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. 2012 May 2;15(5):739-51.
doi: 10.1016/j.cmet.2012.03.002. Epub 2012 Apr 12.

Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity

Lale Ozcan  1 , Catherine C L Wong, Gang Li, Tao Xu, Utpal Pajvani, Sung Kyu Robin Park, Anetta Wronska, Bi-Xing Chen, Andrew R Marks, Akiyoshi Fukamizu, Johannes Backs, Harold A Singer, John R Yates 3rd, Domenico Accili, Ira Tabas
Affiliations

Calcium signaling through CaMKII regulates hepatic glucose production in fasting and obesity

Lale Ozcan et al. Cell Metab. .

Abstract

Hepatic glucose production (HGP) is crucial for glucose homeostasis, but the underlying mechanisms have not been fully elucidated. Here, we show that a calcium-sensing enzyme, CaMKII, is activated in a calcium- and IP3R-dependent manner by cAMP and glucagon in primary hepatocytes and by glucagon and fasting in vivo. Genetic deficiency or inhibition of CaMKII blocks nuclear translocation of FoxO1 by affecting its phosphorylation, impairs fasting- and glucagon/cAMP-induced glycogenolysis and gluconeogenesis, and lowers blood glucose levels, while constitutively active CaMKII has the opposite effects. Importantly, the suppressive effect of CaMKII deficiency on glucose metabolism is abrogated by transduction with constitutively nuclear FoxO1, indicating that the effect of CaMKII deficiency requires nuclear exclusion of FoxO1. This same pathway is also involved in excessive HGP in the setting of obesity. These results reveal a calcium-mediated signaling pathway involved in FoxO1 nuclear localization and hepatic glucose homeostasis.

