Abstract
Background/Aim: The global prevalence of type 2 diabetes (T2D) continues to increase, necessitating the need for understanding the causes of its development. The widespread use of high-fructose corn syrup (HFCS) in drinks and diets is suspected to play a role in metabolic disorders. Although many studies have reported on the effects of excessive HFCS and excessive energy intakes in middle-aged individuals, few have focused on energy restriction. This study aimed to investigate the effects of excessive HFCS drink intake under energy restriction on developing T2D in early middle-aged mice. Materials and Methods: Early middle-aged mice were divided in HFCS and control groups; they were provided either 10% HFCS water or deionized water ad libitum for 12 weeks, respectively. Total energy intake was controlled using a standard rodent diet. Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), tissue weight measurements, serum parameter analyses, and mRNA expression assessments were performed. Results: No increase in body and adipose tissue weight was observed with excessive HFCS intake under energy restriction. Moreover, serum lipid parameters did not differ from those of controls. However, in the OGTT and ITT, the HFCS group showed higher blood glucose levels than the control group. Moreover, the pancreatic weight and insulin II mRNA expression were reduced. Conclusion: The excessive HFCS drink intake under energy restriction did not induce obesity; however, it induced impaired glucose tolerance, indicating its negative effects on the pancreas in early middle-aged mice. When translated in human physiology, our results show that even if one does not become obese, excessive HFCS may affect the overall metabolic mechanism; these effects may vary depending on age.
The global prevalence of diabetes continues to steadily increase annually. As of 2021, approximately 537 million adults between 20 and 79 years old gave diabetes, constituting 10.5% of this age group (1). Projections estimate a further increase to 643 million by 2030 (1), and this global health problem is becoming increasingly serious. Type 2 diabetes (T2D) accounts for approximately 90% of all diabetes cases (2); its etiology is multifaceted, involving genetic predispositions, such as ethnicity and familial history, coupled with environmental factors, such as obesity, aging, imbalanced nutrition, and lack of exercise (1). Among these, obesity has gained significant attention (3-7). For example, the majority of British individuals with T2D are obese, whereas approximately 10%-20% are nonobese (7). In United States, T2D has been found to be more prevalent among people with higher body mass index (BMI) (4). Conversely, in Asia, 70%-80% of individuals with T2D are nonobese (8). Obesity rates considerably vary across nations; although obesity is a factor in diabetes development, it also greatly varies from country to country.
High-fructose corn syrup (HFCS) was developed in the 1960s as a liquid sweetener to counteract the severe shortage of sucrose production; it is widely used in soft drinks (9, 10). HFCS is produced by the isomerization of some of the glucose in corn syrup to fructose. A recent study has implicated HFCS and high-fructose diets in various health risks, including obesity (10-13), metabolic syndrome (14), fatty liver (15, 16), and diabetes (17). Additionally, fructose in HFCS suppresses the production of leptin, an appetite-suppressing hormone (18, 19) and induces leptin resistance (20). Across different age groups, HFCS intake has been associated with weight gain and obesity (10). However, these studies do not consider total energy intake, and the possibility that obesity is not caused by HFCS intake but excess energy intake cannot be ruled out. Thus, elucidating the direct effects of excessive HFCS intake necessitates controlling for total energy intake.
Previously, we examined the effects of excessive soft drink consumption with an unbalanced diet on growing male mice. The results revealed impaired glucose tolerance (IGT) rather than obesity in these growing mice (21). Soft drink consumption has increased worldwide (22), and a certain percentage of individuals, regardless of their age group, habitually consume soft drinks (23). Many studies on the effects of diet have employed relatively young mice and rats. Furthermore, several studies targeting middle-aged individuals have examined conditions, such as high-fat or high-calorie diets (24, 25), and there have been very few studies that have focused on energy intake restriction. In addition, there is a wide range of ages in middle age, and biological mechanisms differ between early and late middle age (26-28).
Therefore, we focused our analysis on early middle-aged mice and investigated the effects of excessive HFCS drink intake under energy restriction in order to draw meaningful conclusions on the effects of consumption of relevant substances in early middle-aged humans.
