We previously reported brown adipocytes can proliferate even after differentiation. To test the involvement of mature adipocyte proliferation in cell number control in fat tissue, we generated transgenic (Tg) mice over-expressing cell-cycle inhibitory protein p27 specifically in adipocytes, using the aP2 promoter. While there was no apparent difference in white adipose tissue (WAT) between wild-type (WT) and Tg mice, the amount of brown adipose tissue (BAT) was much smaller in Tg mice. Although BAT showed a normal cellular morphology, Tg mice had lower content of uncoupling protein 1 (UCP1) as a whole, and attenuated cold exposure-or beta 3-adrenergic receptor (AR) agonist-induced thermogenesis, with a decrease in the number of mature brown adipocytes expressing proliferation markers. An agonist for the beta 3-AR failed to increase the number of proliferating brown adipocytes, UCP1 content in BAT, and oxygen consumption in Tg mice, although the induction and the function of beige adipocytes in inguinal WAT from Tg mice were similar to WT mice. These results show that brown adipocyte proliferation significantly contributes to BAT development and adaptive thermogenesis in mice, but not to induction of beige adipocytes.

We developed electrolyzed water (EW) using carbon electrodes and investigated the ability of the developed EW to inhibit the proliferation of human cervical carcinoma HeLa cells. We observed that EW-containing media inhibited HeLa cell proliferation. Many very small black dots were produced in EW and these were associated with the inhibitory effect on the cell proliferation. Furthermore, the very small black dots that could inhibit cell proliferation were produced only at pH 3 to 3.5 of EW. Additional experiments showed that this inhibition of proliferation is reversible. These results suggest that the effect of EW on HeLa cells is cytostatic and not cytotoxic. Thus, our results indicate that the EW developed in this study may be used to inhibit cell proliretation.

Enhanced activation of phosphoinositide 3-kinase (PI3K) is a hallmark of many human tumors because it promotes cell proliferation and survival through several mechanisms. One of these mechanisms is the phosphorylation of the serine and threonine kinase Akt at the cytosolic side of the plasma membrane by phosphoinositide-dependent protein kinase 1 (PDK1), which is recruited and activated by binding to the phosphoinositides produced by PI3K. We previously demonstrated increased nuclear accumulation of PDK1 in cells with enhanced PI3K activity. We report that nuclear PDK1 promoted cell proliferation by suppressing FOXO3A-dependent transcription of the gene encoding p27(Kip1) (an inhibitor of cell cycle progression), whereas it enhanced cell survival by inhibiting the activation of c-Jun amino-terminal kinase. Cells with nuclear-localized PDK1 showed anchorage-independent growth, and when injected into mice, these cells induced the formation of solid tumors. In human prostate tumors, cytoplasmic localization of PDK1 correlated only with early-stage, low-risk tumors, whereas nuclear PDK1 localization correlated with high-risk tumors. Together, our findings suggest a role for nuclear-translocated PDK1 in oncogenic cellular transformation and tumor progression in mice and humans.

We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK.

We investigated large vessel function in lean Goto-Kakizaki diabetic rats (GK) and Otsuka Long-Evans Tokushima Fatty diabetic rats (OLETF) with possible roles of hyperglycemia/hyperosmolarity and insulin. Both young and old GK showed marked hyperglycemia with normal insulin level and well-preserved endothelium-dependent and endothelium-independent vasodilation in aorta and carotid artery. There were significant elevations in endothelial/inducible nitric oxide synthase (eNOS/iNOS) and inducible/constitutive heme oxygenase (HO-1/HO-2) in GK. The endothelium-dependent vasodilation in GK was inhibited partly by NOS blockade and completely by simultaneous blocking of HO and NOS. In contrast, OLETF showed hyperinsulinemia and mild hyperglycemia but significant endothelium dysfunction beginning at early ages with concomitantly reduced eNOS. Insulin injection corrected hyperglycemia in GK but induced endothelium dysfunction and intima hyperplasia. Hyperglycemia/hyperosmolarity in vitro enhanced vessel eNOS/HO. We suggest that hyperinsulinemia plays a role in endothelium dysfunction in obese diabetic OLETF, while hyperglycemia/hyperosmolarity-induced eNOS/HO upregulation participates in the adaptation of endothelium function in lean diabetic GK.

