Abstract
Background/Aim: Excessive fructose intake reportedly leads to the development of nonalcoholic fatty liver disease (NAFLD). In our previous study, we reported that plasma activities of alkaline phosphatase (ALP) isozymes were markedly changed in rats with excessive fructose intake-induced hepatomegaly. In this study, we examined ALP isozyme activity prior to the occurrence of hepatomegaly, and investigated the effect of the timing of sample collection, to explore its potential as a biomarker. Materials and Methods: After 1-week intake of a 63% high-fructose diet (HFrD), blood samples were collected from male rats during sleep or active phases to analyze biochemical parameters. Results: Body and liver weights were similar between the HFrD and control diet groups, indicating that hepatomegaly due to excessive fructose intake had not occurred. The triglyceride levels and glutamate dehydrogenase (GLDH) activity were significantly elevated to similar degrees at both time points. HFrD intake significantly increased liver-type ALP (L-ALP) activity, stimulating it by 12.7% at the sleep phase and by 124.3% at the active phase. HFrD consumption also significantly decreased intestinal-type ALP (I-ALP) at the active phase, but only showed a decreasing trend during the sleep phase. Conclusion: Measurements of plasma ALP isozyme and GLDH activity, and triglyceride levels are effective early biomarkers of impending NAFLD caused by excessive fructose intake. L-ALP and I-ALP activities during the active phase are particularly sensitive for detection of excessive fructose intake before the occurrence of NAFLD.
Over the past few decades, the human diet has changed dramatically, especially in developed countries. Among these changes, there has been a tremendous shift toward overnutrition, or high-calorie diets (1-3). One of the most important consequences of overnutrition is the metabolic syndrome, a collection of lipid and glycemic abnormalities that includes elevated serum triglycerides, low high-density lipoprotein cholesterol, fatty liver, elevated blood pressure, insulin resistance, elevated serum glucose, and proinflammatory states (4). Home cooking in many developed countries has been replaced in recent years by foods with high-calorie density, in part from the use of high fructose corn syrup in sweetened beverages and foods (5).
It has been reported that excessive fructose intake induces de novo synthesis of fatty acids and triglyceride accumulation in the liver, resulting in the development of nonalcoholic fatty liver disease (NAFLD), hyperlipidemia, and chronic inflammation (6-8). Although NAFLD is a major cause of liver disease, affecting approximately one-third of the world’s population, it is mostly asymptomatic and therefore difficult to diagnose (9, 10). Importantly, approximately 30% of NAFLD patients develop nonalcoholic steatohepatitis (NASH) with necrotic inflammation and fibrosis of the liver, highlighting the urgent need to prevent the various diseases caused by excessive fructose consumption. In addition to advising the public to avoid excessive fructose intake, appropriate tools are needed to diagnose daily fructose overconsumption. To the best of our knowledge, there is no optimal method to identify fructose overdose prior to the onset of NAFLD. Therefore, in our previous study, we evaluated and compared the physiological changes that occurred in rats fed an excessively high-fructose diet (HFrD) versus a control diet for four weeks, with a focus on hepatotoxicity biomarkers (11). We found that the activities of two alkaline phosphatase (ALP) isozymes, liver-type-ALP (L-ALP) and small intestinal-type ALP (I-ALP), were useful indicators for detecting physiological changes caused by excessive fructose consumption in the early stages of NAFLD and, ultimately, the development of metabolic syndrome. However, the development of hepatomegaly at the fourth week of excessive fructose intake indicated the need to follow ALP isozyme activities at earlier stages to be able to verify their potential as biomarkers for NAFLD.
