Novel use of fluorescent glucose analogues to identify a new class of triazine-based insulin mimetics possessing useful secondary effects

Abstract

There is an urgent need to discover new compounds that effectively treat diabetes by mimicking the action of insulin (insulin mimetics). Traditional approaches to studying anti-diabetic agents in cells are inconvenient for screening chemical libraries to identify insulin mimetics. 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-NBDG are fluorescent analogues of glucose that could be employed in screening. However, there are no published data about the use of these analogues to identify new insulin mimetics. We have developed a screening system based on 6-NBDG using 3T3-L1 adipocytes in a 96-well culture plate format. 6-NBDG was found to produce a larger signal than 2-NBDG in this screening system. 6-NBDG uptake in 3T3-L1 adipocytes was sensitive to insulin, known insulin mimetics, inhibitors of glucose transport and insulin-sensitizing compounds. To validate our screening system, a chemical library of 576 tagged, triazine-based small molecules was screened. The screening results were identical to that obtained from a commercial enzyme-based glucose assay. Two inducers of glucose uptake were shown to be non-cytotoxic and confirmed as insulin mimetic compounds by their inhibition of epinephrine-stimulated free fatty acid release from adipocytes. These novel insulin mimetics functioned at a markedly lower concentration than two widely studied insulin mimetics, zinc(ii) complexes and vanadium compounds, and also showed novel, beneficial effects on endothelial cell function (a key determinant of secondary complications in diabetes). The discovery of new insulin mimetics using 6-NBDG validates the use of this probe in the development of large-scale, cell-based screening systems based on the uptake of fluorescent-tagged glucose analogues. This research should aid the development of novel strategies to discover new drugs and drug targets for combating the increasing prevalence of diabetes.

Mitoenergetic Dysfunction Triggers a Rapid Compensatory Increase in Steady-State Glucose Flux

“…However, this approach cannot be used to study the relatively rapid temporal GLC dynamics at the (sub)cellular level. In addition, fluorescently labeled GLC analogs such as 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) and 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-6-deoxyglucose (6-NBDG) have been used to quantify V 1 (56,57). However, these GLC analogs display a different affinity for Glut transporters than GLC (58).…”

Vitamin D Up-regulates Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization Mediated by Cystathionine-γ-lyase (CSE) Activation and H2S Formation in 3T3L1 Adipocytes

“…The glucose utilization values were expressed in nmol/ml per min unit. The glucose uptake assay was performed using 6-NBDG (Invitrogen), a fluorescent analog of 2-deoxyglucose, following the method of Jung et al (16). Briefly, after treatment, cells were incubated with serum-free low glucose medium containing 6-NBDG (20 M) for 30 min.…”

Black ginseng extract exerts anti-hyperglycemic effect via modulation of glucose metabolism in liver and muscle

“…Determination of glucose uptake with 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-glucose(2-NBDG) Glucose uptake in C2C12 myotubes was measured using a fluorescent glucose analog, 2-NBDG (Invitrogen, Carlsbad, CA, USA), as previously described (Jung et al, 2011). Mature C2C12 myotubes were starved with serum-free low-glucose DMEM for 1 h. Subsequently, 100 μM 2-NBDG was added to the culture media for 1 h containing different concentration of GBG05-FF (400 μg/mL and 800 μg/mL, doses were chosen based on preliminary MTS assay (data not shown)) or 100 nM insulin in the presence or absence of selected inhibitors including LY294002 (a potent inhibitor of PI3-K), compound C (an ATP-competitive inhibitor of AMPK), and nicotinamide (a Sirt1 inhibitor).…”