RESUMEN

Objetivos:

Evaluar el rol de la L-carnitina (LC) sobre el estrés oxidativo inducido por fructosa en ratas Holtzman.

Materiales y métodos:

Se realizó un estudio experimental durante 56 días, con cuatro grupos: control, control+LC, fructosa y fructosa+LC. Los grupos con fructosa recibieron el tratamiento durante los 56 días, y los grupos con LC lo recibieron en los últimos 28 días. La fructosa se dio a libre demanda y la LC se administró por vía oral a una dosis de 500 g/kg/24 h. En el hígado se midió la lipoperoxidación (MDA), la actividad de superóxido dismutasa, las proteínas mitocondriales y posmitocondriales, y la LC libre. En el plasma se midió la glicemia, el índice de modelo homeostático para evaluar la resistencia a la insulina (HOMA-IR) e insulina. En el páncreas se midió la insulina y se realizó la histología.

Resultados:

El tratamiento con LC en el hígado mostró disminución (p < 0,05) de MDA frente al grupo control (21,73 ± 5,36 nmol/g tejido vs. 64,46 ± 7,87 nmol/g tejido). Las proteínas mitocondriales y posmitocondriales aumentaron (p < 0,05) frente al grupo control. La insulina pancreática también aumentó frente al control (341,8 ± 42,3 μUI/ml vs. 70,1 ± 9,6 μUI/ml, p<0,05). El rol de LC frente al estrés oxidativo inducido por fructosa no mostró disminución de MDA, pero produjo disminución (p < 0,05) en la actividad de SOD Cu/Zn (9,39 ± 1,5 USOD/mg proteína vs. 13,52 ± 1,5 USOD/mg proteína). En el plasma, se observó que la LC mejora el valor de la HOMA-IR. Histológicamente, la presencia de LC aumentó el número y tamaño de islotes de Langerhans.

Conclusiones:

La LC favorece los cambios del metabolismo oxidativo y ante el consumo de fructosa contribuye con la homeostasis glicémica.

Palabras clave:
Carnitina; Estrés Oxidativo; Fructosa; Antioxidantes; Insulina; Malondialdehído, Superóxido Dismutasa; Glicemia

ABSTRACT

Objectives:

To evaluate the role of L-carnitine (LC) on fructose-induced oxidative stress in Holtzman rats.

Materials and methods:

An experimental study was carried out during 56 days, in patients assigned to 4 groups: control, control+LC, fructose and fructose+LC. Patients in the fructose group received treatment during 56 days, and those in the LC groups were treated during the last 28 days. Fructose was given on demand and LC was administered orally at a dose of 500 g/kg/24 h. Lipid peroxidation (MDA), superoxide dismutase activity, free LC and mitochondrial and post-mitochondrial proteins were measured in liver tissue. Glycemia, insulin and the homeostasis model assessment of insulin resistance (HOMA-IR) were measured in blood plasma. We measured insulin concentration and studied the histology of pancreatic tissue.

Results:

LC treatment showed a decrease (p < 0.05) of MDA when compared to the control group (21.73 ± 5.36 nmol/g tissue vs. 64.46 ± 7.87 nmol/g tissue). Mitochondrial and post-mitochondrial proteins increased (p < 0.05) in comparison to the control group; pancreatic insulin also increased when compared to the control (341.8 ± 42.3 μUI/ml vs. 70.1 ± 9.6 μUI/ml, p<0.05). The role of LC against fructose-induced oxidative stress did not show any decrease of MDA, but decreased (p < 0.05) SOD Cu/Zn activity (9.39 ± 1.5 USOD/mg protein vs. 13.52 ± 1.5 USOD/mg protein). We observed that LC improves HOMA-IR in blood plasma. Histological analysis of the pancreas showed that the presence of LC increased the number and size of the islets of Langerhans.

Conclusions:

LC favors changes in the oxidative metabolism and it also contributes to glycemic homeostasis when fructose is consumed.

Keywords:
L-carnitine; Oxidative stress; Fructose; Antioxidants; Insulin; Malondialdehyde Superoxide Dismutase; Glycemia

INTRODUCTION

In Peru, over the last few years, the number of patients with chronic non-communicable diseases related to inadequate nutrition, such as cardiovascular and respiratory diseases, cancer, and type 2 diabetes mellitus, has increased. Research on experimental animals has shown that a diet rich in fructose causes chronic inflammation, which can lead to obesity, insulin resistance and metabolic syndrome. The evolution of this process can generate diabetes mellitus type 2 ¹1. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN, Mohd Yusof MR, Haji Suhaimi F. The Establishment of Metabolic Syndrome Model by Induction of Fructose Drinking Water in Male Wistar Rats. Biomed Res Int. 2014;2014:263897. doi: 10.1155/2014/263897.
https://doi.org/10.1155/2014/263897... ^,22. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93..