Materials and Methods
Animals. The animal experiments in this study were approved by the Ethics Committee of the Miyazaki University (Miyazaki, Japan; No. 2019-031-2,-3, 2021-014-2), and the protocols were consistent with the committee’s guidelines for the care of animals. Male ICR mice were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan) and individually placed in plastic cages under controlled atmospheric conditions [12-h light/12-h dark (7:00-19:00), 20-22°C]. Following acclimatization to the breeding environment, the 28-week-old mice were randomly divided into two groups and were provided free access to 10% HFCS (55% fructose and 45% glucose) water (HFCS group) (n=6) or deionized water (control group) (n=7). The amount of HFCS water intake was measured daily, and the energy intake was adjusted to be the same as the control group using a standard rodent diet (Rodent LabDiet EQ 5L37, Japan SLC, Inc., 3.38 kcal/g). The body weight of each group was measured weekly. The BMI was calculated by measuring the length from the nose to the anus. The overview of this study is shown in Figure 1.
Overview of study design. The 28-week-old mice were randomly divided into two groups, which were provided either 10% HFCS water (HFCS group) or deionized water (control group) ad libitum. The amount of 10 % HFCS water intake was measured daily, and energy intake was adjusted to be the same as control group using a standard rodent diet. The body weight was measured weekly for 12 weeks. OGTT was performed at 10 weeks, ITT at 11 weeks, and sampling at 12 weeks. Other experiments were performed after the sampling. HFCS: High-fructose corn syrup; OGTT: oral glucose tolerance test; ITT: insulin tolerance test.
Oral glucose tolerance test (OGTT). The measurement was performed using Medisafe Fit blood glucose meter (TERUMO Co., Tokyo, Japan) by fasting mice for 6 h and subsequently administering glucose orally at 2.0 g/kg (body weight). Blood glucose levels were measured 0 (immediately before glucose administration), 15, 30, 60, and 120 min via a cut in the tail. Simultaneously, to obtain serum, blood collected from the tail vein using a heparinized capillary tube was centrifuged (1,700 × g, room temperature, 15 min) and stored at −30°C until measurement. The serum insulin level was measured using a mouse insulin ELISA kit (FUJIFILM Wako Pure Chemical Co., Osaka, Japan).
Insulin tolerance test (ITT). The measurement was performed in a nonfasted state, and insulin (Humulin R; Eli Lilly Japan K.K., Kobe, Hyogo, Japan) was intraperitoneally administered at a dosage of 2.0 U/kg (body weight). Blood glucose levels were measured using the same method as the OGTT.
Tissue weight and serum parameter measurement. Following a 6-h fast, glucose was orally administered to mice at a dose of 2.0 g/kg (body weight). After 60 min, the mice were anesthetized with isoflurane (Abbott Japan LLC., Tokyo, Japan) and euthanized by cardiac blood collection. The pancreas, liver, epididymal, and subcutaneous adipose tissues of the inguinal area were isolated, and the weight of each tissue was measured. Following measurements, only the pancreas segment was quickly frozen in liquid nitrogen and stored at −80°C until used in experiments. To obtain serum, the collected blood was centrifuged at 1,700 × g for 15 min at room temperature and stored at −30°C until measurement. Triglyceride (TG), total cholesterol (T-Cho), uric acid (UA), blood urea nitrogen (BUN), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total protein (TP) levels in the serum were measured using Fuji DRI-CHEM NX700V (FUJIFILM Co., Tokyo, Japan). Serum leptin levels were measured using a mouse leptin ELISA kit (Abcam plc, Cambridge, UK).
Immunohistochemical analyses. The pancreas was extracted and immediately fixed in 10% (w/v) formalin to prevent autodigestion. The paraffin-embedded pancreas was cut into 3 μm-thick sections. After deparaffinization, the sections were incubated with the anti-insulin mouse monoclonal antibody (Abcam plc) in 10% skimmed milk overnight at 4°C. The sections were rinsed in Dulbecco’s phosphate buffered saline with Tween-20 three times for 5 min, and incubated with goat anti-rabbit immunoglobulin horseradish peroxidase (Agilent Technologies, Inc., Santa Clara, CA, USA) for 60 min at room temperature. After rinsing again, immune complexes were visualized by Simple Stain DAB Solution (Nichirei Corp., Tokyo, Japan). Then, after rinsing with distilled water, the nuclei were stained with Carrazzi’s hematoxylin solution (FUJIFILM Wako Pure Chemical Co.). The total pancreas sections and insulin-positive areas, were measured using the image-analysis software ImageJ (https://imagej.net/ij/, accessed on 30 December 2023). The insulin-positive areas were expressed as a percentage of the total pancreas section areas.
