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.

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.

In vitro anti-diabetic effect of flavonoids and pheophytins from Allophylus cominia Sw. on the glucose uptake assays by HepG2, L6, 3T3-L1 and fat accumulation in 3T3-L1 adipocytes.

BACKGROUND AND PURPOSE: Based on ethno-botanical information collected from diabetic patients in Cuba and firstly reported inhibition of PTP1B and DPPIV enzymes activities, Allophylus cominia (A. cominia) was identified as possible source of new drugs that could be used for the treatment of type 2 diabetes mellitus (T2-DM). EXPERIMENTAL APPROACH: in this study, the activity of the characterised extracts from A. cominia was tested on the glucose uptake using HepG2 and L6 cells, 3T3-L1 fibroblasts and adipocytes as well as their effect on the fat accumulation using 3T3-L1 adipocytes. KEY RESULTS: on 2-NBDG glucose uptake assay using HepG2 and L6 cells, extracts from A. cominia enhanced insulin activity by increasing glucose uptake. On HepG2 cells Insulin EC50 of 93 ± 21nM decreased to 13 ± 2nM in the presence of the flavonoids mixture from A.cominia. In L6 cells, insulin also produced a concentration-dependent increase with an EC50 of 28.6 ± 0.7nM; EC50 decreased to 0.08 ± 0.02nM and 5 ± 0.9nM in the presence of 100µg/ml of flavonoids and pheophytins mixtures, respectively. In 3T3-L1 fibroblasts, insulin had an EC50 of >1000nM that decreased to 38 ± 4nM in the presence of the flavonoids extract. However, in adipocytes, insulin produced a significant concentration-dependent increase and an EC50 of 30 ± 8nM was a further confirmation of the insulin responsiveness of the adipocytes to the insulin. At 100µg/ml, flavonoids and pheophytins extracts decreased fat accumulation in 3T3-L1 adipocytes by two folds in comparison to the control differentiated cells (p < 0.05). The crude extract of A. cominia did not show any enhancement of 2-NBDG uptake by 3T3-L1 adipocytes in the presence or absence of 100nM insulin. In addition, in fully differentiated adipocytes, both extracts produced significant decrease in lipid droplets in the cells and no lipid accumulation were seen after withdrawal of the extracts from the cell growth medium. However, there was no effect of both extracts on total protein concentration in cells as well as on Glut-4 transporters. CONCLUSIONS AND IMPLICATIONS: the pharmacological effects of the extracts from A. cominia observed in experimental diabetic models were shown in this study. A. cominia is potentially a new candidate for the treatment and management of T2-DM.

Cyclocarya paliurus extract activates insulin signaling via Sirtuin1 in C2C12 myotubes and decreases blood glucose level in mice with impaired insulin secretion.

Diabetes is caused by the lack of release or action of insulin. Some foods and supplements can compensate for this deficiency; thus, they can aid in the prevention or treatment of diabetes. The aim of this study was to investigate the effects of Cyclocarya paliurus extract (CPE) on insulin signaling and its capacity to correct hyperglycemia in the absence of insulin. To investigate the hypoglycemic effects of CPE, C2C12 cells were exposed to CPE (50 and 100 µg/mL). CPE promoted 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2NBDG) uptake into the cells via translocation of glucose transporter 4 (Glut4) to the plasma membrane. In addition, CPE enhanced tyrosine phosphorylation of insulin receptor substrate and activated phosphatidylinositol 3-kinase and protein kinase B (Akt) via sirtuin1 in C2C12 cells. Moreover, we found that oral administration of CPE (1 g/kg) to streptozotocin-induced hyperglycemic mice produced a progressive decrease in plasma glucose levels at 1 h after single dosing. At that point, CPE significantly increased the expression of skeletal muscle membrane Glut4 and enhanced the phosphorylation of Akt. These results suggest that CPE exerts antidiabetic effects similar to those of insulin, and may be an oral therapeutic alternative for the management of diabetes.

Kazinol B from Broussonetia kazinoki improves insulin sensitivity via Akt and AMPK activation in 3T3-L1 adipocytes.

