Development of a Hyperglycemic Fish Model and Analysis of Bone Metabolism

Abstract

The high plasma glucose induced in glucose metabolism disorders leads to secondary pathologies, including bone disease. Fish scales, similar to mammalian bone, are composed of osteoblasts, osteoclasts, and calcified bone matrix and have been used as a system to analyze hyperglycemia-induced bone abnormalities. Here, we developed a hyperglycemia model in fish to study abnormalities in bone metabolism linked to increased plasma glucose and to analyze the function of calcitonin, the suppressor of osteoclastic activity, while maintaining high glucose levels. Following a 1-d fast and exposure to 5% glucose, plasma glucose concentrations increased significantly. We then examined plasma calcium and osteoclast activity of scales related to bone metabolism in goldfish treated with glucose for 5 d after a 1-d fast. The results showed that glucose treatment significantly increased plasma calcium levels at 3 and 5 d with a decrease in calcium content in the scales of goldfish. Hyperglycemia in glucose-exposed goldfish induced osteoclastic activation in scales, as indicated by the ratio of the osteoclastic activating factor (rankl) to the osteoclast inhibiting factor (osteoprotegerin, opg). Plasma calcitonin was found to be increased in glucose-exposed goldfish, which appears to suppress bone resorption by regulating the rankl/opg ratio. This hyperglycemia model, capable of examining both glucose and bone metabolism, would be valuable for analyzing the mechanism underlying abnormal bone metabolism caused by hyperglycemia.

INTRODUCTION

Diabetes mellitus (DM), commonly known as diabetes, encompasses a group of metabolic diseases defined by chronic hyperglycemia. DM is categorized based on its etiology: either insufficient insulin production by the pancreas (type 1 DM: T1DM) or the body’s cells (muscle, fat, etc.) failing to respond properly to the insulin produced (type 2 DM: T2DM).¹⁾ T2DM is projected to affect over 300 million people by 2025, and currently, 1 in 10 children globally is obese or grossly overweight.²⁾ Moreover, DM leads to other pathologies, including bone disease, diabetic retinopathy, nephropathy, neuropathy, cardiomyopathy, and microangiopathy.¹⁾ In patients with T1DM, reduced bone mineral density (BMD) increases the risk of fracture and osteoporosis.¹⁾ Conversely, T2DM patients exhibit comparable or even higher BMD than healthy individuals.³⁾ Consequently, the mechanism underlying T2DM-induced bone defects, such as fractures and osteoporosis, remains unclear.

Calcitonin, a calcium-regulating hormone that inhibits osteoclastic activity, is used therapeutically for osteoporosis.⁴⁾ Recent reports indicate that calcitonin also regulates energy balance^5–8) and glucose homeostasis,^7,9,10) in addition to its role in bone metabolism. Human studies show that salmon calcitonin inhibits gastric emptying and gastrin release post-meal while dose-dependently relaxing the gallbladder.^5,6) In chronic studies, oral salmon calcitonin reduced food intake and body weight in rat models of obesity and diabetes.^7,8) Oral salmon calcitonin includes the carrier compound N-(5-chlorosalicyloyl)-8-aminocaprylic acid, which protects calcitonin from the acidic gastrointestinal environment and lowers plasma glucose levels compared with the vehicle.⁷⁾ Therefore, calcitonin, particularly fish calcitonin known for its strong affinity with the calcitonin receptor,¹¹⁾ is a therapeutic candidate for T2DM.

In mammals, exogenous calcitonin elicits a better response in diabetic parameters like glucose, insulin, HbA1c, and body weight, likely due to low endogenous calcitonin levels.^4,12,13) Consequently, analyses of calcitonin’s effect on hyperglycemia in mammals have primarily focused on administered calcitonin,¹⁰⁾ and its overall function in mammalian hyperglycemia is not yet fully elucidated. Fish, which can be fasted for a week or more,¹⁴⁾ are suitable models for studying physiological responses after fasting. Here, we developed a hyperglycemia model in goldfish to analyze calcitonin function together with bone metabolism while considering the effects of fasting. We examined the increase in plasma glucose levels following glucose treatment after fasting and analyzed the associated abnormalities in bone metabolism.

MATERIALS AND METHODS

Ethical Treatment of Animals

This study adhered strictly to the ethical guidelines of Kanazawa University. All experimental protocols were approved by the Animal Welfare Committee of Kanazawa University (AP24-010) and were in strict accordance with the ARRIVE guidelines 2.0.¹⁵⁾ Maximum effort was made to avoid causing pain and distress to the experimental animals. Pain was minimized during biological sample collection through preoperative anesthesia, with particular attention paid to its use to alleviate discomfort.

Animals

Goldfish (Carassius auratus) (Supplementary Fig. S1) were purchased from Higashikawa Fish Farm (Yamatokoriyama, Nara, Japan). They were acclimated in freshwater at 25°C for approximately 1 week before being used for experiments. For biological sample collection, fish were anesthetized with MS-222 (Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.) neutralized with sodium bicarbonate.

Determination of Optimal Glucose Exposure Concentration

Goldfish were exposed to glucose solutions ranging from 1% to 5% because goldfish did not survive in a 6% glucose solution in our preliminary experiments. After 1 d of rearing, blood was collected from the caudal blood vessel under anesthesia (MS-222, Sigma-Aldrich) using a heparinized syringe. Plasma was separated by centrifugation (5200 × g for 3 min), and the plasma glucose concentration was measured using a commercial kit (LabAssay™ Glucose, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).

