2025 Volume 50 Issue 12 Pages 661-675
To investigate the effects of reduced food intake on the serum and tissue levels of alkaline phosphatase (ALP) isoenzymes in rats, ALP1, ALP2, ALP3, and ALP5 were analyzed in serum, liver, bone, and small intestine of male and female Sprague-Dawley rats reared under a diet-restricted condition (fed 65% or 45% of the amount of the free-feeding group) for 4 weeks from six-week-old. In addition, to examine the effect of sex hormonal, each ALP isoenzyme level was also analyzed in free-feeding female rats with ovariectomy or testosterone administration. Food restriction was associated with the following changes: an increase of the serum ALP5 level in the females, mainly caused by an increase derived from the small intestine; increases in the liver ALP1 and small intestinal ALP5 levels in both sexes, which represented a compensatory increase of these ALP isoenzyme levels to enhance lipid absorption under the low nutritional condition; a decrease of the bone ALP3 level in females, which was considered reflective of suppressed bone formation. Regarding the sex differences, serum ALP2 and liver ALP1 levels were higher in the males than in the females; the levels in ovariectomized or testosterone-treated females shifted closer to those in the males. These results indicate that food restriction and sex hormonal intervention influenced the serum and tissue ALP isoenzyme levels in rats. These findings provide helpful information for understanding the effects of reduced food intake, often observed in toxicity studies, and sex differences in rats on ALP and help to properly evaluate the effects of test compounds.
Serum alkaline phosphatase (ALP) level is routinely measured in non-clinical toxicity studies as well as in clinical studies and is considered useful as a hepatobiliary or osteogenic marker. ALP is an enzyme that is widely distributed in cell membranes and is abundantly expressed in tissues such as the bone, small intestine, liver, and placenta. Several ALP isoenzymes have been recognized, and in humans, 6 isoenzymes (ALP1 to 6), which are altered by diseases in such as the liver and biliary system and bone, has led to the identification by electrophoresis (Iino, 1995). In non-clinical toxicity studies, analysis of the ALP isoenzymes provides us more accurate information about affected tissues by the test compound in addition to histopathological examination when a serum ALP level is elevated. In rats, which are commonly used in non-clinical toxicity studies, similar ALP isoenzyme separations are used as in humans. Although differences in the serum isoenzyme composition between rats and humans have been reported (Dziedziejko et al., 2005), there are few reports on the tissue-specific composition of ALP isoenzymes and their variations in both serum and tissues. Therefore, it is important to understand the physiological characteristics of the ALP isoenzymes, which are standard examination items in rats, for properly assessing the effects of the test compounds.
In toxicity studies, malnutrition with decreased food intake is often observed at toxic doses of the test compounds. By understanding the changes in the levels of the ALP isoenzymes in animals with decreased food intake, it would be easier to distinguish between a direct effect of the test compound and secondary changes due to reduced food intake. Variations in the levels of the ALP with food restriction have been reported in several animal species, including rats and dogs, but the variations vary between species, increasing in rats and decreasing in dogs (Schwartz et al., 1973; Levin et al., 1993; Seki et al., 1997; Hubert et al., 2000; Morita et al., 2017). In dogs, analysis of the ALP isoenzymes confirmed that the decrease in the serum ALP level was mainly due to a reduction of ALP3 level, which is bone-type ALP (Morita et al., 2017), but it is not clear which ALP isoenzyme is responsible for the increase in the serum ALP level in rats. In addition, there is little information on the sex differences in the serum and tissue levels of the ALP isoenzymes in rats, although sex differences in rat serum ALP have been known (Schwartz et al., 1973; Seki et al., 1997; Hubert et al., 2000; Kurata et al., 2003; Moriyama et al., 2008).
In this study, to obtain detailed information on ALP isoenzymes in rats, six-week-old male and female Sprague-Dawley (SD) rats were reared on a diet that was restricted in amount to 65% or 45% of that in the control rats that were reared on a normal amount of diet for 4 weeks. Data on the general examination items in toxicity studies were collected, and the several ALP isoenzyme levels (ALP1: high-molecular-weight-type ALP, ALP2: liver-type ALP, ALP3: bone-type ALP, and ALP5: intestinal-type ALP), which were considered to be related to the food restriction, in the serum and tissues, including the liver, femur, and small intestine, were analyzed. In addition, ALP enzyme histochemistry was performed in these tissues and the effect of food restriction on the tissue distribution of the ALP enzyme was also examined. In addition, the effects of experimental interventions on the sex hormone levels, such as ovariectomy or testosterone administration in female rats, on the levels of each ALP isoenzyme were analyzed.
