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Glucocorticoids are steroid hormones used as anti-inflammatory treatments. However, this strong immunomodulation causes undesirable side effects that impair bones, such as osteoporosis. Glucocorticoid therapy is a major risk factor for developing steroid-induced osteonecrosis of the femur head (ONFH). Since ONFH is incurable, therapy with mesenchymal stem cells (MSCs) that can differentiate into osteoblasts are a first-line choice. Bone marrow-derived MSCs (BM-MSCs) are often used as a source of stem cell therapy for ONFH, but their proliferative activity is impaired after steroid treatment. Adipose tissue-derived MSCs (AT-MSCs) may be an attractive alternative source; however, it is unknown whether AT-MSCs from steroid-induced ONFH (sAT-MSCs) have the same differentiation ability as BM-MSCs or normal AT-MSCs (nAT-MSCs). In this study, we demonstrate that nAT-MSCs chronically exposed to glucocorticoids show lower alkaline phosphatase activity leading to reduced osteogenic differentiation ability. This impaired osteogenesis is mediated by high expression of Dickkopf1 (Dkk-1) that inhibits wnt/β-catenin signaling. Increased Dkk-1 also causes impaired osteogenesis along with reductions in bone regenerative capacity in sAT-MSCs. Of note, plasma Dkk-1 levels are elevated in steroid-induced ONFH patients. Collectively, our findings suggest that glucocorticoid-induced expression of Dkk-1 could be a key factor in modulating the differentiation ability of MSCs used for ONFH and other stem cell therapies.

Infection-associated pregnancy complications cause premature delivery. Caspase-1 is involved in the maturation of interleukin (IL)-1β, which is activated by the NLRP3 inflammasome. To characterize the significance of the NLRP3 inflammasome pathway in the placenta, the effects of activators and inhibitors on NLRP3-related molecules were examined using isolated primary trophoblasts. Caspase-1 and IL-1β mRNA expression was markedly increased in response to lipopolysaccharide (LPS), a toll-like receptor (TLR)4 ligand. Treatment with the potassium ionophore nigericin significantly increased the level of activated caspase-1. Treatment with either LPS or nigericin stimulated IL-1β secretion, whereas pretreatment with the ATP-sensitive K+ channel inhibitor glibenclamide, the Rho-associated coiled-coil kinase inhibitor Y-27632, or a caspase-1 inhibitor significantly decreased nigericin-induced IL-1β secretion. In addition, dibutyryl-cAMP, which induces trophoblast differentiation, decreased expression of NLRP3, caspase-1, and IL-1β. These findings suggest that trophoblasts can secrete IL-1β through the NLRP3/caspase-1 pathway, which is suppressed by glibenclamide, and that the TLR4-mediated NLRP3 inflammasome pathway is more likely to be stimulated in undifferentiated than differentiated trophoblasts. Our data support the hypothesis that inhibition of the NLRP3 inflammasome can suppress placental inflammation-associated disorders. Keywords: Inflammasome, TLR4, NLRP3, Caspase-1, Trophoblast

The transcription factor GATA2 is an anti-adipogenic factor whose expression is downregulated during adipocyte differentiation. The present study attempted to clarify the molecular mechanism underlying the GATA2 repression and found that the repression is dependent on the activation of the glucocorticoid receptor (GR) during 3T3-L1 preadipocyte differentiation. Although several recognition sequences for GR were found in both the proximal and distal regions of the Gata2 locus, the promoter activity was not affected by the GR activation in the reporter assays, and the CRISPR-Cas9-mediated deletion of the two distal regions of the Gata2 locus was not involved in the GR-mediated Gata2 repression. Notably, the level of histone acetylation was markedly reduced at the Gata2 locus during 3T3-L1 differentiation, and the GR-mediated Gata2 repression was significantly relieved by histone deacetylase inhibition. These results suggest that GR regulates the Gata2 gene by reducing histone acetylation in the early phase of adipogenesis.

Erythropoiesis is strongly influenced by the interactions between stromal cells and erythroid progenitors, as well as by a key regulatory factor, erythropoietin (EPO). We previously generated mice with a knockdown mutation of Hif-2α (referred to as kd/kd) and found that these kd/kd mice exhibited normocytic anemia, even though the EPO expression was not severely affected. However, the VCAM-1 expression in spleen endothelial cells (EC), which is regulated by HIF-2α, was impaired, resulting in defective erythroid maturation. A deficiency of HIF-2α clearly led to pancytopenia. However, the critical level of HIF-2α required for erythropoiesis has not yet been elucidated. In this study, we generated HIF-2α knockdown/knockout heterozygous mice (kd/null). Strikingly, anemia was observed in the kd/null mice, but the red blood cell indices were significantly improved compared to those of kd/kd mice. In the spleens of kd/null mice, higher HIF-1α activity and expansion of the red pulp area were observed compared to those of kd/kd mice. Importantly, EC isolated from kd/null spleens showed high expression of VEGF receptors, FLK-1 and FLT-1, which are regulated by HIF-1α instead of HIF-2α under hypoxic conditions. We also found higher expression of phosphorylated ERK and higher proliferative activity in the EC isolated from kd/null mice compared to those from kd/kd mice. While the HIF-2α expression was diminished, HIF-1α bound to the HRE region in the promoters of genes that are normally regulated by HIF-2α. These results suggest that there is a compensatory pathway involving HIF-1α that regulates the expression of some HIF-2α target genes. J. Cell. Physiol. 230: 1534–1548, 2015. © 2015 Wiley Periodicals, Inc., A Wiley Company

Glucocorticoids cause the delayed wound healing by suppressing inflammation that is required for wound healing process. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) play an important role for wound healing by their cytokine productions including stromal derived factor 1 (SDF-1). However, it has not been clear how glucocorticoids affect the wound healing ability of AT-MSCs. In this study, we found that glucocorticoid downregulated SDF-1 expression in AT-MSCs. In addition, glucocorticoid-treated AT-MSCs induced less migration of inflammatory cells and impaired wound healing capacity compared with glucocorticoid-untreated AT-MSCs. Of note, prostaglandin E2 (PGE2) synthesis-related gene expression was downregulated by glucocorticoid and PGE2 treatment rescued not only SDF-1 expression in the presence of glucocorticoid but also their wound healing capacity in vivo. Furthermore, we found SDF-1-overexpressed AT-MSCs restored wound healing capacity even after treatment of glucocorticoid. Consistent with the results obtained from glucocorticoid-treated AT-MSCs, we found that AT-MSCs isolated from steroidal osteonecrosis donors (sAT-MSCs) who received chronic glucocorticoid therapy showed less SDF-1 expression and impaired wound healing capacity compared with traumatic osteonecrosis donor-derived AT-MSCs (nAT-MSCs). Moreover, the SDF-1 level was also reduced in plasma derived from steroidal osteonecrosis donors compared with traumatic osteonecrosis donors. These results provide the evidence that concomitant application of AT-MSCs with glucocorticoid shows impaired biological modulatory effects that induce impaired wound healing.

