PPP1R6 was originally described as a glycogen-associated PP1 regulatory subunit with wide tissue expression and most common in humans in skeletal muscle and heart . Here we report the lower expression of the PPP1R6 gene in human cultured myotubes than in skeletal muscle tissue. In this regard, PPP1R6 resembles PPP1R3C and differs from the PPP1R3B gene, whose expression is maintained in culture, and also from the muscle-specific PPP1R3A gene, whose expression is almost suppressed . Therefore, on the basis of our data from this and the earlier study , the relative differential gene expression is PPP1R3C > PPP1R3A > PPP1R6 > PPP1R3B in human skeletal muscle tissue and PPP1R3C > PPP1R3B > PPP1R6 > PPP1R3A in cultured human myotubes.
In cultured myotubes, PPP1R6 activated GS and reduced its phosphorylation at Ser-641/0, also referred to as site 3a, in a less powerful manner than PTG, but stronger than GM. GS may be phosphorylated at nine or more sites , but the Ser-641/0 site is crucial for the regulation of GS activity and is extensively regulated by various effectors . The selectivity of PP1 for the different sites of GS phosphorylation has not been established, but it has been proposed that PP1-GTSs may confer such selectivity . Our data provide evidence that the degree of dephosphorylation of the Ser-641/0 site achieved in response to PPP1R6, PTG or GM is tightly associated with that of GS activation, thereby suggesting a similar strong control of enzyme activity through the modulation of the phosphorylation of this site by these PP1-GTSs. The activating effect of PPP1R6 on GS was enhanced by glucose/glycogen depletion, as also observed for GM  but not PTG . In fact, glycogen content is a strong regulator of GS and high glycogen content impairs insulin-stimulated activation of this enzyme, as reviewed in . PPP1R6 did not exert any effect on the cultured myotube GP activity, either the endogenously expressed isoforms, which are predominantly the brain (PYGB) and liver (PYGL) isoforms , or the overexpressed muscle (PYGM) isoform. Like PPP1R6, neither does PTG affect endogenous myotube GP activity , whereas GM inactivates GP . As PPP1R6 markedly activated GS in myotubes, it exerted a potent glycogenic effect. This was seen by the increases in the rate of glycogen synthesis; by an increase in the net glycogen content, as quantified after KOH extraction and ethanol precipitation; and cytochemically, by an increase in the glycogen stained as blue fluorescent PA-SH material.
Glycogen accumulation in response to PPP1R6 was less than that achieved with PTG but higher than with GM. Cellular glycogen is complexed with proteins, including glycogen metabolizing enzymes, PP1 and PP1-GTSs , in the form of particles or granules, also named glycosomes [1, 2]. The morphology of these granules was originally defined by Drochmans  as α-, β- and γ-types. The α particles, commonly found in mammalian liver, are agglomerates of β-particles, have a rosette appearance and large diameters , in the order of 100 nm . Single β-particles are found in most normal tissues, including skeletal muscle , have a spheroid shape and diameters ranging from 15 to 40 nm. The γ-particles are about 3 nm in diameter and are subunits of β- and α-particles . Glycogen particle sizes in skeletal muscle cells have been consistently shown to follow a continuous distribution pattern. In human skeletal muscle, diameters range from 10 to 44 nm and a mean value of 25 nm at rest  and from 8 to 43 nm and a mean particle size of about 13 nm after exercise . In cultured rat myotubes, these particles range from above 15 to above 40 nm (mean value of 29.4 nm) in glycogen-replete cells or from above 10 to below 40 nm (mean value of 24.9 nm) in glycogen-depleted ones . Strikingly, here we show that, despite being in a continuous range, the size distribution pattern of glycogen particles is determined by PP1-GTS scaffolding, with average diameters in the order of PPP1R6 < GM < PTG. In control myotubes most glycogen particles corresponded to the β-type, with a mean diameter of 29.2 nm. No particles smaller than about 20 nm were observed, but a few above 42 nm were found. Notably, the detection limit for small glycogen granules is estimated to be 5-10 nm (at 20000 × magnification) , while the predicted maximum size limit of a granule is 42 nm, consisting of 12 tiers of carbohydrate . PPP1R6 generated small granules, with a mean diameter of 14.4 nm, with most of the granules falling below 20 nm and many between 7 and 15 nm. These granules resembled the γ-type in the formation of filamentous alignments . GM-derived glycogen particles showed an intermediate mean diameter of 28.3 nm and most were between 20 and 40 nm, which corresponds to the β-particle classification. PTG produced the largest particles, with a mean diameter of 36.9 nm and values ranging from 25 to 50 nm. Many of these particles looked like β-type ones, but we observed a few supramolecular clusters with a rosette-like shape that resembled α-particles. In fact, the larger α-type glycogen particles have not been observed in mammalian tissues other than liver . Since PTG is expressed abundantly in liver, in rats  and humans , we speculate that it induces the formation of α-particles in this tissue. In this regard, β particles are suggested to be covalently bonded to form α-particles through a hitherto unsuspected enzyme process operative in the liver , which could be tissue-dependent.
