Masked function of amino acid sensors on pancreatic hormone secretion in ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia.

SUMMARY: In neural regulation of the endocrine pancreas, there is much evidence to suggest that vagal efferents alter insulin and glucagon secretion, but less information on the effects of vagal afferents. In this study, we investigated the role and function of afferent fibers of the vagus nerve in normal and ventromedial hypothalamic (VMH) lesioned rats with marked hyperinsulinemia. In normal rats, hepatic vagotomy was associated with intraperitoneal (ip) arginine-induced enhancement of insulin and glucagon secretion without an accompanying change in blood glucose levels, ip leucine induced enhancement of insulin secretion accompanied by a decrease in blood glucose levels, and ip alanine-induced enhancement of glucagon secretion accompanied by an increase in blood glucose levels. In VMH lesioned rats with marked hyperinsulinemia, none of these amino acids caused significant changes in insulin and glucagon secretion. We conclude that amino acid sensors in normal rats inhibit excess release of pancreatic hormones induced directly by intake of amino acids, such as that in excess protein ingestion, and maintain blood glucose levels within the normal range. In contrast, in VMH lesioned rats with marked hyperinsulinemia, the function of the amino acid sensors is masked due to the marked hyperinsulinemia in these rats.:

Ventromedial hypothalamic lesions enhance small intestinal cell proliferation in mice.

BACKGROUND: We have found previously that ventromedial hypothalamic lesions (VMH) enhance cell proliferation in the visceral organs through vagal hyperactivity in rats. The goal of the current study was to determine the characteristics and nature of cell proliferation in the small intestine in VMH-lesioned mice. METHODS: The weight and length of the small intestine, thickness of the mucosal and muscle layers, number of proliferating cell nuclear antigen (PCNA)-positive cells, and mitotic cell count in the mucosal layer in VMH-lesioned and Sham VMH-lesioned mice were determined at 7 days after the operation. RESULTS: The weight and length of the small intestine in VMH-lesioned mice were significantly greater than those in Sham VMH-lesioned mice, by 11.6% and 15.0%, respectively. The thicknesses of the mucosal and muscle layers of the small intestine in VMH-lesioned mice were also significantly greater than those in Sham VMH-lesioned mice, by 12.7% and 12.5%, respectively. PCNA-positive cells and mitotic cells in the mucosal layer were densely present in crypts in VMH-lesioned mice, and were significantly increased by 31.9% and 71.7%, respectively, compared to Sham VMH-lesioned mice. CONCLUSIONS: These results demonstrate that VMH lesions in mice enhance cell proliferation in the mucosal layers and cause cell hypertrophy or cell proliferation in the muscle layers of the small intestine, which increases the weight and length of the small intestine. VMH lesions in mice may be a new tool for identifying growth factors and related genes involved in enlarging the small intestine mainly through cell proliferation.

Cell proliferation in ventromedial hypothalamic lesioned rats inhibits acute gastric mucosal lesions.

AIM: The role of mucosal layer thickness on prevention of acute gastric mucosal lesions (AGMLs) was examined in ventromedial hypothalamic (VMH)-lesioned rats. MATERIALS AND METHODS: The incidence of AGMLs after 48-h fasting and 60% ethanol injection into the stomach after 24-h fasting, aggressive factors (gastric acid and serum gastrin) and defensive factors [hexosamine, gastric mucosal blood flow (GMBF), serum thiobarbituric acid reacting substances (TBARS), and thickness of the gastric mucosal layer] were evaluated in VMH-lesioned rats. The effects of cell proliferation on the gastric mucosal layer of these rats were evaluated by H-E staining and immunostaining with proliferating cell nuclear antigen (PCNA). RESULTS: After 48-h fasting, no AGMLs were observed in VMH-lesioned and sham VMH-lesioned rats (controls). With 60% ethanol administration after 24-h fasting, the numbers of AGMLs were similar in the two groups, but the ulcer index, a marker of ulcer formation, was lower in VMH-lesioned rats compared to that in sham VMH-lesioned rats. VMH-lesioned rats showed increased gastric acid secretion and serum gastrin compared to sham VMH-lesioned rats, indicating an increase in aggressive factors in VMH-lesioned rats. The two groups had similar levels of gastric mucosal hexosamine, GMBF, and gastric mucosal TBARS, but VMH-lesioned rats had an increased thickness of the mucosal cell layer, indicating an increase in defensive factors in these rats. Histologically, VMH-lesioned rats had an increased total mucosal cell layer, especially for the surface epithelial cell layer, and an increased PCNA-labeling index, a marker of cell proliferation, especially in the proliferative zones of gastric mucosa, indicating increased cell proliferation in the proliferative zone of the gastric mucosa. CONCLUSION: VMH-lesioned rats are resistant to AGML formation due to increased cell proliferation in gastric mucosa through elevating the levels of defensive factors over those of aggressive factors.

