Low Glucose Environment Research Articles

Effect of Lactate Export Inhibition on Anaplastic Thyroid Cancer Growth and Metabolism.

Anaplastic thyroid cancer (ATC) is an aggressive malignancy without effective treatments. ATC cells demonstrate upregulated glycolysis (Warburg effect), generating lactate that is subsequently exported by monocarboxylate transporter 4 (MCT4). This study aims to determine whether MCT4 inhibition can suppress ATC growth. ATC cell lines 8505C, JL30, and TCO1 were grown in low (3 mmol/L; LG) or high (25 mmol/L; HG) glucose medium containing the lactate shuttle inhibitors acriflavine (10-25 μmol/L; ACF), syrosingopine (100 µmol/L; SYR), or AZD3965 (20 µmol/L; AZD). Lactate level and cell proliferation were measured with standard assays. Seahorse analysis was performed to determine glycolytic response. Compared with HG, addition of ACF to LG decreased lactate secretion for both 8505C (p < 10-5) and JL30 (p < 10-4) cells, whereas proliferation was also reduced (p < 10-4 and 10-5, respectively). During Seahorse analysis, addition of oligomycin increased acidification by 84 mpH/min in HG vs 10 mpH/min in LG containing ACF (p < 10-5). Treatment with LG and SYR drastically diminished 8505C and TCO1 growth vs HG (p < 0.01 for both). LG and AZD treatment also led to reduced proliferation in tested cell lines (p ≤ 0.01 for all) that was further decreased by addition of ACF (p < 10-4 vs HG, p ≤ 0.01 vs LG and AZD). Inhibition of lactate shuttles significantly reduced proliferation and glycolytic capacity of ATC cells in a low-glucose environment. Targeting suppression of glycolytic and lactate processing pathways may represent an effective treatment strategy for ATC.

TAZ promotes PDX1-mediated insulinogenesis.

Transcriptional co-activator with PDZ-binding motif (TAZ) is a key mediator of the Hippo signaling pathway and regulates structural and functional homeostasis in various tissues. TAZ activation is associated with the development of pancreatic cancer in humans, but it is unclear whether TAZ directly affects the structure and function of the pancreas. So we sought to identify the TAZ function in the normal pancreas. TAZ defect caused structural changes in the pancreas, particularly islet cell shrinkage and decreased insulin production and β-cell markers expression, leading to hyperglycemia. Interestingly, TAZ physically interacted with the pancreatic and duodenal homeobox 1 (PDX1), a key insulin transcription factor, through the N-terminal domain of TAZ and the homeodomain of PDX1. TAZ deficiency decreased the DNA-binding and transcriptional activity of PDX1, whereas TAZ overexpression promoted PDX1 activity and increased insulin production even in a low glucose environment. Indeed, high glucose increased insulin production by turning off the Hippo pathway and inducing TAZ activation in pancreatic β-cells. Ectopic TAZ overexpression along with PDX1 activation was sufficient to produce insulin in non-β-cells. TAZ deficiency impaired the mesenchymal stem cell differentiation into insulin-producing cells (IPCs), whereas TAZ recovery restored normal IPCs differentiation. Compared to WT control, body weight increased in TAZ-deficient mice with age and even more with a high-fat diet (HFD). TAZ deficiency significantly exacerbated HFD-induced glucose intolerance and insulin resistance. Therefore, TAZ deficiency impaired pancreatic insulin production, causing hyperglycemia and exacerbating HFD-induced insulin resistance, indicating that TAZ may have a beneficial effect in treating insulin deficiency in diabetes.

Lactic acid promotes PD-1 expression in regulatory Tcells in highly glycolytic tumor microenvironments.

The balance of programmed death-1 (PD-1)-expressing CD8+ Tcells and regulatory T (Treg) cells in the tumor microenvironment (TME) determines the clinical efficacy of PD-1 blockade therapy through the competition of their reactivation. However, factors that determine this balance remain unknown. Here, we show that Treg cells gain higher PD-1 expression than effector Tcells in highly glycolytic tumors, including MYC-amplified tumors and liver tumors. Under low-glucose environments via glucose consumption by tumor cells, Treg cells actively absorbed lactic acid (LA) through monocarboxylate transporter 1 (MCT1), promoting NFAT1 translocation into the nucleus, thereby enhancing the expression of PD-1, whereas PD-1 expression by effector Tcells was dampened. PD-1 blockade invigorated the PD-1-expressing Treg cells, resulting in treatment failure. We propose that LA in the highly glycolytic TME is an active checkpoint for the function of Treg cells in the TME via upregulation of PD-1 expression.

