An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementation

Enrique Chávez; Alejandro Rusbel Aparicio-Cadena; Gabriela Velasco-Loyden; María Guadalupe Lozano-Rosas; Mariana Domínguez-López; Amairani Cancino-Bello; Nimbe Torres; Armando R Tovar; Alejandro Cabrera-Aguilar; Victoria Chagoya-de Sánchez

doi:10.1371/journal.pone.0292448

. 2023 Oct 5;18(10):e0292448. doi: 10.1371/journal.pone.0292448

An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementation

Enrique Chávez ^1,^#, Alejandro Rusbel Aparicio-Cadena ^1,^#, Gabriela Velasco-Loyden ¹, María Guadalupe Lozano-Rosas ¹, Mariana Domínguez-López ¹, Amairani Cancino-Bello ¹, Nimbe Torres ², Armando R Tovar ², Alejandro Cabrera-Aguilar ¹, Victoria Chagoya-de Sánchez ^1,^*

Editor: Nobuyuki Takahashi³

PMCID: PMC10553329 PMID: 37796781

Abstract

Metabolic syndrome is a multifactorial disease with high prevalence worldwide. It is related to cardiovascular disease, diabetes, and obesity. Approximately 80% of patients with metabolic syndrome have some degree of fatty liver disease. An adenosine derivative (IFC-305) has been shown to exert protective effects in models of liver damage as well as on elements involved in central metabolism; therefore, here, we evaluated the effect of IFC-305 in an experimental model of metabolic syndrome in rats induced by a high-fat diet and 10% sucrose in drinking water for 18 weeks. We also determined changes in fatty acid uptake in the Huh-7 cell line. In the experimental model, increases in body mass, serum triglycerides and proinflammatory cytokines were induced in rats, and the adenosine derivative significantly prevented these changes. Interestingly, IFC-305 prevented alterations in glucose and insulin tolerance, enabling the regulation of glucose levels in the same way as in the control group. Histologically, the alterations, including mitochondrial morphological changes, observed in response to the high-fat diet were prevented by administration of the adenosine derivative. This compound exerted protective effects against metabolic syndrome, likely due to its action in metabolic regulation, such as in the regulation of glucose blood levels and hepatocyte fatty acid uptake.

Introduction

Metabolic syndrome is a multifactorial pathology that can include obesity, dyslipidemia, glucose intolerance, insulin resistance and arterial hypertension and can be associated with type 2 diabetes mellitus, metabolic dysfunction–associated steatotic liver disease (MASLD), and cardiovascular disease [1, 2].

Metabolic syndrome rates are estimated from those associated with obesity and related diseases. According to the World Obesity Atlas 2022, in 2030, the prevalence of obesity will be one billion people [3]. Overweight and obesity are observed primarily in industrialized or developing countries, such as Mexico [4].

The primary stimulus of metabolic syndrome is an increase in body weight, although insulin resistance associated with an inflammatory response promoted by cytokines and other mediators from adipose tissue and liver is also considered one of the principal causes [5–7]. Moreover, disequilibrium of lipid, glucose, insulin, and proinflammatory cytokine concentrations have been related to the overproduction of reactive oxygen species and mitochondrial dysfunction [7, 8]. There is an increased risk of developing MASLD in patients who have metabolic syndrome [9]. Damage to hepatocytes begins with excessive capture of free fatty acids; furthermore, the synthesis of lipids from carbohydrates is increased because the enzymes involved in these processes are directly regulated by glucose and insulin [10].

Insulin resistance has been reversed using antagonists of adenosine 2b receptor (A2BR), which also diminishes glucose levels in diabetic mice [11]. In addition, A2BR activation induces IL-6 production in macrophages and endothelial cells in diabetic rats [12]. Furthermore, adenosine administration alleviates hyperlipidemia and hyperinsulinemia, increases insulin sensitivity and downregulates the mRNA expression of the A2B receptor [13–15].

IFC-305 or adenosine aspartate is a derivative of adenosine that has been shown to have the same hepatoprotective effects as adenosine (200 mg/kg) at a dose 4 times lower (50 mg/kg) in addition to prolonging the half-life of adenosine. The hepatic maximum concentration of adenosine is at 15 min after its intraperitoneal administration (200 mg/kg), and at 30 min, the concentration is approximately 0.5 μmol/g of tissue, being almost undetectable at 120 min. In contrast, the IFC-305 maximum concentration in the liver tissue (approximately 2.7 μmol/g of tissue) is found 30 min after its administration (50 mg/kg), and at 120 min, the concentration is maintained at 1.7 μmol/g of tissue. This effect may be one of the reasons why a lower dose of IFC-305 than adenosine is needed to obtain the same hepatoprotective effect [16]. Adenosine has been shown to be hepatoprotective by preventing and reversing liver damage in different experimental models [17, 18]. This compound restores hepatic function, including by inducing liver regeneration in a model of experimental cirrhosis; prevents and reverses hepatocellular carcinoma induced by diethylnitrosamine; restores mitochondrial function; induces autophagy [19–22].

According to the abovementioned findings, the aim of this work was to evaluate the effects of IFC-305 on the alterations observed in metabolic syndrome in rats induced by a high-fat diet and 10% sucrose in drinking water for 18 weeks. The high-fat diet and sucrose increased body mass, serum triglycerides and proinflammatory cytokines compared to those in control rats. However, the adenosine derivative significantly prevented these changes, including the histological alterations, and inhibited the alterations in glucose and insulin tolerance, enabling the rats to regulate their glucose levels. The in vitro uptake of fatty acids was also prevented by IFC-305 treatment.

Materials and methods

Chemicals

IFC-305 (adenosine aspartate) was prepared with adenosine free base (MP Biomedicals, LLC, Illkirch, France) and l-aspartic acid (MP Biomedicals, Inc., Eschwege, Germany) as previously described in patent MX 207422 [23].

