Differential pattern of glycogen accumulation after protein phosphatase 1 glycogen-targeting subunit PPP1R6 overexpression, compared to PPP1R3C and PPP1R3A, in skeletal muscle cells

Abstract

Background: PPP1R6 is a protein phosphatase 1 glycogen-targeting subunit (PP1-GTS) abundant in skeletal muscle with an undefined metabolic control role. Here PPP1R6 effects on myotube glycogen metabolism, particle size and subcellular distribution are examined and compared with PPP1R3C/PTG and PPP1R3A/G(M).

Results: PPP1R6 overexpression activates glycogen synthase (GS), reduces its phosphorylation at Ser-641/0 and increases the extracted and cytochemically-stained glycogen content, less than PTG but more than G(M). PPP1R6 does not change glycogen phosphorylase activity. All tested PP1-GTS-cells have more glycogen particles than controls as found by electron microscopy of myotube sections. Glycogen particle size is distributed for all cell-types in a continuous range, but PPP1R6 forms smaller particles (mean diameter 14.4 nm) than PTG (36.9 nm) and G(M) (28.3 nm) or those in control cells (29.2 nm). Both PPP1R6- and G(M)-derived glycogen particles are in cytosol associated with cellular structures; PTG-derived glycogen is found in membrane- and organelle-devoid cytosolic glycogen-rich areas; and glycogen particles are dispersed in the cytosol in control cells. A tagged PPP1R6 protein at the C-terminus with EGFP shows a diffuse cytosol pattern in glucose-replete and -depleted cells and a punctuate pattern surrounding the nucleus in glucose-depleted cells, which colocates with RFP tagged with the Golgi targeting domain of β-1,4-galactosyltransferase, according to a computational prediction for PPP1R6 Golgi location.

Conclusions: PPP1R6 exerts a powerful glycogenic effect in cultured muscle cells, more than G(M) and less than PTG. PPP1R6 protein translocates from a Golgi to cytosolic location in response to glucose. The molecular size and subcellular location of myotube glycogen particles is determined by the PPP1R6, PTG and G(M) scaffolding.

Figure 1

Effect of PPP1R6 overexpression on GS and GP activity. Cultured human myotubes were transduced with Ad-GFP, Ad-R6 or/and Ad-MGP and incubated with 25 mM glucose for 48 h. (A) Immunoblot analyses were performed on extracts (20 μg protein) from cells transduced with Ad-GFP or Ad-R6. Membranes were hybridized with antibody against PPP1R6. Enzyme activities were assessed in cell extracts: (B) GS activity measured with (black columns, total activity) or without (white columns, activity of the active form) glucose 6-phosphate and (C) GP activity measured with (black columns, total activity) or without (white columns, activity of the active form) AMP. Data are means ± SEM from two experiments performed in triplicate. Significance of differences versus cells treated with Ad-GFP under the same incubation conditions: *p < 0.001 and **p < 0.0001.

Figure 2

Immunoblot analysis of GS and PP1 protein content. Cultured human myotubes were transduced with Ad-GFP, Ad-R6, Ad-PTG or Ad-G_M and incubated with 25 mM glucose for 48 h. Immunoblot analyses were performed on cell extracts (20 μg protein for GS and phospho-GS and 5 μg protein for PP1). Membranes were hybridized with antibodies against: (A) phospho-GS (Ser-641/0), (B) muscle GS and (D) PP1α. (A, B, D) A representative image is shown. Bands were quantified with a LAS-3000 (FujiFilm). Data are means ± SEM from three experiments performed in duplicate. Significance of differences versus cells treated with Ad-GFP under the same incubation conditions: *p < 0.05 and **p < 0.01.

Figure 3

GS activity ratio in glucose-replete and -depleted cells. Cultured human myotubes were transduced with Ad-GFP, Ad-R6, Ad-PTG or Ad-G_M and then incubated with (white columns) or without (black columns) 25 mM glucose for 24 h. GS activity ratio (without glucose 6-phosphate/with glucose 6-phosphate) was measured in cell extracts. Data are means ± SEM from five experiments performed in duplicate. Significance of differences: versus cells treated with Ad-GFP under the same incubation conditions, *p < 0.05 and **p < 0.0001; cells without glucose versus with glucose for any viral treatment, †p < 0.01 and ‡p < 0.0001.

Figure 4

Glycogen synthesis and content. Cultured human myotubes were: (A) transduced with Ad-GFP (▲) or Ad-R6 (■) and incubated with glucose-deprived medium for 18 h, then with 10 mM [U-¹⁴C]glucose for the times indicated and harvested to quantify the radioactivity associated with glycogen; (B) transduced with Ad-GFP, Ad-R6, Ad-PTG or Ad-G_M, incubated with 25 mM glucose for 48 h and harvested to assess glycogen content. (A, B) Data are means ± SEM from three experiments performed in duplicate. Significance of differences versus cells treated with Ad-GFP: *p < 0.05 and **p < 0.001. (C) C2C12 cells were transduced with Ad-lacZ, Ad-R6, Ad-PTG or Ad-G_M and incubated with 25 mM glucose for 72 h. Fixed cell monolayers were treated without (-) or with (+) alpha-amylase and glycogen stained with fluorescent PA-SH. A representative image is shown.

Figure 5

Glycogen particles. Cultured human myotubes were transduced with Ad-GFP, Ad-R6, Ad-PTG or Ad-G_M and incubated with 25 mM glucose for 48 h and then fixed and stained for observation of glycogen particles in transmission electronic microscopy. Representative images are shown: (A) image of myotubes obtained at × 40000 magnification, in which glycogen granules appear as dense spheroid particles, and (B) a detail of glycogen granules (arrows). Scale bars = 100 nm.

Figure 6

Diameter of glycogen particle. The graphics plot the diameter of each measured glycogen granule (at least 70 quoted granules for each condition) in cultured human myotubes transduced with Ad-GFP, Ad-R6, Ad-PTG or Ad-G_M and incubated with 25 mM glucose for 48 h.

Figure 7

Cytolocation of R6-EGFP. (A) 293 cells were transfected with 5 μg of plasmids encoding EGFP or R6-EGFP with the aid of GeneJuice, at 48 h post-transfection a Western blotting analysis was performed on cell extracts (30 μg protein) and membranes were hybridized with an antibody against GFP. (B) (a to f) C2C12 myoblasts were cotransfected with pR6-EGFP and pRFP1-N1-GalT, at 48 h post-transfection, cells were incubated with 25 mM glucose (a to c) or without glucose (d to f) for 16 h and a colocation analysis of EGFP and RFP was performed; (g to l) C2C12-mtRFP myoblasts were transfected with pR6-EGFP, at 48 h post-transfection, cells were incubated with 25 mM glucose (g to i) or without glucose (j to l) for 16 h and then colocation analysis of EGFP and RFP was performed. The image shows the fluorescent signal of EGFP (a, d, g, j) and RFP (b, e, h, k) and the colocation of the signal of EGFP and RFP (c, f, i, l).