Intracellular glucose starvation affects gingival homeostasis and autophagy

Human gingival fibroblasts (HGnFs) maintain periodontal tissue homeostasis through active proliferation and migration. Clinically, it is considered that the wound-healing ability of the gingival tissue is maintained even in environments with insufficient supply of nutrients, such as glucose, immediately after periodontal surgery. However, the effects of such glucose-deficient environments on HGnFs remain unclear. This study aimed to investigate the effects of low-glucose environment on HGnFs homeostasis. We evaluated gingival wound healing by examining cell proliferation and migration and collagen synthesis in HGnFs cultured in 100, 50, 25, and 0 mg/dL glucose in vitro. The cellular stress levels were determined by measuring the lactate dehydrogenase (LDH) and reactive oxygen species (ROS) levels. The glucose metabolism of HGnFs in the low-glucose concentrations was studied by measuring glucose transporter type 1 (GLUT1) mRNA expression, glucose uptake assays, lactate and ATP productions. Molecular effects were examined with a focus on the LKB1-AMPK signaling pathway. Autophagy activity in glucose-deprived HGnFs was evaluated by measuring the levels of autophagy-related proteins. Low glucose levels increased cellular stress levels, autophagy activity, and enhanced glucose metabolism through the LKB1-AMPK signaling pathway, providing more ATPs to promote wound healing. Our results regarding glucose transfer suggest the rapid healing of gingival wounds.

Research on the Mechanism of SIRT1/AMPK Inducing Resistance to Imatinib in K562 Cells By Inhibiting Srebp to Promote Lipid Metabolism

Background Leukemia stem cells (LSC) have the characteristics of independent of BCR-ABL kinase activity and metabolic reprogramming to support self-renewal. They are the root cause of the occurrence of chronic myeloid leukemia (CML), mediating TKI resistance and leading to clonal evolution. LSC self-renewal depends on the activity of SIRT1, the enhanced of SIRT1 induces LSC mitochondrial metabolic reprogramming. Studies have shown that SIRT1 and abnormally activated lipid metabolism are important reasons for inducing CML resistance to imatinib.Objective Study the mechanism of SIRT1 regulating lipid metabolism and intervention strategies under imatinib therapy.Method Liquid chromatography coupled with mass spectrometry (LC-MS) were used to analysis the lipid metabolism in imatinib-resistant CML cell line K562-R under low-glucose conditions. RT-qPCR and Western blot were used to detect the key enzymes related to lipid metabolism, and the SIRT1 inhibitor TV-6 was given to study the regulation mechanism of SIRT1 on lipid metabolism.Result Firstly, we found that the glycolysis/gluconeogenesis pathway and fatty acid metabolism were abnormal in K562-R cells by metabolomics, compared with normal culture conditions, the expression of SIRT1 and AMPK were significantly increased, the expression of fatty acid synthesis-related genes (SREBP, FASN, ACC, SCD1) was significantly reduced, and the fatty acid β oxidation rate-limiting enzyme CPT-1 is significantly increased inK562-R cells under low-glucose conditions. Therefore, in the low-glucose environment, the expression of the above genes was reversed after the application of the SIRT1 inhibitor TV-6.Conclusion K562-R imatinib-resistant cells can down-regulate the expression of SREBP through up-regulated SIRT1/AMPK pathway under low-glucose conditions, thereby inhibiting the expression of transcription factors involved in lipogenesis, reducing fatty acid synthesis, and providing the imatinib-resistant cells with the energy which required for proliferation.Keyword Chronic myeloid leukemia; Metabolomics; Lipid metabolism; SIRT1/AMPK DisclosuresNo relevant conflicts of interest to declare.

