SH2-containing inositol 5'-phosphatase 2 selectively impairs hypothalamic insulin signalling and regulation of food intake in mice.

SH2-containing inositol 5'-phosphatase 2 (SHIP2) is a lipid phosphatase that negatively regulates the metabolic signalling of insulin in peripheral tissues; however, the expression of SHIP2 in the hypothalamus and its functional roles are largely unknown. In the present study, immunohistochemical analysis demonstrated that SHIP2 protein exists in neuronal cells expressing neuropeptide Y and pro-opiomelanocortin in the arcuate nucleus of the hypothalamus in C57BL/6J mice. Interestingly, the expression levels of SHIP2 in the hypothalamus were elevated in aged C57BL/6J mice and diabetic db/db mice. To clarify the significance of the increased expression of SHIP2 in the hypothalamus, we examined the central effects of insulin and leptin in transgenic mice overexpressing SHIP2 (SHIP2-Tg). Accumulation of phosphatidylinositol (3,4,5)-trisphosphate and phosphorylation of Akt in the hypothalamus, induced by i.c.v. injection of insulin, were attenuated in SHIP2-Tg compared to wild-type mice, whereas leptin-induced phosphorylation of signal transducer and activator of transcription 3 in the hypothalamus was comparable between them. The suppression of food intake after i.c.v. administration of insulin (but not leptin) was attenuated consistently in SHIP2-Tg. In addition, SHIP2-Tg showed increased food consumption after starvation and become heavier with visceral fat accumulation than wild-type mice, despite normal levels of oxygen consumption and spontaneous movement. These results suggest that SHIP2 contributes to the regulation of food intake mainly via the attenuation of insulin signalling in the hypothalamus of mice.

Age-related insulin resistance in hypothalamus and peripheral tissues of orexin knockout mice.

AIMS/HYPOTHESIS: Orexin/hypocretin is a hypothalamic neuropeptide that regulates motivated behaviours, such as feeding and arousal, and, importantly, is also involved in energy homeostasis. The aim of this study was to reveal the role of orexin in the regulation of insulin sensitivity for glucose metabolism. METHODS: Orexin knockout mice fasted overnight underwent oral glucose tolerance testing and insulin tolerance testing. The impact of orexin deficiency on insulin signalling was studied by Western blotting to measure levels of Akt phosphorylation and its upstream and downstream molecules in the hypothalamus, muscle and liver in orexin knockout mice. RESULTS: We found that orexin deficiency caused the age-related development of impaired glucose tolerance and insulin resistance in both male mice without obesity and female mice with mild obesity, fed a normal chow diet. When maintained on a high-fat diet, these abnormalities became more pronounced exclusively in female orexin knockout mice that developed severe obesity. Insulin signalling through Akt was disrupted in peripheral tissues of middle-aged (9-month-old) but not young adult (2-to-3-month-old) orexin knockout mice fed a normal chow diet. Moreover, basal levels of hypothalamic Akt phosphorylation were abnormally elevated in orexin knockout mice at every age studied, and insulin stimulation failed to increase the level of phosphorylation. Similar abnormalities were observed with respect to GSK3beta phosphorylation in the hypothalamus and peripheral tissues of middle-aged orexin knockout mice. CONCLUSIONS/INTERPRETATION: Our results demonstrate a novel role for orexin in hypothalamic insulin signalling, which is likely to be responsible for preventing the development of peripheral insulin resistance with age.

No evidence to indicate topographic dependency on bone formation around cp titanium implants under masticatory loading.

In vitro studies have proved the topographic dependency upon osteogenesis on titanium plate by investigating the cell-adhesion, -shape, -proliferation, -differentiation, ALP activity and osteocalcin production of osteogenic stem cells, MG36, MC3T3-E1 and wild strains of bone formative cells from animal and human. However, this in vivo study on bone growth around cp titanium dental implants under masticatory loading did not demonstrate significant difference among the different surface roughness in the range of Ra 0.4-1.9 microm, Rz 2.8-11.2 microm, Rmax 3.6-28.1 microm and Sm 2.9-41.0 microm, which was estimated by measuring the bone contacts, bone occupancies and bone bonding strengths at the implant/bone marrow interface. It is revealed that the topographic dependency on the osteogenetic activity is apt to be covered with wide variation in bone healing potential under the clinical condition with functional biting load.

