Biology subjects

Kakinuma, Y.

Publications and source records attributed to Kakinuma, Y..

8 recordsLinked to original sources

bioRxiv2025

Fluorescence analysis of serum lipoproteins using a histidine-imidazole polyacrylamide slab gel electrophoresis method

Polyacrylamide gel electrophoresis (PAGE) has traditionally been used to analyze lipoproteins in human and animal sera. Most conventional PAGE systems for lipoprotein analysis use disc electrophoresis, which limits the number of samples simultaneously analyzed and does not allow the comparison of electrophoretic profiles of multiple samples under the same conditions. Other methods, such as high performance liquid chromatography and agarose gel electrophoresis, are also used for lipoprotein analysis; however, they require specialized equipment and expertise. Here, we report an improved slab PAGE method with high-throughput, cost-effective, simple, rapid, and reproducible analysis of lipoproteins in the human serum. This system uses Tris-histidine as the running buffer and imidazole buffer for the polyacrylamide gel; therefore we named it the histidine-imidazole polyacrylamide gel electrophoresis (HI-PAGE) system. HI-PAGE effectively prevented band distortions of lipoproteins within an hour of electrophoresis and guaranteed precise quantitative analyses of multiple samples containing lipid-protein complexes. Furthermore, we pre-stained the lipoproteins with a fluorescent dye, Nile Red, and applied it to HI-PAGE electrophoresis. Fluorescent HI-PAGE (fHI-PAGE) was applied to the clinical samples and revealed that the method is highly sensitive and allows the quantitative detection of lipoproteins in human serum.

biochemistry↗

bioRxiv2023

Transcriptome analyses of mouse cardiac myocytes and non-cardiomyocytes: postmitotic vs. proliferative cells

Adult heart mostly contains long-lived postmitotic cardiomyocytes and non-cardiomyocytes that have proliferative potential. Here, we isolated cardiomyocytes and non-cardiomyocytes from young and aged mouse heart, and performed transcriptome analyses by RNA sequencing to understand the differences of gene expression in postmitotic and proliferative cells. Gene ontology analyses revealed that genes associated with inflammatory response were upregulated in aged cardiac myocytes, whereas genes including two ATP synthases in mitochondrial respiratory complex V (Atp5l and Atp5J2) and two NADH dehydrogenases in complex I (Ndufa11 and Ndufv3) were significantly downregulated. In aged non-cardiomyocytes, genes related to inflammatory responses were also upregulated, while genes involved in cell cycle and DNA replication process were downregulated. We also found that the expression levels of some small nucleolar RNAs (snoRNAs) are decreased cardiomyocytes with aging. snoRNAs are deeply involved in RNA modification such as pseudouridylation stabilizing ribosomal RNA (rRNA) and mRNA splicing. Therefore, the age-related reduction in snoRNA expression may lead to the destabilization of rRNA, splicing dysfunction, and ultimately a decrease in protein synthesis capacity. A comparison with transcriptome results obtained for non-cardiomyocytes suggests that the decline in the expression of mitochondria-related genes and snoRNAs accompanying aging is specific to cardiomyocytes, implying their potential utility as one of novel aging markers in postmitotic cells.

physiology↗

bioRxiv2023

Positive feedback regulation of Pparγ1sv and Pparγ2 during adipogenesis in 3T3-L1 cells

We have previously identified the novel splicing variant of mouse Ppar{gamma} (Ppar{gamma}1sv) and proposed the synergistic regulation of the early stage of adipocyte differentiation by Ppar{gamma}1sv and Ppar{gamma}2. Here, we report the finding of PPAR{gamma}-binding sites within the Ppar{gamma} gene locus and its importance in adipogenesis and propose the positive feedback regulation of Ppar{gamma}1sv and Ppar{gamma}2 expression during the adipocyte differentiation of 3T3-L1 cells.

cell biology↗

bioRxiv2023

Transcriptome Analysis Reveals Enhancement of Cardiogenesis-Related Signaling Pathways by S-nitroso-N-pivaloyl-D-penicillamine (SNPiP): Implications for Improved Diastolic Function and Cardiac Performance