Mast cells have been suggested to play key roles in adipogenesis. We herein show that the expression of preadipocyte, but not adipocyte, marker genes increases in the white adipose tissue of mast cell-deficient (KitW-sh/W-sh) mice under both obese and non-obese conditions. In vitro culturing with adipogenic factors revealed increased adipocytes differentiated from the KitW-sh/W-sh stromal vascular fraction, suggesting the accumulation of preadipocytes. Moreover, the increased expression of preadipocyte genes was restored by mast cell reconstitution in the KitW-sh/W-sh mice. These results suggest positive effects of mast cells on the preadipocyte to adipocyte transition under both physiological and pathological conditions., Highlights • Mast cell-deficient mice are resistant to diet-induced obesity. • The expression of preadipocyte genes is increased in their white adipose tissue. • The content of preadipocytes is increased in the adipose stromal vascular fraction. • Mast cell reconstitution restores the enhanced expression of preadipocyte genes. • Mast cells may facilitate the preadipocyte–adipocyte transition in white adipose tissue.

The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon- or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre–mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cre–mediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human.

Previous study from our laboratory demonstrated that an increased release of gut glucagon-like immunoreactivity (GLI) following triglyceride digestion was not observed in pancreatectomized dogs. Therefore, in order to clarify the response of gut GLI to triglyceride ingestion, experimental study was carried out in normal and pancreatectomized dogs. When butter was administered to pancreatectomized dogs in combination with pancreatic enzymes, plasma glucagon (IRG) measured by specific antiserum did not change significantly but plasma triglyceride as well as plasma total immunoreactive glucagon (total IRG) measured by nonspecific antiserum increased. Oral administration of glycerol induced no significant increase in plasma IRG but elicited a moderate increase of plasma total IRG. Gastric instillation of palmitic acid induced a remarkable rise of plasma total IRG in normal dogs, whereas plasma IRG did not reveal any changes. Oral administration of triglyceride of medium chain fatty acid, tricaprylin, did not promote any rise of plasma IRG but a slight increase of plasma total IRG. From the present experiment, it is concluded that hydrolysis of triglyceride and its metabolites are important in the release of gut GLI following fat ingestion. Among its metabolites, long chain fatty acid and glycerol promote the GLI secretion, whereas medium chain fatty acid does not.

Glicentin has been demonstrated to be released in response to the intraluminal administration of nutrients, but its biological action remains unknown. To clarify the effect of glicentin on the endocrine function of the pancreas, the present study was performed using an in vivo local circulation system of the canine pancreas. During infusion of 0.5% solution of glucose or arginine, 100 and 400 pmol glicentin and 400 pmol glucagon were administered into the pancreaticoduodenal artery (PA) within 10 minutes at 40-minute intervals successively. During glucose infusion, blood glucose in the femoral artery did not change following administration of 100 pmol glicentin, but slightly increased following 400 pmol glicentin. Plasma insulin (immunoreactive insulin [IRI]) in the pancreaticoduodenal vein (PV) increased significantly only following infusion of 400 pmol glicentin. Plasma glucagon (immunoreactive glucagon [IRG]), measured with a specific antiserum to the C-terminal portion of glucagon, did not change following administration of 100 pmol glicentin, but was slightly elevated following 400 pmol glicentin. Plasma total IRG, measured with a nonspecific antiserum, increased promptly after administration of 100 and 400 pmol glicentin. During arginine infusion, the response of plasma IRI to glicentin was markedly exaggerated both in dosages of 100 and 400 pmol. From the present study it was concluded that human glicentin clearly increases insulin release from the canine pancreas.

Although the insulinotropic action of glucagon is well known, which parts of the glucagon molecule play an importent role in its action remain to be elucidated. To investigate the direct effect of the C-terminal peptides of glucagon on the endocrine function of the pancreas, glucagon(17–29), (21–29), (23–29), and (25–29) were studied using an in situ local circulation system of the canine pancreas. These glucagon fragments, as well as glucagon(1–29), were infused into the superior pancreaticoduodenal artery (PA) in a dosage of 400 pmol for 10 minutes during 0.5% glucose or 0.5% arginine infusion, and plasma insulin (IRI) and glucagon (IRG) levels in the superior pancreaticoduodenal vein (PV) were determined. During the glucose infusion, plasma IRI increased significantly following the administration of glucagon(23–29), (21–29), (17–29), or (1–29), but not glucagon(25–29). In these experiments, plasma IRG increased significantly following infusion of glucagon(21–29), (17–29), or (1–29). During the arginine infusion, all of the glucagon fragments studied enhanced insulin secretion, whereas plasma IRG was increased following the administration of glucagon(21–29), (17–29), or (1–29). In these experiments with glucose or arginine infusion, blood glucose in the femoral artery (FA) did not change significantly except for glucagon(1–29), which increased the blood glucose level. In addition, the administration of graded doses of glucagon(21–29) [50, 150, and 400 pmol] during the glucose infusion elicited an increase in plasma IRI in a dose-related manner. From the present study, it is concluded that the C-terminal peptides of glucagon enhanced insulin release, and that glucagon(21–29) is the minimum structure among the C-terminal glucagon fragments to stimulate insulin secretion.

Midaglizole was introduced as a hypoglycemic agent, but its insulin releasing mechanism remains unknown. In the present study, the effect of midaglizole upon the B cell function of the pancreas was investigated, using an in situ local circulation of the canine pancreas. The graded doses of midaglizole (0.2, 1.0 and 2.0 mg/kg) revealed a dose-related response of plasma insulin. The administration of yohimbine, a classical alpha 2-antagonist (1.11 mg/kg) revealed a similar increase in plasma insulin to that with midaglizole of equimolar amount. During the clonidine infusion midaglizole did not elicit any significant rise in plasma insulin, whereas yohimbine increased plasma insulin significantly. During glucagon infusion plasma insulin increased following midaglizole infusion but not by yohimbine. The simultaneous administration of diazoxide (K-channel opener) suppressed the midaglizole-induced insulin secretion. These results obtained in the present experiments revealed a different mechanism of insulin releasing action of midaglizole from that of yohimbine. Furthermore, the finding with diazoxide administration suggests that midaglizole stimulates insulin release through an interaction of K(+)-channel of the pancreatic B-cell.

OHNEDA, A., OHNEDA, K. and KOIZUMI, F. Insulin Releasing Action of N-Terminal Peptides of Glucagon in Dogs. Tohoku J. Exp. Med., 1992, 167 (4), 287-295-The relationship between the molecular structure and insulin releasing action of glucagon remains unknown. In order to investigate the direct action of N-terminal peptides of glucagon, glucagon (1-14), and glucagon (1-21) were studied using an in situ local circulation of the canine pancreas. These glucagon fragments as well as glucagon (1-29) were infused into the superior pancreaticoduodenal artery in a dose of 400pmol for 10min during the glucose or arginine infusion, and plasma insulin and glucagon in the superior pancreaticoduodenal vein were determined by radioimmunoassay. During the glucose infusion, glucagon (1-14) elicited a slight increase in plasma insulin, whereas glucagon (1-21) and (1-29) revealed significant changes in plasma insulin. In these experiments plasma glucagon did not change significantly following the administration of glucagon (1-14) or (1-21). During the arginine infusion all of the glucagon fragments studied enhanced insulin secretion markedly, whereas glucagon secretion was not affected. Furthermore, graded doses of glucagon (1- 14) (50, 150, and 400pmol) elicited an increase in plasma insulin in a dose-related manner. It is concluded from the present study that the N-terminal peptides of glucagon stimulate insulin release especially during the arginine infusion.- glucagon fragments; canine pancreas; arginine; glucose; insulin