RNA isolation and quantitative PCR (qPCR). Pancreases that had been preserved in a deep-freeze were quickly pulverized at 4°C using Lysing Matrix D (MP Biomedicals Germany GmbH, Eschwege, Germany). Total RNA was extracted using ISOGEN II (Nippon Gene Co., LTD, Tokyo, Japan). cDNA was synthesized from 5.0 μg of the total RNA using ReverTra Ace (Toyobo Inc., Osaka, Japan). mRNA expression levels were quantified using Luna Universal qPCR Master Mix (New England Biolabs, Inc., Ipswich, MA, USA) and specific primer sets (Table I) through real-time PCR using AriaMx Real-Time PCR System (Agilent Technologies, Inc.). The expression ratio relative to the control was calculated using the ΔΔCt method with the housekeeping gene 18S rRNA to standardize the data.
List of primer sequences.
Statistical analysis. All experiments were conducted with n=6-7 mice, and the mean values with ±SEM were presented in the graphs. Data analysis was performed using Microsoft Excel for Microsoft 365 MSO (Version 2201) (Microsoft Co., Redmond, WA, USA). Data were statistically analyzed using Student’s t-test or Welch’s t-test. The significance level was set at p<0.05.
Results
Weekly energy intake and its nutrient ratio. The HFCS group was provided with 10% HFCS water ad libitum for 12 weeks, and the control group was provided with deionized water. Both groups were fed with a normal diet (5L37), and the intake amount of HFCS and the normal diet were measured daily for 12 weeks. The HFCS group was pair-fed so that the energy intake was the same as that of the control group (Figure 2A). In the HFCS group, HFCS energy accounted for more than 50% of the total energy intake over 12 weeks (Figure 2B). The ratio of the three major nutrients in the normal diet was 58.4% carbohydrates, 29.6% protein, and 12.0% fat, and the nutritional ratio ingested by the control group was same to this ratio. In contrast, the HFCS group was 81.3% carbohydrate, including 26.2% HFCS, 13.3% protein, and 5.4% fat (Figure 2C). The HFCS group had a nutritionally unbalanced diet. Serum leptin levels were measured at time points immediately before or 60 min following glucose administration (Figure 2D). The leptin concentration immediately before oral administration was 1.74-fold higher in the HFCS group than that in the control group, and 1.98-fold higher than that in the control group 60 min following administration; however, the difference was not statistically significant.
Energy intake and leptin secretion. (A) Energy intake per week in the HFCS (white triangle) and control groups (black circle). (B) In the HFCS group, the energy intake of feed (white) and HFCS (black) per week is shown. (C) In the HFCS and control groups, the ratio of HFCS, carbohydrate, protein, and lipid intake for 12 weeks is shown. (D) Serum leptin concentration immediately before OGTT (0 min) and 60 min following glucose administration. All data are expressed as means±SEM. n=6-7 mice. HFCS: High-fructose corn syrup; OGTT: oral glucose tolerance test.
Effects of excessive HFCS intake under energy restriction on obesity. Body weights were measured for 12 weeks to evaluate the induction of obesity by excessive HFCS intake under energy restriction. No significant difference in body weight was observed between the two groups (Figure 3A). Additionally, the body weight and BMI after 6 h of fasting at the end of the breeding period were not significantly different (Figure 3B). The weights of epididymal and subcutaneous adipose tissues increased by 1.18- and 1.37-fold in the HFCS group than those in the control group, respectively; however, no significantly difference was noted. Conversely, the liver weight was decreased 0.86-fold that of the control group, which was statistical significantly lower in the HFCS group (Figure 3C). In the histological observation, no differences were observed in the number of adipocytes in the liver, adipocyte size, and morphological changes in the epididymal and subcutaneous adipose tissues (data not shown). These results indicated that excessive HFCS intake under energy restriction did not induce obesity.
No excessive HFCS intake under energy restriction induces obesity. (A) Weekly body weight of the HFCS (white triangle) and control groups (black circle). (B) Body weights and BMI at the end of the breeding period. (C) Epididymal and subcutaneous adipose tissue and liver weight. All data are expressed as means±SEM. n=6-7 mice *p<0.05 vs. control (Student’s t-test). HFCS: High-fructose corn syrup; BMI: body mass index.
Effects of excessive HFCS intake under energy restriction on blood biochemical parameters. Serum TG, T-Cho, UA, BUN, AST, ALT, and TP levels were measured to evaluate the effects of excessive HFCS intake under energy restriction on blood biochemical parameters. The HFCS group had significantly lower BUN and ALT levels; however, no differences were noted in the other parameters (Table II).
Serum biochemical parameters.
