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Liver, Pancreas and Biliary Tract| Volume 52, ISSUE 3, P314-323, March 2020

L-carnitine supplementation attenuates NAFLD progression and cardiac dysfunction in a mouse model fed with methionine and choline-deficient diet

Open AccessPublished:October 10, 2019DOI:https://doi.org/10.1016/j.dld.2019.09.002

      Abstract

      Non-alcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disorder. NAFLD, associated lipotoxicity, fibrosis, oxidative stress, and altered mitochondrial metabolism, is responsible for systemic inflammation, which contributes to organ dysfunction in extrahepatic tissues, including the heart.
      We investigated the ability of L-carnitine (LC) to oppose the pathogenic mechanisms underlying NAFLD progression and associated heart dysfunction, in a mouse model of methionine-choline-deficient diet (MCDD). Mice were divided into three groups: namely, the control group (CONTR) fed with a regular diet and two groups fed with MCDD for 6 weeks. In the last 3 weeks, one of the MCDD groups received LC (200 mg/kg each day) through drinking water (MCDD + LC).
      The hepatic lipid accumulation and oxidative stress decreased after LC supplementation, which also reduced hepatic fibrosis via modulation of α-smooth muscle actin (αSMA), peroxisome-activated receptor gamma (PPARγ), and nuclear factor kappa B (NfƙB) expression. LC ameliorated systemic inflammation, mitigated cardiac reactive oxygen species (ROS) production, and prevented fibrosis progression by acting on signal transducer and activator of transcription 3 (STAT3), extracellular signal-regulated kinase 1-2 (ERK1-2), and αSMA.
      This study confirms the existence of a relationship between fatty liver disease and cardiac abnormalities and highlights the role of LC in controlling liver oxidative stress, steatosis, fibrosis, and NAFLD-associated cardiac dysfunction.

      Keywords

      1. Introduction

      The liver performs essential functions in the regulation of glucose and lipid metabolism during feeding and fasting. Non-alcoholic fatty liver disease (NAFLD), also known as hepatic steatosis, is characterized with an excessive fat deposition in the liver and presents a histological spectrum of conditions ranging from simple steatosis to nonalcoholic steatohepatitis (NASH). Hepatocellular damage, lobular necroinflammation and fibrogenesis are the main features of NASH [
      • Brunt E.M.
      Pathology of nonalcoholic fatty liver disease.
      ]. According to the “Two Hits Hypothesis”, NAFLD development requires the double hit, steatosis and oxidative stress, wherein the latter leads to an increase in lipid peroxidation, inflammation, and fibrosis [
      • Day C.P.
      • James O.F.
      Steatohepatitis: a tale of two “hits”?.
      ]. The initial fat accumulation in the liver results in the activation of multiple cellular stress pathways and induces the generation of reactive oxygen species (ROS), which in turn causes the transdifferentiation of hepatic stellate cells into activated myofibroblasts expressing α-smooth muscle actin (αSMA) [
      • Valenti L.
      • Bugianesi E.
      • Pajvani U.
      • et al.
      Nonalcoholic fatty liver disease: cause or consequence of type 2 diabetes?.
      ,
      • Rotman Y.
      • Sanyal A.J.
      Current and upcoming pharmacotherapy for non-alcoholic fatty liver disease.
      ,
      • Friedman S.L.
      Mechanisms of hepatic fibrogenesis.
      ]. Some authors have recently proposed caspase-2 as a new NASH marker, as its expression increases with the worsening of fibrosis in humans and animal models of NASH [
      • Machado M.V.
      • Kruger L.
      • Jewell M.L.
      • et al.
      Vitamin B5 and N-acetylcysteine in nonalcoholic steatohepatitis: a preclinical study in a dietary mouse model.
      ,
      • Machado M.V.
      • Michelotti G.A.
      • Pereira Tde A.
      • et al.
      Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis.
      ,
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • et al.
      ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P.
      ].
      Together these effects trigger the expression of nuclear factor kappa B (NFκB), a crucial inflammatory signal that may exacerbate NAFLD progression [
      • Leclercq I.A.
      • Farrell G.C.
      • Sempoux C.
      • et al.
      Curcumin inhibits NF-kappaB activation and reduces the severity of experimental steatohepatitis in mice.
      ,
      • Salamone F.
      • Galvano F.
      • Cappello F.
      • et al.
      Silibinin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis.
      ]. Furthermore, activated peroxisome proliferator-activated receptors γ (PPARγ) inhibits NFκB transcription factor [
      • Xu J.
      • Fu Y.
      • Chen A.
      Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth.
      ,
      • Hazra S.
      • Xiong S.
      • Wang J.
      • et al.
      Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells.
      ].
      Evidence suggests that NAFLD not only represents a potentially progressive liver disease but also has systemic consequences [
      • Byrne C.D.
      • Targher G.
      NAFLD: a multisystem disease.
      ]. In particular, recent reports from the Framingham Heart study revealed the significant association between NAFLD and subclinical cardiovascular diseases (CVD) [
      • Long M.T.
      • Fox C.S.
      The framingham heart study—67 years of discovery in metabolic disease.
      ]. Patients with NAFLD showed myocardial metabolism disorders with impaired heart functions and structure, i.e., with cardiovascular insufficiency. Several studies have demonstrated the oxidative stress-induced activation of extracellular signal-regulated kinase (ERK) pathways and the increase in αSMA and collagen expression, which eventually contribute to fibrosis [
      • MacLean J.
      • Pasumarthi K.B.
      Signaling mechanisms regulating fibroblast activation, phenoconversion and fibrosis in the heart.
      ]. Furthermore, recent data have indicated the influence of signal transducer and activator of transcription 3 (STAT3) on the accumulation of fibrotic proteins in the heart [
      • Haghikia A.
      • Ricke-Hoch M.
      • Stapel B.
      • et al.
      STAT3, a key regulator of cell-to-cell communication in the heart.
      ].
      Therefore, innovative NAFLD treatment strategies should focus on both hepatic and cardiovascular damages. Studies have evaluated the effects of nutraceuticals as a co-adjuvant treatment for NAFLD and cardiovascular complications. In some clinical trials, nutraceuticals were found to ameliorate the liver function and histology [
      • Hung C.K.
      • Bodenheimer Jr., H.C.
      Current treatment of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis.
      ]. L-carnitine (LC) is an essential nutrient that converts fats into energy in the mitochondrion and plays an important role in lipid metabolism. It acts as an essential cofactor for β-oxidation by facilitating the transport of long-chain fatty acids. The process of β-oxidation is one of the principal pathways underlying lipid metabolism. The inhibition of β-oxidation leads to the accumulation of lipids within hepatocytes. LC administration was shown to ameliorate or prevent the liver from insult through the augmentation of hepatic mitochondrial β-oxidation [
      • Ishikawa H.
      • Takaki A.
      • Tsuzaki R.
      • et al.
      L-carnitine prevents progression of non-alcoholic steatohepatitis in a mouse model with upregulation of mitochondrial pathway.
      ]. Recent studies have suggested the role of oxidative pathways as favourable targets for the treatment of NAFLD [
      • Li W.
      • Ma F.
      • Zhang L.
      • et al.
      S-propargyl-cysteine exerts a novel protective effect on methionine and choline deficient diet-induced fatty liver via akt/Nrf2/HO-1 pathway.
      ]. LC has been recognized as a nutritional supplement with anti-oxidative properties [
      • Ferraretto A.
      • Bottani M.
      • Villa I.
      • et al.
      L-carnitine activates calcium signaling in human osteoblasts.
      ] and used as an adjunctive therapy in various heart conditions with promising results [
      • Vacante F.
      • Senesi P.
      • Montesano A.
      • et al.
      L-carnitine: an antioxidant remedy for the survival of cardiomyocytes under hyperglycemic condition.
      ]. In particular, Strilakou et al. have described the beneficial effects of LC on a cardiomyopathy rat model fed with a choline-deficient diet [
      • Strilakou A.A.
      • Lazaris A.C.
      • Perelas A.I.
      • et al.
      Heart dysfunction induced by choline-deficiency in adult rats: the protective role of L-carnitine.
      ].
      In the present study, we investigated the antioxidative effects of LC on both liver and cardiac complications of steatohepatitisin a mouse model fed with methionine-choline-deficient diet (MCDD).
      MCDD was used to study the ability of LC to prevent liver fat deposition, oxidative stress, and fibrosis progression and counteract the NAFLD-induced cardiac inflammation and fibrosis.

