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Background: The clinical significance of echocardiographic left ventricular hypertrophy (LVH) in risk stratification of left atrial appendage (LAA) thrombogenic milieu, as a surrogate for cardioembolic risk, in patients with atrial fibrillation (AF) and HA 2 DS 2 -VASc scores of 0-2 is unknown., Methods and Results: We enrolled 707 consecutive patients with AF and CHA 2 DS 2 -VASc scores of 0-2 who underwent transesophageal echocardiography. LAA thrombogenic milieu was defined as the presence of a thrombus, severe spontaneous echo contrast, sludge in the LAA, or LAA flow velocity ≤ 20 cm/s. Alongside conventional parameters, longitudinal strain values for the left ventricle (LV) and left atrium were obtained using transthoracic echocardiography. Among the 707 patients, 77 (10.9 %) exhibited LVH. The LVH group exhibited a significantly higher prevalence of LAA thrombogenic milieu than the non-LVH group (32.5 % vs. 2.5 %, p < 0.001). LVH independently associated with LAA thrombogenic milieu after adjusting for clinical factors (including CHA 2 DS 2 -VASc score, AF type, and serum brain natriuretic peptide levels) and conventional echocardiographic parameters (including LV ejection fraction, LV end-diastolic volume index, and left atrium volume index) (odds ratio [OR]: 7.54, 95 % confidence interval [CI]: 3.49-16.29, p < 0.001 and OR: 7.16, 95 % CI: 3.26-15.73, p < 0.001, respectively). Moreover, LVH provided incremental value for predicting LAA thrombogenic milieu, even when added to the longitudinal strain of the LV and left atrium reservoir strains (p < 0.001)., Conclusion: Echocardiographic LVH significantly improves the prediction of LAA thrombogenic milieu, offering potential utility in further cardioembolic risk stratification for patients with AF and CHA 2 DS 2 -VASc scores of 0-2., Competing Interests: Declaration of competing interest The authors report no relationships that could be construed as a conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

When amino acids are plentiful in the diet, the liver upregulates most enzymes responsible for amino acid degradation. In particular, the activity of urea cycle enzymes increases in response to high-protein diets to facilitate the excretion of excess nitrogen. KLF15 has been established as a critical regulator of amino acid catabolism including ureagenesis and we have recently identified FoxO transcription factors as an important upstream regulator of KLF15 in the liver. Therefore, we explored the role of FoxOs in amino acid metabolism under high-protein diet. Our findings revealed that the concentrations of two urea cycle-related amino acids, arginine and ornithine, were significantly altered by FoxOs knockdown. Additionally, using KLF15 knockout mice and an in vivo Ad-luc analytical system, we confirmed that FoxOs directly regulate hepatic Ass1 expression under high-protein intake independently from KLF15. Moreover, ChIP analysis showed that the high-protein diet increased FoxOs DNA binding without altering the nuclear protein amount. Therefore, FoxOs play a direct role in regulating ureagenesis via a KLF15-independent pathway in response to high-protein intake., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: We hypothesized that if computed tomography (CT) images were used as learning data, we could overcome volume underestimation by echocardiography, improving the accuracy of left ventricular (LV) volume measurements., Methods: We utilized a fusion imaging modality consisting of echocardiography with superimposed CT images for 37 consecutive patients to identify the endocardial boundary. We compared LV volumes obtained with and without CT learning trace-lines (TLs). Furthermore, 3D echocardiography was used to compare LV volumes obtained with and without CT learning for endocardial identification. The mean difference between the echocardiography and CT-derived LV volumes and the coefficient of variation were compared pre- and post-learning. Bland-Altman analysis was used to assess the differences in LV volume (mL) obtained from the 2D pre-learning TL and 3D post-learning TL., Results: The post-learning TL was located closer to the epicardium than the pre-learning TL. This trend was particularly pronounced in the lateral and the anterior wall. The post-learning TL was along the inner side of the high echoic layer in the basal-lateral wall in the four-chamber view. CT fusion imaging determined that the difference in LV volume between 2D echocardiography and CT was small (-25.6 ± 14.4 mL before learning, -6.9 ± 11.5 mL after learning) and that CT learning improved the coefficient of variation (10.9 % before learning, 7.8 % after learning). Significant improvements were observed during 3D echocardiography; the difference in LV volume between 3D echocardiography and CT was slight (-20.5 ± 15.1 mL before learning, 3.8 ± 15.7 mL after learning), and the coefficient of variation improved (11.5 % before learning, 9.3 % after learning)., Conclusions: Differences between the LV volumes obtained using CT and echocardiography either disappeared or were reduced after CT fusion imaging. Fusion imaging is useful in training regimens for accurate LV volume quantification using echocardiography and may contribute to quality control., Competing Interests: Declaration of competing interest This study was funded by Canon Medical Systems, Otawara, Japan. The funders had no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication., (Copyright © 2023. Published by Elsevier Ltd.)

