L-arginine administration exacerbates myocardial injury in diabetics via prooxidant and proinflammatory mechanisms along with myocardial structural disruption

Abstract

BACKGROUND

L-arginine (L-Arg) is one of the most widely used amino acids in dietary and pharmacological products. However, the evidence on its usefulness and dose limitations, especially in diabetics is still controversial.

AIM

To investigate the effects of chronic administration of different doses of L-Arg on the cardiac muscle of type 2 diabetic rats.

METHODS

Of 96 male rats were divided into 8 groups as follows (n = 12): Control, 0.5 g/kg L-Arg, 1 g/kg L-Arg, 1.5 g/kg L-Arg, diabetic, diabetic + 0.5 g/kg L-Arg, diabetic + 1 g/kg L-Arg, and diabetic + 1.5 g/kg L-Arg; whereas L-Arg was orally administered for 3 months to all treated groups.

RESULTS

L-Arg produced a moderate upregulation of blood glucose levels to normal rats, but when given to diabetics a significant upregulation was observed, associated with increased nitric oxide, inflammatory cytokines, and malonaldehyde levels in diabetic rats treated with 1 g/kg L-Arg and 1.5 g/kg L-Arg. A substantial decrease in the antioxidant capacity, superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione concentrations, and Nrf-2 tissue depletion were observed at 1 g/kg and 1.5 g/kg L-Arg diabetic treated groups, associated with myocardial injury, fibrosis, α-smooth muscle actin upregulation, and disruption of desmin cardiac myofilaments, and these effects were not noticeable at normal treated groups. On the other hand, L-Arg could significantly improve the lipid profile of diabetic rats and decrease their body weights.

CONCLUSION

L-Arg dose of 1 g/kg or more can exacerbates the diabetes injurious effects on the myocardium, while 0.5 g/kg dose can improve the lipid profile and decrease the body weight.

Key Words: L-arginine administration; Type 2 diabetes; Cardiac muscle injury; Oxidative stress; Inflammatory cytokines; Desmin disruption

Core Tip: L-arginine (L-Arg) is commonly used amino acid with many physiological effects, but its safety and pharmacological effects are not fully understood and still controversial. This study found that a dose of 0.5 g/kg of L-Arg appears to be the highest dose that can be safely administered without producing cardiac damage in diabetic rats. However, doses of 1 g/kg or higher can worsen myocardial damage by increasing blood glucose levels, inflammation, and oxidative stress. L-Arg can also reduce body weight and improve lipid levels in rats, but these benefits do not outweigh the injurious effects of high doses on the cardiac muscle.

INTRODUCTION

Millions worldwide commonly use L-arginine (L-Arg), a semi-essential amino acid, as a dietary supplement. It plays a vital role in synthesizing various biologically important compounds, including nitric oxide (NO), citrulline, proteins, creatine, L-glutamate, collagen, and agmatine[1,2]. Studies suggest L-Arg supplements might improve athletic performance, explaining its popularity among athletes[3]. L-Arg's significance stems largely from its role as a substrate for NO synthase (NOS) enzymes. These enzymes, neuronal NOS and endothelial NOS (eNOS), which normally produce NO, in addition to, inducible NOS (iNOS) which is activated during inflammation, and significantly increases NO production[4].Administration of a large dose of L-Arg orally has an immunomodulatory impact, this effect can lead to improved clearance of advanced-stage non-enzymatic glycosylation products, and ultimately improving glucose tolerance in diabetic patients[5]. Insightful assessments emerged from molecular dynamic simulations, demonstrated that the combination of L-Arg with metformin can produce notable effects. When L-Arg is coupled with metformin, it is displaced from the NOS activation site, resulting in a decrease in the concentration of NO, this potential benefit of this combination could be advantageous in clinical scenarios when NO could lead to adverse outcomes such as shock and stroke[6]. In human studies, oral short-term L-Arg supplementation increases endothelium-dependent brachial artery vasodilation[7]. However, a prolonged course of oral L-Arg supplementation showed the opposite effect; in which L-Arg administration to patients following myocardial infarction, not only increased mortality but also did not improve assessments of vascular stiffness or ejection fraction, on the contrary of other studies which demonstrated the beneficial effects of L-Arg administration in cardiac patients[7]. Around the world, particularly in developing countries, type 2 diabetes mellitus (T2DM) is a widespread chronic condition. People with T2DM and obesity often experience other metabolic problems, including insulin resistance, high triglycerides (TG), low HDL cholesterol, high blood pressure, excess belly fat, and disrupted release of hormones by fat tissue (adipokines). This combination of comorbidities, known as metabolic syndrome, is linked to a higher risk of developing cardiovascular disease[8,9]. In the inflammatory conditions like diabetes, a dramatic increase in iNOS activity leads to excessive NO production. Studies have shown a positive correlation between NO levels and HbA1c in T2DM, suggesting its crucial role in the development of diabetic complications[10]. Excess NO readily permeates cell membranes and cytoplasm, where it reacts with superoxide to form peroxynitrite. This triggers oxidative stress, damaging DNA, causing lipid peroxidation, and impairing endothelial function. Furthermore, iNOS upregulation is linked to beta cell death and the progression of diabetes. Beta cells, lacking sufficient antioxidant enzymes, are particularly susceptible to oxidative stress. Consequently, the combined elevation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) can readily damage beta cell mitochondria and inhibit insulin secretion[11-13]. Interestingly, research suggests that low NO levels contribute to healthy physiological functions like vascular tone and inflammation regulation[13,14]. However, the effects of L-Arg supplementation, a potential source of NO, in diabetic patients remain controversial. Whereas some studies report its beneficial effects, others show no significant impact[8,15,16]. L-Arg impact on the cardiovascular system, particularly in obese type 2 diabetic patients, remains unclear. To address this knowledge gap, we investigated the effects of long-term L-Arg supplementation using different doses on the cardiac tissue of diabetic rats. We also aimed to identify the potential mechanisms underlying these effects.

MATERIALS AND METHODS

Animals

This study was approved by the Faculty of Pharmacy Ethical Committee at South Valley University (protocol #P.S.V.U 230) for the use of animals. Male Sprague-Dawley rats aged 4-6 weeks, weighing 110-125 g, were obtained from the Egyptian Organization for Biological Products and Vaccines in Giza, Egypt. The rats were housed in cages with standard lighting and ventilation in a temperature-controlled environment (26 ± 3 °C) with a 12-hour light/dark cycle. They had unlimited access to water and standard rodent chow and were handled according to World Health Organization guidelines.