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

Glucose-responsive oral insulin delivery platform for one treatment a day in diabetes

In Silico Study of the Inhibitory Effect of Isoflavones on the Genes Frequently Reported in the Pathophysiology of Breast Cancer

Phytoestrogens are naturally occurring plant-derived active substances found in a wide variety of foods. Numerous health benefits of phytoestrogens include the reduced occurrence of breast tumors. Overexpression of the protein serine-hydroxyl-methyl-transferase-2 (SHMT2) has been reported in breast cancer. In one-carbon metabolism SHMT2 enzyme is present, which is necessary for cancer cell proliferation in a low glucose environment. Hence, this study focused on natural isoflavones that have a toxic effect on the development of breast cancer cells without harming normal cells. The structure of the protein SHMT2 was retrieved from the protein data bank and the structure of isoflavones was determined using the PubChem database. The docking of isoflavones with SHMT2 was done to determine their inhibitory effect on the latter since it causes the rapid development of breast cancer cells. It was identified that two isoflavones (6O-acetyldaidzin and malonyldaidzin) have a high affinity to bind with the protein because of hydrogen bonding. This study highly recommends conducting clinical trials on human beings based on the interaction between phytochemicals and SHMT2 gene to determine the safe dosage level of the former.

Age and sex modify cellular proliferation responses to oxidative stress and glucocorticoid challenges in baboon cells

Aging is associated with progressive loss of cellular homeostasis resulting from intrinsic and extrinsic challenges. Lack of a carefully designed, well-characterized, precise, translational experimental model is a major limitation to understanding the cellular perturbations that characterize aging. Here, we tested the feasibility of primary fibroblasts isolated from nonhuman primates (baboons) as a model of cellular resilience in response to homeostatic challenge. Using a real-time live-cell imaging system, we precisely defined a protocol for testing effects of prooxidant compounds (e.g., hydrogen peroxide (H2O2), paraquat), thapsigargin, dexamethasone, and a low glucose environment on cell proliferation in fibroblasts derived from baboons across the life course (n = 11/sex). Linear regression analysis indicated that donor age significantly reduced the ability of cells to proliferate following exposure to H2O2 (50 and 100µM) and paraquat (100 and 200µM) challenges in cells from males (6.4-21.3years; average lifespan 21years) but not cells from females (4.3-15.9years). Inhibitory effects of thapsigargin on cell proliferation were dependent on challenge duration (2 vs 24h) and concentration (0.1 and 1µM). Cells from older females (14.4-15.9years) exhibited greater resilience to thapsigargin (1µM; 24h) and dexamethasone (500µM) challenges than did those from younger females (4.3-6.7years). The cell proliferation response to low glucose (1mM) was reduced with age in both sexes. These data indicate that donor's chronological age and sex are important variables in determining fibroblast responses to metabolite and other challenges.

Interplay between γδT-Cell Metabolism and Tumour Microenvironment Offers Opportunities for Therapeutic Intervention

Solid tumour targeting using adoptive cell therapy has failed to reproduce the spectacular clinical successes seen with chimeric antigen receptor T cell therapies and B cell malignancies. Low in glucose, oxygen, pH and populated with suppressive cells, the solid tumour microenvironment (TME) remains a formidable obstacle to successful immune targeting. The use of atypical, tissue-tropic lymphocytes, such as γδT cells, may offer enhanced tumour trafficking over canonical αβT cells. Nonetheless, γδT cells too interact with the TME. The consequences of this interaction are poorly understood and of high translational relevance. Lopes and colleagues show that, in a murine context, low glucose environments preferentially retained pro-tumorigenic IL-17-producing γδT cells. Anti-tumorigenic IFN-γ-producing γδT cells, meanwhile, required high ambient glucose to survive and exert effector function. Unexpectedly, this metabolic imprinting was evident in the murine thymus, suggesting that the ontological separation of these functional subsets occurs early in their development. Elucidation of this relationship between TME glucose levels and γδT cell functionality in a human context is likely to carry significant implications for the development of γδT cell-based oncoimmunotherapeutics.

Testing Cellular Proliferation Responses to Oxidative, Glucocorticoid and Metabolic Challenges in the Baboon

