Abstract
Background/Aim: The global prevalence of type 2 diabetes (T2D) continues to rise, with non-obese phenotypes particularly common in East Asian populations. Our previous study in male mice demonstrated that the intake of excessive high-fructose corn syrup (HFCS) under energy restriction impairs glucose tolerance without inducing obesity. This study aimed to elucidate female-specific mechanisms underlying glucose regulation under similar dietary conditions.
Materials and Methods: Early middle-aged female ICR mice were randomly assigned to either the HFCS group or the control group. In the HFCS group, mice were given free access to HFCS water, and the energy intake was adjusted to be the same as that in the control group using a standard rodent diet. After 16 weeks, glucose tolerance and insulin sensitivity were assessed via the oral glucose tolerance and insulin tolerance tests, respectively. Pancreatic morphology, gene expression, and serum biochemical and hormonal parameters were analyzed.
Results: Glucose tolerance and islet size distribution were comparable between the HFCS and control groups. The HFCS group, however, exhibited lower insulin secretion and reduced pancreatic weight relative to controls. mRNA levels of insulin II, pancreatic and duodenal homeobox 1, glucose transporter 2, and glucokinase were similar between groups, whereas ketohexokinase mRNA tended to be elevated in the HFCS group. In addition, the mRNA levels of glutaminase and glutamate dehydrogenase 1 were higher than those in controls. Serum leptin and insulin-like growth factor I showed upward trends in the HFCS group, and glucagon-like peptide-1 levels were significantly increased compared with controls.
Conclusion: Excessive HFCS intake under energy restriction diminished insulin secretion but preserved glucose tolerance in female mice, which might be attributable to extrapancreatic hormonal compensation and adaptive metabolic responses.
Introduction
The continually rising global prevalence of diabetes mellitus is a major international health concern (1, 2). Approximately 90% of diabetes mellitus cases are classified as type 2 diabetes (T2D), a condition whose pathogenesis involves genetic predisposition, obesity, dietary patterns, lifestyle factors, and others (3-6). Obesity induces insulin resistance through adipose tissue hypertrophy and promotes chronic inflammation, thereby impairing β-cell function (7). In contrast, among Asian populations, T2D is considerably prevalent in non-obese individuals (8, 9). Impaired insulin secretory capacity has been identified as a primary pathogenic factor in such cases (8, 10). Thus, although classified under the same disease entity, the mechanisms underlying T2D development vary across regions, diets, and physical variabilities (11).
High-fructose corn syrup (HFCS) is widely used as a liquid sweetener in soft drinks as a substitute for sucrose. Recent studies have suggested that the consumption of HFCS-containing beverages contributes to the onset of T2D and obesity (12, 13). Previously, we investigated the effects of excessive HFCS intake in male mice under controlled energy consumption, simulating a dietary pattern characterized by high intake of sweetened beverages without balanced nutrition (14, 15). We found that excessive HFCS intake did not induce obesity when total energy intake was restricted. However, impaired glucose tolerance (IGT) was observed, and the underlying mechanisms varied with age. Growing male mice exhibited reduced mRNA expression of pancreatic and duodenal homeobox 1 (Pdx1), glucose transporter 2 (Glut2), glucokinase (Gck), and ketohexokinase (Khk) (14), whereas early middle-aged male mice showed a down-regulation of only insulin II (Ins2) (15). These findings suggested that the pathogenesis of IGT might differ by age.
The incidence of T2D is generally higher in men than in women; however, women are more predisposed to this condition after menopause (16, 17). This phenomenon might be related to the effects of estrogen, which enhances insulin secretion and sensitivity (16-18). Therefore, we hypothesized that the pathological mechanisms of diabetes might vary by sex. Nevertheless, relevant studies specifically targeting female models remain fewer than those targeting male models. To better understand the disease mechanisms in females, investigations employing female animals are warranted.
In this study, we investigated the effects of excessive HFCS intake under energy restriction in early middle-aged female mice. Building on our previous findings in male models, this study aimed to elucidate female-specific aspects of IGT under non-obese conditions.
