Glycogenin is considered essential for glycogen synthesis, as it acts as a primer for the initiation of the polysaccharide chain. Against expectations, glycogenin-deficient mice (Gyg KO) accumulate high amounts of glycogen in striated muscle. Furthermore, this glycogen contains no covalently bound protein, thereby demonstrating that a protein primer is not strictly necessary for the synthesis of the polysaccharide in vivo. Strikingly, in spite of the higher glycogen content, Gyg KO mice showed lower resting energy expenditure and less resistance than control animals when subjected to endurance exercise. These observations can be attributed to a switch of oxidative myofibers toward glycolytic metabolism. Mice overexpressing glycogen synthase in the muscle showed similar alterations, thus indicating that this switch is caused by the excess of glycogen. These results may explain the muscular defects of GSD XV patients, who lack glycogenin-1 and show high glycogen accumulation in muscle.

Differently from humans, rodents carry a single Gyg gene, which is expressed in all tissues (). This characteristic makes Mus musculus an ideal model in which to study the impact of glycogenin depletion on glycogen metabolism. To challenge the role of glycogenin, we generated a Gyg knockout mouse model (Gyg KO). Unexpectedly, rather than preventing the synthesis of glycogen, the absence of glycogenin caused glycogen over-accumulation in striated muscles. Remarkably, this glycogen was synthesized without the participation of a substitute protein primer. Furthermore, in spite of the higher muscle glycogen levels, these animals showed impaired endurance muscle performance, a phenotype similar to that of GDS XV patients.

In humans and most mammals, glycogenin is present in two isoforms: GYG1, which is widely expressed, and GYG2, which is predominantly expressed in the liver and to a minor degree in cardiac muscle and the pancreas. Recently, a new form of glycogenosis (GSD XV) resulting from GYG1 loss of function has been described. Patients with this condition present with glycogen accumulation and muscle weakness ().

Glycogen is a branched polymer of glucose residues that is stored and later released to meet energy demands. Skeletal muscle comprises a spectrum of fast-twitch glycolytic fibers, which use glycogen as the main source of energy for anaerobic metabolism to fuel short and intense activity (the extensor digitorum longus [EDL] is a muscle rich in this fiber type), and slow-twitch oxidative fibers, which are used for prolonged low-intensity activity (abundant in the soleus) driven primarily by fuels such as blood glucose and fatty acids (). It is generally accepted that glycogen synthesis is mediated by the action of two enzymes: glycogenin, the primer of the reaction, and glycogen synthase (GS), the elongator of the glucose chain. Glycogenin is a glycosyltransferase that catalyzes the addition of glucose residues to itself. The first glucose is transferred from UDP-glucose to its Tyr195 residue. The following glucose residues are then bound sequentially to form a chain of 10–20 residues (). Further chain elongation is performed by GS, and branches are introduced by the glycogen branching enzyme (GBE). GS interacts directly with the glycosyl-primer chain through the active site and also interacts with the 34 conserved amino acids of glycogenin’s C-terminal domain (). The interaction between GS and glycogenin is considered essential for glycogen synthesis.

The alterations observed in the Gyg KO mouse model could be a result of the absence of glycogenin or the increased accumulation of glycogen in muscles. To discern between the two possibilities, we generated an animal model with skeletal muscle-specific expression of a form of MGS that cannot be inactivated by phosphorylation (9A-MGS) ( Figure 4 A). In resting conditions, these animals accumulated 7-fold more glycogen in this tissue compared to their WT littermates ( Figure 4 B). PAS staining of muscle sections showed that glycogen was uniformly distributed and was mostly degradable by amylase treatment ( Figure 4 C). Interestingly, western blot analyses showed an increase in the quantity of glycogenin, paralleling that of glycogen. To explore whether this increased muscle glycogen affects exercise performance, we subjected mice to forced exercise on a treadmill. 9A-MGSmice reached exhaustion faster than their control littermates, despite the observed mobilization of muscle glycogen during exercise ( Figure 4 D). Indeed, as seen in Gyg KO mice, the net quantity of glycogen degraded after exercise was higher in 9A-MGSanimals than in controls ( Figure 4 B). We next analyzed mitochondrial respiration in fibers extracted from soleus and EDL muscles of 9A-MGSmice. As in the Gyg KO animal, the soleus of 9A-MGSmice showed reduced mitochondrial respiration compared to control littermates ( Figure 4 E). Taken together, the results from the 9A-MGSmice are consistent with the changes observed in the Gyg KO animals and suggest that the over-accumulation of glycogen (rather than the absence of glycogenin) is the underlying cause of the glycolytic switch in oxidative muscles.

