Interaction between inorganic phosphate concentration and glucose metabolism in mild refeeding syndrome model.

Refeeding syndrome is a major clinical problem that leads to fatal complications in patients suffering from malnutrition. Hypophosphatemia inevitably is observed at the onset of refeeding syndrome and therefore is monitored during refeeding; however, the causes of metabolic changes in phosphate concentration during refeeding remain poorly understood. In a previous study, we established a refeeding syndrome model employing total parenteral nutrition with insulin-induced hypophosphatemia, but the symptoms were severe and the metabolic mechanisms in this model may not have been representative of clinical conditions. Therefore, we established a new animal model of mild refeeding syndrome by using a shorter fasting period followed by a single refeeding. These mild refeeding syndrome-model rats exhibited hypophosphatemia without increases in urinary phosphate excretion. Interestingly, administration of the combination of phosphate and insulin during refeeding promoted insulin secretion during refeeding. This model implies that Pi may directly promote insulin secretion in pancreatic cells. These results clarify the interaction between phosphate and glucose metabolism pancreatic cells during refeeding syndrome in a mild refeeding syndrome model.

SLC37A2, a phosphorus-related molecule, increases in smooth muscle cells in the calcified aorta.

Vascular calcification is major source of cardiovascular disease in patients with chronic kidney disease. Hyperphosphatemia leads to increased intracellular phosphorus influx, which leads to an increase in osteoblast-like cells in vascular smooth muscle cell. PiT-1 transports phosphate in vascular smooth muscle cell. However, the mechanism of vascular calcification is not completely understood. This study investigated candidate phosphorus-related molecules other than PiT-1. We hypothesized that phosphorus-related molecules belonging to the solute-carrier (SLC) superfamily would be involved in vascular calcification. As a result of DNA microarray analysis, we focused on SLC37A2 and showed that mRNA expression of these cells increased on calcified aotic smooth muscle cells (AoSMC). SLC37A2 has been reported to transport both glucose-6-phosphate/phosphate and phosphate/phosphate exchanges. In vitro analysis showed that SLC37A2 expression was not affected by inflammation on AoSMC. The expression of SLC37A2 mRNA and protein increased in calcified AoSMC. In vivo analysis showed that SLC37A2 mRNA expression in the aorta of chronic kidney disease rats was correlated with osteogenic marker genes. Furthermore, SLC37A2 was expressed at the vascular calcification area in chronic kidney disease rats. As a result, we showed that SLC37A2 is one of the molecules that increase with vascular calcification in vitro and in vivo.

Effects of dietary fiber on vascular calcification by repetitive diet-induced fluctuations in plasma phosphorus in early-stage chronic kidney disease rats.

Vascular calcification progresses under hyperphosphatemia, and represents a risk factor for cardiovascular disease in chronic kidney disease (CKD) patients. We recently indicated that phosphorus (P) fluctuations also exacerbated vascular calcification in early-stage CKD rats. Dietary fiber intake is reportedly associated with cardiovascular risk. This study investigated the effects of dietary fiber on vascular calcification by repeated P fluctuations in early-stage CKD rats. Unilateral nephrectomy rats were used as an early-stage CKD model. For 36 days, a P fluctuation (LH) group was fed low-P (0.02% P) and high-P (1.2% P) diets alternating every 2 days, and a P fluctuation with dietary fiber intake (LH + F) group was fed low-P and high-P diets containing dietary fiber alternating every 2 days. The effect on vascular calcification was measured calcium content. Effects on uremic toxin were measured levels of indoxyl sulfate (IS) and investigated gut microbiota. The LH + F group showed significantly reduced vessel calcium content compared to the LH group. Further, dietary fiber inhibited increases in blood levels of IS after intake of high-P diet, and decreased uremic toxin-producing intestinal bacteria. Dietary fiber may help suppress progression of vascular calcification due to repeated P fluctuations in early-stage CKD rats by decreasing uremic toxin-producing intestinal bacteria.

Effects of repetitive diet-induced fluctuations in plasma phosphorus on vascular calcification and inflammation in rats with early-stage chronic kidney disease.

Cardiovascular disease is a major cause of death among hemodialysis patients. Hyperphosphatemia induces cardiovascular disease through vascular endothelial dysfunction and calcification. Repetition of a short-term excessive-phosphorus (P) diet causes transient elevations in plasma P and subsequent vascular endothelial dysfunction in normal rats. The purpose of this study was to investigate the effects of the P fluctuation on vascular calcification and inflammation in rats after unilateral nephrectomy as an early-stage chronic kidney disease (CKD) model. Rats were bred for 36 days; CP group, fed a control P (0.6%) diet; HP group, fed a high-P (1.2%) diet; and P fluctuation group, fed low-P (0.02%) and high-P diets alternately every 2 days. Influences on vascular calcification were analyzed using Von Kossa staining and measurement of vessel Ca content. The influence on inflammation was measured as urinary levels of 8-hydroxy-2'-deoxyguanosine. We demonstrated that the P fluctuation group showed similar vascular calcification and inflammation to the HP group, despite having the same total P intake as the CP group. A diet avoiding P fluctuations may be important for patients with early-stage CKD.

