challenging to a large number of patients who often have other debilitating conditions such as severe knee or back pain. Patients are often prescribed multiple drugs to treat various aspects of MetS, so an ideal drug for this illness should improve several diseases associated with this condition.3,4 Evidence from clinical and preclinical studies indicate that type 1 cannabinoid receptor (CB1R) antagonists can produce beneficial effects through a combination of central and peripheral mechanisms.5−8

Cannabinoid receptors belong to the endocannabinoid (EC) system, which consists of receptors, transporters, endocannabinoids, and the enzymes involved in synthesis and degradation of endocannabinoids.2 To date, two different receptors have been identified: CB1R and CB2R. Both CB1R and CB2R are G protein-coupled receptors primarily activating inhibitory G proteins (Gi/o).2 Of these two receptors, CB1R is extensively expressed in the central nervous system (CNS). It is also expressed peripherally on a number of metabolically important tissues such as liver, pancreas, skeletal muscle, and adipose. On the other hand, CB2R is primarily expressed on immune cells. Both receptors are activated by natural and synthetic cannabinoids including (−)-trans-Δ9-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana.

Antagonism of CB1R is a clinically proven strategy to treat obesity and related diseases.4,7,8 The CB1R inverse agonist/antagonist SR141716A (rimonabant) was approved as a drug to treat obesity in Europe (Figure 1).9 However, antagonism of central CB1R receptors with rimonabant was linked to certain dose-dependent adverse psychiatric events including anxiety, depression, and suicidal ideation in a subset of patients, which led to its withdrawal.10 Ongoing research has demonstrated that peripheral populations of CB1R can be targeted selectively to produce beneficial effects including weight loss, reduction of insulin resistance, reduced alcoholic steatosis, blockade of pulmonary fibrosis, and improved glucose regulation.11−15 Some of these effects are independent of weight loss induced by this class of agents. As such, efforts are presently underway to produce antagonists of CB1R with limited brain exposure.

Figure 1.

Figure 1

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Structures of CB1R antagonists.

Several groups are actively pursuing CB1R antagonists with limited brain penetration.16,17 Our group has recently described 3-{1-[8-(2-chlorophenyl)-9-(4-chlorophenyl)-9H-purin-6-yl]piperidin-4-yl}-1-[6-(difluoromethoxy)pyridin-3-yl]urea (RTI1092769), a pyrazole inverse agonist/antagonist of this receptor based on the purine otenabant (CP-945598, Figure 1).18 Otenabant was tested in a Phase III clinical trial by Pfizer as an antiobesity agent. While the compound demonstrated significant clinical efficacy and arguably a better side-effect profile in patients, its development was discontinued by Pfizer due to regulatory concerns.19 The purine urea RTI1092769 has several druglike properties and limited CNS exposure as demonstrated in our previous studies. Further, it also blocked alcoholic steatosis in mice leading us to postulate that the compound might improve MetS associated liver disease.18 Therefore, studies were undertaken in the mouse diet-induced obesity (DIO) model, which has an excellent correlation with human clinical outcome. Treatment with RTI1092769 produced several beneficial effects that are supportive of further clinical development of this compound.

Results

RTI1092769 Inhibited Weight Gain, Normalized Liver Weight, and Decreased Triglyceride Content in the Liver

The DIO model is a clinically relevant model of MetS associated complications. This model was used for efficacy studies with RTI1092769. Male C57BL/6J mice on a high fat diet (HFD) or standard diet (SD) were gavaged once daily with various doses of RTI1092769 or vehicle. As indicated in Figure 2A, RTI-1092769 decreased weight gain in HFD-fed mice in a dose-dependent fashion. Statistical significance was reached for all three dose groups. Treatment with the test agent leads to an ∼9% difference in body weight at the highest dose (1 mpk) compared to HFD controls. This inhibition of weight gain does not appear to be driven by changes in food intake (Figure 2B). Treatment with the test agent also significantly decreased normalized liver weights (Figure 2C) and reduced hepatic triglyceride content (Figure 2D). Taken together, these data suggested that the test agent produced a positive impact on liver steatosis in DIO mice.

Figure 2.

Figure 2

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Studies of RTI1092769 in DIO. (A) Male mice on SD or HFD were treated with vehicle and various doses of RTI1092769 via oral gavage once daily. Statistically significant dose-dependent inhibition of weight gain was noted at each dose (p < 0.0001 vs HFD + vehicle group). (B) RTI1092769 (-769) did not significantly impact food intake. Food intake was measured eight times during the study, and group averages are presented. (C) RTI1092769 (-769) significantly reduced liver weights. Liver weights were normalized to tibia length at the end of the study. (D) Hepatic triglyceride content was measured using an enzymatic assay and normalized to protein concentration per sample. RTI1092769 (-769) decreased hepatic triglyceride content. (Significance: *p < 0.05, ****p < 0.0001; vs HFD + vehicle group.) Data are expressed as mean ± SEM for each analysis.

