Stretch-induced reactive oxygen species contribute to the Frank-Starling mechanism.

Myocardial stretch physiologically activates NADPH oxidase 2 (NOX2) to increase reactive oxygen species (ROS) production. Although physiological low-level ROS are known to be important as signalling molecules, the role of stretch-induced ROS in the intact myocardium remains unclear. To address this, we investigated the effects of stretch-induced ROS on myocardial cellular contractility and calcium transients in C57BL/6J and NOX2-/- mice. Axial stretch was applied to the isolated cardiomyocytes using a pair of carbon fibres attached to both cell ends to evaluate stretch-induced modulation in the time course of the contraction curve and calcium transient, as well as to evaluate maximum cellular elastance, an index of cellular contractility, which is obtained from the end-systolic force-length relationship. In NOX2-/- mice, the peak calcium transient was not altered by stretch, as that in wild-type mice, but the lack of stretch-induced ROS delayed the rise of calcium transients and reduced contractility. Our mathematical modelling studies suggest that the augmented activation of ryanodine receptors by stretch-induced ROS causes a rapid and large increase in the calcium release flux, resulting in a faster rise in the calcium transient. The slight increase in the magnitude of calcium transients is offset by a decrease in sarcoplasmic reticulum calcium content as a result of ROS-induced calcium leakage, but the faster rise in calcium transients still maintains higher contractility. In conclusion, a physiological role of stretch-induced ROS is to increase contractility to counteract a given preload, that is, it contributes to the Frank-Starling law of the heart. KEY POINTS: Myocardial stretch increases the production of reactive oxygen species by NADPH oxidase 2. We used NADPH oxidase 2 knockout mice to elucidate the physiological role of stretch-induced reactive oxygen species in the heart. We showed that stretch-induced reactive oxygen species modulate the rising phase of calcium transients and increase myocardial contractility. A mathematical model simulation study demonstrated that rapid activation of ryanodine receptors by reactive oxygen species is important for increased contractility. This response is advantageous for the myocardium, which must contract against a given preload.

Activation of the Hypoxia Inducible Factor 1α Subunit Pathway in Steatotic Liver Contributes to Formation of Cholesterol Gallstones.

BACKGROUND & AIMS: Hypoxia-inducible factor 1α subunit (HIF1A) is a transcription factor that controls the cellular response to hypoxia and is activated in hepatocytes of patients with nonalcoholic fatty liver disease (NAFLD). NAFLD increases the risk for cholesterol gallstone disease by unclear mechanisms. We studied the relationship between HIF1A and gallstone formation associated with liver steatosis. METHODS: We performed studies with mice with inducible disruption of Hif1a in hepatocytes via a Cre adenoviral vector (inducible hepatocyte-selective HIF1A knockout [iH-HIFKO] mice), and mice without disruption of Hif1a (control mice). Mice were fed a diet rich in cholesterol and cholate for 1 or 2 weeks; gallbladders were collected and the number of gallstones was determined. Livers and biliary tissues were analyzed by histology, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunoblots. We measured concentrations of bile acid, cholesterol, and phospholipid in bile and rates of bile flow. Primary hepatocytes and cholangiocytes were isolated and analyzed. HIF1A was knocked down in Hepa1-6 cells with small interfering RNAs. Liver biopsy samples from patients with NAFLD, with or without gallstones, were analyzed by quantitative reverse-transcription polymerase chain reaction. RESULTS: Control mice fed a diet rich in cholesterol and cholate developed liver steatosis with hypoxia; levels of HIF1A protein were increased in hepatocytes around central veins and 90% of mice developed cholesterol gallstones. Only 20% of the iH-HIFKO mice developed cholesterol gallstones. In iH-HIFKO mice, the biliary lipid concentration was reduced by 36%, compared with control mice, and bile flow was increased by 35%. We observed increased water secretion from hepatocytes into bile canaliculi to mediate these effects, resulting in suppression of cholelithogenesis. Hepatic expression of aquaporin 8 (AQP8) protein was 1.5-fold higher in iH-HIFKO mice than in control mice. Under hypoxic conditions, cultured hepatocytes increased expression of Hif1a, Hmox1, and Vegfa messenger RNAs (mRNAs), and down-regulated expression of AQP8 mRNA and protein; AQP8 down-regulation was not observed in cells with knockdown of HIF1A. iH-HIFKO mice had reduced inflammation and mucin deposition in the gallbladder compared with control mice. Liver tissues from patients with NAFLD with gallstones had increased levels of HIF1A, HMOX1, and VEGFA mRNAs, compared with livers from patients with NAFLD without gallstones. CONCLUSIONS: In steatotic livers of mice, hypoxia up-regulates expression of HIF1A, which reduces expression of AQP8 and concentrates biliary lipids via suppression of water secretion from hepatocytes. This promotes cholesterol gallstone formation. Livers from patients with NAFLD and gallstones express higher levels of HIF1A than livers from patients with NAFLD without gallstones.