Kruppel-like factor 15 (KLF15), a member of the Kruppel-like factor family of transcription factors, has been found to play diverse roles in adipocytes in vitro. However, little is known of the function of KLF15 in adipocytes in vivo. We have now found that the expression of KLF15 in adipose tissue is down-regulated in obese mice, and we therefore generated adipose tissue-specific KLF15 transgenic (aP2-KLF15Tg) mice to investigate the possible contribution of KLF15 to various pathological conditions associated with obesity in vivo. The aP2-KLF15 Tg mice manifest insulin resistance and are resistant to the development of obesity induced by maintenance on a high fat diet. However, they also exhibit improved glucose tolerance as a result of enhanced insulin secretion. Furthermore, this enhancement of insulin secretion was shown to result from down-regulation of the expression of stearoyl-CoA desaturase 1 (SCD1) in white adipose tissue and a consequent reduced level of oxidative stress. This is supported by the findings that restoration of SCD1 expression in white adipose tissue of aP2-KLF15 Tg mice exhibited increased oxidative stress in white adipose tissue and reduced insulin secretion with hyperglycemia. Our data thus provide an example of cross-talk between white adipose tissue and pancreatic beta cells mediated through modulation of oxidative stress.

The details of pharmacological mechanisms of Gorei-san, a traditional Japanese Kampo medicine, remains to be clarified, although it has been used for diuretic and hydrostatic purposes. From these circumstances, the effects of this medicine on the expressions of aquaporin (AQP) 1, 2, 3, 4 and vasopressin 2 receptor (V2R) mRNAs were investigated in relation to diuresis and water balance regulation in the kidney and brain. Gorei-san extract decocted with hot water was given to rats loaded with 50 mL kg-1 volume of physiological saline and AQP1, 2, 3, 4 and V2R mRNAs were measured with real-time polymerase chain reaction (PCR) in the cortex and the medulla of kidney and the forebrain. A low dose of Gorei-san extract (100 mg kg-1) led to an increase in urine excretion and lower AQP3 mRNA expression in the cortex as well as lower expression of AQP2 and AQP3 mRNAs in the medulla of kidney, whereas no change in V2R mRNA expression was observed. AQP1 mRNA expression decreased in the forebrain of rats loaded with an excess volume of physiological saline compared with rats not loaded with excess saline and given no agent. A low dose of Gorei-san extract increased urine excretion volume, probably due to the downregulation of AQP3 mRNA in the cortex and downregulation of AQP2 and AQP3 mRNAs in the medulla of the kidney, in which changes were not related to V2R mRNA expression. An excess volume of physiological saline given to rats caused an inhibition of AQP1 mRNA expression in the forebrain, which probably functioned to maintain the water balance in a hyper-hydrous state.

PDK1 (3-phosphoinositide-dependent protein kinase 1) is a key mediator of signaling by phosphoinositide 3-kinase. To gain insight into the physiological importance of PDK1 in cell proliferation and cell cycle control, we established immortalized mouse embryonic fibroblasts (MEFs) from mice homozygous for a "floxed" allele of Pdk1 and from wild-type mice. Introduction of Cre recombinase by retrovirus-mediated gene transfer resulted in the depletion of PDK1 in Pdk1(lox/lox) MEFs but not in Pdk1(+/+) MEFs. The insulin-like growth factor-1-induced phosphorylation of various downstream effectors of PDK1, including Akt, glycogen synthase kinase 3, ribosomal protein S6, and p70 S6 kinase, was markedly inhibited in the PDK1-depleted (Pdk1-KO) MEFs. The rate of serum-induced cell proliferation was reduced; progression of the cell cycle from the G(0)-G(1) phase to the S phase was delayed, and cell cycle progression at G(2)-M phase was impaired in Pdk1-KO MEFs. These cells also manifested an increased level of p27(Kip1) expression and a reduced level of cyclin D1 expression during cell cycle progression. The defect in cell cycle progression from the G(0)-G(1) to the S phase in Pdk1-KO MEFs was rescued by forced expression of cyclin D1, whereas rescue of the defect in G(2)-M progression in these cells required both overexpression of cyclin D1 and depletion of p27(Kip1) by RNA interference. These data indicate that PDK1 plays an important role in cell proliferation and cell cycle progression by controlling the expression of both cyclin D1 and p27(Kip1).