The ellagitannin trimer rugosin G inhibits recombinant human histidine decarboxylase.

Rugosin G, an ellagitannin trimer, was isolated from the water-soluble fraction of red rose petals, and its inhibitory activity against recombinant human histidine decarboxylase was investigated. Rugosin G showed potent inhibition compared to ellagitannin monomers and a dimer with macrocyclic structure (oenothein B), suggesting the potent inhibition of rugosin G was attributed to its linear oligomeric conformation. Abbreviations: HDC, histidine decarboxylase; Me2CO, acetone; EtOAc, ethyl acetate.

Dietary intake of inorganic phosphorus has a stronger influence on vascular-endothelium function than organic phosphorus.

Phosphorus management through dietetic therapy is vital for the prevention of cardiovascular disease in chronic kidney disease patients. There are two main sources of phosphorus in the diet, organic phosphorus from protein and inorganic phosphorus from food additives. The adverse effects of high phosphorus intake on vascular-endothelium function have been reported; however, the differences in the effects of organic phosphorus versus inorganic phosphorus are not clear. In this study, we examined an acute effect of these high phosphorus meals intake on vascular-endothelium function. This was a randomized, double-blind, cross-over test study design targeting healthy young men. We conducted a food intake test using two test meals, one high in organic phosphorus from organic food sources, and one high in inorganic phosphorus from food additives. Endothelium-dependent vasodilation, phosphorus and calcium in the urine and blood, and phosphorus-related hormones were measured preprandial to 120 min postprandial. The results showed higher serum and urine phosphorus values after the high inorganic phosphorus meal, and a significant reduction in endothelium-dependent vasodilation at 30 min postprandial. These findings are evidence that inorganic phosphorus has a stronger influence on vascular-endothelium function than organic phosphorus.

Hypophosphatemia occurs with insulin administration during refeeding by total parenteral nutrition in rats.

Refeeding syndrome (RFS) is characterized by the metabolic and clinical changes that occur following aggressive nutritional supplementation in malnourished patients. Hypophosphatemia is the hallmark of RFS and is key to its prevention and treatment in clinical practice. However, the mechanism of hypophosphatemia during RFS is unclear because of the lack of an animal model. In this study, we developed a rat RFS model as a first step to clarifying the molecular mechanism. After establishing the parenteral route, rats were fasted for 5 days and refeeding was started using total parenteral nutrition. The animals were infused with a high calorie solution with or without insulin administration. Results showed that plasma phosphate levels did not decrease in rats infused with the high calorie solution alone;in contrast, a 20% reduction compared to baseline was observed in rats administered insulin. In addition, rats infused with the high calorie solution containing added phosphate did not present with hypophosphatemia. Thus, we developed a rat RFS model with hypophosphatemia by tube feeding and insulin administration, and demonstrated the importance of phosphate in preventing refeeding hypophosphatemia. J. Med. Invest. 65:50-55, February, 2018.

Inhibition of Morganella morganii Histidine Decarboxylase Activity and Histamine Accumulation in Mackerel Muscle Derived from Filipendula ulumaria Extracts.

Filipendula ulmaria, also known as meadowsweet, is an herb; its extract was examined for the prevention of histamine production, primarily that caused by contaminated fish. The efficacy of meadowsweet was assessed using two parameters: inhibition of Morganella morganii histidine decarboxylase (HDC) and inhibition of histamine accumulation in mackerel. Ellagitannins from F. ulmaria (rugosin D, rugosin A methyl ester, tellimagrandin II, and rugosin A) were previously shown to be potent inhibitors of human HDC; and in the present work, these compounds inhibited M. morganii HDC, with half maximal inhibitory concentration values of 1.5, 4.4, 6.1, and 6.8 µM, respectively. Application of the extracts (at 2 wt%) to mackerel meat yielded significantly decreased histamine accumulation compared with treatment with phosphate-buffered saline as a control. Hence, F. ulmaria exhibits inhibitory activity against bacterial HDC and might be effective for preventing food poisoning caused by histamine.

Inhibitory activity of Filipendula ulmaria constituents on recombinant human histidine decarboxylase.

