Accumulation of Hypoxanthine : Between Hyperuricemia and Nonalcoholic Fatty Liver — Medical Research Paper @Dr.Tshetiz Dahal

: Framework: An elevated level of plasma Uric Acid has been widely recognized as a risk factor for non-alcoholic fatty liver disease (NAFLD), where oxidative stress and inflammation play an important role in the pathophysiolpgy of the disease. Although the complete molecular mechanisms involved remain unknown, while under physiological conditions uric acid presents antioxidant properties, hyperurecemia has been linked to oxidative stress, chronic low-grade inflammation, and insulin resistance, basic signs of NAFLD.

AIM OF STUDY : Employing in vivo experimentation, we aim to investigate whether a high-fat diet rich in cholesterol (HFD), modifies the metabolism of purines in close relationship to molecular events associated with the development of NAFLD. In vitro experiments employing HepG2 cells are also carried out to study the phenomenon of oxidative stress .

METHODS : Adult male rabbits were fed for 8 weeks an HFD to induce NAFLD. At the beginning of the experiment and every 15 d until the completion of the study, plasma levels of lipids, lipoproteins, and uric acid were measured. Liver tissue was isolated, and histology performed ma followed by the biochemical determination of hypoxanthine, protein expression of xanthineoxidoreductase (XOR) by western blot analysis, and xanthine oxidase (XO) activity using an enzymatickinetic assay. Furthermore, we employed in vitroexperimentation studying HepG2 cells to measure the effect of hypoxanthine and H2O2upon the production of radical oxygen species (ROS), XO activity, and cell viability .

RESULTS AND CONCLUSION — Hepatic tissue from rabbits fed the HFD diet showed signs of NAFLD associated with an increased ROS concentration and an altered purine metabolism characterized by the increase in hypoxanthine, together with an apparent equilibrium displacement of XOR towards the xanthine dehydrogenase (XDH) isoform of the enzyme. This protein shift visualized by a western blot analysis, associated with an increase in plasma uric acid and hepatocyte hypoxanthine could be understood as a compensatory series of events secondary to the establishment of oxidative stress associated with the chronic establishment of fatty liver disease.

INTRODUCTION : According to the World Health Organization, cardiovascular disease (CVD) is the leading worldwide cause of death in the general population .

01. This pathology has been extensively associated with multiple factors such as non-alcoholic fatty liver disease (NAFLD), including hepatic steatosis and non-alcoholic steatohepatitis (NASH), known to contribute to the development of cirrhosis and type 2 diabetes . Several epidemiological studies have shown a positive relationship between the level of uric acid in plasma and the incidence of NAFLD in CVD , where uric acid has been proposed as a predictor of morbidity and mortality . Although the relationship between hyperuricemia and NASH may not be a direct one, there is the possibility that both might be associated because of the establishment of metabolic syndrome. In the human, the catabolism of purines generates uric acid as an oxidative product of hypoxanthine and xanthine due to the enzyme xanthine oxidoreductase (XOR), mainly expressed in the liver, intestine, and vascular endothelial cells. In physiological conditions, this enzyme presents two interconvertible forms by a disulfide bond formation. The first one corresponds to xanthine dehydrogenase (XDH), the most common isoform found in cells, that uses NAD+as an electron acceptor to produce NADH. While xanthine oxidase (XO) transfers electrons directly to molecular oxygen with the concomitant generation of reactive oxygen species (ROS) and particularly , and therefore, considered essential source of reactive peroxides, superoxide, hydroxyl radical, singlet oxygen and α-oxygen . While to date oxidative stress is a key factor in the development of NASH and CVD 18 in the hepatocyte, the molecular mechanisms that correlate the metabolism of purines with a state of oxidative stress following a lipid overload, are still a matter of intensive study. Although uric acid is a risk factor for fatty liver disease and CVD, the molecular mechanisms involved in the way hepatocytes respond to uric acid also remain to be studied, together with the question if oxidative stress potentiates the effects of hyperuricemia. Liver cells from experimental animals fed a high cholesterol/triglyceride diet show an increase in hypoxanthine that promotes an equilibrium displacement of XOR activity towards the XDH form of the enzyme. This phenomenon is most probably associated with the process of activation/inactivation of diverse enzymes involved in the proteolysis of XDH, under an increased hypoxanthine concentration. Considering that it has been shown that under acute cellular oxidative conditions the proteolytic induction of XO is favored, the compensatory mechanisms activated during established chronic metabolic conditions are still a matter of controversy. Therefore, we believe the study of this enzyme in the hepatocyte under conditions of oxidative stress and hypoxanthine accumulation associated with fatty liver disease acquires special relevance in the understanding of how NAFLD and NASH are established as important participants of CVD .