Chronic non-communicable diseases are associated with oxidative stress, as well as fructose consumption. Oxidative stress is the imbalance between the production of reactive oxygen species (ROS) and the defense mechanism, which determines the pathogenesis of several diseases ²2. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93..

L-carnitine (L-3-hydroxy-4-N-N-trimethyl amino-butyrate) facilitates the entry of long chain fatty acids into the mitochondria, for oxidation and production of adenosine triphosphate (ATP) in different tissues ⁽³3. Cao Y, Li X, Shi P, Wang L, Sui Z. Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells. Pharmacology. 2014;94(3-4):123-30. doi: 10.1159/000363062.
https://doi.org/10.1159/000363062... . L-carnitine (LC) is an essential nutrient; 75% obtained from the diet and 25% synthesized endogenously ³3. Cao Y, Li X, Shi P, Wang L, Sui Z. Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells. Pharmacology. 2014;94(3-4):123-30. doi: 10.1159/000363062.
https://doi.org/10.1159/000363062... ^,44. Chang B, Nishikawa M, Nishiguchi S, Inoue M. L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria. Int J Cancer. 2005;113(5):719-29. doi: 10.1002/ijc.20636.
https://doi.org/10.1002/ijc.20636... . Several studies have shown the antioxidant effect of LC in different diseases, either as a scavenger or as a factor that increases the activity of antioxidant enzymes ³3. Cao Y, Li X, Shi P, Wang L, Sui Z. Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells. Pharmacology. 2014;94(3-4):123-30. doi: 10.1159/000363062.
https://doi.org/10.1159/000363062... ^,55. Li J-L, Wang Q-Y, Luan H-Y, Kang Z-C, Wang C-B. Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha. J Biomed Sci. 2012;19:32. doi: 10.1186/1423-0127-19-32.
https://doi.org/10.1186/1423-0127-19-32... ^,66. Agarwal A, Sengupta P, Durairajanayagam D. Role of L-carnitine in female infertility. Reprod Biol Endocrinol. 2018;16(1):5. doi: 10.1186/s12958-018-0323-4.
https://doi.org/10.1186/s12958-018-0323-... .

There are few studies on the effect of LC on oxidative stress in experimental models with high fructose diet. The aim of this research is to evaluate the effect of LC on oxidative stress associated with excessive fructose consumption in an experimental model with Holtzman strain rats.

MATERIALS AND METHODS

Population and sample

This experimental research was carried out on four groups, two of which received water on demand and food with and without LC, and the other two received fructose (40%) on demand and food with and without LC.

Animals and diet

We used 24 two-month-old male Holtzman rats with an approximate weight of 217 ± 40 g, purchased from Instituto Nacional de Salud (Lima, Peru). They were placed in polycarbonate cages with stainless metal lids throughout the study. They were kept for seven days under acclimatization and received tap water on demand as well as food based on a commercial concentrate obtained from Universidad Nacional Agraria La Molina. The experiment was carried out in the vivarium of the Faculty of Medicine of Universidad Nacional Mayor de San Marcos, at room temperature between 23 and 26 °C, and a relative humidity of 60-70% with 12 hours of light/darkness. The LC (500 mg/kg per 24 h) was orally administered by an orogastric cannula.

We formed 4 groups, each with 6 rats randomly assigned with the OpenEpi program. Acclimatization conditions were maintained.

The control group (C) received feed and tap water on demand during the whole experiment; the control + L-carnitine group (C+LC) received feed and tap water on demand during the whole experiment plus L-carnitine at 500 mg/kg/24 h from day 28; the fructose (F) group received feed and fructose (40%) on demand during the whole experiment; and the fructose + L-carnitine group (F+LC) received feed and fructose (40%) on demand during the whole experiment and L-carnitine 500 mg/kg/24 h from day 28.

On the 27th and 56th days of the experiment, all the rats were fasted for the glycemia measurement. On day 57 the rats were euthanized by decapitation, after rapid and deep ether sedation. The flow chart was followed according to figure 1.

Fructose and L-carnitine preparation

The solutions were prepared daily: D-fructose >99% (Omnichem S.A.C, from Wuxi, China) and LC at 10% (Omnichem S.A.C, Ningbo, China). The tap water with fructose (40%) was based on the weight/volume formula.