Histidine decarboxylase (HDC) catalyses the formation of histamine, a bioactive amine. Agents that control HDC activity are beneficial for treating histamine-mediated symptoms, such as allergies and stomach ulceration. We searched for inhibitors of HDC from the ethyl acetate extract of the petal of Filipendula ulmaria, also called meadowsweet. Rugosin D, rugosin A, rugosin A methyl ester (a novel compound), and tellimagrandin II were the main components; these 4 ellagitannins exhibited a non-competitive type of inhibition, with K(i) values of approximately 0.35-1 µM. These K(i) values are nearly equal to that of histidine methyl ester (K(i)=0.46 µM), an existing substrate analogue inhibitor. Our results show that food products contain potent HDC inhibitors and that these active food constituents might be useful for designing clinically available HDC inhibitors.

Genome-wide screen reveals novel mechanisms for regulating cobalt uptake and detoxification in fission yeast.

Cobalt is an essential micronutrient but is toxic when present in excess. To study cobalt homeostasis we performed a genome-wide screen for deletion strains that show sensitivity or resistance to CoCl(2). Among 54 cobalt-sensitive strains, 18 are supersensitive strains, which are involved in histidine biosynthetic process, ubiquitination, mitochondria function, membrane trafficking, transporter and a variety of other known functions or still unknown functions. Furthermore, we identified 56 cobalt-resistant deletion strains, which are mainly involved in mitochondria function, signal transduction, ubiquitination, and gene expression and chromatin remodeling. Notably, deletion of the zhf1(+) gene, encoding a zinc ion transporter, confers supersensitivity to cobalt and overexpression of the zhf1(+) gene confers marked tolerance to cobalt, indicating that Zhf1 play key roles in cobalt detoxification. Interestingly, all the histidine-auxotrophic mutants displayed cobalt sensitivity and deletion of cationic amino acid transporter Cat1, which was shown to be involved in histidine uptake, suppressed the CoCl(2)-sensitive growth defect of the his2 mutants, suggesting that CoCl(2) may be transported into the cell together with histidine via histidine transporters including Cat1. In addition, we obtained results suggesting that the E2 ubiquitin conjugating enzyme Rhp6 and Sty1 stress MAP kinase pathway are involved in the regulation of cobalt homeostasis. Altogether, our genome-wide study demonstrates for the first time the mechanisms of cobalt homeostasis, particularly its uptake and detoxification in fission yeast.

Trp64Arg polymorphism in beta3-adrenergic receptor gene is associated with decreased fat oxidation both in resting and aerobic exercise in the Japanese male.

The purpose of our study was to investigate whether the Trp64Arg polymorphism in beta3-AR gene and the -3826A/G polymorphism in the UCP1 gene were associated with the reduction in energy expenditure and fat oxidation both in resting and aerobic exercise in Japanese. Eighty-six nonobese young healthy Japanese were recruited. Energy expenditure was measured using indirect calorimetry. The subjects performed an aerobic exercise program at 60% of their maximal heart rate for 30 minutes. The level of fat oxidation at rest and aerobic exercise of the male subjects with Trp/Arg of the beta3-AR gene was significantly lower than that of the Trp/Trp genotype. No difference in FO(0-30) was observed in the female subjects. There was no association between UCP-1 polymorphism and energy expenditure during aerobic exercise. It was revealed that the Trp64Arg polymorphism in beta3-AR gene is associated with reduction of fat oxidation both in resting and aerobic exercise in healthy, young Japanese males.

Social support, self-efficacy and psychological stress responses among outpatients with diabetes in Yogyakarta, Indonesia.

We attempted to study whether social support promotes self-efficacy and reduces stress responses of patients with diabetes in Yogyakarta, Indonesia. Diabetic outpatients at Dr. Sardjito Hospital voluntarily participated in a questionnaire survey. Data from 125 patients were subjected to analysis. The questionnaires included the scales and subscales of social support, self-efficacy, psychological stress response, and demographic measure. Data were analyzed by Spearman's rank correlation test to examine the relationships between parameters, Mann-Whitney U-test and Kruskal-Wallis test to compare the scales by characteristics, and structural equation modeling to explore the best-fit model. This study was performed in September 2003. It was found that augmentation of emotional support to patients significantly increased the 'active coping for the disease' and 'controllability of health', and that 'helplessness' was reduced significantly. Behavioral support affected only 'controllably of health'. Self-efficacy reduced stress response of the patients. It was also found that subjects who received support from their children significantly scored higher in perceived availability of social support than those without support from their children. To know their behavioral support better as well as emotional support may be one area to focus on in improving the health status of people with diabetes in Yogyakarta.