HISTOLOGICAL ANALYSIS: At the time of euthanasia, liver samples from the right lobe were collected and a portion of these samples fixed in 10% formal dehyde . Samples were embedded in paraffin and stained with classical hematoxylin-eosin (H&E) to study cell morphology and Masonstrichrome stain that highlights the deposition of collagen fibres characteristic of fibrosis. The remaining tissue was frozen in liquid nitrogen and stored at -80°C until further analysis. Liver sections were evaluated considering histopathological characteristics for NAFLD: Steatosis, inflammation, ballooning, and the presence of fibrosis. Preparations were analyzed using conventional light microscopy and multiphoton microscopy .

Hypoxanthine AND Xanthine Oxidase ACTIVITY MEASUREMENTS : Hypoxanthine levels and XO activity in hepatic tissue were measured with the Amplex Red Xanthine/Xanthine Oxidase Assay kit. This kit provides a sensitive method for detecting xanthine/hypoxanthine, or for monitoring xanthine oxidase activity in the supernatant of homogenized liver tissue samples. Hepatic tissue is homogenized employing saline cold buffer and clarified by centrifugation. A further dilution of the supernatant with the reaction buffer is employed to determine the endpoint fluorescent signal. In the assay, xanthine oxidase catalyzes the oxidation of xanthine/hypoxanthine, to uric acid and superoxide (H2O2), and the H2O2 in the presence of horseradish perooxidase reacts stoichiometrically with the Amplex Red reagent to generate the red-fluorescent oxidation product, resorfin. This compound presents absorption and fluorescence emission maxima of approximately 571 nm and 585, respectively. On the other hand, intracellular hypoxanthine concentrations and XO activity of HepG2 cells were measured with kits from Ray Biotech respectively. The RayBio xanthine/ hypoxanthine assay employs a xanthine enzyme mix that specifically oxidizes xanthine/hypoxanthine to form an intermediate, which reacts with Developer and Probe to form a product that can be measured fluorometrically with absorption and fluorescence maxima at 525/587 nm. The Abcam xanthine oxidase activity assay also employs the oxidation of which reacts stoichiometrically with a probe to generate fluorescence showing an absorption and fluorescence maxima at 535/587 nm. With both kits, standard curves are prepared according to the manufacturer’s instructions and employed in the determination of values from experimental samples. Independently of the kit employed, after different treatments had taken place, culture medium was removed from Petri dishes, and cell monolayers washed three times with PBS at room temperature. Immediately after, cells are detached by using trypsin incubating for 5 min at 37°C, followed by trypsin inactivation using FBS supplemented DMEM, cells are washed once more, centrifuged, and supernatants discarded. For the determination of hypoxanthine concentration and XO activity, the number of cells employed are first standardized, to be further lyzed, clarified by centrifugation, and supernatants used for both measurements.

Xdh Gene Expression by qPCR: Total liver RNA was isolated with Trizol reagent (Thermo Fisher Scientific); 1µg was used to synthesize cDNA using the iScript cDNA synthesis kit (Bio-Rad). The expression of xanthine dehydrogenase (XDH: 5'- CCATCTACGCTTCCAAGGCT- 3' and 5' CAGTGACACACAGGGTGGTGA-3') AND glyceralide dehydrogenase (GAPDH: 5'-TCGGAGTGAACGGATTTGGC-3' and 5'-CCAGCATCACCCCACTTGAT-3') transcripts were determined by qPCR using the PowerUp Sybr Green Master Mix 2X (Applied Biosystems) on an ABI PRISM 7000 Sequence Detection cycler. All samples were analyzed in triplicates and the results are relative levels of mRNA calculated as 2-ΔΔCt .

