Glycogenin Protein Research Articles

Elevated GLUT4 and glycogenin protein abundance correspond to increased glycogen content in the soleus muscle of mdx mice with no benefit associated with taurine supplementation.

Duchenne muscular dystrophy (DMD) patients and the dystrophic mdx mouse have an elevated demand for ATP requiring processes, including Ca2+ regulation and skeletal muscle regeneration. As a key substrate for cellular ATP production, altered glycogen metabolism may contribute significantly to dystrophic pathology and explain reports of mild glucose intolerance. We compare the soleus and extensor digitorum longus (EDL) muscles of the mdx mouse during active muscle necrosis (at 28 days) and at 70 days where pathology is stable. We further investigate the impact of taurine (tau) on dystrophic glycogen metabolism to identify if the benefit seen with tau in a previous study (Barker et al. 2017) was in part owed to altered glycogen handling. The soleus muscle of 28‐ and 70‐day‐old mdx mice had elevated glucose transporter type 4 (GLUT4), glycogenin protein abundances and glycogen content compared to WT (C57BL10/ScSn) controls. Mdx tau mice exhibited modestly reduced glycogen compared to their respective mdx group. The EDL muscle of 28 days mdx tau mice had a ~70% increase in glycogenin protein abundance compared to the mdx but 50% less glycogen content. A twofold greater phosphorylated glycogen synthase (p‐GS) and glycogen phosphorylase (p‐GP) protein abundance was observed in the 70‐day‐old mdx soleus muscle than in the 28‐day‐old mdx soleus muscle. Glycogen debranching enzyme (GDE) protein abundance was elevated in both 28‐ and 70‐day‐old mdx soleus muscles compared to WT controls. We identified an increase in proteins associated with glucose uptake and utilization specific to the predominantly slow‐twitch soleus muscle of mdx mice regardless of age and that taurine affords no obvious benefit to glycogen metabolism in the mdx mouse.

Dysfunctional Muscle and Liver Glycogen Metabolism in mdx Dystrophic Mice

BackgroundDuchenne muscular dystrophy (DMD) is a severe, genetic muscle wasting disorder characterised by progressive muscle weakness. DMD is caused by mutations in the dystrophin (dmd) gene resulting in very low levels or a complete absence of the dystrophin protein, a key structural element of muscle fibres which is responsible for the proper transmission of force. In the absence of dystrophin, muscle fibres become damaged easily during contraction resulting in their degeneration. DMD patients and mdx mice (an animal model of DMD) exhibit altered metabolic disturbances that cannot be attributed to the loss of dystrophin directly. We tested the hypothesis that glycogen metabolism is defective in mdx dystrophic mice.ResultsDystrophic mdx mice had increased skeletal muscle glycogen (79%, (P<0.01)). Skeletal muscle glycogen synthesis is initiated by glycogenin, the expression of which was increased by 50% in mdx mice (P<0.0001). Glycogen synthase activity was 12% higher (P<0.05) but glycogen branching enzyme activity was 70% lower (P<0.01) in mdx compared with wild-type mice. The rate-limiting enzyme for glycogen breakdown, glycogen phosphorylase, had 62% lower activity (P<0.01) in mdx mice resulting from a 24% reduction in PKA activity (P<0.01). In mdx mice glycogen debranching enzyme expression was 50% higher (P<0.001) together with starch-binding domain protein 1 (219% higher; P<0.01). In addition, mdx mice were glucose intolerant (P<0.01) and had 30% less liver glycogen (P<0.05) compared with control mice. Subsequent analysis of the enzymes dysregulated in skeletal muscle glycogen metabolism in mdx mice identified reduced glycogenin protein expression (46% less; P<0.05) as a possible cause of this phenotype.ConclusionWe identified that mdx mice were glucose intolerant, and had increased skeletal muscle glycogen but reduced amounts of liver glycogen.

Glycogen and gold nanoparticle bioconjugates: controlled plasmon resonance via glycogen-induced nanoparticle aggregation

Hybrid nanosystems composed of glycogen biopolymers and gold nanoparticles were prepared by an in situ non-toxic synthetic procedure. Transmission electron microscopy (TEM) and various spectroscopic methods (optical absorption, X-ray photoelectron and photoluminescence spectroscopy) were used for the characterization of the obtained bioconjugates. The gold nanoparticles formed in the presence of glycogen were spherical in shape and approximately 15 nm in diameter. The TEM micrographs show that the nanoparticles aggregated on the surface of the glycogen biomolecules. This effect induced a shift of the surface plasmon resonance band towards higher wavelengths on an increase in gold ion concentration. Effective medium theory calculations were carried out in order to explain the observed redshift of the plasmon band in the absorption spectra of the Au–glycogen colloids. The interactions of the gold nanoparticles with glycogen strongly affected the photoluminescence of the glycogenin protein incorporated into its structure. The Au–glycogen hydrocolloids exhibited good chemical stability in the presence of saline, phosphate buffered saline, alanine, histidine and D-glucose.