Another important aspect of monitoring food factors is the time of day in which the evaluations are conducted. In studies using nocturnal animals, such as rats and mice, samples are commonly collected during the light period (i.e., when rodents are sleeping), largely because it is convenient for the daytime working schedules of researchers. However, it has been recently reported that the sampling time has a dramatic effect on the results, even using the same treatments (12). This is because biological clock regulators, such as aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1) and circadian locomotor output cycles kaput (CLOCK), are found in most biological organisms, including humans and rodents. Accordingly, diurnal variations have been observed for many biological phenomena. For example, compounds such as polyphenols, which are absorbed into the body via transporters with diurnal variations, have different patterns of absorption depending on the time of intake (13). Toxicological expression also differs depending on the time of administration of chemicals, such as ethylnitrosourea (14). Higher melatonin levels in the cerebrospinal fluid have also been noted during the active phase in pigs (15). ALP activity is an indicator of the typical diurnal rhythm, and is higher in the beginning of the sleep phase than in the beginning of the active phase in rats (16). Additionally, a clinical study using diurnally active and nocturnally resting human subjects reported the existence of a circadian rhythm in sister chromatid exchange (17). Taken together, these studies support the possibility that ALP isozymes affected by excessive fructose consumption might also exhibit different activity patterns depending on the time of day of evaluation.
In the present study, we used the rat model described in our previous study (11) to evaluate blood parameters collected during the sleep and active phases after one week of excessive fructose intake.
Materials and Methods
Institutional approval of the study protocol. This study was approved by the Institutional Animal Care and Use Committee of the Yokohama Research Institute of the Central Research Institute of Japan Tobacco Inc. (Protocol Code: 21075, date of approval: 7th December 2021) in accordance with the Law on the Protection and Management of Animals (Law No. 105, 1st October 1973), which defines animal experiments as the use of animals for scientific purposes in consideration of the 3Rs.
Animals and housing environments. In our previous study in six-week-old rats, we measured the activity of ALP isozymes in blood collected during the light period (sleep phase) after four weeks of HFrD feeding (11). The purpose of this study was to evaluate whether variations in ALP isozyme activity occurred between the light (sleep) and dark (active) periods after 1 week of consuming a HFrD. Because animals that are highly active during the dark period exhibit various in vivo fluctuations when they are touched by humans for the first time during that period (18), we sought to minimize these fluctuations by acclimating our rats to humans via daily handling during the active phase. Reportedly, the term required for this habituation is approximately four weeks (18). Therefore, we obtained lactating rats and begun the experiment at the age of six weeks. Briefly, four 18-week-old mother Crl:CD (Sprague-Dawley) rats with twenty-four male two-week-old offspring were purchased from Jackson Laboratory, Inc., (Kanagawa, Japan). Each set of mother and offspring was housed in a plastic cage (W380 mm×D290 mm×H180 mm) with animal bedding in animal housing rooms at a temperature of 23±1°C, relative humidity of 55±5%, 12 h-light/12 h-dark cycle, and ventilation frequency of about 15 times/h. The animals were given free access to the modified AIN-93G purified control diet (CD; D21012002, Research Diets, Inc., New Brunswick, NJ, USA) and tap water. After one week of acclimatization, weaned male rats were separated from their mothers and housed in pairs in plastic cages (W380 mm×D290 mm×H180 mm), and the acclimatization was continued with the CD and tap water for three weeks. Zeitgeber time (ZT) 0 represents the time when the light was turned on at the start of the light period. The light period lasted from ZT0 to ZT12 and the dark period lasted from ZT12 to ZT24. Every daily handling was carried out at ZT2-4 for the sleep-phase group and at ZT14-16 for the active-phase group. Mice are largely insensitive to red light (>650 nm) (19, 20); hence, daily handling maneuvers such as cage exchanges were carried out under a flashlight covered with red film on the active-phase group in accordance with our previous study (18).
Experimental design. After four weeks of acclimatization, two groups of male rats aged six weeks, which had been handled daily at ZT2-4 or ZT14-16, were randomly divided into two groups, as previously described (11): one group continued consuming the CD, and the other began consuming a 63% HFrD (D21012001, Research Diets, Inc.). The detailed composition of the CD and HFrD is shown in Table I. The animals in each group were fed their assigned diets ad libitum for one week. Body weight and food intake were measured daily.
Compositions of the experimental diets.