In order to clarify the relationship between the structure and function of glucagon-like peptide (GLP) 1 in the endocrine function of the pancreas, the response of insulin and glucagon to various synthetic GLP-1-related peptides was investigated in anesthetized dogs. GLP-1-related peptides were administered in a dosage of 400 pmol within 10 min into the pancreatic artery during glucose or arginine infusion and the changes in plasma insulin and glucagon in the pancreatic vein were studied. GLP-1 (7-36) and (7-37), as well as glucagon enhanced insulin release during glucose infusion, whereas neither GLP-1 (1-37), (7-20), (6-37) nor (8-37) stimulated insulin release. The administration of GLP-1 (1-37), (7-36) and (7-37) reduced glucagon release during glucose infusion. When arginine was infused, GLP-1 (7-20), (7-36), (7-37), and glucagon enhanced insulin release. In contrast, glucagon release was increased by the administration of GLP-1 (7-20), (8-37), and (7-37). The present study indicates that histidine at the 7th position of GLP-1 is important in eliciting biological action and that only truncated GLP-1 (7-36), (7-37), and (7-20) showed an insulinotropic action as strong as glucagon in dogs. Furthermore, it is suggested that the response of insulin and glucagon to GLP-1-related peptides is dependent on a background condition.

To elucidate hormonal and metabolic changes in intestinal adaptation, an experimental study was performed in dogs subjected to ileojejunal transposition. Fasting levels of blood glucose, plasma insulin and gastric inhibitory polypeptide (GIP) did not change significantly throughout 12 weeks. Plasma glucagon measured with an antiserum specific to the C-terminal portion of glucagon slightly increased and plasma total glucagon measured with a non-specific antiserum gradually increased. Glucose tolerance deteriorated at the 4th week in the transposed group. The response of plasma insulin and GIP to glucose in the transposed group did not differ from that of the sham operated group. Plasma glucagon increased significantly during glucose loading at the 12th week. The response of plasma total glucagon to glucose was most prominent, reaching a peak of 3,755 +/- 742 pmol/liter. The value was significantly increased, compared with that of the sham group or normal group (p less than 0.01). Insulin-induced hypoglycemia enhanced a larger increment of plasma total glucagon in the transposed group than in the sham group (p less than 0.05). At the 12th week plasma total amino acids were decreased and several amino acids were reduced. It is concluded that ileojejunal transposition elicited an exaggerated response of plasma total glucagon to glucose and several metabolic derangements and that the transposition offers an excellent model for hyperenteroglucagonemia.

Identification of receptor activator of nuclear factor-κB (RANK) and RANK-ligand (RANKL) has provided new insights into the osteoclast differentiation pathway. Osteoclast precursor cells were isolated using monoclonal antibodies against c-Fms and RANK, and the effect of adherence on the in vitro differentiation and proliferation of these cells was examined in 2 different types of stromal-cell–free culture systems: a semisolid culture medium (a nonadherent system) and a liquid culture medium (an adherent system). Osteoclast precursor cells were not able to differentiate into mature osteoclasts efficiently in the semisolid culture system. Trimerized RANKL enhanced osteoclast differentiation in semisolid cultures, but not to the extent seen when cells were allowed to adhere to plastic. Initial precursor cells were capable of differentiating into macrophages or osteoclasts. Once these cells were transferred to adherent conditions, striking differentiation was induced. Multinuclear cells were observed even after they had displayed phagocytic activity, which suggests that cell adhesion plays an important role in the differentiation of osteoclast precursor cells. Integrins, especially the arginine-glycine-aspartic acid (RGD)–recognizing integrins αv and β3, were needed for osteoclast-committed precursor cells to proliferate in order to form multinuclear osteoclasts, and the increase in cell density affected the formation of multinuclear cells. A model of osteoclast differentiation with 2 stages of precursor development is proposed: (1) a first stage, in which precursor cells are bipotential and capable of anchorage-independent growth, and (2) a second stage, in which the further proliferation and differentiation of osteoclast-committed precursor cells is anchorage-dependent.

Osteoclasts are terminally differentiated cells derived from hematopoietic stem cells. However, how their precursor cells diverge from macrophagic lineages is not known. We have identified early and late stages of osteoclastogenesis, in which precursor cells sequentially express c-Fms followed by receptor activator of nuclear factor κB (RANK), and have demonstrated that RANK expression in early-stage of precursor cells (c-Fms+RANK−) was stimulated by macrophage colony-stimulating factor (M-CSF). Although M-CSF and RANKL (ligand) induced commitment of late-stage precursor cells (c-Fms+RANK+) into osteoclasts, even late-stage precursors have the potential to differentiate into macrophages without RANKL. Pretreatment of precursors with M-CSF and delayed addition of RANKL showed that timing of RANK expression and subsequent binding of RANKL are critical for osteoclastogenesis. Thus, the RANK–RANKL system determines the osteoclast differentiation of bipotential precursors in the default pathway of macrophagic differentiation.

BACKGROUND Here we demonstrate the utility of cascade blue (CB), to purify hematopoietic stem cells by flow cytometry. Multicolor immunofluorescence and the sensitivity (signal-to-noise) of the fluorochromes are essential for the identification and isolation of rare stem cell populations. METHODS We isolated hematopoietic stem cells utilizing a 407 nm laser line to excite CB and propidium iodide (PI) in combination with FITC, PE, and Red670 which were excited at 488 nm. RESULTS CB is maximally excited using a 407 nm laser line, when compared to UV or 413 nm excitation. The increase in sensitivity of CB at 407 nm can be contributed to higher absorption of CB and a reduction of autofluorescence at this excitation wavelength (Ropp et al.: Cytometry 21: 309-317, 1995). CONCLUSIONS Despite the fact that the CB antibody conjugate has a tendency to adhere specifically to a B cell subpopulation in bone marrow, we nevertheless could purify stem cells by using CB for the detection and elimination of lineage positive cells. Isolated stem cells from mouse fetal liver (Lin-CD34(+)Sca-1(+)c-Kit(high)) and adult bone marrow (Lin-CD34(-/low)Sca-1(+)c-Kit(+)) were transplanted into lethally irradiated mice, and the sorted stem cells had the ability to efficiently repopulate all mature hematopoietic lineages in recipient mice.

In an initial experiment we observed postprandial changes in plasma concentrations of growth hormone (GH), insulin, glucagon, and somatostatin (SRIF) in sheep. We then examined whether increasing the rumen concentration of volatile fatty acids (VFA) by infusing a VFA mixture at three rates (53.5, 107, and 214 micromol/kg/min for 4 hr) mimicked the postprandial changes in hormone secretion. Feeding significantly (P < 0.05) suppressed the plasma GH concentration for 6 hr, whereas it significantly (P < 0.05) increased plasma concentrations of insulin, glucagon, and SRIF. Plasma glucose levels tended to decrease after feeding but then gradually increased over the prefeeding level (P < 0.05). Intraruminal infusion of the VFA mixture at 107 micromol/kg/min caused similar changes in ruminal VFA concentrations to those seen after feeding. The infusion significantly (P < 0.05) suppressed GH secretion in a dose-dependent manner, whereas it caused a significant (P < 0.05) increase in insulin and glucose concentrations without changing glucagon concentrations. From these results, we conclude that the postprandial change in ruminal VFA concentration may be a physiological signal which modifies GH and insulin secretion in sheep.

