Impact of brown adipose tissue on body fatness and glucose metabolism in healthy humans.

BACKGROUND: Brown adipose tissue (BAT) is involved in the regulation of whole-body energy expenditure and adiposity. Some clinical studies have reported an association between BAT and blood glucose in humans. OBJECTIVE: To examine the impact of BAT on glucose metabolism, independent of that of body fatness, age and sex in healthy adult humans. METHODS: Two hundred and sixty healthy volunteers (184 males and 76 females, 20-72 years old) underwent fluorodeoxyglucose-positron emission tomography and computed tomography after 2 h of cold exposure to assess maximal BAT activity. Blood parameters including glucose, HbA1c and low-density lipoprotein (LDL)/high-density lipoprotein-cholesterol were measured by conventional methods, and body fatness was estimated from body mass index (BMI), body fat mass and abdominal fat area. The impact of BAT on body fatness and blood parameters was determined by logistic regression with the use of univariate and multivariate models. RESULTS: Cold-activated BAT was detected in 125 (48%) out of 260 subjects. When compared with subjects without detectable BAT, those with detectable BAT were younger and showed lower adiposity-related parameters such as the BMI, body fat mass and abdominal fat area. Although blood parameters were within the normal range in the two subject groups, HbA1c, total cholesterol and LDL-cholesterol were significantly lower in the BAT-positive group. Blood glucose also tended to be lower in the BAT-positive group. Logistic regression demonstrated that BAT, in addition to age and sex, was independently associated with BMI, body fat mass, and abdominal visceral and subcutaneous fat areas. For blood parameters, multivariate analysis after adjustment for age, sex and body fatness revealed that BAT was a significantly independent determinant of glucose and HbA1c. CONCLUSION: BAT, independent of age, sex and body fatness, has a significant impact on glucose metabolism in adult healthy humans.

Impact of UCP1 and ß3AR gene polymorphisms on age-related changes in brown adipose tissue and adiposity in humans.

BACKGROUND: Brown adipose tissue (BAT) is involved in the regulation of whole-body energy expenditure and adiposity. The activity and prevalence of BAT decrease with age in humans. OBJECTIVE: To examine the effects of single nucleotide polymorphisms of the genes for uncoupling protein 1 (UCP1) and ß3-adrenergic receptor (ß3AR), key molecules of BAT thermogenesis, on age-related decline of BAT activity and accumulation of body fat in humans. METHODS: One hundred ninety-nine healthy volunteers (20-72 years old (y.o.)) underwent fluorodeoxyglucose-positron emission tomography (FDG-PET) and computed tomography (CT) after 2-h cold exposure to assess BAT activity. The visceral and subcutaneous fat areas at the abdominal level were estimated from the CT images. They were genotyped for -3826 A/G polymorphism of the UCP1 gene and 64 Trp/Arg mutation of the ß3AR gene. RESULTS: BAT was detected in 88 subjects out of 199 (44%), more in younger (30 y.o., 55%) than older subjects (>40 y.o., 15%). BAT prevalence of older subjects tended to be lower in the UCP1 G/G group than the A allele group (A/A and A/G), and also in the ß3AR Arg allele group (Trp/Arg and Arg/Arg) than the Trp/Trp group. When compared subjects who had two or more base substitutions on the two genes (the 2-4 allele group) with those who had less than two base substitutions (the 0-1 allele group), BAT prevalence was comparable in younger subjects (62% vs 50%) but lower in older subjects (0% vs 24%, P<0.05). Visceral fat area of the 2-4 allele group was higher than that of the 0-1 allele group (P<0.05) in older subjects, but not in younger subjects. CONCLUSION: UCP1 -3826 A/G and ß3AR 64 Trp/Arg substitutions accelerate age-related decrease in BAT activity, and thereby may associate with visceral fat accumulation with age.

Quantification of umu genotoxicity level of urban river water.

