Exercise training and enalapril mitigate steatotic liver disease and islet disarrangement but diverge in inflammatory modulation in obese miceAbstract
Metabolic-associated steatotic liver disease (MASLD) is increasingly prevalent worldwide. We previously demonstrated that enalapril and aerobic exercise training (AET) improve steatosis in an obesity model. Here, we investigated the effects of both interventions on pancreatic morphology, oxidative stress, and inflammatory cytokines in the steatotic liver. Male C57BL/6 mice were fed either a standard chow (SC) or a high-fat diet (HF) for 8 weeks. The HF group was divided into enalapril treatment (HF-E, 10 mg/kg/day), AET (HF-T, 300 min/week treadmill running), and both interventions (HF-ET) for 8 additional weeks alongside the HF diet. We measured body mass (BM) gain, plasma cytokines, and pancreatic islet structure. Liver mass, oxidative stress, renin-angiotensin system (RAS), and cytokines were also evaluated. The HF group showed greater BM gain, higher pro-inflammatory cytokines, and hypertrophied pancreatic islets than the SC group. All interventions reduced these parameters, though the HF-E group maintained an enlarged pancreatic islet. In the liver, all interventions improved oxidative stress and reduced RAS overactivation. However, enalapril-treated groups retained elevated IL-6, NFκB, TGF-β, and CD11c protein levels, whereas trained groups exhibited levels comparable to SC. In conclusion, both interventions improved islet structure, oxidative stress, and RAS overactivation, but enalapril failed to reverse the pro-inflammatory cytokine profile in obese mice.
Key words
renin-angiotensin system (RAS); liver; pancreas; inflammation; oxidative stress; aerobic exercise training
INTRODUCTION
Metabolic-associated steatotic liver disease (MASLD) poses a growing health concern, with an estimated prevalence of 25% worldwide. This high prevalence is directly linked to the widespread occurrence of obesity and overweight, particularly in Western countries. A major concern regarding MASLD is its association with other comorbidities such as stroke, cardiovascular disease, and diabetes mellitus (Le et al. 2022). MASLD is considered a spectrum of liver disorders that begins with steatosis, though its development remains a complex process that requires further understanding. Briefly, obesity-associated lipolysis of visceral white adipose tissue and insulin resistance, along with molecular changes in the liver favoring oxidative stress, inflammation, and apoptosis, contribute to the onset of steatosis (Pouwels et al. 2022). Obesity-induced systemic inflammation contributes to insulin resistance by promoting inflammation in pancreatic islets, leading to β-cell dysfunction. As a compensatory mechanism, β-cells increase insulin secretion to counteract systemic insulin resistance. Over time, this compensation fails, resulting in impaired pancreatic insulin secretion, disruption of the insulin signaling pathway, and lipotoxicity in peripheral metabolic organs. Consequently, the fatty liver may progress to lobular inflammation, fibrosis, and cirrhosis through a self-perpetuating cycle that interacts with other obesity- and diabetes-related disorders (Anstee et al. 2019).
The treatment of MASLD still requires better understanding. Without specific approved drugs, most treatments are based on interventions that ameliorate overall obesity symptoms, usually combined with pharmacological treatment and lifestyle changes (Lee et al. 2023). In this context, studies have shown the therapeutic potential of renin-angiotensin system (RAS) inhibitors in obesity comorbidities (Moraes et al. 2021, Connolly et al. 2023, Vernail et al. 2024). Under pathological conditions such as obesity and diabetes, the RAS homeostasis is lost and overexpression of its classical arm (Angiotensin-converting enzyme - ACE/Angiotensin II-Ang/Angiotensin II type 1 receptor - AT1R) to the detriment of its counterregulatory arm (ACE2/Ang-(1-7)/Mas Receptor - MasR) is related to deleterious effects such as inflammation, insulin resistance, apoptosis and fibrosis (Vernail et al. 2024). Therefore, drugs that target this imbalance, such as ACE inhibitors, which favor the counterregulatory arm of the RAS and usually promote protective effects (anti-inflammatory, anti-apoptotic, and anti-fibrotic), are promising options (Frantz et al. 2014). Additionally, exercise training is a crucial lifestyle intervention for managing obesity and its comorbidities (Oppert et al. 2023). In animal obesity models, 150 min/week of aerobic exercise training (AET) was able to improve high systolic blood pressure, insulin resistance, and a disrupted lipid profile caused by a high-fat diet, and 300 min/week was able to shift the RAS towards the counterregulatory arm in skeletal muscle (Frantz et al. 2017).
