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.


Western blot.
L6 rat myoblasts (0.5x10 6 /well) were seeded in 6-well plates in DMEM with 10% FBS. After cell adhesion, cells were washed, cultured overnight at 37°C in DMEM with 5 mM glucose and processed for Western blot and glucose uptake experiments. For Western blot, the supernatant was removed, cells were washed once in Krebs-Ringer HEPES buffer (KRH) and then incubated in KRH with 5 mM glucose for 60 min at 37°C without or with 100 nM ABA. The supernatant was removed and cells were scraped in 100 µL lysis buffer (20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP40) containing a protease inhibitor cocktail. After brief sonication, the protein concentration was determined on an aliquot of each lysate. Western Blot experiments were performed also on quadriceps samples freshly isolated from mice and incubated for 30 and 60 minutes with or without 100 nM ABA; after incubation, muscles were lysed with Tissue Lyser (Qiagen, Milan, Italy), centrifuged for 10 min at 12000 x g and the supernatants were analyzed by Western Blot. SDS-PAGE (on 10% gels) and protein transfer to a nitrocellulose membrane (Bio-Rad, Milan, Italy) were performed according to standard procedures. The membrane was blocked for 1 h with 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Tween 20 (TBST) containing 5% non-fat dry milk and incubated for 1 h at room temperature with the primary antibody (anti LANCL2, anti-phospho AMPK or anti-phospho Akt and anti-vinculin as reference protein). After washing with TBST, the membrane was incubated with an anti-rabbit IgG antibody conjugated with horse radish peroxidase (HRP) (Santa Cruz Biotechnology, Dallas, TX, USA) and developed with Immobilon TM Western Chemiluminescent HRP Substrate (Millipore, Milan, Italy). Band intensity was evaluated with the Chemidoc system (Bio-Rad). After 2 membrane stripping, a second incubation with an anti-AMPK or an anti-Akt primary antibody was performed and the incubation with the secondary HRP-conjugated antibody was repeated.
Specific primers for rat LANCL2, GAPDH and actin were described in [58]. Specific primers for mouse housekeeping genes, ubiquitin and β2-microglobulin were described in [9].
Mice were fasted for 17 hours before the OGTT, then 1 g/Kg BW glucose was administered by gavage in 150 µL water solution. Blood was drawn from the tail vein before gavage (time zero) and 15, 30, 60 and 120 min after gavage: glycemia was immediately measured with a glucometer, each measure being performed in duplicate. The area under the curve (AUC) of glycemia was calculated with the trapezoidal rule, from the blood glucose concentrations measured at the indicated time-points after gavage, relative to the value of glycemia at time 0 (before gavage).

Experimental micro-PET scanning protocol.
In vivo imaging was performed according to a published protocol [61] Daily quality controls always documented a radiochemical purity of in-house produced FDG ≥ 98%. Immediately after anesthesia following gavage (see above), the rats were positioned on the bed of a dedicated micro-PET system (Albira, Bruker, Billerica, MA, USA), centering the scanner field of view on the chest. A dose of 30-45MBq of FDG was injected through a tail vein exactly 15 min after gavage, and a list mode acquisition lasting 50 min was started. Blood was obtained from the tail vein before gavage (time zero), and 15 (immediately before FDG injection), 30 and 60 min after gavage: glycemia was immediately measured with a glucometer, and an aliquot of each blood sample anticoagulated with heparin was immediately centrifuged at 22,000 x g for 30 sec and plasma aliquots were stored at -20°C for the determination of insulinemia.
In a random sequence, each rat underwent an OGTT and an OGTT+ABA, one week apart.
The whole dataset was thus binned using the following framing rate: 10 x 15 sec, 5 x 30 sec, 2 x 150 sec, 6 x 300 sec, 1 x 600 sec. PET data were reconstructed using a maximal likelihood expectation maximization method (MLEM). An experienced observer, unaware of the experimental type of analyzed model, identified a volume of interest (VOI) in the left ventricular chamber. Then, the computer was asked to plot the time-concentration curve within this VOI throughout the whole acquisition to define tracer input function. Whole body FDG clearance (in µL x min -1 x g -1 ) was calculated, using the conventional stochastic approach, as the ratio between injected dose and integral of input function from 0 to infinity, fitting the last 20 min with a mono-exponential function [62]. This value was multiplied by serum glucose level to measure whole body glucose consumption that was normalized for body weight and expressed as nmol x min -1 x g -1 .
Thereafter, all dynamic scans were processed according to the Gjedde-Patlak [63] graphical approach to compartmental analysis by using the routine of a dedicated software (PMOD, Zurich, Switzerland).
Briefly, the software utilizes the input function and transforms the original tissue activity