After a 10-day acclimatization period, the rats were randomly divided into eight groups (12 animals per group): (1) Control group (G1): Rats received a daily dose of 7.5 mL normal saline orally for12 weeks; (2) L-Arg (normal) group (G2): Rats received 0.5 g/kg/day of L-Arg orally for 12 weeks (normal diet)[17]; (3) L-Arg (normal) group (G3): Rats received 1 g/kg/day of L-Arg orally for 12 weeks (normal diet)[18]; (4) L-Arg (normal) group (G4): Rats received 1.5 g/kg/day of L-Arg orally for 12weeks (normal diet)[19]; (5) Diabetic group (G5): Rats were fasted overnight and then injected intraperitoneally with a single dose of 35 mg/kg streptozotocin (STZ) dissolved in citrate phosphate buffer (pH 4.5) to induce diabetes. The oral glucose tolerance test (OGTT) was conducted on two separate days following a two-weeks of STZ injection; blood glucose levels were measured in a fasting state and at 30, 60, 90, and 120 minutes following the oral administration of a 40% glucose solution (3 g/kg of bodyweight). Rats with blood glucose levels over 11.1 mmol/L after 120 minutes from the initiation of the test were considered diabetic, and only uniformly diabetic rats were utilized for subsequent investigations; (6) L-Arg (diabetic) group (G6): Diabetic rats received 0.5 g/kg/day of L-Arg orally for 12 weeks; (7) L-Arg (diabetic) group (G7): Diabetic rats received 1 g/kg/day of L-Arg orally for 12 weeks; and (8) L-Arg (diabetic) group (G8): Diabetic rats received 1.5 g/kg/day of L-Arg orally for 12 weeks.

In this study, rats were fed a specially formulated high-fat diet (HFD) containing 27% protein, 18% carbohydrate, and 55% fat. This HFD comprised lard, casein, a standard chow mix, along with essential vitamins and minerals. Control group received a standard diet.

Throughout the experiment, blood glucose levels were measured twice weekly by taking blood samples from rats’ tail and glucose level was determined using accu-check glucometer, weights were measured weekly and L-Arg doses were adjusted accordingly. Three days before the end of the experiment, an OGTT was performed in triplicate after fasting the animals for 12 hours.

At the end of the experiment, the rats were fasted overnight and weighed. The next morning at 8: 00 a.m., they were sacrificed under ketamine anesthesia (50 mg/kg, i.p.). Blood samples were collected from the inferior vena cava and centrifuged at 3000 rpm for 15 minutes. The serum was collected and stored at −20 °C for further analysis. Cardiac tissues were detached and washed with cold saline (5 °C). A portion of the cardiac tissue was homogenized in potassium phosphate buffer solution (100 mmol) and centrifuged at 3000 rpm for 20 minutes to produce a 10% cardiac tissue homogenate. The produced supernatants were collected and preserved at -80 °C (Figure 1).

Figure 1 Schematic diagram illustrating the major steps of the experiments in the study. HFD: High-fat diet; STZ: Streptozotocin.

Tissue total protein

To quantify the protein content within cardiac tissue, homogenates were prepared and analyzed using the Bradford method[20].

Measurement of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase

In this study, we measured these rum levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and alanine aminotransferase (ALT) using commercially available kits from Abcam (Cambridge, United Kingdom) with catalog numbers ab263883, ab197000, and ab234579, respectively.

Determination of serum creatine kinase-MB

This study investigated the activity of creatine kinase-MB (CK-MB) in the blood serum of rats. Acolorimetric method utilizing a CK-MB assay kit (Abcam) was employed. The technique relies on the reaction between CK-MB, phosphocreatine, and adenosine-diphosphate (ADP), generating a colored intermediate product that absorbs light maximally at a wavelength of 450 nm. As described previously by Wang et al[21].

Determination of the levels of cardiac troponin I

To confirm myocardial damage, we measured cardiac troponin I (cTnI) levels in rat serum using a commercially available ELISA kit (Cloud-Clone Corp #SEB820Ra). Briefly, we collected serum and centrifuged it at 1000 × g for 20 minutes. Then, 100 μL of serum and detection reagents were added to each well of a 96-well plate and incubated for 30 minutes at 37 °C. The plate was washed repeatedly to remove unbound antibodies between incubation steps. Finally, a substrate solution was added for another 30-minute incubation, followed by a stop solution. Absorbance was measured at 450 nm. This method is based on a previously described protocol by Hortmann et al[22].

Determination of serum total antioxidant capacity

The total antioxidant capacity (TAC) in the animal groups was determined in rat serum. This involved measuring the ability of serum antioxidants to neutralize added hydrogen peroxide (H₂O₂). The remaining H₂O₂ was then quantified using a well-established spectrophotometric method described by Apak et al[23]. This method relies on an enzymatic reaction that converts a colorless compound(3,5-dichloro-2-hydroxybenzenesulfonate) into a colored product, allowing for its detection at a specific wavelength (510 nm)[23].

Determination of serum lipids

The blood lipid levels of rats were measured, including high-density lipoprotein (HDL), TG, and total cholesterol (TC), using enzymatic assays based on established protocols[23-25]. Standard assay kits from Abcam (Waltham, MA, United States) were employed following the manufacturer's instructions. Low-density lipoprotein (LDL) levels were calculated using the formula LDL = TC – [HDL + (very low density lipoprotein (VLDL)], where VLDL was estimated as TG/5.

Enzyme-linked immunosorbent assay of inflammatory cytokines

Plasma levels of inflammatory cytokinesinterleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) were measured using ELISA kits (R and D Systems, Minneapolis, MN, United States) following the manufacturer's instructions. Catalog numbers for the kits are DY401-05 for IL-1β and RTA00-1 for TNF-α.

Determination of cardiac catalase activity

The breakdown of H₂O₂ by the catalase (CAT) enzyme. The amount of H₂O₂ decomposed reflects the level of CAT activity in the tissue sample. Finally, the color produced during the reaction is measured at a wavelength of 620 nanometers (nm)[26,27].

