An obligatory requirement for the heterotrimeric G protein G i3 in the antiautophagic action of insulin in the liver

Gohla et al. 10.1073/pnas.0611434104.

Supporting Information

Files in this Data Supplement:

SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Figure 10
SI Table 1
SI Table 2
SI Table 3
SI Table 4
SI Experimental Procedures

SI Figure 6

Fig. 6. Gross morphology of Ga_i3/Ga_i3 mutant embryos at embryonic days 9 and 10 (E9 and E10). Shown are side views of embryos mutant for one allele of Ga_i3 (Upper Left) in comparison to embryos harboring one remaining allele of Ga_i3 (Upper Right) or of Ga_i2 (Lower Left). One allele of Ga_i3 or Ga_i2 is sufficient for development until E9/E10. In contrast, Ga_i2/Ga_i3 double-deficient embryos (Lower Right) are massively retarded by E10.

SI Figure 7A
SI Figure 7B

Fig. 7. Immunocytochemical detection of LC3. (A Upper) Primary mouse hepatocytes in standard culture medium were transfected with GFP-LC3 and stained for LC3 with a goat polyclonal anti-LC3 antibody (LC3 gAb). Overexpressed GFP-LC3 localized to cup- or ring-like (pre)autophagosomes (arrows). The same structures were detected by the goat anti-LC3 antibody. Under these conditions, lysosomes (labeled with LysoTracker) mostly are scattered in the cytosol and show a limited overlap with LC3-positive autophagosomes. (A Lower) Approximately 4-fold magnified view of the dashed areas in Upper. (B Upper) To induce autophagy and the corresponding recruitment of endogenous LC3-II to autophagosomes, mouse hepatocytes were treated with rapamycin (200 nM for 24 h). Autophagosomes were detected with the goat polyclonal anti-LC3 antibody used in A and with a rabbit polyclonal anti-LC3 antibody (LC3 rAb). Both antibodies detect the same structures. Under these conditions, lysosomes are clustered in the vicinity of the nuclei, and some overlap with autophagosomes can be observed. (B Lower) Approximately 4-fold magnified view of the dashed areas Upper. White color in the merged images indicates colocalization. Images are confocal micrographs. n = 2.

SI Figure 8

Fig. 8. Subcellular localization of Ga_i3 in mouse hepatocytes undergoing autophagy. Autophagy was induced by amino acid and growth factor withdrawal. Autophagosomes were visualized with anti-LC3 antibodies, and lysosomes were stained with LysoTracker. Upon starvation, Ga_i3 accumulates both at the plasma membrane and on LC3-positive autophagosomes, which partially overlap with lysosomes. Images are representative confocal micrographs. n = 3.

SI Figure 9

Fig. 9. Ga_i3 localizes to the plasma membrane and to autophagosomal membranes. Plasma membranes and autophagosomal membranes were isolated from mouse hepatocytes by subcellular fractionation as detailed in SI Experimental Procedures. Membrane preparations were solubilized in Laemmli's sample buffer, resolved by SDS/PAGE, blotted onto nitrocellulose, and incubated with the polyclonal Ga_i3 antibody and with the rabbit polyclonal LC3 antibody.

SI Figure 10

Fig. 10. Subcellular localization of Ga_i3 in mouse hepatocytes undergoing autophagy. Primary mouse hepatocytes were starved by growth factor and amino acid withdrawal and were stained as detailed in Experimental Procedures. The following markers were used: LC3 (for autophagosomes), LysoTracker (for lysosomes and other acidic organelles), early endosomal antigen-1 (EEA-1; for early endosomes), and protein disulfide isomerase (PDI; for rough endoplasmic reticulum). Upon starvation, Ga_i3 accumulates both at the plasma membrane and on intracellular vesicular compartments. In addition to a colocalization of Ga_i3 with LC3-positive autophagosomes, the distribution of Ga_i3 overlaps partially with portions of the endoplasmic reticulum and with early endosomes. Images are confocal micrographs. n = 3 for all experiments.

