Leptin can regulate several immune functions. However, the role of leptin on lymphocyte function has not been recognized in vivo. Accordingly, we have investigated the effect of leptin on starvation-induced immune dysfunction using diet-induced obese mice. To induce obesity, C57BL/6J mice were fed a high-fat diet for 14 weeks and control mice were fed a standard diet for the same period. The obese and control groups of mice were then starved for 48 h, and received intraperitoneal injections of recombinant leptin or phosphate-buffered saline four times during starvation. Other control mice in both diet groups were free fed without being starved. Although starvation of the control mice dramatically reduced the weights of the immune organs, cytokine production and increased proliferation of cultured splenocytes, these levels returned to those of the free-feeding groups with exogenous leptin administration. However, these effects of leptin were not observed in obese mice. These findings provide some evidence that leptin can regulate the immune function in vivo. It is also suggested that the action of leptin might not appear in obesity.
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N Mito, H Yoshino, T Hosoda, and K Sato
K Ohtani, H Shimizu, Y Tanaka, N Sato, and M Mori
Abstract
Pioglitazone hydrochloride (AD-4833), one of the thiazolidinedione analogs, is a new anti-diabetic agent which improves peripheral insulin resistance in diabetic patients. We determined the direct effect of AD-4833 on insulin secretion in HIT-T 15 cells. The effects of AD-4833 (10−7 m to 10−5 m) on insulin secretion were examined in 3 and 7 mm glucose-containing F-12 K media. The addition of 10−5 m AD-4833 significantly increased insulin secretion in both media, but its stimulatory effect was more potent in the medium containing 7 mm glucose. The addition of 10−5 m AD-4833 caused an immediate, significant increase in cytosolic free Ca2+ concentration ([Ca2+]i). Nifedipine at all concentrations from 10 to 1000 nm significantly attenuated insulin secretion by 10−5 m AD-4833. In addition, 10−5 m AD-4833 failed to stimulate insulin secretion in the Ca2+-free Kreb's-Ringer bicarbonate buffer. These data indicated that AD-4833 stimulates in vitro insulin secretion in HIT-T 15 cells, perhaps by inducing Ca2+ influx.
Journal of Endocrinology (1996) 150, 107–111
F Ishihara, T Aizawa, N Taguchi, Y Sato, and K Hashizume
Abstract
Insulin release, glucose utilization (3H2O formation from [5-3H]glucose), and glucose oxidation (14CO2 formation from [4C(U)] glucose) were determined in pancreatic islets from 96-h fasted rats at 37 ° C and those from fed rats at 22 ° C, using the islets from fed rats incubated at 37 ° C as controls. In the islets from 96-h fasted rats and those from fed rats incubated at 22 ° C, we could not demonstrate significant insulin release in response to high glucose concentrations of up to 16·7 mmol/l. However, 16·7 mmol/l glucose clearly augmented insulin release caused by a depolarizing concentration (50 mmol/l) of K+ in these islets: i.e. 16·7 mmol/l glucose plus 50 mmol/l K+ produced significantly greater insulin release than 50 mmol/l K alone. Glucose utilization and oxidation by the islet cells were suppressed by 96-h fasting of the rats or by lowering the incubation temperature to 22 ° C, and depolarization with K at 50 mmol/l did not at all augment glucose utilization and oxidation by the islets. Thus we conclude that reduction of glucose metabohsm in islets from fasted rats and in those incubated at low temperature eliminated initiation, but not augmentation, of insulin release by 16·7 mmol/l glucose. The data indicate that the metabolic threshold for the initiation of insulin release is significantly higher than it is for the augmentation of release by glucose.
Journal of Endocrinology (1994) 143, 497–503
T Michigami, H Yamato, H Suzuki, Y Nagai-Itagaki, K Sato, and K Ozono
In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.
MP Wijayagunawardane, A Miyamoto, Y Taquahashi, C Gabler, TJ Acosta, M Nishimura, G Killian, and K Sato
The precise regulatory mechanisms of cyclic oviductal contraction in the cow are unclear. The purpose of this study was to evaluate the effect of luteinizing hormone (LH), steroids, prostaglandins (PGs) and peptides on the oviductal contraction and secretion of PGs and endothelin (ET-1). In addition, the cyclic expression of mRNA for ET-1 and its receptors (ET-R) was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). In the in vitro microdialysis study, an infusion of LH alone or in combination with progesterone (P(4)), estradiol-17beta (E(2)) and/or ET-1 stimulated pronounced release of PGE(2), PGF(2alpha) and ET-1 in the oviducts from cows in the follicular and postovulatory phases. The addition of LH, LH+P(4)+E(2) and/or ET-1 to the medium increased the amplitude of oviductal contraction. However, oxytocin (OT) completely blocked the responses of oviductal secretion and contraction. In contrast, these substances did not show any effect in the oviducts from cows in the mid luteal phase. Similar expression patterns of mRNA encoding for ET-R type A and type B were found, which were highest during the postovulatory phase, lower during the luteal phase, with the lowest expression during the follicular phase. We suggest that the preovulatory LH surge, together with increasing E(2) levels from the Graafian follicle and a basal P(4) from regressing corpora lutea (CL), stimulates maximum oviductal production of PG and ET-1, resulting in oviductal contraction for a rapid transport of gametes. OT released from the newly-formed CL may block these mechanisms, and slow contractions for transport of the embryo to the uterus.