Male and female SD rats were purchased from Jackson Laboratory Japan, Inc. (Kanagawa, Japan). Animals were individually housed in cages with bedding and a wood gnawing block in an air-conditioned animal room controlled at a room temperature of 20°C to 26°C, relative humidity of 30% to 70%, and an artificial lighting cycle of 12/12 hr (lights on from 7:15 to 19:15). Before the group assignment, all the rats were allowed free access to a rodent diet (MF, Oriental Yeast Co., Ltd., Tokyo, Japan) and drinking water. All the experimental procedures involving the animals performed with the approval of the Institutional Animal Care and Use Committee of Taisho Pharmaceutical Co., Ltd., and were in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan, 2006).
Four-week food restriction studySix-week-old male and female rats were divided into 3 groups (8 animals/sex/group) balanced according to the body weights. Rats in the free-feeding (FF) group, as the control group, were allowed free access to the diet for 4 weeks. Rats in the 65% food restriction (65%FR) group and 45%FR group were given 65% and 45% of the average amount of diet fed to the rats of the FF group so as to obtain slight and moderate changes of the body weight, respectively, based on the results of previous studies (Schwartz et al., 1973; Oishi et al., 1979; Ogawa et al., 1985; Levin et al., 1993; Seki et al., 1997; Hubert et al., 2000; Yoshii et al., 2003; Moriyama et al., 2008; Asanuma et al., 2011). The clinical signs and food consumption were monitored daily. Body weight and water consumption were measured 3 or 4 times a week and once or twice a week, respectively. Urinalysis was performed at week 4 of food restriction. At the end of the 4-week food restriction period, blood samples for hematological and blood chemistry examinations were collected from the abdominal aorta under isoflurane anesthesia after the rats were fasted overnight. After they were euthanized, the rats were necropsied, and the organ weights and bone lengths (femur, tibia, and sternum) were measured by a single person using a digital caliper (Mitsutoyo Corp., Kanagawa, Japan). Tissues were collected for histopathological examination, ALP enzyme histochemistry, and/or measurements of the tissue ALP isoenzyme levels.
Ovariectomy studySix-week-old female rats were divided into 2 groups (8 animals/group) balanced according to the body weights. The rats in the ovariectomy (OVX) group were surgically ovariectomized via a dorsal midline incision under isoflurane anesthesia, and the rats in the sham surgery (SS) group, as the control group, underwent sham surgery. For analgesia, the rats received 0.1 mg/kg of buprenorphine (Lepetan injection, Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan) administered subcutaneously once during the surgery and twice daily for 3 days after the surgery; in addition, the rats received 10 mg/head of cefazolin (Cefamezin α, Astellas Pharma Inc., Tokyo, Japan) administered intraperitoneally once after the surgery. The rats were allowed access to food ad libitum for 4 weeks and were subjected to the same examinations as the rats in the 4-week food restriction study described above.
Samples used to examine the effects of testosterone administration on the serum and liver ALP isoenzyme levels in the female ratsA portion of the serum and liver samples were transferred from another study designed to examine the effects of testosterone on the urinary parameters in female rats (our unpublished data). In the study, 5-week-old free-feeding female rats (6 animals) were administered testosterone (vehicle: corn oil) by subcutaneous injection at the dose level of 1 mg/kg/day for 16 days, followed by 2 mg/kg/day for 13 days (for total of 4 weeks). As the control group, 6 female rats were administered corn oil for 4 weeks. The rats were necropsied the day after the last dose, and the serum and tissues, including the liver, were collected.
UrinalysisQualitative and quantitative urinalysis were performed on cumulative 4-hr and 24-hr urine samples, respectively. The urinalysis parameters were determined using a urine chemistry analyzer (CLINITEK Advantus, Siemens Healthcare Diagnostics K.K., Tokyo, Japan), a digital urine specific gravity refractometer (UG-D, Atago Co., Ltd., Tokyo, Japan), and a clinical analyzer (7180, Hitachi High-Tech Corp., Tokyo, Japan).