      2. Materials and methods

      2.1 Materials

      Reagents were purchased from Sigma Chemical Co. (Saint Louis, MO, USA). LC was obtained from Sigma-Aldrich (C0283). Primary antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; sc-25778), NFκB p65 (sc-109), PPARγ (sc-7196), αSMA (sc-53142), phosphor-Ca2+/calmodulin-dependent protein kinase IIα (pCaMKIIα; 22B1), ERK 1-2 (K-23) and pERK 1-2 (E-4), peroxidase-conjugated secondary antibodies for western blot analysis, and fluorescein isothiocyanate (FITC)/rhodamine-conjugated antibodies for immunofluorescence analysis were supplied by Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against STAT3 (#9132), pSTAT3 (#9131), and CaMKIIα (#50049) were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti-caspase-2 (ab179520) and anti-Cytochrome C (COX; ab110325) antibodies were procured from Abcam (Cambridge, UK).
      Cell ROX® Oxidative Stress Reagent Kit (C10443) was purchased from Thermo Fisher Scientific, Life Technologies Italia (Monza-Italy) and Masson-Goldner staining kit (100485), Weigert’s iron haematoxylin kit (115973), Entellan® Neo (107961) were obtained from Merck Millipore, Merck KGaA (Darmstadt, Germany).
      Mayer’s Haematoxylin Solution (MHS16), 0.5% aqueous eosin Y solution (HT110216), Scott’s Tap Water Substitute Concentrate (S5134), 0.5% Oil Red O solution in isopropanol (O1391), glycerol gelatin aqueous slide mounting medium (GG1), 10% formalin (HT501128), and Bouin’s Solution (HT10132) were all procured from Sigma Chemical Co. (Saint Louis, MO, USA).