Beneficial effects of ventromedial hypothalamus (VMH) lesioning on function and morphology of the liver after hepatectomy in rats.

Liver has a high regenerative capacity and restores its mass and function shortly after partial hepatectomy through increased proliferation and metabolic modification of hepatocytes. The proliferation of hepatocytes can be triggered by its mass reduction after hepatectomy or by the neural factors including lesioning of the ventromedial hypothalamus (VMH). In the present study, we examined the effect of VMH lesioning on liver regeneration in hepatectomized rats by evaluating liver function and morphology. We found that functional deficits caused by partial hepatectomy [prolonged prothrombin time (PT), increased indocyanine green (ICG) retention, and decrease in PAS (periodic Acid-Schiff staining)-positive hepatocytes] were restored by VMH lesioning at 1 week after the surgery, whereas these alterations disappeared at 4 weeks. Morphologically, lipid microdroplets, which are considered to be important for maintaining contiguous liver function via supplying fuel for cell proliferation, were found to accumulate in hepatocytes of the hepatectomized rats at early period (1 day) after partial hepatectomy. Interestingly, such lipid microdroplets were also detected in the VMH lesioned rats and the more abundantly in the VMH lesioned, hepatectomized rats up to 1 week after the surgery. In conclusion, our results suggest that VMH lesioning in rats promotes recovery of liver anatomically and functionally after partial hepatectomy by promoting cell proliferation process.

Cell proliferation in visceral organs induced by ventromedial hypothalamic (VMH) lesions: Development of electrical VMH lesions in mice and resulting pathophysiological profiles.

We have found that ventromedial hypothalamic (VMH) lesions produced by electrocoagulation induce cell proliferation in visceral organs through vagal hyperactivity, and also stimulate regeneration of partially resected liver in rats. To facilitate identification of proliferative and/or regenerative factors at the gene level, we developed electrical production of VMH lesions in mice, for which more genetic information is available compared to rats, and examined the pathophysiological profiles in these mice. Using ddy mice, we produced VMH lesions with reference to the previously reported method in rats. We then examined the pathophysiological profiles of the VMH-lesioned mice. Electrical VMH lesions in mice were produced using the following coordinates: 1.6 mm posterior to the bregma, anteriorly; 0.5 mm lateral to the midsagittal line, transversely; and 0.2 mm above the base of the skull, vertically, with 1 mA of current intensity and 10 s duration. The VMH-lesioned mice showed similar metabolic characteristics to those of VMH-lesioned rats, including body weight gain, increased food intake, increased percentage body fat, and elevated serum insulin and leptin. However, there were some differences in short period of hyperphagia, and in normal serum lipids compared to those of VMH-lesioned rats. The mice showed a similar cell proliferation in visceral organs, including stomach, small intestine, liver, and, exocrine and endocrine pancreas. In conclusion, procedures for development of VMH lesions in mice by electrocoagulation were developed and the VMH-lesioned mice showed pathophysiological profiles similar to those of VMH-lesioned rats, particularly in cell proliferation in visceral organs. These findings have not been observed previously in gold thioglucose-induced VMH-lesioned mice. This model may be a new tool for identifying factors involved in cell proliferation or regeneration in visceral organs.

High cardiovascular risk factors among obese children in an urban area of Japan.

SUMMARY: The association between degree of obesity and cardiovascular and related metabolic risk factors were examined in 355 Japanese obese school children from 11 to 12 years old. The parameters evaluated were blood pressure, serum lipids, fasting blood glucose, and serum ALT and AST. ALT, AST and triglycerides were more commonly evaluated in obese boys than in obese girls, while HDL-cholesterol was more commonly lowered in obese girls. Hypercholesterolemia was 2-fold, and abnormal liver functions were 3-fold more common in severely obese than in moderate obese children. Thus, cardiovascular and related metabolic risk factors are present in obesity in school-aged children, particularly in boys.:

The knockdown of endogenous replication factor C4 decreases the growth and enhances the chemosensitivity of hepatocellular carcinoma cells.

