*Article* **Imipramine Accelerates Nonalcoholic Fatty Liver Disease, Renal Impairment, Diabetic Retinopathy, Insulin Resistance, and Urinary Chromium Loss in Obese Mice**

**Geng-Ruei Chang <sup>1</sup> , Po-Hsun Hou 2,3,4, Chao-Min Wang <sup>1</sup> , Jen-Wei Lin <sup>5</sup> , Wei-Li Lin 5,6, Tzu-Chun Lin <sup>1</sup> , Huei-Jyuan Liao <sup>1</sup> , Chee-Hong Chan 7,\* and Yu-Chen Wang 8,9,10,11,\***


**Abstract:** Imipramine is a tricyclic antidepressant that has been approved for treating depression and anxiety in patients and animals and that has relatively mild side effects. However, the mechanisms of imipramine-associated disruption to metabolism and negative hepatic, renal, and retinal effects are not well defined. In this study, we evaluated C57BL6/J mice subjected to a high-fat diet (HFD) to study imipramine's influences on obesity, fatty liver scores, glucose homeostasis, hepatic damage, distribution of chromium, and retinal/renal impairments. Obese mice receiving imipramine treatment had higher body, epididymal fat pad, and liver weights; higher serum triglyceride, aspartate and alanine aminotransferase, creatinine, blood urea nitrogen, renal antioxidant enzyme, and hepatic triglyceride levels; higher daily food efficiency; and higher expression levels of a marker of fatty acid regulation in the liver compared with the controls also fed an HFD. Furthermore, the obese mice that received imipramine treatment exhibited insulin resistance, worse glucose intolerance, decreased glucose transporter 4 expression and Akt phosphorylation levels, and increased chromium loss through urine. In addition, the treatment group exhibited considerably greater liver damage and higher fatty liver scores, paralleling the increases in patatin-like phospholipid domain containing protein 3 and the mRNA levels of sterol regulatory element-binding protein 1 and fatty acid-binding protein 4. Retinal injury worsened in imipramine-treated mice; decreases in retinal cell layer organization and retinal thickness and increases in nuclear factor κB and inducible nitric oxide synthase levels were observed. We conclude that administration of imipramine may result in the exacerbation of nonalcoholic fatty liver disease, diabetes, diabetic retinopathy, and kidney injury.

**Citation:** Chang, G.-R.; Hou, P.-H.; Wang, C.-M.; Lin, J.-W.; Lin, W.-L.; Lin, T.-C.; Liao, H.-J.; Chan, C.-H.; Wang, Y.-C. Imipramine Accelerates Nonalcoholic Fatty Liver Disease, Renal Impairment, Diabetic Retinopathy, Insulin Resistance, and Urinary Chromium Loss in Obese Mice. *Vet. Sci.* **2021**, *8*, 189. https:// doi.org/10.3390/vetsci8090189

Academic Editors: Ana Faustino, Paula A. Oliveira and W. Jean Dodds

Received: 6 July 2021 Accepted: 7 September 2021 Published: 9 September 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Keywords:** fatty liver disease; glucose; imipramine; insulin; obesity; renal impairment; retinal injury

#### **1. Introduction**

Imipramine, derived from dibenzazepine, is a prototypical tricyclic antidepressant (TCA). TCAs have a structure that is close to that of a phenothiazine, and they contain a tricyclic ring system. The central ring contains an alkylamine substituent [1]. On the basis of animal studies, imipramine is known as a candidate therapy for antipredator defensive behavior, sleep deprivation, and related anxiety- and depressive-like behaviors [2–4]. In individuals without depression, imipramine has no impact on arousal or mood; however, it may act as a sedative. In people with depression, their mood is positively affected by imipramine use [5]. Thus, it is among the most effective drugs for the treatment of severe long-term depression [6]. TCAs strongly inhibit the reuptake of norepinephrine and serotonin. 3◦ TCAs (i.e., tertiary TCAs), including amitriptyline and imipramine, have stronger inhibition of serotonin reabsorption than 2◦ TCAs, such as desipramine and nortriptyline [7]. The particular mechanism explaining the treatment-related benefits of imipramine is still not well understood. TCAs are able to blockade muscarinic, histamine H1, and α1-adrenergic receptors, which explains their anticholinergic, sedative, and hypotensive impacts, respectively [8]. The anticholinergic and sedative actions of imipramine are less pronounced than those of other 3◦ TCAs such as clomipramine and amitriptyline. In children, imipramine is used as an antidepressant and as a treatment for bedwetting. Off-label uses include the (1) treatment of panic disorders without or with agoraphobia, (2) second-line treatment of attention deficit hyperactivity disorder in young people, (3) management of bulimia, (4) treatment of postacute and posttraumatic stress, and (5) short-term treatment of acute depression in patients with schizophrenia and bipolar disorder [9–12]. Side effects such as drowsiness, dry mouth, excitement, and weight or appetite alterations may be induced by imipramine [13]. In-depth explorations of the side effects of imipramine have been inconclusive.

