The regulatory-targeting subunit (R GL , also called G M ) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/R GL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of R GL . To investigate the function of the phosphatase, R GL knockout mice were generated. Animals lacking R GL show no obvious defects. The R GL protein is absent from the skeletal and cardiac muscle of null mutants and present at ϳ50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by ϳ50% in the R GL -deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant R GL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by ϳ90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and R GL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that R GL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/R GL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.In recent years, the generality that the activity of the type 1 serine/threonine protein phosphatases (PP1) is dictated by the associated noncatalytic subunits has emerged. These ancillary proteins are thought to target the catalytic component (C1) to distinct subcellular locales in proximity to substrates, to confer specificity, and to regulate activity (10,21,33,41). To date, more than 30 C1-binding polypeptides have been identified that direct the enzyme to a variety of subcellular structures, including glycogen (6,24,25,49,59,60), myosin (2), ribosomes (31), nuclei (4, 13), and neuronal structures (5). A subset of C1-binding proteins includes inhibitory proteins such as inhibitors 1 and 2 (48, 67) and DARPP-32 (46). Four C1-glycogen-targeting subunits are presently known. R GL , also called G M , was the first glycogen-binding subunit of PP1 identified (59), and the corresponding holoenzyme, PP1G/ R GL , consists of the 124-kDa R GL protein (60) in association with C1. R GL is exclusively expressed in skeletal and cardiac muscle (37, 60). The NH 2 -terminal 240 amino acids contain binding sites for glycogen and C1 (64), whereas a hydrophobic region between residues 1063 and 1097 in the COOH terminus anchors the protein to membrane (45,60). Of the other three glycoge...

In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/ R GL (G M ) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J. Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R GL (G M ) knockout (KO) mice similarly to the wild type (WT).To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R GL KO and overexpressors (OE) were subjected to two models of contraction, in vivo treadmill running and in situ electrical stimulation. Both procedures resulted in a 2-fold increase in the GS ؊/؉ glucose-6-P activity ratio in WT mice, but this response was completely absent in the KO mice. The KO mice, which also have a reduced GS activity associated with significantly reduced basal glycogen levels, exhibited impaired maximal exercise capacity, but contraction-induced activation of glucose transport was unaffected. The R GL OE mice are characterized by enhanced GS activity ratio and an ϳ3-4-fold increase in glycogen content in skeletal muscle. These animals were able to tolerate exercise normally. Stimulation of GS and glucose uptake following muscle contraction was not significantly different as compared with WT littermates. These results indicate that although PP1G/R GL is not necessary for activation of GS by insulin, it is essential for regulation of glycogen metabolism under basal conditions and in response to contractile activity, and may explain the reduced muscle glycogen content in the R GL KO mice, despite the normal insulin activation of GS.

Non-fibre carbohydrate (NFC) rich diets are commonly fed in ruminant production systems. The objective was to determine whether NFC challenge affects proteasome activity, or messenger ribonucleic acid (mRNA) expression of proteasome subunits or apoptotic Bcl-2 proteins, in slow- or fast- twitch muscle of sheep. For 12 d prior to slaughter, lambs (n = 8) received either a control diet (28.4% of dry matter as grain), or a diet of increasing amounts of grain up to 79.1% of dry matter. A decrease in urinary pH (P = 0.01), base excess of blood and extracellular fluid (P = 0.01), bicarbonate (P = 0.03) and total carbon dioxide (P = 0.04), and an increase in anion gap (P = 0.07) in NFC lambs are indicative of metabolic acidosis. NFC lambs had significantly lower mRNA expression of the 20S -β subunit (P = 0.05), and a tendency toward lower mRNA expression of the 20S-α subunit (P = 0.11) and the 19S isoform (P = 0.15) in soleus, but not to the same extent in extensor digitorum longus (EDL) muscle. Downregulation of Bad mRNA expression occurred in both soleus (P = 0.10) and EDL (P = 0.08) muscle as a result of NFC challenge. These results indicate that NFC challenge does affect mRNA expression of genes related to the proteasome and apoptosis in a muscle specific manner. Key words: Ubiquitin-mediated proteolytic pathway, non-fibre carbohydrate, apoptosis, sheep