Aging is associated with progressive loss of cellular homeostasis which results from intrinsic and extrinsic challenges. Testing how such challenges relate to the aging process is often limited by the available model systems. We use primary cells (fibroblasts) isolated from baboons as a non-human primate cellular model to address how stressful challenges affect resilience. Using a real-time live-cell imaging system, we characterized a protocol for testing the effects of pro-oxidant compounds (e.g hydrogen peroxide (H2O2), paraquat and thapsigargin), dexamethasone and low glucose environment on cellular proliferation in fibroblasts derived from baboons across the life-course. The inhibitory effect of H2O2 (50 and 100µM), an oxidative stress, on cell proliferation was dose-dependent, with a higher impact in old males (age 14.52-14.80 years; average life span 21 years) compared to young males (6.35-6.39 years). Exposure to a different oxidative stress, paraquat (100 and 200µM) tended to reduce cell proliferation rate with age in males but not females. Inhibitory effects of thapsigargin, an endoplasmic reticulum stress inducer, on cell proliferation were dependent on challenge duration (2 vs 24h), concentration (0.1 and 1µM) and donor age, with greater resilience in young males than young females (4.33-6.70 years). Dexamethasone (100 and 500µM), a glucocorticoid, reduced cell proliferation dose-dependently, with older males exhibiting more resilience than females. In response to low glucose (1mM), cell proliferation reduced with age. Donor’s chronological age and sex are important variables in cellular response to challenge compounds faced during aging, which will guide our on-going studies on the cellular transcriptome and proteome.

Restricting Glycolysis Preserves T Cell Effector Functions and Augments Checkpoint Therapy

Tumor-derived lactic acid inhibits T and natural killer (NK) cell function and, thereby, tumor immunosurveillance. Here, we report that melanoma patients with high expression of glycolysis-related genes show a worse progression free survival upon anti-PD1 treatment. The non-steroidal anti-inflammatory drug (NSAID) diclofenac lowers lactate secretion of tumor cells and improves anti-PD1-induced Tcell killing invitro. Surprisingly, diclofenac, but not other NSAIDs, turns out to be a potent inhibitor of the lactate transporters monocarboxylate transporter 1 and 4 and diminishes lactate efflux. Notably, Tcell activation, viability, and effector functions are preserved under diclofenac treatment and in a low glucose environment invitro. Diclofenac, but not aspirin, delays tumor growth and improves the efficacy of checkpoint therapy invivo. Moreover, genetic suppression of glycolysis in tumor cells strongly improves checkpoint therapy. These findings support the rationale for targeting glycolysis in patients with high glycolytic tumors together with checkpoint inhibitors in clinical trials.

Ketogenic diet combined with antioxidant N-acetylcysteine inhibits tumor growth in a mouse model of anaplastic thyroid cancer

BackgroundAnaplastic thyroid cancer is an aggressive and fatal malignancy. Many advanced cancers are characterized by glucose dependency, leading to oxidative stress and cellular proliferation. Therefore, we sought to determine if a low glucose environment (in vitro) or a ketogenic diet (in vivo) could inhibit anaplastic thyroid cancer tumor growth when combined with the antioxidant N-acetylcysteine. MethodsIn vivo, nude mice were injected with the anaplastic thyroid cancer cell line 8505C (n = 6/group). Group 1 was fed a standard diet; Group 2 was fed a ketogenic diet; Group 3 was given standard diet with N-acetylcysteine (40 mM in the drinking water); and Group 4 was fed ketogenic diet with N-acetylcysteine. Tumor volumes, ketones, and glucose were measured. H&E stains and immunohistochemistry for Ki-67 and Caspase 3 were performed on the tumors. In vitro, 8505C cells were cultured in high glucose (25 mM), low glucose (3 mM), high glucose plus N-acetylcysteine (200 uM), or low glucose plus N-acetylcysteine for 96 hours. We performed CyQUANT proliferation (Thermo Fisher Scientific, Waltham, MA), Seahorse glycolytic stress (Agilent, Santa Clara, CA), and reactive oxidative stress assays. ResultsKetogenic diet plus N-acetylcysteine decreased in vivo tumor volume compared to standard diet (22.5 ± 12.4 mm3 vs 147 ± 54.4 mm3, P < .05) and standard diet plus N-acetylcysteine (P < .05). Blood ketone levels were significantly higher for the mice in the ketogenic diet group compared to standard diet (1.74 mmol/L vs 0.38 mmol/L at week 5, P < .001). However, blood glucose levels were not significantly different between ketogenic diet and standard diet groups. Cells cultured in low glucose plus N-acetylcysteine had significantly reduced proliferation compared to high glucose (98.1 ± 5.0 relative fluorescence units vs 157.8 ± 2.1 relative fluorescence units, P < .001). Addition of N-acetylcysteine to low glucose lowered glycolysis function compared to high glucose (39.0 ± 2.2 mpH/min/cell vs 89.1 ± 13.2 mpH/min/cell, P < .001) and high glucose plus N-acetylcysteine (37.4 ± 2.5 mpH/min/cell vs 70.3 ± 3.3 mpH/min/cell, P < .001). Low glucose plus N-acetylcysteine decreased reactive oxidative stress compared to high glucose (119 ± 34.7 relative fluorescence units vs 277 ± 16.0 relative fluorescence units, P = .014). ConclusionThe combination of a ketogenic diet or glucose restriction with the antioxidant- N-acetylcysteine significantly reduced tumor growth in vivo and in vitro. Further studies are warranted to explore these metabolic therapies in anaplastic thyroid cancer.