Animals

Male Wistar rats weighing 180–220 g (7 to 8 weeks old) were obtained from and housed at the Animal Facility of the Universidad Nacional Autónoma de México (UNAM). The animals were housed in pairs per cage under controlled conditions (22 ± 2°C, 50–60% relative humidity, and 12 h light-dark cycles). The animals were used according to institutional guidelines, and the protocol was approved by the Comité Institucional para el Cuidado y Uso de Animales de Laboratorio del Instituto de Fisiología Celular (VCHH53-14) and the Mexican Official Norm (NOM-062-ZOO-1999). Four experimental groups were created (n = 6) as follows: the control group, the high-fat diet + 10% sucrose in drinking water (HFS) group, the HFS + IFC-305 (HFS+IFC-305) group and the IFC-305 group. The control and IFC-305 groups were fed a standard diet (AIN-93) and were administered saline solution i.p. and IFC-305 (50 mg/kg, daily i.p.), respectively. The HFS and HFS+IFC-305 groups were fed a modified standard diet supplemented with fat from an animal source (HF diet, Table 1) plus sucrose solution 10% ad libitum and were administered saline solution and IFC-305 (50 mg/kg, daily i.p.), respectively. All groups were treated for 18 weeks. The AIN-93 diet components and modifications were established as previously described [24, 25]. The animals were euthanized using sodium pentobarbital (Pisa, Mexico), and the livers were removed and frozen in liquid nitrogen for further analysis. The sections were fixed in 4% paraformaldehyde for histological analysis.

Table 1. HF diet composition.

Ingredients	(g/kg)
Casein	240
L-cystine	3
Corn starch	239.03
Maltodextrin	102.67
Sucrose	77.783
Soy oil	70
Cellulose	50
Mineral Mix	35
Vitamin Mix	10
Choline	2.5
Lard	170
TBHQ	0.013

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The components were first mixed; then, lard and oil were added to obtain a homogeneous mass. It was stored at 4°C until use.

Electron microscopy

Liver samples for electron microscopy were fixed in glutaraldehyde (6%) and stained with osmium tetroxide (1% phosphate-buffered saline solution) as previously reported [21].

Growth curves

Body mass was measured twice a week during treatment for analysis of weight gain.

Glucose and insulin tolerance tests

At the end of the treatment, blood glucose levels were measured. Blood glucose concentrations in tail vein samples were measured using a glucometer (Accu-Chek). Beforehand, the animals were fasted overnight.

Glucose and insulin tolerance tests were performed by gavage administration of glucose solution (3 g/kg) in 3 mL of water and i.p. injection of rapid-acting insulin (100 IU/ml, 0.2 IU/kg), respectively. First, the glucose concentration in the blood was measured at 0, 15, 30, 60, 120 and 180 minutes after glucose solution administration. The insulin tolerance curve was determined using the same fasting conditions.

Serum levels of triglycerides, LDL cholesterol, and insulin

Blood samples were collected by cardiac puncture. Serum samples were obtained by centrifugation at 10,000 rpm for 15 min at 4°C. Triglycerides, LDL cholesterol, and insulin levels were determined using commercial kits (Spinreact^TM refs: 1001310, 41023, EZRMI-13K, respectively). The instructions provided by the manufacturers were followed.

Liver lipid analysis

Liver samples were homogenized in isopropanol (50 mg/mL). The tissue homogenate was centrifuged at 4°C and 10,000 rpm for 10 min. The organic phase of the supernatants was recovered, and triglyceride and total cholesterol levels were determined using commercial kits following the manufacturer’s instructions (Cayman Chemical MT Item No. 10010303, Spinreact^TM ref. no. 1001090).

Histological score for MASLD

Kleiner’s score was calculated by using a histological and semiquantitative scoring system for nonalcoholic fatty liver disease [26].

Measurement of serum cytokines

The levels of IL-1β, TNF-α, IL-6, MCP-1, vascular endothelial growth factor (VEGF), IL-2, IFN-γ, and IL-10 were determined in serum samples using commercially available ELISA kits (PEPROTEC®).

Cell culture and treatment

The human hepatoma-derived cell line Huh-7 was obtained from the American Type Culture Collection (ATCC, US). The cells were grown in high-glucose DMEM (Gibco, US) supplemented with 10% fetal bovine serum (Biowest) and 1% penicillin and streptomycin (Invitrogen, US) at 37°C under 5% CO₂ in a 95% humidified atmosphere. To induce FFA uptake, Huh-7 cells were exposed to FFAs (oleic acid and palmitic acid at a 4:1 volume ratio) for 24 h in the absence or presence of 1 mM IFC-305. Stock solutions of 30 mM FFAs were diluted at the indicated concentrations in serum-free DMEM supplemented with 1% FFA-free BSA as previously described [27].

Oil red O staining and quantification

Huh-7 cells were cultured in 24-well plates at a density of 1.5 X 10⁵ cells/well and treated with different concentrations of FFAs and IFC-305 (1 mM) for 24 hours. The cells were washed two times with PBS and fixed in 4% buffered formalin for 15 min at room temperature. After fixation, the cells were washed twice with PBS and incubated with oil red O solution prepared as previously described for 1 hour at room temperature [28]. The stained cells were then washed in distilled water, isopropanol was added to each sample, and the samples were shaken at room temperature for 10 min. Then, the absorbance of the samples was read at 510 nm on a Cytation 3 Cell Imaging Multi-Mode Reader by BioTek Instruments, Inc., Vermont, U.S.A.

Statistical analysis

The data are expressed as the mean ± standard error of the mean (SEM). Statistically significant differences among the experimental groups were determined by one-way analysis of variance (ANOVA) followed by Tukey’s test for multiple comparisons (except in the weight gain graph, for which the applied statistic is described in the figure legend). Significance was set at p < 0.05. Graphs were created with GraphPad Prism software version 6.0.

Results

Experimental model characterization

We determined weight gain (growth curve), serum triglycerides, LDL cholesterol, serum insulin and glucose and insulin tolerance to characterize the experimental model used in this work (Fig 1). We evaluated the serum levels of AST and ALT in the different experimental groups. There was a tendency toward increased ALT in the HFS group; however, there were no statistically significant differences in the results obtained for both enzymes (S1 Fig). HFS and HFS+IFC groups showed a lower food intake but higher water + sucrose consumption than the rest of the groups (S1 Table). The results suggested that the IFC-305 administration effects are not directly related with a difference in the caloric intake. Sánchez-Tapia et al., determined that even though there are differences in the food and water consumption in rats fed with the high-fat and sucrose diet used in this model, compared with control rats, there are not changes in the caloric or energy intake among the groups [25].

HFS induced an increase of approximately 2-fold in weight compared to that in the control group (Fig 1A); treatment of the rats fed HFS with IFC-305 partially prevented the weight gain (HFS+IFC-305 group). Triglycerides, LDL-C and insulin levels were increased in the HFS group compared to the control group (Fig 1B–1D, respectively). In the HFS+IFC-305 group, a decrease in circulating triglycerides, a small but nonsignificant decrease in LDL cholesterol, and no differences in serum insulin concentration were observed; there was no effect of IFC-305 administration. Fig 1E shows the glucose tolerance curve. In the HFS group, there was a significant increase in circulating glucose 60 min after the administration of glucose solution until 120 min. Interestingly, in the HFS+IFC-305 group, glucose tolerance was equal to that in the control group, demonstrating that IFC-305 regulates glucose levels. This effect was quantified by the area under the curve (AUC) (Fig 1G). The IFC-305 compound did not induce changes in glucose levels in either curve under the same conditions (data not shown).