We have previously demonstrated that although both ACE inhibitor enalapril treatment and AET efficiently reduce steatosis, AET promoted additional benefits, and the enalapril treatment was associated with some hepatic damage (Sepúlveda-Fragoso et al. 2022). To further investigate these outcomes, we assessed crosstalk between the liver and pancreas, given the direct link between β-cell dysfunction and MASLD progression (López-Bermudo et al. 2022). Additionally, we investigated oxidative stress and inflammation markers in the liver to better characterize differences between the two interventions.
Abbreviations
4-HNE: 4-Hydroxynonenal; ACE: Angiotensin Converting Enzyme; Ang: Angiotensin; AET: aerobic exercise training; ANOVA: Analysis of Variance; AT1R: Angiotensin II Type 1 Receptor; BM: Body Mass; SC: Control group; HF: High-Fat group; IL: Interleukin; IκBα: Nuclear Factor of Kappa Light Polypeptide Gene Enhancer in B-cells Inhibitor, Alpha; MasR: Mas Receptor; MERT: Maximal Exercise Running Test; NADPH: Nicotinamide Adenine Dinucleotide Phosphate; NFκB: Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells; Nox: NADPH oxidase; 3-NT: 3-Nitrotyrosine; RAS: Renin-Angiotensin System; ROS: Reactive Oxygen Species; SDS-PAGE: Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis; SD: Standard Deviation; TNF-α: Tumor Necrosis Factor Alpha; Δ: Delta.
MATERIALS AND METHODS
Animals and diet
The Animal Ethics Committee of the Fluminense Federal University previously approved all protocols and animal care in this study (protocol number CEUA 2504060718). Procedures followed the conventional animal experimentation guideline (National Institute of Health Guide, 8th edition, 2011). Male C57BL/6 adult mice, purpose-bred and provided by Fluminense Federal University’s laboratory animals unit, were kept in a room with a 12/12 h light/dark cycle, controlled temperature (23 ± 2°C), relative humidity (60 ± 5%), with access to water and food ad libitum. The sample size was calculated using G*Power software (G*Power Version 3.1.9.2, Germany), and each animal was considered an experimental unit.
At 12 weeks, the animals were randomly divided into two groups: standard chow (SC, 14% protein, 10% fat, and 76% carbohydrates (15 kJ/g); n=10) or high-fat (HF, 14% protein, 50% fat, and 36% carbohydrates (21 kJ/g); n=40) diet. Pragsolucoes (SP, Brazil) manufactured the diets. After 8 weeks of obesity induction, the animals in the HF group were allocated into four experimental groups (n=10 per group) as follows: HF group (HF); HF diet and enalapril treatment (HF-E; 10 mg/kg/day, E6888; Sigma-Aldrich, MO - USA); HF diet and aerobic exercise training protocol (HF-T); HF diet and a combination of enalapril treatment and aerobic exercise training protocol (HF-ET). The treatment with enalapril and aerobic exercise protocol lasted eight weeks. Enalapril was mixed into the manufactured diet. The SC group did not undergo any interventions. Animals were housed in color-labeled cages to reduce potential confounders. Body mass (BM) gain was measured and presented as delta (Final BM - Initial BM).