Preparation of the homogenized products

The liver was washed by perfusion with 0.154 M KCl. The homogenates were prepared at 10% in saline phosphate buffer (SPB) using a Potter-Elvehjem type glass homogenizer. Three centrifugations were carried out at 4 °C (refrigerated centrifuge model MPW380R, MPW Med instruments); the first one was at 700 g for 5 minutes and the precipitate was discarded; the second one, with the supernatant, was at 9,500 g for 15 minutes. The supernatant corresponded to the post-mitochondrial fraction, and the precipitate corresponded to the mitochondrial fraction. The precipitate was washed twice with the SPB buffer at the same speed and for the same time as were needed to obtain the mitochondria. Then it was resuspended with 2 mL of the same buffer. Similarly, we prepared the pancreatic homogenate, which was the supernatant obtained after only one centrifugation at 700 g for 5 minutes.

Measurement of free carnitine, glucose, insulin, and the HOMA-IR

The blood samples were obtained from the tail vein. Glycemia was determined with a glucometer based on the conductometric method (Accu-chek Instant) on day 28 and day 57. Insulin measurement in plasma and pancreatic homogenate was performed with the ELISA kit on day 57 (Sigma-Aldrich, USA). Insulin resistance was evaluated with the insulin resistance homeostatic model assessment: HOMA-IR= [glucose (mg/dL) × insulin (mUI/mL) ] /405. The measurement of free LC in the liver homogenate was performed with the ELISA kit (Sigma-Aldrich, USA).

Superoxide dismutase activity

We measured superoxide dismutase activity on the liver tissue, according to Marklund and Marklund ⁷7. Marklund S, Marklund G. Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem. 1974;47(3):469-74. doi: 10.1111/j.1432-1033.1974.tb03714.x.
https://doi.org/10.1111/j.1432-1033.1974... . The inhibition of pyrogallol autooxidation in alkaline medium was the same for superoxide dismutase (SOD) activity in the mitochondrial fraction (Mn-SOD) and for the post-mitochondrial fraction (Cu/Zn-SOD). The kinetics was followed for three minutes at 420 nm in a spectrophotometer (Thermo Fisher Scientific, G10S UV-Vis). To report the enzymatic activity, the definition of the SOD unit was taken as 1U SOD=Δ of absorbance 0.02/2 × min (±10%).

Measurement of lipoperoxidation

After precipitation with 20% trichloroacetic acid, we measured the action between thiobarbituric acid and the decomposition products of lipoperoxidized species, such as malondialdehyde (MDA) in the hepatic homogenization, and obtained a colored complex that was read at 535 nm. The molar extinction coefficient (ε) was 1.56 × 105 M-1 cm-1 ⁸8. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-10. doi: 10.1016/s0076-6879(78)52032-6.
https://doi.org/10.1016/s0076-6879(78)52... .

Total protein measurement

Total proteins were quantified by the Biuret method ⁹9. Gornall AG, Bardawill CJ, David MM. Determination of serum proteins by means of the biuret reaction. J Biol Chem. 1949;177(2):751-66.; the reading was done after five minutes at 540 nm. We used a 2% albumin solution as a standard and measured total proteins in the mitochondrial and post-mitochondrial fractions obtained from the homogenized liver ⁸8. Buege JA, Aust SD. Microsomal lipid peroxidation. Methods Enzymol. 1978;52:302-10. doi: 10.1016/s0076-6879(78)52032-6.
https://doi.org/10.1016/s0076-6879(78)52... .

Statistical analysis

We used Shapiro Wilk’s test to evaluate normality and Bartlett’s test for variance homogeneity, and the parametric analysis of variance (ANOVA) and Scheffé’s test as post hoc tests for multiple comparisons. Statistical significance was assumed when the value was p < 0.05. We used the statistical program Stata 13.

Ethical aspects

We followed the ethical standards detailed in the Guideline for Handling and Care of Laboratory Animals of Ministerio de Salud - Instituto Nacional de Salud. The chosen type of euthanasia is contemplated in Law 30407, Law of Protection and Welfare of Animals.

RESULTS

Fasting glycemia results and the HOMA-IR scores did not show significant variations. However, the HOMA-IR score increased by 28.3% because of fructose consumption, when compared to the C group. In the F+LC group, it decreased by 25.8% compared to the F group (Table 1).

Free LC, mitochondrial and post-mitochondrial total proteins showed significant group difference in liver tissue. However, the only significant difference in peer evaluation was found in the free LC, which showed an increase of 21.5% in the F+LC group compared to the C group (Table 1).

The administration of LC stimulated production of insulin in the pancreatic tissue. The increase of insulin levels in the C+LC group was highly significant (p < 0.001) compared to the C group; the increase was of 387% (341.8 ± 42.5 vs. 70.1 ± 9.6 µIU/mL). Fructose consumption produced a significant decrease (p < 0.01) in pancreatic insulin (12.6 ± 4.2 µIU/mL). LC administration plus 40% fructose consumption produced a 100% recovery rate (25.8 ± 12.7 vs. 12.6 ± 4.2 µIU/mL), but this value was not like the one obtained from group C (Figure 2).