      2.2 Experimental animal model for NAFLD

      Eight-week-old male C57BL/6 mice (n = 30) were used in this study (Charles River Laboratories, Calco, Bergamo, Italy), which was conducted in compliance with the approved institutional animal care of the Università degli Studi di Milano.
      All animals were maintained in a 12 h light/dark cycle with unlimited access to standard rodent chow, food, and water under controlled temperature (22 ± 2 °C). The study design is shown in Fig. 1A.
      Fig. 1
      Fig. 1Experimental animal model fornon-alcoholic fatty liver disease (NAFLD). (A) Animal experiment scheme. (B) Mice fed with methionine-choline-deficient diet (MCDD) showed a significant loss in body weight as compared with those from the control group (CONTR). This condition remained unchanged despite supplementation with L-carnitine (LC) (n = 10/treatment group: control group, CONTR; methionine-choline deficient diet, MCDD; methionine-choline deficient diet supplemented with L-carnitine, MCDD + LC).
      Data are presented as mean ± SD. ***p < 0.001 as compared with the mice fed with the control diet.
      After two weeks of adaptation, 10-week-old mice were divided into three groups (n = 10 each). One group was fed with 120 g/week of regular diet (CONTR) and two groups were fed with 120 g/week of MCDD (ssniff® EF R/M Induction of fatty liver — Ssniff Spezialdiäten GmbH: 83 KJ% carbohydrate, 8 KJ% fat, 9 KJ% protein, ME calculated with Atwater factor) for 3 weeks. Thereafter, one of the MCDD groups was provided with water-diluted 200 mg/kg of oral LC (MCDD + LC) daily for 3 weeks. Control and MCDD mice received water without LC. The LC dose was adjusted to net body weight. We used CONTR group as an internal control of NAFLD progression induced by MCDD.
      At the end of the 6 weeks, all animals were euthanized for further analysis.

      2.3 Histopathological analysis

      Upon sacrifice, the liver and heart tissues were excised, weighed, and sampled for histological analysis.
      Liver and heart cryosections were examined with different staining techniques. Tissue sections were examined with haematoxylin and eosin staining according to the manufacturer’s instruction. Images were acquired with phase-contrast microscopy and steatosis score was calculated with ImageJ program (http://imagej.nih.gov/ij/).
      Staining for intracellular lipid content was performed with Oil Red O (ORO) technique according to the manufacturer’s instruction.
      Masson-Goldner staining involving a trichrome stain was used to primarily image the connective tissue in organs (nuclei were stained black or blue; cytoplasm and muscle were stained red; collagen fibers were stained blue–green) [
      • Krishna M.
      Role of special stains in diagnostic liver pathology.
      ,
      • Clark I.
      • Torbenson M.S.
      Immunohistochemistry and special stains in medical liver pathology.
      ].

      2.4 Western blot analysis

      Liver and heart protein extracts were obtained from homogenized mouse tissues using radioimmunoprecipitation assay (RIPA) buffer [
      • Senesi P.
      • Montesano A.
      • Luzi L.
      • et al.
      Metformin treatment prevents sedentariness related damages in mice.
      ]. Protein contents were quantified using the Bradford method. Aliquots of quantified 30 μg supernatant proteins were resolved on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and separated protein bands were transferred onto nitrocellulose membranes (Protran®, Whatman® Schleicher & Schuell). The membranes were incubated with specific primary antibodies, followed by treatment with horseradish peroxidase-conjugated species-specific secondary antibodies. To confirm equal protein loading per sample, anti-GAPDH was used. Quantitative measurement of immunoreactive bandintensities was performed with the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Piscataway, NJ, USA) using densitometric analysis with the Scion Image software (Scion Corporation, Frederick, MD, USA). Only caspase-2 and COX bands were visualized with the UVITEC Alliance LD9 gel imaging system (Uvitec, Cambridge, UK). Data were converted into ratio relative to the control.

      2.5 Immunofluorescence analysis

      Liver and heart cryosections were fixed with 4% paraformaldehyde for 30 min at room temperature. The sections were washed with phosphate-buffered saline (PBS), permeabilized with 0.2% Triton X-100, and incubated for 30 min at room temperature with 10% serum. The cryosections were immunostained with specific primary and secondary antibodies (rhodamine/FITC-conjugated) and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) [
      • Codella R.
      • Lanzoni G.
      • Zoso A.
      • et al.
      Moderate intensity training impact on the inflammatory status and glycemic profiles in NOD mice.
      ].
      Cell ROX® oxidative stress reagents are fluorogenic probes designed to reliably measure ROS levels in tissues. These cell-permeable reagents are non-fluorescent or very weakly fluorescent but emit a strong fluorogenic signal under reduced state and upon oxidation. Cell ROX® Orange Reagents are localized in the cytoplasm. [
      • Terruzzi I.
      • Montesano A.
      • Senesi P.
      • et al.
      Erratum to: ranolazine promotes muscle differentiation and reduces oxidative stress in C2C12 skeletal muscle cells.
      ]
      Slides were mounted with Moviol. Tissue sections were observed under a Nikon Eclipse 50I microscope and images were captured using Nis-Elements D 4.00 software (Nikon Instruments Europe BV, Netherlands).

      2.6 Statistical analysis

      All data were presented as the mean ± standard deviation (SD). The Shapiro–Wilk test was used to ascertain normality of data distribution. Every mouse tissue was analyzed thrice.
      A two-way (treatment × time) analysis of variance (ANOVA) followed by Tukey’s multiple comparison test were used to analyse body weight changes throughout the experimental time course.
      For variables with Gaussian distribution, one-way ANOVAwas used to compare the means among groups, followed by Tukey’s post-hoc test. The Kruskal–Wallis test followed by Dunn’s post-hoc test were used for the variables with non-Gaussian distribution.
      A value of p ≤ 0.05 was considered significant. Statistical analysis was performed with GraphPad Prism 7 software (San Diego, CA).