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways., Competing Interests: Declaration of competing interest The authors declare no competing financial and non-financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Brain or B-type natriuretic peptide (BNP) is an objective marker to diagnose the presence of heart failure (HF) and assess its severity. However, the determinants of serum BNP level in elderly patients with severe aortic valve stenosis (AS) referred for transcatheter aortic valve implantation (TAVI) have not been well investigated., Methods: We prospectively studied 106 AS patients who underwent TAVI. Cardiac catheterization, transesophageal echocardiography, and blood collection for plasma BNP level measurements were performed simultaneously just before the TAVI procedures., Results: Ninety-nine patients (83.9±5.0 years, 33% male) were studied. The natural logarithm of BNP (lnBNP) level was 5.4±0.9 pg/mL. Significant correlations with lnBNP level were observed in: 1) the history of syncope, prior HF medication, and New York Heart Association class III or IV (R=0.255, p=0.011) (R=0.210, p=0.037) (R=0.402, p<0.001), 2) albumin and hemoglobin level (R=-0.289, p=0.004) (R=0.263, p=0.009), 3) Left ventricular (LV) ejection fraction and global longitudinal strain (LVGLS) (R=-0.338, p<0.001) (R=0.447, p<0.001), 4) LV end-diastolic volume index (EDVI), LV mass index, and left atrial volume index (R=0.280, p=0.005) (R=0.366, p<0.001) (R=0.337, p<0.001), 5) the catheter-measured pressure gradient across the aortic valve (AVPG) (R=0.365, p<0.001). Note that LV wall stress was not significantly correlated with lnBNP level. LVGLS, AVPG, hemoglobin level, and LVEDVI were independently correlated with ln BNP level (R=0.652, LVGLS; β=0.395, p<0.006, AVPG; β=0.291, p=0.001, hemoglobin level; β=-0.216, p=0.011, and LVEDVI; β=0.203, p=0.016, respectively)., Conclusions: In severe AS patients candidate for TAVI, multiple factors, including the severities of AS and HF conditions and subclinical LV dysfunction determined by LVGLS affects plasma BNP level., Competing Interests: Disclosure The authors declare that there is no conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