Hematology and blood chemistryBlood samples were placed in EDTA (ethylenediamine tetraacetic acid)-2K-treated tubes for hematologic evaluation, in tubes supplemented with 3.2% sodium citrate for evaluation of coagulation parameters, in lithium heparin-treated tubes for the measurement of lactate dehydrogenase (LDH), and in serum separator tubes for the evaluation of blood chemistry parameters other than LDH. Sample tubes other than those treated with EDTA-2K were centrifuged (approximately 1950 ×g, 4°C, 15 min) to obtain plasma or serum. Hematological parameters were determined using a hematology system (ADVIA 2120i, Siemens Healthcare Diagnostics K.K., Tokyo, Japan), coagulation parameters were determined using an automated blood coagulation analyzer (CS-2000i, Sysmex Corp., Hyogo, Japan), blood chemistry parameters were measured using a clinical analyzer, and serum estradiol levels were measured using an Estradiol EIA kit (Cayman Chemical Company, Ann Arbor, MI, USA).
Histopathological examinationsThe testes and epididymides were fixed in a formalin-sucrose-acetic acid mixture and then immersed in 10% neutral buffered formalin. Eyeballs were pre-fixed in formaldehyde-glutaraldehyde solution and then immersed in 10% neutral buffered formalin. Femur, sternum, tibia, and spinal column were fixed in periodate-lysine-paraformaldehyde (PLP) solution under refrigeration and were decalcified with EDTA solution containing zinc sulfate (Lorch, 1947; Miao and Scutt, 2002). Organs other than the bone, eyeballs, testes, and epididymides were fixed in 10% neutral buffered formalin. After fixation, the tissues were embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically.
ALP enzyme histochemistryThe liver, duodenum, jejunum, ileum, femur, and tibia were fixed in PLP solution under refrigeration, and the femur and tibia were decalcified with EDTA solution containing zinc sulfate. The tissues were embedded in paraffin, stained with the TRAP/ALP staining kit (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), and examined microscopically.
Measurements of the ALP isoenzyme levelsThe bones (femur and tibia) were minced with scissors after washing off the bone marrow with 0.01M phosphate-buffered saline (PBS). The small intestine was collected after washing the lumen with PBS, such as that the duodenum, jejunum, and ileum were of similar weights. The liver, bone, and intestine samples were homogenized on ice using an ultrasonic homogenizer (UH-50, SMT Co., Ltd., Tokyo, Japan) in 9 volumes of PBS, and the mixtures were centrifuged (21,600 ×g, 4°C, 15 min). Using an electrophoresis analyzer (Epalyzer 2 Junior, Helena Laboratories Co., Ltd., Saitama, Japan), the serum and supernatants of the tissue mixtures were electrophoresed, and the ratios of each of the ALP isoenzymes (ALP1, ALP2, ALP3 and ALP5) were determined. The levels of the ALP isoenzymes were calculated by multiplying the total ALP level measured with a clinical analyzer by the ratio of each ALP isoenzyme. To correct the values for the total protein content in the tissues, the level of total protein was measured using a clinical analyzer.
Statistical analysisMean values and standard deviations were calculated for each sex and each group. For single comparisons, the homogeneity of variance among the groups was first analyzed using an F-test. Differences between groups were tested for statistical significance using a two-tailed Student’s t-test (if homogeneous) or Aspin-Welch’s t-test (if heterogeneous). For multiple comparisons, the homogeneity of variance among the groups was first analyzed using Bartlett’s test. Differences between groups were tested for statistical significance using Dunnett’s test (if homogeneous) or Steel’s test (if heterogeneous). The significance levels in the F-test and Bartlett’s test set at 5% (two-tailed), while the levels in the other tests were set at 0.1%, 1%, and 5% (two-tailed).
Four weeks of food restriction significantly suppressed the body weight increase in males and females of both the 65%FR and 45%FR groups throughout the food restriction period (Fig. 1). In the 65%FR and 45%FR groups, the mean percent changes in the body weight at the end of the 4-week food restriction period were −29% and −45% in the males, and −27% and −43% in the females, respectively, relative to the values in the FF group. The mean water intake was significantly decreased in both the males and females of the 65%FR and/or 45%FR groups throughout the food restriction period, as compared to with that in the FF group (Supplemental data 1).
