Optimizing a Standard Fasting Time for 2-NBDG Uptake Studies in Murine Breast Cancers

Recently, there has been a larger use of 2-NBDG, a fluorescent glucose analog, to study glucose uptake in different cell types. These cell types have ranged anywhere from bacteria to human cancer cells. However, there has yet to be a standard procedure and practice for using 2-NBDG. In this study, our goal is to create a standard fasting time for the cells before introducing 2-NBDG to them. This study uses 4T07 cells, a murine breast cancer cell line, to help optimize this fasting time. These cells were fasted at different time points in order to find the ideal fasting time. This ideal fasting time ensures the highest uptake of 2-NBDG in the cells. Intensity measurements, resulting from 2-NBDG fluorescence, were calculated to determine the optimal fasting time. The following paper details the procedure used including cell culturing and passaging, microscope setup, fluorescent imaging, and image processing

In vivo multi-parametric imaging of metastatic and non-metastatic breast cancer

A current issue in cancer therapy is the characterization of metastatic tumors, which can increase ease of treatment and patient trials. We present an in vivo study of metastatic (4T1) and non-metastatic (4T1-TWIST KO) breast tumor sister cell lines to understand their metabolic behavior, determine differences in two modes of imaging (reflection & transmission), and observe effect of breathing higher oxygen percentage on vascular hemoglobin oxygen saturation. After injection of 10,000 cells into mice dorsal window chambers, the glucose intake and hemoglobin oxygen saturation was measured using a fluorescent glucose analog (2-NBDG) and hyperspectral trans-illumination imaging from 520-620 nm at 10 nm intervals, respectively. The metastatic tumors exhibited increased oxygen saturation and decreased glucose metabolism than non-metastatic tumors. Reflection mode of imaging was unable to pick intricacies in tumor parameters, and increased inhalation of oxygen caused increase in hemoglobin oxygen saturation

Lactate Regulates Metabolic and Proinflammatory Circuits in Control of T Cell Migration and Effector Functions

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TP53-inducible Glycolysis and Apoptosis Regulator (TIGAR) Metabolically Reprograms Carcinoma and Stromal Cells in Breast Cancer.

A subgroup of breast cancers has several metabolic compartments. The mechanisms by which metabolic compartmentalization develop in tumors are poorly characterized. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a bisphosphatase that reduces glycolysis and is highly expressed in carcinoma cells in the majority of human breast cancers. Hence we set out to determine the effects of TIGAR expression on breast carcinoma and fibroblast glycolytic phenotype and tumor growth. The overexpression of this bisphosphatase in carcinoma cells induces expression of enzymes and transporters involved in the catabolism of lactate and glutamine. Carcinoma cells overexpressing TIGAR have higher oxygen consumption rates and ATP levels when exposed to glutamine, lactate, or the combination of glutamine and lactate. Coculture of TIGAR overexpressing carcinoma cells and fibroblasts compared with control cocultures induce more pronounced glycolytic differences between carcinoma and fibroblast cells. Carcinoma cells overexpressing TIGAR have reduced glucose uptake and lactate production. Conversely, fibroblasts in coculture with TIGAR overexpressing carcinoma cells induce HIF (hypoxia-inducible factor) activation with increased glucose uptake, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKFB3), and lactate dehydrogenase-A expression. We also studied the effect of this enzyme on tumor growth. TIGAR overexpression in carcinoma cells increases tumor growth in vivo with increased proliferation rates. However, a catalytically inactive variant of TIGAR did not induce tumor growth. Therefore, TIGAR expression in breast carcinoma cells promotes metabolic compartmentalization and tumor growth with a mitochondrial metabolic phenotype with lactate and glutamine catabolism. Targeting TIGAR warrants consideration as a potential therapy for breast cancer

Sulforaphane induces adipocyte browning and promotes glucose and lipid utilization

Scope: Obesity is closely related to the imbalance of white adipose tissue storing excess calories, and brown adipose tissue dissipating energy to produce heat in mammals. Recent studies revealed that acquisition of brown characteristics by white adipocytes, termed “browning,” may positively contribute to cellular bioenergetics and metabolism homeostasis. The goal was to investigate the putative effects of natural antioxidant sulforaphane (1-isothiocyanate-4-methyl-sulfonyl butane; SFN) on browning of white adipocytes. Methods and Results: 3T3-L1 mature white adipocytes were treated with SFN for 48 h, and then the mitochondrial content, function, and energy utilization were assessed. SFN was found to induce 3T3-L1 adipocytes browning based on the increased mitochondrial content and activity of respiratory chain enzymes, whereas the mechanism involved the upregulation of nuclear factor E2-related factor 2/ sirtuin1/ peroxisome proliferator-activated receptor gamma coactivator 1 alpha signaling. SFN enhanced uncoupling protein 1 expression, a marker for brown adipocyte, leading to the decrease in cellular ATP. SFN also enhanced glucose uptake and oxidative utilization, lipolysis and fatty acid oxidation in 3T3-L1 adipocytes. Conclusion: SFN-induced browning of white adipocytes enhanced the utilization of cellular fuel, and the application of SFN is a promising strategy to combat obesity and obesity-related metabolic disorder

