Retinoic acid exerts sexually dimorphic effects over muscle energy metabolism and function

Among others, the retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction that converts retinol into retinoic acid (RA), an autacoid that regulates energy balance and suppresses adiposity. Relative to WT, Rdh10+/- males experienced reduced fatty-acid oxidation, glucose intolerance and insulin resistance. Running endurance decreased 40%. Rdh10+/- females increased reliance on fatty acid oxidation and did not experience glucose intolerance nor insulin resistance. Running endurance improved 2.2-fold. Estrogen increased, revealed by a 40% increase in uterine weight. Because skeletal muscle energy use restricts adiposity and insulin resistance, we assessed the mixed fiber type gastrocnemius muscle (GM) to determine the effects of endogenous RA on muscle metabolism in vivo. RA in Rdh10+/- male GM decreased 38% relative to WT. TAG content increased 1.7-fold. Glut1 mRNA and glucose decreased >30%. Rdh10+/- male GM had impaired electron transport chain activity, and a 60% reduction in fasting ATP. The share of oxidative fibers increased, as did expression of the myogenic transcription factors Myog and Myf5. Centralized nuclei increased 5-fold in fibersindicating muscle malady or repair. In Rdh10+/- female GM, RA decreased only 17%, due to a 1.8-fold increase in the estrogen-induced retinol dehydrogenase, Dhrs9. Rdh10+/- female GM did not amass TAG, increase oxidative fibers, decrease Glut1 mRNA or glucose, nor increase centralized nuclei. Expression of Myog and Myf5 decreased. Electron transport chain activity increased, elevating fasting ATP >3-fold. Thus, small decreases in skeletal muscle RA affect whole body energy use, insulin resistance and adiposity, in part through estrogen-related sexual dimorphic effects on mitochondria function.

Insulin-like growth factor 2 mRNA binding protein 2 promotes aerobic glycolysis and cell proliferation in pancreatic ductal adenocarcinoma via stabilizing GLUT1 mRNA

Insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) is a member of the IGF2BP protein family consisting of IGF2BP1~3 with the capacity of binding to many transcripts and regulating RNA stability, localization, and translation. In this study, we discovered that expression of IGF2BP2 was upregulated and led to a poor prognosis in pancreatic ductal adenocarcinoma (PDAC). IGF2BP2 protein was gradually elevated from normal pancreas, pancreatic intraepithelial neoplasia to PDAC in an LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx1-Cre mouse model. Furthermore, we demonstrated that IGF2BP2 promoted aerobic glycolysis and PDAC cell proliferation through directly binding to and stabilizing GLUT1 mRNA. In summary, our study unveiled an important role of IGF2BP2 in PDAC development by modulating aerobic glycolysis and as a potential therapeutic target for PDAC treatment.

Abstract 290: Microrna-199a and Glut1 are Differentially Expressed in Primary Human Arterial and Venous Endothelium and are Deregulated in Culture

Introduction Studies over the last decade have established that the identity of arterial and venous endothelial cells (ECs) is governed by both genetic and environmental factors. Although the ephrin/Eph system is known to be a key determinant of that identity, it is unlikely that system is solely responsible for all the differences between arterial and venous ECs. Furthermore, microRNAs are becoming increasingly recognized for their important role in regulating gene expression and cell fate. We investigate here novel potential markers and regulators of arterial EC identity and the effects of environmental cues on these molecules. Methods ECs were freed from the basement membrane of human umbilical arteries and veins (HUAECs, HUVECs) (n=6) by enzymatic digestion and purified by flow cytometry (CD31+, CD45-) prior to lysis and quantitative PCR or analysis using HPLC-MS/MS. Protein quantification was performed using a proprietary software package (IdentiQuantXL TM ). HUVECs and HUAECs were harvested directly from umbilical cords and placed directly into collagen coated 6-well plates and cultured in standard conditions in EGM2. Results We found that microRNA-199a levels were 23 fold higher in HUVECs than HUAECs (p=0.0001). One of microRNA-199a’s theoretical downstream targets is GLUT1, a facilitative glucose transporter thought to be responsible for basal glucose uptake. GLUT1 mRNA and protein levels were significantly higher (5.5 and 4.3 fold, respectively) in HUAEC than HUVECs (p=0.00002). Using tissue immunohistochemistry (IHC) of we confirmed that GLUT1 expression is restricted to arterial ECs in human umbilical cord and adult peripheral artery and vein sections. Finally, GLUT1 mRNA levels are 2 fold higher in HUAECs than HUVECs in culture at P2 and levels decrease until P5 when they become equal indicating that culture has a homogenizing effect on endothelial heterogeneity. Conclusions MicroRNA-199a levels are higher in HUVECs than HUAECs and GLUT1, its downstream target, is higher in HUAECs both at the mRNA and protein level. Protein expression is restricted to arteries in both the new born umbilical cord and adult peripheral vessels. Importantly, expression in both cell types is downregulated and equilibrated by cell culture.