Determination of Optimal Fasting Period

Goldfish were fasted for 1, 2, 4, or 6 d before a 1-d exposure to a 5% glucose solution. Following the glucose exposure, the goldfish were anesthetized with MS-222 (Sigma-Aldrich), and blood was collected using a heparinized syringe. Plasma was separated by centrifugation (5200 × g for 3 min), and the plasma glucose concentration was measured with a kit (LabAssay™ Glucose, FUJIFILM Wako Pure Chemical Corporation).

Analysis of Plasma Glucose and Calcium Concentrations after Fasting and Glucose Treatment

Goldfish were fasted and subsequently exposed to a 5% glucose solution for 5 d. Blood samples were collected from different groups of goldfish at specific time points: 1 d before glucose exposure; just before glucose exposure (day 0); and at 1, 3, and 5 d after glucose exposure. At each time point, goldfish were anesthetized with MS-222 (Sigma-Aldrich), and blood was collected from the caudal vessels using a heparinized syringe. Plasma was separated by centrifugation (5200 × g for 3 min). Plasma glucose concentration was then measured using the LabAssay™ Glucose kit (FUJIFILM Wako Pure Chemical Corporation), and plasma calcium levels were measured with the AQUAAUTO Ca kit (KAINOS Laboratories, Inc., Tokyo, Japan).

Analysis of Plasma Calcitonin Levels

Plasma calcitonin levels were examined in individual goldfish before and after glucose treatment. The competitive enzyme-linked immunosorbent assay (ELISA) procedure for calcitonin was based on the method described by Suzuki,¹⁶⁾ with modifications. In this study, we used amino group-coated plates (ELISA Plate Amino, Sumitomo Bakelite Co., Ltd., Tokyo, Japan), which allowed us to reduce the preincubation period from 3 to 1 d. In separate glass tubes, 250 µL of diluted antibody (1 : 300000 in diluting solution: 10 mM phosphate-buffered solution containing 0.1% bovine serum albumin and 0.1% NaN₃, pH 7.4) was preincubated with the same volume of serially diluted synthetic salmon calcitonin or plasma samples (diluted 1 : 3 with diluting solution) for 1 d at 4°C. Subsequently, plates coated with salmon calcitonin in sodium bicarbonate buffer were washed four times with washing buffer (10 mM phosphate-buffered saline containing 0.05% Tween 20). Aliquots (100 µL) of the preincubated mixtures were then transferred to the coated plates and incubated at 15°C for 24 h. Color development was carried out after four additional washes with washing buffer. A total of 8 mg of o-phenylenediamine dihydrochloride was added to 12 mL of a solution containing 0.1 M citric acid and 0.2 M Na₂HPO₄ (pH 4.5) and 2.4 µL of 30% hydrogen peroxide (FUJIFILM Wako Pure Chemical Corporation). Aliquots of 100 µL of this solution were added to the wells and incubated with constant agitation at room temperature for 5 min. The reaction was stopped by adding 50 µL of 3 N sulfuric acid. The optical density of the triplicate samples was subsequently measured at 492 nm using a microplate reader (MTP-500, Corona Electric Co. Ltd., Tokyo, Japan). A standard curve was constructed using serially diluted synthetic salmon calcitonin standards in Excel (Microsoft Office, Microsoft Corporation, San Francisco, CA, U.S.A.) to determine the concentration of calcitonin in the experimental sample.

The specificity of the rabbit anti-salmon calcitonin serum (No. 626, Cosmo Bio Co., Ltd., Tokyo, Japan) was evaluated using parathyroid hormone peptide (1–34) (bovine) and human calcitonin gene-related peptide.¹⁶⁾ Because of the high homology (91%) between calcitonin in goldfish and salmon calcitonin,¹⁷⁾ this antibody has been used to investigate changes in plasma calcitonin levels in goldfish.¹⁸⁾

Analysis of Osteoclastic and Osteoblastic Enzyme Marker Activities

Tartrate-resistant acid phosphatase (TRAP) and alkaline phosphatase (ALP) activities, serving as markers for osteoclasts and osteoblasts, respectively, were measured with goldfish scales following glucose exposure as described by Suzuki et al.¹⁹⁾ Goldfish scales, containing both osteoclasts (bone-resorbing) and osteoblasts (bone-forming cells)^20–22) (Supplementary Fig. S1), served as the material for analyzing bone metabolism postglucose exposure.

To measure TRAP activity, 100 µL of acid buffer (20 mM tartrate in 0.1 M sodium acetate buffer, pH 5.3) was added to each well of a 96-well microplate. After glucose exposure, individual scales taken under anesthesia were then placed in separate wells. The microplate was snap-frozen at −80°C and stored at −20°C until use. After thawing, 100 µL of 20 mM para-nitrophenyl phosphate in acid buffer was added to each well. The plate was incubated at 20°C for 20 min with continuous shaking. The reaction was stopped by adding 50 µL of 3 N NaOH solution. An amount of 150 µL of the resulting solution was transferred to a new plate, and absorbance was measured at 405 nm. The amount of produced para-nitrophenol (pNP) was determined using a standard curve. ALP activity was determined under the same conditions as the TRAP assay, with the sole modification being the use of an alkaline buffer system (100 mM Tris–HCl, pH 9.5; 1 mM MgCl₂; 0.1 mM ZnCl₂).