Body weight changes during the 4-week period of food restriction in the males (a) and females (b). White circle: FF group; gray circle: 65%FR group; black circle: 45%FR group. Error bars indicate the standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 for differences vs. the FF group (Dunnett’s test). ## P < 0.01 for differences vs. the FF group (Steel’s test).
Quantitative urinalysis performed at week 4 revealed significant decreases of the urinary specific gravity and urinary excretion levels of electrolytes (sodium, potassium, and chloride) in both the males and females of the 65%FR and/or 45%FR groups as compared with the corresponding values in the FF group (Supplemental data 2). Qualitative urinalysis, on the other hand, revealed no apparent changes (Supplemental data 3).
Hematological tests and testing of the coagulation profile at the end of the 4-week food restriction period revealed the following changes in the experimental groups as compared with the FF group (Supplemental data 4): increases in the erythroid parameters (red blood cell count, hemoglobin level, and/or hematocrit level) in both the males and females of the 65%FR and/or 45%FR groups; a decrease in the reticulocyte count in both the males and females of the 65%RF and 45%RF groups; a decrease in the platelet count in the females of the 45%RF group; a decrease in the mean corpuscular volume of the red blood cell in the females of the 65%FR and 45%FR groups; an increase in the mean corpuscular hemoglobin concentration in the red blood cells in the males of the 45%RF group; decreases in the total white blood cell count and differential white blood cell counts (absolute counts of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and/or large unstained cells) in both the males and females of the 65%FR and/or 45%FR groups; and a prolongation in the prothrombin time in the females of the 45%FR group.
Blood chemistry examination revealed the following changes as compared with those in the FF group (Table 1): increases in the sodium and chloride concentrations in both the males and females of the 65%FR and/or 45%FR groups; increases in the total bilirubin and total cholesterol levels in the males of the 45%FR group; increases in the ALP, γ-GTP, triglyceride, and urea nitrogen levels in the females of the 45%FR group; decreases in the total protein, and calcium levels in both the males and females of the 65%FR and 45%FR groups; a decrease in the triglyceride level in the males of the 65%FR and 45%FR groups; decreases in the creatine phosphokinase (CPK), albumin, free fatty acid, and inorganic phosphorus levels in the females of the 65%FR and/or 45%FR groups. In addition to these changes, a decrease in the aspartate aminotransferase (AST) level was observed in the females of the 65%FR group; however, this change in the AST level was considered as being an incidental change due to the high AST level (123.9 IU/L) observed in the females in the FF group.
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In regard to the organ weights, significant decreases in the weights of the brain, pituitary, heart, lungs, liver, kidneys, spleen, salivary glands, and adrenals in both the males and females, of the prostate in the males, and of the thymus, uterus, and ovaries in the females were observed in the 65%FR and/or 45%FR groups as compared with the FF group (Supplemental data 5). In terms of the bone lengths, significant decreases in the lengths of the femur, tibia, and sternum were observed in the males of the 65%FR and 45%FR groups and females of the 45%FR group as compared with the FF group (Table 2).
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At necropsy, gross decreases in the sizes of the liver, spleen, thymus, and prostate were observed in the 45%FR group, while microscopic examination revealed no remarkable changes. On histopathological examination, a decrease in the trabecular bone in the femur was observed in 2/8 males and 4/8 females of the 45%FR group, and a decrease in the trabecular bone in the tibia was observed in 4/8 males and 6/8 females of the 45%FR group. In the bone marrow, a decrease in hematopoiesis was observed in 2/8 males of the 65%FR group, and in all the males and females of the 45%FR group. In the skin, a decrease in the subcutaneous adipose tissue was observed in 6/8 males and 1/8 females of the 65%FR group, and in all the males and females of the 45%FR group.
Effects of food restriction for 4 weeks on the levels of ALP in the serum, liver, bone, and small intestine in ratsAnalysis of the ALP isoenzyme levels revealed that the predominantly observed ALP isoenzymes in the serum were ALP3 and ALP5 in both the males and females, while the serum level of ALP2 was low and that of ALP1 was barely detectable (Fig. 2). Significant increases in the serum ALP2 and ALP5 levels were observed after 4 weeks of food restriction in the females of the 65%FR and/or 45%FR groups as compared with the FF group, resulting in a significant increase of the serum total ALP level in the females of the 45%FR group. There were no apparent changes of the serum levels in total ALP in males, although a trend was observed towards a higher ALP5 (p = 0.052) level in the 45%FR group as compared with that in the FF group.
