SLC15A4 mediates M1-prone metabolic shifts in macrophages and guards immune cells from metabolic stress.

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR)7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating the mechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage's respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells' ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters.

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

Restoration of the adipogenic gene expression by naringenin and naringin in 3T3-L1 adipocytes.

BACKGROUND: Naringenin and its glycoside naringin are well known citrus flavonoids with several therapeutic benefits. Although the anti-adipogenic effects of naringenin and naringin have been reported previously, the detailed mechanism underlying their anti-adipogenesis effects is poorly understood. OBJECTIVES: This study examined the anti-adipogenic effects of naringenin and naringin by determining differential gene expression patterns in these flavonoids-treated 3T3-L1 adipocytes. METHODS: Lipid accumulation and triglyceride (TG) content were determined by Oil red O staining and TG assay. Glucose uptake was measured using a 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose fluorescent d-glucose analog. The phosphorylation levels of AMP-activated protein kinase (AMPK) and acetyl Co-A carboxylase (ACC) were observed via Western blot analysis. Differential gene expressions in 3T3-L1 adipocytes were evaluated via RNA sequencing analysis. RESULTS: Naringenin and naringin inhibited both lipid accumulation and TG content, increased phosphorylation levels of both AMPK and ACC and decreased the expression level of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) in 3T3-L1 adipocytes. RNA sequencing analysis revealed that 32 up-regulated (> 2-fold) and 17 down-regulated (< 0.6-fold) genes related to lipid metabolism, including Acaca, Fasn, Scd1, Mogat1, Dgat, Lipin1, Cpt1a, and Lepr, were normalized to the control level in naringenin-treated adipocytes. In addition, 25 up-regulated (> 2-fold) and 25 down-regulated (< 0.6-fold) genes related to lipid metabolism, including Acaca, Fasn, Fabp5, Scd1, Srebf1, Hmgcs1, Cpt1c, Lepr, and Lrp1, were normalized to the control level by naringin. CONCLUSIONS: The results indicate that naringenin and naringin have anti-adipogenic potentials that are achieved by normalizing the expression levels of lipid metabolism-related genes that were perturbed in differentiated 3T3-L1 cells.

Intravital Microscopy for the Study of Hepatic Glucose Uptake.

The liver is central in maintaining glucose homeostasis. Indeed, impaired hepatic glucose uptake has been implicated in the development of hyperglycemia in type II diabetes (T2D) and non-alcoholic fatty liver disease (NAFLD). However, current approaches to evaluate glucose mobilization rely on indirect measurements that do not provide spatial and temporal information. Here, we describe confocal-based intravital microscopy (IVM) of the liver that allows the identification of hepatocyte spatial organization and glucose transport. Specifically, we describe a method to fluorescently label hepatic landmarks to identify different compartments within the liver. In addition, we outline an in vivo fluorescent glucose uptake assay to quantitatively measure glucose mobilization in space and time. These protocols allow direct investigation of hepatic glycemic control and can be further applied to murine models of liver disease. © Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Mouse surgical procedure and positioning for liver intravital imaging Basic Protocol 2: Fluorescent labeling and intravital imaging of mouse hepatic compartments Basic Protocol 3: Mouse hepatic glucose uptake assay and intravital imaging analysis.

Molecular Imaging of Glucose Metabolism for Intraoperative Fluorescence Guidance During Glioma Surgery.