In vitro study on bone formation and surface topography from the standpoint of biomechanics.

Effect of surface topography upon cell-adhesion, -orientation and -differentiation was investigated by in vitro study on cellular responses to titanium substratum with different surface roughness. Cell-shape, -function and -differentiation depending upon the surface topography were clarified by use of bone formative group cells (BFGCs) derived from bone marrow of beagle's femur. BFGCs consisted of hematopoietic stem cells (HSC) and osteogenetic stem cells (OSC). Cell differentiation of BFGCs was expressed and promoted by structural changes of cytoskeleton, and cell-organella, which was caused by mechanical stress with cytoplasmic stretching of cell adhesions to the substratum. Phagocytic monocytes of HSC differentiated to osteomediator cells (OMC) by cytoplasmic stretching with cell adhesion to the substratum. The OMC mediated and promoted cell differentiation from OSC to osteoblast through osteoblastic phenotype cell (OBC) by cell-aggregation of nodules with "pile up" phenomenon of OBC onto OMC. The osteogenesis might be performed by coupling work of both cells, OMC originated from monocyte of HSC and OBC originated from OSC, which were explained by SEM, TEM and fluorescent probe investigation on BFGCs on the test plate of cp titanium plates with different topographies. This osteogenetic process was proved by investigating cell proliferation, DNA contents, cell-adhesion, alkaline phosphatase activity and osteocalcine productivity for cells on the titanium plates with different topographies. The study showed increased osteogenic effects for cells cultured on Ti with increased surface roughness. Possible mechanisms were discussed from a biomechanical perspective.

Localization of calcitonin receptor mRNA in the mouse brain: coexistence with serotonin transporter mRNA.

To elucidate the sites of and mechanisms of analgesic effect of centrally injected calcitonin, we examined expression of calcitonin receptor mRNA in the mouse brain by in situ hybridization techniques. Calcitonin receptor mRNA was expressed in various brain regions, including the preoptic area, dorsomedial hypothalamic nucleus, lateral hypothalamic area, periaqueductal gray, dorsal raphe nucleus, locus coeruleus, lateral parabrachial nucleus, gigantocellular reticular nucleus alpha part, lateral paragigantocellular nucleus, raphe magnus nucleus and solitary tract nucleus, which are known to play important roles in pain modulation. In addition, a double in situ hybridization technique demonstrated the intense expression of calcitonin receptor mRNA on serotonergic neurons in some raphe nuclei and the lateral paragigantocellular nucleus, suggesting the involvement of central serotonergic pathways in analgesic effect of calcitonin.

Possible involvement of descending serotonergic systems in antinociception by centrally administered elcatonin in mice.

To elucidate the mechanisms of analgesic action of elcatonin, a synthetic analog of eel calcitonin, the effect of centrally injected elcatonin on acetic acid-induced writhing behavior was examined in mice. Intracisternal or intracerebroventricular injection of elcatonin significantly inhibited acetic acid-induced writhing behavior, while the intrathecal injection of elcatonin did not inhibit it. The inhibitory effect of intracisternal elcatonin was significantly attenuated by subcutaneous pretreatment with methysergide and (+/-)-propranolol or by intrathecal pretreatment with methysergide, (+/-)-propranolol, 1-(2-methoxyphenyl)-4-[4-(2-phthalimido) butyl]-piperazine hydrobromide (NAN-190) and granisetron, but not with (+/-)-atenolol or butoxamine. Further, the depletion of spinal 5-hydroxytryptamine (5-HT, serotonin) by pretreatment with 5,7-dihydroxytryptamine (5,7-DHT) significantly attenuated the inhibitory effect of intracisternally injected elcatonin on acetic acid-induced writhing behavior. These results suggest that the inhibitory descending serotonergic systems may be involved, through 5-HT1A and 5-HT3 receptors, in the production of an antinociceptive effect by centrally injected elcatonin.

Analysis of bilirubin uridine 5'-diphosphate (UDP)-glucuronosyltransferase gene mutations in seven patients with Crigler-Najjar syndrome type II.