Materials and Methods
Animals. The animal experiments in this study were approved by the Ethics Committee of Miyazaki University (Miyazaki, Japan; No. 2022-2), and the protocols were consistent with the committee’s guidelines for animal care. 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]. After acclimatizing to the breeding environment, 28-week-old mice were randomly divided into two groups and given free access to either deionized water (control group, n=12) or 10% HFCS (55% fructose and 45% glucose) (HFCS-55, Nihon Shokuhin Kako Co., Ltd., Tokyo, Japan) water (HFCS group, n=11). The intake of 10% HFCS-water was measured daily, and the energy intake was adjusted to be the same as that in the control group using a standard rodent diet (Rodent Lab Diet EQ 5L37, Japan SLC, Inc.; 3.38 kcal/g). Body weight was measured weekly and over the 16-week breeding period. The study protocol is depicted in Figure 1A.
Energy intake and changes in body weight. (A) Experimental protocol. In the high-fructose corn syrup (HFCS) group, the daily intake of HFCS water was measured, and energy intake was adjusted to match that of the control group using a standard feed. (B) Weekly energy intake. (C) Rate of energy intake from HFCS, carbohydrate, protein, and fat for 16 weeks. (D) Rate of intake of HFCS and standard feed. (E) Weekly body weight. (F) Weights of periovarian adipose tissue, subcutaneous adipose tissue, and liver. All data are expressed as mean±SEM (n=11-12). *p<0.05 and **p<0.01 vs. control.
Oral glucose tolerance test (OGTT). Blood glucose level was measured using the Medisafe Fit blood glucose meter (Terumo Co., Tokyo, Japan) after fasting mice for 6 h, and then 2.0 g/kg (body weight) glucose was orally administered. Blood glucose levels were measured at 0 (before the oral administration), 15, 30, 60, and 120 min post-treatment via a cut in the tail. At the same time points, to obtain serum, blood was collected from the tail vein using a heparinized capillary tube, centrifuged (1,700 × g, room temperature, 15 min), and stored at −30°C until further analysis.
Insulin tolerance test (ITT). Insulin tolerance was measured in the non-fasted state. Insulin (Humulin R; Eli Lilly Japan K.K., Kobe, Hyogo, Japan) was intraperitoneally administered at a dosage of 1.0 U/kg (body weight). Blood glucose levels were measured using the same method employed for OGTT.
Measurement of tissue weight and serum parameters. After 6 h of fasting, glucose was orally administered to the mice at a dose of 2.0 g/kg (body weight). After 15 min, the animals were anesthetized with isoflurane (Abbott Japan LLC., Tokyo, Japan) and euthanized by cardiac blood collection. The pancreas, liver, periovarian, and subcutaneous adipose tissues of the inguinal area were isolated, and their weights were measured. After measurement, a pancreatic segment was quickly frozen in liquid nitrogen for quantitative PCR (qPCR), and fixed in 10% (w/v) formalin solution for histological analysis. To obtain serum, the collected blood was centrifuged (1,700 × g, room temperature, 15 min) and stored at −30°C until further analysis. Triglyceride (TG), total cholesterol (T-Cho), high-density lipoprotein cholesterol (HDLC), 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).
Measurement of serum hormone concentrations. Serum levels of insulin, 17β-estradiol (E2), adiponectin, leptin, insulin-like growth factor 1 (IGF-1), and glucagon-like peptide-1 (GLP-1) were measured using a mouse insulin ELISA kit (FUJIFILM Wako Shibayagi Co., Gunma, Japan), 17 beta estradiol ELISA kit (Abcam plc, Cambridge, UK), adiponectin ELISA kit (R&D Systems, Minneapolis, MN, USA), mouse leptin ELISA kit (Abcam plc, Cambridge, UK), mouse/rat IGF-1 ELISA kit (R&D Systems), and Human/Mouse/Rat GLP-1 EIA kit (RayBiotech, Inc., Peachtree Corners, GA, USA), respectively.