Aerobic exercise protocol and assessment of maximal exercise capacity
Initially, animals were adapted to a motor-driven rodent treadmill (Imbramed®, RS, Brazil) at low velocity (0.05 m/s; 0% slope; 10 min/day; for three consecutive days). After the adaptation, a maximal exercise running test (MERT) was performed to assess individual maximal exercise capacity. For the MERT, the initial velocity was 0.05 m/s, followed by increments of 0.03 m/s every three minutes, at 0% slope. The test ended when the animals reached exhaustion (when they were unable to run and remained at the back of the shocking grid for 5s). The shocking grid bars delivered low electrical currents of ~2 mA, designed to cause minor discomfort without harming the animals.
The MERT was conducted pre-training (8th week), mid-training (12th week, to adjust AET intensity), and post-training (16th week). During the MERT, the maximal time of exhaustion was measured (n=10), and the delta between the 16th and 8th week was calculated. All sedentary mice were also adapted to the treadmill and underwent the MERT (Giori et al. 2020).
AET started in the 8th week of the HF diet protocol. Mice ran 5 days/week, 60 min/day, 0% slope, at moderate intensity (60% of maximal velocity obtained during the MERT), for 8 weeks.
Tissue Extraction and plasma analysis
16 weeks, 72 hours after the last training session, mice were deprived of food for 6 hours and deeply anesthetized with intraperitoneal ketamine (80 mg/kg) and xylazine (10 mg/kg). The blood samples were collected by cardiac puncture, and euthanasia occurred through exsanguination. Plasma was separated by centrifugation (120 g for 20 min) at room temperature and stored individually at -80°C. The liver was collected, weighed, frozen, and stored at -80°C. The pancreas was collected, weighed, fixed in formalin, and embedded in paraffin. Several slices were obtained from the paraffin-embedded samples, and 5-μm-thick sections were obtained.
Plasma concentrations of leptin, resistin, IL-6, and TNF-α were measured using MADKMAG-71K-06 with Luminex xMAP equipment (Millipore, Billerica, MA, USA).
Pancreas morphology and immunohistochemistry
The islet volume density was assessed using a 36-point test system tool in STEPanizer and estimated by point counting using the ratio of points that overlaid the pancreatic islet to the total number of test points. The islet area was estimated using color threshold analysis in ImageJ software by outlining the islet.
Pancreas slices were treated with citrate buffer (96°C, pH 6.0) (EasyPath, SP, Brazil) and then blocked using peroxidase and protein block solution (Novolink Polymer Detection Systems kit, Leica Biosystems, RU). The sections were then incubated overnight at 4°C with insulin and glucagon primary antibodies (1:1000 and 1:2000, respectively; Sigma-Aldrich, USA). Positive immunoreaction was detected by diaminobenzidine, and the sections were then counterstained with hematoxylin. The immunoreactivity areas of the pancreas were evaluated by the ImagePro Plus (Media Cybernetics, Silver Spring, MD, USA) density threshold selection tool. Alpha cell volume density was estimated using the glucagon-positive areas of the islets and represented as a percentage of the islet. Subsequently, alpha cell mass was determined as the product of alpha cell volume density and islet mass (Mandarim-de-Lacerda et al. 2010). Beta cell volume density was determined using the insulin-positive areas of the islets and expressed as a percentage of the islet. Subsequently, beta cell mass was estimated as the product of beta cell volume density and islet mass (Frantz et al. 2011).
Protein carbonylation
Oxidative damage to proteins was assessed in liver homogenates after derivatization of the carbonyl groups with 2,4 dinitrophenylhydrazine followed by absorbance reading at 370 nm according to the protocol described by Wehr and Levine (Wehr & Levine 2013). Results are expressed as nmol/mg protein. Protein content was measured in liver homogenates using the BCA method.