In recent years, the request of environmental safety management for carcinogenic substances, mutagenic substances and/or reproductive toxicity substances (CMR) has increased. This study focused on clarifying the genotoxicity level of environmental water and its release source by using the umu test provided in ISO13829. Although a genotoxicity index "induction ratio (IR)" is used in ISO13829, we normalised it to make it possible to compare various environmental water quantitatively to each other as a new index "genotoxic activity (GA=(IR-1)/Dose)". Sample water was collected and concentrated to 100 times or 1,000 times by a solid phase extraction method. As the test results, it was found that GA level in actual river water varied widely from less than the determination limit of 23 [1/L] to 1,100 [1/L] by quantitative comparison, and the value was also equivalent to more than 50 times the level of tap water. The GA level of household wastewater was not so high, but the levels of treated water from wastewater treatment plant (WTP) were from 220 [1/L] to 3,200 [1/L]. Raw sewage of some WTP shows high level genotoxicity. A part of genotoxicity substances, for example 50%, could be removed by conventional wastewater treatment, but it was not enough to reduce the water environmental load of genotoxicity.

Chlorination by-products of fenitrothion.

The mutagens produced through chemical reaction between chlorine and the insecticide fenitrothion were studied by using a quadrupole GC-MS. The mutagenicity and the mutagen formation potential (MFP) of the identified by-products were evaluated by the Ames assay (preincubation method) using Salmonella typhimurium TA100 without exogenous activation by S9 mix (TA100-S9). Before conducting GC/MS analyses, six compounds were presumed to be produced in chlorinated fenitrothion. These compounds were confirmed to be produced by the GC/MS analyses, but none of them were mutagenic. One of the chlorination by-products, 3-methyl-4-nitrophenol, has 19 times greater MFP than that of fenitrothion. This result suggests that a major mutagen in chlorinated fenitrothion will be produced via a chemical reaction between chlorine and 3-methyl-4-nitrophenol.

Human gastric choriocarcinoma serially transplanted in nude mice.

A gastric choriocarcinoma cell line capable of producing human chorionic gonadotropin (HCG) was studied by sc transplantation and serial passages into nude mice fed under specific pathogen-free (SPF) conditions. The tumor-take rate at the serial heterotransplantation in SPF mice was high (greater than 95%), in contrast with a low rate in coventional animals (35%). The restoration of morphology and function of the original neoplasm were exclusively verified in SPF mice. Multiple lung metastases were found in 2 animals. The production, storage, and excretion of HCG by tumor cells were confirmed by its high content in serum and cystic fluid in tumors and by its intracellular localization. The tumor cells also contained a specific placental alkaline phosphatase in their membranes. The cells were various trophoblastic types ranging from primitive cytotrophoblasts to typical syncytial cells. The hormone effects of the tumor on sex organs of tumor-bearing animals were evident.

Transplantation of human tumors in nude mice.

Ninety-one human tumors, including various common carcinomas, low-grade malignant tumors, and benign tumors, were transplanted into athymic nude mice. Tumor take was confirmed histologically for 22 neoplasms at the initial transplantation, and 14 serially transplantable tumors were established, including some hitherto unestablished or unreported, such as lung and hepatic cell carcinomas. Among the 91 tumors were 21, 14, and 13 carcinomas of the lung, stomach, and breast, respectively. Transplantability was highest in lung carcinomas (10/21), followed by gastric carcinomas (2/14) and breast carcinomas (1/13). Morphology of original tumors was retained well in most transplanted tumors, but desmoplastic or scirrhous tumors, such as gastric and breast carcinomas, tended to become medullary with a decrease in amount of tumor stroma. The ability to produce mucin in gastric carcinomas or melanin in malignant melanoma was maintained in serially transplantable tumors. In addition, ectopic production of adrenocorticotropin and beta melanocyte-stimulating hormone continued in a transplanted small cell carcinoma of the lung. Preliminary results were obtained on hormone dependency of the transplantable breast carcinoma and on alpha1-fetoprotein in the transplantable hepatic cell carcinoma.

A larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction to efficiently determine the toxicity of organic contaminants in river water.