SI Experimental Procedures

Subcellular Fractionation. For each subcellular compartment, hepatocytes were freshly prepared from three 18-h starved male Sv129 mice, giving a yield of ~2 g cellular wet mass.

The isolation of plasma membranes was performed as described with minor modifications [Maeda T, Balakrishnan K, Mehdi SQ (1983) Biochimica et Biophysica Acta 731:115-120]. Briefly, hepatocytes were suspended in homogenization buffer A [HBA; 10 mM sodium phosphate buffer (pH 7.4), 1 mM MgCl₂, 30 mM NaCl, 1 mM DTT, 0.005 mM PMSF, 0.02% NaN₃, and 1-5 mg of DNase] and homogenized by using an UltraTurrax T20b (IKA Labortechnik, Staufen, Germany). The homogenate was layered onto a 41% solution of sucrose and centrifuged at 95,000 ´ g in a TH-641 swinging bucket rotor in a Sorvall ultracentrifuge for 60 min at 4°C. The interface band was diluted in HBA, and the plasma membranes were pelleted by centrifugation at 95,000 ´ g in a type 75 Ti fixed-angle rotor (Beckman Coulter, Krefeld, Germany) for 20 min at 4°C. Plasma membranes were washed twice in HBA.

Autophagosomes were purified according to Seglen et al. with minor modifications [Stromhaug PE, Berg TO, Fengsrud M, Seglen PO (1998) Biochem J 335:217-224]. In brief, freshly isolated hepatocytes were suspended in 20 mM sodium pyruvate, 68.4 mM NaCl, 5.4 mM KCl, 1.1 mM KH₂PO₄, 0.7 mM Na₂SO₄, 0.64 mM MgCl₂ ´6 H₂O, 1.2 mM CaCl₂ ´ 2 H₂O, 30.0 mM Hepes, 30.0 mM TES, 36.3 mM tricine, and 52.5 mM NaOH supplemented with 50 mM vinblastine (Sigma, Deisenhofen, Germany) at 37°C for 2 h to induce autophagosome accumulation. After electrodisruption and homogenization with a Dounce homogenizer, the cell disruptate was diluted in homogenization buffer B [HBB; 0.25 M sucrose, 10 mM Hepes, 1 mM EDTA (pH 7.3)], supplemented with 1% DMSO and 1.5 mM glycyl-L-phenylalanine 2-naphthylamide (Sigma, Deisenhofen, Germany) to destroy lysosomes. The disruptates were centrifuged at 2,000 ´ g in a type 70 Ti rotor (Beckman Coulter, Krefeld, Germany) for 2 min at 4°C to obtain the postnuclear supernatant. The supernatant was divided into three 12-ml aliquots and placed onto a discontinuous Nycodenz gradient (Axis-Shield PoC, Oslo, Norway) prepared by layering 15 ml of 9.5% Nycodenz (1.072 g/ml) in HBB on top of 6 ml of 22.5% Nycodenz (1.127 g/ml) in HBB. After a centrifugation step at 140,000 ´ g in a AH-627 swinging bucket rotor on a Sorvall ultracentrifuge for 1 h at 4°C, the gradient was divided into three fractions: the cytosol, a lighter interface band (fraction A), and a heavier interface band (fraction B), which contained the autophagosomes. Each of these two bands was layered onto a discontinuous Percoll/Nycodenz-gradient [6 ml of 33% Percoll in HBB on top of 2 ml of 22.5% Nycodenz (1.127 g/ml in HBB)] and centrifuged at 72,000 ´g for 30 min at 4°C in a TH-641 swinging bucket rotor on a Sorvall ultracentrifuge. The autophagosomes banded at the upper interface and were solubilized in Laemmli's sample buffer. The purity of the autophagosomal preparation was assessed by immunoblotting for organelle markers. The lysosomal protease cathepsin B, the Golgi matrix protein GM130, and the endoplasmic reticulum-associated protein disulfide isomerase were undetectable in the autophagosomal fraction. In contrast, the autophagosomal fraction contained cytochrome c. The presence of this mitochondrial marker in the autophagosomal fraction likely reflects mitochondria taken up by autophagosomes (data not shown).