We previously reported a novel compound called S-nitroso-N-pivaloyl-D-penicillamine (SNPiP), which was screened from a group of nitric oxide (NO) donor compounds with a basic chemical structure of S-nitroso-N-acetylpenicillamine (SNAP), to activate the non-neuronal acetylcholine (NNA) system. SNPiP-treated mice exhibited improved cardiac output and enhanced diastolic function, without an increase in heart rate. The NNA-activating effects included increased resilience to ischemia, modulation of energy metabolism preference, and activation of angiogenesis. Here, we performed transcriptome analysis of SNPiP-treated mice ventricles to elucidate how SNPiP exerts beneficial effects on cardiac function. A time-course study (24 and 48 h after SNPiP administration) revealed that SNPiP initially induced Wnt and cGMP-protein kinase G (PKG) signaling pathways, along with upregulation of genes involved in cardiac muscle tissue development and oxytocin signaling pathway. We also observed enrichment of glycolysis-related genes in SNPiP-treated samples, indicating a metabolic shift from oxidative phosphorylation to glycolysis following SNPiP administration in the hearts. Additionally, SNPiP significantly upregulated atrial natriuretic peptide (ANP) and sarcolipin (SLN), which play crucial roles in calcium handling and cardiac performance. These findings suggest that SNPiP may have therapeutic potential based on the pleiotropic mechanisms elucidated in this study.

molecular biology↗

bioRxiv2022

Temporal inhibition of electron transport chain attenuates stress-induced cellular senescence by prolonged disturbance of proteostasis in human fibroblasts

We previously developed a stress-induced premature senescence (SIPS) model in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the V-ATPase inhibitor bafilomycin A1 (BAFA1). To elucidate the involvement of mitochondrial function in our SIPS model, we treated cells with an inhibitor of electron transport chain (ETC) complexes I, III, or a mitochondrial uncoupler reagent along with MG132 or BAFA1 and evaluated the induction of premature senescence. SIPS induced by MG132 or BAFA1 was partially attenuated by co-treatment with antimycin A (AA) and rotenone, but not carbonyl cyanide 3-chlorophenylhydrazone (CCCP), in which intracellular reactive oxygen species (ROS) levels, acute mitochondrial unfolded protein responses, and accumulation of protein aggregates were remarkably suppressed. Co-treatment with AA also reversed the temporal depletion of SOD2 in the mitochondrial fraction on day 1 of MG132 treatment. Furthermore, co-treatment with AA suppressed the induction of mitophagy in MG132-treated cells and enhanced mitochondrial biogenesis. These findings provide evidence that the temporal inhibition of mitochondrial respiration exerts protective effects against the progression of premature senescence caused by impaired proteostasis. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/515395v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@165a9eaorg.highwire.dtl.DTLVardef@1766e79org.highwire.dtl.DTLVardef@4ef4beorg.highwire.dtl.DTLVardef@1a5e827_HPS_FORMAT_FIGEXP M_FIG GRAPHICAL ABSTRACT Cellular senescence is induced by prolonged inhibition of proteasome or lysosome function using MG132 and bafilomycin A1 (BAFA1), respectively. When cells were co-treated with a mitochondrial respiration inhibitor, antimycin A, and MG132 or BAFA1, oxidative stress, mitochondrial unfolded protein response (UPRmt), accumulation of protein aggregates, and mitophagy were suppressed whereas mitochondrial biogenesis was enhanced, resulting in the attenuation of stress-induced cellular senescence (SIPS). C_FIG

cell biology↗

bioRxiv2020

Elevated blood pressure in high fat diet-exposed low birth weight rat offspring is most likely caused by elevated glucocorticoid levels