H Shimizu, Y Shimomura, Y Nakanishi, T Futawatari, K Ohtani, N Sato, and M Mori
Abstract
The decrease in estrogen in menopausal women increases body fat. The present studies were undertaken to investigate the involvement of estrogen in leptin production in vivo. In the first study, expression of ob gene mRNA in white adipose tissue was measured at 2 and 8 weeks after ovariectomy in rats. In the second, serum leptin concentration was measured in total body fat of 87 weight-matched human subjects (29 men, 29 premenopausal and 29 postmenopausal women). In the third, changes in serum leptin concentration with the menstrual cycle were determined, ob gene expression decreased in subcutaneous and retroperitoneal white adipose tissue of ovariectomized rats 8 weeks after the operation, while ovariectomy increased ob gene expression in mesenteric white adipose tissue. Serum leptin concentration was decreased by ovariectomy. Estradiol supplement reversed the effect of ovariectomy on ob gene expression and circulating leptin levels. In humans, serum leptin concentration was higher in premenopausal women than in men, and in postmenopausal women it was lower than in premenopausal women, but still higher than in men. In 13 premenopausal women, serum leptin levels were significantly higher in the luteal phase than in the follicular phase. The present studies strongly indicate that estrogen regulates leptin production in rats and human subjects in vivo. Regional variation in the regulation of ob gene expression by estrogen was found.
Journal of Endocrinology (1997) 154, 285–292
J Noguchi, H Hikono, S Sato, G Watanabe, K Taya, S Sasamoto, and Y Hasegawa
The ontogeny of inhibin secretion in the testis of rats was investigated. Testicular localization, content of immunoactive and bioactive inhibin and its molecular size in fetal and neonatal rats (from 16 days of gestation to 5 days of age) were determined. Strong immunostaining with an antiserum against a polypeptide of porcine inhibin alpha-subunit was noted in testicular interstitial cells from 16 days of gestation. Co-localization of inhibin alpha-subunit and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) was observed in the interstitial cells until 2 days of age. Immunoreactive inhibin alpha-subunit in the interstitial tissue had disappeared by 5 days of age, although 3 beta HSD-positive cells were still detected. Weak immunostaining for the inhibin alpha-subunit was detected in the seminiferous tubules, probably in the cytoplasm of Sertoli cells, from 20 days of gestation onward. No inhibin alpha-subunit immunostaining was observed in germ cells throughout the experimental period. Testicular inhibin was detected at 16 days of gestation (49.5 +/- 6.7 pg per testis) by RIA. Testicular immunoreactive inhibin showed a tendency to increase during fetal life and levels were maintained at a similar value after birth (697.0 +/- 46.9 pg per testis at 5 days of age). Inhibin bioactivity and its molecular size in testicular homogenate was examined at 17 days of gestation and 0 and 5 days of age. Although no bioactivity was detected at 17 days of gestation, bioactivity was noted at 0 and 5 days of age (177.7 and 1303.9 pg per testis respectively). Immunoblot analysis with antiserum against inhibin alpha-subunit revealed only approximately 40 kDa molecular masses in the testis at 17 days of gestation, probably inhibin-related proteins, but not inhibin. At 0 and 5 days of age, a protein of 30 kDa molecular mass, possibly inhibin, was detected as well as material of approximately 40 kDa molecular mass. FSH in the plasma was first detected at 19 days of gestation (1197.0 ng/l), increased towards birth, and thereafter decreased (4588.5 +/- 572.3 ng/l at 21 days of gestation and 2400.0 +/- 179.6 ng/l at 5 days of age). These results indicate that Leydig cells in fetal and neonatal rats produce inhibin-related substances with no inhibin bioactivity, whereas Sertoli cells begin to produce inhibin during the perinatal period as a possible regulator of FSH secretion.