Western blot
Total liver proteins were extracted in a homogenizing buffer (Sigma-Aldrich, St Louis, MO, EUA) supplemented with protease and phosphatase inhibitors. After that, protein samples were resolved with an SDS-PAGE 12- 15% gel electrophoresis and transferred to polyvinylidene difluoride membranes (Amersham Biosciences, Piscataway, NJ, USA). The membranes were blocked with 3% bovine serum albumin and thereafter were incubated overnight at 4°C with primary antibodies to evaluate oxidative stress (4-HNE, 3-NT), inflammation (phospho-NFκB, total- NFκB, IκBα, TGF-β, CD11c, IL-6, and IL-10), and the renin-angiotensin system (AT1R, ACE, MasR). The membranes were then incubated with secondary antibodies. We visualized the blot images using ECL Western blot reagents on a ChemiDoc-System (Bio-Rad, Hercules, CA, USA). Cyclophilin served as the loading control, except for 4-HNE and 3-NT, for which stain-free blot images were used. We quantified the intensity of the chemiluminescent bands using ImageJ software, version 1.44 (NIH, imagej.nih.gov/ij, USA).
Statistical analysis
We tested data for normality and homoscedasticity. Values were shown as the mean ± standard deviation. We tested differences among groups using one-way analysis of variance (ANOVA), followed by a Holm-Sidak post hoc test. We performed analyses and created graphs in GraphPad Prism (version 8.0, CA, USA). In all cases, P≤0.05 was considered statistically significant.
RESULTS
Body Mass gain and exhaustion time to MERT
At the end of the 16 weeks of protocol, the BM gain of the HF group was higher in comparison to the SC group (+227.06%, P<0.0001), and all the interventions presented lower BM gain compared to the HF group (HF-E: -90.69%, P<0.0001; HF-T: -53.99%, P=0.0016; HF-ET: -103.22%, P<0.0001; Table I). Additionally, the HF-T group had greater BM gain when compared to the HF-E group (+415.88%, P=0.0237), and the HF-ET group revealed lower BM gain compared to the HF-T group (-100.43%; P=0.0035; Table I).
Before the interventions began, time to exhaustion did not differ significantly among groups (Table I). However, in the 16th week, the trained groups (HF-T and HF-ET) were able to improve their time to exhaustion when compared to the sedentary groups (SC, HF, and HF-E), and compared to their baseline, the HF-T and HF-ET groups also had a higher delta in comparison to the sedentary groups (P<0.05), as shown in Table I.
Plasma cytokines levels
Leptin in plasma showed higher concentrations (+368.52%, P=0.0176; Figure 1) in the HF group compared to the SC group. Resistin levels were higher in the HF and HF-ET groups than in the SC group (+156.97%, P=0.0120; +337.24%, P=0.0172, respectively). The HF group presented greater IL-6 plasmatic levels when compared to the SC group (+5.37%, P=0.0169). However, both groups that underwent the training protocol reduced these results (HF-T: -37.04%, P=0.0488; HF-ET: -12.14%, P=0.0573). When looking at TNF-α, all the interventions were lower in comparison to the HF group (HF-E: -8.55%, P=0.0496; HF-T: -8.26%, P=0.0535; HF-ET: -13.90%, P=0.0138; Figure 1).
Plasma cytokines. (a) Leptin, (b) Resistin, (c) IL-6, and (d) TNF-α. Values presented as mean ± SD, n = 5/group. Significant differences between the groups are indicated by symbols (P≤0.05): a ≠ SC; b ≠ HF, as determined by one-way ANOVA and Holm-Sidak post-test.
Pancreas morphology
The pancreas mass did not differ among groups (data not shown). The HF and HF-E groups showed hypertrophied islets compared to the SC, HF-T, and HF-ET groups (P<0.001 in all cases; Table II). The islet volume density was increased in the HF group compared to the SC group (+203.53%, P<0.001), corroborating the islet hypertrophy. All interventions showed a lower volume of pancreatic islets than HF (HF-E: -50.14%, P<0.05; HF-T: -42.32%, P<0.05; HF-ET: -51.71%, P<0.01). Alpha and beta cell mass were also increased in the HF group compared to SC (+329.13%, P<0.01, +179.25%, P<0.05, respectively). These parameters were all reduced in HF-E (alpha cells: -61.88%, P<0.05; beta cells: -60.45%, P<0.001), HF-T (alpha cells: -59.61%, P<0.05; beta cells: -57.19%, P<0.001) and HF-ET (alpha cells: -67.08%, P<0.01; beta cells: -71.34%, P<0.0001) compared to HF group (Table II).