The number of glycogen particles was increased by the three PP1-GTSs tested. PPP1R6 and PTG induced the greatest increases in particle numbers and quantified glycogen accumulation. The rise in particle numbers per cell area for PPP1R6 was higher than with PTG, although quantification of extracted or cytochemically stained glycogen was not greater, probably because PTG-derived particles have bigger size and tend to form agglomerations of single β-particles. GM caused the smallest increase in the number of particles per cell area, which fits with glycogen quantification data. In fact, early muscle glycogen resynthesis has been shown to also rely on the synthesis of new particles. Elsner et al.  suggested that during early glycogen resynthesis in fasted cultured myotubes, new glycogen molecules are formed while there is a modest increase in glycogen particle diameter. Graham and coworkers  showed that, during recovery from prolonged exercise, resynthesis of muscle glycogen in humans is characterized initially by an increase in number and no change in particle size and later by an increase in particle size but not in number.
The subcellular distribution of glycogen-particles promoted by either PP1-GTS has distinct patterns. In PPP1R6-myotubes, granules were closely associated with cellular components. In GM-myotubes, granules accumulated near the endoplasmic reticulum, which is consistent with GM association with SR via an SR-binding sequence in its C-terminal region [8, 20, 21]; however, granules also appeared in other locations, preferentially subsarcolemmal ones. In fact, two forms of GM, an SR-associated form and a cytosolic glycogen-associated form, can be distinguished in rat skeletal muscle extracts . In contrast, PTG caused the accumulation of glycogen granules in large clusters located in central areas of the cytosol devoid of cellular organelles. In control cells, glycogen granules were distributed throughout the cytosol. Since in control myotubes the PPP1R3C gene is the most expressed gene (of PPP1R3A, PPP1R3B, PPP1R3C and PPP1R6 genes) and PTG is strongly glycogenic, it can be argued that glycogen particles in these cells are mainly PTG-derived, which may explain their preferential cytosolic location. Smaller granules were observed in control myotubes than in PTG-overexpressing cells, probably due to much lower glycogen accumulation in the former. We cannot rule out, however, a role for the still unstudied PPP1R3E protein. In the skeletal muscle tissue of humans [3, 41, 47, 48] and rats , electron microscopy studies have shown compartmental distribution of glycogen particles in three main regions: subsarcolemmal, intermyofibrillar and intramyofibrillar. In the subsarcolemmal location, clustered accumulations are observed at the level of mitochondria and SR , and nuclei and mitochondria . Aneurally cultured human myotubes are not fully differentiated, and likely this influences the pattern of cellular glycogen distribution. However, with PPP1R6 and GM we observed clustering of glycogen granules to the membrane of myotube organelles.
Finally, here we applied computational scanning of the PPP1R6 protein sequence to gain insight into its potential subcellular distribution. These analyses revealed a high probability that PPP1R6 is located at the Golgi complex or imported into the mitochondria. We thus analyzed the subcellular distribution of PPP1R6 tagged at the C-terminus with EGFP. The tagged PPP1R6 showed a diffuse pattern in the cytosol in glucose-replete and -depleted cells and a punctuate pattern clustered around the nucleus in glucose-depleted cells only. Since no colocation of tagged PPP1R6 with the mitochondrial-targeted RFP was observed, irrespective of glucose incubation, no support for the mitochondrial export premise was obtained. Neither was colocation of the tagged PPP1R6 with the Golgi-targeted RFP in glucose-replete C2C12 myoblasts. However, in glucose-depleted myoblasts the two proteins overlapped in the perinuclear region, according to the sequence-based prediction of PPP1R6 location in the Golgi complex. Therefore, our data suggest that PPP1R6 translocates in response to glucose from the Golgi complex to a cytosolic location, where it is associated with glycogen. Along this line of argument, PPP1R6 present in the glycogen/sarcovesicle fraction isolated from rabbit skeletal muscle is specifically associated with glycogen, since it is released by digestion of glycogen with α-amylase . The role that PPP1R6, or PPP1R6-targeted PP1, may exert in the Golgi apparatus in glucose-depleted myotubes is beyond the scope of this study. For instance, PP1 activity was previously reported to be essential in regulating vacuolar fusion and endoplasmic reticulum-to-Golgi and endocytic vesicular trafficking in the yeast Saccharomyces cerevisiae . Noteworthy, in muscle cells GS also shows differential intracellular distribution as a function of glycogen content. In skeletal muscle, GS translocates from a glycogen-enriched membrane fraction to the cytoskeleton as glycogen content is lowered  or it is found associated with spherical structures formed by actin cytoskeleton rearrangement after glycogen-depletion [52, 53]. In cultured C2C12 cells, muscle GS concentrates in the nucleus in the absence of glucose and translocates to the cytosol in response to glucose . These data suggest that after cellular glycogen depletion, some glycogen-associated proteins, such as PPP1R6 and GS, may translocate to diverse cell compartments.