Aims: Renalase expression is regulated by Nuclear Factor (NF)-κB and hypoxia inducible factor (HIF)-1α, and antioxidative stress function in renal cells were reported. However, dynamics of renalase and localizes in intestine were remain unknown. We evaluated the effects of oxidative stress on renalase expression and localization using model of fasting induced oxidative stress and Caco-2 cell, and examined the its physiological effects., Main Methods: 24 male mice were divided into three groups: Control (Con), 72 h fasting (Fast), and 24 h refeeding after fasting (Refeed). Jejunum and ileum were collected respectively. The structure of jejunum and ileum were observed by hematoxylin and eosin (HE) stain. The expression levels of carbonylated protein, renalase, NF-κB p65 and HIF-1α were measured by immunoblotting. Localization of renalase was observed by immunofluorescent. in vitro assay was performed using Caco-2 cell. Renalase was overexpressed using adenovirus. After that, Caco-2 cell was treated with 2 mM H 2 O 2 for 30 min or 24 h., Key Findings: Renalase was increased in Fast and it was localized in crypt. HIF-1α did not increase, but NF-κB p65 increased in Fast. Renalase overexpression protects the Caco-2 cells against H 2 O 2 induced oxidative stress., Significance: Renalase was localized in crypt and increased in Fast. This increase suggested protect response to oxidative stress because undifferenced cells were localized in crypt and need to be protected. Actually, renalase protected Caco-2 cells against H 2 O 2 induced oxidative stress. Small intestinal renalase expression was regulated by NF-κB p65 and was considered to be a defense mechanism against oxidative stress., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Tumor hypoxia drastically changes cancer phenotypes, including angiogenesis, invasion, and cell death. Gangliosides are sialic acid-containing glycosphingolipids that are ubiquitously distributed on plasma membranes and are involved in many biological processes, such as the endoplasmic reticulum stress response and apoptosis. In this study, we investigated the regulation and function of glycosphingolipids, which associate with lipid raft on mammalian plasma membranes under hypoxic condition., Methods: B16F10 melanoma cells were subjected to chemical hypoxia and low pO 2 condition, and the effect of hypoxia on expression of GM3 synthase were analyzed. Cellular resistance to oxidative stress was analyzed in GM3S-KO B16F10 cells., Results: Hypoxia treatment decreased the expression of ganglioside GM3 synthase (GM3S; ST3GAL5), which synthesizes the common substrate of ganglioside biosynthesis. RNA interference of hypoxia inducible factor 1 subunit alpha (HIF-1α) inhibited hypoxia-induced GM3S suppression. Additionally, GM3S deficiency increased cellular resistance to oxidative stress and radiation therapy via upregulation of ERK., Conclusions: Altered synthesis of glycosphingolipids downstream of HIF-1α signaling increased the resistance of melanoma cells to oxidative stress. Furthermore, GM3 has important role on cellular adaptive response to hypoxia., General Significance: This study indicates that tumor hypoxia regulates therapy-resistance via modulation of ganglioside synthesis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: The clinical effect of diabetic microangiopathy on left ventricular (LV) function is still uncertain. The purpose of this study was to assess the relation between diabetic microvascular complications and comprehensive myocardial deformation measurements using three-dimensional (3D) speckle tracking echocardiography., Methods: Seventy-seven asymptomatic patients with type 2 diabetes mellitus (DM) and 35 age-matched healthy control subjects underwent 3D echocardiography. Patients with coronary artery disease or LV ejection fraction <50% were excluded. Presence of proliferative retinopathy, microalbuminuria as nephropathy, and decreased coefficient of variation of R-R intervals (CVRR) <3% as cardiac autonomic neuropathy were defined as diabetic microvascular complications., Results: LV ejection fraction, LV mass index, and global radial strain did not differ between control and DM patients. However, global longitudinal and circumferential strain and endocardial area change ratio were lower in patients with DM than in the controls (-12.0±3.0% vs. -16.2±1.9%, -27.7±7.1% vs. 32.2±5.7%, -37.6±7.6% vs. 44.0±6.2%, respectively, p<0.001). In DM patients, longitudinal strain is related to CVRR (R=0.58, p<0.001), retinopathy stage, and nephropathy stage., Conclusions: Diabetic microangiopathy and its accumulated effects significantly related to subclinical LV dysfunction are characterized by impaired longitudinal shortening., (Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Diphyllobothrium hottai Yazaki, Fukumoto & Abe, 1988 was described based on the morphology of adult worms recovered from golden hamsters that had been experimentally infected with plerocercoids obtained from Japanese surf smelts (Hypomesus pretiosus japonicus) and olive rainbow smelts (Osmerus eperlanus mordax). Although D. hottai was considered to be distinct from Diphyllobothrium ditremum (Creplin, 1825), their taxonomic relationship requires further clarification. In our study, D. hottai and D. ditremum obtained from hamsters experimentally infected with plerocercoids isolated from Japanese surf smelts were compared using morphological and molecular methods. The criterion usually used to differentiate between D. hottai and D. ditremum is the difference in the angle between the long axis of the cirrus sac and that of the seminal vesicle. However, we found variation of the angle within the same individual and, one specimen showed both of the different angles that were supposedly unique to each of the species. Furthermore, phylogenetic analysis of the complete sequences of the mitochondrial cytochrome c oxidase subunit 1 and cytochrome b genes revealed that both species were genetically indistinguishable. Therefore, D. hottai is considered to be a junior synonym of D. ditremum., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objectives: This study clarified the characteristics of intrarenal Doppler ultrasonography (IRD) profiles and their prognostic implications in heart failure (HF)., Background: IRD can assess intrarenal hemodynamics., Methods: Initially, 224 patients with HF were prospectively enrolled; 151 inpatients were enrolled during hospitalization for HF, and 73 were outpatients in our institution. In IRD profiles of interlobar vessels, the arterial resistance index (RI), venous impedance index (VII), and intrarenal venous flow (IRVF) pattern were assessed. Patients were followed to evaluate the associations with 1-year prognosis. Primary endpoints included death from cardiovascular disease and unplanned hospitalization for HF., Results: Finally, 217 patients with adequate IRD images were enrolled. IRD profiles were associated with conventional risk factors for HF. In particular, IRVF was associated with mean right atrial pressure (RAP); 3 IRVF patterns were stratified by RAP (in a continuous pattern: 5.4 ± 2.5; in a biphasic pattern: 9.5 ± 3.5; and in a monophasic pattern: 14.9 ± 4.3 mm Hg; p < 0.001). In addition, the monophasic IRVF pattern had a poorer prognosis than the other patterns (log rank p < 0.001), and prognosis was poorer for the biphasic pattern than for the continuous flow pattern (log rank p = 0.01). Multivariate Cox proportional hazard model analysis revealed that IRVF patterns were associated with the endpoints, independent of other HF risk factors., Conclusions: IRVF patterns, rather than RI, depended on RAP, suggesting a correlation with renal congestion. In addition, IRVF patterns strongly correlated with clinical outcomes independent of RAP and other risk factors and might provide additional information to stratify vulnerable HF patients., (Copyright © 2016 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although Proteus mirabilis is a common human pathogen, bacteremia caused by the organism, especially strains producing extended-spectrum beta-lactamase (ESBL), has rarely been investigated. We examined 64 cases of P. mirabilis bacteremia identified in the Minami Ibaraki Area, Japan, between 2001 and 2010 and compared the characteristics of cases with ESBL-producing and ESBL-non-producing strains (13 and 51 cases, respectively). All ESBL-producing strains with the gene encoding the CTX-M-2-group were genetically nonidentical. Isolation of ESBL-producing strains was significantly associated with onset in a hospital (p = 0.030), receiving hemodialysis (p = 0.0050), and previous antibiotic use within 1 month (p = 0.036; especially penicillin and/or cephalosporin (p = 0.010) and fluoroquinolone (p = 0.0069)). Isolation was also associated with inappropriate antibiotic therapy on the 1st and 4th days (p = 0.011 and 0.032, respectively) but not with mortality on the 30th day. These findings indicate that, for P. mirabilis bacteremia, isolation of ESBL-producing strains causes delay of initiating appropriate antimicrobial therapy but may not be associated with mortality.