Attenuation of Armanni–Ebstein lesions in a rat model of diabetes by a new anti-fibrotic, anti-inflammatory agent, FT011

A key morphological feature of diabetic nephropathy is the accumulation and deposition of glycogen in renal tubular cells, known as Armanni-Ebstein lesions. While this observation has been consistently reported for many years, the molecular basis of these lesions remains unclear. Using biochemical and histochemical methods, we measured glycogen concentration, glycogen synthase and glycogen phosphorylase enzyme activities, and mRNA expression and protein levels of glycogenin in kidney lysates from control and transgenic (mRen-2)27 rat models of diabetes that had been treated with and without a new anti-fibrotic agent, FT011. Diabetic nephropathy was associated with increased glycogen content, increased glycogen synthase activity and decreased glycogen phosphorylase activity. Glycogenin, the key protein responsible for initiating the synthesis of each glycogen particle, had very high levels in the diabetic kidney together with increased mRNA expression compared with control kidneys. Treatment with FT011 did not change glycogen synthase or glycogen phosphorylase enzyme activities but prevented both glycogenin mRNA synthesis and accumulation of Armanni-Ebstein lesions in the diabetic kidney. Armanni-Ebstein lesions found in diabetic nephropathy are due to aberrant glycogenin protein levels and mRNA expression, providing an explanation for the increased glycogen concentration found within the diabetic kidney. FT011 treatment in diabetic rats reduced glycogenin levels and, subsequently, renal glycogen concentration.

Glycogenin protein levels in sub‐cellular fractions of different muscle fiber types from stimulated rodent hind limb following changes in glycogen content

Glycogenin (GN) is bound to glycogen. At rest, GN distribution mimics glycogen (cytosolic/ membrane‐associated). GN may also co‐localize with actin. GN distribution across muscle fiber types following contraction was examined. One hind limb (40 F Sprague Dawley rats) was stimulated for 40 min to lower glycogen. Unstimulated and stimulated soleus (SOL), red (RG), and white (WG) gastrocnemius muscles were separated into sub‐cellular fractions [pellet 1/supernatant 1= P1/S1; S1 separated into S2 (cytosolic) + S3 (membrane/glycogen) + P3 (cytoskeletal)] and examined for total and unglycosylated GN levels. Tissue (corresponding unstimulated and stimulated) was grouped (n=6) by post‐stimulation glycogen level (WGI &lt;40; WGII &lt;100; RGI &lt;100, RGII &gt;100; SOL &gt;100 μmol▸ dry wt.‐1). At rest, ~50% of GN was located in each P1 and S1. In S1, ~4, 87, and 9% of GN was located in S2, S3 and P3 fractions, respectively. Post‐stimulation, GN increased in S2 and decreased in S3 and P3 fractions (P≤0.05). &lt;25% of GN in S2 was unglycosylated, suggesting re‐distribution of mostly small granules. In WGI, however, glycogen content decreased the most and the distribution of total GN (and glycogen synthase) in the P1 fraction increased. Low glycogen content in WG may lead to sequestering of GN (and GS) in the P1 fraction (potentially with actin) for glycogen re‐synthesis following acute stimulation in rodents. Funded by NSERC of Canada.

Glycogenin protein level in muscle fiber types of stimulated rodent hind limb following changes in glycogen content

Glycogenin (GN) may be degraded, may stay bound to partly‐catabolized glycogen or may form unglycosylated GN when glycogen content decreases. Oxidative and glycolytic muscle fibers may respond differently. One hind limb (40 Female Sprague Dawley rats) was stimulated to contract for 40 min to lower glycogen content. The soleus (SOL), red (RG) and white (WG) gastrocnemius muscles from unstimulated and stimulated limbs were examined for total and unglycosylated GN protein levels. Muscle tissue (corresponding unstimulated and stimulated) was grouped (n=6) by post‐stimulation glycogen content (WGI &lt;40; WGII &lt;100; RGI &lt;100, RGII &gt;100; SOL &gt;100 μmol▸g dry wt.−1). GN increased (P≤0.05) in WGI (66%), RGI (33%), and RGII (100%), but was unchanged in WGII and SOL. In WGII, GN decreased in a low‐spin myofibrillar pellet fraction and increased in the soluble fraction (no net change). Unglycosylated GN was detected in stimulated W and RG muscles (up to 25% of total GN) and was most abundant in WGI (lowest glycogen content). In summary, no correlation existed between GN level and glycogen content post‐stimulation either across fiber types or within a fiber type. No net degradation of GN occurred in either W or RG muscles in response to significant reduction in glycogen content; new GN was always synthesized. Pre‐existing GN is likely retained in smaller granules or as unglycosylated protein. Funded by NSERC of Canada.