PURPOSE: This study evaluated the use of molecular imaging of fluorescent glucose analog 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) as a discriminatory marker for intraoperative tumor border identification in a murine glioma model. PROCEDURES: 2-NBDG was assessed in GL261 and U251 orthotopic tumor-bearing mice. Intraoperative fluorescence of topical and intravenous 2-NBDG in normal and tumor regions was assessed with an operating microscope, handheld confocal laser scanning endomicroscope (CLE), and benchtop confocal laser scanning microscope (LSM). Additionally, 2-NBDG fluorescence in tumors was compared with 5-aminolevulinic acid-induced protoporphyrin IX fluorescence. RESULTS: Intravenously administered 2-NBDG was detectable in brain tumor and absent in contralateral normal brain parenchyma on wide-field operating microscope imaging. Intraoperative and benchtop CLE showed preferential 2-NBDG accumulation in the cytoplasm of glioma cells (mean [SD] tumor-to-background ratio of 2.76 [0.43]). Topically administered 2-NBDG did not create sufficient tumor-background contrast for wide-field operating microscope imaging or under benchtop LSM (mean [SD] tumor-to-background ratio 1.42 [0.72]). However, topical 2-NBDG did create sufficient contrast to evaluate cellular tissue architecture and differentiate tumor cells from normal brain parenchyma. Protoporphyrin IX imaging resulted in a more specific delineation of gross tumor margins than intravenous or topical 2-NBDG and a significantly higher tumor-to-normal-brain fluorescence intensity ratio. CONCLUSION: After intravenous administration, 2-NBDG selectively accumulated in the experimental brain tumors and provided bright contrast under wide-field fluorescence imaging with a clinical-grade operating microscope. Topical 2-NBDG was able to create a sufficient contrast to differentiate tumor from normal brain cells on the basis of visualization of cellular architecture with CLE. 5-Aminolevulinic acid demonstrated superior specificity in outlining tumor margins and significantly higher tumor background contrast. Given the nontoxicity of 2-NBDG, its use as a topical molecular marker for noninvasive in vivo intraoperative microscopy is encouraging and warrants further clinical evaluation.

Uptake of fluorescent D- and L-glucose analogues, 2-NBDG and 2-NBDLG, into human osteosarcoma U2OS cells in a phloretin-inhibitable manner.

Mammalian cells take in D-glucose as an essential fuel as well as a carbon source. In contrast, L-glucose, the mirror image isomer of D-glucose, has been considered merely as a non-transportable/non-metabolizable control for D-glucose. We have shown that 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), a D-glucose analogue combining a fluorophore NBD at the C-2 position, is useful as a tracer for monitoring D-glucose uptake through glucose transporters (GLUTs) into mammalian cells. To more precisely evaluate the stereoselectivity of 2-NBDG uptake, we developed an L-glucose analogue 2-NBDLG, the mirror-image isomer of 2-NBDG. Interestingly, 2-NBDLG was taken up into mouse insulinoma MIN6 cells showing nuclear heterogeneity, a cytological feature of malignancy, while remaining MIN6 cells only exhibited a trace amount of 2-NBDLG uptake. The 2-NBDLG uptake into MIN6 cells was abolished by phloretin, but persisted under blockade of major mammalian glucose transporters. Unfortunately, however, no such uptake could be detected in other tumor cell lines. Here we demonstrate that human osteosarcoma U2OS cells take in 2-NBDLG in a phloretin-inhibitable manner. The uptake of 2-NBDG, and not that of 2-NBDLG, into U2OS cells was significantly inhibited by cytochalasin B, a potent GLUT inhibitor. Phloretin, but neither phlorizin, an inhibitor of sodium-glucose cotransporter (SGLT), nor a large amount of D/L-glucose, blocked the 2-NBDLG uptake. These results suggest that a phloretin-inhibitable, non-GLUT/non-SGLT, possibly non-transporter-mediated yet unidentified mechanism participates in the uptake of the fluorescent L-glucose analogue in two very different tumor cells, the mouse insulinoma and the human osteosarcoma cells.

Tyrosine-phosphorylation and activation of glucosylceramide synthase by v-Src: Its role in survival of HeLa cells against ceramide.

Sphingolipids represent a family of cellular lipid-molecules that regulate physiological and pathophysiological processes. Glucosylceramide (GlcCer), the simplest glycosphingolipid (GSL), is synthesized from ceramide and UDP-glucose by GlcCer synthase (GCS). Both GlcCer (and resulting GSLs) and ceramide regulate various cellular functions including cell death and multiple drug resistance. Src family tyrosine kinases are up-regulated in various human cancer cells. We examined the effect of v-Src expression on GCS activity, the formation of 4-nitrobenzo-2-oxa-1,3-diazole (NBD)-labeled GlcCer from NBD-ceramide, and the effect of tyrosine132 mutation in GCS on ceramide-induced cytotoxicity in HeLa cells. Expression of v-Src increased the formation of NBD-GlcCer in both intact cells without marked changes in other sphingolipid metabolites and cell homogenates without changing affinities of NBD-ceramide and UDP-glucose. Expression of v-Src also increased tyrosine-phosphorylated levels in GCS proteins in HeLa and HEK293T cells. In HEK293T cells transiently expressing the GCS mutant, GCS-Y132F-HA, showing replacement of the tyrosine132 residue with phenylalanine, tyrosine-phosphorylated levels in GCS proteins were significantly lower than those in control cells expressing the GCS-wild-type-HA. The formation of NBD-GlcCer in HeLa cells stably expressing GCS-Y132F-HA was significantly lower than that in the control. Ceramide-induced cytotoxicity in HeLa-GCS-Y132F-HA cells was significantly greater than in the control. In this study, we showed for the first time that expression of v-Src up-regulated GCS activity via tyrosine phosphorylation of the enzyme in a post-translational manner. Mechanisms of Src-induced resistance to ceramide-induced cytotoxicity are discussed in relation to the Src-induced up-regulation of GCS activity.