Following the measurement of TRAP and ALP activities, each scale’s size was determined using ImageJ (National Institutes of Health, MD, U.S.A.). These activities were then normalized to the scale’s surface area (cm²).

Analysis of Scale Calcium Concentrations after Fasting and Glucose Treatment

Goldfish were fasted for 1 d and subsequently exposed to 5% glucose solution for 5 d. Scale samples were collected from control and experimental groups of goldfish just prior to glucose exposure (day 0), and at 1, 3, and 5 d following glucose exposure under anesthesia with MS-222 (Sigma-Aldrich). Scale calcium content [mg/dry weight (mg) of scale] was measured using the AQUAAUTO Ca kit (KAINOS Laboratories, Inc., Tokyo, Japan) on a microplate reader after dissolving the dried scale (60°C, 12 h) in nitric acid and neutralization by NaOH.

Analysis of Osteoclast-Osteoblast Gene Interactions in Goldfish Scales

To understand the osteoblast–osteoclast interaction after glucose exposure, we examined the mRNA expression level of the receptor activator of NF-κB ligand (rankl) and osteoprotegerin (opg) in goldfish scales.²³⁾

Following glucose exposure, goldfish scales were collected under anesthesia and placed into 2 mL screw-cap tubes. Each tube contained three 3.0 mm zirconium oxide beads and 400 µL of the provided lysis buffer. Scales were homogenized using a µT-12 bead crusher (Taitec Corporation, Saitama, Japan) at 3000 rpm for 60 s. Total RNA was extracted from the lysate with a total RNA purification kit (NucleoSpin RNA II, TaKaRa Bio Inc., Shiga, Japan). Genomic DNA contamination was removed by treatment with DNase I (RNase-Free DNase Kit, TaKaRa Bio Inc.) for 15 min at room temperature. cDNA was subsequently synthesized using the PrimeScript™ RT Master Mix (Perfect Real Time) (TaKaRa Bio, Inc.).

Quantitative real-time PCR (qPCR) was conducted using a real-time PCR apparatus (QuantStudio™ 1 Real-Time PCR Instrument, Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, U.S.A.).²⁴⁾ Specific primers for the target genes, listed in Supplementary Table S1, were utilized. The annealing temperature for the amplification of rankl, opg, and the housekeeping elongation factor-1α (ef1α) was set at 60°C. The expression level of each target mRNA was normalized to the expression level of ef1α mRNA on the basis of the DeltaDeltaCt method.

Statistical Analysis

Statistical analyses were conducted using R software (v4.3.1, R Core Team, Vienna, Austria). Data were initially assessed for normal distribution using the Shapiro–Wilk test. For comparing two groups, the equality of variances was evaluated with a Levene’s test.

The Student’s t-test was applied for data with equal variances, while Welch’s t-test was used for data with significantly different variances. Nonparametric data that did not show a normal distribution were analyzed using the Mann–Whitney U-test or Wilcoxon signed-rank test.

Multiple comparisons were performed with the Steel test or two-way ANOVA followed by Tukey test. In all analyses, a significance level of p < 0.05 was selected.

RESULTS

Environmental Glucose Influence on Goldfish Plasma

Plasma glucose concentrations of goldfish maintained in rearing water containing 1–5% concentration of glucose for 1 d are presented in Fig. 1. Rearing goldfish in water containing glucose resulted in a concentration-dependent increase in plasma glucose levels. Significant differences were observed in all of glucose treatment groups compared with the control group without glucose treatment. Maintaining goldfish at a 5% glucose concentration led to the seven times increase in plasma glucose concentration.

Fig. 1. Plasma Glucose Concentrations in Goldfish after a 1-d Rearing in Different Glucose Solutions

* and ** denote statistically significant differences from the control: *p < 0.05 and **p < 0.01, n = 6–8 per group. The p-values for the control group without glucose and the groups with 1, 2, 3, 4, and 5% glucose addition were 0.049, 0.0036, 0.0036, 0.0036, and 0.029, respectively.

Influence of Fasting Period on Goldfish Plasma Glucose

Following the results in Fig. 1, a 5% glucose concentration was used for subsequent experiments. We investigated the impact of fasting for 1, 2, 4, and 6 d on plasma glucose concentrations in goldfish exposed to 5% glucose for 1 d (Fig. 2). Plasma glucose was elevated after a 1-d fast followed by 5% glucose exposure. Although plasma glucose also increased after 2 and 4 d of fasting, levels were higher on fasting day 1. After 6 d of fasting, glucose concentration increased to the same level as after 1 d of fasting. Owing to large variations between individuals, no significant changes were observed during the fasting period.

Fig. 2. Plasma Glucose Concentrations in Goldfish by 1-d Glucose (5%) Exposure after Different Fasting Periods

*, ** and *** denote statistically significant differences from the control: *p < 0.05, **p < 0.01, and ***p < 0.001. Control: n = 6–9; Experimental groups: n = 7–9. The p-values for the control group without glucose and the groups addition with 5% glucose at 0, 1, 2, 4, and 6 d were 0.0013, 0.00079, 0.00079, 0.0019, and 0.028, respectively.