      3. Results

      3.1 Changes in body weight

      Animals fed with MCDD showed a loss in body weight, consistent with the histological features of human NASH [
      • Hebbard L.
      • George J.
      Animal models of nonalcoholic fatty liver disease.
      ]. MCD diet is ideal for studying NASH development and, in particular, for the analysis of mechanisms underlying inflammation, fibrosis and, oxidation [
      • Gao D.
      • Wei C.
      • Chen L.
      • et al.
      Oxidative DNA damage and DNA repair enzyme expression are inversely related in murine models of fatty liver disease.
      ].
      The 6-week administration of MCDD resulted in a significant decrease in the body weight of mice from MCDD group as compared with those from the control group (18.8 ± 0.9 g vs 30.6 ± 0.8 g, respectively — p ≤ 0.001) [
      • Kirsch R.
      • Clarkson V.
      • Shephard E.G.
      • et al.
      Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies.
      ]. This difference remained unchanged despite supplementation with LC (19.9 ± 0.4 g — p ≤ 0.001 versus CONTR group) (Fig. 1B).

      3.2 Liver mass, morphology, histology and lipid accumulation

      Based on the effect of MCDD on body weight, hepatic mass was calculated as the percentage of body mass (relative liver mass). Liver mass showed no significant differences among the three groups. The livers from CONTR group were 14.2% and 13% heavier than those from MCDD and MCDD + LC groups, respectively. The livers from MCDD + LC group were 1.4% heavier than those from MCDD group (Table 1). Moreover, the samples from MCDD group appeared paler than those from CONTR and MCDD + LC groups (Fig. 2A).
      Table 1Liver mass analysis and steatosis score. Relative liver mass is expressed as the percentage of body mass. Steatosis score: 0 (<5%), 1 (5–33%), 2 (33–66%), 3 (>66%). Groups: control group-CONTR; methionine-choline deficient diet-MCDD; methionine-choline deficient diet supplemented with L-carnitine-MCDD + LC. n = 5/treatment group. Data are presented as mean ± SD. *p < 0.05 as compared with the mice fed with the control diet.
      CONTRMCDDMCDD + LC
      Liver mass (g)1.65 ± 0.210.90 ± 0.270.94 ± 0.13
      Relative liver mass (as % of body mass)5.46 ± 0.834.68 ± 1.194.75 ± 0.68
      Percentage differences in relative liver mass MCDD and MCDD + LC vs. CONTR (%)14.213.0
      Percentage differences in relative liver mass MCDD vs. MCDD + LC (%)1.4
      Steatosis score0 (0.81% ± 0.51)1 (18.84% ± 4.23)*1 (13.36% ± 3.16)*
      Fig. 2
      Fig. 2Effects of L-carnitine on liver morphology, histology, lipid accumulation, and oxidation. (A) No macroscopic differences were observed in the three mouse groups (n = 10/treatment group: control group, CONTR; methionine-choline-deficient diet, MCDD; methionine-choline-deficient diet supplemented with L-carnitine, MCDD + LC). (B) Haematoxylin and eosin-stained sections showed differences in steatosis and ballooning in the liver of mice fed with methionine-choline-deficient diet (MCDD). n = 5/treatment group. (C) Oil Red O (ORO)-stained sections confirmed hepatic lipid accumulation in the mice from methionine-choline-deficient diet (MCDD) group. LC reduced hepatic fat accumulation. n = 5/treatment group. (D) We used the Cell ROS assay to evaluate the effect of L-carnitine on the liver of mice. Methionine-choline-deficient diet (MCDD) increased the production of reactive oxygen species (ROS). L-carnitine supplementation ameliorated this effect. n = 5/treatment group. (E) Western blot analysis showed that MCDD significantly improved cytochrome C (COX) protein level as compared with control diet. This increase was significantly reduced after L-carnitine supplementation.
      Data are presented as mean ± SD. **p < 0.01 as compared with the mice fed with the control diet. #p < 0.05 as compared with the mice fed with methionine-choline-deficient (MCD) diet.
      Steatosis was scored on HE-stained sections according to Brunt’s criteria [
      • Brunt E.M.
      • Janney C.G.
      • Di Bisceglie A.M.
      • et al.
      Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions.
      ]. As shown in Fig. 2B, 6 weeks of MCDD treatment resulted in the development of hepatocyte steatosis and ballooning (steatosis score: 1–18.84%) while LC supplementation seemed to mitigate this condition (steatosis score: 1–13.36%) (Table 1).
      As shown in Fig. 2C, LC treatment ameliorated hepatic fat accumulation, as evident from ORO staining results.

      3.3 Hepatic antioxidant effects of LC

      Although LC was unable to restore ROS levels to those observed in the CONTR group, it could limit ROS production in mice from MCDD + LC group as compared to those from MCDD group (Fig. 2D). Moreover, we evaluated the protein level of COX involved in ROS production in MCDD mice [
      • Romestaing C.
      • Piquet M.A.
      • Letexier D.
      • et al.
      Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet.
      ]. As reported in Fig. 2E, a a significant increase in COX level was observed in MCDD mice (ratio to CONTR: 1.64 ± 0.2, p ≤ 0.01 as compared with CONTR group), while LC treatment counteracted this effect (ratio to CONTR: 1.27 ± 0.2, p ≤ 0.05 versus MCDD group).