In addition, the effect of insulin administration (100 IU/ml) on circulating glucose is shown in Fig 1F. A significant increase in glucose level was observed 30 min after the administration of glucose solution in the HFS group compared to the control group, with a maximum level observed at 90 min, indicating insulin tolerance. Glucose levels diminished nearly to control-group levels by 120 min. There were no differences between the control and HFS+IFC-305 groups. This result was quantified according to the AUCs (Fig 1H).

IFC-305 prevents histological alterations and hepatic lipid accumulation

Fig 2A shows images of the representative livers for each experimental group as well as their corresponding histological slices with hematoxylin and eosin staining. The images of the control group show the characteristics of a normal liver, such as its color and architecture, without the presence of an inflammatory infiltrate or accumulation of fat in the hepatocytes. In the whole liver picture in the HFS sample, a lighter and mottled color is observed compared to that in the control sample; moreover, an altered hepatic architecture and the presence of macrovesicular steatosis indicated by the ♦ symbol are observed. A representative section of HFS+IFC-305 is shown in Fig 2C, which shows the ability of the compound to prevent macrovesicular steatosis; some lipid droplets can be observed, as indicated by arrowheads. No changes were induced by the administration of IFC-305 alone. Liver triglyceride and cholesterol levels were significantly increased in the HFS group compared to the control group, while the HFS+IFC-305 group showed no significant increase. The IFC-305 group showed that this compound had no effect on these parameters (Fig 2B and 2C). In clinical practice, the steatosis grade (from simple steatosis to MASH) can be determined by Kleiner’s scoring system [26]. According to this score, the group fed an HFS diet showed a hepatic steatohepatitis phenotype, since liver samples had ≥34% parenchymal involvement by steatosis, as well as various inflammatory foci per X20 field and several cells with a ballooning phenotype due to fat accumulation in the hepatocytes. IFC-305 administration prevented the progression of hepatic disease compared to that in the HFS group (Fig 2D).

The results demonstrate the ability of IFC-305 to prevent the increases in cholesterol and triglyceride levels, regulate glucose levels and prevent the accumulation of fat in hepatocytes. For this reason, we evaluated the levels of AMPK, a protein downstream of the adiponectin signaling pathway. The HFS group tended to have increased AMPK levels compared to the control group, and the HFS+IFC-305 group maintained AMPK at a level similar to that in the control group. No significant differences were found between the experimental groups (S2 Fig).

Inflammation induced by HFS and the anti-inflammatory effect of IFC-305

We evaluated the inflammatory state because it is associated with metabolic syndrome and insulin resistance. As shown in Fig 3A–3C, IL-1β, TNF-α and IL-6 levels were increased in the HFS group, but coadministration of IFC-305 prevented this, indicating an anti-inflammatory effect. The chemokine MCP-1 favors the migration of inflammatory macrophages [29]. The level of this cytokine was significantly increased in response to HFS, but IFC-305 prevented this increase (Fig 3D). VEGF is highly expressed in adipose tissue; here, its expression was increased in the HFS group, but IFC-305 decreased it significantly (Fig 3E), showing antiadipogenic activity. The level of IFN-γ, an inflammatory cytokine that favors the differentiation of macrophages, was increased significantly in the HFS group, but IFC-305 treatment prevented this increase, thus decreasing the inflammatory response (Fig 3F). The cytokine IL-10, a typical anti-inflammatory cytokine, decreased with the HFS diet compared to the control diet (Fig 3G). In the HFS+IFC-305 group, the level of the anti-inflammatory cytokine IL-10 was maintained.

Electron microscopy-revealed changes in liver cells induced by HFS and the protective effect of IFC-305

Fig 4A shows a control sample with a normal hepatocyte. It contains mitochondria (MT) associated with rough endoplasmic reticulum (RER). The square in the right inferior border shows a magnified view. Fig 4B shows electron microscopy of a damaged liver (HFS). The mitochondria do not have their typical morphology, and fewer associations are observed with the RER. The presence of ribosomes is reduced, and the number of mitochondria is lower than that in the control group. The number of lipid vesicles was reduced in rats treated with IFC-305 (HFS+IFC-305), corroborating the findings of H&E staining; in addition, the loss of mitochondria was prevented, and the association of mitochondria with the RER was maintained (Fig 4C). Treatment with IFC-305 in rats fed a standard diet did not cause any phenotypic alterations in liver cells (Fig 4D).

In vitro uptake of fatty acids in Huh-7 cells

To corroborate the effect observed in hepatic lipid content, we decided to evaluate the probable inhibitory effect of the IFC-305 compound on fatty acid uptake in vitro. Fig 5 shows the uptake of fatty acids in Huh-7 cells. Incubation with IFC-305 for 24 h partially inhibited fatty acid uptake measured using the oil red method. These results were in accordance with the findings of the lipid content in the liver from the HFS rats shown histologically and by assessment of the hepatic triglyceride and cholesterol content.

Fig 5 — Huh-7 cells were treated with the indicated concentrations of FFAs and IFC-305 for 24 h and then stained with oil red reagent, and the absorbance was read at 510 nm. All results are shown as the mean ± SD (n = 3) from a representative experiment. Statistically significant differences were determined by one-way analysis of variance (ANOVA) with multiple comparisons. “a” indicates statistically significant differences from the control group, P < 0.01. “b” indicates statistically significant differences from the 1 mM FFA group, P < 0.01. “c” indicates statistically significant differences from the 2 mM FFA group, P < 0.01.

Discussion

The compound IFC-305 is an adenosine derivative that has been shown to have the same hepatoprotective effects as adenosine (200 mg/kg) at a dose 4 times lower (50 mg/kg) in addition to exhibited a longer half-life than adenosine. Some of the observed effects of IFC-305 include decreased collagen levels, increased collagenolytic activity, and increased DNA synthesis as well as recovery of liver function in a CCl₄-induced cirrhosis model [16]. In addition, an evaluation of global DNA methylation has revealed decreases in methylation and demethylation and recovery in the presence of IFC-305 in cirrhosis, indicating a double increase in global DNA methylation [30]. In in vitro studies using isolated hepatic stellate cells, IFC-305 has been found to be able to inhibit the TGF-β signaling pathway that induces collagen gene expression (data not published). In a model of hepatocellular carcinoma induced by DEN, IFC-305 has been demonstrated to have chemoprotective effects, having an important effect on the recovery of mitochondrial function [20, 21]. Moreover, previous findings on the hepatoprotective role of IFC-305 and its inhibitory effects on the development of fatty liver prompted us to evaluate the effect of this compound in an experimental model induced by HFS for 18 weeks. The results suggest that IFC-305 is a promising molecule for inhibiting the establishment of steatosis and regulating glucose levels, systemic inflammation and the development of metabolic syndrome.