The effects of biotin on insulin secretion in pair-fed control rats and biotin-deficient rats were investigated using the method of isolated pancreas perfusion. Isolated pancreas perfusion was performed using 20 mM glucose, 10 mM arginine, and 20 mM glucose plus various concentrations of biotin (20 mM glucose + biotin solution) as stimulants of insulin secretion. The insulin response to 20 mM glucose in biotin-deficient rats was approximately 22% of that seen in control rats. The level of the insulin response to 10 mM arginine was also significantly lower in biotin-deficient rats than in control rats. These results indicate that insulin release from the pancreas was disturbed in biotin-deficient rats. The insulin responses to 20 mM glucose + 1 mM biotin in biotin-deficient and control rats increased to 165% and 185%, respectively, of that to 20 mM glucose. These biotin-induced increases in glucose-stimulated insulin release were evident within the first few minutes of the infusion. An enhancement of the arginine-induced insulin response in control rats was not found when arginine and biotin was administered. These results suggest that biotin may play an important role in the mechanism by which glucose stimulates insulin secretion from the beta cells of the pancreatic islets.

Erythroid progenitors have the potential to proliferate rapidly in response to environmental stimuli. This process is referred to as stress erythropoiesis, with erythropoietin (EPO) playing central roles in its promotion. In this study, we wanted to elucidate the molecular mechanisms governing the regulation of stress erythropoiesis and the maintenance of red-cell homeostasis. This was achieved by our development of a noninvasive real-time monitoring system for erythropoiesis using transgenic mouse lines expressing luciferase under the control of the mouse Gata1 hematopoietic regulatory domain (G1-HRD-luc) or human β-globin locus control region (Hbb-LCR-luc). Optical bioluminescence images revealed that the luciferase was specifically expressed in spleen and bone marrow and was induced rapidly in response to anemia and hypoxia stimuli. The G1-HRD-luc activity tracked the emergence and disappearance of proerythroblast-stage progenitors, whereas the Hbb-LCR-luc activity tracked erythroblasts and later stage erythroid cells. Increased plasma EPO concentration preceded an increase in G1-HRD-luc, supporting our contention that EPO acts as the key upstream signal in stress erythropoiesis. Hence, we conclude that G1-HRD-luc and Hbb-LCR-luc reporters are differentially activated during stress erythropoiesis and that the transgenic mouse lines used serve as an important means for understanding the homeostatic regulation of erythropoiesis.

Recent successful synthesis of human glicentin prompted us to establish an immunoassay method for determination of human glicentin in plasma. Human glicentin in plasma was measured using a newly developed sandwich ELISA. The mean fasting levels of human glicentin were 18.6+/-2.4 and 19.7+/-2.1 pM in normal subjects and diabetic patients, respectively. In diabetic patients with renal failure, plasma glicentin was elevated, exceeding 100 pM. In normal subjects, plasma glicentin increased to a peak level of about 130 pM at 60 min after an oral glucose load, and then decreased. In patients who underwent gastrectomy, plasma glicentin rapidly increased to a peak of about 300 pM at 30 min after oral glucose load. In a patient with short bowel syndrome plasma glicentin did not change following an oral glucose load. These results correspond with previous findings for gut glucagon-like immunoreactive materials (GLI) or enteroglucagon. We conclude that glicentin is secreted from the small intestine in response to intraluminal glucose stimulation in humans.

Endothelial cells are an important component of the haemopoietic microenvironment. To investigate how endothelial cells are involved in haemopoiesis, two established murine endothelial cell lines were assayed in stromal cultures with fetal liver haemopoietic cells. Both endothelial cell lines allowed for the proliferation and differentiation of erythroid and monocyte-macrophage precursors, suggesting that support for haemopoiesis is a general property of endothelial cells. Erythropoiesis was dependent on the addition of erythropoietin (Epo), whereas myelopoiesis was independent of added Epo. Haemopoietic colonies developed in close contact with the endothelial cells. Erythroid colonies did not develop when transwell filters were used between the stroma and haemopoietic cells, or when conditioned medium was used in place of stromal cells. In contrast, monocyte-macrophage colonies formed in the presence of transwell filters or conditioned medium. Thus close cell contact is necessary for erythropoiesis but not myelopoiesis under these conditions. These results suggest that different molecular mechanisms are used by endothelial cells to support erythroid development and myeloid development in the mouse fetal liver.

The expression of cerebellin and cerebellin mRNA was studied by radioimmunoassay and Northern blot analysis in the human brain, adrenal gland and the tumour tissues of adrenal tumour, ganglioneuroblastoma and neuroblastoma. Immunoreactive cerebellin was detected in every region of brain studied, with the highest concentrations found in the hemisphere of the cerebellum (424·2 ± 12·6 pmol/g wet weight, n=6, mean ± s.e.m.) and the vermis of the cerebellum (256·8 ± 30·5 pmol/g wet weight). Immuno-reactive cerebellin was also detected in the pituitary (8·2 ± 1·8 pmol/g wet weight), the spinal cord (3·3 ± 0·3 pmol/g wet weight) and the normal parts of adrenal glands (2·98 ± 0·37 pmol/g wet weight, n=9) and some tumour tissues, such as phaeochromocytomas, cortisol-producing adrenocortical adenomas, ganglioneuroblastomas and neuroblastomas. Northern blot analysis showed that cerebellin mRNA was highly expressed in the hemisphere and vermis of the cerebellum. Cerebellin mRNA was also expressed in other regions of the brain and the tumour tissues of phaeochromocytoma, cortisol-producing adrenocortical adenoma, ganglioneuroblastoma and neuroblastoma. Immunocytochemistry of the normal adrenal gland showed that immunoreactive cerebellin was localized in the adrenal medulla. The present study has shown the expression of cerebellin and cerebellin mRNA, not only in the cerebellum but also in other regions of the brain and some tumours, such as cortisol-producing adrenocortical adenoma, phaeochromocytoma and neuroblastoma. These findings suggest possible pathophysiological roles of cerebellin peptides, not only in the cerebellum, but also in the extra-cerebellar tissues. Journal of Endocrinology (1997) 154, 27–34

Four neuropeptides; cerebellin, corticotropin-releasing hormone (CRH), neuropeptide Y and somatostatin were studied by radioimmunoassay in the postmortem human brains obtained from three patients with olivopontocerebellar atrophy (OPCA) and one with Shy-Drager syndrome. Significant decreases in cerebellin and CRH concentrations were found in the cerebellar hemisphere of these diseases compared with controls. These findings suggest important pathophysiological roles of cerebellin and CRH in these cerebellar diseases. Such significant decreases were not found in neuropeptide Y and somatostatin.