Histological analysis. The paraffin-embedded pancreas tissue was cut into 3 μm thick sections. Deparaffinized sections were immersed in Carrazzi’s hematoxylin solution (Fujifilm Wako Pure Chemical Co., Osaka, Japan) and eosin solution (Sakura Finetek Japan Co., Tokyo, Japan). The stained sections were then observed under an inverted microscope IX73 (Olympus Co., Tokyo, Japan). The islet area was assessed using an image analysis software, ImageJ. Islet sizes were defined as follows: small, S (<0.01 mm2); medium, M (0.01-0.05 mm2); and large, L (0.05-0.1 mm2) (19). The islet density was calculated as the number of islets per pancreatic section and calculated as follows: (number of each islet size / total number of islets) × 100.
Total RNA isolation and qPCR. Pancreatic tissue frozen at −80°C was quickly ground at 4°C using Lysing Matrix D (MP Biomedicals Germany GmbH, Eschwege, Germany). Total RNA was isolated using ISOGEN II (Nippon Gene Co., LTD, Tokyo, Japan). cDNA was synthesized using ReverTra Ace (Toyobo Inc., Osaka, Japan), and mRNA levels were determined using Luna Universal qPCR Master Mix (New England Biolabs, Inc., Ipswich, MA, USA) and relevant primer sets (Table I) through real-time PCR using the AriaMx Real-Time PCR System (Agilent Technologies Inc., Santa Clara, CA, USA). 18S rRNA of the housekeeping gene was used for standardization, and relative expression levels were determined using the 2ΔΔ−Ct method.
List of primers for qPCR.
Statistical analysis. All experiments were conducted with 10-12 mice, and the data are presented as the means ± standard error of the mean (SEM). All data analyses were 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 threshold for statistical significance was set at p<0.05.
Results
Dietary composition, energy intake, and body weight. HFCS water intake was monitored daily (Figure 1A), and energy intake of the HFCS group was adjusted to match that of the control group using a standard rodent diet. Consequently, total energy intake was maintained at comparable levels between the groups throughout the 16-week period (Figure 1B). The macronutrient composition of the standard chow for control mice was 58.4% carbohydrates, 29.6% protein, and 12% fat. In contrast, the HFCS group received a diet composed of 90.0% carbohydrates (including 76.0% HFCS), 7.1% protein and 2.9% fat (Figure 1C). The weekly proportion of HFCS intake relative to total energy intake was quantified (Figure 1D). During the experiment, the body weight in the HFCS group fell below that of the control group once HFCS accounted for approximately 80% of total intake (Figure 1E). Moreover, measurements of periovarian adipose tissue, subcutaneous adipose tissue, and liver weights revealed no significant differences between the two groups (Figure 1F).
Effects of excessive HFCS intake under energy restriction on glucose tolerance and pancreatic morphology. The effect of excessive HFCS intake under energy restriction on glucose tolerance was assessed. OGTT revealed that blood glucose levels did not differ significantly between the HFCS and control groups (Figure 2A). In contrast, insulin secretion was lower in the HFCS group than in the control group (Figure 2B). ITT showed similar glucose levels in both groups (Figure 2C). However, pancreatic weight was lower in the HFCS group than in the control group (Figure 2D). Histopathological examination of the pancreas revealed no differences in the total area of islets within pancreatic sections, and islet size distribution did not differ significantly between the two groups (Figure 2E).
Oral glucose tolerance test (OGTT), insulin tolerance test (ITT), and pancreatic morphology. (A) Changes in blood glucose levels assessed via OGTT. (B) Changes in serum insulin levels assessed via OGTT. (C) Changes in blood glucose levels assessed via ITT. (D) Weight of the pancreas. (E) Representative photomicrographs of hematoxylin and eosin-stained pancreatic sections and histological analyses. Islet sizes were defined as follows: Small, S (<0.01 mm2); medium, M (0.01-0.05 mm2); large, L (0.05-0.1 mm2). Scale bar=100 μm. (F) Proportion of each size in total pancreatic islets per pancreatic tissue section. All data are expressed as mean±SEM (n=11-12). **p<0.01 and ***p<0.001 vs. control.