Effects of excessive HFCS intake under energy restriction on glucose tolerance. OGTT was performed 6 h after fasting to evaluate the effects of excessive HFCS intake under energy restriction on glucose tolerance. After 30 min of oral glucose administration, the HFCS group showed significantly increased blood glucose levels compared with the control group (Figure 4A). To evaluate insulin responsiveness in the OGTT, serum insulin levels were measured 0 (immediately before), 30, and 60 min following oral glucose administration; however, no significant differences were noted between the two groups (Figure 4B). Furthermore, ITT was performed under nonfasting conditions to determine whether there was insulin resistance. Blood glucose levels 60 min following the intraperitoneal injection of insulin were significantly lower in the HFCS group than those in the control group (Figure 4C). These suggested that excessive HFCS intake under energy restriction induced IGT.
OGTT and ITT. (A) Changes in blood glucose levels in OGTT of the HFCS (white triangle) and control groups (black circle). (B) Changes in serum insulin concentration in OGTT. (C) Changes in blood glucose levels in ITT. All data are expressed as means±SEM. n=6-7 mice *p<0.05 vs. control (Student’s t-test). OGTT: Oral glucose tolerance test; ITT: insulin tolerance test; HFCS: High-fructose corn syrup.
Effects of excessive HFCS under energy restriction on the pancreas. To investigate the cause of IGT, the effects on the pancreas were evaluated. The HFCS group had significantly lower pancreatic weights than the control group (Figure 5A). However, the percentage of insulin-positive area and the islet morphology in the pancreas were not significantly different between the two groups (Figure 5B). Subsequently, the mRNA expression levels of insulin II (Ins2) and pancreatic and duodenal homeobox 1 (Pdx-1) were measured to examine the insulin secretion-related genes. The mRNA expression of Ins2 in the HFCS group was 0.34-fold that in the control group, which was significantly decreased. Pdx-1 expression in the HFCS group was 0.71-fold that in the control group; however, it was not statistically significant (Figure 5C). The mRNA expression levels of glucose transporter 2 (Glut2) and its downstream enzymes, glucokinase (Gck) and ketohexokinase (Khk), were measured to examine the effects of glucose metabolism-related genes (Figure 5D). Their mRNA expressions were not significantly different between the two groups. These results suggest that excessive HFCS intake under energy restriction induced the decreases in pancreatic weights and Ins2 expression levels.
Effects of HFCS excess on the pancreas. (A) Pancreatic weight. (B) Percentages of insulin immunostaining-positive area per pancreatic tissue section and insulin immunostaining images. Bar=20 μm (C) mRNA expressions of insulin II (Ins2), and pancreatic and duodenal homeobox 1 (Pdx1) (D) mRNA expressions of glucose transporter 2 (Glut2), glucokinase (Gck), and ketohexokinase (Khk). n=6-7 mice **p<0.001, *p<0.05 vs. control (Ins2: Welch’s t-test, all others: Student’s t-test). HFCS: High-fructose corn syrup.
Discussion
This study examined the effects of excessive HFCS drink intake under energy restriction in early middle-aged mice. Although the mice were not obese, IGT was induced. The pancreatic weights and Ins2 mRNA expression levels were decreased, indicating its negative effects on the pancreas. Glucotoxicity refers to the detrimental effects of sustained hyperglycemia on cells and tissues. Prolonged exposure to high glucose levels can lead to β-cell dysfunction and damage (29, 30). This dysfunction impairs the ability of the pancreas to effectively secrete insulin (29). Furthermore, high glucose levels over an extended period can contribute to insulin resistance development (31). The effects of glucotoxicity vary between children and adults. Generally, both age groups are affected; however, the specific mechanisms differ owing to various factors. The growth period requires significant amounts of energy and nutrients for physical development (32); however, adults who have passed the growth period do not require significant amounts of energy.
In this study, early middle-aged mice were fed with an unbalanced diet of low protein, low fat, and high carbohydrate; however, the TP levels in the blood did not decrease and were not different from that of the control group (Table II). This result differs from that observed in growing mice (21). When fed with an unbalanced diet, BUN and ALT levels decreased in both growth age and early middle-aged groups; however, TP levels decreased in the growth age groups. Moreover, its influences on pancreatic β-cells differed between growth and early middle-aged. Although the Ins2 mRNA expression was significantly decreased in the early middle-aged group, no significant differences were noted in glucose metabolism-related genes compared with the control group (Figure 5). However, in the case of growth age, the Pdx-1, Glut2, Gck, and Khk expressions decreased (21). The effects of glucotoxicity on β-cells could be less pronounced in early middle-aged than those in the growth age. A high-fructose diet activates endoplasmic reticulum (ER) stress in the pancreas (33), which causes decreased Ins2 expression (34). Additionally, Akita mice with Ins2 gene mutations are known to exhibit hyperglycemia and β-cell apoptosis, which is believed to be mediated by ER stress (35). Although further studies are needed, it is possible that excessive HFCS intake causes β-cell overload, thereby resulting in chronic ER stress, which in turn reduces Ins2 expression and induces IGT.