Effects of food restriction for 4 weeks on the total ALP levels and levels of each of the ALP isoenzyme in the serum of males (a) and females (b). White bar: FF group; gray bar: 65%FR group; black bar: 45%FR group. Error bars indicate the standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 for differences vs. the FF group (Dunnett’s test).
In the liver, all the ALP isoenzymes were present with a high percentage of ALP1 in the males (Fig. 3a, b). Significant increases in the liver ALP1 and ALP5 levels were observed after 4 weeks of food restriction in both the males and females of the 65%FR and/or 45%FR groups as compared with the FF group, resulting in a significant increase of the liver total ALP levels in both the males and females of the 45%FR group.
Effects of food restriction for 4 weeks on the total ALP levels and levels of each of the ALP isoenzymes in tissues of both males and females. Levels in the liver (a, b), bone (c, d), and small intestine (e, f) of males (a, c, e) and females (b, d, f). White bar: FF group; gray bar: 65%FR group; black bar: 45%FR group. Error bars indicate the standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 for differences vs the FF group (Dunnett’ test). # P < 0.05, ## P < 0.01 for differences vs. the FF group (Steel’s test).
In the bone, ALP3 was the predominant ALP, although ALP2 and ALP5 levels were also high (Fig. 3c, d). Significant decreases in the bone ALP3 and ALP5 levels were observed after 4 weeks of food restriction in the females of the 45%FR group as compared with the FF group, resulting in a significant decrease in the total ALP level.
In the small intestine, the predominant ALP was ALP5, with other ALP isoenzymes being scarcely detected (Fig. 3e, f). A significant increase in the small intestine ALP5 level was observed after 4 weeks of food restriction in the males of the 65%FR and 45%FR groups as well as of the total ALP level in the small intestine. There was a trend towards higher ALP5 levels (p = 0.091) in the females of the 65%FR groups as compared with those in the females of the FF group.
The results of the ALP enzyme histochemistry are summarized in Table 3. In the livers of the FF group, positive staining for ALP was observed in the infiltrating cells in the sinusoids/blood vessels and small bile ducts in both the males and females, and in addition, in the bile canaliculi, in the males (Fig. 4a, b). In the 65%FR and 45%FR groups, the positive ALP staining in the males was comparable to that in the males of the FF group (Fig. 4c). In contrast, 4/8 and 6/8 females in the 65%FR and 45%FR groups, respectively, showed positive staining for ALP in the bile canaliculi as well in addition to the positive staining areas observed in the FF group (Fig. 4d), indicative of increase of the positive staining for ALP after 4 weeks of food restriction in the females. In the bone, strongly positive staining or positive staining for ALP was observed in the osteoblasts and chondrocytes of the femur and tibia in the females of the FF group (Fig. 5a, b). In the 45%FR group, the ALP staining intensity was reduced in the females (positive or weakly positive, Fig. 5c, d). On the other hand, no obvious changes in the intensity of staining for ALP in the bone were observed in the males. In the small intestine, positive staining for ALP was observed in the enterocytes, with a trend towards more intense staining in the upper small intestine (duodenum) and weaker staining in the lower small intestine (ileum) (Supplemental data 6). There was no obvious change in the staining intensities for ALP in either the males or females of the 65%FR and 45%FR groups as compared with those in the FF group.
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Effects of food restriction for 4 weeks on the ALP enzyme histochemistry in the liver. In the FF group, both males (a) and females (b) showed positive staining for ALP in cells infiltrating the sinusoids/blood vessels and small bile ducts (arrowheads), and males also showed positive staining in the bile canaliculi. In males of the 45%FR group (c), the ALP staining intensity was comparable to that in the FF group. In contrast, females of the 45%FR group (d) showed positive staining for ALP in the bile canaliculi in addition to the infiltrating cells in the sinusoids/blood vessels and small bile ducts, indicating an increased staining intensity for ALP after 4 weeks of food restriction. The lower left images in (a) to (d) show the enlarged view. Bars = 50 μm.