      3.4 Role of LC in hepatic fibrosis progression

      To investigate whether LC supplementation could delay fibrosis development, Masson-Goldner staining was performed. Liver fibrosis was higher in MCDD group than in the CONTR group. Blue–green colour, specific for collagen-I, was widespread and corresponded to a strong intensity. Mice supplemented with LC diet showed amelioration in the progression of NAFLD associated with fibrosis as compared with MCDD-fed mice (Fig. 3A).
      Fig. 3
      Fig. 3Effect of L-carnitine on hepatic fibrosis. (A) Masson-Goldner (MG)-trichrome-stained sections showed fibrotic areas in the liver samples from methionine-choline-deficient diet (MCDD) group (magnification 20′). n = 5/treatment group. (B) Immunofluorescence assay revealed the increase in the level of α-smooth muscle actin (α-SMA) protein in the liver samples from mice fed with methionine-choline-deficient diet (MCDD). This increase was attenuated after L-carnitine supplementation. n = 5/treatment group. (C) Caspase-2 protein level was significantly higher in mice from MCDD group. In the other two groups, the levels were similar. We failed to observe any differences in procaspase-2 protein levels. n = 5/treatment group. (D) Western blot data indicated that L-carnitine (LC) supplementation significantly increased the hepatic peroxisome proliferator-activated receptors γ (PPARγ) protein level as compared with the methionine-choline deficient diet (MCDD), and decreased the activation of nuclear factor kappa B (NfκB) p65. n = 5/treatment group.
      Data are expressed as the ratio relative to the control group ± SD. *p < 0.05 as compared to the mice fed with the control diet. ***p < 0.001 as compared to the mice fed with the control diet. #p < 0.05 as compared to the mice fed with methionine-choline-deficient diet (MCDD).
      We evaluated the level of αSMA protein to dermine the rate of hepatic fibrogenesis. As shown in Fig. 3B, 3 weeks of LC supplementation induced a decrease in αSMA level as compared to MCDD diet. Caspase-2 protein level also significantly increased in MCDD mice (ratio to CONTR: 1.52 ± 0.3, p ≤ 0.05 versus CONTR group) but remained low and was similar in CONTR and LC groups (ratio to CONTR: 1.1 ± 0.1) (Fig. 3C). No significant differences were detected in procaspase-2 protein expression between the groups.
      The effect of LC on fibrosis progression was supported by western blot analysis result. LC supplementation caused a significant increase in the level of hepatic PPARγ as compared to CONTR and MCDD treatment (ratio to CONTR: 1.5 ± 0.01, p ≤ 0.05 versus CONTR and MCDD groups) (Fig. 3D).
      We also evaluated NFkB level in inflamed livers and found it to be significantly elevated in mice from MCDD group as compared with those from the CONTR group (ratio to CONTR: 5 ± 0.5, p ≤ 0.001 versus CONTR group) (Fig. 3D). NFkB protein content significantly decreased after LC treatment (ratio to CONTR: 4.1 ± 0.2, p ≤ 0.05 versus MCDD group, p ≤ 0.001 versus CONTR group) (Fig. 3D).

      3.5 Heart morphology and histology

      Among the three groups, cardiac tissues did not showed altered morphology in haematoxylin and eosin staining (Fig. 4A), and differences in the intracellular accumulation of lipids were observed with ORO-staining (Fig. 4B).
      Fig. 4
      Fig. 4Effects of L-carnitine on heart histology, lipid accumulation, and oxidation. (A) Haematoxylin and eosin-stained cardiac tissues showed no diffused vacuolar degeneration in the three groups (control group, CONTR; methionine-choline deficient diet, MCDD; methionine-choline-deficient diet supplemented with L-carnitine, MCDD + LC). Alteration in cardiomyocyte size and morphology were not significant (magnification 20×). n = 5/treatment group. (B) Oil Red O (ORO)-stained sections showed the absence of lipid accumulation in all three groups (magnification 20×). n = 5/treatment group. (C) Cell ROS assay was used to evaluate the effect of L-carnitine (LC) on the heart tissue. Reactive oxygen species (ROS) production increased in the mice fed with methionine-choline-deficient diet (MCDD). L-Carnitine treatment attenuated this effect. n = 5/treatment group. (D) Western blot data showed that methionine-cholinedeficient diet (MCDD) improved the phosphorylated form of Ca2+/calmodulin-dependent protein kinase II (CaMKII). L-carnitine treatment reduced the level of this kinase. n = 5/treatment group.
      Data are expressed as the ratio relative to the control group ± SD. *p < 0.05 as compared with the mice fed with the control diet.

      3.6 Cardiac antioxidant effects of LC

      Oxidant stress is an important feature of CVD. Under stress condition, CaMKII activity (by phosphorylation) increases in the myocardium [
      • Anderson M.E.
      Oxidant stress promotes disease by activating CaMKII.
      ]. ROS level was higher in the heart samples of mice from MCDD group than in those of the mice from the CONTR group (Fig. 4C), and LC supplementation ameliorated this effect. These data were verified with western blotting. CaMKII activation increased in the MCD-fed mice (ratio to CONTR: 1.26 ± 0.01, p ≤ 0.05 versus CONTR group) while LC treatment reduced the phosphorylated form of CaMKII (ratio to CONTR: 1 ± 0.1) (Fig. 4D).