In this study, we evaluated the insulin-sensitizing effect of flavans purified from Broussonetia kazinoki Siebold (BK) on 3T3-L1 adipocytes. Among the tested compounds, kazinol B enhanced intracellular lipid accumulation, gene expression of proliferator-activated receptorγ (PPARγ) and CCAAT/enhancer binding protein-alpha (C/EBPα), and consistently induced PPARγ transcriptional activation. To further investigate the insulin-sensitizing effect of kazinol B, we measured glucose analogue uptake by fully differentiated adipocytes and myotubes. Kazinol B increased 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-d-glucose (2-NBDG) uptake by cells by upregulating the gene expression and translocation of glucose transporter 4 (GLUT-4) into the plasma membrane in adipocytes. Kazinol B stimulated the gene expression and secretion of adiponectin, which is associated with a low risk of types 1 and 2 diabetes mellitus. We also suggested the mechanism of the antidiabetic effect of kazinol B by assaying Akt and AMP-activated protein kinase (AMPK) phosphorylation. In conclusion, kazinol B isolated from BK improved insulin sensitivity by enhancing glucose uptake via the insulin-Akt signaling pathway and AMPK activation. These results suggest that kazinol B might be a therapeutic candidate for diabetes mellitus.

Selaginellin and biflavonoids as protein tyrosine phosphatase 1B inhibitors from Selaginella tamariscina and their glucose uptake stimulatory effects.

As part of an ongoing search for new antidiabetic agents from medicinal plants, the methanol extract of the aerial parts of Selaginella tamariscina was found to possess stimulatory effect on glucose uptake in 3T3-L1 adipocyte cells. Thus, bioassay-guided isolation of this active extract yielded two new compounds (1 and 2) along with five known biflavonoids (3-7). Their structures were elucidated by extensive analysis of spectroscopic and physicochemical data. The absolute configuration of compound 2 was determined by specific rotation and CD data analysis. All isolates exhibited potent inhibitory effects on PTP1B enzyme with IC50 values ranging from 4.5±0.1 to 13.2±0.8µM. Furthermore, the isolates (1-7) showed significant stimulatory effects on 2-NBDG uptake in 3T3-L1 adipocyte cells. Of these, compounds (1, 6, and 7) which exhibited mixed-competitive inhibition modes against PTP1B, showed potent stimulatory effects on 2-NBDG uptake. This result indicated the potential of these biflavonoids as lead molecules for development of antidiabetic agents and the beneficial use of S. tamariscina against hyperglycemia.

Effect of water extracts from edible Myrtaceae plants on uptake of 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose in TNF-α-treated FL83B mouse hepatocytes.

This study investigated the glucose uptake activity of the water extracts from the leaves and fruit of edible Myrtaceae plants, including guava (Psidium guajava Linn.), wax apples [Syzygium samarangense (Blume) Merr. and L.M. Perry], Pu-Tau [Syzygium jambo (L.) Alston], and Kan-Shi Pu-Tau (Syzygium cumini Linn.) in FL83B mouse hepatocytes. The fluorescent dye 2-(n-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose was used to estimate the uptake ability of the cells. Glucose uptake test showed that pink wax apple fruit extract (PWFE) exhibits the highest glucose uptake activity, at an increment of 21% in the insulin-resistant FL83B mouse hepatocytes as compared with the TNF-α-treated control group. Vescalagin was isolated using column chromatography of PWFE. This compound, at the concentration of 6.25 µg/mL, exhibits the same glucose uptake improvement in insulin-resistant cells as PWFE at a 100-µg/mL dose. We postulate that vescalagin is an active component in PWFE that may alleviate the insulin resistance in mouse hepatocytes.

Syzygium aqueum leaf extract and its bioactive compounds enhances pre-adipocyte differentiation and 2-NBDG uptake in 3T3-L1 cells.

The insulin-like and/or insulin-sensitising effects of Syzygium aqueum leaf extract and its six bioactive compounds; 4-hydroxybenzaldehyde, myricetin-3-O-rhamnoside, europetin-3-O-rhamnoside, phloretin, myrigalone-G and myrigalone-B were investigated in 3T3-L1 adipocytes. We observed that, S. aqueum leaf extract (0.04-5 µg/ml) and its six bioactive compounds (0.08-10 µM) at non-cytotoxic concentrations were effectively enhance adipogenesis, stimulate glucose uptake and increase adiponectin secretion in 3T3-L1 adipocytes. Clearly, the compounds myricetin-3-O-rhamnoside and europetin-3-O-rhamnoside showed insulin-like and insulin-sensitising effects on adipocytes from a concentration of 0.08 µM. These compounds were far better than rosiglitazone and the other isolated compounds in enhancing adipogenesis, stimulating 2-NBDG uptake and increasing adiponectin secretion at all the concentrations tested. These suggest the antidiabetic potential of S. aqueum leaf extract and its six bioactive compounds. However, further molecular interaction studies to explain the mechanisms of action are highly warranted.

