Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver

Abstract

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.

Fig. 1.

Accumulation of ADRP in the liver of fasting mice. A: Effects of fasting on ADRP protein levels in the tissues were examined. Male C57BL/6 mice (10 weeks old) were either fed chow diet or fasted for 16 h, euthanized, and then brain, heart, liver, skeletal muscle, and WAT were recovered. Immunoblot analysis of ADRP protein levels in each type of mouse tissue was performed, and the intensity of the detected bands for ADRP and β-actin were quantified by densitometric scanning. The relative index of 1.0 is the ADRP/β-actin in the liver from chow-fed mice. Bar graph shows mean and standard deviation of three mice. B: Fasting altered levels of TIP47, another LD protein, and SCD-1, an enzyme involved in lipid metabolism. Among the PAT family proteins, perilipin was not detectable. The intensity of the detected bands of ADRP, TIP47, SCD-1, and β-actin were quantified by densitometric scanning. ADRP, adipose differentiation-related protein; LD, lipid droplet; SCD-1, stearoyl-CoA desaturase-1; TIP47; tail interacting protein of 47 kDa; WAT, white adipose tissue.

Fig. 2.

ADRP-LDs with intermediate density appeared in the fasted liver. A: The profiles of ADRP, TIP47, MTP, GRP78/BiP, and Raf-1. The liver PNS fractions from mice under feeding conditions and after fasting for 16 h were fractionated by sucrose density gradient centrifugation. MTP and GRP78/BiP are marker proteins for ER; Raf-1 is a marker for cytosol. Each fraction from sucrose density gradient centrifugation was analyzed by immunoblot analysis. MTP seems to increase in the fasted liver, although we have not done a quantitative confirmation yet. B: Distribution of ADRP levels in the density gradient. The intensity of the detected bands of ADRP was quantified by densitometric scanning. The relative index of 1 is defined as the intensity of ADRP in fraction No. 1. C: Distribution of the TG levels in the density gradient. The TG levels of each fraction were quantitatively measured as described in “Experimental Procedures.” The relative index of 1 is the TG level in fraction No. 1. ADRP, adipose differentiation-related protein; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; LD, lipid droplet; MTP, microsomal triacylglycerol transfer protein; PNS, post-nuclear supernatant; TG, triacylglycerol; TIP47; tail interacting protein of 47 kDa.

Fig. 3.

Distribution of ADRP in iLD fractions in the MCD diet-induced fatty liver. Liver homogenates prepared from mice before and after feeding with the MCD diet for 2 weeks were fractionated using sucrose density gradient ultracentrifugation. A: TG levels in the liver homogenates were quantitatively measured by enzymatic assay. B: Distribution of TG in the liver. The TG levels in each fraction were measured. The inset shows detailed pattern of TG distribution in iLD fractions. C: ADRP and GAPDH in the liver homogenates were detected using immunoblot analysis. D, E: Distribution of ADRP after the fractionation was analyzed by immunoblot, and the band intensity was quantified by densitometric scanning (n = 3). The relative index of 1 is defined as the ADRP/GAPDH ratio in the livers of control mice (0-week treatment). ^*P < 0.05, ^**P < 0.01, ^***P < 0.005. ADRP, adipose differentiation-related protein; iLD, intermediate-density lipid droplet; MCD, methione/choline deficient; TG, triacylglycerol.

Fig. 4.