AIMS: To identify differentially expressed genes and thereby detect potential molecular targets for future therapies directed against hepatocellular carcinoma (HCC). METHODS: To isolate differentially expressed genes between HCC and adjacent non-cancerous liver tissues, cDNA microarray and quantitative reverse transcriptase polymerase chain reaction analyses were performed. Gene knockdown experiments in HepG2 cells were also performed using small interfering RNAs (siRNAs). Proteins were detected by immunostaining, and cell proliferation was analysed using the MTT/WST-8 assay. Apoptosis and cell cycle analyses were performed using flow cytometry. RESULTS: After an intensive screening for differentially expressed genes in HCC tissues, we isolated 23 upregulated genes in these lesions. Among these, we focused on the replication factor C4 (RFC4) gene. The expression of endogenous RFC4 proteins in HepG2 cells was found to be significantly reduced by RFC4-specific siRNA. This inhibition of RFC4 expression correlated with a decrease in cellular proliferation, increased levels of apoptosis and a sensitizing of the cells to the DNA-damaging chemotherapeutic agents, doxorubicin and camptothecin. CONCLUSION: The replication factor C4 gene may be a novel target for developing cancer therapeutics, which can enhance the antitumour activity of chemotherapeutic agents that induce DNA damage.

Activation of a system A amino acid transporter, ATA1/SLC38A1, in human hepatocellular carcinoma and preneoplastic liver tissues.

The expression of amino acid transporter (AT) mRNAs including A system (ATA1/SNAT1/SLC38A1, ATA2/SNAT2/SLC38A2 and ATA3/SNAT3/SLC38A4), L system (LAT1/SLC7A5 and LAT2/SLC7A8), and y+ (CAT2/SLC7A2) genes, were compared among hepatocellular carcinoma (HCC) and non-cancerous liver cells. Among them the ATA1 mRNA expression was significantly elevated in all HCC cell lines (HepG2, HLF, HuH7 and JHH4) examined compared with normal liver tissue. We further discovered that the expression of ATA1 mRNA was significantly activated in HCC tissues and also elevated in pre-malignant cirrhotic livers from HCC patients, compared with normal livers from non-HCC patients. The ATA1 protein was extensively accumulated in the cytoplasm of pre-malignant liver and most HCCs, while being weak or undetectably low in normal liver tissues. SiRNA-mediated suppression of endogenous ATA1 lowered the viability of HepG2 cells. Thus, the activation of ATA1 confers growth and survival advantages in pre-malignant and malignant liver lesions.

Transformation-associated gene regulation by ATF6alpha during hepatocarcinogenesis.

We have previously reported that the endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor 6 alpha (ATF6alpha) is implicated in the pathogenesis of hepatocellular carcinomas (HCCs). In order to further identify genes that are regulated by ATF6alpha, the global gene expression profiles of the ATF6alpha-transfected and untransfected HCC cell line, HLF, were analyzed. These results were then compared with the differential gene expression patterns of poorly differentiated HCC and control non-tumorous liver tissue. Our findings demonstrate that at least 18 genes are specifically upregulated by ATF6alpha, while another UPR mediator, XBP1 or ER-stress inducer, thapsigargin could partially stimulate or even repress some of them in HCC cells. Moreover, six of these identified genes contain potential ER stress-responsive elements and/or unfolded protein response elements in their 5' regulatory regions.

Vascular endothelial growth factor mRNA levels quantified by reverse transcription-polymerase chain reaction in microdissected breast carcinoma tissues are correlated with histological type and grade of both invasive and intraductal components.

In breast cancer, vascular endothelial growth factor (VEGF) is a prognostic factor, but the relationship of VEGF mRNA levels with various parameters or tumor progression is unclear. VEGF mRNA levels were measured in 48 cases of invasive ductal carcinoma by using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction (RT-PCR). The mean VEGF mRNA levels were compared among different histological types and grades in 41 and 29 samples of invasive and intraductal components, respectively. VEGF mRNA levels were always higher in cancerous cells than in non-cancerous cells, but mean VEGF mRNA levels were not significantly different between invasive component (3.24 +/- 3.18-fold the value of non-cancerous tissue) and intraductal component (4.14 +/- 4.43-fold). They were higher in papillotubular type than in other types, and higher in grade 2 carcinomas than in grade 3 carcinomas of invasive component, and higher in comedo type than in other types of intraductal component. Mean VEGF mRNA levels were higher in the VEGF-immunopositive group than in the VEGF-immunonegative group. There was no correlation between VEGF mRNA levels and tumor size, nodal status, or hormone receptor status. VEGF expression may play an important role in the development of both invasive and intraductal carcinoma components, especially those carcinoma components of less aggressive histological features.