Development of a cell-based nonradioactive glucose uptake assay system for SGLT1 and SGLT2.

Sodium-dependent glucose cotransporters (SGLT1 and SGLT2), which have a key role in the absorption of glucose in the kidney and/or gastrointestinal tract, have been proposed as a novel therapeutic strategy for diabetes and cardiomyopathy. Here we developed a simple cell-based, nonradioactive method for functional screening of SGLT1 and SGLT2 inhibitors. Stable cell lines expressing human SGLT1 and SGLT2 were established by transfecting HEK293 cells with vectors (pCMV6-Neo) having full-length human SGLT1 and SGLT2 and selecting the positive clones following neomycin treatment. We confirmed the gene expression of SGLT1 and SGLT2 by reverse transcription polymerase chain reaction (RT-PCR) and immunoblotting. Furthermore, to analyze the function of SGLTs, we incubated stable cell lines with 2-deoxyglucose or fluorescent d-glucose analog (2-NBDG) and performed glucose uptake assay. A significant (P<0.001) increase in glucose uptake was observed in both cell lines. The increased glucose uptake in both cell lines was completely inhibited when treated with nonspecific SGLT1/SGLT2 inhibitors and phlorizin (100µM), but not when treated with nonspecific sodium-independent facilitative glucose transporter (GLUT) inhibitors (100µM). Taken together, our data suggest that cell-based methods developed for screening SGLT1/SGLT2 inhibitors are phlorizin sensitive and specific for respective glucose transporters. This assay provides a simple and rapid method for identifying novel and selective SGLT inhibitors.

Antidiabetic effects of Justicia spicigera Schltdl (Acanthaceae).

ETHNOPHARMACOLOGICAL IMPORTANCE: Justicia spicigera is a plant species used for the Teenak (Huesteca Potosina) and Mayan (Yucatan peninsula) indigenous for the empirical treatment of diabetes, infections and as stimulant. AIM OF THE STUDY: To evaluate the cytotoxicity, antioxidant and antidiabetic properties of J. spicigera. MATERIALS AND METHODS: The effects of ethanolic extracts of J. spicigera (JSE) on the glucose uptake in insulin-sensitive and insulin-resistant murine 3T3-F442A and human subcutaneous adipocytes was evaluated. The antioxidant activities of the extract of JSE was determined by ABTS and DPPH methods. Additionally, it was evaluated the antidiabetic properties of JSE on T2DM model. RESULTS: JSE stimulated 2-NBDG uptake by insulin-sensitive and insulin-resistant human and murine adipocytes in a concentration-dependent manner with higher potency than rosiglitazone 1mM. JSE showed antioxidant effects in vitro and induced glucose lowering effects in normoglycemic and STZ-induced diabetic rats. CONCLUSION: The antidiabetic effects of administration of J. spicigera are related to the stimulation of glucose uptake in both insulin-sensitive and insulin-resistant murine and human adipocytes and this evidence justify its empirical use in Traditional Medicine. In addition, J. spicigera exerts glucose lowering effects in normoglycemic and STZ-induced diabetic rats.

Magnolia dealbata Zucc and its active principles honokiol and magnolol stimulate glucose uptake in murine and human adipocytes using the insulin-signaling pathway.

Some Magnolia (Magnoliaceae) species are used for the empirical treatment of diabetes mellitus, but the antidiabetic properties of Magnolia dealbata have not yet been experimentally validated. Here we report that an ethanolic extract of Magnolia dealbata seeds (MDE) and its active principles honokiol (HK) and magnolol (MG) induced the concentration-dependent 2-NBDG uptake in murine 3T3-F442A and human subcutaneous adipocytes. In insulin-sensitive adipocytes, MDE 50 µg/ml induced the 2-NBDG uptake by 30% respect to insulin, while HK and MG, 30 µM each, did it by 50% (murine) and 40% (human). The simultaneous application of HK and MG stimulated 2-NBDG uptake by 70% in hormone-sensitive cells, on which Magnolia preparations exerted synergic effects with insulin. In insulin-resistant adipocytes, MDE, HK and MG induced 2-NBDG uptake by 57%, 80% and 96% respect to Rosiglitazone (RGZ), whereas HK and MG simultaneously applied stimulated 2-NBDG uptake more efficiently than RGZ (120%) in both murine and human adipocytes. Inhibitors of the insulin-signaling pathway abolished the glucose uptake induced by Magnolia dealbata preparations, suggesting that their antidiabetic effects are mediated by this signaling pathway. In addition, MDE, HK and MG exerted only mild to moderate proadipogenic effects on 3T3-F442A and human preadipocytes, although the combined application of HK and MG markedly increased the lipid accumulation in both cell types. In summary, Magnolia dealbata and its active principles HK and MG stimulate glucose uptake in insulin-sensitive and insulin-resistant murine and human adipocytes using the insulin signaling pathway.