It has been reported that there is no precise correlation between the serum level of the ALT enzyme and fatty liver; however, ALT has also been suggested as a possible marker [31]. On the other hand, a relationship between AST and MASLD has not been reported, unlike the case for liver damage associated with alcohol consumption, in which an increase in the AST:ALT ratio is observed [32]. The determination of these parameters in our metabolic syndrome model showed only a slight increase in the ALT level that was not observed in the HF+IFC-305 group, and there was no difference in the AST level in the experimental groups, suggesting a protective effect against the accumulation of fat in the liver.

The growth curve showed weight gain that was partially prevented by IFC-305 administration. In addition, this compound had beneficial effects on triglyceride levels and somewhat beneficial effects on insulin and LDL cholesterol levels. Previous studies have demonstrated the ability of adenosine, the base molecule of IFC-305, to diminish hyperlipidemia in stroke-prone spontaneously hypertensive rats fed a high-fat diet by downregulating the A2B receptor [15].

In this study, the animals fed the HFS diet developed glucose intolerance without developing diabetes. After fasting at time 0, they did not exhibit differences from the control animals, but when exogenous glucose was administered, their levels remained elevated until 120 min. The observed insulin resistance in the HFS group indicates the inability of insulin to increase glucose uptake [33]. Here, animals fed HFS were unable to maintain normal glucose levels, so they maintained high levels until 90 min after glucose intake; this result was directly related to the loss of sensitivity to insulin signaling. Some nucleosides, such as adenosine, have been shown to exert hypoglycemic effects during the postprandial period [14]. The IFC-305 compound demonstrated a similar ability and therefore regulated serum insulin levels probably in the same way as adenosine, which could function as an integral treatment because of its different metabolic effects. For instance, adenosine regulates lipolysis and lipogenesis through the A1 receptor. Additionally, its insulin-like functions are well described [34]. Moreover, this molecule has ubiquitous cell signaling actions on energy homeostasis [35].

In this work, animals fed HFS developed liver damage, which was observed macro- and microscopically. Intrahepatic lipid accumulation results from lipolysis, FFA uptake and very low-density-lipoprotein (VLDL) synthesis as well as reduced FFA oxidation and triglyceride (TG) export; moreover, these processes are frequently associated with the inflammatory response [36]. Adenosine has previously been shown to prevent and reverse fatty liver induced by ethanol administration by increasing ATP levels and stimulating oxidation in mitochondria [37]. Histologically, a large number of lipid vacuoles and the phenomenon of “ballooning” were observed, which has been proposed as a histopathological characteristic of NASH [38]. In this work, this characteristic was observed in the HFS group. In contrast, treatment with IFC-305 significantly prevented the accumulation of lipids in hepatocytes, notably improving macroscopic liver appearance. The cholesterol and triglyceride levels observed in the HFS+IFC-305 group corroborated these results. The assay of fatty acid uptake by Huh-7 cells enabled us to confirm the ability of IFC-305 to reduce the accumulation of lipids in hepatocytes, explaining in part the findings in vivo. Previously, it has been shown that adenosine prevents approximately 50% of the accumulation of triglycerides in fatty liver induced by ethanol [37]. The levels of p-AMPK observed in the HFS group and in the HFS+IFC-305 group suggest that the mechanism of action is not through induction of the adiponectin signaling pathway; however, further research is needed. Interestingly, p-AMPK is related to other cellular processes, such as autophagy, a cellular recycling process. If phosphorylation of AMPK is not induced, AMPK does not activate mTOR, an inhibitor of autophagy. We previously demonstrated that IFC-305 induces autophagy and that this cellular process can maintain lipid droplet homeostasis [39]. In addition, IFC-305 favors mitophagic flux, stimulating mitochondrial activity and oxidative metabolism [21, 22]. Moreover, in one study, adenosine prevented free fatty acid hepatic oxidation by inhibiting extramitochondrial acyl-CoA synthetase, thus diminishing plasma fatty acid uptake [40]; however, this effect has since been considered to be transient, since in another study, it was found that adenosine decreases the generation of ketone bodies in a model of fatty liver induced by cycloheximide, suggesting its ability to induce β-oxidation [18]. Once in hepatocytes, fatty acids that were released from adipocytes need to be activated as acyl-CoA to be metabolized, either via β-oxidation in mitochondria or by being used in TG, phospholipid or cholesterol synthesis. The family of enzymes responsible for the activation of fatty acids are called long-chain acyl-CoA synthetases (ACSLs). In this study, we determined the expression of the ACSL1 and ACSL3 isoforms in an in vitro model of fatty acid incorporation in HepG2 cells. We found that the expression of the ACSL3 isoform was decreased in cells exposed to FFAs compared to control cells, while those incubated with IFC-305 in the presence of FFAs or alone had recovery of ACSL3 expression. Regarding the ACSL1 isoform, no differences were observed, although there was a tendency toward an increase in cells incubated with IFC-305. One possibility is that the slight increases in the expression of ACSL1 and ACSL3 induced by IFC-305 favor the synthesis of acyl-CoA for oxidation and not for the synthesis of TG, since in both the in vivo and in vitro models, a decrease in the accumulation of TG deposits was observed. This is in accordance with another study in which the expression of ACSL3 was induced with oncostatin M in a model of hyperlipidemic hamsters and in HepG2 cells; in that study, greater oxidation of FFAs and a decrease in the accumulation of TG were observed [41]. In another study, in hamsters fed a high-fructose diet, the expression of hepatic ACSL3 was decreased, and re-expression of ACSL3 was accompanied by a decrease in the TG level in the liver, similar to the findings with IFC-305 [42].

According to this, MASLD, obesity and insulin resistance play important roles in lipid disorders, and steatosis has been correlated with insulin resistance [36].

Mitochondria have been reported to undergo structural and functional alterations during the progression of liver disease associated with insulin resistance. Observed changes include strong condensation of structures of the mitochondrial matrix or changes in the matrix density, loss of ridges and a decrease in mitochondrial biogenesis [43, 44]. Liver sections were also analyzed by electron microscopy, which revealed diminished and morphologically altered mitochondria in the animals fed the high-fat diet and 10% sucrose in drinking water. In the group fed the HFS diet supplemented with sucrose, the simultaneous administration of IFC-305 prevented the mitochondrial alterations observed by electron microscopy, as previously demonstrated in hepatocarcinoma [21].

