Protein kinase- and lipase inhibitors of inositide metabolism deplete IP7 indirectly in pancreatic beta-cells: Off-target effects on cellular bioenergetics and direct effects on IP6K activity

Author(s)

Rajasekaran, Subu Surendran ^[1] ; Illies, Christopher ^[1] ; Shears, Stephen B. ^[2] ; Wang, Huanchen ^[2] ; Ayala, Thais S. ^{[1, 3]} ; Martins, Joilson O. ^[3] ; Dare, Elisabetta ^[1] ; Berggren, Per-Olof ^[1] ; Barker, Christopher J. ^[1]

Affiliation

^[1] Karolinska Inst, Rolf Luft Res Ctr Diabet & Endocrinol, SE-17176 Stockholm - Sweden

^[2] NIEHS, Signal Transduct Lab, Inositol Signaling Grp, Bldg 101, Room F239, 111 TW Alexander Dr, Res Triangle Pk, NC 27709 - USA

^[3] Univ Sao Paulo, Fac Pharmaceut Sci, Dept Clin & Toxicol Anal, Lab Immunoendocrinol, Sao Paulo - Brazil

• Document type: Journal article
• Source: CELLULAR SIGNALLING; v. 42, p. 127-133, JAN 2018.
• Web of Science Citations: 1

Abstract

Inositol pyrophosphates have emerged as important regulators of many critical cellular processes from vesicle trafficking and cytoskeletal rearrangement to telomere length regulation and apoptosis. We have previously demonstrated that 5-di-phosphoinositol pentakisphosphate, IP7, is at a high level in pancreatic beta-cells and is important for insulin exocytosis. To better understand IP7 regulation in beta-cells, we used an insulin secreting cell line, HIT-T15, to screen a number of different pharmacological inhibitors of inositide metabolism for their impact on cellular IP7. Although the inhibitors have diverse targets, they all perturbed IP7 levels. This made us suspicious that indirect, off-target effects of the inhibitors could be involved. It is known that IP7 levels are decreased by metabolic poisons. The fact that the inositol hexakisphosphate kinases (IP6Ks) have a high K-m for ATP makes IP7 synthesis potentially vulnerable to ATP depletion. Furthermore, many kinase inhibitors are targeted to the ATP binding site of kinases, but given the similarity of such sites, high specificity is difficult to achieve. Here, we show that IP7 concentrations in HIT-T15 cells were reduced by inhibitors of PI3K (wort-mannin, LY294002), PI4K (Phenylarsine Oxide, PAO), PLC (1173122) and the insulin receptor (HNMPA). Each of these inhibitors also decreased the ATP/ADP ratio. Thus reagents that compromise energy metabolism reduce IP7 indirectly. Additionally, PAO, U73122 and LY294002 also directly inhibited the activity of purified IP6K. These data are of particular concern for those studying signal transduction in pancreatic beta-cells, but also highlight the fact that employment of these inhibitors could have erroneously suggested the involvement of key signal transduction pathways in various cellular processes. Conversely, IP7's role in cellular signal transduction is likely to have been underestimated. (AU)

• FAPESP's process: 14/05214-1 - Investigating the role of insulin in different infections in diabetic and healthy animals
• Grantee: Joilson de Oliveira Martins
• Support Opportunities: Regular Research Grants
• FAPESP's process: 10/02272-0 - Effect of insulin on lung inflammation in animal with sepsis, innate immunit,activation of insulin gene (BGK) and insulin receptors (IR)-A and IR-B
• Grantee: Joilson de Oliveira Martins
• Support Opportunities: Research Grants - Young Investigators Grants