Abstract 1841: Adaptive upregulation of carbamoyl phosphate synthetase-1 (CPS-1) in glucose-deprived metastatic lung adenocarcinoma cells

Abstract Advanced lung cancers frequently metastasize to the brain, where nutrient deprivation from tumour microenvironments at either primary, invading or metastatic sites may exert a selective pressure on tumour cells. Our lab has identified a rare brain metastasis cell line of lung cancer origin: A1115, that possessed heightened basal glycolytic activity. Characterization of the metabolic profiles of A1115 and 4 other lung adenocarcinoma cell lines revealed A1115 to have the highest dependence on glycolysis and the lowest utilization of mitochondrial oxidative phosphorylation (OXPHOS) for metabolism. Thus, we hypothesized that A1115 is hypersensitive to glucose starvation. A1115 relied heavily on glycolysis on survival and propagation, while inhibiting mitochondrial OXPHOS had less effect. Glucose deprivation induced the nutrient sensor AMP-activated protein kinase (AMPK) to be elevated in A1115, while the proliferative capacity of A1115 was drastically reduced by an AMPK activator, AICAR, suggesting AMPK to be detrimental to cell viability of A1115 under glucose deprivation. To understand how such metastatic cell line could survive the low glucose environment in blood circulation and during the invasion into the brain parenchyma, we established clonal A1115 sublines that could propagate under low glucose (LG) environment. LG sublines regained hypersensitivity to glucose deprivation upon exposure to normal glucose concentrations suggest an adaptive response. We hypothesised that tumour cells maintain cell viability under glycolytic stress conditions by undergoing metabolic reprogramming, this involves changes in metabolite utilization and transcriptional activity of key metabolic enzymes. A parallel metabolomics (targeted LC/MS) and transcriptomics (RNAseq) analysis was conducted comparing A1115 LG sublines with the parental control. Metabolomics analysis revealed that LG sublines under glucose deprivation harboured a significant metabolite increase in amino acids: glutamate, arginine, aspartic acid and pyrimidine: cytidine, whereas integrated pathway analysis of metabolomics and transcriptomics revealed upregulation in both pyrimidine metabolism and alanine, aspartate and glutamate metabolism. Moreover, increased carbamoyl phosphate synthetase 1 (CPS-1) expression was observed in LG sublines, an initiating enzyme for pyrimidine biosynthesis and urea cycle, silencing CPS-1 decreased cell viability on LG in normal conditioned media, suggesting that the disruption of pathway could impede survival. Overall, our results suggest that lung adenocarcinoma may undergo reprogramming towards a starkly different constituent of transcriptional activation leading to metabolite flux and signalling activation to allow viable adaption to glucose deprivation in the microenvironment. Citation Format: Kin Lok Wong, Andrew M. Chan. Adaptive upregulation of carbamoyl phosphate synthetase-1 (CPS-1) in glucose-deprived metastatic lung adenocarcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1841.

Flow Cytometry for Evaluation of EGFR-mTOR Signal Way in the Growth Inhibitory Rate of Hela Cell Under Low Glucose and High Lactic Acid Environment