Crigler-Najjar syndrome (CN) type II is caused by a reduction in hepatic bilirubin uridine 5'-diphosphate (UDP)-glucuronosyltransferase activity. Recently, there has been progress in mutation analysis of patients with CN type II. Here, we analyzed both the coding and the promoter regions of the gene in seven Japanese patients with CN type II from five unrelated families. The mutations found in this study were classified into three types. The first type was composed of double homozygous missense mutations (Gly71Arg and Tyr486Asp) in exons 1 and 5. These mutations, which were detected in five patients from three unrelated families, were the commonest. The second type, which was detected in one patient, consisted of a single homozygous missense mutation (Arg209Trp) in exon 1. The third type, which was detected in one patient and was a new type of mutation combination, was composed of a homozygous insertion mutation of the TATAA element and a heterozygous missense mutation (Pro229Gln) in exon 1. Although the first and the second type of mutations are recessive, the third type appears to be dominant with incomplete penetrance, since the allele frequency of the insertion mutation of the TATAA element is very high (40%).

Analysis of relationships between sex hormone dynamics and bone metabolism and changes in bone mass in surgically induced menopause.

A 3-year follow-up study was performed of bone metabolism and bone changes induced by surgical menopause as a consequence of hysterectomy and oophorectomy (OVX) is 52 nonmenopausal women. We investigated 22 bone parameters and determined seven bone indices as indicators of bone mineral content by dual energy X-ray absorptiometry (DXA), quantitative computed tomography (QCT), and microdensitometry (MD). The significant correlations between levels of sex hormones and/or bone parameters and bone indices demonstrated that marked sex-steroid deficiency after surgical menopause induced bone uncoupling during high bone turnover and subsequent rapid bone loss on the early period after OVX. Principal component analysis using correlation coefficients suggested a seven-loading-factor matrix composed of bone parameters and a two-loading-factor matrix composed of bone indices. Two groups of parameters--estradiol and estriol, and androstenedione together, and luteinizing hormone and follicle stimulating hormone together--indicated that the rate of bone loss was greater in the trabecular bone than in the cortical bone. Three other groups of parameters--urine calcium, urine hydroxyproline, and serum bone Gla-protein together; serum alkaline phosphatase, serum calcium, and 1,25-dihydroxycholecalciferol [1,25(OH)2D] together; and plasma tartrate-resistant acid phosphatase--indicated that bone uncoupling, with a prevalence of resorption over formation of bone, was greater in trabecular bone than is cortical bone and also that magnitude and rate of bone loss in the axial vertebrae surpassed those in the appendicular metacarpals after OVX. Two other groups of parameters, namely, trabecular bone mineral density (Dd) and bone mineral content (Dc), both measured by DXA, and bone mineral density (L2, L3), measured by QCT, together; and the cortical thickness index (MCI), cortical bone mineral density (sigmaGS/D), and the ratio of GSmin/max, measured by MD, indicated that the relative rates of bone reduction at the 3-year follow-up were greater in the axial vertebrae than in the appendicular metacarpals. Thus, bone change in the trabecular bone was associated with rapid loss during the early phase after OVX, whereas that in the cortical bone was slow during the late phase.

[Statistic analysis for the association of hormone dynamics and biochemical parameters with bone turnover in surgically induced menopause].