All studies have been conducted in control and Gyg KO mice between 15 and 20 weeks of age, unless otherwise indicated. Data are expressed as mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

(E) Mitochondrial respiration indicated by oxygen consumption during different OXPHOS stages in the soleus muscle of control and 9A-MGS MLC1 mice. WT: n = 5; 9A-MGS MLC1 : n = 6.

(C) Glycogen over-accumulation in skeletal muscle fibers is shown by PAS staining in quadriceps from 9A-MGS MLC1 mice and control mice. A minor degree of glycogen accumulation is shown in cardiac muscle, characterized by lower expression of the MLC1 gene compared to skeletal muscle. Glycogen degradability is shown by PAS-D staining in serial tissue slices embedded in paraffin. Scale bar: 25 μm.

(B) Biochemical measurement of glycogen content in the skeletal muscle of mice in a resting state and mice at the point of exhaustion following the treadmill experiment. The top right panel indicates the decrease in glycogen after exercise. p values for the differences between glycogen values for each genotype/exercise groups and for their interaction were calculated using a general linear model. Rested WT: n = 5; rested 9A-MGS MLC1 : n = 4; exercised: n = 3.

(A) Western blot characterization of the 9A-MGS MLC1 mouse model: GS was detected in total tissue homogenate from skeletal muscle; loading control β-actin. Glycogenin was detected in α-amylase-treated samples from total homogenate of skeletal muscle; loading control β-actin.

To further characterize the metabolism in the two muscles, we measured mitochondrial respiration using high-resolution respirometry in isolated soleus and EDL. Once again, we identified a change in the soleus of Gyg KO mice, which showed lower oxygen consumption affecting all mitochondrial states. These lower levels were comparable to those of fast-twitch muscles (e.g., WT EDL). On the other hand, EDL respiration values were not altered in the Gyg KO, giving oxygen consumption values comparable to those of control muscle ( Figure 3 B). However, the differences observed in the Gyg KO soleus were not due to a decreased number of mitochondria ( Figures 3 C and 3D) or to a specific decrease in OXPHOS proteins ( Figures 3 E and 3F and Figure S3 E). We then quantified the adenylate energy charge in the two muscles. No differences were found for EDL, while Gyg KO soleus presented with lower AMP/ATP and ADP/ATP ratios than WT soleus, approaching those found in the EDL ( Figure 3 E).

We next tested the energy expenditure of the two genotypes in a resting condition by measuring Vand V Figure 3 A and Figure S3 A). Indirect calorimetric measurements indicated lower energy consumption in adult Gyg KO animals. Oxygen consumption was lower over the whole day and was associated with a reduction in glucose oxidation (prominent in the dark phase when mice are awake and more active) and diminished lipid oxidation (especially during the light phase when mice are asleep). We confirmed that the differences observed were not due to anomalies in body weight or the proportion of fat:lean body mass, which were both found to be equally maintained in the two animal models ( Figures S3 B and S3C). Moreover, food intake and locomotor activity were comparable ( Figures S3 A and S3D).

All studies have been conducted in control and Gyg KO mice between 15 and 20 weeks of age, unless otherwise indicated. Data are expressed as mean ± SEM.p < 0.05;p < 0.01;p < 0.001. See also Figure S3

(F) PAS and SDH staining in parallel sections of OCT-embedded soleus and EDL muscle of WT and Gyg KO adult males. SDH is used as a mitochondrial marker.

(D) Immunoblot for mitochondrial proteins from soleus and EDL muscle extracted from Gyg KO and WT adult males. Porin, Mfn2, and Tim 44. Tubulin was used as a loading control.