Obesity is a major risk factor for various conditions related to metabolic syndrome, such as hyperlipidemia, nonalcoholic fatty liver disease (NAFLD), insulin resistance (IR), and type 2 diabetes [14]. An imbalance between expenditure and consumption causes an increase in energy storage within the body, which could lead to weight gain and obesity [15]. Moreover, recent studies have suggested that atypical glucose–insulin homeostasis is linked to various depression severity indicators and could be a neurohormonal mediator of certain depression symptoms, such as neurocognitive impairments, and disorders that are comorbid with depression, such as cardiovascular disease [4,5,16]. Gupta et al. reported a significant association between imipramine administration and increased blood glucose levels of rabbits. They also indicated that hyperglycemic responses increased more when adrenaline and imipramine were administered simultaneously than when they were administered alone [17]. In addition, some scholars have observed the effects that the tricyclic antidepressants trimipramine and amitriptyline have on glucose homoeostasis in rats, indicating that their long-term administration could cause IR and diabetes [18]. Obesity is regularly linked to NAFLD, a group of conditions that can take the form of cirrhosis, nonalcoholic steatohepatitis, and fatty liver [19]. As well as drug-related factors including liver metabolism, daily dose, and chemical structure, different risk factors heighten the likelihood of liver injury induced by drugs [20]. How patients with fatty liver further develop nonalcoholic steatohepatitis is complicated; the mechanism could involve various factors such as the overproduction of reactive oxygen species, lowered reactive oxygen species (ROS) detoxification, and heightened profibrogenic and proinflammatory cytokine release [21]. In addition, tricyclic antidepressants may have adverse health effects associated with kidney damage, diabetes, and FLD, and their prolonged use may impede weight control and aggravate diabetes [22].

Additionally, obesity is a major renal disease risk factor. Similar to hypertension and diabetes, it heightens the risk of end-stage renal disease (ESRD) and chronic kidney disease (CKD) [23]. Moreover, in ESRD or CKD, associations exist between depression and unfavorable quality of life as well as poor health outcomes [24]. One study highlighted that symptoms of depression could be related to reduced kidney function and dialysis commencement; such symptoms should be monitored in all disease stages in CKD patients [25]. Long-term hyperglycemia and IR are among the key factors related to blood– retina barrier dysfunction and retina damage linked to diabetes [26]. The retina is crucial to human vision, and diabetes is, of course, a risk factor for diabetic retinopathy (DR). Heightened inflammation and oxidative stress are suggested to be the major mechanisms behind neural retina damage in diabetes [27,28]. Research has indicated that within 3 years of antipsychotic medication initiation in patients with schizophrenia who have visual disturbances, visual cortex function and retinal thickness gradually deteriorate [29]. Other studies have indicated that processes including vasorelaxation through alpha adrenergic blocking or direct impacts on the retinal vascular endothelium could be the reasons for cystoid macular edema induced by antidepressant use [30]. Thus, we sought to understand whether imipramine affects the retina and to elucidate the possible mechanisms.