Life concerns of elderly people living at home determined as by Community General Support Center staff: implications for organizing a more effective integrated community care system. The Kurihara Project.

BACKGROUND: In Japan, the integrated community care system aims to enable people to continue to live in their homes. Based on the concept, one of the activities of a Community General Support Center (CGSC) is to provide preventive intervention based on a Community Support Program. Currently, a Basic Checklist (BC) is sent to elderly people to identify persons appropriate for a Secondary Prevention Program. METHODS: To find people who had not responded to the BC, CGSC staff evaluated the files of 592 subjects who had participated in the Kurihara Project to identify activities they cannot do that they did in the past, decreased activity levels at home, loss of interaction with people other than their family, and the need for medical interventions. This information was classified, when applicable, into the following categories: (A) 'no life concerns'; (B) 'undecided'; and (C) 'life concerns'. The relationships between these classifications and clinical information, certified need for long-term care, and items on the BC were examined. RESULTS: The numbers of subjects in categories A, B, and C were 291, 42, and 186, respectively. Life concerns were related to scores on the Clinical Dementia Rating, global cognitive function, depressive state, and apathy. Most items on the BC were not associated with classification into category C, but ≥25% of the subjects had life concerns related to these items. DISCUSSION: Assessment of life concerns by the CGSC staff has clinical validity. The results suggest that there are people who do not respond to the checklist or apply for Long-Term Care Insurance, meaning that they 'hide' in the community, probably due to apathy or depressive state. To organize a more effective integrated community care system, the CGSC staff should focus mainly on preventive care.

Epizootiological study of tick-borne encephalitis virus infection in Japan.

Tick-borne encephalitis virus (TBEV) is a zoonotic agent causing severe encephalitis in humans. Rodent species that are potential hosts for TBEV are widely distributed in various regions in Japan. In this study, we carried out large-scale epizootiological surveys in rodents from various areas of Japan. A total of 931 rodent and insectivore sera were collected from field surveys. Rodents seropositive for TBEV were found in Shimane Prefecture in Honshu and in several areas of Hokkaido Prefecture. These results emphasize the need for further epizootiological and epidemiological research of TBEV and preventive measures for emerging tick-borne encephalitis in Japan.

[Influence of protamine sulfate as neutralizer of heparin on a vancomycin assay in serum].

Levels of vancomycin (VCM), measured with cobas 6000 c501(c501), were low when blood-collecting tubes for heparinized blood (SQH) were used. It was determined that the phenomenon was due to the effects of protamine sulfate, a heparin neutralizer. VCM levels decreased by approximately 10% when the concentration of protamine sulfate was 0.01 mg/ml, and were undetectable when the concentration was 0.045 mg/ml. However, the effects of protamine sulfate, with the dose being up to approximately twice, were not seen in the presence of heparin. There were no such effects if specimens in SQH were maintained at a specified volume. The phenomenon was characteristic of the measurement of VCM levels using c501. Measurements of other agents such as valproic acid, which is measured in the same manner as VCM, using the same equipment did not lead to the identification of any effect. In addition, when VCM levels were measured with reagents of INTEGRA 800, no effect was seen. It is difficult to elucidate the mechanisms of action, as the manufacturer has not provided detailed information regarding reagents. Protamine sulfate is estimated to partially influence the antigen-antibody reaction involving anti-VCM antibodies in reagents of c501. Protamine sulfate is also used as an injection, and, hence, the influence of the agent on VCM levels measured with c501 cannot be ruled out even if other blood collecting tubes are used. Attachment to separating mediums presents problems when measuring blood concentrations of drugs, but there has been no report regarding a heparin neutralizer, as seen in this case. This is a new influential factor that requires attention.

[Dapagliflozin, a Sodium-Glucose Co-transporter-2 Inhibitor, Acutely Reduces Energy Expenditure in Brown Adipose Tissue via Neural Signals in Mice].

　Selective sodium glucose transporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i administration. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i on systemic energy expenditure have not been fully elucidated. We investigated the acute effects of dapagliflozin, an SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin administration, oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared with those after vehicle administration. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa), which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression, NE contents in BAT, and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, occurred prior to the suppression of BAT thermogenesis, e.g., 6 h after dapagliflozin treatment. Collectively, these results suggest that SGLT2i acutely suppresses energy expenditure in BAT via regulation of an interorgan neural network consisting of the common hepatic vagal branch and sympathetic nerves.

Olfactory receptors are expressed in pancreatic ß-cells and promote glucose-stimulated insulin secretion.