Determination of superoxide dismutase activity

Superoxide dismutase (SOD) activity was measured using a commercially available kit from Biodiagnostics (Cairo, Egypt). This method relies on SOD's ability to convert tetrazoliumsalts into a colored, water-soluble formazan dye. The intensity of this dye, measured at a wavelength of 450 nanometers (nm)[28,29], is proportional to SOD activity.

Determination of reduced glutathione concentration

The reduced glutathione (GSH) levels in the heart muscle tissue were measured using a standardized kit (Biodiagnostics, Cairo, Egypt). This method relies on GSH's ability to reduce a compound, 5,5'-dithiobis-2-nitrobenzoic acid, which generates a yellow color measurable at 412 nm using a colorimetric technique[30].

Determination of glutathione peroxidase

GSH peroxidase (GSH-Px) activity in homogenized heart tissue was measured using a commercial kit (Biodiagnostics, Cairo, Egypt). The assay monitored the formation of a product at a wavelength of 412 nm, as described by Razygraev et al[31].

Determination of cardiac malondialdehyde content

Cardiac lipid peroxidation was assessed by measuring Thiobarbituric Acid Reactive Substances (TBARS). This method utilizes the reaction between TBARS, a byproduct of lipid peroxidation, and TBARS under acidic conditions (pH 9.5) at 95 °C. This reaction produces a pink chromophore, which is then quantified by measuring its absorbance at 532 nm[32,33].

To assess the extent of lipid peroxidation in heart tissue, researchers measured the levels of TBARS. TBARS are a byproduct formed when lipids in the heart undergo oxidation. Their concentration can be determined using a TBA assay. The TBA assay is based on the reaction of MDA with TBA in acidic pH at 95 °C, which produces a pink color that can be measured at 532 nm. This method has been previously described by Aguilar Diaz De Leon et al[32] and Aboubakr et al[33].

NO determination

The levels of NO in the myocardial tissue homogenate were determined using the Griess reagent assay. This method is based on the reaction of NO with Griess reagent to produce a deep purple color that can be measured at 540 nm. This method has been previously described by Sun et al[34].

The NO levels in homogenized myocardial tissue were measured using the Griess reagent assay. This method detects NO by its reaction with Griess reagent, producing a deep purple color measurable at 540 nm[34].

Histopathological examination

Hematoxylin and eosin (HE) staining: The rats' myocardia were fixed in 10% formalin for 24 hours at 27 °C. They were then embedded in wax and cut into 5-μm-thick sections. The sections were stained with HE according to standard protocol. Haematoxylin (0.8%) was added for 5 minutes, followed by eosin (0.35%) for 3 minutes at 27 °C. The myocardial tissue structures were examined using a light microscope (Olympus BX53) at 200 × magnification. Six fields were randomly selected per slide and analyzed.

Two independent pathologists assessed myocardial injury using a 0-point to 4-pointscoring system. Here's how the scale translates: 0: No signs of tissue damage; 1: Mild cell death (necrosis) and swelling (edema) with somewhat organized cell structure; 2: Moderate localized cell death and swelling with slightly disorganized cell structure; 3: Severecell death, inflammatory cell infiltration, and significant disruption of cell arrangement; 4: Extensive and very advanced cell death, significant inflammatory cell infiltration, and extreme disruption of cell arrangement.

Masson’s trichrome staining

For analysis of perivascular and interstitial fibrosis size fraction, myocardial tissues were paraffin-embedded, sectioned at 5 μm, and stained with blue aniline Masson's trichrome. Light microscopy at 200 × magnification was used to quantify fibrosis according to the method described by Stilli et al[35]. Six random fields were analyzed per slide.

Immunohistochemistry

Dissected tissues were dehydrated, embedded in paraffin wax, and sectioned at 3 µm using a microtome. To ensure proper antigen retrieval, deparaffinized sections underwent heat-mediated retrieval in 85 °C water for 20 minutes. Between each step, washes with 0.1 M PBS were performed. Following the manufacturer's instructions, the sections were incubated with diluted rabbit polyclonal primary antibodies (Abcam, Cambridge, United Kingdom) against iNOS, Nrf-2, α-SMA, and desmin for 16 hours.

Under alight microscope (Leica DM2000 LED Ergonomic System), six random fields per slide were analyzed using Image-Pro Plus 5.0 (National Institutes of Health, Bethesda, MD, United States) software. Staining scores were assigned based on previously established protocols by Weng et al[36] and Liu et al[37]. Two independent pathologists, blinded to the treatment groups, evaluated and scored both histopathological and immunohistochemical staining. The final score representing pathological findings and protein expression represents the mean value across six randomly chosen fields per slide.

Statistical analysis

Statistical analysis was performed using graph-pad prism version 9.2.0, whereas results were represented as mean ± SE, n = 12. One-way analysis of variance was used followed by the Tukey–Kramer test. The differences between values were considered significant for P < 0.05.

RESULTS

Effect on blood glucose level

Giving diabetic rats 0.5 g of L-Arg per kilogram of body weight (0.5 g/kg) led to a small rise in their blood glucose levels at various time points (0, 30, 60, 90, and 120 minutes). These levels were slightly higher compared to untreated diabetic rats at the same time points. However, higher doses of L-Arg (1 g/kg and 1.5 g/kg) significantly increased blood glucose levels in the treated rats compared to the untreated group. This effect is detailed in Table 1, which shows the blood glucose readings for each group at each time point.

Table 1 The effect of oral administration of L-arginine on rats’ blood glucose concentrations at 0, 30, 60, 90, and 120 minutes, mean ± SD.