[Effect of Chaihu Shugan Tang on excitability in different brain regions of pentylenetetrazole-kindled chronic epileptic rats].

OBJECTIVE: To study the effect of the Chinese compound prescription Chaihu Shugan Tang (CHSGT) on the excitability in the cerebral cortex and hippocampus (different brain regions) of pentetrazole (PTZ)-kindled chronic epileptic rats. METHOD: To establish the model of chronic kindling rats intraperitoneal injected with pentylenetet. Fully kindled rats were randomized into control and experimental groups for intragastric administration of normal saline (control, model), Sodium Valproate and CHSGT at the high, medium and low doses for 4 consecutive weeks. The content of 2-NBDG, the glutamate (Glu) and the aspartate (Asp) in different brain regions of rats were detected by fluorescence imaging techniques and HPLC assay respectively. RESULT: CHSGT at the high, medium and low doses all significantly decreased the content of 2-NBDG, the Glu and the Asp in different brain regions of chronic epileptic rats (P < 0.01). CONCLUSION: CHSGT can inhibit the excitability in different brain regions of PTZ-induced epileptic rats, by decreasing the level of excitatory neurotransmitter maybe one of its antiepileptic mechanisms.

Cecropia obtusifolia Bertol and its active compound, chlorogenic acid, stimulate 2-NBDglucose uptake in both insulin-sensitive and insulin-resistant 3T3 adipocytes.

ETHNOPHARMACOLOGICAL IMPORTANCE: Cecropia obtusifolia Bertol (Cecropiaceae) is a plant extensively used for the empirical treatment of type 2 diabetes in México. Although some of its hypoglycemic principles have been described, their mechanisms of action remain unclear. AIM OF THE STUDY: To investigate the anti-diabetic mechanisms of Cecropia obtusifolia aqueous extract (CAE) and its active compound chlorogenic acid (CGA). MATERIALS AND METHODS: Non-toxic concentrations of CAE and CGA were assayed on the adipogenesis and 2-NBDglucose uptake in 3T3-F442A murine adipocytes. RESULTS: Added to adipogenic medium, CAE 70 microg/ml induced a modest increment (20%) in 3T3 adipogenesis whereas CGA did not affect adipogenesis at any of the tested concentrations (0.1-100 microM). Both preparations stimulated 2-NBDG uptake in adipocytes by 51% (CAE) and 176% (CGA) in the absence of insulin, and by 174% (CAE) and 404% (CGA) in the presence of the hormone. CAE and CGA also stimulated the 2-NBDG uptake in insulin-resistant 3T3 adipocytes by 35% and 141%, respectively, compared with the incorporation shown by insulin-sensitive adipocytes stimulated by the hormone. The potency of CGA to stimulate 2-NBDG uptake was comparable to the anti-diabetic drug rosiglitazone. CONCLUSION: Cecropia obtusifolia and CGA exert their anti-diabetic effects stimulating glucose uptake in both insulin-sensitive and insulin-resistant adipocytes without appreciable pro-adipogenic effects.

The anti-diabetic properties of Guazuma ulmifolia Lam are mediated by the stimulation of glucose uptake in normal and diabetic adipocytes without inducing adipogenesis.