Hysteretic control of grid-side current for a single-phase LCL grid-connected voltage source converter

© 2016. This manuscript version is made available under the CC-BY-NC-ND 4.0 license http://creativecommons.org/licenses/by-nc-nd/4.0/This paper proposes a new approach to control the grid-side current of LCL-grid connected voltage source converters using hysteretic relay feedback controllers. The closed loop system is stabilized by designing a local feedback around the relay element. The compensator allows the use of relay feedback controllers by making the controlled plant almost strictly positive real. The article proposes the use of the locus of the perturbed relay system as analysis and design tool and studies orbital stability for several plant and controller conditions. The approach is validated by means of simulation testing.Postprint (author's final draft

Time-lapse 3-D measurements of a glucose biosensor in multicellular spheroids by light sheet fluorescence microscopy in commercial 96-well plates

Light sheet fluorescence microscopy has previously been demonstrated on a commercially available inverted fluorescence microscope frame using the method of oblique plane microscopy (OPM). In this paper, OPM is adapted to allow time-lapse 3-D imaging of 3-D biological cultures in commercially available glass-bottomed 96-well plates using a stage-scanning OPM approach (ssOPM). Time-lapse 3-D imaging of multicellular spheroids expressing a glucose Förster resonance energy transfer (FRET) biosensor is demonstrated in 16 fields of view with image acquisition at 10 minute intervals. As a proof-of-principle, the ssOPM system is also used to acquire a dose response curve with the concentration of glucose in the culture medium being varied across 42 wells of a 96-well plate with the whole acquisition taking 9 min. The 3-D image data enable the FRET ratio to be measured as a function of distance from the surface of the spheroid. Overall, the results demonstrate the capability of the OPM system to measure spatio-temporal changes in FRET ratio in 3-D in multicellular spheroids over time in a multi-well plate format

Recombinant canine single chain insulin analogues: Insulin receptor binding capacity and ability to stimulate glucose uptake

Virtually all diabetic dogs require exogenous insulin therapy to control their hyperglycaemia. In the UK, the only licensed insulin product currently available is a purified porcine insulin preparation. Recombinant insulin is somewhat problematic in terms of its manufacture, since the gene product (preproinsulin) undergoes substantial post-translational modification in pancreatic β cells before it becomes biologically active. The aim of the present study was to develop recombinant canine single chain insulin (SCI) analogues that could be produced in a prokaryotic expression system and which would require minimal processing. Three recombinant SCI constructs were developed in a prokaryotic expression vector, by replacing the insulin C-peptide sequence with one encoding a synthetic peptide (GGGPGKR), or with one of two insulin-like growth factor (IGF)-2 C-peptide coding sequences (human: SRVSRRSR; canine: SRVTRRSSR). Recombinant proteins were expressed in the periplasmic fraction of Escherichia coli and assessed for their ability to bind to the insulin and IGF-1 receptors, and to stimulate glucose uptake in 3T3-L1 adipocytes. All three recombinant SCI analogues demonstrated preferential binding to the insulin receptor compared to the IGF-1 receptor, with increased binding compared to recombinant canine proinsulin. The recombinant SCI analogues stimulated glucose uptake in 3T3-L1 adipocytes compared to negligible uptake using recombinant canine proinsulin, with the canine insulin/cIGF-2 chimaeric SCI analogue demonstrating the greatest effect. Thus, biologically-active recombinant canine SCI analogues can be produced relatively easily in bacteria, which could potentially be used for treatment of diabetic dogs

Optimized Protocol for Measuring 2-NBDG Uptake as a Cellular Marker of Glycolytic Demand

2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) a fluorescently tagged analog of FDG is ideal for in vitro studies and imaging. 2-NBDG has been proven to be an adequate marker for glucose uptake in many different types of cells [1]. However, across the spectrum of 2-NBDG use a lack of consensus is observed for the following questions. What is the ideal time to fast cells to optimize cellular uptake of 2-NBDG? Also, what is the ideal concentration of 2-NBDG to be used when quantifying glucose uptake? Finally, what is the effect of serum on the uptake of 2-NBDG? To answer these questions and understand glucose uptake, the Balb/cfC3H 4T07 murine breast cancer cell line was fasted at varying time points between 0 and 150 minutes. Cell viability was evaluated for these time points using Promega’s (Madison, WI) CellTiter-Glo® luminescent assay. Cells were also plated into 35mm glass bottom dishes, incubated for 24 hours, and fasted for varying times between 0 and 150 minutes. 400µM of 2-NBDG was introduced for 20 minutes and uptake was quantified using fluorescence microscopy. The peak of cell viability and glucose uptake was compared to find the optimal fasting time. Once fasting studies were complete, cells were fasted according to ideal conditions and concentration dependency of 2-NBDG was investigated. It was found that 4T07 cell viability is significantly decreased by 60 minutes of fasting cells in DMEM (-) glucose in the absence of 10% serum. The addition of 10% serum to the DMEM (-) glucose prolongs the fasting range to at least 150 minutes. 2-NBDG uptake is higher with the addition of 10% serum to DMEM (-) glucose in 20 minute fasting conditions. Also, 400µM 2-NBDG is the ideal concentration to optimize cell viability, cost effectiveness, and uptake