RTI1092769 Improved Glucose Utilization

Insulin resistance is associated with MetS and is present in the mouse DIO model. Therefore, the effects of RTI1092769 were assessed by measuring fasting glucose levels and also on glucose utilization following a bolus challenge in the oral glucose tolerance test (oGTT). Treatment with the test agent improved glucose utilization. Fasting glucose levels were lower with RTI1092769 treatment (Figure 3A) as well. Similarly, positive trends were also noted in the oGTT. Area under the curve (AUC) analyses (Figure 3B, C) confirmed statistically significant changes to oral glucose levels following a bolus challenge at the 0.1 and 1 mpk dose levels.

Figure 3.

Figure 3

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Effect of RTI1092769 (-769) on blood glucose. (A) Animals were fasted for 16 h and blood glucose was measured as described under Materials and Methods. (B, C) Animals were administered a bolus oral dose of glucose and glucose levels were monitored over 120 min. Data are expressed as area under the curve (AUC). SD, standard diet; HFD, high fat diet; veh,vehicle; -769, RTI1092769. Significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; vs HFD + vehicle group. Data are expressed as mean ± SEM for each analysis.

RTI1092769 Reduced Liver Steatosis and Associated Biomarkers

Liver diseases including NAFLD and NASH are common comorbid conditions in MetS patients. As such, the effect of RTI1092769 was evaluated in the context of NAFLD in DIO mice.

Semiquantitative histology was performed on liver tissue sections stained with Oil Red O to assess whether treatment with RTI1092769 improved liver steatosis and biochemical end points associated with the disease. As demonstrated in Figure 4A and B, treatment with RTI1092769 improved liver steatosis in a dose dependent manner.

Figure 4.

Figure 4

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Effect of RTI1092769 on hepatic steatosis and biomarkers of cellular injury. (A) Liver sections from animals administered SD + vehicle (SD), HFD + vehicle (HFD), and HFD containing various doses of RTI1092769 (0.1, 0.3, 1 mpk) were stained with Oil Red O. Dose dependent decrease of staining was noted. (B) Staining levels were quantified as described in Materials and Methods. Scale bar = 200 μm. Mice on HFD had elevated levels of AST (C) and ALT (D) in plasma compared to animals on SD. Treatment with various doses of RTI1092769 (-769) reduced circulating levels of these enzymes as assessed using enzymatic assays compared to animals on HFD administered vehicle only. No significant impact was noted on LDH (E). Significance: *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001; vs HFD + vehicle group. Data are expressed as mean ± SEM for each analysis.

Injury to the liver often leads to increased circulating levels of certain prognostic enzymes in blood such as transaminases alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Similarly, cell death leads to increased circulatory levels of lactate dehydrogenase (LDH). Therefore, clinical chemistry assays were used to determine circulating levels of these three enzymes. As demonstrated in Figure 4C and D, DIO was associated with increased circulating levels of ALT and AST compared to lean animals on SD, and treatment with RTI1092769 suppressed these levels. On average, ∼50% less circulating AST and ALT levels were noted following 1 mpk treatment compared to animals on HFD receiving vehicle only. Levels of LDH were decreased as well but statistical significance was not reached.

RTI1092769 Altered Expression of Genes Associated with Metabolic Syndrome in the Liver

The promising phenotypic outcomes associated with RTI1092769 led us to explore molecular end points affected by improved MetS in DIO mice. As such, expression of several genes associated with lipid synthesis, inflammation, cellular stress, and glucose metabolism were evaluated within the liver. Real time RT-PCR analyses indicated that treatment with the test agent altered several but not all genes tested. These discrete changes supported previous phenotypic findings from the study as discussed below.

Sterol regulatory element binding proteins (SREBPs) are transcription factors that regulate lipid homeostasis by controlling a range of enzymes required for endogenous cholesterol, fatty acid (FA), triacylglycerol, and phospholipid synthesis.20 Previous studies indicated that de novo lipogenesis is affected by EC signaling within the liver through the lipogenic transcription factor serum response element binding protein 1c (Srebf1) and some of its targets including fatty acid synthetase (Fasn) and acetyl-CoA carboxylase-1 (Acaca).21 Our data (Figure 5A, B) demonstrate that treatment with RTI1092769 led to reduced expression of both Srebf1 and Srebf2. Expression of Fasn and Acaca (targets of SREBP-1c) were reduced as well (Figure 5C, D).