Olfactory receptors (ORs) mediate olfactory chemo-sensation in OR neurons. Herein, we have demonstrated that the OR chemo-sensing machinery functions in pancreatic ß-cells and modulates insulin secretion. First, we found several OR isoforms, including OLFR15 and OLFR821, to be expressed in pancreatic islets and a ß-cell line, MIN6. Immunostaining revealed OLFR15 and OLFR821 to be uniformly expressed in pancreatic ß-cells. In addition, mRNAs of Olfr15 and Olfr821 were detected in single MIN6 cells. These results indicate that multiple ORs are simultaneously expressed in individual ß-cells. Octanoic acid, which is a medium-chain fatty acid contained in food and reportedly interacts with OLFR15, potentiated glucose-stimulated insulin secretion (GSIS), thereby improving glucose tolerance in vivo. GSIS potentiation by octanoic acid was confirmed in isolated pancreatic islets and MIN6 cells and was blocked by OLFR15 knockdown. While Gα olf expression was not detectable in ß-cells, experiments using inhibitors and siRNA revealed that the pathway dependent on phospholipase C-inositol triphosphate, rather than cAMP-protein kinase A, mediates GSIS potentiation via OLFR15. These findings suggest that the OR system in pancreatic ß-cells has a chemo-sensor function allowing recognition of environmental substances obtained from food, and potentiates insulin secretion in a cell-autonomous manner, thereby modulating systemic glucose metabolism.

MicroRNAs 106b and 222 Improve Hyperglycemia in a Mouse Model of Insulin-Deficient Diabetes via Pancreatic ß-Cell Proliferation.

Major symptoms of diabetes mellitus manifest, once pancreatic ß-cell numbers have become inadequate. Although natural regeneration of ß-cells after injury is very limited, bone marrow (BM) transplantation (BMT) promotes their regeneration through undetermined mechanism(s) involving inter-cellular (BM cell-to-ß-cell) crosstalk. We found that two microRNAs (miRNAs) contribute to BMT-induced ß-cell regeneration. Screening murine miRNAs in serum exosomes after BMT revealed 42 miRNAs to be increased. Two of these miRNAs (miR-106b-5p and miR-222-3p) were shown to be secreted by BM cells and increased in pancreatic islet cells after BMT. Treatment with the corresponding anti-miRNAs inhibited BMT-induced ß-cell regeneration. Furthermore, intravenous administration of the corresponding miRNA mimics promoted post-injury ß-cell proliferation through Cip/Kip family down-regulation, thereby ameliorating hyperglycemia in mice with insulin-deficient diabetes. Thus, these identified miRNAs may lead to the development of therapeutic strategies for diabetes.

Dapagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, Acutely Reduces Energy Expenditure in BAT via Neural Signals in Mice.

Selective sodium glucose cotransporter-2 inhibitor (SGLT2i) treatment promotes urinary glucose excretion, thereby reducing blood glucose as well as body weight. However, only limited body weight reductions are achieved with SGLT2i treatment. Hyperphagia is reportedly one of the causes of this limited weight loss. However, the effects of SGLT2i treatment on systemic energy expenditure have not been fully elucidated. Herein, we investigated the acute effects of dapagliflozin, a SGLT2i, on systemic energy expenditure in mice. Eighteen hours after dapagliflozin treatment oxygen consumption and brown adipose tissue (BAT) expression of ucp1, a thermogenesis-related gene, were significantly decreased as compared to those after vehicle treatment. In addition, dapagliflozin significantly suppressed norepinephrine (NE) turnover in BAT and c-fos expression in the rostral raphe pallidus nucleus (rRPa) which contains the sympathetic premotor neurons responsible for thermogenesis. These findings indicate that the dapagliflozin-mediated acute decrease in energy expenditure involves a reduction in BAT thermogenesis via decreased sympathetic nerve activity from the rRPa. Furthermore, common hepatic branch vagotomy abolished the reductions in ucp1 expression and NE contents in BAT and c-fos expression in the rRPa. In addition, alterations in hepatic carbohydrate metabolism, such as decreases in glycogen contents and upregulation of phosphoenolpyruvate carboxykinase, manifested prior to the suppression of BAT thermogenesis, e.g. 6 hours after dapagliflozin treatment. Collectively, these results suggest that SGLT2i treatment acutely suppresses energy expenditure in BAT via regulation of an inter-organ neural network consisting of the common hepatic vagal branch and sympathetic nerves.

Establishment of a neutralization test involving reporter gene-expressing virus-like particles of tick-borne encephalitis virus.

Previously, a system for packaging tick-borne encephalitis virus (TBEV) subgenomic replicon RNAs into single-round infectious virus-like particles (VLPs) was developed. In the present study, VLPs were applied to measuring the levels of neutralizing antibodies against TBEV as an alternative to performing neutralization tests with live virus. As markers of VLP infection, the genes for GFP and luciferase were inserted into the TBEV replicon, which was then packaged into VLPs. The reporter genes were expressed in cells that were infected with the VLPs, and this infection was inhibited by neutralizing antibodies to TBEV. Serum samples from wild rodents were used to evaluate the neutralization test using VLPs. All the sera that were positive in the conventional neutralization test were also found to be positive in the neutralization test using VLPs, and there were highly significant correlations between the neutralization titres obtained using the native virus and those using VLPs. These results indicate that VLPs that express reporter genes represent a useful and safe alternative to conventional neutralization testing using live virus.