Goldfish Plasma Glucose Changes after 1-d Fast and 5% Glucose Exposure

As shown in Fig. 2, plasma glucose concentrations significantly increased in goldfish fasted for 1 d and then exposed to 5% glucose. We examined changes in plasma glucose concentration under these conditions for 5 d (Fig. 3). Goldfish exposed to 5% glucose for 5 d after a 1-d fast showed a marked increase in plasma glucose. Plasma glucose levels rose sharply after 1 d of exposure and continued to increase gradually over the following days.

Fig. 3. Plasma Glucose Concentrations in Goldfish by Glucose (5%) Exposure after a 1-d Fast

Plasma glucose concentrations at 1 d before fasting are also shown as a reference. ** and *** denote significant differences from the control: **p < 0.01 and ***p < 0.001. Control: n = 7 or 8; Experimental groups: n = 10 or 11. The p-values for the control group without glucose and the groups addition with 5% glucose at 1, 3, and 5 d were 0.0057, 0.000045, and 0.0012, respectively.

Changes in Plasma Calcium Concentration after 1-d Fast and 5% Glucose Exposure

As shown in Fig. 4, plasma calcium concentrations in goldfish increased significantly for 5 d of exposure to a 5% glucose solution after a 1-d fast. No significant changes in plasma calcium were observed after 1 d of glucose exposure. However, at 3 and 5 d postexposure, plasma calcium concentrations were significantly elevated in the glucose-exposed group compared with nonglucose-exposed controls.

Fig. 4. Plasma Calcium Concentrations in Goldfish by Glucose (5%) Exposure after a 1-d Fast

Plasma calcium concentrations at 1 d before fasting are also shown as a reference. *** denotes a significant difference from the control (p < 0.001). Control: n = 7 or 8; Experimental groups: n = 10 or 11. The p-values for the control group without glucose and the groups addition with 5% glucose at 1, 3, and 5 d were 0.66, 0.00024, and 0.00059, respectively.

Changes in Plasma Calcitonin Concentration before and after 5% Glucose Exposure Following a 1-d Fast

Results for changes in plasma calcitonin concentration are indicated in Fig. 5. We compared plasma calcitonin levels before and after glucose treatment in the same individuals. Plasma calcitonin levels significantly increased after 5 d of 5% glucose exposure.

Fig. 5. Plasma Calcitonin Concentrations in Goldfish before and after 5% Glucose Exposure Following a 1-d Fast

* denotes a significant difference from the control (p < 0.05). n = 7 per group. The p-value for the control group without glucose and the group's addition with 5% glucose at 5 d was 0.046.

Changes in TRAP and ALP Activities in Scales after 1-d Fast and 5% Glucose Exposure

TRAP activity in the scales significantly increased after 1 d of glucose exposure (Fig. 6a). However, at 5 d of glucose exposure, TRAP activity decreased to the level of the control group (Fig. 6b), coinciding with the increase in calcitonin (Fig. 5). In the case of ALP activity, glucose exposure induced a decline of 1 d following exposure (p = 0.06) (Fig. 7a). ALP activity recovered to the control level at 5 d after exposure (Fig. 7b).

Fig. 6. TRAP Activity in Goldfish Scales after a 1-d Fast and Subsequent Glucose (5%) Exposure

(a) Activity after 1 d of exposure. (b) Activity after 5 d of exposure. * denotes a significant difference from control scales (p < 0.05). n = 9 for (a); n = 6 for (b). The p-values for the control group without glucose and the group’s addition with 5% glucose at 1 and 5 d were 0.0049 and 0.5571, respectively.

Fig. 7. ALP Activity in Goldfish Scales after a 1-d Fast and Subsequent Glucose (5%) Exposure

(a) Activity after 1 d of exposure. (b) Activity after 5 d of exposure. n = 8 or 9 for (a); n = 6 for (b). The p-values for the control group without glucose and the group’s addition with 5% glucose at 1 and 5 d were 0.060 and 0.68, respectively.

Changes in Scale Calcium Contents after 1-d Fast and 5% Glucose Exposure

Calcium content in goldfish scales did not change at 1 and 3 d following 5% glucose treatment compared with that in control goldfish scales (Fig. 8). Subsequently, at 5 d following glucose treatment, the calcium content of goldfish scales was significantly lower than that of the control group (Fig. 8).

Fig. 8. Changes in Scale Calcium Contents after a 1-d Fast and Subsequent Glucose (5%) Exposure

** denotes a significant difference from the control scales (p < 0.01). n = 8. The p-values for the control group without glucose and the group’s addition with 5% glucose at 0. 1, 3, and 5 d were 0.99, 0.96, 0.97, and 0.0061, respectively.

mRNA Expression Changes of Osteoblast-Osteoclast Interaction Factors in Goldfish Scales

One day after glucose exposure, both rankl and opg expression significantly decreased (Figs. 9a, 9b), but their ratio remained unchanged (Fig. 9c). By day 3 of glucose exposure, rankl expression showed an upward trend (Fig. 10a), while opg expression trended downward (Fig. 10b). This resulted in a significant elevation of the rankl/opg ratio (Fig. 10c). After 5 d of glucose exposure, the trends in rankl and opg mRNA expression were similar to those at day 3 (Fig. 10). In the case of rankl mRNA expression, the rate of increase declined (Fig. 11a). However, the opg mRNA expression continued to show a decrease (Fig. 11b). A significant decline in opg mRNA expression at 5 d after glucose exposure was obtained as compared with nonglucose-exposed controls (Fig. 11b). There was no significant difference in the rankl/opg ratio because of the reduced rate of increase in rankl mRNA expression (Fig. 11c).