Using a larval medaka (Oryzias latipes) acute toxicity assay combined with solid-phase extraction, we proposed a method for efficiently determining the fish toxicity of organic contaminants in river water. Organic toxicants were 10, 20, 50 and 100-fold concentrated from 4 L of the sample with adsorption cartridges. The lethal effect was observed by exposing every ten individuals of 48-72 h old larval medaka to 20 mL of each solution for 48h. The median lethal concentration rate (LCR50) was used as an indicator for the toxicity. With the developed toxicity test method, more than seven times difference was found in the LCR50 of the river water samples. LCR50 distribution profiles were compared with 125 samples in two typical rivers. The result revealed a lower toxicity level in the mainstream than in the confluences, and a lower toxicity level in Sagami River than in Ayase River. LCR50 proved unique as a toxicity indicator, which was impossible to speculate from the conventional water quality indicator of the dissolved organic carbon concentration. As an effective screening test for priority settings, the method can help us with an efficient planning for the environmental investigation and management.

A general transcription initiation factor, human transcription factor IID, overexpressed in human lung and breast carcinoma and rapidly induced with serum stimulation.

A general transcription factor IID which binds to the TATA box promoter element on RNA polymerase II genes regulates and initiates eukaryotic mRNA synthesis. A quantitative polymerase chain reaction procedure was developed and the human transcription factor IID (hTFIID) transcript was measured in normal human tissues, lung carcinomas, lung carcinoma cell lines, and breast carcinomas. In some normal tissues such as liver, fetal lung, and placenta, relatively low to moderate levels of hTFIID mRNA were detected. In contrast, hTFIID transcript was highly expressed in nearly all solid lung carcinomas and cell lines including both small cell lung cancer and non-small cell lung cancer. hTFIID mRNA was present to a greater extent in small cell lung cancer than non-small cell lung cancer in solid tumors and cell lines. In solid carcinomas of breast, overexpression of hTFIID was also detected. A serum induction study using a serum-starved small cell lung cancer cell line, Lu134BS, indicated hTFIID transcription to be rapidly induced at 15 min following stimulation and its response essentially similar to that of protooncogene, c-fos. These results indicate the involvement of the expression of the general transcription factor hTFIID in lung and breast carcinoma, such as being associated with poor differentiation and high mitotic activity.

Hormone dependency of a serially transplantable human breast cancer (Br-10) in nude mice.

The human breast cancer (Br-10) serially transplanted to nude mice (BALB/c-nu/nu) grew well in female mice but very slowly or not at all in untreated male mice and female mice treated with 1 mg of testosterone i.m. twice a week. The growth in female mice was arrested by ovariectomy, and that in male mice was accelerated by 0.1 mg of estradiol i.m. once a week. Tumors in female and estrogenized male mice retained the original histology of duct carcinoma. Tumors in ovariectomized female, androgenized female, and male mice consisted of cells with smaller and more uniform nuclei, forming markedly dilated lumina in the first group and arranged in lobular patterns in the latter two groups. High-affinity 8 S and 4 S estrogen receptors were present in tumors transplanted to female nude mice, but no progesterone receptors were detected. These results provide experimental evidence for the hormone dependency of a human breast cancer in vivo and strongly suggest the important role of estrogen and androgen in the growth regulation of some estrogen receptor-positive human breast cancers.

In vivo and in vitro biological activities of two human cell lines derived from anaplastic lung cancers.

Cell lines Lu-65 and SK-Luci-6 were established from two patients with anaplastic (non-oat cell) lung cancers. These cell lines showed in vivo and in vitro functional activities that could explain the paraneoplastic syndromes which were clinically manifested. In both patients, elevated white blood cell counts occurred in the absence of any evidence of sepsis. Tumor fragments taken directly from one patient and transplanted to nude mice produced a progressive leukocytosis in the mice. Tissue culture-derived cells from both cell lines enhanced white blood cell numbers following heterotransplantation to nude mice. Cell-free extracts from both cell lines were found to enhance granulocyte-macrophage colony formation in soft agar. Greater colony formation was consistently found with the cell line (SK-Luci-6) that was derived from the patient manifesting the more marked leukocytosis. These data suggest that the tumor cells release colony-stimulating activities. Coincidently, one cell line (Lu-65) synthesized and released large amounts of prostaglandin E2 with little or no other prostaglandin product; the other cell line produced no prostaglandins. When the tumor cell lines were cocultured with explanted fetal rat bones, enhanced bone resorption with excessive calcium release occurred. Bone-resorbing activity correlated with tumor prostaglandin synthesis for the cell line releasing prostaglandin E2. An osteolytic factor that was neither prostaglandin nor parathyroid hormone was released by the SK-Luci-6 cell line. Hypercalcemia was a persistent feature only in the patient from whom the latter tumor line was derived.