To elucidate the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in cellular signaling, we constructed and expressed a pseudosubstrate of PDK1, designated as delta AL-PIF, and characterized its properties in cultured cells. delta AL-PIF consists of two fused proteins of the protein kinase C delta (delta PKC) activation loop (delta AL) and PDK1-interacting fragment (PIF). The phosphorylation of delta AL-PIF was detected with anti-delta PKC phospho-Thr(505)-specific antibody and was increased in proportion to the expression level of co-expressed GST-PDK1, indicating that it acts as a pseudosubstrate of PDK1. In cells expressing delta AL-PIF, basal phosphorylation level at the activation loop of PKB alpha, delta PKC and gamma PKC was reduced, compared with that in control cells, suggesting that delta AL-PIF functions as an inhibitory molecule for PDK1. delta AL-PIF affected the stability, translocation and endogenous activity of PKCs. These effects of delta AL-PIF on gamma PKC properties were confirmed by investigation using conditioned PDK1 knockout cells. Furthermore, apoptosis frequently occurred in cells expressing delta AL-PIF for 3 days. These findings revealed that delta AL-PIF served as an effective pseudosubstrate and an inhibitory molecule for PDK1, suggesting that this molecule can be used as a tool for investigating PDK-mediated cellular functions as well as being applicable for anti-cancer therapy.

Krüppel-like zinc finger transcription factors (KLFs) play diverse roles during cell differentiation and development in mammals. We have now shown by microarray analysis that expression of the KLF15 gene is markedly up-regulated during the differentiation of 3T3-L1 preadipocytes into adipocytes. Inhibition of the function of KLF15, either by expression of a dominant negative mutant or by RNA interference, both reduced the expression of peroxisome proliferator-activated receptor gamma (PPARgamma) and blocked adipogenesis in 3T3-L1 preadipocytes exposed to inducers of adipocyte differentiation. However, the dominant negative mutant of KLF15 did not affect the expression of CCAAT/enhancer-binding protein beta (C/EBPbeta) elicited by inducers of differentiation in 3T3-L1 preadipocytes. In addition, ectopic expression of KLF15 in NIH 3T3 or C2C12 cells triggered both lipid accumulation and the expression of PPARgamma in the presence of inducers of adipocyte differentiation. Ectopic expression of C/EBPbeta, C/EBPdelta, or C/EBPalpha in NIH 3T3 cells also elicited the expression of KLF15 in the presence of inducers of adipocyte differentiation. Moreover, KLF15 and C/EBPalpha acted synergistically to increase the activity of the PPARgamma2 gene promoter in 3T3-L1 adipocytes. Our observations thus demonstrate that KLF15 plays an essential role in adipogenesis in 3T3-L1 cells through its regulation of PPAR gamma expression.

<i>Objectives:</i> The dysregulated overexpression of BCL10 that results from a specific chromosomal translocation t(1;14)(p22;q32) in mucosa-associated lymphoid tissue lymphoma has been shown to activate nuclear factor (NF)-ĸB, which may promote growth and survival in tumor cells. Accordingly, the molecular mechanisms underlying NF-ĸB activation may be responsible for lymphomagenesis. The aim of this study was to determine the molecular mechanisms underlying NF-ĸB activation by BCL10 overexpression. <i>Methods:</i> HeLa or COS-1 cells were transfected with BCL10, intracellular localization of BCL10 and the activation of NF-ĸB were analyzed. <i>Results:</i> BCL10 expressed at a high level exhibited a filamentous distribution at the perinuclear region, whereas BCL10 at a low level of expression displayed a diffuse cytoplasmic distribution. Furthermore, the BCL10-mediated NF-ĸB activation was efficiently inhibited by a Ca²⁺ chelating agent or a Ca²⁺ channel blocker. We also found that amino acids (107–119) of BCL10 are required for the formation of filamentous structures at the perinuclear region and NF-ĸB activation. <i>Conclusion:</i> These findings suggest that the filamentous pattern of overexpressed BCL10 at the perinuclear region adjacent to the endoplasmic reticulum is important for the BCL10-mediated NF-ĸB activation.