HFS induced systemic inflammation, and the anti-inflammatory effect of IFC-305 has been demonstrated in a previous study on a cirrhosis model. In that study, IFC-305 reduced IL-1β and IL-6 levels and increased IL-10 levels [45]. IL-1β, TNF-α and IL-6 are inflammatory cytokines correlated with and involved in obesity-related inflammation and insulin resistance, respectively [29, 46]. As we mentioned earlier, systemic inflammation is directly related to metabolic syndrome and may promote NASH. During chronic inflammation, recruitment of M1 macrophages is promoted, and TNF-α and MCP-1 are hypersecreted in both adipose tissue and hepatocytes, which contribute to the establishment of cellular infiltration. In patients with metabolic syndrome, increased serum levels of IL-6 and TNF-α have been found to be associated with the severity of metabolic syndrome development [47]. Previous research has demonstrated an increase in the number of M1 macrophages in an experimental model of cirrhosis induced by CCl₄; in that research, treatment with IFC-305 prevented this increase and favored the production of the anti-inflammatory cytokine IL-10 [45]. According to these findings and the results obtained here regarding the ability of IFC-305 to prevent the increases in the levels of proinflammatory cytokines and a decrease in Kleiner’s score, which reflects lobular inflammation in metabolic syndrome, we propose that this effect is related to a regulation of immune cells. Adipose tissue requires adequate vasculature, and production of the angiogenic factor VEGF is important, but an increase in the VEGF level is a reflection of an increase in the number of adipocytes and therefore obesity [48]. VEGF is proposed to play an important role in weight gain, and a high concentration of this growth factor in serum is positively correlated with increases in body mass index and visceral fat deposition; in contrast, in adipose tissue, VEGF overexpression promotes angiogenesis. Likewise, VEGF dysregulation may be a risk factor for vascular diseases [49].

Altogether, the results presented here demonstrate the beneficial effects of IFC-305 in the experimental model in male rats. However, further studies are required to elucidate the effects with consideration of more variables, since some sex differences have been found in the establishment and progression of MASLD. Such studies will enable us to understand the effects and ensure that they are the same in both sexes [50].

These results demonstrate that IFC-305 prevents the alterations associated with the progression of metabolic syndrome and liver damage associated with excessive fat and sugar consumption in an experimental model. These effects result from the regulation of blood glucose and other serum elements, the preservation of liver structure and the prevention of inflammation. The results indicate that IFC-305 may be a promising molecule for the treatment of metabolic syndrome and related diseases.

Supporting information

S1 Fig. Determination of liver damage by enzymatic transaminases activities.

(A) AST and (B) ALT activity was determined in serum from rats in the control (n = 3), HFS (n = 10), and HFS+IFC-305 (n = 10) groups. The values represent the mean of experiments performed in duplicate assays ± SEM.

(TIFF)

Click here for additional data file.^{(215.2KB, tiff)}

S2 Fig. pAMPK protein level.

pAMPK protein levels in samples of liver tissue determined by Western blot analysis from rats in the control (n = 3), HFS (n = 4), HFS+IFC-305 (n = 4), and IFC-305 (n = 4) groups. GAPDH was used as an internal control. A) The signal intensities were determined by densitometric analysis of treated blots, and the values were calculated as the ratio of p-AMPK to GAPDH. Each bar represents the mean value of experiments ± SEM. B) Representative blot for each group.

(TIFF)

Click here for additional data file.^{(523.5KB, tiff)}

S3 Fig. mRNA expression of ACSL1 and ACSL3 in HepG2 cells.

mRNA expression of ACSL1 and ACSL3 in HepG2 cells. HepG2 cells were cultured in serum-free DMEM supplemented with 1% BSA-free FFAs (control) in the presence of 1 mM IFC-305 or FFA with or without 1 mM IFC-305 for 24 h. Real-time quantitative polymerase chain reaction (qPCR) analysis of the mRNA expression of (A) ACSL1 and (B) ACSL3 isoforms normalized to β-actin. The relative mRNA levels were calculated using the comparative ΔΔCt method. mRNA expression is expressed as the mean value ± SEM from three independent experiments. **P < 0.001.

(TIFF)

Click here for additional data file.^{(511.3KB, tiff)}

S1 Table. Food and water consumption.

Food and water intake (or water+sucrose solution for HFS and HFS+IFC groups) were measured twice a week. Captured data are expressed as mean values ± SEM from 0, 8, 12 and 18 weeks of treatment. Statistical differences were determined with ANOVA followed by a multiple comparison test where “a” indicates a significant difference compared to the control group, P≤0.05.

(DOCX)

Click here for additional data file.^{(17.6KB, docx)}

S1 Data

(ZIP)

Click here for additional data file.^{(681.8KB, zip)}

Acknowledgments

We would like to thank Dr. Rodolfo Paredes Díaz for their support at the imaging unit of the institute; Claudia Rivera Cerecedo, DVM, and Gabriela Xóchitl Ayala Méndez, MSc., from the Bioterium for providing the rats; Elizabeth Morales Sánchez, MSc., Sandra Rodríguez Montaño., MSc., and Leticia Ramírez Lugo, PhD., from the Histology Unit; and Mónica Sánchez-Tapia, PhD, for her support in preparing the diets.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

ARAC is recipient of a Consejo Nacional de Humanidades, Ciencias y Tecnologias (CONAHCYT)-Mexico fellowship 720321. VCdS received financial support from Programa de Apoyo a Proyectos de Investigacion e Innovacion Tecnological-Universidad Nacional Autonoma de Mexico (PAPIIT-UNAM) IN214419. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0292448.r001

Decision Letter 0

Nobuyuki Takahashi

3 Jul 2023

PONE-D-23-08771An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementationPLOS ONE

Dear Dr. Chagoya de Sánchez,

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: I Don't Know

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Reviewer #1: In this paper, the authors examined effects of IFC-305, an adenosine derivative, on metabolic syndrome and nonalcoholic fatty liver disease (NAFLD) using an animal model and cell line. As a result, IFC-305 ameliorated obesity, serum levels of triglyceride and proinflammatory cytokines, glucose tolerance, and hepatic steatosis. The results of the present study are interesting, but the authors should consider the following points.