Objective: To discuss the effect of EGFR-mTOR signal way in the adaption of cervical carcinoma cell in low glucose and high lactic acid. Methods: Hela cells were cultured in four conditions: normal glucose (NG, glucose 10 mmol/L), low glucose (LG, glucose 3 mmol/L), high lactic acid (HL, lactic acid 2.5 mmol/L) and low glucose and high lactic acid (LGHL, glucose 10 mmol/L, lactic acid 2.5 mmol/L). Growth inhibitory rate of Hela cell was determined by CCK-8; Flow cytometry (FCM) was performed to evaluate the cell cycle; Results: Compared with the rate in NG, the rate in low glucose and high lactic acid was significantly increased (P &lt; 0.01). Compared with the rate in NG, the rate of G1/G0 was increased in HL and in LGHL (P &lt; 0.01, P &lt; 0.01); the rate of G1/G0 in LG didn't have significant difference; the rates of G2/M in HL and in LGHL were decreased, and the G2/M rate in LG was increased, but all of them had no statistically significant. The rates of S were decreased in HL, LG and LGHL (P &lt; 0.05, no statistically significant, P &lt; 0.05). The expression level of EGFR mRNA in Hela was decreased and the level of mTOR mRNA was increased in HL (both had no statistically significant), when compared with the levels in NG. The expression level of EGFR mRNA was decreased (P &lt; 0.05) and the level of mTOR mRNA was a little increased (no statistically significant) in LG; while the EGFR and mTOR expression levels were increased in LGHL (P &lt; 0.05). Conclusion: Low glucose and high lactic acid environment is conducive to Hela cell survival, and can promote the expression of EGFR and mTOR.

Bioinformatics Analysis of Gefitinib or Rapamycin on Inhibiting the Survival of Hela in the Low Glucose and High Lactic Acid Environment

Objective: To explore on the antitumor effect of gefitinib and rapamycin and possible mechanism in normal glucose and high lactic acid microenvironment. Methods: Hela cells are cultured in six conditions: the normal glucose group (NG, glucose 3 mmol/L); the normal glucose + gefitinib group (NGG, glucose 3 mmol/L, gefitinib 2.67 μmol/L); the normal glucose + rapamycin group (NGR, glucose 3 mmol/L, rapamycin 2.67 μmol/L); the high lactate group (NGHL, glucose 3 mmol/L, lactic acid 2.5 mmol/L); the normal glucose + high lactate + gefitinib group (NGHLG, glucose 3 mmol/L, lactic acid 2.5 mmol/L, gefitinib 2.67μmol/L); the normal glucose + high lactate + rapamycin group (NGHLG, glucose 10 mmol/L, lactic acid 2.5 mmol/L, rapamycin 2.67μmol/L). Growth inhibitory rate of Hela cell is determined by CCK-8; Flow cytometry (FCM) is performed to evaluate the cell cycle; The annexin V-phycoerythrin/Propidium Iodide (annexin V-PE/PI) staining combined with flow cytometry is used to examine the cell cycle and apoptosis of Hela cells. Results: Under normal glucose with gefitinib or rapamycin environment, the apoptosis rate of Hela cells is higher than that of the normal glucose group. But the cell apoptosis rate of the gefitinib or rapamycin group decreases in high lactic acid and normal glucose, which is lower than that of the normal glucose and high lactate. Combined with the results of cell cycle, compared with the normal glucose group, percentage of Hela cells in G1/G0 phase increases significantly, the proportion of S phase cells decreases significantly in high lactic acid environment. In the normal glucose and gefitinib environment, Hela cells in G1/G0 phase and S phase are slightly higher than the proportion of normal glucose group, and G2/M phase cells are mild lower than the proportion of normal glucose group. Under the environment of high lactate and normal glucose, the percentage of G1/G0 and S phase cells in the gefitinib increase. As for rapamycin, normal glucose and high lactic acid environment makes cells stay in G1/G0 phase. The presence of rapamycin in the environment of normal sugar and high lactate makes more cells stay in G1/G0 or G2/M phase. Conclusion: Normal glucose and high lactic acid environment is conducive to Hela cell survival, and can promote the expression of EGFR and mTOR. Gefitinib is an antagonist of EGFR and rapamycin is an inhibitor of mTOR.

Gibbs Free-Energy Gradient along the Path of Glucose Transport through Human Glucose Transporter 3.