Embryo cryopreservation is a valuable tool for efficient production of animals as well as banking of genetic resources. Even though the laboratory rat is one of the most important experimental animals for various research fields, it has been reported that survival and developmental ability of cryopreserved rat embryos are generally low, especially at the early stages. The aim of the present study was to establish rapid cooling method that can be applied for cryopreservation of rat pronuclear-stage embryos using Cryotops (a device). First, optimal equilibration time was examined. Pronuclear-stage embryos were equilibrated in 7.5% ethylene glycol (EG)+7.5% dimethylsulfoxide (DMSO)+20% fetal calf serum (FCS) for 7, 8 or 9 min at 20-22 degrees C and then 15% EG+15% DMSO+0.5M sucrose+20% FCS for 1 min at 20-22 degrees C, being plunged into liquid nitrogen on Cryotops. This established that development to the 2-cell (82.0+/-9.7% to 96.1+/-3.0%) and blastocyst (36.5+/-2.1% to 40.3+/-10.2%) stages in vitro was not influenced by the equilibration time. Furthermore development to term in vivo (56.0+/-4.9%) was equivalent to the rate (54.8+/-6.6%) obtained with control embryos. Taken together, this demonstrated that this method is suitable for the successful cryopreservation of pronuclear-stage embryos in rats.

A multiplex PCR method was established for the rapid identification of Anisakis simplex sensu stricto, A. pegreffii, A. physeteris, Pseudoterranova decipiens, Contracaecum osculatum and Hysterothylacium aduncum. The sequence alignment of the internal transcribed spacer 1 region (ITS-1) between A. simplex s. str. and A. pegreffii showed a high degree of similarity, but only two C-T transitions were observed. To differentiate A. simplex s. str. from A. pegreffii, an intentional mismatch primer with an artificial mismatched base at the second base from the primer 3' end was constructed. This intentional mismatch primer, which produced a PCR band only from A. pegreffii DNA, was able to differentiate the two morphologically indistinguishable sibling species of A. simplex. Specific forward primers for other anisakid species were also designed based on the nucleotide sequences of the ITS region. The multiplex PCR using these primers yielded distinct PCR products for each of the anisakid nematodes. The multiplex PCR established in this study would be a useful tool for identifying anisakid nematodes rapidly and accurately.

Anisakis simplex complex presently comprises three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. A. simplex is a common parasite in fishes and cephalopods and capable of causing anisakiasis in humans. Therefore, identification of sibling species of A. simplex was important for human health. In this study, one hundred Anisakis type I larvae isolated from eighty five patients with anisakiasis in Hokkaido and Kyushu in Japan were analyzed by adapting the new molecular method that can identify the sibling species of A. simplex complex. Based on the restriction fragment length polymorphism (RFLP) pattern of ITS regions including 5.8 subunit rRNA gene, we identified two sibling species, A. simplex s. str. and A. pegreffii. However, the infection rate of A. simplex s. str. was significantly higher than that of A. pegreffii. Eighty four (98.8%) out of the eighty five patients were infected with A. simplex s. str. On the contrary, one patients (1.2%) in Kyushu infected with A. pegreffii. This study provided basic information about human infection with A. simplex complex. Furthermore, we suggested that A. simplex s. str. is the most important etiological agent in Japan.