ADRP appearance in the iLD fractions were induced by glucagon in HuH-7 human hepatoma cells. A: HuH-7 cells were incubated with or without 0.6 mM OA for 24 h, and in another set of cells, 100 nM glucagon was added for 24 h. Then the cell lysate was fractionated by sucrose ultracentrifugation. The same volume (8 μl) from each fraction was applied to SDS-PAGE and immunoblotted with anti-ADRP antibody. B: HuH-7 cells were treated as above, and the amount of TG in the cell lysates was measured. C: Distribution of TG in the cells incubated with OA with or without glucagon was assessed by dividing them into three fractions: LD fractions (Fr. No. 1–3), iLD fractions (Fr. No. 5–9), and high-density fractions (Fr. No. 11–15). D, E: Distribution of ADRP and TG in the cells incubated with OA with or without glucagon in each fraction separated by sucrose ultracentrifugation. The intensity of the detected bands of ADRP was quantified by densitometric scanning. The relative index of 1 is defined as the intensity of ADRP in fraction No. 1 from the HuH-7 cells treated with OA and glucagon. ADRP, adipose differentiation-related protein; iLD, intermediate-density lipid droplet; OA, oleic acid; TG, triacylglycerol.

Fig. 5.

Distribution of ADRP and other proteins in HuH-7 cells after incubation with OA and glucagon. A: HuH-7 cells were incubated with OA (0.6 mM) and glucagon (100 nM) for 24 h. Then the cell lysate was fractionated by sucrose ultracentrifugation, and the distribution of ADRP, enolase, BiP/GRP78, and calnexin were examined by Western blotting. B: Lysate of HuH-7 cells without treatment with OA and glucagon was fractionated by sucrose ultracentrifugation, and the distribution of BiP/GRP78, and calnexin were examined by Western blotting. ADRP, adipose differentiation-related protein; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; OA, oleic acid.

Fig. 6.

Microscopic views of distribution of ADRP and other proteins in HuH-7 cells after incubation with OA or OA and glucagon. HuH-7 cells were treated with OA (0.6 mM) or with OA plus glucagon (100 nM) for 24 h. A-D: The cells were stained with anti-ADRP IgG (red) in combination with BODIPY493/503, antibodies against calnexin, GRP78/BiP, or enolase (green). The cells were also treated with DAPI to stain the nuclei (blue). The insets in (A) are enlarged images of LD particles in the HuH-7 cells treated with OA plus glucagon showing that ADRP surrounds both large and small LD particles. The cells were visualized and photographed using confocal laser microscopy. The bars indicate 10 μm. ADRP, adipose differentiation-related protein; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; LD, lipid droplet; OA, oleic acid.

Fig. 7.

Microscopic views of distribution of ADRP and other proteins in human primary hepatocytes after incubation with OA or OA and glucagon. Human primary hepatocytes were treated with OA (0.6 mM) or with OA plus glucagon (100 nM) for 24 h. A-D: The cells were stained with anti-ADRP IgG (red), in combination with BODIPY493/503, anti-calnexin, or anti-GRP78/BiP antibodies (green) as in the Fig. 6. The cells were also treated with DAPI to stain the nuclei (blue). The cells were visualized and photographed using confocal laser microscopy. The bars indicate 10 μm. ADRP, adipose differentiation- related protein; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; OA, oleic acid.

Fig. 8.

ADRP appearance in iLD fractions was induced by PKA- and PLC-dependent pathways in HuH-7 human hepatoma cells. A: HuH-7 cells were incubated with 0.6 mM OA and with or without 100 μM dibutyryl cAMP (db-cAMP) for 24 h. Then the cell lysate was fractionated by sucrose ultracentrifugation, and the distribution of ADRP was detected. B: HuH-7 cells were incubated with 0.6 mM OA plus 100 nM glucagon or 100 μM db-cAMP to induce redistribution of ADRP to the iLD fraction. In addition, a PKA inhibitor, either Rp (20 μM) or PKI (50 nM), was added to the cells during the 24 h incubation. B: HuH-7 cells were incubated with 0.6 mM OA plus 100 nM glucagon to induce redistribution of ADRP to the iLD fraction. In addition, the PLC inhibitor U-73122 (5 μM) was added to the cells during the 24 h incubation. ADRP, adipose differentiation-related protein; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; iLD, intermediate-density lipid droplet; OA, oleic acid; PKA, protein kinase A; PKI, PKI14-22amide; PLC, phospholipase C; Rp, Rp-8-Br-cAMPS.