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. 2017 Nov 7;114(45):11926-11931.
doi: 10.1073/pnas.1706489114. Epub 2017 Oct 25.

Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides

Jin Ku Kang  1 , Ok-Hee Kim  1 , June Hur  1 , So Hee Yu  1 , Santosh Lamichhane  1 , Jin Wook Lee  1 , Uttam Ojha  1 , Jeong Hee Hong  1 , Cheol Soon Lee  1 , Ji-Young Cha  2 , Young Jae Lee  2 , Seung-Soon Im  3 , Young Joo Park  4 , Cheol Soo Choi  1 , Dae Ho Lee  1 , In-Kyu Lee  5 , Byung-Chul Oh  6
Affiliations

Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides

Jin Ku Kang et al. Proc Natl Acad Sci U S A. .

Erratum in

Abstract

Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca2+ concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca2+ homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca2+ acts as a negative regulator of insulin signaling. Chronic intracellular Ca2+ overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca2+ inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca2+ abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 Finally, thermodynamic analysis of the binding interaction showed that Ca2+-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca2+ rather than PH domains to PIPs forming Ca2+-PIPs. Thus, Ca2+-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca2+-PIPs. Our findings provide a mechanistic link between intracellular Ca2+ dysregulation and Akt inactivation in insulin resistance.

Keywords: Ca2+-phosphoinositides; PH domain; insulin resistance; intracellular Ca2+ concentration; membrane localization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
High-fat diet (HFD) and palmitate treatment increase intracellular Ca2+ levels and attenuate insulin signaling. (A) Immunoblot analysis of mouse liver extracts after overnight fasting and subsequent refeeding with normal chow or a HFD for 4 h. (B) Representative confocal images of cytosolic free Ca2+ in the hepatocytes expressing adenoviral GCaMP6m from mice fed normal chow or a HFD for 10 wk following 7 d of adenoviral infection. Ex vivo hepatocytes expressing adenoviral GCaMP6m were visualized using confocal microscopy from formalin-fixed liver sections of mice following overnight fasting and subsequent refeeding with normal chow or a HFD for 4 h. (Scale bars: 10 μm.) (Bottom) Images merged with 4,6-diamidino-2-phenylindole (DAPI) staining of nuclei and differential interference contrast (DIC) microscopy. (C and F) Fluorescence intensities of GCaMP6m images (C) and Fluo-3 AM images (F) of cytosolic Ca2+ were quantified with low power field images using ImageJ software. Data represent means ± SEM (n = 3–5, *P < 0.05, **P < 0.01). (D) Immunoblot analysis of HepG2 cells treated with the indicated concentrations of palmitic acid for 24 h followed by treatment with 100 nM insulin for 15 min. (E) Representative Fluo-3 AM images of cytosolic Ca2+ in HepG2 cells treated with the indicated concentrations of palmitic acid for 24 h. Intracellular Ca2+ visualized using confocal microscopy. (Scale bars: 10 μm.)
Fig. 2.
Fig. 2.
The catalytic activity of Akt is modulated by intracellular Ca2+ concentration. (A and D) Immunoblot analysis of the phosphorylation states of Akt, FOXO3A, and AS160, and the total amounts of the indicated proteins in HepG2 cells. Cells were incubated for 30 min with the indicated concentrations of PMA (A) or ionomycin (D), followed by treatment with 100 nM insulin for 15 min. (B, C, E, and F) Representative Fluo-3 AM images (B) and quantification (C) of intracellular Ca2+ in HepG2 cells treated with PMA. Data represent means ± SEM (n = 5, *P < 0.05).
Fig. 3.
Fig. 3.
Higher intracellular Ca2+ concentrations prevent membrane localization of PH domain proteins. (A) Fluorescence images of Akt-PH mCherry. CHO-IR cells were transfected with Akt PH domain-mCherry fusion vector, serum starved for 3 h, and treated with or without ionomycin(10 μM)/PMA (100 nM) for 30 min before a 15-min stimulation with 100 nM insulin. (B) Fluorescence images of PLCδ-PH GFP. CHO-IR cells were transfected with PLCδ-PH GFP fusion vector, followed by incubation with 10 μM ionomycin or 100 nM PMA for 15 min. After 100 nM PMA for 15 min treatment, the cells were incubated with 1 mM EDTA for 5 min to chelate Ca2+. (Scale bars: 10 μm.) (C) Representative fluorescence images of adenoviral Akt-PH mCherry from mice fed normal chow or a HFD for 10 wk following 7 d of adenoviral infection. Ex vivo hepatocytes expressing adenoviral Akt-PH mCherry were visualized using confocal microscopy from formalin-fixed liver sections of mice following overnight fasting and subsequent refeeding with normal chow or a HFD for 4 h. (Scale bars: 5 μm.)
Fig. 4.
Fig. 4.
High Ca2+ concentrations abolishes the electrostatic interactions between PH domains and PIPs through the formation of Ca2+-PIPs. (A) Schematic representation of the various biological phospholipids (PIP strips, Echelon Biosciences), including LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; and S1P, sphingosine 1-phosphate. (B–D) Binding of the PH domains of Akt (B), PLCδ (C), and IRS1 (D) to immobilized phospholipids under the indicated Ca2+ concentrations. (E) Schematic representation of electrostatic interactions between the PH domain of Akt (Protein Data Bank accession code:1H10) and Ins(1,3,4,5)P4. (F–I) ITC results for Ca2+ binding to the PH domain of Akt (F), PI(3,4)P2 (G), PI(4,5)P2 (H), or PI(3,4,5)P3 (I) liposomes. Kd values were determined by curve fitting. (J) Schematic representation of electrostatic repulsion between basic residues in the Akt PH domain and Ca2+-Ins(1,3,4,5)P4. Positive charges of basic residues in the PH domain will repel the positively charged Ca2+-Ins(1,3,4,5)P4 and thus inhibit the electrostatic interactions in E.
Fig. 5.
Fig. 5.
A proposed model of intracellular Ca2+-mediated inhibition of insulin signaling in obesity. Models showing that, under normal physiological conditions, PI(3,4,5)P3 recruits the PH domain of Akt to the plasma membrane, where Akt mediates insulin signaling by phosphorylating GSK3β, FOXO, and AS160. In pathological conditions, such as obesity, however, metabolic stress may lead to elevated intracellular Ca2+ levels, thereby impairing insulin action on carbohydrate and lipid metabolism by blocking membrane localization of the PH domain of Akt through the formation of Ca2+-phosphoinositides.
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