(A) Calorimetric parameters were obtained from metabolic cages in Gyg KO and WT animals. O 2 consumption, glucose oxidation, and lipid oxidation were measured during the light phase, dark phase, and overall. n = 7.

Isometric force generation after applying trains of stimuli at increasing frequency ( Figure 2 G) was recorded in Gyg KO and control muscles. Remarkably, Gyg KO soleus generated a force approximately 2-fold greater than its paired WT control at all the stimulation frequencies tested, while Gyg KO EDL responded with a force comparable to that of control littermates. These results suggest that glycogen accumulation in the absence of glycogenin specifically modifies the performance of the oxidative soleus toward a more glycolytic type, while no significant effect occurs in muscles with a pre-existing glycolytic metabolism, such as the EDL.

We next carried out ex vivo testing of the mechanical properties of isolated muscles representative of the two opposing metabolic and contractile types. Specifically, we tested the slow-twitch soleus muscle (rich in type I fibers, which use oxidative metabolism and serve to cover prolonged resistance activity) and fast-twitch EDL muscle (rich in type II glycolytic fibers, which use anaerobic metabolism and serve to sustain short, high-intensity activity). First, we verified that the muscle mass was maintained and that both soleus and EDL had increased glycogen in the Gyg KO model ( Figures S2 C and S2D). We also ruled out the existence of alterations in the proportion of fiber types in soleus and EDL by immunohistochemical analyses with antibodies specific for myosin heavy chain (MHC) I, IIa, and IIb (MHC IIX were quantified by exclusion of positive staining) ( Figures 2 E and 2F).

Both groups showed a comparable increase in blood glucose and lactate levels at exhaustion ( Figures 2 B and 2C). Although higher levels of glycogen remained in the muscles of Gyg KO mice at exhaustion, skeletal muscle glycogen diminished in both control and Gyg KO mice upon exercise, indicating that the poorer exercise performance in Gyg KO mice is not due to an impaired ability to mobilize glycogen ( Figure 2 D). In fact, the consumption of this polysaccharide was almost 2-fold higher in Gyg KO than in WT animals ( Figure 2 D, insert). This suggests an overall glycolytic shift in muscle metabolism.

To assess the consequences of glycogenin depletion on muscle performance, we subjected Gyg KO and WT mice to forced exercise on a treadmill increasing in speed, until exhaustion. A strong association between muscle glycogen levels and strenuous exercise performance has been previously described (). Unexpectedly, in spite of the higher glycogen level in Gyg KO muscle, these animals reached exhaustion earlier and covered a shorter distance compared to WT animals ( Figure 2 A and Figures S2 A and S2B).

All studies have been conducted in control and Gyg KO mice between 15 and 20 weeks of age, unless otherwise indicated. Data are expressed as mean ± SEM.p < 0.05;p < 0.01;p < 0.001. See also Figure S2

(E) Cross-sections of soleus and EDL muscle were stained for fiber type differentiation using antibodies against MHCs. All staining was performed on frozen sections. Scale bar: 250 μm.

(D) Biochemical measurement of glycogen content in skeletal muscle from mice in resting condition and mice at the point of exhaustion immediately following the treadmill experiment. The insert figure (top right panel) indicates the decrease in glycogen after exercise. p values for the differences between glycogen values for genotype/exercise groups and for their interaction were calculated using a general linear model. n = 6.

(C) Blood lactate was measured just before and after treadmill exercise. Statistical analyses were performed in R using the Wilcoxon/Mann-Whitney U test (non-parametric analog for two-sample t test) to compare variables of interest between WT and KO groups. WT: = 9; Gyg KO: = 8.

(B) Glycemia was measured just before and after treadmill exercise. Statistical analyses were performed in R using the Wilcoxon/Mann-Whitney U test (non-parametric analog for two-sample t test) to compare variables of interest between WT and KO groups. WT: n = 9; Gyg KO: n = 8.