ETHNOPHARMACOLOGICAL IMPORTANCE: Guazuma ulmifolia Lam (Sterculiaceae) is a plant extensively used in México for the empirical treatment of type 2 diabetes. AIM OF THE STUDY: To investigate the anti-diabetic mechanisms of Guazuma ulmifolia. MATERIALS AND METHODS: Non-toxic concentrations of Guazuma ulmifolia aqueous extracts (GAE) were assayed on adipogenesis and 2-NBDglucose uptake in the murine 3T3-F442A preadipose cell line. RESULTS: GAE added to adipogenic medium (AM) did not affect adipogenesis at any of the tested concentrations (1-70 microg/ml), whereas in AM lacking insulin GAE 70 microg/ml induced triglyceride accumulation by 23%. On the other hand, GAE 70 microg/ml stimulated 2-NBDG uptake by 40% in insulin-sensitive 3T3-F442A adipocytes and by 24% in insulin-resistant adipocytes, with respect to the incorporation showed by insulin-sensitive adipocytes stimulated with the hormone. CONCLUSION: Guazuma ulmifolia exerts its anti-diabetic effects by stimulating glucose uptake in both insulin-sensitive and insulin-resistant adipocytes without inducing adipogenesis.

Adjuvant induced glucose uptake by activated T cells is not correlated with increased survival.

Authors contributed equally to this manuscript Natural adjuvants, such as bacterial lipopolysaccharide (LPS), activate antigen presenting cells via Toll-like receptors and, indirectly, increase the survival of antigen-activated T cells. The molecular mechanisms leading to increased survival remain poorly defined. Because T cell clonal expansion leads to high energy demands, we hypothesized that increased glucose uptake and/or utilization in adjuvant-activated T cells could be important molecular event(s) that would lead to adjuvant-associated T cell survival advantage. Using a fluorescent analog of 2-deoxyglucose, 2-NBDG, we measured glucose accumulation and rate of uptake in T cells from mice treated with antigen in the absence or presence of LPS. Although adjuvant activated T cells increased the accumulation of 2-NBDG, the rate of uptake was unchanged compared to cells activated with only antigen. Furthermore, glucose transport inhibitors, cytochalasin B or phloretin, decreased the accumulation of glucose in adjuvant-treated T cells, but this decrease did not impair adjuvant-associated survival advantages. Together, these data indicate that increased glucose uptake through glucose transporters is not required for increased survival of activated T cells.

ATP8B1 requires an accessory protein for endoplasmic reticulum exit and plasma membrane lipid flippase activity.

UNLABELLED: Mutations in ATP8B1 cause progressive familial intrahepatic cholestasis type 1 and benign recurrent intrahepatic cholestasis type 1. Previously, we have shown in mice that Atp8b1 deficiency leads to enhanced biliary excretion of phosphatidylserine, and we hypothesized that ATP8B1 is a flippase for phosphatidylserine. However, direct evidence for this function is still lacking. In Saccharomyces cerevisiae, members of the Cdc50p/Lem3p family are essential for proper function of the ATP8B1 homologs. We have studied the role of two human members of this family, CDC50A and CDC50B, in the routing and activity of ATP8B1. When only ATP8B1 was expressed in Chinese hamster ovary cells, the protein localized to the endoplasmic reticulum. Coexpression with CDC50 proteins resulted in relocalization of ATP8B1 from the endoplasmic reticulum to the plasma membrane. Only when ATP8B1 was coexpressed with CDC50 proteins was a 250%-500% increase in the translocation of fluorescently labeled phosphatidylserine observed. Importantly, natural phosphatidylserine exposure in the outer leaflet of the plasma membrane was reduced by 17%-25% in cells coexpressing ATP8B1 and CDC50 proteins in comparison with cells expressing ATP8B1 alone. The coexpression of ATP8B1 and CDC50A in WIF-B9 cells resulted in colocalization of both proteins in the canalicular membrane. CONCLUSION: Our data indicate that CDC50 proteins are pivotal factors in the trafficking of ATP8B1 to the plasma membrane and thus may be essential determinants of ATP8B1-related disease. In the plasma membrane, ATP8B1 functions as a flippase for phosphatidylserine. Finally, CDC50A may be the potential beta-subunit or chaperone for ATP8B1 in hepatocytes.

Establishment of an in vitro high-throughput screening assay for detecting phospholipidosis-inducing potential.