Experimental groups	0 minutes	30 minutes	60 minutes	90 minutes	120 minutes
Control	80.4 ± 5.4	91.8 ± 3.3	125 ± 5.2	120.2 ± 4.4	88 ± 4.2
L-Arg (0.5 gm/kg)	85.7 ± 6.6	98.8 ± 4.5	133.3 ± 8.2	134 ± 4.5	97.2 ± 5.4
L-Arg (1 gm/kg)	94.2 ± 7	105.6 ± 4.2	148.8 ± 8.7	152.4 ± 6.7	109.8 ± 7.2
L-Arg (1.5 gm/kg)	101.2 ± 7.2	117.3 ± 6.2	160 ± 7.7	162.5 ± 6.4	115.3 ± 6.1
Diabetic	155.2 ± 10.6	215.3 ± 11.3	278.5 ± 10.6	262.8 ± 12.3	198.2 ± 11.3
D + L-Arg (0.5 gm/kg)	168.9 ± 8.4	221.2 ± 10.6	289.3 ± 10.9	272.2 ± 9.8	215.3 ± 10.6
D + L-Arg (1 gm/kg)	174.2 ± 9.5	270.1 ± 9.9	315.4 ± 11.3	330.5 ± 13.3	275.6 ± 12.5
D + L-Arg (1.5 gm/kg)	180.5 ± 9.7	300.1 ± 13.2	366.8 ± 14.1	379.3 ± 14.2	291.1 ± 11.6

L-Arg: L-arginine.

Effect on animals' body weight

After weighing the rats at the end of experiment, the diabetic groups treated with 1 g/kg (234 g ± 31) and 1.5 g/kg (199 ± 18 g) of L-Arg showed significantly lower weights compared to the untreated diabetic group (291.5 ± 35 g). The control group had an average weight of 169.5 ± 12 g. In healthy rats given L-Arg, a slight decrease in weight was observed that corresponded to the amount of L-Arg administered (see Figure 2A).

Figure 2 Analysis of biochemical parameters in normal and treated rat groups, whereas animal weights, alanine aminotransferase, aspartate aminotransferase, serum creatine kinase, serum cardiac troponin I, lactate dehydrogenase. A: Animal weights; B: Alanine aminotransferase; C: Aspartate aminotransferase; D: Serum creatine kinase; E: Serum cardiac troponin I; F: Lactate dehydrogenase. G1 = Control group, G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg), G3 = Normal rats received 1 g/kg/day of L-Arg, G4 = Normal rats received 1.5 g/kg/day of L-Arg, G5 = Diabetic group, G6 = Diabetic rats received 0.5 g/kg/day of L-Arg, G7 = Diabetic rats received 1 g/kg/day of L-Arg, G8 = Diabetic rats received 1.5 g/kg/day of L-Arg. The data presented as means ± SE (n = 12). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; LDH: Lactate dehydrogenase.

Effect of L-Arg on ALT and AST levels

Figure 2 shows that giving L-Arg orally to healthy rats had no significant effect on ALT (Figure 2B) and AST (Figure 2C) levels compared to the control group. However, in diabetic rats, oral L-Arg at a dose of 0.5 g/kg caused a moderate increase in ALT and AST levels (to 85 ± 7 U/L and 50 ± 4 U/L, respectively) compared to untreated diabetic rats (77 ± 8 U/L and 44 ± 3 U/L, respectively). In healthy rats, the control levels of both enzymes were 25.6 ± 3 U/L (ALT) and 19.4 ± 2 U/L (AST). Higher doses of L-Arg (1 g/kg) significantly increased both ALT and AST in diabetic rats (to 97.4 ± 8 U/L and 67.25 ± 7 U/L, respectively). The greatest increase was observed in diabetic rats given 1.5 g/kg of L-Arg, with ALT and AST levels reaching 114 ± 9 U/L and 89.5 ± 8 U/L, respectively.

Effect on cardiac enzymes

This study investigated the effect of L-Arg on cardiac enzymes in diabetic rats. Diabetic rats displayed significantly higher levels of CK, cTnI, and LDH in their serum compared to control rats (184 ± 15 U/L vs 97 ± 8 U/L for CK, 2.6 ± 0.2 ng/mL vs 0.89 ± 0.7 ng/mL for cTnI, and 130 ± 11 IU/L vs 71 ± 5 IU/L for LDH). Interestingly, oral L-Arg supplementation at a dose of 0.5 g/kg had minimal impact on these enzyme levels in diabetic rats. However, a surprising trend emerged at higher doses. Diabetic rats administered 1 g/kg of L-Arg exhibited a further increase in these enzyme levels (215 ± 16 U/L, 3.39 ± 3 ng/mL, and 154 ± 13 IU/L for CK, cTnI, and LDH, respectively). This trend continued with a 1.5 g/kg dose, leading to the highest enzyme levels observed (256 ± 17 U/L, 3.9 ± 0.33 ng/mL, and 173 ± 14 IU/L for CK, cTnI, and LDH, respectively) (Figure 2D-F). Notably, L-Arg administration in healthy rats did not cause any significant changes in these cardiac enzymes as shown in (Figure 2D-F).

Effect on the TAC and GSH

When given to healthy rats, L-Arg at a dose of 0.5 g/kg slightly increased GSH and TAC levels. This effect was not seen with other L-Arg doses. Diabetic rats, on the other hand, had significantly lower GSH and TAC levels (1.63 ± 0.11 mmol and 9.88 ± 0.82 nmol/mg protein, respectively) compared to control rats (2.4 ± 0.15 mmol and 14.02 ± 0.85 nmol/mg protein, respectively). Giving 0.5 g/kg L-Arg orally to diabetic rats did not significantly alter their GSH and TAC levels. Interestingly, higher doses (1 g/kg and 1.5 g/kg) of L-Arg significantly decreased TAC levels in diabetic rats (to 1.1 ± 0.09 mmol and 0.78 ± 0.04 mmol, respectively) and GSH levels (to 8.1 ± 0.7 nmol/mg protein and 5.08 ± 0.44 nmol/mg protein, respectively) (Figure 3A and B).

Figure 3 Effect of L-arginine on the activities of serum total antioxidant capacity, reduced glutathione, low density lipoprotein, very low density lipoprotein, triglyceride, and total cholesterol levels in the normal and treated rat groups. A: Total antioxidant capacity; B: Reduced glutathione; C: Low density lipoprotein; D: Very low density lipoprotein; E: Triglyceride; F: Total cholesterol levels. G1 = Control group, G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg), G3 = Normal rats received 1 g/kg/day of L-Arg, G4 = Normal rats received 1.5 g/kg/day of L-Arg, G5 = Diabetic group, G6 = Diabetic rats received 0.5 g/kg/day of L-Arg, G7 = Diabetic rats received 1 g/kg/day of L-Arg, G8 = Diabetic rats received 1.5 g/kg/day of L-Arg. The data presented as means ± SE (n = 12). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group. TAC: Total antioxidant capacity; GSH: Glutathione; LDL: Low density lipoprotein; VLDL: Very low density lipoprotein.