Nuclear expression of cIAP-1, an apoptosis inhibiting protein, predicts lymph node metastasis and poor patient prognosis in head and neck squamous cell carcinomas.

cIAP-1, an apoptosis inhibiting protein, has been suggested to play important roles in the development of cervical and esophageal squamous cell carcinomas (SCCs). In order to clarify the subcellular localization of cIAP-1 and to investigate its clinicopathological significance in head and neck SCCs (HNSCCs), we examined cIAP-1 expression in four oral SCC cell lines by immunocytochemistry and Western blot. Expressions of nuclear and cytoplasmic cIAP-1, caspase-3, and Smac/DIABLO were also examined immunohistochemically in 57 cases of the HNSCCs. cIAP-1 expression was detected in HSC-2, HSC-3, and HSC-4 cells by immunohistochemistry and Western blot. In HSC-2 and HSC-4 cells, cIAP-1 was detected in both the nuclear and cytoplasmic fractions. Nuclear cIAP-1 expression was positive in 17 (30%) of HNSCCs, was correlated with lymph node metastasis (P=0.020) and advanced disease stage (P=0.032), and tended to be correlated with poor patient prognosis (P=0.059). Cytoplasmic cIAP-1 expression showed similar but weaker clinicopathological correlations. Nuclear cIAP-1 expression was inversely correlated with caspase-3 expression, but was correlated with Smac/DIABLO expression. Nuclear cIAP-1 expression appears to be a useful marker for predicting poor patient prognosis in HNSCCs, and may play roles in HNSCCs through the signaling pathway mediated by Smac/DIABLO and caspase-3.

An inter-subunit disulfide bond affects affinity of human lung extracellular superoxide dismutase to heparin.

Human extracellular superoxide dismutase (EC-SOD) was purified to homogeneity from lung tissue and the nature of the binding of heparin to EC-SOD was investigated. The enzyme was purified using three column chromatographic steps, and 127 microg of purified EC-SOD was obtained. A specific anti-human EC-SOD antibody was obtained by immunization with the purified enzyme. Western blot analysis of the heparin affinity chromatography product indicated that the presence of the inter-subunit disulfide bond affects the affinity of EC-SOD for heparin. The affinity of EC-SOD for heparin is a very important feature of the enzyme because it controls the distribution of the enzyme in tissues. The present study suggests that, not only the processing of the C-terminal region but inter-subunit disulfide bonds also play a role in determining the tissue distribution of EC-SOD. Moreover, the results obtained here also suggest that the redox state of the tissues might regulate the function of the EC-SOD.

Activation of the ATF6, XBP1 and grp78 genes in human hepatocellular carcinoma: a possible involvement of the ER stress pathway in hepatocarcinogenesis.

BACKGROUND/AIMS: We identified the glucose-regulated protein (grp) 78 as a transformation-associated gene in hepatocellular carcinoma (HCC). Grp78 is a molecular chaperone involved in the unfolded protein response, the expression of which can be regulated by the transcription factors ATF6 and XBP1. Thus, we investigated the regulatory mechanisms of the grp78 gene in liver malignancy. METHODS: Expression of grp78, ATF6 and XBP1 was examined by Northern blot, RT-PCR, immunoblot and immunohistochemical analyses. A reporter assay of the grp78 promoter was also performed. RESULTS: Elevation of grp78 and ATF6 mRNAs and the splicing of XBP1 mRNA, resulting in the activation of XBP1 product, occurred in HCC tissues with increased histological grading. Higher accumulation of the grp78 product in the cytoplasm, concomitantly with marked nuclear localization of the activated ATF6 product (p50ATF6), was observed in moderately to poorly differentiated HCC tissues. Cooperation between the distal DNA segment and the proximal endoplasmic reticulum stress response elements was essential for maximum transcription of the grp78 promoter in HCC cells. CONCLUSIONS: The endoplasmic reticulum stress pathway mediated by ATF6 and by IRE1-XBP1 systems seems essential for the transformation-associated expression of the grp78 gene in HCCs.

Nuclear translocation of extracellular superoxide dismutase.

Histochemical examination of mouse tissues showed nuclear staining of extracellular superoxide dismutase (EC-SOD), and the nuclear translocation of EC-SOD was also confirmed in cultured cells that had been transfected with its gene, as shown by immunohistochemistry and Western blot analysis. The EC-SOD which was secreted into the medium was incorporated into 3T3-L1 cells and a significant fraction of the material taken up was localized in the nucleus. Site-directed mutagenesis indicated that the heparin-binding domain of EC-SOD functions as the nuclear localization signal. These results suggest that the mechanism of the nuclear transport of EC-SOD involves a series of N-terminal signal peptide- and C-terminal heparin-binding domain-dependent processes of secretion, re-uptake and the subsequent nuclear translocation. The findings herein provide support for the view that the role of EC-SOD is to protect the genome DNA from damage by reactive oxygen species and/or the transcriptional regulation of redox-sensitive gene expression.