The adverse effects of imipramine likely arise from differences between the conditions under metabolic syndrome that affect body weight and hyperglycemia associated with imipramine. Thus, in our investigation, mice were fed a high-fat diet (HFD) and administered imipramine to mimic obesity; this was conducted to investigate imipramine's effects on patients with obesity concomitant with psychotic disorders. The majority of research on the metabolism-related side effects of imipramine has centered on the central nervous system [4,31,32]. We examined glucose levels, lipid metabolism, and oxidative stress, and then we explored the effects of these on the kidneys caused by abnormal metabolism, particularly related to chromium; this topic is little understood. Chromium is involved in normal lipid, protein, and carbohydrate metabolism and can benefit individuals with diabetes, glucose intolerance, obesity, or nephropathy [22]. These results provide a deeper insight into metabolic impacts and the mechanisms of fatty liver as well as kidney damage stemming from chronic imipramine use as an antipsychotic in humans or animals. We also conducted investigations on whether imipramine exacerbated metabolic abnormalities, chromium changes, oxidative stress, liver and kidney damage, and DR.

#### **2. Materials and Methods**

#### *2.1. Animals with Feed-Induced Obesity Administered Imipramine*

Five-week-old male C57BL/6J mice were procured from Taiwan's National Laboratory Animal Center. Their treatment accorded with the Taiwanese government's Guidelines for the Care and Use of Laboratory Animals. The protocol for using the experimental mice was approved after a review by the institutional animal care and use committee of the first author's university (approval no. 109019). For 12 weeks, the mice were continuously administered an HFD from PMI Nutrition International (St. Louis, MO, USA; diet 58Y1, comprising 23.6% protein, 60.0% of energy from fat, 2.6 µg/g Cr, metabolizable energy: 5.16 kcal/g). To induce obesity, the HFD was administered for 12 weeks (in the literature, 4 weeks was the associated diet duration) [33]. We then categorized the group into two subgroups. During the 8-week treatment, while remaining on the HFD, one group was administered 10 mg/kg of oral imipramine (Sigma-Aldrich, St. Louis, MO, USA) through daily gavage, and the other was administered oral saline. At the start of the treatment, the control mice weighed 28.9 ± 1.48 g, and those receiving imipramine weighed 28.35 ± 1.09 g (*p* > 0.05). The imipramine dose was decided after consultation of the literature on imipramine administration in mouse model studies related to diabetes-like status, neurological behavior observations, antidepressant effects, stress, and memory deficits [34–37]. In addition, obese mice were administered oral imipramine (5 mg/kg/day) in our preliminary investigation; however, differences in weight gain and body weight were not significant between the imipramine-treated and control mice (Figure S1). Accordingly, we adopted 10 mg/kg as the

imipramine dose in this study. Mice were individually housed in micro-isolation cages on HEPA-filtered and ventilated racks. The humidity (55% ± 5%) and temperature (22 ± 1 ◦C) were controlled, and the mice were maintained in a 12 h light/dark cycle. The mice freely accessed water and food. From the commencement of the experiment and on a weekly basis, food consumed and body weight were recorded. When the experiment was terminated, the mice were anesthetized for tissue and blood serum harvesting. Imipramine's impact on food intake, body weight, adipocyte concentration, levels of blood glucose, fatty liver, biochemical alterations, hepatic triglycerides, endocrine profiles, insulin signaling, and renal pathology was investigated. In addition, for urine sample collection, mice were kept for 12 h in individual metabolic cages (SN-783-0; AS ONE, Osaka, Japan) before they were killed.

#### *2.2. Measurement of Food Intake, Body Weight, and Leptin and Insulin Levels*

Every week of the study, we measured food consumed and body weight. To determine food intake, food that remained within each cage dispenser was weighed, as was the food that remained on the cage floor. Furthermore, after tissue and blood samples were harvested, serum leptin and insulin levels were measured following a 12 h fast using an ELISA mouse insulin kit (INSKR020; Crystal Chem, Downers Grove, IL, USA) and a leptin kit (INSKR020; Crystal Chem), respectively.