Glycogenin protein and mRNA expression in response to changing glycogen concentration in exercise and recovery

Glycogenin (GN-1) is essential for the formation of a glycogen granule; however, rarely has it been studied when glycogen concentration changes in exercise and recovery. It is unclear whether GN-1 is degraded or is liberated and exists as apoprotein (apo)-GN-1 (unglycosylated). To examine this, we measured GN-1 protein and mRNA level at rest, at exhaustion (EXH), and during 5 h of recovery in which the rate of glycogen restoration was influenced by carbohydrate (CHO) provision. Ten males cycled (65% VO2 max) to volitional EXH (117.8 +/- 4.2 min) on two separate occasions. Subjects were administered carbohydrate (CHO; 1 g.kg(-1).h(-1) Gatorlode) or water [placebo (PL)] during 5 h of recovery. Muscle biopsies were taken at rest, at EXH, and following 30, 60, 120, and 300 min of recovery. At EXH, total glycogen concentration was reduced (P < 0.05). However, GN-1 protein and mRNA content did not change. By 5 h of recovery, glycogen was resynthesized to approximately 60% of rest in the CHO trial and remained unchanged in the PL trial. GN-1 protein and mRNA level did not increase during recovery in either trial. We observed modest amounts of apo-GN-1 at EXH, suggesting complete degradation of some granules. These data suggest that GN-1 is conserved, possibly as very small, or nascent, granules when glycogen concentration is low. This would provide the ability to rapidly restore glycogen during early recovery.

No limiting role for glycogenin in determining maximal attainable glycogen levels in rat skeletal muscle.

We examined whether the protein level and/or activity of glycogenin, the protein core upon which glycogen is synthesized, is limiting for maximal attainable glycogen levels in rat skeletal muscle. Glycogenin activity was 27.5 +/- 1.4, 34.7 +/- 1.7, and 39.7 +/- 1.3 mU/mg protein in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. A similar fiber type dependency of glycogenin protein levels was seen. Neither glycogenin protein level nor the activity of glycogenin correlated with previously determined maximal attainable glycogen levels, which were 69.3 +/- 5.8, 137.4 +/- 10.1, and 80.0 +/- 5.4 micromol/g wet wt in white gastrocnemius, red gastrocnemius, and soleus muscles, respectively. In additional experiments, rats were exercise trained by swimming, which resulted in a significant increase in the maximal attainable glycogen levels in soleus muscles ( approximately 25%). This increase in maximal glycogen levels was not accompanied by an increase in glycogenin protein level or activity. Furthermore, even in the presence of very high glycogen levels ( approximately 170 micromol/g wet wt), approximately 30% of the total glycogen pool continued to be present as unsaturated glycogen molecules (proglycogen). Therefore, it is concluded that glycogenin plays no limiting role for maximal attainable glycogen levels in rat skeletal muscle.

Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin

Glycogenin, a Mn 2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO 4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO 4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO 4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO 4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO 4-dependent activity. It is concluded that: (1) MnSO 4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO 4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.

Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli.

Glycogenin is a self-glucosylating protein involved in the initiation reactions of glycogen synthesis. Initiation occurs in two stages, requiring first the covalent attachment of a glucose residue to Tyr-194 of glycogenin and then elongation to form an oligosaccharide chain. The latter reaction is known to be catalyzed by glycogenin itself. The glycogenin sequence determined from the protein by Campbell and Cohen (Campbell, D. G., and Cohen, P. (1989) Eur. J. Biochem. 185, 119-125) was used to design oligonucleotide probes to screen a rabbit muscle lambda gt11 library. A cDNA was isolated that predicted an amino acid sequence identical to that of Campbell and Cohen, except that Cys residues replaced Ser-88 and Leu-97. Northern analysis indicated a strongly hybridizing message of 1.8 kilobases, present in most tissues including skeletal muscle, but much weaker in kidney and scarcely detectable in liver. A much weaker 3-kilobase message was also detected in muscle. Polymerase chain reaction was used to isolate DNA fragments encoding a portion of glycogenin from rat and cow. The sequence of this segment was > 90% identical at the amino acid level across the three species, indicating that glycogenin is a highly conserved protein. Using the pET-8c vector, the glycogenin protein was expressed in Escherichia coli. Incubation of the recombinant glycogenin with UDP-[14C]glucose and Mn2+ resulted in labeling of the glycogenin protein, indicating that the recombinant glycogenin was enzymatically active and capable of self-glucosylation. Furthermore, after incubation with UDP-glucose, the recombinant glycogenin could serve as a substrate for glycogen synthase, leading to the production of high M(r) polysaccharide. Therefore, production of functional glycogenin did not require the intervention of any other mammalian protein.

Physiological actions of taurine

Physiological actions of taurine