Blood sampling, and liver necropsy and weight. After one week of diet consumption, blood samples were collected from the abdominal aorta of rats under isoflurane anesthesia (5%) and non-fasting conditions at ZT2-4 or ZT14-16. All blood samples were collected into lithium heparin-coated Vacutainer blood collection tubes (Becton, Dickinson and Company, Auckland, NJ, USA), immediately cooled on ice, and centrifuged at 1,750×g at 4°C for 30 min. The plasma fractions were stored at −80°C until further analysis. Livers were thoroughly examined for gross lesions by a pathologist who was unaware of the type of treatment each animal had received. Liver weight was measured, and the liver weight relative to the final body weight was calculated.
Biochemical analysis. The following plasma parameters were measured at 37°C using an automated analyzer (TBA-120FR, Canon Medical Systems Corporation, Tochigi, Japan) with individual standard reagents in accordance with the manufacturer’s instructions: aspartate aminotransferase (AST, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), alanine aminotransferase (ALT, FUJIFILM Wako Pure Chemical Corporation), ALP (FUJIFILM Wako Pure Chemical Corporation), glutamate dehydrogenase (GLDH, Randox Laboratories Ltd., London, UK), leucine aminopeptidase (LAP, FUJIFILM Wako Pure Chemical Corporation), total cholesterol (Minaris Medical Co., Ltd., Tokyo, Japan), triglycerides (FUJIFILM Wako Pure Chemical Corporation), phospholipids (FUJIFILM Wako Pure Chemical Corporation), and total protein (FUJIFILM Wako Pure Chemical Corporation).
Plasma ALP isozyme analysis. Plasma activities of L-ALP, I-ALP, and bone-type ALP (B-ALP) were measured in a fully automated electrophoresis system (Eparaiza 2, Helena Laboratory, Saitama, Japan) with standard reagents. For B-ALP analysis, plasma was pretreated with neuraminidase (Separator, Helena Laboratory) for 30 min at room temperature as described by Hatayama et al. (21).
Statistical analysis. Data are presented as means±standard deviation. Statistical analyses were conducted using JMP (SAS Institute Inc., Charlotte, NC, USA). Statistical significance among groups was determined using two-way analysis of variance, followed by a Tukey-Kramer post hoc test. Results were considered significant when the possibility of error was <5%.
Results
Growth parameters, liver weight, and necropsy. There were no significant differences in body weight, food intake, or liver weight between the CD and HFrD groups, nor were there differences in sampling at the sleep phase versus the active phase (Table II). No abnormal findings were observed at liver necropsy in any of the groups (Table II).
Effects of fructose consumption on growth parameters, and liver weight and necropsy.
Plasma biochemical parameters. The effects of HFrD consumption on plasma biochemical parameters are summarized in Figure 1. In the CD group, plasma levels of triglycerides, total cholesterol and phospholipids, and ALT activity were significantly lower when sampled during the active phase than during the sleep phase. ALP activity in the CD group was also remarkably decreased in the active phase compared with that in the sleep phase, although not statistically significantly. In the HFrD group, triglycerides and GLDH activity were significantly elevated at both sampling times, and ALP activity was significantly decreased, but only in the active phase. No clear effects of HFrD consumption were observed for any of the other parameters (total cholesterol, phospholipids, total protein, ALT, AST, and LAP levels).
Effects of excessive fructose intake on plasma biochemical parameters. The following parameters were measured at the sleep phase (ZT2-4) or active phase (ZT14-16) in male rats after 1 week on an ad libitum control diet (CD) or high-fructose diet (HFrD): (A) triglycerides; (B) total cholesterol; (C) phospholipids; (D) total protein; (E) alkaline phosphatase (ALP); (F) glutamate dehydrogenase (GLDH); (G) alanine transaminase (ALT); (H) aspartate aminotransferase (AST); (I) leucine aminopeptidase (LAP). Data are expressed as mean±standard deviation (n=6). Significant between-group differences (p<0.05) are indicated by superscripted letters.
Plasma activities of ALP isozymes. In the CD group, L-ALP activity was significantly lower in the active phase than in the sleep phase (Figure 2A), but no such differences according to sampling timing were observed for I-ALP and B-ALP (Figure 2B and C). Furthermore, HFrD intake significantly increased L-ALP activity at both sampling times, with increases of 12.7% in the sleep phase and 124.3% in the active phase. These results indicated that HFrD consumption had a stronger effect on plasma L-ALP activity during the active phase (Figure 2A). By contrast, HFrD consumption significantly decreased I-ALP activity in the active phase, but only led to a decreasing trend in the sleep phase (Figure 2B). There were no effects of the HFrD on B-ALP activity (Figure 2C).