      3.7 Role of LC in cardiac fibrosis progression

      The ratio of pERK 1-2/ERK 1-2 was investigated in cardiac tissues with western blot analysis. As shown in Fig. 5A, MCDD significantly increased ERK activation in mice fed with MCDD as compared with those from the CONTR group (ratio to CONTR: 1.18 ± 0.02, p ≤ 0.05 versus CONTR group). LC supplementation counteracted this effect. pERK 1-2/ERK 1-2 ratio was similar between CONTR and MCDD + LC groups (ratio to CONTR: 1 ± 0.03, p ≤ 0.05 vs MCDD group).
      Fig. 5
      Fig. 5Role of LC in cardiac fibrosis progression. (A) Western blot analysis indicated that methionine-choline-deficient diet (MCDD) significantly increased the activation of extracellular-signal-regulated kinases (ERKs) as compared with the control (CONTR) diet. LC supplementation counteracted this effect. n = 5/treatment group. (B) The ratio of phosphorylated and non-phosphorylated signal transducer and activator of transcription 3 (pSTAT3/STAT3) was similar in the mice from methionine-choline-deficient diet supplemented with L-carnitine (MCDD + LC) and control groups, while MCDD. significantly increased n = 5/treatment group. (C) Masson-Goldner-trichrome stained sections showed fibrotic areas in the heart samples from the mice fed with MCDD. LC treatment ameliorated myocardial fibrosis (magnification 20×). n = 5/treatment group. (D) Immunofluorescence assay for α-smooth muscle actin (αSMA) in the mice fed with methionine-choline-deficient diet (MCDD. LC treatment ameliorated this effect. n = 5/treatment group.
      Data are expressed as ratio relative to the control group ± SD. *p < 0.05 as compared with the mice fed with control diet. # p < 0.05 as compared with the mice fed with methionine-choline-deficient diet (MCDD).
      We also investigated the role of STAT3, a mediator of oxidative stress and/or metabolism in cardiomyocytes. [
      • Haghikia A.
      • Ricke-Hoch M.
      • Stapel B.
      • et al.
      STAT3, a key regulator of cell-to-cell communication in the heart.
      ] As reported in Fig. 5B, LC supplementation significantly decreased pSTAT3/STAT3 ratio (ratio to CONTR: 0.7 ± 0.2, p ≤ 0.05 versus MCDD group), which was significantly higher in MCDD group than in CONTR group (ratio to CONTR: 1.56 ± 0.1, p ≤ 0. versus CONTR group).
      We evaluated fibrosis in heart tissues with Masson-Goldner staining, (Fig. 5C). LC treatment ameliorated myocardial fibrosis and decreased cardiac αSMA signal (Fig. 5D) as compared with MCDD treatment, confirming the delay in the progression of fibrosis.