#### *2.3. Measurement of Serum and Hepatic Triglycerides, Creatinine, Alanine Aminotransferase, Blood Urea Nitrogen, Aspartate Aminotransferase, Serotonin, Soluble Leptin Receptor, and Fibroblast Growth Factor-21*

Serum triglyceride, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine levels were determined from the collected blood samples by means of an automated analyzer (Catalyst One Chemistry Analyzer, IDEXX Laboratories, Westbrook, ME, USA) and commercial kits under the guidance of manufacturer-approved protocols; the variation coefficient between and within analyses was under 2%. Following Folch et al., we extracted hepatic triglycerides in a 2:1 (vol/vol) mixture of chloroform and methanol [38]. Subsequently, extract solubilization was performed; the extracts were twice heated gradually to 90 ◦C over 5 min and subsequently cooled to room temperature. Insoluble materials were removed through centrifugation. Finally, colorimetric assay-based triglyceride analysis was conducted using the supernatant and a BioVision triglyceride quantification kit (Milpitas, CA, USA). Mouse enzyme-linked immunosorbent assay kits were employed to measure serotonin, serum soluble leptin receptor, and fibroblast growth factor-21 (FGF21) levels (EL-M0543, EL-M0545, and EL-M0435, respectively; Zgenebio Biotech, Taipei, Taiwan).

#### *2.4. Histological and Morphometric Analysis*

We weighed the retroperitoneal and epididymal fat pads and kidneys, spleen, liver, and heart; the weights are presented as a proportion (%) of body weight. Hematoxylin and eosin staining (BioTnA, Kaohsiung, Taiwan) was used to reveal hepatic fat infiltration, with scores of 0, 1, 2, 3, and 4 indicating no visible fat, <5% fat infiltration on the liver surface, 5% to 25% infiltration of fat, 25% to 50% fat infiltration, and >50% infiltration of fat, respectively [14,39]. Moreover, many epididymal white adipose tissue (EWAT) and retroperitoneal WAT (RWAT) sections were collected and analyzed in terms of adipocyte number and size. Hematoxylin and eosin staining of sections was conducted. We analyzed ≥10 fields (approximately 100 adipocytes) per slide for every sample [40,41]. Image acquisition was conducted by means of a high-resolution digital microscope (Moticam 2300, Motic Instruments, Canada), and adipocyte size analysis was conducted using Motic Images Plus 2.0. Correlations between the sizes of adipocytes and their distributions (%) were conducted for HFD controls and HFD mice administered imipramine. In addition, the right eyeball was fixed in a solution containing 4% paraformaldehyde, followed by dehydration, which involved passing the eyeball over several graded concentrations of ethanol (70%, 80%, 95%, and 100%). To render them transparent, the eyeballs were

dehydrated and placed in xylene. Subsequent to being embedded in paraffin, mouse eyeballs were sliced into sections for hematoxylin and eosin stains [42].

#### *2.5. Intraperitoneal Glucose Tolerance Test (IPGTT)*

After 7 weeks of the imipramine or saline protocol, we performed an IPGTT on mice with an obesity-like status that were starved overnight but had ad libitum water. The concentration used for the assay was 1 g of glucose per 1 kg of body weight; in animal obesity and diabetes models, this is appropriate for examining antidiabetes activity [22,43]. Blood glucose levels were determined 0, 30, 60, 90, and 120 min after intraperitoneal glucose was administered. We extracted tail vein blood using a One Touch glucose meter (LifeScan, Malpitas, CA, USA). Over the 0–120 min after administration of glucose, we conducted glucose tolerance tests based on area under the curve (AUC) values.

#### *2.6. Insulin Sensitivity (IS) and IR Indexes*

Fasting blood glucose is widely utilized to determine IR and IS indexes [15,33,39]. We therefore used these indexes for assessment of IR and β-cell secretion function following imipramine administration. The homeostasis model assessment-estimated IR (HOMA-IR) value was calculated as HOMA-IR = [fasting insulin (mU/L) × fasting glucose (mmol/L)]/22.5 [23]. The IS index was determined as (1/[fasting insulin (mU/L) × fasting glucose (mmol/L)]) × 1000. The IR and IS models were constructed using fasting values of plasma insulin and glucose levels by using the HOMA approach, which has been validated against clamp measurements [33,43].