Effects of food restriction for 4 weeks on the ALP enzyme histochemistry in the bone (distal end of the femur). Females of the FF group showed strongly positive staining for ALP (a) or positive staining (b) in the osteoblasts and chondrocytes. Females in the 45%FR group showed reduced ALP staining intensity (positive [c] or weakly positive [d]) after 4 weeks of food restriction. Bars = 100 μm.
Analysis of the serum and tissue levels of the ALP isoenzymes in the FF group revealed some differences between the males and females. To investigate the involvement of the sex hormones in these differences, we examined the changes in the serum and tissue levels of the ALP isoenzymes in the serum, liver, bone, and small intestine in ovariectomized females reared under the free-feeding condition. We also examined the changes in the serum and liver levels of the ALP isoenzymes in testosterone-treated females reared under the free-feeding condition.
In the FF group, the serum level of total ALP was significantly higher in the males than in the females, with the difference explained, at least in part, by the higher ALP2 level in the males: the serum level of ALP2 was 3 times higher in the males than in the females (Fig. 6a). In the liver, the total ALP level was markedly higher in the males than in the females, which was accounted for by the differences in the liver level of ALP1: the liver level of ALP1 was 6 times higher in the males than in the females (Fig. 6b). In the bone, the total ALP level was significantly higher in the females, with the difference accounted for by the higher bone levels of ALP2 and ALP3 in the females as compared with the males (Fig. 6c). In the small intestine, ALP5 accounted for most of the ALP, and the total ALP level was significantly higher in females due to the higher ALP5 level of the small intestine in the females than in the males (Fig. 6d). The small-intestinal ALP3 level was also significantly higher in the females, although the ALP3 level in the small intestine was only a small fraction of the total ALP level.
Comparison of the total ALP levels and levels of each of the ALP isoenzymes in the serum (a), liver (b), bone (c), and small intestine (d) between the males and females. White bar: males of the FF group; gray bar: females of the FF group. Error bars indicate the standard deviation. * P < 0.05, ** P < 0.01 for the difference between the males and females (Student’s t-test). # P < 0.05, ## P < 0.01, ### P < 0.001 for the difference between the males and females (Aspin-Welch’s t-test).
After the 4-week study period, the body weight was significantly higher and bone length significantly greater in the ovariectomized females (OVX group) as compared with those in the SS group (Supplemental data 7 and 8). Serum estradiol levels in the OVX group (5.24 ± 1.87 pg/mL) were significantly lower than those in the SS group (9.15 ± 3.60 pg/mL), and the value was comparable to that of the males in the FF group (5.94 ± 3.77 pg/mL) (Supplemental data 9). In the OVX group, the serum ALP2 level was significantly higher than that in the SS group (Fig. 7a). In the liver, the ALP1 level was significantly higher in the OVX group than in the SS group, resulting in the higher total ALP level in the liver of the OVX group (Fig. 7b). No apparent difference in the bone ALP level was observed between the SS group and OVX groups (Fig. 7c). In the small intestine, the ALP5 level was significantly higher in the OVX group compared to that in the SS group, resulting in the higher total ALP level in the small intestine of the OVX group (Fig. 7d).
Comparison of the total ALP levels and levels of each of the ALP isoenzymes in the serum (a), liver (b), bone (c), and small intestine (d) between the sham-surgery females (SS group) and ovariectomized females (OVX group). Gray bar: SS group; dotted pattern bar: OVX group. Error bars indicate the standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001 for the difference between the SS group and OVX group (Student’s t-test).
In the testosterone-treated females, the serum level of ALP2 was significantly higher than that in the corn oil-treated females (Fig. 8a). In the liver, the ALP1 level was markedly higher than that in the corn oil-treated females (Fig. 8b). There was a trend towards higher total ALP level (p = 0.052) in the testosterone-treated females than that in the corn oil-treated females.
Comparison of the total ALP levels and levels of each of the ALP isoenzymes in the serum (a) and liver (b) between corn oil-treated females and testosterone-treated females. Gray bar: corn oil-treated females; checked pattern bar: testosterone-treated females. Error bars indicate the standard deviation. ** P < 0.01 for the difference between the corn oil-treated females and testosterone-treated females (Student’s t-test). # P < 0.05 for the difference between the corn oil-treated females and testosterone-treated females (Aspin-Welch’s t-test).