During the macroscopic evaluation, we observed that fructose increased abdominal fat in the F group and the F+LC group. The increased fat mass included the mesentery and retroperitoneum compared to the C group and the C+LC group.

During the histological evaluation of the pancreatic tissue, we observed that the presence of LC (C+LC group) increased the number and size of the islets of Langerhans, even more than the pancreatic acini, compared to the other groups. In the F group and the F+LC group, the size of the islets of Langerhans increased in some regions near the blood vessels when compared to the C group (Figure 3).

When evaluating the antioxidant effect, we observed a significant decrease of 30.5% of Cu/Zn-SOD activity in the F+LC group when compared to the C group (9.4 ± 1.5 vs. 13.5 ± 1.5 USOD/mg protein, p < 0.05) (Figure 4). However, the compensatory change in both fractions was notorious, while the activity decreased at the post-mitochondrial level, we observed an increase in the mitochondrial activity.

The administration of LC produced a significant decrease in MDA levels (p < 0.01) compared to the C group. Consumption of fructose (40%) (F group) caused a significant increase of 21% (p = 0.03) compared to the C group. LC administration plus fructose consumption did not show a significant decrease of the MDA levels (Figure 5).

DISCUSSION

We have observed that the administration of LC plays an antioxidant role, related to the excessive consumption of fructose in rats of the Holtzman strain.

Fructose is a sugar added to processed foods and its consumption has increased in various societies. Excessive fructose intake is associated with insulin resistance, obesity, dyslipidemia, and metabolic syndrome ¹1. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN, Mohd Yusof MR, Haji Suhaimi F. The Establishment of Metabolic Syndrome Model by Induction of Fructose Drinking Water in Male Wistar Rats. Biomed Res Int. 2014;2014:263897. doi: 10.1155/2014/263897.
https://doi.org/10.1155/2014/263897... ^,22. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93.^,1010. Tappy L, Lê K-A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol Rev. 2010;90(1):23-46. doi: 10.1152/physrev.00019.2009.
https://doi.org/10.1152/physrev.00019.20... ^,1111. Andrade N, Andrade S, Silva C, Rodrigues I, Guardão L, Guimarães JT, et al. Chronic consumption of the dietary polyphenol chrysin attenuates metabolic disease in fructose-fed rats. Eur J Nutr. 2020;59(1):151-165. doi: 10.1007/s00394-019-01895-9.
https://doi.org/10.1007/s00394-019-01895... . L-carnitine is an endogenous aminoacid associated with lipid metabolism; it has also been reported to have antioxidant activity.

The model of fructose-induced oxidative stress was used because of the metabolic changes it produces in serum and tissue. Fructose can generate ROS in vivo and in vitro, as does glucose^(1,2,10,). In this study, fructose (40%) on demand did not modify the fasting plasma glucose levels during eight weeks. Similar results were reported by Andrade et al. ¹¹11. Andrade N, Andrade S, Silva C, Rodrigues I, Guardão L, Guimarães JT, et al. Chronic consumption of the dietary polyphenol chrysin attenuates metabolic disease in fructose-fed rats. Eur J Nutr. 2020;59(1):151-165. doi: 10.1007/s00394-019-01895-9.
https://doi.org/10.1007/s00394-019-01895... who used fructose (10%) as treatment on demand for 18 weeks. However, Mamikutty et al. ¹1. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN, Mohd Yusof MR, Haji Suhaimi F. The Establishment of Metabolic Syndrome Model by Induction of Fructose Drinking Water in Male Wistar Rats. Biomed Res Int. 2014;2014:263897. doi: 10.1155/2014/263897.
https://doi.org/10.1155/2014/263897... demonstrated increased glycemia using fructose at 20% and 25% in Wistar rats for eight weeks. Also, Bulboacă et al. ²2. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93. reported increased glycemia using fructose (10%) in Wistar rats for 12 weeks. It is important to mention that there are genetic differences that express metabolic variations according to each rat strain ¹²12. Conn PM. Animal Models for the Study of Human Disease [Internet]. Elsevier; 2013 [citado el 22 de julio de 2019]. Disponible en: https://linkinghub.elsevier.com/retrieve/pii/C20110052250.
https://linkinghub.elsevier.com/retrieve... .