The SC group showed a core-mantle pattern of islet cell arrangement, with beta cells forming a dense core and alpha cells at the periphery (Figure 2a and f). This organized distribution is crucial to maintaining glucose homeostasis. However, the HF group showed strong glucagon- and insulin-positive areas, and alpha cells in both the core and periphery of the islet were hypertrophied (Figure 2b and g). The treated mice (enalapril and AET) were like the SC mice and showed few infiltrations of alpha cells into the islet core (Figure 2).
Pancreas morphology. Representative photomicrographs showing pancreatic islets with immunoperoxidase (brown)-stained glucagon /alpha cells (first line) and insulin /beta cells (second line), counterstained with hematoxylin. Same magnification in all groups; bar = 50 μm.
Liver Mass
Total liver mass was found to increase in the HF group compared to the SC group (HF: +18.42%, P=0.0526) but decreased in the HF-E and HF-ET groups (HF-E: -20.38%, P=0.0245; HF-ET: -29.26%, P=0.0008). When compared to the HF group, all the interventions were able to reduce liver mass (HF-E: -32.76%, P<0.0001; HF-T: -24.77%, P=0.0001; HF-ET: -40.27%, P<0.0001; Table I). The HF-ET group showed a lower liver mass compared to the HF-T group (-20.60%, P=0.0165; Table I).
Renin-angiotensin system
Regarding the classical arms of RAS in the liver, we found that AT1R protein expression was higher in the HF group in comparison to the SC group (+47.30%, P=0.0497; Figure 3), and all the groups who underwent interventions were able to reestablish the expression of AT1R (HF-E: -38.05%, P=0.0497; HF-T: -42.09%, P=0.0162; HF-ET: -43.88%, P=0.0058). Concerning the SC group, the HF group presented an increase in liver ACE protein expression (+37.99%, P=0.0348). However, all the interventions showed a decrease in ACE expression compared to the HF group (HF-E: -31.84%, P=0.0129; HF-T: -51.61%, P<0.0001; HF-ET: -39.57%, P=0.0018; Figure 3).
RAS components. (a) Representative Western blot bands with numbers on the right indicate molecular weight (MW, kDa). Protein expression of (b) ACE, (c) AT1R, (d) MasR, and (e) AT1R/MasR ratio in hepatic tissue. Data presented as mean ± SD, n=5. Significant differences between the groups are indicated by symbols (P≤0.05): a ≠ SC; b ≠ HF, as determined by one-way ANOVA and Holm-Sidak post-test.
No significant differences were observed among the experimental groups in MasR protein expression in the counterregulatory arm (Figure 3). All interventions (HF-E, HF-T, and HF-ET) reduced the AT1R/MasR protein expression ratio compared with the SC group (HF-E: -44.42%, P=0.0109; HF-T: -57.60%, P=0.0009; HF-ET: -38.53%, P=0.0304). In addition, the HF-T group decreased the AT1R/MasR protein expression ratio (-47.22%, P=0.0304) compared to the HF group (Figure 3).
Liver oxidative stress and inflammation
The HF group showed increased protein oxidation, with carbonyl groups 583.58% higher than in the SC group (P<0.002; Figure 4). All interventions reduced protein carbonyl content compared with the HF group (HF-E: -75.72%, P=0.0048; HF-T: -60.68%, P=0.0248; HF-ET: -82.75%, P=0.0024). However, the 4-HNE and 3-NT protein expression showed no significant differences between groups (Figure 4).
Oxidative stress assessment. Protein expression of (a) nitrotyrosine and (b) 4-HNE, (c) protein carbonyl content, and (d) representative bands with numbers on the left indicate molecular weight (MW, kDa). Data presented as mean ± SD, n=5. Significant differences between the groups are indicated by symbols (P≤0.05): a ≠ SC; b ≠ HF, as determined by one-way ANOVA and Holm-Sidak post-test.