T Takahashi, K Sato, S Kato, T Yonezawa, Y Kobayashi, Y Ohtani, S Ohwada, H Aso, T Yamaguchi, S G Roh, and K Katoh
Ghrelin is a multifunctional peptide that promotes an increase of food intake and stimulates GH secretion. Ghrelin secretion is regulated by nutritional status and nutrients. Although a high-protein (HP) diet increases plasma ghrelin secretion in mammals, the mechanisms and the roles of the elevated ghrelin concentrations due to a HP diet have not been fully established. To clarify the roles of elevated acylated ghrelin upon intake of a HP diet, we investigated the regulation of ghrelin concentrations in plasma and tissues in wethers fed with either the HP diet or the control (CNT) diet for 14 days, and examined the action of the elevated plasma ghrelin by using a ghrelin-receptor antagonist. The HP diet gradually increased the plasma acylated-ghrelin concentrations, but the CNT diet did not. Although the GH concentrations did not vary significantly across the groups, an injection of ghrelin-receptor antagonist enhanced insulin levels in circulation in the HP diet group. In the fundus region of the stomach, the ghrelin levels did not differ between the HP and CNT diet groups, whereas ghrelin O-acyltransferase mRNA levels were higher in the group fed with HP diet than those of the CNT diet group were. These results indicate that the HP diet elevated the plasma ghrelin levels by increasing its synthesis; this elevation strongly suppresses the appearance of insulin in the circulation of wethers, but it is not involved in GH secretion. Overall, our findings indicate a role of endogenous ghrelin action in secretion of insulin, which acts as a regulator after the consumption of a HP diet.
S Yamada, M Komatsu, T Aizawa, Y Sato, H Yajima, T Yada, S Hashiguchi, K Yamauchi, and K Hashizume
When isolated rat pancreatic islets are treated with 16.7 mM glucose, a time-dependent potentiation (TDP) of insulin release occurs that can be detected by subsequent treatment with 50 mM KCl. It has been thought that TDP by glucose is a Ca2+-dependent phenomenon and only occurs when exposure to glucose is carried out in the presence of Ca2+. In contrast to this, we now demonstrate TDP under stringent Ca2+-free conditions (Ca2+-free buffer containing 1 mM EGTA). In fact, under these Ca2+-free conditions glucose caused an even stronger TDP than in the presence of Ca2+. TDP induced by glucose in the absence of extracellular Ca2+ was unaffected by inhibitors of protein kinase C (PKC). However, cerulenin or tunicamycin, two inhibitors of protein acylation, eradicated TDP without affecting glucose metabolism. The TDP by glucose was not associated with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) during subsequent treatment with high K+. Exposure of islets to forskolin under Ca(2+)-free conditions did not cause TDP despite a large increase in the cellular cAMP levels. In conclusion, glucose alone induces TDP under stringent Ca2+-free conditions when [Ca2+]i was significantly lowered. Protein acylation is implicated in the underlying mechanism of TDP.
T Goto, T Endo, H Henmi, Y Kitajima, T Kiya, A Nishikawa, K Manase, H Sato, and R Kudo
Gonadotropin-releasing hormone (GnRH) and its agonist analog (GnRHa) are well known to have luteolytic effects. We previously reported that prolactin (PRL) stimulated matrix metalloproteinase (MMP)-2 activity to degrade collagen type IV as a mechanism of structural luteolysis. The effects of GnRHa treatment on developed corpora lutea are unknown. In this study we assessed the effect of GnRH on MMP expression and induction of structural involution of developed corpora lutea of superovulated rats using GnRHa. Pregnant mare serum gonadotropin-human chorionic gonadotropin (hCG)-synchronized ovulation and luteinization were induced in immature female rats, followed by daily treatment with GnRHa from 5 days after hCG treatment. GnRHa-induced involution of corpora lutea was evident 3 days after the treatment, as shown by their markedly smaller size (60% of the control weight). Nine days after hCG injection, serum progesterone and 20alpha-dihydroprogesterone concentrations were as low as those associated with structural luteolysis. These findings revealed that GnRHa has the ability to induce structural luteolysis in superovulated rats in the same way that PRL does. To gain information on mechanisms of luteal involution induced by GnRHa, we performed gelatin zymography. This showed a significant increase in the active form of MMP-2 in the luteal extract of GnRHa-treated rats (more than twofold that of the control). Activation of pro-MMP-2 by membrane type-MMP (MT-MMP) is reported to be a rate-limiting step for catalytic function. Another function of MT-MMP is to degrade collagen types I and III. The plasma membrane fraction of corpora lutea of GnRHa-treated rats activated pro-MMP-2 of fetal calf serum, resulting in a marked shift of the 68-kDa band to the 62-kDa band in the zymogram. A Northern hybridization study also revealed simultaneous significant increases in expression of MMP-2 mRNA and MT1-MMP mRNA in corpora lutea of GnRHa-treated rats (more than threefold the control level). In summary, hormonal and histological features of corpora lutea of GnRHa-treated superovulated rats correspond to those of structural luteolysis. GnRHa stimulated the expression of MMP-2 and MT1-MMP in developed corpora lutea associated with involution. These findings support the conclusion that MMP-2, activated by MT1-MMP, and MT1-MMP itself, remodel the extracellular matrix during structural luteolysis induced by GnRHa.