Fig. 9. Changes in rankl and opg mRNA Expression and their Ratio in Goldfish Scales after a 1-d Fast and Subsequent 1 d of Glucose (5%) Exposure

(a) rankl expression. (b) opg expression. (c) rankl/opg ratio. * indicates a statistically significant difference from the control scales (p < 0.05). Control group: n = 7; Experimental group: n = 7. The p-values for the control group without glucose and the group’s addition with 5% glucose for rankl and opg expressions and rankl/opg ratio were 0.037, 0.012, and 0.69, respectively.

Fig. 10. Changes in rankl and opg mRNA Expression and Their Ratio in Goldfish Scales after a 1-d Fast and Subsequent 3 d of Glucose (5%) Exposure

(a) rankl expression. (b) opg expression. (c) rankl/opg ratio. * denotes a significant difference from the control scales (p < 0.05). Control group: n = 7; Experimental group: n = 7. The p-values for the control group without glucose and the group's addition with 5% glucose for rankl and opg expressions and rankl/opg ratio were 0.12, 0.30, and 0.034, respectively.

Fig. 11. Changes in rankl and opg mRNA Expression and Their Ratio in Goldfish Scales after a 1-d Fast and Subsequent 5 d of Glucose (5%) Exposure

(a) rankl expression. (b) opg expression. (c) rankl/opg ratio. * denotes a significant difference from the control scales (p < 0.05). Control group: n = 7; Experimental group: n = 8. The p-values for the control group without glucose and the group’s addition with 5% glucose for rankl and opg expressions and rankl/opg ratio were 0.52, 0.031, and 0.70, respectively.

DISCUSSION

This study successfully created a hyperglycemic state in goldfish by rearing them solely in a glucose solution, avoiding chemical treatments. Fasting was effective for hyperglycemia induction, with a 1-d fast promoting faster environmental glucose uptake than other tested fasting durations (0, 2, and 4 d). Elevated plasma glucose levels correlated with increased plasma calcium concentrations, potentially stimulating calcitonin secretion. Consistent with this, we previously reported the presence of the calcium-sensing receptor in the goldfish ultimobranchial gland, the fish calcitonin secretory organ.²⁵⁾ Therefore, increased plasma calcium likely triggered calcitonin release via this receptor. Glucose exposure also tended to decrease ALP activity (an osteoblastic marker) in goldfish scales (p = 0.06). As DM is associated with bone defects like fracture and osteoporosis,^1,3) the bone resorption observed in this hyperglycemic goldfish model mirrors a pathology seen in diabetic conditions.

We previously reported that alloxan treatment induced hyperglycemia in insulin-deficient goldfish.²⁶⁾ However, TRAP and ALP activity remained unchanged in the hyperglycemic goldfish. Because glucose was absorbed from the environmental water instead of being administered as a drug, we imply that in the present study, bone resorption of scales could be induced in goldfish with hyperglycemia by increasing osteoclast activity. Therefore, the hyperglycemic goldfish developed in this study can serve as a good model for diabetes analysis.

The sodium–glucose co-transporter is closely associated with the onset of diabetes, and research on its inhibitors is actively underway.^27–29) When glucose and melatonin were administered into the abdominal cavity of goldfish, melatonin enhanced glucose uptake.³⁰⁾ These findings suggest that glucose transporters may exist in goldfish, as in mammals. We propose that fasting activates glucose transporters, thereby increasing plasma glucose levels.

The RANKL/OPG ratio is a critical regulator of bone resorption and formation balance in mammals.^31,32) Remarkably, DM patients exhibit significantly lower plasma OPG and higher serum RANKL levels compared with healthy individuals.³¹⁾ Therefore, we explored the role of the rankl/opg ratio in goldfish bone resorption. Exposure to 5% glucose for 3 d significantly increased the rankl/opg ratio compared with the controls. As calcitonin directly inhibits osteoclasts (Suzuki, 2021), TRAP activity (an osteoclastic marker) decreased at 5 d after glucose exposure likely due to increased plasma calcitonin levels. Additionally, since calcitonin has been reported to inhibit the Rankl mRNA expression in human bone osteosarcoma epithelial cells (U2OS Line),³³⁾ it is likely that calcitonin decreased the rankl mRNA expression on day 5 of glucose exposure in goldfish. Collectively, our results indicate that hyperglycemia-induced bone resorption in goldfish, similar to humans, involves the RANKL/OPG signaling pathway.^31,32)

In the present study, both TRAP and ALP activities returned to normal levels 5 d after glucose exposure. TRAP activity seems to be associated with calcitonin function. On the contrary, changes in ALP activity may be related to procalcitonin amino-terminal cleavage peptide (N-proCT).³⁴⁾ Accordingly, we identified a novel function of sardine (Sardinops melanostictus) calcitonin in osteoblasts.³⁴⁾ We previously reported that sardine procalcitonin comprises N-proCT, calcitonin, and procalcitonin carboxyl-terminal cleavage peptide.³⁴⁾ In that study, we found that sardine N-proCT (10⁻⁷ M) induced ALP activity, whereas sardine calcitonin had no effect on ALP activity.³⁴⁾ In rats, thyroid levels of calcitonin and N-proCT increase in parallel in vivo,³⁵⁾ suggesting that N-proCT may play a role in osteoblasts in restoring normal conditions in hyperglycemic goldfish.