In case of low fructose, it is converted into glucose and organic acids in the small intestine and is nearly completely metabolized. However, in case of excessive fructose that cannot be completely digested in the small intestine, it is transported to the liver for digestion (36). Fructose is metabolized in the liver, and TGs are easily produced during this process (37). Excessive fructose intake may promote fat accumulation and increase obesity risk (14). However, our results indicated that excessive HFCS intake under energy restriction does not increase weight gain and induce adipose tissue accumulation (Figure 3). Additionally, no differences were observed in lipid parameters compared with the control group (Table II). These results were similar to those observed in growing mice (21). Excessive fructose, sucrose and soft drink intake induce nonalcoholic fatty liver disease (NAFLD) (16, 38). A long-term high-fat, high-cholesterol, high-fructose diet induces NAFLD as well as nodular regenerative hyperplasia in mice (39). However, our results showed that the liver weights were significantly lower in the HFCS group (Figure 3), and cell morphology including steatosis was not changed (data not shown). The decrease in ALT levels is believed to indicate that nutritional balance is inappropriate, such as vitamin B6 deficiency (40). We believe that excessive HFCS intake leads to obesity when the total energy intake exceeds the total daily energy consumption. However, since our experiments focused on intake investigation for a short period of 12 weeks, it is essential to examine intake effects of HFCS in longer period settings.
Moreover, we believed that the leptin levels might be reduced owing to the extremely unbalanced diet wherein most of the energy intake was HFCS dependent. However, the opposite was true. Although no statistical difference was observed, the HFCS group showed an almost two-fold increase in leptin secretion compared with the control group. The reason for this is unknown. It is common in daily life that if we continue to be in a situation where our energy intake is low, we become accustomed to that situation. This may also have an impact on diabetes development in individuals without obesity. As diabetes can develop even if a person is not obese, it is significant to not only pay attention to total energy intake but also to eat a well-balanced diet.
This study focused on the early middle-aged group as the target age range, and provided excessive HFCS drink to 28-week-old mice for 12 weeks. We postulated that the breeding period of these mice corresponded to the period in early middle-aged humans (41-43). Several studies have investigated different methods for correlating the age of mice with humans (41, 42, 44). However, not all of these methods could accurately define the absolute age. Mice have a shorter lifespan than humans, and the maturational rate of mice does not linearly correlate with humans (41, 42). Thus, the exact correlation between mouse and human age remains unclear. Lifestyle factors adopted in middle-aged humans are reported to influence health and functional status in older age (45, 46). As the effects of dietary habits rarely cause immediate problems to the body, investigating them from a long-term perspective is necessary. In the present study, we observed the animals for 12 weeks; however, we believe that an even longer observation is needed. Moreover, estrogen, one of the female hormones, is known to increase insulin sensitivity (47) and prevents insulin deficiency in animal studies (48). Therefore, we expect that the effects of excessive HFCS drink intake under energy restriction differ not only by age, but also by sex. As our study used male mice, the effects of excessive HFCS drink intake on females are unknown. In the future, using female mice to clarify sex-related differences in the effects of excessive HFCS drink intake is required.
Conclusion
The excessive HFCS drink intake under energy restriction did not induce obesity; however, it induced impaired glucose tolerance, indicating its negative effects on the pancreas in early middle-aged mice. As there were no changes in lipid parameters or accumulation of adipose tissue, obesity seems to be related to excessive energy intake rather than excessive HFCS intake. Additionally, while the management of total daily energy intake is important, an imbalance in the sources and types of energy intake adversely affects metabolism. Even if one does not become obese, it affects the metabolic mechanism, and the effects may vary depending on age.
Acknowledgements
The Authors thank Ms. Kurogi for technical assistance with the histological and immunohistochemical analyses.
Footnotes
Authors’ Contributions
HH and YH designed the study; MH and YO performed the experiments; MH and YO analyzed the data; HK advised on histological technique; MH and HH wrote and revised the manuscript; HK and YH reviewed the manuscript. All Authors contributed to the manuscript preparation and approved of the published version of the manuscript.
Conflicts of Interest
The Authors have no relationship with, or financial interest in any commercial companies pertaining to this article.
Funding
This work was supported by JSPS KAKENHI Grant Number (22K11779).
- Received December 1, 2023.
- Revision received January 7, 2024.
- Accepted January 17, 2024.
- Copyright © 2024, 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).