Methods to Characterize Synthesis and Degradation of Sphingomyelin at the Plasma Membrane and Its Impact on Lipid Raft Dynamics.

This chapter will discuss methods for analyses of the rates of sphingomyelin synthesis and turnover associated with lipid rafts or plasma membrane. These methods involve the use of fluorescently (NBD-C6-ceramide or NBD-C6-Sphingomyelin)) or radioactively labeled substrates ([3H-methyl]-phosphatidylcholine, [3H-acyl]-ceramide, [14C-methyl]-sphingomyelin) to quantify in vitro the activity of the sphingomyelin synthase (SMS) (also known as phosphatidylcholine:ceramide phosphocholine transferase), acid sphingomyelinase (the endosomal/lysosomal (L-SMase) and the secretory (S-SMase) forms) and neutral sphingomyelinase-2 (nSMase-2). These methods allow to quantify changes in the activity of enzymes that affect the SM-to-ceramide ratio on the plasma membrane, and consequently, the lipid rafts biophysical properties, dynamics, and raft-associated receptor clustering and signaling events. Specific attention is paid to challenges caused by the fact that SMS and nSMase-2 are integral/membrane bound proteins and how to avoid the use of detergent that suppress their specific activities.

Characterization of water-soluble esters of nitrobenzoxadiazole-based GSTP1-1 inhibitors for cancer treatment.

The 7-nitrobenzo[c][1,2,5]oxadiazole (NBD) derivative NBDHEX (compound 1) and its analogue MC3181 (compound 2) have been found to be potent inhibitors of tumor cell growth in vitro and therapeutically active and safe in mice bearing human melanoma xenografts. To enhance the aqueous solubility of these compounds, we synthesized the hemisuccinate of 1 (compound 3) and the phosphate monoesters of 1 and 2 (compound 4 and 5, respectively). These novel NBD derivatives displayed a solubility in the conventional phosphate-buffered saline up to 150-fold higher than that of 1, and up to 4-fold higher than that of 2. Notably, solubility of phosphates 4 and 5 in a potassium phosphate buffer at pH 7.4, was up to 500-fold higher than that of 1, and ~10-fold higher than that of 2. Compounds 3-5 retained high cytotoxicity towards cultured human melanoma and osteosarcoma cells and were cleaved in vitro by both human and murine hydrolases, thus releasing the corresponding parent compound (i.e., 1 or 2). Interestingly, esters 3-5 displayed high inhibitory activity towards the glutathione transferase (GST) isoform GSTP1-1 and showed a reactivity towards reduced glutathione comparable to that of the respective parent compound. Finally, both 4 and 5 were safe and effective when administered intravenously or orally as an aqueous solution to mice xenografted with A375 human melanoma tumors. Collectively, these results and the previously observed synergistic interaction between 1 and 2 and various approved anticancer drugs, suggest the possible utility of phosphates 4 and 5 as single agents and in combination regimens in cancers with unmet medical need, including melanoma.

Bioenergetic Analyses of In Vitro and In Vivo Samples to Guide Toxicological Endpoints.

Toxicology is a broad field that requires the translation of biochemical responses to xenobiotic exposures into useable information to ensure the safety of the public. Modern techniques are improving rapidly, both quantitatively and qualitatively, to provide the tools necessary to expand available toxicological datasets and refine our ability to translate that data into relevant information via bioinformatics. These new techniques can, and do, impact many of the current critical roles in toxicology, including the environmental, forensic, preclinical/clinical, and regulatory realms. One area of rapid expansion is our understanding of bioenergetics, or the study of the transformation of energy in living organisms, and new mathematical approaches are needed to interpret these large datasets. As bioenergetics are intimately involved in the regulation of how and when a cell responds to xenobiotics, monitoring these changes (i.e., metabolic fluctuations) in cells/tissues post-exposure provides an approach to define the temporal scale of pharmacodynamic responses, which can be used to guide additional toxicological techniques (e.g., "omics"). This chapter will summarize important in vitro assays and in vivo imaging techniques to take real-time measurements. Using this information, our laboratory has utilized bioenergetics to identify significant time points of pharmacodynamic relevance as well as forecast the cell's eventual fate.