There is a significant difference between the absorption process of fructose and glucose. Fructose is absorbed by the GLUT 5 transporter, regardless of the absorption of glucose. After various processes, fructose can enter glycolysis, avoiding the hexokinase and phosphofructokinase-1 regulation points ¹⁰10. Tappy L, Lê K-A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol Rev. 2010;90(1):23-46. doi: 10.1152/physrev.00019.2009.
https://doi.org/10.1152/physrev.00019.20... . Entering glycolysis provides metabolites for lipogenesis and inhibits the beta-oxidation process. This process could explain the increase of visceral fat in the F group and F+LC group that we, macroscopically, observed. On the other hand, fructose is not an accurate way to measure glycemia at the pancreatic level, because beta-pancreatic cells do not have GLUT 5 transporters ¹1. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN, Mohd Yusof MR, Haji Suhaimi F. The Establishment of Metabolic Syndrome Model by Induction of Fructose Drinking Water in Male Wistar Rats. Biomed Res Int. 2014;2014:263897. doi: 10.1155/2014/263897.
https://doi.org/10.1155/2014/263897... ^,1010. Tappy L, Lê K-A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol Rev. 2010;90(1):23-46. doi: 10.1152/physrev.00019.2009.
https://doi.org/10.1152/physrev.00019.20... , so fructose metabolism is independent of insulin and would not increase glycemia ¹⁰10. Tappy L, Lê K-A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol Rev. 2010;90(1):23-46. doi: 10.1152/physrev.00019.2009.
https://doi.org/10.1152/physrev.00019.20... , which would explain the results. Moreover, fructose is related to the conservation of plasma insulin, expressed as HOMA-IR, where we did not observe no significant differences in the groups. HOMA-IR, as a parameter of insulin resistance, showed an increase of 28.3% in the F group compared to the C group; this moderate increase would suggest that the use of fructose for a longer time could generate insulin resistance, as described in other studies¹1. Mamikutty N, Thent ZC, Sapri SR, Sahruddin NN, Mohd Yusof MR, Haji Suhaimi F. The Establishment of Metabolic Syndrome Model by Induction of Fructose Drinking Water in Male Wistar Rats. Biomed Res Int. 2014;2014:263897. doi: 10.1155/2014/263897.
https://doi.org/10.1155/2014/263897... ^,22. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93.^,1010. Tappy L, Lê K-A. Metabolic effects of fructose and the worldwide increase in obesity. Physiol Rev. 2010;90(1):23-46. doi: 10.1152/physrev.00019.2009.
https://doi.org/10.1152/physrev.00019.20... ^,1313. Suwannaphet W, Meeprom A, Yibchok-Anun S, Adisakwattana S. Preventive effect of grape seed extract against high-fructose diet-induced insulin resistance and oxidative stress in rats. Food Chem Toxico. 2010;48(7):1853-7. doi: 10.1016/j.fct.2010.04.021.
https://doi.org/10.1016/j.fct.2010.04.02... . Furthermore, a decrease of 25.8% was observed in the F+LC group compared to the F group. For example, Ringseir et al. ¹⁴14. Ringseis R, Keller J, Eder K. Role of carnitine in the regulation of glucose homeostasis and insulin sensitivity: evidence from in vivo and in vitro studies with carnitine supplementation and carnitine deficiency. Eur J Nutr. 2012;51(1):1-18. doi: 10.1007/s00394-011-0284-2.
https://doi.org/10.1007/s00394-011-0284-... ⁾ reviewed six studies on rats in which LC decreased glycemia and the HOMA-IR.

Fructose consumption produced a significant decrease in the level of insulin in the pancreas. This result, observed in the F group, can be related to the increase of the number and size of adipocytes, which causes the release of MCP-1, which leads to the recruitment of macrophages-M1 and the release of cytokines such as TNF-α, IL1 and IL6, which cause a state of chronic inflammation¹⁵15. Guilherme A, Virbasius JV, Puri V, Czech MP. Adipocyte dysfunctions linking obesity to insulin resistance and type 2 diabetes. Nat Rev Mol Cell Biol. 2008;9(5):367-77. doi: 10.1038/nrm2391.
https://doi.org/10.1038/nrm2391... . Likewise, TNF-α binds to its death receptor, activating the extrinsic pathway and then the intrinsic pathway of apoptosis to finally produce the death of the beta-pancreatic cells ¹⁶16. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004;114(12):1752-61. doi: 10.1172/JCI200421625.
https://doi.org/10.1172/JCI200421625... . Maiztegui et al. ¹⁷17. Maiztegui B, Borelli MI, Madrid VG, Del Zotto H, Raschia MA, Francini F, et al. Sitagliptin prevents the development of metabolic and hormonal disturbances, increased ß-cell apoptosis and liver steatosis induced by a fructose-rich diet in normal rats. Clin Sci. 2011;120(2):73-80. doi: 10.1042/CS20100372.
https://doi.org/10.1042/CS20100372... used 10% fructose on free demand for three weeks and showed the reduction of the number of beta-pancreatic cells due to increased apoptosis. In contrast, during the histological evaluation we observed an increase of the size of the islets of Langerhans in the F group, probably due to compensatory effect of the stimulation of islets’ alpha, delta, F and G cells.