Parasites morphologically consistent with Anisakis simplex sensu lato collected from the coast of Japan and Western North Pacific Ocean were examined by PCR-RFLP of the ITS region (ITS1, 5.8 subunit rRNA gene and ITS2) and mtDNA cox1. The RFLP patterns of rDNA generated by HinfI and HhaI showed that 100% of the larvae collected from Hokkaido and 94% of adults collected from Western North Pacific Ocean were identified as A. simplex sensu stricto. On the other hand, 97% of the larvae collected from Fukuoka prefecture were identified as A. pegreffii. A hybrid genotype was found in adults in Western North Pacific Ocean and larva in Fukuoka prefecture. These findings revealed that A. simplexs. str. is primarily distributed in the North Pacific Ocean and A. pegreffii is primarily distributed in the southern Sea of Japan. RFLP analysis of mtDNA cox1 showed different patterns between A. simplex s. str. and A. pegreffii after digestion with HinfI. This polymorphism obtained by RFLP analysis of mtDNA cox1 proved the usefulness as new genetic markers to distinguish two sibling species.

The sensitivity of adipocytes to lipolytic agents is increased after starvation. In this study, we found that LY294002, an inhibitor of phosphatidylinositol-3 kinase (PI3K), in the concentration of more than 50 microM potentiates lipolysis induced by adenosine deaminase in adipocytes from fed rats (f-adipocytes), but not from starved rats (s-adipocytes). It also enhanced the sensitivity to lipolytic action of isoproterenol in f-adipocytes much more than s-adipocytes. The target of LY294002 may be an anti-lipolytic regulator expressed in response to food intake. Since another PI3K inhibitor, wortmannin, or a phosphodiesterase 3 (PDE3) inhibitor, cilostamide, failed to cause any specific effect to f-adipocytes, the PI3K-PDE3B pathway cannot be a target of LY294002. We found that LY294002 inhibits efficiently the cytoplasmic PDE activity of adipocytes. Rolipram, a specific inhibitor of PDE4, also inhibited the cytoplasmic PDE and caused a preferential increase of lipolysis in f-adipocytes. LY294002 blunted the actions of rolipram on lipolysis and the PDE activity. LY294002 accelerated protein kinase A activation. These data suggest that the rolipram-sensitive PDE4 is an anti-lipolytic enzyme expressed according to food intake. LY294002 may potentiate lipolysis through inhibition of the PDE4.

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.

The effect of intracellular free fatty acid (FFA) accumulation on ob gene expression in adipocytes was examined. In fully differentiated 3T3-L1 adipocytes, triacsin C, a specific acyl CoA synthetase inhibitor with a K(i) of 8.97 microM, inhibited ob gene expression by 20% at 5 x 10(-5)M. At this concentration, triacsin C induced accumulation of intracellular FFA. Treatment with both chylomicron and triacsin C reduced ob gene expression more than treatment with triacsin C alone. Treatment with 2-bromopalmitate, a poorly metabolizable palmitate analog, reduced ob gene expression by 50% at 10(-4)M, but palmitate at the same concentration had no effect. This is the first demonstration that the ob gene is downregulated by intracellular FFA accumulation, thereby raising the possibility that ob product is regulated in response to lipolysis.

Diabetic retinopathy is known to be worsened when hypoglycemia occurs, however the pathogenic mechanisms are not defined. Vascular endothelial growth factor (VEGF) is increased in ocular fluid with diabetic retinopathy and linked with development of retinopathy. In this study, we examined whether glucose deficiency could up-regulate VEGF levels in retinal pigmented cells. Exposure to glucose deprived medium induced 30% up-regulation of VEGF mRNA. In hypoxia, as one of the most well-known inducer of VEGF, its up-regulation mechanism is mainly due to increase of transcription of VEGF gene via hypoxia response element in 5'-untranslated region (5'-UTR). We examined the role of 5'-UTR and 3'-UTR of VEGF gene in glucose deprived conditions using luciferase assay system. 5'-UTR containing reporter vector did not show increase of activity in glucose deprived conditions in contrast to the result in oxygen deprived condition. Both 5'-UTR and 3'-UTR containing vector demonstrated significant increase of activity in glucose deficient conditions compared with 5'-UTR containing vector. These findings suggest 3'-UTR of VEGF gene is important for increasing mRNA level in glucose deprived condition.