Being delivered as a low birthweight (LBW) infant is a risk factor for elevated blood pressure and future problems with cardiovascular and cerebellar diseases. Premature babies have been reported to possess a lower number of nephrons, but the mechanisms by which blood pressure is elevated in full-term LBW infants remain unclear. We generated a fetal low-carbohydrate and calorie-restricted model rat, and some individuals showed postnatal growth failure caused by increased miR-322 expression in the liver and decreased growth hormone receptor expression. Using this model, we examined how a high-fat diet-induced mismatch between prenatal and postnatal environments could elevate blood pressure after growth. Although LBW rats fed standard chow had slightly higher blood pressure than control rats, their blood pressure was significantly higher than controls when exposed to a high-fat diet. Observation of glomeruli subjected to PAM staining showed no difference in number or size. Aortic and cardiac angiotensin II receptor expression was altered with compensatory responses. Blood aldosterone levels were not different between control and LBW rats, but blood corticosterone levels were significantly higher in the latter with high-fat diet exposure. Administration of metyrapone, a steroid synthesis inhibitor, reduced blood pressure to levels comparable to controls. We showed that high-fat diet exposure causes impairment of the pituitary glucocorticoid feedback via miR-449a. These results clarify that LBW rats have increased blood pressure due to high glucocorticoid levels when they are exposed to a high-fat diet. These findings suggest a new therapeutic target for hypertension of LBW individuals.

physiology↗

bioRxiv2020

Prolonged disturbance of proteostasis induces cellular senescence via temporal mitochondrial dysfunction and enhanced mitochondrial biogenesis in human fibroblasts

Proteolytic activities decline with age, resulting in the accumulation of aggregated proteins in aged organisms. To investigate how disturbance in proteostasis causes cellular senescence, we developed a stress-induced premature senescence (SIPS) model, in which normal human fibroblast MRC-5 cells were treated with either the proteasome inhibitor MG132 or the V-ATPase inhibitor bafilomycin A1 (BAFA1) for 5 days. Time-course studies revealed significant increase in intracellular and mitochondrial reactive oxygen species (ROS) during and after drug treatment. Mitochondrial membrane potential initially decreased, but recovered along with PGC-1-mediated mitochondrial biogenesis, especially after drug treatment. Mitochondrial antioxidant enzymes SOD2 and GPx4 were temporally depleted in mitochondria on day 1 of the treatment, which in turn could cause excess production of mitochondrial ROS. Extra-mitochondrial SOD2 colocalized with protein aggregates and lysosomes in MG132-treated cells on day 1. SOD2 partially interacted with HSC70 and LAMP2, implying that dysfunctional SOD2 was degraded through chaperon-mediated autophagy (CMA) and caused SOD2 depletion in mitochondria. SIPS induction by MG132 or BAFA1 was partially attenuated by co-treatment with rapamycin, in which generation of excess ROS and mitochondrial biogenesis were suppressed. Rapamycin co-treatment also augmented the upregulation of HSP70 and decreased protein aggregates after drug treatment. Our study proposes a possible pathway from the disturbance of proteostasis to cellular senescence via functional changes in mitochondria.

cell biology↗

bioRxiv2019

Epigenetic regulation of a mouse PPARγ splicing variant, Pparγ1sv, during adipogenesis in 3T3-L1 cells

We have previously reported the abundant and ubiquitous expression of a newly identified splicing variant of mouse peroxisome proliferator-activated receptor-{gamma} (Ppar{gamma}), namely Ppar{gamma}1sv that encodes PPAR{gamma}1 protein, and plays an important role in adipogenesis. Ppar{gamma}1sv has a unique 5UTR sequence, compared to those of mouse Ppar{gamma}1 and Ppar{gamma}2 mRNAs. This implies the presence of a novel transcriptional initiation site and promoter for Ppar{gamma}1sv. We found that DNA methylation of 42 CpG sites in the proximal promoter region (-733 to -76) of Ppar{gamma}1sv was largely unchanged five days after adipocyte differentiation, whereas chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) using antibodies against H3K4me3 and H3K27ac revealed that these modifications significantly elevated at the transcription start sites of Ppar{gamma}1sv and Ppar{gamma}2 after differentiation.

cell biology↗