By using this model of stress induced by fructose at 40% consumed on free demand, we observed that the administration of LC (F+LC group) induced a recovery of 100% of tissue insulin when compared to the consumption of only fructose (group F), this result, although not significant for this study, is important because it is evidence of the role of LC in the pancreatic tissue. On the other hand, the C+LC group had a different behavior, we observed a 387% increase in the level of insulin, and a greater number and size of the islets of Langerhans (there were even more islets by regions) than pancreatic acini compared to C group.

Several studies show that the administration of LC inhibits apoptosis. Bonomini et al. ¹⁸18. Bonomini M, Zammit V, Pusey CD, De Vecchi A, Arduini A. Pharmacological use of L-carnitine in uremic anemia: has its full potential been exploited? Pharmacol Res. 2011;63(3):157-64. doi: 10.1016/j.phrs.2010.11.006.
https://doi.org/10.1016/j.phrs.2010.11.0... reviewed different studies and suggested that LC could possibly inhibit caspase 3. Agarwal et al. ⁶6. Agarwal A, Sengupta P, Durairajanayagam D. Role of L-carnitine in female infertility. Reprod Biol Endocrinol. 2018;16(1):5. doi: 10.1186/s12958-018-0323-4.
https://doi.org/10.1186/s12958-018-0323-... reported a similar result after analyzing several studies, they found that LC inhibits caspases 3, 7 and 8 and regulates tumor suppressor proteins, which favors oocyte survival. Likewise, Cao et al. ³3. Cao Y, Li X, Shi P, Wang L, Sui Z. Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells. Pharmacology. 2014;94(3-4):123-30. doi: 10.1159/000363062.
https://doi.org/10.1159/000363062... conducted an in vitro study and found that the use of LC favors the decrease of the Bax/Bcl-2 ratio and the production of ROS. In metabolic terms, according to the study by Jiang et al. ¹⁹19. Jiang F, Zhang Z, Zhang Y, Wu J, Yu L, Liu S. L-carnitine ameliorates the liver inflammatory response by regulating carnitine palmitoyltransferase I-dependent PPAR? signaling. Mol Med Rep. 2016;13(2):1320-8. doi: 10.3892/mmr.2015.4639.
https://doi.org/10.3892/mmr.2015.4639... , the presence of LC favors the expression of CPT1 mediated by PPARγ, which increases the process of beta-oxidation. The results of our study lead us to believe that LC could inhibit apoptosis of beta-pancreatic cells, which significantly increased the level of pancreatic insulin in the C+LC group, while in the F + LC group it did not increase as much due to previous damage by fructose. Therefore, the administration of LC (C+LC group) demonstrated the ability to significantly (p < 0.01) stimulate insulin production at the tissue level (Figure 2) without affecting the plasma levels of the hormone.

In the liver, free LC levels increased significantly by 21.5% when it was given as a treatment to F group compared to group C. In addition, we observed that the administration of LC did not produce a significant increase with their peers, probably because LC can act as a scavenger. According to Gülçin ²⁰20. Gülçin I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006;78(8):803-11.doi: 10.1016/j.lfs.2005.05.103.
https://doi.org/10.1016/j.lfs.2005.05.10... the in vitro LC acts as a scavenger of superoxide anion and hydrogen peroxide and favors the chelation of the ferrous ion, due to its carbonyl group, which can stabilize free radicals in alpha carbon by conjugation. It can also be stated that the levels of free LC are stable in physiological situations. However, this changes under physiopathological conditions, such as the consumption of fructose through various mechanisms as reported by Chang et al. ⁴4. Chang B, Nishikawa M, Nishiguchi S, Inoue M. L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria. Int J Cancer. 2005;113(5):719-29. doi: 10.1002/ijc.20636.
https://doi.org/10.1002/ijc.20636... , who stated that the increase of ROS could reduce the expression and function of OCTN-2 (carnitine transporter in the plasma membrane of tissues).