In this study, 6-week-old male and female SD rats were grown under a restricted-diet condition, being fed 65% or 45% of the dietary amounts given to the FF group, for 4 weeks, and the analysis was focused on determining the effects of the 4-weeks’ diet restriction on the ALP isoenzyme levels in the serum, liver, bone, and small intestine. The effects of ovariectomy or testosterone administration on the serum and tissue ALP isoenzyme levels were also investigated.
Four weeks of food restriction was associated with an increase of the serum total ALP level in the females. Similar changes have been reported from previous food restriction studies (Schwartz et al., 1973; Levin et al., 1993; Seki et al., 1997; Hubert et al., 2000). Analysis of the serum ALP revealed that the predominant isoenzymes in the serum were ALP3 and ALP5 in both the males and females of the FF group, in agreement with previous reports (Dziedziejko et al., 2005). Food restriction was associated with increases in the serum ALP2 and ALP5 levels in the females, with a particularly marked increase of the serum ALP5 level. A trend towards increase of the serum ALP5 level was also observed in the males. ALP5 is present in abundance in the small intestine, and food restriction resulted in an increase or trend towards increase of the ALP5 level in the small intestine. Considering that the ALP5 level of the bone, which is a tissue other than the small intestine in which ALP5 is relatively abundant, decreased with food restriction, the increase of the serum ALP5 level observed in the females was thought to be mainly due to an increase of the ALP5 level derived from the small intestine.
Analysis of the hepatic levels of the ALP isoenzymes showed that ALP1 was the predominant ALP isoenzyme in the livers of the males, whereas the levels of all the ALP isoenzymes were comparable in the females. Surprisingly, the level of ALP2, a liver-type ALP isoenzyme, was found to be low in both males and females. Four weeks of food restriction increased the total ALP level in the liver in both sexes. Although positive staining for ALP was observed in the small bile duct in females of FF group, the results of ALP enzyme histochemistry following food restriction showed positive staining not only in the small bile ducts, but also in the bile canaliculi in the females. On the other hand, positive staining for ALP was observed in the small bile ducts and bile canaliculi in the males, consistent with previous reported (Hoshi et al., 1997; Suzuki et al., 2006). Among the liver ALP isoenzymes, the levels of ALP1 and ALP5 following food restriction were increased in both sexes, with a particularly marked increase of the level of ALP1. Since ALP in the liver is involved in bile pH regulation (Alvaro et al., 2000), it is conceivable that the increase in ALP1 level probably represented a compensatory increase to enhance lipid absorption for regulating the bile pH under the low-nutritional condition. On the other hand, ALP1 was barely detectable in the serum, suggesting that even while the ALP1 level increased with food restriction in the liver, which shows no pathological changes, little of it leaks from the liver tissue into the serum.
As for bone, the ALP3, a bone-type ALP, was high, and ALP2 and ALP5 were also relatively abundant. Four weeks of food restriction resulted in changes indicative of suppressed bone formation, such as a shorter bone length and decrease of the trabecular bone. ALP is present in abundance in osteoblasts and chondrocytes, as shown by the results of our ALP enzyme histochemistry and previous reports (Matsuzawa and Anderson, 1971; Weiss et al., 1988; Hoshi et al., 1997; Miao and Scutt, 2002; Orimo, 2010), and is a marker of bone formation associated with bone calcification. In females, food restriction was associated with reduced ALP levels in the bone, and some individuals showed reduced ALP staining of the osteoblasts and chondrocytes in the femur (distal end) and tibia. Analysis of the bone ALP isoenzymes revealed a marked decrease of the level of ALP3, the bone-type ALP. These findings may reflect suppression of differentiation and maturation of osteoblasts and chondrocytes in the bone under the low-nutritional condition.
In the small intestine, ALP is known to be abundant in the intestinal epithelial cells (Lallès, 2014) and to be strongly expressed in the upper small intestine and relatively weakly expressed in the lower small intestine of rats (Wada et al., 2001). The results of the ALP enzyme histochemistry in the current study also stronger staining in the duodenum and jejunum and relatively weak staining in the ileum. Analysis of the ALP isoenzyme showed that ALP5, the intestinal-type ALP, accounted for the majority of the ALP detected in the small intestine. Four weeks of food restriction resulted in an increase of the small-intestinal level of ALP5 in the males and a trend towards increase (p = 0.091) of this isoenzyme in the females. Intestinal ALP is involved in lipid absorption and is transported with lipids from the small intestinal mucosa through the lymphatic duct into the systemic circulation (Zhang et al., 1996). Therefore, it has been suggested that the increase in the level of ALP5 in the small intestine associated with food restriction may represent a compensatory increase to enhance lipid absorption under the low-nutritional condition.