Figure 5.

Figure 5

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Real-time RT-PCR analyses of genes associated with hepatic lipid regulation, hepatic steatosis, and NASH. Samples of RNA were reverse transcribed and amplified using primers specific to (A) Srebf1, (B) Srebf2, (C) Fasn, (D) Acaca, (E) Foxo1, (F) Pparg, (G) Ppargc1a, (H) Ppara, (I) Hnf4a, and (J) Gsk3b along with a housekeeping gene (Rps20) as described in Materials and Methods. Significance: *p < 0.05,**p < 0.01, ***p < 0.001, ****p < 0.0001; vs HFD + vehicle group. Data are expressed as mean ± SEM for each analysis.

Forkhead family transcription factor 1 (Foxo1) can produce pleiotropic effects in the liver, and upregulation of this gene in NAFLD is associated with exacerbation of hepatic steatosis in some studies.22 Treatment of DIO mice with RTI1092769 reduced expression of this gene (Figure 5E), which could be associated with reduced accumulation of cholesterol and triglycerides and decreased steatosis. RTI1092769 also significantly decreased expression of Pparg (Figure 5F), an important regulator in the development of steatosis.23 We noted significant reduction of the transcriptional coactivator PPARG Coactivator 1 alpha (Ppargc1a) and Ppara expression with the 1 mpk dose of RTI1092769 (Figure 5G, H), which aligns with previous reports that suggest cannabinoids increase PPAR activity.24

While the DIO model is not a frank model of NASH with only mild inflammation noted using molecular approaches like RT-PCR, we nevertheless evaluated several markers of NASH and immune response. Treatment with RTI1092769 significantly reduced expression of Hnf4a (Figure 5I), a critical regulator of NASH pathogenesis.25 We also monitored the effect of RTI1092769 on DIO induced upregulation of Gsk3b (Figure 4J), a gene linked to lipoapoptosis in NASH.26 Mice on HFD had significantly higher expression of Gsk3b relative to control animals on SD, and treatment at the 1 mpk dose significantly lowered expression relative to vehicle controls. We investigated two separate biomarkers of macrophage infiltration: MCP-1 (Ccl2) and F4/80 (Adgre1) (Figure S1 A, B). These two genes were not significantly altered in DIO animals compared to lean controls. Expression of Tgfb1 was also not significantly higher in the livers of obese animals (Figure S1C). Markers of neutrophils (Cxcr1, Mpo) and T-cells (Cd69) were not expressed at detectable levels in our liver samples, and we did not observe appreciable Il6 expression. We also noted significant impacts of RTI1092769 on Ide and Hnf1b (Figure S2). We see a significant impact of high fat diet and RTI1092769 on Insulin degrading enzyme (Ide) expression and a significant impact of RTI1092769 on Hnf1b expression.

Discussion

Withdrawal of rimonabant was a setback that led to discontinuation of several advanced CB1R antagonist programs aimed at developing CB1 antagonists as antiobesity agents.10 However, over the past decade or so, considerable data have emerged to suggest that CB1R is still a relevant target for a number of different important diseases. Data from preclinical studies indicate that peripheral populations of CB1R can be targeted for several indications using compounds that have limited brain penetration.10 Indeed, research is underway to develop second and third generation CB1R antagonists that have little to no brain penetration for diverse indications including obesity, liver diseases, pulmonary fibrosis, alcoholic liver disease, and others.16,17 Whether a purely peripheralized compound can be developed remains controversial because the blood-brain barrier is not continuous within the CNS and there are anatomical areas where this protective membrane is absent or discontinuous such as the circumventricular regions.27 Further, studies have also suggested that antagonism of CB1Rs within the brain is a prerequisite for certain effects such as anorexia and associated weight loss.28,29 While these are all very important observations, we argue that antagonism of a subpopulation of central CB1Rs is unlikely to be a major concern in human patients based on significant clinical evidence and preclinical studies. For example, the number of adverse psychiatric effects noted with rimonabant were far less at the 5 mg clinical dose compared to the 20 mg dose over a 2 year period.30 Further, these psychiatric adverse effects emerged early in clinical trials with rimonabant, suggesting the possibility of removing vulnerable patients through proper monitoring and evaluation.30,31 These studies taken together argue in favor of second-generation compounds that have greatly reduced brain penetration, particularly since preclinical studies indicate that antagonism of ∼20% of central CB1Rs might be adequate for eliciting centrally mediated effects through this receptor.32 Another possibility with regard to targeting CB1R is to develop neutral antagonists of this receptor. Most clinically tested compounds to date have been inverse agonists, which can interfere with basal activity of CB1Rs within the CNS. A neutral antagonist, on the other hand, is expected to selectively antagonize CB1R activated by dysregulated EC signaling selectively without interfering with basal activity of the receptor.33−35