Excessive accumulation of phospholipids results in phospholipidosis (PL), which may interfere with cellular functions, leading to acute or chronic disease or even death. Electron-microscopic detection of cytoplasmic lamellar bodies is often used as a diagnostic criterion of PL, but a faster, more convenient procedure is required for high-throughput assay of the PL-inducing potential of candidate drugs. We have developed a 96-well microplate cell-culture method for detecting PL, using a phosphatidylcholine-conjugated dye (NBD-PC) and a fluoro-microplate reader. The fluorescence intensity due to NBD-PC was normalized to that of Hoechst33342, used as an indicator of cell number, to obtain the amount of NBD-PC taken up per living cell. To select a suitable cell type, we examined the PL-detection sensitivity of five cell lines, as well as human and rat primary hepatocyte cultures, with five cationic amphiphilic drugs (CAD) as PL inducers and a negative control compound. The cell lines CHO-K1 and CHL/IU gave the best results. The NBD-PC uptake per CHO-K1 cell showed a high correlation with the pathological score of PL for 24 compounds, including PL-positive and negative compounds. This high-throughput screening assay for PL-inducing potential (HTS-PL assay) offers high sensitivity and accuracy, and it allows simultaneous determination of cytotoxicity.

Induction of ceramide glucosyltransferase activity in cultured human keratinocytes. Correlation with culture differentiation.

Ceramides are the major component of the extracellular lipids that comprise the epidermal permeability barrier. They are derived from glucosylceramides (GlcCer) upon their extrusion from lamellar granules into the extracellular space in the upper layers of the epidermis. To better understand the regulation of the unique pathway for ceramide production in epidermis, we have studied the activity of the enzyme responsible for GlcCer synthesis, ceramide glucosyltransferase (CerGlc transferase), during keratinocyte culture differentiation. Human keratinocyte cultures were expanded in low calcium keratinocyte growth medium (KGM) and then switched to either normal calcium KGM (nKGM) or "complete" Dulbecco's modified Eagle's medium/Ham's F-12 (3:1) supplemented with 10% fetal bovine serum (cDMEM). At 7 and 10 days after the medium switch, electron microscopy revealed that cDMEM cultures were more fully differentiated morphologically and contained numerous lamellar granules. The GlcCer/DNA content of cDMEM cultures increased to 6 times that of day 0 cultures and was nearly 4 times greater than that of nKGM cultures, whereas the total lipid/DNA content of cDMEM cultures increased to only 1.8 times that of day 0 cultures and was approximately 1.2 times that of nKGM cultures. CerGlc transferase activity/DNA increased 6 times in cDMEM cultures but <1.5 times in nKGM cultures. By contrast, beta-glucocerebrosidase activity, which is responsible for the conversion of GlcCer to ceramide, increased to a similar extent in both differentiating culture systems. Treatment of cultures with the reversible CerGlc transferase inhibitor, DL-threo-1-phenyl-2-(palmitoylamino)-3-morpholino-1-propanol, prevented the increase of GlcCer in cDMEM cultures, and blocked conversion of exogenously added ceramide to GlcCer. A low level of CerGlc transferase activity, relative to that in differentiated keratinocytes, was detected in cultures of other human cell types. These results indicate that CerGlc transferase activity is induced during epidermal differentiation and that regulation of this enzyme may be an important determinant of the specialized production and compartmentalization of epidermal sphingolipids.

The use of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipids in determining transmembrane lipid distribution.

Transbilayer lipid distribution of small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs) was measured using 31P-nuclear magnetic resonance (NMR) spectroscopy, chemical modification with 2,4,6-trinitrobenzene sulfonic acid (TNBS) and dithionite reduction of N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)-labeled lipid (NBD-lipid). The dithionite assay was the most reproducible of the three assays, with 1.2% error for SUVs and 3.9% error for LUVs. The dithionite assay also agreed best with theoretical inner:outer leaflet ratios, based on vesicle diameters determined by electron microscopy (Thomas et al. (1989) Biochem. Biophys. Acta 978, 85-90). Dithionite assay measurements were within 2.7% of theoretical ratios for SUVs and 2.3% for LUVs, while the NMR assay for SUVs was 14% lower than theoretical ratios and 23% lower for LUVs. The accuracy of NBD-lipids as markers for total transbilayer lipid was investigated. NBD-labeled phosphatidylserine, phosphatidylcholine and phosphatidylglycerol were accurate markers for total transbilayer lipid distribution, as their distributions were in close agreement with theoretical ratios. However, NBD-labeled phosphatidylethanolamine displayed a slight preference for the inner leaflet at low mole fractions of phosphatidylethanolamine, while native phosphatidylethanolamine showed a preference for the outer leaflet at the same concentration. NBD-labeled phosphatidic acid also showed a slight preference for the inner leaflet. We conclude that although dithionite-based assessment of NBD-labeled lipids across membrane bilayers can be a powerful analytical tool, caution must be used in the interpretation of results.