Glycogen was purified from the skeletal muscle of Gyg KO mice and control littermates by digesting the tissues with 30% KOH under conditions in which the protein that was covalently bound to glycogen were not completely hydrolyzed. The resulting glycogen samples were repeatedly washed in order to remove the non-covalently bound peptides. Glycogen samples, which retained the covalently linked peptides, were then degraded with α-amylase or amyloglucosidase ( Figure S1 H). These two enzymes differ in their ability to act on the covalent bond between an amino acid and a glucose residue. While amyloglucosidase cuts the covalent link (meaning that no hexose should be found in the treated peptides), amylase is not able to cut the covalent amino acid-sugar bond, meaning that all the peptides covalently bound to glycogen should still carry at least one hexose ( Table S2 ). According to our experimental design, criteria for the identification of a glycogenin substitute in the glycogen purified from Gyg KO mice would be as follows: presence of peptide(s) originating from the primer in the samples treated with both amylase and amyloglucosidase, and at least one hexose residue in the sample treated with amylase. We identified the resulting peptides by mass spectrometry. The proteins to which they belong are listed in Table S3 and Figure 1 H. As expected, in the analysis of WT samples, only glycogenin fulfilled the selection criteria (i.e., was present in the two series). Strikingly, in the glycogen extracted from skeletal muscle of Gyg KO mice, no protein corresponding to both selection criteria was identified, indicating that glycogen is synthesized without a protein primer in these animals ( Table S3 ).

The presence of glycogen in the absence of glycogenin implies one of two possibilities: (1) glycogen is synthesized without a priming protein; or (2) another unknown protein replaces glycogenin in the Gyg KO mice. To address these questions, we designed a mass spectrometry-based approach to identify proteins covalently bound to the polysaccharide.

In several glycogen storage diseases (GSDs), such as Lafora disease (LD; EPM2, OMIM254780) and Adult Polyglucosan Bodies disease (APBD, OMIM263570), glycogen is poorly branched and accumulates in the form of amylase-resistant aggregates. However, in the PAS staining of Gyg KO mouse muscle and heart tissue, we observed a uniform distribution of the polysaccharide ( Figure 1 B). Furthermore, amylase treatment resulted in the complete degradation of glycogen (PAS-D). We also measured the degree of branching by analyzing the spectra of the glycogen-iodine complex. Gyg KO glycogen gave the same absorbance peak as commercial glycogen and purified glycogen from WT animals ( Figure 1 D). This indicates that glycogen synthesized by Gyg KO animals shows a normal degree of branching. We also characterized the size of the particles using size-exclusion chromatography (SEC). This analysis revealed that the glycogen granules isolated from Gyg KO muscle were larger than those purified from WT muscle and that they extended over a wide range of sizes, reaching up to a 4-fold greater radius than the particles in the WT animal ( Figure 1 E). Electron microscopy studies also showed large particles, which accumulated in the intermyofibrillar space of Gyg KO muscle ( Figures 1 F and 1G and Figures S1 F and 1G).

To analyze the impact of glycogenin depletion on the two key enzymes of glycogen metabolism, GS and glycogen phosphorylase (GP), we measured their levels and activity in the KO model. In skeletal muscle, the mRNA levels of GS and GP were equal to those found in the controls ( Figure S1 D), while an increase in protein level and activity was detected ( Figure S1 E). This indicates that neither glycogen synthesis nor degradation was impaired.

Unexpectedly, Periodic acid-Schiff staining (PAS) and biochemical measurements revealed that Gyg KO mice were able to synthesize glycogen ( Figures 1 B and 1C). In fact, both the liver and brain contained normal levels of this polysaccharide, while skeletal and cardiac muscle contained four and seven times more glycogen, respectively. Gyg heterozygous mice (carrying one allele of the Gyg gene) showed a moderate increase. Interestingly, the accumulation of glycogen did not increase progressively with age in any tissue ( Figure S1 C).

We generated homozygous Gyg KO mice by mating heterozygous mice (Gyg +/−) for the constitutive disruption on the Gyg gene. The genotype was confirmed by western blot and mRNA expression analysis of glycogenin ( Figure 1 A and Figure S1 A). The number of pups per litter was lower than expected, and the proportion of Gyg KO mice was only 4% (lower than the 25% predicted by Mendelian genetics) ( Figure S1 B). However, the genetic ratio of embryos at E18.5 was in line with Mendelian proportions. Indeed, most of the Gyg KO pups died shortly after birth due to cardiorespiratory failure.