Fish scales serve as a relevant model for mammalian bone, containing osteoblasts, osteoclasts, and a calcified matrix with type I collagen^20,22) (Supplementary Fig. S1). Our previous work focused on scale type I collagen, analyzing its glycation in goldfish.³⁶⁾ This was prompted by the known stimulation of type I collagen glycation by high glucose in DM patients.^37,38) Advanced glycation end products (AGEs), resulting from nonenzymatic glucose-dependent protein modification, are vital risk factors for diabetic complications. Consequently, we developed an in vitro bioassay with goldfish scales to study type I collagen glycation and detected AGEs-induced crosslinking via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE).³⁶⁾ Future plans include examining AGEs-mediated type I collagen crosslinking in the scales of goldfish under in vivo hyperglycemic conditions.

This study focused on calcitonin, demonstrating that hyperglycemia stimulates its secretion. Elevated calcitonin may help suppress the increased plasma calcium levels also observed. In goldfish as well as mammals, parathyroid hormone is known to promote osteoclastogenesis via rankl expression,³⁹⁾ and prostaglandin E₂ (PGE₂) induces hypercalcemia and increases rankl expression.⁴⁰⁾ These factors suggest multiple contributors to hyperglycemia-induced hypercalcemia in vivo. Since serum RANKL is elevated in human DM patients,³¹⁾ other hypercalcemic hormones like parathyroid hormone and PGE₂ may contribute to hypercalcemia in our goldfish model.

In bony fish, including goldfish, the Brockmann body is a structure that plays a crucial role in insulin production, and its physiological function is similar to that of the islets of Langerhans (pancreatic islets) in mammals.^26,41) For example, tilapia Brockmann bodies have been successfully transplanted into diabetic mice, effectively maintaining normal blood glucose levels.⁴¹⁾ By contrast, bony fish have lower glucose tolerance than mammals²⁶⁾ and exhibit sustained hyperglycemia after glucose intake. Therefore, in this study, we developed a hyperglycemia animal model by utilizing the innate hypoglycemia tolerance of goldfish.

Mammalian bones are present inside the body and have a complex internal structure comprising canals called osteons. By contrast, goldfish scales are found on the surface of the body and have a simple layered structure.^20–22) Despite these differences, mammalian bones and goldfish scales both comprise osteoblasts, osteoclasts, osteocytes, and calcified bone matrix, and are involved in regulating blood calcium levels.^21,22) Furthermore, calcitonin is used as an inhibitor of osteoclast activity in both mammalian bones and goldfish scales.^4,25) Because goldfish scales are located on the surface of the body, it is possible to collect them without killing the hyperglycemic goldfish and perform multiple analyses of calcitonin function related to bone cells in the same individual. We plan to conduct a more detailed analysis of calcitonin signaling via the calcitonin receptor and its role in glucose uptake using this model in future.

CONCLUSION

We developed an animal model of hyperglycemia by simply rearing goldfish in water containing glucose without the use of chemicals. It should be emphasized that a 1-d fast prior to glucose treatment was necessary to efficiently induce hyperglycemia. In hyperglycemic fish, plasma calcium level was elevated. Increased plasma calcium was linked to increased activity of osteoclast in the scales. Plasma calcitonin was also found to be increased in the hyperglycemic goldfish model. We speculate that the elevated calcitonin decreases the osteoclast activity and suppresses abnormal bone metabolism induced by hyperglycemia.

Acknowledgments

This study was supported in part by Grants to Nobuo Suzuki (Grant-in-Aid for Scientific Research [C] No. 23K10933 by JSPS), to Yoshiaki Tabuchi (Grant-in-Aid for Scientific Research [C] No. 23K11802 by JSPS), and to Kouhei Kuroda (Support for Pioneering Research Initiated by the Next Generation, No. JPMJSP2135 by JST).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