XOR PROTEIN EXPRESSION by Western Blot Analysis: LIVER SAMPLES were homogenized in cold RIPA buffer (Thermo Fisher Scientific) supplemented with proteaseinhibitors (Roche). Protein concentration was determined using the DC Protein Assay (Bio-Rad, CA, USA), and 100 µg of liver lysates were separated in 8 or 10% polyacrylamidegels (SDS-PAGE) and transferred to PVDF membranes (MilliporeMerck, Burlington, MA, USA). Blots were incubated with specific antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) recognizing XOR (XDH/XO) (sc-398548) and GAPDH (sc-47724). : CELL CULTURE — HepG2 cells (ATCC HB-8065) were grown following ATCC instructions, employing Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum, 10 µg/mL streptomycin, 0.25 µg/mL amphoterecin, and 100 U/mL penicillin at 37°C and 5% CO2. Usually after 72 h of incubation with cells reaching 80–90% confluency, cells are rinsed with PBS and exposed to 80 mMol H2O2 diluted in DMEM. After 1h exposure, cells were trypsinized and reseeded in a proportion of 4.6 × 104cells/cm2in DMEM supplemented medium, with the addition of hypoxanthine from a 1M stock solution solubilized in NaOH. Final concentrations ranged from 25–150 µM hypoxanthine and 156 µM NaOH, for all conditions including negative controls. Under these conditions, no significant changes on medium pH were observed. When indicated, allopurin 10 µM methionine 25 or 50 µM , or Trolox at 25 or 50 µM were added to DMEM, and cells incubated in this medium for 12 h before the oxidative stimulus with H2O2 , and exposure to hypoxanthine takes place.

CELL VIABILITY: The integrity of the plasma membrane was used as a marker of cell viability, measuring Lactic dehydrogenase activity released to the medium. In lysates, results were expressed as a percentage of lactic dehydrogenase activity released to the medium. Collected samples were incubated for 5 min in a Tris/NaCl buffer (pH 7.2) with NADH 0.3 mM, on 96 well plates for UV spectrophotometric readings to follow NADH consumption. The reaction was initiated by adding sodiumpyruvate (10 mM, final concentration) and performed at 30°C. Immediately after starting the reaction, absorbance at 340 nm was measured and changes registered every 30 s for 5 min. Intracellular Reactive Oxygen Species — Cells grown in 96 well plates, at the different study conditions, supplemented and washed with PBS at 37°C were further incubated for 45 min in DMEM and 10 mM dichlorofluoresceindiacetate. Cell suspensions were obtained and transferred to a 96 well dark plate to measure fluorescence at 485/528 nm (excitation/emission). Results were expressed as the percentage of ROS with respect to confluent cells in the absence of the stimulus.

RESULTS :

Hepatic Tissue Examination : At the end of the 60 d of the experiment, animals were sacrificed, and liver samples prepared from both experimental groups (control and HFD). To ensure that tissue damage associated with NAFLD had been established, samples were examined by conventional light microscopy and second-harmonic generation microscopy. ( Figure 1 ) — shows representative images of samples belonging to each one of the two experimental groups studied, displaying the central vein surrounded by layers of hepatocytes. In contrast to rabbits fed the standard diet showing a normal tissue structure and a normal collagen distribution. (Figure 1 — A, C), rabbits fed an HFD show microvesicular steatosis, cellular ballooning, and inflammation, as characteristic signs of NASH (Figure 1 B). Moreover, using Masson’s trichrome stain, it was possible to visualize the presence of extensive collagen fiber deposition, denoting the presence of fibrosis and progressive hepatic disease (Figure 1 — D). The use of second-harmonic generation microscopy, useful in the evaluation of tissue areas with increased deposition of protein shows an extensive network of collagen fibril/fiber structures, supporting the fibrotic feature of livers in the HFD experimental group (Figure 1E, F).

Plasma Metabolite Profiles : To address whether the metabolism of lipid and purines are simultaneously altered during the process of NASH consolidation, the following plasma parameters were measured at the start of the experiment and every two weeks before sacrifice; total cholesterol, triglycerides, C-LDL, C-HDL, and uric acid. As expected, values for lipids such as total cholesterol, triglycerides and C-LDL were increased by the HFD (Figure 2A-C), while the C-HDL levels were to remain close to values observed with control animals (Figure 2D). Interestingly, immediately after the start of the HFD, plasma levels for uric acid dramatically increased in correlation to the plasma levels of cholesterol (Figure 2E).

