Heart disease remains a major cause of death despite advances in medical technology. Heart-regenerative therapy that uses pluripotent stem cells (PSCs) is a potentially promising strategy for patients with heart disease, but the inability to generate highly purified cardiomyocytes in sufficient quantities has been a barrier to realizing this potential. Here, we report a nongenetic method for mass-producing cardiomyocytes from mouse and human PSC derivatives that is based on the marked biochemical differences in glucose and lactate metabolism between cardiomyocytes and noncardiomyocytes, including undifferentiated cells. We cultured PSC derivatives with glucose-depleted culture medium containing abundant lactate and found that only cardiomyocytes survived. Using this approach, we obtained cardiomyocytes of up to 99% purity that did not form tumors after transplantation. We believe that our technological method broadens the range of potential applications for purified PSC-derived cardiomyocytes and could facilitate progress toward PSC-based cardiac regenerative therapy.

Carbohydrates mediate their conversion to triglycerides in the liver by promoting both rapid posttranslational activation of ratelimiting glycolytic and lipogenic enzymes and transcriptional induction of the genes encoding many of these same enzymes. The mechanism by which elevated carbohydrate levels affect transcription of these genes remains unknown. Here we report the purification and identification of a transcription factor that recognizes the carbohydrate response element (ChRE) within the promoter of the L-type pyruvate kinase (LPK) gene. The DNA-binding activity of this ChRE-binding protein (ChREBP) in rat livers is specifically induced by a high carbohydrate diet. ChREBP's DNA-binding specificity in vitro precisely correlates with promoter activity in vivo. Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucoseresponsive regulation. ChREBP is likely critical for the optimal long-term storage of excess carbohydrates as fats, and may contribute to the imbalance between nutrient utilization and storage characteristic of obesity.

Several applications of pluripotent stem cell (PSC)-derived cardiomyocytes require elimination of undifferentiated cells. A major limitation for cardiomyocyte purification is the lack of easy and specific cell marking techniques. We found that a fluorescent dye that labels mitochondria, tetramethylrhodamine methyl ester perchlorate, could be used to selectively mark embryonic and neonatal rat cardiomyocytes, as well as mouse, marmoset and human PSC-derived cardiomyocytes, and that the cells could subsequently be enriched (>99% purity) by fluorescence-activated cell sorting. Purified cardiomyocytes transplanted into testes did not induce teratoma formation. Moreover, aggregate formation of PSC-derived cardiomyocytes through homophilic cell-cell adhesion improved their survival in the immunodeficient mouse heart. Our approaches will aid in the future success of using PSC-derived cardiomyocytes for basic and clinical applications.

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The rs2910164 (G>C) SNP in the miR-146a is associated with susceptibility to GC. In addition, the rs11614913 (C>T) SNP in the miR-196a2 is associated with the degree of H. pylori-induced mononuclear cell infiltration.

Carbohydrate-responsive element-binding protein (ChREBP) is a new transcription factor that binds to the carbohydrate-responsive element of the L-type pyruvate kinase gene (L-PK). The aim of this study was to investigate the mechanism by which feeding high fat diets results in decreased activity of ChREBP in the liver (Yamashita, H., Takenoshita, M., Sakurai, M., Bruick, R. K., Henzel, W. J., Shillinglaw, W., Arnot, D., and Uyeda, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9116 -9121). We cloned the rat liver ChREBP gene for use throughout this study. Acetate, octanoate, and palmitate inhibited the glucose-induced activation of L-PK transcription in ChREBP-overexpressed hepatocytes. In these hepatocytes, the cytosolic AMP concentration increased 30-fold and AMP-activated protein kinase activity was activated 2-fold. Similarly to the fatty acids, 5-amino-4-imidazolecarboxamide ribotide, a specific activator of AMP-activated protein kinase (AMPK) also inhibited the L-PK transcription activity in ChREBPoverexpressed hepatocytes. Using as a substrate a truncated ChREBP consisting of the C-terminal region, we demonstrated that phosphorylation by AMPK resulted in inactivation of the DNA binding activity. AMPK specifically phosphorylated Ser 568 of ChREBP. A S568A mutant of the ChREBP gene showed tight DNA binding and lost its fatty acid sensitivity, whereas a S568D mutant showed weak DNA binding and inhibited L-PK transcription activity even in the absence of fatty acid. These results strongly suggested that the fatty acid inhibition of glucose-induced L-PK transcription resulted from AMPK phosphorylation of ChREBP at Ser 568 , which inactivated the DNA binding activity. AMPK was activated by the increased AMP that was generated by the fatty acid activation.Glucose metabolism in liver is inhibited by administration of fatty acids, the so-called "glucose sparing" effect (1-3). Fatty acids inhibit genes of key enzymes of glycolysis and lipogenesis such as L-pyruvate kinase (L-PK), 1 acetyl-CoA carboxylase, and fatty acid synthetase. L-PK, regulating the flux of metabolites through the pyruvate-phosphoenolpyruvate cycle (4), is known to play an important role in hepatic glucose and lipid metabolism. The activity of L-PK is subject to acute control by covalent modification and allosteric effectors (5). On the other hand, long term control of L-PK is achieved by regulating L-PK gene transcription (5). Fatty acids inhibit transcription of L-PK and other enzymes in glycolysis and lipogenesis pathways, whereas excess glucose induces expression of these genes (6). However, the mechanism of fatty acid inhibition of transcription is not understood. We recently identified a new transcription factor, which binds specifically to the carbohydrate-responsive element of the L-PK gene, and we have termed this new transcription factor, "carbohydrate-responsive element-binding protein" (ChREBP) (7). ChREBP is expressed specifically in liver and is responsive to diet. ChREBP is activated by a high carbohydrate diet and inhibited by a h...

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We have reported that orally administrated acetate contributed to suppression of lipogenesis in the liver and to reduction of lipid accumulation in the adipose tissue of Otsuka Long-Evans Tokushima Fatty (OLETF) rats. The aim of this study was to investigate the effect of acetate on skeletal muscle and adipose tissues. Treatment with acetate showed a higher rate of oxygen consumption and a smaller size of lipid droplets in white adipose and brown adipose tissues. An analysis by Northern blotting revealed that the transcripts of myoglobin and Glut4 genes in the abdominal muscle of the OLETF rats were increased by acetate treatment, while the transcripts of lipolytic genes increased in the white adipose and brown adipose tissues. It is possible that acetate has effects on lipid metabolism in the skeletal muscles and the adipose tissues, and has functions that work against obesity and obesity-linked type 2 diabetes.