      4. Discussion

      Accumulating evidence has shown that NAFLD not only affects the liver, but also the extrahepatic tissues such as the heart. Natural molecules have been successfully used for the prevention and treatment of NAFLD [
      • Del Ben M.
      • Polimeni L.
      • Baratta F.
      • et al.
      The role of nutraceuticals for the treatment of non-alcoholic fatty liver disease.
      ]. The results of the present study demonstrate the favourable effects of LC on hepatocyte steatosis, oxidative stress, and fibrosis development in the liver and myocardium of C57BL/6 male mice fed with MCDD.
      At the cellular level, steatosis is one of the key histological features of NAFLD that evolve into steatohepatitis [
      • Itoh M.
      • Kato H.
      • Suganami T.
      • et al.
      Hepatic crown-like structure: a unique histological feature in non-alcoholic steatohepatitis in mice and humans.
      ]. In our model, LC supplementation decreased the steatosis score (Table 1, Fig. 2B, C), indicative of the recovery from MCD-induced NALFD and prevention of steatohepatitis progression.
      Liver fat accumulation is associated with several pathogenic mechanisms involved in NASH development, including oxidative fat-induced injury [
      • Brunt E.M.
      Pathology of nonalcoholic fatty liver disease.
      ,
      • Friedman S.L.
      Mechanisms of hepatic fibrogenesis.
      ], the major source of ROS [
      • Wei Y.
      • Rector R.S.
      • Thyfault J.P.
      • et al.
      Nonalcoholic fatty liver disease and mitochondrial dysfunction.
      ]. Inhibition of mitochondrial fatty acid oxidation is thought to be a major cause of intrahepatic lipid accumulation and ROS production exacerbation [
      • Brunt E.M.
      Pathology of nonalcoholic fatty liver disease.
      ,
      • Day C.P.
      • James O.F.
      Steatohepatitis: a tale of two “hits”?.
      ,
      • Valenti L.
      • Bugianesi E.
      • Pajvani U.
      • et al.
      Nonalcoholic fatty liver disease: cause or consequence of type 2 diabetes?.
      ]. Considering the role of LC in β-oxidation, studies have suggested the potential use of LC for the treatment of lipotoxicity, under pathological conditions [
      • Benedini S.
      • Perseghin G.
      • Terruzzi I.
      • et al.
      Effect of L-acetylcarnitine on body composition in HIV-related lipodystrophy.
      ], including steatohepatitis [
      • Jun D.W.
      • Cho W.K.
      • Jun J.H.
      • et al.
      Prevention of free fatty acid-induced hepatic lipotoxicity by carnitine via reversal of mitochondrial dysfunction.
      ]. LC has the ability to scavenge free radicals [
      • Montesano A.
      • Senesi P.
      • Luzi L.
      • et al.
      Potential therapeutic role of L-carnitine in skeletal muscle oxidative stress and atrophy conditions.
      ,
      • Terruzzi I.
      • Montesano A.
      • Senesi P.
      • et al.
      L-carnitine reduces oxidative stress and promotes cells differentiation and bone matrix proteins expression in human osteoblast-like cells.
      ] and protect hepatocytes against oxidative damages caused under different conditions including ethanol intoxication [
      • Dobrzyńska I.
      • Szachowicz-Petelska B.
      • Skrzydlewska E.
      • et al.
      Effect of L-carnitine on liver cell membranes in ethanol-intoxicated rats.
      ], obesity [
      • Wu T.
      • Guo A.
      • Shu Q.
      • et al.
      L-carnitine intake prevents irregular feeding-induced obesity and lipid metabolism disorder.
      ], and carcinoma [
      • Jiang F.
      • Zhang Z.
      • Zhang Y.
      • et al.
      L-carnitine ameliorates the liver inflammatory response by regulating carnitine palmitoyltransferase I-dependent PPARγ signaling.
      ]. Here, we observed that LC counteracts COX imbalance caused by MCDD (Fig. 2E). Romestaing et al. [
      • Romestaing C.
      • Piquet M.A.
      • Letexier D.
      • et al.
      Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet.
      ] have demonstrated the increase in COX expression mediated by MCDD that alters mitochondrial metabolism, as demonstrated with elevated ROS level. In addition, other studies have described the capacity of LC to attenuate ROS accumulation and cardiac dysfunctions in ischemic or irradiated hearts [
      • Xue M.
      • Chen X.
      • Guo Z.
      • et al.
      L-carnitine attenuates cardiac dysfunction by ischemic insults through Akt signaling pathway.
      ,
      • Fan Z.
      • Han Y.
      • Ye Y.
      • et al.
      L-carnitine preserves cardiac function by activating p38 MAPK/Nrf2 signalling in hearts exposed to irradiation.
      ] and to decrease oxidative stress in normal and hyperglycemic cardiomyocytes [
      • Mao C.Y.
      • Lu H.B.
      • Kong N.
      • Li
      • et al.
      Levocarnitine protects H9c2 rat cardiomyocytes from H2O2-induced mitochondrial dysfunction and apoptosis.
      ,
      • Vacante F.
      • Senesi P.
      • Montesano A.
      • et al.
      L-carnitine: an antioxidant remedy for the survival of cardiomyocytes under hyperglycemic condition.
      ]. The present study confirmed that LC attenuates ROS production in the liver (Fig. 2D) and heart (Fig. 4C) of mice fed with MCDD. Taken together, our data suggest that LC prevents NAFLD progression and consequently heart damage.
      The activation of hepatic stellate cells and the conversion of cardiac fibroblasts into active myofibroblasts are indicative of the increase in the expression of αSMA in both hepatic and cardiac tissues [
      • Wang J.
      • Fan J.
      • Laschinger C.
      Smooth muscle actin determines mechanical force-induced p38 activation.
      ,
      • Friedman S.L.
      Fibrogenic cell reversion underlies fibrosis regression in liver.
      ,
      • Friedman S.L.
      Mechanisms of hepatic fibrogenesis.
      ]. LC supplementation was found to reduce αSMA expression both in the liver (Fig. 3B) and heart (Fig. 5D), suggestive of its inhibitory effect against fibrogenic αSMA-producing cells. Furthermore, at the hepatic level, the anti-fibrotic effect of LC was confirmed from the level of caspase-2 protein, which is thought to be a marker of NASH [
      • Machado M.V.
      • Kruger L.
      • Jewell M.L.
      • et al.
      Vitamin B5 and N-acetylcysteine in nonalcoholic steatohepatitis: a preclinical study in a dietary mouse model.
      ,
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • et al.
      ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P.
      ]. Caspase-2 level increases based on the stages of fibrosis both in animals and humans. Caspase-2-deficient mice did not develop NASH even after being fed with MCDD [
      • Machado M.V.
      • Michelotti G.A.
      • Pereira Tde A.
      • et al.
      Reduced lipoapoptosis, hedgehog pathway activation and fibrosis in caspase-2 deficient mice with non-alcoholic steatohepatitis.