Pre-diabetic male Zucker diabetic fatty rats (ZDF) become diabetic between 8 and 10 weeks of age. At that time their beta cells exhibit high basal insulin secretion, absent insulin response to glucose and loss of GLUT 2 glucose transporter. Beta-cell volume, which is increased at the onset of non-insulin-dependent diabetes, declines precipitously by age 18 weeks. To determine if expression of this diabetic phenotype was dependent upon the increased food intake of these rats, they were diet-matched to lean littermates for 12 weeks beginning at 6 weeks of age. Untreated control ZDF rats received an unrestricted diet for 3 months. All of the controls became hyperglycaemic by 8 weeks of age, whereas all diet-matched rats remained euglycaemic throughout the 3 months, despite the fact that at 18 weeks of age their mean body weight equaled that of obese rats on an unrestricted diet. In the former rats glucose-stimulated insulin secretion was absent at 12 weeks of age and GLUT-2-positive beta cells had fallen below 30%. The volume fraction of their beta cells was 2.6 times normal at this age but by 18 weeks of age it had declined by 75%. Diet restriction for 3 months prevented the loss of glucose-stimulated insulin secretion and the reduction of beta-cell GLUT-2 and beta-cell volume fraction. However, neither the elevated basal insulin secretion nor the exaggerated arginine-stimulated insulin secretion of the obese rats was reversed or prevented by caloric restriction. We conclude that in diabetic ZDF rats the glucose incompetence of beta cells and the reduction of beta-cell GLUT 2, which coincide with the onset of hyperglycaemia, and the subsequent loss of beta-cell volume, occur only when the caloric intake is excessive. The increased basal insulin secretion and exaggerated insulin response to arginine appear to be relatively independent of caloric intake.

To elucidate the mechanism of the basal hyperinsulinemia of obesity, we perfused pancreata from obese Zucker and lean Wistar rats with substimulatory concentrations of glucose. Insulin secretion at 4.2 and 5.6 mM glucose was ∼10 times that of controls, whereas β-cell volume fraction was increased only 4-fold and DNA per islet 3.5-fold. We therefore compared glucose usage at 1.4, 2.8, and 5.6 mM. Usage was 8-11.4 times greater in Zucker islets at 1.4 and 2.8 mM and 4 times greater at 5.6 mM; glucose oxidation at 2.8 and 5.6 mM glucose was >12 times lean controls. To determine if the high free fatty acid (FFA) levels of obesity induce these abnormalities, normal Wistar islets were cultured with 0, 1, or 2 mM long chain FFA for 7 days. Compared to islets cultured without FFA insulin secretion by FFA-cultured islets (2 mM) perifused with 1.4, 3, or 5.6 mM glucose was increased more than 2-fold, bromodeoxyuridine incorporation was increased 3-fold, and glucose usage at 2.8 and 5.6 mM glucose was increased approximately 2-fold (1 mM FFA) and 3-fold (2 mM FFA). We conclude that hypersecretion of insulin by islets of obese Zucker fatty rats is associated with, and probably caused by, enhanced low Km glucose metabolism and β-cell hyperplasia, abnormalities that can be induced in normal islets by increased FFA.

Hyperinsulinemia, loss of glucose-stimulated insulin secretion (GSIS), and peripheral insulin resistance coexist in non-insulin-dependent diabetes mellitus (NIDDM). Because free fatty acids (FFA) can induce these same abnormalities, we studied their role in the pathogenesis of the NIDDM of obese Zucker diabetic fatty (ZDF-drt) rats from 5 weeks of age (before the onset of hyperglycemia) until 14 weeks. Two weeks prior to hyperglycemia, plasma FFA began to rise progressively, averaging 1.9 +/- 0.06 mM at the onset of hyperglycemia (P < 0.001 vs. controls). At this time GSIS was absent and beta-cell GLUT-2 glucose transporter was decreased. The triacylglycerol content of prediabetic islets rose to 10 times that of controls and was correlated with plasma FFA (r = 0.825; P < 0.001), which, in turn, was correlated with the plasma glucose concentration (r = 0.873; P < 0.001). Reduction of hyperlipacidemia to 1.3 +/- 0.07 mM by pair feeding with lean littermates reduced all beta-cell abnormalities and prevented hyperglycemia. Normal rat islets that had been cultured for 7 days in medium containing 2 mM FFA exhibited increased basal insulin secretion at 3 mM glucose, and first-phase GSIS was reduced by 68%; in prediabetic islets, first-phase GSIS was reduced by 69% by FFA. The results suggest a role for hyperlipacidemia in the pathogenesis of NIDDM; resistance to insulin-mediated antilipolysis is invoked to explain the high FFA despite hyperinsulinemia, and sensitivity of beta cells to hyperlipacedemia is invoked to explain the FFA-induced loss of GSIS.

We sought to explore the emerging concept that malonyl-CoA generation, with concomitant suppression of mitochondrial carnitine palmitoyltransferase I (CPT I), represents an important component of glucose-stimulated insulin secretion (GSIS) by the pancreatic β-cell (Prentki M, Vischer S, Glennon MC, Regazzi R, Deeney JT, Corkey BE: Malonyl-CoA and long-chain acyl-CoA esters as metabolic coupling factors in nutrient-induced insulin secretion. J Biol Chem 267:5802–5810, 1992). Accordingly, pancreases from fed rats were perfused with basal (3 mM) followed by high (20 mM) glucose in the absence or presence of 2 mM hydroxycitrate (HC), an inhibitor of ATP-citrate (CIT) lyase (the penultimate step in the glucose → malonyl-CoA conversion). HC profoundly inhibited GSIS, whereas CIT had no effect. Inclusion of 0.5 mM palmitate in the perfusate significantly enhanced GSIS and completely offset the negative effect of HC. In isolated islets, HC stimulated [1-14C]palmitate oxidation in the presence of basal glucose and markedly obtunded the inhibitory effect of high glucose. Directional changes in 14C incorporation into phospholipids were opposite to those of 14CO2 production. At a concentration of 0.2 mM, 2-bromostearate, 2-bromopalmitate and etomoxir (all CPT I inhibitors) potentiated GSIS by the pancreas and inhibited palmitate oxidation in islets. However, at 0.05 mM, etomoxir did not influence insulin secretion but still caused significant suppression of fatty acid oxidation. The results provide more direct evidence for a pivotal role of malonyl-CoA suppression of CPT I, with attendant elevation of the cytosolic long-chain acyl-CoA concentration, in GSIS from the normal pancreatic β-cell. They also raise the possibility that the tested CPT I inhibitors act, in part, by generating non-metabolizable acyl-CoA species that mimic the effects of natural acyl-CoAs in triggering insulin release.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a novel hypothalamic peptide consisting of 38 amino acids (PACAP1-38) with a potent stimulatory action on adenylate-cyclase in rat pituitary. The presence of PACAP-like immunoreactivity in human brain was studied by radioimmunoassay. Co-localization of PACAP with arginine vasopressin and oxytocin was investigated by immunocytochemistry in the human hypothalamus. Immunoreactive PACAP was detected in all regions of human brain (cortex, thalamus, hypothalamus, pons and hemisphere of cerebellum) with the highest levels found in the hypothalamus (8.5 +/- 1.9 pmol/g wet weight, n = 4, mean +/- S.E.M.). High performance liquid chromatography of the human hypothalamic extract showed that approximately 50% of the immunoreactive PACAP was eluted in the position of PACAP1-38. Immunocytochemical studies showed the presence of PACAP immunoreactive neurons in the paraventricular and supraoptic nuclei of human hypothalamus. PACAP co-localized with arginine vasopressin in magnocellular cells of these nuclei. These findings suggest that PACAP1-38 plays important physiological roles in the human hypothalamus.