Expression of pancreatic genes related to insulin production and glucose metabolism. To clarify the mechanisms underlying diminished insulin secretion in the HFCS group despite preserved glucose tolerance, genes related to pancreatic insulin production and glucose metabolism were analyzed. The expression levels of Ins2, Pdx1, Glut2, and Gck were comparable between the two groups; however, Khk expression tended to be elevated in the HFCS group (Figure 3A and B).
Effects of excessive high-fructose corn syrup (HFCS) intake on the pancreas. Relative gene expression levels of (A) insulin II (Ins2) and pancreatic and duodenal homeobox 1 (Pdx1), (B) glucose transporter 2 (Glut2), glucokinase (Gck), and ketohexokinase (Khk) are presented. All data are expressed as mean±SEM (n=11-12).
Expression of genes related to amino acid metabolism in the pancreas. Because amino acid metabolism–including the glutamine and glutamate pathways–contributes to insulin secretion (20-22), the expression of amino acid metabolism-related genes was examined. The mRNA expression of the amino acid transporter SLC38A3 did not differ significantly between the HFCS and control groups. In contrast, the genes encoding downstream enzymes glutaminase (Gls) and glutamate dehydrogenase 1 (Glud1) were significantly up-regulated in the HFCS group compared to the control group (Figure 4).
mRNA expression levels of amino acid metabolism-related genes in the pancreas; comparison between high-fructose corn syrup (HFCS) intake and control group. Amino acid transporter SLC38A3, glutaminase (Gls), and glutamate dehydrogenase 1 (Glud1). All data are expressed as the mean±SEM (n=11-12). **p<0.01 and ***p<0.001 vs. control.
Serum parameters and hormonal regulation of glucose homeostasis. To investigate extrapancreatic factors potentially involved in maintaining normal blood glucose levels despite reduced insulin secretion, serum parameters and hormones were analyzed. BUN and ALT levels were decreased, and T-Cho and HDLC levels were increased in the HFCS group compared with the control group (Table II). E2 and adiponectin levels did not differ significantly between groups, whereas leptin and IGF-1 levels tended to be higher, and GLP-1 levels were significantly higher in the HFCS group than in controls (Figure 5).
Serum biochemical parameters in the control and high-fructose corn syrup (HFCS) group.
Serum hormone levels. 17β-Estradiol (E2), adiponectin, leptin, insulin-like growth factor 1 (IGF-1), and glucagon-like peptide-1 (GLP-1) levels in the control and high-fructose corn syrup (HFCS) intake group are shown. All data are expressed as the mean±SEM (n=10-12). *p<0.05 vs. control.
Discussion
In this study, we investigated the effects of excessive HFCS intake under energy restriction in early middle-aged female mice. Despite reduced insulin secretion, blood glucose levels were maintained, indicating that extrapancreatic mechanisms likely compensate to sustain glucose tolerance.
Similar to the findings of our previous studies conducted in male mice, excessive HFCS intake under energy restriction did not induce obesity (14, 15). The HFCS group exhibited reduced serum BUN and ALT levels (Table II), potentially reflecting the impact of a low-protein, low-fat, high-carbohydrate diet with insufficient vitamins. These findings were consistent with those observed in male mice (15). Although excessive energy intake is widely recognized as a major risk factor for lifestyle-related diseases (23), and obesity prevention has been emphasized, our results suggest that even under energy restriction and non-obesity, an unbalanced diet can still exert adverse effects on metabolic health.
At the pancreatic level, the expression of Ins2, Pdx1, Glut2, and Gck remained unchanged, whereas Khk expression tended to increase (Figure 3), implying the selective activation of fructose-related metabolic pathways. In addition, Gls and Glud1 were significantly up-regulated in the HFCS group (Figure 4), suggesting that glutamine/glutamate metabolism might contribute to β-cell adaptation. Pancreatic atrophy can result from inflammation, disease, aging, and other causes (24, 25). In the HFCS group, pancreatic weight was reduced (Figure 2D); however, there was no significant inflammatory cell infiltration, adipocyte infiltration, or fibrosis, and islet size remained unchanged (Figure 2E). We suggest that excessive HFCS intake under energy restriction does not directly impair pancreatic function but rather leads to adaptive changes in metabolic pathways specific to female mice.