#### *2.7. Western Blotting*

The mice were killed at the end of the experiment. Their livers and gastrocnemius muscles were rapidly removed, minced roughly, and homogenized immediately. Western blotting was conducted using the approach detailed in another study [44]. We used Akt, phospho-Akt (Ser473), actin, GLUT4, and adiponectin antibodies procured from Cell Signaling Technology (Beverly, MA, USA). Sigma-Aldrich supplied antibodies against patatin-like phospholipid domain containing protein 3 (PNPLA3) and fatty acid synthase (FASN). Enhanced chemiluminescence reagents (Thermo Scientific, Rockford, MA, USA) were employed to obtain immunoreactive signals, which were detected using UVP Chem-Studio (Analytik Jena, Upland, CA, USA). After that, Scion Image (Scion, Frederick, MD, USA) from the National Institute of Health was employed to measure protein expression and phosphorylation.

#### *2.8. RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction (PCR)*

After the mice were sacrificed, total RNA was extracted from their eyeballs and liver tissues using TRIzol reagent (Sigma-Aldrich) per the manufacturer's protocol. We then examined RNA concentration based on absorbance levels of 260 to 280 nm and 230 to 260 nm with a Qubit fluorometer (Invitrogen, Carlsbad, CA, USA). Subsequently, the RNA (1 µg) was subjected to reverse transcription into cDNA by using an iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) following the producer's procedure. Subsequently, realtime polymerase chain reaction (PCR) was performed per the specifications of Bio-Rad's iTaq Universal SYBR Green Supermix kit and with the application of the Bio-Rad CFX Connect Real-Time PCR system. In brief, the cycling conditions were as follows: 95 ◦C for 5 min, 45 cycles at 95 ◦C for 15 s, and 60 ◦C for 25 s. The expression level of every target gene was determined relative to *β-actin* levels, with these levels expressed in the 2 <sup>−</sup>∆∆Ct manner. The primers used for RT-qPCR are listed in Table 1.


**Table 1.** Sequences of primers used for RT-qPCR.

#### *2.9. Analysis of Chromium Content*

After all experiments were complete, blood, urine, and several types of tissue (kidney, blood, liver, bone, fat, and muscle) were collected. Concentrations of chromium in samples were measured according to a prior report [40]. In brief, each sample (0.1 g of tissue and 25 µL of blood and urine) was digested in 65% nitric acid, and then each sample was subjected to digestion with nitric acid overnight (temperature: 100 ◦C). Chromium concentrations were determined using an ICP Mass Spectrometer (NexION 350X, PerkinElmer, MA, USA). Next, distilled water was used to dilute the digested solution to a 5 mL solution prior to measurements. The relative chromium recovery rate was calculated at 10 ppb and 100 ppb of the quantification levels by 5% (*n* = 5) and 8% (*n* = 5), respectively. The absorption data were plotted onto a 1–500 ppb standard curve, and regression analysis was performed to identify the total chromium level in samples (R<sup>2</sup> > 0.996).

#### *2.10. Measurement of Glutathione Peroxidase, Renal Catalase, and Superoxide Dismutase*

We analyzed the antioxidant system's functional activity to determine the enzymatic activity of glutathione peroxidase (GPx), hepatic catalase (CAT), and superoxide dismutase (SOD). Ice-cold saline (0.9% sodium chloride) was used to perfuse kidneys, after which they underwent homogenization in chilled potassium chloride (1.17%) by means of a homogenizer and in accordance with a previous description [22]. Next, the homogenates were gathered for analysis after 5 min of centrifugation at 800× *g* (4 ◦C). Finally, the supernatant was subjected to 20 min centrifugation at 10,500× *g* (also 4 ◦C) to obtain the postmitochondrial supernatant for the kidney samples to measure SOD, catalase, and GPx activity. Commercially available colorimetrical kits were used for these procedures (catalase: #K773-100, #GPx: K762-100, and SOD: #K335-100; BioVision) following manufacturerrecommended protocols.

#### *2.11. Statistical Analysis*

Data are shown as the mean ± standard deviation. The *t*-test was employed for determining intergroup differences (*p* < 0.05 denoted a statistically significant difference). Additionally, we determined contingency data significance by means of Fisher's exact test.