The association between hormone dynamics and bone turnover was statistically examined in 52 women with surgically induced menopause. The mean values of 19 laboratory items which included hormones as well as parameters of bone metabolism were obtained and analyzed by Spearman's rank test. Linear regression models were established. Based on Pearson's correlation matrices, the principal components were analyzed. Matrices were formed from factor loading derived from Varimax's rotation, and their statistical significance was evaluated. 1. Significant correlations were determined from both the examination of the data analyzed separately at the levels of artificial menopause (bilateral and unilateral) and oophorectomy (OVX) method (bilateral or unilateral). For the patients who had bilateral OVX, the pairs showing a positive correlation (0.01 < P, 0.05 < P) were 1-25-(OH)2D with E2 or ASD, 25-(OH)D with E2, E3, Progesterone, or ASD, CT with Progesterone, ASD, or Testosterone, S.A1-P with FSH or LH, and S.Ca with LH. The pairs showing a weaker positive correlation were PTH with E2 or E3, CT with E2, E3 with S.Acid-P or U.Ca/CRN, and S.Ca with FSH. The pairs showing a negative correlation (0.01 < P, 0.05 < P) were S.A1-P with E2 or Progesterone, and S.Ca with Testosterone. The pairs showing a weaker negative correlation were S.A1-P with ASD. For the patients who had unilateral OVX, the pairs showing a positive correlation were 1-25-(OH)2D with E2, ASD, or Testosterone, 25-(OH)D with E2, E3, Progesterone, or ASD, CT with Progesterone, ASD, or Testosterone, and PTH with ASD. The pairs showing a weaker positive correlation were PTH with E2 or E3, and CT with E2. The pairs showing a negative correlation were S.Ca with Testosterone. The pairs showing a weaker negative correlation were S.A1-P with ASD. There was a small but significant difference between the two groups. However, Estrogen and Androgen in sex steroids, and S.A1-P, S.Ca, CT, PTH, 1-25-(OH)2D, and 25-(OH)D in bone parameters, all changed early or late at different rates but participated concomitantly with the high bone turn-over induced by OVX, particularly in bilateral. 2. Significant correlations among all parameters were determined for data analyzed at the level of artificial menopause.(ABSTRACT TRUNCATED AT 400 WORDS)

[A study on tissue inhibitor of metalloproteinases (TIMP) during pregnancy and at delivery].

Metabolism of collagen in the fetal membrane has been proposed as one of causes of premature rupture of the membrane (PROM). It mainly depends on the activities of MMPs and their inhibitor, TIMP. The concentration and localization of TIMP in various body fluids and the tissue of perinatal patients was explored. 1. The TIMP concentration in maternal serum did not show any significant changes during pregnancy, and it was not significantly different from that in non-pregnant serum. In cases of PROM (n = 9) and premature labor (n = 4), there were no differences in the serum levels of TIMP. 2. The mean TIMP value in amniotic fluid was 754 +/- 325 ng/ml. There was no correlation between TIMP and gestational weeks. 3. The level of TIMP at delivery in amniotic fluid was significantly (p < 0.01) higher than that in maternal serum and umbilical serum, but it was not detected in neonatal first voided urine. 4. The mean TIMP values in amnion and chorion were 84 +/- 53 and 48 +/- 22 ng/mg protein respectively, but it was not detected in fetal lung or skin. 5. TIMP was clearly localized in fetal membrane, amniotic epithelium, amniotic fibroblast, chorionic epithelium and decidua, as detected by the immunohistochemical method. These results indicate that the higher level of TIMP in amniotic fluid originates in the fetal membrane.

Investigation of blood group A substance in the circulation of a patient with ovarian cyst.

Blood group substance was investigated in serum, cyst fluid and ascites of a patient with mucinous cystadenoma of the ovary. The patient's blood group was A, Le(a-b+), secretor. The serum contained so abnormally large amounts of A substance that it was difficult to determine the ABO blood of the patient's red cells. Using neutralization of anti-A, the ovarian cyst fluid was estimated to contain about 4,000 and 1,500 times as much substance as the serum before removal of the cyst and ascites, respectively. The A substance in serum reverted to a normal level 13 days after removal of the cyst. Gel infiltration analysis of the serum, ascites and ovarian cyst fluid showed the presence of similar A substance with a molecular weight ranging from one to several millions. These data indicate that A substance from the ovarian cyst entered the blood stream, and disappeared from it after the cyst was removed (half-life: 1 day).

Evidence that alpha-dihydrograyanotoxin II does not bind to the sodium gate.

The basis for the ability of alpha-dihydrograyanotoxin II (alpha-2HG-II) to promote Na+ conductance in axons was sought. The apparent binding of tritiated alpha-2HG-II to neural and other preparations was studied, using equilibrium dialysis, with lobster axon membranes, Torpedo electroplax, housefly head, and rat brain, liver and kidney. In every case the "binding" was nonsaturating and was suggested to involve nonspecific partitioning into the tissue. Supporting evidence was the similarity of extent of "binding" in all tissues and its relative insensitivity to neuropharmacological agents. Alpha-2HG-II did not affect the Na+ conductance of phospholipid bilayers, nor did it permit transport of 22Na into a bulk organic phase. It was concluded that alpha-2HG-II did not bind to the sodium gate, but possibly to a sodium permease present at a frequency of less than one per mu2 of cell membrane.