The Expression Profile of Glycolysis Associated Molecules and Chemoresistance in Acute Myeloid Leukemia

Abstract 4318 Background The drug resistance of leukemic cells remains a major obstacle to chemotherapy in patients with AML, which always leads to treatment failure consequently. However, the complex mechanism in chemoresistance causes a growing concern to looking for some new therapeutic targets and improving the sensitivity of chemotherapy. Recently, researchers have used metabolomics technology to explore the differences of metabolomics between imatinib (IM)-resistant and sensitive chronic myeloid leukemia (CML) cells, and thus to clarify the mechanism of IM resistance, provide new targets to the treatment of IM resistance. The data showed that increased glycolytic activity related with IM drug resistance. As glycolytic rate directlly correlates with tumorigenesis and chemoresistance, it provides a new idea for cancer treatment. Some studies confirmed that there was also abnormal glycolysis in hematopoietic tumor cells. Thus, the study was expected to investigate the relations between the altered energy metabolism and chemoreisitance in AML. Furthermore, the effect of inhibition of glycolysis on chemoresistance in myeloid leukemia cells was evaluated. Methods The bone marrow samples obtained from with newly diagnosed (n=45) and relapsed (n=9) patients with AML (M3 excluded) in our hospitals from October 2010 to November 2011. The AML blasts in every sample was exceed to 80 percent. The total 54 AML cases with average age of 41.6±17.8 years were included in 31 male and 23 female patients. The expression differences of glycolysis associated molecules (e.g. HIF-1α, HK-II, GLUT1, CD147 and LDH) and β subunit of human F1-F0 ATP synthase (ATP5B) in different drug-sensitive AML cells were analyzed. Adriamycin (ADM)-sensitive AML cell line HL-60 and resistant cell line HL-60/ADM were used to evaluate the glycolytic activities and the effect of glycolysis inhibition on cellular proliferation. Results The drug-resistant HL-60/ADM cells exhibited a significantly higher glycolysis rates than the drug-sensitive AML cell line HL-60 (97.4%±0.70% vs. 55.0%±3.16%, P<0.01). The levels of HIF-1α and GLUT1 mRNA expression of the drug-resistant HL-60/ADM cells was higher than that of drug-sensitive HL-60 cells (HIF-1α: 3.70±0.084 vs. 1.0±0.075, P<0.01, GLUT1: 3.36±0.149 vs. 1.0±0.080, P<0.01). Likewise, the levels of HIF-1α, HK-IIand GLUT1 mRNA expression in AML patients with NR were higher than those in groups of health control, CR and PR (P<0.01). Also, the serum level of LDH in AML patients with NR was higher than that in groups of health control, CR and PR (1211.57±456.99 vs. 490.75±278.35 vs. 490.63±213.94 vs. 149.90±31.66, P<0.01). Furthermore, the expression of CD147 on bone marrow blasts from NR group was higher than that in groups of CR and PR (97.75%±4.17% vs. 89.41%±14.22%, P<0.01). Additionally, the expression of ATP5B protein were decreased in the drug-resistant HL-60/ADM cells (0.1388 vs 0.1192), and the level of ATP5B protein in patients with NR was lower than that in CR group (0.0092±0.0042 vs. 0.0540±0.0482, P<0.01). 2-DG or 3BrPA, the glycolysis inhibitors, acts in synergy with ADM in ADM-resistant HL-60/ADM leukemic cells, whereas it didn't increase ADM-induced toxicity in HL-60 cells. Treatment of the cell lines HL-60/ADM and HL-60 with sublethal concentrations of 2-DG or 3 BrPA, either alone or in combination with ADM, resulted in a considerable reduction of glucose uptake compared with non-treated cells. With these treatments for 24h, the cellular apoptosis rate in these two cell lines showed no significant difference (P>0.05). However, the glycolysis inhibitors in combination with ADM increased markedly the necrosis in these cell lines. Conclusion The chemoresistance was associated with increased glycolytic activity and low efficiency of oxidation phosphorylation in AML cells, regarding as the elevated expression of glycolysis associated molecules, i.e. HIF-1α□AHK-II□AGLUT1□ALDH and CD147. Inhibition of glycolysis can reverse the chemoresistance of drug-resistant AML cell line HL-60/ADM. The enhancements of ADM-induced cytotoxicity by 2-DG or 3BrPA may be due to the activation of nonapoptotic, programmed cell death. Disclosures: No relevant conflicts of interest to declare.

