The most important outcome of this study is to demonstrate that RGL is genetically linked to striated muscle glycogen accumulation but is not essential for insulin control of glycogen metabolism. Analyses of the knockout mice demonstrate that RGL directs phosphatase activity towards glycogen-metabolizing enzymes since GS activity is reduced and Ph activity is enhanced in the null mutant mice. Correspondingly, muscle glycogen stores are significantly reduced to only 10% of those in wild-type animals. However, despite the impaired glycogen accumulation, the animals remain normoglycemic and are insulin responsive.
Animals homozygous for disruption of the RGL
gene have a substantial (60%) decrease in overall muscle C1 protein and PP1 activity, indicating that a phosphatase containing RGL
represents a significant proportion of the enzyme in skeletal muscle. Conversely, targeted overexpression of RGL
in the skeletal muscle of mice resulted in an increase in the levels of C1 protein (22
; Y. Suzuki and A. A. DePaoli-Roach, unpublished results). These observations argue that the basal level of C1 protein depends on its association with regulatory subunits. Although the level of the protein can be controlled at different stages, we favor a mechanism involving stabilization of the protein. This hypothesis is supported by the observation that overexpression of C1 in COS1 cells produces mostly insoluble protein associated with lysosomal structures, whereas coexpression with either RGL
or inhibitor 2 (data not shown) generates higher levels of soluble and active enzyme. In fact, the proposed chaperone function of the phosphatase inhibitor 2 (3
) most likely reflects a similar phenomenon whereby formation of a cytosolic form of PP1, the ATP-Mg-dependent phosphatase, also stabilizes C1.
Our results raise some critical questions about the role of muscle glycogen in blood glucose homeostasis (19
). Glycogen deposition in muscle is widely perceived to be the primary fate of ingested glucose. With this premise, one might have predicted that animals impaired in their ability to accumulate glycogen in muscle would become hyperglycemic. This is not the case, suggesting that other mechanisms for glucose homeostasis must be operating. One important consideration, however, is the degree to which studies of mice are an accurate reflection of human metabolism and physiology, and we cannot exclude the possibility that skeletal muscle glycogen accumulation in humans has a different impact on blood glucose levels than in rodents. This caveat, of course, is valid for all of the numerous genetically altered mouse models that have recently been constructed to probe insulin action and whole-body glucose metabolism (for recent reviews see references 35
). Shulman and colleagues (56
) in fact have concluded that muscle glycogen is the predominant fate of ingested glucose in humans, at least under the conditions of hyperglycemic hyperinsulinemic clamps, and that impaired glycogen synthesis correlates with type 2 diabetes. They are also of the opinion that glucose transport is the rate-limiting step for glycogen accumulation, which is defective in the diabetic condition. The RGL
knockout mice, however, have normal basal and insulin-stimulated glucose transport but reduced glycogen content supporting the view that the glycogen-metabolizing enzymes play critical roles in glycogen accumulation (8
). It will be interesting to examine mouse models in which muscle glycogen accumulation is completely defective or in which insuling signaling to GS is specifically ablated. For example, genetic elimination of the novel insulin-stimulated GS phosphatase described in this work might achieve the latter goal.
Glucose is not being accumulated as excess fat, since lean and fat masses as determined by dexascan are similar in wild-type and RGL knockout mice, as is the respiratory quotient (VCO2/VO2) over a 24-h period (data not shown). Liver glycogen is not elevated, so the liver is not compensating. Since food consumption is no different between wild-type and RGL null animals, a simplistic question relates to the fate of the ingested calories. However, the apparent paradox is minimized if one considers that the caloric equivalent of the muscle glycogen in the mouse is less than 2% of a daily ~15-kcal intake, based on a glycogen content of 2.5 mg/g for a 25-g mouse. Therefore, muscle glycogen in mice does not actually represent a significant caloric reserve compared to food consumption. Glycogen is continuously used for local contractile activity and is replaced by incoming glucose, directly after meals, or indirectly from the liver via glycogenolysis or gluconeogenesis during fasting. The RGL null mutant mice appear to have a lower glycogen set point, consistent with GS being inactivated and Ph activated. As demonstrated by the normal basal and insulin-stimulated 2-deoxyglucose uptake, glucose enters the cells normally and presumably is stored as glycogen. However, hyperactive Ph may cause rapid degradation, resulting in a lower steady-state glycogen concentration. Thus, insulin stimulation promotes glucose uptake normally in the RGL knockout mice, and the overall lower glycogen content is most likely due to the altered activities of GS and/or Ph.