Effects of 1 week of excessive fructose intake on plasma activities of three alkaline phosphatase (ALP) isozymes: liver-type ALP (L-ALP), intestinal-type ALP (I-ALP), and bone-type ALP (B-ALP). CD: Control diet; HFrD: high-fructose diet. Data are expressed as means±standard deviation (n=6). Significant between-group differences (p<0.05) are indicated by superscripted letters.
Discussion
Excessive fructose intake has been reported to lead to the development of NAFLD, hyperlipidemia, and chronic inflammation (22, 23). Given that NAFLD is asymptomatic (24), we must take appropriate action to ensure that individuals avoid daily excessive fructose intake, and develop effective tools to diagnose daily excessive fructose intake. Our previous study demonstrated that four weeks of HFrD consumption in rats caused hepatic hypertrophy with lipid accumulation, as well as a significant increase in L-ALP activity and a significant decrease in I-ALP in the blood (11). I-ALP activity decreased from the first week of HFrD consumption, suggesting that the activities of different ALP isozymes in the blood might serve as a specific biomarker of impending fructose-related NAFLD. However, the previous study did not provide a detailed comparison of hepatic damage or the activities of ALP isozymes at an earlier stage. Therefore, in the present study, we further evaluated the activities of three ALP isozymes, in both the active and sleep phases, after just one week of excessive fructose intake.
The body and liver weights of rats after one week of HFrD intake were similar to those of the CD group (Table II). Castro and colleagues reported that fructose intake has a high lipogenic potential in liver tissue, but requires longer than 45 days of consumption to induce a chronic model of NAFLD (25). Indeed, induction periods of 6-16 weeks with excessive fructose intake have been used to model NAFLD and NASH in animals (26-28). Therefore, the hepatomegaly due to fat accumulation that is routinely observed with excessive fructose intake may have not occurred under the one-week HFrD observation period.
As mentioned in the introduction section, given that many biological phenomena are regulated by the biological clock, and hence exhibit diurnal variations in enzyme activity and gene expression (12), the timing of evaluation and treatment administration in research using animal models is important (29-31). In this study, animals were evaluated at two time points: at the beginning of the sleep phase (ZT2-4) and at the beginning of the active phase (ZT14-16). In the CD group, there were no clear differences in body weight or liver weight at these two sampling points (Table II), but the levels of plasma triglycerides, total cholesterol and phospholipids, and ALT activity were significantly lower in the early-active phase than in the early-sleep phase (Figure 1). ALP activity was also lower in the early-active phase than in the early-sleep phase in the CD group, although the difference was not significant. These clear diurnal variations are similar to those reported in several previous studies (16, 32), indicating that the diurnal rhythms of the rats used in the present study were accurately demarcated by the two sampling points. Interestingly, total ALP activity tended to be lower at the early-active phase, but L-ALP activity was significantly and dramatically lower at the early-active phase (Figure 2). By contrast, there were no clear differences in I-ALP and B-ALP activity between the two time points. This might be attributable to the lower intensity of L-ALP activity (about 80 U/l) compared with that of I-ALP (about 700 U/l) and B-ALP (350 U/l). In any case, this study is the first to demonstrate that blood L-ALP activity may have a distinct diurnal variation. To confirm that ALP isozymes have a diurnal rhythm of activity, we plan to collect samples at multiple time points throughout the day, e.g., every 2 h, and compare their activity levels in a future investigation.
Among the nine blood biochemical parameters analyzed in this study, the triglycerides levels and GLDH activity showed increases after just one week of HFrD intake (Figure 1). Although there were no clear differences between the active and sleep phases in these two parameters, similar changes were observed after four weeks of HFrD intake (11), indicating that changes in triglycerides and GLDH activity appear early in the course of excessive fructose intake, before the symptoms of hepatomegaly are observed. In agreement, Castro et al. (25) showed that fructose has a high lipogenic potential, but requires a period of intake longer than one week to induce NAFLD. Additionally, GLDH activity is a more specific marker of cystic injury in the liver than are the activities of AST and ALT (33, 34), suggesting that the increase in its secretion into the blood occurs at a stage earlier than the onset of hepatomegaly. Thus, triglyceride levels and GLDH activity may be useful early biomarkers of possible excessive fructose intake.