Production and molecular size heterogeneity of immunoreactive gastrin-releasing peptide in fetal and adult lungs and primary lung tumors.

Gastrin-releasing peptide (GRP) is known to be a bombesin-like peptide present in mammalian tissues. Using GRP radioimmunoassay specific for carboxyl-terminal portion, the immunoreactive GRP (IR-GRP) content of 5 fetal lungs, 38 adult lungs, and 131 primary lung tumors was determined. All fetal lung extracts contained IR-GRP ranging from 31 to 140 ng/g, wet weight. IR-GRP was present in 7 to 21% of normal adult lungs and lung carcinomas other than small-cell carcinoma; the amount was not very large except in two cases of adenocarcinoma, in which 110 and 140 ng/g of IR-GRP were detected. In the case of small-cell carcinoma, IR-GRP was found in 23 of the 31 cases examined (74%), and nine (29%) of these contained large amounts of IR-GRP (100 to 14,000 ng/g). As for carcinoid tumors, IR-GRP was found in five of the 12 cases examined (42%), and large amounts of IR-GRP were detected in two cases (5,100 to 130,000 ng/g). Immunohistochemically, IR-GRP was found in the neuroendocrine cells of fetal lungs and in the tumor cells of primary lung tumors. When these tissue extracts were examined by bombesin radioimmunoassay that recognizes bombesin but not GRP, they did not contain immunoreactive bombesin, suggesting that IR-GRP in these tissues is more similar to GRP than to bombesin. Sephadex G-50 gel filtration always revealed two peaks of IR-GRP in both fetal lungs and IR-GRP-producing tumors. One was eluted at the position corresponding to that of porcine GRP (Peak 1) and the other, at the position just behind that of porcine GRP (14-27) (Peak 2). In the five fetal lungs, Peak 2 comprised more than 83% of the total IR-GRP. In the 12 IR-GRP-producing tumors examined, the ratio of these two peaks differed from case to case. Our data indicate that IR-GRP, which is present in fetal lung, is often produced by primary lung tumors, especially by small-cell carcinoma and carcinoid tumor, with molecular size heterogeneity.

A human gastric choriocarcinoma cell line with human chorionic gonadotropin and placental alkaline phosphatase production.

A gastric choriocarcinoma cell line synthesizing human chorionic gonadotropin (HCG) was established in 1971 by Oboshi et al. and was found to possess human placental alkaline phosphatase. The present paper also deals with the relationship between the cell growth and HCG secretion and with cellular localization of HCG and human placental alkaline phosphatase by cytochemical and ultrastructural methods. This cell line was found to secrete HCG during cellular proliferation, with the maximum secretion in the stationary phase (about 1 muIU/cell/48 hr), and the hormone could be detected in a small proportion of mono- and/or multinuclear cells in both logarithmic and stationary phases. The organ-specific, heat-stable, L-phenylalanine-sensitive, immunoreactive human placental alkaline phosphatase was localized on the cell membrane of many cells. Ultrastructurally, the line consisted mainly of cytotrophoblastic and intermediate cells in the process of syncytial formation, with more or less squamous metaplasia. From these findings it was concluded that the cell line maintained the properties of trophoblastic cells from morphological and functional aspects, i.e., it was a cell line with two distinct marker substances.

Elevated activities and properties of arylsulfatases A and B and B-variant in human lung tumors.

The activities of arylsulfatases A and B were determined in human primary and secondary tumor tissues (total, 53 cases) of various histological types. Significantly higher activities of these sulfatases were found in almost all the primary lung carcinomas as compared to their corresponding uninvolved tissues. No significant correlation was demonstrated between the enzyme activities and histological figures (stroma amounts, etc.). Lung adenocarcinoma and squamous cell carcinoma showed the presence of an additional arylsulfatase component (B1) which was not detected in normal human lung. The tumor arylsulfatase B1 had an isoelectric point (pI) of 6.7 and was clearly distinguished from arylsulfatase A (pI 4.9) and arylsulfatase B (pI 9.1 to 9.2) in normal lung and lung tumor. The tumor B1 enzyme was demonstrated to be most probably an isoenzyme of arylsulfatase B, since this unusual enzyme was indistinguishable from arylsulfatase B in terms of Ag+ inhibition; its kinetic parameters of Km for p-nitrocatechol sulfate, which was 2.9 mM with B1; optimum pH of 6.3 for B1; heat stability; and substrate specificity for three synthetic and two physiological substrates.