Insulin-independent stimulation of skeletal muscle glucose uptake by low-dose abscisic acid via AMPK activation.

Abscisic acid (ABA) is a plant hormone active also in mammals where it regulates, at nanomolar concentrations, blood glucose homeostasis. Here we investigated the mechanism through which low-dose ABA controls glycemia and glucose fate. ABA stimulated uptake of the fluorescent glucose analog 2-NBDG by L6, and of [18F]-deoxy-glucose (FDG) by mouse skeletal muscle, in the absence of insulin, and both effects were abrogated by the specific AMPK inhibitor dorsomorphin. In L6, incubation with ABA increased phosphorylation of AMPK and upregulated PGC-1α expression. LANCL2 silencing reduced all these ABA-induced effects. In vivo, low-dose oral ABA stimulated glucose uptake and storage in the skeletal muscle of rats undergoing an oral glucose load, as detected by micro-PET. Chronic treatment with ABA significantly improved the AUC of glycemia and muscle glycogen content in CD1 mice exposed to a high-glucose diet. Finally, both acute and chronic ABA treatment of hypoinsulinemic TRPM2-/- mice ameliorated the glycemia profile and increased muscle glycogen storage. Altogether, these results suggest that low-dose oral ABA might be beneficial for pre-diabetic and diabetic subjects by increasing insulin-independent skeletal muscle glucose disposal through an AMPK-mediated mechanism.

Perfusion controls muscle glucose uptake by altering the rate of glucose dispersion in vivo.

These studies test, using intravital microscopy (IVM), the hypotheses that perfusion effects on insulin-stimulated muscle glucose uptake (MGU) are 1) capillary recruitment independent and 2) mediated through the dispersion of glucose rather than insulin. For experiment 1, capillary perfusion was visualized before and after intravenous insulin. No capillary recruitment was observed. For experiment 2, mice were treated with vasoactive compounds (sodium nitroprusside, hyaluronidase, and lipopolysaccharide), and dispersion of fluorophores approximating insulin size (10-kDa dextran) and glucose (2-NBDG) was measured using IVM. Subsequently, insulin and 2[14C]deoxyglucose were injected and muscle phospho-2[14C]deoxyglucose (2[C14]DG) accumulation was used as an index of MGU. Flow velocity and 2-NBDG dispersion, but not perfused surface area or 10-kDa dextran dispersion, predicted phospho-2[14C]DG accumulation. For experiment 3, microspheres of the same size and number as are used for contrast-enhanced ultrasound (CEU) studies of capillary recruitment were visualized using IVM. Due to their low concentration, microspheres were present in only a small fraction of blood-perfused capillaries. Microsphere-perfused blood volume correlated to flow velocity. These findings suggest that 1) flow velocity rather than capillary recruitment controls microvascular contributions to MGU, 2) glucose dispersion is more predictive of MGU than dispersion of insulin-sized molecules, and 3) CEU measures regional flow velocity rather than capillary recruitment.

Nicotine induces insulin resistance via downregulation of Nrf2 in cardiomyocyte.