Major points:

1. The authors should examine serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) to confirm the effects of IFC-305 on hepatocellular injury. Serum levels of adiponectin should also be examined

2. On histopathological evaluation of NAFLD, not only steatosis but also lobular inflammation, hepatocellular ballooning, and fibrosis are important. The authors should evaluate these items semi-quantitatively using Kleiner’s scoring system (Hepatology 2005; 41: 1313-21).

3. The quality of English is insufficient. The authors should seek assistance of a native English speaker.

Minor points

1. Age of the rats at initiation of the experiment should be described.

2. Data of food consumption and calorie intake should be shown if the authors checked them.

3. Line 113: “HFC” should be revised as “HFS.”

4. Line 171, 277, Fig. 3: “INF-γ” should be revised as “IFN-γ.”

5. Line 234: “glucose” should be revised as “insulin.”

6. Line 237: “de” should be revised as “the.”

7. Line 283: “Microscopic” should be revised as “Electron-microscopic.”

8. Line 286: “microscopy” should be revised as “electron microscopy.”

9. Line 304: “red oil” should be revised as “oil red.”

10. Line 347-8: The authors describe that IFC-305 had beneficial effects on insulin and LDL cholesterol levels, but there are no significant differences between the HFS and HFS+IFC-305 groups in Fig. 1(C,D).

11. Line 410: “NAFD” should be revised as “NAFLD.”

12. Fig. 2 (B,C): It seems that there are remarkable differences between the HFS and HFS+IFC-305 groups in the hepatic triglyceride and cholesterol levels. Aren’t the differences statistically significant?

13. Legend of Fig. 4: “N”, “L”, and “GA” are not used in the figure. “MT” and “RER” are defined two times.

Reviewer #2: The authors demonstrated here that an adenosine derivative, IFC-305, ameliorated high-fat-diet-induced hepatic inflammation and steatosis. These findings are very interesting. However, the data presented in this study are only partially convincing. Additional experimental data are needed to confirm the authors' conclusion, as the follows.

1) The authors should examine hepatic macrophage markers such as mRNA levels and/or immunohistochemistry of F4/80. According to the data shown in Fig.3, M1 macrophages would be decreased and/or inactivated. This should be investigated to confirm the authors' conclusion.

2) The authors should measure the mRNA expression levels of lipid metabolism in the liver and Huh-7 hepatocytes. In general, amelioration of inflammation improves the dysfunction of lipid metabolism in the liver. Therefore, it is necessary to examine the mRNA expression levels of fatty acid oxidation and synthesis in the liver of the IFC-305-fed mice and the IFC-305-treated Huh-7 hepatocytes.

3) The authors should measure serum levels of the liver injury markers such as AST and ALT.

4) The authors should check the minor points and English throughout the manuscript. There are many errors.

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Reviewer #1: No

Reviewer #2: No

**********

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PLoS One. 2023 Oct 5;18(10):e0292448. doi: 10.1371/journal.pone.0292448.r002

Author response to Decision Letter 0

4 Sep 2023

We really appreciate all the comments you made about the manuscript we submitted to be considered for publication in Plos One.

All the comments you recommended were considered in order to improve the manuscript.

Major points:

We evaluated the serum levels of AST and ALT as you recommended. There was not statistical significant difference in the results obtained, because of this,we decided to show them as a supplementary figure (S1 Fig).

The phollowing paragraph was added in the Results section

(Line 186-188) We evaluated the serum levels of AST and ALT in the different experimental groups. There was a tendency toward increased ALT in the HFS group; however, there were no statistically significant differences in the results obtained for both enzymes (S1 Fig).

The phollowing paragraph was added in the Discussion section

(Line 336-343) It has been reported that there is no precise correlation between the serum level of the ALT enzyme and fatty liver; however, ALT has also been suggested as a possible marker (33). On the other hand, a relationship between AST and MASLD has not been reported, unlike the case for liver damage associated with alcohol consumption, in which an increase in the AST:ALT ratio is observed (34). The determination of these parameters in our metabolic syndrome model showed only a slight increase in the ALT level that was not observed in the HF+IFC-305 group, and there was no difference in the AST level in the experimental groups, suggesting a protective effect against the accumulation of fat in the liver.

Adiponectin is an important factor that stimulates fatty acid oxidation, inhibits glucose production, and has an anti-inflammatory effect. We thought it was a good idea to determine their levels in the different experimental groups as you suggested. However, due to time constraints, it was not possible for us to acquire the necessary supplies, since we did not have them and we did not want to delay sending the corrected version of the manuscript.

Despite this, we decided to determine by Western blot the levels of p-AMPK, a protein that is found downstream of the adiponectin signaling pathway and that is important for its response. The results are shown in S2 Fig.

The phollowing paragraphs was added in the Results section:

(Line 261-266) The results demonstrate the ability of IFC-305 to prevent the increases in cholesterol and triglyceride levels, regulate glucose levels and prevent the accumulation of fat in hepatocytes. For this reason, we evaluated the levels of AMPK, a protein downstream of the adiponectin signaling pathway. The HFS group tended to have increased AMPK levels compared to the control group, and the HFS+IFC-305 group maintained AMPK at a level similar to that in the control group. No significant differences were found between the experimental groups (S2 Fig).

The phollowing paragraphs was added in the Discussion section

(Line 367-368) Adenosine has previously been shown to prevent and reverse fatty liver induced by ethanol administration by increasing ATP levels and stimulating oxidation in mitochondria (39).

(Line 377-384) The levels of p-AMPK observed in the HFS group and in the HFS+IFC-305 group suggest that the mechanism of action is not through induction of the adiponectin signaling pathway; however, further research is needed. Interestingly, p-AMPK is related to other cellular processes, such as autophagy, a cellular recycling process. If phosphorylation of AMPK is not induced, AMPK does not activate mTOR, an inhibitor of autophagy. We previously demonstrated that IFC-305 induces autophagy and that this cellular process can maintain lipid droplet homeostasis (41). In addition, IFC-305 favors mitophagic flux, stimulating mitochondrial activity and oxidative metabolism (22,42).

The semi-quantitative Kleiner’s score was analyzed according to the reference you recommended.

The following section was added to Methods:

(Line 149-151) Histological score for MASLD

Kleiner’s score was calculated by using a histological and semiquantitative scoring system for nonalcoholic fatty liver disease (27).Panel D has been added to Figure 4 showing the Kleiner’ score.

In addition, the following modifications were made to the manuscript:

- The phollowing sentence was added to the Legend of Fig. 2:

(D) H&E stained liver sections observed at 20X magnification were analyzed according with semiquantitative Kleiner’s criteria.