Spontaneous and dexamethasone-induced noninsulin-dependent diabetes mellitus (NIDDM) in rats is associated with loss of glucose-stimulated insulin secretion (GSIS) and a reduction in both GLUT-2-positive beta cells and high Km glucose transport. To determine if the chronology and correlation of these abnormalities is consistent with a causal relationship, Zucker (fa/fa) rats were studied longitudinally before and during 10 d of dexamethasone-induced (0.4 mg/kg per d i.p.) NIDDM. Within 24 h of dexamethasone treatment blood glucose rose and GSIS declined, becoming paradoxically negative (-87 +/- 12 microU/ml per min) on day 10. Blood glucose was negatively correlated with GSIS (r = -0.92; P < 0.001). 3-0-methyl-D-glucose (3MG) transport was unchanged at 12 h, 23% below normal on day 1, and declined further to a nadir 59% below normal. The GLUT-2-positive beta cell area did not decline until 48 h, reaching a nadir of 35% of normal at 10 d. The area of GLUT-2-positive beta cells was correlated with GSIS (r = 0.77; P < 0.005). We conclude that the chronology and correlation between GSIS loss and hyperglycemia is consistent with a cause-effect relationship, but that the subtotal impairment in glucose transport by itself cannot explain the total loss of GSIS if one assumes that normal beta cells are functionally homogenous.

Fetal liver stromal cells established from 13-day-old embryos support erythropoiesis in vitro in combination with erythropoietin (Epo). Epo stimulated growth of these stromal cells in a dose-dependent manner and mRNA for Epo receptor was detected. These results suggest the possible involvement of Epo in the generation of the fetal erythropoietic microenvironment during development.

To determine if increased secretion of amylin can be implicated in the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) in vitro and in vivo, we studied its relationships to insulin in insulin-resistant rats with and without NIDDM. In obesity-associated and dexamethasone-induced insulin resistance without diabetes, basal and stimulated secretion of amylin and insulin by isolated pancreata were proportionately elevated, leaving the amylin-to-insulin ratio (A/I) unchanged. By contrast, whenever diabetes occurred in dexamethasone-treated rats or in spontaneously diabetic obese insulin-resistant ZDF-drt male rats, a doubling of A/I was invariably observed due to an increase in amylin without a proportional increase in insulin secretion. Correction of dexamethasone-induced hyperglycemia with the glucocorticord receptor antagonist RU-486 was accompanied by a decline in A/I. Longitudinal in vivo studies demonstrated in both spontaneous and dexamethasone-induced models of NIDDM an increase in plasma A/I at the onset of hyperglycemia. In dexamethasone-induced diabetes, the increased A/I was associated with a high proamylin mRNA relative to proinsulin mRNA. We conclude that amylin and insulin expression and secretion rise in concert in compensated insulin-resistant states, but when hyperglycemia is present the increase in amylin exceeds that of insulin. Although a role of an increased A/I in the pathogenesis of NIDDM has not been established directly, these studies indicate that such a role could be possible.

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

The presence of human melanin-concentrating hormone (MCH) was studied in the human brain by radioimmunoassay and immunocytochemistry. Immunoreactive MCH concentrations in the human brain ranged from 0.07 to 19.7 pmol/g wet weight. High performance liquid chromatography of the hypothalamus showed a large immunoreactive peak in the position of human/rat MCH, which was eluted 9 min later than that of salmon MCH. Free-floating sections (40 μm) of the hypothalamus were immunostained. Positive MCH immunostaining was found in perifornical, tuberomammillary, and posterior nuclei. Numerous MCH-immunoreactive nerve fibers were observed throughout the hypothalamus. The presence of high concentrations of MCH in the human brain, in particular in the hypothalamus, suggests that MCH is a neurotransmitter, a neuromodulator, or a neurohormone in man.

The effect of intravenous infusion of acetate, propionate and butyrate (0, 3, 10, 30 mumol kg-1 min-1 over 40 min) on the secretion of growth hormone (GH), insulin and glucagon in response to growth hormone-releasing factor (GRF) injection (0.25 micrograms/kg, 10 min after the onset of acid infusion) was determined in six sheep. The intravenous injection of GRF caused a marked increase in plasma GH at every dose of each acid. The GH response to GRF was unaffected by an intravenous infusion of acetate. The basal plasma levels of insulin, glucagon and glucose were unchanged by acetate infusion. The infusion of propionate markedly suppressed the GH response to GRF in a dose-dependent manner. Propionate produced increases in plasma insulin, glucagon and glucose concentrations. Butyrate infusion also caused a significant attenuation of GRF-induced GH secretion. Butyrate infusion stimulated the secretion of both insulin and glucagon and caused hyperglycemia. After cessation of the infusion of propionate or butyrate plasma GH tended to increase again. Plasma somatostatin concentrations, which were measured only for the highest dose of butyrate, were unchanged during acid infusion, but increased on discontinuing the infusion. It is concluded that propionate and butyrate suppress GH secretion, while stimulating the secretion of insulin and glucagon in sheep.

1. We investigated the usefulness of neuropeptide Y as a plasma marker for phaeochromocytoma, ganglioneuroblastoma and neuroblastoma using a simple and highly sensitive r.i.a. for human neuropeptide Y. 2. Plasma immunoreactive neuropeptide Y concentrations were measured without extraction in plasma samples (100 μl) from patients with various diseases. 3. The plasma immunoreactive neuropeptide Y concentration in patients with phaeochromocytoma (172.3 ± 132.4 pmol/l, mean ± sd, n = 23) was significantly higher than that in healthy adult subjects (40.1 ± 10.1 pmol/l, n = 40, P 4. The plasma immunoreactive neuropeptide Y concentration in patients with essential hypertension (34.0 ± 3.7 pmol/l, n = 18) was within the normal range, but in patients with chronic renal failure undergoing maintenance haemodialysis (192.1 ± 68.0 pmol/l, n = 25) and in non-dialysed patients with chronic renal failure (85.1 ± 23.1 pmol/l, n = 7) it was significantly higher than that in healthy adult subjects (P 5. Eighty-seven per cent of the patients with phaeochromocytoma, 67% of the patients with ganglioneuroblastoma and 80% of the patients with neuroblastoma showed plasma immunoreactive neuropeptide Y concentrations higher than the upper limits in the control subjects [62 pmol/l (adult) and 160 pmol/l (children)]. 6. These results suggest that neuropeptide Y is a useful plasma marker for these tumours in addition to other factors unless the patients have renal failure.