Effect of L-Arg on lipid profile

This study investigated the effects of a HFD and L-Arg supplementation on blood lipid levels in diabetic rats (Figure 3). Diabetic rats fed HFD exhibited significantly (P < 0.05) elevated levels of LDL, VLDL, TG and TC compared to the control group. Specifically, LDL, VLDL, TG, and TC levels increased to 74 ± 6, 76.5 ± 5, 162 ± 9, and 201 ± 16 mg/dL, respectively, compared to 31.6 ± 2, 20 ± 2, 42 ± 4, and 79 ± 6 mg/dL in the control group. Conversely, HDL levels significantly decreased (P < 0.05) to 28.5 ± 2 mg/dL compared to 40.2 ± 4 mg/dL in controls.

Oral L-Arg administration effectively lowered blood LDL, VLDL, TG, and TC levels, while modestly increasing HDL levels. At a dose of 0.5 g/kg, L-Arg slightly reduced LDL, VLDL, TG, and TC levels to 69 ± 5, 57 ± 4, 145 ± 9, and 175 ± 13 mg/dL, respectively, with no significant impact on HDL levels. However, higher doses (1 g/kg and 1.5 g/kg) of L-Arg significantly (P < 0.05) decreased LDL, VLDL, TG, and TC levels. Specifically, the 1 g/kg group showed reductions to 55 ± 5, 41 ± 3, 127 ± 10, and 146 ± 13 mg/dL for LDL, VLDL, TG, and TC as shown in Figure 3C-F respectively, while the 1.5 g/kg group achieved reductions to 43 ± 3, 33 ± 3, 101 ± 8, and 127 ± 9 mg/dL. Additionally, HDL levels rose to 32.5 ± 2 and 34.5 ± 3 mg/dL in the 1 g/kg and 1.5 g/kg groups, respectively. As shown in Figure 3.

Effect on inflammatory mediators

Rats with diabetes fed a HFD had significantly higher levels of inflammatory markers TNF-α and IL-1β compared to control rats. Specifically, diabetic rats on HFD had 48.5 pg/mL and 34 pg/mL of TNF-α and IL-1β, respectively, while control rats had 18.3 pg/mL and 11 pg/mL, respectively.

Giving diabetic rats 0.5 g/kg of L-Arg orally didn't significantly affect these inflammatory markers. However, increasing the L-Arg dose to 1 g/kg and 1.5 g/kg significantly increased TNF-α and IL-1β levels to 70 ± 5 pg/mL and 43.3 ± 3 pg/mL, and 96.7 ± 7pg/mL and 59.4 ± 5 pg/mL, respectively.

Interestingly, giving L-Arg to normal rats at any dose (0.5 g/kg, 1 g/kg, or 1.5 g/kg) didn't cause any significant changes in these inflammatory markers compared to the control group (Figure 4A and B).

Figure 4 Effect of L-arginine on the activities of serum tumor necrosis factor-alpha, interleukin-1 beta, superoxide dismutase and glutathione peroxidase, malondialdehyde, nitric oxide, in the normal and treated rat groups. A: Tumor necrosis factor-alpha; B: Interleukin-1 beta; C: Superoxide dismutase; D: Glutathione peroxidase; E: Malondialdehyde; F: Nitric oxide. G1 = Control group, G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg), G3 = Normal rats received 1 g/kg/day of L-Arg, G4 = Normal rats received 1.5 g/kg/day of L-Arg, G5 = Diabetic group, G6 = Diabetic rats received 0.5 g/kg/day of L-Arg, G7 = Diabetic rats received 1 g/kg/day of L-Arg, G8 = Diabetic rats received 1.5 g/kg/day of L-Arg. The data presented as means ± SE (n = 12). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs to the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-arg 1 g/kg group. TNF-α: Tumor necrosis factor-alpha; IL-1β: interleukin-1 beta; SOD: superoxide dismutase; GSH-Px: Glutathione peroxidase; MDA: Malondialdehyde.

Effect on the antioxidant enzymes

This study investigated the effects of L-Argon antioxidant enzyme levels in diabetic rats. Oral L-Arg administration (0.5 g/kg) in diabetic rats did not significantly alter CAT, SOD, or GSH-Px levels compared to the control group. However, diabetic rats fed a HFD had significantly lower levels of these antioxidant enzymes (CAT: 7.8 ± 0.6 U/mg protein, SOD: 173 ± 15 U/mg protein, GSH-Px: 0.68 ± 0.05 U/mg protein) compared to the control group (CAT: 13.5 ± 1 U/mg protein, SOD: 224 ± 17 U/mg protein, GSH-Px: 0.98 ± 0.1 U/mg protein). Interestingly, higher doses of L-Arg (1 g/kg and 1.5 g/kg) in diabetic rats significantly decreased (P < 0.05) the levels of these enzymes (1 g/kg: CAT: 6.1 ± 0.55 U/mg protein, SOD: 161.5 ± 14 U/mg protein, GSH-Px: 0.48 ± 0.03 U/mg protein; 1.5 ± 0.11 g/kg: CAT: 5.1 ± 0.4 U/mg protein, SOD: 134 ± 10 U/mg protein, GSH-Px: 0.29 ± 0.01 U/mg protein) compared to diabetic controls (Figure 4).

Effect on TBARS

This study investigated how L-Arg supplementation affects tissue damage caused by fat breakdown (lipid peroxidation) in the heart. We measured levels of a biomarker called TBARS in heart tissue from rats. In healthy rats, only the highest dose of L-Arg (1.5 g/kg) significantly increased TBARS levels to 247 ± 17 μmole/g protein (Figure 4). In diabetic rats, however, even a low dose of L-Arg (0.5 g/kg) caused a slight increase (188 ± 16 µmole/g protein) in TBARS compared to untreated diabetic rats (190 ± 16 μmole/g protein). Higher L-Arg doses (1 and 1.5 g/kg) in diabetic rats led to even greater increases in TBARS levels to 198 ± 17 μmole/g protein and 244 ± 19 µmole/g protein respectively as shown in Figure 4.