<i>Objectives:</i> Cardiac ankyrin repeat protein, CARP, is a protein that is restrictedly expressed in the heart but barely expressed in skeletal muscles. Since CARP is induced by pressure overload to the heart, it is proposed to be a genetic marker for cardiac hypertrophy. We recently identified a novel protein, ankyrin repeat protein with PEST and proline-rich region (ARPP), which is homologous to CARP and is preferentially expressed in type 1 skeletal muscle fibers (cf. slow fibers). We also found that both ARPP and CARP expression is induced in experimentally denervated skeletal muscles in mice. Based on these findings, we hypothesized that their expression may be induced in skeletal muscles in neurodegenerating disease. This work aimed to determine the expression pattern of ARPP and CARP in amyotrophic lateral sclerosis (ALS). <i>Methods:</i> In this study, we immunohistochemically analyzed the expression of ARPP and CARP in skeletal muscles of 9 ALS cases. <i>Results:</i> We found that CARP was aberrantly expressed in atrophic skeletal muscle fibers in ALS. Although ARPP-positive fibers were randomly scattered in a checkerboard-like pattern in normal skeletal muscle, this pattern was absent in ALS muscles. Furthermore, we also found that ARPP was expressed in fast myosin heavy chain-positive fibers (cf. type 2 fiber). <i>Conclusion:</i> These findings suggest that type-specific expression patterns of ARPP and CARP are altered in skeletal muscles of ALS.

申請者は、2 型糖尿病の発症に重要な要素の一つである食後高血糖に着目して研究を行っている。特に、食直後の急激な血糖値上昇に肝臓におけるグルカゴン様ペプチド-1 (GLP-1) シグナル系が関与している可能性を見出している。 本研究の目的は、2 型糖尿病モデルマウスを用いて、2 型糖尿病発症の前段階から発症過程において、固形状および粉状といった食餌の形状の違いがもたらす影響について明らかにしていくことである。自然発症 2 型糖尿病モデルマウスである ob/ob マウスを発症前段階の 4 週齢から発症段階の 8 週齢において食餌の形状による血糖値上昇に与える影響を検討するにあたり、医学部での動物実験を予定していたが、新型コロナウィルス感染症の流行拡大により、行うことが困難となったため、細胞レベルでの検討を行うための実験設備を整備した。

Postprandial hyperglycemia is transiently increased blood glucose levels that occur soon after the meal, although fasting blood glucose is normal or boundary range. Thus, it is possible that postprandial hyperglycemia cannot be detected by medical checkup that usually tested fasting blood glucose. We considered that this is one of the causes of the increased diabetic patients and analyzed the mechanism in developing postprandial hyperglycemia. As a results, the hepatic caveolin-1 was decreased and hepatic GLP-1 receptor expression was increased in the ob/ob diabetic model mice at 1 hour after a meal. These results indicated that the GLP-1 receptor dysfunction was associated with the mechanism involved in the development of postprandial hyperglycemia.

Recent studies have shown that high mobility group box1 (HMGB1) plays roles in immune response. We have reported that anti-HMGB1 Ab inhibited the progression of brain infarction and atherosclerosis. However, little is known whether HMGB1 induces the vascular injury, we investigated the involvement of HMGB1 in the endothelial dysfunction and injury using stroke-prone spontaneously hypertensive rats (SHRSP). It is well known that the endothelial dysfunction and injury induces hypertension. Although we could not prove the effect of HMGB1 on the endothelial dysfunction and injury, during this project, we found the mechanism of neutrophil-induced endothelial dysfunction and injury. Moreover, we found a mediator which inhibited the activation of neutrophil. Therefore, we continue the promotion of basic scientific research for formulating drugs for treatment of hypertension.

In this study, we analyzed the gene expression of hormone and stress related factors in liver. We found (1) down-expression of insulin-like growth factor-1 (IGF-1), (2,3) up-expression of IGF binding protein-1 (IGFBP-1) and 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1), (4) the down-expression of ER stress marker and (5) the up-expression of glucagon-like peptide-1 (GLP-1) receptor in liver of type 2 diabetic model mice under the conditions of fasting and refeeding.

Beneficial effects of long-term exercise (EX) may relate to increase endothelial and hematopoietic progenitors, and neuronal stem cells. In EX SHRSP, the number and activity of EPC and hematopoietic cells in bone marrow and peripheral blood cells were increased before and after stroke. The numbers of microvessels and neuronal stem cells around cerebral lesion were increased in EX, coincident with the appearance of EPC. Furthermore, levels of CXCL12, VEGF, and BDNF were higher in the plasma and cerebral homogenates of EX rats than sedentary rats and the signaling molecules, ERK and CREB, were also up-regulated in EX rats.