In different studies, long-term fructose consumption increased the production of ROS ²2. Bulboaca A, D Bolboaca S, Suci S. Protective effect of curcumin in fructose-induced metabolic syndrome and in streptozotocin-induced diabetes in rats. Iran J Basic Med Sci. 2016;19(6):585-93.^,2121. Germoush MO, Elgebaly HA, Hassan S, Mahmoud AM. Anti - Diabetic Effects of Padina Pavonia in Fructose - Induced Diabetic Rats. Aljouf Sci Eng J. 2015;286(3104):1-7. doi: 10.12816/0023935.
https://doi.org/10.12816/0023935... . NADH and FADH₂ are produced when fructose enters glycolysis and the Krebs cycle. These two molecules then go to the electron transport chain in the mitochondria, where there is a large production of superoxide anion. If fatty acids are formed, they can be metabolized by beta-oxidation, which produces ROS and acetyl-CoA, which can generate more NADH and FADH₂²²22. Tangvarasittichai S. Oxidative stress, insulin resistance, dyslipidemia and type 2 diabetes mellitus. World J Diabetes. 2015;6(3):456-80. doi: 10.4239/wjd.v6.i3.456.
https://doi.org/10.4239/wjd.v6.i3.456... . In this sense, Furukawa et al. ¹⁶16. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, et al. Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest. 2004;114(12):1752-61. doi: 10.1172/JCI200421625.
https://doi.org/10.1172/JCI200421625... reported greater activity of the NAPDH-oxidase in the adipocytes of obese people and a decrease in the expression of antioxidant enzymes, which can easily generate oxidative stress.

The higher production of ROS compromises the antioxidant defense mechanisms; at the enzymatic level, SOD is the first one that acts against the univalent reduction of oxygen. As mentioned, LC exerts its main role in the mitochondria, this can explain the 25% increase in mitochondrial enzyme activity on the C group. The LC role in the mitochondria can also explain the slight increase of LC levels (C + LC group). Also, we observed a coupled behavior between cytosolic and mitochondrial isoenzymes. When compared to the C group, the F+LC group showed a 30.5% decrease of the Cu/Zn-SOD activity, while the Mn-SOD activity increased 42%. According to Suzuki et al. ²³23. Suzuki K, Miyazawa N, Nakata T, Seo HG, Sugiyama T, Taniguchi N. High copper and iron levels and expression of Mn-superoxide dismutase in mutant rats displaying hereditary hepatitis and hepatoma (LEC rats). Carcinogenesis. 1993;14(9):1881-1884. doi: 10.1093/carcin/14.9.1881.
https://doi.org/10.1093/carcin/14.9.1881... , excess ROS may lead to inhibition of the Cu/Zn-SOD enzyme and an increase of Mn-SOD, which is probably an adaptive response to ROS production. It should be noted that Mn-SOD is probably the most important enzyme for survival in an oxidative environment ²⁴24. Case AJ. On the Origin of Superoxide Dismutase: An Evolutionary Perspective of Superoxide-Mediated Redox Signaling. Antioxidants (Basel) Switz. 2017;6(4):82. doi: 10.3390/antiox6040082.
https://doi.org/10.3390/antiox6040082... .

In this oxidative environment, derived from mitochondrial activity, LC administration favors the production of large amounts of acetyl-CoA, which generates acetyl groups for protein or histone acetylation processes, and produces post-translational or epigenetic changes ²⁵25. Madiraju P, Pande SV, Prentki M, Madiraju SRM. Mitochondrial acetylcarnitine provides acetyl groups for nuclear histone acetylation. Epigenetics. 2009;4(6):399-403. doi: 10.4161/epi.4.6.9767.
https://doi.org/10.4161/epi.4.6.9767... . In their research, Kerner et al. ²⁶26. Kerner J, Yohannes E, Lee K, Virmani A, Koverech A, Cavazza C, et al. Acetyl-L-carnitine increases mitochondrial protein acetylation in the aged rat heart. Mech Ageing Dev. 2015;145:39-50. doi: 10.1016/j.mad.2015.01.003.
https://doi.org/10.1016/j.mad.2015.01.00... ⁾ observed that acetyl-CoA treatment increased Mn-SOD acetylation. It can be assumed that acetylation could favor increased the activity of this enzyme. On the other hand, we observed in the C+LC group a significant increase of mitochondrial and post-mitochondrial total protein levels, a similar effect was observed in the F group, although it was not significant. These results allow us to presume that the LC would not only act as an activity regulator, but could also be related to protein synthesis, which includes antioxidant enzymes.