The level of the proinflammatory cytokine IL-6 was elevated in both HF and HF-E groups when compared to the SC group in the liver (+312.49%, P=0.0014; +281.29%, P=0.0037, respectively; Figure 5). The HF-T and HF-ET groups were able to reduce the IL-6 concentration when in comparison to the HF group (-59.68%, P=0.011; -59.70%, P=0.011, respectively) and to the HF-E group (-56.39%, P=0.0246; -56.40%, P=0.0246, respectively). The concentration of the anti-inflammatory cytokine IL-10 showed no significant differences between groups (Figure 5).
Inflammatory markers. (a) Representative Western blot bands with numbers on the right indicate molecular weight (MW, kDa). Protein expression of (b) IL-6, (c) IL-10, (d) TGF-β, (e) NFkB (Phospho/Total) ratio, (f) IκB-α, and (g) CD11c in hepatic tissue. Data presented as mean ± SD, n=5. Significant differences between the groups are indicated by symbols (P≤0.05): a ≠ SC; b ≠ HF; c ≠ HF-E, as determined by one-way ANOVA and Holm-Sidak post-test.
Hepatic IkBα protein levels did not differ among groups. However, the NFkB (Phospho/Total) protein expression ratio increased in the HF group compared to the SC group (+73.65%, P=0.001; Figure 5). The HF-T and HF-ET groups showed reduced levels compared to the HF group (-52.59%, P=0.0002; -35.98%, P=0.0055, respectively). In addition, the HF-T group exhibited a reduction compared to the HF-E group (-47.80%, P=0.0119). CD11c concentrations increased in the HF and the HF-E compared to the SC group (+73.58%, P=0.0553; +110.75%, P=0.0021). Furthermore, the HF-T decreased compared to the HF-E group (-38.29%, P=0.0325). TGF-β was increased in both HF and HF-E groups compared to the SC group (+84.86%, P=0.0070; +72.16%, P=0.0251, respectively; Figure 5).
DISCUSSION
This study aimed to investigate the differential effects of enalapril treatment and/or AET on pancreatic and hepatic outcomes in a diet-induced MASLD model. We focused on pancreatic remodeling, hepatic RAS arms, oxidative stress, and inflammation. In summary, all interventions attenuated body mass gain and lowered plasma leptin, resistin, and TNF-α levels, as well as protein carbonyl content and classic RAS components in the liver. Curiously, the enalapril-treated group showed a similar profile to the HF group in some results, maintaining hypertrophied pancreatic islets and higher liver protein levels of IL-6, TGF-β, NF-κB, and CD11c. We previously demonstrated that enalapril treatment in an HF diet model failed to match some of the effects achieved by AET in the steatotic liver and resulted in overall greater liver damage, assessed by hepatocyte binucleation (Sepúlveda-Fragoso et al. 2022). Here, we explored reasons that could help explain these different outcomes, considering the expected shift toward the counter-regulatory arm of the RAS system among all interventions.
Systemically, plasma cytokines showed a similar pattern of results, with all interventions preventing the increased levels found in the HF group compared to the SC group. Our study corroborates the known efficacy of both enalapril treatment and AET in recovering from the high levels of leptin characteristic of obesity (Dinari Ghozhdi et al. 2021, Mitchell et al. 2021). Likewise, resistin and TNFα, pro-inflammatory cytokines strongly related to insulin resistance in obesity, behaved similarly, emphasizing the efficiency of all interventions. In previous studies and here, we have demonstrated that the HF group induced hyperglycemia and hyperglucagonemia, characterized by hypertrophic islets (area and volume), an increased mass of both alpha and beta cells, and alpha cell infiltration into the islet core, all hallmarks of obesity-related insulin resistance (Frantz et al. 2013). AET was more effective than enalapril in restoring glucose homeostasis, consistent with previous findings in which streptozotocin-treated diabetic rats showed larger islet size after enalapril treatment than eutrophic rats (Ahmed et al. 2019). This improved pancreatic remodeling and, consequently, the systemic insulin resistance caused by AET may partially explain the differences observed between the two interventions in the liver, a key regulator of glucose homeostasis that balances hepatic glucose production and storage (Pouwels et al. 2022).
