Abstract
We recently showed that bitter melon-derived triterpenoids (BMTs) activate AMPK and increase GLUT4 translocation to the plasma membrane in vitro, and improve glucose disposal in insulin resistant models in vivo. Here we interrogated the mechanism by which these novel compounds activate AMPK, a leading anti-diabetic drug target. BMTs did not activate AMPK directly in an allosteric manner as AMP or the Abbott compound (A-769662) does, nor did they activate AMPK by inhibiting cellular respiration like many commonly used anti-diabetic medications. BMTs increased AMPK activity in both L6 myotubes and LKB1-deficient HeLa cells by 20–35%. Incubation with the CaMKKβ inhibitor, STO-609, completely attenuated this effect suggesting a key role for CaMKKβ in this activation. Incubation of L6 myotubes with the calcium chelator EGTA-AM did not alter this activation suggesting that the BMT-dependent activation was Ca2+-independent. We therefore propose that CaMKKβ is a key upstream kinase for BMT-induced activation of AMPK.
Highlights
Type 2 diabetes (T2D) accounts for 90% of the world’s escalating diabetes problem, with 439 million people expected to have diabetes by 2030 [1] at a predicted global healthcare cost of US $490 billion per annum [2]
A LiChrospher 100 RP-18 (Merck, USA) column (220625 mm i.d.; particle size 12 mm) was used for isolation. 5-Aminoimidazole4-carboxamide-1-b-D-ribofuranoside (AICAR) was obtained from Toronto Research Chemicals (Ontario, Canada), STO-609 acetate was from Tocris Bioscience (Bristol, UK), EasyTide [c-32P] ATP (10 mCi/ml) was from Perkin Elmer (Boston, MA, USA), AMARA peptide was from Auspep (Vic, Australia); ionomycin, bovine serum albumin (BSA), a-MEM, DMEM, foetal bovine serum (FBS) and 1006antibiotic/antimycotic and Pen/Strep/Glutamine (PSG) were from Invitrogen (Auckland, NZ)
Use of the cell permeable CaMKKb inhibitor STO-609 on both LKB1-deficient HeLa cells and L6 myotubes has demonstrated the key role of CaMKKb in this event (Fig. 6)
Summary
Type 2 diabetes (T2D) accounts for 90% of the world’s escalating diabetes problem, with 439 million people expected to have diabetes by 2030 [1] at a predicted global healthcare cost of US $490 billion per annum [2]. Traditional medicines have been an attractive source of novel anti-diabetic therapeutics One such source is the bitter melon (BM, Momordica charantia) with extracts having been used to treat diabetes-like symptoms in humans for hundreds of years. Bitter melon extracts have been reported to have hypoglycaemic, hypolipidemic, antioxidant and anti-inflammatory effects and improve skeletal muscle insulin signalling in vivo [4,5,6,7]. We have recently demonstrated a sustained efficacy of a triterpenoid in eliminating hyperglycaemia in T2D mice [9].
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