Effect on NO level

This study investigated the effects of L-Arg administration on NO levels in various groups. In healthy rats, none of the administered doses (0.5 g/kg, 1.0 g/kg, or 1.5 g/kg) significantly increased NO levels. However, these doses did raise NO concentrations in a dose-dependent manner, reaching up to 4.9 ± 0.3 μmole/g protein, 5.5 ± 0.4 μmole/g protein, and 7.8 ± 0.6 μmole/g protein, respectively. Interestingly, diabetic rats exhibited significantly higher baseline NO levels (6.8 ± 0.5 μmole/g protein) compared to controls (3.5 ± 0.2 μmole/g protein). Moreover, oral administration of 0.5 g/kg L-Arg further elevated NO in diabetic rats (8.6 ± 0.7μmole/g protein). Increasing the L-Arg dose to 1 g/kg and 1.5 g/kg in diabetic rats resulted in even greater NO level increases, reaching 12.6 ± 1.1 μmole/g protein and 15.4 ± 1.4 μmole/g protein, respectively. Figure 4 illustrates these findings.

Histopathological changes

HE staining: Histopathological analysis of heart tissue under a microscope revealed normal structure in the control group and in L-Arg treated groups receiving 0.5 or 1 g/kg, with minimal inflammation observed in the 1.5 g/kg group (injury score < 1). In contrast, diabetic animals displayed signs of degeneration, including fragmented muscle fibers (myofibrils), shrunken nuclei (pyknosis), widened spaces between heart muscle cells (cardiomyocytes), congested blood vessels, moderate fat accumulation in the heart muscle (myocardial fat deposition), and significant inflammatory cell infiltration (injury score-3). Giving diabetic animals 0.5 g/kg L-Arg daily resulted in minimal changes compared to the untreated diabetic group, although fat deposition appeared slightly reduced. Interestingly, diabetic animals receiving 1 or 1.5 g/kg L-Arg daily showed a dramatic worsening of heart damage with severe inflammatory cell infiltration (injury scores 3.5 and 4, respectively). The higher dose of L-Arg appeared to cause more severe damage, as seen in Figure 5.

Figure 5 Histopathology of rats’ heart hematoxylin eosin. A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Cardiac injury score. Results are presented as mean ± SEM (n = 6). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the L-Arg diabetic + 0.5 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group.

Masson trichrome stain

This study investigated the effects of L-Arg on myocardial fibrosis in healthy and diabetic rats. We found that doses of 0.5, 1, and 1.5 g/kg administered to normal rats caused no significant changes in heart muscle fibrosis (Figure 6). In diabetic rats, 0.5 g/kg of L-Arg orally did not significantly affect fibrosis compared to untreated diabetic animals, which displayed a marked increase in fibrosis compared to the healthy control group. However, higher doses (1 and 1.5 g/kg) of L-Arg given orally to diabetic rats significantly worsened myocardial fibrosis, reaching 1.9% and 2.2%, respectively.

Figure 6 Histopathology of rats’ heart (Masson trichrome stain). A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Cardiac fibrosis. Results are presented as mean ± SEM (n = 6). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group.

Immunohistochemistry

Effect on desmin myofilaments: An examination of heart tissue using both immunohistochemistry and pathology (immunohistopathological analysis) showed strong staining with desmin antibodies in the control group. Additionally, the Z-lines of intercalated discs appeared normal in these animals. This pattern was also seen in normal rats given L-Arg at doses of 0.5, 1, and 1.5 g/kg. In contrast, diabetic animals displayed faint and uneven desmin staining, along with a disruption of the normal banding pattern (cross-striation) and moderate clumping (aggregation) of desmin around the nucleus (perinuclear areas). Interestingly, diabetic rats given L-Arg orally at doses of 1 and 1.5 g/kg showed a significant worsening of these abnormalities, with even greater misalignment of desmin and increased clumping with weak staining (Figure 7).

Figure 7 Immunohistochemistry results of desmin in cardiac tissues. A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Immunostaning area. ^aP < 0.05 vs the control group. Results are presented as mean ± SEM (n = 6). ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group). Results are presented as mean ± SEM (n = 6).

Effect on α-SMA myocardial distribution: This study used α-smooth muscle actin (α-SMA) staining to investigate protein levels. Compared to the normal group, the diabetic group showed a significant increase in α-SMA protein. Giving diabetic rats 0.5 g/kg of L-Arg had no significant effect on α-SMA distribution in the heart. Interestingly, higher doses (1 or 1.5 g/kg) of L-Arg in diabetic rats led to a substantial increase in cardiac α-SMA distribution. In contrast, administering 0.5, 1, or 1.5 g/kg of L-Arg to healthy rats caused no noticeable changes in myocardial α-SMA distribution compared to the control group (Figure 8).

Figure 8 Immunohistochemistry results of alpha smooth muscle actin in cardiac tissues. A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Immunostaning area. Results are presented as mean ± SEM (n = 6). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group).

Effect on Nrf-2 myocardial distribution: We investigated the immune expression of Nrf-2 in heart tissue. The control group displayed strong Nrf-2 expression, similar to healthy rats treated with L-Arg. However, the 1.5 g/kg L-Arg group showed a slight decrease in Nrf-2 compared to the control. Diabetic rats had significantly lower Nrf-2 expression. Interestingly, oral L-Arg (1 g/kg) in diabetic rats further reduced Nrf-2, while a 1.5 g/kg dose resulted in near-complete loss of Nrf-2 expression (Figure 9).

Figure 9 Immunohistochemistry results of nuclear factor erythroid 2 in cardiac tissues. A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Immunostaning area. Results are presented as mean ± SEM (n = 6). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg 05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group.

Effect on iNOS myocardial distribution: In healthy hearts (control group), examination of the muscle tissue (myocardium) revealed a moderate amount of a protein called iNOS. This protein was found to be significantly increased in groups treated with L-Arg, with the level of increase directly related to the amount of L-Arg administered. Diabetic rats also showed increased iNOS levels in their heart muscle, with the greatest increase observed in those given 0.5 g/kg of L-Arg. Even higher doses of L-Arg (1 or 1.5 g/kg) caused a very large rise in iNOS expression in the diabetic rats' heart muscle (Figure 10).