The studies with the RGL
-deficient mice clearly show that the muscle-specific PP1G, although important for basal glycogen accumulation, is dispensable for insulin-induced activation of GS. Together with our finding of an insulin-stimulated phosphatase both in the knockout and wild-type animals, there is compelling evidence that an enzyme distinct from PP1G/RGL
is responsible for activation of GS. Thus, the mechanism of an insulin-induced phosphorylation of RGL
and activation of the associated phosphatase can be discounted, consistent with a recent report (63
) that in rat skeletal muscle phosphorylation of Ser48 is not increased in response to insulin. Furthermore, since our initial communication (22
) that GS was activated by insulin in RGL
-deficient mice, other reports argue against a role for RGL
in insulin control of glycogen metabolism (43
). Our studies are not in agreement with the report that overexpression or depletion of RGL
in L6 cells affected activation of GS and glucose uptake by insulin (50
). The observation that neither insulin activation of GS nor basal or insulin-stimulated glucose uptake is altered in the RGL
null mice indicates that PP1G is not involved in regulation of glucose transport. Rasmussen et al. (51
) were also unable to reproduce the effect of overexpressed RGL
protein on glucose transport.
Recently it has been proposed that RGL
acts as a scaffold that binds both GS and C1 and that this association is required for GS activation and for β-adrenergic control of GS (42
). Our animal studies show that activation of GS can occur in the absence of RGL
, indicating that association with RGL
is not essential for control of GS activity in vivo. Furthermore, even in the RGL
null mutant muscle, all the GS is still bound to glycogen implying that RGL
is not needed for this association.
The work presented suggests that a phosphatase distinct from PP1G/RGL
mediates insulin activation of GS. Although the identity of this insulin-stimulated phosphatase is not known, it is likely to be a novel enzyme. We can exclude the possibility that it is a type 2A, 2B, or 2C enzyme, since it was detected in the presence of 4 nM okadaic acid and 2 mM EGTA, conditions that would inhibit the activity of those forms. Preliminary data indicate that the phosphatase activity was not detectable in the presence of 200 nM okadaic acid, suggesting that it is a type 1 enzyme. Obvious candidates would be enzymes containing the other two glycogen-binding subunits, PTG and R6. No regulatory mechanisms for these proteins have been identified so far. Overexpression of PTG increased both basal and insulin-stimulated GS activity (49
). However, we consider it unlikely that the insulin-stimulated phosphatase activity we detected is associated with PTG or R6, because their expression is not restricted to insulin-responsive tissues. In addition and most importantly, insulin was shown to stimulate the PTG phosphatase activity towards Ph (15
), whereas the insulin-stimulated phosphatase we identified appears to be specific for GS. Insulin does not affect phosphorylase activity, consistent with the inability to detect stimulation of phosphatase activity towards Ph (Fig. ). Neither PTG nor R6 functionally compensates for the loss of RGL
, since loss of RGL
results in low basal muscle glycogen storage and PTG and R6 protein levels are not upregulated in the knockout mice. The implication is that the various glycogen-associated phosphatases are not redundant, even though PTG and R6 are present in skeletal muscle at ~10% of the level of RGL
). Therefore, the insulin-stimulated protein phosphatase may be a form of PP1, involving association of C1 with some other regulatory subunit, either novel or already known in some other context.
Several studies over the last few years have attempted to link mutations in the human RGL
(PPP1R3) gene with type 2 diabetes. However, the frequency of the initially reported polymorphism at codon 905 (29
) was shown to be identical in normal and diabetic populations. More recently, adenovirus-mediated expression of Asp905 and Tyr905 human RGL
in L6 cells indicated no differences in glycogen synthesis (51
). Another substitution at codon 883 and a variant in an ATTTA motif in the 3′ untranslated region of the human RGL
gene have also been found (66
). Polymorphisms in the ATTTA element appear to correlate with lower expression of the protein, insulin resistance, and type 2 diabetes (65
). At least on the basis of our studies showing that mice lacking RGL
are neither diabetic nor insulin resistant, one might question whether subtle modifications of the human protein would cause an aberrant phenotype. Most notably, the small reduction in the level of RGL
observed in subjects carrying the ATTTA spacing polymorphism is unlikely to be responsible for the defective insulin responses associated with diabetes. Similarly, it is not likely that putative alterations of RGL
function in the other coding variants are critically involved in the etiology of type 2 diabetes.
is not required for insulin activation of GS or glucose transport, a key question is whether it is involved in other regulatory processes. Is it necessary or involved in the β-adrenergic control of glycogen metabolism? A main function of glycogen in skeletal muscle is to provide fuel for contraction. Since the glycogen content is extremely low in the RGL
knockout mice, one can ask whether these animals will be able to sustain exercise and whether RGL
is involved in the neuronal activation of GS, such as occurs during contraction and exercise. RGL
has also been proposed to control β-adrenergic-induced phosphorylation of phospholamban in heart (45
). Since this phosphorylation reduces the inhibitory effect of phospholamban on the sarcoplasmic reticulum-associated Ca2+
), it may be that the RGL
knockout mice have an increased cardiac performance, a phenotype observed in the phospholamban null mice (44
). Work is in progress to address these important and interesting questions.