HFrD intake significantly increased L-ALP activity to remarkably different degrees at both the sleep phase and active phase time points: 12.7% increase at the sleep phase and 124.3% increase at the active phase (Figure 2). Excessive fructose intake reduces the diversity in the intestinal microflora and increases lipopolysaccharide (LPS) levels in the intestinal tract (35). LPS disrupts intestinal barrier function, thereby increasing membrane permeability (35, 36), possibly resulting in leakage of LPS into the portal vein, causing fatty liver, inflammation, and hepatocyte death (37). In other words, the increase in plasma L-ALP isozyme activity is thought to result from increased liver production of L-ALP to detoxify endotoxin. The gut microbiota and their metabolites react to normal metabolism regulated by the host circadian clock systems, including the master clock and the peripheral clock (38). Thus, the sensitivity of the gut microbiota to external stimuli such as excessive fructose may be increased during the active phase. Additionally, even under ad libitum feeding, rats show a pattern of eating at the beginning and end of the active phase, and consuming little during the sleep phase (39). Therefore, our observation that the increase in L-ALP activity in blood collected at the beginning of the active phase was more intense than that at the beginning of the sleep phase may be a consequence of natural biological phenomena. Plasma I-ALP isozyme activity showed a decreasing trend in the sleep phase and a significant decrease in the active phase (Figure 2). Reportedly, excessive intake of fructose induces increased intestinal concentrations of inflammatory cytokines (interleukin-1β and interleukin-6) and immunoglobulin G, and infiltration of inflammatory cells into the intestinal tissue (40-42). I-ALP is an important apical brush border enzyme expressed in the gastrointestinal tract and secreted both into the intestinal lumen and bloodstream, and exerts its effects through dephosphorylation of proinflammatory molecules such as lipopolysaccharide (43). Although the mechanism for the reduction in plasma I-ALP isozyme activity observed in the HFrD group in the active phase is unclear, its reduction may increase the risk of disease through changing the microbiome, and causing intestinal inflammation and intestinal permeability (42-45).
In conclusion, we showed that plasma measurements of ALP isozyme and GLDH activity and triglyceride levels were effective early biomarkers of NAFLD development following a week of excessive fructose intake. The time of measurement of these parameters was also found to be important. Measurements of these biomarkers at the beginning of the active phase, which is the dark period for nocturnal animals such as rats and mice, provided an especially clear indication of excessive fructose intake. The activities of the ALP isozymes L-ALP and I-ALP were highly sensitive for the detection of excessive fructose intake before the occurrence of NAFLD, especially during the active phase.
Acknowledgements
The Authors would like to thank the invaluable contributions of Toshiyuki Shoda, Tadakazu Takahashi, Taku Masuyama, Takuya Abe, Iori Tsubakihara, Masumi Takeda, Makoto Saito, Ayaka Iketani and the staff at the Toxicology Research Laboratories, Central Pharmaceutical Research Institute, JAPAN TOBACCO Inc., Japan. The Authors would also like to thank Michelle Kahmeyer-Gabbe, PhD, from Edanz Group (https://en-author-services.edanzgroup.com/) for editing a draft of this manuscript.
Footnotes
Authors’ Contributions
Y.S., A.K. and H.S. conceived the study. Y.S., C.M., K.K., T.S. and K.R. performed the experiments. A.K., D.Y. and H.S. provided scientific advice. Y.S. wrote the paper. D.Y., A.K and H.S. revised the paper.
Funding
This research was partially funded by the Japan Society for the Promotion of Science KAKENHI, grant number 21H03361.
Conflicts of Interest
The Authors declare that they have no conflicts of interest in relation to this study.
- Received May 16, 2023.
- Revision received June 7, 2023.
- Accepted June 8, 2023.
- Copyright © 2023, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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