Quantitative Analysis of Glucose Transporter mRNAs in Endometrial Stromal Cells Reveals Critical Role of GLUT1 in Uterine Receptivity

Recurrent miscarriages affect about 1–2% of couples trying to conceive; however, mechanisms leading to this complication are largely unknown. Most studies focus on the early embryo, but proper development and implantation of the blastocyst are also dependent on optimal endometrial progression into a receptive state. One of the key steps in the uterine preparation for embryo receptivity, known as decidualization, is the differentiation of endometrial stromal cells (ESCs) into decidual cells. During this transition, the ESCs undergo a drastic change in glucose metabolism. The efficiency of glucose uptake is determined by a family of facilitative glucose transporters (GLUTs), and many have been identified in the stroma. The primary focus of this work was to quantify the absolute amount of GLUT mRNAs in this cell type before and after decidualization. We used primary ESCs isolated from murine and human uteri. We developed and validated cDNA-based calibration curves for each GLUT and used these primers to arrive at absolute mRNA copy numbers. Here, we report all the GLUT mRNAs that are present in the ESCs and their abundance under both conditions, control and decidualized. GLUT1 mRNA is the most abundant and critical transporter in ESCs of both species, because knocking down this GLUT with sort hairpin RNA leads to dramatically reduced decidualization. These findings suggest that GLUT1 mRNA expression is essential for decidualization and we are the first to determine a possible mechanism to explain how maternal conditions of abnormal glucose utilization may impair implantation at the level of the ESCs.

Expression and localisation of glucose transporter 1 (GLUT1) in dairy goat mammary gland at different physiological stages

Glucose is the major energy source for mammary epithelial cells, as well as an important substrate for lactose synthesis. Mammary epithelial cells take up glucose from extracellular fluid into the cell through glucose transporter (GLUT). This study was aimed at investigating the expression of GLUT1 glucose transporter in dairy goat mammary gland during puberty, pregnancy, lactation, and involution. Using real-time reverse transcription PCR (qRT-PCR) and Western blotting, we analyzed the expression of GLUT1 mRNA and protein in dairy goat mammary gland. GLUT1 mRNA and protein expression increased during pregnancy and lactation, especially at peak lactation, and decreased strongly after weaning. Furthermore, the location of GLUT1 protein was determined by immunofluorescence laser confocal microscopy. GLUT1 protein localised to the basal and apical plasma membrane of epithelial cells, and also in the cytoplasm. The results from this study showed that GLUT1 is expressed in the dairy goat mammary gland with the greatest expression found in mammary epithelial cells during pregnancy and lactation.Key words: Expression, glucose transporter, goat, mammary gland

Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-cyclins in AML cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and supports the therapeutic potential of mTOR inhibition strategies in leukemias.