All studies have been conducted in control and Gyg KO mice between 15 and 20 weeks of age, unless otherwise indicated. Data are expressed as mean ± SEM.p < 0.05;p < 0.01;p < 0.001. See also Figure S1

(H) Venn diagram reporting the number of proteins detected by mass spectrometry in each subgroup of the analyzed samples of glycogen purified from skeletal muscle (WT and Gyg KO), treated with α-amylase or amyloglucosidase.

(B) Histological localization of glycogen by PAS staining in the absence or presence of diastase (PAS-D) on paraffin-embedded slides of skeletal muscle, heart, liver, and brain from 15- to 20-week-old males. Scale bar: 250 μm.

(A) Immunoblot for glycogenin in the skeletal muscle, heart, liver, and brain of WT, Gyg +/−, and Gyg KO mice. Glycogenin protein is detectable only after treatment with α-amylase, used to degrade covalently bound glycogen and allow entrance in the polyacrylamide gel.

Discussion

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et al. Structural basis for the recruitment of glycogen synthase by glycogenin. Our results unveil new and unexpected aspects of glycogen metabolism and challenge the concept that glycogenin is indispensable for glycogen synthesis (). In addition to showing that glycogen is efficiently synthesized in animals lacking glycogenin, we further demonstrate that a priming protein is unnecessary for the synthesis of this polysaccharide. Furthermore, the finding that the striated muscles of Gyg KO mice display an over-accumulation of glycogen suggests that glycogenin depletion leads to a situation that is exactly the opposite of that expected.

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Hizukuri S. The influence of chain size and molecular weight on the kinetic constants for the span glucose to polysaccharide for rabbit muscle glycogen synthase. One of our most striking findings is that glycogen synthesis in Gyg KO mice proceeds without a priming protein. In fact, this phenomenon can be seen in other organisms, such as bacteria, yeast, and plants (), in which GS homologs act as de novo initiators. Our hypothesis is that glycogen is synthesized by GS in Gyg KO animals, starting from free glucose. In support of this, Salsas and Larner demonstrated that, in the presence of UDP-glucose as a co-substrate, purified muscle GS converts glucose to maltose (). Other authors showed that GS can convert maltose into maltotriose () and that the reaction may subsequently proceed to form oligosaccharides with an increasing number of glucose residues, with the Km decreasing progressively alongside the length of the acceptor chain ().

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Roach P.J. Glycogen biogenesis in rat 1 fibroblasts expressing rabbit muscle glycogenin. Unlike the glycogen aggregates seen to accumulate in Lafora disease (), the glycogen synthesized by the murine Gyg KO model did not form aggregates, demonstrated a regular degree of branching, and was hydrolyzed in vitro by amylase treatment. The latter is in accordance with it being metabolically active in vivo and degraded, at least in part, during exercise. Remarkably, glycogen particles from Gyg KO animals were larger than those from controls, as measured by chromatography and EM. The observation that Gyg KO muscle contains a higher quantity of glycogen than that of the WT control indicates that glycogenin may act as a regulator of glycogen content. There are various possible rationales for this. First, it is known that GS and Gyg interact strongly; therefore, glycogenin may modulate the amount of glycogen synthesized by acting on GS (). This idea is supported by our observation that the skeletal muscle and heart of Gyg heterozygous mice, which have reduced glycogenin expression, also accumulate more glycogen than WT animals. Second, Gyg could be important for the regulation of glycogen particle size, limiting the final volume of the particle. In favor of this hypothesis is our observation that the particles in Gyg KO muscle were larger than those present in WT animals. Furthermore, glycogenin overexpression in rat fibroblasts leads to a greater number of smaller molecules, rather than increasing glycogen production ().