The roles of insulin resistance and beta-cell dysfunction in glucocorticoid-induced diabetes were determined in Wistar and Zucker (fa/fa) rats. All Wistar rats treated with 5 mg/kg per d of dexamethasone for 24 d exhibited increased beta-cell mass and basal and arginine-stimulated insulin secretion, indicating insulin resistance, but only 16% became diabetic. The insulin response to 20 mM glucose was normal in the perfused pancreas of all normoglycemic dexamethasone-treated rats but absent in every diabetic rat. Immunostainable high Km beta-cell transporter, GLUT-2, was present in approximately 100% of beta-cells of normoglycemic rats, but in only 25% of beta cells of diabetic rats. GLUT-2 mRNA was not reduced. All Zucker (fa/fa) rats treated with 0.2-0.4 mg/kg per d of dexamethasone for 24 d became diabetic and glucose-stimulated insulin secretion was absent in all. High Km glucose transport in islets was 50% below nondiabetic controls. Only 25% of beta cells of diabetic rats were GLUT-2-positive compared with approximately 100% in controls. Total pancreatic GLUT-2 mRNA was increased twofold suggesting a posttranscriptional abnormality. We conclude that dexamethasone induces insulin resistance, whether or not it induces hyperglycemia. Whenever hyperglycemia is present, GLUT-2-positive beta cells are reduced, high Km glucose transport into beta cells is attenuated and the insulin response to glucose is absent.

反芻家畜へのアミノ酸投与は,各種の代謝性ホルモンの分泌を介して生産性に寄与すると考えられる.本研究では,グリシン,ロイシン,アスパラギン酸をそれぞれ4日間(96時間),十二指腸内に連続投与した場合の血漿ホルモン濃度について検討した.予め麻酔下に十二指腸カテーテルおよび頚動脈ループを装着した去勢雄成ヒツジ6頭を供試し,各アミノ酸連続投与4日目に,頚動脈より経時的に採血し,GH,インスリン,グルカゴン,IGF-Iの血漿濃度を測定した.アミノ酸の注入量は,0,3.6,7.2,14.4mmol/kg/dayとした.グリシン投与により,血漿インスリンおよびグルカゴン濃度は,投与量に依存して上昇した.最も高い1日平均血漿インスリン濃度は,グリシン14.4mmol/kg/day注入時の26.2±3.4μU/ml(対照12.4±1.6μU/ml)であり,血漿グルカゴン濃度は,グリシン7.2mmol/kg/day注入時の148±20pg/ml(対照27.9±7.1pg/ml)が最大であった.また,グリシン注入によりI/G比(インスリン/グルカゴン:モル比)は,他のアミノ酸注入に比べ,有意に低下した.グリシンは,血漿GH, IGF-I濃度に明かな変動を与えなかった.一方,ロイシン,アスパラギン酸の投与により,血漿インスリン,グルカゴン,IGF-I濃度が各々有意に上昇したが,血漿GH濃度にはほとんど影響を与えなかった.以上の結果から,特定アミノ酸を十二指腸に注入することで,ヒツジの血漿インスリン,グルカゴンおよびIGF-I濃度を調節できることが示唆された.

The effects of intravenous infusion of 17 amino acids, each at a dose of 3 mmol/kg over 30 min, on the secretion of insulin, glucagon, and growth hormone (GH) were studied in 6 castrated male sheep. Insulin-like growth factor I (IGF-I) secretion was also studied using eight of the amino acids. Plasma alpha-amino nitrogen reached a peak at 30 min followed by a gradual decrease thereafter. The greatest increase was obtained using aspartic acid and the smallest with methionine, responses to the remaining amino acids lying between these two. Leucine was the most effective amino acid in stimulating insulin secretion but did not produce any increase in glucagon and GH secretion. Alanine, glycine, and serine induced a greater enhancement of both glucagon and insulin secretion than other amino acids. No amino acid was able to specifically stimulate glucagon secretion without also increasing insulin or GH secretion. With regard to insulin and glucagon secretion, amino acids could be divided into groups according to their R groups. Neutral straight-chain amino acids stimulated both insulin and glucagon secretion, with a greater secretory response to shorter C-chain amino acids. Branched-chain amino acids tended to enhance insulin and suppress glucagon secretion. Acidic amino acids caused an increase in GH secretion. Aspartic acid caused the strongest stimulation of GH secretion, exceeding that induced by arginine. No changes in plasma IGF-I were brought about by any of the amino acids tested.

ODA, S., FUJIMURA, H., SASAKI, Y, and OHNEDA, A. α2-Adrenergic Modulation of Glucagon and Insulin Secretions in Sheep. Tohoku J. Exp. Med., 1991, 163 (2), 101-110 - To investigate the effects of α2-adrenergic receptors on the secretions of pancreatic glucagon and insulin, clonidine, midaglizole and yohimbine were intravenously administered in four conscious sheep. Clonidine infusion at a dosage of 1.0nmol/kg/min produced hyperglucagonemia, hypoinsulinemia and hyperglycemia. Midaglizole or yohimbine was infused for 30min, at doses of 5, 10 and 50nmol/kg/min during the clonidine infusion. The highest yohimbine infusion (50nmol/kg/min) blocked the clonidine-induced responses of glucagon, insulin and glucose. On the other hand, the midaglizole (50nmol/kg/min) infusion brought about no statistical effect on the clonidine-induced responses of glucagon, insulin and glucose. The α2-adrenergic antagonistic effect of midaglizole was clearly less than that of yohimbine in the present experiments. It is concluded that the glucagon secretion is enhanced and the insulin release is inhibited by α2-adrenergic stimulation in conscious sheep.

To investigate the release of neuropeptide Y (NPY) from the pheochromocytomas, we studied the relationship between the plasma and tumor tissue immunoreactive (IR) NPY concentrations in 13 patients with pheochromocytoma and measured the IR-NPY concentration in plasma samples obtained by catheter from several veins (jugular veins, superior vena cava, renal veins, adrenal veins and inferior vena cava) in 2 patients with pheochromocytoma. The plasma IR-NPY concentration in 13 patients with pheochromocytoma ranged from 118 to 1460 pg/ml and the concentration in 10 of 13 patients with pheochromocytoma was above 290 pg/ml (the upper limit of normal range). The tumor tissue IR-NPY ranged from 0.025 to 95.3 micrograms/g wet tissue. Plasma IR-NPY was parallel with tumor tissue IR-NPY in 13 cases of pheochromocytoma (r = 0.76, P less than 0.01). The highest concentration of IR-NPY was found in plasma obtained from the drainage vein from a tumor among the plasma samples obtained from several veins in 2 cases of pheochromocytoma. These findings indicate that in patients with pheochromocytoma, NPY is in most cases excessively released from the tumors into the systemic circulation and plasma IR-NPY in the periphery is increased.

Thirty diabetics who had been receiving ordinary insulin were switched to S-lente insulin, a new mixture of four parts semilente and six parts ultralente insulin. Eight times a day, we measured the glucose, insulin, and C-peptide (CPR) in their blood. Those with more than 250 mg/dl postprandial glucose were designated group A (18 patients) and the other were designated group B (12 patients). Group A diabetics experienced a significant decrease in fasting blood glucose levels whereas group B did not. S-lente improved the daily blood glucose profiles of 72% of group A and 25% of group B. It slightly reduced the sum of the daily blood glucose in group A and did not affect those of group B. The M-value fell significantly in group A but not in group B. Changes in this value correlated significantly with those of blood glucose determination sums. The sums of the determinations of free plasma insulin and CPR remained unaffected by the new insulin. It is concluded that S-lente insulin controls the blood glucose of diabetics whose postprandial blood glucose cannot be controlled by ordinary insulin.