Elevation of glycoprotein galactosyltransferase activity in human lung cancer related to histological types.

Uridine diphosphogalactose:glycoprotein galactosyltransferases were examined in human lung adenocarcinoma and squamous cell carcinoma. The galactosyltransferase activities in tissue homogenates from both carcinomas were higher than in adjacent normal control with asialoagalactofetuin as a substrate. This activity in adenocarcinoma (27 cases) was two times higher than that in squamous cell carcinoma (19 cases) with statistical significance (p less than 0.001). Using Triton-solubilized enzymes from a particulate fraction, similar differences in the activity were observed with ovalbumin, asialoagalactofetuin, and its beta-eliminated derivative as acceptors but not with bovine submaxillary mucin. These observations mean that the higher activity of galactosyltransferase(s) in lung carcinomas (especially in adenocarcinoma) is mainly responsible for galactosylation of carbohydrate chains in N-glycoside-type but not O-glycoside-type glycoproteins.

Production of calcitonin, adrenocorticotropic hormone, and beta-melanocyte-stimulating hormone in tumors derived from amine precursor uptake and decarboxylation cells.

The tumor production of human calcitonin (CT) was examined by radioimmunoassay, and it was found that 50 of 85 (59%) tumor tissues collected at random contained immunoreactive CT. These tumors were grouped as to whether they were derived from the amine precursor uptake and decarboxylation (APUD) series. The group that was derived from APUD cells showed appreciable amounts of CT in 30 of 31 (97%) of these tumors or in 20 of 21 (95%) when the medullary carcinomas of the thyroid were excluded. However, of the non-APUD group of tumors only 20 of 54 (37%) were found to contain CT, so that the difference between these two groups was highly significant (p less than 0.001). Of the tumors with ectopic adrenocorticotropic hormone-melanocyte-stimulating hormone production, 12 of 14 were shown to contain CT. These data indicate that CT is a common product of the APUD tumors and that tumor production of CT is often associated with that of adrenocorticotropic hormone and beta-melanocyte-stimulating hormone.

Leukemic distribution of a human acute lymphocytic leukemia cell line (Ichikawa strain) in nude mice conditioned with whole-body irradiation.

Induction of leukemia in nude mice (BALB/c nu/nu) was attempted by inoculation with a human acute lymphocytic leukemia cell line (Ichikawa strain, maintained in an ascitic form in our institute). Inoculation of the cells i.v. in normal nude mice failed to produce leukemia. However, conditioning with whole-body irradiation (500 rads) resulted in induction of leukemia after i.v. inoculation, especially when such inocluation was performed 3 days after irradiation. The correlation of survival to inoculum size (10(5) to 10(5)) was inversely exponential. Leukemic infiltration was noted in the spleen, lymph nodes, bone marrow, meninges, liver, kidneys, etc., as seen in human leukemia. These cells retained their original cytological characteristics, ultrastructural features, and surface markers and revealed high terminal deoxynucleotidyltransferase activity as T-derived cells. Chromosome analysis revealed aneuploidy in a hypotetraploid range with a mode of 88 chromosomes.

Cool Temperature-Induced Chlorosis in Rice Plants (II. Effects of Cool Temperature on the Expression of Plastid-Encoded Genes during Shoot Growth in Darkness).

It has been proposed that cool temperature-induced chlorosis (CTIC) in Indica cultivars of rice (Oryza sativa L.) is caused by cell growth and plastid development being impeded at cool temperatures. Since it is well known that the overall rate of transcription of plastid-encoded genes changes dramatically during the early phases of plastid development, in this study we focused on the patterns of expression of these genes. Northern blot analysis revealed that the level of 16S rRNA is decreased in a CTIC-sensitive rice cultivar grown at a cool temperature. The expression of the gene for the [beta] subunit of plasmid RNA polymerase (rpoB) was shown to be somewhat disturbed, particularly in terms of its resuppression under cool conditions. The level of transcripts or proteins of plastid-encoded photosynthetic genes was also decreased in a CTIC-sensitive cultivar at a cool temperature. These results suggest that the temperature-dependent inhibition of the onset of gene expression encoding the transcription/translation apparatus may be primarily involved in the mechanism causing CTIC.

Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3.

An increase of intracellular cAMP mediated by prostaglandin E2 (PGE2) has been shown to delay spontaneous apoptosis of neutrophils. It has been demonstrated that a selective agonist for PGE2 receptor subtype 3 (the EP3 receptor) is capable of decreasing cAMP and stimulating phosphoinositide turnover in various types of cells. We investigated the effect of a selective EP3 receptor agonist, ONO-AE-248, on neutrophil viability. ONO-AE-248 rapidly caused a unique form of neutrophil death. The agonist primarily induced morphological changes of the nucleus, including fusion of the lobules, decreased compactness of the chromatin, and blebbing and rupture of the nuclear membrane. This was followed by an increase of plasma membrane permeability and cell lysis. During these processes, neither apoptotic changes such as nuclear condensation, DNA fragmentation, and expression of phospholipid phosphatidylserine on the plasma membrane nor necrotic changes such as chromatin clumping and organelle destruction were apparent in the treated neutrophils. The fatal effect of the agonist night be specific for neutrophils because it failed to promote the rapid death of other types of cells. Although activation of neutrophils by ONO-AE-248 was not evident, experiments using metabolic inhibitors demonstrated that the agonist caused neutrophil death via the activation of protein kinase C in the presence of intracellular ATP. These findings indicated that EP3 receptor-mediated signals might promote a novel form of neutrophil death, which differs from typical apoptosis or necrosis.

Influence of micrometastasis on N stage in gastric cancer and clinical application.

In order to evaluate the real extent of lymph node metastasis (LNM) in gastric cancer, an immunohistochemical (IHC) analysis was performed. We examined 11173 lymph nodes removed from 355 patients with all stages of gastric carcinoma. Tissue preparations were stained with cytokeratin 18, monoclonal antibody against cytokeratin. Micrometastases were found in 2.5% of the lymph nodes and in 31.3% of patients. The incidence of the patients with LNM increased to 9.1% in T(1m) (n = 99), 31.6% in T(1sm) (n = 95, 23.1% in sm1, 34.8% in sm2), 66.7% in T2 (n = 108, 48.8% in mp, 76.5% in ss), 88.1% in T3 (n = 42), and 90.9% in T4 (n = 11) lesions. Upstage was identified in 8.5% of patients: 6.7% in T1 (4.0% in m, 7.7% in sm1, 10.1% in sm2), 14.8% in T2 (20% in mp, 11.8% in ss), 2.4% in T3, and 0% in T4. Factors related to LNM were: tumor size and lymphatic invasion in mucosal lesions; only lymphatic invasion in submucosal lesions; size and depth of tumor, and lymphatic invasion in T2 lesions. In conclusion, the incidence of micrometastasis in regional lymph nodes was higher than we imagined in T1 lesions, more than D1 lymphadenectomy for sm1 and selected cases of mucosal cancer, and D2 lymphadenectomy for sm2 are necessary.

Thyrotropin-producing microadenoma associated with pituitary resistance to thyroid hormone.

A 21-yr-old female with hyperthyroidism is described. Though her serum-free T3 was 17.8 pmol/L and free T4 was 60.2 pmol/L, TSH was as high as 10.7 mU/L. TRH stimulated an increase in TSH from 10.7-91.7 mU/L. T3 administration in gradually increasing doses of 100, 200, and 400 mg/day resulted in gradual reduction in serum TSH. Cranial computed tomography and magnetic resonance imaging revealed a microadenoma of the pituitary gland. Histology of the surgical specimen showed a TSH-producing adenoma with TSH cell cluster islets and decreased numbers of TSH cells in the nonneoplastic pituitary. Cultured cells from the adenoma secreted TSH spontaneously and in response to TRH. This TRH-stimulated TSH secretion was suppressed by T3 in a dose-dependent manner. One year postoperatively, neither residual tumor nor recurrence were seen by computed tomography and magnetic resonance imaging. However TSH, as well as free T3 or T4, was still high and overresponsive to TRH.