Comparison of the serum and tissue ALP levels between males and females revealed some differences. The total serum ALP level was higher in the males than in the females. This sex difference has generally also been observed in previous rat studies (Schwartz et al., 1973; Levin et al., 1993; Seki et al., 1997; Hubert et al., 2000; Kurata et al., 2003; Moriyama et al., 2008). Analysis of the ALP isoenzyme levels in the serum showed higher ALP2 levels in the males as compared with the females, accounting for the sex difference in the total serum ALP level. The total ALP level in the liver was also higher in the males than in the females, however, this was attributed to the higher hepatic ALP1 level in the males. Interestingly, in ovariectomized or testosterone-treated females, the serum ALP2 and liver ALP1 levels increased and shifted closer to the levels in the males. This suggests that the levels of these ALP isoenzymes might be regulated in an inhibitory manner by estrogen and in a promotive manner by testosterone, which could explain, at least in part, the sex differences in the serum and liver ALP levels in rats. In particular, there was a marked difference in the liver ALP1 level between the males and females, with a marked increase of the hepatic ALP1 level in the testosterone-treated females, suggesting that blood testosterone levels may play an important role in this sex difference.
In the bone, the levels of ALP2 and ALP3 were higher in females than in males. Estrogen promotes bone formation, and estrogen deficiency is known to increase bone resorption and decrease bone formation (Manolagas, 2010), which may partially explain the elevated the level of ALP3, a bone-type ALP, in the bones of females, with higher estrogen levels. However, no decrease of the levels of the ALP isoenzymes was observed in the ovariectomized females. It has been reported that rapid loss of estrogen, such as with ovariectomy, results in both increased bone resorption and increased bone formation (Manolagas, 2010), leading to higher serum ALP (Tanizawa et al., 2000, Miyazaki et al., 2004). It is possible that these complex physiological changes in the ovariectomized females were responsible for the observed lack of decrease of the bone ALP3 level.
In the small intestine, ALP5 accounted for most of the ALP, and the small-intestinal ALP5 level was higher in the females than in the males. Surprisingly, ovariectomy resulted in an even further increase of the ALP5 level in the small intestine in the females. The reason for this is not clear, but ovariectomized females also showed increase of the body weight, suggesting that changes in the nutritional requirements related to lipid absorption facilitated by ALP (Zhang et al., 1996) or other factors, not just sex hormones, may have influenced the results.
Apart from the changes in the ALP levels described above, food restriction resulted in changes in several other parameters such as the body weight, water intake, values of the erythrocyte parameters, and electrolyte levels in the urine and serum. These observed changes were basically consistent with those reported from previous studies (Schwartz et al., 1973; Oishi et al., 1979; Ogawa et al., 1985; Levin et al., 1993; Seki et al., 1997; Hubert et al., 2000; Yoshii et al., 2003; Moriyama et al., 2008; Asanuma et al., 2011), and were considered to reflect the low-nutritional condition or hemoconcentration. On the other hand, in non-clinical toxicity studies, the body weight loss comparable to that observed in the food-restricted groups may be considered a severe adverse effect, potentially resulting in an overestimation of toxicity. Considering the typical degree of food intake reduction observed in standard non-clinical toxicity studies, further research conducted under conditions that limited body weight gain suppression to within 10% would be warranted to obtain more meaningful information into the physiological changes associated with malnutrition.
In conclusion, food restriction resulted in changes of the ALP isoenzyme levels in each tissue, and the observed increase of the serum ALP levels was thought to mainly result from an increase of the ALP5 level derived from the small intestine. In addition, differences in the levels of the ALP isoenzymes between the male and female rats could have been influenced by the sex hormones.
We thank Shinsaku Tokumitsu, and Hiroshi Yamasaki for their laboratory assistance, and Haruhiro Yamashita for his kind supervision.
Conflict of interestThe authors declare that there is no conflict of interest.