1. Five sheep were used to investigate the influences of alpha-adrenergic subtype receptor blockade on the secretion of both glucagon and insulin. 2. The glucagon secretion was stimulated through an alpha 2-adrenergic subtype mechanism. 3. The secretion of insulin was inhibited by an alpha 2-adrenergic subtype mechanism in conscious sheep.

Central to cellular responses to hypoxic environment is the hypoxia-inducible factor (HIF) transcriptional control system. A role for HIF-2alpha was investigated in a model of renal ischemia-reperfusion injury (IRI) associated with oxidative stress using HIF-2alpha knockdown mice. In these mice, HIF-2alpha expression was approximately one half that of wild-type mice, whereas HIF-1alpha expression was equivalent. HIF-2alpha knockdown mice were more susceptible to renal IRI, as indicated by elevated blood urea nitrogen levels and semiquantitative histologic analysis. Immunostaining with markers of oxidative stress showed enhanced oxidative stress in the kidney of HIF-2alpha knockdown mice, which was associated with peritubular capillary loss. Real-time quantitative PCR analysis showed decreased expression of antioxidative stress genes in the HIF-2alpha knockdown kidneys. Studies that used small interference RNA confirmed regulation of the antioxidative stress genes in cultured endothelial cells. Although HIF-2alpha knockdown mice were anemic, serum erythropoietin levels were not significantly increased, reflecting inappropriate response to anemia as a result of HIF-2alpha knockdown. Experiments that used hemodiluted mice with renal ischemia demonstrated that anemia of this degree did not affect susceptibility to ischemia. Knockdown of HIF-2alpha in inflammatory cells by bone marrow transplantation experiments demonstrated that HIF-2alpha in inflammatory cells did not contribute to susceptibility to renal IRI. Restoration of HIF-2alpha in endothelium by intercrossing with Tie1-Cre mice ameliorated renal injury by IRI, demonstrating a specific role of endothelial HIF-2alpha. These results suggest that HIF-2alpha in the endothelium has a protective role against ischemia of the kidney via amelioration of oxidative stress.

Although clinical and experimental studies have long suggested a role for the renin-angiotensin system (RAS) in the regulation of erythropoiesis, the molecular basis of this role has not been well understood. We report here that transgenic mice carrying both the human renin and human angiotensinogen genes displayed persistent erythrocytosis as well as hypertension. To identify the receptor molecule responsible for this phenotype, we introduced both transgenes into the AT1a receptor null background and found that the hematocrit level in the compound mice was restored to the normal level. Angiotensin II has been shown to influence erythropoiesis by two means, up-regulation of erythropoietin levels and direct stimulation of erythroid progenitor cells. Thus, we conducted bone marrow transplantation experiments and clarified that AT1a receptors on bone marrow-derived cells were dispensable for RAS-dependent erythrocytosis. Plasma erythropoietin levels and kidney erythropoietin mRNA expression in the double transgenic mice were significantly increased compared with those of the wild-type control, while the elevated plasma erythropoietin levels were significantly attenuated in the compound mice. These results provide clear genetic evidence that activated RAS enhances erythropoiesis through the AT1a receptor of kidney cells and that this effect is mediated by the elevation of plasma erythropoietin levels in vivo.

Adrenomedullin is a potent vasodilator peptide that was isolated from human pheochromocytoma. But the presence of adrenomedullin in the brain has not been clarified. We studied the presence of adrenomedullin in the human brain obtained at autopsy from 6 subjects by radioimmunoassay, as well as in the human adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma. Immunoreactive adrenomedullin was detected in every region of human brain examined (0.26-1.4 pmol/g wet weight) with the highest concentrations found in thalamus (1.40 +/- 0.39 pmol/g wet weight, mean +/- SEM) and hypothalamus (1.28 +/- 0.48 pmol/g wet weight). Reverse phase high performance liquid chromatography showed that the immunoreactive adrenomedullin in the human brain was eluted in the position of synthetic human adrenomedullin 1-52. High concentrations of immunoreactive adrenomedullin were found in human adrenal glands (12.6 +/- 1.0 pmol/g wet weight, n = 7), pheochromocytoma (4.5 +/- 1.5 pmol/g wet weight, n = 11), ganglioneuroblastoma (2.0 +/- 1.3 pmol/g wet weight, n = 4) and neuroblastoma (0.55 +/- 0.21 pmol/g wet weight, n = 3). The present study has shown that adrenomedullin is present in the human brain in high concentrations, suggesting that adrenomedullin acts as a neurotransmitter, neuromodulator or neurohormone in man.

The presence of immunoreactive endothelin (IR-ET) was studied by radioimmunoassay in human kidney with and without clinical renal failure, and the levels were compared with those of three natriuretic peptides: atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). Kidney tissues were obtained at autopsy from three subjects with renal disorders (diabetic nephropathy, renal tubular necrosis, and end-stage kidney disease). Normal renal tissue was obtained at surgery from two patients with renal cell carcinoma. IR-ET was detected in three diseased kidneys obtained at autopsy (0.101 +/- 0.043 pmol/g wet weight, mean +/- SD) but not in tissues of two normal kidneys obtained at surgery (0.015 pmol/g wet weight). Reverse-phase HPLC showed that most of the IR-ET in the kidney (90%) was eluted in the position of ET-1. IR-ANP (0.23 +/- 0.06 pmol/g wet weight), IR-BNP (1.15 +/- 0.94 pmol/g wet weight), and IR-CNP (0.44 +/- 0.16 pmol/g wet weight) were detected in all samples examined. Higher concentrations of IR-BNP were found in three diseased kidneys obtained at autopsy (1.70 +/- 0.83 pmol/g wet weight vs. mean in two normal kidney tissues, 0.32 pmol/g wet weight). Such increases were not observed in IR-ANP or IR-CNP. These findings indicate that IR-ET is present in the human diseased kidney even in end-stage disease, with high concentrations comparable to those of natriuretic peptides. This raises the possibility that the production of ET-1 and BNP is increased in kidneys of patients with renal disorders.

The presence of immunoreactive endothelin (ir-ET) in the tumor tissues of two cases of ectopic ACTH-secreting bronchial carcinoid tumors was studied by radioimmunoassay. The cross-reaction with big ET-1, ET-2, and ET-3 was 5%, 6%, and 6%, respectively. Tumor tissue ir-ET concentrations in two ectopic ACTH-secreting bronchial carcinoid tumors were 920 and 3,370 fmol/g wet weight, which were much higher than those in pheochromocytomas (146 +/- 70 fmol/g wet weight; n = 12, mean +/- SD), adrenocortical tumors (115 +/- 105 fmol/g wet weight, n = 14), and normal parts of adrenal glands (82 +/- 31 fmol/g wet weight, n = 12). High-performance liquid chromatography of the tumor tissue extract showed that the ir-ET was mainly eluted in the position of ET-1. Plasma levels of ir-ET in these two cases were not elevated (1.2 and 1.7 pmol/L). The findings of the present study suggest that ET-1 has local pathophysiologic roles in the tumor tissues of ectopic ACTH-secreting bronchial carcinoid tumors.