The assessment of lipoperoxidation shows the damage made to the membrane by peroxidative reactions of polyunsaturated fatty acids (PUFA); the level of MDA is considered a marker of oxidative stress. The antioxidant properties of LC displayed in liver tissue were also observed in the MDA levels, which significantly decreased (p < 0.01) in the absence of oxidative stress factors such as fructose, which corroborates the scavenger role discussed above ⁽³3. Cao Y, Li X, Shi P, Wang L, Sui Z. Effects of L-Carnitine on High Glucose-Induced Oxidative Stress in Retinal Ganglion Cells. Pharmacology. 2014;94(3-4):123-30. doi: 10.1159/000363062.
https://doi.org/10.1159/000363062... ^,44. Chang B, Nishikawa M, Nishiguchi S, Inoue M. L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria. Int J Cancer. 2005;113(5):719-29. doi: 10.1002/ijc.20636.
https://doi.org/10.1002/ijc.20636... ^,66. Agarwal A, Sengupta P, Durairajanayagam D. Role of L-carnitine in female infertility. Reprod Biol Endocrinol. 2018;16(1):5. doi: 10.1186/s12958-018-0323-4.
https://doi.org/10.1186/s12958-018-0323-... ^,2020. Gülçin I. Antioxidant and antiradical activities of L-carnitine. Life Sci. 2006;78(8):803-11.doi: 10.1016/j.lfs.2005.05.103.
https://doi.org/10.1016/j.lfs.2005.05.10... . Consumption of fructose at 40% on free demand produced an increase in lipoperoxidation by 21%, and the administration of LC could not reverse this change. The consumption of fructose generated a large amount of ROS, so probably a longer treatment time could reduce oxidative stress, expressed as MDA, as has been shown in other studies⁵5. Li J-L, Wang Q-Y, Luan H-Y, Kang Z-C, Wang C-B. Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha. J Biomed Sci. 2012;19:32. doi: 10.1186/1423-0127-19-32.
https://doi.org/10.1186/1423-0127-19-32... ^,2727. Zambrano S, Blanca AJ, Ruiz-Armenta MV, Miguel-Carrasco JL, Revilla E, Santa-María C, et al. The renoprotective effect of L-carnitine in hypertensive rats is mediated by modulation of oxidative stress-related gene expression. Eur J Nutr. 2013;52(6):1649-59. doi: 10.1007/s00394-012-0470-x.
https://doi.org/10.1007/s00394-012-0470-... ^,2828. Lee B-J, Lin J-S, Lin Y-C, Lin P-T. Effects of L-carnitine supplementation on oxidative stress and antioxidant enzymes activities in patients with coronary artery disease: a randomized, placebo-controlled trial. Nutr J. 2014;13:79. doi: 10.1186/1475-2891-13-79.
https://doi.org/10.1186/1475-2891-13-79... . Lipoperoxidation could be diminished by mechanisms that increase the expression of Mn-SOD and Cu/Zn-SOD, which is mediated by the increase of the mRNA expression of PPARα, as reported by Liu et al.²⁹29. Liu X, Jang SS, An Z, Song H, Kim W-D, Yu J-R, et al. Fenofibrate decreases radiation sensitivity via peroxisome proliferator-activated receptor a-mediated superoxide dismutase induction in HeLa cells. Radiat Oncol J. 2012;30(2):88-95. doi: 10.3857/roj.2012.30.2.88.
https://doi.org/10.3857/roj.2012.30.2.88... .

Several studies show that the presence of PPAR activates the expression of Mn-SOD and Cu/Zn-SOD genes through the transcriptional pathway ⁵5. Li J-L, Wang Q-Y, Luan H-Y, Kang Z-C, Wang C-B. Effects of L-carnitine against oxidative stress in human hepatocytes: involvement of peroxisome proliferator-activated receptor alpha. J Biomed Sci. 2012;19:32. doi: 10.1186/1423-0127-19-32.
https://doi.org/10.1186/1423-0127-19-32... ^,2929. Liu X, Jang SS, An Z, Song H, Kim W-D, Yu J-R, et al. Fenofibrate decreases radiation sensitivity via peroxisome proliferator-activated receptor a-mediated superoxide dismutase induction in HeLa cells. Radiat Oncol J. 2012;30(2):88-95. doi: 10.3857/roj.2012.30.2.88.
https://doi.org/10.3857/roj.2012.30.2.88... ^,3030. Kim T, Yang Q. Peroxisome-proliferator-activated receptors regulate redox signaling in the cardiovascular system. World J Cardiol. 2013;5(6):164. doi: 10.4330/wjc.v5.i6.164.
https://doi.org/10.4330/wjc.v5.i6.164... . Then, based on the results of this study, we could propose that a longer treatment with LC would reduce the levels of ROS, which would avoid the lipoperoxidation and its harmful effects at the cellular level.

The availability of resources was one of the limitations of our study, therefore we could not assess the basal concentrations of MDA and SOD and why a longer treatment was not used.

In conclusion, we observed that fructose does not affect glycemia, but it favors lipogenesis and an oxidative environment; in this scenario, administration of LC favors metabolic changes that corroborate its antioxidant function.

Acknowledgements:

he authors wish to thank the following contributors: Dr. Conrad Ortiz, for his help in reviewing the article; Dr. Eddy R. Segura, for his help in statistical consulting; and Lic. Marta Miyashiro, for her help in proofreading.

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