Fourteen glucose transporters (GLUTs) play essential roles in human physiology by facilitating glucose diffusion across the cell membrane. Due to its central role in the energy metabolism of the central nervous system, GLUT3 has been thoroughly investigated. However, the Gibbs free-energy gradient (what drives the facilitated diffusion of glucose) has not been mapped out along the transport path. Some fundamental questions remain. Here we present a molecular dynamics study of GLUT3 embedded in a lipid bilayer to quantify the free-energy profile along the entire transport path of attracting a β-d-glucose from the interstitium to the inside of GLUT3 and, from there, releasing it to the cytoplasm by Arrhenius thermal activation. From the free-energy profile, we elucidate the unique Michaelis–Menten characteristics of GLUT3, low KM and high VMAX, specifically suitable for neurons’ high and constant demand of energy from their low-glucose environments. We compute GLUT3’s binding free energy for β-d-glucose to be −4.6 kcal/mol in agreement with the experimental value of −4.4 kcal/mol (KM = 1.4 mM). We also compute the hydration energy of β-d-glucose, −18.0 kcal/mol vs the experimental data, −17.8 kcal/mol. In this, we establish a dynamics-based connection from GLUT3’s crystal structure to its cellular thermodynamics with quantitative accuracy. We predict equal Arrhenius barriers for glucose uptake and efflux through GLUT3 to be tested in future experiments.

144 - Low glucose environment triggers significant angiogenic response from first trimester (FTM) but not term human umbilical cord perivascular cells (HUCPVCs)

144 - Low glucose environment triggers significant angiogenic response from first trimester (FTM) but not term human umbilical cord perivascular cells (HUCPVCs)

A Disposable Tear Glucose Biosensor-Part 5: Improvements in Reagents and Tear Sampling Component.

A tear glucose (TG) sensor with an integrated tear sampler can provide a noninvasive method for calibrating the continuous TG contact lens and monitoring glucose. Expanding from previous work, an improved TG sensor that implements dried reagents, genetically modified glucose dehydrogenase (GDH), and a tear sampler was developed and compared against the TG sensor prepared with commercial GDH. It was found that neither sensor was affected by the tear interferents: ascorbic acid, acetaminophen, and uric acid. The sensor prepared with commercial GDH generated higher current. This suggests that using enzymes with lower Km may be advantageous when operating in low glucose environments like tears. The improved TG sensor also demonstrated the potential of integrating Schirmer's test strip as a tear sampler for self-monitoring of TG.

Aging impairs mitochondrial respiratory capacity in classical monocytes.

Aging is a critical healthcare concern, with age-related inflammation disposing individuals to a variety of diseases. Monocytes are affected by the aging process, with increased inflammation and impaired cellular functions such as phagocytosis. Mechanisms by which aging alters monocyte function are unknown, but recent research suggests that the balance of metabolic processes determine immune cell phenotype and function. Given the known association between aging and mitochondrial dysfunction in other tissues, we hypothesized that aging would impair mitochondrial function in monocytes. To test this, we isolated classical monocytes from young and older adults and tested mitochondrial function by a Seahorse assay. Aging reduced mitochondrial respiratory capacity and spare capacity in monocytes. Mitochondrial dysfunction is a potential mechanism by which aging alters monocyte phenotype and may impair inflammatory functions, especially in low-glucose environments where oxidative metabolism is necessary to meet energy demands.

Surface-Enhanced Raman Spectroscopy-Based Label-Free Insulin Detection at Physiological Concentrations for Analysis of Islet Performance.

Label-free optical detection of insulin would allow in vitro assessment of pancreatic cell functions in their natural state and expedite diabetes-related clinical research and treatment; however, no existing method has met these criteria at physiological concentrations. Using spatially uniform 3D gold-nanoparticle sensors, we have demonstrated surface-enhanced Raman sensing of insulin in the secretions from human pancreatic islets under low and high glucose environments without the use of labels such as antibodies or aptamers. Label-free measurements of the islet secretions showed excellent correlation among the ambient glucose levels, secreted insulin concentrations, and measured Raman-emission intensities. When excited at 785 nm, plasmonic hotspots of the densely arranged 3D gold-nanoparticle pillars as well as strong interaction between sulfide linkages of the insulin molecules and the gold nanoparticles produced highly sensitive and reliable insulin measurements down to 100 pM. The sensors exhibited a dynamic range of 100 pM to 50 nM with an estimated detection limit of 35 pM, which covers the reported concentration range of insulin observed in pancreatic cell secretions. The sensitivity of this approach is approximately 4 orders of magnitude greater than previously reported results using label-free optical approaches, and it is much more cost-effective than immunoassay-based insulin detection widely used in clinics and laboratories. These promising results may open up new opportunities for insulin sensing in research and clinical applications.