Adipocytes from Munc18c-null mice show increased sensitivity to insulin-stimulated GLUT4 externalization.

Insulin-stimulated glucose uptake in adipocytes is mediated by translocation of vesicles containing the glucose transporter GLUT4 from intracellular storage sites to the cell periphery and the subsequent fusion of these vesicles with the plasma membrane, resulting in the externalization of GLUT4. Fusion of the GLUT4-containing vesicles with the plasma membrane is mediated by a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex consisting of vesicle-associated membrane protein 2 (VAMP2), 23-kDa synaptosomal-associated protein (SNAP23), and syntaxin4. We have now generated mouse embryos deficient in the syntaxin4 binding protein Munc18c and show that the insulin-induced appearance of GLUT4 at the cell surface is enhanced in adipocytes derived from these Munc18c-/- mice compared with that in Munc18c+/+ cells. Wortmannin, an inhibitor of PI3K, inhibited insulin-stimulated GLUT4 externalization, without affecting GLUT4 translocation to the cell periphery, in Munc18c+/+ adipocytes, but it did not affect GLUT4 externalization in Munc18c-/- cells. Phosphatidylinositol 3-phosphate, which induced GLUT4 translocation to the cell periphery without externalization in Munc18c+/+ cells, elicited GLUT4 externalization in Munc18c-/- cells. These findings demonstrate that Munc18c inhibits insulin-stimulated externalization of GLUT4 in a wortmannin-sensitive manner, and they suggest that disruption of the interaction between syntaxin4 and Munc18c in adipocytes might result in enhancement of insulin-stimulated GLUT4 externalization.

Loss of Apm1, the micro1 subunit of the clathrin-associated adaptor-protein-1 complex, causes distinct phenotypes and synthetic lethality with calcineurin deletion in fission yeast.

Calcineurin is a highly conserved regulator of Ca(2+) signaling in eukaryotes. In fission yeast, calcineurin is not essential for viability but is required for cytokinesis and Cl(-) homeostasis. In a genetic screen for mutations that are synthetically lethal with calcineurin deletion, we isolated a mutant, cis1-1/apm1-1, an allele of the apm1(+) gene that encodes a homolog of the mammalian micro1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex. The cis1-1/apm1-1 mutant as well as the apm1-deleted (Deltaapm1) cells showed distinct phenotypes: temperature sensitivity; tacrolimus (FK506) sensitivity; and pleiotropic defects in cytokinesis, cell integrity, and vacuole fusion. Electron micrographs revealed that Deltaapm1 cells showed large vesicular structures associated with Golgi stacks and accumulated post-Golgi secretory vesicles. Deltaapm1 cells also showed the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes and a reduced secretion of acid phosphatase. These phenotypes observed in apm1 mutations were accentuated upon temperature up-shift and FK506 treatment. Notably, Apm1-GFP localized to the Golgi/endosomes, the spindle pole bodies, and the medial region. These findings suggest a role for Apm1 associated with the Golgi/endosome function, thereby affecting various cellular processes, including secretion, cytokinesis, vacuole fusion, and cell integrity and also suggest that calcineurin is involved in these events.

In vivo administration of glucosamine inhibited phosphatidylinositol 3-kinase activity without affecting tyrosine phosphorylation of the insulin receptor or insulin receptor substrate in rat adipocytes.

We examined insulin signaling in rat epididymal adipocytes which developed insulin resistance by the in vivo infusion of glucosamine. Insulin-stimulated 2-deoxyglucose uptake into the adipocytes isolated from rats which were infused glucosamine for 4 hours was diminished by 26%. To analyze insulin signaling in adipocytes, the epididymal fat tissues were harvested 5 minutes after insulin administration (10U/kg), which was administered immediately after glucosamine infusion. Glucosamine had no effect on the insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate (IRS)-1. Glucosamine infusion decreased insulin-stimulated phosphatidylinositol (PI) 3-kinase activity by 66%. Glucosamine infusion also inhibited insulin-stimulated PI 3-kinase activity associated with IRS-1, 2, 3 by 30%, 43%, and 44%, respectively. There was no difference in the association of the 85kDa subunit of PI 3-kinase with the IRS-1 and IRS-2 protein. PI 3-kinase activity in adipocytes from rats treated with glucosamine that were administered platelet derived growth factor (3microg/kg) for 5 minutes was also reduced by 39%. When we measured the kinase activity of protein kinase C (PKC) lamda, which is the downstream effector of PI 3-kinase in isolated adipocytes, we found that glucosamine inhibited insulin stimulated PKClamda kinase activity by 33%. These results suggest that glucosamine infusion contributes to the development of insulin resistance by mainly modulating the PI 3-kinase molecules.