Our group discovered RTI1092769, a peripherally selective CB1R antagonist with reduced brain penetration. Also, this compound has significantly reduced inverse agonist activity compared to rimonabant, which may further limit adverse events.18 In addition, this compound has good druglike properties. In the studies reported, RTI1092769 was successfully tested in a mouse DIO model that recapitulates various aspects of MetS including insulin resistance and NAFLD. Treatment with RTI1092769 produced dose-dependent inhibition of weight gain without significant impact on food intake. Otenabant, the antiobesity agent RTI1092769 was based on, likewise halted weight gain in a mouse model of DIO.36 Otenabant also does not appear to impact rodent feeding behavior at a 1 mpk dose.37 Higher doses of otenabant do impact food intake, though this effect is mostly significant in the first days of dosing and wanes over time, but the metabolic improvements continue arguing for hypophagia-independent effects of this and other CB1R antagonists through inhibition of peripheral receptor populations.36 RTI1092769 also improved glucose utilization and reduced hepatic steatosis as well as triglyceride content in the liver. Circulating biomarkers of liver injury associated enzymes (AST, ALT) were significantly reduced.

Within the liver, obesity and metabolism are associated with enhanced expression of genes related to synthesis of lipids. At the molecular level, treatment with RTI1092769 reduced expression of SREBP genes within the liver, which suggests reduced de novo lipid synthesis in agreement with past results.21 Srebf1 encodes both SREBP-1a and SREBP-1c. SREBP-1a is involved in the synthesis of both FA and triglycerides, whereas SREBP-1c features mostly in the FA synthesis pathway and SREBP2 regulates triglyceride production.20 Our data support previous results showing that CB1R activation leads to increases in Srebf1, Fasn, and Acaca and that CB1R antagonists can mitigate this effect.21

We additionally observed significant impacts on expression of genes that drive steatosis, Foxo1 and Pparg. Blockade of FOXO1 has been shown to reduce phenotypes of NAFLD and NASH, including endoplasmic reticulum stress, ALT, AST, triglycerides, and body weight.38 An impact on FOXO1 phosphorylation has previously been demonstrated after treatment with a CB1R inverse agonist in a DIO mouse model,39 and our results expand on additional impacts of CB1R activity on Foxo1 gene expression level. Therefore, CB1R antagonism appears to reduce de novo lipogenesis associated genes and this observation provides a mechanistic rationale for the development of this class of compounds for liver diseases.

Certain markers of inflammation and NASH were also diminished upon treatment with this antagonist in the liver. Significant reductions of Gsk3b and Hnf4a suggest potential utility of this drug in treating NASH. Hnf4a in particular is suggested to be a central gene in the pathogenesis of NASH.25 In addition to its role in lipoapoptosis, Gsk3b inhibition has also been shown to reduce hepatic apoptosis and leukocyte infiltration into the liver in a model of sepsis.40

RTI1092769 also significantly impacted expression of Ide and Hnf1b. IDE is a rate-limiting enzyme involved in insulin degradation, and previous reports using rodent models of obesity have shown increased Ide expression in the liver.41,42 However, other reports suggest that obesity reduces insulin degradation,43 so this effect may be model-dependent but worthy of follow-up studies. Obesity and steatosis are typically associated with reduced HNF1B, though our data shows a trend upward among animals on a high fat diet.44−46 Additional studies will be necessary to address this discrepancy and to test agreement between gene expression and protein levels in this model.

Together, these results demonstrate that RTI1092769 drives improvement of MetS phenotypes ranging from gene expression changes through clinically relevant biomarkers in the plasma and liver. Future studies will further explore the impact of RTI1092769 in rodent models of alcoholic and nonalcoholic inflammatory liver diseases.