Clinical studies have demonstrated that cigarette smoking is strongly associated with insulin resistance and heart disease. Nicotine is considered the primary toxin constituent associated with smoking. However, the distinct molecular mechanism of nicotine-induced cardiac dysfunction remains unclear. Cardiomyocytes with nicotine-induced insulin resistance are characterized by decreased glucose uptake, as measured by 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG), a fluorescent derivative of glucose, and reactive oxygen species (ROS) generation. Immunoblotting was used to evaluate the expression of nuclear factor erythroid 2-related factor 2 (Nrf2), extracellular signal-related kinase (ERK) and phosphoinositide 3-kinase (PI3K, p85, Y607). We determined the impact of nicotine on insulin resistance and Nrf2, phospho-ERK and phospho-PI3K expression in the myocardial tissue of a mouse model. Nicotine increased ROS production and depressed insulin-induced glucose uptake in cardiomyocytes. Pretreatment with N-acetyl-L-cysteine (NAC), an antioxidant, reversed nicotine-inhibited glucose uptake induced by insulin. Nicotine exposure directly inhibited Nrf2 and increased ERK phosphorylation in cardiomyocytes, which were obstructed by NAC. Further exploration of signaling cascades revealed nicotine-induced ROS involved in inhibiting PI3K/Nrf2 and activating ERK in cardiomyocytes. Moreover, the mouse model treated with nicotine showed glucose intolerance and impaired insulin tolerance accompanied by inhibited PI3K/Nrf2 and increased ERK in myocardial tissues. Thus, nicotine induces insulin resistance via the downregulation of Nrf2 activity in cardiomyocytes, which is a potential mechanism of the pharmacological effects of nicotine. This study identified potential therapeutic targets against nicotine-related cardiovascular diseases.

Anti-Diabetic Effect of a Shihunine-Rich Extract of Dendrobium loddigesii on 3T3-L1 Cells and db/db Mice by Up-Regulating AMPK-GLUT4-PPARα.

The stems of Dendrobium loddigesii, a Chinese herb, are often used to treat diabetes and its polar extract is rich in shihunine, a water-soluble Orchidaceae alkaloid, but little is known about the anti-diabetes effects and mechanism of shihunine. This study investigated the anti-diabetic effect of a shihunine-rich extract of D. loddigesii (DLS) based on 3T3-L1 cells and db/db mice. The underlying mechanisms were primarily explored using Western blot analysis and immunohistochemical staining. The 3T3-L1 cell experiments showed that DLS can reduce the intracellular accumulation of oil droplets as well as triglycerides (p < 0.001) and promote the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2deoxyglucose (2-NBDG) uptake of 3T3-L1 cells (p < 0.001). The animal experiments confirmed that after 8 weeks of DLS treatment, the body weight, fasting blood sugar, and serum lipid levels of mice were significantly lowered, and the oral glucose tolerance test and serum insulin level were significantly improved compared to the no-treatment diabetes mellitus group. Further histomorphology observation led to the conclusion that the quantities of islet cells were significantly increased and the increase in adipose cell size was significantly suppressed. The immunohistochemical test of pancreatic tissue revealed that DLS inhibited the expression of cleaved cysteine aspartic acid-specific protease 3 (cleaved caspase-3). Western blot experiments showed that DLS had agonistic effects on adenosine monophosphate (AMP)-activated protein kinase phosphorylation (p-AMPK) and increased the expression levels of peroxisome proliferator-activated receptor α (PPARα) and glucose transporter 4 (GLUT4) in liver or adipose tissues. These data suggest that the shihunine-rich extract of D. loddigesii is an anti-diabetic fraction of D. loddigesii. Under our experimental condition, DLS at a dose of 50 mg/kg has good anti-diabetic efficacy.

High-throughput Nitrobenzoxadiazole-labeled Cholesterol Efflux Assay.

Atherosclerosis leads to cardiovascular disease (CVD). It is still unclear whether cholesterol-HDL (cHDL) concentration plays a causal role in atherosclerosis development. However, an important factor in early stages of atheroma plaque formation is cholesterol efflux capacity to HDL (the ability of HDL particles to accept cholesterol from macrophages) in order to avoid foam cell formation. This is a key step in avoiding the accumulation of cholesterol in the endothelium and a part of reverse cholesterol transport (RCT) to eliminate cholesterol through the liver. Cholesterol efflux capacity to serum or plasma in macrophage cell models is a promising tool that can be used as biomarker for atherosclerosis. Traditionally, [3H]-cholesterol has been used in cholesterol efflux assays. In this study, we aim to develop a safer and faster strategy using fluorescent labelled-cholesterol (NBD-cholesterol) in a cellular assay to trace the cholesterol uptake and efflux process in THP-1-derived macrophages. Finally, we optimize and standardize the NBD-cholesterol efflux method and develop a high-throughput analysis using 96-well plates.

Aberrant Uptake of a Fluorescent L-Glucose Analogue (fLG) into Tumor Cells Expressing Malignant Phenotypes.