REFERENCES

• 1)  Wu YY, Xiao E, Graves DT. Diabetes mellitus related bone metabolism and periodontal disease. Int. J. Oral Sci., 7, 63–72 (2015).
• 2)  Preshaw PM. Periodontal disease and diabetes. J. Dent., 37, S575–S577 (2009).
• 3)  Wongdee K, Charoenphandhu N. Update on type 2 diabetes-related osteoporosis. World J. Diabetes, 6, 673–678 (2015).
• 4)  Suzuki N.Calcitonin. Handbook of Hormones: Comparative Endocrinology for Basic and Clinical Research, 2nd edition (Ando H., Ukena K., Nagata S. eds) Vol. 1, Academic Press, New York, United States, pp. 405–408 (2021).
• 5)  Jonderko K. Effect of calcitonin on gastric emptying in patients with an active duodenal ulcer. Gut, 30, 430–435 (1989).
• 6)  Jonderko K, Jonderko G, Gołab T. Effect of calcitonin on gastric emptying and on serum insulin and gastrin concentrations after ingestion of a mixed solid-liquid meal in humans. J. Clin. Gastroenterol., 12, 22–28 (1990).
• 7)  Feigh M, Henriksen K, Andreassen KV, Hansen C, Henriksen JE, Beck-Nielsen H, Christiansen C, Karsdal MA. A novel oral form of salmon calcitonin improves glucose homeostasis and reduces body weight in diet-induced obese rats. Diabetes Obes. Metab., 13, 911–920 (2011).
• 8)  Feigh M, Hjuler ST, Andreassen KV, Gydesen S, Ottosen I, Henriksen JE, Beck-Nielsen H, Christiansen C, Karsdal MA, Henriksen K. Oral salmon calcitonin enhances insulin action and glucose metabolism in diet-induced obese streptozotocin-diabetic rats. Eur. J. Pharmacol., 737, 91–96 (2014).
• 9)  Nakamura M, Nomura S, Yamakawa T, Kono R, Maeno A, Ozaki T, Ito A, Uzawa T, Utsunomiya H, Kakudo K. Endogenous calcitonin regulates lipid and glucose metabolism in diet-induced obesity mice. Sci. Rep., 8, 17001 (2018).
• 10)  Mathiesen DS, Lund A, Vilsbøll T, Knop FK, Bagger JI. Amylin and calcitonin: potential therapeutic strategies to reduce body weight and liver fat. Front. Endocrinol., 11, 617400 (2021).
• 11)  Hilton JM, Dowton M, Houssami S, Sexton PM. Identification of key components in the irreversibility of salmon calcitonin binding to calcitonin receptors. J. Endocrinol., 166, 213–226 (2000).
• 12)  Polymeris A, Papapetrou PD, Papandroulaki F, Thanou S. Hyperinsulinemia during oral glucose tolerance test and high normal serum cortisol are associated with increased secretion of calcitonin in normal subjects. Hormones, 10, 304–312 (2011).
• 13)  Feigh M, Andreassen KV, Neutzsky-Wulff AV, Petersen ST, Hansen C, Bay-Jensen AC, Henriksen JE, Beck-Nielsen H, Christiansen C, Henriksen K, Karsdal MA. Oral salmon calcitonin attenuates hyperglycaemia and preserves pancreatic beta-cell area and function in Zucker diabetic fatty rats. Br. J. Pharmacol., 167, 151–163 (2012).
• 14)  Gaucher L, Vidal N, D’Anatro A, Naya DE. Digestive flexibility during fasting in the characid fish Hyphessobrycon luetkenii. J. Morphol., 273, 49–56 (2012).
• 15)  Percie du Sert N, Ahluwalia A, Alam S, et al. Reporting animal research: explanation and elaboration for the ARRIVE guidelines 2.0. PLoS Biol., 18, e3000411 (2020).
• 16)  Suzuki N. Calcitonin-like substance in the plasma of Cyclostomata and its putative role. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 129, 319–326 (2001).
• 17)  Suzuki N, Ueda K, Sakamoto H, Sasayama Y. Fish calcitonin genes: primitive bony fish genes have been conserved in some lower vertebrates. Gen. Comp. Endocrinol., 113, 369–373 (1999).
• 18)  Suzuki N, Yamamoto K, Sasayama Y, Suzuki T, Kurokawa T, Kambegawa A, Srivastav AK, Hayashi S, Kikuyama S. Possible direct induction by estrogen of calcitonin secretion from ultimobranchial cells in the goldfish. Gen. Comp. Endocrinol., 138, 121–127 (2004).
• 19)  Suzuki N, Kitamura K, Omori K, et al. Response of osteoblasts and osteoclasts in regenerating scales to gravity loading. Biol. Sci. Space, 23, 211–217 (2009).
• 20)  Suzuki N, Somei M, Seki A, Reiter RJ, Hattori A. Novel bromomelatonin derivatives as potentially effective drugs to treat bone diseases. J. Pineal Res., 45, 229–234 (2008).
• 21)  Yamamoto T, Ikegame M, Hirayama J, Kitamura K, Tabuchi Y, Furusawa Y, Sekiguchi T, Endo M, Mishima H, Seki A, Yano S, Matsubara H, Hattori A, Suzuki N. Expression of sclerostin in the regenerating scales of goldfish and its increase under microgravity during space flight. Biomed. Res., 41, 279–288 (2020).
• 22)  Hirayama J, Hattori A, Takahashi A, Furusawa Y, Tabuchi Y, Shibata M, Nagamatsu A, Yano S, Maruyama Y, Matsubara H, Sekiguchi T, Suzuki N. Physiological consequences of space flight, including abnormal bone metabolism, space radiation injury, and circadian clock dysregulation: implications of melatonin use and regulation as a countermeasure. J. Pineal Res., 74, e12834 (2023).