Materials and Methods

Efficacy Testing in DIO Mice

All studies were performed in accordance with guidelines established by the Office of Laboratory Animal Welfare (OLAW), and protocols were approved by the institutional animal care and use committee (IACUC). Data reported herein follow ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines for reporting on animal studies. Preconditioned male C57BL/6J mice were purchased from Jackson Laboratories (Bar Harbor, ME) at 18 weeks of age. A total of nine animals per group was used for this study based on power analysis assuming medium effect size to reduce animal usage. These animals were maintained either on a standard diet with 10% fat (D12450B) ad libitum or on 60% fat (D12492) chow from Research Diets (New Brunswick, NJ). Animals were acclimated to the facility for 2 weeks and then randomly assigned to various test groups. Animals were dosed by intragastric gavage once daily with either vehicle (0.5% sodium carboxymethylcellulose with 1% NMP and 0.3% tween 80 in deionized water) or RTI1092769 (0.1, 0.3, or 1 mg/kg) suspended in the vehicle. Dosing volume was maintained at 5 mL/kg. Body weights were recorded at regular intervals. Food intake was measured eight times through the study by measuring the weight of food provided at the time of a cage change and the weight of food left in the cage 2–3 days later. Averages for each group were determined. At termination of study, plasma and vital organs were collected and preserved. Tissue sections (10 μm) from frozen livers were prepared and analyzed following hematoxylin-eosin staining and Oil Red O staining using standard procedures. Images were acquired using an Olympus DP70 camera system mounted to an Olympus IX51 inverted microscope. Digitized images of Oil Red O stained tissues were assessed for degree of steatosis using ImageJ (NIH, Bethesda, MD). Eight images were taken per animal using a 10× objective, and the scale within ImageJ was set using embedded scale bars in each image. The RGB Stack function was used to obtain a three-layered image (Image > Type > RGB Stack). When viewing the blue image, the threshold was set such that Oil Red O staining was highlighted in red (Image > Adjust > Threshold). Next, the red highlighted area of each image was measured (Analyze > Measure). “Area” and “Limit to Threshold” were both selected in the measurement settings (Analyze > Set Measurement). The above steps were combined into a macro for analysis:macro “OilRedO [a]″ {run(“RGB Stack”); setSlice(3);setAutoThreshold(“Default”);//run(“Threshold···″); run(“Measure”);}

Data were graphed using Prism software (GraphPad, La Jolla, CA).

Oral Glucose Tolerance Test (GTT)

Animals were food-deprived for 16 h prior to testing. Animals were orally dosed with a 2 g/kg bolus dose of glucose in water. Using a fine scissors, tails were snipped (<1 mm) once; the same snipped area was used for subsequent blood collection. A very small sample size of approximately 2–5 μL of blood was used. Measurements were performed using a glucometer and standard blood glucose measurement strips at 15, 30,60, 90, and 120 min post challenge.

Biochemical Measurements

Blood samples were collected from all animals at the time of scheduled necropsy via cardiac puncture into SAI Infusion Technologies collection microtubes with lithium heparin as the anticoagulant and centrifuged at approximately 2800g for approximately 10 min at 4 °C to obtain plasma within 1 h of collection. Plasma samples were analyzed for aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels by an independent laboratory (IDEXX Bioanalytics, Sacramento, CA) using good laboratory practices (GLP). Triglyceride content in samples was measured using a commercially available colorimetric kit from Cayman Chemicals (Ann Arbor, Michigan).

RNA Isolation and RT-PCR

Snap frozen tissues were used to isolate DNA-free total RNA using a commercially available kit (RNeasy Plus Mini Kit, Qiagen, Carlsbad, CA). Samples of RNA were reverse transcribed using the Applied Biosystems High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Waltham, MA). Primers for amplification were purchased from Integrated DNA Technologies (Coralville, IA) for genes as described in Table S1. An amount of 50 ng of each cDNA sample was amplified using the Applied Biosystems PowerUp SYBR Green Master Mix (ThermoFisher Scientific, Waltham, MA). The reactions were performed on the LightCycler 96 instrument (Roche, Indianapolis, IN).

The housekeeping gene Rps20 (NCBI Gene ID: 67427) was used for normalization of gene expression between samples. The resulting gene expression was analyzed using the 2–ΔΔCT method.47

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 9.0. Analyses were performed using ANOVA followed by Fisher’s multiple comparisons test, which is appropriate for analyzing such data sets.48,49

Acknowledgments

This research was funded by research grants AA022235, DK124615, DK103625, and DK100414 to R.M. from NIH. N.K. and L.L. contributed equally to this project.

Glossary

Abbreviations

CB1R

type 1 cannabinoid receptor

CNS

central nervous system

MetS

metabolic syndrome

DIO

diet-induced obesity

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

oGTT

oral glucose tolerance test

SD

standard diet

HFD

high fat diet

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.0c00213.

  • Target genes and primer sequences for qPCR analysis and supplemental PCR results (PDF)

Author Contributions

N.K. and L.L. are equal contributors. N.K., L.L., J.W., T.R., K.M., E.G., G.A., and R.M. participated in the design and execution of the experiments described herein. N.K., L.L., and R.M. drafted and edited the manuscript.

The authors declare no competing financial interest.

Supplementary Material

pt0c00213_si_001.pdf (956.7KB, pdf)

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