Figure 10  Immunohistochemistry results of Inducible nitric oxide synthase in cardiac tissues. A: G1 = Control group; B: G2 = Normal rats received 0.5 g/kg/day of L-arginine (L-Arg); C: G3 = Normal rats received 1 g/kg/day of L-Arg; D: G4 = Normal rats received 1.5 g/kg/day of L-Arg; E: G5 = Diabetic group; F: G6 = Diabetic rats received 0.5 g/kg/day of L-Arg; G: G7 = Diabetic rats received 1 g/kg/day of L-Arg; H: G8 = Diabetic rats received 1.5 g/kg/day of L-Arg; I: Immunostaning area. Results are presented as mean ± SEM (n = 6). ^aP < 0.05 vs the control group. ^bP < 0.05 vs the diabetic group. ^cP < 0.05 vs the diabetic + L-Arg05 g/kg group. ^dP < 0.05 vs the diabetic + L-Arg 1 g/kg group.

DISCUSSION

Scientists are divided on the ideal L-Arg supplementation plan, including the best dose and timing. While most research suggests doses between 3 and 6 g daily are safe, some studies report adverse effects exceeding 9 g[38]. Conversely, other research indicates safe administration of up to 40 g per day[39]. This conflicting data highlights the need for further research to establish the optimal dosage and duration of L-Arg supplementation. Additionally, the impact of L-Argon cardiovascular and metabolic health, particularly for individuals with T2DM, remains unclear due to limited data. This study examined the long-term safety of L-Argon heart tissue in normal and type 2 diabetic rats. Three different doses (0.5, 1, and 1.5 g/kg) were administered for 12 weeks. These doses, calculated for 70-kg rats, translate to 35, 70, and 105 g of L-Arg daily. To convert them to human equivalents, we applied the Food and Drug Administration's 2005 conversion ratio (1: 0.16, rats to humans)[40]. This translates to daily human doses of 5.6, 11.13, and 16.66 g of L-Arg for a 70 kg person. T2DM frequently presents with dyslipidemia, characterized by high TG, TC, LDL, and VLDL levels, alongside reduced HDL levels, and this condition can contribute to diabetic cardiomyopathy[29,41]. Our study investigated the effects of orally administered L-Argon blood lipid profiles in both healthy and T2DM animals. We observed significant reductions in TC, TG, LDL, and VLDL levels, accompanied by an increase in HDL levels, in both groups. This finding aligns with previous research demonstrating L-Arg's ability to enhance brown adipose tissue (BAT) mitochondrial function through gene expression stimulation[42,43]. Furthermore, L-Arg's conversion to NO appears to stimulate lipoprotein lipase activity, promoting TG breakdown into usable energy sources[42,43]. Researchers also suggests that L-Arg may increase BAT accumulation and decrease TG, LDL, and VLDL levels through NO-mediated mechanisms. This includes upregulating the expression of peroxisome proliferator-activated receptor gamma and coactivator 1 alpha, ultimately regulating mitochondrial biogenesis and cellular energy distribution[44-46] L-Arg plays a crucial role in the production of NO by an enzyme called NOS. This process occurs in most mammalian cells, including those in the cardiovascular system as described by Zhao et al[47]. Research on NO levels in diabetics has yielded conflicting results. Some studies[48] report elevated NO levels, while others show the opposite. Our study, however, aligns with previous findings[49] by demonstrating a significant increase in NO concentration within the cardiac tissue of diabetic rats. This increase is likely due to hyperglycemia-induced upregulation of genes and protein levels for both iNOS and eNOS forms of NOS. Furthermore, our results showed a significant rise in cardiac NO concentration for both healthy and diabetic groups treated with L-Arg, compared to control and untreated diabetic groups. This increase correlated positively with the L-Arg dose. Immunostaining confirmed elevated iNOS levels, further supporting these findings. iNOS plays a key role in stimulating NO production and reflects the heart tissue's capacity to generate NO[50,51]. Our research suggests that L-Arg, while beneficial in some cases, can react with molecules commonly found in diabetics [like methylglyoxal, a precursor to advanced glycation end products (AGEs)] to generate harmful superoxide radicals that damage cells[6]. Furthermore, elevated NO levels are linked to tissue damage caused by oxidative stress. This occurs when NO reacts with superoxide anions to form peroxynitrite, a potent oxidant and free radical. Peroxynitrite can damage tissues by activating PARP [poly (ADP-ribose) polymerase], forming peroxynitrous acid, hydroxyl radicals, and further increasing superoxide levels[52,53]. In our study, administering L-Arg to diabetic rats significantly increased cardiac NO levels, leading to a substantial increase in oxidative stress damage. This was confirmed by elevated levels of TBARS, a marker of oxidative stress intensity. Nrf2, a transcription factor with a unique basic leucine zipper structure, plays a critical role in the cell's defense against oxidative stress. It achieves this by specifically targeting antioxidant response elements (AREs) found in the promoter regions of certain genes[54,55]. Normally, Nrf2 remains inactive within the cytoplasm, bound to a protein called Kelch-like ECH-associating protein 1 (Keap1). However, when cells experience oxidative stress, Nrf2 breaks free from Keap1 and migrates to the nucleus. There, it combined with small Maf proteins, such as FosB, C-Jun, JunD, ATF2, or ATF4[56,57]. These Nrf2-Maf protein complexes then bind to AREs, triggering the expression of genes involved in the production of antioxidant enzymes like GSH, SOD, and GSH-Px. On the other hand, chronic or intense oxidative stress can result in Nrf2 depletion, which can be produced by excessive NO production[58-61]. In this study we observed a significant decrease in the overall antioxidant capacity of diabetic rat heart tissue. This decline was accompanied by reductions in the levels of antioxidant enzymes (SOD, CAT, and GSH-Px), as well as lower levels of GSH and Nrf-2 protein in the heart. Interestingly, L-Arg administration at doses of 1 g/kg and 1.5 g/kg further worsened these effects in diabetic rats. Notably, the 1.5 g/kg dose nearly eliminated Nrf-2 protein expression in the heart muscle. Conversely, oral L-Arg administration at the same doses (1 g/kg and 1.5 g/kg) caused mild, dose-dependent oxidative stress in the heart tissue of healthy rats. While NO is crucial for our body's defense system, its effects on inflammation can be a double-edged sword. The location and concentration of NO determine whether it promotes or suppresses inflammation, whereas high NO levels, often seen in abnormal situations like diabetes, can trigger inflammation[62]. Studies support this concept, found that blocking NO production with NOS inhibitors reduced inflammation in rats with acute inflammation or arthritis, while giving them L-Arg, a precursor for NO synthesis, worsened it[63,64]. Further research revealed