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Figures

Figure 1
Figure 1. Glucagon and fasting activates hepatic CaMKII
(A) CaMKII enzyme activity was assayed in triplicate wells of primary mouse HCs stimulated with 100 nM glucagon (Gluc) or vehicle control (Veh) for the indicated times (*P < 0.05 and **P < 0.01 vs. Veh; mean ± S.E.M.). (B–J) Extracts of HCs or liver were probed for phospho-CaMKII, total CaMKII, and β-actin by immunoblot assay. (B) HCs were incubated with 100 nM glucagon for the indicated times; (C) Glucagon was added to HCs that were pre-treated for 1 h with vehicle control (Veh) or 5 µM BAPTA-AM; (D) Glucagon was added to HCs that were pre-treated with 0.5 µM xestospongin (XesC) or to HCs from Ip3r1fl/fl mice transduced with adeno-LacZ control or adeno-Cre (bar graph = Ip3r1 mRNA levels); (E) Glucagon was added to HCs that were pre-treated for 1 h with vehicle control (Veh) or 10 µM H89. (F) HCs were incubated with 100 µM 8-bromo-cAMP for the indicated times. (G–J) In vivo experiments. In G, mice were treated for 30 min with 200 µg kg−1 body weight of glucagon i.p., and in H, mice were pre-treated with 10 pmol g−1 xestospongin C or vehicle control i.p. 4 days prior to glucagon treatment. In I–J, mice were fed ad libitum or fasted for 12 h, or fasted for 12 h and then re-fed for 4 h.
Figure 2
Figure 2. CaMKII regulates glucose production and hepatic G6Pc and Pck1 expression in primary HCs
(A) RNA from HCs from 3 WT and 3 Camk2g−/− mice and mouse brain from a WT mouse were probed for the indicated Camk2 isoform mRNAs by RT-PCR. (B) HCs from WT and Camk2g−/− mice were serum-depleted overnight and then incubated with forskolin (10 µm) for 14 h in serum- and glucose-free media, and then glucose in the medium was assayed (**P < 0.01 vs. WT in each group; mean ± S.E.M.). (C) HCs from WT mice were transduced with adenoviral vectors expressing LacZ, CA-CaMKII, or KD-CaMKII at an MOI of 20 and then assayed for glucose production as in (B) (*P < 0.05 and **P < 0.01 vs. LacZ in each group; mean ± S.E.M.). (D–E) HCs similar to those in (B) and (C) were serum-depleted overnight and then incubated for 5 h with 10 µM forskolin or 100 nM glucagon in serum-free media, as indicated. RNA was assayed for G6pc and Pck1 mRNA by RT-qPCR (*P < 0.05 and **P < 0.01 vs. LacZ or WT in each group; mean ± S.E.M.).
Figure 3
Figure 3. CaMKIIγ deficiency or acute inhibition in vivo decreases blood glucose and hepatic G6pc and Pck1
(A) Blood glucose of 12-h-fasted 8-wk/o WT and Camk2g−/− mice (*P < 0.05). (B) As in (A), but the mice were fasted for 18 h and then challenged with 2 mg kg−1 pyruvate (B) (*P < 0.05; **P < 0.01; ***P < 0.005; mean ± S.E.M.). (C) Liver G6pc and Pck1 mRNA in 12-h-fasted WT and Camk2g−/− mice (**P < 0.01; ***P < 0.001; mean ± S.E.M.). (D) WT and Camk2g−/− mice were injected i.p. with glucagon (200 µg kg−1) and sacrificed 30 min later. Liver G6pc mRNA was assayed (*P < 0.05; mean ± S.E.M.). (E–G) 9-wk/o WT mice were administered 1.5 × 109 pfu of adeno-LacZ or KD-CaMKII, and 5 days later the following parameters were assayed in 12-h-fasted mice: E, blood glucose (***P < 0.001; mean ± S.E.M.); F, liver G6pc and Pck1 mRNA (**P < 0.01; ***P < 0.001; mean ± S.E.M.); and G, liver glycogen content and PAS-positive cells (**P < 0.01; mean ± S.E.M.). Panel G also shows liver glycogen content in fasted WT and Camk2g−/− mice (*P < 0.05; mean ± S.E.M.). For all panels, n = 5/group except panel D, where n = 4/group.
Figure 4
Figure 4. CaMKII regulates hepatic FoxO1 subcellular localization
(A) HCs from WT and Camk2g−/− mice were transduced with an adenovirus expressing murine GFP-FoxO1 at an MOI of 2. Cells were serum-depleted overnight and then incubated for 5 h in serum-free media. FoxO1 subcellular localization was assessed by indirect immunofluorescence. Bar, 10 µm. Data are quantified in the right panel. (#P < 0.0005; mean ± S.E.M.). (B) HCs were transduced with adenoviral vectors expressing LacZ, CA-CaMKII, or KD-CaMKII at an MOI of 20 and then transduced 4 h later with adeno-GFP-FoxO1, followed by fluorescence microscopy and quantification as in (A) (#P < 0.005 vs. LacZ; mean ± S.E.M.). Bar, 5 µm. (C) HCs were transduced with adeno-LacZ or CA-CaMKII and then adeno-GFP-FoxO1 as in (B). After incubation in serum-depleted medium o.n. and then serum-free medium for 5 h, the cells were treated with 100 nM insulin for the indicated times. FoxO1 subcellular localization was quantified as in (B) (*P < 0.005 vs. LacZ in each group; mean ± S.E.M.). (D) Nuclear FoxO1 and nucleophosmin were probed by immunoblot in livers from fasted WT mice, Camk2g−/− mice, or WT mice treated with adeno-LacZ or KD-CaMKII; from fed WT mice treated with adeno-LacZ or CA-CaMKII; or from fed WT mice treated for 30 min with 200 µg kg-1 body weight of glucagon i.p. For the glucagon experiment, the average FoxO:Np densitometric ratio values are in the graph (*P = 0.029; blemishes in lanes 6 and 8 were excluded from the densitometry analysis).