The RAS system, present locally in most tissues, is crucial in maintaining homeostasis through the balance of its classical and counterregulatory arms. Conditions such as obesity and MASLD are widely reported to favor activation of the RAS classical arm, which is associated with inflammation, apoptosis, and fibrosis outcomes (Borem et al. 2018). We previously demonstrated the capacity of both enalapril treatment and AET in favoring the counterregulatory arm (Salles et al. 2024), which corresponds to the protein expression of RAS components in the liver found in the present study, where the AT1R/MasR ratio decreased in all treated groups. The oxidative stress analysis found no significant differences in 4-HNE and 3-NT protein expression and reduced protein oxidation in all treated groups compared to the HF group. This suggests that specific oxidative pathways may remain active or that the markers reflect existing damage that requires longer interventions. The interventions may address other biomarkers of oxidative stress, but do not fully affect lipid peroxidation or protein nitration pathways. The reduction in protein oxidation also correlates with lower levels of ACE and AT1R in treated groups compared to the HF group (Alexandre-Santos et al. 2020).
Despite RAS counterregulatory arm shifts in all interventions followed by lower protein oxidation, inflammatory cytokines assessed in the liver showed higher expression in the HF-E group compared to the SC group. Higher hepatic protein levels of IL-6, TGF-β, and CD11c were observed in the HF-E group compared to the SC group, and phosphorylation of NF-κB was not reduced compared to the HF group. These results follow our previous study, which found higher levels of liver damage assessed by hepatocyte binucleation in enalapril-treated groups (Sepúlveda-Fragoso et al. 2022). However, a pro-inflammatory state persists despite the demonstrated benefits of enalapril treatment on obesity and MASLD disruptions. The literature shows that enalapril has been reported to cause drug-induced liver injury in humans and has been characterized as an idiosyncratic hepatotoxicant in rat drug-induced liver injury (Leone et al. 2014, Eun et al. 2015). In this context, the increase in inflammatory markers could reflect a specific liver event due to drug toxicity and therefore would be independent of the beneficial effects promoted by enalapril treatment, both systemically and locally in the fatty liver and pancreas. Furthermore, the anti-inflammatory capacity of AET over the fatty liver has been reported in obesity contexts (Farzanegi et al. 2019). Notably, AET reversed the increased inflammation even in the presence of enalapril.
In conclusion, both interventions effectively addressed the metabolic disruptions caused by obesity, resulting in enhanced islet structure, reduced overactivation of the RAS classical arm in the liver, and a consequent decrease in oxidative stress. However, enalapril alone was insufficient to fully reverse pancreatic islet remodeling, as it failed to restore glucose homeostasis. Given the significant role of insulin resistance in the development of MASLD, these findings - along with the potential hepatotoxicity of enalapril - may explain why it was unable to counteract the increase in pro-inflammatory cytokines in the liver observed in the HF group, despite other metabolic improvements. Finally, combining AET with enalapril treatment not only improved pancreatic islet remodeling but also reduced inflammatory markers compared with the HF and enalapril-treated groups. These results suggest that combining pharmacological treatment for obesity and MASLD with AET may help prevent drug-related pro-inflammatory side effects.
Acknowledgements
The authors thank the laboratory team, Wagner de Souza Rodrigues, Isabelle dos Santos Xavier Dias, and Bernardo Acacio Santini Pereira, for their assistance. Cardiovascular Sciences and Biomedical Science Postgraduate Program, Fluminense Federal University (UFF). This work received financial support from the agencies Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ, www.faperj.br) [grant number E-26/010.001841/2019, EDCF; E-26/201.347/2022, EDCF; E-26/210.725/2021, ACLN; E26/211.280/2019, DCM]; and CAPES [Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior - Brazil - Finance Code 001].
Data availability
References
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