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Saltin B. Diet, muscle glycogen, and endurance performance. MLC1 mice caused a similar defect in muscle performance, consistent with the observation in absence of glycogenin. Although we cannot rule out the different glycogen particle size as a cause of the reduced exercise resistance in the Gyg KO model, the recapitulation of this phenotype in 9A-MGSMLC1 animals supports the key role of glycogen accumulation in the regulation of muscle function. In contrast to the prediction that glycogen accumulation in Gyg KO mice would confer greater resistance to fatigue (), these animals reached exhaustion faster than controls when challenged with strenuous exercise. Importantly, simply generating an over-accumulation of glycogen in the muscle of 9A-MGSmice caused a similar defect in muscle performance, consistent with the observation in absence of glycogenin. Although we cannot rule out the different glycogen particle size as a cause of the reduced exercise resistance in the Gyg KO model, the recapitulation of this phenotype in 9A-MGSanimals supports the key role of glycogen accumulation in the regulation of muscle function.

Our results also support the idea that the greater availability of glycogen in the muscles of both Gyg KO and 9A-MGSMLC1 mice induces oxidative fibers to preferentially use this polysaccharide. This renders them more glycolytic, similar to bona fide type II glycolytic fibers, which primarily use glycogen as a fuel. The aforementioned metabolic switch occurs despite the absence of changes in fiber type proportions (as revealed by MHC analysis) or levels of mitochondrial markers (such as porin, mitofusin 2, Tim44, OXPHOS proteins, and succinate dehydrogenase activity: demonstrated by SDH staining). Nevertheless, Gyg KO animals display lower energy expenditure and low oxygen consumption in a steady rested state, indicating a lower glucose and lipid oxidation capacity. Indeed, significant changes in mitochondrial respiration indicative of impaired functionality were detected by measuring oxygen consumption in permeabilized muscle fibers from the soleus. Our current hypothesis is that the high availability of glycogen in Gyg KO soleus muscle affects energy production, impairing oxidative metabolism. This type of metabolism is particularly important when energy demands are high, such as during endurance exercise. In contrast, the high glycogen content found in the EDL muscle of this mouse model does not lead to any changes in metabolism, likely because these fibers normally utilize glycogen as their main energy source. Thus, a high degree of glycogen accumulation leads to an alteration of muscle function and oxygen consumption in a muscle that is predominantly reliant on oxidative metabolism. This translates into low resistance during prolonged, low-intensity activity.

Despite the high availability of degradable glycogen, both the Gyg KO and 9A-MGSMLC1 models showed a similar, low resistance to fatigue when forced to exercise on a treadmill until exhaustion. An alteration in mitochondrial respiration was also found in the 9A-MGSMLC1 mice, which may explain the poor endurance performance. On the basis of our findings, we conclude that the high glycogen content in the muscles of Gyg KO and 9A-MGSMLC1 mice decreases muscle endurance, against all expectations.

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et al. Cardiomyopathy as presenting sign of glycogenin-1 deficiency-report of three cases and review of the literature. Humans and most mammals carry two isoforms of the glycogenin gene, namely GYG1 and GYG2. GSD XV is a recently described rare human disease caused by GYG1 depletion. As mice express only Gyg1, the Gyg KO mouse constitutively lacks the protein throughout the body. Comparison of GSD XV with Gyg KO mice is therefore beneficial to provide new insights into the role of glycogenin and the physiopathology of GSD XV. Like these patients (), Gyg KO animals also accumulate glycogen in skeletal muscle and show phenotypical muscle weakness. However, while the glycogen in Gyg KO mice is entirely amylase sensitive, the glycogen that accumulates in GSD XV patients is accompanied by polyglucosans. This may contribute to the more severe muscle weakness phenotype in humans. Moreover, the Gyg KO model also shows glycogen accumulation in the heart. This may be of special interest regarding GSD XV patients, some of whom have been shown to suffer from cardiomyopathy and require a heart transplant ().

In conclusion, a lack of glycogenin does not prevent the synthesis of glycogen, but rather causes an over-accumulation of the polysaccharide in striated muscle, reflective of GSD XV patients. Although the over-accumulated glycogen can be mobilized, it leads to functional impairment and metabolic rearrangement. These observations offer a new perspective on glycogen synthesis and the role of the glycogenin-glycogen relationship in muscle physiology.