Serum E2 concentrations were also assessed. Estrogen is known to exert protective effects on insulin secretion and sensitivity (16-18); therefore, it may contribute to the maintenance of glucose tolerance in premenopausal females. Although our data did not indicate a direct role of E2 fluctuations, the basal hormonal environment might provide a background for compensatory mechanisms.
The HFCS groups produced a marked increase in GLP-1, and a tendency for IGF-1 and leptin to increase, whereas adiponectin remained at levels comparable to those in controls (Figure 5). GLP-1 is secreted from intestinal L cells and suppresses the rise in blood glucose by promoting insulin secretion and delaying gastric emptying (26, 27). IGF-1 contributes to enhanced insulin sensitivity and the maintenance of β-cell function (28, 29), and leptin promotes energy expenditure and augments insulin action in skeletal muscle (30, 31). Adiponectin exerts metabolic and antiinflammatory actions that may protect against the development of diabetes (31, 32). The rise in GLP-1 is plausibly attributable to the rapid delivery of HFCS derived monosaccharides to the small intestine, which in turn stimulates L cells. Such an increase in GLP-1 could functionally compensate for reduced insulin secretion via an enhanced incretin effect. In addition, the IGF-1 may have supported insulin sensitivity and β-cell function. The observed trend toward higher leptin despite similar adipose tissues weights suggests either increased leptin secretion per unit adipocyte or contributions from non-adipose tissues sources, such as the gastric mucosa (33). The increase in T-Cho and HDLC despite no significant difference in TG is considered to reflect qualitative alterations in lipoprotein metabolism induced by excessive HFCS intake; however, the fact that mice lack cholesteryl ester transfer protein should also be considered (34). Moreover, sex-differences are known in the secretion and sensitivity of insulin, GLP-1, leptin, and adiponectin (33, 35-37). These findings indicate that excessive HFCS intake under energy restriction elicits metabolic adaptations in females that differ from those in males, and that such female specific compensatory responses may help maintain glucose tolerance despite reduced insulin secretion, thereby influencing whole body glucose homeostasis and systemic metabolism.
Study limitations. First, the study focused on female mice at an early middle-aged stage; however, it remains unclear whether similar compensatory mechanisms operate under conditions of reduced estrogen secretion. In rodents, circulating estrogen does not decline with age as in humans (38). Therefore, experimental models with ovariectomy or pharmacologically suppressed estrogen secretion must be evaluated. Second, we did not evaluate the estrous cycle. Future studies should incorporate cycle monitoring to strengthen the interpretation of sex-specific findings.
Conclusion
Excessive HFCS intake under energy restriction reduced insulin secretion but preserved glucose tolerance in female mice, possibly through extrapancreatic hormonal compensation and coordinated adaptive responses. This phenotype contrasted with the prevailing emphasis on caloric excess as the primary driver of metabolic disease and suggested that diet composition per se can shape glucose regulation even when total energy is constrained. These findings build upon our previous observations in male models and provide additional insights into the sex-dependent mechanisms of glucose regulation. Our results might contribute to a better understanding of non-obese T2D, which is relatively prevalent in Asian populations, and inform risk assessment and preventive strategies targeting lifestyles characterized by high consumption of HFCS-rich beverages.
Acknowledgements
The Authors thank Ms. Kurogi for technical assistance with the histological and immunohistochemical analyses.
Footnotes
Authors’ Contributions
MH and HH designed the study; AN and MH performed the experiments and analyzed the data; YS advised on histological technique; AN and HH wrote and revised the manuscript; HH 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).
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, Microsoft Copilot was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine-learning-based image enhancement tools.
- Received November 30, 2025.
- Revision received December 12, 2025.
- Accepted December 17, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
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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