Glucose, one of the most fundamental sugar elements, has either D- or L-conformation. Of these, most cells preferentially take up D-glucose as an essential energy/carbon source. Such stereoselective uptake of glucose has been explored by fluorophore-bearing D- and L-glucose analogues. 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), the most widely used fluorescent D-glucose analogue, was abundantly taken up into living Escherichia coli cells, whereas no detectable uptake was obtained for 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-L-glucose (2-NBDLG), the antipode of 2-NBDG developed as a fluorescent L-glucose analogue (fLG). Interestingly, we found three-dimensionally accumulating tumor cell aggregates taking up 2-NBDLG when they expressed nuclear heterogeneity, one of the major cytological criteria for cells suspected of high-grade malignancy in clinical diagnosis. 2-NBDLG uptake was not detected in aggregates consisting of homogeneous cells and was specifically abolished by phloretin, a broad-spectrum inhibitor against transporters/channels. Preliminary studies have suggested that a combined use of 2-NBDLG, which emits green fluorescence, with 13-[4-[(2-deoxy-D-glucopyranose-2-yl)aminosulfonyl]-2-sulfonatophenyl]-4,5-trimethylene-7,8-trimethylene-1,2,3,4,6,9,10,11-octahydro-4-aza-6-oxa-8-azoniapentacene (2-TRLG), a membrane-impermeable fLG bearing a large red fluorophore, is effective for discriminating malignant tumor from benign cells both in living biopsy specimens endoscopically dissected from patients with early-stage gastric cancer and in ascites fluid of patients with gynecological cancers. Confocal endomicroscopic imaging of a carcinogen-induced cancer in bile duct of hamsters indicated that the fLG uptake pattern well correlated with pathological diagnosis for carcinoma. Safety tests according to Good Laboratory Practice regulations have been successfully completed so far. fLGs are unique fluorescent glucose analogues for identifying and characterizing living cancer cells based on derangements in their transport function.

Role of ABCA1 on membrane cholesterol content, insulin-dependent Akt phosphorylation and glucose uptake in adult skeletal muscle fibers from mice.

The ATP-binding cassette transporter A1 (ABCA1) promotes cellular cholesterol efflux, leading to cholesterol binding to the extracellular lipid-free apolipoprotein A-I. ABCA1 regulates lipid content, glucose tolerance and insulin sensitivity in adipose tissue. In skeletal muscle, most GLUT4-mediated glucose transport occurs in the transverse tubule, a system composed by specialized cholesterol-enriched invaginations of the plasma membrane. We have reported that insulin resistant mice have higher cholesterol levels in transverse tubule from adult skeletal muscle. These high levels correlate with decreased GLUT4 trafficking and glucose uptake; however, the role of ABCA1 on skeletal muscle insulin-dependent glucose metabolism remains largely unexplored. Here, we evaluated the functional role of the ABCA1 on insulin-dependent signaling pathways, glucose uptake and cellular cholesterol content in adult skeletal muscle. Male mice were fed for 8 weeks with normal chow diet (NCD) or high fat diet (HFD). Compared to NCD-fed mice, ABCA1 mRNA levels and protein content were lower in muscle homogenates from HFD-fed mice. In Flexor digitorum brevis muscle from NCD-fed mice, shABCA1-RFP in vivo electroporation resulted in 65% reduction of ABCA1 protein content, 1.6-fold increased fiber cholesterol levels, 74% reduction in insulin-dependent Akt (Ser473) phosphorylation, total suppression of insulin-dependent GLUT4 translocation and decreased 2-NBDG uptake compared to fibers electroporated with the scrambled plasmid. Pre-incubation with methyl-ß cyclodextrin reestablished both GLUT4 translocation and 2-NBDG transport. Based on the present results, we suggest that decreased ABCA1 contributes to the anomalous cholesterol accumulation and decreased glucose transport displayed by skeletal muscle membranes in the insulin resistant condition.

Chemical Proteomics and Super-resolution Imaging Reveal That Chloroquine Interacts with Plasmodium falciparum Multidrug Resistance-Associated Protein and Lipids.