Figure 5
Figure 5. Impairment of glucose metabolism by CaMKII inhibition is rescued by transduction with constitutively nuclear FoxO1-ADA
(A) HCs from wild-type or L-FoxO1 knockout mice were transduced with adeno-LacZ or CA-CaMKII. The cells were serum-depleted overnight and then incubated for 5 h in the absence or presence of forskolin (10 µm) in serum-free media. RNA was assayed for G6pc mRNA (*P < 0.001; mean ± S.E.M.). (B) HCs from WT and Camk2g−/− mice were administered adeno-LacZ or FoxO1-ADA at an MOI of 0.2. Cells were serum-depleted overnight and then incubated for 5 h with 10 µM forskolin in serum-free media. RNA was assayed for G6pc and Pck1 mRNA (**P < 0.01 vs. WT groups; #P < 0.05 and ##P < 0.01 vs. Camk2g−/−/LacZ group; mean ± S.E.M.). Inset, the nuclei from a parallel set of cells were probed for FoxO1 and nucleophosmin by immunoblot; the average densitometric ratio appears below each pair of lanes. (C–E) 8-wk/o WT mice were administered adeno-LacZ or KD-CaMKII, and then one day later, half of the adeno-KD-CaMKII mice received adeno-FoxO1-ADA, while the other half received adeno-LacZ control. Blood glucose levels were assayed at day 5 after a 12-h fast (*P < 0.05 vs. LacZ/LacZ; #P < 0.05 vs. KD/LacZ; n = 5/group; mean ± S.E.M.), and liver was assayed for nuclear FoxO1 protein (**P < 0.01 vs. KD/LacZ; mean ± S.E.M.); G6pc, Pck1 and Igfbp1 mRNA (*P < 0.05 and **P < 0.01 vs. LacZ/LacZ; #P < 0.05 vs. KD/LacZ; n = 3/group; mean ± S.E.M.). The inset to panel E shows the level of hemagglutinin (HA)-tagged KD-CaMKII protein (anti-HA immunoblot).
Figure 6
Figure 6. The role of non-AKT-phospho-sites of FoxO1 in CaMKII-mediated FoxO1 nuclear localization
(A) HCs from WT and Camk2g−/− mice were transduced with adeno-FLAG-FoxO1 at an MOI of 2. Cells were serum-depleted overnight and then incubated for 5 h in serumfree media. FoxO1 was immunopurified using anti-FLAG, followed by reduction, alkylation, and proteolytic digestion. Phosphorylated peptides were enriched by TiO2 chromatography and then analyzed by LC-MS/MS as described in Experimental Procedures. The table shows spectral count number, Debunker score, and Ascore of phosphorylated peptides in KO and WT samples; Δ in the peptide sequence indicates the phosphorylation site. The cut-off values for spectral count #, Debunker score, and Ascore are set at 5, 0.5 and 12 respectively. The spectra of peptides with scores that are below these values (italics) were checked manually to eliminate uncertain phosphorylation sites (Figures S4A–B for WT peptides 4 and 5; and http://fields.scripps.edu/published/foxo1_Tabas_2012/ for KO peptides 7, 10, and 11). The KO/WT ratio of spectral counts was calculated only for peptides with a combined spectral count in KO and WT > 10. (B) HCs from L-FoxO1 mice were transfected with expression plasmids encoding murine Flag-FoxO1 or Flag-7A-FoxO1 mutant. After 48 h, the cells were serum-depleted overnight and then incubated with glucagon (100 nm) for 4 h in serum-free media. Nuclear extracts were assayed by immunoblot for Flag and nucleophosmin (nuclear loading control), and RNA from a parallel set of cells was probed for Foxo1 mRNA by RT-qPCR. Densitometric quantification of the mRNA and immunoblot data is shown in the graph (*P < 0.005; mean ± S.E.M.) (C) Similar to (B), except the HCs were transduced with adeno-LacZ or CA-CaMKII one day after the transfection with the WT or mutant FoxO1 plasmids (*P = 0.003; mean ± S.E.M.).
Figure 7
Figure 7. The role of CaMKII in hepatic glucose metabolism in obesity
(A) Liver extracts from 10-wk/o WT or ob/ob mice, or WT mice fed a chow or high-calorie diet for 20 wks (diet-induced obesity; DIO), were probed for p- and total CaMKIIγ and β-actin by immunoblot. Densitometric quantification is shown in the bar graph (***P < 0.001; mean ± S.E.M.). Antibody specificity is shown by the absence of p- and total CaMKIIγ bands in liver extracts from DIO Camk2g−/− mice. (B–D) Fasting blood glucose, blood glucose after pyruvate challenge, and liver G6pc, Pck1 and Igfbp1 mRNA in ob/ob mice before or after treatment with adeno-LacZ or KD-CaMKII (n = 5/group; *P < 0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001 vs. LacZ; mean ± S.E.M.).
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