      ]. As shown in Fig. 3C, the levels of pro-caspase-2 and caspase-2 were similar between controls and LC-fed mice, and MCDD significantly elevated caspase-2 protein expression, thereby confirming the anti-fibrotic action of LC.
      Fibrosis is a reversible process, and fibrotic tissues may be spontaneously resorbed upon retraction of the injurious stimulus [
      • Panebianco C.
      • Oben J.A.
      • Vinciguerra M.
      • et al.
      Senescence in hepatic stellate cells as a mechanism of liver fibrosis reversal: a putative synergy between retinoic acid and PPAR-gamma signalings.
      ]. PPARγ, mainly known for its central role in driving adipogenesis and lipid metabolism [
      • Wang Z.
      • Xu J.P.
      • Zheng Y.C.
      • et al.
      Peroxisome proliferator-activated receptor gamma inhibits hepatic fibrosis in rats.
      ], was proposed to perform a putative function in the reversal of liver fibrosis. In fact, the level of in vivo fibrogenesis was found to be decreased in PPARγ-depleted rat livers and increased in PPARγ-overexpressing rats fed with MCDD [
      • Wu C.W.
      • Chu E.S.H.
      • Lam C.N.Y.
      • et al.
      PPARγ is essential for protection against nonalcoholic steatohepatitis.
      ]. Several studies with different nutraceuticals have revealed the increase in hepatic fibrosis upon PPARγ upregulation [
      • Choi J.H.
      • Jin S.W.
      • Choi C.Y.
      • et al.
      Capsaicin inhibits dimethylnitrosamine-induced hepatic fibrosis by inhibiting the TGF-β1/Smad pathway via peroxisome proliferator-activated receptor gamma activation.
      ]. The protective role of PPARγ against liver fibrosis has been widely demonstrated [
      • Xu J.
      • Fu Y.
      • Chen A.
      Activation of peroxisome proliferator-activated receptor-gamma contributes to the inhibitory effects of curcumin on rat hepatic stellate cell growth.
      ,
      • Hazra S.
      • Xiong S.
      • Wang J.
      • et al.
      Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellate cells.
      ]. In particular, Chen et al. [
      • Chen Z.
      • Liu F.
      • Zheng N.
      • et al.
      Wuzhi capsule (Schisandra sphenanthera extract) attenuates liver steatosis and inflammation during non-alcoholic fatty liver disease development.
      ], used MCDD-fed mice and found that although MCDD was unable to modify PPARy level, an extract of Schisandra sphenanthera (medicinal herb) could counteract NASH progression by increasing PPARy expression. Similar to this study, our results showed that the progression of hepatic fibrosis was affected upon LC administration and associated with a significant increase in PPARγ level (Fig. 3D). Further studies are warranted to clarify the effect of PPARγ on NAFLD and NASH progression.
      PPAR-γ activation blocks the NFκB pathway through the repression of the translocation of p65 to the nucleus [
      • Feng X.
      • Weng D.
      • Zhou F.
      • et al.
      Activation of PPARγ by a natural flavonoid modulator, apigenin ameliorates obesity-related inflammation via regulation of macrophage polarization.
      ]. NFκB is known to control liver oxidative stress [
      • Leclercq I.A.
      • Farrell G.C.
      • Sempoux C.
      • et al.
      Curcumin inhibits NF-kappaB activation and reduces the severity of experimental steatohepatitis in mice.
      ,
      • Salamone F.
      • Galvano F.
      • Cappello F.
      • et al.
      Silibinin modulates lipid homeostasis and inhibits nuclear factor kappa B activation in experimental nonalcoholic steatohepatitis.
      ] and the transcription of pro-inflammatory cytokines responsible for the activation of hepatic stellate cells, collagen deposition, and fibrogenesis [
      • Wobser H.
      • Dorn C.
      • Weiss T.S.
      • et al.
      Lipid accumulation in hepatocytes induces fibrogenic activation of hepatic stellate cells.
      ], thereby contributing to the progression of NAFLD and liver fibrosis [
      • Luedde T.
      • Schwabe R.F.
      NF-κB in the liver—linking injury, fibrosis and hepatocellular carcinoma.
      ,
      • Tian Y.
      • Ma J.
      • Wang W.
      • et al.
      Resveratrol supplement inhibited the NF-κB inflammation pathway through activating AMPKα-SIRT1 pathway in mice with fatty liver.
      ]. We found that NFκB p65 protein level increased in MCDD group but decreased in MCDD + LC group. Thus, LC supplementation ameliorated hepatic liver condition by acting on NFκB p65 in a PPARγ-dependent manner (Fig. 3D). Future studies should elucidate how the LC-activated PPAR/NFkB axis could regulate the expression of pro-inflammatory cytokines.
      Hepatic steatosis induces systemic low-grade inflammation, possibly leading to cardiac inflammation and fibrosis [
      • Targher G.
      • Day C.P.
      • Bonora E.
      Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease.
      ]. NAFLD could be ameliorated by LC administration, as evident from the lower inflammatory and oxidant cardiac states and decreased ROS production and CaMKII phosphorylation level (Fig. 4C–D).
      Fibrosis level decreased in the cardiac tissues of mice fed with MCDD + LC, as evident from αSMA staining result (Fig. 5D). Fibrosis was also witnessed from the activation of STAT3 and depended on ERK phosphorylation [
      • Targher G.
      • Day C.P.
      • Bonora E.
      Risk of cardiovascular disease in patients with nonalcoholic fatty liver disease.
      ]. STAT3 is activated in response to several cardiac insults, such as oxidative damage, hypertrophy, and remodeling [
      • Dai B.
      • Cui M.
      • Zhu M.
      • et al.
      STAT1/3 and ERK1/2 synergistically regulate cardiac fibrosis induced by high glucose.
      ,
      • Haghikia A.
      • Ricke-Hoch M.
      • Stapel B.
      • et al.
      STAT3, a key regulator of cell-to-cell communication in the heart.
      ]. The ERKs/STAT3 pathway was inhibited in the LC-supplemented heart tissues (Fig. 5A, B), supporting the hypothesis that LC could prevent cardiac damages observed in NAFLD.

      5. Conclusion

      Our results demonstrate that LC supplementation could mitigate MCDD-induced steatosis and fibrosis in mice and ameliorate the mechanisms underlying NASH development. LC could act on both the “two hits” involved in NAFLD progression. It could regulate hepatic lipid accumulation and oxidative stress, the key phenomena involved in fibrosis. Thus, LC supplementation could serve as an effective nutraceutical adjuvant for the prevention and treatment of NASH.
      Our data suggest that LC may mitigate heart damage associated with NAFLD. Future studies should evaluate the applicability of long-term LC supplementation to control the pathophysiologic evolution of this disease in humans.

      Sources

      This study was partially supported by Ricerca Corrente funding from Italian Ministry of Health to IRCCS Policlinico San Donato.

      Conflicts of interest

      None declared.

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