• 23)  Kuroda K, Srivastav AK, Suzuki A, Rafiuddin MA, Toyota K, Endo M, Honda M, Watanabe K, Maruyama Y, Tabuchi Y, Hattori A, Urata M, Matsubara H, Suzuki N. Stanniocalcin in the corpuscles of Stannius inhibits the osteoclastic activation by regulating the Rankl/Opg expression. J. Biol. Regul. Homeost. Agents, 37, 5141–5149 (2023).
• 24)  Thamamongood TA, Furuya R, Fukuba S, Nakamura M, Suzuki N, Hattori A. Expression of osteoblastic and osteoclastic genes during spontaneous regeneration and autotransplantation of goldfish scale: a new tool to study intramembranous bone regeneration. Bone, 50, 1240–1249 (2012).
• 25)  Kuroda K, Hatano K, Kawamura R, Fukushima A, Sasayama Y, Tabuchi Y, Furusawa Y, Ikegame M, Hattori A, Hirayama J, Fukuda T, Uekura S, Matsubara H, Kawago U, Sekiguchi T, Srivastav AK, Suzuki N. Possible involvement of Calcitonin I and II in calcium metabolism of the female reproductive physiology of goldfish (Carassius auratus). Int. Aquat. Res., 15, 15–26 (2023).
• 26)  Kitamura K, Andoh T, Okesaku W, Tazaki Y, Ogai K, Sugitani K, Kobayashi I, Suzuki N, Chen W, Ikegame M, Hattori A. Effects of hyperglycemia on bone metabolism and bone matrix in goldfish scales. Comp. Biochem. Physiol. A Mol. Integr. Physiol., 203, 152–158 (2017).
• 27)  Samukawa Y, Mutoh M, Chen S, Mizui N. Mechanism-based pharmacokinetic-pharmacodynamic modeling of luseogliflozin, a sodium glucose co-transporter 2 inhibitor, in Japanese patients with type 2 diabetes mellitus. Biol. Pharm. Bull., 40, 1207–1218 (2017).
• 28)  Takasu T, Yokono M, Tahara A, Takakura S. In vitro pharmacological profile of ipragliflozin, a sodium glucose co-transporter 2 inhibitor. Biol. Pharm. Bull., 42, 507–511 (2019).
• 29)  Iketani R, Imai S. Prescription trends for the antidiabetic agents used to treat type 2 diabetes mellitus in Japan from 2012–2020: a time-series analysis. Biol. Pharm. Bull., 46, 592–598 (2023).
• 30)  Watanabe K, Nakano M, Maruyama Y, Hirayama J, Suzuki N, Hattori A. Nocturnal melatonin increases glucose uptake via insulin-independent action in goldfish brain. Front. Endocrinol., 14, 1173113 (2023).
• 31)  Karalazou P, Ntelios D, Chatzopoulou F, Fragou A, Taousani M, Mouzaki K, Galli-Tsinopoulou A, Kouidou S, Tzimagiorgis G. OPG/RANK/RANKL signaling axis in patients with type I diabetes: associations with parathormone and vitamin D. Ital. J. Pediatr., 45, 161 (2019).
• 32)  Xing B, Yu J, Zhang H, Li Y. RANKL inhibition: a new target of treating diabetes mellitus? Ther. Adv. Endocrinol. Metab., 14, 20420188231170754 (2023).
• 33)  Mancini L, Paul-Clark MJ, Rosignoli G, Hannon R, Martin JE, Macintyre I, Perretti M. Calcitonin and prednisolone display antagonistic actions on bone and have synergistic effects in experimental arthritis. Am. J. Pathol., 170, 1018–1027 (2007).
• 34)  Kase Y, Ikari T, Sekiguchi T, Sato M, Ogiso S, Kawada T, Matsubara S, Satake H, Sasayama Y, Endo M, Kitamura KI, Hattori A, Watanabe TX, Maruyama Y, Watanabe Y, Funahashi H, Kambegawa A, Suzuki N. Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish. Comp. Biochem. Physiol. A Mol. Integr. Physiol., 211, 77–83 (2017).
• 35)  Burns DM, Birnbaum RS, Roos BA. A neuroendocrine peptide derived from the amino-terminal half of rat procalcitonin. Mol. Endocrinol., 3, 140–147 (1989).
• 36)  Kitamura K, Hirayama J, Tabuchi Y, Minami T, Matsubara H, Hattori A, Suzuki N. Glyoxal-induced formation of advanced glycation end-products in type 1 collagen decreases both its strength and flexibility in vitro. J. Diabetes Investig., 12, 1555–1559 (2021).
• 37)  Schleicher ED, Wagner E, Nerlich AG. Increased accumulation of the glycoxidation product N(epsilon)-(carboxymethyl)lysine in human tissues in diabetes and aging. J. Clin. Invest., 99, 457–468 (1997).
• 38)  Compston J. Type 2 diabetes mellitus and bone. J. Intern. Med., 283, 140–153 (2018).
• 39)  Suzuki N, Danks JA, Maruyama Y, Ikegame M, Sasayama Y, Hattori A, Nakamura M, Tabata MJ, Yamamoto T, Furuya R, Saijoh K, Mishima H, Srivastav AK, Furusawa Y, Kondo T, Tabuchi Y, Takasaki I, Chowdhury VS, Hayakawa K, Martin TJ. Parathyroid hormone 1 (1-34) acts on the scales and involves calcium metabolism in goldfish. Bone, 48, 1186–1193 (2011).
• 40)  Omori K, Wada S, Maruyama Y, et al. Prostaglandin E₂ increases both osteoblastic and osteoclastic activity in the scales and participates in calcium metabolism in goldfish. Zoolog. Sci., 29, 499–504 (2012).
• 41)  Wright JR Jr, Polvi S, MacLean H. Experimental transplantation with principal islets of teleost fish (Brockmann bodies). Long-term function of tilapia islet tissue in diabetic nude mice. Diabetes, 41, 1528–1532 (1992).