In the Results section, the phollowing paragraph was added:

(243-249) In clinical practice, the steatosis grade (from simple steatosis to MASH) can be determined by Kleiner’s scoring system (27). According to this score, the group fed an HFS diet showed a hepatic steatohepatitis phenotype, since liver samples had ≥34% parenchymal involvement by steatosis, as well as various inflammatory foci per X20 field and several cells with a ballooning phenotype due to fat accumulation in the hepatocytes. IFC-305 administration prevented the progression of hepatic disease compared to that in the HFS group (Fig 2D).

3. The quality of English is insufficient. The authors should seek assistance of a native English speaker.

The corrected versión of the manuscript was sent to AJE.com for English style correction.

Minor points

1. Age of the rats at initiation of the experiment should be described.

The phrase (7 to 8 weeks old) was added in parentheses in the first line of the Animals section.

2. Data of food consumption and calorie intake should be shown if the authors checked them.

In the following table we show the results of the food and water consumption during the time that the experimental model lasted. We include the results as supplementary material (S1 Tab).

The following paragraph was added to the Discussion section

(Line 188-194) HFS and HFS+IFC groups showed a lower food intake but higher water + sucrose consumption than the rest of the groups (S1 Tab). The results suggested that the IFC-305 administration effects are not directly related with a difference in the caloric intake. Sánchez-Tapia et al., determined that even though there are differences in the food and water consumption in rats fed with the high-fat and sucrose diet used in this model, compared with control rats, there are not changes in the caloric or energy intake among the groups (30).

3. Line 113: “HFC” should be revised as “HFS.”

HFC was changed by HFS.

4. Line 171, 277, Fig. 3: “INF-γ” should be revised as “IFN-γ.”

INF-γ was changed by IFN-γ.

5. Line 234: “glucose” should be revised as “insulin.”

The term “glucose” is correct because the way to measure insulin tolerance is to observe how glucose levels behave over a period of time after insulin administration, as we described in the paragraph.

6. Line 237: “de” should be revised as “the.”

The word “de” was changed by “the”.

7. Line 283: “Microscopic” should be revised as “Electron-microscopic.”

“Microscopic” was changed by “Electron-microscopic”

8. Line 286: “microscopy” should be revised as “electron microscopy.”

“Microscopy” was changed by “electron-microscopy”

9. Line 304: “red oil” should be revised as “oil red.”

“red oil” was changed by “oil red”

The phrase “on insulin, triglyceride and LDL cholesterol levels” was changed by “and partially on insulin and LDL”

11. Line 410: “NAFD” should be revised as “NAFLD.”

The acronym NAFLD was changed in the manuscript by MASLD (metabolic dysfunction–associated steatotic liver disease) according to new nomenclature adopted and published in Hepatology. 2023 Jun 24. doi: 10.1097/HEP.0000000000000520.

According to the Tukey’s test for multiple comparisons, as we describe in the statistical analysis in the Method’s section, there is difference statistically significant just in the control group versus the HFS group in both parameters, triglyceride and cholesterol content. This allows us to suggest only a tendency to prevent the accumulation of triglycerides and cholesterol because of the treatment with IFC-305 in fed rats with the HFS diet.

13. Legend of Fig. 4: “N”, “L”, and “GA” are not used in the figure. “MT” and “RER” are defined two times.

“N”, “L”, and “GA” were deleted from the legend of Fig. 4. The repited “MT” and “RER” definition were removed.

It is known that M1 macrophages are increased in metabolic syndrome (DOI: 10.1042/BJ20111708), and as we mentioned in the manuscript according to the cytokine profile, it is suggested that M1 macrophages decreased with the IFC-305 treatment. We agree that exploring how these macrophages behave could provide more detailed information on the effect of IFC-305 on the immune response in this experimental model, and could provide more precise information about if it acts directly limiting this cell population for the development and recovery of the liver.

Unfortunately at this moment we do not have available the F4/80 antibody for immunohistochemistry and it takes approximately 2 months to arrive.

Due to the anti-inflammatory effects of IFC-305 previously resported and that we briefly describe below, and the fact that we evaluate lobular inflammation, hepatocellular ballooning, and fibrosis by using Kleiner’s scoring system (Hepatology 2005; 41: 1313-21), we believe that this information could support our conclusions.

Panel D has been added to Figure 4 showing the Kleiner’ score.

In addition, the following modifications were made to the manuscript:

- The phollowing sentence was added to the Legend of Fig. 2:

(D) H&E stained liver sections observed at 20X magnification were analyzed according with semiquantitative Kleiner’s criteria.

In the Results section, the phollowing paragraph was added:

The phollowing paragraph was added in the Discussion section:

(Line 427-433) Previous research has demonstrated an increase in the number of M1 macrophages in an experimental model of cirrhosis induced by CCl4; in that research, treatment with IFC-305 prevented this increase and favored the production of the anti-inflammatory cytokine IL-10 (48). According to these findings and the results obtained here regarding the ability of IFC-305 to prevent the increases in the levels of proinflammatory cytokines and a decrease in Kleiner’s score, which reflects lobular inflammation in metabolic syndrome, we propose that this effect is related to a regulation of immune cells.

In the present study, we observed an important effect of the hepatoprotective IFC-305 by preventing the accumulation of lipids in the hepatocyte. Your suggestion to evaluate the mRNA of genes involved in lipid metabolism seemed very interesting. The family of enzymes responsible for the activation of fatty acids are called Long-chain acyl-CoA synthetases (ACSL). In this study we determined the expression of the ACSL1 and ACSL3 isoforms in the in vitro model of fatty acid incorporation. The results are shown in S3 Fig.

S3 Fig.

S3 Fig. mRNA expression of ACSL1 and ACSL3 in HepG2 cells. HepG2 cells were cultured in serum-free DMEM supplemented with 1% BSA- free FFA (control) in the presence of 1 mM IFC-305 or FFA with or without 1 mM IFC-305 for 24 h. Real-time quantitative polymerase chain reaction (qPCR) analysis of the mRNA expression of (A) ACSL1 and (B) ACSL3 isoforms normalized to �-actin. Relative mRNA level was calculated using the comparative ΔΔCt method. mRNA expression is expressed as mean values ± SEM of three independent experiments. **P < 0.001

The following paragraph was added to the Discussion section:

(Line 386-406) …however, this effect has since been considered to be transient, since in another study, it was found that adenosine decreases the generation of ketone bodies in a model of fatty liver induced by cycloheximide, suggesting its ability to induce β-oxidation (18). Once in hepatocytes, fatty acids that were released from adipocytes need to be activated as acyl-CoA to be metabolized, either via β-oxidation in mitochondria or by being used in TG, phospholipid or cholesterol synthesis. The family of enzymes responsible for the activation of fatty acids are called long-chain acyl-CoA synthetases (ACSLs). In this study, we determined the expression of the ACSL1 and ACSL3 isoforms in an in vitro model of fatty acid incorporation in HepG2 cells. We found that the expression of the ACSL3 isoform was decreased in cells exposed to FFAs compared to control cells, while those incubated with IFC-305 in the presence of FFAs or alone had recovery of ACSL3 expression. Regarding the ACSL1 isoform, no differences were observed, although there was a tendency toward an increase in cells incubated with IFC-305. One possibility is that the slight increases in the expression of ACSL1 and ACSL3 induced by IFC-305 favor the synthesis of acyl-CoA for oxidation and not for the synthesis of TG, since in both the in vivo and in vitro models, a decrease in the accumulation of TG deposits was observed. This is in accordance with another study in which the expression of ACSL3 was induced with oncostatin M in a model of hyperlipidemic hamsters and in HepG2 cells; in that study, greater oxidation of FFAs and a decrease in the accumulation of TG were observed (44). In another study, in hamsters fed a high-fructose diet, the expression of hepatic ACSL3 was decreased, and re-expression of ACSL3 was accompanied by a decrease in the TG level in the liver, similar to the findings with IFC-305 (45)

3) The authors should measure serum levels of the liver injury markers such as AST and ALT.

The phollowing paragraph was added in the Results section

The phollowing paragraph was added in the Discussion section

4) The authors should check the minor points and English throughout the manuscript. There are many errors.

The corrected versión of the manuscript was sent to AJE.com for English style correction.

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PLoS One. doi: 10.1371/journal.pone.0292448.r003

Decision Letter 1

Nobuyuki Takahashi

18 Sep 2023

PONE-D-23-08771R1An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementationPLOS ONE

Dear Dr. Chagoya de Sánchez,

Please submit your revised manuscript by Nov 02 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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The revised manuscript still contains minor errors as indicated by the reviewer_1. Correct these errors before acceptance.

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Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: In general, the authors revised the manuscript appropriately based on the reviewers’ comments. However, several minor mistakes are remaining.

1. Fig. 3: “INF-γ” should be revised as “IFN-γ.”

2. “N”, “L”, and “GA” are not used in Fig. 4 but explained in the legend of Fig. 4. The authors should either include them in the figure or exclude them form the legend.

Reviewer #2: The authors answered all my questions adequately.

In the future, I would like the authors to use the F4/80 analysis because it is very useful for analyzing inflammation.

**********

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PLoS One. 2023 Oct 5;18(10):e0292448. doi: 10.1371/journal.pone.0292448.r004

Author response to Decision Letter 1

19 Sep 2023

Reviewer #1: In general, the authors revised the manuscript appropriately based on the reviewers’ comments. However, several minor mistakes are remaining.

1. Fig. 3: “INF-γ” should be revised as “IFN-γ.”

Answer: In Fig. 3 “INF-γ” was changed by “IFN-γ.”

2. “N”, “L”, and “GA” are not used in Fig. 4 but explained in the legend of Fig. 4. The authors should either include them in the figure or exclude them form the legend.

Answer: “N”, “L”, and “GA” were removed from the legend of Fig. 4

Reviewer #2: The authors answered all my questions adequately.

In the future, I would like the authors to use the F4/80 analysis because it is very useful for analyzing inflammation.

Thank you very much for the recommendation, we agree that the F4/80 antibody would be very useful to evaluate inflammation.

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PLoS One. doi: 10.1371/journal.pone.0292448.r005

Decision Letter 2

Nobuyuki Takahashi

20 Sep 2023

An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementation

PONE-D-23-08771R2

Dear Dr. Chagoya de Sánchez,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Academic Editor

PLOS ONE

PLoS One. doi: 10.1371/journal.pone.0292448.r006

Acceptance letter

Nobuyuki Takahashi

25 Sep 2023

PONE-D-23-08771R2

An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementation

Dear Dr. Chagoya-de Sánchez:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

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Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Determination of liver damage by enzymatic transaminases activities.

(TIFF)

Click here for additional data file.^{(215.2KB, tiff)}

S2 Fig. pAMPK protein level.

(TIFF)

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S3 Fig. mRNA expression of ACSL1 and ACSL3 in HepG2 cells.

(TIFF)

Click here for additional data file.^{(511.3KB, tiff)}

S1 Table. Food and water consumption.

(DOCX)

Click here for additional data file.^{(17.6KB, docx)}

S1 Data

(ZIP)

Click here for additional data file.^{(681.8KB, zip)}

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

An adenosine derivative prevents the alterations observed in metabolic syndrome in a rat model induced by a rich high-fat diet and sucrose supplementation

Enrique Chávez

Alejandro Rusbel Aparicio-Cadena

Gabriela Velasco-Loyden

María Guadalupe Lozano-Rosas

Mariana Domínguez-López

Amairani Cancino-Bello

Nimbe Torres

Armando R Tovar

Alejandro Cabrera-Aguilar

Victoria Chagoya-de Sánchez

Roles

Abstract

Introduction

Materials and methods

Chemicals

Animals

Table 1. HF diet composition.

Electron microscopy

Growth curves

Glucose and insulin tolerance tests

Serum levels of triglycerides, LDL cholesterol, and insulin

Liver lipid analysis

Histological score for MASLD

Measurement of serum cytokines

Cell culture and treatment

Oil red O staining and quantification

Statistical analysis

Results

Experimental model characterization

Fig 1. Changes in body weight and elements associated with metabolic syndrome induced by HFS and its regulation by IFC-305.

IFC-305 prevents histological alterations and hepatic lipid accumulation

Fig 2. IFC-305 prevents alterations in the hepatic parenchyma and the development of steatosis.

Inflammation induced by HFS and the anti-inflammatory effect of IFC-305

Fig 3. Inflammation induced by a high-fat diet and sucrose was inhibited by IFC-305.

Electron microscopy-revealed changes in liver cells induced by HFS and the protective effect of IFC-305

Fig 4. Morphological changes evaluated by electron microscopy.

In vitro uptake of fatty acids in Huh-7 cells

Fig 5. IFC-305 partially prevented fatty acid uptake by Huh-7 cells.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Nobuyuki Takahashi

Roles

Author response to Decision Letter 0

Decision Letter 1

Nobuyuki Takahashi

Roles

Author response to Decision Letter 1

Decision Letter 2

Nobuyuki Takahashi

Roles

Acceptance letter

Nobuyuki Takahashi

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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