Hypoxanthine and XOR in NASH : In contrast to the control group, the plasma concentration of hypoxanthine in the HFD group showed a significant elevation (Figure 3A). Surprisingly, XO activity that did not show changes between groups fed or not with the HFD (Figure 3B), also, did not present differences in transcript expression of Xdh (Figure 3C).

Nevertheless, a western blot analysis using an anti-XOR antibody that recognizes both isoforms of the enzyme (XDH and XO), shows that liver lysates from control animals present two bands very close to each other at the expected molecular weight region for XOR, a result that most probably corresponds to the presence of the two enzyme isoforms. An upper band containing the molecular form XDH, and a lower molecular weight band reported to be exclusively associated with XO, and normally related to changes in the oxido/reduction equilibrium of cysteines 535 and 992. Although under normal conditions this equilibrium is maintained, it is found that under an adverse cell metabolic condition due to an HFD, a potential oxidative environment promotes an equilibrium shift towards the XDH form of the enzyme (Figure 3D).

Hypoxanthine Effect on Hepatocytes: To further explore if the increase in cell hypoxanthine established in the HFD group is associated to an oxidative environment, we performed a series of in vitro experiments employing 72 h hypoxanthine treated HepG2 cells. Under these conditions, we found the concomitant increase in intracellular hypoxanthine associated to an increment in XO activity (Figure 4A). When the concentration of hypoxanthine is increased up to 125 mM, treated cells show a marked ROS production, with no apparent affectation in cell viability up to 100 mM (Figure 4B). Since during these experiments designed to have acute exposure and response to hypoxanthine, a rise in XO activity has been established, and the potential generation of H 2O 2might be occurring, HepG2 cells were also treated with increasing concentrations of H 2O 2. Under these conditions, a further increase in the intracellular concentration of ROS was observed, associated with a negligible impact on cell viability, again up to 100 mM (Figure 4C).

To further explore if hypoxanthine by itself contributes to the state of oxidative stress, HepG2 cells were incubated with the XO inhibitor allopurinol before treatment with 50 µM hypoxanthine. Under these experimental conditions, oxidative stress was reduced by allopurinol, showing ROS values similar to levels found in control cell cultures (Figure 4D). Uh Furthermore, the addition of Trolox (25 µM) a water-soluble antioxidant molecule analog of vitamin E and methionine known to have an antioxidant effect through induction of the antioxidant protein heme oxygenase-1 . (), prevented the induction of oxidative stress derived from cell exposure to hypoxanthine (Figure4E). Furthermore, the addition of Trolox (25 µM) a water-soluble antioxidant molecule analog of vitamin E (and methionine known to have an antioxidant effect through induction of the antioxidant protein heme oxygenase-1 prevented the induction of oxidative stress derived from cell exposure to hypoxanthine (Figure 4E).

Discussion

Nowadays it has been well established that a high-fat diet that includes cholesterol induces hyperlipidemia associated with metabolic complications such as NASH, hyperglycemia, insulin resistance, dyslipidemia, and even metabolic syndrome. Our study shows that such a diet leads to a significant elevation of plasma triglycerides and cholesterol, simultaneously increasing the concentration of uric acid, suggesting that hyperlipidemia alters the metabolism of purines together with the development of liver steatosis and fibrosis as described in NASH

For some time, it has been known that during specific metabolic states associated with elevated oxidative stress, the metabolism of lipids can be modulated through changes in proteinexpression in such a way that the balance for key proteins can be affected contributing to the development of fatty liver disease and atherosclerosis . Since as shown by our group, the generation of oxidative stress has been also linked with gene processing as well as protein expression . The present study supports the possibility that regulation of nucleotide catabolism in the hepatocyte, might also contribute to modulate the deleterious effects of lipid accumulation in fatty liver disease. Therefore, it will be of special interest employing conditions similar to the ones used in the present study, to directly assay not only XDH and XO activities, but also HGPRT, and seek the specific molecular mechanisms behind the regulation of these enzymes in the hepatocyte during a state of oxidative stress, in an attempt to better understand the development of fatty liver disease associated with CVD.

Originally published at http://doctor8.home.blog on August 25, 2021.