that NO's impact on cytokine release, signaling molecules involved in inflammation, depends on the specific cell type, cytokine involved, and the stimulus triggering it. For instance, NO inhibits the release of MCP-1 but not MIP-2 from peritoneal macrophages stimulated by LPS and IFN-γ, while it increases IFN-β release in RAW264.7 cells[65,66]. Our study investigated the effects of oral L-Arg administration on the inflammatory markers in diabetic animals. While a dose of 0.5 g/kg L-Arg showed no significant impact on TNF-α and Il-1β cytokine levels, higher doses (1 g/kg and 1.5 g/kg) resulted in a significant increase in these cytokines compared to the untreated diabetic group. These findings suggest that L-Arg may exert pro-inflammatory effects in diabetic animals at higher doses, possibly due to increased NO production. In T2DM, high levels of glucose and its breakdown products can lead to the formation of advanced AGEs. These AGEs can impair the function of cardiomyocytes and the cells lining blood vessels (endothelial cells)[41]. Additionally, a lack of effective insulin action (insulin resistance) disrupts metabolism in cardiomyocytes, causing them to take up and burn more fatty acids. However, this process (β-oxidation) can't handle all the extra fatty acids, leading to a buildup of fat within the cells. This fat overload (lipotoxicity) and mitochondrial dysfunction[67] contribute to the increased production of harmful molecules ROS, and RNS, ultimately causing oxidative stress and stress in the endoplasmic reticulum. Together, these detrimental effects can trigger excessive cardiac hypertrophy, inflammation, myocardial remodeling, fibrosis, and even cardiomyocyte death[68], which align with our observations in the current study, whereas we investigated the effects of L-Arg administration to diabetic rats over 12 weeks, and we found that L-Arg administration significantly increased blood glucose levels, which potentially contributed to its detrimental effects on the heart muscle. Consistent with this, diabetic rats showed significant heart damage, including inflammatory cell infiltration, fat accumulation, and scar tissue formation (fibrosis) in the heart muscle. L-Arg administration worsened this damage, and the severity of damage increased with higher L-Arg doses. Desmin is a structural intermediate filament protein that is essential for the normal function of cardiomyocytes. It offers a platform for both intra- and intercellular signaling, and its disruption leads to cardiomyopathy[69]. On the other hand, normal cardiac muscle has fibroblast-like cells that remain deactivated in normal states, however, in the case of an injurious process, these cells can be transformed to activated α-SMA myofibroblasts, which plays a vital role in protecting cardiac muscle from rupture[70], but the excessive production of activated α-SMA-positive myofibroblasts can produce myocardial fibrosis, increasing its myocardial stiffness and leading to cardiac dysfunction[71]. In the present study, we noticed that diabetic rats had disrupted desmin myofilaments structure along with significant upregulation of α-SMA distribution throughout their cardiac tissue. Interestingly, giving diabetic rats L-Arg (at 1 g/kg or 1.5 g/kg) worsened this effect compared to untreated diabetic rats, and this disturbance was not seen in healthy rats given L-Arg. While some studies suggest benefits of L-Arg for diabetics. Conversely, high doses have been linked to potential drawbacks. Research suggests L-Arg may disrupt the structure of pancreatic beta cells[72] and trigger inflammation in the pancreas[73]. Additionally, it may increase activity of the arginase enzyme which is associated with the development of T2DM and insulin resistance[74,75]. Moreover, it was found that the activity of cNOS and iNOS in isolated pancreatic islets are increased in a dose-dependent manner by increasing glucose concentration. However, the activity of cNOS is adjusted more quickly to changes in glucose concentration compared to iNOS activity[76]. Suppressing the activity of cNOS in pancreatic islets enhances GSIS (glucose-stimulated insulin secretion) and eliminates the negative peak that occurs between the first and secondary phases of insulin secretion and this suggests that NO has a negative feedback effect on GSIS and plays a crucial role in determining the pattern of GSIS[77]. The negative feedback mechanism prevents excessive insulin secretion in response to elevated glucose levels, hence preserving β-cells. The mRNA and protein levels of iNOS are increased in the pancreatic islets of individuals with T2DM, and the inhibition of iNOS in these patients improves the GSIS[76]. The primary consequence of NO produced by iNOS is the malfunction of β-cells, leading to decreased insulin production, hyperglycaemia, and ultimately the onset of diabetes[76,78]. The production of NO by iNOS inhibits insulin secretion through a mechanism that is independent of 3′,5′-cGMP, this inhibition occurs by affecting the mitochondrial electron transport chain (specifically complexes I and II) and the activity of mitochondrial aconitase. Additionally, iNOS causes S-nitrosylation of essential thiol groups involved in the secretory process, as well as tyrosine nitration and subsequent downregulation of glucokinase[79]. When considering these results collectively, the present study findings support theses previous findings; thus we noticed a significant upregulation of iNOS myocardial tissue content in diabetic rats, which was dramatically increased by L-Arg administration, and this effect was positively correlated with myocardial tissue damage and inflammation, and this effect was dose-dependent.

CONCLUSION

The safety and effectiveness of L-Arg, a popular amino acid with various physiological effects, remain unclear. While this study suggests a safe dosage of 0.5 g/kg for diabetic rats, higher doses (1 g/kg or more) may worsen cardiac damage by elevating blood glucose, inflammation, and oxidative stress. Although L-Arg may promote weight loss and improve cholesterol in rats, and these benefits are overshadowed by the detrimental effects of high doses on the heart muscle. This research highlights the need for further investigation into the safety of food supplements, often perceived as harmless and consumed in large quantities, particularly by athletes. It also sheds light on the complexities and limitations of L-Arg supplementation. On the other hand, the present study has some limitations which need to be addressed in the future studies including; L-Arg dose optimization by further studies on the human, examine more different doses between 0.5 gm/kg to 1 gm/kg to determine the exact maximum therapeutic dose, examine different doses effect on the pancreatic tissue to determine the exact mechanism by which hyperglycemia was induced and study the effect on Type 1 diabetics which not mentioned in the present study.