It is well established that chloroquine, a quinoline antimalarial, inhibits hemozoin formation in the malaria parasite. Counterintuitively, this archetypal antimalarial is also used in the treatment of diseases in which hemozoin biocrystallization does not play a role. Hence, we decided to investigate whether chloroquine possesses binding targets other than Fe(III) protoporphyrin IX in blood stage Plasmodium falciparum parasites and whether these are related to sites of accumulation within the parasite other than the digestive vacuole. A 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled fluorescent derivative of chloroquine, especially sensitive to regions outside the digestive vacuole and retaining the antiplasmodial pharmacophore, was synthesized to investigate subcellular localization in the parasite. Super-resolution microscopy revealed association with membranes including the parasite plasma membrane, the endoplasmic reticulum, and possibly also the mitochondrion. A drug-labeled affinity matrix was then prepared to capture protein binding targets of chloroquine. SDS-PAGE revealed a single prominent band between 200 and 250 kDa from the membrane-associated fraction. Subsequent proteomic analysis revealed that this band corresponded to P. falciparum multidrug resistance-associated protein (PfMRP1). Intrigued by this finding, we demonstrated pull-down of PfMRP1 by matrices labeled with Cinchona alkaloids quinine and quinidine. While PfMRP1 has been implicated in resistance to quinolines and other antimalarials, this is the first time that these drugs have been found to bind directly to this protein. Based on previous reports, PfMRP1, the only prominent protein found to bind to quinolines in this work, is likely to modulate the activity of these antimalarials in P. falciparum rather than act as a drug target.

A novel biocompatible NiII tethered moiety as a glucose uptake agent and a hit against methicillin-resistant Staphylococcus aureus.

In the efforts to develop a biocompatible transition metal complex as a drug alike for some of the prevailing non-communicable diseases (NCDs) and communicable diseases (CDs), a novel binuclear NiII compound [{NiII(hpdbal-sbdt)}2] (2) has been synthesized by the reaction of Ni(OAc)2.4H2O and H2hpdbal-sbdt (1) [1 is a dibasic tridentate ONS2- donor Schiff base ligand obtained by the condensation of 2-hydroxy-5-(phenyldiazenyl)benzaldehyde (Hhpdbal) and S-benzyldithiocarbazate (Hsbdt)]. Both ligand 1 and compound 2 were structurally characterized in the solid and solution state using various spectroscopic techniques like ATIR, 1H NMR, 13C NMR, TGA, FESEM, EDS and CHNS analysis. The antidiabetic activity of H2hpdbal-sbdt (1) and [{NiII(hpdbal-sbdt)}2] (2) were assessed using 2-NBDG uptake assay. The assay results showed 85% and 95% of fluorescent glucose uptake by insulin resistant HePG2 cells treated with compounds 1 and 2 respectively. The 2-NBDG uptake by the cells treated with the compound 2 was observed to be comparable to the standard antidiabetic drug metformin. Compounds 1 and 2 were also tested against five bacterial and two fungi strains in order to evaluate pathogen killing activity. Compound 2 showed significant inhibitory action towards the methicillin-resistant Staphylococcus aureus (MRSA) strain with an MIC value of 2 µg/mL whereas the ligand 1 was found to be inactive. Furthermore, the interactive nature of compound 2 with a model serum carrier protein bovine serum albumin (BSA) was studied using a multi-spectroscopic approach which provided an insight into the nature and extent of binding, conformational changes and the quenching of amino acid residues of the protein.

Effect of baicalin on GLUT4 expression and glucose uptake in myotubes of rats.

AIMS: Although baicalin could attenuate obesity-induced insulin resistance, the detailed mechanism of baicalin on glucose uptake has not been sufficiently explored as yet. The aim of this study was to survey if baicalin might facilitate glucose uptake and to explore its signal mechanisms in L6 myotubes. MATERIALS AND METHODS: L6 myotubes were treated with 100, 200, 400 µM baicalin for 6 h, 12 h and 24 h in this study. Then 2-NBDG and insulin signal protein levels in myotubes of L6 cells were examined. KEY FINDINGS: We discovered that administration of baicalin enhanced GLUT4, PGC-1α, pP38MAPK, pAKT and pAS160 contents, as well as GLUT4 mRNA and PGC-1α mRNA levels in L6 myotubes. The beneficial metabolic changes elicited by baicalin were abrogated in myotubes of L6 by P38MAPK or AKT inhibitors. SIGNIFICANCE: These results suggest that baicalin promoted glucose uptake in myotubes by differential regulation on P38MAPK and AKT activity. In conclusion, these data provide insight that baicalin is a powerful and promising agent for the treament of hyperglycemia via AKT/AS160/GLUT4 and P38MAPK/PGC1α/GLUT4 pathway.